G1 Arrest In Cells Research Articles

Anti-tumor effect of bisphosphonate (YM529) on non-small cell lung cancer cell lines

BackgroundYM529 is a newly developed nitrogen-containing bisphosphonate (BP) classified as a third-generation BP that shows a 100-fold greater potency against bone resorption than pamidronate, a second-generation BP. This agent is, therefore expected to be extremely useful clinically for the treatment of osteoporosis and hypercalcemia. Recently, YM529 as well as other third-generation BPs have also been shown to exert anti-tumor effects against various types of cancer cells both in vitro or/and in vivo. In this study, we investigate the anti-tumor effect of YM529 on non-small cell lung cancer (NSCLC).MethodsDirect anti-tumor effect of YM529 against 8 NSCLC cell lines (adenocarcinoma: H23, H1299, NCI-H1819, NCI-H2009, H44, A549, adenosquamous cell carcinoma: NCI-H125, squamous cell carcinoma: NCI-H157) were measured by MTS assay and calculated inhibition concentration 50 % (IC50) values. YM529 induced apoptosis of NCI-H1819 was examined by DNA fragmentation of 2 % agarose gel electrophoresis and flowcytometric analysis (sub-G1 method). We examined where YM529 given effect to apoptosis of NSCLC cells in signaling pathway of the mevalonate pathway by western blotting analysis.ResultsWe found that there was direct anti-tumor effect of YM529 on 8 NSCLC cell lines in a dose-dependent manner and their IC50 values were 2.1 to 7.9 μM and YM529 induced apoptosis and G1 arrest cell cycle with dose-dependent manner and YM529 caused down regulation of phospholyration of ERK1/2 in signaling pathways of NSCLC cell line (NCI-H1819).ConclusionOur study demonstrate that YM529 showed direct anti-tumor effect on NSCLC cell lines in vitro, which supports the possibility that third-generation BPs including YM529 can be one of therapeutic options for NSCLC.

Manipulation of cell cycle progression can counteract the apparent loss of correction frequency following oligonucleotide-directed gene repair

BackgroundSingle-stranded oligonucleotides (ssODN) are used routinely to direct specific base alterations within mammalian genomes that result in the restoration of a functional gene. Despite success with the technique, recent studies have revealed that following repair events, correction frequencies decrease as a function of time, possibly due to a sustained activation of damage response signals in corrected cells that lead to a selective stalling. In this study, we use thymidine to slow down the replication rate to enhance repair frequency and to maintain substantial levels of correction over time.ResultsFirst, we utilized thymidine to arrest cells in G1 and released the cells into S phase, at which point specific ssODNs direct the highest level of correction. Next, we devised a protocol in which cells are maintained in thymidine following the repair reaction, in which the replication is slowed in both corrected and non-corrected cells and the initial correction frequency is retained. We also present evidence that cells enter a senescence state upon prolonged treatment with thymidine but this passage can be avoided by removing thymidine at 48 hours.ConclusionTaken together, we believe that thymidine may be used in a therapeutic fashion to enable the maintenance of high levels of treated cells bearing repaired genes.

DNA Degradation at Unprotected Telomeres in Yeast Is Regulated by the CDK1 (Cdc28/Clb) Cell-Cycle Kinase

In the absence of functional telomeric cap protection, the ends of eukaryotic chromosomes are subject to DNA damage responses that lead to cell-cycle arrest and, eventually, genomic instability. However, the controlling activities responsible for the initiation of genome instability on unprotected telomeres remained unclear. Here we show that in budding yeast, unprotected telomeres undergo a tightly cell-cycle-regulated DNA degradation. Ablation of the function of essential capping proteins Cdc13p or Stn1p only caused telomere degradation in G2/M, but not in G1 of the cell cycle. Accordingly, G1-arrested cells with unprotected telomeres remained viable, while G2/M-arrested cells failed to recover. The data also show that completion of S phase and the activity of the S-Cdk1 kinase were required for telomere degradation. These results strongly suggest that after a loss of the telomere capping function, telomere-led genome instability is caused by tightly regulated cellular DNA repair attempts.

The dose-dependent H2O2 stress response promotes increased survival forSchizosaccharomyces pombe cells expressing HIV-1 Vpr

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) exerts multiple effects on viral and host cellular activities during infection, including induction of the cell cycle G2 arrest, and cell death in both human cells and the fission yeast Schizosaccharomyces pombe. We show that treament of exponential-phase wild-type Vpr-expressing S. pombe cells with a low, subinhibitory concentration (0.15 mmol/L) of hydrogen peroxide and 0.1 mmol/L thiamine significantly increased both cell proliferation and survival rates and decreased the number of elongated G2-arrested cells. Short-term, H2O2-induced adaptive stress increased the survival of the cells while acute stress conditions interrupted the Vpr-mediated death of the cells; however, no changes in cell length or cell phase were detected. The results suggest the importance of the oxidative status of the cells in Vpr-mediated processes. Our findings contribute to the development of a new approach via which to investigate the contribution of Vpr to HIV pathogenesis and to reduce the Vpr-mediated effects in HIV-infected patients.

Global Replication-Independent Histone H4 Exchange in Budding Yeast

The eukaryotic genome is packaged together with histone proteins into chromatin following DNA replication. Recent studies have shown that histones can also be assembled into chromatin independently of DNA replication and that this dynamic exchange of histones may be biased toward sites undergoing transcription. Here we show that epitope-tagged histone H4 can be incorporated into nucleosomes throughout the budding yeast (Saccharomyces cerevisiae) genome regardless of the phase of the cell cycle, the transcriptional status, or silencing of the region. Direct comparisons reveal that the amount of histone incorporation that occurs in G(1)-arrested cells is similar to that occurring in cells undergoing DNA replication. Additionally, we show that this histone incorporation is not dependent on the histone H3/H4 chaperones CAF-1, Asf1, and Hir1 individually. This study demonstrates that DNA replication and transcription are not necessary prerequisites for histone exchange in budding yeast, indicating that chromatin is more dynamic than previously thought.

Genome-wide replication profiles of S-phase checkpoint mutants reveal fragile sites in yeast

The S-phase checkpoint kinases Mec1 and Rad53 in the budding yeast, Saccharomyces cerevisiae, are activated in response to replication stress that induces replication fork arrest. In the absence of a functional S-phase checkpoint, stalled replication forks collapse and give rise to chromosome breakage. In an attempt to better understand replication dynamics in S-phase checkpoint mutants, we developed a replication origin array for budding yeast that contains 424 of 432 previously identified potential origin regions. As expected, mec1-1 and rad53-1 mutants failed to inhibit late origin activation. Surprisingly however, 17 early-firing regions were not replicated efficiently in these mutants. This was not due to a lack of initiation, but rather to problems during elongation, as replication forks arrested in close proximity to these origins, resulting in the accumulation of small replication intermediates and eventual replication fork collapse. Importantly, these regions were not only prone to chromosome breakage in the presence of exogenous stress but also in its absence, similar to fragile sites in the human genome.

DNA Damage-induced Down-regulation of Human Cdc25C and Cdc2 Is Mediated by Cooperation between p53 and Maintenance DNA (Cytosine-5) Methyltransferase 1

The Cdc25C phosphatase mediates cellular entry into mitosis in mammalian cells. Cdc25C activates Cdc2 for entry into mitosis by dephosphorylating Thr and Tyr at the site of inhibitory phosphorylation. The Cdc25C gene contains tumor suppressor p53 binding sites and is demonstrated to contribute to the p53-dependent cell cycle arrest upon DNA damage. Here we show that both Cdc25C and Cdc2 were down-regulated in wild-type HCT116 cells but not in p53-null, DNMT1-null or DNMT1and DNMT3b-null cells, upon p53 stabilization following doxorubicin-mediated DNA damage. Furthermore, zebularine, a drug that selectively traps and depletes nuclear DNMT1 and DNMT3b, relieved p53-mediated repression of endogenous Cdc25C and Cdc2. Methylation analysis of the Cdc25C and Cdc2 promoter displayed internal CG methylation proximal to the p53 binding site upon DNA damage in a p53-dependent manner. Chromatin immunoprecipitation of doxorubicin treated wild-type HCT116 cells showed the presence of DNMT1, p53, H3K9me2, and the transcriptional repressor HDAC1 on the Cdc25C and Cdc2 promoters, suggesting their involvement as repressive complexes in Cdc25C and Cdc2 gene silencing. Thus, the general mechanism of p53-mediated gene repression may involve recruitment of other repressive factors.

The Histone Deacetylase Inhibitor Trichostatin A Has Genotoxic Effects in Human Lymphoblasts In Vitro

Histone deacetylase inhibitors (HDACi) are a class of putative chemotherapeutic agents for which the mechanism of toxicity has not been fully identified. To explore the possibility that HDACi are genotoxic, human TK6 lymphoblastoid cells were exposed to trichostatin A (TSA) and genetic damage was measured. TSA caused a dose-dependent increase of G1-arrested cells at 24 h that correlated with increasing levels of p21 and apoptosis. Significantly elevated frequencies of structural chromosomal aberrations in cells exposed to TSA were observed using both the kinetochore-antibody micronucleus assay and nonbanding metaphase chromosome analysis. Increased tail intensities, indicative of elevated levels of DNA damage, were observed using the alkaline comet assay. Elevated levels of phosphorylated histone gammaH2AX protein were observed as early as 3 h following TSA exposure and peaked at 12 h for 200nM TSA. Significant levels of aneuploidy at the 200nM TSA dose were observed using metaphase analysis, but interestingly, kinetochore-positive micronuclei were not detected at any dose using the kinetochore micronucleus assay, suggesting that TSA induces aneuploidy via a nondisjunction event rather than chromosome lagging. Increases in chromosomal loss and breakage were observed using simultaneous FISH metaphase analysis of chromosomes 5, 7, 8, and 21, consistent with data obtained from the micronucleus and metaphase chromosome analyses. We conclude that TSA is both a clastogen and aneugen in the TK6 cell line and propose that the observed cytostatic and apoptotic properties of TSA may partially be due to this genotoxicity.

Differential Binding of Replication Proteins across the Human c-myc Replicator

The binding of the prereplication complex proteins Orc1, Orc2, Mcm3, Mcm7, and Cdc6 and the novel DNA unwinding element (DUE) binding protein DUE-B to the endogenous human c-myc replicator was studied by chromatin immunoprecipitation. In G(1)-arrested HeLa cells, Mcm3, Mcm7, and DUE-B were prominent near the DUE, while Orc1 and Orc2 were least abundant near the DUE and more abundant at flanking sites. Cdc6 binding mirrored that of Orc2 in G(1)-arrested cells but decreased in asynchronous or M-phase cells. Similarly, the signals from Orc1, Mcm3, and Mcm7 were at background levels in cells arrested in M phase, whereas Orc2 retained the distribution seen in G(1)-phase cells. Previously shown to cause histone hyperacetylation and delocalization of replication initiation, trichostatin A treatment of cells led to a parallel qualitative change in the distribution of Mcm3, but not Orc2, across the c-myc replicator. Orc2, Mcm3, and DUE-B were also bound at an ectopic c-myc replicator, where deletion of sequences essential for origin activity was associated with the loss of DUE-B binding or the alteration of chromatin structure and loss of Mcm3 binding. These results show that proteins implicated in replication initiation are selectively and differentially bound across the c-myc replicator, dependent on discrete structural elements in DNA or chromatin.

Non–Small-Cell Lung Cancer and Ba/F3 Transformed Cells Harboring the ERBB2 G776insV_G/C Mutation Are Sensitive to the Dual-Specific Epidermal Growth Factor Receptor and ERBB2 Inhibitor HKI-272

Mutation-specific cancer therapy has shown promising clinical efficacy. In non-small-cell lung cancer (NSCLC), the presence of mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase correlates with clinical response to small-molecule tyrosine kinase inhibitors. Here, we show that cells harboring the G776insV_G/C mutation in the related ERBB2 tyrosine kinase (also known as HER2 or Neu), present in a small percentage of NSCLCs, are sensitive to HKI-272, an irreversible dual-specific kinase inhibitor targeting both EGFR and ERBB2. In the ERBB2-mutant NCI-H1781 cell line, HKI-272 treatment inhibited proliferation by induction of G(1) arrest and apoptotic cell death. Furthermore, HKI-272 abrogated autophosphorylation of both ERBB2 and EGFR. Finally, Ba/F3 murine pro-B cells, engineered to express mutant ERBB2, became independent of interleukin-3 and sensitive to HKI-272. Thus, the subset of NSCLC patients with tumors carrying the ERBB2 G776insV_G/C mutation may benefit from treatment with HKI-272.

Leptin-induced growth of human ZR-75-1 breast cancer cells is associated with up-regulation of cyclin D1 and c-Myc and down-regulation of tumor suppressor p53 and p21WAF1/CIP1

Obesity has been recognized as a risk factor for breast cancer. Adipocyte-derived leptin may play as a paracrine regulator on the growth of breast cancer cells. Expression of both leptin and its OB-Rb receptor was detected in human breast cancer ZR-75-1 cells and further induced by leptin, suggesting that both expression and message mediation of leptin were autoregulated by itself. With cell counting and MTT assay, we had observed leptin stimulated ZR-75-1 growth in dose- and time-dependent manners. To study what steps of cell cycle progression leptin may involve in, we analyzed cell-cycle profile with flow cytometric analysis, mRNA and protein expressions of four cell-cycle regulators with RT-PCR and Western blotting analysis. Under the treatment of leptin, the G1 arrest of cells was reduced accompanied with up-regulation of G1 phase-specific cyclin D1 and proto-oncogene c-Myc, but down-regulation of cyclin-dependent kinase inhibitor p21(WAF1/CIP1) and tumor suppressor p53. Furthermore, JAK2 inhibitor AG490, PI3K/Akt inhibitor Wortmannin, and MEK/ERK1/2 inhibitor PD98059 were efficiently prevented leptin-promoted cell growth. Effect of cooperation between leptin and estrogen on ZR-75-1 growth had been observed. Collectively, the results showed that the proliferative effect of leptin on ZR-75-1 was associated with the up-regulation of cyclin D1 and c-Myc and down-regulation of tumor suppressor p53 and p21(WAF1/CIP1) plausibly through a hypothesized JAK2-PI3K/Akt-MEK/ERK pathway. The leptin- and OB-Rb-expressing capability of ZR-75-1 created a possible autocrine control of leptin, in which signal could be effectively amplified by itself, on cell growth.

L1 retrotransposition in nondividing and primary human somatic cells

Whether long interspersed element-1 (L1 or LINE-1) retrotransposition can occur in quiescent, nondividing, and/or terminally differentiated somatic cells has remained an unanswered fundamental question in human genetics. Here, we used a ubiquitously active phosphoglycerate kinase-1 promoter to drive the expression of a highly active human L1 element from an adenovirus-L1 hybrid vector. This vector system achieved retrotransposition in up to 91% of actively growing immortalized cells, and we demonstrated that L1 retrotransposition can be suppressed by the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine. This adenovirus vector enabled efficient delivery of the L1 element into differentiated primary human somatic cells and G1/S-arrested cells, resulting in retrotransposition in both cases; however, it was not detected in G0-arrested cells. Thus, these data indicate that L1 retrotransposition can occur in nondividing somatic cells.

Growth, Differentiation, and Malignant Transformation of Pre-B Cells Mediated by Inducible Activation of v-Abl Oncogene

The nonreceptor tyrosine kinase, encoded by the v-Abl oncogene of Abelson murine leukemia virus induces transformation of progenitor B cells. The v-Abl oncogene promotes cell cycle progression and inhibits pre-B cell differentiation. The temperature-sensitive form of Abelson murine leukemia virus offers a reversible model to study the role of v-Abl in regulating growth and differentiation. Inactivation of v-Abl elevates p27 and Foxo3a levels and activates NF-kappaB/Rel, which leads to G1 arrest and induction of Ig L chain gene rearrangement, respectively. In turn, v-Abl reactivation reduces p27 and Foxo3a levels, thus permitting G1-arrested cells to reenter the cell cycle. However, the cell lines derived from SCID mice that are defective in the catalytic subunit of DNA-dependent protein kinase retain elevated levels of p27 and Foxo3a proteins despite reactivation of v-Abl. Consequently, these cells are locked in the G1 phase for an extended period of time. The few cells that manage to bypass the G1 arrest become tumorigenic and fail to undergo pre-B cell differentiation induced by v-Abl inactivation. Deregulation of p27, Foxo3a, c-myc, and NF-kappaB/Rel was found to be associated with the malignant transformation of SCID temperature-sensitive form of Abelson murine leukemia virus pre-B cells.

Notch activity opposes ras-induced differentiation during the second mitotic wave of the developing Drosophilaeye

BackgroundEGF receptor acts through Ras and the MAPK cascade to trigger differentiation and maintain survival of most of cell types in the Drosophila retina. Cell types are specified sequentially by separate episodes of EGFR activity. All the cell types differentiate in G1 phase of the cell cycle. Before differentiating, many cells pass through the cell cycle in the "Second Mitotic Wave" in response to Notch activity, but no cell fates are specified during the Second Mitotic Wave. It is not known how fate specification is limited to G1-arrested cells.ResultsCompetence to differentiate in response to activated RasV12 was diminished during the Second Mitotic Wave accounting for the failure to recruit cell fates from cycling cells. Competence was not restored by blocking cell cycle progression, but was restored by reduced Notch activity.ConclusionCompetence to differentiate does not depend on cell cycle progression per se, but on the same receptor activity that also induces cell cycle entry. Dual effects of Notch on the cell cycle and on differentiation help ensure that only G1 phase cells undergo fate specification.

Cdk5 phosphorylates and stabilizes p27kip1 contributing to actin organization and cortical neuronal migration

p27(kip1), a cyclin-dependent kinase (CDK) inhibitor (CKI), generally suppresses CDK activity in proliferating cells. Although another role of p27 in cell migration has been recently suggested in vitro, the physiological importance of p27 in cell migration remains elusive, as p27-deficient mice have not shown any obvious migration-defect-related phenotypes. Here, we show that Cdk5, an unconventional neuronal CDK, phosphorylates and stabilizes p27 as an upstream regulator, maintaining the amount of p27 in post-mitotic neurons. In vivo RNA interference (RNAi) experiments showed that reduced amounts of p27 caused inhibition of cortical neuronal migration and decreased the amount of F-actin in the processes of migrating neurons. The Cdk5-p27 pathway activates an actin-binding protein, cofilin, which is also shown to be involved in cortical neuronal migration in vivo. Our findings shed light on a previously unknown new relationship between CDK and CKI in G0-arrested cells that regulates cytoskeletal reorganization and neuronal migration during corticogenesis.

Involvement of CUL4 Ubiquitin E3 Ligases in Regulating CDK Inhibitors Dacapo/p27Kip1 and Cyclin E Degradation

The CUL4 (cullin 4) proteins are the core components of a new class of ubiquitinE3 ligases that regulate replication and transcription. To examine the roles of CUL4 incell cycle regulation, we analyzed the effect of inactivation of CUL4 in both Drosophilaand human cells. We found that loss of CUL4 in Drosophila cells causes G1 cell cyclearrest and an increased protein level of the CDK inhibitor Dacapo. Co-elimination ofDacapo with CUL4 abolishes the G1 cell arrest. In human cells, inactivation of CUL4Ainduces CDK inhibitor p27Kip1 stabilization and G1 cell cycle arrest which is dependenton the presence of p27, suggesting that this regulatory pathway is evolutionarilyconserved. In addition, we found that the Drosophila CUL4 also regulates the proteinlevel of cyclin E independent of Dacapo. We provide evidence that human CUL4B, aparalogue of human CUL4A, is involved in cyclin E regulation. Loss of CUL4B causesthe accumulation of cyclin E without a concomitant increase of p27. The human CUL4Band cyclin E proteins also interact with each other and the CUL4B complexes canpolyubiquitinate the CUL4B-associated cyclin E. Our studies suggest that the CUL4-containing ubiquitin E3 ligases plays a critical role in regulating G1 cell cycleprogression in both Drosophila and human cells.

A Phase I-II Pilot Study of Divalproex Sodium Alone and in Combination with All-Trans-Retinoic Acid (ATRA) in Relapsed or Refractory Acute Myeloid Leukemia.

Valproic acid (VPA), acetylates histones, induces apoptosis and synergizes with All Trans Retinoic Acid (ATRA), in promoting G1 arrest and leukemic cell differentiation in vitro. Based on these observations we designed a Phase I/II pilot study of VPA alone and in combination with ATRA in patients with relapsed, refractory or poor risk acute myeloid leukemia (AML). VPA was initiated at 15mg/kg/day (day 1 IV followed by daily po) and escalated weekly by 10mg/kg (max 60 mg/kg/day) targeted to reach a serum concentration of 80–120mg/ml. For the combination, ATRA was to be added initially at 25mg/M2/day (first 3 patients) and titrated, in the absence of toxicity, to 45mg/M2/day, in two divided doses, following a minimum of 7 days of VPA therapy alone. Treatment was planned to continue for 45 days following achievement of a VPA level of >80mg/ml on two separate, consecutive measurements. To date we have treated 8 patients (7 tAML/MDS, 1 de novo AML) with VPA alone. Two of 8 additional patients to be treated with VPA + ATRA (1 tAML/ET; 1 tAML/MDS), are too early in their treatment upon which to report. Of the VPA alone treated patients, Median age was 63 (range 52–78). No Prior therapy: 3/8 pts; 5F:3M; ECOG PS 0 (1), 1 (5), 2 (2);Cytogenetic abnormalities: normal (1); t(3;18) (1); complex karyotype (6), including trisomy 8 (3), abnormalities of 11 (2) or monosomy 7 (2). Baseline bone marrow blast percentage > 50%; (8); median bone marrow cellularity 84% (range 35–90). Treatment: Median duration of treatment 45 days (range 19–99). Therapy discontinued before 45 days; withdrawl (2); death at home on study (day 33 of treatment), (1); reversible grade 3 toxicity(2). VPA levels: Serum concentrations of VPA of > 80mg/ml was achieved in all patients; Hematologic effects: Changes in CBC: increase in WBC, ANC and blast count (BLC)(3), followed by decrease in WBC and BLC (2); increase in WBC/ANC &decrease in BLC (1); decrease in WBC, ANC and BLC (3); &initial decrease in BLC &increase in ANC (1). Marrow: decrease in marrow cellularity (2)( 90% to 30%); loss of detectable trisomy 8 population (1); &decrease in bone marrow blasts (1) (64% to 5%) was observed. One patient reverted back to MDS, lasting 5 months. Side Effects: somnolence (grade 2–4) was observed in 4/8 patients, and was the major cause of withholding treatment. We also observed a constellation of symptoms (weight gain, fever, elevated WBC, shortness of breath, elevated LDH, bone pain) and chest x-ray findings consistent with &reminiscent of APL syndrome, within seven days of achieving the targeted serum VPA levels (> 80mg/ml), in 2 pts. Decadron was started two days (1) and immediately (1) following the onset of symptoms: signs and symptoms abated quickly &resolved subsequently within three days. In retrospect, one additional patient had comparable symptoms, for which VPA was stopped, following admission to an outside hospital for fever, pulmonary infiltrates and altered mental status. Correlative studies: serial assessment of bulk histone acetylation (H3, H4) was performed in all 8 patients. 7/8 patients showed evidence of histone hyperacetylation in mononuclear cells from peripheral blood, during treatment with VPA. Additional patients are being treated with VPA + ATRA. Analysis of these studies will be forthcoming.

Hepatocyte Growth Factor Induces Redistribution of p21CIP1 and p27KIP1 through ERK-dependent p16INK4a Up-regulation, Leading to Cell Cycle Arrest at G1 in HepG2 Hepatoma Cells

Hepatocyte growth factor (HGF) has an anti-proliferative effect on many types of tumor cell lines and tumors in vivo. We found previously that inhibition of HGF-induced proliferation in HepG2 hepatoma cells is caused by cell cycle arrest at G1 through a high intensity ERK signal, which represses Cdk2 activity. To examine further the mechanisms of G1 arrest by HGF, we analyzed the Cdk inhibitor p16(INK4a), which has an anti-proliferative function through cell cycle arrest at G1. We found that HGF treatment drastically increased endogenous p16 levels. Knockdown of p16 with small interfering RNA reversed the arrest, indicating that the induction of p16 is required for G1 arrest by HGF. Analysis of the promoter of the human p16 gene identified the proximal Ets-binding site as a responsive element for HGF, and this responded to the high intensity ERK signal. HGF treatment of the cells led to a redistribution of p21(CIP1) and p27(KIP1) from Cdk4 to Cdk2. The redistribution was blocked by the knockdown of p16 with small interfering RNA, which restored the Cdk2 activity repressed by HGF, demonstrating the requirement of p16 induction for the redistribution and eventual repression of Cdk2 activity. Our results reveal a signaling pathway for G1 arrest induced by HGF.

Effect of OA-inhibitor of protein phosphatases PP1 and PP2A — on initiation of DNA replication and mitosis in Vicia faba root meristems

According to the principal control point (PCP) hypothesis, experiments with excised, carbohydrate-starved stationary root meristems of Vicia faba var. minor have demonstrated that cells which previously divided asynchronously were preferentially blocked in G1 (PCP1) and G2 (PCP2) phases. When stationary phase meristems are supplied with exogenous carbohydrate (2 % sucrose), the G1- and G2-arrested cells start out DNA replication and mitotic divisions, respectively. The resumption of DNA synthesis and mitosis is not immediate and the delays of G1- and G2-arrested cells are found different. Using this model, we examined the effects of 4 pulse incubations with okadaic acid (OA), a specific inhibitor of PP1 and PP2A, on the duration of intervals elapsing between the provision of sucrose and the first appearance of S- and M-phase cells. We have found that depending on the period during which OA had been applied, the release from G1 and G2 phase arrest-points becomes prolonged, showing different time-course modifications. The obtained data provide evidence that activation of PP1 and PP2A is required to allow the cells for both PCP1→S and PCP2→M transitions in root meristems of V. faba.

Heterogeneity of dimer excision in young and senescent human dermal fibroblasts

We have examined the relationship between nucleotide excision of the main UV-induced photoproduct, the cyclobutane pyrimidine dimer and in vitro cellular senescence. An in situ semiquantitative immunocytochemical assay has demonstrated that, following a UV-C dose of 15 J m-2, young human dermal fibroblasts maintained in a high level of serum are more efficient than senescent fibroblasts in the removal of dimers. However, in G0-arrested cultures (serum-starved), young fibroblasts are compromised in their ability to remove dimers and are significantly less efficient than senescent cells in this process. Supplementation of the culture medium with 0.1 mm deoxyribonucleosides enhances the removal of dimers in both young and senescent fibroblasts in proliferating or serum-starved cells. These data indicate that overall there is a modest but significant reduction in nucleotide excision of dimer photoproducts in cells as they age in vitro. In addition, G0-arrested young cells exhibit reduced removal of dimers, although this can be complemented by deoxyribonucleoside addition. In addition, this in situ assay has revealed heterogeneity in both susceptibility to UV-C-induced damage and excision. Overall, we provide evidence of reduced UV-induced damage excision in senescent compared with young fibroblasts, and demonstrate modulation of these processes in young and senescent cells under specific growth conditions.