Detection of A 140 KDA nucleolar protein with an anti-PCNA autoantibody

Abstract

To assess the numbers and types of PCNA (proliferating cell nuclear antigen) species, immunoprecipitation studies on HeLa cell, nuclear and nucleolar extracts were performed. A 140 KDa protein from HeLa nucleoli was immunoprecipitated by an autoantibody (E.B.) previously used to detect the proliferating cell nuclear antigens (PCNA). The 140 KDa protein was also detected in the nuclear extract of colon carcinioma cells (Ω) labeled in vitro with 125I-Bolton Hunter reagent. When the growth of the colon carcinoma cells was inhibited by 1% N,N-dimethylformamide for two weeks, the 140 KDa protein was not detected which suggests this protein is associated with cell growth. Several studies have indicated there are nucleolar antigens associated with various stages of the cell cycle. These antigens are referred to as PCNA (Proliferating Cell Nuclear Antigen), SS-B/La (Tan, 1982; Deng et al. , 1981) and RZ-2 (Zweig et al. , 1984). The PCNA antigens were detected in nucleoli of tumor and normal growing cells but not in normal resting tissues (Chan et al. , 1983; Smetana et al. , 1983). Using the immunofluorescence technique, the PCNA antigen was found in nucleoplasm and localized to the nucleoli during the G 1/S phase of the cell cycle (Chan et al. , 1983; Smetana et al. , 1983; Miyachi et al. , 1978; Takasaki et al. , 1981). It was enriched in the nucleoli of rapidly growing tumor cells (Chan et al. , 1983). Several proteins which reacted with the PCNA antibody have been described (Chan et al. , 1983; Yoshinari et al. , 1984; Mathews et al. , 1984). We previously reported that one such protein with a molecular weight of 40 KDa and a pI of 5.0 was immunoprecipitated from total HeLa cell extract (Chan et al. , 1983). Another antigen with molecular weight 33 KDa was partially purified from rabbit thymus extract by Yoshinari et al. , 1984. Mathews et al. (1984) reported that the 35 KDa antigen is related to “cyclin” (Bravo et al. , 1981). The present study was designed to identify these antigens in isolated nucleoli. A 140 KDa protein was detected in nucleoli of HeLa and colon carcinoma cells.