Cross-resistance of dideoxycytidine-resistant cell lines to azidothymidine.

2',3'-Dideoxycytidine (ddC) and azidothymidine (AZT) inhibit HIV-1 replication and currently are used in AIDS therapy. Long-term use of the drugs is associated with the selection of drug-resistant HIV strains, thus limiting their effectiveness. Another mechanism, associated with their altered metabolism in host cells, also can cause "cellular" drug resistance. Human lymphocytic H9 cell lines (H9-ddC0.5w and H9-ddC5.0w) selected for ddC resistance by exposure to 0.5 and 5.0 microM ddC were found to be cross-resistant to AZT. Compared with controls, the thymidine kinase (TK) activities in H9-ddC0.5w and H9-ddC5.0w cells were 56.7 and 51.4% (with thymidine as a substrate) and 50.3 and 42% (with AZT as a substrate). Consequently the cellular incorporation of AZT and thymidine (24-hr incubation) also was reduced to 51.3 and 70.0% in H9-ddC0.5w cells and to 12.1 and 17.3% in H9-ddC5.0w cells. A 3-hr incubation with 25 microM AZT and ddC decreased their cellular incorporation to 50.5 and 76.15% in H9-ddC0.5w cells and to 12.95 and 47.8% in H9-ddC5.0w cells compared with H9 cells. Thus, the change in AZT accumulation did not correlate exactly with the decrease in TK activity and far exceeded the effect on ddC accumulation. Evidence is presented that ddC, in addition to deoxycytidine kinase, affected TK1 activity. The involvement of multidrug resistance proteins in the mechanism of the resistance was ruled out by the failure of trifluoperazine and verapamil to alter cellular accumulations of AZT, ddC, daunorubicin, and rhodamine-123. Development of cellular ddC and AZT cross-resistance may affect the therapeutic efficacy of these antiviral agents.     

3'-Azido-2',3'-dideoxythymidine induced deficiency of thymidine kinases 1, 2 and deoxycytidine kinase in H9 T-lymphoid cells

Continuous cultivation of T-lymphoid H9 cells in the presence of 3′-azido-2′,3′-dideoxythymidine (AZT) resulted in a cell variant cross-resistant to both thymidine and deoxycytidine analogs. Cytotoxic effects of AZT, 2′,3′-didehydro-3′-deoxythymidine as well as different deoxycytidine analogs such as 2′,3′-dideoxycytidine, 2′,2′-difluoro-2′-deoxycytidine (dFdC) and 1-ß-D-arabinofuranosylcytosine (Ara-C) were strongly reduced in H9 cells continuously exposed to AZT when compared to parental cells (>8.3-, >6.6-, >9.1-, 5×10⁴-, 5×10³-fold, respectively). Moreover, anti-HIV-1 effects of AZT, d4T, ddC and 2′,3′-dideoxy-3′-thiacytidine (3TC) were significantly diminished (>222-, >25-, >400-, >200-fold, respectively) in AZT-resistant H9 cells. Study of cellular mechanisms responsible for cross-resistance to pyrimidine analogs in AZT-resistant H9 cells revealed decreased mRNA levels of thymidine kinase 1 (TK1) and lack of deoxycytidine kinase (dCK) mRNA expression. The loss of dCK gene expression was confirmed by western blot analysis of dCK protein as well as dCK enzyme activity assay. Moreover, enzyme activity of TK1 and TK2 was reduced in AZT-resistant cells. In order to determine whether lack of dCK affected the formation of the active triphosphate of the deoxycytidine analog dFdC, dFdCTP accumulation and retention was measured in H9 parental and AZT-resistant cells after exposure to 1 and 10 μM dFdC. Parental H9 cells accumulated about 30 and 100 pmol dFdCTP/10⁶ cells after 4 hr, whereas in AZT-resistant cells no dFdCTP accumulation was detected. These results demonstrate that continuous treatment of H9 cells in the presence of AZT selected for a thymidine analog resistant cell variant with cross-resistance to deoxycytidine analogs, due to deficiency in TK1, TK2, and dCK.     

Role of deoxycytidine kinase in the inhibitory activity of 5-substituted 2'-deoxycytidines and cytosine arabinosides on tumor cell growth

A number of 5-substituted derivatives of dCyd and 1-beta-D-arabinofuranosylcytosine (araC) have been evaluated for their inhibitory effects on the growth of three murine leukemia cell lines (L1210/0, L1210/BdUrd, and L1210/araC). The L1210/BdUrd and L1210/araC cell lines were selected from the parental L1210/0 cell line by their ability to grow at high concentrations of 5-bromo-2'-deoxyuridine and araC, respectively; the L1210/BdUrd cell line was deficient in dThd kinase activity, whereas the L1210/araC cell was deficient in dCyd kinase activity. The most effective inhibitors of L1210/0 cell proliferation were 5-fluoro-dCyd, araC, and 5-fluoro-araC. Their 50% inhibitory dose fell within the 0.001-0.015 micrograms/ml range. The 5-substituted araC analogues were much less inhibitory for L1210/araC cells but equally inhibitory for L1210/BdUrd as for the parental L1210/0 cell line. The role of dCyd kinase in the antitumor activity of the dCyd and araC analogues was further assessed by kinetic studies with dCyd kinase extracted from L1210/0 cells. All dCyd and araC analogues caused a competitive inhibition of dCyd kinase, the most potent inhibitor being 5-fluoro-dCyd (Ki/Km value 0.24). The Km of dCyd kinase from L1210/0 cells for dCyd was 23.1 microM as compared with 50 microM for araC. These values were increased to 53 and 182 microM, respectively, for the dCyd kinase isolated from L1210/araC cells.     

Thymidine and zidovudine metabolism in chronically zidovudine-exposed cells in vitro.

Chronic exposure of H9 cells to 25 microM zidovudine (H9-AZT cells) causes a 2- to 3-fold increase in thymidine kinase (TK) activity (Agarwal RP, Int J Purines Pyrimidine Res, in press). The present study compared thymidine (TdR) and AZT anabolism in H9 and H9-AZT cells. After a 3.5-hr incubation with 10 microM TdR or AZT, the total intracellular accumulations of AZT (48.7 microM in H9 cells and 32.8 microM in H9-AZT cells) were 46.4% of TdR accumulation. Other major differences between TdR and AZT anabolism were: (i) the majority of TdR (84-87%) was incorporated into DNA compared to less than 1% of AZT; and (ii) whereas distribution of TdR in the nucleotides was TTP greater than TMP greater than TDP, zidovudine distributed was AZT-MP much greater than AZT-TP much greater than AZT-DP. Because of the poor substrate activity of AZT-MP for thymidylate kinase (TMP-kinase), most of the AZT (95-98%) remained as AZT-MP. TMP-kinase activities with TMP as substrate were 47.6 +/- 20.3 and 91.4 +/- 28.8 pmol/mg protein/min in H9 and H9-AZT cells, respectively. 5'-Nucleotidase activities with TMP as substrate were 428.9 +/- 37.8 and 255.9 +/- 28.7 pmol/mg protein/min in H9 and H9-AZT cells, respectively. Activities of these enzymes with AZT-MP as a substrate were very low. Despite an increase in TK and TMP-kinase, and a decrease in 5'-nucleotidase activities, the total intracellular accumulations of TdR and AZT were reduced significantly (P less than 0.05) to 67.5% in H9-AZT cells. Thymidine transport (0.66 to 0.68 pmol/sec/10(6) cells) was similar in both the cell lines. The severe reductions of TdR salvage caused by chronic exposure of cells to AZT, if it occurs in AIDS patients on AZT chemotherapy, may explain some of the long-term clinical toxicities of the drug.     

Inhibition by arsenite of anticancer drug cis-diamminedichloroplatinum(II) induced DNA repair and drug resistance in HeLa cells

We have previously reported a cisplatin-resistant HeLa variant cell line (HeLa/CPR) which exhibited an enhancement in repairing cisplatin-DNA adducts (Chao, 1994, Mol. Pharmacol. 45, 1137-1144). In this study, using this cell line, we investigated the modification, by arsenite, of cisplatin-induced cytotoxicity and DNA repair in the resistant cell line. By a sublethal dose of arsenite, cytotoxicity of the resistant cells was enhanced by 2.5-fold, compared to 1.62-fold in the parental cells. Using enzyme-linked immunosorbent assay (ELISA) and a monoclonal antibody specific for cisplatin-DNA adducts, we found that the resistant cells showed a 5.15-fold decrease in the adduct formation compared to the parental cells. However, in the presence of arsenite, the resistant cells showed only a 1.47-fold decrease in the adduct formation, indicating a more than 3-fold modification. Using host cell reactivation of transfected plasmid DNA carrying cisplatin damage (an indirect detection of DNA repair), arsenite also revealed a ～2-fold modification of adduct formation in the resistant cells. In addition, the time-dependent potentiation of cytotoxicity by arsenite in both cell lines was parallel to the increase of adduct formation. These results indicate that arsenite is an effective modifier of cisplatin-induced resistance and enhanced DNA repair in HeLa/CPR cells. The results are consistent with the notion that the cisplatin-resistant phenotype in HeLa cells is mainly mediated by enhancement of DNA repair.     

Collateral sensitivity to azidothymidine in methotrexate resistant human leukemia cells.

Resistance to methotrexate (MTX) is a well established clinical problem and strategies to circumvent this would obviously be beneficial. The human leukemia cell line, CCRF-CEM, was grown in folinic acid to study MTX resistance-reflecting more in vivo conditions. A 15.8-fold resistant subline, CEM/MTX, was evolved by stepwise increases in MTX exposure. The MTX resistant cell line exhibited both increased dihydrofolate reductase (DHFR) activity due to gene amplification as well as impaired MTX uptake. An additional mechanism of resistance to MTX was a 2-fold increase in thymidine kinase (TK). As a result of this increased TK activity, the CEM/MTX cells were collaterally sensitive to the nucleoside analogue, azidothymidine (AZT). CEM cells resistant to MTX possess properties that can be exploited by AZT and these studies may have clinical implications.     

Folate analogues. 32. Synthesis and biological evaluation of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid and related compounds

The chemical synthesis of three close analogues (2-4) of N10-propargyl-5,8-dideazafolate (PDDF) is described. The quinazoline ring of 2 and 4 was constructed from the pivotal intermediate 9 in a novel and unambiguous manner during the final step of the synthesis under very mild conditions. 2-Desamino-2-methyl-N10-propargyl-5,8-dideazafolate (DMPDDF) (2) was a strong inhibitor of human and Lactobacillus casei thymidylate synthases, whereas 2-desamino-2-(trifluoromethyl)-N10-propargyl-5,8-didezafolate (3) and 2-desamino-2,3-dimethyl-N10-propargyl-5,8-dideazafolate (4) were only weak inhibitors of this enzyme. DMPDDF exhibited excellent growth inhibition of Manca human lymphoid leukemia and H35 hepatoma cells in culture. The inhibitor activities of 2 were 43- and 65-fold greater than that of PDDF, respectively, in these cell lines. H35R cells that are resistant to methotrexate by virtue of a transport defect were cross resistant to DMPDDF but not to PDDF. H35FF cells which have 70-fold greater amounts of thymidylate synthase compared to H35N cells were 130-fold resistant to DMPDDF. Furthermore, the toxicity of DMPDDF to H35 hepatoma cells could be completely reversed by thymidine, establishing its locus of action as thymidylate synthase. Transport studies in vitro established that DMPDDF effectively inhibits MTX influx into H35 hepatoma cells, whereas PDDF has no effect on MTX transport in this cell line. These data suggest that the greater activity of DMPDDF relative to PDDF is partly due to the ability of the former compound to enter cells via the MTX/reduced folate transport system. Enzyme inhibition data of 4 suggest that the presence of N3H in DMPDDF is essential for binding to thymidylate synthase.     

The role of human nucleoside transporters in cellular uptake of 4'-thio-beta-D-arabinofuranosylcytosine and beta-D-arabinosylcytosine

4'-Thio-beta-D-arabinofuranosyl cytosine (TaraC) is in phase I development for treatment of cancer. In human equilibrative nucleoside transporter (hENT) 1-containing CEM cells, initial rates of uptake (10 microM; picomoles per microliter of cell water per second) of [3H]TaraC and [3H]1-beta-D-arabinofuranosyl cytosine (araC) were low (0.007 +/- 003 and 0.034 +/- 0.003, respectively) compared with that of [3H]uridine (0.317 +/- 0.048), a highactivity hENT1 permeant. In hENT1- and hENT2-containing HeLa cells, initial rates of uptake (10 microM; picomoles per cell per second) of [3H]TaraC, [3H]araC, and [3H]deoxycytidine were low (0.30 +/- 0.003, 0.42 +/- 0.03, and 0.51 +/- 0.11, respectively) and mediated primarily by hENT1 (approximately 74, approximately 65, and approximately 61%, respectively). In HeLa cells with recombinant human concentrative nucleoside transporter (hCNT) 1 or hCNT3 and pharmacologically blocked hENT1 and hENT2, transport of 10 microM[3H]TaraC and [3H]araC was not detected. The apparent affinities of recombinant transporters (produced in yeast) for a panel of cytosine-containing nucleosides yielded results that were consistent with the observed low-permeant activities of TaraC and araC for hENT1/2 and negligible permeant activities for hCNT1/2/3. During prolonged drug exposures of CEM cells with hENT1 activity, araC was more cytotoxic than TaraC, whereas coexposures with nitrobenzylthioinosine (to pharmacologically block hENT1) yielded identical cytotoxicities for araC and TaraC. The introduction by gene transfer of hENT2 and hCNT1 activities, respectively, into nucleoside transport-defective CEM cells increased sensitivity to both drugs moderately and slightly. These results demonstrated that nucleoside transport capacity (primarily via hENT1, to a lesser extent by hENT2 and possibly by hCNT1) is a determinant of pharmacological activity of both drugs.     

Multiple mechanisms of resistance to methotrexate and novel antifolates in human CCRF-CEM leukemia cells and their implications for folate homeostasis

We determined the mechanisms of resistance of human CCRF-CEM leukemia cells to methotrexate (MTX) vs. those to six novel antifolates: the polyglutamatable thymidylate synthase (TS) inhibitors ZD1694, multitargeted antifolate, pemetrexed, ALIMTA (MTA) and GW1843U89, the non-polyglutamatable inhibitors of TS, ZD9331, and dihydrofolate reductase, PT523, as well as DDATHF, a polyglutamatable glycinamide ribonucleotide transformylase inhibitor. CEM cells were made resistant to these drugs by clinically relevant intermittent 24 hr exposures to 5-10 microM of MTX, ZD1694, GW1843U89, MTA and DDATHF, by intermittent 72 hr exposures to 5 microM of ZD9331 and by continuous exposure to stepwise increasing concentrations of ZD9331, GW1843U89 and PT523. Development of resistance required only 3 cycles of intermittent drug exposure to ZD1694 and MTA, but 5 cycles for MTX, DDATHF and GW1843U89 and 8 cycles for ZD9331. The predominant mechanism of resistance to ZD1694, MTA, MTX and DDATHF was impaired polyglutamylation due to approximately 10-fold decreased folylpolyglutamate synthetase activity. Resistance to intermittent exposures to GW1843U89 and ZD9331 was associated with a 2-fold decreased transport via the reduced folate carrier (RFC). The CEM cell lines resistant to intermittent exposures to MTX, ZD1694, MTA, DDATHF, GW1843U89 and ZD9331 displayed a depletion (up to 4-fold) of total intracellular reduced folate pools. Resistance to continuous exposure to ZD9331 was caused by a 14-fold increase in TS activity. CEM/GW70, selected by continuous exposure to GW1843U89 was 50-fold resistant to GW1843U89, whereas continuous exposure to PT523 generated CEM/PT523 cells that were highly resistant (1550-fold) to PT523. Both CEM/GW70 and CEM/PT523 displayed cross-resistance to several antifolates that depend on the RFC for cellular uptake, including MTX (95- and 530-fold). CEM/GW70 cells were characterized by a 12-fold decreased transport of [3H]MTX. Interestingly, however, CEM/GW70 cells displayed an enhanced transport of folic acid, consistent with the expression of a structurally altered RFC resulting in a 2.6-fold increase of intracellular folate pools. CEM/PT523 cells displayed a markedly impaired (100-fold) transport of [3H]MTX along with 12-fold decreased total folate pools. In conclusion, multifunctional mechanisms of resistance in CEM cells have a differential impact on cellular folate homeostasis: decreased polyglutamylation and transport defects lead to folate depletion, whereas a structurally altered RFC protein can provoke expanded intracellular folate pools.     

Mechanisms of resistance to (2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU) in sensitive and resistant human glioma cells

Resistance to (2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU), an experimental antitumor compound, was investigated in the sensitive SK-MG-1 cells and the 20-fold more resistant SKI-1 human glioma cells [which are 3-fold more resistant to 1,3,bis(2-chloroethyl)-1-nitrosourea (BCNU)]. The transport of SarCNU was examined by utilizing tritiated sarcosinamide. Sarcosinamide uptake into SK-MG-1 cells is via the catecholamine carrier that accommodates epinephrine. Dixon plot analysis of SarCNU inhibition of sarcosinamide uptake reveals that SarCNU is also accommodated by this carrier. The uptake of 0.5 mM [3H]sarcosinamide was temperature dependent, with similar levels of intracellular sarcosinamide accumulating at steady state in both cell lines. The uptake of sarcosinamide in SKI-1 cells obeyed Michaelis-Menten kinetics over a 200-fold range of concentrations with a Km of 1.52 +/- 0.151 mM and Vmax of 0.659 +/- 0.066 nmol/10(6) cells/min. This represents a more than 5-fold decrease in the uptake affinity and a more than 4-fold increase in the transport capacity compared with SK-MG-1 cells (Km = 0.282 +/- 0.041 mM; Vmax = 0.154 +/- 0.024 nmol/10(6) cells/min). The initial rate of sarcosinamide uptake is similar in both cell lines. Dixon plot analysis confirmed that SarCNU is a competitive inhibitor of sarcosinamide transport in SKI-1 cells with a Ki of 17.5 mM, which is more than 5-fold greater than the Ki obtained in SK-MG-1 cells. The steady state accumulation of SarCNU is significantly reduced by 47% in SKI-1 cells compared with the SK-MG-1 cells (cell to medium ratios of 0.65 +/- 0.11 and 1.22 +/- 0.08, respectively) (p less than 0.005). The accumulation of BCNU was comparable in the two cell lines. Since the Vmax of sarcosinamide (SarCNU) uptake is increased in the SKI-1 cells, the decrease in intracellular SarCNU is not related to decreased drug influx via the catecholamine carrier in SKI-1 cells. The efflux of tritiated sarcosinamide was temperature dependent and similar in both cell lines, with 54 and 58% of sarcosinamide being freely exchangeable in SKI-1 and SK-MG-1 cells, respectively. SarCNU efflux may or may not be altered. Since the expression of mdr is higher in the sensitive cells, it is unlikely that increased efflux of SarCNU mediated by the P-glycoprotein is responsible for drug resistance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antiproliferative effects of ZD0473 (AMD473) in combination with 5-Fluorouracil or SN38 in human colorectal cancer cell lines

PURPOSE: ZD0473 (AMD473) [cis-amminedichloro(2-methylpyridine) platinum(II)] is a novel platinum agent of proven activity in vitro against a variety of human tumor-derived cell lines even with intrinsic or acquired resistance to CDDP. The aim of this study is to provide the basis for a rational design of ZD0473-based combination in colon cancer. EXPERIMENTAL DESIGN: We evaluated the cytotoxic effect of ZD0473 administered alone or in combination with 5-Fluorouracil (5FU) or SN38 in a panel of sensitive and 5FU-resistant colorectal cell lines (HT29/HT29-5FUR and LoVo/LoVo-5FUR). We analyzed four sequential schedules of administration: ZD0473 --> 5FU, 5FU --> ZD0473, ZD0473 --> SN38 and SN38 --> ZD0473. MTT-assay and isobologram analyses were performed to determine the synergism/antagonism. RESULTS: The pattern of response towards ZD0473, administered as single agent, was similar in all cases and independent of the 5FU-resistance phenotype (IC50 from 48.1 to 76.6 microM) and/or p53 status. No differences in sensitivity to ZD0473 alone or in combination were observed between DNA-mismatch repair-proficient (HT29/HT29-5FUR) and -deficient (LoVo/LoVo-5FUR) cells. ZD0473 administered prior to 5FU leads to synergistic/additive effect in all cell lines, while the 5FU --> ZD047 schedule was only synergistic in HT29 cells. Exposure to ZD0473 prior to SN38 leads to a synergistic/additive schedule in LoVo/LoVo-5FUR cells, while SN38 --> ZD0473 schedule was only synergistic in parental cell lines. CONCLUSIONS: The combinations of ZD0473 and 5FU or SN38 have shown to be active in sensitive and 5FU-resistant colorectal cell lines when a correct schedule of administration is applied. These results may be further exploited to promote new schedules of administration for advanced colorectal cancer treatment.     

Multidrug resistance in a small cell lung cancer line: rapid selection with etoposide and differential chemosensitization with cyclosporin A.

We developed a multidrug resistant small cell lung cancer line, VPR-2, by exposing H69 parent cells to etoposide (20 microM) for 1 h daily for 3 days every 21-28 days, a schedule similar to that used in the clinic. Resistance (20-fold) to the cytostatic and DNA cleavage activities of etoposide emerged after the third treatment, and this phenotype was stable in the absence of drug exposure for 2.5 years. VPR-2 cells exhibited cross resistance to intercalating agents and vinca alkaloids, but remained sensitive to X-radiation, cisplatin and 5-fluorouracil. The human mdr1 gene was overexpressed in the resistant line, but steady-state concentrations of etoposide were reduced only 1.5-fold. Topoisomerase II catalytic and etoposide stimulated DNA cleavage activity in nuclear extracts from both lines were identical despite retention of a 3-fold level of resistance to etoposide-induced strand breaks in isolated nuclei from VPR-2 cells. Cyclosporin A and verapamil, both of which bind to P-glycoprotein, enhanced accumulation of etoposide in VPR-2 cells, and H69 cells to a lesser extent. Yet only cyclosporin A was effective in differentially enhancing etoposide cytostasis in VPR-2 relative to H69. In VPR-2 whole cells, cyclosporin A enhanced etoposide-induced DNA single-strand break frequency 9-fold but had no effect in isolated nuclei. Rapid selection of this line with a clinically relevant drug exposure schema and stability of the resistant phenotype suggest these cells may have been a steady subpopulation of the parent line through years of serial passage in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Down-regulation of deoxycytidine kinase in human leukemic cell lines resistant to cladribine and clofarabine and increased ribonucleotide reductase activity contributes to fludarabine resistance

Mechanisms of acquired resistance to three purine analogues, 2-chloro-2'-deoxyadenosine (cladribine, CdA), 9-beta-D-arabinofuranosyl-2-fluoroadenine (fludarabine, Fara-A), and 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (clofarabine, CAFdA) were investigated in a human T-lymphoblastic leukemia cell line (CCRF-CEM). These analogues are pro-drugs and must be activated by deoxycytidine kinase (dCK). The CdA and CAFdA resistant cell lines exhibited increased resistance to the other nucleoside analogues activated by dCK. This was also the case for the Fara-A resistant cells, except that they were sensitive to CAFdA and guanosine analogues. The CdA and CAFdA resistant cells displayed a deficiency in dCK activity (to <5%) while the Fara-A resistant cells showed only a minor reduction of dCK activity (20% reduction). The activity of high K(m) 5'-nucleotidase (5'-NT) (cN-II) using IMP as substrate, was 2-fold elevated in the resistant cell lines. The amount of the small subunit R2 of ribonucleotide reductase (RR) was higher in the Fara-A resistant cells, which translated into a higher RR activity, while CdA and CAFdA cells had decreased activity compared to the parental cells. Expression of the recently identified RR subunit, p53R2 full-size protein, in CAFdA cells was low compared to parental cells, but a protein of lower molecular weight was detected in CdA and CAFdA cells. Co-incubation of Fara-A with the RR inhibitor 3,4-dihydroxybenzohydroxamic acid (didox) enhanced cytotoxicity in the Fara-A resistant cells by a factors of 20. Exposure of the cells to the nucleoside analogues studied here also caused structural and numerical instability of the chromosomes; the most profound changes were recorded for CAFdA cells, as demonstrated by SKY and CGH analysis. We conclude that down-regulation of dCK in cells resistant to CdA and CAFdA and increased activity of RR in cells resistant to Fara-A contribute to resistance.     

Induction of thymidylate synthase associated with multidrug resistance in human breast and colon cancer cell lines.

A series of Adriamycin-resistant human breast MCF-7 and human colon DLD-1 cancer cell lines were established by stepwise selection. The concentration of Adriamycin required to inhibit cell proliferation by 50% (IC50) in the parent breast line (MCF-7), Adriamycin-resistant lines (MCF-Ad5 and MCF-Ad10), and a 5-fluorouracil (5-FU)-revertant line (MCF-R) was 0.005, 3.3, 6, and 4.9 microM, respectively. The Adriamycin IC50 value for the resistant colon line (DLD-Ad) was 8.2 microM, 68-fold higher than that for its parent line (DLD-1) (IC50 = 0.12 microM). The MCF-Ad5 and MCF-Ad10 cells were cross-resistant to 5-FU, with respective 5-FU IC50 values of 11.7 and 22.5 microM, or 7.3- and 14-fold less sensitive than their parent MCF-7 (IC50 = 1.6 microM) line. The MCF-R line completely reverted in sensitivity to 5-FU, with an IC50 of 1.7 microM. The resistant DLD-Ad line was 3.5-fold more resistant to 5-FU than was the parent DLD-1 line. Using both the 5-fluoro-2'-deoxyuridine-5'-monophosphate binding and catalytic assays for measurement of thymidylate synthase (TS) activity, there was significantly increased TS activity in the resistant MCF-Ad5 (2.4- and 2.5-fold), MCF-Ad10 (11.5- and 6.8-fold), and DLD-Ad (4.8- and 10.7-fold) lines, for binding and catalytic assays, respectively, compared with their parent MCF-7 and DLD-1 lines. The level of TS in cytosolic extracts, as determined by Western immunoblot analysis, was markedly increased for the resistant MCF-Ad5 (31-fold), MCF-Ad10 (46-fold), and DLD-Ad (52-fold) cells. Measurement of TS mRNA levels by Northern analysis revealed elevation of TS mRNA in the resistant MCF-AD5 (16.7-fold), MCF-Ad10 (31-fold), and DLD-Ad (55-fold) cells. Southern analysis showed that this increase in TS mRNA was not accompanied by any major rearrangements or amplification of the TS gene. Incorporation of 5-FU into the RNA and DNA of the resistant MCF-Ad10 cells was not significantly different, compared with that for parent MCF-7 cells. These studies suggest that exposure of human breast and human colon cancer cells to Adriamycin leads to overexpression of TS, with concomitant development of resistance to 5-FU.     

Antiherpetic activity and mechanism of action of 9-(4-hydroxybutyl)guanine

9-(4-Hydroxybutyl)guanine was synthesized and tested for antiherpes activity. In cell cultures, different strains of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) were inhibited by 50% at 2–14 μM of 9-(4-hydroxybutyl)guanine, while a HSV-1 mutant lacking thymidine kinase (HSV-1 TK^-) was resistant. Linear competitive inhibition of purified HSV-1-induced thymidine kinase (TK) with thymidine as a variable substrate was observed for 9-(4-hydroxybutyl)guanine with an apparent K_i value of 2.06 μM while the corresponding K_i value for the cellular TK was > 250 μM. By using high performance liquid chromatography, the formation of 9-(4-hydroxybutyl)guanine monophosphate by HSV-1 TK was measured and the rate of product formation was found to be about 10% of that found by using thymidine as a substrate. A selective inhibition of HSV-1 DNA synthesis by 9-(4-hydroxybutyl)guanine was observed in infected Vero cells. 9-(4-Hydroxybutyl)guanine had a low cellular toxicity. A weak therapeutic effect on herpes keratitis in rabbits was observed whereas cutaneous HSV-1 infection in guinea pigs and systemic HSV-2 infection in mice were not affected by this compound.     

Biochemical changes associated with a multidrug-resistant phenotype of a human glioma cell line with temozolomide-acquired resistance

Temozolomide (TMZ) is a newly approved alkylating agent for the treatment of malignant gliomas. To investigate resistance mechanisms in a multidrug therapeutic approach, a TMZ-resistant human glioma cell line, SF188/TR, was established by stepwise exposure of human SF188 parental cells to TMZ for approximately 6 months. SF188/TR showed 6-fold resistance to TMZ and cross-resistance to a broad spectrum of other anticancer agents that included 3-5-fold resistance to melphalan (MEL), gemcitabine (GEM), paclitaxel (PAC), methotrexate (MTX), and doxorubicin (DOX), and 1.6-2-fold resistance to cisplatin (CDDP) and topotecan (TPT). Alkylguanine alkyltransferase (AGT) activity was increased significantly in the resistant cell line compared with the parental cell line (P<0.05), whereas no significant differences occurred in the cellular uptake of TMZ and PAC between resistant and parental cells. Depletion of AGT by O(6)-benzylguanine significantly increased the cytotoxicity of TMZ in both the sensitive and resistant cell lines, but did not influence the cytotoxicity of the other drugs tested. Treatment with TMZ caused SF188 cells to accumulate in S phase, whereas SF188/TR cells were unaffected. Expression of Bcl-2 family members in SF188/TR cells compared with SF188 cells indicated that the pro-apoptotic proteins (i.e. Bad, Bax, Bcl-X(S)) were reduced 2-4-fold in the resistant cell line, whereas the anti-apoptotic proteins Bcl-2 and Bcl-X(L) were expressed at similar levels in both cell lines. In conclusion, the mechanism of resistance of SF188/TR cells to TMZ involved increased activity of AGT, a primary resistance mechanism, whereas the broad cross-resistance pattern to other anticancer drugs was due to a common secondary resistance mechanism related to alterations in the relative expression of the pro-apoptotic and anti-apoptotic proteins.