1-2-dielaidoylphosphatidylethanolamine and Urinary-Bladder-Neoplasms

To determine the change of expression of Bcl2 in cisplatin-resistant bladder cancer cell lines and the reversibility of chemoresistance to cisplatin with antisense oligonucleotide against Bcl2, as higher expression of Bcl2 is associated with drug resistance in many different cancer cell lines.. In the cisplatin-resistant bladder tumour cell lines T24R1 and T24R2, the expression of Bcl2 was determined by reverse transcription-polymerase chain reaction and Western blot assay, and antisense oligonucleotide targeting of the Bcl2 coding sequence was administered with lipofectin.. The expression of Bcl2 mRNA and protein was greater in T24R1 and T24R2 cells than in the parent T24 cells. Short-term exposure to cisplatin up-regulated Bcl2 mRNA and protein expression in parent T24 cells. Treatment with antisense oligonucleotide down-regulated Bcl2 protein expression and significantly enhanced the cytotoxicity of cisplatin.. Up-regulation of Bcl2 protein expression might be one of the mechanisms of cisplatin resistance in bladder cancer cells, and antisense Bcl2 oligonucleotide may be helpful in chemotherapy for bladder cancer by reversing cisplatin resistance.

Topics: Antineoplastic Agents; Blotting, Western; Cisplatin; Drug Resistance, Neoplasm; Humans; Oligonucleotides, Antisense; Phosphatidylethanolamines; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured; Up-Regulation; Urinary Bladder Neoplasms

Most antisense oligonucleotide experiments are performed with molecules containing RNase H-competent backbones. However, RNase H may cleave nontargeted mRNAs bound to only partially complementary oligonucleotides. Decreasing such "irrelevant cleavage" would be of critical importance to the ability of the antisense biotechnology to provide accurate assessment of gene function. RNase P is a ubiquitous endogenous cellular ribozyme whose function is to cleave the 5' terminus of precursor tRNAs to generate the mature tRNA. To recruit RNase P, complementary oligonucleotides called external guide sequences (EGS), which mimic structural features of precursor tRNA, were incorporated into an antisense 2'-O-methyl oligoribonucleotide targeted to the 3' region of the PKC-alpha mRNA. In T24 human bladder carcinoma cells, these EGSs, but not control sequences, were highly effective in downregulating PKC-alpha protein and mRNA expression. Furthermore, the downregulation is dependent on the presence of, and base sequence in, the T-loop. Similar observations were made with an EGS targeted to the bcl-xL mRNA.

Topics: 3' Untranslated Regions; bcl-X Protein; Blotting, Western; Down-Regulation; Endoribonucleases; Enzyme Activation; Humans; Isoenzymes; Nucleic Acid Conformation; Oligoribonucleotides; Phosphatidylethanolamines; Protein Kinase C; Protein Kinase C-alpha; Proto-Oncogene Proteins c-bcl-2; Ribonuclease H; Ribonuclease P; RNA Processing, Post-Transcriptional; RNA, Antisense; RNA, Catalytic; RNA, Messenger; RNA, Small Untranslated; RNA, Transfer; Substrate Specificity; Transfection; Tumor Cells, Cultured; Urinary Bladder Neoplasms

For the application of gene therapy to bladder cancer, we examined four in vivo gene transfer methods without viral vectors. For lipofection cationic liposomes (Lipofectin) were instilled into murine bladders. The hemagglutinating virus of Japan (HVJ)-liposomes possessing membrane fusion activity were also injected intraluminally. Using a particle gun, rabbit bladder mucosa was bombarded with DNA-coated gold microcarriers. Electrotransfection was examined in rabbit bladder by pulse direct currents (0.15-0.2 A, 50 msec, repeated 8 times) generated between needle electrodes after submucous injection of DNA solution. beta-galactosidase gene and chloramphenicol acetyltransferase (CAT) gene were used as marker genes. Although lipofection was inefficient in normal urothelium, cancerous urothelium was transfected slightly. HVJ-liposomes more efficiently transfected superficial layers of urothelium with a peak of expression on day 5. The particle gun produced non-uniform but efficient transfection in deeper layers of the urothelium. By electrotransfection, submucous interstitial cells were transfected as well as urothelium. No major complications were observed after these four procedures. HVJ-liposomes are potentially useful for the treatment of carcinoma in situ and the latter two methods may be suitable for the adjuvant therapy of localized bladder tumors.

Topics: Animals; Electroporation; Gene Transfer Techniques; Genetic Therapy; Liposomes; Mice; Mice, Inbred C3H; Phosphatidylethanolamines; Rabbits; Rats; Rats, Sprague-Dawley; Respirovirus; Transfection; Urinary Bladder Neoplasms