Wilms' tumor gene WT1-shRNA as a potent apoptosis-inducing agent for solid tumors

Wilms' tumor gene WT1 is overexpressed in leukemia and various types of solid tumors and plays an important role in leukemogenesis and tumorigenesis. We tested apoptosis-inducing ability of short hairpin RNAs targeting exon 5 (shWTE5), exon10 (shWTE10) and 3'UTR (shWT3U) of the WT1 gene. Among the three WT1-shRNAs, since shWTE5 most effectively induced apoptosis, its ability as an apoptosis-inducing agent was intensively examined. shWTE5 induced mitochondrial damage and resultant apoptosis in five WT1-expressing solid cancer cells originated from gastric (AZ-521), lung (LU99B), ovarian (TYKnuCPr) cancers, fibrosarcoma (HT-1080) and glioblastoma (A172). Moreover, shWTE5 significantly enhanced apoptosis induced by chemotherapeutic agents, doxorubicin (DOX) and etoposide (ETP), or by death ligand TRAIL in all of the four solid tumor cells examined (HT-1080, LU99B, TYK and A172). Transduction of one each of WT1 isoforms with exon 5 [17AA(+)KTS(+) and 17AA(+)KTS(-)] prevented mitochondrial damage induced by ETP or TRAIL and inhibited apoptosis. These results showed that shWTE5 induced apoptosis through the suppression of the WT1 isoform with exon 5. Furthermore, shWTE5 increased expression of proapoptotic Bak and Bax proteins and decreased antiapoptotic Bcl-xL and Bcl-2 proteins in WT1-expressing HT-1080 cells, indicating that WT1 isoforms with exon 5 might play an antiapoptotic role through regulation of Bcl-2 family genes in solid tumor cells. The results presented here demonstrated that WT1-shRNA targeting exon 5 should serve as a potent anti-cancer agent for various types of solid tumors.     

Wilms' tumor gene WT1 17AA(-)/KTS(-) isoform induces morphological changes and promotes cell migration and invasion in vitro

The wild-type Wilms' tumor gene WT1 is overexpressed in human primary leukemia and in a wide variety of solid cancers. All of the four WT1 isoforms are expressed in primary cancers and each is considered to have a different function. However, the functions of each of the WT1 isoforms in cancer cells remain unclear. The present study demonstrated that constitutive expression of the WT1 17AA(-)/KTS(-) isoform induces morphological changes characterized by a small-sized cell shape in TYK-nu.CP-r (TYK) ovarian cancer cells. In the WT1 17AA(-)/KTS(-) isoform-transduced TYK cells, cell-substratum adhesion was suppressed, and cell migration and in vitro invasion were enhanced compared to that in mock vector-transduced TYK cells. Constitutive expression of the WT1 17AA(-)/KTS(-) isoform also induced morphological changes in five (one gastric, one esophageal, two breast and one fibrosarcoma) of eight cancer cell lines examined. No WT1 isoforms other than the WT1 17AA(-)/KTS(-) isoform induced the phenotypic changes. A decrease in alpha-actinin 1 and cofilin expression and an increase in gelsolin expression were observed in WT1 17AA(-)/KTS(-) isoform-transduced TYK cells. In contrast, co-expression of alpha-actinin 1 and cofilin or knockdown of gelsolin expression by small interfering RNA restored WT1 17AA(-)/KTS(-) isoform-transduced TYK cells to a phenotype that was comparable to that of the parent TYK cells. These results indicated that the WT1 17AA(-)/KTS(-) isoform exerted its oncogenic functions through modulation of cytoskeletal dynamics. The present results may provide a novel insight into the signaling pathway of the WT1 gene for its oncogenic functions.     

导入Rb、p53、p16和H-ras反义RNA对人胃癌细胞恶性增殖的影响

构建Rb、p16、p53、H-ras反义RNA的逆转录病毒载体,利用脂质体介导和病毒感染的方法将其分别转入—存在Rb基因部分缺失、p16低表达、p53缺失和H-ras点突变的胃癌细胞系.观察这些基因对该细胞生长及致癌性的影响,探讨肿瘤相关基因在人胃癌发生、发展过程中的作用.结果表明,通过基因重组技术将多种基因分别重组至逆转录病毒载体PLXSN中,经鉴定获得正插及反插有该基因的重组质粒.将这些质粒分别用脂质体介导和病毒感染的方法转染BGC823细胞,G418筛选2～3周后,获得较高的转染效率.对转染后细胞DNA、RNA进行分析,证明外源性基因已整合人BGC823细胞并获稳定表达.表达有外源性p16、p53基因和H-ras反义RNA的裸鼠致癌性均有明显的抑制作用,而Rb基因对BGC823的恶性增殖能力没有明显抑制.这一结果表明p16、p53、H-ras几个基因的异常可能是导致这一个细胞癌变及最终发展成胃癌的主要原因.本实验进一步明确了胃癌的发生、发展是多基因变异累积的结果.     

WT1 and WT1-AS genes are inactivated by promoter methylation in ovarian clear cell adenocarcinoma

BACKGROUND: Ovarian clear cell adenocarcinoma is associated with one of the poorest prognoses among human epithelial ovarian cancers. The authors hypothesized that Wilms tumor suppressor 1 gene (WT1) sense and antisense (WT1-AS) expression and their promoter methylation status could characterize ovarian clear cell adenocarcinoma from ovarian serous adenocarcinoma. METHODS: To test this hypothesis, ovarian cancer cell lines and 42 cancer tissues (17 clear cell and 25 serous adenocarcinoma) were analyzed for expression and methylation of WT1 and WT1-AS genes. RESULTS: These experiments demonstrated that all serous adenocarcinoma tissues expressed both WT1 and WT1-AS genes, although expression of these genes was lacking in clear cell adenocarcinoma. The WT1 and WT1-AS promoter were significantly methylated in clear cell adenocarcinoma (88.2% and 88.2%, respectively) compared with serous adenocarcinoma (24.0% and 20.0%, respectively). Significant correlation between methylation and mRNA expression status was observed for each gene. Also in agreement with these data, WT1 and WT1-AS negative ovarian cancer cell lines reexpressed these genes after treatment with the demethylating agent, 5-aza-2'-deoxycytidine. CONCLUSIONS: The current study shows that CpG hypermethylation is an important mechanism of WT1 and WT1-AS gene inactivation in ovarian clear cell adenocarcinoma. This is the first report that has demonstrated differential expression and methylation of WT1-AS in ovarian clear cell and serous adenocarcinomas. This study presents new molecular characterizations between these two types of adenocarcinoma and may provide insight as to why clear cell adenocarcinoma has a poorer prognosis than serous adenocarcinoma of the ovary.     

p53 gene mutations in gastric cancer metastases and in gastric cancer cell lines derived from metastases

Structural alterations of the p53 gene were investigated in tissue specimens of gastric and cervical cancers and in cell lines of gastric, esophageal, and cervical cancers, by polymerase chain reaction-single-strand conformation polymorphism analysis. Two of the four gastric cancer metastases and four of the eight cell lines originally established from gastric cancer metastases were found to have p53 gene alterations in the exon 5 to 11 region; point mutations and amino acid replacements were detected in a liver and an ovary metastasis at exon 7, in the TMK1 and MKN1 cell lines at exon 5, and in the OKAJIMA cell line at exon 10. The normal allele was not found in these cell lines. In the KATO-III cell line, gross deletion and rearrangement of the p53 gene were noted. However, no p53 mutations were identified in 19 primary lesions of gastric cancer, suggesting that the p53 gene abnormality preferentially occurs in the advanced stages of gastric cancer. In contrast to the gastric cancer, none of the 13 esophageal cancer cell lines, including two cell lines established from metastases, and none of the four cervical cancer cell lines showed any aberration in exons 5 to 11 of the p53 gene. During the course of the study, a novel polymorphism in intron 7 of the p53 gene was found, which can be recognized by restriction enzyme digestions of the polymerase chain reaction product.     

Inhibitory effect of endothelin A receptor blockade on tumor growth and liver metastasis of a human gastric cancer cell line

Background With metastatic progression, gastric cancer is incurable. Using a DNA microarray, we performed differential gene expression analysis of established highly metastatic gastric cancer cell lines and compared the findings with those from a low-metastatic parental cell line. The results demonstrated that the endothelin A receptor (ET-A) gene was the only one from the highly metastatic cell lines that was generally up-regulated. Methods To investigate the role that ET-A plays in gastric cancer metastasis, we studied the effect of an ET-A-selective antagonist, YM598, on cell proliferation, tumor growth, and liver metastasis of the highly liver metastatic cell line AZ-H5c, established from the low metastatic human gastric cancer cell line AZ-521. Results An in vivo study using nude mice demonstrated that YM598 had a significant growth inhibition effect on AZ-H5c at doses of 0.5–10.0 mg/kg. The liver metastatic rate was also significantly reduced by YM598: control, 83.3%; 1 mg/kg dosage, 16.7%; 10 mg/kg, 20%; and pretreatment at 1 mg/kg, 16.7%. There was no evidence of gross toxicity resulting from the YM598 treatment. Conclusion The ET-A blockade by YM598 had a strong inhibitory effect against tumor growth and liver metastasis of the gastric cancer cell lines. These data suggest that YM598 has potential as a novel therapeutic agent for inhibiting liver metastasis of gastric cancer.     

Mutational analysis of the hMSH2 gene in a wide variety of tumors

The hMSH2 gene participates in DNA mismatch repair and its mutation can result in genetic instability of the human genome which is an important feature of tumorigenesis. In this study, genetic alterations of the hMSH2 gene were examined in 43 ovarian, 36 non-small cell lung (NSCL), 31 poorly differentiated gastric, 15 endometrial, and 11 colon cancers, nine gastric cancer cell lines, 41 adult T-cell leukemias (ATLs), two ATL cell lines, and 37 non-Hodgkin's lymphomas (NHLs), using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique. Microsatellite instability (MSI) was also investigated for ovarian, NSCL, and colon cancers. The incidence of MSI was 1/36 (3%) for NSCL, 2/23 (9%) for ovarian, and 1/11 (9%) for colon cancers. Missense base changes of the hMSH2 gene were identified in two gastric cancer patients (ATG to ATA resulting in Met changing to Ile at codon 688 in exon 13 and ACA to GCA resulting in Thr changing to Ala at codon 803 in exon 14). These mutations were found in samples with no MSI. One ovarian and one gastric cancer, and six ATL samples showed two types of polymorphisms of hMSH2 (CTT to TTT resulting in Leu changing to Phe at codon 390 in exon 7 and CAG to AAG resulting in Gin to Arg at codon 419 in exon 7). Our data suggest that MSI and hMSH2 mutations are uncommon in sporadic tumors.     

p16 mutations/deletions are not frequent events in prostate cancer.

Cyclin-dependent kinase-4 inhibitor gene (p16INK4) has recently been mapped to chromosome 9p21. Homozygous deletions of this gene have been found at high frequency in cell lines derived from different types of tumours. These findings suggested therefore, that p16INK4 is a tumour-suppressor gene involved in a wide variety of human cancers. To investigate the frequency of p16INK mutations/deletions in prostate cancer, we screened 20 primary prostate tumours and four established cell lines by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) analysis for exon 1 and exon 2. In contrast to most previous reports, no homozygous deletions were found in prostate cancer cell lines, but one cell line (DU145) has revealed to a mutation at codon 76. Only two SSCP shifts were detected in primary tumours: one of them corresponds to a mutation at codon 55 and the other one probably corresponds to a polymorphism. These data suggest that mutation of the p16INK4 gene is not a frequent genetic alteration implicated in prostate cancer development.     

Low Wilms' tumor gene expression in tumor tissues predicts poor prognosis in patients with non-small-cell lung cancer

We elucidated the relationship between prognosis of non-small-cell lung cancer (NSCLC) and Wilms' tumor gene (WT1) mRNA expression in tumor tissue. The WT1 mRNA expression levels of the fatal cases were lower as compared with those of the survival cases. Overall survival (OS) and disease-free survival (DFS) of the high WT1 expression group were longer than of the low expression group. As for squamous cell lung cancer (SQLC), low WT1 expression was significantly associated with lymph node metastasis. Cox analysis revealed that the gene level was a significant prognostic factor in OS and DFS. Low WT1 expression predicted poor prognosis in patients with NSCLC.     

WT1 is a tumor-associated antigen in colon cancer that can be recognized by in vitro stimulated cytotoxic T cells

The Wilms' tumor suppressor gene (WT1) has been shown to be overexpressed in acute and chronic leukemias and in a variety of solid human malignancies, including cancers of the breast and lung. In our present study, we investigated the potential role of WT1 gene in human colon cancer. WT1 mRNA and protein expression was analyzed in a panel of human colon cancer cell lines and primary colon carcinomas by RT-PCR and Western blot analysis, respectively. A mutational screen of WT1' zinc-finger region was carried out by sequence analysis. Finally, using peptide-stimulated cytotoxic T cells it was investigated whether WT1-expressing colon tumor cells are a potential target for antigen-specific immunotherapy. Medium to high abundant levels of WT1 mRNA were detected by RT-PCR in 10 of 12 (83%) colon cell lines and by quantitative, real-time RT-PCR in 13 of 15 (87%) primary tumors, whereas only very low levels of expression were found in 2 primary tumors. Interestingly, however, low levels of WT1 mRNA were also detected in all samples derived from normal colon mucosa. When RT-PCR products were examined by sequence analysis, both +KTS and -KTS splice isoforms but no zinc-finger mutations were found, suggesting that the wild-type form of the WT1 gene is expressed. To determine whether the WT1 protein can serve as a target antigen for immunotherapy, 2 HLA-A2.1-restricted WT1 peptides (Db126 and WH187) were used for the in vitro induction of WT1-specific cytotoxic T lymphocytes (CTLs). The WH187-specific CTLs not only lysed target cells pulsed exogenously with cognate peptide but also WT1-expressing colon tumor cells in a HLA-restricted manner. These findings identify the WT1 protein as an attractive target for the development of antigen-specific immunotherapy in human colon cancer.     

14株肿瘤细胞P53基因突变的检测

目的：检测14株肿瘤细胞P53基因的突变情况。方法：根据P53基因5～8外显子之间的内含子序列设计引物,分别对人肺癌、脑胶质瘤、肝癌、胃癌、前列腺癌、乳腺癌、结肠癌、脉络膜恶性黑素瘤、视网膜成细胞瘤等9种肿瘤14株细胞的P53基因5、6、7、8的外显子进行PCR扩增反应。琼脂糖凝胶电泳鉴定PCR产物;DNA测序,并与正常的DNA序列比较;抽提肿瘤细胞蛋白进行Western blotting检测P53蛋白的表达。结果：14株肿瘤细胞P53基因热变区5、6、7、8外显子的扩增产物经电泳鉴定与预期相同。DNA测序结果表明,14株肿瘤细胞中8株存在P53基因5～8外显子的突变,其中肺癌细胞H1299、肝癌细胞Hep3B、肝癌细胞SMMC7721、脉络膜恶性黑素瘤细胞OCM-1检测到以前未报道过的突变;突变主要发生在外显子的编码序列,多数为单个碱基替换导致的错义突变,也有部分表现为同义突变;2株正常细胞未检测到突变。Western blotting检测显示,有基因突变的8株肿瘤细胞中仅6株有P53蛋白表达;所有P53基因未突变的肿瘤细胞株和正常细胞株均未检测到P53蛋白。结论：检测的14株肿瘤细胞中有8株细胞P53基因热变区存在突变,其中4株检测到以前未报道过的突变;2株正常细胞未发现P53基因突变。     

新基因1A6定位及在肿瘤中的双色荧光原位杂交分析

目的 了解定位新基因1A6在不同组织来源肿瘤中的状态。方法 用单色FISH法将载于质粒的1A6基因小片段cDNA定位于C组某一染色体长臂,根据该基因测序结果设计8对引物,通过PCR在PAC文库中筛选目的的基因的基因组DNA。用切口翻译法分别用红绿荧光直接标记12号α－卫星重复序列和1A6基因组DNA,采用双色荧光共杂交的方法定位1A6基因,并对其及12号染色体进行了数量分析。结果 1A6基因定位于12q23.2-23.3。1A6基因在乳腺癌、卵巢癌及胃癌细胞株中双色FISH的绿红信号比（R值）为0．96－1．01,在胃癌组织印片中R值为0．93－1．11。12号染色体在乳腺癌细胞株BMI中的双体率87．7％,三体率7．4％;在卵巢癌细胞株SKOV3中的多体率100％,其中五倍体67．6％;在胃癌细胞株BGC823中的多体率为83．1％,其中三体率为71％。在实体瘤胃癌组织印片中,86．4％（19／22）的病例12号染色体以双拷贝为主。结论 1A6基因在实体瘤胃癌组织及肿瘤细胞株（BGC823、SKOV3、BMI）中均无明显扩增和缺失,推测其在肿瘤中的作用方式不是扩增或缺失。12号染色体数目在不同组织来源肿瘤细胞株中各异,意义有待探讨。