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Genomic-scale measurement of mRNA turnover and the mechanisms of action of the anti-cancer drug flavopiridol.

Lam LT, Pickeral OK, Peng AC, Rosenwald A, Hurt EM, Giltnane JM, Averett LM, Zhao H, Davis RE, Sathyamoorthy M, Wahl LM, Harris ED, Mikovits JA, Monks AP, Hollingshead MG, Sausville EA, Staudt LM - Genome Biol. (2001)

Bottom Line: We were therefore able to use flavopiridol to measure mRNA turnover rates comprehensively and we found that different functional classes of genes had distinct distributions of mRNA turnover rates.The observation that transcriptionally inducible genes often have short mRNA half-lives demonstrates that cells have a coordinated strategy to rapidly modulate the mRNA levels of these genes.In addition, the present results suggest that flavopiridol may be more effective against types of cancer that are highly dependent on genes with unstable mRNAs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Metabolism Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. lstaudt@box-l.nih.gov

ABSTRACT

Background: Flavopiridol, a flavonoid currently in cancer clinical trials, inhibits cyclin-dependent kinases (CDKs) by competitively blocking their ATP-binding pocket. However, the mechanism of action of flavopiridol as an anti-cancer agent has not been fully elucidated.

Results: Using DNA microarrays, we found that flavopiridol inhibited gene expression broadly, in contrast to two other CDK inhibitors, roscovitine and 9-nitropaullone. The gene expression profile of flavopiridol closely resembled the profiles of two transcription inhibitors, actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), suggesting that flavopiridol inhibits transcription globally. We were therefore able to use flavopiridol to measure mRNA turnover rates comprehensively and we found that different functional classes of genes had distinct distributions of mRNA turnover rates. In particular, genes encoding apoptosis regulators frequently had very short half-lives, as did several genes encoding key cell-cycle regulators. Strikingly, genes that were transcriptionally inducible were disproportionately represented in the class of genes with rapid mRNA turnover.

Conclusions: The present genomic-scale measurement of mRNA turnover uncovered a regulatory logic that links gene function with mRNA half-life. The observation that transcriptionally inducible genes often have short mRNA half-lives demonstrates that cells have a coordinated strategy to rapidly modulate the mRNA levels of these genes. In addition, the present results suggest that flavopiridol may be more effective against types of cancer that are highly dependent on genes with unstable mRNAs.

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Similar gene expression patterns of OCI-Ly3 cells treated with flavopiridol, actinomycin D and DRB. (a) OCI-Ly3 cells were treated with flavopiridol (1 μM), actinomycin D (10 μg/ml), or DRB (100 μM) for 0.5, 1, 2, 4 and 8 h. These concentrations were chosen to give roughly equivalent (approximately 50%) cytotoxicity at 24 h without significant loss of cell viability at 8 h. The treated samples were labeled red (Cy5) and untreated samples were labeled green (Cy3). Genes were categorized according to their half-lives by the scheme as described in Materials and methods. (b) Northern blot analysis of selected genes. (c) Comparison of turnover rate of selected genes from microarray data and Northern blot analysis (percentage of control versus time).
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Figure 2: Similar gene expression patterns of OCI-Ly3 cells treated with flavopiridol, actinomycin D and DRB. (a) OCI-Ly3 cells were treated with flavopiridol (1 μM), actinomycin D (10 μg/ml), or DRB (100 μM) for 0.5, 1, 2, 4 and 8 h. These concentrations were chosen to give roughly equivalent (approximately 50%) cytotoxicity at 24 h without significant loss of cell viability at 8 h. The treated samples were labeled red (Cy5) and untreated samples were labeled green (Cy3). Genes were categorized according to their half-lives by the scheme as described in Materials and methods. (b) Northern blot analysis of selected genes. (c) Comparison of turnover rate of selected genes from microarray data and Northern blot analysis (percentage of control versus time).

Mentions: To determine whether flavopiridol affects transcription globally, we compared the gene expression profile of flavopiridol treatment with the gene expression profiles induced in OCI-Ly3 cells by treatment with two well-studied transcriptional inhibitors, actinomycin D and DRB. Actinomycin D inhibits transcription initiation broadly by intercalating into DNA whereas DRB inhibits the transcription elongation factor P-TEFb [12]. P-TEFb is a complex of cyclins with CDK9, and flavopiridol can also inhibit this kinase [11]. However, it was not known whether P-TEFb regulates transcriptional elongation of all cellular genes, nor was it known whether flavopiridol would inhibit the same set of genes as DRB. OCI-Ly3 cells were treated with each inhibitor, and the resultant changes in gene expression were found to be virtually indistinguishable (Figure 2a). For the majority of well-measured genes on the Lymphochip, the mRNA abundance decreased with first-order kinetics (Figure 3) and could therefore be used to measure mRNA turnover rates (see Materials and methods). Genes are grouped according to mRNA half-life in Figure 2a. The turnover rates of representative mRNAs were verified by northern blot analysis in Figure 2b and quantitated in Figure 2c. These findings demonstrate that all three compounds behave as global inhibitors of transcription, and demonstrate that P-TEFb regulates the transcriptional elongation of most cellular genes. Further, the similar effects of flavopiridol and DRB argue that the predominant effect of flavopiridol on gene expression results from its ability to inhibit P-TEFb.


Genomic-scale measurement of mRNA turnover and the mechanisms of action of the anti-cancer drug flavopiridol.

Lam LT, Pickeral OK, Peng AC, Rosenwald A, Hurt EM, Giltnane JM, Averett LM, Zhao H, Davis RE, Sathyamoorthy M, Wahl LM, Harris ED, Mikovits JA, Monks AP, Hollingshead MG, Sausville EA, Staudt LM - Genome Biol. (2001)

Similar gene expression patterns of OCI-Ly3 cells treated with flavopiridol, actinomycin D and DRB. (a) OCI-Ly3 cells were treated with flavopiridol (1 μM), actinomycin D (10 μg/ml), or DRB (100 μM) for 0.5, 1, 2, 4 and 8 h. These concentrations were chosen to give roughly equivalent (approximately 50%) cytotoxicity at 24 h without significant loss of cell viability at 8 h. The treated samples were labeled red (Cy5) and untreated samples were labeled green (Cy3). Genes were categorized according to their half-lives by the scheme as described in Materials and methods. (b) Northern blot analysis of selected genes. (c) Comparison of turnover rate of selected genes from microarray data and Northern blot analysis (percentage of control versus time).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC57796&req=5

Figure 2: Similar gene expression patterns of OCI-Ly3 cells treated with flavopiridol, actinomycin D and DRB. (a) OCI-Ly3 cells were treated with flavopiridol (1 μM), actinomycin D (10 μg/ml), or DRB (100 μM) for 0.5, 1, 2, 4 and 8 h. These concentrations were chosen to give roughly equivalent (approximately 50%) cytotoxicity at 24 h without significant loss of cell viability at 8 h. The treated samples were labeled red (Cy5) and untreated samples were labeled green (Cy3). Genes were categorized according to their half-lives by the scheme as described in Materials and methods. (b) Northern blot analysis of selected genes. (c) Comparison of turnover rate of selected genes from microarray data and Northern blot analysis (percentage of control versus time).
Mentions: To determine whether flavopiridol affects transcription globally, we compared the gene expression profile of flavopiridol treatment with the gene expression profiles induced in OCI-Ly3 cells by treatment with two well-studied transcriptional inhibitors, actinomycin D and DRB. Actinomycin D inhibits transcription initiation broadly by intercalating into DNA whereas DRB inhibits the transcription elongation factor P-TEFb [12]. P-TEFb is a complex of cyclins with CDK9, and flavopiridol can also inhibit this kinase [11]. However, it was not known whether P-TEFb regulates transcriptional elongation of all cellular genes, nor was it known whether flavopiridol would inhibit the same set of genes as DRB. OCI-Ly3 cells were treated with each inhibitor, and the resultant changes in gene expression were found to be virtually indistinguishable (Figure 2a). For the majority of well-measured genes on the Lymphochip, the mRNA abundance decreased with first-order kinetics (Figure 3) and could therefore be used to measure mRNA turnover rates (see Materials and methods). Genes are grouped according to mRNA half-life in Figure 2a. The turnover rates of representative mRNAs were verified by northern blot analysis in Figure 2b and quantitated in Figure 2c. These findings demonstrate that all three compounds behave as global inhibitors of transcription, and demonstrate that P-TEFb regulates the transcriptional elongation of most cellular genes. Further, the similar effects of flavopiridol and DRB argue that the predominant effect of flavopiridol on gene expression results from its ability to inhibit P-TEFb.

Bottom Line: We were therefore able to use flavopiridol to measure mRNA turnover rates comprehensively and we found that different functional classes of genes had distinct distributions of mRNA turnover rates.The observation that transcriptionally inducible genes often have short mRNA half-lives demonstrates that cells have a coordinated strategy to rapidly modulate the mRNA levels of these genes.In addition, the present results suggest that flavopiridol may be more effective against types of cancer that are highly dependent on genes with unstable mRNAs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Metabolism Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. lstaudt@box-l.nih.gov

ABSTRACT

Background: Flavopiridol, a flavonoid currently in cancer clinical trials, inhibits cyclin-dependent kinases (CDKs) by competitively blocking their ATP-binding pocket. However, the mechanism of action of flavopiridol as an anti-cancer agent has not been fully elucidated.

Results: Using DNA microarrays, we found that flavopiridol inhibited gene expression broadly, in contrast to two other CDK inhibitors, roscovitine and 9-nitropaullone. The gene expression profile of flavopiridol closely resembled the profiles of two transcription inhibitors, actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), suggesting that flavopiridol inhibits transcription globally. We were therefore able to use flavopiridol to measure mRNA turnover rates comprehensively and we found that different functional classes of genes had distinct distributions of mRNA turnover rates. In particular, genes encoding apoptosis regulators frequently had very short half-lives, as did several genes encoding key cell-cycle regulators. Strikingly, genes that were transcriptionally inducible were disproportionately represented in the class of genes with rapid mRNA turnover.

Conclusions: The present genomic-scale measurement of mRNA turnover uncovered a regulatory logic that links gene function with mRNA half-life. The observation that transcriptionally inducible genes often have short mRNA half-lives demonstrates that cells have a coordinated strategy to rapidly modulate the mRNA levels of these genes. In addition, the present results suggest that flavopiridol may be more effective against types of cancer that are highly dependent on genes with unstable mRNAs.

Show MeSH
Related in: MedlinePlus