Perturbed Hemopoiesis and the Generation of Multipotential Stem

Transcription

1 MOLECULAR AND CELLULAR BIOLOGY, Mar. 1986, p /86/ $02.00/0 Copyright 1986, American Society for Microbiology Vol. 6, No. 3 Perturbed Hemopoiesis and the Generation of Multipotential Stem Cell Clones in src-infected Bone Marrow Cultures Is an Indirect or Transient Effect of the Oncogene JOHN A. WYKE,l* ANDREW W. STOKER,' SIAN SEARLE,' ELAINE SPOONCER,2 PAUL SIMMONS,2 AND T. MICHAEL DEXTER2 Imperial Cancer Research Fund Laboratories, St. Bartholomew's Hospital, Dominion House, Bartholomew Close, London ECIA 7BE,' and Paterson Laboratories, Christie Hospital and Holt Radium Institute, Withington, Manchester M20 9BX2 United Kingdom Received 23 August 1985/Accepted 5 December 1985 Multipotential stem cell lines, derived speciscaily from long-term bone marrow cultures infected with a recombinant retrovirus carrying v-src, lack v-src. Stable consequences thus result from transient actions or indirect effects of v-src on other cells, with the latter possibility being favored by its mosaic expression in marrow cultures. During long-term bone marrow culture (LTBM) in vitro an adherent stroma supports hemopoietic islands, in which self-renewal of multipotential stem cells (CFU-S), differentiation to committed granulocyte-macrophage colonyforming cells (CFC), and amplification and maturation of the CFC occur for several months (6, 8). Perturbation of mouse LTBM, by infection with a recombinant Moloney murine leukemia virus, (MoMuLV) carrying the Rous sarcoma virus (RSV) v-src oncogene [src(momulv) (1)] causes a gradual transformation of the stroma, increases in CFU-S and granulocyte-macrophage CFC, and a decrease in mature granulocytes (3). Reduced granulocyte maturation and an eventual decline in CFU-S production seems an extrinsic consequence of stromal transformation and its ensuing degeneration, because transfer of nonadherent cells to uninfected stroma mitigates these effects. However, at least some CFU-S and granulocyte-macrophage CFC acquire intrinsic alterations that increase their capacity for self-renewal in vitro and in vivo without affecting their response to growth factors, their maturation in vitro, and reconstitution of normal hemopoiesis in irradiated mice (3, 18). To investigate the role of v-src in these intrinsic effects we cloned nonadherent cells, commencing 20 weeks after src(momulv) infection in LTBM, by plating them in soft agar containing hemopoietic cell growth factor (HCGF or interleukin 3) (18). Individual colonies were expanded by liquid culture in Fischers medium plus 20% (vol/vol) horse serum-hcgf. Most colonies from three sets of infected LTBMs produced continuously growing, diploid, nonleukemic lines that had a multipotential nature in a CFC-mix assay (18), producing erythroid cells, granulocytes, and macrophages. Their growth, in the absence of stroma, depends on HCGF, hence their designation FDCP-Mix (factordependent cells, Paterson Laboratories, Mixed colonyforming potential). On one occasion colonies formed in agar in the absence of HCGF; the colonies comprised aneuploid leukemic cells that bore the macrophage-specific marker recognized by monoclonal antibody F4/80 (2). These cells were designated FICP-src-1 (factor-independent cells from src-infected cultures). Virus assays on the cell-free supematants of these lines * Corresponding author. (16, 23) showed that LTBM and FICP-src-1 macrophages produced both src(momulv) and helper MoMuLV, whereas six factor-dependent lines produced helper virus only (Table 1). In addition, supernatant from FDCP-Mix-1 trasmitted MoMuLV to LTBM X27 (Table 1). Such helperinfected LTBM tend to show slightly prolonged hemopoiesis, as described by others (9), but do not show the same patterns of hemopoiesis as seen after infection with src(momulv) (3, 18) and generate no multipotential factor- TABLE 1. Virus production by cell lines derived from bone marrow infected with src(momulv) virus Cella Focus-forming unitsb PFUc Infected LTBM 3.6 x 102; 1.3 x x 103 Cell lines' FDCP-Mix-1 <1; <1 4 x 103 FDCP-Mix-2 <1; <1 7 x 103; 1.2 x 103 FDCP-Mix-3 <1 7 x 103 FDCP-Mix-4 <1 5 x 102 FDCP-Mix-5 <1 7 x 102 FDCP-Mix-6 <1 6 x 102 FDCP-Mix-1, <1 8 x 102 irradiated (1)' FDCP-Mix-1, <1 5 x 102 irradiated (9)' FICP-src-1 9 x x 104 X27f <1 1x103 Uninfected LTBM <1 <1 Infected NIH 3T3 1.3 x 105; 8 x 104 >104 fibroblasts a Al marrow-derived cells were from B6D2F1 mice. b src(momulv) in cell-free supernatant was assayed for focus formation on Rat-1 cells (22). Duplicate figures are for supernatants obtained at different times during the culture of the cell lines. c Helper MoMuLV in cell-free supernatant assayed for plaque formation on XC rat cells (16). Duplicate figures are for supematants obtained at different times during the culture of the cell lines. I Cell lines were derived from colonies obtained after LTBM culture was plated in soft agar in the presence of HCGF (18). ' FDCP-Mix-1 cells were passaged on irradiated, noninfected long-term culture adherent layers (numbers in parentheses are passage number). Under these conditions the multipotential nature of the cells was expressed, and myeloid cells representing all stages of differentiation were produced (18). f An LTBM culture infected with cell-free supernatant fluid from FDCP- Mix

2 960 NOTES MOL. CELL. BIOL. A Eco RI LTR probe I M l 0 a. B Hind III - LTR probe 1 ' am ~ ~~~~* Om C Eco RI - sr. probe D Eco RI - PSRA-2 probe M FIG. 1. Detection of RSV-specific sequences in cell DNA. In panels A, C, and D DNA was digested with EcoRI; in panel B DNA was digested with Hindlll. Panels A and B were hybridized with the nick-translated 0.32-kilobase long terminal repeat- (LTR) specific probe. Panel C is the same blot shown in panel A, from which the probe was removed and hybridized with the 0.8-kilobase src-specific probe. Panel D was hybridized with a probe comprising the whole of psra-2. Closed arrowheads indicate fragments containing the RSV-specific insert of src(momulv). (A and C) Lane 1, LTBM infected with src(momulv); lane 2, uninfected age-matched LTBM; lane 3, NIH 3T3 cells infected with src(momulv); lane 4, Rat-i cells infected with src(momulv); lane 5, uninfected NIH 3T3 cells; lane 6, FDCP-Mix-1; lane 7, FICP-src-1; lane 8, FDCP-Mix-6; lane 9, FDCP-Mix-2. (B) Lane 1, uninfected NIH 3T3 cells; lane 2, NIH 3T3 cells infected with src(momulv); lane 3, FDCP-Mix-7; lane 4, FDCP-Mix-8; lane 5, FDCP-Mix-9; lane 6, FDCP-Mix-6; lane 7, FICP-src-1; lane 8, FDCP-Mix-1. (D) Lane 1, FDCP-Mix-9; lane 2, uninfected NIH 3T3 cells; lane 3, NIH 3T3 cells infected with src(momulv); lane 4, FDCP-Mix-7; lane 5, FDCP-Mix-8; lane 6, FDCP-Mix-6; lane 7, FICP-src-1; lane 8, FDCP-Mix-1. In each panel lane M is bacteriophage X DNA digested with HindIll to give markers of 23.5, 9.59, 6.64, 4.45, 2.29, and 1.95 kilobases. M m m0 p dependent cell lines. This shows that helper MoMuLV alone does not perturb normal LTBM, as confirmed with supernatant from FDCP-Mix-2 and in other experiments (data not shown). Moreover, FDCP-Mix-1 does not produce any other agent that is able to mediate these effects. Because FDCP-Mix cell lines are induced at high frequency only in LTBM infected with src(momulv) (Table 1 [18]), the absence of transforming virus from these lines is a paradox. To investigate this we examined cell DNA by standard methods (12) for the presence of the RSV insert of src(momulv) using various probes derived from the psra- 2 clone of RSV (5). A long terminal repeat-specific probe, which had no cross-reaction with normal mouse DNA, recognized src(momulv) in infected LTBM and FICP-src-1 but not in FDCP-Mix cell lines (Fig. 1A and B) (the minor bands in DNAs of FDCP-Mix-1, -2, and -6 and FICP-src-1 are contaminating plasmid DNA). To check for any rearranged src(momulv) that might lack long terminal repeats, we used probes that recognized src or the whole RSV insert (Fig.1C and D). Because of homology with mouse c-src, these probes revealed a more complex pattern of faintly hybridizing fragments, but they confirmed that FDCP-Mix cultures do not contain src(momulv) in native or rearranged form. Which cells, then, contain and express v-src in src(momulv)-infected LTBM? At 20 weeks and more postinfection the adherent cells were mainly macrophagelike or fibroblastoid (Fig. 2b), with the former expressing pp6fovsrc, as judged by indirect immunofluorescence using anti-pp60vsrc monoclonal JB492 (a gift from J. Brugge [11]),

3 VOL. 6, 1986 NOTES 961 a FIG. 2. Localization of pp6ov-src by immunofluorescence in the adherent layer of a culture 40 weeks after infection. (a) Cells treated with monoclonal antibody JB492. (b) View of the field shown in panel a by phase-contrast microscopy. Note the mosaicism of expression of pp60vsrc. The rounded macrophagelike cells are strongly positive, while the smaller, more stellate cells are negative. Magnification, x 172. The specificity of the monoclonal antibody for recognition of pp6ov-src was demonstrated by the fact that (i) control uninfected cultures showed no positivity, although the adherent layers were rich in macrophages (7) and (ii) immunoglobulin was used to block Fc receptors; nonspecific binding to Fc receptors is therefore unlikely. Ie~ ^ -v 'i,xs'> w.w,r. _,%i, e., b N I N / N I N N N N I N / N I FIG. 3. Kinase activity of pp6ov-src in cells infected with src(momulv). Immune complex assays were performed as described previously (20). In each pair of lanes, lane I shows immune precipitation with a polyclonal anti-pp60v-src serum, and lane N shows the normal serum control. Arrowheads indicate the position of the immunoglobulin heavy chain. Lane 1, NIH 3T3 cells infected with src(momulv); lane 2, FDCP-Mix-7; lane 3, FDCP-Mix-8; lane 4, FDCP-Mix-9; lane 5, FICP-src-1; lane 6, FDCP-Mix-6; lane 7, FDCP-Mix-2; lane 8, FDCP-Mix-1; lane 9, LTBM infected with src(momulv).

4 962 NOTES and with the latter being negative (Fig. 2a). Likewise, the nonadherent fraction contained blast cells that rarely expressed pp6 v-src (data not shown) and transformed stromal cells that expressed the macrophage surface antigen recognized by F4/80 antibody and pp6ov-src', which is in agreement with previously reported data (3). Figure 3 shows pp6ov-src kinase activity (assayed as described previously [20]) in lines derived from these nonadherent cells. As expected, all FDCP-Mix cell lines were negative, but the HCGFindependent macrophage line FICP-src-1 was strongly positive. The malignancy of FICP-src-1 may be related to v-src activity, but because only one such ling arose in five different experiments, the frequent expression of pp6ov-src in macrophagelike cells of infected LTBM (Fig. 2a) is presumably insufficient to induce their growth factor independence and malignancy. These findings raise two important questions: Why is src(momulv) consistently absent from FDCP-Mix cell lines and, consequently, by what mechanism do these lines acquire increased self-renewal capacity? The 10-fold or greater excess of helper over transforming virus in LTBM (Table 1) may answer the first question, but it is also possible that the presence of src(momulv) affects CFU-S self-renewal or differentiation, thus reducing the persistence of any infected cells in LTBM or their clonogenic ability in agar. Expression of pp60v-src may even be lethal, because precedents, exist for the expression of viral oncogenes that kill certain cell types (14, 21, 24). However, pp6gvsrc expression was clearly not deleterious for FICP-src-1, and others have shown that v-src can be expressed in transformed erythroid and lymphoid cells (10, 13, 15). The fact that intrinsically altered FDCP:Mix cell lines are only obtained after infection of LTBM with src(momulv) is crucial to the second question. The helper virus may contribute to these effects but cannot induce them by itself (Table 1; unpublished data). v-src must thus play a role, either directly but transiently or indirectly. The former mechanism seems unlikely because FDCP-Mix lines arise frequently and reproducibly from infected LTBM (18), yet in our experience loss of an integrated provirus was rare, even when selection should have favored it (4, 22). Moreover, an example that originally invoked "hit-and-run" effects by a transforming virus is currently thought to involve insertional mutagenesis by the helper virus, and the transforming component may not play any role (17); this result is unlike those reported here. However, cytotoxicity mediated by v-abl favors the efficient selection of cell lines that lack Abelson murine leukemia virus (24), so src(momulv) might also act by a hit-and-run mechanism if v-src is essential in early and irreversible stages of FDCP-Mix evolution but is subsequently cytotoxic. An indirect induction of intrinsic change by v-src because of transformation or death of another cell type seems more plausible to us at present. Such a mechanism is in accord with the mosaic pattern of v-src expression, the activity of which is mainly in macrophagelike cells (Fig. 2 and 3), with the known extrinsic influence of the stroma (3) and with the concept of hemopoiesis occurring in an environment that is regulated by interacting cell populations (7, 8, 19). Our findings stress the importance of (i) distinguishing transient direct activity of v-src from indirect effects; (ii) characterizing cells that express src(momulv), whether they are stromal components or more mature hemopoietic cells; and (iii) identifying the mechanism of any feedback-regulating CFU-S and the nature of the resultant intrinsic changes in the stem cells. MOL. CELL. BIOL. This work was supported in part by the Cancer Research Campaign. T.M.D. is a Life Fellow of the Cancer Research Campaign. We thank G. 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