Search

Tumor cell resistance to chemotherapeutic agents of diverse structure and mechanism of action is thought to be due to efflux of drug by P-glycoprotein, which is overexpressed in tumor cells with the multidrug-resistant phenotype. Agents generally associated with the multidrug-resistant phenotype include inhibitors of topoisomerase II, e.g., doxorubicin, etoposide, and the microtubule poisons such as vinblastine, vincristine (VCR), and taxol. The antiepileptic drug phenytoin (DPH), an inhibitor of tubulin polymerization, potentiates (P < 0.05) the cytotoxicity of the chemotherapeutically useful microtubule poison VCR in tumor cells with the wild-type or multidrug-resistant phenotype. Among agents associated with the multidrug-resistant phenotype, the modulation of cytotoxicity by DPH was selectively effective with the microtubule poison VCR but not the topoisomerase II inhibitor doxorubicin. The potentiation of vincristine cytotoxicity by DPH was not due to binding to P-glycoprotein or by increasing VCR accumulation. We thus propose a novel mechanism for the modulation of resistance based on evidence that DPH at noncytotoxic concentrations can selectively enhance the cytotoxic potential of vincristine without interfering with P-glycoprotein function. Thus, studies with phenytoin could assist in characterizing other molecular determinants of multidrug resistance and the design of trials to modulate drug efficacy.

The effect of thaliblastine (TBL, NSC-68075), a plant alkaloid, in over-coming multidrug resistance was investigated in doxorubicin (ADR)-resistant murine leukemic P388/R-84 cells. In the soft agar clonogenic assay, a nontoxic concentration of TBL (2 microM) reduced the 50% inhibitory dose of ADR (1-h exposure) from 10.8 to 1.4 microM with a dose modification factor of 7.7. Continuous treatment of P388/R-84 cells with ADR and TBL for 24 h further lowered the 50% inhibitory dose from 3.5 to 0.07 microM, the resistance level being decreased from 233-fold in the absence of TBL to 4.7-fold in the presence of TBL as compared to the parental P388 cells. Although ADR or TBL individually had no detectable effects on cell cycle traverse, the combination of the two drugs caused a significant G2 block. Flow cytometric analysis showed that TBL enhanced ADR retention in P388/R-84 cells in a dose- and time-dependent manner. TBL partially blocked the photolabeling of P-glycoprotein with [3H]azidopine, and this blocking effect was further enhanced in combination with ADR. Our results indicate that TBL can reverse multidrug resistance by direct interaction with P-glycoprotein, thereby increasing cellular ADR retention.

The phagolysosome is perhaps the most effective antimicrobial site within macrophages due both to its acidity and to its variety of hydrolytic enzymes. Few species of pathogens survive and multiply in these vesicles. However, one strategy for microbial survival would be to induce a higher pH within these organelles, thus interfering with the activity of many lysosomal enzymes. Altering the intravesicular milieu might also profoundly influence antigen processing, antimicrobial drug delivery, and drug activity. Here we report the first example of an organism proliferating within phagolysosomes that maintain a relatively neutral pH for a sustained period of time. We inoculated P388D1 macrophages with fluorescein isothiocyanate (FITC)-labeled Histoplasma capsulatum or zymosan. Using the ratio of fluorescence excitations at 495 and 450 nm, we determined that vesicles containing either virulent or avirulent FITC-labeled H. capsulatum yeasts had a pH one to two units higher than vesicles containing either zymosan or methanol-killed H. capsulatum. The difference in pH remained stable for at least 5.5 h postinoculation. Longer-term studies using cells preincubated with acridine orange indicated that phagolysosomes containing live Histoplasma continued to maintain a relatively neutral pH for at least 30 h. Many agents raise the pH of multiple vesicles within the same cell. In contrast, H. capsulatum affects only the phagolysosome in which it is located; during coinoculation of cells with unlabeled Histoplasma and labeled zymosan, organelles containing zymosan still acidified normally. Similarly, unlabeled zymosan had no influence on the elevated pH of vesicles housing labeled Histoplasma. Thus, zymosan and Histoplasma were segregated into separate phagolysosomes that responded independently to their phagocytized contents. This localized effect might reflect an intrinsic difference between phagosomes housing the two particle types, active buffering by the microbe, or altered ion transport across the phagolysosomal membrane such that acidification is inhibited.

Annamycin (AN) is an anthracycline antibiotic with high affinity for lipid membranes which is being developed for clinical studies formulated in liposomes. We studied the in vitro cytotoxicity, cellular pharmacology, and DNA damage induced by AN in P388 cells sensitive and resistant to doxorubicin (DOX). AN was as cytotoxic as DOX against P388-sensitive cells and about 50 times more cytotoxic than DOX against P388-resistant cells (resistance index 5 for AN versus 250 for DOX). Cellular uptake of AN by sensitive cells was 2-3-fold higher than that of DOX. In resistant cells, cellular uptake of AN and DOX was approximately 65% and 30%, respectively, of the cellular uptake in sensitive cells. As a result, cellular uptake of AN by resistant cells was higher than uptake of DOX by sensitive cells. DOX was fully retained in sensitive cells while it was effluxed rapidly from resistant cells. In contrast, efflux of AN was similar in sensitive and resistant cells, thus suggesting that it is not mediated by P-glycoprotein. AN was more effective than DOX in inducing single DNA breaks, double DNA breaks, and DNA-protein cross-links, both in sensitive and resistant cells, although DNA damage was lower in resistant cells than in sensitive cells. DNA lesions induced by AN in resistant cells were similar to or greater than those induced by DOX in sensitive cells. These studies indicate that the lack of cross-resistance between DOX and AN appears to be related, at least in part, to the relatively higher cellular uptake of AN compared with DOX and is associated with the ability of AN to induce significant DNA damage in resistant cells.

In independently derived drug-resistant sublines of the mouse lymphoid tumor P388, multidrug resistance is associated with the exclusive overexpression of the mdr3 gene. In P388/VCR cells, mdr3 overexpression occurs in the absence of gene amplification, while in P388/ADM-2 cells overexpression is associated with mdr3 gene amplification. The mechanism underlying mdr3 overexpression in these cells was investigated. Measurement of the rate of transcription by nuclear "run-on" assays showed that increased mdr3 expression in P388/VCR cells was caused by transcriptional activation of the gene. Analysis of the 5' end of mdr3 mRNA transcripts by primer extension indicated that in P388/VCR cells, these mRNAs extended approximately 200 nucleotides upstream exon 2, about 60 nucleotides longer than their counterparts expressed in normal tissues from the known transcription start site of the gene (TS1). Northern blotting experiments using discrete exon and intron probes derived from the 5' end of the gene near TS1, together with ribonuclease protection using a complementary RNA probe from the same region, demonstrated that transcriptional activation in P388/VCR cells occurred from a novel transcription start site named TS3, located either upstream of TS1 or within intron 1 at a site immediately upstream a novel exon. In P388/ADM-2 cells, Northern blotting and ribonuclease protection identified overexpressed mdr3 mRNAs initiating near TS1 and a large partially spliced mdr3 mRNA species initiating upstream of TS1 at a novel initiation site designated TS2. Therefore, mdr3 overexpression in independently derived multidrug-resistant isolates of P388 cells is associated with the appearance of novel transcription start sites in the gene and novel sequences at the 5' end of the overexpressed mRNAs.

Reserpine, the crystalline active substance isolated from the Rauvolfia plant, produces a characteristic vasodepressor effect in hypertensive patients. Apart from its antihypertensive property, reserpine also possesses transquillising and vasodepressor action, hence it is employed as supportive therapy in the treatment of cardiac disorders. Doxorubicin is a potent anticancer agent, the use of which is limited by its cumulative dose-dependent cardiotoxicity. Epirubicin is a derivative of doxorubicin having more favourable therapeutic index than doxorubicin and possessing less hematologic and cardiac toxicity at comparable doses. The data presented in this paper show the effect of reserpine as a chemosensitizer, when used in combination with epirubicin on P388 murine leukemia cells sensitive (P388/S) and resistant to doxorubicin (P388/DOX) cells. Inhibition of 3H-TdR incorporation into DNA was used as an index of the cytotoxic effects of drug when used alone or in combination. The combination of reserpine (1 microM) and epirubicin (1.7, 8.6 and 17.2 microM) indicated a significant enhancement in the DNA biosynthesis inhibition in P388/S and P388/DOX cell lines. The most prominent feature of the multidrug-resistant cell is the reduced accumulation of the drug intracellularly. P388/DOX cells showed less accumulation of epirubicin in the cell as compared to that of the parental cell line. Further studies demonstrated that reserpine significantly enhanced the intracellular accumulation of epirubicin in both the cell lines. The nature of DNA damage caused by the combination of reserpine and epirubicin was irreversible when studied in P388/DOX cell line. The combination of reserpine (5mg/kg) and epirubicin (1mg/kg) significantly potentiated the antitumor activity of epirubicin in P388/DOX tumor bearing mice. These studies suggest that reserpine can be used as an adjuvant in the cancer chemotherapy to potentiate the antiproliferative activity of anticancer drugs.

We synthesized and developed a new tumor imaging agent, 111In-labeled metalloporphyrin (111In-ATN-10) which consists of a carrier (ATN-10) of the tumor imaging possessing both a non-radioactive manganese complex in the porphyrin ring and a bifunctional chelating group (DTPA) attached to its side chain. The images of the three kinds of tumors were delineated more clearly by 111In-ATN-10 than 67Ga-citrate. Moreover, there was no photosensitivity in ATN-10. 111In-ATN-10 is studied as a new tumor positive scintigraphic agent instead of 67Ga-citrate.

KW-2149 is a new derivative of mitomycin C (MMC). The plasma concentrations, distribution, metabolism, and excretion of [3H]-KW-2149 in normal and tumor-bearing mice after i.v. administration of 16.6 mg/kg were investigated. The plasma radioactivity decreased biexponentially after i.v. administration in normal mice. However, the unchanged drug disappeared rapidly, showing a half-life (t1/2) of 9.7 min, which was shorter than MMC's (18 min). The radioactivity was excreted in mouse urine (33%) and feces (58%) within 144 h. High radioactivity was distributed in the gallbladder, liver, kidney, pancreas, and lung at 1 h after i.v. administration to normal mice. The tumor concentration was lower than the plasma or blood concentration. The lowest radioactivity was observed in the brain. The metabolic rate of KW-2149 was very rapid. The methyl sulfide form (M-16), the symmetrical disulfide dimer (M-18), and the albumin conjugate were detected in plasma, which possessed anticellular activity. The specific anticellular activity of these compounds against uterine carcinoma (HeLa S3) was 1/100, 1, and 1/20 respectively, as compared with that of KW-2149.

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 approximately 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.

KW-2149, a new mitomycin C (MMC) derivative, inhibited the growth of murine P388 leukemia in vitro at 20-fold lower concentrations than those of MMC. KW-2149 was also effective in inhibiting the growth of MMC-resistant P388 (P388/MMC) cells. To elucidate these characteristics of KW-2149, its uptake and efflux were compared with those of MMC in MMC-sensitive and -resistant P388 cells. Both MMC and KW-2149 accumulated rapidly in P388 cells after incubation at the concentration of 0.47 and 0.024 microM, respectively, which were the IC50 values at 1-h exposure. Although this concentration of KW-2149 was 20 times lower than that of MMC, its intracellular concentration was little more than that of MMC, suggesting that KW-2149 accumulated in the cells quite efficiently. The accumulated KW-2149 in the cells after 1-h treatment remained for as long as 24 h after the incubation of the cells in drug-free medium, suggesting that most of the intracellular KW-2149 or MMC was bound to cellular components. The ratios of resistance of P388/MMC cells to MMC and KW-2149 were 34 and 8.8, respectively, at 1-h exposure, suggesting that P388/MMC cells were partially resistant to KW-2149 in vitro. P388/MMC cells also showed partial resistance to cisplatin, Adriamycin, m-AMSA, and etoposide. The accumulation of MMC in P388/MMC cells was lower than that in P388 cells, although the size of the former cells was almost equal to that of the latter. As a result, the amount of DNA-bound MMC was lower in P388/MMC cells than in P388 cells, suggesting its involvement in the mechanisms of MMC resistance in P388/MMC cells.

Inhibition by staurosporine derivatives of cyclic AMP-dependent protein kinase (A-kinase) and protein kinase C (C-kinase), and drug resistance has been investigated. The substitution of an acetyl or an ethoxycarbonyl group for the amine N-ethoxycarbonyl-7-oxostaurosporine moiety on the tetrahydropyran ring of staurosporine decreased inhibition of both protein kinases, but increased selectivity for C-kinase by further modification of the lactam moiety to the imide (NA-382). The activities of SF-2370 on protein kinases were decreased by decarboxylation and hydroxyalkylation. These staurosporine derivatives enhanced accumulation of vinblastine in adriamycin-resistant P388 (P388/ADR) cells in a dose-dependent manner. The potency for the drug accumulation of these compounds was correlated with their inhibitory activity on the drug efflux, but was not correlated with their activity on protein kinases. Staurosporine and NA-382, with high potency for vinblastine accumulation, inhibited the photolabelling of [3H]azidopine on 140 kDa P-glycoprotein in the plasma membrane. The tetrahydrofuran compounds and NA-357, which had low potency for the drug accumulation, hardly interacted with azidopine on P-glycoprotein. Most of these compounds were highly cytotoxic by themselves, and only NA-382 was less cytotoxic among them and completely reversed the vinblastine-resistance of P388/ADR cells at a non-cytotoxic concentration. These results suggest that staurosporine derivatives can enhance drug accumulation and inhibit drug resistance through their direct action on the P-glycoprotein.

The biochemical mechanism of the synergy of 5-fluorouracil (FUra) and cisplatin (CDDP) was studied using transplantable tumors in rodents in vivo. The reduced folate 5,10-methylenetetrahydrofolate (CH2FH4) and its precursor tetrahydrofolate (FH4) are essential cofactors for the formation of a tight ternary complex of thymidylate synthase (TS) and 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) derived from FUra. Intraperitoneal administration of CDDP (5 mg/kg) inhibited the incorporation of exogenous L-methionine into ascitic tumor cells and increased the levels of CH2FH4 and FH4 in ascitic Yoshida sarcoma and P-388 cells transplanted into rats and mice to levels about 2-3 times those measured in cells from animals that were not treated with CDDP. Preincubation with 10(-6) M FUra in Hanks' medium inhibited [6-3H]-2'-deoxyuridine incorporation into DNA of tumor cells from CDDP-treated rats 3 times more than that into cells from untreated rats, indicating that the inhibition of TS by FdUMP derived from FUra was enhanced in the presence of CH2FH4. Intraperitoneal administration of CDDP on day 1 and continuous infusion of FUra from day 1 to day 6 had synergistic effects in inhibiting tumor growth in Yoshida sarcoma-bearing rats. Oral administration of UFT, a combined form of 1 M tegafur and 4 M uracil, for 7 consecutive days beginning at 24 h after tumor implantation and a single i.p. injection of CDDP on day 1 had a significantly greater effect than did either agent alone. These results suggest that CDDP significantly enhances FUra cytotoxicity by inhibiting intracellular L-methionine metabolism and consequently increasing the reduced folate pool in mammalian tumor models in vivo.

BMY 25282 and BMY 25067, analogues of mitomycin C (MMC), were synthesized in an attempt to increase the therapeutic potential of the parent drug. The present studies were undertaken to determine if the cytotoxicity of MMC or its analogues is affected by cellular glutathione (GSH) and/or GSH transferase (GST) levels by using sensitive (P388/S) and multidrug resistant (P388/R-84) mouse leukemia cells as a model. P388/R-84 cells were cross-resistant to all three drugs. BMY 25067 was > 100 times more cytotoxic than MMC in both the cells. MMC and BMY 25282 produced significantly lower DNA interstrand cross-links (ISC) in P388/R-84 cells, whereas BMY 25067 induced ISC formation was comparable in these cells. GSH depletion with D,L-buthionine-S,R-sulfoximine (BSO) increased sensitivity to MMC, BMY 25282, and BMY 25067 by 3.4-, 4.1-, and 1.8-fold, respectively, in the resistant cells. Pretreatment of P388/R-84 cells with a nontoxic concentration of ethacrynic acid (EA) (10 micrograms/ml for 1 h), an inhibitor of GST activity, also resulted in a significant increase in the cytotoxic activities of MMC and BMY 25282 (3.8- and 3.1-fold, respectively), but not of BMY 25067. Combined pretreatment of P388/R-84 cells with BSO and EA caused further increase in the cytotoxic activities of both MMC and BMY 25282. Potentiation of BMY 25067 cytotoxicity by combined BSO and EA pretreatments was similar to that observed by BSO pretreatment alone. The ISC formation by MMC and BMY 25282 were also increased significantly by BSO or EA pretreatment in these cells. Whereas BSO treatment increased BMY 25067 induced ISC formation, it was not affected by EA pretreatment. These results suggest that (a) a potentiation of the cytotoxic activity of MMC or BMY 25282 can be achieved by GSH depletion and/or GST inhibition, (b) the enhanced cytotoxicity may be caused at least in part by the increased formation of drug-DNA cross-links, and (c) the mechanism of BMY 25067 cytotoxicity may be different from the other two drugs. The results of the present study also suggest that BMY 25067 may be seriously considered for further clinical development because it is much more active than MMC, and unlike the parent drug cytotoxicity of BMY 25067 does not seem to be affected by GST levels, which have been suggested to play an important role in cellular resistance to several cancer chemotherapy drugs.

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.

A new triazinoaminopiperidine derivative, Servier 9788 (S9788), was investigated for its ability to increase Adriamycin (ADR) accumulation and retention in two rodent (P388/ADR and DC-3F/AD) and three human (KB-A1, K562/R and COLO 320DM) cell lines displaying the P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) phenotype. Depending on the cell line S9788 was shown to be two to five times more active and five to 15 times more potent than Verapamil (VRP) in increasing ADR accumulation in resistant cells. ADR retention in KB-A1 cells maintained in a concentration of 10 microM S9788 was twice that in VRP-treated cells, and similar to that measured in the untreated sensitive KB-3-1 cells. Although 5 microM S9788 and 50 microM VRP gave the same values of ADR uptake in KB-A1 cells, S9788 was shown to induce a greater ADR retention following cell wash and post-incubation in resistance modifier- and ADR-free medium. Taking into account that S9788 had no effects on ADR accumulation and retention in sensitive KB-3-1 cells, it can be suggested that S9788 inhibits specifically the P-gp dependent ADR efflux, and in a manner less reversible than that observed with VRP. Moreover, [3H]azidopine photolabeling of P-gp, in P388/ADR plasma membranes, was completely inhibited by 100 microM S9788. Although S9788, as VRP, had no effect on the cell cycle of P388 cells, 5 microM S9788 increased 700-fold the efficacy of ADR to block P388/ADR cells in the G2+M phase of the cell cycle. Together, these results show that the sensitization, by S9788, of cell lines resistant to ADR is mainly due to an increase in ADR accumulation and retention, leading to an increase in the number of resistant cells blocked in the G2+M phase.

Taking into account the possible regulatory influences of the beta-adrenergic system on lymphocyte proliferation as well as the proposed role of cyclic 3'-5'-adenosine monophosphate (cAMP) in the modulation of multidrug resistance (MDR) in tumour cells, we have tried to assess the status of the interactions between the beta-adrenergic system and a mouse lymphocytic leukemia, the P388, both as a doxorubicin-sensitive (P388) and -resistant (MDR) variant (P388/DXR). P388 showed a low total number of high affinity 125I-pindolol binding sites (340 +/- 33/cell, Kd 108 pM) when compared with normal splenocytes (1221 +/- 67 sites/cell, Kd 97 pM). The number of beta-adrenergic receptors was even lower in P388/DXR cells (230 +/- 41 sites/cell, Kd 101 pM). In addition, these receptors were subnormally expressed on the cell surface: only 26% and 52% of the total receptors were surface receptors in P388 and P388/DXR, respectively, whereas it was 87% in normal splenocytes. Isoproterenol slightly (less than 1-fold) stimulated cAMP accumulation in P388 and P388/DXR; the stimulation observed in splenocytes was 2.5-fold. In addition, the basal levels of cAMP appeared to be low (0.48 +/- 0.05 pmoles/10(6) cells in P388 and 0.71 +/- 0.08 pmoles/10(6) cells in P388/DXR; 3.47 +/- 0.28 pmoles/10(6) cells in splenocytes) in the two leukemias and they were only slightly (less than 2-fold) increased by forskolin, which otherwise stimulated about 15-fold cAMP accumulation in splenocytes; thus, P388 and P388/DXR were probably also defective in their adenylate cyclase activity. It can be concluded that, owing to multifactorial mechanisms, the lymphocytic leukemia P388, also as an MDR variant, is minimally sensitive to the direct influences of the beta-adrenergic system, probably including any effect of this system on drug-sensitivity.

This paper refers to some of the chemical and biological properties of a new platinum (II) complex where the aromatic amino group of procaine is involved in the coordination with platinum and whose structure was defined by UV, IR, 1H-NMR, and elemental analysis. This new cationic platinum-triamine complex (DPR) displays excellent solubility (> 50 mg/ml) and stability in water. DPR has significant in vitro cytotoxicity against murine P388 leukemic cell line, human K562 erythroleukemic cell line and human Jurkat T cell line. The in vitro cytotoxic effects of DPR on P388 and Jurkat leukemic cells were comparable to those of cis-diamminedichloroplatinum (II) (DDP), while its activity on K562 cells was significantly better than that of DDP [IC50 = 1.07 +/- 0.36 (SD) microM vs 2.62 +/- 0.23 (SD) microM, P < 0.01]. The in vitro Pt accumulation rate for P388 cells was twice as rapid after DPR than after DDP exposure, while no difference in cellular platinum efflux was observed. The antitumor activity of DPR was tested in vivo against P388 leukemic cells in BDF1 mice and gave a % ILS value (75%) similar to that of the maximum tolerated dose (MTD) of DDP (8 mg/Kg). A comparative study of plasma urea nitrogen (PUN) levels and kidney morphological analysis in tumor-bearing mice receiving the LD50 dose of both drugs (39.3 mg/Kg and 16.5 mg/Kg for DPR and DDP, respectively), showed DPR to be less nephrotoxic than DDP. These results indicate that this new cationic platinum-triamine complex containing primary amine ligand is surprisingly active both in vitro and in vivo. In summary, the good characteristics of DPR in terms of high solubility, encouraging anticancer activity and absence of nephrotoxic effects make DPR a promising new platinum anticancer agent for preclinical development.

P388 (murine) and CEM (human) leukemia cells were exposed in vitro to a serine-deprived medium. Cultivation was carried out at 37 degrees C, 5% CO2. Proliferation assay was conducted with a RPMI 1640 medium (control) and a serine-deprived medium for 3 days. The deprivation of serine reduced the proliferation of both cells, and the necessity of serine for the cell proliferation was thus recognized. The effects of the substance on the level and pattern of intracellular amino acids were observed. P388 cells exposed to serine-deprived medium for 3 h were then transferred to the control medium. The cellular amino acid levels were determined at the time of medium change and 1, 2, 3 h thereafter. Serine-deprivation improved intracellular amino acids in comparison with those from control, and the medium change to control reduced their levels. Therefore, extracellular serine appeared to regulate the efflux of amino acids from cells. This suggests that serine-deprivation may be useful for anticancer drug retention in the cells.

We studied the effects of etoposide on the influx, intracellular accumulation and efflux of ara-C in P388 leukemic cells. Etoposide inhibited the active influx of ara-C in a dose dependent manner, and the inhibition was reversible. Etoposide also affected the intracellular accumulation of ara-C, though its inhibitory effect for the intracellular accumulation was weaker than that for the influx of ara-C. It was also shown that etoposide inhibited the active efflux of ara-C. Furthermore, etoposide inhibited the active transport of ara-C bidirectionally but the insensitive route of etoposide existed especially at high concentration of ara-C. The effect of inhibition of the transport of ara-C on the accumulation of ara-C was more significant at lower concentration of ara-C. Judging from the drug concentrations used in this study, an interaction between etoposide and ara-C could occur in the clinical treatment.

The biochemical activity of cepharanthine and the possible mechanism by which it reverses the resistance to doxorubicin in P388 leukemia cells were examined in vitro. The microfluorometric analysis of the cellular level of doxorubicin in drug-resistant cells showed that cepharanthine markedly enhanced the sensitivity of doxorubicin against resistant cells in the cellular level. Cepharanthine also enhanced the inhibitory effect of doxorubicin on the incorporation of thymidine into DNA in resistant cells. The analysis of DNA histogram obtained by flow cytometry showed that doxorubicin exerted its growth-inhibitory effect by blocking the cell cycle at the G2 phase in P388 cells. At higher concentrations, doxorubicin prolonged the S phase and inhibited cell cycle progression to the G2/M phase in cells. The treatment with cepharanthine potentiated these blocking effects induced by doxorubicin in cells. It seems that the modifications of the biological effect of doxorubicin by cepharanthine are due to the change of their ability to induce DNA damage in cells.

This study was undertaken to elucidate the mechanism of cross-resistance to mitomycin C (MMC) in a multi-drug resistant mouse leukemia cell line (P388/R-84). The P388/R-84 cells were 6.2-fold more resistant to MMC as compared to the sensitive cells (P388/S). Since oxygen radical formation has been suggested to be important in MMC cytotoxicity, MMC-dependent peroxidation of membrane lipids (an index of oxygen radical formation) was compared in these cell lines. MMC-dependent lipid peroxidation was 2.56-fold higher using P388/S cell extract when compared to that of P388/R-84 cells. Depletion of cellular glutathione (GSH) levels in the resistant cells produced a 1.76-fold increase in MMC-dependent lipid peroxidation. MMC cytotoxicity was also increased significantly by GSH depletion in P388/R-84 cells. These results suggest that cellular resistance to MMC in P388/R-84 cells may, at least in part, be due to the reduced formation of oxyradicals in these cells.

The overexpression of P-glycoprotein (PGP) and alterations in DNA topoisomerase II (TOPO II) were evaluated in mouse leukemia P388 cells selected in vivo for mitoxantrone (MTT) resistance (P388/MTT) and compared to doxorubicin (DOX) resistant (P388/DOX) or vincristine (VCR) resistant (P388/VCR) models. Among a panel of TOPO II inhibitors which included etoposide (VP-16), DOX, MTT and 4'-[(9-acridinyl)-amino]methanesulfon-m-anisidide (m-AMSA), the relative resistance compared to parental sensitive P388/S cells was: P388/DOX greater than P388/MTT greater than P388/VCR. All the resistant sublines exhibited minimal cell kill (less than 20%) at vincristine concentrations greater than 100-fold the IC50 for P388/S cells. In a soft-agar colony-forming assay, the modulation of cytotoxicity in P388/MTT cells by the calmodulin inhibitor trifluoperazine following a 3-hr drug treatment demonstrated a marked potentiation in cell kill with MTT, VP-16, DOX and m-AMSA but not VCR. Immunoblotting data revealed that while PGP was not detectable in P388/S cells, the overexpression of PGP was apparent in P388/MTT cells and the relative expression between the resistant sublines was: P388/DOX greater than P388/MTT greater than P388/VCR. Although the amount and DNA cleavage activity of TOPO II in nuclear extracts from P388/VCR cells were comparable to those in P388/S cells, they were markedly lower in both P388/DOX and P388/MTT cells. However, decatenation activity of TOPO II in nuclear extracts was comparable between the sensitive (P388/S) and resistant sublines (P388/MTT, P388/DOX, and P388/VCR). Results from the present study demonstrated that P388 cells selected for resistance to mitoxantrone exhibit changes in TOPO II and overexpression of PGP similar to P388/DOX cells, while vincristine resistant cells only overexpress PGP. Since therapeutic strategies are primarily designed to interfere with PGP-mediated drug efflux, the choice of agents for modulating resistance in tumors which overexpress PGP versus tumors which overexpress PGP with altered TOPO II could be different.

Vermixocins A and B, 3-(1'-hydroxy-3'-methylbutyl)- and 3-(1'-acetoxy-3'-methylbutyl)-11-hydroxy-4-methoxy-9-methyl-5H,7H- dibenzo[c,f][1,5]dioxocin-5-one, respectively, were isolated from the mycelium of Penicillium vermiculatum. Both metabolites showed cytotoxic effects on lympholeukemia cells P388.

The sugar boronated thymidine nucleoside, 5' -0-[(triphenylphosphine-boryl) carbonyl]-3'-0-acetyl thymidine 1, and the boron-modified nucleoside phosphotriester, 5'-(diethylphosphite- cyanoborane)-3'-acetylthymidine 2, were successfully synthesized. Both compounds demonstrated differential activity when tested against eight cell lines, with significant cytotoxic activity against the growth of human Tmolt3 leukemia, colon adenocarcinoma, HeLa S3 uterine carcinoma, and osteosarcoma cells. In in vivo studies these agents were found to be active against the growth of Ehrlich ascites carcinoma at 8 mg/kg/day I.P. and to be marginally active against the growth of L1210 and Lewis lung cancers in mice. The mode of action of these thymidine derivatives in Tmolt3 cells was the inhibition of DNA and protein synthesis. Compound 2 was highly effective in inhibiting DNA polymerase alpha and m-RNA, r-RNA and t-RNA polymerase activities. Both compounds inhibited ribonucleoside reductase activity. The de novo purine pathway appeared to be the major site of inhibition of the agents, with IMP dehydrogenase, PRPP amido transferase, and dihydrofolate reductase activities being significantly inhibited. In the pyrimidine pathway, carbamyl phosphate synthetase and aspartate transcarbamylase activities were inhibited by 1. As expected, d[NTP] levels were significantly reduced by treatment with the agents. DNA strand scission was evident after incubating Tmolt3 cells for 24 hr with the agents.

Pyrularia thionin and snake venom cardiotoxin are strongly basic peptides which induce hemolysis, depolarization of muscle cells and activation of endogenous phospholipase A2. An earlier study of the hemolysis reaction indicated that the two peptides bind to and compete for the same site on human erythrocytes. A recent study examined the hemolysis induced by both peptides as the phosphate and Ca2+ content of the reaction mixture was varied. The results of the recent study (VERNON, L. P. and ROGERS, A., Toxicon 30, 701-709) agree with this companion study on the binding of 125I-labeled pyrularia thionin and cardiotoxin to erythrocytes under the same conditions. Added phosphate ion at 5 mM and removal of membrane-bound Ca2+ by treatment with 10 mM EGTA make more binding sites of the same affinity available to both peptides, which are shown to bind in a competitive fashion to the same site. Addition of 10 mM Ca2+ to the medium decreases peptide binding due to competitive binding of Ca2+ to the same site on the membrane. For human erythrocytes the number of binding sites/cell for the thionin ranged from 0.7 to 1.7 x 10(5) and for cardiotoxin from 0.82 to 1.6 x 10(5). The calculated dissociation constants (Kd) from the Scatchard plots ranged from 0.43 to 1.1 microM for the thionin and from 0.40 to 0.98 microM for the cardiotoxin. The binding sites for thionin and cardiotoxin with sheep erythrocytes were 1.7 and 2.0 x 10(4) sites/cell, respectively, and both cow and horse erythrocytes demonstrated 2.7 x 10(4) sites/cell for the thionin. Binding studies with murine P388 cells showed 7.0 and 9.5 x 10(6) sites per cell for Pyrularia thionin and cardiotoxin, respectively.

The main purpose of the present investigation was to study the effect of cloturin on aerobic glycolysis, endogenous and exogenous respiration and the level of ATP in both Ehrlich ascites carcinoma (EAC) and P388 murine leukaemia cells incubated in vitro. Also its effect on the level of total (T-SH) and non-protein (NP-SH) thiol groups was investigated. A significant inhibition of aerobic glycolysis was found only in P388 cells after 60 min of cloturin action. Cloturin inhibited both endogenous and exogenous respiration of EAC with succinate as substrate. Cloturin decreased the level of ATP after 2 h incubation in both types of tumour cell. The level of NP-SH was decreased more than that of T-SH in both types of cell.

We investigated the effects of seven isoquinoline derivatives in overcoming resistance to vinblastine in Adriamycin-resistant mouse leukemia P388/ADR cells and human myelogeneous leukemia K562/ADR cells. N-(2-Methylpiperazyl)-5-isoquinoline-sulfonamide (H-7), N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), and N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) did not reverse resistance to vinblastine in these resistant cells. N-[2-[N-[3-(4-Chlorophenyl)-2-propenyl]amino]ethyl]-5- isoquinolinesulfonamide (H-86) and N-[2-[N-[3-(4-chlorophenyl)-1-methyl-2-propenyl]- amino]ethyl]-5-isoquinolinesulfonamide (H-87) caused significant accumulation of intracellular vinblastine and marked reversal of the resistance to vinblastine in both resistant cell lines. Addition of a formyl group at the terminal amino group of H-86 (H-85) or addition of an aminoethyl group to the nitrogen atom at the sulfonamide group of H-86 (W-66) reduced those activities. The activity on vinblastine accumulation seems to correlated with the hydrophobicity of the compounds. The compounds that effectively reversed resistance to vinblastine inhibited [3H]vinblastine efflux and photoaffinity labeling of P-glycoprotein with a photosensitive analogue of vinblastine, N-(p-azido-(3-[125I]iodo)-salicyl)-N'-beta-aminoethylvindesine. Although these isoquinoline derivatives inhibited protein kinase A and protein kinase C with various potencies, these inhibitory activities did not correlate with the reversal of drug resistance. These results indicate that hydrophobic isoquinoline derivatives reverse multidrug resistance due to the suppression of drug binding to P-glycoprotein, without involvement of their activities on protein kinase A and protein kinase C.

Ninety quinolones were evaluated to determine whether their ability to induce mammalian topoisomerase II mediated DNA cleavage in vitro correlated with their antitumor activity in vivo. Ten quinolones generated linear DNA at a yield of more than 10% of substrate supercoiled DNA in the mammalian topoisomerase II mediated DNA cleavage assay. All of these compounds showed a significant increase in life span (greater than 20%) in the murine leukemia P388 model. These antitumor quinolones have closely related structures: two halogens at C-6 and C-8; and cyclopropyl at N-1 of quinolone skeleton. In contrast, many analogues of the above quinolones, as well as new quinolones used clinically as an antibacterial drug, did not induce the cleavable complex in vitro or show antitumor activity in vivo. These findings indicate that quinolone derivatives can be a promising new class of antitumor agent targeting mammalian topoisomerase II.

It was the aim of this study to investigate whether leukemia P388 being an important murine transplantation tumor may alter the plasma concentration-time profiles of the alkylating antineoplastic agent bendamustine (1) in mice. In an advanced tumor stage the rapid decline of 1 plasma levels was found to be retarded in tumor-bearing in comparison to tumor-free animals both after i.v. and p.o. drug administration. These changes cannot be explained by the neoplasia-related depression of drug metabolism whereas the 1-containing ascitic fluid may be a possible reason for the prolonged drug levels in plasma. After p.o. administration of 1, the bioavailability of the drug was found to be increased in the leukemia-bearing animals.

31P NMR was used to study the systemic effects of a tumor on a host organism by monitoring the phosphate metabolite content in freshly excised mouse liver at 0-4 degrees C and in ethanolic liver extracts of animals suffering from La, L1210 and P388 leukemias and Ehrlich ascites tumor (EAT). The progression of murine leukemia is characterized by increases in the intensities of the resonances of Pi and phosphomonoesters (PME), in particular, phosphorylethanolamine, in liver; phosphodiester (PDE) signals increase two- to four-fold during the period of rapid tumor growth and decline to undetectable levels in the terminal stage. There were no reliable alterations detected in the ATP content and intracellular pH throughout the course of the leukemia. The kinetics of intracellular phosphates are similar in various kinds of leukemia but quite different in EAT. The reduction of inoculum causes the appearance of maxima in the Pi and PME profiles in the latent period of La leukemia, but the profiles of liver PDE considered from the end of the latent period are independent of inoculum. Possible mechanisms for the changes in PDE concentrations and their biochemical role are discussed. NMR spectroscopy of liver may be used to indirectly monitor the progression of tumors unavailable for direct NMR assay.

The initial stages of the interaction of daunomycin (DNM) with drug-sensitive (P388/S) and drug-resistant (P388/100) cells have been defined by a rapid kinetics stopped-flow procedure. The process can be described by two kinetic components. The faster component accounts for rapid occupation of cell surface sites by DNM, as supported by experiments with liposomes with different surface charge. On the other hand, the effect of verapamil in the assays, suggests that the slower component is involved in the transport of the drug into the cells. Our observations are consistent with a loss in the control of the passive permeability to the drugs in the drug-resistant tumor cells.

The effect of galactose-6-mustard (G-6-M) on cell growth and cell cycle kinetics was studied in murine P388 leukemia and Chinese hamster ovary (CHO) cells in vitro and compared with the effect of L-phenylalanine mustard (L-PAM). The IC50 values of G-6-M for the P388 and CHO cells were 10 and 100 microM, respectively. No difference of the IC50 value of L-PAM (2 microM) between the two cell lines was found. The effect of G-6-M and L-PAM on cell kinetics was similar for the two cell lines at IC50 doses. The relative cell outflow from the G2 stage was inhibited to a higher extent than the relative cell outflow from the S phase. The relative cell outflow from the G1 stage was only partly inhibited. These results are discussed in relation to growth conditions, differences in DNA repair capacity, and cellular uptake of G-6-M between P388 and CHO cells.

Numerous soluble factors and their receptors contribute to the regulation of immune responses. An important area of investigation concerns defining the regulation of expression of such receptor/ligand pairs, since understanding such events are central in the quest to manipulate immune responses. Receptors for the neuropeptide, substance P, are present on a variety of leukocytes, and these receptor positive cells respond to in vitro stimulation with substance P in a variety of ways. Unfortunately, little is known about the regulation of expression of substance P or its receptor in leukocytes. Here we begin to address this question by examining the ability of macrophages to express mRNAs which encode substance P and its receptor. A radiolabeled oligonucleotide probe complementary to the mRNA which encodes substance P (i.e., preprotachykinin mRNA) hybridized to a 1.3 kb RNA species present in rat macrophages. In addition, the expression of this RNA could be upregulated 6 to 8 fold when macrophages were stimulated with LPS. The ability of macrophages to synthesize and secrete immunoreactive-substance P was demonstrated by incorporation of L-[35S]methionine into material from macrophage cultures which could be recognized by a monoclonal antisubstance antibody. Macrophage RNA of approximately 3.1 kb in size was capable of hybridizing with an oligonucleotide probe complementary to rat brain substance P receptors. In addition, this RNA could be upregulated when cells were exposed to LPS. Taken together, these studies suggest that the genes used by neuronal cells and macrophages to encode substance P and its receptor are similar if not identical.(ABSTRACT TRUNCATED AT 250 WORDS)

We have examined the cytotoxicity and accumulation of (-)-(R)-2-aminomethylpyrrolidine(1,1-cyclobutanedicarboxylato++ +)platinum(II) monohydrate (DWA2114R) in parent and cisplatin-resistant mouse P388 leukemia cells (P388 and P388/DDP), in comparison with those of cisplatin (CDDP) and carboplatin (CBDCA). The degrees of resistance to CDDP and CBDCA, expressed as the ratio of IC50 for P388/DDP cells to IC50 for P388 cells, were 75-33 and 100-27, respectively, under the conditions of 2-24 h exposure to each drug at a density of 10(6) cells/ml. The corresponding values (25-7) for DWA2114R were relatively low. Accumulations of CDDP and CBDCA were reduced in P388/DDP cells; however, no reduction in accumulation of DWA2114R was observed at various exposure periods and concentrations of the drugs. The accumulations of CDDP in P388 and P388/DDP cells at drug concentrations corresponding to the IC50 values for drug exposure periods of 2-24 h were 0.41-0.97 and 13.1-33.7 ng Pt/10(7) cells, respectively, suggesting that an intracellular mechanism of resistance against CDDP could be activated in P388/DDP cells. P388/DDP cells also showed relatively low resistance to DWA2114R via this mechanism in comparison with CDDP and CBDCA. From the relationship between structure and activity of several Pt-complexes, these different properties of DWA2114R compared with CDDP and CBDCA could be due not only to the differences in carrier ligand structure but also to the properties of the whole molecule associated with the carrier ligand and leaving group.

When the anthracycline daunomycin (DNM) is incorporated into isolated plasma membranes from P388 murine leukemia cells, the drug partitions between 'deep' and 'surface' membrane domains. Such domains have been characterized on the basis of: (1) fluorescence resonance energy transfer between 1,6-diphenylhexa-1,3,5-triene or 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene as energy donors, which are well known in their positioning within the membrane, and daunomycin as the energy acceptor, and (2) quenching of the fluorescence of the membrane-associated drug by the water-soluble quencher iodide. The distribution of DNM between the two plasma membrane domains is different depending on the cellular phenotype. Thus, in membranes from drug-sensitive cells, DNM is preferentially confined to 'surface' domains, while in membranes from drug-resistant cells, the drug distributes more homogeneously between 'surface' and 'deep' domains. Experiments using artificial lipid vesicles suggest that differences in the relative levels of certain lipids in the plasma membranes from drug-sensitive and drug-resistant cells, namely phosphatidylserine and cholesterol, are partly responsible for the observed differences in the distribution of DNM. Since drug-membrane interactions are important in anthracycline cytotoxicity, it is possible that our observations on a different membrane distribution of daunomycin, may be related to the different sensitivity to the drug exhibited by these cells.

We have established in vivo cisplatin-resistant mouse leukemia cell lines, L-1210/DDP and P388/DDP, in order to elucidate the mechanism of acquired resistance to cisplatin. Resistance indices were 22 and 14, respectively, when the cells were exposed to cisplatin for 48 h. Uptake of cisplatin by both resistant lines was significantly reduced, compared with values for the respective parent lines (17% for L-1210/DDP and 27% for P388/DDP, at 100 microM for 1 h). While glutathione contents in the resistant cells were 1.7-1.9 times higher than those in the sensitive ones, their reduction by preincubation with buthionine sulfoximine did not influence the sensitivity of the cells to cisplatin. In addition, the resistant lines did not show lower sensitivity to CdCl2 than the respective sensitive ones, suggesting that intracellular SH groups might contribute little to the mechanism of cisplatin resistance in these cells. Postincubation with DNA repair inhibitors, caffeine and aphidicolon, did not selectively enhance the sensitivity of the resistant cells to cisplatin. These results suggested that reduced drug uptake would be a primary mechanism of cisplatin resistance in L-1210/DDP and P388/DDP. Cross-resistance patterns to platinum complexes were quite different between L-1210/DDP and P388/DDP. Colon 26/DDP, another cisplatin-resistant mouse tumor showed a different pattern from those observed with L-1210/DDP and P388/DDP. In the development of new platinum complexes we should use plural resistant lines for examining cross-resistance patterns to candidate platinum complexes.

The main purpose of the present investigation was to study the effect of 9-hydroxybenfluron (HBF) on aerobic glucose consumption, lactic acid formation, content of total (T-SH) and non-protein thiol groups (NP-SH), endogenous respiration and levels of ATP in both Ehrlich ascites and P388 murine leukemia cells. The lowest concentrations of HBF significantly stimulated both glucose consumption and lactate formation in Ehrlich ascites cells. HBF decreased the level of both T-SH and NP-SH in Ehrlich cells. However, the decrease in the level of NP-SH was significantly higher. Both respiration and ATP levels were inhibited more markedly in Ehrlich than in P388 cells. In P388 cells a significant decrease in ATP level (67%) was noted only at the highest concentration of HBF (100 mumol/l).

The interaction between interleukin IL-1 alpha and PGE2 on P388D1 cells has been investigated. Preincubation of murine macrophage-like cells, P388D1, with IL-1 alpha (0-73 pM) reduced the binding of PGE2 to these cells in a concentration-dependent manner. Scatchard analysis showed that IL-1 alpha decreased the PGE2 binding by lowering both the high and low affinity receptor binding capacities (from 0.31 +/- 0.02 to 0.12 +/- 0.01 fmol/10(6) cells for the high affinity receptor binding sites and from 2.41 +/- 0.12 to 1.51 +/- 0.21 fmol/10(6) cells for the low affinity receptor binding sites). However, the dissociation constants of the receptors of the IL-1 alpha-treated cells remained unchanged. Inhibition of PGE2 binding by IL-1 alpha did not involve changes in either protein phosphorylation or intracellular cyclic AMP levels. Our data clearly show that IL-1 alpha inhibits the binding of PGE2 to monocytes/macrophages and may thereby counter the immunosuppressive actions of PGE2.

The antitumor effects of mitoxantrone (MITO) and the various mechanisms involved therein were investigated in the adriamycin sensitive (P388/S) and resistant (P388/ADR) P388 leukemia cells. Utilizing the MTT (3-[4,5/dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay, MITO concentration less than 100 ng elicited 50% inhibition of P388/S tumor cell survival, while a 10 times greater dose of MITO was required to inhibit the P388/ADR cell survival by 50%. A MITO dose dependent inhibition of DNA, RNA and protein biosynthesis was observed in the sensitive cells, while MITO elucidated a negligible effect on the macromolecular biosynthesis in the resistant tumor cells. Induction of DNA strand scission was observed in P388/S cells exposed to 0.1 and 1 microgram/ml MITO, while a minimal formation of DNA lesions was evident in the P388/ADR cells treated with 5 micrograms/ml MITO. These strand breaks were found to be not associated with proteins in either P388/S or P388/ADR cells. Generation of free radicals due to MITO and formation of alkylating metabolites of MITO were found to be not involved in the cytotoxic response of MITO against P388/S and P388/ADR cells. MITO did not affect the glutathione based detoxification mechanism of the sensitive and resistant tumor cells. Results indicate that in spite of reduced intracellular drug retention and induction of DNA strand breaks in P388/ADR cells other hitherto unknown mechanisms besides DNA binding might be involved in the antitumorigenic potential of MITO.

The method of 31P nuclear magnetic resonance was used to study in vivo the level of phosphorus-containing metabolites in P388 leukemia cells sensitive or resistant to rubomycin (daunomycin) and its nitroxyl analog-emoxyl. It was shown that decreased content of phosphomonoesters (PME) is characteristic of the resistant strains in comparison with the parent cells. Rubomycin and emoxyl were established not to affect practically the pool of phosphorus-containing metabolites in the cells of the resistant strains, but caused considerable increase of PME level in the cells of the parent strain.

This paper presents data on the effects of benfluron and its two metabolites DBF and NOBF on both endogenous and exogenous, respiration in the presence of succinate as substrate, of both P388 murine leukemia and Ehrlich ascites carcinoma cells. The most efficient inhibitors of endogenous and exogenous respiration were benfluron and DBF. NOBF did not interfere with respiratory processes in Ehrlich cells, even at quite high concentration. BF and DBF exert an almost identical inhibitory effect on exogenous respiration, the least effective being NOBF (Ehrlich cells). The decrease in the respiratory rates in cancer cells might be due to the effects of benfluron and its metabolites on the cell membrane. P388 murine leukemia cells are less "sensitive" than Ehrlich ascites cells.

The distribution and excretion of [57Co]Bleomycin, dissolved in saline or encapsulated in liposomes, was studied in normal or tumor-bearing [P388 leukemia, reticulum cell sarcoma (RS)] mice. The free substance is cleared relatively quickly from the organism both after intravenous and intraperitoneal administration (t1/2 0.17 and 2.41 h, respectively), and is excreted predominantly via the urinary tract. In contrast, following entrapment in small unilamellar vesicles (SUV) or multilamellar vesicles (MLV), the 57Co radioactivity remains 7- to 30-fold longer in the blood stream and is detectable in considerable amounts in liver, spleen, lung and tumor of the RS model even after 48 h. Concomitantly, the renal excretion is diminished to about 50% of the free drug and the feces excretion is slightly increased, possibly due to the higher concentrations in the liver. Whereas the renal levels of radioactivity were similar with all application forms of [57Co]Bleomycin, there were marked differences in all the other tissues studied. After administration of SUV there was a higher activity in liver, brain and tumor, whereas MLV were more concentrated in spleen and lung. Therapeutic experiments confirmed the favorable results obtained with liposomes. While the free Bleomycin in the P388 leukemia had only a moderate influence on the lifetime of the animals with a treated/control value of 111%, encapsulation of the drug in SUV or MLV improved the results to 194 and 167%, respectively. In the intramuscular transplanted RS model, the SUV in a day 1 schedule had the same effect on tumor growth as the free drug in a day 1-4 schedule.(ABSTRACT TRUNCATED AT 250 WORDS)

Multidrug-resistant P388 cells were found to be resistant also to a variety of ammonium, phosphonium and arsonium compounds. As previously shown for anthracyclines and vinca alkaloids, the resistance to the permanently charged lipophilic cationic compounds could be circumvented by verapamil. Relative to drug-sensitive cells, K+ uptake and plasma membrane Mg-ATPase activity in multidrug-resistant cells are ouabain resistant. The intracellular K+ concentration in drug-resistant cells is maintained at a normal level by increased activity of the furosemide sensitive transport system. It is suggested that the reduced activity of the electrogenic Na(+)-K+ pump in multidrug-resistant, cells could result in a lower transmembrane potential and therefore reduced accumulation of cationic lipophilic compounds.

In order to assess mechanisms for acquired resistance to 5-fluorouracil (5-FU) of P388 cells on a cellular basis, we compared sensitivities of P388 and its 5-FU-resistant subline (P388/5-FU) cells to 5-FU, 5-fluorouridine (FUrd) and 5-fluoro-2'-deoxyuridine (FdUrd). P388/5-FU cells exhibited an approximately 10-fold resistance to 5-FU and 170-fold cross-resistance to FUrd but not to FdUrd when they were exposed to each agent for 5 h in vitro. 5-FU-induced growth inhibition was hardly reversed with thymidine, suggesting its ribonucleic acid (RNA)-directed effect. This was supported by the fact that similar amounts of 5-FU were incorporated into cellular RNA in P388 and P388/5-FU when these cells were incubated with equitoxic concentrations of 5-FU. Furthermore, incorporation of 5-FU and FUrd into cellular RNA in P388/5-FU cells were significantly lower than in P388 cells when cells were exposed to them at the same concentration. These results suggest a major action of 5-FU is directed toward RNA in these cells at least under the present experimental condition, and 5-FU resistance of this cell line is closely associated with reduced uridine kinase activity among various enzymatic changes previously observed.

Fluorimetric techniques were used to examine accumulation of fluorescent probes by the P388 murine leukemia and an anthracycline-resistant subline, P388/Adriamycin(ADR), which expresses the multidrug-resistant phenotype. P388 could be differentiated from P388/ADR on the basis of fluorescence intensity measurements using 3 classes of cationic dyes that are sensitive to membrane potential differences: rhodamine esters, cyanines, and styrylpyridinium dyes. But fluorescence intensity differences were also observed with potential-insensitive dyes: zwitterionic rhodamines and an acridine orange derivative. In all cases, fluorescence intensity differences were caused by impaired dye accumulation, and could be eliminated by treatment of P388/ADR cells with verapamil. Moreover, fluorescence signals from 2 anionic potential-sensitive dyes, merocyanine 540 and a bis-oxonol, were identical in P388 and P388/ADR. None of these dyes could be used to delineate CCRF-CEM, a lymphoblastic leukemia of human origin from the CEM/VM-1 subline that exhibits a markedly atypical drug resistance pattern not based on an enhanced outward transport. But accumulation of both neutral and cationic dyes was impaired in CEM/VLB100, a subline of CCRF-CEM expressing mdr. These studies show that many cationic and neutral fluorescent probes are substrates for the enhanced outward drug transport system associated with P388/ADR cells, and cannot be used to probe membrane-potential differences in cells expressing the mdr phenotype. With several dyes, differences in fluorescence intensity were sufficient so that flow cytometry could be used to delineate P388 from P388/ADR and CCRF-CEM from CEM-VLB100. The latter technique may be useful for identifying malignant cell populations expressing multidrug resistance in patients with neoplastic disease.

The pharmacokinetics of a radiolabelled analog of helenalin, [3H]-plenolin ([3H]-11,13-dihydrohelenalin), was determined in BDF1 mice following intravenous, intraperitoneal, and oral administration. A two-compartment pharmacokinetic model predicted that the maximum terminal (beta) half-life of [3H]-plenolin was 57.3 hours. Urinary excretion accounted for 40.3% to 64.4% of the administered radioactivity, while fecal excretion accounted for 9.3% to 39.7%. The fecal excretion data also suggested that [3H]-plenolin was secreted in the bile. Following intraperitoneal administration of [3H]-plenolin, no radioactivity was sequestered in the major organs. However, radioactivity was sustained in the carcass and skin for 24 days. [3H]-Plenolin was rapidly taken up by murine tumor cells and human fibroblasts. The drug did not significantly associate with DNA, RNA, or protein of P388 leukemia or human fibroblast cells.

Girodazole, a new marine compound has been isolated from the sponge Pseudaxinyssa cantharella. Girodazole is active in vivo on several murine grafted tumors including leukemias (P388, L1210, i.p./i.p.) and solid tumors (MA 16/C mammary adenocarcinoma, M5076 histiocytosarcoma, s.c./i.v.). In addition, girodazole has identical cytotoxic properties in vitro on P388 and P388/DOX cells and retains antitumor activity in vivo on P388/DOX. Girodazole has a unique chemical structure different from those of known anticancer agents and of new compounds undergoing clinical trials. Biochemical studies indicate that girodazole inhibits protein synthesis during the elongation/termination steps. Toxicological studies have been done in mice and in dogs and did not reveal any major toxic effect which could preclude administration in patients. Girodazole is now undergoing phase I clinical studies.

As part of the exploration of the mechanism of platinum(II) complex-induced growth inhibition and/or cytotoxicity, we studied the intracellular levels of several nucleotides during treatment of mouse leukemia P388/D1 at selected concentrations of 1 microM cis-diamminedichloroplatinum(II) (cis-DDP) and 20 microM of its trans-isomer (trans-DDP). The effects and their time-dependences are correlated with those on cell growth parameters previously published (Just G and Holler E, Cancer Res 49: 7072-7078, 1989). The effects of cis-DDP are strong and irreversible, whereas those of trans-DDP are marginal and reversible, in parallel with similar effects on cell growth parameters. Concentrations of nucleoside 5'-di- and 5'-triphosphates increase in parallel with cellular DNA and protein content by three- to four-fold after 60 hr of treatment. The nucleoside monophosphates dAMP, dGMP and dTMP reveal concentration maxima during exponential cell growth that are two- to six-fold higher than in the control cultures. Levels of cyclic AMP, adenosine(5')tetraphospho(5')adenosine (Ap4A) and CDP rise three- to five-fold above those in the control cultures within a few hours of the start of treatment. The level of coenzyme NAD+ falls below that of the control, concomitantly with an arrest of cells in the G2 phase of the cell cycle and with the appearance of giant cells. Due to the high reactivity of cis-DDP and the continuous concentration increase during the treatment, purine nucleoside 5'-triphosphates provide a possibility for the acquisition of resistance to cis-DDP. The correlation of responses of metabolically and regulatory active nucleotides with biological effects suggests their function in antitumorigenesis.