Multidrug-resistant Lines Research Articles

Combination of cyclosporin A and buthionine sulfoximine (BSO) as a pharmacological strategy for circumvention of multidrug resistance in small cell lung cancer cell lines selected for resistance to doxorubicin : Larsson R, Bergh J, Nygren P. Department of Clinical Pharmacology, University Hospital, Uppsala University, S-751 85 Uppsala. Anti-cancer Res 1991;11:455-9

The small cell lung cancer (SCLC) cell lines U-1285 and U-1690 were adapted to growth in continuous presence of doxorubicin (Dox). The resulting cell lines U-1285R and U-1690R were investigated with respect to sensitivity to the glutathione (GSH) depleting agent buthionine sulfoximine (BSO) and the immunosuppressant cyclosporin A (CsA) as well as the Dox resistance modifying ability of these agents. The parental U-1285 cells were more sensitive to BSO compared to parental U-1690 and the multidrug resistant (MDR) sublines, whereas no difference in sensitivity to CsA was observed between parental and MDR lines. BSO (10 microM) or CsA (1 microgram/ml) alone were able to partially reverse Dox resistance in the MDR cell lines, CsA being only marginally active in U-1285R cells. However, the combination of these two drugs at the same concentrations completely reversed Dox resistance in the MDR U-1690R cells whereas the combination was less effective in the U-1285R cells. The results demonstrate that a combination of low concentrations of BSO and CsA, only partially active by themselves in modifying Dox resistance, may be used as a pharmacological strategy to increase Dox sensitivity in some MDR SCLC cells.

Distinct patterns of phorbol ester-induced downregulation of protein kinase C activity in adriamycin-selected multidrug resistant and parental murine fibrosarcoma cells

Specific activators of protein kinase C (PKC), including the phorbol-ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), can reduce the chemosensitivities of a variety of mammalian tumor cell lines and their cytotoxic drug-selected multidrug resistant (MDR) variants to MDR-linked drugs, thus implicating PKC in the MDR phenotype. Previously, we reported that the adriamycin-selected MDR murine fibrosarcoma cell line UV-2237M-ADR R has approximately twice as much PKC activity as the parental UV-2237M line. In this report, we show that the level of [ 3H]phorbol-12,13-dibutyrate specific binding activity was elevated 3.5-fold in the MDR cells, thus establishing that phorbol-ester responsive PKC is overexpressed in the MDR line. Phorbol esters mediate downregulation of PKC by stimulating proteolysis of the enzyme, without altering the rate of PKC synthesis. We report that the kinetics of TPA-induced downregulation of PKC activity differ markedly in parental and MDR UV-2237M cells, providing evidence that the overexpression of phorbol-ester responsive PKC in adriamycin-selected MDR UV-2237M-ADR R cells results, at least in part, from a reduced rate of PKC degradation in the cells.

Overcoming Murine Tumor Cell Resistance to Vinblastine by Presentation of the Drug in Multilamellar Liposomes Consisting of Phosphatidylcholine and Phosphatidylserine

We determined whether vinblastine (VLB) encapsulated within multilamellar vesicle-liposomes (MLV) would reverse target cell resistance to the drug exhibited by the UV-2237M murine fibrosarcoma and its Adriamycin (ADR)-selected multidrug resistant (MDR) variants. Resistant fibrosarcoma cells were grown in medium containing 1 and 10 micrograms/ml ADR to yield the MDR lines UV-2237M/ADRR (ADR-1) and UV-2237M/ADRRR (ADR-10), respectively. VLB encapsulated in MLV composed of phosphatidylcholine (PC) and phosphatidylserine (PS) (7:3 molar ratio) was hydrophobic, occupied an internal space equivalent of 6.13 microliters/mumol, and was stable in medium at 37 degrees C for up to 6 days. The 50% inhibitory concentrations (IC50) of VLB were 2, 25, and 70 ng/ml for the parent, ADR-1, and ADR-10 cell lines, respectively. VLB in MLV significantly enhanced sensitivity of tumor cells to VLB. The respective IC50 of liposomal VLB were 0.5, 5.7, and 12 ng/ml for the parent, ADR-1, and ADR-10 lines. MLV containing saline were not toxic to the cells. These data indicate that presentation of VLB entrapped in PC:PS MLV provides a method to overcome tumor cell resistance to this drug.

Increased [formula omitted] gene expression and decreased drug accumulation in a human colonic cancer cell line resistant to hydrophobic drugs

An adriamycin-resistant human colonic cancer cell line was characterized. This clone exhibits the classical multidrug resistance (MDR) phenotype, being cross-resistant to hydrophobic drugs such as colchicine, and vinblastine. In contrast, this clone shows a normal response to DNA-damaging agents. The appearance of MDR in these cells was linked to a decreased accumulation of the drug [ 3H]colchicine as compared to the drug-sensitive cells. This MDR line expressed 80–100 fold increased levels of the specific 4.5-kb mdr mRNA, and a gene amplification. Our results indicate that MDR in human colonic cancer cells can result from increased expression of at least one member of the mdr gene family.

Chemosensitisation by verapamil and cyclosporin A in mouse tumour cells expressing different levels of P-glycoprotein and CP22 (sorcin)

The relationships between resistance to adriamycin, vincristine, colchicine and etopside, expression of P-glycoprotein and CP22 (sorcin), and resistance modification by verapamil and cyclosporin A have been studied in a panel of multidrug-resistant (MDR) mouse tumour cell lines. Whereas there was a generally good correlation between the degree of resistance and the amount of P-glycoprotein, no relationship between resistance and CP22 expression was seen. At 3.3 microM verapamil, the sensitisation of the MDR cell lines was no greater than that of the parent line. At 6.6 microM verapamil, however, sensitisation of the MDR lines generally exceeded that of the parent line, although the line CR 2.0, expressing very high levels of P-glycoprotein was an exception. Little sensitisation to etoposide was seen in any of the lines. When cyclosporin A was used as the sensitiser at either 2.1 or 4.2 microM, there was a greater effect in lines expressing moderate to high levels of P-glycoprotein than in the parent line, although this tendency was less for adriamycin than for the other cytotoxics. Sensitisation to etoposide was much greater with cyclosporin A than with verapamil. At low levels (less than 1 microM) of CsA, however, sensitisation to colchicine was greater in the parent line than in cell line CR 2.0. These studies indicate that chemosensitisation by verapamil and cyclosporin A is extremely complex, depending upon sensitiser dose, the particular cytotoxic and the cell line. At low doses of the sensitisers, the sensitisation may be greater in lines expressing low levels of P-glycoprotein than in lines showing high levels.

Design of NDA Intercalators To Overcome Topoisomerase II-Mediated Multidurg Resistance

Murine P388 (P) leukemia cell lines resistant to amsacrine (P/AMSA), dactinomycin (P/DACT), and doxorubicin (P/DOX) were compared with the parental strain in their sensitivity to a number of derivatives of amsacrine. The P/DACT cell line, which shows the characteristics of a transport-mediated multidrug-resistant cell line, was cross-resistant to vincristine, doxorubicin, etoposide, and a number of acridine-substituted amsacrine derivatives, but was sensitive in vitro and in vivo to amsacrine and its analog CI-921. The P/DOX cell line was cross-resistant to amsacrine but showed a similar pattern of cross-resistance to that of P/DACT in its in vitro response to amsacrine derivatives. In contrast, the P/AMSA line was substantially cross-resistant (from 27- to 146-fold) to all acridine-substituted amsacrine derivatives. However, when the substituents on the anilino side chain of amsacrine were changed, the in vitro cross-resistance of the P/AMSA line could be substantially reduced and even overcome. Derivatives with low cross-resistance ratios were tested in vivo against the P/AMSA leukemia and, in contrast to amsacrine and CI-921, were found to be active. Since the target enzyme for amsacrine action, topoisomerase II, is thought to be structurally modified in the P/AMSA line as well as in some other multidrug-resistant lines, these results suggest the feasibility of tailoring topoisomerase II-directed drugs specifically for the altered enzymes in resistant cells. New drug design approaches are therefore available for overcoming two major types of multidrug resistance.

Improved cellular accumulation is characteristic of anthracyclines which retain high activity in multidrug resistant cell lines, alone or in combination with verapamil or cyclosporin A

We have examined the cellular accumulation of anthracycline compounds, alone or in conjunction with resistance modifiers, in an attempt to identify mechanisms by which multidrug resistance (MDR) can be circumvented. This was facilitated by using the EMT6 mouse mammary tumour cell line EMT6/P and its MDR subline EMT6/AR1.0 with 30-fold resistance to Adriamycin® (ADM), and the human small cell lung cancer line H69/P together with its MDR subline H69/LX4 with 100-fold resistance to ADM. Both MDR lines hyperexpress membrane P-170 glycoprotein. The accumulation of ADM was compared to that seen for the anthracycline analogues aclacinomycin A (ACL), Ro 31–1215 and 4' deoxy-4'-iodo-Adriamycin® (iodo-ADM). These analogues were selected because of their high activity against MDR sublines, including H69/LX4 and EMT6/AR1.0. Both MDR cell lines exhibited a deficiency in ADM accumulation compared to the parent lines. Smaller differentials were seen using Ro 31–1215 or iodo-ADM. Both resistant sublines were able to accumulate ACL in identical amounts to their respective parental sublines. Improved drug accumulation is likely to contribute to the improved activity of the analogues against MDR cell lines. However, the relative accumulation defects in the resistant lines did not correlate exactly with the degree of resistance to a particular compound. Cyclosporin A (5 μg/ml) or verapamil (3.3 μg/ml) caused a preferential increase in uptake in both MDR sublines, with a small or negligible effect for the parental line. A smaller effect was observed with iodoADM and Ro 31–1215, and levels of ACL were unchanged in the MDR lines in the presence of either resistance modifier. These results indicate two mechanisms for circumventing drug resistance due to reduced drug accumulation. Structurally modified derivatives can partially or completely eliminate uptake differentials between parent and drug resistant cell lines. Any residual uptake can be eliminated using resistance modifiers. The two mechanisms may both operate via inhibition or circumvention of P170 mediated efflux. The situation is complex, however, and this study indicates the possible involvement of additional resistance mechanisms.

Resistance of multidrug-resistant lines to natural killer-like cell-mediated cytotoxicity.

Multidrug resistance (MDR) refers to a complex phenotype that describes a number of features characterized primarily by resistance to a wide range of structurally unrelated drugs. In this paper we investigated the relationship between drug resistance and resistance to NK-mediated cytotoxicity. Studies with two independently selected multidrug-resistant cell lines indicated that increased drug resistance was associated with both an increased resistance to NK-mediated cytotoxicity and increased levels of membrane P-glycoprotein expression. This resistance to cytotoxicity appears to result partly from an alteration in the membrane structure of the target cells inasmuch as there was a reduction in effector:target cell recognition. Resistance to NK-mediated cytotoxicity should be included with the numerous pleiotropic changes associated with the multidrug resistance phenotype.

Specific gene amplification associated with consistent chromosomal abnormality in independently established multidrug-resistant Chinese hamster ovary cells.

Multidrug-resistant (MDR) Chinese hamster ovary (CHO) cell lines were established by selection for resistance to the toxicities of vinblastine (VB) and Adriamycin (AD) in progressively increasing drug concentrations. These cell lines have amplified the DNA sequence that has previously been shown to be amplified in another MDR CHO cell line which was selected with vincristine (VC). An overproduced 4.5 kb mRNA was detected in these MDR cell lines. We report here that the levels of DNA amplification and the 4.5 kb transcript do not correlate with the levels of drug resistance, suggesting that either translational control for the expression of the amplified gene is involved or multiple genes are participating in conferring drug resistance in these cell lines. The amplified DNA sequence was used as a probe and localized by in situ hybridization to chromosome 1q 26-28 (middle portion of the long arm) in the drug-sensitive CHO line, but proximal to the telomere of chromosome 1q in both VB- and AD-selected MDR cell lines. This is consistent with results that have been previously reported for the VC-selected MDR cell lines. Cytogenetic analyses revealed abnormal chromosomal banding patterns or homogeneously staining regions (HSR) between 1q 26-28 and the 1q ter in these independently established MDR lines. These results, taken together, suggest that chromosomal rearrangements leading to gene translocation have consistently accompanied gene amplification in these MDR cell lines. The mechanisms of translocation and its implication in multidrug resistance in these cell lines are discussed.

Multiple drug-resistant human KB carcinoma cells independently selected for high-level resistance to colchicine, adriamycin, or vinblastine show changes in expression of specific proteins.

We have established four cell lines derived from the human KB carcinoma cell line which express high-level multiple drug resistance. One of these lines was selected for resistance to colchicine, one was selected for resistance to colchicine in the presence of the tumor promoter, mezerein, one for resistance to vinblastine, and one for resistance to adriamycin. All of these cell lines are cross-resistant to the other selective agents. The development of multidrug resistance in these cultured human carcinoma cells is associated with a limited number of specific protein alterations revealed by high resolution two-dimensional gel electrophoresis and Western blot analysis. These protein alterations in multidrug-resistant lines include the decreased prevalence of members of a family of proteins of molecular mass 70,000 to 80,000 daltons, pI 4.8-5.0, the increased synthesis of a protein of molecular mass 21,000 daltons, pI 5.0, in the colchicine-resistant cell lines only, and the increased expression of a 170,000-dalton protein in membrane preparations from all of the resistant cells. The loss of the 70,000- to 80,000-dalton proteins in the multidrug-resistant lines, which can also be demonstrated by immunoprecipitation of these proteins with specific antisera, is associated with a loss of translatable mRNA for these proteins. These studies suggest that only a limited number of protein changes occur in multidrug-resistant cell lines.