Adenovirus-mediated in vivo B7-1 gene transfer induces anti-tumor immunity against pre-established primary tumor and pulmonary metastasis of rat osteosarcoma

We have previously reported that an osteosarcoma vaccine generated by ex vivo transfection of B7-1 cDNA induces protective as well as curative immunity against B7-1-negative parental osteosarcoma. Because establishment of human osteosarcoma cell lines, which is a prerequisite for ex vivo gene transfer, is rarely successful, we, in the present study, investigated the therapeutic efficacy of adenovirus-mediated in vivo B7-1 gene transfer to pre-established primary tumor as well as pulmonary metastasis of osteosarcoma. Adenovirus-mediated rat B7-1 gene transfer induced (a) expression of B7-1 molecules in osteosarcoma cells by both in vitro and in vivo infection procedures, (b) curative immunity against pre-established primary osteosarcoma and, subsequently, hosts gained protection against additional challenge of parental B7-1-negative osteosarcoma cells, (c) systemic immunity against pre-established pulmonary metastasis, and (d) activation of regional lymph node CD4(+) T cells, expansion of dendritic cells and natural killer cells and the secretion of interferon-gamma. These findings collectively support the therapeutic value of adenovirus-mediated in vivo gene transfer on osteosarcoma, which is of greater simplicity than cell-based B7-1 vaccine, and represent an attractive strategy for therapy of patients with metastatic osteosarcama who acquired resistance to current therapeutic protocols.     

In vivo immunogenicity of osteosarcoma cells that express B7-1a,an alternatively spliced form of B7-1

BACKGROUND: B7 family members play a central costimulatory role in T cell activation. We have previously identified B7-1a, an alternatively spliced form of B7-1. The function of B7-1a in induction of anti-tumor immunity remains uncertain. MATERIALS AND METHODS: The cDNAs of murine B7-1, B7-1a and B7-2 were introduced into a murine osteosarcoma cell line, LM8. The ability of B7 transfectants to elicit in vivo anti-tumor immunity was comparatively analyzed with respect to tumorigenecity, pulmonary metastasis and survival time. RESULTS: LM8 cells expressing B7-1, B7-1a, or B7-2 all elicited immunological responses in immunocompetent C3H/He mice. Notably, the anti-tumor effects were most obvious in mice inoculated with B7-1a-transfected LM8 cells. Such a difference among B7-transfectants became indistinguishable in immunodeficient nude mice. CONCLUSION: These findings indicate that B7-1a serves as a more efficacious costimulatory molecule than B7-1 or B7-2 in the induction and maintenance of anti-tumor immune responses against a poorly immunogenic osteosarcoma cell line.     

Prostate cancer gene therapy: comparison of adenovirus-mediated expression of interleukin 12 with interleukin 12 plus B7-1 for in situ gene therapy and gene-modified, cell-based vaccines.

We have documented previously that adenovirus-mediated interleukin 12 (IL-12) gene therapy is effective for orthotopic tumor control and suppression of pre-established metastases in a preclinical prostate cancer model (Y. Nasu et al., Gene Ther., 6: 338-349, 1999). In this report, we directly compare the effectiveness of an adenovirus that expresses both IL-12 and the costimulatory molecule B7-1 (AdmIL12/B7) with one that expresses IL-12 alone (AdmIL-12) using the poorly immunogenic RM-9 orthotopic murine model of prostate cancer. We document AdmIL-12/B7-mediated secretion of IL-12 and increased surface expression of B7-1 in infected RM-9 tumor cells. A significant reduction in orthotopic tumor size and increased survival was demonstrated in mice treated with a single orthotopic injection of AdmIL-12/B7 compared with AdmIL-12 or controls. Six of 19 animals treated with AdmIL-12/B7 survived long term with apparent eradication of the primary tumor in contrast to one of 38 animals in the AdmIL-12-treated group. Orthotopic treatment of tumors with both vectors led to an infiltration of both CD4+ and CD8+ immunoreactive cells, with AdmIL-12/B7 treatment having a more prolonged infiltration of CD8+ cells. AdmIL-12/B7 was also more effective than AdmIL-12 or controls at suppression of pre-established metastases. We further developed a vaccine model based on s.c. injection of infected, irradiated RM-9 cells and found that both AdmIL-12 and AdmIL-12/B7 are effective at suppressing the development and growth of challenge orthotopic tumors using this protocol.     

B7-1基因转染对骨肉瘤细胞生物学行为的影响

目的:研究B7-1真核表达载体pcDNA3-B7-1转染对骨肉瘤细胞株LM8的生物学行为的影响。方法:用脂质体(lipofectamine)介导B7-1基因和空载体pcDNA3转染LM8细胞,G418筛选得到抗性克隆,分别命名为LM8/B7-1和LM8/pcDNA3,用聚合酶链反应(RT-PCR)和流式细胞仪(FCM)鉴定转染基因的表达,采用四甲基偶氮唑蓝微量酶反应比色法(MTT)和FCM检测细胞体外增殖活性,并观察转染细胞在C3h小鼠体内致瘤能力。结果:RT-PCR和FCM显示B7-1基因在骨肉瘤细胞株LM8中呈高表达,LM8/B7-1体外增殖活性与LM8/pcDNA3和LM8细胞差异并无显著性(P>0.05),但LM8/B7-1的致瘤能力显著下降(P<0.01)。结论:B7-1基因对骨肉瘤细胞的体内增殖能力产生明显抑制作用,可以作为骨肉瘤基因治疗的理想目的基因之一。     

Increased Fas expression reduces the metastatic potential of human osteosarcoma cells.

PURPOSE: The process of metastasis requires the single tumor cell that seeds the metastatic clone to complete a complex series of steps. Identifying factors responsible for these steps is essential in developing and improving targeted therapy for metastasis. Resistance to receptor-mediated cell death, such as the Fas/Fas ligand pathway, is one mechanism commonly exploited by metastatic cell populations. EXPERIMENTAL DESIGN AND RESULTS: LM7, a subline of the SAOS human osteosarcoma cell line with low Fas expression, was selected for its high metastatic potential in an experimental nude mouse model. When transfected with the full-length Fas gene (LM7-Fas), these cells expressed higher levels of Fas than the parental LM7 cells or LM7-neo control-transfected cells. These cells were also more sensitive to Fas-induced cell death than controls. When injected intravenously into nude mice, the LM7-Fas cell line produced a significantly lower incidence of tumor nodules than control cell lines. Lung weight and tumor nodule size were also decreased in those mice injected with LM7-Fas. Levels of Fas were quantified in osteosarcoma lung nodules from 17 patients. Eight samples were Fas negative, whereas the remaining 9 were only weakly positive compared with normal human liver (positive control). CONCLUSIONS: Our results demonstrate that altering Fas expression can impact the metastatic potential of osteosarcoma cells. We conclude that the increase of Fas on the surface of the LM7 osteosarcoma cells increased their sensitivity to Fas-induced cell death in the microenvironment of the lung, where Fas ligand is constitutively expressed. Thus, loss of Fas expression is one mechanism by which osteosarcoma cells may evade host resistance mechanisms in the lung, increasing metastatic potential. Fas may therefore be a new therapeutic target for osteosarcoma.     

IL-12协同B7-1诱导机体抗肿瘤免疫的实验研究

目的研究白细胞介素-12(Interleukin-12,IL-12)协同共刺激分子B7-1,联合诱导机体抗肿瘤免疫对大鼠卵巢上皮癌的治疗作用并探讨其机理.方法用携带有小鼠IL-12和B7-1的逆转录病毒表达载体感染大鼠卵巢癌细胞株NuTu-19,建立高表达细胞株NuTu-19/IL-12、NuTu-19/B7-1及双基因共表达细胞株NuTu-19/IL-12-B7-1,并以转染空载体pLXSN的细胞NuTu-19/Neo为对照.用经丝裂霉素C处理的各种基因修饰的肿瘤细胞免疫动物,观察卵巢癌腹腔转移模型动物生存期,及其诱导细胞毒性T淋巴细胞(cytotoxic lymphocyte,CTL)杀伤活性的作用.结果经各种基因修饰的肿瘤细胞免疫后,大鼠脾淋巴细胞增殖能力有不同程度的提高,CTL杀伤同源肿瘤细胞的活性明显增强,IL-12和B7-1基因联合修饰的肿瘤细胞免疫动物对模型动物生存期的延长具有显著意义(P＜0.05).结论IL-12和B7-1基因联合修饰的肿瘤细胞可以刺激机体CTL增殖、成熟,增强CTL对肿瘤细胞的识别和杀伤活性等,两者联合具有明显的协同效应.联合免疫基因治疗作为一种新的卵巢癌治疗方法,值得深入研究.     

In situ expression of soluble B7-1 in the context of oncolytic herpes simplex virus induces potent antitumor immunity

In vivo delivery of immunomodulatory genes is a promising strategy for solid tumor vaccination. A drawback is that it necessitates induction of a large effect from transgene expression in a small percentage of tumor cells. Although the B7 family is known to be the most potent of the costimulatory molecules, gene transduction of B7 alone has not been effective in inducing antitumor immunity in nonimmunogenic tumors by ex vivo methods, much less in vivo. We have developed a novel approach where a gene encoding soluble B7-1, a fusion protein of the extracellular domain of murine B7-1 and the Fc portion of human IgG1, is delivered to tumor cells in vivo in the context of an oncolytic replication-competent herpes simplex virus, and the gene product is secreted by tumor cells rather than expressed on the cell surface. Defective herpes simplex virus vectors containing the B7-1-immunoglobulin (B7-1-Ig) fusion transgene (dvB7Ig) were generated using G207 as a helper virus and tested in the poorly immunogenic murine neuroblastoma, Neuro2a, in syngeneic A/J mice. Intraneoplastic inoculation of dvB7Ig/G207 at a low titer successfully inhibited the growth of established s.c. tumors, despite the expression of B7-1-Ig being detected in only 1% or fewer of tumor cells at the inoculation site, and prolonged the survival of mice bearing intracerebral tumors. Immunohistochemistry of dvB7Ig/G207-inoculated tumors revealed a significant increase in CD4+ and CD8+ T-cell infiltration compared with control tumors inoculated with defective vector expressing alkaline phosphatase (dvAP/G207). The antitumor effect of dvB7Ig/G207 was not manifested in athymic mice. In vivo depletion of immune cell subsets in A/J mice further revealed that CD8+ T cells, but not CD4+ T cells, were required. Animals cured of their tumors by dvB7Ig/G207 treatment were protected against rechallenge with a lethal dose of Neuro2a cells but not SaI/N cells. The results demonstrate that the use of soluble B7-1 for immune gene therapy is a potent and clinically applicable means of in situ cancer vaccination.     

Humanization of predicted T-cell epitopes reduces the immunogenicity of chimeric antibodies: new evidence supporting a simple method

Genetic engineering has provided several approaches to reduce immunogenicity of murine antibodies. We described previously a new method based on the humanization of the linear epitopes presented to T cells. In brief, potential immunogenic epitopes in the variable region were identified and subjected to point mutations to make them human and/or to modify amphipatic motifs. The resulting recombinant antibody retained its antigen binding affinity and was less immunogenic in monkeys than their murine or chimeric predecessors are. The present study provides two new examples of this T-cell epitope humanization approach: ior-t1A murine monoclonal antibody (mMAb), which recognizes the human-CD6 molecule, and ior-C5 mMAb, which recognizes a novel glycoprotein expressed on the surface of malignant colorectal cells. Seven amino acids were substituted in ior-C5 and eleven residues in ior-t1A, by the corresponding residues from the highest homologous human sequences. Surprisingly, the homology between re-shaped chimeric antibody variable region frameworks and human sequences was 80-90%. Experiments in monkeys showed that T1AhT and C5hT "detopes" antibodies were less immunogenic than their chimeric analogues while they retained 30-50% of antigen binding affinities. The proposed method might be of general applicability to reduce immunogenicity of chimeric antibodies with therapeutic potential.     

Protein transfer of glycosyl-phosphatidylinositol-B7-1 into tumor cell membranes: a novel approach to tumor immunotherapy.

Modification of tumor cells with one or more costimulatory adhesion molecules has been proposed as a means to develop therapeutic cancer vaccines for use in human immunotherapy. Expression of B7-1 (CD80) in tumors by gene transfer creates an immunogenic tumor cell that induces antitumor immunity and protects mice from further challenge with wild-type tumor cells. In this report, we demonstrate that protein transfer of glycosyl-phosphatidylinositol (GPI)-anchored costimulatory molecules into tumor cell membranes could be used as an alternative to gene transfer for tumor immunotherapy. Incubation of isolated tumor membranes with purified GPI-anchored B7-1 results in stable incorporation of B7-1 on tumor cell membranes within a few hours. Immunization of C57BL/6 mice with EG7 tumor membranes modified to express GPI-B7-1 by protein transfer induces tumor-specific T-cell proliferation and CTLs. In addition, immunization with these EG7 membranes protects mice from parental tumor challenge. The protein transfer approach used here does not require foreign vectors or live tumor cells and is completed within a matter of hours. Irradiated cells or membrane preparations from fresh or frozen tumor tissue can be used. Therefore, protein transfer of glycolipid-anchored molecules provides an efficient and novel approach to modify tumor membranes for human immunotherapy. This approach is not limited to costimulatory molecules because any cell surface protein can be converted to a GPI-anchored form by recombinant techniques.     

肿瘤靶向DNA载体--转铁蛋白-多聚乙烯亚胺体外介导小鼠IL-12基因转染小鼠骨肉瘤细胞的实验研究

目的 评价转铁蛋白-多聚乙烯亚胺(Transferrin-polyethylenimine，Tf-PEI)靶向性转染小鼠骨肉瘤细胞的可行性。以Tf-PEI为载体，将小鼠IL-12基因导入小鼠骨肉瘤细胞。方法 以Tf-PEI为载体，将荧光素酶基因和小鼠IL-12基因分别导入小鼠骨肉瘤细胞，并检测其转染效率。以游离转铁蛋白竞争性拮抗Tf-PEI，并观察其对转染效率的影响。结果 Tf-PEI可以高效的、靶向性的转染小鼠骨肉瘤细胞。当N/P比值为5时，Tf-PEI的转染效率最高。游离转铁蛋白能够显著的拮抗Tf-PEI的转染。Tf-PEI还可以成功地将小鼠IL-12基因导入小鼠骨肉瘤细胞。结论 Tf-PEI是一种高效、低毒、肿瘤靶向性的基因转染载体，它能成功地将mlL-12基因导入骨肉瘤细胞，广泛应用于体内外恶性肿瘤基因治疗的实验。     

Stable expression of chimeric anti-CD3 receptors on mammalian cells for stimulation of antitumor immunity

Expression of CD80 or CD86 costimulatory molecules on tumor cells can produce rejection of immunogenic but not poorly immunogenic tumors. We have previously shown that anti-CD3 single-chain antibodies expressed on the surface of cells can directly activate T cells. We therefore investigated whether anti-CD3 "receptors" could enhance CD86-mediated rejection of poorly immunogenic tumors. Expression of anti-CD3 receptors on cells was increased by introduction of membrane-proximal "spacer" domains containing glycosylation sites between the single-chain antibody and the transmembrane domain of the chimeric receptors. Removal of glycosylation sites in the spacer reduced surface expression due to increased shedding of chimeric receptors from the cell surface. Induction of T-cell proliferation by anti-CD3 receptors did not correlate with the expression level of chimeric protein, but rather depended on the physical properties of the spacer. Anti-CD3 receptors effectively induced T-cell cytotoxicity, whereas coexpression with CD80 or CD86 was required for generating T-cell proliferation and IL-2 secretion. Although expression of CD86 did not significantly delay the growth of poorly immunogenic B16-F1 tumors, expression of anti-CD3 receptors with CD86 produced complete tumor rejections in 50% of mice and induced significant protection against wild-type B16-F1 tumor cells. Our results show that spacer domains can dramatically influence the surface expression and the biological activity of chimeric antibody receptors. The strong antitumor activity produced by anti-CD3 receptors and CD86 on tumor cells indicates that this strategy may be beneficial for the gene-mediated therapy of poorly immunogenic tumors.     

B7-1基因转染骨肉瘤细胞诱导抗骨肉瘤主动免疫的实验研究

目的:探讨B7-1基因转染骨肉瘤细胞能否诱导抗骨肉瘤主动免疫作用。方法:利用脂质体将B7-1真核表达载体pcDNA3-B7-1和空载体pcDNA3分别导入骨肉瘤细胞株LM8中,G418筛选出阳性克隆(分别命名为LM8/B7-1和LM8/pcDNA3),通过RT-PCR、Western blot和流式细胞仪检测B7-1基因与蛋白的表达;通过软琼脂克隆形成实验观察转基因细胞株LM8/B7-1的体外增殖能力;用LM8、LM8/B7-1和LM8/pcDNA3分别经腹腔免疫小鼠,得到腹腔浸润淋巴细胞和致敏脾细胞,MTT法检测其体外杀伤活力;将LM8、LM8/B7-1和LM8/pcDNA3细胞分别接种到C3H雄性小鼠前腋下,观察其致瘤能力;观察LM8/B7-1致敏的小鼠对骨肉瘤细胞LM8是否具有免疫保护作用。结果:B7-1基因在转基因细胞LM8/B7-1中能得到高表达;LM8/B7-1体外增殖能力与LM8和LM8/pcDNA3无明显差异(P>0.05);LM8/B7-1诱导的CTL的杀伤活力显著高于LM8和LM8/pcDNA3诱导的CTL对相同靶细胞的杀伤活力,LM8/B7-1诱导的CTL对LM8/B7-1的杀伤活力也明显高于对LM8和LM8/pcDNA3的杀伤活力(P<0.05);LM8/B7-1致瘤能力较LM8和LM8/pcDNA3细胞明显下降(P<0.01);LM8/B7-1致敏的小鼠对骨肉瘤细胞LM8具有免疫保护作用。结论:B7-1基因转染骨肉瘤细胞能诱导抗骨肉瘤主动免疫作用,为利用B7-1基因进行骨肉瘤免疫基因治疗提供了实验依据。     

PD-L1/B7H-1 inhibits the effector phase of tumor rejection by T cell receptor (TCR) transgenic CD8+ T cells

Although increased circulating tumor antigen-specific CD8(+) T cells can be achieved by vaccination or adoptive transfer, tumor progression nonetheless often occurs through resistance to effector function. To develop a model for identifying mechanisms of resistance to antigen-specific CTLs, poorly immunogenic B16-F10 melanoma was transduced to express the K(b)-binding peptide SIYRYYGL as a green fluorescent protein fusion protein that should be recognized by high-affinity 2C TCR transgenic T cells. Although B16.SIY cells expressed high levels of antigen and were induced to express K(b) in response to IFN-gamma, they were poorly recognized by primed 2C/RAG2(-/-) T cells. A screen for candidate inhibitory ligands revealed elevated PD-L1/B7H-1 on IFN-gamma-treated B16-F10 cells and also on eight additional mouse tumors and seven human melanoma cell lines. Primed 2C/RAG2(-/-)/PD-1(-/-) T cells showed augmented cytokine production, proliferation, and cytolytic activity against tumor cells compared with wild-type 2C cells. This effect was reproduced with anti-PD-L1 antibody present during the effector phase but not during the priming culture. Adoptive transfer of 2C/RAG2(-/-)/PD-1(-/-) T cells in vivo caused tumor rejection under conditions in which wild-type 2C cells or CTLA-4-deficient 2C cells did not reject. Our results support interfering with PD-L1/PD-1 interactions to augment the effector function of tumor antigen-specific CD8(+) T cells in the tumor microenvironment.     

转染人CD80基因的黑色素瘤细胞生长特性及免疫原性探讨

】 ObjectivesTo investigate the effects of the CD80 costimulatory molecule expression on the immunogenicity and biological characteristics of poorly immunogenic B16 melanoma cells,explore the relationship between the immunogenicity of tumor cells and cell surface molecules. MethodsHuman CD80 cDNA was transfected into B16 melanoma cells by lipofectin-mediated gene transfer before the expansion of tumor cells were tested by MTT method in vitro;C57BL/6 mice were inculated subcutaneously either mock-transfected B16 cells (B16-neo)or CD80-transfected B16 cells (B16-CD80),then the latency,survival times and tumor mass growth were investigated.The lymphocytes were examined for both proliferation indices (PI) and specific cytotoxic activity by MTT method and improved LDH-releasing method,after syngenic Mixed Lymphocyte tumor cultures (MLTCs). ResultsIn the B16-CD80 cells,in which CD80 expression were detected by SABC method, demonstrated slower growth rate than B16-neo Clles in vivo (P<0.05),though they didn't in vitro. Compared with B16-wt and B16-neo cells,B16-CD80 cells more effectively induced the proliferation of effector lymphocytes and the generation of specific lytic activity against B16-wt cells. ConclusionsThe CD80 (B7-1) expression in poorly immunogenic tumor cells can increase their immunogenicity and decrease their tumorigenicity,the effects are associated with the expression of ICAM-1 and MHC molecules on tumor cells surface.     

B7-1/GFP基因真核表达载体的构建及其在骨肉瘤细胞中的表达

Objective： To construct eukaryotic expression vector containing B7-1/GFP geneand study its expression in osteosarcoma cell line LM8. Methods： By using gene cloning technique, eukaxyotic expression vector pEGFP-C1 was used to construct the murine B7-1 recombinant plasmid （pEGFP-C1/B7）. Recombinant plasmid was transfected into LM8 cells with liposome and was confirmed by restriction endonuclease digestion and DNA sequencing. The expression of the fusion protein was detected using fluorescence microscope and Western blot analysis. Results： The recombinant eukaryotic expression plasmid pEGFP-C1/B7 was successfully constructed, which was confirmed by DNA sequencing, RT-PGR and restriction enzymes analysis. The green fluorescent protein could be detected in the transfected LM8 with fluorescence microscope. The expected B7-1 and green fluorescent protein （GFP） fusion protein was detected by RT-PCR and Western blot. Conclusion： The eukaryotic expression vector containing B7-1/GFP gene was constructed successfully, and it could be expressed in LM8 after transfection.     

Intranasal therapy with an adenoviral vector containing the murine interleukin-12 gene eradicates osteosarcoma lung metastases.

The purpose of these studies was to determine the effect of adenovirus-mediated interleukin-12 (IL-12) gene transfer on the growth and development of osteosarcoma (OS) lung metastases in nude mice. A nude mouse model was produced by repetitive cycling of human SAOS OS cells through the lung. The resultant SAOS-LM6 cell line produced microscopic lung metastases by 5-6 weeks after i.v. injection of the tumor cells, with visible lung metastases present 8 weeks after injection. Transfection of SAOS-LM6 cells with a plasmid containing the murine IL-12 gene resulted in a decrease in metastatic potential. Animals injected with IL-12-transfected clones had fewer metastases compared with mice injected with SAOS-LM6 cells transfected with a control plasmid. Furthermore, nasal delivery of an adenoviral vector containing the murine IL-12 gene resulted in the inhibition of pulmonary metastases. Together, these data indicate that IL-12 may be an effective agent against OS and that nasal delivery may offer a unique way to deliver the gene to the local tumor environment, potentially decreasing systemic toxic effects.     

Systemic genetic transfer of p21WAF-1 and GM-CSF utilizing of a novel oligopeptide-based EGF receptor targeting polyplex

Based on the fact that aberrant overexpression of some growth factor receptors was observed in a variety of human cancer cells, a novel nonviral gene delivery system GE7, which contains a 16-amino-acid ligand for identifying EGF receptor was constructed for tumor-targeted gene therapy. Intravenous administration of GE7 system revealed that it has the ability to target beta-galactosidase (beta-gal) reporter gene into murine hepatoma (Hepa) cells. Owing to the limited antitumor effects elicited by a single-gene transfer, recent efforts to treat malignancy using combined gene therapy have been accomplished with varying degrees of success. In this study, the human cyclin-dependent kinase inhibitor gene p21(WAF-1) and the murine cytokine gene granulocyte-macrophage colony-stimulating factor (GM-CSF) were used simultaneously for in vivo gene therapy through systemic injection of the EGF R targeted GE7/DNA complex into murine hepatoma-bearing mice. The results demonstrated that combined administration of p21(WAF-1) and GM-CSF could remarkably inhibit the growth of subcutaneously transplanted hepatoma Hepa cells, and significantly increase the survival rate of tumor-bearing mice. The activities of natural killer (NK) cells and specific cytotoxic T lymphocytes (CTL) were clearly enhanced after combined gene therapy. In vitro experiments showed that p21(WAF-1) gene transfer exhibited a suppressive function on the growth of Hepa cells and the expression of H-2K(b) and B7-1 molecules on Hepa cells increased significantly after combined genes delivery. All these results suggested that the GE7 system was able to target therapeutic genes efficiently to cancer cells, which showed high EGF R expression. The cotransfer of p21(WAF-1) and GM-CSF genes apparently inhibited the growth of tumors through (a) the arrest of tumor cell growth and (b) the enhancement of systemic antitumor immunity.     

Interleukin (IL)-12 and IL-12 gene transfer up-regulate Fas expression in human osteosarcoma and breast cancer cells

Expression of Fas (CD95, APO-1), a cell surface receptor capable of inducing ligand-mediated apoptosis, is involved in tissue homeostasis and elimination of targeted cells by natural killer and T cells. Corruption of this pathway, such as reduced Fas expression, can allow tumor cells to escape elimination and promote metastatic potential. In this study, the status of Fas expression has been examined in the parental SAOS human osteosarcoma cells that do not metastasize and in selected variants that cause lung metastases in 16 weeks (LM2) or 8 weeks (LM6) after i.v. injection into nude mice. Fas expression correlated with the metastatic potentials of the three cell lines. Northern and fluorescence-activated cell-sorting analyses indicated that LM6 cells expressed Fas at a lower level than seen in the parental cells. Infection of the LM6 cells with an adenoviral vector containing the murine interleukin (IL)-12 gene (AD:mIL-12) or treatment with recombinant murine IL-12 resulted in a dose-dependent up-regulation of FAS: The up-regulation of Fas by IL-12 was also demonstrated in human etoposide-resistant MDA-MB-231 breast cancer cells. [(3)H]Thymidine growth inhibition studies indicated that the cell surface Fas induced after IL-12 exposure was functional and able to mediate cell death on cross-linking with anti-FAS: We also demonstrate that this effect is independent of IFN-gamma. Whereas these cell lines are sensitive to IFN-gamma, incubation with IFN-gamma does not increase susceptibility to Fas-mediated cell death, nor do these cells produce IFN-gamma with or without IL-12 treatment. We hypothesize that expression of Fas may play a role in the elimination of metastatic tumor cells in the lung, an organ in which Fas ligand is expressed. The antitumor activity of IL-12 may be secondary in part to its ability to up-regulate Fas expression on tumor cells, which subsequently increases immune-mediated destruction of osteosarcoma cells.     

mB7－1基因转染小鼠黑色素瘤细胞诱导的抗瘤效应研究

目的：研究共刺激Ｂ７－１（ＣＤ６０）在诱导有效的抗肿瘤免疫反应反应中所起的作用。方法：在体外,三种肿瘤细胞与同上鼠脾淋巴细胞混合培养（ＭＬＴＣＳ）后,测定淋巴细胞增殖指数（ＭＴＴ法）和特异杀伤活性（ＬＤＨ释放改良法）;ＭＴＴＩ地测定肿瘤细胞体外增殖能力后,将Ｂ１６－ｎｅｏ和Ｂ１６－ｍＢ７－１妆种于Ｃ５７ＢＬ／６小鼠皮下后观察成瘤期、荷瘤小鼠存活以及肿瘤结节生长速度。结果：与野生型Ｂ１６细胞和模拟转