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Promoter methylation-mediated silencing is a hallmark of many established tumor suppressor genes. This review focuses on the methylation and suppression of a receptor-type tyrosine phosphatase gene, PTPRO, in a variety of solid and liquid tumors. In addition, PTPRO exhibits many other characteristics of a bona fide tumor suppressor. Reactivation of genes silenced by methylation using inhibitors of DNA methyltransferases and histone deacetylases, and the potential application of PTPRO as a molecular target for cancer therapy have been discussed.Cancer Gene Therapy (2005) 12, 665–672. doi:10.1038/sj.cgt.7700828 Published online 1 April 2005 [ABSTRACT FROM AUTHOR]

A diet lacking folic acid and choline and low in methionine (folate/methyl deficient diet, FMD diet) fed to rats is known to produce preneoplastic nodules (PNNs) after 36 weeks and hepatocellular carcinomas (tumors) after 54 weeks. FMD diet-induced tumors exhibit global hypomethylation and regional hypermethylation. Restriction landmark genome scanning analysis with methylation-sensitive enzyme NotI (RLGS-M) of genomic DNA isolated from control livers, PNNs and tumor tissues was performed to identify the genes that are differentially methylated or amplified during multistage hepatocarcinogenesis. Out of the 1250 genes analysed, 2 to 5 genes were methylated in the PNNs, whereas 5 to 45 genes were partially or completely methylated in the tumors. This analysis also showed amplification of 3 to 12 genes in the primary tumors. As a first step towards identifying the genes methylated in the PNNs and primary hepatomas, we generated a rat NotI-EcoRV genomic library in the pBluescriptKS vector. Here, we describe identification of one methylated and downregulated gene as the rat protein tyrosine phosphatase receptor type O (PTPRO) and one amplified gene as rat C-MYC. Methylation of PTPRO at the NotI site located immediate upstream of the trancription start site in the PNNs and tumors, and amplification of C-MYC gene in the tumors were confirmed by Southern blot analyses. Bisulfite genomic sequencing of the CpG island encompassing exon 1 of the PTPRO gene revealed dense methylation in the PNNs and tumors, whereas it was methylation free in the livers of animals on normal diet. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed significant decrease in the expression of PTPRO in the tumors and in a transplanted rat hepatoma. The expression of PTPRO mRNA in the transplanted hepatoma after demethylation with 5-azacytidine, a potent inhibitor of DNA methyltransferases, further confirmed the role of methylation in PTPRO gene expression. These results demonstrate alteration in methylation profile and expression of specific genes during tumor progression in the livers of rats in response to folate/methyl deficiency, and further implicate the potential role of PTPRO as a novel growth regulatory gene at least in the hepatocellular carcinomas.Oncogene (2003) 22, 6319-6331. doi:10.1038/sj.onc.1206750 [ABSTRACT FROM AUTHOR]

Metallothionein-I (MT-I) gene is silenced by methylation of CpG islands in mouse lymphosarcoma P1798 cells but not in the thymus, the cell type from which the tumor was derived. Bisulfite genomic sequencing revealed that all 21 CpG dinucleotides present within -216 bp to +1 bp with respect to transcription start site are methylated in the tumor cell line, but none is methylated in the thymus. The lymphosarcoma cells induced MT-I in response to heavy metals only after demethylation with 5-azacytidine (5-AsaC). The electrophoretic mobility shift assay using specific oligonucleotide probes showed that the key transcription factors regulating MT-I gene (e.g., MTF-1, Sp 1 and MLTF/USF) are active in P1798 cells. In vivo footprinting of the proximal promoter region showed that none of the metal regulatory elements (MREs) or MLTF/USF are occupied in response to heavy metals. Demethylation of the lymphosarcoma cells with 5-AzaC resulted in constitutive footprinting at MLTF/ARE, and zinc-inducible footprinting at MRE-c, MRE-d and MRE-e sites. Demethylation of just 10 – 20% of the CpG islands was sufficient to render the gene inducible by cadmium or zinc. The MT-I induction persisted in the cancer cells for several generations even after withdrawal of 5-AzaC from the culture medium. [ABSTRACT FROM AUTHOR]

We have previously purified and characterized a rat liver protein C′BP-1 that is either identical or closely related to C/EBPδ (). The mouse metallothionein-I (MT-I) promoter contains two C′BP-1 binding sites, one of which includes the MRE-c′ region (-135 to -110). The C′BP-1 binding activity was detected by EMSA as a major activity for MRE-c′ in nonproliferating adult liver cells but not in rat hepatoma cells. ln this study, we purified and characterized a factor, C′BP-2, which had a dominant binding activity for MRE-c′ in Morris hepatoma 3924A, a poorly differentiated, fast-growing tissue. C′BP-2 is a 28 kDa protein which exists in solution as a monomer. As observed for C′BP-1, affinity-purified C′BP-2 stimulated transcription from the mMT-I gene promoter. DNase I footprinting revealed two C′BP-2 binding sites in the regions that overlap with the CCAAT homologies of the C′BP-1 binding sites on the mMT-I promoter. C′BP-2 made essential contacts with the CCAAT homology and in the region upstream of this sequence. Competition electrophoretic mobility shift assay and methylation interference analysis revealed that C′BP-2 is a protein closely related, but not identical, to CP2. These data suggest that C′BP-1 and C′BP-2 may play a role in hepatocyte proliferation and/or differentiation. [ABSTRACT FROM AUTHOR]

We have previously demonstrated that the core promoter of rat ribosomal RNA gene (rDNA) contains an E-box-like sequence to which the core promoter binding factor CPBF binds and that the 44 kDa subunit of this protein is immunologically related to USF1, the helix – loop – helix-zipper DNA binding protein. Further, we showed that RNA polymerase I (pol I) transcription in vitro is competed by oligonucleotides containing USF-binding site, which suggested a key role for USF in rDNA transcription. To prove the potential role of USF in pol I transcription in vivo, USF1 and USF2 homodimers and USF1/USF2 heterodimer were overexpressed in CHO cells by transfection of the respective cDNAs. Co-transfection of a plasmid containing rDNA followed by primer extension analysis showed that overexpression of USF1 and USF2 as homodimers resulted in inhibition of rDNA transcription by as much as 85 – 90% whereas overexpression of USF1/USF2 in the heterodimeric form activated transcription approximately 3.5-fold. Transfection of mutant USF2 cDNA that is devoid of the basic DNA-binding domain produced only minimal inhibition of rDNA transcription. These data show that USF can modulate transcription of rRNA gene in vivo by functioning as a repressor (homodimer) or activator (heterodimer) of pol I transcription in vivo and suggest that inhibition of rDNA transcription may be responsible for the antiproliferative action of USF homodimers. [ABSTRACT FROM AUTHOR]