The effect of alkylating agents on the activity of adenosine 3',5'-monophosphate-dependent protein kinase in Walker carcinoma cells.

The effect of some alkylating agents on the activity of the adenosine 3′,5′-monophosphate (cyclic AMP)-dependent protein kinase has been studied in Walker cells sensitive and resistant to the cytotoxic action of such agents. Chlorambucil (5 μg/ml) caused an activation of the cAMP-dependent protein kinase in sensitive Walker carcinoma cells which reached a maximum 1.5 hr after drug addition. Sephadex gel chromatography indicated that during this activation, the catalytic subunit of the protein kinase was released from the holoenzyme to the same extent as that measured in the crude supernatant of the tumour cells. The degree of activation was equivalent to that produced by 100 μg/ml of N⁶O^2′-dibutyryl cAMP. In contrast, the monofunctional N-ethyl analogue of chlorambucil had no effect on the cAMP-dependent protein kinase at a dose of 250 μg/ml. The protein kinase activity ratio in sensitive cells increased with increasing doses of chlorambucil and reached a maximal activation at a concentration of 5 μg/ml, which was sufficient to cause complete inhibition of tumour cell growth. A much larger dose of chlorambucil (100 μg/ml) was required to cause activation of the kinase in Walker cells resistant to this agent. Chlorambucil (25 μg/ml) also caused an activation of the cAMP-dependent protein kinase in TLX5 cells, though the time scale of the activation differed for that found in Walker cells. Both merophan and 5-aziridinyl-2,4-dinitrobenzamide (CB 1954) caused an increase in the protein kinase activity ratio of sensitive Walker cells. The increase caused by CB 1954 could be abolished by 4-amino-2-phenylimidazole-5-carboxamide (2-phenyl-AlC). which reverses the tumour growth inhibitory action of CB 1954. The degree of stimulation of the cytosolic protein kinase by saturating concentrations of cAMP, and the apparent dissociation constant for cAMP bound to protein kinase decreased with increasing resistance of the cell lines to alkylating agents. These results sugest that the biological effect of the increase in cAMP in sensitive Walker cells induced by the alkylating agents is mediated through a protein kinase.     

Identification of a glutathione S-transferase associated with microsomes of tumor cells resistant to nitrogen mustards

Walker 256 rat mammary carcinoma cells resistant to chlorambucil (WR) exhibited an approximate 4-fold increase in glutathione S-transferase (GST) activity using 1-chloro-2,4-dinitrobenzene as compared to the sensitive parent cell line (WS). WR cells maintained without biannual exposure to chlorambucil (WRr) reverted to the sensitive phenotype and possessed GST levels equivalent to WS. Mitochondria, microsomes and cytosol were isolated from WS, WR and WRr cell lines and analyzed for their GST composition. GST activity in each subcellular compartment of resistant cells was increased over the sensitive cells. Antibodies raised against total rat liver cytosolic GST crossreacted in resistant cells with two microsomal proteins (25.7 kD and 29 kD). The 29 kD protein was not detected in microsomal fractions from either WS or WRr and this protein was found to be dissimilar from cytosolic GST subunits in its isoelectric point (pI 6.7) and migration on two-dimensional polyacrylamide gels. In addition, the 29 kD microsome-associated GST from WR cells was immunologically distinct from a 14 kD GST subunit previously identified in rat liver microsomes. These data implicate the induction of a specific microsomal GST subunit in WR cells following drug selection and suggest its potential involvement in the establishment of cellular resistance to chlorambucil.     

Tumours from a cell strain with a high content of metallothionein show enhanced resistance against cis-dichlorodiammineplatinum

Cultured cells with a high content of the extremely cysteine-rich protein metallothionein (MT) have previously been shown to exhibit resistance when exposed to otherwise lethal doses of cis-dichlorodiammineplatinum (cis-DDP), chlorambucil or prednimustine, and to have a significant proportion of intracellular drug associated with MT after such treatment. In order to study this protective mechanism in vivo, cells from a MT-rich variant of a murine fibroblast line resistant to cis-DDP and its parent sensitive line with only trace amounts of MT, were injected subcutaneously into nude mice (24 animals in each group), and tumour growth was compared during cis-DDP treatment. Animals in the treatment groups received 3 intravenous doses of either 4 mg/kg or 8 mg/kg of cis-DDP on day 12, 26 and 33 after inoculation of cells, whereas the control groups received saline. The 8 mg/kg dose produced an almost complete growth inhibition of the tumours derived from the parent cells, as well as from the MT-rich variant. However, following the injections with 4 mg/kg of cis-DDP, tumour volume was reduced by approximately 80% in tumours from the parent cells, whereas the tumours from MT-rich cells were almost completely resistant. This study provides for the first time evidence that metallothionein-conferred protection against cis-DDP toxicity also is mediated in tumours in vivo.     

Uptake and decomposition of chlorambucil by L5178Y lymphoblasts in vitro

The uptake of [¹⁴C]chlorambucil by L5178Y lymphoblasts was studied using thin-layer chromatography to identify the various radioactive components that enter or leave cells. Theoretical calculations predicted that entry of chlorambucil into cells by simple diffusion would be rapid and essentially complete in 45 sec or less. Uptake of intact chlorambucil was rapid, reaching a cell/medium ratio of approximately 1.5 in less than 15 sec at both 37° and 4°, consistent with a simple diffusion mechanism. In cells treated with [¹⁴C]chlorambucil for 60 min, the intracellular level of intact drug decreased with time, and this decay was attributed to hydrolysis and alkylation. The level of intact drug in the medium decreased at a similar rate resulting in a nearly constant cell/medium distribution ratio. Intact chlorambucil in the cells was found to be entirely ethanol- and trichloroacetic acid-soluble. Efflux of intact chlorambucil was very rapid and temperature-insensitive. These findings suggest that chlorambucil efflux, as well as influx, is by a simple diffusion mechanism. A derivative of chlorambucil was found in ethanol solutions of the drug. This derivative, which may be the ethyl ester of the drug, is highly concentrated in cells and may interfere with pharmacological investigations of chlorambucil.     

Comparison of the Cytotoxic Effect of Prednimustine in Cultured Cells with High or Low Levels of Metallothionein

Abstract: The purpose of this investigation was to evaluate the ability of the cysteinyl-rich protein metallothionein (MT) to protect cells against the cytotoxic effects of prednimustine, an ester of chlorambucil and prednisolone. The cells studied were MT-rich substrains of murine fibroblasts (CI 1D₁₀₀) and human epithelial cells (HE₁₀₀), both demonstrated in an earlier report to exhibit an approximate 3-fold increase in resistance to chlorambucil compared to their parent lines (C) 1D and HE) (Endresen et al. 1983). Both in cloning and in growth rate studies the MT-rich strains proved to be significantly more resistant also to predinimustine. E.g. in cloning studies D₀ for C11D cells (D₀ = the dose of drug reducing survival to 1/e) was 8.7 μg/ml prednimustine, whereas D₀ for C11D ₁₀₀ was 12.4 μg/ml, representing an approximate 1.5-fold increase in resistance, P<0.001, t-test. Other cloning studies revealed that prednimustine had a significantly higher cell killing activity in the resistant cells than equimolar concentrations of its components, single or in combination. Following treatment with ³H, ^l4C-prednimustine (³H in the prednisolone moiety, ¹⁴C in the chlorambucil moiety) and subsequent gel filtration, about 40% of the cytosolic chlorambucil eluted with MT. However, no intact prednimustine was recovered in the MT fractions. The data indicate that the MT-rich cells possess increased resistance to prednimustine due to a sequestration by MT of the alkylating moiety. Since the interaction probably does not take place until after hydrolysis, it is possible that the intact conjugate may bypass this cellular defence mechanism.     

Human renal UOK130 tumor cells: a drug resistant cell line with highly selective over-expression of glutathione S-transferase-pi isozyme

Drug resistance remains one of the primary causes of suboptimal outcomes in cancer chemotherapy. This study reports the development of a drug resistant cell line with over-expression of glutathione S-transferase (GST). The resistant tumor cell model was established by continuous exposure of UOK130, a human renal tumor cell line, to escalating concentrations of cisplatin. By immunoblotting the cisplatin-resistant cells (UOK(CR)) were found to express an elevated level of GST-pi isozyme. Neither alpha nor mu isozyme was detected by the corresponding polyclonal antibodies. A significant increase in cellular glutathione (GSH) was also observed in UOK(CR) cells comparative to the parental cells. In addition, the continuous exposure to cisplatin resulted in decreased cell susceptibility not only to cisplatin (resistant factor: 5.7) but also to melphalan (resistant factor: 2.9) and chlorambucil (resistant factor: 2.3). A transgenic cell line was developed by transfecting of UOK130 cells with GST-pi cDNA. The transfection of the GST-pi virus into UOK130 cell apparently increased its intracellular GST-pi activity. The resistance of the transfectants to cisplatin was consistently increased, compared with that of mock transfectants. A haloenol lactone (HEL) derivative known as a selective inhibitor of GST-pi was applied to evaluate the suitableness of the cell model for GST-pi-mediated drug resistance studies. The inhibitor was found to potentiate the cytoxicity of cisplatin to both UOK130 and UOK(CR) cell lines and to reverse their resistance to cisplatin. In conclusion, we developed a multiple drug resistant tumor cell line with selective over-expression of GST-pi. The cell model provides a unique tool for mechanistic studies of drug resistance mediated by over-expression of GST-pi.     

The use of Tris-lipidation to modify drug cytotoxicity in multidrug resistant cells expressing P-glycoprotein or MRP1

1. Increasing the lipophilicity is a strategy often used to improve a compound's cellular uptake and retention but this may also convert it into a substrate for an ATP-dependent transporter such as P-glycoprotein or the multidrug resistance-associated protein (MRP1), which are involved in cellular efflux of drugs. Tris-Lipidation of compounds is a convenient way of modifying drug lipophilicity and generating an array of derivatives with diverse properties. 2. To determine the effect of Tris-Lipidation on a drug's cytoxicity in multidrug resistant cells, various glycyl-Tris-mono- (GTP1), di- (GTP2) and tri-palmitate (GTP3) derivatives were prepared of the cancer chemotherapeutic drugs chlorambucil and methotrexate, and of the anti-HIV drug AZT. The cytotoxicity of these derivatives and their parent compounds was determined in the CEM/VLB(100) cells with increased P-glycoprotein expression, the CEM/E1000 cells that overexpress MRP1 and the parent, drug-sensitive CCRF-CEM cells. 3. Increasing the lipophilicity of AZT increased its cytotoxicity in the sensitive CCRF-CEM parental cell line while decreased cytotoxicity was observed for the methotrexate derivatives. For the chlorambucil derivatives, both increased (GTP1) and decreased (GTP2) cytotoxicity occurred in the CCRF-CEM cells. With the exception of AZT-GTP1, all GTP1 and GTP2 derivatives of chlorambucil, methotrexate and AZT had decreased cytotoxicity in the P-glycoprotein-expressing CEM/VLB(100) cells while chlorambucil-GTP1, methotrexate-GTP2 and methotrexate-GTP3 were the only compounds with decreased cytotoxicity in the MRP1-overexpressing CEM/E1000 cells. 4. The number of palmitate residues, the position of derivatisation and the type of linkage all may affect the P-glycoprotein and MRP1 substrate properties. 5. Tris-Lipidation may therefore provide a useful way of manipulating the pharmacokinetic properties of drugs.     

Relationships between resistance to cross-linking agents and glutathione metabolism, aldehyde dehydrogenase isozymes and adenovirus replication in human tumour cell lines

In a panel of 10 human tumour cell lines with no prior exposure to drugs in vitro, resistance to cisplatin correlated with resistance to the nitrogen mustard derivatives Asta Z-7557 (mafosfamide, an activated form of cyclophosphamide), melphalan and chlorambucil. Simultaneous treatment with ,R-sulfoximine did not enhance the toxicity of cisplatin or Asta Z-7557, and no correlation was found between drug resistance and cellular levels of metallothioneins (as judged by sensitivity to cadmium chloride), glutathione (GSH), GSH reductase, GSH transferase, or γ-glutamyltranspeptidase. The two cell lines most resistant to Asta Z-7557 expressed aldehyde dehydrogenase cytosolic isozyme 1, found also in normal ovary, but not isozyme 3. Treatment of resistant cells with cisplatin or Asta Z-7557 inhibited cellular DNA synthesis and replication of adenovirus 5 to a lesser extent than in sensitive cells. The virus could be directly inactivated by both drugs prior to infection, subsequent replication being inhibited to the same extent in sensitive and resistant cells. In contrast to Asta Z-7557 and other DNA damaging agents, cisplatin was much more toxic to adenovirus ( ₃₇ 0.022–0.048 μM) than to cells ( ₃₇ 0.25–2.5 μM). The adenovirus 5 mutant Ad5ts125 having a G → A substitution was even more sensitive to cisplatin ( ₃₇ 7–8 nM) than wild type virus and another mutant. Cisplatin was detoxified less by sonicated resistant cells than sensitive cells, as judged by inactivation of Ad5ts125 added to the reaction mixture. It can be inferred that (i) the major differences in cellular resistance to cisplatin and Asta Z-7557 in the present material did not involve enhanced DNA repair or protection by metallothioneins or GSH, but were associated with the ability to continue cellular and viral DNA synthesis during treatment, (ii) resistance was not associated with less template damage, and (iii) the adenovirus genome may be a suitable probe for predicting tumour resistance to cisplatin and for elucidating the DNA sequence dependence of cisplatin toxicity.     

Inhibitors of p-glycoprotein--lead identification and optimisation

The membrane bound drug efflux pump P-glycoprotein (P-gp) transports a wide variety of functionally and structurally diverse cytotoxic drugs out of tumour cells. Overexpression of P-glycoprotein is one of the predominant mechanisms responsible for development of multiple drug resistance in tumour therapy. Thus, inhibition of P-gp represents a promising approach for treatment of multidrug resistant tumours. This review highlights concepts for identification and optimization of new inhibitors of P-glycoprotein.     

Prolidase-activated prodrug for cancer chemotherapy cytotoxic activity of proline analogue of chlorambucil in breast cancer MCF-7 cells

Although prolidase [EC 3.4.13.9] is found in normal cells, substantially increased levels are found in some neoplastic tissues. Because prolidase possesses the ability to hydrolyse imido bonds of various low molecular weight compounds coupled to L-proline, we hypothesized that coupling of L-proline through an imido bond to anticancer drugs might create prodrugs which would be locally activated by tumour-associated prolidase and consequently would be less toxic to normal cells that evoke lower prolidase activity. To test this concept we have synthesized a conjugate of chlorambucil-proline (CH-pro) as a possible prodrug. Treatment of this prodrug with prolidase generated the L-proline and the free drug, demonstrating its substrate susceptibility to prolidase. We have compared several aspects of biological actions of chlorambucil (CH) and its prodrug in breast cancer MCF-7 cells. IC50 values for chlorambucil and for CH-pro in DNA synthesis were found to be 54 and 16 microM, respectively. CH-pro also exhibited a lesser ability to inhibit collagen biosynthesis in breast cancer MCF-7 cells compared to the free drug. The IC50 values for chlorambucil and for CH-pro in collagen biosynthesis were found to be about 32 and 80 microM, respectively. This suggests that the targeting of prolidase may serve as a potential strategy for converting antineoplastic prodrugs.     

Concentration and time-dependent inter-relationships for cytotoxicities of nitrogen mustard drugs against lymphoblasts in-vitro

Peripheral lymphoblasts were exposed either to different initial concentrations of the alkylating agents (melphalan, chlorambucil or phenylacetic acid mustard) using a fixed incubation time or a constant [3H]methylthymidine incorporation into the trichloracetic acid-insoluble fraction of the cells. The concentration-time relationships were evaluated by calculating the amount of drug which had chemically reacted in the incubation system. Melphalan showed lower cytotoxicity at short exposure times and high drug concentrations, while chlorambucil exhibited higher cytotoxicity at longer exposure times. In the latter case the effect could be accounted for by the cytotoxic activity of monohydroxy chlorambucil which was formed in the incubation system.     

Hemisynthesis of antineoplastic conjugates with nitrogen-mustard proteins

The synthesis of new conjugates with inhibitory action on tumour growth is investigated by linking amino functions of proteins compounds (lysozyme and alpha s-casein) through an amide linkage at the carboxylic function of nitrogen mustards (chlorambucil and melphalan). The polychlorambucil amides of lysozyme and alpha s-casein derivatives prepared showed experimental antitumour activity when these conjugates were screened against the experimental P388 leukemia. In the case of the conjugates lysozyme-melphalan, an antitumour activity is observed when the amino function of the drug is combined with the carboxylic functions of the protein contrary to the situation of the free amino function of the drug described into the literature.     

Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists, 1994: HYPOXIA-ACTIVATED PRODRUGS AS ANTITUMOUR AGENTS: STRATEGIES FOR MAXIMIZING TUMOUR CELL KILLING

1. Hypoxia arises in solid tumour because of inefficient blood supply. While hypoxic cells are resistant to radiotherapy and probably to many chemotherapeutic drugs they can, in principle, be turned to advantage through the development of hypoxia-activated cytotoxic drugs (bioreductive drugs). 2. Three general approaches to exploiting tumour hypoxia are discussed. The first relies on fluctuating blood flow in tumours and the consequent cycling of cells through the hypoxic compartment. The second incorporates a prodrug approach in which drug activation gives rise to cytotoxic metabolites which diffuse out of hypoxic zones. The third utilizes selective inhibitors of tumour blood flow to induce additional hypoxia and thus enhance bioreductive drug activation. 3. The latter two approaches are illustrated by recent studies with the dinitrobenzamide nitrogen mustard class of bioreductive drugs and their combination with the tumour blood flow inhibitor 5,6-dimethylxanthenone-4-acetie acid.     

Resistance to Fas-mediated apoptosis in malignant tumours is rescued by KN-93 and cisplatin via downregulation of c-FLIP expression and phosphorylation

1. The purpose of the present study was to investigate the molecular mechanisms that control tumour cell resistance and to search for molecules that could overcome Fas ligand (FasL) or CH-11 resistance in certain tumours, including glioma and melanoma. 2. Twelve tumour cell lines were examined for their sensitivity to CH-11-induced apoptosis and then two of each of the CH-11-sensitive and -resistant tumour cell lines were analysed for Fas-mediated death-inducing signalling complex (DISC). The calmodulin kinase II (CaMKII) inhibitor KN-93 and the chemotherapeutic drug cisplatin were used to treat resistant cells; the effects of these two drugs on CH-11-resistant tumour cells were investigated. 3. In CH-11-sensitive tumour cells, apoptosis-initiating caspase 8 and caspase 10 were recruited to the DISC, where they became activated through autocatalytic cleavage, leading to apoptosis through cleavage of downstream substrates, such as caspase 3 and DNA fragmentation factor 45. 4. In CH-11-resistant cells, cellular Fas-associated death domain-like interleukin-1b-converting enzyme inhibitory protein (c-FLIP) proteins were recruited to the DISC, resulting in inhibition of caspase 8 and caspase 10 cleavage. The c-protein expression and phosphorylation of FLIP and CaMKII protein and enzyme activity were upregulated in resistant cells. Treatment of resistant cells with 100 micromol/L KN-93 and 10 microg/mL cisplatin downregulated c-FLIP expression, inhibited c-FLIP phosphorylation and rescued CH-11 sensitivity. 5. In conclusion, KN-93 and cisplatin inhibit c-FLIP protein expression and phosphorylation restores CH-11-induced apoptosis in tumour cells. tHe present study provides evidence for the use of a new combination therapeutic strategy in the treatment of malignant tumours.     

Characterization of a chlorambucil-resistant human ovarian carcinoma cell line overexpressing glutathione S-transferase mu.

Ovarian carcinoma cells 10-fold resistant to the alkylating agent chlorambucil (CBL) were isolated after repeated exposure of the parent cells to gradually escalating concentrations of the drug. The resistant variant, A2780(100), was highly cross-resistant (9-fold) to melphalan and showed lower-level resistance to other cross-linking agents. The resistant A2780(100) cells had almost 5-fold higher glutathione S-transferase (GST) activity than the parental A2780 cells with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The pi-class GST(s) was the major isoform(s) in both cell lines. However, the resistant A2780(100) cells had at least 11-fold higher GST mu as compared with the parental cells, in which this isoform was barely detectable. A significant induction of GST mu was observed in A2780 cells, but not in the resistant cells, 18 hr after a single exposure to 100 microM CBL. The induction of GST mu by CBL was both time- and concentration-dependent. Assays of the conjugation of CBL with GSH showed that the human mu-class GST had 3.6- and 5.2-fold higher catalytic efficiency relative to the pi- and alpha-class GSTs, respectively. This difference was reflected in the relatively higher (about 6-fold) efficiency of CBL conjugation in A2780(100) cells as compared with the parental cells. These results have demonstrated for the first time a near-linear correlation between CBL resistance and overexpression of mu-class GSTs and suggest that this overexpression maybe responsible, at least in part, for the acquired resistance of ovarian carcinoma cells to CBL, and possibly the other bifunctional alkylating agents. Consistent with this hypothesis, we found evidence for decreased formation of DNA lesions in A2780(100) compared with the drug-sensitive A2780 cells after exposure to CBL.     

Resistance in cancer: a target for drug discovery

Resistance remains a major problem in the clinical utility of cancer chemotherapy. However, it also represents a tumour cell phenotype that is in many ways different, and thus distinguishable, from the majority of normal cells. Two approaches to the targeting of resistant cells are described involving intratumoral P450 expression, mechanisms of drug-efflux and defective DNA repair. It is suggested that the view of the solid tumour as a complex organ rather than a collection of individual cells will inform future drug development and both overcome and target multiple resistance mechanisms.     

Drug delivery to tumours: recent strategies

Despite several advancements in chemotherapy, the real therapy of cancer still remains a challenge. The development of new anti-cancer drugs for the treatment of cancer has not kept pace with the progress in cancer therapy, because of the nonspecific drug distribution resulting in low tumour concentrations and systemic toxicity. The main hindrance for the distribution of anti-cancer agents to the tumour site is the highly disorganized tumour vasculature, high blood viscosity in the tumour, and high interstitial pressure within the tumour tissue. Recently, several approaches such as drug modifications and development of new carrier systems for anti-cancer agents have been attempted to enhance their tumour reach. Approaches such as drug delivery through enhanced permeability and retention (EPR) effect have resulted in a significant improvement in concentration in tumours, while approaches such as drug-carrier implants and microparticles have resulted in improvement in local chemotherapy of cancer. This review discusses different strategies employed for the delivery of anti-cancer agents to tumours, such as through EPR effect, local chemotherapeutic approaches using drug delivery systems, and special strategies such as receptor-mediated delivery, pH-based carriers, application of ultrasound and delivery to resistant tumour cells and brain using nanoparticles.     

Expression of human mu or alpha class glutathione S-transferases in stably transfected human MCF-7 breast cancer cells: effect on cellular sensitivity to cytotoxic agents.

Increased expression of certain glutathione S-transferase (GST) isoenzymes has frequently been associated with the development of resistance to alkylating agents and other classes of antineoplastic drugs in drug-selected cell lines. The question arises whether this phenomenon is causal or is a stress-induced response associated with drug resistance in these cell lines. We have constructed mammalian expression vectors containing the human GST mu and GST alpha 2 (Ha2) cDNAs and stably transfected them into the human breast cancer cell line MCF-7. Whereas the parental and pSV2neo-transfected cell lines display low GST activity, three individual transfected clones were identified in each group that expressed either GST mu or GST alpha 2. The range of GST activities was similar to those observed in cells selected for anticancer drug resistance. The GST mu specific activities were 56, 150, and 340 mlU/mg, compared with 10 mlU/mg of endogenous GST mu in control lines. Specific activities in GST alpha 2-transfected clones were 17, 28, and 52 mlU/mg, compared with no detectable alpha class GST in control lines. These clonal lines and the parental and pSV2neo-transfected control lines were tested for sensitivity to antineoplastic agents and other cytotoxic compounds. The clones with the highest activity in each group were 1.7-fold (GST alpha 2) to 2.1-fold (GST mu) resistant to the toxic effects of ethacrynic acid, a known substrate for GSTs. However, the GST-transfected cell lines were not resistant to doxorubicin, L-phenylalanine mustard, bis(2-chloroethyl)-1-nitrosourea, cisplatin, chlorambucil, or the GST substrates 1-chloro-2,4-dinitrobenzene or tert-butyl hydroperoxide. Thus, although L-phenylalanine mustard, bis(2-chloroethyl)-1-nitrosourea, chlorambucil, tert-butyl hydroperoxide, and 1-chloro-2,4-dinitrobenzene are known to be metabolized by glutathione-dependent GST-catalyzed reactions, there was no protection against any of these agents in MCF-7 cell lines overexpressing GST mu or GST alpha 2. We conclude that, at the levels of GST obtained in this transfection model system, overexpression of GST mu or GST alpha 2 is not by itself sufficient to confer resistance to these anticancer agents. These studies do not exclude the possibility that GST may be a marker of drug resistance or that other gene products not expressed in MCF-7 cells might cooperate with GST to confer drug resistance.     

Effects of adriamycin and etoposide on the replication of adenovirus 5 in sensitive and resistant human tumour cells

Adenovirus is a potential probe for identifying and understanding drug sensitivity in primary, nonproliferating cultures of human normal and tumour cells but the scope and limitations of such an approach first need to be evaluated in established cell lines. For this purpose we have identified an ovarian tumour cell line (CI-80-13S) with natural resistance to adriamycin, etoposide and crosslinking agents compared with other human tumour lines. Resistance to adriamycin correlated poorly with resistance to etoposide in these cell lines (r = 0.05). Adenovirus replication in drug-treated cells (viral capacity) was found to be differentially inhibited in sensitive cells when the drug was administered to cells simultaneously with infection (adriamycin) or 20 hr after infection (etoposide). Viral capacity could not be inhibited by more than 90% in sensitive cells. In contrast, no such plateau was exhibited in the dose-responses of cell survival or inhibition of cellular DNA synthesis, both of which distinguished sensitive from resistant cells. Adenovirus was not inactivated by preincubation with high doses of adriamycin or etoposide, thus confirming that no functionally-relevant damage is directly induced by these agents in DNA. Uptake of adriamycin and etoposide was similar in sensitive and resistant cells and both agents blocked cells in the G2 phase of the cell cycle. Protein-linked DNA was induced in sensitive cells. The results indicate that (a) these drugs have two dose-dependent effects in cells, one of which does not inhibit replication of adenovirus; and (b) inhibition of adenovirus replication could in principle be used to predict sensitivity to adriamycin and etoposide.     

Amidine analogue of chlorambucil is a stronger inhibitor of protein and DNA synthesis in breast cancer MCF-7 cells than is the parent drug

A novel amidine analogue of chlorambucil-N-(2-(4-(4-bis(2-chloroethyl)aminophenyl)butyryl)aminoethyl)-5-(4-amidinophenyl)-2-furancarboxamide hydrochloride (AB(1)) and the parent drug were compared for their effects on collagen and DNA biosynthesis in breast cancer MCF-7 cells. IC(50) values for chlorambucil and AB(1) for collagen biosynthesis were found to be about 33 and 13 microM, respectively. The greater potency of AB(1) to suppress collagen synthesis was found to be accompanied by a stronger compared with chlorambucil inhibition of prolidase activity and expression. The phenomenon was related to inhibition of beta(1)-integrin and IGF-I receptor-mediated signaling caused by this compound. The expression of beta(1)-integrin receptor, as well as Src, son of sevenless protein (SOS) and phosphorylated mitogen activated protein (MAP) kinases (MAPK), extracellular-signal-regulated kinase 1 (ERK(1)) and kinase 2 (ERK(2)) but not focal adhesion kinase pp125(FAK) (FAK), Shc, and Grb-2 was significantly decreased in cells incubated for 24 h with 10 microM AB(1) compared to the control, whereas in the same conditions chlorambucil did not evoke any changes in expression of all these signaling proteins, as shown by Western immunoblot analysis. Furthermore, AB(1) induced a stronger down-regulation of the expression of IGF-I receptor and evoked a higher antiproliferative effect. During 12 and 24 h of incubation AB(1) decreased DNA biosynthesis by about 33 % and 51 % of the control, whereas chlorambucil decreased it by about 19 % and 35 %, respectively. These data suggest that the amidine analogue of chlorambucil is a stronger inhibitor of protein and DNA synthesis in MCF-7 cells than is the parent drug.