Reversible Activation of c-Myc in Skin Stella Pelengaris, Trevor Littlewood, Michael Khan, George Elia, Gerard Evan  Molecular Cell  Volume 3, Issue 5, Pages 565-577 (May 1999) DOI: 10.1016/S1097-2765(00)80350-0

Figure 1 Generation and Characterization of Involucrin-c-mycERTM Transgenic Mice (a) Transgene construction. The cDNA encoding human c-myc fused to the modified estrogen receptor ligand-binding domain (ERTM) (Littlewood et al. 1995) was cloned, along with SV40 splice and polyA sites, downstream of the human involucrin promoter (Carroll et al. 1993). Three founder lines of transgenic animals were established, and the transgene copy number was estimated as described in Experimental Procedures. (b) Transgene protein expression. Western blot analysis was performed using an antibody specific for human c-Myc protein (mouse monoclonal, 9E10) (Evan et al. 1985). A 95 kDa protein band (corresponding to c-MycERTM) was detected in the epidermis of all three transgenic founder lines (lanes 2, 3, and 4), but not in a wild-type littermate (lane 1). The transgene copy number (indicated in parentheses) correlates with the level of c-MycERTM protein expression. Control lysates were prepared from Rat-1 fibroblasts infected with empty retroviral vector pBabepuro (lane 5) or pBabepuro expressing c-MycERTM protein (Littlewood et al. 1995) (lane 6). Molecular Cell 1999 3, 565-577DOI: (10.1016/S1097-2765(00)80350-0)

Figure 2 c-MycERTM Protein Expression Is Restricted to the Suprabasal Keratinocytes Frozen dorsal skin sections from transgenic (1348D) and wild-type mice were stained with Myc (PM [a–d]) and estrogen receptor (HL7 [e and f]) antibodies. c-MycERTM protein expression is restricted to the nuclei of the suprabasal epidermis and inner root sheath of hair follicles of transgenic animals either untreated (a) or after topical administration of 4OHT for 4 days (c and e). In contrast, no staining was observed in sections from wild-type littermates with PM (b) antibody. Detection of c-MycERTM protein was specifically blocked by preincubation of the PM and ER antibodies with their immunogenic peptides ([d] and [f], respectively). IRS, inner root sheath; ba, basal layer; su, suprabasal layer. Size bar, 50 μm. Molecular Cell 1999 3, 565-577DOI: (10.1016/S1097-2765(00)80350-0)

Figure 3 Topical Administration of 4OHT Activates c-MycERTM In Vivo Analysis of the transcriptional induction of the c-Myc target gene, ODC. Dorsal skin sections from transgenic (1348D) (b, c, e, and f) and wild-type littermate (a and d) animals were analyzed for ODC mRNA expression by in situ hybridization using a probe specific for ODC. Photographs of bright field (a–c) and corresponding dark field (d–f) sections are shown. No detectable ODC staining was present in epidermis or hair follicles of wild-type skin after 21 days treatment with 4OHT (d), whereas intense staining was evident in suprabasal epidermis and hair follicles of transgenic skin, 8 hr (e) and 21 days (f) following topical administration of 4OHT. hf, hair follicles; su, suprabasal layer. Size bar, 50 μm. Molecular Cell 1999 3, 565-577DOI: (10.1016/S1097-2765(00)80350-0)

Figure 4 Activation of c-Myc in Suprabasal Skin Results in Hyperplasia, Dysplasia, and Papillomatosis Activation of c-Myc leads to profound histological changes in the skin and marked proliferation of suprabasal keratinocytes. Hematoxylin and eosin (H and E)–stained skin sections from transgenic (1348D) (c, e, and g) and wild-type (a) mice either untreated (c) or treated topically with 4OHT for 7 (e) or 21 (a and g) days. Untreated transgenic skin (c) shows no histological difference to that of wild-type skin treated with 4OHT for 21 days (a). In contrast, topical administration of 4OHT to transgenic skin for 7 days leads to hyperplasia with associated parakeratosis, and focal areas of dysplasia (e). After 21 days, papillomatosis is evident, associated with prominent angiogenesis and parakeratosis (g). Immunohistochemical staining of dorsal skin sections with Ki-67, an antibody specific for a nuclear protein expressed in proliferating cells. Dorsal skin sections from untreated transgenic (1348D) (d) and wild-type mice treated with 4OHT for 21 days (b) demonstrates that proliferation is confined to the basal keratinocytes of the epidermis. Hyperplastic skin from transgenic mice following 7 days topical treatment with 4OHT (f) shows a high proportion of proliferating suprabasal keratinocytes, while basal cell proliferation remains unchanged. After 21 days of 4OHT treatment (h), when papillomatous skin is evident, the majority of keratinocytes of the suprabasal compartment and inner root sheath of hair follicles are proliferating. ba, basal layer; su, suprabasal layer; pa, parakeratosis; an, angiogenesis; gr, granular cell; hf, hair follicle. Size bar, 50 μm. Molecular Cell 1999 3, 565-577DOI: (10.1016/S1097-2765(00)80350-0)

Figure 5 Activation of c-Myc Disrupts Normal Keratinocyte Differentiation Dorsal skin sections from transgenic mice (1348D) treated topically with 4OHT for 21 days (b, d, f, and i) or wild-type mice treated with 4OHT for 21 days (a, c, and e) were stained for keratins 1 (a and b), 6 (e and f), and 14 (c and d) and α6 integrin (i). A normal pattern of immunostaining for suprabasal-specific K1 is present in wild-type epidermis (a) but is weak in focal areas of the suprabasal compartment of transgenic papillomatous skin (b). Conversely, K14, which is normally confined to basal keratinocytes (c), extends into the suprabasal epidermis in transgenic papillomatous skin (d). Keratin 6 is absent from wild-type epidermis (e) but is expressed in suprabasal keratinocytes of the transgenic hyperplastic skin (f). Keratin 6–expressing cells (g) maintain expression of MycERTM (h). In addition, expression of α6 integrin is detected in the suprabasal keratinocytes (i). ba, basal layer; su, suprabasal layer; hf, hair follicle. Size bar, 50 μm. Molecular Cell 1999 3, 565-577DOI: (10.1016/S1097-2765(00)80350-0)

Figure 6 c-Myc Activation in Transgenic Keratinocytes In Vitro Leads to an Increase in VEGF Secretion Keratinocytes were isolated from transgenic (1348D) and wild-type skin and cultured in low calcium medium as described in Experimental Procedures. Aliquots of culture medium were collected at various times after the addition of 100 nM 4OHT and assayed for VEGF protein. Open circle, wild type; closed diamond, transgenic. Molecular Cell 1999 3, 565-577DOI: (10.1016/S1097-2765(00)80350-0)

Figure 7 c-Myc Induces Apoptosis in Keratinocytes In Vitro Keratinocytes isolated from wild-type and transgenic (1348D) mice were cultured in 1% or 5% FBS in the presence of 100 nM 4OHT. The number of cell divisions and apoptotic cell deaths from a field of 100 cells was determined by time-lapse video microscopy over 48 hr as described previously (Evan et al. 1992). Keratinocytes from wild-type animals proliferated slowly in the presence of both 1% and 5% FBS, with little evidence of apoptosis. In contrast, activation of c-MycERTM in keratinocytes from transgenic animals resulted in increased proliferation in both 1% and 5% FBS and marked apoptosis in 1% FBS but not in 5% FBS. Molecular Cell 1999 3, 565-577DOI: (10.1016/S1097-2765(00)80350-0)

Figure 8 The c-Myc-Induced Phenotype in the Epidermis of Transgenic Animals Is Reversible Dorsal skin of transgenic mice (1348D) was treated topically with 4OHT daily for 21 days to induce papillomatosis (a and d). Topical treatment with 4OHT was stopped (day 0) and punch biopsies of the dorsal skin taken after 14 (b and e) and 25 (c and f) days were examined for histological changes by H and E staining (a–c) and expression of the proliferation marker Ki-67 (d–f). The papillomatous phenotype that develops after 21 days of continuous 4OHT treatment gradually regresses to normal after 25 days. Concomitant with this, expression of Ki-67 becomes limited to the proliferating basal keratinocytes, a pattern characteristic of untreated and wild-type epidermis. su, suprabasal layer; ba, basal layer; an, angiogenesis; pa, parakeratosis; gr, granular cell. Size bar, 50 μm. Molecular Cell 1999 3, 565-577DOI: (10.1016/S1097-2765(00)80350-0)