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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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Analysis of genes inhibited by flavopiridol. OCI-Ly3 cells were treated with flavopiridol (1 μM) for 0.5, 2, 3, 4, 6 or 8 h. Total RNA was prepared for microarray analysis on Lymphochips with approximately 17,000 microarray elements. The treated samples were labeled red (Cy5) and untreated samples were labeled green (Cy3). Genes were first categorized according to their half-lives using the scheme as described in the Materials and methods section. These genes were then clustered according to their function. (a) Proliferation genes with half-lives of less than 4 h. The cell-cycle phase in which the particular gene is expressed is indicated. (b) Anti-apoptotic genes with short half-lives. (c) Percentages of induced or not-induced microarray elements as categorized by their half-lives. (d) Induced genes with half-lives of less than 4 h. See text for details.
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Figure 4: Analysis of genes inhibited by flavopiridol. OCI-Ly3 cells were treated with flavopiridol (1 μM) for 0.5, 2, 3, 4, 6 or 8 h. Total RNA was prepared for microarray analysis on Lymphochips with approximately 17,000 microarray elements. The treated samples were labeled red (Cy5) and untreated samples were labeled green (Cy3). Genes were first categorized according to their half-lives using the scheme as described in the Materials and methods section. These genes were then clustered according to their function. (a) Proliferation genes with half-lives of less than 4 h. The cell-cycle phase in which the particular gene is expressed is indicated. (b) Anti-apoptotic genes with short half-lives. (c) Percentages of induced or not-induced microarray elements as categorized by their half-lives. (d) Induced genes with half-lives of less than 4 h. See text for details.

Mentions: Given the demonstration that flavopiridol is a global transcriptional inhibitor, we wished to understand the selective activity of flavopiridol against certain cancer types in vivo. Clearly, genes with short mRNA half-lives would be most rapidly and quantitatively inhibited by flavopiridol, and thus we wished to assign the genes with short half-lives in OCI-Ly3 cells to functional classes. First, we searched for genes involved in cellular proliferation that have unstable mRNAs, as decreased expression of these genes might provide a mechanism by which flavopiridol preferentially targets proliferating cancer cells. Microarray gene expression analysis can readily identify a proliferation 'signature' consisting of hundreds of genes that vary in expression between proliferating and quiescent cells [1,25]. Genes can be assigned to the proliferation signature on the basis of high expression in diverse cell lines and low expression in chronic lymphocytic leukemia (CLL), a malignancy in which the leukemic cells are primarily in G0/G1 phase of the cell cycle [1]. Figure 4a presents 23 genes with half-lives of less than 4 hours for which the average expression in six cell lines was fourfold greater than the average expression in three CLL samples. This selection criteria identified c-myc, a gene previously known to have a short-lived mRNA, and an obviously important target of flavopiridol given its role in G1/S phase progression [26]. Interestingly, roughly half of these genes (11/23) are expressed preferentially in M phase and/or encode proteins involved in M-phase events. These genes represent important targets of flavopiridol action as they encode M-phase regulatory kinases (PLK, aurora/IPL1-related kinase, and ckshs2), M-phase checkpoint proteins (CHK1), and chromosome segregation proteins (CENP-E, CENP-F) (Figure 4a). Given the relatively short duration of M phase in most cells, the short half-lives of these mRNAs may be important to ensure that key M-phase regulators are not expressed as the cell progresses from M to G1 phase.


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)

Analysis of genes inhibited by flavopiridol. OCI-Ly3 cells were treated with flavopiridol (1 μM) for 0.5, 2, 3, 4, 6 or 8 h. Total RNA was prepared for microarray analysis on Lymphochips with approximately 17,000 microarray elements. The treated samples were labeled red (Cy5) and untreated samples were labeled green (Cy3). Genes were first categorized according to their half-lives using the scheme as described in the Materials and methods section. These genes were then clustered according to their function. (a) Proliferation genes with half-lives of less than 4 h. The cell-cycle phase in which the particular gene is expressed is indicated. (b) Anti-apoptotic genes with short half-lives. (c) Percentages of induced or not-induced microarray elements as categorized by their half-lives. (d) Induced genes with half-lives of less than 4 h. See text for details.
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Related In: Results  -  Collection

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Figure 4: Analysis of genes inhibited by flavopiridol. OCI-Ly3 cells were treated with flavopiridol (1 μM) for 0.5, 2, 3, 4, 6 or 8 h. Total RNA was prepared for microarray analysis on Lymphochips with approximately 17,000 microarray elements. The treated samples were labeled red (Cy5) and untreated samples were labeled green (Cy3). Genes were first categorized according to their half-lives using the scheme as described in the Materials and methods section. These genes were then clustered according to their function. (a) Proliferation genes with half-lives of less than 4 h. The cell-cycle phase in which the particular gene is expressed is indicated. (b) Anti-apoptotic genes with short half-lives. (c) Percentages of induced or not-induced microarray elements as categorized by their half-lives. (d) Induced genes with half-lives of less than 4 h. See text for details.
Mentions: Given the demonstration that flavopiridol is a global transcriptional inhibitor, we wished to understand the selective activity of flavopiridol against certain cancer types in vivo. Clearly, genes with short mRNA half-lives would be most rapidly and quantitatively inhibited by flavopiridol, and thus we wished to assign the genes with short half-lives in OCI-Ly3 cells to functional classes. First, we searched for genes involved in cellular proliferation that have unstable mRNAs, as decreased expression of these genes might provide a mechanism by which flavopiridol preferentially targets proliferating cancer cells. Microarray gene expression analysis can readily identify a proliferation 'signature' consisting of hundreds of genes that vary in expression between proliferating and quiescent cells [1,25]. Genes can be assigned to the proliferation signature on the basis of high expression in diverse cell lines and low expression in chronic lymphocytic leukemia (CLL), a malignancy in which the leukemic cells are primarily in G0/G1 phase of the cell cycle [1]. Figure 4a presents 23 genes with half-lives of less than 4 hours for which the average expression in six cell lines was fourfold greater than the average expression in three CLL samples. This selection criteria identified c-myc, a gene previously known to have a short-lived mRNA, and an obviously important target of flavopiridol given its role in G1/S phase progression [26]. Interestingly, roughly half of these genes (11/23) are expressed preferentially in M phase and/or encode proteins involved in M-phase events. These genes represent important targets of flavopiridol action as they encode M-phase regulatory kinases (PLK, aurora/IPL1-related kinase, and ckshs2), M-phase checkpoint proteins (CHK1), and chromosome segregation proteins (CENP-E, CENP-F) (Figure 4a). Given the relatively short duration of M phase in most cells, the short half-lives of these mRNAs may be important to ensure that key M-phase regulators are not expressed as the cell progresses from M to G1 phase.

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