腺病毒介导的p21基因联合顺铂对胃癌细胞的影响

目的：探讨腺病毒介导的p21基因^WAF1/CIP1基因（p21基因）在人胃癌细胞系SGC0－7901中的表达及其联合顺铂化疗对胃癌细胞生长和荷瘤小鼠的作用。方法：采用腺病毒载体携带p21基因感染人胃癌细胞系，用流式细胞仪测定p21基因对SGC－7901细胞周期的影响，并与DDP联合应用，观察p21基因和（或）DDP对SGC－7901细胞系和荷瘤小鼠的作用。结果：腺病毒介导的p21基因可在胃癌细胞系SGC－7901中过度表达，使胃癌细胞系S期含量下降，抑制其生长，并可诱导胃癌细胞的凋亡；p21基因联合DDP作用使SGC－7901细胞G2／M含量明显下降，并可抑制荷瘤小鼠肿瘤细胞的生长，且联合作用比单一用药作用更明显。结论：腺病毒介导的p21基因可抑制人胃癌细胞系的生长，抑制瘤体的生长，结合化疗药物其作用更强，为临床应用提供了依据。     

Evidence for a p53-independent pathway for upregulation of SDI1/CIP1/WAF1/P21 RNA in human cells

SDI1 is an inhibitor of DNA synthesis that we isolated by expression screening cDNAs prepared from senescent, terminally nondividing human cells. Other groups then cloned this gene as a cyclin-dependent kinase (cdk)-interacting protein (CIP1, p21) that inhibits cdks; the gene was also isolated by screening for genes transactivated by p53 (WAF1). p53 levels are low in senescent and quiescent contact-inhibited or serum-deprived normal human cells, which we have found express high levels of SDI1 mRNA. This indicates that alternate pathways for upregulation of message level of this gene may exist. We therefore proceeded with the study presented here, treating human cells with a variety of growth-arrest-inducing agents, including some that damaged DNA, and found that RNA levels of SDI1 were increased in all cases that resulted in growth inhibition. More important, with the exception of γ-radiation, most of these agents were able to elevate SDI1 message levels in cells lacking wild-type p53. At least two distinct kinetic profiles for RNA induction were observed, one that implicated p53 transactivation and occurred early enough to cause arrest, and another that clearly was p53 independent and suggested a role for the SDI1 gene product in the maintenance rather than in the cause of inhibition of DNA synthesis. ©1994 Wiley-Liss, Inc.     

p21WAF1/CIP1 protein expression in primary ovarian cancer

p21WAF1/CIP1 protein is a cyclin-dependent kinase inhibitor, able to prevent the CDK2/cyclin E induced retinoblastoma protein (pRB) phosphorylation, thus inhibiting cell cycle progression at G1 phase. p21WAF1/CIP1 protein levels were examined in a series of 102 ovarian tissue samples including normal ovary, primary ovarian tumors, omental metastasis, recurrent disease and residual tumor after chemotherapy exposure, by Western blot analysis. The association of p21WAF1/CIP1 status with clinicopathological parameters and clinical outcome was also investigated. p21WAF1/CIP1 protein was detectable in 76 out of 102 (74%) ovarian tissue samples. We observed a significant trend of p21 levels to gradually increase from normal ovarian tissues (median 0 a.u.) through primary ovarian cancers (median 0.19 a.u.), omental metastases (median 0.33 a.u.) and recurrence of disease (median 0.44 a.u.) (p=0.015). In the group of stage III-IV ovarian cancer patients, p21-positive cases showed a more favourable prognosis with respect to p21-negative cases: the 3-year time to progression (TTP) rate was 58% for p21-positive compared with 33% of p21-negative cases (p=0.036). In conclusion, p21WAF1/CIP1 expression levels seem to be correlated with tumor status at the time of diagnosis and can predict TTP in a selected group of patients.     

Mutational analysis of the p21/WAF1/CIP1/SDI1 coding region in human tumor cell lines

p21/WAF1/CIP1/SDI1 is an important cell-cycle mediator with tumor suppressor gene capabilities, and its inactivation could potentially lead to tumor progression. Because tumor suppressor genes are commonly inactivated by somatic and germline mutations, we analyzed a variety of human tumor cell lines for p21 mutations. We used single-strand conformational analysis and direct sequencing to identify possible mutations in the p21 coding region. Two base-alterations were observed in 41 immortalized human tumor cell lines. A previously reported polymorphism that results in a serine-to-arginine amino-acid substitution at codon 31 was found in 24% (10 of 41) of the tumor cell lines but was also found in 10% (six of 62) of normal parental DNAs tested and 7% (three of 43) of normal DNAs from patients with primary endometrial tumors. Another nucleotide substitution found at codon 80 resulted in the replacement of threonine with methionine. Codon 80 changes were found in 7% (three of 41) of the tumor cell lines (all endometrial) and in 2% (one of 62) of the normal parental DNAs. (This article is a US Government work and, as such, is in the public domain in the United States of America.)     

p53和p21WAF1/CIP1基因在上皮性卵巢癌中的表达及临床意义

目的探讨联合检测p53和p21蛋白表达与上皮性卵巢癌预后的关系.方法 108例上皮性卵巢癌标本用于研究,每个标本同时用免疫组化的方法检测p53和p21蛋白表达.结果 p53(－)和p53( )患者的5年生存率分别为60.47%和29.43%,差异有显著性(P=0.0228).p21(－)和p21( )患者的5年生存率分别为31.58%和47.14%,差异有显著性(P=0.0246).p53(－)而p21( )患者的预后明显优于其他患者(P=0.0013).多因素分析显示p53、p21蛋白联合表达状态是判断上皮性卵巢癌预后的独立因子. 53蛋白表达与p21蛋白表达呈负相关(P=0.0003).结论联合检测p53、p21蛋白表达在判断上皮性卵巢癌预后上的意义优于单纯检测p53或p21蛋白表达,对临床有指导意义.     

Endogenous p21WAF1/CIP1 status predicts the response of human tumor cells to wild-type p53 and p21WAF1/CIP1 overexpression

Expression of exogenous wild-type (wt) p53 protein can suppress the growth and/or induce apoptosis in different tumor cells. The effect of exogenous p21(WAF1/CIP1) expression is more controversial: while it can induce apoptosis in some cells, it can protect against p53-mediated apoptosis in others. We used adenoviral vectors to introduce p53 and p21(WAF1/CIP1) genes into human tumor cell lines with different p53 and/or p21(WAF1/CIP1) status. The cell growth inhibition and the induction of apoptosis were measured. Overexpression of wt p53 induced more efficient growth inhibition and apoptosis in SW 620 (mutant p53) and HeLa (inactivated p53 protein) than in MCF-7 (wt p53) and CaCo-2 cell line, which was the most resistant to p53 overexpression despite the p53 mutation. Unlike HeLa and SW 620 cells, the basal p21 protein level was readily detected in CaCo-2 and MCF-7 cells. Overexpression of p21(WAF1/CIP1) gene induced somewhat less pronounced growth inhibition of all cell lines tested, but it also induced apoptosis in HeLa and SW 620 cells. These results suggest that the basal, but not the inducible, levels of p21(WAF1/CIP1) protein in tumor cells could protect from p53-mediated apoptosis. On the other hand, overexpression of p21(WAF1/CIP1) gene itself can induce apoptosis in cells with no basal p21(WAF1/CIP1) protein level. Possible mechanisms of the differential response to these genes are discussed.     

Flavopiridol inversely affects p21(WAF1/CIP1) and p53 and protects p21-sensitive cells from paclitaxel

Resting cells are relatively resistant to microtubule-active drugs including paclitaxel (PTX). By causing p53-mediated arrest, pretreatment with low concentrations of doxorubicin (DOX) protected HCT116 cells from the cytotoxicity caused by PTX. Unlike DOX, flavopiridol (FL) did not protect HCT116 cells. Low concentrations of FL (50 nM) induced p21 but not p53. High concentrations of FL (500 nM) decreased levels of p21 and Mdm-2 but dramatically induced p53. Thus, FL reciprocally affects p21 and p53. In LNCaP, a prostate cancer cell line which is highly sensitive to p21-induced growth arrest (p21-sensitive), low concentrations of FL (50 nM) induced p21 (without induction of p53) and caused G1 and G2 arrest. This precluded mitotic arrest, Bcl-2 and Raf-1 phosphorylation, and diminished cell death caused by PTX. In contrast, FL did not protect PC3M, arrest-resitant and highly aggressive prostate cancer cells. Like LNCaP, HL60 and SKBr3 cells are known to be p21-sensitive. As predicted, low concentrations of FL antagonized PTX-mediated cytotoxicity in HL60 and SKBr3 cell lines. In summary, only low concentrations of FL can induce p21, and, in turn, only p21-sensitive cells are protected from PTX.     

ANLN plays a critical role in human lung carcinogenesis through the activation of RHOA and by involvement in the phosphoinositide 3-kinase/AKT pathway

Gene expression profile analysis of non-small cell lung cancers (NSCLC) and subsequent functional analyses revealed that human ANLN, a homologue of anillin, an actin-binding protein in Drosophila, was transactivated in lung cancer cells and seemed to play a significant role in pulmonary carcinogenesis. Induction of small interfering RNAs against ANLN in NSCLC cells suppressed its expression and resulted in growth suppression; moreover, treatment with small interfering RNA yielded cells with larger morphology and multiple nuclei, which subsequently died. On the other hand, induction of exogenous expression of ANLN enhanced the migrating ability of mammalian cells by interacting with RHOA, a small guanosine triphosphatase, and inducing actin stress fibers. Interestingly, inhibition of phosphoinositide 3-kinase/AKT activity in NSCLC cells decreased the stability of ANLN and caused a reduction of the nuclear ANLN level. Immunohistochemical staining of nuclear ANLN on lung cancer tissue microarrays was associated with the poor survival of NSCLC patients, indicating that this molecule might serve as a prognostic indicator. Our data imply that up-regulation of ANLN is a common feature of the carcinogenetic process in lung tissue, and suggests that selective suppression of ANLN could be a promising approach for developing a new strategy to treat lung cancers.     

胃肠道类癌中生长抑素和p21^WAF1/CIP1蛋白表达的意义

目的探讨生长抑素和p21WAF1/CIP1蛋白阳性表达与胃肠道类癌的组织分化、浸润和转移的关系.方法采用免疫组化S-P法对36例胃肠道类癌组织生长抑素和p21WAF1/CIP1蛋白的表达进行检测.结果 36例类癌组织中,生长抑素和p21WAF1/CIP1蛋白较多表达于高分化类癌组(P＜0.05),随着肿瘤的浸润和淋巴结转移,生长抑素阳性表达率显著降低(P＜0.01),p21WAF1/CIP1阳性表达差异有显著性(P＜0.05).结论生长抑素和p21WAF1/CIP1低表达在类癌的组织分化和发展中起着重要作用,可用于临床对患者进行预后判断.     

Topological analysis of p21WAF1/CIP1 expression in esophageal squamous dysplasia.

In the normal stratified squamous epithelium of the esophagus, only the third to the fifth layers of cells express the cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21). Using immunohistochemical staining, we examined the topological distribution of cells expressing p21, p53, Ki67, and cytokeratin 10 (CK10), a differentiation marker of esophageal squamous cell carcinoma (SCC), in 25 superficial SCCs and 72 dysplastic lesions of the esophagus. Image analysis of p21, p53, and Ki67 expression was also performed in 48 dysplastic lesions. In superficial SCCs, although Ki67- and p53-expressing cells were mainly distributed in the deep layers of tumors despite tumor differentiation, the distribution of p21 correlated with tumor differentiation. In dysplastic lesions, p53- and Ki67-coexpressing cells tended to locate in the same layers and expand in the lower layers of epithelium with the progression of dysplasia. p21-expressing cells shifted to the upper layers of the epithelium with the progression of dysplasia. However, this change was heterogeneous; in some lesions, p21-expressing cells were confined to the superficial layers of atypical cells (confined type), whereas in others, p21-overexpressing cells were scattered among atypical cells (scattered type). CK10 expression was observed in 25% of dysplastic lesions, and the frequency of CK10 expression was significantly higher in the scattered than in the confined type. Our results suggest that esophageal squamous dysplasia represents the earliest pathological process in esophageal squamous carcinogenesis. Our results also suggest that differentiation of esophageal SCC is determined at the stage of dysplasia, and that p21 plays a critical role in the differentiation process.     

Relationship between expression of p21WAF1/CIP1 and radioresistance in human gliomas.

The role of p21WAF1/CIP1 (p21) in DNA repair and apoptosis following gamma-irradiation remains controversial. In this study the influence of p21 on the radiosensitivity of human brain tumors was investigated. Resected tumors were stained immunohistochemically for p21. Expression of p21 in astrocytic tumors was high, but it was low in medulloblastomas, germinomas, and primary malignant lymphomas. Glioma and medulloblastoma cell lines were transfected with pcDNA/p21 to cause p21 overexpression, then tumor-cell colony formation and apoptosis were assessed following gamma-irradiation of the transfected and nontransfected cells. Overexpression of p21 enhanced clonogenic survival and suppressed apoptosis after gamma-irradiation in human brain tumor cell lines with or without p53 protein deficiency. Radioresistance was acquired when p21 was overproduced in the glioma cell lines irrespective of p53 status.     

Correlation between reduced p21(WAF1/CIP1) expression and abnormal p53 expression in esophageal carcinomas

Direct gene transfer into somatic tissue iii vivo is a developing technology with potential application for cancer gene therapy. In this study, recombinant vaccinia virus encoding human IL-2 gene (rVV-IL-2) was used as a candidate vector in mediating iii vivo gene therapy. After rVV-IL-2 was expanded in VERO cells for 72 h, high titer (10(8)-10(10) PFU/ml) rVV-IL-2 were harvested. When 10(6) murine melanoma cells (F16-F10) were infected with rVV-IL-2, about 200 U/ml IL-2 activity was detected in the supernatants at 8 h, and the up-regulation of ICAM-1 and MHC-I expressions on the melanoma cells were observed. The treatment of murine melanoma model by local injection of rVV-IL-2 into the tumor site showed that rVV-IL-2 transfection significantly inhibited the tumor growth and prolonged the survival time of tumor-bearing mice. The splenocytes from rVV-IL-2 treated mice showed higher cytotoxicities of NK, LAK and CTL in comparison with those from the controls. These results suggest that in vivo transfection mediated by rVV-IL-2 has potential effectiveness in enhancing host immunity and would be a useful approach to cancer gene therapy.     

Exogenous expression of p21(WAF1/CIP1) exerts cell growth inhibition and enhances sensitivity to cisplatin in hepatoma cells

Lipid peroxidation (as malondialdehyde; MDA), activities of some antioxidant enzymes (as superoxide dismutase; SOD, glutathione peroxidase; GPx, glutathione reductase; GR), glutathione status, and oxidative DNA damage (as 8-hydroxy-2'-deoxyguanosine; 8-OHdG) were investigated in the lungs of rats exposed to hematite (Fe(2)O(3); 3 mg), benzo(a)pyrene (B(a)P; 3 mg), or B(a)P (3 mg)-coated onto Fe(2)O(3) particles (3 mg). Approximately 2-fold increases in MDA production were seen in animals exposed to Fe(2)O(3), B(a)P, or B(a)P-coated onto Fe(2)O(3) particles (P<0.01). Decreases in SOD activities were observed in rats treated with Fe(2)O(3) (1.66-fold, P<0.01), B(a)P (1.66-fold, P<0.001) or B(a)P-coated onto Fe(2)O(3) particles (1.43-fold, P<0.01). GPx and GR activities could not be detected. No alteration of the glutathione status was observed. Significant increases in the 8-OHdG formation occurred in response to exposure to B(a)P (2.0-fold, P<0.01) or B(a)P-coated onto Fe(2)O(3) particles (23.7-fold, P<0.001). Our results demonstrate also that Fe(2)O(3) generates free radical (FR)-induced lung injury and is not an inert carrier. We established that exposure to B(a)P or B(a)P-coated onto Fe(2)O(3) particles resulted in lipid peroxidation and SOD inactivation, thereby leading to oxidative damages in DNA. The main findings of this work was that B(a)P-coated onto Fe(2)O(3) particles caused higher lung concentrations of 8-OHdG than B(a)P by itself. Hence, our data may explain why exposure to B(a)P-coated onto Fe(2)O(3) particles resulted in a decreased latency and an increased incidence of lung tumors in rodents compared to exposure to B(a)P.     

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.