Expression of drug resistance-associated mdr-1, GST pi, and topoisomerase II genes during cell cycle traverse.

The expression of drug resistance-associated mdr-1, GST pi, and topoisomerase II genes was analyzed in cell cycle phase enriched populations of doxorubicin-resistant murine leukemic P388/R-84 cells. Flow cytometric analysis of bromodeoxyuridine (BrdU) incorporation and staining with anti-BrdU antibodies was used to confirm the purity of cell cycle phase enriched populations obtained by centrifugal elutriation. Doxorubicin (DOX) and daunorubicin (DNR) accumulation was significantly lower in S-phase cells, and coincubation with verapamil (VPL) or chlorpromazine (CPZ) enhanced DOX and DNR accumulation more in S-phase than in G1- and G2/M-phase cells. While the cellular content of mdr-1 and topoisomerase II mRNAs changed, GST pi mRNA content remained constant during the cell cycle. S-phase cells had about 3-fold higher mdr-1 mRNA content than G1- and G2/M-phase cells. In G1 cells, P-glycoprotein expression, as determined by C219 monoclonal antibody, was 12% less than that of S and G2/M cells. Topoisomerase II mRNA content increased with the progression of cell cycle and peaked in G2/M cells. These observations suggest that cell cycle stage related changes in expression of drug resistance markers may have a major bearing on chemosensitivity of drug-resistant cells.

Prochlorperazine as a doxorubicin-efflux blocker: phase I clinical and pharmacokinetics studies.

Doxorubicin (DOX) efflux in drug-resistant cells is blocked by phenothiazines such as trifluoperazine (TFP) and prochlorperazine (PCZ) in vitro. The present phase I study was conducted in 13 patients with advanced, incurable, nonhematologic tumors to determine whether PCZ plasma levels high enough to block DOX efflux could be achieved in vivo. The treatment schedule consisted of prehydration and i.v. administration of 15, 30, 50, and 75 mg/m2 PCZ followed by a standard dose of 60 mg/m2 DOX. The hematologic toxicities attributable to DOX were as expected and independent of the PCZ dose used. Toxicities attributable to PCZ were sedation, dryness of the mouth, cramps, chills, and restlessness. The maximal tolerated dose (MTD) of PCZ in this schedule was 75 mg/m2. Pharmacokinetic analysis indicated a large interpatient variation in peak plasma PCZ levels that ranged from 95 to 1100 ng/ml. The three plasma half-lives of PCZ were: t1/2 alpha (+/- SE), 20.9 +/- 5.3 min; t1/2 beta, 1.8 +/- 0.3 h; and t1/2 gamma, 21.9 +/- 5.3 h. The volume of distribution (Vd), total clearance (ClT), and area under the curve (AUC) for PCZ were 2254 +/- 886 l/m2, 60.2 +/- 13.5 l m-2 h-1, and 1624 +/- 686 ng ml-1 h, respectively. DOX retention in tumor cells retrieved from patients during the course of therapy indicated the appearance of cells with enhanced DOX retention. The combination of DOX and high-dose i.v. PCZ appeared to be safe, well tolerated, and active in non-small-cell lung carcinoma.

Flow cytometric studies on modulation of cellular adriamycin retention by phenothiazines.

Phenothiazines (chlorpromazine and trifluoperazine) enhance the cellular retention and cytotoxicity of adriamycin in P388/S and P388/R cells. Effects on the resistant cells are more pronounced than those on the sensitive cells. Multiparametric flow-cytometric determination of cellular Adriamycin fluorescence shows that the phenothiazine effects are not uniform in a cell population, and cells in later part of the cell cycle traverse (S and G2/M), mitogen-stimulated lymphocytes (as compared to nonstimulated lymphocytes), and ascites (as compared to splenocytes) have greater sensitivity to phenothiazine effects on Adriamycin retention.

Suppressor activity of splenocytes from mice treated with Ecteinascidia turbinata extract.

Extracts of a marine tunicate, Ecteinascidia turbinata (Ete) were previously shown to be capable of suppressing humoral and cellular immune responses in vivo and in vitro. In the present work we have examined the mechanisms of suppression in Ete-treated DBA/2 and BALB/c mice. Treatment with Ete resulted in a significant splenomegaly accompanied by a diminished response to mitogenic stimulation, reaching coincident maxima of effect at days 4 to 6. Spleen size and blastogenic reaction returned to normal at 12 to 21 days. Cells from enlarged spleens inhibited blastogenic responses of normal splenocytes to Con A. Marker studies indicated that the suppressor activity was exerted by cells possessing T lymphocyte characteristics.

Ecteinascidia turbinata extracts inhibit DNA synthesis in lymphocytes after mitogenic stimulation by lectins.

Aqueous ethanol extract of a tunicate which was previously found to exert antitumor and immunosuppressive activities in vivo was tested for its effect on normal human lymphocytes in vitro. The extract suppressed the uptake of tritiated thymidine by lymphocytes stimulated with mitogen. This suppressive effect did not require continuous presence of the extract. Treatment of lymphocytes prior to mitogenic stimulation resulted in suppressive effect. The fact that suppression by the extract could also be achieved 24 hr after exposure to mitogen, an interval which was found to suffice for the attainment of maximal commitment for blastogenic transformation indicates that Ete can act at a stage subsequent to the binding of the lectin and elicitation of a mitogenic signal(s).