Knockdown of Simulated-Solar-Radiation-Sensitive miR-205-5p Does Not Induce Progression of Cutaneous Squamous Cell Carcinoma In Vitro.

Solar radiation is the main risk factor for cSCC development, yet it is unclear whether the progression of cSCC is promoted by solar radiation in the same way as initial tumorigenesis. Additionally, the role of miRNAs, which exert crucial functions in various tumors, needs to be further elucidated in the context of cSCC progression and connection to solar radiation. Thus, we chronically irradiated five cSCC cell lines (Met-1, Met-4, SCC-12, SCC-13, SCL-II) with a custom-built irradiation device mimicking the solar spectrum (UVB, UVA, visible light (VIS), and near-infrared (IRA)). Subsequently, miRNA expression of 51 cancer-associated miRNAs was scrutinized using a flow cytometric multiplex quantification assay (FirePlex®, Abcam). In total, nine miRNAs were differentially expressed in cell-type-specific as well as universal manners. miR-205-5p was the only miRNA downregulated after SSR-irradiation in agreement with previously gathered data in tissue samples. However, inhibition of miR-205-5p with an antagomir did not affect cell cycle, cell growth, apoptosis, or migration in vitro despite transient upregulation of oncogenic target genes after miR-205-5p knockdown. These results render miR-205-5p an unlikely intracellular effector in cSCC progression. Thus, effects on intercellular communication in cSCC or the simultaneous examination of complementary miRNA sets should be investigated.

Enrichment of Human Dermal Stem Cells from Primary Cell Cultures through the Elimination of Fibroblasts.

Dermal stem cells (DSCs), which are progenitor cells of melanocytes, are isolated from human foreskin and cultivated as mixed cultures containing both DSCs and fibroblasts in varying proportions. These contaminating fibroblasts may have an impact on the results of experimental studies and are a serious limitation for certain applications. The aim of the present study was to purify or enrich DSCs-an indispensable step towards future investigations. Applying different methods, we demonstrated that highly enriched DSCs with a good recovery rate can be obtained through positive selection with MACS® immunomagnetic cell sorting. These DSCs remain vital and proliferate constantly in culture, maintaining a high level of purity after enrichment. Other approaches such as treatment with Geneticin or selective detachment were not suitable to purify DSC-fibroblast co-cultures. Overall, enriched DSCs represent a novel and unique model to study the effects of UV radiation on the differentiation of DSCs into melanocytes and their potential relevance in the genesis of malignant melanoma.

UVA-induced epigenetic regulation of P16(INK4a) in human epidermal keratinocytes and skin tumor derived cells.

UVA-radiation (315-400 nm) has been demonstrated to be capable of inducing DNA damage and is regarded as a carcinogen. While chromosomal aberrations found in UVA-irradiated cells and skin tumors provided evidence of the genetic involvement in UVA-carcinogenesis, its epigenetic participation is still illusive. We thus analysed the epigenetic patterns of 5 specific genes that are involved in stem cell fate (KLF4, NANOG), telomere maintenance (hTERT) and tumor suppression in cell cycle control (P16(INK4a), P21(WAFI/CIPI)) in chronically UVA-irradiated HaCaT human keratinocytes. A striking reduction of the permissive histone mark H3K4me3 has been detected in the promoter of P16(INK4a) (4-fold and 9-fold reduction for 10 and 15 weeks UVA-irradiated cells, respectively), which has often been found deregulated in skin cancers. This alteration in histone modification together with a severe promoter hypermethylation strongly impaired the transcription of P16(INK4a) (20-fold and 40-fold for 10 weeks and 15 weeks UVA-irradiation, respectively). Analysis of the skin tumor-derived cells revealed the same severe impairment of the P16(INK4a) transcription attributed to promoter hypermethylation and enrichment of the heterochromatin histone mark H3K9me3 and the repressive mark H3K27me3. Less pronounced UVA-induced epigenetic alterations were also detected for the other genes, demonstrating for the first time that UVA is able to modify transcription of skin cancer associated genes by means of epigenetic DNA and histone alterations.

Adjunctive Bright Light Therapy for Non-Seasonal Major Depressive Disorder: A Randomized Controlled Trial.

This double-blind, randomized controlled trial assessed bright light therapy (BLT) augmentation efficacy compared with placebo light in treating non-seasonal major depressive disorder. The study participants belonged to a subtropical area (24.5°-25.5°N) with extensive daylight and included outpatients who had received stable dosages and various regimens of antidepressive agents for 4 weeks before enrollment. The outcomes were the 17-item Hamilton Depression Rating Scale, Montgomery-Asberg Depression Rating Scale, and Patient Health Questionnaire-9, which were assessed at weeks 1, 2, and 4. A total of 43 participants (mean age 45 years, ranging from 22-81) were randomized into the BLT [n = 22] and placebo light groups [n = 21]. After a 4-week administration of morning light therapy (30 min/day), depressive symptoms did not reduce significantly, which might be due to the small sample size. Nonetheless, this study had some strengths because it was conducted in warmer climates, unlike other studies, and examined diverse Asians with depression. Our findings suggest that several factors, such as poor drug response, different antidepressive regimens, duration of BLT, and daylength variability (i.e., natural daylight in the environment) may influence the utility of add-on BLT. Researchers may consider these important factors for future non-seasonal depression studies in subtropical environments.

Characterisation of the novel spontaneously immortalized and invasively growing human skin keratinocyte line HaSKpw.

We here present the spontaneously immortalised cell line, HaSKpw, as a novel model for the multistep process of skin carcinogenesis. HaSKpw cells were established from the epidermis of normal human adult skin that, without crisis, are now growing unrestricted and feeder-independent. At passage 22, clonal populations were established and clone7 (HaSKpwC7) was further compared to the also spontaneously immortalized HaCaT cells. As important differences, the HaSKpw cells express wild-type p53, remain pseudodiploid, and show a unique chromosomal profile with numerous complex aberrations involving chromosome 20. In addition, HaSKpw cells overexpress a pattern of genes and miRNAs such as KRT34, LOX, S100A9, miR21, and miR155; all pointing to a tumorigenic status. In concordance, HaSKpw cells exhibit reduced desmosomal contacts that provide them with increased motility and a highly migratory/invasive phenotype as demonstrated in scratch- and Boyden chamber assays. In 3D organotypic cultures, both HaCaT and HaSKpw cells form disorganized epithelia but only the HaSKpw cells show tumorcell-like invasive growth. Together, HaSKpwC7 and HaCaT cells represent two spontaneous (non-genetically engineered) "premalignant" keratinocyte lines from adult human skin that display different stages of the multistep process of skin carcinogenesis and thus represent unique models for analysing skin cancer development and progression.

UV-type specific alteration of miRNA expression and its association with tumor progression and metastasis in SCC cell lines.

PURPOSE: UV exposure is the main risk factor for development of cutaneous squamous cell carcinoma (cSCC). While early detection greatly improves cSCC prognosis, locally advanced or metastatic cSCC has a severely impaired prognosis. Notably, the mechanisms of progression to metastatic cSCC are not well understood. We hypothesized that UV exposure of already transformed epithelial cSCC cells further induces changes which might be involved in the progression to metastatic cSCCs and that UV-inducible microRNAs (miRNAs) might play an important role. METHODS: Thus, we analyzed the impact of UV radiation of different quality (UVA, UVB, UVA + UVB) on the miRNA expression pattern in established cell lines generated from primary and metastatic cSCCs (Met-1, Met-4) using the NanoString nCounter platform. RESULTS: This analysis revealed that the expression pattern of miRNAs depends on both the cell line used per se and on the quality of UV radiation. Comparison of UV-induced miRNAs in cSCC cell lines established from a primary tumor (Met-1) and the respective (un-irradiated) metastasis (Met-4) suggest that miR-7-5p, miR-29a-3p and miR-183-5p are involved in a UV-driven pathway of progression to metastasis. This notion is supported by the fact that these three miRNAs build up a network of 81 potential target genes involved e.g. in UVA/UVB-induced MAPK signaling and regulation of the epithelial-mesenchymal transition. As an example, PTEN, a target of UV-upregulated miRNAs (miR-29a-3p, miR-183-5p), could be shown to be down-regulated in response to UV radiation. We further identified CNOT8, the transcription complex subunit 8 of the CCR4-NOT complex, a deadenylase removing the poly(A) tail from miRNA-destabilized mRNAs, in the center of this network, targeted by all three miRNAs. CONCLUSION: In summary, our results demonstrate that UV radiation induces an miRNA expression pattern in primary SCC cell line partly resembling those of metastatic cell line, thus suggesting that UV radiation impacts SCC progression beyond initiation.

UVA and UVB irradiation differentially regulate microRNA expression in human primary keratinocytes.

MicroRNA (miRNA)-mediated regulation of the cellular transcriptome is an important epigenetic mechanism for fine-tuning regulatory pathways. These include processes related to skin cancer development, progression and metastasis. However, little is known about the role of microRNA as an intermediary in the carcinogenic processes following exposure to UV-radiation. We now show that UV irradiation of human primary keratinocytes modulates the expression of several cellular miRNAs. A common set of miRNAs was influenced by exposure to both UVA and UVB. However, each wavelength band also activated a distinct subset of miRNAs. Common sets of UVA- and UVB-regulated miRNAs harbor the regulatory elements GLYCA-nTRE, GATA-1-undefined-site-13 or Hox-2.3-undefined-site-2 in their promoters. In silico analysis indicates that the differentially expressed miRNAs responding to UV have potential functions in the cellular pathways of cell growth and proliferation. Interestingly, the expression of miR-23b, which is a differentiation marker of human keratinocytes, is remarkably up-regulated after UVA irradiation. Studying the interaction between miR-23b and its putative skin-relevant targets using a Luciferase reporter assay revealed that RRAS2 (related RAS viral oncogene homolog 2), which is strongly expressed in highly aggressive malignant skin cancer, to be a direct target of miR-23b. This study demonstrates for the first time a differential miRNA response to UVA and UVB in human primary keratinocytes. This suggests that selective regulation of signaling pathways occurs in response to different UV energies. This may shed new light on miRNA-regulated carcinogenic processes involved in UV-induced skin carcinogenesis.

A homolog of ScRAD5 is involved in DNA repair and homologous recombination in Arabidopsis.

Rad5 is the key component in the Rad5-dependent error-free branch of postreplication repair in yeast (Saccharomyces cerevisiae). Rad5 is a member of the Snf2 ATPase/helicase family, possessing as a characteristic feature, a RING-finger domain embedded in the Snf2-helicase domain and a HIRAN domain. Yeast mutants are sensitive to DNA-damaging agents and reveal differences in homologous recombination. By sequence comparisons we were able to identify two homologs (AtRAD5a and AtRAD5b) in the Arabidopsis thaliana genome, sharing about 30% identity and 45% similarity to yeast Rad5. AtRad5a and AtRad5b have the same kind of domain organization with a higher degree of similarity to each other than to ScRad5. Surprisingly, both genes differ in function: whereas two independent mutants of Atrad5a are hypersensitive to the cross-linking agents mitomycin C and cis-platin and to a lesser extent to the methylating agent, methyl methane sulfonate, the Atrad5b mutants did not exhibit any sensitivity to all DNA-damaging agents tested. An Atrad5a/Atrad5b double mutant resembles the sensitivity phenotype of the Atrad5a single mutants. Moreover, in contrast to Atrad5b, the two Atrad5a mutants are deficient in homologous recombination after treatment with the double-strand break-inducing agent bleomycin. Our results suggest that the RAD5-dependent error-free branch of postreplication repair is conserved between yeast and plants, and that AtRad5a might be functionally homologous to ScRad5.

The transcriptional response of Arabidopsis to genotoxic stress - a high-density colony array study (HDCA).

A genome-wide transcription profiling of Arabidopsis upon genotoxic stress has been performed using a high-density colony array (HDCA). The array was based on a library of 27 000 cDNA clones derived from Arabidopsis cells challenged with bleomycin plus mitomycin C. The array covers more than 10 000 individual genes (corresponding to at least 40% of Arabidopsis genes). After hybridisation of the HDCA with labelled cDNA probes obtained from genotoxin-treated (bleomycin plus mitomycin C, 6 h) and untreated seedlings, 39 genes revealed an increased and 24 genes a decreased expression among the 3200 highly expressed clones (representing approximately 1200 individual genes because of redundancy of the cDNA library). Of the 4900 clones with a low transcriptional level, the expression of 500 clones was found to be altered and 57 genes with increased and 22 genes with decreased expression were identified by sequence analysis of 135 identified clones. The HDCA results were validated by real-time PCR analysis. For about 80% of genes (34 out of 42), alteration in expression was confirmed, indicating the reliability of the HDCA for transcription profiling. DNA damage and stress-responsive genes encoding, for instance transcription factors (myb protein and WRKY1), the ribonucleotide reductase small subunit (RNR2), thymidine kinase (TK), an AAA-type ATPase, the small subunit of a DNA polymerase and a calmodulin-like protein were found to be strongly upregulated. Also, several genes involved in cell cycle regulation revealed significant alteration in transcription, as detected by real-time PCR analysis, suggesting disturbance of cell cycle progression by mutagen treatment.

The Rad17 homologue of Arabidopsis is involved in the regulation of DNA damage repair and homologous recombination.

Rad17 is involved in DNA checkpoint control in yeast and human cells. A homologue of this gene as well as other genes of the pathway (the 9-1-1 complex) are present in Arabidopsis and share conserved sequence domains with their yeast and human counterparts. DNA-damaging agents induce AtRAD17 transcriptionally. AtRAD17 mutants show increased sensitivity to the DNA-damaging chemicals bleomycin and mitomycin C (MMC), which can be reversed by complementation, suggesting that the loss of function of Rad17 disturbs DNA repair in plant cells. Our results are further confirmed by the phenotype of a mutant of the 9-1-1 complex (Rad9), which is also sensitive to the same chemicals. AtRAD9 and AtRAD17 seem to be epistatic as the double mutant is not more sensitive to the chemicals than the single mutants. The mutants show a delay in the general repair of double-strand breaks (DSBs). However, frequencies of intrachromosomal homologous recombination (HR) are enhanced. Nevertheless, the mutants are proficient for a further induction of HR by genotoxic stresses. Our results indicate that a mutant Rad17 pathway is associated with a general deregulation of DNA repair, which seems to be correlated with a deficiency in non-homologous DSB repair.