Interferon Regulatory Factor 1 (IRF-1) induces p21(WAF1/CIP1) dependent cell cycle arrest and p21(WAF1/CIP1) independent modulation of survivin in cancer cells

We have shown that the ectopic expression of Interferon Regulatory Factor 1 (IRF-1) results in human cancer cell death accompanied by the down-regulation of the Inhibitor of Apoptosis Protein (IAP) survivin and the induction of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1). In this report, we investigated the direct role of p21 in the suppression of survivin. We show that IRF-1 down-regulates cyclin B1, cdc-2, cyclin E, E2F1, Cdk2, Cdk4, and results in p21-mediated G1 cell cycle arrest. Interestingly, while p21 directly mediates G1 cell cycle arrest, IRF-1 or other IRF-1 signaling pathways may directly regulate survivin in human cancer cells.     

Differential sensitivity of normal and malignant breast epithelial cells to genistein is partly mediated by p21(WAF1).

Genistein, a soy metabolite, is a potential chemopreventive agent against various types of cancer. There are several studies documenting molecular alterations leading to cell cycle arrest and induction of apoptosis in a variety of cancer cells; however, no studies, to date, have shown the effect of genistein in isogenic normal and malignant breast epithelial cells. In this study, we investigated whether genistein shows any differential sensitivity to normal (MCF10A and MCF12A) and malignant (MCF10CA1a and MDA-MB-231) breast epithelial cells. We found that genistein causes a greater degree of G(2)-M arrest and induces apoptosis in malignant cell lines compared with normal breast epithelial cells. After genistein treatment, flow cytometric analysis revealed a hyperdiploid population in malignant cells that was not observed in normal cells. Cell cycle regulator p21(WAF1), which is known to be up-regulated by genistein treatment, was greatly induced at RNA and protein levels in normal cells, whereas its level was only slightly induced in malignant MDA-MB-231 cells and not detectable in malignant MCF10CA1a cells. Therefore, we investigated the causal role of p21(WAF1) in the differential sensitivity of genistein among these cell lines. We examined the effects of genistein on p21(WAF1) -/- and p21(WAF1) +/+ HCT116 cells, which were used as controls prior to studies on breast cancer cells. We found that there was a greater degree of cell cycle arrest and apoptosis in p21(WAF1) -/- cells compared with p21(WAF1) +/+ HCT116 cells after genistein treatment. Flow cytometric analysis after genistein treatment showed a significant number of p21(WAF1) -/- cells in the hyperdiploid population, which are probably programmed to die through apoptotic processes. To further confirm the causal role of p21(WAF1) in genistein-mediated cell cycle arrest and apoptosis, we down-regulated p21(WAF1) by antisense p21(WAF1) cDNA transfection experiments. We found that both normal and malignant p21(WAF1) antisense (AS)-expressing clones became more sensitive to G(2)-M arrest after genistein treatment. Flow cytometric analysis showed an increase in the hyperdiploid population in the AS clones. Further evaluation showed an increase in apoptosis in malignant AS clones but not in normal breast epithelial AS clones. These results suggest that p21(WAF1) may play an important role in determining the sensitivity of normal and malignant breast epithelial cells to genistein.     

Galectin-3 mediates genistein-induced G(2)/M arrest and inhibits apoptosis

Many recent studies have focused on potential chemopreventive activities of dietary genistein, a natural isoflavonoid compound found in soy products. Genistein has been implicated in anticancer activities, including differentiation, apoptosis, inhibition of cell growth and inhibition of angiogenesis. In previous studies, genistein was shown to induce apoptosis and cell cycle arrest at G(2)/M in several cancer cell lines in vitro, which is associated with induction of p21(WAF1/CIP1), a universal inhibitor of cyclin-dependent kinases. At present, the molecular basis for diverse genistein-mediated cellular responses is largely unknown. In the present study, we investigated whether galectin-3, an anti-apoptotic gene product, regulates genistein-mediated cellular responses. We show that genistein effectively induces apoptosis without detectable cell cycle arrest in BT549, a human breast epithelial cell line which does not express galectin-3 at a detectable level. In galectin-3 transfected BT549 cells, genistein induced cell cycle arrest at the G(2)/M phase without apoptosis induction. Interestingly, genistein induces p21(WAF1/CIP1) expression in galectin-3-expressing BT549 cells, but not in control BT549 cells undergoing apoptosis. Collectively, the results of the present study suggest that galectin-3, at least in part, is a critical determinant for genistein-mediated cell cycle arrest and apoptosis, and genistein induction of p21(WAF1/CIP1) is associated with cell cycle arrest, but not required for apoptosis induction.     

Phenoxodiol, a novel isoflavone, induces G1 arrest by specific loss in cyclin-dependent kinase 2 activity by p53-independent induction of p21WAF1/CIP1

Phenoxodiol, an isoflavone derivative of genistein with unknown mechanism of action, is currently being evaluated in early human cancer clinical trials. To determine the mechanism of antiproliferative effects of phenoxodiol, we examined its effects in a battery of human cell lines. Although we observed caspase-dependent apoptosis in HN12 cells as early as 24 hours after exposure, clonogenic death occurred only after 48-hour exposure despite caspase blockade by the general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (ZVAD)-fmk. Moreover, clear evidence of cell death as determined by nuclear morphology and plasmatic membrane damage occur despite ZVAD, suggesting that another mechanism besides caspase-dependent apoptosis is required for clonogenic death induced by phenoxodiol. In search for other potential antiproliferative effects, we assessed the effects of phenoxodiol in the cell cycle progression of human carcinoma cell lines. A significant G(1)-S arrest was observed by 12 hours of exposure in HN12 cell lines at concentrations > or =5 microg/mL. Cell cycle arrest occurred several hours (approximately 12 hours) before induction of apoptosis. Analysis of in vitro purified cyclin-dependent kinase (cdk) activity showed that phenoxodiol did not inhibit cdk activity. In contrast, cellular cdk2 activity obtained from HN12 cell lines exposed to phenoxodiol for 12 hours decreased by 60%, whereas cdk6 activity remained unaltered, suggesting that the loss of cdk2 activity was specific. Loss in cdk2 activity was preceded by the accumulation of the endogenous cdk inhibitor p21(WAF1). To assess the role of p21(WAF1) induction by phenoxodiol, we used HCT116 isogenic cell lines and showed that phenoxodiol induced G(1) arrest together with p21(WAF1) expression in wild-type clones. In contrast, p21(-/-) variants failed to show G(1) arrest. Finally, induction of p21 by phenoxodiol is p53 independent, as phenoxodiol induced p21 in HCT116 lacking p53. These data therefore indicate that phenoxodiol promotes G(1)-S arrest by the specific loss in cdk2 activity due to p53-independent p21(WAF1) induction. This novel feature of phenoxodiol may have clinical implications, as the majority of human malignancies have aberrations in cell cycle progression regulation.     

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.     

Genistein induced molecular changes in a squamous cell carcinoma of the head and neck cell line

Epidemiological studies have shown lower incidence of breast and prostate cancers in Asian populations consuming a traditional diet rich in soy. Protection from these cancers was attributed to the isoflavones, particularly genistein and daidzein found in vivo as the major metabolites of soy isoflavones. However, the role of isoflavones in head and neck cancer is less clear. In our previous studies we reported that genistein can induce cell growth inhibition by arresting the cells at S/G2-M phases, and also induces apoptosis in HN4 squamous cell carcinoma of the head and neck cell line (HNSCC). In this report we show that these changes are accompanied by the down-regulation of Cdk1, and CyclinB1, and up-regulation of the cyclin dependent kinase (Cdk) inhibitor p21WAF1, which may be responsible for the induction of cell cycle arrest and apoptosis. The evidence for the induction of apoptosis was supported by the appearance of DNA ladder as reported previously, and further supported by our current results on the cleavage of poly-ADP-ribose polymerase (PARP), hallmark of apoptosis. This was also accompanied by the up-regulation of Bax, with modest down-regulation of Bcl-2 protein expression, which changes the balance between pro- and anti-apoptosis molecules in favor of pro-apoptosis. Furthermore, we also observed down-regulation and degradation of Cdc25C, which is a marker of cell proliferation, and plays important role in CyclinB-Cdk1 complex activation. The down-regulation followed by the degradation of Cdc25C is an indicator of G2/M arrest and anti-proliferation effects of genistein. Collectively, these data provide strong molecular evidence for the anti-tumor activity of genistein in HNSCC cells.     

Apigenin induces cell cycle arrest and p21/WAF1 expression in a p53-independent pathway

Apigenin, a common dietary flavonoid, has been shown to induce cell growth-inhibition and cell cycle arrest in many cancer cell lines. One important effect of apigenin is to increase the stability of the tumor suppressor p53 in normal cells. Therefore, apigenin is expected to play a large role in cancer prevention by modifying the effects of p53 protein. However, the mechanisms of apigenin's effects on p53-mutant cancer cells have not been revealed yet. We assessed the influence of apigenin on cell growth and the cell cycle in p53-mutant cell lines. Treatment with apigenin resulted in growth-inhibition and G2/M phase arrest in two p53-mutant cancer cell lines, HT-29 and MG63. These effects were associated with a marked increase in the protein expression of p21/WAF1. We have shown that p21/WAF1 mRNA expression was also markedly increased by treatment with apigenin in a dose- and time-dependent manner. However, we could not detect p21/WAF1 promoter activity following treatment with apigenin. Similarly, promoter activity from pG13-Luc, a p53-responsive promoter plasmid, was not activated by treatment with apigenin with or without p53 protein expression. These results suggest that there is a p53-independent pathway for apigenin in p53-mutant cell lines, which induces p21/WAF1 expression and growth-inhibition. Apigenin may be a useful chemopreventive agent not only in wild-type p53 status, but also in cancer with mutant p53.     

Diallyl trisulfide-induced G(2)-M phase cell cycle arrest in human prostate cancer cells is caused by reactive oxygen species-dependent destruction and hyperphosphorylation of Cdc 25 C

Molecular mechanism of cell cycle arrest caused by diallyl trisulfide (DATS), a garlic-derived cancer chemopreventive agent, has been investigated using PC-3 and DU 145 human prostate cancer cells as a model. Treatment of PC-3 and DU 145 cells, but not a normal prostate epithelial cell line (PrEC), with growth suppressive concentrations of DATS caused enrichment of the G(2)-M fraction. The DATS-induced cell cycle arrest in PC-3 cells was associated with increased Tyr(15) phosphorylation of cyclin-dependent kinase 1 (Cdk 1) and inhibition of Cdk 1/cyclinB 1 kinase activity. The DATS-treated PC-3 and DU 145 cells also exhibited a decrease in the protein level of Cdc 25 C and an increase in its Ser(216) phosphorylation. The DATS-mediated decrease in protein level and Ser(216) phosphorylation of Cdc 25 C as well as G(2)-M phase cell cycle arrest were significantly attenuated in the presence of N-acetylcysteine implicating reactive oxygen species (ROS) in cell cycle arrest caused by DATS. ROS generation was observed in DATS-treated PC-3 and DU 145 cells. DATS treatment also caused an increase in the protein level of Cdk inhibitor p21, but DATS-induced G(2)-M phase arrest was not affected by antisense-mediated suppression of p21 protein level. In conclusion, the results of the present study indicate that DATS-induced G(2)-M phase cell cycle arrest in human prostate cancer cells is caused by ROS-mediated destruction and hyperphosphorylation of Cdc 25 C.     

Up-regulation of p21WAF1/Cip1 by saRNA induces G1-phase arrest and apoptosis in T24 human bladder cancer cells

Very recent studies have reported that chemically synthesized small duplex RNAs complementary to the promoters of target genes can activate gene expression in different cancer cell lines. Such dsRNA have been referred to as saRNA for small activating RNA. The present study was conducted to evaluate the potential of p21(WAF1/Cip1) (p21) induction by small activating RNA targeting the p21 promoter in the treatment of bladder cancer. Using T24 human bladder cancer cells, we found that p21 saRNA caused dose- and time-dependent inhibition of cell proliferation and viability which was associated with induced G1-phase cell cycle arrest and apoptosis. The decreased anti-apoptotic protein Bcl-xL and activation of caspase-3 and PARP also supported the efficacy of the treatment. These data suggest that up-regulation of p21 by saRNA may be an effective way for treating human bladder and other types of cancers.     

Downregulation of c-Jun expression and cell cycle regulatory molecules in acute myeloid leukemia cells upon CD44 ligation

In the present study, we investigated the mechanism of CD44 ligation with the anti-CD44 monoclonal antibody A3D8 to inhibit the proliferation of human acute myeloid leukemia (AML) cells. The effects of A3D8 on myeloid cells were associated with specific disruption of cell cycle events and induction of G0/G1 arrest. Induction of G0/G1 arrest was accompanied by an increase in the expression of p21, attenuation of pRb phosphorylation and associated with decreased Cdk2 and Cdk4 kinase activities. Since c-Jun is an important regulator of proliferation and cell cycle progression, we analysed its role in A3D8-mediated growth arrest. We observed that A3D8 treatment of AML patient blasts and HL60/U937 cells led to the downregulation of c-Jun expression at mRNA and protein level. Transient transfection studies showed the inhibition of c-jun promoter activity by A3D8, involving both AP-1 sites. Furthermore, A3D8 treatment caused a decrease in JNK protein expression and a decrease in the level of phosphorylated c-Jun. Ectopic overexpression of c-Jun in HL60 cells was able to induce proliferation and prevent the antiproliferative effects of A3D8. In summary, these data identify an important functional role of c-Jun in the induction of cell cycle arrest and proliferation arrest of myeloid leukemia cells because of the ligation of the cell surface adhesion receptor CD44 by anti-CD44 antibody. Moreover, targeting of G1 regulatory proteins and the resulting induction of G1 arrest by A3D8 may provide new insights into antiproliferative and differentiation therapy of AML.     

Genetic status of p53 and induction of apoptosis by radiation or isoflavones in human gastric carcinoma cell lines

We have shown previously that various human cancer cell lines undergo morphological changes and internucleosomal DNA fragmentation characteristic of apoptosis after exposure to ionizing radiation or isoflavones. Here, we assessed the role of p53 gene in cell cycle and apoptosis following treatment of 11 gastric carcinoma cell lines with gamma-rays, genistein, biochanin A, or daidzein. Cell survival was measured by trypan blue staining, and apoptosis was assessed by fluorochrome staining. The rate of cell survival and apoptosis of the cells by gamma-irradiation or isoflavones did not correlate with p53 gene abnormalities. Flow cytometric measurement of DNA content demonstrated that while gamma-irradiation and genistein induced G(2) arrest, biochanin A and daidzein blocked the cell cycle of all carcinoma cells at G(1) phase. At multiple time points following irradiation, G(2) arrest was observed at 12-16 h in the wild-type and mutant p53 cell lines. Induction of p53 and p21 proteins was not observed in wild-type p53 lines after exposure to gamma-irradiation or isoflavones by Western blotting. Moreover, transfection of the wild-type p53 gene into MKN-1 cells failed to induce G(1) arrest by gamma-irradiation and genistein. Based on these results, we hypothesize that gastric cancer cells may possess a signal pathway which is different from the usual mechanisms of the p53-mediated DNA damage response in normal or hematopoietic tumor cells.     

Inhibition of myeloma cell growth by all-trans retinoic acid is associated with upregulation of p21WAF1 and dephosphorylation of the retinoblastoma protein

Retinoic acid and dexamethasone, in combination, inhibit the growth of human myeloma cell lines in a synergistic manner. Previously, we observed that all-trans retinoic acid (ATRA) caused G1 arrest and inhibited clonogenic growth of the OPM-2 human myeloma cell line. This was associated with downregulation of the IL-6 receptor (IL-6R) gp80 protein, while autocrine IL-6 production and gp130 were not affected. Growth inhibition was not reversed by the addition of exogenous IL-6 or forced, constitutive expression of the IL-6 receptor gp80 protein, suggesting that the mechanism of action of ATRA may be due to effects on the post-receptor pathway. Therefore, in this study we have investigated whether growth arrest was associated with changes in the level of phosphorylation of the RB protein. ATRA decreased the level of phosphorylation of the RB protein at doses > 5 x 10(-9) M and also induced a five fold increase in p21WAF1, while levels of p27KIP1 and CDK2 were unchanged. The ATRA-mediated increase in p21 preceded the change in RB phosphorylation and G1 arrest and was not reversed by the addition of exogenous IL-6. The levels of CDK2 activity were inhibited approximately 60% in ATRA-treated cells, suggesting that the increased p21 levels were sufficient to inhibit CDK activity and cause RB hypophosphorylation. Increased levels of p21 have recently been observed in human myeloma cells exposed to dexamethasone, and we suggest that the common ability of these two agents to inhibit myeloma cell growth depends on their induction of p21.     

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.