Radiation response and cell cycle regulation of p53 rescued malignant keratinocytes

Mutations in the tumor suppressor gene p53 were found in more than 90% of all human squamous cell carcinomas (SCC). To study the function of p53 in a keratinocyte background, a tetracycline-controlled p53 transgene was introduced into a human SCC cell line (SCC15), lacking endogenous p53. Conditional expression of wild-type p53 protein upon withdrawal of tetracycline was accompanied with increased expression of p21(WAF1/Cip1) resulting in reduced cell proliferation. Flow-cytometric analysis revealed that these cells were transiently arrested in the G1/S phase of the cell cycle. However, when SCC15 cells expressing p53 were exposed to ionizing radiation (IR), a clear shift from a G1/S to a G2/M cell cycle arrest was observed. This effect was greatly depending on the presence of wild-type p53, as it was not observed to the same extent in SCC15 cells lacking p53. Unexpectedly, the p53- and IR-dependent G2/M cell cycle arrest in the keratinocyte background was not depending on increased expression or stabilization of 14-3-3sigma, a p53-regulated effector of G2/M progression in colorectal cancer cells. In keratinocytes, 14-3-3sigma (stratifin) is involved in terminal differentiation and its cell cycle function in this cell type might diverge from the one it fulfills in other cellular backgrounds.     

Cell cycle arrest and cell death are controlled by p53-dependent and p53-independent mechanisms in Tsg101-deficient cells

Our previous studies have shown that cells conditionally deficient in Tsg101 arrested at the G(1)/S cell cycle checkpoint and died. We created a series of Tsg101 conditional knock-out cell lines that lack p53, p21(Cip1), or p19(Arf) to determine the involvement of the Mdm2-p53 circuit as a regulator for G(1)/S progression and cell death. In this new report we show that the cell cycle arrest in Tsg101-deficient cells is p53-dependent, but a null mutation of the p53 gene is unable to maintain cell survival. The deletion of the Cdkn1a gene in Tsg101 conditional knock-out cells resulted in G(1)/S progression, suggesting that the p53-dependent G(1) arrest in the Tsg101 knock-out is mediated by p21(Cip1). The Cre-mediated excision of Tsg101 in immortalized fibroblasts that lack p19(Arf) seemed not to alter the ability of Mdm2 to sequester p53, and the p21-mediated G(1) arrest was not restored. Based on these findings, we propose that the p21-dependent cell cycle arrest in Tsg101-deficient cells is an indirect consequence of cellular stress and not caused by a direct effect of Tsg101 on Mdm2 function as previously suggested. Finally, the deletion of Tsg101 from primary tumor cells that express mutant p53 and that lack p21(Cip1) expression results in cell death, suggesting that additional transforming mutations during tumorigenesis do not affect the important role of Tsg101 for cell survival.     

Inactivated tumor suppressor Rb by nitric oxide promotes mitosis in human breast cancer cells

Inactivation of tumor suppressor protein retinoblastoma (Rb) is important mechanism for the G1/S transition during cell cycle progression. Human breast cancer cells T47D release great amount of nitric oxide (NO), but its relation to tumor suppressor Rb is unknown. In this study, it is shown that NO induces phosphorylation and inactivation of Rb tumor suppressor protein, increasing G2/M phase and cell proliferation of breast cancer cells T47D. NO did not induce changes in p53 ser-15 phosphorylation, the most phosphorylated site of p53 during its activation. These data indicate that NO induces cell proliferation through the Rb pathway. NO phosphorylates and inactivates tumor suppressor protein Rb inducing mitosis by the p53 independent pathway in breast cancer cell.     

Electron microscopy evidence that cytoplasmic localization of the p16INK4A "nuclear" cyclin-dependent kinase inhibitor (CKI) in tumor cells is specific and not an artifact. A study in non-small cell lung carcinomas

It is well established that p16^INK4A protein acts as a cell cycle inhibitor in the nucleus. Therefore, cytoplasmic localization of p16 ^INK4A usually is disregarded by investigators as nonspecific. Three recent studies reported findings that differ from the current view concerning p16^INK4A immunohistochemical localization. All three demonstrated that breast and colon cancers expressing cytoplasmic p16^INK4 represent distinct biological subsets. We previously detected in a percentage of non-small cell lung carcinomas simultaneous nuclear and cytoplasmic p16^INK4A staining. In view of the reports concerning breast and colon carcinomas, we conducted an ultrastructural re-evaluation of our cases to clarify the specificity of p16^INK4A cytoplasmic expression. We observed p16 ^INK4A immunolocalization in both the nucleus and the cytoplasm of a proportion of tumor cells. Diffuse dense nuclear staining was detected in the nucleoplasm, whereas weaker granular immunoreactivity was observed in the cytoplasm near the rough endoplasmic reticulum. Negative tumor cells also were visible. In the tumor-associated stromal, cells p16^INK4A immunoreactivity was detected only in the nuclei. We have demonstrated that p16^INK4A cytoplasmic staining is specific and suggest that it represents a mechanism of p16^INK4A inactivation similar to that observed in other tumor suppressor genes.     

Effects of Iejimalide B, a marine macrolide, on growth and apoptosis in prostate cancer cell lines

Iejimalide B, a marine macrolide, causes growth inhibition in a variety of cancer cell lines at nanomolar concentrations. We have investigated the effects of Iejimalide B on cell cycle kinetics and apoptosis in the p53+/AR+ LNCaP and p53-/AR- PC-3 prostate cancer cell lines. Iejimalide B, has a dose and time dependent effect on cell number (as measured by crystal violet assay) in both cell lines. In LNCaP cells Iejimalide B induces a dose dependent G0/G1 arrest and apoptosis at 48 h (as measured by Apo-BrdU staining). In contrast, Iejimalide B initially induces G0/G1 arrest followed by S phase arrest but does not induce apoptosis in PC-3 cells. qPCR and Western analysis suggests that Iejimalide B modulates the steady state level of many gene products associated with cell cycle (including cyclins D, E, and B and p21(waf1/cip1)) and cell death (including survivin, p21B and BNIP3L) in LNCaP cells. In PC-3 cells Iejimalide B induces the expression of p21(waf1/cip1), down regulates the expression of cyclin A, and does not modulate the expression of the genes associated with cell death. Comparison of the effects of Iejimalide B on the two cell lines suggests that Iejimalide B induces cell cycle arrest by two different mechanisms and that the induction of apoptosis in LNCaP cells is p53-dependent.     

rBTI通过上调PTEN的表达抑制Hep G2细胞增殖

磷酸酶及张力蛋白的同源基因(PTEN) 是一种抑癌基因,可以调控细胞的增殖,与癌症的发生和发展息息相关。本研究采用MTT法和流式细胞术分别检测了重组荞麦胰蛋白酶抑制剂(rBTI)对人肝癌细胞株Hep G2细胞的增殖以及周期的影响。免疫荧光及Western印迹法检测了PTEN和p PTEN的亚细胞定位及蛋白表达的变化。采用qRT-PCR及Western印迹法检测了周期相关蛋白的表达。旨在探究PTEN和p PTEN在rBTI抑制Hep G2细胞增殖和周期阻滞中的作用。结果表明,rBTI能显著抑制Hep G2细胞增殖,将细胞周期阻滞在G0/G1期,并呈时间和剂量依赖性;rBTI作用于Hep G2后,可显著上调PTEN和p-PTEN的表达。同时发现,p-PTEN主要分布于细胞核中,能与核仁发生共定位;周期相关蛋白检测表明,细胞内p53、p21转录水平和蛋白水平均增加。综上所述,rBTI通过上调PTEN的表达,使得细胞周期阻滞于G0/G1期,进而抑制Hep G2细胞的增殖。     

Kruppel-like factor 4 mediates p53-dependent G1/S cell cycle arrest in response to DNA damage

The tumor suppressor p53 is required for the maintenance of genomic integrity following DNA damage. One mechanism by which p53 functions is to induce a block in the transition between the G(1) and S phase of the cell cycle. Previous studies indicate that the Krüppel-like factor 4 (KLF4) gene is activated following DNA damage and that such activation depends on p53. In addition, enforced expression of KLF4 causes G(1)/S arrest. The present study examines the requirement of KLF4 in mediating the p53-dependent cell cycle arrest process in response to DNA damage. We show that the G(1) population of a colon cancer cell line, HCT116, that is null for the p53 alleles (-/-) was abolished following gamma irradiation compared with cells with wild-type p53 (+/+). Conditional expression of KLF4 in irradiated HCT116 p53-/- cells restored the G(1) cell population to a level similar to that seen in irradiated HCT116 p53+/+ cells. Conversely, treatment of HCT116 p53+/+ cells with small interfering RNA (siRNA) specific for KLF4 significantly reduced the number of cells in the G(1) phase following gamma irradiation compared with the untreated control or those treated with a nonspecific siRNA. In each case the increase or decrease in KLF4 level because of conditional induction or siRNA inhibition, respectively, was accompanied by an increase or decrease in the level of p21(WAF1/CIP1). Results of our study indicate that KLF4 is an essential mediator of p53 in controlling G(1)/S progression of the cell cycle following DNA damage.     

Targeted p21(WAF1/CIP1) Activation by RNAa Inhibits Hepatocellular Carcinoma Cells

RNA activation (RNAa) is a mechanism of gene activation triggered by promoter-targeted small double-stranded RNA (dsRNA), also known as small activating RNA (saRNA). p21(WAF1/CIP1) (p21) is a putative tumor suppressor gene due to its role as a key negative regulator of the cell cycle and cell proliferation. It is frequently downregulated in cancer including hepatocellular carcinoma (HCC), but is rarely mutated or deleted, making it an ideal target for RNAa-based overexpression to restore its tumor suppressor function. In the present study, we investigated the antigrowth effects of p21 RNAa in HCC cells. Transfection of a p21 saRNA (dsP21-322) into HepG2 and Hep3B cells significantly induced the expression of p21 at both the mRNA and protein levels, and inhibited cell proliferation and survival. Further analysis of dsP21-322 transfected cells revealed that dsP21-322 arrested the cell cycle at the G(0)/G(1) phase in HepG2 cells but at G(2)/M phase in Hep3B cells which lack functional p53 and Rb genes, and induced both early and late stage apoptosis by activating caspase 3 in both cell lines. These results demonstrated that RNAa of p21 has in vitro antigrowth effects on HCC cells via impeding cell cycle progression and inducing apoptotic cell death. This study suggests that targeted activation of p21 by RNAa may be explored as a novel therapy for the treatment of HCC.     

Liriodenine induces G1/S cell cycle arrest in human colon cancer cells via nitric oxide- and p53-mediated pathway

Liriodenine is an aporphine alkaloid compound extracted from the leaves of Michelia compressa var. lanyuensis. It had been reported to have an anti-colon cancer effect, but the mechanism remains unclear. In the present study, the antiproliferative mechanisms of liriodenine were investigated in the human colon cancer SW480 cells. Flow cytometry analysis indicated that liriodenine notably induced the G1/S phase arrest. The G1/S phase cycle-related proteins analysis illustrated that the expressions of cyclin-dependent kinase (CDK) 2, CDK4 and CDK6, and of cyclin D1 and A, as well as the phosphorylation of retinoblastoma tumor suppressor protein (ppRB) were found to be markedly reduced by liriodenine, whereas the protein levels of the CDK inhibitors (CKIs), p21 and p27 were increased. Moreover, the intracellular nitric oxide (NO) production, protein levels of inducible NO synthase (iNOS) and, p53 were increased. The p53 overexpression was a downstream event of NO production in liriodenine-induced G1/S-arrested SW480 cells, and the up-regulation of p21 and p27 was found to be mediated by a p53-dependent pathway. The inhibition of p53 by pifithrin-α (PFT-α), down-regulation of p21 and p27 by siRNA, or NO reduction by S-ethylisothiourea (ETU) entirely abolished the liriodenine-induced G1/S phase arrest. We concluded that liriodenine potently inhibited the cell cycle of SW480 cancer cells via NO- and p53-dependent G1/S phase arrest pathway. These results suggest that liriodenine might be a powerful agent against colon cancer.     

Mutant p53 tumor suppressor alleles release ras-induced cell cycle growth arrest.

Overexpression of an activated ras gene in the rat embryo fibroblast line REF52 results in growth arrest at either the G1/S or G2/M boundary of the cell cycle. Both the DNA tumor virus proteins simian virus 40 large T antigen and adenovirus 5 E1a are able to rescue ras induced lethality and cooperate with ras to fully transform REF52 cells. In this report, we present evidence that the wild-type activity of the tumor suppressor gene p53 is involved in the negative growth regulation of this model system. p53 genes encoding either a p53Val-135 or p53Pro-193 mutation express a highly stable p53 protein with a conformation-dependent loss of wild-type activity and the ability to eliminate any endogenous wild-type p53 activity in a dominant negative manner. In cotransfection assays, these mutant p53 genes are able to rescue REF52 cells from ras-induced growth arrest, resulting in established cell lines which express elevated levels of the ras oncoprotein and show morphological transformation. Full transformation, as assayed by tumor formation in nude mice, is found only in the p53Pro-193-plus-ras transfectants. These cells express higher levels of the ras protein than do the p53Val-135-plus-ras-transfected cells. Transfection of REF52 cells with ras alone or a full-length genomic wild-type p53 plus ras results in growth arrest and lethality. Therefore, the selective event for p53 inactivation or loss during tumor progression may be to overcome a cell cycle restriction induced by oncogene overexpression (ras). These results suggest that a normal function of p53 may be to mediate negative growth regulation in response to ras or other proliferative inducing signals.     

The role of p21(CIP1/WAF1) in growth of epithelial cells exposed to hyperoxia

Previous studies have shown that hyperoxia inhibits proliferation and increases the expression of the tumor suppressor p53 and its downstream target, the cyclin-dependent kinase inhibitor p21(CIP1/WAF1), which inhibits proliferation in the G1 phase of the cell cycle. To determine whether growth arrest was mediated through activation of the p21-dependent G1 checkpoint, the kinetics of cell cycle movement during exposure to 95% O2 were assessed in the Mv1Lu and A549 pulmonary adenocarcinoma cell lines. Cell counts, 5-bromo-2'-deoxyuridine incorporation, and cell cycle analyses revealed that growth arrest of both cell lines occurred in S phase, with A549 cells also showing evidence of a G1 arrest. Hyperoxia increased p21 in A549 but not in Mv1Lu cells, consistent with the activation of the p21-dependent G1 checkpoint. The ability of p21 to exert the G1 arrest was confirmed by showing that hyperoxia inhibited proliferation of HCT 116 colon carcinoma cells predominantly in G1, whereas an isogenic line lacking p21 arrested in S phase. The cell cycle arrest in S phase appears to be a p21-independent process caused by a gradual reduction in the rate of DNA strand elongation. Our data reveal that hyperoxia inhibits proliferation in G1 and S phase and demonstrate that p53 and p21 retain their ability to affect G1 checkpoint control during exposure to elevated O2 levels.     

Sp1 plays a critical role in the transcriptional activation of the human cyclin-dependent kinase inhibitor p21(WAF1/Cip1) gene by the p53 tumor suppressor protein

In the present study we present evidence for the critical role of Sp1 in the mechanism of transactivation of the human cell cycle inhibitor p21(WAF1/Cip1) (p21) gene promoter by the tumor suppressor p53 protein. We found that the distal p53-binding site of the p21 promoter acts as an enhancer on the homologous or heterologous promoters in hepatoma HepG2 cells. In transfection experiments, p53 transactivated the p21 promoter in HaCaT cells that express Sp1 but have a mutated p53 form. In contrast, p53 could not transactivate the p21 promoter in the Drosophila embryo-derived Schneider's SL2 cells that lack endogenous Sp1 or related factors. Cotransfection of SL2 cells with p53 and Sp1 resulted in a synergistic transactivation of the p21 promoter. Synergistic transactivation was greatly decreased in SL2 cells and HaCaT cells by mutations in either the p53-binding site or in the -82/-77 Sp1-binding site indicating functional cooperation between Sp1 and p53 in the transactivation of the p21 promoter. Synergistic transactivation was also decreased by mutations in the transactivation domain of p53. Physical interactions between Sp1 and p53 proteins were established by glutathione S-transferase pull-down and coimmunoprecipitation assays. By using deletion mutants we found that the DNA binding domain of Sp1 is required for its physical interaction with p53. In conclusion, Sp1 must play a critical role in regulating important biological processes controlled by p53 via p21 gene activation such as DNA repair, cell growth, differentiation, and apoptosis.     

The lack of G1/S arrest of teratocarcinoma F9 cells is determinated by degradation of cyclin-dependent kinases inhibitor p21waf1/cip1

We have studied the ability of F9 teratocarcinoma cells to arrest in G1/S and G2/M checkpoints following gamma-irradiation. Wild-type p53 protein is rapidly accumulated in F9 cells after gamma-irradiation, however this is not followed by G1/S arrest; there is just a 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 levels of regulatory cell cycle proteins: G1-cyclins, cyclin dependent kinases and kinase inhibitor p21WAF1/CIP1. We have shown that in spite of p53-dependent activation of p21WAF1/CIP1 promoter, p21WAF1/CIP1 protein is not revealed by different polyclonal and monoclonal antibodies, either by immunoblotting or by immunofluorescent staining. However, when cells are treated with specific proteasome inhibitor lactacystin, p21WAF1/CIP1 protein is revealed. We therefore suggest that p21WAF1/CIP1 protein is subjected to proteasome degradation in F9 cells and probably the lack of G1/S arrest after gamma-irradiation is due to this degradation. Thus, it is the combination of functionally active p53 with low level expression of p21WAF1/CIP1 that causes a short delay of the cell cycle progression in G2/M, rather than the G1-arrest after gamma-irradiation of F9 cells.     

Apoptin® specifically causes apoptosis in tumor cells and after UV-treatment in untransformed cells from cancer-prone individuals: A review

Tumor formation is caused by an imbalance between cell replication and apoptosis, which is a physiological form of cell death. For instance, UV damage can result in tumor formation due to mutations of the tumor-suppressor gene p53, a major apoptosis-inducing protein. Over-expression of the proto-oncogene Bcl-2, due to chromosomal translocation, can also inhibit apoptosis resulting in, e.g., lymphomas and leukemias. Anti-tumor therapies are often based on induction of apoptosis mediated via p53 and/or inhibited by Bcl-2, which explains the frequently poor results of anti-tumor treatment. The avian-virus-derived protein ‘Apoptin’, induces apoptosis in a p53-independent way, is stimulated by Bcl-2 and is insensitive to BCR-ABL, another inhibitor of chemotherapeutic agents. Apoptin induces apoptosis in human transformed/tumorigenic cells but not in normal diploid cells. Co-synthesis of SV40 large T antigen and Apoptin results in induction of apoptosis, illustrating that the establishment of a stable transformed state is not required. UV-irradiation causes an aberrant SOS-response in primary diploid cells from cancer-prone individuals and renders such cells susceptible to Apoptin-induced apoptosis. All these features make Apoptin a potential candidate as a therapeutic and diagnostic tool in cancer treatment.