Adenovirus-mediated p53 gene therapy in nasopharyngeal cancer

EBV-associated nasopharyngeal cancer (NPC) occurs with high frequency in China and is a major cause of morbidity and mortality. To explore the potential use of adenovirus-mediated tumor suppressor p53 gene therapy In NPC, we first examined the in vitro effects of p53 introduced into the NPC cell lines RPMI 2650, Fadu and Detroit 562. p21(WAF1/CIP1) induction by chemotherapy was used as a functional assay which revealed that RPMI 2650 expresses wild-type p53 whereas Fadu and Detroit 562 encode mutant p53. Infection with p53-expressing adenovirus (Ad-p53) induced apoptosis and inhibited cell growth in all three NPC cell lines, regardless of the endogenous p53 status. Adenovirus infectivity was greatest in RPMI 2650 cells, with 100% of the cells expressing beta-galactosidase following Ad-LacZ infection using an MOI of 100, as compared to 20-30% infectivity with the other NPC lines. Using RPMI 2650 cells injected into nude mice, we developed an animal model for nasopharyngeal cancer. Established tumors (0.6-0.8 cm) were injected with 5x10(9) PFU Ad-LacZ, Ad-p53 or PBS in a 100 mu l volume. We found evidence for in vivo expression of beta-galactosidase or p53 and p21 up to two weeks following Ad-LacZ or Ad-p53 virus injection respectively. Objective regression of tumor size was observed at two weeks in 4/6 Ad-p53-treated tumors, but not in Ad-LacZ or PBS-treated tumors. The results provide an animal model for human nasopharyngeal cancer, and indicate a potential use of p53 in its therapy in vivo.     

Adenovirus-mediated transfer of wild-type p53 gene results in apoptosis or growth arrest in human cultured gastric carcinoma cells.

We examined the susceptibility of six human gastric carcinoma cell lines to infection with recombinant p53 adenovirus vector (AxCA-p53). AxCA-p53 infection at a muliplicity of infection (MOI) of 50 resulted in apoptotic cell death (MKN-1 cells), growth arrest (MKN-45, MKN-74 and KATO-III cells), or non-effectiveness (TMK-1 and OCUM-2M cells). Western blot analysis revealed increasing expression levels of p21/WAF1 protein after infection with AxCA-p53 in all the cell lines. After infection with AxCA-p53, the expression levels of bax or bcl-XL protein changed in MKN-1, but not in the other cell lines. These results suggest that the apoptotic pathway (dependence on the expressions of bcl-2 family proteins) dominates the growth arrest pathway (dependence on the expressions of p21/WAF1 protein) after infection with AxCA-p53. Thus, the bcl-2 family might play a crucial role in p53-mediated growth arrest and apoptosis in human gastric carcinoma cells.     

Adenoviral p53 gene therapy promotes heat-induced apoptosis in a nasopharyngeal carcinoma cell line

Background: It has previously been demonstrated that Ad5CMV-p53 gene transfer, either used alone or delivered concomitantly with ionizing radiation, resulted in cytotoxicity mediated by apoptosis in nasopharyngeal carcinoma (NPC) cell lines. In this study, a novel approach was evaluated of combining Ad5CMV-p53 gene therapy with hyperthermia (HT), in the CNE-1 NPC cell line, which harbours a mutation in codon 249 of the p53 gene. Materials and methods: CNE-1 cells were infected using either Ad5CMV-p53 or Ad5CMV-B-gal, followed, 24 h later, by HT (43°C x 0-2h). Protein was extracted for Western blot analysis, and apoptosis was evaluated using acridine-orange ethidium bromide staining, followed immediately by fluorescent microscopy examination for the proportion of cells displaying morphologic features of apoptosis. Results: Ad5CMV-p53 gene therapy combined with HT resulted in a dose-dependent cytotoxicity with less than 1% clonogenic survival when 10 pfu/cell of Ad5CMV-p53 was combined with 2h heating at 43°C. Western blotting demonstrated that treatment with Ad5CMV-p53 resulted in the rapid expression of p53, which was minimally affected by HT. The inducible form of hsp70 was maximally expressed at 48h post-HT, with minimal effect when cells were additionally treated with Ad5CMV-p53. Clonogenic cytotoxicity was associated with the development of apoptosis, with up to 70% of CNE-1 cells displaying morphologic features of apoptosis after the combination treatments. Conclusion: Based on the shapes of the clonogenic survival curves, Ad5CMV-p53 gene therapy and HT appear to interact in an additive manner, suggesting the therapeutic potential of this combined treatment approach for patients with NPC.     

Adenovirus-mediated PEDF expression inhibits prostate cancer cell growth and results in augmented expression of PAI-2

PEDF is one of the most potent inhibitor of angiogenesis. Loss of PEDF was found in human prostate tumors and associated with the progression toward a metastatic phenotype. To test the therapeutic potential of PEDF, we constructed a replication-defective adenoviral vector capable of efficient transduction and expression of PEDF (Ad-PEDF) in PC-3 prostate carcinoma cells. As controls, we used adenoviruses expressing beta-galactosidase (Ad-LacZ). We showed that overexpression of PEDF inhibited proliferation of cells and augmented apoptosis in serum-starved cells, in comparison with Ad-LacZ. Furthermore, Ad-PEDF suppresses anchorage-independent growth cells in soft agar and tumor formation in athymic nude mice associated with decreased microvessel density. Microarray analysis showed that 56 out of 8464 genes were found to be upregulated and downregulated in the PC-3 infected with Ad-PEDF as compared with Ad-LacZ. The differentially expressed genes cover a broad range of functional activities including catalytic activity, protein binding, signal transduction activity and cell invasion. Among them, PAI-2 and DRHC were confirmed to be upregulated with real-time PCR and Western blot, suggesting a possible association with PEDF-induced signaling for cell motility and metastasis. These results can contribute to our understanding of the molecular mechanisms of treatment strategies of PEDF for prostate cancer.     

Adenovirus-mediated p16INK4a gene expression radiosensitizes non-small cell lung cancer cells in a p53-dependent manner

We examined the influence of adenovirus-mediated wild-type p16INK4a (Ad/p16) expression on the radiation sensitivity of NSCLC cell lines, all of which lacked constitutive p16INK4a but each of which varied in p53 status: A549 (-p16INK4a/ +pRb/wt-p53), H322 (-p16INK4a/ +pRb/mt-p53), and H1299 (-p16INK4a/ +pRb/deleted-p53). The in vitro clonogenic survival results indicate that Ad/p16 enhanced the radiosensitivity of A549 but not H322 or H1299. Further analysis indicated that the apoptosis induced by combination therapy using Ad/p16 plus irradiation was dependent on the endogenous p53 status of the cancer cells. We performed Western blotting to analyse the p53 protein expression of A549 cells treated with either Ad/p16 or Ad/Luc. Endogenous p53 protein levels were higher in A549 cells transfected with Ad/p16 than in those transfected with Ad/Luc. Furthermore, when wt-p53 protein expression was restored in H1299 using Ad/ p53, Ad/p16 stabilized p53 protein expression and radiosensitized the cells. These results suggest that Ad/ p16-induced stabilization of p53 protein may play an important role in Ad/p16 mediated radiosensitization by enhancing or restoring apoptosis properties. Thus, Ad/ p16 plus radiation in combination may be a useful gene therapy strategy for tumors that have wt-p53 but nonfunctional p16INK4a.     

p53 Regulates insulin-like growth factor-I receptor gene expression in uterine serous carcinoma and predicts responsiveness to an insulin-like growth factor-I receptor-directed targeted therapy

The role of the insulin-like growth factors (IGF) in endometrial cancer has been well established. The IGF-I receptor (IGF-IR), which mediates the biological actions of IGF-I, is usually overexpressed in endometrial tumours. Uterine serous carcinoma (USC) constitutes a defined histological category among endometrial cancers. Mutation of the p53 gene appears early in the course of the disease and is considered a key event in the initiation of USC. The aim of the present study was to evaluate the potential interactions between p53 and the IGF-IR in USC. In addition, we investigated the role of p53 as a biomarker in IGF-IR targeted therapies. Immunohistochemical analysis in a collection of 35 USC specimens revealed that IGF-IR is highly expressed in primary and metastatic USC. Likewise, p53 was expressed in 85.7% of primary tumours and 100% of metastases. A significant negative correlation between p53 expression and survival was noticed. In addition, using USC-derived cell lines we provide evidence that p53 regulates IGF-IR gene expression via a mechanism that involves repression of the IGF-IR promoter. We show that the mechanism of action of p53 involves interaction with zinc finger protein Sp1, a potent transactivator of the IGF-IR gene. Finally, we demonstrate that USC tumours overexpressing p53 are more likely to benefit from anti-IGF-IR therapies. In summary, we provide evidence that p53 regulates IGF-IR gene expression in USC cells via a mechanism that involves repression of the IGF-IR promoter. The interplay between the p53 and IGF-I signalling pathways is of major basic and translational relevance.     

Adenoviral p53 gene therapy promotes heat-induced apoptosis in a nasopharyngeal carcinoma cell line.

BACKGROUND: It has previously been demonstrated that Ad5CMV-p53 gene transfer, either used alone or delivered concomitantly with ionizing radiation, resulted in cytotoxicity mediated by apoptosis in nasopharyngeal carcinoma (NPC) cell lines. In this study, a novel approach was evaluated of combining Ad5CMV-p53 gene therapy with hyperthermia (HT), in the CNE-1 NPC cell line, which harbours a mutation in codon 249 of the p53 gene. MATERIALS AND METHODS: CNE-1 cells were infected using either Ad5CMV-p53 or Ad5CMV-B-gal, followed, 24 h later, by HT (43 degrees C x 0-2 h). Protein was extracted for Western blot analysis, and apoptosis was evaluated using acridine-orange ethidium bromide staining, followed immediately by fluorescent microscopy examination for the proportion of cells displaying morphologic features of apoptosis. RESULTS: Ad5CMV-p53 gene therapy combined with HT resulted in a dose-dependent cytotoxicity with less than 1% clonogenic survival when 10 pfu/cell of Ad5CMV-p53 was combined with 2 h heating at 43 degrees C. Western blotting demonstrated that treatment with Ad5CMV-p53 resulted in the rapid expression of p53, which was minimally affected by HT. The inducible form of hsp70 was maximally expressed at 48 h post-HT, with minimal effect when cells were additionally treated with Ad5CMV-p53. Clonogenic cytotoxicity was associated with the development of apoptosis, with up to 70% of CNE-1 cells displaying morphologic features of apoptosis after the combination treatments. CONCLUSION: Based on the shapes of the clonogenic survival curves, Ad5CMV-p53 gene therapy and HT appear to interact in an additive manner, suggesting the therapeutic potential of this combined treatment approach for patients with NPC.     

p53 expression and p21 expression are mutually exclusive in esophageal squamous cell carcinoma

The accumulation of p53 protein, which is considered to be caused by a p53 gene mutation, is closely associated with poor prognosis in patients with certain types of carcinomas. The progression of esophageal squamous cell carcinoma (ESCC) is also suspected to depend on p53 gene status. We analyzed the relationship between p53 and p21 protein accumulation in ESCC, and simultaneously analyzed the frequency of apoptosis. Formalin-fixed paraffin-embedded sections were taken from 46 patients who underwent esophagectomy for ESCC. These sections were examined by immunostaining with monoclonal antibodies PAb1801 and EA10 to determine p53 and p21 protein accumulation, respectively. We also analyzed the frequency of apoptosis by TdT-mediated dUTP-biotin nick end-labeling (TUNEL). For estimation of the proportion of stained cells, we used computer analysis with NIH image analysis software. p21 protein accumulation showed an almost inverse distribution to that of p53 protein. In areas where both p53 and p21 proteins were accumulated, few apoptotic cells were observed. Particularly in cases of mucosal tumors, p53 protein was prominently accumulated in the lower layer of the tumor, whereas p21 protein accumulation was confined to the upper layer. Our results suggest that progression of esophageal squamous cell carcinoma is controlled by a p53-dependent pathway.     

Adenovirus-mediated p53 gene therapy in osteosarcoma cell lines: sensitization to cisplatin and doxorubicin

The poor prognosis for patients with metastatic osteosarcoma (OS) indicates that new therapeutic options should be explored. Studies with adenoviral-mediated p53 gene transfer have been conducted in many cancer types including cervical, ovarian, prostatic and head and neck tumors. However, limited work has been carried out with pediatric cancers, including OS. Using three viral constructs containing cDNA for wild-type p53, mutant p53 (Cys135Ser) and lacZ, we studied the effect of adenoviral-mediated gene therapy in four OS cell lines: Saos-2 (p53-/-), HOS (R156P), KHOS/NP (R156P) and MNNG (R156P, F270L). We demonstrated that the virus efficiently enters the cells using the beta-galactosidase assay. Using the MTT assay, we have shown a dose-dependent decrease in cell viability 72 h post-treatment that occurs with Ad-wtp53 but not with Ad-mutp53. We have also shown that treatment with Ad-wtp53 significantly increases sensitivity of the cell lines to cisplatin and doxorubicin, chemotherapeutic agents commonly used in the treatment of OS. Our results indicate that restoration of wt p53 function in OS cells provides a basis for novel approaches to treatment of this disease.     

子宫内膜增生及癌变组织中增殖细胞核抗原与p16和p53基因表达的相关性研究

目的探讨增殖细胞核抗原（PCNA）及基因p16、p53在单纯性子宫内膜增生（ESH）、子宫内膜不典型增生（EAH）和子宫内膜癌（EC）组织中表达情况。方法应用免疫组化法分别对26例EC、28例EAH、24例ESH组织进行PCNA和p16、p53基因蛋白定位及表达程度检测,并对比其差异,分析它们之间的关系。结果PCNA在ESH、EAH和EC3组病例中强阳性表达分别为16．7％、82．1％和96．2％,ESH与EAH、EC比较差异有统计学意义（P〈0．01）,而EAH与EC比较差异无统计学意义（P〉0．05）。p16在ESH、EAH和EC3组病例中阳性表达分别为95．8％、46．4％和50．0％,ESH与EAH、Ec组比较差异有统计学意义（P〈0．05）;EAH与EC组比较差异无统计学意义（P〉0．05）。p53在ESH组无阳性表达病例,EAH组阳性率25％,EC组阳性率65．4％,3组病例之间比较差异均有统计学意义（P〈0．01）。结论PCNA、p53表达在子宫内膜增殖性病变、细胞恶性转化的进程中呈递增趋势,而p16趋于递减,提示细胞的过度增殖和增殖-凋亡相关基因的表达失衡在子宫内膜恶性转化过程中起重要作用。PCNA、p16和p53的异常表达可作为子宫内膜增生性疾病不同发展阶段的诊断及预测其生物学行为的参考指标。     

子宫内膜增生及癌变组织中增殖细胞核抗原与p16和p53基因表达的相关性研究

目的探讨增殖细胞核抗原(PCNA)及基因p16、p53在单纯性子宫内膜增生(ESH)、子宫内膜不典型增生(EAH)和子宫内膜癌(EC)组织中表达情况。方法应用免疫组化法分别对26例EC、28例EAH、24例ESH组织进行PCNA和p16、p53基因蛋白定位及表达程度检测,并对比其差异,分析它们之间的关系。结果PCNA在ESH、EAH和EC 3组病例中强阳性表达分别为16．7％、82．1％和96．2％,ESH与EAH、EC比较差异有统计学意义(P＜0．01),而EAH与EC比较差异无统计学意义(P＞0．05)。p16在ESH、EAH和EC 3组病例中阳性表达分别为95．8％、46．4％和50．0％,ESH与EAH、EC组比较差异有统计学意义(P＜0．05);EAH与EC组比较差异无统计学意义(P＞0．05)。p53在ESH组无阳性表达病例,EAH组阳性率25％,EC组阳性率65．4％,3组病例之间比较差异均有统计学意义(P＜0．01)。结论PCNA、p53表达在子宫内膜增殖性病变、细胞恶性转化的进程中呈递增趋势,而p16趋于递减,提示细胞的过度增殖和增殖-凋亡相关基因的表达失衡在子宫内膜恶性转化过程中起重要作用。PCNA、p16和p53的异常表达可作为子宫内膜增生性疾病不同发展阶段的诊断及预测其生物学行为的参考指标。     

Adenovirus-mediated gene transfer of wild-type p53 results in melanoma cell apoptosis in vitro and in vivo

Gene transfer techniques may provide efficient treatment for a variety of malignant neoplasms. A replication-deficient adeno-virus (Ad) vector which carries the cDNA for wild-type p53 (AdCMV.p53) was tested for its in vitro and in vivo effects on the growth of murine melanoma cell line B16-G3.26 and human melanoma cell line SK-MEL-24. The growth of B16-G3.26 cells infected with AdCMV.p53 was inhibited when compared to the uninfected cells or cells infected with the control vector AdCMV.NLSβgal. Similarly, the growth of SK-MEL-24 cells infected with AdCMV.p53 was also below that of AdCMV.NLSβgal-infected and uninfected controls. DNA laddering using agarose gel electrophoresis and in situ labeling of DNA fragmentation (TUNEL) showed that AdCMV.p53-infected murine and human melanoma cells underwent apoptosis. Nude mice injected s.c. either with B16-G3.26 cells or with SK-MEL-24 cells developed localized tumors. These tumors were subsequently infiltrated with either AdCMV.p53, AdCMV.NLSβgal or saline alone. One week after infection, B16-G3.26 tumors exposed to AdCMV.p53 were 2.5 times smaller than control tumors and exhibited DNA fragmentation. A similar growth-inhibitory effect of AdCMV.p53 was observed with SK-MEL-24 tumors. Thus, Ad-mediated wild-type p53 overexpression resulted in melanoma cell apoptosis and inhibition of melanoma growth in vitro and in vivo. These gene therapy approaches may be useful in targeting rapidly growing, malignant melanomas in a clinical setting. © 1995 Wiley-Liss, Inc.     

胃癌中p21WAF1与p53基因的相关性研究

目的　探讨胃癌中 p2 1WAF 1与 p5 3基因的关系。方法　采用 S- P法检测 p2 1WAF 1和 p5 3在胃癌中的表达。结果　 p2 1与 p5 3在正常胃黏膜、不典型增生、胃癌中的阳性表达率分别为 10 0 .0 %与 0 %、92 .5 %与 15 .0 %、39.8%与 5 6 .5 % ;40例不典型增生 ,p5 3阳性而 p2 1阴性者为 5 .0 % ,p5 3阴性而 p2 1阳性为 82 .5 % (P<0 .0 5 ) ;p2 1、p5 3阳性高分化腺癌各为 6 3.3%与 36 .7% ,与低分化腺癌 32 .7%与 78.2 %比较 ,差异有显著性 (P<0 .0 5 ) ;48.1%的胃癌 p2 1、p5 3蛋白表达相反 (P>0 .0 5 ) ;p2 1表达在侵犯浅肌层为 6 0 .0 %显著高于深肌层的 35 .2 % (P<0 .0 5 ) ;p2 1、p5 3在原发与转移癌中一致表达为 35 .3%与 70 .6 % ,皆与未转移癌 6 2 .5 %与 42 .5 %有显著性差异 (P<0 .0 5 ) ;p2 1在 TNM 期 6 0 .0 %、 期 5 6 .2 %显著高于 期 2 7.8%、 期 2 2 .2 % (P<0 .0 5 )。结论　正常胃黏膜和不典型增生 p2 1蛋白表达大部分依赖 p5 3蛋白 ,而胃癌在相当程度上不依赖。p2 1失表达和 p5 3异常表达与胃癌发生、分化程度、转移有关 ,而 p2 1失表达还与侵袭、临床分期关系密切     

Cisplatin-induced p53-independent growth arrest and cell death in cancer cells

Classical Kaposi's sarcoma (CKS) is a rare indolent proliferative disease which is particularly prevalent among Jews of Ashkenazi and Mediterranean origin. To define guidelines for its comprehensive management, we conducted a retrospective analysis of 123 patients, focusing mainly on treatment modalities. The CKS-related mortality was 4% (5 patients). Of the 39 patients for whom observation only was the primary approach, 15 (38%) remained progression-free for 1-83 months (median, 4 months). Twenty-nine of the 52 (56%) patients who underwent surgery as the primary approach remained recurrence-free for 1-162 months (median, 15 months). Radiotherapy achieved an objective response in 74 courses (85%), including 50 (58%) complete responses. Symptomatic relief was reported in 95% of the patients. Vinblastine (27 series) achieved an objective response in 73% of series, including 22% complete responses. Multivariate analysis of time to progression with observation alone identified immunosuppression as the only significant independent factor that predicted disease progression. Our study suggests that observation alone may be sufficient for immunocompetent asymptomatic patients; symptomatic resectable lesions are suitable for simple excision; and more advanced disease or unresectable lesions require radiotherapy. If disease is extensive or the other approaches fail, chemotherapy is appropriate. Tailoring the treatment for CKS is an integrative process, requiring good understanding of the role of each available modality in the different clinical disease settings.     

Adenovirus-mediated delivery of the PTEN gene inhibits cell growth by induction of apoptosis in endometrial cancer

PTEN, a gene encoding a dual specificity phosphatase, is frequently altered in endometrial carcinoma. Moreover, these alterations are observed even in atypical hyperplasia of the endometrium. This evidence suggests that mutation of PTEN is an early genetic alteration involved in endometrial carcinogenesis. Adenovirus-mediated gene transfer was carried out using Ishikawa 3 H 12 and RL95-2, the endometrial cancer cell lines with completely inactivated PTEN, together with endometrial cancer cell lines HEC1-A and KLE expressing wild-type PTEN as the control. The PTEN transgene significantly suppressed cell growth in vitro through induction of apoptosis in cells lacking wild-type PTEN. Furthermore, the ex vivo tumor formation by Ishikawa 3 H 12 cells was completely inhibited by the introduction of wild-type PTEN. However, neither regression nor progression was observed in inoculated tumors of either cell line by in vivo introduction of the PTEN gene. These results suggest that PTEN may be a good candidate for gene therapy in patients with endometrial carcinoma.     

Adenovirus-mediated p53 gene therapy in pediatric soft-tissue sarcoma cell lines: sensitization to cisplatin and doxorubicin

Sarcomas, or tumors of connective tissue, represent roughly 20% of childhood cancers. Although the cure rate for sarcomas in general has significantly improved in the last 10 years, there continue to be subgroups that are difficult to treat. High-grade or metastatic soft-tissue sarcomas and rhabdomyosarcomas (RMS) of the extremities remain therapeutic challenges and their prognosis is often poor. The future of sarcoma therapy will likely include molecular approaches including gene/protein expression profiling and gene-based therapy. Most sarcomas harbor defects in the p53 or pRb pathways. The tumor suppressor p53 is central to regulation of cell growth and tumor suppression and restoring wild-type p53 function in pediatric sarcomas may be of therapeutic benefit. Studies with adenoviral-mediated p53 gene transfer have been conducted in many cancer types including cervical, ovarian, prostatic and head and neck tumors. Studies of this approach, however, remain limited in pediatric cancers, including sarcomas. Using three viral constructs containing cDNA for wild-type p53, mutant p53 (C135S) and lacZ, we studied the effect of adenoviral-mediated gene therapy in four pediatric sarcoma cell lines, RD and Rh4 (RMS), Rh1 (Ewing's sarcoma) and A204 (undifferentiated sarcoma). Using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, we have shown a dose-dependent decrease in cell viability 72 h post-treatment that occurs with Ad-wtp53 but not with Ad-mutp53. Cells treated with Ad-wtp53 show upregulation of the p53 downstream targets, p21(CIP1/WAF1) and bax. Growth curves demonstrate suppression of cell growth over a period of 4 days and cells treated with Ad-wtp53 demonstrate a significant increase in sensitivity to the chemotherapeutic agents, cisplatin and doxorubicin. Our results indicate that restoration of wild-type p53 function in pediatric sarcoma cells could provide a basis for novel approaches to treatment of this disease.     

Adenoviral p53 gene therapy in head and neck squamous cell carcinoma cell lines

We reconstructed the recombinant p53-expressing adenovirus and examined its infections and effects in head and neck squamous cell carcinoma cell lines. Eight human head and neck squamous cell carcinoma cell lines were infected by the recombinant adenovirus harboring the lacZ gene (AxCAiLacZ) or the wild-type p53 gene (AxCAip53), and the effects were investigated. The eight cell lines were successfully infected by AxCAiLacZ at a level of more than 50%. The survival of all 8 squamous cell lines were inhibited in the range from 8 to 26.7% by only one treatment of the AxCAip53 infection. This result suggested that p53 gene therapy might become a useful tool in head and neck squamous cell carcinoma treatment.     

Restoration of RUNX3 enhances transforming growth factor-beta-dependent p21 expression in a biliary tract cancer cell line

RUNX3 is a candidate tumor suppressor gene localized in 1p36, a region commonly inactivated by deletion and methylation in various human tumors. To elucidate the role of RUNX3 in transforming growth factor (TGF)-beta signaling in biliary tract cancer, we transfected Mz-ChA-2 cells, which do not express RUNX3 but have intact TGF-beta type II receptor and SMAD4 genes, with the RUNX3 expression plasmid pcDNA3.1/RUNX3 or with the vector pcDNA3.1 as a control. Four Mz-ChA-2/RUNX3 clones and one control clone were obtained. Although TGF-beta1 only slightly inhibited growth of the control cells, growth inhibition and TGF-beta-dependent G(1) arrest were significantly enhanced in the RUNX3-transfected clones. None of the clones, however, exhibited apoptosis. The slightly increased TGF-beta1-induced p21 expression in the control clone was strongly enhanced in the RUNX3-transfected clones, and was accompanied by augmented decreases in the expression of cyclins D1 and E. When RUNX3 small interfering RNA was added, TGF-beta-dependent induction of p21 was reduced in the RUNX3-transfected clones. Xenografts of the clones in nude mice demonstrated that tumorigenicity was significantly decreased in the RUNX3-transfected clones in inverse proportion to the expression levels of RUNX3. Based on these results, RUNX3 is involved in TGF-beta-induced expression of p21 and the resulting induction of TGF-beta-dependent G(1) arrest.     

Effects of p53 gene therapy in radiotherapy or thermotherapy of human head and neck squamous cell carcinoma cell lines

We report herein the effects of p53 gene therapy in the radiotherapy or thermotherapy of eight human head and neck squamous cell carcinoma (SCC) cell lines. The discrepancy between radiosensitivity combined with p53 gene therapy than that without p53 gene therapy increased among the eight SCC cell lines. The discrepancy increased in the thermosensitivity at 43 degrees C and decreased in that at 44 degrees C among the eight SCC cell lines. Thus, the p53 gene therapy did not always improve the discrepancy between radiosensitivity and thermosensitivity in the eight SCC cell lines. In the radiotherapy combined with adenoviral p53 gene therapy, the survival rates of three of eight SCC cell lines decreased, and that of only one cell line increased compared with radiotherapy alone. In thermotherapy combined with p53 gene therapy, the survival rates of three at 44 degrees C and five at 43 degrees C of the eight SCC cell lines decreased, although only one cell line at 43 degrees C increased its survival rate compared with thermotherapy alone. The p53 gene therapy decreased the survival rates of both radiotherapy and thermotherapy in three of eight SCC cell lines. Further, the distribution of plots on the basis of the time for 10% survival of radiotherapy and the dose for 10% survival of thermotherapy with p53 gene therapy shifted to the lower left side of the plots compared with those without p53 gene therapy. These findings indicated that p53 gene therapy improves the effects of both radiotherapy and thermotherapy.