ABCG2 encodes for a multidrug efflux transporter called the mitoxantrone resistance protein (MXR, BCRP) that mediates the efflux of substrates out of the cell and is important in detoxification. The present study was focused on the expression and function of MXR amino acid variants, activity of the ABCG2 promoter and promoter variants, characterization of ABCG2 locus cis-regulatory elements and variant enhancers, and examination of DNA methylation around ABCG2. MXR expression, localization and activity were characterized using whole cells and inside-out vesicles. The Q141K variant had reduced expression. MXR I206L had increased efflux of pheophorbide A and both V12M and D620N had increased ATPase activity. The activity of ABCG2 regulatory elements was tested in in vitro and in vivo luciferase assays. Two promoter SNPs (rs76656413 and rs59370292) had decreased in vivo liver enhancer activity. Six regions with in vivo liver enhancer activity and several enhancer SNPs (rs9999111, rs12508471, rs72873421, rs149713212 and rs2725263) with altered activity in vivo were identified. Association of these SNPs with ABCG2, PPM1K or PDK2 expression in different tissues was detected. In vitro assays were used to identify nuclear receptor response elements. The ABCG2 promoter responded to multiple nuclear receptor ligands, and the promoter SNP rs66664036 had a significantly increased response to 17β-estradiol. Nine rifampin, six 17β-estradiol and three dexamethasone responsive regions were identified. Enhancer SNP rs12508471 had decreased response to 17β-estradiol and increased response to dexamethasone, while rs573519157 had an increased and rs190754327 had a decreased response to 17β-estradiol. Finally, methylation of CpG islands in the ABCG2 locus was correlated with the expression of ABCG2 in human liver and kidney tissues. There was no correlation of whole CpG island methylation with ABCG2 expression. However, a CpG site within CpG4 correlated with ABCG2 expression in the kidney, and part or all of select CpG islands had significantly lower methylation in liver than in kidney. The genetic and epigenetic regulation of the ABCG2 gene locus described in this dissertation may contribute to clinical variation in ABCG2 expression.

Since the emergence of the HIV epidemic it has been recognized that complications to HIV infection and variations in drug response and toxicity are influenced by patient genetics. Identification of genetic predictors of HIV infection complications and variation in drug response and toxicity will lead to better treatment options for patients and reduce HIV-related mortality and morbidity. This dissertation contains research that uses candidate gene and genome-wide approaches to identify and characterize novel genetic predictors of nevirapine pharmacokinetics, nucleoside reverse transcriptase inhibitor-induced peripheral neuropathy and HIV-induced peripheral neuropathy. This research demonstrates that nevirapine pharmacokinetic properties are heritable in European and African patients and characterizes the significant effects of CYP2B6 516G>T, CYP2B6 983T>C and ABCC10 rs2125739 on nevirapine Cmin concentrations in a Ugandan HIV+ population. It also highlights the importance of considering all three polymorphisms for prediction of nevirapine Cmin. This dissertation also explores the genetic predictors of NRTI-SN using whole genome and candidate gene approaches in a Ugandan HIV+ population. A polymorphism in VAMP4, rs188298690, was identified in the whole genome study and bioinformatic analyses found that this marker is in an active regulatory region and also a population specific eQTL locus. The candidate gene analysis found that polymorphisms in SLC28A1 and ABCC4 are predictive of the development of NRTI-SN. Finally, this dissertation describes research to identify genetic predictors of HIV-SN. Several polymorphisms in the FOLH1 region were identified in a whole genome study and bioinformatic analyses support a role for these polymorphisms in determining FOLH1 expression. Analysis of the top FOLH1 polymorphism in additional samples showed a trend towards significance and a meta-analysis of the discovery and replication cohorts had improved statistical significance. The research obtained in this dissertation increases the understanding of the role of genetic variation in determining antiviral pharmacokinetics and toxicity and in complications to HIV infection.

ATP binding cassette (ABC) transporters are a family of proteins whose activity is vital to cell detoxification, protection against xenobiotics and oxidative stress, and maintenance of homeostasis of endogenous compounds. Subtle changes in endogenous expression level of these transporters can have significant clinical implications. In particular, the impact of such variation on the role of ABC transporters in transport of pharmaceutical agents is of interest; inter-individual differences in expression of ABC transporters can result in changes in exposure to pharmaceutical agents or their metabolites, leading to altered drug efficacy or drug-induced toxicities. Variation in gene expression can be caused by a number of factors that modulate the "normal" activity during transcription or translation. In this study, mechanisms that regulate mRNA or protein expression of ATP-binding cassette transporters were characterized in human tissues. First, the impact of DNA sequence variation on mRNA expression across individuals was evaluated in the human kidney. Several expression quantitative trait loci (eQTLs) were identified for ABC transporters in the kidney, and one eQTL for ABCG2 (BCRP) was validated in an in vitro reporter-gene assay. Next, the role of alternative splicing in regulation of transporter expression was explored using transcriptome sequencing data from multiple individuals in several human tissues. Examples of splicing events that were found to modulate transporter expression across individuals include alternate 5 prime untranslated regions (5' UTRs) in the genes ABCC5 and ABCA8. Further, a splicing event in the gene ABCC6 was identified that produces a premature termination codon, triggering the nonsense mediated decay process; this event may be responsible for regulating inter-tissue expression of ABCC6. Finally, transcription factor regulators of ABC transporters were identified by searching for transcription factor binding motif enrichment in sets of genes co-expressed with ABC transporters in several human tissues. A number of potential transcriptional regulators of transporters were identified.

The ATP-binding cassette (ABC) superfamily consists of energy-dependent transporters which in many cases play crucial roles in physiological processes; indeed, mutations in several ABC transporter genes has been shown to cause inherited diseases such as cystic fibrosis.  The involvement of ABC transporters in systemic xenobiotic protection has led to an examination of the influence of these transporters on drug pharmacokinetics (plasma levels), including a number of clinical studies in which transporter expression levels were associated with drug plasma levels in healthy subjects.  Researchers have also demonstrated correlations between the presence of genetic polymorphisms in ABC transporters and altered drug pharmacokinetics.  The impact of ABC transporters and their naturally occurring genetic variants on drug pharmacodynamics (pharmacological action) has been characterized to a lesser extent, in part because of the difficulties of quantifying the pharmacological actions of many drugs.  In this dissertation, we focus on the pharmacodynamic effects of ABC transporter expression and function in peripheral, non-pharmacokinetic tissues such as adipose and lymphocytes, especially with respect to anti-HIV therapies.First, we describe the expression of a putative splice variant of the ABCB1 (P-glycoprotein) transporter in lymphocytes.  This half-sized protein functioned similarly to the classic full-size P-glycoprotein, but displayed altered immunoreactivity.  ABCB1 RNA transcripts of approximately half the length of normal ABCB1 transcripts were found in lymphocytes from healthy subjects.  The putative P-glycoprotein splice variant was detected in both HIV-negative and -positive subjects, indicating that it could influence the pharmacodynamics of anti-HIV drugs which have been classified as substrates of P-glycoprotein.  We also investigated the effects of exposure to the HIV protease inhibitors atazanavir and saquinavir on the expression of lymphocyte P-glycoprotein.  We found that in several subjects, the amount of P-glycoprotein expression increased substantially, suggesting that while there is no detectable generalized change in lymphocyte P-glycoprotein expression following atazanavir/saquinavir exposure, there may be genetic or environmental influences which affect the extent of lymphocyte P-glycoprotein induction in certain individuals.We also examine the roles that polymorphisms in candidate genes play in patient response to anti-HIV medications.  These genes include the drug-metabolizing enzyme cytochrome P450 2B6, the xenobiotic transporters ABCB1 and ABCC4, and the inflammatory cytokine TNF.  We performed this study in HIV-infected populations in San Francisco and Uganda, and found no significant associations between any of the polymorphisms investigated and patient virologic or immunologic response to antiretroviral therapies.  We also characterized the ancestral admixture of the San Francisco cohort and determined that the minor allele frequencies of several of the polymorphisms differed between ethnicities.Last, we describe the effects of the adipose RNA expression of the transporter genes ABCC4 and SLC29A1, which regulate cellular exposure to the nucleoside analogue-based HIV reverse transcriptase inhibitors, on the development of lipodystrophy, a fairly common side effect of this class of anti-HIV therapies.  Lipodystrophy is thought to result from mitochondrial toxicity, specifically the inhibition of the mitochondrial DNA polymerase.  Indeed, we found that the nucleoside analogue fialuridine inhibited mitochondrial DNA synthesis in vitro; the effects of transporters in this system remain inconclusive.  While we did not see any correlation between adipose transporter expression and the occurrence of lipodystrophy, we identified a genetic polymorphism in ABCC4 that was significantly associated with the development of lipodystrophy following stavudine treatment in a San Francisco population of HIV-infected individuals.  It is possible that screening for this polymorphism may help to predict which patients will develop lipodystrophy following nucleoside analogue exposure.In general, the results described in this dissertation indicate that ABC transporters in lymphocytes and adipocytes may have important pharmacodynamic functionalities, as they modulate the intracellular concentrations of certain drugs.  In particular, we have focused on HIV antiretroviral therapies because they exert pharmacological effects (beneficial or detrimental) in these cell types.  Finally, we show that genetic variation in ABC transporters may be an important factor which determines how a patient responds to an antiretroviral regimen.  Future studies should expand on this work with mechanistic studies to further unravel the role that transporters play in antiretroviral action, as well as carefully designed clinical studies to ascertain the validity of the pharmacogenetic associations reported here.

Bevacizumab is a vascular endothelial growth factor-specific angiogenesis inhibitor indicated as an adjunct to chemotherapy for the treatment of several types of cancer. Hypertension is commonly observed during bevacizumab treatment, and high-grade toxicity can limit therapy and lead to other cardiovascular complications. The factors that contribute to interindividual variability in blood pressure response to bevacizumab treatment are not well understood. To identify novel mechanisms of bevacizumab-induced hypertension, the research in this dissertation explored genetic variation associated with the toxicity. A sequencing analysis of whole exomes and candidate gene regulatory regions identified a genomic region between SLC29A1 and HSP90AB1 containing several variants associated with hypertension in colorectal cancer patients with extreme toxicity phenotypes. Functional experiments in human endothelial cells provide evidence that variation in SLC29A1 (ENT1) expression is associated with altered adenosine signaling that modulates the synthesis of vasodilatory molecules during bevacizumab treatment. These results suggest that increased basal expression of SLC29A1 and low extracellular adenosine levels may sensitive patients to a rise in blood pressure during bevacizumab exposure. Additionally, a genome-wide association study of a larger, independent cohort of breast cancer patients identified variants associated with cumulative bevacizumab dose at the first occurrence of hypertension. Several of these SNPs are located within or near genes of biological interest (MSH6, SDC4, ASB5, SMYD5) and may highlight additional mechanisms important in the pathogenesis of the toxicity. Collectively, the studies in this dissertation identified novel genetic loci that potentially modify the risk of developing bevacizumab-induced hypertension. The results of this research will advance understanding of the biological mechanism of this adverse drug reaction and should be considered in the future use and development of angiogenesis inhibitors.

It is important to accurately model drug disposition in the preclinical setting before novel drug compounds are introduced into humans. However, it is evident from current statistics on the drug development process that there is much room for improvement in the preclinical assessment of drug candidates in order to avoid clearance or toxicity issues when the drug is used in the clinic. One organ that plays a significant role in drug disposition and is therefore important to model is the kidney, which filters blood and is responsible for elimination of over one third of all drugs. The focus of this dissertation was to improve in vitro modeling of the kidney. First, multiple proximal tubule cell lines were systematically compared in order to identify the most physiologically relevant line. It was found that while the human cell lines hold some promise, non-human transfected cell lines remain more robust models of proximal tubule drug transport. Compared to the human cell lines, non-human transfected cell lines have superior monolayer formation, higher levels of transporter expression and function and are easier to grow in culture. Second, the role of one particular aspect of the in vivo environment, shear stress, was studied to better understand the effect on drug transporters and the possible biological pathways involved. Results indicated that shear stress affects active transport of organic cations and expression of relevant transporters in renal cells in a cilia dependent manner. Lastly, the design of a 1:1000 scaled model of the kidney that can be incorporated into a 1:1000 scaled human-on-a-chip system is presented along with the results of initial prototyping and future directions. In summary, the studies in this dissertation provide insight into the limitations of existing renal cell models, expand our current knowledge of the interaction between the cellular environment and drug transport functionality and provide a foundation for the future development of a scaled kidney on a chip module.

Lysosomes are a type of functional compartment, or organelle, that is responsible for crucial cellular processes such as protein homeostasis, nutrient sensing, molecular signaling, and secretion. These biochemical pathways ultimately dictate the intricate balance between health and diseases, including neurodegeneration and cancer. Despite decades of research on lysosomes, their role in devastating disorders such as Alzheimer’s disease remains largely unknown. In order to achieve a better understanding of lysosomes, specifically in the context of therapeutics for neurodegeneration, my thesis aimed to (1) describe the exciting field of organelle probe development for phenotypic discovery campaigns, (2) engineer a novel biochemical probe that measures lysosomal pH, a crucial aspect of lysosomal function in cells and (3) launch a high-throughput drug screen focused on identifying small molecules and possible molecular pathways that govern lysosomal function. Ultimately, this work contributes to the actively expanding field of phenotypic drug discovery, with specific focus on lysosomal pH and development of cell-based quantitative techniques.