| Summary: | 碩士 === 國防醫學院 === 生物及解剖學研究所 === 93 === Six isoforms of p63, TAp63α, TAp63β, TAp63γ, ΔNp63α, ΔNp63β and ΔNp63γ, have been identified. p63 is considered as a keratinocyte stem cell maker. During the differentiation of keratinocyte, down-regulation of ΔNp63α and keratin 14 in keratinocyte could be observed. Over expression of TAp63 isofroms could block calcium induced keratinocytes differentiation. TAp63 isoforms induced K14 expression in some simple squamous carcinoma cell lines without p63 and K14 expression. In our previous studies, TPA inhibited K14 expression in human ovary teratocarcinoma cells (HOTC) - C9 cells and promoted differentiation. In order to investigate how TAp63 regulated K14 expression and against to TPA response, a TAp63α expression vector was cloned and then transfected to C9 cells. Our results indicated that K14 was up-regulated by TAp63α in mRNA and protein levels as well as K14 promoter reporter activity. In addition, ΔNp63α didn’t influence the enhancement of K14 reporter activity by TAp63α in C9 cell. TAp63α enhancement activity also found in Hela cell which had no ΔNp63α. TAp63α regulated K14 expression through (-1~-280) K14 reporter region which contained three half-site of p53 binding sites and an AP2 binding site. According to co-immunoprecipitation data, it had ruled out that TAp63α enhanced K14 expression had to medicate through AP2α associated response. TAp63α enhanced K14 reporter activity also found in HepG2 cell which had no AP2α. An electrophoretic mobility shift assay (EMSA) was used to analyze which half-site of p53 binding site is responsible for the regulation of K14 expression by TAp63α. The bands of -209~-176 and -143~-124 in K14 reporter region could be retarded from the nuclear protein of different cell lines, and p53 half-site consensus sequence oligomer could compete these retarded bands. We also found enrichment the retarded bands in Hela cell and H1299 cell overexpression with TAp63α. Thus, in these research we find two p53 half-site -204~-195 and -138~-129 in K14 promoter for binding of TAp63α.
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