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R S Paules

Publications and source records attributed to R S Paules.

18 recordsLinked to original sources
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↗
PubMed1992

pp39mos is associated with p34cdc2 kinase in c-mosxe-transformed NIH 3T3 cells.

We investigated the possible interactions between pp39mos and p34cdc2 kinase in NIH 3T3 cells transformed by c-mosxe. pp39mos is coprecipitated with p34cdc2 when using either anti-PSTAIR antibody or p13suc1-Sepharose beads. Likewise, p34cdc2 is coprecipitated with pp39mos when using anti-mos antibody. However, pp39mos was not present in histone H1 kinase-active p34cdc2 complexes precipitated with anti-p34cdc2 C-terminal peptide antibody even during metaphase of the cell cycle. The molar ratio of p34 to pp39mos in the p13suc1 complex is approximately 2:1. Consistent with the tight association between pp39mos and tubulin, tubulin was also present in equivalent amounts with pp39mos and p34 in the p13suc1 complex. This pp39mos-p34cdc2-tubulin complex may be important in transformation by the mos oncogene.

3T3 Cells↗
PubMed1992

Patterns of neoplasia in c-mos transgenic mice and their relevance to multiple endocrine neoplasia.

We have previously described a neurological phenotype for transgenic mice carrying the c-Mos proto-oncogene. Pheochromocytomas and C-cell thyroid neoplasms occur in these transgenic lines in patterns that are similar to those seen in multiple endocrine neoplasia type 2 (MEN 2). Characterization of the pathological lesions via immunohistochemistry underscores similarities between MEN 2 and these transgenic mice. When transgenic mice that do not display the MEN 2 phenotype are crossed to a different background, the progeny display the MEN 2 phenotype. Thus the interaction of the background with the transgene is such that it can suppress tumor information. This observation bears special relevance to the human syndrome in that this model system may be used to study the question of penetrance of phenotype.

Adrenal Gland Neoplasms↗
PubMed1992

Characterization of activated and normal mouse Mos gene in murine 3T3 cells.

We have characterized the mouse Mos proto-oncogene product, pp39Mos, in murine fibroblasts. When expressed in NIH3T3 cells under the influence of the long terminal repeat regulatory element from Moloney murine sarcoma virus [NIH(pTS-1) cells], the Mos protein was present in low levels and had a half-life of about 30 min. In extracts from NIH(pTS-1) cells, we detected additional forms of Mos protein that apparently arose from internal initiation codons (p24Mos and p29Mos) or from upstream non-AUG initiation codons (p42Mos and p44Mos). The Mos protein was found to exist in these cells as a phosphoprotein, pp39Mos, and, when immunoprecipitated with an antiserum specific for the Mos N-terminus [anti-Mos(6-24)], had autophosphorylating kinase activity. We found that anti-Mos(6-24) also detected non-Mos protein kinase activity and non-Mos phosphoproteins in addition to p39Mos. We present evidence, on both the RNA and protein levels, that non-transformed mouse 3T3 cells do not express endogenous Mos.

3T3 Cells↗
PubMed1991

Ability of the c-mos product to associate with and phosphorylate tubulin.

The mos proto-oncogene product, pp39mos, is a protein kinase and has been equated with cytostatic factor (CSF), an activity in unfertilized eggs that is thought to be responsible for the arrest of meiosis at metaphase II. The biochemical properties and potential substrates of pp39mos were examined in unfertilized eggs and in transformed cells in order to study how the protein functions both as CSF and in transformation. The pp39mos protein associated with polymers under conditions that favor tubulin oligomerization and was present in an approximately 500-kilodalton "core" complex under conditions that favor depolymerization. beta-Tubulin was preferentially coprecipitated in pp39mos immunoprecipitates and was the major phosphorylated product in a pp39mos-dependent immune complex kinase assay. Immunofluorescence analysis of NIH 3T3 cells transformed with Xenopus c-mos showed that pp39mos colocalizes with tubulin in the spindle during metaphase and in the midbody and asters during telophase. Disruption of microtubules with nocodazole affected tubulin and pp39mos organization in the same way. It therefore appears that pp39mos is a tubulin-associated protein kinase and may thus participate in the modification of microtubules and contribute to the formation of the spindle. This activity expressed during interphase in somatic cells may be responsible for the transforming activity of pp39mos.

Amino Acid Sequence↗
PubMed1991

A characterization of cytostatic factor activity from Xenopus eggs and c-mos-transformed cells.

In Xenopus oocytes, the mos proto-oncogene product is required during meiosis I for the activation of maturation promoting factor (MPF) and the subsequent breakdown of the germinal vesicle (GVBD). In addition, the mos product has been shown to be a candidate "initiator" of meiotic maturation and is an active component of cytostatic factor (CSF), an activity responsible for metaphase II arrest. Here we demonstrate that pp39mos is required throughout oocyte maturation. We found that in progesterone stimulated oocytes, depletion of mos RNA immediately before GVBD terminally decreased MPF. Likewise, oocytes depleted of mos RNA and induced to mature with crude MPF proceeded through GVBD but lacked the MPF activity required to arrest mature oocytes at metaphase II. Thus, during maturation the mos product is required, directly or indirectly, to sustain MPF activity. On the other hand, mouse NIH/3T3 cells transformed by the constitutive expression of pp39mosxc possessed CSF activity but lacked constitutive levels of MPF or its associated histone H1 kinase activity. Moreover, cytosols prepared from transformed NIH/3T3 cells or Xenopus eggs had similar levels of CSF activity, but pp39mos levels were greater than 40-fold higher in the transformed cell extract. These analyses show that maintenance of CSF during interphase does not result in the maintenance of MPF.

Animals↗
PubMed1989

Mouse Mos protooncogene product is present and functions during oogenesis.

We have identified the mouse Mos-encoded protein product, p39mos, in maturing mouse oocytes and have shown that it is indistinguishable from the product expressed in Mos-transformed NIH 3T3 cells. p39mos is detected in oocytes arrested in the first meiotic prophase, during germinal-vesicle breakdown, metaphase I, anaphase I, and in ovulated eggs. We show that microinjection of three different Mos antisense (but not sense) oligodeoxyribonucleotides into germinal vesicle-stage oocytes prevents first polar-body emission and therefore interrupted the normal progression of meiosis. These results show that in mouse oocytes, as in the amphibian Xenopus [Sagata, N., Oskarsson, M., Copeland, T., Brumbaugh, J. & Vande Woude, G.F. (1988) Nature (London) 335, 519-525], the product of Mos is necessary for normal meiotic maturation.

Animals↗
PubMed1988

Benzo[alpha]pyrene diol epoxide I binds to DNA at replication forks.

The distribution of lesions in DNA caused by (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo [alpha]pyrene (B[alpha]P diol epoxide-I) was studied in synchronized C3H/10T1/2 cells treated in S phase. Sites of carcinogen modification of DNA were identified by polyclonal rabbit antibodies elicited against DNA modified with B[alpha]P diol epoxide-I in vitro. This antigenic DNA contained trans-(7R)-N2-[10-(7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene)-yl]- deoxyguanosine; other adducts were not detected by liquid chromatography. In this study, DNA replication forks with antibodies bound to B[alpha]P diol epoxide-I adducts were detected by electron microscopy. The frequency of replication forks containing carcinogen adducts associated with the fork junction was found to be 8-fold higher than expected for an average distribution. The proportion of replication forks that were apparently blocked at the site of the DNA damage increased when replication was allowed to occur after carcinogen exposure. These results support the conclusions that the fork junction is particularly vulnerable to adduction by B[alpha]P diol epoxide-I and that B[alpha]P diol epoxide-I adducts block the displacement of replication forks during DNA synthesis in intact cells.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1985

Quantitation by electron microscopy of the binding of highly specific antibodies to benzo[a]pyrene-DNA adducts.

Highly specific antibodies bound to carcinogen adducts in DNA modified with (+/-)7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE I) were quantitated by electron microscopy (EM) visualization and these observations were compared with quantitation of adducts by enzyme-linked immunosorbent assay (ELISA). The antiserum, elicited in rabbits following inoculation with BPDE I-modified DNA, has been found to be highly specific in its recognition of BPDE I-deoxyguanosine moieties. Parallel DNA samples prepared for analysis by ELISA and EM quantitation were randomized, encoded, and analyzed to determine extents of carcinogen modification in double-blind studies. After levels of modification were determined by immunoassays, DNA samples were prepared for EM analysis by incubation with amounts of anti-BPdG-DNA serum in excess of that necessary for complete binding of antibody to antigenic sites. At equilibrium, samples were enzymatically digested with papain in order to cleave anti-BPdG-DNA IgG molecules into Fab fragments in situ. Following column exclusion chromatography, BPdG-DNA-Fab complexes were incubated with ferritin-labeled Fab' fragments of goat [anti-rabbit F(ab')2] IgG in amounts in excess of those necessary for complete binding. When DNA samples were modified to between 0 and 40 fmol adduct/micrograms DNA, excellent agreement was obtained between ELISA quantitation and visualization by EM of antibodies bound to adducts.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗