Treatment of human renal cell carcinoma by a conditionally replicating herpes vector G207

PURPOSE: Surgical removal remains the only potentially curative therapy for renal cell carcinoma. In this study we evaluated the inhibitory effect of the replication competent engineered herpes simplex virus type 1, G207, for renal cell carcinoma in vitro and in vivo. MATERIALS AND METHODS: The nature of G207 enables it to replicate within cancer cells, thus, causing cytolysis, but replication is restricted within normal cells. The susceptibility of the human renal cancer cell lines ACHN and A498 to G207 at a multiplicity of infection of 0.1 was examined. In addition, the growth characteristics of G207 was assessed. In vivo athymic mice bearing subcutaneous tumors were inoculated with 1 x 10(7) plaque forming units of G207 intra-neoplastically. For pathological analysis subcutaneous tumors were stained with X-gal. RESULTS: Two cell lines were efficiently destroyed by G207 within 1 week. The viral yields of G207 increased in a time dependent manner. In vivo the intra-neoplastic inoculation of G207 caused significantly decreased tumor growth in athymic mice harboring subcutaneous human renal cancer cells. On day 14 the mean growth ratio of ACHN and A498 lesions was significantly inhibited in G207 treated compared to control tumors (p <0.005 and <0.0001, respectively). CONCLUSIONS: These results suggest that G207 should be considered another potential therapeutic agent for renal cell carcinoma.     

Effective treatment of pancreatic tumors with two multimutated herpes simplex oncolytic viruses

Pancreatic cancer is an aggressive, rapidly fatal disease against which current nonsurgical therapy has minimal impact. This study evaluates the efficacy of two novel, replication-competent, multimutated herpes viruses (G207 and NV1020) in an experimental model of pancreatic cancer. Four human pancreatic carcinoma cell lines were exposed to G207 or NV1020, and cell survival and viral progeny production were determined. Flank tumors in athymic mice were subjected to single or multiple injections of 1 X 10⁷ G207 or NV1020, and tumor volume was evaluated over time. For all of the cell lines, G207 and NV1020 produced infection, viral replication, and cell lysis (P <0.05). NV1020 resulted in a higher production of viral progeny compared to G207. The efficacy of viral tumor cell kill was greatest in those cells with the shortest in vitro doubling time. For flank tumors derived from hs766t, single or multiple injections of both viruses were equally effective and significantly reduced flank tumor burden (P <0.05). Complete hs766t flank tumor eradication was achieved in 25% (5 of 20) of animals treated with G207 and 40% (8 of 20) of animals treated with NV1020. In vivo efficacy correlated with in vivo tumor doubling time. There were no adverse effects related to viral administration observed in any animal. NV1020 and G2O7 effectively infect and kill human pancreatic cancer cells in vitro and in vivo. Given the lack of effective nonoperative treatments for pancreatic cancer, oncolytic herpes viruses should be considered for clinical evaluation. Presented in part at the Forty-First Annual Meeting of The Society for Surgery of the Alimentary Tract, San Diego, Calif., May 21–24, 2000.     

Bmi-1基因诱导人胰腺导管上皮细胞恶性转化

【摘要】 目的 探讨原癌基因B细胞特异性莫洛氏鼠白血病病毒插入位点-1(Bmi-1)过表达对人正常胰腺上皮细胞株hTERT-HPNE恶性转化的影响。方法〓采用逆转录病毒介导转染方法将携带原癌基因Bmi-1的质粒或空质粒稳定转染hTERT-HPNE,通过Real-time PCR及Western blot在mRNA及蛋白水平鉴定转染效果。采用MTS法、Traswell小室法、软琼脂克隆形成试验检测稳定转染Bmi-1对hTERT-HPNE细胞增殖、迁移、侵袭和软琼脂克隆形成能力的影响。结果〓Real-time PCR及Western blot结果均表明成功建立稳定转染Bmi-1基因的hTERT-HPNE细胞株。过表达Bmi-1基因使hTERT-HPNE细胞增殖、迁移、侵袭和软琼脂克隆形成能力明显增高。结论〓在细胞水平过表达Bmi-1基因恶性转化hTERT-HPNE细胞。     

Carcinoembryonic antigen directed herpes viral oncolysis improves selectivity and activity in colorectal cancer

BACKGROUND: G207 is an oncolytic herpes virus whose replicative cycle requires cellular ribonucleotide reductase (RR) for viral DNA synthesis. We attempt to enhance viral cytotoxicity in carcinoembryonic antigen (CEA)-producing colorectal cancer (CRC) cells through CEA-driven RR production. METHODS: CEA enzyme-linked immunosorbent assay was performed on LS174T and HCT-8 human CRC cells. The CEA enhancer-promoter (CEA E-P) was functionally assessed by luciferase assay. CEA E-P was cloned upstream of UL39, the gene encoding the large subunit of RR. Cells were transfected with CEA E-P/UL39 and infected with G207 for cytotoxicity assays. LS174T, with or without CEA E-P/UL39, were implanted into athymic mouse flanks (n = 28) and treated with G207. RESULTS: CEA levels were 7-fold higher in LS174T versus HCT-8 ( P <.00001). CEA E-P increased luciferase expression 7.5-fold in LS174T ( P <.01), with no increase in HCT-8. G207 cytotoxicity of'CEA E-P/UL39-transfected LS174T cells increased 69% by day 10 versus nontransfected cells ( P <.001), with no significant increase in HCT-8. Combining CEA E-P/UL39 with G207 in LS174T flank tumors resulted in a 65% decrease in tumor volume versus G207, phosphate-buffered saline, or'CEA E-P/UL39 alone ( P <.0001). CONCLUSIONS: CEA-driven RR production by CEA-secreting CRC cells significantly improves oncolytic viral cytotoxicity and specificity in vitro, and reduces tumor burden in vivo.     

Utilizing tumor hypoxia to enhance oncolytic viral therapy in colorectal metastases

OBJECTIVE: To determine the effects of hypoxia-induced ribonucleotide reductase (RR) production on herpes oncolytic viral therapy. SUMMARY BACKGROUND DATA: Hypoxia is a common tumor condition correlated with therapeutic resistance and metastases. Attenuated viruses offer a unique cancer treatment by specifically infecting and lysing tumor cells. G207 is an oncolytic herpes virus deficient in RR, a rate-limiting enzyme for viral replication. METHODS: A multimerized hypoxia-responsive enhancer was constructed (10xHRE) and functionally tested by luciferase assay. 10xHRE was cloned upstream of UL39, the gene encoding the large subunit of RR (10xHRE-UL39). CT26 murine colorectal cancer cells were transfected with 10xHRE-UL39, incubated in hypoxia (1% O2) or normoxia (21% O2), and infected with G207 for cytotoxicity assays. CT26 liver metastases, with or without 10xHRE-UL39, were created in syngeneic Balb/C mice (n = 40). Livers were treated with G207 or saline. Tumors were assessed and stained immunohistochemically for G207. RESULTS: 10xHRE increased luciferase expression 33-fold in hypoxia versus controls (P < 0.001). In normoxia, 10xHRE-UL39 transfection did not improve G207 cytotoxicity. In hypoxia, G207 cytotoxicity increased 87% with 10xHRE-UL39 transfection versus nontransfected cells (P < 0.001). CT26 were resistant to G207 alone. Combining 10xHRE-UL39 with G207 resulted in a 66% decrease in tumor weights (P < 0.0001) and a 65% reduction in tumor nodules (P < 0.0001) versus G207 monotherapy. 10xHRE-UL39-transfected tumors demonstrated greater viral staining. CONCLUSIONS: Hypoxia-driven RR production significantly enhances viral cytotoxicity in vitro and reduces tumor burden in vivo. G207 combined with RR under hypoxic control is a promising treatment for colorectal cancer, which would otherwise be resistant to oncolytic herpes virus alone.     

Gene transfer into rat renal cells using adeno-associated virus vectors

Adeno-associated virus (AAV) vectors have a number of attractive features, including lack of cytotoxicity, ability to transduce nondividing cells, and long-term transgene expression. We investigated whether rat renal cells could be efficiently transduced with AAV vectors. Rat glomerular mesangial cells were transduced with AAV-lacZ vector containing beta-galactosidase gene in vitro, and the expression of beta-galactosidase was evaluated by X-gal staining and ELISA. For ex vivo experiments, sections of rat kidneys were incubated with AAV-lacZ, and then evaluated by X-gal histochemical staining. The level of beta-galactosidase expression in cultured rat mesangial cells increased in a dose-dependent manner (ranging from 1 x 10(5) to 5 x 10(6) particles/cell). When transduced with 5 x 10(6) vector particles/cell of AAV-lacZ, about 50% of mesangial cells were stained positively with X-gal, and the level of beta-galactosidase expression reached 9.9 +/- 1.5 ng/mg protein. Expression was detectable during the culture period for at least 7 days. X-gal histochemical examination of the ex vivo transduced renal tissue revealed tubular cell and interstitial tissue staining. However, gene transfer was not clearly observed in glomeruli. These findings suggest that AAV vectors have the potential for gene therapy of renal diseases.     

Utilizing alpha-fetoprotein expression to enhance oncolytic viral therapy in hepatocellular carcinoma

OBJECTIVE: To determine whether alpha-fetoprotein (AFP)-regulated ribonucleotide reductase (RR) production would promote more vigorous and specific viral killing in AFP-expressing hepatocellular carcinoma (HCC). BACKGROUND: AFP is expressed in over 70% of primary HCC but not in normal adult liver. AFP production by HCC can be exploited to target viral killing of tumor cells. G207 is an oncolytic herpes virus lacking UL39, the gene encoding RR. RR is an enzyme required for viral DNA synthesis and cytotoxicity. METHODS: Enzyme-linked immunosorbent assay (ELISA) was performed for AFP levels on Hep3B and PLC5 human HCC cells. An AFP-albumin enhancer-promoter complex (AFP-alb) was constructed in a luciferase vector to assess function. AFP-alb was cloned upstream of UL39 (AFP-alb/UL39) and transfected into HCC cells for G207 cytotoxicity assays. Viral plaque forming assays evaluated G207 replication. Hep3B flank tumors, with and without AFP-alb/UL39 transfection, were established in athymic mice (n = 28) and treated with G207. RESULTS: Hep3B had 5-fold higher AFP levels than PLC5 (P < 0.00001). AFP-alb increased luciferase expression 72-fold in Hep3B (P < 0.001) and 3-fold in PLC5 (P < 0.001). AFP-alb/UL39 transfection increased G207 cytotoxicity 93% in Hep3B (P < 0.0005), with no significant increase in PLC5. Peak viral titers were 46-fold higher in Hep3B transfected with AFP-alb/UL39 versus mock-transfected cells (P < 0.01), with no significant change in PLC5. Flanks tumors transfected with AFP-alb/UL39 and treated with G207 demonstrated a 76% volume reduction versus mock-transfected tumors infected with G207 (P < 0.0001). CONCLUSIONS: AFP-driven RR production by hepatoma cells significantly enhances herpes viral cytotoxicity and specificity in vitro and reduces tumor burden in vivo.     

Transduction of human islets with the lentiviral vector

Establishment of an efficient gene delivery system for human pancreatic beta cells is important for the development of diabetes-targeted cell therapies. The human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vector is well documented to be an effective gene transfer tool for various types of cells. Thus, we examined the efficiency of lentivirus-mediated gene delivery for human islets. Human islets were isolated using defined protocols for enzymatic dissociation and purification using discontinuous Ficoll gradients with a refrigerated Cobe 2991 machine. Isolated islets were shipped to Japan, cryopreserved for 3 months, and then subjected to transduction. A vesicular stomatitis virus G-protein (VSV-G)-pseudotyped lentiviral vector LtV-NLS/LacZ was produced in 293T cells under the Fugene6 method. 804G extracellular matrices were applied for monolayer formation of islets. Detection of NLS/LacZ expression was performed using X-gal staining. Lentiviral transduction was effective in these monolayer islets.     

Glucose-stimulated insulin production in mice deficient for the PAS kinase PASKIN

The Per-ARNT-Sim (PAS) domain serine/threonine kinase PASKIN, or PAS kinase, links energy flux and protein synthesis in yeast and regulates glycogen synthase in mammals. A recent report suggested that PASKIN mRNA, protein, and kinase activity are increased in pancreatic islet beta-cells under hyperglycemic conditions and that PASKIN is necessary for insulin gene expression. We previously generated Paskin knockout mice by targeted replacement of the kinase domain with the beta-geo fusion gene encoding beta-galactosidase reporter activity. Here we show that no 5-bromo-4-chloro-3-indolyl-ss-d-galactopyranoside (X-gal) staining was observed in islet beta-cells derived from Paskin knockout mice, irrespective of the ambient glucose concentration, whereas adenoviral expression of the lacZ gene in beta-cells showed strong X-gal staining. No induction of PASKIN mRNA could be detected in insulinoma cell lines or in islet beta-cells. Increasing glucose concentrations resulted in PASKIN-independent induction of insulin mRNA levels and insulin release. PASKIN mRNA levels were high in testes but undetectable in pancreas and in islet beta-cells. Finally, blood glucose levels and glucose tolerance after intraperitoneal glucose injection were indistinguishable between Paskin wild-type and knockout mice. These results suggest that Paskin gene expression is not induced by glucose in pancreatic beta-cells and that glucose-stimulated insulin production is independent of PASKIN.     

Adenovirus-mediated wild-typep53 tumor suppressor gene therapy induces apoptosis and suppresses growth of human pancreatic cancer

Background: Thep53 tumor suppressor gene is mutated in up to 70% of pancreatic adenocarcinomas. We determined the effect of reintroduction of the wild-typep53 gene on proliferation and apoptosis in human pancreatic cancer cells using an adenoviral vector containing the wild-typep53 tumor suppressor gene. Methods: Transduction efficiencies of six p53-mutant pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, CFPAC-1, MIA PaCa-2, and PANC-1) were determined using the reporter gene construct Ad5/CMV/-gal. Cell proliferation was monitored using a³H-thymidine incorporation assay, Western blot analysis forp53 expression was performed, and DNA laddering and fluorescence-activated cell sorter analysis were used to assess apoptosis.p53 gene therapy was tested in vivo in a subcutaneous tumor model. Results: The cell lines varied in transduction efficiency. The MIA PaCa-2 cells had the highest transduction efficiency, with 65% of pancreatic tumor cells staining positive for beta-galactosidase (-gal) at a multiplicity of infection (MOI) of 50. At the same MOI, only 15% of the CFPAC-1 cells expressed the -gal gene. Adenovirus-mediatedp53 gene transfer suppressed growth of all human pancreatic cancer cell lines in a dose-dependent manner. Western blot analysis confirmed the presence of the p53 protein product at 48 hours after infection. DNA ladders demonstrated increased chromatin degradation, and fluorescence-activated cell sorter analysis demonstrated a four-fold increase in apoptotic cells at 48 and 72 hours following infection with Ad5/CMV/p53 in the MIA PaCa-2 and PANC-1 cells. Suppression of tumor growth mediated by induction of apoptosis was observed in vivo in an established nude mouse subcutaneous tumor model following intratumoral injections of Ad5/CMV/p53. Conclusions: Introduction of the wild-typep53 gene using an adenoviral vector in pancreatic cancer withp53 mutations induces apoptosis and inhibits cell growth. These data provide preliminary support for adenoviral mediatedp53 tumor suppressor gene therapy of human pancreatic cancer.Presented at the 51st Annual Cancer Symposium of The Society of Surgical Oncology, San Diego, California, March 26–29, 1998.     

Myxoma Virus Is Oncolytic for Human Pancreatic Adenocarcinoma Cells

Background  Viral oncolytic therapy, which seeks to exploit the use of live viruses to treat cancer, has shown promise in the treatment of cancers resistant to conventional anticancer therapies. Among the most difficult to treat cancers is advanced pancreatic adenocarcinoma. Our study investigates the ability of a novel oncolytic agent, myxoma virus, to infect, productively replicate in, and kill human pancreatic cancer cells in vitro. Methods  The myxoma virus vMyxgfp was tested against a panel of human pancreatic adenocarcinoma cell lines. Infectivity, viral proliferation, and tumor cell kill were assessed. Results  Infection of tumor cells was assessed by expression of the marker gene enhanced green fluorescent protein (e-GFP). vMyxgfp had the ability to infect all pancreatic cancer cell lines tested. Killing of tumor cells varied among the 6 cell lines tested, ranging from >90% cell kill at 7 days for the most sensitive Panc-1 cells, to 39% in the most resistant cell line Capan-2. Sensitivity correlated to replication of virus, and was found to maximally exhibit a four-log increase in foci-forming units for the most sensitive Panc-1 cells within 72 h. Conclusion  Our study demonstrates for the first time the ability of the myxoma virus to productively infect, replicate in, and lyse human pancreatic adenocarcinoma cells in vitro. These data encourage further investigation of this virus, which is pathogenic only in rabbits, for treatment of this nearly uniformly fatal cancer.     

Specific gene expression and therapy for pancreatic cancer using the cytosine deaminase gene directed by the rat insulin promoter

Suicide gene therapy has been shown to be an effective means of destroying pancreatic cancer cells, but cell-specific delivery of the gene is required to limit host toxicity. The objective of this study is to determine whether the rat insulin promoter (RIP) will permit cell-specific gene delivery and subsequent cell death in human pancreatic cancer cells. The RIP DNA was amplified using polymerase chain reaction (PCR), and the purified fragment was inserted into pCR-Blunt II-TOPO plasmid at the SpeI site, which contains the coding sequence of yeast cytosine deaminase (CD). Transfection assays were carried out using both RIP-lacZ and RIP-CD DNA constructs in two human pancreatic cancer cell lines, PANC-1 and MIA PaCa-2. Reporter assays using X-gal staining were performed, and the in vitro cytotoxicity was examined in RIP-CD-transfected cells treated with 5-.ucytosine for 5 days. The expression levels of CD protein in the transfected cells were determined 2 days after transfection by Western blot analysis. The expression levels of insulin promoter factor (IPF-1/PDX-1) in these human pancreatic cell lines, as well as in freshly isolated human pancreatic cancer specimens, were determined using in situ immunohistochemistry analysis. After transfection with RIP-lacZ, only PANC-1 cells, but not MIA PaCa-2 cells, were positive for RIP-lacZ expression, indicating that RIP-directed reporter gene expression occurred only in PANC-1 cells. After transfection with RIP-CD and treatment with 5-flucytosine, PANC-1 cells had a significantly increased cell death rate compared with that of MIA PaCa-2 cells, suggesting that RIP-directed suicide gene expression occurred only in PANC-1 cells. Western blot analysis demonstrated that only PANC-1 cells were able to express the CD protein and that significantly increased levels of PDX-1 were found in PANC-1 but not in Mia PaCa-2 cells. In situ immunohistochemical analysis of both cell lines showed that PDX-1 was only expressed in the nuclei of PANC-1 cells and not in MIA PaCa-2 cells. Furthermore, two freshly isolated human pancreatic cancer specimens had significantly increased levels of PDX-1. The RIP is activated in PANC-1 cells, but not in Mia PaCa-2 cells, and the mechanism of activation is via PDX-1. Pancreatic cancer-specific cytotoxicity can be achieved with the use of RIP-CD and 5-flucytosine treatment in vitro. Significantly increased levels of PDX-1 have been found in human pancreatic cancer specimens. These results suggest that RIP could be used for cell-specific suicide gene therapy to target human pancreatic tumors. Presented at the Forty-Fourth Annual Meeting of The Society for Surgery of the Alimentary Tract, Orlando, Florida May 18–21, 2003 (oral presentation). Supported by an Advances in Technology Transfer Grant (ATP1176) from the State of Texas Department of Education and by grant NCI R01 (CA95731).     

Oncolytic herpes viral therapy is effective in the treatment of hepatocellular carcinoma cell lines

The rising incidence of hepatocellular carcinoma (HCC) in western countries, along with the poor prognosis offered by present-day treatment modalities, makes novel therapies for this disease necessary. Oncolytic herpes simplex viruses (HSV) are replication-competent viruses that are highly effective in the treatment of a wide variety of experimental models of human malignancies. This study seeks to investigate the effectiveness of oncolytic herpes viruses in the treatment of primary HCC cell lines. Sixteen commercially available human HCC cell lines were studied. G207 is an attenuated, replication-competent, oncolytic HSV engineered to selectively replicate within cancer cells. Cell lines were tested for viral sensitivity to G207 and their ability to support viral replication using standard cytotoxicity and viral replication assays. Eleven of 16 cell lines were moderately to highly sensitive to G207 viral oncolysis. HCC cell lines additionally demonstrated the ability to support viral replication in vitro with as high as 800-fold amplification of the administered viral dose observed. G207 is cytotoxic to, and efficiently replicates within, HCC cell lines in vitro. From these data, we suggest that oncolytic HSV therapy may have a role in the treatment of HCC, and in vivo studies are warranted. Presented in part at the 2005 American Hepato-Pancreato-Biliary Association Congress, Hollywood, Florida, April 14–17, 2005. Supported by grants R01CA75461 and R01CA72632 from the National Institutes of Health, and by grant MBC-99366 from the American Cancer Society (Yuman Fong).     

Kidney cysts, pancreatic cysts, and biliary disease in a mouse model of autosomal recessive polycystic kidney disease

Mutations in PKHD1 cause autosomal recessive polycystic kidney disease (ARPKD). We produced a mouse model of ARPKD by replacing exons 1–3 of Pkhd1 with a lacZ reporter gene utilizing homologous recombination. This approach yielded heterozygous Pkhd1 ^lacZ/+ mice, that expressed β-galactosidase in tissues where Pkhd1 is normally expressed, and homozygous Pkhd1 ^lacZ/lacZ knockout mice. Heterozygous Pkhd1 ^lacZ/+ mice expressed β-galactosidase in the kidney, liver, and pancreas. Homozygous Pkhd1 ^lacZ/lacZ mice lacked Pkhd1 expression and developed progressive renal cystic disease involving the proximal tubules, collecting ducts, and glomeruli. In the liver, inactivation of Pkhd1 resulted in dilatation of the bile ducts and periportal fibrosis. Dilatation of pancreatic exocrine ducts was uniformly seen in Pkhd1 ^lacZ/lacZ mice, with pancreatic cysts arising less frequently. The expression of β-galactosidase, Pkd1, and Pkd2 was reduced in the kidneys of Pkhd1 ^lacZ/lacZ mice compared with wild-type littermates, but no changes in blood urea nitrogen (BUN) or liver function tests were observed. Collectively, these results indicate that deletion of exons 1–3 leads to loss of Pkhd1 expression and results in kidney cysts, pancreatic cysts, and biliary ductal plate malformations. The Pkhd1 ^lacZ/lacZ mouse represents a new orthologous animal model for studying the pathogenesis of kidney cysts and biliary dysgenesis that characterize human ARPKD.     

苯乙酸抑制胰腺癌BXPC-3细胞增殖及RNA编辑酶表达的研究

目的 探讨苯乙酸对胰腺癌诱导分化作用及RNA编辑在胰腺癌细胞增殖分化过程中的作用机理.方法 采用MTT比色法和流式细胞术检测苯乙酸对胰腺癌BXPC-3细胞系增殖抑制作用及对BXPC-3细胞周期的影响.RT-PCR方法检测RNA编辑酶ADAR mRNA在胰腺癌细胞系和胰腺癌组织中的表达以及应用苯乙酸后ADAR mRNA表达的变化.结果 人胰腺癌组织及胰腺癌BXPC-3细胞中均可检测到编辑酶ADAR2.应用1.0及2.0mmol/L苯乙酸后24h及72 h,BXPC-3细胞增殖抑制率增加,G0/G1期比例下降,S期比例显著升高.ADAR2 mRNA表达降低,差异均有统计学意义(P<0.01).结论 ADAR2可能在胰腺细胞增殖分化过程中发挥作用,且苯乙酸可能通过调控ADAR2 mRNA的表达来发挥诱导分化作用.     

反义K-ras基因对胰腺癌细胞增殖和凋亡的影响

目的 探讨反义K-ras癌基因对胰腺癌细胞增殖和凋亡及Fas/FasL、Bcl-2、Bax表达的影响.方法 将重组逆转录病毒载体pLXSN转染包装细胞PT-67,获得重组逆转录病毒.在细胞和动物水平将该重组逆转录病毒分别感染PC-3和BxPC-3胰腺癌细胞,经G418筛选获得稳定细胞系,应用MTT、流式细胞仪和免疫组化法分别研究其增殖、凋亡及其相关基因表达情况.结果 成功制备含反义K-ras基因的重组逆转录病毒.感染含反义K-ras基因重组病毒后,胰腺癌PC-3细胞和移植瘤(有K-ras基因第12密码子点突变GGT→GTT)增殖受到抑制,G<,0/1>期细胞增加而S期细胞明显减少,细胞凋亡增加.免疫组化显示含反义K-ras癌基因逆转录病毒感染后PC-3细胞Bax/Bcl-2比值升高,Fas表达上调.结论 反义K-ras基因可使胰腺癌细胞及移植瘤增殖受到抑制,并可通过上调Bax/Bcl一2比值和(或)促进Fas表达诱导细胞凋亡.     

基因枪转导突变特异性K-ras siRNA对胰腺癌细胞生长的抑制作用

目的探讨基因枪转导突变特异性K-ras siRNA对胰腺癌细胞生长的抑制作用。方法根据胰腺癌细胞系突变特征，设计突变特异性K-ras siRNA；应用基因枪将siRNA转导入胰腺癌细胞系，提取总RNA和胞浆蛋白；应用逆转录-聚合酶链反应（RT-PCR）和免疫印迹法（Western blot）方法。检测基因枪转导突变特异性K，ras siRNA对突变型K-ras基因表达的干扰作用；行细胞爬片K-ras p21蛋白免疫组织化学染色，探讨siRNA对K-ras p21蛋白表达的抑制作用；采用CCK-8（cell countingkit，8）活细胞计数法，绘制转基因前后细胞生长曲线，观察siRNA的抑瘤效果。结果RT-PCR结果显示，K-ras突变特异性siRNA，能有效抑制K-ras基因表达（P〈0．05）；Western blot和免疫组织化学结果表明，K-ras p21蛋白的表达明显降低（P〈0．05）；细胞生长曲线显示，转导siRNA组较对照组细胞生长明显受抑制（P〈0.05）。结论基因枪可以有效地对小片段双链siRNA进行细胞内转导；突变特异性K-ras siRNA，可显著下调胰腺癌细胞系突变型K-ras mRNA及K-ras p21蛋白表达，并能有效抑制肿瘤细胞的增殖。