Drug-sensitive P388 Research Articles

Comparison of cyclosporin A, verapamil, PSC-833 and cremophor EL as enhancing agents of VP-16 in murine lymphoid leukemias

Although verapamil, cyclosporin A, cremophor EL and PSC-833 are active as multidrug resistance modulators, there has been limited study of these compounds as possible chemotherapy enhancing agents against drug-sensitive tumors. We compared these agents as modifiers of VP-16 cytotoxicity in vitro and modifiers of VP-16 efficacy in vivo against drug-sensitive P388 and L1210 leukemias. Our study indicates that cyclosporin A enhances VP-16 cytotoxicity to a significantly greater extent than equimolar concentrations of verapamil or PSC-833. Although cremophor EL shows significantly greater activity than verapamil in VP-16 cytotoxicity enhancement in vitro, it is ineffective when added to VP-16 therapy of mice bearing L1210 leukemia.

INDUCTION OF RESISTANCE TO DAUNORUBICIN IN DRUG-SENSITIVE LEUKEMIA P388 CELLS - A ROLE OF PKC-BETA-I ISOZYME

A role of protein kinase C (PKC) has been suggested in multidrug resistance (MDR). Because of the molecular and biochemical heterogeneity of PKC, we examined a role of PKC beta isozyme in drug sensitive murine leukemia P388 cell line. Drug sensitive P388 and MDR P388/ADR cells were treated with various concentrations of 12-deoxyphorbol-13-O-phenylacetate 20 acetate (DPPA, an agonist of PKC beta I isozyme) and examined for its effect on daunorubicin (DNR) accumulation and sensitivity to DNR. dPPA increased DNR resistance and decreased DNR accumulation in P388 cells but had no effect in P388/ADR cells. The reduced dPPA-induced DNR accumulation was due to decreased uptake without any effect on DNR efflux. Furthermore, treatment of P388 cells with dPPA was associated with translocation of PKC beta isozyme from cytosol to plasma membrane. These data suggest that PKC beta I isozyme plays a role in acquired drug resistance.

EFFECT OF 12-DEOXYPHORBOL 13-PHENYLACETATE ON DAUNORUBICIN RESISTANCE AND CALCIUM-INDEPENDENT PROTEIN-KINASE-C ISOZYMES IN DRUG-SENSITIVE MURINE LEUKEMIA P388 CELLS

Because of a suggested role of protein kinase C (PKC) in multidrug resistance (MDR) and recent. demonstration of molecular and biochemical heterogeneity of PKC, we examined the effect of 12-deoxyphorbol 13-phenylacetate (dPP) on intracellular accumulation and drug sensitivity of daunorubicin (DNR) in drug sensitive P388 murine leukemia cell line. In addition, effect of dPP on Ca++-independent PKC (delta, epsilon and zeta) isozymes was examined with Western blot, using laser densitometry. dPP induced significant resistance to DNR in P388 cells, however had no significant effect on DNR accumulation. In addition, dPP increased the levels of plasma membrane associated PKCdelta and epsilon isozymes in P388 cells and but had no effect on plasma membrane-associated PKCzeta isozyme. This study demonstrates a role of Ca++-independent PKCdelta and epsilon isozymes in drug resistance.

Possible coexistence of two independent mechanisms contributing to anthracycline resistance in leukaemia P388 cells

Murine leukaemia P388 and L1210 cell sublines with varying degrees of resistance to the anthracycline daunomycin (DNM) have been used to monitor (i) intracellular accumulation of DNM, (ii) expression of the drug efflux pump P-glycoprotein (pgp) and (iii) cytoplasmic pH changes. Drug-resistant L1210 65 cells (65-fold resistance), overexpress pgp, and display decreased intracellular accumulation of DNM and identical intracellular pH as compared to the parental drug-sensitive L1210 cell line. On the other hand, moderately drug-resistant P388 20 cells (20-fold resistance), which also exhibit a decreased intracellular drug accumulation with respect to drug-sensitive P388 S cells, display only moderate pgp-encoding mdr1 gene transcription without detectable levels of pgp protein, and undergo cytoplasmic alkalinisation (up to ∼0.2 pH units). A further increase in the level of drug resistance ( P388 100 cells, 100-fold resistance), results in a more pronounced decrease in drug accumulation, significant pgp expression and slightly higher intracellular alkalinisation. Alterations in the degree of protonation of DNM have been shown previously to influence processes such as the rate of uptake and the intracellular accumulation of the drug. On this basis, we propose that the changes in intracellular pH, observed at low levels of drug resistance ( P388 20 cells), could constitute an early cellular response aimed at decreasing the intracellular accumulation of ionisable anti-neoplastics. As the level of resistance increases ( P388 100 ), the cells seem to require more efficient mechanisms of defense against the drug, such as that represented by the expression of pgp. Since there is no apparent correlation between the extent of the changes in intracellular pH and the level of pgp expression in DNM-resistant P388 cell sublines, it is suggested that these two cellular responses contributing to drug resistance could operate independently.

Verapamil reverses the ultrastructural alterations in the plasma membrane induced by drug resistance

Two P388 cell sublines with different levels of resistance to daunomycin (DNM), P388/20 and P388/100 cells (∼ 20- and 100-fold resistance, respectively), undergo a significant (∼ 2-fold) increase in the number of intramembrane particles (IMPS) present at their plasma membrane, as compared to that exhibited by the parental, drug-sensitive P388 (P388/S) cell line. Regardless of the level of resistance, incubation of drug-resistant cells with verapamil, a well known reverting agent of anthracycline resistance, restores the morphology of the plasma membrane in these cells, yielding a pattern in which the number and size distribution of IMPs at both leaflets of the bilayer, become undistinguishable from those displayed by drug-sensitive celIs. Furthermore, verapamil did not affect the ultrastructural organization of the plasma membrane of drug-sensitive cells. It is possible that the alterations in the structural organization of the plasma membrane of the antineoplastic-resistant tumor cells, might represent a reliable ‘marker’ for early diagnosis of drug resistance.

Protein kinase C isoforms in multidrug resistant P388/ADR cells: a possible role in daunorubicin transport

To identify the role of protein kinase C (PKC) isoforms in multidrug resistance in tumor cells, we examined the PKC isoform pattern in the multidrug resistant P388/ADR cell line and studied the effect of down regulation of PKC isoforms on intracellular daunorubicin accumulation and P-glycoprotein expression. Using monoclonal antibodies to PKCα, β and γ and flow cytometry technique we showed that P388/ADR cells overexpressed PKCα and β as compared to drug sensitive P388 cells. Prolonged treatment of P388/ADR cells with phorbol myristate acetate (PMA), a procedure that is known to down regulate PKC, resulted in the down regulation of total PKC activity and the PKCβ isoform (at the protein level) that was accompanied by the correction of daunorubicin accumulation in P388/ADR cells. The level of expression of P-glycoprotein in PMA treated cells was similar to that of untreated cells. These results suggest that PKCβ regulates the drug efflux function of P-glycoprotein.

Improvement of flow-cytometric detection of multidrug-resistant cells by cell-volume normalization of intracellular daunorubicin content.

To improve the ability of flow cytometry to detect multidrug-resistant cells, we studied the extent to which cell volume heterogeneity accounts for the variance of intracellular daunorubicin (DNR) content. For P388 murine or HL-60 human leukemia cells exposed to DNR (1 micrograms/ml, 60 min), log intracellular DNR content varied in direct proportion to log cell volume measured by flow cytometry, with a correlation coefficient of .9. This relationship was confirmed by cell sorting based on intracellular DNR content with subsequent volume determination of the sorted cells. Normalization of intracellular DNR content for cell volume (thus obtaining intracellular DNR concentration) was accomplished by subtracting log cell volume from log intracellular DNR content for each cell. This resulted in a 34% decrease (range 23-58%) in standard deviation compared to DNR content measurements without volume normalization for all cell types tested. Following exposure to DNR (as above), intracellular DNR content of drug-sensitive P388 or HL-60 cells measured by flow cytometry was 12- and 8-fold greater than that of the multidrug-resistant sublines P388/ADR and HL-60/AR, respectively. However, because of the variance of intracellular DNR content, the predictive value of flow-cytometric determination of intracellular DNR content as a discriminant assay for detecting the frequency of drug-resistant cells in a mixed population was acceptable only when the frequency of resistant cells in the population exceeded 10%. In contrast, volume normalization of intracellular DNR content enhanced the ability of the flow-cytometric assay to discriminate resistant cells by 10-fold for P388 cells and 100-fold for HL-60 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of drug efflux blockers on vital staining of cellular DNA with hoechst 33342

The present study shows that staining of certain live cells, e.g., adriamycin-resistant P388 cells, by Hoechst 33342 is difficult because of the presence of a rapid efflux pump, which reduces intracellular dye concentration. Coincubation of these refractory cells in the presence of efflux blockers such as phenothiazines (trifluoperazine) or Ca++ channel blockers (verapamil) enhances dye retention and thus leads to generation of normal DNA distribution histograms. Laser flow cytometric data is confirmed by fluorometric assays, which show that P388/R cells retain one-third the amount of Hoechst 33342, and coincubation with efflux blockers increases Hoechst retention to values similar to those of drug-sensitive P388 cells. DNA histograms of mouse splenocytes incubated with Hoechst 33342 alone have a bimodal distribution possibly because of the presence of subpopulations that do not retain the fluorochrome owing to rapid efflux. Coincubation with an efflux blocker results in the generation of unimodal DNA histograms from these cells. These preliminary studies suggest that reduced retention of Hoechst 33342 in certain cell types (because of rapid efflux) can be blocked by efflux blockers, thus leading to generation of typical DNA distribution histograms.