Development of bruneomycin and rubomycin resistance in mouse lymphadenosis tumor cells and Staphyloccus when these preparations are used separately or together

Two sublines of mouse lymphadenosis (L-5178) resistant to bruneomycin and rubomycin used alone, as well as a subline with induced resistance to the combination of these drugs were employed in the study. The studies showd that in separate use of rubomycin and bruneomycin the tumor cell resistance to the respective drug was evident at the 10th passage. After 30 passages neither bruneomycin nor rubomycin produced reliable inhibition of the tumor growth in the respective subline of lyphadenosis. When the antibiotics were used in combination, no significant decrease in sensitivity of the tumor cells to either of the drugs or their combination was observed. The experiment with Staph. aureus also showed that the rate of the resistance increase to the drug combination was lower than that to the drugs used alone. Therefore, it was shown that the combined use of bruneomycin and rubomycin provided a means for preventing to a significant extent of development of the resistance in lymphadenosis tumor cells and Staph. aureus. This may be considered as an indication for clinical trials of the above combination.     

Results of an experimental study of the antitumor activity of 1-asparaginase from E. coli and Erw. carotovora]

The antitumour activity of the preparations of L-asparaginase from E. coli and Erw. carotovora with respect to lymphadenosis L-5178 and Yorker's carcinosarcoma (ascitic cariants) has been established. No difference in antitumour efficacy of the preparation of L-asparaginase obtained from E. coli and Erw. carotovora was noted.     

The contribution of alkylation to the activity of quinone antitumor agents

Studies have shown that the quinone group can produce tumor cell kill by a mechanism involving active oxygen species. This cytotoxic activity can be correlated with the induction of DNA double strand breaks and is enhanced by the ability of the quinone compound to bind to DNA by alkylation. The cytotoxic activity and the production of DNA damage by model quinone antitumor agents were compared in L5178Y cells, sensitive and resistant to alkylating agents, to assess the contribution of alkylation to the activity of these agents. The resistant L5178Y/HN2 cells were found to be two fold and six fold more resistant to the alkylating quinones, benzoquinone mustard and benzoquinone dimustard, respectively, than parent L5178Y cells. In contrast, the L5178Y/HN2 cells showed no resistance to the nonalkylating quinones, hydrolyzed benzoquinone mustard and bis(dimethylamino)benzoquinone. The alkylating quinones produced approximately two fold less cross-linking in L5178Y/HN2 cells compared with L5178Y sensitive cells. DNA double strand break formation by hydrolyzed benzoquinone mustard and bis(dimethylamino)benzoquinone was not significantly different in sensitive and resistant cells. However, the induction of double strand breaks by the alkylating quinones benzoquinone mustard and benzoquinone dimustard was reduced by 5-fold and 15-fold, respectively, in L5178Y/HN2 cells. These results show that the alkylating activity of the alkylating quinones cannot directly explain all of the enhanced cytotoxic activity of these agents. Furthermore, they provide strong evidence that the enhanced formation of DNA double strand breaks by alkylating quinone agents is directly related to the ability of these agents to bind to DNA. This increased formation of strand breaks may account for the enhanced cytotoxic activity of the alkylating quinones.     

Actively induced experimental allergic orchitis in Lewis-resistant (Le-R) rats: reversibility of disease resistance by immunization with Bordetella pertussis

Differential susceptibility to the induction of experimental allergic orchitis (EAO) was examined in Lewis/NCr and Le-R subline rats. Lewis/NCr rats were found to be fully susceptible to the induction of EAO whereas Le-R subline rats were not. Disease resistance exhibited by Le-R rats could be overcome by including Bordetella pertussis in the immunization protocol. However, reversal of resistance with B. pertussis was dependent on the dose of rat testicular homogenate in the inoculum and found to be effective only at lower doses of antigen (10 mg/rat). Disease resistance in Le-R rats as well as B. pertussis-induced reversal of resistance did not appear to be associated with either (1) a significant difference in the number of mast cells in the ductus efferentes, the anatomic location of the earliest inflammatory infiltrates, or (2) an alteration in the phenotypic expression of either innate or B. pertussis-induced sensitivity to vasoactive amines. The results are discussed in the context of the role of B. pertussis in other animal models of organ-specific autoimmune diseases. It is proposed that the phenotypic expression of resistance to EAO in Le-R rats is a result of a mutation in a common regulatory locus affecting susceptibility to multiple autoimmune diseases and whose immunoregulatory action is normally exerted during the sensitization phase of the immune response.     

Development of resistance in Fisher lymphadenosis cells to dipin and bruneomycin when these preparations are used separately and in combination]

It was shown that dipin and bruneomycin resistant tumor cells appeared in mice with transplanted lymphadenosis after 10 passages on single use of the drugs. When the drugs were used in combination, no lymphadenosis cells resistant either to bruneomycin, or to dipin and their combination were found. The combined use of the drugs prevented development of resistance to them in the lymphadenosis cells at least during 10 passages on mice. The data on the possible prevention of the resistance development in the mouse lymphadenosis cells by means of combined use of low doses of dipin and bruneomycin provided an assumption that it is expedient to test the combination in clinics.     

Radiation could induce p53-independent and cell cycle--unrelated apoptosis in 5-fluorouracil radiosensitized head and neck carcinoma cells

The effect of chemoresistance induction in radiosensitivity and cellular behavior after irradiation remains misunderstood. This study was designed to understand the relationship between radiation-induced cell cycle arrest, apoptosis, and radiosensitivity in KB cell line and KB3 subline selected after 5-fluorouracil (5FU) exposure. Exposure of KB cells to 5FU led to an increase in radiosensitivity. G2/M cell cycle arrest was observed in the two cell lines after irradiation. The radioresistant KB cell line reached the maximum arrest two hours before KB3. The cellular exit from this arrest was found to be related to the wild type p53 protein expression induction. After irradiation, only KB3 cell line underwent apoptosis. This apoptosis induction seemed to be independent of G2/M arrest exit, which was carried out later. The difference in radiosensitivity between KB and KB3 subline may result therefore from both a difference in apoptosis induction and a difference in G2/M arrest maximum duration. Moreover, 5FU exposure has led to an increase in constitutive p53 protein expression, which may be associated with an increase in basal apoptosis cell fraction. Given the existing correlation between radiosensitivity and the percentage of basal apoptosis, the constitutive p53 protein expression may be related to intrinsic radiosensitivity in our cellular model.     

DNA repair,cell cycle progression and cell death following camptothecin treatment in two murine lymphoma L5178Y sublines

The processes involved in cell response to camptothecin (CPT) were investigated in two sublines of L5178Y (LY) murine lymphoma; LY-R, resistant and LY-S, sensitive to X-irradiation, which are inversely cross-sensitive to the drug. The cells were pulse-treated with 2 μM CPT for 1 h; this resulted in equal numbers of replication-related DNA double-strand breaks (DSBs) in both sublines.¹ After drug removal, at different time points up to 24 h, the levels of DSBs were measured by using field inversion gel electrophoresis (FIGE) and comet assay at neutral pH. Both methods revealed faster DSBs repair in LY-S than in LY-R cells, in contrast with X-ray-induced DSBs. This however, was followed by the appearance of secondary breaks in the former subline. The cell cycle arrest was at S/G₂ phase and comprised equal numbers of cells in LY-S and LY-R populations. In both sublines formation of giant cells took place, as well as delayed apoptosis starting about 20 h post-CPT incubation and proceeding with similar intensity. At the same time, the total number of necrotic cells appearing during post-exposure incubation in the LY-R subline exceeded that in the LY-S subline. We suggest that, beside previously documented higher susceptibility of topoisomerase I (Topo I) from LY-R cells to CPT,^2,3 a higher initial rate of replication-related DSBs repair, but not lower propensity to apoptosis, may contribute to the relative CPT resistance of LY-S versus LY-R cells. Copyright © 1998 John Wiley & Sons, Ltd.     

Simultaneous changes in various mechanisms that mediate the cell incorporation of folate compounds account for low levels of resistance to methotrexate.

Changes in the mechanisms of folate incorporation were studied in cells treated with low concentrations of methotrexate in order to evaluate their contribution to the development of resistance to antifolate drugs. The uptake of methotrexate via reduced-folate system, the membrane-associated high-affinity folate binding capacity and the activity, levels and affinity for methotrexate of dihydrofolate reductase were measured in L5178 murine leukemic lymphoblasts and in a subline, MTX/R16, 16 times more resistant to methotrexate which was isolated after a short exposure to the antifolate. Various simultaneous changes were characterized in MTX/R16 cells which co-participated in the development of resistance: a decreased affinity of the carrier for methotrexate uptake via the reduced-folate system of entry, the increase of dihydrofolate reductase activity and levels and a two-fold increased expression of a membrane-associated high-affinity folate-binding protein (mFBP). The increase of the mFBP expression, besides ensuring the growth of resistant cells by its contribution to the reduced folate intake, also participates in the methotrexate resistance by the internalization of folate cofactor which would compete with methotrexate hindering the effective inhibition of dihydrofolate reductase by the antifolate.     

Hydrogen peroxide induced reproductive and interphase death in two strains of L5178Y murine lymphoma differing in radiation sensitivity

Summary L5178Y-R (LY-R) and L5178Y-S (LY-S) cells, differing in radiation sensitivity and susceptibility to the radiosensitizing effect of benzamide (Bz) were examined for susceptibility to hydrogen peroxide. Survival and chromatid aberration frequency indicated that LY-R cells were considerably more sensitive to H₂O₂ than LY-S cells. So, LY strains were found to be inversely crosssensitive to X/ rays and H₂O₂. The relative resistance to H₂O₂ corresponded with the previously found twofold difference in catalase activity (Jaworska et al. 1987). At higher concentrations H₂O₂ treatment caused interphase death, that was delayed by benzamide (Bz, 2 mM), an inhibitor of po1y(ADP-ribosylation), to a lesser extent in the more resistant cell subline (LY-S). From the examination of the H₂O₂ induced increase in the free Ca²⁺ concentration (with or without 2 mM Bz treatment) with the use of Fura-2 it followed, that the cells responded to the oxidative stress by Ca²⁺ release. The Ca²⁺ concentration increase was neither directly related to the killing effect of H₂O₂ treatment, nor did it correspond with the twofold difference in catalase activity in LY strains.     

Effects of a contractile prostaglandin antagonist (L-640,035) upon allergen-induced bronchoconstriction in hyperreactive rats and conscious squirrel monkeys

The effects of an antagonist of contractile prostanoids, L-640,035 (3-hydroxymethyl-dibenzo[b,f]thiepin-5-dioxide) upon antigen-induced bronchoconstriction have been studied in inbred rats with non-specific bronchial hyperreactivity and in conscious squirrel monkeys. L-640,035 was a potent inhibitor of antigen-induced dyspnea (ED50 3.1 mg/kg p.o.) in inbred rats pretreated with methysergide (3 micrograms/kg i.v.) but produced no significant inhibition in untreated rats. Administration of L-640,035 (10 mg/kg p.o.) to conscious squirrel monkeys exposed to aerosols of ascaris antigen markedly inhibited changes in pulmonary resistance (RL) and dynamic compliance (CDYN). At a lower dose (1 mg/kg p.o.) the inhibition of changes in CDYN were similar but the effects on RL were reduced. It was concluded first that contractile prostanoids may be important mediators of antigen-induced bronchoconstriction and secondly that L-640,035 may be effective in human allergic asthma.     

Chromosomal instability in mutagen-sensitive mutants isolated from mouse lymphoma L5178Y cells. II. Abnormal induction of sister-chromatid exchanges and chromosomal aberrations by mutagens in an ionizing radiation-sensitive mutant (M10) and an alkylating agent-sensitive mutant (MS1)

To determine the mutual relationships between cell survival and induction of sister-chromatid exchanges (SCEs) as well as chromosomal aberrations (CAs), mutagen-induced SCEs and CAs were analyzed in an ionizing radiation-sensitive mutant (M10) and an alkylating agent-sensitive mutant (MS 1) isolated from mouse lymphoma L5178Y cells. The levels of CA induction in both mutants strictly corresponded to the sensitivity to lethal effects of mutagens, except that caffeine-induced CAs in M10 are considerably lower than those in L5178Y. The results clearly indicate that except for caffeine-induced CAs in M10, mutagen-induced lethal lesions are responsible for CA induction. In contrast, SCE induction in mutants was complicated. In M10, hypersensitive to killing by gamma-rays, methyl methanesulfonate (MMS), and 4-nitroquinoline 1-oxide (4NQO), but not sensitive to UV or caffeine, the frequency of SCEs induced by gamma-rays was barely higher than that in L5178Y, and the frequencies of MMS- and UV-induced SCEs were similar to those in L5178Y, but 4NQO- and caffeine-induced SCEs were markedly lower than those in L5178Y. MS 1, which is hypersensitive to MMS and caffeine, but not sensitive to UV or 4NQO, responded to caffeine with an enhanced frequency of SCEs and had a normal frequency of MMS-induced SCEs, but a reduced frequency of UV- and 4NQO-induced SCEs. Thus, susceptibility to SCE induction by mutagens is not necessarily correlated with sensitivity of mutants to cell killing and/or CA induction by mutagens. Furthermore, the spontaneous levels of SCEs are lower in M10 and higher in MS 1 than that in L5178Y (Tsuji et al., 1987). Based on these results, we speculate that M10 may be partially defective in the processes for the formation of SCEs caused by mutagens. On the other hand, MS 1 may modify SCE formation-related lesions induced by UV and 4NQO to some repair intermediates that do not cause SCE formation. In addition, MMS-induced lethal lesions in MS 1 may not be responsible for SCE induction whereas caffeine-induced lethal lesions are closely correlated with SCE induction. Thus, the lesions or mechanisms involved in SCE production are in part different from those responsible for cell lethality or CA production.     

The effect of dose rate on X-radiation-induced mutant frequency and the nature of DNA lesions in mouse lymphoma L5178Y cells

The induction of mutants at the heterozygous tk locus by X radiation was found to be dose-rate dependent in L5178Y-R16 (LY-R16) cells, but very little dose-rate dependence was observed in the case of strain L5178Y-S1 (LY-S1), which is deficient in the repair of DNA double-strand breaks. Induction of mutants by X radiation at the hemizygous hprt locus was dose-rate independent for both strains. These results are in agreement with the hypothesis that the majority of X-radiation-induced TK-/- mutants harbor multilocus deletions caused by the interaction of damaged DNA sites. Repair of DNA lesions during low-dose-rate X irradiation would be expected to reduce the probability of lesion interaction. The results suggest that in contrast to the TK-/- mutants, the majority of mutations at the hprt locus in these strains of L5178Y cells are caused by single lesions subject to dose-rate-independent repair. The vast majority of the TK-/- mutants of strain LY-R16 showed loss of the entire active tk allele, whether the mutants arose spontaneously or were induced by high-dose-rate or low-dose-rate X irradiation. The proportion of TK-/- mutants with multilocus deletions (in which the products of both the tk gene and the closely linked gk gene were inactivated) was higher in the repair-deficient strain LY-S1 than in strain LY-R16. However, even though the mutant frequency decreased with dose rate, the proportion of mutants showing inactivation of both the tk and gk genes increased with a decrease in dose rate. The reason for these apparently conflicting results concerning the effect of DNA repair on the induction of extended lesions is under investigation.     

Cell-cycle dependency of BUdR-induced mutation for six genetic markers in cultured mammalian cells.

Synchronized mouse L5178Y cells were treated with BUdR during each one of four sequential periods of the cell cycle (M-G1, early S, middle S and late S-G2). Among the 6 markers examined, asparagine independence (Asn+), 6-thioguanine resistance (TGr) and excess thymidine resistance (TdRr) showed maximal induction of mutation rates in the early S period, methotrexate resistance (MTXr) gave maximal induction during the middle S period, and two other markers [arabinosylcytosine resistance (Ara-Cr) and ouabain resistance (Ouar)] showed little mutation induction in any period under the experimental conditions. These results suggest that (i) genes responsible for Asn+, TGr and TdRr activity may be replicated in the early S period and the gene for MTXr activity replicated in the middle S period, and (ii) the mechanisms of mutation induction for the Ouar and Ara-Cr markers may be essentially different from those for the Asn+, TGr, TdRr and MTXr markers.     

The mutagenicity of 5-azacytidine and other inhibitors of replicative DNA synthesis in the L5178Y mouse lymphoma cell

The mutagenic potential of the cytidine analog, 5-azacytidine (Aza Cyd), was tested at the thymidine kinase (TK) gene locus of L5178Y mouse lymphoma cells. 3-h exposure to as little as 20 ng/ml Aza Cyd yielded a substantial increase in TK-deficient L5178Y cells as measured by drug-induced resistance to trifluorothymidine (TFTres) 48 h later. This mutagenic effect was diminished up to 75% when Aza Cyd was tested in the presence of either enzymatically active or heat-denatured 9000 X g supernatant prepared from rat liver homogenate. The mutagenicity of Aza Cyd was also decreased in the presence of 1-5 X 10(-3) M thymidine and eliminated in the presence of greater than 1 X 10(-5) M cytidine. Two L5178Y TK-deficient cell lines had no selective survival advantage compared to TK-competent L5178Y cell stock when plated in soft-agar medium that contained Aza Cyd. Four other specific inhibitors of scheduled DNA synthesis in mammalian cells, deoxyadenosine, aphidicolin, 1-beta-D-arabinofuranosylcytosine, and hydroxyurea were also L5178Y/TK mutagens. These data along with other published results suggest that chemicals known to disrupt nucleotide biosynthesis, alter deoxyribonucleotide pools, or directly inhibit DNA polymerase can cause stable, heritable increases in TFT resistance through mechanisms dependent upon altered replicative DNA synthesis, yet not necessarily dependent upon DNA incorporation or the binding of these mutagenic agents to nuclear DNA.     

Genetic study of the resistance of mouse somatic cells to colchicine (high resistance level)

The studies of the high level of colchicine resistance of mouse L cells have shown that two mutagens (EMS and NMM) do not induce cell variants resistant to 8 microgram/ml of colchicine in the population of mouse heteroploid L-53 cells (subline of L cells, the level of colchicine resistance 140) and that colchicine resistance of L-53 cells gradually diminishes when cells are propagated in non-selective conditions: after 1 month it diminishes 2-fold, after 3 month--9-fold. The extent of the decrease of the drug resistance was the same in 6 independent cultures obtained from the inoculum of 200 cells and in control cultures propagated by large quantities of cells. These data coincide with the results of the previous studies of lower level of colchicine resistance. In both studies the frequency of the occurrence of colchicine resistant variants in selective medium was about 2.10(-4). These data are consistent with the hypothesis that colchicine resistance of mouse L cells is not due to a gene mutation.     

Low level of mitochondrial deoxyguanosine kinase is the dominant factor in acquired resistance to 9-beta-D-arabinofuranosylguanine cytotoxicity

9-beta-D-arabinofuranosylguanine (Ara-G) is an important and relatively new guanosiue analog with activity in patients with T-cell malignancies. The biochemical and molecular events leading to resistance to Ara-G are not fully understood. Therefore we generated two Ara-G-resistant human MOLT-4 leukemic cell lines with different levels of resistance. The mitochondrial enzyme deoxyguanosine kinase (dGK) and the nuclear/cytosol enzyme deoxycytidine kinase (dCK) are key enzymes in the activation of Ara-G. Decreased levels of dGK protein and mRNA were found in both resistant cell sublines. The activity of dCK was decreased in the subline with higher resistance to Ara-G and these cells were highly cross-resistant to other nucleosides activated by dCK. Increased activity of the mitochondrial enzyme thymidine kinase 2 was observed in both resistant sublines and this could be related to the dGK deficiency. In search for other resistance mechanisms it was found that the resistant cells overexpress the mdr1 gene, while no changes were detected in the levels of multidrug resistance-associated protein 1 through 6, lung resistance-associated protein or topoisomerase IIalpha or IIbeta. Taken together, our findings demonstrate that multiple mechanisms are involved in the acquired resistance to Ara-G. However, low expression of dGK is the most apparent alteration in both resistant cell lines. Partial deficiency of dCK was found in the subline cells with higher resistance to Ara-G. Furthermore, Ara-G may select for high expression of the multidrug resistance (mdr1) which could be a specific resistance mechanism but more likely part of an overall cellular stress response.     

The mutagenic potency of 1,8-dinitropyrene in cultured mouse lymphoma cells

Although non-toxic, 1,8-dinitropyrene (1,8-DNP) was mutagenic for mouse lymphoma L5178Y cells when assayed for induced resistance to 6-thioguanine, methotrexate, ouabain and 1-β-D-arabinofuranosyl cytosine. In bacteria, nitropyrenes are potent inducers of frame-shift mutations, and the induction of ouabain-resistant mutants, believed to be due to base-pair substitutions, suggests that the mechanism of action may be different in mouse cells and bacteria. Long treatment times were required to detect 1,8-DNP-induced mutants in L5178Y cells, suggesting the possibility of an inducible activation system. 4-Nitroquinoline 1-oxide was both toxic and mutagenic to these same 4 mutation assays after short (2 h) treatment times. The dilemma that exists when comparing the mutagenic potential of test chemicals when concentration of mutagen, treatment times and toxicity are markedly different, is discussed.     

Actinomycin D induction of acid phosphatase in synchronized 15178Y mouse leukemia cells

Actinomycin D was found to induce synthesis of acid phosphatase activity in L5178Y mouse leukemic cells. The degree of induction was dose-dependent. Furthermore, other RNA synthesis inhibitors (Lucanthone and Daunomycin) were also found to cause an increase in enzyme activity. Such induction of enzyme activity was also found to be dependent on cell cycle position. For up to 5 h after release from Colcemid synchronized cells treated with actinomycin D showed little or no variation from controls. Only when cells were in late S (5th to 8th hours of the cell cycle) could enzyme activity be induced. At this time full induction occurred within 1 h.     

Mutation induction in a mouse lymphoma cell mutant sensitive to 4-nitroquinoline 1-oxide and ultraviolet radiation

The mutant mouse lymphoma cell Q31, which is sensitive to 4-nitroquinoline 1-oxide and ultraviolet radiation (UV), was compared with the parental L5178Y cell for the effect of caffeine and mutation induction after UV irradiation. Caffeine potentiated the lethal effect of UV in both cell strains to a similar extent, indicating that the defective process in Q31 cells was caffeine-insensitive. UV-induced mutation to 6-thioguanine resistance was determined in L5178Y and Q31 cells. The maximal yield of mutants was obtained 7 days post-irradiation in L5178Y cells and 14 days in Q31 cells for higher UV doses. It appears that a much longer time is required for the mutant cells than for the parental cells for full expression of the resistance phenotype even at equitoxic UV doses. A substantially higher frequency in induced mutations was observed in Q31 cells than in L5178Y cells at a given dose of UV. A plot of induced mutation frequency as a function of logarithm of surviving fraction again indicates hypermutability of Q31 cells as compared with the parental strain. In contrast, X-rays induced a similar frequency of mutations to 6-thioguanine resistance in L5178Y and Q31 cells.