Modulation of Retinoblastoma Gene in Normal Adult Hematopoiesis: Peak Expression and Functional Role in Advanced Erythroid Differentiation G

Proc. Natl. Acad. Sci. USA Vol. 92, pp. 4808-4812, May 1995 Cell Biology

Modulation of retinoblastoma gene in normal adult hematopoiesis: Peak expression and functional role in advanced erythroid differentiation G. L. CONDORELLI*t, U. TESTAt, M. VALTIERIt, L. VITELLIt, A. DE LUCA*, T. BARBERI4, E. MONTESORO4, S. CAMPISI4, A. GIORDANO*t, AND C. PESCHLE*t *Thomas Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107-5541;and tDepartment of Hematology-Oncology, Istituto Superiore di Sanita, Rome, Italy Communicated by Sidney Weinhouse, Thomas Jefferson University, Philadelphia, PA, January 11, 1995 (received for review December 14, 1994)

ABSTRACT The retinoblastoma (RB) gene specifies a pRb is un- or hypophosphorylated through the early G1 stage, nuclear phosphoprotein (pRb 105), which is a prototype reaches different levels of hyperphosphorylation from late G1 tumor suppressor inactivated in a variety of human tumors. through mitosis, and is again hypophosphorylated after the Recent studies suggest that RB is also involved in embryonic end of mitosis. The phosphorylation state of pRb correlates development of murine central nervous and hematopoietic with its growth-suppressive potential, the hypophosphorylated systems. We have investigated RB expression and function in form being the growth suppressive one; introduction of un- human adult hematopoiesis-i.e., in liquid suspension culture phosphorylated pRb into cells inhibits cell cycle progression of purified quiescent hematopoietic progenitor cells (HPCs) (9) and reverts the tumor phenotype in RB- cells (10). induced by growth factor stimulus to proliferation and uni- Furthermore, the unphosphorylated form of pRb binds onco- lineage differentiation/maturation through the erythroid or proteins of DNA tumor viruses-e.g., adenovirus ElA, simian granulocytic lineage. In the initial HPC differentiation stages, virus 40 large T antigen, and human papillomavirus E6 the RB gene is gradually induced at the mRNA and protein (11-13). The ability of these oncoproteins to immortalize level in both erythroid and granulopoietic cultures. In late mammalian cells correlates with their capacity to complexwith HPC differentiation and then precursor maturation, RB gene unphosphorylated pRb, thus suggesting that their growth- expression is sustained in the erythroid lineage, whereas it is promoting activity is mediated by pRb inactivation. In human sharply downmodulated in the granulocytic series. Functional cells, pRb binds to a number of nuclear proteins (14), including studies were performed by treatment of HPC differentiation the transcription factor E2F (15, 16); upon pRb hyperphos- culture with phosphorothioate antisense oligomer targeting phorylation, E2F is released and transactivates a variety of cell Rb mRNA; coherent with the expression pattern, oligomer cycle-related genes (17, 18). treatment of late HPCs causes a dose-dependent and selective Recent evidence strongly suggests a role for pRb in onto- inhibition of erythroid colony formation. These observations genetic development of the hematopoietic system. Mice ren- suggest that the RB gene plays an erythroid- and stage- dered RB by gene targeting of embryonic stem cells die in specific functional role in normal adult hematopoiesis, par- earlygestation because ofgross defects ofboth central nervous ticularly at the level of late erythroid HPCs. and hematopoietic systems (19-21). The latter abnormalities involve reduced formation of hepatic blood islands coupled Hematopoiesis is a multistep cell proliferation and differen- with a marked increase of immature nucleated erythroid cells; tiation process, which is sustained by a pool of hematopoietic in line with these observations, in vitro clonogenetic studies on stem cells (HSCs) (1-3). HSCs are capable of extensive embryonic liver RB- cells showed a normal number of late self-renewal and feed into lineage-committed hematopoietic erythroid HPCs (CFU-E), which are hampered in their dif- progenitor cells (HPCs). The HPCs are functionally defined as ferentiation/maturation capacity (19-21). However, these ob- colony- or burst-forming units (CFUs, BFUs)-i.e., HPCs of servations do not provide insight into the role of RB in adult the erythroid (BFU-E, CFU-E), granulomonocytic (CFU- hematopoiesis, which is still unknown. GM, CFU-G, CFU-M) lineage and multipotent HPCs for We have investigated RB gene expression and function in erythroid, granulomonocytic, and megakaryocytic series human adult HPCs induced to proliferate, differentiate, and (CFU-GEMM). Early HPCs (BFU-E, CFU-GM, CFU- mature through terminal stages along the erythroid or granu- GEMM) are stimulated by multilineage hematopoietic growth lopoietic lineage. factors HGFs-i.e., interleukin 3 (IL-3), GM colony- stimulating factor (GM-CSF)-while late HPCs (CFU-E, MATERIALS AND METHODS CFU-G, CFU-M) are stimulated by unilineage HGFs [erythropoietin (Ep), G-CSF, and M-CSF, respectively]. HPCs dif- Hematopoietic Growth Factors and Cell Culture Medium. ferentiate into morphologically recognizable precursors that Recombinant human growth factors were obtained from stan- mature to terminal elements circulating in peripheral blood dard commercial sources (22-24). Iscove's medium (IMDM; (PB). Early-late HPCs are also present in PB (3, 4). GIBCO) was freshly prepared on a weekly basis. The retinoblastoma (RB) gene is a tumor suppressor gene; inactivation ofboth RB loci results in retinoblastoma and other Abbreviations: HSC, hematopoietic stem cell; HPC, hematopoietic progenitor cell; BFU-E, erythroid burst-forming unit; CFU-E, ery- adult tumors (5). The product of the RB gene is a nuclear throid colony-forming unit; CFU-GM, granulomonocytic CFU; CFU- phosphoprotein (pRb; 105 kDa) that is ubiquitously expressed GEMM, erythroid, granulomonocytic, and megakaryocytic CFU; in vertebrates (6). The functional properties of pRb relate to HGF, hematopoietic growth factor; IL-3, interleukin 3; GM-CSF, GM its phosphorylation state, which is cell cycle dependent (7, 8). colony-stimulating factor; Ep, erythropoietin; PB, peripheral blood; mAb, monoclonal antibody; RT, reverse transcriptase; ca-Rb, antisense retinoblastoma. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Thomas Jefferson payment. This article must therefore be hereby marked "advertisement" in Cancer Institute, Bluemle Life Sciences Building, Room 528, Locust accordance with 18 U.S.C. §1734 solely to indicate this fact. and 10th Street, Philadelphia, PA 19107-5541. 4808 Cell Biology: Condorelli et at Proc. Natl. Acad Sci. USA 92 (1995) 4809 G-CSF (10 ng) as described (24). Cells were periodically counted and analyzed for morphology, membrane phenotype, L.iJ and RB gene expression. 107 Immunofluorescence and Western Blot Analysis. Indirect immunofluorescence was performed as described (24) with the XZ55 monoclonal antibody (mAb) (26), which was selected a 106 after testing on Rb+ U20S and Rb- SAOS-2 cells. Nuclear extracts, prepared according to ref. 27, were normalized for E cell number and loaded onto a 7% polyacrylamide gel. A mixture of XZ55, -56, and -77 anti-Rb mAbs (26) was used to probe the blotted membrane for pRb. Bands were visualized 10 by enhanced chemiluminescence (Amersham). Reverse Transcriptase RT-PCR mRNA Analysis. Method- 0 5 10 15 20 0 5 10 15 20 ology for semiquantitative RT-PCR analysis has been reported (23, 24, 28-30). cDNAs were normalized by the p32-mi- *a BLASTS.MYELOBLASTS croglobulin gene (29). RT-RNA from CEM cell line was used -o- * BLASTS + PROERYTH. PROMYELOCYTES as an internal positive control. Rb primers and probe were as o BASOPHIUC ERYTH. -a-- MYELOCYTES a POLYCHROM. ERYTH. METAMYELOCYTES follows: 5' primer, 5'-GCTATGTCAAGACTGTTG-3'; 3' i * ORTHOC. ERYTH. I& BAND GRANULOCYTES primer, 5'-TTGAGCAACATGGGAGGT-3'; internal probe, -a*-MONOCYTES 5'-TTGGTGCTAAAAGTTTCTTGGATC-3' (6). The am- 100 plification procedure included denaturation at 95°C for 30 sec, annealing at 54°C for 30 sec, and extension at 72°C during 40 80 PCR cycles-i.e., within the range of linear amplification. Meth- odology for c-myb mRNA RT-PCR has been reported (31). 60 HPC Oligomer Treatment and Clonogenetic Assay. Treat- 40 ment was as described (24, 29-31). Oligomers. The following phosphorothioate oligodeoxynucle- 20 otides were used: antisense Rb (a-Rb), 5'-GTGAACGA- CATCTCATCTAGG-3' complementary to 21 nucleotides start- 0l I ing from 465- to 485-bp Rb mRNA coding sequence (6); scrambled a-Rb, 5'-TACTGGCJAAGCCTAGCATGA-3'. 0 5 10 15 20 0 5 10 15 20 Oligomer treatment. Early HPC treatment: Step IIIP cells (5 Days Dcws X 103 cells per ml) in standard erythroid or granulopoietic liquid-phase culture (see above) were supplemented with (i) FIG. 1. Liquid-phase culture of step IIIP HPCs undergoing eryth- the standard erythroid- or granulopoietic-specific HGF com- ropoietic (E) or granulopoietic (G) differentiation and maturation bination (see above) and (ii) a-Rb or scrambled a-Rb (25, 50, induced by appropriate HGF stimuli, as evaluated in terms of cell or 100 ,ug/ml). In some experiments, the erythroid or granu- number (Upper) and morphology (Lower). Mean ± SEM values from lopoietic liquid-phase culture was supplemented with saturat- at least seven independent experiments are shown. ing HGF stimulus (i.e., IL-3 at 100 units/ml and GM-CSF at Adult PB HPC Purification and 10 ng with or without 3 units of Ep) and a-Rb or scrambled Clonogenetic Assay. HPCs a-Rb (25, 50, or 100 ,ug). Cells were were purified from the PB incubated from day 0 buffy coat according to the method through day 1; mock-treated controls were included. Cells in ref. as 22 modified described (23, 24). Purified HPC were then plated in FCS- clonogenetic culture (see above) clonogenetic cultures with or without fetal calf serum (FCS+ supplemented with the same HGF combination used in the or FCS-) were prepared as described (23-25). liquid-phase culture. Late HPC treatment: Step IIIP cells were HPC Erythroid and Granulopoietic Liquid Suspension diluted in standard liquid-phase erythroid and granulopoietic Culture. Step IIIP HPCs were seeded (5 x 104 cells per ml) and cultures. At day 6 a-Rb or scrambled a-Rb oligomer (25, 50, grown in liquid FCS- medium (see ref. 25) supplemented with or 100 ,ug) was added; mock-treated controls were included. HGFs-i.e., in erythropoietic culture, very low doses of IL-3 After 24-48 h of incubation, cells from erythroid or granulo- (0.01 unit/ml) and GM-CSF (0.001 ng) and a saturating level poietic culture were plated in FCS- clonogenetic culture with of Ep (3 units); in granulopoietic culture, low amounts of IL-3 either the erythroid- or granulopoietic-specific HGF combi- (1 unit) and GM-CSF (0.1 ng) and saturating amounts of nation or the erythroid or granulopoietic saturating HGF

G w~~~~~~~~~~~~~~~0 0I 0 Lu a .LLI a 0 1 3 5 7 10 1214 17z Day 00u 3 5 710121417 z

Rb NE__-s*269bp c-myb _ ___;230bp

f2m __IP-amM a IS..I4 4- 253 bp

FIG. 2. Rb and c-myb mRNA expression in purified HPCs and their progeny grown in liquid-phase erythroid (E) or granulopoietic (G) culture, as evaluated by semiquantitative RT-PCR. 132-Microglobulin (32m) was used for normalization. Human T lymphoblastic cell line CEM was the internal positive control. A negative control (N. cont.) and CD34- control cells are also included. One representative experiment of three independent experiments is shown. Rb and c-myb filters were autoradiographed for 48 h and 132m filters were autoradiographed for 3 h. N.E., not evaluated. 4810 Cell Biology: Condorelli et al Proc. Natl. Acad ScL USA 92 (1995) In FCS- the A Day 0 liquid suspension culture, purified HPCs, L-- triggered into cycling by HGFs, undergo proliferation and gradual differentiation along either the erythroid -pathway (upon addition of very low amounts of IL-3 and GM-CSF and Day 2 a saturating level of Ep) or the granulocytic lineage (upon treatment with saturating amounts of IL-3 + GM-CSF) (28). However, two drawbacks are inherent in the HPC granulopoietic differentiation system. Thus, (i) at advanced matura- Day 6 tion stages .10% contaminant monocytes are present, while granulopoietic cells are mostly eosinophilic, and (ii) the saturating IL-3 + GM-CSF dosage triggers HPCs into rapid Day 8 proliferation, in contrast to the slower cycling induction of HPCs in the erythroid differentiation culture. To avoid these limitations, we have developed a granulopoietic culture system Day 12 based on low-dose IL-3 + GM-CSF and saturating G-CSF stimulus (ref. 24; see Fig. 1). In the first week of culture, the HPCs are gradually induced into cycling; amplification of their

B number is associated with progressive differentiation along the 100 granulopoietic pathway, as shown by the decrease of (i) the size of granulocytic colonies generated by HPCs, (ii) CD34 antigen expression, and (iii) percent blast number (Fig. 1; data not 60 shown; see also ref. 24). In the second week, the differentiated 40 granulopoietic precursors show increasing expression of lin- a eage-specific markers (e.g., coupled with a converse n CD15), Q drop of CD34+ cell frequency to undetectable levels (data not shown); morphology analysis shows a gradual wave of differentiation along the granulopoietic pathway to terminal differ- 0 2 5 7 9 12 entiation (Fig. 1 Lower Right). The maturing cells exhibit Days normal morphology and consist of .98-99% granulopoietic precursors (>90% of neutrophilic type) with s1-2% mono- C _ ppRb cytic cells. Comparative results on erythroid differentiation 105 M.W.St.- culture of the same HPC populations are shown in Fig. 1 Left (see also ref. 28). .o 100- Expression of Rb mRNA in Erythroid or Granulopoietic CD 80- HPC Cultures. RNA samples were generated from liquid

.U 60 suspension culture at sequential days (representative results , 40t -3 are shown in Fig. 2). Rb mRNA, barely expressed in the E starting quiescent HPCs, is rapidly induced and expressed 20]L through the first culture week in both differentiation systems. In the second week, we observed that (i) in the erythroid Day 0 6E 12E 6G 12G pathway high-level Rb mRNA expression is sustained through terminal maturation; (ii) in the granulopoietic pathway initial FIG. 3. (A) Immunofluorescence labeling with anti-Rb mAb of Rb mRNA expression is downregulated at the stage of early purified HPCs and their progeny grown in liquid-phase erythroid (E) granulopoietic precursors (days 8-10 of culture; see Fig. 1 or granulopoietic (G) culture, as evaluated at sequential days. Fluo- Lower) and is apparently abolished through terminal matura- rescent cells were examined under an Axiophot Zeiss microscope. tion (days 14 and 17). As a control, c-myb mRNA is induced (x630.) (B) pRb+ cells, as determined by immunofluorescence label- upon HGF stimulus and expressed through advanced ery- ing in HPC erythroid (E) or granulopoietic (G) culture. Mean ± SEM throid and granulocytic maturation (Fig. values of pRb+ cells from four separate experiments are shown. (C) 2). Western blot analysis of pRb in nuclear extracts of HPCs freshly Expression of pRb in Differentiating HPC Culture. In purified (day 0) or grown for 6 or 12 days in erythroid (E) or parallel we evaluated (i) pRb expression by indirect immuno- granulopoietic (G) culture. (Upper) Representative autoradiogram. fluorescence analysis and (ii) pRb expression and phosphor- Molecular weight standard (Mr 105,000) is indicated on the left. pRb, ylation level by Western blot. Immunofluorescence analysis: Hypophosphorylated Rb protein; ppRb, hyperphosphorylated Rb Day 0 quiescent HPCs do not exhibit detectable pRb (Fig. 3A). protein. (Lower) Densitometric analysis of Western blot autoradio- The initial differentiation of HPCs along the erythroid or gram. granulopoietic pathway is associated with the induction of pRb. In the second culture week, pRb expression is still stimulus (see above). Fluoresceinated oligomer uptake was elevated in differentiating erythroblasts but abruptly declines evaluated as described (29). and almost completely disappears in granulopoietic precursors (Fig. 3A andB). Western blot analysis (Fig. 3C): In day 0 HPCs RESULTS pRb is expressed at a low level in its fast-migrating, hypophosphorylated state. At day 6, induction of HPC proliferation and HPC Purification and Unilineage Differentiation/Matura- differentiation induces a sharp increase of pRb levels, coupled tion Culture. The step IIIP cell population obtained by the with the appearance of the hyperphosphorylated, slow- modified purification procedure (23, 24) is characterized by migrating form(s) ofpRb in both erythroid and granulopoietic 90% CD34+ cells and 80% HPC frequency coupled with 45% cultures. At day 12, the pRb level is still elevated in erythro- HPC recovery (in 12 separate purification experiments, we blasts, whereas it is sharply downmodulated in granulopoietic obtained 90.6% ± 1.2% CD34+ cells, 81.3% ± 1.2% HPCs precursors. including BFU-E + CFU-GM + CFU-GEMM, and 44.9% Effect of a-Rb Oligonucleotide in HPC Differentiation 3.8% HPC recovery as related to the HPC number in the Ficoll Culture. In a first set of experiments, purified HPCs were fraction). seeded in liquid-phase erythroid or granulopoietic culture; Cell Biology: Condorelli et aL Proc. Natt Acad Sci USA 92 (1995) 4811

50jig lO0 gg Rb l _ _w - 269 bp v 100- -" 100 - 0 _2m - 253 bp u o 80- 0 80 - CD0 C S at S at __ 60 60 - 0 6 z Z 40 >. 40 - c O 20- Darn~~~~~~~~~~~~- O 20- u O 0- Control 25 ig 50 100 25 gg 50 100 Control 100 jg 100 jg Scrambled ct-Rb Scrambled a-Rb FIG. 4. Treatment of HPC erythroid or granulopoietic differentiation liquid culture with a-Rb or scrambled a-Rb oligomer (25, 50, or 100 ,ug/ml) at days 6-8. Effect on HPC erythroid (E) or granulocytic (G) colony formation in semisolid culture induced by erythroid- or granulopoietic-specific HGF stimulus, as applied in liquid-phase culture, is shown. Mock-treated controls are also presented. Mean SEM values from three independent experiments, each performed in triplicate dishes, are shown. *, P < 0.05; **, P < 0.01 when compared to corresponding "scrambled" group. (Inset) Representative RT-PCR experiment showing the effect of 50- and 100-,ug dosages of either a-Rb (a) or scrambled a-Rb (S) oligomers on Rb and 132-microglobulin (132m) mRNA expression; mock-treated controls (C) are also presented. alternatively, they were seeded in the same liquid-phase me- G-CSF, respectively) (ref. 24 and the present results). These dium supplemented with saturating levels of IL-3 and GM- culture systems allow sequential collection and molecular CSF with or without Ep. All cultures were treated with a-Rb analysis of discrete subsets of HPCs and hematopoietic pre- or scrambled a-Rb oligomer from day 0 through day 1. HPCs cursors at a homogenous stage of differentiation/maturation were then plated in semisolid culture supplemented with the along a particular lineage (23, 24, 28, 29). same HGF combination used in the corresponding liquid In HPC erythroid and granulopoietic cultures the RB gene culture. Addition of a-Rb had no influence on erythroid is modulated at both mRNA and protein levels. In the freshly colony formation and only a slight, barely significant effect on purified, largely quiescent HPCs Rb mRNA and protein is the number of CFU-GM colonies upon saturating IL-3 and detected at a low level by RT-PCR and Western blot (the lack GM-CSF treatment (data not shown). In a second set of ofpRb by immunofluorescence may be attributed to the higher experiments purified HPCs were first grown for 6 days in liquid threshold of detection). After HGF-induced HPC prolifera- suspension culture under either erythroid or granulopoietic tion, the RB gene is gradually activated, as in phytohemag- differentiation conditions, treated with a-Rb or scrambled glutinin-activated T cells (36). The pRb, while hypophosphor- oligomers from day 6 through day 7 or 8, and finally plated in ylated in the quiescent HPCs, becomes prevailingly hyperphos- semisolid cultures supplemented with either the same HGF phorylated in the proliferating HPCs, thus in line with the stimuli used in liquid suspension culture (Fig. 4) or saturating phosphorylation pattern observed in other cell types in qui- amounts of IL-3 and GM-CSF with or without Ep (data not escent and cycling status (7, 8). The modulation of RB ex- shown). In both cases, a-Rb treatment caused a dose- pression during HPC differentiation and precursor maturation dependent decline of erythroid colony number, as compared is typically biphasic: (i) during HPC differentiation Rb mRNA with that in scrambled a-Rb or untreated controls. Conversely, and protein are induced in both erythroid and granulopoietic a-Rb treatment did not significantly modify the number of cultures and (ii) during precursor maturation sustained ex- CFU-GM colonies in either Ep+ or Ep- culture conditions. pression of Rb mRNA and protein in the erythroid series Control experiments showed that a-Rb treatment causes a contrasts with marked downmodulation in the granulopoietic marked decrease of Rb mRNA, particularly at 100 ,ug, while pathway. it does not modify the level of f32-microglobulin mRNA (Fig. Functional experiments were based on Rb mRNA suppres- 4 Inset). Further control studies showed a >95% fluorescein- sion by antisense oligomers. Addition of a-Rb on early HPCs- ated oligomer uptake in both a-Rb- and scrambled a-Rb- i.e., at day 0-1 of culture-exerted no effect on erythroid treated cells (results not shown). colony formation but slightly enhanced CFU-GM clonogenetic capacity in the presence of an elevated level of IL-3 and DISCUSSION GM-CSF, thus in line with a previous report (37); the latter phenomenon may suggest that Rb mRNA inhibition in quies- The discrete molecular events underlying early hematopoiesis cent CFU-GM mildly facilitates their response to a saturating are still poorly understood, primarily because of the extreme growth factor stimulus. More important, treatment with a-Rb rarity of HPCs and HSCs in human bone marrow (1) and PB of late HPCs-i.e., addition at days 6-8 of erythroid or (4, 22). Therefore, we have developed methodology for (i) granulopoietic culture-induces a dose-dependent inhibition HPC purification and (ii) unilineage differentiation in liquid- of erythroid but not granulopoietic colony formation. These phase culture. (i) The purification methodology (22) has been results indicate that RB plays a lineage-restricted role in late recently improved to allow both stringent purification and erythroid HPC differentiation. abundant recovery of HPCs from adult PB (3, 23, 24, 29). The Altogether, these expression/function results consistently purified step IIIP HPCs (CFU-GEMM, BFU-E, and CFU- indicate that the RB gene is modulated in adult hematopoiesis GM) are a homogenous population of highly undifferentiated according to a lineage- and stage-specific pattern with peak HPCs (3, 22-24, 28), as compared to primitive bone marrow pRB expression and a key functional role at the level of late HPCs (32-35). (ii) Furthermore, we have developed a FCS- erythroid HPCs. liquid suspension culture for gradual differentiation of the The developmental defect of hematopoiesis in RB- mice purified step IIIP HPCs along the erythroid or granulopoietic may be explained by the ability of pRb to associate with and neutrophilic lineage (comprising <2-3% nonerythroid cells or potentiate the activity of erythroid-specific transcription fac- - 1-2% monocytes at late culture times, respectively) by tors (20). Along these lines, biochemical association of pRb addition of low-level multilineage HGFs (IL-3, GM-CSF) with products of the MyoD gene family in skeletal muscle cells combined with saturating amounts of a unilineage HGF (Ep, is essential for terminal differentiation (38). In adult HPC 4812 Cell Biology: Condorelli et al Proc. NatL Acad ScL USA 92 (1995) differentiation culture, the expression patterns of RB (these 18. Ewen, M. E., Sluss, H. K., Sherr, C. J., Matsushime, H., Kato, studies) and GATA-1 (24, 28) genes are remarkably similar, J. Y. & Livingston, D. M. (1993) Cell 73, 487-497. while addition of antisense oligomers targeting Rb (this re- 19. Clarke, A. R., Maandag, E. R., van Roon, M., van de Lugt, port) or GATA-1 (24) mRNA induces selective suppression of N. M. T., van der Valk, M., Hooper, M. L., Berns, A. & te Riele, erythropoiesis. Knockout studies indicate that both RB (19- H. (1992) Nature (London) 359, 328-330. are for devel- 20. Jacks, T., Fazeli, A., Schmitt, E. M., Bronson, R. T., Goodell, 21) and GATA-1 (39) genes required erythroid M. A. & Weinberg, R. A. (1992) Nature (London) 359, 295-300. opment; differentiation of RB- embryonic liver cells is 21. Lee, E. Y.-H. P., Chang, C. Y., Hu, N., Wang, Y. C. J., Lai, C. C., blocked at the level of late erythroid HPCs/early erythroblasts Herrup, K., Lee, W. H. & Bradley, A. (1992) Nature (London) (19-21), and GATA-1- embryonic stem cells differentiate 359, 288-294. only to the proerythroblast stage (40). In view of these striking 22. Gabbianelli, M., Sargiacomo, M., Pelosi, E., Testa, U., Isacchi, G. similarities between RB and GATA-1 expression/function in & Peschle, C. (1990) Science 249, 1561-1564. erythropoiesis, the hypothesis may be considered that RB and 23. Labbaye, C., Valtieri, M., Testa, U., Giampaolo, A., Meccia, E., GATA-1 proteins directly or indirectly interact at biochemical Sterpetti, P., Parolini, I., Pelosi, E., Bulgarini, D., Cayre, Y. E. & and/or functional levels, particularly in late erythroid HPCs. Peschle, C. (1994) Blood 83, 651-656. 24. Labbaye, C., Valtieri, M., Barberi, T., Meccia, E., Masella, B., This work was in part supported by National Institutes of Health Pelosi, E., Condorelli, G. L., Testa, U. & Peschle, C. (1995) J. Grant R01 CA60999 and by the Council for Tobacco Research and the Clin. Invest., in press. 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