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Development of new therapy for uterine cervical carcinoma

宫颈癌新疗法的开发

基本信息

批准号：
08557092
负责人：
WAKE Norio
金额：
$ 5.76万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08557092/
关键词：
cervical cancer SM-alphaactin cell morphology alteration Molecular diagnosis of metastasis E6 E7 psolaren-conjugate antisense oligo DNA cell growth suppression SM-αactn psolaren conjugate antisense oligo DNA HPV Cytoskeleton Gene d

项目摘要

1. Alterations in cell morphology induced by HPV16E7 We have shown that SM-alpha actin transcription and translation are completely inhibited in the presence of HPV16E7 protein expressed in Rat embryonal fibroblasts. To explore the mechanism involved in the transcriptional silencing of SM-alpha actin, we have made the C24G (Sub-stitution of codon 24 glycine for cystein) and the C91G mutant E7 protein as Rb-and C-jun binding domains are important for E7-mediated cell transformation. A Wild-type E7 and C24G mutant had a potential to inhibit SM-alpha actin transcription. In turn, aC91G mutant abrogated its function to silence the SM- alpha transcription, suggesting the importnatce of c-ter region of E7. In addition, suppression of MyoD expression by wild-type and 024G mutants were in contrast to the absent of MyoD suppression by 091 G.These suggested that modulation of MyoD expression by E7 and E7-c-jun binding are important for the SM-alpha actin silencing in the HPV16E7 expressing cells … More .2. Molecular diagnosis of cervical cell carcinoma progression We have amplified HPV E6/E7 DNAs obtained from lymph nodes of 64 patients by the use of consensus primers targeted to the E6/E7 and amplified DNAs were subjected to southern blot analyses. Histo-pathological examinations indicated the positive lymph node metastasis in 10 out of 64 patients. However, the PCR-southern blot examination showed the presence of HPV16 E6/E7 DNAs in the lymph nodes from 45 patients out of the remaining lymph node metastasis negative 54 patients. These suggested the sensitivity of PCR-Southern methods to detect lymph node metastasis.3. Development of antisense oligo nucleotides (AS) with enhanced functions. We have developed the psolaren-conjugated AS that is targeted to HPV16 or 18 E6 (PS-P1, PS-K3 and PS-K4). A scramble PS-1scr was used as a control. In the presence of UV irradiation, PS-Pi suppressed the growth of cervical cancer cells, that was dependent on the E6 gene sequence. C4 II cervical cancer cells maybe encoded the endogenous mRNA targeted by both PS-K3 and PS-K4. PS-AS had aAS potential to inhibit the cancer cell growth, that was more than 80 folds, compared to phospborothioate AS.PS-AS did not exhibit the toxic effects to normal keratinocyte growth. Less

1. HPV 16 E7诱导的细胞形态学改变我们已经证明，在大鼠胚胎成纤维细胞中表达的HPV 16 E7蛋白存在时，SM-α肌动蛋白的转录和翻译被完全抑制。为了探索SM-α肌动蛋白转录沉默的机制，我们将C24 G（24号密码子甘氨酸取代半胱氨酸）和C91 G突变体E7蛋白作为Rb和C-jun结合结构域，对E7介导的细胞转化是重要的。野生型E7和C24 G突变体具有抑制SM-α肌动蛋白转录的潜力。而C91 G突变体则使SM-α转录沉默功能丧失，提示E7的c-ter区的重要性。此外，野生型和024 G突变体对MyoD表达的抑制与091 G对MyoD表达的不抑制形成对比。这些表明E7和E7-c-jun结合对MyoD表达的调节对于HPV 16 E7表达细胞中SM-α肌动蛋白沉默是重要的 ...更多信息 .2.宫颈细胞癌进展的分子诊断我们通过使用靶向HPV E6/E7的共有引物扩增从64名患者的淋巴结中获得的HPV E6/E7 DNA，并对扩增的DNA进行Southern印迹分析。64例患者中10例组织病理检查显示淋巴结转移阳性。而在54例淋巴结转移阴性的患者中，45例患者的淋巴结中存在HPV 16 E6/E7 DNA。提示PCR-Southern方法检测淋巴结转移的敏感性.具有增强功能的反义寡核苷酸的开发。我们已经开发了靶向HPV 16或18 E6的psolaren缀合的AS（PS-P1、PS-K3和PS-K4）。使用加扰PS-1 scr作为对照。在紫外线照射的情况下，PS-Pi抑制宫颈癌细胞的生长，这依赖于E6基因序列。C4 Ⅱ宫颈癌细胞可能编码PS-K3和PS-K4共同靶向的内源性mRNA。PS-AS对癌细胞生长的抑制作用是硫代磷酸AS的80倍以上，对正常角质形成细胞的生长无毒性作用。少