Efficacy of the GemOx-R regimen leads to the identification of Oxaliplatin as a highly effective drug against Mantle Cell Lymphoma.

Mantle Cell Lymphoma (MCL) is an aggressive lymphoma subtype that accounts for 6-8% of non-Hodgkin lymphomas. The disease is mostly incurable and characterized by a continuous pattern of relapse. Major changes have recently been implemented in the management of MCL, but continuous relapses still mark this disease as a challenge for clinicians. We previously reported the efficacy of GemOx-R (Gemcitabine, Oxaliplatin and Rituximab) in patients with refractory and relapsing MCL. We present results for a larger series with longer follow-up and including high-risk frontline patients, showing an overall response rate of 83%. The efficacy of each component of GemOx-R was evaluated in a panel of MCL cell lines. Also, patient-derived primary cells were used in ex vivo experiments. The results show that oxaliplatin has a profound effect on cellular viability and is the most effective drug within this regimen. We further present synergistic efficacy of oxaliplatin combined with cytarabine in MCL cells. Interestingly, this synergistic effect was not seen when cisplatin and cytarabine were combined, indicating that among the platinum-derived agents oxaliplatin may be the preferred approach. Taken together our findings suggest that oxaliplatin alone or combined with cytarabine could constitute an alternative backbone for MCL regimens.

Cell uptake and localization studies of squaramide based fluorescent probes.

Cell internalization is a major issue in drug design. Although squaramide-based compounds are receiving much attention because of their interesting bioactivity, cell uptake and trafficking within cells of this type of compounds are still unknown. In order to monitor the cell internalization process of cyclosquaramide compounds we have prepared two fluorescent probes by covalently linking a fluorescent dye (BODIPY derivative or fluorescein) to a noncytotoxic cyclosquaramide framework. These two probes (C2-BDP and C2-FITC) rapidly internalize across live cell membranes through endocytic receptor-mediated mechanisms. Due to its higher fluorescence and photochemical stability, C2-BDP is a superior dye than C2-FITC. C2-BDP remains sequestered in late endosomes allowing their fast and selective imaging in various live cell types. Cyclosquaramide-cell membrane interactions facilitate cell uptake and have been investigated by binding studies in solution as well as in live cells. Cyclosquaramide 1 (C2-BDP) can be used as a highly fluorescent probe for the rapid and selective imaging of late endosomes in live cells.

Retama monosperma n-hexane extract induces cell cycle arrest and extrinsic pathway-dependent apoptosis in Jurkat cells.

BACKGROUND: Retama monosperma L. (Boiss.) or Genista monosperma L. (Lam.), locally named as "R'tam", is an annual and spontaneous plant belonging to the Fabaceae family. In Morocco, Retama genus is located in desert regions and across the Middle Atlas and it has been widely used in traditional medicine in many countries. In this study, we show that Retama monosperma hexane extract presents significant anti-leukemic effects against human Jurkat cells. METHODS: Human Jurkat cells, together with other cell lines were screened with different concentrations of Retama monosperma hexane extract at different time intervals. Growth inhibition was determined using luminescent-based viability assays. Cell cycle arrest and apoptosis were measured by flow cytometry analysis. Combined caspase 3 and 7 activities were measured using luminometric caspase assays and immunoblots were performed to analyze expression of relevant pro- and anti-apoptotic proteins. GC-MS were used to determine the chemical constituents of the active extract. RESULTS: Retama monosperma hexane extract (Rm-HE) showed significant cytotoxicity against Jurkat cells, whereas it proved to be essentially ineffective against both normal mouse fibroblasts (NIH3T3) and normal lymphocytes (TK-6). Cytometric analysis indicated that Rm-HE promoted cell cycle arrest and apoptosis induction accompanied by DNA damage induction indicated by an increase in p-H2A.X levels. Rm-HE induced apoptosis was partially JNK-dependent and characterized by an increase in Fas-L levels together with activation of caspases 8, 3, 7 and 9, whereas neither the pro-apoptotic nor anti-apoptotic mitochondrial membrane proteins analyzed were significantly altered. Chemical identification analysis indicated that α-linolenic acid, campesterol, stigmasterol and sitosterol were the major bioactive components within the extract. CONCLUSIONS: Our data suggest that bioactive compounds present in Rm-HE show significant anti leukemic activity inducing cell cycle arrest and cell death that operates, at least partially, through the extrinsic apoptosis pathway.

The tumour suppressor FOXO3 is a key regulator of mantle cell lymphoma proliferation and survival.

The FOXO3 (Forkhead/winged helix box class O 3) transcription factor is a crucial regulator of haematopoietic cell fate that controls proliferation and apoptosis, among other processes. Despite the central role of FOXO3 as a tumour suppressor and phosphatidylinositol 3-kinase (PI3K)/AKT effector, little is known about its involvement in mantle cell lymphoma (MCL) biology. This study investigated the expression and activity of FOXO3 in MCL cell lines and in primary cultures. We analysed the expression of key FOXO regulators and targets, and studied the effect of modulators of FOXO function on cell viability and apoptosis. FOXO3 was constitutively inactivated in MCL cell lines, and showed cytoplasmic localization in patient-derived cells. PI3K and AKT, but not mammalian target of rapamycin (mTOR), inhibitors induced FOXO3 nuclear translocation and activation in correlation with their impact on MCL proliferation and survival. Moreover, FOXO3-defective cells were resistant to PI3K/AKT inhibitors. Reactivation of FOXO function with a nuclear export inhibitor had a profound effect on cell viability, consistent with FOXO3 nuclear accumulation. Interestingly, inhibition of FOXO3 nuclear export enhanced the effect of doxorubicin. Taken together, our results confirm that FOXO3 is a relevant regulator of proliferation and apoptosis in MCL, and suggest that reactivation of FOXO3 function might be a useful therapeutic strategy in MCL patients.

Rnd3 Is a Crucial Mediator of the Invasive Phenotype of Glioblastoma Cells Downstream of Receptor Tyrosine Kinase Signalling.

Enhanced invasiveness is one of the defining biological traits of glioblastoma cells, which exhibit an infiltrative nature that severely hinders surgical resection. Among the molecular lesions responsible for GBM aggressiveness, aberrant receptor tyrosine kinase (RTK) signalling is well-characterised. Enhanced RTK signalling directly impacts a myriad of cellular pathways and downstream effectors, which include the Rho GTPase family, key regulators of actin cytoskeletal dynamics. Here, we have analysed the functional crosstalk between oncogenic signals emanating from RTKs and Rho GTPases and focused on the specific contribution of Rnd3 to the invasive phenotype of GBM in this context. We found that RTK inhibition with a panel of RTK inhibitors decreased cell motility and cell invasion and promoted dramatic actin cytoskeleton reorganisation through activation of the RhoA/Rho-associated protein kinase 1 (ROCK) axis. RTK inhibition also significantly decreased Rnd3 expression levels. Consistently, shRNA-mediated Rnd3 silencing revealed that Rnd3 depletion promoted substantial changes in the actin cytoskeleton and reduced cell motility and invasion capacity, recapitulating the effects observed upon RTK inhibition. Our results indicate that Rnd3 is a crucial mediator of RTK oncogenic signalling involved in actin cytoskeletal reorganisation, which contributes to determining the invasive phenotype of GBM cells.

Glioblastoma Embryonic-like Stem Cells Exhibit Immune-Evasive Phenotype.

BACKGROUND: Glioma stem cells (GSCs) have self-renewal and tumor-initiating capacities involved in drug resistance and immune evasion mechanisms in glioblastoma (GBM). METHODS: Core-GSCs (c-GSCs) were identified by selecting cells co-expressing high levels of embryonic stem cell (ESC) markers from a single-cell RNA-seq patient-derived GBM dataset (n = 28). Induced c-GSCs (ic-GSCs) were generated by reprogramming GBM-derived cells (GBM-DCs) using induced pluripotent stem cell (iPSC) technology. The characterization of ic-GSCs and GBM-DCs was conducted by immunostaining, transcriptomic, and DNA methylation (DNAm) analysis. RESULTS: We identified a GSC population (4.22% ± 0.59) exhibiting concurrent high expression of ESC markers and downregulation of immune-associated pathways, named c-GSCs. In vitro ic-GSCs presented high expression of ESC markers and downregulation of antigen presentation HLA proteins. Transcriptomic analysis revealed a strong agreement of enriched biological pathways between tumor c-GSCs and in vitro ic-GSCs (κ = 0.71). Integration of our epigenomic profiling with 833 functional ENCODE epigenetic maps identifies increased DNA methylation on HLA genes' regulatory regions associated with polycomb repressive marks in a stem-like phenotype. CONCLUSIONS: This study unravels glioblastoma immune-evasive mechanisms involving a c-GSC population. In addition, it provides a cellular model with paired gene expression, and DNA methylation maps to explore potential therapeutic complements for GBM immunotherapy.

RhoE inhibits 4E-BP1 phosphorylation and eIF4E function impairing cap-dependent translation.

The Rho GTPase family member RhoE inhibits RhoA/ROCK signaling to promote actin stress fiber and focal adhesion disassembly. We have previously reported that RhoE also inhibits cell cycle progression and Ras-induced transformation, specifically preventing cyclin D1 translation. Here we investigate the molecular mechanisms underlying those observations. RhoE inhibits the phosphorylation of the translational repressor 4E-BP1 in response to extracellular stimuli. However, RhoE does not affect the activation of mTOR, the major kinase regulating 4E-BP1 phosphorylation, as indicated by the phosphorylation levels of the mTOR substrate S6K, the dynamics of mTOR/Raptor association, and the observation that RhoE, as opposed to rapamycin, does not impair cellular growth. Interestingly, RhoE prevents the release of the eukaryotic initiation factor eIF4E from 4E-BP1, inhibiting cap-dependent translation. Accordingly, RhoE also inhibits the expression and the transcriptional activity of the eIF4E target c-Myc. Consistent with its crucial role in cell proliferation, we show that eIF4E can rescue both cell cycle progression and Ras-induced transformation in RhoE-expressing cells, indicating that the inhibition of eIF4E function is critical to mediate the anti-proliferative effects of RhoE.

Therapeutic concepts in mantle cell lymphoma.

Mantle cell lymphoma has been considered an incurable disease with current chemotherapy regimens. Recent intense chemoimmunotherapy induction regimens with or without consolidation with autologous stem cell transplantation procedures are showing a potential for cure in a sizable fraction of patients. Similarly, in the salvage setting, preliminary experience with non-myeloablative allogeneic transplant may cure some patients even after multiple therapeutic failures. However, the recent knowledge of the three basic biologic derangements that are integrated in the disease may change the therapeutic approach of the disease in the near future. In fact, new drugs that target more specifically the major molecular alterations of the disease are being progressively incorporated into the therapeutic armamentarium of the disease. In the near future, more individualized approaches that will take into account not only risk factors present at diagnosis but also biomarkers representative of the molecular alterations present in the disease are foreseen. In this review, we are going to discuss the current therapeutic approaches and the main new drugs that target more specifically the major molecular pathways alterations of the disease.

Toward a Rational Design of Polyamine-Based Zinc-Chelating Agents for Cancer Therapies.

In vitro viability assays against a representative panel of human cancer cell lines revealed that polyamines L1a and L5a displayed remarkable activity with IC50 values in the micromolar range. Preliminary research indicated that both compounds promoted G1 cell cycle arrest followed by cellular senescence and apoptosis. The induction of apoptotic cell death involved loss of mitochondrial outer membrane permeability and activation of caspases 3/7. Interestingly, L1a and L5a failed to activate cellular DNA damage response. The high intracellular zinc-chelating capacity of both compounds, deduced from the metal-specific Zinquin assay and ZnL2+ stability constant values in solution, strongly supports their cytotoxicity. These data along with quantum mechanical studies have enabled to establish a precise structure-activity relationship. Moreover, L1a and L5a showed appropriate drug-likeness by in silico methods. Based on these promising results, L1a and L5a should be considered a new class of zinc-chelating anticancer agents that deserves further development.

Increase in Fru-2,6-P(2) levels results in altered cell division in Schizosaccharomyces pombe.

Mitogenic response to growth factors is concomitant with the modulation they exert on the levels of Fructose 2,6-bisphosphate (Fru-2,6-P2), an essential activator of the glycolytic flux. In mammalian cells, decreased Fru-2,6-P2 concentration causes cell cycle delay, whereas high levels of Fru-2,6-P2 sensitize cells to apoptosis. In order to analyze the cell cycle consequences due to changes in Fru-2,6-P2 levels, the bisphosphatase-dead mutant (H258A) of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase enzyme was over-expressed in Schizosaccharomyces pombe cells and the variation in cell phenotype was studied. The results obtained demonstrate that the increase in Fru-2,6-P2 levels results in a defective division of S. pombe, as revealed by an altered multisepted phenotype. The H258A-expressing cells showed impairment of cytokinesis, but normal nuclear division. In order to identify cellular mediators responsible for this effect, we transformed different S. pombe strains and observed that the cytokinetic defect was absent in cells defective for Wee1 kinase function. Therefore, in S. pombe, Wee1 integrates the metabolic signal emerging from changes in Fru-2,6-P2 content, thus coupling metabolism with cell proliferation. As the key regulators of the cell cycle checkpoints are conserved throughout evolution, these results may help to understand the experimental evidences obtained by manipulation of Fru-2,6-P2 levels in mammalian cells.

Hydrogen peroxide regulates the mitochondrial content of uncoupling protein 5 in colon cancer cells.

Uncoupling protein-2 (UCP2) and uncoupling protein-5 (UCP5) are ion carriers located in the inner mitochondrial membrane that mediate a regulated discharge of the proton gradient generated by the respiratory chain and are possibly involved in the protection against free radical production. Mitochondrial dysfunction is involved in the pathogenesis of colon cancer for which UCP2 has been proposed as a potential therapeutic target. The object of this study was to investigate the possible effects of mitochondrial dysfunction and oxidative stress on the mitochondrial content of UCP2 and UCP5 in colonic cells. Proliferation rate/index, antioxidant systems, reactive oxygen species, oxidative damage, mitochondrial markers and protein expression of UCP2 and UCP5 were analysed in non-metastatic HT-29 cells, metastatic SW-620 cells, and in a HT-29 differentiated derivative (Glu-R). In this study, we found a clear relationship between mitochondrial dysfunction in colonic epithelial cells and the levels of UCP2 and UCP5 proteins. Furthermore, hydrogen peroxide was responsible for the coordinated activation of the antioxidant response and the increase of UCP5 protein levels. Our findings suggest that uncoupling activity could act as a local feedback mechanism to limit the enhanced steady-state H(2)O(2) production in dysfunctional mitochondria of colonic cells.

FOXO3a mediates the cytotoxic effects of cisplatin in colon cancer cells.

Cisplatin is a conventional chemotherapeutic agent that binds covalently to purine DNA bases and mediates cellular apoptosis. A better understanding of the downstream cellular targets of cisplatin will provide information on its mechanism of action and help to understand the mechanism of drug resistance. In this study, we have investigated the effects of cisplatin in a panel of colon carcinoma cell lines and the involvement of the phosphoinositide-3-kinase/forkhead/winged helix box class O (FOXO) pathway in cisplatin action and resistance. Cisplatin-sensitive and cisplatin-resistant cell lines have been characterized in cell viability, flow cytometry, and clonogenic assays. The main components of the phosphoinositide-3-kinase/protein kinase B pathway, particularly FOXO3a, have been analyzed in sensitive and resistant cells on cisplatin treatment. Interestingly, in sensitive cells, cisplatin induces FOXO3a dephosphorylation and nuclear translocation, and expression of its target genes, whereas in resistant cells the effect of cisplatin on FOXO3a is incomplete. Consistent with this, protein kinase B/FOXO signaling axis modulators triciribine and psammaplysene A sensitize the resistant HT29 cells to cisplatin treatment. Critically, knockdown of FOXO3a expression using small interfering RNA rescues sensitive SW620 cells from cisplatin-induced short- and long-term cell death. Together, our findings suggest that FOXO3a is a relevant mediator of the cytotoxic effects of cisplatin in colon cancer cells.

The tumor suppressor FOXO3a mediates the response to EGFR inhibition in glioblastoma cells.

PURPOSE: Although EGFR activation is a hallmark of glioblastoma (GBM), anti-EGFR therapy has so far not yielded the desired effects. Targeting PI3K/Akt has been proposed as a strategy to increase the cellular sensitivity to EGFR inhibitors. Here we evaluated the contribution of FOXO3a, a key Akt target, in the response of GBM cells to EGFR inhibition. METHODS: FOXO3a activation was assessed by immunofluorescence and gene reporter assays, and by evaluating target gene expression using Western blotting and qRT-PCR. Cellular effects were evaluated using cell viability and apoptosis assays, i.e., Annexin V/PI staining and caspase 3/7 activity measurements. Drug synergism was evaluated by performing isobolographic analyses. Gene silencing experiments were performed using stable shRNA transfections. RESULTS: We found that EGFR inhibition in GBM cells led to FOXO3a activation and to transcriptional modulation of its key targets, including repression of the oncogene FOXM1. In addition, we found that specific FOXO3a activation recapitulated the molecular effects of EGFR inhibition, and that the FOXO3a activator trifluoperazine, a FDA-approved antipsychotic agent, reduced GBM cell growth. Subsequent isobolographic analyses of combination experiments indicated that trifluoperazine and erlotinib cooperated synergistically and that their concomitant treatment induced a robust activation of FOXO3a, leading to apoptosis in GBM cells. Using gene silencing, we found that FOXO3a is essential for the response of GBM cells to EGFR inhibition. CONCLUSIONS: Our data indicate that FOXO3a activation is a crucial event in the response of GBM cells to EGFR inhibition, suggesting that FOXO3a may serve as an actionable therapeutic target that can be modulated using FDA-approved drugs.

Cell cycle inhibition by FoxO forkhead transcription factors involves downregulation of cyclin D.

The FoxO forkhead transcription factors FoxO4 (AFX), FoxO3a (FKHR.L1), and FoxO1a (FKHR) represent important physiological targets of phosphatidylinositol-3 kinase (PI3K)/protein kinase B (PKB) signaling. Overexpression or conditional activation of FoxO factors is able to antagonize many responses to constitutive PI3K/PKB activation including its effect on cellular proliferation. It was previously shown that the FoxO-induced cell cycle arrest is partially mediated by enhanced transcription and protein expression of the cyclin-dependent kinase inhibitor p27(kip1) (R. H. Medema, G. J. Kops, J. L. Bos, and B. M. Burgering, Nature 404:782-787, 2000). Here we have identified a p27(kip1)-independent mechanism that plays an important role in the antiproliferative effect of FoxO factors. Forced expression or conditional activation of FoxO factors leads to reduced protein expression of the D-type cyclins D1 and D2 and is associated with an impaired capacity of CDK4 to phosphorylate and inactivate the S-phase repressor pRb. Downregulation of D-type cyclins involves a transcriptional repression mechanism and does not require p27(kip1) function. Ectopic expression of cyclin D1 can partially overcome FoxO factor-induced cell cycle arrest, demonstrating that downregulation of D-type cyclins represents a physiologically relevant mechanism of FoxO-induced cell cycle inhibition.

FoxO3a and BCR-ABL regulate cyclin D2 transcription through a STAT5/BCL6-dependent mechanism.

Cell cycle arrest by FoxO transcription factors involves transcriptional repression of cyclin D, although the exact mechanism remains unclear. In this study, we used the BCR-ABL-expressing cell line BV173 as a model system to investigate the mechanisms whereby FoxO3a regulates cyclin D2 expression. Inhibition of BCR-ABL by STI571 results in down-regulation of cyclin D2 expression, activation of FoxO3a activity, and up-regulation of BCL6 expression. Using reporter gene assays, we demonstrate that STI571, FoxO3a, and BCL6 can repress cyclin D2 transcription through a STAT5/BCL6 site located within the cyclin D2 promoter. We propose that BCR-ABL inhibition leads to FoxO3a activation, which in turn induces the expression of BCL6, culminating in the repression of cyclin D2 transcription through this STAT5/BCL6 site. This process was verified by mobility shift and chromatin immunoprecipitation analyses. We find that conditional activation of FoxO3a leads to accumulation of BCL6 and down-regulation of cyclin D2 at protein and mRNA levels. Furthermore, silencing of FoxO3a and BCL6 in BCR-ABL-expressing cells abolishes STI571-mediated effects on cyclin D2. This report establishes the signaling events whereby BCR-ABL signals are relayed to cyclin D2 to mediate cell cycle progression and defines a potential mechanism by which FoxO proteins regulate cyclin D2 expression.

Progestins regulate the expression and activity of the forkhead transcription factor FOXO1 in differentiating human endometrium.

Menstruation, or cyclic shedding of nonpregnant endometrial tissue with associated bleeding, occurs only in humans and a few other species. This breakdown of the endometrium in response to falling ovarian progesterone levels is a complex process, characterized by local leukocyte infiltration, expression and activation of matrix metalloproteinases, and apoptosis. Spontaneous decidualization (differentiation) of the stromal compartment precedes the cyclic shedding of the endometrium in various menstruating species but the mechanisms that link these processes are not understood. In this study, we identified FOXO1 as a key transcription factor responsible for mediating apoptosis of decidualized human endometrial stromal cells (HESCs) in response to progesterone withdrawal. We demonstrate that medroxyprogesterone acetate (MPA, a synthetic progestin) enhances the expression of FOXO1 in differentiating HESCs while simultaneously inducing cytoplasmic retention and inactivation of FOXO1. Withdrawal of MPA from decidualized HESCs results in rapid nuclear accumulation of FOXO1, increased BIM expression, a proapoptotic FOXO1 target gene, and cell death. Conversely, silencing of FOXO1 expression completely abolishes cell death induced by MPA withdrawal. In summary, the observation that differentiating HESCs become dependent on progesterone signaling for survival through induction and reversible inactivation of FOXO1 suggests a novel mechanism that links decidualization of the endometrium to menstruation.

Direct transcriptional regulation of Bim by FoxO3a mediates STI571-induced apoptosis in Bcr-Abl-expressing cells.

In this study, we have used the human BV173 and the mouse BaF3/Bcr-Abl-expressing cell lines as model systems to investigate the molecular mechanisms whereby STI571 and FoxO3a regulate Bim expression and apoptosis. FoxO3a lies downstream of Bcr-Abl signalling and is constitutively phosphorylated in the Bcr-Abl-positive BV173 and BaF3/Bcr-Abl cells. Inhibition of Bcr-Abl kinase by STI571 results in FoxO3a activation, induction of Bim expression and apoptosis. Using reporter gene assays, we demonstrate that STI571 and FoxO3a activate Bim transcription through a FoxO-binding site (FHRE) located within the promoter. This was verified by DNA pull-down and chromatin immunoprecipitation analyses. We find that conditional activation of FoxO3a leads to induction of Bim expression and apoptosis. Conversely, silencing of FoxO3a in Bcr-Abl-expressing cells abolishes STI571-mediated Bim induction and apoptosis. Together, the results presented clearly confirm FoxO3a as a key regulator of apoptosis induced by STI571, and show that Bim is a direct transcriptional target of FoxO3a that mediates the STI571-induced apoptosis. Thus, STI571 induces an accumulation of FoxO3a activity which in turn binds directly to an FHRE in the promoter to activate Bim expression and apoptosis.

N-(2-methyl-indol-1H-5-yl)-1-naphthalenesulfonamide: A novel reversible antimitotic agent inhibiting cancer cell motility.

A series of compounds containing the sulfonamide scaffold were synthesized and screened for their in vitro anticancer activity against a representative panel of human cancer cell lines, leading to the identification of N-(2-methyl-1H-indol-5-yl)-1-naphthalenesulfonamide (8e) as a compound showing a remarkable activity across the panel, with IC50 values in the nanomolar-to-low micromolar range. Cell cycle distribution analysis revealed that 8e promoted a severe G2/M arrest, which was followed by cellular senescence as indicated by the detection of senescence-associated ß-galactosidase (SA-ß-gal) in 8e-treated cells. Prolonged 8e treatment also led to the onset of apoptosis, in correlation with the detection of increased Caspase 3/7 activities. Despite increasing γ-H2A.X levels, a well-established readout for DNA double-strand breaks, in vitro DNA binding studies with 8e did not support interaction with DNA. In agreement with this, 8e failed to activate the cellular DNA damage checkpoint. Importantly, tubulin staining showed that 8e promoted a severe disorganization of microtubules and mitotic spindle formation was not detected in 8e-treated cells. Accordingly, 8e inhibited tubulin polymerization in vitro in a dose-dependent manner and was also able to robustly inhibit cancer cell motility. Docking analysis revealed a compatible interaction with the colchicine-binding site of tubulin. Remarkably, these cellular effects were reversible since disruption of treatment resulted in the reorganization of microtubules, cell cycle re-entry and loss of senescent markers. Collectively, our data suggest that this compound may be a promising new anticancer agent capable of both reducing cancer cell growth and motility.

An intronic AP-1 sequence mediates the transcriptional activation of the F-type 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by serum.

The expression of F-type 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is rapidly induced by growth factors. We report here that an AP-1 intragenic sequence located at position +612 of the F-type 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase transcription initiation site is involved in the transcriptional activation of this gene by serum. We have demonstrated in vitro DNA-protein interaction on this AP-1 site of the F-promoter. Indeed, this element was recognized by c-Fos and JunD in vitro, and mutation or deletion of this element reduced the early response to serum stimulation by 60%. We conclude that the serum response of the F-type 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene requires the co-ordinated function of ets, E2F and AP-1 binding sites.