Reinitiation Of Cell Cycle Research Articles

Cell Cycle Status Influences Resistance to Apoptosis Induced by Oxidative Stress in Human Breast Cancer Cells, Which Is Accompanied by Modulation of Autophagy.

Cancer cells are characterised by uncontrolled cell proliferation; however, some of them can temporarily arrest their cell cycle at the G0 or G1 phase, which could contribute to tumour heterogeneity and drug resistance. The cell cycle status plays a critical role in chemosensitivity; however, the influence of G0- and G1-arrest has not been elucidated. To study the cell cycle arrest-mediated resistance, we used MCF-7 cells and generated three populations of cells: (1) cells arrested in the G0-like phase, (2) cells that resumed the cell cycle after the G0-like phase and (3) cells arrested in early G1 with a history of G0-like arrest. We observed that both the G0-like- and the G1-arrested cells acquired resistance to apoptosis induced by oxidative stress, accompanied by a decreased intracellular reactive oxygen species and DNA damage. This effect was associated with increased autophagy, likely facilitating their survival at DNA damage insult. The cell cycle reinitiation restored a sensitivity to oxidative stress typical for cells with a non-modulated cell cycle, with a concomitant decrease in autophagy. Our results support the need for further research on the resistance of G0- and G1-arrested cancer cells to DNA-damaging agents and present autophagy as a candidate for targeting in anticancer treatment.

Role of the Cell Cycle Re-Initiation in DNA Damage Response of Post-Mitotic Cells and Its Implication in the Pathogenesis of Neurodegenerative Diseases.

Neurodegenerative diseases are often associated with both normal and premature aging. Resumption of the cell cycle by neurons induced by DNA damage may lead to their apoptosis, which contributes to the degeneration of neuronal tissue. Cell cycle and DNA replication proteins are frequently found in patients with neurodegenerative diseases. Oxidative stress, which is considered to play an important role in aging and pathogenesis of many neurodegenerative diseases, can induce DNA damage and stimulate cell cycle re-entry by neuronal cells. DNA damage activates ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), breast cancer 1 (BRCA1), E2F transcription factor 1 (E2F1), and other proteins that regulate the cell cycle, DNA damage repair, and apoptosis. Because the E2F complexes associate with histone-modifying enzymes, histone modifications, including histone acetylation and methylation, are required for cell cycle re-entry and may play a regulatory role in DNA repair or apoptosis. Aberrant cell cycle regulation has been shown to play a role in age-related macular degeneration (AMD) in which retinal cells are affected and in inclusion body myositis, which is characterized by muscle cell dysfunction. There is also evidence to suggest that cytostatic chemotherapy could decrease dementia in Alzheimer's disease and multiple myeloma, supporting the use of cell cycle inhibitors in the therapy of degenerative diseases.

Identification of N-Terminally Truncated Stable Nuclear Isoforms of CDC25B That Are Specifically Involved in G2/M Checkpoint Recovery

CDC25B phosphatases must activate cyclin B-CDK1 complexes to restart the cell cycle after an arrest in G2 phase caused by DNA damage. However, little is known about the precise mechanisms involved in this process, which may exert considerable impact on cancer susceptibility and therapeutic responses. Here we report the discovery of novel N-terminally truncated CDC25B isoforms, referred to as ΔN-CDC25B, with an exclusively nuclear and nonredundant function in cell cycle re-initiation after DNA damage. ΔN-CDC25B isoforms are expressed from a distinct promoter not involved in expression of canonical full-length isoforms. Remarkably, in contrast to the high lability and spatial dynamism of the full-length isoforms, ΔN-CDC25B isoforms are highly stable and exclusively nuclear, strongly suggesting the existence of two pools of CDC25B phosphatases in the cell that have functionally distinct properties. Using isoform-specific siRNA, we found that depleting full-length isoforms, but not ΔN-CDC25B isoforms, delays entry into mitosis. Thus, in an unperturbed cell cycle, the full-length isoforms are exclusively responsible for activating cyclin B-CDK1. Strikingly, in the late response to DNA damage, we found a CHK1-dependent shift in accumulation of CDC25B isoforms toward the ΔN-CDC25B species. Under this physiological stress condition, the ΔN-CDC25B isoform was found to play a crucial, nonredundant function in restarting the cell cycle after DNA damage-induced G2 phase arrest. Our findings reveal the existence of a previously unrecognized CDC25B isoform that operates specifically in the nucleus to reinitiate G2/M transition after DNA damage.

KSR1 Is Required for Cell Cycle Reinitiation Following DNA Damage

KSR1 (kinase suppressor of Ras 1) is a molecular scaffold and positive regulator of the Raf/MEK/ERK phosphorylation cascade. KSR1 is required for maximal ERK activation induced by growth factors and by some cytotoxic agents. We show here that KSR1 is also required for maximal ERK activation induced by UV light, ionizing radiation, or the DNA interstrand cross-linking agent mitomycin C (MMC). We further demonstrate a role for KSR1 in the reinitiation of the cell cycle and proliferation following cell cycle arrest induced by MMC. Cells lacking KSR1 underwent but did not recover from MMC-induced G(2)/M arrest. Expression of KSR1 allowed KSR1(-/-) cells to re-enter the cell cycle following MMC treatment. However, cells expressing a mutated form of KSR1 unable to bind ERK did not recover from MMC-induced cell cycle arrest, demonstrating the requirement for the KSR1-ERK interaction. In addition, constitutive activation of ERK was not sufficient to promote cell cycle reinitiation in MMC-treated KSR1(-/-) cells. Only cells expressing KSR1 recovered from MMC-induced cell cycle arrest. Importantly, MMC-induced DNA damage was repaired in KSR1(-/-) cells, as determined by resolution of gamma-H2AX-containing foci. These data indicate that cell cycle reinitiation is not actively signaled in the absence of KSR1, even when DNA damage has been resolved. These data reveal a specific role for the molecular scaffold KSR1 and KSR1-mediated ERK signaling in the cellular response to DNA interstrand cross-links.

A non-canonical Grb2–PLC-γ1–Sos cascade triggered by lipovitellin 1, an apolipoprotein B homologue

The injection of the Grb2 adapter in Xenopus oocytes promotes G2/M transition without stimulation from a receptor only the first day after the oocytes removal from the ovaries. This cell cycle reinitiation is Ras-dependent and requires the SH2 and SH3 domains of Grb2. The SH2 domain of Grb2 binds the tyrosine phosphorylated lipovitellin1, a homologue of the human apolipoprotein B. The N-SH3 domain of Grb2 is linked to a proline-rich sequence of the C2 domain of PLC-γ1, PLC-γ1 itself is linked, through its SH3 domain, to the C-terminal proline-rich region of Sos. When Grb2–PLC-γ1–Sos is associated, PLC-γ1 is not phosphorylated on Y783 but shows a phospholipase activity. Inhibition of lipovitellin 1 or PLC-γ1 avoids Grb2-induced cell cycle reinitiation. Therefore, the Grb2–lipovitellin 1 association is the starting point of a novel signaling pathway, where PLC-γ1 binds Grb2 and recruits Sos.

Plasma membrane destination of the classical Xenopus laevis progesterone receptor accelerates progesterone‐induced oocyte maturation

Xenopus laevis oocyte maturation is induced by the steroid hormone progesterone through a non-genomic mechanism initiated at the cell membrane. Recently, two Xenopus oocyte progesterone receptors have been cloned; one is the classical progesterone receptor (xPR-1) involved in genomic actions and the other a putative seven-transmembrane-G-protein-couple receptor. Both receptors are postulated to be mediating the steroid-induced maturation process in the frog oocyte. In this study, we tested the hypothesis that the classical progesterone receptor, associated to the oocyte plasma membrane, is participating in the reinitiation of the cell cycle. Addition of a myristoilation and palmytoilation signal at the amino terminus of xPR-1 (mp xPR-1), increased the amount of receptor associated to the oocyte plasma membrane and most importantly, significantly potentiated progesterone-induced oocyte maturation sensitivity. These findings suggest that the classical xPR-1, located at the plasma membrane, is mediating through a non-genomic mechanism, the reinitiation of the meiotic cell cycle in the X. laevis oocyte.

P53-independent G1 cell cycle arrest of human colon carcinoma cells HT-29 by sulforaphane is associated with induction of p21CIP1 and inhibition of expression of cyclin D1

Isothiocyanate sulforaphane (SFN) is a potent cancer chemopreventive agent. We investigated the mechanisms underlying the anti-proliferative effects of SFN in the human colon carcinoma cell line, HT-29. We demonstrate that SFN inhibits the growth of HT-29 cells in a dose- and time-dependent manner. Treatment of serum-stimulated HT-29 cells with SFN suppressed the re-initiation of cell cycle by inducing a G(1) phase cell cycle arrest. At high doses (>25 microM), SFN dramatically induces the expression of p21(CIP1) while significantly inhibits the expression of the G(1) phase cell cycle regulatory genes such as cyclin D1, cyclin A, and c-myc. This regulation can be detected at both the mRNA and protein levels as early as 4 h post-treatment of SFN at 50 microM. Additionally, SFN activates MAPKs pathways, including ERK, JNK and p38. Exposure of HT-29 cells with both SFN and an antioxidant, either NAC or GSH, completely blocked the SFN-mediated activation of these MAPK signaling cascades, regulation of cyclin D1and p21(CIP1) gene expression, and G(1)phase cell cycle arrest. This finding suggests that SFN-induced oxidative stress plays a role in these observed effects. Furthermore, the activation of the ERK and p38 pathways by SFN is involved in the upregulation of p21(CIP1) and cyclin D1, whereas the activation of the JNK pathway plays a contradictory role and may be partially involved in the downregulation of cyclin D1. Because cyclin D1 and p21(CIP1) play opposing roles in G(1) phase cell cycle progression regulation, blocking the activation of each MAPK pathway with specific MAPK inhibitors, is unable to rescue the SFN-induced G(1) phase cell cycle arrest in HT-29 cells.

Intracellular pH and intracellular free calcium responses to protein kinase C activators and inhibitors in Xenopus eggs

Cell activation during fertilization of the egg of Xenopus laevis is accompanied by various metabolic changes, including a permanent increase in intracellular pH (pHi) and a transient increase in intracellular free calcium activity ([Ca2+]i). Recently, it has been proposed that protein kinase C (PKC) is an integral component of the Xenopus fertilization pathway (Bement and Capco, J. Cell Biol. 108, 885-892, 1989). Indeed, activators of PKC trigger cortical granule exocytosis and cortical contraction, two events of egg activation, without, however, releasing the cell cycle arrest (blocked in second metaphase of meiosis). In the egg of Xenopus, exocytosis as well as cell cycle reinitiation are supposed to be triggered by the intracellular Ca2+ transient. We report here that PKC activators do not induce the intracellular Ca2+ transient, or the activation-associated increase in pHi. These results suggest that the ionic responses to egg activation in Xenopus do not appear to depend on the activation of PKC. In addition, in eggs already pretreated with phorbol esters, those artificial activators that act by releasing Ca2+ intracellularly, triggered a diminished increase in pHi. Finally, sphingosine and staurosporine, two potent inhibitors of PKC, were found to trigger egg activation, suggesting that a decrease in PKC activity might be an essential event in the release of the metaphase block, in agreement with recent findings on the release of the prophase block in Xenopus oocytes (Varnold and Smith, Development 109, 597-604, 1990).

The influence of insulin in the cell cycle of human aging cells

Normal human IMR 90 fibroblasts at population doubling levels (PDL) 28 approximately 33 had an increased incorporation of 3H-thymidine into DNA when the cells were pretreated with physiologic concentrations of insulin (5 approximately 10 ng/ml) prior to re-initiation of the cell cycle. Insulin stimulation was not observed with fibroblasts at PDL 50 nor with SV40 virus transformed cells. In all cells treated with supra-physiologic concentrations of insulin (1000 approximately 6000 ng/ml) prior to the re-initiation of cell cycle, the incorporation of thymidine into DNA was greatly diminished. Autoradiographic studies of the effect of insulin suggest that a larger fraction of cells was found to enter S phase than untreated cultures. This effect was evident in younger cells rather than senescent cells.