PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-3 (3)
 

Clipboard (0)
None

Select a Filter Below

Journals
Authors
more »
Year of Publication
Document Types
1.  Targeted detection of in vivo endogenous DNA base damage reveals preferential base excision repair in the transcribed strand 
Nucleic Acids Research  2011;40(1):206-219.
Endogenous DNA damage is removed mainly via base excision repair (BER), however, whether there is preferential strand repair of endogenous DNA damage is still under intense debate. We developed a highly sensitive primer-anchored DNA damage detection assay (PADDA) to map and quantify in vivo endogenous DNA damage. Using PADDA, we documented significantly higher levels of endogenous damage in Saccharomyces cerevisiae cells in stationary phase than in exponential phase. We also documented that yeast BER-defective cells have significantly higher levels of endogenous DNA damage than isogenic wild-type cells at any phase of growth. PADDA provided detailed fingerprint analysis at the single-nucleotide level, documenting for the first time that persistent endogenous nucleotide damage in CAN1 co-localizes with previously reported spontaneous CAN1 mutations. To quickly and reliably quantify endogenous strand-specific DNA damage in the constitutively expressed CAN1 gene, we used PADDA on a real-time PCR setting. We demonstrate that wild-type cells repair endogenous damage preferentially on the CAN1 transcribed strand. In contrast, yeast BER-defective cells accumulate endogenous damage preferentially on the CAN1 transcribed strand. These data provide the first direct evidence for preferential strand repair of endogenous DNA damage and documents the major role of BER in this process.
doi:10.1093/nar/gkr704
PMCID: PMC3245927  PMID: 21911361
2.  Pharmacodynamic Modeling of Anti-Cancer Activity of Tetraiodothyroacetic Acid in a Perfused Cell Culture System 
PLoS Computational Biology  2011;7(2):e1001073.
Unmodified or as a poly[lactide-co-glycolide] nanoparticle, tetraiodothyroacetic acid (tetrac) acts at the integrin αvβ3 receptor on human cancer cells to inhibit tumor cell proliferation and xenograft growth. To study in vitro the pharmacodynamics of tetrac formulations in the absence of and in conjunction with other chemotherapeutic agents, we developed a perfusion bellows cell culture system. Cells were grown on polymer flakes and exposed to various concentrations of tetrac, nano-tetrac, resveratrol, cetuximab, or a combination for up to 18 days. Cells were harvested and counted every one or two days. Both NONMEM VI and the exact Monte Carlo parametric expectation maximization algorithm in S-ADAPT were utilized for mathematical modeling. Unmodified tetrac inhibited the proliferation of cancer cells and did so with differing potency in different cell lines. The developed mechanism-based model included two effects of tetrac on different parts of the cell cycle which could be distinguished. For human breast cancer cells, modeling suggested a higher sensitivity (lower IC50) to the effect on success rate of replication than the effect on rate of growth, whereas the capacity (Imax) was larger for the effect on growth rate. Nanoparticulate tetrac (nano-tetrac), which does not enter into cells, had a higher potency and a larger anti-proliferative effect than unmodified tetrac. Fluorescence-activated cell sorting analysis of harvested cells revealed tetrac and nano-tetrac induced concentration-dependent apoptosis that was correlated with expression of pro-apoptotic proteins, such as p53, p21, PIG3 and BAD for nano-tetrac, while unmodified tetrac showed a different profile. Approximately additive anti-proliferative effects were found for the combinations of tetrac and resveratrol, tetrac and cetuximab (Erbitux), and nano-tetrac and cetuximab. Our in vitro perfusion cancer cell system together with mathematical modeling successfully described the anti-proliferative effects over time of tetrac and nano-tetrac and may be useful for dose-finding and studying the pharmacodynamics of other chemotherapeutic agents or their combinations.
Author Summary
Clinical treatment protocols for specific solid cancers have favorable response rates of 20%–25%. Cancer cells frequently become resistant to treatment. Therefore, novel anti-cancer drugs and combination regimens need to be developed. Conducting enough clinical trials to evaluate combinations of anti-cancer agents in several regimens to optimize treatment is not feasible. We showed that tetrac inhibits the growth of various cancer cell lines. Our newly developed in vitro system allowed studying the effects of tetrac over time in various human cancer cell lines. Our mathematical model could distinguish two effects of tetrac and may be used to predict effects of other than the studied dosage regimens. Human breast cancer cells were more sensitive to the effect on success of replication than the effect on growth rate, whereas the maximum possible effect was larger for the latter effect. Nanoparticulate tetrac, which does not enter into cells, had a larger effect than unmodified tetrac. The combinations of tetrac and resveratrol, tetrac and cetuximab (Erbitux), and nano-tetrac and cetuximab showed approximately additive effects. Our in vitro perfusion system together with mathematical modeling may be useful for dose-finding, translation from in vitro to animal and human studies, and studying effects of other chemotherapeutic agents or their combinations.
doi:10.1371/journal.pcbi.1001073
PMCID: PMC3033367  PMID: 21304935
3.  Cloning the human and mouse MMS19 genes and functional complementation of a yeast mms19 deletion mutant 
Nucleic Acids Research  2001;29(9):1884-1891.
The MMS19 gene of the yeast Saccharomyces cerevisiae encodes a polypeptide of unknown function which is required for both nucleotide excision repair (NER) and RNA polymerase II (RNAP II) transcription. Here we report the molecular cloning of human and mouse orthologs of the yeast MMS19 gene. Both human and Drosophila MMS19 cDNAs correct thermosensitive growth and sensitivity to killing by UV radiation in a yeast mutant deleted for the MMS19 gene, indicating functional conservation between the yeast and mammalian gene products. Alignment of the translated sequences of MMS19 from multiple eukaryotes, including mouse and human, revealed the presence of several conserved regions, including a HEAT repeat domain near the C-terminus. The presence of HEAT repeats, coupled with functional complementation of yeast mutant phenotypes by the orthologous protein from higher eukaryotes, suggests a role of Mms19 protein in the assembly of a multiprotein complex(es) required for NER and RNAP II transcription. Both the mouse and human genes are ubiquitously expressed as multiple transcripts, some of which appear to derive from alternative splicing. The ratio of different transcripts varies in several different tissue types.
PMCID: PMC37259  PMID: 11328871

Results 1-3 (3)