Rates of Mutation to Growth Factor Autonomy and Tumorigenicity. Differ in Hematopoietic Stem and Precursor Cells Expressing the Multilineage ...
MOLECULAR AND CELLULAR BIOLOGY, Dec. 1989, p. 5746-5749
Vol. 9, No. 12
0270-7306/89/125746-04$02.00/0 Copyright © 1989, American Society for Microbiology
Rates of Mutation to Growth Factor Autonomy and Tumorigenicity Differ in Hematopoietic Stem and Precursor Cells Expressing the Multilineage Colony-Stimulating Factor Gene C. LAKER,' N. KLUGE,' C. STOCKING,' U. JUST,' M. J. FRANZ,' W. OSTERTAG,1* J. F. DELAMARTER,2 M. DEXTER,3 AND E. SPOONCER3
Heinrich-Pette-Institut fur Experimentelle Virologie und Immunologie an der Universitat Hamburg, Martinstrasse 52, 2000 Hamburg 20, Federal Republic of Germany'; Glaxo Institute for Molecular Biology, S.A., 1211 Geneva 24, Switzerland; and The Paterson Institute for Cancer Research, Manchester M20 9BX, United Kingdom3 Received 25 April 1989/Accepted 29 July 1989
At least two separate but interdependent events are required to attain autonomous growth as a consequence of ectopic expression of the multilineage colony-stimulating factor gene in hematopoietic progenitor cells. The rate at which the second event occurs is more than 3 orders of magnitude higher in precursor cell lines (FDC-Pl or FDC-P2) than in stem cell lines (FDC-Pmix). Autonomous, but not density-dependent, growth is tightly coupled to tumorigenicity in precursor cells; however, neither growth-factor-independent nor autonomously growing stem cell lines are tumorigenic.
Several steps are involved in the development of leukemia. The nature of the multiple genetic changes necessary to convert normal hematopoietic cells into leukemic cells, however, remains an enigma. The discovery that oncogenes may be related to normal growth factor genes or receptors (for reviews, see references 6 and 11) prompted studies of the oncogenic potential of ectopic growth factor expression in hematopoietic cells. Both the multilineage colony-stimulating factor (multi-CSF; interleukin-3) and the granulocytemacrophage-CSF support proliferation of multilineage stem cells and progenitors in addition to stimulating the function of more mature cells (10). Studies have shown that the introduction and expression of genes encoding one of these CSFs into myeloid precursor cell lines elicits growth autonomy (abrogation of the requirement for external CSF stimulation) and tumorigenicity (5, 9, 16). Our own work has shown that the acquisition of growth autonomy after the autogenous production of granulocytemacrophage CSF in established precursor cells could be dissected into two or more interdependent events (8). Immediately after introduction of the granulocyte-macrophage CSF gene, cells proliferate independently of an exogenous source of CSF but are not truly autonomous, since they still require external stimulation by the autogenous CSF (autocrine stimulation). After an unknown secondary event, these cells exhibit true growth autonomy characterized by densityindependent clonability and cell proliferation in the presence of neutralizing anti-CSF antisera. It remains unresolved whether tumorigenic growth of these cells is acquired during the first step or as a consequence of growth factor autonomy. Part of this work was designed to answer this question. Past studies have been limited to committed progenitors and thus may not reflect the alterations that occur in the stem cell during the preleukemic stage of stem cell leukemias. The work presented here was thus additionally designed to determine if the acquisition of autonomous growth and tumorigenicity as a consequence of ectopic CSF expression would occur at the same frequency in multipotent stem cells as in progenitor cells. *
For this study, both multipotent stem cell lines (FDCPmix) (13) and progenitor cell lines (FDC-P1 and FDC-P2) (3) were used. Unlike the progenitor lines, FDC-Pmix can be induced to undergo differentiation into the myeloid and erythroid lineages under the appropriate culture conditions and generally retain a normal karyotype. Cells were infected with either a retroviral vector carrying the multi-CSF gene (M3-MuV) or a control vector without the growth factor gene (Neor Mos-) (Fig. 1). Infection was carried out either by cocultivation of the hematopoietic cells with irradiated, virus-producing PA317 or %f2 cells or by exposure to viral supernatant as previously described (8). Threefold serial dilutions of infected cells were cloned either with G418 or in the absence of an externally added growth factor at concentrations ranging from 105 to 101 cells per ml immediately after infection. Both progenitor and stem cell lines infected with M3-MuV showed a disproportionate increase in colony formation with increasing cell numbers in the absence of exogenous growth factor. Low but detectable levels (up to 10 U/ml) of multiCSF in supernatants of M3-MuV-infected cells (26 clones analyzed) but not in supernatants of uninfected or of control infected cells were measured by monitoring the proliferation of CSF-dependent cell lines. Furthermore, the proliferation of FDC-Pmix cells infected with the M3-MuV vector could be inhibited with anti-multi-CSF antiserum (Fig. 2). These results are consistent with our earlier findings with FDC-P1 cells expressing granulocyte-macrophage CSF (8) and support a model of autostimulation in which the autogenous multi-CSF must be secreted to interact with its receptor. A total of nine precursor and three stem cell clonal lines established after infection with M3-MuV were monitored over several weeks to determine if an increase in the fraction of autonomously growing cells occurred. All cell lines were cultured with exogenous multi-CSF at levels shown to stimulate maximum proliferation. Cells were then cloned at different densities in the presence and absence of an external source of CSF to determine the proportion of autonomous clones in the total cell population. After 3 to 4 weeks in culture, recloning of all of the infected FDC-Pmix and three of nine FDC-P1/P2 clones retained a strict density-depen-
Corresponding author. 5746
NOTES
VOL. 9, 1989
5747
No. of colonies
neo
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FIG. 1. Genetic map of cells infected with a control vector (Neor Mos-) or M3-MuV. All vectors used were based on the myeloproliferative sarcoma virus and contained the TnS gene for Neor (2). Two vectors that did not contain the multi-CSF gene were used as control vectors and are depicted as Neor Mos-. They are the Neor Mos- vector (14) and a similar vector in which the myeloproliferative sarcoma virus long terminal repeat has been replaced by that of the Moloney murine leukemia virus (M. Grez, unpublished results). The M3-sMuV, depicted as M3-MuV, was constructed by inserting an HpaII-NcoI fragment encoding the normal leader peptide and the mature multi-CSF protein from a cDNA clone (4) into a derivative of Neor Mos-, M3neo (8). Splice donor (s.d.) and splice acceptor (s.a.) signals are indicated.
dent growth pattern (Fig. 3A). In striking contrast, the majority of infected FDC-P1/P2 clones showed a dramatic shift toward autonomous growth, as demonstrated by linear or near-linear clonabiity as a function of density in six of nine clones tested (Fig. 3B). The rate at which the infected clones progressed to an altered factor requirement was calculated. M3-MuV-infected clones selected in medium containing multi-CSF were expanded and maintained for 56 (FDC-P1 and FDC-P2) or 34 (FDC-Pmix) cell generations in the presence of multi-CSF and were then recloned under the 3H-Thymidine
1012 103
1o4
104
103
102
No. of cells plated 0
FIG. 3. Recloning of MuV-infected cultures. Clones were picked from primary infections as previously described (8) and maintained in the presence of Wehi 3B conditioned medium (CM). Cells were washed and threefold serial dilutions of cells ranging from 3 x 10' to 3 x 101 cells were plated in 3 ml of semisolid medium in triplicate either in the presence of G418 and WEHI CM (@) or in the absence of WEHI CM (0). The cloning efficiency as a function of cell density of two representative MuV-infected FDC-P2 clones is plotted. (A) Factor-independent, density-dependent clone (FDC-P2 129); (B) density-independent, autonomous clone (FDC-P2 127).
conditions described in the legend to Fig. 3. The proportion of infected colonies that grew without factor at the lowest cell density was determined. This number was divided by the calculated number of cell generations during the cloning experiment to obtain the rate of mutation to autonomy. The calculation is only an approximation of the true rate of mutation to growth factor autonomy and is based on two assumptions. The first is that the cells growing at low densities are, indeed, mutants that grow autonomously. This could be shown by picking clones at a low density and replating them with or without multi-CSF; all replated clones were density independent or had a much-reduced require-
A
TABLE 1. Rates of mutation to growth factor autonomy in uninfected and infected progenitor and stem cell lines 100
Cell type
FDC-P1
80
Vector
None Neor Mos M3-MuV
60
FDC-P2
40
None Neor MosM3-MuV
20
Clone no.
mb
