Genetic alterations disrupting the nuclear localization of the retinoblastoma-related gene RB2/p130 in human tumor cell lines and primary tumors

• Published in: Cancer research (Chicago, Ill.) Ill.), 2000-01, Vol.60 (2), p.383-389
• Main Authors: CINTI, C, CLAUDIO, P. P, HOWARD, C. M, NERI, L. M, YAN FU, LEONCINI, L, TOSI, G. M, MARALDI, N. M, GIORDANO, A
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Biological and medical sciences; Burkitt Lymphoma - genetics; Cell Nucleus - metabolism; Cell Nucleus - pathology; Cell physiology; Cell transformation and carcinogenesis. Action of oncogenes and antioncogenes; Exons; Female; Frameshift Mutation; Fundamental and applied biological sciences. Psychology; Humans; Immunohistochemistry; Jurkat Cells; Leukemia; Lymphoma; Male; Molecular and cellular biology; Mutagenesis, Site-Directed; Mutation; p105 gene; p107 gene; p130 gene; Phosphoproteins - analysis; Phosphoproteins - genetics; Phosphoproteins - metabolism; Point Mutation; Polymorphism, Single-Stranded Conformational; Proteins; RB gene; RB2 gene; Recombinant Fusion Proteins - analysis; Recombinant Fusion Proteins - biosynthesis; Retinoblastoma; Retinoblastoma-Like Protein p130; Transfection; Tumor Cells, Cultured; Tumor Stem Cell Assay
• ISSN: 0008-5472; 1538-7445

The prototypic tumor suppressor gene, the retinoblastoma gene (RB/ p105), is mutated in a variety of human tumors. However, to date, mutational data on retinoblastoma family members p107 and RB2/p130 in tumors is lacking. We studied the expression of pRb2/p130 by immunocytochemistry and Western blot analysis in a panel of human osteosarcoma and lymphoid cell lines. Only the lymphoid cell lines showed an abnormal cytoplasmic localization of pRb2/p130, suggesting possible alterations within the region of nuclear localization signaling. We screened these cell lines for genetic alterations of the RB2/p130 gene in the region of the putative bipartite nuclear localization signal (NLS). This region is highly homologous with that of the RB/p105 gene. In addition, we screened four primary Burkitt's lymphomas for genetic alterations in the RB2/p130 gene. Naturally occurring mutations, which disrupt the putative bipartite NLS, were found in lymphoma cell lines and primary tumors, but not in the osteosarcoma cell lines, where normal nuclear localization of the protein was detectable. Site-directed mutagenesis and transfection assay using NLS mutants displayed markedly reduced biological activity as measured by flow cytometric analysis. This study clearly describes RB2/ p130 as an important target for mutations and subsequent inactivation in lymphoma pathogenesis, thus validating that RB2/p130 is a classical tumor suppressor gene.