Growth stimulation of UV-induced DNA damage retaining epidermal basal cells gives rise to clusters of p53 overexpressing cells.

Ultraviolet (UV) radiation induces cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts ((6-4)PPs) in DNA, which may give rise to clusters of cells expressing mutant p53 ('p53 patches') and eventually to skin carcinomas. We have previously reported that some basal cells in murine skin accumulate CPDs upon chronic low-level UV exposure and that these CPD-retaining basal cells (CRBCs) encompass epidermal stem and progenitor cells. Through replication of their damaged DNA CRBCs may become mutagenic foci from which tumors might form. We therefore investigated whether CRBCs may give rise to p53 patches after forced proliferation by repeated applications of 12-O-tetradecanoylphorbol-13-acetate (TPA). CRBCs, induced in SKH-1 hairless mice by chronic low-level UV exposure (70 J/m(2) daily for 40 days), disappeared in the TPA-induced epidermal hyperplasia within 2 weeks and numerous clusters of epidermal cells with overexpressed p53 appeared after 4 weeks. Neither mutant p53 patches nor any foci of pErk1/2-overexpressing cells that could have caused reactive wild type p53 expression were found. In skin exposed to a single high UV dose (2.8 kJ/m(2)) no CRBCs occurred, and no p53 clusters were observed after TPA treatment. These experiments suggest that CRBCs are a prerequisite for the formation of clusters of p53-overexpressing cells. The high frequency of these clusters (about 1 for every 3 CRBCs) precludes mutations in p53 as a likely cause. We surmise that forced proliferation of CRBCs gives rise to genomic instability that is propagated in daughter cells and evokes wild type p53 overexpression, signifying a potentially oncogenic process different from classic UV carcinogenesis involving mutant p53.

CD34 expression by hair follicle stem cells is required for skin tumor development in mice.

The cell surface marker CD34 marks mouse hair follicle bulge cells, which have attributes of stem cells, including quiescence and multipotency. Using a CD34 knockout (KO) mouse, we tested the hypothesis that CD34 may participate in tumor development in mice because hair follicle stem cells are thought to be a major target of carcinogens in the two-stage model of mouse skin carcinogenesis. Following initiation with 200 nmol 7,12-dimethylbenz(a)anthracene (DMBA), mice were promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 20 weeks. Under these conditions, CD34KO mice failed to develop papillomas. Increasing the initiating dose of DMBA to 400 nmol resulted in tumor development in the CD34KO mice, albeit with an increased latency and lower tumor yield compared with the wild-type (WT) strain. DNA adduct analysis of keratinocytes from DMBA-initiated CD34KO mice revealed that DMBA was metabolically activated into carcinogenic diol epoxides at both 200 and 400 nmol. Chronic exposure to TPA revealed that CD34KO skin developed and sustained epidermal hyperplasia. However, CD34KO hair follicles typically remained in telogen rather than transitioning into anagen growth, confirmed by retention of bromodeoxyuridine-labeled bulge stem cells within the hair follicle. Unique localization of the hair follicle progenitor cell marker MTS24 was found in interfollicular basal cells in TPA-treated WT mice, whereas staining remained restricted to the hair follicles of CD34KO mice, suggesting that progenitor cells migrate into epidermis differently between strains. These data show that CD34 is required for TPA-induced hair follicle stem cell activation and tumor formation in mice.

Epidermal stem and progenitor cells in murine epidermis accumulate UV damage despite NER proficiency.

Ultraviolet (UV) radiation induces cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs) in DNA, which through gene mutations (e.g. in P53) may lead to skin carcinogenesis. Upon chronic low-level UV exposure, certain basal cells in mouse epidermis were reported to accumulate CPDs. These observations raised questions on whether these cells were fully DNA-repair deficient, and whether they were stem or progenitor cells, as suggested by their long residence time. We found that CPD-retaining basal cells (CRBCs) in SKH-1 hairless mice were repair proficient as accumulation of (6-4)PP, which is a hallmark for complete nucleotide excision repair-deficiency in rodents, was not observed. Accumulation of 6-4PP as well as CPD did, however, occur in basal cells in the epidermis of DNA repair-deficient Xpc-/- mice. Chronic UV exposure of DDB2 transgenic mice and DDB2 knockout mice revealed that the occurrence of CRBCs was inversely correlated with DDB2-expression, indicating that a boost in DNA repair lowered CPD accumulation. Stem cells are quiescent cells and can be identified as 5-bromo-2'-deoxyuridine-label retaining cells (BrdU-LRCs). Induction of BrdU-LRCs followed by chronic UV irradiation showed that all BrdU-label retaining stem cells were also CPD-retaining cells. As most CRBCs were not BrdU-labeled we surmized that these cells must include BrdU-negative stem cells and early progenitor cells. In confirmation of the latter, we found that CRBCs occurred among MTS24+ hair follicle progenitor cells. These findings provide the first evidence that epidermal stem and progenitor cells are prone to the accumulation of UV-induced DNA-damage and can be a prominent target in skin carcinogenesis.

The cell-surface marker MTS24 identifies a novel population of follicular keratinocytes with characteristics of progenitor cells.

We describe a novel murine progenitor cell population localised to a previously uncharacterised region between sebaceous glands and the hair follicle bulge, defined by its reactivity to the thymic epithelial progenitor cell marker MTS24. MTS24 labels a membrane-bound antigen present during the early stages of hair follicle development and in adult mice. MTS24 co-localises with expression of alpha6-integrin and keratin 14, indicating that these cells include basal keratinocytes. This novel population does not express the bulge-specific stem cell markers CD34 or keratin 15, and is infrequently BrdU label retaining. MTS24-positive and -negative keratinocyte populations were isolated by flow cytometry and assessed for colony-forming efficiency. MTS24-positive keratinocytes exhibited a two-fold increase in colony formation and colony size compared to MTS24-negative basal keratinocytes. In addition, both the MTS24-positive and CD34-positive subpopulations were capable of producing secondary colonies after serial passage of individual cell clones. Finally, gene expression profiles of MTS24 and CD34 subpopulations were compared. These results showed that the overall gene expression profile of MTS24-positive cells resembles the pattern previously reported in bulge stem cells. Taken together, these data suggest that the cell-surface marker MTS24 identifies a new reservoir of hair follicle keratinocytes with a proliferative capacity and gene expression profile suggestive of progenitor or stem cells.