Search PubMed⌕ Search

Biomedical subjects

William K Kaufmann

Publications and source records attributed to William K Kaufmann.

18 recordsLinked to original sources
PubMed2006

Clonal evolution of lymphoblastoid cell lines.

Lymphoblastoid cell lines represent Epstein-Barr virus (EBV)-immortalized B lymphocytes, which are typically prepared by in vitro culture of normal blood cells. In this study, we evaluated the kinetics of clonal evolution in lymphoblastoid cell lines (LCLs) established from five different donors. Immunoglobulin heavy chain (IGH) and T-cell receptor gamma (TRG) gene rearrangements were tracked over time using PCR, while EBV clonality was tracked using Southern blot analysis of the viral terminal repeat fragment. All five cultures evolved towards monoclonal B cells within 8 weeks, while T lymphocytes disappeared over the same period. No significant association was found between the rapidity of clonal emergence and either the proliferation rate or the size of the EBV terminal repeat fragment, suggesting the random nature of clonal selection. Our results suggest that EBV-driven B lymphocytes rapidly progress from polyclonal to virtually monoclonal, which has implications for the pace at which lymphoma might evolve in vivo. In addition, our findings indicate that established lymphobastoid cell lines are not an ideal 'normal control' for human B lymphocytes because they do not represent the spectrum of polyclonal B cells found in healthy humans; instead, they primarily represent the progeny of a single B lymphocyte.

B-Lymphocytes↗
PubMed2006

Checkpoint regulation of replication dynamics in UV-irradiated human cells.

At any moment during S phase, regions of genomic DNA are in various stages of replication (i.e., initiation, chain elongation, and termination). These stages may be differentially inhibited after treatment with various carcinogens that damage DNA such as UV. We used visualization of active replication units in combed DNA fibers, in combination with quantitative analyses of the size distributions of nascent DNA, to evaluate the role of S-checkpoint proteins in UV-induced inhibition of DNA replication. When HeLa cells were exposed to a low fluence (1 J/m(2)) of 254 nm UV light (UVC), new initiation events were severely inhibited (5-6-fold reduction). A larger fluence of UVC (10 J/m(2)) resulted in stronger inhibition of the overall rate of DNA synthesis without decreasing further the frequency of replicon initiation events. Incubation of HeLa cells with caffeine and knockdown of ATR or Chk1 kinases reversed the UVC-induced inhibition of initiation of new replicons. These findings illustrate the concordance of data derived from different experimental approaches, thus strengthening the evidence that the activation of the intra-S checkpoint by UVC is dependent on the ATR and Chk1 kinases.

Ataxia Telangiectasia Mutated Proteins↗
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

Dangerous entanglements.

The promise of stem-cell therapy is long-term repair of damaged organs by transplantation. Inoculum size might be a determinant of successful repair, and efforts are being made to improve conditions for ex vivo expansion of stem cells prior to transplantation. A recent article by Damelin and colleagues raised concerns about this approach, demonstrating that stem cells lack the decatenation G2 checkpoint that preserves genetic stability. Cells that fail to disentangle chromatids prior to mitosis are prone to the types of chromosomal aberrations that are observed in cancer cells. Ex vivo expansion of stem cells might have the unintended consequence of encouraging malignant progression.

Animals↗
PubMed2006

ATM requirement in gene expression responses to ionizing radiation in human lymphoblasts and fibroblasts.

The heritable disorder ataxia telangiectasia (AT) is caused by mutations in the AT-mutated (ATM) gene with manifestations that include predisposition to lymphoproliferative cancers and hypersensitivity to ionizing radiation (IR). We investigated gene expression changes in response to IR in human lymphoblasts and fibroblasts from seven normal and seven AT-affected individuals. Both cell types displayed ATM-dependent gene expression changes after IR, with some responses shared and some responses varying with cell type and dose. Interestingly, after 5 Gy IR, lymphoblasts displayed ATM-independent responses not seen in the fibroblasts at this dose, which likely reflect signaling through ATM-related kinases, e.g., ATR, in the absence of ATM function.

Ataxia Telangiectasia↗
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↗
PubMed2005

Standardizing global gene expression analysis between laboratories and across platforms.

To facilitate collaborative research efforts between multi-investigator teams using DNA microarrays, we identified sources of error and data variability between laboratories and across microarray platforms, and methods to accommodate this variability. RNA expression data were generated in seven laboratories, which compared two standard RNA samples using 12 microarray platforms. At least two standard microarray types (one spotted, one commercial) were used by all laboratories. Reproducibility for most platforms within any laboratory was typically good, but reproducibility between platforms and across laboratories was generally poor. Reproducibility between laboratories increased markedly when standardized protocols were implemented for RNA labeling, hybridization, microarray processing, data acquisition and data normalization. Reproducibility was highest when analysis was based on biological themes defined by enriched Gene Ontology (GO) categories. These findings indicate that microarray results can be comparable across multiple laboratories, especially when a common platform and set of procedures are used.

Gene Expression Profiling↗
PubMed2005

Coupling of human circadian and cell cycles by the timeless protein.

The Timeless protein is essential for circadian rhythm in Drosophila. The Timeless orthologue in mice is essential for viability and appears to be required for the maintenance of a robust circadian rhythm as well. We have found that the human Timeless protein interacts with both the circadian clock protein cryptochrome 2 and with the cell cycle checkpoint proteins Chk1 and the ATR-ATRIP complex and plays an important role in the DNA damage checkpoint response. Down-regulation of Timeless in human cells seriously compromises replication and intra-S checkpoints, indicating an intimate connection between the circadian cycle and the DNA damage checkpoints that is in part mediated by the Timeless protein.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Cell-type-specific responses to chemotherapeutics in breast cancer.

Recent microarray studies have identified distinct subtypes of breast tumors that arise from different cell types and that show statistically significant differences in patient outcome. To gain insight into these differences, we identified in vitro and in vivo changes in gene expression induced by chemotherapeutics. We treated two cell lines derived from basal epithelium (immortalized human mammary epithelial cells) and two lines derived from luminal epithelium (MCF-7 and ZR-75-1) with chemotherapeutics used in the treatment of breast cancer and assayed for changes in gene expression using DNA microarrays. Treatment doses for doxorubicin and 5-fluorouracil were selected to cause comparable cytotoxicity across all four cell lines. The dominant expression response in each of the cell lines was a general stress response; however, distinct expression patterns were observed. Both cell types induced DNA damage-response genes such as p21(waf1), but the response in the luminal cells showed higher fold changes and included more p53-regulated genes. Luminal cell lines repressed a large number of cell cycle-regulated genes and other genes involved in cellular proliferation, whereas the basal cell lines did not. Instead, the basal cell lines repressed genes that were involved in differentiation. These in vitro responses were compared with expression responses in breast tumors sampled before and after treatment with doxorubicin or 5-fluorouracil/mitomycin C. The in vivo data corroborated the cell-type-specific responses to chemotherapeutics observed in vitro, including the induction of p21(waf1). Similarities between in vivo and in vitro responses help to identify important response mechanisms to chemotherapeutics.

Antineoplastic Combined Chemotherapy Protocols↗
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↗
PubMed2002

ATR enforces the topoisomerase II-dependent G2 checkpoint through inhibition of Plk1 kinase.

An ATR-dependent G(2) checkpoint responds to inhibition of topoisomerase II and delays entry into mitosis by sustaining nuclear exclusion of cyclin B1-Cdk1 complexes. Here we report that induction of this checkpoint with ICRF-193, a topoisomerase II catalytic inhibitor that does not cause DNA damage, was associated with an ATR-dependent inhibition of polo-like kinase 1 (Plk1) kinase activity and a decrease in cyclin B1 phosphorylation. Expression of constitutively active Plk1 but not wild type Plk1 reversed ICRF-193-induced mitotic delay in HeLa cells, suggesting that Plk1 kinase activity is important for the checkpoint response to ICRF-193. G(2)/M synchronized normal human fibroblasts, when treated with ICRF-193, showed a decrease in cyclin B1 phosphorylation and Plk1 kinase activity despite high cyclin B1-Cdk1 kinase activity. G(2) fibroblasts that were treated with caffeine to override the checkpoint response to ICRF-193 displayed a high incidence of chromosomal aberrations. Taken together, these results suggest that ATR-dependent inhibition of Plk1 kinase activity may be one mechanism to regulate cyclin B1 phosphorylation and sustain nuclear exclusion during the G(2) checkpoint response to topoisomerase II inhibition. Moreover, the results demonstrate an important role for the topoisomerase II-dependent G(2) checkpoint in the preservation of human genomic stability.

Alleles↗
PubMed2002

DNA damage responses protect xeroderma pigmentosum variant from UVC-induced clastogenesis.

Lack of DNA polymerase eta and the attendant defect in bypass replication of pyrimidine dimers induced in DNA by ultraviolet light (UV) underlie the enhanced mutagenesis and carcinogenesis observed in xeroderma pigmentosum variant (XP-V). We investigated whether diploid XP-V fibroblasts growing in culture are also more susceptible to UV-induced clastogenesis than normal human fibroblasts (NHF). This study utilized diploid fibroblasts immortalized by the ectopic expression of human telomerase. The cell lines displayed checkpoint responses to DNA damage comparable with those measured in the parental strains. Shortly after exposure to low doses of UVC (< or =4 J/m2), XP-V cells accumulated daughter strand gaps in excess of normal controls (>25-fold). Daughter strand gaps generated in UV-irradiated S phase cells are potential precursors of chromatid-type chromosomal aberrations. Nonetheless, chromatid-type chromosomal aberrations were only 1.5 to 2 times more abundant in XP-V than in NHF exposed to the same UVC dose. XP-V cells, however, displayed S phase delays at lower doses of UVC and for longer periods of time than NHF. These results support the hypothesis that aberrant DNA structures activate S phase checkpoint responses that increase the time available for postreplication repair. Alternatively, cells that cannot be properly repaired remain permanently arrested and never reach mitosis. These responses protect human cells from chromosomal aberrations, especially when other pathways, such as accurate lesion bypass, are lost.

Carcinogenicity Tests↗
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↗