We have isolated a multiprotein complex for DNA synthesis, designated the DNA synthesome, from human breast cancer (MDA MB-468) cells, biopsied human breast tumor tissue and xenografts from nude mice injected with the human breast cancer cell line MCF-7. The breast cell DNA synthesome was shown to fully support the in vitro replication of simian virus 40 (SV40) origin-containing DNA in the presence of the viral large T-antigen. Moreover, our results obtained from a forward mutagenesis assay indicate that the DNA synthesome isolated from malignant breast cells possesses a lower fidelity for DNA replication in vitro than the complex from a nonmalignant breast cell line. The proteins and enzymes found to copurify with the breast cell DNA synthesome include: DNA polymerases alpha, delta, and epsilon, DNA primase, proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), replication protein A (RP-A), DNA ligase, DNA topoisomerases I and II and poly(ADP-ribose) polymerase. To begin to determine the organization of these DNA synthetic proteins within the breast cell DNA synthesome, we performed co-immunoprecipitation experiments with antibodies directed against DNA polymerases alpha, delta and PCNA. We found that DNA polymerases alpha, delta, DNA primase, RF-C and PCNA tightly associate with each other in the complex, whereas DNA polymerase epsilon, PARP and several other components interact with the synthesome via an interaction with only PCNA or DNA polymerase alpha. Furthermore, we employed the breast cell DNA synthesome as a model to study the mechanisms of action of two anti-breast cancer agents that target the DNA synthetic process, irinotecan (CPT-11/SN-38) and etoposide (VP-16). We obtained novel data suggesting that both SN-38 and VP-16 stabilized cleavable complexes represent blocks to replication fork progression, as each agent caused an accumulation of short DNA products during synthesome mediated in vitro replication. Overall, our results indicate that breast cancer cells utilize an asymmetric multiprotein complex to mediate DNA synthesis and that utilization of the DNA synthesome as a drug model may provide important new insights into the mechanisms of action of SN-38 and VP-16.
The development of a malignancy is a multistep process that is not clearly understood. Nonmalignant cells undergo a transformation process resulting in aberrantly proliferating malignant cells suggesting that the replication machinery of malignant cells is altered. In this study the DNA replication apparatus (the DNA synthesome) was examined to identify proteins altered in malignant cells. Analysis of malignant and nonmalignant cells by two dimensional gel electrophoresis (2D PAGE) demonstrated that many malignant cell types contain a unique, acidic form of proliferating cell nuclear antigen (PCNA). This protein is a 36 kD nuclear protein required for DNA replication and DNA repair. The unique form of PCNA was found in malignant breast, prostate, colon, cervical, brain and leukemia cell lines and in malignant human breast tumors and chronic myelogenous leukemia specimens. Serum collected from a breast cancer patient was analyzed and found to contain the cancer specific form of PCNA. Analysis of nonmalignant breast tissue and serum collected from cancer free individuals demonstrated that the cancer specific form of PCNA was not present. Further experiments were performed to characterize the unique form of PCNA. 2D PAGE analysis was performed on nonmalignant, transformed breast cell lines overexpressing the oncogenes c-myc (A1N4 myc) and SV40T (A1N4T). The cancer specific form of PCNA was present in these transformed cells. It was also determined that the acidic form of PCNA was not the result of growth stimulation or genetic mutation, suggesting that differential post-translational modification may be responsible. Although PCNA does undergo poly (ADP-ribosylation), 2D PAGE analysis demonstrated that the cancer specific form of PCNA was unmodified. The cancer specific form of PCNA appears to be a fundamental characteristic of malignancy and its role in tumorigenesis needs to be examined. These results suggest that epigenetic changes may contribute to the development of cancer.
The physiological changes leading to exercise-mediated inhibition of hepatic cytochromes P450 have not been characterized. There is clinical evidence of increased release of cytokines (mediators of the immune system) after exercise. In addition, the modulation of P450 enzymes by nitric oxide (NOdot), another mediator of the immune system, has been demonstrated both in vivo and in vitro. Therefore, a series of experiments were designed to characterize the involvement of the immune system in exercise-mediated changes in P450. These experiments further evaluated the independent effects of exercise on the components of phase I-mediated drug metabolism. In vitro studies demonstrated that Spermine/NO (an NOdot donor) dramatically inhibited microsomal activity towards several P450 isozyme-specific substrates. In vivo studies with rats showed that treadmill running also resulted in an inhibition of P450 enzymatic activity towards substrates specific for P4502 family. These inhibitions were associated with a significant decrease in P450 reductase enzymatic activity and only small decreases in enzyme expression. Treatment of exercising rats with low levels of the antiinflamatory dexamethasone resulted in partial reversal of the inhibition. Dexamethasone alone produced no significant changes on the parameters measured. In addition, exercise was associated with an increase in the hepatic expression of the inducible form of nitric oxide synthase. These data support our hypothesis that the immune system is activated as a result of exercise. Further, the data support that exercise-induced alterations in immune mediators (i.e. NOdot) inhibit specific P450 metabolic pathways.
The Chinese hamster lung cell line DC-3F8/A55 has a 4,500-fold increase in resistance to methotrexate over the parental cell line DC-3F. Although DC- 3F8/A55 cells have a 4.5-fold increase in a mutant form of dihydrofolate reductase, this does not fully account for the high level of resistance to methotrexate. The purpose of this study is to determine the molecular basis for the inability of DC-3F8/A55 cells to accumulate methotrexate, and to identify the mechanism of transport of folate compounds in DC-3F8/A55 cells. This work has revealed that DC-3F8/A55 cells harbor a debilitating mutation to the reduced folate carrier gene, resulting in the loss of reduced folate carrier function. A nonsense mutation changes an arginine at amino acid 88 to a STOP codon, resulting in a non-functional protein. The parental cell line DC-3F is heterozygous at this locus, possessing one mutant and one wild-type allele of the RFC gene, thus retaining reduced folate carrier activity. These facts are supported by the kinetics of folate transport in both of these cell lines. The parental cell line DC-3F has a Kt for folinic acid of 10.69 +/- 0.67 muM and for methotrexate of 8.88 +/- 0.82 muM, values characteristic of a cell expressing a reduced folate carrier. DC-3F8/A55 cells were found to have a Kd for MTX of 3.16 +/- 1.03 nM, for folinic acid of 7.75 +/- 2.16 nM, and for folic acid of 1.42 +/- 0.54 nM. The high affinity of DC-3F8/A55 cells for folic acid, with a Kd for folic acid in the nM range, suggests that these cells are expressing a folate receptor. Northern blot analysis revealed a 1.6 kb transcript with low homology to FR-alpha and FR-gamma in DC-3F8/A55 cells. Overall, these studies suggest that the methotrexate transport-defective cell line DC-3F8/A55 expresses a previously unidentified folate receptor which may be a new member of the folate receptor family.
The goal of this dissertation was to investigate three new curve comparison metrics, the Rescigno Index, fl, and the Chinchilli Metric as tools to compare pharmacokinetic profiles for the assessment of assess relative bioavailability (BA) and bioequivalence (BE). The specific objectives were to (1) compare the relative sensitivity of the new metrics to detect differences in AUC and Cmax as a function of the pharmacokinetics of the drug products, and (2) to estimate relative bioavailability and bioequivalence. Methods. Retrospective analysis of experimental data and Monte Carlo simulations of bioequivalence trials were used to evaluate the relative sensitivity of the metrics to detect profile differences. The experimental data study involved determining the degree of discordance with typical criteria when judging individual profiles to be the same or different, and then examining the relationship between the degree of discordance and the pharmacokinetics of the drug product. The simulation studies involved determining the proportion of clinical studies failing bioequivalence under different pharmacokinetic models. Product bioequivalence was estimated using data from 35 typical 2 treatment-2 period bioequivalence study experimental datasets. Three different bioequivalence limits were applied to the curve metrics. Results. The new metrics more effectively detect differences in absorption time lags but less effectively detect differences in Cmax under some conditions. The relative sensitivity to Cmax depends on the shape of the curve, where increasing the ka(ref)/ke(ref) increases the disparity across the metrics. The curve metrics show increased sensitivity to variability in disposition, elimination, and random residual error, but comparable sensitivity to differences in bioavailability. Fourteen of the 35 studies failed typical criteria (AUC and Cmax). Applying bioequivalence limits of 25%, 21 and 26 studies failed the Chinchilli and fl criteria respectively. At the 30% limit, 14 and 20 studies failed the Chinchilli and fl criteria respectively. The specific studies failing each criterion varied. The within-subject variability of the Chinchilli Metric was higher than Cmax. Both the Chinchilli Metric and fl showed a tendency toward extreme values. Conclusions. The metrics differ in pharmacokinetic sensitivities and differ in statistical properties. There are advantages and disadvantages associated with these differences.
The objective of this work was to examine the use of tissue slices as an in vitro model for the study of integrated drug metabolism (biotransformation). Liver slices are more representative of the in vivo situation with respect to cellular architecture and diversity than hepatocytes or subcellular fractions. This work has demonstrated the effects of model inducing agents on hepatic phase I-phase II integrated drug metabolism using alkoxycoumarin derivatives. Additionally, this work detailed the development of an in vitro model for novel compound screening using acetaminophen and paraquat as model toxicants. Dynamic incubation of liver slices was found to be superior to a linear incubation over a 2 hour incubation. Biotransformation was assessed with the use of 7-methoxy-, 7-ethoxy- and 7-hydroxycoumarin (7-HC). The major metabolite of O-dealkylase activities of cytochrome P 450, 7-HC, was conjugated with glucuronic acid or sulfate moieties in a capacity limited fashion. Total phase II activity was assessed using 7-HC as the primary substrate and found to be 7-fold higher than total phase I activity. Phenobarbital pretreatment induced liver slice O-demethylase and O-deethylase activities 3.1- and 3.6-fold over control values, respectively. Glucuronosyl transferase activity for 7-HC was found to be increased over sulfation in all metabolite profiles by pretreatment with phenobarbital. 3-methylcholanthrene pretreatment showed a novel induction profile with a 1.8-fold increase in O-demethylase activity and 9-fold increase in O-deethylase activity over control rat liver slices. Increased levels of free 7-HC from O-dealkylase activities indicated possible substrate competition from persistence of 3-methylcholanthrene or its hydroxylated metabolites in the liver slice. The utility of liver slices as a paradigm to investigate cytotoxicity by intracellular enzyme release was detailed with the use of acetaminophen and paraquat. Phenobarbital was shown to potentiate mitochondrial damage by oxidative mechanisms. These studies highlight the utility and versatility of liver slices in the analysis of biotransformation and bioactivation. Implementation of the tissue slice system as a routine screen may facilitate drug discovery and development.
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