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EP0450048A4 - Stable mammalian cell lines that express aromatase - Google Patents

Stable mammalian cell lines that express aromatase

Info

Publication number
EP0450048A4
EP0450048A4 EP19900916561 EP90916561A EP0450048A4 EP 0450048 A4 EP0450048 A4 EP 0450048A4 EP 19900916561 EP19900916561 EP 19900916561 EP 90916561 A EP90916561 A EP 90916561A EP 0450048 A4 EP0450048 A4 EP 0450048A4
Authority
EP
European Patent Office
Prior art keywords
aromatase
cell
cell lines
plasmid
mammalian cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19900916561
Other languages
English (en)
Other versions
EP0450048A1 (fr
Inventor
Shiuan Chen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
City of Hope
Original Assignee
City of Hope
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by City of Hope filed Critical City of Hope
Publication of EP0450048A1 publication Critical patent/EP0450048A1/fr
Publication of EP0450048A4 publication Critical patent/EP0450048A4/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • This invention relates to mammalian cell lines which express aromatase.
  • the cell lines are useful to screen aromatase inhibitors useful as anti-breast cancer drugs.
  • Aromatase catalyzes the formation of C-18 estrogenic steroids from C-19 androgens. Aromatase inhibitors are useful in breast cancer therapy because of the central importance of estrogens to the development of such malignancies.
  • Aromatase has been expressed in yeast. Pompon, D., et al.. Molecular Endrocrinoloqy 3:1477-1487 (1989). However, the yeast model is not preferred for screening drugs for humans. The need for a reliable and a rapid method for the primary screening of aromatase inhibitors is apparent. Such a method requires stable mammalian cell lines which contain high levels of aromatase. Corbin, C.J., et al., Proc.Natl.Acad.Sci.USA 85:8948-8952 (1988) report the expression of human aromatase cDNA in mammalian COS cells through a transient expression method. Because the enzyme is expressed for only a short period of time, the Corbin cell lines are impractical for screening anti-breast cancer drugs.
  • This invention provides stable mammalian cell lines which contain high levels of aromatase useful for the primary screening of aromatase inhibitors.
  • the uptake efficiency of such inhibitors can be evaluated because aromatase activity is measured using intact cells.
  • the effect on cell growth is apparent from a comparison of growth rate in the presence and absence of inhibitors.
  • this invention includes the construction of expression plasmids containing aromatase cDNA and mammalian cells transfected with such plasmids which express a functional aromatase protein.
  • the aromatase expression product has enzymatic properties substantially identical to the enzyme in human placenta.
  • the expressed enzyme has the same Michales-Menken constant (Km) as the wild type enzyme and is coupled efficiently with the endogenous NADPH-cytochrome P-450 reductase.
  • Km Michales-Menken constant
  • the activity of the expressed enzyme is inhibited by known aromatase inhibitors with Ki values similar to those reported in the literature. Accordingly, an important aspect of this invention includes methods to screen aromatase inhibitors as drugs to treat, inter alia, estrogen dependent breast cancer.
  • a full-length human placental aromatase cDNA clone "Aro 2" was isolated upon screening a human placental cDNA library with an aromatase cDNA probe and an oligonucleotide probe whose sequence was derived from a human aromatase genomic clone.
  • Expression plasmids containing the aromatase cDNA clone were constructed. The enzyme was expressed at high levels in transfected mammalian cell lines. The expressed enzyme activity was inhibited by a known aromatase inhibitor, 4-hydroxyandrostenedione.
  • the transfected cell lines are useful in known assay procedures to screen aromatase inhibitors. Such assays may be performed directly on cultured cells without purifying the expressed enzyme.
  • Figure 1 depicts the nucleotide sequence of cDNA clone Aro 2 and the deduced amino acid sequence.
  • the peptide sequence data confirms that the Aro 2 clone encodes for human placental aromatase. Regions corresponding to peptides determined by microsequencing methods are underlined.
  • Figure 2 shows the structure of an aromatase expression plasmid pH ⁇ -hro useful to express aromatase in mammalian cell lines.
  • Figure 3 shows that aromatase expressed in CHO cells transfected with the plasmid of Figure 2 has activity following the normal Michales-Menken kinetics.
  • FIG 4 shows that the aromatase expressed in CHO cells transfected with the plasmid of Figure 2 is inhibited by 4-hydro ⁇ yandrostenedione.
  • yeast S. cerevisiae is a useful host for the expression of mammalian genes particularly cytochrome P-450s because the cells contain microsomal membranes, cytochrome P-450 reductase and cytochrome bs to allow membrane integration and catalytic stability.
  • Gene expression in yeast can be accomplished by two ways. The first involves the stable integration of the foreign gene into the yeast nuclear DNA. This gives stably transformed cells expressing the heterologous protein at a low level.
  • the alternative approach is to include the foreign gene in an autonomous multicopy replicate plasmid which is similar to, but more complex to that used to transform bacteria like Esherichia coli. Therefore, the transformed strain can express high levels of a heterologous protein. In addition, the presence of plasmids is stabilized by the maintenance of a constant selection pressure attributed by a marker being constructed into the plasmids.
  • the second approach was used to express aromatase in yeast.
  • Figure 5 of Pompon, D. , et al., supra is the diagram for the construction of the aromatase expression plasmids. An adaptor with the following sequences
  • Plasmids (i.e. PYeDPll) bearing the insert in the orientation in which the BamHI site just flanking the GALIO-CYCI promoter was destroyed were selected. Plasmid pYeDPll, therefore, has a Bgl II site and a new unique BamHI site.
  • the EcoRI fragment corresponding to the full-length aromatase cDNA was excised from the ⁇ gtll vector by limited EcoRI digestion and cloned into the EcoRI site of pYeDPll in the orientation that the 5' end of the cDNA is next to the GALIO-CYCI segment of the vector giving pExxl.
  • An adaptor-primer with the sequence 5'-GATCAGATCTATGGTTTTGGAAATGCTG-3', was hybridized with the single stranded phase DNA and was elongated using deoxynucleotide-triphosphates and the Klenow fragment of E. coli DNA polymerase I. This newly synthesized single stranded was purified, and the Ml3 reverse sequencing primer was then added to initiate the synthesis of the complementary strand.
  • a double stranded DNA including a synthetic Bgl II site immediately flanking the transduction initiation codon of the aromatase cDNA was obtained.
  • pAroX17 was digested with restriction enzymes, Bgl II and Stu I, and the previously described 1.9 Kb fragment containing aromatase cDNA was purified.
  • the end of the fragment created through restriction by Bgl II has a 3'-OH recessed end, and it was filled in to form blunt end by the addition of Klenow enzyme and the appropriate deoxynucleotides.
  • This aromatase cDNA fragment was then ligated to a Sal I and Hind III restricted and bluntly ended expression vector, pH ⁇ Apr-1-neo (see Gunning, P., et al., Proc.Natl.Acad.Sci. USA 84:4831-4835 (1987)).
  • Aromatase was expressed by human breast cancer cell lines MCF-7 and BT-20 and one non-cancerous human cell line HBL-100 transfected in known manner with the expression plasmid pH -Aro. Aromatase was also expressed by the Chinese hamster ovary (CHO) cell line transfected with the pH y9-Aro plasmid.
  • the transfected cell lines express high level of aromatase as indicated by activity measurement.
  • the enzyme assay was performed directly on cultured cells without purifying the enzyme. Cells are grown to confluence on six-well cell culture plates. Cells are washed twice with serum free cell culture medium before assay.
  • the substrate, androst-4-ene-3, 17-dione [l ,2y ⁇ - 3 H(N) ] (specific activity, 43.1 Ci/mol) is added into each well. After 30 min incubation at 37°C and followed by 5 min incubation on ice, l ml of culture medium is withdrawn from each well.
  • the culture medium is initially mixed with equal volume of chloroform to extract unused substrate, and further mixed with dextran treated charcoal. Charcoal is removed by brief centrifugation, and the supernatant containing the product, tritiated water, is counted.
  • the protein concentration is determined after dissolving cells with 0.5 N NaOH.
  • Figure 3 serves as an example and shows that the aromatase expressed in CHO cells has activity following normal Michales-Menken kinetics. Table I shows that aromatase expressed in these cell lines has Michales-Menken constant (K m ) and maximum velocity (V max ) similar to those calculated for aromatase in human placental microsomes.
  • FIG. 4 shows that the expressed aromatase is inhibited by 4-hydroxyandrostenedione, a well-known aromatase inhibitor. A 50% inhibition of the activity would be achieved by the addition of 30 nM of the inhibitor, a concentration similar to that reported in the literature. This result indicates that this system will be useful to screen aromatase inhibitors as drugs to treat breast cancer.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Bidet-Like Cleaning Device And Other Flush Toilet Accessories (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Lignées cellulaires mammifères stables exprimant l'aromatase humaine, utiles pour sélectionner les médicaments contre le cancer du sein.
EP19900916561 1989-10-18 1990-10-12 Stable mammalian cell lines that express aromatase Withdrawn EP0450048A4 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US42290489A 1989-10-18 1989-10-18
US422904 1989-10-18

Publications (2)

Publication Number Publication Date
EP0450048A1 EP0450048A1 (fr) 1991-10-09
EP0450048A4 true EP0450048A4 (en) 1992-12-02

Family

ID=23676900

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19900916561 Withdrawn EP0450048A4 (en) 1989-10-18 1990-10-12 Stable mammalian cell lines that express aromatase

Country Status (5)

Country Link
EP (1) EP0450048A4 (fr)
JP (1) JPH04502261A (fr)
AU (1) AU6645790A (fr)
CA (1) CA2044249A1 (fr)
WO (1) WO1991005794A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL98506A (en) * 1990-06-18 1996-09-12 Fujisawa Pharmaceutical Co Cyclic peptide antibiotics processes for the preparation thereof and pharmaceutical compositions containing them
US5882871A (en) * 1996-09-24 1999-03-16 Smithkline Beecham Corporation Saliva binding protein
AU2958099A (en) 1998-03-26 1999-10-18 Kyowa Hakko Kogyo Co. Ltd. Method for searching steroid sulfatase inhibitors

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4985352A (en) * 1988-02-29 1991-01-15 The Trustees Of Columbia University In The City Of New York DNA encoding serotonin 1C (5HT1c) receptor, isolated 5HT1c receptor, mammalian cells expressing same and uses thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOLOGICAL ABSTRACTS, vol. 84 Philadelphia, PA, US; abstract no. 100057, (R. W. BRUEGGEMEIER ET AL.): 'Effects of the aromatase inhibitor 7-alpha-4' aminophenylthio-4-androstene-3 17-dione in MCF-7 human mammary carcinoma cell culture' *
CANCER RES. vol. 50, no. 21, 1990, pages 6949 - 6954 (D. ZHOU ET AL.): 'Stable expression of human aromatase complementary DNA in mammalian cells is a useful system for aromatase inhibitor screening' *
FASEB JOURNAL. MEETING HELD JUNE 4-7, 1990. vol. 4, no. 7, 26 April 1990, BETHESDA, MD, US; page A2238 (D. ZHOU ET AL.): 'Stable expression of human aromatase cDNA in mammalian cells' *
See also references of WO9105794A1 *

Also Published As

Publication number Publication date
CA2044249A1 (fr) 1991-04-19
AU6645790A (en) 1991-05-16
WO1991005794A1 (fr) 1991-05-02
JPH04502261A (ja) 1992-04-23
EP0450048A1 (fr) 1991-10-09

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