Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.21 (CPT)
 4,580 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This study provides evidence for the importance of p21(CDKN1A) for the repair of replication-mediated DNA double-strand breaks (DSBs) induced by topoisomerase I. We report that defects of p21(CDKN1A) and p53 enhance camptothecin-induced histone H2AX phosphorylation (gammaH2AX), a marker for DNA DSBs. In human colon carcinoma HCT116 cells with wild-type (wt) p53, gammaH2AX reverses after camptothecin removal. By contrast, gammaH2AX increases after camptothecin removal in HCT116 cells deficient for p53 (p53-/-) or p21(CDKN1A) (p21-/-) as the cells reach the late-S and G2 phases. Since p21-/- cells exhibit similar S-phase arrest as wt cells in response to camptothecin and aphidicolin does not abrogate the enhanced gammaH2AX formation in p21-/- cells, we conclude that enhanced gammaH2AX formation in p21-/- cells is not due to re-replication. The cell cycle checkpoint abrogator and Chk1/Chk2 inhibitor 7-hydroxystaurosporine (UCN-01) also increases camptothecin-induced gammaH2AX formation and inhibits camptothecin-induced p21(CDKN1A) upregulation in HCT116 wt cells. TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling) assays demonstrate that gammaH2AX formation in late S and G2 cells following CPT treatment corresponds to DNA breaks. However, these breaks are not related to apoptotic DNA fragmentation. We propose that p21(CDKN1A) prevents the collapse of replication forks damaged by stabilized topoisomerase I cleavage complexes.
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PMID:p21CDKN1A allows the repair of replication-mediated DNA double-strand breaks induced by topoisomerase I and is inactivated by the checkpoint kinase inhibitor 7-hydroxystaurosporine. 1640 43

We examined the benefit of gene expression analysis on peripheral blood cellular subsets of different radiosensitivity to elucidate their utility as biodosimeters for estimation of dose in irradiated individuals. Peripheral mononucleated cells were isolated from 18 healthy volunteers employing density separation in a CPT-NH tube. Peripheral mononucleated cells were cultured in RPMI 1640 medium containing 10% autologous serum and were irradiated with 0.1-1 Gy (240 kV, 13 mA, X rays at 1 Gy/min). A low-dose study was performed with isolated peripheral mononucleated cells from one healthy donor in three independent experiments. Peripheral mononucleated cells were irradiated at 0 (sham), 1, 2.5 and 5 cGy (70 kV, 13 mA X rays at 1 cGy/min) and gene expression was measured 24 and 48 h after irradiation. After irradiation, CD4(+) or CD8(+) cells were isolated by magnetic beads in independent experiments. RNA from lymphocyte subsets and peripheral mononucleated cells was isolated after 24 and 48 h and converted into cDNA. Gene expression of GADD45, CDKN1A, DDB2, PCNA, BAX and ATF3 were determined using RTQ-PCR. Data were analyzed employing linear and logistic regression analysis. The same examinations were performed in 5 individuals either diagnosed using CT scans (up to 4.3 cGy) or by administering (F-18)-fluoro-2-deoxy-d-glucose (F-18 FDG, 0.6 cGy). Methodological, intra- and inter-individual variability in 90-95% of measurements did not exceed the introduced twofold change over sham-irradiated control values in peripheral mononucleated cells and CD4(+) cells, and therefore no false positive results were observed. Dose reconstruction in peripheral mononucleated cells in opposite to CD4(+) lymphocytes required fewer genes and appeared more efficient (R-square = 84.8% compared to 51.8%). In vitro samples exposed to 10 cGy could be completely discriminated from sham-irradiated samples without individual pre-exposure controls, which coincided with our preliminary in vivo results. However, in vitro differential gene expression was measured relative to control values and did not differ significantly at 24 and 48 h after irradiation in contrast to our preliminary in vivo data. In addition, below 5 cGy in vitro data did not show reproducible significant changes in gene expression, which was opposite to our preliminary in vivo data. Therefore a twofold change in gene expression over control sufficiently controls for different sources of variance, and measuring gene expression in peripheral mononucleated cell for biological dosimetry purposes appears superior over measurements in lymphocyte subsets. The increased gene expression measured after low absorbed doses in vivo and in vitro might indicate a particular applicability of this method for a low-level radiation scenario in the absence of individual pre-exposure controls. However, the constant gene expression values measured up to 48 h in our in vitro model at doses >10 cGy, and the absence of reproducible and statistically significant gene expression changes below 5 cGy contrast to the preliminary in vivo results performed at similar doses. Therefore, measurements with our in vitro models should be interpreted cautiously.
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PMID:Gene expression comparisons performed for biodosimetry purposes on in vitro peripheral blood cellular subsets and irradiated individuals. 2276 26