CA2566041A1 - T-type calcium channel splice variant compositions and methods - Google Patents

Abstract

Cell proliferative conditions are associated with expression of a previously unknown Cav3.1 T-type calcium channel splice variant, Cav3.1ac. Also, it has been determined that the Cav3.1ac T-type splice variant interacts with annexin III (ANX III). This interaction is useful in identifying substances to treat cell proliferation. Also, diagnostics and prognostics of cell proliferative disorders and methods for treating such disorders are disclosed.

Description

')C'-TYF~ CALCIUM CHANNEL SPLhCE YARIA~iT ~I~MP(~SrTIUNS AhTI~
M~THI~DS
Cross~ksference to Related Application [00111] This application claims benefit of U.S. F3tavisiQnal Application 601549,879 filed 10 lvxay 2004. The contents of this application are incorporated herein by reference in their entirety.
Technical Field [0002] The invention relates to Cav3.l calciunn channel splice variants and their involvement in cell proliferative c4nditions, and to a nov$1 interaction between a newly discovered c~a,,3.1 calcium channel splice variant and annexin Bf. The invention also relates to methods and composition for pragnosing, diagnosing and treating cell proliferative conditions.
Sacksround [OD03] 1~"rimary l7rain tumors are classified according to the presurrted cell of origin. Glia-derived brain tumors are categorized as glianna and acCOUnt far the majority of primary brain tu~nQrs (Sutherland et al., 1987). High-grade gliorna are often associated with a poor pxognosis, as the tumor may proliferate rapidly, invade neighboring areas of the brain and often recurs following surgical resection andlor radiotherapy (reviewed in Walker and I~ye, 2001).
Tumor fornnation alters genies involved in cell proliferation, difFexentiation, migration arid apoptosis. Ion cf~annels are important mediators of such functions and recent findings eoni~irmed are, altered regulation and expression of chloride, patassiurn arid sodium channels in gliorna (0lsen et al., 2003, Ransom et al., 20(12, Schrey er al , 20D2).
[0004] Calcium channels, mostly T-types, are also regulated during cell differentiation and tumor fbnnation (l3ertolesi et al., 2.003, Chemin et cxl, 2002, I~iroaka ea aL, 2002, Mariot er al" 20U2, Toyota et al.,1999). To date, tkucee different genes encoding distinct cx!
subunits of the T-type channel have been identified (Ga"3.1, Ca"3.2 and Ca,,3.3) (Cribbs et al., 1998, Lee et a~, 1999, l~cltory et rcl., 2001, Perez-Reyes et al., 1998). Alternative splicing generates additional isafaxms of these channels with distinct biophysical properties such as kinetics and voltage-dependence of activation, inactivation and deactivation (Chemin et al., 2f~ia, Monteil ez al., 2000}.

[OOUS~ Ca"3.1 channels ara expressed abundantly in CN5 neurons and were recently shown to be ptpsent in 2~strocytes (Klugl~auer et aL,1999, Latour et at., 2Cl113).
However, their function in astrocytes remains poorly understood.
[OU06j The function and rnetnbrane expression of a nutttber of higlx voltage-activated calcium, channels is regulated by interaction with other Cellular proteins. FQr example, N- and P!Q-type channel function is regulated by synaptic proteins, such as syntaxin, SNAP-25 or cysteine string protein (88,89), (U.S Pat Nos. 5,23,051 and 6,Q94,631). L.-type channels are rztQdulated through interactions witk~ calmadulin (Liang, H., DeMaria, C.D., Erickson, M.C'.r., lVXori, M.X., Alseikhan, B.A., and'Yue, D.T. 2003. I3nified mechanisms of Ca''"' regulation across the ~az'~ channel family.
,Neuron 39, 951-9ti0.) as well as A-Kinase anchoring proteins (Alder, C., Dubel, S.J., Earrexe, C., Jarvis, S.E., Statz, a,~., Spaetgens, R.L., Scott, .T.D., Carnet, Y., De 'Waard, M. ~amponi, G.W., NargeQt, J., and Baurinet, E. 2002 Traf>:<aking of ~.-type calcium channels mediated by the gostsynaptic scaffolding pz~otein AR.Ai'79. J. ,i3ia1 Chem. 277 33f98-336113), The close association of calmadulin with the channels 2~Llows channels to directly activate calciumlcalznodulin activated signaling cascades (Dalmetsch, It"E., Pajvani,1(3., Fife, I~., Spans, J.M. and Cn-eenbert, Irr.C.E. 21101.
Signaling to the nucleus by an L-type calciuttt channel-calmodulin complex through the MAP kinase pathway. Science 294, 333-3~9.). To date, with the exception of G protean ~3z subunit interactions with ~a"3.2 (Wolfe, J.T., Wang, kL, Howard, f., Garrison, J.C., and Barren, P.C,). 20U3. T-type calcium channel regulation by speck g-protein betagamma subunits. Nature 424, 2119-213.), proteins interacting with T-type channels have not been clearly identified.
Disclosure of the Invention tOtl~~l] In humans, three isofoems of the T Type (Cav3.1) calcium channel a, subunit have been reported as a result of alternate splicing of exans 25 and 26 in the ITt-N
linacer region (Cav3.1a, Ca,,3.lb or ~Ca"3.lbc). According to the invention, human gliomas express Cav3.1 channels in situ, splicing of these exons is uniquely regulated, and there is expression of a glioma-specific novel T-type variant (Gav3.lac). As discussed below, seven human glionta, samples were. collected at the time of sutgexy, ftNA was extracted and cDNA was produced forl~.T-1'CIt analysis. In addition, three glioma cell lines (U87, IJ~63 and. U251N), primary cultures of humian fetal astrocytes as well as adult and Fetal human brain cDN'A were used. Previously described Ca"3.1 splice varianks were present in glioma samples, cultured cells and whole brain. The results xevealed that in the normal adult brain, Cav3.la transexipts predominate while Ca"3.1b is ntostiy fetal-specil~e_ 'V'itT-PCR

results an gtlama and gliotna cell lines showed that Ca"3.1 expression in tumor cells resembles fetal brain expression pattern as Cav3.lbc is pcedorninantly expressed. In addition, a novel splice variant, Cay3.1 ac, was identified, as expressed in three glioma biopsies and one glioma cell line, but not in normal brain or fetal astrocytes. Transient expression of this variant revealed that Ca,,3.lac displays similar current-voltage and steady-state inactivation properties compared to Ca"3.1b, but a slower recavexy from inaCLlvatlan. Io addition, a navel interaetio~n between the Cav3.lae splice variant and annexin III was discovered. Thus, provided is a xnechanisrn which can be targeted fc~r tumor growkh inhibition.
(OOOSj It has been discovered that Ca,,3.1 splice variants axe associated with cell praliferative conditions, such as brain cancers, gliatna, breast cancers, eye cancers and retinablastoma.. Far example, Cay3.1 splice variant rr~NA and protein are present in glioma sales and giioma cell lines. It alsr~ has been discovered that gliainas express a splice variant of Cav3.1 that is normally mainly found in fetal cells (Ca"3.1b~, as well as a. navel, gliama-specific Cav3_1 splirx isaform (Ca~3.lac). l;lectmphysiological oharacteriaation of Ca,,3.lac indicates similar gating characteristics to other ~av~.1 splice isofonns. In addition, it was discovered that the protein annexin 1II (ANA III interacted with a T-type calcium channel.
[0009] Thus, provided herein is a method for screening cells irr a human for abnarnral expression of Cav3. 1. T~type isoforms, thus deterniining the turnorigenic gatential of the cells. one aspect is a method far detecting a risk of, or the presence of, a cell prolifexative disorder in a subject, which oonlprises determining the presence ar absence of an abnormal expression of Ca"3.1 T-type isafoxms, whereby said abnormal expression indicates the risk or ~resenae of this disorder. ~y "expression" of a Ca"~.1 splice variant or isofonn is meant the presence of nxR.l~A encoding the variant or isofarzn and/or the presence of the protein itself. The expression levels of these splice variants can be datermi~aed in sannples comprising cells taken from the subject. The simples may be biopsies, tissues, ar circulating cells. The presence of rnRNA carr he detected try a variety of methods, including prlar formation of cDNAvnd hybridizatia~n or I~T-P'CR.
Narthvern Blot rnay also be USed. ~~e presence 4f the pratain may be dcteated in sorr~c instances by iruuaunoreaction with antibodies specific far the isaform.
[Qpl4] At least three indicaiior~s of abnormal expression may be assessed. In one aspect, the determination is rr~de of ttte presence car absence of an isofarm uniquely associated with abnornsally proliferative cells, such as gliornas, i.e., the Cav3.iac form. In another method to determine abnormal expression, the distribution of isaforms is determined whereby the frresence of an abnormally high percentage of the Cav3.lbc form is indicative of the risk or presence of a cellular proliferative condition. The presence of the Cav~.lb fo~n is also an indication. zn these, embodiments, the cell proliferative disorder sometimes is selected from the gz°oup consisting oi= brain cancer, glioma, breast saucer, eye cancer and retinoblastoma. °
[0011.] The methods described above sometimes further comprise determining whether thez'e is increased cell pmliferatio~n within a subject identified as having the presence of a nucleotide sequence that encodes a Cav3.1 ae calcium channel or the presettce of a Cav~.1 ac calcium channel protein in cells. The presence or absence of increased cell proliferation sometimes is detected in a tissue biopsy front the subject, and sametirnes increased cell proliferation is detected ire vlvo. The previously described methods sometimes further comprise administering a molecule that reduces cell proliferation in a subject identified as having the presence of a nucleotide sequence that encodes a Ca"3, l ac calcium channel, the presence of a Cav3.1 ac calcium channel protein in cells, andlar the presence of increased cell proliferation, in an amount effective to reduce the cell proliferation. The molecule that reduces cell proliferation in the subject sometimes is a Cav3.lac calcium channel antagonist molecule andlcr a molecule that inhibits an interaction between a Cav3.iac Calcium channel and ,AhT~~ III.
[0012] ,Also provided is a rr~ethaci. foc creating a cell proliferative disorder chaxaeteri~ed by expression Ca"3.1 ae, which comprises adnninistering a T-type calcium channel blocker to a subject in need thereof in an amount effective tv treat the cell proliferative disorder. Specific embodiments are directzd to a metttad for treating a cell proliferative disorder in a subject, which comprises administering a Cav3.1 ac calcium channel antagonist molecule or a molecule that inhibits the interaction of a Cav3.lac calcium channel with ANX iir to a subject in need thereof in an amount effc~tive to treat tyre cell proliferative disvrdef. These substances may be used in the preparation of rnedicaxnents for such treatment as well, [QQL3] Provided also is a composition which comprises a cancer cell in combination with an antibody that specifically binds to a Ca~r3.lac calcium channel protein, or a synthetic nucleic acid C4mpPfSi1'l~ a nucleotide Sequence complementary to a polynueleotide sequence in a cancer cell nucleic acid that encodes a Cav3.lac calcium channel. The synthetic nucleic acid sometimes is linked to a sokid sugpor<, and the nucleic acid sometimes is arranged on the solid support in an arxay.
.A.lsa provided is a composition which comprises a cancer cell in combination with a Cav3.lae calcium channel antagonist molecule or a molecule that inhibits the interaction of a Ca,,3.1 ac calcium channel with AhTX iII. Also featured is a composition which comprises an isolated Ca~,3.lac calcium channel-encadir~g nucleic acid or protein and 2rzt isolated ANX 17I-encoding nucleic acid or protein.

[004] Also provided are a variety of methods and compositions related to screening cornpaunds for the ability to inhibit tl~ interaction between Ca~'J.Iac arid ANX
IIh(e.g., by a compound's ability to bind to a selected Cav3.lac-like peptide). One aspect is a. method for identifying a molecule that inhibits cell proliferation, which comprises contactirAg ozte or mare cells comprising a Ca~3.lac calcium channel encoding nucleic acid andlor a Ca"3.l.aa calcium channel polypeptide with a test molecule, and determining whether the test molecule decreases cell proliferation, whereby a test molecule that decreases cell proliferation is identified a.~ a molecule that inhibits cell prcsliferation.
Another aspect is a zrzethad of screening for a molecule that it~tiEaits cell proliferation, one of which comprises: (a) incubating a Ca"3.lac palypeptide or substantially identical polypeptide thereof with a test molecule under conditions sufficient to permit binding between tlae polypeptide and the test molevule irr a reaction mixture, (b) contacting A~ III with the xeaotion mixture antler conditions sufficient to permit binding between the polypeptide and ANX T.>I, and (c) detecting the laresence or absence of deat~ased binding between the polypeptide and ANX 1TI, whereby the preseneE of decreased binding between the polypeptide and ,A.l~l~ III identifies the test molecule as a molecule that inhibits tl~e interaction between Cav3.I ac and ANX III. Analogous methods are described for detecting compounds that inhibit the intet'action of ~-type calcium channels with syntaxin and 51'TAP-25 in U.S. Pat Nas. 5,623,051. and 6,090,631. A ~a"~.lac polypeptide is a polypeptide that is unique to this isoforrn. The Car3.lac polypeptide sometimes comprises ~5 ox more sequential amino acids selected from a region spanning amino acid 156 to amino acid 1570 of a Ca"3.Iac T-type calcium channel, and the polypegtide sometimes consists of the amino acid s~uence SK.E1~(~MAIaL.h~C.DDYIAS~S$ASAAS.
~1101~j ri~ethods and compositions described herein sometimes pertain to a Ca,,3.lb calcium channel instead of, or sometimes in addition ta, a ~aV3.lac calcium channel.
For example, methods for detecting a risk or presence of a cell proliferative condition in a subject sometimes is perforEned by detecting the pz~esence of a Ca"3.1b calcium channel encoding nucleotide sequence or Cay3.lb calcium channel protein in a sample instead of, or in addition to, detecting the presence of a Cay3. l ac calcium channel component. The Ca~3. i b calcium cha~anel sotr~titnes is targeted in other eutbodiments, such as in a method for treating a cell prolifexative condition by administering a Cav3. x b calcium channel antagonist to a subject in need there~af in an amount cuff eient to treat the cell praliferative condition instead af, ar in addition to, administering a Ca"3.lac calcium channel antagonist.
[p01~6] These and other aspects of the present invention are evident upon reference to the following detailed description and attached drawings.
Brief Description of the Drawinss [iJa~.7] Figures 1A td lE depict immunofiuorescen( a studies showing Ca"3.1 calcium channel expression in glioma. Irnmunocytochemistry of Ca"3.1 channels on T~251N
glian~a. cells is assessed by confoca! microscopy at40~ (Figure lA) and lkl0~'. (Figure 1B). A paraffin-embedded section of a malignant astracytorna showing OFAZy expression is,in Figure 1C and showing Ca,,3.1 channel expression is in Figure 1D. Coexpressian of T-type channels on GFAF-stained astroeytes is shown in Figure 1E.
loty~.~) Figures 2A and 2.f3 shaves that specific isQforms of the Cav3.l ~I-IV
linl~er generated by alternative splicing of exon 25 and 26 are differentially expmssed in the human brain at various develapme~ntal stages and in glioma. Figure 2A is a schematic rept'esentation of alternative splicing mechanisms in the Ca"3.1 FII-ATV linker. Inh~an-exon boundaries and splice donor and acceptor sites are shown in lower case letters, artlino acid sequence is shaven in upper case lettering. The sequences at tlae beginning of exans 27 and 26 are shown. The entire axon 2G
contains ttae amino acid sequence LML.DDVIASGSSASAAS, such that inclusion of this exon results in a 50 by increase, as shown in Figure 2B. Fxan 2S can either produce a long (CaV3.la) ar short isofarm (Ca"3.1b), wktile exon 26, when present, gives rise t4 two additiox~a.!
variants (Cav3.lac or Ca~3.lbc). Figure 2B is an agamse gel showing Iii-N linker 1tT-PCIZ results in the normal adult (lane 1), fetal brain (lane 2), u251.'bT gliorna cells (lane ~) and a glioma sample (lane 4). The first lane represents the molecular weight marker (MW weight is indicated by armws)_ [~Ox9~ Figures 3A to 3D show biophysical properties of Ca"3.lac (~r) and Cav3.lb (4). Error bars reflect standard errors. Figure 3A shows current-voltage relationships far Ca"3.lac (n=13) and Ca.V3. l b (n=10) vax-iants. The data were fitted with the Boltzmanri relation (solid line). Figure 3l~
shows steady-state inactivation relationships, fitted with the Baitztx~ann relation. Far CaY3.lac (n=11) and Ca"3.1b (n=8). Note that there is no difference in slope or half inactivation potential with, these two channel isafarms. Figure 3C shawl time constants far inactivation at various test potentials. The time constants were obtained by tnonaexpaztential fits to fla;
raw data. Only at +2U
mV is there a statistical differez~oe between the two channel isofarnrs (asterisk). A total of 13 and 10 experiments are included far the ac and b variants, respectively. Inset:
Current records obtained from CaY3.16 and Cav3.lac, elicited by a 150 ms step depolarization. The cumenks were scaled to overlap at peak, the peak curxent amplitudes of the raw two currents, were, respectively 6$0 pA and 574 pA. Note the similar inactivation kinetics, Figure 3D depicts a time constant of recovery from inactivarion. The data were obtained by using an inactivating prepulse followed by a test depolarization at various recovery intervals. The data normalized and fitted rnonoexpanetially. Note that the ac variant shows an increase in the time canstar~t of recovery from inactivation. Ca"3.1b:
°r=1~0C1.(IOrns(n= 9), Cav3.lac: ~15A~.52rr~s (n=9).
~acrza] 1~igures ~4A antl X13 illustrate Ca,,3.1 III-IY linker splice isaform distribution in normal brain, gliama cell lines and glioma samples. lligure 4A shows the nutxaber of clones far each canditiota corresponding to the various isoforms. Figure ~4B is a schematic representation a~ the percentage of each isaform in fetal and adult brain, fetal astracytes and gliarrta (gliazna cell lines and ,glioma samples combined).
[0021] Figure 5 shows the identification and confirmation of annexin IIf as a liinding partner of tk~e Ca"3.lac splice isoform IIl-kV' linker.
Detailed Description ' [0022] The present invention offers several strategies for pmgnosing, diagnosing and treating cell prolifez~ative conditions. .1'n prognostic and diagnostic methods, Cav3.
l splice variant-encoding nucleotide sequences ar proteins are detected in a sample from the subject, and such methods sometimes are coupled with furthex diagnostic inforrxLatian andlor procedures, andlor treatment pxacEdures. In methods of treating cell proliferative disorders, T-type calcium channel bkyckers can be used to prevent entry of eaiciurn into mitogenic cells, thus preventing initiation of cell proliferation. Alternatively, a molecule that inhibits the interaction between Cav3.lac and ANA III
can tae administered to reduce cell proliferation. Ca"3.1 splice variant antagonists, such as antisense molecules, ribozyrnes, si~IA molecules, compounds or antibodies directed specifically to Cav3.lac ar C:av3.l,b splice variants, can also be adtrAinistered to reduce cell proliferation. Alternatively, A1VX
ZXI antagonists cane be administered to reduce cell proliferation and treat Gel1 praliferative disorders.

Itx certain embodiments, the cancer cell is from a brain tumor ar glioma, and in other embodiments the cancer cell is Pram a breast cancer tumor or eye cancer tumor (e.g., retinablastanraa).
[0023] ,A characteristic of the cells of the invention that exhibit undesired proliferation is the presence trf abnormal expression of at least one Cav3. l calcium channel splice variant. This abnormal expression can manifest itself in a number of ways so as to meet the definition of "abnormal expression.°' 1~irst, the presence of the sglice variant Cav3.lac, either in the farm of m~NA or protein or bath, is indicative of such cells and of such abnartttal expression. Second, the presence of the splice variant Cav3.lb also indicates abnormal expression;
such expression is represented by either dr both the presence of tnRiV'.A ar protein corresponding to this splice variant.
In addition, the proportion of the total CaY3.1 expression attributable to Cav3.lbc indicates abnormal expressxnn if the cells are non-fetal cells and the percentage measured as a mole percent o;F mRlwdA
or pr~rtein is greater than 20%n of the total Cav3.1 calcium channel mRNA or protein level. i These percentages are an a male basis.
Cantaositions [U02~j liven the association of certain Cav3.1 splice variants with cell praliferative disorders, provided is a composition which comprises a cancer cell in combination with an anti>~vdy that specifically binds to a Cav3.1 calcium channel protein splice variant associated with the cancer (s.y., a ~Ca."3.1 ac ar ('av3.lla caloium channel protein). Examples of antibodies are described hereafter. In some embodiments, the cancer cell is intact, is in association with Other cells (e.g., the cancer cell is in a cell culture or in a tissue) or is separated from other cells (e.g., the cancer cell is dispersed in a liquid medium). In other embodiments, the cancer cell is not intact (e.g., it is a cell lysate). The cancer cell sometimes is isolated from a subject having a'cell praliferative condition, sometimes is isolated from a tissue of the subject, and sometimes is isolated from ar is part of a cell Lire Established from a subject having a cell praliferative condition.
X11025] Cell prolifexative disorders also include but are not limited to cancers of the calarectum, bmast, lung, liver, pancreas, lymph node, colon, prostate, brain, head and r~eclc, skin, liver, kidney, and heart. The cell praliferative condition sometimes is a brain cancer such as glioma., sometimes is an eye cancer such as rctinablastoma, and sametimcs is breast cancer. A cell prali~Grative condition sometimes is a hematopoietic neaplastic disorder, which is a disease involving hyperplastic/neoplastic cells of hematopaietic origin (e.g., arising from tltyeloid, Lymphoid or erythroid lineages, or precursor cells thereof). The disease can arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryabIastic leukemia.
,Additional myeloid disorders include, but ate oat limited ta, acute promyeiaid leukemia (APML), acute myela,genaus leukemia (AML) aid chronic myelogenous leukemia (CML) (reviewed in Vaickus, Grit. l~ev_ in OncolJHernatol. 11:267..97 (1991)); lymphoid malignancies include, but are oat limited to acute lymphoblastic leukemia (ALL), which includes B-lineage ALL and T-lineage ALL, chronic lymphacytic leukemia (~LL~, gralymphacytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrorn's macroglobulinemia (WM). Additional farms of malignant lymphomas include, but are riot limited to non-.~Iodgkin lymphoma and va~ciants thereof, peripheral T
cell tymphaxnas, adult T cell leukemia/iymphoma (ATL), eutaneous T-cell lymphasna (CTCL)> urge granular lymphacytic leukemia (Ltig'), Hadghin's disease and Reed-Sternherg disease.
[O(126~ Tn certain embodiments, the composition catnprises a cancer cell and a synthetic nucleic acid comgnising a nucleotide sequence complementary to a palynucleatide ser~uence in a cancer cell nucleic acid that encodes a Ca,~3.1 calcium channel splice variaa~t associated with a cancer (e.,g., a Ca,,3.lac or Ca~3.l~ calcium channel). As used herein, the term "nucleic acid"
includes DNA
molecules (e_~., a complementary DNA (eDNA) and genamic DN'A (gDNA)), RNA
molecules (~.g., rr~RNA ar siRNA) az~d analogs c~f DNA ar RNA (e.g., RNA or DNA comprising or consisting of nucleotide analogs). The ~nueleic acid molecule sometimes is single-stranded and often is dnuble-stranded. The nucleic acid sometimes is a fragment, which sometimes is 50,100, ar 200 or more base pairs in length, and is sometimes about 300, 40f?, 500, 600, 700, 800, 900, 1400, 1 i 00,1200, 1300, ar 1400 base pairs in length. An example of a nucleic acid fragment is a~n oligonucleatide. As used herein, the term "oli~cnttcleotide" refers to a nucleic acid ca;mprising about $ to about S0 covalently linked nucleotides, often eornprising from about $ to about 35 nucleotides, and mare often Exam about 10 to about 25 nucleotides. The backbone and nucleotides within an aligonucleatide rnay be the same as those of naturally occurring nucleic acids, ar analogs ar derivatives of naturally occurring nucleic acids, provided that oligonucleatides having-such analogs ar derivatives retain the ability to hybridize specifically to a nucleic acid comprising a targeted polytrzorphisrn. c~ligc~nucleatides described herein may be used as hybridization probes or as components of prognostic or diagnostic assays, for example, as described herein.
[O~D~7] Oligonuclentides often are syxwchesiaxd using standard methods and equipment, such as the A13ITM39001-Tigh Ttn~oughput DNA Synthesizer and the EDTTETM89o9 N'uafeic Acid Synthesizer, bath of which one available from. Applied Biosystems (Foster City, CA). Analogs and derivatives are exemplified its LT.S. Fat. Nos. 4,469,863; 5,535,$21;
5,541,306; 5,637,683;
5,637,fr84; ~,70U,922; S,7f'7,~83; 5,719,262; 5,739,308; 5,773,401; 5,886,1 b5; 5,929,26;
5,977,296; 6,140,482; WO QD/56746; Wp 01114398, and related puf~licatians.
Methods fox synthesizing aligonucleatides comprising such analogs or derivatives are disclosed, for example, in the patent publications cited above and in iT.S. Ixat. Nos. 5,614,622;
5,739,314; 5,955,599;
5,962,674.; 6,117,992; in WO 00175372; and in related publications.
(3ligt~nt~cleotides sometimes are linked to a second moiety. The second moiety may be an additional nucleotide Sequence such as a tail sequence (e.g., a polyadenosine tail), an adapter sequence (e.g.,~phage M13 universal tail sequence), arid others. Alternatively, the second moiety nnay be a non-nual~eotide moiety such as a moiety which facilitates linkage to a solid support or a label to facilitate detection of the oligocrucleotide, ~'he second moiety may be attached to any position of the oligonucleotide, and labels include but are not limited to a radioactive label, a fluorescent label, a chemiluminescent label, a paramagnetic Label, and the Like.
[(11128] The synthetic nucleic acid may be linked to a solid support, and the nucleic acid may be arranged on the solid support in an array. An array, sometimes referred to as a "microarray,"
sometimes includes an oligonucleotides described herein, and methods for txraking aced using oligonucleotide microarrays are disclosed in ~fJ.S. Pat. Nos. 5,492,$06;
5,525,464; 5,589,330;
5,695,90; 5,849,4$3; 6,01$,041; 6,1145,996; 6,136,541; 6,142,681; 6,156,501;

6,197,506;
6,223,127; 6,22.5,625; 6,22,9,911; 6,239,273: WO 00152625; WA 01!254$5; and W~J 01129259. The microarray typically comprises a solid support and the oligonucleokides sometimes are linked to the solid support by covalent or non-covalent inter~.ctions. ~'he oligonucleatides sometimes are linked to the solid support directly or by a spacer molecule.
[1ID29~ The invention further includes cotrepositinns wllieh comprise an isolated Cav3.lac calcium channel-encoding nucleic acid or proCeirr. The term "isolated" refers to substances that are separated from their natural environments or from the materials present in the natural source. For example, with rEgard to genoxnic »NA, the term "isolated" includes nucleic acids which are separated from the chromosome with which the genomic DNA is naturahy associated. An "isolated" nucleic acid is often free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' andlor 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various ennbodiments, the isolated nucleic acid molecule can contain less than about S kb, 4 kb, 3 kb, 2 kb, l kb, 0.5 kb or O.l kb of 5' and/or 3" nucleotide sequences whick~ flank the nucleic acid molecule in genomic DNA
of the cell from which the nucleic acid is derived. An "isolated" nucleic acid molecule, such as a cl:)NA molecule, sometimes is substantially fi~e of other cellular material, ar culture medium when produced by recombinant techniques, car substantially free of chemical precursors or other chemicals when chemically syritl~esixed. An "isolated" polypeptide car protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or~srtbstantially free frown chemical precursors or other chemicals when chemically synthesized. "Substantially free" means a preparation of a substance having less than about 30°la, 20/0, 10°lo and mare preferably 5~'0 (by dry weight), of material from its source of derivation. Wkren the palypeptide or a biologically active portion thereof is produced recaznbinantly, it often is substantially free of culture medium, specifically, where culture medium represents less thin about 20%, sometimes less than about 10~, arid often less than about 5°!a of the volume of the polypeptide preparation.
((130] Alsa provided is a composition which comprises a cancer cell in combination with a molecule that antagonizes a Ca~3.1 oalcium channel splice variant associated with a cell proliferative disorder (e.g., antagonizes a t'av3.lac ox Ca~3.Ik~ calcium channel) ar a molecule that inhibits ttte interaction of a Ga"3.1 calcium channel splice variant with ANA
III (e.g., Ca~3.lac).
Examples c~f such molecules include but tire not limited to cotnpaunds, antisense nucleic acids, ribozyme nucleic acids, inhibitory ItNA, and antibodies, which are deserlbed in greater detail h~~~~.
Compounds (40.31] Compounds can he obtained using any of the numerous approaches iz~
combinatorial library methods la~own in the art, including biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a xtovel, hors-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain biaactive (see, e.g., 2uckexmann et al., J. Med. Chem37: 2b78-SS (1994)); spatially addressable parallel solid phase ox solution phase libraries; synthetic library methods requiring decanvalution; "one-bead one-compound" library methods; and synthetic library methods using affinity chromatography selection. Biological library and peptoid library approaches are typically limited to peptide libraries, while the other approaches~~
am applicable to peptide, non-peptide rrligomer ar small molecule libraries of compounds (LJam, Anticancer Drug Ides. 12: 145, ( 1997)). Examples of methods for synthesizing molecular libraries 'ara described, far example, in DeWitt et al., Proc. Nod. Acad. Sci. U'.S.A.
9(1: 69CI9 (1993); Exb er ai:, Froc. Natl. Acad. Sci. USA 91: 11422 (1994); 2;uckermaivi et al., J. Med.
Chem. 37: 267$
(l994); Cho et ad., Science 261: 1303 (1993); Carrell et aL, Angew. Gksem.
lot. Ed. )~~ngl. 33: 2059 (1994); Care!! er al., Angew. Ohem. Irrt. Ed. Engl. 33: 2061 (1994); and in Gallop et aL, J. Med.
Cbem. 37: 1233 ( 1994).
~00~2] Libraries of compounds may be presented itl solution (e.g., Houghten, Bioteahniques 1.3:
41.2-421 (1992)), or on beads (Lam, Nature 354: $2-$4 (19~9i)), chips (Fodor, Nature 364: SSS-556 (1993)), bacteria ar spores (Ladner, United States Patent No. 5,223,409), plasmids (Cuh er al., l'roc.
Natl. Acad. Sci. USA 89: 1865-1869 (1992)) or on phage (Scott and Smith, Science 249: 3$6-390 (1994}; l~evlin, Science 249: JL04-4.t?~6 (19917): Cv~rirla ed al., Proc.
~atl. Aced. Sci. 87: 4378-6382 (1990}; Feiici, J. Mol. Blol. 2?2: 301-310 (1991}; l.~:dner supra.).
[4033] A compound sometimes modulates expression or activity of a palypeptides and often is a small molecule. Small molecules include, but are not litrated to, peptides, peptidomimetics {e.g., peptoids}, amino acids, atnina acid analogs, pofynucleotides, polynucleatide analogs, nucleotides, nucleotide analogs, organic ar ina~rganic compounds (e.e., including heceroarganic and organometallic compounds} having a molecular w~lght less than about 14,000 grams per mole, organic or inorganic cax~c~paunds having a molecular weight less than about 5,000 grams per mole, organic ar inorganic compounds having a molecular weight less than about 1,000 grams prer mote, organic or inorganic compounds having a molecular weight less Chart about 500 grams per male, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
10434] Fxamgles of'f'-type calcium channel small molecule antagonists and methods for determining their effect on calcium channel funetir~n are disclosed in CJ~.S.
patent application publication no. fJ'S-2fl04-0034035-A1 published February 19, 2004; U.~. patent application publication no, us-2004-0044004-A1 published March 4, 2004; T.T.~. patent application no.
10/763,9?4 filed January 22, 2004 and U.B. patent application tta. 6111474,$64 filed May 30, 2003.
Antisense. Ribazyme~R~Ai, silitVA and Modified Nucleic Acid Molecules [11035] An "antise~nse" nucleic acid refers to a nucleotide sequence complementary to a "sense"
nucleic acid encoding a palypeptide, e.g., complementary to the codartg strand of a double-stranded a'I~NA molecule or complerne~ntary to an mRNA sequence. 'fhe antisense nucleic acid Cart be complementary to an ex~kire coding strand, or to only a portion thereof. In another embodiment, the antisense nucleic acid molecule is ancisense to a "noncading zegiori' of the coding strand of a nucleotide sequence encoding the calciut:n channel ar AN'~ III protein.
[0031i] An antisense nucleic acid can be designed such that it is complementary to the entire.
coding region, and often the antiset~se nucleic acid is an aligonucleatide ant3sense to only a gartion of a coding ar noneoding region of rnRNA. Far example, the antisense of igoz~ucleatide can be complementary to the region surrounding the translation start site of tnRNA, e.g., between the -10 and f10 regioins of tt~e target gene nucleotide sequence of interest. An antisense aligonucleatide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, ~0, GS, 70, 75, $0, or mare nucleotides in length. The antisense nucleic acids, which include the rihozynnes described hereafter, can kre designed to target calcium chanxxel a»d AhIX IF nucleic acids.
[Q0~3T] An antisense nucleic acid can he constructed using chemical synthesis and enzymatic ligatian reactions using standard procedures. k'or example, an antisense nucleic acid {e.~ , an antisense aliganucleatide} can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the m~ol~ules yr to increase the physics! stability of the duplex formed between the antisensE and sense zlucleie acids, e.g., phasphorathiaate derivatives and acridine substituted nucleotides can be used. Antisense nucleic acid else can be produced biologically using an expression vector into whick~ a nucleic acid has beon subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense arAez~tation to a target nucleic acid of interest, described further in the following suf~section).
~fl038] When utilized as therapeutics, antisense nucleic acids often are administered to a subject (e.g.,1~y direct injection at a tissue site) err generated in situ such tl~at,they hybridize with or bind to cellulax rnltLTA andlor genornic DNA, encoding a polypeptide and thefeby inhibit expression of the palypeptide, for example, by inhibiting transcription a~ndlor translation.
Alternatively, anti~nse nucleic acid molecules can be tnodifle~i to target selected cells and then are administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed an a selected ce1!
surface, for e~arnple, by linking antisense nucleic acid molecules tc~ peptides or antibodies which bind to cell surface receptors ar antigens. Antisense nucleic acid molecules can also be delivered to cells using the vectors described herein, Sufficient intraceliuiar concentrations of antisense molecules are achieved by incorporating a strong promoter, such as a poi 11 or poi III promoter, in the vector construct.
m [0039 Atttisensc nucleic acid molecules sometimes are alpha-auomeric nucleic acid molecules.
An alpha-anameric nucleic acid molecule forms speeif a double-stranded hybrids with complementary RNA in which, contrary to the usual beta-units, the strands run parallel to each Other (Cxaultier et al., Nucleic Acids. Res.15; 6625-6641 ( 19$7)). Antisense nucleic acid molecules can also comprise a 2'-o-methylribantrcleotide (Inane et al., Nucleic Acids Res.
15: X131-6I4$ (19$7)7 or a chirneric ItNA-3~NA analogue (moue e~r al., FI~S ~,ett. X15: 327-33U
(1987)). A,ntisense nucleic acids sometimes are composed of DNA or PNA or any other nucleic acid derivatives described previously.
[OiI~IU] In another embodiment, an antisense nucleic acid Is a ribazyxtae. A, ribazyme having speci~~city ~or a calcium channel or ANX III-encoding nucleic acid can include one or more sequences complementary to the nucleotide sequence of a calcium channel or ANX
Zli sequence, and a sequence having a ltttawn catalytic region responsible for mRNA cleavage (see e.,g., U.S. Pat.
No. 5,093,246 or Haselhoff and Oerlach, Nature 334: 5$5-591 (1988)). For ercample, a derivative of a TeGrahymena L-19 IVS RNA is sometimes utilized in which the nucleotide sequeraie of the active site is complementary to the nucleotide sequence to be cleaved in the target rnRNA (see e,g., Gech et ar. U.S. Patent 1~1'a. 4,987,071; and Leah er al. U.S. Patent No. 5,11G,74Z).
Also, target mRNA can be eased to select a catalytic RNA having a specific ribanuclease activity from x pawl of RNA
molecules see e; g., Barrel & Szostak, science 261: 14i i-1418 (1993)).
[t1Q41] Antagonists include in certain embodiments nucleic acids that can farm triple helix structures with a calcium chaxanel nncleotitle sequence, especially ore that inoludas a regulatory region that controls calcium channel expression. Caldittm channel gene expressicm can be inhibited 1~y targeting nucleotide sequences complementary to the regulatory region c~f the channel (e.g., promoter andlor erthancers) to form triple helical structures that prevent transcription of the channel gene in target cells (see e.g., Ilelene, Antioat~cer f7rug I?es. 6(6): 669-84 (1991); Helene at r~l., Ann.
N.Y. Aced. Sci. 6b0: 27-36 (1992); amdMaher, Bioassays 14(12): 807-i~ (1992).
Potential sequences that can be targeted for triple helix formation can be increased by creating a so-palled "switehback"" nuoleic acid rnaleoule. Switchback nnalecules are synthesized in an alternating S'-3', 3'-~' manner, such that they base pair with first one strand of a duplex and then tl~e other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand caf a duplex.
[Ot142~ Caloium channel antagonists also include RNAi and siRNA nucleic acids.
Gene expression may be inhibited by the introduction of double-stranded 1~NA
(dsltNA), which induces potent and specific gene silencing, a phenomenon called RNA interference nor RNAi. See, e.g., Fire et aL, US Patent Number G,5(IG,559; Tuschl et aL PCT >:nternatianal Publication Na. WO 01175164;
day et rxL PCf International Publication Na. WO 03/0101 St7Al; or Basher dM, Labduesse, Nat Cell 13io12000 Feb;2(2):831-6. This process has been improved by decreasing the size of the dauble-stranded RNA to 20-24 base pairs (to create small-interfering RNAs ar sil~NAs) that "switched off"
genes in marnz3nalian cells without initiating an acute phase response, i. e., a host defense mechanism that often results in cell death (see, e.g., Caplen et ett. Proc Natl Aced Sci U ~ .A.. 2()01 Aug 14;98(17):9742-'7 and Elbashir et at. Methods 2bIl2 Fe6;26(Z):199-213). There is increasing evidence of past-transcriptianal gene silencing by RNA interference (I~NAi) for inhibiting targeted expression in mammalian cells at the mltNA level, in human cells. There is additional evidence of effective methods for inhibiting the proliferation and migration of tumax Dells in human patients, and far inhibiting metastatic cancer development (,see, e,g,, U.S. Patent .Application No.
US2001000993183; Gaplen et al. Prat Natl Acad Sci U S A; and Ahderrahmani ~t at. Mo1 Cell Bial 2,OOi Nov21(21):7256-67).
[01143 An ''siRNA'" or "RNAi" refers to a nucleic acid that forms a double stranded )2.NA and has the ability to reduce or inhibit expr~essian of a gene or target gene when the siRNA is delivered to or expressed in the same cell as the gene or target gene. "siRNA" refers to short double-stranded Rl'iA fanned by the complementary strands. Complementary portions of the siltNA tltat hybridize to form the double stranded molecule often have substantial or complete identity to the target molecule sequence. In one embodiment, azt siF~IriA refers to a nucleic acid that has substantial or complete identity to a target gene and farms a double stranded siRNA, such as a calcium channel encoding nucleotide sequence, far example.
j0044] When designing the siRNA molecules, the targeted region often; is selected from a given I1N'A sequence beginning 54 to lOty nucleotides downstream of the start codan.
See, e.g., Elbashir et ai,. Methods 2b;199-213 (2002), hnitially, 5' or 3' L3TRs and regions nearby the start cadan were avoided assuming that UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP or RISC endr~nuclease complex_ Sometimes regions of the target 23 nucleotides in length carifarming to the secluenee motif AA(N19)TT' (N, axe nucleotide), and raglans with approximately 309to to 70% G/C-content (often about 50% C'x!C-content) often are selected. If na suitable sequences are found, the search often is extended using the motif NA(N21). The sequence of the sense siRIVA sometimes corresponds to (N19) TT ar N21 (position 3 to 23 of the 23-nt motif), respectively. In the latter case, the 3' end of the sense siRNA
often is converted to TT. , The rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense 3' overhangs. 'T'he antisense SiRNA is synthesized as the complement to position 1. to 2I of the 23-nt motif because position 1.
of the 23-nt motif is not recognized sequence-specifically by the antisense sirtNA, the 3'-mast nucleotide residue of the antisense siRNA can be chosen deliberately. However, the penultimate nucleotide of the antisense siltNA (complementary to position 2 of the 23,nt motif) often is complementary to the targeted sequence. For simpiifyi~ng chemical synttzesis, TT' often is utilized, siRl'rTAs corresponding to the target motif NAR(N17)YNN, where R is patina (A,Cr) and Y is pyrimidine (C,C.~, often are selected.
Respective 21 nucleotide sense and antisense siRNAs often begin with a patina nucleotide and can also be expressed from pol IlI expression vectors without a change in targeting site. Expression of 'RNAs from pal III promoters often is efficient when the first transcribed nucleotide is a patina.
j004S] The sequence of the sil2NA can correspond to the full length target gene, or a subsequenee thereof: Often, the sit~NA is about 15 to about 50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is 15-50 nucleotides in lengtth, and the double stranded siRNA is about I ~-50 base pairs in length, sometimes about 20-30 nucleotides in length or about 2(1-25 nucleotides in length., e.g., 2U, 21, 22, 23, 24, 25, 2~6, 27, 2$, 2q, or 30 nucleotides in length. ~'he siRhTA sometimes is about 21 nucleotides in length. Methods of using siRl~'~1. are well laxovcm in the axt, and specific siRNA molecules ~.y be purchased from a number of companies including Dhaxsnacon Rcseareh, Inc.
j0046] Antisense, ribazyme, RNAi and siRNA nucleic acids can be altered to form modified nucleic acid molecules. The nucleic acids can be altered at base moieties, sugar moieties or phosphate backbone moieties to improve stability, hybridization, ar salability of the molecule. Far 1.5 example, the deoxyribose phosphate backbone of nucleic said nr~olec~tles can be modified to generate peptide nucleic acids (see Hyrup et at., Bioorganic & Medicinal Chemistry 4 (1}: 5-23 (1996}). AS used herein, the farms "pegtide nucleic acid" ar ''PNA" refers to a nucleic acid mimic such as a DIVA n~inaxo, in which the deoxyribase phosphate bactcbane is replaced by a pseudopeptide Backbone and only the fou~c natural nucleaBases are retained. The neutral Backbaae of a PICA can allow far specific hybridization to DNA and RNA under conditions of law ionic strength. Synthesis of PNA otigotners can be performed using standard solid phase peptide synthesis protocols as described, for example, inn Hyrup et al., (1996) supr~x and'Perry-O'Kee~
etal., Prat. Nati. Aced. Sci.
93: 14470-675 (1996).
[4~D47j PNAs of nucleic acids can be used in pragnc~stic, diagnostic, and tlzerapeatie applications.
For example, PNAs can be used as antisense or antlgene agents fQr sequence-specific modulation of gene expression by, for example, inducitlg transcription or translation arrest or inhibiting replication.
>?lVAs of nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed 1'CR clamping); as "artificial restriction enzymes" when used in cornbinatian wikh other ez~yEnes, (e.g., S1 nucleases (Hyrup (t~9G} supra));
or as probes Qr primers far DNA sequencing ar hybridization (Hyrup et at, (1996} supra; Pet~ry-C1'Keefe supra}.
[U048j fn other embodiments, aliganucleotides may include other appended groups such as peptides (e.g., for targeting' host cell receptors in viva), or agents facilitating transport across cell membranes (see e.g., Letsinger et ul., Proc. Natl. Aced. Sci. USA 86:
6553~655b (19$9); T emaitre ei al., F'xac. Natl. Aced. Sci. rl"SA. 84: 6~I8-652 (198'7); I~T P'ublicatian NQ.
WU881t?981U) or the blood-brain barrier (sue, e.g., FCT' Publication No. W089f Ifl134). In addition, oligonucleatides can be modified with hybrldi~ation-triggered cleavage agents (see, e.g., Krol et .al., Bio-.'T'echniques 6:
~5$-976 (1988)) ar intercalating agents. (See, e.g., Zan, Pharm, Res. 5: 539-54~ (19$8) }. To this end, the oligonucieotide may be conjugated t0 another molecule, (e.g., a peptide, hybridization triggered crass-linking agent, transport agent, or hybridization-triggered cleavage agent).
Anti-Calcium Channel and A'NX in Antibodies X0049] 'file term "antibody" as used herein refers t0 an imsmunoglobulin molecule aF
immunologically active portion thereof, i.e., an antigen-binding portion.
Examples of irtununologically active portions of irrimunaglobulin molecules include Flab) and 1~(ab')2 fragments which can 6e generated by treating the ~ntiBody with an enzyme such as pepsin.
An ~ntibvdy sometimes is a palyclonal, monoclan,al, recombinant (e.g., a chimeric or humanized}, fully human, non-hurnan (e.g., marine), or a single chain antibody. A,n antibody rnay have effeCtor fu;action and can i'wx complement, and is sometimes caapled to a toxin ar imaging agent.

[$050] A full-length calcium channel or ANX III polypeptide or antigenic peptide fragment can be used as an immunogezl or can be used to identify anti-calcium channel or ANA III antibodies made with other imnnunogens, e.g., cells, membrane preparations, and the like.
Arr antigenic peptide often includes at least 8 amino acid residues of the target protein and encompasses an epitape of the protein. Antigenic peptides sometimes include 10 or more amino acids, 15 or snore amino acids, ZO
ar mare amino acids, or 30 or more amino acids. Hydrophilic and hydrophobic fragments of target polypeptides sometimes are used as irnmunogens. Epitopes encompassed by the, antigenic peptide often are regit~ns located on the surface of tk~e polypeptide (e.g., hydrophilic regions) and regions with high antigenioity. Por example, an Emini suxface probability analysis of human polypeptide sequences can be used to indicate the regions that have a particularly high probability of being localized to the surface of calcium channel and ANX ILI polypeptide and are thus likely to constitute surface residues u.~eful four targeting antibody production. The antibody may bind an epitope on any domain or region of calcium channel ox ANA !~ poiypeptides. An antibody can be made by immunizing with a purified calcium channel or ANX lII antigen, or a fragment thereof, a membrane associated antigen, tissues, e.g., crude tissue preparations, whole cells (e.g., living cells), lysed cells, or cell fractions.
[0051] Chimeric, humanized, and completely human antibodies are useful for applications which include repeated administration to subjects. Chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, can be Wade using standard recombinant DNA
techniques. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et a1 International Application hlo. PC'I'fI~'S86I02z~9; Akira, et al European Patent Application 1$4,1.$7;
Taniguchi, M., European Patent Application 171,4~b; Morrison et al European Patent Application 173,494; Neuberger et ai FC"T International Fublicatlan No. WO 8b/01533;
Cabilly et al U.S. Patent No. 4,$15,57; Cabilly et aI European Patent Application 125,023; Better et al., Science 240: 1041-1043 (1988); Liu .et ad., F'roc_ Natl. Acad. Sci. USA $4: 3439-3443 (1987; Liu et ai., r. Immunol.
139: 3521-3526 (19$7); Sun et tel" Froc:. Natl. Acad. Sci. USA 84: 214-218 (t987~; Nishimura et al., Canc. Res. 47: 999-1005 (1987; Wood et ad., Nature 314: 446-449 (19$5); az~d Shaw et ul., Y. Natl.
CaneerTnst. $0: 1553-1559 (1988); Mon7son, S. L., Science 229: 1202-1207 (19$5); Cti eta~, BivTechniques 4: 214 (198b); Winter I:I.S. Patent 5,225,539; Jones er al., Nature 321: 552-SZ5 (195; ~'erttoeyan et al_, Science 239: 1534; and Beidler et al., J. Imntunol.
141: 4053-405U (1988).
[OOS2) Completely human antibodies are desirable for therapeutic treatment of human patients.
Such antibodies can be produced using transgenio mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can e~cpress human heavy and light chain genes. See, fox example, Lonberg and Huszar, Int. Rev. Ixnmuz~ol. 13: 65-93 (1995); and U.S. Patent Nas. 5,625,126; 5,633,425; 5,569,825; 5,661,016; and 5,545,800. In addition, companies such as Abgenix, hnc. (Fre~nont, CA) and Medarex, Inc. (F~rincetan, NJ}, can be engaged to provide human antibodies directed against a selected antigen using tecltnolagy similar to that described above-Campletely l~ur~n antibodies that recognize a selected epitape also can be gexterated using a technique referred to as "guided selection:' In !isle approach a selected non-human ~nnanaclonal antibody (e.g., a marine aatibady) is used to guide the selection of a completely human antibody recognizing the same epitape. This technology is described far example by Jespers et al., »iofi'echnolagy 12: 899-903 (1994).
[U05~] Aa anti-calcium channel ar AblX rII antibody can be a sitxgle chain antibody. A single chain antibody (scFV) can be engineered (see, c_g., Calcher et rzl., Axon. N
~l Acad. Sci. 880: 263-80 (19990; and L;Geitsr, Clin. Catxcer P.es. 2: 245-52 (1996)). Single chain antibodies can be dimerized ~ar multimerized to generate multivalent antibodies having speci:facities for different epitapes of the same target palypeptide.
[11054] Antibodies also may be selected ar nwdified so that they exhibit reduced or no ability to bind an Fc receptor. 1~ar example, an antilyady !nay be an isotype or subtype, fragment ar ether rnuta~at, which does not support binding to an Fc receptor (e.g., it has a mutagenized ar deleted 1~c receptor binding region). ' [~U05Sa Also, an antibody (ar fragment thereof) may be conjugated to a therapeutic tzraiety such as a c~rtatoxin, a therapeutic agent ar a radioactive mete! ion. A cytotoxin ar cytotaxic agent includes any agent that is detrimental to cells, Examples include taxol, aytochalasin B, gramicidin D, etltidium bromide, emetinc, mitomycix~, etopasirle, tenoposide, vincristine, viublastine, calchicin, daxarubicin, daunorubicin, dihydraxy anthracin diane, mitoxantrone, mitlaz~mycin, acti~namycin D, 1 dehydratestostero~ne, glucocorticaids, procaine, tetxacaine, lidocaine, propranolol, arid puramycin and analogs ar homalags thereof. Therapeutic agents include, but are not limited to, antimetabalites (e.,~., raetborrexate, 6-mercaptapurine, 6-thiaguaniae, cytarabiae, 5-fluorouracil ~leca3cbazine), alkylating agents (e.g., tkaeGhlorethamine, thiotepa. chlorambucil, melphalan, carmustine (BCNU}
and lamustine (~CNU), cyclophosphamide, busuifan, dibromornannitol, streptazatacin, znitamycin C, and cis-dichloradiamine platl~num (fl} (DDl'} cisplatia), anthracyclines (e.g., dauaorubicin (formerly ~daunomycin} and doxorubicin), antibiotics (e.~., dactinamycin (formerly actinornycin), bleort~ycin, mithramycin, and anthramyoi~n (AMC)}, and anti-mitotic agents (e.g., vincristine and vinblastine).
[01156] Antibody conjugates can be used far modifying a given biological response. For example, the drug moiety may be a protein ar palypeptide possessing a desired biological activity.
Such proteins tray include, for example, a toxin such as abrin, ricin A, Qseudomonas exataxia, ar diphtheria t4xin; a polypeptide such as tumor necrosis factor, ~y-interferon, a-interferon, nerve 1$

growth factor, platelet derived growth factor, tissue plasminogen activator;
or, biological response modifiers such as, far example, lyxnphokines, interleukiz~-1 ("IL-1"), interls;ukin-2 ("IIr~,"), interieukin-6 ("IL,-b"), granulocyte macrophage colony stimulating factor ~"GM-CSF'), granuiocyte colony stimulating factor ("G-CSF'), or other growth factors. Also, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Sega!
in U.S.1'atent No.
4,b76,980, for example.
[0057] An anti-calciunn channel ar AN3~ III antibody (e.g., monoclonal antibody) can be used to isolate calcium channel or AN'~ III polypeptides by standard techniques, such as affinity chromatography or immunaprecipitatian. An anti-calciutn~ channel or ANX rII
antibody can be used to detect a calcium channel or AN'X III polypeptide (~.g., in a cellular lysate or cell supernatant) to evaluate the abundance and pattern of e~eprcssion of the polypeptide. Anti-calcium channel or AIVX
III antibodies can be used diagnostically to monitor polypeptide levels in tissue as part of a clinical testing procedure, e. g., to determine the efficacy of a given treatmezrt regimen. Detection can be facilitated by coupling (i.e., physically linieing) the antibody to a detectable substance (i.e., antibody labeling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent nnaterials, bioluminescent materials, and radioactive materials.
Examples ol'suitable enzymes Include horseradish pem~cidase, alkaline phosphakase, ~-gaiactosidase, ar aeetylchviinesterase; examples of suita#~le prosthetic group complexes include streptavidinlbiatin and avidin/biotin; examples of suitable fluorescent materials include umbelliferane, ftuorescein, fluoreseein isothiocyanate, rhodamine, dichlorotriazinylamine fluarescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ~~I, is~I, 35S or 3I~ or any radioactive molecule suitable for andlor used in in vaVa nuclear medicine proeedu~nes. Also, an anti-calcium channel or A1VX III antibody can be utilized as a test molecule for deternvning whether it can txeat a cell praliferative disordez~, and as a therapeutic for administration to a subject for treat3x~g a cell praliferative disorder.
r0(iS~] Included x~re antlbadies that bind only a native calcium channel splice variant or ANX III
l~lyPePtide, only denatured or otherwise non-native calcium channel or A1V'X
III polypeptide, or which bind both, as well as those having linear or canfarmaxional epitopes.
Conformational epitopes sometimes can be identified by selecting antibodies that bind to native but not deztatured polypeptide.

Methods For l7etermininQ a Disk of or i"resence of a Ceii FraliferativE
Disorder [U059] Frovided herein are methods for detecting a risk of, ar the presence of, a cell proliferative disorder in a subject, which methods comprise determining whether an abnormal patterns of expression of Cav~.1 caleiutn channel m'f~TA, ar protein exists. l;xpressian of a particular splice variant can be determined by detecting the presence of m~t'A encoding the splice variant andlor by detecting the protein splice variant itself. As demonstrated below, cells that undergo abnornnal proliferation uniquely produce the splice variant Cav3.lae, and thus the presence or absence of expression of this splice variant is indicative of the propensity of the cei!
for proliferation. The presence of this splice variant expression when found in the cells in a sample from a subject indicates that the subject is at risk for, or is afflicted with, a cell praliferative disorder. Similarly, the presence of Cav3.1 b splice variant is characteristic of hyperproliferative cells and the presence of this splice variant expression i;n cells of a sample derived from a subject indicates that the subject is at risk far or is afflicted with a cell pmliferative disorder. Also diagnostic is the propartian of the splice variant Ca~3.l bc, where the presence of more than about 2c~9~, especially more than about 3096, in particular more than about 4~'0, 5~%, or 60%n of Cav3.lbc as a percentage of the total erav3.1 expression in the cells indicates the presence of a cell prolifetative disorder. As noted, expression levels tnay be measured either at the mRI~A level or the protein level.
[Oi~60j The mRNA or protein is detected in a biological sample from the subject. For example, the biological sample may be bleed, saliva, sputum, urine, cell scrapings, andlar biopsy tissue. The tez~n "subject" as used herein refers primarily to a human but else refers tv other mammals such as aog$~ Cats, and ungulates (e.g., cattle, sheep, and swine). Subjects also include avians (e.g., chickens and turkeys), reptiles, and fish (e-g., salmon), as embodixments described herein can be adapted to nucleic acid samples isolated frarn any of these organisms. A nucleic acid, protein or biological sample may be isolated from the subject and then directly utilized in a method for determining the presence of a calcium channel splice variant associated with a cell praliferative condition, or alternatively, the sample may be isaiated and then stored (e.g., frozen) for a period of time before being subjected to analysis.
t0I161] The nucleotide sequence or polypeptide sequence detected sometimes is substantially identical to the nucleotide sequence ar amino acid sequence of the calcium channel associated with a cell proliferative condition. Jn some embodiments, the n~ucleatirle sequence or pc~lypeptide sequence detected is substantially identical to a Ca~3.iao calcium cba~nnel or Cav3.lb calcium channel encoding nucleotide sequence ar polypeptide sequence, as allelic variants may occur- The sequence detected at ti~t7,es is $0%a or snare, 81 % or znore...85% ar more... $9% Qr rnore, or 9Q% tar more identical to $ Cav3.lac calcium channel or Cav3.lb calcium channel encoding nucleotide sequence ar palypeptide sequence, and often is 91~'o ar more, 92% or more...9a% or mare...9'~~ ar more, 98% or more, or 999'o ar more identical to a Ca,,3.lac calcium channel or Cav3.lb calcium channel encoding nucleotide sequancc or poIypeptide sequence. Also, an intracellular loop region bet~coveer~
conserved transmetnbrane zegions, such as an intracellular it~og between region I and iZ, between II
and fEl or between lIi and N in a detected nucleotide sequence or pcrlypeptide sequence sametinnes bears less sequence identity to a corresponding loop in a Cav3.lac calcium channel Qr Cav3.lb calcium channel as compared to the rest of the sequence. In such loop regions, the detected nucleotide sequence ar palypeptide sequence st~metimes bears SO% ar mare, 51%'0 or more...,fiU% or rnare...70% or rnare...8Q9O car more...90%Q ar rnore...9~% ar more_ ..97% ar more, 98% or more or 999'0 or snore sequence identity to a corresponding loop sequence in a Cav3.1 ac calcium channel ar Cav3.I~a calcium channel.
(0(rf2] Any suitable method for detecting the Ca,,~.1 calcium channel splice variant nucleotide Sequence or amino acid sequence is utilised. Far examrple, irt a process far detecting a specific Ca"3.1 calcium chatmel splice variant protein, a biological sample from a subject sometimes is processed (e.g" processed to disrupt cell membranes) and then contacted with an antibody that specifically binds to the particular Cav3.1 calcium channel splice variant being detected (e.g., a Cav3.lac or Cav3.1 b caiciutn channtel splice variant). A variety of methods are knov~n f~,r detecting floe presence or absence of a particular Cav3.1 calcium channel splice variant nucleotide sequence assaciaced with a cell praliferative condition, v~rhich include the ti.T-PCIZ, techniques descriherl hem,.after.
(~lHS3j xl= it is determined that a subject is at risk of a particular cell praliferative disorder, such as one described previously, the risk may be expressed in any lazown and useful man~rter. Far example, risk of a cell pmliferative disorder sorrtetimes is expressed as a probability, such as an odds ratio, percentage. or risk factor, The risk assessment is based upon the presence or absence c~f a Ca"~.1 calcium channel nucleotide sequence or polypeptide associated with ttie cell proliferative condition (e.~,, x Ca"3.lac or Cav3.lh calcium channel splice variant), and also may be teased in part upon phenotypic traits of the individual being tested. Ivlethads for calculating risks based upon patient data ate known (see, ~.,~., Agresti, Categorical Data Analysis, 2nd ~d. 2Q02.
Wiley).
j00fr4] I~rocesses for identifying a nucleotide sequence encoding a Cay3.I
calciurct chanr~ei splice variant associated with a sell praliferative disorder or the encoded protein in a sample from the subject sametitx~s is combined with other infarcnatiort ar processes.
1~ombining with other information or frrocesses often enhances the abilit~r of a health care provider to diagnose ar treat the cell pxoliferative disorder. Other information sotnetitxces is the presence or absence of another calcium cltannei splice variant nucleotide sequence or palypeptide in a sample frarn the subject;
phenotypic information pertaining to the subject (e.g., family history of a cell proliferative disorder and personal history of a teal proliferative disorders); andlor information from a further process far diagnosing a cell proliferative disorder, For example, another process for diagrnasing a cell proliferative disorder sometimes is detecting tha presence or absence of increased eel! protiferatian within a subject ittentified as having the presence of a nucleotide sequence that encodes a Ca~3.1 calcium channel or the presence of a CaY3.I calcium channel protein assaoiated with a cell pxaliferative disorder. The presence or absence of increased cell praliferatian sametitnes is detected in a tissue biopsy from the su6jeet. In other embodiments, the presence 4r absence of increased. cell proliferation is detected in viva, such as by nuclear medicine procedures, far example.
[~fh~] In ether embodiments. the processes described above satnetimes are combined with a further process far treating the cell proliferative disorder. Any treatment of the cell praliferative disorder can be adr~aenisterec~ to the subject- A general rJtethad for treating a cell praliferative disorder sometimes is prescribed, such as administering a chemotherapy or radiation therapy regimen to the subject that reduces cell pratifaratian. l4lethads that specifically target tha cell praliferative condition also may he prescribed, such as removing one ar more tumors frarn the subject in surgery, andlar treatment with an antagonist of a specific rr~alecule that causes the cell proliferative disorder. Compositions and methods for treating cell proliferative disorders by antagonizing a T-type calcium chaxmet (e.,g" a Ca"3.1 cai~cium channel splice variant associated with a cell proliferative disorder) are described in greater detail hereafter.
~,ethods to Inhibit Cellular Proliferatiari [01156] Cellular pxaliferation may be inhibited itt cells that express abnormal patterns of Ca~,~.i calcium channel splice variants using a number of techniques. As noted above, the abnormal pattern ctzay reflect itself in the ratio of the various splice variants or the presence of Cav3.lb or the presence of Cav3.lac.
[b067] In orte approach, bioclcers of T-kype calcium channels rnay be used;
however, a more nuartced approach focuses an the T-type channels that are abnormally expressed - e.g., Ca,.3, i bt Cav3.lac, and Cav3.lbc, rnhibltion of either expression or activity of these chatanels or both t~.y be effective. Expression of these splice variants rnay be negatively affected using, for example, 2~

antisense oligonucleotides, inb;ibitory lx,~TA, ar ribozyrnes, all pf which are designed tp be speck for these sQlice variants to be targeted. Alternatively, specific antagonists, such as antibodies that are direceed toward the splice variant in question are useful. In the ease c~f Ca"3.lac, inhibitors of its ixtteractian with the ANX III protein or substances that bind specifically to AhTX ~ protein may also be used.
[006$] As dempttstrated below, the spline variants differ in the tinker sequence between regions III attd TV' of the ai protein characteristic of Cav3.I calcium chanrteIs (see Pig. 2). Thus, ribo~ymes, inhibitory RIBA, and antisense ~quences targeted to the mRl'fA
zegion specific for the sequences characteristic of the undesired splice variant in t,~cis raglan rnay be used, as wail as antibodies specifically itnmunoreactive with the protein encoded in this region.
[0069] As further described below, these approaches to inhibiting the ptnliferation of abnormally proliferating cells e~.hibiting abnormal ~a"3.1 splice variant exptesssian can be applied to cells that exist in a subject as well as cells in cultetre or biopsy.
Cell proliferative Treatment Cannposit~rws and Methods [007(1] Provided are zr~thods for treating a cell pr4liferative disorder characterized by expression of a Ca"3.1 calcinrn channel splice variant assaciatcd with the cell praliferative disorder (e.g., Cav3. tar andlor Cav3.Ib and/or Ca"3.lbc~, which cpmprise administering a T-type calcium channel bfocker to a subject in need thereof in an atrtount effective to treat the oell proliferative disorder. An antagonist molecule specific far a Cav3. t calcium channel splice variant associated with the cell pratife~'ative disorder (e.g., Ca"3.lac andic~r Ca"3,1b andlor Cav3.lbc) is preferred, or a rnatecerl$
that inhibits the interaction between AbTX IIi with a. Ca"3. t calcium channel associated with the cell proliferative disorder (e.,g., Cav3.lac), t4 a subject in need thereof in an amount effective to treat the cell praliferative disorder.
[00?1] In addition, expression of undesired splice variants n toy be inhibited by an antisense, ribo~yme, Rt~FAi, siIthTA, or triple helix-forming nucleic acid. Activity may be inhibited by an antibody ax compound small molecule type. The antagonist sometis blocks the calcium channel and sometimes interferes with and inhibits an interaction between a Cav3.1 calcium channel splice variant and Ahl'X III (e.g., inhibiting biztdirig l,~etween a Cav3.1 aalciurn channel splice variant and AhTX TII), Inhibition of binding between a Ca"3.1 calcium channet splice variant and AbIX zIf sometitr~s is effected by binding of the antagpnist to the calcium, channel, to ANX IZI or to a complex fornned by the calcium channel and ANX ItI. In some embodiments, a nnolecule ~3 administered is a compound described in U.S. patent application publication no. US-20Q4-0034~35-l , A1 published February 19, 2t7Q4; U.$. patent apglicatun publication no. U5-2(14-00~4004~A1 published March 4, 2004; U.S. patent application no. 101763,974 filed Ianuary 22, 2004 and U.S.
patent application no. ~01474,~64 filed May 30, x.003. In other embodiments, a rnafecuta administered is art antibody that specifically binds to a Ca"3.1 aatcium charuael associated with a cell proliferative disorder (~ ~., Ca"3..t ac andior Cav3.l b), ar an antibody that specifically binds to .ANA
lII. An antiba~ly that binds to a calcium channel often intribits or blocks the function of the molecule andlor inhibits the interaction between the calcium channel and A1VX
III. An antibody that binds to ANX III often inhibits tire interaction between tfre calcium channel and ANX III.
j0U72~ The arttaganist often is formulated as a pharmaceutical cant~rsition with a pharmaceutically ~acceptat~(e carrier. As used herein, the terrrt "pharmaceutically acceptat~le carrier"
inc;;adc.s sofveras, ulnpesmiun u~dia, coatings, antibacterial and antifungat agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical adrrrinistratian.
Supplementary active compounds sometimes are incorporated into the compositions.
[0073] A phsrfnaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, ~.g., intravenous, intradenxraf, sut~utanenus, oral (~.g., inhalation), transdermal (topical), transmucosal, and rectal adrrtinistratian. Solutions or suspensions used for patr~nteral, intradern~al, or subcutaneous apglicatian can include the following eaxrarponents: a sterile difuent such as water far injection, saline solution, f red ails, polyethylene glycals, glycerin, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid ar sodium bisulfate; chelating agents such as ethylcnediarrtinetetraacetic acid;
buffers such as acetates.
citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride ar dextrose.
gI~ can be adjusted with acids or bases, such as hydrtichlaric acrd or sodium hydroxide. The Qarenteral preparation can be enclosed in ampoules, disposable syringes ar multiple dose vials made of glass ar plastic.
~0474J t,~ra1 catrrpasitians generally include an inert diluent ar an edible carrier. 1~or the ptupc~se of oral ttrerapeutic administration, the active compound can be incorporated with excipieiats and used in the farm of tablets, troches, or capsules, e.g., gelatin capsules.
Oral compositions can else be preparred using a fluid carrier for use as a rrroutlewasft, k'harrrAaceutically compatible binding agents, andlar adjuvant materials can be included as part of the composition.
The tablets, hills, capsules, r~roches and the Like cart contain any of the following ingredients, ar eompourxds of a similar nature: a binder such as micracrystalline cellulose, gum tragacantll or gelatin; an excipient such as starch or lacdyse, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotss; a glidant such as colloidal siliean dioxide; a sweetening agent such as sucrose or saccharin; or a Elevating agent such as peppermint, nnethyl salicylate, ar orarfge flavoring.
[OO~Sj Pheutical comgasitions suitable fdr inject~ble use include sterile aqueous solutions (where warEr soluble) or dispersions and sterile powders for the exGerx~paraneaus preparation of sterile injectabla solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, baeteriostatic water, Cremophor FL'rM (I~ASF, Parsippany, lad) or phosphate buffered saline (PAS). In alt cases, the camposidan must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under tile conditzans of manufacture and starags and must be pzeserved against the contaminating action of microorganisms such.
as bacteria and fungi. The carrier can be a solvexit or dispersion r»edium ac~ntaining, far example, water, ethanol, polyol (for e~cample, glycerol, propylene glycol, and liquid galyethylene glycol, and the like), and suitable nxixtures there Proper fluidity can be maintained. for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of micrat~rganisms can be achieved by various antibacterial and antifungai agents, far example, parabens, Chloxabutanol, phenol, ascorbic acid, thimsrosal, and the like. In many cases, it is preferable to include isotonic agents, for exarnpie, sugars, polyslcahols such as mattnital, sarbitol, sodium chloride in the campositic~n" Prc~tanged absorption of the injectahle compositions can be brought about by including in the composition an ag$nt which delays absorption, for example, alun~inunrt moriostearate and gelatin.
[007~~ Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a cambinatian of ingredients described shave, as required, fallawed by filtered sterilization_ Generally, dispersions are prepared by incorporating the active compound into a sterile Vehicle which contains a basic dispersion medium and the required ether ingredian~ from those described. In the case of sterile powders for the preparation of stirrile injectable solutions, the methods of preparation often utili2ed a~
vacuum drying and freexe-drying which yieids a powder of the active ingredient plus any additional desired ingredient from a previausIy steriIe~filtered solution thereof.
[Oif~'T.i Far administration by inhalstion, the compounds are delivered in the form of err aerosol spray from pressured container ar dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, ar a nebulizer. Systemic administration can else be by transmucosal or transdermaI
rneans. Far trazzsmucosal or transderntal administration, penetrants appropriate to the barrier to be petymeated are used in tile formuiation_ Such penet~rants aro generally Known in the art, and include, far example, far transmucasal administration, detergents, bile salts, and fusidic acid derivatives.
Transmucosal administration can be accomplished through the use of nasal sprays ar suppositories.

For transdermal administration, the active compounds are formulated into ointrraents, salves, gels, ar creates (e.g., sunscreen) as generally known in the art. N~ol~cules can also be prepared in the farm of suppositories (e.g., with conventional suppository bases such as cocoa butter and ether glyceridas) or retention srtemas far rectal delivery.
[Df1?g] Itt one embodir~aent, active molecules are prepared vrith carriers that will protect the campCVnd against tepid elimination Pram the body, such as a controlled release formulation, including implants and tnicroencapsulated delivery systems, biodegradable, laiacornpatible polymers can he used., such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, cc~~iagen, polyarthaesters, and polylactie acid. Methods for preparation of such forrr~ulations will lee apparent to those skilled a the art. Materials can also lx; obtained camrnercially from Alza Corpo~xatian anal Nova Pharmaceuticals, hoc. Liposamal suspensions (including liposames targeted to infected cells with znanoclonal antibodies to viral antigens) can also be used as pharzna~ceutically acceptable carriers. These cart be prepared according to methods known to those skilled in. the art, far example, as descrilaed in U.S. l~atettl hTo, 4,~22,$i i.
[0079] It is advantageous zo formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit faun as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated, where each unit aantains a predetermined quantity of active compound calculated to produce the desh~d therapeutic effect in association with the required pharmaceutical carrier, [OU80] Toxicity and therapeutic efficacy of molecules and formulations can be determined dy standard phartr~ceuticxl procedures in cell cultures Qr experimental animals in which ~,Dso values (the dose lethal to 50'0 of the poputation) and Eb~, values (the dose therapeutically effective in 5!~
pf the population) sometirnss era determined. The dose ratio between toxic arid therapeutic effects is the therapeutic index and it can be expressed as ttwe ratio ~.D~~D~.
MoleGUles which exhibit high therapeutic indices often are utilized. 'While molecules that exhibit toxic side effects may be used, care should be taken to design a delivery systettt that targets such compounds to the site of affected tissue for minimizing potential clamagr~ to uninfected cells and reducing side effects.
[OOgI,3 Data attained Pram cell culture assays and animal studies can be used irt.formuIating a range 4f dosages far use in humans. A dosage of such molecules Lies ~ferably within a range of circulating coneer~tratinns that include the ):DSO ~vitla little or na toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
Par any mt~lecules used in the method, the therapeutically effective dose can be estimated initially from cell culture assays. .A dace may be.forn~ulated in animal models to achieve a circulating plasma concentration range that includes the ICSO (i.e., the concentration of the test compound which achieves a half maxixral inhibition of sytnptams) as determined in cell culture. Such ittfarrraation can be used to rn~are accurately determine useful doses in humans. Levels in plasma naay be measured, far example, by high performance liquid ehromatography_ @452] A therapeukically effective amount of protein ar polypeptide (i.e., an effective dosage) ranges from abaat U.001 to 3p ~/kg body weight, sometimes about 0.0 t to 25 mglkg body weight, often about Q.1 to 20 tnglkg body weight, and more often about 1 to lU mglkg, 2 to 9~ mglkg, 3 to 8 mgtkg, 4 to 7 mglkg, or 5 to ~r mglkg body weight. The prc~tain or polypeptide can be administered one tune per week for between about I to 10 weeks, sometimes between 2 to S
weeks, often between about 3 to 7 weeks, and more often for about ~l, 5, or 6 weeks. The skilled artisan will xpprecia~
that certain factors may influence the dQSage and tuning reguired to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health audlor age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, palypeptide, ar antibody cats include a single treatment or can include a series of treatments.
[OQ85] Iu fotrnulation ambodirnents comprising an antibody, a dosage of 0.1 rngrlc,8 of body weight (,generally 10 mglkg to 20 mg/kg) is often utilized. If the antibody is to act in the brain, a dosage of 50 mglkg to 1 t» mgtkg often is appropriate. Generally, partially human antibodies and fully human antibodies have a Longer half life within the human body than other antibodies.
Accordingly, lower dosages and less frerluent adnninistration is often possible. Modifications such as lipidatiatt can be used to stabilize antibodies and to enhance uptake and tissue penetration (e,g., into the brain). .,4 method far lipidatian of antibodies is desciabed by Cruikslaank et al., 1. Acquired Immune Deficiency Syndxames and.I3uman Retravirolagy I4: X~3 (197).
00084] Antibody conjugates can be used for modifying a given biological response, and the drug moiety is not to.be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, far example, a toxin such as abrin, ricin A, pseudarnonas exotoxin, ar diphtheria toxin;
a polypeptide such as tumor necrosis factor, .alpha.-.interferon, .beta.-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; ar, biological response rrtodifiers sucte as, for example, lymphokines, interleukin-1 ("Il;-1»}, interleukin-2 ("Ih-2"), interleukin-~ ("IL-f"), granulocyte macrophage colony stimulat4~ng factor ("GM-CS)~''~, granulocyte colony stimulating factor ("CS-CSF'~, or other growth factors. Alternatively, an antibody can be conjugated to a se,.cond antibody to form an antibody heteroconj~tgate as descKibed in rJ.S. Patent No.
A.,b7b,g80.
[d055] Far compounds, exemplary doses include milligram or microgram amounts of the compound per kilogram of subject or sarr~ple weight, for example, about i nrricrograsn per kilogram to about 5011 m~illigraens per l~ilogram, about I00 micrograms per kilogram to about 5 milligrams per kilagraFn, or about 1 microgram per kilogram to about 51~ micrograms per kilogram. It is understood z~

that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
[0086] Pharmaceutical compositions of active ingredients can be administered by any of the paths described herein for therapeutic and prophylactic methods for treating a cell proliferative disorder. With regard to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from pharmacogenomic analyses. As used herein, the term "treatment" is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic molecule to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward the disease.
Screening Methods [0087] Provided herein are methods for identifying a molecule that inhibits cell proliferation, which comprise contacting one or more cells comprising a Ca~3.1 calcium channel encoding nucleic acid and/or a Ca~3.1 calcium channel polypeptide with a test molecule, and determining whether the test molecule decreases cell proliferation, whereby a test molecule that decreases cell proliferation is identified as a molecule that inhibits cell proliferation. The Ca~3.1 calcium channel utilized in the process is associated with a cell proliferative disorder, and sometimes is a Ca~3.lac and/or Ca~3.lb and/or an excess of CaV3.lbc calcium channel splice variant. Thus, in one form of these assays, compounds may be evaluated by their effects on expression of splice variants characteristic of abnormal cellular proliferation or by their ability to inhibit the activity of these splice variants in transport of calcium ion. Also provided are methods of screening for a molecule that inhibits the interaction between Cav3.lac and ANX III. In one embodiment, this method comprises detecting SUBSTITUTE SHEET (RULE 26) the 1ev$I of interaction of the calcium channel splice variant (lac) and AN.~
~I protein the presence arid absence of the substance to be tested. A decrease in the interaction in the presence as compared to~ the absence of the test substance indicates that the test substance is a successful candidate molecule for treatment of cell proliferative disorders and fox inhibiting, cell proliferation generally.
In lieu of using the splice variant ,Fer se, a poiypeptide representative of the splice variant, i.e., that encoded by the diFferentiatin~ portion c~f the rnRN,A encoding the 1~ N
linlaer, can be used. As hated in .Figure ~, the sequence SKI;xCt7MAD,per.se is unique to Ca,,3.lac and the extended faun of this segmept into exan 2~5 includes sequences shared with the abnormally hi ftxly expressed Ca,,3.lbc. xn one spec:ifie embodiment, rlxe method comprises: (a) incubating a Cav3. lac palypeptide or substantially identical polypeptide thereof with a test molecule under conditions suf~tcie~at to pernnit binding between the polypeptide and the test molecule in a reaction mixture, (b) contacting AlrtJ~ 'flI with the reaction mixture under conditions sufficient to permit binding betwc~n the polypeptide and Abl'X III, and (e) detecting the presence or absence of decreased binding between the palypeptide and AI~1'.~~ IfI, whereby the presence of decreased binding between the polypeptide and ANA IlI identifies the test molecule as a nnolecule that inhibits the interaction between t~aV3.lac and ANA III. In such methods, the CaY3.lac polypeptide soznetinxes camprisEs ?5 ar more sequential anxino acids selected from a region spanning amino acid 1545 to amino acid 1570 of a Ca,~3.I ac T-type calcium channel, and in certain embodiments, the poiypeptide consists of the amint~ acid sequence S1~EKC~MAI7LMLI7I~VL4SGSSASAAS.
[t108$a A reaction mixture or system soexietimes is a cell free in vitro environment and sometimes is a celi~based environment such as a collection of cells, a tissue, as organ, or an organism. A
system is "contacted" with a test molecule in a variety of manners, including adding' rnalecules in solution and allowing theta to interact with one anotlt~er by diffusion, cell in3ection, and any administration routes in an animal. As used herein, the terns "interaction"
refers to an effect of a test molecule on a calcium channel nucleic acid or paiygeptide, At~t~ III nucleic acid or palypeptide~ ar complex between a calcir~m channel and ANX III, svltere the effect is sometimes binding between the test malececle and the nucleic acid or polypeptide, and sometiKnes is an observable change in cells, tissue, or an organism.
~OtlB~a Any rnethcxl far determining whether a calcium channel is inhibited or blocked can be utilized, and exarnpies of such processes are described in U.S, patent application publication na. US-2004-0034035-A1 published February 19, ~OU4; LT.S. patent application publication rta. US-2.t70~-004hOC14-A 1 published lVlarch 4, 2(IQ4; I3.S. patent application na.
101763,9"74 filed January ~2, 2Cx?4 and U.S. fratent application no. 60!474,864 filed May 30, 203. In an embodiment, a standard patch clamp technique is etnplayed td identify blockers of'I'-type calcium channel curFenrs. Briefly, 2~

HEK cell tines stably expressing a human a1G T-type channel are used for recordings (passage #: Q.
20, 37"C, 5°lu COQ. To obtain T type currents, plastic dishes containing semi confluent cells axe positioned on the stage of a ZEhSS A~rUVERT 5100 microscope after replacing the cntture rxxedium with external solution. Whale cell patches are obtained using pipettes (borosilicate glass with filament, tJ.D.: 1.5 mm, LD.: 0.86 mm, 10 cm length), fabricated on a SLITTER F 97 pullet with resistance values of ~S Nls2. increases or decreased in currents ors detected, to determine whether a test nnolecuIe modulates the si,~nal of a T-type calcium channel.
Examples of other aSSays are described in Example 4 hereafter.
[It090) Any other z~nethod for determining whether the test molecule interaucts with a calcium channel or for determining whether an interaction between a ealciucn channel splice variant and ANA III is inhibited can be utilized, Examples of suoh methods include, for example, titrametric, acidirnetric, radiometric, NMft" txeonolayer, polarographic, spectrophotvarretric, fEuoresaent, and ESR assays. Specific embodiments include fluorescence resonance energy transfer 1:FRE'T) assays, surface plasmon resonance assays, and certain heterogeneous assays.
[OU91] In FRET assay embodirr~nts, a fluorvphore label on a first, "donor"
molecule (s.g., the calcium channel) is selected such that its emitted fluorescent energy is absorbed by a fluorescent label an a second, "acceptor" molecule (e.~., ANX IIIy, which in turn is able to fluoresce due to the absorbed energy frvrn the donor (e.g., ):,afcvwiez er al., U.S. P'atent No, 5,631,169; Stavrianopoulos et cal_ I3.S. Patent No. 4,868, lU3). Alternately, a donor of a non derivatized palypsptide may be natural fluorescence energy of tryptoph~an residues. When Iabels are utilized, they are chosen to emit a different wa~eleztgth of light, such that the acceptor label may be differentiated from that of the donor. Since the efficien~ey of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can i~ assessed. In a situation in which 'binding occurs between the molecules, the fluorescence emission of the °'acceptox" rnalecule label in the assay should be maximal. ,A h~ET
>~inding event can be conveniently measured by standard fluvrometrac detectors.
[092] rn surface plastnon resonance assay embodiments, biospeCific interactions are detected in real time without labeling any of the ,interactants with detectable chemicals (e.g., Sjvlander &
TJrbarwic2k, .A,nal. them. 63: 233$-2345 ( 1991) and S2abo et at., Ctur. Opin.
Struct, Bial. 5: 8~9~-705 (1995)). Changes in mass at the binding surface, which axe indicative of binding events, result in aiteratians ~f the reftactlve index of light near the surface (the optical phenvmentsn of surface plasmon resonance (SFR)), which is a detectable signal used to monitor real-time interactions between biological mQleeules. This type of assay sometimes is referred to as biomolecular interaction analysis (BIA). In such assays, the calcium channel splice variant protein or ANA ILI
protein sometimes i$ IinlcEd to a solid support surface and the effect of a test molecule an the binding of the other added binding partner (e.g., the added binding partner is ANX III
where the calcium channel is linked to the solid support) is determined by detecting changes in SPR.
[0093] In other embodiments, the calcium channel, ANX III or test molecule is anchored to a solid surface in a heterogeneous assay. The target calcium channel or ANX III
molecule often is anchored to a solid surface, and the non-anchored molecule sometimes is directly labeled and sometimes is indirectly labeled. The anchored molecule may be linked to any suitable solid surface, examples of which include a surface of a microtiter plate, test tube, micro-centrifuge tube or silicon chip. In certain embodiments, the molecule to be anchored may be produced by recombinant processes as a product that includes a contiguous and heterologous polypeptide region, where the heterologous region is capable of binding to a molecule linked to a solid surface. For example, a calcium chamiel splice variant polypeptide or ANX III polypeptide may be fused to glutathione-S-transferase and then adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione-derivatized microtiter plates. The molecule to be conjugated to the solid surface (e.g., a calcium channel splice variant) may be linked before, during or after the other molecules (e.g., ANX
III and/or a test molecule) are added to the system, which are combined under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH).
Other techniques for immobilizing a calcium channel or ANX III molecule on a solid support include using biotin and streptavidin. For example, a biotinylated calcium channel or ANX III
polypeptide can be prepared from biotin-NHS (N-hydroxy-succinimide) using known techniques (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in wells of a streptavidin-coated microtiter plate (Pierce Chemical).
[0094] In heterogeneous assay embodiments, one or more non-immobilized components are added to the coated surface containing the anchored component or components under conditions conducive to binding. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions allowing for any molecules bound to the anchored molecule or molecules to remain immobilized on the solid surface. The detection of molecules anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed.
Where a previously non-irninobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface. In certain embodiments, an indirect label is a labeled antibody that specifically binds to the immobilized, non-anchored component, or an antibody that specifically binds to the immobilized, non-anchored component, which in turn is labeled directly, or is labeled indirectly with a labeled anti-Ig antibody, for example. In the latter embodiments, the label sometimes is an enzyme utilized in an enzyme-linked format. In embodiments incorporating an antibody, the antibody often specifipally binds to a calcium channel splice variant or ANX III

SUBSTITUTE SHEET (RULE 26) molecule without significantly interfering W th binding o~ the catciura channel to ANA III
polypeptide or binding oaf the test molecule, Such antibodies can be anchored to the solid. support.
j1109S] In alternative embodiments, a hotttogeneous assay conducted in a liquid. phase without a solid support can be utilized. In such an assay, the reaction products are separated from unreacted components by standard techniques that include but are not lirr~ited ea differential centrifugation (see, for example, Itivas, G., and Mirlton, A. P., Trends Biachem act Aug;1.8(~): 284-7 (1993));
chrarnatograptty (~ ~ , gel filtration ekxromatagraphy, ion-exchange chrornstography);
el~ectr~aphoresis (e.g., Ausubel et crL, eds. Current Frotocals in Molecular Biology , J. Wiley: htew 'York (1999)); irrurtunopreeipitation (e.g., A,usubel, F. et aL, eds. Current Protocols in lwloIecular Biology , d. 'Whey: New York (1999)) and mass sgectraxrtetry (e.g., r.T.S.
Pat. lVos. 5,547,$35;
~,605,7~5; 5,691.141; 5,849,542; 5,8fi9,2A.2; .x,928,906; 6,043,031, and x,194,144).
[i1p,96] Tlxe assay embodiments described above sometimres are conducted in a direct format or a compeEitive format. It1 the latter farnnat, an interaction betweetl a calcium channel splice variant, AN'X lII andlor a test molecule is determined by detecting an interaction of one component with a target component in the presence of another carnpnnent that interacts with the target component. In an emhodimertt, a competitive assay sometimes is conducted by monitoring the amount of an antibody bound to a calcium channel splice variant in the presence of ANA ~
andlor a test rnniecule, where the atztilxady and AY~IX III compete for binding to the calcium channel splice variant. In the assay embodiments described herein, a convenient measure of binding affinity can be calculated from signals generated by the assays using known methods. For example, a .K~, I~;, I~m, pre-steady sta#e kinetic constant, ICS, LDSO or Eb~ parameter can be utili~d to rank tesk molecules in the assays.
[pi197~ In certain entbodimetuts, modulators of calcium channel sptice variant or ANX III
expression are identified. For example, a cell or cell-free mixture is contacted with a rest molecule anal the expression of tlxe calcium channel splice variant ar A1~X III mRNA or poEypeptide is evaluated relative to the Eevel of such products in the absence of the candidate compound. When expt~ssian of the calcium channel splice variant or AlII mh.~'A or polypeptide is less in the presence of the test molecule than iri its absence, the candidate compH~und is identified as a stimulator of calcium channel or ANA III expression, for ex2~mpie. Calcium channel splice variant or ANX IIx mR.N'A~. or palypeptide expression levels can be determined by standard methods, such as thane described hereirk.
[UQ913J Where membrane-hound farms of a calcium channel era used, it trAay tae desirable to utilize a solubiIizing agent. Examples of such solubilizing agents include non-ionic detergents such a5 n-octylgiucoside, n-dodeeylglueasida, n-dodecylzt~alt4side, actanayl-N-metltylglucarnide, decanoyl-N-methylglucamide, Tritons ~~-104, Tritons ~-1,14,'1'~hesit~, Isotridecypoly(Ethylene glycol ethEr)n, 3-[(3-cholaxrtidapropyl)dixnethylamminio3-1-propane sulfonate (CHAPS), 3-[{3-cholamidaprapYltItYlammittiol-~-hydmxy-1-propane sultanate (CHAP~Q), ar N'-dadecyl-~t,N-diznethyf-3-atrumanio-1-propane sulfonate.
[0099] The foliowin~ examples are intended to Illustrate hut not tcs Iirnit the invention.
Exarno_le 1 fiissue samples [0100] Glioma samples were obtained with informed consent from seveat patients who underwent craniQt~amy far brain tumor resection. The extent of tk~e resection and standard neurosurgical methods were not modified for the purpose of this study. Part of tlxe specimen was removed, quickly frozen in liquid nitrageta and stared at-$0°C until RNA preparation. Another part ores seat for routine neurapathalogicai evaluation. The clinicapathalagical data of the patients are presented in Table 1. fix of the tumors were eategarized as astrocytoma and One was aligodendrogliarna (case 01 ). The astraocytocnas ranged in World Health OrgartizatiQn (WHU) grade (Kleihe~es et csl. 1~~4) from ZI (increased aelluIarity and xniid atypic without mitoses, endothelial proliferation and necrosis) to IV (marked cellutar heteragenity, cytaplasmic and nuclear pleomorphism, mitotic figures including atypical farcKts, endothelial proliferation and necrosis) Grade ITI tumors were differentiated fxom Grade fV by the absence by endothelial proliferation amd necroses. The dligadendroglioma was anaplastic WHO Orade III (high cellularity, widespread nuclear atylaia and pleamorphisrn, high mytotic index, endothelial prolifaratian and necrosis).
Table x. Clinuopat~~olrrgical d$t~a of patients undergoing xesechau of a glivrita p'atien~t 'Vi~tO Olassi$cationbest Recurrence 01 III M Yes 02 III F ~ Yes 43 III M ' 'Yes 04 IV 142 Na 0~ III M Na O8 IEx M ~'es .-11 1T M hla * World Health organization {I~leihues et ,~L, 189).
Fxam_pte ~
Cell culture and tran~xent tratl5fecticxn [01(il~ primary cultures of human fetal astracytes (~99~'o purity) are prepared and maintained as described ict Corley et ctl. (2000). The glioma cell lines LT87, L?2Sl.N
and U56~ are prepared and u$ed as previously reported (l3esson and'Yong, 200x). These cells were cultured iz~ I7ulbecco's modified Eagle's nzediurn (l~MEhri) containing I O%a (vJv) fetal calf serum with L-glutaxnine (~~, radium pyruvate (t~ and 1% (viv) null-essential amino acids. They were maintained at 37°C in a~hurnidified atmosphere with 9~% air and 5% CC7~andgrawn until cvnfluency.
The medium was changed every two days. Tissue culture and transfection of tsA-X01 cells is described. in detail in l~eedle et aL (2002). ~ricfly,1~EI~ tails were ,grown to 85% contluency at 37°C (5~'o Cc~27 in »ivIEIVI (tl0~ro fetal bovine serum, 20(li1/rnI penicillin and f).2mgerrU
strept~amycin, Life Technoltrgies, Tnc.). Cells were dissociated with txypsin (4.~~%)-BDTA and plated an glass caverslips. All of the above solutions used far cell culture were purchased from Gibca~~RL. The Ca,,3.iac and Ca~3.lb variants (~pg) and green fluorescent ~rt~tein (I~,g) DNA
were trarlsfected into teals using the calcium phosphate method. Cells were transferred to 28RC 2~1.
hours after transfectian and recordings were performed two days later.
Example 3 RNA, isofatian and RT-PCR
[~Ia~;] Total RNA from gliaxna sazreples and cell tines as wet! as from primary cultures of fetal astrocytes was extracted following a ~'rizol protocol (Life Technolagles), l7Nase treated, extracted using phendtlchloroform extraction and resuspended irt bFPC-treated water. The RT-13CR forward (5'CA. ,t"rTTACCGGTC'x~GGT~CCGCACAA_3') and reverse (5'GAATG"FGGGGGCTGCTGfiiTA..t",.TG~.'fCCAT3°) primers were directed to the I~I-TV' lintker and were based on the hun~arl CAChiIG sequence (Genbank accession number A~1269f5). The detailed protocol for RT-F'CR is described irt Latour et aZ. (2f'l03).
[d7,(I~] As illustrated in Figure ~A, splicing of axon 25 to axon 27 results in CaV3.la or the Ca,,3.ib variant. Splicing of axon 25 to axon 26 ~esuit~s in tire Ca"3.lac ar Cav3.lbc variant. To determine Ca"3.1 gene expzussion in human whole brain, glioxna samples and.
glioma call lines, RT-PCIt analysis was performed using printers directed to the Ill-IV Iinker regiaxr of Ca"3,1 which contains these axons. Agarose gel analysis ofRT-PCR results can hurnxa adult brain (Figure 2B lane 1) reveals the expression c~f two different Ca,,3_1 mRNA transcripts. The Iawer molecular weight prodr~ct likely carresgonds tc~ CaV3.1 a while the higher weight product probably aamespands t4 the Ca,,~,lbc isaform. ftesuits obtained with RT-Pof fetal brain (Figure 2~ lane 2) suggest expression of Ca"3.lbc (top band), but not of Ca,.3.I a. The faint lower band indicates mRNA
expressxan of Ca,,3.lb in fetal tissue. These results are carlsisterxt with previous findings by {i) showing mRl~TA expression ~7f Ca"3.1a, b and be in the brain and (ii) slaawing differential expression of exam 25 variants, with Cav3.lb being stra~n~ly fetal-specific and Cav3.la being mire abundant in the adult brain (Manteil et aL, 2Ci00). To determine Ga"3.1 expression in glionna, RT-I'~It, analysis was parfornxed an U251N giiama cells (Figure 2B lane 3) and gliozna biopsies (Figure 2B lane 4) using the same primer pairs. Agarase gel analysis revealed the presence of a higher molecular weight ~rocluct in lanes 3 and 4, suggesting iha presence of a novel, longer isoform of the II1-IV
linlter expressed in glxoma, Sequencing of the 1t~'-1'CR product confirmed the presence of a novel.
variant, ~av3.lac. These results are evidence of tumor specific expression of a Cav3.1 channel splice variant in human trrain.
[01U4] To further investigate the differential Ca~3.1 gene expression in, adult and fetal normal brain and glic~rna, ,ten clones were sequenced from each RT FCR reaction and the relative abundance of Cav3.la, b, be aztd ac were determined for each condition, pfistributiort of the variants is shown in Figure 4A. Consistent with the literature (ManEeil et al., 2()CIO), sequencing revealed the exclusive expression of Ca"3.lbc in, fetal brain and a predominant expression of Cav3.la in adult brain. since glioma frequently arises from astroeytes, RT-l~CR analysis was performed on human fetal astro~cyte cultures to evaluate Ca"3.1 gene expression in normal astracytes. As illustrated in Figure ~, Cav3.11~ is predo~minaritly expressed in fetal astrocytes, consistent with fetal whole brain RT-PCIt results. Sequencing analysis of the IT563, U$7 and IJ2~lhl' gliama cell lines demonstrated that these cells mostly express Cav3,lb and Cav3.tbc. Since these cells are derived from adult 6rair~ tissue, a predarninant expression of Cav3.la was expected. The presence of the Cav3.lac variant in the LTZS1N Dell line and in three giioma sarreples was identified. 'his variant was observed only nn gliorna and not in normal tisS~ue, suestic,g a gliaxna-specific expression of Cav3.lac, which is evidence pf a differential gene expression of Cav3. I in gliorna, as gliotnas mostly express fetal isoforms and the Cav3.lac spline variant.
Examale 4 ~lectJah,~ol~x (llxOS] Glass coverslips carrying transfected ce(Is were transferred to a 3 cm culture dish containing the recording solution (2IimM BaCl2, 1mM MgCl2, ltfmM I~EF'E~, 4UnnM
tetraethylammanium chloride,1t?mtvl gluctsse, 65miM CsCt, pH 7.2 with TEA-Qki). Calcium channel activity in tratwsfeeted tsA~201 cells was characterized via whole-cell patch clamp recordings using an .Axapatch 2(fOB amEplif'~r (Axon Instrmnents, Faster City, t:A) linked to a persanai computer equipped with pC~.AMP v8Ø Patch pipettes (Sutler barasilicate glass, EF15U-SG-15) were pulled (Sutler P'-87 micraelectrode pullet), fire polished (Narishige) and showed typical resistances of 3 to 4 MS2 when Tilted wish pipette Solution (in cnM: 108 CsMeSQ~, h lVlg~lx, 9 EG~'A, pH 7.2). All data fig~erss, fits and statistics were completed using SigrnaPlat 2004 (51'SS
Inc).
[QlOGj Splicing of the III-.TV linlter has prGViausly been shown to alter the biophysical properties of T-type channels (Chemin et aL., 2001a). The IfI-fV linker region of CaW3.l ac was subclaned into a full-length Ca"3.1 channel, and transiently expressed the variant in tsA-2ta1 Bells for electraphysialogical characterization. Both clones expz~essed well in HEK
cells arLd produced typical fi-type eurxent densities and wavefarrns. Figure 3 compares the biophysical pxapertie~ of Cav3.lac to the Cav3,lb variant (Beedle et al., 2002). As shown in Figure 3, there was no statistical difference in the position of the current voltage-relations (Figure 3A), the voltage-dependences Qf inactivation (Figure 3$), oar the majority of time constants of inactivation (Figure 3C). Current densities also were similar fox bath variants. "there was, however, a statistically significant slowing in the time course of recovery from inactivation far the Cav3.lac variant (1~igure 3D). These observations suggest that the gIiama-.specifio Ca~3.lac variant displays electraphysiological cha~,Cte~stics that are similar to those observed with Cav3.lb.
[0107j ?here results pxavide evidence far law voltage-activated calcium channels in human gliama. Liven that a range of different calcium-mediated intracellular cascades are involved in cancers, this suggests that tha activation of those channels and subsequent calcium influx likely contribute to calcium signaling irt gtioma cells. Since Car,3.1 ac is Likely ea contribute to tumor ,growth, it is interesting that the laiophysical properties of this channel are virtually indistinguishable from Ca~3.lb expressed in tsA-2A1 ceps. However, it has been spawn that Ca"3.3 T-type calcium channels expressed in a nenranal cellular baclcgraund can show different electraphysialagical characteristics compared tQ channels expressed in tsA-201 cells (Chemin er r~L, ZOClIb). xhis difference can be due to the presence of neuron-specific xnteraeting proteins.
The ~a~3.lac variant contains the longest amino acid seqt.~ence of all domain l~-IV linker splice variants, thus it is possible that sptioittg of this region could lead to the creation of an intcractian site far neuronigtial specific regulatory proteins which may affect channel function, aT be involved in intracellular signaling events mediated by channel activity. Tt zs spawn in Example G that a Cav3.lac variant interacts with annexin T1I while other Cav3.I isafoz~ms da r<ot.

Exam le S
Invuunofluarescence ~l To determine iP gliama cells express T-type calcium clsannels, irnrraunostaining was performed an gliarna samples and on a gliom~a cell line using a rabbit polyclonal Cav3.l antibody directed to the rZ-11I Iirnlcer region 4f the oc, subunit (Latour ~x al"
2003). The presence of T-type channels art U251x3 ,gliarua cells was assessed using Cy~3-conjugated anti-rabbit secondary antibody. As shown in representative 40X (Figure I,A) and 100X (Figure 1B) cactfacal images. mast of the r3251N cells display abun~:iant expression of the Ca~3.1 channel.
bauble-imrnunc~staining ~svas performed on paraffin-ezni~r3ded glictma sections using the giial ~brillary aaidlc protein (CxFAF) mouse monoclonal antibody in addition to the T-type channel antibody. The fluorescent anti-manse CyTM2-con,~ugated secondary anti6ady was used against the manse primary (CxFAP) antibody. This dual staining canf'n-med the expression of ~ay3. I channels vn astracytes fn sine. A confocal image of T-typE channel inamunafluarescance in a malignant astrocytama is shown in Figuze 1C, The staining reveals clear expression of ~a~3.1 chatutels in the tumoz. The pt~senee c~f astraeytes in the tissue was canfirtned by GrFAP itnmunoiabeling (Figure 1b). The ca-expccessiaxt of GFAP and T-type char;nals is assessed in Figure lE. CelIs were pre-incubated with the Ca"3.1 aantrol peptide and stained following the same pratoool to car4~'trm antibody specificity. 'this procedure significantly decreased the brightness of the immunofluorescence signal in glioma. ~,s an additional caritrol, gliatna cells and sections also were ir~ubated in the presence of the fluorescent secondary an~,tibody alone and no signal was detected. This evidence shows expression of a voltage-Baked talciurn channel in human gliatna cells and demonstrates that glioma and cultured glianna cells abundantly eMpress ~av3.1 channels, [Oi~j The U251N glioma cells tvez~ grwwn until contluencp in a culture dish and detached fxam the dish with a 3-5 min. 37°'C incabatian with trypsin. culture medium was added to the resuspension sc~le~ian and cells were plated directly onto glass Coverslips in 2~F-well plates. Cells were grown far an additional 24h, fixed with a 15 rain, irteubatian period. in 4~'o paraforrnaldehyde at 4° C and stained using a pratQCal described in Latour et al., 0003).
Paraf~r~-embedded glioma sections obtained from tlm Foothills I~nspitai pathology services wets deparafiFirdzed in xylene and taktydrated irt a descending ethanol ,series (1(IQ~o, gStYo, SO~o and 70'0).
Tutttar sections were then washed twice itt ~'BS and incubated for Ih at room temperature in a fcnsh f''BS solution coatainlng 59'o normal donkey serum (3acksan Tmmunol~esearch). Slides were ttaen transferred to a fresh >?BS
solution containing 0.0'1°~o BaA (Sigma), mrtuse anti-gNal fibrillary acidic prcaiein (GFA,P) (1:3000) and rabbit anti-~ocla (1:5000) overnight at 4°C. The next clay, the sections wexe washed tluee effnes in PBS and were incubated at 4°C overnight in a firesh FBS solution containing Cy~"°'3-conjugated donkey anki-rabbit 1gG (1:2UD0, Jackson .Isnmuna~esearch) and Cy~'"2-conjugated donkey anti-rnause Igt:'x (1:2!700, Jaeksan rmmunaResearch). The next day, the sections were washed again tlvee tim~a with cold P$S and xnaunted with Fiuarsave (~~lbiochem).
Example 6 Interaction of Ca,,3.lac with Annexin Z1I
[01,10] liT PCR was used tc~ examine the presence of Cay3..! T-type channels in human gtlotrta.
Using this approach, a splice variant of Cav3.1 (Cav3.lac) was identified that contains both crops 25 and 26 in the intracellular loop connecting domains Ill and IV (Figure 2).
This variant was seen in surgery samples from gliama patients, and in the LT251N human gtioma cell line. Moreover, the variant was detected in the human retinoblastama cell line Y 79, rn contrast, this variant was pat present in normal human brain ar auitured human astrocytes, which expressed predominantly the previously identified Cav3.la anti CaV3.lbc variants. The full length cl~hTA
of Ca"3.lac was assembled and functionally characterized in tsA-~O1 cells. In a pul! dawn assay using GST-fusion proteins Qf various da~ain ).lI-IV linker splice variants, the ac variant was selectively able to pull down a protein band in the 30-40 kDa range (Figure 5). This band was excised, digested and subjected to fingerprinting via mass spectrometry, with a positive identification of the band as annexln ITI (AI'T~ lIi). The identifiaatian and specificity for Cav3.lac was subsequently aonf~u~ed via additional pul! downs and Western blotting with an ANX Ill antibody (Figure 5). Her~ee, a Ca"3_1 splice variant that appears to be selectively expressed in mitagenic cells can form a complex wills ANX IfI.
[Olli~ Human anxxexins are a family of 13 di~'ererlt calcium binding proteins with wide distribution across different tissues (Oerl~, V. and Moss, S.B. 2002.
Annexins: prom sttretare to function. FhysioZ. Rev. 82, 331-371). They appear to share an ability to bind lrhasphaligids (Raynal, P., arid Follard, H.B.199d.. Annexins: the problem of assessing the biological role far a gene family oi' multi~unctianal calcium- and phosphalipids-binding proteiats.
Bivchim. Biaphys.
Actor 1197, ~3-93. Swairjo, M.A., and Season, B.A. 1,994.. Annexin structure and membrane interactions; a tnalecular perspective. Annec, l~ev. Biophys. Biomol. Srruct, 23,193-213. F'erron, B., Lewi-T3entley. A., ~"xeny,13.. and Russa-Marie, F.1997. Can, enzymatic activity, ar otherwise, be inferred fCOrn structural studies of annexin 1II? ,l. Biol. Chetrz. 272,11321-LI32~_ Sapkova, J., ltaguenes-Nicoi., C., Vincent, M., Chevalier, A., Lewit-Bentley, A., Retsso-Marie, 'F., and Gaitay, J.
20x2. c:a(2+) and membrane binding to annexin 3 modulate; the structure and dyn~nics of its N
3$

terminus and domain Iii. l~ratei» ~'ei. xi,161~-1625.), and some ;members of the annexin family have been shown to form calcium perrr~able pares in bilayers, while others have been linked to cell signaling in tumors llfygaaxd, S.T., Flaugiand, H.I~., Kwistoffersen, E.K., Lund-datZansen, M., Laerum, t~.D., an;d Tysnes, C>.1~.1998. Expression of artnexin II in gliotna cell lines and in brain tumor biopsies. J. Neuro~o»ca~ a8,11-18.). ANA-I, IV and VI have been Shown to modulate calcium, potassium and/or chloride channel activity (I~aciff, J.M., Behbehani, M.M., Kaetzel, M.A., and Dedman,1~~ t 996. Annexin YI Enadulatcs Ca~~ and I~.+ conductances of spinal cord and dorsal rootganglion,neurons, Am, T. Fhysioh Cell Fhysiol. 271, C2()04-C2CJ15.
Kaet2el, M.A,., Chang Chars, .Ii.> Dubinsky, W .P., I)edman> J.R., and Nelson, D.J. 1994. A role for annexin IV in epithelial cell function. Inhibition of calcium-activated chloride conductance. ~: Bial, Cherra. 2b9, 5297-53(15.). Although not neuron specific, A,NX III is expressed in DRS
neurons and in astrocytes (Naciff, J.M., Kastzle, M.A,> Behbehani, M.M. and Dedman, J.It. 1996.
Differential expression of annexin,s I-IV in rat dorsaE root ganglia and spinal oord. J Comp, ,NeurQl, 368, 356-37fi.), and has been linked to a host of intracellular signaling events, such as inhibition of phpspholipase A2 activity (itaynal, P., and I?otlard, ?~LB. X994. Annexins: the problem of assessing the biological role for a gene family of multifunctional calcium- and phospholipids-binding proteins. ~iochim.
Biophys. Acts 1197, 63-93.). AbrX III is comprised of faun homologous domains and has a predicted molecular weight of about ~6 kDa (Favier-pearon,'E_, Lewit-.Bentley, ~A~., and Russo-Marie, F.199~6. The high-resolution crystal structure a~ humane anttexin III
shows subtle differences with annexin V. Biochemistry 35, 1740-1744.x. ANX III snay regulate calcium chanuael activity, and calcium influx through Cav3,1ac channels may stimulate cell praliferati<nn via ANX III.
[01X2] Calcium entry via L-type channels mediates the dissociation of preassociated calmadulin from the channel, wlxich results in a downstream activation of CREB
mediated gene transcription. Considering that AriTX-III is also a calcium binding protein, its association with CaV3.1 channels is similar to this mechanisrrt. As tire expression of Ca,,3. l ac channel isofarm is specific to znitogetxic cells, it is possible that Cav3.1 ac is re,~uired far proliferation, and that the ,ANA-IIIICav~.lac interaction plays a role in proliferation. It has been shown that T-type channel blockers and knockdown of T-type ahan,nels can inhibit prali~eration of Y-79 cells (Bertotesi, G.E., Shi, C., Elbaum, L., 3o11icnore, C., Rozenberg, G_, Barnes, 5., Kelly, M.E.
2Q02. The Ca(2+) ehazlnei antaganist5 mibefradil and pimozide ii~liibit cell ,growth via different cytoxic naeChaniszns.
.A~foL .lslurmrntu~ol. 62, 210-219.).

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Overexpressaan of an alpha i~I (Ca"3.~) T-type calcium channel daring neuroenttocrine differentiation of hunnan prostate cancer cells. J $iol Cherxi 2'77:10824-33.
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Cysteine stritx~
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Neuron '8,195-ZU4.
Nucleotide Sectuenre and Amino Acid Sectuence $mbodime~2ts z3uman aZpha~.G ao splia~ variant (SEQ ZD N0, 11 ~p,~~GGACGArGAGGAGGATGGAGCGGGCC',,CCGAGGAGTCGGGFICAC',,GCCCGGAGCTTCAfiCCGGC'1"C
AACGACCT
GTC GGGCCGGCCGGGGCCGGGGTCAGCAGAAAAGGACC!CGGGCAGCGCGGACTCCGAGGCCGAGG
GGCTGCCG1'ACCCGGCGCTGGCCCCGGTGGT'i'TTCTTCTACfiTG~I.GCCAGGACAGCCGCCCGCGGAGCfiGGT
GT
CTCCGCACGGTCTG'1'AACCCCTGGTfiTGAGCGCA'z'CAGC~.1,',C'G7:
I'GGTCATCCT'.T'CTGAACTGCG'T'GACCCTGGG
CATG'.~TCCGGCCATGCGt~GGACATCGCCTGTGACTCCCAGCGCTGCGGGATCCTGCACGCCTTTGAfiGACTTCA
TCTTTGCCTTCT'I'TC~CCGmGGP~.GATGGTGGTGAA,GATGGTGGCCTTGGGCATCTTTGGGAAAAAGTGTTAGCT
G
C~,SAGACACT~'GGA,ACCGGCTTGACT'TTTTCATCGTC,ATCGCAGGGATGCfiGGI~GTACTCGCTGGACCTGCA
GAA
CGTCAGCTZ'CTCAGCTGTCAGGACAGTCCGTGTGCTGCGACCGCTCAGGGCCATTAACGGGGTGCCCAGCA2'GC
GCATCCTTGTCACGTTGCTGCTGGATACGCTGCCCA't'GCTGGGCAACGTCCTGCTGC't'CTGCTTCTTCGTCTTC
'L'TCATCT'TCGGCATCGTCGGCGTCCAt~CTGTGGGCAGGGCfiGCTTCGGAACCGAfiGCTTCC~'ACCTGAGAAT
TT
CAGCCTCCCCCTGA4s'CGTGGA,G:CTGOA,GCGCTZi,'LTACC~'sGAGA(",'TAGAACGAGGA'X'GAGAC7CC
CC'x'TCATCTGCT
CGCAGCCACGCGAGA1~CGGCAfiGCGG2'CCTGC~.GAAGCGTGCCCACGCTGCGCGGGGACGGGGGCGGTGGCCCA
CCTTGGGGTC'TGGACTA'z'GAGGCCTACAACAGGTCCAGCAACACCACCTGTGTCAACTGG13ACCAGTACTACAC
C,AACTGC'T,'CAGCGGGGGAGCACAACCCGTTCAACaGGCGCCA'~CAACT'I'TGACAACAT'Z'GGCTATGGCTG
GATCG
CCATC'I'TCCAGGTC.~1TCACGC'T'GGAGGGCTGGGTCGACATGATGTACTT'TGTGATGC,,ATGCTCATTCCTZ
'CTAG
AATTTCATCTAGTTCATCCTCCTCATCATCGTGGGCTCCTTC'I~TCATGATCAACG2'GTC~CGTGGTGGTGATTGC
CACGCAGTTCAGTGAfsA,CCAAGCA,raCGGGAAAGCCAGCTGATGCGGGAGCAGCGfiGTGCGGTTCCTGTCCAACG

CCAGCACGCTGGC~'AGCTTCTCTGAGCCCGGCAGCTGCTATGAGGAGCTGC'~CAA,c'.,TACCTGGTGTACATCCT
fi CGTA1~GGCAGCCCGCAGGCTGGCTCAGG'I'CTCTCGGGCAGCAGGZ'GTGCGGGTTGGGCTGCTCAGCAGCCCAGC
ACCCCTC
,CxGGGGCGAGGAG1~CCCAGCCCAGC.?~GCAGCTGCTCTCGCTCCCACC(~CCGCCT.ATCCG'x'CCACCACC
TGGTGCACCACCACCACCACCA'x'CACCACCAGTACCACGTGGGCAATGGGACGCx'CAGGGCGCCCGGGGCCAGC
CCGGAGATCCAGGAGAGGGATGCCAATGGG'~CCCGCA.GGCTCATGCTGCCACCACCC'~CGAGGCGTGCCCTCTC
CGGGGCCCCCCCTGGTGGCGCAGAGTCTGTGCACAGCTTCTAGGATGGCGACTGCCAGTTAGAGCCAGTCCGCT
GCCAGGCGCCCCCTCCCAGGTCCCCATGTGAGGGATCCGGCAGGAC"t'GTGGGCAGCGGGAAGC",TGTATCGCACC
GTCC.A,CACCP,GCCCTCGACCGGAGACGCTGAh~GGAGAAGGCACTAGTAGA(',GTG
.~,aC'!,'GC4"AGCTt,~~'1,'GCiGCCCCC
AAGCCTCACCAGCCTCAACATCCCACCGGGGCCCTACAGCTCCA,TGCACAAGCTGCTGGAGACACAGAGTAC.t~G
GTGCCTGCCAAAGCTCTTGCAAGATCTCCAC~,CCCTTGCTTGAAAGCAGACAGTGGAGCCTGTGGTCCF1GACAGC
TGCCCCTACTGTGCCCGGGCCGGGGCAGGGGAGGTGGAGCTGGGCGACCGTf",AA23Te;CCfiGACTCAGACAGCGA

GGCAGTTTATGAGTTCACACAGGATGGCCAGCACAGCGACCTGCGGGACCCCCACAGCCGGCGGCA~,CGGAGCC
TGGGCCCAGATGCAGAGCCCA.GCTCTGTGCTGGCCTTCTCSGAGGC'tAATC°t'GTGACACCTTCC,(', A~1AGATTGTG
GACAGGAAGTAC'~TTGGCCGGGGAATCATGATCGCCATGCTGGTCAACACACTCAGGATGGGCATC.n.~AATACCA
CGAGCAGCCCGAGGAGCTTACCAACGCCCTAGAAATGAGCAACATCGTCTTG,ACCAGCCTCTTTGCCG'tGGAGA
TGCTGCTGAA,GCTGCTmGTGTATGGTCCCTTTGGC'i'ACATCAAGAATCCCTACAACATCTmCGATGGTGTCATT
GTGGmCATCAGCGTG'~C,GGAGATCGTGGGCC~1,GCAGGGGGGCGGCCTGTCGCTGCTGCGGACCTfiGCGCCTGAT

CGTCACTGTCT'I'TCAGATCCTGACCCAGGAGGAC'.PGGAACAAAGTCCTCTACAATGGTATGGCCTCCACGT'CGT

CCTGGGCGGCCCT'.~T,bITT'z'CAT'1'GCCCTCATGACCTTGGGCAACTACGTGCTCTTCAA~'TTGCTGGTCGC
CATT
CT
'GG'TGGAGGGCT2'CCAGGCGGAC7GGAGfITGCGA.~1CAAGr'I'CCGAATCA,rrAGCCCGATT'.~CTTCTCAC
GCAGCC'f' GGATGGTGA'rGGGGACAGGAAGAAGTGGTfiGc,CCTTGGTGTCCCTGGGAGAGCACCCGGACyCTGCGGAAGAGCC
TGGTGCCGCCTCTCATGATCCACACGGCCGCGACACCCATC~'CGGZ'GCCCAAGAGCACCAGCACGGGCCTGGGC
GAGGCGCTGGGCCCTGCGTCGCGCCGCACCAGCAGCAGCGGGTCGGCAGAGCCTGGGGCGGCCCACGAGATGA.~, GTCACCGCCCAGCGCCCGCAGCTCTCCGGACAGCCCCTGGAGCGCTGCAAGCAGCTGGACCAGCAGGCGCTGCA

GCCGGAACA~'',CCTCGGCCGTGCACCCAGCCTCaAAGCGGAGAAGCCCAAGTGG2aGAGCGGCGGTCG'CTGTTGTC
G
GGAGAAGGCCAC,GAGAGCCAGGATGAAGAGGAGAGCTCAGAAGAGGAGCGGGCCAGCGCfiGCGGGCAGTGAt~CA
fiCGCCACAGt3GGGTCCCTGGAGCGGGAGGCCAAGAGTTCCfiTTGrr,CCTGCCAGACACACTtAGGTGCCAGGGC
TGCA'~CCaCACT
,~"aCCAG'~C~GCCGAGGGTCTGCTTCTGAGCACCP,GGACTGCAATGGCAAGTCGGC'I'TL~AGGC'zCGC
CTGGCGCGGGCCCTGGGGCCTGA~'GACCCCCCACT'GGATGGGGATGACCCCGATGACGAGGGGAACCT'GAGCAA
AGGGGAACGGGTCCGCGCG'I~aGATCCGAGCCCGACTCCCTGCCTGCTGCCTCGAGCGAGACTCCTGGTCAGCCT
ACATCTTCCCTCC~'CAGTCCAGGTTCCGCCTCCTGTGTCACGGGATCAfiCACCCACAAGATGT'T'CGAGCACGTG
GTCCTTGTCATCAfiCTTCCTTAACTGCATCACCATCGCCATGGZ.1GCGCCCCAAAATTGACCCCC3?,CAGCGCTGA

ACGCA.TCTTCGTGACGCTCTCGAATTACRTCTTC.fi,CCGCAGTCTTTCTGGCTGAAATGACAGTGAAGGTGGTGCs i<ACTGGGCTGGTGCTTCGGGGAGCAGGCGTACCTGGGGAGCAGTTGGAACGTGCTGGACGGGCTGTTGGTGCTC
ATCTCCGTCA'T'CGAvCA'.tTCTGGTGTCCATGGTCTCTGACAGCGGCACCAAGATCCTGGt;CATGCTGAGGG'~G
CT
GCGGC'Z'GCTGCGGACCCTGCGCCCGGTCAGGGTGATCAGCCGGGCGCAGGGGCTGAAGCTGGTGGTGGAGACGC
TGATGTCCTCACTGAAACCCATCGGGAACATTGTAGTCATCTGCTGTGCCTTCTTCATCATTTTCGGCATCTTG
GGGGTC'aC~.GCTC'.I'TCAAP~GGGAAGTTTTTCGTGT6CCAGGGCGAGGATACGAGGAACATCACCAATAAATCG
GA
C',L'GTGCCGAGGCCAGTTACCGGTGGGTCCGGCACAAGTACAACTTTGACAACCTTGGCCAGGCCCTGATGfiCCC
TGTTC
,t'"xTTTTGGCCTGGAAGGATGGTTGGGTGGACA'.G'CATGTACGATGGGGTGGATGCZ'GTC'aGGCGTGGACCA.
G
CAGCCCA,TC.'~TGAACCACAACCCCTGGATGCTGCTGTAC'1'~'CATC'~'CGTTCCTGC'1"CATTGTGGCCTTC
T'~'TGT
CGTGAACA'rGTT'3,'GTGGG'~'GTGG'x'GGTGGAGAACTTCCACAAGTGTCGGCAGCACCAGGAGGAAGAGGAGG
CCC
GGCGGCGGGAGGAGAAGCGCCTACGAA,GACTGGAGAAAAAGA,GAAGGAGTAAGGAGAAGCA,t',7ATGGCTGATC' .l'A
ATGCTGGACGATGTAATTGC'I'fiCCGGC1~1GCTCAGCCAGGGCTGGGTCAGAAGCCCAGTGCAAACCTTACTAC'I
~
CGACTAGTCCCGCTTCCGGCTCCTCCa~~CCACGACfiTGTGCACGAGCCACfiACCTGGACCTCTTCATCACAGGTG
TCA,TCGGGC'TGAACGTGG'1'CACCATGGCCATGGAGCA.CTACCAGCAGCCCCA,GATTGTGGATGP.GGCTCTGA
AG
ATCTGCAACTACATCT'TC~.C'I'GTCAT'CTTTGTGTTGGAGTCAGTT~"TCAAACTTGTCiGCCTTTGGTTTCCC;
Z'CG
GTTCxTCCA~GGACAGG'I'GGAACCAGCTGGACCTGGCCAT'~GTGCTGCTGTCCATCAfiGGGCATCACGCTGGAGG

AAATCGAGGTCAACGCC'.C'CGCTGCCCATCAACCCCACCA'rCATCCGCATCATGAG(~GTGCTGCGCA'~'TGCCC
GA
GTGCTGAAGCTGCTGAAGATGGCTGTGGGCATGGc~GGCCCTGCTGGACACGGTGATGCAGGCCCTGCCCCAGGT
GGGfiAACCfiGGGAC~i'CTCTTGATGTTGTTGTTTTTGATCTTTGCAGGTCTGGGCGTGGAGC1'CTT'~GGAGI3.
CC
TGGAGTGTGACGAGAGACACGCCTGTGAGGGGCTGGGCCGTCATGCCACCT'T'TCGGAACTfiTGGCATGGCCTTC
CTAACCCTCTTCCGAGTCTCCACAGGTCaACAAT~,'GGAATGGCATTATC~AAGGAC,ACCGfiCCGGGACTGTGACC
A
Gts'T;GTCCACC."TGC'PACAA,CACGGTCATC'x'CGCC'.C'ATCTI'.CT'I'TGTGTCCTTCGTGCTGACGGC
CCAG'~'TCGTGC
TAGfiCAACGTGGT'GATGGCCGTGCTGATGAAC',CACCTGGAGGAGAGCAACA,AGGAGGCCAAGGAGGAGGCCGAG

CTAGAGGCTGAGGTGGAGCTGGAGA'f'GAP,GACCCTCAGCCCCGAGCCCCACTGGCCAC'I'GGGCAGGCCCTTCCT

CTGGCCTGGGGTCGAGGGCCGCGACAGCCCCGACAGCC4CAAGCCTGt"aGGCTCTGCACCCAGCGGCGCACGCGA
GATCAGCCTCCCAC2~TTTCCC7,'GGAGGACCCGACGATGCAGCCCCACCCCACGGAGCTGCCAGGACCAGAC2~A
CTGACTGTGCGGAAGTCTGGtaGTCAGCCGAACGCACTCTCTGCCCAATGACAGCTACATGTGTCGGCATGGGAG
CACTGCCGAGGGGCCCCTGGGACACAGGGGCTGGGGGCTCCCCAAAGCTCAGTCAGGCTCCGTCTfiGTCCGTTC
ACTCCCAGCCAGCAGATACCAGCTACkITCCTGCAGCTTCCCAAAGATGCACCTGATC~'GCTCCAGCCCCACAGC
GCCCCAACC'~'GGGGCACCAT'CCCCAAACTGCCCCCACCAGGACGCTCCCCTT~'GGCTCAGAGGCCACTG.AGGCG

CCAGGCAGCa~IA.TAAGGACTGACTCCT'x'GGACGTTCAGGGTCTGGGCAGCCGGG.~lAGACCTGCTGGC'AC"sA
Gfx'TGA
CxTGGGCCCTCCCCGCCCCTGC,CCCrGGCCTACTC~'~'TCTGGGGCCAGTCAAGTACCCAGGCACAGCAGCACTCC
CGCAGCCACAGCAAGATCTGCAAGCACATGACCCCGCCAGCCCCTTGGCGAGGCCCAGAACCCAACTGGGGCAA
GGGCCCTCCAGAGAr''CAGAAGC.AGCTTAGAG'x"TGGG'ACAC
.t'aGAGCTGAGC'~'GGATTTCAGGAK~ACCTCC'TGCCCC
CTGGCGGCCAGGAGGA,GCCCCCATCCCCACGGGACCTGAAGAAGTGCTACAGCGTGGAGGCCCAGAGCTGCCAG
CGCCGGGCTACGTCC'1'GGCTGGATGAGCAGAGGAGACACTCTATCGCCGTCAGCTGCCT'GGACAGCGGCTCCCA.
ACCCCACCTGGGGACAGACCCCTCTAACCTTGGGGGCCAGCCTCTTGGGGGGGCTGGGAGCCGGCCCAAGAAZaA
AACTCAGCCCGGCTAG'Y'ATCACCATAGACCCCCCCGAGAGCCAAGG2'CCTCGGACCCCGCCCAGCCCTCiGTATG
~'GGCTCGGGAGGAGGGCTCCGTCCAGCGACTCCAAGGATCCCTTGGCCTCTGGCCCCCCTGACAGCATGGCTGG
CTCGCCCTCCCCAAAGAAAc~ATGfiGCTGAG'Z'CTCTCCGGTTTATCCTCTGACCCAGCAGACCTGGACCGG"xGA
i~Iuma~z alphalG ac splice ~tra~iant Witk~ amino arid txanslatio~, tSEQ TD
A1C: 21 atrgg~acgag9'aggag'9'atggacrcg9gc3c~ga.ggagtcg9gacag~cccggag~tx~atg !~I D E E E D G A G A E E S G Q P R S F M
~9gotcaaag~,cctgtcgggggc~ggg9'3~c99eCg9fggG~ggggtcagcagaaaae~gac R 2~ N D ~ S G A G G R ~' G P G s A E K D

acgggcagcgcggactecgaggcggaggggctgcagtaccoggcgatggaaceggtggtt P G S A D S E A E G L P Y P A L .~. P V 'V
ttettctacttgagccaggacagccgcccgcggagCtggtgtctccgcacggtctgtaac F F ~'' L 8 ~ t1 S R P R fiu W ~.' Tr R 'T V C lIi ecctggtttgagcgcatcagcatgttg9tcatccttctcaactgcgtgaCCCtgggcatg pWFE~,'.I SMLVZLIrNCV2'LC~M
ttecggceatgcgaggacatcgcctgtgactcaaagcgatgccggatcctgcaggccttt F Ft Q C E D I A C I7 S Q ~t C ~t I L S~ A F
gatgacttcatctttgccttctttgccgtggagatggtggtgaagats~gtggcciagggc D D F t F A F P A V E I~ V V If M V A L C,t atetttgggaa~.aagtgttacctgggac~acacttggaaccg~gettgactttttcatcgtc 2 F G kt K C Y L G D T W N R L D F F z V
atagcaggcfatgctggagtactcgctggacCtgcac~aacgtcagcttctcagctgtcagg r A G M ?a .E Y S L h L t~7 N V S F $ A V R
acagtccgtgtgctgcgacogctcag$gccaetaaccgg9t9'cacagcatgcgcaCcatt T V R V L R P L R A I N R V P S M R I L
gtcaec~ttgatgctgga,tacgctg~cccatgetgggaaacg'teetgetgctctgettcttc 'V '.~' L L T., D T L P ~i L G N U T~ z. L ~ C F F
gtcttcttcatcttcggcatcgtcggcgtcCagctgtgggcagg9ct9cttcggaaccga V F F I F G I V G V Q L W A G L L R N R
tgcttcetaeatgagaatttcagcotccccctqagcgtggacctggagcgctattaacag C F L P E N F S L F L $ V D L E R $
acagagaaegagc~atgagagcaccttcatotgetcGCagccacgcgagaaeggaatgcgg T E 1V &: I7 'E S P F Z C S Q P R E N G I~ R
tactgcagaagcgtgcccacgctgrcgcgg9gacS'Sgg9'cJStg9cccacettgcggtctg S G R S V P T L 1~ ~'r D G G G G P P C G I.
gactatgaggactacaacagatacagcaacaccaactgtgtcaactggaacc~.gt~.ctaa D Y E .A Y N S S S N T T C V N W N' Q Y Y
accaactgc~.oagcgggggagcacaaccccttcaag~ggcg~acataaa.cttt.gacaacatt T ~3 C.' S A G ~.'' H N Y' F K G A I I3 F D I3 I
ggctatgcctggatagccatGttccaggtcatcacgctggagggctgggtcgaoatcacg G Y A tn1! T A I F ~ '~ I T L E G W V D I M
tactttgtgatggatgctcattcottctacaatttcatctaattaatectcateG.tcatc F V M D A I3 6 ~' Y' N F° I Y F I L r, I I
gtgggctccttcttca.t$atCaacCtgtgCCtgg'tggtc,~attgCaacgcagttaa.gtgag V G $ F F M I I3 L C L V V I A T ~ F S E
aceaagcagegggaaagccagctgatgcgggag~cagc9'tgtgcB9ttcatgtccaaagcc TKQRES QLMREQ~tVI~FL SN.A
agcaccatggctagcttctctgagccCCfgsagCtgctatga.ggagctgatcaagtacatc,~

gtgtaaatGCtGCgtaaggcagCCCgcagg~atggctCaggtCtctCgggCagcagc,~tgtg V Y' I L R K A A R R L A Q V' S R h. A G V
aJggttgggctgCtCagaagcGCac~'cacCCetcgggggacaggagaaccagcacagcagc R V G L L S S P A. P L G 6 Q E 'x" Q P 5 S
agetgetGtegctcceacegccgcctatecgtcCa~caacctggtgcaaeaccaccaccac a~ C B R S FI R R L S V H H T~ V H H ti H H
catcaccaccaetaecacatgggaaatggga~getGagggcec~eegggccagcccggag H H H FI Y H L G N G T L R A p R A S P E
atccaggacagggatgacaatgggtcccgcaggctcatgctgacaccaccatagacgcat I Q D R D A N G S R R L M L P P P $ T P
gacctatccggggcceaccctggtggcgGagagtatgtgcacagcttctaccatgccga,c AtrSt'aAI~P~"aGAESVH~"FYHAD
tgGGa4ttagagCCaC~tcGgCtr~CCaggCC~CGCCCtCecaggt~CCcatCtgclggCaCcc C H L E P V R C Q A P P P 1~ S F S E A, S
g JCagg~;1.ctcftgggCa9'Cgg9aaggtgtatcCGacCgt GCacaGCagCC ctCCa4Cc~ga.g G R T V G S G it V ~'' P T V H 'i' S P P F E
acgcCgaaggagaac~gcactagtagaggGg9ctgccagctctgggcccecaaccctaacc T L k E K A L V E V A A $ 5 G P P T L T

agcctcaacatCCCacCCgggccctacagctccatgcacaagctgctggagacacagagt SLNI1~~PGFYS SMIiIi.LLETS~S
acaggtgcctgccaaagetcttgcaagatctccagcccttgcttgaaagcagacagtgga T G A c Q s s a K z s s P c L ~e A D s G
gectgtggtacagacagctgcccctactgt9ccCgJgecJg9gca39JgaJgt~'gagctc A C G P D S C P Y C A R A G A G E V E L
gccgaccgtgaaatgactc~actca.gaeagcgaggcagtttatg~agttcacacaggatgcc A D Vii. L M P D S D S E A V Y E F T Q D A
eageaeagcgacctccgggaccaecacagccggcggcaaeggagCCtgggCCCag'atgca Q 'H S If L R D P H S F2 R Q R S L G P D A
gagcccagctctgtgctggccttctggaggctaat~tgtgacaccttcCgaaagattgtg E P S S V L A F W R T~ I C D T F R K . I V
gaCagcaagtaetttggccggggaatcatgatcgccatcctc~gtaaacacactcagcatg D S R Y F G R G 2 M I A T T~ V N T L S M
ggcatcgaataccaagagcag4ccgaggagcttaccaacgcectagaaatcagcaacatc G~ I E Y H E Q P E E L. T ~T A L E Y S N I
gtcttcaccagcctctttgccctggagatgctgctgaagctgcttgtgtatggtcecttt V F T S L F A L E M L L EC L L V Y G P F
ggctac~.tcaagaatCCCtacaacatcttegatggtgteattgkggtcatea.gcg~tgtgg G Y Z If ~T P X N I F L1 G V I V V I S V W
gaga.tcgtgggcCagCagggsgggeggcctgtaggtgctgcgga.CattccgcCtg~atgcgt E I V 6 Q Q G G G L S V L R 't' F R L M R
gtgctgaagctggtgcgattcctgccggcgctgcageggaagctggtggtgctcatgaag V L K L V R F Ta P A L Q R Q L V V L I~2 K
accatggacaacgtggccaccttetgcatgctgcttatgctcttcatCttcatcttcage fi M D N V A '~ F C M L L M L F I F I F S
atCCtgggcatgcatctcttcggCtgcaagtttgcCtCtgag~g'ggatggggaeaccCtg I L G M H L F G C TC F A S E R D G ~7 T L
ccagaccggaagaattttgactccttgctctgggccatcgtcactgtctttcagatcctg D R IC N k' D S L L W A T V T V F Q I L
acccaggaggactggaacaaagtcctctacaatggtatggcctccacgtcgtCCtgggcg '1'QEDWNKVLYf3GMAS~'SSWA
gccctttatttcattgecctcatgacctteggcaacta.cgtgcttttcaatttgctggta A L Y F I A L M T F G N Y V L F N L L V
gccattctggtggagggcCtccaggcggaggg~agatgccaacaagtacgaatcagagcce A I J~ V E G F Q A E G D ,,~ N K S E S E P
gatttcttctcacccagcctggatggtgatggggacage,~aagaagtgcttggccttggtg D F F S P S L D G 1~ G D R K K C L A L V
tccctgggagagcacccggagctgcggaagagectgctgccgcctctcatcatccacacg S L G E F~ P E L R K S L L p P ~r I 2 H T .
gccgccacacccatgtcgctgcccaagagcaccagaacgggcctgggcgagc~cJct9ggc A A T P M S L P K S '~' S T G L fr E A L Ca CctgcgtCgcgccgcaccagcagcagcgggtCggcagagcctggggcggcccacgagatg P A S R R T S S S G S A E P G A A H E M
aagtcaccgcccagcgcocgcagctctccgcaoagcccctggagcgctc~Gaagcagctgg K S P p S A R S S P H S P W S A A S S W
accagcaggcgctccagccggaacagcctcggccgtgcaccaagcctgaagcggagaagc T S R R S S kt N S L G R A 1~ S L T~ R R S
cc~gtggagagcggcg9'tccctgttgtcgB Jagaaggcc~.9'gagagccaggatgaaga.g P S G E R R S T, L S G E G Q E S Q D E E
gagagctcagaagaggagcgggccagccaCgcgggcac~tgaccatC~ccacagggggtcc E S S E E E R ,A $ P' A G S 17 H R H R G S
Gts~gagegggaggcaaagagttcctttga,CCtgccagaaaeactgcaggtgCCagggCtg L E R E A K S S F D L P D T L c~ V P G ~, catcgcactgccagtggccgagggtctgattctgagcaccaggactgcaatggcaagtcg H R T A S G R G S A S E H Q D C N G K S
gcttaagggcgcctggccegggccctgcggcctgatgacaccccactggaCggggatgac A S G R L A R A L R P D p p p L p G D p gcagatgacgagggcaacctgagcaaaggggaacgggtccgcgegtggatcCgagccGga A D D E G N L S K G E R V R A W I !2 A R
etaectgcatgctgcctcgagccgagactcctggtcagcctacatcttccetcctcagtcc L F A C C L E R. D S W S A '~' I F ~' P S2 S
aggttcegcctcctgtgtcaccggateatcaccoa.caagatgttcgaccacgtggteett R F R L L C H R I I T H K M F D H V V L
gtoatcatcttcvttaactgcatcaccatcgcCatggagcgcoccaaaattgaccccaac V I Z F L N C I T I A M E R P Ti I D P H
agcgatgaaagcatattcctgaccctctcoaattacatcttcaccgcagtctttctggct S ~,, E ft I F' h Z' L S P1 Y I ~' T A V F L A
gaaa.tgacagtgaaggtggtggcactgggct99't8attcggg3agcaggc3tacotgagg E M T V K V V ,A L G W C F G E Q A Y L R
ag~cagtt9~gaacgtg'ctggaagggctgttggtgatcatetccgtca,tcgacaCtatggtg S S V~I Id 'tt' L D G L L V L I S V I D I L V
tccatggtctctgacagcggcaccaagatcctgg'gcatgctgagggtgctgcggctgctg S M V S D S G T K I L G M Ir R V L Ti L L
Cggacca~.gegcccgctcagggtgatcagccgg~tcgca9'gggctgaagctggt3gtggag R T L R F' L R V I S R A Q ~G L IC L V V E
acgctgatgtaatcactgaaacccatCggcaaCc~ttgtagtcatctgctgtgccttcttc '1' L M S S L K P I G N I V V' I C C A F F
atcattttcggcatctY~gggggtgcagctattcaaagggaagGtttxcgtgtgccagg9c t I F' G I L G V Q L F K G I~ F F V C Q G
gaggataccaggaacatcaecaataaatcggaetgtgccgac~gccagttaccggtgggtc E D T R N I T D1 K S D C ~ E A S Y, kt W V
cggcaeaagt~acaaCtttgacaaccttggGCaggccctgatgtocctgttcgttttggcc R H K 'Y N F D N L G Q A L M S L F V IJ A
tccaaggat9'Jttgggtcgacatcatgtacgatgggat9'9'at9ct9'tgggcgtggacCag S K D G W V D I M 'Y D G L D A V G V D Q
cagcccatcatgaaccacaacccctggatgatgctgtacttcatetcgttcctgctcatt Q P I M N H N P W M L L Y F I S F L L x gtc~gccttctttgtcetgaacatgtttgtgggtgtggtggtggagaacttccacaagGgt V A F F V L N N! F V' G V V V ~.' N F H K C
cggcagcaccag~'aggaagaggaggcccggcggagggaggagaagcgcctacgaagactg R Q H Q E E E E A R R R E E K R L R R L
gagaaaaagagaaggagtaaggagaagc~.gatggctgatetaatgctggaagatgtaatt E K K R R S iC E IC Q M A D L M L i! D Y I
gcttccggcagctcagacagcgaCgcgtcagaagccGa,gtgcaaaccttaotactccgac A 8 1~a ~ S A G A A S E A Q G Ff J.' Y Y S D
tactcacgcttcaggctcctcgtacaecacttgtgcaccagcCactacctggacctcttc Y S R F R L L V H H L C T S H Y L D L F
ateaoaggtgtcatcgggctgaacgtggtaacca.tggacatggagca.ctaccagaagcac I T G V I G L N V V T M. A M E H Y Q Q P
cagattctggatgaggctctgaa.gatctgcaactacatattcactgtaa.tctttgtcttg Q I L D E A L fC 2 C N Y I F T V 2' F V L
gagtcagttttcaaacttgtggcctttggtttacgtcggttctCacaggacaggtggaaa E S V F Tt L V A F G F R R F F Q D R W, N
cagetggacctggccattgtgctgctgtccatcatgggcatcacgctggaggaaatcgag Q L D L A I V L L S I M G I T L E E I E
gtcaact~cctagctgcecatcaaCCCCaacatcatCCgcatcatgagggtgctgcgcatt V N A S L P I N P T Z I R z M R V' L R I
gcccgagtgctgaagctgctgaagatggctgtgggcatgcgggcgctgctggacacggtg A Ii V L K L L ff M A V G M R A L L D T V
atgcaggccctgccccaggtggggaacctgggacttctattcatgtCgttgtttttcatc M i,~ A L P f~ V G N L G L L F M L L F F Z
tttgcagctetgggcgtggagctctttggagacctggagtgtgacgagacacacccctgt F A A L G V E .r. F G D L E C b E T I3 P C
g'agggcctgggcogtcatgccacctttcggaactttggcatggtcttactaaccctct~tc EGLGRf~AT~'RN~'GMA~'LTLF

cgagtctCCacaggtgacaattggaatggcattatgaagg~.caccctocgggaatgtgac R V S T G D 13 W 1~T G I M K D T xa R I1 .~'y' D
caggaS-tacacctgctacaacaaggtcatctcgcctatctactttgtgtacttcgtgctg QESTC'~N~'V I SPIYk'VSFVL
acggeccagttcgtgctagtcaacgtggtgat:egccgtgotJatgaagcacctggaggag T A Q fi V fa V L3 V V I A V L M IC H L E E
dt~Caacdag~a.ggCaaaggaggaggocgagctagaggctgagctggagctggag2ttgaag S N YC E A K E E A E L E A E L E Ir E M K
accctcagcocCCagCCCCactcgccactgg9cagccacttcctotggcetggggteJa9 T L 8 P Q P ~j S P L G S P k~ L W P G V E
ggccccg~cagccccgacagccccaag~ctggggctctgCacccagcggcccaagcgaga G P I1 S P D S P K P G A Ta H P A A H A R
tcagactccaacttttacctggagcaccccaogatgcagceccaccccacggagctgc~a S A S H F S L E ti P ~' M Q F H P 'f E L F
ggaccagacttactgactgtgcggaagtctggggtcagccgaacgcaGtctctgcacaat G P D L L T V R K S G V S It T H S L F R
gacagctacatgtgtcggeatgggagcactgocgaggggcccctgggacacaggggctgg D S Y M C R $ G S T A E G P L. G" 'ri R G W
gggCtCCCCaaagCCCagtC;~r~gCtccgtottgtcCg~.tcactCCCagecagCagat~CC
G 7r P 1'C A Q $ G S V L S Y kI S ~,,7 P A D 'P
agatacat.cotgcagettcccaaagatgcaoctcat:ctgatccagccccacagcgcccca $ Y I L Q L P K 3:1 A F ~I L L Q P #i S A 1?
acetggggcacCatccocaaactgcccccaccaggaegctcccctttggctca,gaggcca T Tnl' G T I F K L P P F G R S P L t1 Q R P
ctcaggcgccaggcagcaata~.9'gactgaatcettggacgttca~~gtctgggcagccgg L R R Q A A I R T D S L '17 V Q G L G S R
gaagaCatgctggcagaggtgagtggJccctccccgccectggcccgBgceta.ctctttc E 17 L L A 'E V S G P S P P L A R A '1' S F
tggggccagtoaagt.aeacaggcacageagcactcacgcagcc.~cagcaagatctceaag W G ~ S S T Q A Q ø H S R S H S ~~ z S It cacatgaccccgccagccccCCgcccaggGccagaacacaactggggc~.agggccatcca YL M T P P A P C $ G P E P N W G K ,ra P P
gagacaagaagcagattagagttggacacggag~ctgagctggatttaaggagacctcctg~
E T 'R S 9 L 8 L D T E L S W I S G D L L
ccccctggcggccag9'aggagaccacatcaccacgggacCtgaagaagtgrcta.cagcgtg $ P G G Q E E F P S F R D L K K C Y S V
gaggccCagagctgccagcs~cCggcctacgtcvt9Jct9gatgaJcagaggagacactat E A Q S C Q R R P ~' S G~1 L D E Q R R H S
atagcegtcag4tgcctgga.cagaggctcccaaccccacctgggcacagaccectctaac I A V S c L D $ G 8 Q P H L G T D .L~ S N
C~tgg~J9gCCagCCtCt'~g95<JgJ'catg9'gagCCggCCCa~gaaaaaaC~caC3CCCgc.ct LGGt,~PLGC'aP .(~xS~P~Fi.KKLS PP
agtatC?.CCa'tags'i..'cCCCCCG~$gagCCl,aggt:CCtC JCJa.CCG'CtgCCCa~GCGtC,~gCat'.G
s z m I n ~ F ~ s Q ~ ~ R T P F s ~ c ~
tgcatccggaggagggcteagtccag~gactcCaaggatccattggactCtggC~C~cCt C L R R R A P S S L1 S K D $ L A S G P P
gacagcatggctgcctcgccctacecaaagaaagatgtgctgagtctctccggtttatcc D S M .'~ A S P S P K K D V L S L 8 G L S
tctgaccaagcagacctggacccatga S D F' A D ,T, D P -X0113] Modifieatians rnay 1>e r~r~ade to the foregoing without degarrit~p Pram the basic aspects of the invention. Although the invention has l5eeit described in substantial detail with reference to one or more specific em6oditnents, those of skill in the art will recc~nize Shat chat~~e$ may be made to the embodiments specifically disclosed in this application, yet these madi~icatians aid irnprtrvements are within the scope and spirit of the invention, as set forth in the clainr~s which follow. Citatir~n of the above publications and documents is not intended as an admission that any of the foregoing is pertinent pria~r art, rior.does it constitute any admission as to the contents' or date of these pu'blieations or documents. Each patent, patent application, document and publication referenced herein is hereby incorporated by reference in its entirety.
4$

Claims (25)

1. A method for detecting a risk of, or presence of, a cell proliferative disorder in a subject, which comprises determining the presence or absence of abnormal expression of at least one Ca v3.1 calcium channel splice variant in a sample comprising cells of said subject, whereby the presence of abnormal expression of a Ca v3.1 calcium channel splice variant determines that the subject is at risk of, or has, a cell proliferative disorder.

2. The method of claim 1, wherein said abnormal expression is determined as the presence or absence of mRNA encoding Ca v3.1ac calcium channel or of said Ca v3.1ac calcium channel protein.

3. The method of claim 1, wherein said abnormal expression is determined as the presence or absence of mRNA encoding Ca v3.1b calcium channel or of said Ca v3.1ac calcium channel protein.

4. The method of claim 1, wherein said abnormal expression is determined as the presence or absence of a percentage of expression of Ca v3.1bc calcium channel of greater then 20%
of the expression of Ca v3.1 calcium channel on a per mole basis.

5. The method of claim 4, wherein said percentage is measured in terms of mRNA
or protein.

6. The method of any of claims 2-5, wherein the presence or absence of protein is determined by contacting said sample with an antibody that specifically binds to the protein.

7. The method of any of claims 1-6, wherein the cell proliferative disorder is selected from the group consisting of brain cancer, glioma, breast cancer, eye cancer and retinoblastoma.

8. The method of any of claims 1-7, which further comprises determining the presence or absence of increased cell proliferative within a subject identified as having a risk of or presence of a cell proliferative disorder.

9. A method to inhibit the proliferation of cells that exhibit abnormal Ca v3.1 calcium channel splice variant expression, which comprises contacting said cells with a substance that inhibits the expression or activity of at least one Ca v3.1 calcium channel splice variant.

10. The method of claim 9, wherein said abnormal expression is determined as the presence or absence of mRNA encoding Ca v3.1ac calcium channel or of said Ca v3.1ac calcium channel protein.

11. The method of claim 9, wherein said abnormal expression is determined as the presence or absence of mRNA encoding Ca v3.1b calcium channel or of said Ca v3.1ac calcium channel protein.

12. The method of claim 9, wherein said abnormal expression is determined as the presence or absence of a percentage of expression of Ca v3.1bc calcium channel of greater than 20%
of the expression of Ca v3.1 calcium channel on a per mole basis.

13. The method of any of claims 9-12, wherein the substance is an antisense, ribozyme, RNAi, or triple helix-forming nucleic acid.

14. The method of any of claims 9-12, wherein the substance is an antibody.

15. The method of claim 10 wherein the substance is one that inhibits interaction between Ca v3.1ac calcium channel and ANX III.

16. A method for identifying a substance that inhibits cell proliferation, which comprises contacting one or more cells that exhibit abnormal Ca v3.1 calcium channel expression with a test substance, and determining whether the test substance decreases cell proliferation, whereby a test substance that decreases cell proliferation is identified as a substance that inhibits cell proliferation.

17. The method of claim 16, wherein said abnormal expression is determined as the presence or absence of mRNA encoding Ca v3.1ac calcium channel or of said Ca v3.1ac calcium channel protein.

18. The method of claim 16, wherein said abnormal expression is determined as the presence or absence of mRNA encoding Ca v3.1b calcium channel or of said Ca v3.1ac calcium channel protein.

19. The method of claim 16, wherein said abnormal expression is determined as the presence or absence of a percentage of expression of Ca v3.1bc calcium channel of greater than 20%
of the expression of Ca v3.1 calcium channel on a per mole basis.

20. A method of identifying a substance that inhibits cellular proliferation, which method comprises incubating a Ca v3.1ac calcium channel polypeptide or substantially identical polypeptide and ANX III thereof with a test substance and determining whether the presence of said substance decreases the binding of polypeptide to ANX III, whereby a substance that decreases said binding is identified as a substance that inhibits cell proliferation, or incubating a Ca v3.1ac calcium channel polypeptide with said substance and determining whether said substance binds to the polypeptide, whereby a substance that binds said polypeptide is identified as a substance that inhibits cellular proliferation.

21. The method of claim 20, wherein the Ca v3.1ac calcium channel polypeptide comprises 25 or more sequential amine acids selected from a region spanning amino acid 1546 to amino acid 1570 of a Ca v3.1ac T-type calcium channel.

22. The method of claim 21, wherein the polypeptide consists of the amine acid sequence SKEKQMADLMLDDVIASGSSASAAS.

23. An isolated mRNA or protein that encodes or has the amino acid sequence of Ca v3.1ac calcium channel splice variant.

24. A cell that exhibits abnormal expression of at least one Ca v3.1 calcium channel splice variant in contact with or containing a substance that inhibits the expression or activity of said Ca v3.1 calcium channel splice variant.

25. The cell of claim 24 wherein the splice variant is Ca v3.1ac Ca v3.1b or Ca v3.1bc.