[CANCER RESEARCH 56. 2003-2008. May 1. 1996] Advances in Brief Functional Analysis of pRb2/pl30 Interaction with Cyclins1 Pier Paolo Claudio,2 Antonio De Luca,3 Candace M. Howard, Alfonso Baldi,2 Eduardo J. Firpo, Andrew Koff, Marco G. Paggi,4 and Antonio Giordano5 Departments of Microbiology/Immunology and Pathology. Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 ¡P.P. C.. A. D. L, C. M. H., A. B., A. G.]: Department of Basic Science, Fred Hutchinson Cancer Research Center. Seattle, Washington 98104 ¡E.J. F.I; Memorial Sloan-Kettering Cancer Center, New York, New York 10021 ¡A.K.¡.and Regina Elena Institute far Cancer Research. CRS 00158 Rome. Italy [M. G. P.I Abstract The retinoblastoma (Rb) family consists of the tumor suppressor pRb and related proteins pl07 and pRb2/pl30. Ectopie expression of pRb and p 1117results in a growth arrest of sensitive cells in the G, phase of the cell cycle. We demonstrated here that the growth-suppressive properties of pRb2/pl30 were also specific for the G, phase. The A-, E-, and D-type cyclins as well as transcription factor E2F1 and the EIA viral oncoprotein were able to rescue the pRb2/pl30-mediated G, growth arrest in SAOS-2 cells. The rescue with cyclins A and E correlated with their physical interaction with pRb2/pl30, which surprisingly has been found to occur over all phases of the cell cycle. The phosphorylation status as well as the kinase activity associated with pRb2/pl30 dramatically increased near the G,-S-phase transition. This suggests that, like the other Rb family mem bers, pRb and p 107, the phosphorylation of pRb2/pl30 is controlled by the cell cycle machinery and that pRb2/pl30 may indeed be another key G,-S-phase regulator. Introduction Since the discovery of the first tumor suppressor gene, much energy has been directed toward the identification and characterization of such genes which serve as negative regulators of the cell cycle machinery and the elucidation of their involvement in human malig nancies. According to Knudson's "two-hit" hypothesis (1), the devel opment of several human cancers is thought to involve loss of het- erozygosity of putative tumor suppressor genes, several of which are yet to be identified (2). One of the most extensively studied tumor suppressor genes is the retinoblastoma susceptibility gene (RB), whose gene product (pRb) is a nuclear phosphoprotein that has been shown to serve as a negative regulator of cell cycle progression through G|-S-phase transition, at least in some specific cell types (3). G, growth arrest by pRb is dependent on the sequences necessary for its physical interaction with the E2F family of transcription factors as well as with a number of oncoproteins from human DNA viruses (4-7). Binding to these proteins occurs at a specific "pocket region" in pRb, shared by two additional ElA-associated proteins, which has led to the identification of two members of the Rb family, p 107 and pRb2/pl30 (8-11). pRb2/pl30 maps to human chromosome 16ql2.2, Received 2/14/96; accepted 3/19/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was supported by Sbarro Institute for Cancer Research and Molecular Medicine. NIH Grant RO1CA60999-01A1, and The Council for Tobacco Research (A. G.). P. P. C. was supported by a fellowship from the American-Italian Cancer Foundation. M. G. P. was supported by grants from Associazione Italiana per la Ricerca sul Cancro and Consiglio Nazionale delle Ricerche-Applicazioni Cliniche della Ricerca Oncologica. 1 On leave of absence from the Departments of Maxillo-Facial Surgery and Anatomic Pathology, School of Medicine, University of Naples "Federico II," Naples, Italy. ' Recipient of an "Advanced Fellowship NATO-CNR". 4 Recipient of a United States (NIH)-Italy (Consiglio Nazionale delle Ricerche) Short- term Scientist Exchange Program fellowship. 5 To whom requests for reprints should be addressed, at Departments of Microbiology/ Immunology and Pathology, Thomas Jefferson University, Philadelphia. PA 19107. Phone: (215) 503-0781; Fax: (215) 923-9626; E-mail: agiordan@lac.jci.tju.edu. an area in which deletions have been found in several human neopla- sias including breast, ovarian, hepatic, and prostatic cancers (12). Moreover, the gene responsible for familial cylindromatosis, a rare autosomal dominant disease characterized by multiple neoplasms originating from the skin appendages, has been recently localized to chromosome 16ql2-ql3, thereby further lending credence to the hy pothesis that the Rb2/pl30 gene is a tumor suppressor gene (13). The human Rb2/pl30 gene, recently characterized by us, consists of 22 exons, spanning over 50 kb of genomic DNA (14). Recent functional studies of p 107 and pRb2/pl30 indicated that although the Rb family members may be able to complement each other, the proteins are not fully functionally redundant (15, 16). Like pRb, ectopie expression of either p 107 or pRb2/pl30 is able to suppress the growth of the osteosarcoma cell line SAOS-2 which lacks a functional pRb (7, 15, 16). The T98G human glioblastoma cell line is refractory to the effects of pRb and p 107 but undergoes growth arrest from pRb2/pl30, indicating that pRb2/p 130 is not merely a surrogate for either pRb or pl07 and that there are differences in the specific mechanisms of growth inhibition employed by the three family members (15). The interplay between the Rb6 family and the E2F family is hypothesized to regulate transcription and progression of the cell cycle and offers unique models for the growth suppression by the Rb family members. Several sources of data suggest that pRb, p 107, and pRb2/pl30 associate with distinct E2F species that modulate their activity in a sequential manner (17-21). The Rb-E2F complexes may in turn be modulated by the cell cycle machinery through the phos phorylation of Rb family members by cdks, resulting in the release of active E2F species to stimulate the transcription of important genes involved in DNA synthesis and progression of the cell cycle. The ability of pRb and p 107 to negatively regulate E2F correlates strongly with their ability to arrest cell growth (6, 7, 22, 23). The phosphoryl ation of pRb and p 107 has been shown to be cell cycle regulated (24-26). At this point, it is uncertain as to how and whether the cell cycle machinery regulates the growth suppressive action of pRb2/ p 130. pRb2/pl30 binds to cyclins A and E in vivo and to cyclins Dl, D2, and D3 in vitro (9, 10). We present data herein that support a cell cycle regulation for the phosphorylation of pRb2/pl30. We have found pRb2/pl30 to be highly phosphorylated at the G,-S transition. We have also investigated the effects of known components of the cell cycle machinery on pRb2/pl30-mediated G, growth arrest. We have compared and contrasted our rescue analysis of pRb2/pl30 with that previously obtained by others for pRb and p 107 in SAOS-2 cells (16, 27) in an attempt to decipher the actual mechanisms of inhibition by the family members. Our studies have been furthered by delving into the regulatory processes involving pRb2/p 130 throughout the various stages of the cell cycle. 6 The abbreviations used are: cdk, cyclin-dependent kinase; FBS, fetal bovine serum; FACS, fluorescence-activated cell sorting; Rb, retinoblastoma; PVDF. polyvinylidene difluoride. 2003 Research. on August 17, 2015. © 1996 American Association for Cancer cancerres.aacrjournals.org Downloaded from