Unr, a cytoplasmic RNA-binding protein with cold-shock domains, is involved in control of apoptosis in ES and HuH7 cells.

Unr (upstream of N-ras) is a cytoplasmic RNA-binding protein involved in the regulation of messenger RNA stability and internal initiation of translation. We have used Unr-deficient murine embryonic stem (ES) cells to analyse Unr role in cell proliferation and response to stress. Disruption of both unr gene copies had no effect on ES cell proliferation. However, after ionizing radiation (IR), clonogenic survival of unr(-/-) ES cells was approximately 3-fold enhanced as compared to unr(+/+) cells. We further determined that IR-induced apoptosis was decreased in unr(-/-) ES cells, and that reintroduction of the unr gene in unr(-/-) cells restored normal IR-induced apoptosis. Three pro-apoptotic genes, p53, caspase-3 and Gadd45gamma, were downregulated in unr(-/-) ES cells, indicating that Unr, as other cytoplasmic RNA-binding proteins, regulates a complex genetic program, promoting cell death after IR. In contrast, in the human hepatoma cell line HuH7, Unr knockdown using unr-specific small interfering RNAs induced apoptosis, both in untreated and gamma-irradiated cells. Thus, our results establish that Unr acts as a positive or negative regulator of cell death, depending on the cell type. Manipulating the level of Unr may constitute a specific approach to sensitize cancer cells to anticancer treatments.

Multiple resistance mechanisms in Chinese hamster cells resistant to 9-hydroxyellipticine.

We have previously shown that Chinese hamster lung cells resistant to 9-hydroxyellipticine, DC-3F/9-OH-E, display multiple phenotypical alterations including cross-resistance to a variety of drugs as well as loss of tumorigenicity. We now analyze a DC-3F/9-OH-E subline that has been maintained for a prolonged period of time in drug-free medium in order to clarify the relationships between the various phenotypic traits. The absence of selection resulted in a partial recovery of the ability to form colonies in soft agar as well as of the tumorigenicity in nude mice. In contrast, no change was observed with respect to population-doubling time. Our results also show that the resistance to 9-hydroxyellipticine, which is associated with an altered topoisomerase II activity, is stable in the absence of drug for more than 1 year. In contrast, the cross-resistance to doxorubicin is partially reversible and the cross-resistance to vincristine is totally reversible in the absence of selection. The cross-resistance to vincristine and doxorubicin is accompanied by a decreased drug uptake. Northern blot analysis shows that the multidrug resistance-associated Mr 170,000-180,000 glycoprotein is overexpressed in the DC-3F/9-OH-E cells and that the overexpression is lost in the absence of selection. We conclude that (a) the DC-3F/9-OH-E cells exhibit multiple mechanisms of resistance which can be dissociated, (b) the tumorigenicity and the altered topoisomerase activity are independent biochemical events whereas the oncogenic potential appears to follow the expression of the multidrug resistance, and (c) the multidrug resistance phenotype may be induced by a drug which is not itself recognized by the multidrug resistance mechanism such as 9-hydroxyellipticine.

Role of topoisomerase II beta in the resistance of 9-OH-ellipticine-resistant Chinese hamster fibroblasts to topoisomerase II inhibitors.

In the Chinese hamster lung cell line DC-3F/9-OH-E, made resistant to 9-OH-ellipticine and cross-resistant to other topoisomerase II inhibitors, the amount of topoisomerase II alpha is 4-5-fold lower than in the parental DC-3F cells. A mutation in position 1710 of topoisomerase II beta cDNA, generating a stop codon, completely abolishes the expression of this isoform in DC-3F/9-OH-E cells. To analyze the contribution of the loss of topoisomerase II beta to the resistance phenotype, DC-3F/9-OH-E cells were cotransfected with two plasmids, one conferring the resistance to G418, the other carrying the topoisomerase II beta cDNA. Among 200 G418-resistant clones, one was found to contain a topoisomerase II beta activity similar to that in the parental cells. These cells constitute an in vivo mammalian model to study the pharmacological role of topoisomerase II beta. In the transfected cells, different levels of cleavable complex formation and resistance reversion were observed with each topoisomerase II inhibitor examined. This work demonstrates that topoisomerase II beta is a pharmacological target for 9-OH-ellipticine, etoposide, or 4'-(9-acridinylamino)methanesulfon-m-anisidide and plays a role in the cytotoxicity of these agents. Furthermore, topoisomerase II beta is the preferential target for 4'-(9-acridinylamino)methanesulfon-m-anisidide. The loss of topoisomerase II beta activity in the DC-3F/9-OH-E cells is then in part responsible for their resistance to topoisomerase II inhibitors.

Expression of drug sensitivity and tumorigenicity in intraspecies hybrids between 9-hydroxyellipticine-sensitive and -resistant cells.

Development of resistance to 9-hydroxyellipticine in Chinese hamster lung cells is associated with a loss of oncogenic potential. In order to determine whether these phenotypical traits are the consequences of the same biochemical modification, or whether they can be dissociated, we have studied their expression in intraspecies hybrids between sensitive and resistant cells. Thirteen hybrid clones were thus examined. At early passages after fusion, they displayed drug sensitivities intermediate to those of parental cells, but relatively close to that of the sensitive parent. Upon injection into nude mice, these clones exhibited variable oncogenic potentials. These variations were independent of the drug sensitivity. After further growth in the presence of 9-hydroxyellipticine, the hybrid cells recovered the drug resistance of the resistant parent. Yet, most of them remained clearly tumorigenic. Finally, the tumors produced by injection of the initial hybrids to nude mice were explanted in culture and then tested again for their drug sensitivity and tumorigenicity. These cells displayed both the tumorigenicity of the sensitive parent and the resistance of the resistant parent. Karyological analyses at the different steps of this work did not reveal any significant change which could be related to modifications of the expression of the characters studied. Our results show that the loss of tumorigenicity and the resistance to 9-hydroxyellipticine can be expressed independently and cannot be accounted for by a unique biochemical alteration.

Effects of 9-OH-ellipticine on cell survival, macromolecular syntheses, and cell cycle progression in sensitive and resistant Chinese hamster lung cells.

In an effort to understand the mechanism of action of the DNA-intercalating antitumor agent 9-hydroxyellipticine (9-OH-E), we have examined the effects of this drug on the cell survival, macromolecular syntheses, and cell cycle progression in sensitive and resistant cells. Our results show that 9-OH-E toxicity on sensitive and resistant cells involves different mechanisms of action: the drug toxicity in the sensitive cells appears to result from lethal lesions mediated through the interaction of the drug with an intracellular protein, independently of any effect of the drug on the macromolecular syntheses; in the resistant cells, the cell death occurs concomitantly with the inhibition of these syntheses. Cell cycle progression analysis after 9-OH-E treatment showed that, in the sensitive cells, the drug is inducing a G1 and a G2 block, which are both released in the presence of 1 mM caffeine, without any effect on the 9-OH-E toxicity. In the resistant cells, a G2 block was also observed but only when the cells were resuming their growth after about a 30- to 40-h growth arrest. Caffeine release of this block, which again had no effect on 9-OH-E toxicity, was only observed when it was added from 40 to 60 h after 9-OH-E treatment, when the cells resumed their growth. Finally in the sensitive cells, cycloheximide exerted an inhibitory effect on 9-OH-E toxicity when it was added before and during the cell exposure to the drug. This effect was interpreted as indicating that 9-OH-E toxicity in the sensitive cells relies on a protein which is not induced by the drug but has to be present in the cells when the drug is added. The possible implication of DNA topoisomerases in 9-OH-E toxicity mechanism is discussed.

Effects of new antitumor bifunctional intercalators derived from 7H-pyridocarbazole on sensitive and resistant L 1210 cells.

The antitumor properties of 7H-pyridocarbazole dimers, a new series of bifunctional intercalators, have recently been described (Pelaprat, D. Delbarre, A., Le Guen, I., Roques, B. P., and Le Pecq, J. B. J. Med. Chem., 23: 1336-1343, 1980; and Roques, B. P., Pelaprat, D., Le Guen, I., Porcher, G., Gosse, C., and Le Pecq, J. B. Biochem. Pharmacol., 28: 1811-1815, 1979). In order to study the mechanism of action of these compounds, an L1210 subline was made resistant to one dimer (NSC 335153; ditercalinium). Selection of resistant cells was based on an in vitro-in vivo procedure as follows. Ascitic cells were taken from a leukemic mouse and incubated in vitro with the dimer for 1 hr. They were then injected into mice. After the development of the ascites, L1210 cells were collected and the process was repeated 13 times, until establishment of the resistance. Cloned resistant cells have maintained their resistance for 18 months of in vitro culture. The effects of two dimers (NSC 335153 and NSC 335154) on cell viability, growth, colony formation, and cell cycle progression were investigated on parental and resistant L1210 cells. The cross-resistance of these two L1210 cell lines to several cytotoxic agents was estimated. Several observations indicate that the mechanism of action of these dimers might be different from that of monointercalating agents: (a) these drugs induce a delayed toxicity (growth arrest occurring five generations after drug exposure) in sensitive but not in resistant cells; (b) cells exposed to the dimers arrested almost randomly in all phases of the cell cycle, whereas the corresponding monomer provokes a block in the G2 + M phase. Resistant cells were cross-resistant to 7H-pyridocarbazole monomer, Adriamycin, and vincristine but not to 6H-pyridocarbazole monomer derivatives, actinomycin D, and methotrexate.

Attempts to target antitumor drugs toward opioid receptor-rich mouse tumor cells with enkephalin-ellipticinium conjugates.

Human colorectal and pulmonary carcinomas have been shown to contain high levels of opioid peptides and their corresponding membrane-bound receptors. Therefore possible targeted drugs, consisting of modified enkephalins linked to cytotoxic drugs, were designed. Such conjugates were expected to be specifically internalized within opioid receptor-bearing cells. As a model to this approach, we have synthesized enkephalin-ellipticinium conjugates in which the D-Ala2-D-Leu5-enkephalin (DADLE) was coupled to the 2-nitrogen of either ellipticine or 9-hydroxyellipticine, two drugs acting through different mechanisms of cytotoxicity. These conjugates, DADLE-ellipticinium (NME) and DA-DLE-9-hydroxyellipticinium (NMHE), respectively, were previously shown to retain in vitro both opioid receptors and DNA affinities close to those of the parent compounds. In this paper, we first show that each individual moiety in the complexes remains capable of recognizing its cellular targets. Thus, pretreatment of NG108-15 cells containing delta-opioid receptors by the DADLE-ellipticinium conjugates induced a loss of opioid receptor (down-regulation), while the smaller peptide conjugates, tyrosinyl-D-alanylglycine-ellipticinium, prepared as control, do not. On the other hand, peptide-NMHE conjugates were able to induce DNA topoisomerase II-associated DNA strand breaks suggesting that they have a mode of action similar to that of their parent molecule, NMHE. We then examined whether or not these molecules could exert a specific toxicity on opioid receptor-bearing cells. However, when tested on NG108-15 tumor cells and L-fibroblasts as control, the enkephalin-ellipticinium conjugates (DADLE-NME and DADLE-NMHE) proved to be similarly more cytotoxic on both cell lines than their ellipticinium (NME and NMHE) precursors. In order to understand this apparent lack of specificity we examined the cellular accumulation and distribution of DADLE-NME by fluorescence techniques. These experiments revealed that an important intracellular overconcentration caused by a nonspecific process is probably masking the specific targeted effect of the conjugates. Hence, the project of linking DADLE to highly cytotoxic molecules which cannot cross the plasma membrane without site-directed targeting is discussed.

Transfection of 9-hydroxyellipticine-resistant Chinese hamster fibroblasts with human topoisomerase IIalpha cDNA: selective restoration of the sensitivity to DNA religation inhibitors.

In the Chinese hamster lung cell line DC-3F/9-OH-E, selected for resistance to 9-OH-ellipticine and cross-resistant to other topoisomerase II inhibitors, the amount of topoisomerase IIalpha is 4-5-fold lower than in the parental DC-3F cells, whereas topoisomerase IIbeta is undetectable. Cloning and sequencing of topoisomerase IIalpha cDNAs from DC-3F and DC-3F/9-OH-E cells revealed an allele polymorphism, one allele differing from the other by the presence of seven silent mutations and three mutations in the noncoding region. In addition, the mutated allele contains three missense mutations located close to the ATP binding site (Thr371Ser) or to the catalytic site (Ala751Gly; Ile863Thr). To analyze the contribution of these topoisomerase IIalpha alterations to their resistance phenotype, DC-3F/9-OH-E cells were transfected with an eukaryotic expression vector containing the human topoisomerase IIalpha cDNA. In one transfected clone, the amount of topoisomerase IIalpha isoform and the catalytic activity were similar to that in the parental DC-3F cells. These cells, which contain only topoisomerase IIalpha, are then a unique mammalian cell line to analyze the physiological and pharmacological properties of this enzyme. However, the restoration of a nearly normal topoisomerase IIalpha activity in the DC-3F/9-OH-E cells did not have the same effect on their sensitivity to different enzyme inhibitors; a 75% reversion of the resistance, associated with a 2-3-fold increased stabilization of the cleavable complex, was observed with both etoposide and m-AMSA, two drugs that inhibit the DNA religation step in the enzyme catalytic cycle; in contrast, the transfected cells remained fully resistant to ellipticine derivatives that did not induce the stabilization of the cleavable complex. We hypothesized that a trans-acting factor, inhibiting the induction of cleavable complex formation by drugs that are not religation inhibitors, might be present in the resistant cells. However, such a factor was not detected in in vitro experiments, and other hypotheses are discussed.

Reduced formation of protein-associated DNA strand breaks in Chinese hamster cells resistant to topoisomerase II inhibitors.

DNA intercalating drugs and the epipodophyllotoxins etoposide and teniposide interfere with the action of mammalian DNA topoisomerase II by trapping an intermediate complex of the enzyme covalently linked to the 5'-termini of DNA breaks. This effect can be observed in intact cells by alkaline elution measurement of protein-associated DNA strand breaks. To assess the cytotoxic role of this effect, we have studied a subline of DC3F Chinese hamster lung cells selected for resistance to the intercalating agent 9-hydroxyellipticine. This subline (DC3F/9-OHE) was cross-resistant to other intercalators as well as to etoposide. Resistance to Adriamycin was associated with reduced uptake. However, resistance to 4'-(9-acridinylamino)methanesulfon-m-aniside and 2-methyl-9-hydroxyellipticinium was observed in the absence of changes in drug uptake, suggesting a second mode of resistance. DC3F/9-OHE cells formed fewer protein-associated DNA strand breaks in response to 4'-(9-acridinylamino)methanesulfon-m-aniside, 2-methyl-9-hydroxyellipticinium, or etoposide than did the sensitive parental cells. The same was true for isolated nuclei from these cells, which is consistent with a mode of resistance unrelated to drug uptake through the plasma membrane. These data suggest that resistance to DNA topoisomerase II inhibitors exhibited by DC3F/9-OHE cells is due in part to a modification of topoisomerase II activity.

Inhibitory effects of the tyrosine kinase inhibitor genistein on mammalian DNA topoisomerase II.

Tyrosine phosphorylation plays a crucial role in cell proliferation and cell transformation which suggests that tyrosine kinase-specific inhibitors might be used as anticancer agents. When the cytotoxic effect of the potent tyrosine kinase inhibitor genistein on various cell lines was studied, we observed that 9-hydroxyellipticine-resistant Chinese hamster lung cells (DC-3F/9-OH-E) were markedly more resistant to genistein than the parental cell line (DC-3F). The DC-3F/9-OH-E cells have been shown to have an altered DNA topoisomerase II activity. We therefore examined the effects of genistein on DNA topoisomerase II-related activities of nuclear extracts from DC-3F cells as well as on purified DNA topoisomerase II from calf thymus. Our results show that genistein (a) inhibits the decatenation activity of DNA topoisomerase II and (b) stimulates DNA topoisomerase II-mediated double strand breaks in pBR322 DNA on sites different from those of 4'-(9-acridinylamino)methanesulfon-m-anisidide, etoposide, and 2-methyl-9-hydroxyellipticinium. Structure-activity studies with six chemically related compounds show that only genistein has an effect on the cleavage activity of DNA topoisomerase II in the concentration range studied. Finally, genistein treatment of DC-3F cells results in the occurrence of protein-linked DNA strand breaks as shown by DNA filter elution. Viscometric (lengthening) studies demonstrate that genistein is not a DNA intercalator. Genistein is therefore an interesting compound because it induces cleavable complexes without intercalation. Taken together, our results show that genistein is an inhibitor of both protein tyrosine kinases and mammalian DNA topoisomerase II. This could be accounted for by the sharing of a common structure sequence between the two proteins at the ATP binding site.

Cloning and characterization of full-length cDNAs coding for the DNA topoisomerase II beta from Chinese hamster lung cells sensitive and resistant 9-OH-ellipticine.

DNA topoisomerase II beta cDNAs from Chinese hamster lung cells sensitive (DC-3F) and resistant to 9-OH-ellipticine (DC-3F/9O-HE) were isolated. In the sensitive cells, the sequence defines an open reading frame of 4839 nucleotides, and extends over a 323 nucleotides untranslated region up to the putative polyadenylation site. The deduced amino acid sequence predicts a protein with 1612 amino acids in length and a calculated molecular mass of approx. 182 kDa. The cDNAs from the resistant cells only differs by one mutation at position length and a calculated molecular mass of approx. 182 kDa. The cDNAs from the resistant cells only differs by one mutation at position 1710 which converts a Trp codon (TGG) to a stop codon (TGA). This mutation accounts for the loss of DNA topoisomerase II beta in the 9-OH-ellipticine resistant cells.

Alteration in p53 pathway and defect in apoptosis contribute independently to cisplatin-resistance.

The accumulation of molecular genetic defects selected during the adaptation process in the development of cisplatin-resistance was studied using progressive cisplatin-resistant variants (L1210/DDP2, L1210/DDP5, L1210/DDP10) derived from a murine leukemia cell line (L1210/0). Of these cell lines, only the most resistant L1210/DDP10 was cross-resistant to etoposide and deficient in apoptosis induced by these two drugs, indicating that resistance to DNA-damaging agents correlates with a defect in apoptosis. This defect was tightly associated with the loss of a Ca2+/Mg2+-dependent nuclear endonuclease activity present in the less cisplatin-resistant cells. Evidence is presented that p53-dependent function (a) is lost not only in the apoptosis defective L1210/DDP10 cells, but also in the apoptosis susceptible L1210/DDP5 cells; (b) is unrelated to drug-induced cell cycle perturbations. These results suggest that deficiency in the p53 pathway and resistance to DNA-damaging agents due to a defect in apoptosis are independent events.

Synthesis and biological activity of new dimers in the 7H-pyrido[4,3-c] carbazole antitumor series.

Ditercalinium (NSC 366241) is a 7H-pyrido[4,3-c]carbazole dimer with a diethylbipiperidine rigid chain linking the two heterocyclic rings. Ditercalinium is characterized by a high DNA affinity and bisintercalating ability, associated with potent antitumor properties, involving an original mechanism of action. Unfortunately as ditercalinium is hepatotoxic, its clinical evaluation has been interrupted. In order to eliminate or at least minimize the serious drawbacks related to its toxic effects, several chemical modifications have been made to the structure of ditercalinium, and their influence has been evaluated by measuring the DNA affinities, intercalation properties, and toxicity toward leukemia cells of the newly synthesized dimers. Reduction of the pyridinic moieties of ditercalinium, in order to suppress the permanent charges provided by the quaternizing chain, led to an almost complete loss of activity, although the DNA bisintercalating property of the dimer was preserved. Dimerization of the 7H-pyrido[4,3-c]carbazole rings by introduction of the rigid spacer on the N7- or C6-positions corresponding to the convex face of the pyridocarbazole, instead of the N2-position in ditercalinium, led to DNA bisintercalating dimers practically devoid of antitumor properties. However after quaternarization of the N2 atoms, the dimer linked by the N7 atoms exhibited a very high DNA affinity (greater than 10(9) M-1) and recovered antitumor activity, supporting the requirement of positive charges for the emergence of antitumor activity in these dimers. Introduction on the C6 of the 7H-pyridocarbazole ring of an aminomethyl or carboxyl group, a sugar residue, or C or N free amino acids such as Lys or Glu has also been carried out, in order to increase the hydrophilic properties of the molecules or to enable them to use amino acid transport systems. Although some of these compounds were active, none of them exhibited the pharmacological potency of ditercalinium.

Ellipticine derivatives with an affinity to the estrogen receptor, an approach to develop intercalating drugs with a specific effect on the hormone-dependent breast cancer.

In order to obtain breast tumor directed agents, we have prepared mixed compounds using estradiol or (E)-clomiphene as specific vectors of the breast tissue and a DNA intercalator from the ellipticine series as the cytotoxic agent. Among the newly synthesized ellipticine derivatives, only the 2-[3-aza-5-(3,17 beta-dihydroxy-1,3,5-estratrien-17 alpha-yl)-4-oxopentamethylene]ellipticinium bromide shows the desired properties, DNA intercalation and affinity for estrogen receptor. Competition experiments with estradiol on the hormone-dependent human MCF-7 breast cancer cell line demonstrate that a transport by the estrogen receptor system is not involved in the antitumor activity of derivative 24.

Epidermal growth factor receptor signaling cascade as target for tyrphostin (RG 50864) in epithelial cells. Paradoxical effects on mitogen-activated protein kinase kinase and mitogen-activated protein kinase activities.

Tyrphostins are synthetic compounds that have been described as in vitro inhibitors of epidermal growth factor receptor (EGF-R) tyrosine kinase activity. The inhibitory effect of tyrphostins in intact cells has been shown only after prolonged treatment. However, these compounds appear to be readily incorporated, which suggests that tyrphostin acts indirectly on EGF-R. We studied the effects of a tyrphostin derivative, RG 50864, without preincubation in intact epithelial cells. We selected two human cell lines differing in degree of expression of the p185erbB2 protein, which is closely related to EGF-R. We showed that tyrphostin (RG 50864) had no effect on EGF-dependent EGF-R tyrosine phosphorylation in the parental cell line. On the contrary, it prolonged the EGF-dependent EGF-R and p185erbB2(V-E) tyrosine phosphorylation in p185erbB2(V-E)-expressing cells. Because tyrphostin has been shown to be an inhibitor of p185erbB2 and EGF-R in vitro, this finding indicates that the tyrphostin effect on p185erbB2(V-E) and EGF-R was the result of an indirect mechanism in transfected cells. Tyrphostin treatment alone led to the activation of mitogen-activated protein (MAP) kinase kinase or MAP kinase or extracellular signal-regulated kinase kinase (MEK), suggesting that one of the tyrphostin targets was upstream of MEK1. MAP kinase, however, was not activated after tyrphostin treatment. This finding indicates that tyrphostin had another target in intact cells because MEK1 activation by tyrphostin alone did not correlate with MAP kinase activation. In the two cell lines, tyrphostin modified the time course of EGF-dependent MEK and MAP kinase activation. We conclude that whereas tyrphostins were designed to inhibit EGF-R tyrosine kinase activity, under our conditions EGF-R is not a physiological target for tyrphostin, nor is one of its related protein tyrosine kinases, p185erbB2(V-E). On the contrary, our results show that tyrphostin targets are multiple, leading to complex effects on receptor signaling in these epithelial cells.

Induction of pgp3 expression and reversion of the multidrug resistance phenotype in 9-OH-ellipticine-resistant Chinese hamster lung fibroblasts transfected with the MYC oncogene.

Chinese hamster lung cells resistant to the DNA topoisomerase II inhibitor 9-OH-ellipticine (DC-3F/9-OH-E) are cross resistant to various drugs through the expression of the MDR phenotype. The myc oncogene was approximately 10-fold amplified and 20-fold overexpressed in parental DC-3F cells as compared with DC-3F/9-HO-E cells. Transfection of the resistant cells with a mouse c-myc gene did not alter the resistance to topoisomerase II inhibitors and, in cells with a low multidrug (MDR) expression, reversed this phenotype. Northern and Western blot analyses revealed an increased expression of pgp1 in the DC-3F/9-OH-E cells, which was not modified in the myc-transfected clones. However, myc expression in these clones resulted in an increased expression of pgp3, roughly in proportion to the level of myc expression. Transfection of the DC-3F/9-OH-E cells with the human MDR3 gene, homologous to pgp3, also resulted in the reversion of the MDR phenotype. These results show that (1) expression of the transfected myc gene positively regulates pgp3 expression but has no effect on pgp1; (2) when observed, reversion of the MDR phenotype is proportional to the levels of myc and pgp3 expression; and (3) this reversion, resulting from pgp3 expression, is associated with a decreased functional activity of the pgp1 protein and might require an appropriate balance of pgp1 and pgp3 expression.

Uptake and cytofluorescence localization of ellipticine derivatives in sensitive and resistant Chinese hamster lung cells.

Uptake of two ellipticine derivatives, 2-N-methyl-ellipticinium (NME) and 2-N-methyl-9-hydroxy-ellipticinium, by sensitive and resistant Chinese hamster lung cells was studied. The results show that uptake and retention of these molecules by both types of cells were identical, thus indicating that the resistance to ellipticines, in this system, is not related to an impaired permeability of the cells to the drugs. However, influx and efflux kinetics, as well as experiments at increasing external concns, showed that both drugs accumulate within the cells in different ways. A cellular overconcentration of the drugs, which does not require an energy-dependent process, is observed. Fluorescence microscopy showed that, in both sensitive and resistant cells, NME is mainly, if not entirely, located in the cytoplasm.

Effects of verapamil on the cellular accumulations and toxicity of several antitumor drugs in 9-hydroxy-ellipticine-resistant cells.

9-OH-Ellipticine (9-OH-E)-resistant cells are not only resistant to the DNA topoisomerase II inhibitors, but also to some other antitumor agents, such as actinomycin D (AD), adriamycin (ADM), daunorubicin and vincristine. It was previously shown that a decreased uptake accounts for the cross-resistance of these cells to AD and ADM which then suggested that the 9-OH-E-resistant cells might display some of the properties usually associated with the multidrug resistance phenotype. In this work, we have examined the effects of verapamil, a drug which is known to overcome the multidrug resistance, on the toxicity and the cellular accumulation of four cytotoxic agents: 9-OH-E, 2N-methyl-9-hydroxy-ellipticinium (NMHE), AD and ADM, either on 9-OH-E resistant cells or on a multidrug resistant subline derived from the same sensitive parental cells. Verapamil inhibited the cellular accumulation of the ellipticine derivatives in the sensitive DC-3F cells, and the toxicity of these drugs on these cells was correspondingly decreased. On either one of the resistant cell lines, verapamil had no effect on the toxicity and the cellular accumulation of 9-OH-E. In contrast, in the presence of verapamil, the cellular accumulation of NMHE by the 9-OH-E and the multidrug resistant cells was about 50% and 300% increased, respectively. The increased NMHE cellular concentration in the multidrug resistant cells was associated with an 8-fold increased toxicity. The major structural characteristics which might account for this difference between the sensitivities of both ellipticine derivatives to the effects of verapamil on the multidrug resistant cells is the presence of a positive charge on the nitrogen in position 2 of the 6H-pyridocarbazole molecule. Finally, verapamil circumvented partially the cross-resistance of DC-3F/9-OH-E cells to AD and ADM by increasing the accumulation of these drugs inside the cells.

Reduced DNA topoisomerase II activity and drug-stimulated DNA cleavage in 9-hydroxyellipticine resistant cells.

We have isolated a Chinese hamster lung cell line resistant to 9-hydroxyellipticine (DC-3F/9-OH-E) which is also cross-resistant to topoisomerase II inhibitors such as amsacrine and etoposide. In this work we have studied quantitatively both DNA topoisomerase II activity by decatenation of kinetoplast DNA and drug-stimulated DNA cleavage of pBR 322. DNA topoisomerase II activity of DC-3F/9-OH-E nuclear extract was reduced by 3.5-fold as compared to that from DC-3F (sensitive parent) nuclear extract. We also found that DC-3F/9-OH-E nuclear extracts have a reduced capacity to induce in vitro topoisomerase II-mediated DNA cleavage upon stimulation by etoposide and amsacrine (7- and 10-fold respectively). Besides, mixing nuclear extracts from both sensitive and resistant cells indicates that either the enzyme in resistant cells is modified or a modulating factor is associated to it. Our results suggest that the resistance of the DC-3F/9-OH-E cell line to topoisomerase II inhibitors might be due to both a reduced amount of the enzyme and its reduced ability to form the cleavable complex in the presence of drugs.

Effects of 7H-pyridocarbazole mono and bifunctional DNA-intercalators on Chinese hamster lung cells in vitro.

The effects of two 7H-pyridocarbazole dimers, PyDi1 and PyDi2, on Chinese hamster lung cells in culture in vitro, were compared to those of the corresponding monomers, PyMo1 and PyMo2, by measuring the rates of macromolecule syntheses, the growth kinetics of the drug-treated cells, and the cell cycle progression. The dimers, which are endowed with a very high DNA affinity, were about 10- and 40-fold more cytotoxic than the monomers from which they markedly differ in the following ways: in contrast to monomers, the dimers do not provoke the arrest of cell cycle progression in the G2 + M phase; after a transitory exposure to either one of the dimers, the cell growth arrest was delayed for 6-8 generations. Therefore, the 7H-pyridocarbazole dimers express their cytotoxicity through a mechanism of action different from that of their mono-intercalating counterparts. They might then constitute a new series of antitumour drugs.