Resistance mechanisms in three human small cell lung cancer cell lines established from one patient during clinical follow-up

Mechanisms for resistance were studied in three classic type, human small cell lung cancer cell lines, GLC14, GLC16, and GLC19, that were established from one patient during clinical follow-up. Clinically the tumor changed from sensitive (GLC14) to completely resistant to (chemo)therapy (GLC19) during this period. The stain with JSB-1 antibody, detecting the Mr 170,000 multidrug resistance associated glycoprotein, was most pronounced in GLC16 and absent in GLC19. Intracellular Adriamycin (Adr) concentrations were decreased in GLC16 and GLC19 versus GLC14. Glutathione levels were 12.9, 15.5, and 16.6 micrograms/mg protein; total sulfhydryl groups were 36.5, 45.7, and 48.8 micrograms/mg protein; and glutathione S-transferase activity was 13, 29, and 43 nmol I-chloro-2,4-dinitrobenzene/min/mg protein for GLC14, GLC16, and GLC19, respectively. Incubation with DL-buthionine-S,R-sulfoximine increased Adr and cisplatin induced cytotoxicity, whereas X-ray induced cytotoxicity remained the same. Catalase activity increased from 0.88 to 1.73 to 3.83 mumol H2O2/min/mg protein in, respectively, GLC14, GLC16, and GLC19. Compared to GLC14 and GLC16, Adr induced a higher amount of DNA strand breaks in GLC19. In none of the three cell lines could Adr induced DNA strand breaks be repaired. X-ray induced a comparable amount of DNA strand breaks in all three cell lines but all cell lines were capable of repairing the X-ray induced DNA strand breaks within 90 min. It is concluded that a number of different mechanisms are operative and that some but not all of the observed changes in mechanisms for drug resistance in these lines correlate with the clinical data.     

Combined in vitro modulation of adriamycin resistance.

In a P-glycoprotein-negative cell line, GLC4-Adr90, a 75-fold acquired Adriamycin (Adr) resistance coincided with a reduced cellular Adr level, an increased detoxifying capacity (glutathione (GSH) and glutathione S-transferase (GST) elevated), and a reduced topoisomerase-II (topo-II) activity compared with the parent cell line GLC4. The effect on Adr resistance of buthionine sulfoximine (BSO, GSH synthesis inhibitor), was studied alone or in combination with verapamil (drug-efflux inhibitor), docosahexaenoic acid (membrane lipid domain affector), ethacrynic acid (GST inhibitor), aphidicolin (DNA-polymerase-alpha inhibitor) or novobiocin (NOV, topo-II inhibitor). Cytotoxicity was tested using a microculture tetrazolium assay. In GLC4-Adr90, BSO and NOV increased Adr-induced cytotoxicity 12.9-fold and 1.8-fold respectively. The combination of BSO plus NOV showed an additive effect, decreasing the Adr resistance factor from 75 to 2.7. Combination of modulators of Adr resistance directed at different resistance mechanisms appears promising in vitro.     

In vitro and in vivo modulation of multi-drug resistance with amiodarone.

The modulating effect on drug resistance of amiodarone (AM) and its metabolite desethylamiodarone (DEA) was studied in a P-glycoprotein-positive human colon carcinoma cell line COLO 320, and a human small-cell lung carcinoma cell line GLC4 and its adriamycin (Adr)-resistant subline GLC4-Adr (both P-glycoprotein-negative). AM, DEA and verapamil induced an increase in cytotoxicity of Adr, vincristine and etoposide (VP16) in COLO 320 cells, while in the GLC4 and GLC4-Adr cell line no effect was seen. In the COLO 320 cell line, AM caused more intracellular, and especially intranuclear, fluorescence of Adr and more Adr-induced DNA strand breaks as compared to Adr alone. Moreover, an increase in VP16-induced topoisomerase II-DNA complexes was observed when AM was added. Competition between AM and Adr for the same efflux pump was suggested in efflux studies. The colony-forming unit granulocyte macrophage (CFU-GM) assay showed no increase in cytotoxicity of Adr when AM was added. Fourteen patients with Adr-resistant tumors were treated with Adr and AM. In these patients, peak serum levels of AM plus DEA of 10 microM were reached. Patient serum (20%) obtained after the first i.v. AM infusion induced in vitro significantly more cell kill of Adr in COLO 320 cells. Apart from a transient first-degree AV block in one patient, no cardiac toxicity was observed with the combination of Adr and AM. Bone-marrow toxicity was the same as expected from Adr alone in these patients. One of the 13 evaluable patients obtained a partial remission.     

Multifactorial drug resistance in an adriamycin-resistant human small cell lung carcinoma cell line

In a human small cell lung carcinoma cell line, GLC4, Adriamycin (ADR) resistance was induced. In the resistant cell line, GLC4/ADR, a 45% decreased intracellular ADR level was found compared to GLC4, but this could not fully explain the resistance. Evaluation of DNA damage in both cell lines after incubation with ADR by alkaline and neutral elution revealed single-strand breaks, DNA-protein cross-links, and double-strand breaks (DSB). At all incubation concentrations there was a decreased amount of all types of DNA damage in GLC4/ADR. The number of DSB was decreased also when corrected for the decreased intracellular concentration. This can at least partly be explained by the decreased stability of ADR induced DSB. After removal of ADR, 80% of DSB was repaired in 1 h in GLC4/ADR against no repair in GLC4. X-ray induced DSBs were also repaired faster: in GLC4/ADR t1/2 = 10 min and in GLC4 t1/2 = 23 min. Ratios for single strand breaks/DSBs and single strand breaks/DNA-protein cross-links between GLC4 and GLC4/ADR after exposure to ADR differed; these differences were compatible with differences in the distribution of the various types of DNA damage induced in the cell lines due to an altered ADR-topoisomerase II interaction. In this human small cell lung carcinoma cell line the resistance is multifactorial with decreased intracellular ADR levels, increased DNA repair, and altered ADR-topoisomerase interaction.     

Indomethacin-induced activation of the death receptor-mediated apoptosis pathway circumvents acquired doxorubicin resistance in SCLC cells

Small-cell lung cancers (SCLCs) initially respond to chemotherapy but are often resistant at recurrence. A potentially new method to overcome resistance is to combine classical chemotherapeutic drugs with apoptosis induction via tumour necrosis factor (TNF) death receptor family members such as Fas. The doxorubicin-resistant human SCLC cell line GLC4-Adr and its parental doxorubicin-sensitive line GLC4 were used to analyse the potential of the Fas-mediated apoptotic pathway and the mitochondrial apoptotic pathway to modulate doxorubicin resistance in SCLC. Western blotting showed that all proteins necessary for death-inducing signalling complex formation and several inhibitors of apoptosis were expressed in both lines. The proapototic proteins Bid and caspase-8, however, were higher expressed in GLC4-Adr. In addition, GLC4-Adr expressed more Fas (3.1x) at the cell membrane. Both lines were resistant to anti-Fas antibody, but plus the protein synthesis inhibitor cycloheximide anti-Fas antibody induced 40% apoptosis in GLC4-Adr. Indomethacin, which targets the mitochondrial apoptotic pathway, induced apoptosis in GLC4-Adr but not in GLC4 cells. Surprisingly, in GLC4-Adr indomethacin induced caspase-8 and caspase-9 activation as well as Bid cleavage, while both caspase-8 and caspase-9 specific inhibitors blocked indomethacin-induced apoptosis. In GLC4-Adr, doxorubicin plus indomethacin resulted in elevated caspase activity and a 2.7-fold enhanced sensitivity to doxorubicin. In contrast, no effect of indomethacin on doxorubicin sensitivity was observed in GLC4. Our findings show that indomethacin increases the cytotoxic activity of doxorubicin in a doxorubicin-resistant SCLC cell line partly via the death receptor apoptosis pathway, independent of Fas.     

The role of glutathione in resistance to cisplatin in a human small cell lung cancer cell line

The role of glutathione (GSH) in resistance to cisplatin (CDDP) was studied in a human small cell lung carcinoma cell line (GLC4) and a CDDP-resistant subline (GLC4-CDDP). In addition to studying the steady state of GSH, the kinetics of this defence system were also studied via the monitoring of the GSH status of the cells under continuous pressure of CDDP. GLC4-CDDP maintained its elevated GSH level whereas GLC4 (under pressure of CDDP) quickly synthesised GSH to about twice its initial level, corresponding with 80% of the GSH level of GLC4-CDDP. D,L-buthionine-S,R-sulphoximine (BSO) was used to analyse the role of GSH in resistance to CDDP. Pretreatment with BSO (48 h, 50 microM, GSH not detectable) increased the CDDP-induced cytotoxicity 2.8-fold in GLC4-CDDP and 1.7-fold in GLC4. In GLC4 no changes in the amount of platinum (Pt) bound to DNA could be observed after GSH depletion. Changes in formation of interstrand cross-links or the main Pt-containing intrastrand cross-link in digested DNA, the Pt-GG adduct, were also not observed. In GSH depleted GLC4-CDDP cells, an increase in the amount of Pt bound to DNA and in the Pt-GG adduct was observed. Pretreatment with BSO substantially reduced the repair of Pt bound to DNA in both cell lines. We conclude that an increased GSH level and GSH synthesis capacity were demonstrated in CDDP resistant cells. The observations after BSO treatment suggest two roles for GSH in CDDP resistance, namely that of a cytosolic elimination resulting in less DNA platination and a nuclear effect on the formation and repair of DNA platinum adducts.     

Characterisation of adriamycin- and amsacrine-resistant human leukaemic T cell lines

Cell lines resistant to adriamycin and amsacrine were derived from cloned sublines of the human T cell line Jurkat. Most of the lines resemble atypical MDR cells (Danks et al., 1987; Beck et al., 1987). Thus, resistant Jurkat sublines were cross resistant to several topoisomerase II inhibiting drugs but had low or no resistance to other classes of drugs, resistance was not reversed by verapamil, Pgp was not overexpressed, and drug accumulation was unaltered in resistant compared to parental (control) sublines. Other findings were that anthracycline metabolism differed between resistant and parental sublines, and that resistant sublines displayed altered expression of small polypeptides (less than 20K MW) and an 85K MW protein. Drug resistant cells showed resistance to the production of drug induced cytogenetic aberrations, DNA breaks, and protein-DNA complexes. Resistance was not mediated by altered binding of drugs to DNA or by increased repair of DNA damage. Indirect evidence suggests that the resistant cells had an altered drug-DNA-topoisomerase II association. The study highlights the complex relationships between DNA breaks, cytogenetic aberrations, protein-DNA complexes and drug cytotoxicity, and shows that the relationships differ for adriamycin and amsacrine, suggesting some differences in the modes of action and/or resistance for the drugs and cell lines.     

Indomethacin induces apoptosis via a MRP1-dependent mechanism in doxorubicin-resistant small-cell lung cancer cells overexpressing MRP1

Small-cell lung cancers (SCLCs) initially respond to chemotherapy, but are often resistant at recurrence. The non-steroidal anti-inflammatory drug indomethacin is an inhibitor of multidrug resistance protein 1 (MRP1) function. The doxorubicin-resistant MRP1-overexpressing human SCLC cell line GLC(4)-Adr was highly sensitive for indomethacin compared with the parental doxorubicin-sensitive line GLC(4). The purpose of this study was to analyse the relationship between hypersensitivity to indomethacin and MRP1 overexpression. The experimental design involved analysis of the effect of MRP1 downregulation on indomethacin-induced cell survival and apoptosis in GLC(4)-Adr and GLC(4), using siRNA. In addition the effect of indomethacin on glutathione levels and mitochondrial membrane potential was investigated. Small interfering RNAs directed against MRP1 reduced MRP1 mRNA levels twofold and reduced efflux pump function of MRP1, which was reflected by a 1.8-fold higher accumulation of MRP1 substrate carboxyfluorescein, in si-MRP1 versus si-Luciferase-transfected GLC(4)-Adr cells. Multidrug resistance protein 1 downregulation decreased initial high apoptosis levels 2-fold in GLC(4)-Adr after indomethacin treatment for 24 h, and increased cell survival (IC(50)) from 22.8+/-2.6 to 30.4+/-5.1 microM following continuous indomethacin exposure. Multidrug resistance protein 1 downregulation had no effect on apoptosis in GLC(4) or on glutathione levels in both lines. Although indomethacin (20 microM) for 2 h decreased glutathione levels by 31.5% in GLC(4)-Adr, complete depletion of cellular glutathione by L-buthionine (S,R)-sulphoximine only resulted in a small increase in indomethacin-induced apoptosis in GLC(4)-Adr, demonstrating that a reduced cellular glutathione level is not the primary cause of indomethacin-induced apoptosis. Indomethacin exposure decreased mitochondrial membrane potential in GLC(4)-Adr cells, suggesting activation of the mitochondrial apoptosis pathway. Indomethacin induces apoptosis in a doxorubicin-resistant SCLC cell line through an MRP1-dependent mechanism. This may have implications for the treatment of patients with MRP1-overexpressing tumours.     

The role of methoxymorpholino anthracycline and cyanomorpholino anthracycline in a sensitive small-cell lung-cancer cell line and its multidrug-resistant butP-glycoprotein-negative and cisplatin-resistant counterparts

The cytotoxic action of two morpholino anthracyclines, methoxymorpholino anthracycline (MRA-MT, FCE 23 762) and cyanomorpholino anthracycline (MRA-CN), was compared with the cytotoxicity of doxorubicin (DOX), the topoisomerase II inhibitor etoposide (VP-16), the topoisomerase I inhibitor camptothecin, methotrexate, and cisplatin in GLC4, a human small-cell lung-cancer cell line, in GLC4-Adr, its P-glycoprotein (Pgp)-negative, multidrug-resistant (MDR; 100-fold DOX-resistant) subline with overexpression of the MDR-associated protein (MRP) and a lowered topoisomerase II activity, and in GLC4-CDDP, its cisplatin-resistant subline. GLC4-Adr was about 2-fold cross-resistant for the morpholino anthracyclines and GLC4-CDDP was, relative to GLC4, more resistant for the morpholino anthracyclines than for DOX. Overall, MRA-CN was about 2.5-fold more cytotoxic than MRA-MT. The cytotoxicity profile of the morpholino anthracyclines in these cell lines mimicked that of camptothecin.     

Multifactorial mechanisms associated with broad cross-resistance of ovarian carcinoma cells selected by cyanomorpholino doxorubicin.

The cyanomorpholino derivative of doxorubicin (MRA-CN) is a DNA intercalator and alkylator that is a highly potent cytotoxin, non-cross-resistant in multidrug-resistant cells, and noncardiotoxic in comparison with doxorubicin. To further examine mechanisms of action and resistance to MRA-CN, a cell line resistant to MRA-CN, ES-2R, was established by growing a human ovarian carcinoma cell line, ES-2, in increasing concentrations of the drug. The resistant subline was 4-fold resistant to MRA-CN and cross-resistant to other DNA cross-linking agents, cisplatin (7-fold) and carmustine (3-fold), as well as to the DNA strand-breaking agents etoposide (6-fold), doxorubicin (2-fold), bleomycin (5-fold), and ionizing radiation (2-fold). In contrast, ES-2R cells were not cross-resistant to vinblastine. Several months of additional growth of ES-2R cells in MRA-CN did not yield higher, stable levels of drug resistance. A low level of P-glycoprotein was detectable in the ES-2R cells. However, the extent of intracellular accumulation of [3H]MRA-CN by this resistant cell line was identical to that of the sensitive line. The number of DNA cross-links formed by cisplatin in ES-2R was only 50% of that of the ES-2 cells and was associated with a 50% increase in the rate of repair of these cross-links in the resistant cells. Ionizing radiation induced similar amounts of single- and double-strand breaks in the ES-2 line as well as in the ES-2R cells. There was no apparent difference between the two cell lines in the rate and extent of repair of these DNA breaks. Thus, enhanced DNA repair cannot explain the phenomenon of cross-resistance to radiation. Comparisons of glutathione (GSH) content and the enzymes involved in GSH homeostasis showed significant differences. Resistant cells contained 1.5-fold more GSH, a 2.2-fold increase in gamma-glutamyltranspeptidase activity, and a 2.4-fold increase in GSH reductase compared with ES-2 cells (all P less than 0.05). Total glutathione-S-transferase (GST) activity was 2.6-fold higher (P less than 0.01) in the ES-2R line. The pi-class GST subunit by Western blotting and GST activity toward ethacrynic acid were increased 2-fold in the resistant cells. Depletion of GSH levels in ES-2R cells by buthionine sulfoximine restored the sensitivity of ES-2R to MRA-CN. These findings implicate a role for GSH metabolism in the resistance phenotype of ES-2R cells. We have previously reported that these cells have an increased generation time and decreased topoisomerase II content. Thus, the ES-2R cell line exhibits a complex phenotype of broad cross-resistance, which is likely to involve multiple mechanisms, and includes enhanced DNA repair and increased GSH content and GST activity.     

Further characterization of two distinct adriamycin-resistant sublines from LoVo human colon carcinoma cells.

We previously reported that two multidrug resistant sublines, AdR1.2 and SRA1.2, derived from LoVo human colon carcinoma cells, apparently expressed different resistance phenotypes including differential expression of p-glycoprotein (Pgp). Here, we further examined and compared other potential resistance mechanisms between AdR1.2 and SRA1.2 resistant cells. Our results showed that the Pgp-mediated AdR1.2 cells possessed an activated drug efflux pump and decreased nucleus binding of Adriamycin, while the non-Pgp-mediated SRA1.2 cells only held the second feature. Verapamil, however, partially reversed resistance in both sublines. Although glutathione-s-transferase was overexpressed in AdR1.2 but not in SRA1.2, both sublines had lower susceptibilities to drug-induced DNA strand breaks and greater capacities to repair such damage than did LoVo cells. These data suggest that, despite the differences in multidrug resistance phenotypes, the features of decreased susceptibility to DNA damage and enhanced DNA repair capacities may represent the common mechanisms responsible for drug resistance in both Pgp- and non-Pgp-mediated multidrug resistant cells.     

A study of human small-cell lung carcinoma (hSCLC) cell lines with different sensitivities to detect relevant mechanisms of cisplatin (CDDP) resistance

The cisplatin(CDDP)-resistant cell line GLC4-CDDP shows a variety of differences from the parent line GLC4. The aim of this study was to determine which of the observed changes correlated with the degree of resistance and was therefore relevant to the phenomenon of CDDP resistance. For these experiments we used cells of the sensitive hSCLC cell line GLC4 and the in vitro-acquired CDDP-resistant sublines GLC4-CDDP3 and GLC4-CDDP11, with a resistance factor (RF) of 3 and 11 respectively for CDDP and of 1.8 and 7.4 respectively for carboplatin. Carboplatin was used, in addition to CDDP in seeking relevant mechanisms. No consistency was found between the RF and the growth pattern or antigen expression, cellular volume, doubling time, cellular or nuclear platinum (Pt) content or the level of Pt-non-histone chromatin protein (NHCP) binding. A correlation was found between the RF and the level of glutathione (GSH), and a trend was found for the level of Pt-DNA binding, Pt-GG adduct content and the amount of interstrand cross-links (ISC). These changes might therefore be relevant for the development of resistance. These findings are compatible with a GSH-induced reduction of the amount of reactive Pt in the resistant cell, resulting in a lower net platination and toxic Pt-DNA adduct formation.     

“彗星” 法分析人肿瘤细胞DNA放射损伤和修复

探讨“彗星”分析法 (CA)在检测体外培养人肿瘤细胞DNA放射损伤与修复和放射敏感性的关系。方法　采用碱性CA检测鼻咽高分化鳞癌上皮细胞系 (CNE 1)、食管鳞癌上皮细胞系 (Ec 10 9)、肺腺癌细胞系 (GLC)和胃低分化腺癌细胞系 (BGC 82 3)经 6MVX射线照射后单细胞内DNA单链断裂 (SSB)程度及其与放射剂量的关系 ,以及CNE 1和GLC细胞系在分别接受了 16GyX射线照射后的自身修复情况 ,同时与相应的细胞存活曲线比较。结果　碱性CA可以检测出 4个细胞系DNA的SSB ,并与放射剂量呈良好的线性关系。 16Gy剂量下各细胞系的SSB程度依次为CNE 1>Ec 10 9>GLC >BGC 82 3。CNE 1,Ec 10 9,GLC和BGC 82 3细胞系的Do值分别为 1.2 5 ,1.2 7,1.88和 1.5 6。 16Gy诱导CNE 1和GLC细胞系的DNA损伤后 ,其半修复时间分别为 7.4,17.8min。结论　采用碱性CA能确切反映 4种人肿瘤细胞系DNA的定量损伤程度及其与放射剂量的关系 ,并能确切反映辐射损伤后自身修复能力 (CNE 1和GLC)在受测时间中的定量变化情况。自身修复能力的大小在鳞癌和腺癌细胞系间与放射敏感性的大小 (Do值 )有较好的对应关系。     

The formation and removal of cisplatin (CDDP) induced DNA adducts in a CDDP sensitive and resistant human small cell lung carcinoma (HSCLC) cell line

In DNA digested samples of CDDP sensitive (GLC4) and an 11-fold resistant (GLC4-CDDP) hSCLC line, the CDDP induced DNA adducts Pt-GG (Pt-(NH3)2d (pGpG], Pt-AG (Pt-(NH3)2d (pApG], G-Pt-G (Pt-(NH3)2d (GMP)2) and Pt-GMP (Pt-(NH3)3d GMP), were measured with polyclonal antibodies. The total amount of platinum (Pt) bound to DNA was also measured but with the help of atomic absorption spectroscopy (AAS). An increased net formation in GLC4 compared with GLC4-CDDP is found for the total Pt bound to DNA, Pt-GG and Pt-AG adducts after a 2 h 100 microM CDDP treatment. No significant difference is detected in the net formation of the Pt-GMP and G-Pt-G adducts. A slow Pt-AG adduct formation, with a maximum reached 10 h after CDDP composition, is found for both cell lines. In the 22 h period after the 2 h 100 microM CDDP treatment, a significant removal in GLC4 is measured for the Pt-GG, Pt-AG and the Pt-GMP adducts. For GLC4-CDDP a significant removal is detected in the total Pt bound to DNA, the Pt-AG and the Pt-GMP adducts. The removal of the total Pt bound to DNA in GLC4-CDDP cannot be explained by an adduct measured with the immunochemical method. In conclusion, no evidence is found that CDDP resistance is based upon the repair of the Pt-GG, Pt-AG, G-Pt-G and Pt-GMP adducts.     

Cellular resistance to chloroethylnitrosoureas, nitrogen mustard, and cis-diamminedichloroplatinum(II) in human glial-derived cell lines

We investigated the cytotoxic and cytogenetic effects of 3-(4-amino-2-methyl-5-pyrimidinyl)methyl-1-(2-chloroethyl)-1-nitrosourea and 1,3-bis(2-chloroethyl)-1-nitrosourea on five cell lines established from human glioma biopsy specimens. Compared to the sensitive cell line SF-126, SF-188 cells are 3- to 6.5-fold more resistant to the cytotoxic effects and 8- to 14-fold more resistant to the induction of sister chromatid exchanges. Cytotoxic effects and induction of sister chromatid exchanges are intermediate for SF-210 and SF-295 cell lines compared with SF-126 and SF-188. There is a good correlation between susceptibility to the cytotoxic effects and formation of DNA interstrand cross-links for cells treated with 3-(4-amino-2-methyl-5-pyrimidinyl)methyl-1-(2-chloroethyl)-1-nitrosourea . We quantitated the extent of repair of O6-methylguanine after treatment of these cell lines with [3H]methylnitrosourea. SF-126 cells showed no detectable repair of O6-methylguanine, SF-210 and SF-295 had intermediate levels of repair, and SF-188 had very high levels of repair. We conclude that the cellular capacity to repair O6-chloroethylguanine adducts in DNA, which is reflected in the methyl repair process, is an important factor in determining cytotoxic response, and that increased repair of O6-chloroethylguanine decreases cytotoxicity and causes fewer sister chromatid exchanges and DNA interstrand cross-links to form in cells treated with chloroethylnitrosoureas. We studied the effects of cis-diamminedichloroplatinum(II) and nitrogen mustard in these cell lines. cis-Diamminedichloroplatinum(II) was equally cytotoxic and induced the same number of sister chromatid exchanges and DNA interstrand cross-links in all five cell lines. In contrast to the results obtained by treatment with chloroethylnitrosoureas, SF-126 cells treated with nitrogen mustard are 7.6-fold more resistant to the cytotoxic effects, 2-fold more resistant to the induction of sister chromatid exchanges, and 3-fold more resistant to the induction of DNA interstrand cross-links than are SF-188 cells. The results of this investigation with five human glial-derived cell lines clearly indicate that the molecular mechanisms of cellular resistance to alkylating chemotherapeutic agents are highly specific. Cellular resistance to chloroethylnitrosoureas does not result in cross-resistance to nitrogen mustard or cis-diamminedichloroplatinum(II).     

Relationship between cytotoxicity,drug accumulation,DNA damage and repair of human ovarian cancer cells treated with doxorubicin: modulation by the tiapamil analog RO11-2933

Summary The effect ofN-(3,4-dimethoxyphenyl)N-methyl-2-(naphthyl)-m-dithiane-2-propylamine hydrochloride (RO11-2933), an analog of the calcium channel blocker tiapamil, on doxorubicin (DOX)-induced cytotoxicity and DNA damage in human ovarian cancer cells sensitive and resistant to DOX was investigated. A2780-DX2, A2780-DX3, and A2780-DX6 cell sublines were characterized by 7-, 26-, and 48-fold resistance after 2 h DOX exposure and 30-, 50-, and 500-fold resistance after 72 h DOX exposure, respectively. Increased drug efflux resulting in a lower intracellular drug accumulation, decreased DOX-induced DNA single-strand breaks (DNA SSBs), and rapid DNA repair correlated with the degree of resistance. In addition, DNA SSBs were rapidly repaired within 8 h in A2780-DX3 cells, whereas no significant repair of DNA SSBs was observed in sensitive cells. In comparison with verapamil, RO11-2933 was found to reverse DOX resistance at lower and nontoxic concentrations (2 M as compared with 10 M verapamil). This reversion was complete in cells with a low degree of resistance (A2780-DX1 and A2780-DX2) but partial in highly resistant cells (A2780-DX3 and A2780-DX6), and continuous exposure to RO11-2933 was essential for optimal reversal of drug resistance. Interestingly, RO11-2933 was found to inhibit the repair of DNA SSBs induced by DOX but not those induced by X-ray. These results suggest that the potentiation of DNA SSBs and the specific inhibition of DNA repair by RO11-2933 in multidrug-resistant cells could be of particular value in overcoming MDR in the clinic.Abbreviations RO11-2933 N-(3,4-dimethoxyphenethyl)-N-methyl-2-(2-naphthyl)-m-dithiane-2-propylamine hydrochloride - DOX doxorubicin-HCl - SSBs single-strand breaks - MDR multidrug resistance This work was supported in part by CA 18420 and CA 21071     

Flow cytometric analysis of the cell cycle phase specificity of DNA damage induced by radiation, hydrogen peroxide and doxorubicin

We have optimized a flow cytometric DNA alkaline unwinding assay to increase the sensitivity in detecting low levels of DNA damage (strand breaks and alkali-labile sites) and to permit the measurement of the extent of DNA damage within each cell cycle compartment. The lowest gamma radiation dose that induced detectable DNA damage in each cell cycle phase of HeLa and CEM cells was 10 cGy. The lowest H(2)O(2) concentration that induced detectable DNA damage in each cell cycle phase was 0.5 microM in HeLa cells, and 1-2.5 TmicroM in CEM cells. For both HeLa cells and CEM cells, DNA damage in each cell cycle compartment increased approximately linearly with increasing doses of gamma radiation and H(2)O(2). Although untreated HeLa and CEM cells in S phase consistently exhibited greater DNA unwinding than did G(1) or G(2) cells (presumably due to DNA strand breaks associated with replication forks), there was no difference between the susceptibility of G(0)/G(1), S and G(2)/M phase cells to DNA damage induced by gamma radiation or H(2)O(2), or in the rate of repair of this damage. In each cell cycle phase, the susceptibility to gamma radiation-induced DNA damage was greater in CEM cells than in HeLa cells. In contrast to the lack of cell cycle phase-specific DNA damage induced by exposure to gamma radiation or H(2)O(2), the cancer chemotherapeutic drug doxorubicin (adriamycin) predominantly induced DNA damage in G(2) phase cells.     

In vitro multidrug resistance of P388 murine leukemia selected for resistance to diaziquone

P388 leukemia sublines were isolated from leukemia-cell-bearing CD2F1 mice that had been treated in vivo with increasing amounts of diaziquone (AZQ). The sublines isolated for in vitro studies were AZQ19 and AZQ30 which corresponded to the 19th and 30th in vivo passages, respectively. The AZQ19 subline displayed a very low degree of resistance to AZQ (1.5-fold), whereas the AZQ30 subline was sensitive. Both sublines, however, had much higher degrees of resistance to Adriamycin than to AZQ (24-fold for AZQ30 cells and 10-fold for AZQ19 cells). Both cell lines were also more resistant to actinomycin D, colchicine, and vincristine than to AZQ. The AZQ19 line was resistant to the alkylator thio-TEPA to the same degree that it was to AZQ, but the AZQ30 line was sensitive to thio-TEPA. On the other hand, AZQ30 cells were resistant to hydrogen peroxide with a very low degree of resistance (1.27-fold, P less than 0.05), whereas the AZQ19 line was sensitive. Drug accumulation experiments indicated that AZQ-resistant cells differed from the parental line in that they did not accumulate Adriamycin or vinblastine. In the case of AZQ, however, resistant and parental lines accumulated the same amounts of exchangeable AZQ. Using the immunoblotting technique, no P-glycoprotein was found in resistant cells. The resistant lines consumed oxygen at greater rates than the parental line. Oxygen consumption (Mean +/- SD) in sensitive cells was 2.0 +/- 0.4% O2 consumed/min, whereas in resistant cells it was nearly 3.1 +/- 0.6% O2 consumed/min. The increase in oxygen consumption with drug resistance was statistically significant (P less than 0.01). The kinetics of production of hydroxyl free radicals and of AZQ free radicals were faster in the resistant lines reflecting, in essence, their increased oxygen consumption. It appears that the two sublines analyzed here show resistance mechanisms that may have been elicited by the two distinct chemical constituents of AZQ. Therefore, in the AZQ19-resistant line, the alkylating aspect of AZQ was emphasized, whereas in the AZQ30 line, the quinone and, thus, free radical aspect was emphasized. This is consistent with AZQ30 cells being sensitive to the alkylator thio-TEPA and resistant to hydrogen peroxide, and the AZQ19 line being resistant to thio-TEPA and sensitive to hydrogen peroxide. In addition, the AZQ30 cell line was relatively more resistant than the AZQ19 line to Adriamycin.     

Mechanisms of drug resistance to the platinum complex ZD0473 in ovarian cancer cell lines

Acquired drug resistance to the sterically hindered platinum drug ZD0473 (formerly known as JM473 and AMD473) and currently being tested in phase I clinical trials, has been studied in two human ovarian carcinoma cell lines (CH1 and A2780) where previously, acquired cisplatin resistance has been described. Common mechanisms of resistance were observed in A2780 acquired cisplatin and ZD0473R (resistant) lines (including reduced drug transport and DNA platination, increased glutathione (GSH) and loss of the MLH1 DNA mismatch repair gene). However, contrasting mechanisms were observed in the CH1 sublines. While ZD0473 retained activity against the acquired cisplatin resistant sublines, cisplatin did not circumvent acquired ZD0473 resistance. The trans platinum complex JM335 circumvented resistance in CH1cisR and A2780ZD0473R lines, but not in A2780cisR or CH1ZD0473R cells. Overexpression of metallothionein (MT) in A2780 cells by stable gene transfection resulted in protection from the growth-inhibitory effects of cadmium chloride (3. 8-fold) and a range in protection with platinum drugs (from 7-fold with cisplatin, but only 1.3-fold with ZD0473). Overall, the results show that some mechanisms of resistance to ZD0473 are shared with those previously described in the same parental lines for cisplatin (e.g. in A2780), but in the CH1 lines, differing mechanisms were apparent. Moreover, ZD0473 possesses distinct cellular pharmacological properties in comparison with cisplatin with respect to reduced interactions with MTs, a thiol-containing species associated with tumour resistance to cisplatin.     

Radiation-induced transient cisplatin resistance in murine fibrosarcoma cells associated with elevated metallothionein content.

Cisplatin resistant mouse fibrosarcoma cells were isolated after fractionated irradiation in the absence of any drug treatment. Several sublines have been established; clone SSK-rad1 was used for further studies. These cells exhibit unchanged radiosensitivity and are compared to cisplatin resistant sublines, SSK-cis2, previously induced by low dose cisplatin exposure. Both resistant sublines are characterised by reduced CdCl2 sensitivity, indicating enhanced metallothionein content; loss of cisplatin resistance occurs after 10 to 25 generations and correlates with rising CdCl2 toxicity. Increase of MT is demonstrated directly by 109Cd binding to the MT containing region after FPLC. Both sublines differ in GSH level, which is increased only in SSK-rad1 cells, and in cellular platinum content, which is reduced in SSK-cis2 cells compared to the parental SSK cell line. These factors may contribute to cisplatin resistance but are not the main cause responsible for the transient nature of the drug resistance observed. Our results indicate that transient cisplatin resistance in SSK cells can be induced not only by the drug itself but also by gamma-irradiation and is based on the same mechanism of increased cellular MT content.