FGF inhibits the activity of the cyclin B1/CDK1 kinase to induce a transient G2 arrest in RCS chondrocytes

Fibroblast growth factors (FGFs) negatively regulate long bone development by inhibiting the proliferation of chondrocytes that accumulate in the G1 phase of the cycle following FGF treatment. Here we report that FGF also causes a striking but transient delay in mitotic entry in RCS chondrocytes by inactivating the cyclin B1-associated CDK1(CDC2) kinase. As a consequence of this inactivation, cells accumulate in the G2 phase of the cycle for the first 4-6 hours of the treatment. Cyclin B1/CDK1 activity is then restored and cells reach a G1 arrest. The reduced cyclin B1/CDK1 activity was accompanied by increased CDK1 inhibitory phosphorylation, likely caused by increased activity and expression of the Myt1 kinase. FGF1 also caused dephosphorylation of the CDC25C phosphatase, that however appears due the inactivation of cyclin B1/CDK1 complex in the CDK1 feedback loop, and not the activation of specific phosphatases. The inactivation of the cyclin B1/CDK1 complex is a direct effect of FGF signaling, and not a consequence of the G2 arrest as it can be observed also in cells blocked at mitosis by Nocodazole. The Chk1 and ATM/ATR kinase are known to play essential roles in the G2 checkpoint induced by DNA damage/genotoxic stress, but inhibition of Chk1 or ATM/ATR not only did not prevent, but rather potentiated the FGF-induced G2 arrest. Additionally our results indicate that the transient G2 arrest is induced by FGF in RCS cell through mechanisms that are independent of the G1 arrest, and that the G2 block is not strictly required for the sustained G1 arrest but may provide a pausing mechanism that allows the FGF response to be fully established.     

FGF inhibits the activity of the cyclin B1/CDK1 kinase to induce a transient G2 arrest in RCS chondrocytes

Fibroblast growth factors (FGFs) negatively regulate long bone development by inhibiting the proliferation of chondrocytes that accumulate in the G₁ phase of the cycle following FGF treatment. Here we report that FGF also causes a striking but transient delay in mitotic entry in RCS chondrocytes by inactivating the cyclin B1-associated CDK1(CDC2) kinase. As a consequence of this inactivation, cells accumulate in the G₂ phase of the cycle for the first 4–6 hours of the treatment. Cyclin B1/CDK1 activity is then restored and cells reach a G₁ arrest.The reduced cyclin B1/CDK1 activity was accompanied by increased CDK1 inhibitory phosphorylation, likely caused by increased activity and expression of the Myt1 kinase. FGF1 also caused dephosphorylation of the CDC25C phosphatase. That, however, appears due the inactivation of cyclin B1/CDK1 complex in the CDK1 feedback loop and not the activation of specific phosphatases. The inactivation of the cyclin B1/CDK1 complex is a direct effect of FGF signaling and not a consequence of the G₂ arrest as can be observed also in cells blocked at mitosis by Nocodazole. The Chk1 and ATM/ATR kinase are known to play essential roles in the G₂ checkpoint induced by DNA damage/genotoxic stress, but inhibition of Chk1 or ATM/ATR not only did not prevent, but rather potentiated the FGF-induced G₂ arrest.Additionally, our results indicate that the transient G₂ arrest is induced by FGF in RCS cell through mechanisms that are independent of the G₁ arrest, and that the G₂ block is not strictly required for the sustained G₁ arrest but may provide a pausing mechanism that allows the FGF response to be fully established.Key words: fibroblast growth factor, chondrocyte, G₂/M arrest, Myt1, cyclin B1, CDK1     

Inhibition of S/G2 phase CDK4 reduces mitotic fidelity

Cyclin-dependent kinase 4 (CDK4)/cyclin D has a key role in regulating progression through late G(1) into S phase of the cell cycle. CDK4-cyclin D complexes then persist through the latter phases of the cell cycle, although little is known about their potential roles. We have developed small molecule inhibitors that are highly selective for CDK4 and have used these to define a role for CDK4-cyclin D in G(2) phase. The addition of the CDK4 inhibitor or small interfering RNA knockdown of cyclin D3, the cyclin D partner, delayed progression through G(2) phase and mitosis. The G(2) phase delay was independent of ATM/ATR and p38 MAPK but associated with elevated Wee1. The mitotic delay was because of failure of chromosomes to migrate to the metaphase plate. However, cells eventually exited mitosis, with a resultant increase in cells with multiple or micronuclei. Inhibiting CDK4 delayed the expression of the chromosomal passenger proteins survivin and borealin, although this was unlikely to account for the mitotic phenotype. These data provide evidence for a novel function for CDK4-cyclin D3 activity in S and G(2) phase that is critical for G(2)/M progression and the fidelity of mitosis.     

Akt-induced promotion of cell-cycle progression at G2/M phase involves upregulation of NF-Y binding activity in PC12 cells

Akt is a key downstream effector of the PI3K signaling pathway and plays a role in cell growth and survival. Expression of a myristoylated constitutively active form of Akt (myr-Akt) in PC12 cells could override cell-growth arrest at G2/M phase and apoptosis that were induced by etoposide treatment. On the other hand, inactivation of Akt by expression of its dominant negative mutant form (km-Akt) inhibited cell proliferation by arresting the cells at G2/M phase. Expression of myr-Akt also led to an increase in the protein and mRNA levels of CDK1 and cyclin B1. Furthermore, EMSA data revealed that expression of myr-Akt promoted the binding of NF-Y to the consensus CCAAT promoter sequence, whereas expression of km-Akt almost completely abolished it. Moreover, the Akt activity was minimal in the cells that were arrested at G2/M phase by nocodazole treatment, but reached to a maximal level as the cells progressed to mitosis and G1 phase upon removal of the drug. Treatment with Akt inhibitors, but not with those of MEK or p70S6K, blocked the release of the cells from the nocodazole-induced G2/M arrest, further revealing that the Akt activity is required for G2/M phase transition. These results suggest that Akt facilitate cell-cycle progression at G2/M phase in PC12 cells and this Akt activity is correlated with upregulation of NF-Y DNA-binding activity and cyclin B1/CDK1 gene expression.     

The molecular mechanisms of vitamin C on cell cycle regulation in B16F10 murine melanoma

Vitamin C has inconsistent effects on malignant tumor cells, which vary from growth stimulation to apoptosis induction. It is well known that melanoma cells are more susceptible to vitamin C than any other tumor cells, but the precise mechanism remains to be elucidated. In the present study, the proliferation of B16F10 melanoma cells was suppressed by vitamin C, which induced growth arrest in a dose-dependent manner without cytotoxic effects. Therefore, we investigated the changes in cell cycle distribution of B16F10 melanoma cells by staining DNAs with propidium iodide (PI). The growth inhibition of B16F10 melanoma by vitamin C was associated with an arrest of cell cycle distribution at G1 stage. In addition, the levels of p53-p21Waf1/Cip1 increased during G1 arrest, which were essential for vitamin C-induced cell cycle arrest. The increased p21Waf1/Cip1 inhibited CDK2. Moreover, the activity of p53-p21Waf1/Cip1 pathway was closely related with the activation of checkpoint kinase 2 (Chk2). Inhibitor of the PI3K-family, LY294002 and the ATM/ATR inhibitor, caffeine, blocked vitamin C-induced growth arrest in B16F10 melanoma cells. These results suggest that vitamin C might be a potent agent to inhibit proliferative activity of melanoma cells via the regulation of Chk2-p53-p21Waf1/Cip1 pathway.     

Regulation of Polo-like kinase 1 by DNA damage in mitosis. Inhibition of mitotic PLK-1 by protein phosphatase 2A

DNA damage triggers multiple checkpoint pathways to arrest cell cycle progression. Polo-like kinase 1 (Plk1) is an important regulator of several events during mitosis. In addition to Plk1 functions in cell cycle, Plk1 is involved in DNA damage check-point in G2 phase. Normally, ataxia telangiectasia-mutated kinase (ATM) is a key enzyme involved in G2 phase cell cycle arrest following DNA damage, and inhibition of Plk1 by DNA damage during G2 occurs in a ATM/ATR-dependent manner. However, it is still unclear how Plk1 is regulated in response to DNA damage in mitosis in which Plk1 is already activated. Here, we show that treatment of mitotic cells with doxorubicin and gamma-irradiation inhibits Plk1 activity through dephosphorylation of Plk1, and cells were arrested in G2 phase. Treatments of the phosphatase inhibitors and siRNA experiments suggested that PP2A pathway might be involved in regulating mitotic Plk1 activity in mitotic DNA damage. Finally, we propose a novel pathway, which is connected between ATM/ATR/Chk and protein phosphatase-Plk1 in DNA damage response in mitosis.     

A preliminary study: the anti-proliferation effect of salidroside on different human cancer cell lines

Salidroside (p-hydroxyphenethyl-beta-d-glucoside), which is present in all species of the genus Rhodiola, has been reported to have a broad spectrum of pharmacological properties. The present study, for the first time, focused on evaluating the effects of the purified salidroside on the proliferation of various human cancer cell lines derived from different tissues, and further investigating its possible molecular mechanisms. Cell viability assay and [³H] thymidine incorporation were used to evaluate the cytotoxic effects of salidroside on cancer cell lines, and flow cytometry analyzed the change of cell cycle distribution induced by salidroside. Western immunoblotting further studied the expression changes of cyclins (cyclin D1 and cyclin B1), cyclin-dependent kinases (CDK4 and Cdc2), and cyclin-dependent kinase inhibitors (p21^Cip1 and p27^Kip1). The results showed that salidroside inhibited the growth of various human cancer cell lines in concentration- and time-dependent manners, and the sensitivity to salidroside was different in those cancer cell lines. Salidroside could cause G1-phase or G2-phase arrest in different cancer cell lines, meanwhile, salidroside resulted in a decrease of CDK4, cyclin D1, cyclin B1 and Cdc2, and upregulated the levels of p27^Kip1 and p21^Cip1. Taken together, salidroside could inhibit the growth of cancer cells by modulating CDK4-cyclin D1 pathway for G1-phase arrest and/or modulating the Cdc2-cyclin B1 pathway for G2-phase arrest.     

TGF-beta induced G(1) cell cycle arrest requires the activity of the proteasome pathway. Transforming growth factor

Transforming growth factor-beta (TGF-beta) induces a potent G(1)/S-phase cell cycle arrest of epithelial cells by inhibiting the activities of cyclin D- and cyclin E-associated kinase complexes. Downregulation of the kinase activities is mediated by induction of cyclin dependent kinase (CDK) inhibitor p15(Ink4b) which blocks CDK4 and CDK6 kinases and leads to binding of p27(Kip1) to CDK2-cyclin E complex. Levels of several of these factors are controlled by the ubiquitin-proteasome pathway. We demonstrate here that proteasomal inhibitors release the cells from TGF-beta imposed G(1)-phase arrest and instigate the entry of the cells into S-phase. Proteasomal inhibitors are shown to specifically increase the activity of the cyclin D-kinase complex by increasing the levels of p27(Kip1) and cyclin D and by maintaining CDK4/6 protein levels leading to phosphorylation of the retinoblastoma protein without increasing cyclin E-associated kinase activity. The results indicate caution in the potential therapeutic use of the proteasome inhibitors due to unscheduled initiation of DNA replication in the presence of a physiological growth inhibitor.     

Parvovirus B19 infection of human primary erythroid progenitor cells triggers ATR-Chk1 signaling, which promotes B19 virus replication

Human parvovirus B19 (B19V) infection is restricted to erythroid progenitor cells of the human bone marrow. Although the mechanism by which the B19V genome replicates in these cells has not been studied in great detail, accumulating evidence has implicated involvement of the cellular DNA damage machinery in this process. Here, we report that, in ex vivo-expanded human erythroid progenitor cells, B19V infection induces a broad range of DNA damage responses by triggering phosphorylation of all the upstream kinases of each of three repair pathways: ATM (ataxia-telangiectasi mutated), ATR (ATM and Rad3 related), and DNA-PKcs (DNA-dependent protein kinase catalytic subunit). We found that phosphorylated ATM, ATR, and DNA-PKcs, and also their downstream substrates and components (Chk2, Chk1, and Ku70/Ku80 complex, respectively), localized within the B19V replication center. Notably, inhibition of kinase phosphorylation (through treatment with either kinase-specific inhibitors or kinase-specific shRNAs) revealed requirements for signaling of ATR and DNA-PKcs, but not ATM, in virus replication. Inhibition of the ATR substrate Chk1 led to similar levels of decreased virus replication, indicating that signaling via the ATR-Chk1 pathway is critical to B19V replication. Notably, the cell cycle arrest characteristic of B19V infection was not rescued by interference with the activity of any of the three repair pathway kinases.     

Cell cycle and biochemical effects of PD 0183812. A potent inhibitor of the cyclin D-dependent kinases CDK4 and CDK6

Progression through the G1 phase of the cell cycle requires phosphorylation of the retinoblastoma gene product (pRb) by the cyclin D-dependent kinases CDK4 and CDK6, whose activity can specifically be blocked by the CDK inhibitor p16(INK4A). Misregulation of the pRb/cyclin D/p16(INK4A) pathway is one of the most common events in human cancer and has lead to the suggestion that inhibition of cyclin D-dependent kinase activity may have therapeutic value as an anticancer treatment. Through screening of a chemical library, we initially identified the [2,3-d]pyridopyrimidines as inhibitors of CDK4. Chemical modification resulted in the identification of PD 0183812 as a potent and highly selective inhibitor of both CDK4 and CDK6 kinase activity, which is competitive with ATP. Flow cytometry experiments showed that of the cell lines tested, only those expressing pRb demonstrated a G1 arrest when treated with PD 0183812. This arrest correlated in terms of incubation time and potency with a loss of pRb phosphorylation and a block in proliferation, which was reversible. These results suggest a potential use of this chemical class of compounds as therapeutic agents in the treatment of tumors with functional pRb, possessing cell cycle aberrations in other members of the pRb/cyclin D/p16(INK4A) pathway.     

CDK4 inhibition restores G₁-S arrest in MYCN-amplified neuroblastoma cells in the context of doxorubicin-induced DNA damage

Relapse with drug-resistant disease is the main cause of death in MYCN-amplified neuroblastoma patients. MYCN-amplified neuroblastoma cells in vitro are characterized by a failure to arrest at the G?-S checkpoint after irradiation- or drug-induced DNA damage. We show that several MYCN-amplified cell lines harbor additional chromosomal aberrations targeting p53 and/or pRB pathway components, including CDK4/CCND1/MDM2 amplifications, p16INK4A/p14ARF deletions or TP53 mutations. Cells with these additional aberrations undergo significantly lower levels of cell death after doxorubicin treatment compared with MYCN-amplified cells, with no additional mutations in these pathways. In MYCN-amplified cells CDK4 expression is elevated, increasing the competition between CDK4 and CDK2 for binding p21. This results in insufficient p21 to inhibit CDK2, leading to high CDK4 and CDK2 kinase activity upon doxorubicin treatment. CDK4 inhibition by siRNAs, selective small compounds or p19^INK4D overexpression partly restored G?-S arrest, delayed S-phase progression and reduced cell viability upon doxorubicin treatment. Our results suggest a specific function of p19^INK4D, but not p16^INK4A, in sensitizing MYCN-amplified cells with a functional p53 pathway to doxorubicin-induced cell death. In summary, the CDK4/cyclin D-pRB axis is altered in MYCN-amplified cells to evade a G?-S arrest after doxorubicin-induced DNA damage. Additional chromosomal aberrations affecting the p53-p21 and CDK4-pRB axes compound the effects of MYCN on the G? checkpoint and reduce sensitivity to cell death after doxorubicin treatment. CDK4 inhibition partly restores G?-S arrest and sensitizes cells to doxorubicin-mediated cell death in MYCN-amplified cells with an intact p53 pathway.     

Middle Infrared Radiation Induces G2/M Cell Cycle Arrest in A549 Lung Cancer Cells

There were studies investigating the effects of broadband infrared radiation (IR) on cancer cell, while the influences of middle-infrared radiation (MIR) are still unknown. In this study, a MIR emitter with emission wavelength band in the 3–5 µm region was developed to irradiate A549 lung adenocarcinoma cells. It was found that MIR exposure inhibited cell proliferation and induced morphological changes by altering the cellular distribution of cytoskeletal components. Using quantitative PCR, we found that MIR promoted the expression levels of ATM (ataxia telangiectasia mutated), ATR (ataxia-telangiectasia and Rad3-related and Rad3-related), TP53 (tumor protein p53), p21 (CDKN1A, cyclin-dependent kinase inhibitor 1A) and GADD45 (growth arrest and DNA-damage inducible), but decreased the expression levels of cyclin B coding genes, CCNB1 and CCNB2, as well as CDK1 (Cyclin-dependent kinase 1). The reduction of protein expression levels of CDC25C, cyclin B1 and the phosphorylation of CDK1 at Thr-161 altogether suggest G₂/M arrest occurred in A549 cells by MIR. DNA repair foci formation of DNA double-strand breaks (DSB) marker γ-H2AX and sensor 53BP1 was induced by MIR treatment, it implies the MIR induced G₂/M cell cycle arrest resulted from DSB. This study illustrates a potential role for the use of MIR in lung cancer therapy by initiating DSB and blocking cell cycle progression.     

Induction of apoptotic death by curcumin in human tongue squamous cell carcinoma SCC-4 cells is mediated through endoplasmic reticulum stress and mitochondria-dependent pathways

Curcumin from the rhizome of the Curcuma longa plant has been noted for its chemo-preventative and chemo-therapy activities, and it inhibits the growth of many types of human cancer cell lines. In this study, the mechanisms of cell death involved in curcumin-induced growth inhibition, including cell cycle arrest and induction of apoptosis in human tongue cancer SCC-4 cells, were investigated. Herein, we observed that curcumin inhibited cell growth of SCC-4 cells and induced cell death in a dose-dependent manner. Treatment of SCC-4 cells with curcumin caused a moderate and promoted the G(2) /M phase arrest, which was accompanied with decreases in cyclin B/CDK1 and CDC25C protein levels. Moreover, curcumin significantly induced apoptosis of SCC-4 cells with a decrease of the Bcl-2 level, reduction of mitochondrial membrane potential (ΔΨ(m) ), and promoted the active forms of caspase-3. Curcumin also promoted the releases of AIF and Endo G from the mitochondria in SCC-4 cells by using confocal laser microscope. Therefore, we suggest that curcumin induced apoptosis through a mitochondria-dependent pathway in SCC-4 cells. In addition, we also found that curcumin-induced apoptosis of SCC-4 cells was partly through endoplasmic reticulum stress. In conclusion, curcumin increased G(2) /M phase arrest and induced apoptosis through ER stress and mitochondria-dependent pathways in SCC-4 cells.     

Characterization of adriamycin-induced G2 arrest and its abrogation by caffeine in FL-amnion cells with or without p53

We investigated the effect of Adriamycin on FL-amnion (FL) cells. After treatment with the drug, the cells arrested at G2, but we did not detect an increase in the p21 levels. We established a p53-deficient derivative of these cells, in which G2 arrest also occurred after treatment with Adriamycin, suggesting that the arrest we observed in these cells is independent of the p53 pathway. Low doses of Adriamycin (100-200 ng/ml) induced G2 arrest, while late S-phase arrest was observed at high doses (500-1000 ng/ml) in both FL and p53-deficient FL cells. Accumulation of cyclin B1 was detected only in cells arrested at G2, and not in those arrested at S phase, suggesting that the S-phase checkpoint functioned efficiently even in p53-deficient FL cells. In both cell lines, caffeine-induced activation of CDC2 kinase was detected only in cells arrested at G2 and CDC2 kinase-activated cells died exhibiting features of apoptosis. CDC2 kinase activation was inhibited by cycloheximide. Furthermore, cycloheximide inhibited activation of CDK2:cyclin A, which normally precedes CDC2 kinase activation in caffeine-treated cells. These results suggest that p53 and p21 do not have special roles in the S- and G2-phase checkpoints and that CDK2:cyclin A could be the target of the G2-phase DNA damage checkpoint.     

Fbxw8 is involved in the proliferation of human choriocarcinoma JEG-3 cells

Fbxw8 is the F-box component of a SCF-like E3 ubiquitin ligase complex. Mice lacking Fbxw8 exhibit pathological defects in placenta and embryo similar to fetal growth retardation, suggesting a role of Fbxw8 in placentation. Proliferative capacity of trophoblast cells is very important in placental development. In this context, we revealed that Fbxw8 was expressed in four different human trophoblast cell lines. Silencing of Fbxw8 expression by siRNA inhibited the growth of choriocarcinoma JEG-3 cells. By Western blotting, cell cycle analysis, we showed that down-regulation of Fbxw8 by RNAi induced cell-growth arrest at G2/M phase through decreasing the levels of CDK1, CDK2, cyclin A and cyclin B1 and up-regulation of p27 at protein level. Conversely, over-expression of Fbxw8 led to the opposite effect. These results suggest that Fbxw8 plays an essential role in the proliferation of human trophoblast cells, especially JEG-3 cells, via G2/M phase transition in association with regulation of CDK1, CDK2, cyclin A, cyclin B1 and p27 expression.     

4-Hydroxynonenal Induces G2/M Phase Cell Cycle Arrest by Activation of the Ataxia Telangiectasia Mutated and Rad3-related Protein (ATR)/Checkpoint Kinase 1 (Chk1) Signaling Pathway

4-Hydroxynonenal (HNE) has been widely implicated in the mechanisms of oxidant-induced toxicity, but the detrimental effects of HNE associated with DNA damage or cell cycle arrest have not been thoroughly studied. Here we demonstrate for the first time that HNE caused G₂/M cell cycle arrest of hepatocellular carcinoma HepG2 (p53 wild type) and Hep3B (p53 null) cells that was accompanied with decreased expression of CDK1 and cyclin B1 and activation of p21 in a p53-independent manner. HNE treatment suppressed the Cdc25C level, which led to inactivation of CDK1. HNE-induced phosphorylation of Cdc25C at Ser-216 resulted in its translocation from nucleus to cytoplasm, thereby facilitating its degradation via the ubiquitin-mediated proteasomal pathway. This phosphorylation of Cdc25C was regulated by activation of the ataxia telangiectasia and Rad3-related protein (ATR)/checkpoint kinase 1 (Chk1) pathway. The role of HNE in the DNA double strand break was strongly suggested by a remarkable increase in comet tail formation and H2A.X phosphorylation in HNE-treated cells in vitro. This was supported by increased in vivo phosphorylation of H2A.X in mGsta4 null mice that have impaired HNE metabolism and increased HNE levels in tissues. HNE-mediated ATR/Chk1 signaling was inhibited by ATR kinase inhibitor (caffeine). Additionally, most of the signaling effects of HNE on cell cycle arrest were attenuated in hGSTA4 transfected cells, thereby indicating the involvement of HNE in these events. A novel role of GSTA4-4 in the maintenance of genomic integrity is also suggested.     

Apigenin causes G(2)/M arrest associated with the modulation of p21(Cip1) and Cdc2 and activates p53-dependent apoptosis pathway in human breast cancer SK-BR-3 cells

We studied the effects of apigenin on the cell cycle distribution and apoptosis of human breast cancer cells and explored the mechanisms underlying these effects. We first investigated the antiproliferative effects in SK-BR-3 cells exposed to between 1 and 100 microM apigenin for 24, 48 and 72 h. Apigenin significantly inhibited cell proliferation at concentrations over 50 microM, regardless of exposure time (P<.05), and resulted in significant cell cycle arrest in the G(2)/M phase after 48 h of treatment at high concentrations (50 and 100 microM; P<.05). To investigate the regulatory proteins of cell cycle arrest affected by apigenin, we treated cells with 50 and 100 microM apigenin for 72 h. Apigenin caused a slight decrease in cyclin D and cyclin E expression, with no change in CDK2 and CDK4. In addition, the apigenin-induced accumulation of the cell population in the G(2)/M phase resulted in a decrease in CDK1 together with cyclin A and cyclin B. In an additional study, apigenin also increased the accumulation of p53 and further enhanced the level of p21(Cip1), with no change in p27(Kip1). The expression of Bax and cytochrome c of p53 downstream target was increased markedly at high concentration treatment over 50 microM apigenin. Based on our findings, the mechanism by which apigenin causes cell cycle arrest via the regulation of CDK1 and p21(Cip1) and induction of apoptosis seems to be involved in the p53-dependent pathway.     

Butylene fipronil induces apoptosis in PC12 murine nervous cells via activation of p16‐CDK4/6‐cyclin D1 and mitochondrial apoptotic pathway

Butylene fipronil (BFPN) is a phenylpyrazole insecticide, acting at the γ‐aminobutyric acid (GABA) receptor. Here, we show that BFPN inducedcytotoxicity in PC12 murinenervous cells, which lacks GABA receptor. Treatment with BFPN for 48 hours significantly enhanced G0/G1 arrest and induced apoptosis. BFPN decreased the expression of cyclin‐dependent kinase (CDK4 and CDK6) and increased P16 and cyclin D1. Simultaneously, Bcl‐2 protein was declined while Bax and cytochrome c were significantly enhanced in BFPN‐treated groups. The apoptotic enzymes caspase‐8, ‐9, and ‐3 were also activated by BFPN. Furthermore, treatment with BFPN significantly stimulated reactive oxygen species (ROS) generation, and pretreatment with antioxidant diphenyleneiodonium, substantially reduced cell death. Overall, these results suggest that BFPN is effective to induce G0/G1‐phase arrest and apoptosis in PC12 murine nervous cell. Stimulating ROS generation and activation of P16‐CDK4/6‐cyclin D1 and mitochondrial apoptotic pathway may participate in the cytotoxicity of BFPN.     

Study of the cytolethal distending toxin-induced cell cycle arrest in HeLa cells: involvement of the CDC25 phosphatase

HeLa cells exposed to Escherichia coli cytolethal distending toxins (CDT) arrest their cell cycle at the G2/M transition. We have shown previously that in these cells the CDK1/cyclin B complex is inactive and can be reactivated in vitro using recombinant CDC25 phosphatase. Here we have investigated in vivo the effects of CDC25 on this cell cycle checkpoint. We report that overexpression of CDC25B or CDC25C overrides an established CDT-induced G2 cell cycle arrest and leads the cells to accumulate in an abnormal mitotic stage with condensed chromatin and high CDK1 activity. This effect can be counteracted by coexpression of the WEE1 kinase. In contrast, overexpression of CDC25B or C prior to CDT treatment prevents G2 arrest and allows most of the cells to progress through mitosis with only a low percentage of cells arrested in abnormal mitosis. The implications of these results on the biochemical nature of the CDT-induced cell cycle arrest are discussed.     

Induction of cell cycle arrest and apoptosis in HT-29 human colon cancer cells by the dietary compound luteolin

Luteolin is 3',4',5,7-tetrahydroxyflavone found in celery, green pepper, and perilla leaf that inhibits tumorigenesis in animal models. We examined luteolin-mediated regulation of cell cycle progression and apoptosis in the HT-29 human colon cancer cell line. Luteolin decreased DNA synthesis and viable HT-29 cell numbers in a concentration-dependent manner. It inhibited cyclin-dependent kinase (CDK)4 and CDK2 activity, resulting in G1 arrest with a concomitant decrease of phosphorylation of retinoblastoma protein. Activities of CDK4 and CDK2 decreased within 2 h after luteolin treatment, with a 38% decrease in CDK2 activity (P < 0.05) observed in cells treated with 40 micromol/l luteolin. Luteolin inhibited CDK2 activity in a cell-free system, suggesting that it directly inhibits CDK2. Cyclin D1 levels decreased after luteolin treatment, although no changes in expression of cyclin A, cyclin E, CDK4, or CDK2 were detected. Luteolin also promoted G2/M arrest at 24 h posttreatment by downregulating cyclin B1 expression and inhibiting cell division cycle (CDC)2 activity. Luteolin promoted apoptosis with increased activation of caspases 3, 7, and 9 and enhanced poly(ADP-ribose) polymerase cleavage and decreased expression of p21(CIP1/WAF1), survivin, Mcl-1, Bcl-x(L), and Mdm-2. Decreased expression of these key antiapoptotic proteins could contribute to the increase in p53-independent apoptosis that was observed in HT-29 cells. We demonstrate that luteolin promotes both cell cycle arrest and apoptosis in the HT-29 colon cancer cell line, providing insight about the mechanisms underlying its antitumorigenic activities.