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R S Paules

Publications and source records attributed to R S Paules.

At least 19 recordsLinked to original sources
PubMed2001

The human decatenation checkpoint.

Chromatid catenation is actively monitored in human cells, with progression from G(2) to mitosis being inhibited when chromatids are insufficiently decatenated. Mitotic delay was quantified in normal and checkpoint-deficient human cells during treatment with ICRF-193, a topoisomerase II catalytic inhibitor that prevents chromatid decatenation without producing topoisomerase-associated DNA strand breaks. Ataxia telangiectasia (A-T) cells, defective in DNA damage checkpoints, showed normal mitotic delay when treated with ICRF-193. The mitotic delay in response to ICRF-193 was ablated in human fibroblasts expressing an ataxia telangiectasia mutated- and rad3-related (ATR) kinase-inactive ATR allele (ATR(ki)). BRCA1-mutant HCC1937 cells also displayed a defect in ICRF-193-induced mitotic delay, which was corrected by expression of wild-type BRCA1. Phosphorylations of hCds1 or Chk1 and inhibition of Cdk1 kinase activity, which are elements of checkpoints associated with DNA damage or replication, did not occur during ICRF-193-induced mitotic delay. Over-expression of cyclin B1 containing a dominant nuclear localization signal, and inhibition of Crm1-mediated nuclear export, reversed ICRF-193-induced mitotic delay. In combination, these results imply that ATR and BRCA1 enforce the decatenation G(2) checkpoint, which may act to exclude cyclin B1/Cdk1 complexes from the nucleus. Moreover, induction of ATR(ki) produced a 10-fold increase in chromosomal aberrations, further emphasizing the vital role for ATR in genetic stability.

Ataxia Telangiectasia↗
PubMed2001

The Ataxia telangiectasia gene product is required for oxidative stress-induced G1 and G2 checkpoint function in human fibroblasts.

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by neuronal degeneration accompanied by ataxia, telangiectasias, acute cancer predisposition, and sensitivity to ionizing radiation (IR). Cells from individuals with AT show unusual sensitivity to IR, severely attenuated cell cycle checkpoint functions, and poor p53 induction in response to IR compared with normal human fibroblasts (NHFs). The gene mutated in AT (ATM) has been cloned, and its product, pATM, has IR-inducible kinase activity. The AT phenotype has been suggested to be a consequence, at least in part, of an inability to respond appropriately to oxidative damage. To test this hypothesis, we examined the ability of NHFs and AT dermal fibroblasts to respond to t-butyl hydroperoxide and IR treatment. AT fibroblasts exhibit, in comparison to NHFs, increased sensitivity to the toxicity of t-butyl hydroperoxide, as measured by colony-forming efficiency assays. Unlike NHFs, AT fibroblasts fail to show G(1) and G(2) phase checkpoint functions or to induce p53 in response to t-butyl hydroperoxide. Treatment of NHFs with t-butyl hydroperoxide activates pATM-associated kinase activity. Our results indicate that pATM is involved in responding to certain aspects of oxidative damage and in signaling this information to downstream effectors of the cell cycle checkpoint functions. Our data further suggest that some of the pathologies seen in AT could arise as a consequence of an inability to respond normally to oxidative damage.

Adolescent↗
PubMed2001

Assessing gene significance from cDNA microarray expression data via mixed models.

The determination of a list of differentially expressed genes is a basic objective in many cDNA microarray experiments. We present a statistical approach that allows direct control over the percentage of false positives in such a list and, under certain reasonable assumptions, improves on existing methods with respect to the percentage of false negatives. The method accommodates a wide variety of experimental designs and can simultaneously assess significant differences between multiple types of biological samples. Two interconnected mixed linear models are central to the method and provide a flexible means to properly account for variability both across and within genes. The mixed model also provides a convenient framework for evaluating the statistical power of any particular experimental design and thus enables a researcher to a priori select an appropriate number of replicates. We also suggest some basic graphics for visualizing lists of significant genes. Analyses of published experiments studying human cancer and yeast cells illustrate the results.

Computational Biology↗
PubMed2001

MAPS: a microarray project system for gene expression experiment information and data validation.

SUMMARY: MAPS is a MicroArray Project System for management and interpretation of microarray gene expression experiment information and data. Microarray project information is organized to track experiments and results that are: (1) validated by performing analysis on stored replicate gene expression data; and (2) queried according to the biological classifications of genes deposited on microarray chips.

Computer Communication Networks↗
PubMed2000

Oxidative stress and cell cycle checkpoint function.

Oxidative stress and the damage that results from it have been implicated in a wide number of disease processes including atherosclerosis, autoimmune disorders, neuronal degeneration, and cancer. Reactive oxygen species (ROS) are ubiquitous and occur naturally in all aerobic species, coming from both exogenous and endogenous sources. ROS are quite reactive and readily damage biological molecules, including DNA. While the damaging effects of ROS on DNA have been intensively studied, the effects of oxidative damage on cell cycle checkpoint function have not. Here will we review several biologically important ROS and their sources, the cell cycle, checkpoints, and current knowledge about the effects of ROS on initiating checkpoint responses.

Aerobiosis↗
PubMed1999

Lack of involvement of ataxia telangiectasia mutated (ATM) in regulation of nuclear factor-kappaB (NF-kappaB) in human diploid fibroblasts.

It has been suggested that the cellular response to exposure to ionizing radiation involves activation of the transcription factor nuclear factor-kappaB (NF-kappaB) and that this response is defective in cells from individuals with ataxia telangiectasia (AT). In one study, it was found that SV40 large T-transformed cells derived from a patient null for the AT mutated (ATM) gene exhibited constitutive activation of NF-kappaB and that in those cells, inhibition of NF-kappaB by expression of a modified form of IkappaBalpha led to correction of the radiosensitivity associated with the AT phenotype [M. Jung et al., Science (Washington DC), 268: 1691-1621, 1995]. From those data, it was suggested that NF-kappaB played a role in the AT phenotype. We show here that normal diploid cells derived from AT patients do not exhibit constitutive activation of NF-kappaB. Furthermore, we provide data that the transformation process associated with SV40 large T antigen expression in AT-/- cells leads to aberrant cellular responses. Our studies highlight the importance of using diploid, nontransformed AT-/- cells for in vitro studies relevant to the AT phenotype whenever possible.

Ataxia Telangiectasia↗
PubMed1999

Cell cycle control, checkpoint mechanisms, and genotoxic stress.

The ability of cells to maintain genomic integrity is vital for cell survival and proliferation. Lack of fidelity in DNA replication and maintenance can result in deleterious mutations leading to cell death or, in multicellular organisms, cancer. The purpose of this review is to discuss the known signal transduction pathways that regulate cell cycle progression and the mechanisms cells employ to insure DNA stability in the face of genotoxic stress. In particular, we focus on mammalian cell cycle checkpoint functions, their role in maintaining DNA stability during the cell cycle following exposure to genotoxic agents, and the gene products that act in checkpoint function signal transduction cascades. Key transitions in the cell cycle are regulated by the activities of various protein kinase complexes composed of cyclin and cyclin-dependent kinase (Cdk) molecules. Surveillance control mechanisms that check to ensure proper completion of early events and cellular integrity before initiation of subsequent events in cell cycle progression are referred to as cell cycle checkpoints and can generate a transient delay that provides the cell more time to repair damage before progressing to the next phase of the cycle. A variety of cellular responses are elicited that function in checkpoint signaling to inhibit cyclin/Cdk activities. These responses include the p53-dependent and p53-independent induction of Cdk inhibitors and the p53-independent inhibitory phosphorylation of Cdk molecules themselves. Eliciting proper G1, S, and G2 checkpoint responses to double-strand DNA breaks requires the function of the Ataxia telangiectasia mutated gene product. Several human heritable cancer-prone syndromes known to alter DNA stability have been found to have defects in checkpoint surveillance pathways. Exposures to several common sources of genotoxic stress, including oxidative stress, ionizing radiation, UV radiation, and the genotoxic compound benzo[a]pyrene, elicit cell cycle checkpoint responses that show both similarities and differences in their molecular signaling.

Animals↗
PubMed1997

Deregulation of specific E2F complexes by the v-mos oncogene.

The product of the c-mos proto-oncogene is a protein kinase that is normally expressed in germ cells and functions during oocyte maturation. It has been shown, however, that inappropriate expression of either the viral or cellular mos gene can induce neoplastic progression in somatic cells. Furthermore, v-mos-transformed NIH3T3 cells will undergo arrest of proliferation in early G1 upon serum withdrawal but are unable to appropriately down-regulate cell cycle regulatory proteins, such as cyclin and cdc2 proteins, that normally are down-regulated in quiescent, untransformed NIH3T3 cells. Since the levels of these proteins are partially transcriptionally controlled, we investigated whether there were alterations in the expression of E2F and AP-1 transcription factor complexes. Indeed, the putative G0/G1-specific p130-E2F complex that is normally observed during low serum-induced cell cycle arrest in NIH3T3 cells is not present in serum starved v-mos-transformed cells. Instead, G1-phase arrested v-mos-transformed cells stably express two E2F protein complexes that are normally observed only during S-phase in untransformed cells. The elevation of these complexes in arrested v-mos-transformed cells may be the cause of the transcriptional activation of the E2F-regulated genes cdc2, DHFR, cyclin A, and E2F1 seen in serum starved v-mos-transformed cells. In addition, there are high levels of AP-1 DNA binding activity in serum starved v-mos-transformed cells compared to very low amounts in nontransformed cells. This altered regulation of transcription factor complexes and cell cycle control proteins upon serum withdrawal may provide a mechanism for the uncontrolled cell growth associated with neoplastic transformation induced by certain proto-oncogenes.

3T3 Cells↗
PubMed1997

Serum starved v-mos-transformed cells are unable to appropriately downregulate cyclins and CDKs.

Serum deprived v-mos-transformed NIH3T3 cells are unable to enter a true quiescent state, but instead, arrest in the early G1 phase of the cell cycle. We have analysed several cell cycle regulatory proteins in these G1 arrested cells and show altered regulation in the expression and activity of certain cyclins and cyclin-dependent kinases. In particular, p34cdc2, cyclin A, cyclin D and cyclin E are not appropriately down-regulated in serum starved, G1 arrested, v-mos-transformed cells as compared with quiescent NIH3T3 cells. Furthermore, serum starved v-mos-transformed cells have elevated histone H1 kinase activity associated with cyclin A, cyclin E, p33cdk2, and p34cdc2. Using a metallothionein-inducible c-mos(mu) expression system, we show that c-mos(mu) induction in quiescent NIH3T3 cells causes elevated expression of p34cdc2. However, this induction of c-mos(mu) and subsequent expression of p34cdc2 was not sufficient to promote significant entry of cells into S phase. Analysis of extracts from serum starved v-H-ras, v-src, and tpr-met transformed NIH3T3 cells demonstrates that these oncogene-transformed cells also contain elevated levels of p34cdc2. We propose that the altered regulation of these critical cell cycle regulatory molecules, and specifically the inability to fully downregulate their activity, contributes significantly to neoplastic transformation and subsequent unregulated growth of tumor cells.

3T3 Cells↗
PubMed1997

Inactivation of G2 checkpoint function and chromosomal destabilization are linked in human fibroblasts expressing human papillomavirus type 16 E6.

Chromosomal stability was linked to G2 checkpoint function in human fibroblasts expressing the human papillomavirus type 16 E6 oncoprotein. Soon after expression of E6, cells displayed an undamaged, diploid karyotype and normal mitotic delay after gamma-irradiation. As the E6-expressing cells aged through their in vitro life span, G2 checkpoint function diminished progressively. After 30-70 population doublings, 60-86% of the E6 cells displayed defective G2 checkpoint response. This attenuation of G2 checkpoint function was also associated with radiation-resistant cyclin B1/CDK1 protein kinase activity. Numerical and structural abnormalities of chromosomes developed in unirradiated E6 cells with kinetics that mirrored the loss of G2 checkpoint function. A significant correlation between inactivation of the G2 checkpoint and acquisition of chromosomal abnormalities was found, suggesting that the G2 checkpoint represents a barrier to genetic instability in cells lacking G1 checkpoint function.

CDC2 Protein Kinase↗
PubMed1996

DNA damage and cell cycle checkpoints.

DNA is prone to numerous forms of damage that can injure cells and impair fitness. Cells have evolved an array of mechanisms to repair these injuries. Proliferating cells are especially vulnerable to DNA damage due to the added demands of cellular growth and division. Cell cycle checkpoints represent integral components of DNA repair that coordinate cooperation between the machinery of the cell cycle and several biochemical pathways that respond to damage and restore DNA structure. By delaying progression through the cell cycle, checkpoints provide more time for repair before the critical phases of DNA replication, when the genome is replicated, and of mitosis, when the genome is segregated. Loss or attenuation of checkpoint function may increase spontaneous and induced gene mutations and chromosomal aberrations by reducing the efficiency of DNA repair. Defects in checkpoint control have been seen in certain hereditary cancer syndromes and at early stages of cell transformation. Mutations in checkpoint control genes therefore may contribute to the genetic instability that appears to drive neoplastic evolution.

Animals↗
PubMed1995

p21CIP1 is not required for the early G2 checkpoint response to ionizing radiation.

We have previously reported that the immediate G2 checkpoint delay of normal human fibroblasts in response to ionizing radiation is correlated with inhibition of p34CDC2/cyclin B kinase activity. Here, we observed increased amounts of the cyclin-dependent protein kinase inhibitor p21CIP1 associated with p34CDC2/cyclin B protein complexes from irradiated normal human fibroblasts. Since wild-type p53 function is not required for the early G2 checkpoint response to ionizing radiation, we investigated whether a p53-independent induction of p21CIP1 was required for the G2 checkpoint. Early passage human fibroblasts expressing the E6 oncoprotein of human papilloma virus-type 16 (NHF4 E6) were analyzed. It has been demonstrated earlier than inactivation of wild-type p53 function in these cells by E6 protein does not alter their intact early G2 checkpoint response to gamma-rays. p21CIP1 was found to be undetectable in p34CDC2/cyclin B protein complexes and in total extracts from the E6-expressing cells, with or without exposure to ionizing radiation. These data indicate that p21CIP1 is not required for the immediate G2 checkpoint response and is not induced by a p53-independent pathway in G2 phase following exposure to gamma-rays.

Cell Cycle↗
PubMed1995

Defective G2 checkpoint function in cells from individuals with familial cancer syndromes.

The early events in the G2 checkpoint response to ionizing radiation (IR) were analyzed in diploid normal human fibroblasts (NHFs) and fibroblasts from patients with two heritable cancer syndromes. Exposure to gamma-radiation of asynchronously growing NHFs resulted in a rapid reduction in the number of cells in mitosis (G2 delay) and was accompanied by a quantitatively similar reduction in the p34CDC2/cyclin B in vitro histone H1 kinase activity as compared with sham-treated controls. This G2 delay was strong by 1 h following exposure to IR, maximal by 2 h, and was accompanied by an accumulation of tyrosine-phosphorylated p34CDC2 molecules. In contrast, fibroblasts from individuals with ataxia telangiectasia displayed significantly less reduction of the mitotic index or histone H1 kinase activity after IR. Low passage fibroblasts from individuals with Li-Fraumeni syndrome having one wild-type and one mutated p53 allele were similar to NHFs in their immediate G2 checkpoint response to IR, as were NHFs expressing the human papilloma virus type 16 E6 gene product (functionally inactivating p53) and low passage cells from p53-deficient mouse embryos. However, the p53-deficient fibroblasts were genomically unstable and became defective in their early G2 checkpoint response to IR. Furthermore, immortal Li-Fraumeni syndrome fibroblasts lacking wild-type p53 displayed an attenuated G2 checkpoint response. These results link the early events in G2 checkpoint response to IR in NHFs with a rapid inhibition of p34CDC2/cyclin B protein kinase activity and demonstrate that while not required for this immediate G2 delay, lack of p53 can lead to subsequent genetic alterations that result in defective G2 checkpoint function.

Ataxia Telangiectasia↗
PubMed1995

Attenuation of G2 checkpoint function precedes human cell immortalization.

We have investigated the hypothesis that attenuation of the G2 checkpoint, which delays entry into mitosis in response to damage to DNA and protects against clastogenesis, may contribute to the genetic instability of immortal human cell lines. IMR-90 normal human fibroblasts displayed stringent G2 checkpoint response to gamma-radiation-induced DNA damage. Irradiation with 1.5 Gy induced 98% inhibition of mitosis and 79% inhibition of cyclin B1/p34CDC2 kinase activity within 2 h. SV40-transformed IMR-90 cells with extended in vitro proliferative lifespan and immortal derivative cells displayed significantly less radiation-induced G2 delay (60-70%) and less inhibition of cyclin B1/p34CDC2 protein kinase activity (43-46%) than was seen in normal cells. Two other SV40-transformed lines and a fibrosarcoma line displayed a similar attenuation of G2 checkpoint function. The attenuation of G2 checkpoint function in SV40 transformed IMR-90 cells was associated with elevated levels of expression of cyclin B1 (8-fold greater) and p34CDC2 (2.5-fold greater). By allowing cells with damaged chromatids to enter mitosis, an attenuation of G2 checkpoint function in finite lifespan cells may promote the genetic alterations necessary for the conversion to immortality.

Cell Cycle↗
PubMed1994

v-mos-transformed cells fail to enter quiescence but growth arrest in G1 following serum withdrawal.

The product of the mos protooncogene normally functions in the induction of meiosis and regulation of cell-cycle progression in oocytes. Here we have investigated the cell-cycle progression of NIH3T3 cells transformed by the v-mos gene. Flow cytometric analysis showed that logarithmically growing v-mos-transformed cells do not differ from their nontransformed counterparts in the distribution of cells in the G1, S, and G2/M phases. Likewise, after serum withdrawal for 48 h, both normal and v-mos-transformed NIH3T3 cells have essentially ceased proliferation, as analyzed by flow cytometry, [3H]thymidine and BrdU incorporation into newly synthesized DNA, and mitotic indexes. However, while the normal NIH3T3 cells are arrested in a quiescent state, the v-mos-transformed cells are arrested in early to mid G1, prior to the point where cells require certain amino acids for proliferation (V point). In agreement with these different arrest points, the v-mos-transformed cells enter S phase following serum stimulation within about 8 h, without the additional 4- to 6-h lag period characteristically displayed by the parental NIH3T3 cells. In addition, we show a lack of expression of a growth arrest-specific gene product, gas1, in the serum-arrested v-mos-transformed cells. These data demonstrated that v-mos-transformed cells display growth characteristics that differ fundamentally from those of normal cells or cells transformed by overexpression of myc [1]. Our results suggest that the v-mos oncoprotein transforms cells, at least in part, by preventing exit from the cell cycle into quiescence.

3T3 Cells↗
PubMed1992

Pheochromocytomas and C-cell thyroid neoplasms in transgenic c-mos mice: a model for the human multiple endocrine neoplasia type 2 syndrome.

Transgenic mice carrying and expressing a mos protooncogene, linked to the Moloney murine sarcoma virus long terminal repeat, develop severe neurological defects and lens abnormalities. Here we report that after long latent periods, mice in three of four of these mos transgenic lines develop a high frequency of multicentric pheochromocytomas and/or medullary thyroid neoplasms. The pattern of tumor formation is remarkably similar to the human autosomal dominantly inherited neoplastic syndrome, multiple endocrine neoplasia type 2 (MEN 2), and tumors from these transgenic animals display the same neuroendocrine marker staining pattern as seen in MEN 2. The similarity between the tumor pathologies and presentation patterns of MEN 2 patients and mos transgenic mice suggests that they may arise through related pathways. The type of tumor presentation varies in a line-dependent manner indicating that there is interaction between the transgene and the genetic background. Moreover, when the non-tumor-bearing mos transgenic line is crossed to a different mouse background, the F1 offspring display the MEN 2 phenotype. These studies indicate that penetrance of the autosomal dominant mos transgenic phenotype is dependent on both integration site and background.

Adrenal Gland Neoplasms↗