Delayed biological consequences induced by gamma-radiation in human tumorigenic diploid line SK-UT-1B

The progeny of SK-UT-1B cells that survived gamma-irradiation with 4 Gy up to the 80th passage was examined. Descendants of irradiated cells lost p53 transactivation properties. Simultaneously, in the presence of nocodazole coordination between M and S phases was disrupted. Meanwhile, descendants of irradiated cells maintained the accurate spindle assembly checkpoint. These data suggest that p53 transactivation function may be required for coordination of M and S phases, rather than for spindle assembly checkpoint. Since it is known that p53 regulates both these processes on the basis of data obtained, we suggest that functions of p53 required for coordination of M and S-phases and for spindle assembly checkpoint are separated. Besides, the data obtained indicate that radiation-induced chromosomal rearrangements are associated with activation of DNA recombination process.     

MCT-1 oncogene downregulates p53 and destabilizes genome structure in the response to DNA double-strand damage

Tumor suppressor p53 protein mediates checkpoint controls and the apoptotic program that are critical for maintaining genomic integrity and preventing tumorigenesis. Forced-induction of MCT-1 decreased p53 expression before and after genomic insults. While inhibiting protein synthesis, the levels of ubiquinated-p53 and the phospho-MDMA2 were significantly increased in ectopic MCT-1 cells. Abrogation of the proteosome degradation process attenuated p53 destabilization and p21 down-regulation by MCT-1. Concomitantly, MCT-1 overexpression enhanced the phosphorylation status of MAPK (ERK1/ERK2). While MCT-1 gene knockdown or MEK/ERK pathway inhibition dramatically reduced MAPK phosphorylation, the genotoxin-induced p53 and p21 production were noticeably elevated. Upon Etoposide treatment, ectopic MCT-1 cells relaxed S-phase and G2/M checkpoints followed by G1 phase progressing. Moreover, cells inducing with MCT-1 abridged accumulations of G2/M populations in the response to gamma-irradiation. The polyploidy (DNA content>4N) populations were increased in association with p53 loss in MCT-1 oncogenic cells. Alkaline comet assay validated that ectopic MCT-1 cells were less susceptibility to the genotoxicity. Furthermore, the allocation of nuclear MCT-1 induced by the genotoxic stress was moderately coincided with gamma-H2AX appearances. Throughout damage-repairing process, ectopic MCT-1 cells displayed many larger chromosomes and multiple chromosomal fusions compared to the controls that showed increase in chromosomal breaks/gaps and minute chromosomal fragments. Spectral karyotyping analysis precisely identified the acquisition of a single extra copy of chromosome 14 together with a complex genome organizations in ectopic MCT-1 cells, including extra copies of chromosome segments that had been translocated to derivative chromosomes 6 [der(6)] and 9 [der(9)]. In conclusion, MCT-1 deregulates p53-p21 network and impairs the damage checkpoints those are robustly connected to oncogenic chromosomal abnormalities.     

Requirement of Krüppel-like factor 4 in preventing entry into mitosis following DNA damage

Previous studies indicate that Krüppel-like factor 4 (KLF4 or GKLF) controls the G1/S cell cycle checkpoint upon DNA damage. We present evidence for an equally important role of KLF4 in maintaining the integrity of the G2/M checkpoint following DNA damage. HCT116, a colon cancer cell line with wild type p53 alleles, underwent sustained G2 arrest up to 4 days after gamma-irradiation. In contrast, HCT116 cells null for p53 were able to enter mitosis following irradiation. Western blot analyses of irradiated HCT116 cells showed increased levels of p53, KLF4, and p21WAF1/CIP1 and decreased levels of cyclin B1 when compared with unirradiated controls. In contrast, the levels of cyclin B1 increased in irradiated HCT116 p53-/- cells, in which KLF4 failed to increase due to the absence of p53. When KLF4 was inhibited by small interfering RNA, irradiated HCT116 cells exhibited increased mitotic indices and a rise in cyclin B1 levels. Conversely, irradiated HCT116 p53-/- cells that were infected with KLF4-expressing adenoviruses demonstrated a concurrent reduction in mitotic indices and cyclin B1 levels. In each case, Cdc2 kinase measurements showed an inverse correlation between Cdc2 kinase activities and KLF4 levels. Co-transfection experiments showed that KLF4 repressed the cyclin B1 promoter through a specific GC-rich element. Moreover, chromatin immunoprecipitation experiments demonstrated that both KLF4 and HDAC were associated with the cyclin B1 promoter in irradiated HCT116 cells. We conclude that KLF4 is essential in preventing mitotic entry following gamma-irradiation and does so by inhibiting cyclin B1 expression.     

p53 Regulates Cellular Responses to Environmental Carcinogen Benzo[a]pyrene-7,8-diol-9,10-epoxide in Human Lung Cancer Cells

The p53 tumor suppressor is a mutational target of environmental carcinogen anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE). We now demonstrate that p53 plays an important role in regulation of cellular responses to BPDE. Exposure of p53-null H1299 human lung cancer cells to BPDE resulted in S and G2 phase cell cycle arrest, but not mitotic block, which correlated with induction of cyclin B1 protein expression, down-modulation of cell division cycle 25C (Cdc25C) and Cdc25B protein levels, and hyperphosphorylation of Cdc25C (S216), cyclin-dependent kinase 1 (Cdk1; Y15), checkpoint kinase 1 (Chk1; S317 and S345) and Chk2 (T68). The BPDE-induced S phase block, but not the G2/M phase arrest, was significantly attenuated by knockdown of Chk1 protein level. The BPDE-mediated accumulation of sub-diploid fraction (apoptotic cells) was significantly decreased in H1299 cells transiently transfected with both Chk1 and Chk2 specific siRNAs. The H460 human lung cancer cell line (wild-type p53) was relatively more sensitive to BPDE-mediated growth inhibition and enrichment of sub-diploid apoptotic fraction compared with H1299 cells. The BPDE exposure failed to activate either S or G2 phase checkpoint in H460 cells. Instead, the BPDE-treated H460 cells exhibited a nearly 8-fold increase in sub-diploid apoptotic cells that was accompanied by phosphorylation of p53 at multiple sites. Knockdown of p53 protein level in H460 cells attenuated BPDE-induced apoptosis but enforced activation of S and G2 phase checkpoints. In conclusion, the present study points towards an important role of p53 in regulation of cellular responses to BPDE in human lung cancer cells.     

p53-dependent Chk1 phosphorylation is required for maintenance of prolonged G2 Arrest

Targeting checkpoint kinases has been shown to have a potential chemosensitizing effect in cancer treatment. However, inhibitors of such kinases preferentially abrogate the DNA damage-induced G2 checkpoint in p53-/- as opposed to p53+/+ cells. The mechanisms by which p53 (TP53) can prevent abrogation of the G2 checkpoint are unclear. Using normal human diploid p53+/+ and p53-/- fibroblasts as model systems, we have compared the effects of three checkpoint inhibitors, caffeine, staurosporine and UCN-01, on gamma-radiation-induced G2 arrest. The G2 arrest in p53+/+ cells was abrogated by caffeine, but not by staurosporine and UCN-01, whereas the G2 arrest in p53-/- cells was sensitive to all three inhibitors. Chk2 (CHEK1) phosphorylation was maintained in the presence of all three inhibitors in both p53+/+ and p53-/- cells. Chk1 phosphorylation was maintained only in the presence of staurosporine and UCN-01 in p53+/+ cells. In the presence of caffeine Chk1 phosphorylation was inhibited regardless of p53 status. The pathway of Chk1 phosphorylation --> Cdc25A degradation --> inhibition of cyclin B1/Cdk1 activity --> G2 arrest is accordingly resistant to staurosporine and UCN-01 in p53+/+ cells. Moreover, sustained phosphorylation of Chk1 in the presence of staurosporine and UCN-01 is strongly related to phosphorylation of p53. The present study suggests the unique role of Chk1 in preventing abrogation of the G2 checkpoint in p53+/+ cells.     

Zinc-induced G2/M blockage is p53 and p21 dependent in normal human bronchial epithelial cells

The involvement of p53 and p21 signal pathway in the G2/M cell cycle progression of zinc-supplemented normal human bronchial epithelial (NHBE) cells was examined using the small interferring RNA (siRNA) approach. Cells were cultured for one passage in a different concentration of zinc: <0.4 microM (ZD) as zinc deficient; 4 microM as normal zinc level (ZN) in culture medium; 16 microM (ZA) as normal human plasma zinc level; and 32 microM (ZS) as the high end of plasma zinc attainable by oral supplementation. Nuclear p21 protein and mRNA levels as well as promoter activity in ZS cells, but not in ZD cells, were markedly elevated to almost twofold compared with ZN control cells. G2/M blockage in ZS cells was coupled with the observation of elevated p21 gene expression. In ZS cells, the abrogation of p21 protein induction by the transfection of p21 siRNA was shown to alleviate the G2/M blockage, demonstrating the positive linkage of p21 elevation and G2/M blockage. Abolishment of the increase in p53 protein in ZS cells with transfection of p53 siRNA normalized the elevated p21 protein to a similar level as in ZN control cells, which demonstrated that the p21 induction is p53 dependent. Furthermore, the normalization of p53 protein by siRNA treatment in ZS cells alleviated cell growth depression and G2/M blockage, which demonstrated that p53 was involved in the high zinc status-induced G2/M blockage and growth depression. Thus high zinc status in NHBE cells upregulates p53 expression which in turn elevates p21 that eventually induces G2/M blockage.     

Effect of inactivating various components of the signal pathways of the tumor suppressor p53 on genomic stability

To evaluate the role of different p53-regulated signaling pathways in the control of genomic integrity, we studied the frequency of changes in chromosome number and structure of cells of the sublines of mouse primary embryonic fibroblasts with the "knocked-out" genes for proteins p53, p21WAF, pRb, and p19ARF. Protein p21WAF is transactivated by p53 and is responsible for the cell block in the G1 phase of the damaged cells; protein pRb is a target for p21WAF which controls the G1-S-phase transition; and p19ARF protein is responsible for p53 activation in cells with certain anomalies. Inactivation of either of the studied genes proved to increase significantly the frequency of changes in the karyotype. However, the resultant chromosome instability differed: the frequency of the chromosome breaks, both spontaneous and induced with ethylmethane sulfonate (EMS), was in cells with inactivated p53 and lowest in cells with inactivated pRb. These distinctions were not caused by a different effect of various gene inactivation on the cell cycle progression: in all sublines, the cell block in G1 was abolished and the checkpoint function in G2 remained normal. However, the induction of apoptosis in EMS-treated cells differed in the studied sublines. The lowest number of apoptotic nuclei were determined in p53-/- cultures, whereas the highest were in the Rb-/- cultures. It is apparent that the degree of genetic instability is determined by a combined effect of apoptosis and abnormal regulation of the cell-cycle checkpoints.     

Dual and opposing roles of ERK in regulating G(1) and S-G(2)/M delays in A549 cells caused by hyperoxia

This study explores the role of ERK activation in regulating G(1) and S-G(2)/M delays during hyperoxia. We demonstrate here that exposing A549 human alveolar type 2 adenocarcinoma cells to hyperoxia (95% O(2)) for 0.5-24 h time-dependently increases phospho-ERK, phospho-p53(Ser15), p53, and p21(CIP1) protein levels. Decreasing phospho-ERK with the pharmacological inhibitors, PD98059 and U0126, markedly suppresses hyperoxia-stimulated phospho-p53(Ser15), p53, and p21(CIP1), and also restores the hyperoxia-reduced kinase activities of cyclin D1/E1-Cdks. Our results suggest that ERK activation during hyperoxia contributes to the p53/p21-mediated G(1) checkpoint. However, inhibition of ERK signaling during hyperoxia further delays S-phase entry and progression. Hyperoxia induces significant expression of cyclin A/B1 and translocation of cyclin A into nuclei while marginally decreasing cyclin A/B1-Cdks kinase activities, which may be related to nuclear association with p21. Interestingly, inhibition of ERK signaling markedly suppresses the elevation of cyclin A/B1 proteins and cyclin A/B1-Cdks kinase activities during hyperoxia. Taken together, the results presented here suggest that hyperoxia-activated ERK acts upstream of p53 and p21 to suppress G(1)-Cdk activities; however, it is also required for induction of cyclin A/B1 and maintenance of cyclin A/B1-Cdk activities that oppose delays in S-phase entry and progression.     

p53 checkpoint-defective cells are sensitive to X rays, but not hypoxia

X-ray-induced damage leads to cell-cycle "checkpoint" arrest by p53-dependent induction of the cyclin-dependent kinase inhibitor p21 (Waf1/Cip1/Sdi1). Human tumor cells that lack this response fail to arrest after exposure to DNA-damaging agents, undergo multiple rounds of endoreduplicative DNA synthesis, and eventually commit to an apoptotic cell death. Since low oxygen tension can also induce p53 protein accumulation, and can lead to cell-cycle arrest or apoptosis, we examined the expression of p21 in tumor cells under normoxic and hypoxic conditions. In a survey of cells, mRNA for the p21 gene was induced two- to threefold in response to hypoxia in a seemingly p53-independent manner. We therefore examined genetically matched cells that differ in their p21 and p53 status for response to ionizing radiation and hypoxia. We found that both p21-deficient and p53-deficient cells exhibit an increase in chromosome instability, an increased level of apoptosis, and a failure to arrest after exposure to ionizing radiation. However, cells that lack either p21 or p53 exhibit no increase in chromosome instability or elevated apoptosis and still arrest in response to hypoxia. Thus, the mechanism responsible for the differential response to either hypoxia or X rays presumably lies in the control of cell-cycle progression in response to stress and its dependence on p21. Since the loss of a DNA-damage-dependent checkpoint does not sensitize cells to killing by stresses that elicit a DNA-damage-independent checkpoint, targeting the function of p21 pharmacologically will not kill tumor cells in situ in the absence of a DNA damage signal.     

BCR/ABL Recruits p53 Tumor Suppressor Protein to Induce Drug Resistance

Tumors expressing the ABL oncoproteins (BCR/ABL, TEL/ABL, v-ABL) can avoidapoptosis triggered by DNA damaging agents. The tumor suppressor protein p53 is animportant activator of apoptosis in normal cells; conversely its functional loss may causedrug resistance. The ABL oncoprotein - p53 paradigm represents the relationship between anoncogenic tyrosine kinase and a tumor suppressor gene. Here we show that BCR/ABLoncoproteins employ p53 to induce resistance to DNA damage in myeloid leukemia cells.Cells transformed by the ABL oncoproteins displayed accumulation of p53 upon DNAdamage. In contrast, only a modest increase of p53 expression followed by activation ofcaspase-3 were detected in normal cells expressing endogenous c-ABL. Phosphatidylinositol-3 kinase-like protein kinases (ATR and also ATM) -dependent phosphorylation of p53-Ser15residue was associated with the accumulation of p53, and stimulation of p21Waf-1 andGADD45, resulting in G2/M delay in BCR/ABL cells after genotoxic treatment. Inhibition ofp53 by siRNA or by the temperature-sensitive mutation reduced G2/M accumulation anddrug resistance of BCR/ABL cells. In conclusion, accumulation of the p53 proteincontributed to prolonged G2/M checkpoint activation and drug resistance in myeloid cellsexpressing the BCR/ABL oncoproteins.     

p53-independent regulation of cyclin B1 in normal human fibroblasts during UV-induced G2-arrest

Recently we demonstrated, using normal human fibroblasts (NHFs), that UVc radiation induces a G2/M arrest which was even more pronounced when p53 expression was inhibited. So, the aim of this study was to evaluate in NHFs the relationship between UV-induced G2/M arrest and cyclin B1 regulation and to investigate if p53 could contribute to the cyclin B1 regulation in these conditions. Following exposure of asynchronous NHFs to UV light, we showed that the induced G2/M arrest was accompanied by a dose-dependent down-regulation of cyclin B1 mRNA as evaluated by RT-PCR. Concomitantly, using flow cytometric analysis, we observed a strong accumulation of cyclin B1 protein which was correlated to the apparition of the G2/M arrest. In order to study the contribution of p53 to the cyclin B1 accumulation in response to UV exposure, we inhibited p53 induction using p53 antisense oligonucleotides. We found that the inhibition of p53 protein induction after UV exposure had no effect on the level of cyclin B1 mRNA. Moreover, although inhibition of p53 protein induction increased the number of the cells in the G2-M phase, the mean content of cyclin B1 protein was not augmented in these cells. These results indicate clearly that the induction of p53 protein following UV exposure does not regulate the level of cyclin B1 mRNA or protein in normal cells.     

Hepatitis B virus X protein differentially regulates cell cycle progression in X-transforming versus nontransforming hepatocyte (AML12) cell lines.

Hepatitis B virus (HBV) X protein (pX) is implicated in hepatocarcinogenesis of chronically infected HBV patients. To understand mechanism(s) of pX-mediated cellular transformation, we employed two tetracycline-regulated, pX-expressing cell lines, constructed in AML12 immortalized hepatocytes: one a differentiated (3pX-1) and the other a de-differentiated (4pX-1) hepatocyte cell line. Only 3pX-1 cells undergo pX-mediated transformation, via sustained Ras-Raf-mitogen-activated protein kinase pathway activation. pX-nontransforming 4pX-1 cells display sustained, pX-dependent JNK pathway activation. To understand how pX mediates different growth characteristics in 3pX-1 and 4pX-1 cells, we report, herein, comparative cell cycle analyses. pX-transforming 3pX-1 cells display pX-dependent G(1), S, and G(2)/M progression evidenced by cyclin D(1), A, and B(1) induction, and Cdc2 kinase activation. pX-nontransforming 4pX-1 cells display pX-dependent G(1) and S phase entry, followed by S phase pause and absence of Cdc2 kinase activation. Interestingly, 4pX-1 cells exhibit selective pX-induced expression of cyclin-dependent kinase inhibitor p21(Cip1), tumor suppressor p19(ARF), and proapoptotic genes bax and IGFBP-3. Despite the pX-mediated induction of growth arrest and apoptotic genes and the absence of pX-dependent Cdc2 activation, 4pX-1 cells do not undergo pX-dependent G(2)/M arrest or apoptosis. Nocodazole-treated, G(2)/M-arrested 4pX-1 cells exhibit pX-dependent formation of multinucleated cells, similar to human T-cell lymphotropic virus type I Tax-expressing cells. We propose that in 4pX-1 cells, pX deregulates the G(2)/M checkpoint, thus rescuing cells from pX-mediated apoptosis.     

The effect of dibenzo[a,1]pyrene and benzo[a]pyrene on human diploid lung fibroblasts: the induction of DNA adducts, expression of p53 and p21(WAF1) proteins and cell cycle distribution

Polycyclic aromatic hydrocarbons (PAHs) present in ambient air are considered as potential human carcinogens, but the detailed mechanism of action is still unknown. Our aim was to study the in vitro effect of exposure to dibenzo[a,l]pyrene (DB[a,l]P), the most potent carcinogenic PAH ever tested, and benzo[a]pyrene (B[a]P) in a normal human diploid lung fibroblast cells (HEL) using multiple endpoints. DNA adduct levels were measured by 32P-postlabelling, the expression of p53 and p21(WAF1) proteins by western blotting and the cell cycle distribution by flow cytometry. For both PAHs, the DNA adduct formation was proportional to the time of exposure and dependent on the stage of cell growth in culture. DNA binding was detectable even at the lowest concentration used (24h exposure, 0.01 microM for both PAHs). The highest DNA adduct levels were observed after 24h of exposure in near-confluent cells (>90% of cells at G0/G1 phase), but DNA damage induced by DB[a,l]P was approximately 8-10 times higher at a concentration one order of magnitude lower as compared with B[a]P (for B[a]P at 1 microM and for DB[a,l]P at 0.1 microM: 237+/-107 and 2360+/-798 adducts/10(8) nucleotides, respectively). The induction of p53 and p21(WAF1) protein occurred subsequent to the induction of DNA adducts. The DNA adduct levels correlated with both p53 (R=0.832, P<0.001 and R=0.859, P<0.001, for DB[a,l]P and B[a]P, respectively) and p21(WAF1) levels (R=0.808, P<0.001 and R=0.797, P=0.001, for DB[a,l]P and B[a]P, respectively), regardless of the PAH exposure and the phase of cell growth. The results showed that a detectable increase of p53 and p21(WAF1) proteins (> or = 1.5-fold as compared with controls) requires a minimal DNA adduct level of approximately 200-250 adducts/10(8) nucleotides for both PAHs tested and suggest that the level of adducts rather than their structure triggers the p53 and p21(WAF1) responses. The cell cycle was altered after 12-16h of treatment, and after 24h of exposure to 0.1 microM DB[a,l]P in growing cells, there was approximately 24% increase in S phase cells accompanied by a decrease in G1 and G2/mitosis (G2/M) cells. Cell treatment with 1.0 microM B[a]P resulted in more subtle alterations. We conclude that DB[a,l]P, and to a lesser degree B[a]P, are able to induce DNA adducts as well as p53 and p21(WAF1) without eliciting G1 or G2/M arrests but rather an S phase delay/arrest. Whether the S phase delay observed in our study is beneficial for the survival of the cells remains to be further established.     

Loss of p53 induces M-phase retardation following G2 DNA damage checkpoint abrogation

Most cell lines that lack functional p53 protein are arrested in the G2 phase of the cell cycle due to DNA damage. When the G2 checkpoint is abrogated, these cells are forced into mitotic catastrophe. A549 lung adenocarcinoma cells, in which p53 was eliminated with the HPV16 E6 gene, exhibited efficient arrest in the G2 phase when treated with adriamycin. Administration of caffeine to G2-arrested cells induced a drastic change in cell phenotype, the nature of which depended on the status of p53. Flow cytometric and microscopic observations revealed that cells that either contained or lacked p53 resumed their cell cycles and entered mitosis upon caffeine treatment. However, transit to the M phase was slower in p53-negative cells than in p53-positive cells. Consistent with these observations, CDK1 activity was maintained at high levels, along with stable cyclin B1, in p53-negative cells. The addition of butyrolactone I, which is an inhibitor of CDK1 and CDK2, to the p53-negative cells reduced the floating round cell population and induced the disappearance of cyclin B1. These results suggest a relationship between the p53 pathway and the ubiquitin-mediated degradation of mitotic cyclins and possible cross-talk between the G2-DNA damage checkpoint and the mitotic checkpoint.     

Diminished G1 checkpoint after gamma-irradiation and altered cell cycle regulation by insulin-like growth factor II overexpression

High levels of insulin-like growth factor II (IGFII) mRNA expression are detected in many human tumors of different origins including rhabdomyosarcoma, a tumor of skeletal muscle origin. To investigate the role of IGFII in tumorigenesis, we have compared the mouse myoblast cell line C2C12-2.7, which was stably transfected with human IGFII cDNA and expressed high and constant amounts of IGFII, to a control cell line C2C12-1.1. A rhabdomyosarcoma cell line, RH30, which expresses high levels of IGFII and contains mutated p53, was also used in these studies. IGFII overexpression in mouse myoblast C2C12 cells causes a reduced cycling time and higher growth rate. After gamma-irradiation treatment, C2C12-1.1 cells were arrested mainly in G0/G1 phase. However, C2C12-2.7 and RH30 cells went through a very short G1 phase and then were arrested in an extended G2/M phase. To verify further the effect of IGFII on the cell cycle, we developed a Chinese hamster ovary (CHO) cell line with tetracycline-controlled IGFII expression. We found that CHO cells with high expression of IGFII have a shortened cycling time and a diminished G1 checkpoint after treatment with methylmethane sulfonate (MMS), a DNA base-damaging agent, when compared with CHO cells with very low IGFII expression. It was also found that IGFII overexpression in C2C12 cells was associated with increases in cyclin D1, p21, and p53 protein levels, as well as mitogen-activated protein kinase activity. These studies suggest that IGFII overexpression shortens cell cycling time and diminishes the G1 checkpoint after DNA damage despite an intact p53/p21 induction. In addition, IGFII overexpression is also associated with multiple changes in the levels and activities of cell cycle regulatory components following gamma-irradiation. Taken together, these changes may contribute to the high growth rate and genetic alterations that occur during tumorigenesis.     

Involvement of p21 in the PKC-induced regulation of the G2/M cell cycle transition

Activation of protein kinase C (PKC) inhibits cell cycle progression at the G1/S and G2/M transitions. We found that phorbol 12-myristate 13-acetate (PMA) induced upregulation of p21, not only in MCF-7 cells arrested in the G1 phase as previously shown, but also in cells delayed in the G2 phase. This increase in p21 in cells accumulated in the G1 and G2/M phases of the cell cycle after PMA treatment was inhibited by the PKC inhibitor GF109203X. This indicates that PKC activity is required for PMA-induced p21 upregulation and cell cycle arrest in the G1 and G2/M phases of the cell cycle. To further assess the role of p21 in the PKC-induced G2/M cell cycle arrest independently of its G1 arrest, we used aphidicolin-synchronised MCF-7 cells. Our results show that, in parallel with the inhibition of cdc2 activity, PMA addition enhanced the associations between p21 and either cyclin B or cdc2. Furthermore, we found that after PMA treatment p21 was able to associate with the active Tyr-15 dephosphorylated form of cdc2, but this complex was devoid of kinase activity indicating that p21 may play a role in inhibition of cdc2 induced by PMA. Taken together, these observations provide evidence that p21 is involved in integrating the PKC signaling pathway to the cell cycle machinery at the G2/M cell cycle checkpoint.     

Chk1, but not Chk2 , is Involved in the Cellular Response to DNA Damaging Agents: Differential Activity in Cells Expressing,or not,p53

Mammalian Chk1 and Chk2 protein kinases are two important components of the G2DNA damage checkpoint. They are activated by upstream kinases (ataxia telangectasiamutated gene and ATM and Rad 3 related gene) and interfere with the activity of thecdc2/cyclinB1 complex, necessary for the G2-M transition, through the inactivation of thecdc25 phosphatases (cdc25A and cdc25C). To understand the role of Chk1 and Chk2 in thecellular response to different anticancer agents, we knocked down the expression of eachprotein or simultaneously of both proteins by using the small interfering RNA techniquein the HCT-116 colon carcinoma cell line and in its isogenic systems in which p53 and p21have been inactivated by targeted homologous recombination. We here show thatinhibition of Chk1 but not of Chk2 in p21-/- and p53-/- cells caused a greater abrogationof G2 block induced by ionizing radiation and cis-diamine-dichloroplatinum treatmentsand a greater sensitisation to the same treatments than in the parental cell line with p53and p21 wild type proteins. These data further emphasise the role of Chk1 as a moleculartarget to inhibit in tumors with a defect in the G1 checkpoint with the aim of increasingthe selectivity and specificity of anticancer drug treatments.