Development of new therapy for uterine cervical carcinoma

1. HPV16E7蛋白在大鼠胚胎成纤维细胞中表达时，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C24G（半胱氨酸密码子24甘氨酸替代）和C91G突变体E7蛋白作为rb和C-jun结合域，在E7介导的细胞转化中起重要作用。野生型E7和C24G突变体具有抑制sm - α肌动蛋白转录的潜力。反过来，aC91G突变体取消了其沉默SM- α转录的功能，表明E7的c-ter区域的重要性。此外，野生型和024G突变体对MyoD的表达有抑制作用，而091 g突变体对MyoD的表达没有抑制作用，这表明E7和E7-c-jun结合对MyoD表达的调节对于HPV16E7表达细胞中sm - α肌动蛋白的沉默是重要的。我们利用针对E6/E7的一致引物扩增了64例患者淋巴结中的HPV E6/E7 dna，扩增后的dna进行了southern blot分析。64例患者组织病理检查显示10例淋巴结转移阳性。然而，PCR-southern blot检查显示，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HPV16 E6/E7 dna。提示PCR-Southern检测淋巴结转移的敏感性。具有增强功能的反义寡核苷酸（AS）的开发。我们已经开发了针对HPV16或18e6 （PS-P1， PS-K3和PS-K4）的psolaren2偶联AS。PS-1scr作为对照。在紫外线照射下，PS-Pi抑制宫颈癌细胞的生长，这种抑制作用依赖于E6基因序列。C4 II宫颈癌细胞可能同时编码PS-K3和PS-K4靶向的内源性mRNA。PS-AS对癌细胞生长的抑制作用是磷硫酸as的80倍以上，对正常角质细胞生长没有毒性作用。少

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