Inactivation of Cyclin D2 Gene in Prostate Cancers by Aberrant Promoter Methylation

4730 Vol. 9, 4730–4734, October 15, 2003 Clinical Cancer Research

Asha Padar, Ubaradka G. Sathyanarayana, from our laboratory (R. Maruyama et al., Clin. Cancer Res., Makoto Suzuki, Riichiroh Maruyama, 8: 514–519, 2002)] studied in the same set of samples. The concordances between methylation of Cyclin D2 and the Jer-Tsong Hsieh, Eugene P. Frenkel, ␤ 1 methylation of RAR , GSTP1, CDH13, RASSF1A, and APC John D. Minna, and Adi F. Gazdar were statistically significant, whereas methylation of P16, Hamon Center for Therapeutic Oncology Research [A. P., U. G. S., DAPK, FHIT, and CDH1 were not significant. The differ- M. S., R. M., J. D. M., A. F. G.], The Simmons Cancer Center ences in methylation index between malignant and nonma- [E. P. F.], and Departments of Pathology [U. G. S., A. F. G.], Urology [J-T. H.], Internal Medicine [J. D. M.], and Pharmacology [J. D. M.], lignant tissues for all 10 genes were statistically significant The University of Texas Southwestern Medical Center, Dallas, (P < 0.0001). Among clinicopathological correlations, the Texas 75390 high Gleason score group had significantly greater methyl- Although the .(0.004 ؍ ation frequency of Cyclin D2 (42%; P high preoperative serum prostate-specific antigen (PSA) ABSTRACT group did not have significantly greater methylation fre- Purpose: Loss or abnormal expression of Cyclin D2,a quency, methylation of Cyclin D2 had higher mean PSA crucial cell cycle-regulatory gene, has been described in value. Also, the prostate cancers in the high Gleason score human cancers; however, data for prostate tumors are lack- group had high mean values of PSA. ing. We investigated the epigenetic silencing of Cyclin D2 Conclusions: Our results indicate that methylation of gene in prostate cancers and correlated the data with clini- Cyclin D2 in prostate cancers correlates with clinicopatho- copathological features. logical features of poor prognosis. These findings are of Experimental Design: Cyclin D2 promoter methylation biological and potential clinical importance. was analyzed in 101 prostate cancer samples by methylation-specific PCR. In addition, we analyzed 32 nonmalignant INTRODUCTION prostate tissue samples, which included 24 samples of benign In mammalian cells, progression through the cell cycle is disease, benign prostatic hypertrophy, or prostatitis and 7 2 normal tissues adjacent to cancer. The methylation status of governed by a family of Cdks, whose activity is regulated by Cyclin D2 was correlated with the methylation of nine other phosphorylation, activated by binding of cyclins, and inhibited tumor suppressor genes published previously from our lab- by Cdk inhibitors (1). The D-type cyclins (cyclins D1, D2, and D3) are involved in the regulation of transition from G to S oratory on the same set of samples (R. Maruyama et al., 1 Clin. Cancer Res., 8: 514–519, 2002). The methylation index during the cell cycle (2, 3). The INK4 family of cell cycle was determined as a reflection of the methylated fraction of inhibitors (P16INK4a, P15INK4b, P18INK4c, and P19INK4d) the genes examined. negatively regulates the activity of Cdk4/6 by preventing cyclin Results: The frequency of methylation of Cyclin D2 D binding. Cdks are protein kinases that require association with promoter was significantly higher in prostate cancers (32%) D-type cyclins and phosphorylation for their activity. One of -and their substrates is believed to be the retinoblastoma gene prod ,(0.004 ؍ than in nonmalignant prostate tissues (6%; P uct, pRb, and a critical negative regulator of the G -S transition. it was not age related. Aberrant methylation was present at 1 insignificant levels in peripheral blood lymphocytes (8%). Phosphorylation of pRb by Cdks inactivates it and allows cells We also compared methylation of cyclin D2 with methyla- to enter S phase (2). tion of nine tumor suppressor genes [published previously Transcription and protein synthesis of D-type cyclin mRNAs are highest in middle-late G1 and lowest during S phase (4). Because they play a critical role in cell cycle regulation, their abnormal or untimely expression or loss of expression could disrupt the cell cycle and therefore render them oncogenes Received 12/16/02; revised 4/21/03; accepted 4/28/03. or TSGs. Cyclin D1 has been proposed as a proto-oncogene The costs of publication of this article were defrayed in part by the because its derangement contributes to the uncontrolled cell payment of page charges. This article must therefore be hereby marked growth characteristic of tumors (5). Overexpression and rear- advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. rangement of the Cyclin D1 gene have been reported to be This work was supported by Grant U01CA84971 from the Early De- associated with tumor progression and/or poor prognosis in tection Research Network National Cancer Institute (Bethesda, MD) and awards from the Nasher Family Cancer Program and the Amon Carter Foundation. 1 To whom requests for reprints should be addressed, at Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern 2 The abbreviations used are: Cdk, cyclin-dependent kinase; MSP, meth- Medical Center at Dallas, 6000 Harry Hines Boulevard, Dallas, TX ylation-specific PCR; MI, methylation index; TSG, tumor suppressor 75390-8593. Phone: (214) 648-4921; Fax: (214) 648-4940; E-mail: adi. gene; GS, Gleason score; PSA, prostate-specific antigen; BPH, benign [email protected]. prostatic hypertrophy.

many different tumor types, including carcinomas of the breast Table 1 Frequency of Cyclin D2 promoter methylation in prostate (6), esophagus (7), pancreas (8), and head and neck (9) and cancers and control tissues mantle cell lymphomas (10). Aberrant expression of Cyclin D2 Median age has been noted in human ovarian granulosa cell tumors and (range) Sample size Methylation testicular germ cell tumor cell lines (11). It has been reported Sample designation (yrs) (n) (%) that Cyclin D2 is a direct target of Myc and that accumulation of Prostate cancersa 63 (43–81) 101 32 (32) Cyclin D2 contributes to sequestration of the cell cycle inhibitor Nonmalignant 32 2 (6) prostatic tissuesa p27 and to cell cycle entry (12, 13). Overexpression of Cyclin BPHb 67 (49–86) 24 1 (4) D2 has been reported in gastric cancer and was shown to Prostatitisb NAc 1 0 (0) correlate with disease progression and poor prognosis (4, 14). Adjacent to cancerb 65 (54–76) 7 1 (14) b Previous studies have reported that Cyclin D2 mRNA and Lymphocytes 37 (32–65) 13 1 (8) protein were absent in almost all breast cancer cell lines exam- a The median age differences by Mann-Whitney nonparametric U ϭ ined, whereas cultured normal breast epithelial cells had abun- statistical test were insignificant (P 0.061). b From patients without cancer. dant expression (15–18). In tumorigenesis, multiple mecha- c NA, not applicable. nisms of inactivating TSGs such as loss of heterozygosity, point mutations, homozygous deletions, and aberrant promoter methylation have been reported (19). Recently, it has been reported that promoter methylation is a mechanism for the loss of Cyclin 37°C. Aliquots of 10 mM hydroquinone (30 ␮l; Sigma Chemical D2 expression in breast cancers (20). Inactivation of many TSGs Co., St. Louis, MO) and 3 M sodium bisulfite (pH 5.0; 520 ␮l; by epigenetic phenomenon has been reported in prostate cancers Sigma Chemical Co.) were added, and the solution was incu- (21). To investigate whether Cyclin D2 is silenced by epigenetic bated at 52°C for 16 h. Treated DNA was purified by use of a phenomenon in prostate cancers, we studied the methylation Wizard DNA Purification System (Promega Corp., Madison, status of Cyclin D2 promoter in prostate cancers and nonmalig- WI). Modified DNA was stored at –80°C until use. Treatment nant tissue samples. We report here that inactivation of Cyclin of genomic DNA with sodium bisulfite converts unmethylated D2 in prostate cancers is associated with the hypermethylation cytosines (but not methylated cytosines) to uracil, which is then of the Cyclin D2 promoter. converted to thymidine during subsequent PCR (25). Thus, after bisulfite treatment, alleles that were originally methylated have DNA sequences different from those of their corresponding MATERIALS AND METHODS unmethylated alleles, and these differences can be used to de- Clinical Samples. The 101 prostate samples used in this sign PCR primers that are specific for methylated or unmethyl- study were collected from patients with prostate cancer. Among ated alleles. PCR was performed using primer sequences essen- these samples, seven of them had adjacent nonmalignant tissue tially as described previously (20). The primers used for from the same patients. We also obtained nonmalignant tissues amplification of the methylated form of the Cyclin D2 promoter from 24 patients with BPH or prostatitis. The patients underwent were 5Ј-TACGTGTTAGGGTCGATCG-3Ј (sense; Ϫ1427 to radical prostatectomy or transurethral resection at University of –1409) and 5Ј-CGAAATATCTACGCTAAACG-3Ј (antisense; Texas Southwestern Medical Center-affiliated hospitals in Dal- Ϫ1152 to –1171), which yielded a 276-bp PCR product (20). las, Texas between 1994 and 2000, after Institutional Review Reactions were hot started at 94°C for 12 min, and temperature Board approval and signed consent were obtained. The tissues conditions for PCR were as follows: 5 cycles of 94°C for 20 s, were maintained frozen in the urology tissue bank at Ϫ70°C 55°C for 55 s, and 72°C for 20 s; and 30 cycles of 90°C for 20 s, until use. The histological grading was performed according to 55°C for 40 s, and 72°C for 40 s; followed by 1 cycle of 72°C GS (22), and the stage of the disease was assessed by using the for 4 min. P16 unmethylated primer was used as control in MSP clinical tumor-node-metastasis (TNM) classification of the to check the integrity of bisulfite-treated DNA in tissue samples American Joint Committee on Cancer (23). The clinicopatho- (25). DNA from peripheral blood lymphocytes (n ϭ 13) was logical features of cancer patients are described elsewhere (21). used as negative controls for MSP assays. DNA from lympho- Briefly, the median age was 63 years (range, 43–81 years), and cytes of healthy volunteers treated with SssI methyltransferase there were mainly white (n ϭ 69), black (n ϭ 15), and Hispanic (New England Biolabs, Beverly, MA) and then subjected to (n ϭ 5) races. Median ages (and the range in years) of patients bisulfite treatment was used as a positive control for methylated from whom malignant and nonmalignant prostate tissue samples alleles. Water blanks and PCR mixtures (without template) were were collected are presented in Table 1. The TNM staging used as negative controls in each assay. PCR products were consisted mainly of three groups (I, n ϭ 12; II, n ϭ 14; and III, visualized on 2% agarose gels stained with ethidium bromide. n ϭ 25). Peripheral blood lymphocytes from 13 healthy volun- Results were confirmed by repeating bisulfite treatment and teers were also obtained. MSP assays for all samples. DNA Extraction. Genomic DNA was extracted from Data Analysis. The frequencies of methylation between malignant and nonmalignant tissues by digestion with protein- two groups were compared using ␹2 test and Fisher’s exact test ase K (Life Technologies, Inc.) for 1 day at 50°C, followed by with continuity correction. To compare the overall extent of two extractions with phenol:chloroform (1:1; Ref. 24). methylation for the panel of genes examined, we calculated the MSP. The MSP uses an initial bisulfite reaction to mod- MI. The MI is defined as the total number of genes methylated ify the DNA as described previously (25). Briefly, 1 ␮gofDNA divided by the total number of genes analyzed. The MI of was denatured by incubation with 0.2 M NaOH for 15 min at different groups was compared using the Mann-Whitney non-

Fig. 1 Representative examples of MSP of Cyclin D2 (276 bp) gene in prostatic tumors and nonmalignant tissues. N1/T1 and N2/T2, two pairs of nonmalignant prostatic tissues and corresponding tumors; T3–T10, prostate tumor samples; L, normal lymphocyte DNA as an example of negative control; NC, negative control (water blank or PCR mixture without template); P, positive control (normal lymphocyte DNA treated with SssI methyl transferase). The unmethylated form of P16 (151 bp) was run as a control for DNA integrity.

parametric U test. For all of the tests, P Ͻ 0.05 was considered Table 2 Concordances between methylation of Cyclin D2 and the statistically significant. All of the statistical tests were two methylation of other known TSGs sided. All data were analyzed using Stat View program. Gene name Concordance (%) P GSTP1 82 Ͻ0.0001a RESULTS RAR␤ 66 0.0001a RASSF1A 64 0.0007a Promoter Methylation of Cyclin D2 in Prostatic Tissues. CDH13 69 0.004a To examine the methylation pattern of Cyclin D2 promoter in APC 67 0.03a prostatic tissues, we used the MSP assay as described in “Ma- P16INK4a 69 0.2 terials and Methods.” Results of aberrant methylation in malig- FHIT 65 0.5 nant prostatic tissues and control tissues are detailed in Table 1, CDH1 55 0.5 and representative examples are illustrated in Fig. 1. The un- a Statistically significant. methylated form of P16, run as a control for DNA integrity, was present in all samples. The frequency of methylation of Cyclin D2 promoter was significantly higher in prostate cancers (32 of 101, 32%) than in nonmalignant prostate tissues (2 of 32, 6%; respectively. Tumor staging information was available for 60 of P ϭ 0.004). The percentage of methylation in BPH (1 of 24, the patients. Because of the relatively small numbers in each of 4%), prostatitis (0 of 1, 0%), and samples from tissues adjacent the four grades, we pooled patients into low stage (stages I and ϭ ϭ to cancer (1 of 7, 14%) were found to be at very low frequency. II; n 26) or high stage (stages III and IV; n 34) groups. Aberrant methylation was present at low levels in peripheral The high GS group had significantly greater methylation ϭ blood lymphocytes (1 of 13, 8%). We also compared methyla- frequency of Cyclin D2 (42%; P 0.004), whereas methylation tion of Cyclin D2 with methylation of 9 TSGs (published of Cyclin D2 was not significant in the high stage group. previously from our laboratory; Ref. 21) studied in the same set Although the high PSA group did not have significantly greater of samples, and the concordances are presented in the Table 2. methylation frequency, methylation of Cyclin D2 had higher Ϯ Ϯ The concordances between methylation of Cyclin D2 and the mean PSA (mean SE: 14 3 for methylation-positive sam- Ϯ methylation of RAR␤ (67 of 101, 66%; P ϭ 0.0001), GSTP1 (83 ples and 12 1.7 for methylation-negative samples) value. of 101, 82%; P Ͻ 0.0001), CDH13 (70 of 101, 69%; P ϭ Also, the prostate cancers in high GS group had high mean Ϯ Ϯ Ϯ 0.004), RASSF1A (65 of 101, 64%; 0.0007), and APC (68 of values of PSA (mean SE: 15 2 for high GS group and 8 101, 67%; P ϭ 0.032) were significant, whereas the methylation 1 for low GS group). of P16, DAPK, FHIT, and CDH1 were not significant. The Patients from whom the nonmalignant prostate tissues were differences in MI between malignant and nonmalignant tissues collected had slightly higher median ages and very low level of for all 10 genes were statistically significant (P Ͻ 0.0001). frequency of methylation compared with those from whom Correlation between Methylation of Cyclin D2 and malignant tissues were collected (Table 1). Mann-Whitney non- Clinicopathological Features. We compared the methylation parametric U statistical test for the median age differences frequency of Cyclin D2 with the GS, the preoperative serum between malignant and nonmalignant prostate samples revealed ϭ PSA, and the tumor stage, three known clinicopathological that the differences are not statistically significant (P 0.061). features that were available for prostate cancer patients. It was Survival data were available for 44 prostate cancer patients with reported that prostatic tumors with GS values of 5–6 have a a median follow-up period of 27 months. Although methylation significantly better clinical course than those with values of Ն7 status of Cyclin D2 did not correlate with survival, no deaths (26). Hence we divided our tumors into those with values of Յ6 were noted until 48 months after surgery. Thus, a lengthy (low GS group; n ϭ 36) and those with values of Ն7 (high GS follow-up period will be required to determine whether methyl- group; n ϭ 65). The median value of preoperative serum PSA in ation is a prognostic factor for survival. our cancer patients was 7.5 ng/ml. To obtain an approximately equal number of patients in each category, we divided our DISCUSSION patients into a “low PSA” group (Յ8 ng/ml; n ϭ 47), and a Lack of growth control is one of the hallmarks of trans- “high PSA” group (Ͼ8 ng/ml; n ϭ 45). The median values of formed cells (27). In addition to the role of cyclins in cell cycle PSA in the low and high PSA groups were 5.6 and 14.2 ng/ml, regulation, the D-type cyclins have been implicated in differen-

tiation and neoplastic transformation (2, 20, 28). It has been there was a high degree of concordance with the methylation of reported that Cyclin D2 overexpression correlated with gastric APC, RAR␤, CDH13, RASSF1A, and GSTP1 genes. Association cancer progression and prognosis, suggesting that Cyclin D2 is of methylation of multiple TSGs in the same set of samples may a proto-oncogene (4, 14). However, a recent report on associa- help in using these markers in prognosis. tion of loss of Cyclin D2 expression with promoter methylation suggests a different role as a TSG (20). These differences suggest that Cyclin D2 may function as an oncogene or TSG in REFERENCES a tumor type-dependent manner. Thus, we studied the methyl- 1. Tsihlias, J., Kapusta, L., and Slingerland, J. The prognostic signifi- ation pattern of Cyclin D2 in malignant and nonmalignant cance of altered cyclin-dependent kinase inhibitors in human cancer. Annu. Rev. Med., 50: 401–423, 1999. prostate tissue samples and correlated the data to clinicopatho- 2. Zhang, P. The cell cycle and development: redundant roles of cell logical features. cycle regulators. Curr. Opin. Cell Biol., 11: 655–662, 1999. Multiple mechanisms of inactivating TSGs such as loss of 3. Sherr, C. J. D-type cyclins. Trends Biochem. Sci., 20: 187–190, heterozygosity, point mutations, homozygous deletions, and ab- 1995. errant promoter methylation have been reported in various types 4. Takano, Y., Kato, Y., Masuda, M., Ohshima, Y., and Okayasu, I. of cancers (19). Aberrant methylation of CpG islands was iden- Cyclin D2, but not cyclin D1, over expression closely correlates with tified as an epigenetic mechanism for the transcriptional silenc- gastric cancer progression and prognosis. J. Pathol., 189: 194–200, 1999. ing of TSGs in many cancer types, and the number of methyl- 5. Keyomarsi, K., and Pardee, A. B. Redundant cyclin over expression ated genes in individual cancers is estimated to be very high and gene amplification in breast cancer cells. Proc. Natl. Acad. Sci. (29–31). Inactivation of multiple TSGs by promoter hyper- USA, 90: 1112–1116, 1993. methylation has been reported in prostate cancers (21). Tran- 6. Gillett, C., Fantl, V., Smith, R., Fisher, C., Bartek, J., Dickson, C., scriptional silencing of Cyclin D2 gene by promoter methylation Barnes, D., and Peters, G. Amplification and over expression of cyclin has been reported in breast cancers, whereas normal breast D1 in breast cancer detected by immunohistochemical staining. Cancer epithelium expressed Cyclin D2 (20). Previous studies have Res., 54: 1812–1817, 1994. shown low frequency of epigenetic inactivation of p16 (21, 32), 7. Gramlich, T. L., Fritsch, C. R., Maurer, D., Eberle, M., and Gansler, T. S. Differential polymerase chain reaction assay of cyclin D1 gene which negatively controls progression of the cell cycle through amplification in esophageal carcinoma. Diagn. Mol. Pathol., 3: 255– the G1 phase of the cell cycle, in prostate cancers. It has been 259, 1994. suggested that abrogation of the Rb pathway in prostate cancer 8. Gansauge, S., Gansauge, F., Ramadani, M., Stobbe, H., Rau, B., is achieved by down-regulation of RB protein (or mutation) Harada, N., and Beger, H. G. Overexpression of cyclin D1 in human rather than by p16 changes (33). pancreatic carcinoma is associated with poor prognosis. Cancer Res., 57: 1634–1637, 1997. The methylation of Cyclin D2 was significantly higher in 9. Michalides, R. J., van Veelen, N. M., Kristel, P. M., Hart, A. A., malignant prostate samples than in nonmalignant samples Loftus, B. M., Hilgers, F. J., and Balm, A. J. Overexpression of cyclin (which include BPH and tissues adjacent to cancer) and lym- D1 indicates a poor prognosis in squamous cell carcinoma of the head phocytes, indicating that methylation of Cyclin D2 may play a and neck. Arch. Otolaryngol. Head Neck Surg., 123: 497–502, 1997. role in tumorigenesis. Tumor suppressor role for Cyclin D2 in 10. Nakamura, S., Yatabe, Y., and Seto, M. Cyclin D1 overexpression breast cancers has been reported previously (20). We also cor- in malignant lymphomas. Pathol. Int., 47: 421–429, 1997. related the methylation of Cyclin D2 with clinicopathological 11. Sicinski, P., Donaher, J. L., Geng, Y., Parker, S. B., Gardner, H., features such as GS and preoperative serum PSA and observed Park, M. Y., Robker, R. L., Richards, J. S., McGinnis, L. K., Biggers, J. D., Eppig, J. J., Bronson, R. T., Elledge, S. J., and Weinberg, R. A. that methylation of Cyclin D2 was significantly associated with Cyclin D2 is an FSH-responsive gene involved in gonadal cell prolif- high GS group and had higher mean PSA value. This might help eration and oncogenesis. Nature (Lond.), 384: 470–474, 1996. in prognosis. Cyclin D2 methylation might play a role in in- 12. Bouchard, C., Thieke, K., Maier, A., Saffrich, R., Hanley-Hyde, J., creased proliferation and poor differentiation that is reflected by Ansorge, W., Reed, S., Sicinski, P., Bartek, J., and Eilers, M. Direct high GS. To address whether the observed methylation fre- induction of cyclin D2 by Myc contributes to cell cycle progression and sequestration of p27. EMBO J., 18: 5321–5333, 1999. quency for Cyclin D2 in malignant prostate samples is age 13. Perez-Roger, I., Kim, S. H., Griffiths, B., Sewing, A., and Land, H. dependent (i.e., a higher methylation frequency in older people), Cyclins D1 and D2 mediate myc-induced proliferation via sequestration we compared the median ages (and age range) of patients from of p27Kip1 and p21Cip1. EMBO J., 18: 5310–5320, 1999. whom malignant and nonmalignant prostate tissues were col- 14. Takano, Y., Kato, Y., van Diest, P. J., Masuda, M., Mitomi, H., and lected. We found that those patients from whom the nonmalig- Okayasu, I. Cyclin D2 overexpression and lack of p27 correlate posi- nant prostate tissues were collected had slightly higher median tively and cyclin E inversely with a poor prognosis in gastric cancer ages (and age range) than those from whom the malignant cases. Am. J. Pathol., 156: 585–594, 2000. tissues were collected, ruling out the possibility of age-depen- 15. Buckley, M. F., Sweeney, K. J., Hamilton, J. A., Sini, R. L., Manning, D. L., Nicholson, R. I., deFazio, A., Watts, C. K., Musgrove, dent methylation of Cyclin D2 in prostate cancer patients. The E. A., and Sutherland, R. L. Expression and amplification of cyclin differences in median ages between malignant and nonmalig- genes in human breast cancer. Oncogene, 8: 2127–2133, 1993. nant prostate samples were statistically insignificant. Also, lym- 16. Tam, S. W., Theodoras, A. M., Shay, J. W., Draetta, G. F., and phocytes from healthy volunteers had a very low level of meth- Pagano, M. Differential expression and regulation of cyclin D1 protein ylation frequency, further supporting the observed Cyclin D2 in normal and tumor human cells: association with Cdk4 is required for cyclin D1 function in G progression. Oncogene, 9: 2663–2674, 1994. methylation frequency as tumor specific and a potential tumor 1 17. Lukas, J., Bartkova, J., Welcker, M., Petersen, O. W., Peters, G., suppressor function of Cyclin D2. We also compared the meth- Strauss, M., and Bartek, J. Cyclin D2 is a moderately oscillating ylation of Cyclin D2 with methylation of other TSGs studied in nucleoprotein required for G1 phase progression in specific cell types. the same set of samples (21) in our laboratory and observed that Oncogene, 10: 2125–2134, 1995.

18. Sweeney, K. J., Sarcevic, B., Sutherland, R. L., and Musgrove, 7 prostate cancer be considered a unique grade category? Urology, 53: E. A. Cyclin D2 activates Cdk2 in preference to Cdk4 in human breast 372–377, 1999. epithelial cells. Oncogene, 14: 1329–1340, 1997. 27. Hanahan, D., and Weinberg, R. A. The hallmarks of cancer. Cell, 19. Shapiro, G. I., Park, J. E., Edwards, C. D., Mao, L., Merlo, A., 100: 57–70, 2000. Sidransky, D., Ewen, M. E., and Rollins, B. J. Multiple mechanisms of 28. Ortega, S., Malumbres, M., and Barbacid, M. Cyclin D-dependent p16INK4A inactivation in non-small cell lung cancer cell lines. Cancer kinases, INK4 inhibitors and cancer. Biochim. Biophys. Acta, 1602: Res., 55: 6200–6209, 1995. 73–87, 2002. 20. Evron, E., Umbricht, C. B., Korz, D., Raman, V., Loeb, D. M., Niranjan, B., Buluwela, L., Weitzman, S. A., Marks, J., and Sukumar, S. 29. Baylin, S. B., Herman, J. G., Graff, J. R., Vertino, P. M., and Issa, Loss of cyclin D2 expression in the majority of breast cancers is associated J. P. Alterations in DNA methylation: a fundamental aspect of neopla- with promoter hypermethylation. Cancer Res., 61: 2782–2787, 2001. sia. Adv. Cancer Res., 72: 141–196, 1998. 21. Maruyama, R., Toyooka, S., Toyooka, K. O., Virmani, A. K., 30. Costello, J. F., Fruhwald, M. C., Smiraglia, D. J., Rush, L. J., Zochbauer-Muller, S., Farinas, A. J., Minna, J. D., McConnell, J., Robertson, G. P., Gao, X., Wright, F. A., Feramisco, J. D., Peltomaki, Frenkel, E. P., and Gazdar, A. F. Aberrant promoter methylation profile P., Lang, J. C., Schuller, D. E., Yu, L., Bloomfield, C. D., Caligiuri, of prostate cancers and its relationship to clinicopathological features. M. A., Yates, A., Nishikawa, R., Su Huang, H., Petrelli, N. J., Zhang, X., Clin. Cancer Res., 8: 514–519, 2002. O’Dorisio, M. S., Held, W. A., Cavenee, W. K., and Plass, C. Aberrant 22. Gleason, D. F., and Mellinger, G. T. Prediction of prognosis for CpG-island methylation has non-random and tumour-type-specific pat- prostatic adenocarcinoma by combined histological grading and clinical terns. Nat. Genet., 24:132–138, 2000. staging. J. Urol., 111: 58–64, 1974. 31. Esteller, M. CpG island hypermethylation and tumor suppressor 23. Beahrs, O. H., Henson, D. E., and Hutter, R. V. P. American Joint genes: a booming present, a brighter future. Oncogene, 21: 5427–5440, Committee on Cancer: Manual for Staging Cancer, 4th ed. Philadelphia: 2002. Lippincott, 1992. 32. Jarrard, D. F., Bova, G. S., Ewing, C. M., Pin, S. S., Nguyen, S. H., 24. Herrmann, B. G., and Frischauf, A. M. Isolation of genomic DNA. Baylin, S. B., Cairns, P., Sidransky, D., Herman, J. G., and Isaacs, W. B. Methods Enzymol., 152: 180–183, 1987. Deletional, mutational, and methylation analyses of CDKN2 (p16/ 25. Herman, J. G., Graff, J. R., Myohanen, S., Nelkin, B. D., and MTS1) in primary and metastatic prostate cancer. Genes Chromosomes Baylin, S. B. Methylation-specific PCR: a novel PCR assay for meth- Cancer, 19: 90–96, 1997. ylation status of CpG islands. Proc. Natl. Acad. Sci. USA, 93: 9821– 33. Nguyen, T. T., Nguyen, C. T., Gonzales, F. A., Nichols, P. W., Yu, 9826, 1996. M. C., and Jones, P. A. Analysis of cyclin-dependent kinase inhibitor 26. Tefilli, M. V., Gheiler, E. L., Tiguert, R., Sakr, W., Grignon, D. J., expression and methylation patterns in human prostate cancers. Prostate, Banerjee, M., Pontes, J. E., and Wood, D. P., Jr. Should Gleason score 43: 233–242, 2000.

Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2003 American Association for Cancer Research. Inactivation of Cyclin D2 Gene in Prostate Cancers by Aberrant Promoter Methylation

Asha Padar, Ubaradka G. Sathyanarayana, Makoto Suzuki, et al.

Clin Cancer Res 2003;9:4730-4734.

Updated version Access the most recent version of this article at: http://clincancerres.aacrjournals.org/content/9/13/4730

Cited articles This article cites 32 articles, 9 of which you can access for free at: http://clincancerres.aacrjournals.org/content/9/13/4730.full#ref-list-1

Citing articles This article has been cited by 5 HighWire-hosted articles. Access the articles at: http://clincancerres.aacrjournals.org/content/9/13/4730.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/9/13/4730. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.