Pass H I, Mew D J, Carbone M, Matthews W A, Donington J S, Baserga R, Walker C L, Resnicoff M, Steinberg S M
Thoracic Oncology Section, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.
Cancer Res. 1996 Sep 1;56(17):4044-8.
We evaluated the effect of antisense insulin-like growth factor (IGF) receptor transcripts on the proliferation and tumorigenicity in an SV40-induced, immunocompetent hamster mesothelioma model (H9A). Expression of IGF-1 and IGF-1 receptor (IGF-1R) genes was identified from H9A RNA using reverse transcription-PCR and Northern analysis. H9A cells were electroporated with inducible expression vectors (under the transcriptional control of heat shock promoter HSP70) containing a cDNA fragment corresponding to base pairs 1-309 of IGF-1R in the sense or antisense orientation to generate the respective clones A3 sense or B9 antisense. The expression vector in genomic DNA was detected with PCR analysis as a 173-bp fragment on ethidium bromide gels. The effects of the expression vectors were then evaluated in vitro under active (at 39 degrees C) or inactive (at 34 degrees C) conditions. At 39 degrees C, the B9 antisense transfectants demonstrated significantly less proliferation than A3 sense transfectants (P2 < 0.02). At 34 degrees C, cell growth of A3 sense- and B9 antisense-transfected cells was not significantly different. In vivo tumorigenicity was evaluated in hamsters inoculated with 10(5) A3 sense- or B9 antisense-transfected cells. The A3 sense clones resulted in greater numbers of tumors in vivo compared to the B9 antisense clone (P2 = 0.0001). When genomic DNA from tumors that developed in A3 sense and B9 antisense animals was analyzed for the expression vectors, a 173-bp fragment amplified from the expression vector was identified in the sense tumors but not in antisense B9 or wild-type H9A tumors, indicating a loss of the vector from the antisense clones that proliferated in vivo. The inhibitory effect of IGF-1R antisense transcripts on hamster mesothelioma demonstrated in this study by decreased growth and tumorigenicity in vitro and in vivo may have implications for the therapy of human mesothelioma.
我们在一个由SV40诱导的、具有免疫活性的仓鼠间皮瘤模型（H9A）中评估了反义胰岛素样生长因子（IGF）受体转录本对细胞增殖和致瘤性的影响。使用逆转录聚合酶链反应（RT-PCR）和Northern分析从H9A RNA中鉴定IGF-1和IGF-1受体（IGF-1R）基因的表达。用含有与IGF-1R第1至309个碱基对相对应的cDNA片段的可诱导表达载体（在热休克启动子HSP70的转录控制下）对H9A细胞进行电穿孔，该cDNA片段以正义或反义方向构建，以产生相应的克隆A3正义或B9反义。通过PCR分析在溴化乙锭凝胶上检测基因组DNA中的表达载体，其为一个173碱基对的片段。然后在活跃（39℃）或不活跃（34℃）条件下在体外评估表达载体的作用。在39℃时，B9反义转染细胞的增殖明显低于A3正义转染细胞（P2<0.02）。在34℃时，A3正义和B9反义转染细胞的生长没有显著差异。在用10(5)个A3正义或B9反义转染细胞接种的仓鼠中评估体内致瘤性。与B9反义克隆相比，A3正义克隆在体内产生的肿瘤数量更多（P2 = 0.0001）。当分析来自A3正义和B9反义动物体内形成的肿瘤的基因组DNA中的表达载体时，在正义肿瘤中鉴定出从表达载体扩增出的173碱基对片段，而在反义B9或野生型H9A肿瘤中未鉴定出，这表明在体内增殖的反义克隆中载体丢失。本研究通过体外和体内生长及致瘤性降低所证明的IGF-1R反义转录本对仓鼠间皮瘤的抑制作用可能对人类间皮瘤的治疗具有启示意义。
