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

Alleman, Wade G.; Tabios, Ray; Chandramouli, Gadisetti V.R.; Aprelikova, Olga; Torres‐Cabala, Carlos A.; Mendoza, Arnulfo; Rodgers, Craig; Sopko, Nikolai A.; Linehan, W. Marston; Vasselli, James R.

doi:10.1158/1078-0432.ccr-04-0516

Cited by 47 publications

(30 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Human sporadic clear cell renal carcinoma cell lines UOK121, UOK143, and UOK181 were kindly provided by M. W. Linehan (NCI, Bethesda, MD). The methylation status of UOK121 and UOK143 has been described previously (1,23). UOK121 and UOK143 had lost one VHL allele and retained a single heavily methylated allele.…”

Section: Methodsmentioning

confidence: 83%

Aberrant Promoter Methylation of the ABCG2 Gene in Renal Carcinoma

Zhan

Bates

2006

Molecular and Cellular Biology

113

101

View full text Add to dashboard Cite

ABCG2 is a ubiquitous ATP-binding cassette transmembrane protein that is important in clinical drug resistance. Little is known about the mechanism(s) regulating the expression of ABCG2. We hypothesized that DNA methylation could play a role in the epigenetic regulation of ABCG2 gene expression. The promoter methylation status of three renal carcinoma cell lines was assessed with restriction enzyme digestion-coupled PCR and bisulfite genomic sequencing. Both UOK121 and UOK143, with known methylation of the VHL promoter, showed induction of ABCG2 expression after 5-aza-2-deoxycytidine (5-aza-dC) treatment, suggesting that aberrant methylation of the ABCG2 gene was associated with gene silencing. In vitro methylation of the ABCG2 promoter-driven luciferase reporter vector resulted in a significant inhibition of transcription. Our data suggested that the ABCG2 gene is regulated coordinately at both histone and DNA levels. A chromatin immunoprecipitation assay demonstrated that the methylated promoter in UOK121 and UOK143, but not the unmethylated one in UOK181, is associated with the methyl CpG binding domain proteins (MBDs), MBD2 and MeCP2. Histone deacetylase 1 and a corepressor, mSin3A, were identified binding to the promoter region containing the CpG island, thereby suppressing ABCG2 transcription. Reactivation of ABCG2 was achieved by treatment with 5-aza-dC, a demethylating agent, concomitant with the release of MBDs from the promoter. Furthermore, the association of methylated lysine 9 on histone H3, a hallmark of promoter methylation, with the promoter was reduced following 5-aza-dC treatment. These data suggest that DNA methylation-dependent formation of a repressor complex in the CpG island contributes to inactivation of ABCG2.ABCG2 (previously named BCRP, MXR, or ABCP) is an ATP-binding cassette half-transporter, originally identified as a multidrug resistance transporter (2, 15, 39). It is highly expressed in many normal tissues, including the epithelium of the small intestine and the liver canalicular membrane (36). In addition to conferring resistance in cancer cells to chemotherapeutic agents such as mitoxantrone, topotecan, and methotrexate (14, 56), ABCG2 has been shown to mediate apically directed drug transport and to play a significant role in absorption, distribution, and elimination of its substrates (8,55).To date, little is known about the molecular mechanisms controlling ABCG2 expression. Characterization of the ABCG2 gene promoter revealed that it is a TATA-less promoter with several Sp1, AP1, and AP2 sites and a CCAAT box downstream from a putative CpG island. Both positive and negative cis-regulatory elements have been suggested in the ABCG2 promoter (4). Although the role of these cis-elements in ABCG2 transcription has not been assessed, recent studies have demonstrated functional hormone (16) and hypoxia (32) response elements in the ABCG2 promoter. Early observations in our laboratory revealed that ABCG2 expression in some renal clear cell carcinoma (RCC) cell lines could be upregula...

show abstract

Section: Methodsmentioning

confidence: 83%

Aberrant Promoter Methylation of the ABCG2 Gene in Renal Carcinoma

Zhan

Bates

2006

Molecular and Cellular Biology

113

101

View full text Add to dashboard Cite

show abstract

“…The resulting loss of VHL protein function is thought to stabilize HIF-1α, leading to the induction of VEGF expression. Re-expression of VHL by 5-aza-CdR can modulate the expression of VEGF, indicating that the promoter remains otherwise intact (29). Zeb treatment globally changes the methylation of the genome and, therefore, it is likely that in addition to affecting VHL it affects the expression of many other genes.…”

Section: Discussionmentioning

confidence: 99%

Zebularine-induced reduction in VEGF secretion by HIF-1α degradation in oral squamous cell carcinoma

Suzuki

Shinohara

Rikiishi³

2008

Mol Med Report

View full text Add to dashboard Cite

Abstract. Vascular endothelial growth factor (VEGF) is a potent inducer of angiogenesis in oral squamous cell carcinoma (OSCC). In this study, we used the novel DNA methyltransferase inhibitor zebularine (Zeb) to investigate epigenetic influences on the secretion of VEGF-A in the OSCC cell line HSC-3. Under normoxic conditions, we found that Zeb inhibited secretion in a dose-dependent manner by reducing the activity of hypoxia-inducible factor-1α (HIF-1α). Treatment of the cells with the proteasome inhibitor MG132 protected the HIF-1α protein from Zeb-mediated epigenetic regulation. In addition, our study revealed that neither the PI3K/Akt nor the p53 signaling pathway is required for Zeb-induced HIF-1α degradation. In short, Zeb influenced the stability of the HIF-1α protein and the activity of its targets, such as VEGF, in HSC-3 cells in normoxic conditions. This study has laid the foundation for a novel anti-cancer approach, which may find applications in molecular staging.

show abstract

“…To test this hypothesis, in addition to the renal cell carcinoma cell line RCC4, we used another renal cancer cell line, UOK121, in which VHL is epigenetically silenced, along with the RCC4 + VHL and UOK121 + VHL cell lines, which were stably transfected with functional VHL. We also treated UOK121 with 5-aza-2′-deoxycytidine (5-Aza-dCyd) for 10 days to demethylate its genomic DNA and restore VHL expression [9]. All cells were assayed under normoxic (21 % oxygen) and hypoxic (1 % oxygen) conditions, and the results are shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

VHL-deficient renal cancer cells gain resistance to mitochondria-activating apoptosis inducers by activating AKT through the IGF1R-PI3K pathway

2016

View full text Add to dashboard Cite

We previously developed (2-deoxyglucose)-(ABT-263) combination therapy (2DG-ABT), which induces apoptosis by activating Bak in the mitochondria of highly glycolytic cells with varied genetic backgrounds. However, the rates of apoptosis induced by 2DG-ABT were lower in von Hippel-Lindau (VHL)-deficient cancer cells. The re-expression of VHL protein in these cells lowered IGF1R expression in a manner independent of oxygen concentration. Lowering IGF1R expression via small interfering RNA (siRNA) sensitized the cells to 2DG-ABT, suggesting that IGF1R interfered with the activation of apoptosis by the mitochondria. To determine which of the two pathways activated by IGF1R, the Ras-ERK pathway or the PI3K-AKT pathway, was involved in the impairment of mitochondria activation, the cells were treated with a specific inhibitor of either PI3K or ERK, and 2DG-ABT was added to activate the mitochondria. The apoptotic rates resulting from 2DG-ABT treatment were higher in the cells treated with the PI3K inhibitor, while the rates remained approximately the same in the cells treated with the ERK inhibitor. In 2DG-ABT-sensitive cells, a 4-h 2DG treatment caused the dissociation of Mcl-1 from Bak, while ABT treatment alone caused the dissociation of Bcl-xL from Bak without substantially reducing Mcl-1 levels. In 2DG-ABT-resistant cells, Mcl-1 dissociated from Bak only when AKT activity was inhibited during the 4-h 2DG treatment. Thus, in VHL-deficient cells, IGF1R activated AKT and stabilized the Bak-Mcl-1 complex, thereby conferring cell resistance to apoptosis.Electronic supplementary materialThe online version of this article (doi:10.1007/s13277-016-5260-2) contains supplementary material, which is available to authorized users.

show abstract

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

Cited by 47 publications

References 53 publications

Aberrant Promoter Methylation of the ABCG2 Gene in Renal Carcinoma

Aberrant Promoter Methylation of the ABCG2 Gene in Renal Carcinoma

Zebularine-induced reduction in VEGF secretion by HIF-1α degradation in oral squamous cell carcinoma

VHL-deficient renal cancer cells gain resistance to mitochondria-activating apoptosis inducers by activating AKT through the IGF1R-PI3K pathway

Contact Info

Product

Resources

About