Different Responses of RT‐PCR Amplifications after Tumor Necrosis alpha (TNFa) Induction at Nuclear Factor kappa B (NFkB) Gene Silenced LNCaP Cells

Abstract

Prostate cancer is the second leading reason of morbidity and mortality in men in the Western World. Androgen receptor positive and androgen responsive lymph‐node metastasis of prostate cancer cell line LNCaP was used in all further experiments. Western with NFkB specific antibody was done after TNFa induction at LNCaP cells and a significant increase was taken.LNCaP cells transfected with siNFkB construct for 1,2 and 4 days and induced with TNFa for 24 hours. RNAisolation, cDNA conversion and RT‐PCR amplifications were done with apoptosis related gene primers such as p53, Bcl‐2, STAMP family primers. Chromatine immunoprecipitation (ChIP) assay was done with NFkB specific antibody before and after TNF induction at LNCaP and normal prostate cell line WPMY1. Since both cell lines express STAMP1 and 2, promoter region located NFkB response‐elements were identified and shown with specific primers. However, no change was shown at STAMP2 RT‐PCR amplification at −/+ TNFa native/siNFkB; STAMP1 showed a significant decrease with TNFa induction, while a significant increase with siNFkB.Survival gene NFkB silencing caused anti‐apoptotic STAMP1 increase that repress p53, together with MDM2.Gene silencing differed a panel of cancer regulatory genes.