Expression and role of PML gene in normal adult hematopoiesis: functional interaction between PML and Rb proteins in erythropoiesis C Labbaye 1 , M Valtieri 1 , F Grignani 3 , R Puglisi 1 , L Luchetti 2 , B Masella 2 , M Alcalay 4 , U Testa 1 and C Peschle* ,1,2 1 Department of Hematology and Oncology, Istituto Superiore di Sanita Á, Rome, Italy; 2 Thomas Jeerson University, Kimmel Cancer Center, Philadelphia, Pennsylvania, USA; 3 Department of Clinical Medicine, University of Perugia, Perugia, Italy; 4 Department of Experimental Oncology, European Institute of Oncology, Milan, Italy The expression of the PML gene was investigated in puri®ed early hematopoietic progenitor cells (HPCs) induced to unilineage erythroid or granulocytic dier- entiation. PML mRNA and protein, while barely detectable in quiescent HPCs, are consistently induced by growth factor stimulation through the erythroid or granulocytic lineage. Thereafter, PML is downmodu- lated in late granulocytic maturation, whereas it is sustainably expressed through the erythroid pathway. In functional studies, PML expression was inhibited by addition of antisense oligomers targeting PML mRNA (a-PML). Interestingly, early treatment (day 0 HPCs) with a-PML reduced the number of both erythroid and granulocytic colonies, whereas late treatment (day 5 culture) reduced erythroid, but not granulocytic, clono- genesis. These ®ndings suggest that PML is required for early hematopoiesis and erythroid, but not granulocytic maturation. The pattern of PML expression in normal hematopoiesis mimics that of retinoblastoma pRb 105. Combined treatment of HPCs with a-PML and a-Rb oligomers inhibited both PML and Rb protein expression and completely blocked erythroid colony development. Furthermore, PML and pRb 105 were co-immunopreci- pitated in cellular lysates derived from erythroid precursors indicating that this functional interaction may have a biochemical basis. These results suggest a key functional role of PML in early hematopoiesis and late erythropoiesis: the latter phenomenon may be related to the molecular and functional interaction of PML with pRb 105. Keywords: HPCs; PML; RB; erythropoiesis Introduction Hematopoiesis involves the coordinate activation of genetic programs that control the processes of lineage commitment and maturation (Shivdasani and Orkin, 1996). The genes that take part in chromosome rearrange- ments of human acute leukemias are most likely involved in the control of dierentiation and prolifera- tion. Many of these genes are important regulators of normal hematopoiesis (Sawyers et al., 1991; Rabbitts 1991). The t(15;17) chromosomal translocation, found at molecular level in over 95% of patients with acute promyelocytic leukemia (APL), involves the retinoic acid receptor a (RAR a) gene on chromosome 17 and the PML gene on chromosome 15, generating the PML/ RARa fusion gene that encodes fusion proteins (Warrell et al., 1993; Grignani et al., 1994). The PML/RARa protein plays a major role in the pathogenesis of APL (Alcalay et al., 1992). Expression of PML/RARa in myeloid cell lines blocks dierentiation and decreases sensitivity to apoptotic cell death (Grignani et al., 1993, 1995; Rousselot et al., 1994). In addition, PML/RARa transgenic mice develop an APL-like leukemia (Grisolano et al., 1997; He et al., 1997b; Brown et al., 1997). The PML protein-dimerization and RARa DNA binding domains must interact to ensure the biological activity of PML/RARa (Perez et al., 1993; Grignani et al., 1996). This ®nding suggests that the biological eects of the fusion protein depend on the alteration of both RARa and PML function, the latter possibly mediated by protein-protein interactions (Grignani et al., 1996). A role in normal hematopoietic differentia- tion has been established for RARa (Kastner et al., 1992); however, little is known about the physiological function of PML. Ablation of murine PML protein by homologous recombination revealed a possible role for PML in the control of cell proliferation and differentia- tion of hematopoietic precursors, possibly mediated via interaction with the RA pathway (Wang et al., 1998). Therefore, the study of PML protein expression and role in normal hematopoiesis is relevant for the comprehension of both the pathogenesis of APL and the physiology of hematopoiesis. PML is a phosphoprotein associated with the nuclear matrix and expressed in most tissues (Daniel et al., 1993; Chang et al., 1995). It belongs to a large family of proteins with common structural features, including a RING motif, two additional Cys/His-rich regions, named B-boxes and a coiled coil region, through which PML is able to form homodimers and, in APL cells, heterodimers with PML/RAR (Kastner et al., 1992; Goddard et al., 1991; Reddy et al., 1992; Borden et al., 1995, 1996). Within the nucleus, the PML protein is mainly localized in speci®c subdomains, the PML nuclear bodies (PML- NBs) (Dyck et al., 1994). In APL cells the PML-NBs are disrupted and PML colocalizes with PML/RARa in novel structures called microspeckles (Dyck et al., 1994; Koken et al., 1994; Weis et al., 1994). The delocalization of PML by PML-RARa, is associated with cellular transformation and can be reversed by treatment of cells with RA, that induces reaggregation *Correspondence: C Peschle, T Jeerson University, Kimmel Center, Bluemle Life Science Building, 233 South 10th Street, Philadelphia, PA 19107-5541, USA Received 26 May 1998; revised 5 November 1998; accepted 13 January 1999 Oncogene (1999) 18, 3529 ± 3540 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc