RESUMO
Ultraviolet (UV) radiation is one of the most important external stimuli that affects skin by inducing cancer, inflammation and cell death. To identify the regulation of genes regulated by UV during transformation, normal human keratinocyte cell line, HaCaT, was exposed to multiple doses of UVA+B (UVA - 150-200 mJ/cm2 and UVB - 15-20 mJ/cm2 x 6). Malignant transformation was confirmed by formation of colonies on soft agar and DNA methylation assay. To identify the genes involved in this process, random amplification of polymorphic DNA using RNA from unexposed and multiple exposed cells was performed after each exposure. A few up-regulated genes were identified, cloned and sequenced. One of the genes had homology to EDD (E3 identified by differential display) that was up-regulated at second exposure but was down-regulated in colony-forming cells (cells that received six or more exposures) as determined by RT-PCR. This is a progesterone-induced gene and progesterone treatment reduced the extent of colony formation on soft agar plate. It is possible that hormone therapy may have some effects on skin cancer in vivo.
Assuntos
Dano ao DNA , Queratinócitos/citologia , Queratinócitos/efeitos da radiação , Ubiquitina-Proteína Ligases/genética , Raios Ultravioleta , Linhagem Celular Transformada , Clonagem Molecular , Primers do DNA , Relação Dose-Resposta à Radiação , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Queratinócitos/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Human keratinocytes (HaCaT) were exposed to UV (A+B) (UVA-350-400 mJ/cm2 and UVB-30 mJ/cm2) which induces apoptosis as evidenced by MTT assay, DNA laddering, Bax and Fas up-regulation. UV induced apoptotic conditioned media (6 h or earlier) did not cause apoptosis in unexposed cells. However, treatment with conditioned medium collected post UV exposure (1 h) induced Bax in unexposed cells as observed by RT-PCR. The induction of cell death was initiated by conditioned medium collected 12 h after UV exposure and the extent of death was increased progressively when conditioned medium collected 24 or 72 h post UV exposure was used. Medium collected 24 h after UV exposure also increased mitochondrial membrane permeability as determined by rhodamine uptake. Conditioned medium induced apoptosis did not involve reactive oxygen species (ROS) unlike UV induced apoptosis indicating that the apoptosis pathway could be different. Interestingly, at high dilution apototic conditioned medium did not induce apoptosis but actually protected cells from UV insult. The role of nerve growth factor (NGF) in UV induced bystander effects are also discussed.
Assuntos
Apoptose , Efeito Espectador , Dano ao DNA , Queratinócitos/fisiologia , Raios Ultravioleta , Permeabilidade da Membrana Celular , Meios de Cultivo Condicionados , Formazans/farmacologia , Regulação da Expressão Gênica , Humanos , Mitocôndrias , Espécies Reativas de Oxigênio , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Sais de Tetrazólio/farmacologia , Regulação para CimaRESUMO
The normal human keratinocyte cell line, HaCaT, was transformed using multiple doses of ultraviolet (UV)A+B (UVA, 150-200 mJ/cm(2) and UVB, 15-20 mJ/cm(2) x 6). Malignant transformation was confirmed by upregulation of Cyclin D1 (mRNA) and formation of colonies on soft agar. To identify the genes involved in this transformation process, we have done rapid amplification of polymorphic DNA using RNA from unexposed and multiple-exposed cells. Six percent PAGE showed several differentially regulated genes in exposed cells compared with unexposed cells. Total 19 genes were identified, cloned and sequenced. Three of these 19 cloned genes showed 99% homology at both DNA and protein levels to a stretch of 540 bp (180 aa) of long interspersed element (LINE)-1 reverse transcriptase (RT) open reading frame (ORF-2). Colonies from soft agar showed upregulation of this gene compared with non-colonized (lawn on soft agar) cells as detected by RT-PCR. This data implicates LINE-1 RT (ORF-2) in UV-induced malignancy and can possibly be used as a marker for the diagnosis of UV-induced skin cancer.
Assuntos
Apoptose/genética , Apoptose/efeitos da radiação , Queratinócitos/enzimologia , Queratinócitos/efeitos da radiação , Elementos Nucleotídeos Longos e Dispersos/genética , Elementos Nucleotídeos Longos e Dispersos/efeitos da radiação , Raios Ultravioleta , Biomarcadores Tumorais/metabolismo , Linhagem Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/genéticaRESUMO
Differential gene regulation during UVB induced apoptosis of human keratinocyte cell line (HaCaT) has been investigated. Rapid amplification of polymorphic DNA (RAPD)-PCR was done to identify novel/unique genes in the purified apoptotic and non-apoptotic populations. Two genes were identified and cloned in pGemT vector. One of these genes (apgene-1) was upregulated in UV induced apoptotic cells and in the non apoptotic cells exposed to UV. The other gene (apgene-2) was not detected in apoptotic cells but expressed in non-apoptotic/non necrotic cells that had been exposed to UV. The presence of apgene-1 mRNA was not detected in camptothecin induced apoptotic as well as non apoptotic cells. Apgene-2 was not detected in camptothecin induced apoptotic cells but expressed in non-apoptotic/non necrotic cells. This data indicates differential regulation of these two genes during UV and chemical induced apoptosis in human keratinocytes. Additionally, since apgene-2 was upregulated in the non necrotic/non apoptotic population could be involved in protection.
