miRNA expression in colon polyps provides evidence for a multihit model of colon cancer

Changes in miRNA expression are a common feature in colon cancer. Those changes occurring in the transition from normal to adenoma and from adenoma to carcinoma, however, have not been well defined. Additionally, miRNA changes among tumor subgroups of colon cancer have also not been adequately evaluated. In this study, we examined the global miRNA expression in 315 samples that included 52 normal colonic mucosa, 41 tubulovillous adenomas, 158 adenocarcinomas with proficient DNA mismatch repair (pMMR) selected for stage and age of onset, and 64 adenocarcinomas with defective DNA mismatch repair (dMMR) selected for sporadic (n = 53) and inherited colon cancer (n = 11). Sporadic dMMR tumors all had MLH1 inactivation due to promoter hypermethylation. Unsupervised PCA and cluster analysis demonstrated that normal colon tissue, adenomas, pMMR carcinomas and dMMR carcinomas were all clearly discernable. The majority of miRNAs that were differentially expressed between normal and polyp were also differentially expressed with a similar magnitude in the comparison of normal to both the pMMR and dMMR tumor groups, suggesting a stepwise progression for transformation from normal colon to carcinoma. Among the miRNAs demonstrating the largest fold up- or down-regulated changes (≥4), four novel (miR-31, miR-1, miR-9 and miR-99a) and two previously reported (miR-137 and miR-135b) miRNAs were identified in the normal/adenoma comparison. All but one of these (miR-99a) demonstrated similar expression differences in the two normal/carcinoma comparisons, suggesting that these early tumor changes are important in both the pMMR- and dMMR-derived cancers. The comparison between pMMR and dMMR tumors identified four miRNAs (miR-31, miR-552, miR-592 and miR-224) with statistically significant expression differences (≥2-fold change).     

Mismatch repair

Specific repair systems are activated in response to the DNA damage. Mismatch repair protects the genome of prokaryotic and eukaryotic cells from lesions that appear during process of DNA replication or are induced by mutagenic factors. The methyl directed mismatch repair distinguishes the new strand from the old strand by the hemi-methylated state of the DNA and controls the fidelity of genetic information after homologous recombination. The very short patch repair restores the mismatches at the sites with nucleotide sequence CC(W/T)GG. The "8-oxoG" pathway is independent of the hemi-methylated state of the DNA, and removes the oxidated nucleotides from the genome of prokaryotes and eukaryotes. Mutations in genes of mismatch repair enhance the process of mutagenesis in prokaryotic cell, and are the reason for the development of the colon cancer in humans. The mechanisms of mismatch repair and the role of defective repair proteins in mutagenesis and carcinogenesis are discussed in this review.     

Different mechanisms underlie DNA instability in Huntington disease and colorectal cancer.

Two recent lines of evidence raise the possibility that instability in germ-line or somatic cells arises by a common mechanism that involves defective mismatch repair. Mutations in mismatch-repair proteins are known to cause instability in hereditary nonpolyposis colorectal cancer, instability that is physically similar to germ-line instability observed in Huntington disease (HD). Furthermore, both germ-line and somatic-cell instability are likely to be mitotic defects, the former occurring early in embryogenesis. To test the hypothesis that defective repair is a common prerequisite for instability, we have utilized two disease groups that represent different instability "conditions." Germ-line instability within simple tandem repeats (STR) at 10 loci in 29 HD families were compared with somatic instability at the same loci in 26 colon cancer (CC) patients with identified or suspected defects in mismatch-repair enzymes. HD is known to be caused by expansion within the CAG repeat of the locus, but the extent or pattern of STR instability outside this region has not been examined systematically. We find a distinctly different pattern of STR mutation in the two disease groups, suggesting different mechanisms. Instability in HD is generally confined to a single locus, whereas instability is widespread for the same loci in CC. Our data do not support a causative role for defective mismatch-repair enzymes in instability associated with HD; rather, our data are consistent with a model in which DNA structure may inhibit normal mismatch repair at the expansion site.     

Antimutagenicity of cinnamaldehyde and vanillin in human cells: Global gene expression and possible role of DNA damage and repair

Vanillin (VAN) and cinnamaldehyde (CIN) are dietary flavorings that exhibit antimutagenic activity against mutagen-induced and spontaneous mutations in bacteria. Although these compounds were antimutagenic against chromosomal mutations in mammalian cells, they have not been studied for antimutagenesis against spontaneous gene mutations in mammalian cells. Thus, we initiated studies with VAN and CIN in human mismatch repair-deficient (hMLH1(-)) HCT116 colon cancer cells, which exhibit high spontaneous mutation rates (mutations/cell/generation) at the HPRT locus, permitting analysis of antimutagenic effects of agents against spontaneous mutation. Long-term (1-3 weeks) treatment of HCT116 cells with VAN at minimally toxic concentrations (0.5-2.5mM) reduced the spontaneous HPRT mutant fraction (MF, mutants/10(6) survivors) in a concentration-related manner by 19-73%. A similar treatment with CIN at 2.5-7.5microM yielded a 13-56% reduction of the spontaneous MF. Short-term (4-h) treatments also reduced the spontaneous MF by 64% (VAN) and 31% (CIN). To investigate the mechanisms of antimutagenesis, we evaluated the ability of VAN and CIN to induce DNA damage (comet assay) and to alter global gene expression (Affymetrix GeneChip) after 4-h treatments. Both VAN and CIN induced DNA damage in both mismatch repair-proficient (HCT116+chr3) and deficient (HCT116) cells at concentrations that were antimutagenic in HCT116 cells. There were 64 genes whose expression was changed similarly by both VAN and CIN; these included genes related to DNA damage, stress responses, oxidative damage, apoptosis, and cell growth. RT-PCR results paralleled the Affymetrix results for four selected genes (HMOX1, DDIT4, GCLM, and CLK4). Our results show for the first time that VAN and CIN are antimutagenic against spontaneous mutations in mammalian (human) cells. These and other data lead us to propose that VAN and CIN may induce DNA damage that elicits recombinational DNA repair, which reduces spontaneous mutations.     

DNA mismatch repair and Lynch syndrome

The evolutionary conserved mismatch repair proteins correct a wide range of DNA replication errors. Their importance as guardians of genetic integrity is reflected by the tremendous decrease of replication fidelity (two to three orders of magnitude) conferred by their loss. Germline mutations in mismatch repair genes, predominantly MSH2 and MLH1, have been found to underlie the Lynch syndrome (also called hereditary non-polyposis colorectal cancer, HNPCC), a hereditary predisposition for cancer. Lynch syndrome affects predominantly the colon and accounts for 2–5% of all colon cancer cases. During more than 30 years of biochemical, crystallographic and clinical research, deep insight has been achieved in the function of mismatch repair and the diseases that are associated with its loss. We review the biochemistry of mismatch repair and also introduce the clinical, diagnostic and genetic aspects of Lynch syndrome.     

Checkpoint signaling, base excision repair, and PARP promote survival of colon cancer cells treated with 5-fluorodeoxyuridine but not 5-fluorouracil

The fluoropyrimidines 5-fluorouracil (5-FU) and FdUrd (5-fluorodeoxyuridine; floxuridine) are the backbone of chemotherapy regimens for colon cancer and other tumors. Despite their widespread use, it remains unclear how these agents kill tumor cells. Here, we have analyzed the checkpoint and DNA repair pathways that affect colon tumor responses to 5-FU and FdUrd. These studies demonstrate that both FdUrd and 5-FU activate the ATR and ATM checkpoint signaling pathways, indicating that they cause genotoxic damage. Notably, however, depletion of ATM or ATR does not sensitize colon cancer cells to 5-FU, whereas these checkpoint pathways promote the survival of cells treated with FdUrd, suggesting that FdUrd exerts cytotoxicity by disrupting DNA replication and/or inducing DNA damage, whereas 5-FU does not. We also found that disabling the base excision (BER) repair pathway by depleting XRCC1 or APE1 sensitized colon cancer cells to FdUrd but not 5-FU. Consistent with a role for the BER pathway, we show that small molecule poly(ADP-ribose) polymerase 1/2 (PARP) inhibitors, AZD2281 and ABT-888, remarkably sensitized both mismatch repair (MMR)-proficient and -deficient colon cancer cell lines to FdUrd but not to 5-FU. Taken together, these studies demonstrate that the roles of genotoxin-induced checkpoint signaling and DNA repair differ significantly for these agents and also suggest a novel approach to colon cancer therapy in which FdUrd is combined with a small molecule PARP inhibitor.     

Effects of the lncRNA ENST00000623984 on colon cancer and the biological characteristics of colon cancer cells

The aim of this study was to explore the effects of the lncRNA ENST00000623984 on colorectal cancer. In this study, the expression levels of ENST000000623984 were first examined in tumor tissue and adjacent normal tissue from 40 patients with colorectal cancer and LoVo cells using quantitative real-time PCR. By siRNA transfection, ENST00000623984 expression was knocked down. Using flow cytometry, cell cycle progression and cell viability were examined in basal and knockdown LoVo cells. The CCK-8 assay was used to assess the cell proliferation rate, and the Transwell assay was used to determine the migration and invasion abilities. The ENST000000623984 expression level was increased in colorectal cancer. Knockdown of ENST000000623984 reduced cell viability, proliferation rate, cell migration and invasion. These results suggested that lncRNA ENST000000623984 may be involved in colorectal cancer development.Key words: LncRNA, ENST00000623984, colorectal cancer, expression, PCR     

Molecular cloning and chromosomal localization of DNA sequences associated with a human DNA repair gene.

The genes and gene products involved in the mammalian DNA repair processes have yet to be identified. Toward this end we made use of a number of DNA repair-proficient transformants that were generated after transfection of DNA from repair-proficient human cells into a mutant hamster line that is defective in the initial incision step of the excision repair process. In this report, biochemical evidence is presented that demonstrates that these transformants are repair proficient. In addition, we describe the molecular identification and cloning of unique DNA sequences closely associated with the transfected human DNA repair gene and demonstrate the presence of homologous DNA sequences in human cells and in the repair-proficient DNA transformants. The chromosomal location of these sequences was determined by using a panel of rodent-human somatic cell hybrids. Both unique DNA sequences were found to be on human chromosome 19.     

Short-patch correction of C/C mismatches in human cells

We examined whether the human nucleotide excision repair complex, which is specialized on the removal of bulky DNA adducts, also displays a correcting activity on base mismatches. The cytosine/cytosine (C/C) lesion was used as a model substrate to monitor the correction of base mismatches in human cells. Fibroblasts with different repair capabilities were transfected with shuttle vectors that contain a site-directed C/C mismatch in the replication origin, accompanied by an additional C/C mismatch in one of the flanking sequences that are not essential for replication. Analysis of the vector progeny obtained from these doubly modified substrates revealed that C/C mismatches were eliminated before DNA synthesis not only in the repair-proficient background, but also when the target cells carried a genetic defect in long-patch mismatch repair, in nucleotide excision repair, or when both pathways were deleted. Furthermore, cells deficient for long-patch mismatch repair as well as a cell line that combines mismatch and nucleotide excision repair defects were able to correct multiple C/C mispairs, placed at distances of 21–44 nt, in an independent manner, such that the removal of each lesion led to individual repair patches. These results support the existence of a concurrent short-patch mechanism that rectifies C/C mismatches.     

Mismatch repair proteins are activators of toxic responses to chromium-DNA damage

Chromium(VI) is a toxic and carcinogenic metal that causes the formation of DNA phosphate-based adducts. Cr-DNA adducts are genotoxic in human cells, although they do not block replication in vitro. Here, we report that induction of cytotoxicity in Cr(VI)-treated human colon cells and mouse embryonic fibroblasts requires the presence of all major mismatch repair (MMR) proteins. Cr-DNA adducts lost their ability to block replication of Cr-modified plasmids in human colon cells lacking MLH1 protein. The presence of functional mismatch repair caused induction of p53-independent apoptosis associated with activation of caspases 2 and 7. Processing of Cr-DNA damage by mismatch repair resulted in the extensive formation of gamma-H2AX foci in G(2) phase, indicating generation of double-stranded breaks as secondary toxic lesions. Induction of gamma-H2AX foci was observed at 6 to 12 h postexposure, which was followed by activation of apoptosis in the absence of significant G(2) arrest. Our results demonstrate that mismatch repair system triggers toxic responses to Cr-DNA backbone modifications through stress mechanisms that are significantly different from those for other forms of DNA damage. Selection for Cr(VI) resistant, MMR-deficient cells may explain the very high frequency of lung cancers with microsatellite instability among chromate workers.     

RecB-dependent mutator phenotype in Neisseria meningitidis strains naturally defective in mismatch repair

Several invasive serogroup B meningococcal strains phylogenetically related to the lineage III (ET-24) exhibited a mutator phenotype as shown by mutagenicity assay using rifampicin-resistance as a selection marker. Hypermutation was associated to the presence of defective mutL alleles that were genetically characterized. Interestingly, the mutator phenotype was suppressed when a non-functional recB(ET-37) allele, derived from ET-37 meningococcal strains, replaced the functional recB allele in a lineage III strain. In contrast, the same gene replacement did not affect mutation frequencies in a mismatch repair-proficient strain. These results suggested that in MutL-deficient strains spontaneous mutations mostly arise from post-replicative DNA synthesis associated to the activity of the RecBCD recombination pathway.     

Dimerization of MLH1 and PMS2 limits nuclear localization of MutLalpha

DNA mismatch repair maintains genomic stability by detecting and correcting mispaired DNA sequences and by signaling cell death when DNA repair fails. The mechanism by which mismatch repair coordinates DNA damage and repair with cell survival or death is not understood, but it suggests the need for regulation. Since the functions of mismatch repair are initiated in the nucleus, we asked whether nuclear transport of MLH1 and PMS2 is limiting for the nuclear localization of MutLalpha (the MLH1-PMS2 dimer). We found that MLH1 and PMS2 have functional nuclear localization signals (NLS) and nuclear export sequences, yet nuclear import depended on their C-terminal dimerization to form MutLalpha. Our studies are consistent with the idea that dimerization of MLH1 and PMS2 regulates nuclear import by unmasking the NLS. Limited nuclear localization of MutLalpha may thus represent a novel mechanism by which cells fine-tune mismatch repair functions. This mechanism may have implications in the pathogenesis of hereditary non-polyposis colon cancer.     

Comparative study of the antimutagenic potential of Vitamin E in different E. coli strains

The antimutagenic potential of Vitamin E due to its antioxidative properties was studied. The new Escherichia coli K12 assay-system designed in our laboratory was employed in order to detect the antimutagenic potential of Vitamin E and to determine its molecular mechanisms of action. The assay is composed of three tests. In Test A, we examine the influence of the antioxidant on induced oxidative mutagenesis in a repair-proficient strain. Spontaneous mutagenesis is monitored in Test B, which is performed with two mutator strains, one mismatch repair-deficient (mutS) and another deficient in 8-oxo-dGTP-ase activity (mutT). In Test M, a repair-proficient strain and its mismatch repair-deficient counterpart (mutH), both carrying a plasmid with microsatellite sequences, are used to measure the level of microsatellite instability. To examine the antimutagenic potential of Vitamin E we also used the WP2 antimutagenicity test. Protective properties of Vitamin E against oxidative mutagenesis were detected in all tests with the E. coli K12 assay-system as well as in the WP2 antimutagenicity test. This study confirms that mismatch repair is essential for repair of oxidative DNA damage. The results obtained indicate that Vitamin E prevents the formation of DNA adducts by lipid peroxidation products rather than those formed by direct oxidation of DNA bases. Moreover, it can reduce microsatellite instability. After further validation, the new E. coli K12 assay-system can be used to test the antimutagenic potential of antioxidants.     

Transformation depending on intermolecular homologous recombination is stimulated by UV damage in transfected DNA

DNA-mediated gene transfer (DMGT) was performed in DNA repair-proficient and UV-hypersensitive, repair-deficient Chinese hamster ovary (CHO) cell lines using the UV-irradiated thymidine kinase gene from herpes simplex virus (HSV-TK). Transformation frequencies in repair-deficient CHO cell lines declined relative to repair-proficient cells with increasing UV damage in transfected DNA; approximately 3-fold higher UV fluence was required to inactivate 50% of irradiated HSV-TK plasmid molecules in repair-proficient cells. In cotransfection experiments performed with pairs of HSV-TK plasmids containing linker insertion mutations in TK coding sequences, moderate UV damage in plasmid DNA enhanced the yield of TK+ transformants resulting from homologous recombination between HSV-TK sequences up to 4-fold. These results suggest that UV damage in DNA can stimulate transformation of mammalian cells dependent on intermolecular DNA homology.     

Colon cancer cells maintain low levels of pyruvate to avoid cell death caused by inhibition of HDAC1/HDAC3

Human colon cancer cells and primary colon cancer silence the gene coding for LDH (lactate dehydrogenase)-B and up-regulate the gene coding for LDH-A, resulting in effective conversion of pyruvate into lactate. This is associated with markedly reduced levels of pyruvate in cancer cells compared with non-malignant cells. The silencing of LDH-B in cancer cells occurs via DNA methylation, with involvement of the DNMTs (DNA methyltransferases) DNMT1 and DNMT3b. Colon cancer is also associated with the expression of pyruvate kinase M2, a splice variant with low catalytic activity. We have shown recently that pyruvate is an inhibitor of HDACs (histone deacetylases). Here we show that pyruvate is a specific inhibitor of HDAC1 and HDAC3. Lactate has no effect on any of the HDACs examined. Colon cancer cells exhibit increased HDAC activity compared with non-malignant cells. HDAC1 and HDAC3 are up-regulated in colon cancer cells and in primary colon cancer, and siRNA (small interfering RNA)-mediated silencing of HDAC1 and HDAC3 in colon cancer cells induces apoptosis. Colon cancer cells silence SLC5A8, the gene coding for a Na(+)-coupled pyruvate transporter. Heterologous expression of SLC5A8 in the human colon cancer cell line SW480 leads to inhibition of HDAC activity when cultured in the presence of pyruvate. This process is associated with an increase in intracellular levels of pyruvate, increase in the acetylation status of histone H4, and enhanced cell death. These studies show that cancer cells effectively maintain low levels of pyruvate to prevent inhibition of HDAC1/HDAC3 and thereby to evade cell death.     

A role for Pms2 in the prevention of tandem CC --> TT substitutions induced by ultraviolet radiation and oxidative stress

DNA mismatch repair (MMR) is important for preventing base-pair substitutions caused by spontaneous or damage-related DNA polymerase errors. We have used a reversion assay based on mouse Aprt to investigate the role of MMR in preventing ultraviolet radiation (UV) and oxidative stress induced tandem CC --> TT base pair substitutions in cultured mammalian cells. The reversion construct used for this assay can detect both C --> T and CC --> TT mutational events. Most spontaneous mutations in Pms2-deficient cells were single C --> T substitutions (88%), with the remainder being tandem CC --> TT substitutions (12%). The percentage of tandem CC --> TT substitutions rose to 64% and 94% for Pms2-deficient cells exposed to UV and a mixture of hydrogen peroxide and metals (Cu/Fe), respectively. Exposure to hydrogen peroxide alone or metals alone did not induce the tandem substitutions, nor did treatment of the cells with the alkylating agent ethylmethane sulfonate, which induces G --> A substitutions on the opposite strand. Tandem CC --> TT substitutions were also induced by UV irradiation and the hydrogen peroxide/metal mixture in Pms2-proficient cells, but at frequencies significantly lower than those observed in the Pms2-deficient cells. We conclude that mismatch repair plays an important role in preventing tandem CC --> TT substitutions induced by certain genotoxin exposures.     

左右半结肠癌多组学分子特征差异的研究进展

结肠癌(colon cancer,CC)是一种常见的恶性肿瘤,其发病率和死亡率均占癌症前列.根据解剖学位置,CC可分为左半结肠癌(left-sided colon cancer,LCC)和右半结肠癌(right-sided colon cancer,RCC),两者在临床特征上表现出较大的差异.近些年来,随着生物学技术和...     

CC to TT mutation in the mitochondrial DNA of normal skin: relationship to ultraviolet light exposure

We have previously reported that ultraviolet (UV)-specific (CC to TT) mutations in p53 gene can be detected in normal skin. This, however, cannot be used as a cumulative marker of UV exposure, since cells with the p53 mutation acquire a clonal growth advantage. Moreover, a large skin biopsy is necessary for each assay. In order to circumvent these problems, we have measured mitochondrial (Mt) DNA mutations; there are more than 1000 copies of the Mt genome per cell, and Mt genes are not directly involved in cell growth. We have established a sensitive allele-specific polymerase chain reaction (AS-PCR) assay capable of detecting one CC to TT mutation in Mt DNA among 10(7) wild-type genes using a mismatch allele-specific primer. With this assay, we found no mutation-positive samples from internal non-exposed tissue (stomach, colon, and blood) (0/50). In contrast, 17 out of 111 skin samples were positive: the mutation frequency in positive samples was around 10(7)-10(-6) (10-100 copies of mutant in 10(8) wild-type Mt DNA). In normal skin tissue, the prevalence of positive samples was higher in those from exposed sites (13/51) than in those from less-exposed sites (1/26) (p<0.05). However, a quantitative correlation between sunlight exposure and the accumulation of mutations was not found. We conclude that the UV exposure-associated CC to TT mutation in Mt DNA can be detected in normal skin, but that further studies are required to develop this as a quantitative marker for UV exposure.