RESUMEN
PURPOSE: To evaluate γ-H2AX foci as a pharmacodynamic marker for DNA damage induced by DNA interstrand cross-linking drugs. EXPERIMENTAL DESIGN: γ-H2AX foci formation was validated preclinically in comparison with the Comet assay, and evaluated pharmacodynamically in two phase I studies of different dosing schedules of the novel cross-linking agent SJG-136 (SG2000). RESULTS: The measurement of γ-H2AX foci in human fibroblasts and lymphocytes in vitro was more than 10-fold more sensitive than Comet assay measurement of cross-linking, with peak γ-H2AX response 24 hours after the peak of cross-linking. In lymphocytes from a phase I study (every three week schedule), γ-H2AX foci were detectable 1 hour following the end of administration, and in all patients, maximum response was observed at 24 hours. Significant levels of foci were still evident at days 8 and 15 consistent with the known persistence of the DNA damage produced by this agent. In two tumor biopsy samples, foci were detected 4 hours postinfusion with levels higher than in lymphocytes. Extensive foci formation was also observed before the third dose in cycle 1 in lymphocytes from a second phase I study (daily × 3 schedule). These foci also persisted with a significant level evident before the second cycle (day 21). An increased γ-H2AX response was observed during the second cycle consistent with a cumulative pharmacodynamic effect. No clear relationship between foci formation and administered drug dose was observed. CONCLUSION: This is the first use of γ-H2AX as a pharmacodynamic response to a DNA cross-linking agent in a clinical trial setting.
Asunto(s)
Benzodiazepinonas/farmacología , Daño del ADN/efectos de los fármacos , Histonas/genética , Pirroles/farmacología , Ensayo Cometa , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Histonas/metabolismo , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , FarmacogenéticaRESUMEN
BACKGROUND: Radiotherapy-induced DNA double-strand breaks (DSBs) are critical cytotoxic lesions. Inherited defects in DNA DSB repair pathways lead to hypersensitivity to ionising radiation, immunodeficiency and increased cancer incidence. A patient with xeroderma pigmentosum complementation group C, with a scalp angiosarcoma, exhibited dramatic clinical radiosensitivity following radiotherapy, resulting in death. A fibroblast cell line from non-affected skin (XP14BRneo17) was hypersensitive to ionising radiation and defective in DNA DSB repair. AIM: To determine the genetic defect causing cellular radiation hypersensitivity in XP14BRneo17 cells. METHODS: Functional genetic complementation whereby copies of human chromosomes containing genes involved in DNA DSB repair (chromosomes 2, 5, 8 10, 13 and 22) were individually transferred to XP14BRneo17 cells in an attempt to correct the radiation hypersensitivity. Clonogenic survival assays and gamma-H2AX immunofluorescence were conducted to measure radiation sensitivity and repair of DNA DSBs. DNA sequencing of defective DNA repair genes was performed. RESULTS: Transfer of chromosome 8 (location of DNA-PKcs gene) and transfection of a mammalian expression construct containing the DNA-PKcs cDNA restored normal ionising radiation sensitivity and repair of DNA DSBs in XP14BRneo17 cells. DNA sequencing of the DNA-PKcs coding region revealed a 249-bp deletion (between base pairs 3656 and 3904) encompassing exon 31 of the gene. CONCLUSION: We provide evidence of a novel splice variant of the DNA-PKcs gene associated with radiosensitivity in a patient with xeroderma pigmentosum and report the first double mutant in distinct DNA repair pathways being consistent with viability.
Asunto(s)
Proteína Quinasa Activada por ADN/fisiología , Neoplasias de Cabeza y Cuello/radioterapia , Hemangiosarcoma/radioterapia , Proteínas Nucleares/fisiología , Tolerancia a Radiación/genética , Neoplasias Cutáneas/radioterapia , Xerodermia Pigmentosa/genética , Empalme Alternativo/fisiología , Secuencia de Aminoácidos , Supervivencia Celular/genética , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Proteína Quinasa Activada por ADN/genética , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Hemangiosarcoma/genética , Hemangiosarcoma/patología , Humanos , Isoenzimas/genética , Isoenzimas/fisiología , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Traumatismos por Radiación/genética , Cuero Cabelludo , Homología de Secuencia de Aminoácido , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Xerodermia Pigmentosa/patologíaRESUMEN
PURPOSE: This phase I dose-escalation study was undertaken to establish the maximum tolerated dose of the sequence-selective minor groove DNA binding agent SJG-136 in patients with advanced solid tumors. The study also investigated antitumor activity and provided pharmacokinetic and pharmacodynamic data. EXPERIMENTAL DESIGN: Sixteen patients were assigned sequentially to escalating doses of SJG-136 (15-240 microg/m(2)) given as a 10-minute i.v. infusion every 21 days. The dose was subsequently reduced in incremental steps to 45 microg/m(2) due to unexpected toxicity. RESULTS: The maximum tolerated dose of SJG-136 was 45 microg/m(2). The main drug-related adverse event was vascular leak syndrome (VLS) characterized by hypoalbuminemia, pleural effusions, ascites, and peripheral edema. Other unexpected adverse events included elevated liver function tests and fatigue. The VLS and liver toxicity had delayed onset and increased in severity with subsequent cycles. Disease stabilization was achieved for >6 weeks in 10 patients; in 2 patients this was maintained for >12 weeks. There was no evidence of DNA interstrand cross-linking in human blood lymphocytes with the use of the comet assay. Evidence of DNA interaction in lymphocytes and tumor cells was shown through a sensitive gamma-H2AX assay. SJG-136 had linear pharmacokinetics across the dose range tested. CONCLUSIONS: SJG-136 was associated with dose-limiting VLS and hepatotoxicity when administered by short injection every 21 days. DNA damage was noted, at all dose levels studied, in circulating lymphocytes. The etiology of the observed toxicities is unclear and is the subject of further preclinical research. Alternative clinical dosing strategies are being evaluated.
Asunto(s)
Antineoplásicos/uso terapéutico , Benzodiazepinonas/uso terapéutico , Neoplasias/tratamiento farmacológico , Pirroles/uso terapéutico , Adulto , Anciano , Benzodiazepinonas/efectos adversos , Benzodiazepinonas/farmacocinética , ADN/metabolismo , Histonas/análisis , Humanos , Dosis Máxima Tolerada , Persona de Mediana Edad , Pirroles/efectos adversos , Pirroles/farmacocinéticaRESUMEN
Neutrophil elastase is a serine protease stored in the azurophilic granules of leukocytes. It has been implicated in the pathology of several lung diseases and is generally presumed to contribute to the tissue destruction and extracellular matrix damage associated with these conditions. To delineate the role of neutrophil elastase in pulmonary inflammation and fibrosis, neutrophil elastase-null mice were intratracheally instilled with bleomycin. In neutrophil elastase-null mice, biochemical and morphological characteristics of pulmonary fibrosis were attenuated for at least 60 days after bleomycin administration despite a typical response to bleomycin as evidenced by assessment of indices of DNA and cell damage. Neutrophil burden of bleomycin-treated wild-type and neutrophil elastase-null mice was comparable, and marked neutrophilic alveolitis was manifest in bleomycin-treated neutrophil elastase-null mice. An absence of immunostaining for active transforming growth factor (TGF)-beta in lung tissue from bleomycin-treated neutrophil elastase-null mice suggested a defect in TGF-beta activation, which was confirmed by biochemical assessment of TGF-beta levels in bronchoalveolar lavage fluid and lung tissue. These data point to novel and unexpected fibrogenic consequences of neutrophil elastase activity in the inflamed lung.
Asunto(s)
Bleomicina/toxicidad , Elastasa de Leucocito/genética , Fibrosis Pulmonar , Animales , Líquido del Lavado Bronquioalveolar , Daño del ADN/efectos de los fármacos , Resistencia a Medicamentos/genética , Elastasa de Leucocito/deficiencia , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/inmunología , Fibrosis Pulmonar/patología , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Xeroderma pigmentosum (XP) is characterised by defects in nucleotide excision repair, ultraviolet (UV) radiation sensitivity and increased skin carcinoma. Compared to other complementation groups, XP-F patients show relatively mild cutaneous symptoms. DNA interstrand cross-linking agents are a highly cytotoxic class of DNA damage induced by common cancer chemotherapeutics such as cisplatin and nitrogen mustards. Although the XPF-ERCC1 structure-specific endonuclease is required for the repair of ICLs cellular sensitivity of primary human XP-F cells has not been established. In clonogenic survival assays, primary fibroblasts from XP-F patients were moderately sensitive to both UVC and HN2 compared to normal cells (2- to 3-fold and 3- to 5-fold, respectively). XP-A fibroblasts were considerably more sensitive to UVC (10- to 12-fold) but not sensitive to HN2. The sensitivity of XP-F fibroblasts to HN2 correlated with the defective incision or 'unhooking' step of ICL repair. Using the comet assay, XP-F cells exhibited only 20% residual unhooking activity over 24 h. Over the same time, normal and XP-A cells unhooked greater than 95% and 62% of ICLs, respectively. After HN2 treatment, ICL-associated DNA double-strand breaks (DSBs) are detected by pulse field gel electrophoresis in dividing cells. Induction and repair of DNA DSBs was normal in XP-F fibroblasts. These findings demonstrate that in primary human fibroblasts, XPF is required for the unhooking of ICLs and not for the induction or repair of ICL-associated DNA DSBs induced by HN2. In terms of cancer chemotherapy, people with mild DNA repair defects affecting ICL repair may be more prevalent in the general population than expected. Since cellular sensitivity of primary human fibroblasts usually reflects clinical sensitivity such patients with cancer would be at risk of increased toxicity.
Asunto(s)
Reactivos de Enlaces Cruzados/farmacología , Daño del ADN/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Xerodermia Pigmentosa/patología , Técnicas de Cultivo de Célula , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , ADN/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Humanos , Mecloretamina/farmacología , Rayos Ultravioleta/efectos adversosRESUMEN
SJG-136, a pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer, is a highly efficient interstrand crosslinking agent that reacts with guanine bases in a 5'-GATC-3' sequence in the DNA minor groove. SJG-136 crosslinks form rapidly and persist compared to those produced by conventional crosslinking agents such as nitrogen mustard, melphalan or cisplatin which bind in the DNA major groove. A panel of Chinese hamster ovary (CHO) cells with defined defects in specific DNA repair pathways were exposed to the bi-functional agents SJG-136 and melphalan, and to their mono-functional analogues mmy-SJG and mono-functional melphalan. SJG-136 was >100 times more cytotoxic than melphalan, and the bi-functional agents were much more cytotoxic than their respective mono-functional analogues. Cellular sensitivity of both SJG-136 and melphalan was dependent on the XPF-ERCC1 heterodimer, and homologous recombination repair factors XRCC2 and XRCC3. The relative level of sensitivity of these repair mutant cell lines to SJG-136 was, however, significantly less than with major groove crosslinking agents. In contrast to melphalan, there was no clear correlation between sensitivity to SJG-136 and crosslink unhooking capacity measured using a modified comet assay. Furthermore, repair of SJG-136 crosslinks did not involve the formation of DNA double-strand breaks. SJG-136 cytotoxicity is likely to result from the poor recognition of DNA damage by repair proteins resulting in the slow repair of both mono-adducts and more importantly crosslinks in the minor groove.
Asunto(s)
Antineoplásicos/toxicidad , Benzodiazepinonas/toxicidad , Reactivos de Enlaces Cruzados/toxicidad , Reparación del ADN , Proteínas de Unión al ADN/fisiología , Endonucleasas/fisiología , Melfalán/análogos & derivados , Pirroles/toxicidad , Recombinación Genética , Animales , Antineoplásicos/química , Benzodiazepinas/química , Benzodiazepinas/toxicidad , Benzodiazepinonas/química , Células CHO , Supervivencia Celular , Cricetinae , Cricetulus , Reactivos de Enlaces Cruzados/química , Daño del ADN , Melfalán/química , Melfalán/toxicidad , Pirroles/químicaRESUMEN
SJG-136 (NSC 694501) is a rationally designed pyrrolobenzodiazepine dimer that binds in the minor groove of DNA. It spans 6 bp with a preference for binding to purine-GATC-pyrimidine sequences. The agent has potent activity in the National Cancer Institute (NCI) anticancer drug screen with 50% net growth inhibition conferred by 0.14 to 320 nmol/L (7.4 nmol/L mean). Sensitive cell lines exhibit total growth inhibition and 50% lethality after treatment with as little as 0.83 and 7.1 nmol/L SJG-136, respectively. COMPARE and molecular target analysis of SJG-136 data versus that of >60,000 compounds tested in the NCI 60 cell line screen shows that, although the agent has similarity to other DNA binding agents, the pattern of activity for SJG-136 does not fit within the clusters of any known agents, suggesting that SJG-136 possesses a distinct mechanism of action. Testing in the NCI standard hollow fiber assay produced prominent growth inhibition in 20 of 24 i.p. and 7 of 24 s.c. test combinations with 5 of 12 cell lines exhibiting cell kill. In addition, SJG-136 produced antitumor activity in mice bearing CH1 and CH1cisR xenografts, a cisplatin-resistant human ovarian tumor model, and also in mice bearing LS174T xenografts, a human colon tumor model. SJG-136 produces DNA interstrand cross-links between two N-2 guanine positions on opposite strands and separated by 2 bp. In human tumor cell lines, the cross-links form rapidly and persist compared with those produced by conventional cross-linking agents such as nitrogen mustards. In mice bearing the LS174T human colon xenograft, DNA interstrand cross-links can be detected in tumor cells using a modification of the single cell gel electrophoresis (comet) assay after administration of a therapeutic dose. Cross-links in the tumor increase with dose and are clearly detectable at 1 hour after i.v. administration. The level of cross-linking persists over a 24-hour period in this tumor in contrast to cross-links produced by conventional cross-linking agents observed over the same time period.
Asunto(s)
Antineoplásicos/farmacología , Benzodiazepinonas/farmacología , Reactivos de Enlaces Cruzados/farmacología , ADN/efectos de los fármacos , Pirroles/farmacología , Animales , Benzodiazepinas/farmacología , Línea Celular Tumoral , Ensayo Cometa , ADN/metabolismo , Perros , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The anticancer drug cisplatin reacts with DNA leading to the formation of interstrand and intrastrand cross-links that are the critical cytotoxic lesions. In contrast to cells bearing mutations in other components of the nucleotide excision repair apparatus (XPB, XPD, XPG and CSB), cells defective for the ERCC1-XPF structure-specific nuclease are highly sensitive to cisplatin. To determine if the extreme sensitivity of XPF and ERCC1 cells to cisplatin results from specific defects in the repair of either intrastrand or interstrand cross-links we measured the elimination of both lesions in a range of nucleotide excision repair Chinese hamster mutant cell lines, including XPF- and ERCC1-defective cells. Compared to the parental, repair-proficient cell line all the mutants tested were defective in the elimination of both classes of adduct despite their very different levels of increased sensitivity. Consequently, there is no clear relationship between initial incisions at interstrand cross-links or removal of intrastrand adducts and cellular sensitivity. These results demonstrate that the high cisplatin sensitivity of ERCC1 and XPF cells likely results from a defect other than in excision repair. In contrast to other conventional DNA cross-linking agents, we found that the repair of cisplatin adducts does not involve the formation of DNA double-strand breaks. Surprisingly, XRCC2 and XRCC3 cells are defective in the uncoupling step of cisplatin interstrand cross-link repair, suggesting that homologous recombination might be initiated prior to excision of this type of cross-link.