Interaction Between HIV-1 Tat and Prb2/P130: a Possible Mechanism in the Pathogenesis of AIDS-Related Neoplasms

Interaction between HIV-1 Tat and pRb2/p130: a possible mechanism in the pathogenesis of AIDS-related neoplasms

Giulia De Falco1,2, Cristiana Bellan1, Stefano Lazzi1, PierPaolo Claudio2, Domenico La Sala1,3, Caterina Cinti2,3, Piero Tosi1, Antonio Giordano*,2 and Lorenzo Leoncini1

1Department of Human Pathology and Oncology, University of Siena, Siena, Italy; 2Sbarro Institute for Cancer Research and Molecular Medicine, College of Science & Technology, Temple University, Philadelphia, PA, 19122, USA; 3ITOI-CNR, Bologna unit, Bologna, Italy

Tat protein is an early nonstructural protein necessary for proliferating cells may be one reason (Dalgleish and virus replication, which is secreted by infected cells and O’Byrne, 2002), while the roles of cytokines and tumor taken up by uninfected cells. Extensive evidence indicates suppressor genes are other areas of active research that Tat may be a cofactor in the development of AIDS- (Clark et al., 2000; Mahieux et al., 2001). Tat protein of related neoplasms. The molecular mechanism underlying HIV is also a likely candidate to contribute to tumor Tat’s oncogenic activity may include deregulation of pathogenesis in HIV-infected patients (Ensoli et al., cellular genes. Among these genes, it has recently been 1994; Altavilla et al., 1999; Fiorelli et al., 1999; Kundu shown that pRb2/p130 oncosuppressor protein is one of et al., 1999). Tat protein is an early nonstructural the targets in the interaction between HIV gene product protein necessary for virus replication, which is secreted Tat and host proteins. However, whether the HIV-1 gene by infected cells and taken up by uninfected cells product Tat may inactivate the oncosuppressive function (Rubartelli et al., 1998). Extensive evidence indicates of pRb2/p130 has not yet been elucidated. Here, we show that Tat is a cofactor in the development of AIDS- that mRNA levels of pRb2/p130 increase in the presence related neoplasms (Noonan and Albini 2000) and the of Tat, whereas no change in the phosphorylation status of protein has also been found to have an oncogenic role in pRb2/p130 is observed. In addition, Tat can inhibit the vitro and in vivo (Kim et al., 1992; Corallini et al., 1993, growth control activity exerted by pRb2/p130 in the T98G 1996; Kundu et al., 1999). However, the molecular cell line. Finally, Tat does not compete with E2F-4 in mechanism of Tat-mediated tumorigenesis is not clear at binding to pRb2/p130. The interaction between Tat and present. There is experimental evidence suggesting a pRb2/p130 seems to result in the deregulation of the potential role of Tat-mediated chemotaxis and invasion control exerted by pRb2/p130 on the cell cycle. Taken in the pathogenesis of AIDS-related malignancies together, these results open a window on the role of pRb2/ (Barillari and Ensoli, 2002). Deregulation of cellular p130 in AIDS-related oncogenesis. genes and functions by Tat can also cause abnormalities Oncogene (2003) 22, 6214–6219. doi:10.1038/sj.onc.1206637 that may contribute to AIDS pathogenesis and to the development of AIDS-associated disorders (Kundu Keywords: pRb2/p130; Tat; HIV; lymphoma; cell cycle et al., 1997, 1998; Kashanchi et al., 2000). Extracellular Tat is able to regulate many cellular genes that are involved in cell signaling and translation and ultimately controls the host proliferation and Introduction differentiation signals (Chang et al., 1995; Li et al., 1995; Zauli et al., 1995; De La Fuente et al., 2002). The Since the emergence of the HIV pandemic, a close molecular mechanism underlying Tat’s pleiotropic association between HIV infection and the development activity may include the generation of functional of a selected group of cancers has been brought to light heterodimers of Tat with cell cycle proteins (Kashanchi (Boshoff and Weiss, 2002). Several mechanisms of et al., 2000). In particular, Tat protein of HIV has pathogenesis have been reported; yet the reason why also recently been shown to interact physically with neoplasia, in particular non-Hodgkin’s lymphomas RB2/p130tumor suppressor gene product (Lazzi et al., (NHL), is more common in the HIV than in other 2002). forms of immunodepression is not completely under- RB2/p130tumor suppressor gene belongs to the stood (Gaidano et al., 1998; Knowles, 1999). The failure retinoblastoma gene family together with pRb and of CD4-helper T cells to recognize clones of abnormal p107 (Stiegler et al., 1998). Many of the sequence similarities among these genes reside in a homologous functional domain known as the pocket region. *Correspondence: A Giordano, College of Science & Technology, This particular region mediates the interaction with Temple University, BioLife Science Bldg., Suite 333, 1900 N. 12th Street, Philadelphia, PA 19122, USA; E-mail: [email protected] E2F/DP members and viral oncoproteins. There are Received 7 March 2003; revised 4 April 2003; accepted 7 April 2003 fundamental differences in the specific mechanisms Physical interaction between pRb2/p130 and Tat GDeFalcoet al 6215 of growth inhibition employed by the proteins Transfections (Claudio et al., 1994). An example can be seen in the Cells were transfected with the calcium phosphate kit T98G cell line, which is refractory to the effects of (Invitrogen, CA, USA), using 10 mg of each plasmid for pRb and p107 but undergoes growth arrest caused RNA and protein extraction, and 2 mg for the luciferase assay. by pRb2/p130. Additionally, when associated with At 48 h from transfection, cells were harvested for RNA, certain viral oncoproteins, the phosphorylation status protein extraction or for the luciferase assay. of the retinoblastoma family members varies from one family member to another. Besides mutations Half-life measurement of the gene (Cinti et al., 2000b; Claudio et al., After transfection with Tat, transfected and untransfected cells 2000a, b), interaction with viral oncoproteins is were treated with cycloheximide and dissolved in ethanol, at a another important mechanism of inactivation of pRb2/ concentration of 25 mg/ml, for times ranging between 0and p130, as oncoviral disruption of E2F/DP complexes 96 h. As a negative control, cells treated with ethanol alone reinduces site-dependent transcription and cell were used. cycle progression (Fattaey et al., 1993; De Luca et al., 1997). RNA extraction The aim of the present paper is to elucidate whether RNA was extracted using the Trizol reagent (Invitrogen, CA, the physical interaction between Tat protein of HIV and USA), following the manufacturer’s instructions. pRb2/p130is able to inactivate the oncosuppressive properties of the latter. The understanding of this basic RT–PCR information may be of significance for early molecular 1 diagnosis, prognosis and the implementation of future RNA. (400 ng) was reverse transcribed for 1 h at 42 C, using Avian Myeloblastosis Virus (AMV) (Promega, WI, USA) and therapeutic regimes, including the design of new RNAsin (Promega, WI, USA) as enzymes. The cDNA (1 ml) therapeutic approaches. was amplified by real-time PCR using the apparatus supplied by Roche. The DNA master SYBR green 1 kit (Roche Diagnostics, Germany) was used according to the manufacturer’s conditions. The oligonucleotides used for pRb2/p130have been previously Materials and methods described (Cinti et al., 2000a and b). G3PDH was used as a control gene and mRNA levels for pRb2/p130were rationalized Plasmids to those for G3PDH. Primer sequences for G3PDH have been The pRb2/p130coding vectors have been previously described previously described (Wu et al., 1999). (Lazzi et al., 2002). pcDNA-3 Tat and GST-Tat were obtained from M Giacca (International Center Engineering in Biotech- Recombinant Tat nology, Trieste, Italy). The artificial E2F promoter containing three consecutive E2F consensus binding sites linked to a The recombinant Tat HIV-1 IIIB (aa 1–86) from Dr J Raina luciferase reporter gene has been previously described (Claudio was obtained through the EU Programme EVA/MRC et al., 2001). Centralised Facility for AIDS Reagents, NIBSC, UK (Grant numbers QLK2-CT-1999-00609 and GP828102). The stock solution was diluted in saline citrate buffer as recommended, and aliquots were stored at À801C until use. The concentration Cell lines of endotoxin was below 0.01 endotoxin unit (EU)/mg of The human 293 and T98G cell lines were obtained from protein. Extracellular Tat (50ng/ml) was added to the medium American Type Culture Collection (ATCC) (Rockville, MD, culture of 293 cells for 48 h. 293 cells, grown in the absence of USA). Cells were cultured in DMEM supplemented with 10% Tat, were used as a control. FBS, 1% l-glutamine, and penicillin/streptomycin, with 5% CO2 at 371C. Luciferase assay For the luciferase assay, 2 mg of each plasmid was transfected. Cells were harvested 48 h from transfection and subjected to a Colony-formation assay luciferase assay. Reactions were normalized by using beta- For the colony-formation assay, T98G cells were transfected galactosidase. The luciferase assay was performed in triplicate, with 10 mg of each vector and selected with 600 mg/ml G-418 as previously described (De Falco et al., 2000). (Sigma), starting at 48 h from transfection, for 3 weeks. Colonies were then washed with PBS and stained with 1% methylene blue in 50% ethanol. Results

Immunoprecipitation and western blotting Tat and pRb2/p130 interact in vivo through the pocket Cells were harvested and lysed in EBC buffer (50m m Tris-HCl region of pRb2/p130 m pH.8. 0, NaCl 120 m , 0.5% NP40 and fresh inhibitors). The Previously we demonstrated that Tat and pRb2/p130 extracts were immunoprecipitated with a polyclonal anti-Tat antibody (Immunodiagnostics, MA, USA). Western blotting interact both in vitro and in vivo (Lazzi et al., 2002). (WB) was performed with both monoclonal anti-pRb2/p130 Here, we mapped the site of this interaction occurring in antibody (Transduction Laboratory, UK) at a dilution of vivo in the 293 cell line. Brieﬂy, 293 cells were transfected 1 : 400 and polyclonal anti-pRb2/p130 (Rockland) at a dilution with expression vectors coding for Tat and pRb2/p130 of 1 : 400. mutants, lacking the amino- and carboxyl-terminal

Oncogene Physical interaction between pRb2/p130 and Tat GDeFalcoet al 6216

Figure 2 RB2/p130mRNA increases in the presence of Tat, In 293 cells transfected with Tat, mRNA levels for pRb2/p130 dramatically increase if compared to the wild-type cells, suggesting that Tat alters the transcription/translation rate for pRb2/p130

Figure 3 The phosphorylation status of pRb2/p130is not altered in the presence of Tat. 293 cells treated with extracellular Tat were Figure 1 Tat and pRb2/p130interact through the pocket region monitored for the phosphorylation status of pRb2/p130. No of p130. (a) The product of pRb2/130deleted of the carboxyl- significant difference was observed after Tat treatment if compared terminal domain is still able to bind to Tat. (b) The protein deleted to the untreated cells of the amino-terminus of pRb2/p130still retains its ability to interact with Tat. (c) WB with an anti-Tat antibody of the sample from transfection and RNA was extracted. The mRNA immunoprecipitated with anti-Tat. DC: pRb2/p130deleted of the carboxyl-terminal domain. DN: pRb2/p130deleted of the amino- was reverse transcribed and then amplified by a real- terminal domain. NRS: normal rabbit serum; CE: cell extract; IP: time PCR, using specific primers for RB2/p130. The immunoprecipitated sample results indicate that the transcription level for pRb2/ p130increases dramatically in the presence of Tat (Figure 2).

domains, respectively. At 48 h from transfection, cells Tat does not alter the phosphorylation status of pRb2/ were harvested and cell extracts were immunoprecipi- p130 tated with an anti-Tat antibody, followed by WB with two different anti-pRb2/p130antibodies, directed In order to assess whether Tat may influence pRb2/p130 against either the amino- or the carboxyl-terminal phosphorylation status, 293 cells were grown in the domains, depending on the region to be identified. The presence of extracellular Tat for 48 h. Wild-type cells results are shown in Figure 1. In both cases, Tat and cultured in the absence of Tat were also used as a pRb2/p130still interact, even though p130was deleted. control. We compared the phosphorylation status of This suggests that the carboxyl- and the amino-terminal pRb2/p130in both the cell lines by using an anti-pRb2/ domains are not necessary for this interaction, which p130antibody in WB. The result shows that no occurs through the pocket region of pRb2/p130. difference is observed in terms of pRb2/p130phosphor- ylation status (Figure 3). In addition, 293 cells were also transfected with an expression vector coding for Tat, Tat induces an increase in pRb2/p130 mRNA levels then harvested and subjected to WB with anti-pRb2/ We monitored the levels of mRNA for pRb2/p130, p130antibody. The result obtained using this approach either in the presence or in the absence of Tat. 293 cells also confirms that no significant difference in terms of were transfected with an expression vector coding for pRb2/p130phosphorylation is observed (data not Tat or with the empty vector. Cells were harvested 48 h shown). Half-life of pRb2/p130was also monitored

Oncogene Physical interaction between pRb2/p130 and Tat GDeFalcoet al 6217

Figure 4 Tat inhibits the growth control exerted by pRb2/p130. T98G cells transfected with Tat and pRb2/p130, alone or in combination, were positively selected for resistance to G-418. The addition of Tat to the culture determines the loss of the growth control mediated by pRb2/p130. The number of colonies observed in the sample, which overexpress both Tat and pRb2/p130, was Figure 5 Tat does not compete with E2F-4 in binding to pRb2/ comparable to those of the negative control containing the empty p130. A luciferase assay was performed on samples transfected with vector Tat and pRb2/p130, alone or in combination. Cotransfection of pRb2/p130and Tat did not result in an increase in the activation of a promoter responsive to E2F-4, suggesting that E2F-4 is not released in the presence of Tat alone both in the presence and absence of Tat, and in this case no differences were observed (data not shown).

Discussion Tat inhibits pRb2/p130 control on cell cycle To evaluate whether Tat is able to inhibit the HIV Tat, a nonstructural protein essential for viral oncosuppressive effect of pRb2/p130, T98G cells were replication, is a potent transactivator of the HIV LTR transfected with expression vectors coding for pRb2/ (Gatignol et al., 2000). Tat also transactivates several p130, for Tat, and for both vectors together. The empty endogenous cellular genes, including cytokines, extra- vector was also transfected as a positive control. T98G cellular matrix proteins, and proto-oncogenes (Vene cells were used because this cell line contains normal et al., 2000). Besides its nuclear localization and pRb/p105 and is resistant to the suppression effect of function, Tat protein can also be released into the both pRb/p105 and p107. Cells were then selected with extracellular environment by HIV-infected cells (Ru- G-418 and cultured for approximately 3 weeks in order bartelli et al., 1998). Exogenous Tat has been suggested to allow resistant cells to form colonies. The result is to have a wide range of activities due to the interaction shown in Figure 4. Cells transfected with pRb2/p130 of specific Tat domains with cellular receptors and alone formed few colonies compared to the wild-type cellular genes (Ensoli et al., 1993; Frankel and Pabo, cells, containing the empty vector. In the presence of 1988). Extracellular Tat activities seem to mediate the Tat, pRb2/p130seems to lose its growth-suppressive majority of Tat effects in vivo and are implicated in the properties, as demonstrated by the increase in the pathogenesis of HIV-related diseases (Vene et al., 2000). number of colonies formed (Figure 4), in comparison Transgenic approaches provided evidence that there is a to cells transfected with pRb2/p130alone. This result correlation between Tat protein expression and the suggests that Tat binding to pRb2/p130is not just a insurgence of a variety of AIDS-related neoplasms physical interaction, but may lead to the deregulation of including Kaposi’s sarcoma (KS) and NHLs (Vogel cell cycle control activities mediated by pRb2/p130, as et al., 1988; Corallini et al., 1993; Kundu et al., 1999). demonstrated by the loss of its growth-suppressive The expression of Tat protein in tissues of AIDS-related properties. lymphomas in humans has recently been demonstrated (Lazzi et al., 2002). Interestingly, the same tumors The interaction with Tat does not release E2F-4 from overexpressed pRb2/p130tumor suppressor protein by et al pRb2/p130 immunohistochemistry (Lazzi ., 2002). The overexpression of pRb2/p130in tumors with high prolif- To evaluate whether the interaction between Tat and erative activity such as AIDS-related lymphomas pRb2/p130disrupts the E2F4/p130complexes, an suggested that the oncosuppressive properties of pRb2/ artificial E2F promoter containing three consecutive p130could be inactivated (Lazzi et al., 2002). In E2F consensus binding sites (Claudio et al., 2001) linked addition, an interaction between pRb2/p130and Tat to the luciferase reporter gene was expressed in 293 cells proteins of HIV was also detected in living cells (Lazzi in the presence of either pRb2/p130or Tat, or both et al., 2002). The aim of the present paper is to together. At 48 h from transfection, cells were harvested investigate the effect of such an interaction on the and processed for luciferase assay. The results are shown oncosuppressive properties of pRb2/p130. in Figure 5 and demonstrate that E2F-4 is not released First, we mapped the interaction of Tat and pRb2/ from pRb2/p130in the presence of Tat, as no significant p130 in vivo, demonstrating that it occurs through the variation in promoter activation is observed. This pocket region of pRb2/p130. We also monitored the suggests that Tat does not compete with E2F-4 in levels of mRNA for pRb2/p130, either in the presence or binding to pRb2/p130. in the absence of Tat and found that in the presence of

Oncogene Physical interaction between pRb2/p130 and Tat GDeFalcoet al 6218 Tat, the transcription level for pRb2/p130increases stimulation, E2F-4 is released from pRb2/p130, accu- dramatically. Furthermore, the results presented here mulates in free form, and initiates a cascade of events show that no significant difference in terms of pRb2/ leading to progression of the cell cycle (Paggi et al., p130phosphorylation status is observed in the presence 1996). The interaction of Tat with pRb2/p130could or absence of Tat, suggesting that the protein is not thus interfere with the binding with E2F-4. Our results, stabilized in the presence of Tat. These observations are however, show that the presence of Tat alone is not in line with the overexpression of pRb2/p130observed sufficient for the release of E2F-4, since no significant by immunohistochemistry in AIDS-related lymphomas variation in an E2F-responsive promoter activation is (Lazzi et al., 2002), and suggest that pRb2/p130 observed. A similar mechanism has been described for overexpression may be due to an increased transcrip- other oncoproteins. The SV 40large T antigen in fact tion/translation for pRb2/p130stimulated by Tat, binds to pRb2/p130and E2F-4, but the release of E2F-4 rather than to an altered turnover of the protein, which from this complex is an active process, which is ATP- requires pRb2/p130phosphorylation (Stiegler et al., dependent and mediated by a chaperone (Sullivan et al., 1998). 2000). Secondly, we evaluated whether the previously The data described here demonstrate that Tat and demonstrated interaction of Tat with pRb2/p130may pRb2/p130interact through the pocket region of pRb2/ have consequences on the ability of pRb2/p130to p130, resulting in the inhibition of pRb2/p130 oncosup- control cell proliferation. The results of the colony pressive properties and uncontrolled cell proliferation. formation assay demonstrate that, in T98G cells, the Whether this can occur through an ATP-dependent presence of Tat determines the loss of growth inhibition chaperone model remains to be determined. In addition, of pRb2/p130. This result suggests that Tat binding to the interaction of Tat and pRb2/p130alone may not be pRb2/p130is not just a physical interaction, but leads to sufficient for neoplastic transformation in vivo and other the deregulation of cell cycle control activities by pRb2/ cofactors may be required. This is also consistent with p130and the loss of its growth-suppressive properties. the finding that Tat cannot induce cell growth unless Our next aim was to determine through which cells are previously activated with inflammatory cyto- molecular mechanism Tat inactivates the pRb2/p130 kines (Ensoli et al., 1990; Barillari et al., 1992; Albini oncosuppressive function. There is increasing evidence et al., 1995; Fiorelli et al., 1999). Further studies are that the pleiotropic activities of Tat are cell cycle related necessary to elucidate completely the molecular mechan- (Kashanchi et al., 2000; Ambrosino et al., 2002). ism underlying the Tat-pRb2/p130interaction. Never- Progression through the cell cycle requires an ordered theless, these results open a window on the role of array of biochemical interactions, illustrated by the pRb2/p130in AIDS-related oncogenesis and suggest a cyclin-Cdk complexes and their Cdk-inhibitory regula- re-evaluation of HIV itself as an oncogenic virus. tors (Sherr and Roberts, 1999). The regulation of basal This may result in the implementation of future and upstream activator transcription factors also plays therapeutic regimes and the design of new therapeutic an important role in the coordinated cascade of the cell approaches. cycle. For example, the activity of the cellular transcription factor E2F is tightly regulated by the Rb proteins (Stein et al., 1999). However, each Rb protein has a Acknowledgements We thank Professor Cosima Baldari, Department of Evolu- different temporary profile of interaction with different tionary Biology of the University of Siena, for her critical E2F members. pRb/p105 preferentially binds to a subset review of the manuscript. This work is supported by grants of E2F members E2F-1, -2, and -3, whereas pRb2/p130 from CNR and MURST-LAG-CO3 (CC), NIH (R21- and p107 binds to E2F-4 and -5. In G0, E2F-4 and -5 CA099955), and Sbarro Health Research Organization (AG), are associated with pRb2/p130in a transcriptionally short-term mobility 2001 from CNR, and ‘Progetto Giovani inactive complex (Paggi et al., 1996). Upon mitogenic Ricercatori’, University of Siena (GDF).

References

Albini A, Barillari G, Benelli R, Gallo RC and Ensoli B. Chang HK, Gallo RC and Ensoli B. (1995). J. Biomed. Sci., 2, (1995). Proc. Natl. Acad. Sci. USA, 92, 4838–4842. 189–202. Altavilla G, Trabanelli C, Merlin M, Caputo A, Lanfredi M, Cinti C, Claudio PP, Howard CM, Neri LM, Fu Y, Leoncini Barbanti-Brodano G and Corallini A. (1999). Am. J. Pathol., L, Tosi GM, Maraldi NM and Giordano A. (2000a). Cancer 154, 1231–1244. Res., 60, 383–389. Ambrosino C, Palmieri C, Puca A, Trimboli F, Schiavone M, Cinti C, Leoncini L, Nyongo A, Ferrari F, Lazzi S, Bellan C, Olimpico F, Ruocco MR, Di Leva F, Toriello M, Quinto I, Vatti R, Zamparelli A, Cevenini G, Tosi GM, Claudio PP, Venuta S and Scala G. (2002). J. Biol. Chem., 277, Maraldi NM, Tosi P and Giordano A. (2000b). Am. J. 31448–31458. Pathol., 156 (3), 751–760. Barillari G, Buonaguro L, Fiorelli V, Hoffman J, Michaels F, Clark E, Santiago F, Deng L, Chong S, de La Fuente C, Wang Gallo RC and Ensoli B. (1992). J. Immunol., 149, L, Fu P, Stein D, Denny T, Lanka V, Mozafari F, Okamoto 3727–3734. T and Kashanchi F. (2000). J. Virol., 74, 5040–5052. Barillari G and Ensoli B. (2002). Clin. Microbiol. Rev., 15, Claudio PP, Howard CM, Baldi A, De Luca A, Fu Y, 310–326. Condorelli G, Sun Y, Colburn N, Calabretta B and Boshoff C and Weiss R. (2002). Nat. Rev. Cancer, 2, 373–382. Giordano A. (1994). Cancer Res., 54, 5556–5560.

Oncogene Physical interaction between pRb2/p130 and Tat GDeFalcoet al 6219 Claudio PP, Howard CM, Fu Y, Cinti C, Califano L, Micheli Kashanchi F, Agbottah ET, Pise-Masison CA, Mahieux R, P, Mercer EW, Caputi M and Giordano A. (2000a). Cancer Duvall J, Kumar A and Brady JN. (2000). J. Virol., 74, Res., 60, 8–12. 652–660. Claudio PP, Howard CM, Pacilio C, Cinti C, Romano G, Kim CM, Vogel J, Jay G and Rhim JS. (1992). Oncogene, 7, Minimo C, Maraldi NM, Minna JD, Gelbert L, Leoncini L, 1525–1529. Tosi GM, Hicheli P, Caputi M, Giordano GG and Knowles DM. (1999). Mod. Pathol., 12, 200–217. Giordano A. (2000b). Cancer Res., 60, 372–382. Kundu M, Guermah M, Roeder RG, Amini S and Khalili K. Claudio PP, Stiegler P, Howard CM, Bellan C, Minimo C, (1997). J. Biol. Chem., 272, 29468–29474. Tosi GM, Rak J, Kovatich A, De Fazio P, Micheli P, Caputi Kundu M, Sharma S, De Luca A, Giordano A, Rappaport J, M, Leoncini L, Kerbel R, Giordano GG and Giordano A. Khalili K and Amini S. (1998). J. Biol. Chem., 273, (2001). Cancer Res., 61, 462–468. 8130–8136. Corallini A, Altavilla G, Pozzi L, Bignozzi F, Negrini M, Kundu RK, Sangiorgi F, Wu LY, Pattengale PK, Hinton DR, Rimessi P, Gualandi F and Barbanti-Brodano G.. (1993). Gill PS and Maxson R. (1999). Blood, 94, 275–282. Cancer Res., 53, 5569–5575. Lazzi S, Bellan C, De Falco G, Cinti C, Ferrari F, Nyongo A, Corallini A, Campioni D, Rossi C, Albini A, Possati L, Claudio PP, Tosi GM, Vatti R, Gloghini A, Carbone A, Rusnati M, Gazzanelli G, Benelli R, Masiello L, Sparacciari Giordano A, Leoncini L and Tosi P. (2002). Hum. Pathol., V, Presta M, Mannello F, Fontanini G and Barbanti- 33, 723–731. Brodano G. (1996). Aids, 10, 701–710. Li CJ, Wang C, Friedman DJ and Pardee AB. (1995). Proc. Dalgleish AG and O’Byrne KJ. (2002). Adv. Cancer Res., 84, Natl. Acad. Sci. USA, 92, 5461–5464. 231–276. Mahieux R, Lambert PF, Agbottah E, Halanski MA, De Falco G, Bagella L, Claudio PP, De Luca A, Fu Y, Deng L, Kashanchi F and Brady JN. (2001). J. Virol., 75, Calabretta B, Sala A and Giordano A. (2000). Oncogene, 19, 1736–1743. 373–379. Noonan D and Albini A. (2000). Adv. Pharmacol., 48, De La Fuente C, Santiago F, Deng L, Eadie C, Zilberman I, 229–250. Kehn K, Maddukuri A, Baylor S, Wu K, Lee C, Pumfery A Paggi MG, Baldi A, Bonetto F and Giordano A. (1996). J. and Kashanchi F. (2002). BMC Biochem., 3, 14. Cell. Biochem., 62, 418–430. De Luca A, Baldi A, Esposito V, Howard CM, Bagella L, Rubartelli A, Poggi A, Sitia R and Zocchi MR. (1998). Rizzo P, Caputi M, Pass HI, Giordano GG, Baldi F, Immunol. Today, 19, 543–545. Carbone M and Giordano A. (1997). Nat. Med., 3, 913–916. Sherr CJ and Roberts JM. (1999). Genes Dev., 13, Ensoli B, Barillari G, Salahuddin SZ, Gallo RC and Wong- 1501–1512. Staal F. (1990). Nature, 345, 84–86. Stein GS, Baserga R and Giordano A et al. (1999). The Ensoli B, Buonaguro L, Barillari G, Fiorelli V, Gendelman R, Molecular Basis of Cell Cycle and Growth Control. Wiley- Morgan RA, Wingﬁeld P and Gallo RC. (1993). J. Virol., Liss: New York. 67, 277–287. Stiegler P, Kasten M and Giordano A. (1998). J. Cell. Ensoli B, Gendelman R, Markham P, Fiorelli V, Colombini S, Biochem. Suppl, 30–31, 30–36. Raffeld M, Cafaro A, Chang HK, Brady JN and Gallo RC. Sullivan CS, Cantalupo P and Pipas JM. (2000). Mol. Cell. (1994). Nature, 371, 674–680. Biol., 20, 6233–6243. Fattaey AR, Harlow E and Helin K. (1993). Mol. Cell. Biol., Vene R, Benelli R, Noonan DM and Albini A. (2000). Clin. 13, 7267–7277. Exp. Metast., 18, 533–538. Fiorelli V, Barillari G, Toschi E, Sgadari C, Monini P, Sturzl Vogel J, Hinrichs SH, Reynolds RK, Luciw PA and Jay G. M and Ensoli B. (1999). J. Immunol., l, 62, 1165–1170. (1988). Nature, 335, 606–611. Frankel AD and Pabo CO. (1988). Cell, 55, 1189–1193. Wu A, Ichihashi M and Ueda M. (1999). Cancer, 86, Gaidano G, Carbone A and Dalla-Favera R. (1998). Am. J. 2038–2044. Pathol., 152, 623–630. Zauli G, Gibellini D, Caputo A, Bassini A, Negrini M, Monne Gatignol A and Jeang KT. (2000). Adv. Pharmacol., 48, M, Mazzoni M and Capitani S. (1995). Blood, 86, 209–227. 3823–3834.

Oncogene