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PubMed1987

Role of arachidonic acid release in the G2 delay induced by tumor promoter TPA in HeLa cells.

The tumor promoter TPA2 (12-O-tetradecanoylphorbol-13-acetate) has been shown to exhibit a radiomimetic activity on the cell cycle of HeLa cells (V. Kinzel, J. Richards, and M. Stöhr (1980) Science 210, 429). The response includes a delay of cells in G2 phase. The relation between TPA-induced release of arachidonic acid (AA) and the inhibition in G2 phase was studied. Exogenous AA (greater than 10(-4) M; in presence of 10% serum) is shown to delay HeLa cells in G2 and to enhance the effectiveness of TPA in this respect. The inhibition of the TPA-induced AA liberation by fluocinolone acetonide, however, does not influence the TPA-effected G2 delay. The diacylglycerols 1,2-dioctanoyl-glycerol and 1-oleoyl-2-acetylglycerol delay HeLa cells in G2 but without major stimulation of AA liberation. On the basis of the data it is concluded that AA released from HeLa cells due to the action of TPA is not involved in the TPA-induced delay of cells in G2 phase.

Arachidonic Acid↗
PubMed1980

Nuclear division cycle in Neurospora crassa hyphae under different growth conditions.

Treatment with picolinic acid blocked Neurospora crassa nuclei in G1, and recovery from the treatment allowed a synchronous wave of deoxyribonucleic acid synthesis to occur. Nuclei, which appeared as compact globular bodies during the period of blockage, assumed a ring shape during the following S phase, which was also maintained in the G2 phase. The proportion of compact globular nuclei was much higher in hyphae growing at lower rates, whereas that of ring nuclei increased when the hyphae were growing at higher rates. Horseshoe nuclei (probably mitotic nuclei) and double ring nuclei were also observed in growing hyphae, but their frequencies were low and fairly independent of the rate of growth. The length of the S phase of the Neurospora nuclear division cycle was determined to be about 30 min. From the frequencies of the phase-specific nuclear shapes, the durations of the G1 phase and the combined S plus G2 phases were calculated. The results showed that variations in the growth rates of the mycelia were mainly coupled with variations in the G1 phase of the nuclear division cycle. For mycelia growing in minimal sucrose, the lengths of all of the phases of the nuclear division cycle were estimated.

Cell Division↗
PubMed1994

The Dictyostelium cell cycle and its relationship to differentiation.

The dictyostelium vegetative cell cycle is characterized by a short mitotic period followed immediately by a short S-phase (less than 30 min) and a long and variable G2 phase. The cell cycle continues during differentiation despite a decrease in cell mass: DNA replication and mitosis occur early in development and also at the tipped aggregate stage. Cells that are in mitosis, S-phase or early G2, when starved differentiate into prestalk cells and cells that are in the middle of G2 differentiate into prespore cells. We postulate that there is a restriction point late in the G2 phase, about 1-2 h before mitosis, where the cells can be arrested either by starvation and the initiation of development, by growing into stationary phase, or by prolonged incubation at low temperature. During development, this block persists to the tipped aggregate stage, where it is specifically released in prespore cells, and these cells then go through one more round of cell division. Genes encoding components of the cell cycle machinery have recently been isolated and attempts to specifically block the cell cycle by reverse genetics to study the effects on differentiation have been initiated.

Animals↗
PubMed1981

Effects of the L isomer (+)-1,2-bis(3,5-dioxopiperazine-1-yl)propane on cell survival and cell cycle progression of cultured mammalian cells.

The effects of the L isomer (+)-1,2-bis(3,5-dioxopiperazine-1-yl)propane (ICRF 159; NSC 169780) on cell viability, growth, and progression through the cell cycle were investigated in suspension cultures of murine leukemia (Friend leukemia and L1210) cells and normal human lymphocytes stimulated with phytohemagglutinin and in adherent cultures derived from human neuroblastoma and Chinese hamster ovary (CHO) cells. CHO cell colony formation was inhibited by 50% following either an 8.5-hr exposure of exponentially growing cells to 10 micrograms ICRF 159 per ml or a 24-hr exposure to 3 micrograms ICRF 159 per ml. This effect was cell cycle phase specific; early G1- and G2-phase cells were more sensitive than were late-G1- or early and mid-S-phase CHO cells. Stationary-phase CHO cells were unaffected by the drug at concentrations up to 500 micrograms/ml. Incubation of L1210 cells with 3 micrograms ICRF 159 per ml for 24 hr or with 10 micrograms ICRF 159 per ml for 6 hr inhibited cell growth by 50%. In contrast, 24-hr incubation of human lymphocytes with up to 50 micrograms ICRF 159 per ml had no effect on their viability or on their ability to be stimulated by phytohemagglutinin. Constant exposure of Friend leukemia, L1210, human neuroblastoma, and phytohemagglutinin-stimulated human lymphocytes to 10.0 to 50 micrograms ICRF 159 per ml resulted in inhibition of cell division which led to cell growth at higher ploidy levels. Thus, proliferating human cells of normal or tumor origin and murine leukemic cell lines all had a similar sensitivity to the drug. Detailed analysis of cell cycle progression in L1210 cells in the presence of the drug determined that cell progression through G1 phase (G1A to G1B transition) was slowed by approximately 50%. The rate of traverse of cells through S phase was also slowed. However, the most pronounced effect was the accumulation of cells in G2 phase occurring almost immediately after addition of the drug. The data suggest that the L isomer has a range of cytotoxicity and identical cytokinetic effects similar to that of the clinically tested racemate (+/-)-ICRF 159 (NSC 129943) and, therefore, that the more soluble L isomer may have increased clinical applicability.

Animals↗
PubMed2000

The AgNOR proteins: qualitative and quantitative changes during the cell cycle.

AgNOR proteins are a set of argyrophilic nucleolar proteins that accumulate in highly proliferating cells whereas their expression is very low in non-proliferating cells. Some of these proteins remain associated with the nucleolar organizer regions (NORs) during mitosis. In situ, the expression of AgNOR proteins is measured globally by quantification of the level of silver staining using morphometry and image analysis. To go deeper into the understanding of the relationship between the cell cycle and quantity of AgNOR proteins, it was necessary to determine the phases of cell cycle during which expression of AgNOR varies and what are the most variable proteins in each phase. To answer these questions, we set up the protocol permitting to detect and quantify AgNOR proteins on protein samples electrophoresed and transferred onto nitrocellulose membranes. This approach makes it possible to quantitatively evaluate individual AgNOR proteins and identify them, using nucleolar, nuclear and whole interphasic cell extracts, and chromosome-associated protein extracts. By this means, we identified nucleolin and protein B23 as the two major AgNOR proteins in the nucleolus during interphase and subunits of RNA polymerase I and transcription factor UBF as AgNOR proteins remaining associated with NORs during mitosis. We also observed that the increase in the level of nucleolin and protein B23 in rat liver seems to be linked with the cell cycle and not exclusively with stimulation of ribosomal gene (rDNA) transcription. Similarly in synchronized cells, the amount of nucleolin rapidly increases when cells enter the S phase (1.6-fold of the value of serum-deprived cells at 9 h, and 2.35-fold at 12 h after refeeding). The amount of protein B23 exhibits a lower and progressive increase with a maximum when the percentage of cells in G2 phase increased, i.e. after 24 h of cell cycle stimulation. We consider that the amount of AgNOR proteins can be a marker of proliferation, because this amount is related to cell cycle phases, schematically low for G1 phase and high for S-G2 phase. Thus, it is a measure of the relative proportion of cells in each phase, and consequently of the timing of each phase. The higher value indicates that the major part of the cells are in the S-G2 phase and correlatively few are in the G1 phase, and this characterizes a rapid cell cycle.

Animals↗
PubMed2002

A novel indolocarbazole, ICP-1, abrogates DNA damage-induced cell cycle arrest and enhances cytotoxicity: similarities and differences to the cell cycle checkpoint abrogator UCN-01.

DNA damaging agents such as cisplatin arrest cell cycle progression at either G1, S, or G2 phase, although the G1 arrest is only seen in cells expressing the wild-type p53 tumor suppressor protein. We have reported that 7-hydroxystaurosporine (UCN-01) overcomes S and G2 phase arrest and enhances the cytotoxicity of cisplatin. Abrogation of arrest appears to be selective for cells defective in p53 and therefore provides a potential, tumor-targeted therapy. Unfortunately, UCN-01 binds avidly to human plasma proteins, limiting access to the tumor. A screen of related indolocarbazoles identified analogues with both beneficial and undesirable properties. This led to a synthetic program to develop a novel analogue rationally designed to overcome the obstacles observed with the other analogues. We report the synthesis and analysis of a novel analogue, ICP-1. This analogue abrogated S and G2 phase arrest and enhanced cytotoxicity induced by cisplatin only in p53 defective cells. ICP-1 also abrogated arrest and enhanced cell killing induced by the topoisomerase I inhibitor SN38. Analysis of proteins that regulate cell cycle arrest suggest both drugs inhibit checkpoint kinases Chk1 and/or Chk2. In contrast to UCN-01, checkpoint abrogation by ICP-1 was only slightly inhibited by human plasma. UCN-01 and ICP-1 differed significantly in other regards. UCN-01 potently enhanced the activity of 1-beta-D-arabinofuranosylcytosine in both p53 wild-type and mutant cells, whereas ICP-1 was inactive in this combination. This property of UCN-01 was independent of its ability to inhibit protein kinase C because more specific inhibitors of protein kinase C failed to enhance cell killing induced by 1-beta-D-arabinofuranosylcytosine. High concentrations of UCN-01 also inhibit C-TAK1 that results in S phase-arrested cells directly entering mitosis, but this property was not observed with ICP-1. Hence, ICP-1 appears to be a more selective inhibitor of the S and G2 cell cycle checkpoint than previously studied analogues and is worthy of study in preclinical tumor models.

Alkaloids↗
PubMed2003

Down-regulation of proliferating cell nuclear antigen gene expression occurs during cell cycle arrest induced by human fecal water in colonic HT-29 cells.

Cancer is a disease in which the cell cycle is altered, and the elucidation of the mechanisms by which constituents of human fecal water influence the cell cycle can lead to noninvasive measurement of colon cancer risk. The purpose of the present study was to investigate the effect of human fecal water on HT-29 cell-cycle progression with sodium selenite as a reference for comparison. Both human fecal water (2.5-5.0%) and selenite (3-4 micro mol/L) significantly inhibited cell growth. Cell-cycle analysis revealed that human fecal water decreased the proportion of S + G2 phase cells and increased that of G1 phase cells. In contrast, selenite decreased G1 phase cells and increased proportions of S and G2 phase cells. Both 5% human fecal water and 4 micro mol/L selenite greatly increased the mRNA level of the cyclin-dependent kinase inhibitor gene p21(waf1). Interestingly, the mRNA levels of cyclin A and proliferating cell nuclear antigen (PCNA) were dramatically decreased by 69 and 62%, respectively, in HT-29 cells treated with fecal water but not selenite. In contrast, the mRNA level of DNA damage-inducible transcript 1, gadd45, was significantly increased by 2.28-fold in HT-29 cells treated with selenite but not fecal water. Furthermore, a PCNA gene promoter was cloned into a luciferase reporter construct and its activity was significantly reduced in a dose-dependent manner in cells treated with fecal water but not selenite. Collectively, these results suggest that human fecal water and selenite can differentially induce growth arrest genes, and that PCNA gene expression is uniquely and highly sensitive to human fecal water.

Body Water↗
PubMed2004

SCE analysis in G2 lymphocyte prematurely condensed chromosomes after exposure to atrazine: the non-dose-dependent increase in homologous recombinational events does not support its genotoxic mode of action.

Several studies have been carried out to evaluate the mutagenic and carcinogenic potential of atrazine, the most prevalent of triazine herbicides classified as a "possible human carcinogen". The majority of these studies have been negative but positive responses have been also reported including mammary tumors in female Sprague-Dawley rats. Sister chromatid exchanges (SCEs) caused by the presence of DNA lesions at the moment of DNA replication have been extensively used for genotoxicity testing, but for non-cytotoxic exposures to atrazine controversial results have been reported. Even though exposures to higher concentrations of atrazine could provide clear evidence for its genotoxicity, conventional SCE analysis at metaphase cells cannot be used because affected cells are delayed in G2-phase and do not proceed to mitosis. As a result, the genotoxic potential of atrazine may have been underestimated. Since clear evidence has been recently reported relating SCEs to homologous recombinational events, we are testing here the hypothesis that high concentrations of atrazine will cause a dose-dependent increase in homologous recombinational events as quantified by the frequency of SCEs analyzed in G2-phase. Towards this goal, a new cytogenetic approach is applied for the analysis of SCEs directly in G2-phase prematurely condensed chromosomes (PCCs). The methodology enables the visualization of SCEs in G2-blocked cells and is based on drug-induced PCCs in cultured lymphocytes. The results obtained for high concentrations of atrazine do not demonstrate a dose-dependent increase in homologous recombinational events. They do not support, therefore, a genotoxic mode of action. However, they suggest that an important part in the variation of SCE frequency reported by different laboratories when conventional SCE analysis is applied after exposure to a certain concentration of atrazine, is due to differences in cell cycle kinetics of cultured lymphocytes, rather than to a true biological variation in the cytogenetic end point used.

Adult↗
PubMed1992

[Flow cytometric analysis of the effect of DWA2114R on the cell cycle traverse of RPMI 8402 cells--comparison with CDDP].

The (-) - (R) - 2-aminomethylpyrrolidine (1, 1-cyclobutanedicarboxylato) platinum(II) monohydrate, DW A2114R, is a new derivative of cis-dichlorodiammineplatinum(II), CDDP, which is reported to have a lower nephrotoxicity than CDDP. In this report, we investigated the effects of DW A2114R, in comparison with CDDP, on the cell growth, the cell cycle traverse and the colony-forming ability of human leukemia RPMI 8402 cells in vitro. The inhibitory effects of DW A2114R on the cell growth and the colony-forming ability were about 1/10 and 1/10-1/30, respectively, of the CDDP dose. And DW A2114R showed a higher time-dependency than CDDP in the experiment comparing exposure time to the drugs. Analysis of DNA histograms obtained by flow cytometry revealed that DW A2114R had a similar effect on the cell cycle traverse to that of CDDP. Both drugs exerted their growth-inhibitory effect by blocking cells at G2 phase. At higher concentrations, they prolonged the traverse of cells through S phase and completely inhibited them throughout the cell cycle. The comparative study on the colony-forming ability suggested that the G2 phase accumulation was irreversible and that these drugs are lethal, so the G2 phase accumulation could be considered a cytocidal effect of the drugs. In early clinical studies, however, the dose of DW A2114R was put at about 15-fold higher than the therapeutic dose of CDDP. This was comparable with our results in vitro and DW A2114R showed a much lower toxicities, with special regard to nephrotoxicity, than CD DP. These clinical data and our results suggest that DW A2114R could be clinically effective as an antitumor agent.

Antineoplastic Agents↗
PubMed1981

Cation transport and growth regulation in neuroblastoma cells. Modulations of K+ transport and electrical membrane properties during the cell cycle.

Cation transport and membrane potential were studied during the cell cycle of neuroblastoma cells (clone Neuro-2A) to investigate the role of these parameters in growth regulation. The cells were synchronized by selective detachment of mitotic cells. The membrane potential and intracellular K+ activity were measured with conventional and K+-selective microelectrodes respectively. Both the membrane potential and K+ activity were high in mitosis, decreased to half maximal in G1 phase, and rose again during S phase. K+ efflux across the plasma membrane was studied with 42K+ as a radioactive tracer using a washing method for cells grown in monolayer and a continuous efflux method for mitotic cells in suspension. The intracellular K+ content and unidirectional K+ efflux rate obtained from these measurements showed modulations during the cell cycle similar to those of the membrane potential. Using equations of electrodiffusion theory the membrane permeabilities to K+ and Na+ were calculated. These permeabilities were high in mitosis, decreased rapidly in G1 phase and increased during S phase, followed by a transient decrease in G2 phase. A rapid increase was observed between G2 phase and the next mitosis. A similar pattern was obtained for the K+ conductance. K+ resistance changes during the cell cycle were similar to changes in the specific membrane resistance, measured by microelectrodes, except for the early cell cycle phases (mitosis and G1). These studies clearly demonstrate large modulations of the passive membrane permeability properties during the cell cycle. These modulations can be correlated with physicochemical membrane variations during the cell cycle, such as membrane fluidity and lateral mobility of lipids.

Animals↗
PubMed1985

Cell cycle kinetic measurements in an irradiated rat rhabdomyosarcoma using a monoclonal antibody to bromodeoxyuridine.

Cell cycle kinetics after X-irradiation were studied in a solid rat rhabdomyosarcoma using a monoclonal antibody to bromodeoxyuridine (BrdUrd) in cells in which the DNA was labeled by BrdUrd. It could be shown that this tumor was composed of about 80% diploid host cells, and only 20% of the cells in the dissociated tumor were actually tetraploid tumor cells. When rats were injected intraperitoneally with BrdUrd to label S-phase cells in the tumor, only a fraction of both types of cells became labeled with BrdUrd during S-phase, even 24 h after injection. The diploid BrdUrd-labeled cells progressed rapidly into cycle; 4 h after injection of BrdUrd, labeled diploid G1-phase cells could be observed. Only 25% of the tetraploid S-phase cells could be labeled by a single injection of BrdUrd (160 mg/kg body weight). These labeled tetraploid cells progressed through the cell cycle with similar velocities as did labeled diploid cells. Using a "Mini Osmotic Pump" containing bromodeoxycytidine (BrdCyd) at high concentration (0.3 mol/L) that released BrdCyd continuously into the organism where it was converted to BrdUrd, it could be shown that after 2 days about 60% of cells in S-phase and 70% of cells in G2-phase were labeled. The fraction of labeled G2-phase cells in irradiated tumors (D = 10 and 20 Gy) was enhanced between 10 and 50 h after irradiation due to a radiation-induced G2 block in cycling tetraploid tumor cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

The effect of triethylene glycol dimethacrylate on the cell cycle of mammalian cells.

The induction of DNA damage by a genotoxic agent is a signal leading to cell cycle delay, and thereby enables and induces DNA repair prior to cell cycle progression. Triethylene glycol dimethacrylate (TEGDMA), a monomer of dental resinous materials, caused mutagenic effects in mammalian cells probably as a consequence of DNA damage. Therefore, we hypothesized that TEGDMA will induce a cell cycle delay in mammalian cells. Here, cell lines deficient and proficient of a functional p53 tumor suppressor protein were used to study the effects of TEGDMA on the various phases of the cell cycle. V79 Chinese hamster lung fibroblasts (p53 deficient), N1 human skin fibroblasts (p53 proficient), and primary human pulp fibroblasts (p53 proficient) were exposed to increasing TEGDMA concentrations (0-3 mmol/l). Cell survival and vitality were determined after a 24-h exposure period and a 24-h recovery period, and the distribution of cells between the phases of the cell cycle in untreated and TEGDMA-treated cultures was analyzed by flow cytometry. The majority of the TEGDMA-treated V79 cells accumulated in G2 phase. In contrast, about 30% of human N1 fibroblasts were reversibly blocked in G1 phase by 0.5-3.0 mmol/l TEGDMA. The fraction of G2-phase cells was increased only by high TEGDMA concentrations. The percentage of human pulp cells in G1 phase increased very slightly with 1 mmol/l TEGDMA, but cell numbers in G1 phase were reduced by 10-20% by 1.5-3 mmol/l TEGDMA. The percentage of pulp cells in G2 phase increased about 2-fold without any obvious effect of a 24-h recovery period. Therefore, TEGDMA caused cell cycle delays through p53-dependent and independent pathways in the various cell lines. From these results, we conclude that TEGDMA may influence physiological processes like cell growth and differentiation of human pulp cells in vivo.

Animals↗
PubMed1985

Cell cycle-dependent variations in RNA polymerase activity in the nuclear and cytoplasmic fractions of Physarum polycephalum.

The RNA polymerase activities of the soluble enzymes from the nuclear and cytoplasmic fractions from synchronous macroplasmodia of Physarum polycephalum were measured. Activity in the nuclear fraction prepared from cells in the G2 phase was several times higher than that at metaphase, and activity in the cytoplasmic fraction was much higher at metaphase than in the G2 phase. The amount of activity was nearly constant during the late G2 phase and early prophase, then rose abruptly just before metaphase, becoming tenfold higher at metaphase then decreasing rapidly during anaphase-telophase. The cytoplasmic activity at metaphase was attributed almost exclusively to the type II (or B) enzyme as shown by alpha-amanitin inhibition and chromatographic analysis of the enzyme. Results indicate that at metaphase RNA polymerase II molecules are loosely associated with condensed chromosomes or that they move from the mitotic nuclei into the cytoplasm.

Cell Cycle↗
PubMed1995

Cell cycle-dependent regulation of RNA polymerase II basal transcription activity.

Regulation of transcription by RNA polymerase II (pol II) in eukaryotic cells requires both basal and regulatory transcription factors. In this report we have investigated in vitro pol II basal transcription activity during the cell cycle by using nuclear extracts from synchronized HeLa cells. It is shown that pol II basal transcription activity is low in the S and G2 phases and high in early G1 phase and TFIID is the rate limiting component of pol II basal transcription activity during the cell cycle. Further analyses reveal that TFIID exists as a less active form in the S and G2 phases and nuclear extracts from S and G2 phase cells contain a heat-sensitive repressor(s) of TATA box binding protein (TBP). These results suggest that pol II basal transcription activity is regulated by a qualitative change in the TFIID complex, which could involve repression of TBP, during the cell cycle.

Cell Cycle↗
PubMed1990

Mathematical model analysis of mouse epidermal cell kinetics measured by bivariate DNA/anti-bromodeoxyuridine flow cytometry and continuous [3H]-thymidine labelling.

In a previous study the epidermal cell kinetics of hairless mice were investigated with bivariate DNA/anti-bromodeoxyuridine (BrdU) flow cytometry of isolated basal cells after BrdU pulse labelling. The results confirmed our previous observations of two kinetically distinct sub-populations in the G2 phase. However, the results also showed that almost all BrdU-positive cells had left S phase 6-12 h after pulse labelling, contradicting our previous assumption of a distinct, slowly cycling, major sub-population in S phase. The latter study was based on an experiment combining continuous tritiated thymidine [( 3H]TdR) labelling and cell sorting. The purpose of the present study was to use a mathematical model to analyse epidermal cell kinetics by simulating bivariate DNA/BrdU data in order to get more details about the kinetic organization and cell cycle parameter values. We also wanted to re-evaluate our assumption of slowly cycling cells in S phase. The mathematical model shows a good fit to the experimental BrdU data initiated either at 08.00 hours or 20.00 hours. Simultaneously, it was also possible to obtain a good fit to our previous continuous labelling data without including a sub-population of slowly cycling cells in S phase. This was achieved by improving the way in which the continuous [3H]TdR labelling was simulated. The presence of two distinct subpopulations in G2 phase was confirmed and a similar kinetic organization with rapidly and slowly cycling cells in G1 phase is suggested. The sizes of the slowly cycling fractions in G1 and G2 showed the same distinct circadian dependency. The model analysis indicates that a small fraction of BrdU labelled cells (3-5%) was arrested in G2 phase due to BrdU toxicity. This is insignificant compared with the total number of labelled cells and has a negligible effect on the average cell cycle data. However, it comprises 1/3 to 1/2 of the BrdU positive G2 cells after the pulse labelled cells have been distributed among the cell cycle compartments.

Animals↗
PubMed1988

Influence of cis-diamminedichloroplatinum(II) on DNA synthesis and cell cycle progression in excision repair proficient and deficient Chinese hamster ovary cells.

DNA has been implicated as the critical intracellular target for cis-diamminedichloroplatinum(II) (cis-DDP) action. Inhibition of DNA synthesis is a consequence of platination and has become accepted as the critical step in cis-DDP-induced toxicity. We have previously demonstrated that, following incubation with cis-DDP, murine leukemia L1210 cells progress through synthesis only to arrest in the G2 phase of the cell cycle. The G2 arrest was transient at low drug concentrations and was persistent at higher concentrations with a concomitant loss of viability. Chinese hamster ovary cell lines both proficient and deficient for DNA excision repair have been used to analyze the relationship between inhibition of DNA synthesis and toxicity and to determine whether DNA repair is necessary for cell cycle progression. Two repair-deficient cell lines were hypersensitive to cis-DDP and demonstrated a marked arrest in the G2 phase. The arrest was transient over only a small range of concentrations. At higher concentrations, the arrest was persistent and the cells subsequently died. Incorporation of [3H]thymidine into macromolecules demonstrated no inhibition of DNA synthesis while these cells progressed through the S phase. In contrast, at higher, but nontoxic, concentrations of cis-DDP, the repair-proficient cells exhibited inhibition of DNA synthesis while in S. At toxic concentrations, these cells also arrested in G2. Therefore, direct inhibition of DNA synthesis correlated only with the concentration of drug and not with the different sensitivities of the cell lines. Arrest of cells in G2 did correlate with toxicity. In every cell line, the appearance of G2-arrested cells preceded cell disintegration. It is proposed that the G2-arrested cells preceded cell disintegration. It is proposed that the G2 arrest results from the inability of the cells to transcribe genes required for passage into mitosis. Cells proficient in DNA repair can circumvent this arrest by repairing the damaged DNA and permitting transcription to proceed. These results support the hypothesis that inhibition of DNA synthesis is not the critical step in cis-DDP-induced cytotoxicity.

Animals↗
PubMed1996

Competency in mismatch repair prohibits clonal expansion of cancer cells treated with N-methyl-N'-nitro-N-nitrosoguanidine.

The phenomenon of alkylation tolerance has been observed in cells that are deficient in some component of the DNA mismatch repair (MMR) system. An alkylation-induced cell cycle arrest had been reported previously in one MMR-proficient cell line, whereas a MMR-defective clone derived from this line escapes from this arrest. We examined human cancer cell lines to determine if the cell cycle arrest were dependent upon the MMR system. Growth characteristics and cell cycle analysis after MNNG treatment were ascertained in seven MMR-deficient and proficient cell lines, with and without confirmed mutations in hMLH1 or hMSH2 by an in vitro transcription/translation assay. MMR-proficient cells underwent growth arrest in the G2 phase of the cell cycle after the first S phase, whereas MMR-deficient cells escaped an initial G2 delay and resumed a normal growth pattern. In the HCT116 line corrected for defective MMR by chromosome 3 transfer, the G2 phase arrest lasted more than five days. In another MMR-proficient colon cancer cell line, SW480, cell death occurred five days after MNNG treatment. A competent MMR system appears to be necessary for G2 arrest or cell death after alkylation damage, and this cell cycle checkpoint may allow the cell to repair damaged DNA, or prevent the replication of mutated DNA by prohibiting clonal expansion.

Alkylating Agents↗