Effects of miltefosine on various biochemical parameters in a panel of tumor cell lines with different sensitivities.

We investigated endocytosis activity, uptake of miltefosine (hexadecylphosphocholine), phospholipid and cholesterol content, the cell cycle, and apoptosis in 13 tumor cell lines (MCF7, MCF7/ADR, KB-3-1, KB-8-5, KB-C1, HeLa, HeLa-MDR1-G185, HeLa-MDR1-V185, CCRF/CEM, CCRF/VCR1000, CCRF/ADR5000, HL-60, HL-60/AR) with different sensitivities to treatment with the antitumor phospholipid analogues miltefosine and D-21266 (octadecyl-(N,N-dimethyl-piperidino-4-yl)-phosphate). In this panel of cell lines, MDR1 (multidrug resistance gene 1)- and MRP1 (multidrug resistance-associated protein)-expressing cells were found to be slightly more resistant to both compounds than sensitive parental cells. No correlation was found between resistance to miltefosine and endocytosis, intracellular concentration of miltefosine, the phospholipid and cholesterol content, induction of apoptosis, or cell cycle alterations in all the cell lines tested. Wild-type p53 containing WMN Burkitt's lymphoma cells and wild type p53-deficient CA46 exhibited similar sensitivities to miltefosine. The low percentage of apoptosis induced in MCF7 cells lacking caspase 3 indicated that caspase 3 seems to play an essential role in miltefosine-induced apoptosis.

Regulation of phospholipase D isoenzymes by transforming Ras and atypical protein kinase C-iota.

The activation of phospholipase D (PLD) by transforming Ras is well documented. Although two distinct PLD isoforms, PLD1 and PLD2, have been cloned from mammalian cells, it has remained unclear whether both isoenzymes are activated by Ras and, if this is the case, whether they are stimulated by a common mechanism. In the present study we show that expression of transforming Ras in HC11 mouse mammary epithelial cells enhanced the activity of endogenous PLD. Co-expression of Ras with either PLD1b or PLD2 resulted in elevated activities of both PLD isoenzymes in HC11 cells, indicating that transforming Ras was capable of activating both PLD isoforms in vivo. Ras-induced activation of PLD was resistant to the protein kinase C (PKC) inhibitor GF109203X, which preferentially affects conventional- and novel-type PKCs, but sensitive to Ro-31-8220, which inhibits atypical PKCs more effectively. Co-transfection of atypical PKC-iota with either PLD1b or PLD2 led to a selective activation of PLD2 by PKC-iota, whereas PLD1b was not affected. PLD1b, however, was found to be a potent activator of PKC-iota, whereas PLD2 was less effective in this respect. The data suggest that PKC-iota acts upstream of PLD2 and that PLD1b is implicated in the activation of PKC-iota. The data are discussed as indicating a putative signalling cascade comprising Ras-->PLD1b-->PKC-iota-->PLD2. Evidence for the implication of this pathway in the transcriptional regulation of cyclin D1 is also presented.

Protein kinase C isoforms involved in the transcriptional activation of cyclin D1 by transforming Ha-Ras.

Transcriptional activation of the cyclin D1 by oncogenic Ras appears to be mediated by several pathways leading to the activation of multiple transcription factors which interact with distinct elements of the cyclin D1 promoter. The present investigations revealed that cyclin D1 induction by transforming Ha-Ras is MEK- and Rac-dependent and requires the PKC isotypes epsilon, lambda, and zeta, but not cPKC-alpha. This conclusion is based on observations indicating that cyclin D1 induction by transforming Ha-Ras was depressed in a dose-dependent manner by PD98059, a selective inhibitor of the mitogen-activated kinase kinase MEK-1, demonstrating that Ha-Ras employs extracellular signal-regulated kinases (ERKs) for signal transmission to the cyclin D1 promoter. Evidence is presented that PKC isotypes epsilon and zeta, but not lambda are required for the Ras-mediated activation of ERKs. Expression of kinase-defective, dominant negative (DN) mutants of nPKC-epsilon or aPKC-zeta inhibit ERK activation by constitutively active Raf-1. Phosphorylation within the TEY motif and subsequent activation of ERKs by constitutively active MEK-1 was significantly inhibited by DN aPKC-zeta, indicating that aPKC-zeta functions downstream of MEK-1 in the pathway leading to cyclin D1 induction. In contrast, TEY phosphorylation induced by constitutively active MEK-1 was not effected by nPKC-epsilon, suggesting another position for this kinase within the cascade investigated. Transformation by oncogenic Ras requires activation of several Ras effector pathways which may be PKC-dependent and converge on the cyclin D1 promoter. Therefore, we investigated a role for PKC isotypes in the Ras-Rac-mediated transcriptional regulation of cyclin D1. We have been able to reveal that cyclin D1 induction by oncogenic Ha-Ras is Rac-dependent and requires the PKC isotypes epsilon, lambda, and zeta, but not cPKC-alpha. Evidence is presented that aPKC-lambda acts upstream of Rac, between Ras and Rac, whereas the PKC isotypes epsilon and zeta act downstream of Rac and are required for the activation of ERKs.

MDR1 causes resistance to the antitumour drug miltefosine.

Miltefosine (hexadecylphosphocholine) is used for topical treatment of breast cancers. It has been shown previously that a high percentage of breast carcinomas express MDR1 or MRP. We investigated the sensitivity of MDR1 -expressing cells to treatment with miltefosine. We show that cells overexpressing MDR1 (NCI/ADR-RES, KB-8-5, KB-C1, CCRF/VCR1000, CCRF/ADR5000) were less sensitive to miltefosine treatment when compared to the sensitive parental cell lines. HeLa cells transfected with MDR1 exhibited resistance to the compound, indicating that expression of this gene is sufficient to reduce the sensitivity to miltefosine. The resistance of MDR1-expressing cells to miltefosine was less pronounced than that to adriamycin or vinblastine. Expression of MDR2 did not correlate with the resistance to miltefosine. As shown by a fluorescence quenching assay using MIANS-labelled P-glycoprotein (PGP), miltefosine bound to PGP with a K(d)of approximately 7 microM and inhibited PGP-ATPase activity with an IC(50)of approximately 35 microM. Verapamil was not able to reverse the resistance to miltefosine. Concentrations of miltefosine up to approximately 60 microM stimulated, whereas higher concentrations inhibited the transport of [3H]-colchicine with an IC(50)of approximately 297 microM. Binding studies indicated that miltefosine seems to interact with the transmembrane domain and not the cytosolic nucleotide-binding domain of PGP. These data indicate that expression of MDR1 may reduce the response to miltefosine in patients and that this compound interacts with PGP in a manner different from a number of other substrates.

Unique structural and functional properties of the ATP-binding domain of atypical protein kinase C-iota.

Atypical protein kinase C-iota (aPKCiota) plays an important role in mitogenic signaling, actin cytoskeleton organization, and cell survival. Apart from the differences in the regulatory domain, the catalytic domain of aPKCiota differs considerably from other known kinases, because it contains a modification within the glycine-rich loop motif (GXGXXG) that is found in the nucleotide-binding fold of virtually all nucleotide-binding proteins including PKCs, Ras, adenylate kinase, and the mitochondrial F1-ATPase. We have used site-directed mutagenesis and kinetic analysis to investigate whether these sequence differences affect the nucleotide binding properties and catalytic activity of aPKCiota. When lysine 274, a residue essential for ATP binding and activity conserved in most protein kinases, was replaced by arginine (K274R mutant), aPKCiota retained its normal kinase activity. This is in sharp contrast to results published for any other PKC or even distantly related kinases like phosphoinositide 3-kinase gamma, where the same mutation completely abrogated the kinase activity. Furthermore, the sensitivity of aPKCiota for inhibition by GF109203X, a substance acting on the ATP-binding site, was not altered in the K274R mutant. In contrast, replacement of Lys-274 by tryptophan (K274W) completely abolished the kinase activity of PKCiota. In accordance with results obtained with other kinase-defective PKC mutants, in cultured cells aPKCiota-K274W acted in a dominant negative fashion on signal transduction pathways involving endogenous aPKCiota, whereas the effect of the catalytically active K274R mutant was identical to the wild type enzyme. In summary, aPKCiota differs from classical and novel PKCs also in the catalytic domain. This information could be of significant value for the development of specific inhibitors of aPKCiota as a key factor in central signaling pathways.

Novel membrane-targeted ERK1 and ERK2 chimeras which act as dominant negative, isotype-specific mitogen-activated protein kinase inhibitors of Ras-Raf-mediated transcriptional activation of c-fos in NIH 3T3 cells.

Expression of constructs encoding fusion proteins of ERK1 and ERK2 containing a C-terminal farnesylation motif (CAAX) is predominantly localized at the cell membrane and was activated by coexpression of constitutively active Ha-RasL61 and epidermal growth factor. Both fusion proteins significantly inhibit the transcriptional activation of a c-fos-chloramphenicol acetyltransferase reporter induced by RasL61, constitutively active MEK1, or constitutively active RafBXB. The corresponding SAAX chimeras or overexpression of the wild-type ERKs did not interfere with the transcriptional activation of c-fos. The inhibition of the Ras-mediated c-fos induction by ERK2-CAAX can in part be rescued by coexpression of a wild-type ERK2 but not by wild-type ERK1. We find that ERK1-CAAX acts in the same fashion, indicating that mitogen-activated protein kinase (MAPK)-CAAX chimeras interact in an isotype-specific manner. It is demonstrated that both ERK1-CAAX and ERK2-CAAX associate with the corresponding endogenous ERKs, which explains the isotype-specific inhibitory effects of the ERK-CAAX chimeras. Evidence is presented that expression of ERK-CAAX fusion proteins inhibits the nuclear translocation of the corresponding endogenous ERKs. Disruption of MAPK translocation by membrane targeting provides additional, independent proof that nuclear translocation of ERKs is essential for the transcriptional activation of c-fos.

Age-dependent deamidation of H1(0) histones in chromatin of mammalian tissues.

The composition of the H1(o) histone subfractions was examined in different rat and mouse tissues. Using reverse-phase HPLC and hydrophilic-interaction liquid chromatography we have found that the relative proportions of all four forms of H1(o) differ from tissue to tissue and from species to species. In principle, we observed an age-dependent increase in the amount of both the N-terminally acetylated (H1(o)a Asn-3 and H1(o)a Asp-3) and the deamidated forms of H1(o) (H1(o)a Asp-3 and H1(o)b Asp-3). Compared with the proportion of N-terminally acetylated H1(o) forms in liver, kidney and brain of rats and mice 20 days of age, we found an increase in these H1(o) subfractions of up to 30% in the corresponding organs of 300-day-old animals. The proportion of deamidated H1(o) forms was 1.6- to 4-fold higher in the livers and 8- to 12-fold higher in the brains of 300-day-old mice and rats, respectively, than in 20-day-old animals. The tissue-specific nature of the ratio of H1(o) subfractions suggests that the different forms of histone H1(o) have specific individual functions. The possible biological significance of age-related accumulation of N-terminal acetylated and deamidated histone H1(o) forms is discussed in the light of our results.

Evidence that atypical protein kinase C-lambda and atypical protein kinase C-zeta participate in Ras-mediated reorganization of the F-actin cytoskeleton.

Expression of transforming Ha-Ras L61 in NIH3T3 cells causes profound morphological alterations which include a disassembly of actin stress fibers. The Ras-induced dissolution of actin stress fibers is blocked by the specific PKC inhibitor GF109203X at concentrations which inhibit the activity of the atypical aPKC isotypes lambda and zeta, whereas lower concentrations of the inhibitor which block conventional and novel PKC isotypes are ineffective. Coexpression of transforming Ha-Ras L61 with kinase-defective, dominant-negative (DN) mutants of aPKC-lambda and aPKC-zeta, as well as antisense constructs encoding RNA-directed against isotype-specific 5' sequences of the corresponding mRNA, abrogates the Ha-Ras-induced reorganization of the actin cytoskeleton. Expression of a kinase-defective, DN mutant of cPKC-alpha was unable to counteract Ras with regard to the dissolution of actin stress fibers. Transfection of cells with constructs encoding constitutively active (CA) mutants of atypical aPKC-lambda and aPKC-zeta lead to a disassembly of stress fibers independent of oncogenic Ha-Ras. Coexpression of (DN) Rac-1 N17 and addition of the phosphatidylinositol 3'-kinase (PI3K) inhibitors wortmannin and LY294002 are in agreement with a tentative model suggesting that, in the signaling pathway from Ha-Ras to the cytoskeleton aPKC-lambda acts upstream of PI3K and Rac-1, whereas aPKC-zeta functions downstream of PI3K and Rac-1. This model is supported by studies demonstrating that cotransfection with plasmids encoding L61Ras and either aPKC-lambda or aPKC-zeta results in a stimulation of the kinase activity of both enzymes. Furthermore, the Ras-mediated activation of PKC-zeta was abrogated by coexpression of DN Rac-1 N17.

Epidermal growth factor (EGF) receptor blockade inhibits the action of EGF, insulin-like growth factor I, and a protein kinase A activator on the mitogen-activated protein kinase pathway in prostate cancer cell lines.

Epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) are potent mitogens that regulate proliferation of prostate cancer cells via autocrine and paracrine loops and promote tumor metastasis. They exert their action through binding to the corresponding cell surface receptors that initiate an intracellular phosphorylation cascade, leading to the activation of mitogen-activated protein kinases (MAPKs), which recruit transcription factors. We have studied the effects of EGF, IGF-I, and the protein kinase A (PKA) activator forskolin on the activation of p42/ extracellular signal-regulated kinase (ERK)2, which is a key kinase in mediation of growth factor-induced mitogenesis in prostate cancer cells. The activity of p42/ERK2 was determined by immune complex kinase assays and by immunoblotting using a phospho p44/p42 MAPK-specific antibody. EGF, IGF-I, and forskolin-induced PKA activity stimulate intracellular signaling pathways converging at the level of p42/ERK2. In the androgen-insensitive DU145 cell line, there is a constitutive basal p42/ ERK2 activity that is not present in androgen-sensitive LNCaP cells. Constitutive p42/ERK2 activity is abrogated by blockade of the EGF receptor. Hence, it is obviously caused by an autocrine loop involving this receptor. The effects of EGF on p42/ERK2 are potentiated by forskolin in both cell lines. The blockade of PKA by the specific inhibitor H89 attenuates this synergism. This finding is in contrast to those obtained in several other systems studied thus far, in which PKA activators inhibited MAPKs. p42/ERK2 in DU145 cells is highly responsive to IGF-I stimulation, whereas no effect of IGF-I on p42/ERK2 can be measured in LNCaP cells. Moreover, our results demonstrate that selective blockade of the EGF receptor in prostate cancer cells does not only inhibit the action of EGF, but also IGF-I-induced activation of the MAPK pathway and the interaction with the PKA pathway. In conclusion, these findings offer new possibilities for a therapeutical intervention in prostate cancer by targeting signaling pathways of growth factors and PKA.

The involvement of protein kinase C isoenzymes alpha, epsilon and zeta in the sensitivity to antitumor treatment and apoptosis induction.

In order to obtain additional information on the involvement of protein kinase C (PKC) isoenzymes in the resistance of cells to anticancer drugs and in the induction of apoptosis, we employed antisense oligonucleotides to PKC alpha and PKC zeta, CGP 53506, a new inhibitor of PKC alpha, and cells overexpressing PKC alpha, PKC epsilon and PKC zeta. We found that in HeLa cells which express PKC alpha and zeta, down-modulation of either PKC alpha or PKC zeta with antisense oligonucleotides induced apoptosis. The PKC alpha selective inhibitor CGP 53506 reduced the proliferation rate of PKC alpha overexpressing NIH3T3 cells more than that of wild-type cells and induced apoptosis, indicating that such a PKC alpha inhibitor may be useful in the treatment of tumors overexpressing PKC alpha such as glioblastomas. NIH3T3 cells overexpressing PKC alpha were more resistant, whereas NIH3T3 cells overexpressing PKC epsilon or PKC zeta were more sensitive to treatment with cis-platin, adriamycin or gamma-irradiation compared to parental NIH3T3 wild-type cells. The observed resistance and sensitization corresponded to the extent of apoptosis induced by these treatments. Alterations in the expression of p53, bcl-2 and bax in the PKC isoenzyme overexpressing cells indicate that these proteins may be involved in the different sensitivities of these cells.

PKC-independent modulation of multidrug resistance in cells with mutant (V185) but not wild-type (G185) P-glycoprotein by bryostatin 1.

Bryostatin 1 is a new antitumor agent which modulates the enzyme activity of protein kinase C (PKC, phospholipid-Ca2+-dependent ATP:protein transferase, EC 2.7.1.37). Several reports have suggested that the pumping activity of the multidrug resistance gene 1 (MDR1)-encoded multidrug transporter P-glycoprotein (PGP) is enhanced by a PKC-mediated phosphorylation. It was shown here that bryostatin 1 was a potent modulator of multidrug resistance in two cell lines over-expressing a mutant MDR1-encoded PGP, namely KB-C1 cells and HeLa cells transfected with an MDR1-V185 construct (HeLa-MDR1-V185) in which glycine at position 185 (G185) was substituted for valine (V185). Bryostatin 1 is not able to reverse the resistance of cells over-expressing the wild-type form (G185) of PGP, namely CCRF-ADR5000 cells and HeLa cells transfected with a MDR1-G185 construct (HeLa-MDR1-G185). Treatment of HeLa-MDR1-V185 cells with bryostatin 1 was accompanied by an increase in the intracellular accumulation of rhodamine 123, whereas no such effect could be observed in HeLa-MDR1-G185 cells. HeLa-MDR1-V185 cells expressed the PKC isoforms alpha, delta and zeta. Down-modulation of PKC alpha and delta by 12-O-tetradecanoylphorbol-13-acetate (TPA) did not affect the drug accumulation by bryostatin 1. Bryostatin 1 depleted PKC alpha completely and PKC delta partially. In HeLa-MDR1-V185 cells, short-term exposure to bryostatin 1, which led to a PKC activation, was as efficient in modulating the pumping activity of PGP as long-term exposure leading to PKC depletion. Bryostatin 1 competed with azidopine for binding to PGP in cells expressing the MDR1-V185 and MDR1-G185 forms of PGP. It is concluded that bryostatin 1: i) interacts with both the mutated MDR1-V185 and the wild-type MDR1-G185; ii) reverses multidrug resistance and inhibits drug efflux only in PGP-V185 mutants; and iii) that this effect is not due to an interference of PKC with PGP. For gene therapy, it is important to reverse the specific resistance of a mutant in the presence of a wild-type transporter and vice versa. Our results show that it is possible to reverse a specific mutant PGP.

Transcriptional activation of c-fos by oncogenic Ha-Ras in mouse mammary epithelial cells requires the combined activities of PKC-lambda, epsilon and zeta.

The implication of protein kinase C (PKC) isoforms cPKC-alpha, nPKC-epsilon, aPKC-lambda and aPKC-zeta in the transcriptional activation of a c-fos promoter-driven CAT-reporter construct by transforming Ha-Ras has been investigated. This was achieved by employing antisense constructs encoding RNA directed against isoform-specific 5' sequences of the corresponding mRNA, and expression of PKC mutants representing either kinase-defective, dominant negative, or constitutively active forms of the PKC isoforms. The data indicate that in HC11 mouse mammary epithelial cells, transforming Ha-Ras requires the activities of the three PKC isozymes: aPKC-lambda, nPKC-epsilon and aPKC-zeta, not, however, of cPKC-alpha, for the transcriptional activation of c-fos. Co-expression of oncogenic Ha-Ras with combinations of kinase-defective, dominant negative and constitutively active mutants of the various PKC isozymes are in agreement with a tentative model suggesting that, in the signaling pathway from Ha-Ras to the c-fos promoter, aPKC-lambda acts upstream whereas aPKC-zeta functions downstream of nPKC-epsilon.

Reversal of multidrug resistance by the staurosporine derivatives CGP 41251 and CGP 42700.

It has been shown previously that the staurosporine derivative CGP 41251, a specific inhibitor of protein kinase C (IC50 = 50 nM), exhibits antitumor activity and reverses mdr1 mediated multidrug resistance. At present, the compound is evaluated as an anticancer drug in clinical phase I trials. We compared the effects of CGP 41251 with CGP 42700, another staurosporine derivative, which exhibits low protein kinase C inhibiting activity (IC50 = > 100 microM). We found that in contrast to CGP 41251, CGP 42700 does not show antiproliferative activity in HeLa and KB cells in tissue culture (up to a concentration of 10 microM). We compared both compounds for their ability to reverse mdr1-mediated resistance in KB-C1 and in HeLa-MDR1 cells (transfected with the mdr1 gene). CGP 42700 is able to reverse mdr1-mediated resistance to a similar extent as CGP 41251. The intracellular accumulation of rhodamine 123 in KB-C1 cells following pretreatment with CGP 41251 for 30 min was higher than that following treatment with CGP 42700 if determined in medium without serum. However, quantitation of rhodamine efflux in an ex vivo assay using human CD8+ cells in serum showed that CGP 42700 is more effective in inhibiting the efflux of rhodamine 123 than CGP 41251. We conclude from our results that (1) CGP 42700 is more effective in reversal of multidrug resistance in serum than CGP 41251, indicating that the compound may be useful for treatment of patients, and (2) CGP 42700 does not inhibit protein kinase C and cell proliferation and, therefore, may be less toxic and elicit less side effects in humans than other chemosensitizers.

Molecular basis of targeted chemotherapy: novel concepts with special reference to the treatment of Hodgkin's disease.

Concepts for the treatment of Hodgkin's lymphomas based on novel insights of the molecular mechanisms responsible for the maintenance of the transformed phenotype of Reed-Sternberg cells, their proliferation and sensitivity to radiation and anti-tumor agents are discussed. The potentials of some recently developed new signal transduction inhibitors for the treatment of Hodgkin's lymphomas are discussed in greater detail and comprise agents directed against Janus kinase 2 (JAK 2); Signal Transducers and Activators of Transcription (STAT factors); agents directed against SH 2-domains: the fes/fps oncogene, Ras; protein kinase C (PKC) isotypes and means of inducing radiation or drug-induced apoptosis.

Conventional PKC-alpha, novel PKC-epsilon and PKC-theta, but not atypical PKC-lambda are MARCKS kinases in intact NIH 3T3 fibroblasts.

Phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) in intact cells has been employed as an indicator for activation of protein kinase C (PKC). Specific PKC isoenzymes responsible for MARCKS phosphorylation under physiological conditions, however, remained to be identified. In our present study using stably transfected NIH 3T3 cell clones we demonstrate that expression of constitutively active mutants of either conventional cPKC-alpha or novel nPKC-epsilon increased phosphorylation of endogenous MARCKS in the absence of phorbol 12,13-dibutyrate in intact mouse fibroblasts, implicating that each of these PKC isoforms itself is sufficient to induce enhanced MARCKS phosphorylation. Similarly, ectopic expression of a constitutively active mutant of PKC-theta significantly increased MARCKS phosphorylation compared to vector controls, identifying PKC-theta as a MARCKS kinase. The PKC-specific inhibitor GF 109203X (bisindolylmaleimide I) reduced MARCKS phosphorylation in intact cells at a similar dose-response as enzymatic activity of recombinant isoenzymes cPKC-alpha, nPKC-epsilon, and nPKC-theta in vitro. Consistently, phorbol 12,13-dibutyrate-dependent MARCKS phosphorylation was significantly reduced in cell lines expressing dominant negative mutants of either PKC-alpha K368R or (dominant negative) PKC-epsilon K436R. The fact, that the constitutively active PKC-lambda A119E mutant did not alter the MARCKS phosphorylation underscores the assumption that atypical PKC isoforms are not involved in this process. We conclude that under physiological conditions, conventional cPKC-alpha and novel nPKC-epsilon, but not atypical aPKC-lambda are responsible for MARCKS phosphorylation in intact NIH 3T3 fibroblasts.

Resistance to the new anti-cancer phospholipid ilmofosine (BM 41 440).

The thioether phospholipid ilmofosine (BM 41 440) is a new anti-cancer drug presently undergoing phase II clinical trials. Because resistance to anti-tumour drugs is a major problem in cancer treatment, we investigated the resistance of different cell lines to this compound. Here we report that the multidrug-resistant cell lines MCF7/ADR, CCRFNCR1000, CCRF/ADR500, CEM/VLB100 and HeLa cell lines transfected with a wild-type and mutated (gly/val185) multidrug resistance 1 gene (MDR1) are cross-resistant to ilmofosine compared with the sensitive parental cell lines. In CEMNM-1 cells, in which the resistance is associated with an altered topoisomerase II gene, no cross-resistance to ilmofosine was observed. Ilmofosine is not capable of modulating multidrug resistance and neither does it reduce the labelling of the P-glycoprotein (P-gp) by azidopine nor alter ATPase activity significantly. The resistance to ilmofosine in multidrug-resistant CCRF/VCR1000 cells cannot be reversed by the potent multidrug resistance modifier dexniguldipine-HCI (B8509-035). A tenfold excess of ilmofosine does not prevent the MDR-modulating effect of dexniguldipine-HCl. Treatment of cells with ilmofosine does not alter the levels of MDR1 mRNA. Long-term treatment of an ilmofosine-resistant Meth A subline with the drug does not induce multidrug resistance, indicating that ilmofosine does not increase the level of P-gp. Determination of the MDR2 mRNA levels in the cells revealed that the resistance pattern to ilmofosine is not correlated with the expression of this gene. It is concluded, therefore, that multidrug-resistant cells are cross-resistant to ilmofosine and that the compound is not a substrate of Pgp. No association between the expression of the MDR2-encoded P-gp and resistance to ilmofosine was observed. It is supposed that MDR1-associated alterations in membrane lipids cause resistance to ilmofosine.