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Cytoskeletal active drugs modulate signal transduction in the protein kinase C pathway
G K Pavlath1, Y Shimizu, N Shimizu
1Department of Molecular and Cellular Biology, University of Arizona, Tucson 85721.
Abstract:
The cytoskeletal network of cells is postulated to play a role in the signal transduction pathways of growth promoting stimuli. We show here that cytoskeletal active drugs modulate the mitogenic signal transduction pathway of the tumor promoter TPA in 3T3-L1 cells. Compounds which act on microtubules (vinblastine sulfate) and microfilaments (cytochalasin B) have opposite effects on DNA synthesis. Vinblastine sulfate leads to stimulation, whereas cytochalasin B causes potent inhibition of DNA synthesis in response to TPA. These drugs are cell cycle specific and apparently exert their regulatory action distal to activation of protein kinase C by TPA. The expression of four genes necessary for DNA synthesis in response to tumor promoters was examined: two nuclear proto-oncogenes (c-myc and c-fos), a transcription factor (c-jun/AP-1) and a key enzyme involved in polyamine synthesis (ornithine decarboxylase). c-jun mRNA levels are not modulated during the action of cytoskeletal disrupting drugs on TPA-mediated mitogenesis, whereas c-myc and c-fos mRNA levels are similarly enhanced. Expression of ornithine decarboxylase mRNA and protein is increased by vinblastine sulfate but decreased by cytochalasin B in TPA treated cells. These data indicate that changes in cytoskeletal organization may play a role in regulating the levels of an enzyme critical for DNA synthesis.
Insights
Cellular cytoskeleton organization impacts cell growth signals. Disrupting microtubules stimulates DNA synthesis, while disrupting microfilaments inhibits it, affecting key gene expression.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The cellular cytoskeleton is implicated in signal transduction pathways for growth-promoting stimuli.
- Tumor promoters like TPA (12-O-tetradecanoylphorbol-13-acetate) activate mitogenic signaling pathways.
Purpose of the Study:
- To investigate how cytoskeletal drugs modulate the mitogenic signal transduction pathway activated by the tumor promoter TPA in 3T3-L1 cells.
- To determine the effects of microtubule and microfilament disruption on DNA synthesis and gene expression in response to TPA.
Main Methods:
- Treatment of 3T3-L1 cells with TPA and cytoskeletal drugs (vinblastine sulfate for microtubules, cytochalasin B for microfilaments).
- Measurement of DNA synthesis rates.
- Analysis of mRNA and protein expression levels for c-myc, c-fos, c-jun/AP-1, and ornithine decarboxylase.
Main Results:
- Vinblastine sulfate stimulated TPA-induced DNA synthesis, while cytochalasin B potently inhibited it.
- c-myc and c-fos mRNA levels were enhanced by both drugs, while c-jun mRNA levels remained unchanged.
- Vinblastine sulfate increased ornithine decarboxylase mRNA and protein, whereas cytochalasin B decreased them in TPA-treated cells.
Conclusions:
- Cytoskeletal organization plays a regulatory role in TPA-mediated mitogenesis.
- Differential effects of microtubule and microfilament disruption suggest distinct roles in regulating DNA synthesis and gene expression, potentially through enzymes like ornithine decarboxylase.