melphalan and tallimustine

We investigated whether recombinant human (rHu-IFN-beta) (IFN-beta) could counteract the inhibition of natural killer (NK) activity caused by antitumor agents. Peripheral blood lymphocytes (PBL) were incubated with different antitumor agents alone or in combination with IFN-beta for 3 days and then tested in a cytotoxicity assay against the K562 cell line. The following drugs were used, all of which caused a dose-dependent inhibition of NK activity: etoposide, camptothecin, doxorubicin, cis-DDP, tallimustine, and L-PAM. Concomitant treatment with (1000 U/ml) IFN-beta counteracted the inhibitory effect of etoposide and camptothecin but had no consistent effect on the inhibition mediated by the other drugs. Mean values of inhibition of NK activity at 1 microM camptothecin was 48%+/-3.4% and with IFN-beta was 10%+/-4.9%. With 100 microM etoposide, mean value of inhibition was 78%+/-3.3%, and with IFN-beta, it was 18%+/-1.5%. Cell viability, assessed by vital dye exclusion, and drug uptake, assessed with radiolabeled etoposide, were similar in cells treated with or without IFN-beta. The protective effect of IFN-beta on NK function was rather selective, as IFN-beta did not counteract the drug-mediated inhibition of PBL proliferation when stimulated by phytohemagglutinin (PHA). Other cytokines, IFN-alpha, IFN-gamma, and interleukin-2 (IL-2), had similar protective effect, although IFN-beta, was slightly more potent. On the other hand, IL-6, a cytokine sharing some properties with IFNs was ineffective. Camptothecin inhibited the expression of mRNA for granzyme B, a lytic protein involved in lymphoid-mediated cytotoxicity. Combined treatment with IFN-beta restored-at least in part-the transcription of granzyme B mRNA. These results show that the immunosuppressive effect of some antitumor agents could be partly counteracted by treatment with IFN-beta.

Topics: Antineoplastic Agents; Camptothecin; Cisplatin; Cytotoxicity Tests, Immunologic; Distamycins; Doxorubicin; Etoposide; Immunosuppressive Agents; Interferon-beta; Killer Cells, Natural; Melphalan; Nitrogen Mustard Compounds; Recombinant Proteins

The benzoyl nitrogen mustard derivative of distamycin A, tallimustine, belongs to a new class of alkylating agents, known as DNA minor groove alkylating agents. It alkylates adenine N3 with high sequence specificity, causing no alkylation of guanine N7, the main site of alkylation of clinically used nitrogen mustards such as L-PAM. The present study investigated the in vivo antitumour activity of a combination of tallimustine and melphalan (L-PAM). Two murine tumours were used: i.p. (intraperitoneally) transplanted L1210 leukaemia and i.m. (intramuscularly) transplanted M5076 ovarian reticulum cell sarcoma (M5). In L1210, which is only marginally sensitive to tallimustine, the combination of tallimustine 3 mg/kg i.p. with L-PAM 10 mg/kg i.p. was as effective as 20 mg/kg L-PAM, which is the maximum tolerated dose. In M5, which is sensitive to both drugs, the combination was superior to either drug alone. The results suggest that the combination of tallimustine and L-PAM--or possibly in general, minor groove alkylators and major groove alkylators--may be therapeutically advantageous and therefore should be investigated clinically.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Distamycins; Female; Leukemia L1210; Lymphoma, Large B-Cell, Diffuse; Male; Melphalan; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Neoplasm Transplantation; Nitrogen Mustard Compounds; Ovarian Neoplasms; Survival Rate

Some new alkylating agents which bind to the minor groove of DNA and have sequence-specific patterns of alkylation have shown anti-neoplastic activity in pre-clinical systems. Two of them, carzelesin and tallimustine, are now in phase II. Considering the severe dose-limiting bone marrow toxicity of both these drugs in clinical use, it was of interest to investigate the mechanism of their myelotoxicity in a detailed pre-clinical study and compare it with a conventional alkylating agent, such as melphalan. The origin and progression of the myelotoxicity of carzelesin, tallimustine and melphalan were investigated comparatively in mice, combining data on bone marrow and peripheral blood cellularity with data on the proliferative activity of bone marrow cells, obtained by in vivo administration of bromodeoxyuridine. Significant differences were found between the hematopoietic response to the 3 drugs, though all caused severe leukopenia. Carzelesin induced a short-term increase in myeloid proliferative activity, which prevented the high leukocytopenia on day 3 observed with the other drugs. However, when this effect was exhausted, a second nadir was seen in peripheral blood, with a new wave of cell proliferation of all lineages in the bone marrow. Reconstruction of the lymphoid lineage was slow for all 3 drugs but particularly difficult with high-dose tallimustine. In general, the hematopoietic system response to tallimustine was dampened, with no overshoots, suggesting either lasting effects or extensive cytotoxicity from the early to late precursors of all lineages.

Topics: Animals; Antineoplastic Agents, Alkylating; Benzofurans; Body Weight; Bone Marrow Cells; Cell Cycle; Cell Division; Distamycins; Duocarmycins; Flow Cytometry; Indoles; Leukocyte Count; Male; Melphalan; Mice; Mice, Inbred Strains; Neutropenia; Nitrogen Mustard Compounds; Survival Rate; Thrombocytopenia

MEN 10710 is a new synthetic distamycin derivative possessing four pyrrole rings and a bis-(2-chloroethyl)aminophenyl moiety linked to the oligopyrrole backbone by a flexible butanamido chain. Its biological properties have been investigated in comparison with the structurally related compound, tallimustine (FCE24517), and the classical alkylating agent, melphalan (L-PAM). Cytotoxic potency of MEN 10710 was increased from 10- to 100-fold, as compared to tallimustine or L-PAM in murine L1210, human LoVo and MCF7 tumor cell lines. MEN 10710 was still active against L1210/L-PAM leukemic cells, while a partial cross-resistance was observed in LoVo/DX and in MCF7/DX cells selected for resistance to doxorubicin and expressing a MDR phenotype. Treatment with verapamil (VRP) reduced the resistance to tallimustine, but not to MEN 10710, in MCF7/DX cells. The cytotoxic effects reflect in vivo antitumor potency and toxicity in the treatment of human tumor xenografts. MEN 10710 was more effective in A2780/DDP, an ovarian carcinoma selected for resistance to cisplatin. On the other hand, the IC30 for inhibiting murine granulocyte/macrophage colony formation was 50 times higher for MEN 10710 than for tallimustine, suggesting a lower myelotoxic potential. In conclusion, the particular biological profile of MEN 10710 characterized by a marked cytotoxic potency, an interesting antitumor efficacy and a reduced in vitro myelosuppressive action may represent a further improvement in the rational design of a novel distamycin-related alkylating compound.

Topics: Animals; Antineoplastic Agents; Antineoplastic Agents, Alkylating; Calcium Channel Blockers; Cell Survival; Distamycins; Humans; Leukemia L1210; Melphalan; Mice; Neoplasm Transplantation; Neoplasms, Experimental; Neoplastic Stem Cells; Nitrogen Mustard Compounds; Organ Size; Tumor Cells, Cultured; Verapamil

The activities and the expression of 3-methyladenine glycosylase (3-meAde gly) and O6-alkylguanine-DNA-alkyltransferase (O6 ATase) were investigated in ten human cancer cell lines. Both 3-meAde gly and O6 ATase activities were variable among different cell lines. mRNA levels of the O6 ATase gene, appeared to be related to the content of O6 ATase in different cell lines, whereas no apparent correlation was found between mRNA of 3-meAde gly and the enzyme activity. No correlation was found between the activity of the two enzymes and the sensitivity to alkylating agents of different structures such as CC-1065, tallimustine, dimethylsulphate (DMSO), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), cis-diamminedichloroplatinum (cDDP) and melphalan (L-PAM). The most striking finding of this study is that a correlation exists between the activity of O6 ATase and 3-meAde gly in the various cell lines investigated (P<0.01), suggesting a common mechanism of regulation of two DNA repair enzymes.

Topics: Alkylating Agents; Antineoplastic Agents, Alkylating; Carcinogens; Cell Division; Cisplatin; Distamycins; DNA Glycosylases; Humans; Melphalan; Methylnitronitrosoguanidine; Methyltransferases; N-Glycosyl Hydrolases; Neoplasms; Nitrogen Mustard Compounds; O(6)-Methylguanine-DNA Methyltransferase; RNA, Messenger; Sensitivity and Specificity; Sulfuric Acid Esters; Tumor Cells, Cultured

Alkylating distamycin derivative FCE-24517 (l) is the prototype of a novel class of alkylating agents. In the present study we have investigated the effect of further chemical modifications introduced in the alkylating distamycin-derived molecule with the aim of improving their ability to bind DNA. The new compound, MEN 10710 (II), has a four pyrrolecarboxamide backbone linked at its N-terminus and through a butanamido residue to a 4-[bis(chloroethyl)amino]phenyl moiety. We have demonstrated that the presence of the flexible trimethylene chain confers to the novel distamycin derivative a peculiar mode of interaction with DNA as compared with I or melphalan. In fact, interstrand cross-links are detected in DNA samples treated even with low concentrations of II (being 200-fold more efficient than melphalan) but not with I. Similar results were obtained with a related compound of II containing a three pyrrole ring backbone. Compound II induces a conformational change in the DNA structure as deduced from the inhibition of T4 DNA ligase activity. In alkylation experiments, unlike melphalan, both I and II induce DNA breaks at bases closely located to AT-rich tracts, however II was more potent than I in producing greater amount of covalent adducts. These data suggest that the new compound shows a different and peculiar mechanism of interaction with DNA.

Topics: Alkylation; Base Sequence; Cross-Linking Reagents; Distamycins; DNA; DNA Adducts; DNA Ligases; DNA, Superhelical; Enzyme Inhibitors; Melphalan; Molecular Sequence Data; Nitrogen Mustard Compounds; Nucleic Acid Conformation

Nucleotide excision repair (NER) is one of the major DNA repair systems in mammalian cells, able to remove a broad spectrum of unrelated lesions. In this report the role of ERCC (excision repair cross-complementing) 1, ERCC2, ERCC3, ERCC4, and ERCC6 genes in removing the lesions caused by alkylating agents with different structures and mechanisms of action has been studied using UV-sensitive DNA repair-deficient mutant CHO cell lines. We confirmed that ERCC1 and ERCC4 play a role in the repair of cis-diamminedichloroplatinum (DDP)- and Melphalan (L-PAM)-induced DNA damage, while a marginal role of ERCC2 and ERCC3 in the cellular response to DDP and L-PAM treatment has been observed. Treatment with methylating agents (DM and MNNG) showed a lack of a preferential cytotoxicity between the parental and the different NER. deficient cell lines, emphasizing the importance of other repair systems such as 3-methyladenine glycosylase and O6 alkyl-guanine-DNA-alkyl-transferase. ERCC1, ERCC2, ERCC3 and ERCC4, but not ERCC6 gene products seem to be involved in removing the lesions caused by Tallimustine and CC1065, minor groove alkylating agents that alkylate N3 adenine in a sequence-specific manner. ERCC6-deficient cells were as sensitive as the parental cell line to all the cytotoxic drugs tested, except DDP. These data emphasize the importance of the CHO mutant cell lines with specific defects in the DNA repair system for investigating the mechanism of action of different anti-cancer agents.

Topics: Animals; Antineoplastic Agents, Alkylating; CHO Cells; Cisplatin; Cricetinae; Cricetulus; Distamycins; DNA Damage; DNA Helicases; DNA Repair; DNA-Binding Proteins; DNA, Neoplasm; Drosophila Proteins; Duocarmycins; Endonucleases; Indoles; Leucomycins; Melphalan; Methylnitronitrosoguanidine; Nitrogen Mustard Compounds; Nucleotides; Proteins; Radiation Tolerance; Sulfuric Acid Esters; Transcription Factors; Ultraviolet Rays; Xeroderma Pigmentosum Group D Protein

By using electromobility shift assay (EMSA), we have identified a protein able to recognize the DNA only if it was previously reacted with minor groove binders. This protein binds with very high affinity AT containing DNA treated with minor groove binders such as distamycin A, Hoechst 33258 and 33342, CC-1065 and ethidium bromide minor groove intercalator, but not with major groove binders such as quinacrine mustard, cisplatin or melphalan, or with topoisomerase I inhibitor camptothecin or topoisomerase II inhibitor doxorubicin. This protein was found to be present in different extracts of human, murine and hamster cells, with the human protein which appears to have a molecular weight slightly lower than that of the other species. This protein was found to be expressed both in cancer and normal tissues. By using molecular ultrafiltration techniques as well as southwestern analysis it was estimated that the apparent molecular weight is close to 100 kDa. We can exclude an identity between this protein and other proteins, with a similar molecular weight previously reported to be involved in DNA damage recognition/repair, such as topoisomerase I, mismatch repair activities such as the prokaryotic MutS protein and its human homologue hMSH2 or proteins of the nucleotide excision repair system such as ERCC1, -2, -3 and -4.

Topics: Animals; Antineoplastic Agents; Binding Sites; Bisbenzimidazole; Cell Line; Cricetinae; Distamycins; DNA; DNA Damage; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Humans; Intercalating Agents; Jurkat Cells; Melphalan; Mice; Mice, Inbred C57BL; Nitrogen Mustard Compounds; Tumor Cells, Cultured

Tallimustine or N-deformyl-N-[4-N-N,N-bis(2-chloroethylamino)benzoyl], a distamycin-A derivative (FCE 24517), is a novel anti-cancer agent which alkylates N3 adenine in the minor groove of DNA. The cell-cycle phase perturbations induced by the drug were investigated and compared with those caused by melphalan (L-PAM) in SW626 human ovarian-cancer cells. By coupling bromodeoxyuridine (BUdR) immunoreaction with biparametric flow-cytometric (FCM) analysis, we investigated the cell-cycle phase perturbation induced by tallimustine or L-PAM, considering separately the cells which, during the 1-hr treatment, were in the S phase or in G1-G2/M phases of the cell cycle. L-PAM delayed the S-phase progression of cells exposed to the drug when they were in S phase, with a consequent accumulation of cells as soon as they reached the G2 phase. In contrast, the S-phase cells treated with tallimustine were not perturbed during the DNA-synthesis phase progression, and were blocked in G2 only after they had passed through the G1/S transition of a new cell cycle. In cells which were in G1 or G2/M phases during drug treatment, tallimustine and L-PAM caused similar accumulation in G2. The differences in the cell-cycle perturbation caused by tallimustine and L-PAM may well be related to the different DNA damage the 2 drugs produced. These findings emphasize the different properties of DNA-minor-groove alkylating agents and conventional ones.

Topics: Alkylating Agents; Bromodeoxyuridine; Cell Cycle; Distamycins; DNA; DNA, Neoplasm; Female; Flow Cytometry; Humans; In Vitro Techniques; Melphalan; Nitrogen Mustard Compounds; Nucleic Acid Conformation; Ovarian Neoplasms; Tumor Cells, Cultured

In two human cancer cell lines, the breast mcf-7 and the T-cell leukemia MOLT4, we investigated the cytotoxicity of four antineoplastic agents having different mechanisms of action. We selected doxorubicin as a DNA-topoisomerase II inhibitor, FCE24517 (a Distamycin A derivative) as a DNA minor groove binder with specificity for AT bases, melphalan as an alkylating agent and cis-platinum as an alkylating agent able to form DNA-intrastrand crosslinks. From the cytotoxicity experiments a moderately toxic (less than 10% of growth inhibition) and a highly toxic (about 75% growth inhibition) dose were selected to evaluate the expression of genes involved in cell proliferation and in cell response to extracellular insults. The expression was evaluated at early times (60 min.) and 24 hrs. after treatment. At the concentrations utilized in both cell lines we could not find any alteration in the expression of p53, gas-1 and heat shock 70. After melphalan treatment down regulation of c-myc and of the H2A histone was seen at high doses, while no significant alteration of their expression was seen with the other drugs.

Topics: Antineoplastic Agents; Blotting, Northern; Cell Division; Cisplatin; Distamycins; DNA Damage; DNA, Complementary; Doxorubicin; Female; Gene Expression; Genes, myc; Genes, p53; Heat-Shock Proteins; Histones; Humans; Melphalan; Nitrogen Mustard Compounds; Proto-Oncogene Proteins c-myc; RNA, Neoplasm; RNA, Ribosomal, 28S; Tumor Cells, Cultured; Tumor Suppressor Protein p53