[CANCER RESEARCH 41, 537-545, February 1981] 0008-5472/81 /0041-OOOOS02.00 Purification and Characterization of a Nuclear DMA-binding Phosphoprotein in Fetal and Tumor Tissues1 Egon Durban, David Roll,2 Gregory Beckner, and Harris Busch Department of Pharmacology, Baylor College of Medicine. Houston. Texas 77030 ABSTRACT The basic nonhistone phosphoprotein 110/8.4 (M.W. x 10~3/pl) was found in 0.35 M NaCI nuclear extracts of four tumor tissues, i.e., fast-growing Novikoff hepatoma, Morris hepatoma 3924A, HeLa cells, and Namalwa cells; it was also found in fetal rat liver. This protein was not detected in normal or regenerating liver and thus may represent an "oncofetal" protein of potential interest as a cancer "marker." Protein 110/8.4 was purified approximately 4000- to 5000-fold under nondenaturing condition from 0.35 M NaCI nuclear extracts of Novikoff hepatoma cells or Namalwa cells by ammonium sulfate fractionation, calcium phosphate gel treatment, and phospho- cellulose chromatography. Sodium dodecyl sulfate:polyacryl- amide gel electrophoresis of the purified native protein revealed a single polypeptide chain with a molecular weight of approxi mately 110,000. The pi of the protein was estimated to be 8.4 by nonequilibrium pH gradient electrophoresis in 9 M urea; accordingly, this protein was designated 110/8.4. Amino acid analysis showed that Protein 110/8.4 had an acidic:basic amino acid ratio of 1.25 and a high lysine and serine content; approximately 20% of the serine residues were found to be phosphorylated. Hydrazinolysis indicated that the carboxyl-ter- minal amino acid was serine; the amino terminus appeared to be blocked. Binding of Protein 110/8.4 to DNA was studied by the nitrocellulose filter assay. High-affinity binding occurred at ionic strength equal to or below 0.15 M. INTRODUCTION Various lines of evidence indicate that nonhistone phospho- proteins may be involved in gene regulation (14, 32). Such proteins have been reported to cause alterations in the rate and pattern of in vitro gene transcription in many different systems [references tabulated in a review by Kleinsmith (14)]. In addition, they have been found to bind preferentially to homologous DNA in vitro (13, 15, 18, 33). Proteins with these characteristics are present in a phosphoprotein-enriched subfraction of 0.35 M NaCI chromatin extracts (15). While extracting chromatin of Novikoff ascites cells (labeled in vivo with 32Pi) with NaCI solution of the same ionic strength (0.35 M), a phosphoprotein of an estimated molecular weight of 110,000 and an estimated pi of 8.4 was found on autoradi- ograms of 2-dimensional nonequilibrium pH gradient and SDS3: ' These studies were supported by Cancer Research Grant CA 10893, P. 1, the Taub Foundation, the DeBakey Foundation, and the Pauline Sterne Wolff Memorial Foundation. 2 Present address: Chemistry Department, Roberts Wesleyan College, Roch ester, N. Y. 14524. 3 The abbreviations used are: SDS, sodium dodecyl sulfate; NKM, 0.13 M NaCI:5 rnw KCI: 8 mm MgCI2:0.1 mM PMSF; RSB buffer, 10 mw Tris-HCI (pH 7.4): 10 mM NaCI: 1.5 mM MgCb; PMSF, phenylmethylsulfonyl fluoride: HMG, high-mobility group; LMG, low-mobility group. Received May 23, 1980; accepted October 20, 1980. polyacrylamide gels (41). By ammonium sulfate treatment of 0.35 M NaCI extracts, Protein 110/8.4 was also found by Coomassie blue staining in rat hepatoma 3924A, in 2 human tumor lines (HeLa and Namalwa), in a Burkitt's lymphoma- derived line, and in fetal liver. It could not be detected in normal or regenerating liver. The present study reports on 2 methods of purification of this "oncofetal" nuclear phosphoprotein. The first, in the presence of urea, led to higher yields, which is useful for chemical characterization. The second method was carried out under nondenaturing conditions to provide Protein 110/8.4 in native form for studies on its biological function. MATERIALS AND METHODS Materials. Proteins used for calibration of molecular weights were obtained from Bio-Rad Laboratories, Richmond, Calif. Ampholines were purchased from LKB Instruments, Inc., Rock- ville, Md. Calcium phosphate gel, Escherichia coli DNA, and B! nyclease were obtained from Sigma Chemical Co., St. Louis, Mo., and phosphocellulose P-11 was obtained from Whatman, Inc., Clifton, N. J. Nonidet P-40 (Shell) was purchased from Particle Data, Inc., Elmhurst, III. and [3H]dCTP was purchased from New England Nuclear, Boston, Mass. Animals and Tumor Cells. Novikoff hepatoma ascites cells were transplanted i.p. in 200-g male albino rats (Holtzman Co., Madison, Wis.). After 6 days, the animals were sacrificed, the ascites fluid was filtered through cheesecloth, and the cells were washed 3 times at 4°with NKM (23). Normal, fetal, and regenerating rat liver and Morris hepatoma 3924A were minced in NKM and rinsed 3 times in NKM. Fetal livers were obtained from 19- to 20-day-old fetuses. HeLa S3 cells were grown in Eagle's medium (Grand Island Biological Co.), washed twice with phosphate-buffered saline (0.01 M sodium phosphate buffer, pH 7.2:0.145 M NaCI), and washed once with RSB buffer. The Namalwa line is a Burkitt tumor line generously provided by Dr. John Douros and Fred Klein of the Frederick Cancer Research Center. Isolation of Nuclei. All tissues or cells, except HeLa and Namalwa cells, were homogenized in 0.5% Nonidet P-40:0.01 M NaCI:1.5 mM MgCI2:0.1 mM PMSF:10 mM Tris-HCI, pH 7.6 (26), by 15 strokes in a 200-ml Teflon:glass homogenizer with 0.01-inch standard clearance (Glenco Scientific, Inc., Houston, Texas). The homogenate was centrifuged at 12,000 x g for 10 min. The crude nuclear pellet was resuspended by homog- enization with a loose-fitting pestle (>0.01-inch clearance) in 1 M sucrose:NKM. Nuclei were collected by centrifugation at 1000 x g for 10 min. The HeLa cells washed once with RSB buffer were resuspended in RSB buffer containing 0.5% Non idet P-40 and homogenized in a Dounce homogenizer with 20 up-and-down strokes. After centrifugation at 600 x g for 8 min, pellets were resuspended in 0.25 M sucrose:0.5 mw FEBRUARY 1981 537 on May 27, 2016. © 1981 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from