Adenovirus-mediated p14(ARF) gene transfer in human mesothelioma cells

BACKGROUND: The p14(ARF) protein encoded by the INK4a/ARF locus promotes degradation of the MDM2 protein and thus prevents the MDM2-mediated inhibition of p53. Homozygous deletion of the INK4a/ARF locus is common in human mesothelioma and may result in the loss of p14(ARF) and the inactivation of p53. We designed this study to evaluate the biologic and potential therapeutic roles of p14(ARF) expression in mesothelioma cells. Methods and Results: We constructed Adp14, an adenoviral vector carrying human p14(ARF) complementary DNA, and used it to transfect human mesothelioma cell lines H28, H513, H2052, and MSTO-211H. Overexpression of p14(ARF) led to increased amounts of p53 and the p21(WAF) proteins and dephosphorylation of the retinoblastoma protein. The growth rate of mesothelioma cells was inhibited markedly by infection with Adp14 compared with mock infection or infection with a control adenovirus vector, AdCtrl. Overexpression of p14(ARF) induced G(1)-phase cell cycle arrest and apoptotic cell death. Cytotoxicity assays showed that Adp14 had a statistically significantly (P =.002) greater effect on colon cancer (HCT116) cell lines containing two copies of the wild-type p53 gene than on p53-null cells, suggesting that functional p53 is a critical determinant of p14(ARF)-mediated cytotoxicity. CONCLUSIONS: The transfection of p14(ARF) into mesothelioma cells led to the overexpression of p14(ARF), which resulted in G(1)-phase arrest and apoptotic cell death. These results suggest that this gene therapy-based approach may be of use in the treatment of mesothelioma.     

Adenovirus-mediated overexpression of Fas induces apoptosis of gliomas

Gliomas express a higher amount of Fas than normal brain tissue. It is of interest to know whether expression of the Fas receptor is unfavorable to the antiapoptotic pathways in gliomas. In this study, we introduced the Fas gene via an adenovirus vector (Adeno-Fas) into the A-172, U251, and U-373 MG glioma cell lines, each of which expresses Fas on the cell surface. Infection of Adeno-Fas induced apoptosis in each glioma cell line. In U251 cells and A-172 cells that express the same level of Fas as a result of infection with Adeno-Fas, a much higher percentage of U251 cells underwent apoptosis than did A-172 cells. This suggests that each glioma cell line has its own threshold of Fas expression, above which apoptosis is induced, and that the constitutive expression of Fas is below the level of this threshold. It was found that the constitutive expression of the anti-apoptotic gene Bcl-X(L) is higher in A-172 cells than in U251 cells. Adenovirus-mediated transduction of the Bcl-X(L) gene into U251 cells effectively suppressed Adeno-Fas-mediated apoptosis. These data indicate that the Bcl-X(L) gene is one of the important determinants of the threshold for Fas-mediated apoptosis. When U251 and U-373 MG cells were transduced with the Fas gene controlled by the myelin basic protein promoter, which had been shown to be active in gliomas but not in neural tissues, the cells underwent markedly enhanced apoptosis. Taken together, these results indicate that the overexpression of Fas alone induced apoptosis in each glioma cell line. The degree of Fas-mediated apoptosis was attenuated by the expression of an anti-apoptotic gene, Bcl-X(L). The adenovirus-mediated induction of Fas gene controlled by a tissue-specific promoter (e.g., myelin basic protein promoter) would be a promising therapeutic approach for malignant glioma.     

p14ARF induces G2 arrest and apoptosis independently of p53 leading to regression of tumours established in nude mice

Until recently, the ability of ARF (human p14(ARF), murine p19(ARF)) tumour-suppressor protein, encoded by the INK4A/ARF locus, to inhibit cell growth in response to various stimuli was related to its ability to stabilize p53 through the so-called ARF/MDM2/p53 pathway. However, recent data have demonstrated that ARF is not implicated in this unique p53-dependent pathway. By use of transient and stable expression, we show here that human p14(ARF) inhibits the growth of human tumoral cells lacking functional p53 by inducing a transient G(2) arrest and subsequently apoptosis. This p14(ARF)-induced G(2) arrest was correlated with inhibition of CDC2 activity, inactivation of CDC25C phosphatase and induction of the CDK inhibitor p21(WAFI). Apoptosis was demonstrated using Hoechst 33352 staining, proteolytic activation of caspase-3 and PARP cleavage. Similar results were obtained in experiments with cells synchronized by hydroxyurea block. Importantly, we were able to reproduce these effects 'in vivo' by showing that p14(ARF) inhibits the growth of p53 nullizygous human tumours in nude mice and induces the regression of p53 -/- established tumours. In these experiments, tumoral regression was associated with inhibition of cell proliferation as well as induction of apoptosis confirming the data obtained in cell lines.     

E2F-1 up-regulates c-Myc and p14(ARF) and induces apoptosis in colon cancer cells.

Although overexpression of E2F-1 can induce apoptosis in a variety of tumor cell lines, the mechanisms by which E2F-1 induces apoptosis remain ambiguous. In this study, we examine the ability of E2F-1 to induce apoptosis in colon cancer and the molecular mechanisms underlying E2F-1-mediated apoptosis. HT-29 and SW-620 colon adenocarcinoma cells (both mutant p53) were treated by mock infection or adenoviral vectors Ad5CMV (empty vector), Ad5CMVLacZ (beta-galactosidase), and Ad5CMVE2F-1 (E2F-1) at multiplicity of infection of 100. Western blot analysis confirmed marked overexpression of E2F-1 in both cell lines. By 5 days after infection, E2F-1 overexpression resulted in >25-fold reduction in cell growth and >90% loss of cell viability in both cell lines. Cell cycle analysis of Ad-E2F-1-infected cells revealed an increase in G(2)/M and sub-G(1) populations. By in situ terminal deoxynucleotidyl transferase (Tdt)-mediated nick end labeling analysis, evidence of apoptosis was observed including internucleosomal DNA fragmentation and the formation of apoptotic bodies. In addition, caspase-3 and poly(ADP-ribose) polymerase apoptotic fragments were detected by 48 h after treatment with Ad-E2F-1. Of mechanistic importance, overexpression of E2F-1 caused a G(2)/M arrest followed by increased levels of c-Myc and p14(ARF) proteins. Additionally, expression of the antiapoptotic Bcl-2 family member Mcl-1 was down-regulated in E2F-1-overexpressing cells. In conclusion, E2F-1 overexpression initiates apoptosis and suppresses growth in HT-29 and SW620 colon adenocarcinoma cells. Overexpression of E2F-1 triggers apoptosis and is associated with up-regulation of c-Myc and p14(ARF) proteins and down-regulation of Mcl-1. Therefore, E2F-1 is a potentially active gene therapy agent for the treatment of colon cancer.     

p14ARF homozygous deletion or MDM2 overexpression in Burkitt lymphoma lines carrying wild type p53

The hallmark of Burkitt lymphoma (BL) is a constitutively activated c-myc gene that drives tumor cell growth. A majority of BL-derived cell lines also carry mutant p53. In addition, the p16INK4a promoter is hypermethylated in most BL biopsies and BL cell lines, leading to silencing of this gene. Activation of c-myc and/or cell cycle dysregulation can induce ARF expression and p53-dependent apoptosis. We therefore investigated the p14ARF-MDM2-p53 pathway in BL cell lines. p14ARF was expressed and localized to nucleoli in all BL carrying mutant p53. Three out of seven BL carrying wt p53 had a homozygous deletion of the CDKN2A locus that encodes both p14ARF and p16INK4a. Three BL carrying wild type p53 retained the CDKN2A locus and overexpressed MDM2. DNA sequencing revealed a point mutation in CDKN2A exon 2 in one of these BL, Seraphine. However, this point mutation did not affect p14ARF's nucleolar localization or ability to induce p53. The Bmi-1 protein that negatively regulates the p14ARF promoter and co-operates with c-myc in tumorigenesis was expressed at low to moderate levels in all BL analysed. Our results indicate that inactivation of the ARF-MDM2-p53 pathway is an essential step during the development of Burkitt lymphoma, presumably as a mechanism to escape c-myc induced apoptosis.     

Expression of p14ARF overcomes tumor resistance to p53

Tumors without p53 mutation are often resistant to p53 gene therapy. We examined the mechanism using p53-resistant A549 cells and p53-sensitive H1299 cells. We found that p53 delivered by adenovirus is poorly expressed in A549 (ARF-null) cells but efficiently expressed in H1299 cells (ARF-positive). Strong p53 expression and apoptosis can be achieved in A549 cells using a p53 mutant resistant to degradation by MDM2 or by coexpression of ARF. The results suggest that enhanced MDM2 activity attributable to loss of ARF contributes to p53 resistance. Surprisingly, tumor cell lines with MDM2 gene amplification are still deficient for ARF expression, suggesting that MDM2 amplification does not substitute for ARF inactivation during tumor development.     

p14ARF在宫颈癌中的表达及其与p53表达相关性的研究

目的探讨宫颈癌组织p14ARF蛋白的表达及其与p53表达的相关性.方法应用免疫组化方法检测p14ARF、p53基因在41例宫颈癌及20例正常宫颈组织中的表达.结果p14ARF在正常宫颈组织中不表达,41例宫颈癌中35例表达阳性,占85.4%.病理分级为G1、G2级的宫颈癌的p4ARF阳性表达率为68.4%,G3级为100%,两者比较,有显著性差异(P＜0.05).宫颈癌组织中p53蛋白表达阳、阴性者中p14ARF蛋白表达阳性率分别为75.0%(12/16)和92.0%(23/25),两者比较,无显著性差异,p14ARF与p53表达不相关.结论p14ARF在宫颈癌中高表达有一定的诊断和估测预后价值,可能是宫颈癌新的肿瘤标志物.     

Hypermethylation-associated inactivation of p14(ARF) is independent of p16(INK4a) methylation and p53 mutational status

The INK4a/ARF locus encodes two cell cycle-regulatory proteins, p16INK4a andp14ARF, which share an exon using different reading frames. p14ARF antagonizes MDM2-dependent p53 degradation. However, no point mutations in p14ARF not altering p16INK4a have been described in primary tumors. We report that p14ARF is epigenetically inactivated in several colorectal cell lines, and its expression is restored by treatment with demethylating agents. In primary colorectal carcinomas, p14ARF promoter hypermethylation was found in 31 of 110 (28%) of the tumors and observed in 13 of 41 (32%) colorectal adenomas but was not present in any normal tissues. p14ARF methylation appears in the context of an adjacent unmethylated p16INK4a promoter in 16 of 31 (52%) of the carcinomas methylated at p14ARF. Although p14ARF hypermethylation was slightly overrepresented in tumors with wild-type p53 compared to tumors harboring p53 mutations [19 of 55 (34%) versus 12 of 55 (22%)], this difference did not reach statistical significance. p14ARF aberrant methylation was not related to the presence of K-ras mutations. Our results demonstrate that p14ARF promoter hypermethylation is frequent in colorectal cancer and occurs independently of the p16INK4a methylation status and only marginally in relation to the p53 mutational status.     

Human p14(ARF)-mediated cell cycle arrest strictly depends on intact p53 signaling pathways

The tumor suppressor ARF is transcribed from the INK4a/ARF locus in partly overlapping reading frames with the CDK inhibitor p16(Ink4a). ARF is able to antagonize the MDM2-mediated ubiquitination and degradation of p53, leading to either cell cycle arrest or apoptosis, depending on the cellular context. However, recent data point to additional p53-independent functions of mouse p19(ARF). Little is known about the dependency of human p14(ARF) function on p53 and its downstream genes. Therefore, we analysed the mechanism of p14(ARF)-induced cell cycle arrest in several human cell types. Wild-type HCT116 colon carcinoma cells (p53(+/+)p21(CIP1+/+) 14-3-3sigma(+/+)), but not p53(-/-) counterparts, underwent G(1) and G(2) cell cycle arrest following infection with a p14(ARF)-adenovirus. In p21(CIP1-/-) cells, p14(ARF) did not induce G(1) or G(2) arrest, while 14-3-3sigma(-/-) counterparts were mainly arrested in G(1), pointing to essential roles of p21(CIP1) in G(1) and G(2) arrest and cooperative roles of p21 and 14-3-3sigma in ARF-mediated G(2) arrest. Our data demonstrate a strict p53 and p21(CIP1) dependency of p14(ARF)-induced cell cycle arrest in human cells.     

p14ARF和p53在子宫内膜癌组织中的表达及其临床意义

目的研究子宫内膜癌组织中p14ARF和p53的表达及意义.方法采用免疫组化LSAB法,检测118例子宫内膜癌及30例正常宫内膜组织中p14ARF及p53的表达.结果p14ARF阴性、弱阳性、中度阳性和强阳性表达率在子宫内膜癌中分别为25.4%(30/118)、38.1%(45/118)、14.4%(17/118)和22.0%(26/118),与正常内膜0(0/30)、56.7%(17/30)、33.3%(10/30)和10.0%(3/30)相比差异有统计学意义,P<0.001,子宫内膜癌中p14ARF表达缺失的比例高于正常宫内膜;p14ARF表达水平与手术病理分期、宫颈间质受累和淋巴结转移相关;与病理分级、组织学类型和肌层侵犯深度无关;不同p14ARF表达水平患者生存率差异无统计学意义,P=0.578 1.p53阴性、弱阳性、中度阳性和强阳性表达率在子宫内膜癌中分别为13.6%(16/118)、40.1%(48/118)、28.0%(33/118)和17.8%(21/118);与正常内膜相比分别为36.7%(11/30)、36.7%(11/30)、16.2%(5/30)和10.0%(3/30)差异有统计学意义,P=0.01,子宫内膜癌中p53阳性表达的比例高于正常宫内膜;p53表达与肿瘤分期、盆腔淋巴结转移和肌层侵犯深度相关,与病理分级、组织学类型和宫颈间质受累无关;不同p53表达水平患者生存率差异无统计学意义,P=0.416 6;在子宫内膜癌组织中p14ARF与p53表达呈负相关,r＝-0.243,P＝0.008.结论p14ARF和p53异常表达与子宫内膜癌发生密切相关,但对其预后的影响有待进一步的研究.     

P14ARF sensitizes human osteosarcoma cells to cisplatin-induced apoptosis in a p53-independent manner

The tumor suppressor p14ARF, encoded by the INK4a/ARF locus, is often disrupted in human cancers, p14ARF triggers cell cycle arrest and sensitizes cells to apoptosis in the presence of collateral signals. To investigate the role of p14ARF in chemotherapeutic drugs-induced apoptosis, p14ARF was overexpressed by stable transfection in human osteosarcoma cell lines, U2OS (p53-wt/p14ARF-null) and MG63 (p53-mt/p14ARF-null). The results showed that ectopic p14ARF sensitized both cell lines to cisplatin-induced cytotoxicity and apoptosis. This sensitization of cisplatin-induced apoptosis was associated with upregulation of p53, Bax and p21 in U2OS cells. Conversely, such a result was not observed in MG63 cells. Moreover, the sensitization of cisplatin-induced cytotoxicity in U2OS cells was unaltered by p53 siRNA. Together, we show here p14ARF sensitizes human osteosarcoma cells to cisplatin-induced apoptosis in a p53-independent manner. Proper combinations of p14ARF gene transfer and conventional chemotherapy may be a valuable strategy in human osteosarcoma treatment.     

p14(ARF) modulates the cytolytic effect of ONYX-015 in mesothelioma cells with wild-type p53

ONYX-015 has been reported to kill selectively tumor cells lacking functional p53. Genetic alterations of INK4a/ARF locus, which is a predominant event in malignant pleural mesothelioma, may result in loss of p14(ARF) and subsequent disruption of p53 pathway in cancer cells. In the present study, ONYX-015 was able to kill three mesothelioma cell lines (H28, H513, and 211H) with wild-type p53 but lacking p14(ARF). In contrast, MS-1 mesothelioma cells, which expressed both p53 and p14(ARF), were resistant to ONYX-015. Introducing p14(ARF) gene into the H28 cell, a mesothelioma cell without p14(ARF) expression, significantly increased the resistance of this cell line to the cytolytic effect of ONYX-015. Our results suggest that human mesotheliomas with wild-type p53 yet lacking p14(ARF) are potential candidates for ONYX-015 therapy.     

Adenovirus-mediated tBid overexpression results in therapeutic effects on p53-resistant hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with a very high mortality. Because the success of the conventional therapies is limited, gene therapy may represent an alternative for HCC management. Our earlier study has shown that Bid plays a role in the development of HCC. The aim of our study is to evaluate the possibility of using truncated Bid (tBid) as a novel therapy for HCC treatment. Two HCC cell lines, Hep3B and PLC/PRF/5, were used in the experiment. Hep3B was a p53-resistant while PLC/PRF/5 a p53-sensitive. A recombinant adenovirus-Ad/AFPtBid, which contained a tBid gene driven by an alpha-fetoprotein (AFP) promoter, was constructed. Both Hep3B and PLC/PRF/5 cells infected with Ad/AFPtBid showed a significant decrease in cell viability. The decrease in cell viability by Ad/AFPtBid resulted from apoptosis of HCC cells, evident by enhanced activity of caspases and increased release of cytochrome c. In vivo experiment was performed by the intratumor injection of Ad/AFPtBid in nude mice inoculated with Hep3B. Ad/AFPtBid injection significantly inhibited tumor growth, and tumor tissues showed a marked increase in TUNEL-positive cells. Our experiment also demonstrated that Ad/AFPtBid only targeted AFP-producing cells but not those non-AFP producing cells. In conclusion, these results indicate that the introduction of Ad/AFPtBid can not only significantly but specifically kill HCC cells that produce AFP. The cell death induced by Ad/AFPtBid in HCC cells is via an apoptotic pathway that can be independent of p53 status.     

Wild-type p53 overexpression and its correlation with MDM2 and p14ARF alterations: an alternative pathway to non-small-cell lung cancer.

PURPOSE: We found a relatively reduced frequency of p53 mutation with a much greater frequency of p53 protein overexpression, which reflected stabilization of p53 protein in the absence of p53 gene mutation. Therefore, we investigated the possibility of alternative mechanisms leading to p53 protein stabilization. PATIENTS AND METHODS: We performed gene and protein alteration studies on p53 and its upstream effectors, MDM2 and p14ARF, in tumors from 94 non-small-cell lung cancer (NSCLC) patients. RESULTS: Immunohistochemical and sequencing analyses indicated that 37 tumors showed overexpression of wild-type p53. An absence of nuclear staining of MDM2 protein was found in 95% of these tumors (35 of 37; P < .001). The tumors with negative MDM2 staining showed a significantly high concordance of loss of Akt activity and low MDM2 mRNA expression (P < .001). Sequencing analysis revealed five distinct MDM2 splicing variants disrupting the conserved p53 binding domain. Corresponding variant proteins were detected in three lung cancer cell lines using the Western blot analysis. Our results also indicated that among the tumors with overexpression of the wild-type p53, 92% (34 of 37) showed immunoreactivity to p14ARF (P = .001). In addition, the deregulation of p53 and MDM2 genes was significantly associated with squamous lung cancer (P < .05) and was correlated with advanced stages (P < .05) and poor prognosis (P < .05). CONCLUSION: Our data suggest that immunopositivity of p14ARF together with a low expression of MDM2 contributes to accumulation of the wild-type p53, and that deregulation of the p53-MDM2-p14ARF pathway is important in the pathogenesis and outcome of a subset of NSCLC.     

Adenovirus-mediated transfer of p53 and Fas ligand drastically enhances apoptosis in gliomas

Various therapeutic approaches toward killing glioma cells by inducing apoptosis have been developed, but these approaches are often hampered by anti-apoptotic mechanisms. In this study, we attempted to develop a technique that overrides the resistance toward apoptosis in glioma cells. To date, p53- and Fas-mediated apoptotic pathways have been shown to be different. Therefore, we carried out a gene therapy that combines the pro-apoptotic effect of these two different pathways. The recombinant adenoviruses (Advs) for p53 and Fas ligand (FL) (Adv-p53 and Adv-FL, respectively) were constructed. Transfecting the p53 gene into glioma cell lines (A-172 and U251 glioma cells) led to overexpression of Bax, a protein that induces permeability transition; at the same time, this transfection brought about an overexpression of Fas. To intensify Fas-mediated apoptosis, we transferred the FL gene together with the p53 gene by Adv-mediated gene transduction into A-172 and U251 cells. Coinfecting Adv-p53 and Adv-FL into A-172 cells, which are relatively resistant to apoptosis by infection with Adv-p53 or Adv-FL alone (Adv-p53, multiplicity of infection (MOI) of 100: 8.5 +/- 0.7%; Adv-FL, MOI of 100: 3.0 +/- 0.1%) resulted in a drastic enhancement of the percentage of apoptotic cells (Adv-p53 and Adv-FL, each at an MOI of 30: 24.2 +/- 0.9%). Coinfection with Adv-p53 and Adv-FL in U251 cells resulted in a similar enhancement of the percentage of apoptotic cells (Adv-p53 and Adv-FL, each at an MOI of 30: 59.0 +/- 2.3%) compared with that seen in U251 cells transfected with Adv-p53 or Adv-FL alone (Adv-p53, MOI of 30: 3.1 +/- 0.3%; Adv-FL, MOI of 30: 18.1 +/- 0.3%). Regardless of whether a cell line is resistant or sensitive to FL- and p53-mediated apoptosis, coinfection with Adv-p53 and Adv-FL dramatically enhanced the degree of apoptosis of glioma cells. Our results indicate that the coinfection approach can be used as a modality for the gene therapy of gliomas, overriding the apoptosis-resistant mechanisms in glioma cells.     

p14ARF induces the relocation of HDM2 and p53 to extranucleolar sites that are targeted by PML bodies and proteasomes

Background  

p14ARF is a protein product of the alternative reading frame of the human INK4a locus. It functions as a tumor suppressor protein. p14ARF suppresses growth through p53-dependent and p53-independent pathways.     

Mdm2 overexpression and p14(ARF) inactivation are two mutually exclusive events in primary human lung tumors

Pathways involving p53 and pRb tumor suppressor genes are frequently deregulated during lung carcinogenesis. Through its location at the interface of these pathways, Mdm2 can modulate the function of both p53 and pRb genes. We have examined here the pattern of expression of Mdm2 in a series of 192 human lung carcinomas of all histological types using both immunohistochemical and Western blot analyses and four distinct antibodies mapping different epitopes onto the Mdm2 protein. Using Immunohistochemistry (IHC), Mdm2 was overexpressed as compared to normal lung in 31% (60 out of 192) of all tumors analysed, whatever their histological types. Western blotting was performed on 28 out of the 192 tumoral samples. Overexpression of p85/90, p74/76 and p57 Mdm2 isoforms was detected in 18% (5 out of 28), 25% (7 out of 28) and 39% (11 out of 28) of the cases respectively. Overall, overexpression of at least one isoform was observed in 14 out of 28 (50%) lung tumors and concomittant overexpression of at least two isoforms in 7 out of 28 (25%) cases. A good concordance (82%) was observed between immunohistochemical and Western blot data. Interestingly, a highly significant inverse relationship was detected between p14(ARF) loss and Mdm2 overexpression either in NSCLC (P=0.0089) or in NE lung tumors (P<0.0001). Furthermore, a Mdm2/p14(ARF) >1 ratio was correlated with a high grade phenotype among NE tumors overexpressing Mdm2 (P=0.0021). Taken together, these data strongly suggest that p14(ARF)and Mdm2 act on common pathway(s) to regulate p53 and/or pRb-dependent or independent functions and that the Mdm2 : p14(ARF) ratio might act as a rheostat in modulating the activity of both proteins.