Amino acid sequence of the BSC-1 cell growth inhibitor (polyergin) deduced from the nucleotide sequence of the cDNA.

Abstract

The complete amino acid sequence of the BSC-1 cell growth inhibitor, including its precursor polypeptide, is reported. The sequence was deduced from the nucleotide sequence of the cDNA. The N-terminal amino acid sequence of the mature bioactive BSC-1 cell growth inhibitor is identical with the N-terminal sequences of the factors that have been called type j32 transforming growth factor and cartilageinducing factor B, suggesting that these are identical. The complete amino acid sequence of the mature BSC-1 cell growth inhibitor differs from that of human type 13 transforming growth factor in 32 of the 112 amino acids. Polyergin is proposed as the name for the BSC-1 cell growth inhibitor.A growth inhibitor (GI) produced by BSC-1 African green monkey kidney epithelial cells has been isolated from serumfree growth medium exposed to high-density cultures of the cells (1). The GI is very active in arresting the growth of a variety of cells in culture (2) and it can inhibit the growth of a human mammary carcinoma in vivo in nude mice (2). The BSC-1 cell GI has been found to be functionally related to human platelet type 8 transforming growth factor (TGF-83) (3). TGF-p is one of a number of structurally related growth and differentiation factors (4). The present paper reports the complete amino acid sequence of BSC-1 GI, and its precursor polypeptide, as deduced from the nucleotide sequence of the cDNA.t MATERIALS AND METHODSIsolation of the BSC-1 Cell GI. High-density cultures of BSC-1 cells (5) were maintained in roller bottles (Corning, 490 cm2) in 125 ml of Dulbecco's modified Eagle's medium with 1% calf serum. To prepare conditioned medium, the culture medium was decanted from the bottles and the bottles were filled with Dulbecco's modified Eagle's medium without serum (pH 7.2-7.4, 37°C) and the bottles were incubated for 24 hr at 37°C without rolling (6). The conditioned medium was collected and was concentrated in a 2.5-liter Amicon unit with a 150-mm YM10 ultrafiltration membrane. Each group of nine roller bottles gave 12 liters of conditioned medium and the inhibitor was obtained at the conclusion of the concentration procedure in a total of 30 ml of 1 M acetic acid washes of the YM10 membrane (1, 7). Six sets of 30-ml washes were combined and concentrated to 2 ml in a 400-ml Amicon unit, using a 76-mm YM10 membrane. The concentrate was diluted with 30 ml of 1 M acetic acid and reconcentrated to 2 ml. The concentrate was combined with two 2 ml of 1 M acetic acid washes of the YM10 membrane and the combined solution was subjected to gel filtration through a column of Bio-Rad P60 (2 x 30 cm) in 1 M acetic acid (1, 8). After assay (1) the fractions with highest specific activities (fractions from 60-84 ml of the effluent, with 2.5 Ag of bovine serum albumin added per ml) were lyophilized, the residues were transferred to a single tube using small volumes of 1 M acetic acid, and the solution was relyophilized. The final residue was dissolved in 200 p.l of 0.1% trifluoroacetic acid and the solution was injec...

Adenine nucleotides stimulate migration in wounded cultures of kidney epithelial cells.

“…As BSC-1 cells constitutively release PDGF and TGF-fl2 (15,29), we were particularly interested in finding out if peptide growth factors contribute to control of cell migration. The Heparin (gig/ml) ADP induces rapid reorganization of cytokeratin-8 filaments and alters cell shape (23), which suggests a direct effect.…”

Selective growth arrest and phenotypic reversion of prostate cancer cells in vitro by nontoxic pharmacological concentrations of phenylacetate.

“…RNA transfer, hybridization with 32P-labeled specific probes, and autoradiography (XAR5 X-ray film, Eastman Kodak Co., Rochester, NY) were performed according to standard procedures (34). The probes, HLA-A3 HindIII/EcoRI DNA fragment (35), 1.2-kb rat TGF-O31 cDNA (36), and 1.2-kb simian TGF-f32 cDNA (37), were labeled with [ 32p]dCTP (New England Nuclear) using random primed DNA labeling kit (Boehringer Mannheim, GmbH, Mannheim, FRG).…”

C-CAM1 expression: Differential effects on morphology, differentiation state and suppression of human PC-3 prostate carcinoma cells

“…The following cDNA clones were used as probes: for C-CAM, a 218 bp EcoRI ± HindIII fragment of pBS ± BglII/NsiI containing a 2.0 kb C-CAM1 cDNA ; for CK-8, a 1.25 kb PstI fragment of mouse endoA, the homolog of human CK-8 (Duprey et al, 1983) kindly provided by Dr Adam Glick (Laboratory of Cellular Carcinogenesis, National Cancer Institute); for TGF-b1, a 0.9 kb XbaI ± HindIII fragment of rat TGF-b1 in plasmid pRTGFb1 kindly provided by Dr Su Wen Qian (Laboratory of Chemoprevention, National Cancer Institute) (Qian et al, 1990); for TGF-b2, a 1.2 kb XbaI ± EcoRI fragment of simian TGF-b2 in plasmid pSB2-ORF kindly provided by Dr Robert W Holley (Salk Institute, San Diego, CA, USA) (Hanks et al, 1988); for TGF-b3, a 1.2 kb EcoRI ± BamHI fragment of mouse TGF-b3 in pBluescript kindly provided by Dr Denhez (Laboratory of Chemoprevention, National Cancer Institute) (Denhez et al, 1990); for TGF-b type I receptor, a 1.6 kb XbaI ± BamHI fragment from pCMV5-TbRI-HA kindly provided by Dr Je Wrana (The Hospital for Sick Children, University of Toronto, Canada); for TGFb type II receptor, a 1.7 kb BamHI ± HindIII fragment from H2-3FF kindly provided by Dr Robert A Weinberg (Whitehead Institute, MIT, Cambridge, MA, USA) and for PSA, the entire cDNA excised by HindIII and XhoI from pGEM72f(+) kindly provided by Dr Donald J Tindall (Mayo Clinic, Rochester, MN, USA). In addition to the cDNA probes, a probe speci®c for the 3' untranslated region (UTR) of human BGPI was constructed in our laboratory by RT ± PCR using a forward primer of sequence 5'-AAAAAAGCAGTAATGAAACCTGTC-3' (nucleotides 1650 ± 1673) and a reverse primer of sequence 5'-CCTACA-GACTCCCAATGTACTTTC-3' (nucleotides 1844 ± 1867).…”