RESUMEN
<p><b>OBJECTIVE</b>To study the effect of gene expression of mouse uroplakin II (UPII) promoter on human bladder cell cancer cell line.</p><p><b>METHODS</b>The mRNA expression of different cell lines was quantified by RT-PCR. Green fluorescent protein (GFP) and luciferase (Luc) were used as reporter genes. The plasmids carrying UPII or GFP were constructed and transfected into human cell lines of bladder transitional cell cancer (BIU-87), kindey cancer (GRC-1), vascular endothelium (EC), lung cancer cell line (A549) and skin fibroblast cell line (Hs27). GFP activity of cells was detected by confocual microscopy and flow cytometry (FCM). Luciferase value was measured by luminometer (microplate) and luciferase to beta-galactosidase ratios (L/G values) were used for evaluating transfection efficiency.</p><p><b>RESULTS</b>RT-PCR showed high expression level of UPII mRNA in bladder cancer cell line BIU-87, whereas low level or no expression in nonbladder cancer cell lines. The activity of GFP in bladder cancer (BIU-87) cell was higher than that in the other cell lines (5 - 10/HP versus 0 - 2/HP), with 4.34% positive cells in BIU-87 detected by FCM, but no positive cell was found in the other cell lines. L/G values indicated that the luciferase expression in human bladder cancer cells transfected with mouse UPII promoter was 1.8 - 8.2-fold as high as that in the nonbladder cell lines.</p><p><b>CONCLUSION</b>Mouse UPII promoter gene can be expressed in a tissue-specific fashion in human urinary bladder cancer. It is capable of initiating transcription of reporter genes in human bladder cancer cell line.</p>
Asunto(s)
Animales , Humanos , Ratones , Línea Celular Tumoral , Citometría de Flujo , Terapia Genética , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes , Genética , Proteínas de la Membrana , Genética , Especificidad de Órganos , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Neoplasias de la Vejiga Urinaria , Genética , Terapéutica , Uroplaquina IIRESUMEN
<p><b>OBJECTIVE</b>To construct and screen the suppression subtractive hybridization (SSH) library of human renal cell carcinoma (RCC).</p><p><b>METHODS</b>Poly A(+) RNA was isolated from RCC lines 786-O (tester) and renal cell (RC) lines HK-2 (driver), respectively. SSH procedure was performed according to the protocol of the PCR-Select cDNA Subtraction Kit (Clontech), and PCR products were cloned into pT-Adv vector and transformed E. coli TOP10F'. All positive clones picked out were digested and some of which were sequenced.</p><p><b>RESULTS</b>The SSH library contained 362 clones with SSH cDNA fragments distributed mainly from 0.3 to 0.9 kb. Among 50 clones sequenced randomly, 2 represented unknown genes and the other 48 derived from 36 known genes.</p><p><b>CONCLUSION</b>The quality of the SSH library of human RCC is reliable and its construction is the basis for further screening differentially expressed genes of RCC.</p>