Cell, Vol. 62, 697-706,

August

24, 1990, Copyright

0 1990 by Cell Press

Skin Hyperkeratosis and Papilloma Formation in Transgenic Mice Expressing a ras Oncogene from a Suprabasal Keratin Promoter Bernard Bailleul:# M. Azim Surani,t Stephen White:* Sheila C. Barton,t Kenneth Brown: Manfred Blessing,5 Jose Jomano,ll and Allan Balmain’ Beatson Institute for Cancer Research Garscube Estate, Switchback Road, Bearsden Glasgow G61 1BD Scotland tAFRC Institute of Animal Physiology and Genetics Research Department of Molecular Embryology Babraham Hall Cambridge, CB2 4AT England *Department of Dermatology University of Glasgow Glasgow G12 600 Scotland SDepartment of Cell and Tumor Biology German Cancer Research Center Im Neuenheimer Feld 260 D-6900 Heidelberg Federal Republic of Germany 11CIEMAT lnstituto Pryma Ciudad Universitaria 29040-Madrid Spain l

The promoter region of the auprabasal keratin 10 gene has been used to direct expression of a mutant human Harvey-ras oncogene to the differentiating cells of the mouse epidermis. fransgenic animals develop hyperkeratoais of the skin and forestomach-the two sites known to express high levels of the keratin 10 polypeptide in vivo. Papillomas subsequently develop on the skin surface, initially at sites subject to biting or scratching such as the base of the tall or behind the ears. The results suggest that the “second event” involved in tumor development in these transgenic animals is the local induction of a mild wounding stimulus. Furthermore, because the H-ras transgene is expressed in suprabasal cells, it appears that cells which have left the stem cell compartment can be induced to form at least benign tumors in vivo. Introduction The development of transgenic mice has provided important new avenues for the study of specific gene functions in differentiation, embryogenesis, and neoplastic development. The consequences of introduction of oncogenes into the mouse germline have been the subject of particularly intense investigation (for reviews, see Palmiter and Brinster, 1966; Hanahan, 1966; Groner et al., 1967). Tu-

#Present address: Place de Verdun,

U124 Inserm, lnstitut de Recherches 59045 Lille Cedex, France.

sur le Cancer,

mors of the mammary gland have been observed in transgenie mice expressing the ras, myc, or neu oncogenes, either alone or in combination, from the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) (Stewart et al., 1964; Sinn et al., 1967; Muller et al., 1966). Expression of the c-myc gene in B lymphocytes by virtue of linkage to an enhancer element from the immunoglobulin heavy chain gene (Eu) produced tumors of the B cell lineage in a substantial proportion of the transgenic animals (Adams et al., 1965; Langdon et al., 1966). A frequent conclusion from these studies is that expression of an oncogene in the target tissue is not sufficient in itself for tumor formation. Highly variable latency periods are Observed, and in those cases where it has been possible to investigate tumor clonality, it was concluded that a single cell within the population of oncogene-expressing cells underwent additional events leading to malignancy (Adams et al., 1965; Suda et al., 1967; Stewart et al., 1964). This was observed even when two cooperating oncogenes were expressed in the same cell (Sinn et al., 1967). An exception appears to be the very rapid induction of tumors in the mammary gland (Muller et al., 1986) induced by tissue-specific expression of the neu gene. In this case, oncogene expression may be sufficient for tumor induction, although the possibility that secondary changes were elicited by the transgene and contributed to the rapidity of the response has not been excluded. A puzzling feature of most reports is that aberrant expression of ras oncogenes does not cause gross changes in tissue development or differentiation. In the mammary gland, a tissue that undergoes both proliferation and differentiation, expression of ras under the control of the MMTV LTR or the whey acidic protein gene promoter, which should induce different temporal patterns of expression, nevertheless gives rise to glands of essentially normal morphology (Sinn et al., 1967; Andres et al., 1967). Expression of a mutant fas allele in cells of apparently normal morphology has been observed in the lung and spleen of mice bearing a transgenic ras gene driven by an immunoglobulin enhancer (Suda et al., 1967). On the other hand, expression of the same gene in the Harderian gland epithelial cells (Sinn et al., 1967) or in the pancreas (Quaife et al., 1967) has profound proliferation-inducing effects. It therefore appears that the tissue microenvironment is a very important determinant of the response to ras oncogene expression. In this laboratory, we are attempting to use transgenic mice as a tool to investigate the mechanisms of multistage carcinogenesis. The mouse skin model system (Boutwell, 1974; Hecker et al., 1962) has been invaluable in formulating the principles of initiation, promotion, and progression of tumors (for review, see Yuspa and Poirier, 1966). A major advantage of this system is that discrete premalignant stages are observed before the onset of malignancy. Of the many premalignant lesions (papillomas) that develop in initiated and promoted animals, only a small proportion progresses to invasive tumors (carcinomas). Such a sys-

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tern might appear ideal for the application of the transgenie mouse approach, since early lesions can be easily seen without killing the animals or performing extensive surgery, and the roles of cooperating oncogenes at different stages of carcinogenesis are amenable to study either by breeding “double transgenics;’ expressing two different oncogenes (Sinn et al., 1967), or by direct application of retroviruses, carrying oncogenes to the skin of individual transgenic animals (Brown et al., 1966). Molecular analysis of the stages of chemical carcinogenesis in the skin has implicated the Harvey-ras (H-ras) gene as a major target for a mutational event that takes place at the time of initiation (for review, see Balmain and Brown, 1966). Mutated H-ras genes are found in a high proportion of premalignant tumors, indicating that the mutation is an early event (Balmain et al., 1964). Furthermore, the specific mutations observed depend on the nature of the initiating carcinogen (Zarbl et al., 1965; Quintanilla et al., 1966; Bizub et al., 1966; Wiseman et al., 1966; Brown et al., 1990) suggesting a direct interaction between the carcinogen and the H-ras gene. However, the number of initiated cells, their localization and state of differentiation within the epidermis, and the critical events involved in clonal selection to form papillomas are not presently known. In previous experiments it was shown that treatment with the initiating chemical can be replaced by direct application of a retrovirus encoding a mutant ras gene (Brown et al., 1966). Papillomas developed in treated animals, but only after subsequent application of the tumor promoter 120tetradecanoylphorbol 1%acetate (TPA), indicating that ras and TPA have a strong synergistic action in promoting outgrowth of papillama cells. Others have described a similar phenomenon in vitro: isolated cells harboring mutant ras genes can become transformed either by TPA treatment (Hsiao et al., 1964; Dotto et al., 1965) or, significantly, by removal of surrounding normal cells (Land et al., 1966). Such observations, together with previous results on the effects of TPA on intercellular communication (Yotti et al., 1979; Newbold and Amos, 1961) have led to the emergence of the hypothesis that normal cells may exert an inhibitory effect on isolated initiated cells, which can be removed by TPA treatment. We attempted to address some of these questions by directing the expression of a mutant H-ras gene to the epidermis of transgenic mice by linkage to a promoter of one of the keratin gene family members (Lehnert et al., 1964). Using such promoters, it should be possible to engineer oncogene expression not only in the appropriate tissue, the epidermis, but also at a particular stage of differentiation. Some keratin genes are expressed in the basal cells of stratified epithelia but are switched off at the transcriptional level after differentiation, whereas others are expressed only when the basal cells become committed to terminal differentiation and exit from the basal cell compartment (Fuchs and Green, 1960; Moll et al., 1962; Schweizer et al., 1964). It should therefore be feasible to target all of the cells in a particular compartment of the epidermis with the mutant ras gene. By definition, in such a situation there should be no “normal cells;’ and it may

consequently be possible to induce papillomas without TPA treatment. We report here that transgenic mice expressing a mutant ras gene from a keratin 10 (KlO) promoter develop generalized hyperkeratosis of the skin and the forestomach-both known to be sites of KlO expression in vivo (Schweizer et al., 1966). Animals that survived to the age of 7-6 weeks developed highly differentiated papillomas initially at body sites exposed to mechanical irritation. We conclude that the ras gene causes a dramatic change in the differentiation pattern of the whole epidermis without inducing hyperplasia, but also sensitizes the epidermal cells to the effects of a wounding stimulus, resulting in preneoplasia.

Construction of the Keratin-ras Transgene A number of genes of the keratin family of intermediate filament proteins have been isolated (for reviews, see Steinert and Roop, 1966; Fuchs et al., 1966; Fuchs, 1966). In some cases, the promoters of these genes have been characterized by transfection into appropriate target cells after linkage to indicator plasmids (Blessing et al., 1967, 1969; Giudice and Fuchs, 1967) or in transgenic mice (Vassar et al., 1969). These experiments have confirmed the overall tissue specificity in expression directed by keratin promoters, but the features responsible for localization of expression at particular body sites or at defined stages of epithelial differentiation have not yet been identified. The KlO gene is expressed suprabasally in the epidermis of rodents and humans and at low concentration in some other stratified epithelia (Mall et al., 1962; Schweizer et al., 1966). A minor population of cells within the basal layer has also been shown to express this keratin gene, but detailed immunofluorescence studies suggest that these cells represent postcommitment cells that are in transit from the basal to the suprabasal layer (Schweizer et al., 1964). Synthesis of this keratin is therefore an early marker for terminal differentiation of the epidermis. The bovine equivalent of the human KlO gene (bovine keratin VI) has been cloned and the promoter region identified (Blessing et al., 1967,1969; J. Jorcano, unpublished data). To investigate the effects of oncogenic ras expression in the epidermis, a construct was prepared (pBK10 ras) in which the promoter region of the bovine KVI gene was ligated to the human H-ras oncogene originally cloned from T24 (EJ) bladder carcinoma cells (Reddy et al., 1962). An outline of this construct is shown in Figure 1A. Generation of Transgenic Mice Carrying and Expressing the Keratin-ras Gene Fertilized eggs from (C57BU60 x CBAQ)F~ females x CFLP males were injected with purified insert DNA obtained from pBK10 ras. In the first set of experiments, a total of 35 pups was born, 2 of which, 1 male and 1 female, carried the intact transgene, as determined by probing DNA from tail tip biopsies (data not shown). The female founder mouse, although carrying an apparently intact

Hyperkeratosis 699

A

in ras Transgenic

pBK10m

Mice

Figure Mouse

1. Papilloma 10.2 Carrying

Formation the pBKl0

in Founder ras Transgene

(A) Structure the pBKlOras construct. The promoter region from the bovine equivalent of human KlO gene was ligated to the coding region of the mutant human H-ms gene. The insert was excised with EcoRl and injected into fertilized eggs. (B) Founder mouse 10.2 showing the appearante of papillomas at multiple sites. Some papillomas also appeared on the ventral skin. (C) and (D) Histological appearance of papillomas from founder 10.2. Magnification x34 (C) or x55 (D).

Figure 2. Papilloma Mechanical Irritation er Mouse 10.2

Formation in Response to in Male Progeny of Found-

(A) Inheritance pattern of the transgene. Male progeny of founder male 10.2 are indicated by squaresand female progeny by circles. Hatched squares represent mice carrying the transgene. of 23 males tested, 5 were positive, including males 7.3 and 5.1 described in the text. None of 21 female progeny inherited the transgene. (B) Papillomas at specific body sites in pBKl0 rss transgenic mice. The transgenic mouse on the right developed papillomas at the base of the tail, behind the ears, on the back skin, and the footpad (indicated by arrows). The mouse on the left is a normal litter mate. (C) Histological section through the ear of a normal mouse showing the cut edge of the punch hole. Magnification, x34. (0) Similar section through the ear of a transgenie mouse, showing a small papilloma adjacent to the edge of the ear punch. Magnification, x34.

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copy of the inserted gene, developed no unusual phenotype and also failed to transmit the gene to any progeny. Progeny were obtained from the male founder mouse (10.2) which inhibited the phenotype of skin hyperkeratosis and stunted growth (see below). That this phenotype was not due to insertional mutagenesis of a cellular gene was shown by the observation of similar properties in the progeny of an independently generated male founder mouse (T2). In this case, the hyperkeratosis and stunted growth were extremely pronounced at birth, and animals carrying the transgene survived only 2-3 days. Progeny of a third founder mouse (T6) appeared to exhibit a milder form of the same phenotype. Further studies of this line are in progress. The overall yield of transgenic mice from these experiments was very low in comparison with that seen after injection of other constructs into fertilized eggs (unpublished data). Only 6 transgenic founder mice were obtained after injection of over 1000 embryos, 3 of which transmitted the gene through the germline. The reasons for this are not completely clear, but it may be due to embryonic lethality caused by the high levels of ras expression generatad from the strong keratin promoter. The successful cases reported here may be due to down-regulation of expression to an acceptable level. The pBKl0 ras Transgene Is Located on Different Chromosomes in the Founder Mice 10.2 and T2 Initial attempts to breed from 10.2 were unsuccessful. In vitro fertilization was therefore carried out using sperm derived from 10.2 and eggs from superovulated (C57BU6o x CBAQ)Fl females. After reimplantation of ml00 fertilized eggs into recipient females, a total of 44 pups (23 male, 21 female) was born. Several males died before being weaned at 3-4 weeks. Analysis by Southern blotting showed that all of these had inherited the transgene. In contrast, none of the 21 females analyzed at this time had inherited the gene, and only 4 of the remaining 19 males. We conclude that the original founder mouse 10.2 was a germline chimera and that the transgene had integrated into the Y chromosome. The inheritance pattern of the transgene is summarized in Figure 2A. Founder mouse T2 was also chimeric and transmitted the transgene at relatively low frequency to both male and female offspring. This inheritance pattern shows conclusively that these two independent founder mice had integrated the transgene at different chromosomal locations. Spontaneous Development of Papillomas in Founder Mouse 10.2 Male 10.2 developed papillomas spontaneously at the age of about 10 weeks. These were first noticed on the dorsal skin but were subsequently observed also behind one ear, on the flank and ventral skin, and on the penis. By the time the animal was sacrificed for autopsy at the age of 12 weeks, a large number of papillomas were observed at multiple body sites (Figure 1B). Many of the smaller lesions were pedunculated papillomas, whereas others

were flatter and more broad based in appearance. Histological examination indicated that the tumors were highly differentiated, some resembling keratoacanthomas in morphology (Figure 1C). No abnormalities were detected in any other tissues examined, including the uninvolved dorsal skin (data not shown; see also Discussion below). Papillomas Develop Preferentially at Sites Exposed to Mechanical Irritation Positive male progeny of 10.2 that survived beyond 6-7 weeks developed papillomas, but in contrast to founder male 10.2, these occurred preferentially at specific body sites. For example, progeny 7.3 and 6.1 first developed papillomas at the base of the tail (Figure 28). Subsequently, lesions were also seen behind the ears, on the footpads, and on the dorsal skin (Figure 28). Observation of the affected animals indicated that they frequently scratched themselves at the base of the tail and behind the ears. The papillomas that arose in the dorsal skin of the longest surviving mouse (8.1) were located in two patches on either side of the spine (Figure 28). Although no scratching of this region was obvious, mechanical irritation may have taken place by rubbing on the cage roof during feeding. Eventually, mouse 8.1 developed papillomas at other body sites including the ventral skin (data not shown). These observations that papillomas developed initially at sites subjected to some form of mild irritation lead to the conclusion that the presence of the pBK10 ras transgene sensitizes the epidermal cells to the effects of a wounding stimulus. This interpretation is supported by histological examination of sections through the ears of the mice, which had been tagged using an ear punch at the time of weaning. Sections through the ears of normal littermates showed a slightly thickened epidermis at the site of the punch hole (Figure 2C), whereas in the transgenic mice, some small papillomas were visible adjacent to the cut edge (Figure 2D). These occurred on the outer surface of the ear and were probably induced partly by scratching. It is obvious from this comparison that the expression of the ras transgene in the skin causes an abnormally strong response to a stimulus that induces wound healing. Skin of Transgenic Mice Exhibits Hyperkeratosis without Marked Hyperplasia Previous studies on transgenic mice expressing a ras oncogene in specific tissues have shown that the gene can induce either minimal changes in tissue morphology (Sinn et al., 1987; Suda et al., 1967) or pronounced hyperplasia (Sinn et al., 1987; Quaife et al., 1987). We therefore inspected the nonpapillomatous skin of pBKlOras transgenic mice for evidence of changes in epidermal morphology. In contrast to founder mouse 10.2, which was chimeric for the transgene and exhibited normal skin morphology (Figure 3A), transgenic progeny of 10.2 and T2 had pronounced epidermal hyperkeratosis (Figure 3). This was particularly evident over the dorsal and ventral skin, which had a thickened scaly appearance at 8-10 weeks of age in progeny from 10.2 and was easily visible shortly after birth in off-

Hyperkeratosis 701

in ras Transgenic

Figure 3. Hyperkeratosis

Mice

of the Skin in pBKl0

ras Transgenic

Mice

(A) Section through the dorsal skin of founder mouse 10.2 showing normal skin morphology. Magnification, x76. (6) Section through nonpapillomatous skin of transgenic mouse 8.1 showing the large increase in the number of keratin layers present. Map nification, x76. (C) Section through a region showing an area of focal epidermal hyperplasia in skin of transgenic mouse 8.1. Magnification, x76. (D) Transgenic (left) and nontransgenic (right) progeny of founder mouse T2 2 days after birth, showing the early stunted growth and flaky skin.

spring of male founder T2 (Figure 3D). Hyperkeratosis was also evident in the footpads (Figures 4A and 46). Over most of the sections examined of dorsal skin, there was very little evidence of hyperplasia, although focal hyperplastic areas could occasionally be detected (Figure 3C). It seems most likely that the proliferative areas are those that eventually give rise to macroscopically visible papillomas. Hyperkeratosis Is Also Observed in the Forestomach Histological examination of transgenic mice that developed papillomas demonstrated that no gross morphological changes could be detected in most of the internal tissues such as the lung, liver, colon, muscle, trachea, spleen, kidney, brain, testes, eye, or esophagus (data not shown). However, hyperkeratosis was observed in the forestomach (Figures 4C and 4D). During the course of these studies, it was reported that KlO is normally expressed in suprabasal cells in the forestomach and, to a lesser extent, in

Figure 4. Hyperkeratosis Transgenic Mice

of the Footpad

and Forestomach

in pSKl0 tas

(A) and (B) show the footpads and (C) and (0) the forestomachs of normal (A and C) or transgenic (6 and D) mice. (C) and (0) show the junction behwen the squamous and glandular (G) parts of the stomach. K indicates the keratinizing part of the forestomach. Magnification for all sections, x76.

the tongue of the mouse (Schweizer et al., 1988). The histopathological pattern we observed therefore correlates with the known tissue specificity of KlO expression. We conclude that the 3.4 kb fragment, containing the KlO promoter and 5’flanking region has all of the elements necessary to direct the expression of a heterologous gene correctly in particular subpopulations of epithelial cells. Keratin-ras Transgenic Mice Have Stunted Growth An additional feature of the keratin-ras transgenic mice was their relatively slow growth rate in comparison to normal littermates (Figures 2B and 3D). This stunted growth appears to be associated with development of hyperkeratosis rather than tumor formation, since it was evident before the appearance of papillomas in progeny of 10.2, and newborn mice derived from T2 exhibited an extreme phenotype of hyperkeratosis and growth retardation but no evidence of tumor formation. It is not at present clear whether the stunted growth is caused by hyperkeratosis of the forestomach, which leads to inefficient processing of the food intake. Alternatively, it may be due to induction of factors that influence digestive processes in the glandular stomach or intestine. A possible candidate for such a factor could be transforming growth factor-a (TGFa), which

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Figure 5. RNAase Protection Transgene Expression

pBSBK6 ras (HUMAN)

A

p65GEBl

ras (MOUSE) pSP65

P"",,

pSP65

i-----i

6 7 8

179nt

9 10 11 12 13 14

EXON2 MOUSE+

EXONI HUMAN+

AT5

IDIA

LUNG 10.2

Analysis

of pBK10

(A) Constructs used to generate human or mouse ras-specific probes for RNAase protection analysis. Black boxes represent exon sequences of the human or mouse f-f-ms genes. The sizes of the diagnostic protected fragments are indicated for each construct. RNA from cells expressing the pBKl0 ras transgene gives a 145 nucleotide protected fragment, whereas RNA from control cells expressing the intact human T24 H-ras gene gives a 134 nucleotide fragment. Transcription of the mouse H-ras gene is indicated by a fragment of 179 nucleotides generated using p65GEBl ras. (6) Autoradiograph showing RNAase protection analysis to demonstrate expression of human or mouse H-ms genes. Numbers on the right indicate the sizes in nucleotides of the markers in lane 14 (194 and 116 nucleotides) or of the protected fragments expected (179 for mouse H-ms. 145 or 134 for human H-me.). Letters below the autoradiograph indicate whether the probe used was for human (H), mouse (M), or both (H + M) ras genes. Lanes l-6 show results obtained using controls consisting of the RNA from mouse cell lines AT5 or lDlA, which were transfected with a human T24 H-ras gene. The expected fragments of 179 nucleotides (mouse) or 134 nucleotides (human) are seen. Lanes lo-12 show a similar analysis with papilloma RNA from founder mouse 10.2. Expression of the pBK10 ras transgene is indicated by the presence of a protected fragment of 145 nucleotides. Lung (lanes 7-9) or kidney (lane 13) RNA from the same mouse shows only expression of the endogenous mouse H-ras gene.

PAP 10.2

is known to be secreted by cells expressing activated oncogenes (Marquardt et al., 1983; Derynck, 1988) and can also markedly influence acid secretion in the glandular stomach (Gregory et al., 1988). An interesting parallel is that epidermal growth factor (EGF), which is structurally similar to TGF-a, is known to induce epidermal differentiation both in vivo and in vitro (Carpenter and Cohen, 1979) and can also affect gastric acid secretion (Rhodes et al., 1988). In addition, early experiments showed that EGF causes stunted growth when injected into newborn animals (Cohen, 1982). Further studies would be required to determine whether TGF-a secretion is responsible for the stunted growth of keratin-ras transgenic animals. Expression of the Transgene in Hyperkeratotic Skin and Paplllomas The abnormal morphology of the skin of transgenic animals and the subsequent appearance of papillomas suggested that the human ras gene is expressed in a tissue-

specific manner under the control of the KlO promoter. An RNAase protection assay was devised to test whether transcription of the human ras gene could be correlated with the histopathology of the skin and the other main tissues from which RNA could be isolated. Constructs were prepared that would allow the unambiguous determination of relative transcript levels of the endogenous mouse Was gene and the human transgene (Figure 5A). In control experiments (Figure 56) it was shown that in mouse cells expressing the human T24 ras gene, probe H detects a band of 134 nucleotides corresponding to exon 1 of the human T24 gene (lanes 1 and 4) while probe M detects a protected band of 179 nucleotides derived from the endogenous mouse H-ras gene transcript (lanes 2 and 5). The relative levels of both transcripts was assayed simultaneously by mixing the two probes (lanes 3 and 8). When this analysis was carried out on tumors from founder mouse 10.2, the transgene was expressed in papillomas (lanes 10-12) but not in lung tissue from the same mouse (lanes

Hyperkeratosis 703

in ras Transgenic

Mice

A 1 2

3 4 5

6 7 8 9 10 11 12 13

: *‘

i ‘.

.‘,i; :

.. 234

EXON 2 MOUSE

EXON 1 HUMAN

B 1 2 3

4 5 6 7 8 9 10 11 12 13 14 15

Figure 6. Tissue-Specific Expression of the pSKl0 ras Transgene Papillomas and Hyperkeratotic Skin of Transgenic Mice

in

(A) Expression of the transgene in papillomas of founder mouse 10.2. Shown is an autoradiograph of an RNAase protection experiment using probes that will detect expression of both human and mouse H-ras genes (H + M; see legend to Figure 5). RNA samples used were from papillomas (lanes 2-5) adjacent skin (lane 6) and ear, lung, spleen, kidney, brain, and muscle (lanes 7-12, respectively). Some degradation is seen of the spleen RNA sample. Lanes 6-12 showed no bands corresponding to expression of the transgene, even after much longer exposure periods. Lanes 1 and 13 contain the size markers. (B) Expression of the transgene in transgenic mouse 7.3. Shown is an autoradiograph of an RNAase protection analysis on tissues from mouse 7.3. A mixture of human and mouse probes was used (H + M; see legend to Figure 5). RNA samples used were derived from control mouse ceil lines (lanes l-3) two individual papillomas (lanes 4 and

7-9). The band corresponding to the protected fragment of human exon 1 from the transgenic mice is slightly longer (145 nucleotides) than the corresponding band from cells expressing the T24 ras oncogene because of the insertion of the keratin promoter (see Figure 5A). Further analysis of individual papillomas from mouse 10.2 showed a similar expression pattern (Figure 6A, lanes 2-5), but no transcripts of the human gene were detected in any other tissues, including the adjacent skin which was morphologically normal in appearance (lane 6). Expression of the transgene was, however, detected in the skin of transgenic progeny of 10.2 that developed hyperkeratosis. Nonpapillomatous hyperkeratotic skin showed expression of the transgene, as did papillomas from the same mouse (Figure 6B, lanes 12 and 13) but no protected bands of the appropriate size were observed using RNA from other tissues. Localization of Transgene Expression in Suprabasal Epidermal Cells The KlO gene is expressed in the suprabasal cells of mouse and human epidermis (Schweizer et al., 1988; Steinert and Roop, 1988; Moll et al., 1982). Because the construct used in the present experiments used the bovine equivalent of the KlO gene promoter, it was necessary to investigate the expression pattern of the transgene within the various epidermal layers of the transgenic mice. In situ hybridization was therefore carried out on a variety of tissue sections using the human-specific probe described in the legend to Figure 5. Preliminary control experiments showed that this probe hybridized specifically to NIH 3T3 cells transformed by the mutant human H-ras gene, but not to similar cells transformed by an activated mouse H-ras gene (data not shown). In situ hybridizations to sections through small papillomas on the ear of mouse 7.3 are shown in Figure 7. Hybridization was predominantly to the epithelial cells of the section (Figures 7A and 7B). At higher magnification (Figures 7C and 7D), it could clearly be seen that most of the silver grains were located above the suprabasal, differentiating epidermal cells, with only background levels above most of the basal cells. Similar sections hybridized with a probe for the endogenous mouse H-ras gene did not display this specificity (Figures 7E and 7F). The overall expression level of the endogenous H-ras gene was somewhat lower than of the transgene, and grains were distributed over basal and some suprabasal cells. Discussion ras Oncogene Expression in Transgenic Mice: Contrasting Effects in Different Tissues The results described in this paper illustrate the dramatic effects of targeting ras oncogene expression to a differen12) a control mouse cell line expressing the human T24 ras gene (lane 5) spleen, lung, kidney, brain, liver (lanes 7-11, respectively), hyperkeratotic skin (lane 13) and muscle (lane 14). The samples showing expression of the transgene are the papillomas and the hyperkeratotic skin. Size markers are in lanes 6 and 15.

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Figure 7. Suprabasal man ras Transgene Mouse 7.3

Expression of the Huin Ear Papillomas from

(A), (C), and (E) are light-field and (Et), (D), and (F) the respective dark-field photographs of in situ hybridizations using probes specific for the human ras transgene (A-D) or the mouse H-ras gene (E and F). The square in (A) denotes the area shown at higher magnification in (C) and (D). The white curves in (D) and (F) show the position of the basement membrane separating the epidermal layers (Ep) from the stromal cells (S). Some intense nonspecific fluorescence can be seen over the stromal cells in (6) and(D) that is not associated with silver grains. It can clearly be seen that the basal cells of the epidermis show only background levels of hy bridization (C and D), with most of the silver grains localized over the suprabasal cells. A more uniform pattern is seen using the probe for the mouse H-res gene. Magnification: (A) and (6) x140; (C)-(F), x360.

tiating cell compartment within the epidermis. In contrast to some other systems in which ras has been expressed in transgenic mice, the epidermis undergoes gross morphological changes as a consequence of induction of differentiation, apparently without a substantial increase in the number of proliferative cell layers. The strong induction of differentiation rather than hyperplasia was surprising in view of the fact that the ras p21 is associated with stimulation of DNA synthesis on microinjection into fibroblast cells (Mulcahy et al., 1985). Similarly, overexpression of ras p21 in transgenic mice led to hyperproliferation of the Harderian gland when expressed from the MMTV promoter (Sinn et al., 1987) and to hyperplasia with early tumor development in the pancreas when expressed using the elastase promoter (Quaife et al., 1987). In the mammary gland, p21 ras expression under the control of the whey acidic protein gene promoter inhibits the functional differentiation of the epithelial cells and decreases the synthesis of milk proteins during lactation (Andres et al., 1987, 1988). It therefore appears that the response of par-

ticular cell types to expression of a ras oncogene in vivo is highly variable, resulting in either repression of differentiation or hyperplasia. Similarly disparate effects have been observed in vitro: ras p21 induces proliferation and morphological transformation of fibroblasts (Stacey and Kung, 1984; Mulcahy et al., 1985), but can inhibit DNA synthesis in Schwann cells (Ridley et al., 1988) and cause differentiation in PC12 pheochromocytoma cells (Noda et al., 1985) or hematopoietic cells (Hankins and Scolnick, 1981). Even within a single tissue, there may be different consequences of ras oncogene expression, depending on the differentiation state of the target cell. For example, Yuspa et al. (1985) have shown that introduction of a viral ras gene into primary mouse keratinocytes in culture, which are assumed to arise from the basal compartment, inhibits their response to inducers of differentiation. In this paper, we show that expression of mutant ras in suprabasal cells in vivo actually increases the apparent degree of differentiation. This analogy is somewhat artificial, since the comparison is between cells growing in vitro or in vivo,

Hyperkeratosis 705

in fas Transgenic

Mice

but it serves to illustrate the point that ras expression may have different consequences for cells within the same lineage, depending on their relative position during development and their state of differentiation. An alternative interpretation of these results, which cannot be excluded at present, is that the mutant ras expression does not directly induce differentiation but actually increases the proliferation rate or lifespan of the committed suprabasal cells, giving the appearance of hyperkeratos/s. Further detailed studies on the kinetics of epidermal turnover are necessary to resolve this question. Secondary Events Leading to Tumor Formation Tumors that develop in transgenic animals harboring oncogenes are generally clonal in origin (Adams et al., 1985; Suda et al., 1987; Stewart et al., 1984; Hanahan, 1988) which suggests that they arise from single cells in which secondary events have taken place. Transgenic mice with the KlO-ras construct develop hyperkeratotic skin in which the mutant ras gene is expressed, but papillomas arise initially at specific sites, apparently as a consequence of irritation as a result of scratching or biting. This is graphically demonstrated by the sections through the ear punch holes, which show that the proliferative response induced in the KlO-ras transgenic mice is much stronger than in normal littermates. The “second event” leading to tumor formation in these animals may therefore be a wound stimulus giving rise to increased growth factor production at the site of injury. An interesting parallel exists between these experiments and previous studies in which papillomas were initiated by direct application of Harvey murine sarcoma virus to mouse skin. Some epidermal cells were infected with the virus, but no papillomas developed unless the animals were subsequently treated with the tumor promoter TPA (Brown et al., 1986). TPA elicits a type of wound response when applied to mouse skin, and indeed, wounding itself was shown many years ago to be an efficient promoting stimulus (for review, see Argyris, 1982). It therefore appears that in both situations where an exogenous ras oncogene is introduced into epidermal cells, either sporadically using a retrovirus or uniformly as in the transgenic mice, a localized proliferative stimulus is required to generate benign tumors. The development of papillomas in transgenic mice is preceded by generalized hyperkeratosis of the skin. Some similar properties can be seen in humans with a particular variant of the skin disorder keratoderma (Howell-Evans syndrome; Howell-Evans et al., 1958). These patients show an autosomal-dominant inheritance pattern and develop hyperkeratosis of the palms and soles, particularly at pressure sites. Affected individuals have a tendency to develop carcinomas later in life, sometimes in the hyperkeratotic skin but also at other body sites, particularly the esophagus and stomach (Howell-Evans et al., 1958; Yesudian et al., 1980). Transgenic mice in which oncogenes or growth factor genes are expressed in the skin may therefore provide useful models for the study of human skin disorders.

The Nature of the Target Cell for Initiation of Skin Carcinogenesis The tumors that arise in epithelial cells as a consequence of treatment with chemical carcinogens vary markedly in morphology and degree of differentiation (Klein-Szanto, 1989). The nature of the target cell(s) for the initiating event is unknown, but it is generally thought to be a stem cell residing in the basal cell layer (Morris et al., 1986). The observation that transgenic mice expressing an oncogene under the control of asuprabasal keratin promoter develop papillomas suggests that cells within the epidermis that have already progressed to this stage may also constitute a target for the initiating chemical. It is noteworthy that most of the papillomas on the transgenie mice were highly differentiated, and some resembled keratoacanthomas in morphology. Similar tumors are occasionally seen after chemical initiation and promotion of mouse skin (Klein-Szanto, 1989) and it is possible that these arise as a consequence of a mutation in a differentiated cell that has left the stem cell compartment. Pellicer and co-workers have reported cases of human or rabbit keratoacanthomas with activated H-ras genes (Leon et al., 1988). These tumors do not progress to carcinomas, nor do the majority of mouse skin papillomas that also contain activated ras genes (Burns et al., 1976; Quintanilla et al., 1986). Because of the relatively early death of the transgenie mice, it is unknown whether the papillomas that developed on them would have progressed. These experiments, however, suggest that the expression of oncogenes from promoters active at specific epidermal differentiation stages may lead to the formation of tumors of distinct histological types and consequently help to identify target cells for initiation. Preliminary experiments have already been carried out to determine whether less differentiated, more aggressive tumors develop in animals expressing the same ras oncogene from a basal cell-specific keratin promoter (keratin 5). No transgenic progeny have as yet been derived from these experiments (unpublished data), possibly because the basal keratin promoter used is widely expressed in a variety of stratified epithelia (Mall et al., 1982). Expression of a ras oncogene during the development of these epithelia may be lethal. Other approaches involving the use of chimeric mice may have to be used to address this question. The Use of Keratin Promoters for Targeted Gene Expression The keratins comprise a family of some 20-30 proteins encoded by distinct genes that display both tissue-specific and differentiation stage-specific patterns of expression (Mall et al., 1982). This fact, together with the high abundance levels in most epithelial cells, makes the promoters of these genes potentially useful tools for directing the expression of other genes to specific cell types. Because most human tumors arise in epithelial cells, the keratin promoters could provide a valuable means of Investigating the roles of oncogenes or growth factor genes in multistage carcinogenesis in vivo. The results described here

Cell 706

demonstrate that the bovine KlO promoter, when linked to a ras oncogene, exerts profound effects on the differentiation and susceptibility to tumor development of the two main sites of expression of the KlO polypeptide-the epidermis and the forestomach. The specificity exhibited in the expression pattern of the keratin-ras transgene mirrors the regional localization of the endogenous mouse KIO polypeptide in vivo (Schweizer et al., 1988). The results augur well for the possibility of using keratin promoters in gene therapy to deliver growth factors or drugs to specific body sites by means of skin grafting. During the preparation of this paper, a report appeared that the human K14 gene is faithfully expressed in stratified epithelia of transgenic mice (Vassar et al., 1989). Taken together with advances in techniques for gene transfer into keratinocytes (Morgan et al., 1987), it appears that the use of skin grafts with keratin promoters to provide a high abundance of important gene products may be a viable alternative to other delivery systems. Experimental

Plocedums

Piasmid Construction The plasmid pEKl0 ras was obtained by insertion of the bovine KlO promoter (described as bovine keratin VI in Blessing et al., 1989) into a r&s cassette (pRS-T24) (Baiileui et al., 1988). A 4.8 kb fragment containing the KlO promoter, extending from a Sail site lo the Kpnl site 20 bp downstream of the TATA box (Blessing e1 al., 1989), was introduced as a blunt-ended fragment into the Xbai site of pUC18, which had previously been rendered blunt ended with Klenow DNA poiymerase. A 3.4 kb fragment from this plasmid, from the Bgili site to the Smal site in the pUC 18 poiylinker, was inserted into pR8-l24 cu1 with Bglll and Nrul. The resultant pBK10 res plasmid had the keratin promoter in the correct orientation with respect to the ras coding sequence. The complete insert was excised with EcoRi and prepared for microinjection into fertilized eggs. Plasmids used to prepare antisense RNA probes for human and mouse H-ras were prepared as follows. A Hincll-Xhol fragment containing 1.6 kb of the bovine keratin 6 gene (referred to as bovine keratin IV in Blessing et al., 1989) was ligated into pRET24 digested with Xhol and EcoRV. A 1.1 kb fragment of the resultant plasmid, extending from an EcoRi site 750 bp upstream of the CAP site to the Kpnl site within intron 1 of the H-res gene, was subcloned into Kpnl/EcoRi-digested vector pBS (Stratagene inc., San Diego). This piasmid (pBSBK6 ras) contained exon 1 of the human H-ras gene in an antisense orientation with respect to the T3 promoter. To generate an antisense probe for the endogenous mouse H-ras gene, a 598 bp Hindili-Pstl fragment of the genomic c-H-ras clone pUC.NPR (Brown et al., 1989) was rendered blunt ended with Kienow polymerase and ligated into the Smal site of pSP65. The resultant plasmid p65GEBl ras was kindly provided by Dr. Jing-De Zhu. Pronuclear Injections Injection of purified insert from plasmid pBKl0 ras into fertilized eggs was carried out as previously described (Allen et al., 1987). The 7.3 kb insert was excised with EcoRi and purified by gel electrophoresis on low-melting agarose; this was followed by passage through a NACS-52 Prepac column and ethanol precipitation as previously described (Allen et al., 1987). Fertilized eggs were obtained following superovulation of (C57BU6a x CBAQ)Fl females followed by mating to CFLP males. Of ~500 eggs injected in the first series of experiments. 350 were transferred at the 2-cell stage into oviducts of pseudopregnant recipient females on day 1 of gestation. Thirty-six live newborn animals were obtained, of which 2 carried the transgene. Subsequent injections gave rise lo 17 newborn animals, 4 of which carried the human H-ras gene. Histology Tissues for histological

examination

were fixed in 4% buffered

formalin

overnight, dehydrated, and embedded in paraffin ods. Sections (5 pm) were stained in hematoxylin

by standard and eosin.

meth-

Blot Hybridization DNA was extracted from 1 cm portions of mouse tails and removed under general anesthetic as previously described (Allen et al., 1987). Aliquots (10 pg) of the DNA samples were digested with the appropriate restriction enzymes, run on 0.8% agarose gels, and transferred to nitrocellulose filters (Southern, 1975). The presence of the transgene was detected by hybridization with a BamHl insert from the human H-ras plasmid pT24C3. Hybridization and washing conditions were as previously described (Balmain et al., 1984). Analysis of Transgene Expression Total RNA was prepared from mouse tissues using the guanidinium thiocyanate-cesium chloride method as previously described (Balmain et al., 1984). Aiiquots (20 wg) of RNA were used for each assay. Randomly labeled antisense RNA probes were prepared according to the Bluescribe system (Stratagene. San Diego). A human H-res-specific probe was prepared using piasmid pBSBK6 ras after linearization with EcoRl and incubation with T3 polymerase in the presence of =P-labeled CTP (Amersham, England). A probe that recognized mouse H-ras exon 2 was prepared in a similar way from plasmid p65GEBi ras linearized with Pvull and transcribed using SP6 polymerase. The final reaction mix contained 0.5 bg of linearized template and 7.5 U of polymerase in a volume of 20 ~1 containing Tris (40 mM), MgClp (6 mM), spermidine (2 mM), and DTT (10 mM) at pH 7.5, with RNAsin (12.5 U), GTP, ATP, and UTP (each 0.5 mM), [&P]CTP and cold CTP (each 250 pmol). The reaction was incubated at 4oOC for 90 min, and the probe was purified by treatment with RNAase-free DNAase. phenol/ chloroform extraction, and chromatography on Biogel A (1.5 M). Approximately 5 x lo5 cpm of probe was hybridized with 20 pg of RNA in 80% formamide, 400 mM NaCI, 40 mM PIPES, 1 mM EDTA (pH 6.4) for 5 min at 85OC, 2 hr at 45OC, and 8 hr at 25’C. The hybrids were digested with RNAase A (10 pg/ml) and RNAase Tl (150 U/ml), treated with proteinase K (100 pglml), phenol extracted, and run on 8% poiyacrylamide (0.4% bisacrylamide) gel in 8 M urea at 1200 V/40 mA for 2 hr. The gel was dried down and exposed to Kodak X-OMAT film in the presence of intensifying screens. In Situ Hybridization Tissues were fixed overnight in fresh 4% paraformaldehyde at 4OC, dehydrated, and embedded in paraffin wax for sectioning. Sections (7 wm) were hybridized with human or mouse H-ras-specific probes prepared as described above for RNAase protection experiments, but incorporating [35S]UTP (Amersham, England) into the reaction mix. In situ hybridization was essentially as described by Wilkinson et al. (1987) except that hybridization was in 60% formamide at 52oC. Sections were exposed to autoradiographic emulsion (Ilford K5) for periods of 2-21 days and developed according to the manufacturer’s instructions. Acknowledgments The continuing support of the Cancer Research Campaign of Great Britain is gratefully acknowledged. B. B. was supported by fellowships from the European Medical Research Councils and European Science Foundation and by the Association pour la Recherche sur le Cancer. The authors are grateful to Dr. Federico Baeza for useful discussions, and to Rosemary Akhurst, Liz Duffie, and Marlyn Turbitt for invaluable assistance with in situ hybridizations. 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

July 26, 1989; revised

April 4. 1990.

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