Experimental cancer gene therapy by multiple anti-survivin hammerhead ribozymes.

To improve the efficacy of gene therapy for cancer, we designed four hammerhead ribozyme adenoviruses (R1 to R4) targeting the exposed regions of survivin mRNA. In addition to the in vitro characterization, which included a determination of the sequence specificity of cleavage by primer extension, assays for cell proliferation and for in vivo tumor growth were used to score for ribozyme efficiency. The resulting suppression of survivin expression induced mitotic catastrophe and cell death via the caspase-3-dependent pathway. Importantly, administration of the ribozyme adenoviruses inhibited tumor growth in a hepatocellular carcinoma xenograft mouse model. Co-expression of R1, R3 and R4 ribozymes synergistically suppressed survivin and, as this combination targets all major forms of the survivin transcripts, produced the most potent anti-cancer effects. The adenoviruses carrying the multiple hammerhead ribozymes described in this report offered a robust gene therapy strategy against cancer.

A novel set of DNA methylation markers in urine sediments for sensitive/specific detection of bladder cancer.

PURPOSE: This study aims to provide a better set of DNA methylation markers in urine sediments for sensitive and specific detection of bladder cancer. EXPERIMENTAL DESIGN: Fifty-nine tumor-associated genes were profiled in three bladder cancer cell lines, a small cohort of cancer biopsies and urine sediments by methylation-specific PCR. Twenty-one candidate genes were then profiled in urine sediments from 132 bladder cancer patients (8 cases for stage 0a; 68 cases for stage I; 50 cases for stage II; 4 cases for stages III; and 2 cases for stage IV), 23 age-matched patients with noncancerous urinary lesions, 6 neurologic diseases, and 7 healthy volunteers. RESULTS: Despite six incidences of four genes reported in 3 of 23 noncancerous urinary lesion patients analyzed, cancer-specific hypermethylation in urine sediments were reported for 15 genes (P < 0.05). Methylation assessment of an 11-gene set (SALL3, CFTR, ABCC6, HPR1, RASSF1A, MT1A, RUNX3, ITGA4, BCL2, ALX4, MYOD1, DRM, CDH13, BMP3B, CCNA1, RPRM, MINT1, and BRCA1) confirmed the existing diagnosis of 121 among 132 bladder cancer cases (sensitivity, 91.7%) with 87% accuracy. Significantly, more than 75% of stage 0a and 88% of stage I disease were detected, indicating its value in the early diagnosis of bladder cancer. Interestingly, the cluster of reported methylation markers used in the U.S. bladder cancers is distinctly different from that identified in this study, suggesting a possible epigenetic disparity between the American and Chinese cases. CONCLUSIONS: Methylation profiling of an 11-gene set in urine sediments provides a sensitive and specific detection of bladder cancer.

Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis.

BACKGROUND: Hepatocellular carcinoma (HCC) presents one of the major health threats in China today. A better understanding of the molecular genetics underlying malignant transformation of hepatocytes is critical to success in the battle against this disease. The methylation state of C5 of the cytosine in the CpG di-nucleotide that is enriched within or near the promoter region of over 50 % of the polymerase II genes has a drastic effect on transcription of these genes. Changes in the methylation profile of the promoters represent an alternative to genetic lesions as causative factors for the tumor-specific aberrant expression of the genes. METHODS: We have used the methylation specific PCR method in conjunction with DNA sequencing to assess the methylation state of the promoter CpG islands of twenty genes. Aberrant expression of these genes have been attributed to the abnormal methylation profile of the corresponding promoter CpG islands in human tumors. RESULTS: While the following sixteen genes remained the unmethylated in all tumor and normal tissues: CDH1, APAF1, hMLH1, BRCA1, hTERC, VHL, RARbeta, TIMP3, DAPK1, SURVIVIN, p14ARF, RB1, p15INK4b, APC, RASSF1c and PTEN, varying degrees of tumor specific hypermethylation were associated with the p16INK4a, RASSF1a, CASP8 and CDH13 genes. For instance, the p16INK4a was highly methylated in HCC (17/29, 58.6%) and less significantly methylated in non-cancerous tissue (4/29. 13.79%). The RASSF1a was fully methylated in all tumor tissues (29/29, 100%), and less frequently methylated in corresponding non-cancerous tissue (24/29, 82.75%). CONCLUSIONS: Furthermore, co-existence of methylated with unmethylated DNA in some cases suggested that both genetic and epigenetic (CpG methylation) mechanisms may act in concert to inactivate the p16INK4a and RASSF1a in HCC. Finally, we found a significant association of cirrhosis with hypermethylation of the p16INK4a and hypomethylation of the CDH13 genes. For the first time, the survey was carried out on such an extent that it would not only provide new insights into the molecular mechanisms underscoring the aberrant expression of the genes in this study in HCC, but also offer essential information required for a good methylation-based diagnosis of HCC.