Effects of Mg2+ and adenine nucleotides on thymidylate synthetase from different mouse tumors

Summary Magnesium ions variably influenced activity of highly purified thymidylate synthetase preparations from different mouse tumors, activating the enzyme from Ehrlich ascites carcinoma (EAC) cells and inhibiting the enzyme from L1210 and L5178Y cells and from 5-fluorodeoxyuridine (FdUrd)-resistant EAC cells. In the presence of Mg²⁺ in a concentration resulting in either maximum activation or inhibition (25–30 mM) the enzymes from both the sensitive and FdUrd-resistant EAC lines and L5178Y cells were activated by ATP. Under the same conditions of Mg²⁺ concentration ADP and AMP inhibited the enzyme from the parental but not from the FdUrd-resistant EAC cells.     

Thymidylate synthetase overproduction in 5-fluorodeoxyuridine-resistant mouse fibroblasts.

We describe the isolation and characterization of a series of 5-fluorodeoxyuridine (FdUrd)-resistant mouse 3T6 cell lines that overproduce thymidylate synthetase (TS) by up to 50-fold compared with the parental cells. The resistant cells were selected by growing 3T6 cells or a methotrexate-resistant 3T6 cell line (M50L3, isolated previously in our laboratory) in gradually increasing concentrations of FdUrd. Uridine and cytidine were included in the culture medium to reduce toxicity from metabolic products of FdUrd. Cells that were resistant to the drug by virtue of loss of thymidine kinase activity were eliminated by selection in medium containing hypoxanthine, methotrexate, and thymidine. M50L3 cells were found to adapt to FdUrd more readily than 3T6 cells. A number of clones were isolated that were able to grow in the presence of 3 microM (M50L3 derived) or 0.3 microM (3T6 derived) FdUrd. Several were found to overproduce TS by 10 to 50-fold compared with normal 3T6 cells. All were found to have thymidine kinase activity, although the enzyme level was significantly reduced in some clones. The overproduced TS was inactivated by 5-fluorodeoxyuridylic acid at the same concentration as the enzyme from 3T6 cells. TS was purified from the LU3-7 clone (50-fold overproducer) by affinity chromatography on methotrexate-polyacrylamide. The monomer molecular weight was about 38,000, which was the same as the molecular weight of the monomer in 3T6 cells. The overproduction trait was gradually lost (half-life, 3 weeks) when LU3-7 cells were grown in the absence of FdUrd. The overproducing cells will provide an abundant supply of TS and (very likely) its mRNA and may serve as a convenient model system for detailed studies of the regulation of TS gene expression during the cell cycle.     

A Strain of Rosa damascena Cultured Cells Resistant to Ultraviolet Light

A strain of cultured cells of Rosa damascena Mill. which showed unusual resistance to damage by short wave (254 nanometers) ultraviolet radiation was isolated. The resistant cells were 2.2 to 2.8 times larger and had about twice the amount of DNA and more chromosomes than the parental, sensitive cells. The resistant cells also produced larger quantities of polyphenolic compounds, principally flavonoids, during the later phases of culture growth. At 10 days, resistant cells had 4 times more nonflavonoid polyphenolics and 14 times more flavonoids than parental cells. The resistance, which was also observed only in the later phases of culture growth, was best correlated with the production of polyphenolics, which apparently shielded ultraviolet-sensitive target molecules from damage.     

Replication of herpes simplex virus type 1 on hydroxyurea-resistant baby hamster kidney cells.

Hydroxyurea-resistant (HUr) baby hamster kidney cells were isolated, subcloned, and characterized. One clonal line, which contained elevated levels of ribonucleotide reductase, lost its HU resistance during passage in the absence of the inhibitor, whereas another clonal line was stably resistant. The replication of herpes simplex virus type 1 on these cells was compared with that of the parvovirus minute virus of mice. Herpes simplex virus type 1 was found to be as sensitive to HU on both lines of HUr baby hamster kidney cells as it was on parental (HU-sensitive) cells, whereas parvovirus replication was about eight times more resistant on HUr baby hamster kidney cells compared with the parental cells. The results suggest that herpes simplex virus type 1 cannot use the cellular reductase and may code for its own.     

The role of the BCG dose and the mouse strain in the inhibition of development of neoplasms susceptible and nonsusceptible to the action of natural cytotoxic cells

The susceptibility of Ehrlich Ascites Carcinoma (EAC) cells to the action of natural cytotoxic cells of DBA/2 and Balb/c mice in vitro was established. Leukaemia L 1210 cells proved insensitive to the in vitro action of natural cytotoxic cells of DBA/2 mice, but not to those of Balb/c ones. BCG, one of the inductors of cytotoxic NK cells, when administered to DBA/2 or Balb/c mice before introduction of EAC cells inhibited the growth of this tumour but did not retard the development of leukaemia L 1210 in DBA/2 mice. The change in the number of peritoneal exsudate cells (PEC) in DBA/2 mice after intraperitoneal injection of BCG was demonstrated to be dependent on the dose and the time elapsed after bacilli introduction. The antitumour action of BCG does not depend on changes in the number of PEC caused by the bacilli. Both large (3.0 mg) and small (0.02 mg) doses of BCG inhibit the development of EAC in Balb/c mice ("sensitive" to BCG), notwithstanding the time of administration of the bacilli. In DBA/2 mice ("resistant" to BCG) development of EAC can be inhibited only by the large dose of BCG since small one is sometimes ineffective.     

The role of a novel copper complex in overcoming doxorubicin resistance in Ehrlich ascites carcinoma cells in vivo

One of the important pathways of resistance to anthracyclines is governed by elevated levels of glutathione (GSH) in cancer cells. Resistant cells having elevated levels of GSH show higher expression of multidrug-resistant protein (MRP); the activity of glutathione S-transferases (GSTs) group of enzymes have also been found to be higher in some drug-resistant cells. The general mechanism in this type of resistance seems to be the formation of conjugates enzymatically by GSTs, and subsequent efflux by active transport through MRP (MRP1-MRP9). MRPs act as drug efflux pump and can also co-transport drugs like doxorubicin (Dox) with GSH. Depletion of GSH in resistant neoplastic cells may possibly sensitize such cells, and thus overcome multidrug resistance (MDR). A number of resistance modifying agents (RMA) like DL-buthionine (S, R) sulfoxamine (BSO) and ethacrynic acid (EA) moderately modulate resistance by acting as a GSH-depleting agent. As most of the GSH-depleting agents have dose-related toxicity, development of non-toxic GSH-depleting agent has immense importance in overcoming MDR. The present study describes the resistance reversal potentiality of novel copper complex, viz., copper N-(2-hydroxy acetophenone) glycinate (CuNG) developed by us in Dox-resistant Ehrlich ascites carcinoma (EAC/Dox) cells. CuNG depletes GSH in resistant (EAC/Dox) cells possibly by forming conjugate with it. Depletion of GSH results in higher Dox accumulation that may lead to enhanced rate of apoptosis in EAC/Dox cells. In vivo studies with male Swiss albino mice bearing ascitic growth of EAC/Dox showed tremendous increase in life span (treated/control, T/C = 453%) for the treated group with apparent regression of tumor. Resistance to Dox in EAC/Dox cells is associated with over expression of GST-P1, GST-M1 (enzymes involved in phase II detoxification) and MRP1 (a transmembrane ATPase efflux pump for monoglutathionyl conjugates of xenobiotics). CuNG causes down regulation of all these three proteins in EAC/Dox cells. The effect of CuNG as RMA is better than BSO in many aspects.     

Transient inhibition of DNA synthesis by 5-fluorodeoxyuridine leads to overexpression of dihydrofolate reductase with increased frequency of methotrexate resistance

Transient but incomplete suppression of DNA synthesis by a single exposure of an asynchronous population of cells to 5-fluoro-2'-deoxyuridine (FdUrd) increases the frequency of appearance of methotrexate (MTX)-resistant colonies. This increase was greater than 10-fold following a 6-h incubation of cells with 3 microM FdUrd prior to selection in MTX, an interval one-half the normal L1210 cell cycle time. During this period of exposure to FdUrd, DNA synthesis decreased to 25% of control rates and cells accumulated at the G1/S interface. The 6-h incubation with FdUrd resulted in greater than a 2.5-fold increase in the dihydrofolate reductase protein level in the treated cell population, which was accounted for, at least in part, by increased de novo synthesis of the enzyme as assessed by [35S]methionine labeling. This increase in dihydrofolate reductase was associated with a decrease in growth inhibition by MTX. A brief reversal (2 h) of FdUrd-induced DNA synthesis inhibition by the addition of thymidine eliminated the amplification of dihydrofolate reductase and the enhanced emergence of MTX-resistant clones. Beyond this, an analysis of clones that survive MTX selection indicates that the dihydrofolate reductase gene copy in cells spontaneously resistant to 50 nM MTX and those which resulted after the additional pretreatment with FdUrd for 6 h are comparable with a 2-4-fold amplification of enzyme in most clones. These studies demonstrate that FdUrd enhancement of dihydrofolate reductase expression can have a profound effect upon the incidence and expression of MTX resistance and that dihydrofolate reductase gene amplification may be another basis for antagonism between these agents.     

Genetic control of resistance to Coccidioides immitis: a single gene that is expressed in spleen cells determines resistance

We have previously reported that inbred mice vary widely in their resistance to Coccidioides immitis peritonitis. To investigate the number of genes controlling resistance, (susceptible X resistant)F1 X susceptible backcross mice were tested for resistance to infection. A 1:1 ratio of resistant:susceptible offspring was observed, which is consistent with a single dominant gene determining resistance. To find out whether this gene, which we designated Cms, is expressed in the immune and/or the inflammatory responses, radiation chimeras were constructed by transplanting spleen cells from the resistant F1 mice into the susceptible parental strain. These chimeras were consistently more resistant to infection than the susceptible parental strain. We concluded that resistance to C. immitis is determined primarily by a single gene, and that this gene is expressed by spleen cells.     

Proliferation-dependency of gamma-glutamyl hydrolase activity in various mouse cells.

The activity of gamma-glutamyl hydrolase, the enzyme which deglutamylates folyl and antifolyl polyglutamates, changed significantly in mouse cells during different phases of growth, being about two times lower in actively proliferating mice splenocytes and fibroblasts than in nondividing cells. In EAC cells growing in vivo the lowest activity was observed in cells in the logarythmic phase. Methotrexate treatment of mice in a dose of 500 mg/kg body weight increased the activity of the enzyme in EAC cells about 1.5 times. We suggest that gamma-glutamyl hydrolase is a proliferating dependent enzyme which together with folypolyglutamate synthetase ensures in cells an appropriate amount of folates in the form of polyglutamates necessary for optimizing folate-dependent biosynthetic activities.     

Catabolism of 5-fluoro-2'-deoxyuridine by isolated rat intestinal epithelial cells

The kinetics of conversion of 5-fluoro-2'-deoxyuridine (FdUrd) to 5-fluorouracil (FUra) by isolated rat intestinal epithelial cells was investigated. Also, the effects of potential inhibitors of this reaction, which is catalyzed by uridine phosphorylase and thymidine phosphorylase, were determined. A 2.5% suspension of isolated cells was incubated with FdUrd or FUra, and at specific times cells were lysed with perchloric acid and fluoropyrimidines were determined by high-performance liquid chromatography. During a 25-min incubation with either FdUrd or FUra, the amount of drug in the incubation system (total volume 0.8 ml) fell by less than 5%. However, in the presence of FdUrd, the amount of FUra increased linearly over 25 min. The apparent Vmax and Km for FUra formation were 17-27 nmole/mg DNA/min and 1.6-2.5 mM, respectively. With each nucleoside phosphorylase inhibitor, the apparent Km increased but Vmax was unaffected. The apparent Ki values were as follows (in mM): 5-nitrouracil (an inhibitor of both uridine phosphorylase and thymidine phosphorylase), 0.12; 4-thiothymine (a uridine phosphorylase-selective inhibitor), 1.52; and 6-benzyl-2-thiouracil (a thymidine phosphorylase-selective inhibitor), 0.73. It was concluded that intestinal epithelial cells are capable of degrading FdUrd to FUra and that the cells possess both uridine phosphorylase and thymidine phosphorylase activity.     

Synthesis and one-electron reduction characteristics of radiation-activated prodrugs possessing two 5-fluorodeoxyuridine units

Two molecules of an antitumor agent, 5-fluorodeoxyuridine (5-FdUrd), were connected by a 2-oxoalkyl linker (Oxo-linker) at the N(3) position to obtain radiation-activated prodrugs, FdUrd(2) A and FdUrd(2) B. The prodrugs in this study released 5-FdUrd via one-electron reduction initiated by hypoxic X-irradiation. The release of 5-FdUrd from FdUrd(2) A and FdUrd(2) B proceeded more efficiently than that of previous prodrug, Oxo-FdUrd, which possessed one molecule of 5-FdUrd. FdUrd(2) A exhibited increased cytotoxicity against A549 cells when the FdUrd(2) A solution had been irradiated with a large dose of X-rays before administration to the cells. However, we observed no effect on cytotoxicity when the cells were X-irradiated under hypoxic conditions in the presence of FdUrd(2) A because the amount of 5-FdUrd released in the cells seemed to be too low to induce cytotoxic activity.     

Respiration characteristics of mitochondria in parental and giant transformed cells of the murine Nemeth-Kellner lymphoma

Respiration characteristics of mitochondria of the parental and giant cells of murine NK/Ly (Nemeth-Kellner lymphoma) were studied. The giant cell-enriched ascites were obtained by serial intraperitoneal injections of vinblastine in tumour-bearing mice. Ascites containing >70% giant cells were used. Their diameter of was over 17 μm (~2800 μm(3)), while the diameter of the parental cells was 12.7 μm (1100 μm(3)). The respiration rate of mitochondria in situ was measured by oxygen consumption in intact and digitonin-permeabilized NK/Ly cells. Endogenous respiration of intact giant NK/Ly cells was three times higher compared to the parental ones, roughly in agreement with the volume change. The giant NK/Ly cells were far more resistant to permeabilization with digitonin than the parental cells, as shown by Trypan Blue and LDH (lactate dehydrogenase) release tests. After digitonin permeabilization, oxygen consumption was reduced to a minimal level (0.06 ng atom O/(s × 106 cells) in both types of cells. Addition of α-ketoglutarate or succinate to the incubation medium increased oxygen consumption in the parental cells by 46 and 164% respectively. In the giant NK/Ly cells, the corresponding increases were 164 and 276%. Addition of ADP to α-ketoglutarate- or succinate-supplemented medium further stimulated oxygen consumption of the permeabilized NK/Ly cells; however, the effect of ADP was more pronounced in the giant cells. In addition, indices of respiratory control were significantly higher in the giant cells. Oligomycin suppressed considerably the respiration of the intact giant cells but had a much weaker effect on parental cells. Thus, giant NK/Ly cells possess much higher respiration rates and show tighter coupling between the respiration and oxidative phosphorylation compared with parental cells.     

Selection of aphidicolin-resistant CHO cells with altered levels of ribonucleotide reductase

Chinese hamster ovary cells were initially selected for resistance to aphidicolin at 0.3 microgram/ml. Serial cultivation with aphidicolin at concentrations up to 5.0 micrograms/ml yielded a series of mutants with increasing resistance. The most resistant mutant isolated was 44 times more resistant to aphidicolin than the parental CHO. The alpha-polymerases, assayed in the cytoplasmic extracts of the mutants, did not increase in specific activity or differ from the parental CHO in their sensitivity to aphidicolin. When cultured in the presence of deoxythymidine, deoxyadenosine, and 1-beta-D-arabinofuranosyl cytosine (araC) the mutants showed considerably more resistance to these inhibitors than did the parental CHO. The intracellular pools of all four deoxynucleoside triphosphates (dNTPs) in the mutants increased with increasing resistance to aphidicolin. The elevated dNTP pools in the mutant most resistant to aphidicolin appear to be the result of a 4- to 8-fold increase in the level of ribonucleotide reductase (2'-deoxyribonucleoside diphosphate:oxidized thioredoxin 2'-oxidoreductase, EC 1.17.4.1).     

Iron N-(2-hydroxy acetophenone) glycinate (FeNG), a non-toxic glutathione depletor circumvents doxorubicin resistance in Ehrlich ascites carcinoma cells in vivo

Multidrug resistance-associated protein 1 (MRP1) reduces intracellular anticancer drug accumulation either by co transporting them with glutathione (GSH) or extruding drug-GSH conjugates outside of the cell. Thus, MRP1 confers multidrug resistance (MDR) and worsen successful chemotherapeutic treatment against cancer. Although the exact mechanism of MRP1 involved in MDR remains unknown, the elevated level of intracellular GSH is considered as a key factor responsible for MDR in cancer. Hence the quest for non-toxic molecules that are able to deplete intracellular GSH has profound importance to subdue MDR. The present preclinical study depicts the resistance reversal potentiality of an iron complex; viz. Ferrous N-(2-hydroxy acetophenone) glycinate (FeNG) developed by us in doxorubicin resistant Ehrlich ascites carcinoma (EAC/Dox) cells. FeNG potentiate cytotoxic effect of doxorubicin on EAC/Dox cells ex vivo and also increases the survivability EAC/Dox bearing Swiss albino mice in vivo as well. Moreover, in vivo administration of FeNG significantly depletes intracellular GSH with ensuant increase in doxorubicin concentration in EAC/Dox cells without alternation of MRP1 expression. In addition, intra-peritoneal (i.p.) application of FeNG in normal or EAC/Dox bearing mice does not cause any systemic toxicity in preliminary trials in mouse Ehrlich ascites carcinoma model. Therefore, the present report provides evidence that FeNG may be a promising new resistance modifying agent against drug resistant cancers.     

Metabolism of 5-fluorouracil in sensitive and resistant Novikoff hepatoma cells.

N1-S1/FdUrd Novikoff hepatoma cells, which lack thymidine kinase activity, are resistant to 5-fluorouracil (FUra) as well as 5-fluorodeoxyuridine (FdUrd), suggesting that the pathway, FUra leads to FdUrd leads to FdUMP, is utilized for the conversion of FUra to FdUMP. However, the inhibition of thymidylate synthetase activity, the presumed target of FdUMP, by 1 X 10(-4) M FUra in intact N1-S1 Novikoff hepatoma cells, which have significant levels of thymidine kinase activity, is completely eliminated by 5 X 10(-4) M hydroxyurea, which is a potent inhibitor of ribonucleotide reductase. These results imply that the formation of ribonucleotides and does not involve thymidine kinase. This apparent dichotomy can be explained by the fact that, in addition to the well known lack of thymidine kinase activity, [14C]FUra conversion to ribonucleotides is greatly depressed in the N1-S1/FdUrd cells. Hence, the formation of FdUMP from FUra in Novikoff hepatoma cells apparently proceeds primarily via the intermediate formation of ribonucleotides. The decreased conversion of FUra to ribonucleotides in N1-S1/FdUrd cells decreases not only the ability of the analog to inhibit DNA synthesis, but also its effect on RNA metabolism. FUra, at a concentration of 1 X 10(-5) M, inhibits rRNA maturation in N1-S1 cells, but not in N1-S1/FdUrd cells. Since N1-S1/FdUrd cells are completely resistant to 1 X 10(-5) M FUra, whereas N1-S1 cells are completely inhibited by 1 X 10(-5) M FUra, even in the presence of 1 X 10(-4) M thymidine, the effects of FUra on RNA metabolism appear to contribute significantly to the cytotoxicity of the analog at higher drug concentrations.     

Circadian influence on the immunization of mice with live Bacillus Calmette-Guérin (BCG) and subsequent challenge with Ehrlich ascites carcinoma

Non-specific immunostimulation with Bacillus Calmette-Guérin (BCG) is of current interest in the treatment of cancer. The main objective of the series of experiments described in this paper was to evaluate the influence the host's circadian system has on a. the stimulation of the immune system with BCG and b. the subsequent efficiency of that stimulated immune system against the Ehrlich Ascites Carcinoma (EAC). There was a circadian rhythm in the length of survival time in non-immunized mice challenged with the EAC. Mice receiving an EAC challenge during the middle of the light period survived significantly longer than those challenged with the EAC around the time of transition from dark to light. Mice immunized with BCG and challenged with EAC also demonstrated a circadian rhythm in the length of survival 30 days after EAC challenge with 86% survivors in the mice treated at 10(00) and 60% survivors in the mice treated at 07(00). The same relationship was also observed 70 and 80 days after EAC challenge. Eighty days after EAC challenge, a circadian rhythm was apparent in the frequency of solid tumors at the site of the initial EAC injection. The highest incidence of solid tumors occurred at 13(00). A circadian rhythm was found in the increase in body weight between the first and second BCG or saline injections. Rectal temperatures recorded on the 8th, 12th and 16th day after EAC challenge were characterized by circadian rhythmicity. In the mice without development of ascites, the peak temperature consistently occurred at 01(00). In the mice with ascites there was a phase advance in the rectal temperature rhythm of 3 h so that the peak in the rhythm consistently occurred at 22(00). In the mice with ascites a further finding was an increasing hypothermia as the ascites continued to develop; however, this hypothermia was not detectable during the time of the peak (10(00)) in the temperature rhythm. The mice which did not die by the 80th day after EAC challenge were challenged again with 5.0 x 10(6) EAC cells, and during the next 46 days circadian variations were observed in the numbers of mice which survived. Similar changes were observed during an additional 46 days after a third EAC challenge of 41.5 x 10(6) cells.     

Dynamics of toxin and lectin receptors on a lymphoma cell line and its toxin-resistant variant using ferritin-conjugated, 125I-labeled ligand

The dynamics of the toxin Ricinus communis agglutinin II (RCAII or ricin) on cells of a murine lymphoma line (BW5147) and a toxin-resistant variant line (BW5147RicR.3) that is 200 times more resistant than the parent to direct RCAII cytotoxicity were examined using ferritin-conjugated, affinity purified, 125I-labeled RCAII (ferritin-125I-RCAII). Ferritin-125I-RCAII was indistinguishable from native RCAII in quantitative binding and cytotoxicity experiments. When RCAII-sensitive BW5147 and -resistant BW5147RicR.3 cells were labeled with ferritin-125I-RCAII at various toxin concentrations (1--10 microgram/ml), no differences in toxin binding were observed. These same cells were examined by electron microscopy. At low ferritin-125I-RCAII concentrations (1-3 microgram/ml RCAII) where only the parental BW5147 cells were significantly more sensitive to RCAII, toxin receptors were internalized by ferritin-125I-RCAII-induced endocytosis. In parallel experiments, ferritin-125I-RCAII that bound to the resistant BW5147RicR.3 cells remained relatively dispersed or clustered, and there was little evidence of transport into cells via endocytosis. At higher ferritin-125I-RCAII concentrations (greater than 7 microgram/ml RCAII) where both parental and resistant variant cells are sensitive to the cytotoxic effects of RCAII, more ferritin-conjugated toxin was bound, and subsequent endocytosis occurred to a similar degree in both cell types. Endocytosis of ferritin-conjugated concanavalin A was indistinguishable on RCAII-sensitive parental and resistant variant cells at all concentrations tested. The results suggest that a specific defect on the selected BW5147RicR.3 cells prevents RCAII entry into these cells a low toxin concentrations, rendering them more resistant to the cytotoxic effects of RCAII.     

Human leukemic cells resistant to 5-fluoro-2'-deoxyuridine contain a thymidylate synthetase with lower affinity for nucleotides

A line of human lymphocytic leukemia cells (CCRF-CEM) has been obtained which is 140-fold resistant to the potent cell growth inhibitor 5-fluoro-2'-deoxyuridine (FdUrd). The cells were also 11-fold cross-resistant to 5-fluorouracil. In contrast to several previous studies involving FdUrd-resistant mouse cells, thymidylate synthetase levels were not substantially elevated in these FdUrd-resistant human leukemic cells. Thymidine kinase activity was also unchanged in the resistant cells, although the levels of 5-fluoro-2'-deoxyuridylate (FdUMP), the potent inhibitor of thymidylate synthetase, generated at equimolar doses of FdUrd were about 40% lower than in the sensitive cells. Studies of the kinetics of FdUMP binding to thymidylate synthetase isolated from the FdUrd-resistant cells disclosed a considerably higher dissociation constant (Kd = 1.0 X 10(-9) M) for the ternary covalent enzyme . FdUMP . 5,10-methylene tetrahydrofolate complex compared to the value obtained with enzyme from sensitive cells (Kd = 4.4 X 10(-11) M). The thymidylate synthetase from the FdUrd-resistant cells also showed 17-fold weaker binding of 2'-deoxyuridylate, even though the Km value for 2'-deoxyuridylate was 3-fold lower compared to the enzyme from FdUrd-sensitive cells. The turnover number of the altered enzyme was 1.8-fold higher than that for the normal enzyme but the rate constants for the release of FdUMP from the ternary complex, which is also an enzyme-catalyzed reaction, were identical for both enzymes. Electrophoresis of the radiolabeled ternary complexes on nondenaturing gels showed small but reproducible differences in migration rates. These results demonstrate that the mechanism of resistance to FdUrd in this cell line involves an alteration in the target enzyme, thymidylate synthetase, which causes it have a lower affinity for nucleotides.     

Increased gene-specific repair of cisplatin interstrand cross-links in cisplatin-resistant human ovarian cancer cell lines.

We have studied several aspects of DNA damage formation and repair in human ovarian cancer cell lines which have become resistant to cisplatin through continued exposure to the anticancer drug. The resistant cell lines A2780/cp70 and 2008/c13*5.25 were compared with their respective parental cell lines, A2780 and 2008. Cells in culture were treated with cisplatin, and the two main DNA lesions formed, intrastrand adducts and interstrand cross-links, were quantitated before and after repair incubation. This quantitation was done for total genomic lesions and at the level of individual genes. In the overall genome, the initial frequency of both cisplatin lesions assayed was higher in the parental than in the derivative resistant cell lines. Nonetheless, the total genomic repair of each of these lesions was not increased in the resistant cells. These differences in initial lesion frequency between parental and resistant cell lines were not observed at the gene level. Resistant and parental cells had similar initial frequencies of intrastrand adducts and interstrand cross-links in the dihydrofolate reductase (DHFR) gene and in several other genes after cisplatin treatment of the cells. There was no increase in the repair efficiency of intrastrand adducts in the DHFR gene in resistant cell lines compared with the parental partners. However, a marked and consistent repair difference between parental and resistant cells was observed for the gene-specific repair of cisplatin interstrand cross-links. DNA interstrand cross-links were removed from three genes, the DHFR, multidrug resistance (MDR1), and delta-globin genes, much more efficiently in the resistant cell lines than in the parental cell lines. Our findings suggest that acquired cellular resistance to cisplatin may be associated with increased gene-specific DNA repair efficiency of a specific lesion, the interstrand cross-link.