Histone deacetylases 1, 2 and 3 are highly expressed in prostate cancer and HDAC2 expression is associated with shorter PSA relapse time after radical prostatectomy.

High activity of histone deacetylases (HDACs) causes epigenetic alterations associated with malignant cell behaviour. Consequently, HDAC inhibitors have entered late-phase clinical trials as new antineoplastic drugs. However, little is known about expression and function of specific HDAC isoforms in human tumours including prostate cancer. We investigated the expression of class I HDACs in 192 prostate carcinomas by immunohistochemistry and correlated our findings to clinicopathological parameters including follow-up data. Class I HDAC isoforms were strongly expressed in the majority of the cases (HDAC1: 69.8%, HDAC2: 74%, HDAC3: 94.8%). High rates of HDAC1 and HDAC2 expression were significantly associated with tumour dedifferentiation. Strong expression of all HDACs was accompanied by enhanced tumour cell proliferation. In addition, HDAC2 was an independent prognostic marker in our prostate cancer cohort. In conclusion, we showed that the known effects of HDACs on differentiation and proliferation of cancer cells observed in vitro can also be confirmed in vivo. The class I HDAC isoforms 1, 2 and 3 are differentially expressed in prostate cancer, which might be important for upcoming studies on HDAC inhibitors in this tumour entity. Also, the highly significant prognostic value of HDAC2 clearly deserves further study.

High expression of RelA/p65 is associated with activation of nuclear factor-kappaB-dependent signaling in pancreatic cancer and marks a patient population with poor prognosis.

Activation of nuclear factor-kappaB (NF-kappaB) signaling was observed in pancreatic adenocarcinoma cell lines and tumours. However, information on the expression of RelA/p65, the major transcription activating NF-kappaB subunit, in these carcinomas and possible correlations thereof with NF-kappaB activation and patient survival is not available. To provide this missing translational link, we analysed expression of RelA/p65 in 82 pancreatic adenocarcinomas by immunohistochemistry. Moreover, we measured activation of the NF-kappaB pathway in 11 tumours by quantitative PCR for NF-kappaB target genes. We observed strong cytoplasmic or nuclear expression of RelA/p65 in 42 and 37 carcinomas, respectively. High cytoplasmic and nuclear expression of RelA/p65 had negative prognostic impact with 2-year survival rates for patients without cytoplasmic or nuclear RelA/p65 positivity of 41 and 40% and rates for patients with strong cytoplasmic or nuclear RelA/p65 expression of 22 and 20%, respectively. High RelA/p65 expression was correlated to increased expression of NF-kappaB target genes. The observation that high expression of RelA/p65 is correlated to an activation of the NF-kappaB pathway and indicates poor patient survival identifies a patient subgroup that might particularly benefit from NF-kappaB-inhibiting agents in the treatment of pancreatic cancer. Based on our findings, this subgroup could be identified by applying simple immunohistochemical techniques.

Polo-like kinase isoform expression is a prognostic factor in ovarian carcinoma.

The Polo-like kinase (PLK) family comprises three serine/threonine kinases, functionally involved in signal transduction pathways essential for the accomplishment of mitosis in both normal and malignant cells. Moreover, certain PLKs have been functionally linked to cytoskeletal reorganisation. In this study, the expression of PLK1 and PLK3 was determined immunohistochemically in tissue specimen of normal ovaries (n=9), cystadenomas (n=17), borderline tumours (n=13) and ovarian carcinomas (n=77). PLK 1 and PLK3 expression was low in normal ovarian surface epithelium and borderline tumours, with moderately higher expression levels in cystadenomas. In ovarian carcinomas, 26% of cases were PLK1 positive and 50.6% of cases were PLK3 positive. A positive correlation of both PLK1 and PLK3 expression with indicators of mitotic frequency could be established. The overexpression of either isoenzyme had an impact on patient prognosis with shortened survival time for patients with tumours positive for PLK1 (P=0.02) and PLK3 (P=0.02), but only PLK1 expression remained a prognostic factor in multivariate survival analysis (P=0.03). The results of this study, if interpreted in the context of recently published functional data, suggest that inhibition of PLKs might represent an interesting new targeted approach for chemotherapy of epithelial ovarian cancer. Furthermore, this study suggests that PLK1 is a novel independent prognostic marker in ovarian carcinomas.

Anticancer drug-mediated induction of multidrug resistance-associated genes and protein kinase C isozymes in the T-lymphoblastoid cell line CCRF-CEM and in blasts from patients with acute lymphoblastic leukemias.

The major determinants mediating drug resistance in acute lymphoblastic leukemias (ALL) unresponsive to chemotherapy, are still unclear. For example, it is still unknown whether selection or induction processes are responsible for drug resistance here or whether protein kinase C (PKC) isozymes contribute to the resistant phenotype. Therefore, inducibility of resistance factors or PKC isozymes genes was examined in CCRF-CEM cells treated with diverse anticancer drugs--adriamycin, camptothecin, etoposide or vincristine--at sublethal concentrations for 24 h. MDR1, MRP1, LRP and PKC isozyme alpha, beta(1), beta(2), epsilon, iota, eta, theta, zeta gene expression was determined by cDNA-PCR. We found significant dose-dependent, mostly combined, induction of the MDR1, MRP1 and LRP genes. Significantly enhanced gene expression of the majority of PKC isozyme genes was found after treatment with camptothecin. PKCzeta was upregulated throughout by each anticancer drug applied in this setting. A series of selected CCRF-CEM-derived multidrug resistance (MDR) sublines also showed enhanced expression of the PKC isozymes compared to the parental cell line. MDR1 and PKCeta gene expression levels were correlated highly significantly. Blasts from two patients with ALL during the first week of monotherapy with steroids revealed combined induction of the MDR1, multidrug resistance-associated protein 1 (MRP1), lung cancer resistance-related protein (LRP) and most PKC isozymes, predominantly PKCzeta. Another patient with T-ALL, who failed to respond to four months of intensive chemotherapy, showed an enhanced MRP1 gene expression combined with markedly overexpression of PKCeta and PKCtheta. Furthermore, the camptothecin and etoposide-mediated induction of resistance factors in the CCRF-CEM cell line could be suppressed by staurosporine, a rather unspecific inhibitor of protein kinases. However, selective inhibitors of PKC isozymes (bisindolylmaleimide GO 6850, indolocarbazole GO 6976) produced no significant effects here. Therefore, the PKC isozymes eta, theta and zeta are of interest as potential targets to overcome drug resistance in ALL.

Dexamethasone and cyclosporin A do not inhibit interleukin-15 expression in the human lung carcinoma cell line A549.

A549 cells constitutively expressed IL-15 mRNA which could be upregulated by stimulation with TNF-alpha- or IL-1beta. Constitutive and induced levels of IL-15 mRNA were not decreased in the presence of 10- 6 M dexamethasone. Control experiments revealed that 10- 6 M dexamethasone inhibited the TNF-alpha- or IL-1beta-mediated increase of IL-8 mRNA in A549 cells, which showed that the glucocorticoid was functional. A549 cells did not secrete relevant amounts of IL-15 protein. The constitutive expression and the TNF-alpha- or IL-1beta-mediated upregulation of intracellular IL-15 protein was not inhibited by dexamethasone, in contrast, the release of IL-8 protein was inhibited. Also, cyclosporin A at 250 ng/ml did not inhibit the TNF-alpha-induced upregulation of IL-15 mRNA and intracellular IL-15 protein. The data suggest that the synthesis of IL-15 mRNA and protein is not influenced by immunosuppressive glucocorticoids or by cyclosporin A.

Functional analysis of P-glycoprotein and multidrug resistance associated protein related multidrug resistance in AML-blasts.

Despite the high effectiveness of various P-glycoprotein (P-gp) modulating substances in vitro their clinical value e.g. for combination treatment of acute myelogenous leukemias (AML) remains still unclear. This might be explainable by recent findings that other factors than P-gp (e.g. the multidrug resistance associated protein (MRP)) may also be involved in clinical occurring drug resistance. To study P-gp and MRP mediated MDR in AML blasts from patients with relapses at the functional level we measured rhodamine 123 (RHO) efflux in combination with a P-gp specific (SDZ PSC 833) or a MRP specific (MK571) modulator, respectively. Furthermore, direct antineoplastic drug action was monitored by determination of damaged cell fraction of a blast population using flow cytometry. We generally found strongly modulated RHO efflux by SDZ PSC 833 but slight RHO-efflux modulation by MK571 in blasts from relapsed states of AML expressing MDR1 or MRP mRNA at various levels. We could not demonstrate, though, significant PSC 833 or MK571 mediated modulation of the cytotoxic effects of etoposide. The results point to the possibility that combination of etoposide and a modulator might not improve responses to chemotherapy by targeting P-gp or MRP exclusively.

Rhodamine 123 efflux modulation in the presence of low or high serum from CD56+ hematopoietic cells or CD34+ leukemic blasts by B9309-068, a newly designed pyridine derivative.

The newly designed pyridine derivative B9309-068 and a series of structurally different compounds were tested for their ability to modulate rhodamine 123 (RHO) efflux from CD56+ hematopoietic cells in the presence of either 10% fetal calf serum or undiluted human AB serum. Furthermore, efflux modulation was investigated on CD34+ blast populations obtained from four patients with relapsed state AML. Target cells were specified throughout by labeling with peridinine chlorophyll protein (PerCP)-conjugated monoclonal antibodies, allowing clear differentiation from RHO emission spectrum by flow cytometry. In the presence of low serum each compound efficiently modulated RHO efflux without significant differences in the range of final concentrations (1.0-3.0 microM). At 0.1 microM, however, RHO efflux was differentially modulated following the series GF120918 approximately B9309-068 > PSC 833 > DNIG approximately DVER. With CD56+ cells in the presence of undiluted human AB serum at a final modulator concentration of 0.1 microM, all chemosensitizers tested were found to be inefficient. At final concentrations of 0.3 microM or higher, distinct RHO efflux modulation was found with the following efficacies: B9309-068 approximately GF120918 > PSC 833 >> DVER approximately DNIG. The efficacies seen in undiluted human AB serum at 3.0 microM were comparable to those seen on CD56+ cells at final modulator concentrations of 0.1 microM in low serum. Our results identify the pyridine derivative B9309-068 as a promising compound for modulating P-glycoprotein-mediated drug resistance under conditions resembling the clinical setting. Nonetheless, modulation potencies of a series of structurally very different chemosensitizers was revealed to be substantially diminished at high serum concentrations in vitro.

Induction of IL-15 mRNA and protein in A549 cells by pro-inflammatory cytokines.

Interleukin-15 is a recently discovered cytokine which is functionally similar to IL-2. In order to learn more about possible targets for modulation of the expression of IL-15 we investigated the expression of IL-15 mRNA and protein in the A549 (human lung carcinoma) cell line. Constitutive expression of IL-15 mRNA was detected in A549 cells. Treatment with TNF-alpha or IL-1 beta (10 ng/ml each) induced an about 2-fold increase of IL-15 mRNA; IFN-gamma induced significant effects only at 100 ng/ml. Stimulation with a combination of TNF-alpha and IFN-gamma was not superior to stimulation with TNF-alpha alone. EGF, KGF and the combination thereof were without effects. IL-15 protein was detected in cellular lysates of unstimulated cells and was increased by stimulation with TNF-alpha or IL-1 beta. No significant amounts of IL-15 protein were detected in cellular supernatants.

Expression analysis of protein kinase C isozymes and multidrug resistance associated genes in ovarian cancer cells.

We recently demonstrated a correlation between the expression levels of the PKC eta isozyme and the MDR1 or MRP genes in blasts from AML patients, and in primary breast cancers. In order to extend these findings we analysed ovarian cancer cells from 14 ascites aspirates from 8 patients using a cDNA-PCR approach. 5 patients were examined in follow up studies. 4 out of these 5 patients received continuous chemotherapy. The relative increases in MDR1, MRP, LRP or PKC eta mRNA expression levels were monitored. In one of these patients combined significant increase in MDR1, MRP, LRP and PKC was seen. One follow up sample was obtained after chemotherapy was discontinued. In this case significant relative decreases of MDR1, LRP and PKC eta mRNA expression levels were found. Furthermore, a significant positive correlation was determined for the relative mRNA expression levels of MRP and PKC eta. These results point to a multifactorial emergence of MDR in this type of tumor with a possible involvement of the PKC eta isozyme.

Phosphodiesterase profile of human B lymphocytes from normal and atopic donors and the effects of PDE inhibition on B cell proliferation.

1. CD19+ B lymphocytes were purified from the peripheral blood of normal and atopic subjects to analyse and compare the phosphodiesterase (PDE) activity profile, PDE mRNA expression and the importance of PDE activity for the regulation of B cell function. 2. The majority of cyclic AMP hydrolyzing activity of human B cells was cytosolic PDE4, followed by cytosolic PDE7-like activity; marginal PDE3 activity was found only in the particulate B cell fraction. PDE1, PDE2 and PDE5 activities were not detected. 3. By cDNA-PCR analysis mRNA of the PDE4 subtypes A, B (splice variant PDE4B2) and D were detected. In addition, a weak signal for PDE3A was found. 4. No differences in PDE activities or mRNA expression of PDE subtypes were found in B cells from either normal or atopic subjects. 5. Stimulation of B lymphocytes with the polyclonal stimulus lipopolysaccharide (LPS) induced a proliferative response in a time- and concentration-dependent manner, which was increased in the presence of interleukin-4 (IL-4). PDE4 inhibitors (rolipram, piclamilast) led to an increase in the cellular cyclic AMP concentration and to an augmentation of proliferation, whereas a PDE3 inhibitor (motapizone) was ineffective, which is in accordance with the PDE profile found. The proliferation enhancing effect of the PDE4 inhibitors was partly mimicked by the cyclic AMP analogues dibutyryl (db) cyclic AMP and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3',5'-cyclic monophosphorothioate, Sp-isomer (dcl-cBIMPS), respectively. However, at concentrations exceeding 100 microM db-cyclic AMP suppressed B lymphocyte proliferation, probably as a result of cytotoxicity. Prostaglandin E2 (PGE2, 1 microM) and forskolin (10 microM) did not affect B cell proliferation, even when given in combination with rolipram. 6. Inhibition of protein kinase A (PKA) by differentially acting selective inhibitors (KT 5720, Rp-8-Br-cyclic AMPS) decreased the proliferative response of control cells and reversed the proliferation enhancing effects of rolipram. 7. Importantly, PDE4 activity in LPS/IL-4-activated B lymphocytes decreased by about 50% compared to unstimulated control values. 8. We conclude that an increase in cyclic AMP, mediated by down-regulation of PDE4 activity, is involved in the stimulation of B cell proliferation in response to LPS/IL-4. B cell proliferation in response to a mitogenic stimulus can be further enhanced by pharmacological elevation of cyclic AMP.

Multiple gene expression analysis reveals distinct differences between G2 and G3 stage breast cancers, and correlations of PKC eta with MDR1, MRP and LRP gene expression.

A possible link between protein kinase C (PKC) and P-glycoprotein (P-gp)-mediated-multidrug resistance (MDR) was assumed from studies on MDR cell lines selected in vitro. The functional relevance of PKC for the MDR phenotype remains unclear, and the involvement of a particular PKC isozyme in clinically occurring drug resistance is not known. Recently, we have demonstrated significant correlations between the expression levels of the PKC eta isozyme and the MDR1 or MRP (multidrug resistance-associated protein) genes in blasts from patients with acute myelogenous leukaemia (AML) and in ascites cell aspirates from ovarian cancer patients. To extend these findings to further types of human tumours we analysed specimens from 64 patients with primary breast cancer for their individual expression levels of several MDR-associated genes (MDR1, MRP, LRP (lung cancer resistance-related protein), topoisomerase (Topo) II alpha/IIbeta, cyclin A and the PKC isozyme genes (alpha, beta1, beta2, eta, theta, and mu) by a cDNA-PCR approach. We found significantly enhanced mean values for MRP, LRP and PKC eta gene expression, but significantly decreased Topo II alpha and cyclin A gene expression levels in G2 tumours compared with G3. Remarkably, significant positive correlations between the MDR1, MRP or LRP gene expression levels and PKC eta were determined: MDR1/PKC eta (rs = +0.6451, P < 0.0001) n = 62; MRP/PKC eta (rs = +0.5454, P < 0.0001) n = 63; LRP/PKC eta (rs = +0.5436, P < 0.0001) n = 62; MRP/LRP (rs = +0.7703, P < 0.0001) and n = 62, MDR1/MRP (rs = +0.5042, P < 0.0001) n = 62. Our findings point to the occurrence of a multifactorial MDR in the clinics and to PKC eta as a possible key regulatory factor for up-regulation of a series of MDR-associated genes in different types of tumours.

A novel method for direct and fluorescence independent determination of drug efflux out of leukemic blast cells.

Multi drug resistance (MDR) is often due to an increased efflux of anti cancer drugs out of leukemic blast cells. Efflux assays are used to get an impression of functional resistance in those cells. Dyes like rhodamine 123 or 3'3'-diethyloxocarbocyanine iodide are commonly used for this purpose. A major known disadvantage is that dyes do not behave like cytotoxic drugs in efflux experiments. Assays using the self fluorescence of drugs like anthracyclines can not reveal a real impression of intracellular or effluxed drug due to quenching of the drug fluorescence in the nuclei of the cells. We have developed a reproducible and sensitive assay for direct and quantitative determination of drug efflux out of blast cells. This was done by a novel double radioactive labelling using a 3H-labelled drug and 14C-labelled sucrose as extracellular marker. So this assay can be applied to every drug of interest. Quenching of fluorescence is also by-passed with this technique as well as protracting washing or silicon oil procedures. As a model system we used the T-lymphoblastoid cell line CCRF CEM and its resistant sublines vincristine 100 and adriamycin 5000. The results were also transferable to clinical specimens of leukemic patients. In conclusion our assay may be used for precise and direct efflux measurement of a broad range of anti-cancer drugs in clinical MDR evaluation.

Phosphodiesterase profiles of highly purified human peripheral blood leukocyte populations from normal and atopic individuals: a comparative study.

BACKGROUND: Several previous reports have suggested an increased activity of cAMP phosphodiesterases (PDEs) and a higher sensitivity of these enzymes toward PDE inhibitors in leukocytes of patients suffering from atopic dermatitis. OBJECTIVE: The purpose of the present study was to comprehensively analyze and compare the PDE expression and activity profile of highly purified populations of leukocytes from normal and atopic blood donors. In addition, the influence of PDE inhibitors on function of leukocytes from normal and atopic individuals was investigated. METHODS: Density gradient centrifugation, elutriation, and magnetic cell sorting techniques were used to purify eosinophils, monocytes, and B and T lymphocytes from peripheral human blood. Complementary DNA-polymerase chain reaction was used to analyze PDE4 subtype messenger RNA (mRNA) expression levels in addition to PDE isoenzyme activities. PDE4 inhibitor sensitivity was determined in monocyte homogenates from both groups. Functionally, suppression of lipopolysaccharide-induced synthesis of tumor necrosis factor-alpha in monocytes as well as phytohemagglutinin-induced T cell proliferation in peripheral blood mononuclear cell fractions by PDE4 and PDE3/4 inhibitors was compared. RESULTS: Identical PDE activities and mRNA expression profiles were found in all cells from normal and atopic donors except that there was an increase in the mRNA levels of PDE4A and PDE4B2 in atopic T cells, which was, however, not reflected in overall PDE4A activity. In addition, no differences in sensitivity of the functional responses to PDE inhibitors were noted. The mixed PDE3/4 inhibitor zardaverine was a more potent inhibitor of T cell proliferation than rolipram, a selective PDE4 inhibitor. CONCLUSION: No evidence for alterations of PDE activities in atopy is provided by our findings.

The dihydropyridine dexniguldipine hydrochloride inhibits cleavage and religation reactions of eukaryotic DNA topoisomerase I.

Dexniguldipine hydrochloride (B859-35, a dihydropyridine with antitumor and multidrug resistance-reverting activity) inhibits both the DNA cleavage and religation reactions of purified human DNA topoisomerase I at concentrations >1 microM, whereas at concentrations <1 microM it inhibits selectively the religation step and stabilizes the covalent topoisomerase I-DNA intermediate in a similar fashion as camptothecin. Inhibition of religation by camptothecin can be overcome by increasing the concentration of the DNA substrate in the religation reaction, indicating a competitive type of inhibition. In contrast, dexniguldipine hydrochloride decreases rate constants of topoisomerase I-mediated DNA religation independently of the concentration of the DNA substrate, suggesting a noncompetitive mechanism of inhibition, which is different from that of camptothecin.

Superantigen-mediated cellular cytotoxicity is dependent on antigen expression, but independent of the P-glycoprotein multidrug resistance phenotype.

Superantigen-activated T cells can be targeted by monoclonal antibodies (mAb) to lyse MHC class II negative tumour cells. In this study we determined the susceptibility of the T-lymphoblastoid leukaemic cell line CCRF-CEM and its multidrug resistant sublines CCRF VCR100, CCRF VCR1000 and CCRF ADR5000 to lysis by monoclonal antibody-targeted and superantigen-activated T cells (superantigen-dependent cellular cytotoxicity, SDCC). A recombinant fusion protein of protein A and the superantigen Staphylococcus enterotoxin A (SEA) was used together with the mAbs anti-CD7, anti-CD38, anti-CD45RA and 4E3 (anti-P-glycoprotein) to correlate susceptibility to SDCC with expression of the MDR1-gene product. Our results demonstrated SDCC to be independent of MDR1-gene expression. This was further confirmed by blocking the function of Pgp in the leukaemic cell lines with a cyclosporine A derivative, which had no influence on SDCC. As expected, expression of the respective cell surface antigens on target cells had a strong impact on SDCC, although other factors seem to influence efficiency of SDCC as well.

Effects of the selective bisindolylmaleimide protein kinase C inhibitor GF 109203X on P-glycoprotein-mediated multidrug resistance.

Inhibition of protein kinase C (PKC) is discussed as a new approach for overcoming multidrug resistance (MDR) in cancer chemotherapy. For evaluation of this concept we applied the bisindolylmaleimide GF 109203X, which shows a highly selective inhibition of PKC isozymes alpha, beta 1, beta 2, gamma, delta and epsilon in vitro. The efficacy of this compound in modulation of MDR was examined using several P-glycoprotein (P-gp)-overexpressing cell lines including a MDR1-transfected HeLa clone, and was compared with the activities of dexniguldipine-HCI (DNIG) and dexverapamil-HC1 (DVER), both of which essentially act via binding to P-gp. As PKC alpha has been suggested to play a major role in P-gp-mediated MDR, cell lines exhibiting different expression levels of this PKC isozyme were chosen. On crude PKC preparations or in a cellular assay using a cfos(-711)CAT-transfected NIH 3T3 clone, the inhibitory qualities of the bisindolylmaleimide at submicromolar concentrations were demonstrated. At up 1 microM final concentrations of the PKC inhibitor GF 109203X, a concentration at which many PKC isozymes should be blocked substantially, no cytotoxic or MDR-reversing effects whatsoever were seen, as monitored by 72 h tetrazolium-based colorimetric MTT assays or a 90 min rhodamine 123 accumulation assay. Moreover, depletion of PKC alpha by phorbol ester in HeLa-MDR1 transfectants had no influence on rhodamine 123 accumulation after 24 or 48 h. MDR reversal activity of GF 109203X was seen at higher final drug concentrations, however. Remarkably, [3H]vinblastine-sulphate binding competition experiments using P-gp-containing crude membrane preparations demonstrated similar dose dependencies as found for MDR reversion by the three modulators, i.e. decreasing efficacy in the series dexniguldipine-HCl > dexverapamil-HCl > GF 109203X. Similar interaction with the P-gp in the micromolar concentration range was revealed by competition of GF 109203X with photoincorporation of [3H]azidopine into P-gp-containing crude membrane preparations. No significant effect of the PKC inhibitor on MDR1 expression was seen, which was examined by cDNA-PCR. Thus, the bisindolylmaleimide GF 109203X probably influences MDR mostly via direct binding to P-gp. Our work identifies the bisindolylmaleimide GF 109203X as a new type of drug interacting with P-gp directly, but does not support the concept of a major contribution of PKC to a P-gp-associated MDR, at least using the particular cellular model systems and the selective, albeit general, PKC inhibitor GF 109203X.

MDR1, MRP, topoisomerase IIalpha/beta, and cyclin A gene expression in acute and chronic leukemias.

Gene expression was analyzed by cDNA-PCR at the mRNA level in bone marrow samples (>80% blasts) from ALL (28 primary, 22 first relapses, 10 recurrent relapses), from AML (14 primary, 23 relapses), In peripheral blood lymphocytes from CLL (five untreated, 10 treated), in one CML in blast crisis in the course of the disease (four samples), and in bone marrow samples from healthy donors (12 specimens). We found low mean MDR1 expression in primary ALL, first relapses of ALL, and primary AML. Significantly higher mean relative MDR1 expression levels were seen in recurrent relapses of ALL, and in the group of relapsed state AML. MDR1 expression measured intermediate in bone marrow samples from healthy donors. The CLL lymphocytes showed generally relatively high MDR1 expression levels. MRP gene expression measured very similar in primary ALL, first relapses of ALL, primary AML, and normal bone marrow. Significantly increased MRP mRNA levels were observed in the groups of recurrent ALL and relapsed state AML. CLL lymphocytes also showed high MRP expression levels. A combined increase of MDRI (about 20-fold) and MRP (about four-fold) was monitored in samples obtained from the CML in blast crisis after chemotherapy. While no significant differences of the mean topoisomerase IIbeta mRNA levels were found throughout, a significantly decreased topoisomerase IIalpha gene expression was measured in first and recurrent relapses of ALL. In CLL lymphocytes either the expression of the topoisomerase IIalpha gene was not detectable by cDNA-PCR, or it measured very low. Topoisomerase IIalpha gene expression was correlated to cyclin A gene expression in the samples of acute leukemias, Indicating the link of topoisomerase IIalpha expression to the proliferative activity of these leukemic blast cells. Our results point to a potentially multifactorial emergence of multidrug resistance in particular states and types of leukemias.

Structural requirements for activity of propafenone-type modulators in P-glycoprotein-mediated multidrug resistance.

The sodium channel blocker propafenone and a series of analogs have been identified as effective modulators of P-glyco-protein-mediated multidrug resistance in human tumor cells. A series of closely related structural homologues showed a highly significant correlation between lipophilicity and pharmacological effect. Reduction of the carbonyl group as well as conversion to a methylether led to a remarkable decrease in activity, whereby lipophilicity lost its predictive character as the main determinant for modulator potency. Similarly, the relative positioning of the acyl- and propanolamine side chains also influences activity, so the distance between carbonyl group and nitrogen atom seems important.

Modulation of P-glycoprotein mediated drug accumulation in multidrug resistant CCRF VCR-1000 cells by chemosensitisers.

P-glycoprotein (PGP) mediated transport of cytostatic drugs out of resistant cancer cells is a major cause of experimental and probably also of clinical multidrug resistance, which often leads to treatment failure during chemotherapy. The broad substrate specificity of PGP strongly restricts effective chemotherapy and diminishes the patients' prognosis. Inhibition of PGP's pumping function by chemosensitisers is one way to restore cellular responsiveness to otherwise ineffective cytostatics. Clinical trials with several chemosensitisers are under way. To date, it is not clear whether a certain chemosensitiser potentiates the action of different cytostatic drugs, transported by PGP equally well, or whether the chemosensitising potency is dependent on the cytostatic drugs used. Therefore, we compared the effects of five potent chemosensitisers on cellular accumulation using [3H]daunomycin, [3H]vincristine and rhodamine-123 as substrates for PGP. The acridonecarboxamide derivative GF 120918 was the most potent compound and a half-maximal effect was seen at concentrations ranging from 5 nM for rhodamine-123 accumulation to 14 and 19 nM for [3H]vincristine or [3H]daunomycin accumulation, respectively. The new chemosensitiser B9203-016 was slightly less effective than GF 120918 in all three test systems. Dexniguldipine was of intermediate potency with half-maximal effects at concentrations between 62 and 194 nM. The cyclic undecapeptide SDZ PSC 833 showed somewhat lower potency ranging from 151 to 331 nM. Cyclosporin A was less potent than SDZ PSC 833. Furthermore, enhancement of drug accumulation produced by each chemosensitiser was similar, regardless of which PGP substrate was measured, that is, the rank order of potency to increase accumulation was the same in each of the assays used. Our data point to similar, if not identical, mechanisms of drug transport by PGP and inhibition of drug transport by chemosensitisers at least for the substrates rhodamine-123, vincristine and daunomycin.

Expression of PKC isozyme and MDR-associated genes in primary and relapsed state AML.

For investigation of relative differences in mRNA expression levels and of correlations in the expression of genes possibly involved in multidrug resistance (MDR) of acute myelogenous leukemias (AML), a complementary DNA polymerase chain reaction (cDNA-PCR) analysis was established for the genes encoding MDR1/P-glycoprotein, the multidrug resistance-associated protein (MRP), topoisomerase II alpha, topoisomerase II beta, topoisomerase I, glutathione S-transferase pi, protein kinase C (PKC) isozymes alpha, beta 1, beta 2, epsilon, eta, theta and cyclin A. In a first descriptive study comprising samples of childhood or adult AML we calculated the mean values from primary (n=14) or relapsed (n=23) states of the diseases, respectively. We found in the latter significant increases of MDR1, MRP, gst pi, and PKC theta gene expression. MDR1 and MRP gene expression levels were generally correlated (rs= +0.4128, P<0.02, n=37), as well as topoisomerase II alpha and cyclin A gene expression levels (rs= +0.8727, P<0.0001, n=35). Within the group of relapsed state AML a significant negative correlation between the gene expression levels of MDR1 and topoisomerase II alpha (rs= -0.5500, P<0.01, n=22) was observed. Remarkably, highly significant positive correlations were found for MDR1/PKC eta (rs= +0.5560, P<0.001, n=32), MRP/PKC theta (rs= +0.6573, P<0.0001, n=34) and MRP/PKC eta (rs= +0.5241, P<0.005, n=32).