siRNA-mediated downregulation of MMP-9 and uPAR in combination with radiation induces G2/M cell-cycle arrest in Medulloblastoma

Our previous work and that of other investigators strongly suggest a relationship between the upregulation of metalloproteinase-9 (MMP-9) and urokinase-type plasminogen activator receptor (uPAR) in tumor angiogenesis and metastasis. In this study, we evaluated the role of MMP-9 and uPAR in medulloblastoma cancer cell resistance to ionizing irradiation (IR) and tested the antitumor efficacy of siRNA (short interfering RNA) against MMP-9 [plasmid siRNA vector for MMP-9 (pM)] and uPAR [plasmid vector for uPAR (pU)] either alone or in combination [plasmid siRNA vector for both uPAR and MMP-9 (pUM)]. Cell proliferation (BrdU assay), apoptosis (in situ TUNEL for DNA fragmentation), and cell-cycle (FACS) analyses were carried out to determine the effect of siRNA either alone or in combination with IR on G2/M cell-cycle arrest in medulloblastoma cells. IR upregulated MMP-9 and uPAR expression in medulloblastoma cells; pM, pU, and pUM in combination with IR effectively reduced both MMP-9 and uPAR expression, thereby leading to increased radiosensitivity of medulloblastoma cells. siRNA treatments (pM, pU, and pUM) also promoted IR-induced apoptosis and enhanced IR-induced G2/M arrest during cell-cycle progression. While IR induces G2/M cell-cycle arrest through inhibition of the pCdc2- and cyclin B-regulated signaling pathways involving p53, p21/WAF1, and Chk2 gene expression, siRNA (pM, pU, and pUM) alone or in combination with IR induced G2/M arrest mediated through inhibition of the pCdc2- and cyclin B1-regulated signaling pathways involving Chk1 and Cdc25A gene expression. Taken together, our data suggest that downregulation of MMP-9 and uPAR induces Chk1-mediated G2/M cell-cycle arrest, whereas the disruption caused by IR alone is dependent on p53- and Chk2-mediated G2/M cell-cycle arrest.     

Flavokawain B induces apoptosis of non-small cell lung cancer H460 cells via Bax-initiated mitochondrial and JNK pathway

Flavokawain B (FKB) possesses strong anti-neoplastic activity against many cancer cells. Here we assessed its antitumor activity and molecular mechanisms in lung cancer H460 cells in vitro. FKB significantly inhibited cell proliferation and caused arrest of the cell cycle G2-M of H460 cells in a dose-dependent manner. FKB also inducted apoptosis, which was associated with cytochrome c release, caspase-7 and caspase-9 activation and Bcl-xL/Bax dys-regulation. FKB significantly down-regulated survivin and XIAP, and the inhibitory effect induced by FKB was greatly attenuated by through over-expression of survivin or Bax(-/-) MEFs. Furthermore, FKB activated the mitogen-activated protein kinases and the JNK inhibitor SP600125 significantly decreased the growth-inhibitory and apoptotic effects of FKB. Together, these results suggest the anti-lung cancer potential of flavokawain B for the prevention and treatment of lung cancer.     

Induction of G2/M phase arrest and apoptosis by a novel enediyne derivative, THDA, in chronic myeloid leukemia (K562) cells

We studied the effect of 2-(6-(2-thieanisyl)-3(Z)-hexen-1,5-diynyl)aniline(THDA), a newly developed anti-cancer agent, on cell proliferation, cell cycle progression, and induction of apoptosis in K562 cells. THDA was found to inhibit the growth of K562 cells in a time-and dose-dependent manner. Cell cycle analysis showed G2/M phase arrest and apoptosis in K562 cells following 24 h exposure to THDA. During the G2/M arrest, cyclin-dependent kinase inhibitors (CDKIs), p21 and p27 were increased in a time-dependent manner. Analysis of the cell cycle regulatory proteins demonstrated that THDA did not change the steady-state levels of cyclin B1, cyclin D3 and Cdc25C, but decreased the protein levels of Cdk1, Cdk2 and cyclin A. THDA also caused a marked increase in apoptosis, which was associated with activation of caspase-3 and proteolytic cleavage of poly (ADP-ribose) polymerase. These molecular alterations provide an insight into THDA-caused growth inhibition, G2/M arrest and apoptotic death of K562 cells.     

Critical role of cyclin B1/Cdc2 up-regulation in the induction of mitotic prometaphase arrest in human breast cancer cells treated with 2-methoxyestradiol

Earlier studies showed that 2-methoxyestradiol (2ME(2)), an endogenous nonpolar metabolite of estradiol-17β, is a strong inducer of G(2)/M cell cycle arrest (based on analysis of cellular DNA content) in human cancer cell lines. The present study sought to investigate the molecular mechanism underlying 2ME(2)-induced cell cycle arrest. We found that 2ME(2) can selectively induce mitotic prometaphase arrest, but not G(2) phase arrest, in cultured MDA-MB-435s and MCF-7 human breast cancer cells. During the induction of prometaphase arrest, there is a time-dependent initial up-regulation of cyclin B1 and Cdc2 proteins, occurring around 12-24h. The strong initial up-regulation of cyclin B1 and Cdc2 matches in timing the 2ME(2)-induced prometaphase arrest. The 2ME(2)-induced prometaphase arrest is abrogated by selective knockdown of cyclin B1 and Cdc2, or by pre-treatment of cells with roscovitine, an inhibitor of cyclin-dependent kinases, or by co-treatment of cells with cycloheximide, a protein synthesis inhibitor that was found to suppress the early up-regulation of cyclin B1 and Cdc2. In addition, we provided evidence showing that MAD2 and JNK1 are important upstream mediators of 2ME(2)-induced up-regulation of cyclin B1 and Cdc2 as well as the subsequent induction of mitotic prometaphase arrest. In conclusion, treatment of human cancer cells with 2ME(2) causes up-regulation of cyclin B1 and Cdc2, which then mediate the induction of mitotic prometaphase arrest.     

RNA interference-mediated silencing of the PAR gene inhibits the growth of PC3 cells via the induction of G2/M cell cycle arrest and apoptosis

BACKGROUND: The prostate androgen-regulated (PAR) gene is ubiquitously overexpressed in prostate cancer (PCa) cells and is involved in proliferation of PCa. However, the mechanism by which the modulation of PAR gene expression elicits its biological effects on PCa cells is not well documented. Here, we investigate the mechanism of PAR depletion inhibiting PCa cell growth. METHODS: PAR expression was depleted by small interfering RNA (siRNA) and its subsequent effects on proliferation of PC3 cells were determined by the trypan blue exclusion assay. Flow cytometric analysis provided the evidence for the progression of cell cycle and the induction of apoptosis which was further confirmed by the observation of cleavage of poly(ADP-ribose) polymerase. Western blot analysis was performed to investigate the involvement of critical molecular events known to regulate the cell cycle and the apoptotic machinery. RESULTS: siRNA transfection results in a dose-dependent inhibition of cell growth in PC3 cells by causing G2/M phase cell cycle arrest and apoptosis. The G2/M arrest by PAR depletion was associated with decreased levels of cyclin B1, pCdc2 (Tyr15), Cdc2 and Cdc25C. PAR depletion also was found to result in inhibition of procaspases 9, 8, 6 and 3 with significant increase in the ratio of Bax : Bcl-2. CONCLUSIONS: Our data indicate that PAR depletion induces G2/M arrest via the Cdc25C-Cdc2/cyclin B1 pathway. Furthermore, the results of the present study point toward involvement of pathways mediated by both caspase 8 and caspase 9 in apoptosis induction by PAR depletion.     

Phosphorylation of Mcl‐1 by CDK1–cyclin B1 initiates its Cdc20‐dependent destruction during mitotic arrest

The balance between cell cycle progression and apoptosis is important for both surveillance against genomic defects and responses to drugs that arrest the cell cycle. In this report, we show that the level of the human anti‐apoptotic protein Mcl‐1 is regulated during the cell cycle and peaks at mitosis. Mcl‐1 is phosphorylated at two sites in mitosis, Ser64 and Thr92. Phosphorylation of Thr92 by cyclin‐dependent kinase 1 (CDK1)–cyclin B1 initiates degradation of Mcl‐1 in cells arrested in mitosis by microtubule poisons. Mcl‐1 destruction during mitotic arrest requires proteasome activity and is dependent on Cdc20/Fizzy, which mediates recognition of mitotic substrates by the anaphase‐promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. Stabilisation of Mcl‐1 during mitotic arrest by mutation of either Thr92 or a D‐box destruction motif inhibits the induction of apoptosis by microtubule poisons. Thus, phosphorylation of Mcl‐1 by CDK1–cyclin B1 and its APC/C^Cdc20‐mediated destruction initiates apoptosis if a cell fails to resolve mitosis. Regulation of apoptosis, therefore, is linked intrinsically to progression through mitosis and is governed by a temporal mechanism that distinguishes between normal mitosis and prolonged mitotic arrest.     

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.     

Brefeldin A Is a Potent Inducer of Apoptosis in Human Cancer Cells Independently of p53

Brefeldin A (BFA) is a natural product that affects the structure and function of the Golgi apparatus and is in development for cancer chemotherapy. We observed that a wide range of cancer cells could undergo DNA fragmentation associated with apoptosis after BFA treatment. This DNA fragmentation was induced within 15 h in HL60 leukemia cells and after 48 h in K562 leukemia and HT-29 colon carcinoma cells with BFA concentrations as low as 0.1 μM.The DNA fragmentation had the typical internucleosomal pattern in HL60 and HT-29 cells. Apoptotic cells were also detected by microscopy. BFA-induced apoptosis is p53-independent as HL60 and K562 cells are p53 null and HT-29 are p53 mutant cells. BFA could potentiate UCN-01 and staurosporine-induced DNA fragmentation in HL60 cells. Cyclin B1/Cdc2 kinase activity decreased after BFA treatment in HL60 cells, indicating that BFA-induced DNA fragmentation was independent of a cyclin B1/Cdc2 kinase upregulation pathway. Cycloheximide could not prevent BFA-induced DNA fragmentation in HL60 cells, suggesting that protein synthesis is not needed for HL60 cells to undergo apoptosis. On the contrary, cycloheximide blocked BFA-induced DNA fragmentation in HT-29 cells, indicating that apoptosis in HT-29 cells requires macromolecular synthesis. Cell-free system experiments suggested that cytosolic proteins play an important role in triggering DNA fragmentation during apoptosis induced by BFA. Our results show that transduction signaling pathways play central roles in apoptotic regulation.     

Involvement of Cyclin-Dependent Kinase Activities in CD437-Induced Apoptosis

A novel synthetic retinoid, 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437), is a selective ligand of the RARgamma nuclear receptor. We examined the in vitro effects of CD437 and found that CD437 induces S phase arrest within 24 to 48 h, followed by cell death, in the p53-negative Hep3B and the p53-positive HepG2 human hepatoma cell lines. Based on observations of cellular and nuclear fragmentation, chromatin condensation, and DNA fragmentation, the CD437-mediated cell-killing effect appears to be due to apoptosis. On morphological examination, a number of CD437-treated cells were found to have increased 5- to 10-fold in size and persisted as single giant cells without cell division, while the remainder underwent nuclear division (multiple nuclei) but were unable to complete cytokinesis, and finally all died by apoptosis. In HepG2 cells that possessed wild-type p53, CD437-induced S phase arrest and apoptosis were accompanied by the up-regulation of cyclin A, cyclin B, p53, p21(CIP1/Waf1), Bad, and Bcl-Xs proteins and by a decrease in Bcl-2 protein levels. In Hep3B cells, CD437-mediated S phase arrest and apoptosis were also associated with a concomitant up-regulation of cyclin A, cyclin B, Bad, and Bcl-Xs. However, Hep3B cells did not express p53 or Bcl-2 messages. Olomoucine and roscovitine, the potent p34(cdc2) and CDK2 inhibitors, effectively blocked CD437-mediated cyclin A- and B-dependent kinase activation and prevented CD437-induced cell death. Furthermore, antisense oligonucleotide complementary to cyclin A and B mRNA significantly rescued CD437-induced apoptosis. These findings suggest that activation of cyclin A- and B-dependent kinases is a critical determinant of apoptotic death mediated by CD437.     

Cdc2 and Cdk2 kinase activated by transforming growth factor-beta1 trigger apoptosis through the phosphorylation of retinoblastoma protein in FaO hepatoma cells.

The signaling pathway leading to TGF-beta1-induced apoptosis was investigated using a TGF-beta1-sensitive hepatoma cell line, FaO. Cell cycle analysis demonstrated that the accumulation of apoptotic cells was preceded by a progressive decrease of the cell population in the G(1) phase concomitant with a slight increase of the cell population in the G(2)/M phase in response to TGF-beta1. TGF-beta1 induced a transient increase in the expression of Cdc2, cyclin A, cyclin B, and cyclin D1 at an early phase of apoptosis. During TGF-beta1-induced apoptosis, the transient increase in cyclin-dependent kinase (Cdk) activities coincides with a dramatic increase in the hyperphosphorylated forms of RB. Treatment with roscovitine or olomoucine, inhibitors of Cdc2 and Cdk2, blocked TGF-beta1-induced apoptosis by inhibiting RB phosphorylation. Overexpression of Bcl-2 or adenovirus E1B 19K suppressed TGF-beta1-induced apoptosis by blocking the induction of Cdc2 mRNA and the subsequent activation of Cdc2 kinase, whereas activation of Cdk2 was not affected, suggesting that Cdc2 plays a more critical role in TGF-beta1-induced apoptosis. In conclusion, we present the evidence that Cdc2 and Cdk2 kinase activity transiently induced by TGF-beta1 phosphorylates RB as a physiological target in FaO cells and that RB hyperphosphorylation may trigger abrupt cell cycle progression, leading to irreversible cell death.     

TNFalpha-mediated cell death is independent of cdc25A

Tumor necrosis factor (TNFs) have been shown to be synthesized by ovarian carcinomas, and may therefore affect tumor cells in an autocrine manner. Therefore, we investigated the effects of recombinant TNFs on ovarian carcinoma cells N.1 and examined expression of the proto-oncogenes c-myc and cdc25A which are known to play a prominent role in apoptosis. TNFalpha elicited apoptosis in N.1 cells within 72 h which was shown by typical morphological changes, DNA fragmentation and signature type cleavage of poly(ADP-ribose) polymerase into a 89 kDa proteolytic peptide. TNFalpha-induced apoptosis was accompanied by constitutive c-Myc expression, although the mRNA level of phosphatase cdc25A was suppressed within 24 h of TNFalpha treatment and the protein level decreased after 48 h. Cdc25A tyrosine phosphatase is an activator of the cdk2-cyclin E complex which allows for cell cycle progression. As expected, we found TNFalpha-mediated Cdc25A down-regulation to inhibit Cdk2 activity. Cdc25A suppression was related to TNFalpha-induced apoptosis but not to a TNFalpha-induced G0 arrest because cyclin D1 expression was unaffected and the gene gas6 (growth arrest specific 6) was not induced. Arresting cells by treatment with genistein prevented TNFalpha-triggered apoptosis and inhibited c-myc expression. TNFalpha-induced apoptosis is not accompanied by cell cycle arrest which may be due to constitutive c-Myc expression, although Cdc25A and Cdk2 activity is also down-regulated. High c-Myc and low Cdc25A activity might present conflicting signals to the cell cycle machinery which are incompatible with cell survival.     

Anticancer activities of chalcone flavokawain B from Alpinia pricei Hayata in human lung adenocarcinoma (A549) cells via induction of reactive oxygen species-mediated apoptotic and autophagic cell death

Chalcones found in fruits and vegetables have promising cancer chemopreventive properties. This study attempts to identify the anticancer efficacies of chalcone flavokawain B (FKB) in the rhizomes of Alpinia pricei Hayata by examining key molecular events in non-small-cell lung cancer (A549) cells. Our results indicated that in human A549 cells, FKB (0–15 μg/ml) decreases cell viability and colony formation, dysregulates the Bax:B-cell lymphoma 2 ratio and increases apoptotic DNA fragmentation. Mitochondrial (caspase-9/-3 and poly ADP ribose polymerase [PARP]) signaling was found to be involved in FKB-induced apoptosis. In addition, FKB-induced reactive oxygen species (ROS) generation, and N-acetylcysteine attenuated FKB-induced apoptotic cell death. Moreover, FKB triggered autophagy, as evidenced by the improved acidic vesicular organelle formation, lipidated light chain 3 (microtubule-related light chain 3) accumulation, and ATG7 expression and the decreased mammalian target of rapamycin phosphorylation. Furthermore, FKB suppressed ROS-mediated ATG4B expression. Inhibiting autophagy using 3-methyladenine/chloroquine diminished FKB-induced cell death, indicating that autophagy is triggered as a death mechanism by FKB. In summary, FKB has a crucial role in the execution and propagation of ROS-mediated apoptotic and autophagic cell death of lung adenocarcinoma cells.     

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.     

Anti-proliferative effects of evodiamine on human prostate cancer cell lines DU145 and PC3

Prostate carcinoma is one of the most common malignant tumors and has become a more common cancer in men. Previous studies demonstrated that evodiamine (EVO) exhibited anti-tumor activities on several cancers, but its effects on androgen-independent prostate cancer are unclear. In the present study, the action mechanisms of EVO on the growth of androgen-independent prostate cancer cells (DU145 and PC3 cells) were explored. EVO dramatically inhibited the growth and elevated cytotoxicity of DU145 and PC3 cells. The flow cytometric analysis of EVO-treated cells indicated a block of G2/M phase and an elevated level of DNA fragmentation. The G2/M arrest was accompanied by elevated Cdc2 kinase activity, an increase in expression of cyclin B1 and phosphorylated Cdc2 (Thr 161), and a decrease in expression of phosphorylated Cdc2 (Tyr 15), Myt-1, and interphase Cdc25C. TUNEL examination showed that EVO-induced apoptosis was observed at 72 h. EVO elevated the activities of caspase 3, 8, and 9 in DU145 cells, while in PC3 cells only the activities of caspase 3 and 9 were elevated. EVO also triggered the processing of caspase 3 and 9 in both DU145 and PC3 cells. We demonstrate that roscovitine treatment result in the reversion of G2/M arrest in response to EVO in both DU145 and PC3. However, inhibitory effect of roscovitine on EVO-induced apoptosis could only be observed in DU145 rather than PC3. In DU145, G2/M arrest might be a signal for initiation of EVO-triggered apoptosis. Whereas EVO-triggered PC3 apoptosis might be independent of G2/M arrest. These results suggested that EVO inhibited the growth of prostate cancer cell lines, DU145 and PC3, through an accumulation at G2/M phase and an induction of apoptosis.     

Induction of apoptosis in vascular cells by plasminogen activator inhibitor-1 and high molecular weight kininogen correlates with their anti-adhesive properties

Plasminogen activator inhibitor-1 (PAI-1) and two-chain high molecular weight kininogen (HKa) exert anti-adhesive properties in vitronectin-dependent cell adhesion. Here, the hypothesis was tested that these anti-adhesive components promote apoptosis in vascular cells. PAI-1 or HKa induced a 2- to 3-fold increase in apoptosis of human umbilical-vein endothelial cells (HUVEC) and vascular smooth muscle cells (VSMC) adherent to vitronectin, as determined by annexin V-FACS assay, similar to alphav-integrin inhibitor cyclo-(Arg-Gly-Asp-D-Phe-Val)-peptide (cRGDfV). Apoptosis occurred after 12 h incubation and was attributable to caspase 3 activation that in turn induced DNA fragmentation. Induction of apoptosis strongly correlated with the anti-adhesive effect of PAI-1 and HKa on these cells. In contrast, PAI-1 and HKa did not affect fibronectin-dependent adhesion or cell survival. uPA did not influence apoptosis in vitronectin- or fibronectin-adherent cells. In atherosclerotic vessel sections, congruent distribution of vitronectin, PAI-1, HK, and of components of the urokinase plasminogen activator/receptor system with apoptotic cells lining foam cell lesions was demonstrated by immunostaining. These results indicate that inhibition of vitronectin-dependent cell adhesion through PAI-1 and HKa correlates with apoptosis induction in vascular cells mediated through the caspase 3 pathway. Co-distribution of apoptosis with plasminogen activation system components in atherosclerosis exemplifies the significance of anti-adhesive mechanisms and apoptosis for tissue remodeling, such as in neointima development.     

Role of cyclin B1/Cdc2 up-regulation in the development of mitotic prometaphase arrest in human breast cancer cells treated with nocodazole

Background

During a normal cell cycle, the transition from G₂ phase to mitotic phase is triggered by the activation of the cyclin B1-dependent Cdc2 kinase. Here we report our finding that treatment of MCF-7 human breast cancer cells with nocodazole, a prototypic microtubule inhibitor, results in strong up-regulation of cyclin B1 and Cdc2 levels, and their increases are required for the development of mitotic prometaphase arrest and characteristic phenotypes.

Methodology/Principal Findings

It was observed that there was a time-dependent early increase in cyclin B1 and Cdc2 protein levels (peaking between 12 and 24 h post treatment), and their levels started to decline after the initial increase. This early up-regulation of cyclin B1 and Cdc2 closely matched in timing the nocodazole-induced mitotic prometaphase arrest. Selective knockdown of cyclin B1or Cdc2 each abrogated nocodazole-induced accumulation of prometaphase cells. The nocodazole-induced prometaphase arrest was also abrogated by pre-treatment of cells with roscovitine, an inhibitor of cyclin-dependent kinases, or with cycloheximide, a protein synthesis inhibitor that was found to suppress cyclin B1 and Cdc2 up-regulation. In addition, we found that MAD2 knockdown abrogated nocodazole-induced accumulation of cyclin B1 and Cdc2 proteins, which was accompanied by an attenuation of nocodazole-induced prometaphase arrest.

Conclusions/Significance

These observations demonstrate that the strong early up-regulation of cyclin B1 and Cdc2 contributes critically to the rapid and selective accumulation of prometaphase-arrested cells, a phenomenon associated with exposure to microtubule inhibitors.     

Pro-apoptotic Role of Cdc25A: ACTIVATION OF CYCLIN B1/Cdc2 BY THE Cdc25A C-TERMINAL DOMAIN*

Cdc25A is a dual specificity protein phosphatase that activates cyclin/cyclin-dependent protein kinase (Cdk) complexes by removing inhibitory phosphates from conserved threonine and tyrosine in Cdks. To address how Cdc25A promotes apoptosis, Jurkat cells were treated with staurosporine, an apoptosis inducer. Upon staurosporine treatment, a Cdc25A C-terminal 37-kDa fragment, designated C37, was generated by caspase cleavage at Asp-223. Thr-507 in C37 became dephosphorylated, which prevented 14-3-3 binding, as shown previously. C37 exhibited higher phosphatase activity than full-length Cdc25A. C37 with alanine substitution for Thr-507 (C37/T507A) that imitated the cleavage product during staurosporine treatment interacted with Cdc2, Cdk2, cyclin A, and cyclin B1 and markedly activated cyclin B1/Cdc2. The dephosphorylation of Thr-507 might expose the Cdc2/Cdk2-docking site in C37. C37/T507A also induced apoptosis in Jurkat and K562 cells, resulting from activating cyclin B1/Cdc2 but not Cdk2. Thus, this study reveals that Cdc25A is a pro-apoptotic protein that amplifies staurosporine-induced apoptosis through the activation of cyclin B1/Cdc2 by its C-terminal domain.     

A novel indoloquinoline derivative, IQDMA, induces S-phase arrest and apoptosis in promyelocytic leukemia HL-60 cells

N′-(11H-indolo[3,2-c]quinolin-6-yl)-N,N-dimethylethane-1,2-diamine (IQDMA), an indoloquinoline compound, was identified in our laboratory as a novel antineoplastic agent with a broad spectrum of antitumor activity against many human cancer cells. Cell cycle analysis showed S-phase arrest and induction of apoptosis in HL-60 cells following 24 h exposure to IQDMA. Analysis of the cell cycle regulatory proteins demonstrated that IQDMA did not change the steady-state levels of cyclin B1, cyclin D3, and p21, but decreased the protein levels of Cdk1, Cdk2, and cyclin A. IQDMA also caused a marked increase in apoptosis, which was accompanied by increased levels of Bax, activated caspase-3, -8, and -9, and cleaved PARP. These molecular alterations provide an insight into IQDMA-caused growth inhibition, S-phase arrest, and apoptotic death of HL-60 cells.     

Cdc2 and Cdk2 play critical roles in low dose doxorubicin-induced cell death through mitotic catastrophe but not in high dose doxorubicin-induced apoptosis

In Huh-7 hepatoma cells, low dose (LD) doxorubicin treatment induces cell death through mitotic catastrophe accompanying the formation of large cells with multiple micronuclei, whereas high dose (HD) doxorubicin induces apoptosis. In this study, we investigated the role of Cdc2 and Cdk2 kinase in the regulation of the two modes of cell death induced by doxorubicin. During HD doxorubicin-induced apoptosis, the histone H1-associated activities of Cdc2 and Cdk2 both progressively declined in parallel with reductions in cyclin A and cyclin B protein levels. In contrast, during LD doxorubicin-induced cell death through mitotic catastrophe, the Cdc2 and Cdk2 kinases were transiently activated 1 day post-treatment, with similar changes seen in the protein levels of cyclin A, cyclin B, and Cdc2. Treatment with roscovitine, a specific inhibitor of Cdc2 and Cdk2, significantly blocked LD doxorubicin-induced mitotic catastrophe and cell death, but did not affect HD doxorubicin-induced apoptosis in Huh-7, SNU-398, and SNU-449 hepatoma cell lines. Our results demonstrate that differential regulation of Cdc2 and Cdk2 activity by different doses of doxorubicin may contribute to the induction of two distinct modes of cell death in hepatoma cells, either apoptosis or cell death through mitotic catastrophe.