Mutation rates and mechanisms of resistance to etoposide determined from fluctuation analysis.

BACKGROUND: The major known mechanisms of resistance to etoposide include altered expression of its target enzyme, topoisomerase II (Topo II), and the multidrug-resistant phenotypes encoded by the mdr1 and MRP (multidrug resistance-associated protein) genes. There is little information regarding the distribution, frequency, and origin of these mechanisms in cancer cells. PURPOSE: We performed fluctuation analysis experiments with the human sarcoma cell line, MES-SA, to assess 1) if selection or induction mechanisms are involved in resistance to etoposide, 2) mutation rates for cellular resistance to etoposide, and 3) the nature of the single-step selected surviving clones. METHODS: Three groups of 10 flasks were seeded with more than 2000 cells each and allowed to grow to near confluence (approximately 3 x 10(6) cells per flask). After reseeding, each group received etoposide for 1 week at a final concentration of 0.5 microM (group A), 1.0 microM (group B), and 5.0 microM (group C). Surviving colonies in each of the 30 populations were scored and individually harvested. RESULTS: Mutation rates were estimated at 2.9 x 10(-6) (group A), 5.7 x 10(-7) (group B), and 1.7 x 10(-7) (group C) per cell generation. Of 61 propagated colonies, four of 26 from group A, five of 19 from group B, and none of 16 from group C were stably resistant. Analysis of variance supported the hypothesis of spontaneous mutations rather than induction, conferring etoposide resistance in groups A and B. Five of the stably resistant clones were cross-resistant to doxorubicin. Analysis by polymerase chain reaction failed to detect the expression of the multidrug-resistant gene mdr1 messenger RNA (mRNA) in any of the clones. No increase in expression of the MRP gene was observed. However, a significant decrease in both Topo II alpha and II beta mRNA (30%-70%) was found in six of seven stably resistant and six of six unstably resistant mutants. CONCLUSIONS: Our study demonstrates that resistance to etoposide arises spontaneously, with most clones surviving either stochastically or through very labile mechanisms of resistance. The experimental design has derived a set of resistant mutants from a single-step selection. In those clones, decreased expression of Topo II is the predominant mechanism selected. IMPLICATIONS: These findings suggest that stable resistance to etoposide chemotherapy may be acquired by selection of spontaneously arising mutants rather than induction by drug exposure. The stably resistant clones may represent descendants from a single mutational event in each population.

Modulation of subcellular distribution of doxorubicin in multidrug-resistant P388/ADR mouse leukemia cells by the chemosensitizer ((2-isopropyl-1-(4-[3-N-methyl-N-(3,4-dimethoxy-beta- phenethyl)amino]propyloxy)-benzenesulfonyl))indolizine.

The impact of the novel chemosensitizer ((2-isopropyl-1-(4-[3-N-methyl-N-(3,4-dimethoxy-beta- phenethyl)amino]propyloxy)benzenesulfonyl))indolizine (SR33557) on the intracellular distribution of doxorubicin (DOX) within the multidrug-resistant murine P388/ADR leukemia cell line was studied by fluorescence microscopy. We found that under conditions which modulated multidrug-resistant (30 microM SR33557 for 1 h), P388/ADR cells presented an original sequestration of DOX in large intracellular vesicles, where SR33557 is itself sequestered, as seen by colocalization studies. Colocalization experiments with lysosomal and mitochondrial probes suggest that these vesicles are neither mitochondrial in nature nor functional lysosomes. To investigate the biochemical basis for this effect, we studied the impact of SR33557 on the sphingolipid metabolism of P388/ADR cells. We observed that although P388/ADR cells normally catabolized exogenous [3H]sphingomyelin, when pretreated with SR33557 they showed almost complete inhibition of sphingomyelin breakdown. Finally, in order to demonstrate that the inability of P388/ADR cells to degrade sphingomyelin in the presence of SR33557 (which is a potent inhibitor of acid lysosomal sphingomyelinase) leads to phospholipid accumulation, we performed electron microscopy where we observed laminated inclusions. These morphological modifications are similar to those observed in Niemann-Pick disease lymphoblastoid cell lines which are inherently deficient in acid sphingomyelinase activity. The observation that, in the absence of SR33557, these Niemann-Pick disease cell lines presented similar DOX sequestration to that of SR33557-treated P388/ADR cells strongly suggests that DOX accumulates in SR33557-induced myeloid bodies. The redistribution of DOX within these vesicles, perhaps by preventing its expulsion by P-glycoprotein, may be a key in discovering the mechanism of action of SR33557.

The protein kinase C activators phorbol esters and phosphatidylserine inhibit neutral sphingomyelinase activation, ceramide generation, and apoptosis triggered by daunorubicin.

To address the role of protein kinase C (PKC) in the regulation of ceramide production, we evaluated the impact of the PKC activators 12-O-tetradecanoylphorbol-13-acetate and phosphatidylserine on the apoptotic signaling pathway triggered by the chemotherapeutic drug daunorubicin. Treatment of U937 and HL-60 cells with 0.5-1 microM daunorubicin induced a greater than 30% activation of neutral sphingomyelinase activity within 4-10 min with concomitant sphingomyelin hydrolysis and ceramide generation. Activation of PKC by 12-O-tetradecanoylphorbol-13-acetate and phosphatidylserine inhibited daunorubicin-induced neutral sphingomyelinase activation, sphingomyelin hydrolysis, ceramide generation, and apoptosis. The apoptotic response could be restored by the addition of 25 microM cell-permeant C6-ceramide. In conclusion, PKC emerges as a potentially critical negative regulator of the anthracycline-activated sphingomyelin-ceramide apoptotic pathway.

Cell surface-directed interaction of anthracyclines leads to cytotoxicity and nuclear factor kappaB activation but not apoptosis signaling.

Anthracyclines are, above all, DNA intercalators, which induce genetic damage leading to cell death. However, increasing evidence firmly suggests that the underlying mechanism for anthracycline cytotoxicity is the induction of apoptosis through intracellular-mediated signaling pathways. Whether drug/DNA interaction is necessary for such apoptosis signaling is unknown. We investigated the cellular effects of the anthracyclines daunorubicin (DNR) and doxorubicin (DOX) using the myeloid leukemia cell line U937. By comparing free drug against agarose bead-immobilized drug iDNR and iDOX (which cannot accumulate within the cell), we observed that whereas both free and immobilized anthracyclines were cytotoxic, only the former induced apoptosis; the latter induced necrosis. Indeed, we did not observe ceramide generation, neutral sphingomyelinase activation, poly (ADP-ribose) polymerase cleavage, or other apoptotic events with iDNR or iDOX. However, both free and immobilized drug were similarly capable of triggering nuclear factor kappaB activation. These observations demonstrate that whereas activation of certain cellular signaling pathways can be achieved solely through membrane interaction, apoptosis signaling requires anthracycline internalization. These results also show that the initiation of cell survival pathways (illustrated by nuclear factor kappaB activation) is independent of intracellular drug/target interaction.

Enhancement of ricin A chain immunotoxin activity by perhexiline on established and fresh leukemic cells.

In the perspective of increasing the clinical potential of ricin A chain immunotoxins (RTA-ITs), perhexiline (Pex) and four structural analogues (Pex 2, Pex 3, Pex 7, and Pex 11) were evaluated for their ability to enhance RTA-IT activity in vitro. Only perhexiline significantly enhanced the cytotoxic activity of anti-CD5 RTA-ITs, T101 and T101-F(ab')2, on CEM III cell line (30- to 2000-fold), and of anti-HLA-DR RTA-IT, HNC-241, on both RAJI cell line (greater than 100-fold) and two immortalized cell lines originating from patients suffering from B-cell chronic lymphocytic leukemia, EHEB and FS2 D5 (10-fold). On 16 consecutive fresh B-cell chronic lymphocytic leukemia cell samples, significant T101-F(ab')2 RTA-IT and HNC-241 RTA-IT enhancement was observed with perhexiline which was comparable to that of NH4Cl and monensin. Perhexiline almost completely blocked RTA-IT intracellular degradation and profoundly modified its routing. These observations were linked to perhexiline-induced lipidosis via inhibition of sphingomyelinase activity. In conclusion, since the concentrations used are relevant with the pharmacokinetics of this agent, perhexiline appears to be a promising agent for in vivo enhancement of ricin A chain immunotoxins.

Prevalence of multidrug resistance related to activation of the mdr1 gene in human sarcoma mutants derived by single-step doxorubicin selection.

Fluctuation analysis experiments were performed in the human sarcoma cell line MES-SA to assess whether selection or induction mechanisms determine resistance to doxorubicin (DOX), mutation rates, and the nature of the surviving clones. Thirteen flasks were seeded with 2000 cells/flask and grown to confluent populations of approximately 3.3 x 10(6) cells. After reseeding in 96-well plates, each population was treated with 40 nM DOX for 2 weeks. Surviving colonies were scored and harvested. Clones were propagated and analyzed for drug resistance phenotype. Expression of the mdr1, mrp, and topoisomerase II alpha and II beta genes was analyzed by reverse transcription-polymerase chain reaction. Accumulation of the P-glycoprotein substrate rhodamine-123 was measured by flow cytometry, with and without the cyclosporin D analogue SDZ PSC 833. Cellular glutathione levels were measured by flow cytometry, and M(r) 110,000 vesicular protein (p110) expression was detected by immunohistochemistry. Analysis of variance supported the hypothesis of spontaneous mutations rather than induction conferring DOX resistance. At this stringent level (5-6 log cell killing) of drug exposure, the mutation rate was estimated at 1.8 x 10(-6) per cell generation. All 30 propagated clones demonstrated cross-resistance to vinblastine, etoposide, and paclitaxel (Taxol), but not to cisplatin or bleomycin. Increased mRNA levels of mdr1 were observed in all 27 clones tested, including at least 1 from each of the 13 populations. No alterations were found in expression or level of topoisomerase II alpha or II beta, mrp, glutathione, and p110. Expression of P-glycoprotein was confirmed by flow cytometry using the monoclonal antibody UIC2. In almost all tested clones, decreased intracellular rhodamine-123 accumulation was modulated by 2 microM SDZ PSC 833, and the vinblastine resistance in all examined clones was completely reversed by SDZ PSC 833 and verapamil. Our study demonstrates that survival of cells exposed to DOX in a single step occurs as a result of a stochastic process consistent with mutational events. Activation of the mdr1 gene is the predominant mechanism selected by DOX in these resistant clones.

In vitro and in vivo enhancement of ricin-A chain immunotoxin activity by novel indolizine calcium channel blockers: delayed intracellular degradation linked to lipidosis induction.

With regard to increasing the clinical potential of ricin A-chain immunotoxins (RTA-ITs), a novel class of calcium channel blockers, indolizines SR33557 [2-isopropyl-1-[4-(3-N-methyl-N-(3,4-dimethoxy-beta- phenethyl)amino)propyloxy)benzenesulfonyl))indolizine] and SR33287 [isopropyl-2-((1-butylamino-3-propyl)oxy-4-benzoyl)-3-indolizine], were evaluated for their ability to enhance RTA-IT activity in vitro and in vivo. Five microM SR33287 and 5 microM SR33557 were potent enhancers of both anti-Thy 1.2 AT15E RTA-IT (84- and 64-fold, respectively) on T2 cells and anti-CD5 T101 (622- and 538-fold) and T101 F(ab')2 RTA-IT (34- and 28-fold) on CEM III cells. This was superior to the effect achieved by both 10 microM verapamil and 10 mM NH4Cl, albeit slightly inferior to that of 50 nM monensin and 5 microM perhexiline. Murine T2 lymphoma cells bearing the Thy 1.2 antigen were injected i.v. in Thy 1.2 (-) BL. 1.1 mice (median survival time, 17.7 days). Intravenous treatment with 10 micrograms of AT15E RTA-IT prolonged the survival of mice (median survival time, 26.8 days). When 400 micrograms of SR33287 were coinjected i.v. with 10 micrograms of AT15E RTA-IT, mouse survival was further increased, with 5 of 6 mice surviving, disease free, over 42 days. SR33287 had a significant impact on the intracellular routing of 125I-AT15E RTA-IT, which induced a greater than 2-fold increase in intracellular intact AT15E RTA-IT at 90 min. This effect on RTA-IT half-life was distinctly different from that observed with either NH4Cl or monensin and may be linked to the inhibition of acid lysosomal sphingomyelinase by SR33287, leading to cellular lipidosis. In conclusion, indolizines appear to be promising agents not only for immunotoxin enhancement but also for increasing the activity of any number of targeted therapeutic agents where modifying either the intracellular routing or increasing the intracellular half-life of the ligand would be beneficial to its cytotoxic activity.

Signalling sphingomyelinases: which, where, how and why?

A major lipid signalling pathway in mammalian cells implicates the activation of sphingomyelinase (SMase), which upon cell stimulation hydrolyses the ubiquitous sphingophospholipid sphingomyelin to ceramide. This review summarizes our current knowledge on the nature and regulation of signalling SMase(s). Because of the controversy on the identity of this(these) phospholipase(s), the roles of various SMases in cell signalling are discussed. Special attention is also given to the subcellular site of action of signalling SMases and to the cellular factors that positively or negatively control their activity. These regulating agents include lipids (arachidonic acid, diacylglycerol and ceramide), kinases, proteases, glutathione and other proteins.

Inhibition of lysosomal acid sphingomyelinase by agents which reverse multidrug resistance.

An increasing body of evidence appears to implicate the lipid bilayer of multidrug resistant (MDR) cells with P-glycoprotein activity. Several cationic amphiphilic drugs (CADs) have been extensively described as modulators of MDR. These same agents are also known to (1) inhibit lysosomal acid sphingomyelinase (ASmase), a phospholipid degrading enzyme, and/or (2) induce phospholipidosis in animal tissues or cultured cell lines. In this report, we randomly selected 17 CADs and evaluated their potency in modulating MDR in the murine MDR P388/ADR leukemia cell line. We compared these results with their ability to inhibit ASmase and observed a significant dose-dependent linear relationship (95% central confidence interval), between ASmase inhibition and MDR reversal. This approach permitted us to identify three new modestly potent chemosensitizers: trimipramine, desipramine, and mianserine. Modulation of MDR was not cell line specific, since CADs at 10 microM increased doxorubicin (DOX) and vinblastine (VBL) (but not methotrexate, MTX) cytotoxicity in both P388/ADR and the human MDR cell lines MES-SA/Dx5 and K562/R7, but not in the parental drug-sensitive cells. Although all chemosensitizing CADs at 10 microM significantly increased Rhodamine-123 (Rho-123) accumulation in the human leukemia MDR cell line K562/R7 and most presented significant displacement of the photoaffinity labelling probe iodoarylazidoprazosin, no correlation between these observations and the ability of CADs to sensitize MDR cells to DOX and VBL was found. In conclusion, our study strongly suggests that the chemosensitizing potency of agents such as CADs may be due to a dual mechanism of action: direct antagonism of P-gp activity and indirect modulation of P-gp activity through the disruption of cellular lipid metabolism.

Lack of ceramide generation in TF-1 human myeloid leukemic cells resistant to ionizing radiation.

The mechanism(s) by which ionizing radiation (IR) induces cell death is of fundamental importance in understanding cell sensitivity and resistance. Here we evaluated the response to IR of two subclones of the autonomous human erythromyeloblastic cell line TF-1: TF-1-34 (which expresses CD34) and TF-1-33 (which lacks CD34). In clonogenic assays, TF-1-34 cells were found to be relatively less sensitive to IR compared to TF-1-33 cells based on the D0 determination (3.01 vs 1.56 Gy). Furthermore, after IR at 12 Gy, TF-1-33 cell viability decreased by approximately 50% within 24 h, whereas TF-1-34 cell growth was unaffected during this time. Gradual loss of TF-1-34 cell viability was observed only after 48 h. Morphological and molecular analysis revealed that TF-1-33 cells died of apoptosis, and TF-1-34 cells of delayed reproductive cell death. While IR produced comparable amounts of DNA double strand breaks (DSB) in both cell lines, TF-1-34 retained DSB much longer than TF-1-33 suggesting that radioresistance and the defective apoptotic response of TF-1-34 cells was not related to a higher DNA repair capacity. However, the lack of an apoptotic response in TF-1-34 was correlated to the absence of a sphingomyelin (SM)-ceramide (CER) signaling pathway. Indeed, IR triggered in TF-1-33 cells but not in TF-1-34, SM hydrolysis (25% at 12 Gy) and CER generation (>50%) through the activation of neutral but not acid sphingomyelinase. Synthetic cell permeate CER induced apoptosis in both TF-1-33 and TF-1-34 cells. This study indicates that alterations of the SM-CER signaling pathway can significantly influence the cell death process as well as the survival of acute myeloid leukemia cells after IR exposure.

Effect of 5637-conditioned medium and recombinant cytokines on P-glycoprotein expression in a human GM-CSF-dependent leukemic myeloid cell line.

This study was aimed at evaluating the influence of 5637-conditioned medium (5637-CM) and human recombinant cytokines on both expression and function of P-glycoprotein (P-gp) in TF-1, a GM-CSF/IL-3-dependent acute myeloid leukemia cell line which constitutively expresses functional P-gp. P-gp expression was measured by flow cytometry using MRK16 monoclonal antibody. P-gp function was measured by rhodamine 123 (Rh 123) efflux kinetics. When TF-1 cells were cultured with 5637-CM (50% v/v), both P-gp expression and P-gp efflux capacity were increased in a time-dependent manner with a 4-fold increase in P-gp expression level at day 6 whereas TF-1 cell differentiation status remained unchanged as assessed by morphological studies, phenotypical and cytochemistry analysis. Recombinant cytokines including GM-CSF, G-CSF, IL-1 beta, IL-6, stem cell factor, LIF, erythropoietin, and IL-3 had no effect on P-gp expression whereas TNF alpha induced dose- and time-dependent P-gp and mdr-1 gene overexpression. However, TNF alpha-induced P-gp overexpression had no influence on P-gp efflux capacity. Furthermore, when TF-1 cells were exposed to IL-3 for periods longer than 1 month, we found that P-gp efflux capacity was increased as compared to cells cultured with GM-CSF whereas P-gp expression was unchanged. Both TNF alpha and IL-3 did not induce TF-1 differentiation. Collectively, these results suggest that cytokines may influence both expression and function of P-gp in TF-1 cells without interfering with their differentiation status. In contrast to cytokines, phorbol esters enhanced expression and efflux capacity of P-gp in parallel with TF-1 cell monocytic differentiation. Finally, our study suggests that paracrine and/or autocrine secretion of cytokines may interfere with P-gp activity in some acute myeloid leukemia cells.

Lack of correlation between expression and function of P-glycoprotein in acute myeloid leukemia cell lines.

In a panel of acute myeloblastic leukemia (AML) cell lines, representative of distinct differentiation stages, we investigated the possible correlation between drug-resistance and both expression and function of the multidrug resistance (MDR)-related P-glycoprotein (P-gp). The AML cell lines were KG1a, KG1, TF1, HEL, ML1, and two non drug-selected P-gp positive subclones originating from HL-60 (HL-60JD) and U937 (U937AQ). All these cells overexpressed the mdr1 gene (analyzed by RT-PCR) and displayed variable levels of P-gp expression. Flow cytometric semi-quantitative evaluation of P-gp with two P-gp specific monoclonal antibodies (MRK16 and UIC2) showed the following P-gp expression hierarchy: TF1 < KG1a < HEL < KG1 < HL-60JD < ML1 < U937AQ; the latter expressing 13 times more P-gp than TF1. When P-gp function was assessed by Rhodamine 123 (Rh123) efflux kinetics, we found that only KG1a and KG1 cells, which have an early (immature) CD34+ CD33- CD38- phenotype, and to a lesser extent TF1, with an intermediate (CD34+ CD33+ CD38+) phenotype, displayed significant P-gp activity which could be inhibited by both verapamil and SDZ PSC 833. In contrast, the other more mature CD33+ CD34- AML cell lines presented no Rh123 efflux capacity although they expressed higher P-gp levels. Daunorubicin (DNR) accumulation studies showed that inhibitors of P-gp increased DNR accumulation only in the immature AML cells whereas they had no impact on the mature AML cell lines. MTT drug cytotoxicity assay confirmed that the immature AML cells were 10-15-fold more resistant to DNR than the mature AML cells. Although P-gp inhibitors were able to increase the cytotoxicity of DNR in AML cells which displayed functional P-gp, they could not increase DNR cytotoxicity to levels comparable to that of the CD34- CD33+ cells, suggesting that DNR resistance of immature AML cells may not solely be related to P-gp. With drug-selection, AML subclones displayed higher levels of P-gp expression and higher extruding capacities, and therefore chemoresistance, and this independently of their initial differentiation phenotype. Finally, this study provides evidence for a lack of correlation between expression and function of P-gp in AML cells; this relationship being dependent upon leukemic cell differentiation in unselected myeloid leukemic cells.

Antisense inhibition of myeloperoxidase increases the sensitivity of the HL-60 cell line to vincristine.

Myeloperoxidase (MPO), a heme-peroxidase found in the HL-60 myeloblastic cell line, is involved in vincristine (VCR) metabolism and the inactivation of this drug. We have examined whether decreased MPO activity correlated with increased sensitivity to VCR toxicity in myeloid leukemia cells. We have used MPO antisense RNA to reduce 60% of the MPO activity in the HL-60 cells. The MPO-deficient HL-60 cell line, C15, was significantly more sensitive to VCR than the parental MPO-positive cell line. Both cell lines were negative for P170-glycoprotein expression. Conversely, an MPO-positive C15 subclone was more resistant to VCR than the MPO-deficient C15 cell line. No significant differences in cytotoxic effects were observed between MPO-positive and MPO-deficient cells, following treatment with either daunorubicin or actinomycin D, two multidrug resistance-related drugs. These results strongly support an important role for MPO in VCR resistance in HL-60 cells. Antisense manipulation of the MPO content of myeloid cells could be of potential interest in leukemia treatment.

Effects of H-7 and staurosporine on proliferation and self-renewal of acute myeloid leukemia progenitors.

In this study, we compared the impact of two protein kinase (PK) inhibitors, H-7 and staurosporine, on the normal myeloid progenitors (CFU-GM) and acute myeloid leukemia progenitors (AML-CFU) proliferation measured by in vitro clonogenic assay. H-7 and staurosporine displayed a biphasic dose-effect on both CFU-GM and AML-CFU recovery. At the lowest concentration range (0.1 microM to 20 microM for H-7 and 0.1 nM to 1 nM for staurosporine), we observed growth stimulation whereas higher concentrations induced dose-dependent growth inhibition. Moreover, AML-CFU proved to be significantly more sensitive to the inhibitory effect of both H-7 and staurosporine than CFU-GM (3.16- and 2.12-fold, respectively). These results were further confirmed with comparable murine cell line models (FDC-P1, a hematopoietic cell line generated from normal bone marrow and WEHI, a myelomonocytic leukemia cell line). Furthermore, we report that both H-7 and staurosporine present similar inhibitory effects on proliferation (PE1) as on self-renewal (PEs) of AML-CFU. In an attempt to understand more fully the mechanism of action of H-7 and staurosporine, we investigated their impact (when used at their D50) on the human myelogenous leukemia cell line, K562. H-7 and staurosporine induced a transient decrease of cell growth, between 0 and 24 hours, and produced a transient blockade of K562 cells in the S-phase, either 24 or 48 hours after the addition of staurosporine and H-7, respectively.

Doxorubicin induces slow ceramide accumulation and late apoptosis in cultured adult rat ventricular myocytes.

OBJECTIVES: Anthracyclines cause apoptotic death in many cell types through activation of the ceramide pathway. We tested the hypothesis that doxorubicin induces cardiac myocyte apoptosis through ceramide generation. METHODS: Adult rat ventricular myocytes were grown in the presence of 10% fetal calf serum, and exposed to 0.5 microM doxorubicin (Dox) for 1 h on the day of cell isolation (day 0). We used the membrane-permeant ceramide analog C2-ceramide (C2-cer) to mimic the effects of endogenous ceramide and PDMP to induce endogenous ceramide accumulation. Apoptosis was assessed upon morphological criteria and DNA fragmentation by the TUNEL method and agarose gel electrophoresis. Ceramide concentration was assessed using the DAG kinase assay. RESULTS: Myocyte exposure to Dox was associated with cellular and nuclear alterations typical of apoptosis on day 7 but not on day 3. At day 7, the percentage of TUNEL-positive myocytes was markedly increased in Dox-treated cultures compared to control (Cl) cultures (82 +/- 3 vs. 12 +/- 1%, n = 7; p < 0.001); internucleosomal DNA fragmentation was confirmed by the observation of DNA ladders. These alterations were associated with an increase in the intracellular ceramide concentration (1715 +/- 243 vs. 785 +/- 99 pmol/mg prot, n = 5; p < 0.01), a phenomenon also detected on day 3 (731 +/- 59 vs. 259 +/- 37 pmol/mg prot, n = 5; p < 0.001). Incubation of myocytes at day 0 with 50 microM C2-cer induced rapid cell shrinkage and DNA fragmentation (45 +/- 3 vs. 10 +/- 1% TUNEL-positive myocytes on day 1 in C2-cer-treated and Cl cultures, respectively; n = 6, p < 0.001). Myocyte exposure to 10 microM PDMP for 7 days (n = 5), caused ceramide accumulation (1.7-fold increase vs. Cl, p < 0.01), and a marked increase in the percentage of TUNEL-positive myocytes (62 +/- 6 vs. 11 +/- 3% in Cl cultures, p < 0.001). Ventricles of rats injected intraperitoneally with a cumulative dose of 14 mg/kg Dox over a period of 2 weeks also showed an increased ceramide concentration 2 weeks later (11.01 +/- 0.64 vs. 5.24 +/- 0.88 pmol/mg prot, n = 6; p < 0.001). CONCLUSION: Our study confirms the existence of a functional ceramide pathway related to apoptosis in cardiac myocytes, and points to its possible involvement in doxorubicin-induced cardiomyopathy.

The intriguing link between modulation of both multidrug resistance and ligand-toxin conjugate cytotoxicity.

Pharmacological agents which possess a chemosensitizing activity (i.e. the ability to modulate the multidrug resistance phenotype) can equally enhance ligand-toxin conjugate cytotoxicity. By confronting results obtained in both fields of research it appears that quite a number of agents, which are structurally unrelated, possess this bilateral effect. We have therefore attempted to provide a brief review of the literature and to discuss a hypothesis by which a common mechanism such as modifications in intracellular vesicle sorting and/or lipid metabolism may be implicated. We believe that these observations may provide clues for future research.

p38 MAPK mediates the regulation of alpha1(I) procollagen mRNA levels by TNF-alpha and TGF-beta in a cell line of rat hepatic stellate cells(1).

The role of members of the mitogen-activated protein kinase (MAPK) family on tumor necrosis factor alpha (TNF-alpha)-mediated down-regulation of col1a1 gene was studied. TNF-alpha increased extracellular-regulated kinase and Jun-N-terminal kinase phosphorylation, but these effects were not related to its inhibitory effect on alpha1(I) procollagen (col1a1) mRNA levels. Phosphorylation of p38 MAPK was decreased in response to TNF-alpha, and the specific p38 MAPK inhibitor SB203580 mimicked the effect of TNF-alpha on col1a1 mRNA levels. Transforming growth factor beta (TGF-beta) increased p38 MAPK phosphorylation and SB203580 prevented the induction of col1a1 mRNA levels by TGF-beta. These results suggest that p38 MAPK plays an important role in regulating the expression of col1a1 in hepatic stellate cells in response to cytokines.

Sensitivity of fresh leukemic cells to T101 ricin A-chain immunotoxin: a comparative study between Fab fragment and whole Ig conjugates.

We compared the cell killing potency of a whole Ig ricin A-chain immunotoxin (T101 IgG-RTA) against its Fab fragment counterpart (T101 Fab-RTA) on both CEM cells and fresh malignant lymphoid cells. A dye exclusion assay (DEA), was used to evaluate the kinetics of leukaemia cell viability mediated in vitro by each immunotoxin (IT). This study found that in the absence of ammonium chloride (NH4Cl), used as an enhancer agent, T101 Fab-RTA was significantly more toxic to both CEM and fresh leukaemia cells than T101 IgG-RTA. In the presence of NH4Cl (10(-2) M), while no differences could be found between the two IT on CEM cells, T101 Fab-RTA was clearly superior to T101 IgG-RTA on fresh leukaemia cells. These results suggest that T101 Fab-RTA may offer an excellent alternative to T101 IgG-RTA for IT treatment of CD5 positive leukaemia patients.

Effects of an anti HLA-DR immunotoxin on leukaemia cells and hematopoietic progenitors.

An anti HLA-class II immunotoxin has been prepared by coupling to ricin A-chain (RTA) and anti-DR-DP monoclonal antibody (2G5-MoAb). Protein synthesis inhibition assays showed that 2G5-RTA immunotoxin is: highly cytotoxic to B-cell lymphoid neoplastic cells, and variable so to ALL cells, while AML cell lines display a generally poor susceptibility. Toxicity of 2G5-RTA on normal hematopoietic cells (HPC) was found to be dose-dependent, and to increase significantly with the addition of NH4Cl, used as an activating agent. After 4 h of incubation with 2G5-RTA (10(-8) M), without NH4Cl, percentages of CFU-GM and CFU-GEMM (colony forming units-granulo-monocytes and -multipotent, respectively) progenitor cell recovery were in the order of 50% and 30% respectively. In the same treatment conditions, 2G5-RTA induced a 6 log kill on RAJI cells--measured by clonogenic assay. Finally, this study shows that anti-DR immunotoxins may represent an original, efficient, and relatively safe approach for bone marrow purging of DR positive malignant B-cell populations; while their clinical potential pertaining to ALL and AML remains uncertain.

The acid sphingomyelinase inhibitor SR33557 counteracts TNF-alpha-mediated potentiation of IL-1beta-induced NF-kappaB activation in the insulin-producing cell line Rinm5F.

Cytokines induce nitric oxide (NO) production and cell death in insulin-producing cells in vitro but the signaling pathways mediating the cytokine effects are not well characterized. The aim of this study was to determine whether sphingomyelinase (SMase) participates in cytokine signaling leading to NF-kappaB activation, iNOS induction and cell death in insulin-producing cells. Acute exposure to IL-1beta or TNF-alpha did not affect SMase activities in rat insulinoma (RINm5F) cells. TNF-alpha activated NF-kappaB in gel shift experiments without inducing iNOS--as assessed by nitrite formation--whereas IL-1beta stimulated both NF-kappaB activation and iNOS induction. Natural ceramide did not activate NF-kappaB or iNOS. However, both ceramide and TNF-alpha potentiated IL-1beta- induced activation of NF-kappaB and iNOS. Moreover, the potentiating effects of TNF-alpha were counteracted by the acid SMase inhibitor SR33557. The combination of IL-1beta and IFN-gamma induced apoptosis in RINm5F cells, which was paralleled by a modest increase in acid SMase, whereas ceramide mainly induced necrosis. It is concluded that cytokine-induced beta-cell signaling is associated with the induction of iNOS but not with enhanced SMase activities. However, TNF-alpha-mediated potentiation of the IL-1beta effect may involve an increased sensitivity to basal acid SMase activity. An increased acid SMase activity may participate in the execution of cytokine-induced beta-cell apoptosis.