In vivo enhancement of 5-fluorouracil cytotoxicity to AKR leukemia cells by thymidine in mice.

The spleen colony assay was used to examine the effect of thymidine (dThd) on 5-fluorouracil (FUra) cytotoxicity in two transplantable leukemias, AKR (in AKR mice) and L1210 [in (BALB/c x DBA/2)F1 mice], in vivo. A large dose of dThd (10 mg/mouse) could not rescue these cell lines from FUra toxicity. Instead, when dThd was given within 1 hour before FUra, it enhanced FUra cytotoxicity by a factor between 100 and 1,000 in AKR leukemia. That dThd increased the cytotoxicity of FUra only by a factor of 3 in L1210 leukemia suggested a different mechanism of interaction of the two drugs in the two cell lines. Examination in hybrid mice capable of supporting the growth of both leukemias showed the enhancement to be tumor related rather than host related. We also demonstrated a dose-dependent effect of dThd injection 15 minutes before FUra in AKR leukemia. Concerning the kinetics of killing of AKR leukemia colony-forming units (LCFU) following the administration of dThd 15 minutes before FUra, LCFU survival continued to decrease for 24--36 hours following drug administration.

In vivo potentiation of 5-fluorouracil cytotoxicity against AKR leukemia by purines, pyrimidines, and their nucleosides and deoxynucleosides.

The effect of various natural pyrimidines and purines and their nucleosides and deoxynucleosides on 5-fluorouracil (FUra) cytotoxicity was examined against the transplantable AKR leukemia in female AKR mice. The spleen colony assay was used for a quantitative evaluation of the antitumor activity of the combination treatments. The base or nucleoside was administered 15 minutes before each mouse received an injection of 0.6 mg FUra. All of the compounds tested, with the exception of adenine, potentiated the cell-killing effect of FUra. On a molar basis (16-30 mumol/mouse), thymine, uracil, thymidine, and uridine enhanced FUra cytotoxicity more than a hundredfold. 2'-Deoxyuridine and the purine nucleosides and deoxynucleosides had similar potentiating activities between tenfold and fortyfold at 30 mumol per mouse. Finally, glucose, also administered 15 minutes before each mouse received an injection of 0.6 mg FUra, enhanced the antitumor activity of the drug by a factor of about five.

Schedule-dependent cytotoxicity of 5-fluorouracil in mice.

The cytotoxic effect of 5-fluorouracil (FUra) given as a 24-hour infusion against both AKR and L1210 leukemias was quantitated by spleen colony assays. The antitumor effect was enhanced in AKR leukemia but was reduced in L1210 leukemia when FUra was given as a 24-hour infusion compared to when the drug was given as an iv bolus injection. No difference in cytotoxicity was noted for these two schedules against normal hematopoietic stem cells in either AKR or (BALB/c X DBA/2)F1 mice. The pharmacokinetics of FUra given as an infusion was similar in the mice bearing these two transplantable leukemias, which ruled out the possibility that the pharmacokinetics of FUra was the cause of the difference observed. Finally, correlation was good between the spleen colony assays and the therapeutic effect defined by life-span assays for both leukemias.

MOPC-315 murine plasmacytoma as a model anticancer screen for human multiple myeloma.

The murine tumor MOPC-315 plasmacytoma was studied as a model for human multiple myeloma. Plasmacytoma cells (10(6)) were injected iv into BALB/c mice, and 14 days later a single ip dose of the anticancer agent to be tested was administered at a dose that would result in 10% toxicity within 30 days (LD10). Increases in life-span and cures resulting from the LD10 dose were the parameters assessed. The response of this MOPC-315 plasmacytoma model to a variety of anticancer agents demonstrated good correlation with clinically active agents. A number of investigational agents were found to be highly active and potential candidates for clinical phase II studies.

Potentiation of cytotoxicity of anticancer agents by several different polyene antibiotics.

The ability of several different polyenes to potentiate the cytotoxicity of carmustine, lomustine (LOM), and doxorubicin (DX) against AKR mouse leukemia was quantitated. All polyenes could potentiate the cytotoxicity of the anticancer agents; however, the methyl ester derivatives of the heptaenes amphotericin B and candicidin and the triene trienine were most effective with potentiations in the order of ten thousandfold for DX and LOM. A steep dose-response relationship for the polyenes was also noted. Neither the toxicity of the polyenes nor their level of potentiation could be correlated with structure or class.

Cellular quantitation of in vivo effects of 1-beta-D-arabinofuranosylcytosine on leukemia L1210.

We derived a cellular model for the use of the cytidine analogue 1-beta-D-arabinofuranosylcytosine (ara-C) against L1210 leukemia in vivo from dose- and time-survival studies. We employed a quantitative assay for leukemia colony-forming cells to construct dose- and time-survival curves for single, divided, and infused doses of ara-C. Time-survival curves for a large dose range of ara-C indicated not only cell killing but also progression delay effects in vivo. Divided dose studies showed the extent of cell killing (optimum effect) to be dependent upon both the dose and the interval of time between administration of the drugs. When the drug was given as an infusion, the extent of cell killing was as great as that produced by the best fractionation schedule, an effect which was verified in terms of therapeutic efficacy in leukemic mice.

Potentiation of anticancer agents by amphotericin B.

Amphotericin B (AmB) has been shown to potentiate the cytotoxicity of several different anticancer agents in AKR mice. The extent of potentiation, as assessed quantitatively by a spleen colony assay, varied from a factor of approximately 3 for 1,3-bis(2-chlorethyl)-1-nitrosourea (BCNU) to over 100 for a number of alkylating agents and to over 1,000 for 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. Most phase-specific agents showed little potentiation of cytotoxicity by AmB. A dose-response effect was noted with both BCNU and adriamycin, with increased potentiation being observed with increasing doses of the anticancer agents. The effect on increase in life-span was also studied and for most of the combinations it correlated with the potentiating effects disclosed by the spleen colony assay. Such a correlation was not found for AmB combined with either BCNU or vincristine.

Cytotoxicity of adriamycin combined with methotrexate against L1210 leukemia in mice.

The combination of adriamycin and methotrexate was found in mice to be synergistic in cytotoxicity to L1210 leukemia cells for a range of time intervals between the two agents. Cytotoxicity for normal hematopoietic stem cells was less than additive. Spleen colony assays were used to quantitate both cell populations. Increase in life-span assays yielded similar results. Also, the extent of synergy was dependent on the dose of adriamycin only.

Schedule-dependent potentiation of lomustine cytotoxicity by amphotericin B in mice.

The sensitivity of leukemia cells of AKR/J mice to subsequent lomustine [1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU)] exposure was found to vary with time following the ip administration of a single dose or multiple doses of amphotericin B (AmB) (0.5 mg/mouse/dose). Following 1 dose of AmB, leukemia cell sensitivity to CCNU, as measured by a spleen colony assay, reached a maximum by 24 hours with a tenfold increase in cell killing noted over cell killing with CCNU alone (0.15 mg/mouse). Two pretreatments with AmB separated by 24 hours led to a hundredfold increase in cell killing, whereas 4 pretreatments, each separated by 24 hours, gave more than a thousandfold increase in cytotoxicity by 8 hours, gave more than a thousandfold increase in cytotoxicity by 8 hours. Increasing the AmB dose, given as a single injection 24 hours before CCNU, resulted in increased potentiation of CCNU cytotoxicity, which reached a maximum at 0.5 mg/mouse. The kinetics of cell killing following either CCNU alone or the combination of AmB and CCNU were similar, although the extent of cell killing was greater with the combination. The AmB plasma pharmacokinetics for single doses showed a dose-dependent serum peak level and a half-life of about 24 hours for doses up to 1 mg/mouse. Nonlinearity was noted at the dose level of 2 mg/mouse because of saturation of absorption from the peritoneal cavity. These data are of importance for the optimal sequencing and dosing of these drugs for future clinical trials.

A murine model for central nervous system leukemia and its possible relevance to human leukemia.

Treatment of a transplantable leukemia in AKR mice with both amphotericin B and 1,3-Bis(2-chloroethyl)-1-nitrosourea cured a significant percentage of animals with advanced disease. Some long-term survivors developed paralysis, and they invariably demonstrated central nervous system (CNS) leukemia. Some of these animals had a systemic relapse of their leukemia, and the CNS appeared to act as a focus for systemic dissemination. The occurrence patterns and histopathologic features of the CNS leukemia in the long-term survivors were strikingly similar to those observed in humans with acute lymphoblastic leukemia.

Response of transplanted AKR leukemia to combination therapy with amphotericin B and 1,3-bis(2-chloroethyl)-1-nitrosourea: dose and schedule dependency.

A number of different amphotericin B (AmB)-1,3-bis(2 chloroethyl)-1-nitrosourea (BCNU) treatment regimens were evaluated with our model of transplantable AKR leukemia. We found that dose levels and treatment schedules were critical in determining the number of survivors. A 4-day treatment regimen of 0.5 mg AmB/mouse on days 1, 2, 3, and 4 and 0.2 mg BCNU/mouse on day 4 was found to be the most effective and has been chosen as our standard regimen. The efficacy of the treatment regimen depended on the presence of a large tumor burden, and the response was abolished when the mice were preirradiated or treated with the immunosuppressive agent, cyclophosphamide. These results, as well as others which we discuss, supported our notion that AmB affected host immune response to the tumor.

Protective effects of amphotericin B against spontaneous and transplantable murine tumors.

Two tumor systems were used to test prophylactic effects of amphotericin B (AmB). When 0.5 mg AmB was given ip every 2 weeks to AKR mice beginning at 8 weeks of age, the 50% tumor incidence for spontaneous lymphoma development was delayed 2-3 months. In the second tumor system, BALB/c mice received injections of either 20 or 50 mug AmB before receiving MOPC-315-C cells sc. The mice given the low dose of AmB demonstrated a decreased tumor incidence and a reduced tumor growth rate, when compared with controls. Opposite effects were found for the group administered the high dose; tumor incidence and rate of growth were increased.

Marked histoincompatibility between and within sublines of AKR mice used in a syngeneic leukemia model.

Sublines of AKR mice differed significantly in their histoincompatibility to AKR/J mice, as indicated by their ability to reject AKR/J skin grafts and an AKR transplantable leukemia. Further, all sublines tested demonstrated some degree of heterogeneity, because isografts were rejected in 5-97% of the mice, depending on the subline. The presence of excessive heterogeneity among and within sublines poses a serious problem for experimental oncologists using these sublines in their tumor models.

Amphotericin B potentiation of the cytotoxicity of anticancer agents against both normal hematopoietic and leukemia cells in mice.

Dose-response curves in AKR or CD2F1 mice were obtained for AKR leukemia, L1210 leukemia, and hematopoietic stem cells following in vivo administration of one of the following anticancer agents alone or in combination with amphotericin B (AmB): doxorubicin, lomustine, or melphalan. AmB potentiated drug cytotoxicity of all agents against both leukemias. Potentiation of all three agents was greatest with the AKR leukemia demonstrating dose-modifying factor (DMF) values of 1.8, 3.0, and 2.7, respectively, for lomustine, melphalan, and doxorubicin. L1210 leukemia was somewhat less sensitive to drug potentiation by AmB with respective DMF values of 1.8, 1.8, and 1.3. AmB did not potentiate the action of any agent against hematopoietic stem cells. The increased life-span assays generally confirmed the clonogenic assays supporting the validity of the latter results. The specificity of AmB potentiation of anticancer agent cytotoxicity for tumor cells indicates that AmB may be a useful addition to antitumor drug combinations.

Survival of mice bearing a transplanted syngeneic lymphoma following treatment with cyclophosphamide, 5-fluorouracil, or 1,3-bis(2-chloroethyl)-1-nitrosourea.

SUMMARY-A model has been derived for the effect of the chemotherapeutic agents cyclophosphamide (CY), 5-fluorouracil (5-FU), and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on the survival of mice bearing a transplantable lymphoma. The model is based on assumptions that 1) failures in treatment result from the survival of viable malignant cells, 2) the lymphoma cells in the host increase exponentially with time, 3) the host dies when the lymphoma cell population reaches a critical value, and 4) the number of lymphoma cells in the host decreases exponentially as a function of the infected drug dose. Parameters were found for the growth of lymphoma cells in vivo and for the survival of lymphoma cells in the host following the administration of CY, 5-FU, and BCNU. When these parameters were incorporated into the model, predictions were obtained concerning the survival time of lymphoma-bearing mice as well as the fraction of long-term survivors as a function of the injected dose. It was found that the model and experimental data were in excellent agreement for low doses of the drug, i.e., doses which reduced the survival of the lymphoma cell population to the order of 10(-4) survival. However, discrepancies were observed above these dose levels. The discrepancies are discussed in terms of the assumptions of the model.

Growth and rejection of leukemia cells in individual mice after combined treatment with amphotericin B and 1,3-bis(2-chloroethyl)-1-nitrosourea.

We assayed the femoral marrows of individual AKR mice for leukemia colony-units (LCFU) after treatment with amphotericin B (AmB) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or with BCNU alone. No differences between the groups were noted in the first 7 days after treatment. All the mice treated with BCNU alone were dead by day 8, and all the survivors among the animals receiving AmB and BCNU retained high levels of LCFU for 2 more days; these LCFU were subsequently rejected by the host. By day 12, LCFU were undetectable. Histologic examination of organs from the same mice on day 5 showed fewer leukemia cells in the mice treated with the combination of agents. In all treatment groups, mice dying of leukemia early (by day 9) had systemic leukemia and most had central nervous system (CNS) involvement. All animals dying between days 10 and 14 had CNS leukemia, but few had systemic leukemia; at later times, though few animals died, they invariably had CNS leukemia without systemic involvement.

Growth characteristics of MOPC-315 plasmacytoma and response to anticancer agents.

We examined the growth characteristics and response to anticancer agents of the murine plasmacytoma MOPC-315, a rapidly growing and widely disseminating tumor syngeneic in BALB/c mice. The doubling time for the tumor was approximately 24 hours, and about 1 in 100 cells was tumorigenic. We developed a spleen colony assay for the clonogenic component of the tumor and used it to define the dose-response relationships for various anticancer agents, including melphalan, cyclophosphamide, 1,3-bis(chloroethyl)-1-nitrosourea, 5-fluouracil, gamma-radiation, methotrexate, methylprednisolone, cytosine arabinoside, and vincristine.

Chemotherapy of L1210 leukemia with 5-azacytidine in combination with vincristine, adriamycin, or beta-cytosine arabinoside.

The cytotoxicity of vincristine (VCR), beta-cytosine arabinoside (Ara-C), or adriamycin (ADRIA) in combination with 5-azacytidine (Aza-CR) to L1210 leukemia in vivo was measured to determine if schedule-dependent synergistic or antagonistic drug interaction occurred. Two dose levels of Aza-CR were studied (0.1 and 0.5 mg/mouse), and cytotoxicity was measured by the spleen colony assay. For the combination of Aza-CR plus VCR, cytotoxicity was essentially additive or antagonistic when VCR preceded Aza-CR and additive or synergistic when VCR followed Aza-CR. When Aza-CR was combined simultaneously with either Ara-C or ADRIA, cytotoxicity was markedly antagonistic but was additive if drugs were given sequentially. When Ara-C was given as a 24-hour infusion before Aza-CR, the resultant cell kill was antagonistic (although slightly greater than that obtained for Ara-C alone). However, antagonism was even more marked when Aza-CR was given before the 24-hour infusion of Ara-C; cell kill was less than that observed with Ara-CR alone. Synergistic cytotoxicity was observed only with VCR administered after a low dose of of Aza-CR. Therefore, scheduling of drugs in combination with Aza-CR may be critical in the determination of the antileukemic cytotoxic effects.

Contrasting cytotoxicity kinetics of 5-azacytidine and dihydro-5-azacytidine hydrochloride in L1210 leukemia in mice.

For a comparison of the kinetics of the cytotoxicity of dihydro-5-azacytidine hydrochloride (DHAzaCR) and 5-azacytidine (AzaCR) in L1210 leukemia, the spleen colony assay was used to determine the surviving fraction of normal hematopoietic colony-forming units (NCFU) and leukemia colony-forming units (LCFU). Increasing doses of DHAzaCR above 1 mg per mouse enhanced cytotoxicity to LCFU but not to NCFU. The NCFU dose-survival curve showed a plateau with DHAzaCR, such that increasing the dose from 1 to 80 mg per mouse produced no further decrease in NCFU survival. In contrast, AzaCR produced a biphasic dose-NCFU survival curve without a plateau. Although DHAzaCR produced less cytotoxicity on a milligram basis than did AzaCR, both DHAzaCR and AzaCR elicited a biphasic dose-survival curve for LCFU. An infusion of DHAzaCR was less cytotoxic than was a similar dose of DHAzaCR administered as an iv bolus. Although high doses of AzaCR administered as an iv bolus. Although high doses of AzaCR delayed LCFU repopulation, both low and high doses of DHAzaCR were associated with prompt LCFU repopulation. Confirming this prompt repopulation of LCFU, there was a good correlation between the increase in life-span of mice with leukemia predicted by LCFU data following DHAzaCR treatment, compared to the discrepancy between predicted survival and observed survival following AzaCR. Therefore, the kinetics of cytotoxicity of DHAzaCR differ from those of AzaCR.

Effects of a low-fat diet on levels of oxidative damage to DNA to human peripheral nucleated blood cells.

Fat in the diet has been associated with increased breast cancer risk. In this study, blood samples were obtained from 21 women at high risk for breast cancer who had been randomly assigned to either a nonintervention diet or a low-fat diet. Oxidative damage was examined in the DNA from nucleated peripheral blood cells. The levels of oxidized thymine, specifically 5-hydroxymethyluracil, were threefold higher in the nonintervention diet group than in the low-fat diet group. Without regard to diet arm, there also was a significant linear relationship between daily total fat intake and 5-hydroxymethyluracil level. These results suggest that oxidative damage to DNA may be a marker of dietary fat intake. In addition, oxidative DNA damage may be a mechanistic link between fat in the diet and cancer risk, since such damage is associated with the process of tumor promotion.