ABSTRACT
Immunofluorescent analysis is a standard method for detecting DNA virus oncoproteins in transformed cells. Here we demonstrate the detection of DNA virus nuclear oncoproteins by flow cytometry of unfixed cells, after saponin permeabilization. This method could to be of value in the evaluation and quantitation of oncogene products in transformed cells.
Subject(s)
Flow Cytometry , Oncogene Proteins, Viral/analysis , Saponins , Tumor Suppressor Protein p53/analysis , Adenovirus E1 Proteins/analysis , Animals , Antigens, Polyomavirus Transforming/analysis , Cell Line, Transformed , Cell Membrane Permeability/drug effects , Cell Nucleus/chemistry , Mice , Propidium , Saponins/pharmacologyABSTRACT
The early region 1 (E1) of human adenovirus (Ad) type 12 represses the expression of major histocompatibility (MHC) Class I genes in transformed primary rodent cells. In this paper we show that both NF-kappa B and KBF1 (p50 dimer) binding activity to the H2TF1 element in the Class I promoter is reduced in Ad12-13S-E1A-transformed cells compared to Ad5E1- or Ad12-12S-E1A-transformed cells. Consistently, in Ad12E1A-13S-transformed cells the H2TF1 element does not contribute to transcriptional activity in transient expression assays, whereas it does contribute in Ad12E1A-12S-transformed cells. Therefore, the most likely explanation is that reduced binding of NF-kappa B and KBF1 to the H2TF1 element accounts for the down-regulation of MHC Class I expression in Ad12E1- and Ad12E1A-13S-transformed cells.
