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Clinical Trials Unit, Developmental Therapeutic Program, National Cancer Institute, NIH, Bethesda, Maryland 20892 [G. M., E. A. S., K. C., T. L., A. M. S.], and Laboratory of Molecular Biology, Institute G. Gaslini, Genoa 16157, Italy [L. V.]
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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VEGF is an angiogenic factor produced by normal and transformed cells. VEGF plays an important role in the acquisition of a metastatic phenotype by cancer cells (7) . The induction of VEGF mRNA by hypoxia occurs at the transcriptional and posttranscriptional level. Forsythe et al. (8) have demonstrated that a HIF-1 binding site of the VEGF promoter was required for responsiveness to hypoxia of a VEGF promoter-luciferase construct in the human hepatoblastoma cell line Hep3B, indicating that HIF-1 mediates transcriptional activation of the VEGF gene under hypoxic conditions. However, hypoxia-mediated increased stabilization of the VEGF mRNA has also been demonstrated, and putative RNA-binding proteins and RNA instability regions of the VEGF 3' flanking region have been identified (5 , 9, 10, 11) .
Flavopiridol, a novel protein kinase inhibitor with selectivity for CDKs, has been tested recently in a Phase I clinical trial at the National Cancer Institute (12) and is presently under investigation in Phase I/II clinical trials at other institutions in the United States and abroad. In vitro studies have demonstrated that flavopiridol acts by competitive inhibition of the ATP-binding site of CDKs (13 , 14) . Flavopiridol blocks cell proliferation and exerts antitumor activity in vitro and in vivo (15, 16, 17) ; flavopiridol also induces apoptosis in several human tumor cell lines and in normal and transformed lymphoid cells (18 , 19) .
An unexpected finding in the Phase I clinical trial (12) , in which flavopiridol was administered as continuous infusion to patients with refractory neoplasms, was the appearance of a proinflammatory syndrome including fever, chills, and tumor pain, along with alteration in acute phase reactants, suggesting that flavopiridol might affect gene expression. However, whether flavopiridol plays a role in the regulation of gene expression is presently unknown.
In this report, we show that flavopiridol caused down-regulation of VEGF mRNA and protein expression induced by hypoxia in human monocytes. Flavopiridol did not affect hypoxia-induced transcriptional activation of VEGF but significantly decreased VEGF mRNA half-life. These data provide the first evidence for a novel mechanism of action of flavopiridol in the down-regulation of gene expression. This finding also suggests that flavopiridol may have an antiangiogenic activity that might contribute to its therapeutic potential. Additional studies are warranted to investigate the antiangiogenic activity of flavopiridol in clinical trials.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Northern Blot Analysis.
Total cellular RNA, gel electrophoresis, and blotting were performed as described previously (6)
. The cDNA probe specific for human VEGF gene was kindly provided by Dr. Marsha Merril (NIH, Bethesda, MD) and was radiolabeled as described (6)
. The blot was hybridized for 1 h at 68°C in ExpressHyb (Clontech, Palo Alto, CA) with the radiolabeled probes (1 to 2 x 106 cpm/ml). The blot was washed four times at room temperature in wash solution 1 (2x SSC/0.05% SDS) and two times at 50°C in wash solution 2 (0.1x SSC/0.1% SDS), according to manufacturer’s instruction, and autoradiographed. Densitometry analysis was performed using the UN-SCAN-IT software (Silk Scientific, Orem, Utah). For each sample, results were normalized to the corresponding level of 28S.
ELISA.
The content of VEGF protein in culture supernatants of human monocytes was determined by using a commercially available kit following the manufacturer’s instruction (Quantikine Human VEGF Immunoassay; R&D Systems, Minneapolis, MN).
Transient Transfection of ANA-1 and Luciferase Assay.
ANA-1 macrophages were transfected by a modification of the DEAE-dextran method, as described (6)
. Cells were transfected with plasmid VEGF-P7 containing 1005 bp of the human VEGF 5'-flanking region linked to the luciferase reporter gene, or plasmid p11W, which contained only 47 bp of the VEGF 5'-flanking sequence between -985 and -939, encompassing the HIF-1 binding site at -975 (5'-TACGTGGG-3'), linked to the luciferase reporter gene (both plasmids were kindly provided by Gregg L. Semenza, Johns Hopkins University, Baltimore, MD). Cells were lysed with 1x Reporter Lysis buffer (Promega Corp., Madison, WI), and luciferase activity was assayed according to manufacturer’s recommendations using a Packard LumiCount luminometer. The protein content was determined as described by Bradford, using the Bio-Rad Protein Assay.
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Results and Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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, human monocytes cultured under normal oxygen levels expressed low but detectable levels of VEGF mRNA that were significantly augmented by incubation under hypoxic conditions. The levels of VEGF mRNA were already increased after 3 h of incubation in hypoxia, peaked at 6 h (4.5-fold relative to normoxic cells), and were still elevated at 16 h, demonstrating that human monocytes exposed to low oxygen tension do in fact express higher levels of VEGF mRNA. To investigate whether the increase in VEGF mRNA levels was paralleled by an increase in the amount of protein, ELISA experiments were performed on supernatants from monocytes cultured under normoxic or hypoxic conditions for 16 h. In the experiment shown in Fig. 1B
, human monocytes expressed low levels of VEGF protein (22 pg/ml) constitutively. Consistent with the Northern blot data, a 3.3-fold increase in the levels of VEGF (73 pg/ml) protein was observed in cells cultured for 16 h under hypoxic conditions. We have observed a donor-to-donor variability in the baseline levels of VEGF mRNA and protein expression in human monocytes cultured under normoxic conditions. Among five independent experiments performed with human monocytes harvested from different donors, we have observed up to 5-fold changes in the baseline levels of VEGF mRNA expression, ranging from minimal to low but detectable levels. In three separate ELISA experiments, we have observed a range of baseline expression of VEGF protein from 22 to 120 pg/ml (5.4-fold). These results indicate that fresh human monocytes exhibit a significant donor-to-donor variability in the baseline levels of VEGF expression. However, irrespective of the different levels of baseline expression in human monocytes from different donors, we have consistently observed in each separate experiment a hypoxic induction of VEGF mRNA ranging from 4-fold up to 10-fold above the level expressed in monocytes cultured under normoxic conditions. At the level of VEGF protein, we have observed in each independent experiment a consistent hypoxic induction above the baseline ranging from 2.7-fold up to 4.9-fold (range, 73–352 pg/ml). Thus, hypoxia is a stimulus for the induction of VEGF expression in human monocytes.
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, human monocytes cultured under normoxic conditions expressed detectable basal levels of VEGF mRNA that were slightly inhibited by the addition of flavopiridol (100 nM). Monocytes cultured under hypoxia expressed a 5.8-fold higher level of VEGF mRNA relative to monocytes cultured under normoxic conditions. However, the addition of flavopiridol dramatically and completely abolished hypoxia-induced VEGF mRNA expression (Fig. 2A
, Lane 4). In five independent experiments performed with fresh human monocytes harvested from different donors, we have consistently observed in the presence of flavopiridol a decrease of VEGF mRNA expression ranging from 80 to 99% relative to the levels induced by hypoxia. Similar results were also obtained using the human monocytic cell line THP-1 (data not shown). The inhibition of hypoxia-induced VEGF expression by flavopiridol was not restricted to cells of the myeloid lineage. In fact, flavopiridol also potently inhibited the constitutive and hypoxia-induced expression of VEGF in the human adenocarcinoma DLD-1 cell line (data not shown). The DLD-1 cell line represents an interesting model because of the genetic alteration and constitutive activation of the ras pathway, which has been implicated in the up-regulation of VEGF expression (21)
. Although the main focus of our investigation was to study the effects of flavopiridol in normal human monocytes, these data indicate that our findings may be extrapolated to tumor cell lines in which the role of oncogenic transformation and genetic abnormalities must be considered.
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, human monocytes cultured under normoxic conditions expressed detectable basal levels of VEGF protein (120 pg/ml) that were not significantly affected by addition of flavopiridol (100 nM). In contrast, the hypoxic induction of VEGF protein was decreased, in the presence of flavopiridol, from 2.75-fold (330 pg/ml) to 1.25-fold (152 pg/ml) above the levels present in human monocytes cultured under normoxic conditions, which represents an 86% inhibition of the hypoxic induction above the background levels. In three separate ELISA experiments, the hypoxic induction of VEGF protein ranged from 2.75- to 4.8-fold above the baseline level. In the presence of flavopiridol, the hypoxic induction of VEGF protein was decreased to a range of 1.1–1.4-fold above the levels detected in untreated human monocytes. In conclusion, irrespective of the baseline levels of VEGF expression in human monocytes cultured under normal oxygen conditions, we have consistently observed an inhibitory effect of flavopiridol relative to the hypoxic induction of VEGF above the baseline levels ranging from 80 to 99% at the mRNA level and from 75 to 90% at the protein level. Our data then demonstrate for the first time that flavopiridol is a potent inhibitor of hypoxia-induced VEGF expression in human monocytes.
To test the range of concentrations where flavopiridol was effective, human monocytes cultured under hypoxic conditions were exposed to increasing concentrations of flavopiridol, ranging from 25 to 100 nM. As shown in Fig. 3A
, a concentration-dependent inhibition of VEGF mRNA expression by flavopiridol was observed. Flavopiridol at 25 nM inhibited by 
50% the hypoxia-induced expression of VEGF (Lane 5), as assessed by densitometry analysis, whereas flavopiridol at 50 nM caused a 75% inhibition (Lane 4). Complete inhibition was observed at doses of 100 nM (Lane 3). Of note, flavopiridol did not significantly affect the expression of glyceraldehyde-3-phosphate dehydrogenase, which also is a hypoxia-inducible gene, or the expression of rRNA 28S and 18S. The effective concentration of flavopiridol that inhibited hypoxia-induced VEGF expression (50–100 nM) is slightly lower than the IC50 for the inhibition of CDK activity (100–300 nM; Ref. 14
). Whether the flavopiridol inhibition of gene expression and CDK activity share a common target and/or mechanism of action remains to be elucidated.
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Protein Synthesis Is Not Required for the Inhibition of VEGF mRNA Expression by Flavopiridol.
Next, we performed experiments to establish whether protein synthesis was required for the down-regulation of hypoxia-induced VEGF mRNA expression by flavopiridol. Human monocytes were pretreated with CHX or medium for 30 min and then were cultured for additional 5 h under either normoxic or hypoxic conditions in the presence or absence of flavopiridol. As shown in Fig. 3B
, untreated monocytes cultured under normoxic conditions expressed detectable levels of VEGF mRNA that were significantly augmented by the addition of CHX under normoxic conditions. Flavopiridol decreased the constitutive levels of VEGF mRNA expression and, more importantly, completely blocked CHX-dependent induction of VEGF. In addition, flavopiridol inhibited hypoxia-induced VEGF mRNA expression in the absence or the presence of CHX. In contrast, CHX did not augment the induction of VEGF by hypoxia. These data demonstrate that protein synthesis is not required for the inhibitory effects of flavopiridol on the induction of VEGF expression by hypoxia.
Flavopiridol Does Not Affect Hypoxia-induced Transcriptional Activation of VEGF Promoter.
To investigate whether flavopiridol affects the transcriptional activation of VEGF, we tested the effects of flavopiridol on the hypoxia-induced expression of a VEGF promoter-luciferase reporter plasmid. Because human monocytic cell lines are difficult to transfect for reporter gene assays, we used the ANA-1 murine macrophage cell line that has been well characterized for transient transfection studies. Preliminary experiments indicated that ANA-1 macrophages expressed VEGF mRNA after exposure to hypoxia and that flavopiridol inhibited the hypoxia-induced expression, consistent with the data obtained in human monocytes (data not shown). ANA-1 cells, transiently transfected with plasmid VEGF-P7, containing 1005 bp of the VEGF 5'-flanking region linked to the luciferase reporter gene, were cultured under normoxic or hypoxic conditions in the presence or absence of flavopiridol, and luciferase levels were measured after 24 h of incubation. As shown in Fig. 4A
, the VEGF promoter was constitutively active at low levels in ANA-1 macrophages. Culture under hypoxic conditions caused a 3- to 5-fold induction of luciferase expression above the baseline. Flavopiridol did not significantly affect either the basal or the hypoxia-induced expression of VEGF-luciferase reporter plasmid in ANA-1 macrophages. Because the HIF-1 binding site at -975 of the VEGF promoter is required for hypoxia-induced activation of the VEGF promoter and also mediates hypoxia-induced transcription of other genes, we tested whether flavopiridol affected HIF-1-transcriptional activation induced by hypoxia in macrophages. A luciferase reporter plasmid containing 47 bp of the VEGF promoter encompassing the HIF-1 binding site at -975 (plasmid P11W) was transiently transfected in ANA-1 macrophages. Hypoxia-treated macrophages expressed between 5- and 10-fold higher levels of luciferase than cells cultured under normoxic conditions (Fig. 4B)
. Flavopiridol did not affect either the basal or the hypoxia-inducible expression of the HIF-1-luciferase reporter construct in macrophages. Taken together, these data suggest that flavopiridol does not affect the transcriptional activation of VEGF induced by hypoxia in macrophages. In addition, the finding that protein synthesis was not required for flavopiridol inhibition of VEGF expression is consistent with the data that HIF-1 is not a likely target of flavopiridol activity. In fact, increased stability of HIF-1 protein is the primary mechanism by which HIF-1 accumulates in hypoxic cells (22)
. The lack of inhibition of HIF-1-mediated transcription also indicates that flavopiridol does not act as a general inhibitor of the hypoxia-dependent pathway.
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4 h (Fig. 4C)
. In contrast, the half life of VEGF mRNA in THP-1 cells treated with hypoxia in the presence of flavopiridol was dramatically reduced to 2 h. Similar results were consistently observed in at least three separate experiments. The half-life of the VEGF mRNA that we have observed in THP-1 cells is longer than that reported in the literature for other cell types (1.5–2 h; Ref. 5
). However, to the best of our knowledge, there is no data available on the half-life of VEGF mRNA in human monocytes cultured under hypoxic conditions. This discrepancy in the half-life of VEGF mRNA between human monocytes and other cell types may be due to a "tissue specific" regulation present in cells of the myeloid lineage. These data demonstrate that flavopiridol destabilizes hypoxia-induced VEGF mRNA expression and decreases VEGF mRNA half-life, providing the first evidence that flavopiridol can control gene expression by acting at a posttranscriptional level. The VEGF mRNA 3'-UTR contains instability regions rich in adenylate-uridylate (AU) sequences, which have been demonstrated to mediate the rapid turnover of multiple cytokine mRNAs. A hypoxia-inducible protein complex that binds to the AU-rich element in the 3'-UTR of the VEGF mRNA has been described recently (10)
. Interestingly, the tyrosine kinase inhibitor genistein, although at relatively high concentrations (500 µM), blocked the hypoxia-induced stabilization of VEGF 3'-UTR transcripts and inhibited hypoxia-induced protein binding to the VEGF 3'-UTR (5)
. Although the mechanism by which flavopiridol inhibits hypoxia-induced stabilization of VEGF mRNA remains to be elucidated, our results are consistent with a model in which flavopiridol might prevent the binding of a hypoxia-induced RNA-binding protein to elements of the VEGF mRNA 3'-UTR. Flavopiridol is a protein kinase inhibitor and might interfere with the phosphorylation of the hypoxia-induced RNA trans-activating factor. This possibility is consistent with our data indicating that protein synthesis was not required for the inhibitory effects of flavopiridol to occur, making it less likely that flavopiridol might block the expression and/or production of the RNA binding protein. Studies are ongoing to identify the target of flavopiridol in the hypoxia-induced signaling cascade. In this study, we report that flavopiridol, a known CDK inhibitor, potently inhibits VEGF expression in human monocytes. More importantly, flavopiridol blocks the expression of VEGF induced by hypoxia, a component of many solid tumors, which represents a pathophysiological stimulus for the induction of angiogenesis in vivo. The identification of molecular target(s) by which pharmacological agents inhibit hypoxic induction of VEGF expression may lead to the successful manipulation of VEGF production. Macrophages are a suitable target of a novel therapeutic strategy aimed at decreasing tumor progression and metastasis (1) . Our data may have important therapeutic implications in particular because flavopiridol is presently under investigation in several Phase I and II clinical trials. The potential for antiangiogenic activity of flavopiridol, which is implicit in these experiments, warrants further investigation in future clinical trials.
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at DTP-Tumor Hypoxia Program, Building 432, Room 218, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702-1201. E-mail:melillo{at}dtpax2.ncifcrf.gov 
2 The abbreviations used are: HIF, hypoxia inducible factor; VEGF, vascular endothelial growth factor; CDK, cyclin-dependent kinase; CHX, cycloheximide; UTR, untranslated region.
Received 5/27/99. Accepted 9/21/99.
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is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc. Natl. Acad. Sci. USA, 95: 7987-7992, 1998.This article has been cited by other articles:
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