RESUMO
The radiomimetic enediyne C-1027 induces almost exclusively DNA double-strand breaks (DSB) and is extremely cytotoxic. Unique among radiomimetics, ataxia-telangiectasia mutated (ATM) is dispensable for cellular responses to C-1027-induced DNA damage. This study explores the biological activity of three recently bioengineered C-1027 analogues: 7''-desmethyl-C-1027 (desmethyl), 20'-deschloro-C-1027 (deschloro), and 22'-deshydroxy-C-1027 (deshydroxy). Each compound maintains the characteristic ability of radiomimetics to cleave DNA in cell-free systems, varying in activity from 2-fold (deschloro) to 55-fold (desmethyl) less than C-1027. The induction of cellular DNA breaks based on pulsed field gel electrophoresis, comet analysis, and gammaH2AX activation was in the same rank order as cell-free DNA break induction, although the amount of breaks induced by desmethyl is greatly reduced compared with the other analogues. Despite the disparity in inducing DNA DSBs, all of the analogues produced G2-M cell cycle arrest and activated DNA DSB damage response proteins, such as p53-Ser15 and Chk2-Thr68, at concentrations in concordance with their ability to inhibit cell growth. Interestingly, of the three analogues, only the desmethyl-induced DNA damage response was similar to C-1027, as it did not cause hypersensitive cell growth inhibition in the absence of ATM nor require the kinase to phosphorylate p53 or Chk2. These findings show that simple modifications of the chromophore of C-1027 can result in varied induction of, and cellular response to, DNA DSBs.
Assuntos
Aminoglicosídeos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/genética , Dano ao DNA , Proteínas de Ligação a DNA/genética , DNA/efeitos dos fármacos , Enedi-Inos/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/genética , Aminoglicosídeos/química , Antibióticos Antineoplásicos/química , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular , Sistema Livre de Células , DNA/metabolismo , Enedi-Inos/química , Fibroblastos , Humanos , Mutação , Relação Estrutura-AtividadeRESUMO
This study examined the extent of chromosome instability induced in cultured human colon carcinoma HCT116 cells by the antitumor radiomimetic enediyne antibiotic C-1027. Spectral karyotype analysis showed frequent intrachromosomal fusions and fragmentations 26 hours after addition of as little as 0.035 nmol/L C-1027. When the concentration was increased to 0.14 nmol/L C-1027, 92% of cells showed chromosomal aberrations compared with only 2.9% after treatment with an equivalent growth inhibitory dose of ionizing radiation (20 Gy). Thus, chromosome misrejoining was associated to a much greater extent with C-1027-induced than with ionizing radiation-induced cell growth inhibition. Despite these aberrations, a large fraction of C-1027-treated cells progressed into G1. Comet analysis showed that these extensive chromosomal anomalies were not due to increased induction or reduced repair of C-1027-induced compared with ionizing radiation-induced strand breaks. Fluorescence in situ hybridization analysis showed that misrejoining of telomere repeats (i.e., chromosomes joined end to end at their telomeres or fused together after complete loss of telomere sequences) was observed within 26 hours of C-1027 addition. The extreme cytotoxicity of C-1027 may reflect both induction and erroneous repair of DNA double-strand break in the whole genome and/or in subgenomic targets such as telomere sequences.
Assuntos
Aminoglicosídeos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Aberrações Cromossômicas/induzido quimicamente , Telômero/efeitos dos fármacos , Ensaio Cometa , Enedi-Inos , Células HCT116 , Humanos , Cariotipagem , Telômero/fisiologiaRESUMO
Fluorescent microgonotropens (FMGTs) are A/T selective, minor groove-binding bisbenzimidazole ligands. Basic side chains extending from these agents electrostatically contact the major groove side of the phosphodiester backbone of DNA, endowing them with high binding affinity. Here, we evaluate the potential of these agents as inhibitors of transcription factor (TF) binding to DNA and explore whether their ability to contact both grooves enhances their inhibitory activity. A series of FMGTs (L2-L5), with polyamine tails of varying lengths and degrees of branching, were compared to an analog lacking these basic side chains (L1), and the classical bisbenzimidazole Hoechst 33342 for effects on TF complex formation on the c-fos serum response element (SRE). Although L1 could not inhibit TF/SRE interactions, L2-L5 did so at submicromolar concentrations. Moreover, the FMGTs were up to 50 times more potent than Hoechst 33342 in inhibiting TF complex formation in electrophoretic mobility shift assays. The FMGTs also inhibited c-fos promoter-driven cell-free transcription and topoisomerase II activity in nuclei. These studies establish the potential of FMGTs as TF/DNA complex inhibitors in cell-free systems, provide insight into the relationship between their structure and biological activities, and demonstrate the benefits of functionalizing minor groove binding-agents with major groove-contacting groups.
Assuntos
Benzimidazóis/farmacologia , Proteínas de Ligação a DNA , DNA/química , Distamicinas/farmacologia , Pirróis/farmacologia , Fatores de Transcrição/química , Células 3T3 , Animais , Sistema Livre de Células , Desenho de Fármacos , Ensaio de Desvio de Mobilidade Eletroforética , Fluorescência , Camundongos , Proteínas Proto-Oncogênicas/química , Elemento de Resposta Sérica , Relação Estrutura-Atividade , Fatores de Transcrição/antagonistas & inibidores , Proteínas Elk-1 do Domínio etsRESUMO
The ability of the radiomimetic anti-tumor enediyne C-1027 to induce DNA inter-strand crosslinks (ICLs), in addition to the expected DNA strand breaks, is unique among traditional DNA targeted cancer therapies. Importantly, radiation therapy and most radiomimetic drugs have diminished effect in hypoxic environments due to decreased induction of DNA strand breaks, which is an oxygen requiring process. However, C-1027's induction of ICLs is enhanced under hypoxia and it is actually more potent against hypoxic cells, overcoming this common tumor resistance mechanism. In this study, an analog of C-1027, 20'-deschloro-C-1027 was examined for its ability to induce DNA ICLs under hypoxic conditions. Deschloro-induced ICLs were detected under hypoxic cell-free conditions, with a concomitant reduction in the induction of DNA strand breaks. In cells deschloro behaved similarly, inducing cellular ICLs under hypoxic conditions with a reduction in DNA breaks. The cytotoxicity of deschloro treatment was similar in normoxic and hypoxic cells, suggesting that the ICL induction allows deschloro to retain its cytotoxic activity under hypoxia. It appears that rational engineering of the C-1027 family of radiomimetics holds promise toward overcoming the radioresistance associated with the hypoxic environment associated with solid tumors.
Assuntos
Aminoglicosídeos/toxicidade , Antibióticos Antineoplásicos/toxicidade , Reagentes de Ligações Cruzadas/toxicidade , Quebras de DNA , Enedi-Inos/toxicidade , Tolerância a Radiação , Aminoglicosídeos/uso terapêutico , Hipóxia Celular , Enedi-Inos/uso terapêutico , Células HCT116 , HumanosRESUMO
The hypoxic nature of cells within solid tumors limits the efficacy of anticancer therapies such as ionizing radiation and conventional radiomimetics because their mechanisms require oxygen to induce lethal DNA breaks. For example, the conventional radiomimetic enediyne neocarzinostatin is 4-fold less cytotoxic to cells maintained in low oxygen (hypoxic) compared with normoxic conditions. By contrast, the enediyne C-1027 was nearly 3-fold more cytotoxic to hypoxic than to normoxic cells. Like other radiomimetics, C-1027 induced DNA breaks to a lesser extent in cell-free, or cellular hypoxic, compared with normoxic environments. However, the unique DNA interstrand cross-linking ability of C-1027 was markedly enhanced under the same hypoxic conditions that reduced its DNA break induction. Although the unique chemistry of C-1027 allows it to concurrently generate both DNA breaks and cross-links in normoxic cells, a low oxygen environment represses the former and promotes the latter. Thus, treatment with C-1027 offers a facile approach for overcoming the radioresistance associated with poorly oxygenated cells.