Induction of apoptosis and down regulation of cell cycle proteins in mantle cell lymphoma by flavopiridol treatment

Typical mantle cell lymphoma (MCL) is a distinct B-cell non-Hodgkin's lymphoma associated with over-expression of cyclin D1 related to translocation between the IgH and BCL-1 genes. Due to the important functional interaction between cyclin D1 and cyclin dependent kinases, cyclin dependent kinase inhibitors such as flavopiridol are under consideration for treatment of patients with MCL. The present study investigated the in vitro effects of flavopiridol on the MCL cell line (JeKo-1). Flavopiridol at a dose of 10nmol/L induced apoptosis by 6h of treatment as noted by flow cytometric analysis, morphologic examination and Western blotting. The cleavage of procaspase-3 and PARP and the decrease of flavopiridol-induced apoptosis by pan-caspase inhibition suggested that the caspase pathway serves an important role in the apoptotic process. Furthermore, MCL cells exposed to flavopiridol showed down regulation of key cell cycle proteins acting at the restriction point control between the G1 and S phases. The onset of flavopiridol-induced apoptosis also coincided with the down regulation of Mcl-1, anti-apoptotic protein. Collectively, our data indicates that flavopiridol may have significant therapeutic potential in the context of MCL.     

Growth inhibition and apoptosis of myeloma cells by the CDK inhibitor flavopiridol.

Although myeloma shows responsiveness in intensive chemotherapy, overall survival remains less than 40% at 2 years. Since myeloma appears to be dependent on cytokines, such as IL-6, we hypothesized that targeting signal transduction molecules could effectively treat myeloma. Two myeloma cell lines U266 and RPMI-8226 and CD38+ myeloma cells were studied by immune complex kinase assay or anti-phosphotyrosine blot for evidence of constitutive activation of tyrosine kinases. Growth arrest and apoptosis were evaluated in these two cell lines following their treatment with specific kinase inhibitors. We found that a variety of Src and Janus kinases were present and constitutively active in U266 and RPMI-8226 cells. Inhibitors of both Src and Janus kinases were inferior to the cyclin-dependent kinase inhibitor, flavopiridol, in inducing both growth arrest with GI50 of 100 nM and apoptosis in both cell lines and CD38+ myeloma cells. Although, flavopiridol did not affect cyclin D1 and cyclin A levels, it inhibited Mcl-1 and Bcl-2 protein levels and cyclin-dependent kinase 2 activity. Flavopiridol is a well-tolerated drug, currently in phase I-II trials for a variety of tumors. A clinical trial using flavopiridol should be performed in patients with myeloma. Its mechanism of action may involve targets other than the cyclin-dependent kinases.     

E2F4 deficiency promotes drug-induced apoptosis

E2F1 and E2F4 are known to have opposing roles in cell cycle control. In the present work, we examine the role of both E2F1 and E2F4 in apoptosis induced by three cyclin-dependent kinase inhibitors (roscovitine, BMS-387032, and flavopiridol) as well as by three established chemotherapeutic drugs (VP16, cisplatin and paclitaxel). We find that E2F4 levels are diminished following treatment with cyclin dependent kinase inhibitors (flavopiridol, roscovitine and BMS-387032) or with DNA damaging drugs (cisplatin and VP16). In contrast, each of these drugs induced E2F1. We find that mouse fibroblasts nullizygous for the E2F4 gene are more sensitive to apoptosis induced by roscovitine, flavopiridol, cisplatin, and VP16, whereas E2F1-deficient fibroblasts are less sensitive. Likewise, we find that RNAi-mediated reductions in E2F4 in human cancer cells results in increased drug sensitivity. Taken together, these results support a model in which E2F1 and E2F4 play opposing roles during drug-induced apoptosis.     

Flavopiridol, a cyclin-dependent kinase inhibitor, prevents spindle inhibitor-induced endoreduplication in human cancer cells.

Defects in cell cycle checkpoints can lead to chromosome abnormality, aneuploidy, and genomic instability, all of which can contribute to tumorigenesis. Recent studies and data presented in this study indicate that cells with compromised G1 checkpoint endoreduplicate and become polyploid in response to microtubule inhibitors. Previous studies have shown that polyploid cells are unstable and lose chromosomes randomly to give aneuploidy. In this study, we show that endoreduplication and polyploidation can be prevented by inhibiting the cyclin-dependent kinases (Cdks) by flavopiridol, a synthetic flavone presently undergoing phase II clinical trials. In our initial studies, we treated MCF-7 cells with paclitaxel, which results in the arrest of cells in G1 with 4n DNA content (pseudo G1). This was coincident with increased p53 and p21 protein expression and decreased cyclin E/Cdk2 kinase activity. In contrast, G1 checkpoint-compromised MDA-MB-468 (p53-/- and pRb-/-) and p21-/- HCT116 do not arrest in the pseudo G1 state after exposure to microtubule inhibitors and enter in the S phase with 4n DNA content. More than 60% of MDA-MB-468 cells accumulate with >4n DNA content after 72 h of nocodazole treatment. The MPM-2 labeling showed that 8n cells also undergo mitosis. These cells display deregulated and persistent activation of cyclin E/Cdk2 and cyclin B1/cdc2 kinase activity. Administration of flavopiridol after mitotic block results in the arrest of cells in the pseudo G1 state and the dramatic decrease in cells containing >4n DNA content in MDA-MB-468 cells. The cyclin E/Cdk2 and cyclin B1/cdc2 kinase activities remained low after exit from mitosis. Furthermore, pRb was hypophosphorylated after the addition of flavopiridol in p21-deficient HCT116 cells, indicating the arrest of cells at the pseudo G1 state. Based on these studies, we propose that flavopiridol preserves the genomic stability by preventing endoreduplication and polyploidy and thus has the potential to be used as a chemopreventive agent to prevent the occurrence of neoplasia.     

Cyclin B and E2F-1 expression in prostate carcinoma cells treated with the novel retinoid CD437 are regulated by the ubiquitin-mediated pathway

E2F-1 and cyclin B are important regulators of the cell cycle, and their expressionand degradation are tightly regulated. Proteolysis of both molecules is mediated by the ubiquitin degradation pathway involving the activation of specific E3 ubiquitin ligases. Treatment of prostate carcinoma cells with the novel retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437/AHPN) results in the enhanced expression of E2F-1 and rapid degradation of cyclin B in the absence of the modulation of mRNA levels; this is accompanied by the S phase arrest of the cells and subsequent apoptosis. The elevated level of E2F-1 is because of the enhanced stability of the molecule, as indicated by pulse-labeling studies, demonstrating a prolonged half-life. The enhanced E2F-1 stability is associated with the concomitant acetylation of E2F-1, the disassociation of E2F-1 from the E2F-1 E3 ligase p45(SKP2), and decreased E2F-1 ubiquitination, suggesting CD437 inhibition of E-3 E2F-1 ligase activity. Exposure of the cells to CD437 also results in the enhanced association of the cyclin B E3 ligase APC with cyclin B and the rapid proteolysis of cyclin B. The CD437-enhanced proteolysis of cyclin B is blocked in the presence of the ubiquitin proteolysis inhibitor N-acetyl-leu-leu-norleu-al. Thus, CD437 modulates the expression of E2F-1 and cyclin B through the simultaneous stimulation and inhibition of the cyclin B and E2F-1 E3 ligases, respectively.     

Inhibition of cyclin A kinase activity in E2F-1 chemogene therapy of colon cancer.

Adenoviral-mediated gene transfer of the apoptotic gene E2F-1 has been shown to induce apoptosis in a variety of tumor cells and acts in an additive or cooperative fashion with several specific chemotherapeutic agents to induce tumor cell death. The apoptotic function of E2F-1 is dependent on its ability to bind DNA; cyclin A kinase activity has been shown to negatively regulate the DNA-binding capacity of E2F-1. In the present study, we sought to determine whether cyclin A kinase activity is involved in mediating the interaction between E2F-1 and chemotherapeutic agents in colon cancer cells. Therefore, human colon adenocarcinoma (SW620) cells were treated with an adenovirus expressing E2F-1 (Ad-E2F-1, multiplicity of infection 20). Immediately following infection, a panel of conventional chemotherapeutic agents with varying modes of cytotoxic action were administered at LD(25 )doses. Three days following treatment, viability and growth inhibition were determined by trypan blue exclusion assay. Apoptosis was confirmed using cellular morphology, poly (ADP-ribose) polymerase cleavage, and flow-cytometric analysis. E2F-1 overexpression and cyclin A protein expression were monitored by immunoblot, and cyclin A kinase activity was determined by kinase assay. Vincristine (VIN), camptothecin (CPT), and actinomycin D were found to have a cooperative (>38% over the additive single therapy values) effect on E2F-1-mediated apoptosis. Etoposide, cisplatin (CIS), and 5-fluorouracil (5-FU) showed the least cooperation ( 0.1) compared to Ad-E2F-1 treatment alone. Combination of Ad-LacZ/5-FU and Ad-LacZ/actinomycin D significantly inhibited cyclin A kinase activity compared to Ad-LacZ treatment alone (p < 0.005). No other Ad-LacZ/drug combinations significantly affected cyclin A kinase activity (p > 0.05). In conclusion, combinations of E2F-1 adenovirus and VIN, CPT, or actinomycin D at LD(25 )had significant cooperative effects on colon cancer apoptotic cell death in vitro. Although inhibition of cyclin A kinase activity was observed in most Ad-E2F-1/drug combination treatments compared to Ad-E2F-1 treatment alone, there was no consistent correlation between degree of inhibition of cyclin A kinase activity and the cooperative effect. Nonetheless, inhibition of cyclin A kinase activity may be an important mechanism by which the chemogene therapy effects involving E2F-1 are modulated.     

BCL-x(L) and BCL2 delay Myc-induced cell cycle entry through elevation of p27 and inhibition of G1 cyclin-dependent kinases

The anti-apoptotic molecules BCL-x(L) and BCL2 delay cell cycle entry from quiescence. We used serum induction and induction of a Myc-estrogen receptor fusion protein (MycER) in quiescent fibroblasts to investigate the mechanisms underlying the cell cycle activity of BCL-x(L) and BCL2. We demonstrate for the first time that BCL-xL and BCL2 delayed serum-induced and Myc-induced, but not E2F-induced, cell cycle entry. The cyclin-dependent kinase inhibitor p27 was elevated during serum deprivation and cell cycle entry in BCL-x(L) or BCL2-expressing NIH3T3 cells and a Rat1MycER cell line. Activation of cyclin-dependent kinase 2 (cdk2) and cyclin-dependent kinase 4 (cdk4) were delayed during progression to S phase, while the induction of cyclin D1 protein, as well as the levels of cyclin E, cdk2, and cdk4 were unaltered by BCL-x(L) or BCL2. Inhibition of cyclin/cdk activities in BCL-x(L) or BCL2 expressing cells was associated with excess p27 in the cyclin/cdk complexes. Neither BCL-x(L) nor BCL2 delayed S phase entry in cells deficient in p27, thus p27 is required for the cell cycle function of BCL-x(L) and BCL2. The cell cycle effects of BCL-x(L) and BCL2 were more profound in Myc-induced than in serum-induced cell cycle entry. Our results suggest that one possible mechanism by which BCL-x(L) and BCL2 delay cell cycle entry may be the inhibition of Myc activity through the elevation of p27.     

Flavopiridol,a cyclin dependent kinase (CDK) inhibitor,induces apoptosis by regulating Bcl-x in oral cancer cells

Flavopiridol is a synthetic flavone that inhibits tumor growth by suppressing cyclin-dependent kinases (CDKs). We have investigated effects of flavopiridol in oral squamous cell carcinoma (OSCC). Flavopiridol was found to inhibit the growth of OSCC cells in a time- and dose-dependent manner. Induction of apoptosis was observed in all cells showing accumulated cells with sub-G(1) DNA contents, DNA fragmentations, and PARP cleavages. While Bcl-2 and Bax expression did not change, Bcl-x(L) was down regulated and Bcl-xs was up-regulated after being exposed to flavopiridol. Flavopiridol treatments also resulted in remarkable reductions of cyclin A, cyclin B, and cyclin D1 expressions. We also found that expression levels of CDK activation kinase and CDC25C were reduced, and p34 inactive form CDK2 were up-regulated. Our data indicate that flavopiridol has growth inhibition activities against OSCC. Flavopiridol not only inhibits CDKs directly, but it also inhibits the CDKs activation pathway and activates the Bcl-x apoptotic pathway.     

Adhesion-regulated G1 cell cycle arrest in epithelial cells requires the downregulation of c-Myc

Adhesion to the extracellular matrix is required for the expression and activation of the cyclin-cyclin-dependent kinase (CDK) complexes, and for G1 phase progression of non-transformed cells. However, in non-adherent cells no molecular mechanism has yet been proposed for the cell adhesion-dependent up-regulation of the p27 cyclin-dependent kinase inhibitor (CKI), and the associated inhibition of cyclin E-CDK2. We now show that in epithelial cells the expression of c-Myc is tightly regulated by cell-substrate adhesion. When deprived of adhesion, two independently derived mammary epithelial cell lines, 184A1N4 and MCF-10A, rapidly decrease their level of c-Myc mRNA and protein. This decrease in levels of c-Myc correlates with G1 phase arrest, as indicated by hypophosphorylation of pRb and inhibition of the activity of the cyclin E-CDK2 complex. In 184A1N4 cells, cell-substrate adhesion is required for the suppression of p27, and induction of cyclin E, E2F-1, but not cyclins D1 and D3. Enforced expression of c-Myc in non-adherent 184A1N4 and MCF-10A cells reverses the adhesion-dependent inhibition of cell cycle progression. Restoration of c-Myc in non-adherent cells induces the expression of E2F-1, and hyperphosphorylation of pRb in response to EGF treatment. In addition, expression of c-Myc results in the anchorage-independent activation of the CDK2 complex, the associated upregulation of cyclin E, and the destabilization and degradation of p27 by the ubiquitin-proteasome pathway. Our study thus suggests that c-Myc is the link between cell adhesion and the regulation of p27 and cyclin E-CDK2. Furthermore, we describe a role for c-Myc in adhesion-mediated regulation of E2F-1.     

Cell type-specific responses of human cells to inhibition of replication licensing

Replication origins are 'licensed' for a single initiation event by loading Mcm2-7 complexes during late mitosis and G1. Licensing is blocked at other cell cycle stages by the activity of cyclin-dependent kinases and a small protein called geminin. Here, we describe the effects of over-expressing a non-degradable form of geminin in various cell lines. Geminin expression reduced the quantity of Mcm2 bound to chromatin and blocked cell proliferation. U2OS (p53+/Rb+) cells showed an early S phase arrest with high cyclin E and undetectable cyclin A levels, consistent with the activation of an intra-S checkpoint. Saos2 (p53-/Rb-) cells showed an accumulation of cells in late S and G2/M with approximately normal levels of cyclin A, consistent with loss of this intra-S phase checkpoint. Geminin also induced apoptosis in both these cell lines. In contrast, IMR90 primary fibroblasts over-expressing geminin arrested in G1 with reduced cyclin E levels and no detectable apoptosis. A 'licensing checkpoint' may therefore act in primary cells to prevent passage into S phase in the absence of sufficient origin licensing. These results suggest that inhibition of the licensing system may cause cancer-specific cell killing and therefore represent a novel anti-cancer target.