Genomic-scale measurement of mRNA turnover and the mechanisms of action of the anti-cancer drug flavopiridol
- Lloyd T Lam1,
- Oxana K Pickeral1,
- Amy C Peng2,
- Andreas Rosenwald1,
- Elaine M Hurt1,
- Jena M Giltnane1,
- Lauren M Averett1,
- Hong Zhao1,
- R Eric Davis1,
- Mohan Sathyamoorthy1,
- Larry M Wahl3,
- Eric D Harris4,
- Judy A Mikovits4,
- Anne P Monks4,
- Melinda G Hollingshead4,
- Edward A Sausville4 and
- Louis M Staudt1Email author
© Lam et al., licensee BioMed Central Ltd 2001
Received: 27 April 2001
Accepted: 25 July 2001
Published: 13 September 2001
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.
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-β-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.
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.
DNA microarrays have proven very useful in establishing molecular definitions of human cancer subtypes [1,2,3]. In some cases, cancers that were assigned to a single diagnostic category by conventional morphological diagnostic methods have been found to have different gene expression profiles and belong to distinct molecular subtypes. In particular, diffuse large B-cell lymphoma (DLBCL) was shown to consist of at least two molecular subtypes that differed in the expression of over several hundred genes . Furthermore, patients with these two DLBCL subtypes had strikingly different long-term outcomes following conventional multi-agent chemotherapy. Patients with germinal center B-like DLBCL had a favorable prognosis, with an apparent cure rate of 75%. On the other hand, patients with activated B-like DLBCL had a poor prognosis, with less than a quarter of these patients alive five years following therapy. Therefore, for patients with activated B-like DLBCL, alternative therapeutic agents must be identified.
For this reason, we have begun a survey of novel cancer agents in order to identify drugs with significant activity against activated B-like DLBCL. One drug identified in this screen, flavopiridol, was found to be significantly cytotoxic for cell lines derived from activated B-like DLBCL. Flavopiridol is a member of the growing family of cyclin-dependent kinase (CDK) inhibitors that have varying activities against the multiple CDK family members (CDK1, CDK2, CDK4, CDK6 and CDK7) by competitively blocking their ATP-binding pocket [4,5,6,7,8]. In addition to arresting cells in vitro at the G2-to-M and the G1-to-S transitions, flavopiridol decreases the rate of progression through S phase. Since flavopiridol is being evaluated in multiple cancer clinical trials [7,9], and has been shown to be highly active in inducing apoptosis in hematopoietic neoplasms , we were interested in whether flavopiridol might be effective against activated B-like DLBCL.
Although it is clear that flavopiridol inhibits CDKs, whether flavopiridol inhibits other cellular targets is not known. Recently, flavopiridol was found to inhibit the activity of a transcription elongation factor P-TEFb, a complex of cyclins with CDK9 . P-TEFb phosphorylates the carboxy-terminal domain of the RNA polymerase II complex , facilitating transcription elongation. It was not known, however, whether P-TEFb regulates transcriptional elongation of all cellular genes or whether other factors promote transcriptional elongation on subsets of cellular genes.
Using DNA microarrays, we found that flavopiridol inhibited gene expression broadly in a manner highly related to other transcription inhibitors such as actinomycin D and 5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole (DRB). We were therefore able to use flavopiridol to study the turnover rate of mRNA on a genomic-scale. mRNA turnover is regulated by a variety of cellular factors acting on cis-elements in mRNA molecules. For many labile mRNAs, adenylate uridylate-rich elements (AREs) are required for their rapid degradation. Three different classes of ARE have been defined . A conserved motif present in two of these classes, AUUUA, plays an important role in transcript stability for many cytokines and early-response genes (ERGs) . However, there is now increasing evidence that other motifs may determine the stability of transcripts .
By measuring mRNA turnover rates comprehensively, we identified unexpected relationships between the function of a gene and its mRNA stability. In addition, we found that genes that were transcriptionally inducible were disproportionately represented in the class of genes with labile mRNAs, thus revealing a coordinated strategy to rapidly modulate the mRNA levels of these genes. This understanding of mRNA turnover on a genomic scale provides fresh insights into the effectiveness of flavopiridol as an anti-cancer agent.
Results and discussion
Flavopiridol is a member of the growing family of CDK inhibitors that have varying activities against the multiple CDK family members. As most of the CDK inhibitors bind to the ATP-binding pocket of the CDKs , and all CDKs have similar ATP-binding pockets, it was possible that other CDK inhibitors also inhibit transcription. We determined whether the inhibition of transcription is specific to flavopiridol by comparing gene expression profiles of flavopiridol-treated cells with those of cells treated with the CDK inhibitors roscovitine and 9-nitropaullone. Roscovitine is a purine derivative that inhibits CDK1, CDK2 and CDK5 and blocks cell-cycle progression both in late G1/early S and in M phase [18,19,20,21]. 9-nitropaullone, the most potent member of a novel class of the paullone CDK inhibitor family, inhibits CDK1/cyclin B at 10-fold lower concentrations than flavopiridol and roscovitine [22,23,24]. As shown in Figure 1c, the gene expression changes caused by roscovitine and 9-nitropaullone were different from those of flavopiridol, and only flavopiridol globally reduced mRNA levels.
To investigate whether the mRNA degradation rates were first-order, we fitted a linear regression model for the three drugs for each gene (see Materials and methods). The adjusted R-squared statistic, which is a measure of correlation between the observed data values and the predicted values from the linear model, shows how well a first-order kinetics model would describe the mRNA degradation rates. Figure 3 shows a histogram of the adjusted R-squared statistics obtained from fitting a model for each of the 2,794 genes. The median adjusted R-squared was 0.86, and 79.7% of the genes had an adjusted R-squared statistic over 0.7, showing that most of the genes had first-order mRNA degradation rates.
Another important class of genes that might contribute to the efficacy of flavopiridol would include anti-apoptotic genes with rapid mRNA turnover (Figure 4b). Two anti-apoptotic Bcl-2 family members that were expressed in OCI-Ly3 cells - A1 and MCL1 - had mRNA half-lives shorter than 3 hours. Three members of the IAP family (c-IAP1, c-IAP2, NAIP) had very short mRNA half-lives and encode anti-apoptotic proteins that act by directly binding to caspases 3, 7, and 9 . The NF-κ B target gene A20, an inhibitor of tumor necrosis factor (TNF)-induced apoptosis , is expressed in OCI-Ly3 cells and had a very short mRNA half-life. As TNF-α is expressed by OCI-Ly3 cells, inhibition of A20 expression by flavopiridol treatment may contribute to its cytotoxic effect on these cells. In accord with these results, flavopiridol treatment was recently shown to downregulate two of these anti-apoptotic proteins, MCL1 and Bag-1, and induce apoptosis in B-CLL cells treated with flavopiridol .
The majority of the genes with labile mRNAs, however, did not fall into the cell cycle or apoptosis categories but instead included many genes that are known to be inducible by cellular signaling. For example, transcription factors belonging to the NF-κB family are activated by a wide variety of cellular signaling events, and several known NF-κB target genes (A1, A20, c-IAP1, c-IAP2, IκB alpha, IκB epsilon, CD54, IL-6, IL-8, TNF-α, IRF-4) were found to have unstable mRNAs (Figure 4d). This observation led us to investigate the possibility that the class of genes that are transcriptionally induced by cellular signaling events is enriched for genes with labile mRNAs. We used Lymphochip microarrays to profile the changes in gene expression that occurred in response to a wide variety of signaling events: B-cell lymphoma cell lines stimulated by crosslinking the B-cell antigen receptor or by treatment with phorbol ester and ionomycin (PI), resting peripheral blood T cells stimulated with PI, and peripheral blood monocytes stimulated with lipopolysaccharide. Figure 4d shows genes with relatively short mRNA half-lives (<2 hours and 2-4 hours) that were also transcriptionally induced at least threefold in one of the cellular activation experiments described above. Roughly one third of the inducible genes in OCI-Ly3 cells were in this rapid mRNA turnover category (Figure 4c). By contrast, 16.8% of the genes that were not induced by these stimuli had mRNA half-lives of less than 4 hours (P < 0.001).
We next investigated the functional characteristics of the genes assigned to each mRNA turnover class (Figure 5b). More than one third of genes regulating apoptosis were both inducible and had mRNA half-lives of less than 2 hours. By contrast, only 10% of the genes encoding cell-surface proteins were in this class. The apoptosis regulators included several anti-apoptotic genes discussed previously (A20, MCL1, c-IAP2), as well as potentially pro-apoptotic genes such as those for Fas ligand and TNF receptor type II. PHLDA1 is the human homolog of TDAG51, a pleckstrin-homology domain protein that may modulate apoptosis by regulating Fas ligand expression following stimulation through the T-cell receptor. It is well known that apoptosis in T lymphocytes is an elaborately orchestrated process following lymphocyte activation that allows clonal expansion of the activated T cells while eventually eliminating the clone through the process of activation-induced cell death . Thus, anti-apoptotic gene products are required early during lymphocyte activation, whereas attenuation of the clonal burst eventually requires pro-apoptotic signaling through Fas and the TNF receptors. This change in apoptotic responsiveness may be accomplished rapidly by combining transcriptional regulation with rapid mRNA turnover of key apoptosis-regulatory genes.
Transcriptional reprogramming following lymphocyte activation involves the coordinate induction of transcription factors  and many of these were found to have labile mRNAs (Figure 5b). Several of these genes are known to encode immediate early transcription factors (for example, FosB, NAB2, ATF-3, NOT1) that are induced by a wide range of cellular stimuli, and this class of transcription factors is well known to have unstable mRNAs . Other inducible transcription factors with labile mRNAs have more lymphocyte-specific functions, such as IRF-4, a factor that is required for lymphocyte activation but is not required in other lineages .
Not surprisingly, the most inducible class of genes in PBMCs encoded cytokines, chemokines and their receptors (Figure 5c). Within this class, more than 25% of genes had mRNA half-lives of less than 2 hours. As expected, many cytokine mRNAs were both rapidly inducible and rapidly degraded (Figure 5d). Unexpectedly, the chemokine genes had much more stable mRNAs than the cytokine genes, even though they were strongly induced by PI treatment (Figure 5d). The mRNAs for the cytokine and chemokine receptors also differed in their stability. The inducible chemokine receptors CCR1 and CXCR5 had labile mRNAs, whereas most of the inducible cytokine receptors (IL-2, IL-3, IL-6 receptor α chains) had relatively stable mRNAs. The differential mRNA stabilities of cytokines, chemokines and their receptors reflect their divergent biological functions. Cytokines potently induce activation and differentiation of target cells in a tightly regulated temporal fashion during immune responses . To limit inappropriate effects of cytokines both temporally and spatially on bystander cells, the levels of these factors must be modulated rapidly, which can be achieved given their short mRNA half-lives. On the other hand, chemokines are chemoattractants that signal leukocyte migration by setting up concentration gradients that are recognized by chemokine receptors . The inducible chemokines are used in inflammatory responses and wound healing to attract the appropriate complement of immune cells to the affected tissues. These cellular reactions need to be rapidly initiated but are slowly resolved, a kinetic profile that can be understood in light of the relative stability of chemokine mRNAs.
The systematic determination of mRNA degradation rates described in the present report revealed an underlying logic that relates gene function and mRNA stability. The largest category of genes found to have unstable mRNAs included genes that were inducible by various cellular signaling events. The inducibility of many of these genes is known to be due to transcriptional activation of their promoters. Thus, the rapid degradation of these mRNAs allows their levels to rapidly drop when transcriptional activation is terminated. By categorizing genes using the functional properties of their encoded proteins, we found that certain functional classes were relatively enriched for unstable mRNAs. Two such classes, the cytokine genes and immediate early transcription factor genes, were previously known to have unstable mRNAs. The class of apoptosis regulators, however, was not previously known to be enriched for labile mRNAs. However, given the need to rapidly modulate apoptosis during development, cellular differentiation, and immune responses, this property of the apoptotic genes can be readily understood. Another unanticipated finding was that many critical regulators of M phase of the cell cycle had rapidly degraded mRNAs. In light of the short duration of M phase, these labile M-phase regulators may need to be eliminated rapidly to allow correct entry into G1 phase of the cell cycle.
The initial impetus of this research was to understand the mechanism of action of the anti-cancer drug flavopiridol. The regulatory logic of mRNA degradation revealed in this study gives us insight into how this general transcriptional inhibitor can have selective toxicity for tumor cells. In particular, the short half-lives of mRNAs encoding M-phase proteins and other key cell-cycle regulators (for example, c-Myc) may contribute to the cytotoxicity of flavopiridol for cancer cells but may also contribute to the dose-limiting toxicity of flavopiridol for gut epithelium, leading to secretory diarrhea . However, the present results also suggest that flavopiridol may be more effective in treating certain types of cancer cells that are highly dependent on genes with unstable mRNAs. For example, OCI-Ly3 lymphoma cells constitutively express two anti-apoptotic BCL-2 family members, A1 and MCL1, which are not expressed at high levels in normal lymphocytes. The instability of the A1 and MCL1 mRNAs therefore may contribute to the toxicity of flavopiridol for these lymphoma cells. Gene expression profiling demonstrated that the OCI-Ly3 cell line is an excellent model for the activated B-like DLBCL subtype . Indeed, fresh lymphoma biopsies from such patients revealed high expression of A1 and MCL1 in most cases . These results suggest that activated B-like DLBCL might be a particularly attractive candidate disease for new clinical trials of flavopiridol.
Materials and methods
Chemicals and cells
Flavopiridol and 9-nitropaullone were obtained from the NCI Developmental Therapeutics Program. DRB, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and ionomycin were purchased from Sigma. Actinomycin D, roscovitine, phorbol ester and lipopolysaccharide (LPS) were purchased from Calbiochem. Anti-human IgM was purchased from Jackson ImmunoResearch Laboratories, Inc.
Primary T cells and monocytes were purified from human blood by magnetic cell sorting. PBMCs were purified by Ficoll density gradient centrifugation. OCI-Ly1 and OCI-Ly3 were maintained in Iscove's modified essential medium with β-mercaptoethanol (55 μM), penicillin (50 units/ml), streptomycin (50 μg/ml), and 20% heparinized normal human plasma. SUDHL-5, SUDHL-6, WSU-1, Jurkat, MCF-7, PC3, BJAB, T cells, monocytes and PBMCs were maintained in RPMI 1640 medium with L-glutamine, HEPES, penicillin, streptomycin and 10% fetal bovine serum. Cells were grown in a 37°C incubator in the presence of 5% carbon dioxide. PBMCs (except the activation time course) and T cells were stimulated for 3 h with phorbol ester (50 ng/ml) and ionomycin (1.5 μM). B cell lines were stimulated with anti-IgM antibody (50 μg/ml). Monocytes were stimulated for 3 h with LPS (0.2 μg/ml). CLL samples and cell lines used for clustering in Figure 4a were prepared according to Alizadeh et al. . For all experiments, 1 μM flavopiridol was used (except the titration experiment in Figure 1a). PBMCs were first activated at 5 × 106 cells/ml with PI for 3 h before flavopiridol treatment. The activation of the PBMCs by PI treatment was confirmed by FACS analysis of CD69 and CD44 expression.
MTT assays were done as described . In brief, OCI-Ly3 cells grown in 96-well plates were treated with increasing concentration of flavopiridol (0 to 2 μM) for 8, 24 or 48 h. MTT was added to the cells 2 h before harvesting. Cells were lysed completely in isopropanol with 1% hydrochloric acid. The plate was read with a 96-well spectrometer using a 570-nm filter. The background was subtracted using a dual-wavelength setting of 570 and 630 nm.
Microarray analysis and northern blotting
Microarray analysis was performed as described . Total RNA was prepared using Trizol reagent (Gibco BRL) according to the manufacturer's instruction. For each sample, 40 μg of total RNA was used for the labeling reaction. All primary microarray data are available at our website .
Ten micrograms of total RNA were used for northern blot analysis. The probes were prepared from PCR amplification of the cDNA clones used for printing the Lymphochip.
The raw data from each cDNA array were normalized as described . Data extracted must fulfill the following criteria. Spot size must be >25 μm and expression levels for all flavopiridol studies must be >500 relative fluorescent units (RFU) for the Cy3 channel, and >0 RFU for the Cy5 channel; PBMC induction studies: >50 RFU for both channels.
The expression data for array elements representing the same gene were averaged. Hierarchical clustering was performed using Cluster software and visualized using the Tree View software .
The accession numbers of transcripts with one to five copies of AUUUA were downloaded from the ARE-mRNA database . These transcripts were categorized by their half-lives according to the PBMC database.
Mathematical analysis of mRNA half-lives
A first-order kinetic model would imply that the log-ratio of Cy5 to Cy3 would decrease linearly with time. For each gene, we fitted a linear regression model of the log-ratios to the time points, where the model included a separate intercept and coefficient term for each of the three drugs:
log2(Cy5/Cy3) = (αFlav + βFlavt) * I[Flav] + (αActD + βActDt) * I[ActD] + (αDRB + βDRBt) * I[DRB],
where t denotes the time in hours; I[.] denotes an indicator function for the treatment drug, taking the values 0 or 1, depending on whether or not that treatment drug was used for the Cy5 measurement of that log-ratio; and αFlav, βFlav, αActD, βActD, αDRB, and βDRB are model parameters to be estimated.
Although each drug was allowed to have a distinct first-order degradation rate in the model, the three drugs were assumed to have identical error distributions in their assay variability. The mRNA half-life for each drug treatment was computed as the negative of the inverse of the coefficient for each drug:
tFlav = -1/βFlav, tActD = -1/βActD, tDRB = -1/βDRB.
The following test , was used to evaluate whether the proportion of induced genes with half-life less than 2 h is statistically different from the proportion of not induced genes with half-life less than 2 h, for both the OCI-Ly3 (Figure 4c) and PBMC (Figure 5a):
z = (| p2 - p1 | - 1/2 * (1/n1 + 1/n2)) / (p * q * (1/n1 + 1/n2)) 1/2,
where n1 = number of the induced genes, n2 = number of the not induced genes, p1 = proportion of induced genes with half-life less than 2 h, p2 = proportion of not-induced genes with half-life less than 2 h, p = overall proportion of genes with half-life less than 2 hours, and q = 1 - p.
Additional data files
The following additional data files containing the primary data for the figures are included with this article: Figure 1a, flavopiridol titration (Excel csv file); Figure 1c, Cdk inhibitors (Excel csv file); Figure 2a, flavopiridol, actinomycin D and DRB (Excel csv file); Figure 3, half-lives of well-measured genes (Excel csv file); Figure 4a, proliferation genes (Excel csv file); Figure 4b, anti-apoptotic genes (Excel csv file); Figure 4d, induced genes in OCI -Ly3 cells (Excel csv file); Figure 5c, highly induced genes in PBMC (Excel csv file); Figure 5d, highly induced chemokines, cytokines and their receptors (Excel csv file).
We would like to thank members of the Staudt laboratory for helpful discussions.
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