Effects of inhibition of RNA or protein synthesis on CHO cell cycle progression

Chinese hamster ovary (CHO) cells, synchronized by selective detachment at mitosis, were treated with various concentrations of actinomycin D (AMD) or cycloheximide (CHX) either immediately, or 1, 2, or 3 hr after mitosis. Since the minimum duration of G₁ phase in these cultures was 3.4 hr, the addition of RNA or protein synthesis inhibitors took place at the beginning, first third, second third, or end (G₁–S boundary) of G₁ phase. The kinetics of exit from G₁ phase, the rate and extent of traverse of S phase, and the reaccumulation of RNA were estimated under each set of growth conditions by flow cytometry of acridine orange-stained cells. A mathematical model was constructed to describe the trajectories of the cell populations with respect to their increase in RNA and DNA content in the absence or presence of the inhibitor. The chronologic synchrony imposed on the CHO cell population began to decay within 3 hr, resulting in stochastic entrance of cells into S phase in the absence of inhibitor. Addition of AMD or CHX at 0, 1, 2, or 3 hr after mitosis, regardless of the inhibitor concentration, did not provide evidence of a critical restriction point in G₁ beyond which cells were committed to enter S phase and were no longer sensitive to moderate suppression of RNA or protein synthesis. The observed kinetics of cell entrance into and traverse of S phase were consistent with an inherently heterogenous response to serum stimulation occurring at or just after cell division.     

Hydrogen peroxide inhibits non-small cell lung cancer cell anoikis through the inhibition of caveolin-1 degradation

Anoikis or detachment-induced apoptosis plays an essential role in the regulation of cancer cell metastasis. Caveolin-1 (Cav-1) is a key protein involved in tumor metastasis, but its role in anoikis and its regulation during cell detachment are unclear. We report here that Cav-1 plays a key role as a negative regulator of anoikis through a reactive oxygen species (ROS)-dependent mechanism in human lung carcinoma H460 cells. During cell detachment, Cav-1 is downregulated, whereas ROS generation is upregulated. Hydrogen peroxide and hydroxyl radical are two key ROS produced by cells during detachment. Treatment of the cells with hydrogen peroxide scavengers, catalase and N-acetylcysteine, promoted Cav-1 downregulation and anoikis during cell detachment, indicating that produced hydrogen peroxide plays a primary role in preventing anoikis by stabilizing Cav-1 protein. Catalase and N-acetylcysteine promoted ubiquitination and proteasomal degradation of Cav-1, which is a major pathway of its downregulation during cell anoikis. Furthermore, addition of hydrogen peroxide exogenously to the cells inhibited Cav-1 downregulation by preventing the formation of Cav-1-ubiquitin complex, supporting the inhibitory role of endogenous hydrogen peroxide in Cav-1 degradation during cell detachment. Together, these results indicate a novel role of hydrogen peroxide as an endogenous suppressor of cell anoikis through its stabilizing effect on Cav-1.     

N-acetyl cysteine inhibits cell cycle progression in pancreatic carcinoma cells

The antioxidant N-acetyl cysteine (NAC) is a precursor of intracellular glutathione (GSH) and is also a well known as one of the chemopreventive agents which act through a variety of cellular mechanisms. We examined the effects of NAC on cell cycle progression in the pancreatic carcinoma cell lines, SW1990 and JHP1. Cells were incubated with or without NAC. Cell cycle distribution was analyzed by flow cytometry and immunoblotting. NAC suppressed cell proliferation in a concentration-dependent manner, whereas NAC increased intracellular glutathione content significantly in a dose-dependent manner. The percentage of cells in the G1 phase after treatment with NAC was significantly higher than the percentage seen for control cells. Cyclin D1 expression of carcinoma cells treated with NAC decreased remarkably compared with cells without NAC treatment. Thus, the antiproliferative effect of NAC by prolongation of the G1 phase in human pancreatic carcinoma cells shows its possible utility as an antitumor agent.     

Vesicular stomatitis virus inhibits mitotic progression and triggers cell death

Vesicular stomatitis virus (VSV) infects and kills a wide range of cell types; however, the mechanisms involved in VSV‐mediated cell death are not fully understood. Here we show that VSV infection interferes with mitotic progression, resulting in cell death. This effect requires the interaction of VSV matrix (M) protein with the Rae1–Nup98 complex in mitosis, which is associated with a subset of ribonucleoproteins (RNPs). VSV displaced Rae1 from spindle poles, caused spindle abnormalities and triggered substantial cell death during metaphase. These effects were attenuated in cells infected with VSV expressing a mutant M protein that does not bind efficiently to the Rae1–Nup98–RNP complex. In cells that progressed to late mitosis, M protein prevented proper nuclear formation and chromatin decondensation. VSV is an oncolytic (anti‐tumour) agent as it preferentially replicates and kills tumour cells. As tumour cells have a high mitotic index, VSV‐mediated mitotic cell death probably contributes to its oncolytic activity.     

Immunolocalization of the 100 kDa nucleolar protein during the mitotic cycle in CHO cells

The localization of a major nucleolar protein with a molecular weight of 100,000 has been followed during mitosis in Chinese hamster ovary CHO cells using specific antibodies to this protein and immunocytochemical techniques. The 100 kDa protein was visualized at discrete sites on metaphase chromosomes, corresponding to nucleolus organizer regions, and in large, immunostained nucleolar remnants that are discarded in the cytoplasm after nucleolar disintegration. After mitosis, the 100 kDa protein was shown to play an early role in nucleolar reformation. It was first detected in small deposits around the anaphase chromosomes. In telophase, the protein accumulated simultaneously in prenucleolar bodies and in the reforming nucleoli. The early presence of the 100 kDa protein in the telophase nucleus suggests that it is essential for the reestablishment of nucleolar function after mitosis. Thus this protein is present throughout the CHO cell cycle, an observation which supports the hypothesis that it plays a fundamental role in cell organization.     

Acetylcholine inhibits cell cycle progression in rat Schwann cells by activation of the M2 receptor subtype

Cultures of Schwann cells from neonatal rat sciatic nerves were treated with acetylcholine agonists and the effects on cell proliferation evaluated. (3)[H]-thymidine incorporation shows that acetylcholine (ACh) receptor agonists inhibit cell proliferation, and FACS analysis demonstrates cell-cycle arrest and accumulation of cells in the G1 phase. The use of arecaidine, a selective agonist of muscarinic M2 receptors reveals that this effect depends mainly on M2 receptor activation. The arecaidine dependent-block in G1 is reversible because removal of arecaidine from the culture medium induces progression to the S phase. The block of the G1-S transition is also characterized by modulation of the expression of several cell-cycle markers. Moreover, treatment with ACh receptor agonist causes both a decrease in the PCNA protein levels in Schwann cell nuclei and an increase in p27 and p53 proteins. Finally, immuno-electron microscopy demonstrates that M2 receptors are expressed by Schwann cells in vivo. These results indicate that ACh, by modulating Schwann cell proliferation through M2 receptor activation, might contribute to their progression to a more differentiated phenotype.     

Effects of growth media on cell cycle progression in CHO cells exposed to the radioprotector WR-1065

Abstract. WR-1065 (2-[(aminopropyl)amino]ethanethiol) reduces cytotoxic and mutagenic effects caused by exposure of cells to radiation and chemotherapeutic drugs, but the mechanisms involved are not fully known. We have observed an accumulation of cells in G, in WR-1065 treated Chinese hamster ovary cells grown in a-minimal essential medium, while others have found no cell cycle effects in WR-1065 treated Chinese hamster ovary cells grown in McCoy's 5A medium. To determine if the two types of media had an effect on cells treated with WR-1065, we examined survival and cell cycle progression. Population doubling times of 12 h were observed for cells grown in both media. Incubation of AA8 cells grown in McCoy's 5A medium with 4 mM WR-1065 30 min prior to and during irradiation with ^13'Cs gamma-rays resulted in a protection factor of 2.2, in close agreement with the value of 2.0 we previously obtained for AA8 cells grown in α-minimal essential medium. Treatment with WR-1065 caused an alteration in the cell cycles of cells grown in both media. An increase in the G₂ population and a decrease in the G₁ population was observed in cells incubated up to 3 h in the presence of 4 mM WR-1065, with a redistribution of the cells throughout the cell cycle occurring following removal of the drug. These data suggest that exposure of cells to WR-1065 is the cause of perturbations in cell cycle progression, and is not affected by the type of medium the cells are grown in.     

Hydrogen peroxide inhibits insulin signaling in vascular smooth muscle cells

Both insulin resistance and reactive oxygen species (ROS) have been reported to play essential pathophysiological roles in cardiovascular diseases, such as hypertension and atherosclerosis. However, the mechanistic link between ROS, such as H2O2 and insulin resistance in the vasculature, remains undetermined. Akt, a Ser/Thr kinase, mediates various biological responses induced by insulin. In this study, we examined the effects of H2O2 on Akt activation in the insulin-signaling pathway in vascular smooth muscle cells (VSMCs). In VSMCs, insulin stimulates Akt phosphorylation at Ser473. Pretreatment with H2O2 concentration- and time-dependently inhibited insulin-induced Akt phosphorylation with significant inhibition observed at 50 microM for 10 min. A ROS inducer, diamide, also inhibited insulin-induced Akt phosphorylation. In addition, H2O2 inhibited insulin receptor binding partially and inhibited insulin receptor autophosphorylation almost completely. However, pretreatment with a protein kinase C inhibitor, GF109203X (2 microM), for 30 min did not block the inhibitory effects of H2O2 on insulin-induced Akt phosphorylation, suggesting that protein kinase C is not involved in the inhibition by H2O2. We conclude that ROS inhibit a critical insulin signal transduction component required for Akt activation in VSMCs, suggesting potential cellular mechanisms of insulin resistance, which would require verification in vivo.     

Chlorsulfuron inhibition of cell cycle progression and the recovery of G1 arrested cells by Ile and Val

Cultured pea root tips were treated with 14 nM chlorsulfuron (CS) for up to 60 h. The progression of cells from G₁ into S was monitored by measuring³H-thymidine (³H-Thy) incorporation into DNA. Chlorsulfuron treatment decreased the amount of³H-Thy incorporated; however, this amount never reached zero. Short-term labeling experiments indicate that the cells are arrested in a narrow band within G₁. Cell progression recovered from the CS treatment when roots were transferred to either White's medium or White's supplemented with isoleucine (Ile) and valine (Val). Lag time before recovery began in the White's or White's plus Ile and Val medium was 12 and 4 h, respectively. The initial slope of the recovery curves was similar irrespective of the type of recovery media. Increasing the duration of CS treatment did not change the length of the lag or initial slopes of the recovery curves. The level of³H-Thy incorporation decreased with increasing duration of CS treatment for root segments transferred to Ile and Val recovery medium.     

Inhibition of cell proliferation and cell cycle progression by specific inhibition of basal JNK activity: evidence that mitotic Bcl-2 phosphorylation is JNK-independent

The c-Jun NH(2)-terminal kinase (JNK) subgroup of mitogen-activated protein kinases has been implicated largely in stress responses, but an increasing body of evidence has suggested that JNK also plays a role in cell proliferation and survival. We examined the effect of JNK inhibition, using either SP600125 or specific antisense oligonucleotides, on cell proliferation and cell cycle progression. SP600125 was selective for JNK in vitro and in vivo versus other kinases tested including ERK, p38, cyclin-dependent protein kinase 1 (CDK1), and CDK2. SP600125 inhibited JNK activity and KB-3 cell proliferation with the same dose dependence, suggesting that inhibition of proliferation was a direct consequence of JNK inhibition. Inhibition of proliferation by SP600125 was associated with an increase in the G(2)-M and apoptotic fractions of cells but was not associated with p53 or p21 induction. Antisense oligonucleotides to JNK2 but not JNK1 caused highly significant inhibition of cell proliferation. Wild-type mouse fibroblasts responded similarly with proliferation inhibition and apoptosis induction, whereas c-jun(-/-) fibroblasts were refractory to the effects of SP600125, suggesting that JNK signaling to c-Jun is required for cell proliferation. Studies in synchronized KB-3 cells indicated that SP600125 delayed transit time through S and G(2)-M phases. Correspondingly, JNK activity increased in late S phase and peaked in late G(2) phase. During synchronous mitotic progression, cyclin B levels increased concomitant with phosphorylation of c-Jun, H1 histone, and Bcl-2. In the presence of SP600125, mitotic progression was prolonged, and c-Jun phosphorylation was inhibited, but neither H1 nor Bcl-2 phosphorylation was inhibited. However, the CDK inhibitor roscovitine inhibited mitotic Bcl-2 phosphorylation. These results indicate that JNK, and more specifically the JNK2 isoform, plays a key role in cell proliferation and cell cycle progression. In addition, conclusive evidence is presented that a kinase other than JNK, most likely CDK1 or a CDK1-regulated kinase, is responsible for mitotic Bcl-2 phosphorylation.     

Endoplasmic reticulum stress inhibits cell cycle progression via induction of p27 in melanoma cells

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), a stress signaling pathway. The UPR coordinates the induction of ER chaperones with decreased protein synthesis and growth arrest in G1 phase of the cell cycle. However, the molecular mechanism underlying UPR-induced G1 cell cycle arrest remains largely unknown. Here we report that activation of the UPR response by tunicamycin (TM), an ER stress inducer, leads to accumulation of p27 and G1 cell cycle arrest in melanoma cells. This accumulation of p27 is due to the inhibition on its polyubiquitination and subsequent degradation upon TM treatment. Correlated with p27 stabilization, the levels of Skp2, an E3 ligase for p27, are decreased in response to TM treatment. More importantly, knockdown of p27 greatly reduces TM-induced G1 cell cycle arrest. Taken together, these data implicate p27 as a critical mediator of ER stress-induced growth arrest.     

Spindle assembly checkpoint regulates mitotic cell cycle progression during preimplantation embryo development

Errors in chromosome segregation or distribution may result in aneuploid embryo formation, which causes implantation failure, spontaneous abortion, genetic diseases, or embryo death. Embryonic aneuploidy occurs when chromosome aberrations are present in gametes or early embryos. To date, it is still unclear whether the spindle assembly checkpoint (SAC) is required for the regulation of mitotic cell cycle progression to ensure mitotic fidelity during preimplantation development. In this study, using overexpression and RNA interference (RNAi) approaches, we analyzed the role of SAC components (Bub3, BubR1 and Mad2) in mouse preimplantation embryos. Our data showed that overexpressed SAC components inhibited metaphase-anaphase transition by preventing sister chromatid segregation. Deletion of SAC components by RNAi accelerated the metaphase-anaphase transition during the first cleavage and caused micronuclei formation, chromosome misalignment and aneuploidy, which caused decreased implantation and delayed development. Furthermore, in the presence of the spindle-depolymerizing drug nocodazole, SAC depleted embryos failed to arrest at metaphase. Our results suggest that SAC is essential for the regulation of mitotic cell cycle progression in cleavage stage mouse embryos.     

Indomethacin inhibits cell growth of medullary thyroid carcinoma by reducing cell cycle progression into S phase

Indomethacin, a non-steroidal anti-inflammatory drug (NSAID), has been reported to inhibit the growth of medullary thyroid carcinoma (MTC) cells in vitro. However, the mechanism of inhibition of MTC cell growth by indomethacin and its potency have yet to be revealed. We examined the effect of indomethacin on three different MTC cell lines (TT cells, DRO 81-1 cells and HRO 85-1 cells) and two non-MTC cells. The mechanism of indomethacin action in MTC cells was investigated by analyzing intracellular prostaglandin level, apoptosis, and cell cycle in TT cells. Indomethacin inhibited cell growth of all three MTC cell lines but not normal thyroid cells or anaplastic thyroid carcinoma cells. Indomethacin at 10 microM or greater showed a dose response inhibition of cell growth. Indomethacin at 25 muM, a putative therapeutic serum indomethacin level, showed potency similar to 100 to 200 nM sunitinib, a receptor tyrosine kinase inhibitor. To examine whether prostaglandin depletion might determine the inhibition of MTC cell growth, we created different prostaglandin E2 (PGE2) levels in TT cells using three different NSAIDs. A profound PGE2 depletion by indomethacin-ester, a potent cyclooxygenase (COX) II inhibitor, showed the least inhibition of cell growth. Indomethacin did not increase apoptosis of TT cells. Indomethacin, but not naproxen or indomethacin-ester, reduced cell cycle progression into S phase; this was unrelated to the degree of PGE2 depletion. The expression of phosphorylated retinoblastoma (pRb) protein that shifts cells from G(1) to S phase was reduced after exposure to indomethacin. In conclusion, indomethacin has specific anti-tumor effect on MTC cells, probably by reducing cell cycle progression into S phase rather than by prostaglandin depletion. Since no drug therapy is currently available for MTC, indomethacin may be one of the therapeutic candidates.     

Transport of thymidine during the cell cycle in mitotically synchronized CHO cells

The kinetics of total uptake of thymidine into the cell were determined for cells which had been mitotically synchronized, plated into scintillation vials and pulsed with five concentrations of [³H]-thymidine at various times during the cell cycle. From Lineweaver-Burk plots of these rates, V_max and K_m values were determined for the transport of thymidine. The V_max values ranged from a low of 2.0 pmoles/ min/10⁶ cells in mid-G1 to a high of 99.7 in mid-S before a decline in late S and G2. K_m values displayed only a 5-fold range in values.     

Characterization of the deoxynucleoside-dependent reversal of hypoxia-induced inhibition of cell cycle progression in Ehrlich ascites tumor cells

Studies on Ehrlich ascites tumor cells enriched in different cell cycle compartments by centrifugal elutriation have shown that after exclusion of oxygen, G1- and early S-phase cells are restricted from further cell cycle progression. On addition of a balanced mixture of deoxypyrimidine and deoxypurine nucleosides to these anaerobic cultures (70% in G1-phase, as determined by flow cytometry), the cells resumed DNA synthesis and passed through the cycle. After 24 h, 19% of the cell population was still in G1. This cell cycle traverse does not seem to depend on RNA and protein synthesis. Using colcemid, mitotic activities became more evident in the presence of deoxynucleosides 20 h after establishing the protective atmosphere (argon/CO2). In the absence of colcemid binucleate cells could be detected. This was not observed in unsupplemented anaerobic cells. The ultrastructural changes of mitochondria in anaerobic cells resembled those in nucleoside-stimulated anaerobic cells: enlargement in profiles is accompanied by a simplification of cristae and a pallor of the intramatrical compartment. In addition, two different appearances of mitochondrial structures were visible if cells were cultured in the presence of deoxynucleosides.     

RPS27a promotes proliferation,regulates cell cycle progression and inhibits apoptosis of leukemia cells

Ribosomal protein S27a (RPS27a) could perform extra-ribosomal functions besides imparting a role in ribosome biogenesis and post-translational modifications of proteins. The high expression level of RPS27a was reported in solid tumors, and we found that the expression level of RPS27a was up-regulated in advanced-phase chronic myeloid leukemia (CML) and acute leukemia (AL) patients. In this study, we explored the function of RPS27a in leukemia cells by using CML cell line K562 cells and its imatinib resistant cell line K562/G01 cells. It was observed that the expression level of RPS27a was high in K562 cells and even higher in K562/G01 cells. Further analysis revealed that RPS27a knockdown by shRNA in both K562 and K562G01 cells inhibited the cell viability, induced cell cycle arrest at S and G2/M phases and increased cell apoptosis induced by imatinib. Combination of shRNA with imatinib treatment could lead to more cleaved PARP and cleaved caspase-3 expression in RPS27a knockdown cells. Further, it was found that phospho-ERK(p-ERK) and BCL-2 were down-regulated and P21 up-regulated in RPS27a knockdown cells. In conclusion, RPS27a promotes proliferation, regulates cell cycle progression and inhibits apoptosis of leukemia cells. It appears that drugs targeting RPS27a combining with tyrosine kinase inhibitor (TKI) might represent a novel therapy strategy in TKI resistant CML patients.