Ultraviolet B irradiation induces expansion of intraepithelial tumor cells in a tissue model of early cancer progression.

Ultraviolet B irradiation is thought to enable skin cancer progression as clones of genetically damaged keratinocytes escape apoptosis and expand at the expense of adjacent normal cells. Mechanisms through which potentially malignant cells in human skin undergo clonal expansion, however, are not well understood. The goal of this study was to characterize the role of ultraviolet B irradiation on the intraepithelial expansion of early stage human tumor cells in organotypic skin cultures. To accomplish this, we have studied the effect of ultraviolet B irradiation on organotypic cultures that were fabricated by mixing normal human keratinocytes with beta-galactosidase-marked, intraepithelial tumor cells (HaCaT-ras, clone II-4), which bear mutations in both p53 alleles and harbor an activated H-ras oncogene. We found that when organotypic mixtures were exposed to an ultraviolet B dose of 50 mJ per cm2, intraepithelial tumor cells underwent a significant degree of proliferative expansion compared to nonirradiated cultures. To understand this response, organotypic cultures of nor-mal keratinocytes were exposed to ultraviolet B and showed a dose-dependent increase in numbers of sunburn cells and TUNEL-positive cells although their proliferation was suppressed. In contrast, neither the apoptotic nor the proliferative response of II-4 cells was altered by ultraviolet B in organotypic cultures. The differential response of these cell types suggested that II-4 cells were resistant to ultraviolet-B-induced alterations, which allowed these intraepithelial tumor cells to gain a selective growth and survival advantage relative to neighboring normal cells. These findings demonstrate that ultraviolet B exposure can induce the intraepithelial expansion of apoptosis-resistant, p53-mutant, and ras-activated keratinocytes, suggesting that this agent can act to promote the early stages of epithelial carcinogenesis.

Polyacrylamide Gel Polymerization: Ascorbic Acid-ferrous Sulfate-ammonium Persulfate Initiator for Acid System.

A new initiator system for polyacrylamide gel polymerization at low pH is developed. The system consists of ascorbic acid, ferrous sulfate and ammonium persulfate (AP). It is effective under acid conditions even if the concentration of the initiators is very low. To get high incorporation efficiency of acrylamide, the ratio of the AP, ascorbic acid and ferrous sulfate must be in a suitable range. The conversion efficiency has been assessed as a function of the pH of the gelling solution, in pH2.0--6.58 range. New system is able to guarantee>98% conversion in the pH 2.0--5.0 range. In contrast to the hydrogen peroxide-ascorbic acid-ferrous sulfate system widely used under acid conditions, new system gives a much higher incorporation efficiency of acrylamide, lower gel pore size at the same concentration and cross linkage and better gel mechanical property that made the gel easy handle for general purpose. The recommended concentrations of the initiators are: AP, 2.2-3.3 mmol/L, ascorbic acid, 0.91-1.82mmol/L, FeSO(4), 0.046-0.092 mmol/L.

Escape from microenvironmental control and progression of intraepithelial neoplasia.

We previously reported that normal human keratinocytes controlled neoplastic progression of tumor cells at an early stage of transformation in stratified squamous epithelium. We now studied if cells at a more advanced stage of transformation were also subject to such microenvironmental control. To accomplish this, 3D human tissues that mimic intraepithelial neoplasia were fabricated by mixing genetically marked (beta-gal), early-stage (II-4 cells) or advanced-stage (SCC13) transformed keratinocytes with normal keratinocytes, and tumor cell fate and phenotype were monitored in organotypic culture and after surface transplantation to nude mice. In vivo, SCC13 cells evaded local growth suppression to undergo connective tissue invasion at significantly lower tumor cell volumes (12:1, 50:1 normal:tumor cells) than II-4 cells. This behavior was explained by the growth suppression of II-4 cells, while advanced-stage tumor cells escaped this control and continued to undergo clonal expansion in mixed cultures to form large, intraepithelial tumor clusters. These communities of tumor cells underwent autonomous growth that was associated with altered expression of markers of differentiation (keratin 1) and cell-cell communication (connexin-43). Furthermore, significantly greater numbers of SCC13 cells expanded into a basal position after low-calcium stripping of suprabasal cells of mixed cultures compared to II-4 cells, suggesting that expansion of these cells enabled tumor cell invasion after transplantation. These findings demonstrated that early tumor development in human stratified squamous epithelium required escape from microenvironmental growth control that was dependent on the transformation stage of intraepithelial tumor cells during the premalignant stage of cancer progression.