Tumorigenic conversion of immortal human skin keratinocytes (HaCaT) by elevated temperature

Abstract

UV-radiation is a major risk factor for non-melanoma skin cancer causing speci®c mutations in the p53 tumor suppressor gene and other genetic aberrations. We here propose that elevated temperature, as found in sunburn areas, may contribute to skin carcinogenesis as well. Continuous exposure of immortal human HaCaT skin keratinocytes (possessing UV-type p53 mutations) to 408C reproducibly resulted in tumorigenic conversion and tumorigenicity was stably maintained after recultivation of the tumors. Growth at 408C was correlated with the appearance of PARP, an enzyme activated by DNA strand breaks and the level corresponded to that seen after 5 Gy g-radiation. Concomitantly, comparative genomic hybridization (CGH) analyis demonstrated that chromosomal gains and losses were present in cells maintained at 408C while largely absent at 378C. Besides individual chromosomal aberrations, all tumor-derived cells showed gain of chromosomal material on 11q with the smallest common region being 11q13.2 to q14.1. Cyclin D1, a candidate gene of that region was overexpressed in all tumor-derived cells but cyclinD1/ cdk4/cdk6 kinase activity was not increased. Thus, these data demonstrate that long-term thermal stress is a potential carcinogenic factor in this relevant skin cancer model, mediating its e ect through induction of genetic instability which results in selection of tumorigenic cells characterized by gain of 11q.

Tumor Progression of Skin Carcinoma Cells in Vivo Promoted by Clonal Selection, Mutagenesis, and Autocrine Growth Regulation by Granulocyte Colony-Stimulating Factor and Granulocyte-Macrophage Colony-Stimulating Factor

“…The stress was exerted either by repeated single-cell cloning (HaCaTp) 36 or by growth at 40°C instead of 37°C (HaCaT40°). 35 Under both stress conditions HaCaT cells were converted to tumorigenicity. HaCaTp cells formed noninvasive, slowly growing benign cysts, 36 whereas HaCaT40°c ells gave rise to well-differentiated SCCs.…”

“…This in vivo progression of HaCaT clones, which had been converted into tumorigenicity without the influence of the H-ras oncogene to an enhanced malignant tumor phenotype, has been reproducibly observed. 35,36 …”

“…The tumors formed were either noninvasive, slowly growing benign cysts HaCaTp 36 or locally invasive SCCs HaCaT40°. 35 The following cell lines were established after in vivo passage by recultivation of subcutaneously transplanted tumors: HaCaTpT, HaCaT40°RT, II-3RT, II-4RT, A-5RT1, and A-5RT3 (Figure 1). …”

“…26 Because of their genetic make-up and primarily maintained phenotypic normality, they are considered to represent a very early stage in skin tumorigenesis. 4 Their tumorigenic conversion was achieved by transfection with the mutated val-12 Harvey-ras oncogene 34 but also by growth under elevated temperature (sunburn conditions) 35 or extremely stressful culture conditions such as repeated single cell cloning 36 or adaptation to autotrophy. 37 In epithelial skin tumors, an activated ras oncogene has been detected in squamous and basal cell carcinomas, suggesting its causal role at least in a subset of epithelial skin tumors.…”

Alloreactive microenvironment after human hematopoietic cell transplantation induces genomic alterations in epithelium through an ROS-mediated mechanism: in vivo and in vitro study and implications to secondary neoplasia

“…For western blotting, 50 mg protein extract was analyzed using the following antibodies, as described: 22 rabbit anti-MSH3, anti-PMS1 and anti-PMS2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti-MSH6 and anti-MLH1 (BD Pharmingen, Heidelberg, Germany); mouse anti-MSH2 (Calbiochem, San Diego, CA, USA); and mouse antip53 (DakoCytomation, Glostrup, Denmark). Intracellular reactive oxygen species (ROS) generation was measured using the fluorescent probe H 2 -DCFDA (Molecular Probes Inc., Eugene, OR, USA) and analyzed by flow cytometry, as described.…”