Two Distinct E2F Transcriptional Modules Drive Cell Cycles and Differentiation.

Orchestrating cell-cycle-dependent mRNA oscillations is critical to cell proliferation in multicellular organisms. Even though our understanding of cell-cycle-regulated transcription has improved significantly over the last three decades, the mechanisms remain untested in vivo. Unbiased transcriptomic profiling of G0, G1-S, and S-G2-M sorted cells from FUCCI mouse embryos suggested a central role for E2Fs in the control of cell-cycle-dependent gene expression. The analysis of gene expression and E2F-tagged knockin mice with tissue imaging and deep-learning tools suggested that post-transcriptional mechanisms universally coordinate the nuclear accumulation of E2F activators (E2F3A) and canonical (E2F4) and atypical (E2F8) repressors during the cell cycle in vivo. In summary, we mapped the spatiotemporal expression of sentinel E2F activators and canonical and atypical repressors at the single-cell level in vivo and propose that two distinct E2F modules relay the control of gene expression in cells actively cycling (E2F3A-8-4) and exiting the cycle (E2F3A-4) during mammalian development.

PCNA damage caused by antineoplastic drugs.

Structurally diverse chemotherapeutic and chemopreventive drugs, including camptothecin, doxorubicin, sanguinarine, and others, were found to cause covalent crosslinking of proliferating cell nuclear antigen (PCNA) trimers in mammalian cells exposed to fluorescent light. This PCNA damage was caused by both nuclear and cytoplasmically localizing drugs. For some drugs, the PCNA crosslinking was evident even with very brief exposures to laboratory room lighting. In the absence of drugs, there was no detectable covalent crosslinking of PCNA trimers. Other proteins were photo-crosslinked to PCNA at much lower levels, including crosslinking of additional PCNA to the PCNA trimer. The proteins photo-crosslinked to PCNA did not vary with cell type or drug. PCNA was not crosslinked to itself or to other proteins by superoxide, hydrogen peroxide or hydroxyl radicals, but hydrogen peroxide caused monoubiquitination of PCNA. Quenching of PCNA photo-crosslinking by histidine, and enhancement by deuterium oxide, suggest a role for singlet oxygen in the crosslinking. SV40 large T antigen hexamers were also efficiently covalently photo-crosslinked by drugs and light. Photodynamic crosslinking of nuclear proteins by cytoplasmically localizing drugs, together with other evidence, argues that these drugs may reach the nucleoplasm in amounts sufficient to photodamage important chromosomal enzymes. The covalent crosslinking of PCNA trimers provides an extremely sensitive biomarker for photodynamic damage. The damage to PCNA and large T antigen raises the possibility that DNA damage signaling and repair mechanisms may be compromised when cells treated with antineoplastic drugs are exposed to visible light.

Overcoming resistance to interferon-induced apoptosis of renal carcinoma and melanoma cells by DNA demethylation.

Epigenetic editing of gene expression by aberrant methylation of DNA may help tumor cells escape attack from the innate and acquired immune systems. Resistance to antiproliferative effects and apoptosis induction by interferons (IFNs) was postulated to result from silencing of IFN response genes by promoter hypermethylation. Treatment of human ACHN renal cell carcinoma (RCC) and A375 melanoma cells with the DNA demethylating nucleoside analog 5-AZA-2'-deoxycytidine (5-AZA-dC) synergistically augmented antiproliferative effects of IFN- alpha (alpha) 2 and IFN-beta (beta). Either 5-AZA-dC or an antisense to DNA methyltransferase 1 (DNMT1) overcame resistance to apoptosis induction by IFNs with up to 85% apoptotic cells resulting from the combinations. No similar potentiation occurred in normal kidney epithelial cells. IFN response genes were augmented more than 10 times in expression by 5-AZA-dC. Demethylation by 5-AZA-dC of the promoter of the prototypic, apoptosis-associated IFN response gene XAF1 was confirmed by methylation-specific polymerase chain reaction. siRNA to XAF1 inhibited IFN-induced apoptosis; conversely, overexpression of XAF1 overcame resistance to apoptosis induction by IFN-beta. As occurred with apoptosis-resistant melanoma cells in vitro, tumor growth inhibition in the nude mouse of human A375 melanoma xenografts resulted from treatment with 5-AZA-dC in combination with IFN-beta, an effect not resulting from either single agent. The importance of epigenetic remodeling of expression of immune-modifying genes in tumor cells was further suggested by identifying reactivation of the cancer-testis antigens MAGE and RAGE in ACHN cells after DNMT1 depletion. Thus, inhibitors of DNMT1 may have clinical relevance for immune modulation by augmentation of cytokine effects and/or expression of tumor-associated antigens.

Expression of RASSF1A, an epigenetically silenced tumor suppressor, overcomes resistance to apoptosis induction by interferons.

Resistance of human renal cell carcinoma (RCC) and melanoma to the apoptosis-inducing effects of IFNs was postulated to result from epigenetic silencing of genes by DNA methylation, a common feature of human cancers. To reverse silencing, 5-AZA-deoxycytidine (5-AZA-dC) or selective depletion of DNA methyltransferase 1 (DNMT1) by phosphorothioate oligonucleotide antisense (DNMT1 AS) were employed in cells resistant (<5% terminal deoxynucleotidyl transferase-mediated nick-end labeling positive) to apoptosis induction by IFN-alpha2 and IFN-beta (ACHN, SK-RC-45, and A375). 5-AZA-dC and DNMT1 AS similarly depleted available DNMT1 protein and, at doses that did not cause apoptosis alone, resulted in apoptotic response to IFNs. The proapoptotic tumor suppressor RASSF1A was reactivated by DNMT1 inhibitors in all three cell lines. This was associated with demethylation of its promoter region. IFNs augmented RASSF1A protein expression after reactivation by DNMT1 inhibition. In IFN-sensitive WM9 melanoma cells, expression of RASSF1A was constitutive but also augmented by IFNs. RASSF1A small interfering RNA reduced IFN-induced apoptosis in WM9 cells and in DNMT1-depleted ACHN cells. Conversely, lentiviral expression of RASSF1A but not transduction with empty virus enabled IFN-induced apoptosis. IFN induced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and TRAIL-neutralizing antibody inhibited apoptotic response to IFN in RASSF1A-expressing ACHN cells. Accordingly, RASSF1A markedly sensitized to recombinant TRAIL. Normal kidney epithelial cells, although expressing RASSF1A, did not undergo apoptosis in response to IFN or TRAIL but had >400-fold higher TRAIL decoy receptor 1 expression than transduced ACHN cells (real-time reverse transcription-PCR). Results identified RASSF1A as regulated by IFNs and participating in IFN-induced apoptosis at least in part by sensitization to TRAIL.

Silencing and CpG island methylation of GSTP1 is rare in ordinary gastric carcinomas but common in Epstein-Barr virus-associated gastric carcinomas.

BACKGROUND: The GSTPI gene encodes for glutathione S-transferase pi (GST-pi), which protects cells from cytotoxic agents. The carcinogenic role of this enzyme is at issue because functional polymorphisms have been shown to be a risk factor of human cancer. Moreover, GST-pi protein loss has frequently been reported in various human cancers. MATERIALS AND METHODS: The expression of GST-pi and the methylation status of the promoter area of GST-pi were investigated in gastric carcinomas. Eleven human SNUgastric cancer cell lines, PC-3 prostate cancer cell lines, various cancer tissues and normal gastric mucosa tissues were analyzed by immunohistochemistry, in situ hybridization, Western blot and methylation specific PCR. RESULTS: Only 22 (2.0%o) out of 1081 cases showed loss of GST-pi expression. Interestingly, 16 out of 22 GST-pi-negative cases were Epstein-Barr virus (EBV)-associated gastric carcinomas. The loss of expression of GST-pi among EBV-associated gastric carcinomas was found to be 27.1% (16/59), but to be 0.6% (6/1022) in EBV-negative gastric carcinomas (p < 0.001). Eight out of 16 cases with loss of GST-pi expression showed CpG island methylation in the GSTP1 promoter region, while none of the normal gastric mucosa or EBV-negative gastric carcinomas showed methylation (p < 0.001). CONCLUSION: These findings demonstrate that the loss of GST-pi expression is clustered in a subset of gastric carcinomas with EBV incorporation, and that the methylation of the promoter of the GSTP1 gene is correlated with this loss of GST-pi expression. Our results suggest that GST-pi abrogation by CpG island hypermethylation may account for EBV-associated gastric carcinoma.

Loss of caspase-1 gene expression in human gastric carcinomas and cell lines.

The caspases are a family of aspartic acid-specific proteases that fulfill varied and often critical roles in mammalian apoptosis or in the proteolytic activation of cytokines. Caspase-1 (interleukin-1beta-converting enzyme) is a member of the cysteine protease family, which cleaves target proteins following aspartic acid residues. We investigated caspase-1 expression in stomach cancer, tissues and cell lines. Of 301 consecutive gastric carcinomas, 58 cases (19.3%) showed the expressional loss of caspase-1. Loss of caspase-1 expression was significantly associated with pTNM stage (p=0.03), lymph node metastasis (p=0.01) and patient survival (p<0.01). Caspase-1 expression was also significantly correlated in an inverse manner with p53 expression (p<0.01). Among the 11 gastric cancer cell lines examined, three cell lines showed loss of expression at the protein and mRNA levels. On treatment with 5-aza-2'-deoxycytidine (5-aza-C), and/or trichostatin A (TSA), all three cell lines re-expressed caspase-1 mRNA. The above findings suggest that epigenetic events such as DNA methylation and histone deacetylation play important roles in the regulation of caspase-1, and that loss of caspase-1 expression is associated with poor survival in gastric carcinoma.

Manganese superoxide dismutase expression correlates with chemosensitivity in human gastric cancer cell lines.

PURPOSE: The purpose is to investigate the potential correlation between antioxidant enzyme (AOE) levels and resistance to anticancer drugs in human gastric carcinoma cell lines. EXPERIMENTAL DESIGN: Protein contents of AOEs such as manganese superoxide dismutase (MnSOD), copper/zinc superoxide dismutase, catalase, glutathione S-transferase-pi, p-glycoprotein, and multidrug resistance-associated protein were observed by Western blot analysis, and MnSOD activity was measured in six Korean gastric cancer cell lines. The direct correlation between AOEs and the chemosensitivity to doxorubicin (DOX), mitomycin C, 5-fluorouracil, and vinblastine was analyzed by cytotoxicity test. MnSOD was overexpressed by transient transfection of human MnSOD cDNA. RESULTS: Expressions of AOEs in gastric cancer cell lines were variable. MnSOD expression was related with the resistance to DOX and mitomycin C but not with that to 5-fluorouracil and vinblastine. In comparison, expressions of other AOEs, p-glycoprotein and multidrug resistance-associated protein, were not correlated with tumor sensitivity to any of the drugs used. Cell lines with a high MnSOD protein content showed higher MnSOD activity than those with a low MnSOD protein content. In addition, MnSOD overexpression increased the resistance of gastric carcinoma cells to DOX. CONCLUSIONS: MnSOD is an important factor of drug response to reactive oxygen species-generating anticancer drugs in the gastric cancer cells. Thus, measurement of MnSOD levels in clinical samples may provide an indication of subsequent treatment response of gastric cancer patients.

Alteration of O6-methylguanine-DNA methyltransferase in colorectal neoplasms in sporadic and familial adenomatous polyposis patients.

DNA repair failure is known to be a critical event during carcinogenesis of colorectal cancers. To investigate whether O(6)-methylguanine-DNA methyltransferase (MGMT) is altered during colorectal carcinogenesis, we performed immunohistochemical staining on 265 sporadic colorectal cancers, 113 sporadic adenomas, 33 familial adenomatous polyposis (FAP) colorectal cancers, and 93 FAP adenomas. Sixty-seven of 265 sporadic colorectal cancer cases and five of 113 sporadic adenoma cases showed loss of MGMT expression (P < 0.001). Among FAP patients, four of 33 cancers and six of 93 adenomas showed loss of MGMT protein expression. When we compared the association between MGMT promoter hypermethylation and protein expression, almost all cases without a methylated allele were positive for the expression of MGMT. In contrast, cases with promoter methylation frequently showed loss of MGMT expression (P < 0.01). Loss of MGMT was correlated with some clinicopathological characteristics, i.e., tumor invasion (P = 0.013) and stage (P = 0.035) in sporadic colorectal cancer, and degree of atypism (P = 0.042) in sporadic adenoma. Our results show that loss of expression of MGMT occurs more frequently in cancer than in adenoma in both sporadic and FAP patients, and that loss of expression of MGMT is associated with hypermethylation of the promoter area of MGMT gene.