A retrovirus vector expressing the putative mammary oncogene int-1 causes partial transformation of a mammary epithelial cell line

Cell, Vol. 46, 1001-1009,

September

26, 1986, Copyright

0 1986 by Cell Press

A Retrovirus Vector Expressing the Putative Mammary Oncogene int-1 Causes Partial Transformation of a Mammary Epithelial Cell Line Anthony M. C. Brown,’ Robert S. Wildin:* Thomas J. Prendergast:§ and Harold E. Varmus’t Department of Microbiology and immunology t Department of Biochemistry and Biophysics University of California Medical Center San Francisco, California 94143 l

Summary In mammary tumors induced by the mouse mammary tumor virus (MMTV), the int-1 gene is frequently activated by adjacent proviral insertions and is thereby strongly implicated in tumorigenesis. To seek a direct biological effect of inf-1 that would validate its proposed role as an oncogene, we constructed a retrovirus vector containing the gene and examined its effects on tissue culture cells. Expression of inf-1 in a mammary epithelial cell line caused striking morphological changes, unrestricted growth at high cell density, and focus formation on a monolayer, although the cells were not tumorigenic in vivo. This partial transformation induced by inf-1 was not observed in cells infected by an otherwise identical virus bearing a frameshift mutation in the gene. These findings strongly support the hypothesis that inf-1 plays a functional role in MMTV-induced mammary tumorigenesis. Introduction Proto-oncogenes are normal cellular genes that can be converted to active components of a neoplastic process by mutations that alter their expression or protein products. Over 40 such genes have been identified thus far, on the basis of several criteria that vary in genetic rigor. On the one hand, the status of many proto-oncogenes is based upon the functional criteria that their mutant forms are the transforming genes of highly oncogenic retroviruses (Bishop and Varmus, 1985) or that their mutant alleles can transform cultured fibroblasts (Weinberg, 1982). On the other hand, the identification of other candidate proto-oncogenes relies on circumstantial evidence, most commonly the repeated occurrence of structural rearrangements in tumors (chromosomal translocations, gene amplifications, or proviral insertions), often in the absence of demonstrated biological activity (Varmus, 1984). The in&l gene was one of the first candidate protooncogenes of the latter type. The gene was implicated in carcinogenesis by the mouse mammary tumor virus (MMTV) because as many as three-fourths of mammary tumors in C3H mice contain MMTV proviral DNA within 15 kb on either side of the transcribed region of the in&l locus $ Present address: Department of Pediatrics, University of Washington Affiliated Hospitals, Seattle, Washington 98195. 5 Present address: School of Medicine, University of California, San Francisco, California 94143.

(Nusse and Varmus, 1982). Two additional facts have been used to argue for a functional role for in&l in mammary tumorigenesis: first, tumors with MMTV insertion mutations in the int-1 locus contain an average l-10 copies per cell of in&l mRNA, whereas no in&l RNA is detectable in normal mammary glands (Nusse and Varmus, 1982; Nusse et al., 1984); and second, proviral insertions may be within the transcription unit of in&l, but they never interrupt the protein coding sequence (van Ooyen and Nusse, 1984). Together, these findings imply that mammary tumors are clonal outgrowths of cells selected for insertion mutations that initiate production of in&l protein. Furthermore, the in&l gene shows other features compatible with its classification as a proto-oncogene, such as strong conservation during vertebrate evolution (Nusse et al., 1984; van Ooyen et al., 1985) and temporal regulation during mouse embryogenesis (Jakobovits et al., 1988). Similar arguments have been made for the proto-oncogenic character of int-2, another mouse gene insertionally activated during MMTV-induced carcinogenesis (Peters et al., 1983, 1986; Dickson et al., 1984; Jakobovits et al., 1986). Since their predicted protein products display no homologies with known proto-oncogene proteins or with each other (van Ooyen and Nusse, 1984; Fung et al., 1985; Moore et al., 1986), in&l and M-2 appear to be novel, if provisional, members of the proto-oncogene family. Confirmation of the claims that genes such as in&l are proto-oncogenes that can contribute to tumorigenesis requires direct demonstration that activated alleles have transforming activity in appropriate target cells. Since insertionally activated in&l genes in mammary tumors produce abnormal amounts of mRNA without alteration in protein coding sequence (Fung et al., 1985), we have tested an in&l gene, derived from a recombinant DNA library of mouse genomic DNA, for biological effects when it is efficiently expressed in cultured cells infected by a retrovirus vector transporting the gene. Unlike many oncogenes that are activated in a variety of tumor types and able to transform heterologous cells, the in&l gene has been implicated thus far only in mammary tumorigenesis. We show here that while expression of the gene does not alter the appearance of several fibroblast lines, it produces striking phenotypic changes in an established line of mouse mammary epithelial cells. The effects include several properties characteristic of transformed cells from other lineages, and the results strongly support claims that in&l is a novel proto-oncogene with the potential to contribute to neoplasia. Results Production of Recombinant Retroviruses Expressing inf-1 To investigate the phenotypic effects of an expressed in&l gene in a variety of cell types, we constructed a murine leukemia virus (MLV)-based recombinant retrovirus containing the in&l gene along with a neomycin phos-

Cell 1002

pMXint-l.neo

(MXIN)

pMXfs Int-l.neo

Barn HI KlWlOW

(MXfslN)

B.

virus

DNA

VlvJS

i!L!il 92

:’,:’ G+----+ -6.3kb1

C.

2

4.7kb3

4

c4.7kb

Figure

1. Retroviral

Vectors

Carrying

in&l and neo

(A) Structures of the plasmids pMXint-l.neo and pMXfsint-l.neo, used to generate the virus stocks MXIN and MXfslN, respectively. A Stul fragment of C3l-l mouse genomic DNA was ligated with Bglll linkers and then inserted into the retroviral vector plasmid pMX1122neo (see Experimental Procedures) to give pMXint-l.neo. To generate the frameshift mutation in in&l, pMXint-l.neo was cleaved at the unique BamHl site, treated with Klenow polymerase to fill in the Bamlil ends, and reclosed by ligation. The resulting construct, pMXfsint-l.neo, contains a 4 bp insertion within exon 2 of h-G-1 and an additional Clal recognition sequence at the site of the mutation. The exons of int-1 are represented by rectangular boxes, with solid black areas indicating the coding region, and the neo gene is shown by a stippled box. The sequence of the 4 bp insertion in pMXfsint-l.neo is indicated. Jagged lines indicate short segments of genomic DNA that flank the long terminal repeats (LTRs) and that are derived from the original pZlP MLV clone (see Experimental Procedures). Restriction sites: Bg, Bglll; Xb, Xbal; Ba, BamHI; E, EcoRI. So and SA mark the positions of the retroviral splice donor and acceptor sites, respectively. (6) Scheme for production of helper-free MXIN and MXfslN virus stocks. DNA of pMXint-1 .neo or pMXfsiWI .neo was introduced into PA12 cells by CaPO., transfection, and the cells were selected for resistance to G418. Amphotropic helper-free virus from these cells was then used to infect ~2 cells, again with selection for G418 resistance, and single colonies of infected ~2 cells were expanded into lines for production of ecotropic virus stocks. The expected structures of the proviral DNAs integrated after transfection or infection are shown, the latter having lost the introns of in&l, together with the predicted sizes of the respective LTR-LTY7 Xbal (X) fragments (see ]A]). The in61 coding region is represented by solid black boxes. (C) Southern blot analysis of proviral DNA in transfected and infected cells, showing loss of the introns of int-1 from the retroviral genome. Total cellular DNA was digested with Xbal, separated by agarose gel electrophoresis, transferred to nitrocellulose, and probed with 32P-

photransferase gene (neo), which confers resistance to the antibiotic G418 and can serve as a selectable marker. A 3.4 kb Stul fragment of cloned C3H mouse genomic DNA encompassing the entire open reading frame of in&l, but lacking the extreme 5’ and 3’ untranslated regions of the transcription unit, was inserted into the vector pMXl122nec to give a recombinant named pMX/nt-l.neo (Figure 1A). The pMXl122neo vector, constructed by M. Scott in this laboratory, retains the viral splice donor and acceptor sites normally used for production of env mRNA; hence, the proviral form of pMXin&l.neo should express in&l from a viral genomic-length RNA, whereas neo should be expressed from a spliced subgenomic message. We also made a control construct, pMXfsint-l.neo, in which a 4 bp insertion was introduced at the unique BamHl site in exon 2 of int-1 so as to cause a frameshift mutation close to the 5’ end of the protein coding region (Figure 1A). With the exception of this 4 bp insertion, pMXfsinr-1 .neo is identical to pMXinr-1 .neo. Helper-free virus stocks were derived from these reconstructed plasmids using the packaging cell lines PA12 and ~2, as shown in Figure 1B. Amphotropic viruses produced from transfected PA12 cells were used to infect ~2 packaging cells, and ecotropic virus stocks were ultimately obtained from clonal lines derived from single colonies of infected ~2 cells. Virus stocks derived from the plasmids pMXint-l.neo and pMXfsinr-l.neo were named MXIN and MXfslN, respectively, and they gave titers of 0.5-2 x lo5 colony-forming units per milliliter when assayed for transduction of G418 resistance. Confirmation of the Structure and Coding Potential of the Proviruses Since the inr-1 sequence introduced into the retroviral vectors was of genomic origin and contained three introns, we anticipated that the introns would be removed from the viral genomic RNA by splicing (Cepko et al., 1984) and hence would be absent from the integrated proviral DNA in infected cells. To investigate whether or not such splicing had occurred, we performed Southern analysis of the proviral DNA in transfected PA12 cells and the infected ~2 cells (Figure 1C). Annealing of an W-1 probe to Xbal digestion products of transfected PA12 cell DNA shows the expected 6.3 kb fragment, representing the intact recombinant provirus in the transfected pMXinr-l.neo plasmid (Figure lB), as well as a larger fragment from the endogenous in&l locus (Figure lC, lanes 1 and 2). In Xbal digests of DNA from a clonal line of MXIN-infected ~2 cells, however, the only novel inr-l-related fragment is 4.7 kb in length, the size expected for a Xbal fragment of a provirus from which the three introns of W-1 have been removed (Figure lC, lanes 3 and 4). Digests made with other restriction enzymes support this interpretation (data not shown). labeled in&l cDNA. Lanes 1 and 3, DNA of untransfected PA12 and uninfected ~2 cells, respectively, showing the endogenous genomic in&l fragment; lane 2, DNA from pooled PA12 cells transfected with pMXint-1 .neo; lane 4, DNA from a single colony of q12 infected with MXIN virus from the transfected PA12 cells. Arrows indicate the 6.3 kb and 4.7 kb proviral fragments corresponding to those diagramed in (6).

Transformation 1003

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proviral DNA from an infected ~2 line and then subjecting it to a direct test of coding capacity. Cloning of the proviral DNA was achieved by using the neo gene in the provirus as a selectable marker in Escherichia coli, and the irtt-1 coding region of the recovered clone was transferred to an SP6 transcription vector (see Experimental Procedures). When transcribed and translated in vitro, the resulting plasmid directed synthesis of a 37 kd polypeptide that comigrated on SDS-polyacrylamide gels with the translation product of a full-length in&l cDNA clone obtained after the initial construction of these viral vectors (Fung et al., 1985; data not shown). In addition, the products of both clones were recognized with similar efficiency in immunoprecipitations using antisera raised against synthetic jot-1 peptides (unpublished data). These results strongly support the conclusion that the introns were correctly spliced from the MXIN provirus, and they suggest that cells expressing MXIN RNA are also capable of expressing jot-1 protein.

Figure 2. Morphological Changes C57MG Mammary Epithelial Cells

Caused

by Expression

of in&l in

After infection with MXIN or MXfslN virus stocks, the cells were selected in G418 and were subsequently grown as pools of 50-100 infected colonies. (A) Uninfected C57MG cells. (8) C57MG cells infected with MXIN virus. (C) C57MG cells infected with MXfslN, a frameshift mutant control virus. All cells are shown at confluence.

A 4.7 kb Xbal fragment is also present in the DNA of the transfected PA12 culture; we assume that this is caused by infection of PA12 cells with the virus that they themselves produced, since the PA12 line is only weakly resistant to superinfection (Miller et al., 1986). Similar digests of genomic DNA from cells infected with the frameshift mutant control virus MXfslN indicated that the introns had also been removed from these proviruses (data not shown). Since the M-1 coding potential of the virus stocks produced from the infected ~2 cells is dependent on the precise removal of the three introns, we wished to test the fidelity of the splicing more rigorously by cloning the MXIN

Infection with MXIN Causes Morphological Changes in C57MG Cells Having verified that the spliced provirus within the infected ~2 lines had the expected genetic structure and coding capacity, we used virus stocks harvested from these cells to infect other cell lines. No morphological effects were evident following infection of ~2, NIH 3T3, BALE/c 3T3, or Rat-2 fibroblasts with either the virus containing wild-type inf-1 or that containing the frameshift mutant gene. Since the only neoplasms in which in&1 RNA has yet been detected are mammary carcinomas, it seemed appropriate to test for phenotypic effects of in&l in mammary epithelial cells. Accordingly, we infected the mammary epithelial cell line C57MG, derived from the normal mammary gland of a C57Bl/6 mouse (Vaidya et al., 1978). After infection with either MXIN or MXfslN, cells were selected for resistance to G418, and 50-100 resistant colonies were grown together as a mass culture. Uninfected C57MG cells form a monolayer with a regular and cuboidal appearance at confluence (Figure 2A). The cells infected with MXIN, however, exhibit a strikingly different morphology. The cells are more elongated and refractile, and they grow in a disordered pattern at confluence, with cells overgrowing one another (Figure 28). These morphological changes are dependent on the presence of a functional int-1 allele in the virus, since C57MG cells infected in parallel with the MXfslN frameshift mutant virus and selected under the same conditions are morphologically indistinguishable from the uninfected cells (Figures 2A and 2C). Similarly, when individual G418resistant colonies were picked and grown separately, the elongated and refractile morphology was evident in more than 90% of those infected with MXIN, but not in any of those infected with the control virus. Although no effects on morphology were evident after infection with MXIN of two other mammary epithelial cell lines, NMG (Owens et al., 1974) and Cl27 (Lowy et al., 1978), the morphological changes shown in Figure 2 were obtained using either of two different sublines of C57MG as the target cells. These

Cd 1004

Er

C57MG

A.

I

6.

$2

-II

12

34

Xbal

5

6

8

Xbal + BamHl+

Figure 3. Southern C57MG Cells

7

Blot Analysis

9

IO

nm

Xbal

EcoRl of Proviral

DNA in Infected

qr2 and

Cellular DNA was digested with Xbal (lanes l-4 and E-10) or Xbal. BamHI, and EcoRl (lanes 5-7); separated on 0.7% agarose gels; transferred to nitrocellulose; and hybridized with an in&l cDNA probe. The DNA was from MXIN-infected y2 cells (lane l), uninfected C57MG cells (lanes 2 and 5) or C57MG cells infected with MXIN virus (lanes 3 and 6) or with MXfslN virus [lanes 4, 7, and 10). The proviral Xbal fragment of 4.7 kb is indicated, as are the heavy bands of 14 kb and 2.7 kb derived from the endogenous int-1 alleles. The 3.2 kb and 1.5 kb fragments in lane 6 are produced by additional digestion of the MXIN proviral Xbal fragment with BamHI; these fragments are absent from lane 7, since the BamHl site in MXfslN was destroyed by the frameshift mutation. EcoRl was included in the digests shown in lanes 5-7 to prevent the major endogenous int-I fragment from comigrating with the proviral fragment of 3.2 kb. There are no sites for EcoRl within the MXIN provirus. Lane 6, DNA from C57MG cells infected with a stock of MXIN virus that produced a proportion of deletion variants giving a 3.7 kb Xbal fragment. DNA from a subculture containing only this deleted form of the provirus is shown in lane 9.

morphological changes represent the first phenotypic fects that can be ascribed directly to int-1.

ef-

Morphologically Transformed C57MG Cells Contain MXIN Proviruses and Express Proviral Transcripts of the Expected Sizes To confirm that the infected C57MG cells contain inr-1 proviruses of the expected structure, genomic DNA of pooled infected colonies was analyzed by Southern blotting (Figure 3). As expected, Xbal digests of DNA from the infected C57MG cells contained proviral fragments of the same size as those present in the ~2 virus-producer lines (Figure 3, lanes 1, 3, and 4). Additional digestion with BamHl confirmed that the BamHl site within in&l exon 2 in the MXIN proviruses was absent from the proviruses in cells infected with the frameshift mutant control virus MXfslN (Figure 3, lanes 6 and 7) the recognition sequence for BamHl having been disrupted by the 4 bp insertion that generated the frameshift. Although analysis of DNA from pooled cultures of MXIN-infected cells indicated that the majority of cells contained proviruses of the expected size, in some cases

1

12345 int-1

2 neo

probe

Figure 4. Northern and C57MG Cells

C57MG

$2

C57MG

nI

Blot Analysis

of Proviral

Transcripts

3

4

5

probe in Infected

~2

One-half microgram of poly(A)+ RNA was separated on a 1% agarose gel containing 2.2 M formaldehyde, transferred to a nylon membrane, and hybridized with an M-1 cDNA probe (A). After removal of the annealed in&l probe, the filter was rehybridized with a neo gene probe (B). RNA was from MXIN-infected ~2 producer cells (lane l), uninfected C57MG cells (lane 2), or C57MG cells infected with two different stocks of MXIN virus (lanes 3 and 4) or with MXfslN virus (lane 5). The 4.7 kb viral genomic transcript and 2.2 kb subgenomic mRNA are indicated, as is the minor 4.0 kb transcript discussed in the text. The 3.7 kb RNA in lane 4 probably originates from the partially deleted provirus detected in the same cell population (see Figure 3, lane 6).

we detected smaller Xbal fragments, of approximately 3.7 kb, in submolar quantities (see Figure 3, lane 8). This indicated that a small proportion of the cells had been infected with a deletion variant of the virus, or that deleted forms arose at low frequency during reverse transcription of viral RNA in the C57MG cells. From these pooled populations we subsequently isolated cell cultures showing only the 3.7 kb proviral Xbal fragment on Southern blots (Figure 3, lane 9); these cells appeared morphologically indistinguishable from uninfected C57MG cells. Although we have not determined the boundaries of the deletion precisely within the proviral DNA, further mapping suggested that it includes part of the int-1 coding region (data not shown). Thus, the morphologically transformed phenotype of infected cells appears again to require an intact inr1 coding region in the integrated provirus. We next studied by Northern blotting the RNA transcripts expressed from the proviruses in the infected C57MG cells. All the infected cultures contain a 4.7 kb poly(A)+RNA species that hybridizes with the in&l probe and comigrates with the major proviral transcript detected in the ~2 producer lines (Figure 4A). The size of this RNA is that predicted for the MXIN and MXfslN viral genomiclength transcripts. In addition, all of the infected cultures contain a less abundant class of in&l RNA that is approximately 0.7 kb smaller than the major species. While the smaller species was not investigated further, we suggest that it may consist of transcripts initiated downstream of the TATA sequence present at the extreme 5’ end of the

:ra6Tformation

by Mammary

Oncogene

Table 1. Saturation Cell Densities MXIN or MXfslN Retroviruses

int-1

of C57MG

Cells

Infected

Cell Line

Cells/cm2

C57MG uninfected C57MG/MXfslN, pooled colonies C57MGIMXfslN clone 2 C57MG/MXIN, pooled colonies C57MGIMXIN clone 486

1.5 1.4 1.4 5.1 7.4

x x x x x

with

105 10s 10s 1cP 10s

Cells were plated at a density of 104/cm2 in 5 cm dishes and were allowed to grow to confluence without a change of medium. The cells in two dishes of each cell line were counted at daily intervals after confluence until the numbers were no longer increasing. Standard deviations for the results shown were less than 5% in each case.

DNA in the retroviral constructs (van Ooyen and Nusse, 1984). The assumption that one or both of the 4.7 kb and 4.0 kb RNA species serves as a functional mRNA for in&l protein in C57MG ceils has recently been confirmed by immunoprecipitation studies with appropriate antisera (unpublished results). One of the cell populations infected with MXIN contained a third M-1 RNA species, approximately 3.7 kb in length (Figure 4A, lane 4). These were the same cells in which partially deleted proviruses were detected by Southern blotting (Figure 3, lane 8), and the extra RNA species corresponds in size to that predicted for a transcript expressed from these deletion variants. All of the RNA species that reacted with the M-1 probe were also detected when the filter was hybridized with a neo probe (Figure 48). In addition, this probe revealed a 2.2 kb RNA species in all of the virus-infected cells; the size of this species suggests that it is the expected subgenomic neo RNA produced by splicing between the retroviral splice donor and acceptor sites in the vector. The 4.7 kb viral genomic transcript and 2.2 kb putative subgenomic message are present in roughly equimolar quantities, which suggests that the splicing efficiency for these recombinant viral RNAs is comparable to that typically observed for wild-type MLV RNA in infected cells. in&l

Other Transformation Parameters Are Affected by W-1 Expression We next examined the morphologically altered MXINinfected C57MG cells for other changes in growth parameters and properties associated with neoplastic transformation. While we were unable to detect a difference in the growth rate of subconfluent cultures of C57MG cell8 after infection with MXIN virus (data not shown), the disordered appearance of these cells at confluence suggested that they were overgrowing one another and perhaps achieving a higher cell density than the monolayer formed by uninfected or MXfslN-infected cells. To test this, cells were grown to confluence in medium containing 10% serum, and their saturation densities were determined by counting cells on successive days. As shown in Table 1, the cells infected with MXIN grew to a final density 3-to 5-fold higher than that of uninfected C57MG cell8 or of cells infected with MXfslN. We also studied the relative levels of DNA synthesis in

Figure 5. C57MG Cells Infected with thesize DNA after Confluence while Quiescent

MXIN Virus Continue to SynUninfected Control Cells Are

Two days after reaching confluence the cells were allowed to incorporate 13H]thymidine for 24 hr, and labeled nuclei were subsequently detected by autoradiography. (A) Uninfected C57MG cells. (B) MXINinfected C57MG cells. Each panel shows a representative field of cells photographed under bright-field illumination. At the start of the labeling period, the cells shown in (6) were at a 3-fold higher density than those in (A), in which cells had already reached their saturation density.

these cells after they reached confluence by labeling with [3H]thymidine over 24 hr periods. Autoradiography of the labeled dishes showed striking differences in thymidine incorporation; a large fraction of cells infected with MXIN continued to synthesize DNA at least 3 days after confluence, while the uninfected control cells were quiescent (Figure 5). Together these data indicate that C57MG cell8 expressing in&l continue to replicate and divide under conditions that induce growth arrest in control cells. The cells infected with MXIN also acidified their growth medium more quickly than control cells did, and they were more easily detached from the culture dishes. In assays for growth in soft agar, however, the cells showed no significant anchorage-independence, nor did they show increased tumorigenicity in syngeneic or athymic mice. This was investigated by injecting 2 x 105-2 x lo6 cells subcutaneously in newly weaned C57Bl/8 females that were subsequently allowed to breed. No tumors were observed at 25 injection sites for either cell type after 7 months, although a different uninfected subline of C57MG cells previously shown to be tumorigenic gave palpable tumors within 2 months in the same assay. Similar injections of C57MG cells into athymic nulnu mice of either sex failed

Cell 1006

Figure 6. Focus Formation by MXIN Virus on a Monolayer of C57MG Mammary Epithelial Cells C57MG cells were infected with MXIN or MXfslN at low multiplicity of infection, grown without selection for G418 resistance, and allowed to remain at confluence for 2-3 days. The figure shows a typical focus in a dish infected with MXIN. No such foci were visible in dishes infected with the MXfslN control virus.

to show an increase in tumorigenicity MXIN (data not shown).

due to infection with

A Focus Assay for inf-1 Expression The morphological effects of an active i&-l gene in C57MG cells shown in Figure 2 were observed after MXIN-infected cells had been selected by virtue of their resistance to G418. To show that these changes were not dependent on removal of the surrounding uninfected cells, we performed experiments in which 102-lo3 MXINinfected cells were mixed with lo5 uninfected C57MG cells. Two days after the mixed population reached confluence, morphologically distinct foci were detectable amid the flat cuboidal monolayer of uninfected cells (data not shown). This result prompted us to test the effect of infecting C57MG cells with the in&l viruses without selecting for G418 resistance. The cells were infected at a density of 2.5 x lOYcm2 at low multiplicities of infection (1O-3 to 1O-2 G418-resistant colony-forming units per cell) and then allowed to grow to confluence without a change of medium. Two days after the dishes infected with MXIN reached confluence, morphologically transformed foci were again visible; they contained refractile cells that appeared to be piled up in relation to the surrounding monolayer (Figure 6). The number of foci per dish was within an order of magnitude of the titer of neo transducing virus used in each infection. No such foci were visible in dishes infected with similar titers of the control virus MXfslN, demonstrating that focus formation is dependent upon expression of a functional in&l allele. Discussion In this report we have shown that infection of a mouse mammary epithelial cell line with a retrovirus vector carrying an intact coding sequence for in&l protein produces

several changes in morphology and growth control that are characteristic of neoplastic transformation. This is the first demonstration that an oncogene identified solely by repeated rearrangements in tumor DNA (see Introduction) has transforming activity in cultured cells. As such, the results should encourage similar tests of other putative proto-oncogenes, such as int-2 (Dickson et al., 1984), pim1 (Cuypers et al., 1984) pvf/mis-1 (Villeneuve et al., 1986), bcl-1 (Tsujimoto et al., 1984), and others for which no direct biological assays are yet available. Our findings provide strong support for the hypothesis that in&l contributes to mammary tumorigenesis when the normally dormant gene is transcriptionally activated by proviral insertion mutations by MMTV In addition, the biological consequences of infection of this cell line by a virus bearing in&l have enabled us to design a convenient focus assay that should facilitate a systematic analysis of the gene. Virus Vectors for inf-1 To introduce in&l efficiently into a variety of cell types, we used a retrovirus vector containing the in&l gene together with a bacterial neomycin resistance gene, which was used as a selectable marker. Although the starting construct contained W-1 sequences of genomic origin, the three introns of the gene were accurately and efficiently spliced out of the RNA packaged into viral particles by transfected packaging cells (Figure l), as has been similarly reported by Rijsewijk et al. (1986). The retrovirus vector used here to express M-1 and neo retained the viral splice donor and acceptor sequences; hence, neo should be expressed from a spliced subgenomic message. While some other combinations of genes have impeded dual expression from related retroviral vectors (M. Scott, personal communication; S. Gerondakis, personal communication), the MXIN retrovirus described here expressed both in&l and neo in a very high percentage of cells selected for expression of the lat-

Transformation 1007

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in&l

ter marker, and the two expected transcripts were present at similar levels in infected cells (Figure 48). Although we observed some genetic instability of the recombinant retroviruses, in the form of provirus variants containing deletions in the W-1 region (Figure 3), these mutant forms typically represented less than 10% of the total virus in any one stock. A control virus, MXfslN, bearing a frameshift mutation close to the amino terminus of the int-1 coding region, as well as the spontaneous deletion variants of MXIN, failed to produce any detectable morphological changes or alterations in growth properties in infected cells, thereby confirming that the observed effects of the MXIN virus were absolutely dependent upon the production of int-1 protein. Transforms Mammary Epithelial Cells Infection of several rodent fibroblast cell lines with MXIN produced no discernable morphological change, despite documented expression of int-1 in such cells. Moreover, we have made numerous unsuccessful attempts to cotransform primary rat embryo cells with long terminal repeat (LTR)-driven int-1 and either a mutant c-Ha-ras gene or an efficiently expressed c-myc gene (unpublished results of G. Shackleford and H. E. V.). In view of such negative findings with cell types commonly used for assays of neoplastic activity in vitro, we tested three established lines of mammary epithelial cells as hosts for MXIN virus and observed transformation in one of them, regardless of whether the cells were initially selected for G418 resistance. The dramatic changes that followed infection of C57MG cells with MXIN are reminiscent of those traditionally associated with transformation of fibroblasts: alterations of cell shape and refractility; change from an orderly monolayer of cells to a multilayered array of randomly oriented and loosely adherent cells; unrestrained growth when cells are confluent, producing higher cell numbers at maximum density as well as acidification of the medium; and formation of dense foci of altered cells against a background of unaffected cells. These properties are not characteristic of primary cultures of mouse mammary tumor cells, but such cultures have very limited growth potential in vitro, whereas C57MG is an established line. Irrespective of the interpretation of the transformed phenotype caused by W-1 in C57MG cells, the apparent tissue specificity of the effect merits further investigation by infecting other cell types, including primary mammary cell cultures, with MXIN virus. Moreover, our results should stimulate efforts to use epithelial cell lines as recipients in gene transfer experiments designed to identify active oncogenes in carcinomas, particularly those for which fibroblast transformation assays have been unrewarding. MXIN-infected C57MG cells differ from fully transformed fibroblasts in that they fail to form colonies after suspension in agar, and they show no increased tumorigenie potential in either syngeneic or athymic mice. Since cells form MMTV-induced mouse mammary carcinomas are highly tumorigenic when transplanted, these findings imply that int-1 is itself not sufficient to induce the phenotype of a mammary carcinoma cell, despite levels of int-1 int-1

RNA in MXIN-infected cells that are at least lo-fold higher than those encountered in MMTV-induced tumors. This interpretation is consistent with the widely held view that MMWinduced tumorigenesis is a multistep phenomenon that requires the participation of genetic, hormonal, and perhaps environmental factors, as well as the virus itself (Nandi and McGrath, 1973; Hilgers and Bentvelzen, 1978). However, infection of primary mammary epithelial cells with MXIN virus and more widespread experience with epithelial cell transformation systems will be required to define the contribution of in&l to mammary tumorigenesis more rigorously. Our findings afford opportunities to pursue two important additional issues. First, the high efficiency of focus formation by MXIN on monolayers of C57MG provides a convenient test for biological activity after site-directed mutagenesis of W-1 DNA. Second, if MMTWnduced tumorigenesis is indeed a multistep pathway involving additional genetic changes beyond the activation of i&l, then conversion of MXIN-infected C57MG cells to a tumorigenic state might be an appropriate assay for detection of other active oncogenes in mouse mammary tumor DNA. Recent evidence for insertional activation of both in&l and M-2 in a substantial number of mammary tumors in BR6 mice (Peters et al., 1986) suggests that the two genes may cooperate in tumorigenesis. Introduction of a transcriptionally activated M-2 locus into MXIN-infected C57MG cells might provide a direct test of this notion. Experimental Procedures Production of Retroviruses Carrying id-1 and IWO To generate pMXin&l.neo a 3.4 kb Stul fragment containing the in&l coding region was isolated from a 1 phage clone of mouse strain C3l-l genomic DNA, ligated with Bgiii linkers, and inserted into the retroviral vector plasmid pMX112Pneo (provided by M. Scott). Ail MLV sequences in this plasmid are derived from the Moloney MLV clone pZlP (Hoffman et al., 1982; M. Scott and H. E. V., unpublished results). The vector retains the normal viral splice donor and acceptor sites, and it carries a transposon Tn5 neo gene in place of the viral env sequences. The initiator AUG of p65sag, together with most of the MLV gag@ region, is deleted, leaving cloning sites for Xhol and Bglii in this position. The C3H genomic clone of h-1 was provided by G. Shackleford. Helper-free stocks of the viruses MXIN and MXfsiN were derived from the plasmids pMXint-l.neo and pMXfs.int-l.neo, respectively. The plasmids were introduced into the amphotropic packaging cell line PA12 (Miller et al., 1985) by CaP04 transfection (Graham and van der Eb, 1973) followed by selection for resistance to G418. Helper-free virus produced by a pool of these transfected ceils was then used to infect the ecotropic packaging line ~2 (Mann et al., 1983), again with selection for G418 resistance. Single colonies of infected ry2 cells were expanded into lines, and the helper-free virus produced was harvested for subsequent infection of other cells. Virus Infections and Cell Culture PA12 and ~2 cells were obtained from A. Miller and R. Mulligan, respectively, and were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum. For infection of ~2 or C57MG cells, virus-containing culture medium supernatants were diluted with fresh medium, polybrene was added to 8 flglmi, and 2 ml of the mixture was applied to 3 x lo5 cells in a 5 cm dish for 2 hr. One day later the cells were split into medium containing 200-400 pg/ml (net) G418. The resulting G4Wresistant colonies were either grown as a mixed pool of 50-100 colonies or were picked individually with cloning cylinders and grown independently. Virus stocks were titered by determining their G418-resistant transduction frequency upon infection of susceptible target cells.

Cell 1008

Clone D2 of the C57MG mammary epithelial line (Vaidya et al., 1978) was obtained from A. Vaidya and was recloned to minimize the frequency of spontaneous morphological variants. Clones were chosen that were nontumorigenic in syngeneic mice. The cells were maintained in DMEM containing 4.5 g/l glucose, 10% fetal calf serum, and 10 pg/ml insulin, and they were typically passaged every 5 days. For saturation cell density determinations, 2 x IO5 cells were plated in 5 cm dishes and were left to grow to confluence without additional changes of medium. On the first day that the cells appeared confluent, two dishes of each cell line were rinsed in EDTA/saline, and total cell numbers were determined using a Coulter counter. Additional dishes were then counted on subsequent days until the cell numbers no longer increased. Focus formation was achieved by infecting 5 x IO4 C57MG cells in a 5 cm dish with 10*-l@ MXIN virus particles for 2 hr and allowing the cells to continue growing in the same dish in the absence of G418. Foci were visible to the naked eye 2 days after the monolayer appeared confluent. Growth in soft agar of C57MG cells infected with MXIN or MXfslN virus was assayed by embedding 103-IO6 cells in 0.35% agar (Difco) in DMEM plus supplements, over a substratum of 0.5% agar. No significant colony formation was observed after 6 weeks of incubation; as a positive control, mouse fibroblasts transformed with v-src produced visible colonies within 1 week. Autoradiography Cells were grown as described for saturation density determinations. At various times after confluence, 13H]thymidine (Amersham; 86 Cilmmol) was added to the existing culture medium to a final concentration of 10 &i/ml. Twenty-four hours later the cells were rinsed with Tris-buffered saline, fixed in 3.7% formaldehyde for 10 min. and washed sequentially with 5% trichloroacetic acid, water, and 70% ethanol. After drying, the dishes were coated with Kodak NT62 photographic emulsion, dried again, and left in the dark for 2 days. The emulsion was then developed, and the cells were stained briefly with 2% methyl green. The total cell numbers in dishes grown in parallel were determined before and after the labeling period. Hybridization Procedures Southern blotting and hybridization of nitrocellulose filters with nicktranslated int-1 cDNA probes were performed by standard procedures (Maniatis et al., 1982). For Northern blots, RNA was separated on agarose-formaldehyde gels and then transferred to a nylon membrane (Amer$ham Hybond-N). By the procedure of Church and Gilbert (1984), the RNA was cross-linked to the membrane using ultraviolet radiation and then hybridized to nick-translated probes at 65°C in a solution containing 0.5 M NaHP04 (pH 7.2), 7% SDS, 1% bovine serum albumin, and 1 mM EDTA. Washes were performed in 40 mM NaHP04, 1% SDS, and 1 mM EDTA at 55OC. Before rehybridization of the membrane, the annealed probe was removed by boiling in distilled water. Cloning of MXIN Provlral DNA and In Vitro Synthesis of inf-1 Protein Cloning of the proviral DNA lacking the introns of in&l was achieved by using the neo gene in the provirus as a selectable marker in E. coli: size-fractionated Xbal genomic DNA fragments were cloned directly into a plasmid vector, and the desired clone was selected in medium containing kanamycin. Genomic DNA from a ~2 cell line infected with MXIN was digested with Xbal, and a fraction enriched for fragments of the desired molecular weight was obtained by preparative gel electrophoresis in 0.6% low-melting-temperature agarose (SeaPlaque). One microgram of this fraction was ligated to 1.5 pg of pMXOO2 vector DNA linearized with Xbal and treated with alkaline phosphatase. pMX002 is a pBR322 deletion derivative carrying a solo MLV LTR (M. Scott, unpublished experiments); the unique Xbal site in the vector is therefore equivalent to the Xbal site in each LTR of the MXIN provirus. Competent E. coli HE101 (1.25 ml), sufficient to give 2.5. x IO7 ampicillin-resistant colonies from 1 w of supercoiled pMXOO2 DNA, were transformed with 1 m of the ligation products, divided into aliquots of different volumes, and incubated at high cell density for 4 hr at 3PC to allow expression of kanamycin resistance. The bacteria were then diluted into a total volume of 700 ml of L broth containing 50 fig/nil

ampicillin and 7.5 pglml kanamycin sulphate, and shaken at JIOC. The bacterial promoter of the neo gene is not present in the pMX1122neo vector or in pMXint-l.neo, but in E. coli both plasmids nevertheless confer resistance to low levels of kanamycin (5-10 pglml) that are sufficient for selection. From the cultures that grew to saturation density within 24 hr, we recovered clones of the expected neo-containing MXIN proviral fragment in the pMX002 vector, and in all cases the inserted fragment was in the correct orientation to reconstitute a two-LTR retroviral vector plasmid suitable for reintroduction into mammalian cells. One of these proviral clones was designated pMXIN. The plasmid exhibited genetic instability in HElOl, but it was stable when introduced into the recA deletion strain DKl. The int-1 coding region of pMXlN was transferred to pSP65 on a Bglll fragment, and the resulting clone was transcribed and translated in vitro as previously described (Fung et al., 1985). Acknowledgments We wish to thank Mike Scott, Greg Shackleford, Larry Donehower, and Mike Verderame for helpful advice and gifts of materials, and Akhil Vaidya for supplying the C57MG cell line. We are particularly grateful to Mike Scott for the pMX1122neo vector. A. M. C. B. was supported by a Medical Research Council Travelling Fellowship and is now a Special Fellow of the Leukemia Society of America. H. E. V. is an American Cancer Society Research Professor. This work was supported by grants from the National Institutes of Health. 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

May 30, 1986; revised

July 14, 1986.

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and chemically

in-