ABSTRACT
DNA damage can lead to either DNA repair with cell survival or to apoptotic cell death. Although the biochemical processes underlying DNA repair and apoptosis have been extensively studied, the mechanisms by which cells determine whether the damage will be repaired or the apoptotic pathway will be activated is largely unknown. We have studied the role of nucleotide excision repair (NER) in cisplatin DNA damage-induced apoptotic cell death using both normal human fibroblasts and NER-defective xeroderma pigmentosum (XP) XPA and XPG cells. The caspase-3 activation experiment demonstrated a greatly increased casapse-3 activation in the NER-defective cells following cisplatin treatment. The flow cytometry experiment revealed an altered cell cycle arrest pattern of the NER-defective cells following cisplatin treatment. The results obtained from the Western blot experiment showed that NER defects resulted in enhanced CHK1 phosphorylation and p21 induction after cisplatin treatment. The cisplatin treatment-induced ATM phosphorylation, however, was attenuated in NER-defective cells. The results obtained from our immunoprecipitation experiment further demonstrated that the ATM protein interacted with the TFIIH basal transcription factor and the XPG protein of the NER pathway. It also showed that a functional XPC protein was required for the association of the ATM protein to genomic DNA. These results suggest that the NER process may prevent the cisplatin treatment-induced apoptosis by activating the ATM protein, and that the presence of the XPC protein is essential for recruiting the ATM protein to the DNA template.
Subject(s)
Cell Cycle Proteins/chemistry , Cisplatin/pharmacology , DNA Repair , DNA-Binding Proteins/chemistry , Nucleotides/chemistry , Protein Serine-Threonine Kinases/chemistry , Tumor Suppressor Proteins/chemistry , Antineoplastic Agents/pharmacology , Apoptosis , Ataxia Telangiectasia Mutated Proteins , Caspase 3 , Caspases/metabolism , Cell Cycle Proteins/metabolism , Cell Survival , Checkpoint Kinase 1 , DNA Damage , DNA-Binding Proteins/metabolism , Enzyme Activation , Fibroblasts/metabolism , Humans , Phosphorylation , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolismABSTRACT
Polymethylmethacrylate bone cement beads impregnated with antibiotic are a common treatment for patients with persistent articular joint infections or osteomyelitis. They also are used as a prophylaxis for infection in patients with large soft tissue wounds. The current study was designed to evaluate the relationship between bead geometry and elution of the antibiotic tobramycin by methodically varying the shape of the beads for a given set of volumes. Beads of five shapes (spherical to ovoid) and two volumes were prepared and studied. Only 0.9% to 3.3% of the total amount of tobramycin present actually eluted from the beads in a 96-hour period and of this amount, approximately 1/3 eluted within the first 4 hours. The elution mass data indicate the benefit of numerous, small and elliptically shaped beads for maximal antibiotic availability. Additionally, a mathematical model is presented that describes these findings and can be used to predict tobramycin delivery rates from bone cement beads. This model assumes that the antibiotic is delivered through two mechanisms: fast dissolution of tobramycin initially adhering to the bead surface and slow release by diffusion through the polymer. The results generate diffusion coefficients for tobramycin in polymethylmethacrylate bone cement on the order of 2 x 10 cm/s.
