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Multidrug resistance (MDR) is one of the foremost causes of treatment failure in childhood malignancies. MDR is a multifactorial process, but classic resistance to cytotoxic drugs has most often been associated with over-expression of one or more MDR transporter proteins in malignant cells, conferring on them the ability to extrude an extraordinarily diverse array of endo- and xenobiotics out of the cell. The best characterized multidrug transporters, P-glycoprotein (Pgp) and the Multidrug Resistance-associated Protein (MRP), belong to the ATP-binding cassette (ABC) gene superfamily, and have been previously implicated in the development of drug resistance in the clinical context. The work described herein examined the various aspects of the MDR genotype and phenotype in the childhood malignancies acute lymphoblastic leukaemia (ALL) and neuroblastoma. The first series of studies tested the hypothesis that morphine, a potential Pgp substrate, might influence the efficacy and/or toxicity of clinically used chemotherapy agents which are substrates for Pgp. The results, however, provided no evidence in a variety of human tumour cell lines of morphine influencing response to selected chemotherapeutic drugs. This finding is particularly important as morphine remains to be the opioid of choice for the treatment of cancer pain in the clinic. The second series of studies examined the effect of single nucleotide polymorphisms (SNPs) in the MDR1 gene, encoding Pgp, and in the MRP1 gene, on patient outcome in childhood ALL or neuroblastoma, with a view to identifying novel prognostic markers for these malignancies. It was found that two of the examined SNPs in the MRP1 gene were associated with improved outcome in neuroblastoma, which had not previously been demonstrated in this disease. Moreover, each of the relevant MRP1 SNPs were associated with lower MRP1 gene expression in both patient samples and tumour cell lines, supporting previous studies indicating that low MRP1 expression in neuroblastoma is strongly associated with improved patient outcome. Importantly, the results of this study suggest a role for selected MRP1 polymorphisms in predicting clinical response in neuroblastoma. Finally, a series of studies were undertaken, using both in vitro and in vivo model systems, to test the efficacy of putative small molecule inhibitors of the MRP1 gene and its transcriptional regulator, the MYCN oncogene, in neuroblastoma. These studies demonstrated for the first time the efficacy of a novel compound, 4H10, at reversing multidrug resistance either in cultured neuroblastoma cells, or in the MYCN transgenic mice, which develop neuroblastoma that closely mirror the human disease. The results indicate that inhibition of MRP1 function has potential clinical importance in the treatment of neuroblastoma, and therefore warrant further research in this area. In contrast, the results failed to provide evidence of the in vivo efficacy of the novel putative small molecule MYCN inhibitors analysed in these studies. Collectively, the findings of these studies contribute to a better understanding of the mechanisms of clinical drug resistance, and may help in the development of new approaches for risk assessment and treatment of aggressive childhood malignancies and thereby improve the long-term outlook of children diagnosed with these debilitating diseases.

Multidrug resistance (MDR) is one of the foremost causes of treatment failure in childhood malignancies. MDR is a multifactorial process, but classic resistance to cytotoxic drugs has most often been associated with over-expression of one or more MDR transporter proteins in malignant cells, conferring on them the ability to extrude an extraordinarily diverse array of endo- and xenobiotics out of the cell. The best characterized multidrug transporters, P-glycoprotein (Pgp) and the Multidrug Resistance-associated Protein (MRP), belong to the ATP-binding cassette (ABC) gene superfamily, and have been previously implicated in the development of drug resistance in the clinical context. The work described herein examined the various aspects of the MDR genotype and phenotype in the childhood malignancies acute lymphoblastic leukaemia (ALL) and neuroblastoma. The first series of studies tested the hypothesis that morphine, a potential Pgp substrate, might influence the efficacy and/or toxicity of clinically used chemotherapy agents which are substrates for Pgp. The results, however, provided no evidence in a variety of human tumour cell lines of morphine influencing response to selected chemotherapeutic drugs. This finding is particularly important as morphine remains to be the opioid of choice for the treatment of cancer pain in the clinic. The second series of studies examined the effect of single nucleotide polymorphisms (SNPs) in the MDR1 gene, encoding Pgp, and in the MRP1 gene, on patient outcome in childhood ALL or neuroblastoma, with a view to identifying novel prognostic markers for these malignancies. It was found that two of the examined SNPs in the MRP1 gene were associated with improved outcome in neuroblastoma, which had not previously been demonstrated in this disease. Moreover, each of the relevant MRP1 SNPs were associated with lower MRP1 gene expression in both patient samples and tumour cell lines, supporting previous studies indicating that low MRP1 expression in neuroblastoma is strongly associated with improved patient outcome. Importantly, the results of this study suggest a role for selected MRP1 polymorphisms in predicting clinical response in neuroblastoma. Finally, a series of studies were undertaken, using both in vitro and in vivo model systems, to test the efficacy of putative small molecule inhibitors of the MRP1 gene and its transcriptional regulator, the MYCN oncogene, in neuroblastoma. These studies demonstrated for the first time the efficacy of a novel compound, 4H10, at reversing multidrug resistance either in cultured neuroblastoma cells, or in the MYCN transgenic mice, which develop neuroblastoma that closely mirror the human disease. The results indicate that inhibition of MRP1 function has potential clinical importance in the treatment of neuroblastoma, and therefore warrant further research in this area. In contrast, the results failed to provide evidence of the in vivo efficacy of the novel putative small molecule MYCN inhibitors analysed in these studies. Collectively, the findings of these studies contribute to a better understanding of the mechanisms of clinical drug resistance, and may help in the development of new approaches for risk assessment and treatment of aggressive childhood malignancies and thereby improve the long-term outlook of children diagnosed with these debilitating diseases.

Multidrug resistance protein 1 (MRP1) is an integral membrane protein belonging to the ATP-binding cassette (ABC) superfamily that utilizes ATP binding and hydrolysis to transport various endogenous substrates and/or xenobiotics across membranes against a concentration gradient. The overall goal of my research was to examine the nucleotide and protein interactions of MRP1 using various biochemical methods. In the first study, Cu2+(Ph)3 which promotes cross-linking of two nearby Cys residues and limited proteolysis were used to study conformational changes of MRP1 at different stages of ATP binding and hydrolysis at the nucleotide binding domains (NBDs). The limited trypsin digestion patterns indicated that some Cys residues of MRP1 could be cross-linked in the nucleotide-free state and that the Cys cross-linked MRP1 was more susceptible to trypsinolysis. Furthermore, binding of ATP, AMP-PNP, and trapping of ADP by MRP1 prevented the cross-linking events from occurring, but binding of ATPγS did not. However, the ATPγS-bound MRP1, like nucleotide-free MRP1, showed enhanced sensitivity towards trypsinolysis. These studies show that the two ATP analogs, AMP-PNP and ATPγS, interact with MRP1 in different ways. In the second study, the interaction of MRP1 with other cellular proteins was examined. An in vivo chemical cross-linking approach combined with affinity purification and MS analysis was initially used to identify protein partners directly interacting with MRP1. When this approach proved unsuccessful, a second approach involving immunoaffinity purification of MRP1-containing complexes followed by MS analysis was adopted. Six potential candidate interacting protein partners of MRP1 were identified via this approach and two of them, FUS and drebrin, were further characterized by co-immunoprecipitation and colocalization experiments. FUS seems unlikely to be an important binding partner of MRP1 since confocal and subcellular fractionation studies showed it to be exclusively localized in the nucleus. On the other hand, drebrin depletion by siRNA knock-down resulted in a moderate decrease in MRP1 overall expression levels although the membrane localization of MRP1 remained unchanged. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2008-12-08 17:44:52.767

Multidrug resistance (MDR) is one of the foremost causes of treatment failure in childhood malignancies. MDR is a multifactorial process, but classic resistance to cytotoxic drugs has most often been associated with over-expression of one or more MDR transporter proteins in malignant cells, conferring on them the ability to extrude an extraordinarily diverse array of endo- and xenobiotics out of the cell. The best characterized multidrug transporters, P-glycoprotein (Pgp) and the Multidrug Resistance-associated Protein (MRP), belong to the ATP-binding cassette (ABC) gene superfamily, and have been previously implicated in the development of drug resistance in the clinical context. The work described herein examined the various aspects of the MDR genotype and phenotype in the childhood malignancies acute lymphoblastic leukaemia (ALL) and neuroblastoma. The first series of studies tested the hypothesis that morphine, a potential Pgp substrate, might influence the efficacy and/or toxicity of clinically used chemotherapy agents which are substrates for Pgp. The results, however, provided no evidence in a variety of human tumour cell lines of morphine influencing response to selected chemotherapeutic drugs. This finding is particularly important as morphine remains to be the opioid of choice for the treatment of cancer pain in the clinic. The second series of studies examined the effect of single nucleotide polymorphisms (SNPs) in the MDR1 gene, encoding Pgp, and in the MRP1 gene, on patient outcome in childhood ALL or neuroblastoma, with a view to identifying novel prognostic markers for these malignancies. It was found that two of the examined SNPs in the MRP1 gene were associated with improved outcome in neuroblastoma, which had not previously been demonstrated in this disease. Moreover, each of the relevant MRP1 SNPs were associated with lower MRP1 gene expression in both patient samples and tumour cell lines, supporting previous studies indicating that low MRP1 expression in neuroblastoma is strongly associated with improved patient outcome. Importantly, the results of this study suggest a role for selected MRP1 polymorphisms in predicting clinical response in neuroblastoma. Finally, a series of studies were undertaken, using both in vitro and in vivo model systems, to test the efficacy of putative small molecule inhibitors of the MRP1 gene and its transcriptional regulator, the MYCN oncogene, in neuroblastoma. These studies demonstrated for the first time the efficacy of a novel compound, 4H10, at reversing multidrug resistance either in cultured neuroblastoma cells, or in the MYCN transgenic mice, which develop neuroblastoma that closely mirror the human disease. The results indicate that inhibition of MRP1 function has potential clinical importance in the treatment of neuroblastoma, and therefore warrant further research in this area. In contrast, the results failed to provide evidence of the in vivo efficacy of the novel putative small molecule MYCN inhibitors analysed in these studies. Collectively, the findings of these studies contribute to a better understanding of the mechanisms of clinical drug resistance, and may help in the development of new approaches for risk assessment and treatment of aggressive childhood malignancies and thereby improve the long-term outlook of children diagnosed with these debilitating diseases.

Thesis (Ph. D.)--Colorado State University, 2004. / Includes bibliographical references.

Cancer remains the most cause death disease in Taiwan at least ten years. Liver cancer, which consists predominantly of hepatocellular carcinoma (HCC), is the most common cause of cancer mortality in men and the second most in women. Not only liver section and liver transplantation are used in HCC therapy but also local ablation therapy and transarterial therapy. Transarterial chemoembolization (TACE) is one of the local therapies that inject chemotherapeutic drugs directly into liver tumor. However, drug resistance is the mainly restriction in patient after chemotherapy. Moreover, it is known that drug resistance was associated to over-expression of certain ABC transporter genes, especially ABCB1, ABCG2, and ABCC family in cancer cell and those ABC transporters were also expressed in liver. Base on clinical study, they use 5-fluororuacil, cisplatin and mitomycin-C for liver cancer treatment. In this study, we hypothesized that cancer therapies may be augmented through blocked the drug efflux ABC channels with the ABC transporter inhibitors such as verapamil. The associations among drug treatments, inhibitor incorporation and the expression of ABC transporters were evaluated in HepG2 and Hep3B cells. MTT assay demonstrated that the cell viability was considerable decreased by treating triple drugs with verapamil. RT-PCR data showed that ABC transporters mRNA expression has no significantly change. However, membrane ABCB1 and ABCG2 were induced after drugs and inhibitors treatment either 1 or 24 hours by flow cytometry analysis. P-glycoprotein functional assay also showed p-glycoprotein was inhibited by verapamil, and hence Rhodamine 123 retention was increased. Taken together, there are different response of ABC transporters in HepG2 and Hep3B after drugs and inhibitors treatment. Membrane ABCB1 and ABCG2 were induced by drugs and inhibitors treatment. However, p-glycoprotein¡¦s function was restrained simultaneously by inhibitors treatment. Therefore, verapamil can enhance cell death by inhibiting ABC transporters and its cytotoxic effect rather than the increased expression of ABC transporters. This finding might provide a better way in liver cancer therapy.

The ATP-binding cassette (ABC) transporter superfamily consists of 49 members, to which both Multidrug Resistance Protein 1 (MRP1/gene symbol: ABCC1) and MRP4 (ABCC4) belong. Single nucleotide polymorphisms (SNPs) in drug metabolizing genes have been shown to affect individual responses to drugs and toxins. However, the role of SNPs in modulating the activity of drug transporters, such as MRP1 and MRP4, is poorly characterized. The overall goal of my thesis was to determine the effects of SNPs in the promoter regions of human ABCC1 and ABCC4. For MRP1/ABCC1, two proximal promoter SNPs (-275A>G, -260G>C) were identified in the literature and recreated in vitro, and the activity of the mutant ABCC1 promoter constructs was measured in five human cell lines using a dual luciferase assay. The activity of the -275A>G promoter was comparable to the wild-type ABCC1 promoter. On the other hand, the -260G>C substitution decreased ABCC1 promoter activity in HepG2, MCF-7 and HeLa (40 - 60%) cells. A 1706 bp fragment containing the 5’-flanking and untranslated regions of ABCC4 were isolated from two bacterial artificial chromosome clones and six serially deleted ABCC4 promoter reporter constructs generated. Luciferase assays of the basal promoter constructs of ABCC4 in HEK293T cells revealed the presence of one or more negative regulatory regions between -1706 and -876, between -876 and -641, and one or more positive regulatory regions between -641 and -356, and between -356 and -17. Also, the ABCC4 promoter displayed differential activity in MDCKI and LLC-PK1 cells than in HEK293T cells. One SNP (-523G>C) was identified from an online database and its activity tested. However, -523G>C SNP did not cause any significant change in the ABCC4 promoter activity in both HEK293T and HepG2 cells (80 – 130%). In summary, the data obtained suggest that the promoter SNPs studied may affect the transcriptional activity of ABCC1 or ABCC4, but it seems likely that this is not true in all cell types. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2007-09-28 10:03:16.119