Collateral sensitivity between methylene dimethane sulfonate and halogenated methotrexate derivatives in the Yoshida sarcoma in vivo and in vitro.

A Yoshida lymphosarcoma line (YMDR8) resistant to methylene dimethane sulfonate (MDMS) showed collateral sensitivity against three halogenated methotrexates: 3'-bromomethotrexate (NSC-98580), 3'-bromo-5'-chloromethotrexate (NSC-98579), and 3',5'-dichloromethotrexate (NSC-29630); however, it was cross-resistant to methotrexate itself. Two other independently derived MDMS-resistant cell lines, YMDR7 and YMDR9, also demonstrated collateral sensitivity against 3'-bromomethotrexate, but with the latter, the origin of the "induced" sensitivity probably was not due to interference with antigenic or oncogenic properties of the cell line. When these agents were used in vivo (in Wistar rats) and in vitro, subpopulation changes within the tumor lines could be observed. The possible importance of this parameter in the development of such sensitivity is discussed.

DNA-DNA interstrand crosslinking by dimethyanesulphonic acid esters. Correlation with cytotoxicity and antitumour activity in the Yoshida lymphosarcoma model and relationship to chain length.

Members of the homologous series of nine antitumour dimethanesulphonic acid esters, with the exception of ethylene dimethanesulphonate (EDMS), were found to cause DNA-DNA interstrand crosslinking in cells derived from the transplantable rodent Yoshida lymphosarcoma. The ability of the series to induce interstrand crosslinks was compared with in vitro cytotoxicity and in vivo antitumour activity and is discussed in relation to their chain length and ability to span critical target distances in DNA.

The role of metallothionein, glutathione, glutathione S-transferases and DNA repair in resistance to platinum drugs in a series of L1210 cell lines made resistant to anticancer platinum agents.

The glutathione contents, glutathione S-transferase activities and metallothionein contents have been measured in a series of L1210 cell lines which show decreased sensitivities to platinum drugs. Resistance to cisplatinum cisDDP, cis-diamminedichloroplatinum (II)] and chip [ioproplatin, cisdichloro-bis-isopropylamine-trans dihydroxy platinum IV] was found to correlate with glutathione levels but not metallothionein. Conversely, resistance to tetraplatin was found to be correlated with metallothionein but not glutathione levels. However, depletion of glutathione by buthionine 1-sulphoximine sensitizes all cell lines to the effects of cisDDP, chip and tetraplatin [d,1-trans-tetrachloro-1,2-diamino-cyclohexanplatinum (IV)]. Inhibition of DNA repair by aphidicholin or caffeine also partially restored sensitivity to these platinum drugs. These results indicate the complexity of the changes occurring upon the development of drug resistance.

The effect of ifosfamide and its metabolites on intracellular glutathione levels in vitro and in vivo.

The effect of ifosfamide and its metabolites on intracellular levels of glutathione in P388 cells in vitro has been studied. It is demonstrated that glutathione depletion occurs only in the presence of 4-hydroperoxyifosfamide and chloroacetaldehyde. In contrast isophosphoramide mustard had no effect on glutathione levels in intact cells. The concentration of 4-hydroperoxyifosfamide required to reduce glutathione levels by 50% was approximately 1 mM and this represents a concentration far in excess of that achievable in patients receiving the drug. However the concentration of chloroacetaldehyde (approximately 100 microM) required to reduce intracellular levels of glutathione to a similar extent is attained in patients receiving ifosfamide. The glutathione levels in lymphocytes isolated from a patient undergoing an eight hour infusion of ifosfamide showed a marked decrease to about 30% of their original value. We conclude that ifosfamide causes glutathione depletion in vivo and the majority of this can be accounted for by the production of chloroacetaldehyde.

Cross-linking between histones and DNA following treatment with a series of dimethane sulphonate esters.

The cross-linking of the nucleohistone to the associated DNA by a series of methanesulphonates of the busulphan series (CH3SO2O(CH2)nOSO2CH3) from n = 4-9 has been studied by gel electrophoresis, by the use of 35S labelling and by pyrenemaleimide competition. It has been shown that all the dimethanesulphonates react with the Cys 114 of histone H3, and that octamethylene dimethanesulphonate (n = 8) cross-links histone H3 with DNA via the sulphydryl group, as well as forming H3-H3 dimers.

Differential cytotoxicity of combretastatins A1 and A4 in two daunorubicin-resistant P388 cell lines.

Combretastatin A4, a novel anti-mitotic agent was effective against two P388 cell lines with acquired resistance to daunorubicin. In contrast, Combretastatin A1, a close structural analogue of A4, showed a high degree of cross-resistance. Combretastatin A1 was also more efficient at increasing intracellular daunorubicin concentrations in both resistant cell lines. Neither agent was capable of altering anthracycline accumulation in the parental (sensitive) cell line. We propose that the cross-resistance to Combretastatin A1 occurs, at least in part, as a result of the increased affinity of the drug-efflux process operative in these resistant cells for Combretastatin A1 vs Combretastatin A4. Hence, Combretastatin A4 may play a role in the treatment of tumours with acquired resistance to the anthracycline antibiotics.

Repair of DNA interstrand crosslinks after busulphan. A possible mode of resistance.

The technique of alkaline elution has been used to study the interaction between the antineoplastic drug busulphan and the DNA of cells derived from the transplantable rodent Yoshida sarcoma. A dose-dependent proteinase-resistant filter retention was observed after drug treatment, which indicated the presence of DNA interstrand cross-links. Such cross-links were removed after 6 h in cells resistant to busulphan but not in the busulphan-sensitive parent cells, even after 24 h. Such temporal differences in DNA cross-linking could be correlated with cell survival and also with the level of anaphase chromosome aberrations, which was found to be four-fold higher in the sensitive line than in the resistant line.

A proposed mechanism of resistance to cyclophosphamide and phosphoramide mustard in a Yoshida cell line in vitro.

A Yoshida sarcoma cell line (YR/cyclo) showing decreased sensitivity to metabolically activated cyclophosphamide in vitro has been shown to be cross-resistant to phosphoramide mustard, the ultimate alkylating agent formed from cyclophosphamide. Resistance to these alkylating agents has been shown to be associated with increased activity of the glutathione S-transferase group of enzymes, and with elevated levels of glutathione, the cosubstrate of the enzyme. The resistant cell line shows lower levels of cellular damage, as measured by alkaline elution following treatment with phosphoramide mustard, than the parental (YS) line. The mechanism of resistance is ascribed to increased deactivation of potentially damaging metabolites of cyclophosphamide by the glutathione S-transferase enzymes, resulting in decreased cellular damage in the resistant cell line.

Comparative studies of DNA cross-linking reactions following methylene dimethanesulphonate and its hydrolytic product, formaldehyde.

The technique of alkaline elution was employed to study the interactions of methylene dimethane sulphonate (MDMS) and formaldehyde (HCHO) with DNA from Yoshida lymphosarcoma cells treated with these agents. MDMS and HCHO produced a proteinase sensitive filter retention which indicated the presence of DNA-protein cross-links. MDMS also produced some proteinase K-resistant filter retention which was believed to indicate DNA-interstrand cross-linking, whilst only single-strand breaks could be detected following treatment with HCHO. Co-incubation with semicarbazide prevented all DNA-protein cross-links induced by MDMS and HCHO as well as single-strand breaks, most obvious following HCHO treatment. Semicarbazide also reduced HCHO-induced cytotoxicity in the Yoshida lymphosarcoma cell line, while no significant alteration in MDMS-induced cytotoxicity was observed. These results suggest that HCHO-induced DNA-protein cross-links and single-strand breaks do not contribute to MDMS-induced cytotoxicity, and therefore the small but significant level of MDMS-induced DNA-interstrand cross-links is the most likely cytotoxic lesion of this agent.

Benzamide potentiation of the cytotoxicity of bifunctional galactitol [correction of galacticol] in resistant P388 leukemia correlates with inhibition of DNA ligase II.

Benzamide (BA) enhances the cytotoxicity of 1,2:5,6-dianhydrogalactitol (DAG) in resistant P388 leukemia cell lines but not in the sensitive parent line. To examine the reason for this difference in response, we carried out an alkaline elution assay using proteinase K to study DNA interstrand cross-linking. At early time points, equal concentrations of DAG produced the same level of interstrand cross-linking (ICL) in the resistant and sensitive P388 leukemic cells, although marked differences were observed in their cytotoxicity toward the two cell lines. In the sensitive cells, neither the amount of DNA cross-linking nor the cytotoxicity changed during the observation period (38 h) in either the presence or the absence of BA. In contrast, the elution rate of the DNA of DAG-treated resistant cells increased with time and had reached the control levels by 38 h. However, when these cells were postincubated with BA for 38 h, the elution rate of DNA was much faster than that observed for the untreated resistant cells, indicating an accumulation of DNA single-strand breaks (SSB). The SSB accumulation caused by BA was associated with an inhibition of the activity of ligase II enzyme, which was stimulated when resistant cells were treated with DAG alone. The potentiating effect of BA on the resistant cells can thus be related to the inhibiting action of BA on the DNA-rejoining enzyme, ligase II. The lack of sensitization by BA of the DAG-treated parent cell line may be attributable to the absence of DNA-SSB formation, which is necessary for ligase II activation through the stimulation of poly(ADP-ribose) synthesis.

Comparative studies of the uptake of daunorubicin in sensitive and resistant P388 cell lines by flow cytometry and biochemical extraction procedures.

The intracellular accumulation of daunorubicin as determined by flow cytometry correlates well with that as determined by extraction of the drug from cell homogenates. Two P388 mouse leukaemia cell lines showing differential sensitivity to the drug have been used to investigate the transport changes associated with resistance. Resistance to daunorubicin in these cell lines occurs through an alteration in the intracellular accumulation of the drug, resulting from the increased efflux of the anthracycline from the resistant cells. The effect of temperature, drug concentration, pH, and metabolic inhibitors on this process have been investigated. Uptake by a carrier-mediated process of the un-ionised form of the drug (pK = 8.25), coupled with an energy-dependent efflux process, is proposed as the mechanism of cellular accumulation in the case of the resistant cell line.

The use of a fluorescent methotrexate probe to monitor the effects of three vinca alkaloids on a mixed population of parental L1210 and gene-amplified methotrexate-resistant cells by flow cytometry.

Cells resistant to methotrexate (L1210/R7A) and possessing an increased level of dihydrofolate reductase due to gene amplification can be detected by the technique of flow cytofluorimetry using a new fluorescent derivative of methotrexate (F-MTX) based on a putrescine linker. Comparative studies of dihydrofolate reductase enzyme and cell growth inhibition following treatment with methotrexate and F-MTX suggest that the two agents possess similar modes of action. In an artificially mixed population of cells sensitive and resistant to methotrexate it is possible, using F-MTX, to recognise and separate distinct cell subpopulations showing differential fluorescence using a fluorescence-activated cell sorter (FACS IV). The selective removal of the resistant cells within a mixed population of sensitive and resistant cells has been demonstrated for 5 X 10(-8) M vinblastine by means of flow cytometry. The effectiveness of the vinca alkaloids decreases in the order vinblastine greater than vindesine greater than vincristine, which previously was shown to be the order of effectiveness in producing collateral sensitivity.

Collateral sensitivity of a methotrexate-resistant L1210 cell line to the vinca alkaloids.

L1210 mouse leukaemia cell lines showing a 20,000-fold differential sensitivity to methotrexate have been shown to exhibit some collateral sensitivity to at least two of the vinca alkaloids, vinblastine and vindesine. Vinblastine is the more cytotoxic for both cell lines. The extent of the collateral sensitivity decreases in the order vindesine greater than vinblastine greater than vincristine. Total cellular uptake studies with radiolabelled methotrexate showed only a two- to three-fold greater incorporation in the sensitive line. On the other hand, a two-fold greater incorporation of labelled vincristine occurred in the resistant line. No significant difference in the uptake occurred following labelled vinblastine treatment by the two cell lines. It is unlikely that differences in uptake account for the altered drug responses observed in the two cell lines.

The effect of vinca alkaloids in enhancing the sensitivity of a methotrexate-resistant (L1210/R7A) line, studied by flow cytometric and chromosome number analysis.

Two L1210 murine lymphoma cell lines sensitive and resistant to methotrexate (L1210 and L1210/ R7A , respectively) and previously shown to exhibit collateral sensitivity to the vinca alkaloids have been studied by flow cytofluorimetric techniques following propidium iodide staining of the DNA. Following treatment with a range of concentrations of vincristine, both cell lines showed a build-up of fluorescence in the 4n position. However, the methotrexate-resistant cell line exhibited this effect at lower doses of vincristine. On an equimolar basis, the vinca alkaloids ranked for intensity of this effect in the order vinblastine greater than vindesine greater than vincristine. DNA fluorescent histograms following various times of continuous exposure to vincristine showed an accumulation of material at the 8n position, which was shown by chromosome analysis to be due to polyploidy. It was concluded that methotrexate-resistant cells (L1210/ R7A ) experience difficulty in traversing mitosis and this difficulty is enhanced by the vinca alkaloids.

Effect of verapamil on daunorubicin accumulation in lymphocytes isolated from patients undergoing chemotherapy.

Verapamil was shown to be capable of increasing intracellular daunorubicin levels in normal lymphocytes isolated from patients undergoing chemotherapy for epithelial ovarian cancer. The extent of the increase in daunorubicin accumulation was variable, occurring in the range of 0-123% as compared with intracellular daunorubicin levels attained in the absence of verapamil. No similar effect was seen in lymphocytes isolated from healthy volunteers. A tentative explanation of these data may be the induction of multidrug resistance (mdr)-like characteristics in normal lymphocytes following cytotoxic chemotherapy.

An autoradiographic study of the intrarenal localisation and retention of cisplatin, iproplatin and paraplatin.

The intrarenal localisation of platinum following the intravenous administration of platinum-195m-labelled cisplatin, iproplatin and paraplatin was studied using autoradiography. Following injection of cisplatin, platinum was distributed throughout the kidney even up to 14 days after treatment. In the case of iproplatin and paraplatin rapid platinum clearance was noted from the glomeruli, blood vessels and renal medulla within 2 h of administration. Relative cortical and medullary platinum radionuclide concentrations for all three agents were determined by Chalkley grid analysis. This showed greater relative concentrations of platinum in the cortex at increasing times following iproplatin and paraplatin compared with cisplatin. It is suggested that the lower renal toxicity of iproplatin and paraplatin than of cisplatin may be due to reduced platinum retention within the pars recta.

Blood clearance of radioactively labelled cis-diammine 1,1-cyclobutane dicarboxylate platinum (II) (CBDCA) in cancer patients.

The blood and urinary clearances of cis-diammine 1,1-cyclobutane dicarboxylate platinum(II) (CBDCA, JM8) were determined in four patients with malignancy. A 40 muCi iv injection of 191 Pt/193 Pt (3:1)-labelled CBDCA was followed by serial blood and urine sampling to 72 h. The blood clearance was triphasic, mean values for the fast, intermediate, and slow phases being 10.8 min, 2.5 h, and 125 h, respectively. The fraction of activity excreted in the urine within the first 6 h had a mean value of 66.7%, contrasting with 26.4% for cisplatin. There was only a small fraction of CBDCA excreted by the slow phase (1.5%) as against an average of 20% for CHIP and 27% for cisplatin. The early and rapid renal clearance of CBDCA may account for reduced nephrotoxicity.

Bryostatin 1 induces productive Epstein-Barr virus replication in latently infected cells: implications for use in immunocompromised patients.

Bryostatin 1 is a novel anti-tumor agent currently undergoing clinical trial. We investigated the effect of this drug on B-lymphocyte cell lines that carry the Epstein-Barr virus and found that it induces these latently infected cells into the production of transforming virus particles over a wide range of concentrations. These results may have clinical implications, particularly with regard to the use of the drug in the immunocompromised patient.

Blood clearance of three radioactively labelled platinum complexes: cis-dichlorodiammine platinum II, cis, trans-dichlorodihydroxy-bis-(isopropylamine) platinum IV, and cis-dichloro-bis-cyclopropylamine platinum II, in patients with malignant disease.

The blood clearances of three platinum compounds, cis-dichlorodiammine platinum II (DDP), cis, trans-dichloro-dihydroxy-bis-(isopropylamine) platinum IV (CHIP), and cis-dichloro-bis-cyclopropylamine platinum II (CP), were determined in nine patients with malignant disease. The complexes were prepared using radioactive platinum (191Pt and 193Pt). A 10-mu Ci dose of each complex, containing the equivalent of 1-2 mg elemental platinum, was injected IV into groups of three patients. Serial blood and urine samples were collected over 72 h. No obvious difference was found between the three complexes for blood clearance, median t1/2a being 16.8 (range 11.2-23.5) min and median t1/2 beta 89 (range 63.7-127) h. The urinary excretion was greatest for CHIP, 60% of injected dose as against 42.6% for CP and 38.8% for DDP. Differences in renal excretion of DDP analogues could indicate potentially less nephrotoxic agents. The use of radioactive Pt will allow in vivo dynamic imaging of the distribution of platinum compounds in areas of interest.

Suitability of cisplatin solutions for 14-day continuous infusion by ambulatory pump.

The stability of cisplatin (DDP) solutions (1 and 1.6 mg/ml in saline-mannitol) in plastic infusion bags was studied for up to 14 days at 25 degrees C, 37 degrees C and 60 degrees C. Small changes in the solution were observed, but no evidence of any decomposition product was seen. Some precipitation of DDP was seen in the 1.6-mg/ml solution at the lower temperatures. Fluid loss from the bags was significant at the higher temperatures.