Reversal of etoposide resistance in non-P-glycoprotein expressing multidrug resistant tumor cell lines by novobiocin.

Previous reports from this laboratory have demonstrated that novobiocin produces supraadditive cytotoxicity and increases the formation of drug-stabilized topoisomerase II-DNA covalent complexes in WEHI-3B myelomonocytic leukemia and A549 lung carcinoma cells when combined with etoposide (VP-16). Inhibition of the efflux of VP-16 by novobiocin is responsible for the increase in VP-16 accumulation, which in turn leads to increased formation of VP-16-stabilized topoisomerase II-DNA covalent complexes and increased cytotoxicity. We now report that novobiocin synergistically enhanced the sensitivity of the multidrug resistant variants, WEHI-3B/NOVO and A549(VP)28, to VP-16, causing almost complete reversal of the resistance to the epipodophyllotoxin. These two tumor cell variants are resistant to several topoisomerase II-targeted drugs, particularly VP-16, but not to Vinca alkaloids; this finding corresponds to the fact that they do not overexpress the P-glycoprotein. The effects of novobiocin in these resistant sublines are mediated through the intracellular accumulation of VP-16, resulting in an increase in the formation of lethal VP-16-induced topoisomerase II-DNA covalent complexes. In the P-glycoprotein expressing multidrug resistant HCT116(VM)34 colon carcinoma and L1210/VMDRC0.06 leukemia cell lines, the latter being transfected with the human mdr-1 gene, novobiocin did not potentiate the cytotoxic activity of VP-16 nor increase the intracellular accumulation of VP-16 and the formation of covalent complexes, whereas their normal counterparts were sensitive to the potentiating activity of novobiocin when used in combination with VP-16. These results indicate that the action of novobiocin on the intracellular transport of VP-16 is not directed at the level of the P-glycoprotein, but that the action of novobiocin is antagonized by the presence of the P-glycoprotein. Since novobiocin is a clinically available antibiotic, has numerous structural analogues available for comparative studies, and has a relatively low toxicity profile, this drug, as well as structurally related agents, would appear to have significant clinical potential in combination with an epipodophyllotoxin for the treatment of non-P-glycoprotein expressing multidrug resistant tumors.

Development and characterization of a WEHI-3B D+ monomyelocytic leukemia cell line resistant to novobiocin and cross-resistant to other topoisomerase II-targeted drugs.

A novobiocin-resistant subline of WEHI-3B D+ murine monomyelocytic leukemia cells was developed by the continuous exposure of cells to this agent in vitro. Sensitive (WEHI-3B/S) and novobiocin-resistant (WEHI-3B/NOVO) sublines were cloned in vitro. WEHI-3B/NOVO cells were stable in the absence of novobiocin for more than 3 months, and the sensitive and resistant clones displayed the same growth rate, cell cycle distribution, cell size, DNA and protein content, and cloning efficiency. Novobiocin has been shown to compete with ATP for the ATP-binding site of topoisomerase II; therefore, intracellular ATP levels can influence the cellular sensitivity to novobiocin. High-performance liquid chromatographic analysis of total cell extracts demonstrated that no difference exists between WEHI-3B/S and WEHI-3B/NOVO cells in the content of ATP. Furthermore, exposure of both cell lines to novobiocin did not affect intracellular ATP levels. In addition to an approximately 2-fold level of resistance to novobiocin, the WEHI-3B/NOVO subline was also 7- and 11-fold cross-resistant to the topoisomerase II-targeted drugs, teniposide and etoposide (VP-16), respectively. A lower level of cross-resistance, comparable to that of novobiocin, was observed in WEHI-3B/NOVO cells for the intercalating topoisomerase II-reactive drugs, doxorubicin, 4'-(9-acridinylamino)methanesulfon-m-anisidide and aclacinomycin A, while the sensitivity to the cytotoxic action of the non-topoisomerase II-acting agents, camptothecin and vincristine, was not altered. After 3-6 h of exposure to 1 microM VP-16, WEHI-3B/S cells accumulated in the S and G2 + M phases of the cell cycle. Similar changes were detected in WEHI-3B/NOVO cells only after exposure to a 10-fold higher concentration of VP-16. Exposure to 150 microM novobiocin caused an accumulation of WEHI-3B/S cells in the G0-G1 phase of the cell cycle but did not affect the cell cycle distribution of WEHI-3B/NOVO cells, while camptothecin induced the same type and extent of changes in the cell cycle distribution of both cell lines. Although the WEHI-3B/NOVO subline appeared to be less responsive to the differentiation-inducing activity of novobiocin and teniposide, the capacity of WEHI-3B/NOVO cells to respond to the differentiation-inducing agent 13-cis-retinoic acid was not significantly different from that of WEHI-3B/S cells. A slight decrease in the accumulation of VP-16 occurred in the resistant cell line, which did not appear to be of sufficient magnitude to account for the 11-fold increase in the degree of resistance to this agent.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of the differentiation of WEHI-3B D+ monomyelocytic leukemia cells by inhibitors of topoisomerase II.

Topoisomerase II has been suggested to have a role in the early events of differentiation. This possibility was evaluated by measuring the effects of inhibitors of topoisomerase II on the induction of the differentiation of WEHI-3B D+ monomyelocytic leukemia cells. Differentiation of this cell line was induced along the granulocytic pathway by treatment with the topoisomerase II inhibitors novobiocin (150-300 microM), teniposide (20-50 nM), etoposide (0.1 microM), elsamicin (0.5 microM), and doxorubicin (40 nM). Maturation was assessed by the morphological appearance of mature forms of the granulocytic lineage, an increase in cell surface Fc receptors, the ability to reduce nitroblue tetrazolium, and the loss of proliferative capacity. In contrast, the non-topoisomerase II-reactive agent cisplatin and the topoisomerase I-reactive drug camptothecin did not cause the maturation of WEHI-3B D+ cells. Aclacinomycin A and retinoic acid, which are known efficacious inducers of the differentiation of this cell line, affected topoisomerase II extracted from WEHI-3B D+ cells in vitro, causing concentration-dependent inhibition of the strand-passing activity of the enzyme. Treatment of WEHI-3B D+ cells with novobiocin at 150 microM for 3 h or with teniposide at 50 nM for 24 h resulted in a 2- to 3-fold increase in etoposide-induced protein-DNA cross-links. Nuclear proteins in 0.35 M NaCl extracts from cells treated with novobiocin at 150 microM for 3 h or with teniposide at 50 nM for 24 h showed a slight increase in topoisomerase II activity compared to untreated cells. No changes in topoisomerase II levels, as measured by immunoblotting, were detected after treatment of WEHI-3B D+ cells with 150 microM novobiocin or 50 nM teniposide during the first 2 days of treatment. At day 3 of treatment, however, a decrease in topoisomerase II was observed in cells treated with either drug, possibly due to decreased cellular proliferation consequent to cell differentiation. The findings support the conclusion that topoisomerase II may have a role in the induction of granulocytic differentiation of WEHI-3B D+ leukemia cells.

Evidence that the multidrug resistance protein (MRP) functions as a co-transporter of glutathione and natural product toxins.

The MRP (multidrug resistance protein) gene, a member of the ubiquitous superfamily of ATP-binding cassette transporters, is associated with the multidrug resistance of mammalian cells to natural product anticancer agents. We have previously shown that abrogation of MRP expression by gene targeting leads to hypersensitivity to several drugs. In two independently produced MRP double knockout clones, the baseline export of glutathione (GSH) was one-half that of wild-type embryonic stem (ES) cells. The export of GSH from wild-type ES cells, but not from the MRP double knockout clones, increased in the presence of etoposide (VP-16) and sodium arsenite, accompanied by equivalent decreases in intracellular levels of GSH. In the two MRP double knockout clones, the intracellular steady-state concentration of etoposide was twofold greater than that in wild-type cells. Depletion of intracellular GSH by D,L-buthionine sulfoximine increased the intracellular accumulation of radiolabeled etoposide in parental ES cells up to the level present in the two MRP knockout clones but did not change etoposide levels in the MRP knockout clones. These observations provide evidence that: (a) MRP exports GSH physiologically, presumably in association with an endogenous compound(s); (b) baseline MRP expression protects cells from the toxic effects of xenobiotics by effluxing the xenobiotics and GSH from the intracellular compartment into the extracellular medium by a co-transport mechanism; and (c) disruption of the gene encoding MRP abrogates the cotransport of xenobiotics and GSH.

Double knockout of the MRP gene leads to increased drug sensitivity in vitro.

Overexpression of the multidrug resistance-associated protein (MRP) gene has been implicated in the resistance of tumor cell lines to a wide array of chemotherapeutic agents, but its normal physiological function(s) remains unknown. We have compared the sensitivity to chemotherapeutic drugs and toxins of wild-type W9.5 embryonic stem cells (ES) and of single and double MRP gene knockout cells derived therefrom. MRP expression was totally abrogated in the double knockout cell line and partially abrogated in the single knockout cell line. Reverse transcription-PCR analyses demonstrated that the MDR1, MDR2, and MDR3 genes were not expressed in either wild-type or MRP knock-out cells. The cytotoxic activities of etoposide, teniposide, vincristine, doxorubicin, daunorubicin, and sodium arsenite were significantly greater in double knockout cells than in parental wild-type ES cells; single knockout ES cells displayed an intermediate level of sensitivity. In contrast, no difference in sensitivity to colchicine and 1-beta-D-arabinofuranosylcytosine existed between the cell lines. Etoposide accumulation in double knockout ES cells was 2-fold higher than in wild-type ES cells. These findings indicate that baseline MRP expression has the capacity to exert a protective role against the toxicity of multiple chemotherapeutic agents and natural toxins.

Disruption of the murine MRP (multidrug resistance protein) gene leads to increased sensitivity to etoposide (VP-16) and increased levels of glutathione.

The mrp (multidrug resistance protein) gene has been associated with the multidrug resistance of cancer cells in vitro and in vivo. To gain information on its physiological role, embryonic stem cells were used to generate mice homozygous for a disruption of the mrp gene, resulting in complete abrogation of mrp expression. No physiological abnormalities were observed, at least up to 4 months of age. Viability, fertility, and a range of histological, hematological, and serum-chemical parameters were similar in mrp(+/+) and mrp(-/-) mice. mrp(-/-) mice displayed an increased sensitivity to etoposide phosphate (2-fold) accompanied by greater bone marrow toxicity, whereas the acute toxicity of sodium arsenite was equivalent in mrp(+/+) and mrp(-/-) mice. Tissue levels of glutathione (GSH) were elevated in breast, lung, heart, kidney, muscle, colon, testes, bone marrow cells, blood mononuclear leukocytes, and blood erythrocytes of mrp(-/-) mice and were unchanged in organs known to express little if any mrp, such as the liver and small intestine. The increase in GSH was not due to an increase in the activity of gamma-glutamylcysteine synthetase, the rate-limiting enzyme for GSH synthesis. The findings demonstrate that mrp is dispensable for development and growth but exerts a role in drug detoxification and GSH metabolism.

Accumulation of DNA strand breaks in cells exposed to methotrexate or N10-propargyl-5,8-dideazafolic acid.

N10-Propargyl-5,8-dideazafolic acid (CB 3717), a new antifolate which directly inhibits thymidylate synthase and which is now under early clinical investigation, was compared with methotrexate (MTX) for its antiproliferative activity and mode of action on M14 human melanoma cell line and NIH/3T3 murine fibroblasts transfected with human c-Ha-ras oncogene (NIH/3T3R). CB 3717 was as active as MTX on both cell lines in inhibiting colony formation, but 20-100 times less potent. After 24 h of exposure both drugs caused an accumulation of cells in the G1 phase of the cell cycle, probably because of inhibition of DNA synthesis and blockage at the G1-S boundary. In NIH/3T3R treated for 16 h with 2 microM MTX or 200 microM CB 3717, we found DNA single-strand breaks amounting to approximately 130 and 140 rad equivalents, respectively, and a considerable number of DNA double-strand breaks, far more than expected if they had been the result of the proximity of single-strand breaks on the two complementary DNA strands. No DNA-protein cross-links were detected. When cells were incubated in drug-free medium for 8 h, there was a further accumulation of single-strand breaks, possibly due to the effects of the drug retained intracellularly as polyglutamyl derivative. Simultaneous treatment with 1.77 microM cycloheximide prevented DNA damage produced by both drugs. Thymidine (10 microM), renewed in the culture medium every 24 h, also prevented DNA damage and cytotoxicity. Since after 16 h treatment with MTX or CB 3717 cells were completely viable, as assessed by [3H]thymidine release, trypan blue exclusion test, and 51Cr release, DNA damage appears to be an early event preceding cell death and may be a feature of the killing ability of the drugs. The involvement of a protein in the formation of DNA breaks is suggested by the fact that when protein synthesis was inhibited with cycloheximide DNA damage was no longer seen.

Increased rate of adenosine triphosphate-dependent etoposide (VP-16) efflux in a murine leukemia cell line overexpressing the multidrug resistance-associated protein (MRP) gene.

WEHI-3B/NOVO is a cloned murine leukemia cell line selected for resistance to novobiocin that is cross-resistant to the cytotoxic action of etoposide (VP-16) and to a lesser extent to a variety of other topoisomerase II (topo II)-reactive drugs. We have reported previously (Cancer Res. 52: 2782-2790, 1992) that WEHI-3B/NOVO cells exhibit a pronounced decrease in VP-16 induced DNA-topo II cross-link formation compared to the parental WEHI-3B/S cell line in intact cells, in the absence of a significant difference in the P4 unknotting activity of topo II assayed in nuclear extracts. Because the pattern of cross-resistance was suggestive of a topo II-mediated mechanism, we have ascertained whether a change in topo II can account for the multidrug-resistant phenotype of WEHI-3B/NOVO cells. No differences existed between WEHI-3B/S and WEHI-3B/NOVO cells in topo II mRNA and protein levels, as well as in the amount of topo II associated with the nuclear matrix. Neither sensitive nor resistant cells expressed detectable levels of the MDR1 gene; however, VP-16 accumulation in WEHI-3B/NOVO cells was 3-4-fold less than that present in WEHI-3B/S cells, whereas doxorubicin accumulation was the same in both cell lines. Over the first 60 s, no difference existed in the rate of uptake of VP-16 between parental and resistant cells; however, beyond the first 60 s of incubation, [3H]VP-16 accumulated to a greater extent in parental sensitive cells. Thus, an increased rate of efflux of VP-16 was responsible for the lower steady-state concentration of the drug in resistant cells. The efflux Km for VP-16 in WEHI-3B/NOVO cells was 254.7 microM and the Vmax was 10.4 pmol/s/10(7) cells. In the presence of the inhibitors of energy metabolism, sodium azide and deoxyglucose, the efflux of VP-16 was markedly inhibited; readdition of glucose restored the original efflux rate. Northern blot analyses using the human 10.1 probe for the 3'-terminal region of the multidrug-resistance protein (MRP) cDNA revealed a mRNA species of approximately 6 kb in WEHI-3B/NOVO cells but not in WEHI-3B/S cells. Overexpression was associated with amplification of the cognate gene. To ascertain whether the overexpressed gene in WEHI-3B/NOVO cells was the murine MRP or a different member of the same superfamily of ATP-binding ABC cassette transporters, a 341-bp MRP cDNA probe was generated from a murine genomic library.(ABSTRACT TRUNCATED AT 400 WORDS)

Novel bicistronic retroviral vector expressing gamma-glutamylcysteine synthetase and the multidrug resistance protein 1 (MRP1) protects cells from MRP1-effluxed drugs and alkylating agents.

We have constructed two retroviral vectors, one expressing multidrug resistance protein 1 (MRP1) alone (SF91MRP) and the other expressing MRP1 and gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme of glutathione biosynthesis (SF91GCS-MRP). We have utilized the hybrid FMEV (Friend mink cell focus-forming/murine embryonic stem cell virus) backbone, previously shown to be efficient in early hematopoietic cells, even when coexpressing two distinct genes. In SF91GCS-MRP, the cDNAs were combined via an internal ribosomal entry site (IRES) sequence from poliovirus, resulting in a bicistronic mRNA produced via the long terminal repeat (LTR). Producer Fly-eco clones were established by trans-infection with vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped retroviral supernatants. Drug-resistant producer clones were subsequently selected with antimony potassium tartrate, a nonmutagenic MRP1 substrate. By RNA slot-blot and transduction of 3T3 fibroblasts, titers of both SF91MRP and SF91GCS-MRP were found to be greater than 10(6) viral particles/ml. The correct viral integration in the genome was established by Southern blotting. By flow cytometry, both MRP1 and bicistronic clones showed an increase in expression of the MRP1 protein. The bicistronic producer clones, as well as 3T3 cells transduced with SF91GCS-MRP, presented an increase in intracellular glutathione levels, compared with the parental counterparts. Producer cells, 3T3 fibroblasts transduced with either SF91MRP or SF91GCS-MRP, and primary murine myeloid progenitor cells transduced with SF91GCS-MRP were resistant to MRP1-effluxed drugs. However, only bicistronic producers, 3T3 fibroblasts transduced with SF91GCS-MRP, and primary murine myeloid progenitor cells transduced with SF91GCS-MRP were also resistant to alkylating agents. We conclude that the retrovirus SF91GCS-MRP has features that make it a suitable vector to induce bone marrow resistance to multiple classes of chemotherapeutic agents. The strategy of coexpressing gamma-GCS and MRP1 may help to design an effective in vivo selection for various clinical protocols of gene therapy.

Retroviral transfer of MRP1 and gamma-glutamyl cysteine synthetase modulates cell sensitivity to L-buthionine-S,R-sulphoximine (BSO): new rationale for the use of BSO in cancer therapy.

MRP1 (multidrug resistance protein 1) co-exports glutathione (GSH) and drug(s) and exports GSH, glucuronide, and sulphate-conjugated drugs. Human Fly-eco fibrosarcoma cells producing the MRP1-expressing retrovirus SF91MRP (Fly-eco MRP1), as well as 3T3 cells transduced with SF91MRP (3T3/MRP1), presented a decrease in intracellular GSH levels, as measured by two different methods. The enhanced export of GSH caused by the overexpression of MRP1 was partially counterbalanced by an increased rate of GSH synthesis. Fly-eco MRP1 and 3T3/MRP1 were hypersensitive to the GSH-depleting and cytotoxic activities of L-buthionine-S,R-sulphoximine (BSO), compared with their parental counterparts. In addition, the potentiation by BSO of the cytotoxic activity of chlorambucil and doxorubicin in Fly-eco MRP1 cells was greater than in parental Fly-eco cells. Although the turnover time of GSH, i.e. the theoretical time in which the entire GSH pool is resynthesised, was approximately 50% faster in Fly-eco MRP1 cells than in parental cells, this was not sufficient to fully restore the intracellular GSH level. In addition, mrp1 (-/-) mice were resistant to the GSH-depleting activity of intraperitoneally (i.p.) injected BSO, compared with mrp1 (+/+) mice. Co-transfer of the cDNAs for MRP1 and the heavy subunit of gamma-glutamyl cysteine synthetase (GCS) resulted in increased intracellular GSH levels and in high-level resistance to the GSH-depleting and cytotoxic activities of BSO. These data, and in particular the elevated single-agent cytotoxicity of BSO, provide a new rationale for the use of BSO in the treatment of MRP1-overexpressing tumours.

Efficient expansion and gene transduction of mouse neural stem/progenitor cells on recombinant fibronectin.

Neural stem/progenitor cells (NSCs) are commonly grown as floating neurospheres in medium containing basic fibroblast growth factor and epidermal growth factor. Under these conditions, about 1% of the cells retain multipotentiality. We developed a protocol based on culture of NSCs in adherence on recombinant fibronectin (rFN) to transduce up to 90% NSCs at a multiplicity of infection of 2 with no need for viral concentration or production of serum-free retroviral supernatants. NSCs grew faster on rFN than as neurospheres on tissue culture plastic and did not lose their stem cell nature or multipotentiality. Furthermore, retroviral-mediated transgene expression was sustained with time in culture and upon differentiation into neurons and astrocytes. These experimental conditions may be utilized to study the function of various genes in NSCs, and to manipulate NSCs for gene and cell therapy of several neurological diseases.

New insights into the biology and pharmacology of the multidrug resistance protein (MRP) from gene knockout models.

Growing interest in the MRP (multidrug resistance protein) gene stems from its importance in multidrug resistance to chemotherapy, its possible use in gene therapy, and its relationship with the glutathione system. The recent generation of mrp gene knockout models in vitro and in vivo is providing information on the mechanism of action and the physiological function(s) of mrp. The importance of mrp in protection of normal tissues from the toxicity of the anticancer agent etoposide has been established. A total block of mrp has been found to be compatible with life, suggesting that MRP inhibitors can be safely used for treating cancer patients. In some sub-classes of leukocytes, mrp contributes to the transport of leukotriene C4, an endogenous glutathione-S-conjugate. However, the baseline expression of mrp does not appear to contribute to the export of glutathione-S-conjugates of alkylating agents, and thus does not exert a protective role against their toxicity. Besides being capable of exporting certain glutathione-S-conjugates, mrp also catalyzes the co-transport of GSH and drug and, presumably, a presently unknown endogenous metabolite(s).

Inhibitors of ribonucleotide reductase. Comparative effects of amino- and hydroxy-substituted pyridine-2-carboxaldehyde thiosemicarbazones.

A new series of alpha-(N)-heterocyclic carboxaldehyde thiosemicarbazones (HCTs) was studied for their effects on L1210 cell growth in culture, cell cycle transit, nucleic acid biosynthesis and ribonucleotide reductase activity. 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) and 3-amino-4-methylpyridine-2-carboxaldehyde thiosemicarbazone (3-AMP) were the most active compounds tested with respect to inhibition of cell growth and ribonucleotide reductase activity. 5-Aminopyridine-2-carboxaldehyde thiosemicarbazone (5-AP) and 4-methyl-5-aminopyridine-2-carboxaldehyde thiosemicarbazone (5-AMP) were slightly less active. 3-AP, 3-AMP, 5-AP and 5-AMP inhibited the incorporation of [3H]thymidine into DNA without affecting the rate of incorporation of [3H]uridine into RNA. The uptake and incorporation of [14C]cytidine into cellular ribonucleotides and RNA, respectively, were not decreased by 3-AP or 3-AMP; however, the incorporation of cytidine into DNA via ribonucleotide reductase was inhibited markedly. Thus, a pronounced decrease in the formation of [14C]deoxyribonucleotides from radioactive cytidine occurred in the acid-soluble fraction of 3-AP- and 3-AMP-treated L1210 cells. Consistent with an inhibition of DNA replication that occurred at relatively low concentrations of 3-AP and 3-AMP, cells gradually accumulated in the S-phase of the cell cycle; at higher concentrations of 3-AP and 3-AMP, a more rapid accumulation of cells in the G0/G1 phase of the cell cycle occurred, with the loss of the S-phase population, implying that a second less sensitive metabolic lesion was created by the HCTs. N-Acetylation of 3-AMP resulted in a compound that was 10-fold less active as an inhibitor of ribonucleotide reductase activity and 8-fold less active as an inhibitor of L1210 cell growth. N-Acetylation of either 5-AP or 5-AMP did not alter the inhibitory properties of these compounds. The results obtained provide an experimental rationale for the further development of the HCTs, particularly 3-AP and 3-AMP, as potential drugs for clinical use in the treatment of cancer.

Overexpression of the multidrug resistance genes mdr1, mdr3, and mrp in L1210 leukemia cells resistant to inhibitors of ribonucleotide reductase.

L1210 MQ-580 is a murine leukemia cell line resistant to the cytotoxic activity of the alpha-(N)-heterocyclic carboxaldehyde thiosemicarbazone class of inhibitors of ribonucleotide reductase. The line is cross-resistant to etoposide, daunomycin, and vinblastine. L1210 MQ-580 cells expressed 8-fold resistance to 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP), a relatively newly developed inhibitor of ribonucleotide reductase. The accumulation of [14C]3-AP by L1210 MQ-580 cells was 5- to 6-fold less than by parental L1210 cells. An increased rate of efflux of 3-AP was responsible for the lower steady-state concentration of 3-AP in resistant cells. In reverse transcription-polymerase chain reaction assays, L1210 MQ-580 cells were found to overexpress the multidrug resistance genes mdr1, mdr3, and mrp, but not the mdr2 gene, compared with parental L1210 cells. Measurement of the steady-state concentration of doxorubicin, a potential substrate for both the mdr and mrp gene products, demonstrated that L1210 MQ-580 cells accumulated 4-fold less anthracycline than parental cells. These findings indicate that drug efflux is a major determinant of the pattern of cross-resistance of L1210 MQ-580 cells. To extrapolate these observations to the human homologues of the mdr1, mdr3, and mrp murine genes, the effects of 3-AP were measured in L1210/VMDRC0.06 and NIH3T3 36-8-32 cells transfected with human MDR1 and MRP cDNAs, respectively. The transfectants were 2- to 3-fold resistant to the cytotoxic effects of 3-AP and accumulated less [14C]3-AP than their parental mock-transfected counterparts. Moreover, the cytotoxic activity of 3-AP was significantly greater in two double mrp gene knockout cell lines than in parental W 9.5 embryonic stem cells. Thus, the results suggest that 3-AP is a substrate for both the P-glycoprotein and MRP and that baseline MRP expression has the capacity to exert a protective role against the toxicity of this agent.

Structure-function relationships for a new series of pyridine-2-carboxaldehyde thiosemicarbazones on ribonucleotide reductase activity and tumor cell growth in culture and in vivo.

The synthesis of a new series of pyridine-2-carboxaldehyde thiosemicarbazones (HCTs) that have amino groups in the 3- and 5-positions has allowed the comparison of the structure/function relationships with regard to inhibition of ribonucleotide reductase activity, L1210 cell growth in culture and L1210 leukemia in vivo. 3-Aminopyridine-2-carboxaldehyde thiosemicarbazones are more active than the corresponding 3-hydroxy-derivatives. The 3-amino-2-pyridine carboxaldehyde thiosemicarbazones were also more active then the 5-amino-2-carboxaldehyde thiosemicarbazones in inhibiting ribonucleotide reductase activity and L1210 cell growth in culture and in vivo. N-Acetylation of the 3-amino derivative resulted in a compound that was much less active both in vitro and in vivo; N-acetylation of the 5-amino derivative did not alter the in vitro inhibitory properties, but did eliminate the antitumor properties in vivo. When the most active HCTs were studied in more detail, it was found that the incorporation of [3H]thymidine into DNA was inhibited completely without the inhibition of [3H]uridine incorporation into RNA. Further, the conversion of [14C]cytidine to deoxycytidine nucleotides and incorporation into DNA was inhibited by the HCTs without an effect on the incorporation of cytidine into RNA. These data support the conclusion that ribonucleotide reductase is the major site of action of these HCTs. The 3-aminopyridine-2-carboxaldehyde thiosemicarbazones emerge as strong candidates for development for clinical trials in cancer patients.

Structure-activity studies of novobiocin analogs as modulators of the cytotoxicity of etoposide (VP-16).

We have previously reported that the antibiotic novobiocin enhanced the toxicity of the anticancer agent etoposide (VP-16) to several drug-sensitive and -resistant tumor cell lines. The increase in VP-16 cytotoxicity produced by novobiocin was not due to the combined effects of these agents on topoisomerase II, but to inhibition by novobiocin of VP-16 efflux, which in turn led to increased accumulation of VP-16 and increased formation of potentially lethal VP-16-stabilized topoisomerase II-DNA covalent complexes. We have now identified novobiocin analogs that are essentially equivalent to novobiocin as inhibitors of the activity of topoisomerase II, but that are more potent than novobiocin (a) as modulators of the cytotoxicity of VP-16 to WEHI-3B leukemia and A549 lung carcinoma cells and (b) in increasing VP-16 accumulation in these cell lines. Thus, removal of the sugar moiety of novobiocin to form novobiocic acid enhanced the potency of the antibiotic as a modulator of VP-16, whereas the substituted coumarin ring alone (U-7587) was devoid of VP-16 modulatory activity. Modifications of the side chain of novobiocin significantly influenced modulatory activity, with cyclonovobiocic acid, which was formed from novobiocic acid by acid-catalyzed cycloaddition, being the most active in enhancing the cytotoxicity of VP-16. The increased potency of novobiocic acid and cyclonovobiocic acid as modulators of VP-16 activity was achieved with no change from novobiocin in the capacity of these analogs to inhibit the catalytic activity of mammalian topoisomerase II, indicating a change in the specificity of these analogs.

Altered efflux properties of mouse leukemia L1210 cells resistant to 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone.

A mouse leukemia L1210 cell line, denoted MQ-580, that was selected for resistance to the ribonucleotide reductase inhibitor, 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (MAIQ), in addition to having altered properties at the ribonucleotide reductase site had other alterations that contributed to its resistant phenotype; these included the expression of p-glycoprotein and the multi-drug resistance associated protein (MRP). The efflux of rhodamine 123 (Rh-123) or daunomycin (Dau) was greatly increased in MQ-580 cells compared to parental wild-type (WT) cells. The effluxes of Rh-123 and Dau were ATP- and temperature-dependent. The p-glycoprotein inhibitors, verapamil, cyclosporin A and reserpine blocked the efflux of both Rh-123 and Dau. In contrast, the inhibitors of MRP, MK571, BSO-treatment, arsenite and genistein did not block the efflux of either Rh-123 or Dau from MQ-580 cells. These findings suggest that the p-glycoprotein is the major transporter involved in effluxing Rh-123 and Dau from MQ-580 cells.