Tumorigenesis by mouse mammary tumor virus: Evidence for a common region for provirus integration in mammary tumors

Cell, Vol. 33, 369-377,

June 1963, Copyright

0 1963 by MIT

0092.8674/83/060369-09

$02.00/O

Tumorigenesis by Mouse Mammary Tumor Virus: Evidence for a Common Region for Provirus Integration in Mammary Tumors Gordon Peters, Sharon Brookes, Rosalind Smith, and Clive Dickson Department of Viral Carcinogenesis imperial Cancer Research Fund Laboratories P.O. Box 123, Lincoln’s Inn Fields London, WC2A 3PX, England

Summary We have prepared specific probes for unique-sequence cellular DNA adjacent to each of the newly integrated proviruses in tumors induced by mouse mammary tumor virus (MMTV). The use of such probes to screen a large number of independent mammary tumors in the BR6 strain of mouse has indicated that in at least 17 out of the 40 tumors examined so far, an MMTV provirus has integrated into a common chromosomal domain. A 10 kb Eco RI fragment of single copy DNA from this region has been isolated and partially characterized by restriction enzyme mapping. Of the proviruses located within this fragment in different tumors, all but one are complete, in the same orientation, and clustered within about 3 kb of cellular DNA. These findings are consistent with an insertional mutagenesis model for tumorigenesis by MMTV, in which the integration of a provirus in a particular region of cellular DNA may activate a neighboring oncogene. The region we describe here appears to be different from that reported for mammary tumors in the C3H strain of mouse. Introduction Mouse mammary tumor virus (MMTV) is a milk-borne, Btype retrovirus that has been identified as at least one of the causative agents in the high incidence of mammary carcinomas sustained by several inbred strains of mouse (Moore et al., 1979; Cardiff and Young, 1980; Michalides et al., 1981). Based on our present understanding of retroviruses, the most tenable model for the tumorigenic potential of MMTV is that of insertional mutagenesis (Jenkins et al., 1981; Noori-Daloii et al., 1981; Varmus et al., 1981; Payne et al., 1982). Such a model envisages that the chance integration of a DNA provirus in the vicinity of a specific cellular gene may perturb the expression of that gene. The existence of potential oncogenes in the DNA of normal somatic cells has been amply demonstrated, both by analyses of the transforming sequences acquired by the acutely oncogenic retroviruses, and by DNA transfection experiments (Weinberg, 1981; Bishop and Varmus, 1982). Thus neoplasia is thought to result from aberrant expression of such a gene, either at abnormally high levels or in some altered or mutated form (Hayward et al., 1981; Payne et al., 1982; Reddy et al., 1982; Tabin et al., 1982; Taparowsky et al., 1982). As well as attempting to verify whether an insertional mutagenesis model might apply to

MMTV, the present study was motivated by the prospect that any gene implicated in mammary carcinogenesis may prove to be novel, and could play an important role in the proliferation of mammary epithelial cells. Although MMTV differs from other retroviruses in several respects, particularly its tissue specificity, some features of the system encourage the belief that it may act via a cellular oncogene in a similar fashion to that originally described for avian leukosis virus (ALV) and the cellular gene c-myc (Fung et al., 1981; Hayward et al., 1981; Neel et al., 1981; Payne et al., 1981). For example, the viral genome does not appear to contain any specific oncogenie sequences, as evidenced by its inability to transform cells in culture. The viral replicative genes (gag, pal, and env) and the additional coding capacity unique to the long terminal repeat (LTR) of the MMTV provirus (indicated as off in Figure I, Dickson and Peters, 1981; Donehower et al., 1981; Sen et al., 1981; Fasel et al., 1982; Kennedy et al., 1982; Peters et al., 1982) may contribute to some aspects of tumorigenesis. It should be noted, however, that while a high percentage of cells in the mammary gland may be productively infected by the virus and express these genes, the vast majority of these cells remain phenotypically normal (Cohen et al., 1979; Cardiff and Young, 1980). Virally induced tumors arise only after a latency of around 6 to 9 months, and as a general rule, appear to contain one or more additional proviruses as compared to uninfected tissues (Cohen et al., 1979; Cohen and Varmus, 1980; Fanning et al., 1980; Groner et al., 1980; Morris et al., 1980; Nusse and Varmus, 1982). The fact that new proviral elements can be detected against the background of endogenous MMTV sequences indicates that tumors are derived from one, or at most a few, transformed cells (Cohen et al., 1979; Moore et al., 1979; Cardiff and Young, 1980). A prediction of the insertional mutagenesis model is that in all virally induced tumors, at least one of the proviral elements should reside in a specific region of chromosomal DNA. Our aim was therefore to isolate recombinant DNA clones containing the cellular DNA adjacent to the proviruses in one or more tumors, and to use these as probes for screening a large number of independent, spontaneous tumors. Using this strategy, we have obtained evidence for a region in the mouse chromosome, extending for approximately 24 kb, in which an MMTV provirus has integrated in 17 out of the 40 tumors examined.

Results Cloning of Virus-Cell Junctions We initially chose to examine tumors induced in the BALB/ c strain of mouse, because of the relatively simple and well documented pattern of endogenous MMTV sequences present in the germ line of this strain (Cohen and Varmus, 1979; Morris et al., 1980; Traina et al., 1981). DNA was extracted from a number of tumors, digested with the restriction enzyme Eco RI, and analyzed by agarose gel electrophoresis and Southern blotting (Southern, 1975;

Cell 370

Maniatis et al., 1982). Eco RI has a single recognition site close to the midpoint of the MMTV provirus (Cohen et al., 1979; and Figure 1) so that each complete proviral unit yields two characteristic fragments, representing the 5’ and 3’ virus-cell junctions, the sizes of which will be determined by the position of the neighboring Eco RI sites in the cellular DNA. The two fragments can be distinguished on the basis of hybridization to the 5’- or 3’specific probes illustrated in Figure 1. As an example, Figure 2 shows the analysis of DNA from four independent BALB/c tumors using a 3’specific probe. In non-tumor tissue, in this case the spleen (lane S), MMTV sequences are detected corresponding to the endogenous proviral units common to all tissues of this strain. In BALB/c’mice, these are represented by 3’ junction fragments of 6.2 kb (Unit Il/mtv-8) and 9.2 kb (Unit Ill/mtv-9) (Traina et al., 1981). In the mammary tumors, on the other hand, additional fragments are clearly discernible, superimposed on the endogenous pattern. That such bands are detected at all is evidence for the clonality of the cells making up the tumor. Ideally, we hoped to find a tumor in which there was only a single new provirus which could be implicated in tumorigenesis, but in this experiment and in the subsequent screening of a large number of spontaneous tumors, such a situation proved to be extremely rare. Most tumors contained between two (Figure 2, lanes 2 and 4) and six (Figure 2, lane 3) newly acquired proviruses. In view of its relative simplicity, and the fact that the 3’ end fragments were in the optimal size range for cloning into particular X vectors, we chose to concentrate on tumor 4, although similar, less complete studies have been conducted with the others. DNA from tumor 4 was digested to completion with Eco RI and the resultant fragments were ligated into the separated arms of the EK2 vector Xgt

WES. XB (Maniatis et al., 1982). After in vitro packaging, the recombinant phages were plated on E. coli LE392, and screened, using an MMTV LTR probe (Figure 1) to detect both 5’ and 3’ ends of the provirus. From a total of approximately 8 x 1 O5 plaques, in two separate experiments, we obtained 13 authenticated clones of virus-cell junctions, of which seven were endogenous 3’ ends, and one an endogenous 5’ end. In agreement with the reports from other laboratories (Groner et al., 1980; Ucker et al., 1981; Donehower et al., 1981; Majors and Varmus, 1981) no clones corresponding to the 5’ ends of exogenously transmitted MMTV proviruses were recovered in this experiment. The other positive recombinants corresponded to three and two copies, respectively, of the 6.2 kb and 7.0 kb 3’ junction fragments characteristic of the newly acquired proviruses in tumor 4. In Figure 2, the 6.2 kb fragment was obscured by endogenous sequences.

MS1234

23

9.4 6.6

4.4

2.3 2.0 .

Figure 1. Restriction the MMTV Provirus

Enzyme

Map and Derivation

of Specific

Probes

for

The organization of the MMTV genome RNA and its relationship to the double-stranded DNA provirus is depicted schematically-the boxed sections represent regions from the 5’ and 3’ ends of the viral RNA, which are duplicated in the formation of the long terminal repeat (LTR) segments. The location of cleavage sites for the enzymes Eco RI (RI) and Pst I (P), relevant to the derivation of 5’, 3’. and LTR-specific probes, are indicated. The respective Pst I to Pst I or Eco RI to Pst I fragments were obtained from unintegrated viral DNA from the GR strain of milk-borne MMTV, and cloned into the appropriate sites in the drug-resistant plasmid vector pAT153 (unpublished data).

Figure 2. Analysis of MMTV Proviruses Induced in BALB/c Mice

in the Spleen and Mammary

Tumors

High molecular weight DNA (15 pg) from the spleen (lane S) and four virally induced mammary tumors (lanes 1 through 4) from BALB/c mice was digested with Eco RI, fractionated by electrophoresis in a 0.6% agarose gel, and transferred to nitrocellulose (Southern, 1975). The nitrocellulose filter was then hybridized with a “P-labeled plasmid probe specific for the 3’ end of the MMTV provirus (Figure 1). Each band on the autoradiograph should therefore correspond to a single provirus. except in sttuations where similarly sized fragments comigrate (e. g.. the 6.2 kb fragment in tumor 4). Fragments of bacteriophage A DNA digested with Hind III were used as molecular weight markers (lane M). The sizes of these standard fragments are indicated in kilobases.

MMTV Integration 371

Site in Tumors

Characterization

of Virus-Cell Junctions

Each of the cloned junction fragments was transferred into the plasmid vector pAT153 (Maniatis et al., 1982) to facilitate preparation of the DNA, and characterized by restriction enzyme digestion and Southern blot analysis with MMTV-specific probes. This permitted the construction of restriction maps for the two prototype clones (401 and 408) of the newly integrated proviruses (Figure 3). Such analyses confirmed that the viral sequences present in each junction fragment were intact, corresponding to the exogenous virus used to induce the original tumor, and clearly different from either of the endogenous proviral units (unpublished data). In addition, these analyses identified restriction fragments specific for the cellular DNA flanking the virus in each clone. Selected fragments of the cellular DNA were then isolated, and screened for the presence of repetitive sequence elements by hybridization

to total BALE/c spleen DNA. In the case of 401, a 0.8 kb Sau 3A fragment of unique-sequence DNA was chosen and subcloned into the Barn HI site of pAT153 to generate plasmid 411. With 408, no repetitive elements were detected, and a subclone of the 1.75 kb Eco RI to Sac I fragment was prepared in an appropriate pAT153-based vector, pSP6 (J. Jenkins, personal communication), to generate plasmid clone 418 (Figure 3). As a demonstration that these probes recognized unique-sequence DNA, plasmids 411 and 418 were labeled by nick translation, and hybridized to an Eco RI digest of DNA from tumor 4 and from the spleen of the same animal. As shown in Figure 3, 418 recognizes a 9IO kb Eco RI fragment (accurately measured as 10 kb in later experiments) in spleen DNA, representing the normal, unoccupied site in the chromosomal DNA before provirus integration (lane S). In the tumor (lane T), this fragment is s

r

9.4KUnoccupied 401

site

4.4-

2.32.o0

I1

2I

3I

4I

5

6

:

kb S

T

23-

408

b$j-+

9.46.&

s

CI

-Unoccupied

site

-408

4.4-

410

2.32.0-

Figure 3. Characterization

of Cloned Virus-Cell Junction

Fragments

from Tumor 4

The cloned DNA fragments spanning the virus-cell junctions in tumor 4, represented by the prototype clones 401 and 408, were isolated and mapped by digestion with various combinations of the following restriction enzymes: Eco RI (RI), Hind Ill (H), Ram HI (B), Pst I (P), Sac I (S), Cla I (C), and Bgl It (Bg). No sites were detected for the enzymes Xho 1, Xba I, Sat I, and Kpn I. The size of each digestion product was calculated, relative to a series of standard fragments derived from plasmid DNA of known sequence, and viral-specific fragments were identified by blot hybridization using a cDNA probe. Viral-specific sequences are represented by the heavier line to distinguish them from cellular DNA (thin line) and the LTFt segment (boxed). Restriction sites mapped within viral sequences were in perfect agreement with those predicted from the DNA sequence of this region of the MMTV provirus (Fasel et al., 1982; Redmond and Dickson, 1983). Probes specific for unique-sequence cellular DNA flanking each provirus were prepared by subcloning a 0.8 kb Sau 3A fragment from 401, and a 1.75 kb Eco RI to Sac I fragment from 408 (indicated as stippled bars in the figure) into appropriate plasmid vectors, The specificity of these probes, designated 411 and 418, respectively, was assessed by hybridization to Eco RI digests of spleen (S) and tumor 4 (T) DNA as shown in the insets. As predicted, each probe recognized fragments of cellular DNA corresponding both to the novel virus-cell junction and to the unoccupied site as it occurs on the other chromosome or in uninfected cells.

Cell 372

present on only one of the two chromosomes, the other having been interrupted by the provirus to generate the new 7.0 kb fragment, corresponding to clone 408. If the tumor were truly clonal, the intensity of the virus-cell junction band on one chromosome should be equivalent to that of the unoccupied site on the other. The presence of any normal tissue (for example, stroma) in the initial tumor biopsy would obviously reduce the relative intensity of the interrupted site, as appears to be the case with the 418 probe. With the 411 probe, no such conclusions can be drawn, since both the occupied and unoccupied sites yielded Eco RI fragments of the same size (Figure 3).

Screening of Multiple Tumors If an insertional mutagenesis model were to apply, then one of the proviruses represented by clones 401 and 408 must presumably have been a causative agent in the induction of tumor 4. Thus either the 6.2 kb Eco RI fragment detected by the 411 probe or the 10 kb Eco Ri fragment detected by 418 should correspond to a region of chromosomal DNA in which one might expect to find integrated MMTV in other virally induced tumors. To test this prediction, we analyzed a total of 39 spontaneous mammary tumors obtained from the BR6 strain of mice (kindly provided by Dr. Audrey Lee, of this institute). The BR6 strain was originally derived from a cross between C57BL and Rlll mice, and has since been maintained as an inbred colony (Foulds, 1949). Females of this strain develop a very high incidence of both hormone-dependent and hormone-independent mammary tumors (Foulds, 1949) presumably as a result of the milk-borne infectious MMTV characteristic of the Rlll strain. High molecular weight DNA was prepared from each of the tumors, digested with Eco RI, and examined by blot hybridization with either 411 or 418 as probe. With the 411 probe, no fragments other than the normal 6.2 kb unoccupied site were detected in any tumor (data not shown). However, with the 418 probe, in addition to the 10 kb unoccupied site, novel bands were observed in 11 out of the 39 BR6 tumors examined (see below for examples). The use of the enzyme Eco RI restricted the range of the 418 probe to only 10 kb of cellular DNA. By testing a number of alternative enzymes, it became clear that the range could be extended to around 24 kb by using Xba I to digest the DNA, prior to Southern blotting. By effectively doubling the scope of our analyses in this way, five more examples of provirus integration in this region were identified, bringing the total number of positive tumors to 17 out of the 40 tested.

identified in addition to the two original copies of 408. The DNA insert was isolated and characterized by restriction enzyme mapping, as indicated in Figure 4. The entire 10 kb fragment proved to be free of repetitive sequence elements, and was therefore used as a hybridization probe to examine the organization of MMTV proviruses within this region in different tumors.

Organization of MMTV Proviruses in the 418 Region As shown in Figure 5, Southern blots were prepared of Eco RI-digested DNA from 11 mammary tumors in which an MMTV provirus was shown to have integrated within the 10 kb region recognized by the 418 probe. Lane A in each panel corresponds to the original tumor 4 from a BALB/c mouse, whereas lanes B through K represent independent, spontaneous tumors from BR6 mice. This strain difference is reflected in the patterns of endogenous MMTV sequences, detected using either 3’specific (Figure 5a) or 5’specific (Figure 5c) viral probes. In BALB/c mice, the endogenous units are present on 3’specific fragments of 9.2 kb and 6.2 kb, and 5’specific fragments of 7.6 kb and 7.2 kb (Traina et al., 1981). BR6 mice, on the other hand, contain two 7.8 kb and one 6.2 kb 3’specific fragments, and 9.8 kb, 7.9 kb, and 7.6 kb 5’specific fragments. These virus-specific probes also indicated that each tumor contained one or more newly acquired MMTV proviruses superimposed on the respective endogenous patterns (Table 1). The same nitrocellulose filters that were used for Figures 5a and 5c were rinsed in 30 mM NaOH to remove residual viral probe, and rehybridized to either 418 (Figure 5b) or to the entire 10 kb Eco RI fragment representing the unoccupied 418 site (Figure 5d). As shown in 5b, probe 418 identified the expected 10 kb fragment in the uninterrupted chromosome in each tumor, whether of BALB/c or BR6 origin, plus at least one novel fragment. Most of these novel fragments were reproducibly smaller than the IO kb unoccupied site and therefore did not reflect incomplete digestion of the tumor DNA. Moreover, with the exception of the sample shown in lane H, all of the novel fragments also contained

0

I?

I.

5

6

78

0

Cloning and Characterization of the Unoccupied Integration Site

Figure 4. Restriction

Having established that the chromosomal site recognized by the 418 probe is frequently interrupted by integration of MMTV DNA, we decided to characterize the region further, particularly the unoccupied, 10 kb Eco RI fragment. The original recombinant phage library from tumor 4 was rescreened with the 418 probe, and a positive clone

The 10 kb Eco RI fragment recognized by the 418 probe was isolated from the original recombinant phage library from tumor 4, and characterized by restriction mapping. The recognition sites for the enzymes Eco RI (RI), Pst I (P), Sac I (S), Barn HI (B), Bgl II (Bg), and Kpn I (K) are indicated; no sites were detected for Xba I, Xho I, Cla I, and Sal I. The orientation and location of the proviruses in ten of the tumors from Figure 5 are shown above the map. The letters refer to the respective lanes in Figure 5.

Map and Location

of Proviruses

‘:

Lb

in the 418 Region

MMTV Integration 373

Sate in Tumors

a A

b 6

C

D

JK

EFGHl

A

B

C

DEFGHIJK

-2.3 -2.0 418 Probe

3’ Probe

d

C A

B

C

D

E

ABCDEFGH

FGHIJK

1

J

K

-23 a WYam

-9.4 urn -6.6

-4.4

-2.3 -2.0 5’ Probe Figure 5. Analysis

of Tumors Containing

10kb an MMTV Provirus

Probe

within the 418 Region

DNA from 11 selected mammary tumors was dtgested with Eco RI, fractronated by electrophoresis in agarose gels, and transferred to nitrocellulose filters. The filters were then hybridized successively to probes for 3’specific viral sequences (a), the 418 fragment (b). 5’specific viral sequences (c), or the 10 kb Eco RI fragment representing the unoccupied site for the 408 provirus (d). Lane A in each panel corresponds to the onginal tumor 4 from a BALB/c mouse (see Figure 2), whereas lanes B through K represent independent, spontaneous tumors from BR6 mice. The sizes of the various fragments were calculated relative to a Hind III digest of X DNA, indicated in kilobases on the right of b and d.

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Table 1. Sizes of Novel Restriction Independent Tumors

Fragments

Detected

Approximate Sizes (in kb) of Novel Fragments the Indicated Probes

in 11

Detected

with

Tumor

3’Specific Virus Probe Fig. 5a)

A

7.0, 6.2

13.0, 10.0

13.0, 7.0

B

7.4

7.4

12.5

12.5, 7.4

C

7.0, 6.4

6.4

13.5, 11.0

13.5,6.4

D

4.7

7.8’

11.9

I I .9, 7.8’

E

4.8

14.7, 5.8

14.7,4.a

F

5.6, 4.9

5.6

14.2, 5.9

14.2, 5.6

G

ND

7.8”

11.7, 6.2

11.7,7.a’

H

7.0, 4.6

11.0”

I

9.4, 7.2

7.2

12.5

12.5, 7.2

J

5.8, 4.2

5.8

13.8, 11.3

13.8, 5.8

K

9.0, 6.4, 4.9 4.6, 4.4

6.4

13.2, 6.3, 5.9

13.2, 6.4

416 Probe (Fig. 5b)

14.7,4.ab

5’Specific Virus Probe (Fig. 5c)

10 kb Probe (Fig. 5d)

7.2, 6.5, 6.3

11.0”

The approximate sizes of the various restriction fragments observed In Figure 5 were calculated relative to the Hind Ill-digested X DNA standards. The table lists only Eco RI fragments present in the tumors but not in normal tissue. With the 10 kb probe, two novel fragments, in addition to the endogenous 10 kb band, were detected in every tumor except H. The sum of these fragments, 19.6 f 0.3 kb, was consistent with the insertion of a complete 9.6 kb provirus with a single Eco RI site into the 10 kb region. In each case, the larger of the two fragments was found to correspond in size to a new 5’.specrfic virus-host junction while the smaller, 416-positive fragments correlated with one of the new 3’specific junctions. “The 7.6 kb, 4lSpositive fragments were presumably obscured by the comigrating endogenous sequences. b The presence of two novel bands with this probe indicated that provrrus integration had taken place withrn the 416 fragment. c The novel 11 .O kb fragment detected in tumor H did not hybridize to either 3’. or 5’.specific viral probes but was later found to be positive for MMTV LTR sequences (not shown). ND: none detected.

viral sequences, suggesting that they did not result from restriction site polymorphism in this region of the chromosome. Interestingly, the correlation was invariably with one of the 3’ viral fragments (Table l), indicating that all of the proviruses in question must be in the same orientation. This correlation was strengthened by the fact that with the complete 10 kb fragment as a hybridization probe, every tumor (except H) contained two novel fragments (Figure 5d). In each case, the smaller fragment correlated with 3’ viral sequences as before, and the larger fragment with 5’ viral sequences (Table 1). From the combined sizes of the two fragments, it would appear that (within the limits of this type of analysis) each of the proviruses is probably complete. Thus the integration of a full-sized provirus into this region has introduced a new Eco RI site and approximately 9.8 kb of additional DNA. Making the assumption that the viral sequences are intact, we can calculate roughly where in the 10 kb region the integration event has occurred. Thus as shown in Figure 4, ten of the proviruses analyzed in Figure 5 are clustered within about 3 kb of cellular DNA. The location of the provirus in lane E

within the Eco RI to Sac I region covered by the 418 probe therefore accounts for the appearance of two novel fragments with this sample in Figure 5b as well as in 5d. The one exception in the foregoing discussion (Figure 5, lane H) can be partly resolved by the finding that although the novel fragment detected with 418 was negative for both 5’ and 3’ viral sequences, it can be detected using an LTR-specific probe (data not shown). This is the one example thus far of a deleted form of provirus being present in these tumors, and the precise extent of this deletion is presently under investigation.

Discussion The experiments described were instigated in an attempt to test some of the predictions of the insertional mutagenesis model, as it might apply to MMTV-induced mammary carcinomas. To this end, we prepared cloned DNA probes representative of regions of cellular DNA into which an MMTV provirus had integrated in tumors induced in the BALB/c strain of mice. One such tumor (Figure 2, lane 4) contained two apparently independent, newly acquired proviruses and yielded the unique-sequence cellular DNA probes 411 and 418. These recognized Eco RI fragments of 6.2 kb and 10 kb, respectively, in the normal tissues of BALB/c and BR6 mice (Figure 3). With the 411 probe, we found no evidence for integration of an MMTV provirus within the 6.2 kb region in any tumor other than the one from which the clone was derived. Although this admittedly represents a very small region of DNA relative to the total mouse genome, we feel that these negative observations are significant in the light of the positive results obtained with the 418 probe. The latter probe, which was subsequently shown to recognize a 24 kb Xba I fragment in normal cellular DNA, provided a clear indication that a provirus had integrated in this region of the chromosome in 17 out of the 40 tumors examined. The bulk of current evidence in the MMTV and other retrovirus systems favors the notion that, at least in the absence of clonal selection, provirus integration can take place at multiple, possibly random sites (Cohen et al., 1979; Morris et al., 1979; Varmus and Swanstrom, 1982). The detection of MMTV DNA sequences within a unique 24 kb region of cellular DNA in over 40% of the mammary tumors examined in this study is therefore unlikely to be a chance occurrence. Although it is conceivable that particular regions of chromosomal DNA act as preferred target sites for provirus integration (Lemons et al., 1978; Cohen and Murphey-Corb, 1983) to explain our results on this basis would require that 418 correspond to a preferred site and that 411 would not. Moreover, in tumors where integration has occurred within the 418 region, only one of the two available chromosomal domains is interrupted, and most of these tumors also contain proviruses at other sites. Thus a more likely explanation would be that integration is indeed essentially random, and that among the large numbers of MMTV-induced cells in each lactating mammary gland, any one cell in which a provirus happens to integrate in this particular chromosomal domain may ac-

MMTVlntegratlonSitein Tumors 375

quire a selective growth advantage and proliferate into a monoclonal, neoplastic nodule. We would therefore argue that interruption of the cellular DNA detected by the 418 probe may have been a cause rather than an effect of tumorigenesis. How the provirus is able to exert an influence on the phenotype of such a cell remains an open question. Until we obtain evidence for an RNA transcript from the region specified by the 418 probe, we are reluctant to draw conclusions or to speculate as to the presence of any functional gene in the vicinity. In a preliminary screen, we examined the 10 kb Eco RI fragment that was recognized by 418 by restriction mapping (Figure 4) and hybridization (data not shown), and detected no homology to the known viral oncogenes src. erb, myc, myb, abl, mos, Ki-ras, Haras, fes, and fms (Coffin et al., 1981). One piece of evidence that may prove relevant is that of ten complete proviruses located within the 10 kb region (shown in Figure 5), all are in the same orientation and clustered within about 3 kb of cellular DNA (Figure 4). However, although this configuration would be consistent with direct “downstream promotion” as originally described for ALV and cmyc in chicken lymphomas (Hayward et al., 1981; Neel et al., 1981; Payne et al., 1981), there is no evidence for hybrid mRNA species in virally induced mammary tumors (Robertson and Varmus 1981; and unpublished data). While our data are entirely compatible with an insertional mutagenesis model for MMTV-induced tumorigenesis, the fact that to date only 40% of the tumors examined have proviruses within the 418 region requires some explanation. One obvious possibility is that the chromosomal domain in question is larger than that detected by our available probes. We are therefore attempting to isolate clones of the cellular DNA on either side of the 10 kb region described here, in order to extend the range of our analyses. A consequence of this conclusion would be that whatever influence the provirus might be exerting on neighboring DNA, it may be capable of doing so over a distance in excess of 24 kb. An alternative possibility is that other integration regions may exist with the potential of contributing to tumorigensis, and the tumors that scored negatively with the 418 probe may have proviral DNA in one of these other regions. During the course of our studies, Nusse and Varmus (1982) reported the results of parallel experiments on mammary tumors in the C3H mouse. In their case, over 70% of the tumors examined were found to have a provirus within a unique 25 kb region, which they have termed MMTV intl. However, on the basis of restriction enzyme mapping, cross hybridization of probes, and preliminary results of chromosome mapping (unpublished results, and R. Nusse, personal communication), the region detected with our 418 probe, which we now propose to call int2, is quite distinct from intl. It is, then, conceivable that different chromosomal domains may be implicated in mammary tumorigenesis, possibly influenced by either the strain of mouse or the strain of virus in question. In our case, the tumor from which the 418 probe was derived was induced by injection of C3H virus into a

BALB/c mouse, and may therefore represent a completely different situation from the milk-borne transmission of C3H virus in C3H mice pertaining in the Nusse and Varmus study. Subsequent screening of the BR6 mice, in which infection is presumably by milk-borne Rlll virus, revealed no significant differences in the proportion of hormonedependent and independent tumors showing integration in the int2 region. The relationship between the strains of either virus or host, the pathology of the tumor, and the provirus integration site clearly require further investigation. Nevertheless, the results, as they stand, might suggest that activation of any one of a number of different cellular genes may result in the same neoplastic disease. There is some precedent for this in ALV-induced lymphomas where the region of DNA identified from analysis of integrated proviruses is apparently different from that implicated by DNA transfection experiments (Cooper and Neiman, 1981). Similarly, Lane et al. (1981) have identified cellular DNA in virally induced mouse mammary tumors that is capable of transforming cultured NIH/3T3 cells but does not appear to be linked to any integrated MMTV sequences. How this DNA compares with the intl or int2 regions described here remains to be established. It will be interesting to determine if these different regions of DNA represent distinct cellular genes, and whether they function independently or as parts of a single biochemical pathway. Experimental

Procedures

Source of Tumors The original mammarytumors from which molecular clones were derived were obtained by injecting newborn BALE/c mice with virus purified from the &sue culture fluid from either the GR3A or Mm5MT/Cl cell lines (Dickson and Peters, 1961) corresponding to the GR and C3H strains of milk-borne MMTV, respectively. Subsequently, the tumors used for screenIng with molecularly cloned probes were all spontaneous mammary tumors ansing in the 6R6 strain of mice, and were generously supplied by A. Lee, Imperial Cancer Research Fund, London. The BR6 mice were originally developed from a C57BL x Rlll cross, and suffer a high incidence of mammary tumors, presumably as a result of the milk-borne MMTV characterlstic of the Rlll strain of mice. Isolation of High Molecular Weight DNA Freshly dissected tissues, normally tumor and spleen, were disrupted by Dounce homogenization in 10 mM Tris-HCI (pH 7.6). 100 mM NaCI, and 1 mM EDTA, and filtered through muslin to remove nondisrupted tissue. Pronase (1 mg/ml) and SDS (1 %) were added, and the mixture was incubated for a minimum of 16 hr at 37°C before being deproteinized by successive extractions with phenol, phenol:chloroform:isoamyl alcohol (2424:l). and chloroform:isoamyl alcohol (24:l). Ethanol-precipitated DNA was recovered by spooling, redissolved In 10 mM Tris-HCI (pH 7.Q 1 mM EDTA, and dialyzed exhaustively against the same buffer.The nucleicacid concentration was calculated from the absorbance at 260 nm. Restriction Enzyme Digestion and Analysis of DNA Restriction enzymes were purchased from New England B~olabs and Boehringer, Mannheim, and all digestions were performed In a standard buffer containing 20 mM Tris-HCI (pH 7.6), 50 mM NaCI, 10 mM magnesium acetate. 5 mM 2-mercaptoethanol. and 100 pg/ml bovine serum albumln. High molecular weight tissue DNA (10 to 15 pg) was digested with an excess of enzyme, in a total volume of 400 pl, and ethanol precipitated. The resultant DNA fragments were fractionated by electrophoresis in horizontal agarose gels in 40 mM Tris-acetate (pH 7.5), 5 mM sodtum acetate, 1 mM EDTA contatning 0.5 fig/ml ethidium bromide, and vlsuallzed

Cell 376

under UV light (Maniatis et al., 1982). Fragments of bacteriophage h DNA, generated by digestion with Hind III, were used as molecular weight standards. Where necessary, these markers were 3’.end-labeled with a%P-dCTP and Klenow DNA polymerase for detection by autoradiography. The fractionated DNAs were then denatured and transferred to nitrocellulose filters by blotting in 6x SSC (1 x SSC is 0.015 M sodium citrate and 0.15 M NaCI), essentially as described by Southern (1975). Specific fragments of DNA immobilized on the nitrocellulose were detected by hybridization with 32P-labeled nucleic acid probes. Hybridization was performed at 42°C in a mixture of 56% fonamide, 3x SSC, 50 ag/ml yeast RNA, and 20 pg/ml denatured calf thymus DNA in Denhardt’s buffer (0.02 % each of ficoll, polyvinylpyrrolidone, and bovine serum albumin), containing approximately 5 x 105 cpm of 32P-labeled nucleic acid per ml. After 24 to 48 hr of hybridization, the filters were washed in 0.1x SSC, 0.1% SDS at 65’C for 1 hr, rinsed extensively in the same buffer at room temperature, and exposed to preflashed x-ray film at -70°C in the presence of intensifying screens. Preparation of Specific Probes Radioactive probes, specific for different segments of the MMTV provirus, were prepared using molecularly cloned restriction fragments of viral DNA as shown in Figure 1. Probes for cellular DNA flanking the various integrated proviruses were described in the text. In some instances, specific cloned fragments were excised from the plasmid vector prior to labeling, and punfied by agarose gel electrophoresis. Required fragments were recovered from gels by electroelution in dialysis tubing, and further purified by DEAEcellulose chromatography and ethanol precipitation (Maniatis et al., 1982). Recombinant plasmids or purified DNA fragments (0.5 cg) were labeled by nick translation in reactions containing 20 mM Tris-HCI (pH 8.3) 40 mM KCI, 10 mM magnesium acetate, 5 mM dithiothreitol, 5 mg/ml phosphocreatine, 10 pg/ml creatine phosphokinase, 200 pM each of dATP, dGTP, and dTTP, 50 &I a-“P-dCTP (2-3000 Ci/mmol Amersham International) 10 ng DNAase I, and 20 U of E. coli DNA polymerase I in a total volume of 50 ~1. After incubation at 15°C for 4 hr, unincorporated nucleotides were removed by gel filtration. The specific activity of the resultant probes was between 5 x IO’ and 1 x I@ cpm per pg of DNA. Nick-translated probes were denatured at 100°C for 2 min prior to use in hybridization. In some experiments, cDNA probes representative of the entire MMTV genome were prepared by reverse transcription of MMTV vinon RNA, in the presence of an excess of random oligonucleotide primers (Taylor et al., 1976). Conditions were essentially as above, but using 1 pg of viral RNA with 500 pg of primers, and AMV reverse transcriptase in place of DNAase I and DNA polymerase. Molecular Cloning Procedures and Analysis of Clones The majority of techniques employed in the isolation of molecular clones were as described by Maniatis et al (1982). High molecular weight tumor DNA was digested to completion with Eco RI and ligated into the separated DNA arms of the bacteriophage vector XgtWES.XB. After packaging in vitro, the recombinant phages were plated on the E. coli host LE392, and screened, using the hybridization conditions described above. Phages scoring positively with an MMTV LTR probe were plaque-purified and DNA was prepared from small-scale (5 ml) liquid cultures. After cutting with Eco RI, the phage DNA was ligated directly into the Eco RI site of the drugresistant plasmid vector pAT153, and transfected into E. coli HBlOl. High density screening of drug-resistant colonies was carried out using a representative MMTV cDNA probe. Recombinant plasmid DNAs were prepared by a scaled up (400 ml) modification of the procedure of Birnboim and Doly (1979) and equilibrium centrifugation in cesium chloride gradients. The required cloned Eco RI fragments were excised from the plasmids and purified by agarose gel electrophoresis and electroelution. Restriction maps were established by digestion with various combinations of enzymes. The sizes of the various digestion products were calculated using a series of plasmid-derived standards of known sequence, ranging from 6.5 kb down to 0.15 kb.

Varmus for communication of results prior to publication. Our thanks also go to J. Wyke and M. Parker for helpful discussions, and to Audrey Gibson for preparation of the manuscript. 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

February 7, 1983; revised

March 14, 1983

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R. A., Papageorge,