RESUMEN
Mutations in the FERMT1 gene, encoding the focal adhesion protein kindlin-1 underlie the Kindler syndrome (KS), an autosomal recessive skin disorder with a phenotype comprising skin blistering, photosensitivity, progressive poikiloderma with extensive skin atrophy, and propensity to skin cancer. The FERMT1 mutational spectrum comprises gross genomic deletions, splice site, nonsense, and frameshift mutations, which are scattered over the coding region spanning exon 2-15. We now report three KS families with mutations affecting the promoter region of FERMT1. Two of these mutations are large deletions (â¼38.0 and 1.9 kb in size) and one is a single nucleotide variant (c.-20A>G) within the 5' untranslated region (UTR). Each mutation resulted in loss of gene expression in patient skin or cultured keratinocytes. Reporter assays showed the functional relevance of the genomic regions deleted in our patients for FERMT1 gene transcription and proved the causal role of the c.-20A>G variant in reducing transcriptional activity.
Asunto(s)
Vesícula/genética , Epidermólisis Ampollosa/genética , Proteínas de la Membrana/genética , Mutación , Proteínas de Neoplasias/genética , Enfermedades Periodontales/genética , Trastornos por Fotosensibilidad/genética , Regiones Promotoras Genéticas , Adolescente , Biomarcadores , Vesícula/diagnóstico , Preescolar , Análisis Mutacional de ADN , Epidermólisis Ampollosa/diagnóstico , Humanos , Masculino , Enfermedades Periodontales/diagnóstico , Fenotipo , Trastornos por Fotosensibilidad/diagnóstico , Piel/patología , Adulto JovenRESUMEN
The poly(ADP-ribose) polymerase (PARP) is involved in cell recovery from DNA damage, such as methylation of N3-adenine, that activates the base excision repair process. In the present study we demonstrated that MeOSO(2)(CH(2))(2)-lexitropsin (Me-Lex), a methylating agent that almost exclusively produces N3-methyladenine, induced different modalities of cell death in human leukemic cell lines, depending on the presence of PARP inhibitor. Growth inhibition, provoked by the combination of Me-Lex and PARP inhibitor, was associated with a marked down-regulation of c-myc, increased generation of single strand breaks and apoptosis. When used as single agent, at concentrations that saturated cell repair ability, Me-Lex induced mainly cell death by necrosis. Surprisingly, addition of a PARP inhibitor enhanced apoptosis and reduced the early appearance of necrosis. Telomerase activity was completely suppressed in cells exposed to Me-Lex alone, by 24 h after treatment, whereas it did not change when Me-Lex was combined with PARP inhibitor. Thereafter, inhibition of telomerase was observed with both treatments. The results suggest new insights on different modalities of cell death induced by high levels of N3-methyladenine per se, or by the methylated base in the presence of PARP inhibitor.
Asunto(s)
Apoptosis/efectos de los fármacos , Daño del ADN/efectos de los fármacos , ADN Glicosilasas , Metilación de ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/efectos de los fármacos , Necrosis , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Alquilantes/farmacología , Apoptosis/fisiología , División Celular/efectos de los fármacos , División Celular/genética , Daño del ADN/fisiología , Reparación del ADN/fisiología , ADN de Cadena Simple/efectos de los fármacos , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Humanos , Células Jurkat/citología , Células Jurkat/efectos de los fármacos , Células Jurkat/metabolismo , N-Glicosil Hidrolasas/metabolismo , Netropsina/análogos & derivados , Netropsina/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Proteínas Proto-Oncogénicas c-myc/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/genética , Telomerasa/efectos de los fármacos , Telomerasa/metabolismoRESUMEN
Mutations or transcriptional silencing of mismatch repair genes have been linked with tumour cell resistance to O(6)-guanine methylating agents, 6-thioguanine, cisplatin, doxorubicin and etoposide. Recently, it has been demonstrated that overexpression of the MSH3 protein is associated with depletion of the mismatch binding factor MutSalpha, and then with a marked reduction in the efficiency of base/base mismatch repair. In the present study we evaluated sensitivity of the HL-60 cell line and its methotrexate-resistant subline HL-60R, which overexpresses the hMSH3 gene, to a panel of chemotherapeutic agents. Cell growth inhibition induced by temozolomide, 6-thioguanine and N-methyl-N'-nitro-N-nitrosoguanidine was significantly lower in the hMSH3-overexpressing HL-60R cell line as compared with the HL-60 parental line. Moreover, HL-60R cells were more resistant than HL-60 cells to chromosome aberrations induced by either N-methyl-N'-nitro-N-nitrosoguanidine or temozolomide, and to apoptosis triggered by the latter drug. Both cell lines were equally susceptible to growth inhibition induced by cisplatin, etoposide or doxorubicin. In addition, HL-60 and HL-60R cells showed comparable sensitivity to the clastogenic and apoptotic effects of cisplatin and etoposide. These results further confirm that loss of base/base mismatch repair is the most important molecular mechanism involved in cell resistance to O(6)-guanine methylating agents and 6-thioguanine. However, the status of the mismatch repair system could still influence tumour cell sensitivity to cisplatin, etoposide and doxorubicin, depending on the specific component of the system that is lost, and on the genetic background of the cell.
Asunto(s)
Antineoplásicos/farmacología , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , División Celular/efectos de los fármacos , Aberraciones Cromosómicas , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos/genética , Células HL-60 , Humanos , Proteína 3 Homóloga de MutS , MutaciónRESUMEN
Mismatch repair deficiency contributes to tumor cell resistance to O6-guanine methylating compounds and to other antineoplastic agents. Here we demonstrate that MeOSO2(CH2)2-lexitropsin (Me-Lex), a DNA minor groove alkylating compound which generates mainly N3-methyladenine, has cytotoxic and clastogenic effects in mismatch repair-deficient leukemic cells. Moreover, MT-1 cells, which express p53 upon drug treatment and possess low levels of 3-methylpurine DNA glycosylase activity, are more susceptible to cytotoxicity induced by Me-Lex, with respect to p53-null and 3-methylpurine DNA glycosylase-proficient Jurkat cells. In both cell lines, the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide, which inhibits base excision repair capable of removing N-methylpurines, increases cytotoxicity and clastogenicity induced by Me-Lex or by temozolomide, which generates low levels of N3-methyl adducts. The enhancing effect is more evident at low Me-Lex concentrations, which induce a level of DNA damage that presumably does not saturate the repair ability of the cells. Nuclear fragmentation induced by Me-Lex + 3-aminobenzamide occurs earlier than in cells treated with the single agent. Treatment with Me-Lex and 3-aminobenzamide results in augmented expression of p53 protein and of the X-ray repair cross-complementing 1 transcript (a component of base excision repair). These results indicate that N3-methyladenine inducing agents, alone or combined with poly(ADP-ribose) polymerase inhibitors, could open up novel chemotherapeutic strategies to overcome drug resistance in mismatch repair-deficient leukemic cells.
Asunto(s)
Antineoplásicos/farmacología , ADN de Neoplasias/efectos de los fármacos , Mutágenos/farmacología , Netropsina/análogos & derivados , Apoptosis , Aberraciones Cromosómicas , Proteínas de Unión al ADN/genética , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Células HT29 , Humanos , Células Jurkat , Netropsina/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Proteína p53 Supresora de Tumor/biosíntesis , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos XRESUMEN
Methylating triazenes have shown marked antileukemic effects, possibly through generation of a variety of DNA adducts. Cells tolerant to O6-methylguanine due to a defect in the mismatch repair system (MRS), might become sensitive to other methyl adducts, by inhibiting the N-methylpurine repair, which requires base excision repair (BER) and poly(ADP-ribose) polymerase (PADPRP). Therefore, MRS-deficient Jurkat leukemic cells resistant to methylating triazenes, have been treated with temozolomide (TZM) and PADPRP inhibitors. Expression of PADPRP or molecules involved in the BER system [3-methylpurine-DNA glycosylase (MPG) and X-ray repair cross-complementing 1 (XRCC1)], have been explored. Cytotoxic effects of TZM associated with PADPRP inhibitors are evident shortly after treatment, suggesting that completion of cell division is not required for the lethal effect of the drug combination. Increase of PADPRP or MPG transcripts was found after treatment with TZM alone or combined with PADPRP inhibitor. XRCC1 transcript was positively modulated only in the case of drug combination. This could suggest that in the presence of PADPRP inhibitor, persistence of DNA damage triggers XRCC1 transcription. Our results suggest that association of TZM and PADPRP inhibitors might be of benefit for MRS-deficient malignancies unresponsive to the methylating agent.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Apoptosis , Benzamidas/farmacología , ADN Glicosilasas , Reparación del ADN/efectos de los fármacos , Dacarbazina/análogos & derivados , Inhibidores Enzimáticos/farmacología , Leucemia/tratamiento farmacológico , Inhibidores de Poli(ADP-Ribosa) Polimerasas , ADN Ligasas/análisis , ADN Ligasas/genética , Dacarbazina/farmacología , Interacciones Farmacológicas , Resistencia a Antineoplásicos , Guanina/análogos & derivados , Guanina/farmacología , Humanos , Células Jurkat , Leucemia/enzimología , Leucemia/genética , N-Glicosil Hidrolasas/fisiología , ARN Mensajero/análisis , Temozolomida , Transcripción Genética/efectos de los fármacos , Triazenos/farmacologíaRESUMEN
Cell killing by monofunctional methylating agents is due mainly to the formation of adducts at the O6 position of guanine. These methyl adducts are removed from DNA by the O6-alkylguanine DNA alkyltransferase (OGAT). The mechanism by which O6-methylguanine (O6meG) induces cell death in OGAT-deficient cells requires a functional mismatch repair system (MRS). We have previously reported that depletion of OGAT activity in the human T-cell leukemic urkat line does not sensitize these cells to the cytotoxic and apoptotic effects of the methylating triazene temozolomide (Tentori et al., 1995). We therefore decided to establish whether the tolerance of Jurkat cells to O6meG could be associated with a defect in MRS. The results of mismatch repair complementation studies indicated that Jurkat cells are defective in hMutSalpha, a heterodimer of the hMSH2 and hMSH6 proteins. Cytogenetic analysis of two Jurkat clones revealed a deletion in the short arm of chromosome region 2p15-21, indicating an allelic loss of both hMSH2 and hMSH6 genes. DNA sequencing revealed that exon 13 of the second hMSH2 allele contains a base substitution at codon 711, which changes an arginine to a termination codon (CGA-->TGA). In addition, a (C)8-->(C)7 frameshift mutation in codon 1085-1087 of the hMSH6 gene was also found. Although both hMSH2 and hMSH6 transcripts could be detected in Jurkat clones, the respective polypeptides were absent. Taken together, these data indicate that tolerance of Jurkat cells to methylation damage is linked to a loss of functional hMutSalpha.
Asunto(s)
Disparidad de Par Base , Metilación de ADN/efectos de los fármacos , Reparación del ADN , Proteínas de Unión al ADN/genética , Leucemia de Células T/genética , Mutación/genética , Proteínas Proto-Oncogénicas/genética , Resistencia a Antineoplásicos , Humanos , Células Jurkat , Leucemia de Células T/tratamiento farmacológico , Proteína 2 Homóloga a MutS , Células Tumorales CultivadasRESUMEN
A study of phenotypic and genotypic characteristics was carried out on 182 strains isolated from soil of different geographical areas; the type strains were B. licheniformis, B. subtilis, B. pumilus, B. cereus and B. coagulans. The results showed, primarily on the basis of phenotypic features, that all the isolates belonged to the B. licheniformis species, however DNA relatedness studies revealed only 161 to be genetically related to B. licheniformis, the DNA relatedness levels ranging from 66 to 100%. The other 21 isolates appeared to be genetically distinct not only from B. licheniformis but also from B. subtilis and B. pumilus, where there were low levels of DNA relatedness (from 4 to 37%). Nevertheless the ARDRA results indicate that the 21 atypical isolates were phylogenetically related to B. licheniformis. Our data and the phenotypic homogeneity found suggest the presence of three different genomovars.