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1.  MSH3-Deficiency Initiates EMAST without Oncogenic Transformation of Human Colon Epithelial Cells 
PLoS ONE  2012;7(11):e50541.
Elevated microsatellite instability at selected tetranucleotide repeats (EMAST) is a genetic signature in certain cases of sporadic colorectal cancer and has been linked to MSH3-deficiency. It is currently controversial whether EMAST is associated with oncogenic properties in humans, specifically as cancer development in Msh3-deficient mice is not enhanced. However, a mutator phenotype is different between species as the genetic positions of repetitive sequences are not conserved. Here we studied the molecular effects of human MSH3-deficiency.
HCT116 and HCT116+chr3 (both MSH3-deficient) and primary human colon epithelial cells (HCEC, MSH3-wildtype) were stably transfected with an EGFP-based reporter plasmid for the detection of frameshift mutations within an [AAAG]17 repeat. MSH3 was silenced by shRNA and changes in protein expression were analyzed by shotgun proteomics. Colony forming assay was used to determine oncogenic transformation and double strand breaks (DSBs) were assessed by Comet assay.
Despite differential MLH1 expression, both HCT116 and HCT116+chr3 cells displayed comparable high mutation rates (about 4×10−4) at [AAAG]17 repeats. Silencing of MSH3 in HCECs leads to a remarkable increased frameshift mutations in [AAAG]17 repeats whereas [CA]13 repeats were less affected. Upon MSH3-silencing, significant changes in the expression of 202 proteins were detected. Pathway analysis revealed overexpression of proteins involved in double strand break repair (MRE11 and RAD50), apoptosis, L1 recycling, and repression of proteins involved in metabolism, tRNA aminoacylation, and gene expression. MSH3-silencing did not induce oncogenic transformation and DSBs increased 2-fold.
MSH3-deficiency in human colon epithelial cells results in EMAST, formation of DSBs and significant changes of the proteome but lacks oncogenic transformation. Thus, MSH3-deficiency alone is unlikely to drive human colon carcinogenesis.
PMCID: PMC3507781  PMID: 23209772
2.  PARP inhibition potentiates the cytotoxic activity of C-1305, a selective inhibitor of topoisomerase II, in human BRCA1-positive breast cancer cells☆ 
Biochemical Pharmacology  2012;84(10):1318-1331.
Graphical abstract
Two cellular proteins encoded by the breast and ovarian cancer type 1 susceptibility (BRCA1 and BRCA2) tumor suppressor genes are essential for DNA integrity and the maintenance of genomic stability. Approximately 5–10% of breast and ovarian cancers result from inherited alterations or mutations in these genes.
Remarkably, BRCA1/BRCA2-deficient cells are hypersensitive to selective inhibition of poly(ADP-ribose)polymerase 1 (PARP-1), whose primary functions are related to DNA base excision repair; PARP-1 inhibition significantly potentiates the cytotoxicity of various anti-cancer drugs, including inhibitors of topoisomerase I and II.
In the present study, we examined the anti-proliferative and pro-apoptotic effects of C-1305, a selective inhibitor of topoisomerase II, on human breast cancer cell lines with different BRCA1 and p53 statuses. BRCA1-competent breast cancer cell lines exhibited different responses to topoisomerase II inhibition. BT-20 cells that express high levels of BRCA1 levels were most resistant to C-1305 than other tested cells. Surprisingly, pharmacological interference with PARP-1 activity strongly inhibited their proliferation and potentiated the efficacy of C-1305 treatment. In contrast, PARP-1 inhibition only weakly affected the proliferation of BRCA1-deficient SKBr-3 cells and was not synergistic with the effects of C-1305. Further experiments revealed that the inhibition of PARP-1 in BT-20 cells caused the accumulation of DNA strand breaks and induced caspase-3 dependent apoptosis. These results seem to indicate that PARP-1 inhibition can potentiate the cytotoxicity of anti-cancer drugs in cancer cells with functional BRCA1 and suggest that mutations in other DNA repair proteins may render cancer cells more sensitive to interference with PARP-1 activity.
PMCID: PMC3494830  PMID: 22906755
BER, base excision repair; BRCA1, breast cancer type 1 susceptibility protein; HMC, Hoffman modulation contrast; HR, homologous recombination; NER, nucleotide excision repair; NHEJ, non-homologous end joining; MMP, mitochondrial membrane potential; PARP-1, poly(ADP-ribose)polymerase-1; PD, Petri dish; PVDF, polyvinylidene difluoride; TOPO, topoisomerase; WCL, whole cell lysate; WT, wild-type; DNA strand breaks; Caspase-3 activation; Apoptosis; Cell cycle; G2 arrest; Potential of mitochondrial membrane
3.  Inhalative Exposure to Vanadium Pentoxide Causes DNA Damage in Workers: Results of a Multiple End Point Study 
Environmental Health Perspectives  2008;116(12):1689-1693.
Inhalative exposure to vanadium pentoxide (V2O5) causes lung cancer in rodents.
The aim of the study was to investigate the impact of V2O5 on DNA stability in workers from a V2O5 factory.
We determined DNA strand breaks in leukocytes of 52 workers and controls using the alkaline comet assay. We also investigated different parameters of chromosomal instability in lymphocytes of 23 workers and 24 controls using the cytokinesis-block micronucleus (MN) cytome method.
Seven of eight biomarkers were increased in blood cells of the workers, and vanadium plasma concentrations in plasma were 7-fold higher than in the controls (0.31 μg/L). We observed no difference in DNA migration under standard conditions, but we found increased tail lengths due to formation of oxidized purines (7%) and pyrimidines (30%) with lesion-specific enzymes (formamidopyrimidine glycosylase and endonuclease III) in the workers. Bleomycin-induced DNA migration was higher in the exposed group (25%), whereas the repair of bleomycin-induced lesions was reduced. Workers had a 2.5-fold higher MN frequency, and nucleoplasmic bridges (NPBs) and nuclear buds (Nbuds) were increased 7-fold and 3-fold, respectively. Also, apoptosis and necrosis rates were higher, but only the latter parameter reached statistical significance.
V2O5 causes oxidation of DNA bases, affects DNA repair, and induces formation of MNs, NPBs, and Nbuds in blood cells, suggesting that the workers are at increased risk for cancer and other diseases that are related to DNA instability.
PMCID: PMC2599764  PMID: 19079721
comet assay; cytokinesis-block micronucleus assay; DNA damage; occupational exposure; vanadium pentoxide

Results 1-3 (3)