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PubMed1996

Expression and subcellular localization of CDK2 and cdc2 kinases and their common partner cyclin A in thyroid epithelial cells: comparison of cyclic AMP-dependent and -independent cell cycles.

Dog thyroid epithelial cells in primary culture constitute a model of positive control of DNA synthesis initiation and G0-S prereplicative phase progression by cyclic AMP as a second messenger for TSH. In tis early steps, this mitogenic control is quite distinct from cyclic AMP-independent mitogenic cascades elicited by growth factors. We demonstrate here that TSH (cyclic AMP) and EGF+serum (cyclic AMP-independent) stimulations cooperate and finally converge on proteins that control the cell cycle machinery. This convergence included a common induction of the expression of cyclin A and p34cdc2, and to a lesser extent of p33/38cdk2, which was already expressed in quiescent thyroid cells, and common changes of cdc2 and CDK2 phosphorylations as evidenced by electrophoretic mobility shifts. Kinetic differences in these processes after stimulation by TSH or EGF+serum or by these factors in combination correlated with differences in cell cycle kinetics. Moreover, an immunofluorescence analysis of these proteins using the double labeling of PCNA as a marker of each cell cycle phase shows: (1) a previously undescribed nuclear translocation of CDK2 before S phase initiation; (2) a sudden increase of cdc2 nuclear immunoreactivity at G2/mitosis transition. These data support the roles of CDK2 and cdc2 at G1/S and G2/mitosis transitions, respectively. (3) We were unable to demonstrate in individual cells a strict association between the nuclear appearance of cyclin A and G1/S transition, and an association of cyclin A and CDK2 with PCNA-stained DNA replication sites. On the other hand, the lengthening of G2 phase in the TSH/cyclic AMP-dependent thyroid cell cycle was associated with a stabilization of Tyr15 inhibitory phosphorylation of cdc2 and an especially high nuclear concentration of cyclin A and CDK2. We hypothesize that high nuclear accumulation of cyclin A and CDK2 during G2 phase could be causative in the cyclic AMP-dependent delay of mitosis onset.

Animals↗
PubMed1990

Actin gene expression in murine erythroleukemia cells treated with cytochalasin D.

The expression of cytoskeletal protein genes may be linked to both cell growth and the status of the cytoskeleton. Actin gene expression was examined in murine erythroleukemia cells treated with the microfilament disrupting agent, cytochalasin D (CD), at a concentration which was determined to inhibit cell growth and arrest cells in the S and G1 phase of the cell cycle. Levels of actin mRNA and protein synthesis were elevated eight- and sixfold, respectively, after 9 h in CD. This increase was reflected in levels of nuclear run-on actin transcripts and prevented by actinomycin D, suggesting that enhanced transcription of the actin gene was responsible for the increase. Removal of CD resulted in immediate resumption of cell cycle progression with the accumulation of a G2-phase-enriched population and a rapid return of actin mRNA and protein synthesis to control levels (half-life 4.8 h). These results are consistent with a model linking actin gene expression to cell growth by regulating transcription during the G1 and mRNA decay during the G2 phase of the cell cycle.

Actin Cytoskeleton↗
PubMed1988

Analysis of cell division among subpopulations of lymphoid cells in sheep. I. Thymocytes.

The number, distribution and surface phenotype of dividing cells in the thymus, and differences between the cell cycle status of thymocyte subpopulations, were studied in fetal and post-natal lambs using double-labelling techniques. Dividing cells were labelled in vivo for various periods with 5-bromo-2-deoxyuridine (BrdU). The proportions of constituent thymocyte subpopulations that had synthesized DNA during the labelling period were measured by flow cytometry or immunohistochemistry using a panel of monoclonal antibodies (mAbs) specific for sheep lymphocyte differentiation antigens and MHC class I and class II antigens in conjunction with an anti-BrdU mAb. The proportion of thymocytes that incorporated BrdU during a 1-hr labelling period varied with age, and levels of 30%, 13% and 9% were measured, respectively, in 40- and 125-day-old fetuses and 8-week-old lambs. Eight percent of the thymocytes in lambs were synthesizing DNA, with 4% entering the G2 phase per hour, and a substantial number of thymocytes (21%) had a G2 + M phase DNA content. A small subset of thymocytes (1-3%) recognized by mAb E-79 localized to the subcapsular region of the cortex and displayed the highest level of BrdU incorporation. Cortical-type thymocytes (CD1+) comprised 50-70% of thymocytes; however, few of these incorporated BrdU and the proportion in the G2 + M phase of the cell cycle was higher than for other thymocyte subpopulations. The 197+CD4-CD8- T cells also showed no evidence of in vivo division.

Animals↗
PubMed1989

Cell kinetic evidence suggests elevated oxidative stress in cultured cells of Bloom's syndrome.

Bromodeoxyuridine/Hoechst flow cytometry was used to analyse disturbed cell proliferation of fibroblasts and lymphoblastoid cells from Bloom's syndrome (BS). Fibroblasts show poor activation, arrest in the G2 phase of the cell cycle along with a prolongation of the G1 phase. This pattern of perturbed cells proliferation is akin to that elicited in normal fibroblasts by 4-hydroxy-nonenal, a breakdown product of lipid peroxides. Treatment with vitamin E improved growth of BS fibroblasts more strongly than growth of normal fibroblasts. Lymphoblastoid cells from BS, to the contrary, experience only a minor arrest in the G2 phase after one round of bromodeoxyuridine incorporation, but are strongly inhibited during and after the second S phase. Thus, their cell cycle arrest is dependent upon BrdU incorporation, as has been found previously in normal cells exposed to elevated concentrations of oxygen or paraquat, a superoxide generating compound. These results suggest that BS cells may suffer from an elevated, endogenous generation of oxygen free radicals.

Bisbenzimidazole↗
PubMed1985

Differential effects of ellipticine and aza-analogue derivatives on cell cycle progression and survival of BALB/c 3T3 cells released from serum starvation or thymidine double block.

10-[Diethylaminopropylamino]-6-methyl-5H-pyrido[3',4':4,5] pyrrolo[2,3-g]isoquinoline (BD-40) (NSC-327471D) is an aza-ellipticine derivative with a promising antitumor activity (M. Marty, C. Jasmin, P. Pouillard, C. Gisselbrecht, G. Gouvenia, and H. Magdalainat, 17th Annual Meeting of the American Society of Clinical Oncology, C-108, 1981) and less toxicity than ellipticine. We have compared the effects of ellipticine, several of its analogues, and two aza-analogue ellipticine derivatives (BD-40 and BR-1376) on cell cycle progression of BALB/c 3T3 mouse cells under different growth conditions. Both drug series were found to stop cell growth and block cells in G2 phase in exponentially growing cultures and cultures released from a thymidine double block. Long-term viability of these cells was completely suppressed after a short exposure to the drugs. In contrast, while ellipticine and its derivatives caused identical effects in cells recovering from serum starvation, BD-40 and BR-1376 did not block cells in G2 phase and did not prevent the completion of the first division round occurring after serum addition to quiescent cells. This transient refractory state was accompanied by a total conservation of long-term viability of these cells at least for the next 6 h following serum and drug addition. This lack of effect was not related to an impaired drug uptake by cells recovering from serum starvation or by a dramatic change in drug distribution inside the cells. These results indicate that the nitrogen substitution in the ellipticine heterocycle is an important if not unique feature for the particular effect of the aza-analogues of ellipticine. Furthermore, they suggest that, in contrast to ellipticine derivatives, these compounds require an activation step before exhibiting cytotoxicity.

Alkaloids↗
PubMed2002

Fission yeast chk1 mutants show distinct responses to different types of DNA damaging treatments.

BACKGROUND: Chk1 kinase is activated by phosphorylation at serine-345 by Rad3 checkpoint kinase and is required for DNA damage checkpoint in late S and G2 phase of S. pombe cell cycle. We studied the ability of two chk1 mutants, chk1-1 and chk1-2, to undergo phosphorylation and to delay cell cycle progression in response to different types of DNA lesions. RESULTS: Both the Chk1-1 and Chk1-2 mutant proteins are phosphorylated to various extents when DNA is damaged in early G2 phase of cell cycle by either UV irradiation or gamma irradiation. However, chk1-2 mutant does not delay cell cycle progression in a dose dependent manner specifically upon gamma irradiations. This defect is not associated with an important loss of survival. Furthermore, both chk1 mutants survive to Camptothecin treatment despite undetectable Chk1-1 or Chk1-2 phosphorylated forms. We show that both mutant proteins are not phosphorylated in cds1 devoid cells treated with ribonucleotide reductase inhibitor hydroxyurea or when the replisome is affected by a thermosensitive mutation in DNA polymerase delta. This inability is associated with the loss of checkpoint function. We found that an increased level of Crb2/Rhp9 protein specifically complements the defect of the chk1-1 mutant allowing Chk1-1 phosphorylation upon treatment with hydroxyurea of dcds1 cells. CONCLUSIONS: Mutants chk1-1 and chk1-2 behave differently according to the type of lesion generated on DNA.

Alleles↗
PubMed2003

Differentiation of Trypanosoma brucei may be stage non-specific and does not require progression of cell cycle.

Ubiquitination and proteasomal degradation of cell cycle regulatory proteins are known to play a pivotal role in controlling the progression of the eukaryotic cell cycle. Using the technique of RNA interference (RNAi) on the bloodstream form of Trypanosoma brucei, we were able to knock down expression of each of the 11 non-ATPase regulatory subunit proteins (Rpns) in the 19S regulatory complex of the 26S proteasome. In each case, the knock-down led to arrest of cells within the G1 and G2 phases, suggesting blockage of cell cycle progression at both G1/S and G2/M boundaries. This finding differs from that observed previously in the procyclic form of T. brucei, in which loss of individual Rpns blocks only passage across the G2/M boundary. Thus, proteasomal degradation of additional regulatory protein(s) may be required for exiting from G1 phase in the bloodstream form. In vitro differentiation of each of the 11 Rpn-depleted bloodstream form cell lines into the procyclic form was monitored. Each cell line proceeded to completion of the differentiation process like the wild-type cells with the total percentage of differentiated cells about equivalent to the sum of G1 and G2 cells. Thus, cells trapped in either G1 or G2 phase can apparently still enter and complete the process of differentiation, which is probably neither stage specific nor dependent on the progression of the T. brucei cell cycle. The process is probably a simple pattern change of gene expression in the trypanosome induced by a temperature decrease from 37 degrees C to 26 degrees C in the presence of citrate and cis-aconitate.

Animals↗
PubMed1984

Relation between flow cytometric DNA distribution and pathology in human bladder cancer. A report on 69 cases.

High-resolution flow cytometric measurements of cellular DNA content have been performed in 69 patients with transitional cell carcinomas of the urinary bladder. By selective sampling of cells at cystoscopy, mapping of the whole bladder could easily be performed, including the tumor as well as different areas of the surrounding normal-appearing mucosa. The cell-cycle distribution showed an increasing fraction of cells with S- and G2-phase content parallel to the World Health Organization (WHO) grade of the tumors, ranging from 1.7% S-phase cells in normal subjects to nearly 20% in tumors of WHO grade 3. WHO grade 2 tumors could be divided into two populations: (1) diploid, with a cell-cycle distribution similar to grade 1 tumors and low frequency of infiltration; and (2) aneuploid, with a high rate of infiltration and high fractions of S- and G2-phase cells. Furthermore, the aneuploid tumors could be subdivided into two main classes, one hypertriploid with high frequency of involvement of the surrounding normal appearing mucosa; and one hypotetraploid, with less frequency of such involvement and infiltration. It is concluded that by selective sampling and mapping of the bladder mucosa using flow cytometry, subclassification of this type of tumor can be performed rapidly, giving a reliable measure of the involvement of the bladder in the neoplastic disease and a biologic subclassification based on the type of DNA aberrations.

Adult↗
PubMed1994

Effects of ultraviolet irradiation on the cell cycle.

Cultured mouse Cloudman melanoma cells, EMT6 breast carcinoma cells, and 3T3 fibroblasts all accumulated in the G2/M phase of the cell cycle when exposed to UVB radiation. The effects of UVB were maximal at 20-30 mJ/cm2 for all three cell lines, and could be observed by flow cytometry as early as 12 hr post irradiation. It has been known since the mid-1970s that MSH receptor binding activity is highest on Cloudman melanoma cells when they are in the G2/M phase of their cycle. Here we show that either UVB irradiation or synchronization of Cloudman cells with colchicine results in a stimulation of MSH binding within 24 hr following treatment, a time when both treatments have resulted in accumulation of cells in the G2/M phase of the cycle. Furthermore, the two treatments performed together on the melanoma cells stimulated MSH receptor activity to the same extent as either treatment performed separately, suggesting that each may be influencing MSH receptor activity solely through a G2/M accumulation of cells. Together, these results raise the possibility that an increase in the number of cells in the G2 phase of the cell cycle is a generalized cellular response to injury, such as UV irradiation. However, in the case of pigment cells this response includes a mechanism for increasing melanin formation, i.e., increased MSH receptor activity. Should this be the case, similar G2/M "injury responses" of other cell types might be expected, consistent with their differentiated phenotypes.

Animals↗
PubMedDate not supplied

Radiation-induced cell cycle response in lymphocytes is not related to clinical side-effects in breast cancer patients.

BACKGROUND: We examined whether development of radiation-induced lung injury after irradiation for breast cancer correlates with lymphocyte radiosensitivity (LRS) in vitro. MATERIALS AND METHODS: Patients were selected from a cohort of 177 patients, who were treated with adjuvant postoperative radiotherapy (RT) for loco-regional breast cancer, and included 14 patients who had severe early lung injury measured as respiratory symptoms caused by RT and treated with corticosteroids (i.e. "cases") and a corresponding 14 control patients without such symptoms. LRS was measured as the postirradiation fraction of mitogen-stimulated blood lymphocytes in S- and G2-phase. Plasma levels of TGF-beta 1 and thiols, both suggested to be involved in the pathogenesis of radiation-induced lung injury, were also analysed. RESULTS: The result showed that cells from the controls responded to a higher extent to mitogen stimulation than cells from the cases (p < 0.05). Analysis of the fraction of S- and G2-phase cells after irradiation showed no significant difference between the two groups (p = 0.57 and 0.31, respectively). There was no difference in plasma levels of TGF-beta 1 and thiols in patients who did or did not develop pulmonary injury after RT. Tamoxifen administered before or at RT did not influence the incidence of pulmonary reactions. CONCLUSION: No differences in LRS were found between breast cancer patients with lung complications after RT and matched control patients without complications.

Adult↗
PubMed1978

In vitro induction of uncoiled chromosomes with inhibitors of RNA and protein synthesis.

When inhibitors of cellular DNA, RNA or protein synthesis are added to the growth medium of human lymphoid cells in G2 phase, these agents produce, within narrow ranges of concentrations, G banded or uncoiled chromosomes in the treated metaphase cells. It is suggested that the induced structural alteration is a consequence of an inhibition of the normal chromosome condensation and fusion of bands taking place between prometaphase and metaphase. This inhibition seems to be due to a partial inhibition of G2 phase synthesis of RNA and protein molecules, necessary for normal chromosome condensation in metaphase. It is found that this condensation can be reversed during a resting period after metaphase.

Amanitins↗
PubMed1991

[From ovocyte to biochemistry of the cell cycle].

The cell division cycle in eukaryotes contains up to three major transition points; the conversion of quiescent cells to a stage of active proliferation, the initiation of DNA synthesis (S phase) and the induction of mitosis in cells with newly replicated genome (M phase). Within the past years two strategies, have converged to identify, genetically and biochemically a key protein kinase p34 cdc2 that governs the entry into mitosis. In the fission yeast Schizosaccharomyces pombe a number of mutants in the mitotic regulatory circuit have been isolated. A central gene in the network is cdc2 which is essential for the proper execution of mitosis. The cdc2 gene interacts with a number of other genes for correct mitotic control. The Amphibian oocyte, the oocyte from Xenopus laevis particularly, is arrested at the G2 phase of the first meiotic division; when it enters M phase, it contains a dominant regulatory factor known as MPF (M-phase or maturation promoting factor). Purified MPF is an heterodimer formed of two polypeptides p34cdc2 an homologue of the product of the gene cdc2 and p45cdc13 or cyclin an homologue of the product of the gene cdc13. Biochemical studies have revealed that p34cdc2 is a phosphotyrosine protein during the G2 phase of the cell cycle, both mitotic and meiotic. The tyrosine phosphorylation of p34cdc2 is regulated by the gradual accumulation of cyclin. At the onset of M phase, the complex p34cdc2/cyclin is activated as an histone H1 kinase, and p34cdc2 is tyrosine dephosphorylated. The mechanism of activation of p34cdc2 is negatively regulated by a form of protein phosphatase 2A. Ovulated vertebrate oocytes are arrested at metaphase of the second meiotic division (M II) under the control of the proto-oncogene c-mos a protein kinase. The exit of M II phase and the initiation of early embryonic mitotic cell cycles are physiologically induced by the spermatozoa at the time of fertilization. They requires the degradation of c-mos by a Ca2+ dependent proteolytic enzyme and the destruction of cyclin by an ubiquitin dependent pathway. The Xenopus oocyte has led to the molecular elucidation of MPF and identified links between cell cycle control, protein phosphorylation and proto-oncogenes. Despite the impresive progess of recent years, there is still much to be learned about the control of meiosis in Xenopus oocytes.

Animals↗
PubMed1980

An in vivo double labelling study of the subsequent fate of cells arrested in metaphase by vincristine in the JB-1 mouse ascites tumour.

The fate of cells arrested by Vincristine (VCR) in metaphase is of interest because of the wide use of this substance in cancer chemotherapy and, particularly, in relation to its use in so-called 'synchronization' therapy. The present study was designed to answer the question of whether cells blocked in metaphase by VCR subsequently proliferate further or whether they become infertile and die. By means of a double labelling technique with [3H] and [14C]thymidine (TdR) it was shown that all VCR-arrested metaphases in the JB-1 ascites tumour subsequently became necrotic. These cells did not re-enter a viable G2 phase following arrest and thus could not take part in a wave of synchronous proliferation. In agreement with earlier studies, VCR was found to lead to arrest in metaphase, not only of cells in or shortly prior to mitosis at the time of VCR administration, but also of the majority of cells which had at this time been in the S and G2 phase.

Animals↗
PubMed1996

The plant cell cycle: conserved and unique features in mitotic control.

Somatic plant cells can use a hormone checkpoint in late G2 phase. Here cytokinin stimulates removal of phosphotyrosine from p34cdc2 kinase and concurrently capacity for activation of the kinase by Cdc25 phosphatase declines while activity of the kinase increases and cells enter mitosis. Processes unique to plant mitosis are driven by the mitotically active kinase since the enzyme taken from plant cells in metaphase, when injected, can disassemble the preprophase band microtubules that form in G2 phase at the site of the future cross wall. This action is specific, since microtubules are not depolymerised when in interphase cytoplasmic array, or spindle, or phragmoplast. Plant metaphase kinase acts as MPF by accelerating chromosome condensation and nuclear envelope breakdown.

Amino Acid Sequence↗
PubMed1979

G2 chromosomal radiosensitivity in Fanconi's anemia.

Both the peripheral lymphocytes from 4 patients affected with the inherited disease Fanconi's anemia (FA), and tissue-culture fibroblasts from skin biopsies from 3 patients similarly affected were found to be about twice as sensitive to the induction of chromatid-type chromosomal aberrations by X-rays administratered in the G2 phase of the cell cycle as cells from normal controls. Using tritiated thymidine labelling of peripheral lymphocytes and of cultured fibroblasts, it was determined that 3 affected patients and 3 normal controls all had similar percent labeled mitoses (PLM) curves, so the increased induced aberration yields seen in the FA cells do not appear to be simply a consequences of a longer than normal G2 phase of the cell cycle.

Anemia, Aplastic↗
PubMed1980

Effects of 9,10-anthracenedione, 1,4-bis[[2-[(2-hydroxyethyl)amino]-ethyl]amino]-, diacetate on cell morphology and nucleic acids of friend leukemia cells.

Treatment of Friend leukemia cells for 18 hours with 9,10-anthracenedione, 1,4-bis[[(2-hydroxyethyl)amino]ethyl]amino]-, diacetate (ANT) at concentrations up to 1.0 microgram/ml induced significant changes in cell metabolism and structure. Alterations in cell nucleic acid content were detected in cells stained with acridine orange under conditions such that DNA and RNA contents could be measured simultaneously by flow cytometry. Cells treated for 18 hours with ANT at concentrations of 0.05-0.1 microgram/ml became partially blocked at the G2 phase. In addition, about 30% of the cells became polyploid and demonstrated diplochromosomes at the 8C level of mitosis. The nuclear chromatin of blocked cells had an altered structure as reflected by a change in sensitivity of DNA in situ to denaturation induced by low pH. All viable cells treated with ANT for 18 hours at concentrations of 0.4-1.0 microgram/ml were blocked in G2 phase. These cells had significantly more RNA than did untreated cells. Transmission electron microscopic observations of thin-sectioned cells suggested that this increased RNA content in ANT-treated cells was mostly due to an approximately 50% increased cell diameter and partly due to a disproportionate increase in nucleolar size. In addition, electron microscopy revealed that ANT caused increased chromatin condensation and granulation. The drug had no apparent effect on production of the endogenous Friend murine leukemia virus.

Animals↗
PubMed1996

p21waf1/cip1 protein associates with the detergent-insoluble form of PCNA concomitantly with disassembly of PCNA at nucleotide excision repair sites.

Evidence is presented that after exposure of normal human fibroblasts to UV-C light, nuclear binding of the proliferating cell nuclear antigen (PCNA) required for nucleotide excision repair, was rapidly triggered in the G1 and G2 phases of the cell cycle. Association to repair sites of the detergent-insoluble form of PCNA reached a peak 15-30 min after irradiation, and then decreased to basal levels within 24-48 h. In contrast, the nuclear association of p21 protein showed a slower kinetics, reaching maximal levels between 24 and 48 h but, similarly to PCNA, occurring only in G1 and G2 phases. Although the two proteins are known to be associated as detergent-soluble proteins, it is unknown whether they associate also in the detergent-insoluble form. To address this question, the chromatin-bound form of PCNA was released by using DNAse I. DNA digestion resulted in the almost complete release of PCNA from its binding sites, while only about 60% of nuclear-bound p21 could be solubilized. Immunoprecipitation of PCNA and p21 released by enzymatic digestion showed that p21 was associated with PCNA bound to late DNA repair sites. These results indicate that during nucleotide excision repair, nuclear binding of PCNA precedes that of p21 protein, and suggest that temporal association of p21 with the detergent-insoluble fraction is coincident with the disassembly of PCNA from DNA repair sites.

Cell Cycle↗
PubMed1985

Functional and structural heterogeneity of nucleoli: the dependence of the activity of transport of newly synthesized rRNA on nucleolar size and phase of the cell cycle.

On the basis of radioactivity of interphase cells in the antheridial filaments of Chara vulgaris after a 20 min. incubation with 3H-uridine and after a 40 min. postincubation the activity of synthesis and transport of newly synthesized RNA has been studied in different-sized nucleoli. Investigations of their ultrastructure have been also performed. It was found that the smallest nucleoli from 0.2-0.5 micron in diameter indicate the structure typical of nucleoli inactive in RNA synthesis. Nucleoli ranging from 0.5-2.0 microns in diameter which constitute the most numerous population were evidenced to be active both in synthesis and transport of newly synthesized RNA. Their structure is characterized by nucleoloneme-like appearance with small quantity of granular components. The largest nucleoli which are present in small numbers at the onset of interphase gradually increase in number till G2 phase. They indicate similar transcriptional activity as the latter category of nucleoli but a lower activity of transport of newly synthesized RNA; moreover, they abound in granular components surrounding the nucleoloneme. Nucleoli from all size-classes increase their transcriptional activity in middle S and middle G2 phases. Transport of newly synthesized RNA, however, is differentiated and it depends on both the phase of the cell cycle and nucleolar size.

Cell Cycle↗