The retinoblastoma gene (RB/p105) is the prototypic tumor suppressor gene, whose inactivation has been related to several human neoplasia. Based on structural and functional similarity to pRb/p105, the pRb2/p107 and the pRb2/p130 proteins are considered to form the Rb protein family. These nuclear proteins display a cell-cycle-related phosphorylation, form complexes with the E2F family of transcription factors, and act as negative regulators of cell cycle progression, blocking cells in the G1 phase. The Rb proteins present cell-type-specific growth suppressive properties, so that they are considered not functionally redundant, but complementary, pRb2/p130 expression in lung cancer appears inversely related to tumor malignancy and RB2/p130 maps to a chromosome area in which loss of heterozygosity has been found in several human tumors, suggesting an involvement of RB2/ p130 mutation in human carcinogenesis. Taking advantage of the knowledge of the complete genomic sequence of the RB2/p130 gene, the expression and the functional role of pRb2/p130 have been investigated in two clusters of tumor lymphoid and osteosarcoma cell lines. Since all the examined lymphoid tumor cell lines showed pRb2/ p130 localized at the cytoplasm level, while normal lymphocytes and the osteosarcoma cell lines all exhibited an exclusive nuclear localization, possible mutations occurring at the nuclear localization signal (NLS) in the C-terminus of the protein have been screened. The mutations, mainly found at exons 21 and 22, all resulted in the alteration of NLS in lymphoid tumor cell lines, as well as in EBV-positive primary Burkitt's lymphomas. This ruled out the possibility that mutations were a product of cell growth in culture. On the other hand, the point mutations found in exons 19-21 in the osteosarcoma cell lines did not alter the NLS, in agreement with the nuclear localization of pRb2/p 130 in these cells. Besides the loss of functional NLS, the mutations occurring in lymphoid tumor cell lines caused a premature stop codon and a shorter transcription product, as confirmed by Western blot analysis of pRb2/p130 protein, which showed a 116 kD mutant form. The mutations observed in osteosarcoma cells, causing alterations in the two putative phosphorylation sites, resulted in either unphosphorylated or persistent hypophosphorylated forms of the protein. The functional consequences of NLS disruption were analyzed by recreating the NQ point mutations by PCR-based site-directed mutagenesis of the full-length pRb2/p130 protein in an expression vector that drives gene expression by the constitutive CMV promoter. The EGFP fluorescent chimerical protein has been found to be localized at the cytoplasm in transiently transfected osteosarcoma Saos-2 cells that normally show an exclusive nuclear localization when the pRb2/p130 protein has been mutated at both NLS. The effect on cell proliferation of these mutant NLS was determined in transfected Saos-2 cells by FACS analysis and colony assay. The results indicated that only the disruption of both NLS almost completely abolished the growth suppressive activity of pRb/p130 protein and that this effect was not due to the toxicity of the over-expressed protein. In conclusion, the genetic alterations of the RB2/p130 gene found in some primary tumors suggest that, as in case of the RB2/p105 gene, pRb2/p130 is involved in cell cycle control and that it can act as a tumor suppressor gene. Moreover, the non-redundancy of growth-suppressive properties of the Rb protein family is further demonstrated by the presence of specific mutations in the NLS sequences of the pRb2/p130 in lymphoid tumor cells that prevent a nuclear localization of the protein with an obvious loss of its function, while in osteosarcoma cells this can occur by mechanisms affecting the phosphorylation of the protein or its interaction with viral oncoproteins.

Maraldi, N.M., Giordano, A., Manzoli, L., Falconi, M., Pol, A.D., Cinti, C. (2001). Genetic alterations at the nuclear localization signal of the RB2/p130 gene occur in lymphoid tumor but not in osteosarcoma cell lines. ADVANCES IN ENZYME REGULATION, 41(1), 31-55 [10.1016/S0065-2571(00)00006-6].

Genetic alterations at the nuclear localization signal of the RB2/p130 gene occur in lymphoid tumor but not in osteosarcoma cell lines

Giordano A.;
2001-01-01

Abstract

The retinoblastoma gene (RB/p105) is the prototypic tumor suppressor gene, whose inactivation has been related to several human neoplasia. Based on structural and functional similarity to pRb/p105, the pRb2/p107 and the pRb2/p130 proteins are considered to form the Rb protein family. These nuclear proteins display a cell-cycle-related phosphorylation, form complexes with the E2F family of transcription factors, and act as negative regulators of cell cycle progression, blocking cells in the G1 phase. The Rb proteins present cell-type-specific growth suppressive properties, so that they are considered not functionally redundant, but complementary, pRb2/p130 expression in lung cancer appears inversely related to tumor malignancy and RB2/p130 maps to a chromosome area in which loss of heterozygosity has been found in several human tumors, suggesting an involvement of RB2/ p130 mutation in human carcinogenesis. Taking advantage of the knowledge of the complete genomic sequence of the RB2/p130 gene, the expression and the functional role of pRb2/p130 have been investigated in two clusters of tumor lymphoid and osteosarcoma cell lines. Since all the examined lymphoid tumor cell lines showed pRb2/ p130 localized at the cytoplasm level, while normal lymphocytes and the osteosarcoma cell lines all exhibited an exclusive nuclear localization, possible mutations occurring at the nuclear localization signal (NLS) in the C-terminus of the protein have been screened. The mutations, mainly found at exons 21 and 22, all resulted in the alteration of NLS in lymphoid tumor cell lines, as well as in EBV-positive primary Burkitt's lymphomas. This ruled out the possibility that mutations were a product of cell growth in culture. On the other hand, the point mutations found in exons 19-21 in the osteosarcoma cell lines did not alter the NLS, in agreement with the nuclear localization of pRb2/p 130 in these cells. Besides the loss of functional NLS, the mutations occurring in lymphoid tumor cell lines caused a premature stop codon and a shorter transcription product, as confirmed by Western blot analysis of pRb2/p130 protein, which showed a 116 kD mutant form. The mutations observed in osteosarcoma cells, causing alterations in the two putative phosphorylation sites, resulted in either unphosphorylated or persistent hypophosphorylated forms of the protein. The functional consequences of NLS disruption were analyzed by recreating the NQ point mutations by PCR-based site-directed mutagenesis of the full-length pRb2/p130 protein in an expression vector that drives gene expression by the constitutive CMV promoter. The EGFP fluorescent chimerical protein has been found to be localized at the cytoplasm in transiently transfected osteosarcoma Saos-2 cells that normally show an exclusive nuclear localization when the pRb2/p130 protein has been mutated at both NLS. The effect on cell proliferation of these mutant NLS was determined in transfected Saos-2 cells by FACS analysis and colony assay. The results indicated that only the disruption of both NLS almost completely abolished the growth suppressive activity of pRb/p130 protein and that this effect was not due to the toxicity of the over-expressed protein. In conclusion, the genetic alterations of the RB2/p130 gene found in some primary tumors suggest that, as in case of the RB2/p105 gene, pRb2/p130 is involved in cell cycle control and that it can act as a tumor suppressor gene. Moreover, the non-redundancy of growth-suppressive properties of the Rb protein family is further demonstrated by the presence of specific mutations in the NLS sequences of the pRb2/p130 in lymphoid tumor cells that prevent a nuclear localization of the protein with an obvious loss of its function, while in osteosarcoma cells this can occur by mechanisms affecting the phosphorylation of the protein or its interaction with viral oncoproteins.
2001
Maraldi, N.M., Giordano, A., Manzoli, L., Falconi, M., Pol, A.D., Cinti, C. (2001). Genetic alterations at the nuclear localization signal of the RB2/p130 gene occur in lymphoid tumor but not in osteosarcoma cell lines. ADVANCES IN ENZYME REGULATION, 41(1), 31-55 [10.1016/S0065-2571(00)00006-6].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11365/1129270
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