Steady-state levels of human DNA mismatch repair (MMR) transcripts and proteins were measured in MMR-proficient and -deficient cell lines by the newly developed competitive quantitative reverse transcription-polymerase chain reaction and Western analysis normalized with purified proteins. In MMR-proficient cells, hMSH2 is the most abundant MMR protein and is expressed 3 to 5 times more than hMLH1. The hMLH1 protein was expressed 1.5 to 2.5 times more than hPMS2. Steady-state levels of mRNA expression correlated well with protein expression. hMSH2-mutated LoVo cells did not express detectable hMSH3 or hMSH6 proteins. Similarly, hMLH1-mutated HCT116 cells did not express detectable hMLH1 or hPMS2 protein, whereas in hMLH1-restored HCT116؉ch3 cells, hPMS2 protein was reexpressed. In hMSH6-mutated HCT15 cells, both hMSH3 protein and mRNA were increased. In SV40-transformed lung fibroblasts, all MMR mRNAs and proteins examined were expressed at levels 1.5-5-fold higher than in their nontransformed counterpart. The steady-state levels of MMR proteins indicate that substantially more hMutS proteins, which are involved in DNA mismatch recognition, are present in comparison with the hMutL proteins. Stability of hMSH3 and hMSH6 proteins appears to depend upon the presence of the hMSH2 protein, and, similarly, the stability of the hPMS2 protein depends upon hMLH1. When the hMSH6 is mutationally inactivated, hMSH3 increases by both transcriptional up-regulation and enhanced protein stability. A balanced up-regulation of all of the components was seen after viral transformation in a fibroblast model. Quantitative changes of the MMR components are a potential mechanism to modify the DNA MMR capabilities of a cell. The human mismatch repair (MMR)1 system ensures replication fidelity by correcting postreplication errors that have escaped the DNA proofreading function of DNA polymerase. Defects in the MMR system result in the development of a genetically unstable mutator phenotype and render the cell more susceptible to neoplastic transformation (reviewed in Refs. 1-3). The MMR system functions through the interactions between several proteins, such as hMSH2, hMSH3, hMSH6, hMLH1, hPMS2, and recently discovered hMLH3 (1-4). hMutS-␣ is a heterodimer of hMSH2 and hMSH6 that binds to mismatched nucleotides or single insertion/deletion loops (5-7). In addition, hMSH2 can dimerize with hMSH3 creating the hMutS-␤ complex, which binds larger DNA insertion/deletion mispaired loops (5,7,8). Subsequently, activated hMutS-␣ or hMutS-␤ interacts with hMutL-␣ (a heterodimer of hMLH1 with hPMS2) to direct DNA repair (9, 10).Recent studies suggest that maintaining an appropriate balance of the components of the human MMR system is critical for its proper repair function. In hMSH6-defective HCT15 cells, hMSH3 protein is highly expressed, whereas in hMSH2-defective LoVo cells, hMSH3 and hMSH6 proteins are not detected (11). The suggested cause for the absence of the hMSH3 and hMSH6 proteins is that they are relatively unstable without forming a h...

Oxidative stress inactivates the human DNA mismatch repair system. Am J Physiol Cell Physiol 283: C148-C154, 2002; 10.1152/ajpcell.00422.2001.-In the human DNA mismatch repair (MMR) system, hMSH2 forms the hMutS␣ and hMutS␤ complexes with hMSH6 and hMSH3, respectively, whereas hMLH1 and hPMS2 form the hMutL␣ heterodimer. These complexes, together with other components in the MMR system, correct single-base mismatches and small insertion/deletion loops that occur during DNA replication. Microsatellite instability (MSI) occurs when the loops in DNA microsatellites are not corrected because of a malfunctioning MMR system. Low-frequency MSI (MSI-L) is seen in some chronically inflamed tissues in the absence of genetic inactivation of the MMR system. We hypothesize that oxidative stress associated with chronic inflammation might damage protein components of the MMR system, leading to its functional inactivation. In this study, we demonstrate that noncytotoxic levels of H2O2 inactivate both single-base mismatch and loop repair activities of the MMR system in a dose-dependent fashion. On the basis of in vitro complementation assays using recombinant MMR proteins, we show that this inactivation is most likely due to oxidative damage to hMutS␣, hMutS␤, and hMutL␣ protein complexes. We speculate that inactivation of the MMR function in response to oxidative stress may be responsible for the MSI-L seen in nonneoplastic and cancer tissues associated with chronic inflammation.

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JC virus (JCV), along with other members of the polyomavirus family, encodes a class of highly conserved proteins, T antigens, that are capable of inducing aneuploidy in cultured cells. We have previously isolated T-antigen DNA variants of JCV from both colon cancer tissues and the corresponding nonneoplastic gastrointestinal tissues, raising new questions about the role of JCV in the development of chromosomal instability of the colon. Based on the sequence of the transcriptional control region (TCR), JCV can be classified as archetype or tandem repeat variants. Among the latter, Mad-1, the prototype virus first isolated from a patient with progressive multifocal leukoencephalopathy, is characterized by lacking the 23-and 66-bp sequences that are present in the archetype and by duplication of a 98-bp sequence. In this study, we evaluated differences in the TCR of JCV isolated from colon cancer tissues and nonneoplastic epithelium. To characterize JCV variants, we first treated eight pairs of DNA samples from colon cancers and noncancerous tissue with topoisomerase I and then amplified and cloned the JCV TCR. We obtained 285 recombinant clones from the JCV TCR, 157 from nonneoplastic samples, and 128 from colon cancer tissues. Of these clones, 262 spanned the length of the JCV Mad-1 TCR: 99.3% from nonneoplastic samples and 82.8% from colon cancer tissues. In sequencing 54 clones in both directions, we did not find archetype JCV either in the nonneoplastic tissue or in the cancer samples. From all colon cancer tissues, 18 clones had a deletion of one 98-bp tandem repeat. This deleted strain was not detected in any of the nonneoplastic tissues (14 versus 0% [ 2 ‫؍‬ 23.6; P < 0.001]). Our study demonstrates that the only JCV strain present in the human colon is Mad-1, and the variant with a single 98-bp sequence is found exclusively in the cancer tissues. This strain may be involved in the development of chromosomal instability.The human polyomavirus JC virus (JCV) infects a large proportion of the population worldwide (23). The initial infection is unapparent, but subsequently JCV establishes a latent infection, becoming reactivated when the immune system is impaired (8). The JCV genome is double-stranded, negatively supercoiled circular DNA that consists of early and late coding regions separated by two noncoding regions at both the 5Ј and 3Ј ends. Within the noncoding sequences is the transcriptional control region (TCR), which contains the promoter and enhancer for the early and late proteins; the intergenic region separates the 3Ј regions of T antigen and the capsid protein, VP1.T antigen is an oncogenic protein capable of transforming mammalian cells by interacting with cellular proteins such as p53 and the Rb family proteins (5, 21). T antigen also has ATPase and helicase activities, the latter of which may eventually contribute to chromosomal breakage and recombination (7). We have recently reported the presence of JCV T-antigen DNA sequences in nonneoplastic gastrointestinal tissues, colon cancer samples,...

Frame-shift mutations at microsatellites occur as a time-dependent function of polymerase errors followed by failure of postreplicational mismatch repair. A cell-culture system was developed that allows identification of intermediate mutant cells that carry the mutation on a single DNA strand after the initial DNA polymerase errors. A plasmid was constructed that contained 13 repeats of a poly(dC-dA)⅐poly(dGdT) oligonucleotide immediately after the translation initiation codon of the enhanced GFP (EGFP) gene, shifting the EGFP gene out of its proper reading frame. The plasmid was introduced into human mismatch repair-deficient (HCT116, hMLH1-mutated) and mismatch repair-proficient (HCT116؉chr3, hMLH1 wild type) colorectal cancer cells. After frame-shift mutations occurred that restored the EGFP reading frame, EGFP-expressing cells were detected, and two distinct fluorescent populations, M1 (dim cells) and M2 (bright cells), were identified. M1 cell numbers were stable, whereas M2 cells accumulated over time. In HCT116, single M2 cells gave rise to fluorescent colonies that carried a 2-bp deletion at the (CA)13 microsatellite. Twenty-eight percent of single M1 cells, however, gave rise to colonies with a mixed fluorescence pattern that carried both (CA)13 and (CA)12 microsatellites. It is likely that M1 cells represent intermediate mutants that carry (CA)13⅐(GT)12 heteroduplexes. Although the mutation rate in HCT116 cell clones (6.2 ؋ 10 ؊4 ) was 30 times higher than in HCT116؉chr3 (1.9 ؋ 10 ؊5 ), the proportion of M1 cells in culture did not significantly differ between HCT116 (5.87 ؋ 10 ؊3 ) and HCT116؉chr3 (4.13 ؋ 10 ؊3 ), indicating that the generation of intermediate mutants is not affected by mismatch-repair proficiency. mismatch repair ͉ frame-shift mutation ͉ microsatellite instability ͉ mutation rate

Purpose: Notch signaling has been implicated to play a critical role in the tumorigenesis of neuroblastoma (NB) and can modulate calreticulin (CRT) expression that strongly correlates with tumor differentiation and favorable prognosis of NB. We thus sought to determine how Notch regulates CRT expression and affects NB tumor behavior.Experimental Design: The Notch-dependent regulation of CRT expression in cultured NB cells was analyzed by confocal microscopy and Western blotting. Notch1 protein expression in 85 NB tumors was examined by immunohistochemistry and correlated with the clinicopathologic/biological characters of NB patients. The progression of NB tumors in response to attenuated Notch signaling was examined by using a xenograft mouse model.Results: We showed that CRT is essential for the neuronal differentiation of NB cells elicited by inhibition of Notch signaling. This effect was mediated by a c-Jun-NH 2 -kinase-dependent pathway. Furthermore, NB tumors with elevated Notch1 protein expression were strongly correlated with advanced tumor stages, MYCN amplification, an undifferentiated histology, as well as a low CRT expression level. Most importantly, the opposing effect between Notch1 and CRT could reciprocally affect the survival of NB patients. The administration of a γ-secretase inhibitor into a xenograft mouse model of NB significantly suppressed the tumor progression.Conclusions: Our findings provide the first evidence that a c-Jun-NH 2 -kinase-CRT-dependent pathway is essential for the neuronal differentiation elicited by Notch signaling blockade and that Notch1 and CRT can synergistically predict the clinical outcomes of NB patients. The present data suggest that Notch signaling could be a therapeutic target for NB.

Nucleotide changes in the nifH gene of Klebsiella pneumoniae were identified by DNA cloning and sequencing of six selected mutant strains. The strains were UN60, C-640-GC----TGC; UN116, C-67-TC----TTC; UN117, G-688-AG----AAG; UN1041, CG-302-C----CAC; UN1678, GC-713-C----GTC; and UN1795, G-439-AG----AAG. Their corresponding amino acid substitutions were UN60, Arg-214----Cys; UN116, Leu-23----Phe; UN117, Glu-230----Lys; UN1041, Arg-101----His; UN1678, Ala-238----Val; and UN1795, Glu-147----Lys. Results from Western and Northern blots of the mutant strains showed significant reductions in both steady-state levels of the accumulated Fe protein and nifH mRNA during derepression in the presence of serine. The relative specific activities of the nitrogenases in strains UN60, UN1041, and UN1795 were less than 2% of the wild type, whereas those in UN116, UN117, and UN1678 were between 28 and 40% of the wild type during enhanced derepression with serine. The residues of Arg-101 (UN1041), Glu-147 (UN1795), and Arg-214 (UN60) were invariant in sequences of a dozen diazotrophs that have been examined thus far. In UN1041, in which Arg-101 of the Fe protein was replaced by His, the Fe protein had a larger apparent molecular weight than that of the other strains on sodium dodecyl sulfate-gel electrophoresis, as detected with rabbit antiserum raised against the C-terminal peptide of the wild-type Fe protein. The reduced levels of nifH mRNA in point mutant strains suggests that nifH (the gene or gene product) may be involved in self-regulation. mRNA transcripts of different sizes were detected when a nifH-specific probe, CCKp2003, was used in the Northern blot hybridization.