Constitutive hyperexpression of p21(WAF1) in human U266 myeloma cells blocks the lethal signaling induced by oxidative stress but not by Fas.

p21(WAF1/CIP1) is expressed in a majority of myeloma cells. To investigate the role of p21 in myeloma cell death, comparative studies using two clones of myeloma cells, Fas-sensitive RPMI8226, and Fas-resistant U266 were performed. These latter cells were also resistant to H(2)O(2) up to 100 microM, whereas the former cells were not. SAPK/JNK was found to be a common mediator of RPMI8226 cell death induced by both H(2)O(2) and Fas. Interestingly, the concentrations of H(2)O(2) which activated SAPK/JNK in RPMI8226 cells failed to do so in U266 cells. In contrast, Fas ligation activated SAPK/JNK in both cells almost equally. U266 cells expressed p21 to levels much higher than in RPMI8226 cells. When the p21 levels were reduced using its antisense, H(2)O(2) killed U266 cells by activating SAPK/JNK. However, the reduction in p21 levels neither rendered the U266 cells susceptible to Fas-mediated cell death, nor significantly influenced Fas-induced SAPK/JNK activation. Overall, our data suggest that the p21 hyperexpression in U266 cells blocks the lethal signaling that is induced by H(2)O(2), but not by Fas. The mechanism whereby U266 cells resist Fas-mediated cell death is discussed.     

Gamma delta T cells kill myeloma cells by sensing mevalonate metabolites and ICAM-1 molecules on cell surface

We evaluated the mechanism of recognition of myeloma cells by γδT cells. The expanded γδT cells killed RPMI8226 and U266 myeloma cells in a γδT-cell dose-dependent manner. Pretreatment of myeloma cells with zoledronic acid or mevastatin showed that γδT cells kill myeloma cells by recognizing the mevalonate metabolites. The expression level of intercellular cell adhesion molecule-1 (ICAM-1) on myeloma cells correlates with the cytotoxicity by γδT cells. Pretreatment of RPMI8226 and U266 with an anti-ICAM-1 monoclonal antibody inhibited their cytolysis. Moreover, AMO-1 myeloma cells transfected with of human ICAM-1 cDNA were susceptible to γδT cells compared to parental AMO-1 cells. In conclusion, γδT cells recognize the mevalonate metabolites and ICAM-1 on myeloma cells.     

FLIP is Constitutively Hyperexpressed in Fas-resistant U266 Myeloma Cells, but Is Not Induced by IL-6 in Fas-sensitive RPMI8226 Cells

Despite the expression of Fas, some clones of myeloma cells are resistant to Fas-mediated apoptosis. To define a cellular factor involved in the resistance, we performed a comparative study using two clones of myeloma cells, RPMI8226 and U266. These cells were reported to express cell surface Fas at similar levels, but only RPMI8226 cells lost their viability upon anti-Fas treatment. The resistance of U266 cells to anti-Fas did not appear to reflect dysregulation of Bcl-2, Bcl-X^L, and Bax, because these proteins were expressed in both RPMI8226 and U266 cells to similar levels. Moreover, levels of those proteins were not significantly altered by treating RPMI8226 cells with IL-6, a cytokine which suppresses the Fas-mediated death of RPMI8226 cells. Interestingly, mRNA levels of FLIP^L, an endogenous inhibitor of Fas signaling, were constitutively elevated in U266 cells. Consistent with this observation, U266 cells expressed both FLIP^L protein and its truncated 43 kDa product which is seen in FLIP^L-overexpressing cells. The truncated form of FLIP^L protein was not detected in RPMI8226. Moreover, the levels of truncated FLIP^L in U266 cells were considerably higher than those of pro-FLIP^L in RPMI8226. The overall data indicate that FLIP^L is constitutively hyperexpressed in U266 cells. However, IL-6 failed to enhance the protein levels of FLIP molecules in either of the tested cells. It appears, therefore, that FLIP^L plays a role in the intrinsic resistance of U266 cells to the apoptotic action of Fas, but is not involved in the protective action of IL-6.     

Extracellular calcium sensing in rat aortic vascular smooth muscle cells

Extracellular calcium (Ca(2+)(o)) can act as a first messenger in many cell types through a G protein-coupled receptor, calcium-sensing receptor (CaR). It is still debated whether the CaR is expressed in vascular smooth muscle cells (VSMCs). Here, we report the expression of CaR mRNA and protein in rat aortic VSMCs and show that Ca(2+)(o) stimulates proliferation of the cells. The effects of Ca(2+)(o) were attenuated by pre-treatment with MAPK kinase 1 (MEK1) inhibitor, as well as an allosteric modulator, NPS 2390. Furthermore, stimulation of the VSMCs with Ca(2+)(o)-induced phosphorylation of ERK1/2, but surprisingly did not cause inositol phosphate accumulation. We were not able to conclusively state that the CaR mediates Ca(2+)(o)-induced cell proliferation. Rather, an additional calcium-sensing mechanism may exist. Our findings may be of importance with regard to atherosclerosis, an inflammatory disease characterized by abnormal proliferation of VSMCs and high local levels of calcium.     

Inactivation of the calcium sensing receptor inhibits E-cadherin-mediated cell-cell adhesion and calcium-induced differentiation in human epidermal keratinocytes

Extracellular Ca(2+) (Ca(2+)(o)) is a critical regulator that promotes differentiation in epidermal keratinocytes. The calcium sensing receptor (CaR) is essential for mediating Ca(2+) signaling during Ca(2+)(o)-induced differentiation. Inactivation of the endogenous CaR-encoding gene CASR by adenoviral expression of a CaR antisense cDNA inhibited the Ca(2+)(o)-induced increase in intracellular free calcium (Ca(2+)(i)) and expression of terminal differentiation genes, while promoting apoptosis. Ca(2+)(o) also instigates E-cadherin-mediated cell-cell adhesion, which plays a critical role in orchestrating cellular signals mediating cell survival and differentiation. Raising Ca(2+)(o) concentration ([Ca(2+)](o)) from 0.03 to 2 mm rapidly induced the co-localization of alpha-, beta-, and p120-catenin with E-cadherin in the intercellular adherens junctions (AJs). To assess whether CaR is required for the Ca(2+)(o)-induced activation of E-cadherin signaling, we examined the impact of CaR inactivation on AJ formation. Decreased CaR expression suppressed the Ca(2+)(o)-induced AJ formation, membrane translocation, and the complex formation of E-cadherin, catenins, and the phosphatidylinositol 3-kinase (PI3K), although the expression of these proteins was not affected. The assembly of the E-cadherin-catenin-PI3K complex was sensitive to the pharmacologic inhibition of Src family tyrosine kinases but was not affected by inhibition of Ca(2+)(o)-induced rise in Ca(2+)(i). Inhibition of CaR expression blocked the Ca(2+)(o)-induced tyrosine phosphorylation of beta-, gamma-, and p120-catenin, PI3K, and the tyrosine kinase Fyn and the association of Fyn with E-cadherin and PI3K. Our results indicate that the CaR regulates cell survival and Ca(2+)(o)-induced differentiation in keratinocytes at least in part by activating the E-cadherin/PI3K pathway through a Src family tyrosine kinase-mediated signaling.     

Cellular "sensing" of extracellular calcium (Ca(2+)(o)): emerging roles in regulating diverse physiological functions

The extracellular Ca(2+) (Ca(2+)(o))-sensing receptor (CaR) critically influences Ca(2+)(o) homeostasis by regulating parathyroid hormone (PTH) secretion and renal Ca(2+) handling. Moreover, its expression in intestinal and bone cells suggests roles in all of the organs involved in maintaining systemic Ca(2+)(o) homeostasis. This G-protein coupled receptor is also expressed in a wide variety of additional cells throughout the body. While our understanding of its role(s) outside of the system governing Ca(2+)(o) metabolism remains rudimentary, the CaR will probably emerge as a versatile regulator of diverse cellular functions, including proliferation, differentiation, apoptosis, gene expression and maintenance of membrane potential. Finally, the recently developed, "calcimimetic" CaR activators, exemplified by a NPS R-467 and NPS R-568, provide novel approaches to treating diseases that previously had no effective medical therapies: topic likewise covered in this review.     

Karyotypic variability of human multiple myeloma cell lines

Cytogenetic analysis of human multiple myeloma (MM) cell lines L363, Karpas 707, RPMI 8226, and U-266 was carried out. During long-term existence in vitro, the number of chromosomes in the cell lines was shown to be preserved at the near-diploid level (L363, Karpas 707, U-266) or to increase up to the hypotriploid level (RPMI 8226). There were complexly rearranged karyotypes with abnormalities of chromosomes of all pairs in all cell lines; however, no identical chromosomal translocations have been revealed. Loci of chromosomes involved in structural rearrangements in these cell lines often coincided with sites of DNA copy number imbalances characteristic for MM in vivo. Distinct types of the karyotypic structure of cell populations differing in the combination of cells with the main and additional structural variants of karyotype and of cells with nonclonal chromosome rearrangements were found in MM cell lines. In general, the karyotypic variability of the MM cell lines corresponds to the dynamics of karyotype of myeloma cells in vivo and, hence, has a tumor-specific character.     

Biochemistry, physiology and pathophysiology of the extracellular calcium-sensing receptor

Calcium (Ca(2+)) has long been recognized as a physiologically indispensable ion owing to its numerous intra- and extracellular roles. More recently, it has become apparent that extracellular calcium (Ca(2+)(o)) also serves as an extracellular first messenger following the cloning of a Ca(2+)(o)-sensing receptor (CaR) that belongs to the superfamily of G protein-coupled receptors (GPCR). The CaR probably functions as a dimer in performing its central role of "sensing" minute alterations in Ca(2+)(o) and adjusting the secretion of parathyroid hormone (PTH) so as to normalize Ca(2+)(o) through the actions of PTH on the effector elements of the mineral ion homeostatic system (e.g., kidney, bone and intestine). Several inherited human conditions are caused by inactivating or activating mutations of this receptor, and mice have been generated with targeted disruption of the CaR gene. Characteristic changes in the functions of parathyroid and kidney in patients with these conditions and in CaR-deficient mice have proven the physiological importance of the CaR in mineral ion homeostasis. An accumulating body of evidence, however, suggests that the CaR also plays numerous roles outside the realm of systemic mineral ion homeostasis. The receptor regulates processes such as cellular proliferation and differentiation, secretion, membrane polarization and apoptosis in a variety of tissues/cells. Finally, the availability of specific "calcimimetic", allosteric CaR activators - which are currently in clinical trials - will probably have therapeutic implications for diseases caused by malfunction of the CaR in tissues not only within, but also outside, the mineral ion homeostatic system.     

Xanthohumol exhibits anti-myeloma activity in vitro through inhibition of cell proliferation,induction of apoptosis via the ERK and JNK-dependent mechanism,and suppression of sIL-6R and VEGF production

BackgroundXanthohumol (XN, a hop-derived prenylflavonoid) was found to exert anticancer effects on various cancer types. However, the mechanisms by which XN affects the survival of multiple myeloma cells (MM) are little known. Therefore, our study was undertaken to address this issue.MethodsAnti-proliferative activity of XN towards two phenotypically distinct MM cell lines U266 and RPMI8226 was evaluated with the MTT and BrdU assays. Cytotoxicity was determined with the LDH method, whereas apoptosis was assessed by flow cytometry and fluorescence staining. The expression of cell cycle- and apoptosis-related proteins and the activation status of signaling pathways were estimated by immunoblotting and ELISA assays.ResultsXN reduced the viability of RPMI8226 cells more potently than in U266 cells. It blocked cell cycle progression through downregulation of cyclin D1 and increased p21 expression. The marked apoptosis induction in the XN-treated RPMI8226 cells was related to initiation of mitochondrial and extrinsic pathways, as indicated by the altered p53, Bax, and Bcl-2 protein expression, cleavage of procaspase 8 and 9, and elevated caspase-3 activity. The apoptotic process was probably mediated via ROS overproduction and MAPK (ERK and JNK) activation as N-acetylcysteine, or specific inhibitors of these kinases prevented the XN-induced caspase-3 activity and, hence, apoptosis. Moreover, XN decreased sIL-6R and VEGF production in the studied cells.ConclusionsERK and JNK signaling pathways are involved in XN-induced cytotoxicity against MM cells.General Significance: The advanced understanding of the molecular mechanisms of XN action can be useful in developing therapeutic strategies to treat multiple myeloma.     

Allosteric modulation of the calcium-sensing receptor by gamma-glutamyl peptides: inhibition of PTH secretion, suppression of intracellular cAMP levels, and a common mechanism of action with L-amino acids

γ-Glutamyl peptides were identified previously as novel positive allosteric modulators of Ca(2+)(o)-dependent intracellular Ca(2+) mobilization in HEK-293 cells that bind in the calcium-sensing receptor VFT domain. In the current study, we investigated whether γ-glutamyl-tripeptides including γ-Glu-Cys-Gly (glutathione) and its analogs S-methylglutathione and S-propylglutathione, or dipeptides including γ-Glu-Ala and γ-Glu-Cys are positive allosteric modulators of Ca(2+)(o)-dependent Ca(2+)(i) mobilization and PTH secretion from normal human parathyroid cells as well as Ca(2+)(o)-dependent suppression of intracellular cAMP levels in calcium-sensing receptor (CaR)-expressing HEK-293 cells. In addition, we compared the effects of the potent γ-glutamyl peptide S-methylglutathione, and the amino acid L-Phe on HEK-293 cells that stably expressed either the wild-type CaR or the double mutant T145A/S170T, which exhibits selectively impaired responses to L-amino acids. We find that γ-glutamyl peptides are potent positive allosteric modulators of the CaR that promote Ca(2+)(o)-dependent Ca(2+)(i) mobilization, suppress intracellular cAMP levels and inhibit PTH secretion from normal human parathyroid cells. Furthermore, we find that the double mutant T145A/S170T exhibits markedly impaired Ca(2+)(i) mobilization and cAMP suppression responses to S-methylglutathione as well as L-Phe indicating that γ-glutamyl peptides and L-amino acids activate the CaR via a common mechanism.     

In vitro sensitization of human lymphocytes to a myeloma cell-related antigen

Peripheral blood lymphocytes from normal human donors were cocultivated with cells from two established human multiple myeloma cell lines, RPMI 8226 and K-737, and with lymphoblastoid cells from a third B cell line, RAMM. After a comparison of three methods of lymphocyte sensitization, a 6-day incubation protocol with equal numbers of normal lymphocytes and mitomycin C-treated tumor cells was selected. Cells from the RPMI 8226 myeloma line stimulated the differentiation of lymphocytes into cytotoxic effector cells as measured by ⁵¹Cr release from labeled target cells. The RPMI 8226-sensitized lymphocytes were cytotoxic for myeloma cells (RPMI 8226 and K-737) and for lymphoblastoid cells (RAMM) but not for cells from human lung tumor lines (A549, A427, MB9812), a breast carcinoma line (ALAB), a normal diploid fibroblast line (HSBP), or normal lymphocytes.     

Activation of p42/44 and p38 mitogen-activated protein kinases by extracellular calcium-sensing receptor agonists induces mitogenic responses in the mouse osteoblastic MC3T3-E1 cell line

Recently, substantial evidence has accumulated that the G-protein-coupled, extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) is expressed in bone marrow-derived cells, including osteoblasts, stromal cells, monocytes-macrophages, and osteoclast precursor cells. Our previous studies have shown that the mouse osteoblastic MC3T3-E1 cell line also expresses the CaR and exhibits mitogenic responses when exposed to various CaR agonists. In this study, in order to understand the signaling pathway(s) mediating this response, we studied the effects of CaR agonists on the phosphorylation of p42/44 mitogen-activated protein kinase (MAPK) (Erk1/2), p38 MAPK, and c-Jun N-terminal kinase (JNK) in MC3T3-E1 cells. Raising the level of Ca(2+)(o) (4.5 mM) or addition of the polycationic CaR agonists, gadolinium (Gd(3+)) (25 microM), neomycin (300 microM) or spermine (1 mM), each stimulated phosphorylation of both p42/44 and p38 MAPKs, but not JNK, as assessed using phospho-specific antibodies to the respective MAPKs. Furthermore, phosphorylation of p42/44 and p38 MAPK were markedly inhibited by their selective and potent inhibitors, PD98059 (50 microM) and SB203580 (10 microM), respectively. Finally, the two inhibitors suppressed [(3)H]thymidine incorporation into DNA in MC3T3-E1 cells at a normal level of Ca(2+)(o) (1.8 mM) as well as when stimulated by high (4.5 mM) Ca(2+)(o), Gd(3+), or neomycin. Thus, in mouse osteoblastic MC3T3-E1 cells, both the p42/44 and p38 MAPK cascades play pivotal roles in CaR-stimulated mitogenic responses.     

Impaired NHEJ function in multiple myeloma

Multiple myeloma (MM) is characterized by multiple chromosomal aberrations. To assess the contribution of DNA repair to this phenotype, ionizing radiation was used to induce DNA double strand breaks in three MM cell lines. Clonogenic survival assays showed U266 (SF4 = 15.3 + 6.4%) and RPMI 8226 (SF4 = 12.6.0 + 1.7%) were radiation sensitive while OPM2 was resistant (SF4 = 78.9 + 4.1%). Addition of the DNA-PK inhibitor NU7026 showed the expected suppression in radiation survival in OPM2 but increased survival in both radiation sensitive cell lines. To examine non-homologous end joining (NHEJ) repair in these lines, the ability of protein extracts to support in vitro DNA repair was measured. Among the three MM cell lines analyzed, RPMI 8226 demonstrated impaired blunt ended DNA ligation using a ligation-mediated PCR technique. In a bacterial based functional assay to rejoin a DNA break within the β-galactosidase gene, RPMI 8226 demonstrated a 4-fold reduction in rejoining fidelity compared to U266, with OPM2 showing an intermediate capacity. Ionizing radiation induced a robust γ-H2AX response in OPM2 but only a modest increase in each radiation sensitive cell line perhaps related to the high level of γ-H2AX in freshly plated cells. Examination of γ-H2AX foci in RPMI 8226 cells confirmed data from Western blots where a significant number of foci were present in freshly plated untreated cells which diminished over 24 h of culture. Based on the clonogenic survival and functional repair assays, all three cell lines exhibited corrupt NHEJ repair. We conclude that suppression of aberrant NHEJ function using the DNA-PK inhibitor NU7026 may facilitate access of DNA ends to an intact homologous recombination repair pathway, paradoxically increasing survival after irradiation. These data provide insight into the deregulation of DNA repair at the site of DNA breaks in MM that may underpin the characteristic genomic instability of this disease.     

Three adjacent serines in the extracellular domains of the CaR are required for L-amino acid-mediated potentiation of receptor function

The extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) is a key player in Ca(2+)(o) homeostasis. The activity of CaR can be potentiated by various l-amino acids. In this study, we examined whether conserved amino acid residues involved in the binding of glutamate to metabotropic glutamate receptors (mGluRs) also participate in the potentiation of the activity of CaR by l-phenylalanine. Ser-170 corresponding to Thr-188 in rat mGluR1a appears to be important for the modulating actions of phenylalanine. In the presence of phenylalanine, a mutant CaR with a single mutation S170A showed no significant decrease in its EC(50) for stimulation by Ca(2+)(o) and a modest increase in its maximal activity. In addition, mutating Ser-169 and Ser-171 together with Ser-170 yielded a more complete block of the phenylalanine modulation than did the single mutation. The presence of the triple mutation, S169A/S170A/S171A, also eliminated phenylalanine potentiation of the activities of heterodimeric receptors in which one of the monomeric receptors had intact triple serines (A877Stop). The putative amino acid binding site of the CaR is probably close to or structurally dependent on the Ca(2+)(o) binding sites of the receptor, because mutant CaRs with mutations in the putative amino acid binding site exhibited severely reduced responses to Ca(2+)(o).     

Molecular mechanisms underlying antitumor activity of camel whey protein against multiple myeloma cells

Treating drug-resistant cancer cells is a clinical challenge and it is also vital to screen for new cancer drugs. Multiple myeloma (MM) is a plasma cell clonal cancer that, despite many experimental therapeutics, remains incurable. In this study, two MM cell line lines U266 and RPMI 8226 were used to determine the impact of camel whey protein (CWP). The CWP IC₅₀ was calculated by MTT examination, while the flow cytometry analysis was used to investigate the chemotaxis responses of MM cells in relation to CXCL12 and the pro-apoptotic effect of CHP. MM cells were treated with CWP and Western blot analysis was used to determine the underlying molecular mechanisms. Dose and time based on the impact of CWP on the cell viability of MM cells with IC₅₀ of 50 μg/ml, without affecting the viability of normal healthy PBMCs. CWP reduced chemotaxis of MM cells significantly from the CXC chemokine ligand 12 (CXCL12). Using Western blot analysis, we found that CWP decreased the activation of AKT, mTOR, PLCβ3, NFαB and ERK, which was mechanistically mediated by CXCL12/CXCR4. In both U266 and RPMI 8226, CWP induced apoptosis by upregulating cytochrome C expression. In addition, CWP mediated the growth arrest of MM cells by robustly decreasing the expression of the anti-apoptotic Bcl-2 family members Bcl-2, Bcl-XL and Mcl-1. Conversely, the expression of pro-apoptotic Bcl-2 family members Bak, Bax and Bim was increased after treatment with CWP. Our data indicates CWP's therapeutic potential for MM cells.     

Calcium sensing receptor activation by a calcimimetic suggests a link between cooperativity and intracellular calcium oscillations

Activation of the calcium sensing receptor (CaR) by small increments in extracellular calcium (Ca(2+)(e)) induces intracellular calcium (Ca(2+)(i)) oscillations that are dependent on thapsigargin-sensitive intracellular calcium stores. Phenylalkylamines such as NPS R-568 are allosteric modulators (calcimimetics) that activate CaR by increasing the apparent affinity of the receptor for calcium. We determined, by fluorescence imaging with fura-2, whether the calcimimetic NPS R-568 could activate Ca(2+)(i) oscillations in HEK-293 cells expressing human CaR. NPS R-568 was more potent than Ca(2+)(e) at eliciting Ca(2+)(i) oscillations, particularly at low [Ca(2+)](e) (as low as 0.1 mm). The oscillation frequencies elicited by NPS R-568 varied over a 2-fold range from peak to peak intervals of 60-70 to 30-45 s, depending upon the concentrations of both Ca(2+)(e) and NPS R-568. Finally, NPS R-568 induced sustained (>15 min after drug removal) Ca(2+)(i) oscillations, suggesting slow release of the drug from its binding site. We exploited the potency of NPS R-568 for eliciting Ca(2+)(i) oscillations for structural studies. Truncation of the CaR carboxyl terminus from 1077 to 886 amino acids had no effect on the ability of Ca(2+) or NPS R-568 to induce Ca(2+)(i) oscillations, but further truncation (to 868 amino acids) eliminated both highly cooperative Ca(2+)-dependent activation and regular Ca(2+)(i) oscillations. Alanine scanning within the amino acid sequence from Arg(873) to His(879) reveals a linkage between the cooperativity for Ca(2+)-dependent activation and establishment and maintenance of intracellular Ca(2+) oscillations. The amino acid residues critical to both functions of CaR may contribute to interactions with either G proteins or between CaR monomers within the functional dimer.     

Bone marrow stroma confers resistance to Apo2 ligand/TRAIL in multiple myeloma in part by regulating c-FLIP

Apo2 ligand (Apo2L)/TRAIL induces apoptosis of cancer cells that express the specific receptors while sparing normal cells. Because the tumor microenvironment protects myeloma from chemotherapy, we investigated whether hemopoietic stroma induces resistance to Apo2L/TRAIL apoptosis in this disease. Apo2L/TRAIL-induced death was diminished in myeloma cell lines (RPMI 8226, U266, and MM1s) directly adhered to a human immortalized HS5 stroma cell line but not adhered to fibronectin. In a Transwell assay, with myeloma in the upper well and HS5 cells in the lower well, Apo2L/TRAIL apoptosis was reduced when compared with cells exposed to medium in the lower well. Using HS5 and myeloma patients' stroma-conditioned medium, we determined that soluble factor(s) produced by stroma-myeloma interactions are responsible for a reversible Apo2/TRAIL apoptosis resistance. Soluble factor(s) attenuated procaspase-8, procaspase-3, and poly(ADP-ribose) polymerase cleavage and diminished mitochondrial membrane potential changes without affecting Bcl-2 family proteins and/or Apo2L/TRAIL receptors. Soluble factor(s) increased the baseline levels of the anti-apoptotic protein c-FLIP in all cell lines tested. Inhibition of c-FLIP by means of RNA interference increased Apo2/TRAIL sensitivity in RPMI 8226 cells. Unlike direct adhesion to fibronectin, soluble factor(s) have no impact on c-FLIP redistribution within cellular compartments. Cyclohexamide restored Apo2L/TRAIL sensitivity in association with down-regulation of c-FLIP, suggesting that c-FLIP synthesis, not intracellular traffic, is essential for soluble factor(s) to regulate c-FLIP. Additionally, IL-6 conferred resistance to Apo2L/TRAIL-mediated apoptosis in association with increased c-FLIP levels. In conclusion, the immune cytotoxic effect of Apo2L/TRAIL can be restored at least in part by c-FLIP pathway inhibitors.