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.     

2ME and 2OHE2 exhibit growth inhibitory effects and cell cycle arrest at G2/M in RL95-2 human endometrial cancer cells through activation of p53 and Chk1

Evidence is accumulating that estradiol (E2) may play a dual role in carcinogenic and anticarcinogenic effects by different metabolic pathways. It has been shown that some metabolites of E2 exert proliferative and others anti-proliferative properties on human cancer cells. In the present study, the effects of E2 and its four primary metabolites including 2-hydroxyestradiol (2OHE2), 4-hydroxyestradiol (4OHE2), 2-methoxyestradiol (2ME), and 4-methoxyestradiol (4ME) on proliferation and cell cycle in RL95-2 human endometrial cells were investigated. Our results indicate that 2ME and 2OHE2, but not E2, 4ME, and 4OHE2, exhibit the inhibitory effect through cell cycle arrest at G2/M. 2ME and 2OHE2-induced G2/M cell cycle arrest associated with activation of p53 (Ser15), upregulation of p21(WAF1/Cip1) (p21) and GADD45, inactivation of Cdc2 (Tyr15), as well as downregulation of Cyclin B1. 2ME and 2OHE2-mediated cell cycle arrest at G2/M was also related to activation of protein kinase Chk1 which is associated with p53 (Ser20) activation and downstream responses.     

Reprimo, a new candidate mediator of the p53-mediated cell cycle arrest at the G2 phase

A novel gene, Reprimo, in which induction in cells exposed to X-irradiation is dependent on p53 expression, has been isolated. Ectopic p53 expression results in the induction of its mRNA. Reprimo is a highly glycosylated protein and, when ectopically expressed, it is localized in the cytoplasm and induces G(2) arrest of the cell cycle. In the arrested cells, both Cdc2 activity and nuclear translocation of cyclin B1 are inhibited, suggesting the involvement of Reprimo in the Cdc2.cyclin B1 regulation pathway. Thus, Reprimo may be a new member involved in the regulation of p53-dependent G(2) arrest of the cell cycle.     

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.     

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.     

CUGBP2 downregulation by prostaglandin E2 protects colon cancer cells from radiation-induced mitotic catastrophe

Prostaglandin E(2) (PGE(2)) is a potent inhibitor of ionizing radiation (IR)-induced cell death. Exposure of colon cancer cells to IR leads to increased CUGBP2 expression. Therefore, we tested the hypothesis that PGE(2) radioprotects colon cancer cells by inhibiting CUGBP2 expression. Exposure of HCT-116 cells to gamma-IR (0-12 Gy) resulted in a dose-dependent reduction in cell growth and an increase in the G(2)-M phase of the cell cycle. Western blot analyses demonstrated increased levels of activated caspase 9 and caspase 3. In addition, whereas Bax expression is increased, that of Bcl-2 and Bcl-x(L) was reduced. Further analyses demonstrated increased activation of Chk1 and Chk2 kinases, coupled with higher levels of nuclear cyclin B1 and Cdc2. Pretreatment with PGE(2) suppressed the activation of caspase 3 and caspase 7 and inhibited Bax expression. In addition, PGE(2) treatment restored growth and colony formation to control levels. IR significantly upregulated the expression of CUGBP2 in the cells, which was suppressed when cells were pretreated with PGE(2). Ectopic overexpression of CUGBP2 also induced apoptosis. Furthermore, it reversed the PGE(2)-mediated protection from IR-induced mitotic catastrophe. Furthermore, there was an increase in nuclear localization of cyclin B1 and Cdc2 coupled with increased phosphorylation of p53, Chk1, Chk2, and Cdc25c proteins. Cell cycle analysis also demonstrated increased G(2)-M transition. In contrast, siRNA-mediated suppression of CUGBP2 expression restored normal cell cycle progression and decreased IR-induced apoptosis. Taken together, these data demonstrate that PGE(2) protects colon cancer cells from IR-induced mitotic catastrophe in part through suppression of CUGBP2 expression.     

Sulforaphane-induced G2/M phase cell cycle arrest involves checkpoint kinase 2-mediated phosphorylation of cell division cycle 25C

Previously, we showed that sulforaphane (SFN), a naturally occurring cancer chemopreventive agent, effectively inhibits proliferation of PC-3 human prostate cancer cells by causing caspase-9- and caspase-8-mediated apoptosis. Here, we demonstrate that SFN treatment causes an irreversible arrest in the G(2)/M phase of the cell cycle. Cell cycle arrest induced by SFN was associated with a significant decrease in protein levels of cyclin B1, cell division cycle (Cdc) 25B, and Cdc25C, leading to accumulation of Tyr-15-phosphorylated (inactive) cyclin-dependent kinase 1. The SFN-induced decline in Cdc25C protein level was blocked in the presence of proteasome inhibitor lactacystin, but lactacystin did not confer protection against cell cycle arrest. Interestingly, SFN treatment also resulted in a rapid and sustained phosphorylation of Cdc25C at Ser-216, leading to its translocation from the nucleus to the cytoplasm because of increased binding with 14-3-3beta. Increased Ser-216 phosphorylation of Cdc25C upon treatment with SFN was the result of activation of checkpoint kinase 2 (Chk2), which was associated with Ser-1981 phosphorylation of ataxia telangiectasia-mutated, generation of reactive oxygen species, and Ser-139 phosphorylation of histone H2A.X, a sensitive marker for the presence of DNA double-strand breaks. Transient transfection of PC-3 cells with Chk2-specific small interfering RNA duplexes significantly attenuated SFN-induced G(2)/M arrest. HCT116 human colon cancer-derived Chk2(-/-) cells were significantly more resistant to G(2)/M arrest by SFN compared with the wild type HCT116 cells. These findings indicate that Chk2-mediated phosphorylation of Cdc25C plays a major role in irreversible G(2)/M arrest by SFN. Activation of Chk2 in response to DNA damage is well documented, but the present study is the first published report to link Chk2 activation to cell cycle arrest by an isothiocyanate.     

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.     

Human T-lymphotropic virus type 1 oncoprotein tax promotes unscheduled degradation of Pds1p/securin and Clb2p/cyclin B1 and causes chromosomal instability

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia. The HTLV-1 transactivator, Tax, is implicated as the viral oncoprotein. Na?ve cells expressing Tax for the first time develop severe cell cycle abnormalities that include increased DNA synthesis, mitotic arrest, appearance of convoluted nuclei with decondensed DNA, and formation of multinucleated cells. Here we report that Tax causes a drastic reduction in Pds1p/securin and Clb2p/cyclin B levels in yeast, rodent, and human cells and a loss of cell viability. With a temperature-sensitive mutant of the CDC23 subunit of the anaphase-promoting complex (APC), cdc23(ts); a temperature-sensitive mutant of cdc20; and a cdh1-null mutant, we show that the diminution of Pds1p and Clb2p brought on by Tax is mediated via the Cdc20p-associated anaphase-promoting complex, APC(Cdc20p). This loss of Pds1p/securin and Clb2p/cyclin B1 occurred before cellular entry into mitosis, caused a G(2)/M cell cycle block, and was accompanied by severe chromosome aneuploidy in both Saccharomyces cerevisiae cells and human diploid fibroblasts. Our results support the notion that Tax aberrantly targets and activates APC(Cdc20p), leading to unscheduled degradation of Pds1p/securin and Clb2p/cyclin B1, a delay or failure in mitotic entry and progression, and faulty chromosome transmission. The chromosomal instability resulting from a Tax-induced deficiency in securin and cyclin B1 provides an explanation for the highly aneuploid nature of adult T-cell leukemia cells.     

p21-Mediated nuclear retention of cyclin B1-Cdk1 in response to genotoxic stress

G2 arrest of cells suffering DNA damage in S phase is crucial to avoid their entry into mitosis, with the concomitant risks of oncogenic transformation. According to the current model, signals elicited by DNA damage prevent mitosis by inhibiting both activation and nuclear import of cyclin B1-Cdk1, a master mitotic regulator. We now show that normal human fibroblasts use additional mechanisms to block activation of cyclin B1-Cdk1. In these cells, exposure to nonrepairable DNA damage leads to nuclear accumulation of inactive cyclin B1-Cdk1 complexes. This nuclear retention, which strictly depends on association with endogenous p21, prevents activation of cyclin B1-Cdk1 by Cdc25 and Cdk-activating kinase as well as its recruitment to the centrosome. In p21-deficient normal human fibroblasts and immortal cell lines, cyclin B1 fails to accumulate in the nucleus and could be readily detected at the centrosome in response to DNA damage. Therefore, in normal cells, p21 exerts a dual role in mediating DNA damage-induced cell cycle arrest and exit before mitosis. In addition to blocking pRb phosphorylation, p21 directly prevents mitosis by inactivating and maintaining the inactive state of mitotic cyclin-Cdk complexes. This, with subsequent degradation of mitotic cyclins, further contributes to the establishment of a permanent G2 arrest.     

Nuclear Localization of Cyclin B1 Controls Mitotic Entry After DNA Damage

Mitosis in human cells is initiated by the protein kinase Cdc2-cyclin B1, which is activated at the end of G2 by dephosphorylation of two inhibitory residues, Thr14 and Tyr15. The G2 arrest that occurs after DNA damage is due in part to stabilization of phosphorylation at these sites. We explored the possibility that entry into mitosis is also regulated by the subcellular location of Cdc2-cyclin B1, which is suddenly imported into the nucleus at the end of G2. We measured the timing of mitosis in HeLa cells expressing a constitutively nuclear cyclin B1 mutant. Parallel studies were performed with cells expressing Cdc2AF, a Cdc2 mutant that cannot be phosphorylated at inhibitory sites. Whereas nuclear cyclin B1 and Cdc2AF each had little effect under normal growth conditions, together they induced a striking premature mitotic phenotype. Nuclear targeting of cyclin B1 was particularly effective in cells arrested in G2 by DNA damage, where it greatly reduced the damage-induced G2 arrest. Expression of nuclear cyclin B1 and Cdc2AF also resulted in significant defects in the exit from mitosis. Thus, nuclear targeting of cyclin B1 and dephosphorylation of Cdc2 both contribute to the control of mitotic entry and exit in human cells.     

Induction of G2/M phase arrest by squamocin in chronic myeloid leukemia (K562) cells

Squamocin is one of the annonaceous acetogenins and has been reported to have anticancer activity. Squamocin was found to inhibit the growth of K562 cells in a time- and dose-dependent manner. Cell cycle analysis showed G2/M phase arrest in K562 cells following 24 h exposure to squamocin. During the G2/M arrest, cyclin-dependent kinase inhibitors (CDKIs), p21 and p27 were increased in a dose-dependent manner. Analysis of the cell cycle regulatory proteins demonstrated that squamocin did not change the steady-state levels of Cdk2, Cdk4, cyclin A, cyclin B1, cyclin D3 and cyclin E, but decreased the protein levels of Cdk1 and Cdc25C. These results suggest that squamocin inhibits the proliferation of K562 cells via G2/M arrest in association with the induction of p21, p27 and the reduction of Cdk1 and Cdc25C kinase activities.     

Cyclic AMP delays G2 progression and prevents efficient accumulation of cyclin B1 proteins in mouse macrophage cells

In mouse macrophage cells, the increase of the intracellular cAMP level activates protein kinase A (PKA) and results in inhibition of cell cycle progression in both G1 and G2/M phases. G1 arrest is mediated by a cdk inhibitor, p27Kip1, which prevents G1 cyclin/cdk complexes from being activated in response to colony stimulating factor-1, whereas inhibition of G2/M progression has not been fully elucidated. In this report we analyzed the effect of cAMP on G2/M progression in a mouse macrophage cell line, BAC1.2F5A. Flow cytometric analysis and mitotic index measurement using both synchronized and asynchronized cells revealed that addition of cAMP-elevating agents (8-bromoadenosine 3':5'-cyclic monophosphate and 3-isobutyl-methyl-xanthine), although they did not affect S phase progression or M/G1 transition, temporarily arrested cells in G2 but eventually the cells proceeded to M phase, resulting in about 4 hours delay of G2 progression. Timing of cyclin B1/Cdc2 kinase activation was also retarded by about 4 hours, which was accompanied by inhibition of efficient accumulation of cyclin B1 proteins. Initial induction and accumulation of cyclin B1 mRNA were not hampered, but the half life of cyclin B1 proteins was significantly shorter during G2 phase in the presence of cAMP-elevating agents compared with that of the cells blocked from progressing through M phase by nocodazole. These results imply that the cAMP/PKA pathway regulates G2 phase progression by altering the stability of a crucial cell cycle regulator.     

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.     

Human Parvovirus B19 Induces Cell Cycle Arrest at G2 Phase with Accumulation of Mitotic Cyclins

Human parvovirus B19 infects specifically erythroid progenitor cells, which causes transient aplastic crises and hemolytic anemias. Here, we demonstrate that erythroblastoid UT7/Epo cells infected with B19 virus fall into growth arrest with 4N DNA, indicating G(2)/M arrest. These B19 virus-infected cells displayed accumulation of cyclin A, cyclin B1, and phosphorylated cdc2 and were accompanied by an up-regulation in the kinase activity of the cdc2-cyclin B1 complex, similar to that in cells treated with the mitotic inhibitor. However, degradation of nuclear lamina and phosphorylation of histone H3 and H1 were not seen in B19 virus-infected cells, indicating that the infected cells do not enter the M phase. Accumulation of cyclin B1 was persistently localized in the cytoplasm, but not in the nucleus, suggesting that B19 virus infection of erythroid cells raises suppression of nuclear import of cyclin B1, resulting in cell cycle arrest at the G(2) phase. The B19 virus-induced G(2)/M arrest may be the critical event in the damage of erythroid progenitor cells seen in patients with B19 virus infection.     

Geldanamycin induces cell cycle arrest in K562 erythroleukemic cells

Geldanamycin (GA), a benzoquinone ansamycin, is one of the specific inhibitors of 90-kDa heat shock protein and induces growth inhibition and apoptosis in certain cancer cell lines. We have investigated the mechanism of GA-induced growth inhibition in K562 erythroleukemic cells. DNA flow-cytometric analysis indicated that GA-induced growth arrest was associated with G2/M phase arrest of the cell cycle. GA treatment down-regulated the expression of cyclin B1 and inhibited phosphorylation of Cdc2 protein, both key regulatory proteins at the G2/M boundary. GA also markedly inhibited the Cdc2 kinase activity, which may be in part a result of up-regulation of p27KIP1 by GA. The present results suggest a novel mechanism that p27KIP1 could be involved in the regulation of G2 to M phase transition.     

In vitro effects of 2-methoxyestradiol on cell morphology and Cdc2 kinase activity in SNO oesophageal carcinoma cells

The effects of 1 x 10(-6) M exogenous 2-methoxyestradiol (2 ME) were determined on cell morphology and cell division cycle (Cdc) 2 kinase activity in SNO oesophageal carcinoma cells. Mitotic indices revealed an increase in metaphase cells (11.2%) when compared to the 0.5% vehicle-treated cells after 18 h of exposure to 2 ME. Vehicle-treated control cells did not show any hallmarks of apoptosis after 18 h of exposure to dimethyl sulphoxide. Only 0.5% of 2 ME-treated cells showed characteristics of apoptosis. Conversely, increased morphological hallmarks of apoptosis were observed in SNO-treated cells after 21.5 h of 2 ME exposure. When compared to the 0.5% in vehicle-treated cells, 4.7% of cells were in apoptosis. Furthermore, 34.1% of cells were blocked in metaphase after 21.5 h of 2 ME exposure compared to 0.6% of vehicle-control cells. In addition, Cdc2 kinase activity was statistically significantly increased (1.3-fold) (p<0.005) in 2 ME-treated cells when compared to vehicle-treated controls. The present preliminary study suggests that the accumulation observed in metaphase cells and the increase in Cdc2 kinase activity caused by 2 ME are consistent with morphological hallmarks of mitotic arrest and disrupted mitotic spindle formation, thus leading to induction of apoptosis in SNO cells.     

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.