| Summary: | 碩士 === 國立成功大學 === 藥理學研究所 === 81 === (12S)-Hydroxyeicosatetraenoic acid is biosynthesized from
arachidonic acid by the microsomal fraction of human epidermal
carcinoma A431 cells, and the microsomal 12-lipoxygenase
activity is enhanced by about 2 folds by epidermal growth
factor(EGF)with a 10-h lag period. In this thesis, the
regulation and signal transduction of 12-lipoxygenase
expression induced by EGF was studied. The enzyme was immuno-
precipitated by a monoclonal antibody against human platelet
12-lipoxygenase but not by that against porcine leukocyte
enzyme. A 3.1-kb mRNA was detected by Northern blot analyses
using cDNA probe of human platelet 12- lipoxygenase. In term of
immunogenicity and cDNA hybridization, the microsomal
12-lipoxygenase was a platelet-type enzyme. EGF could increase
the 12-lipoxygenase mRNA level by about 2 folds with a lag
period of 4-8 h and 2-6 h ahead of the increase in the enzyme
activity.The induction completely blocked by cycloheximide
,indicating that a de novo protein biosynthesis was essential
for EGF-induced 12-lipoxygenase mRNA experssion. Whether PKC is
involved in this induction was then studied. Staurosporine and
calphostin C, two PKC inhibitors, inhibited the EGF-induced
enzyme activity and mRNA expression of 12-lipoxygenase. Effect
of two PKC activators on 12-lipoxygenase expression was then
investigated. PMA and DiC8 significantly induced the enzyme
activity and the mRNA expression. The formation of IP3 and DAG
could be detected when cells were treated with EGF, indicating
that EGF induced the hydrolysis of PIP2. In order to know what
subspecies of PKC exists in A431 cells, six cDNA probes of PKC
were used for Northern blot analyses. Expression of mRNA of PKC
.alpha.,.delta.and.zeta. was detected in A431 cells.In this
study , the results provide the evidences for the inducibility
of human 12-lipoxygenase gene expression by a growth factor and
the involvement of PKC in this signal transduction.
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