Impairment of red blood cell deformability by tumor growth.

Red blood cell deformability is a major determinant of capillary blood flow. In mice with L1210 leukemia and with Lewis lung carcinoma, red blood cell deformability was significantly decreased during tumor growth. In mice with L1210 leukemia, deformability was significantly decreased by day 5 after transplantation and progressively decreased through day 9. Terminally, red blood cell deformability returned to normal or above normal values. In mice with Lewis lung carcinoma, significant decreases in deformability were noted 21--28 days after transplantation and persisted throughout the remainder of the tumor course. Impaired capillary blood flow, secondary to abnormal red blood cell deformability, is therefore associated with advanced cancer.     

Enhancement of the antitumor effect of 1,3-bis(2-chloroethyl)-l-nitrosourea (BCNU) by phenylethylbiguanide (phenformin).

Phenlethylbiguanide (phenformin) a commonly used antidiabetic medication has been found to enhance the antitumor effect of 1,3-bis(2-chloroethyl)-l-nitrosourea (BCNU) in advanced subcutaneously implanted murine L1210 leukemia. Enhancement required two doses of phenformin given twelve to 18 hours apart, the treatment starting either before or after BCNU administration. With BCNU alone median survival (MS) was 18 days with 5% cures. BCNU plus phenformin, in optimal dose and schedule, gave a MS of 25 days with 29% cures. Th mechanism of action of phenformin is unknown but may involve several established metabolic effects of this drug.     

Treatment of primary radiogenic C57BL mouse cell leukemia/lymphoma by 1,3-bis(2-chloroethyl)-1-nitrosourea chemotherapy and adjuvant cellular therapy

Primary radiation-induced or radiation leukemia virus (RadLV)-induced T-leukemias/lymphomas were treated in vivo in an early to advanced state by using 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). BCNU was given at various times after the tumor induction procedure. Death from RadLV lymphomas which had been initiated in 33 +/- 3 day old C57BL mice by intrathymic injection of RadLV was scored in untreated, BCNU-treated or BCNU and cellular adjuvant treated mice. Intrathymic RadLV injection in 33 +/- 3 day old mice produced tumors in 98% of injected mice. Median survival time (MST) was increased by BCNU and by BCNU plus bone marrow cell therapy whether done 33 or 47 days after RadLV. There was increased in MST from 108 days to 171 days by BCNU and bone marrow cell therapy given 33 days after tumor initiation and to 195 days when therapy was given 47 days after initiation. In radiation-induced lymphomas produced by 190 rad every week X 4 of 33 +/- 3 day old mice, spleen cell (X 1) therapy or BCNU treatment increased the MST of treated mice from 142 days to 177 days after iv spleen cells or to 195 days after iv-ip spleen cells, and this protocol produced 31% long-term cures. Cellular adjuvant therapy combined with BCNU chemotherapy was effective for curing the lymphomas but cellular adjuvant therapy alone was also highly effective for therapy.     

Chemotherapy-induced pulmonary toxicity in mice bearing L1210 leukemia

Fatal pulmonary toxicity can be consistently produced in L1210 leukemia-bearing mice by single therapeutic doses of cyclophosphamide, BCNU, and mitomycin C but not by adriamycin. Lung toxicity is principally determined by an existing tumor burden at the time of drug administration. Thus when any of the four chemotherapeutic agents was given 5 days after L1210 transplantation there was no mortality. Pulmonary pathology in these mice was equivalent to that noted in normal mice receiving identical drug treatment or to that noted in untreated L1210-bearing mice sacrificed 7, 8, or 10 days after tumor transplantation. When chemotherapy was delayed to day 7 after L1210 transplantation for mitomycin C or to day 8 after transplantation for BCNU and cyclophosphamide, more severe pulmonary toxicity was found. Mortality within the first 5 days of treatment was 38, 50, and 80%, respectively. Pulmonary pathology included moderate to severe vascular congestion and interstitial pneumonitis, diffuse pulmonary hemorrhage often involving the entire pulmonary parenchyma, pulmonary edema, and alveolar cell metaplasia. A unique finding, associated with cyclophosphamide treatment, was the occurrence of perivascular-intramural edema of the walls of medium-size pulmonary vessels. It is hypothesized that stasis within the pulmonary capillary circulation, resulting from advanced tumor growth and from drug treatment, may contribute to the development of chemotherapy-related toxicity.     

Combination chemotherapy with AMSA on L1210 leukaemia and B16 melanoma

Clinical interest in AMSA, [4′-9(acridinylamino)methanesulfon-m-anisididel has grown recently because it has shown remarkable activity in the treatment of adult myelogenous acute leukaemia. In the present investigation on two experimental murine tumours, namely L1210 leukaemia and B16 melanoma, m-AMSA was administered in association with BCNU, melphalan (MLP) and DTIC. The therapeutic activity of the AMSA-MLP-BCNU combination in murine L1210 leukaemia was compared to that of the MLPBCNU-DTIC combination. These two associations proved to be very active (30% cures were recorded on day 60). The dose levels of the drugs used in the combination represent only 30–50% of the optimal dose of the same drugs given separately. When DTIC was added to the AMSA-MLP-BCNU combination, considerable lengthening of survival time as well as up to 7 cures out of 10 mice on day 60 were recorded. However, none of the combination chemotherapy was able to induce 10 cures (out of 10 mice) like treatment with BCNU, at optimal dose.     

Correlation of the in vitro cytotoxicity of ethyldeshydroxysparsomycin and cisplatin with the in vivo antitumour activity in murine L1210 leukaemia and two resistant L1210 sublcones

Summary The cultured murine leukaemia L1210 cell populations used in the present study were derived from L1210 cells that had been grown in vivo. Subclones resistant to sparsomycin (L1210/Sm) or cisplatin (L1210/CDDP) were also developed in vivo. The doubling times of the cultured cell populations were identical. Fractions surviving after durg treatment in vitro were determined by colony formation in soft agar. The results, based on the differential sensitivity of the cell populations to ethyldeshydroxysparsomycin (EdSm) and CDDP, indicated that after a short exposure, cultured L1210/CDDP cells were cross-resistant to EdSm. L1210/Sm cells, however, were not cross-resistant to CDDP. The results obtained in cultured cell populations were confirmed in vivo. CD2f₁ mice bearing i.p. implants of 1×10⁵ tumour cells were given EdSm or CDDP and a combination of the two agents. Drugs were given once daily every 4 days for 3 doses starting at 24 h after tumour implantation. Treatment of mice bearing L1210/wt leukaemia with combined EdSm and CDDP caused strongly synergistic amtitumour activity. In animals bearing the two resistant subclones, however, combined drug treatment did not improve the antitumour activity. The corresponding median survival of mice receiving combined drug treatment was 60 days in each group containing 6 mice bearing L1210/wt, with 4–6 cures being noted; 19 days in animals harbouring L1210/Sm, with 2 cures being recorded among 6 mice; and 11 days in mice bearing L1210/CDDP, with no cure being obtained. The results of this study indicate that the synergism resulting from combined treatment with CDDP and EdSm is a function of the cellular properties of the target tumour-cell populations and is independent of host factors.     

An experimental model for meningeal leukemia in rats (L5222). Effect of treatment with BCNU and cyclophosphamide

An experimental model of meningeal leukemia in rats is developed by intracerebral (IC) inoculation of leukemic cells from the transplantable acute leukemia L5222. The L5222 proliferates exponentially in the central nervous system (CNS) and the disease becomes systemic 2 days following IC inoculation. Chemotherapeutic studies with BCNU and cyclophosphamide yielded cures in a high percentage of cases when treatment began at an early stage of meningeal leukemia. When treatment was started at the advanced stage, only BCNU showed a large number of cures. However, cyclophosphamide resulted in a marked increase of life-span. The activity of cyclophosphamide against meningeal leukemia, which is in contrast to the results obtained by Skipper et al. (1961) in the L1210 mouse leukemia, suggests that cyclophosphamide crosses in part the blood—brain barrier in a rat bearing meningeal leukemia. After subcutaneous inoculation, BCNU and cyclophosphamide showed the same rate of cures.     

Experimental chemotherapy (L1210) with 5-aza-2'-deoxycytidine in combination with pyran copolymer (MVE-4), an immune adjuvant

The life-span of CDF1 (BALB/c X DBA/2)F1 mice that received intraperitoneal implants with 10(5) L1210 tumor cells was prolonged to 23 days (compared to 8 days in L1210 tumor-implanted, untreated mice) when 5-aza-2'-deoxycytidine (DAC) was given to the mice after the tumor cells were allowed to metastasize (3 days after implant); DAC, however, resulted in no cures (survival beyond 48 days). When the pyran copolymer MVE-4, an immune adjuvant, was given the day after DAC, 25% of the mice treated were cured and the life-span of dying mice was increased by 7 days. When MVE-4 was repeated weekly for 4 weeks, 79% of treated mice were cured. Cured mice were able to resist a subsequent challenge of approximately 2 logs of L1210 cells. This combination of DAC plus MVE-4 was more effective than DAC alone only if the tumor cells and MVE-4 were given intraperitoneally. When this combination was repeated weekly, it became lethally toxic after 3 weeks, but only to L1210-tumor-bearing mice and not to normal mice. When DAC alone was given 2 days before tumor implant, it induced an apparent immune effect so that mice could resist a subsequent challenge of approximately 1.5-2 logs of L1210 cells. Support for part of the antitumor action of DAC exerted through the immune system was given by data that show that later treatment with noncurative doses of DAC is superior to early treatment in mice with large L1210 tumor burdens.     

Eradication of a disseminated mouse lymphoma by 1,3-bis(2-chloroethyl)-1-nitrosourea is immunologically mediated and accompanied by de novo generation of anti-tumor cytotoxicity

The anti-tumor effect of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was examined in BALB/c mice bearing increasing burdens of a syngeneic lymphoma (YC8). A single i.p. injection of the drug resulted in over 75% of cures when given at day 3, 5, 7 or 10 after an i.v. inoculum of 10(4) YC8 cells. The efficacy of BCNU on mice bearing large tumor burdens (from day 5 on) was not only due to its tumoricidal activity, but was immunologically mediated. Residual tumorigenic cells could be recovered in the livers of 5-day tumor bearers (TB) up to 2 weeks after BCNU treatment and only a low percentage of cures could be achieved when BCNU was administered to nude mice. In addition, BCNU-cured mice specifically rejected a lethal YC8 challenge and their splenocytes developed anti-tumor cytotoxicity in response to in vitro stimulation with YC8 cells. During kinetic experiments a 2-week period elapsed after BCNU injection before an anti-tumor cytotoxic T-lymphocyte (CTL) response could be generated by spleen cells of BCNU-treated 5-day TB. This period was characterized by immunosuppression as evaluated from impairment in the generation of lymphokine-activated killer (LAK) cells or of allospecific primary CTL responses by spleen cells from BCNU-treated 5-day TB and BCNU-treated normal mice. LAK cells first recovered and could be generated 7 days later, whereas primary allospecific CTL responses could only be detected by day 14, concomitantly with the generation of anti-tumor cytotoxicity by 5-day TB. The development of secondary in vitro CTL responses, however, was permanently abrogated. Spleen cells from BALB/c mice immunized either with YC8 or with DBA/2 minor histocompatibility antigens and treated with BCNU 1 week after the last immunization failed to mount an in vitro CTL response to their immunizing antigen, even when the cultures were supplemented with recombinant interleukin-2.     

Superiority ofMicrococcus lysodeikticus to BCG in chemo-immunotherapy of advanced L1210 leukaemia

When CDF₁ mice initially inoculated with10⁵ leukaemic cells were treated once by12 mg/kg of nitroso-urea BCNU onday 6.5 (when the tumour burden reaches >10⁸ cells) and by1 mg Micrococcus injections ondays 8, 11, 14, 17 and20 or ondays 8, 9, 10, 11 and12, 30% of cured mice were recorded. Immunotherapy with1 mg injections of BCG,B. subtilis orMicrococcus lysodeikticus given ondays 8, 11, 14, 17 and20 after BCNU chemotherapy (one12 mg/kg injection onday 6.5) respectively cured5, 10 and50% of leukaemic mice after a graft of4000 L1210 cells onday 0 and0, 10 and50% of leukaemic mice after a graft of25,000 cells. Using this chemo-immunotherapy combination, Micrococcus immunotherapy proved to be significantly effective (x² test, P < 0.005) against >10⁸ L1210 cells whereasB. subtilis or BCG immunotherapy was not statistically significant. Mice cured by chemo-immunotherapy (BCNU + Micrococcus) rejected new local as well as systemic grafts of10⁴ L1210 cells (onday 120).     

局部与常规化疗联合放疗治疗颅内恶性肿瘤的探讨

目的 :分析颅内恶性肿瘤术后局部化疗加放疗与常规化疗加放疗的疗效。方法 :随机分两组 ,局部化疗组 2 2例。两组放疗DT60～ 70Gy。局部化疗组术中放置化疗泵 ,术后将卡氮芥 (BCNU) 12 5mg注入化疗泵 ;常规组将BCNU 12 5mg与生理盐水混合后静脉输入。两组病人均为术后 6～ 12天开始化疗 ,连用 2天 ,9～ 13天后行放疗 ,放疗结束后再化疗 1～ 3次。结果 :局部化疗组平均生存期为 2 9 5个月 ,常规组 2 1 3个月 ,其中胶质母细胞效果最好 ,局部化疗副作用轻微。结论 :局部化疗加放疗比常规加放疗治疗颅内恶性肿瘤效果更满意 ,并且局部化疗更直接 ,副作用更小     

Severe depletion of cellular thiols and glutathione-related enzymes of a carmustine-resistant L1210 strain associates with collateral sensitivity to cyclophosphamide

Cyclophosphamide (CPA) increased the life span of both carmustine (BCNU)-resistant (L1210/BCNU) and BCNU-sensitive L1210 (L1210/0) leukaemic mice; their sensitivity to CPA, however, was extremely different. The BCNU-resistant strain was much more sensitive (collaterally) to CPA than was its sensitive counterpart. The collateral sensitivity was accompanied by a severe reduction in the activity of glutathione-related enzymes and in protein thiol (SH) and non-protein SH levels in BCNU-resistant cells. The activity of glutathione reductase (GSSG-R) was 2 times higher in the L1210/0 cells than in the L1210/BCNU cells. Glutathione-S-transferase (GST) was also almost 2 times more active in the sensitive cells than in the resistant strain. To develop resistance against CPA with a single treatment (60 mg/kg) per passage, the L1210/BCNU strain needed 26 passages, whereas the L1210/0 strain required significantly fewer. The resistance developed against CPA was associated with a moderate elevation of thiols in the L1210/CPA cells, whereas this elevation was approximately 3 times more pronounced in the L1210/BCNU/CPA cells. The severely reduced activity of GST in the L1210/BCNU strain was markedly increased when these cells were made resistant to CPA; the GSSG-R activity, however, remained low, suggesting an irreversible injury of this enzyme by BCNU.     

Severe depletion of cellular thiols and glutathione-related enzymes of a carmustine-resistant L1210 strain associates with collateral sensitivity to cyclophosphamide

Cyclophosphamide (CPA) increased the life span of both carmustine (BCNU)-resistant (L1210/BCNU) and BCNU-sensitive L1210 (L1210/0) leukaemic mice; their sensitivity to CPA, however, was extremely different. The BCNU-resistant strain was much more sensitive (collaterally) to CPA than was its sensitive counterpart. The collateral sensitivity was accompanied by a severe reduction in the activity of glutathione-related enzymes and in protein thiol (SH) and non-protein SH levels in BCNU-resistant cells. The activity of glutathione reductase (GSSG-R) was 2 times higher in the L1210/0 cells than in the L1210/BCNU cells. Glutathione-S-transferase (GST) was also almost 2 times more active in the sensitive cells than in the resistant strain. To develop resistance against CPA with a single treatment (60 mg/kg) per passage, the L1210/BCNU strain needed 26 passages, whereas the L1210/0 strain required significantly fewer. The resistance developed against CPA was associated with a moderate elevation of thiols in the L1210/CPA cells, whereas this elevation was approximately 3 times more pronounced in the L1210/BCNU/CPA cells. The severely reduced activity of GST in the L1210/BCNU strain was markedly increased when these cells were made resistant to CPA; the GSSG-R activity, however, remained low, suggesting an irreversible injury of this enzyme by BCNU.     

Augmentation of Cancer Chemotherapy by Preinjection of Human Macrophage Colony-stimulating Factor in L1210 Leukemic Cell-inoculated Mice

Human macrophage colony-stimulating factor (hM-CSF) is a potent stimulator of the effector functions of monocytes/macrophages. We investigated the antitumor effects of this factor in CDF₁ male mice inoculated with L1210 cells, a mouse B-cell leukemia line. Mice preinoculated with various numbers of L1210 cells on day 0 were given intravenous injections of vehicle (human serum albumin; HSA) (100μg/kg/day) or hM-CSF (20μg/kg/day) for 3 days from day 1. In mice preinoculated with 10² L1210 cells but not with 10³ or more L1210 cells, a marked increment in survival rate was observed with hM-CSF treatment. We next examined the effect of hM-CSF treatment combined with chemotherapy on the survival of mice that had been preinoculated with 10⁵ L1210 cells. In our system, the administration of 4.9 mg/kg adriamycin (ADM) alone slightly prolonged survival of the tumorbearing mice, but all of the mice died within 20 days. When hM-CSF was injected for 3 days before this ADM treatment, the invasion and proliferation of tumor cells in the liver and spleen were markedly inhibited and 50% of the mice were still alive at day 50. We detected inhibitory activity toward L1210 growth in serum of mice administered with hM-CSF, and the degree of the inhibitory activity was correlated with the level of nitrite (NO₂) in the serum. When L1210 cells were co-cultured with peritoneal macrophages from mice intraperitoneally injected with hM-CSF, the uptake of [³H]thymidine in L1210 cells was inhibited. The inhibition was abolished by the addition of N^G-monomethyI-L-arginine, an inhibitor of NO₂− synthesis, suggesting that the reactive nitrogen oxide intermediate is involved in hM-CSF-induced inhibition of L1210 growth.     

Participation of T-lymphocytes in the curative effect of a novel synthetic polyamine analogue, N,N'-bis[3-(ethylamino)propyl]-1,7-heptanediamine, against L1210 leukemia in vivo

We have recently established that combination therapy with N,N'-bis[3-(ethylamino)propyl]-1,7-heptanediamine (BEPH), a synthetic polyamine analogue, and N,N'-bis-2,3-butadieneyl-putrescine, a polyamine oxidase inhibitor, eradicated L1210 leukemia in mice and induced resistance to a subsequent L1210 challenge. We now demonstrate that BEPH treatment alone, given on a more frequent schedule (5 mg/kg, day 3, 4, 5) or at a higher dose (10 mg/kg, day 3, 4), cures 100% of L1210 leukemic mice. These treated animals were subsequently immune to a second challenge with L1210 tumor cells. However, mice cured with BEPH did not reject P388 leukemic cells, although their mean survival time was slightly prolonged. In an in vivo tumor neutralization assay, splenocytes from cured mice and L1210 cells were injected into naive mice; 80% did not develop L1210 leukemia. Coculturing lymphocytes from cured mice with L1210 cells in vitro generated a potent tumor-specific cytolytic response against L1210 target cells, whereas lymphocytes from naive mice did not generate any significant cytolytic activity. Both the in vitro and in vivo activities were completely eliminated by pretreating the splenic lymphocyte population with anti-Thy-1.2 monoclonal antibodies and complement, indicating T-cells as the effector population. In T-cell-deficient nude mice BEPH treatment was not curative, increasing survival time by approximately 2-fold. We conclude from these studies that T-cell-mediated immunity plays a pivotal role in the mechanism by which synthetic polyamine analogues, such as BEPH, prevent neoplastic growth.     

Equivalent chemotherapy efficacy against leukemia in mice treated with topical vasoconstrictors to prevent cancer therapy side effects

Topically applied vasoconstrictor is a new strategy to prevent oral mucositis and alopecia, two complications of chemotherapy and stem‐cell transplant. We sought to determine whether mice treated with topical vasoconstrictor minutes before chemotherapy to suppress L1210 leukemia would develop a vasoconstrictor‐induced L1210 cell sanctuary, and with it, significantly worse survival outcomes. B6D2F1 mice received 10⁴ mouse L1210 leukemia cells via retro‐orbital intravenous injection and were then divided into treatment groups, which included: (i) no further treatment, (ii) a single, sub‐curative, intraperitoneal dose of cyclophosphamide (90 µg/gm bw) 24 hr after L1210 cell inoculation, (iii) topical epinephrine (25–400 mM) to clipped dorsal backs 20 min before cyclophosphamide or (iv) orotopical phenylephrine (16–130 mM), epinephrine (10 mM) or norepinephrine (25 mM) 20 min before cyclophosphamide. All mice were then followed until day of death. Differences in median survival time and percent survival between mice receiving cyclophosphamide alone and mice treated with either orotopical phenylephrine, epinephrine or norepinephrine; or topical epinephrine before cyclophosphamide were not significantly different. A discernible leukemia sanctuary was not created by topical vasoconstrictor treatment prior to chemotherapy; there was no significant difference in leukemia progression between untreated mice and those treated with either orotopical or topical vasoconstrictor before chemotherapy. We have opened a Phase I/IIa dose escalation trial to evaluate the safety and efficacy of orotopical phenylephrine in preventing oral mucositis in subjects undergoing hematopoietic stem cell transplant conditioning with cyclophosphamide plus total body irradiation. This could provide a cost‐effective and convenient method to prevent oral mucositis.     

In vitro and in vivo methazolastone-induced DNA damage and repair in L-1210 leukemia sensitive and resistant to chloroethylnitrosoureas

DNA damage caused by methazolastone [an analogue of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide which does not require metabolic activation] was investigated in L-1210 leukemia which is sensitive to this drug and in a L-1210 subline [L-1210/N,N-bis(2-chloroethyl)-N-nitrosourea (BCNU)] which is resistant to both chloroethylnitrosoureas and methyltriazenes. Both in vitro and in vivo metazolastone caused formation of DNA alkali-labile sites (assessed by alkaline elution techniques) which were present in similar amounts and repaired at a similar rate in L-1210 and L-1210/BCNU. This suggests that these lesions are not crucial to methyltriazenes activity. DNA alkali-labile sites may be due to the removal of 7-methylguanine by 7-methylguanine-DNA glycosylase which showed the same activity in L-1210 and L-1210/BCNU. Flow cytometry studies revealed that in L-1210 but not in L-1210/BCNU methazolastone induced an arrest of cells in SL-G2-M phases. This blockade was delayed, occurring after at least two cell divisions after drug treatment and therefore appeared temporally unrelated to the presence of DNA alkali-labile sites. There was three times more O6-methylguanine-DNA methyltransferase in L-1210/BCNU than in L-1210 suggesting that methylation of O6-guanine is an important lesion for methyltriazenes activity and resistance to this drug may be linked to its repair.     

Direct reversal of DNA damage by mutant methyltransferase protein protects mice against dose-intensified chemotherapy and leads to in vivo selection of hematopoietic stem cells

Direct reversal of O6 adducts caused by chemotherapy agents is accomplished in mammalian cells by the protein O6-methylguanine DNA methyltransferase (MGMT). Some tumors overexpress MGMT and are resistant to alkylator therapy. One future approach to treatment of these tumors may rely on concurrent pharmacological depletion of tumor MGMT with O6-benzylguanine (6-BG) and protection of sensitive tissues, such as hematopoietic stem and progenitor cells, using genetic modification with 6-BG-resistant MGMT mutants. We have used retroviral-mediated gene transfer to transduce murine hematopoietic bone marrow cells with MGMT point mutants showing resistance to 6-BG depletion in vitro. These mutants include proline to alanine and proline to lysine substitutions at the 140 position (P140A and P140K, respectively), which show 40- and 1000-fold resistance to 6-BG compared with wild-type (WT) MGMT. Lethally irradiated mice were reconstituted with murine stem cells transduced with murine stem cell virus retrovirus expressing each mutant, WT MGMT, or mock-infected cells and then treated with a combination of 30 mg/kg 6-BG and 10 mg/kg 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or with 40 mg/kg BCNU alone. Compared with mice treated with BCNU alone, significant myeloid toxicity and death occurred in mice reconstituted with mock-infected or WT MGMT (<0.1 probability of survival) or the P140A mutant (0.13 probability of survival) MGMT cDNAs. In contrast, after an initial period of mild cytopenia, mice reconstituted with the P140K mutant (0.83 probability of survival) recovered nearly normal blood counts, even during continued treatment. Comparison of peripheral blood neutrophils after completion of 5 weekly treatments in these animals showed a direct correlation between the treatment and in vivo selection for progeny of transduced cells (pretreatment, approximately 8-12% transduced cells; no treatment, approximately 6% transduced cells; BCNU only, 51% transduced cells; 6-BG/BCNU, 93% transduced cells). To determine whether this selection occurred at the stem cell level, bone marrow from each treatment group was infused into secondary recipients. Whereas animals that received bone marrow from untreated animals reconstituted with 2% transduced cells, animals receiving marrow from 6-BG/BCNU-treated animals reconstituted with 94% transduced cells, demonstrating nearly complete selection for stem cells in the primary animals. Mice reconstituted with marrow from animals treated with BCNU only demonstrated 23% transduced cells, consistent with partial selection of stem cells in the primary mice. The levels of transduced cells also correlated with survival during a second round of intensive combination chemotherapy (probability of survival: 6-BG/BCNU, 1.0; BCNU alone, >0.70; no treatment, <0.1). These data demonstrate that mutant MGMT expressed in the bone marrow can protect mice from time- and dose-intensive chemotherapy and that the combination of 6-BG and BCNU leads to uniform selection of transduced stem cells in vivo in mice.     

Sister chromatid exchange induction in patients with anaplastic gliomas undergoing treatment with radiation plus diaziquone or 1,3-bis(2-chloroethyl)-1-nitrosourea

Diaziquone (AZQ) (NSC 182986), a lipid-soluble benzoquinone derivative, is presently being tested in a Phase III clinical trial to determine its efficacy in patients with anaplastic gliomas compared to the more standard 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment following whole-brain irradiation. These patients on single-drug chemotherapy allowed us to evaluate the effects of each agent on sister chromatid exchange (SCE) induction in vivo. Eight weeks following the final radiation treatment, patients were randomly assigned to one of two groups: (a) 200 mg BCNU/m2, i.v., every 8 weeks; of (b) 15 mg AZQ/m2/day, i.v., for 3 consecutive days, every 4 weeks. Blood (5-10 ml) was drawn by venipuncture before treatment, within 10 h after treatment, and for two BCNU-treated patients at various other times. Peripheral blood lymphocytes were cultured by standard techniques for analysis of SCE. Eight weeks after irradiation but before chemotherapy, the mean SCE frequency in the patients' peripheral blood lymphocytes was 9.6 SCEs/metaphase. Following treatment with AZQ or BCNU, the baseline SCE frequency was increased more than 2-fold or 3-fold, respectively. Two months after BCNU treatment, there was less than a 25% reduction in SCE levels compared to samples taken and cultured within 10 h after treatment. These data show that lesions leading to SCE in human peripheral blood lymphocytes are relatively longlived, and that on a mg/m2 basis, AZQ is a more potent inducer of SCE in vivo than is BCNU.     

In vivo response of KHT sarcomas to combination chemotherapy with radiosensitizers and BCNU.

Female C3H/HeJ mice bearing intramuscularly transplanted KHT sarcomas were treated with a single dose of 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU, 30 mg/kg, i.p.) alone or in combination with a single dose of misonidazole (MISO, 1.0 mg/g, i.p.) or its desmethylated metabolite Ro-05-9963 (2.0 mg/g, i.p.). The effectiveness of drug therapy was assessed by a tumour growth-delay assay (i.e. measuring the median time required for tumours to grow to treatment size x 4). The relative efficacy of administering the nitroimidazoles in various schedules ranging from 12 h before to 12 h after BCNU administration also was evaluated. Untreated control KHT tumours grew to the initial size x 4 in a median time of 4 days. No significant growth delay was seen in mice treated with either nitroimidazole alone, whilst treatment with BCNU alone produced a median growth delay of 7 days. Combination chemotherapy with 9963 administration 3 h after BCNU significantly increased the median tumour growth delay to 9 days. However, no significant growth delay was produced in any of the other combinations of these agents. The median growth delay was significantly reduced to 5 days when MISO was administered 3 h before BCNU, whereas MISO administered simultaneously 3,6, or 12 h after BCNU significantly enhanced delays ( 9 days). These results indicate that both MISO and 0063 may be combined with conventional therapeutic agents, in this particular case a nitrosourea, to produce an enhanced tumour response. The production of such a response appears to be nitroimidazole as well as schedule dependent.