A tumor-associated 120 kDa nuclear protein: characterization using a monoclonal antibody and a partial cDNA clone.
1. We have established a murine hybridoma (F86) that secretes a monoclonal antibody (MoAb) specific for a 120 kDa nuclear protein (p120). p120 is expressed in all human cell lines investigated, whether of tumor or normal cell origin. 2. However, expression of p120 is significantly higher in neoplastic cells than in normal cells. The amount of p120 is relatively constant through the cell cycle and does not appear to be modulated by 72 hr serum starvation. 3. These results suggest that p120 plays some role in nuclear events associated with neoplastic phenotypes rather than in cell proliferation. 4. In situ immunofluorescence analyses indicate that p120 is located exclusively in nuclei of interphase cells. It is not present in nucleoli. 5. During mitosis, p120 is distributed in the cytoplasm and is not associated with condensed chromosomes which, together with RNAse experiments, suggests that it may be associated with hnRNA or hnRNP particles. 6. Western blot analyses indicate that p120 consists of two molecular weight forms which differ by 2-3 kDa in reduced SDS-PAGE, and several isoelectric variants in the acidic range. 7. Fractionation studies indicate that p120 has accessible free sulfhydryl group(s) and can bind ssDNA and heparin. 8. A partial cDNA clone, encoding the carboxyl terminus of p120, was isolated from a lambda gt11 library which had been prepared from human hepatoma cells (KYN-1). 9. Sequence analysis of the open reading frame revealed two possible nuclear localization sequences and several clusters of acidic amino acid residues, including a continuous run of 11 glutamic acid residues. 10. Northern blot analyses of human hepatoma RNA revealed hybridization to three transcripts which are about 4.1, 3.6, and 0.6 kb in size. 11. Dot blot analyses show that these transcripts are about 10-fold more abundant in KYN-1 hepatoma cells than in normal liver cells.
Fukuda
K
K
Department of Biochemistry, University of Vermont, College of Medicine, Burlington 05405.
Chiu
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D004277:DNA, Single-Stranded / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D051379:Mice; D008938:Mitosis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012334:RNA, Neoplasm / Q000032:analysis; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D004277:DNA, Single-Stranded; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D012334:RNA, Neoplasm; D011994:Recombinant Proteins; D004247:DNA