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Molecular cloning of gene sequences regulated by tumor promoters and mitogens through protein kinase C
M D Johnson1, G M Housey, P T Kirschmeier
1Cancer Center, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Abstract:
cDNA clones representing genes whose expression is modulated by treatment with mitogens and tumor promoters were isolated and characterized. TPA-S1 corresponds to an mRNA species whose abundance was increased markedly within 1 h of exposure to the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), and TPA-R1 represents an mRNA that was decreased in TPA-treated cells. The induction of TPA-S1 was blocked by actinomycin D but was not affected by cycloheximide, and it was specific for phorbol esters with tumor-promoting activity. The role of protein kinase C in the induction of TPA-S1 is supported by the following lines of evidence. (i) Agents that activated protein kinase C (TPA, platelet-derived growth factor, and diacylglycerol) also increased TPA-S1 mRNA levels. (ii) A potent PKC inhibitor blocked the induction of TPA-S1. (iii) Down-regulation of PKC activity, by treatment of cells with TPA for 24 h, resulted in a loss of responsiveness to TPA-S1 induction by subsequent TPA treatment. DNA sequence analysis of TPA-S1 predicts a cysteine-rich, secreted protein with a molecular weight of 22.6 X 10(3) that exhibits homology with sequences representing a protein with human erythroid-potentiating activity and protease inhibitory activity.
Insights
Tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) rapidly increases TPA-S1 mRNA levels, a process dependent on protein kinase C (PKC) activity. This suggests PKC plays a key role in regulating gene expression in response to tumor promoters.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Gene expression modulation is critical in cellular responses to external stimuli.
- Tumor promoters, like 12-O-tetradecanoyl phorbol-13-acetate (TPA), significantly alter cellular pathways.
Purpose of the Study:
- To isolate and characterize cDNA clones responsive to mitogens and tumor promoters.
- To investigate the mechanism of TPA-induced gene expression, focusing on TPA-S1 mRNA.
Main Methods:
- cDNA library screening
- Northern blot analysis
- Actinomycin D and cycloheximide treatments
- Protein kinase C (PKC) activation and inhibition studies
- DNA sequencing
Main Results:
- TPA treatment rapidly increased TPA-S1 mRNA abundance.
- TPA-S1 induction was blocked by actinomycin D but not cycloheximide, indicating a transcriptional regulation mechanism.
- TPA-S1 induction was dependent on protein kinase C (PKC) activity, as shown by activation, inhibition, and down-regulation studies.
- DNA sequence analysis predicted TPA-S1 encodes a secreted protein with homology to erythroid-potentiating and protease inhibitory activities.
Conclusions:
- Protein kinase C (PKC) is a key mediator in the induction of TPA-S1 mRNA by TPA.
- TPA-S1 represents a novel gene regulated by tumor-promoting phorbol esters via a PKC-dependent pathway.
- The predicted protein product of TPA-S1 may have roles in cell growth, differentiation, or protease regulation.