F9 embryonal carcinoma cells fail to stop at G1/S boundary of the cell cycle after gamma-irradiation due to p21WAF1/CIP1 degradation

We studied the ability of F9 teratocarcinoma cells to arrest in G1/S and G2/M checkpoints after gamma-irradiation. Wild-type p53 protein was rapidly accumulated in F9 cells after gamma-irradiation, however, this was followed not by a G1/S arrest but by a short and 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 expression of p53 downstream target Cdk inhibitor p21WAF1/CIP1. In spite of p53-dependent activation of p21WAF1/CIP1 gene promoter and p21WAF1/CIP1 mRNA accumulation upon irradiation, the p21WAF1/CIP1 protein was not detected by either immunoblot or immunofluorescence techniques. However, the cells treated with a specific proteasome inhibitor lactacystin revealed the p21WAF1/CIP1 protein both in non-irradiated and irradiated cells. Therefore we suggest that p21WAF1/CIP1 protein is degraded by a proteasome-dependent mechanism in F9 cells and the lack of G1/S arrest after gamma-irradiation is due to this degradation. We also examined the expression and activity of cell cycle regulatory proteins: G1- and G2-cyclins and cyclin-dependent kinases. In the absence of functional p21WAF1/CIP1 inhibitor, the activity of G1 cyclin/Cdk complexes was insufficiently inhibited to cause a G1 arrest, whereas a decrease of cdc2 and cyclin B1-associated kinase activities was enough to contribute to a reversible G2 arrest following gamma-irradiation. After gamma-irradiation, the majority of F9 cells undergo apoptosis implying that wt-p53 likely triggers pro-apoptotic gene expression in DNA damaged cells. Elimination of defected cells might ensure maintenance of genome integrity in the remaining cell population.     

Effects of genistein and structurally related phytoestrogens on cell cycle kinetics and apoptosis in MDA-MB-468 human breast cancer cells

We have studied the effects of phytoestrogens (genistein, quercetin, daidzein, biochanin A and kaempferol) on proliferation, cell cycle kinetics, and apoptosis of MDA-MB-468 breast cancer cells. Genistein and quercetin inhibited cell growth with IC50 values of 8.8 and 18.1 muM, respectively, while the other phytoestrogens were less effective. Flow cytometric analysis showed G2/M cell cycle arrest with 25 muM and higher concentrations of genistein. At 100 muM, genistein, quercetin and kaempferol caused accumulation of 70, 60 and 35% of cells, respectively, in G2/M phase by 24 h. In contrast, biochanin A and daidzein were ineffective. APO-BRDU analysis revealed apoptosis with 10 muM genistein (19.5%), reaching 86% at 100 muM. Apoptosis by genistein was confirmed by Hoechst 33342 staining and fluorescence microscopy. With 100 muM quercetin, 47% of the cells were apoptotic, while the other bioflavonoids had little effect. Genistein treatment resulted in a biphasic response on cyclin B1: 70% increase in cyclin B1 level at 25 muM, and 50 and 70% decrease at 50 and 100 muM, respectively. In contrast, the action of quercetin involved an increase in cyclin B1 level. Genistein had no effect on cdc2 level up to 50 muM concentration; however, there was a decrease in the phosphorylated form of the protein at 100 muM. Quercetin had no effect on cdc2 levels. Our results suggest that the action of genistein and quercetin involves G2/M arrest and apoptosis in MDA-MB-468 cells. Biochanin A and daidzein, although structurally related to genistein, did not share this mechanism. Thus, structurally related phytoestrogens have discrete target sites and mechanisms in their growth inhibitory action on breast cancer cells.     

Constitutive activation of cyclin B1-associated cdc2 kinase overrides p53-mediated G2-M arrest

Recent studies have suggested that p53 regulates the G2 checkpoint in the cell cycle and that this function is required for the maintenance of genomic integrity. In this study, we investigated a regulatory role of p53 specifically in G2-M transition. Human bladder carcinoma cells lacking functional p53 were synchronized at G1-S, which is preceded by p53-mediated G1 arrest. p53 expression in the synchronized cells was induced by infection with a recombinant adenovirus that encodes p53. After release from the G1-S arrest, the cells progressed to S-phase and G2 but failed to enter mitosis. Biochemical analysis showed that p53 inhibits cell cycle-dependent expression of cdc2 and cyclin B1 and consequently inhibits cdc2 kinase. The role of cyclin B1-associated cdc2 kinase in p53-mediated G2-M arrest was further investigated by expression of both cyclin B1 and cdc2AF, in which inhibitory phosphorylation sites were substituted. The cells expressing both cdc2AF and cyclin B1 showed a constitutive activation of cdc2 kinase during cell cycle progression and passed through G2-M regardless of p53 expression. Therefore, inactivation of cdc2 kinase through cdc2 and cyclin B1 repression is an essential step in p53-mediated G2-M arrest.     

Inhibition of p34cdc2 kinase activation, p34cdc2 tyrosine dephosphorylation, and mitotic progression in Chinese hamster ovary cells exposed to etoposide.

p34cdc2 kinase, an enzyme essential for mitosis in mammalian cells, may play a role in etoposide-induced G2 phase arrest of Chinese hamster ovary cells. In this study, etoposide is shown to cause inhibition of a specific p34cdc2 kinase activation pathway, that of tyrosine dephosphorylation. Exposure of asynchronously dividing cells to etoposide caused a simultaneous rapid decline of both mitotic index and p34cdc2 kinase activity, suggesting that the kinase was not activated and that the arrest point was in late G2 phase. Using synchronized cells, p34cdc2 kinase exhibited maximal activity at the G2/M transition. Activation of the kinase and the onset of mitosis were accompanied by increased electrophoretic mobility and tyrosine dephosphorylation of the p34cdc2 protein. A 1-h exposure to etoposide during early G2 phase inhibited p34cdc2 kinase activation, its shift in electrophoretic mobility, and its tyrosine dephosphorylation, all of which correlated with a delay in mitotic progression. The interaction between the p34cdc2 and cyclin B proteins appeared unaffected under etoposide exposure conditions which resulted in greater than 70% inhibition of p34cdc2 kinase activity and almost complete cessation of transition into mitosis. These data suggest that mammalian cells express a DNA damage-responsive mechanism which controls mitotic progression at the level of p34cdc2 tyrosine dephosphorylation.     

Induction of p21 by p53 following DNA damage inhibits both Cdk4 and Cdk2 activities

DNA damage often activates the p53-p21 pathway and causes G(1)-phase arrest in mammalian cells. Although there is ample evidence that p21 induction by p53 leads to Cdk2 inhibition, it is unclear whether this checkpoint event also leads to Cdk4 inhibition. Diaminocyclohexane(trans-diacetato)(dichloro) platinum(IV) (DAP), a platinum-based coordination complex, is a DNA-damaging agent that is effective against a variety of tumor cells resistant to the parental drug cisplatin. Our previous studies established that treatment of human cancer cells with low effective concentrations of DAP specifically activates the G(1)-phase checkpoint and simultaneously inhibit Cdk4 and Cdk2 activities. Here we demonstrate that DAP treatment of human cancer cells activates the p53-p21 pathway without activating other known mechanisms that inhibit Cdk4 and Cdk2 activities. The induced p21 binds to both the Cdk4/cyclin D and Cdk2/cyclin E complexes and inhibits both of their kinase activities. Conversely, inhibition of p21 induction by cycloheximide or by p21 gene deletion prevents DAP-induced inhibition of Cdk4 and Cdk2 activities. Attenuated p53 expression and p21 induction also eliminates DAP-induced G(1)-phase arrest and inhibition of Cdk4 and Cdk2 activities. Together, these findings establish that activation of the p53-p21 pathway is responsible for the DAP-induced G(1)-phase checkpoint response and provide the first solid evidence that p21 induction by p53 during a DNA damage-induced G(1)-phase checkpoint response inhibits both Cdk4 and Cdk2 activities.     

p27-Associated G1 arrest induced by hinokitiol in human malignant melanoma cells is mediated via down-regulation of pRb,Skp2 ubiquitin ligase,and impairment of Cdk2 function

Increasing evidence has confirmed that hinokitiol (β-thujaplicin), a tropolone-related compound, exhibits anticancer activity in a variety of cancers through inhibition of cell proliferation. The present study indicates that hinokitiol selectively inhibits cell growth and DNA synthesis in FEM human melanoma cells. Hinokitiol-induced growth inhibition was associated with strong G1 cell cycle arrest. Consistent with blocking the G1–S-phase transition, hinokitiol markedly increased p27 protein levels, but caused only a moderate increase in p21, in addition to a decrease in Cdk2, cyclin E, and phosphorylated Rb. In addition, hinokitiol increased the stability of the p27 protein by inhibiting p27 phosphorylation at Thr¹⁸⁷ and by down-regulating Skp2 expression. siRNA knockdown of p27 abrogated hinokitiol-mediated growth inhibition, while knockdown of Skp2 exacerbated the G1 arrest. In addition to increasing Cdk inhibitor levels and decreasing cyclin A expression, hinokitiol also impaired Cdk2 function by inhibiting Cdk2 kinase activity, impeding cyclin E or A/Cdk2 binding, and inducing translocation of the Cdk2 protein complex. Taken together, our data demonstrate that the novel anticancer mechanism of hinokitiol involves accumulation of p27, down-regulation of Skp2, and impairment of Cdk2 function in FEM human melanoma cells. The therapeutic potential of hinokitiol may lead to novel cell-cycle-based anticancer strategies for malignant melanoma.     

Arsenic trioxide-mediated growth inhibition in MC/CAR myeloma cells via cell cycle arrest in association with induction of cyclin-dependent kinase inhibitor, p21, and apoptosis

We investigated the in vitro effect of As2O3 on proliferation, cell cycle regulation, and apoptosis in human myeloma cell lines. As2O3 significantly inhibited the proliferation of all of eight myeloma cell lines examined in a dose-dependent manner with IC50 of approximately 1-2 microM. DNA flow cytometric analysis indicated that As2O3 (2 microM) induced a G1 and/or a G2-M phase arrest in these cell lines. To address the mechanism of the antiproliferative effect of As2O3, we examined the effect of As2O3 on cell cycle-related proteins in MC/CAR cells in which both G1 and G2-M phases were arrested. Western blot analysis demonstrated that treatment with As2O3 (2 microM) for 72 h did not change the steady-state levels of CDK2, CDK4, cyclin D1, cyclin E, and cyclin B1 but decreased the levels of CDK6, cdc2, and cyclin A. The mRNA and protein levels of CDKI, p21 were increased by treatment with As2O3, but those of p27 were not. In addition, As2O3 markedly enhanced the binding of p21 with CDK6, cdc2, cyclin E, and cyclin A compared with untreated control cells. Furthermore, the activity of CDK6-associated kinase was reduced in association with hypophosphorylation of Rb protein. The activity of cdc2-associated kinase was decreased, which was accompanied by the up-regulation of cdc2 phosphorylation (cdc2-Tyr15 phosphorylation) resulting from reduction of cdc25B and cdc25C phosphatases. As2O3 also induced apoptosis in MC/CAR cells as evidenced by flow cytometric detection of sub-G1 DNA content and annexin V binding assay. This apoptotic process was associated with down-regulation of Bcl-2, loss of mitochondrial transmembrane potential (delta psi(m)), and an increase of caspase-3 activity. These results suggest that As2O3 inhibits the proliferation of myeloma cells, especially MC/CAR cells, via cell cycle arrest in association with induction of p21 and apoptosis.     

Cdc25-dependent activation of cyclin A/cdk2 is blocked in G2 phase arrested cells independently of ATM/ATR

Cyclin A/cdk2 is active during S and G2 phases of the cell cycle, but its regulation and function during G2 phase is poorly understood. In this study we have examined the regulation of cyclin A/cdk2 activity during normal G2 phase progression and in genotoxin-induced G2 arrest. We show that cyclin A/cdk2 is activated in early G2 phase by a cdc25 activity. In the G2 phase checkpoint arrest initiated in response to various forms of DNA damage, the cdc25-dependent activation of both cyclin A/cdk2 and cyclin B1/cdc2 is blocked. Ectopic expression of cdc25B, but not cdc25C, in G2 phase arrested cells efficiently activated both cyclin A/cdk2 and cyclin B1/cdc2. Finally, we demonstrate that the block in cyclin A/cdk2 activation in the G2 checkpoint arrest is independent of ATM/ATR. We speculate that the ATM/ATR-independent block in G2 phase cyclin A/cdk2 activation may act as a further layer of checkpoint control, and that blocking G2 phase cyclin A/cdk2 activation contributes to the G2 phase checkpoint arrest.     

The involvement of active DNA synthesis in camptothecin-induced G2 arrest: altered regulation of p34cdc2/cyclin B.

Cell cycle arrest in G2 phase is a common response to a variety of DNA-damaging agents. The coupling between DNA damage and G2 arrest was studied in synchronized HeLa cells using camptothecin, a highly specific inhibitor of topoisomerase I that damages DNA through the formation of reversible topoisomerase I-DNA cleavable complexes. Brief camptothecin treatment of early S-phase HeLa cells caused arrest at G2 phase and abolished the activation of p34cdc2 protein kinase. Both tyrosine dephosphorylation of p34cdc2 and cyclin B accumulation were altered. These cell cycle-dependent changes were not observed when DNA replication was inhibited by aphidicolin during the brief camptothecin treatment. Our results suggest that to produce G2 arrest, active DNA synthesis is required at the time of camptothecin treatment, as was previously shown for camptothecin-induced cytotoxicity. Furthermore, our results suggest that the interaction of the replication fork with DNA damage may ultimately trigger altered regulation of p34cdc2/cyclin B, leading to cell cycle arrest at the G2 phase.     

EGFR and Src are involved in indole-3-carbinol-induced death and cell cycle arrest of human breast cancer cells

Indole-3-carbinol (I3C), a dietary chemopreventive compound, induced marked reduction in epidermal growth factor receptor (EGFR) prior to cell death in cells representing three breast cancer subtypes. Signalling pathways, linking these events were investigated in detail. I3C modulated tyrosine phosphorylation from 30 min in four cell lines. In MDA-MB-468 and HBL100 cells, it induced Src activation after 5 h. In MDA-MB-468 cells, I3C induced signalling between 4.5 and 7 h, which involved sequential activation of Src, EGFR, STAT-1 and STAT-3, followed by EGFR degradation. It also induced physical association between activated Src and EGFR. In MCF7 and MDA-MB-231 cells, I3C modulated expression of cell cycle-related proteins, p21Cip1, p27Kip1, cyclin E, cyclin D1 and CDK6, with upregulation of p21Cip1 and cyclin E being dependent on Src. Inhibition of EGFR by specific inhibitors PD153035 or ZD1839 increased susceptibility to I3C-induced apoptosis of MCF7, MDA-MB-468 and MDA-MB-231 cells. Inhibition of Src sensitized MDA-MB-468 and MDA-MB-231 cells to I3C, whereas overexpression of c-Src increased resistance to I3C in MDA-MB-468 and HBL100 cells. Modulation of Src in MDA-MB-468 cells influenced the basal level of EGFR expression and cell viability; the latter being positively correlated with EGFR activation levels. Therefore, EGFR and Src activities are essential for I3C-induced cell cycle arrest and death; however, I3C-induced pathways depend on specific features of breast cancer cells. The cancer types, which rely on 'EGFR addiction' or Src deregulation, are likely to be susceptible to I3C.     

Altered cell cycle response of drug-resistant lung carcinoma cells to doxorubicin

The effect of doxorubicin treatment on cell cycle parameters in asynchronous populations of multidrug-resistant human lung carcinoma cell lines was investigated. A sensitive (DLKP-SQ) and three resistant (DLKP-SQ A250 10p#7, DLKP-A2B and DLKP-A5F) variants of a human lung carcinoma cell line DLKP were exposed to equitoxic concentrations of doxorubicin. The latter three were 8-fold, 30-fold and 300-fold resistant to doxorubicin, respectively. Irreversible G2/M arrest in sensitive (DLKP-SQ) cells was observed 24 h after initiation of doxorubicin treatment. In resistant variants, G2/M arrest occurred at 12-16 h with a subsequent bypass of the G2/M arrest to re-emerge and accumulate in G1. This transient G2/M arrest and subsequent progression into G1 indicated an inefficient checkpoint for monitoring DNA damage induced by doxorubicin treatment. Caffeine treatment could bypass the G2/M block in DLKP-SQ cells. Doxorubicin treatment did not alter cyclin B or cdc2 protein levels, the ability of cdc2 to form complexes with cyclin B or the levels of cyclin B bound to cdc2. The G2/M arrest seen in sensitive cells was associated with an increase in inhibitory phosphorylation of Tyr15 on cdc2. In contrast, tyrosine 15 phosphorylation did not change in resistant variants after drug treatment and a general increase in cdc2 kinase activity was seen. Cdc25C levels were not altered following drug treatment.     

Transduced p16INK4a peptides inhibit hypophosphorylation of the retinoblastoma protein and cell cycle progression prior to activation of Cdk2 complexes in late G1.

Progression of cells through the G1 phase of the cell cycle requires cyclin D:Cdk4/6 and cyclin E:Cdk2 complexes; however, the duration and ordering of these complexes remain unclear. To address this, we synthesized a peptidyl mimetic of the Cdk4/6 inhibitor, p16INK4a that contained an NH2-terminal TAT protein transduction domain. Transduction of TAT-p16 wild-type peptides into cells resulted in the loss of active, hypophosphorylated pRb and elicited an early G1 cell cycle arrest, provided cyclin E:Cdk2 complexes were inactive. We conclude that cyclin D:Cdk4/6 activity is required for early G1 phase cell cycle progression up to, but not beyond, activation of cyclin E:Cdk2 complexes at the restriction point and is thus nonredundant with cyclin E:Cdk2 in late G1.     

The carcinogen 2-acetylaminofluorene inhibits activation and nuclear accumulation of cyclin-dependent kinase 2 in growth-induced rat liver

Growth arrest in G(1) is a common cellular response to DNA damage. In the present study, liver regeneration was combined with continuous exposure for 2-acetylaminofluorene (AAF) to study mechanisms of carcinogen-induced growth arrest in vivo. Growth arrest of uninitiated hepatocytes is central for AAF-induced promotion of premalignant lesions in rat liver. To characterize this growth arrest, we examined the activity of cyclin-dependent kinase (Cdk) 2 in unexposed liver and in AAF-exposed liver after growth induction by partial hepatectomy (PH). Rats were fed either a control diet or an AAF-supplemented diet. After 7 d, a two-third PH was performed and the animals were killed after 0, 12, 18, 24, and 36 h. Kinase assays showed that cyclin E- and Cdk2-associated activities were lower in AAF-exposed liver than in unexposed liver after PH. Although the total cellular levels of cyclin E and Cdk2 were similar, cyclin E-Cdk2 assembly was markedly reduced. In unexposed hepatocytes, Cdk2 translocated to the nuclei after PH. Much of the nuclear Cdk2 was in a rapidly migrating form, presumably representing the Thr160-phosphorylated form of Cdk2. In contrast, in AAF-exposed liver both nuclear Cdk2 accumulation and Thr160-phosphorylation of Cdk2 were reduced. Although p53 and p21(waf1/cip1) were induced by AAF, the binding of p21 to cyclin E and Cdk2 was not increased in growth arrested liver. In conclusion, hepatocyte growth arrest caused by AAF exposure was characterized by a lowered Cdk2 activity that was accompanied by a reduced assembly of cyclin E-Cdk2 complexes but not by binding of p21.     

p16INK4a, but not constitutively active pRb, can impose a sustained G1 arrest: molecular mechanisms and implications for oncogenesis.

p16ink4 and pRb, two components of a key G1/S regulatory pathway, and tumor suppressors commonly targeted in oncogenesis, are among the candidates for gene therapy of cancer. Wild-type p16 and a constitutively active pRb(delta cdk) mutant both blocked G1 in short-term experiments, but only p16 imposed a sustained G1 arrest. Unexpectedly, cells conditionally exposed to pRb(delta cdk) entered S phase after 2 days, followed by endoreduplication between days 4-6. The distinct phenotypes evoked by p16 vs pRb(delta cdk) appear mediated by cyclin E/CDK2 which, while active in the pRb(delta cdk)-expressing cells, became rapidly inhibited through restructuring diverse cyclin/CDK/p21 complexes by p16. These results provide novel insights into the roles of p16, pRb and cyclin E in G1/S control and multistep oncogenesis, with implications for gene therapy strategies.     

Potent inhibitors of cyclin-dependent kinase 2 induce nuclear accumulation of wild-type p53 and nucleolar fragmentation in human untransformed and tumor-derived cells

The cdk2 gene has been identified as a human cdc2/CDC28-related gene that encodes a protein kinase essential for the G1/S transition in mammalian cells, but not for the G2/M transition, which requires Cdk1, another p34cdc2/CDC28 homolog. Novel potential functions of Cdk2 have been uncovered by using two potent and specific inhibitors of its kinase activity, roscovitine and olomoucine, on human wt p53-expresser untransformed and tumor-derived cells. At concentrations equal or superior to respectively 30- and 20-fold their in vitro IC50 values for cyclin B/Cdk1, cyclin A/Cdk2 and cyclin E/Cdk2, the Cdk inhibitors precipitately induce a dramatic nuclear accumulation of wt p53 and a delocalization of nucleolin from the nucleolus in all interphase cells, whatever their cell cycle status, acting in this way like the DNA-damaging drug, mitomycin C (7 microg/ml). These early events are soon followed by a nucleolar fragmentation in both normal and tumor cells in the presence of the Cdk inhibitors but not in the presence of the DNA-damaging drug. Yet, treatment with either type of compounds eventually triggers rapidly the death of the tumor cells and, much more slowly, that of the normal cells. The Cdk inhibitors, however, stimulate cell death from any stage of the cell cycle, whereas the DNA-damaging drug kills more efficaciously S phase cells. These observations provide a hint that the Cdk2 kinase might be involved in controlling the nuclear levels of the tumor suppressor wt p53 protein and in maintaining the nucleolar integrity and function, linking in this way the cell division cycle machinery to survival functions and overall cell metabolism via the control of nucleocytoplasmic transport and of ribosome production.     

Cell-cycle arrest at G2/M and growth inhibition by apigenin in human colon carcinoma cell lines

Apigenin, a common dietary flavonoid, has been shown to induce cell cycle arrest in both epidermal and fibroblast cells and inhibit skin tumorigenesis in murine models. The present study assessed the influence of apigenin on cell growth and the cell cycle in the human colon carcinoma cell lines SW480, HT-29, and Caco-2. Treatment of each cell line with apigenin (0-80 microM) resulted in a dose-dependent reduction in both cell number and cellular protein content, compared with untreated control cultures. DNA flow cytometric analysis indicated that treatment with apigenin resulted in G2/M arrest in all three cell lines in a time- and dose-dependent manner. Apigenin treatment (80 microM) for 48 h produced maximum G2/M arrest of 64%, 42%, and 26% in SW480 cells, HT-29 cells, and Caco-2 cells, respectively, in comparison with control cells (15%). The proportion of S-phase cells was not altered by apigenin treatment in each of the three cell lines. The G2/M arrest was reversible after 48 h of apigenin treatment in the most sensitive cell line SW480. The degree of G2/M arrest by apigenin was inversely correlated with the corresponding inhibition of cell growth measurements in all three cell lines (r = -0.626 to -0.917, P相似文献   

Removal of Cdk inhibitors through both sequestration and downregulation in zearalenone-treated MCF-7 breast cancer cells

Treatment of MCF 7 cells with the fungal estrogen zearalenone induced cyclin E-associated kinase activity transiently within 9-12 h; total cyclin-dependent kinase (Cdk) 2 activity was elevated for 24 h and beyond. This increased cyclin E/Cdk2 activity was associated with sequestration of the Cdk inhibitor p27 Cdk inhibitor 1B (p27(KIP1)) by newly formed cyclin D1/Cdk4 complexes and with downregulation of p27(KIP1) expression. The activation of cyclin A/Cdk2 activity corresponded with virtual elimination of p27(KIP1). The activity of cyclin E/Cdk2 complexes from zearalenone-treated lysates was inhibited in vitro by recombinant p27(KIP1), and this inhibition was relieved by the addition of recombinant cyclin D1/Cdk4 complexes. Thus, sequestration of p27(KIP1) by cyclin D1/Cdk4 resulted in activation of Cdk2 in vitro. Cdk inhibitory activity in lysates of zearalenone-treated cells was depleted by anti-p27(KIP1) and anti-Cdc2 interacting protein (p21(CIP1)) antibodies. Overexpression of the Cdk4/6-specific Cdk inhibitor of Cdk4 p16(INK4A) was associated with increased association of p27(KIP1) with Cdk2, concomitant with disruption of D cyclin/Cdk4 complexes. The proteasome inhibitor 2-leu-leu-leu-H aldehyde (MG-132) was relatively ineffective in inhibiting the initial, sequestration-dependent activation of cyclin E/Cdk2 yet was as effective as p16(INK4A) in inhibiting activation of cyclin A/Cdk2 later in G(1). Downregulation of p27(KIP1) proceeded in p16(INK4A)-expressing cells after zearalenone treatment, and G(1) arrest afforded by p16(INK4A) expression was reversible upon prolonged treatment with zearalenone. Zearalenone treatment of MCF-7 cells elicited expression of F-box protein S phase kinase-associated protein 2 (p45(SKP2)), a substrate-specific component of the ubiquitin-ligase complex that targets p27(KIP1) for degradation in the proteasome. These studies suggest that both sequestration of Cdk inhibitors by cyclin D1/Cdk4 complexes and downregulation of p27(KIP1) play major roles in the induction of Cdk2 activity and S phase entry elicited by estrogens in MCF-7 cells.     

Correlation between gamma-ray-induced G2 arrest and radioresistance in two human cancer cells.

PURPOSE: The correlation between radioresistance and gamma-ray-induced G2 arrest was examined in two human cancer cell lines, HeLa (cervical carcinoma) and MeWo (melanoma). METHODS AND MATERIALS: Cellular radioresistance was examined by a colony formation assay and Hoechst 33342 staining. G2 arrest induced by gamma-rays was examined by flow cytometry, and the accumulation of cyclin B1 and cdc2 proteins was analyzed using Western blotting. RESULTS: HeLa was more resistant (10% survival dose[D10] = 10 Gy) than MeWo (D10 = 4 Gy) to gamma-rays. In HeLa, cell cycle analysis showed that G2 arrest was induced 10 or 24 h after irradiation of 10 or 4 Gy, respectively. In contrast, no clear G2 arrest in MeWo was observed after irradiation. Western blot analysis showed that cell cycle regulators, cyclin B1 and cdc2, were accumulated in HeLa but not in MeWo. The accumulation of cyclin B1 and cdc2 reached peak levels 24-34 h after irradiation of 10 Gy, and 24 h after irradiation of 4 Gy. In addition, Hoechst staining revealed similar increase in apoptotic bodies with time after irradiation in HeLa and MeWo at isosurvival doses. CONCLUSION: Radioresistance of these human cancer cells is closely correlated with gamma-ray-induced G2 arrest, and cyclin B1 and cdc2 are possible regulators of G2 arrest.