Vol. 10, 7011–7021, October 15, 2004 Clinical Cancer Research 7011 The In vitro and In vivo Effects of Re-Expressing Methylated von Hippel-Lindau Tumor Suppressor Gene in Clear Cell Renal Carcinoma with 5-Aza-2؅-deoxycytidine Wade G. Alleman,1,2 Ray L. Tabios,2 Well described phenotypic changes of VHL expression in- Gadisetti V. R. Chandramouli,3 cluding decreased invasiveness into Matrigel, and decreased Olga N. Aprelikova,3 Carlos Torres-Cabala,2 vascular endothelial growth factor and glucose transport- 4 5 er-1 expression were observed in the treated lines. VHL Arnulfo Mendoza, Craig Rodgers, methylated ccRCC xenografted tumors were significantly 2 2 Nikolai A. Sopko, W. Marston Linehan, and reduced in size in mice treated with 5-aza-dCyd. Mice bear- James R. Vasselli2 ing nonmethylated but VHL-mutated tumors showed no 1Howard Hughes Medical Institute, Chevy Chase, MD; 2Urology tumor shrinkage with 5-aza-dCyd treatment. Branch, 3Laboratory of Biosystems and Cancer, and 4Pediatric Conclusion: Hypo-methylating agents may be useful in Oncology Branch, Center for Cancer Research, National Cancer 5 the treatment of patients having ccRCC tumors consisting of Institute, Bethesda, Maryland; The Brady Urologic Institute, The cells with methylated VHL. Johns Hopkins Hospital, Baltimore, Maryland INTRODUCTION ABSTRACT An estimated 31,900 people are diagnosed annually with Purpose: Clear cell renal carcinoma (ccRCC) is cancer of the kidney in the United States, of which the majority strongly associated with loss of the von Hippel-Lindau (VHL) are clear cell type. In sporadic clear cell renal carcinoma tumor suppressor gene. The VHL gene is functionally lost (ccRCC), 50–85% of patients are found to have biallelic loss of through hypermethylation in up to 19% of sporadic ccRCC the von Hippel-Lindau (VHL) tumor suppressor gene (1–3), cases. We theorized that re-expressing VHL silenced by located on chromosome 3p25 (4, 5). It has been reported that in methylation in ccRCC cells, using a hypo-methylating agent, up to 19% of sporadic ccRCC, VHL function is lost because of may be an approach to treatment in patients with this type hyper-methylation of a CpG island in the promoter region of the of cancer. We test the ability of two hypo-methylating agents VHL gene (3, 6). DNA methylation is the result of DNA meth- to re-express VHL in cell culture and in mice bearing human yltransferase enzymatic activity transferring a methyl group ccRCC and evaluate the effects of re-expressed VHL in these from S-adenosyl-methionine to the C-5 position of cytosine (7). models. The majority of available hypo-methylating agents are Experimental Design: Real-time reverse transcription- structural variations of cytidine with a modified position 5 of the PCR was used to evaluate the ability of zebularine and pyrimidine ring. After phosphorylation, the compound is incor- 5-aza-2؅-deoxycytidine (5-aza-dCyd) to re-express VHL in porated into DNA where it forms an irreversible covalent bond four ccRCC cell lines with documented VHL gene silencing with DNA methyltransferase. Total enzyme levels decrease after through hypermethylation. We evaluated if the VHL re- DNA replication, leading to global hypo-methylation (8, 9). The expressed after hypo-methylating agent treatment could re- hypo-methylating agent with the greatest selectivity for DNA is create similar phenotypic changes in ccRCC cells observed 5-aza-2Ј-deoxycytidine (5-aza-dCyd), first synthesized in 1964 when the VHL gene is re-expressed via transfection in cell (10). It has been shown to induce expression of silenced genes culture and in a xenograft mouse model. Finally we evaluate (11) and suppress growth of tumor cells in vitro (12). Despite global gene expression changes occurring in our cells, using some efficacy as a treatment for leukemia, minimal success has microarray analysis. been seen in the treatment of solid tumors (13–15). Zebularine, Results: 5-Aza-dCyd was able to re-express VHL in our a cytidine deaminase inhibitor, has been described recently as an cell lines both in culture and in xenografted murine tumors. effective hypo-methylating agent (16). Functioning in a similar manner to 5-aza-dCyd, zebularine has the advantage of being stable in aqueous solution up to a pH of 12 (17, 18) and having oral bioavailibility (16). Received 3/15/04; revised 6/24/04; accepted 7/7/04. In this report we evaluate the ability of 5-aza-dCyd and The costs of publication of this article were defrayed in part by the zebularine to re-express VHL in several human hyper-methyl- payment of page charges. This article must therefore be hereby marked ated VHL ccRCC cell lines. Furthermore, we assess the ability advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. of the re-expressed VHL to cause biochemical and phenotypic Note: Supplementary data for this article can be found at Clinical alterations in the cells observed when the VHL gene is trans- Cancer Research Online (http://clincancerres.aacrjournals.org). fected back into the cells. We also document the ability of Requests for reprints: W. Marston Linehan, Chief, Urologic Oncology 5-aza-dCyd to re-express VHL in xenografted, VHL-methyl- Branch, CCR National Cancer Institute, Building 10, Room 2B47, Bethesda, Maryland 20892-1501. Phone: 301-496-6353; Fax: 301-402- ated, ccRCC cells that have formed small tumors in mice. 0922; E-mail: [email protected]. We then evaluate the effects of re-expressing VHL in ©2004 American Association for Cancer Research. established tumors consisting of VHL-negative cells. Finally, Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2004 American Association for Cancer Research. 7012 Re-Expressing Methylated VHL with 5-Aza-dCyd using cDNA microarrays, we evaluate the changes in global The technique used for bisulfite modification and subsequent gene expression in our cell lines after they are exposed to RT-PCR analysis has been described previously (23). Briefly, 5-aza-dCyd. we subjected 1 ␮g of sample DNA to sodium bisulfite modifi- cation, using the CpGenome DNA Modification Kit (Chemicon, Temecula, CA). We used universally methylated DNA to gen- MATERIALS AND METHODS erate a standard curve, based on which we calculated the amount Cells and Cell Culture. Six cell lines from sporadic of DNA in the samples. White blood cell DNA was used as a clear cell renal carcinomas were analyzed: UOK 108, UOK 121, negative control (unmethylated). Amplification of MYOD1 was UOK 127, UOK 143, UOK 171, and 786–0. The methylation used to determine total amount of amplifiable DNA in the status of UOK 108, UOK 121, UOK 127, and UOK 143 has samples. Our PCR reaction mix contained 1 ␮L of DNA in 25 been described previously (3). UOK 121 and UOK 143 have one ␮L of total volume, which also included 2.5 ␮Lof10ϫ PCR ␮ hyper-methylated copy and one silent copy of VHL. UOK 127 buffer II, 5 mmol/L MgCl2, 250 mol/L deoxynucleoside has two hyper-methylated copies of VHL. UOK 108 has one triphosphate, 1 ␮mol/L of each primer, 0.2 ␮mol/L probe, 1.25 ␮ silent and one partially methylated copy of VHL, indicating units Taq gold, and 10.25 LH2O. The PCR buffer, MgCl2, and cellular heterogeneity within the culture. UOK 171 is a non- Taq gold are parts of the “AmpliTaq Gold with GeneAmp” kit methylated VHL ϩ/ϩ cell line, whereas 786–0 is a nonmethy- from Applied Biosystems (Roche, Basel, Switzerland). The lated VHL Ϫ/Ϫ line, exhibiting loss of one VHL allele and PCR was done at 95°C for 8 minutes, followed by 45 cycles of inactivation of the other by truncation after amino acid 104. 95°C for 15 seconds, 60°C for 1 minute, and 72°C for 1 minute. Additionally, UOK 121 wild-type, is the UOK 121 cell line with Every sample was run in triplicate. A “methylation index” was a stably transfected VHL (19). The 786–0 ccRCC cell line is calculated that is the ratio between amount of DNA for the VHL VHL negative through deletion of one allele and nonsense gene and amount of MYOD1 that allowed us to calculate the mutation causing a shortened protein (amino acid 1–104) in the relative levels of VHL gene methylation between 5-aza-dCyd- second allele (20). Cells were grown in DMEM containing 10% treated and untreated cells. fetal bovine serum (FBS) and incubated at 37°C in 5% CO2. Western Blot Analysis. To evaluate if we could detect UOK 121 wild-type was grown in the same media with 1 mg/ml VHL protein re-expression in the UOK 121 cell line after neomycin antibiotic. 5-aza-dCyd exposure, we did a Western blot analysis. The UOK Real-Time Reverse Transcription-PCR Analysis. For 121 cell line stably transfected with VHL and UOK 121 trans- the studies on the renal cancer lines, two million viable cells fected with empty plasmid were each plated into several 15-cm counted by trypan blue exclusion method were plated in 15-cm plates. One plate of each cell line was exposed to 3 ␮mol/L tissue culture dishes. 5-Aza-dCyd (Sigma, St. Louis, MO) and 5-Aza-dCyd as described above for the RT-PCR experiments, zebularine (a generous gift of Dr. Victor E. Marquez) were and the remaining plates were allowed to grow normally without dissolved in water. Cells were incubated in zebularine at 50 treatment. At the appropriate time points the cells were washed ␮mol/L, 500 ␮mol/L, and 1,000 ␮mol/L for 24 or 48 hours and with ice cold PBS and trypsinized into a pellet.