The neurofibromatosis type 1 (Nf1) tumor suppressor is a modifier of carcinogen-induced pigmentation and papilloma formation in C57BL/6 mice

Abstract

There is increasing evidence implicating the human NF1 gene in epithelial carcinogenesis. To test if NF1 can play a part in skin tumor formation, we analyzed effects of the skin cancer initiator dimethylbenz-anthracene and/or the tumor promoter 12-O-tetradecanoyl-13-acetylphorbol on mice heterozygous for null mutations in Nf1 (Nf1+/-). Mice were on the C57BL/6 background, noted for resistance to chemical carcinogens. Nf1+/- mice (18 of 24) developed papillomas after treatment with dimethylbenzanthracene and 12-O-tetradecanoyl-13-acetylphorbol; papillomas did not develop in wild-type C57BL/6 mice nor Nf1+/- mice treated with 12-O-tetradecanoyl-13-acetylphorbol alone. All papillomas analyzed (six of six) had mutations in codon 61 of H-ras, demonstrating strong cooperation between the Nf1 GTPase activating protein for Ras, neurofibromin, and Ras-GTP. After exposure to 12-O-tetradecanoyl-13-acetylphorbol, Nf1+/- keratinocytes showed significant, sustained, increases in proliferation, implicating Nf1 in phorbol ester responsive pathways. Thus, Nf1 levels regulate the response of keratinocytes to 12-O-tetradecanoyl-13-acetylphorbol. Nf1+/- mice also showed a 2-fold increase in the development of pigmented skin patches stimulated by dimethylbenzanthracene; patches were characterized by hair follicles in anagen phase, implicating keratinocytes in the aberrant hyperpigmentation. Our results show that mutation in the Nf1 gene causes abnormal keratinocyte proliferation that can be revealed by environmental assaults such as carcinogen exposure. The data support a plausible role for NF1 mutation in human epithelial carcinogenesis.

Figure 1. Gross appearance and histology of DMBA treated wild-type and Nf1+/− skin

(A–C) Show gross photographs of shaved dorsum of mice 2 mo after initiation with two doses of 40 µg of DMBA. Treated areas are outlined in white. (A) Representative of a wild-type animal after initiation; this animal shows no large pigmented spot. Other, affected, animals with large pigmented spots are shown in B and C. The mouse shown in B is wild type; in C is a heterozygous mouse. Pigmented areas are designated (P) and unpigmented areas designated (U). (D–F) Hematoxylin and eosin-stained sections of skin, all at the same magnification. (D) Initiated skin from an unpigmented area of Nf1+/− skin, identical in histology to wild-type skin. (E,F) Sections from pigmented region of wild-type and Nf1+/− animals shown in B and C, respectively. White arrows point to hair follicles filled with pigment; sweat glands are pointed out with black arrowheads. (F) A section from a pigmented region with dermal pigmentation that was not cell-associated (black arrow). (G–I) Anti-proliferating cell nuclear antigen immunostaining (brown precipitate shown by black arrowheads) with hematoxlyin counterstain of sections from the regions shown in D–F,H,I melanin is present in follicles (black arrows). Scale bar: (D–F) 50 µm; (G–I) 10 µm.

Figure 2. Gross and histologic appearance of skin and papillomas from Nf1+/− mice after tumor promotion

(A) Gross appearance of a representative affected Nf1+/− mouse after initiation and 24 wk of tumor promotion with 0.8 µg of TPA three times per week. Hair was gently shaved to reveal pigmented areas, P and unpigmented areas, U. White arrowheads point to a papilloma on the dorsum of this mouse. (B–F) Photographs of hematoxylin and eosin-stained sections. (B,C) The histologic appearance of unpigmented, B and pigmented, C skin from the mouse shown in A. Note the marked expansion of hair follicles in C. (D) A section through a typical exophytic papilloma with epidermal hyperplasia, centrally located follicular cysts and trapped sweat glands. Black arrows indicate the interface between the papilloma and adjacent normal skin. At higher magnification, keratinocyte hyperplasia (e) is shown in E and follicular cysts (c) in F, as are the sebaceous glands (s) prominent in most of the papillomas generated. Scale bar. (B, C) 50 µm; (D) 125 µm; (E, F) 10 µm.

Figure 3. Papilloma formation in Nf1+/− mice

(A) The number of animals that developed papillomas after the indicated number of weeks of promotion with TPA. In this experiment, 12 animals of each genotype were treated. Similar results were obtained in a second experiment. Squares represent papillomas in Nf1+/− mice; diamonds represent wild-type mice, which never developed papillomas. (B) Volumes of papillomas in Nf1+/− mice 24 wk after promotion with TPA. *80 mm³ papilloma.

Figure 4. Mutational analysis of codon 61 from the H-ras gene in treated skin and papillomas

(A,B) PCR amplification of the A→T transversion at codon 61 from the H-ras gene. Presence of the top band (162 bp) indicates the presence of the mutation (arrow). (A) Pigmented skin (lanes 1 and 3) and unpigmented skin (lanes 2 and 4) from the treated area of wild-type (lanes 1 and 2) and Nf1+/− (lanes 3 and 4) animals. Lanes 5 and 6 represent negative control and positive controls, respectively, and M = marker. Data are representative of that collected from wild-type (n = 4) and Nf1+/− (n = 5) individual mice. (B) Papillomas were isolated 24 wk after tumor promotion with TPA. Lanes 1–4 show results of four representative papillomas analyzed from individual animals. Lane 5 shows a water control, lanes 6 and 7 represent negative and positive controls, respectively. M = marker. (C) Autoradiogram showing sequencing analysis, showing a mutation (A to T transversion) within the second base of codon 61 (asterisk) of the H-ras gene. All six papillomas that tested positive for mutations in the PCR screen were confirmed by sequence analysis.

Figure 5. TPA exposure results in enhanced epidermal labeling index in Nf1+/− mice

Anti-BrdU staining of typical skin sections from wild-type (A) and Nf1+/− (B) mice 48 h after the last of four treatments with TPA (4.0 µg). Arrows designate positively stained basal keratinocytes. Scale bar. 10 µm. (C) Data from mice in two individual experiments are shown. The percent of BrdU-labeled basal keratinocytes was counted. Each bar (hatched = wild type; solid = Nf1+/−) represents the mean percent labeled cells in two to three sections from each mouse. Error bars show standard error. Wild-type and mutant mice were significantly different (p < 0.0001; Student’s t-test).