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WO1993008701A1 - Partenaires de fixation de l'adn de c-myc, motifs, dosage de criblage et utilisations - Google Patents

Partenaires de fixation de l'adn de c-myc, motifs, dosage de criblage et utilisations Download PDF

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Publication number
WO1993008701A1
WO1993008701A1 PCT/US1992/008603 US9208603W WO9308701A1 WO 1993008701 A1 WO1993008701 A1 WO 1993008701A1 US 9208603 W US9208603 W US 9208603W WO 9308701 A1 WO9308701 A1 WO 9308701A1
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myc
complex
dna
seq
protein
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PCT/US1992/008603
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Robert E. Kingston
Ophelia Papoulas
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The General Hospital Corporation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • This invention is directed to methods for the purification of mammalian Myc protein, and methods for the identification of compounds that inhibit c-Myc transcriptional activity.
  • Myc is a nuclear oncogene whose aberrant expression is associated with many different types of human cancers in many different tissues (Cole, M.D., Ann. Rev. Genet. 20:361-384 (1986)). While the mechanism of c-Myc oncoprotein action remains unknown, it clearly plays a role in the control of cell growth and differentiation (Luscher and Eisenman, Genes & Dev. 4:2025-2035 (1990); Penn et al. , Sem. Cancer Biol. 1:69 (1990)). One plausible mechanism of Myc action is as a regulator of transcription in a pathway directly controlling proliferation and differentiation. This model is consistent with several observations. First, Myc has long been known as a nuclear protein with a general affinity for DNA (Abrams et al.
  • Myc contains two domains that suggest it oligomerizes, perhaps as a dimer, and binds specifically to DNA: a leu ⁇ ne zipper domain and a basic- helix-loop-helix (B-HLH) domain.
  • the leucine zipper is an ⁇ -helical structure found in sequence specific DNA-binding proteins such as Fos and Jun where it mediates homo- or heterodimerization via a coiled-coiled interaction (Landschulz et al, Science 240:1759-1764 (1988); O'Shea et al, Science 243:538-542 (1989); and reviewed in Busch and Sassone-Corsi, TIG 6:36-40 (1990)).
  • HLH region also appears to mediate oligomerization necessary for DNA binding in several developmentally important proteins (Murre et al., Cell 58:537-544 (1989); Murre et al., Cell 56:777-783 (1989)).
  • HLH proteins form a large and growing family and include the products of the achaete-scute and
  • a cellular transcription factor (USF or MLTF) which binds to the USE has recently been cloned and also contains a B-HLH domain adjacent to a leucine zipper (Gregor et al., Genes & Dev. 4:1730-1740 (1990)).
  • B-HLH or leucine zipper proteins have been found to form not only homodimers but heterodimers with other proteins having like dimerization motifs (reviewed in Busch and Sassone-Corsi, TIG 6:36-40 (1990); Jones, N., Cell 67:9-11 (1990)).
  • Heterodimerization between specific groups of B-HLH or leucine zipper proteins can alter their DNA binding properties. While homodimers might bind weakly, heterodimers with the appropriate partner can bind with increased affinity and in some cases with a new specificity (Jones, N., Cell 61:9-11 (1990); Blackwell and Weintraub, Science 250:1104-1110 (1990); Wright et al., Mol. Cell. Biol.
  • Myc is capable of forming a homo-oligomer at high concentrations in vitro (Dang et al., Nature 337:664-666 (1989); Kerkhoff and Bister, Oncogene 6:93-102 (1991)), although it is not clear whether that homo-oligomer actually forms in vivo (Dang et al., Mol. Cell. Biol. 11:954-962 (1991)). It seems likely that Myc directly interacts with other cellular protein(s) to form hetero-oligomer(s), and indeed one such partner" protein, designated Max, has recently been identified (Blackwood and Eisenmann, Science 251:1211-1217 (1991)). The effect that such partner proteins have on Myc DNA-binding specificity is likely to be central to understanding the function of Myc.
  • Myc will be determined in large part by hetero-oligomerization with Max and perhaps with other, as yet unidentified, factors.
  • C1 complexes homo-oligomer complexes
  • C2 complexes hetero-oligomer complexes formed by heterodimerization of at least two peptides, at least one of which is not the c-Myc peptide, and specifically hetero-oligomerization between c-Myc and a 26-29 kd factor
  • C2' complex c-Myc-dependent hetero-oligomeric complexes
  • the invention is directed to a reliable and accurate method for the purification of Myc from a mammalian source.
  • the invention is further directed to the use of oligomers containing the DNA motif 5'-CACGTG-3 ' , in its double stranded DNA form, as a reliable and accurate method for the detection of the presence of C1 complexes in a sample.
  • the invention is further directed to the use of the DNA motif 5'- CACGTG-3', in its double stranded DNA form, as a reliable and accurate method for the detection of C2 complexes in a sample.
  • the invention is further directed to the use of the DNA motif 5'- CAGCTG-3% in its double stranded DNA form, as a reliable and accurate method for the detection of C2 ' complexes in a sample.
  • the invention is further directed to a 26-29 kD protein fraction purified from Chinese hamster ovary (CHO) cells or baculovirus, such protein fraction containing at least one peptide capable of forming C2 complex oligomers with c-Myc.
  • CHO Chinese hamster ovary
  • baculovirus such protein fraction containing at least one peptide capable of forming C2 complex oligomers with c-Myc.
  • the invention is further directed to a 40-50 kD protein fraction purified from CHO cells, such protein fraction containing at least one peptide capable of forming C2' complex oligomers in the presence of c-Myc.
  • the invention is further directed to a reliable and accurate methodfor objectively classifying compounds, including human pharmaceuticals, as inhibitors of c-Myc activity, and especially as an inhibitor of C1 complex formation, C2 complex formation or C2' complex formation.
  • the invention is further directed to a reliable and accurate method for objectively classifying compounds, including human pharmaceuticals, as inhibitors of c-Myc activity, and especially as an inhibitor of C1 complex DNA binding, C2 complex DNA binding, or C2' complex DNA binding.
  • the invention further provides a method for identifying and classifying the mechanism of action of a bioactive c-Myc-inhibiting compound.
  • the invention further provides an assay for the monitoring of the isolation and/or purification of a peptide capable of forming a C2 or C2 ' complex, or a mixture of such peptides from a crude preparation.
  • the invention further provides an assay for the monitoring of the isolation and/or purification of an c-Myc-inhibiting compound or mixture of such compounds from a crude preparation of such compounds.
  • Fig. 1 Purified c-Mvc Protein.
  • Fig. 2 DNA Binding of Purified c-Mvc Proteins.
  • the EMSA was carried out as described in materials and methods using equal amounts (approximately 2 ng) of the following probes and 0.5 ⁇ g of either purified
  • Fig. 3. C1 Binding Activity is Present in Myc containing Slices of SDS Gels. 400 ⁇ g of CHO produced c-Myc or 163 ⁇ g of baculovirus produced c-Myc was separated on an SDS-PAGE gel. Proteins from 0.5 cm slices were recovered, renatured as described in materials and methods, and analyzed by EMSA using the (USE)3 probe. 0.4 ⁇ g of the CHO Myc load and 5 ⁇ l of the protein from the CHO Myc-containing slice were analyzed on the same gel (left panel). 0.37 ⁇ g of the baculovirus Myc load and 5 ⁇ l of the protein from the baculovirus Myc slice were analyzed on the same gel (right panel). Slices from other molecular weight ranges of the same gel showed no binding (data not shown).
  • Fig. 4 Activity is Formed bv c-Myc and a 26-29 kD Factor.
  • Lanes 1-4 represent proteins from the same gel loaded with baculovirus produced Myc described for Fig. 5. These lanes contain 0.37 ⁇ g of the loaded material (lane 1), 0.75 ⁇ g BSA with 7.5 ⁇ l of proteins from either a Myc slice (lane 2) or a 26-29 kD slice (lane 3), or 7.5 of each slice used for lanes 1 and 2 plus 0.2 ⁇ g of BSA (lane 4). Lanes 5-8 and 10 contain proteins from gels loaded with Myc purified from CHO cells.
  • Lanes 9-12 utilize the bacterially expressed Protein A-Myc fusion proteins containing either the Myc B-HLH and leucine zipper domains (amino acids 353-439) or lacking the basic region and containing Myc amino acids 372-439. These were expressed and purified as described in materials and methods.
  • Lane 9 contains 0.5 ⁇ g of Protein A-Myc(353-439) and lane 10 contains the same plus 7 ⁇ l of the 26-29 kD slice.
  • Lane 11 contains 1 ⁇ g of Protein A- Myc(372-439) and lane 12 contains 0.5 ⁇ g of Protein A-Myc(372-439) plus 7 ⁇ l of the 26-29 kD slice.
  • Fig. 5. C2' Binding Activity Requires a 40-50 Kd Factor.
  • EMSA samples contained 0.3 ⁇ g of the SDS gel load (lane 1), 7.5 ⁇ l of the proteins from the Myc slice Oane 2), or the 40-50 kD slice (lane 3), or 7.5 ⁇ l of both slices renatured together (lane 4).
  • EMSA samples contained 0.9 ⁇ g purified baculovirus produced c-Myc (lane 5), 3 ⁇ l of protein from the 40-50 kD slice of a gel loaded with 400 ⁇ g CHO produced c-Myc (lane 6), or both renatured together (lane 7,.
  • the probe was ERP1/2.
  • EMSA samples contained 10 ⁇ l (0.9 ⁇ g) of bacterially produced c-Myc fusion protein containing Myc amino acids 353-439 (lane 8), 0.47 ⁇ g of CHO produced c-Myc Oane 9), 5 ⁇ l of protein from the 40-50 kD slice of a gel loaded with 400 ⁇ g of the CHO Myc shown in lane 9 Oane 10), or 5 ⁇ l of the same 40-50 kD material renatured in the presence of either 0.9 ⁇ g of the baculovirus produced Myc shown in lane 5 Oane 11), 2 ⁇ l (0.18 ⁇ g) of the bacterially produced Myc fusion protein containing Myc amino acids 353-439 (lane 8), 0.47 ⁇ g of CHO produced c-Myc Oane 9), 5 ⁇ l of protein from the 40-50 kD slice of a gel loaded with 400 ⁇ g of the CHO Myc shown in lane 9 Oane 10), or 5 ⁇ l of the same 40-50
  • EMSA reactions were set up with the indicated Myc protein preparations (0.37 ⁇ g baculovirus produced c-Myc or 0.47 ⁇ g of CHO produced c-Myc). These reactions were preincubated 30 min on ice in the presence of the indicated antibody ( ⁇ -Myc monoclonal 1F7 or a
  • Oligonucleotides Selected from Random Sequence after 8 Rounds of EMSA. Sequences were selected from oligonucleotides containing 20 base pairs of random sequence using a reiterative EMSA procedure described in materials,and methods. Underlined nucleotides are from the PCR primer sites. Tables below the aligned sequences tabulate the frequency of each base in the 6 flanking positions surrounding the CACGTG motifs.
  • Fig. 8 Selected Sites form Predicted Complexes.
  • EMSA was carried out using either 2.8 ng of the SMS probe or equal amounts (1 ng) of probes 1-11 indicated in Fig. 7. Probes 1-11 were labeled and gel isolated in parallel and had approximately equal specific activities. Binding reactions contained either no additional protein (-), 0.37 ⁇ g of baculovirus produced c-Myc (B) or 0.47 ⁇ g of CHO produced c-Myc (C). Free probe is visible at the bottom of the gel.
  • EMSA reaction was scaled up for 11 samples containing 0.4 ⁇ g of purified baculovirus produced c-Myc per sample. Probe and competitor were (USE) 3 . After allowing 20 min for binding 20 ⁇ l was loaded on a prerun EMSA gel as a measure of the starting amount of complex (ST) and enough cold competitor was added to the remaining sample to achieve a
  • an "oligomer of interest” refers to any of the following types of oligomeric proteins: first, Myc-containing oligomers including homo-oligomers of Myc peptides (a C1 complex), and hetero-oligomers containing at least one peptide of Myc and one peptide of a Myc "partner" (a C2 complex); second, oligomers that form in the presence of Myc-containing homo-oligomers or Myc-containing hetero-oligomers but which themselves do not contain the Myc peptide, such oligomers including non-Myc-containing homo-oligomers that associate in the presence of Myc and non-Myc-containing hetero-oligomers that associate in the presence of Myc (a C2' complex).
  • Oligomer as it refers to proteins, means a protein composed of more than one peptide subunit, such as dimers, trimers, tetramers, etc. Such oligomeric protein may be a homo-oligomer, that is, composed entirely of two or more identical subunits; alternatively, such oligomeric protein may be a hetero-oligomer, that is, composed of at least two different peptides. Oligomers containing three or more peptides may contain more than one copy of a peptide.
  • a "C2 ' protein” is a protein or peptide that is a member of the second class of the "oligomers-of-interest,” that is, a protein that forms oligomers in the presence of Myc, c-Myc homo-oligomers or Myc-containing hetero-oligomers so as to bind to a specific DNA sequence, but which does not contain a Myc peptide, such oligomers including non-Myc-containing homo-oligomers that associate in the presence of Myc and non-Myc-containing hetero-oligomers that associate in the presence of Myc.
  • two macromolecular elements are operably-linked when the two macromolecular elements are physically arranged such that factors which influence the activity of the first element cause the first element to induce an effect on the second element.
  • factors which influence the activity of the first element cause the first element to induce an effect on the second element.
  • the transcription of a coding sequence which is operably-linked to a promoter element is induced by factors which "activate” the promoter's activity; transcription of a coding sequence which is operably-linked to a promoter element is inhibited by factors which "repress" the promoter's activity.
  • a promoter region would be operably-linked to the coding sequence of a protein if transcription of the coding sequence activity was influenced by the activity of the promoter.
  • response is intended to refer to a change in any parameter which can be used to measure, indicate or otherwise describe c-Myc action or oligomer (homo-oligomer (C1 complex) or hetero-oligomer (C2 complex)) formation, including c-Myc dependent hetero-oligomerization (C2' complex) formation.
  • the response may be revealed as a physical change (such as a change in phenotype) or, it may be revealed as a molecular change (such as a change in a reaction rate or affinity constant). Detection of the response may be performed by any means appropriate. "Detecting” refers to any method by which such response may be evaluated- so as to provide a meaningful indicia of whether the event has occurred.
  • Compound is intended to refer to a chemical entity, whether in the solid, liquid, or gaseous phase.
  • the term should be read to include synthetic compounds, natural products and macromolecular entities such as polypeptides, polynucleotides, or lipids, and also small entities such as neurotransmitters, ligands, hormones or elemental compounds.
  • Bioactive Compound is intended to refer to any compound which induces a detectable or measurable response in the methods of the invention.
  • promoter is a DNA sequence located proximal to the start of transcription at the 5' end of the transcribed sequence.
  • the promoter may contain multiple regulatory elements which interact in modulating transcription of the operably-linked gene.
  • Expression is the process by which the information encoded within a gene is revealed. If the gene encodes a protein, expression involves transcription of the DNA into mRNA, the processing of mRNA (if necessary) into a mature mRNA product, and translation of the mature mRNA into protein.
  • a nucleic acid molecule such as a DNA or gene is said to be
  • a polypeptide if the DNA contains the coding sequences for the polypeptide and expression control sequences which, in the appropriate host environment, provide the ability to transcribe, process and translate the genetic information contained in the DNA into a protein product, and if such expression control sequences are operably-linked to the nucleotide sequence which encodes the polypeptide.
  • Cloning vehicle is any molecular entity that is capable of delivering a nucleic acid sequence into a host cell for cloning purposes.
  • Examples of cloning vehicles include plasmids or phage genomes.
  • a plasmid that can replicate autonomously in the host cell is especially desired.
  • a nucleic acid molecule that can insert into the host cell's chromosomal DNA is especially useful.
  • Cloning vehicles are often characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the vehicle, and into which DNA may be spliced in order to bring about its replication and cloning.
  • the cloning vehicle may further contain a marker suitable for use in the identification of cells transformed with the cloning vehicle. Markers, for example, are tetracycline resistance or ampicillin resistance.
  • Expression vehicle is a vehicle or vector similar to a cloning vehicle but is especially designed to provide sequences capable of expressing the cloned gene after transformation into a host.
  • the gene to be cloned is usually operably-linked to certain control sequences such as promoter sequences.
  • Expression control sequences will vary depending on whether the vector is designed to express the operably-linked gene in a prokaryotic or eukaryotic host and may additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites.
  • host any organism that is the recipient of a cloning or expression vehicle. a. Isolation of c-Myc Protein From Mammalian Cells and
  • Myc protein preparations described therein, and the methods used to isolate that protein failed to achieve the requisite amount of yield needed to sequence characterize Myc action in mammalian sources.
  • the inventors have overcome this problem and describe, for the first time, a unique and useful method for the isolation of highly purified mammalian c-Myc protein which provides the requisite high degree of quantity of mammalian c-Myc protein needed for the characterization of c-Myc directed DNa binding and biological action.
  • the inventors have also been able to purify large quantities of Myc from a recombinant insect cell system.
  • the purified Myc protein of the invention exhibits the only known biochemical activity of c-Myc, an ability to bind the sequence CACGTG.
  • the inventors were able to identify peptides that naturally associate with c-Myc in a hetero-oligomers, or peptides that naturally associate with each other as a result of the action of c-Myc, such peptides found to be present in certain column
  • mammalian source is preferably achieved utilizing a mammalian cell line that overexpresses either recombinant or non-recombinant c-Myc and is performed completely on ice or equivalent temperatures of 0-5°C, using reagents and buffers at the same temperature.
  • a mammalian cell line that overexpresses either recombinant or non-recombinant c-Myc and is performed completely on ice or equivalent temperatures of 0-5°C, using reagents and buffers at the same temperature.
  • CHO cell line 5A is useful for such purification.
  • recombinant mouse c-Myc is under the control of a regulatable promoter, and has been integrated and amplified in the genome of the parent CHO cell line for maximum stability and production.
  • the native or recombinant Myc should include at least the two coding exons of Myc.
  • the cells After collecting the cells by centrifugation using techniques known in the art, and prior to lysis of the outer cell membrane, the cells should be washed at least once in a low salt neutral buffer such as 0.9% NaCl in 10- 50 mM phosphate, pH 7.0-7.5 (phosphate buffered saline, PBS) to remove remaining growth medium.
  • a low salt neutral buffer such as 0.9% NaCl in 10- 50 mM phosphate, pH 7.0-7.5 (phosphate buffered saline, PBS) to remove remaining growth medium.
  • Lysis of the washed cells is also achieved in a low salt, neutral to mildly acidic lysis buffer, preferably about pH 6.8, containing at least one protease inhibitor, such as aprotinin or phenylmethylsulfonyl fluoride
  • PMSF preferably containing a combination of such inhibitors.
  • Salts such as potassium (in the KCl form) and magnesium (in the MgCl 2 form) are also preferably added.
  • nonionic detergents such as NP40 (0.5% v/v) and Na-deoxycholate (0.1 %) should be added.
  • Cell outer membrane lysis should be performed under conditions that lyse the host cell without lysing the nucleus, or induce significant leakage from the nuclear membrane.
  • the cells may be allowed to sit for a short period of time, for example, 10 minutes, in the detergent-containing lysis buffer before mechanical intervention is utilized in the lysis step.
  • Mechanical intervention is best performed with a gentle disruption of the detergent treated cells, for example, utilizing 40 strokes in a Dounce homogenizer with a type A pestle, or the equivalent of such treatment.
  • Nuclei may be collected from the lysed cell preparation using techniques known in the art, such as, for example, centrifugation at 1000xg for 5 min at 4°C and washed at least once in the same low salt lysis buffer used to lyse the outer cell membrane.
  • Nuclei are then resuspended in the low salt lysis buffer that additionally contains sufficient DNAse I and incubated for a time sufficient to efficaciously degrade the DNA in such nuclei to a size and viscosity that allows subsequent purification of the c-Myc from this preparation as described below.
  • the sample is diluted with a high salt neutral buffer that brings the salt (as NaCl) concentration of the sample to at least 2 M.
  • a high salt neutral buffer that brings the salt (as NaCl) concentration of the sample to at least 2 M.
  • Such high salt buffer preferably additionally also contains amounts MgCl 2 sufficient to maintain the same concentration of this, salt in the final diluted preparation, and also additional detergent NP40so as to retain efficacious levels after sample dilution.
  • c-Myc In mammalian host cells, c-Myc is generally tightly associated with the nuclei. Accordingly, it is necessary to solubilize c-Myc in a manner that does not destroy its biological activity Or its ability to renature into a biologically active form. The residual nuclear material is first removed by centrifugation and then the pellet resuspended for solubilization of the c-Myc. Solubilization of the c-Myc protein in a manner that destroys this association may be achieved with either sodium dodecyl sulfate (SDS) or urea at concentrations greater than 4 M. Preferably, 5M urea is utilized.
  • SDS sodium dodecyl sulfate
  • urea Preferably, 5M urea is utilized.
  • Residual non-lysed nuclei may also be solubilized at this time by vigorous stirring for about 30 min. The solution is then centrifuged to pellet any remaining insoluble material prior to the subsequent chromatography steps, for example, at 5000xg for about 10 min.
  • the supernatant fraction recovered from the centrifugation step is applied to a DEAE Sepharose CL-6B column equilibrated in the urea-containing buffer as described above, and the column thoroughly washed with such buffer to remove unbound protein. A second wash was performed with the addition of an intermediate amount of NaCl, 0.1M NaCl to the buffer. Finally, Myc protein was eluted by raising the salt concentration in the buffer to 0.35M.
  • Myc may be identified in the column eluent by any technique that specifically recognizes Myc protein or its activity.
  • a monoclonal antibody such as 1F7 may he used in an immunoassay for the presence of Myc protein.
  • DNA binding activity to an oligonucleotide containing the sequence 5'-CACGTG-3' may be used to monitor the purification.
  • Monoclonal antibody 1F7 is directed against the peptide sequence of amino acids 305-317 in murine c-Myc.
  • Other Myc monoclonal antibodies useful in such assays are commercially available.
  • Pools of fractions from this column contained the C2 and C2' binding activities described below, and the presence of peptides capable of entering into C2 and C2' hetero-oligomers, and especially C2 and C2' hetero-oligomers, may be assayed by the ability of such hetero-oligomers to bind to the DNA sequences 5'-CACGTG-3' and 5'-CAGCTG-3', respectively.
  • Myc purified from the CHO cells appeared as multiple bands by immunoblot.
  • c-Myc Purification of c-Myc and Its Partners From a Baculovirus Source Human c-Myc may also been purified using the baculovirus overexpression system.
  • Sf9 cells that had been infected with recombinant baculovirus carrying the c-Myc gene, using techniques known in the art were harvested just prior to the onset of lysis ( ⁇ 48 hours post infection). Solubilization and purification of the recombinant c-Myc were carried out as with the CHO produced Myc resulting in a yield of 2.5 mg/8x10 8 cells.
  • Myc purified from these insect cells was apparently homogeneous by silver staining, and ran on electrophoresis as a single diffuse band of ⁇ 60kD. This was in contrast to the multiple bands observed with mammalian Myc by immunoblot (Fig. 1B).
  • the above preparations contain two sequence specific DNA-binding activities that both contain Myc protein.
  • the first activity contains only Myc (i.e., forms the Myc homo-oligomer) and binds very weakly to sequences with the core CACGTG.
  • the binding is assayed by determining the off rate and by competitor assays, both techniques known in the art.
  • a binding site selection procedure may be used to determine the optimal binding site for Myc. Sites may be selected from a pool of random oligomers, such as 20-mers, in order to decrease bias in determining an optimal binding site.
  • [SEQ ID No. 1] may be used, with the central E box core of CACGTG appearing to be most conserved.
  • Halazonetis and Kandil (Halazonetis and Kandil, Proc. Natl. Acad. Sci. USA 88:6162-6166 (1991)) assumed that the flanking sequences might be symmetric, and reported an optimal sequence of GACCACGTGGTC [SEQ ID No. 2].
  • This sequence is quite similar to the consensus that is preferred here, differing in only the 10th position (where predominantly a C was utilized in the invention, although G is significantly represented Fig. 7, Group I). Accordingly to the invention, it is possible to select a 12 base consensus sequence from a pool of predicted complexity of 4 20 ( ⁇ 10 12 ) thus indicating that Myc has a strong sequence preference despite its apparent weak binding affinity.
  • the second Myc containing DNA-binding complex provided in the preparations of the invention also binds to sequences with a core of CACGTG, but binds significantly more tightly than Myc alone.
  • This complex (the C2 complex) requires a 26-29 kD factor in addition to Myc.
  • This additional factor copurified with Myc, presumably because of similar chromatographic properties and not via association with Myc since the chromatography performed in 5M urea would denature such association.
  • This additional factor resembles Max, a protein whose gene was recently isolated from mammalian cells, in that it does not bind efficiently to DNA by itself but can hetero-oligomerize with Myc to bind tightly to the sequence CACGTG.
  • Max is reported to migrate at 21 kD).
  • the Myc/Max hetero-oligomer appears to migrate at least as slowly as a Myc only complex in EMSAs, while the C2 complex of the invention migrates more rapidly than Myc alone.
  • C2' complexes contained a CAGCTG core (the ⁇ E2 sequence motif) as well as flanking sequences which bear a striking resemblance to a recently reported binding site for myogenin homo- oligomers (Wright et al., Mol. Cell. Biol. 77:4104-4110 (1991)).
  • Myogenin is an HLH containing protein of predicted molecular weight 32.5 kD whose optimal binding site is AACAGT/CTGTT [SEQ ID No. 3]. None of the sites (0/36) selected by the C2 or C2' complexes of the invention contained a CAGTTG motif while roughly half of the myogenin selected sites contained such core sequences. d. Assay for a Compound that Inhibits Myc Action
  • C1 complex association and/or DNA binding, C2 complex association and/or DNA binding, and C2' complex association and/or DNA binding are all referred to as c-Myc activity.
  • Assays for c-Myc activity may be performed in vitro or in vivo. In vitro assays may be performed as described in the Examples, for example, by evaluating the effects the desired compound or various amounts of such compound on the results of the electrophoretic mobility shift assay and site selection techniques that will reveal whether binding of the oligomer of interest to a specific DNA sequence motif has occurred in the presence of the compound.
  • For the in vivo assay of a compound that inhibits the desired Myc activity at least two genetic constructs are utilized. First is required a recombinant construct capable of expressing Myc is required; second is required a reporter gene whose expression is operably linked to the Myc activity and especially to the binding of the desired oligomer to the specific
  • a recombinant construct capable of expressing a C2 complex protein or C2' complex protein may also be used.
  • a host may be chosen may be chosen that naturally expresses such protein.
  • Recombinant constructs that are capable of expressing Myc protein may be constructed utilizing the guidelines as described below or purchased commercially.
  • the desired DNA binding sequence may be operably linked to any gene which confers a selectable marker in the host system.
  • a marker gene which allows phenotypic selection in yeast, and especially in Saccharomyces cerevisiae is used.
  • Yeast that have been co-transformed with both an expressible myc gene and with the desired DNA binding sequence may be used to (1) identify the presence or absence of endogenous host proteins that interact with Myc in a C2 or C2 'complex (2) classify a protein as a C1 complex protein or as a C2' complex protein; and (3) identify and classify compounds as agents which disrupt such Myc activity.
  • C2 complex proteins have previously also been termed Myc "partner" proteins.
  • Hosts that have been co-transformed with both an expressible c-Myc gene and with the desired DNA binding sequence may be used to assay for the presence or absence of endogenous host proteins that interact with c- Myc activity. If such analyses reveal that the host contains c-Myc binding proteins, or c-Myc dependent oligomers which, in the presence of c-Myc specifically bind to a desired DNA sequence, such c-Myc partner protein or dependent-oligomer protein may be isolated using techniques known in the art such as gel mobility shift analysis, cDNA expression cloning vectors such as, for example, ⁇ gt10 and ⁇ gt11, or other cloning systems
  • yeast such as, for example, pG1 and pTRP56, all of which are commercially available (Clontech, Palo Alto, California).
  • C2 complex proteins c-Myc partner proteins
  • C2' complex proteins C2 complex proteins
  • reporter gene transcription from endogenous partner proteins may be negligible, or of such low amount that it does not otherwise alter the utility of the methods of the invention.
  • the levels of c-Myc will be high enough to overcome a low level background and such c-Myc constructs may be used to analyze the ability of cloned c-Myc partners to influence c-Myc DNA binding.
  • One of ordinary skill in the art can adapt the expression system to the level of expression desired using methods known in the art.
  • the C2 complex protein (the partner protein), or the C2 ' complex protein, if supplied as a recombinant construct to the host cell, should be capable of expressing at levels comparable to that of the c-Myc protein.
  • C2 complex proteins may be identified by utilizing a phage plaque assay, as described in the commonly-owned, copending U.S. patent application entitled “Protein Partner Screening Assays and Uses Thereof," Application No. 510,254, filed April 19, 1990, and incorporated herein by reference. Proteins identified by such screening assay can be subcloned into
  • the genetic constructs of the invention may be placed on different plasmids, or combined on one plasmid.
  • a construct may also be inserted into the genome of a host cell.
  • the construct coding for the c- Myc protein and the construct coding for the C2 complex protein or the C2' complex protein are provided to the host on two different plasmids.
  • the desired DNA binding motif may be located at any site in the transcription cassette of the reporter gene which allows for the transcription of that gene to be operably-linked to binding of the desired oligomer.
  • such motif may be located 5' to the transcriptional start site or 3' to the .transcriptional start site, for example, in an intron, similar to its location relative to the promoter region in the immunoglobulin genes.
  • the reporter gene whose expression is operably linked to c-Myc activity and especially to oligomer DNA binding may be any gene whose expression can be monitored. Any detectable phenotype change may serve as the basis for the methods of the invention.
  • the reporter gene is a gene not normally expressed by the host, or a gene that replaces the host's endogenous gene. Any reporter gene which is capable of being operably-linked to a promoter capable of responding to the binding of the oligomer of interest to the specific target DNA sequence may be used.
  • genes that endow the host with an ability to grow on a selective medium are useful.
  • yeast use of the yeast LEU2 gene as a reporter gene in strains that normally lack LEU2 allows such yeast to grow on leucine as a sole carbon source. Expression the reporter gene is monitored by merely observing whether the host possesses the ability to grow on leucine.
  • suc2 gene as a reporter gene would allow growth of the a suc2- yeast host on sucrose to be used as the detection method.
  • growth on the indicated substrate could be used to indicate specific DNA binding of the oligomer of interest and lack of such growth could be used to indicate lack of binding or lack of oligomer formation.
  • a construct (and host) which is gall + gal10- would respond to galactose in the medium; a construct (and host) which is lac2 + gal1 + would be lactose sensitive.
  • Other reporter genes include his3, ura3 and trp5.
  • Reporter constructs in which the desired DNA sequence motif and the lacZ reporter gene are operably linked will express ⁇ -galactosidase in response to binding of a c-Myc activity induced oligomer binding to such DNA sequence. Such expression can be easily scored by monitoring the ability of the host to produce ⁇ -galactosidase (Maniatis, T. et al.,
  • ⁇ -galactosidase may be visually monitored by detecting its activity to reduce the chromophoric dye, X-gal (commercially available from International Biotechnologies, Inc., New Haven, CT). ⁇ -galactosidase reduces X-gal to a form which possesses a blue color.
  • CAT chloramphenicol acetyltransferase
  • any detection method that can identify expression of the reporter gene may be used.
  • levels of the product of the reporter gene may be directly assayed with an immunoassay.
  • immunoassays include those wherein the antibody is in a liquid phase or bound to a solid phase carrier.
  • the reporter gene can be detectably labeled in various ways for use in immunoassays.
  • the preferred immunoassays for detecting a reporter protein using the include radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), or other assays known in the art, such as immunofluorescent assays, chemiluminescent assays, or
  • yeast strains that express such the desired peptide or peptides and which contain the related DNA binding sequence motif may be plated and grown as lawns and the compound to be tested may be applied to the plates on a filter paper disk that is impregnated with such compound.
  • the compound may be incorporated into the media within which the host cells are growing.
  • the methods of the invention can be used to screen compounds in their pure form, at a variety of concentrations, and also in their impure form.
  • the methods of the invention can also be used to identify the presence of such inhibitors in crude extracts, and to follow the purification of the inhibitors therefrom.
  • the methods of the invention are also useful in the evaluation of the stability of the inhibitors identified as above, to evaluate the efficacy of various preparations.
  • the permeability of cells to various compounds can be enhanced, if necessary, by use of a mutant cell strain which possess an enhanced permeability or by using compounds which are known to increase permeability.
  • a mutant cell strain which possess an enhanced permeability or by using compounds which are known to increase permeability.
  • compounds which are known to increase permeability for example, in yeast compounds such as polymyxin B nonapeptide may be used to increase the yeast's permeability to small organic compounds.
  • DMSO dimethyl sulfoxide
  • Analogs of such compounds which are more permeable across yeast membranes may also be used. For example, dibutyryl derivatives often display an enhanced permeability.
  • the genetic constructs and the methods for using them are utilized in eukaryotic hosts, and especially in yeast, insect and mammalian cells.
  • the introduced sequence is incorporated into a plasmid or vector capable of either autonomous replication or integrative activity.
  • the DNA sequence of the desired gene may be chemically constructed if it is not desired to utilize a clone of the genome or mRNA as the source of the genetic information.
  • Methods of chemically synthesizing DNA are well known in the art (Oligonucleotide Synthesis, A Practical
  • a cloned protein encoding DNA sequence obtained through the methods described above, (preferably in a double-stranded form), may be operably-linked to sequences controlling transcriptional expression in an expression vector, and introduced, for example by transformation, into a host cell to produce recombinant proteins useful in the methods of the invention, or functional derivatives thereof.
  • Such techniques are well known in the art (Recombinant DNA Methodology, Wu, R. et al., eds., Academic Press, (1989); Maniatis, T. et al., Molecular Cloning (A
  • Transcriptional initiation regulatory signals can be selected which allow for repression or activation of the expression of the c-Myc construct or construct of the recombinant C2 complex peptide (or the C2' peptide), or both, so that expression of such constructs can be modulated, if desired.
  • regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical regulation, for example, by a metabolite, salt, or substrate added to the growth medium.
  • sequences functional in the host cell may be substituted.
  • constructs of the invention may result in different post-translational modifications which may alter the properties of the proteins expressed by these constructs. It is necessary to express the proteins in a host wherein the ability of the protein to retain its biological function is not hindered. Expression of proteins in yeast hosts is preferably achieved using yeast regulatory signals.
  • the vectors of the invention may contain operably-linked regulatory elements such as upstream activator sequences in yeast, or DNA elements which confer species, tissue or cell-type specific expression on an operably-linked gene.
  • expression vectors containing transcriptional regulatory sequences are used in connection with a host These sequences facilitate the efficient transcription of the gene fragment operably-linked to them.
  • expression vectors also typically contain discrete DNA elements such as, for example, (a) an origin of replication which allows for autonomous replication of the vector, or, elements which promote insertion of the vector into the host's chromosome in a stable manner, and (b) specific genes which are capable of providing phenotypic selection in transformed cells.
  • Eukaryotic expression vectors may also contain elements which allow it to be maintained in prokaryotic hosts; such vector are known as shuttle vectors.
  • yeast are used as the host cells.
  • the elements necessary for transcriptional expression of a gene in yeast have been recently reviewed (Struhl, K. Ann. Rev. Biochem. 58:1051-1077
  • promoters In yeast, most promoters contain three basic DNA elements: (1) an upstream activator sequence (UAS); (2) a TATA element; and, (3) an initiation (I) element Some promoters also contain operator elements.
  • UAS upstream activator sequence
  • TATA TATA
  • I initiation element
  • mammalian cells are used as the host cells.
  • a wide variety of transcriptional and translational regulatory signals can bederived for expression of proteins in mammalian cells and especially from the genomic sequences of viruses which infect eukaryotic cells.
  • Genetically stable transformants may be constructed with episomal vector systems, or with integrated vector systems whereby the fusion protein DNA is integrated into the host chromosome. Such integration may occur de novo within the cell or be assisted by transformation with a vector which functionally inserts itself into the host chromosome, for example, with retroviral vectors, transposons or other DNA elements which promote integration of DNA sequences in chromosomes.
  • Cells which have been transformed with the DNA vectors of the invention are selected by also introducing one or more markers which allow for selection of host cells which contain the vector, for example, the marker may provide biocide resistance, e.g., resistance to antibiotics, or heavy metals, such as copper, or the like.
  • the marker may provide biocide resistance, e.g., resistance to antibiotics, or heavy metals, such as copper, or the like.
  • the transformed host cell can be fermented or cultured according to means known in the art to achieve optimal cell growth, and also to achieve optimal expression of the cloned protein sequence fragments. As described hereinbelow, a high level of recombinant protein expression for the cloned sequences coding for the proteins can be achieved according to a preferred procedure of this invention.
  • the methods of the invention are not intended to be limited to c-Myc and possess utility for the characterization of inhibitors against any Myc protein, such as, for example, N-Myc and L-Myc.
  • the C2 complex peptides of the invention may interact with more than one Myc protein and the C2' complex peptides of the inventions may form as the result of the activity of more than one Myc protein.
  • Cell Growth and Myc Overexpression The 5 A cell line was maintained in spinner culture under selection with 80 ⁇ M methotrexate. Protein purification started with roughly 6 liters of cells at 8x10 5 /ml grown up without selection. Heat shock promoter induction was achieved by resuspension in preheated fresh media (43 °C) at 1/3 the original volume. Cells were incubated with stirring at 43°C for 1 h. To allow translation of the accumulated mRNA, cells were transferred to 37°C culture conditions for 3 h. Cells were then subjected to the purification described below.
  • the baculovirus overexpression vector was constructed by insertion of the BamHl/Bcll fragment of pGEMMycB [Halazonetis and Kandil, Proc Natl Acad. Sci. USA 88:6162-6166 (1991)] into the BamHl site of a baculovirus expression vector, pVL941, obtained from the laboratory of
  • the resulting plasmid contained the entire coding sequence of the human Myc gene including 6 nudeotides 5' of the initiation codon and 3' untranslated sequence extending to the genomic Rsal site.
  • Sf9 cells were grown and infected with recombinant baculovirus according to the methods of
  • the Protein A-c-Myc fusion protein was expressed in the E. coli
  • AR68 strain from a previously published pRIT2T vector [Dang, C.V., Anal. Biochem. 174:313-317 (1988)] which fused the Ig binding portion of protein A to either amino acids 353-439 or amino acids 372-439 of c-Myc. Growth and induction of the cells was as per Dang et al. ⁇ Anal. Biochem. 174:313-317 (1988)].
  • KCl 5 mM MgCl 2 , 0.5% NP40, 0.1 % Na-deoxycholate, 1 ⁇ g/ml aprotinin, and 0.1 mM PMSF
  • KCl 5 mM MgCl 2 , 0.5% NP40, 0.1 % Na-deoxycholate, 1 ⁇ g/ml aprotinin, and 0.1 mM PMSF
  • the residual nuclear material (including the c-Myc protein) was pelleted (2000xg, 10 min, 4oC) and resuspended for solubilization at 5.5x10 7 nucleus equivalents/ml in Buffer A (50 mM Tris, pH 8.0, 2 mM EDTA, 5 % glycerol, .1 mM DTT, and .1 mM PMSF) [Watt et al., Mol. Cell. Biol. 5:448-456 (1985)] containing 5 M urea (referred to as 5 M urea Buffer A) achieved by dilution of a freshly deionized stock of 6 M urea.
  • Buffer A 50 mM Tris, pH 8.0, 2 mM EDTA, 5 % glycerol, .1 mM DTT, and .1 mM PMSF
  • the protein containing fractions of this 0.35 M NaCl step were pooled and diluted with fresh 5 M urea Buffer A to 0.1 M NaCl and loaded onto a 1 ml FPLC Mono-Q column (Pharmacia) run at 0.5 ml/min.
  • the Mono-Q column was eluted with a programmed gradient of 5 ml spanning
  • the Myc containing fractions were pooled based on purity and dialyzed against buffer containing 20 mM Tris, pH 7.8, 50 mM KCl, 10 % glycerol, 0.1 mM DTT, and 0.1 mM PMSF (referred to as Dialysis Buffer) in bags of SpectroPor 2 membrane for 3 changes, 2 liters each, for a minimum of 3 h each. Pools of fractions prepared this way contained C1 and C2 (and C2') binding activities. To obtain pure C1 binding activity the Myc-containing Mono Q fractions were assayed by EMSA and those free of C2 binding activity were pooled and dialyzed separately.
  • buffer containing 20 mM Tris, pH 7.8, 50 mM KCl, 10 % glycerol, 0.1 mM DTT, and 0.1 mM PMSF (referred to as Dialysis Buffer) in bags of SpectroPor 2 membrane for 3 changes, 2 liters each,
  • the bacterially produced Protein A-c-Myc fusion protein was partially purified by differential centrifugation and solubilized in 5 M urea according to Watt et al. [Bagchi et al., Mol. Cell. Biol. 7:4151-4158
  • Protease inhibitors were present in the initial lysis buffer (10 ⁇ g/ml pepstatin, 1 mM PMSF, 50 ⁇ g/ml aprotinin, 2 ⁇ g/ml leupeptin, 10 mM Na-metabisulfite, and 1 mM benzamidine) and cells were sheared by 6 bursts of 15 s each in a Cuisinart MiniMate on ice. The urea solubilized material was cleared of insoluble material by centrifugation (10,000xg, 10 min, 4°C) and dialyzed into Dialysis Buffer containing 0.5 mM DTT.
  • initial lysis buffer 10 ⁇ g/ml pepstatin, 1 mM PMSF, 50 ⁇ g/ml aprotinin, 2 ⁇ g/ml leupeptin, 10 mM Na-metabisulfite, and 1 mM benzamidine
  • Precipitated material was removed by centrifugation (15,000xg, 20 min, 4°C).
  • Protein A-Myc fusion protein was purified from the supernatant by IgG affinity essentially according to Nilsson et al. [EMBO J. 4:1075-1080 (1985)].
  • a 1 ml aliquot of supernatant was incubated with 0.1 ml of a 50% slurry of IgG Sepharose 6 fast flow (Pharmacia) rocking for 1 h at 4°C.
  • the pellet was washed twice with Buffer A and the fusion protein eluted with 0.3 M lithium diiodosalicylate (LIS).
  • LIS lithium diiodosalicylate
  • Antibodies The monoclonal antibody, 1F7 (a generous gift of R.
  • Electrophoretic Mobility Shift Assay Radiolabeled probes were produced via a Klenow fill in of annealed oligonucleotides containing 4 base 5' overhangs at each end (see table below for sequences).
  • Binding reactions took place in a final volume of 20 ⁇ l containing 2 ng of labeled probe, 125 ng poly d(IC), an indicated amount of protein, and the following final buffer conditions: 10 mM Tris, pH 7.5, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 1 mM MgCl 2 and 5% glycerol. Binding reactions were allowed to proceed for 20 min at room temperature and were then loaded immediately on a 4% polyacrylamide gel which had been prerun at least 1 h at 10V/cm. Electrophoresis was for 1.5 h at 10V/cm in 0.5xTBE.
  • the initial round of binding site selection by EMSA utilized 200 ng of this pool and other 0.37 ⁇ g of baculovirus produced c-Myc or 0.5 ⁇ g of CHO produced c-Myc. Other parameters were as previously described for EMSA. Lanes containing randomer probes were alternated with reference lanes containing 2 ng (USE) 3 probe and 0.37 ⁇ g of baculovirus c-Myc. The completed EMSA gel was electroblotted onto NA45 membrane (200 mA, 2.5 hrs) and the wet membrane was wrapped in plastic wrap and exposed for at least 1.5 hrs.
  • the regions of the randomer lanes corresponding to the visible C1 and C2 complexes of the reference lanes were excised and eluted with 100 of elution solution (10 mM Tris, pH 8.0, 1 mM EDTA, 1 M NaCl) 30 min at 68°C.
  • the liquid was transferred to a fresh tube and the membrane was rinsed with 100 ⁇ l TE which was added to this eluate.
  • the DNA was precipitated with the addition of 10 ⁇ g glycogen, 2 ⁇ l 1 M MgCl 2 and 2.5 volumes of ethanol. The pellet was rinsed with 70% ethanol, dried, and the recovery assessed by scintillation counter.
  • the products were gel purified on 10% acrylamide and precipitated using 10 ⁇ g glycogen as carrier. Recovery was measured by scintillation counter and after resuspension in the EMSA reaction buffer (10 mM Tris, pH 7.5, 50 mM KCl, 1 mM EDTA, 1 mM MgCl 2 , and 5% glycerol) this probe was used for the next round of EMSA selection. Subsequent cycles were primarily as above, however, 50 ng of probe was used. Eight rounds of selection and amplification were completed for the baculovirus c-Myc and seven rounds for the CHO c-Myc. After the final PCR reaction the
  • Oligonucleotide sequences that were used are shown below, with the E-Box core sequences underlined:
  • SEQ ID NO. 9 MLC-A 5 ' TCGACGTCGCAGCAGGTGCAG 3 ' ;
  • ERP3/4 5' AGCTTTAAAATCCCCACCAGCTGGCGAAGCAACAGGTGCA 3 ' ;
  • a primary goal of this work was to purify and characterize Myc from a mammalian source.
  • the resulting products were phosphoproteins of 60, 62, and 72kD which were immunoprecipitable with Myc-specific monoclonal antibodies (Wurm et al., Proc. Natl. Acad. Sci. USA 83:5414-5418 (1986)).
  • the c-Myc produced was tightly associated with the nuclei and attempts to solubilize it using a number of detergents, salts, and reducing agents were unsuccessful (data not shown). Significant solubilization was achieved however with either SDS or with urea at concentrations greater than 4 M.
  • the Myc was solubilized with 5 M urea and chromatographed on DEAE resin and FPLC Mono-Q as described in materials and methods.
  • Human c-Myc has also been purified using the baculovirus overexpression system.
  • Sf9 cells that had been infected with recombinant virus were harvested just prior to the onset of lysis ( ⁇ 48 hours post infection).
  • Myc produced using the baculovirus system has been previously reported to be both phosphorylated and tightly associated with the nucleus (Miyamoto et al., Mol. Cell. Biol. 5:2860-2865 (1985)). Solubilization and purification were carried out as with the CHO produced Myc resulting in a yield of 2.5 mg/8x10 8 cells.
  • Myc purified from these insect cells was apparently homogeneous by silver staining, and ran as a single diffuse band of ⁇ 60kD (Fig. 1B). This was in contrast to the multiple bands observed with mammalian Myc by immunoblot (Fig. 1B).
  • Myc was purified to near homogeneity from overexpressing mammalian cells and baculovirus infected cells.
  • the mammalian derived protein appears to be highly modified in contrast to Myc expressed in and purified from insect cells.
  • Up to 19 distinct species of c-Myc can be identified by two dimensional gel electrophoresis (Fig. 1). These species differ both in size (approximate MRs of 60,000, 62,000 and 72,000, although this estimate of size can vary with different gel conditions) and in pi. These differences in pi might in part be attributed to differences in phosphorylation, as c-Myc is known to be phosphorylated and the change in pi of the species is consistent with incremental additions of phosphate.
  • Myc produced by the baculovirus overexpression system does not demonstrate the same molecular weight heterogeneity as the mammalian protein, it too is phosphorylated (Miyamoto et al., Mol. Cell Biol. 5:2860- 2865 (1985)). The specific sites of phosphorylation have not been determined for either Myc preparation and other as yet unidentified modifications may distinguish these two Myc preparations.
  • CACGTG Adenovirus major late promoter upstream element
  • MLC myosin light chain
  • the immunoglobulin enhancer site and the B site (CAGCTG) which has the same core sequence as the ⁇ E2 site.
  • the heat shock element (HSE) served as a control since its sequence does not resemble an E-Box core.
  • C1 USE specific
  • C2 USE specific
  • C2' ⁇ E2 specific
  • CHO and baculovirus Myc preparations were similar with regard to the C1 complex, however they differed with regard to the faster migrating complexes.
  • the C2' complex formed on the ⁇ E2 site of the immunoglobulin enhancer and the is ⁇ E2-like sequence of the MLC-B site (Fig. 2, lanes 1 and 4).
  • Baculovirus Myc contained no binding activity with this specificity (Fig. 2, lanes 7 and 10). In contrast, formation of the C2 complex was detected using either Myc preparation.
  • Myc might not be the only protein involved in formation of the three complexes.
  • Myc was also purified from a bacterial overexpression system.
  • the expression system and purification method used were those of Chi Dang and colleagues (see materials and methods).
  • the bacterially produced protein contains the IgG binding segment of protein A fused to the C-terminal 85 amino acids of Myc, the segment of Myc which contains the B-HLH and leucine zipper motifs.
  • the small region of the protein containing the B-HLH motif is not only necessary but fully sufficient for DNA binding if the correct oligomerization partner is present.
  • This protein was able to form the C1 complex on tlie USE probes (Fig. 4, lane 9) and to combine with the 26-29 kD factor to create the C2 complex (Fig. 4, lane 10).
  • This protein contains most of the HLH domain and the entire leucine zipper domain but no basic region. Although this protein is capable of forming homo-oligomers in solution (Gentz et al., Sdence 243:1695-1699 (1989)), it was unable to bind to DNA to form the C1 complex and was also unable to combine with the 26-29 kD factor to create any USE binding activity (Fig. 4, lane 12). These data argue that the role of Myc in the C2 hetero-oligomer requires an intact basic region, the region responsible for specific DNA contacts in other B-HLH proteins.
  • the Myc preparations were incubated with a Myc-specific monoclonal antibody prior to EMSA.
  • the probe used in this experiment contained a single site with the USE core sequence, CACGTG.
  • the Myc-specific antibody eliminated both the C1 and C2 complexes and produced a prominent complex of slower mobility (Fig. 6). It is not clear from these data which of the two complexes was supershifted but the presence of one predominant shifted complex when antibody is present and two complexes in the absence of antibody argues that the Myc-specific antibody also completely disrupted one of the original complexes. There was no effect of a control monoclonal antibody on the formation of either the C1 or C2 complex. The Myc-specific antibody did not alter the C2 ' complex, suggesting that Myc is not present in this complex.
  • the C1 complex is formed by Myc alone, mat the C2 complex contains Myc and a 26-29 kd factor and that the C2 ' complex contains a 40-50 kd factor but does not contain Myc. It is interesting that the C2' complex requires the presence of Myc for formation, but apparently does not contain Myc. Myc therefore appears capable of affecting the 40-50 kd factor's ability to form the C2' complex without being a member of the complex. Whatever the
  • Max protein can be immunoprecipitated from avian and human cells and low stringency Southern analysis has suggested that a single Max gene or a small family of genes exist in other vertebrates as well (Blackwood and Eisenmann, Science 251:1211-1217 (1991)). It is possible that hamster and insect cells have an equivalent of Max. The recovery of a Max-like activity from insect cells is particularly interesting since no Myc homologs have been found in insects to date. Drosophila dearly uses B-HLH heterodimers to regulate aspects of development and the possibility remains that the natural partner for the 26-29 kD protein in insect cells is an as yet unidentified B-HLH protein which functions like Myc. The presence of the 26-29 kD factor in these preparations might limit their usefulness for certain experiments. By pooling Myc containing fractions based on an EMSA assay, one may obtain fractions that contain only the C1 activity and that do not contain the C2 activity, although this modification reduces the final yield by approximately 80%.
  • the DNA that ran at the position of the C1 or C2 (and comigrating C2 ') complexes was isolated, amplified by the polymerase chain reaction (PCR), and used in a second round of EMSA selection. Either seven (CHO preparation) or eight (baculovirus
  • Group I contains sequences that were selected by the C1 complex from either mammalian or baculovirus preparations. These sequences were pooled for analysis because with both preparations formation of the C1 complex requires only Myc protein, and because the two sets of sequences (that isolated with mammalian Myc and that isolated with baculovirus Myc) were similar to each other. Most of the selected sequences in this group contained the sequence CACGTG (21/27 of sequenced subclones). By aligning all of the sequences that contained this central core sequence, it was found that the sequences flanking this core were also nonrandom. A 12 base consensus sequence of
  • GACCACGTGCTC [SEQ ID. No. 1] was determined for sites selected by the C1 complex (see table in Fig. 7 for frequencies at each position; for a base to be included in the consensus it had to be found in at least 10 out of the 21 sequences with a CACGTG core).
  • baculovirus preparations selected sequences similar to those selected by the C1 complex (Fig. 7, Group II). Most of these selected sequences also contained the CACGTG core (19/22). These sequences had similar flanking sequences adjacent to the core hexamer to those found with the C1 complex, although there was a slight preference for GCC over CTC in the 3' flank (see table for Group II in Fig. 7).
  • yeast host cells are transformed with plasmids carrying a c-Myc expression vector (host 'a'); or the c-Myc expression vector and a 26-29 kilodalton C2 complex protein identified as above (host 'b').
  • all yeast strains are cotransformed with a plasmid that contains the coding sequence for ⁇ -galactosidase operably-linked to the CACGTG sequence motif as described above.
  • a lawn of each of the transformed yeast strains is spread on agar plates containing X-gal in the medium and small filter disks containing compound W, X, Y, or z are placed on the lawns.
  • the yeast are allowed to grow and the plates are monitored for colony growth and colony color by visui 1 observation. Typical results from such an experiment arc shown in Table 1.
  • Compound Y does not prevent induction of ⁇ -galactosidase activity in the 'b' host cells. Therefore, compound Y is not an inhibitor of C2 complex hetero-oligomer formation.
  • Compound Z shows an interesting effect of inducing ⁇ -galactosidase activity in the 'a' host cells which does not contain the C2 complex protein used in the 'b' hosts, rather than preventing hetero-oligomer formation. This suggests that compound Z may induce synthesis of a partner protein which is not otherwise present in the yeast host cells or that it may be (or mimic) such a protein.
  • compound W would be identified as an inhibitor of C2 complex formation and/or DNA binding and thus of c-Myc transcriptional activity in vivo.
  • yeast host cells are transformed with two plasmids, each plasmid carrying a C2' complex expression vector encoding at least one 40-50 kilodalton C2' peptide (host 'a'); or the c-Myc expression vector in addition to the vectors encoding the C2' complex proteins identified as above (host 'b').
  • all yeast strains are cotransformed with a plasmid that contains the coding sequence for ⁇ -galactosidase operably- linked to the CAGCTG sequence motif as described above.
  • W is an inhibitor of C2' complex hetero-oligomer formation and an inhibitor of the c-Myc biological activity that is directed towards promoting such C2' complex hetero-oligomer formation.
  • Compound X inhibits the growth of yeast per se and thus would not be a compound of interest.
  • Compound Y does not prevent induction of ⁇ -galactosidase activity in the 'b' host cells. Therefore, compound Y is not an inhibitor of C2 complex hetero-oligomer formation.
  • Compound Z shows an interesting effect of inducing ⁇ -galactosidase activity in the 'a' host cells which does not contain the Myc protein used in the 'b' hosts, rather than preventing hetero-oligomer formation. This suggests that compound Z may induce synthesis of a protein that can substitute for Myc in promoting formation of the C2' complex which is not otherwise present in the yeast host cells or that it may be (or mimic) such a protein.
  • compound W would be identified as an inhibitor of C2' complex formation and/or DNa binding activity and thus of c-Myc transcriptional activity in vivo. All references cited herein are fully incorporated by reference.
  • ADDRESSEE Sterne, Kessler, Goldstein & Fox
  • NAME Cimbala, Michele A

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  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Hospice & Palliative Care (AREA)
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Abstract

L'invention décrit le développement d'une lignée de cellules de mammifère surexprimant Myc, ainsi que la purification de quantités importantes de c-Myc à partir desdites cellules. L'invention décrit également trois types de formation d'oligomérisation (ou complexe) de protéines provoqués par c-Myc: (1) des complexes homo-oligomères (dénommés complexes C1) constitués par l'association d'au moins deux peptides de c-Myc, (2) des complexes hétéro-oligomères (dénommés complexes C2) constitués par l'hétérodimérisation d'au moins deux peptides, dont au moins une n'est pas le peptide de c-Myc et par l'hétéro-oligomérisation spécifique entre c-Myc et un facteur de 26-29 kd, (3) des complexes hétéro-oligomères dépendant de c-Myc (dénommés complexes C2') constitués en présence de c-Myc, de telles protéines hétéro-oligomères ne contenant cependant pas de peptides qui sont c-Myc. L'invention se rapporte de plus à un procédé fiable et précis servant à classifier objectivement des composés, y compris des produits pharmaceutiques s'adressant à l'homme, en tant qu'inhibiteurs de l'activité de c-Myc.
PCT/US1992/008603 1991-10-30 1992-10-09 Partenaires de fixation de l'adn de c-myc, motifs, dosage de criblage et utilisations WO1993008701A1 (fr)

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US78556791A 1991-10-30 1991-10-30
US07/785,567 1991-10-30

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996017960A3 (fr) * 1994-12-07 1996-08-29 Scriptgen Pharm Inc Procedes d'inhibition de l'activite amplificatrice de transcription de la proteine x du virus de l'hepatite b
US5759776A (en) * 1995-06-05 1998-06-02 California Pacific Medical Center Targets for breast cancer diagnosis and treatment
US5776683A (en) * 1996-07-11 1998-07-07 California Pacific Medical Center Methods for identifying genes amplified in cancer cells
WO1998039483A1 (fr) * 1997-03-04 1998-09-11 Ventana Genetics, Inc. Methodes d'identification de sequences d'acide nucleique codant des agents qui influent sur des phenotypes cellulaires
US6566057B1 (en) 1997-02-14 2003-05-20 Deltagen Proteomics, Inc. Methods and compositions for peptide libraries displayed on light-emitting scaffolds
US6623922B1 (en) 1997-02-14 2003-09-23 Deltagen Proteomics Methods for identifying, characterizing, and evolving cell-type specific CIS regulatory elements
EP1469870A4 (fr) * 2001-11-08 2005-11-02 Sagres Discovery Inc Nouvelles compositions et methodes pour le traitement du cancer

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
ANN. REV. GENET., Volume 120, issued 1986, M.D. COLE, "The Myc Oncogene: Its Role in Transformation and Differentiation", pages 361-384. *
MOLECULAR AND CELLULAR BIOLOGY, Volume 5, Number 3, issued March 1985, R.A. WATT et al., "Expression and Characterization of the Human C-Myc DNA Binding Protein", pages 448-456. *
NATURE, Volume 296, issued 18 March 1982, P. DONNER et al., "Nuclear Localization and DNA Binding of the Transforming Gene Product of Avian Myelocytomatosis Virus", pages 262-266. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES UNITED STATES OF AMERICA, Volume 81, issued December 1984, G. RAMSEY et al., "The Protein Encoded by the Human Proto-Oncogene C-Myc", pages 7742-7746. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES UNITED STATES OF AMERICA, Volume 83, issued August 1986, F.M. WURM, "Inducible Overproduction of the Mouse C-Myc Protein in Mammalian Cells", pages 5414-5418. *
SCIENCE, Volume 225, issued 17 August 1984, H. PERSSON et al., "Nuclear Localization and DNA Binding Properties of a Protein Expressed by Human C-Myc Oncogene", pages 718-721. *
SCIENCE, Volume 250, issued 23 November 1990, T.K. BLACKWELL et al., "Sequence-Specific DNA Binding by the C-Myc Protein", pages 1149-1151. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996017960A3 (fr) * 1994-12-07 1996-08-29 Scriptgen Pharm Inc Procedes d'inhibition de l'activite amplificatrice de transcription de la proteine x du virus de l'hepatite b
US6051373A (en) * 1994-12-07 2000-04-18 Scriptgen Pharmaceuticals, Inc. Methods for screening for inhibitors of the transcription-enhancing activity of the X protein of hepatitis B virus
US5759776A (en) * 1995-06-05 1998-06-02 California Pacific Medical Center Targets for breast cancer diagnosis and treatment
US5776683A (en) * 1996-07-11 1998-07-07 California Pacific Medical Center Methods for identifying genes amplified in cancer cells
US5955275A (en) * 1997-02-14 1999-09-21 Arcaris, Inc. Methods for identifying nucleic acid sequences encoding agents that affect cellular phenotypes
US6566057B1 (en) 1997-02-14 2003-05-20 Deltagen Proteomics, Inc. Methods and compositions for peptide libraries displayed on light-emitting scaffolds
US6579675B2 (en) 1997-02-14 2003-06-17 Deltagen Proteomics, Inc. Methods for identifying nucleic acid sequences encoding agents that effect cellular phenotypes
US6623922B1 (en) 1997-02-14 2003-09-23 Deltagen Proteomics Methods for identifying, characterizing, and evolving cell-type specific CIS regulatory elements
WO1998039483A1 (fr) * 1997-03-04 1998-09-11 Ventana Genetics, Inc. Methodes d'identification de sequences d'acide nucleique codant des agents qui influent sur des phenotypes cellulaires
EP1469870A4 (fr) * 2001-11-08 2005-11-02 Sagres Discovery Inc Nouvelles compositions et methodes pour le traitement du cancer

Also Published As

Publication number Publication date
PT101014A (pt) 1994-02-28
AU2808392A (en) 1993-06-07

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