Development of mammary hyperplasia and neoplasia in MMTV-TGFα transgenic mice

Cell, Vol. 61, 1147-1155,

June

15, 1990, Copyright

0 1990 by Cell Press

Development of Mammary in MMTV-TGFa Transgenic Yasuhisa Matsui,” Susan A. Halter,t* Jeffrey T. Holt,“t Brigid L. M. Hogan, and Robert J. Coffey’s* * Department of Cell Biology t Department of Pathology 5 Department of Medicine Vanderbilt University Medical School *Veterans Administration Medical Center Nashville, Tennessee 37232

Summary To study the role of transforming growth factor a (TGFa) in normal mammary development and mammary neoplasia in vivo, we have generated transgenic mice in which a human TGFa cDNA is expressed under the control of the MMTV enhancer/promoter. Overexpression of TGFa in the mammary epithelium, as confirmed by in situ hybridization and immunohistochemistry, is associated with hyperplasia of alveoli and terminal ducts in virgin female and pregnant transgenie mice. A range of morphologic abnormalities including lobular hyperplasia, cystic hyperplasia, adenoma, and adenocarcinoma is seen in mammary tissue of transgenic females. In contrast, no morphologic abnormalities are seen in transgenic males in spite of TGFa overexpression in salivary glands and reproductive organs. TGFa can therefore act as an oncogene in vivo and appears to predispose mammary epithelium to neoplasia and carcinoma. Introduction Transforming growth factor a (TGFa) is a 50 amino acid, 5.6 kd secreted polypeptide that is cleaved from a larger integral membrane glycoprotein (Derynck et al., 1987). Recent work from two independent groups has shown that the membrane-bound proTGFa can be biologically active in the absence of processing (Brachmann et al., 1989; Wong et al., 1989). The mature molecule shares 35% sequence homology with epidermal growth factor (EGF), binds to the same receptor, and has similar (although not identical) biologic effects (for review see Derynck, 1988; Derynck et al., 1989). A commonly cited action of both TGFa and EGF is that they act as potent mitogens in a number of epithelial cell systems (Carpenter and Cohen, 1979). In addition, TGFa stimulates epithelial cell migration (Bade and Feindler, 1988; Barrandon and Green, 1987), promotes angiogenesis (Schreiber et al., 1986), induces bone resorption (Ibbotson et al., 1985), and inhibits gastric acid secretion (Rhodes et al., 1986). TGFa was originally isolated from conditioned medium of virally transformed 3T3 cells (DeLarco and Todaro, 1978) and later from conditioned medium of human carcinoma cells (Todaro et al., 1980). It was initially postulated

Hyperplasia Mice

and Neoplasia

that TGFa acted in an autocrine manner to induce a malignant phenotype. According to this hypothesis, TGFa secreted by the malignant cell binds to specific EGF receptors (EGFRs) on the cell surface and promotes proliferation, thus conferring a growth advantage to this cell over its nontransformed neighbors @porn and Todaro, 1980). Transfection of TGFa cDNA constructs into nontransformed Rat-l fibroblasts resulted in a transformed phenotype that was reversed by the addition of antibodies to TGFa (Rosenthal et al., 1986). However, NIH 3T3 cells transfected with similar TGFa constructs did not exhibit features of transformation (Finzi et al., 1987). Additional uncertainty as to the role of TGFa in neoplasia stems from the observation that TGFa expression is not restricted to neoplasia. It is expressed in embryonic tissues (Twardzik et al., 1982; Wilcox and Derynck, 1988; Rappolee et al., 1988) as well as in a wide range of normal cells and tissues, including keratinocytes (Coffey et al., 1987), activated macrophages (Madtes et al., 1988), gastric mucosa (Beauchamp et al., 1989), and mammary epithelium (Liu et al., 1987; Smith et al., 1989). To better understand the role of TGFa in the development of neoplasia, we have examined the consequences of overproduction of TGFa in mice bearing a human TGFa cDNA transgene under the contol of the mouse mammary tumor virus (MMTV) enhancer/promoter. We report that overproduction of TGFa in transgenic females is associated with a range of histologic abnormalities in the mammary glands, ranging from simple hyperplasia to clear adenocarcinoma. In contrast, prolonged overproduction of TGFa in a number of organs and tissues in transgenic males is not associated with any morphologic changes. These results point to a selective role for TGFa in the development of mammary neoplasia. Results Construction of MMTV-TGFa Transgenic Mice Transgenic mice expressing human TGFa under the control of the complete MMTV long terminal repeat (LTR) were generated by injecting one-cell embryos with the DNA construct shown in Figure 1. The vector pMMTV-TGFa contains exons 2 and 3 of the rabbit P-globin gene and thus provides a splicing event upstream of the human TGFa cDNA. The biologic activity of this MMTV-TGFa fusion gene was tested by transfection into the human breast cancer cell line Hs578T. The.production of a 1.4 kb mRNA was induced >200-fold by 1 PM dexamethasone (data not shown). A total of ten founder transgenic mice (three males and seven females) were generated and used to produce lines. To date, females from three of these lines (29, 64, and 257) have developed histologic abnormalities. One of the lines (29) has been studied in detail. None of the females of this line has been able to suckle her young successfully.

Cell 1146

MMTV-TGFa xhd

Figure

1. Structure

of MMTV-TGFa

A

Construct

The cross-hatched region corresponds to 1.5 kb of the MMTV LTR. The filled region corresponds to the second and third exons of the rabbit f3-globin gene. The open region corresponds to the second intron and 3’flanking region of the gene. A 930 bp human TGFa cDNA (stippled region) was inserted into the third exon of the 6-globin gene.

Tissue Specificity of Transgene Expression Transgene expression was assayed by hybridizing total RNA extracted from various tissues from male and female mice of line 29 with either a rabbit 3-globin or a human TGFa cDNA probe with identical results. A 1.4 kb transcript from the transgene was detectable in the testis, seminal vesicle, salivary gland, and lung of the male (Figure 2), but only in the mammary gland of the female (Figure 3 and data not shown). High levels of expression were seen in normal mammary glands from both virgin and pregnant, multiparous females and in neoplastic mammary tissue (Figure 3). No signal was detected when either probe was hybridized to RNA isolated from the mammary glands of nontransgenic females. Consequences of MMTV-TGFa Transgene Expression in Female Mice Line 29 Virgin Females Whole-mount preparations of the mammary

glands from

B

Figure

2. MMW-TGFa

Gene

Expression

in Male Tissues

Total cellular ANA (IO pg) from various tissues of transgenic male mouse 29-7 (4 months old) was hybridized with 3zP-labeled human TGFu cDNA (A) and mouse glyceraldehyde-3-phosphate dehydrogenase cDNA (8). A 1.4 kb transcript was observed consistent with the size of the chimeric MMN-TGFa construct. The higher molecular weight species detected likely represents readthrough transcription. Hybridization to a 32P-labeled rabbit 6-globin gene probe (EcoRI-Xhol 573 bp fragment) showed the identical two transcripts. The filter was exposed to Kodak X-AR5 film for 9 days (A) and for 3 days (B).

28S-

18S-

4

28S-

l.4kb

4

l8S-

Figure 3. MMTV-TGFa Mammary Gland

Gene

Expression

in Normal

and Tumorigenic

Total cellular RNA (10 ug) from various mammary tissues was hybridized with 32P-labeled human TGFu cDNA (A), rat EGFR cDNA (B), and mouse glyceraldehyde-d-phosphate dehydrogenase cDNA (C). Tumor 29, mammary tumor from the founder female of line 29 (9% months old); tumor/normal 29-7-9, mammary tumor and uninvolved mammary gland from a progeny transgenic female 29-7-g (4 months old); tralnon pregnant, mammary glands from a pregnant progeny female 29-9-12 (2 months old) and a nontransgenic pregnant female ICR (2 months old); tralnon virgin, mammary glands from a virgin transgenie progeny female 29-9-29 (3 months old) and a nontransgenic female 29-9-7 (3 months old). The arrowheads show the 1.4 kb transgene transcript (A) and the 10, 6, and 2.4 kb transcripts of the EGFR gene (B). The filter was exposed for 1.5 hr (A), 7 days (B), and 2 days (C).

nontransgenic (Figure 4A) and transgenic (Figure 48) virgin females at 104 and 92 days, respectively, showed significant alveolar hyperplasia throughout all the mammary glands in the transgenic compared with the nontransgenie mouse. This was confirmed with routine histologic sections (Figures 4C and 4D). In the transgenic mouse (Figure 4D) there were abundant alveolar glands and numerous terminal ducts as compared with the nontransgenie animal (Figure 4C), which had simple duct development only. In contrast, both whole-mount and histologic examination of the mammary tissue from transgenic and nontransgenic virgin females at 4 weeks of age showed no alveolar development in either mouse (data not shown). This suggests that sexual maturity and hormonal influences are necessary for hyperplastic changes to occur in the epithelial cells of the transgenic mice. Line 29 Multiparous Females Histologic examination of mammary glands from pregnant

Mammary 1149

Figure

Neoplasia

4. Morphologic

in MMTV-TGFa

Appearance

Ransgsnic

of Transgenic

Mice

and Nontransgenic

Mammary

Glands

(A-D) Whole mounts of mammary glands from nontransgenic 29-9-7 (A) and transgenic 29-9-29 (6) female virgin littermates (3 months old). Magnification 18x. Histologic sections from these animals are shown in (C)and (D), respectively. Note the alveolar hyperplasia in the transgenic mouse compared with the nontransgenic control. (E and F) Histologic sections from the mammary gland of a pregnant nontransgenic ICR mouse (2 months old) (E) and from a pregnant transgenic mouse 29-9-12 (2 months old) (F) at the same gestational stage (day 20). Note the marked proliferation of the stromal cells and the eosinophilic secretions in the lumen of the glands of the transgenic mouse compared with the nontransgenic tissue. Magnification 55x.

nontransgenic (Figure 4E) and transgenic (Figure 4F) 2-month-old mice at 20 days of gestation showed marked proliferation of the stromal cells in the transgenic mouse. In addition, there was more secretion in the alveoli of the transgenic mouse, and the nuclei of the epithelial cells were more prominent. After 3 months, a transgenic female that had been pregnant three times developed a mass in the inguinal mammary fat pad, and this gave rise to a circumscribed, firm tumor. Histologic examinination showed fibrous encapsulation of the tumor with no evidence of local invasion. The tumor was composed of numerous well-formed alveoli with rare mitotic figures (Figure 5A). The glands were small with minimal secretion in the lumen. The stroma around the glands was markedly abundant, but mitoses were not seen, and there was no evidence of dysplasia in either the epithelial or stromal cells. In areas away from the tumor, mammary tissue was composed of hyperplastic glandular epithelium containing secretions (Figure 58).

The stroma between the glands was also markedly hyperplastic. At 5 months of age, after multiple pregnancies, two female mice developed bilateral swollen, “lumpy” thoracic mammary glands. Examination of the tissue showed abundant alveolar and ductular proliferation in all mammary glands, which contained abundant secretions (Figures 5C and 5D). In one animal, squamous metaplasia with prominent keratinization was present (data not shown). In the second animal, in addition to numerous areas of cystic hyperplasia (Figure 5C), there were dysplastic changes in the proliferative epithelium with one focus of marked atypia and mitotic figures (Figure 5D). In addition to the epithelial proliferation, there was a marked stromal hyperplasia between the glands in both animals. These cells were spindle shaped and resembled fibroblasts or myoepithelial cells. The founder mouse developed a mass in the inguinal mammary gland during pregnancy but this later regressed.

Cdl 1150

Figure

5. Histopathology

of Transgenic

Mammary

Glands

Histologic section from an adenoma (A) that arose in a 3-month-old transgenic female 29-7-9. Note abundant loose stroma and well-formed alveoli. Mammary tissue from another mammary gland (6) in the same animal has numerous glands containing eosinophilic secretions. Papillary cystic hyperplasia (C)was noted in another transgenic female 29-6-W (6 months old) with marked hyperplasia and dysplasia (D). The founder mouse of this line 29 (9% months old) developed an adenocarcinoma (E) characterized by anaplastic glands surrounded by proliferative stroma. Dysplastic areas (arrowhead) were found in several mammary glands in this animal adjacent to uninvolved mammary tissue (F). Magnification 55x.

However, at 264 days of age she developed a mass in the inguinal fat pad and in the soft tissues of the upper thorax. At sacrifice at 9% months, she was found to have nodules in the mammary glands in both of these areas. The mass in the inguinal fad pad was circumscribed and firm. On histologic examination, it was an adenocarcinoma composed of glands with small cystic spaces containing malignant epithelial cells with hyperchromatic nuclei and abundant mitotic figures (Figure 5E). Although stromal proliferation was present, it was not as striking as the epithelial proliferation. The epithelial cells appeared to be invading the adjacent benign mammary tissue. The mass in the upper thorax consisted of malignant mammary epithelial glands invading the surrounding muscle and soft tissue. This mammary adenocarcinoma was not as well circumscribed as the inguinal tumor. No metastases were found. Although examination of the remaining mammary tissue revealed a few areas of normal lactating epithelium, there was a spectrum of changes ranging from slightly increased epithelial glandular formation to cystic formation

and epithelial dysplasia. In some areas normal glandular epithelium was accompanied by scattered atypical, hyperplastic glands with a prominent stromal component (Figure 5F). Several of these areas were consistent with hyperplastic alveolar nodules seen in virus- and carcinogen-treated mouse mammary epithelium. Line 64 and 257 Females To date one female of transgenic line 64 has developed mammary abnormalities at 3 months of age, after only one pregnancy. Histologic examination of the mammary tissue showed clear dysplasia with large bizarre nuclei, epithelial hyperplasia, and stromal proliferation. A virgin female of line 257 showed cystic abnormalities of the ducts but little alveolar development at 2% months. Evidence for Expression of the TGFa Transgene in Transgenic Mammary Epithellum Expression of the transgene was examined by in situ hybridization using a 35S-labeled rabbit P-globin cRNA probe. In histologic sections of mammary tissue from both

Mammary 1151

Figure

Neoplasia

in MMTV-TGFa

6. In Situ Hybridization

Transgenic

of Mammary

Mice

Gland

from Virgin

and Pregnant

Transgenic

Mice

(A and C) Bright-field photomicrograph of a section of mammary gland derived from a virgin transgenic mouse 29-9-12 (C). Corresponding dark-field photomicrographs are shown in (B) and (D), respectively. in mammary epithelial cells in alveoli and small ducts, but not in larger ducts (arrowheads). Mammary was hybridized with the same probe, but no specific signal was detected (not shown). Magnification

3-month-old virgin (Figures 6A and 6B) and P-month-old pregnant (Figures 6C and 6D) females, hybridization grains localized to the small ducts and alveoli. No hybridization was seen to cells in the large ducts (Figures 6B and 6D). Examination of the TGFa protein produced by the transgene was carried out with a polyclonal antibody to recombinant human TGFa (R9; Triton Biosciences). In sections adjacent to those in Figure 6C, the pattern of TGFa immunostaining was similar to the results obtained by in situ hybridization. TGFa immunoreactivity was observed in the small ducts and alveoli of the lobular unit, and staining was much reduced in the large ducts (Figure 7A). Weak staining was noted in the adjacent nonhyperplastic epithelium (data not shown). Staining was abolished by preincubation with excess recombinant human TGFa (Figure 78). Within dysplastic mammary tissue, there was regional variation in TGFa immunostaining. Results from a section adjacent to that in Figure 5C are representative (Figure 7C). Certain glands demonstrated strong immunoreactivity with positively staining secretions, whereas other glands had little staining. Stromal staining was also observed. Focal stromal staining was seen in the hyperplastic areas of all transgenic mice (data not shown), suggesting that in some regions TGFa secreted by the epithelial cells is made available to stromal elements. Thus, evidence is presented that in mammary tissue the TGFa transgene is expressed and translated. Evidence for Up-Regulation of EGFR mRNA Expression in Transgenic Mammary Gland We next studied whether the TGFa protein produced

by

mouse 29-9-29 (A) and a pregnant transgenic High level transgene expression is detected gland from a nontransgenic pregnant female 110x.

the transgene exhibited any known biologic effects. Since EGF and TGFa up-regulate EGFR mRNA expression in vitro (Clark et al., 1985; Kudlow et al., 1986; Earp et al., 1986; R. J. C., unpublished data), mouse mammary tissues were examined for EGFR mRNA expression. In mammary tissues that express high levels of the transgene, there is a corresponding increase in expression of the 10, 6, and 2.4 kb endogenous EGFR transcripts, indicated by the three arrowheads to the right of Figure 38. This finding provides additional evidence that the product of the TGFa transgene is a functional molecule that is able to act through its cognate receptor and to exert biologic effects in these transgenic mice. Consequences of Transgene Expression in Male Mice Two line 29 male transgenic mice were examined at 4 and 6 months. Despite expression of the transgene, no histologic abnormalities of the mammary glands, salivary glands, seminal vesicles, or testes were found. Discussion We present evidence that overproduction of TGFa in the mammary epithelium of mice bearing a MMTV-TGFa transgene results in a wide range of histologic abnormalities, including adenocarcinoma (Figure 5). Within hyperplastic mammary epithelium, TGFa production is localized to the terminal ducts and alveoli by in situ hybridization (Figure 6) and immunohistochemistry (Figure 7). Functional consequences of this TGFa overproduction are seen, i.e., upregulation of EGFR mRNA (Figure 38). We submit that in

Cell 1152

Figure

7. TGFa

lmmunoreactivity

in Mouse

Mammary

Tissue

A polyclonal antiserum to recombinant human TGFa was used at a dilution of 1:500 as described in Experimental Procedures. Brown staining represents TGFa immunoreactivity using diaminobenzidine as substrate for the avidin-biotinylated horseradish peroxidase complex. (A) and (B) are sections from pregnant transgenic female 29-9-12 (see Figure 6C). Arrowhead in (A) indicates the large duct. In (B) the antibody was incubated overnight at 4% with recombinant human TGFa (10 uglml). (C) is a dysplastic region from pregnant transgenic female 29-7-11 (see Figure 5C). No staining was seen without the addition of primary antibody. Under these experimental conditions, no staining was seen in nontransgenic mammary tissues. Magnifications: 260x in (A) and (B), 70x in (C).

the appropriate environmental milieu, overproduction of TGFa can result in transformation and in this in vivo context it acts as an oncogene. Overexpression of TGFa in the mammary epithelium of

transgenic virgin female mice leads to widespread hyperplasia of the terminal ducts and alveolar glands between 4 weeks and 3 months of age (Figure 4). This is consistent with the observation that the proliferation of normal mammary epithelial cell lines is stimulated by TGFa in vitro (Zajchowski et al., 1988; Smith et al., 1989) and that local application of TGFa in slow-release form to mammary glands of 5-week-old mice is associated with local alveolar and ductal growth (Vonderhaar, 1987). In our transgenic mice, hyperplasia is not seen until some time after 4 weeks of age, suggesting that some additional hormonal stimulus or effect due to puberty is required to elicit the stimulatory effect of TGFa on mammary epithelium. One possibility is that a hormonal stimulus is required to increase the activity of the MMTV enhancer/promoter in these cells and so increase local TGFa secretion above a threshold level. Other possibilities cannot, however, be eliminated at this time. For example, in the studies of Vonderhaar (1987) the growth-stimulating effects of slow-release TGFa on C&week-old mammary epithelium were enhanced by supplemental estrogen/progesterone. Overexpression of TGFa in mammary epithelium in vivo appears to predispose the hyperplastic tissue to dysplasia and malignant transformation. Although a progression of states from hyperplasia through dysplasia to adenocarcinoma has not been formally proven, it is suggested by the coexistence of all three morphologies in the mammary glands of the founder mouse 29 and the time course of appearance of morphologic changes in other transgenic mice of the same line. In any case, it is clear that overexpression of TGFa is not sufficient to cause the malignant phenotype and, as in most in vivo models of mammary oncogenesis, a secondary event is required (Sinn et al., 1987; reviewed in Hanahan, 1988, 1989). Our MMN-TGFa transgenic model, in which neoplastic change is preceded by widespread alveolar and ductal hyperplasia, differs in several respects from other transgenie models in which cellular proto-oncogenes are expressed under the control of the MMTV enhancer/promoter. In MMTV-in&l transgenic mice, for example, neoplastic change is preceded by widespread alveolar hyperplasia in virgin mice (Tsukamoto et al., 1988). However, in these transgenic animals, mammary hyperplasia and adenocarcinoma are also seen in male mice, while in our lines all males are thus far normal. In MMTV-c-myc (Stewart et al., 1984) and one class of MMTV-c-neu (Bouchard et al., 1989) transgenic lines, development of adenocarcinoma is also a stochastic process, requiring cell proliferation associated with multiple pregnancies. However, in contrast to our MMTV-TGFa transgene (Figures 4 and 5), expression of the c-myc and c-neu transgene apparently does not stimulate growth of the alveoli and terminal ducts in virgin females, nor result in hyperplasia and morphologic abnormalities in pregnant females. In MMTV-in&P transgenic mice, mammary hyperplasia, but not adenocarcinoma, was observed (Muller et al., 1990). Our MMTWTGFa transgenic model also differs from that in which TGFa is expressed under the control of the metallothionein (MT) promoter (Sandgren et al., 1990). In these transgenic animals, high levels of TGFa expression

Mammary 1153

Neoplasia

in MMTV-TGFa

Transgenic

Mice

were detected in a wide range of tissues, with nonneoplastic phenotypic abnormalities detected most notably in the pancreas and coagulation gland. Despite low levels of TGFa expression in breast tissue, mammary adenocarcinoma was observed in a lCmonth-old founder female and dysplastic changes were noted in younger offspring. The site of expression of TGFa in these mice was not localized to either the epithelium or stroma, raising the possibility that in the MT-TGFa females the effect on mammary epithelium is mediated through the stroma. However, the study of Sandgren et al. (1990) coupled with the present report suggests that mammary epithelium is particularly susceptible to the transforming properties of TGFa At present we can only speculate about the nature of the secondary event(s) that results in the development of neoplasia in our mice. One possibility is up-regulation of EGFR. Recent in vitro transfection studies indicate that marked up-regulation of TGFa production may contribute to neoplasia if sufficient numbers of EGFR are present (Di Fiore et al., 1987; Di Marco et al., 1989; Shankar et al., 1989; McGeady et al., 1989) and it has been suggested that an abundance of both ligand and its cognate receptor is required to achieve a critical threshold in terms of the mitogenic signal cascade to induce a malignant phenotype (Di Marco et al., 1989). In this context, it is interesting to note that mammary tissues harboring histologic abnormalities expressed high levels of the TGFa transgene and displayed increased expression of the endogenous EGFR mRNA. EGF/TGFa-mediated up-regulation of EGFR expression is a well-described in vitro phenomenon (Clark et al., 1985; Kudlow et al., 1988; Earp et al., 1988; R. J. C., unpublished data). Other, as yet undetermined secondary events are likely to occur. It is inviting to speculate that overexpression of TGFa within the mammary gland results in a hyperproliferative epithelium that is more susceptible to such secondary events. The MMTV-TGFa transgenic model reported here provides a very useful system for exploring this and other possibilities. Experimental

Procedures

Generation of Transgenlc Ylce To construct pMMTV-TGFQ the SV40 early promoter region in the vector pKCA (O’Hare et al., 1961; Nishi et al., 1988) was replaced by the complete MMTV LTR. A 925 bp human TGFa cDNA from the plasmid phTFGl-10-925 (kindly provided by Dr. Graeme Bell, University of Chicago) was then inserted into the EcoRl site of (3-globin exon 3. The 3.6 kb Xhol fragment was purified by CsCl centrifugation, as described (Hogan et al., 1966) and microinjected into (C57BL x DBA)FS fertilized eggs. Transgenic mice were identified either by Southern blot analysis of tail DNA using a 526 bp EcoRI-Xhol fragment of the rabbit 6-globin exon 3 or by polymerase chain reaction analysis. Transgenic lines were generated by mating founder animals to (C57BL x DBA)FI males and females. Yorphologlc Assessment of Mammary Glands The skin containing the mammary fat pads was fixed in 10% buffered formalin for at least 24 hr. The mammary glands were then dissected free from the skin and processed as a whole mount, using a modification of the method of Medina (1973). They were stained with hematoxytin and examined with a dissecting microscope. Routine sections of tumor and mammary tissue were prepared after fixation in 10% buffered formalin by embedding in paraffin, sectioning at 5 pm, and staining in hematoxylin and eosin.

Northern Hybrldlzatlon Ten micrograms of total RNA, isolated as described (Krumlauf et al., 1987) was electrophoresed in 1% agarose-formaldehyde gels, transferred to Zetabind membrane, and baked. The 925 bp EcoRl human TGFa cDNA or a 526 bp EcoRI-Xhol fragment of rabbit 6-globin exon 3 was used as a hybridization probe under described conditions (Jenkins et al., 1982). The filters were rehybridized with a 1.6 kb rat EGFR cDNA (Earp et al., 1988) and a plasmid containing a 280 bp insert from a murine glyceraldehyde+phosphate dehydrogenase cDNA. In Situ Hybrldlzetion A 526 bp EcoRI-Xhol fragment of rabbit 6-globin exon 3 was inserted into pSP73, and single-stranded antisense RNA probe was labeled with 35S-lJTP as described (Lyons et al., 1989). In situ hybridization and washes were carried out at high stringency as described (Lyons et al., 1989) with the following modifications. After hybridization, sections were washed for 20 min in 50% formamide at 85OC, treated with RNAase A at a concentration of 20 uglml for 20 min at 3PC, and washed at 85OC for 30 min in 2x SSC, and 30 min in 0.1x SSC. lmmunohistochemistry lmmunohistochemical localization was carried out using a rabbit antihuman recombinant TGFa primary antibody at a dilution of 1:500 and the Vectastain Elite Peroxidase Kit (Vector Laboratories, Burlingame, CA). The sections were fixed in 4% paraformaldehyde, deparaffinized and rehydrated, incubated in PBS containing 10% normal goat serum, and then treated with diluted primary antibody alone or with antibody that had been preincubated overnight at 4°C with recombinant human TGFa (10 uglml). Sections were treated with affinity-purified biotinylated anti-rabbit IgG and then with avidin-biotinylated horseradish peroxidase complex. Color was developed by treatment with 0.05% diaminobenzidine and 0.01% H202 in 50 mM Tris-HCI (pH 7.4). Sections were counterstained with hematoxylin. Acknowledgments We thank Karen Oldham, Ramona Graves-Deal, and Carol McCutchen for excellent technical assistance, Peter Dempsey, Karen Lyons, and Lynn Matrisian for helpful comments, and Lisa Hall for typing the manuscript. This work was supported in part by grant CA 48799 to B. L. M. H. and grant CA 46413 to R. J. C., who is also supported by the Department of Veterans Affairs (VA) and the Culpeper Foundation. The TGFa cDNA, EGFR cDNA, and R9 TGFa antibody were kindly provided by Drs. Graeme I. Bell (University of Chicago), H. Shelton Earp (University of North Carolina), and Rick Harkins (Triton Biosciences), respectively. 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

April 24, 1990.

References Bade, E. G., and Feindler, S. (1988). Liver epithelial cell migration induced by epidermal growth factor or transforming growth factor alpha is associated with changes in the gene expression of secreted proteins. In Vitro Cell. Dev. Biol. 24 149-154. Barrandon, Y., and Green, H. (1987). Cell migration is essential tained growth of keratinocyte colonies: the roles of transforming factor-a and epidermal growth factor. Cell 50, 1131-1137.

for susgrowth

Beauchamp, R. D., Barnard, J. A., McCutchen, C. M., Cherner, J. A., and Coffey, R. J. (1989). Localization of transforming growth factor a and its receptor in gastric mucosal cells. J. Clin. Invest. 84, 1017-1023. Bouchard, L., Lamarre, L., Tremblay, P. J., and Jolicoeur, I? (1969). Stochastic appearance of mammary tumors in transgenic mice carrying the MMTVlc-neu oncogene. Cell 57; 931-936. Brachmann, R., Lindquist, P B., Nagashima, M., Kohr, W., Lipari, T., Napier, M., and Derynck, R. (1989). Transmembrane TGF-a precursors activate EGFfTGF-a receptors. Cell 56, 891-700.

Cdl 1154

Carpenter, G., and Cohen, S. (1979). Rev. Biochem. 48, 193-216.

Epidermal

growth

factor.

Annu.

Clark, A. J. L., Ishi, S., Richer& N., Merlino, G. T., and Pastan, I. (1965). Epidermal growth factor regulates the expression of its own receptor, Proc. Natl. Acad. Sci. USA 82, 8374-6378. Coffey, Ft. J., Derynck, Ft., Wilcox, J. N., Bringman, T. S., Goustin. A. S., Moses, H. L., and Pittelkow, M. R. (1967). Production and autoinduction of transforming growth factor-a in human keratinocytes. Nature 328, 617-620. DeLarco, J. E., and Todaro, sarcoma virus-transformed 4001-4005. Derynck,

R. (1966).

G. J. (1976). Growth factors from murine cells. Proc. Natl. Acad. Sci. USA 75,

Transforming

growth

factor

Derynck, R., Roberts, A. B., Winkler, M. E., Chen, D. V. (1967). Human transforming growth factor-a: and expression in E. coli. Cell 38, 267-297.

a. Cell 54, 593-595. E. Y., and Goeddel, precursor structure

Derynck, R., Lindquist, P B.. Bringman, T. S., Wilcox, J. N.. Elder, J. T., Fisher, G. L., Voorhees, J. J., Moses, H. L., Pittelkow, M. R., and Coffey, R. J. (1969). Expression of the transforming growth factor-a gene in tumor cells and normal cells. In Cancer Cells 7, M. Furth and M. Greaves, eds. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press), pp. 297-301. Di Fiore, P P., Pierce, J. H., Fleming, T. P., Hazan, R., Ullrich, A., King, C. R., Schlessinger, J.. and Aaronson, S. A. (1967). Overexpression of the human EGF receptor confers an EGF-dependent transformed phenotype to NIH 3T3 cells. Cell 51, 1063-1070. Di Marco, E., Pierce, J. H., Fleming, Aaronson, S. A., and Di Fiore, l? P tween TGFu and the EGF-receptor: duction of the malignant phenotype.

T. P., Kraus, M. H., Molloy, C. J., (1969). Autocrine interaction bequantitative requirements for inOncogene 4, 631-836.

Earp, H. S., Austin, K. S., Blaisdell, J., Rubin, R. A., Nelson, K. G., Lee, L. W., and Grisham, J. W. (1966). Epidermal growth factor (EGF) stimulates EGF receptor synthesis, J. Biol. Chem. 267, 4777-4780. Earp, H. S., Hepler, J. R., Petch, L. A., Miller, A., Berry, A. R., Harris, J., Raymond, V. W., McCune, B. K., Lee, L. W., Grisham, J. W., and Harden, T. K. (1966). Epidermal growth factor (EGF) and hormones stimulate phosphoinositide hydrolysis and increase EGF receptor protein synthesis and mRNA levels in rat epithelial cells. J. Biol. Chem. 263, 13866-13674. Finzi, E., Fleming, T., Segatto, O., Pennington, C. Y., Bringman, T. S.. Derynck, R., and Aaronson, S. A. (1967). The human transforming growth factor type a coding sequence is not a direct-acting oncogene when overexpressed in NIH 3T3 cells. Proc. Natl. Acad. Sci. USA 84, 3733-3737. Hanahan, D. (1986). Dissecting multistep mice. Annu. Rev. Genet. 22, 479-519. Hanahan, D. (1969). Transgenic Science 246, 1265-1275.

tumorigenesis

mice as probes

in transgenic

into complex

systems.

Hogan, B. L. M., Costantini, F., and Lacy, E. (1966). Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press). Ibbotson, K. J., Twardzik, D. R., Souza, S. M., Hargreaves, W. R., Todaro, G. J., and Mundy, G. R. (1965). Stimulation of bone resorption in vitro by synthetic transforming growth factor-alpha. Science 228, 1007-1009. Jenkins, N. A., Copeland, N. G., Taylor, B. A., and Lee, B. K. (1962). Organization, distribution and stability of endogenous ectopic murine leukemia virus DNA sequences in chromosomes of Mus mu~ctfl~s. J. Virol. 43, 26-36. Krumlauf, R., Holland, P W. H., McVey, J. H., and Hogan, B. L. M. (1967). Developmental and spatial expression of the mouse homeobox gene, Hox 2.1. Development 99, 603-617. Kudlow, J. E., Cheung, M., and Bjorge, J. D. (1966). Epidermal growth factor stimulates the synthesis of its own receptor in a human breast cancer cell line. J. Biol. Chem. 267, 4134-4136. Liu, S. C., Sanfilippo, B., Perroteau, I., Derynck, R., Salomon, D. S., and Kidwell, W. R. (1967). Expression of transforming growth factor a (TGFa) in differentiated rat mammary tumors: estrogen induction of TGFa production. Mol. Endocrinol. 1, 683-692.

Lyons, K. M., Pelton, R. W., and Hogan, B. L. M. (1969). Patterns of expression of murine VgR-1 and BMP-2a suggest that transformina growth factor+like genes coordinately regulate aspects of embryonic development. Genes Dev. 3, 1657-1666. Madtes, D. K., Raines, E. W., Sakariassen, K. S., Assoian. R. K., Sporn, M. B., Bell, G. I., and Ross, R. (1966). Induction of transforming growth factor-a in activated human alveolar macrophages. Cell 53, 285-293. McGeady, M. L., Kerby, S., Shankar, V., Ciardiello, F., Salomon, D., and Seidman, M. (1969). Infection with a TGF-a retroviral vector transforms normal mouse mammary epithelial cells but not normal rat fibroblasts. Oncogene 4, 1375-1382. Medina, D. (1973). Preneoplastic lesions genesis. In Methods in Cancer Research, ademic Press), pp. 3-53.

in mouse H. Busch,

mammary tumoried. (New York: Ac-

Muller, W. J., Lee, F. S.. Dickson, C., Peters, G., Pattengale, Leder, l? (1990). The inf-2 gene product acts as an epithelial factor in transgenic mice. EMBO J. 9, 907-913.

P., and growth

Muller, W. J., Sinn, E., Pattengale, l? K., Wallace, R., and Leder, P (1966). Single-step induction of mammary adenocarcinoma in transgenie mice bearing the activated c-neu oncogene. Cell 54, 105-115. Nishi, M., Ishida, Y., and Honjo, T. (1966). Expression of functional interleukin-2 receptors in human light chain/Tat transgenic mice. Nature 331, 267-269. O’Hare, K., Benoist, C., and Breathnach, R. (1981). Transformation of mouse fibroblasts to methotrexate resistance by a recombinant plasmid expressing a prokaryotic dihydrofolate reductase. Proc. Natl. Acad. Sci. USA 76, 1527-1531. Rappolee, D.A., Brenner, C. A., Schultz, R., Mark, D., and Werb, Z. (1966). Developmental expression of PDGF, TGF-a and TGF-6 genes in preimplantation mouse embryos. Science 247, 1823-1625. Rhodes, J. A., Tam, J. f?, Finke, U., Sanders, M., Bernanke, J., Silen, W., and Murphy, R. A. (1966). Transforming growth factor a inhibits secretion of gastric acid. Proc. Natl. Acad. Sci. USA 83, 3844-3646. Rosenthal, A., Lindquist, P B., Bringman, T. S., Goeddel, D. V., and Derynck, R. (1966). Expression in rat fibroblasts of a human transforming growth factor-a cDNA results in transformation. Cell 46, 301-309. Sandgren, E. P, Luetteke, N. C., Palmiter, R. D.. Brinster, R. L. and Lee, D. C. (1990). Overexpression of TGFa in transgenic mice: induction of epithelial hyperplasia, pancreatic metaplasia, and carcinoma of the breast. Cell 67, this issue. Schreiber, A. B., Winkler, M. E., and Derynck, R. (1966). Transforming growth factor-a: a more potent angiogenic mediator than epidermal growth factor. Science 232, 1250-1253. Shankar, V., Ciardiello, F., Kim, N., Derynck, R., Liscia, D. S., Merlo, G., Langton, B. C., Sheer, D., Callahan, R., Bassin, R. H., Lippman, M. E., Hynes, N., and Salomon, D. S. (1969). Transformation of an established mouse mammary epithelial cell line following transfection with a human transforming growth factor alpha cDNA. Mol. Carcinog. 2, l-11. Sinn, E., Muller, W., Pattengale, P, Tepler, I., Wallace, R., and Leder, P. (1967). Coexpression of MMTVlv-Ha-ras and MMTVlc-myc genes in transgenic mice: synergistic action of oncogenes in vivo. Cell 49, 465-475. Smith, J. A., Barraclough, R., Fernig, D. G.. and Rudland, P S. (1989). Identification of alpha transforming growth factor as a possible local trophic agent for the mammary gland. J. Cell. Physiol. 747, 362-370. Sporn, M. B., and Todaro, G. J. (1980). Autocrine secretion and malignant transformation of cells. N. Engl. J. Med. 303, 878-880. Stewart, T. A., Pattenglae, P K., and Leder, P (1964). Spontaneous mammary adenocarcinomas in transgenic mice that carry and express MTVlmyc fusion genes, Cell 38, 627-637. Todaro, G. J., Fryling, C., and DeLarco, J. E. (1960). Transforming growth factors produced by certain human tumor cells: polypeptides that interact with epidermal growth factor receptors. Proc. Natl. Acad. Sci. USA 77, 5256-5262. Tsukamoto, A. S., Grosschedl, R., Guzman, R. C., Parslow, T., and Varmus, H. E. (1968). Expression of the inr-1 gene in transgenic mice

Mammary 1155

Neoplasia

in MMTV-TGFu

Transgenic

is associated with mammary gland hyperplasia in male and female mice. Cell 55, 619-625.

Mice

and adenocarcinomas

Twardzik, D. Ft., Ranchalis, J. E., and Todaro, G. J. (1962). Mouse embryonic transforming growth factors related to those isolated from tumor cells. Cancer Res. 42, 590-593. Vonderhaar, 8. K. (1967). Local effects of EGF, a-TGF, and EGF-like growth factors on lobuloalveolar development of the mouse mammary gland in viva. J. Cell. Physiol. 732, 581-584. Wilcox, J. N., and Derynck, transforming growth factors Biol. 8, 3415-3422.

R. (1988). Developmental expression of alpha and beta in mouse fetus. Mol. Cell.

Wong, S. T., Winchell, L. F., McCune, B. K., Earp, H. S., Teixido, J., Massague, J., Herman, B., and Lee, D. C. (1989). The TGFa precursor expressed on the cell surface binds to the EGF receptor on adjacent cells, leading to signal transduction. Cell 56, 495-508. Zajchowski, D., Band, V., Pauzie, N., Tager, A., Stampfer, M., and Sager, R. (1988). Expression of growth factors and oncogenes in normal and tumor-derived human mammary epithelial cells. Cancer Res. 48. 7041-7047. Notes

Added

in Proof

To date, 9 of 20 (45%) female progeny of line 29 have developed mammary lesions, and a founder female of a fourth line (351) has a firm mammary tumor at 123 days of age. We thank

Ron Pelton for help with in situ hybridization