ABSTRACT
The polyamine transport system (PTS), hyperactive in cancer cells, can constitute a gate to deliver F14512, a novel spermine epipodophyllotoxin conjugate recently selected for clinical development in AML phase I. We investigated in vitro the high antiproliferative effect of F14512 against 13 leukemia cell lines, and demonstrated a statistically significant correlation with the level of PTS activity, using a novel fluorescent marker F96982. This labelling protocol was then adapted for clinical applications for blood, bone marrow and AML samples with CD45 gating. Within the patient samples, the PTS activity varied significantly in AML cells, as compared to normal lymphocytes. In conclusion, the identification of PTS-positive AML with F98982 probe offers new perspectives to select patients prone to respond to F14512.
Subject(s)
Biogenic Polyamines/metabolism , Fluorescent Dyes/metabolism , Leukemia, Myeloid, Acute/drug therapy , Oxadiazoles/metabolism , Podophyllotoxin/analogs & derivatives , Spermine/analogs & derivatives , Animals , Antigens, CD34/analysis , Biological Transport , CHO Cells , Cell Line, Tumor , Cricetinae , Cricetulus , Flow Cytometry , Humans , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Podophyllotoxin/pharmacokinetics , Podophyllotoxin/therapeutic use , Spermine/metabolismABSTRACT
Acridine derivatives, such as amsacrine, represent a well known class of multi-targeted anti-cancer agents that generally interfere with DNA synthesis and inhibit topoisomerase II. But in addition, these tricyclic molecules often display secondary effects on other biochemical pathways including protein metabolism. In order to identify novel anti-cancer drugs, we evaluated the mechanism of action of a novel series of bis- and tetra-acridines. As expected, these molecules were found to interact with DNA and inhibit the topoisomerase II-mediated DNA decatenation. Interestingly when tested on human tumour cells either sensitive (HL-60) or resistant (HL-60/MX2) to topoisomerase II inhibitors, these molecules proved equicytotoxic against the two cell lines, suggesting that they do not only rely on topoisomerase II inhibition to exert their cytotoxic effects. In order to identify alternative targets, we tested the capacity of acridines 1-9 to inhibit the proteasome machinery. Four tetra-acridines inhibited the proteasome in vitro, with IC(50) values up to 40 times lower than that of the reference proteasome inhibitor lactacystin. Moreover, unlike peptide aldehydes used as reference inhibitors for the proteasome, these new acridine compounds demonstrated a good selectivity towards the proteasome, when tested against four unrelated proteases. A cellular assay based on the degradation of a proteasome protein substrate indicated that at least two of the tetra-acridines maintained this proteasome inhibition activity in a cellular context. This is the first report of tetra-acridines that demonstrate dual topoisomerase II and proteasome inhibition properties. This new dual activity could represent a novel anti-cancer approach to circumvent certain forms of tumour resistance.
Subject(s)
Acridines/pharmacology , Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Proteasome Inhibitors , Topoisomerase II Inhibitors , Acridines/chemical synthesis , Acridines/chemistry , Acridines/classification , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/classification , Cell Culture Techniques , Cell Line, Tumor , Cell Proliferation/drug effects , DNA, Neoplasm/metabolism , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/classification , HL-60 Cells , Humans , Inhibitory Concentration 50 , Leukemia, Promyelocytic, Acute/drug therapy , Models, Biological , Molecular Structure , Proteasome Endopeptidase Complex/analysis , Sensitivity and Specificity , Structure-Activity RelationshipABSTRACT
PURPOSE: Vinflunine (VFL) is a novel Vinca alkaloid with markedly superior experimental in vivo antitumour activity to its parent molecule, vinorelbine (Navelbine, NVB), against a panel of murine and human tumours. The aim of this study was to establish whether there are differences in the rate and extent of development of resistance, both in vivo and in vitro, to these two newer Vinca alkaloids under identical selection conditions. METHODS: Using P388 leukaemia cells in vivo, it was evident that VFL induced drug resistance far less readily than NVB, as shown by the number of passages required to select for total resistance. Under in vitro conditions, using A549 human lung carcinoma cells, it was also clearly shown by drug sensitivity determinations that VFL was a less-potent inducer of drug resistance than NVB. Resistance resulting from either in vivo or in vitro selection was associated with a classic multidrug resistance profile. Further characterization of the drug-resistance phenotype of the most highly resistant A549 sublines showed that the level of total beta-tubulin expression appeared to be modified exclusively in the NVB-resistant cells. CONCLUSION: The clear demonstration that resistance to VFL developed far less readily than resistance to NVB both in vivo and in vitro may have potential clinical implications.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Vinblastine/analogs & derivatives , Vinblastine/pharmacology , Animals , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Female , Leukemia P388/drug therapy , Mice , Mice, Inbred DBA , Tubulin/analysis , Tumor Cells, Cultured , VinorelbineABSTRACT
DNA damage induced in V79 cells by F 11782, or 2",3"-bis pentafluorophenoxyacetyl-4',6'-ethylidene-beta-D glucoside of 4'-phosphate-4'-demethylepipodophyllotoxin 2N-methyl glucamine salt, a novel dual catalytic inhibitor of topoisomerases I and II, was investigated using both alkaline and neutral versions of the comet assay methodology. A comparison was then made of the DNA damage induced by F 11782 with that induced by either etoposide or camptothecin under comparable experimental conditions. The results revealed that F 11782 initially induced less DNA strand breaks that either etoposide or camptothecin and rejoined such breaks more slowly. However, unlike these other drugs, the extent of DNA damage induced by F 11782 increased linearly with time of incubation. F 11782 produced both DNA single- and double-strand breaks without any clear specificity relative to phase of the cell cycle, although proliferating cells were preferentially damaged. The marked time-dependency of induction of DNA strand breaks by F 11782 may serve to explain, at least in part, its major in vivo antitumour activities.
Subject(s)
DNA Damage , DNA/drug effects , Naphthalenes/pharmacology , Pyrans/pharmacology , Topoisomerase I Inhibitors , Animals , Camptothecin/pharmacology , Cell Line , Comet Assay , DNA/genetics , DNA Topoisomerases, Type I/metabolism , Dose-Response Relationship, Drug , Etoposide/pharmacology , Spheroids, Cellular/drug effects , Spheroids, Cellular/metabolism , Time FactorsABSTRACT
PURPOSE: F 11782 (2",3"-bis pentafluorophenoxyacetyl-4",6"-ethylidene-beta-D-glucoside of 4'-phosphate-4'-dimethylepipodophyllotoxin, di-N-methyl glucamine salt) is a newly synthesized dual catalytic inhibitor of topoisomerases I and II with major in vivo antitumour activity. In this study, we compared and contrasted F 11782 with three other known inhibitors of both these nuclear enzymes, namely aclarubicin. intoplicin and TAS-103, and established its novel mechanism of action. METHODS: In vitro growth-inhibitory effects against a panel of murine and tumour cell lines were measured by cell counting, clonogenicity or tetrazolium metabolic dye (MTT) assays. In vivo antitumour activities were evaluated against two murine tumour models (i.v. P388 leukaemia and s.c. B16 melanoma). Finally, interactions with either DNA or DNA-topoisomerases were determined using various methodologies: DNA-intercalator displacement, pBR322 DNA relaxation, kDNA decatenation, topoisomerase II extractability measurements, stabilization of topoisomerase-induced cleavable complexes (CC) in vitro and in cells, and gel retardation assays. RESULTS: F 11782 had a different profile of sensitivities and proved generally less cytotoxic than the other dual inhibitors tested in vitro, while showing significantly superior antitumour activity in vivo. F 11782, which did not stabilize CC either in vitro or in cells, was the only compound of this series capable of inhibiting the catalytic activity of both DNA-topoisomerases without interacting with DNA, and of completely impairing the binding of these nuclear proteins to DNA. Moreover, only cotreatment of cells in vitro with F 11782 enhanced the cytotoxic activity of etoposide. CONCLUSION: These results emphasize the novel mechanism of action of F 11782 vis-a-vis the other dual inhibitors of topoisomerases I and II and so augur well for its future clinical development.
Subject(s)
Antineoplastic Agents/toxicity , Leukemia P388/drug therapy , Melanoma, Experimental/drug therapy , Naphthalenes/toxicity , Pyrans/toxicity , Topoisomerase I Inhibitors , Topoisomerase II Inhibitors , Aclarubicin/toxicity , Aminoquinolines/toxicity , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Division , Enzyme Inhibitors/pharmacology , Etoposide/therapeutic use , Humans , Indenes/toxicity , Indoles/toxicity , Leukemia L1210 , Mice , Mice, Inbred C57BL , Naphthalenes/pharmacology , Naphthalenes/therapeutic use , Pyrans/pharmacology , Pyrans/therapeutic use , Pyridines/toxicity , Tumor Cells, Cultured , Urinary Bladder NeoplasmsABSTRACT
PURPOSE: Vinflunine (20'-20'-difluoro-3',4'-dihydrovinorelbine), a novel derivative of vinorelbine characterized by marked antitumour activity in vivo in a series of experimental murine and human tumours is currently undergoing phase I evaluation. To investigate its potential for inclusion in combination chemotherapy regimens, this preclinical study was undertaken. The in vitro cytotoxicity of vinflunine incubated simultaneously with one of the following drugs was investigated: camptothecin, cisplatin, doxorubicin, etoposide, 5-fluorouracil, gemcitabine, mitomycin C, paclitaxel or vinorelbine. METHODS: The combinations were first evaluated in vitro against the A549 human non-small-cell lung cancer cell line using median-effect analyses. RESULTS: The results revealed synergistic cytotoxicity when vinflunine was combined with cisplatin, mitomycin C, doxorubicin or 5-fluorouracil. Synergy was also observed when testing similar combinations against CCRF-CEM human leukaemia cells. Finally, these findings were comparable with those resulting from such combinations involving vinorelbine instead of vinflunine. CONCLUSION: Vinflunine appears a promising candidate for combining with other anticancer drugs.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacokinetics , Vinblastine/analogs & derivatives , Camptothecin/pharmacology , Cell Division/drug effects , Cisplatin/pharmacology , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Dose-Response Relationship, Drug , Doxorubicin/pharmacology , Drug Evaluation , Drug Synergism , Drug Therapy, Combination , Etoposide/pharmacology , Fluorouracil/pharmacology , Humans , Mitomycin/pharmacology , Models, Chemical , Paclitaxel/pharmacology , Tumor Cells, Cultured , Vinblastine/pharmacokinetics , Vinblastine/pharmacology , Vinorelbine , GemcitabineABSTRACT
F 11782, a novel epipodophylloid, proved a potent inhibitor of the catalytic activities of both topoisomerases I and II. Unlike classical inhibitors such as camptothecin or etoposide, F 11782 did not stabilise cleavable complexes induced by either topoisomerases I or II nor did it preferentially inhibit the religation step of the catalytic cycle of either enzyme. F 11782 neither intercalated DNA nor bound in its minor groove, and showed only weak inhibition of the ATPase activity associated with topoisomerase II. F 11782 appeared to act by inhibiting the binding of topoisomerases I and II to DNA in a manner dependent both on drug and enzyme concentrations, via a mechanism not previously described or shared by other known topoisomerase 'poisons' or inhibitors. In contrast, F 11782 had only a weak effect or none at all on various other DNA-interacting enzymes. In conclusion, F 11782, as a non-intercalating, specific catalytic inhibitor of both topoisomerases I and II with an original mechanism of action, may be considered to represent the first of a new class of topoisomerase-interacting agents.
Subject(s)
Enzyme Inhibitors/pharmacology , Naphthalenes/pharmacology , Pyrans/pharmacology , Topoisomerase I Inhibitors , Topoisomerase II Inhibitors , Adenosine Triphosphatases/drug effects , Adenosine Triphosphatases/metabolism , Animals , Antineoplastic Agents, Phytogenic/pharmacology , Camptothecin/pharmacology , Catalysis , Cattle , Cell Nucleus/enzymology , DNA/metabolism , DNA Topoisomerases, Type I/metabolism , DNA Topoisomerases, Type II/metabolism , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Etoposide/pharmacology , Evaluation Studies as Topic , Humans , Saccharomyces cerevisiaeABSTRACT
F 11782, or 2", 3"-bis pentafluorophenoxyacetyl-4',6'-ethylidene-beta-D glucoside of 4'-phosphate-4'-dimethylepipodophyllotoxin 2N-methyl glucamine salt, is a novel fluorinated lipophylic epipodophylloid which has proven cytotoxic activity in vitro and has shown markedly superior antitumour activity in vivo compared to etoposide in various experimental tumour models. However, the precise mechanism(s) of cytotoxicity of F 11782 remains to be defined. In this study, the DNA damaging activity of F 11782 was investigated in GCT27 and C6S cells using, respectively the fluorescence enhancement assay and the technique of DNA alkaline elution. All the results obtained were consistent with induction of DNA damage by F 11782. No evidence of any stabilisation of DNA-topoisomerase cleavable complexes though was obtained with this catalytic inhibitor. Furthermore, such induction of DNA damage has not been reported with other known catalytic topoisomerase inhibitors and so it appears to be unique to F 11782.
Subject(s)
Antineoplastic Agents/pharmacology , DNA Damage , DNA, Neoplasm/drug effects , Enzyme Inhibitors/pharmacology , Naphthalenes/pharmacology , Pyrans/pharmacology , Topoisomerase I Inhibitors , Vinblastine/analogs & derivatives , Animals , Antineoplastic Agents, Phytogenic/pharmacology , DNA Repair , DNA, Neoplasm/radiation effects , Etoposide/pharmacology , Leukemia P388/drug therapy , Leukemia P388/genetics , Nucleic Acid Synthesis Inhibitors/pharmacology , Radiation-Sensitizing Agents/pharmacology , Rats , Tumor Cells, Cultured/drug effects , Vinblastine/pharmacology , VinorelbineABSTRACT
F 11782 is a newly identified catalytic inhibitor of topoisomerases I and II, without any detectable interaction with DNA. This study aimed to establish whether its catalytic inhibition of topoisomerase II was mediated by mechanisms similar to those identified for the bisdioxopiperazines. In vitro combinations of F 11782 with etoposide resulted in greater than additive cytotoxicity in L1210 cells, contrasting with marked antagonism for combinations of etoposide with either ICRF-187 or ICRF-193. All three compounds caused a G2/M blockade of P388 cells after an 18-h incubation, but by 40 h polyploidization was evident only with the bisdioxopiperazines. Gel retardation data revealed that only F 11782, and not the bisdioxopiperazines, was capable of completely inhibiting the DNA-binding activity of topoisomerase II, confirming its novel mechanism of action. Furthermore, unlike ICRF-187 and ICRF-193, the cytotoxicity of F 11782 appeared mediated, at least partially, by DNA damage induction in cultured GCT27 human teratoma cells, as judged by a fluorescence-enhancement assay and monitoring p53 activation. Finally, the major in vivo antitumor activity of F 11782 against the murine P388 leukemia (i.v. implanted) and the B16 melanoma (s.c. grafted) contrasted with the bisdioxopiperazines' general lack of activity. Overall, F 11782 and the bisdioxopiperazines appear to function as quite distinctive catalytic topoisomerase II inhibitors.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Naphthalenes/pharmacology , Piperazines/pharmacology , Pyrans/pharmacology , Razoxane/pharmacology , Topoisomerase II Inhibitors , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Cell Cycle/drug effects , DNA Damage , DNA Topoisomerases, Type II/metabolism , DNA, Neoplasm/metabolism , Diketopiperazines , Drug Screening Assays, Antitumor , Enzyme Inhibitors/administration & dosage , Etoposide/administration & dosage , Female , Humans , Leukemia P388/drug therapy , Melanoma, Experimental/drug therapy , Mice , Mice, Inbred C57BL , Naphthalenes/administration & dosage , Neoplasm Transplantation , Piperazines/administration & dosage , Pyrans/administration & dosage , Razoxane/administration & dosage , Tumor Cells, CulturedABSTRACT
A series of molecules with apurinic/apyrimidic (AP) endonuclease activity targeted to abasic sites in DNA, which incorporate an intercalating moiety linked to a purine base by a polyamino chain and recognize and cleave abasic sites in DNA with high efficiency, has been studied. The aim was first to establish whether these compounds were inhibitors of base excision DNA repair, since abasic sites are generated during this process. Using an extension of a recently established methodology, two members of this series have been identified as definite repair inhibitors. Secondly, the potential of using such compounds as sensitizers of alkylating agents has been investigated by determining the cytotoxic activity of these compounds on L1210 cells in culture. A concentration-dependent potentiation of nitrosoureas has been demonstrated, but interpretation is complicated by the inherent cytotoxic properties of the test compounds themselves. Such molecules, however, provide interesting lead compounds for new strategies aimed at enhancing the cytotoxic potential of clinically useful DNA-damaging agents.
Subject(s)
Antineoplastic Agents, Alkylating/pharmacology , DNA Repair/drug effects , Endonucleases/pharmacology , Leukemia L1210/drug therapy , Animals , Antibiotics, Antineoplastic/pharmacology , Base Pairing , Carmustine/pharmacology , DNA, Neoplasm/biosynthesis , DNA, Neoplasm/drug effects , Drug Screening Assays, Antitumor , Inhibitory Concentration 50 , Leukemia L1210/genetics , Methyl Methanesulfonate/pharmacology , Mice , Protein Biosynthesis , Proteins/drug effects , RNA/biosynthesis , RNA/drug effects , Streptozocin/pharmacology , Thiotepa/pharmacology , Tumor Cells, CulturedABSTRACT
Vinflunine, a newly synthesized derivative, possesses marked in vivo antitumor properties and, like other alkaloids, inhibits in vitro tubulin assembly at microM concentrations. However, in contrast to other vinca alkaloids, vinflunine exhibits relatively low in vitro cytotoxic potency. The aim of this report was to investigate whether the action(s) of vinflunine on the microtubule cytoskeleton could account for its cytotoxicity or if its cellular action requires another molecular target. Four vinca alkaloids used in cancer therapy and vinflunine were studied using PtK2 cells. Their activities on the most dynamic microtubules were investigated in mitosis and in interphase by evaluating the disturbance of the metaphase plate and the splitting of the diplosome, respectively. No correlation was observed between the cellular accumulation of these compounds and either their cytotoxicity or their action(s) on the microtubule cytoskeleton. In contrast, cytotoxicity, mitotic disturbance and diplosome splitting were observed in the nM range for vinblastine, vincristine, vindesine and vinorelbine, although these events occurred at 10 times higher concentrations in the case of vinflunine. Hence, dynamic modifications of both the mitotic and interphasic microtubule cytoskeleton are compatible with in vitro cytotoxicity of vinflunine, raising questions about the conventional biochemical screening of these vinca alkaloids.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Cytoskeleton/drug effects , Microtubules/drug effects , Vinblastine/analogs & derivatives , Vinca Alkaloids/pharmacology , Animals , Antineoplastic Agents, Phytogenic/pharmacokinetics , Antineoplastic Agents, Phytogenic/toxicity , Cell Division/drug effects , Cell Survival/drug effects , Chromosomes/drug effects , Chromosomes/ultrastructure , Cytoskeleton/ultrastructure , Fluorescent Antibody Technique , Indicators and Reagents , Macropodidae , Microtubules/ultrastructure , Tumor Cells, Cultured , Vinblastine/pharmacokinetics , Vinblastine/pharmacology , Vinblastine/toxicity , Vinca Alkaloids/pharmacokinetics , Vinca Alkaloids/toxicityABSTRACT
Vinflunine (VFL) is a novel derivative of vinorelbine (NVB, Navelbine), which has shown markedly superior antitumor activity to NVB, in various experimental animal models. To establish whether this new Vinca alkaloid participates in P-glycoprotein (Pgp)-mediated multidrug resistance (MDR), VFL-resistant murine P388 cells (P388/VFL) were established in vivo and used in conjunction with the well established MDR P388/ADR subline, to define the in vivo resistance profile for VFL. P388/VFL cells proved cross-resistant to drugs implicated in MDR (other Vinca alkaloids, doxorubicin, etoposide), but not to campothecin or cisplatin and showed an increased expression of Pgp, without any detectable alterations in topoisomerase II or in glutathione metabolism. The P388/ADR cells proved cross-resistant to VFL both in vivo and in vitro, and this VFL resistance was efficiently modulated by verapamil in vitro. Cellular transport experiments with tritiated-VFL revealed differential uptake by P388 sensitive and P388/ADR resistant cells, comparable with data obtained using tritiated-NVB. In various in vitro models of human MDR tumor cells, whilst full sensitivity was retained in cells expressing alternative non-Pgp-mediated MDR mechanisms, cross resistance was identified in Pgp-overexpressing cells. Differences were, however, noted in terms of the drug resistance profiles relative to the other Vinca, with tumor cell lines proving generally least cross-resistant to VFL. Overall, these results suggest that VFL, like other Vinca alkaloids, participates in Pgp-mediated MDR, with tumor cells selected for resistance to VFL overexpressing Pgp, yet MDR tumor cell lines proved generally less cross resistant to VFL relative to the other Vinca alkaloids.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Antineoplastic Agents, Phytogenic/pharmacology , Drug Resistance, Multiple , Vinblastine/analogs & derivatives , Animals , Antineoplastic Agents, Phytogenic/chemistry , Calcium Channel Blockers/pharmacology , Doxorubicin/pharmacology , Drug Resistance, Neoplasm , Female , Humans , Leukemia P388 , Mice , Mice, Inbred BALB C , Mice, Inbred DBA , Phenotype , Tumor Cells, Cultured , Verapamil/pharmacology , Vinblastine/chemistry , Vinblastine/pharmacologyABSTRACT
Podophyllotoxin and epipodophyllotoxin react with tubulin at the same binding site as colchicine, but in contrast to colchicine, do not appear to exert their cytotoxicities by mechanisms dependent on P-glycoprotein (Pgp) expression. To investigate structural requirements for Pgp recognition a series of podophyllotoxin and epipodophyllotoxin derivatives have been synthesized. Their interactions with the multidrug resistance-related protein Pgp have been studied by evaluating their relative cytotoxicities versus P388-sensitive murine leukemic cells and a classic multidrug-resistant (MDR) Pgp-overexpressing subline (P388/ADR), and their relative tubulin polymerization inhibitory activities against microtubular proteins have been determined. Based on tridimensional structure-activity relationships within this series of compounds, structural requirements for Pgp recognition have been identified. Moreover, proposals are made for extending these criteria to other chemical classes of anticancer drugs.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Podophyllotoxin/analogs & derivatives , Podophyllotoxin/pharmacology , Animals , Mice , Microtubule Proteins/chemistry , Models, Molecular , Podophyllotoxin/chemical synthesis , Structure-Activity Relationship , Tubulin/chemistry , Tumor Cells, CulturedABSTRACT
This study aimed to define the mechanism of action of vinflunine, a novel Vinca alkaloid synthesised from vinorelbine using superacidic chemistry and characterised by superior in vivo activity to vinorelbine in preclinical tumour models. In vitro vinflunine cytotoxicity proved dependent on concentration and exposure duration, with IC50 values (72-hr exposures) generally ranging from 60-300 nM. Vinflunine induced G2 + M arrest, associated with mitotic accumulation and a concentration-dependent reduction of the microtubular network of interphase cells, accompanied by paracrystal formation. These effects, while comparable to those of vincristine, vinblastine or vinorelbine, were achieved with 3- to 17-fold higher vinflunine concentrations. However, vinflunine and the other Vincas all inhibited microtubule assembly at micromolar concentrations. Vinflunine, like vinblastine, vincristine and vinorelbine, appeared to interact at the Vinca binding domain, as judged by proteolytic cleavage patterns, and induced tubulin structural changes favouring an inhibition of GTP hydrolysis. However, vinflunine did not prevent [3H]vincristine binding to unassembled tubulin at concentrations < or = 100 microM, and only weakly inhibited binding of [3H]vinblastine or [3H]vinorelbine. Indeed, specific binding of [3H]vinflunine to tubulin was undetectable by centrifugal gel filtration. Thus, the comparative capacities of these Vincas to bind to or to interfere with their binding to tubulin could be classified as: vincristine > vinblastine > vinorelbine > vinflunine. By monitoring alkylation of sulfhydryl groups, differential effects on tubulin conformation were identified with vinflunine and vinorelbine acting similarly, yet distinctively from vinblastine and vincristine. Overall, vinflunine appears to function as a definite inhibitor of tubulin assembly, while exhibiting quantitatively different tubulin binding properties to the classic Vinca alkaloids.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Mitosis/drug effects , Tubulin/drug effects , Vinblastine/analogs & derivatives , Alkylation , Animals , Cell Cycle/drug effects , Cells, Cultured , Guanosine Triphosphate/metabolism , Hydrolysis , Iodoacetamide/pharmacology , Leukemia L1210/metabolism , Leukemia L1210/pathology , Mice , Protein Binding , Rats , Tubulin/metabolism , Tumor Cells, Cultured , Vinblastine/pharmacology , Vincristine/pharmacology , VinorelbineABSTRACT
The 1990s have already heralded an enormous expansion of our knowledge of DNA repair. Gene by gene, protein by protein, each partner in the molecular processes of DNA repair is being identified and characterized, not only in bacteria and yeast, but also in mammalian cellular systems. Several distinctive mechanisms are now explained at a molecular level, even if certain specific parts still remain to be elucidated fully. The techniques used to study DNA repair have also profited from this progress with a plethora of novel in vitro assays, specific antibodies, together with DNA or RNA probes becoming available. The increased use of these tools has permitted a multiplicity of studies on DNA repair which are now not exclusively mechanistically based. Thus, certain studies have now implicated DNA repair processes as likely to be involved in the multifactorial phenomenon of drug resistance to anticancer drugs. Under these circumstances, DNA repair mechanisms should provide useful pharmacological targets to attack with novel inhibitors, with the aim of reducing and/or sensitizing tumor cells to anticancer drugs which damage DNA. Our increased knowledge of the molecular mechanisms associated with DNA repair permits us now to consider such new pharmacological targeting. In this article, we review the present status of these DNA-repair-related pharmacological studies, and discuss both the likely and possible approaches which might have potential therapeutic applications.
Subject(s)
DNA Repair/genetics , DNA Repair/immunology , Drug Resistance, Neoplasm/genetics , Drug Resistance, Neoplasm/immunology , Antineoplastic Agents/pharmacology , DNA Ligases/metabolism , DNA, Neoplasm/drug effects , HumansABSTRACT
A recently derived in vitro chemiluminescence assay (Salles et al. [1995] Anal. Biochem., 232, 37-42) has been used to investigate the effects of a panel of twenty-two anticancer drugs and certain antibiotics on the excision repair activity of cell-free extracts from the human cell line, HeLa. This methodology, termed the 3D (Damaged DNA Detection) assay, based on the in vitro excision repair assay previously developed (Wood et al. [1988] Cell, 53, 97-106) has provided data indicating definite in vitro inhibition of DNA repair by actinomycin D, aphidicolin, doxorubicin, distamycin A and mithramycin A. This assay therefore offers the potential for identifying agents with the ability to inhibit DNA repair.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antineoplastic Agents/pharmacology , DNA Repair/drug effects , DNA, Bacterial/drug effects , DNA, Bacterial/radiation effects , Dose-Response Relationship, Drug , HeLa Cells , Humans , Luminescent Measurements , Methods , Plasmids , Ultraviolet RaysABSTRACT
OBJECTIVE: To evaluate the tuberculin reactivity among medical students and the utility of RT-23 (2 tuberculin unit [TU] purified protein derivative [PPD]) produced in Mexico. DESIGN: A blind and comparative study was conducted in a school of medicine using simultaneously RT-23 and an American product (5 TU PPD) randomly assigned to each forearm and read 48 hours later. Ninety eight volunteers were included, 69 from the first year and 29 from the fifth year as medical students. RESULTS: Using 5 TU PPD, 16% of first year students and 41% of fifth grade students were positive (induration > or = 10 mm). The RT-23 worked well and showed a sensitivity of 87% and a specificity of 92%. There was a large difference in the positivity of fifth versus first year students (x2 = 6.00, P = 0.014) with an estimated annual conversion of 5.1%. The high tuberculin conversion rate urges the need to establish preventive and early diagnosis programs for tuberculosis. The utility of RT-23 was found to be comparable to 5 TU PPD in our population.
Subject(s)
Tuberculin Test/methods , Tuberculin , Adolescent , Adult , Cross-Sectional Studies , Female , Humans , Male , Mexico/epidemiology , Sensitivity and Specificity , Students, Medical , Tuberculosis/epidemiologyABSTRACT
Nucleotide excision repair (NER) activity was investigated in lymphocytes from patients with chronic lymphocytic leukemia (CLL). The NER process consists of two broad stages: incision/excision of the damaged oligonucleotide and resynthesis of the repair patch. NER in CLL lymphocytes was monitored with the use of in vitro biochemical assays, allowing the determination of either the extent of repair synthesis or the incision activity on damaged plasmid DNA during incubation with whole-cell protein extracts. Fresh CLL tumor cells were purified from the blood of 7 untreated patients and 11 patients who had been treated with chemotherapy. No repair activity was found in 14 extracts (7 treated and 7 untreated) or in normal blood peripheral lymphocytes. The defect was at the level of both repair synthesis and incision/excision activity of DNA damage. In contrast, 4 of the extracts exhibited 25-60% of the repair activity measured in an extract from a control repair-proficient cell line. A linear relationship was found between the values of DNA-repair synthesis and incision activities, which indicates that the extent of significant incision was the limiting factor in these protein extracts. All of the extracts that exhibited DNA-repair activity were purified from lymphocytes of treated patients. These data suggest that chemotherapy might exert an effect on the status of repair activity in the lymphoid tumor cells of patients.
Subject(s)
DNA Repair , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Aged , B-Lymphocytes/physiology , Cells, Cultured , DNA Damage , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Middle Aged , Plasmids , T-Lymphocytes/physiologyABSTRACT
DNA repair activity in human peripheral blood lymphocytes (PBL) has been investigated by various techniques. Here, we report the use of an in vitro assay in order to assess nucleotide excision repair activity (NER). The mechanism of this major repair process relies on two broad steps: first, recognition, incision and excision of the damaged DNA; second, repair synthesis on the gapped DNA. Briefly, damaged plasmids were incubated with whole cell extracts which allows one to quantify DNA repair synthesis. When NER was determined on plasmid DNA damaged with UV-light or cisplatin, PBL extracts showed no repair synthesis for unstimulated lymphocytes. Using a new in vitro assay measuring only the damage-specific DNA incision activity in cell extracts, we found that the incision step in the repair reaction was blocked in unstimulated PBL. By mixing PBL with XP (group A, B, C, D) extracts, no restoration of NER activity was observed. In addition, these lymphocytes also lacked DNA replication activity as determined with pre-incised plasmid substrate. However, a phytohemagglutinin treatment of PBL led to an extent of repair synthesis similar to that observed with extracts from lymphoblastoid cells. When lymphocytes were incubated in 20% serum medium with and without phytohemagglutinin, the repair activity increased dramatically after 24 h. During the activation of lymphocytes, the extent of repair synthesis was proportional to the percentage of cells in S phase of the cell cycle. Our results suggest that the blockage of the cell cycle in G0/G1 in PBL may be responsible for their lack of NER activity.
Subject(s)
Blood Proteins/metabolism , DNA Repair , Lymphocytes/physiology , Nucleotides/genetics , Nucleotides/metabolism , Cell Extracts , Cell Line , DNA/blood , Genetic Complementation Test , Humans , Kinetics , Lymphocyte Activation , Lymphocytes/drug effects , Lymphocytes/metabolism , Phytohemagglutinins/pharmacology , Stimulation, ChemicalABSTRACT
cis-Dichlorodiammineplatinum(II) (CDDP) resistance in L1210/10 murine leukemia cells is multifactorial and involves decreased drug uptake, increased glutathione content, and enhanced DNA repair activity. We show here that 0.35 M NaCl nuclear extracts from L1210/10 cells possess an approximately 3-fold increase in DNA topoisomerase II activity, compared with parental L1210 cells, as measured by decatenation of kinetoplast DNA. No difference in topoisomerase I activity is observed between the two cell lines. Immunoblot analysis of topoisomerase II protein in resistant and sensitive cells suggests that the observed differences in topoisomerase II activity cannot be explained by differences in the level of protein expressed. L1210/10 cells are 2.5-fold more sensitive than L1210 cells to the cytotoxic effects of the topoisomerase II inhibitor 4'-(9-acridylamino)methane-sulfon-m-anisidide. Sequential treatment with 4'-(9-acridyl-amino)methanesulfon-m-anisidide and CDDP leads to an additive cytotoxic effect of the two drugs in sensitive L1210 cells, as determined by colony formation in semi-solid medium. In contrast, the same treatment leads to a supra-additive effect in L1210/10 cells, which strongly suggests a role for topoisomerase II in the CDDP resistance of this cell line.