The Tumor Suppressor Gene p 53 Can Mediate Transforming The Growth Factor 31-induced Differentiation of Leukemic Cells Independently of Activation of the Retinoblastoma Protein 1

Although the involvement of the tumor suppressor gene p53 in normal hematopoiesis is uncertain, it can give rise to differentiation signals in leukemic cells. It is not clear, however, whether differentiation merely is a consequence of the ability of p53 to arrest cell proliferation or whether hitherto unknown molecular mechanisms are responsible for the p53-mediated differentiation. To further explore the role of p53 in leukemic cell differentiation, we investigated whether transforming growth factor flu (TGF-131), a cytokine involved in cell cycle control at several levels, can cooperate with wild-type p53 to induce differentiation of monoblastic U-937 and erythroleukemic K562 cells. Indeed, wild-type p53-expressing cells were found to be more sensitive to TGF31 -induced differentiation than control cells, lending support to the idea that p53 is of importance for differentiation induction of leukemic cells. In addition, it is shown that TGFI can suppress p53-mediated cell death, thus reinforcing the differentiation response. The cyclin-dependent kinase inhibitor p21 and the retinoblastoma protein (pRb) are downstream effectors of p53-mediated growth arrest. Therefore, the roles for these molecules in p53mediated differentiation were examined. The p53dependent signals of differentiation were associated with induction of p21 in both cell lines investigated. However, activation of pRb by induced hypophosphorylation and concomitant decreased growth rate on p53-mediated differentiation was observed only in U-937 cells expressing an inducible, temperature-sensitive form of p53 but not in K562 cells constitutively expressing p53. Thus, our data suggest a Received 5/15/97; revised 7/23/97; accepted 8/20/97. 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 mdicate this fact. 1 Supported by the Swedish Cancer Society; the Swedish Society for Pediatric Cancers; the Georg Danielsson Foundation; the Gunnar, Arvid and Elisabeth Nilsson Foundation; the Thelma zoega Foundation; the Tobias Foundation; and the John and Augusta Persson Foundation. 2 To whom requests for reprints should be addressed, at Department of Hematology, Research Department 2, E-block, University Hospital, S-221 85 Lund, Sweden. Phone: 4646/1 73550; Fax: 4646/184493. role for p53 in the regulation of differentiation in leukemic cells that can be independent of its ability to activate pRb and arrest cell proliferation. Introduction The tumor suppressor gene p53 is dearranged in a wide variety of human tumors, suggesting an important role for maintaining the benign phenotype (1). In leukemias, however, deletions or mutations of the p53 gene are comparatively rare and have mostly been associated with worsening or relapsing of the disease, such as blast crisis of chronic myeloid leukemia (2-i 0). It is possible to restore a capacity for differentiation by transgenic expression of p53 in leukemic cell lines, many of which lack p53 (11 , 12). For instance, ectopic expression of wild-type p53 in Ieukemic Li 2 pre-Bcells, erythroleukemic K562 cells, promyelocytic HL-60 cells, or Friend virus-transformed erythroleukemic cells induces signs of differentiation (i 3-i 7). Moreover, K562 or monoblastic U-937 cells engineered to express wild-type p53 have been shown to be more sensitive to induction of differentiation by tumor necrosis factor and i ,25-dihydroxycholecalciferol (Vit D3),3 respectively, than control cells lacking p53 (15, 18). Notably, by interference with wild-type p53 activity in leukemic 32D cells, it is possible to abrogate induced differentiation in response to granulocyte colony-stimulating factor (1 9). Taken together, these findings indicate that wildtype p53 is of importance for induced differentiation of leukemic cells. How these p53-dependent differentiation signals are mediated is unknown. Wild-type p53 can transactivate the gene for the CDK inhibitor p21 , an important regulator of the cell cycle (20, 2i). p2i inhibits the activity of the CDK/cyclin complexes implicated in phosphorylation and inactivation of the retinoblastoma protein, pRb (21-23). Active, hypophosphorylated pRb keeps the cell in the G1 phase of the cell cycle by sequestering E2F-1 and related proteins, a family of transcription factors necessary for progression from G1 to S phase (24). The importance of p21 as a regulator of the G1-S transition is underscored by the fact that p21-deficient cells have an impaired ability to arrest in G1 (25). Besides regulating the cell cycle, p21 is likely to be of importance for differentiation in several tissues (26-28). In certain leukemic cells, induction of p21 correlates with induction of differentiation in a p53-independent manner (283 The abbreviations used are: ‘lit D3, vitamin D3; CDK, cyclin-dependent kinase; TGF, transforming growth factor; NBT, nitro blue tetrazolium; PMA, phorbol 1 2-myristate 13-acetate; CMV, cytomegalovirus; FBS, fetal bovine serum.