Restricted replication-competent adenovirus: a novel strategy for cancer gene therapy

Mutant adenoviruses that are selectively replication-competent in tumor cells have been developed as a novel strategy for gene therapy of cancer. Such selectively replicating viruses may overcome the limitations of the gene transfer of adenoviral vectors. The replication of such viruses in a small fraction of the tumor cells leads to amplification and extension of the antitumor effect, with cell killing being caused exclusively by viral replication and cell lysis. Replication-competent adenoviruses have several theoretical advantages over replication-defective adenoviruses for cancer gene therapy. It has been demonstrated that replication-competent adenoviruses are not only a strong weapon themselves but that they are also useful carriers of genes for anti-tumor factors, acting as virus vectors specific to tumors without normal p53 function or an intact retinoblastoma gene (RB) pathway. Clinical trials will clarify these advantages in future. Received: March 15, 2000     

以EGFR为靶点的肿瘤反义基因治疗策略

表皮生长因子受体(EGFR)在许多肿瘤细胞中超量表达,借助其介导的信号传导可使细胞生长失控与恶性转化,因此它是良好的肿瘤治疗靶点.现主要针对EGFR基因治疗中的反义策略作一综述.     

血管抑素基因转染抑制胆囊癌细胞的实验研究

目的:观察血管抑素基因转染对体外人胆囊癌细胞系GBC-SD细胞株增殖及血管抑素表达和裸鼠致瘤体积的影响,初步探讨血管抑素基因转染抑制胆囊癌细胞生长的可能性.方法:应用pcDNA3.1( )-angiostatin 基因转染GBC-SD胆囊癌细胞.观察细胞生长情况,制作细胞生长曲线;Western-blot法分析血管抑素的表达情况;裸鼠种植瘤模型观察肿瘤的体积和质量.结果:pcDNA3.1( )-angiostatin 基因转染的GBC-SD胆囊癌细胞生长明显受到抑制,血管抑素的蛋白表达也明显增加.转染的肿瘤细胞在裸鼠种植瘤明显小于阴性组和空白组.结论:血管抑素基因转染可能具有抑制胆囊癌细胞增殖及生长的作用.     

Angiostatin generation by human tumor cell lines: involvement of plasminogen activators

Angiostatin is a tumor-derived angiogenesis inhibitor consisting of an internal fragment of plasminogen. Little is known about the production of angiostatin by human tumors. In this study, we examined the in vitro angiostatin-generating capacities of a panel of human tumor cell lines (total n = 75) and the proteolytic molecule(s) involved. Angiostatin formation was determined by assessing the level of plasminogen digestion in conditioned medium by Western-blot analysis. We found that the capacity to produce angiostatin is a common feature of many cell lines, depending on the tumor type. All 6 bladder-carcinoma and 6 out of 7 prostate-carcinoma cell lines showed intermediate to potent angiostatin-generating activity. In contrast, only 2 out of 7 colon-carcinoma and 2 out of 9 renal-cell carcinoma cell lines were able to generate angiostatin at intermediate levels. Out of 25 melanoma cell lines, only one line failed to generate angiostatin. In the other cell-line groups (cervix, breast and ovary), angiostatin formation varied. Remarkably, angiostatin bands were not of equal size in all plasminogen digests. Since reported data have indicated that plasminogen activators (uPA and tPA) were able to excise the angiostatin fragment from the plasminogen parent molecule via plasmin generation, we determined levels of uPA and tPA and PAI-1 antigen in the conditioned media, and correlated the results with angiostatin-generating capacity. Whereas prostate- and bladder-carcinoma lines capable of generating high levels of angiostatin showed high uPA levels, angiostatin generation in melanoma cell lines was correlated with tPA levels. Generally, angiostatin non-producers did not express uPA or tPA. In 6 out of 75 cell lines, however, we found angiostatin generation combined with low or absent levels of plasminogen activator, suggesting the involvement of alternative proteolytic pathways in the generation of angiostatin.     

血管抑素基因转染抑制胆囊癌细胞的实验研究

目的：观察血管抑素基因转染对体外人胆囊癌细胞系GBC—SD细胞株增殖及血管抑素表达和裸鼠致瘤体积的影响，初步探讨血管抑素基因转染抑制胆囊癌细胞生长的可能性。方法：应用pcDNA3．1（＋）-angiostatin基因转染GBC—SD胆囊癌细胞。观察细胞生长情况，制作细胞生长曲线；Western—blot法分析血管抑素的表达情况；裸鼠种植瘤模型观察肿瘤的体积和质量。结果：pcDNA3．1（＋）-angiostatin基因转染的GBC—SD胆囊癌细胞生长明显受到抑制，血管抑素的蛋白表达也明显增加。转染的肿瘤细胞在裸鼠种植瘤明显小于阴性组和空白组。结论：血管抑素基因转染可能具有抑制胆囊癌细胞增殖及生长的作用。     

工程厌氧菌靶向肿瘤基因治疗新策略

实体瘤内存在低氧代谢区，厌氧菌具有趋低氧的特点，因而对实体瘤组织具有良好的靶向件，成为肿瘤基因治疗的转移载体。运用基因工程技术获得的一系列工程厌氧菌，提高了载体的安全性，因而更具临床应用前景。现综述工程厌氧菌作为基因治疗载体及与其他治疗方法联合运用方面的研究进展。     

Growth of human melanoma xenografts is suppressed by systemic angiostatin gene therapy

The effect of local and systemic delivery of the angiostatin gene on human melanoma growth was studied in nude mice. Liposome-coated plasmids carrying the cDNA coding for murine and human angiostatin (CMVang and BSHang) were injected weekly, locally or systemically, in mice transplanted with melanoma cells. The treatment reduced melanoma growth by 50% to 90% compared to that occurring in control animals treated with liposome-coated plasmid carrying the lacZ gene or in untreated controls. The growth of both locally injected and controlateral uninjected tumors in mice bearing two melanoma grafts was significantly suppressed after intratumoral treatment. Tumor growth inhibition was also observed in mice treated by intraperitoneal delivery, suggesting that angiostatin gene therapy acts through a systemic effect. Both melanoma growth suppression and delay in the onset of tumor growth were observed in treated mice. PCR performed on tumors and normal tissues showed that the lipofected DNA was present in tissues from treated mice, and angiostatin expression was demonstrated by RT-PCR. Histopathological analysis of melanoma nodules revealed an increase in apoptotic cells and a reduction in vessel density in tumors from treated mice. Our results suggest that systemic, liposome-mediated administration of genes coding for antiangiogenic factors represents a promising strategy for melanoma treatment in humans.     

Combining radiotherapy with gene therapy (from the bench to the bedside): a novel treatment strategy for prostate cancer

Combined radiotherapy and gene therapy is a novel therapeutic approach for prostate cancer. There are various potential benefits in combining ionizing radiation with gene therapy to achieve enhanced antitumor effects: A) ionizing radiation improves transfection/ transduction efficiency, transgene integration, and possibly, the "bystander effect" of gene therapy; B) gene therapy, on the other hand, may interfere with repair of radiation-induced DNA damage and increase DNA susceptibility to radiation damage in cancer cells, and C) radiotherapy and gene therapy target at different parts of the cell cycle. Preclinical data have demonstrated the enhanced antitumor effects of this combined approach in local tumor control, prolongation of survival, as well as systemic control. This combined radio-gene therapy is under study in an ongoing clinical trial in prostate cancer. Our study adds gene therapy to the standard of care therapy (radiotherapy). These treatment modalities have different toxicity profiles. The goal of this combined approach is to enhance cancer cure without an increase in treatment-related toxicity. This approach also offers a new paradigm in spatial cooperation, whereby two local therapies are combined to elicit both local and systemic effects. Early clinical results showed the safety of this approach.     

A novel apoptotic pathway induced by the drs tumor suppressor gene

The drs gene was originally isolated as a suppressor against v-src transformation. Expression of drs mRNA was markedly downregulated in a variety of human cancer cell lines and tissues, suggesting that the drs gene acts as a tumor suppressor. In this study, we found that ectopic expression of the Drs protein induced apoptosis in human cancer cell lines. Analyses using deletion mutants of drs revealed that both the C-terminal region and the three consensus repeats in the N-terminal region are essential for the induction of apoptosis. Caspase-12, -9, and -3 were sequentially activated by drs, and specific inhibitors of caspase-3 and -9 suppressed drs-induced apoptosis. The release of cytochrome c from the mitochondria into the cytoplasm was not observed in apoptosis by drs, suggesting that the mitochondrial pathway does not mediate drs-induced apoptosis. Furthermore, we found that the Drs protein can interact with ASY/Nogo-B/RTN-x(S), an apoptosis-inducing protein localized in the endoplasmic reticulum, and that coexpression of these genes increased the efficiency of apoptosis. These results indicated that Drs induces apoptosis by a novel pathway mediated by ASY/Nogo-B/RTN-x(S), caspase-12, -9, and -3.     

Pleiotrophic inhibition of pericellular urokinase-type plasminogen activator system by endogenous tumor suppressive maspin.

Maspin is a novel serine protease inhibitor with tumor suppressive activity, inhibiting tumor invasion and metastasis. To date, the underlying molecular mechanism of maspin remains elusive. Recombinant maspin has been shown to specifically inhibit cell surface-associated urokinase-type plasminogen activator (uPA) and fibrinogen-bound tissue-type plasminogen activator. However, the role of endogenous maspin in plasminogen activation is totally unknown. To address this issue, we generated stable maspin-expressing transfectants using prostate carcinoma cells DU145 as the parental cell line. We report here that endogenous maspin exerts pleiotropic inhibitory effects on the pericellular uPA system. Maspin expression led to a significantly reduced level of cell surface-bound uPA and uPA receptor proteins without altering the steady-state levels of the respective mRNAs. Treatment with receptor-associated protein (RAP), a specific inhibitor of low-density lipoprotein receptor-related protein, lead to a significantly increased level of secreted uPA and cell surface uPAR in maspin transfectants but not in the mock control cells. A combination of enzymatic and molecular analyses revealed that maspin inhibits the cell surface-mediated plasminogen activation by forming an SDS-resistant complex with cell surface-bound uPA. In addition, maspin expression led to a dramatic reduction in the release of active uPA, both high molecular weight and the low molecular weight, into the conditioned culture medium. Consistently, the conditioned medium of maspin transfectant clones had a significantly reduced activity in converting plasminogen to plasmin. The inhibitory effect of maspin on pericellular uPA correlates with significantly decreased cell invasion potential and motility in vitro. The maspin-neutralizing antibody (Abs4A) reversed the subdued invasive potential of maspin transfectant cells in a dose-dependent manner. In summary, this study provides the first evidence that endogenous maspin is a potent inhibitor of pericellular uPA. Furthermore, our results support a current hypothesis that maspin blocks tumor invasion and motility by inhibiting localized pericellular proteolysis.     

Wild-type p53 gene transfer is not detrimental to normal cells in vivo: implications for tumor gene therapy

The p53 oncosuppressor is strictly maintained in an inactive form under normal conditions, while it is post-translationally activated by a variety of stresses, enacting different protective biological functions. Since one critical issue in cancer gene therapy is tumor specificity, we asked whether the tight p53 regulation applies also to exogenously transferred p53. In principle, this type of regulation could allow p53 gene transfer in both normal and tumor cells to produce detrimental effects only in the latter ones. Here, we report that primary bone marrow cells infected with a p53 recombinant retrovirus and transplanted into irradiated mice reconstitute the hematopoietic system, with no detectable alterations in any of its compartments. Furthermore, simultaneous infection of leukemia and bone marrow cells depleted the neoplastic contamination, allowing lifelong, disease-free survival of 65% of the transplanted animals. These results show that exogenous p53 is controlled as tightly as the endogenous one, and opens the way to p53 gene therapy, without requiring tumor targeting.     

肿瘤基因治疗的腺病毒策略

 腺病毒载体是目前肿瘤临床试验中使用最为广泛的载体之一。在过去的十几年里，随着人们对腺病毒活动周期的逐步了解，已经开发出了一系列专门针对恶性肿瘤细胞的靶向腺病毒载体。这些载体可以在特定的细胞中表达治疗性基因或进行自主复制，显示了其在肿瘤治疗方面的独特优势。但目前对腺病毒进行的各种改进方式都存在不同程度的缺陷，因此仍需进一步研究。文章讨论腺病毒载体用于肿瘤基因治疗方面的一些基本情况及其在临床应用的潜力。     

肿瘤相关基因NDRG2的研究进展

NDRG2隶属于NDRG家族（N-myc down-stream regulated gene family）,是一种与细胞增殖和分化相关的基因,参与了肿瘤的发生、发展和转归,目前功能定位于抑癌候选基因。将NDRG2基因转染入U373和U138胶质瘤细胞系后,明显抑制了胶质瘤细胞的增殖;在结肠癌及高危腺瘤中,NDRG2mRNA表达量明显低于正常组织,同时随着Dukes′分级的增高,NDRG2表达有下降趋势。随着相关研究的不断深入,该基因的其他功能也逐渐被揭示：与组织胚胎的发育和细胞的分化密切相关,与神经系统的发育及其疾病的发生相关,参与了醛固酮对肾远曲小管和集合管的水钠代谢调节作用,以及多种应激反应例如：DNA损伤、缺氧等。目前其转录调控机制及其相互作用分子研究表明,NDRG2受c-Myc负调控且该调控需要Miz-1参与,同时NDRG2还是HIF-1的靶基因。维尔姆斯肿瘤基因（WT1）可以直接或间接诱导NDRG2表达等。但NDRG2生物学功能至今还尚未完全明确,值得进一步探索。     

In vivo generation of angiostatin isoforms by administration of a plasminogen activator and a free sulfhydryl donor: a phase I study of an angiostatic cocktail of tissue plasminogen activator and mesna.

PURPOSE: Angiostatin4.5 (AS4.5), the endogenous human angiostatin, is derived from plasminogen in a two-step process. A plasminogen activator converts plasminogen to plasmin, then plasmin undergoes autoproteolysis to AS4.5. A free sulfhydryl donor can mediate plasmin autoproteolysis. To translate this process to human cancer therapy, we conducted a phase I trial of administration of a tissue plasminogen activator (tPA) with a free sulfhydryl donor (mesna). PATIENTS AND METHODS: Fifteen patients with advanced solid tumors were treated. The dose of tPA was escalated (cohorts; 1, 2, 3, 5, and 7.5 mg/h for 6 hours). Mesna was administered as a 240 mg/m2 bolus followed by an infusion of 50 mg/h, concurrent with tPA. Both tPA and mesna were administered 3 consecutive days every 14 days. RESULTS: No dose-limiting toxicity was observed. Two AS4.5 isoforms were generated, Lys-AS4.5 and Glu-AS4.5. Mean baseline Lys-AS4.5 level was 20.4 nmol/L (SE, 2.9). In the 5 mg/h tPA cohort, Lys-AS4.5 levels increased by an average of 143% or 24 nmol/L (SE, 4.9) above baseline. Glu-AS4.5 (M(r) approximately 62,000) was also generated (additional 77 amino acids at amino terminus compared with Lys-AS4.5). Glu-AS4.5 level at baseline was undetectable in four of five patients in the 5 mg/h tPA cohort, but at end of infusion, was approximately 67 nmol/L (SE, 20). Two patients in the 5 mg/h tPA cohort experienced decreases in tumor markers with treatment, although no clinical objective responses were observed. CONCLUSION: This study shows that in vivo generation of AS4.5 is safe in humans and may provide a practical approach to achieve antiangiogenic therapy.