Evaluation Of The Biological Characteristics Of Reversible Immortalized Melanocytes Induced By SV40T Antigen And Cre/loxP System

Usually life span of normal human cells cultured in vitro was limited.After a period of time proliferation cells then grew slowly, mitosis stagnated, and finally died.This phenomenon of loss of cellular growth and proliferation capacity was named replicative senescence.So,it was clear that the cells cultured in vitro could not generate enough amount to meet fully with requirements for experimental research and clinical application.It was observed that tumor cells had ability of a continuing proliferation and could be subcultured for a long time, this phenomenon was defined as immortalization.With further study, it was found that some immortalized tumor cells contained the virus gene in their genome.Therefore it could be possible to transform the normal cells to the immortalized cells by transducing and integrating the virus gene into the genome of cells cultured in vitro. Now such transgenic technology was proved feasible and became more and more common and popular in medical research.Simian virus 40 (SV40),was a very important member of DNA viruses as tools for the cellurar tranformation.In the 1960s,SV40 was firstly found and separated from the monkey kidney cells, and its natural hosts was human being.SV40 genome could encode two important proteins,T (large T) antigen and t (small t) antigen.T antigen was one kind protein of ATP enzyme and DNA helicase, which led to phosphorylation of serine/threonine residues,ADP ribosylation and acylation.T antigen also could activate host cells ribosomal gene and promote DNA synthesis, Increase the activity of transcription factor by modification of phosphorylation.Small t antigen could activate of the RNA polymerase gene promoter,as well as c-myc and c-fos oncogene transcription,repress cytoplasmic actin transcription and decrease cellular adhesion.T antigen played a decisive role in the transformation and its continuous expression was necessary for maintaining phenotype of the transformed cell.Small t antigen was non-essential for transformation,but could strengthen it.The process of immortalization was complex and still not entirely understood. Generally it was postulated that T antigen could affect cellular growth through regulating gene expression and DNA replication .It was proved T antigen could interacte with the tumor suppressores such as pRB and p53,affecting their functions of regulating cell cycle and apoptosis.And it was also found in some immortalized cells that T antigen could promote the activity of telomerase in cells to maintain telomere length.So far, T antigen gene has been extensively used to establish many immortalizated cells ,which was very useful for investigating mechanism of cellular transformation and could bring hopes for the cellular transplantation.But immortalization was considered as an indispensable stage for tumorigenesis,thus clinical application would be challenged because of its biological security.Cre/LoxP was a genetic recombination system which was extensily used in the research area of gene local integration,knocking out of a gene in particular tissues, conditional genetic recombination and chromosome translocation. In our early experiments reversible immortalized melanocytes was successfully established by using site-specific recombinant enzyme Cre/loxP system joint T antigen gene. The cell phenotype S-100,HMB-45 of the reversible immortalized melanocytes was not changed.And tumorigenicity was not found by soft-agar cloning experiment and nude mice transplanted experiment. In order to study more biological characteristics and functions of the reversible immortalized melanocytes, in particular its possibility of oncogenicity,the passage 15th immortalized cells chromosome were analysed, expressions of HLA-DR and hTERT protein were tested by immunohistochemistry and hTERT mRNA was teste by RT-PCR.Then the passage 20th immortalized cells and reversible immortalized cells proliferations were tested by MTT,cell cycle and DNA ploidy were surveyed by flow cytometry.Expressions of hMLH1 and hMSH2 mismatch repair gene were measured by semiquantitative RT-PCR.And possibility of microsatellite instability occurred in p22 (D1S187),5q14 (D5S107),and 18q12.2-12.3 (D18S34) ,three separate microsatellites localized to specific chromosome region was detected by PCR and Urea-PAGE.At the same time,tyrosinase activity and melanin content in the reversible immortalized melanocytes were compared with normal ones.The results showed that immortalized cells and reversible immortalized cells maintained normal karyotype; Expression of HLA-DR was still negative,T antigen did not activite hTERT to mediated the process of immortalization;The immortalized cells grew faster than the normal melanocytes and had the larger percentage of S+G2 stages in cell cycle. Semiquantitative RT-PCR analysis showed mRNA of hMLH1 and hMSH2 was up-regulated compared to the normal cells, but microsatellite instability(MI) was not detected in the immortalizated cells.After excision of the T antigen gene in the genome,it was found thet apoptosis occurred in some the reversible immortalized melanocytes.The cells then grew slowly like normal cells. Tyrosinase activity and melanin content of the reversible immortalized cells had no significant difference with the normal ones.The conclusions:the potential malignant transformation of the established reversible immortalized melanocytes was not detected, and normal phenotype and biological functions were maintained. These results indicated the reversible immortalized cell line would be used as ideal cell seeds in the treatment of vitiligo and could offer large quantity of melanocytes for scientific research.