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Comparison of p53 Mutational Status with mRNA and Protein Expression in a Panel of 24 Human Breast Carcinoma Cell Lines

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Abstract

We analyzed the p53 mutational status, mRNA and protein expression in 24 human breast carcinoma cell lines. Following measurement of their DNA content with flow cytometry, we ascertained the copy numbers of the centromere of chromosome 17 (cen17) and p53 with fluorescence in situ hybridization (FISH). A functional yeast assay (FASAY) was used to screen for inactivating mutations. Positive results were subsequently verified by DNA sequencing. Finally, we assessed the mRNA expression with a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay and the protein expression with immunocytochemical staining, western blot, and quantitative flow cytometry. The DNA content of the cell lines ranged from 0.85 to 2.58. Nine cell lines had concordant copy numbers (between two and four) of p53 and cen17, whereas 12 had more, and three less cen17 than p53 copies. The FASAY was successful in all but one cell line and revealed the presence of mutated alleles in 16 of them, 13 cell lines expressed only the mutated, and three both the mutated and the wild-type alleles. The mutations were comprised of 11 missense, two nonsense, and three frameshift mutations. Immunocytochemical staining, western blot and quantitative flow cytometry yielded comparable p53 protein expression results. However, both the mRNA and the protein expression levels varied considerably in the different cell lines and no consistent pattern with regard to the respective p53 mutational status became evident. The results obtained in these breast carcinoma cell lines indicate that no clear-cut linear relationship exists between the p53 mutational status and the extent of its respective mRNA and protein expression. Therefore, direct DNA analyses and functional assays remain the only methods for the reliable detection of p53 mutations.

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References

  1. Lane DP: Cancer: p53, guardian of the genome. Nature 258: 15–16, 1992

    Google Scholar 

  2. Zambetti GP, Bargonetti J, Walker J: Wild-type p53 mediates positive regulation of gene expression through a specific DNA sequence element. Genes Dev 6: 1143–1152, 1992

    Google Scholar 

  3. Kastan MB, Onyekwere O, Sidransky D, Vogelstein B, Craig RW: Participation of p53 protein in cellular response to DNA damage. Cancer Res 51: 6304–6311, 1991

    Google Scholar 

  4. Hartwell IH, Kastan MB: Cell cycle control and cancer. Science 266: 1821–1828, 1994

    Google Scholar 

  5. Hollstein M, Sidransky D, Vogelstein B, Harris CC: p53 mutations in human cancers. Science 253: 49–53, 1991

    Google Scholar 

  6. Eliopoulos AG, Kerr DJ, Herod J, Hodgkins I, Krajewski S, Reed JC, Young LS: The control of apoptosis and drug resistance in ovarian cancer: influence of p53 and bcl-2. Oncogene 11: 1217–1228, 1995

    Google Scholar 

  7. Lowe SW, Ruley HE, Jacks T, Housman DE: p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 74: 957–967, 1993

    Google Scholar 

  8. Blaszyk H, Hartmann A, Cunningham JM, Schaid D, Wold IE, Kovach JS, Sommer SS: A prospective trial of midwest breast cancer patients: a p53 gene mutation is the most important predictor of adverse outcome. Int J Cancer 89: 32–38, 2000

    Google Scholar 

  9. Powell BL, Bydder S, Grieu F, Gnanasampathan G, Elsaleh H, Sashadri R, Berns EM, Iacopetta B: Prognostic value of TP53 gene mutation in adjuvant treated breast cancer patients. Breast Cancer Res Tr 69: 65–68, 2001

    Google Scholar 

  10. Reed W, Hannisdal E, Boehler PJ, Gundersen S, Host H, Marthin J: The prognostic value of p53 and c-erb B-2 immunostaining is overrated for patients with lymph node negative breast carcinoma: a multivariate analysis of prognostic factors in 613 patients with a follow-up of 14-30 years. Cancer 88: 804–813, 2000

    Google Scholar 

  11. Powell B, Soong R, Iacopetta B, Seshadri R, Smith DR: Prognostic significance of mutations to different structural and functional regions of the p53 gene in breast cancer. Clin Cancer Res 6: 443–451, 2000

    Google Scholar 

  12. Gupta M, Fan S, Zhan Q, Kohn KW, O'Connor PM, Pommier Y: Inactivation of p53 increases the cytotoxicity of camptothecin in human colon HCT116 and breast MCF-7 cancer cells. Clin Cancer Res 3: 1653–1660, 1997

    Google Scholar 

  13. Caron de Fromentel C, Nardeux PC, Soussi T, Lavialle C, Estrade S, Carloni G, Chandrasekaran K, Cassingena R: Epithelial HBL-100 cell line derived from milk of an apparently healthy woman harbours SV40 genetic information. Exp Cell Res 160: 83–94, 1985

    Google Scholar 

  14. Ziche M, Gullino PM: Angiogenesis and neoplastic progression in vitro. J Natl Cancer I 69: 483–487, 1982

    Google Scholar 

  15. Flaman JM, Frebourg T, Moreau V, Charbonnier F, Martin C, Chappuis P, Sappino AP, Limacher JM, Bron I, Benhattar J, Tada M, Van Meir EG, Estreicher A, Iggo RD: A simple p53 functional assay for screening cell lines, blood, and tumors. P Natl Acad Sci USA 92: 3963–3967, 1995

    Google Scholar 

  16. Sjogren S, Inganas M, Norberg T, Lindgren A, Nordgren H, Holmberg I, Bergh J: The p53 gene in breast cancer: prognostic value of complementary DNA sequencing versus immunohistochemistry. J Natl Cancer I 88: 173–182, 1996

    Google Scholar 

  17. Tong D, Schneeberger C, Leodolter S, Zeillinger R: Quantitative determination of gene expression by competitive reverse transcription-polymerase chain reaction in degraded RNA samples. Anal Biochem 251: 173–177, 1997

    Google Scholar 

  18. Hsu S, Raine I, Fanger H: The use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase technique-a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29: 577–580, 1981

    Google Scholar 

  19. Kohler MF, Kerns BJ, Humphrey PA, Marks JR, Bast RC, Berchuck A: Mutation and overexpression of p53 in earlystage epithelial ovarian cancer. Obstet Gynecol 81: 643–650, 1993

    Google Scholar 

  20. Greenblatt MS, Bennett WP, Hollstein M, Harris CC: Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res 54: 4855–4878, 1994

    Google Scholar 

  21. Harris N, Brill E, Shohat O, Prokocimer M, Wolf D, Arai N, Rotter V: Molecular basis for heterogeneity of the human p53 protein. Mol Cell Biol 6: 4650–4656, 1986

    Google Scholar 

  22. Runnebaum IB, Nagarajan M, Bowman M, Soto D, Sukumar S: Mutations in p53 as potential molecular markers for human breast cancer. P Natl Acad Sci USA 88: 10657–10661, 1991

    Google Scholar 

  23. Knudson AG: Hereditary cancer, oncogenes, and antioncogenes. Cancer Res 45: 1437–1443, 1985

    Google Scholar 

  24. Lavarino C, Corletto V, Mezzelani A, Della Torre G, Bartoli C, Riva C, Pierotti MA, Rilke F, Pilotti S: Detection of TP53 mutation, loss of heterozygosity and DNA content in fine-needle aspirates of breast carcinoma. Brit J Cancer 77: 125–130, 1998

    Google Scholar 

  25. Thompson AM, Anderson TJ, Condie A, Prosser J, Chetty U, Carter DC, Evans HJ, Steel CM: p53 allele losses, mutations and expression in breast cancer and their relationship to clinico-pathological parameters. Int J Cancer 50: 528–532, 1992

    Google Scholar 

  26. Bergh J, Norberg T, Sjögren S, Lindgren A, Holmberg L: Complete sequencing of the p53 gene provides prognostic information in breast cancer patients, particularly in relation to adjuvant systemic therapy and radiotherapy. Nat Med 1: 1029–1034, 1995

    Google Scholar 

  27. Cross SM, Sanchez CA, Morgan CA, Schimke MK, Ramel S, Idzerda RÖ, Raskind WH, Reid BJ: A p53-dependent mouse spindle checkpoint. Science 267: 1353–1356, 1995

    Google Scholar 

  28. Fukasawa K, Choi T, Kuriyama R, Rulong S, Vande Woude GF: Abnormal centrosome amplification in the absence of p53. Science 271: 1744–1747, 1996

    Google Scholar 

  29. Cahill DP, Lengauer C, Yu J, Riggins GJ, Willson JK, Markowitz SD, Kinzler KW, Vogelstein B: Mutations of mitotic checkpoint genes in human cancers. Nature 392: 300–303, 1998

    Google Scholar 

  30. Nayak BK, Das BR: Mutation and methylation status of p53 gene promoter in human breast tumors. Tumor Biol 20: 341–346, 1999

    Google Scholar 

  31. Pogribny IP, Pogribna M, Christman JK, James SI: Singlesite methylation within the p53 promoter region reduces gene expression in a reporter gene construct: possible in vivo relevance during tumorigenesis. Cancer Res 60: 588–594, 2000

    Google Scholar 

  32. Bartek J, Bartkova J, Vojtesek B, Saskova Z, Rejthar A, Kovarik J, Lane DP: Patterns of expression of the p53 tumor suppressor in human breast tissues and tumors in situ and in vitro. Int J Cancer 40: 839–844, 1990

    Google Scholar 

  33. Varley JM, Brammar WJ, Lane DP, Swallow JE, Dolan C, Walker RA: Loss of chromosome 17p13 sequences and mutation of p53 in human breast carcinomas. Oncogene 6: 413–421, 1991

    Google Scholar 

  34. Midgley CA, Lane DP: p53 protein stability in tumor cells is not determined by mutation but is dependent on Mdm2 binding. Oncogene 15: 1179–1189, 1997

    Google Scholar 

  35. Fu I, Minden MD, Benchimol S: Translational regulation of human p53 gene expression. EMBO J 15: 4392–4401, 1996

    Google Scholar 

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Authors and Affiliations

  1. Molecular Oncology Group, Department of Gynecology and Obstetrics, Vienna, Austria

    Nicole Concin, Christa Zeillinger, Dan Tong, Margit Stimpfl, Lukas Hefler, Christian Kainz, Sepp Leodolter & Robert Zeillinger

  2. Children's Cancer Research Institute, St. Anna Children's Hospital, Vienna, Austria

    Margit König, Dieter Printz & Oskar A. Haas

  3. Department of Gynecology and Obstetrics, Division of Endocrinology, University of Vienna Medical School, Vienna, Austria

    Felix Stonek & Christian Schneeberger

  4. Ludwig Boltzmann Institute for Gynecological Oncology and Fertility Treatment, Vienna, Austria

    Sepp Leodolter

  5. Ludwig Boltzmann Institute for Cytogenetic Diagnosis, Vienna, Austria

    Oskar A. Haas

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  1. Nicole Concin
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  2. Christa Zeillinger
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  3. Dan Tong
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Concin, N., Zeillinger, C., Tong, D. et al. Comparison of p53 Mutational Status with mRNA and Protein Expression in a Panel of 24 Human Breast Carcinoma Cell Lines. Breast Cancer Res Treat 79, 37–46 (2003). https://doi.org/10.1023/A:1023351717408

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