Biomedical subjects

E A Thompson

Publications and source records attributed to E A Thompson.

At least 55 records · Page 3Linked to original sources

PubMed1997

Glucocorticoid inhibition of fibroblast proliferation and regulation of the cyclin kinase inhibitor p21Cip1.

Glucocorticoids inhibit the proliferation of fibroblastic cells in vivo and in culture; however, the molecular mechanism that accounts for this effect has remained obscure. We have undertaken to elucidate the mechanism whereby glucocorticoids decrease the rate of proliferation of mouse L929 fibroblastic cells. Addition of dexamethasone to mid-log phase fibroblasts prolongs G1 phase. This increase in the G1 interval is associated with, and probably due to, inhibition of phosphorylation of the product of the Rb-1 tumor suppressor gene, pRb. Inhibition of pRb phosphorylation by cyclin D-dependent kinases can be demonstrated in vitro. Nevertheless, there is no detectable change in the expression of cyclin D1, cyclin D2, or cyclin D3. Cyclin-dependent kinase-4 (Cdk4) and Cdk6 are not down-regulated in L929 cells after addition of glucocorticoids, and the abundance of cyclin D/Cdk4 complexes does not change. Inhibition of pRb kinase activity is associated with an increase in the abundance of one of the Cdk inhibitors, p21Cip1. The abundance of another cyclin kinase inhibitor, p27Kip1, remains constant. The amount of Cdk4 that is bound to p21Cip1 increases rapidly after addition of dexamethasone, and the activity of Cdk4-pRb kinase decreases in parallel. These results indicate that glucocorticoid inhibition of fibroblast proliferation is due to induction of p21Cip1, which binds to and inactivates cyclinD/Cdk4 complexes. The abundance of p21 mRNA increases about 5-fold within 2 h after addition of dexamethasone. This effect does not obtain in L929 mutants that are null for the glucocorticoid receptor, and a variant that expresses the glucocorticoid receptor from a tetracycline-repressible expression vector demonstrates induction of p21 mRNA only in the absence of tetracycline. Cycloheximide does not block induction of p21 mRNA, and dexamethasone has no detectable effect on the apparent rate of degradation of p21 mRNA. Nuclear run-on transcription of the Cip1 gene increases within 2 h after addition of dexamethasone. This effect can be blocked by tetracycline-mediated repression of the glucocorticoid receptor.

Animals↗

PubMed1997

The checkpoint control for anaphase onset does not monitor excess numbers of spindle poles or bipolar spindle symmetry.

Exit from mitosis in animal cells is substantially delayed when spindle assembly is inhibited, spindle bipolarity is disrupted, or when a monopolar spindle is formed. These observations have led to the proposal that animal cells have a 'spindle assembly' checkpoint for the metaphase-anaphase transition that monitors bipolar spindle organization. However, the existence of such a checkpoint is uncertain because perturbations in spindle organization can produce unattached kinetochores, which by themselves are known to delay anaphase onset. In this study we have tested if cells monitor bipolar spindle organization, independent of kinetochore attachment, by analyzing the duration of mitosis in sea urchin zygotes and vertebrate somatic cells containing multipolar spindles in which all kinetochores are attached to spindle poles. We found that sea urchin zygotes containing tripolar or tetrapolar spindles progressed from nuclear envelope breakdown to anaphase onset with normal timing. We also found that the presence of supernumerary, unpaired spindle poles did not greatly prolong mitosis. Observation of untreated PtK1 cells that formed tripolar or tetrapolar spindles revealed that they progressed through mitosis, on average, at the normal rate. More importantly, the interval between the bipolar attachment of the last monooriented chromosome and anaphase onset was normal. Thus, neither of these cell types can detect the presence of gross aberrations in spindle architecture that inevitably lead to aneuploidy. We conclude that animal cells do not have a checkpoint for the metaphase-anaphase transition that monitors defects in spindle architecture independent of the checkpoint that monitors kinetochore attachment to the spindle. For dividing cells in which spindle microtubule assembly is not experimentally compromised, we propose that the completion of kinetochore attachment is the event which limits the time of the metaphase-anaphase transition.

Anaphase↗

PubMed1997

Enhancing outcomes in an indicated drug prevention program for high-risk youth.

This study examined the net effects of refining a high school-based indicated drug prevention program. The Personal Growth Class (PGC), tailored to meet the needs of high-risk youth, was designed to increase control of drug use, school performance, and emotional well-being. The program integrates social support and life-skills training. Process evaluation revealed the need for program enhancements to address underestimated levels of depression, anger, and suicidal behaviors prevalent among high-risk youth and to ensure that core content was being emphasized consistently. Youth participating in Late cohorts received the refined PGC with enhanced skills training. Study participants (N = 280) were youth, primarily ages fifteen to-seventeen, at high-risk for school failure or dropout. Indicators of emotional well-being (e.g., depression, stress, anger, self-esteem, personal control), drug involvement, and school performance were compared for Late versus Early cohorts. Regression analyses revealed the Late versus Early cohorts showed significantly greater decreases in hard drug use, depression, perceived stress, and anger, and greater increases in self-esteem. The results support arguments that effective indicated prevention programs should target specific high-risk youth employing strategies to counteract the multifaceted risk factors they experience and enhance needed protective factors.

Adolescent↗

PubMed1997

N-(4-hydroxyphenyl)retinamide induces apoptosis in T lymphoma and T lymphoblastoid leukemia cells.

We demonstrate that N-(4-hydroxyphenyl)-all-trans-retinamide (4-HPR), a synthetic retinoic acid (RA) derivative, is a potent and selective inducer of apoptosis in malignant T lymphoid cells, but has little effect on normal lymphoid cells of the thymus or spleen. 4-HPR and its stereoisomer, 9-cis-4-HPR, are 50 to > 150 times more potent than 7 other retinoids in killing CEM-C7 human T lymphoblastoid leukemia cells and P1798-C7 murine T lymphoma cells. 4-HPR's apoptotic action requires the intact molecule bearing both the retinoid moiety and the hydroxyphenol ring; 4-HPR remains unmetabolized after uptake into CEM-C7 and P1798-C7 cells for up to 24 hours. We also show that glucocorticoid (GC)-resistant variants are equally susceptible to 4-HPR as are GC-sensitive cells. Thus, 4-HPR may be potentially important as a new chemotherapeutic drug for use as alternative to, or in combination with, conventional drugs for treating lymphoid malignancies.

Aminophenols↗

PubMed1997

Allelic disequilibrium and allele frequency distribution as a function of social and demographic history.

Allelic disequilibrium between closely linked genes is a common observation in human populations and often gives rise to speculation concerning the role of selective forces. In a previous treatment, we have developed a population model of the expected distribution of rare variants (including private polymorphisms) in Amerindians and have argued that, because of the great expansion of Amerindian numbers with the advent of agriculture, most of these rare variants are of relatively recent origin. Many other populations have similar histories of striking recent expansions. In this treatment, we demonstrate that, in consequence of this fact, a high degree of linkage disequilibrium between two nonhomologous alleles <0.5 cM apart is the "normal" expectation, even in the absence of selection. This expectation is enhanced by the previous subdivision of human populations into relatively isolated tribes characterized by a high level of endogamy and inbreeding. We also demonstrate that the alleles associated with a recessive disease phenotype are expected to exist in a population in very variable frequencies: there is no need to postulate positive selection with respect to the more common disease-associated alleles for such entities as phenylketonuria or cystic fibrosis.

Alleles↗

PubMed1996

Human cytomegalovirus inhibits cellular DNA synthesis and arrests productively infected cells in late G1.

Human embryonic lung fibroblasts (LU) can be productively infected with human cytomegalovirus (HCMV). During the course of productive infection, the virus elicits a number of responses that resemble certain aspects of G1 cell cycle progression. The virus activates cyclin E/Cdk2 kinase in both subconfluent, serum-arrested, and density-arrested cultures. Activation of cyclin E-dependent kinase is due, in part, to induction of cyclin E and, in part, to inhibition of the cyclin kinase inhibitors, Cip1 and Kip1. However, G1 progression is incomplete in HCMV-infected cells. Neither cyclin A nor cyclin D is induced, and cellular DNA synthesis does not occur if one takes care to avoid addition of fresh serum to serum-starved cultures. The data indicate that the virus induces a state of late G1 arrest, in which cyclin E/Cdk2 activates nucleotide metabolism and other biosynthetic processes that are necessary for viral replication. Failure to activate host cell DNA synthesis ensures that the virus will have uncompleted access to such precursors.

CDC2-CDC28 Kinases↗

PubMed1996

Private polymorphisms: how many? How old? How useful for genetic taxonomies?

The data on the distribution and frequencies of private polymorphisms in the tribal populations of Central and South America are used to address the question of the extent to which such data can be used to address questions of phylogenetic history. It is shown that due to the great increases in population number that accompanied agricultural development, most private polymorphisms have arisen since population settlement and tribal differentiation. Conversely, the absence of Amerindian variants of wide distribution confirms the small size of the hemispheric population until relatively recent times. Patterns of recent population decline and recovery that accompanied European contact since 1492 have also had a strong impact on the age distribution of extant variants, eliminating many that were relatively young in 1492. The majority of surviving variants that have achieved polymorphic frequencies in a tribe or group of tribes are from 100 to 400 generations old (2500 to 10,000 years). Such genetic variants thus characterize tribes, or groups of closely related tribes, but do not provide a greater time depth of phylogenetic history.

Alleles↗

PubMed1996

Distribution of genome shared IBD by half-sibs: approximation by the Poisson clumping heuristic.

The distribution of the proportion of genome shared identical by descent (IBD) by c half-sibs is calculated for a general c. With data availability the consideration of the chromosome as a whole rather than discrete loci becomes of increasing practical importance. Assuming the genomic continuum model, which allows for recombination, the crossover process in a chromosome pedigree is viewed as a continuous-time Markov random walk on the vertices of a hypercube with time parameter map distance along the chromosome. The desired distribution corresponds to the distribution of sojourn times of the process in a small set of vertices. The sojourn times are well approximated via the Poisson clumping heuristic. It is shown explicitly that there is no "equivalent" number of independently segregating loci that will yield the same results as with the genomic continuum model. Results are given for the human genome.

Crossing Over, Genetic↗

PubMed1996

The probability distribution of the amount of an individual's genome surviving to the following generation.

The probability that at least p% of an individual's genome is passed on collectively to his children is calculated. With data availability the consideration of the chromosome as a whole rather than discrete loci becomes of increasing practical importance. Assuming the genomic continuum model, which allows for recombination, the crossover process in a chromosome pedigree is viewed as a continuous-time Markov random walk on the vertices of a hypercube with time parameter map distance along the chromosome. The desired probability corresponds to the probability of sojourn times of the process in a small set of vertices, which are well approximated via the Poisson clumping heuristic. Results are given for the human genome. It is very likely that an individual with at least four children passes on at least 90% of his genome. There exists no "equivalent" number of independently segregating loci for this distribution.

Genetics, Population↗

PubMed1996

Intestinal cell cycle regulations. Interactions of cyclin D1, Cdk4, and p21Cip1.

OBJECTIVE: The p21Cip1 protein is a potent stoichiometric inhibitor of cyclin-dependent kinase activity, and p21Cip1 mRNA expression is localized to the nonproliferative compartment of the intestinal villus, suggesting an in vivo growth-inhibitory role in the gut. The authors determined whether nontransformed rat intestinal epithelial cells (IECs) underwent reversible cell cycle arrest by contact inhibition, and determined whether increases in the relative amount of p21 associated with cyclin D/Cdk4 protein complexes were associated with cell growth arrest. METHODS: Density arrest was achieved by prolonged culture IEC-6 in confluent conditions (5 or more days). Release from density arrest was achieved by detaching the cells from the culture plate and reseeding them at a 1:4 ratio. The DNA synthesis was estimated by [3H]-thymidine incorporation and expressed as mean plus or minus standard error of the mean (n = 4). Cyclin D1, Cdk4, and p21 mRNA and protein levels were determined by standard Northern and Western blot analyses, respectively. Cyclin D1, Cdk4, and p21 protein complex formation was analyzed by immunoprecipitating the complexes from cell lysates with an antibody to one of the constituents, followed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of the precipitated complexes using antibodies to the other proteins. The kinase activity of the immunoprecipitated Cdk4 was determined using recombinant Rb as substrate. RESULTS: The IEC-6[3H]-thymidine incorporation was decreased 7.5-fold from day 1 confluence to day 7 of confluence. Twenty-four hours after release from density arrest, there was a 43-fold increase in [3H]-thymidine incorporation. Cyclin D1 and Cdk4 mRNA levels remained relatively constant during contact inhibition, whereas immunoblotting showed that the levels of cyclin D1 and Cdk4 proteins decreased by 70.9% and 68.7%, respectively, comparing day 3 with day 9 during density arrest. The levels of cyclin D1 increased 5.8-fold and Cdk4 increased by 4.4-fold by 24 hours after reseeding the day 9 density-arrested cultures, coincident with the increase in DNA synthesis. The amount of p21 associated with the cyclin D1 and Cdk4 complex in the density-arrested cells was 170% of that observed in the reseeded, proliferating cells. More important, the p21::Cdk4 ratio was 6.4-fold higher in the density-arrested (quiescent) cells as compared with rapidly proliferating cells by 24 hours after release from growth arrest. Recovery of Cdk4-dependent kinase activity occurred by 4 hours after release from growth arrest, coincident with decreased binding of p21 to the complex. CONCLUSIONS: Intestinal epithelial cells in culture can undergo density-dependent growth arrest. This process involves downregulation of cyclin D1 and Cdk4 at the level of protein expression, whereas the mRNA levels remain relatively unchanged. Further, during contact inhibition, there is more p21 associated with cyclin D1/Cdk4, which further contributes to the inhibition of the kinase complex. The authors also have shown that the process of contact inhibition is reversible, which may explain partly the ability of the intestinal epithelium to increase proliferative activity in response to injury.

Animals↗

PubMed1996

Cell cycle protein suppression and p21 induction in differentiating Caco-2 cells.

Despite intensive efforts, the exact cellular mechanisms leading to gut differentiation and development remain largely undefined. The cyclins, the cyclin-dependent kinases (Cdks), and the Cdk inhibitors (e.g., p21 and p27) are proteins that are important for cell cycle progression, subsequent growth inhibition, and differentiation of various cell types. The purpose of our study was to better define the role of these cell cycle proteins in gut differentiation using the Caco-2 human cell line, which spontaneously differentiates to a small bowel phenotype, as demonstrated by induction of sucrase-isomaltase (SI) gene expression. We found that protein levels of the cyclins (both D- and E-type) and the Cdks (both Cdk2 and Cdk4) progressively decreased in postconfluent Caco-2 cells. Moreover, cyclin E-associated histone H1 kinase activity decreased in an analogous fashion as the cyclins and Cdks. In contrast, induction of the Cdk inhibitor p21 occurred by 3 days postconfluency, which was before the increase in SI mRNA levels. These changes in the cell cycle proteins, which include a progressive decrease of the cyclins and Cdks and a concomitant induction of p21, suggest an important role for these proteins in Caco-2 cell differentiation. Identifying the cell cycle mechanisms responsible for intestinal cell differentiation will be important to our understanding of both normal gut development as well as gut neoplasia, which involves aberrant regulation of cell cycle arrest.

CDC2-CDC28 Kinases↗

PubMed1996

Glucocorticoid regulation of a transcription factor that binds an initiator-like element in the murine thymidine kinase (Tk-1) promoter.

Glucocorticoids inhibit transcription of the murine cytoplasmic thymidine kinase gene (Tk-1). Glucocorticoid regulation of Tk-1 transcription can be demonstrated in cells that are arrested in late G1. This observation indicates that inhibition of Tk-1 expression is not dependent upon redistribution within the cell cycle but is due to glucocorticoid regulation of this gene. Transfection studies have been carried out using chimeric genes in which restriction fragments of the Tk-1 promoter were fused to chloramphenicol acetyltransferase or neomycin phosphotransferase. These chimeric reporters were assayed for stable expression and glucocorticoid regulation in P1798 lymphoma cells. A 140-bp fragment, extending from -143 to -3 bp with respect to the thymidine kinase translational start site, was capable of both basal and glucocorticoid-regulated transcription of reporter genes. The extent of inhibition by glucocorticoids was similar to that observed for the endogenous gene, and no increase in basal expression or the extent of inhibition was observed with constructs containing additional 5'-flanking DNA. The 140-bp Tk-1 core promoter fragment binds to transcription factors in extracts from P1798 cells. Control cell extracts contain factors that bind to and protect (from deoxyribonuclease I) a distal promoter element from -106 to -87 bp, relative to the translational start site. A second, proximal element was protected at -43 to -36 bp. The proximal element of the Tk-1 promoter resembles an RNA polymerase II initiator element. No other elements were protected. Glucocorticoids inhibit the amount or activity of the transcription factor that binds to this initiator-like element within the Tk-1 promoter. This element, when fused to upstream activation sequences from the herpes simplex virus thymidine kinase promoter, conveys glucocorticoid sensitivity in cis.

Administration, Topical↗

PubMed1996

The Drug Involvement Scale for Adolescents (DISA).

This article specifically addressed the need for a multidimensional approach to measuring adolescents' drug involvement. The Drug Involvement Scale for Adolescents (DISA) was theoretically specified and its measurement properties were tested using Confirmatory Factor Analyses and traditional procedures with 705 high-risk and typical high school students. Five first-order dimensions, Drug Access, Alcohol Use, Other Drug Use, Drug Use Control Problems and Adverse Drug Use Consequences, and a hierarchical model of Drug Involvement demonstrated a good fit between model and data. Further, the DISA demonstrated good internal consistency (alpha = .91); correlated as expected with known correlates of adolescent drug use; discriminated drug involvement between high-risk and typical high school students; and predicted later drug involvement and known drug-related consequences among adolescents. The results suggest the DISA should be useful for capturing a multidimensional view of adolescent drug involvement in both etiologic and prevention studies. A major advantage of the DISA is its brevity: twenty-two indicators constructed from twenty-nine items.

Achievement↗

PubMed1996

Cyclin E/Cdk2 activity is controlled by different mechanisms in the G0 and G1 phases of the cell cycle.

The experiments described in this report were undertaken to define the parameters that regulate cyclin E/cyclin-dependent kinase 2 (Cdk2) kinase activity in mitotically quiescent, serum-starved fibroblastic cells and in cells that had been stimulated to enter the cell cycle and progress through G1 into S phase. We have analyzed the expression of cyclin E and Cdk2, the extent to which these two proteins form complexes, and the enzymatic activity of cyclin E/cdk2 kinase. Particular attention was focused upon subcellular localization and the effect of compartmentalization on the association between cyclin E and Cdk2. In addition, we have examined the interaction of cyclin E/Cdk2 complexes with two well-characterized inhibitors of Cdk2 kinase activity, Cip1 and Kip1. This represents the first report in which all of these parameters have been measured simultaneously in a single, normal diploid cell line. In G0 cells, there is abundant cyclin E and Cdk2, yet there is little or no detectable Cdk2-dependent histone H1 kinase activity. After serum stimulation, there is a rapid increase in the amount of cyclin E that is bound to Cdk2, although there is no significant change in the abundance of either the cyclin or the Cdk. Immunocytochemical data indicate that cyclin E, Cip1, and Kip1 are located within the nuclei of cell in G0, but very little Cdk2 is observed within the nuclei of serum-starved cells. Cdk2 rapidly enters the nucleus upon serum stimulation. The abundance of the cyclin E/Cdk2 complex increases to the extent that the binding capacity of Cip1 is exceeded about 8-12 h after serum stimulation. The abundance of Kip1 decreases at the same time that the Cip1 threshold is exceeded, so that cyclin E/Kip1-containing complexes decrease by 90% within 8-12 h. Cyclin E/Cdk2 kinase activity begins to increase rapidly thereafter, reaching a maximum level about 16 h after serum stimulation. We have been unable to detect histone H1 kinase activity in complexes that contain cyclin E bound to Kip1 or Cip1. We conclude that compartmentalization is the predominant barrier to activation of cyclin E-dependent kinases in quiescent cells. Cip1 and Kip1 serve to prevent premature activation of cyclin E/Cdk2 complexes that form during G0 or early G1.

CDC2-CDC28 Kinases↗

PubMed1995

c-Myc and cyclin D3 (CcnD3) genes are independent targets for glucocorticoid inhibition of lymphoid cell proliferation.

Glucocorticoids inhibit the expression of critical cell cycle-regulatory genes. The G1 cyclin gene CcnD3, which encodes cyclin D3, is inhibited by dexamethasone in P1798 murine T lymphoma cells. Glucocorticoids also inhibit expression of the catalytic partner of cyclin D3, Cdk4. Inhibition of these two genes results in a decrease in the ability to phosphorylate the Rb-1 tumor suppressor gene product. Stable transformation with SV40 T antigen expression vectors prevents glucocorticoid-mediated cell cycle arrest, which is consistent with the conclusion that glucocorticoids inhibit Rb-1 phosphorylation. Overexpression of cyclin D3 suffices to restore Rb-kinase activity in glucocorticoid-treated cells. Nevertheless, overexpression of cyclin D3 does not prevent glucocorticoid inhibition of cell proliferation. Cells transformed with Cdk4 expression vectors, with or without cyclin D3 expression vectors, also undergo G0 arrest in the presence of dexamethasone. Glucocorticoids inhibit c-Myc expression in lymphoid cells, and transient expression of c-Myc protein attenuates the lytic response in glucocorticoid-treated human leukemia cells (R. Thulasi, D. V. Harbour, and E. B. Thompson, J. Biol. Chem., 268: 18306-16312, 1993). However, P1798 cells stably transfected with c-Myc expression vectors are sensitive to glucocorticoid-mediated G0 arrest. Such transformants withdraw from the cell cycle when treated with dexamethasone. P1798 cells were transformed so as to express both c-Myc protein and cyclin D3 in the presence of glucocorticoids. These Myc/D3 cells continue to proliferate in the presence of dexamethasone, and virtually all of these cells are capable of entering S phase in the presence of the steroid. Rapid apoptotic cell death occurs when wild-type P1798 cells are treated with dexamethasone in serum-free medium. Myc-transformed and cyclin D3-transformed cells also die rapidly when treated with glucocorticoids in the absence of serum. T antigen transformants are resistant to glucocorticoid-mediated apoptosis in serum-free medium. Double transformants that express both cyclin D3 and c-Myc are also resistant to apoptosis in the presence of dexamethasone. We conclude that inhibition of both CcnD3 and c-Myc genes is critical to glucocorticoid-mediated G0 arrest. Furthermore, those genes that convey resistance to growth arrest also convey resistance to cell death.

Animals↗

PubMed1995

Transforming growth factor-beta 1 inhibits cyclin D1 expression in intestinal epithelial cells.

Transforming growth factor-beta 1 (TGF-beta 1) inhibits most epithelial cell types by blocking cell cycle progression during the G1 phase. D cyclins are normally expressed during G1 and are regulators of G1 progression. One of the crucial functions of D cyclins is their ability to bind to a cyclin-dependent kinase (Cdk4). In mink lung epithelial cells, TGF-beta 1 inhibits Cdk4 expression. We have measured cell cycle progression and D cyclins and Cdk4 expression in non-transformed rat intestinal epithelial cell lines (IEC-6 and RIE-1) after TGF-beta 1 treatment. In exponentially growing cultures, TGF-beta 1 blocked DNA synthesis and suppressed cyclin D1 mRNA and protein expression, whereas the levels of cyclins D2, D3 and Cdk4 remained relatively unchanged. TGF-beta 1 was also added to G0-synchronized IEC-6 cells after serum stimulation. TGF-beta 1 prevention of G1 progression was associated with an inhibition of cyclin D1 protein expression. Cyclin D3 levels were not affected by TGF-beta 1 during G1 traverse. Our results suggest that cyclin D/Cdk4 is a crucial target of TGF-beta 1 and that regulation of this kinase is mediated through cyclin D1 in intestinal epithelial cells.

Animals↗

PubMed1995

Nuclear envelope breakdown is under nuclear not cytoplasmic control in sea urchin zygotes.

Nuclear envelope breakdown (NEB) and entry into mitosis are though to be driven by the activation of the p34cdc2-cyclin B kinase complex or mitosis promoting factor (MPF). Checkpoint control mechanisms that monitor essential preparatory events for mitosis, such as DNA replication, are thought to prevent entry into mitosis by downregulating MPF activation until these events are completed. Thus, we were surprised to find that when pronuclear fusion in sea urchin zygotes is blocked with Colcemid, the female pronucleus consistently breaks down before the male pronucleus. This is not due to regional differences in the time of MPF activation, because pronuclei touching each other break down asynchronously to the same extent. To test whether NEB is controlled at the nuclear or cytoplasmic level, we activated the checkpoint for the completion of DNA synthesis separately in female and male pronuclei by treating either eggs or sperm before fertilization with psoralen to covalently cross-link base-paired strands of DNA. When only the maternal DNA is cross-linked, the male pronucleus breaks down first. When the sperm DNA is cross-linked, male pronuclear breakdown is substantially delayed relative to female pronuclear breakdown and sometimes does not occur. Inactivation of the Colcemid after female NEB in such zygotes with touching pronuclei yields a functional spindle composed of maternal chromosomes and paternal centrosomes. The intact male pronucleus remains located at one aster throughout mitosis. In other experiments, when psoralen-treated sperm nuclei, over 90% of the zygote nuclei do not break down for at least 2 h after the controls even though H1 histone kinase activity gradually rises close to, or higher than, control mitotic levels. The same is true for normal zygotes treated with aphidicolin to block DNA synthesis. From these results, we conclude that NEB in sea urchin zygotes is controlled at the nuclear, not cytoplasmic, level, and that mitotic levels of cytoplasmic MPF activity are not sufficient to drive NEB for a nucleus that is under checkpoint control. Our results also demonstrate that the checkpoint for the completion of DNA synthesis inhibits NEB by acting primarily within the nucleus, not by downregulating the activity of cytoplasmic MPF.

Animals↗

PubMed1995

Glucocorticoid regulation of cyclin D3 gene transcription and mRNA stability in lymphoid cells.

Glucocorticoids cause G0/G1 arrest of lymphoid cells. This is due, at least in part, to a decrease in the abundance of the G1 progression factor, cyclin D3. The mRNA encoding cyclin D3 (CcnD3 mRNA) is rapidly down-regulated when dexamethasone is added to P1798 murine T lymphoma cells. Fifty percent maximum inhibition is observed within about 2 h. Maximum inhibition of 75-85% obtains within 4-5 h. Cyclin D3 protein has a half-life of about 0.5 h in P1798 cells. Consequently, the abundance of cyclin D3 protein decreases in parallel with the abundance of CcnD3 mRNA. The effects of glucocorticoids are reversible. CcnD3 mRNA returns to near control levels within 2-3 h after removal of dexamethasone. Cyclin D3 protein recovers somewhat more slowly. The data indicate that glucocorticoids regulate the abundance of cyclin D3 mRNA. There is no significant decrease in nuclear run-on transcription of CcnD3 within 6 h after addition of glucocorticoids, although transcription is inhibited more than 80% after 24 h in the presence of dexamethasone. CcnD3 mRNA is very stable in mid-log phase P1798 cells, with a half-life of more than 8 h. The half-life of CcnD3 mRNA in glucocorticoid-treated cells is less than 1 h. Actinomycin D blocks the effects of glucocorticoids, suggesting that dexamethasone induces a substance that increases the turnover rate of CcnD3 mRNA. Regulation of CcnD3 mRNA abundance and of CcnD3 transcription has been studied in cells arrested at the G1/S interface by thymidine blockade. Glucocorticoids down-regulate CcnD3 mRNA in the absence of cell cycle progression. This observation indicates that glucocorticoid inhibition of cyclin D3 expression is not a secondary consequence of cell cycle arrest. However, glucocorticoids have no significant effect on transcription of CcnD3 in G1/S phase-arrested cells. Inhibition of transcription of CcnD3 is a delayed response and probably reflects withdrawal into a G0 state, rather than any proximal consequence of glucocorticoid action. Destabilization of CcnD3 mRNA appears to be a direct effect of glucocorticoids, independent of cell cycle progression, and mediated by a glucocorticoid-induced protein(s) that accelerates the degradation of CcnD3 mRNA.

Animals↗