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PubMed2026

LiCl induces GSK-3β mediated autophagy, DNA damage, and cell cycle arrest in HPV driven cervical cancer cells.

High-risk HPV infections induce cervical cancer progression by disrupting cellular homeostasis and survival pathways, including autophagy. Targeting autophagy represents a promising therapeutic strategy. Lithium chloride (LiCl), extensively studied for its neuroprotective properties, can be investigated for its potential anticancer effects in HPV-driven cervical cancer cells. Treatment with 30 mM LiCl induced significant phosphorylation of glycogen synthase kinase-3β (GSK-3β) at Ser9, inducing functional inhibition and downstream signal alterations. This modulation of GSK-3β activity compromised genomic integrity, validated by increased double strand DNA breaks, increased oxidative and cellular stress, and reduced antioxidant enzyme activity. Consequently, LiCl treated cells exhibited significant G2/M phase arrest, indicating disruption in cell cycle progression. Interestingly, the observed cytotoxicity occurred independently of classical apoptotic pathways, suggesting the activation of alternative cell death mechanisms. Mechanistic studies revealed a robust autophagic flux, with GSK-3β mediated autophagy, validated through siRNA mediated knockdown experiments. These findings highlight a novel cytotoxic mechanism of LiCl and propose its potential repurposing from neurobiology to targeted cancer therapeutics.

Humans↗
PubMed2007

Constitutively high dNTP concentration inhibits cell cycle progression and the DNA damage checkpoint in yeast Saccharomyces cerevisiae.

In eukaryotic cells the concentration of dNTP is highest in S phase and lowest in G1 phase and is controlled by ribonucleotide reductase (RNR). RNR activity is eliminated in all eukaryotes in G1 phase by a variety of mechanisms: transcriptional regulation, small inhibitory proteins, and protein degradation. After activation of RNR upon commitment to S phase, dATP feedback inhibition ensures that the dNTP concentration does not exceed a certain maximal level. It is not apparent why limitation of dNTP concentration is necessary in G1 phase. In principle, dATP feedback inhibition should be sufficient to couple dNTP production to utilization. We demonstrate that in Saccharomyces cerevisiae constitutively high dNTP concentration transiently arrests cell cycle progression in late G1 phase, affects activation of origins of replication, and inhibits the DNA damage checkpoint. We propose that fluctuation of dNTP concentration controls cell cycle progression and the initiation of DNA replication.

Alleles↗
PubMed2004

Combining DNA damaging agents and checkpoint 1 inhibitors.

During the cell cycle that leads to mitosis, checkpoints are activated in response to DNA damage. The checkpoints control the ability of cells to arrest the cell cycle allowing time to repair the DNA. In more than 50% of cancer cells, the G1 checkpoint is inactive due to mutations of p53. Therefore, the combination of a DNA damaging agent with a G2 checkpoint inhibitor should force selectively cancer cells into a premature and lethal mitosis. This approach which has recently drawn considerable interest is discussed in this paper.

Antineoplastic Agents↗
PubMed2000

Activation of Xenopus Chk1 by mutagenesis of threonine-377.

Xenopus Chk1 (Xchk1) is required for the checkpoint-associated delay of the cell cycle in frog egg extracts containing unreplicated or UV-damaged DNA. Phosphorylation of Xchk1 at multiple sites in the SQ/TQ domain (residues 314-366) in response to unreplicated or UV-damaged DNA results in elevation of its kinase activity. We have found that mutagenesis of Thr-377 in the conserved Thr-Arg-Phe (TRF) motif of Xchk1 also leads to a substantial increase in kinase activity. Thr-377 does not appear to be a site of phosphorylation in Xchk1. These findings suggest that Thr-377 may play a role in suppressing the activity of Xchk1.

Amino Acid Sequence↗
PubMed1999

The ATM protein is required for sustained activation of NF-kappaB following DNA damage.

Cells lacking an intact ATM gene are hypersensitive to ionizing radiation and show multiple defects in the cell cycle-coupled checkpoints. DNA damage usually triggers cell cycle arrest through, among other things, the activation of p53. Another DNA-damage responsive factor is NF-kappaB. It is activated by various stress situations, including oxidative stress, and by DNA-damaging compounds such as topoisomerase poisons. We found that cells from Ataxia Telangiectasia patients exhibit a defect in NF-kappaB activation in response to treatment with camptothecin, a topoisomerase I poison. In AT cells, this activation is shortened or suppressed, compared to that observed in normal cells. Ectopic expression of the ATM protein in AT cells increases the activation of NF-kappaB in response to camptothecin. MO59J glioblastoma cells that do not express the DNA-PK catalytic subunit respond normally to camptothecin. These results support the hypothesis that NF-kappaB is a DNA damage-responsive transcription factor and that its activation pathway by DNA damage shares some components with the one leading to p53 activation.

Adolescent↗
PubMed2002

Growth factor requirements and basal phenotype of an immortalized mammary epithelial cell line.

Carcinogenesis involves a multistep process whereby a normal healthy cell undergoes both immortalization and oncogenesis to become fully transformed. Immortalization results from the subversion of critical cell cycle regulatory checkpoints, thereby allowing a cell to extend its finite life span and to maintain telomeric length. Oncogenesis is the manifestation of additional genetic events that are capable of conferring upon the cell an actual growth advantage. Such an advantage may relieve a cell of its normal requirements for a particular growth factor or may enhance the ability of a cell to proliferate outside of its normal microenvironment. To further investigate this multistep process, we developed an immortalized mammary epithelial cell line by overexpressing the catalytic subunit of telomerase (human telomerase reverse transcriptase) in primary human mammary epithelial cell lines. We present evidence that the overexpression of human telomerase reverse transcriptase was sufficient to extend the life span of the cells and allow for additional events that lead to immortalization. The result was the establishment of an IMEC line. Biochemical analysis of these cells indicates a basal epithelial phenotype with expression of high molecular weight cytokeratins. We show that continued growth of the IMECs is rigorously dependent upon both insulin and epidermal growth factor, and that the mitogenic effects of these factors on the IMECs are mediated in part by AKT. In addition, IMECs express the p53 family member DeltaN-p63-alpha, which is found in basal epithelial cells of many tissues and has been implicated as playing an essential role in normal epithelial development. Our studies suggest that the immortalization of basal epithelial cells of the mammary gland may be an early step in the initiation of a subset of breast cancers with a basal epithelial phenotype.

Breast↗
PubMed1998

Sum1, a highly conserved WD-repeat protein, suppresses S-M checkpoint mutants and inhibits the osmotic stress cell cycle response in fission yeast.

The S-M checkpoint ensures that entry into mitosis is dependent on completion of DNA replication. In the fission yeast Schizosaccharomyces pombe, the SM checkpoint mutant cdc2-3w is thought to be defective in receiving the checkpoint signal. To isolate genes that function in the checkpoint pathway, we screened an S. pombe cDNA library for genes that, when overexpressed, could suppress the checkpoint defect of cdc2-3w. Using this approach, we have identified a novel gene, sum1+ (suppressor of uncontrolled mitosis). sum1+ encodes a highly conserved WD-transducin repeat protein with striking sequence similarity to the human transforming growth factor (TGF)-beta-receptor interacting protein TRIP-1 and to the translation initiation factor 3 subunit eIF3-p39, encoded by the TIF34 gene in Saccharomyces cerevisiae. S. pombe sum1+ is an essential gene, required for normal cell growth and division. In addition to restoring checkpoint control, overexpression of sum1+ inhibits the normal cell cycle response to osmotic stress. Furthermore, we demonstrate that inactivation of the stress-activated MAP kinase pathway, required for cell cycle stress response, restores the S-M checkpoint in cdc2-3w cells. These results suggest that Suml interacts with the stress-activated MAP kinase pathway and raise the possibility that environmental conditions may influence the checkpoint response in fission yeast.

Amino Acid Sequence↗
PubMed2001

Checkpoint genes in cancer.

The mammalian cell cycle is exquisitely controlled by a 'machinery' composed of cyclin-dependent kinases and their binding partners, the cyclins. These kinases regulate transitions into DNA synthesis and mitosis, and their inactivity contributes to cellular quiescence, differentiation and senescence. Cell cycle transitions are, in turn, controlled by checkpoints that monitor ribonucleotide pools, oxygen tension, the extracellular environment, growth signalling programmes, the status of DNA replication, and the mitotic spindle apparatus. Genes positively controlling cell cycle checkpoints can be targets for oncogenic activation in cancer, whereas negative regulators, such as tumour suppressor genes, are targeted for inactivation. Understanding the molecular details of cell cycle regulation and checkpoint abnormalities in cancer offers insight into potential therapeutic strategies.

CDC2 Protein Kinase↗
PubMed1999

Ascorbic acid-dehydroascorbate induces cell cycle arrest at G2/M DNA damage checkpoint during oxidative stress.

Reactive oxygen species induce cellular damage and have been implicated as mediators for cellular signaling pathways. However, a linkage between the cellular redox status and cell cycle progression has not been demonstrated. We previously demonstrated, using the Chinese hamster ovary cell line AS52, that the cytotoxic and mutagenic effects of oxidative stress is prevented by ascorbic acid (AA), but only when cells are treated with AA prior to treatment with the stressor. To elucidate the mechanism(s) responsible for this effect, we determined the effect of AA on cell cycle progression during oxidative stress. Flow cytometric analyses demonstrated that treatment of AS52 cells with AA (50 microM), prior to treatment with a radical generating system (RGS), enhanced cell cycle arrest at the G2/M DNA damage checkpoint when compared to cells treated with RGS. AA had no effect on cell cycle progression in the absence of oxidative stress. Furthermore, under conditions that prevent the reduction of dehydroascorbate (DHA), the oxidized form of AA, cell cycle arrest was also induced at the G2/M DNA damage checkpoint. These observations demonstrate that during periods of oxidative stress, AA functions as an antioxidant and DHA enhances transient arrest at the G2/M checkpoint by delaying the activation of cyclin B-cdc2. These results suggest the presence of a unique redox mechanism for the regulation of cell cycle progression and also demonstrate a novel mechanism by which AA protects cells from damage due to oxidative stress.

Animals↗
PubMed2005

Pharmacodynamics of cytarabine alone and in combination with 7-hydroxystaurosporine (UCN-01) in AML blasts in vitro and during a clinical trial.

Chk1 and Akt signaling facilitate survival of cells treated with nucleoside analogues. Activation of Chk1 in response to cytarabine (ara-C) induced an S-phase checkpoint characterized by the inhibition of Cdk2, cell cycle arrest, no change in constitutively active Akt, or low-stress kinase signaling in ML-1 cells. However, inhibition of Chk1 by UCN-01 in S-phase-arrested cells resulted in an abrogation of the checkpoint, inhibition of Akt, activation of JNK, and a rapid induction of apoptosis. Similarly, primary acute myelogenous leukemia (AML) blasts exposed to ara-C and UCN-01 demonstrated a selective loss in cloning potential when compared with normal progenitors. Therefore, we evaluated a pilot clinical trial of ara-C in combination with UCN-01 in patients with relapsed AML. Blasts from some patients demonstrated a previously activated Chk1-Cdk2 DNA damage response pathway that decreased during therapy. Constitutively phosphorylated Akt kinase declined on addition of UCN-01 to the ara-C infusion, an action accompanied by an activation of JNK and reduction in absolute AML blast counts. Thus, use of UCN-01 in combination with ara-C decreases Chk1 phosphorylation, inhibits the Akt survival pathway, and activates JNK during the course of therapy, offering a rationale for the cytotoxic action of this combination during AML treatment.

Adolescent↗
PubMed2002

Chemosensitivity of human prostate cancer cells PC3 and LNCaP to genistein isoflavone and beta-lapachone.

A wide spectrum of anti-cancer activity of genistein and beta-lapachone in various tumors has been reported in single treatments. In this study the combined effects of genistein and beta-lapachone on the chemosensitivity of LNCaP and PC3 human prostate cancer cells was determined in vitro, using 3-[4,5-dimethylthiazol-2-yl]-2-,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) to study treatment-induced growth inhibition and cytotoxicity and, annexin V-fluoresceine (FI) and terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-propidium iodide (PI) assays to determine potential treatment-induced apoptosis and/or necrosis. The results showed: i) that both PC3 and LNCaP are sensitive to single and combination treatments regardless of hormone sensitivity status, ii) that treatment induced dual death pathways (apoptosis and necrosis) in both cell types, iii) that growth inhibition in both cell types correlated positively with cell death via apoptosis at lower drug concentrations and necrosis at higher concentrations, iv) that combination of genistein and beta-lapachone had synergistic inhibitory effects on growth and proliferation in both cell types. The synergistic inhibitory effect was correlated positively with treatment-induced cell death via apoptosis and necrosis. The overall results indicate that combination treatments with beta-lapachone and genistein are more potent in killing both PC3 and LNCaP cancer cells than treatment with either genistein or beta-lapachone alone. beta-lapachone acts at the G1 and S phase checkpoints in the cell cycle, while genistein induces cell cycle arrest at the G2-M stage. The current results are therefore in agreement with the hypothesis that drug combinations that target cell cycles at different critical checkpoints would be more effective in causing cell death. This result provides a rationale for in vivo studies to determine whether beta-lapachone-genistein combination will provide effective chemotherapy for prostate cancer, regardless of the tumor sensitivity to hormone.

Adenocarcinoma↗
PubMed1999

Poly(ADP-ribose) polymerase activity is not affected in ataxia telangiectasia cells and knockout mice.

Poly(ADP-ribose) polymerase (PARP) is a constitutive factor of the DNA damage surveillance network in dividing cells. Based on its capacity to bind to DNA strand breaks, PARP plays a regulatory role in their resolution in vivo. ATM belongs to a large family of proteins involved in cell cycle progression and checkpoints in response to DNA damage. Both proteins may act as sensors of DNA damage to induce multiple signalling pathways leading to activation of cell cycle checkpoints and DNA repair. To determine a possible relationship between PARP and ATM, we examined the PARP response in an ATM-null background. We demonstrated that ATM deficiency does not affect PARP activity in human cell lines or Atm-deficient mouse tissues, nor does it alter PARP activity induced by oxidative damage or gamma-irradiation. Our results support a model in which PARP and ATM could be involved in distinct pathways, both effectors transducing the damage signal to cell cycle regulators.

Animals↗
PubMed1999

Unique checkpoints during the first cell cycle of fertilization after intracytoplasmic sperm injection in rhesus monkeys.

Intracytoplasmic sperm injection has begun an era of considerable improvements in treating male infertility. Despite its success, questions remain about the dangers of transmitting traits responsible for male infertility, sex and autosomal chromosome aberrations and possible mental, physical and reproductive abnormalities. We report here the first births of rhesus monkeys produced by intracytoplasmic sperm injection at rates greater or equal to those reported by clinics. Essential assumptions about this process are flawed, as shown by results with the preclinical, nonhuman primate model and with clinically discarded specimens. Dynamic imaging demonstrated the variable position of the second meiotic spindle in relation to the first polar body; consequently, microinjection targeting is imprecise and potentially lethal. Intracytoplasmic sperm injection resulted in abnormal sperm decondensation, with the unusual retention of vesicle-associated membrane protein and the perinuclear theca, and the exclusion of the nuclear mitotic apparatus from the decondensing sperm nuclear apex. Male pronuclear remodeling in the injected oocytes was required before replication of either parental genome, indicating a unique G1-to-S transition checkpoint during zygotic interphase (the first cell cycle). These irregularities indicate that the intracytoplasmic sperm injection itself might lead to the observed increased chromosome anomalies.

Animals↗
PubMed1999

NOS gene transfer inhibits expression of cell cycle regulatory molecules in vascular smooth muscle cells.

The mechanisms of nitric oxide (NO)-mediated inhibition of vascular smooth muscle (VSM) cell proliferation are still obscure. Cyclins A and E in association with cyclin-dependent kinase 2 (cdk2) serve as positive regulators for mammalian cell cycle progression through the G1/S checkpoint of the cell cycle and subsequent cell proliferation. Therefore, we have tested the effect of adenovirus-mediated transfection of the endothelial nitric oxide synthase (eNOS) gene into guinea pig coronary VSM cells on platelet-derived growth factor (BB homodimer) (PDGF-BB)-stimulated cell proliferation and the expression of cell cycle regulatory molecules. Transfection of the eNOS gene (eNOS) into VSM cells significantly inhibited (P < 0.05) [3H]thymidine incorporation into the DNA in response to PDGF-BB stimulation compared with lacZ-transfected control cells. The eNOS transfer significantly inhibited (P < 0.05) PDGF-BB-induced proliferating cell nuclear antigen (PCNA) and cyclin A expression in VSM cells compared with cells transfected with the control vector. The time course of cyclin E expression in response to PDGF-BB stimulation was delayed in eNOS-transfected cells. Levels of cyclin-dependent kinase inhibitors p21 and p27 were not significantly affected by eNOS transfer. eNOS transfer did not decrease PDGF-beta receptor number, affinity, and autophosphorylation measured by radioreceptor assay and Western analysis. These results suggest that inhibition of PDGF-stimulated expression of cyclin A, cyclin E, and PCNA is the target of NO action. These findings could explain, at least in part, NO-mediated inhibition of VSM cell proliferation.

Adenoviridae↗
PubMed2002

Genetic basis of pancreatic cancer.

Because in the normal state, cells of the pancreas show a very low rate of proliferation, entering the cell cycle is assumed to be the initial event during tumorigenesis. So-called checkpoints monitor cell cycle progression and guarantee the proper duplication of the entire genome. Loss of one or more checkpoints causes subsequent accumulation of genetic alterations which finally results in cancer. Cancer cells are characterized by unrestricted growth, invasion of adjacent tissue and metastasis. All of these features can be explained in terms of genetic changes and the functional consequence of these changes. Activation of the proto-oncogene K-Ras and inactivation of the tumour suppressor gene loci INK4a, p53 and SMAD4 are characteristic for pancreatic cancer. The progression model of pancreatic cancer proposes that pancreatic intraepithelia neoplasia is the pre-cancerous lesion. A preferred genetic pathway has started to evolve. Germ-line mutations in specific genes are responsible for cases in which there is a familial predisposition to pancreatic cancer.

Humans↗
PubMed1997

Chk1 is a wee1 kinase in the G2 DNA damage checkpoint inhibiting cdc2 by Y15 phosphorylation.

The G2 DNA damage checkpoint ensures maintenance of cell viability by delaying progression into mitosis in cells which have suffered genomic damage. It is controlled by a number of proteins which are hypothesized to transduce signals through cell cycle regulators to delay activation of p34cdc2. Studies in mammalian cells have correlated induction of inhibitory tyrosine 15 (Y15) phosphorylation on p34cdc2 with the response to DNA damage. However, genetic studies in fission yeast have suggested that the major Y15 kinase, p107wee1, is not required for the cell cycle delay in response to DNA damage, although it is required for survival after irradiation. Thus, the target of the checkpoint, and hence the mechanism of cell cycle delay, remains unknown. We show here that Y15 phosphorylation is maintained in checkpoint-arrested fission yeast cells. Further, wee1 is required for cell cycle arrest induced by up-regulation of an essential component of this checkpoint, chk1. We observed that p107wee1 is hyperphosphorylated in cells delayed by chk1 overexpression or UV irradiation, and that p56chk1 can phosphorylate p107wee1 directly in vitro. These observations suggest that in response to DNA damage p107wee1 is phosphorylated by p56chk1 in vivo, and this results in maintenance of Y15 phosphorylation and hence G2 delay. In the absence of wee1, other Y15 kinases, such as p66mik1, may partially substitute for p107wee1 to induce cell cycle delay, but this wee1-independent delay is insufficient to maintain full viability. This study establishes a link between a G2 DNA damage checkpoint function and a core cell cycle regulator.

Blotting, Western↗
PubMed2006

Rad4TopBP1, a scaffold protein, plays separate roles in DNA damage and replication checkpoints and DNA replication.

Rad4TopBP1, a BRCT domain protein, is required for both DNA replication and checkpoint responses. Little is known about how the multiple roles of Rad4TopBP1 are coordinated in maintaining genome integrity. We show here that Rad4TopBP1 of fission yeast physically interacts with the checkpoint sensor proteins, the replicative DNA polymerases, and a WD-repeat protein, Crb3. We identified four novel mutants to investigate how Rad4TopBP1 could have multiple roles in maintaining genomic integrity. A novel mutation in the third BRCT domain of rad4+TopBP1 abolishes DNA damage checkpoint response, but not DNA replication, replication checkpoint, and cell cycle progression. This mutant protein is able to associate with all three replicative polymerases and checkpoint proteins Rad3ATR-Rad26ATRIP, Hus1, Rad9, and Rad17 but has a compromised association with Crb3. Furthermore, the damaged-induced Rad9 phosphorylation is significantly reduced in this rad4TopBP1 mutant. Genetic and biochemical analyses suggest that Crb3 has a role in the maintenance of DNA damage checkpoint and influences the Rad4TopBP1 damage checkpoint function. Taken together, our data suggest that Rad4TopBP1 provides a scaffold to a large complex containing checkpoint and replication proteins thereby separately enforcing checkpoint responses to DNA damage and replication perturbations during the cell cycle.

Cell Cycle↗
PubMed1996

Tyrosine kinases wee1 and mik1 as effectors of DNA replication checkpoint control.

Cell cycle studies have revealed mechanisms that prevent cell division if DNA fails to be completely replicated or sustains damage. Here we focus on the evidence from yeast genetics that the wee1 and mik1 tyrosine kinases cooperate in the inhibitory phosphorylation of cdc2p, and the possibility that these kinases function in pathways that ensure the integrity of the genome prior to cell division. We also review the progress in cloning and analysing wee1-like tyrosine kinases from higher eukaryotes, and the evidence for and against their functioning in ensuring DNA replication prior to mitosis. Finally, we discuss the genes involved in these feedback controls and suggest that wee1p and mik1p might be the ultimate effectors that prevent mitosis when a checkpoint is triggered.

Animals↗