All-trans retinoic acid (ATRA)-induced apoptosis is preceded by G1 arrest in human MCF-7 breast cancer cells.

In this study the effects of all-trans retinoic acid (ATRA) on cell cycle and apoptosis of MCF-7 human breast cancer cells were investigated to elucidate the mechanisms underlying the antineoplastic potential of this retinoid in breast cancer. The antiproliferative effect of ATRA was evaluated by DNA content measurements and dual-parameter flow cytometry of bromodeoxyuridine (BrdU) incorporation and of the expression of cell cycle-related proteins (Ki-67 as proliferation marker and statin as quiescence marker) vs DNA content. Apoptosis was also studied by flow cytometry of either DNA content or Annexin V labelling. After 10(-6) M ATRA treatment, the fraction of S-phase cells decreased significantly, and cells accumulated in the G0/G1 range of DNA contents. Dual-parameter flow cytograms showed a decrease in the percentage of Ki-67-labelled cells (after 10 days, only 20% of the cells were still positive for Ki-67 compared with 95% in controls), while the fraction of statin-positive cells increased slightly. From 3 days of treatment onwards, apoptosis was found to occur. These results show that ATRA-induced inhibition of MCF-7 cell growth is related to two mechanisms, i.e. the block of cell proliferation, mostly in a pre-S phase, and the induction of apoptosis. These results should be taken into account when attempting to design treatment programmes that associate ATRA with antineoplastic compounds of different cell cycle specificity.     

p53/p21(WAF1/CIP1) expression and its possible role in G1 arrest and apoptosis in ellagic acid treated cancer cells.

Ellagic acid is a phenolic compound present in fruits and nuts including raspberries, strawberries and walnuts. It is known to inhibit certain carcinogen-induced cancers and may have other chemopreventive properties. The effects of ellagic acid on cell cycle events and apoptosis were studied in cervical carcinoma (CaSki) cells. We found that ellagic acid at a concentration of 10(-5) M induced G arrest within 48 h, inhibited overall cell growth and induced apoptosis in CaSki cells after 72 h of treatment. Activation of the cdk inhibitory protein p21 by ellagic acid suggests a role for ellagic acid in cell cycle regulation of cancer cells.     

p21WAF1/CIP1 antisense therapy radiosensitizes human colon cancer by converting growth arrest to apoptosis

Substantial evidence suggests that loss of cellular p21WAF1/CIP1 results in increased apoptotic killing by ionizing radiation. We hypothesized that a p21 antisense (AS) oligodeoxynucleotide (ODN) could be used to sensitize cancer cells to radiotherapy. In vitro treatment of colon cancer cells (HCT116/p21+/+) with p21 AS ODN (200 nM) led to inhibition of radiation-induced p21 expression (>95% inhibition, 0-30 Gy), resulting in a loss of G1 arrest and an enhancement of apoptosis to comparable levels and with similar kinetics to HCT116/p21-/- cells (approximately 60% apoptotic cells at 96 h after 10 Gy). In vivo, p21 AS ODN in combination with radiation (i.p. ODN for 6 days at 20 mg/kg/day and 15 Gy) increased apoptosis in s.c. p21+/+ tumors in nude mice to levels similar to those of p21-/- tumors (2-fold at 24 h postirradiation) and improved radiocurability of p21+/+ tumors to levels comparable to those of p21-/- tumors (p21+/+, two of eight cures versus p21-/-, two of nine cures). Our findings suggest that p21 AS treatment may be a rational approach to improve conventional radiotherapy outcomes.     

Adriamycin induced G2/M cell cycle arrest in transitional cell cancer cells with wt p53 and p21(WAF1/CIP1) genes

Adriamycin (ADM), widely used for systemic and local treatment of bladder tumors, triggers apoptosis in bladder cancer cells. Here we investigated the effect of ADM on cell cycle progression and expression of cell cycle regulating proteins in bladder cancer cell lines with various p53 and p21(WAF1/CIP1) status. Flowcytometric analysis was used to estimate the cell cycle distribution of T24, HT-1376, RT4, and SCaBER bladder cancer cell lines. Cell cycle regulating proteins were analyzed by Immunoblot. Treatment of RT4 cells, bearing wild type p53 and p21(WAF1/CIP1), with ADM induced expression of both proteins and cell cycle arrest, not in G1, as was anticipated, but in the G2 phase. Simultaneously, Retinoblastoma (Rb) protein expression was decreased. Expression of PCNA, which is a target gene of E2F, was not changed. The results suggest that even if the tumor cells bear wild type (wt) p53 and wt p21(WAF1/CIP1) and both proteins accumulate due to genotoxic stimuli, the cell cycle arrest might happen not in the G1 but in the G2 phase.     

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.     

丁酸钠诱导MCF-7细胞凋亡过程中端粒酶活性的变化

目的　研究丁酸钠诱导MCF 7细胞凋亡过程中端粒酶活性变化及其机制。方法　采用MTT法和倒置相差显微镜观察不同浓度丁酸钠对MCF 7细胞的抑制作用 ,流式细胞仪和琼脂糖凝胶电泳检测 2 .5mmol/L丁酸钠作用后的细胞凋亡情况 ,TRAP ELISA法检测端粒酶活性变化 ,RT PCR分析端粒酶各组分的mRNA表达情况。结果　丁酸钠对MCF 7细胞的抑制作用具有时间和剂量依赖性 ,AnnexinV/PI双染法显示 ,2 .5mmol/L丁酸钠作用 72h后 ,细胞凋亡率为 84 .3% ,琼脂糖凝胶电泳可见间隔为 180bp的DNA梯状条带。 2 .5mmol/L丁酸钠作用 2 4和 4 8h后 ,端粒酶活性分别下降为1.82± 0 .2 2和 1.6 1± 0 .0 9。RT PCR显示 ,端粒酶逆转录酶 (hTERT)表达下降 ,而端粒酶RNA模板(hTR)和端粒酶相关蛋白 (hTP1)表达无明显改变。结论　丁酸钠诱导MCF 7细胞凋亡过程中端粒酶活性下降 ,其机制可能与丁酸钠下调hTERT转录水平有关。     

Induction of cell cycle arrest and p21(CIP1/WAF1) expression in human lung cancer cells by isoliquiritigenin

Isoliquiritigenin is a natural flavonoid isolated from licorice, shallot and bean sprouts. The effect of isoliquiritigenin on cell proliferation and cell cycle progression was examined in the A549 human lung cancer cell line. Isoliquiritigenin significantly inhibited the proliferation of lung cancer cells in a dose- and time-dependent manner. Flow cytometric analysis demonstrated that isoliquiritigenin restrained the cell cycle progression at G2/M phase. Further examinations using cDNA arrays and real-time quantitative RT-PCR revealed that isoliquiritigenin enhanced the expression of p21(CIP1/WAF1), a universal inhibitor of cyclin-dependent kinases. These results suggest that isoliquiritigenin will be a promising agent for use in chemopreventive or therapeutics against lung cancer.     

POST-TRANSCRIPTIONAL REGULATION OF P21~(WAF1/CIP1) BY P53

The tumor suppressive activity of p53 protein is mediated at least in part through the induction of p21WAF1/CIP1 gene expression leading to cell cycle arrest. The p21WAF1/CIP1 gene codes a 21-kd protein that is known as Waf1, Cip1, mda-6 or Sdi1. It is now known that p21WAF1/CIP1 plays a role in a variety of other biological processes such as differentiation and DNA repair. p21WAF1/CIP1 is induced by p53 following DNA damage and is capable of inhibiting cdks that are active in dif…     

硒对乳腺癌细胞株p21^WAF1/CIP1启动子的调控作用

目的:研究硒对p21的转录调控及其调控位点.方法:通过向转染了重组质粒pGL3- p21p的乳腺癌细胞株MCF7先后加入不同的p21因子启动子的负调节因子和乳酸硒,对比分析荧光素酶表达活性,以确定硒对p21的转录调控及调控位点,并验证硒对癌细胞的生长的负调控作用.结果:perifosine、depsipeptide、apicidin、butyrate与硒共同诱导荧光素酶,酶活性表达无显著差异;而C-Myc与醋酸硒先后诱导酶活性表达差异显著.结论:硒对癌细胞具有诱导凋亡的作用,转录调节位点在p21启动子的sp1结合位点.     

Unique induction of p21(WAF1/CIP1)expression by vinorelbine in androgen-independent prostate cancer cells

To study the mechanisms of the development of hormone refractory prostate cancer, we established an androgen-independent (AI) prostate cancer cell line derived from hormone-dependent (AD) LNCaP cells. Our previous studies have demonstrated that AI cells are deficient in expression of p21(WAFl/CIP1) (p21) due to overexpressed AR and are resistant to apoptosis. In this study, the induction of p53 and p21 expression by vinorelbine (Navelbine) was compared between AD and AI cells in an attempt to understand the difference(s) in apoptotic signalling pathways in these cells. Using a series of deletion of p21 reporter constructs, we found that vinorelbine mediated p21 induction in a p53-dependent manner in AD cells. In contrast, p21 expression restored by vinorelbine in AI cells was found to be through both p53-dependent and-independent pathways. In the absence of two p53 binding sites, Spl-3 and Spl-4 sites, in the promoter of human p21 gene, were found to be required for vinorelbine-mediated p21 activation. No p21 induction was observed by paclitaxel in AI cells. Exposure of AI cells to paciltaxel followed by vinorelbine produced synergism. Our data, thus, provide a basis for the synergistic combination of vinorelbine and paclitaxel for the treatment of advanced prostate cancer.     

P21(WAF1/CIP1) may be a tumor suppressor after all

The cyclin-dependent kinase inhibitor p21WAF1/CIP1 was originally considered to be a tumor-suppressor because it was identified as a key mediator of p53-dependent cell cycle arrest. However, it has been suggested that p21 may also act as an oncogene because it often inhibits apoptosis. For example, deletion of p21 from p53-deficient mice resulted in longer survival and in a significantly reduced number of thymic lymphomas that was explained by higher apoptotic rates in these mice. However, recently it has been shown that a p53 mutant that had lost its ability to induce apoptosis, but retained its ability to induce p21 and cell cycle arrest, was able to suppress lymphomagenesis in different cancer models. Tumor suppression by this p53 mutant was modulated by p21, which induced senescence and preserved chromosomal stability. These data suggest that the ability of p21 to induce cell-cycle arrest may lead to tumor suppression in some types of cancer.     

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.     

Evidence of a functional role for the cyclin-dependent kinase-inhibitor p21WAF1/CIP1/MDA6 in promoting differentiation and preventing mitochondrial dysfunction and apoptosis induced by sodium butyrate in human myelomonocytic leukemia cells (U937).

The impact of dysregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1/MDA6 has been examined in U937 human monocytic leukemia cells in relation to cell cycle arrest and differentiation following treatment with the histone deacetylase inhibitor sodium butyrate (SB). Cells stably transfected with a p21WAF1/CIP1/MDA6 antisense construct, in marked contrast to their wild-type counterparts, failed to up-regulate p21WAF1/CIP1/MDA6, undergo G1 arrest, or express the maturation marker CD11b when exposed to 1 or 3 mM SB. However, antisense-expressing cells were significantly more susceptible to SB-mediated mitochondrial injury and apoptosis, manifested by increased cytosolic translocation of cytochrome c, activation of pro-caspase 3, and degradation of PARP. Dysregulation of p21WAF1/CIP1/MDA6 did not modify the extent of SB-induced histone acetylation, but did result in cleavage of p27KIP1, Bcl-2 and pRb, as well as diminished levels of full-length underphosphorylated pRb. Finally, dysregulation of p21WAF1/CIP1/MDA6 did not modify SB-mediated down-regulation of E2F-1 or c-Myc, but was associated with enhanced down-regulation of cyclins D1 and E. Together, these findings indicate that in U937 leukemia cells, p21WAF1/CIP1/MDA6 plays a critical functional role in SB-mediated G1 arrest and maturation, and suggest that cells displaying dysregulation of this CDKI respond to SB by engaging a default apoptotic program.     

TGF-beta-induced cell-cycle arrest through the p21(WAF1/CIP1)-G1 cyclin/Cdks-p130 pathway in gastric-carcinoma cells.

Transforming growth factor-beta1 (TGF-beta) inhibits cell-cycle progression of many types of cells by arresting them in G(1)/S phase through inhibition of the active cyclin-Cdk complexes that lead to inhibition of Rb phosphorylation. In gastric-cancer cells, SNU16, TGF-beta treatment induced enhanced expression of p21(WAF1/CIP1) (p21), which inhibited the kinase activity of cyclin-D- and cyclin-E-associated Cdks and blocked p130 phosphorylation. TGF-beta also enhanced the stability of p130, suggesting that hypophosphorylation of p130 and increased stability of p130 contribute to p130-mediated G(1) arrest in gastric-cancer cells. Our results demonstrate that p21 and p130 are major downstream targets of TGF-beta in gastric-cancer cells and that a p21-G(1) cyclin/Cdks-p130/E2F pathway mediates growth inhibition by TGF-beta in these cells.