ERK1/2 Signaling Plays an Important Role in Topoisomerase II Poison-Induced G2/M Checkpoint Activation

Topo II poisons, which target topoisomerase II (topo II) to generate enzyme mediated DNA damage, have been commonly used for anti-cancer treatment. While clinical evidence demonstrate a capability of topo II poisons in inducing apoptosis in cancer cells, accumulating evidence also show that topo II poison treatment frequently results in cell cycle arrest in cancer cells, which was associated with subsequent resistance to these treatments. Results in this report indicate that treatment of MCF-7 and T47D breast cancer cells with topo II poisons resulted in an increased phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and an subsequent induction of G2/M cell cycle arrest. Furthermore, inhibition of ERK1/2 activation using specific inhibitors markedly attenuated the topo II poison-induced G2/M arrest and diminished the topo II poison-induced activation of ATR and Chk1 kinases. Moreover, decreased expression of ATR by specific shRNA diminished topo II poison-induced G2/M arrest but had no effect on topo II poison-induced ERK1/2 activation. In contrast, inhibition of ERK1/2 signaling had little, if any, effect on topo II poison-induced ATM activation. In addition, ATM inhibition by either incubation of cells with ATM specific inhibitor or transfection of cells with ATM specific siRNA did not block topo II poison-induced G2/M arrest. Ultimately, inhibition of ERK1/2 signaling greatly enhanced topo II poison-induced apoptosis. These results implicate a critical role for ERK1/2 signaling in the activation of G2/M checkpoint response following topo II poison treatment, which protects cells from topo II poison-induced apoptosis.     

Quantitative immunofluorescence and immunoelectron microscopy of the topoisomerase IIα associated with nuclear matrices from wild-type and drug-resistant Chinese hamster ovary cell lines

Topo IIα is considered an important constituent of the nuclear matrix, serving as a fastener of DNA loops to the underlying filamentous scaffolding network. To further define a mechanism of drug resistance to topo II poisons, we studied the quantity of topo IIα associated with the nuclear matrix in drug-resistant SMR₁₆ and parental cells in the presence and absence of VP-16. Nuclear matrices were prepared from nuclei isolated in EDTA buffer, followed by nuclease digestion with DNase II in the absence of RNase treatment and extraction with 2 M NaCl. Whole-mount spreading of residual structures permits, by means of isoform-specific antibody and colloidal-gold secondary antibodies, an estimate of the amount of topo IIα in individual nuclear matrices. There are significant variations in topo IIα amounts between individual nuclear matrices due to the cell cycle distribution. The parental cell line contained eight to ten times more nuclear matrix–associated topo IIα than the resistant cell line matrices. Nuclear matrix–associated topo IIα from wild-type and resistant cell lines correlated well with the immunofluorescent staining of the enzyme in nuclei of intact cells. The amount of DNA associated with residual nuclear structures was five times greater in the resistant cell line. This quantity of DNA was not proportional to the quantity of topo IIα in the same matrix; in fact they were inversely related. In situ whole-mount nuclear matrix preparations were obtained from cells grown on grids and confirmed the results from labeling of isolated residual structures. J. Cell. Biochem. 67:112–130, 1997. © 1997 Wiley-Liss, Inc.     

Adenovirus-mediated expression of both antisense ornithine decarboxylase and s-adenosylmethionine decarboxylase induces G1 arrest in HT-29 cells

To evaluated the effect of recombinant adenovirus Ad-ODC-AdoMetDCas which can simultaneously express both antisense ornithine decarboxylase (ODC) and Sadenosylmethionine decarboxylase (AdoMetDC) on cell cycle distribution in colorectal cancer cell and investigated underlying regulatory responses, human colorectal cancer cells HT-29 were cultured in RPMI 1640 medium and infected with Ad-ODC-AdoMetDCas. Cell cycle progression was detected by flow cytometry analysis. The expression levels of cell cycle regulated proteins were measured by Western blot analysis. The mRNA level of cyclin D1 was measured by RT-PCR. And a luciferase reporter plasmid of cyclin D1 promoter was constructed to observe the effect of Ad-ODC-AdoMetDCas on cyclin D1 promoter activity. The results showed that recombinant adenovirus Ad-ODC-AdoMetDCas significantly induced G1 arrest, decreased levels of cyclin D1 protein and mRNA and suppressed the promoter activity. Ad-ODC-AdoMetDCas also inhibited nuclear translocation of beta-catenin. In conclusion, downregulation of ODC and AdoMetDC mediated by Ad-ODC-AdoMetDCas transfection induces G(1) arrest in HT-29 cells and the arrest was associated with suppression of cyclin D1 expression and inhibition of beta-catenin nuclear translocation. As a new anticancer reagent, the recombinant adenovirus Ad-ODC-AdoMetDCas holds promising hope for the therapy of colorectal cancers.     

Enhanced sensitivity of G1 arrested human cancer cells suggests a novel therapeutic strategy using a combination of simvastatin and TRAIL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in tumor cells over normal cells. To study the relationship between cell cycle progression and TRAIL-induced apoptosis, SW480 colon cancer and H460 lung cancer cell lines were examined for their sensitivity to TRAIL after arrest in different cell cycle phases. Cells were synchronized in G0/G1, S, and G2/M phase by serum starvation, aphidicolin, or nocodazole treatment, respectively. We found that arrest of cells in G0/G1 phase confers significantly higher susceptibility to TRAIL-induced apoptosis as compared to cells in late G1, S, or G2/M phase. To determine if cell cycle phase could be harnessed for therapeutic gain in the presence of TRAIL, we used the HMG-CoA reductase inhibitor, Simvastatin and lovastatin, to enrich a cancer cell population in G0/G1. Both simvastatin and lovastatin significantly augmented TRAIL-induced apoptosis in tumor cells, but not in normal keratinocytes. The results indicate that TRAIL, in combination with a HMG-CoA reductase inhibitor, may have therapeutic potential in the treatment of human cancer.     

NF-kappaB pathway is involved in griseofulvin-induced G2/M arrest and apoptosis in HL-60 cells

Griseofulvin (GF), an oral antifungal agent, has been shown to exert antitumorigenesis effect through G2/M cell cycle arrest in colon cancer cells. But the underlying mechanisms remained obscure. The purpose of this study is to test the cytotoxic effect of GF on HL-60 and HT-29 cells and elucidate its underlying molecular pathways. Dose-dependent and time-course studies by flow cytometry demonstrated that 30 to 60 microM GF significantly induced G2/M arrest and to a less extend, apoptosis, in HL-60 cells. In contrast, only G2/M arrest was observed in HT-29 cells under similar condition. Pretreatment of 30 microM TPCK, a serine protease inhibitor, completely reversed GF-induced G2/M cell cycle arrest and apoptosis in HL-60 cells but not in HT-29 cells. The GF-induced G2/M arrest in HL-60 cells is reversible. Using EMSA and super-shift analysis, we demonstrated that GF stimulated NF-kappaB binding activity in HL-60 cells, which was completely inhibited by pretreatment of TPCK. Treatment of HL-60 with 30 microM GF activated JNK but not ERK or p38 MAPK and subsequently resulted in phosphorylation of Bcl-2. Pretreatment of TPCK to HL-60 cells blocked the GF-induced Bcl-2 phosphorylation but not JNK activation. Time course study demonstrated that activation of cdc-2 kinase activity by GF correlated with Bcl-2 phosphorylation. Taken together, our results suggest that activation of NF-kappaB pathway with cdc-2 activation and phosphorylation of Bcl-2 might be involved in G2/M cell cycle arrest in HL-60 cells.     

Cycloalkyl-substituted aryl chloroethylureas inhibiting cell cycle progression in G0/G1 phase and thioredoxin-1 nuclear translocation

1-(2-Chloroethyl)-3-(4-cyclohexylphenyl)urea (cHCEU) has been shown to abrogate the presence of thioredoxin-1 into the nucleus through its selective covalent alkylation. In the present letter we have evaluated the structure-activity relationships of the substituents at positions 3 and 4 of the phenyl ring of cHCEU derivatives on cell cycle progression and thioredoxin-1 nuclear translocation. Active CEU derivatives exhibited GI(50) ranging from 1.9 to 49muM on breast carcinoma MCF-7, skin melanoma M21, and colon carcinoma HT-29 cells. On one hand, compounds 1, 2, 9c, 10c, 13, and 14 arrested the cell cycle in G(2)/M phase while CEUs 3, 4, 5c, 6c, 11c, and 12c blocked the cell division in G(0)/G(1) phase. On the other hand, CEUs 2-4, 5c, 7c, 8c, 11c, and 12c abrogated the translocation of thioredoxin-1 while the other CEU derivatives were inactive in that respect. Our results suggest that CEU substituted on the phenyl ring at position 3 or 4 by lower cycloalkyl or cycloalkoxy groups arrest cell progression in G(0)/G(1) phase through mechanism of action different from their antimicrotubule counterparts, presumably via thioredoxin-1 alkylation and modulation of its activity. The mechanism of action of these new molecules is still undetermined. However, the significant accumulation of cells in G(0)/G(1) phase suggests that these molecules may act similarly to known chemopreventive agents against cancers. In addition, the inhibition of Trx-1 nuclear localization also suggests the abrogation of an important chemoresistance mechanism towards a variety of chemotherapeutic agents.     

Inhibition of DNA topoisomerase II by ICRF-193 induces polyploidization by uncoupling chromosome dynamics from other cell cycle events

ICRF-193, a novel noncleavable, complex-stabilizing type topoisomerase (topo) II inhibitor, has been shown to target topo II in mammalian cells (Ishida, R., T. Miki, T. Narita, R. Yui, S. Sato, K. R. Utsumi, K. Tanabe, and T. Andoh. 1991. Cancer Res. 51:4909-4916). With the aim of elucidating the roles of topo II in mammalian cells, we examined the effects of ICRF-193 on the transition through the S phase, when the genome is replicated, and through the M phase, when the replicated genome is condensed and segregated. Replication of the genome did not appear to be affected by the drug because the scheduled synthesis of DNA and activation of cdc2 kinase followed by increase in mitotic index occurred normally, while VP-16, a cleavable, complex-stabilizing type topo II inhibitor, inhibited all these processes. In the M phase, however, late stages of chromosome condensation and segregation were clearly blocked by ICRF-193. Inhibition at the stage of compaction of 300-nm diameter chromatin fibers to 600-nm diameter chromatids was demonstrated using the drug during premature chromosome condensation (PCC) induced in tsBN2 baby hamster kidney cells in early S and G2 phases. In spite of interference with M phase chromosome dynamics, other mitotic events such as activation of cdc2 kinase, spindle apparatus reorganization and disassembly and reassembly of nuclear envelopes occurred, and the cells traversed an unusual M phase termed "absence of chromosome segregation" (ACS)-M phase. Cells then continued through further cell cycle rounds, becoming polyploid and losing viability. This effect of ICRF-193 on the cell cycle was shown to parallel that of inactivation of topo II on the cell cycle of the ts top2 mutant yeast. The results strongly suggest that the essential roles of topo II are confined to the M phase, when the enzyme decatenates intertwined replicated chromosomes. In other phases of the cycle, including the S phase, topo II may thus play a complementary role with topo I in controlling the torsional strain accumulated in various genetic processes.     

Cell cycle stage-specific phosphorylation of the Epstein-Barr virus immortalization protein EBNA-LP.

EBNA-LP is a viral nuclear oncoprotein implicated in the immortalization of B lymphocytes by Epstein-Barr virus. An analysis of EBNA-LP migration on polyacrylamide gels was performed with protein derived from the X50-7 lymphoblastoid cell line blocked by hydroxyurea or aphidicolin at the G1/S phase of the cell cycle or by nocodazole at the G2/M phase. More slowly migrating species of EBNA-LP were detected in G2/M phase-arrested cell extracts. Release from nocodazole G2/M block or treatment with phosphatase caused the more slowly migrating species of EBNA-LP to disappear. Analyses of 32PO(4)(3-)-labeled EBNA-LP protein immunoprecipitated from the drug-synchronized cells showed that phosphorylated EBNA-LP was present throughout the cell cycle but that phosphorylation increased in G2 and was maximal at G2/M. Phosphoamino acid analysis revealed that all phosphorylation was on serine residues only. The ability of EBNA-LP to be phosphorylated by p34(cdc2) kinase and casein kinase II exclusively on serines implicates these enzymes as being potentially involved in EBNA-LP phosphorylation.     

Enhanced Sensitivity of G1 Arrested Human Cancer Cells Suggests a Novel Therapeutic Strategy Using a Combination of Simvastatin and TRAIL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in tumor cells over normal cells. To study the relationship between cell cycle progression and TRAIL-induced apoptosis, SW480 colon cancer and H460 lung cancer cell lines were examined for their sensitivity to TRAIL after arrest in different cell cycle phases. Cells were synchronized in G0/G1, S, and G2/M phase by serum starvation, aphidicolin, or nocodazole treatment, respectively. We found that arrest of cells in G0/G1 phase confers significantly higher susceptibility to TRAIL-induced apoptosis as compared to cells in late G1, S, or G2/M phase. To determine if cell cycle phase could be harnessed for therapeutic gain in the presence of TRAIL, we used the HMG-CoA reductase inhibitor, Simvastatin and lovastatin, to enrich a cancer cell population in G0/G1. Both simvastatin and lovastatin significantly augmented TRAIL-induced apoptosis in tumor cells, but not in normal keratinocytes. The results indicate that TRAIL, in combination with a HMG-CoA reductase inhibitor, may have therapeutic potential in the treatment of human cancer.

Key Words

TRAIL, Synchronization, Simvastatin, Cancer Therapy, Lovastatin, Cell Cycle, Apoptosis     

Nuclear phospholipase C-beta1b activation during G2/M and late G1 phase in nocodazole-synchronized HL-60 cells

In this study, the activity of nuclear phosphatidylinositol-specific phosholipase C (PI-PLC) was investigated in HL-60 cells blocked at G(2)/M phase by the addition of nocodazole, and released into medium as synchronously progressing cells. Two peaks of an increase in the nuclear PI-PLC activities were detected; an early peak reached a maximum at 1 h after release from the nocodazole block, and a second increase was detected at 8.5 h after the release. Immunoprecipitation studies indicated that the increase in the activity was due to the activation of the nuclear PI-PLC-beta(1). Western blot analysis demonstrated no changes in the level of both a and b splicing variants of PI-PLC-beta(1) in the nuclei of cells isolated at either 1 h or 8.5 h after the block. However, an increase in the serine-phosphorylation of PI-PLC-beta(1b) was detected in the nuclei of HL-60 cells isolated at 1 and 8.5 h after the block, and the presence of MEK-inhibitor PD98059 completely inhibited both the serine phosphorylation and the increase in the PI-PLC activities in vitro. The presence of PI-PLC inhibitor prevented the progression of HL-60 cells through the G(1) into S phase of the cell cycle. These results demonstrate that two peaks of nuclear PI-PLC activities, which are due to a PD98059-sensitive phosphorylation of nuclear PLC-beta(1b) on serine, occur at the G(2)/M and late G(1) phase and are necessary for the progression of the cells through the cell cycle.     

Trichomonas vaginalis: Dominant G2 Period and G2 Phase Arrest in a Representative of an Early Branching Eukaryotic Lineage

ABSTRACT. Eukaryotic mitotic cell cycles have been extensively studied in yeasts and vertebrate cells but little is known about cell cycle mechanisms in early branches of the eukaryotic lineage. Trichomonas vaginalis represents one of the earliest branching eukaryotic lineages available for study. In contrast with most yeasts and vertebrate cells, the T. vaginalis G2 period was prolonged, comprising 50 to 58% of the cell population. Hydroxyurea, aphidicolin, and excess thymidine, all of which arrest yeasts and vertebrate cells at the G1/S phase boundary, had no effect on the T. vaginalis cell cycle, probably due to the known absence of synthetic pathways. The antimicrotubule mitotic inhibitors, colchicine and nocodazole, induced G2 phase synchrony. Metronidazole, a therapeutic reagent, also caused G2 phase arrest. These observations suggest that T. vaginalis is similar to yeasts and vertebrate cells in G2 and M phases, but the parasite's G1/S phase transition is distinctive. The results also suggest potentially therapeutic, anti-trichomonad activity of microtubule inhibitors such as nocodazole. The cultured parasite may prove useful as a model for the mitotic cell cycle in the absence of G1/S phase transitional activities universal in yeasts and vertebrate cells.     

Human cytomegalovirus infection inhibits G1/S transition.

Cell cycle progression during cytomegalovirus infection was investigated by fluorescence-activated cell sorter (FACS) analysis of the DNA content in growth-arrested as well as serum-stimulated human fibroblasts. Virus-infected cells maintained in either low (0.2%) or high (10%) serum failed to progress into S phase and failed to divide. DNA content analysis in the presence of G1/S (hydroxyurea and mimosine) and G2/M (nocodazole and colcemid) inhibitors demonstrated that upon virus infection of quiescent (G0) cells, the cell cycle did not progress beyond the G1/S border even after serum stimulation. Proteins which normally indicate G1/S transition (proliferating cell nuclear antigen [PCNA]) or G2/M transition (cyclin B1) were elevated by virus infection. PCNA levels were induced in infected cells and exhibited a punctate pattern of nuclear staining instead of the diffuse pattern observed in mock-infected cells. Cyclin B1 was induced in infected cells which exhibited a G1/S DNA content by FACS analysis, suggesting that expression of this key cell cycle function was dramatically altered by viral functions. These data demonstrate that contrary to expectations, cytomegalovirus inhibits normal cell cycle progression. The host cell is blocked prior to S phase to provide a favorable environment for viral replication.     

A two-step computer-assisted method for deriving steady-state rate equations

Solid tumors commonly contain regions with glucose-starved and hypoxic conditions. Tumor cells under the adverse conditions can survive through the stress response, such as cell cycle arrest. In this study, we found that the stress conditions stimulated nuclear accumulation of proteasomes, large multicatalytic protease complexes, in human colon cancer HT-29 cells. The nuclear proteasome levels both in amount and in activity were increased approximately 4 and 2 times by glucose starvation and hypoxia, respectively. No changes were detected in the total expression levels of proteasome. The nuclear proteasome accumulation was also observed in ovarian cancer A2780 cells under glucose starvation, suggesting that this response was regardless of the origin of cancer cells. Our results indicate that the nuclear proteasome distribution is enhanced by glucose starvation and hypoxia, and suggest that the proteolysis by proteasome in the nucleus may play roles in the stress response of solid tumor cells.     

The lack of G1/S arrest of teratocarcinoma F9 cells is determinated by degradation of cyclin-dependent kinases inhibitor p21waf1/cip1

We have studied the ability of F9 teratocarcinoma cells to arrest in G1/S and G2/M checkpoints following gamma-irradiation. Wild-type p53 protein is rapidly accumulated in F9 cells after gamma-irradiation, however this is not followed by G1/S arrest; there is just a reversible delay of the cell cycle in G2/M. In order to elucidate the reasons of the lack of G1/S arrest in F9 cells we investigated the levels of regulatory cell cycle proteins: G1-cyclins, cyclin dependent kinases and kinase inhibitor p21WAF1/CIP1. We have shown that in spite of p53-dependent activation of p21WAF1/CIP1 promoter, p21WAF1/CIP1 protein is not revealed by different polyclonal and monoclonal antibodies, either by immunoblotting or by immunofluorescent staining. However, when cells are treated with specific proteasome inhibitor lactacystin, p21WAF1/CIP1 protein is revealed. We therefore suggest that p21WAF1/CIP1 protein is subjected to proteasome degradation in F9 cells and probably the lack of G1/S arrest after gamma-irradiation is due to this degradation. Thus, it is the combination of functionally active p53 with low level expression of p21WAF1/CIP1 that causes a short delay of the cell cycle progression in G2/M, rather than the G1-arrest after gamma-irradiation of F9 cells.     

MEKK1 is essential for DT40 cell apoptosis in response to microtubule disruption

Vinblastine and other microtubule-damaging agents, such as nocodazole and paclitaxel, cause cell cycle arrest at the G2/M transition and promote apoptosis in eukaryotic cells. The roles of these drugs in disrupting microtubule dynamics and causing cell cycle arrest are well characterized. However, the mechanisms by which these agents promote apoptosis are poorly understood. We disrupted the MEKK1 kinase domain in chicken bursal B-cell line DT40 by homologous recombination and have shown that it is essential for both vinblastine-mediated apoptosis and vinblastine-mediated c-Jun N-terminal protein kinase activation. In addition, our data indicate that vinblastine-mediated apoptosis in DT40 cells requires new protein synthesis but does not require G2/M arrest, suggesting that vinblastine-mediated cell cycle arrest and apoptosis are two independent processes.     

Effect of etoposide and amsacrine on mitotic progression of GM-130 and Hep-2 cell lines. The flow cytometry assay

It has been shown that inhibitors of topoisomerase II (topo II) etoposide and amsacrine results in accumulation of GM-130 and Hep-2 cells with 4c DNA amount. The differential analysis based on flow cytometry (Zenin et al., 2001) and enabled us to discriminate cells with 4c DNA--G2, M, including metaphase and anaphase cells and cells in pseudo-G1. 1 microM etoposide evoked cell accumulation in G2 phase, while 40 microM etoposide blocked cell proliferation, which was confirmed by a complete absence of both mitotic cells and 4c DNA cell accumulation. GM-130 and Hep-2 cells that were first blocked and then washed from nocodazole, and after that treated with 50 microM etoposide or 20 microM amsacrine, were shown to enter pseudo-G1 with 4c DNA amount per cell. In the presence of nocodazole, 4 and 40 microM amsacrine evoked transition of all mitotic cells to pseudo-G1 within 1 h. 15 or 30 minutes pulse treatments of GM-130 cells with 40 microM amsacrine in the presence of nocodazole, followed by incubation in drug-free medium, resulted in the similar transition of cells to pseudo-G1.     

Skp2 regulates G2/M progression in a p53-dependent manner

Targeted proteasomal degradation mediated by E3 ubiquitin ligases controls cell cycle progression, and alterations in their activities likely contribute to malignant cell proliferation. S phase kinase-associated protein 2 (Skp2) is the F-box component of an E3 ubiquitin ligase complex that targets p27^Kip1 and cyclin E1 to the proteasome. In human melanoma, Skp2 is highly expressed, regulated by mutant B-RAF, and required for cell growth. We show that Skp2 depletion in melanoma cells resulted in a tetraploid cell cycle arrest. Surprisingly, co-knockdown of p27^Kip1 or cyclin E1 failed to prevent the tetraploid arrest induced by Skp2 knockdown. Enhanced Aurora A phosphorylation and repression of G2/M regulators cyclin B1, cyclin-dependent kinase 1, and cyclin A indicated a G2/early M phase arrest in Skp2-depleted cells. Furthermore, expression of nuclear localized cyclin B1 prevented tetraploid accumulation after Skp2 knockdown. The p53 status is most frequently wild type in melanoma, and the tetraploid arrest and down-regulation of G2/M regulatory genes were strongly dependent on wild-type p53 expression. In mutant p53 melanoma lines, Skp2 depletion did not induce cell cycle arrest despite up-regulation of p27^Kip1. These data indicate that elevated Skp2 expression may overcome p53-dependent cell cycle checkpoints in melanoma cells and highlight Skp2 actions that are independent of p27^Kip1 degradation.