Abstract
One of the most important components of G1 checkpoint is the retinoblastoma protein (pRB110). The activity of pRB is regulated by its phosphorylation, which is mediated by genes such as cyclin D1 and p16/MTS1. All three genes have been shown to be commonly altered in human malignancies. We have screened a panel of 26 oral squamous cell carcinomas (OSCC), nine premalignant and three normal oral tissue samples as well as eight established OSCC cell lines for mutations in the p16/MTS1 gene. The expression of p16/MTS1, cyclin D1 and pRB110 was also studied in the same panel. We have found p16/MTS1 gene alterations in 5/26 (19%) primary tumours and 6/8 (75%) cell lines. Two primary tumours and five OSCC cell lines had p16/MTS1 point mutations and another three primary and one OSCC cell line contained partial gene deletions. Six of seven p16/MTS1 point mutations resulted in termination codons and the remaining mutation caused a frameshift. Western blot analysis showed absence of p16/MTS1 expression in 18/26 (69%) OSCC, 7/9 (78%) premalignant lesions and 8/8 cell lines. One cell line, H314, contained a frameshift mutation possibly resulting in a truncated p16/MTS1 protein. pRB was detected in 14/25 (56%) of OSCC but only 11/14 (78%) of these contained all or some hypophosphorylated (active) pRB. In premalignant samples, 6/8 (75%) displayed pRB, and all three normal samples and eight cell lines analysed contained RB protein. p16/MTS1 protein was undetectable in 10/11 (91%) OSCCs with positive pRB. Overexpression of cyclin D1 was observed in 9/22 (41%) OSCC, 3/9 (33%) premalignant and 8/8 (100%) of OSCC cell lines. Our data suggest p16/MTS1 mutations and loss of expression to be very common in oral cancer cell lines and less frequent in primary OSCC tumours. A different pattern of p16/MTS1 mutations was observed in OSCC compared to other cancers with all the detected p16/MTS1 mutations resulting in premature termination codons or a frameshift. The RB protein was expressed in about half (44%) of OSCCs and its expression inversely correlated with p16/MTS1 expression. In conclusion, we show that abnormalities of the RB pathway are a common mechanism of oral carcinogenesis.
Similar content being viewed by others
Article PDF
Change history
16 November 2011
This paper was modified 12 months after initial publication to switch to Creative Commons licence terms, as noted at publication
References
Andl, T., Kahn, T., Pfuhl, A., Nicola, T., Erber, R., Conradt, C., Klein, W., Helbig, M., Dietz, A., Weidauer, H. & Bosch, F. X. (1998) Etiological involvement of oncogenic human papillomavirus in tonsillar squamous cell carcinomas lacking retinoblastoma cell cycle control. Cancer Res 58: 5–13.
Arap, W., Knudsen, E. S., Wang, J. Y., Cavenee, W. K. & Huang, H. J. (1997) Point muatations can inactivate in vitro and in vivo activities of p16 (INK4a) /CDKN2A in human glioma. Oncogene 14: 603–609.
Bagchi, S., Weinmann, R. & Raychaudhuri, P. (1991) The retinoblastoma protein copurifies with E2F-I, an E1A-regulated inhibitor of the transcription factor E2F. Cell 65: 1063–1072.
Bartkova, J., Lukas, J., Muller, H., Strauss, M., Gusterson, B. & Bartek, J. (1995a) Abnormal patterns of D-type cyclin expression and G1 regulation in human head and neck cancer. Cancer Res 55: 949–956.
Bartkova, J., Lukas, J., Strauss, M. & Bartek, J. (1995b) Cyclin D1 oncoprotein aberrantly accumulates in malignancies of diverse histogenesis. Oncogene 10: 775–778.
Berenson, J. R., Yang, J. & Mickel, R. A. (1989) Frequent amplification of the bcl-1 locus in head and neck squamous cell carcinomas. Oncogene 4: 1111–1116.
Chellappan, S. P., Hiebert, S., Mudryj, M., Horowitz, J. M. & Nevins, J. R. (1991) The E2F transcription factor is a cellular target for the RB protein. Cell 55: 1053–1061.
Chen, P. L., Scully, P., Shew, J. Y., Wang, J. Y. & Lee, W. H. (1989) Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation. Cell 55: 1193–1198.
Cheng, J. Q., Jhanwar, S. C., Klein, W. M., Bell, D. W., Lee, W. C., Altomare, D. A., Nobori, T., Olopade, O. I., Buckler, A. J. & Testa, J. R. (1994) p16 alterations and deletion mapping of 9p21-p22 in malignant mesothelioma. Cancer Res 54: 5547–5551.
Elamin, F., Steingrimsdottir, H., Warnakulasuriya, S., Johnson, N. W. & Tavassoli, M. (1998) Prevalence of human papillomavirus infection in premalignant and malignant lesions of the oral cavity in U.K. subjects: A novel method of detection. Eur J Cancer 34: 191–197.
Ewen, M. E., Sluss, H. K., Sherr, C. J., Matsushime, H., Kato, J. & Livingston, D. M. (1993) Functional interactions of the retinoblastoma protein with mammalian D-type cyclins. Cell 73: 487–497.
Farrell, W. E., Simpson, D. J., Bicknell, J. E., Talbot, A. J., Bates, A. S. & Clayton, R. N. (1997) Chromosome 9p deletions in invasive and noninvasive nonfunctional pituitary adenomas: the deleted region involves markers outside of the MTS1 and MTS2 genes. Cancer Res 57: 2703–2709.
Fountain, J. W., Karayiorgou, M., Ernstoff, M. S., Kirkwood, J. M., Vlock, D. R., Titus Ernstoff, L., Bouchard, B., Vijayasaradhi, S., Houghton, A. N., Lahti, J., Kidd, V. J., Housman, D. E. & Dracopoli, N. C. (1992) Homozygous deletions within human chromosome band 9p21 in melanoma. Proc Natl Acad Sci USA 89: 10557–10561.
Gonzalez-Zulueta, M., Bender, C. M., Yang, A. S., Nguyen, T., Beart, R. W., Van Tornout, J. M. & Jones, P. A. (1995) Methylation of the 5′ CpG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing. Cancer Res 55: 4531–4535.
Goodrich, D. W. & Lee, W. H. (1993) Molecular characterization of the retinoblastoma susceptibility gene. Biochim Biophys Acta 1155: 43–61.
Hagemeier, C., Caswell, R., Hayhurst, G., Sinclair, J. & Kouzarides, T. (1994) Functional interaction between the HCMV IE2 transactivator and the retinoblastoma protein. EMBO J 13: 2897–2903.
Hannon, G. J. & Beach, D. (1994) p15INK4B is a potential effector of TGF-beta-induced cell cycle arrest [see comments]. Nature 371: 257–261.
Hara, E., Smith, R., Parry, D., Tahara, H., Stone, S. & Peters, G. (1996) Regulation of p16 (CDKN2) expression and its implications for cell immortalization and senescence. Mol Cell Biol 16: 859–867.
Hunter, T. & Pines, J. (1994) Cyclins and cancer. II: Cyclin D and CDK inhibitors come of age [see comments]. Cell 79: 573–582.
Jiang, W., Kahn, S. M., Zhou, P., Zhang, Y. J., Cacace, A. M., Infante, A. S., Doi, S., Santella, R. M. & Weinstein, I. B. (1993) Overexpression of cyclin D1 in rat fibroblasts causes abnormalities in growth control, cell cycle progression and gene expression. Oncogene 8: 3447–3457.
Johnson, N. W. (1991). A global view of the epidemiology of oral cancer. In Oral Cancer, Johnson NW (ed), pp. 3–26 Cambridge University Press: Cambridge
Kamb, A., Gruis, N. A., Weaver Feldhaus, J., Liu, Q., Harshman, K., Tavtigian, S. V., Stockert, E., Day, RSIII, Johnson, B. E. & Skolnick, M. H. (1994) A cell cycle regulator potentially involved in genesis of many tumor types [see comments]. Science 264: 436–440.
Kamijo, T., Zindy, F., Roussel, M. F., Quelle, D. E., Downing, J. R., Ashmun, R. A., Grosveld, G. & Sherr, C. J. (1997) Tumour suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Cell 91: 649–659.
Koh, J., Enders, G. H., Dynlacht, B. D. & Harlow, E. (1995) Tumour-derived p16 alleles encoding proteins defective in cell-cycle inhibition. Nature 375: 506–510.
Kyomoto, R., Kumazawa, H., Toda, Y., Sakaida, N., Okamura, A., Iwanaga, M., Shintaku, M., Yamashita, T., Hiai, H. & Fukumoto, M. (1997) Cyclin-D1-gene amplification is a more potent prognostic factor than its protein over-expression in human head-and-neck squamous-cell carcinoma. Int J Cancer 74: 576–581.
Kyritsis, A. P., Zhang, B., Zhang, W., Xiao, M., Takeshima, H., Bondy, M. L., Cunningham, J. E., Levin, V. A. & Bruner, J. (1996) Mutations of the p16 gene in gliomas. Oncogene 12: 63–67.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
Li, Y., Graham, C., Lacy, S., Duncan, A. M. & Whyte, P. (1993) The adenovirus E1A-associated 130-kD protein is encoded by a member of the retinoblastoma gene family and physically interacts with cyclins A and E. Genes Dev 7: 2366–2377.
Liu, Q., Neuhausen, S., McClure, M., Frye, C., Weaver Feldhaus, J., Gruis, N. A., Eddington, K., Allalunis Turner, M. J., Skolnick, M. H. & Fujimura, F. K. (1995) CDKN2 (MTS1) tumor suppressor gene mutations in human tumor cell lines. Oncogene 11: 2455
Lukas, J., Parry, D., Agard, L., Mann, D. J., Bartkova, J., Strauss, M., Peters, G. & Bartek, J. (1995) Retinoblastoma-protein-dependent cell-cycle inhibition by the tumour suppressor p16. Nature 375: 503–506.
Martin, K., Trouche, D., Hagemeier, C., Sorensen, T. S., La Thangue, N. B. & Kouzarides, T. (1995) Stimulation of E2F/DP1 transcriptional activity by MDM2 oncoprotein. Nature 375: 691–694.
Mayol, X., Grana, X., Baldi, A., Sang, N., Hu, Q. & Giordano, A. (1993) Cloning of a new member of the retinoblastoma gene family (pRb2) which binds to the E1A transforming domain. Oncogene 8: 2561–2566.
Medema, R. H., Herrera, R. E., Lam, F. & Weinberg, R. A. (1995) Growth suppression by p16ink4 requires functional retinoblastoma protein. Proc Natl Acad Sci USA 92: 6289–6293.
Mori, T., Miura, K., Aoki, T., Nishihira, T., Mori, S. & Nakamura, Y. (1994) Frequent somatic mutation of the MTS1/CDK41 (multiple tumor suppressor / cyclin-dependent kinase 4 inhibitor) gene in esophageal squamous cell carcinoma. Cancer Res 54: 3396–3397.
Ohtsubo, M. & Roberts, J. M. (1993) Cyclin-dependent regulation of G1 in mammalian fibroblasts. Science 259: 1908–1912.
Olshan, A. F., Weissler, M. C., Pei, H., Conway, K., Anderson, S., Fried, D. B. & Yarbrough, W. G. (1997) Alterations of the p16 gene in head and neck cancer: frequency and association with p53, PRAD-1 and HPV. Oncogene 14: 811–818.
Otterson, G. A., Khleif, S. N., Chen, W., Coxon, A. B. & Kaye, F. J. (1995) CDKN2 gene silencing in lung cancer by DNA hypermethylation and kinetics of p16INK4 protein induction by 5-aza 2´ deoxycytidine. Oncogene 11: 1211–1216.
Papadimitrakopoulou, V., Izzo, J., Lippman, S. M., Lee, J. S., Fan, Y. H., Clayman, G., Ro, J. Y., Hittelman, W. N., Lotan, R., Hong, W. K. & Mao, L. (1997) Frequent inactivation of p16INK4a in oral premalignant lesions. Oncogene 14: 1799–1803.
Parry, D., Bates, S., Mann, D. J. & Peters, G. (1995) Lack of cyclin D-Cdk complexes in Rb-negative cells correlates with high levels of p16INK4/MTS1 tumour suppressor gene product. EMBO J 14: 503–511.
Patel, V., Jakus, J., Harris, C. M., Ensley, J. F., Robbins, K. C. & Yeudall, W. A. (1997) Altered expression and activity of G1/S cyclins and cyclin-dependent kinases characterize squamous cell carcinomas of the head and neck. Int J Cancer 73: 551–555.
Pomerantz, J., Schreiber Agus, N., Liegeois, N. J., Silverman, A., Alland, L., Chin, L., Potes, J., Chen, K., Orlow, I., Lee, H. W., Cordon Cardo, C. & Depinho, R. A. (1998) The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2’s inhibition of p53. Cell 92: 713–723.
Quelle, D. E., Zindy, F., Ashmun, R. A. & Sherr, C. J. (1995) Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest. Cell 83: 993–1000.
Quelle DE Cheng, M., Ashmun, R. A. & Sherr, C. J. (1997) Cancer-associated mutations at the INK4a locus cancel cell cycle arrest by p16INK4a but not by the alternative reading frame protein p19ARF. Proc Natl Acad Sci USA 94: 669–673.
Reed, A. L., Califano, J., Cairns, P., Westra, W. H., Jones, R. M., Koch, W., Ahrendt, S., Eby, Y., Sewell, D., Nawroz, H., Bartek, J. & Sidransky, D. (1996) High frequency of p16 (CDKN2/MTS-1/INK4A) inactivation in head and neck squamous cell carcinoma. Cancer Res 56: 3630–3633.
Ruas, M. & Peters, G. (1998) The p16/INK4a/CDKN2A tumor suppressor and its relatives. Biochim Biophys Acta 1378: 115–177.
Serrano, M., Hannon, G. J. & Beach, D. (1993) A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4 [see comments]. Nature 366: 704–707.
Serrano, M., Lee, H., Chin, L., Cordon-Cardo, C., Beach, D. & Depinho, R. A. (1996) Role of the INK4a locus in tumour suppression and cell mortality. Cell 85: 27–37.
Shapiro, G. I., Park, J. E., Edwards, C. D., Mao, L., Merlo, A., Sidransky, D., Ewen, M. E. & Rollins, B. J. (1995) Multiple mechanisms of p16INK4A inactivation in non-small cell lung cancer cell lines. Cancer Res 55: 6200–6209.
Sherr, C. J. (1994) G1 phase progression: cycling on cue [see comments]. Cell 79: 551–555.
Smith, C. J. & Pindborg, J. J. (1969). Histological Grading of Oral Epithelial Atypia by the Use of Photographic Standards. C Hamburgers Bogtrykkeri: Copenhagen
Steingrimsdottir, H., Penhallow, J., Farzeneh, F., Johnson, N. & Tavassoli, M. (1997) Detection of p53 mutations in oral cancer samples using a sensitive PCR-based method. DNA Damage Mutagenesis 25: 315–318.
Stone, S., Jiang, P., Dayananth, P., Tavtigian, S. V., Katcher, H., Parry, D., Peters, G. & Kamb, A. (1995) Complex structure and regulation of the P16 (MTS1) locus. Cancer Res 55: 2988–2994.
Suzuki, T., Kitao, S., Matsushime, H. & Yoshida, M. (1996) HTLV-1 Tax protein interacts with cyclin-dependent kinase inhibitor p16INK4A and counteracts its inhibitory activity towards CDK4. EMBO J 15: 1607–1614.
Tam, S. W., Shay, J. W. & Pagano, M. (1994) Differential expression and cell cycle regulation of the cyclin-dependent kinase 4 inhibitor p16Ink4. Cancer Res 54: 5816–5820.
Timmermann, S., Hinds, P. W. & Munger, K. (1997) Elevated activity of cyclin-dependent kinase 6 in human squamous cell carcinoma lines. Cell Growth Differ 8: 361–370.
Towbin, H., Staehelin, T. & Gordon, J. (1992) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. 1979 [classical article]. Biotechnology 24: 145–149.
Van Der Riet, P., Nawroz, H., Hruban, R. H., Corio, R., Tokino, K., Koch, W. & Sidransky, D. (1994) Frequent loss of chromosome 9p21–22 early in head and neck cancer progression. Cancer Res 54: 1156–1158.
Wang, H. G., Draetta, G. & Moran, E. (1991) E1A induces phosphorylation of the retinoblastoma protein independently of direct physical association between the E1A and retinoblastoma products. Mol Cell Biol 11: 4253–4265.
Weinberg, R. A. (1995) The retinoblastoma protein and cell cycle control. Cell 81: 323–330.
Welcker, M., Lukas, J., Strauss, M. & Bartek, J. (1996) Enhanced protein stability: a novel mechanism of D-type cyclin over-abundance identified in human sarcoma cells. Oncogene 13: 419–425.
Wong, D. T., Todd, R., Tsuji, T. & Donoff, R. B. (1996) Molecular biology of human oral cancer. Crit Rev Oral Biol Med 7: 319–328.
Xiao, Z. X., Chen, J., Levine, A. J., Modjtahedi, N., Xing, J., Sellers, W. R. & Livingston, D. M. (1995) Interaction between the retinoblastoma protein and the oncoprotein MDM2. Nature 375: 694–697.
Xiong, Y., Hannon, G. J., Zhang, H., Casso, D., Kobayashi, R. & Beach, D. (1993) p21 is a universal inhibitor of cyclin kinases [see comments]. Nature 366: 701–704.
Yeudall, W. A., Paterson, I. C., Patel, V. & Prime, S. S. (1995) Presence of human papillomavirus sequences in tumour-derived human oral keratinocytes expressing mutant p53. Eur J Cancer B Oral Oncol 31B: 136–143.
Yoo, G. H., Xu, H. J., Brennan, J. A., Westra, W., Hruban, R. H., Koch, W., Benedict, W. F. & Sidransky, D. (1994) Infrequent inactivation of the retinoblastoma gene despite frequent loss of chromosome 13q in head and neck squamous cell carcinoma. Cancer Res 54: 4603–4606.
Yoshida, S., Todoroki, T., Ichikawa, Y., Hanai, S., Suzuki, H., Hori, M., Fukao, K., Miwa, M. & Uchida, K. (1995) Mutations of p16Ink4/CDKN2 and p15Ink4B/MTS2 genes in biliary tract cancers. Cancer Res 55: 2756–2760.
Zhang, S. Y., Klein Szanto, A. J., Sauter, E. R., Shafarenko, M., Mitsunaga, S., Nobori, T., Carson, D. A., Ridge, J. A. & Goodrow, T. L. (1994) Higher frequency of alterations in the p16/CDKN2 gene in squamous cell carcinoma cell lines than in primary tumors of the head and neck. Cancer Res 54: 5050–5053.
Zhang, Y., Xiong, Y. & Yarbrough, W. G. (1998) ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways. Cell 92: 725–734.
Zhou, P., Jiang, W., Weghorst, C. M. & Weinstein, I. B. (1996) Overexpression of cyclin D1 enhances gene amplification. Cancer Res 56: 36–39.
Zhou, X., Tarmin, L., Yin, J., Jiang, H. Y., Suzuki, H., Rhyu, M. G., Abraham, J. M. & Meltzer, S. J. (1994) The MTS1 gene is frequently mutated in primary human esophageal tumors. Oncogene 9: 3737–3741.
Author information
Authors and Affiliations
Rights and permissions
From twelve months after its original publication, this work is licensed under the Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/
About this article
Cite this article
Sartor, M., Steingrimsdottir, H., Elamin, F. et al. Role of p16/MTS1, cyclin D1 and RB in primary oral cancer and oral cancer cell lines. Br J Cancer 80, 79–86 (1999). https://doi.org/10.1038/sj.bjc.6690505
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/sj.bjc.6690505
Keywords
This article is cited by
-
RB1 and TP53 pathways in radiation-induced sarcomas
Oncogene (2007)
-
p400 function is required for the adenovirus E1A-mediated suppression of EGFR and tumour cell killing
Oncogene (2007)
-
Immunohistochemical detection of p16INK4a in dysplastic lesions of the oral cavity
Modern Pathology (2006)
-
RB family members as predictive and prognostic factors in human cancer
Oncogene (2006)
-
Estudio de la expresión de ciclina D1, p16, MIB-1 y p53 en lesiones precancerosas orales
Clinical and Translational Oncology (2004)