Development and characterisation of an irradiation device for biomedical studies covering the solar spectrum with individual regulated spectral bands.

To understand the importance of terrestrial solar exposure on human skin, not only individual spectral components need to be considered in biomedical studies, but also the relevance of the combined action profile of the complete solar spectrum (cSS) must be established. We therefore developed a novel irradiation device that combines the emission of four individual lamps (UVB, UVA, VIS and nIR) to achieve exposure from 280 to 1400 nm with individual controllable lamps. The integrated irradiance of each spectral band is similar to the solar spectrum. The lamps can be utilised individually or in any desired combination. Here we present the design, realisation, and validation of this irradiation device as well as biological results on cellular metabolism (MTT assay), cell cycle alterations, and clonogenic growth in HaCaT cells after exposures to the individual spectral bands as well as their simultaneous combinations. Thereby, we demonstrate that UVB combined with UVA is the main determinant for the metabolic activity within cSS. Also, UVB-dependent effects dominate cell cycle regulation in cSS, whilst UVA and nIR have little influence. Lastly, also clonogenic growth is dominated by the UVB action profile in cSS, despite nIR showing modulatory activity when applied in combination with UVB. Together, this highlights the regulatory influence of the different spectral bands on the three biological endpoints and demonstrates their modulation when being part of the complete solar spectrum.

CCHCR1 interacts with EDC4, suggesting its localization in P-bodies.

Coiled-coil alpha-helical rod protein 1 (CCHCR1) is suggested as a candidate biomarker for psoriasis for more than a decade but its function remains poorly understood because of the inconsistent findings in the literature. CCHCR1 protein is suggested to be localized in the cytoplasm, nucleus, mitochondria, or centrosome and to regulate various cellular functions, including steroidogenesis, proliferation, differentiation, and cytoskeleton organization. In this study, we attempted to find a consensus between these findings by identifying the interaction partners of CCHCR1 using co-immunoprecipiation with a stable cell line expressing EGFP-tagged CCHCR1. Out of more than 100 co-immunoprecipitants identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), the enhancer of mRNA-decapping protein 4 (EDC4), which is a processing body (P-body) component, was particularly found to be the major interacting partner of CCHCR1. Confocal imaging confirmed the localization of CCHCR1 in P-bodies and its N-terminus is required for this subcellular localization, suggesting that CCHCR1 is a novel P-body component. As P-bodies are the site for mRNA metabolism, our findings provide a molecular basis for the function of CCHCR1, any disruption of which may affect the transcriptome of the cell, and causing abnormal cell functions.

"Trans-differentiation" from epidermal to mesenchymal/myogenic phenotype is associated with a drastic change in cell-cell and cell-matrix adhesion molecules.

Cells of the human keratinocyte line HaCaT were shifted to a mesenchymal/myogenic phenotype (DTHMZ cells) by MyoD1 transfection, 5-aza-2' deoxycytidine treatment, and selection for reduced adhesion on plastic. Since this correlated with loss of stratification (inability to form a multilayered tissue), we determined the status of cell-cell and cell-matrix adhesion molecules involved in epidermal morphogenesis. Expression of desmosomal proteins (plakoglobin, desmoglein, desmoplakin) and uvomorulin was no longer detectable at the mRNA and protein level in the DTHMZ cells while both HaCaT cells and malignant variants (transfected with c-Ha-ras oncogene) expressed uvomorulin in vitro and in transplants in vivo, the latter even in invasively growing tumor nodules. Furthermore, HaCaT cells stained positive for the integrin subunits beta 1, alpha 2, alpha 3, and alpha 5, typical for cultured keratinocytes. In contrast, the putative fibronectin receptor alpha 5 beta 1, common also in fibroblasts, was the only integrin showing strong staining in DTHMZ cells. The integrin subunits alpha v and a6, clearly expressed at the mRNA level, weakly stained HaCaT cultures and led to a dotlike fluorescence in DTHMZ cells, possibly representing focal adhesion plaques. The respective integrin status correlated well with the growth behavior on different matrices. While HaCaT cells readily attached and proliferated on collagen (type I), fibronectin-coated, and laminin-coated collagen gels, DTHMZ cells formed monolayers only on fibronectin-coated collagen. This was, however, not sufficient to allow stratification in vivo. Altogether, the status of adhesion molecules in DTHMZ cells more likely reflects that seen in mesenchymal cells as compared to the pattern of keratinocytes displayed by HaCaT cells. Moreover, since the DTHMZ cells were clearly HaCaT descendants, the results support our hypothesis of a "trans-differentiation" process from an epidermal (HaCaT) to a mesenchymal/myogenic phenotype (DTHMZ).

Progressive stages of "transdifferentiation" from epidermal to mesenchymal phenotype induced by MyoD1 transfection, 5-aza-2'-deoxycytidine treatment, and selection for reduced cell attachment in the human keratinocyte line HaCaT.

The ability of the myogenic determination gene (MyoD1) to convert differentiating human keratinocytes (HaCaT cell-line) to the myogenic pathway and the effect of MyoD1 on the epidermal phenotype was studied in culture and in surface transplants on nude mice. MyoD1 transfection induced the synthesis of myosin, desmin, and vimentin without substantially altering the epidermal differentiation properties (morphology, keratin profile) in vitro nor epidermal morphogenesis (formation of a complex stratified squamous epithelium) in surface transplants, demonstrating the stability of the keratinocyte phenotype. 5-Aza-CdR treatment of these MyoD1-transfected cells had little effect on the cultured cells but a morphologically unstructured epithelium was formed with no indications of typical cell layers including cornification. Since prevention of epidermal strata in transplants was not accompanied by blocked epidermal differentiation markers (keratins K1 and K10, involucrin, and filaggrin), the dissociation of morphogenesis and expression of these markers argues for independently controlled processes. A subpopulation of less adhesive cells, isolated from the 5-aza-CdR treated MyoD1-transfectants, had lost most epithelial characteristics in culture (epidermal keratins, desmosomal proteins, and surface-glycoprotein Gp90) and had shifted to a mesenchymal/myogenic phenotype (fibroblastic morphology, transactivation of Myf3 and myogenin, expression of myosin, desmin, vimentin, and Gp130). Moreover, the cells had lost the ability to stratify and remained as a monolayer of flat elongated cells in transplants. These subsequent changes from a fully differentiated keratinocyte to a mesenchymal/myogenic phenotype strongly argue for a complex "transdifferentiation" process which occurred in the original monoclonal human epidermal HaCaT cells.

Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.

In contrast to mouse epidermal cells, human skin keratinocytes are rather resistant to transformation in vitro. Immortalization has been achieved by SV40 but has resulted in cell lines with altered differentiation. We have established a spontaneously transformed human epithelial cell line from adult skin, which maintains full epidermal differentiation capacity. This HaCaT cell line is obviously immortal (greater than 140 passages), has a transformed phenotype in vitro (clonogenic on plastic and in agar) but remains nontumorigenic. Despite the altered and unlimited growth potential, HaCaT cells, similar to normal keratinocytes, reform an orderly structured and differentiated epidermal tissue when transplanted onto nude mice. Differentiation-specific keratins (Nos. 1 and 10) and other markers (involucrin and filaggrin) are expressed and regularly located. Thus, HaCaT is the first permanent epithelial cell line from adult human skin that exhibits normal differentiation and provides a promising tool for studying regulation of keratinization in human cells. On karyotyping this line is aneuploid (initially hypodiploid) with unique stable marker chromosomes indicating monoclonal origin. The identity of the HaCaT line with the tissue of origin was proven by DNA fingerprinting using hypervariable minisatellite probes. This is the first demonstration that the DNA fingerprint pattern is unaffected by long-term cultivation, transformation, and multiple chromosomal alterations, thereby offering a unique possibility for unequivocal identification of human cell lines. The characteristics of the HaCaT cell line clearly document that spontaneous transformation of human adult keratinocytes can occur in vitro and is associated with sequential chromosomal alterations, though not obligatorily linked to major defects in differentiation.

Novel roles of NM23 proteins in skin homeostasis, repair and disease.

Keratinocyte growth factor (KGF) is an important regulator of epidermal homeostasis and repair. Therefore, the identification of KGF target genes in keratinocytes should contribute to our understanding of the molecular mechanisms underlying these processes. In a search for KGF-regulated genes, we identified the gene encoding the nucleoside diphosphate kinase NM23-H1. Apart from a housekeeping function, NM23 proteins are involved in the regulation of many cellular processes as well as in tumor metastasis, but their functions in epidermal homeostasis and repair are largely unknown. Here, we show a high expression of NM23-H1 and NM23-H2 in the KGF-responsive keratinocytes of the hyperproliferative epidermis of mouse skin wounds and of patients suffering from the skin disease psoriasis. To determine if this overexpression is functionally important, we generated HaCaT keratinocyte cell lines overexpressing NM23-H1 and/or -H2. Whereas the enhanced levels of NM23 did not affect cell proliferation in monoculture, NM23-H2 and double transfectants but not NM23-H1 transfectants formed a strongly hyperthickened epithelium in three-dimensional organotypic cultures. The abnormal epithelial morphology resulted from enhanced proliferation, reduced apoptosis and alterations in the differentiation pattern. These findings suggest that epidermal homeostasis depends on a tight regulation of the levels of NM23 isoforms.

Telomere dysfunction and loss of p53 cooperate in defective mitotic segregation of chromosomes in cancer cells.

Aneuploidy is a fundamental principle of many cancer cells and is mostly related to defects in mitotic segregation of chromosomes. Many solid tumors as well as some preneoplastic lesions have been shown to contain polyploid chromosome numbers. The exact mechanisms behind whole-genome duplications are not known but have been linked to compromised mitotic checkpoint genes. We now report that the telomere checkpoint plays a key role for polyploidy in colon cancer cells. Telomerase suppression by a dominant-negative mutant of hTERT and consecutive telomere dysfunction in wild-type HCT116 colon cancer cells resulted in only minor stable chromosomal alterations. However, higher ploidy levels with up to 350 chromosomes were found when the cell-cycle checkpoint proteins p53 or p21 were absent. These findings indicate that telomere dysfunction in the absence of cell-cycle control may explain the high frequency of alterations in chromosome numbers found in many solid tumors.

Gain of 11q/cyclin D1 overexpression is an essential early step in skin cancer development and causes abnormal tissue organization and differentiation.

Non-melanoma skin cancers, in particular keratoacanthomas (KAs) and squamous cell carcinomas (SCCs), have become highly frequent tumor types especially in immune-suppressed transplant patients. Nevertheless, little is known about essential genetic changes. As a paradigm of 'early' changes, that is, changes still compatible with tumor regression, we studied KAs by comparative genomic hybridization and show that gain of chromosome 11q is not only one of the most frequent aberration (8/18), but in four tumors also the only aberration. Furthermore, 11q gain correlated with amplification of the cyclin D1 locus (10/14), as determined by fluorescence in situ hybridization, and overexpression of cyclin D1 protein (25/31), as detected by immunohistochemistry. For unraveling the functional consequence, we overexpressed cyclin D1 in HaCaT skin keratinocytes. These cells only gained little growth advantage in conventional and in organotypic co-cultures. However, although the control vector-transfected cells formed a well-stratified and orderly differentiated epidermis-like epithelium, they showed deregulation of tissue architecture with an altered localization of proliferation and impaired differentiation. The most severe phenotype was seen in a clone that additionally upregulated cdk4 and p21. These cells lacked terminal differentiation, exhibited a more autonomous growth in vitro and in vivo and even formed tumors in two injection sites with a growth pattern resembling that of human KAs. Thus, our results identify 11q13 gain/cyclin D1 overexpression as an important step in KA formation and point to a function that exceeds its known role in proliferation by disrupting tissue organization and thereby allowing abnormal growth.

Id proteins: novel targets of activin action, which regulate epidermal homeostasis.

Activin is a member of the transforming growth factor beta (TGF-beta) family, which plays a crucial role in skin morphogenesis and wound healing. To gain insight into the underlying mechanisms of action, we searched for activin-regulated genes in cultured keratinocytes. One of the identified target genes encodes Id1, a negative regulator of helix-loop-helix transcription factors. We show that Id1, Id2, and Id3 are strongly downregulated by activin in keratinocytes in vitro and in vivo. To determine the role of Id1 in keratinocyte biology, we generated stable HaCaT keratinocyte cell lines overexpressing this protein. Our results revealed that enhanced levels of Id1 do not affect proliferation of keratinocytes in monoculture under exponential culture conditions or in response to activin or TGF-beta1. However, in three-dimensional organotypic cultures, Id1-overexpressing HaCaT cells formed a hyperthickened and disorganized epithelium that was characterized by enhanced keratinocyte proliferation, abnormal differentiation, and an increased rate of apoptosis. These results identify an important function of Id1 in the regulation of epidermal homeostasis.

Phenotypic and genotypic characteristics of a cell line from a squamous cell carcinoma of human skin.

A human cell line (SCL-1) from a poorly differentiating cutaneous squamous cell carcinoma (SCC) ws studied through 20 passages during 2 years. Cells maintained their original morphology with low degree of keratinization, as indicated by light and electron microscopy. The keratin peptide pattern resembled the type in SCC tumors, and the corresponding filaments were detected by immunofluorescence at all passage levels. Cells did not grow in soft agar but formed tumor-like nodules in an "organotypic" culture assay (on lifted collagen gels) and grew invasively after transplantation to immunosuppressed inbred C3H mice. After injection into BALB/c nu/nu mice, tumors of SCC morphology were formed. The hypodiploid tumor stem-line was maintained for about 10 passages, when a shift to hyperploidy started, as determined by chromosome and DNA flow microfluorometric analyses. Two stable marker chromosomes (in 100 and 70% of the metaphases, respectively), involving chromosomes 7 and 9, strongly indicated a monoclonal origin of this cell line.

Environmental modulation of the expression of differentiation and malignancy in six human squamous cell carcinoma cell lines.

Cell lines from six human squamous cell carcinomas exhibiting different degrees of differentiation and malignancy were studied under in vitro and in vivo growth conditions. The stability of phenotypic traits of these carcinoma cells and their sensitivity to environmental influences were analyzed to further elucidate the interdependency of differentiation and malignancy expressed under experimental conditions. In conventional (submerged) cultures the cell lines exhibited unique growth patterns with an individual but generally poor expression of differentiation (stratification). In a new organotypical culture assay where the cells grew on lifted collagen gels at the air-medium interface, three-dimensional structures were formed exhibiting organizational features and degrees of differentiation similar to those of the respective tumors. Both in tumors formed after s.c. injection of cells and in transplants (performed with silicone chambers on the dorsal muscle fascia) in nude mice, an enhancement of the individually distinct pattern of differentiation was observed. While anchorage independent growth was an unreliable marker for malignancy, all six lines were tumorigenic after s.c. injection into nude mice. However, the tumor yield (20 to 100%) and latency period (2 to 12 weeks) varied considerably. In contrast all lines exhibited (within 1 to 2 weeks) invasive growth in 100% of animals after transplantation onto the dorsal muscle fascia. All tumors (squamous carcinomas) and invading cells were identified as epithelial and as human by specific antibodies. The two new test systems, the organotypical culture assay in vitro and the transplantation assay in vivo, proved to be reliable and sensitive models also for human squamous carcinoma cells to analyze their differentiative and malignant potential. In comparing the individually maintained degrees of differentiation and malignancy in the different test systems, it was apparent that, opposite to the prevailing opinion, cell lines with the highest differentiation potential were at least as malignant as were the least differentiated ones.

Telomere erosion varies during in vitro aging of normal human fibroblasts from young and adult donors.

The life span of normal fibroblasts in vitro (Hayflick limit) depends on donor age, and telomere shortening has been proposed as a potential mechanism. By quantitative fluorescence in situ hybridization and Southern blot analysis, we show progressive telomere loss to about 5 kb mean telomere restriction fragment length in fibroblasts from two adult donors within 40 population doublings, whereas in fibroblasts from two infant donors, telomere erosion is reduced, leaving a mean telomere restriction fragment length of approximately 7 kb at senescence (after approximately 60 population doublings). Aging of fibroblasts from both infant and adult donors was not accompanied by chromosomal abnormalities but was correlated with increased telomere repeat-binding factor 2 expression at both the protein and transcriptional level.

Epidermal morphogenesis and keratin expression in c-Ha-ras-transfected tumorigenic clones of the human HaCaT cell line.

Several tumorigenic (benign and malignant) clones have been raised from the human epidermal cell line HaCaT after transfection with the c-Ha-ras oncogene (val 12) (P. Boukamp et al., Cancer Res., 50: 2840-2847, 1990). In culture, these HaCaT-ras clones expressed epidermal differentiation markers, such as keratins K1 and 10, at high density or upon depletion of retinoic acid. Accordingly, as HaCaT cells, the clones formed well-differentiated stratified epithelia synthesizing K1 and 10 in surface transplants, while simple and internal epithelial keratins seen in culture were suppressed (as upon retinoic acid depletion in vitro). In transplants of HaCaT cells, in contrast to those of normal keratinocytes, K1 appeared prematurely already in basal cells, while K10 localized rather normally in the suprabasal position. Keratins 1 and 10 were also synthesized in transplants of HaCaT-ras clones (again K1 preceding K10), but both generally shifted toward upper layers. This was particularly evident in thicker transplants of malignant clones. Staining for both keratins persisted "suprabasally" in invasive tissue masses, and this corresponded to their marked expression in solid carcinomas (after s.c. injection), seen by immunofluorescence and two-dimensional gel electrophoresis. Thus, notwithstanding some variations, differentiation potential was not significantly reduced in these clones disregarding levels of ras oncogene expression and malignant properties.

Preservation of morphological, functional, and karyotypic traits during long-term culture and in vivo passage of two human skin squamous cell carcinomas.

Two cell lines (SCL-I and SCL-II) derived from squamous cell carcinomas of human skin were investigated during 4 years in culture. Both lines were tumorigenic in nude mice, and cells could be recultivated from xenografts. Growth in agar remained poor, but both cell lines developed abnormal stratified epithelial structures in organotypical cultures. The morphological and particularly ultrastructural characteristics remained typical in both cultures and xenografts. Keratinization slightly decreased, but nude mouse tumors differentiated as the original tumors, and this was reflected in keratin expression. Six major polypeptides (Mr 61,000, 57,000, 54,000, 51,000, 49,000, and 45,000) were similarly identified in both tumors and cell lines, also after animal passage, which was further substantiated by two-dimensional gel electrophoresis, but quantitative variations were found with different growth conditions. A distinct keratin cytoskeletal network was visualized in both lines by immunofluorescence, but only a few cells in SCL-II also expressed vimentin. Flow cytometry demonstrated 2c DNA stem lines for original tumors and derived lines. Early passages were hypodiploid by cytogenetic analysis of banded chromosomes. In SCL-I, a shift to tetraploidy occurred before passage 20 and remained stable throughout. In SCL-II, an incomplete shift to near tetraploidy and a stem line deviation were apparent, but later passages, nude mouse tumors, and cells recultured therefrom were hypodiploid (2c) again. Chromosome studies further revealed distinct stable marker chromosomes which showed additional structural aberrations with time in culture and after animal passage. Thus, phenotypically and genotypically, each squamous cell carcinoma and its derived cell line were distinct, and characteristics were preserved over long time periods in vitro and through in vivo passage.

c-Ha-ras oncogene expression in immortalized human keratinocytes (HaCaT) alters growth potential in vivo but lacks correlation with malignancy.

Spontaneously immortalized human skin keratinocytes (HaCaT) were transfected with the c-Ha-ras (EJ) oncogene via a plasmid construct which also contained the selectable neomycin gene. Clones were selected on the basis of G418 resistance. Those clones that had stable integrants of Ha-ras fell into 3 classes with respect to tumorigenicity. Class I clones were nontumorigenic, i.e., formed nodules which rapidly regressed. This phenotype is identical to that seen with parental HaCaT cells. Class II clones formed slowly growing, highly differentiated cystic or papillomatous-type benign tumors, and class III clones formed highly differentiated, locally invasive squamous cell carcinomas. The clones of all three classes exhibited similar morphology and growth potential in culture and retained the ability to reconstitute an epidermis-like stratified epithelium in transplantation experiments. Only the malignant clones showed locally invasive growth. Both the benign and the malignant clones exhibited higher levels of ras integration and variable levels of mutated p21 protein product. Thus, expression of the cellular Ha-ras oncogene in these human epithelial cells significantly altered growth regulation, resulting in varying degrees of growth potential in vivo, ranging from benign to malignant tumors. However, no direct correlation was seen between high levels of p21 expression and malignant growth.

Expression of epithelial antigens Exo-1 and EPM-1 in human epidermal keratinocyte maturation and benign and malignant neoplasia.

Exo-1, a polar neutral glycolipid, and EPM-1, a high molecular weight glycoprotein, are developmental antigens of human epithelial cells, initially described as components both on the cell surface and in secretions of gastrointestinal epithelial and respective tumors. In order to assess the biological significance of both antigens for epithelial cell differentiation and neoplastic transformation, their expression during human skin development and benign and malignant neoplasia was analyzed in fresh frozen tissue specimens of skin biopsies and of human epidermal keratinocytes growing in experimental model systems. Antigen expression was assessed immunohistochemically with specific monoclonal antibodies. During fetal development Exo-1 was temporarily expressed in intermediate cells but was absent in normal adult human skin. Exo-1 expression reemerged in neoplasias, both benign and malignant, but was restricted to spinous-like differentiated cells. Similarly, Exo-1 was not expressed in transplants of normal keratinocytes mimicking the normal epidermis but was clearly visible in differentiated areas of transplants of malignantly transformed keratinocytes. EPM-1 appeared first in basal epidermal cells in the second half of gestation and remained detectable in the stratum basale of adult skin. While squamous cell carcinomas continued to express EPM-1, it was not detectable in basal cell epitheliomas and in normal epidermis after invasion by neuroectodermal tumor cells. In experimental models, EPM-1 was present in the basal layers of normal human keratinocytes and of transformed keratinocytes with benign growth characteristics whenever a well stratified and keratinized epidermis-like epithelium had formed in transplants. In transformed keratinocytes with malignant growth behavior, EPM-1 was expressed irregularly, as in squamous cell carcinomas in situ. Thus, expression of Exo-1 is a marker for an early embryonic differentiation pathway of human keratinocytes and in adult tissue reveals abnormal differentiation associated with certain stages of hyperproliferation. EPM-1 expression is part of developmental programs and is influenced by microenvironmental interactions and alterations of tissue homeostasis.

Step-wise progression in human skin carcinogenesis in vitro involves mutational inactivation of p53, rasH oncogene activation and additional chromosome loss.

Two mechanisms relevant for skin carcinogenesis in man are mutational inactivation of p53 and oncogenic activation of c-rasH gene. Previously, we transfected c-rasH oncogene into human skin keratinocytes (HaCaT) with u.v.-typic mutations in both p53 alleles, which produced benign and malignant tumorigenic clones, expressing similar amounts of mutant Ras protein. Here we show that neither the ras integration site nor the karyotypic changes affects the formation of the benign or malignant tumorigenic phenotype. From the original malignant HaCaT-ras clone we took single human chromosomes, carrying the c-rasH oncogene and transferred them by microcell mediated chromosome transfer into genetically different untransfected nontumorigenic HaCaT cells. This novel approach identified the genetic background of the recipient cell as a critical determinant for the resulting tumor phenotype. Exhibiting similar oncogene expression, microcell hybrids from early passage cells remained nontumorigenic or formed benign tumors, while those with more cytogenetic aberrations (later passages) and loss of > 1 copy of chromosome 15 became malignant. Since aberrations in chromosome 15 were also detected in three of five human skin carcinoma lines this study provides evidence that p53 and c-rasH mutations are early events of human skin carcinogenesis, while loss of gene(s) on chromosome 15 is a late event.

Molecular and cytogenetic analysis of immortalized human primary keratinocytes obtained after transfection with human papillomavirus type 16 DNA.

A proliferating population of human foreskin keratinocytes (presently in the sixtieth passage) has been obtained after transfection with human papillomavirus (HPV) type 16 DNA. In contrast, the control cultures did not survive beyond the sixth passage. Cytogenetic analysis of cells taken from the twelfth passage revealed a heteroploid male karyotype. In approximately 50% of the cells a common marker chromosome was found, suggesting a clonal origin for at least part of the population. This is further substantiated by Southern blot analysis of cellular DNA which revealed oligomeric HPV 16 genomes integrated at a single site within the host DNA. RNA transcribed from the early region of the HPV 16 genome was identified in the cytoplasm. The immortalizing effect of HPV 16 DNA on human keratinocytes could be reproduced in a second experiment. Such cell lines represent an unique system to study the interaction of HPV with its natural target cell in vitro.

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

UV-radiation is a major risk factor for non-melanoma skin cancer causing specific 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 40 degrees C reproducibly resulted in tumorigenic conversion and tumorigenicity was stably maintained after recultivation of the tumors. Growth at 40 degrees C was correlated with the appearance of PARP, an enzyme activated by DNA strand breaks and the level corresponded to that seen after 5 Gy gamma-radiation. Concomitantly, comparative genomic hybridization (CGH) analyis demonstrated that chromosomal gains and losses were present in cells maintained at 40 degrees C while largely absent at 37 degrees C. 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 effect through induction of genetic instability which results in selection of tumorigenic cells characterized by gain of 11q.

Growth and differentiation characteristics of transformed keratinocytes from mouse and human skin in vitro and in vivo.

The altered phenotypic expression and chromosomal characteristics of mouse and human malignant keratinocyte lines have been studied in vitro and in vivo (in comparison with normal primary cultures). The cell lines exhibited different morphologic aspects that are probably more related to their respective degree of differentiation than to different stages in malignancy. Although all cell lines studied were deficient in some aspects of keratinization, certain basic structural and biochemical features were maintained, and these may serve as valid criteria for the identification of their epithelial nature. The altered expression of keratin proteins and morphologic differentiation can be modulated under in vivo growth conditions, but they cannot be reverted toward normality. Chromosomal alterations (in number and structure) occur early and are highly indicative criteria for malignancy, even though no tumor-specific aberrations have been identified. Two new approaches for evaluating characteristics of abnormal growth and differentiation in vitro, and of invasiveness in vivo, have been developed and have proved sensitive test methods for identifying malignant cells. While several abnormalities in growth and differentiation of cell lines in vitro are highly indicative of their malignant nature, the final proof that they are tumor cells still requires in vivo assay. The transplantation assay for studying cellular invasiveness not only improves the sensitivity of in vivo malignancy tests but has also proved to be a valuable model system for elucidating the modulation of differentiation by external influences.