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. 1988 Aug 1;107(2):791–799. doi: 10.1083/jcb.107.2.791

Two independent mechanisms for escaping epidermal growth factor- mediated growth inhibition in epidermal growth factor receptor- hyperproducing human tumor cells

PMCID: PMC2115197  PMID: 2458359

Abstract

Human squamous cell carcinoma cell lines often possess increased levels of epidermal growth factor (EGF) receptor. The growth of these EGF receptor-hyperproducing cells is usually inhibited by EGF. To investigate the mechanism of EGF-mediated inhibition of cell growth, variants displaying alternate responses to EGF were isolated from two squamous cell carcinoma lines, NA and Ca9-22; these cell lines possess high numbers of the EGF receptor and an amplified EGF receptor (EGFR) gene. The variants were isolated from NA cells after several cycles of EGF treatment and they have acquired EGF-dependent growth. Scatchard plot analysis revealed a decreased level of EGF receptor in these ER variants as compared with parental NA cells. Southern blot analysis and RNA dot blot analysis demonstrated that the ER variants had lost the amplified EGFR gene. One variant isolated from Ca9-22 cells, CER-1, grew without being affected by EGF. CER-1 cells had higher numbers of EGF receptor than parental Ca9-22 but similar EGFR gene copy number. Flow cytometric analysis indicated an increase in ploidy and cell volume which may give rise to the increase in receptor number per cell. The EGF receptors on both Ca9-22 and CER-1 cells were autophosphorylated upon EGF exposure in a similar manner suggesting no obvious alteration in receptor tyrosine kinase. However, very efficient down-regulation of the EGF receptor occurred in CER-1 cells. These data suggest two independent mechanisms by which EGF receptor-hyperproducing cells escape EGF-mediated growth inhibition: one mechanism is common and involves the loss of the amplified EGFR genes, and another is novel and involves the efficient down-regulation of the cell-surface receptor.

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Selected References

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  1. Barnes D. W. Epidermal growth factor inhibits growth of A431 human epidermoid carcinoma in serum-free cell culture. J Cell Biol. 1982 Apr;93(1):1–4. doi: 10.1083/jcb.93.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beemon K., Hunter T. Characterization of Rous sarcoma virus src gene products synthesized in vitro. J Virol. 1978 Nov;28(2):551–566. doi: 10.1128/jvi.28.2.551-566.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Behzadian M. A., Shimizu N. Monoclonal antibody that immunoreacts with a subclass of human receptors for epidermal growth factor. Cell Struct Funct. 1985 Sep;10(3):219–232. doi: 10.1247/csf.10.219. [DOI] [PubMed] [Google Scholar]
  4. Behzadian M. A., Shimizu N. Variant of A431 cells isolated by ricin A-conjugated monoclonal antibody directed to EGF receptor: phosphorylation of EGF receptor and phosphatidylinositol. Somat Cell Mol Genet. 1985 Nov;11(6):579–591. doi: 10.1007/BF01534723. [DOI] [PubMed] [Google Scholar]
  5. Bravo R., Burckhardt J., Curran T., Müller R. Stimulation and inhibition of growth by EGF in different A431 cell clones is accompanied by the rapid induction of c-fos and c-myc proto-oncogenes. EMBO J. 1985 May;4(5):1193–1197. doi: 10.1002/j.1460-2075.1985.tb03759.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bravo R. Epidermal growth factor inhibits the synthesis of the nuclear protein cyclin in A431 human carcinoma cells. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4848–4850. doi: 10.1073/pnas.81.15.4848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Buss J. E., Chouvet C., Gill G. N. Comparison of protein phosphorylations in variant A431 cells with different growth responses to epidermal growth factor. J Cell Physiol. 1984 Jun;119(3):296–306. doi: 10.1002/jcp.1041190307. [DOI] [PubMed] [Google Scholar]
  8. Buss J. E., Kudlow J. E., Lazar C. S., Gill G. N. Altered epidermal growth factor (EGF)-stimulated protein kinase activity in variant A431 cells with altered growth responses to EGF. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2574–2578. doi: 10.1073/pnas.79.8.2574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Carpenter G., Cohen S. Epidermal growth factor. Annu Rev Biochem. 1979;48:193–216. doi: 10.1146/annurev.bi.48.070179.001205. [DOI] [PubMed] [Google Scholar]
  10. Cohen S., Carpenter G., King L., Jr Epidermal growth factor-receptor-protein kinase interactions. Co-purification of receptor and epidermal growth factor-enhanced phosphorylation activity. J Biol Chem. 1980 May 25;255(10):4834–4842. [PubMed] [Google Scholar]
  11. Cohen S., Ushiro H., Stoscheck C., Chinkers M. A native 170,000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles. J Biol Chem. 1982 Feb 10;257(3):1523–1531. [PubMed] [Google Scholar]
  12. Crissman H. A., Steinkamp J. A. Rapid, simultaneous measurement of DNA, protein, and cell volume in single cells from large mammalian cell populations. J Cell Biol. 1973 Dec;59(3):766–771. doi: 10.1083/jcb.59.3.766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Downward J., Parker P., Waterfield M. D. Autophosphorylation sites on the epidermal growth factor receptor. Nature. 1984 Oct 4;311(5985):483–485. doi: 10.1038/311483a0. [DOI] [PubMed] [Google Scholar]
  14. Fabricant R. N., De Larco J. E., Todaro G. J. Nerve growth factor receptors on human melanoma cells in culture. Proc Natl Acad Sci U S A. 1977 Feb;74(2):565–569. doi: 10.1073/pnas.74.2.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Filmus J., Trent J. M., Pollak M. N., Buick R. N. Epidermal growth factor receptor gene-amplified MDA-468 breast cancer cell line and its nonamplified variants. Mol Cell Biol. 1987 Jan;7(1):251–257. doi: 10.1128/mcb.7.1.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gamou S., Hirai M., Rikimaru K., Enomoto S., Shimizu N. Biosynthesis of the epidermal growth factor receptor in human squamous cell carcinoma lines: secretion of the truncated receptor is not common to epidermal growth factor receptor-hyperproducing cells. Cell Struct Funct. 1988 Feb;13(1):25–38. doi: 10.1247/csf.13.25. [DOI] [PubMed] [Google Scholar]
  17. Gamou S., Hunts J., Harigai H., Hirohashi S., Shimosato Y., Pastan I., Shimizu N. Molecular evidence for the lack of epidermal growth factor receptor gene expression in small cell lung carcinoma cells. Cancer Res. 1987 May 15;47(10):2668–2673. [PubMed] [Google Scholar]
  18. Gamou S., Kim Y. S., Shimizu N. Different responses to EGF in two human carcinoma cell lines, A431 and UCVA-1, possessing high numbers of EGF receptors. Mol Cell Endocrinol. 1984 Sep;37(2):205–213. doi: 10.1016/0303-7207(84)90053-4. [DOI] [PubMed] [Google Scholar]
  19. Gamou S., Shimizu N. Change in metabolic turnover is an alternate mechanism increasing cell surface epidermal growth factor receptor levels in tumor cells. J Biol Chem. 1987 May 15;262(14):6708–6713. [PubMed] [Google Scholar]
  20. Gill G. N., Lazar C. S. Increased phosphotyrosine content and inhibition of proliferation in EGF-treated A431 cells. Nature. 1981 Sep 24;293(5830):305–307. doi: 10.1038/293305a0. [DOI] [PubMed] [Google Scholar]
  21. Gill G. N., Weber W., Thompson D. M., Lin C., Evans R. M., Rosenfeld M. G., Gamou S., Shimizu N. Relationship between production of epidermal growth factor receptors, gene amplification, and chromosome 7 translocation in variant A431 cells. Somat Cell Mol Genet. 1985 Jul;11(4):309–318. doi: 10.1007/BF01534689. [DOI] [PubMed] [Google Scholar]
  22. Haigler H. T., Maxfield F. R., Willingham M. C., Pastan I. Dansylcadaverine inhibits internalization of 125I-epidermal growth factor in BALB 3T3 cells. J Biol Chem. 1980 Feb 25;255(4):1239–1241. [PubMed] [Google Scholar]
  23. Haigler H., Ash J. F., Singer S. J., Cohen S. Visualization by fluorescence of the binding and internalization of epidermal growth factor in human carcinoma cells A-431. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3317–3321. doi: 10.1073/pnas.75.7.3317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hunter T., Cooper J. A. Epidermal growth factor induces rapid tyrosine phosphorylation of proteins in A431 human tumor cells. Cell. 1981 Jun;24(3):741–752. doi: 10.1016/0092-8674(81)90100-8. [DOI] [PubMed] [Google Scholar]
  25. Hunts J. H., Shimizu N., Yamamoto T., Toyoshima K., Merlino G. T., Xu Y. H., Pastan I. Translocation chromosome 7 of A431 cells contains amplification and rearrangement of EGF receptor gene responsible for production of variant mRNA. Somat Cell Mol Genet. 1985 Sep;11(5):477–484. doi: 10.1007/BF01534841. [DOI] [PubMed] [Google Scholar]
  26. Hunts J., Gamou S., Hirai M., Shimizu N. Molecular mechanisms involved in increasing epidermal growth factor receptor levels on the cell surface. Jpn J Cancer Res. 1986 May;77(5):423–427. [PubMed] [Google Scholar]
  27. Iwashita S., Fox C. F. Epidermal growth factor and potent phorbol tumor promoters induce epidermal growth factor receptor phosphorylation in a similar but distinctively different manner in human epidermoid carcinoma A431 cells. J Biol Chem. 1984 Feb 25;259(4):2559–2567. [PubMed] [Google Scholar]
  28. Kawamoto T., Mendelsohn J., Le A., Sato G. H., Lazar C. S., Gill G. N. Relation of epidermal growth factor receptor concentration to growth of human epidermoid carcinoma A431 cells. J Biol Chem. 1984 Jun 25;259(12):7761–7766. [PubMed] [Google Scholar]
  29. Lifshitz A., Lazar C. S., Buss J. E., Gill G. N. Analysis of morphology and receptor metabolism in clonal variant A431 cells with differing growth responses to epidermal growth factor. J Cell Physiol. 1983 Jun;115(3):235–242. doi: 10.1002/jcp.1041150304. [DOI] [PubMed] [Google Scholar]
  30. Lin C. R., Chen W. S., Kruiger W., Stolarsky L. S., Weber W., Evans R. M., Verma I. M., Gill G. N., Rosenfeld M. G. Expression cloning of human EGF receptor complementary DNA: gene amplification and three related messenger RNA products in A431 cells. Science. 1984 May 25;224(4651):843–848. doi: 10.1126/science.6326261. [DOI] [PubMed] [Google Scholar]
  31. Maron R., Jackson R. A., Jacobs S., Eisenbarth G., Kahn C. R. Analysis of the insulin receptor by anti-receptor antibodies and flow cytometry. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7446–7450. doi: 10.1073/pnas.81.23.7446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Merlino G. T., Xu Y. H., Ishii S., Clark A. J., Semba K., Toyoshima K., Yamamoto T., Pastan I. Amplification and enhanced expression of the epidermal growth factor receptor gene in A431 human carcinoma cells. Science. 1984 Apr 27;224(4647):417–419. doi: 10.1126/science.6200934. [DOI] [PubMed] [Google Scholar]
  33. Miskimins W. K., Ferris W. R., Shimizu N. Genetics of receptors for bioactive polypeptides: a variant of Swiss/3T3 fibroblasts resistant to a cytotoxic insulin accumulates lysosome-like vesicles. J Supramol Struct Cell Biochem. 1981;16(1):105–113. doi: 10.1002/jsscb.1981.380160110. [DOI] [PubMed] [Google Scholar]
  34. Miskimins W. K., Shimizu N. Genetics of cell surface receptors for bioactive polypeptides: variants of Swiss/3T3 fibroblasts resistant to a cytotoxic chimeric insulin. Proc Natl Acad Sci U S A. 1981 Jan;78(1):445–449. doi: 10.1073/pnas.78.1.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Miskimins W. K., Shimizu N. Uptake of epidermal growth factor into a lysosomal enzyme-deficient organelle: correlation with cell's mitogenic response and evidence for ubiquitous existence in fibroblasts. J Cell Physiol. 1984 Mar;118(3):305–316. doi: 10.1002/jcp.1041180314. [DOI] [PubMed] [Google Scholar]
  36. Mullaney P. F., Dean P. N. The small angle light scattering of biological cells. Theoretical considerations. Biophys J. 1970 Aug;10(8):764–772. doi: 10.1016/S0006-3495(70)86334-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Müller R., Bravo R., Burckhardt J., Curran T. Induction of c-fos gene and protein by growth factors precedes activation of c-myc. Nature. 1984 Dec 20;312(5996):716–720. doi: 10.1038/312716a0. [DOI] [PubMed] [Google Scholar]
  38. Sawyer S. T., Cohen S. Enhancement of calcium uptake and phosphatidylinositol turnover by epidermal growth factor in A-431 cells. Biochemistry. 1981 Oct 13;20(21):6280–6286. doi: 10.1021/bi00524a057. [DOI] [PubMed] [Google Scholar]
  39. Schechter Y., Hernaez L., Schlessinger J., Cuatrecasas P. Local aggregation of hormone-receptor complexes is required for activation by epidermal growth factor. Nature. 1979 Apr 26;278(5707):835–838. doi: 10.1038/278835a0. [DOI] [PubMed] [Google Scholar]
  40. Schlessinger J., Shechter Y., Willingham M. C., Pastan I. Direct visualization of binding, aggregation, and internalization of insulin and epidermal growth factor on living fibroblastic cells. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2659–2663. doi: 10.1073/pnas.75.6.2659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shimizu N., Shimizu Y., Miskimins W. K. EGF-ricin A conjugates: kinetic profiles of cytotoxic effects and resistant cell variants. Cell Struct Funct. 1984 Sep;9(3):203–212. doi: 10.1247/csf.9.203. [DOI] [PubMed] [Google Scholar]
  42. Ullrich A., Coussens L., Hayflick J. S., Dull T. J., Gray A., Tam A. W., Lee J., Yarden Y., Libermann T. A., Schlessinger J. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. 1984 May 31-Jun 6Nature. 309(5967):418–425. doi: 10.1038/309418a0. [DOI] [PubMed] [Google Scholar]
  43. White B. A., Bancroft F. C. Cytoplasmic dot hybridization. Simple analysis of relative mRNA levels in multiple small cell or tissue samples. J Biol Chem. 1982 Aug 10;257(15):8569–8572. [PubMed] [Google Scholar]
  44. Yamamoto T., Kamata N., Kawano H., Shimizu S., Kuroki T., Toyoshima K., Rikimaru K., Nomura N., Ishizaki R., Pastan I. High incidence of amplification of the epidermal growth factor receptor gene in human squamous carcinoma cell lines. Cancer Res. 1986 Jan;46(1):414–416. [PubMed] [Google Scholar]

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