Relaxed cell-cycle arrests and propagation of unrepaired chromosomal damage in cancer cell lines with wild-type p53

The role of the p53 protein in mediating G₁ and G₂ cell-cycle arrests after genotoxic insult has been clearly and reproducibly established in primary diploid fibroblasts, but data obtained from p53 wild-type (wt) cancer cell lines are inconsistent. Furthermore, a large proportion of human tumors have p53 wt genotypes but present genetic aberrations that may result from defective cell-cycle checkpoints. We therefore investigated the integrity of G₁/S and G₂/M cell-cycle arrests in p53 wt cancer cell lines. In the study presented here, we showed that in most cancer cells tested, G₁ arrest was relaxed or absent in comparison with arrest in normal diploid fibroblasts, despite seemingly normal p53 and p21 responses. Two cell lines (MCF7 and HCT116) were synchronized in G₀/G₁ by leucine starvation and subjected to genotoxic stress to determine more precisely the relative proportion of cells arresting in G₁ and G₂. Whereas the MCF7 cells showed consistent G₁ arrest, the HCT116 cells showed none at all. Furthermore, cell-cycle arrests in G₁ and G₂ in response to γ irradiation and bleomycin treatment were transient, as the cells resumed cycling after 48–72 h. The cells resuming proliferation suffered massive apoptosis, but a proportion of the cells were rescued and showed normal doubling times. These cells retained a p53 wt genotype but presented gross chromosomal aberrations in 15–20% of the analyzed metaphases. The aberrations were not clonal. These data show that p53 wt cancer cells have relaxed cell-cycle controls after genotoxic insult and tolerate unrepaired chromosomal damage, despite normal p53 function. Mol. Carcinog. 23:1–12, 1998. © 1998 Wiley-Liss, Inc.     

Jaceosidin induces p53-dependent G2/M phase arrest in U87 glioblastoma cells

Flavonoid compounds have been shown to trigger cell cycle arrest at G0/G1, S and G2/M checkpoints, allowing cells to repair DNA damage before entry into mitosis. Jaceosidin, a flavonoid compound, has been reported to induce apoptosis in various cancer cell lines. In our previous study, we established that jaceosidin induces apoptosis in U87 glioblastoma cells through G2/M phase arrest. However the molecular mechanisms oremained unclear. In the present study, mRNA and protein expression levels of major cell cycle regulatory genes were analyzed by semi-quantitative RT-PCR and Western blot studies respectively. The results demonstrated that jaceosidin-induced G2/M phase arrest in U87 cells is associated with DNA fragmentation, up-regulation of p53 and p21 and subsequent down-regulation of cyclin B1 and CDK1 expression at mRNA as well as at protein level. These findings provide insights into jaceosidin-induced G2/M phase arrest in U87 glioblastoma cells.     

Inhibition of Id proteins by a peptide aptamer induces cell-cycle arrest and apoptosis in ovarian cancer cells

Background:

Inhibitors of DNA-binding proteins (Id1-4), lacking the basic DNA-binding domain, function as dominant inhibitors of cell-cycle regulators. Overexpression of Id proteins promotes cancer cell proliferation and resistance against apoptosis. Level of Id protein expression, especially of Id1, correlates with poor differentiation, enhanced malignant potential and more aggressive clinical behaviour of ovarian tumours. Although overexpression of Ids has been found and shown to correlate with poor clinical outcome, their inhibition at protein level has never been studied.

Methods:

A peptide aptamer, Id1/3-PA7, targeting Id1 and Id3, was isolated from a randomised combinatorial expression library using yeast and mammalian two-hybrid systems. Id1/3-PA7 was fused, expressed and purified with a cell-penetrating protein transduction domain.

Results:

Intracellular-delivered Id1/3-PA7 colocalised to Id1 and Id3. It induced cell-cycle arrest and apoptosis in ovarian cancer cells ES-2 and PA-1. It activated the E-box promoter and increased the expression level of cyclin-dependent kinase inhibitor (CDKN2A) in a dose-dependent manner that is paralleled by the cleavage of poly-ADP ribose polymerase. These effects were counteracted by ectopically overexpressed Id1 and Id3.

Conclusion:

Id1/3-PA7 could represent an exogenous anti-tumour agent that can significantly trigger cell-cycle arrest and apoptosis in ovarian cancer.     

Direct interaction with and activation of p53 by SMAR1 retards cell-cycle progression at G2/M phase and delays tumor growth in mice

The tumor-suppressor p53 is a multifunctional protein mainly responsible for maintaining genomic integrity. p53 induces its tumor-suppressor activity by either causing cell-cycle arrest (G(1)/S or G(2)/M) or inducing cells to undergo apoptosis. This function of wild-type p53 as "guardian of the genome" is presumably achieved by forming molecular complexes with different DNA targets as well as by interacting with a number of cellular proteins, e.g., Mdm2, Gadd45, p21, 14-3-3sigma, Bax and Apaf-1. Upon activation, p53 activates p21, which in turn controls the cell cycle by regulating G(1) or G(2) checkpoints. Here, we report SMAR1 as one such p53-interacting protein that is involved in delaying tumor progression in vivo as well as in regulating the cell cycle. SMAR1 is a newly identified MARBP involved in chromatin-mediated gene regulation. The SMAR1 gene encodes at least 2 alternatively spliced variants: SMAR1(L) (the full-length form) and SMAR1(S) (the shorter form). We report that expression of SMAR1(S), but not of SMAR1(L), mRNA was decreased in most of the human cell lines examined, suggesting selective silencing of SMAR1(S). Overexpression of SMAR1(S) in mouse melanoma cells (B16F1) and their subsequent injection in C57BL/6 mice delays tumor growth. Exogenous SMAR1(S) causes significant retardation of B16F1 cells in the G(2)/M phase of the cell cycle compared to SMAR1(L). SMAR1(S) activates p53-mediated reporter gene expression in mouse melanoma cells, breast cancer cells (MCF-7) and p53 null cells (K562), followed by activation of its downstream effector, p21. We further demonstrate that SMAR1 physically interacts and colocalizes with p53. These data together suggest that SMAR1 is the only known MARBP that delays tumor progression via direct activation and interaction with tumor-suppressor p53.     

低氧条件下食管癌细胞中PRRX1通过p53介导线粒体通路诱导细胞凋亡的研究

目的:探究低氧条件下配对相关同源框1（PRRX1）通过p53介导的线粒体通路诱导食管癌细胞凋亡。方法:GEPIA2数据库分析PRRX1基因在食管癌组织和正常食管组织中的表达变化。RT-qPCR和Western blotting分别检测HEEC、Eca-109、TE-1细胞中PRRX1的mRNA和蛋白表达。二氧化钴处理模拟低氧微环境,在常氧和低氧条件下检测Eca-109、TE-1细胞中PRRX1的mRNA和蛋白表达情况。采用小干扰RNA（Si-PRRX1）转染TE-1细胞,设置分组为Hypoxia-Si-NC、Hypoxia-Si-PRRX1。流式细胞术检测TE-1细胞凋亡,RT-qPCR检测p53 mRNA表达,Western blotting检测p53、BCL-2、BAX、Cleaved-Caspase3等蛋白表达,在TE-1-PRRX1 KO细胞系中过表达p53,检测 BCL-2、BAX、Cleaved-Caspase3等蛋白表达。结果:PRRX1在食管癌组织及细胞系中均高表达。在TE-1细胞中敲低PRRX1,抑制细胞凋亡,下调p53的mRNA及蛋白表达,抑制BAX、Cleaved-Caspase3蛋白表达,促进BCL-2蛋白表达;过表达PRRX1则上调p53蛋白表达。在TE-1-PRRX1 KO细胞系中过表达p53显著抑制BCL-2蛋白表达,促进BAX、Cleaved-Caspase3蛋白表达。结论:低氧条件下,PRRX1通过p53介导的线粒体通路诱导食管癌细胞凋亡。     

Caffeic acid phenethyl ester induced cell cycle arrest and growth inhibition in androgen-independent prostate cancer cells via regulation of Skp2, p53, p21Cip1 and p27Kip1

Prostate cancer (PCa) patients receiving the androgen ablation therapy ultimately develop recurrent castration-resistant prostate cancer (CRPC) within 1–3 years. Treatment with caffeic acid phenethyl ester (CAPE) suppressed cell survival and proliferation via induction of G1 or G2/M cell cycle arrest in LNCaP 104-R1, DU-145, 22Rv1, and C4–2 CRPC cells. CAPE treatment also inhibited soft agar colony formation and retarded nude mice xenograft growth of LNCaP 104-R1 cells. We identified that CAPE treatment significantly reduced protein abundance of Skp2, Cdk2, Cdk4, Cdk7, Rb, phospho-Rb S807/811, cyclin A, cyclin D1, cyclin H, E2F1, c-Myc, SGK, phospho-p70S6kinase T421/S424, phospho-mTOR Ser2481, phospho-GSK3α Ser21, but induced p21^Cip1, p27^Kip1, ATF4, cyclin E, p53, TRIB3, phospho-p53 (Ser6, Ser33, Ser46, Ser392), phospho-p38 MAPK Thr180/Tyr182, Chk1, Chk2, phospho-ATM S1981, phospho-ATR S428, and phospho-p90RSK Ser380. CAPE treatment decreased Skp2 and Akt1 protein expression in LNCaP 104-R1 tumors as compared to control group. Overexpression of Skp2, or siRNA knockdown of p21^Cip1, p27^Kip1, or p53 blocked suppressive effect of CAPE treatment. Co-treatment of CAPE with PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or Bcl-2 inhibitor ABT737 showed synergistic suppressive effects. Our finding suggested that CAPE treatment induced cell cycle arrest and growth inhibition in CRPC cells via regulation of Skp2, p53, p21^Cip1, and p27^Kip1.     

Mammary gland-specific ablation of focal adhesion kinase reduces the incidence of p53-mediated mammary tumour formation

Background:

Elevated expression of focal adhesion kinase (FAK) occurs in numerous human cancers including colon-, cervix- and breast cancer. Although several studies have implicated FAK in mammary tumour formation induced by ectopic oncogene expression, evidence supporting a role for FAK in spontaneous mammary tumour development caused by loss of tumour suppressor genes such as p53 is lacking. Alterations in the tumour suppressor gene p53 have been implicated in over 50% of human breast cancers. Given that elevated FAK expression highly correlates with p53 mutation status in human breast cancer, we set out to investigate the importance of FAK in p53-mediated spontaneous mammary tumour development.

Methods:

To directly assess the role of FAK, we generated mice with conditional inactivation of FAK and p53. We generated female p53^lox/lox/FAK^+/+/WapCre, p53^lox/lox/FAK^flox/+/WapCre and p53^lox/lox/FAK^flox/−/WapCre mice, and mice with WapCre-mediated conditional expression of p53^R270H, the mouse equivalent of human p53^R273H hot spot mutation, together with conditional deletion of FAK, P53^R270H/+/FAK^lox/+/WapCre and p53^R270H/+/FAK^flox/−/WapCre mice. All mice were subjected to one pregnancy to induce WapCre-mediated deletion of p53 or expression of p53 R270H, and Fak genes flanked by two loxP sites, and subsequently followed the development of mammary tumours.

Results:

Using this approach, we show that FAK is important for p53-induced mammary tumour development. In addition, mice with the mammary gland-specific conditional expression of p53 point mutation R270H, the mouse equivalent to human R273H, in combination with conditional deletion of Fak showed reduced incidence of p53^R270H-induced mammary tumours. In both models these effects of FAK were related to reduced proliferation in preneoplastic lesions in the mammary gland ductal structures.

Conclusions:

Mammary gland-specific ablation of FAK hampers p53-regulated spontaneous mammary tumour formation. Focal adhesion kinase deletion reduced proliferative capacity of p53 null and p53^R270H mammary epithelial cells but did not lead to increased apoptosis in vivo. Our data identify FAK as an important regulator in mammary epithelial cell proliferation in p53-mediated and p53^R270H-induced mammary tumour development.     

Simultaneous phosphorylation of p53 at serine 15 and 20 induces apoptosis in human glioma cells by increasing expression of pro-apoptotic genes

Understanding the mechanism underlying p53’s ability to induce cell cycle arrest versus apoptosis is critical to treating human gliomas, 70% of which contain wild-type p53. Although N-terminal phosphorylation results in activation of p53, the role of N-terminal phosphorylation, particularly at serines 15 and 20, in p53’s ability to induce cell cycle arrest versus apoptosis remains controversial. Here we test the hypothesis that phosphorylation of serine 15 and/or 20 is causally related to p53-mediated apoptosis in human gliomas. Introduction of p53 plasmids containing alanine mutations at serine 15 or/and serine 20 (which block phosphorylation) or aspartate mutations (which mimic phosphorylation) at the same sites, implicated simultaneous phosphorylation of both sites in the induction of apoptosis. When a double phosphorylation-mimicking adenoviral p53 vector (Ad-p53-15D20D) was compared with an unphosphorylated p53 vector (Ad-p53), treatment with Ad-p53 resulted in G1-arrest, whereas Ad-p53-15D20D induced apoptosis. These effects occurred independent of phosphorylation of other N-terminal serine (i.e., serines 6, 9, 33, 37, 46) indicating that phosphorylation of S15 and S20 is sufficient for inducing apoptosis. Mechanistically, Ad-p53 was capable only of increasing the expression of p21/CIP, whereas Ad-p53-15D20D increased the binding to and expression of the pro-apoptotic genes Fas, Puma and PIG3. However, Ad-p53-15D20D did not alter the expression of Noxa, Bid, IGFBP3, PERP and Killer/DR5, suggesting that phosphorylation of S15 and S20 resulted in the expression of specific pro-apoptotic gene. In conclusion, simultaneous phosphorylation of S15 and S20 is causally associated with apoptosis, resulting in increased expression of specific p53-responsive pro-apoptotic genes.     

Arsenic trioxide induces apoptosis and G2/M phase arrest by inducing Cbl to inhibit PI3K/Akt signaling and thereby regulate p53 activation

Arsenic trioxide (ATO) strongly induces apoptosis in acute promyelocytic leukemia (APL), but it induces cell cycle arrest in most solid tumors. In this study, we investigated the mechanism of ATO action on APL-derived NB4 cells and gastric cancer cell lines. ATO decreased the viability of both cell lines, but gastric cancer cells were much less susceptible. ATO-induced G2/M phase arrest and p53 degradation in gastric cancer MGC803 cells. In contrast, ATO-induced apoptosis in NB4 cells without degradation of p53. Both processes were accompanied by transient activation of Akt. The PI3K/Akt inhibitor LY294002 significantly increased the amount of p53 protein and ATO-induced apoptosis in both cell lines and decreased G2/M phase arrest of MGC803 cells. In addition, ATO up-regulated the expression of Cbl proteins in both cell lines. Inhibition of Cbl with the proteasome inhibitor Ps341 decreased apoptosis in NB4 cells and increased the G2/M phase arrest of MGC803 cells, and it also prolonged the activation of PI3K/Akt by ATO. Consistent results with those in MGC803 cells were showed in gastric cancer cell BGC823 and SGC7901 after ATO treatment. These results demonstrate that inhibition of PI3K/Akt signaling by Cbl is involved in both ATO-induced apoptosis of NB4 cells and ATO-induced G2/M phase arrest of gastric cancer cells. Cbl achieved these effects probably via its regulating PI3K/Akt pathway, and thereby modulated p53 activation.     

Inhibitor of cyclooxygenase-2 induces cell-cycle arrest in the epithelial cancer cell line via up-regulation of cyclin dependent kinase inhibitor p21

Cyclooxygenase-2 is the rate-limiting enzyme in synthesis of prostaglandins and other eicosanoids. Prior reports have shown that inhibition of cyclooxygenase-2 activity, either by selective inhibitors or by antisense oligonucleotide, results in suppression of growth of squamous cell carcinoma cell lines which express high cyclooxygenase-2 levels, such as NA, a cell line established from a squamous cell carcinoma of the tongue. To investigate the mechanisms by which cyclooxygenase-2 inhibitors suppressed growth of these cells, the effects of NS-398, the selective cyclooxygenase-2 inhibitor, on cell-cycle distribution were examined. NS-398 induced G0/G1 cell-cycle arrest in NA cells which expressed cyclooxygenase-2. G0/G1 arrest induced by NS-398 was accompanied by up-regulation of cyclin-dependent kinase inhibitor p21, but not by up-regulation of the other cyclin-dependent kinase inhibitors. Transfection with p21 antisense oligonucleotide inhibited cell-cycle arrest induced by NS-398. Accumulation in G0/G1 was also observed in NA cells transfected with cyclooxygenase-2 antisense oligonucleotide. On the other hand, NS-398-treated NA cells showed a loss of plasma membrane asymmetry, a marker of early events in apoptosis. However, NS-398 did not induce other morphological and biochemical changes related to apoptotic cell death. These results suggest that cyclooxygenase-2 inhibitor induces G0/G1 cell-cycle arrest in NA cells by up-regulation of p21. Our results also suggest that NS-398 is not sufficient to complete the whole process of apoptosis in NA cells, although it induces an early event in apoptosis.     

Molecular mechanisms of G0/G1 cell-cycle arrest and apoptosis induced by terfenadine in human cancer cells

Terfenadine (TF), a highly potent histamine H1 receptor antagonist, has been shown to exert no significant central nervous system side effects in clinically effective doses. In this study, we demonstrated that TF induced significant growth inhibition of human cancer cells, including Hep G2, HT 29, and COLO 205 cells, through induction of G(0)/G(1) phase cell-cycle arrest. The minimal dose of TF induced significant G(0)/G(1) arrest in these cells was 1-3 microM. The protein levels of p53, p21/Cip1, and p27/Kip1 were significantly elevated, whereas the kinase activities of cyclin-dependent kinase 2 (CDK2) and CDK4 were inhibited simultaneously in the TF-treated cells. On the other hand, significant apoptosis, but not G(0)/G(1) arrest, was induced in the HL 60 (p53-null) or Hep 3B (with deleted p53) cells when treated with TF (3-5 microM). To clarify the roles of p21/Cip1 and p27/Kip1 protein expression, which was involved in G(0)/G(1) arrest and apoptosis induced by TF in human cancer cells, antisense oligodeoxynucleotides (ODNs) specific to p21/Cip1 and p27/Kip1 were used, and the expression of the p21/Cip1 and p27/Kip1 were monitored by immunoblotting analysis. Our data demonstrated that the percentage of the apoptotic cells detected by annexin V/PI analysis in the TF-treated group was clearly attenuated by pretreatment with p27/Kip1-specific ODNs. These results indicated that p27/Kip1 (but not p21/Cip1) protein indeed played a critical role in the TF-induced apoptosis. We also demonstrated that the TF-induced G(0)/G(1) cell-cycle arrest effect was not reversed by TF removal, and this growth inhibition lasted for at least 7 d. Importantly, the occurrence of apoptosis and cell growth arrest was not observed in the TF-treated normal human fibroblast, even at a dose as high as 25 microM. Our study showed the molecular mechanisms for TF-induced cell growth inhibition and the occurrence of apoptosis in human cancer cells.     

Overexpression of the p53-inducible brain-specific angiogenesis inhibitor 1 suppresses efficiently tumour angiogenesis

The brain-specific angiogenesis inhibitor 1 gene has been isolated in an attempt to find fragments with p53 "functional" binding sites. As reported herein and by others, brain-specific angiogenesis inhibitor 1 expression is present in some normal tissues, but is reduced or lost in tumour tissues. Such data and its particular structure prompted the hypothesis that brain-specific angiogenesis inhibitor 1 may act as a mediator in the local angiogenesis balance. We herein demonstrate that brain-specific angiogenesis inhibitor 1 over-expression suppresses tumour angiogenesis, delaying significantly the human tumour growth in immunodeficient mice. The inhibitory effect of brain-specific angiogenesis inhibitor 1 was documented using our intravital microscopy system, strongly implicating brain-specific angiogenesis inhibitor 1 as a mediator in the control of tumour angiogenesis. In contrast, in vitro tumour cell proliferation was not inhibited by brain-specific angiogenesis inhibitor 1 transfection, whereas some level of cytotoxicity was assessed for endothelial cells. Immunohistochemical analysis of tumour samples confirmed a reduction in the microvessel density index in brain-specific angiogenesis inhibitor 1-overexpressing tumours. At messenger level, moderate changes could be detected, involving the down-regulation of vascular endothelial growth factor and collagenase-1 expression. Furthermore, brain-specific angiogenesis inhibitor 1 expression that was lost in a selection of human cancer cell lines could be restored by wild-type p53 adenoviral transfection. Brain-specific angiogenesis inhibitor 1 should be considered for gene therapy and development of efficient drugs based on endogenous antiangiogenic molecules.     

p53核输入功能在鼠双微体2调节的p53蛋白降解和泛素化中的作用

目的研究p53核输入功能在鼠双微体2(murine double minute2,MDM2)调节的p53降解与泛素化中的作用。方法由直接点突变的方法构建质粒,将决定p53-GFP核位置信号(NLS)的两个部分5个基本氨基酸(赖氨酸和精氨酸)单一替换为丙氨酸,构建了p53-NLS突变型053KRKKK-GFP,并且经DNA序列确定。用DNA克隆技术将p53KRKKK与pEGF-Nuc融合,构建了p53KRKKK-NLS-GFP,然后分别转染U2OS细胞,在荧光显微镜下直接观察活细胞的蛋白表达部位;同时用间接免疫荧光染色、Western blot和泛素化分析的方法,观察p53KRKKK与MDM2野生型或MDM2-NLS突变型共转染U2OS细胞时的蛋白表达部位、降解和泛素化现象。结果p53KRKKK-GFP蛋白表达完全在细胞浆,并且不能被野生型和NLS突变型的MDM2降解,但是可以被泛素化。p53KRKKK-NLS-GFP可以使p53蛋白表达重新回到细胞核,此时能被MDM2野生型和NLS突变型降解和泛素化。p53野生型和NLS突变型均可被MDM2野生型和NLS突变型泛素化,当p53KRKKK和MDM2-NLS同时表达在细胞浆时,p53蛋白出现了更高效率的泛素化,但不能被降解。结论p53核输入对由MDM2调节的p53降解是必须的;MDM2调节的p53泛素化可以出现在细胞浆,而且效率更高。