禽白血病/肉瘤病毒肿瘤基因及其与致肿瘤机制的关系

根据致瘤机制的差异,禽白血病/肉瘤病毒可以分为两大类,其差异主要在于所表达肿瘤基因的不同。慢性转化型病毒通过插入启动机制间接激活细胞原癌基因,肿瘤形成速度缓慢;急性转化型病毒则是直接转录表达病毒自身基因组中的病毒肿瘤基因,从而诱导肿瘤的快速形成。作者拟对禽白血病/肉瘤病毒肿瘤基因及其与致肿瘤机制的关系进行综述。

Expression of Epstein-Barr virus genes in EBV-associated gastric carcinomas

AIM: To understand the expression of latent and lytic genes of Epstein-Barr virus (EBV) in EBV-associated gastric carcinoma (EBVaGC) and to explore the relationship between EBV-encoded genes and development of EBVaGC at molecular level, METHODS: One hundred and seventy-two gastric carcinoma tissues and 172 corresponding para-carcinoma tissues were tested for EBV genome by polymerase chain reaction (PCR)-Southern blotting. EBV-encoded small RNA (EBER) 1 of the PCR positive specimens was detected by in situ hybridization (ISH). Gastric carcinomas with positive EBER1 signals were classified as EBVaGCs. RT-PCR and Southern hybridization were applied to the detection of expression of nuclear antigen (EBNA) promoters (Qp, Wp and Cp), EBNA 1 and EBNA 2, latent membrane proteins (LMP) 1, 2A and 2B and lytic genes (immediate early genes BZLF1 and BRLF1, early genes BARF1 and BHRF1, late genes BcLF1 and BLLF1) in EBVaGCs. RESULTS: Eleven EBV positive samples existed in gastric carcinoma tissues (6.39%). No EBV positive sample was found in corresponding para-carcinoma tissues. The difference between EBV positivity in carcinoma tissues and corresponding para-carcinoma tissues was significant (x2 = 9.0909, P = 0.0026). Transcripts of Qp and EBNA1 were detected in all the 11 EBVaGCs, while both Wp and Cp were silent. EBNA2, LMP1 and LMP2B mRNA were absent in all the cases, while LMP2A mRNA was detected in 4 of the 11 cases. Of the 11 EBVaGCs, 7 exhibited BcLFl transcripts and 2 exhibited BHRF1 transcripts. The transcripts of BZLF1 and BARF1 were detected in 5 cases, respectively. No BLLF1 and BRLF mRNA were detected. CONCLUSION: The latent pattern of EBV in gastric carcinoma corresponds to the latency I/II. Some lytic infection genes are expressed in EBVaGCs tissues. BARF1 and BHRF1 genes may play an important role in tumorigenesis of gastric carcinoma.

Hepatitis B virus genotypes and hepatocellular carcinoma in Thailand

AIM: The role of hepatitis B virus (HBV) genotypes on the clinical features and prognosis of patients with hepatocellular carcinoma (HCC) is currently unknown. The aim of the present study was to evaluate the distribution of HBV genotypes and their clinical relevance in Thai patients.METHODS: HBV genotypes were determined by PCR-RFLP in stored sera of 93 asymptomatic carriers, 103 patients with chronic hepatitis, 60 patients with cirrhosis and 76patients with HCC. The clinical data were analyzed in relation to the HBV genotype.RESULTS: HBV genotypes C and B were predominant in Thailand, accounting for 73% and 21%, respectively. The distributions of genotypes B and C were similar in HCC patients compared to the other groups. Genotype C was significantly more common in HCC patients who were under 40 years old than genotype B (18% vs 0%, P= 0.03), but was significantly less common in patients older than 60 years (26% vs 56.5%, P= 0.01). The positive rate of hepatitis B e antigen (HBeAg) in patients with genotype C was significantly higher than that in patients with genotype B (71.6% vs 44.4%, P = 0.03 in chronic hepatitis; 56.8% vs 11.1%,P = 0.01 in cirrhosis). There were no differences between HCC patients with genotypes B and C regarding tumor staging by CLIP criteria and the overall median survival. Multivariate analyses showed that HBV genotype was not an independent prognostic factor of survival in HCC patients.CONCLUSION: Patients with genotype C had a higher positive rate of HBeAg and exhibited earlier progression of cirrhosis and HCC than those with genotype B. However,there were no differences in the risk of developing HCC and its prognosis between patients with these genotypes.

Adenoviral gene therapy in gastric cancer: A review

Gastric cancer is one of the most common malignancies worldwide. With current therapeutic approaches the prognosis of gastric cancer is very poor, as gastric cancer accounts for the second most common cause of death in cancer related deaths. Gastric cancer like almost all other cancers has a molecular genetic basis which relies on disruption in normal cellular regulatory mechanisms regarding cell growth, apoptosis and cell division. Thus novel therapeutic approaches such as gene therapy promise to become the alternative choice of treatment in gastric cancer. In gene therapy, suicide genes, tumor suppressor genes and anti-angiogenesis genes among many others are introduced to cancer cells via vectors. Some of the vectors widely used in gene therapy are Adenoviral vectors. This review provides an update of the new developments in adenoviral cancer gene therapy including strategies for inducing apoptosis, inhibiting metastasis and targeting the cancer cells.

Gene-viral vectors: a promising way to target tumor cells and express anticancer genes simultaneously

OBJECTIVE: To develop a new kind of vector system called gene-viral vector, which combines the advantages of gene and virus therapies. METHODS: Using recombinant technology, an anti-tumor gene was inserted into the genome of replicative virus specific for tumor cells. The cell killing effect, reporter gene expression of the green fluorescence protein, anti-tumor gene expression of mouse interleukin-12 (mIL-12) and replication of virus were observed by the methods of cell pathology, fluorescence microscopy, ELISA and electron microscopy, respectively. RESULTS: A new kind of gene-viral vector system of adenovirus, in which the E1b-55 kD gene was deleted but the E1a gene was preserved, was constructed. The vector system, like the replicative virus ONYX-015, replicated and proliferated in tumor cells but not in normal ones. Our vector had an advantage over ONYX-015 in that it carried different kinds of anti-tumor genes to enhance its therapeutic effect. The reporter gene expression of the green fluorescence protein in tumor cells was much better than the adenovirus vector employed in conventional gene the rapy, and the expression in our vector system was as low as or even less than that in the conventional adenovirus gene therapy system. Similar results were observed in experiments with this vector system carrying the anti-tumor gene mIL-12. Replication and proliferation of the virus carrying the mIL-12 gene in tumor cells were confirmed by electron microscopy. CONCLUSIONS: Gene-viral vectors are new vectors with an anti-tumor gene inserted into the genome of replicative virus specific for tumor cells. Because of the specific replication and proliferation of the virus in tumor cells, expression of the anti-tumor gene is increased hundreds to thousands of times. This approach takes full advantages of gene therapy and virus therapy to enhance the effect on the tumor. It overcomes the disadvantages of conventional gene therapy, such as low transfer rate, low gene expression, lack of target tropism, and low anti-tumor activity. We believe that this is a promising means for future tumor treatment.

肌内注射rAAV2/rTNFR：Fc对大鼠胶原性类风湿关节炎模型的治疗作用

目的观察肌肉注射2型重组腺相关病毒/肿瘤坏死因子受体:IgG1FC融合基因(rAAV2/rTNFR:Fc)对大鼠类风湿关节炎模型的治疗作用。方法皮内注射Ⅱ型胶原乳剂,诱发胶原性大鼠类风湿关节炎模型。将大鼠随机分为5组,每组10只,包括:正常对照组、模型对照组、载体对照组、地塞米松对照组和rAAV2/rTNFR:Fc肌肉注射给药组。观察各组大鼠的关节炎指数(AI)、关节肿胀程度、踝关节的X线变化、组织病理改变与动物血清肿瘤坏死因子(TNF)-α及其受体(TNFR)水平。结果与模型对照和载体对照组比较,肌肉注射rAAV2/rTNFR:Fc可使关节炎症明显减轻,AI降低、关节肿胀程度减轻;X线检查显示rAAV2/rTNFR:Fc给药组关节结构清晰,无明显骨质破坏。组织病理检查结果也表明,给药组关节腔内滑膜增生和炎性细胞浸润程度减轻,增生的血管翳肉芽组织减少;此外,与模型对照组和载体对照组比较,给药组血清中TNF-α水平显著下降(P<0.05),而TNFR水平明显上升(P<0.05)。结论肌肉注射给予rAAV2/rTNFR:Fc对大鼠胶原性类风湿关节炎模型具有明显的抗炎作用。

Effects of anti-HPV16E6-ribozyme on phenotype and gene expression of a cervical cancer cell line

OBJECTIVE: To investigate the effects of anti-HPV16E6-ribozyme (HRz) on phenotype and gene expression of a cervical cancer cell line. METHODS: HRz was designed by computer programs. HRz's activity was identified by cleavage experiments in vitro. HRz and empty eukaryotic plasmids were transfected into CaSKi cells with lipofectin, then renamed CaSKi-R and CaSKi-P, respectively. The expression of ribozyme in transfected cells was observed by RNA dot blot. The amounts of E6 mRNA in three kinds of cells lines were detected by Northern blot. Cell growth curves and soft agar forming ability were studied. The ability of each cell line to form tumors was assessed in nude mice. Apoptosis rates and expression of c-myc, bcl-2, p53 and Fas were detected by flow cytometry (FCM). Antigens of tumor cells, HLA-1, HLA-2, B7-1 and B7-2 were also detected. NK, LAK, and CD(3)AK cells were induced. Their cytotoxicities were detected in CaSKi-R, CaSKi-P, and CaSKi cells. RESULTS: In vitro cleavage reaction demonstrated that HRz could cleave HPV16E6 mRNA in a site-specific manner. HRz could be expressed stably in transfected CaSKi cells. Northern blot analysis showed that E6 mRNA levels were lower in CaSKi-R than in CaSKi. The growth rate of CaSKi-R was slower than those of CaSKi and CaSKi-P. The soft agar-forming rate of CaSKi-R was lower compared with those of CaSKi and CaSKi-P cells. The ability of CaSKi-R to form tumors in nude mice was also poor. The apoptosis rate of CaSKi-R cells was much higher than those of CaSKi and CaSKi-P. HRz could reduce the expression of E6, c-myc and bcl-2 proteins, and increase the expression of p53 as well. HRz could increase the expression of HLA-2, B7-1 and B7-2 antigens. The cytotoxicity of NK, LAK and CD(3)AK cells was much higher in CaSKi-R than in CaSKi-P and CaSKi cells. CONCLUSION: HRz not only reverses the malignant phenotype of CaSKi cells partially, but also induces apoptosis in the cells, and increases sensitivity of CaSKi cells to immune cells.

Effects of anti-HPV16E6-ribozyme on phenotype and gene expression of a cervical cancer cell line

To investigate the effects of anti HPV16E6 ribozyme (HRz) on phenotype and gene expression of a cervical cancer cell line Methods HRz was designed by computer programs HRz’s activity was identified by cleavage experiments in vitro HRz and empty eukaryotic plasmids were transfected into CaSKi cells with lipofectin, then renamed CaSKi R and CaSKi P, respectively The expression of ribozyme in transfected cells was observed by RNA dot blot The amounts of E6 mRNA in three kinds of cells lines were detected by Northern blot Cell growth curves and soft agar forming ability were studied The ability of each cell line to form tumors was assessed in nude mice Apoptosis rates and expression of c myc, bcl 2, p53 and Fas were detected by flow cytometry (FCM) Antigens of tumor cells, HLA 1, HLA 2, B7 1 and B7 2 were also detected NK, LAK, and CD 3AK cells were induced Their cytotoxicities were detected in CaSKi R, CaSKi P, and CaSKi cells Results In vitro cleavage reaction demonstrated that HRz could cleave HPV16E6 mRNA in a site specific manner HRz could be expressed stably in transfected CaSKi cells Northern blot analysis showed that E6 mRNA levels were lower in CaSKi R than in CaSKi The growth rate of CaSKi R was slower than those of CaSKi and CaSKi P The soft agar forming rate of CaSKi R was lower compared with those of CaSKi and CaSKi P cells The ability of CaSKi R to form tumors in nude mice was also poor The apoptosis rate of CaSKi R cells was much higher than those of CaSKi and CaSKi P HRz could reduce the expression of E6, c myc and bcl 2 proteins, and increase the expression of p53 as well HRz could increase the expression of HLA 2, B7 1 and B7 2 antigens The cytotoxicity of NK, LAK and CD 3AK cells was much higher in CaSKi R than in CaSKi P and CaSKi cells Conclusion HRz not only reverses the malignant phenotype of CaSKi cells partially, but also induces apoptosis in the cells, and increases sensitivity of CaSKi cells to immune cells