Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 26 (1), 249-57

UVB-induced Apoptosis Drives Clonal Expansion During Skin Tumor Development

Affiliations

UVB-induced Apoptosis Drives Clonal Expansion During Skin Tumor Development

Wengeng Zhang et al. Carcinogenesis.

Abstract

The mechanism by which a single mutant cell clonally expands is usually assumed to involve an additional mutation in a cell cycle regulatory gene. An alternative mechanism for driving clonal expansion is apoptosis, which might create vacant stem cell compartments that can be repopulated by mutant cells. This model predicts that in a mouse with reduced apoptotic capacity (i) more mutated cells will appear initially but (ii) these cells will expand into clones more slowly than in wild-type animals. To test this hypothesis for ultraviolet B (UVB)-induced skin carcinogenesis, we examined UVB-induced p53 mutant clones and tumors in a transgenic (Tg) mouse (K14-Survivin) with skin-specific expression of the apoptosis inhibitor Survivin. To limit the effects of Survivin on apoptosis, without affecting epidermal proliferation or differentiation, we used Survivin expression levels and UVB doses that resulted in a 2-fold reduction in keratinocyte apoptosis. After 5 weeks of chronic UVB irradiation, newly created p53 mutant keratinocyte clones (indicative of initial mutation frequency) were 1.4-fold more frequent in K14-Survivin mice (P = 4 x 10(-6)). As predicted, this effect was reversed for clones growing by clonal expansion, which were rarer in Tg skin by 1.7-fold (P = 0.047). At 10 weeks large expanding Tg clones were rarer by a magnitude approaching the apoptosis differential (approximately 2-fold, P = 4 x 10(-5)). Survivin expression also retarded clonal expansion at later stages of tumor development. By 20 weeks 95% of animals carried tumors (primarily papillomas), which were 1.6-fold rarer in apoptosis-defective Tg mice (P = 0.03). In contrast, the rate of tumors attaining large size (> or =3 mm, P = 0.048) and converting to carcinoma was increased approximately 2-fold in Tg mice. Thus, Survivin-regulated apoptosis appears to suppress two stages that involve new mutations, initiation and malignant conversion, yet drives clonal expansion of existing p53 mutant cells.

Figures

Fig. 1
Fig. 1
Survivin expression suppresses UVB-induced apoptosis but does not perturb epidermal differentiation or proliferation. (a) K14-Survivin (open bars) and non-Tg (filled bars) littermates were exposed to the indicated dose of UVB. The figure shows sunburn cell formation as detected histologically in skin sections harvested 24 h following UVB exposure. Data are expressed as sunburn cell number per linear cm of skin. Error bars denote SEM. Actual numbers of mice in each group are indicated in parentheses above the bars. P values for comparisons between groups of K14-Survivin and non-Tg mice are indicated. (b) Histological sections of Survivin Tg and non-Tg skin stained with hematoxylin and eosin, demonstrating similar epidermal thickness and absence of hyperkeratosis in Tg skin. (c) Staining of a skin section from Tg and non-Tg mice for BrdU, indicating comparable rates of BrdU incorporation. Dark staining cells have incorporated BrdU.
Fig. 2
Fig. 2
Representative UVB-induced tumors and visualization of Survivin staining and p53 mutant clones. Immunostaining of (a) small (arrow) and (b) large p53 mutant clones at 13 weeks in K14-Survivin mouse. Clinical and histological photographs of (c) isolated papilloma on K14-Survivin mouse, (d) atypical papillomas (arrows) on non-Tg mouse, (e) SCC on K14-Survivin mouse and (f) cyst on non-Tg mouse. Atypical papilloma from K14-Survivin mouse demonstrating (g) in situ staining of Survivin and (h) control staining in the absence of the riboprobe. Original magnifications of histological photographs: (a)–(e), ×200; (f), ×100; (g)–(h), ×400.
Fig. 3
Fig. 3
Survivin expression enhances creation of new p53 mutations. K14-Survivin (open bars) and non-Tg littermates (filled bars) were UVB-irradiated for (a) 5 or (b) 7 weeks. Skin was harvested and analyzed for p53 mutant clones by immunohistochemistry. Data are expressed as number of clones per cm2 of skin for various clone size bins. Bins represent ≤1 stem cell compartment (1–16 cells), and multiples of 2, 3, 4–10 and 11–31 compartments. The P values for comparisons of clonal densities from Tg and non-Tg mice are two-sided and significant values are indicated above the bars. For statistical analysis the assumption was made that the random observed number of clones of a given size follows a Poisson distribution, with mean proportional to the total area.
Fig. 4
Fig. 4
Survivin expression impedes expansion of existing p53 mutnt clones. K14-Survivin (open bars) and non-Tg littermates (filled bars) were UVB irradiated for (a) 10 or (b) 13 weeks. Skin was harvested, and p53 mutant clones were analyzed as in Figure 3. Fewer intermediate and large clones are found in Tg skin.
Fig. 5
Fig. 5
Tumor development and histological type induced by chronic UVB treatment. K14-Survivin (open circles and bars) and non-Tg (filled circles and bars) littermates were irradiated with UVB as described for 20 weeks. (a) Percent animals that did not form tumors. The number of animals in each group is indicated in parentheses. The P value for comparison of survival curves is 0.18. (b) Histological analysis of UVB-induced tumors. Tumors <2 mm in diameter were assumed to be papillomas. Percentages of total tumors formed at the end-point for each group are shown. The actual numbers of tumors are indicated above bars. (c) Mitotic index in UVB-induced tumors. Representative papillomas, atypical papillomas and SCC were stained for PCNA and the mitotic index was determined by calculating percent positive cells in multiple fields. The actual numbers of tumors analyzed in each group and P values are indicated above bars.
Fig. 6
Fig. 6
Schematic model for the role of apoptosis in multi-step UVB carcinogenesis, showing the effects of apoptosis suppression resulting from transgenic expression of Survivin. Under normal conditions (top) UVB induces a p53 mutation in a keratinocyte stem cell, which then populates its own stem cell compartment (black hexagon). Further UVB exposures delete neighboring compartments by apoptosis (white hexagons) and these compartments can become repopulated by mutant cells (third panel). Continued UVB promotes expansion of the premalignant clone. Large clones will become papillomas, and in some papillomas cells acquire additional mutations (stippled black hexagons) that result in conversion to SCC. Under conditions of apoptosis suppression (bottom) resulting from Survivin expression, the creation of initial p53 mutations is enhanced (first panel). However, diminished apoptosis (second panel) impedes clonal expansion resulting in fewer large clones (third panel) and ultimately fewer papillomas. Reduced apoptosis promotes mutations in additional genes, as with p53, and there is enhanced malignant conversion (double arrows) of papillomas to SCC.

Similar articles

See all similar articles

Cited by 31 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

Substances

Feedback