Multiple mechanisms of resistance in a series of human testicular teratoma cell lines selected for increasing resistance to etoposide

Mechanisms of resistance to VP-16 were monitored in a series of sublines of the human testicular teratoma cell line (SuSa) derived following exposure either to fractionated X-irradiation (DXR-10) or to VP-16 using pulsed 24-hr exposures (VP 10) or continuous exposure conditions (VPC2, VPC3 and VPC4). Orders of resistance expressed (ranging from 3- to 33-fold based on ICS0 values derived from colony forming assays) were comparable with those likely to be encountered clinically, All of these resistant sublines showed some cross -resistance to VCR, and the 3 drug-selected sublines tested also proved cross-resistant to ADR. Resistance was not associated with modified ³H-VP-16 accumulation. However, decreased VP-16-induced SSBs were detectable in all the resistant sublines and a strong positive correlation was noted between the extent of SSB formation and VP-16 resistance by linear regression analysis. Topo IIα protein content, as judged by Western blotting, was significantly decreased only in the sublines derived by continuous exposure to VP-16, but this was not progressive with increasing levels of resistance expressed. RNase protection assays also showed no significant differences in Topo IIα expression in the low-level resistant DXR-10 and VP 10 sublines, contrasting with the 2-fold decreases identified in the VPC2, VPC3 and VPC4 sublines. Significantly, however, mRNA levels of two alternately spliced Topo IIβ mRNAs were markedly decreased (2- to 9-fold) in all the drug-selected resistant sublincs. No mutations in consensus ATP-binding sequences or in the DNA-binding region of Topo Ma were detected by single strand conformations I polymorphism analysis. Significant Pgp over express ion was only identified in the most highly resistant sublines VPC3 and VPC4, which both showed 4-fold cross-resistance to VCR. Decreased ³H-VCR accumulation and partial reversal of resistance by VPM (6.6 μM) addition was also identified, consistent with a functional Pgp being overexpressed in these sublines. Modifications of Topo II expression therefore appear to precede Pgp overexpression in this series of sequentially derived VP-16 resistant sublines and to represent the predominate mechanism underlying low level (< 10-fold) resistance. © 1994 Wiley-Liss, Inc.     

Comparison of glutathione S-transferase activity between drug-resistant and-sensitive human tumor cells: Is glutathione S-transferase associated with multidrug resistance?

Summary We have studied the levels of glutathione S-transferase in drug-resistant and-sensitive human tumor cell lines to examine a possible involvement of glutathione S-transferase (GST) in multidrug resistance mechanisms. No increase in the activity of glutathione S-transferase was detected in myelogenous leukemia K562 resistant to adriamycin (K562/ADM), ovarian carcinoma cell line A2780 resistant to adriamycin (2780AD), or acute lymphoblastic leukemia cell line CCRF-CEM resistant to vinblastine (CEM-VLB100), compared with the drug-sensitive parent tumor cells. The human breast cancer cell lines Hattori and MCF-7 had a 12- to 63-fold lower level of glutathione S-transferase activity than K562, A2780, CCRF-CEM, and their drug-resistant sublines. Induction of ADM resistance in Hattori did not increase the activity of glutathione S-transferase. However, induction of colchicine resistance in MCF-7 resulted in a 70-fold increase in the activity of glutathione S-transferase. A revertant of the colchicine-resistant MCF-7 contained a level of glutathione S-transferase activity similar to that of the resistant subline. The increase of glutathione S-transferase activity did not alter the sensitivity of the cell to cytotoxic drugs. The increased activity was due to the appearance of glutathione S-transferase , as shown by enzyme inhibition using anti-glutathione S-transferase antibody. Our findings indicate that increased cellular glutathione S-transferase activity is not associated with the development of multidrug resistance.Abbreviations GST glutathione S-transferase - ADM adriamycin - VCR vincristine - VLB vinblastine     

Comparative effectiveness of mitoxantrone and doxorubicin in overcoming experimentally induced drug resistance in murine and human tumour cell lines in vitro

Summary Using a range of cell lines of murine and human tumour origin in which relatively modest levels (2- to 17-fold) of drug resistance have been selected in vitro by exposure to a range of standard antitumour drugs, we compared the cytotoxic effects of doxorubicin (DOX) and mitoxantrone (MITO). In general, significantly lower concentrations of MITO than of DOX were required to achieve comparable cytotoxicity, confirming previously published data. MITO appears more generally effective against the murine L5178Y drug-resistant sublines than DOX, although there was no expression of collateral sensitivity to this newer agent. In the various human tumour lines there was a lack of cross-resistance to both DOX and MITO in two 5-fluorouracil (FU)-resistant lines and one of two cisplatin (CDDP)-resistant cells, but cross-resistance was expressed in one subline resistant to vincristine (VCR) and two etoposide (VP-16)-resistant sublines. One murine and two human DOX-resistant sublines were effectively killed by MITO, whilst DOX proved effective against the human MITO-resistant subline. This apparent lack of cross-resistance between DOX and MITO in these resistant sublines expressing low levels of resistance in vitro therefore appears to contrast with previous reports involving highly multidrug-resistant DOX-selected sublines. However, since the latter lines generally exhibited profound cross-resistance to VCR and definite cross-resistance to VP-16, this may at least in part dictate their responses to MITO. Therefore, attempts to use experimentally derived drug-resistant sublines for preclinical drug screening should be approached with caution, since patterns of drug response appear to be influenced by the level of drug resistance expressed. The need remains to determine which type of model system provides the most relevant clinical information.     

Over-expression of P-glycoprotein and glutathione S-transferase pi in MCF-7 cells selected for vincristine resistance in vitro.

This study has provided evidence that exposure of the wild-type MCF-7 human breast carcinoma cell line to the mutagen ethyl methane sulphonate (EMS), followed by selection in vincristine (VCR), resulted in a stably-resistant subline, designated VCREMS, which expressed an approximately 14-fold level of resistance to VCR. This VCREMS subline showed cross-resistance (3-fold) to adriamycin (ADR) and to etoposide (3-fold), but not to cisplatin. The addition of a non-toxic concentration of verapamil (6.6 microM) significantly enhanced VCR cytotoxicity only in the resistant subline. This resistance was associated with over-expression of P-glycoprotein (Pgp), but without a concomitant increase in Pgp mRNA or gene amplification. In addition, activities of total glutathione S-transferases (GST) and glutathione peroxidase were elevated in this resistant subline, with over-expression of the GST-pi isozyme and its associated mRNA being identified, without gene amplification. This VCR-selected resistant MCF-7 cell line therefore provides another example of a breast carcinoma subline in which there is co-ordinate over-expression of both Pgp and GST-pi, without attributing a causal relationship to either event, and extends the range of anti-tumour drugs known to elicit modifications in glutathione metabolism.     

DNA strand breaks produced by etoposide (VP-16,213) in sensitive and resistant human breast tumor cells: implications for the mechanism of action

Pleotropic resistant human breast cancer cells (MCF-7), selected for resistance to Adriamycin, were used to study the production of DNA strand breaks by etoposide (VP-16) and its relationship to drug cytotoxicity. It was shown that the resistant MCF-7 cell line was cross-resistant to VP-16, and the degree of resistance was found to be 125-200-fold. Alkaline elution studies indicated that the parental cell line was very sensitive to VP-16 which caused extensive DNA strand breakage. In contrast, little DNA strand breakage was detected in the resistant MCF-7 cells, even at very high drug concentrations, indicating a good agreement between strand breaks and cytotoxicity. Further studies indicated that the nuclei isolated from the parental cell line were more resistant to VP-16-induced DNA strand breaks than the intact cells, while the opposite was found in the resistant cell line. In addition, the alkaline elution studies in isolated nuclei showed only a 2-fold reduction of VP-16-induced DNA breaks in nuclei from the resistant cells. In agreement with this result, it was found that nuclear extract from the resistant cells produced 2-3-fold less VP-16-induced DNA breaks than that from the sensitive cells in 32P-end-labeled SV40 DNA. VP-16 uptake and efflux studies indicated that there was a 2-3-fold decrease in net cellular accumulation of VP-16 in the resistant cells. Although the reduced uptake of VP-16 and decreased drug sensitivity of topoisomerase II appear to contribute to the mechanism of action and the development of resistance to VP-16, they do not completely explain the degree of resistance to VP-16 in this multidrug-resistant MCF-7 cell line indicating that other biochemical factors, such as activation of VP-16, are also involved in drug resistance and suggesting that the resistance is multifactorial.     

Glutathione S-transferase isoenzymes in human tumours and tumour derived cell lines

An increasing body of evidence indicates that glutathione S-transferases play a role in the intrinsic and acquired resistance of tumours to anticancer drugs. In view of the wide use of tumour cell lines to understand the factors which confer either sensitivity or resistance to chemotherapeutic agents we have determined glutathione S-transferase (GST) activity and isozyme composition in nine human cell lines. These data have been compared with the values obtained in solid tumours. In most cases overall GST activity was higher in the tumours than in the cell lines. This was most pronounced for the breast tumour samples relative to MCF7 cell line. The pi class GST subunit was present at similar concentration in the cell lines and the tumours, and in most cases was the most abundant subunit present. The alpha and mu class GST were expressed in most of the cell lines but at much lower concentration than the pi class subunit. Also considerable variability particularly in the expression of the mu subunits was observed. This was also the case for the expression of these subunits in the solid tumour samples. The levels of these GSTs (when expressed) in the solid tumours was invariably higher than that observed in the cell lines. There are therefore several similarities but also some significant differences in GST expression in solid tumours and cell lines. Whether the differences are because expression is lost during the generation of the cell lines or whether it reflects the individuality of human tumours remains to be clearly established.     

Hypophosphorylation of topoisomerase IIalpha in etoposide (VP-16)-resistant human carcinoma cell lines associated with carboxy-terminal truncation.

Topoisomerase IIalpha is a target for many chemotherapeutic agents in clinical use. To define mechanisms of resistance and regions crucial for the function of topoisomerase IIalpha, drug-resistant cell lines have been isolated following exposure to topoisomerase II poisons. Two resistant sublines, T47D-VP and MCF-7-VP, were isolated from human carcinoma cell lines following exposure to 300 or 500 ng / ml etoposide (VP-16). Cytotoxicity studies confirmed resistance to etoposide and other topoisomerase II poisons. KCl-sodium dodecyl sulfate (K-SDS) precipitation assays using intact cells showed reduced DNA-topoisomerase II complex formation following VP-16 or amsacrine (m-AMSA). RNAse protection analysis identified a deletion of 200 base pairs in the topoisomerase IIalpha cDNA of T47D-VP and rising dbl quote, left (low)AA insertion" in the topoisomerase IIalpha cDNA of MCF-7-VP. Reduced topoisomerase IIalpha mRNA and protein levels were observed in both cell lines. It was somewhat surprising to find that nuclear extracts from T47D-VP and MCF-7-VP cells had comparable topoisomerase II activity to that of parental cells. Analysis of the extent of phosphorylation demonstrated that topoisomerase IIalpha from the resistant cells was relatively hypophosphorylated compared to that of parental cells. In these cell lines, hypophosphorylation secondary to loss of a portion of the C-terminal domain of topoisomerase IIalpha mediated the restored activity, despite a fall in topoisomerase IIalpha mRNA and protein, and this resulted in cross resistance to topoisomerase II poisons.     

Glutathione, glutathione S-transferases, and related redox enzymes in Adriamycin-resistant cell lines with a multidrug resistant phenotype

Friend erythroleukemia cells (FLC) selected by exposure to Adriamycin (doxorubicin) express an approximate 2.5-fold (ARN1) or 13-fold (ARN2) resistance to the drug with various degrees of cross-resistance to other anthracyclines, vinca alkaloids, and epipodophyllotoxins. Because the redox cycling of the quinone moiety of Adriamycin is known to produce oxidative stress, however, an analysis of glutathione (GSH) and related enzyme systems was undertaken in the wild-type and selected resistant cells. In ARN1 and ARN2, superoxide dismutase (SOD) and catalase activities were slightly decreased, intracellular GSH and GSH reductase were essentially unchanged, and total GSH peroxidase, glutathione S-transferase (GST), and DT-diaphorase activities were slightly elevated. In each case there was no stoichiometric relationship between degree of resistance and level of activity. GST isozymes were purified from each cell line by HPLC GSH affinity column chromatography. Two-dimensional gel electrophoresis and western blot immunoreactivity against a battery of GST isozyme polyclonal antibodies determined that both the resistant and sensitive cells expressed isozymes of the alpha, pi, and mu classes (alternative murine nomenclature: M1, M2, M3). Of significance, both ARN1 and ARN2 cell lines expressed a unique alpha subunit which was absent from the parent FLC cell line. This isozyme presumably accounted for the increased GSH peroxidase activity (cumene hydroperoxide as substrate) found in ARN1 and ARN2 and may play a role in the small incremental resistance to melphalan found for both resistant lines. Expression of the isozyme was not stoichiometric with respect to degree of resistance. The presence of this isozyme may contribute to the resistant phenotype or may be the consequence of a more general cellular response to oxidative stress.     

Differential expression of steroid receptors,hsp27, and pS2 in a series of drug resistant human breast tumor cell lines derived following exposure to antitumor drugs or to fractionated X-irradiation

Summary This study examined whether levels of estrogen receptor (ER), progesterone receptor (PR), and expression of estrogen regulated pS2 and/or heat shock protein (hsp) 27 were associated with drug resistance in a series of MCF-7 sublines expressing modest (i.e. 3- to 14-fold), yet clinically relevant, levels of resistance to vincristine (VCR). These sublines were variously derived following pulsed exposures to VCR, to fractionated X-irradiation, or to alternating drug and X-ray treatments. This selection procedure more closely reflects the clinical treatment of breast tumors than the use of continuous drug exposures. The drug-selected sublines exhibited the classical multidrug resistance phenotype (MDR) characterized by cross-resistance to vinblastine (VLB), etoposide (VP-16), and Adriamycin (ADR), overexpression of P-glycoprotein (Pgp), impaired accumulation of [³H]-VCR and of Rhodamine-123 (Rh 123), and altered activities of certain drug detoxification enzymes. This classic MDR phenotype was associated with a lack of mitogenic response to estrogen or antiestrogen, related to loss of detectable ER and PR; consistent with these data, neither pS2 nor hsp27 expression was detectable. In contrast, X-ray-pretreated VCR-resistant cells (MCF/DXR-10) cells exhibited a distinctive resistance phenotype proving cross-resistant to VLB and VP-16 but not to ADR, and Pgp overexpression was not detectable. Furthermore, these VCR-resistant DXR-10 cells retained parental levels of ER and PR, exhibited sensitivity to estrogen and 4-hydroxytamoxifen, and expressed detectable levels of pS2 and hsp27. Comparable characteristics to these MCF-7/DXR-10 cells were also identified in a similarly-derived X-ray-pretreated VCR-resistant subline of the ZR-75-1 human breast tumor cell line. These data therefore indicate that functional ER are frequently, but not invariably, modified in tumor cells which express resistance to multiple drugs.     

Modulation of resistance to alkylating agents in cancer cell by gossypol enantiomers

Several cell lines resistant to alkylating agents possess increased activity of glutathione-S-transferase (GST) drug detoxifying enzymes. Inhibition of certain enzymes of the glutathione redox system may affect cellular sensitivity to alkylators. We report that the (-.)enantiomer of gossypol is a potent and selective inhibitor of GST alpha and GST pi isozymes, and that in combination with buthionine sulfoximine (BSO), causes the enhanced modulation of alkylator resistance in two drug resistant cell lines with increased GST activity. The use of (-)gossypol alone had no effect on the 2-5-fold resistance of MCF-7 Adr and Walker resistant cells to chlorambucil, melphalan and BCNU. Cellular depletion of glutathione with BSO resulted in a 2-4-fold modulation of cell sensitivity to these alkylators. However, the combination of (-)gossypol with BSO resulted in a markedly greater modulation of alkylator sensitivity than with either inhibitor alone. Therefore, the complementary inhibition of glutathione and GST by BSO and (-)gossypol, respectively, produced a synergistic modulation of alkylator cytotoxicity in these drug resistant cell lines. The favorable clinical pharmacokinetics of (-)gossypol suggest its further evaluation for use in combination with BSO and alkylating agents in clinical trials.     

Hypophosphorylation of Topoisomerase IIα in Etoposide (VP-16)-resistant Human Carcinoma Cell Lines Associated with Carboxy-terminal Truncation

Topoisomerase Ilα is a target for many chemotherapeutic agents in clinical use. To define mechanisms of resistance and regions crucial for the function of topoisomerase IIα, drug-resistant cell lines have been isolated following exposure to topoisomerase II poisons. Two resistant sublines, T47D-VP and MCF-7-VP, were isolated from human carcinoma cell lines following exposure to 300 or 500 ng/ml etoposide (VP-16). Cytotoxicity studies confirmed resistance to etoposide and other topoisomerase II poisons. KCl-sodium dodecyl sulfate (K-SDS) precipitation assays using intact cells showed reduced DNA-topoisomerase II complex formation following VP-16 or amsacrine (m-AMSA). RNAse protection analysis identified a deletion of 200 base pairs in the topoisomerase Ilα cDNA of T47D-VP and "AA insertion" in the topoisomerase Ilα cDNA of MCF-7-VP. Reduced topoisomerase Ila mRNA and protein levels were observed in both cell lines. It was somewhat surprising to find that nuclear extracts from T47D-VP and MCF-7-VP cells had comparable topoisomerase II activity to that of parental cells. Analysis of the extent of phosphorylation demonstrated that topoisomerase Ila from the resistant cells was relatively hypophosphorylated compared to that of parental cells. In these cell lines, hypophosphorylation secondary to loss of a portion of the C-terminal domain of topoisomerase Ilα mediated the restored activity, despite a fall in topoisomerase Ila mRNA and protein, and this resulted in cross resistance to topoisomerase II poisons.     

Increased phosphorylation of DNA topoisomerase II in etoposide-resistant mutants of human cancer KB cells.

We have isolated two etoposide (VP16)-resistant cell lines, KB/VP-1 and KB/VP-2, from human cancer KB cells after stepwise exposure to increasing doses of VP16. KB/VP-1 and KB/VP-2 showed 30- and 50-fold higher resistance to VP16 and also 20- and 30-fold higher resistance to teniposide than the parent cell line. Furthermore, both resistant cell lines showed more than 2-fold cross-resistance to Adriamycin and daunomycin than KB cells. The levels of accumulation and outward transport of radioactive VP16 were similar in KB/VP-1, KB/VP-2, and KB. The activity of nuclear extracts of DNA topoisomerase II for both KB/VP-1 and KB/VP-2 assayed by decatenation of kinetoplast DNA was consistently similar to that of KB. However, in both immunoblot assay with specific anti-topoisomerase II antibody and Northern blot analysis with specific human DNA topoisomerase II complementary DNA, cellular levels of topoisomerase II in both resistant cell lines were less than one-tenth the level in KB. The cellular levels of DNA topoisomerase I, however, were similar between the mutants and their parent. A quantitative precipitation assay of covalent DNA-topoisomerase II complexes showed greatly reduced VP16-induced cleavages of 3'-32P-DNA by nuclear extracts of KB/VP-1 or KB/VP-2 cells in comparison with KB cells. The relative specific phosphorylation of DNA topoisomerase II was about 14- to 18-fold higher in the mutants than in the parental cells. Phosphoamino acid analysis of DNA topoisomerase II showed that serine was the phosphorylated amino acid in all three cell lines, KB, KB/VP-1, and KB/VP-2. These data suggest that reduced expression of DNA-topoisomerase II might account for the acquired VP16 resistance and reduced VP16-induced cleavages of DNA-topoisomerase II complexes in both VP16-resistant variants.     

Human breast cancer cell lines resistant to pure anti-estrogens are sensitive to tamoxifen treatment

The pure steroidal anti-estrogens ICI 164,384 and ICI 182,780 are very potent growth inhibitors of the estrogen receptor-positive human breast cancer cell line MCF-7. However, long-term treatment of MCF-7 cells with 10^?7 M concentrations of these compounds results in selection of proliferating colonies of resistant cells. Our report describes 4 ICI 164,384- and 3 ICI 182,780-resistant MCF-7 sublines established after long-term treatment. Resistant sublines are estrogen receptor-positive, and all sublines have lost expression and estrogen inducibility of the progesterone receptor protein. Based on IC₅₀ concentrations, all tested resistant sublines had a reduced sensitivity to pure anti-estrogens on the order of 100- to 1000-fold compared with parent MCF-7 cells. All resistant cell lines have survived propagation for more than 15 subcultivations in the presence of 10^?7 M pure anti-estrogen. The MCF-7/182^R-6 subline has been tested for stability of resistance and appeared to be stably resistant after 13 weeks of propagation without the selective pressure of ICI 182,780. Cell lines resistant to the ICI 182,780 compound are cross-resistant to the ICI 164,384 compound and vice versa. However, the sublines resistant to pure anti-estrogens are sensitive to tamoxifen. Our results show that although the pure steroidal anti-estrogens are very potent growth inhibitors, they do not circumvent development of resistance. © 1995 Wiley-Liss, Inc.     

Establishment and Characterization of 6-[[2-(Dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one dihydrochloride (TAS-103)-resistant Cell Lines

6-[[2-(Dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1- c ]quinol in-7-one dihydrochloride (TAS-103) is a novel anticancer agent that was developed to target both topoisomerase (Topo) I and Topo II. To elucidate its mechanism of action, we have established and characterized TAS-103-resistant cells, derived from mouse leukemia (P388), human colon cancer (DLD-1), and human lung adenocarcinoma (A549) cell lines, by exposure to stepwisely increasing concentrations of TAS-103 in the culture medium. P388/TAS cells showed only cross-resistance to VP-16 and adriamycin (ADR). The Topo II activity in these cells was decreased to below one-fourth of that in the parental cells, while the Topo I activity remained unchanged. DLD/TAS cells appeared to be cross-resistant to VP-16, ADR, camptothecin (CPT), SN-38 and vincristine (VCR). The enzymatic activities of both Topo I and Topo II in these cells were decreased to one-fourth of that observed in the parental cells. Furthermore, the decreased activities were accompanied by lower expression at the mRNA and protein levels. A549/TAS cells acquired cross-resistance to VP-16, ADR and VCR, though the Topo activities were virtually unchanged. In this cell line, the intracellular accumulation of TAS-103 was significantly decreased and the expression of multidrug resistance associated protein (MRP) was elevated when compared with the parental cells. The results indicate that the affected activities of Topo I and/or Topo II, and in some instances decreased accumulation of TAS-103, are associated with the development of resistance to TAS-103, although the main mechanism of resistance to TAS-103 varied among cell lines.     

Antineoplastic drug sensitivity of human MCF-7 breast cancer cells stably transfected with a human alpha class glutathione S-transferase gene

Studies have suggested that the alpha class glutathione S-transferase (GST) may protect cells from the chemotherapeutic drugs chlorambucil and melphalan. In order to further define the function of human alpha class GST, a complementary DNA which encodes it was ligated into an expression vector under the direction of the human metallothionein-IIA promoter and stably transfected into human MCF-7 breast cancer cells in conjunction with the G418-selectable plasmid pSV2neo. Clonal cell lines were identified which expressed increased levels of GST enzyme activity (2.2- to 5.6-fold). The transfected cell lines also had increased peroxidase activity using cumene hydroperoxide as the substrate (1.9- to 3.8-fold) which is consistent with the intrinsic peroxidase activity of alpha class GSTs. Southern blot analysis indicated that genomic DNA from these cells contained a fragment indistinguishable from the transfected alpha class GST complementary DNA (850 base pairs); Northern blot analysis of total cellular RNA indicated that these cells contained appropriately sized alpha class GST RNA (980 nucleotides); and Western blot analysis indicated that, while MCF-7 cells contained no detectable alpha class GST protein, the transfected cells contained markedly elevated levels of alpha class GST but no detectable mu or pi class GST. These alpha class GST transfected cells had increased resistance to ethacrynic acid (2.1- to 3.0-fold). However, the transfected cells failed to show any increased resistance measured at the drug dosage which inhibited 50% of the colony formation to the chemotherapeutic drugs chlorambucil, melphalan, Adriamycin, or cisplatin under conditions of either continuous or 1-h drug exposure. Neither was there any change in sensitivity to the cytotoxins benzo(a)pyrene, benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (anti), or 1-chloro-2,4-dinitrobenzene. These studies indicate that expression of this human alpha class GST by itself in MCF-7 human breast cancer cells does not confer resistance to the chemotherapeutic drugs tested under the conditions used in these studies.     

Studies on glutathione transferases belonging to class pi in cell lines with different capacities for conjugating (+)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

The glutathione transferases (GST) belonging to class pi are primarily responsible for the intracellular detoxification of the highly mutagenic and carcinogenic compound (+)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). The aim of the present investigation was to study the nature and function of the GST pi gene in relation to the mutagenicity of BPDE in different cell lines. The studies were performed on three cell lines commonly used in toxicological studies, i.e. rat hepatoma cells (H4IIE), human mammary carcinoma cells (MCF-7) and Chinese hamster lung fibroblasts (V79). Western blotting with antisera against GST pi revealed a high level of reaction with cytosol from V79 and H4IIE cells. Furthermore, cytosol from the V79 cells demonstrated low levels of GSTs belonging to the alpha and mu classes, suggesting that a considerable portion of the total capacity of these cells to conjugate chlorodinitrobenzene (CDNB) was provided by GST pi. The level of mRNA for GST pi, as measured by Northern blots, was high in V79 and H4IIE and undetectable in the MCF-7 cell line. Analysis of the DNA fragment patterns using a series of restriction enzymes, revealed that all three cell lines have the pi class gene, although with different band patterns. The findings with H4IIE and MCF-7 cells with respect to their expression of the GST pi gene and their ability to conjugate BPDE were in agreement with the mutagenic effects of BPDE, produced by metabolic activation of (-)-7 beta, 8 alpha-dihydroxybenzo[a]-pyrene in the cells. In contrast, V79 cells although expressing high levels of GST pi, showed no ability to conjugate BPDE or to inhibit the mutagenicity of this compound. Based on these results, we suggest that V79 Chinese hamster lung cells contain a GST pi with a different substrate specificity from those of the human and rat GST pi enzymes.     

Establishment and characterization of 6-[[2-(Dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one dihydrochloride (TAS-103)-resistant cell lines.

6-2-(Dimethylamino)ethyl?mino-3-hydroxy-7H-indeno2, 1-cquinolin-7-one dihydrochloride (TAS-103) is a novel anticancer agent that was developed to target both topoisomerase (Topo) I and Topo II. To elucidate its mechanism of action, we have established and characterized TAS-103-resistant cells, derived from mouse leukemia (P388), human colon cancer (DLD-1), and human lung adenocarcinoma (A549) cell lines, by exposure to stepwisely increasing concentrations of TAS-103 in the culture medium. P388 / TAS cells showed only cross-resistance to VP-16 and adriamycin (ADR). The Topo II activity in these cells was decreased to below one-fourth of that in the parental cells, while the Topo I activity remained unchanged. DLD / TAS cells appeared to be cross-resistant to VP-16, ADR, camptothecin (CPT), SN-38 and vincristine (VCR). The enzymatic activities of both Topo I and Topo II in these cells were decreased to one-fourth of that observed in the parental cells. Furthermore, the decreased activities were accompanied by lower expression at the mRNA and protein levels. A549 / TAS cells acquired cross-resistance to VP-16, ADR and VCR, though the Topo activities were virtually unchanged. In this cell line, the intracellular accumulation of TAS-103 was significantly decreased and the expression of multidrug resistance associated protein (MRP) was elevated when compared with the parental cells. The results indicate that the affected activities of Topo I and / or Topo II, and in some instances decreased accumulation of TAS-103, are associated with the development of resistance to TAS-103, although the main mechanism of resistance to TAS-103 varied among cell lines.     

Combined modalities of resistance in etoposide-resistant human KB cell lines

The alkaloid derivative 4'-demethylepipodophyllotoxin 9-(4,6-O-ethylidene)-beta-D-glucopyranoside (etoposide, VP-16) is believed to exert cytotoxicity by causing double-stranded DNA breaks through interruption of the breaking-resealing reaction of topoisomerase II (topo II). Thus it was conceivable that cells could become resistant to VP-16 by a decrease in topo II enzyme level, since this would lead to fewer DNA breaks. As well, given the structure of VP-16, it was also possible that a pleiotropic mechanism of resistance could decrease sensitivity to this drug. To study these possibilities, a series of VP-16-resistant human KB cell lines was established by stepwise selection. The concentrations of VP-16 required to inhibit cell proliferation by 50% in the parent line and KB/1c, KB/7d, KB/20a, and KB/40a lines were, respectively, 0.16, 4.7, 24, 31, and 47 microM. These cell lines expressed cross-resistance to 4'-(9-acridinylamino)methanesulfon-m-anisidide, doxorubicin, vincristine, and methotrexate, although the pattern of relative drug sensitivity was quite different from that of pleiotropic resistant cell lines reported elsewhere. The resistance to vincristine and methotrexate did not increase above the level of the KB/1c cells, and resistance to VP-16, doxorubicin, and especially vincristine was unstable in VP-16-resistant cells cultured in the absence of drug. Although the drug resistance marker Mr 180,000 glycoprotein could not be detected in any of our cell lines, cellular accumulation of [3H]VP-16 was reduced 50-75% in the resistant lines compared with parent KB. With increasing VP-16 resistance, the level of topo II protein, detected by antibody staining, decreased at each step of selection, concomitant with a general decrease in topo II unknotting activity. Sensitivity of the topo II unknotting assay to inhibition by VP-16 was the same for the parent and all resistant cell lines. The level of topo I activity and enzyme increased slightly in the resistant cells. Thus, these cell lines are resistant to VP-16 by virtue of at least two mechanisms: (a) reduced levels of topo II, which confers cross-resistance to other compounds which are topo II-dependent cytotoxic agents; and (b) reduced accumulation of drug, which is likely also responsible for vincristine and methotrexate resistance. However, the possible existence of other mechanisms of resistance cannot be ruled out.     

Contribution of glutathione and glutathione-dependent enzymes in the reversal of adriamycin resistance in colon carcinoma cell lines.

Four human colon cancer cell lines (SW620, LS 180, DLD-I, and HCT-15) and sub-lines isolated in vitro by selection with Adriamycin were studied for reversal of intrinsic and acquired Adriamycin resistance, using buthionine sulfoximine (BSO) to deplete cellular glutathione alone and in combination with the P-glycoprotein antagonist verapamil. GSH levels varied among the parental cell lines but did not increase with resistance. In the parental SW620, DLD-I and HCT-15 and their drug-resistant derivatives, there was no relation between the effect of the glutathione-depleting agent BSO, the mRNA expression of both selenium-dependent glutathione peroxidase (GPx) and glutathione S-transferase pi (GST pi), bulk glutathione S-transferase (GST) activity, and the degree of resistance. However, in LS 180 and its derivative sub-lines, which do not principally rely on P-glycoprotein (Pgp) for Adriamycin resistance, treatment with BSO demonstrated a relatively diminished GSH depletion and enhanced recovery. In comparison with the other acquired cell lines, BSO specifically reversed acquired resistance in the LS 180 Adriamycin-resistant subline (LS 180 Ad150) after short-term drug exposure. Furthermore, the LS 180 Ad150 cells demonstrated an increase in both GPx and GST pi mRNA expression. These observations suggest that glutathione-mediated detoxification of Adriamycin may play a role in the resistance of this sub-line. Verapamil enhanced Adriamycin cytotoxicity 1.2- to 12-fold in the intrinsically resistant cells and as much as 15-fold in cell lines with acquired resistance. Combination of BSO with verapamil resulted in additive, but not synergistic, reversal of resistance. The results underscore the complex nature of Adriamycin resistance, and suggest a role for drug-resistance-modulating agents in the treatment of colon carcinoma.     

Differential patterns of anti-tumour drug responses and mechanisms of resistance in a series of independently-derived VP-16-resistant human tumour cell lines

Resistance to etoposide, which was expressed following exposure of a human tumour cell line (HN-I) to fractionated X-irradiation (II fractions to a total dose of 50Gy), was found to be exhibited after delivery of only 5 fractions (total dose of 22.5Gy). In addition, 2 new etoposide-resistant sublines of these HN-I cells have been developed by continuous exposure in vitro to sublethal drug concentrations. No significant differences in growth characteristics were shown between all these resistant sublines and the parental line. The drug-treated line, HN-I/VP-2, expressed cross resistance to vincristine, adriamycin and daunomycin, and marginal cross resistance to vinblastine and cisplatin. The X-irradiation-treated subline (HN-I/DXR-II) also proved cross-resistant to vincristine and marginally cross-resistant to vinblastine, but showed unaltered responses to adriamycin and daunomycin, and expressed marginal collateral sensitivity to cisplatin. Comparisons of drug-uptake characteristics showed that only the HN-I/VP2 cells and not the HN-I/DXR-II cells had reduced uptake of vincristine, vinblastine and daunomycin. However, etoposide uptake was not altered in either resistant subline. Further investigations have shown that the approximately 4-fold level of resistance to etoposide in these HN-I/VP-2 and HN-I/DXR-II cells was associated with a reduction in etoposide-induced DNA single-strand breakage. However, repair of these lesions, after drug removal, was rapid and similar in the parental and drug-resistant sublines, with 50% having resealed within 20-26 min. Resistance to etoposide was also associated with significantly elevated (p less than 0.01) glutathione peroxidase activity in both sublines, whilst glutathione S-transferase activity was marginally elevated (117%) only in the HN-I/DXR-II cells. There were no significant alterations in total glutathione levels. These results suggest that not only do patterns of response to anti-tumour drugs differ depending upon the agent employed to "induce" resistance, but that multiple mechanisms appear to be associated with these altered responses.