Clinical Cancer Research Molecular Diagnostics in Cancer Therapeutic Development: Fulfilling the Promise of Personalized Medicine Infection and Cancer: Biology, Therapeutics, and Prevention
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Alleman, W. G.
Right arrow Articles by Vasselli, J. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Alleman, W. G.
Right arrow Articles by Vasselli, J. R.
Clinical Cancer Research Vol. 10, 7011-7021, October 15, 2004
© 2004 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

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. Alleman1,2, Ray L. Tabios2, Gadisetti V. R. Chandramouli3, Olga N. Aprelikova3, Carlos Torres-Cabala2, Arnulfo Mendoza4, Craig Rodgers5, Nikolai A. Sopko2, W. Marston Linehan2 and James R. Vasselli2

1 Howard Hughes Medical Institute, Chevy Chase, MD; 2 Urology Branch, 3 Laboratory of Biosystems and Cancer, and 4 Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland; 5 The Brady Urologic Institute, The Johns Hopkins Hospital, Baltimore, Maryland


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Clear cell renal carcinoma (ccRCC) is strongly associated with loss of the von Hippel-Lindau (VHL) tumor suppressor gene. The VHL gene is functionally lost through hypermethylation in up to 19% of sporadic ccRCC cases. We theorized that re-expressing VHL silenced by methylation in ccRCC cells, using a hypo-methylating agent, may be an approach to treatment in patients with this type of cancer. We test the ability of two hypo-methylating agents to re-express VHL in cell culture and in mice bearing human ccRCC and evaluate the effects of re-expressed VHL in these models.

Experimental Design: Real-time reverse transcription-PCR was used to evaluate the ability of zebularine and 5-aza-2'-deoxycytidine (5-aza-dCyd) to re-express VHL in four ccRCC cell lines with documented VHL gene silencing through hypermethylation. We evaluated if the VHL re-expressed after hypo-methylating agent treatment could recreate similar phenotypic changes in ccRCC cells observed when the VHL gene is re-expressed via transfection in cell culture and in a xenograft mouse model. Finally we evaluate global gene expression changes occurring in our cells, using microarray analysis.

Results: 5-Aza-dCyd was able to re-express VHL in our cell lines both in culture and in xenografted murine tumors. Well described phenotypic changes of VHL expression including decreased invasiveness into Matrigel, and decreased vascular endothelial growth factor and glucose transporter-1 expression were observed in the treated lines. VHL methylated ccRCC xenografted tumors were significantly reduced in size in mice treated with 5-aza-dCyd. Mice bearing nonmethylated but VHL-mutated tumors showed no tumor shrinkage with 5-aza-dCyd treatment.

Conclusion: Hypo-methylating agents may be useful in the treatment of patients having ccRCC tumors consisting of cells with methylated VHL.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An estimated 31,900 people are diagnosed annually with cancer of the kidney in the United States, of which the majority are clear cell type. In sporadic clear cell renal carcinoma (ccRCC), 50–85% of patients are found to have biallelic loss of the von Hippel-Lindau (VHL) tumor suppressor gene (1, 2, 3) , located on chromosome 3p25 (4 , 5) . It has been reported that in up to 19% of sporadic ccRCC, VHL function is lost because of hyper-methylation of a CpG island in the promoter region of the VHL gene (3 , 6) . DNA methylation is the result of DNA methyltransferase enzymatic activity transferring a methyl group from S-adenosyl-methionine to the C-5 position of cytosine (7) .

The majority of available hypo-methylating agents are structural variations of cytidine with a modified position 5 of the pyrimidine ring. After phosphorylation, the compound is incorporated into DNA where it forms an irreversible covalent bond with DNA methyltransferase. Total enzyme levels decrease after DNA replication, leading to global hypo-methylation (8 , 9) . The hypo-methylating agent with the greatest selectivity for DNA is 5-aza-2'-deoxycytidine (5-aza-dCyd), first synthesized in 1964 (10) . It has been shown to induce expression of silenced genes (11) and suppress growth of tumor cells in vitro (12) . Despite some efficacy as a treatment for leukemia, minimal success has been seen in the treatment of solid tumors (13, 14, 15) . Zebularine, a cytidine deaminase inhibitor, has been described recently as an 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) .

In this report we evaluate the ability of 5-aza-dCyd and zebularine to re-express VHL in several human hyper-methylated VHL ccRCC cell lines. Furthermore, we assess the ability of the re-expressed VHL to cause biochemical and phenotypic alterations in the cells observed when the VHL gene is transfected back into the cells. We also document the ability of 5-aza-dCyd to re-express VHL in xenografted, VHL-methylated, ccRCC cells that have formed small tumors in mice.

We then evaluate the effects of re-expressing VHL in established tumors consisting of VHL-negative cells. Finally, using cDNA microarrays, we evaluate the changes in global gene expression in our cell lines after they are exposed to 5-aza-dCyd.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Cell Culture.
Six cell lines from sporadic clear cell renal carcinomas were analyzed: UOK 108, UOK 121, UOK 127, UOK 143, UOK 171, and 786–0. The methylation status of UOK 108, UOK 121, UOK 127, and UOK 143 has been described previously (3) . UOK 121 and UOK 143 have one hyper-methylated copy and one silent copy of VHL. UOK 127 has two hyper-methylated copies of VHL. UOK 108 has one silent and one partially methylated copy of VHL, indicating cellular heterogeneity within the culture. UOK 171 is a nonmethylated VHL +/+ cell line, whereas 786–0 is a nonmethylated VHL –/– line, exhibiting loss of one VHL allele and inactivation of the other by truncation after amino acid 104. Additionally, UOK 121 wild-type, is the UOK 121 cell line with a stably transfected VHL (19) . The 786–0 ccRCC cell line is VHL negative through deletion of one allele and nonsense mutation causing a shortened protein (amino acid 1–104) in the second allele (20) . Cells were grown in DMEM containing 10% fetal bovine serum (FBS) and incubated at 37°C in 5% CO2. UOK 121 wild-type was grown in the same media with 1 mg/ml neomycin antibiotic.

Real-Time Reverse Transcription-PCR Analysis.
For the studies on the renal cancer lines, two million viable cells counted by trypan blue exclusion method were plated in 15-cm tissue culture dishes. 5-Aza-dCyd (Sigma, St. Louis, MO) and zebularine (a generous gift of Dr. Victor E. Marquez) were dissolved in water. Cells were incubated in zebularine at 50 µmol/L, 500 µmol/L, and 1,000 µmol/L for 24 or 48 hours and were given 24, 48, and 72 hours for cell division. Incubation with 5-aza-dCyd at 0.5, 3.0, and 5.0 µmol/L concentrations occurred for 24 hours; cells were then allowed 72 hours for cell division. We also exposed a bladder cancer cell line, T24, that has a known methylated p16 gene, to 500 µmol/L zebularine for 24 hours and then gave 72 hours for cell division (21) . At the appropriate times, total RNA was isolated with Trizol reagent (Invitrogen, Carlsbad, CA) and purified with three phenolchloroform extractions. Using TaqMan Reverse Transcription kit (ABI, Foster City, CA), 2 µg of total RNA were used to form cDNA in a 25 µL solution, following the manufacturer’s suggested protocol. Real-time quantitative PCR was carried out with ABI Prism 7000 machine. Assays-on-demand probe and primer master mixes from ABI were used for all reverse transcription (RT)-PCR reactions except for the probe and primers for VHL that were described previously (22) . The comparative CT (threshold cycle) method described in the manufacturer’s protocol was used to do relative quantitation of expression, comparing treated samples to concordant untreated controls and the internal control, ß-actin. Each sample was run in triplicate and statistical analysis done using Microsoft Excel and one-sided student t tests.

DNA Methylation Analysis.
We isolated DNA with the Puregene DNA Isolation kit (Puregene, Ashby Park, United Kingdom) from untreated UOK 121 cells or cells treated with 500 µmol/L 5-aza-dCyd for 24 hours than grown for 72 hours. The technique used for bisulfite modification and subsequent RT-PCR analysis has been described previously (23) . Briefly, we subjected 1 µg of sample DNA to sodium bisulfite modification, using the CpGenome DNA Modification Kit (Chemicon, Temecula, CA). We used universally methylated DNA to generate a standard curve, based on which we calculated the amount of DNA in the samples. White blood cell DNA was used as a negative control (unmethylated). Amplification of MYOD1 was used to determine total amount of amplifiable DNA in the samples. Our PCR reaction mix contained 1 µL of DNA in 25 µL of total volume, which also included 2.5 µL of 10x PCR buffer II, 5 mmol/L MgCl2, 250 µmol/L deoxynucleoside triphosphate, 1 µmol/L of each primer, 0.2 µmol/L probe, 1.25 units Taq gold, and 10.25 µL H2O. The PCR buffer, MgCl2, and Taq gold are parts of the "AmpliTaq Gold with GeneAmp" kit from Applied Biosystems (Roche, Basel, Switzerland). The PCR was done at 95°C for 8 minutes, followed by 45 cycles of 95°C for 15 seconds, 60°C for 1 minute, and 72°C for 1 minute. Every sample was run in triplicate. A "methylation index" was calculated that is the ratio between amount of DNA for the VHL gene and amount of MYOD1 that allowed us to calculate the relative levels of VHL gene methylation between 5-aza-dCyd-treated and untreated cells.

Western Blot Analysis.
To evaluate if we could detect VHL protein re-expression in the UOK 121 cell line after 5-aza-dCyd exposure, we did a Western blot analysis. The UOK 121 cell line stably transfected with VHL and UOK 121 transfected with empty plasmid were each plated into several 15-cm plates. One plate of each cell line was exposed to 3 µmol/L 5-Aza-dCyd as described above for the RT-PCR experiments, and the remaining plates were allowed to grow normally without treatment. At the appropriate time points the cells were washed with ice cold PBS and trypsinized into a pellet. Whole cell extracts were generated with lysis buffer containing 1% Igepal, 0.25% sodium deoxycholate, 1% SDS, 50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 10 mmol/L EDTA, 1 mmol/L sodium orthovanadate, 1 mmol/L sodium fluoride, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µmol/L leupeptin, and 0.10 µg/ml aprotinin. The lysate was incubated at 4°C on a rotator for 30 minutes and centrifuged to recover the total protein. Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) was used to quantify the protein, and equal amounts of protein were fractionated on 4 to 20% Tris-Glycine Bio-Rad Gels and transferred to polyvinylidene difluoride membranes. The VHL antibody Ig32 (BD Biosciences-Phramingen, San Diego, CA) at a 1:250 v/v concentration was used to probe for the VHL protein. Protein bands were detected with horseradish peroxidase conjugated goat antirabbit or goat antimouse secondary antibodies (Amersham Pharmacia, Piscataway, NJ).

In vitro Proliferation Analysis.
In vitro proliferation was measured for UOK 121, UOK 121 stably transfected with VHL, and 786–0 cell lines. Cells were treated with 3 µmol/L 5-aza-dCyd for 24 hours at 37°C, and then collected with CellStripper (CellGro, Herndon, VA). The trypan blue exclusion method was used to count cells, and 10,000 cells suspended in 100 µL of 10% FBS media added to each well of a 96-well plate. Eight identical wells were measured for each cell line, and a different 96-well plate was used for each day of measurement. The CellTiter 96 nonradioactive cell proliferation assay (Promega, Madison, WI) was used according to the manufacturer’s protocol. This assay is based on the conversion of a tetrazolium salt, and represents a measure of the in vitro metabolism of the cells. Promega Aqueous One, the indicator dye, was added to each well (20 µL/well) and allotted three hours to develop. The absorbance was then measured at 490 nm by a SpectraMax Plus 96-well plate reader (Molecular Devices, Sunnyvale, CA), every 24 hours for three total measurements.

Branching Morphogenesis Assays.
A description of the branching morphogenesis assay has been described previously (24) . Briefly, UOK 108, UOK 127, and 786–0 cells growing in log phase were either treated with 3 µmol/L 5-aza-dCyd for 24 hours or kept in standard 10% FBS media (controls), then collected with CellStripper and resuspended in 10% FBS media. Cells were counted with the trypan blue exclusion method, and two million cells were plated into wells of a 96-well plate in a 50:50 mixture of Matrigel and 10% FBS media in a volume of 130 µL. The Matrigel cellular mixture was allowed to harden at 37°C for 30 minutes. Once the Matrigel hardened, 130 µL of media alone or media with 100 ng/mL recombinant hepatocyte growth factor (HGF, R&D systems, Minneapolis, MN) was added to the appropriate wells. All experiments were carried out in triplicate. Cells were incubated at 37°C for 72 hours before being photographed.

Microarray Assays.
Total RNA from each of the six cell lines exposed to 3 µmol/L 5-aza-dCyd, analyzed in the real-time quantitative PCR experiments described previously, was also used for the microarray analysis. The gene expression from the 5-aza-dCyd-exposed cells was compared with total RNA isolated from the matched cell lines grown under similar conditions but not exposed to 5-aza-dCyd. Reverse transcription was carried out with 5 µg of total RNA with the Superscript II Reverse Transcription kit (Invitrogen); the T7-d(T)24 primer was used in place of the oligodeoxythymidylic acid primer supplied in the kit. Purification was done with the Genechip Sample Clean-up Module (Qiagen, Valencia, CA). In vitro transcription was carried out to form biotin-labeled cRNA with BioArray High Yield RNA Transcription Labeling kit (Enzo, New York, NY) and purified with the Qiagen Cleanup Module kit. Fragmentation was done with 5x fragmentation buffer at 95°C for 35 minutes, and the reaction was chilled on ice. The fragmented cRNA was then applied to HG-U133A array chips (Affymetrix, Santa Clara, CA) and hybridized to the array probes for 16 hours. After washing and staining with streptavidin-phycoerythrin, the chip was scanned with the GeneArray Scanner (Hewlett-Packard, Palo Alto, CA). Duplicate microarray experiments were done for each cell line, both with and without 5-aza-dCyd exposure; thus a total of 24 microarray experiments were done for this analysis.

Microarray Data and Statistical Analysis.
The protocols for statistical analysis of Affymetrix microarray data have been described previously (25 , 26) . Microarray Suite version 5.0 (MAS5) by Affymetrix [Statistical Algorithms Reference Guide, Part No. 701110 Rev 1, Affymetrix Inc. (2001) Santa Clara, CA 95051]5 was used to determine signal values (one-step Tukey’s Biweight estimate) and detection calls based on Wilcoxon’s signed rank test. The signal values were normalized to a trimmed (excluding 2% lowest and 2% highest signals) mean level of 500 within each array. Expression ratios of post- to pre-5-aza-dCyd exposure and statistical significance of expression change as "increase," "decrease," and "no change" were determined by comparison analysis of MAS5. Because two separate arrays were done for all data points, we had two gene profiles pre- and two gene profiles post-5-aza-dCyd treatment in each cell line tested. We thus were able to make four comparisons for each cell line; for example, we compared pretreatment gene profile A to post-treatment profile A, then pretreatment profile A to post-treatment profile B and so on. To be included in the analysis, a gene had to increase or decrease calls. We considered a gene to be affected by 5-aza-dCyd treatment if its mean expression had changed by 2-fold using the criteria listed above in at least three of the six cell lines tested. We categorized the above significant genes by Gene Ontologies using Expression Analysis Systematic Explorer software (27) . The number of genes in the significant list was compared with the population of all genes on the microarray. Over-representation analysis was done with a modified Fisher’s test to estimate the probability of finding significant genes in a Gene Ontology term from the gene population on array. Expression Analysis Systematic Explorer score, which is upper boundary of the distribution of Jackknife Fisher exact probabilities, was calculated for each Gene Ontology term in significant list, and the Gene Ontology terms were ranked by significance.

Murine Tumor Xenograft Experiments.
Immunocompromised male SCID/beige mice were used to develop a subcutaneous tumor xenograft model with UOK 121 cells. In the first murine study, ten million cells were suspended in 250 µL of Hank’s balanced salt solution and injected subcutaneously into the left inguinal region of 6-week-old mice. The tumors were allowed to grow for 15 days at which time the mean tumor diameter was 15 mm. Three mice received one intraperitoneal dose (2.5 mg/kg) of 5-aza-dCyd whereas two mice received saline injections and after five days were sacrificed. Tumors were frozen to –70°C and crushed to a fine powder with mortar and pestle while on dry ice; the powder from the treated mice and the powder from the untreated mice were combined to form two samples. Total RNA was isolated with TRIzol and real-time quantitative PCR carried out in quadruplicate to determine VHL message levels.

In the second murine study, 20 mice received injection with five million UOK 121 cells, and 10 mice received injection with five million 786–0 cells in the same manner described above. Subcutaneous tumors were allowed to develop for 15 days (UOK 121) and 20 days (786–0) before starting the 5-aza-dCyd injections. For the 20 mice bearing UOK 121 tumors, 10 received an intraperitoneal injection of 2.5 mg/kg 5-aza-dCyd dissolved in PBS, and 10 received only PBS, once a week for 4 consecutive weeks. Five mice with 786–0 tumors received 2.5 mg/kg 5-aza-dCyd injection, and the remaining 5 received PBS placebo injections at the same dosing schedule as the mice with UOK 121 tumors. Mice were weighed and tumors measured in the two largest dimensions every 3 to 4 days by a technician blinded to the experiment. Tumor volume (TV) was calculated according to the following equation: TV = (D)(d2)({pi}/6), where D is the larger diameter and d is the smaller diameter (28) . After 28 days of treatment (1 week after the last 5-aza-dCyd dose), all mice were sacrificed, and tumors were dissected away from normal tissue and weighed. The tumors were formalin fixed and paraffin embedded.

Histologic and Terminal Deoxynucleotidyl Transferase Assay Evaluation.
Sequential thin (6 µm) tissue sections from the UOK 121 cell line tumors of the 5-aza-dCyd-treated and -untreated mice were mounted on noncharged slides for terminal deoxynucleotidyl transferase (TUNEL) assay and H&E staining. The TUNEL assay was performed using the Dead End TUNEL assay kit (Promega) as described by the manufacturer, without modification.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of 5-Aza-dCyd and Zebularine on VHL Expression.
Treatment with 0.5 µmol/L, 3.0 µmol/L, and 5.0 µmol/L of 5-aza-dCyd significantly increased VHL expression in the four hyper-methylated VHL lines (UOK 108, UOK 121, UOK 127, and UOK 143) as determined by real-time RT-PCR (Fig. 1A)Citation .



View larger version (48K):
[in this window]
[in a new window]
  		Fig. 1. Effects of 5-aza-dCyd on gene expression. A, real-time quantitative PCR analysis of VHL expression for six cell lines. The 1st column for each cell line represents the control sample with no 5-aza-dCyd; the remaining three represent increasing doses of the drug at 0.5, 3, and 5 µmol/L. B, expression analysis of VEGF. C, expression analysis of Glut-1. B and C represent downstream products of HIF, and decreased expression is hypothetically a result of increased VHL expression. All experiments were done in triplicate and error bars represent SEM in all three graphs.

 
The bisulfite modification analysis showed that VHL methylation became half as prevalent in the UOK 121 line after 3 µmol/L 5-aza-dCyd treatment as described above (supplemental Fig. A). UOK 121 showed a 160-fold increase in VHL transcript expression that was the largest increase of all cell lines tested. In each of the four cell lines, VHL expression significantly increased at all three doses, increasing by as much as 7.8-fold in 127, 11.6-fold in 143, and 1.9-fold in 108 (P < 0.05 in all four lines). VHL protein re-expression was clearly evident on Western blot in the UOK 121 cell line after exposure to 5-aza-dCyd (Fig. 2)Citation . Exposure to 5-aza-dCyd did not significantly change VHL expression in the non-VHL–methylated UOK 171 or 786–0 cell lines. Zebularine was similarly tested for its ability to increase VHL expression in UOK 121 and UOK 127 at 50 µmol/L, 500 µmol/L, and 1,000 µmol/L concentrations and at six different time points after exposure. However, zebularine failed to consistently increase VHL expression at any dose or time point in the renal lines. To establish that our zebularine treatment regimen could re-express a methylated gene in a cell line, we showed an up-regulation of the expression of p16 in the T24 cell line after the cells were exposed to 500 µmol/L of zebularine for 24 hours (supplemental Fig. B).



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 2. Western blot of UOK 121 cell lysates stably transfected with empty plasmid or VHL into the cells. The cells were exposed to 5-aza-dCyd as described in the text.

 
5-Aza-dCyd is an S-phase-specific agent; thus, the duration of VHL re-expression is dependent upon the speed at which each cell line divides in vitro. UOK 127 was treated with 4 µmol/L of 5-Aza-dCyd for 24 hours; then, every two days, RNA was isolated from the treated cells and VHL expression was measured. VHL expression increased rapidly, reaching a plateau at 3 days from removal of the agent. Expression remained relatively constant until 7 days, followed by a steady decline until 15 days, when the expression of VHL returned to control levels (data not shown).

Re-expressed VHL Effects on Vascular Endothelial Growth Factor and Glut-1 Expression.
To establish whether VHL re-expression resulting from 5-aza-dCyd exposure is sufficient to decrease expression of the downstream hypoxia-inducible factor (HIF) targets, transcript levels of Glut-1 and vascular endothelial growth factor (VEGF; Fig. 1B and CCitation ) were evaluated. Glut-1 expression significantly decreased across all four methylated cell lines: UOK 108 expression decreased to 83% of the original, UOK 121 to 33%, UOK 127 to 67%, and UOK 143 to 63% (P < 0.05 in all cases). Glut-1 expression also decreased in 786–0, with expression after treatment down to 15% of control levels (P < 0.005). VEGF expression decreased in all six cell lines as well. In the hyper-methylated VHL lines, UOK 108 expression decreased to 72% of control, UOK 121 to 34%, UOK 127 to 68%, and UOK 143 to 53% (P < 0.05). In UOK 171 and 786–0, the VEGF expression significantly decreased to 52% and 48%, respectively, of control levels (P < 0.005).

In vitro Proliferation and Branching Morphogenesis.
We evaluated the effects of VHL re-expression on UOK 121 proliferation, using a colorimetric assay based on the metabolism of a tetrazolium compound by viable cells. The non-VHL–methylated 786–0 cells were used as a control. The in vitro proliferation of the hyper-methylated VHL line UOK 121 was not significantly inhibited post-5-aza-dCyd exposure (compared with non-5-aza-dCyd exposed cells). For comparison we also show that the growth of UOK 121 stably expressing VHL had no effect on proliferation rate compared with the parental UOK 121 line. Treatment with 5-aza-dCyd did not significantly alter the proliferation of 786–0 cells in culture (Fig. 3)Citation .



View larger version (32K):
[in this window]
[in a new window]
  		Fig. 3. Effects of 5-aza-dCyd on cell proliferation. Cell proliferation at 24 and 72 hours after plating into 96-well plates measured via absorption at 490 nm after addition of an indicator dye with and without 5-aza-dCyd treatment. A, the proliferation rates of UOK 121 hyper-methylated VHL lines alone, exposed to 3 µmol/L 5-aza-dCyd for 24 hours before plating and UOK 121 stably transfected with VHL. B, proliferation of 786–0 cells, a nonmethylated VHL –/– control untreated and exposed to 5-aza-dCyd for 24 hours before plating in the 96-well plates. Error bars represent SEM.

 
When VHL –/– RCC cell lines, such as 786–0, are transfected with the VHL gene, the ability of the HGF-Met signaling cascade to cause in vitro branching morphogenesis into Matrigel is prevented (24) . We evaluated whether re-expression of VHL by 5-aza-dCyd is adequate to prevent branching morphogenesis in the VHL-methylated UOK 108 and UOK 127 cell lines. Substantial branching morphogenesis was observed 72 hours after HGF exposure in UOK 108 and UOK 127 cells whereas these cells pretreated with 5-aza-dCyd remained spherical and showed no branching, similar to HGF-negative controls (Fig. 4)Citation . The 786–0 cells continued to exhibit significant branching even after exposure to 5-aza-dCyd, strongly suggesting that the inhibition of branching in the UOK 108 and UOK 127 cell lines was a result of increased VHL expression and not a product of toxicity from 5-aza-dCyd.



View larger version (105K):
[in this window]
[in a new window]
  		Fig. 4. Effects of 5-aza-dCyd treatment on branching morphogenesis. Comparison of branching morphogenesis in two ccRCC VHL-methylated cell lines, UOK 108 and UOK 127, and the ccRCC VHL-negative but non-VHL–methylated 786–0 cell line. The cell lines are organized into three rows across, and each column shows a different treatment condition. Column A, cells growing in Matrigel for 72 hours with no treatment and no HGF. Column B, cells exposed to 100 µg of HGF for 72 hours and no prior treatment with 5-aza-dCyd. Column C, cells exposed to 100 µg of HGF for 72 hours and exposed to 3 µmol/L 5-aza-dCyd for 24 hours before plating into Matrigel. All photos taken on inverted microscope at 200x magnification.

 
Gene Expression Analysis of 5-aza-dCyd–Exposed RCC Cell Lines.
The microarray assays were done to identify other genes, besides VHL, re-expressed by 5-aza-dCyd in the clear cell RCC lines. We began by looking at the overall (global) expression changes associated with exposure to 5-aza-dCyd in each of our 6 cell lines. The Affymetrix HG-U133A oligonucleotide microarray has 22,283 probe-sets with 13,903 unique genes on each chip. Treatment with 3 µmol/L 5-aza-dCyd for 24 hours resulted in a >2-fold up-regulation in 1,043 (7.5%) of 13,900 genes evaluated in the UOK 121 cell line, 242 (1.7%) in the 127 line, 26 (0.2%) in the UOK 108 and 140 (1.0%) in the UOK 143 line. In the cell lines where VHL is not methylated, UOK 171 and 786–0, 258 (1.9%) and 774 (5.6%) of 13,900 genes are up-regulated, respectively. Because the re-expression of some genes can down-regulate others, we next identified the genes 2-fold down-regulated in our cell lines. Three hundred and thirty-seven (2.4%) genes are >2-fold down-regulated in the UOK 121 cell line, 501 (3.6%) genes in the UOK 127 line, 1 gene in the 108, and 12 (0.1%) genes in the UOK 143 line. In the non-VHL–methylated lines, UOK 171 and 786–0, 132 (0.9%) and 394 (2.8%) of 13,900 genes are down-regulated. The UOK 108 cell line has only 26 genes that are ≥2-fold up-regulated and one gene down-regulated after 5-aza-dCyd exposure. Only two genes, {alpha}1 collagen and matrix metalloproteinase 1, were ≥2-fold up-regulated and no genes were ≤2-fold down-regulated across all six cell lines after 5-aza-dCyd treatment, as defined by the array analysis. The VHL signal is too low across all of the arrays for us to make any determination of the VHL expression status by array analysis, but the RT-PCR had already verified the VHL re-expression. In a few cell lines (e.g., UOK 108 and UOK 143), many genes have low expression change after 5-aza-dCyd exposure. Therefore to establish a larger cohort of genes with altered expression after exposure to 5-aza-dCyd in our ccRCC lines by array analysis, we reduced our criteria for inclusion and identified genes with >2-fold expression change post-5-aza-dCyd treatment in three or more of the six cell lines. Using the criteria outlined in the methods, we identified 151 (unique of 173) genes ≥2-fold up-regulated and four genes ≥2 down-regulated in at least three of the six cell lines (supplemental information, Table A). We chose a set of 15 genes from the 157 identified above that affect a broad spectrum of cellular processes and confirmed the increased expression found by array analysis using real-time quantitative RT-PCR analysis (Table 1)Citation . Eight of the 15 genes listed in Table 1Citation were found to have CpG islands in the promotor region of the gene by performing a combination of literature search and internet gene analysis tools (Genomatix and CpG Island Searcher6 ; ref. 29, 30, 31, 32, 33, 34, 35, 36, 37 ). The RNA used for the RT-PCR was isolated from two of the three cell lines where the gene was found up-regulated by the array analysis. All samples tested show an increased expression >2-fold, equal to or exceeding the results of the microarray data. The Expression Analysis Systematic Explorer analysis described in the methods was done on the array data to find "classes" of genes disproportionately up- or down-regulated compared with other classes of gene represented on the array, post-5-aza-dCyd exposure. Unexpectedly, we found that the major categories of genes up-regulated after 5-aza-dCyd exposure are the nucleosome and nucleosome assembly class of genes, which includes many histone genes (Table 2)Citation . This suggests that 5-aza-dCyd may disproportionately increase the expression of several classes of histone genes as compared with other classes of genes in our cell lines. A list of all Gene Ontologies identified by the Expression Analysis Systematic Explorer analysis is included in the supplementary information (Table B).


View this table:
[in this window]
[in a new window]
  		Table 1 List of genes up-regulated by 2-fold or higher after 5-aza-dCyd exposure in at least three of the six cell lines chosen and confirmed by RT-PCR

 

View this table:
[in this window]
[in a new window]
  		Table 2 The nucleosome assembly cohort of genes as identified by EASE analysis

 
Effects of 5-aza-dCyd in Murine Xenograft Models.
It has been shown previously that VHL-negative cell lines lose their ability to form tumors in vivo with reintroduction of wild-type VHL gene (38 , 39) before injection into mice; this work was done with 786–0 cells. Our series of experiments evaluates the biological behavior of VHL-negative cells that have already established tumors in the mouse at the time of VHL gene re-expression in the tumor cells.

Our first experiment verified the in vivo ability of 5-aza-dCyd to re-express VHL in established tumors. Tumor bearing mice were given a single intraperitoneal injection of 5-aza-dCyd and sacrificed 5 days later as described in "Materials and Methods." Tumors were dissected and surrounding normal tissue cleared from the tumor. Total RNA was extracted from the tumor specimen and real-time quantitative PCR performed. Treated mice demonstrated approximately 1.5-fold higher VHL expression than PBS controls (P < 0.05; Fig. 5ACitation ).



View larger version (23K):
[in this window]
[in a new window]
  		Fig. 5. In vivo tumor effects with IP 5-aza-dCyd administration. A, real-time quantitative PCR comparing in vivo expression of VHL from mRNA isolated out of UOK 121 cell line tumor xenografts in mice receiving a dose of 5-aza-dCyd to PBS (control). B, comparison of the xenografted tumor masses in mice treated with 5-aza-dCyd to mice given PBS for tumors arising from UOK 121 and 786–0 cell lines. C, comparison of xenografted tumor volumes in mice treated with 5-aza-dCyd to mice given PBS for tumors arising from UOK 121 cells. The difference in tumor volume becomes significant (P < 0.05) by day 20 after 5-aza-dCyd was started (day 35 after the cells were injected) P values obtained with one-sided t tests. All error bars represent SEM. D, comparison of 786–0 xenografted tumor volumes exactly as done for UOK 121 in C above. There is no significant difference in 786–0 tumor volume.

 
The second murine experiment evaluated the in vivo effects of 5-aza-dCyd and re-expression of VHL on the established VHL-methylated cell line tumors. UOK 121 and 786–0 cells were injected as described, and tumors were allowed to develop for 15 days for UOK 121 and 20 days for 786–0 at which time the approximate mean tumor volumes were 200 mm3 (UOK 121) and 150 mm3 (786–0). Mice were then given intraperitoneal injections of 5-aza-dCyd (2.5 mg/kg) or PBS placebo every 7 days, for a total of four injections. Tumor growth was significantly reduced in the treatment group of UOK 121 compared with the control group by 20 days after the 5-aza-dCyd injections started (P < 0.05), and tumor weight was significantly decreased at 28 days (P < 0.05; Fig. 5B and CCitation ). In vivo, 5-aza-dCyd treatment did not have a significant effect on either tumor growth or tumor weight in the 786–0 tumors (Fig. 5B and DCitation ; P > 0.05).

Histology and TUNEL Assay.
There were clear differences in the histology from the UOK 121 cell line tumors of the 5-aza-dCyd-treated mice versus the untreated mice. The tumors from the untreated mice showed solid sheets of poorly differentiated clear cells with very few areas of necrosis or fibrosis. The tumors from the treated mice had more extensive areas of fibrosis and necrosis (Fig. 6)Citation . The TUNEL assay showed no difference in the amounts of apoptosis in the tumors of the treated and untreated mice. Both groups showed only very small amounts of apoptosis compared with positive controls.



View larger version (89K):
[in this window]
[in a new window]
  		Fig. 6. Histology of tumors in mice. Histology of UOK 121 cell line tumors grown in SCID/beige mice. Representative H&E-stained 6 µm-thick sections from UOK 121 cell tumors excised 43 days after 5 million UOK 121 cells were injected subcutaneously (original magnification, x200). A, section from a mouse that received PBS. B, section from a mouse that received 5-aza-dCyd at 2.5 mg/kg once a week for 4 weeks.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Re-expression of the VHL gene in VHL-negative ccRCC cell lines is associated with a constellation of biochemical and phenotypic changes in vitro and in vivo. The effects of re-expressing VHL in VHL-negative ccRCC cells have been defined previously by stably transfecting the VHL gene into cells and then comparing phenotypic characteristics of the transfected cells with the nontransfected cells. In this report, we used the hypo-methylating agent 5-aza-dCyd to re-express VHL in ccRCC cell lines having methylated VHL, both in vitro cell culture and in vivo murine models.

We began by showing that hyper-methylated VHL can be re-expressed in cell lines exposed to 5-aza-dCyd with real-time RT-PCR analysis. Four cell lines from the tumors of patients with clear cell RCC in which the VHL gene is hyper-methylated were used in this study. Exposure to 5-aza-dCyd re-expresses VHL in all four hyper-methylated cell lines in vitro; the largest increase occurred in the UOK 121 cell line with over 100-fold increase in VHL expression at the 3 and 5 µmol/L 5-aza-dCyd concentrations. The smallest increase of VHL expression occurred in the 108 cell line with approximately 2-fold increase at the highest 5-aza-dCyd concentration. The incomplete nature of the methylation of the VHL gene in the UOK 108 line may account for the relatively small increase in VHL expression after 5-aza-dCyd exposure as compared with the other three cell lines.

We were also able to re-express VHL in the UOK 121 line after these cells had formed small tumors in mice. The VHL expression levels attained were 1.5- to 1.8-fold higher than in untreated mice with UOK 121 cell line tumors of similar size. The reason for the lower levels of VHL expression in the mice compared with what we saw in vitro may be attributable to a lower concentration of 5-aza-dCyd delivered to the UOK 121 cells in the mice. Also, the mouse tumors were grossly dissected from the mice, and other cells, contributed by the mouse such as vasculature and interstitial stromal cells, were included in our samples.

We next showed that 5-aza-dCyd re-expressed VHL can modulate the expression of the HIF-regulated genes, VEGF and glucose transporter (GLUT)-1. Under normoxic conditions, the VHL gene product forms a multimeric complex with elongin B, elongin C, Cul2, and Rbx1 (40, 41, 42, 43, 44) , which in turn acts as a ubiquitin ligase targeting HIF for proteosomal degradation (45 , 46) . Loss of normal VHL function leads to a failure to degrade HIF, resulting in constitutive expression of HIF target genes (20 , 38 , 47, 48, 49, 50) . The message levels of VEGF and GLUT-1 significantly decreased in the four methylated cell lines after 5-aza-dCyd exposure correlating to the levels of VHL re-expression noted in these lines. The largest decreases in expression occurred in the UOK 121 cell line where the VEGF and GLUT-1 expression was decreased to one third the pre-5-aza-dCyd message levels; this coincides with the UOK 121 cell line showing the largest increase in VHL expression after 5-aza-dCyd exposure. We cannot directly attribute the decreases in these HIF-regulated genes to VHL re-expression alone because VEGF and GLUT-1 expression also decrease in the non-VHL–methylated lines. Other genes beyond HIF may regulate the expression of VEGF and GLUT-1, and 5-aza-dCyd may be causing the hypo-methylation of other regulatory genes causing the decreased expression noted in the UOK 171 and 786–0 lines.

The next series of studies evaluates the phenotypic changes in our cell lines after 5-aza-dCyd exposure. We chose the phenotypic characteristics evaluated in this study because they represent well-described characteristics observed in VHL-negative lines when VHL function is re-established by gene transfection. It is well documented that 5-aza-dCyd has a generalized, nonspecific cytotoxic effect on cells attributable, in part, to the formation of enzyme-DNA adducts (51) . To document that there are no cytotoxic effects of 5-aza-dCyd at the concentrations used, we control each experiment by exposing the non-VHL–methylated 786–0 ccRCC cell line to 5-aza-dCyd. The 786–0 line is an excellent control because it is VHL-negative through allele loss and mutation, and it shows similar phenotypic changes observed in the VHL-methylated lines after VHL plasmid transfection. When 786–0 and UOK 121 cells are grown in Matrigel and exposed to HGF they become highly invasive into the Matrigel 3-dimensional matrix, and their morphology changes extensively to that of a highly branched form. When VHL is transfected into these lines, HGF exposure has little to no effect on either cell line. The UOK 121 line did not invade into Matrigel after 5-aza-dCyd exposure whereas the 786–0 continued to branch after 5-aza-dCyd. We next evaluated the proliferation rates of cells with 5-aza-dCyd exposure. The re-expression of VHL in the 786–0 and UOK 121 cell lines did not change their proliferation rates in vitro. Similarly, the proliferation rate of the 786–0 and UOK 121 line was unaffected by 5-aza-dCyd treatment. The above experiments show that the ability of 5-aza-dCyd to re-express VHL in our methylated lines can recreate the phenotypic changes shown in these cells that occur when the VHL gene is transfected into them. Furthermore, the cytotoxic effects of 5-aza-dCyd played little role in causing the observed phenotypic changes because the 3 µmol/L concentration had no effect on the non-VHL–methylated 786–0 line.

Perhaps a most notable aspect of the role of the VHL gene as a tumor suppressor gene is its ability to inhibit VHL-negative cells from forming tumors in mice when these cells are transfected with the VHL gene before injection into the mouse. In this study we now show that re-expressing VHL in cells that have established a tumor in a mouse can stop the growth of the tumor. Mice were injected with the UOK 121 VHL-methylated line or the 786–0 non-VHL–methylated controls. After small tumors had grown, the mice were treated with 5-aza-dCyd as described and the tumor size and weight at sacrifice were measured. We based our 5-aza-dCyd dosing in the mice on what had been used in previous studies and chose the lowest doses that had been shown to be effective (52, 53, 54) . We picked a low dose to minimize the cytotoxic effects of 5-aza-dCyd on tumor growth. Whereas the 786–0 cells produced tumors of similar weight in mice treated with 5-aza-dCyd as they did in mice receiving no treatment, the UOK 121 VHL-methylated line tumors were significantly reduced in size and weight in the treated mice. The mice did not seem to have any morbidity from receiving 5-aza-dCyd at the dosages given because there were no remarkable differences between the treated and untreated mice. At the time the 5-aza-dCyd was begun in the mice, the UOK 121 VHL-methylated tumors were approximately 200 mm3 in size. Over the course of the 25-day 5-aza-dCyd-treatment period, the tumors in the treated mice remained 200 mm3 whereas the untreated tumors more than doubled in volume. Although VHL inhibits xenografted tumor growth in mice it does not slow the growth of cells in vitro. However, the re-expression of VHL is associated with a decrease in expression of growth factors such as VEGF, transforming growth factor-{alpha}, and platelet-derived growth factor in the tumor cells, which may enable the tumor to grow to sizes that require neo-vascularization allowing adequate oxygenation. Thus we would expect there to be areas of necrosis and fibrosis in the 5-aza-dCyd-treated tumors (where the VHL gene is re-expressed), which was observed in these tumors without significant amounts of apoptosis. The observation that the VHL-methylated UOK 121 lines were significantly smaller with 5-aza-dCyd treatment whereas the 786–0 lines were virtually unaffected suggests that the cytotoxic effects of 5-aza-dCyd had little role in the inhibition of tumor growth.

The array analysis was done to identify global changes in gene expression and categorize the major classes of genes with expression changes after 5-aza-dCyd exposure in our cell lines. Five of the genes listed in Table 1Citation that were confirmed by RT-PCR, E-Cadherin, TFPI2, Stratifin, S100A4 and prostasin are silenced by methylation in other solid tumors; one of these genes, E-cadherin was previously shown to be methylated in the 786–0 line (34) . In RCC a larger percentage of higher staged cancers had methylated E-cadherin compared with lower staged tumors and the same is true for TFPI-2 in glioblastoma (31) . The entire list of genes up- and down-regulated in our cell lines after 5-aza-dCyd exposure is available as supplemental information. The most notable finding from the microarray analysis may be that many histone genes were found to be disproportionately up-regulated by5-aza-dCyd. It has been described that 5-aza-dCyd can modify histone protein methylation but not that the levels of histone RNA message increase after 5-aza-dCyd exposure (55) . 5-aza-dCyd has been shown to decrease lysine-9 methylation whereas increasing lysine-4 methylation on histone 3. It is not known how 5-aza-dCyd modifies histone methylation at present. Whether histone hypo-methylation by 5-aza-dCyd is related to the observed increase in histone message level is unknown. The significance of having a disproportionate up-regulation of histone genes occurring across our cell lines after 5-aza-dCyd exposure will require additional investigation. Clearly the interaction of promoter hypo-methylation and alterations in histone methylation is complex, and genotypic and phenotypic changes occurring from this interaction is difficult to correlate. Large effects on gene expression might be attributable to the presence of multiple gene copies. However, of the cohort of histone genes listed in Table 2Citation , only the TSPY gene is known to exist in multiple copies with multiple CpG islands (56) .

Therapy based on the re-expression of the VHL tumor suppressor gene in the tumor cells of patients that have lost VHL function is a logical hypothesis, if it could be accomplished in vivo in an effective manner. The use of 5-aza-dCyd in the mice is, to our knowledge, the first experimental test of such a model in which the VHL gene is re-expressed in an established ccRCC VHL-negative tumor. VHL hyper-methylation occurs in up to 19% of patients with sporadic clear cell RCC, and the use of a hypo-methylating agent in these patients affords us an unusual opportunity to achieve VHL re-expression in the tumor cells.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article can be found at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org).

Requests for reprints: W. Marston Linehan, Chief, Urologic Oncology Branch, CCR National Cancer Institute, Building 10, Room 2B47, Bethesda, Maryland 20892-1501. Phone: 301-496-6353; Fax: 301-402-0922; E-mail: UOB{at}nih.gov

5 http://www.affymetrix.com/support/technical/technotes/statistical_reference_guide.pdf Back

6 Genomatix, www.genomatix.de/; CpG Island Searcher, www.uscnorris.com/cpgislands/cpg.cgi. Back

Received 3/15/04; revised 6/24/04; accepted 7/ 7/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Gnarra JR, Tory K, Weng Y, et al Mutations of the VHL tumor suppressor gene in renal carcinoma. Nat Genet 1994;7:85-90.[CrossRef][Medline]
  2. Foster K, Prowse A, van den BA, et al Somatic mutations of the von Hippel-Lindau disease tumour suppressor gene in non-familial clear cell renal carcinoma. Hum Mol Genet 1994;3:2169-73.[Abstract/Free Full Text]
  3. Herman JG, Latif F, Weng Y, et al Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. Proc Natl Acad Sci USA 1994;91:9700-4.[Abstract/Free Full Text]
  4. Latif F, Tory K, Gnarra J, et al Identification of the von Hippel-Lindau disease tumor suppressor gene. Science (Wash D C) 1993;260:1317-20.
  5. Seizinger BR, Rouleau GA, Ozelius LJ, et al Von Hippel-Lindau disease maps to the region of chromosome 3 associated with renal cell carcinoma. Nature (Lond) 1988;332:268-9.[CrossRef][Medline]
  6. Clifford SC, Prowse AH, Affara NA, Buys CH, Maher ER Inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene and allelic losses at chromosome arm 3p in primary renal cell carcinoma: evidence for a VHL-independent pathway in clear cell renal tumourigenesis. Genes Chromosomes Cancer 1998;22:200-9.[CrossRef][Medline]
  7. Robertson KD DNA methylation, methyltransferases, and cancer. Oncogene 2001;20:3139-55.[CrossRef][Medline]
  8. Santini V, Kantarjian HM, Issa JP Changes in DNA methylation in neoplasia: pathophysiology and therapeutic implications. Ann Intern Med 2001;134:573-86.[Abstract/Free Full Text]
  9. Lubbert M DNA methylation inhibitors in the treatment of leukemias, myelodysplastic syndromes and hemoglobinopathies: clinical results and possible mechanisms of action. Curr Top Microbiol Immunol 2000;249:135-64.[Medline]
  10. Pliml I, Sorm F Synthesis of 2'-deoxy-D-ribofuranosyl-5-aza-cytosine. Coll Czech Chem Commun 1964;29:2576-8.
  11. Jones PA Altering gene expression with 5-azacytidine. Cell 1985;40:485-6.[CrossRef][Medline]
  12. Bender CM, Pao MM, Jones PA Inhibition of DNA methylation by 5-aza-2'-deoxycytidine suppresses the growth of human tumor cell lines. Cancer Res 1998;58:95-101.[Abstract/Free Full Text]
  13. Braakhuis BJ, van Dongen GA, van Walsum M, Leyva A, Snow GB Preclinical antitumor activity of 5-aza-2'-deoxycytidine against human and neck cancer xenografts [corrected]. Investig New Drugs 1988;6:299-304.[Medline]
  14. Abele R, Clavel M, Dodion P, et al The EORTC Early Clinical Trials Cooperative Group experience with 5-aza-2'-deoxycytidine (NSC 127716) in patients with colo-rectal, head and neck, renal carcinomas and malignant melanomas. Eur J Cancer Clin Oncol 1987;23:1921-4.[CrossRef][Medline]
  15. Goffin J, Eisenhauer E DNA methyltransferase inhibitors-state of the art. Ann Oncol 2002;13:1699-1716.[Abstract/Free Full Text]
  16. Cheng JC, Matsen CB, Gonzales FA, et al Inhibition of DNA methylation and reactivation of silenced genes by zebularine. J Natl Cancer Inst (Bethesda) 2003;95:399-409.[Abstract/Free Full Text]
  17. Barchi J, Musser S, Marquez VE The decomposition of 1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine) in alkali - mechanism and products. J Org Chem 1992;57:536-41.[CrossRef]
  18. Kelley JA, Driscoll JS, McCormack JJ, Roth JS, Marquez VE Furanose-pyranose isomerization of reduced pyrimidine and cyclic urea ribosides. J Med Chem 1986;29:2351-8.[CrossRef][Medline]
  19. Kuzmin I, Geil L, Ge H, et al Analysis of aberrant methylation of the VHL gene by transgenes, monochromosome transfer, and cell fusion. Oncogene 1999;18:5672-9.[CrossRef][Medline]
  20. de Paulsen N, Brychzy A, Fournier MC, et al Role of transforming growth factor-alpha in von Hippel–Lindau (VHL)(–/–) clear cell renal carcinoma cell proliferation: a possible mechanism coupling VHL tumor suppressor inactivation and tumorigenesis. Proc Natl Acad Sci USA 2001;98:1387-92.[Abstract/Free Full Text]
  21. Cheng JC, Weisenberger DJ, Gonzales FA, et al Continuous zebularine treatment effectively sustains demethylation in human bladder cancer cells. Mol Cell Biol 2004;24:1270-8.[Abstract/Free Full Text]
  22. Maranchie JK, Vasselli JR, Riss J, Bonifacino JS, Linehan WM, Klausner RD The contribution of VHL substrate binding and HIF1-alpha to the phenotype of VHL loss in renal cell carcinoma. Cancer Cell 2002;1:247-55.[CrossRef][Medline]
  23. Yegnasubramanian S, Kowalski J, Gonzalgo ML, et al Hypermethylation of CpG islands in primary and metastatic human prostate cancer. Cancer Res 2004;64:1975-86.[Abstract/Free Full Text]
  24. Koochekpour S, Jeffers M, Wang PH, et al The von Hippel-Lindau tumor suppressor gene inhibits hepatocyte growth factor/scatter factor-induced invasion and branching morphogenesis in renal carcinoma cells. Mol Cell Biol 1999;19:5902-12.[Abstract/Free Full Text]
  25. Lockhart DJ, Dong H, Byrne MC, et al Expression monitoring by hybridization to high-density oligonucleotide arrays. Nat Biotechnol 1996;14:1675-80.[CrossRef][Medline]
  26. Golub TR, Slonim DK, Tamayo P, et al Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science (Wash D C) 1999;286:531-7.
  27. Hosack DA, Dennis G, Jr., Sherman BT, Lane HC, Lempicki RA Identifying biological themes within lists of genes with EASE. Genome Biol 2003;4:R70[CrossRef][Medline]
  28. Tomayko MM, Reynolds CP Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol 1989;24:148-54.[CrossRef][Medline]
  29. Dao D, Walsh CP, Yuan L, et al Multipoint analysis of human chromosome 11p15/mouse distal chromosome 7: inclusion of H19/IGF2 in the minimal WT2 region, gene specificity of H19 silencing in Wilms’ tumorigenesis and methylation hyper-dependence of H19 imprinting. Hum Mol Genet 1999;8:1337-52.[Abstract/Free Full Text]
  30. Hube F, Reverdiau P, Iochmann S, Rollin J, Cherpi-Antar C, Gruel Y Transcriptional silencing of the TFPI-2 gene by promoter hypermethylation in choriocarcinoma cells. Biol Chem 2003;384:1029-34.[CrossRef][Medline]
  31. Konduri SD, Srivenugopal KS, Yanamandra N, et al Promoter methylation and silencing of the tissue factor pathway inhibitor-2 (TFPI-2), a gene encoding an inhibitor of matrix metalloproteinases in human glioma cells. Oncogene 2003;22:4509-16.[CrossRef][Medline]
  32. Lehmann U, Langer F, Feist H, Glockner S, Hasemeier B, Kreipe H Quantitative assessment of promoter hypermethylation during breast cancer development. Am J Pathol 2002;160:605-12.[Abstract/Free Full Text]
  33. Lodygin D, Yazdi AS, Sander CA, Herzinger T, Hermeking H Analysis of 14-3-3sigma expression in hyperproliferative skin diseases reveals selective loss associated with CpG-methylation in basal cell carcinoma. Oncogene 2003;22:5519-24.[CrossRef][Medline]
  34. Nojima D, Nakajima K, Li LC, et al CpG methylation of promoter region inactivates E-cadherin gene in renal cell carcinoma. Mol Carcinog 2001;32:19-27.[CrossRef][Medline]
  35. Schwienbacher C, Angioni A, Scelfo R, et al Abnormal RNA expression of 11p15 imprinted genes and kidney developmental genes in Wilms’ tumor. Cancer Res 2000;60:1521-5.[Abstract/Free Full Text]
  36. Wu AH, Low WC Molecular cloning and identification of the human interleukin 13 alpha 2 receptor (IL-13Ra2) promoter. Neuro-oncol 2003;5:179-87.[Abstract]
  37. Yu JX, Chao L, Ward DC, Chao J Structure and chromosomal localization of the human prostasin (PRSS8) gene. Genomics 1996;32:334-40.[CrossRef][Medline]
  38. Gnarra JR, Zhou S, Merrill MJ, et al Post-transcriptional regulation of vascular endothelial growth factor mRNA by the product of the VHL tumor suppressor gene. Proc Natl Acad Sci USA 1996;93:10589-94.[Abstract/Free Full Text]
  39. Iliopoulos O, Kibel A, Gray S, Kaelin WG, Jr. Tumour suppression by the human von Hippel-Lindau gene product. Nat Med 1995;1:822-6.[CrossRef][Medline]
  40. Duan DR, Pause A, Burgess WH, et al Inhibition of transcription elongation by the VHL tumor suppressor protein. Science (Wash D C) 1995;269:1402-6.
  41. Kibel A, Iliopoulos O, DeCaprio JA, Kaelin WG, Jr. Binding of the von Hippel-Lindau tumor suppressor protein to Elongin B and C. Science (Wash D C) 1995;269:1444-6.
  42. Pause A, Lee S, Worrell RA, et al The von Hippel-Lindau tumor-suppressor gene product forms a stable complex with human CUL-2, a member of the Cdc53 family of proteins. Proc Natl Acad Sci USA 1997;94:2156-61.[Abstract/Free Full Text]
  43. Pause A, Peterson B, Schaffar G, Stearman R, Klausner RD Studying interactions of four proteins in the yeast two-hybrid system: structural resemblance of the pVHL/elongin BC/hCUL-2 complex with the ubiquitin ligase complex SKP1/cullin/F-box protein. Proc Natl Acad Sci USA 1999;96:9533-8.[Abstract/Free Full Text]
  44. Kamura T, Koepp DM, Conrad MN, et al Rbx1, a component of the VHL tumor suppressor complex and SCF ubiquitin ligase. Science (Wash D C) 1999;284:657-61.
  45. Cockman ME, Masson N, Mole DR, et al Hypoxia inducible factor-alpha binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein. J Biol Chem 2000;275:25733-41.[Abstract/Free Full Text]
  46. Ohh M, Park CW, Ivan M, et al Ubiquitination of hypoxia-inducible factor requires direct binding to the beta-domain of the von Hippel-Lindau protein. Nat Cell Biol 2000;2:423-7.[CrossRef][Medline]
  47. Kourembanas S, Hannan RL, Faller DV Oxygen tension regulates the expression of the platelet-derived growth factor-B chain gene in human endothelial cells. J Clin Investig 1990;86:670-4.
  48. Siemeister G, Weindel K, Mohrs K, Barleon B, Martiny-Baron G, Marme D Reversion of deregulated expression of vascular endothelial growth factor in human renal carcinoma cells by von Hippel-Lindau tumor suppressor protein. Cancer Res 1996;56:2299-301.[Abstract/Free Full Text]
  49. Iliopoulos O, Levy AP, Jiang C, Kaelin WG, Jr, Goldberg MA Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein. Proc Natl Acad Sci USA 1996;93:10595-9.[Abstract/Free Full Text]
  50. Semenza G Signal transduction to hypoxia-inducible factor 1. Biochem Pharmacol 2002;64:993-8.[CrossRef][Medline]
  51. Karpf AR, Jones DA Reactivating the expression of methylation silenced genes in human cancer. Oncogene 2002;21:5496-503.[CrossRef][Medline]
  52. Covey JM, Zaharko DS Comparison of the in vitro cytotoxicity (L1210) of 5-aza-2'-deoxycytidine with its therapeutic and toxic effects in mice. Eur J Cancer Clin Oncol 1985;21:109-17.[CrossRef][Medline]
  53. Momparler RL Molecular, cellular and animal pharmacology of 5-aza-2'-deoxycytidine. Pharmacol Ther 1985;30:287-99.[CrossRef][Medline]
  54. Vesely J, Cihak A Incorporation of a potent antileukemic agent, 5-aza-2'-deoxycytidine, into DNA of cells from leukemic mice. Cancer Res 1977;37:3684-9.[Abstract/Free Full Text]
  55. Kondo Y, Shen L, Issa JP Critical role of histone methylation in tumor suppressor gene silencing in colorectal cancer. Mol Cell Biol 2003;23:206-15.[Abstract/Free Full Text]
  56. Dechend F, Williams G, Skawran B, et al TSPY variants in six loci on the human Y chromosome. Cytogenet Cell Genet 2000;91:67-71.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Mol. Cell. Biol.Home page
K. K. W. To, Z. Zhan, and S. E. Bates
Aberrant Promoter Methylation of the ABCG2 Gene in Renal Carcinoma
Mol. Cell. Biol., November 15, 2006; 26(22): 8572 - 8585.
[Abstract] [Full Text] [PDF]

Home page
 J Biomol ScreenHome page
G. M. Woldemichael, J. R. Vasselli, R. S. Gardella, T. C. Mckee, W. M. Linehan, and J. B. McMahon
Development of a Cell-Based Reporter Assay for Screening of Inhibitors of Hypoxia-Inducible Factor 2-Induced Gene Expression
J Biomol Screen, September 1, 2006; 11(6): 678 - 687.
[Abstract] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Alleman, W. G.
Right arrow Articles by Vasselli, J. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Alleman, W. G.