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Phorbol esters regulate preprogastrin-releasing peptide messenger RNA in small cell lung cancer cells
1Department of Biochemistry and Molecular Biology, George Washington University Medical Center, Washington, D.C. 20037.
Abstract:
The expression of preprogastrin-releasing peptide (GRP) mRNA was studied using human small cell lung cancer (SCLC) cells. By Northern analysis, preproGRP mRNA was stimulated by 4 beta-phorbol 12-myristate 13 alpha-acetate (PMA) in a concentration- and time-dependent manner in these cells. In cell line NCI-H209, the addition of 10(-6) M PMA increased a 0.9-kb mRNA after 8 h. An inactive phorbol ester, 4 alpha-PMA, had little effect on preproGRP mRNA. A nuclear run-on assay indicated that 10(-6) M PMA increased preproGRP transcription 3-fold, whereas beta-actin and glyceraldehyde 3-phosphate dehydrogenase transcription was unaltered. In contrast, PMA had little effect on beta-actin mRNA expression. PMA (1 microM) in the presence of 100 microM 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), a protein kinase C inhibitor, had little effect on preproGRP mRNA. Addition of PMA after protein kinase C down-regulation did not alter preproGRP mRNA. PMA (1 microM) caused translocation of protein kinase C from the cytosol to the membrane of SCLC cells. Also, PMA (10(-6) M) stimulated and H7 (10(-4) M) reduced SCLC growth in vitro. When new synthesis of preproGRP mRNA was blocked by the addition of actinomycin D, preproGRP mRNA remained stable for 15 h. These data suggest that PMA induces transcription of GRP mRNA in SCLC cells.
Insights
Phorbol ester (PMA) stimulates gastrin-releasing peptide (GRP) mRNA transcription in small cell lung cancer (SCLC) cells, mediated by protein kinase C. This finding offers insights into SCLC gene regulation.
Area of Science:
- Molecular Biology
- Oncology
- Cell Biology
Background:
- Gastrin-releasing peptide (GRP) is a key peptide in small cell lung cancer (SCLC).
- Understanding GRP gene regulation is crucial for SCLC therapeutic strategies.
Purpose of the Study:
- To investigate the effect of 4 beta-phorbol 12-myristate 13 alpha-acetate (PMA) on preprogastrin-releasing peptide (GRP) mRNA expression in SCLC cells.
- To elucidate the underlying molecular mechanisms, including the role of protein kinase C (PKC).
Main Methods:
- Northern analysis to quantify preproGRP mRNA levels.
- Nuclear run-on assays to assess transcriptional activity.
- Protein kinase C inhibition and down-regulation studies.
- Cell growth assays in vitro.
Main Results:
- PMA significantly increased preproGRP mRNA expression in a dose- and time-dependent manner.
- PMA enhanced preproGRP gene transcription by approximately 3-fold, without affecting beta-actin or glyceraldehyde 3-phosphate dehydrogenase transcription.
- The effect of PMA on preproGRP mRNA was dependent on active protein kinase C.
- PMA stimulated SCLC cell growth, while the PKC inhibitor H7 reduced it.
Conclusions:
- Phorbol ester (PMA) induces the transcription of GRP mRNA in SCLC cells.
- Protein kinase C activation is essential for PMA-induced GRP gene expression in SCLC.
- These findings highlight a potential therapeutic target for modulating GRP in SCLC.