Interactions of BCNU, low pH, glucose and hyperthermia in cultured RIF cells

The interactions of BCNU (1,3-bis[2-chloroethyl]-1-nitrosourea) with low pH, glucose and hyperthermia were studied in cultured RIF tumor cells. The effect of a mild heat treatment of 43 degrees C, 1 h at pH 7.4 on cell killing [surviving fraction (S) = 0.27 +/- 0.05, standard error of the mean (S.E.)] was significantly enhanced by pH 6.5 (S = 0.11 +/- 0.02, S.E.) and 50 mM D-glucose (S = 0.14 +/- 0.01, S.E.). When heat (43 degrees C, 1 h) was added to BCNU, cytotoxicity was increased approximately 14-fold over BCNU alone. Moreover, pH 6.5 increased killing with BCNU and heat by an additional factor of 28. The presence of glucose at 37 degrees C at either pH 6.5 or 7.4 reduced BCNU toxicity in a dose dependent fashion. However, the presence of glucose did not reduce cell killing by BCNU at 43 degrees C. As a result BCNU cytotoxicity was enhanced by approximately 2 orders of magnitude when tumor cell acidification (glucose and low pH) was combined with BCNU and heat.     

Cell cycle age response of 9L cells to 1,3-bis(2-chloroethyl)-1-nitrosourea and modification by alpha-difluoromethylornithine

We have determined the cell cycle age response of 9L rat brain tumor cells to 1,3-bis(2-chloroethyl)-1-nitrosourea using centrifugal elutriation to obtain populations of cells enriched in G1, S, and G2-M phases. While cells in all phases of the cell cycle were killed by 20 or 40 microM 1,3-bis(2-chloroethyl)-1-nitrosourea, cells in G1 and G2-M phases were more sensitive than cells in S phase. The differential sensitivity was more pronounced at the higher dose, which will markedly alter the distribution of cells through the cell cycle. In a clinical setting, this factor could affect the efficacy of either fractionated or multimodality protocols. Treatment with alpha-difluoromethylornithine, a polyamine biosynthesis inhibitor, potentiated the cytotoxic effects of 20 microM 1,3-bis(2-chloroethyl)-1-nitrosourea against G1- and G2-M- but not against S-phase cells; however, at a higher dose of 1,3-bis(2-chloroethyl)-1-nitrosourea (40 microM), the cytotoxicity was potentiated for cells in all phases of the cell cycle. In alpha-difluoromethylornithine-treated cells, the phenomenon could be reversed by adding 1 mM putrescine 24 hr before treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea. Therefore, the potentiation of 1,3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity appears to be related to polyamine depletion.     

Myeloprotective effect of diethyldithiocarbamate treatment following 1,3-bis(2-chloroethyl)-1-nitrosourea, adriamycin, or mitomycin C in mice

The effect of diethyldithiocarbamate (DDTC) on myelotoxicity induced by 1,3-bis(2-chloroethyl)-1-nitrosourea, Adriamycin, or mitomycin C in C57BL/6J x DBA/2J mice is reported here. All drugs were administered i.v. Myelotoxicity was assessed, 24 h after administration of the myelotoxic drug, using bone marrow stem cell (spleen colony-forming unit) and granulocyte/macrophage progenitor cell (granulocyte/macrophage colony-forming unit in culture) clonogenic assays. Administration of DDTC alone had no effect on spleen colony-forming units or granulocyte/macrophage colony-forming units in culture. 1,3-Bis(2-chloroethyl)-1-nitrosourea showed a dose-dependent toxicity for both cell types, and subsequent treatment with DDTC (300 mg/kg i.v. 3 h after 1,3-bis(2-chloroethyl)-1-nitrosourea) ameliorated this toxicity. The same dosing regimen of DDTC ameliorated Adriamycin-induced toxicity to bone marrow stem cells at the two higher doses tested. However, the myelosuppressive effects of mitomycin C were not altered by DDTC administration (300 mg/kg i.v. 3 h after or 30 min before mitomycin C). These results demonstrate that DDTC ameliorates myelotoxicity induced by several, but not all, chemotherapeutic agents and suggest a broad role for DDTC in cancer chemotherapy.     

Formation of DNA interstrand cross-links by the novel chloroethylating agent 2-chloroethyl(methylsulfonyl)methanesulfonate: suppression by O6-alkylguanine-DNA alkyltransferase purified from human leukemic lymphoblasts

The formation of DNA interstrand cross-links was compared in DNA treated with either 1,3-bis(2-chloroethyl)-1-nitrosourea or 2-chloroethyl(methylsulfonyl)methanesulfonate. DNA that was pulse treated briefly with either of these drugs continued to form cross-links at 37 degrees C for over 8 h after drug removal, indicating that such DNA contained stable precursors of cross-links. When human O6-alkylguanine-DNA alkyltransferase was added to the drug-treated DNA further cross-link formation was prevented at all points during this protracted time course, indicating that these stable cross-link precursors also remained substrates for this repair enzyme. Although the pattern of 2-chloroethyl(methylsulfonyl)methanesulfonate-induced cross-link formation and susceptibility to suppression by O6-alkylguanine-DNA alkyltransferase resembled that for 1,3-bis(2-chloroethyl)-1-nitrosourea, quantitative differences in the rates of cross-link formation and in the amounts of O6-alkylguanine-DNA alkyltransferase required to suppress cross-link formation suggest that critical differences exist between these agents.     

Differential sensitivities of five rat hepatoma cell lines to anticancer drugs.

Five permanent tumor cell lines derived originally from either a solid or an ascites biopsy of rat hepatoma exhibited differential sensitivities to bleomycin, Adriamycin, 1-beta-D-arabinofuranosylcytosine, hydroxyurea, 1-trans-(2)-chloroethyl)-3-(4-methoylcyclohexyl)-1-nitrosourea, and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. The cells were least sensitive to hydroxyurea and 1-beta-D-arabinofurano-sylcytosine, with some cell lines being almost totally resistant to these drugs. However, from 25- to 700-fold differences in survival were obtained between cell lines treated with either bleomycin or Adriamycin.     

Mechanism of uptake of nitrosoureas by L5178Y lymphoblasts in vitro

The mechanism of uptake of nitrosoureas by L5178Y cells in vitro was investigated. A time course of the uptake of radioactivity on incubation of L5178Y lymphoblast with [14C]-1,3-bis(2-chloroethyl)-1-nitrosourea was linear for 30 min and then entered a plateau phase; it was markedly temperature dependent. A similar time course for cells incubated with [14C]ethylene-labeled 1-(2-chlorethyl)-3-cyclohexyl-1-nitrosourea reached equilibrium rapidly, was temperature independent, and resulted in a relatively low level of uptake of radioactivity. However, cells treated with 3-[cyclohexyl-14C]-1-(2-chlorethyl)-1-nitrosourea had a time course that was linear for 30 min, resulted in much higher levels of uptake of radioactivity, and was strongly temperature dependent. These findings, at least for 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, suggest that some drug decomposition precedes uptake. The percentage of radioactivity found in the cell sap fraction was at least 85% of total cell activity when cells were incubated with any of the three 14C-labeled nitrosoureas. Furthermore, thin-layer chromatography of the cell sap fraction revealed the presence of free intact drug. These findings indicate that intracellular uptake of intact nitrosoureas occurred. A time course of uptake of intact 1,3-bis(2-chloroethyl)-1-nitrosourea reached equilibrium rapidly with cell/medium distribution ratios of 0.2 to 0.6 and was temperature independent. The addition of excess unlabeled 1,3-bis(2-chlorethyl)-1-nitrosourea or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea had no effect on uptake of [14C]-1,3-bis(2-chloroethyl)-1-nitrosourea, These findings suggest that uptake of intact 1,3-bis(2-chloroethyl)-1-nitrosourea was by passive diffusion. A time course of the uptake of intact 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea with either [14C]ethylene- or ring-labeled drug rapidly reached equilibrium, was temperature independent, and attained a cell/medium ratio greater than unity. Uptake of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea was sodium independent and was unaffected by the metabolic inhibitors (sodium fluoride, sodium cyanide, or 2,4-dinitrophenol) or by urea, a potential physiological competitor. Furthermore, addition of unlabeled 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea or 1,3-bis(2-chlorethyl)-1-nitrosourea had no effect on uptake of labeled 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. These findings suggest that uptake of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea also occurs by passive diffusion.     

The oncogenic potential of a combination of hyperthermia and chemotherapy agents

The modulating effect of 43 degrees C hyperthermia on the induction of oncogenic transformation by the antineoplastic agents, actinomycin D, mitomycin C, and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was examined using the C3H 10T1/2 cell line. For any given level of cytotoxicity, cells exposed to the three chemotherapy agents at 37 degrees C showed similar frequencies of transformation. Transformation frequencies induced by all three drugs were reduced by hyperthermia. The reduction was most pronounced for cells exposed to BCNU, and to a lesser extent, by cells exposed to actinomycin D and mitomycin C. The modulating effects of heat on drug-induced transformation incidence appeared to be independent of whether application of heat and drug was concurrent or sequential.     

In vivo and in vitro mechanisms of drug resistance in a rat mammary carcinoma model

Many in vitro tumor models have been examined to help understand the precise mechanisms responsible for drug resistance. The importance of these results in vivo remains uncertain. MatB 13762 is a rat mammary adenocarcinoma cell line that can be grown both in vitro and as a solid tumor in Fischer 344 rats, thus permitting the examination of tumor cell drug resistance under both conditions. Two cell lines have been selected in vitro for resistance to Adriamycin (AdrR) and melphalan (MlnR), respectively. Each subline has the following features: AdrR, increased mdr-1 messenger RNA, a high level of cross-resistance to vincristine and atypical low level resistance to melphalan and 1,3-bis(2-chloroethyl)-1-nitrosourea, decreased cellular glutathione content, and increased expression of Yc and Yp glutathione S-transferase isozymes; MlnR, low level drug resistance to melphalan and cross-resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea, Adriamycin, and vincristine; increased cellular concentration of glutathione; elevated glutathione S-transferase activity; and greatly increased messenger RNA specific to the Yc and Yp glutathione-S-transferase subunits. Most of the biochemical and molecular features described above are present but significantly less prominent in tumors grown in vivo. This model provides the opportunity to examine the magnitude of expression and the clinical significance of in vitro resistance in an in vivo model.     

Differential inhibition of the rejoining of X-ray-induced DNA strand breaks in normal and transformed human fibroblasts treated with 1,3-bis(2-chloroethyl)-1-nitrosourea in vitro.

The effects of 1,3-bis(2-chloroethyl)-1-nitrosourea on the rejoining of X-ray-induced DNA strand breaks were examined in normal human fibroblasts (WI-38) and a simian virus 40-transformed derivative (VA-13) with the use of alkaline sucrose sedimentation. 1,3-Bis(2-chloroethyl)-1-nitrosourea was capable of partially inhibiting repair of X-ray-produced DNA strand breaks in both cell types when the drug was added to the culture medium immediately after X-irradiation. However, when 1,3-bis(2-chloroethyl)-1-nitrosourea exposure preceded X-ray by 1 hr, DNA repair was inhibited to a much greater extent than it was when 1,3-bis(2-chloroethyl)-1-nitrosourea followed X-ray. The inhibition of DNA repair by 1,3-bis(2-chloroethyl)-1-nitrosourea appeared to be complete in the transformed VA-13 cells, while only partial inhibition of repair was observed in the normal WI-38 cells.     

Chemical structure of carbamoylating groups and their relationship to bone marrow toxicity and antiglioma activity of bifunctionally alkylating and carbamoylating nitrosoureas

Although the antitumor effects of chloroethylnitrosoureas have been shown to be due primarily to DNA-DNA cross-linking by the alkylating moieties of these agents, the basis of the often accompanying bone marrow toxicity has been more controversial. We report on the relative bone marrow toxicity of four model nitrosoureas with different alkylating and carbamoylating activities: 1,3-bis(2-chloroethyl)-1-nitrosourea; 1,3-bis(trans-4-hydroxycyclohexyl)-1-nitrosourea; chlorozotozin, (2-[3-(2-chloroethyl)-3 -nitrosoureido]-2-deoxy-D-glucopyranose); and -3-(beta-D-glucopyranosyl)-1-nitrosourea. Inhibitions of DNA, RNA, and protein synthesis in murine bone marrow cells and of colony growth of myeloid precursor cells (granulocyte-macrophage colony-forming units) were used as in vitro end points of myelotoxicity. Further, we determined the antiglioma activity of the four nitrosoureas on two human gliomas in a clonogenic tumor cell assay and studied the effect of the non-nitrosourea carbamoylators potassium cyanate, chloroethyl isocyanate, cyclohexyl isocyanate, ethyl isocyanate, and ethyl isothiocyanate on granulocyte-macrophage colony-forming units. The results show that, at equivalent drug exposures, clonogenic glioma cell kill was significant and comparative for 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-(beta-D-glucopyranosyl)-1-nitrosourea, and chlorozotocin; 1,3-bis(trans-4-hydroxycyclohexyl)-1-nitrosourea showed little activity. In contrast, granulocyte-macrophage colony-forming unit toxicity was low with chlorozotocin and 1-(2-chloroethyl)-3-(beta-D-glucopyranosyl)-1-nitrosourea and very high with 1,3-bis(2-chloroethyl)-1-nitrosourea and 1,3-bis(trans-4-hydroxycyclohexyl)-1-nitrosourea. Of the isocyanates, bone marrow toxicity was highest with chloroethyl isocyanate and cyclohexyl isocyanate, intermediate with ethyl isocyanate, and lowest with KOCN and ethyl isothiocyanate. Our results indicate that (a) bifunctional alkylation is essential for antiglioma activity of nitrosoureas and (b) myelosuppression is at least partly linked with carbamoylation but that structural entities in the carbamoylating isocyanate rather than a quantitative degree of carbamoylation determine the degree of potential myelotoxicity.     

Effects of antineoplastic agents and hyperthermia on cytotoxicity toward chronically hypoxic glioma cells.

The effects of hyperthermia and antineoplastic agents on the cytotoxicity to normally oxygenated and chronically hypoxic glioma cells were investigated in vitro. Exposure to temperatures above 43.0 degrees C was less cytotoxic to hypoxic cells which predominantly accumulated in the G0/G1 phase fraction. On the other hand, mitomycin C (MMC) and adriamycin (ADM) were preferentially cytotoxic to hypoxic cells not only at 37 degrees C but also at elevated temperatures (42 degrees C and 43 degrees C). These two agents showed marked synergistic effects with hyperthermia under both oxygenated and hypoxic conditions. In contrast, bleomycin (BLM), cis-diamminedichloroplatinum(II) (CDDP), and vincristine (VCR) were preferentially cytotoxic to oxygenated cells at both 37 degrees C and elevated temperatures. CDDP showed cytotoxic synergism with hyperthermia that appeared to be oxygen-dependent. A nitrosourea derivative, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU), showed no major preferential toxicity under either oxygenated or hypoxic conditions. This study suggests that hyperthermia in combination with MMC or ADM would have a greater cytotoxic effect on hypoxic cell subpopulations of malignant gliomas.     

Effects of dicyclohexylamine sulfate, a spermidine synthase inhibitor, in 9L rat brain tumor cells

Growth characteristics, polyamine levels, and distribution of cells in the cell cycle were determined for 9L rat brain tumor cells treated for various periods with 1 mM dicyclohexylamine sulfate (DCHA). Continuous treatment of cells with DCHA caused growth inhibition at 2 days of treatment. After 2 days of treatment the growth rate of cells increased to approximately the same rate as control cells, even though treatment was continuous. Levels of spermidine were depleted to less than 10% of control levels, spermine levels were essentially unchanged, and putrescine levels were elevated to more than 350% of control levels after 9L cells were treated with DCHA for 2 days. In contrast to results found for the polyamine biosynthesis inhibitor alpha-difluoromethylornithine, treatment of 9L cells with DCHA did not potentiate the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea. To mimic the effects on polyamine levels caused by treatment with DCHA, 9L cells were treated with 5 mM putrescine alone or with 5 mM putrescine and 1 mM DCHA after treatment with 1 mM alpha-difluoromethylornithine. Results of these experiments suggest that treatment with DCHA alone does not potentiate the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea because elevated levels of putrescine caused by treatment counteract the effects of decreased spermidine levels.     

Combined modality therapy with bleomycin, hyperthermia, and radiation

In an attempt to develop better combination therapies for use with local radiation, the interaction between bleomycin and hyperthermia +/- radiation was studied in the FSaIIC tumor system. In cells exposed in vitro to bleomycin at 37 degrees C and at pH 7.40, the drug was substantially more toxic toward normally oxygenated than hypoxic cells. At hyperthermic temperatures (42 degrees or 43 degrees C), however, the differential killing between the normally oxygenated and hypoxic cells disappeared and bleomycin became significantly more toxic. Exposure to bleomycin at pH 6.45 did not substantially alter the cytotoxicity of the drug at 42 degrees or 43 degrees C. In tumor growth delay experiments, combining bleomycin, hyperthermia, and radiation induced long delays, and the more successful sequences were bleomycin----radiation----hyperthermia or bleomycin----hyperthermia----radiation. If radiation was given prior to drug and hyperthermia, however, the sequence was significantly less effective. In tumor excision experiments performed 24 h after treatment, increasing doses of bleomycin produced a shallow, log-linear increase in tumor cell kill at 37 degrees C, but bleomycin followed by hyperthermia (43 degrees C, 30 min) led to about 1 log more cell killing. Administration of bleomycin just prior to treatment with a single dose of radiation was cytotoxically additive. In this assay the most effective trimodality treatment sequence was bleomycin----hyperthermia----radiation. In tumor subpopulations defined by Hoechst 33342 dye staining, bleomycin at 37 degrees C was about two-fold more toxic toward the bright (presumably well-oxygenated) cells than toward the dim (presumably hypoxic) cell subpopulation. The addition of hyperthermia following bleomycin produced nearly a log more tumor cell killing in both the bright and dim tumor cells. The combination of bleomycin followed by hyperthermia and then radiation was at least additive in the bright cells and caused a large cell kill, but in comparison, there was marked sparing of the dim cells. These results indicate that treatment with bleomycin and hyperthermia in conjunction with radiation can add substantially to tumor cell killing. This combination is significantly less effective in the hypoxic than oxic tumor regions, however, in spite of in vitro data which demonstrate that the cytotoxicity of bleomycin at hyperthermic temperatures is not oxygen-dependent.     

Effects of chemotherapeutic drugs on platelet and metastatic tumor cell-endothelial cell interactions as a model for assessing vascular endothelial integrity

An in vitro assay for examining the sublethal effects of chemotherapeutic agents on vascular endothelial integrity is described. Using vascular endothelial cell monolayers, the kinetics of binding of radiolabeled platelets or metastatic tumor cells were altered when endothelial cells were pretreated for 2 hr with low, clinically relevant concentrations of certain drugs. Electron microscopic examination by scanning electron microscopy revealed that these same drugs caused endothelial cell retraction and exposure of subendothelial matrix. Platelets and tumor cells were found bound only to the exposed areas of subendothelial matrix. Some drugs (bleomycin, 1,3-bis(2-chloroethyl)-1-nitrosourea, vincristine) induced rapid endothelial cell retraction and increased platelet and tumor cell binding to exposed subendothelial matrix, while one of the drugs tested (Adriamycin) caused delayed (1 to 3 days after a 2-hr drug treatment) endothelial cell retraction and increased cell binding. Of the drugs tested, only 5'-fluoro-2'-deoxyuridine which interferes with DNA replication failed to induce endothelial cell retraction and increased tumor cell and platelet binding. The results suggest that certain drug effects on the vascular endothelium can be assessed using the vascular endothelial cell monolayer model.     

Ultrasound enhanced drug toxicity on Chinese hamster ovary cells in vitro

Chinese hamster ovary cells (HA1) were exposed to therapeutic ultrasound (F = 2.025 MHz) in the presence of various drugs at temperatures of 37-43 degrees C. The space averaged intensities used were 0.5-2 W/cm2. The survival of these cells was subsequently tested using the clonogenic assay. Marked enhancement by ultrasound of the cytotoxicity of Adriamycin and amphotericin B was observed. For Adriamycin, the potentiation was dependent upon the intensity of sonication (exposure duration being 30 min). At 0.5 W/cm2, there was enhancement of cytotoxicity above 41 degrees C. At 1 W/cm2, there was a 3-order increase in cytotoxicity at 37 degrees C. Thus an increase in intensity resulted in a decrease in "threshold" temperature. The effect with Adriamycin could be explained in part by an increase in net uptake of drug into the cells. Further, ultrasound was observed to increase the sensitivity of cells to Adriamycin. For amphotericin B, the enhancement was observed only at exposure durations greater than 30 min and at 43 degrees C. There was no enhancement observed for cisplatin and etoposide. From these results, it appears that ultrasound potentiates the cytotoxicity of drugs the mode of action of which (at least in part) involves the plasma membrane.     

Flow cytometry analysis of DNA damage and the evaluation of cytotoxicity of alkylating agents

Monoclonal antibody (MAb) F7-26 generated against nitrogen mustard (HN2)-treated DNA (O.S. Frankfurt, Exp. Cell Res., 170: 369-380, 1987) reacted with regions of local DNA denaturation (distortion) induced by DNA alkylation. The relationship between immunoreactivity of cellular DNA with MAb F7-26 and cytotoxic effects of HN2, L-phenylalanine mustard (L-PAM), and 1,3-bis(2-chloroethyl)-1-nitrosourea was studied in HeLa S3 cultures. Cells were treated with drugs for 1 h and assessed for cell survival by colony formation assay and for DNA immunoreactivity by flow cytometry. Cells were fixed in ethanol, exposed to MAb, and stained with fluorescein-labeled anti-mouse immunoglobulin. Immunofluorescence (IF) intensity was measured on a flow cytometer. For each drug the cell killing and the binding of MAb to DNA appeared in the same dose ranges. A strong correlation (r = 0.96) between cell survival (log10 surviving fraction) and IF was observed when data for HN2, L-PAM, and 1,3-bis(2-chloroethyl)-1-nitrosourea were combined. This correlation was apparent in the range of 1-5 log10 cell killing. Enhancement of L-PAM cytotoxicity by buthionine sulfoximine (BSO) or hyperthermia was accompanied by a proportional increase of DNA immunoreactivity with MAb F7-26. The enhancement factors calculated from survival curves (a ratio of the dose decreasing cell survival by 1 log10 for L-PAM alone to that for L-PAM combined with modulating factor) were 1.67, 1.58, and 3.07 for BSO, hyperthermia, and BSO plus hyperthermia, respectively. For the same treatment regimens the enhancement factors calculated from drug dose-IF curves were 1.73, 1.34, and 3.79. A strong correlation between log10 surviving fraction and IF intensity (r = 0.93) was observed when data for L-PAM alone or L-PAM combined with BSO and/or hyperthermia were considered together in the range of 1-6 log10 cell killing. The cytotoxicity of alkylating agents and nitrosoureas and the effectiveness of factors modulating chemotherapeutic effects can be predicted by flow cytometry analysis of DNA immunoreactivity with MAb F7-26.     

Sensitization of 9L rat brain gliosarcoma cells to 1,3-bis(2-chloroethyl)-1-nitrosourea by alpha-difluoromethylornithine, an ornithine decarboxylase inhibitor

alpha-Difluoromethylornithine, a known inhibitor of polyamine biosynthesis, significantly enhanced the cytotoxic effect of 1,3-bis(2-chloroethyl)-1-nitrosourea, a cell cycle-nonspecific agent, in 9L rat brain gliosarcoma cells in vitro. Administered as a single agent, alpha-difluoromethylornithine was not cytotoxic to 9L cells and, compared to untreated control cells, caused no perturbation of cell cycle kinetics. alpha-Difluoromethylornithine-induced depletion of intracellular polyamine levels appears to have caused the observed sensitization of 9L cells to 1,3-bis(2-chloroethyl)-1-nitrosourea. Restoration of intracellular polyamine levels by the addition of exogenous putrescine to treated 9L cells reversed this phenomenon.     

Effect of novobiocin on the antitumor activity and tumor cell and bone marrow survivals of three alkylating agents

Our previous in vitro studies demonstrated marked synergy with alkylating agents when novobiocin was present during and after alkylating agent exposure. To determine whether this effect is observed in vivo, novobiocin was administered daily for 3 days prior to alkylating agent treatment, during alkylating agent treatment, and for 2 days after completion of alkylating agent treatment. When combined with cis-diamminedichloroplatinum(II), 1,3-bis(2-chloroethyl)-1-nitrosourea, or cyclophosphamide, there was significant enhancement of the growth delay of the FSaIIC fibrosarcoma implanted s.c. in C3H mice when compared with alkylating agents alone. In a second assay using ex vivo studies of tumor cells exposed in vivo, single doses of 100 mg/kg of novobiocin followed by cis-diamminedichloroplatinum(II) resulted in a 3- to 4-fold increase in tumor cell killing by cis-diamminedichloroplatinum(II). At a dose of 100 mg/kg of 1,3-bis(2-chloroethyl)-1-nitrosourea there was about a 7-fold increase in tumor cell kill upon addition of novobiocin. Cyclophosphamide showed a dose response effect with novobiocin, reaching 13-fold at a dose of 300 mg/kg of cyclophosphamide. In all cases bone marrow elements were affected less than were neoplastic cells, suggesting that the combination of novobiocin and alkylating agents may be a clinically useful strategy.     

The sensitivity of cells in exponential and stationary phases of growth to bleomycin and to 1,3-bis(2-chloroethyl)-1-nitrosourea

Studies of EMT6 mouse tumour cells growing in vitro have shown that these cells become less sensitive to bleomycin as they pass from exponential growth into stationary phase. This result is the opposite of that recently reported by two other groups of workers using different cell systems, but in agreement with the results of a third group of workers. Results for 1,3-bis(2-chloroethyl)-1-nitrosourea, however, confirm the findings of other workers that cells become more sensitive to this agent as they pass into stationary phase.     

Pharmacokinetics of nitrosoureas: comparison of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) after oral and intravenous administration to rats

Differential pulse polarographic assay of intact nitrosoureas revealed the lower bioavailability of CCNU (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea) in stomach and liver after oral administration to rats in comparison to BCNU (1,3-bis-(2-chloroethyl)-1-nitrosourea); blood levels of CCNU were below the detection limit of the method (20 ng). After i.v. bolus the CCNU concentration in plasma fell faster than that of BCNU. The rate of CCNU decomposition during incubation with blood at 37 degrees C was 3 times lower than that of BCNU.