Escaping the stem cell compartment: sustained UVB exposure allows p53-mutant keratinocytes to colonize adjacent epidermal proliferating units without incurring additional mutations - PubMed (original) (raw)

Escaping the stem cell compartment: sustained UVB exposure allows p53-mutant keratinocytes to colonize adjacent epidermal proliferating units without incurring additional mutations

W Zhang et al. Proc Natl Acad Sci U S A. 2001.

Abstract

Once mutated, a single cell must expand into a clone before becoming significant for carcinogenesis. The forces driving clonal expansion and the obstacles that must be overcome are poorly understood. In a genetic mechanism, acquiring a second mutation conferring a proliferative advantage would enable the cell to expand autonomously. If carcinogen exposure instead induced a physiological change, clonal expansion would require the carcinogen's continued presence. To determine which is the case, we studied microscopic clones of keratinocytes mutated in the p53 tumor suppressor gene. Carcinogen exposure was controlled by irradiating mice with 280-320 nm UV radiation (UVB), sunlight's principal carcinogenic component; expansion of mutant clones was observed in epidermal sheets. p53-mutant clones grew only during chronic UVB exposure. Therefore, clonal expansion was not triggered by a proliferative mutation but was instead continually driven by UVB. Unexpectedly, the clone size distribution showed periodicity with maxima at estimated intervals of 16 +/- 6 cells, the size of the epidermal proliferating unit in murine dorsal skin. In the absence of UVB, rare "imprisoned clones" increased in cell number without increasing in area. We conclude that: stem cell compartments act as physical barriers to clonal expansion of a p53-mutant keratinocyte; a rate-limiting step in clonal expansion is the colonization of an adjacent compartment; and sustained UVB enables the p53-mutant keratinocyte to colonize without incurring an additional mutation.

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Figures

Figure 1

Epidermal domains and _p53_-mutant clusters in epidermal sheets from UVB-irradiated mice. (A) Polygonal domains of nonoverlapping superficial corneocytes; counterstain is diaminobenzamide. (B) Keratinocyte nucleus doublets and triplets (arrows) regularly spaced amid single nuclei; hematoxylin staining. (C–F) Morphology of _p53_-mutant clusters. UVB dose was 1,250 J/m2 daily for 5 wks except (F) 11 wks. Mutant clusters (c) and follicles (f) are indicated. Digital photomicrography was ≈×400, except (F) ×200.

Figure 2

Number and size of _p53_-mutant clones increase with UVB irradiation. Clone number is a visual assay for initial mutational events; clone size is an assay of subsequent clonal expansion. (A) Time course. Mice were irradiated daily with 1,250 J/m2 UVB for the indicated number of weeks. Clone frequency and size were scored in epidermal sheets immunostained with CM5 antibody for normal and mutant p53 protein. (B) Data normalized to percentage of total clone number, showing the increased proportion of large _p53_-mutant clones with irradiation.

Figure 3

Sustained UVB is required for clonal expansion. (A) Clone number declines rapidly after cessation of daily 1,250 J/m2 irradiation (dashed line); ≈20% of _p53_-mutant clones stably resist regression for up to 13 wks. Mean clone number ± SEM. (B) Clone area does not grow after UVB ends. Each point on the graph has a broad distribution of clone sizes, as in Fig. 1; mean clone size is plotted. The difference between the two branches of the curve is statistically significant (P = 0.003 at 9 wks and P = 0.0002 for 11 vs. 13 wks, Wilcoxon rank sum test two-sided). One of two similar experiments is presented.

Figure 4

Quantized distribution of sizes of _p53_-mutant clones. (A) In a qualitative analysis of 868 clones grouped into size bins three cells in width, maxima occurred in the clone size distribution at intervals of ≈12–16 cells/clone. Clones larger than 100 cells are not shown. (B) Statistical analysis of clone size frequencies as the product of the aperiodic Γ distribution and a periodic series of evenly spaced normal distributions (see Methods) leads to an estimated spacing parameter μ of 16 ± 6 cells (P < 10−16). Because an epidermal stem cell compartment in murine dorsal skin contains ≈12–14 nucleated keratinocytes, these peaks correspond to individual mutant stem cell compartments. The height of successive peaks decreased in ratios of 0.5–0.8, implying that a clone of size n compartments has an ≈2/3 probability of expanding to size _n_ + 1 under these irradiation conditions. All clones were included in the analysis; not shown because of space are clones >100 cells and three points corresponding to high-frequency small clones.

Figure 5

Imprisoned clones. In the absence of UVB, rare _p53_-mutant clones achieved high cell density without a corresponding increase in clone area. Clonal expansion was thus spatially restricted in the absence of UVB exposure. UVB (1,250 J/m2) daily for 5 wks, followed by 0 J/m2 for 8 wks (×400).

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Escaping the stem cell compartment: sustained UVB exposure allows p53-mutant keratinocytes to colonize adjacent epidermal proliferating units without incurring additional mutations - PubMed (original) (raw)