Alterations of the retinoblastoma‐related gene RB2/p130 in lung adenocarcinomas induced by N‐nitrosobis(2‐hydroxypropyl)amine in rats

Abstract

Alteration of the retinoblastoma-related gene RB2/p130 was investigated in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in male Wistar rats. At 6 wk of age, 21 animals were given 2000 ppm of BHP in their drinking water for 12 wk and then maintained without further treatment until they were killed at the end of week 25. A total of 21 lung adenocarcinomas were obtained, and total RNAs were extracted from each for mutation analysis of RB2/p130 by the reverse transcription-polymerase chain reaction-single-strand comformation polymorphism approach. No mutations were found in exons 19-22. However, examination of the expression of the RB2/p130 gene by Northern blot analysis showed mRNA levels to be significantly lower than those of normal lung tissues. Western blot analysis showed reduction of the pRb2/p130 protein in all of the adenocarcinomas examined. These results suggest that alteration of the RB2/p130 gene may play important roles in the development of lung adenocarcinomas induced by BHP in rats.

Molecular Aspects during Multi-step Chemical Induced Carcinogenesis in the Lung and Pancreas

“…Portions of the tumors were also fixed in 10% neutrally buffered formalin at 4°C, routinely processed for embedding in paraffin, sectioned, and stained with hematoxylin and eosin for histological examination. For mutational analysis, polymerase chain reactionsingle strand conformation polymorphism (PCR-SSCP) analysis and reverse transcription (RT)-PCR-SSCP analysis were performed [7][8][9][10][11][12][13] . For the investigation of aberrant transcription and gene expression, RT-PCR, northern blot analysis and ribonuclease protection assay (RPA) were carried out [9][10][11][12][13][14][15][16][17] .…”

“…For the investigation of aberrant transcription and gene expression, RT-PCR, northern blot analysis and ribonuclease protection assay (RPA) were carried out [9][10][11][12][13][14][15][16][17] . Western blot analysis was also used for assessment of protein expression 9,12 . The genes and the specific methods used for the analysis of the alterations in rats are summarized in Table 1.…”

“…4. Histologically, adenocarcinoma developed through sequential changes of duct epithelium from hyperplasia, atypical hyperplasia, intraductal PCR-SSCP mutation Ki-ras (7) <20> , β-catenin (8) <21> , APC (8) , p53 (7) <22> , Ha-ras (7) , FHIT (9) RT-PCR-SSCP mutation Smad2 (11) , Smad4 (11) , TGFβRII (10) , M6P/IGF2R (13) , RB2/p130 (12) RT-PCR expression TGFβRII (10) , TGFα <23> , VEGF <23> aberrant transcription FHIT (9) <24> , M6P/IGF2R (13) RPA expression TGFβs (16) , Interleukins (17) Southern blot shorten Telomere <25> Northern blot expression VEGF (15) , VEGFRs (15) , midkine (14) <27> , RB2/p130 (12) , COX-2 < * > , MMPs <26>…”

“…MT-MMP <26> , TIMPs <26> , nm23 <28> , c-jun <28> , bax <29> , bcl2 <29> Western blot expression FHIT (9) , pRB2/p130 (12) TRAP assay activation Telomerase <25> Immunohistochemistry expression MMP-2 <26> , β-catenin <21> , midkine <27>…”