Expression of a hemopoietic growth factor cDNA in a factor-dependent cell line results in autonomous growth and tumorigenicity

Cell, Vol. 43, 531-542,

December

1985 (Part

I), Copyright

0092-8674/85/l

0 1985 by MIT

20531-l

2 $02.0010

Expression of a Hemopoietic Growth Factor cDNA in a Factor-Dependent Cell Line Results in Autonomous Growth and Tumorigenicity Richard A. Lang,” Donald Metcalf,+ Nicholas M. Gough: Ashley Ft. Dunn: and Thomas J. Gonda’ * Melbourne Tumour Biology Branch Ludwig Institute for Cancer Research fWalter and Eliza Hall Institute of Medical l? 0. Royal Melbourne Hospital Victoria 3050, Australia

Research

Summary Production of a growth factor by a cell that responds to this factor has been termed “autocrine” stimulation of proliferation. Considerable experimental data have suggested that tumor cells often exhibit autocrine growth stimulation and that this may contribute to the process of malignant transformation. To experimentally approach the relationship of autocrine growth stimulation to the malignant transformation of hemopoietic cells, we have used a retroviral vector to express sequences encoding a hemopoietic growth factor, granulocyte-macrophage colony stimulating factor (GM-CSF) in a factor-dependent murine cell line (FDC-Pl). Virally infected cells synthesized and secreted GM-CSF, grew independently of exogenous CSF, and-unlike the parental FDC-Pl cells-produced tumors in syngeneic mice. We have thus experimentally induced autocrine growth regulation in a factor-dependent hemopoietic cell line and have shown that this results in tumorigenicity. Introduction The concept of autocrine stimulation of cellular proliferation requires that a cell can both produce and respond to a particular growth factor (9odaro et al., 1977; Sporn and Todaro, 1980). Under certain conditions, such a cell might then become independent of external growth regulation in a manner that would lead to malignant transformation, and indeed such examples have been described (for review see Sporn and Roberts, 1985). These include production of tumor growth factors (TGFs) by certain transformed cells (De Larco and Todaro, 1978; Kaplan et al., 1982) which, like their nontransformed progenitors, respond to the TGFs. Another important example is malignant transformation by the v-s& oncogene of simian sarcoma virus (SSV), which encodes a growth factor that is very similar or identical to platelet-derived growth factor (PDGF) (Waterfield et al., 1983; Doolittle et al., 1983). Thus, transformation of cells infected with SSV may be attributed, at least in part, to the autocrine production of a PDGF-like growth factor which stimulates their continuous proliferation. Normal hemopoietic cells show an absolute dependence on a group of specific growth regulators, the colony stimulating factors (CSFs), for survival, proliferation, and

differentiation when cultured in vitro (Metcalf, 1984). Like normal hemopoietic cells, primary leukemias of granulocyte-macrophage populations (myeloid leukemias) are dependent on exogenous CSF for proliferation (Metcalf, 1984). However, myeloid leukemias that have been maintained for prolonged periods by transplantation or continuous culture eventually become autonomous and in many cases can be shown to constitutively synthesize one of the CSFs (Metcalf and Nicola, 1985). A number of laboratories have established immortalized cell lines derived from murine hemopoietic cells (Dexter et al., 1980; Greenberger et al., 1983) which are characterized by absolute dependency on exogenous CSF, usually Multi-CSF (IL-3). However, such cell lines are nonleukemogenic when transplanted to syngeneic recipient mice. There are two reports in which acquisition by such cell lines of a capacity to synthesize CSF, and subsequently to grow autonomously in vitro, coincided with the acquisition of leukemogenicity (Hapel et al., 1981; Schrader and Crapper, 1983). It has been proposed that autocrine synthesis of a CSF (in this case Multi-CSF) was responsible for conversion to a malignant phenotype (Schrader and Crapper, 1983). To directly test the possibility that autonomous synthesis of CSF by factor-dependent cell lines can result in leukemogenicity, we have used a retroviral vector to introduce and express sequences encoding a CSF in one such cell line. Specifically, we inserted a cDNA encoding the granulocyte-macrophage colony stimulating factor (GM-CSF) (Burgess et al., 1977) into a retroviral vector and used the resulting virus (GMV) to infect FDC-Pl (FD) cells (Dexter et al., 1980; Hapel et al., 1984) which are GM-CSFdependent. This resulted in the development of cells that synthesized GM-CSF, proliferated in the absence of exogenous GM-CSF, and produced transplanted leukemias in recipient syngeneic mice. Furthermore, the growth properties of the GMV-infected FD cells raise the possibility that the GM-CSF produced by these cells may be acting internally rather than interacting with receptors on the cell surface after secretion into the culture medium. Results Construction and Properties of a Retroviral Vector for Expressing Murine GM-CSF The pGMV plasmid was constructed by inserting a cDNA clone of the murine GM-CSF mRNA (Gough et al., 1984; Gough et al., 1985) into the Moloney murine leukemia virus-based vector pZIPNeoSV(X)l (Cepko et al., 1984) as shown in Figure 1. The GM-CSF cDNA clone used, pGM5’A7, was chosen because of the high levels of GMCSF produced when it was expressed in COS cells using an SV40-based vector (Gough et al., 1985). In the GMV construct, GM-CSF should be translated from a viral genome-length mRNA transcribed under the control of the promoter/enhancer in the viral long terminal repeat (LTR), while the neo sequences, conferring resistance to

Cell 532

Figure

pZIPneoSV(X)I BarnI-

3’LTR GM-CSFA7 cONA

the antibiotic G418 (Davies and Jimenez, 1980; Colb&eGarapin et al., 1981; Southern and Berg, 1982), should be translated from a spliced subgenomic messenger RNA (Cepko et al., 1984). Infectious, helper-free GM-CSF virus (GMV) was produced by transfecting the GM-CSF viral construct into the packaging cell line ~2 (Mann et al., 1983). Culture media from expanded G418-resistant ~2 clones were assayed for infectious virus and GM-CSF activity as described in Experimental Procedures. Titres of virus ranged between 1 x 10’ and 2 x lo5 G418-resistant colony forming units per milliliter when assayed on BALB/c 3T3 fibroblasts. BALB/c 3T3 fibroblasts infected with virus from several ~2 producer cell lines were selected for G418 resistance and tested for GM-CSF secretion (Figure 2A). The conditioned media from these GMV-infected cells stimulated the proliferation of FD cells (responsive to either GM-CSF or Multi-CSF; Hapel et al., 1984) but not the proliferation of 32D cells (responsive only to Multi-CSF; Hapel et al., 1984; data not shown). We therefore concluded that the GMV-infected cells were producing GM-CSF. Thus, GMV could both induce G418 resistance and direct the synthesis of GM-CSF by infected fibroblasts. As expected, the ZlPNeo virus induced G418 resistance (data not shown) but not GM-CSF production in 3T3 fibroblasts (Figure 2A). Cells infected with defective virus produced by ~2 cells do not themselves produce infectious virus (Mann et al., 1983). Thus, the assay data genuinely reflect CSF production by the 3T3 cells and not infection and transformation to factor independence of the indicator cells used in the GM-CSF assay. Infection of Factor-Dependent Hemopoietic Cells with GMV We next attempted to infect FD cells with GMV. FD is a continuous hemopoietic cell line with some characteristics of myeloid progenitor cells (Dexter et al., 1980; Hapel et al., 1984; Metcalf, 1985). Since FD cells are absolutely dependent on either GM-CSF or Multi-CSF (IL-3) for both survival and proliferation, we reasoned that infection of these cells with GMV might lead to autocrine growth stimulation and factor independence. FD cells were infected either by exposure to supernatants from virus-producing ~2 clones or by cultivation with these ~2 cells. The FD cells were then selected in soft agar cultures for G418 resistance, growth in the absence of added CSF, or both. The results of some of these experiments are given in Table 1. These data show that both GMV and ZlPNeo viruses could confer G418 resistance in

lkb

1. Construction

of pGMV

Cleavage sites of Sac I, Barn HI, and Hint II within the proviral DNA are indicated, as are 5’ and 3’ splice site (SS) positions. Large arrowheads indicate the direction of transcription. pZIPNeoSV(X)l is described by Cepko et al. (1984).

’

20( -CSF Standard 16C \i

WGMV-Infected

12c

120

80 40

114096 1:1024 Dilution Figure

2. Assays

1:256

1:64

of conditioned

of CSF Activity

1:16

1:4

1:l

medium assayed

in Media from GMV-Infected

Cells

The level of GM-CSF in conditioned media from (A) GMV-infected 3T3 cultures and (B) eight cloned cell lines derived from GMV-infected FD cells was determined using microwell assays. Each point represents the mean cell counts from duplicate wells. Each of the virally infected cell lines is identified according to the experiment from which it was derived and the ~2 cell line used as a source of virus (see Table 1).

clonogenic FD cells and that infection with GMV, but not with ZlPNeo virus, allowed colony formation in the absence of added CSF. The frequencies of infection of FD cells with GMV were much higher following cultivation with the virus producer cells than following infection with virus-containing culture supernatants. This agrees with other results (G. Johnson and T. J. G., unpublished observations) which indicate that cocultivation is a more efficient mode of infection for hemopoietic cells. A total of 32 discrete G418-resistant and CSF-independent FD colonies were isolated and established as clonal cell lines. These lines were readily established and maintained in the absence of CSF. Since in no case did an autonomous colony fail to establish a continuous cell line, it appears that GMV can transform FD cells to factor inde-

CSF Retrovirus 533

Table

1. Infection

Experiment A

Transforms

Factor-Dependent

of FD Cells with Retroviral

Cells

Vectors:

Incidence

of G416Resistant

Mode of Infection

Virus Producer

Virus

Titrea

culture supernatant

GMVl

GM-CSF

9 x IO”

and Factor-Independent

Selectionb GM-CSF

G410

+ +

+ +

culture supernatant

GMV5

cocultivation

GMVl

GM-CSF

2 x 105

+ +

+ +

D

cocultivation

GMVS

GM-CSF

GM-CSF

9 x 10’

2 x 105

+ +

+

-

+

+ +

+ +

E

cocultivation

T2

ZlPNeo

5 x 10’

+ +

+

-

+

Colonies Colonies/lOS Cells Plated

Relative Plating EfficiencyC

5.6 x 10’ 1.5 0.7 0.5

100 ,003 ,001 .0009

6.0 5 1 2

x 10’

100 ,008 ,002 .003

6.6 x 10’ 3.7 x 103 5.2 x lo3 4.4 x 103

100 5.4 7.6 6.5

7.6 x 10’ 4.0 x 10’ 7.3 x 105 5.4 x 10’

100 5.1 9.4 6.9

2.6 x 10” 4.5 x 10’ 0 0

100 1.7 0 0

a Titre expressed as number of G41Eresistant colony forming units per milliliter of producer clone culture supernatant assayed on 3T3 fibroblasts. b Cells were plated in soft agar medium supplemented with GM-CSF (400 units/ml) and/or G416 (1 mglml) as indicated. c The relative plating efficiency is the ratio of colony formation under selective conditions to colony formation under nonselective conditions (with GM-CSF and without G416) expressed as a percentage.

pendence. The culture media from such factor-independent lines were assayed for the presence of GM-CSF using microwell cultures of factor-dependent FD cells (Figure 2B). Although all lines synthesized GM-CSF, the exact levels produced by individual cell lines varied slightly with successive passages. The medium from GMV-infected FD cell lines also stimulated granulocyte and/or macrophage colony formation in semi-solid cultures of adult bone marrow ceils, the resultant colonies being typical of those stimulated by purified GM-CSF (Burgess et al., 1977). From these assays the levels of GMCSF production were calculated to vary from 1500 to 4000 units/2 x lo6 cells/ml in 5 day conditioned media. The CSF activity in such media could be neutralized by an anti-GM-CSF serum (see Figure 78) confirming the conclusions reached from the FD assays that the active factor produced by GMV-infected cells was GM-CSF. Analysis of Viral Integration and Transcription in GMV-Infected FD Cells Proviral integration and viral gene transcription in GMVinfected FD cells were demonstrated using Southern and Northern blotting, respectively. Analysis of genomic DNA digested with Sac I by Southern blotting and hybridization to a GM-CSF cDNA probe (Figure 3A) revealed an extra fragment in the GMV-infected cells compared with ZIPNeo-infected cells (lane 1) or mouse embryo DNA (lane 20): the latter two DNAs showed only the 7.8 kb Sac I fragment corresponding to the murine GM-CSF gene. The extra fragment in DNA from the GMV-infected cells (lanes 2 to 19) was in most cases 5.5 kb long. It thus corresponds to the GMV provirus, since Sac I cleaves the GMV con-

struct only once in each LTR (see Figure 1). Hybridization of these DNAs to a neoR probe (Figure 36) revealed, as expected, the same sized proviral fragments in the GMVinfected FD cells (lanes 2-19), while mouse embryo DNA gave no neo%pecific fragments (lane 20). The cell line 85.3 (lane 8) shows a Sac I fragment of 5.1 kb which hybridized to both GM-CSF and neo probes. As this cell line is both factor-independent and G418-resistant, it is likely that a deletion of approximately 400 bp between the LTFis of the provirus has reduced the size of the hybridizing fragment. In addition, DNA from the ZIPNeo-virus-infected cells contained the expected 4.2 kb Sac I fragment (lane 1). The level of hybridization to the proviral Sac I fragments in Figure 3A compared with that of the endogenous (7.8 kb) fragment suggested that in most cases only one copy of proviral DNA was present in each cell. Densitometric analysis revealed that cell lines Cl.1 (lane 12) and D5.4 (lane 19) were exceptions, having two and three proviral insertions respectively. These results were confirmed by Southern analysis of Hint II digests of DNA from the various cell lines using a neoR probe. Since Hint II cleaves the proviral genome only once, a single fragment which hybridizes with the neoR probe should result for each proviral integration (see Figure 1). Figure 3C shows that, as expected, DNA from most of the infected cell lines gave one hybridizing band and therefore have a single proviral integration. Cell line Cl.1 (lane 5) gave two hybridizing fragments in agreement with Sac I digest analysis. Since DNA from cell lines D5.1 and D5.3 has an identically sized hybridizing fragment, it is possible they were derived from the same GMV-infected FD cell.

Cdl

534

A 1 2

3

4

5

6

7

6

9

10 11

12 13

14 15

16

17 18

Figure 3. Analysis of Proviral Virus-Infected FD Cells

19 20

-

23.5

-

9.6

-

6.7

-

4.4

’ -

2.3 2.0

‘

*

B 123

4

567

8

9

10111213

Integration

in

High molecular weight DNA from the following cell lines was cleaved with Sac I and analyzed by Southern blotting using (A) a GM-CSF probe and (E) a neoR gene probe: lane 1, ZIPNeoinfected cell line E2.4; lanes 2-19, GMVinfected cells lines Al.l, A1.3, 85.1, 85.2, B5.3, 05.4, 85.5, 85.7, 85.6, 135.10, C1.l, C1.2, C1.3, C1.4, D5.1, D5.2, D5.3, and D5.4, respectively; lane 20, BALBk mouse embryo DNA. (C) High molecular weight DNA from the following cell lines was cleaved with Hint II and analyzed as for Figure 38: lane 1, BALBlc mouse embryo DNA; lanes 2-9, GMV-infected cell lines D5.3, D5.1, C1.3, C1.l, 85.6, 85.7, A1.3, and Al.l, respectively; lanes 10 and 11, ZIPNeo-infected cell lines E2.4 and E2.3, respectively.

14151617l.61920

I 4

‘I L

i

4.

-

23.5

-

9.6

-

6.7

-

4.4

6

2.3 2.0

C 123456769

I,

“*I

-

23.5

-

9.6

-6.7 +

4.4

-

2.3 2.0

Polyadenylated RNA from the infected FD cells contained two species that hybridized to the neoR probe (Figure 4A). The larger species, of approximately 4.5 kb in ZIPNeo-infected cells (lanes 10-12) and 5.6 kb in GMVinfected cells (lanes 2-9), corresponds to the expected genomic RNAs of the two viruses, whereas the smaller species, of approximately 4.2 kb, in each case corresponds to the spliced subgenomic neoR gene mRNA. For reasons that are unclear, the levels of viral RNA in the GMV-infected cells (lanes 2-9) were significantly lower than in the ZIPNeo-infected cells (lanes 10 and 11); furthermore, the ratios of the levels of the subgenomic RNA to the genomic RNA were lower in the GMV-infected cells. This latter phenomenon has been observed with other ZlPNeo constructs (T. J. G., unpublished observations; S. Cory, 0. Bernard, and J. Schrader, personal communication). Hybridization to a GM-CSF cDNA probe (Figure 48) re-

vealed only one RNA species in GMV-infected cells, which corresponded to the viral genomic RNA of approximately 5.6 kb. Polyadenylated RNA from concanavalin A-stimulated LB3 cells (lane 1) contained the expected 1.2 kb GMCSF transcript (Gough et al., 1964). No hybridization was detected in ZIPNeo-infected FD cells (lanes 10 and 11) under conditions where one copy of GM-CSF mRNA per cell could be detected. Thus, the only GM-CSF mRNA in the GMV-infected FD cells is the viral genomic RNA, and therefore any GM-CSF produced by these cells must be encoded by the viral genome. Autonomous Growth of GMV-Infected FD Cells The growth properties of the GMV-infected cell lines were explored using three experimental approaches. First, the influence of cell density on the frequency of colony formation was determined. For each of eight cell lines tested, a linear relationship was observed between the number of

CSF Retrovirus 535

A

1

2

Transforms

3

4

5

Factor-Dependent

6

7

8

Cells

9 10 11

12

-5.6 -4.5 -4.2

*

61

2

3

4

5

6

7

8

91011

-‘i. -5.6

1.2 Figure 4. Detection CSF and the neoa

of Virus-Specific Gene Product

Messenger

RNAs Encoding

GM-

Polyadenylated RNA from the following cell lines was fractionated on a denaturing gel and analyzed by Northern blotting using (A) a neoR gene probe and (6) a GM-CSF probe: lane 1, LB3 cells stimulated with concanavalin A; lanes 2-9, GMV-infected cell lines D5.3, D5.1, C1.3, (X.1, B5.8, 85.7, Al.3, and Al.l, respectively; lanes 10 and 11, ZIPNeoinfected cell lines E2.4 and E2.3, respectively. Lane 12 of (A) is a shorter autoradiographic exposure of lane 11. Each lane contains 1.5 rg of RNA except lane 1, which contains 0.1 pg.

3612

25 50 Number of cells cultured

Figure 5. Colony Formation in Agar Lines of GMV-Infected FD Cells

cells cultured and the number of colonies developing: two examples are shown in Figure 5. Furthermore, colony size was not influenced by colony crowding (data not shown). This indicates that colony formation was not dependent on CSF or other growth factor8 that might have been released by other cells or colonies in the culture. The effects of an addition of a high concentration (400 units/ml) of GM-CSF to such cultures was also investigated. With some cell lines no effect of exogenous GM-CSF was observed on colony numbers or size (e.g. Figure 5A); however, as illustrated in Figure 5B, other cell lines showed a moderate increase in colony numbers. This shows that some cell lines remained responsive to additional stimulation by exogenous GM-CSF, suggesting that factor production by these lines might be insufficient to result in maximal proliferative stimulation. Furthermore, these data also suggest that at least some of the GMV-infected FD cell lines display functional cell surface receptors for GM-CSF, in agreement with ‘*%GM-CSF binding studies, which showed that

by Cells from

100 Two Cloned

Cell

Cell lines (A) 85.13 and (B) D5.2 were cultured at the indicated densities in the presence of either 400 units of GM-CSF or an equivalent saline volume and colonies counted after 7 days incubation.

most of the cell lines retained GM-CSF receptor levels similar to those of the parental FD cells (F. Walker and A. W. Burgess, personal communication). We next determined whether single GMV-infected FD cells could proliferate in large culture volumes in the absence of exogenous CSF. Proliferative stimulation of FD cells in agar cultures requires use of a minimum concentration of 20 units of GM-CSF per ml (D. Metcalf, unpublished data). From the measured rate of accumulation of GM-CSF in medium conditioned by GMV-infected FD cells (1500-4000 units/2 x lo8 cells/ml/5 days) a single cell could not be expected to accumulate more than 0.002 units of GM-CSF. In a 1 ml culture, this is unlikely to result in a sufficiently high GM-CSF concentration to ensure receptor binding and stimulation of proliferation. However,

Cell 536

DT=lS~Shr 160

k

Control serum

00

I

DT=14,5hr Al.3 /

n/

A13CM+Control

Al.2 DT=16hr

serum

160

Al3CM+GM-CSF 120

:: 0

24 Hoursof

Figure 6. Progressive Proliferation ferred to 1 ml Agar Cultures (A) Uninfected FD cells cultured in CSF or an equivalent saline volume. infected cell lines in cultures lacking mean values f standard deviations

48

80

incubation of Washed

Single

Cells

Trans-

the presence of 400 units of GM(6) Cells from two different GMVadded GM-CSF. Points represent of 40 individual clones per group.

55,13CM+GM-CSF

40

10,240

2560 Flnal

single GMV-infected FD cells could in fact proliferate with no initial lag in 1 ml agar cultures in the absence of exogenous GM-CSF (Figure 6B), whereas single FD cells were unable to survive or proliferate under these conditions (Figure 6A). In experiments using six different lines, 5%-53% of cells had completed their first cell division within 6 hr of transfer of the cell into the culture dish. Thereafter clone sizes increased exponentially with doubling times of 14-16, hr similar to those of clones of uninfected FD cells stimulated by high levels of GM-CSF. In a third series of experiments, addition of serial dilutions of anti-GM-CSF serum to microwell cultures of 50 GMV-infected cells produced no significant inhibition of cell proliferation in excess of the minor nonspecific effect noted with the lowest serum dilution, an effect seen also with the control pre-immunization rabbit serum (Figure 7A). In control experiments (Figure 78, Figure 28) normal FD cells were stimulated by conditioned medium from cultures of the same GMV-infected FD cells used in the experiments shown in Figure 7A. Calculations showed that the amount of CSF added in the experiment shown in Figure 78 would be equivalent to that accumulated over 5 days in cultures of 1000 GMV-infected cells. Although the anti-GM-CSF serum effectively blocked stimulation of FD cells by this amount of CSF, it was unable to significantly impair proliferation of only 50 GMV-FD cells.

serum

Figure 7. Effect of GM-CSF Antiserum Infected and Uninfected FD Cells

640

160 00

dllutlon on Microwell

Cultures

of GMV-

(A) Failure of antiserum to significantly inhibit the proliferation of 50 Al.3 cells. (6) Ability of antiserum to inhibit stimulation of FD cell proliferation by conditioned medium (CM) from GMV cell lines B5.13 and A1.3. Cultures were incubated for 46 hr. Each point represents mean cell counts from duplicate wells.

Tumorigenicity of GMV-Infected FD Cells In view of the autocrine hypothesis, we tested the GMVinfected FD cell lines for their ability to cause tumors in mice. To test the leukemogenicity of both uninfected and GMV-infected FD cells, 1 x lo6 cells were injected subcutaneously into syngeneic DBA mice. Additional control mice were injected with cells from three CSF-dependent cloned lines that were G416-resistant, derived by infection of FD cells with the ZlPNeo virus. As shown in Table 2, no mice injected with FD cells developed transplanted leukemias during an observation period of 26 weeks. Similarly, no mice injected with CSF-dependent cell lines infected with the ZlPNeo virus developed transplanted leukemias. By contrast, all but one of 72 mice injected with nine different GMV-infected FD cell lines developed progressively growing transplanted leukemias, the mice dying within 4-6 weeks of transplantation. Mice with transplanted tumors developed large diffusely infiltrating tumor masses at the site of injection

CSF Retrovirus 537

Transforms

Table 2. Production

Factor-Dependent

of Transplanted

Ceils

Leukemias

by GMV-Transformed

FD Cell Lines

Mice with Tumors/Number Injected

Weeks to Death

Karyotype of Autonomous Colony Ceils Grown from Transplanted Tumors

0122

NA

NA

B/8 710 8/8

5-6 6-6 6

85.3

016

6-8

85.4 85.5 85.13

I318 8/6 6/8

5-7 4-6 7

B5.1

818

5-7

85.2 Cl.1

8/E 616

6-8 4-6

Cl.2 D5.3

818 018

4-7 4-6

D5.4

018

4-8

8 metacentrics, 24 or 25 acrocentrics 8 metacentrics, 24 or 25 acrocentrics 8 metacentrics, 24 or 25 acrocentrics, majority tetraploid 8 metacentrics, 24 or 25 acrocentrics, many tetraploid All tetraploid with metacentrics 8 metacentrics. 24 acrocentrics 8 metacentrics, 24 acrocentrics, most tetraploid 8 metacentrics, 24 acrocentrics. a few tetraploid 8 metacentrics, 23-30 acrocentrics 8 metacentrics, 24 or 25 acrocentrics. a few tetraploid 8 metacentrics, 24 acrocentrics 8 metacentrics, 24 or 25 acrocentrics, a few tetraploid 8 metacentrics, 24 or 25 acrocentrics, many tetraploid

018 O/8 018

NA NA NA

Cells

Infected

Uninfected

FD

GMV-Infected Al.1 Al .2 Al .3

FD

ZIPNeo-Infected E2.1 E2.4 E2.5

FD Cells

a Each syngeneic DBA/P b All mice were observed NA: not applicable.

Figure

8. Tumors

Caused

mouse was injected subcutaneously for a minimum period of 12 weeks

by GMV-Infected

NA NA NA with 10’ cells. (26 weeks for mice injected

with uninfected

FD cells).

FD Cells

(A) Mouse with transplanted leukemia following subcutaneous injection of 1 x lo8 GMV-infected rows). (8) Liver showing infiltrating tumor cells (arrow). (C) Metaphase from autonomous colony eight metacentric chromosomes (arrows) identifying FD-derived cells.

FD cells. Note infiltrating subcutaneous tumor (argrown from transplanted tumor tissue showing the

(Figure 8A) with histological evidence of infiltration of the spleen and liver (Figure 86) and cytological evidence of bone marrow invasion. Agar cultures of cells from the tumor mass, spleen, and bone marrow of these animals all developed typical colonies of GMV-infected FD cells able to grow in the absence of added GM-C% The FD line exhibits eight metacentric marker chromosomes with 24 or 25 acrocentric chromosomes; occasional cells are tetraploid again with metacentric marker chromosomes. Since this is a cloned cell line, the karyotype appears to be unstable. Karyotypic analysis of dividing cells in CSF-independent colonies grown from the tumors of transplanted mice (Figure 8C, Table 2) revealed that all exhibited the metacentric marker chromosomes and either 24 or 25 acrocentrics. These observations documented that the transplanted tumors arose from the transformed FD cells that were originally injected. With some of the cloned lines varying proportions of tetraploid cells were also present. In most cases the transplanted cells initiating the original CSF-independent colony were likely to have been euploid, but the karyotype of many of the lines, like that of the parental FD cells, appeared unstable. The karyotypic variation seen in the tumor-derived cells was not significantly different from that in the parental lines, making it unlikely that further karyotypic aberrations contributed to their tumorigenicity. Discussion We report here the use of a retroviral vector to infect a CSF-dependent cell line and express GM-CSF. We have thus experimentally created a situation in which autocrine stimulation of cellular proliferation could, and in fact does, occur. The following evidence supports this conclusion. First, whereas the parental FD cells require either exogenous Multi-CSF or exogenous GM-CSF for survival and proliferation, the lines derived by infection with GMV do not. Second, the GMV-infected lines both produce GMCSF, and retain receptors for this factor. Third, GM-CSF production by GMV-infected cells is clearly a consequence of expression of the viral GM-CSF mRNA, as indicated by the absence of detectable nonviral GM-CSF RNA species in the GMV-infected lines. These data, and the high incidence of factor-independent lines following infection with GMV, discount the possibility that we have selected cells that either spontaneously, or as a consequence of viral infection per se, express the cellular GMCSF gene. Two major issues raised by our results need to be addressed: the relationship between growth factor dependence and malignant transformation, and the site and mechanism of action of the GM-CSF produced by the GMV-infected cells. Factor Independence and Tumorigenicity As outlined above, our data imply that the only change relevant to the tumorigenesis of the GMV-infected FD cells is the synthesis of GM-CSF; thus this appears to be sufficient to convert the FD cells to a tumorigenic phenotype.

It is of interest to compare these results with recent reports demonstrating that Abelson murine leukemia virus (AbMLV) can, like GMV, convert FD cells (Cook et al., 1985), mast cells (Pierce et al., 1985), and Friend murine leukemia virus-immortalized factor-dependent cell lines (Oliff et al., 1985) to factor independence and tumorigenicity. In view of our data, we suggest that in these systems transformation by AbMLV to a tumorigenic phenotype is a direct consequence of the loss of factor dependence of these cells; we need not invoke any other changes to the cells. Another experimental system that warrants comparison with ours is that described by Adkins et al. (1984) who superinfected chicken “myeloblasts” transformed by the v-myb-containing retrovirus E28 with retroviruses carrying src-related oncogenes. The myeloblasts, which are dependent on an avian CSF, cMGF (Beug et al., 1982), became independent of added factor following superinfection. Furthermore, the factor-independent cells both secreted CSF, unlike the AbMLV-infected murine cells (Oliff et al., 1985; Cook et al., 1985; Pierce et al., 1985) and appeared to require the secreted CSF for growth. It will be of interest to compare the in vivo tumorigenicity of the v-myb-transformed cells with that of the factorindependent cells derived by superinfection. In what sense, then, does the GM-CSF gene represent a proto-oncogene-or less dramatically-to what extent can autocrine CSF production contribute to malignant transformation? Preliminary results indicate that GMV does not transform hemopoietic cells from normal fetal liver or bone marrow, although it can stimulate the proliferation and differentiation of myeloid progenitor cells (G. Johnson, R. A. L., andT J. G., unpublished observations). However, in at least some circumstances, several known oncogenes fail individually to transform primary fibroblasts but rather require the action of a second, complementing oncogene (Ruley, 1983; Land et al., 1983). Since FD cells are immortalized cells that resemble myeloid progenitors they can be viewed as being “blocked” from terminally differentiating. Thus it is possible that GMV-infection and autocrine CSF production may complement known oncogenes in the transformation of primary hemopoietic cells. Moreover, the susceptibility of FD cells to transformation by GMV may be due to the fact that FD cells, being an immortalized cell line, have already undergone one step in the transformation process. Indeed, most leukemias, like most other cancers, are probably multi-stage diseases; the acquisition of independence of exogenous growth factors may comprise one such stage, as in the in vitro generation of tumorigenic cells in retrovirus-infected long term bone marrow cultures (Heard et al., 1984). Moreover, the spontaneous acquisition of factor independence, often paralleled by increased autocrine production of Multi-CSF (“PSF”) has been reported to correlate with the development of tumorigenicity in factordependent mast cell lines (Hapel et al., 1981; Schrader and Crapper, 1983) although these cells did not produce sufficient factor to allow autonomous proliferation in large culture volumes (Schrader and Crapper, 1983). Thus, our results, taken together with others, suggest that loss of

CSF Retrovirus 539

Transforms

Factor-Dependent

Cells

factor dependence can be an important stage in the malignant transformation of hemopoietic cells and may in some cases occur by autocrine synthesis of the factor. The correlation between tumorigenicity and factor independence of hemopoietic cell lines discussed above contrasts with the observation that primary myeloid leukemias (both murine and human) are dependent on exogenous CSFs for their proliferation in vitro (Metcalf, 1984). That such cells can behave as leukemic cells in vivo and still be dependent on exogenous CSF is apparently inconsistent with the failure of factor-dependent continuous hemopoietic cell lines to behave as leukemic cells when injected in vivo. However, it may be that primary leukemic cells proliferate in microenvironments that contain adequate levels of growth factors; these may be CSFs or other, as yet unidentified, factors. Subcutaneously injected FD cells, on the other hand, may not have access to sufficient CSF to ensure their continued growth. Also, it is probably significant that establishment of primary myeloid leukemias in continuous culture in vitro eventually results in loss of dependence on exogenous CSF and usually in endogenous synthesis of CSF (Metcalf, 1984; Metcalf and Nicola, 1985). This suggests that most primary myeloid leukemias are able to behave as malignant cells at what is actually a relatively early stage in their biological progression (Furth, 1953) and that acquisition of factor independence, continuous in vitro growth, and ready transplantability represent later stages in the evolution of leukemic cell populations. The only cases other than that described here, in which constitutive autocrine synthesis of a growth factor has been shown directly to cause malignant transformation, is that involving the sis oncogene. The v-sis oncogene of Simian sarcoma virus (SSV) encodes a factor that is either very similar or identical to PDGF (Waterfield et al., 1983; Doolittle et al., 1983). This factor appears to be secreted by SSV-transformed cells (Owen et al., 1984; Huang et al., 1984) although the proportion of the v-sis gene product secreted into the medium is low (Robbins et al., 1985). Furthermore, Gazit et al. (1984) and Clarke et al. (1984) have shown that the human c-sis gene can transform NIH3T3 cells when coupled to appropriate transcriptional and translational control elements. Most in vitro transformation studies with SSV or c-sis have used established fibroblast lines such as NIH-3T3. Although SSV can induce morphological transformation of primary fibroblasts (Aaronson, 1973), it does not appear capable of immortalizing such morphologically altered primary cells (S. Aaronson, personal communication). These observations closely resemble our results with GMV; GMV can transform to malignancy an immortalized cell line but not normal hemopoietic cells. Thus, autocrine stimulation of proliferation does not appear to result in immortalization. This is consistent with the idea that autocrine stimulation by growth factors may represent only one stage or component in the process of transformation to malignancy. Mechanism of Autocrine Stimulation by GM-CSF One somewhat surprising implication of the data

presented here is that the GM-CSF secreted by the GMVinfected cell lines may not be responsible for the independence of these cells of exogenous CSF. This is suggested by two characteristics of the GMV-infected FD cells. First, single cells could proliferate in liquid culture under conditions where the concentration of GM-CSF in the medium (secreted by the infected cells) would be too low to support proliferation of uninfected FD cells, and second, an antiGM-CSF serum failed to inhibit the growth of the GMVinfected FD cell lines. This latter result is in stark contrast to the work of Adkins et al. (1984) in which antibodies to cMGF inhibited the growth of virally transformed, factorindependent chicken cells that secreted cMGF. Similarly, antibodies to bombesin, a factor able to stimulate cells from many human small cell lung cancers, inhibit the proliferation of these cells (Cuttitta et al., 1985). We currently have no explanation for the different results obtained in these systems but suggest that an explanation will require a detailed understanding of the kinetics and mechanisms of processing of the factors and their receptors. Our observations are somewhat similar to those for certain SSV-transformed cells and an osteosarcoma cell line (U-2 OS C16) that secreted PDGF (Betsholtz et al., 1984; Huang et al., 1984). Huang et al. (1984) found that antiPDGF antiserum could only partially inhibit the growth of SSV-transformed NIH-3T3 and NRK cells that secrete the PDGF-like growth factor and failed to inhibit the SSV-NPl cells (which apparently do not secrete the factor). The importance of secreted v-sis gene product to the transformation of SSV-infected cells is also questioned by the recent findings of Robbins et al. (1985) that less than lo/o-2% of the total v-sis gene product is secreted. Moreover, antiPDGF antiserum did not inhibit the growth of U-2 OS Cl6 osteosarcoma cells, which both produce a PDGF-like factor and have functional PDGF receptors (Betsholtz et al., 1984). These results could imply either that synthesis of PDGF is irrelevant to the growth and/or transformed phenotype of these cells or that secretion of the factor into the medium is unnecessary for autocrine growth. Since the first alternative appears unlikely, especially in the case of the virally transformed cells, we will confine our discussion to the second. There are three possible explanations for the notion that growth factor secretion is not required for cellular growth and maintenance of the transformed phenotype. One is that the factor acts intracellularly and independently of normal receptors. This seems unlikely in view of available data on growth factor action and the apparent restriction of autocrine growth to cells possessing appropriate receptors. The second possibility is that the secreted growth factor interacts with the receptor immediately after the factors appearance at the cell surface, and that this interaction is favored over the interaction of the factor with antibody molecules. A third, and rather intriguing, explanation is that the growth factor binds to its receptor in an intracellular compartment before the receptor reaches the cell surface, as also suggested by Betscholtz et al. (1984). Since secreted and integral membrane proteins can be present in the same subcellular membrane compartments

Cell 540

(Strous et al., 1983; Goding, 1982) the ligand-receptor complex may be activated within the cell and, in the environment of an intracellular membrane, may be fully active. Alternatively, the ligand-receptor complex may be transported to the plasma membrane where it could function before being internalized. Finally, it is worth considering, in the light of the possible interaction of GM-CSF (or any growth factor) with its receptor inside the cell, the potential for differences between the effects of a growth factor synthesized within a responding cell and a factor present outside the cell. If a receptor can be occupied by its ligand within the cell, the capacity of the cell to restrict the formation of ligandreceptor complexes by internalizing the receptors is lost. Thus, one level of negative modulation of the cellular response to a growth factor would be abrogated in cells that exhibit autocrine stimulation of growth; this may have further implications for the growth regulation of such cells. Experimental

Procedures

Maintenance of Cell Lines Cells were routinely grown in Dulbeccos modified Eagles medium (DME) supplemented with 10% fetal calf serum. Where applicable for growth and clone selection, medium was supplemented with G418 at 400 rglml for ~2 cells and BALE/c 3T3 fibroblasts and at 1 mglml for FD cells. FD cells (Dexter et al., 1980; Hapel et al., 1984) were maintained with Multi-CSF as WEHI-3B(D-) conditioned medium at 10% culture volume. GM-CSF Assays Assays for GM-CSF levels in conditioned media were performed using microwell assays containing 200 FD cells (Gough et al., 1985) in 10 PI volumes of DME containing 10% FCS. Viable cell counts were performed after 48 hr of incubation at 3pC. Assays were performed in replicate wells using 5 JJI of serial twofold dilutions of the test material. Control cultures contained serial dilutions of GM-CSF purified from mouse lung-conditioned medium (Burgess et al., 1977). Assays for GM-CSF were also performed in 1 ml agar cultures of 75,000 C57BL bone marrow cells in DME containing a final concentration of 20% FCS and 0.3% agar. Serial dilutions of 0.1 ml of test material were added to the cultures prior to addition of the cell suspension in agar medium. The formation of granulocyte and/or macrophage colonies was scored after an incubation period of 7 days at 3PC (Metcalf, 1984). Units of CSF were calculated from the linear portion of the dose-response curve, assigning 50 units to the concentration stimulating the formation of half-maximal numbers of colonies. Clonal Culture of GMV-Infected FD Cells Suspensions of GMV-infected FD cells were washed and cultured in 1 ml agar medium cultures containing DME and a final concentration of 20% FCS and 0.3% agar. Cell concentrations cultured ranged from 1OJ-lo5 per ml. Replicate cultures contained the following combinations of agents: 400 units GM-CSF, 400 units GM-CSF plus 1 mg G418, 1 mg G418, or no addition. After 7 days of incubation, cultures were scored for colony formation. These colonies were compact and usually contained more than 1000 cells. No surviving cells were observed in intercolony areas, except were cells had previously been cocultivated with ~2 cells when occasional fibroblast-like aggregates were observed in the agar cultures. Individual G418-resistant and CSF-independent colonies were removed intact using a fine pipette and transferred to 1 ml cultures of DME. When the transferred colonies had generated a population of 0.5-1.0 x 10’ cells, the cloned cell population was transferred to 20 ml flat bottomed culture bottles and maintained with once or twice weekly medium changes. Control cloned cell lines were generated by removing G418-resistant colonies from cultures of FD cells infected with the

ZlPNeo virus GM-CSF.

and

maintained

Single Cell Cultures Individual uninfected or in pre-gelled 1 ml agar transfer pipette (Metcalf, clone sizes recorded at

in cultures

containing

GMV-infected cells were washed cultures over a reference circle 1984). Cultures were incubated intervals.

400 units/ml

then placed using a fine at 3pC and

Cell Growth Inhibition Experiments Rabbit anti-GM-CSF serum was prepared using recombinant GM-CSF (DeLamarter et al., 1985) and kindly supplied by Dr. C.-M. Liang (Biogen SA, Geneva). Fifty GMV-infected FD cells were put into a series of microwell cultures (as described for GM-CSF assays) containing different dilutions of the anti-GM-CSF serum. After 2 days of incubation, cell numbers were counted. Transplantation Tests GMV-infected FD cells were washed and 1 x 10e cells injected subcutaneously to groups of eight, 3-month-old DBAR mice. Mice were monitored twice weekly and, when moribund, killed and sectioned. Cells from tumor tissue, spleen, and bone marrow were cultured in 1 ml agar medium cultures lacking added GM-CSF to determine the frequency of clonogenic autonomous FD cells. For karyotype analysis 20 ~1 of a 1 mg/ml solution of vinblastine was added to the surface of 7 day cultures of transplanted tumor cells. After 3 hr of further incubation, colonies were removed using a fine pipette, pooled, and metaphase preparations made from the dispersed colony cells. DNA Transfections ~‘2 cells (Mann et al., 1983) were transfected with 1 rg of vector DNA and 8 Kg of high molecular weight carrier DNA per 50 mm petri dish using the calcium phosphate coprecipitation technique (Graham and van der Eb, 1973) as modified by Parker and Stark (1979) and Wigler et al. (1979). Forty-eight hours after transfection, cells were trypsinized and replated at a 1:20 dilution using medium containing G418. G418resistant colonies were picked and expanded after approximately 2 weeks. Viral Infections Infectious virus was collected as a 24 hr culture supernatant from the virus producing ~2 cells. To assay for infectious virus, 20% confluent 50 mm dishes of BALBlc 3T3 fibroblasts were exposed to 0.5 ml of a 1:lOO dilution of virus-containing culture supernatant in growth medium containing 4 rglml polybrene for 2 hr at 37X. The volume of growth medium was then increased to 4 ml for a 48 hr expression period after which the cells were trypsinized and replated using a I:20 dilution into G418containing medium. Resistant colonies were counted after approximately 2 weeks and titres expressed as G418-resistant cfulml of supernatant The nonadherent FD cells were infected either by exposure to w2 clone culture supernatant or by cultivation with the adherent ~2 cells. Infections of FD cells with virus-containing supernatants were carried out as for 3T3 infections, except that culture supernatant was used undiluted and supplemented with 10% WEHI-3B(D-) conditioned medium. After the 48 hr expression period cells were plated in soft agar as described below. Infections bycocultivation were implemented by adding 5 x 105 FD cells to lo%-20% confluent 50 mm dishes of the virus producer cells in DME supplemented with 10% FCS and 10% WEHI-3B(D-) conditioned medium in the presence of 4 pglml polybrene. After 48 hr, FD cells were collected, washed to remove residual Multi-CSF, and plated in soft agar as described below. Plasmid Construction pGMV was constructed by first ligating Barn HI linkers to the GM-CSF cDNAclone pGM517 (Gough et al., 1985) and subsequent ligation into the Barn HI site of pZIPNeo. Enzymic manipulations of DNA and preparation of plasmid DNA was performed according to standard procedures (Maniatis et al., 1982). Transcription Polyadenylated

Analysis RNA was isolated

and analyzed

using agarose-form-

CSF Retrovirus 541

Transforms

Factor-Dependent

Cells

aldehyde gel electrophoresis according to Gonda et al. (1982). RNA was transferred to nitrocellulose and hybridized with DNA fragments labeled with a[“P]dATP by nick translation (Rigby et al., 1977). Hybridization was performed in 6x SSC, 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, 0.2% ficoll, and 50 pglml denatured salmon sperm DNA for 16 hr to 20 hr at 65%. Northern filters were washed at 68% for 30 min in 0.2x SSC and 0.1% SDS, then for 30 min in 0.1x SSC prior to autoradiography. The neo probe used was the Hind Ill to Sma I fragment of pSV2neo (Southern and Berg, 1962). The GM-CSF probe was the Pst I to Eco RI fragment of a pUCB subclone of GM-CSF cDNA clone 3.2 (Gough et al., 1965). Analysis of Proviral Integration Cellular DNA was isolated according to Hughes et al. (1979) and digested with restriction endonucleases according to the manufacturer’s recommendations. Southern analysis (Southern, 1975) was performed using the hybridization conditions and probes described above for transcription analysis. Southern filters were washed at 68% in 2x SSC and 0.1% SDS for 30 min, then 0.2x SSC for 30 min prior to autoradiography. Acknowledgments We are indebted to Richard Mulligan (Whitehead Institute for Biomedical Research, Cambridge, Massachusetts) and his colleagues for providing us with the pZlPNeo SV(x) plasmid and with the ~2 cell line. We also thank: C.-M. Liang (Biogen SA, Geneva) for anti-GM-CSF serum, J. De Blaquiere, C. Quilici, and Y. Wiluszynski for excellent technical assistance; A. W. Burgess (Ludwig Institute, Victoria, Australia) for helpful discussion and with F. Walker (Ludwig Institute), for receptor binding data. S. Blackford, S. Belan, F! Smith, and A. May provided invaluable help in the preparation of the manuscript. D. M. was supported by the Anti-Cancer Council of Victoria, the National Health and Medical Research Council, Canberra, and the National Institutes of Health, Bethesda, Maryland, Grant No. 22556. R. A. L. was supported in part by a Commonwealth Post-Graduate Research Award. 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 indicate this fact. Received

August

Colbere-Garapin, F, Horoduiceanu, F., Kennilsky, P, and Garapin, A. C. (1981). A new dominant hybrid selective marker for higher eukaryotic cells. J. Mol. Biol. 750, 1-14. Cook, W. D., Metcalf, D., Nicola, N. A., Burgess, A. W., and Walker, F. (1985). Malignant transformation of a growth factor-dependent myeloid cell line by Abelson virus without evidence of an autocrine mechanism. Cell 41, 677-683. Cuttitta, F., Carney, D. N., Mulshire, J.. Moody, T W., Fedorko, J., Fischler, A., and Minna, J. D. (1985). Bombesin-like peptides can function as autocrine growth factors in human small-cell lung cancer, Nature 316. 823426. Davies, cloning

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factors

and

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