NOTES
Dephosphorylation of the retinoblastoma protein during differentiation of HL60 cells1 PETERW H Y T E ~AND ROBERT N. EISENMAN
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Fred Hutchinson Cancer Research Center, I124 Columbia St., Seattle, WA 98104, U.S.A. Received April 3, 1992 WHYTE,P., and EISENMAN, R. N. 1992. Dephosphorylation of the retinoblastoma protein during differentiation of HL60 cells. Biochem. Cell Biol. 70: 1380-1384. Immunoprecipitated retinoblastoma protein from HL60 cells migrated as a series of bands during electrophoresis. The heterogeneity appeared to be generated by phosphorylation of the retinoblastoma protein. Treatment of the cells with the phorbol ester, tetradecanoyl phorbol acetate (TPA), resulted in both a loss of the heterogeneity of the pRB species and a significant decrease in the level of pRB phosphorylation. These changes accompanied differentiation of the HL60 cells into macrophages. Treatment of the cells with dibutyryl CAMP also resulted in dephosphorylation of pRB as well as cell cycle arrest, although no recognizable differentiation occurred. These results are consistent with a model in which TPA and dibutyryl cAMP dependent pathways can activate pRB by altering its phosphorylation. Key words: retinoblastoma, cell cycle, differentiation, leukemia. WHYTE,P., et EISENMAN, R. N. 1992. Dephosphorylation of the retinoblastoma protein during differentiation of HL60 cells. Biochem. Cell Biol. 70 : 1380-1384. La protkine du rCtinoblastome immunoprCcipitCe des cellules HL60 migre sous forme d'une sCrie de bandes lors de 1'Clectrophorbe. L'hCtCrogCnCite semble gCnCrCe par la phosphorylation de la protCine du retinoblastome. Le traitement des cellules avec un phorbolester, le tCtradCcanoyl phorbol acktate (TPA), provoque a la fois une perte de I'hCtCrogtnCite de I'espbce pRB et une diminution importante du taux de phosphorylation de la pRB. Ces changements accompagnent la differenciation des cellules HL60 en macrophages. Le traitement des cellules avec le dibutyryl CAMPentrdne Cgalement la dCphosphorylation de la pRB de m&mequ'un arr&tdu cycle cellulaire, mais pas de diffkrenciation reconnaissable. Ces rCsultats sont compatibles avec un modkle ou les voies dkpendantes du TPA et du dibutyryl cAMP peuvent activer la pRB en alterant sa phosphorylation. Mots clPs : rktinoblastome, cycle cellulaire, differenciation, leuckmie. [Traduit par la rkdaction]
Introduction Regulation of cellular proliferation is thought to be controlled by both positively and negatively acting events (reviewed in Bishop 1991). Studies on growth factors and oncogenes have provided insight into the pathways that promote cellular proliferation (reviewed in Cantley et al. 1991). Less is known about pathways causing cell cycle arrest and (or) terminal differentiation. Extracellular factors promoting arrest or differentiation, such as the members of the TGFP family, and the tumour suppressor proteins provide potential opportunities to study pathways restricting proliferation. In recent years, several tumour suppressor genes have been identified and cDNAs cloned (reviewed in Marshall 1991; Weinberg 1991). These genes are frequently mutated or deleted during tumourigenesis, suggesting that they are involved in negatively regulating growth. The best characterized of the tumour suppressor genes is the retinoblastoma gene, which was originally identified as a gene disrupted or deleted in hereditary retinoblastomas (reviewed ABBREVIATIONS: TPA, tetradecanoyl phorbol acetate; TGFP, transforming growth factor-beta; kDa, kilodalton(s); MEM, minimal essential medium; DMSO, dimethyl sulfoxide; NP40, Nonidet P-40. he substance of this paper was presented at the Fifth Biennial Rossiter Research Conference "The Eukaryotic Genome in Human Disease," held September 27-29, 1991, at Geneva Park, Lake Couchiching, Ont. 2 ~ r e s e n taddress: Institute for Molecular Biology and Biotechnology and Department of Pathology, McMaster University, 1280 Main St. W., Hamilton, Ont., Canada L8S 4K1. Pr~ntedin Canada / lmpr~rneau Canada
in Benedict et al. 1990; Weinberg 1991). This gene is frequently disrupted not only in retinoblastoma, but also in many other tumour types (Benedict et al. 1990). pRB, the protein encoded by this gene, is a nuclear phosphoprotein of approximately 105 kDa and is expressed ubiquitously (Lee et al. 1987). Several DNA tumour viruses produce transforming proteins that target pRB by forming specific complexes with it (Whyte et al. 1988; DeCaprio et al. 1988; Dyson et al. 1989b). The adenovirus E l A proteins, the papovavirus large T antigens, and the human papillomavirus E7 proteins are each capable of forming complexes with pRB and it has been suggested that this results in the inactivation or blockage of the normal function of pRB. Several observations have suggested that the pRB protein product of the retinoblastoma gene plays an important role in regulating cellular proliferation either by restricting progress through the G, stage of the cell cycle or arresting cells in a Go state. During periods of cellular proliferation, pRB appears to be negatively regulated by phosphorylation. Phosphorylation of pRB takes place in a cell cycle dependent manner with high levels of phosphate accumulating in late GI and remaining until mitosis (Buchkovich et al. 1989; Chen et al. 1989; DeCaprio et al. 1989; Xu et al. 1989; Mihara et al. 1989). Recently evidence has been presented that p34cdc2 or a related kinase is responsible for phosphorylation of pRB (Lin et al. 1991; Lees et al. 1991). Because SV40 T antigen forms complexes with only the underphosphorylated species of pRB, it has been postulated that pRB is active only when in the underphosphorylated state (Ludlow et al. 1989). These observations are consistent
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with it acting as a restriction point at a Go-G1 position in the cell cycle. Although the precise role of pRB as a tumour suppressor is not clear, several pieces of evidence point to a role in terminal differentiation. First, it is at the Go-G1 position that cells frequently enter into an arrested state, the same position where pRB is thought to function (Buchkovich et al. 1989; Chen et al. 1989; DeCaprio et al. 1989; Ludlow et al. 1989; Mihara et al. 1989; Xu et al. 1989). Second, viruses such as adenovirus rely on their transforming proteins to stimulate Go arrested cells into actively cycling cells that are more permissive for viral replication. A role for pRB in maintaining the Go state is consistent with these viral proteins binding to pRB and blocking its function. Third, it has recently been shown that pRB is a component of the E2F-DRTF transcriptional factor complex (Bagchi et al. 1991; Bandera and LaThangue 1991; Chellappan et al. 1991; Chittendan et al. 1991). This sequence-specific transcriptional activity is thought to be inhibited by pRB. This is consistent with the observation that DRTF activity is down regulated during differentiation of F9 embryo carcinoma cells. To examine a possible role for pRB during differentiation we have used the HL60 promyelocytic leukemia cell line as a model. Here we describe changes in pRB phosphorylation following induction of differentiation. The results presented here are consistent with pRB being a component of a pathway leading to growth arrest. Materials and methods Cell culture HL60 cells were grown in Dulbecco's MEM (Gibco) sup~ was carried plemented with 10% fetal bovine serum. 3 S labelling out in methionine-deficient medium for 4 h. 3 2 labelling ~ was for 2 h in phosphate-deficient medium. TPA (1 mg/mL in DMSO) was added to cultures to a final concentration of 0.1 pg/mL. Dibutyryl CAMP (Sigma; lo-' M in culture medium without serum) was added to a final concentration of M. Retinoic acid was made up as a stock solution of M in ethanol and was added to cultures to a final concentration of M. Immunoprecipitations Immunoprecipitations of pRB were performed using the C36 monoclonal antibody (Whyte et al. 1988) as has been previously described (Harlow et al. 1986). Control immunoprecipitations used the monoclonal antibody MAb419, which recognizes the SV40 T antigen (Harlow et a/. 1981). Briefly, cells were lysed in a buffer containing 0.1% NP40, 125 mM NaCI, and 50 mM HEPES (pH 7.0). Insoluble material was cleared from the lysate by centrifugation and aliquots of the lysate were incubated with the appropriate antibody plus protein A - Sepharose for 60 min with continuous agitation. The protein A - Sepharose beads were washed with RIPA buffer (150 mM NaCI, 1.0% NP40, 0.5% sodium deoxycholate, 0.1 VO sodium dodecyl sulfate, 50 mM Tris (pH 8.0)) and pelleted by centrifugation three times, and then resuspended in Laemmli sample buffer and separated by electrophoresis (Laemmli 1970).
Results When pRB was immunoprecipitated from asynchronously growing HL60 cells, it migrated as a heterogeneous group of bands (Fig. 1). This heterogeneity is due to varying degrees of phosphorylation of the pRB protein (Buchkovich et al. 1989; Chen et al. 1989; DeCaprio et al. 1989; Ludlow et al. 1989; Mihara et al. 1989; Xu et al. 1989). Because pRB
0
Hours of TPA treatment 5 10 24 48
72
I -
C -Rb C Rb C Rb C Rb C Rb C Rb -=
m y
%.:-Y.,"v
-8-
FIG. 1. Altered mobility of pRB proteins following TPA stimulation. HL60 cells were labelled with [35~]methioninefor 4 h, lysed, immunoprecipitated with either anti-pRB specific monoclonal antibody (Rb) or a control antibody (C), and then run on a 7.5% SDS-polyacrylamide gel. Cells were treated with TPA for the indicated times prior to lysis.
is presumed to act in a manner restrictive to cellular proliferation, changes to pRB were examined as cells were induced to withdraw from the cell cycle into a terminally differentiated state. HL60 cells were induced to differentiate into macrophages using the phorbol ester TPA, a known stimulator of protein kinase C enzymes (Rovera et al. 1979; Nishizuka 1986). At various times after TPA addition to the culture, pRB was immunoprecipitated and run on a polyacrylamide gel (Fig. 1). The heterogeneity of pRB bands began to disappear between 10 and 24 h after treatment of the cells with TPA, suggesting a reduction in the phosphorylation of pRB. This coincides with the onset of morphological differentiation and time of cell cycle arrest (Rovera et al. 1979; Yen et al. 1987; data not shown). By 48 h, all of the slower migrating forms of pRB were no longer present and pRB migrated as a single species. At this time, all cells had morphologically differentiated into macrophages. The relative intensity of labelling suggests that the biosynthesis of pRB was similar throughout the time course of the experiments and that the loss of the slowly migrating species was due only to a change in the phosphorylation pattern. To examine the phosphorylation state of pRB, this experiment was repeated using 32~-labelledextracts t o immunoprecipitate pRB. In asynchronously growing HL60 cells, pRB was labelled relatively well with 3 2 and ~ the pRB migrated as a broad heterogeneous band 3'~-label~ed (Fig. 2). Following treatment with TPA, the amount of 3 2 immunoprecipitated as pRB was significantly reduced and the protein migrated as one distinct band, The half-life of phosphate moieties on pRB has been found to be substantially shorter than the protein half-life, indicating that the phosphorylation state is a dynamically maintained state; consequently, 32~-labelled pRB should be representative of protein synthesized at all times and not just newly syn-
~
BIOCHEM. CELL BIOL. VOL. 70, 1992
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Days of TPA treatment
FIG. 2. TPA-induced dephosphorylation of pRB. HL60 cells were labelled with jE~]phasphate for 2 h, lysed. immuno-
precipitated with either the C36 anti-pRB monoclonal antibody or a control antibody, and then run on a 7.5% SSD-polyacrylamide gel. CelIs were treated with TPA For the indicated number of days prior to lysis.
thesized protein (Chen et al. 1989). Together, the results from the 3 5 ~ and 32~-labelling experiments (Figs. 1 and 2) indicate that the level of phosphorylation of pRB is substantially reduced after TPA treatment. The above results are consistent with a pathway stimulated by protein kinase C resulting in dephosphoryIation of pRB. To determine if other agents that cause cell cycle arrest and (or) terminal differentiation affect the phosphorylation state of pRB, HL60 cells were treated with several compounds for 48 h, and then they Iabelled with 3 5 and ~ pRB was irnmunoprecipitated (Fig. 3). In addition to TPA, dibutyry1 CAMP, a stimulator of cAMPdependent kinase, also appeared to cause a decrease in pRB phosphorylation. Following the addition of dibutyryl cAMP to the culture medium, the cells underwent cell cycle arrest suggesting that, like TPA, dibutyryl CAMP can stimulate a pathway eventually leading to both activation of pRB and growth arrest. Although these cells underwent cell cycle arrest they did not appear to morphologically differentiate. Another compound tested, retinoic acid, is known to induce HL60 cells to differentiate into granulocytes after 7-10 days; however, after 48 h of treatment no changes in the phosphorylation state of pRB was observed (Fig. 3). It is possible that differentiation and cell cycle arrest by retinoic acid occurs through a mechanism distinct from TPA and does not directly influence pRB phosphorylation because, in contrast to TPA and cAMP analogues, retinoic acid is not known to directly affect phosphorylation pathways. After longer periods of treatment with retinoic acid pRB migrated as a single electrophoretic species, suggesting that loss of phosphoryIation of pRB eventually occurs, but in this case it may be only a consequence of cell cycle arrest (data not shown).A similar conclusion was reached by another group examining retinoic acid induced differentiation of HL60 cells (Mihara et al. 1989).
FTG. 3. Loss of phosphorylated forms of pRB after stimulation of cells with various compounds. HL60 cells were treated with the indicated compounds for 48 h, labelled with ["~]methionine, lysed, and immunoprecipitated with either the C36 monoclonal antibody (Rb) or a control antibody (C). Immunoprecipitated proteins were run on a 7.5% SDS-polyacrylamide gel.
Discussion The results presented in this report raise the possibility that pRB, a tumour suppressor protein, plays a role in one or more pathways leading to cell cycle arrest. HL60 cells exposed to either the phorbol ester TPA or dibutyryl CAMP underwent cell cycle arrest and a concommitant decrease in the phosphorylation of pRB. As outlined above, phosphorylation of pRB is believed to negatively regulate the activity of pRB. Thus, the loss of pRB phosphorylation observed in our experiments is consistent with a function for pRB in arresting cells in a Go state. These results are also consistent with the results of several other groups who have shown that in differentiated or senescent cells pRB remains in an underphosphorylated state (Chen et al. 1989; Mihara et al. 1989;Akiyama and Toyoshima 1990;Stein et al. 1990). TPA-mediated changes in the phosphorylation state of pRB are suggestive of a pathway mediated by protein kinase C. This protein kinase is frequently an intermediate in pathways stimulated by extracellular ligands, especially those mediated by tyrosine kinase receptors (Nishizuka 1986). A model in which pRB lies on a pathway stimulated by an extracellular factor is presented in-Fig. 4. In this model, protein kinase C mediates the activation of pRB
SIGNAL PROTEIN KINASE C
1 ACTIVATION OF PRB 1 CELL CYCLE ARREST
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TPA NONRESPONSIVE LINES
E l A, T antigens, E7
FIG.4. Model of RB in a signal transduction pathway resulting in cell cycle arrest. A schematic diagram of events involving the retinoblastoma protein and its regulation via signal transduction. At the right are positions in the pathway where certain cell lines are blocked and where viral transforming proteins function to block the pathway.
through an unknown mechanism, resulting in a decrease in the overall phosphorylation of pRB and eventually leading to cell cycle arrest. A signal transduction pathway would contain multiple points where potentially oncogenic events could disrupt the pathway. Some known oncogenic events are consistent with disruption of this putative pathway at several positions. One is the mutation or disruption of the pRB gene as occurs in retinoblastomas and various other tumours (reviewed in Benedict et al. 1990). Transforming proteins from DNA tumour viruses such as the adenovirus E1A protein, papovavirus T antigens, and papillomavirus E7 proteins may also block the pathway through their interactions with pRB. In HL60 cells, a pathway resulting in the loss of phosphorylation of pRB can be activated by TPA. Similar results have been found for several other cell lines capable of differentiating in response to TPA (Chen et at. 1989; Akiyama and Toyoshima 1990; P. Whyte and R.N. Eisenman, unpublished data). One possibility is that normal regulation of these cells may have been disrupted by mutations in a signal transduction pathway upstream of protein kinase C. Another part of the pathway that could be disrupted in transformed cells lies between protein kinase C and the dephosphorylation of pRB. Many transformed cell lines do not differentiate in response to TPA. In these cells, it is possible that other oncogenic events have disrupted the pathway downstream of protein kinase C, rendering the cells nonresponsive to TPA. Characterization of the mechanism through which pRB becomes dephosphorylated may allow tumour-derived cell lines to be examined for potentially oncogenic events affecting this pathway. It is possible that more than one signal transduction pathway leads to the activation of pRB. Addition of dibutyryl CAMP to cell culture of HL60 cells resulted in a loss of phosphorylation of pRB and cell cycle arrest. This may be representative of a second pathway influencing the phosphorylation state of pRB and others may also exist. pRB may be representative of a family of related proteins, including other cellular proteins that interact with the same region of E1A as does pRB. This is supported by the recent
cloning of a cDNA for p107, a cellular protein that binds to the pRB binding domain of E1A and SV40 T antigen (Egan et al. 1988; Whyte et al. 1989; Dyson et al. 1989a; Ewen et al. 1989, 1991). pRB and p107 share regions of homology and appear to be members of the same family of proteins. It will be of interest to see if phosphorylation of p107 is affected during differentiation. Acknowledgements P.W. was supported by a Damon Runyon-Walter Winchell Cancer Research Fund Fellowship (DRG-973). This work was supported by National Institutes of Health grant R01 CA20525 awarded to R.N.E. Akiyama, T.. and Toyoshima, K. 1990. Marked alteration in phosphorylarion of the RB protein during differentiation of human promyelocytic HLbO cells. Oncogene, 5: 179-183. Bagchi, S . , Weinmann, R., and Raychaudhuri, P. 1991. The rednobIastoma protein copurifies with E2F-1, an E l A-regulated inhibitor of transcription Factor E2F. Cell, 65: 1063-1072. Bandara, L . R . , and La Thangue, N.R. 1991. Adenovirus EIA prevents the retinoblastoma gene product from complexing with acellular transcription factor. Nature (London), 351: 494-497. Benedict, W.F., Xu, H.-J., Hu, S.-X., and Takahashi, R. 1990. Role of the retinoblastoma gene in the initiation and progression of human cancer. J. Clin. Invest. 85: 988-993. Bishop, J.M. 1991. Molecular themes in oncogenesis. Cell, 64: 235-248. Buchkovich, K., Duffy, L.A., and Harlow, E. 1989. The retinoblastoma protein is phosphorylated during specific phases of the cell cycle. Cell, 58: 1097-1105. Cantley, L.C., Auger, K.R., Carpenter, C., Duckworth, B., Graziani, A., Kapeller, R., and Soltoff, S. 1991. Oncogenes and signal transduction. Cell, 64: 281-302. Chellappan, S.P., Hiebert, S., Mudryj, M., Horowitz, J.M., and Nevins, J.R. 1991. The E2F transcription factor is a target for the RB protein. Cell, 65: 1053-1061. Chen, P.-L., Scully, P., Shew, J.-y., Wang, J.Y.J., and Lee, W.-H. 1989. Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation. Cell, 58: 1193-1 198. Chittendan, T., Livingston, D.M., and Kaelin, W.G., Jr. 1991. The T/EIA binding domain of the retinoblastoma product can interact selectively with the a sequence specific DNA-binding protein. Cell, 65: 1073-1082. DeCaprio, J.A., Ludlow, J.W.. Figge, J., Shew,J.-Y.,Huang, C.-M., Lee,W.-H., Marsilio, E.,Paucha, E., and Livingston. D.M. 1988. SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell, 54: 275-283. DeCaprio, J.A., Ludlow, J.W., Lynch, D., Furakawa, Y., Griffin, J., Piwinica-Worms, H.,Huang, C.-M., and Livingston, D.M. 1989. The product of the retinoblastoma susceptibility gene has propmies of a cell cycIe regulatory element. Cell, 58: 3085-1095. Dyson, N., Buchkovich, K.J.,Whyte, P., and Harlow, E, 1989a. The cellular 107K protein that binds to adenovirus ELA also associates with large T antigens of SV40 and J C virus. Cell, 58: 249-255. Dyson, N., Howley, P.M., Munger, K., and Harlow, E. 19896. The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science (Washington, D.C.), 243: 934-937. Egan, C., Jelsma, T.N., Howe, J.A., Bayley, S.T., Ferguson, B., and Branton, P.E. 1988. Mapping of cellular protein-binding sites on the products of early-region 1A of human adenovirus type 5. Mol. Cell. Biol. 8: 3955-3959. Ewen, M.E., Ludlow, J.W., Marsilio, E., DeCaprio, J.A., Millikan, R.C., Chen, S.H., Paucha, E., and Livingston, D.M.
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1989. An N-terminal transformation governing sequence of SV40 large T antigen contributes to both pl lORb and a second protein, p.120. Cell, 58: 257-267. Ewen, M.E., Xing, Y., Lawrence, J.B., and Livingston, D.M. 1991. Molecular cloning, chromosomal mapping and expression of the cDNA for p107, a retinoblastoma gene product-related protein. Cell, 66: 1155-1 164. Harlow, E., Crawford, L.V., Pim, D.C., and Williamson, N.M. 1981. Monoclonal antibodies specific for simian virus 40 tumor antigens. J. Virol. 39: 861-869. Harlow, E., Whyte, P., Franza, B.R., Jr., and Schley, C. 1986. Association of adenovirus early-region 1A proteins with cellular polypeptides. Mol. Cell. Biol. 6: 1579-1589. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 278: 680-685. Lee, W.-H., Shew, J.-Y., Hong. F., Sery, T.W., Donoso, L.A., Young, L.-J., Bookstein, R., and Lee, E.Y.-H.P. 1987. The retinoblastoma susceptibility gene encodes a nuclear phosphoprotein associated with DNA binding activity. Nature (London), 329: 642-645. Lees, J.A., Buchkovich, K.J., Marchak, D.R., Anderson, C.W., and Harlow, E. 1991. The retinoblastoma protein is phosphorylated on multiple sites by human cdc2. EMBO J. 10: 4279-4290. Lin, B.T.-Y., Gruenwald, S., Morla, A.O., Lee, W.-H., and Wang, J.Y. J. 1991. Retinoblastoma cancer suppressor gene product is a substrate of the cell cycle regulator cdc2 kinase. EMBO J. 10: 857-864. Ludlow, J.D., DeCaprio, J.A., Huang, C.-M., Lee, W.-H., Paucha, E., and Livingston, D.M. 1989. SV40 large T antigen
binds preferentially to an underphosphorylated member of the retinoblastoma susceptibility gene product family. Cell, 56: 57-65. Marshall, C.J. 1991. Tumor suppressor genes. Cell, 64: 313-326. Mihara, K., Cao, X.-R., Yen, A., Chandler, S., Driscoll, B., Murphree, A.L., T'Ang, A., and Fung, Y.-K.T. 1989. Cell cycledependent regulation of phosphorylation of the human retinoblastoma gene product. Science (Washington, D.C.), 246: 1300-1303. Nishizuka, Y. 1986. Studies and perspectives of protein kinase C. Science (Washington, D.C.), 233: 305-312. Rovera, G., O'Brien, T.G., and Diamond, L. 1979. Induction of differentiation in human promyelocytic leukemia cells by tumor promoters. Science (Washington, D.C.), 204: 868-870. Stein, G.H., Beeson, M., and Gordon, L. 1990. Failure to phosphorylate the retinoblastoma gene product in senescent human fibroblasts. Science (Washington, D.C.), 249: 666-669. Weinberg, R.A. 1991. Tumor suppressor genes. Science (Washington, D.C.), 254: 1138-1 145. Whyte, P., Buchkovich, K.J., Horowitz, J.M., Friend, S.H.. Raybuck, M., Weinberg, R.A., and Harlow, E. 1988. Association between an oncogene and an antioncogene: the adenovirus ElA proteins bind to the retinoblastoma gene product. Nature (London), 334: 124-129. Yen, A., Brown, D., and Fishbaugh, J. 1987. Control of HL60 monocytic differentiation. Exp. Cell. Res. 173: 80-84. Xu, H.-J., Hu, S.-X., Hashimoto, T., Takahashi, R., and Benedict, W.F. 1989. The retinoblastoma susceptibility gene product: a characteristic pattern in normal cells and abnormal expression in malignant cells. Oncogene, 4: 807-812.
Dephosphorylation of the retinoblastoma protein during differentiation of HL60 cells1 PETERW H Y T E ~AND ROBERT N. EISENMAN
Biochem. Cell Biol. 1992.70:1380-1384. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 12/27/14. For personal use only.
Fred Hutchinson Cancer Research Center, I124 Columbia St., Seattle, WA 98104, U.S.A. Received April 3, 1992 WHYTE,P., and EISENMAN, R. N. 1992. Dephosphorylation of the retinoblastoma protein during differentiation of HL60 cells. Biochem. Cell Biol. 70: 1380-1384. Immunoprecipitated retinoblastoma protein from HL60 cells migrated as a series of bands during electrophoresis. The heterogeneity appeared to be generated by phosphorylation of the retinoblastoma protein. Treatment of the cells with the phorbol ester, tetradecanoyl phorbol acetate (TPA), resulted in both a loss of the heterogeneity of the pRB species and a significant decrease in the level of pRB phosphorylation. These changes accompanied differentiation of the HL60 cells into macrophages. Treatment of the cells with dibutyryl CAMP also resulted in dephosphorylation of pRB as well as cell cycle arrest, although no recognizable differentiation occurred. These results are consistent with a model in which TPA and dibutyryl cAMP dependent pathways can activate pRB by altering its phosphorylation. Key words: retinoblastoma, cell cycle, differentiation, leukemia. WHYTE,P., et EISENMAN, R. N. 1992. Dephosphorylation of the retinoblastoma protein during differentiation of HL60 cells. Biochem. Cell Biol. 70 : 1380-1384. La protkine du rCtinoblastome immunoprCcipitCe des cellules HL60 migre sous forme d'une sCrie de bandes lors de 1'Clectrophorbe. L'hCtCrogCnCite semble gCnCrCe par la phosphorylation de la protCine du retinoblastome. Le traitement des cellules avec un phorbolester, le tCtradCcanoyl phorbol acktate (TPA), provoque a la fois une perte de I'hCtCrogtnCite de I'espbce pRB et une diminution importante du taux de phosphorylation de la pRB. Ces changements accompagnent la differenciation des cellules HL60 en macrophages. Le traitement des cellules avec le dibutyryl CAMPentrdne Cgalement la dCphosphorylation de la pRB de m&mequ'un arr&tdu cycle cellulaire, mais pas de diffkrenciation reconnaissable. Ces rCsultats sont compatibles avec un modkle ou les voies dkpendantes du TPA et du dibutyryl cAMP peuvent activer la pRB en alterant sa phosphorylation. Mots clPs : rktinoblastome, cycle cellulaire, differenciation, leuckmie. [Traduit par la rkdaction]
Introduction Regulation of cellular proliferation is thought to be controlled by both positively and negatively acting events (reviewed in Bishop 1991). Studies on growth factors and oncogenes have provided insight into the pathways that promote cellular proliferation (reviewed in Cantley et al. 1991). Less is known about pathways causing cell cycle arrest and (or) terminal differentiation. Extracellular factors promoting arrest or differentiation, such as the members of the TGFP family, and the tumour suppressor proteins provide potential opportunities to study pathways restricting proliferation. In recent years, several tumour suppressor genes have been identified and cDNAs cloned (reviewed in Marshall 1991; Weinberg 1991). These genes are frequently mutated or deleted during tumourigenesis, suggesting that they are involved in negatively regulating growth. The best characterized of the tumour suppressor genes is the retinoblastoma gene, which was originally identified as a gene disrupted or deleted in hereditary retinoblastomas (reviewed ABBREVIATIONS: TPA, tetradecanoyl phorbol acetate; TGFP, transforming growth factor-beta; kDa, kilodalton(s); MEM, minimal essential medium; DMSO, dimethyl sulfoxide; NP40, Nonidet P-40. he substance of this paper was presented at the Fifth Biennial Rossiter Research Conference "The Eukaryotic Genome in Human Disease," held September 27-29, 1991, at Geneva Park, Lake Couchiching, Ont. 2 ~ r e s e n taddress: Institute for Molecular Biology and Biotechnology and Department of Pathology, McMaster University, 1280 Main St. W., Hamilton, Ont., Canada L8S 4K1. Pr~ntedin Canada / lmpr~rneau Canada
in Benedict et al. 1990; Weinberg 1991). This gene is frequently disrupted not only in retinoblastoma, but also in many other tumour types (Benedict et al. 1990). pRB, the protein encoded by this gene, is a nuclear phosphoprotein of approximately 105 kDa and is expressed ubiquitously (Lee et al. 1987). Several DNA tumour viruses produce transforming proteins that target pRB by forming specific complexes with it (Whyte et al. 1988; DeCaprio et al. 1988; Dyson et al. 1989b). The adenovirus E l A proteins, the papovavirus large T antigens, and the human papillomavirus E7 proteins are each capable of forming complexes with pRB and it has been suggested that this results in the inactivation or blockage of the normal function of pRB. Several observations have suggested that the pRB protein product of the retinoblastoma gene plays an important role in regulating cellular proliferation either by restricting progress through the G, stage of the cell cycle or arresting cells in a Go state. During periods of cellular proliferation, pRB appears to be negatively regulated by phosphorylation. Phosphorylation of pRB takes place in a cell cycle dependent manner with high levels of phosphate accumulating in late GI and remaining until mitosis (Buchkovich et al. 1989; Chen et al. 1989; DeCaprio et al. 1989; Xu et al. 1989; Mihara et al. 1989). Recently evidence has been presented that p34cdc2 or a related kinase is responsible for phosphorylation of pRB (Lin et al. 1991; Lees et al. 1991). Because SV40 T antigen forms complexes with only the underphosphorylated species of pRB, it has been postulated that pRB is active only when in the underphosphorylated state (Ludlow et al. 1989). These observations are consistent
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with it acting as a restriction point at a Go-G1 position in the cell cycle. Although the precise role of pRB as a tumour suppressor is not clear, several pieces of evidence point to a role in terminal differentiation. First, it is at the Go-G1 position that cells frequently enter into an arrested state, the same position where pRB is thought to function (Buchkovich et al. 1989; Chen et al. 1989; DeCaprio et al. 1989; Ludlow et al. 1989; Mihara et al. 1989; Xu et al. 1989). Second, viruses such as adenovirus rely on their transforming proteins to stimulate Go arrested cells into actively cycling cells that are more permissive for viral replication. A role for pRB in maintaining the Go state is consistent with these viral proteins binding to pRB and blocking its function. Third, it has recently been shown that pRB is a component of the E2F-DRTF transcriptional factor complex (Bagchi et al. 1991; Bandera and LaThangue 1991; Chellappan et al. 1991; Chittendan et al. 1991). This sequence-specific transcriptional activity is thought to be inhibited by pRB. This is consistent with the observation that DRTF activity is down regulated during differentiation of F9 embryo carcinoma cells. To examine a possible role for pRB during differentiation we have used the HL60 promyelocytic leukemia cell line as a model. Here we describe changes in pRB phosphorylation following induction of differentiation. The results presented here are consistent with pRB being a component of a pathway leading to growth arrest. Materials and methods Cell culture HL60 cells were grown in Dulbecco's MEM (Gibco) sup~ was carried plemented with 10% fetal bovine serum. 3 S labelling out in methionine-deficient medium for 4 h. 3 2 labelling ~ was for 2 h in phosphate-deficient medium. TPA (1 mg/mL in DMSO) was added to cultures to a final concentration of 0.1 pg/mL. Dibutyryl CAMP (Sigma; lo-' M in culture medium without serum) was added to a final concentration of M. Retinoic acid was made up as a stock solution of M in ethanol and was added to cultures to a final concentration of M. Immunoprecipitations Immunoprecipitations of pRB were performed using the C36 monoclonal antibody (Whyte et al. 1988) as has been previously described (Harlow et al. 1986). Control immunoprecipitations used the monoclonal antibody MAb419, which recognizes the SV40 T antigen (Harlow et a/. 1981). Briefly, cells were lysed in a buffer containing 0.1% NP40, 125 mM NaCI, and 50 mM HEPES (pH 7.0). Insoluble material was cleared from the lysate by centrifugation and aliquots of the lysate were incubated with the appropriate antibody plus protein A - Sepharose for 60 min with continuous agitation. The protein A - Sepharose beads were washed with RIPA buffer (150 mM NaCI, 1.0% NP40, 0.5% sodium deoxycholate, 0.1 VO sodium dodecyl sulfate, 50 mM Tris (pH 8.0)) and pelleted by centrifugation three times, and then resuspended in Laemmli sample buffer and separated by electrophoresis (Laemmli 1970).
Results When pRB was immunoprecipitated from asynchronously growing HL60 cells, it migrated as a heterogeneous group of bands (Fig. 1). This heterogeneity is due to varying degrees of phosphorylation of the pRB protein (Buchkovich et al. 1989; Chen et al. 1989; DeCaprio et al. 1989; Ludlow et al. 1989; Mihara et al. 1989; Xu et al. 1989). Because pRB
0
Hours of TPA treatment 5 10 24 48
72
I -
C -Rb C Rb C Rb C Rb C Rb C Rb -=
m y
%.:-Y.,"v
-8-
FIG. 1. Altered mobility of pRB proteins following TPA stimulation. HL60 cells were labelled with [35~]methioninefor 4 h, lysed, immunoprecipitated with either anti-pRB specific monoclonal antibody (Rb) or a control antibody (C), and then run on a 7.5% SDS-polyacrylamide gel. Cells were treated with TPA for the indicated times prior to lysis.
is presumed to act in a manner restrictive to cellular proliferation, changes to pRB were examined as cells were induced to withdraw from the cell cycle into a terminally differentiated state. HL60 cells were induced to differentiate into macrophages using the phorbol ester TPA, a known stimulator of protein kinase C enzymes (Rovera et al. 1979; Nishizuka 1986). At various times after TPA addition to the culture, pRB was immunoprecipitated and run on a polyacrylamide gel (Fig. 1). The heterogeneity of pRB bands began to disappear between 10 and 24 h after treatment of the cells with TPA, suggesting a reduction in the phosphorylation of pRB. This coincides with the onset of morphological differentiation and time of cell cycle arrest (Rovera et al. 1979; Yen et al. 1987; data not shown). By 48 h, all of the slower migrating forms of pRB were no longer present and pRB migrated as a single species. At this time, all cells had morphologically differentiated into macrophages. The relative intensity of labelling suggests that the biosynthesis of pRB was similar throughout the time course of the experiments and that the loss of the slowly migrating species was due only to a change in the phosphorylation pattern. To examine the phosphorylation state of pRB, this experiment was repeated using 32~-labelledextracts t o immunoprecipitate pRB. In asynchronously growing HL60 cells, pRB was labelled relatively well with 3 2 and ~ the pRB migrated as a broad heterogeneous band 3'~-label~ed (Fig. 2). Following treatment with TPA, the amount of 3 2 immunoprecipitated as pRB was significantly reduced and the protein migrated as one distinct band, The half-life of phosphate moieties on pRB has been found to be substantially shorter than the protein half-life, indicating that the phosphorylation state is a dynamically maintained state; consequently, 32~-labelled pRB should be representative of protein synthesized at all times and not just newly syn-
~
BIOCHEM. CELL BIOL. VOL. 70, 1992
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Days of TPA treatment
FIG. 2. TPA-induced dephosphorylation of pRB. HL60 cells were labelled with jE~]phasphate for 2 h, lysed. immuno-
precipitated with either the C36 anti-pRB monoclonal antibody or a control antibody, and then run on a 7.5% SSD-polyacrylamide gel. CelIs were treated with TPA For the indicated number of days prior to lysis.
thesized protein (Chen et al. 1989). Together, the results from the 3 5 ~ and 32~-labelling experiments (Figs. 1 and 2) indicate that the level of phosphorylation of pRB is substantially reduced after TPA treatment. The above results are consistent with a pathway stimulated by protein kinase C resulting in dephosphoryIation of pRB. To determine if other agents that cause cell cycle arrest and (or) terminal differentiation affect the phosphorylation state of pRB, HL60 cells were treated with several compounds for 48 h, and then they Iabelled with 3 5 and ~ pRB was irnmunoprecipitated (Fig. 3). In addition to TPA, dibutyry1 CAMP, a stimulator of cAMPdependent kinase, also appeared to cause a decrease in pRB phosphorylation. Following the addition of dibutyryl cAMP to the culture medium, the cells underwent cell cycle arrest suggesting that, like TPA, dibutyryl CAMP can stimulate a pathway eventually leading to both activation of pRB and growth arrest. Although these cells underwent cell cycle arrest they did not appear to morphologically differentiate. Another compound tested, retinoic acid, is known to induce HL60 cells to differentiate into granulocytes after 7-10 days; however, after 48 h of treatment no changes in the phosphorylation state of pRB was observed (Fig. 3). It is possible that differentiation and cell cycle arrest by retinoic acid occurs through a mechanism distinct from TPA and does not directly influence pRB phosphorylation because, in contrast to TPA and cAMP analogues, retinoic acid is not known to directly affect phosphorylation pathways. After longer periods of treatment with retinoic acid pRB migrated as a single electrophoretic species, suggesting that loss of phosphoryIation of pRB eventually occurs, but in this case it may be only a consequence of cell cycle arrest (data not shown).A similar conclusion was reached by another group examining retinoic acid induced differentiation of HL60 cells (Mihara et al. 1989).
FTG. 3. Loss of phosphorylated forms of pRB after stimulation of cells with various compounds. HL60 cells were treated with the indicated compounds for 48 h, labelled with ["~]methionine, lysed, and immunoprecipitated with either the C36 monoclonal antibody (Rb) or a control antibody (C). Immunoprecipitated proteins were run on a 7.5% SDS-polyacrylamide gel.
Discussion The results presented in this report raise the possibility that pRB, a tumour suppressor protein, plays a role in one or more pathways leading to cell cycle arrest. HL60 cells exposed to either the phorbol ester TPA or dibutyryl CAMP underwent cell cycle arrest and a concommitant decrease in the phosphorylation of pRB. As outlined above, phosphorylation of pRB is believed to negatively regulate the activity of pRB. Thus, the loss of pRB phosphorylation observed in our experiments is consistent with a function for pRB in arresting cells in a Go state. These results are also consistent with the results of several other groups who have shown that in differentiated or senescent cells pRB remains in an underphosphorylated state (Chen et al. 1989; Mihara et al. 1989;Akiyama and Toyoshima 1990;Stein et al. 1990). TPA-mediated changes in the phosphorylation state of pRB are suggestive of a pathway mediated by protein kinase C. This protein kinase is frequently an intermediate in pathways stimulated by extracellular ligands, especially those mediated by tyrosine kinase receptors (Nishizuka 1986). A model in which pRB lies on a pathway stimulated by an extracellular factor is presented in-Fig. 4. In this model, protein kinase C mediates the activation of pRB
SIGNAL PROTEIN KINASE C
1 ACTIVATION OF PRB 1 CELL CYCLE ARREST
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TPA NONRESPONSIVE LINES
E l A, T antigens, E7
FIG.4. Model of RB in a signal transduction pathway resulting in cell cycle arrest. A schematic diagram of events involving the retinoblastoma protein and its regulation via signal transduction. At the right are positions in the pathway where certain cell lines are blocked and where viral transforming proteins function to block the pathway.
through an unknown mechanism, resulting in a decrease in the overall phosphorylation of pRB and eventually leading to cell cycle arrest. A signal transduction pathway would contain multiple points where potentially oncogenic events could disrupt the pathway. Some known oncogenic events are consistent with disruption of this putative pathway at several positions. One is the mutation or disruption of the pRB gene as occurs in retinoblastomas and various other tumours (reviewed in Benedict et al. 1990). Transforming proteins from DNA tumour viruses such as the adenovirus E1A protein, papovavirus T antigens, and papillomavirus E7 proteins may also block the pathway through their interactions with pRB. In HL60 cells, a pathway resulting in the loss of phosphorylation of pRB can be activated by TPA. Similar results have been found for several other cell lines capable of differentiating in response to TPA (Chen et at. 1989; Akiyama and Toyoshima 1990; P. Whyte and R.N. Eisenman, unpublished data). One possibility is that normal regulation of these cells may have been disrupted by mutations in a signal transduction pathway upstream of protein kinase C. Another part of the pathway that could be disrupted in transformed cells lies between protein kinase C and the dephosphorylation of pRB. Many transformed cell lines do not differentiate in response to TPA. In these cells, it is possible that other oncogenic events have disrupted the pathway downstream of protein kinase C, rendering the cells nonresponsive to TPA. Characterization of the mechanism through which pRB becomes dephosphorylated may allow tumour-derived cell lines to be examined for potentially oncogenic events affecting this pathway. It is possible that more than one signal transduction pathway leads to the activation of pRB. Addition of dibutyryl CAMP to cell culture of HL60 cells resulted in a loss of phosphorylation of pRB and cell cycle arrest. This may be representative of a second pathway influencing the phosphorylation state of pRB and others may also exist. pRB may be representative of a family of related proteins, including other cellular proteins that interact with the same region of E1A as does pRB. This is supported by the recent
cloning of a cDNA for p107, a cellular protein that binds to the pRB binding domain of E1A and SV40 T antigen (Egan et al. 1988; Whyte et al. 1989; Dyson et al. 1989a; Ewen et al. 1989, 1991). pRB and p107 share regions of homology and appear to be members of the same family of proteins. It will be of interest to see if phosphorylation of p107 is affected during differentiation. Acknowledgements P.W. was supported by a Damon Runyon-Walter Winchell Cancer Research Fund Fellowship (DRG-973). This work was supported by National Institutes of Health grant R01 CA20525 awarded to R.N.E. Akiyama, T.. and Toyoshima, K. 1990. Marked alteration in phosphorylarion of the RB protein during differentiation of human promyelocytic HLbO cells. Oncogene, 5: 179-183. Bagchi, S . , Weinmann, R., and Raychaudhuri, P. 1991. The rednobIastoma protein copurifies with E2F-1, an E l A-regulated inhibitor of transcription Factor E2F. Cell, 65: 1063-1072. Bandara, L . R . , and La Thangue, N.R. 1991. Adenovirus EIA prevents the retinoblastoma gene product from complexing with acellular transcription factor. Nature (London), 351: 494-497. Benedict, W.F., Xu, H.-J., Hu, S.-X., and Takahashi, R. 1990. Role of the retinoblastoma gene in the initiation and progression of human cancer. J. Clin. Invest. 85: 988-993. Bishop, J.M. 1991. Molecular themes in oncogenesis. Cell, 64: 235-248. Buchkovich, K., Duffy, L.A., and Harlow, E. 1989. The retinoblastoma protein is phosphorylated during specific phases of the cell cycle. Cell, 58: 1097-1105. Cantley, L.C., Auger, K.R., Carpenter, C., Duckworth, B., Graziani, A., Kapeller, R., and Soltoff, S. 1991. Oncogenes and signal transduction. Cell, 64: 281-302. Chellappan, S.P., Hiebert, S., Mudryj, M., Horowitz, J.M., and Nevins, J.R. 1991. The E2F transcription factor is a target for the RB protein. Cell, 65: 1053-1061. Chen, P.-L., Scully, P., Shew, J.-y., Wang, J.Y.J., and Lee, W.-H. 1989. Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation. Cell, 58: 1193-1 198. Chittendan, T., Livingston, D.M., and Kaelin, W.G., Jr. 1991. The T/EIA binding domain of the retinoblastoma product can interact selectively with the a sequence specific DNA-binding protein. Cell, 65: 1073-1082. DeCaprio, J.A., Ludlow, J.W.. Figge, J., Shew,J.-Y.,Huang, C.-M., Lee,W.-H., Marsilio, E.,Paucha, E., and Livingston. D.M. 1988. SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell, 54: 275-283. DeCaprio, J.A., Ludlow, J.W., Lynch, D., Furakawa, Y., Griffin, J., Piwinica-Worms, H.,Huang, C.-M., and Livingston, D.M. 1989. The product of the retinoblastoma susceptibility gene has propmies of a cell cycIe regulatory element. Cell, 58: 3085-1095. Dyson, N., Buchkovich, K.J.,Whyte, P., and Harlow, E, 1989a. The cellular 107K protein that binds to adenovirus ELA also associates with large T antigens of SV40 and J C virus. Cell, 58: 249-255. Dyson, N., Howley, P.M., Munger, K., and Harlow, E. 19896. The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science (Washington, D.C.), 243: 934-937. Egan, C., Jelsma, T.N., Howe, J.A., Bayley, S.T., Ferguson, B., and Branton, P.E. 1988. Mapping of cellular protein-binding sites on the products of early-region 1A of human adenovirus type 5. Mol. Cell. Biol. 8: 3955-3959. Ewen, M.E., Ludlow, J.W., Marsilio, E., DeCaprio, J.A., Millikan, R.C., Chen, S.H., Paucha, E., and Livingston, D.M.
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