Biomedical subjects

Dennis A Simpson

Publications and source records attributed to Dennis A Simpson.

9 recordsLinked to original sources

PubMed2006

Radiation clastogenesis and cell cycle checkpoint function as functional markers of breast cancer risk.

BACKGROUND: Familial breast cancer is associated with mutations in several genes (BRCA1, BRCA2, p53, ATM) whose protein products protect against radiation-induced genotoxicity. This study tested whether sporadic breast cancer was associated with constitutive radiation hypersensitivity. METHODS: Blood lymphocytes and EBV-transformed lymphoblasts from patients with newly diagnosed breast cancer and controls without cancer were evaluated for ionizing radiation (IR)-induced chromosomal aberrations and cell cycle delays. Lymphoblasts from patients with ataxia telangiectasia (AT) and heterozygous AT carriers were tested as positive controls for radiation hypersensitivity. RESULTS: Lymphoblasts from AT patients and AT carriers displayed G2-irradiation, chromosomal hypersensitivity (GICH). Irradiated G2 phase lymphocytes from breast cancer cases and controls displayed 3-fold inter-individual variation in frequencies of chromatid damage. However, the percentage of breast cancer cases with damage frequencies in excess of 2 SD of the control mean (8/102 or 8%) was not significantly elevated compared to controls (2/48 or 4%, P=0.5). Lymphoblasts sampled 24 h after 3 Gy of IR also varied in the ratios of cells with 4N and 2N DNA content (4N/2N ratio), as a measure of cell cycle checkpoint function. 4N/2N ratios in irradiated lymphoblasts were strongly correlated with the fractions of S phase cells in un-irradiated control cultures (Pearson's correlation coefficient, r=0.87). After normalization to S fraction, the radiation-induced increment in the 4N/2N ratio was significantly elevated in AT lymphoblasts but not in lymphoblasts from AT carriers. The fraction of breast cancer cases with reduced checkpoint function (2/45 or 4%) was equal to the control fraction (2/45 or 4%). For breast cancer cases and controls, GICH in primary lymphocytes was not associated with reduced cell cycle checkpoint function in lymphoblasts. CONCLUSION: Constitutive radiation hypersensitivity in blood lymphocytes and lymphoblasts was not a useful biomarker for identifying women at increased risk of breast cancer.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2006

Profiles of global gene expression in ionizing-radiation-damaged human diploid fibroblasts reveal synchronization behind the G1 checkpoint in a G0-like state of quiescence.

Cell cycle arrest and stereotypic transcriptional responses to DNA damage induced by ionizing radiation (IR) were quantified in telomerase-expressing human diploid fibroblasts. Analysis of cytotoxicity demonstrated that 1.5 Gy IR inactivated colony formation by 40-45% in three fibroblast lines; this dose was used in all subsequent analyses. Fibroblasts exhibited > 90% arrest of progression from G2 to M at 2 hr post-IR and a similarly severe arrest of progression from G1 to S at 6 and 12 hr post-IR. Normal rates of DNA synthesis and mitosis 6 and 12 hr post-IR caused the S and M compartments to empty by > 70% at 24 hr. Global gene expression was analyzed in IR-treated cells. A microarray analysis algorithm, EPIG, identified nine IR-responsive patterns of gene expression that were common to the three fibroblast lines, including a dominant p53-dependent G1 checkpoint response. Many p53 target genes, such as CDKN1A, GADD45, BTG2, and PLK3, were significantly up-regulated at 2 hr post-IR. Many genes whose expression is regulated by E2F family transcription factors, including CDK2, CCNE1, CDC6, CDC2, MCM2, were significantly down-regulated at 24 hr post-IR. Numerous genes that participate in DNA metabolism were also markedly repressed in arrested fibroblasts apparently as a result of cell synchronization behind the G1 checkpoint. However, cluster and principal component analyses of gene expression revealed a profile 24 hr post-IR with similarity to that of G0 growth quiescence. The results reveal a highly stereotypic pattern of response to IR in human diploid fibroblasts that reflects primarily synchronization behind the G1 checkpoint but with prominent induction of additional markers of G0 quiescence such as GAS1.

Cell Line↗

PubMed2005

Telomerase expression is sufficient for chromosomal integrity in cells lacking p53 dependent G1 checkpoint function.

BACKGROUND: Secondary cultures of human fibroblasts display a finite lifespan ending at senescence. Loss of p53 function by mutation or viral oncogene expression bypasses senescence, allowing cell division to continue for an additional 10-20 doublings. During this time chromosomal aberrations seen in mitotic cells increase while DNA damage and decatenation checkpoint functions in G2 cells decrease. METHODS: To explore this complex interplay between chromosomal instability and checkpoint dysfunction, human fibroblast lines were derived that expressed HPV16E6 oncoprotein or dominant-negative alleles of p53 (A143V and H179Q) with or without the catalytic subunit of telomerase. RESULTS: Cells with normal p53 function displayed 86-93% G1 arrest after exposure to 1.5 Gy ionizing radiation (IR). Expression of HPV16E6 or p53-H179Q severely attenuated G1 checkpoint function (3-20% arrest) while p53-A143V expression induced intermediate attenuation (55-57% arrest) irrespective of telomerase expression. All cell lines, regardless of telomerase expression or p53 status, exhibited a normal DNA damage G2 checkpoint response following exposure to 1.5 Gy IR prior to the senescence checkpoint. As telomerase-negative cells bypassed senescence, the frequencies of chromosomal aberrations increased generally congruent with attenuation of G2 checkpoint function. Telomerase expression allowed cells with defective p53 function to grow >175 doublings without chromosomal aberrations or attenuation of G2 checkpoint function. CONCLUSION: Thus, chromosomal instability in cells with defective p53 function appears to depend upon telomere erosion not loss of the DNA damage induced G1 checkpoint.

Journal Article↗

PubMed2005

Overproduction of DNA polymerase eta does not raise the spontaneous mutation rate in diploid human fibroblasts.

Telomerase-immortalized lines of diploid xeroderma pigmentosum variant (XP-V) fibroblasts (XP115LO and XP4BE) were complemented for constitutive or regulated expression of wild-type human DNA polymerase eta (hpol eta). The ectopic gene was expressed from a retroviral LTR at a population average of 34- to 59-fold above the endogenous (mutated) mRNA and high levels of hpol eta were detected by immunoblotting. The POLH cDNA was also cloned downstream from an ecdysone-regulated promoter and transduced into the same recipient cells. Abundance of the wild-type mRNA increased approximately 10-fold by addition of ponasterone to the culture medium. Complemented cell lines acquired normal resistance to the cytotoxic effects of UVC, even in the presence of 1mM caffeine. They also tolerated higher levels of UVC-induced template lesions during nascent DNA elongation when compared to normal fibroblasts (NHF). UVC-induced mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus were measured in the XP115LO+XPV cell line overproducing hpol eta constitutively (E. Bassett, N.M. King, M.F. Bryant, S. Hector, L. Pendyala, S.G. Chaney, M. Cordeiro-Stone, The role of DNA polymerase eta in translesion synthesis past platinum-DNA adducts in human fibroblasts, Cancer Res. 64 (2004) 6469-6475). Induced mutation frequencies were significantly reduced, even below those observed in NHF; however, the average mutation frequency in untreated cultures was about three-fold higher than in the isogenic vector-control cell line. In this study, spontaneous HPRT mutation frequencies were measured at regular intervals, as isogenic fibroblasts either lacking or overproducing hpol eta were expanded for 100 population doublings. The mutation rates estimated from these results were not significantly increased in XP115LO cells expressing abnormal levels of hpol eta, relative to the cells lacking this specialized polymerase. These findings suggest that diploid human fibroblasts with normal DNA repair capacities and intact checkpoints are well protected against the potential mutagenic outcome of overproducing hpol eta, while still benefiting from accurate translesion synthesis of UV-induced pyrimidine dimers.

Blotting, Western↗

PubMed2003

Cell cycle checkpoint function in bladder cancer.

BACKGROUND: Cell cycle checkpoints function to maintain genetic stability by providing additional time for repair of DNA damage and completion of events that are necessary for accurate cell division. Some checkpoints, such as the DNA damage G1 checkpoint, are dependent on p53, whereas other checkpoints, such as the decatenation G(2) checkpoint, are not. Because bladder transitional cell carcinomas (TCCs) often contain numerous chromosomal aberrations and appear to have highly unstable genomes, we analyzed cell cycle checkpoint functions in a panel of TCC lines. METHODS: Cell cycle arrest was induced in normal human fibroblasts (NHF1-hTERT) and normal human uroepithelial cells (HUCs), and TCC lines and checkpoint functions were quantified using flow cytometry and fluorescence microscopy. The inducers and checkpoints were ionizing radiation (i.e., DNA damage) (G1 and G2 checkpoints), the mitotic inhibitor colcemid (polyploidy checkpoint), or the topoisomerase II catalytic inhibitor ICRF-193 (decatenation G2 checkpoint). Four of the five TCC lines expressed mutant p53. RESULTS: HUCs had an effective G1 checkpoint response to ionizing radiation, with 68% of cells inhibited from moving from G1 into S phase. By contrast, G1 checkpoint function was severely attenuated (<15% inhibition) in three of the five TCC lines and moderately attenuated (<50% inhibition) in the other two lines. NHF1-hTERT had an effective polyploidy checkpoint response, but three of five TCC lines were defective in this checkpoint. HUCs had effective ionizing radiation and decatenation G2 checkpoint responses. All TCC lines had a relatively effective G2 checkpoint response to DNA damage, although the responses of two of the TCC lines were moderately attenuated relative to HUCs. All TCC lines had a severe defect in the decatenation G2 checkpoint response. CONCLUSION: Bladder TCC lines have defective cell cycle checkpoint functions, suggesting that the p53-independent decatenation G2 checkpoint may cooperate with the p53-dependent G1 checkpoints to preserve chromosomal stability and suppress bladder carcinogenesis.

Carcinoma, Transitional Cell↗

PubMed2003

Caffeine and human DNA metabolism: the magic and the mystery.

The ability of caffeine to reverse cell cycle checkpoint function and enhance genotoxicity after DNA damage was examined in telomerase-expressing human fibroblasts. Caffeine reversed the ATM-dependent S and G2 checkpoint responses to DNA damage induced by ionizing radiation (IR), as well as the ATR- and Chk1-dependent S checkpoint response to ultraviolet radiation (UVC). Remarkably, under conditions in which IR-induced G2 delay was reversed by caffeine, IR-induced G1 arrest was not. Incubation in caffeine did not increase the percentage of cells entering the S phase 6-8h after irradiation; ATM-dependent phosphorylation of p53 and transactivation of p21(Cip1/Waf1) post-IR were resistant to caffeine. Caffeine alone induced a concentration- and time-dependent inhibition of DNA synthesis. It inhibited the entry of human fibroblasts into S phase by 70-80% regardless of the presence or absence of wildtype ATM or p53. Caffeine also enhanced the inhibition of cell proliferation induced by UVC in XP variant fibroblasts. This effect was reversed by expression of DNA polymerase eta, indicating that translesion synthesis of UVC-induced pyrimidine dimers by DNA pol eta protects human fibroblasts against UVC genotoxic effects even when other DNA repair functions are compromised by caffeine.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2003

An attenuating mutation in nsP1 of the Sindbis-group virus S.A.AR86 accelerates nonstructural protein processing and up-regulates viral 26S RNA synthesis.

The Sindbis-group alphavirus S.A.AR86 encodes a threonine at nonstructural protein 1 (nsP1) 538 that is associated with neurovirulence in adult mice. Mutation of the nsP1 538 Thr to the consensus Ile found in nonneurovirulent Sindbis-group alphaviruses attenuates S.A.AR86 for adult mouse neurovirulence, while introduction of Thr at position 538 in a nonneurovirulent Sindbis virus background confers increased neurovirulence (M. T. Heise et al., J. Virol. 74:4207-4213, 2000). Since changes in the viral nonstructural region are likely to affect viral replication, studies were performed to evaluate the effect of Thr or Ile at nsP1 538 on viral growth, nonstructural protein processing, and RNA synthesis. Multistep growth curves in Neuro2A and BHK-21 cells revealed that the attenuated s51 (nsP1 538 Ile) virus had a slight, but reproducible growth advantage over the wild-type s55 (nsP1 538 Thr) virus. nsP1 538 lies within the cleavage recognition domain between nsP1 and nsP2, and the presence of the attenuating Ile at nsP1 538 accelerated the processing of S.A.AR86 nonstructural proteins both in vitro and in infected cells. Since nonstructural protein processing is known to regulate alphavirus RNA synthesis, experiments were performed to evaluate the effect of Ile or Thr at nsP1 538 on viral RNA synthesis. A combination of S.A.AR86-derived reporter assays and RNase protection assays determined that the presence of Ile at nsP1 538 led to earlier expression from the viral 26S promoter without affecting viral minus- or plus-strand synthesis. These results suggest that slower nonstructural protein processing and delayed 26S RNA synthesis in wild-type S.A.AR86 infections may contribute to the adult mouse neurovirulence phenotype of S.A.AR86.

Animals↗

PubMed2002

An ATR- and Chk1-dependent S checkpoint inhibits replicon initiation following UVC-induced DNA damage.

Inhibition of replicon initiation is a stereotypic DNA damage response mediated through S checkpoint mechanisms not yet fully understood. Studies were undertaken to elucidate the function of checkpoint proteins in the inhibition of replicon initiation following irradiation with 254 nm UV light (UVC) of diploid human fibroblasts immortalized by the ectopic expression of telomerase. Velocity sedimentation analysis of nascent DNA molecules revealed a 50% inhibition of replicon initiation when normal human fibroblasts were treated with a low dose of UVC (1 J/m(2)). Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and AT-like disorder fibroblasts, which lack an S checkpoint response when exposed to ionizing radiation, responded normally when exposed to UVC and inhibited replicon initiation. Pretreatment of normal and AT fibroblasts with caffeine or UCN-01, inhibitors of ATR (AT mutated and Rad3 related) and Chk1, respectively, abolished the S checkpoint response to UVC. Moreover, overexpression of kinase-inactive ATR in U2OS cells severely attenuated UVC-induced Chk1 phosphorylation and reversed the UVC-induced inhibition of replicon initiation, as did overexpression of kinase-inactive Chk1. Taken together, these data suggest that the UVC-induced S checkpoint response of inhibition of replicon initiation is mediated by ATR signaling through Chk-1 and is independent of ATM, Nbs1, and Mre11.

Ataxia Telangiectasia Mutated Proteins↗

PubMedDate not supplied

Degradation of ATM-independent decatenation checkpoint function in human cells is secondary to inactivation of p53 and correlated with chromosomal destabilization.

DNA topoisomerase II is required in the cell cycle to decatenate intertwined daughter chromatids prior to mitosis. To study the mechanisms that cells use to accomplish timely chromatid decatenation, the activity of a catenation-responsive checkpoint was monitored in human skin fibroblasts with inherited or acquired defects in the DNA damage G2 checkpoint. G2 delay was quantified shortly after a brief incubation with ICRF-193, which blocks the ability of topoisomerase II to decatenate chromatids, or treatment with ionizing radiation (IR), which damages DNA. Both treatments induced G2 delay in normal human fibroblasts. Ataxia telangiectasia fibroblasts with defective G2 checkpoint response to IR displayed normal G2 delay after treatment with ICRF-193, demonstrating that ATM kinase was not required for signaling when chromatid decatenation was blocked. The G2 delay induced by ICRF-193 was reversed by caffeine, indicating that active checkpoint signaling was involved. ICRF-193-induced G2 delay also was independent of p53 function, being evident in cells expressing HPV16E6 to inactivate p53. However, as fibroblasts expressing HPV16E6 aged in culture, they lost the ability to delay entry to mitosis, both after DNA damage and when decatenation was blocked. This age-related loss of G2 delay in response to ICRF-193 and IR in E6-expressing cells was blocked by induction of telomerase. Expression of telomerase also prevented chromosomal destabilization in aging E6-expressing cells. These observations lead to a new model of genetic instability, in which attenuation of G2 decatenatory checkpoint function permits cells to enter mitosis with insufficiently decatenated chromatids, leading to aneuploidy and polyploidy.

Ataxia Telangiectasia Mutated Proteins↗