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[Cancer Research 60, 383-389, January 15, 2000]
© 2000 American Association for Cancer Research

Molecular Biology and Genetics

Genetic Alterations Disrupting the Nuclear Localization of the Retinoblastoma-related Gene RB2/p130 in Human Tumor Cell Lines and Primary Tumors1

Caterina Cinti, Pier Paolo Claudio, Candace M. Howard, Luca Maria Neri, Yan Fu, Lorenzo Leoncini, Gian Marco Tosi, Nadir Mario Maraldi and Antonio Giordano2

Istituto di Citomorfologia Normale e Patologica, CNR, 40136 Bologna, Italy [C. C., L. M. N., N. M. M.]; Departments of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 [P. P. C., C. M. H., Y. F., A. G.]; Dipartimento di Scienze Odontostomatologiche e Maxillo-Facciali, Università di Napoli "Federico II," 80100 Naples, Italy [P. P. C.]; Istituto di Anatomia Umana Normale, Università di Ferrara, 44100 Ferrara, Italy [L. M. N.]; Laboratorio di Biologia Cellulare e Microscopia Elettronica, Istituti di Ricerca "Codivilla Putti," IOR, 40100 Bologna, Italy [N. M. M.]; and Istituto di Anatomia e Istologia Patologica [L. L.] and Dipartimento di Scienze Oftalmologiche e Neurochirurgiche [G. M. T.], Università di Siena, 53100 Siena, Italy

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/p130as an important target for mutations and subsequent inactivation in lymphoma pathogenesis, thus validating that RB2/p130 is a classical tumor suppressor gene.




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