Our research includes molecular biological studies designed to further define gene control and gene function that are responsible for the excessive growth and invasive and metastatic characteristics of cancer cells. These studies are correlated with studies on the responsiveness of cancer cells in animals and patients to a variety of new and potentially useful agents that interfere with their synthetic and metabolic functions. In the evaluation of new and improved therapeutic approaches to the treatment of cancer, ultrastructural and biochemical analyses are being made on both animal and human tumor cells, as well as their products. We previously purified a 110 kilodalton nuclear phosphoprotein with pI 8.4 from chromatin extracts of Novikoff hepatoma which was subsequently identified as a type I topoisomerase. By two-dimensional gel analysis, a similar protein was detected in fetal rat liver, HeLa, and Namalwa cells, but it could not be detected in chromatin extracts of adult rat liver. Using a monospecific IgG fraction against Novikoff topoisomerase I in an ELISA competition assay, a 70 kilodalton, pI 8.4 protein was identified in normal rat liver. Changes in phosphorylation will change the enzymatic activity of topoisomerase I. To determine the sites of phosphorylation, purified topoisomerase I was labeled in vitro with gamma-?32?P-ATP by nuclear protein kinase NII, digested with trypsin, and chromatographed on a C18 reverse-phase HPLC column. Protein B23 (1 mg/ml) was partially cleaved by S. aureus V8 protease (10 micrograms per ml) in 0.1 M NH?4?HCO?3?, pH 7.8, 30 min at 37~C. An antigenic peptide with molecular weight about three to four kilodaltons was identified by the immunoblot assay. The antigenic peptide was purified by DEAE cellulose chromatography. The sequence of the first thirty amino acids is KFKKQEKTPKTPKGPSSVEDIKAKPQADIE. The total nuclear extract of HeLa cells or human colon carcinoma cells was labeled with ?35?S-met or ?125?I-Bolton-Hunter reagent. The nuclear extracts were immunoprecipitated with PCNA antibody (EB) and protein A-Sepharose 4B. A major antigen with molecular weight 120 kilodaltons and a minor antigen with molecular weight 40 kilodaltons were identified on 10% SDS gels. Preliminary experiments are in progress on isolation and purification of the DNA-bound nuclear antigen 86 kilodaltons. (V)
| Rosenberg, N L (1986) Isolation of an MSP1-derived transcriptionally active nucleolar particle. Exp Cell Res 165:41-52 |
