GLP-1 receptor expression in human tumors and human normal tissues: potential for in vivo targeting

Peptide hormone receptors overexpressed in human tumors, such as somatostatin receptors, can be used for in vivo targeting for diagnostic and therapeutic purposes. A novel promising candidate in this field is the GLP-1 receptor, which was recently shown to be massively overexpressed in gut and lung neuroendocrine tumors--in particular, in insulinomas. Anticipating a major development of GLP-1 receptor targeting in nuclear medicine, our aim was to evaluate in vitro the GLP-1 receptor expression in a large variety of other tumors and to compare it with that in nonneoplastic tissues. METHODS: The GLP-1 receptor protein expression was qualitatively and quantitatively investigated in a broad spectrum of human tumors (n=419) and nonneoplastic human tissues (n=209) with receptor autoradiography using (125)I-GLP-1(7-36)amide. Pharmacologic competition experiments were performed to provide proof of specificity of the procedure. RESULTS: GLP-1 receptors were expressed in various endocrine tumors, with particularly high amounts in pheochromocytomas, as well as in brain tumors and embryonic tumors but not in carcinomas or lymphomas. In nonneoplastic tissues, GLP-1 receptors were present in generally low amounts in specific tissue compartments of several organs--namely, pancreas, intestine, lung, kidney, breast, and brain; no receptors were identified in lymph nodes, spleen, liver, or the adrenal gland. The rank order of potencies for receptor binding--namely, GLP-1(7-36)amide = exendin-4 >> GLP-2 = glucagon(1-29)--provided proof of specific GLP-1 receptor identification. CONCLUSION: The GLP-1 receptors may represent a novel molecular target for in vivo scintigraphy and targeted radiotherapy for a variety of GLP-1 receptor-expressing tumors. For GLP-1 receptor scintigraphy, a low-background signal can be expected, on the basis of the low receptor expression in the normal tissues surrounding tumors.

Finite element study on fracture patterns in human scaphoid wrist bone due to free fall

Scaphoid is one of the 8 carpal bones found adjacent to the thumb supported proximally by Radius bone. During the free fall, on outstretched hand, the impact load gets transferred to the scaphoid at its free anterior end. Unique arrangement of other carpal bones in the palm is also one of the reasons for the load to get transferred to scaphoid. About half of the total load acting upon carpal bone gets transferred to scaphoid at its distal pole. There are about 10 to 12 clinically observed fracture pattern in the scaphoid due to free fall. The aim of the study is to determine the orientation of the load, magnitude of the load and the corresponding fracture pattern. This study includes both static and dynamic finite element models validated by experiments. The scaphoid model has been prepared from CT scans of a 27 year old person. The 2D slices of the CT scans have been converted to 3D model by using MIMICS software. There are four cases of loading studied which are considered to occur clinically more frequently. In case (i) the load is applied at the posterior end at distal pole whereas in case (ii), (iii) and (iv), the load is applied at anterior end at different directions. The model is given a fixed boundary condition at the region which is supported by Radius bone during the impact. Same loading and boundary conditions have been used in both static and dynamic explicit finite element analysis. The site of fracture initiation and path of fracture propagation have been identified by using max principal stress / gradient and max principal strain / gradient criterion respectively in static and dynamic explicit finite element analysis. Static and dynamic impact experiments were performed on the polyurethane foam specimens to validate the finite element results. Experimental results such as load at fracture, site of fracture initiation and path of fracture propagation have been compared with the results of finite element analysis. Four different types of fracture patterns observed in clinical studies have been identified in this study.

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) is closely associated with the chondrocyte nucleus in human articular cartilage

OBJECTIVE: Insulin-like growth factor-I (IGF-I) is critically involved in the control of cartilage matrix metabolism. It is well known that IGF-binding protein-3 (IGFBP-3) is increased during osteoarthritis (OA), but its function(s) is not known. In other cells, IGFBP-3 can regulate IGF-I action in the extracellular environment and can also act independently inside the cell; this includes transcriptional gene control in the nucleus. These studies were undertaken to localize IGFBP-3 in human articular cartilage, particularly within cells. DESIGN: Cartilage was dissected from human femoral heads derived from arthroplasty for OA, and OA grade assessed by histology. Tissue slices were further characterized by extraction and assay of IGFBPs by IGF ligand blot (LB) and by enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry (IHC) for IGF-I and IGFBP-3 was performed on cartilage from donors with mild, moderate and severe OA. Indirect fluorescence and immunogold-labeling IHC studies were included. RESULTS: LBs of chondrocyte lysates showed a strong signal for IGFBP-3. IHC of femoral cartilage sections at all OA stages showed IGF-I and IGFBP-3 matrix stain particularly in the top zones, and closely associated with most cells. A prominent perinuclear/nuclear IGFBP-3 signal was seen. Controls using non-immune sera or antigen-blocked antibody showed negative or strongly reduced stain. In frozen sections of human ankle cartilage, immunofluorescent IGFBP-3 stain co-localized with the nuclear 4',6-diamidino-2-phenyl indole (DAPI) stain in greater than 90% of the cells. Immunogold IHC of thin sections and transmission electron immunogold microscopy of ultra-thin sections showed distinct intra-nuclear staining. CONCLUSIONS: IGFBP-3 in human cartilage is located in the matrix and within chondrocytes in the cytoplasm and nuclei. This new finding indicates that the range of IGFBP-3 actions in articular cartilage is likely to include IGF-independent roles and opens the door to studies of its nuclear actions, including the possible regulation of hormone receptors or transcriptional complexes to control gene action.

Methods for identification of Staphylococcus aureus isolates in cases of bovine mastitis

A total of 272 staphylococcal isolates from cases of bovine mastitis (159 Staphylococcus aureus) belonging to 12 different species were identified with ID32 STAPH galleries, and 51 of them were confirmed by 16S rRNA gene (rrs) sequencing. The same isolates were examined for their hemolytic activity on sheep blood agar, DNase activity, and coagulase activity and with two rapid identification kits (Slidex Staph Plus kit and RAPIDEC Staph from Bio-Merieux). The results of this study confirm those obtained by other groups with hemolysis, DNase, and coagulase. Only 50% of S. aureus isolates from mastitis cases show coagulase activity after 4 h of incubation, and a 24-h incubation is necessary for the full sensitivity of this test. In contrast to results from other studies with human isolates, the Slidex Staph Plus kit was not sensitive enough for the identification of S. aureus from bovine mastitis samples. The aurease test of the RAPIDEC Staph kit showed 100% sensitivity and 100% specificity. Used in conjunction with hemolysis patterns, the RAPIDEC Staph kit is therefore very well adapted to rapid, efficient, and cost-effective identification of S. aureus in cultures from bovine mastitis samples. Sequencing of rrs genes also proved very efficient in identifying the Staphylococcus species encountered in these samples and confirming phenotypical identification results with unsatisfactory scores. With continuously improving technologies and decreasing costs, genetic identification methods like rrs gene sequencing will soon find a place in routine veterinary diagnostics.

Differential regulation of human 3β-hydroxysteroid dehydrogenase type 2 for steroid hormone biosynthesis by starvation and cyclic AMP stimulation: studies in the human adrenal NCI-H295R cell model

Human steroid biosynthesis depends on a specifically regulated cascade of enzymes including 3β-hydroxysteroid dehydrogenases (HSD3Bs). Type 2 HSD3B catalyzes the conversion of pregnenolone, 17α-hydroxypregnenolone and dehydroepiandrosterone to progesterone, 17α-hydroxyprogesterone and androstenedione in the human adrenal cortex and the gonads but the exact regulation of this enzyme is unknown. Therefore, specific downregulation of HSD3B2 at adrenarche around age 6-8 years and characteristic upregulation of HSD3B2 in the ovaries of women suffering from the polycystic ovary syndrome remain unexplained prompting us to study the regulation of HSD3B2 in adrenal NCI-H295R cells. Our studies confirm that the HSD3B2 promoter is regulated by transcription factors GATA, Nur77 and SF1/LRH1 in concert and that the NBRE/Nur77 site is crucial for hormonal stimulation with cAMP. In fact, these three transcription factors together were able to transactivate the HSD3B2 promoter in placental JEG3 cells which normally do not express HSD3B2. By contrast, epigenetic mechanisms such as methylation and acetylation seem not involved in controlling HSD3B2 expression. Cyclic AMP was found to exert differential effects on HSD3B2 when comparing short (acute) versus long-term (chronic) stimulation. Short cAMP stimulation inhibited HSD3B2 activity directly possibly due to regulation at co-factor or substrate level or posttranslational modification of the protein. Long cAMP stimulation attenuated HSD3B2 inhibition and increased HSD3B2 expression through transcriptional regulation. Although PKA and MAPK pathways are obvious candidates for possibly transmitting the cAMP signal to HSD3B2, our studies using PKA and MEK1/2 inhibitors revealed no such downstream signaling of cAMP. However, both signaling pathways were clearly regulating HSD3B2 expression.

Co-activation of JAK/STAT and p38 MAPK pathways by type I interferon enhances apoptosis in human basophils

It has previously been published that interferon-α (type I IFN) improves clinical symptoms of asthma patients. Since human basophils are major inflammatory cells in maintaining chronic allergic asthma we investigate whether type I IFN affect human blood basophils. Furthermore, previous studies have shown that spontaneous apoptosis of human basophils is slow due to constitutive expression of anti-apoptotic BCL-2 family members. In addition, IL-3 exceptionally promotes survival of basophils by enhancing constitutive expression of BCL-2 family members and by inducing de-novo expression of Pim-1 kinase. Thus, we also assessed whether type I IFN might overcome IL-3-induced survival of human basophils. Our data show that type I IFN enhances apoptosis in purified human blood basophils compared to spontaneous apoptosis of controls or type II IFN treated cells. Furthermore, we demonstrate that both type I IFN and FasL enhance apoptosis in human basophils with similar efficiency in a rather additive than synergistic way. Analyses of signaling pathways reveal that type I IFN promote prolonged phosphorylation of STAT1/STAT2. By using a pan-JAK inhibitor the phosphorylation of STAT1/STAT2 is inhibited and most importantly the pro-apoptotic effect of type I IFN is abolished. On the other hand, type I IFN do not reduce IL-3-induced de novo expression of Pim-1 and BCL-2. This is in line with our observation that IL-3-induced survival is dominant over type I IFN-enhanced apoptosis. In addition, phosphorylation of p38 MAPK in type I IFN treated cells is comparable to non-treated cells. Particularly however, inhibition of this p-p38 activity abrogates apoptosis as well. We conclude that type I IFN-enhanced apoptosis is tightly regulated by the cooperation of JAK/STAT and p38 MAPK pathways. Our study identifies a so far unknown effect of type I IFN and may explain the improved clinical symptoms of asthma patients treated with type I IFN.

Antisense-mediated inhibition of papillomavirus type-18 in human squamous cell carcinoma

Numerous co-factors, genetic, environmental and physical, play an important role in development and prognosis of cancer. Each year in the USA, more than 31,000 cases of oral and 13,000 cases of cervical cancer are diagnosed. Substantial epidemiological data supports a high correlation between development of these cancers and the presence of specific types of human papillomaviruses (HPV). Molecular biological studies show that not only are several of the viral genes necessary and sufficient to cause transformation but they also function synergistically with other co-factors. Evidence suggests that prevention of infection or inhibition of viral gene expression may alter the course of malignant transition. The main objective of this project was to test the hypothesis that some human carcinoma cells, containing HPV, behave in malignant manner because the viral genes function in the maintenance of some aspect of the transformed phenotype.^ The specific aims were (1) to select oral and cervical cancer cell lines which were HPV-negative or which harbored transcriptionally active HPV-18, (2) to construct and determine the effects of recombinant sense or antisense expressing vectors, (3) to test the effects of synthetic antisense oligodeoxynucleotides on the transformed behavior of these cells.^ To screen cells, we performed Southern and Northern analysis and polymerase chain reactions. When antisense-expressing vectors were used, cells harboring low numbers of HPV-18 where unable to survive transfection but they were readily transfected with all other constructs. Rare antisense transfectants obtained from HPV-positive cells showed significantly altered characteristics including malignant potential in nude mice. The HPV-negative cells showed no differences in transfection efficiencies or growth characteristics with any construct.^ In addition, treatment of the HPV-positive cells with antisense, but not random oligodeoxynucleotides, resulted in decreased cell proliferation and even cell death. These effects were dose-dependent, synergistic and HPV-specific.^ These results suggest that expression of viral genes play an important role in the maintenance of the transformed phenotype which implies that inhibition of expression, by antisense molecules, may be therapeutic in HPV-induced tumors. ^

Taking personalized medicine seriously: Biomarker approaches in phase IIb/III studies in major depression and schizophrenia

The success rate in the development of psychopharmacological compounds is insufficient. Two main reasons for failure have been frequently identified: 1) treating the wrong patients and 2) using the wrong dose. This is potentially based on the known heterogeneity among patients, both on a syndromal and a biological level. A focus on personalized medicine through better characterization with biomarkers has been successful in other therapeutic areas. Nevertheless, obstacles toward this goal that exist are 1) the perception of a lack of validation, 2) the perception of an expensive and complicated enterprise, and 3) the perception of regulatory hurdles. The authors tackle these concerns and focus on the utilization of biomarkers as predictive markers for treatment outcome. The authors primarily cover examples from the areas of major depression and schizophrenia. Methodologies covered include salivary and plasma collection of neuroendocrine, metabolic, and inflammatory markers, which identified subgroups of patients in the Netherlands Study of Depression and Anxiety. A battery of vegetative markers, including sleep-electroencephalography parameters, heart rate variability, and bedside functional tests, can be utilized to characterize the activity of a functional system that is related to treatment refractoriness in depression (e.g., the renin-angiotensin-aldosterone system). Actigraphy and skin conductance can be utilized to classify patients with schizophrenia and provide objective readouts for vegetative activation as a functional marker of target engagement. Genetic markers, related to folate metabolism, or folate itself, has prognostic value for the treatment response in patients with schizophrenia. Already, several biomarkers are routinely collected in standard clinical trials (e.g., blood pressure and plasma electrolytes), and appear to be differentiating factors for treatment outcome. Given the availability of a wide variety of markers, the further development and integration of such markers into clinical research is both required and feasible in order to meet the benefit of personalized medicine. This article is based on proceedings from the "Taking Personalized Medicine Seriously-Biomarker Approaches in Phase IIb/III Studies in Major Depression and Schizophrenia" session, which was held during the 10th Annual Scientific Meeting of the International Society for Clinical Trials Meeting (ISCTM) in Washington, DC, February 18 to 20, 2014.

Angiogenesis-related ultrastructural changes to capillaries in human skeletal muscle in response to endurance exercise

The ultrastructure of capillaries in skeletal muscle was morphometrically assessed in vastus lateralis muscle (VL) biopsies taken before and after exercise from 22 participants of two training studies. In study 1 (8 wk of ergometer training), light microscopy revealed capillary-fiber (C/F) ratio (+27%) and capillary density (+16%) to be higher (P ≤ 0.05) in postexercise biopsies than in preexercise biopsies from all 10 participants. In study 2 (6 mo of moderate running), C/F ratio and capillary density were increased (+23% and +20%; respectively, P ≤ 0.05) in VL biopsies from 6 angiogenesis responders (AR) after training, whereas 6 nonangiogenesis responders (NR) showed nonsignificant changes in these structural indicators (-4%/-4%, respectively). Forty capillary profiles per participant were evaluated by point and intersection counting on cross sections after transmission electron microscopy. In study 1, volume density (Vv) and mean arithmetic thickness (T) of endothelial cells (ECs; +19%/+17%, respectively) and pericytes (PCs; +20%/+21%, respectively) were higher (P ≤ 0.05), whereas Vv and T of the pericapillary basement membrane (BM) were -23%/-22% lower (P ≤ 0.05), respectively, in posttraining biopsies. In study 2, exercise-related differences between AR and NR-groups were found for Vv and T of PCs (AR, +26%/+22%, respectively, both P ≤ 0.05; NR, +1%/-3%, respectively, both P > 0.05) and BM (AR, -14%/-13%, respectively, both P ≤ 0.05; NR, -9%/-11%, respectively, P = 0.07/0.10). Vv and T of ECs were higher (AR, +16%/+18%, respectively; NR, +6%/+6%, respectively; all P ≤ 0.05) in both groups. The PC coverage was higher (+13%, P ≤ 0.05) in VL biopsies of individuals in the AR group but nonsignificantly altered (+3%, P > 0.05) in those of the NR group after training. Our study suggests that intensified PC mobilization and BM thinning are related to exercise-induced angiogenesis in human skeletal muscle, whereas training per se induces EC-thickening.

Mechanisms of Ad-p53 induced apoptosis in human malignant glioma

Malignant brain tumors are one of the most challenging cancers affecting society today. In a recent survey, an estimated 17,000 annual cases were recorded with a staggering total of 13,300 deaths. A unique degree of heterogeneity typifies glial tumors and presents a challenge for solitary anti-neoplastic treatments. Tumors subsist as heterogeneous masses that progress through dysplasia to astrocytomas, mixed glioma and glioblastoma multiforme. Although traditional therapeutic approaches have provided increments of success, the median survival time remains 12 months. The urgency to improve upon current clinical protocols has encouraged alternative experimental strategies such as p53 adenoviral gene therapy (Ad-p53). This study addresses the efficacy of Ad-p53 for the treatment of glioma. Our model presents a tumor response that is unique among human cancers. Ad-p53 effectively induces apoptosis in mutant p53 expressing cells yet fails to do so in those with wildtype p53. In order to adopt Adp53 as a standard anti-cancer modality, we characterized the role of the tumor suppressor gene p53 in mediating apoptosis. We demonstrate that altering cellular p53 status through the introduction of a dominant negative mutant p53 (175H, 248W, 273H) sensitized cells to Ad-p53. We discovered that wild-type p53 expressing glioma cells retain the apoptotic machinery necessary to accomplish cell death, but have developed mechanisms that interfere with p53 signaling. Earlier studies have not addressed the mechanisms of Ad-p53 apoptosis nor the resistance exhibited by wild-type p53 glioma. To explain the divergent phenotypes, we identified apoptotic pathways activated and effectors of the response. We illustrated that modulation of the death receptor Fas/APO-1 is a principal means of Ad-p53 signaling that is impaired in wild-type p53 glioma. Moreover, the apoptotic response was found to be a multi-faceted process that engaged several caspases, most notably caspases -1, -3 and -8. Lastly, we assessed the ability of anti-apoptotic molecules Bcl-2 and CrmA to inhibit Ad-p53 apoptosis. These studies revealed that Ad-p53 is a powerful tool for inducing apoptosis that can be delayed but not inhibited by anti-apoptotic means. This work is critical for understanding the development of glioma and the phenotypic and genotypic alterations that account for tumor resistance. ^

Regulation of vascular endothelial growth factor expression in human pancreatic cancer

Pancreatic adenocarcinoma is currently the fifth-leading cause of cancer-related death in the United States. Like with other solid tumors, the growth and metastasis of pancreatic adenocarcinoma are dependent on angiogenesis. Vascular endothelial growth factor (VEGF) is a key angiogenic molecule that plays an important role in angiogenesis, growth and metastasis of many types of human cancer, including pancreatic adenocarcinoma. However, the expression and regulation of VEGF in human pancreatic cancer cells are mostly unknown. ^ To examine the hypothesis that VEGF is constitutively expressed in human pancreatic cancer cells, and can be further induced by tumor environment factors such as nitric oxide, a panel of human pancreatic cancer cell lines were studied for constitutive and inducible VEGF expression. All the cell lines examined were shown to constitutively express various levels of VEGF. To identify the mechanisms responsible for the elevated expression of VEGF, its rates of turnover and transcription were then investigated. While the half-live of VEGF was unaffected, higher transcription rates and increased VEGF promoter activity were observed in tumor cells that constitutively expressed elevated levels of VEGF. Detailed VEGF promoter analyses revealed that the region from −267 to +50, which contains five putative Sp1 binding sites, was responsible for this VEGF promoter activity. Further deletion and point mutation analyses indicated that deletion of any of the four proximal Sp1 binding sites significantly diminished VEGF promoter activity and when all four binding sites were mutated, it was completely abrogated. Consistent with these observations, high levels of constitutive Sp1 expression and DNA binding activities were detected in pancreatic cancer cells expressing high levels of VEGF. Collectively, our data indicates that constitutively expressed Sp1 leads to the constitutive expression of VEGF, and implicates that both molecules involve in the aggressive pathogenesis of human pancreatic cancer. ^ Although constitutively expressed in pancreatic cancer cells, VEGF can be further induced. In human pancreatic cancer specimens, we found that in addition to VEGF, both inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were overexpressed, suggesting that nitric oxide might upregulate VEGF expression. Indeed, a nitric oxide donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) significantly induced VEGF mRNA expression and protein secretion in pancreatic adenocarcinoma cells in a time- and dose-dependant manner. Using a luciferase reporter containing both the VEGF promoter and the 3′ -UTR, we showed that SNAP significantly increased luciferase activity in human pancreatic cancer cells. Notwithstanding its ability to induce VEGF in vitro, pancreatic cancer cells genetically engineered to produce NO did not exhibit increased tumor growth. This inability of NO to promote tumor growth appears to be related to NO-mediated cytotoxicity. The balance between NO mediated effects on pro-angiogenesis and cytotoxicity would determine the biological outcome of NO action on tumor cells. ^ In summary, we have demonstrated that VEGF is constitutively expressed in human pancreatic cancer cells, and that overexpression of transcription factor Sp1 is primarily responsible. Although constitutively expressed in these cells, VEGF can be further induced by NO. However, using a mouse model, we have shown that NO inhibited tumor growth by promoting cytotoxicity. These studies suggest that both Sp1 and NO may be important targets for designing potentially effective therapies of human pancreatic cancer and warrant further investigation. ^

Integrin mediated growth and apoptosis in human gastric adenocarcinoma

Integrins are important as the primary cell adhesion molecule providing information about the extracellular microenvironment to the interior of the cell to influence cellular behavior such as differentiation, proliferation and apoptosis. Apoptotic death due to loss of adhesion is termed anoikis. In this study we have obtained a parental human gastric adenocarcinoma cell line that yielded two variant lines that had differing responses to lack of adhesion. The STAD.APO cell line undergoes apoptosis when denied adherence and the STAD.ARR cell line enters into cell cycle arrest under the identical suspended conditions. We have shown that cyclin A and cyclin D mRNA and protein are down regulated when cells are denied adherence for 24 hours in tissue culture wells previously coated with poly-HEMA. To test whether cyclin A was able to rescue cells from cell cycle arrest and/or anoikis by overriding the cell cycle machinery we transfected the full length cDNA in to each cell type. Surprisingly we found that anoikis and cell cycle arrest due to suspended conditions was not affected by overexpression of cyclin A protein, but that growth under adhered conditions was reduced compared to vector alone control transfectants. Further, we transfected other cell lines; ST7, gastric cancer, MDA-MB-4.35, breast cancer, and HPB T-cell leukemic and in no case were suspended culturing conditions overcome by cyclin A. This result indicates an additional level of regulation for the cell cycle machinery. Additionally, soluble collagen was shown to be able to save from anoikis and also from cell cycle arrest while the β1 specific mAb 33B6 was only able to save from anoikis. Immunofluorescent studies show that soluble collagen creates clusters of β1 with FAK and also β1 with actin in the STAD.ARR cells but does not in the STAD.APO cells. This result indicates that the phenotypes under suspended conditions between these cell lines may diverge at their requirements for integrin ligation. Additionally we characterized the nature of anoikis by showing cytochrome c release, caspase 3, p21 and p53 activation in STAD.APO cells. Thus, our results have implications in the understanding of integrin biology and neoplastic progression. ^

Theoretical and experimental studies of linkage disequilibrium in human populations

Linkage disequilibrium (LD) is defined as the nonrandom association of alleles at two or more loci in a population and may be a useful tool in a diverse array of applications including disease gene mapping, elucidating the demographic history of populations, and testing hypotheses of human evolution. However, the successful application of LD-based approaches to pertinent genetic questions is hampered by a lack of understanding about the forces that mediate the genome-wide distribution of LD within and between human populations. Delineating the genomic patterns of LD is a complex task that will require interdisciplinary research that transcends traditional scientific boundaries. The research presented in this dissertation is predicated upon the need for interdisciplinary studies and both theoretical and experimental projects were pursued. In the theoretical studies, I have investigated the effect of genotyping errors and SNP identification strategies on estimates of LD. The primary importance of these two chapters is that they provide important insights and guidance for the design of future empirical LD studies. Furthermore, I analyzed the allele frequency distribution of 26,530 single nucleotide polymorphisms (SNPs) in three populations and generated the first-generation natural selection map of the human genome, which will be an important resource for explaining and understanding genomic patterns of LD. Finally, in the experimental study, I describe a novel and simple, low-cost, and high-throughput SNP genotyping method. The theoretical analyses and experimental tools developed in this dissertation will facilitate a more complete understanding of patterns of LD in human populations. ^

Bortezomib modulates TRAIL sensitivity in human bladder and prostate cancer

Tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is a member of the TNF superfamily of cytokines that can induce cell death through engagement of cognate death receptors. Unlike other death receptor ligands, it selectively kills tumor cells while sparing normal cells. Preclinical studies in non-human primates have generated much enthusiasm regarding its therapeutic potential. However, many human cancer cell lines exhibit significant resistance to TRAIL-induced apoptosis, and the molecular mechanisms underling this are controversial. Possible explanations are typically cell-type dependent, but include alterations of receptor expression, enhancement of pro-apoptotic intracellular signaling molecules, and reductions in anti-apoptotic proteins. We show here that the proteasome inhibitor bortezomib (Velcade, PS-341) produces synergistic apoptosis in both bladder and prostate cancer cell lines within 4-6 hours when co-treated with recombinant human TRAIL which is associated with accumulation of p21 and cdk1/2 inhibition. Our data suggest that bortezomib's mechanism of action involves a p21-dependent enhancement of caspase maturation. Furthermore, we found enhanced tumor cell death in in vivo models using athymic nude mice. This is associated with increases in caspase-8 and caspase-3 cleavage as well as significant reductions in microvessel density (MVD) and proliferation. Although TRAIL alone had less of an effect, its biological significance as a single agent requires further investigations. Toxicity studies reveal that the combination of bortezomib and rhTRAIL has fatal consequences that can be circumvented by altering treatment schedules. Based on our findings, we conclude that this strategy has significant therapeutic potential as an anti-cancer agent. ^

The effects of proteasome inhibition on angiogenesis and autophagy in human prostate cancer cells

The proteasome degrades approximately 80% of intracellular proteins to maintain homeostasis. Proteasome inhibition is a validated therapeutic strategy, and currently, proteasome inhibitor bortezomib is FDA approved for the treatment of MM and MCL. Specific pathways affected by proteasome inhibition have been identified, but mechanisms of the anti-tumor effects of proteasome inhibition are not fully characterized and cancer cells display marked heterogeneity in terms of their sensitivity to proteasome inhibitor induced cell death. ^ The antitumor effects of proteasome inhibition involve suppression of tumor angiogenesis and vascular endothelial growth factor (VEGF) expression, but the mechanisms involved have not been clarified. In this dissertation I investigated the mechanisms underlying the effects of two proteasome inhibitors, bortezomib and NPI-0052, on VEGF expression in human prostate cancer cells. I found that proteasome inhibitors selectively downregulated hypoxia inducible factor 1alpha (HIF-1α) protein and its transcriptional activity to inhibit VEGF expression. Mechanistic studies demonstrated that proteasome inhibitors mediate the induction of the unfolded protein response (UPR) and that downregulation of HIF-1α is caused by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation and translation repression. Importantly, I showed that proteasome inhibitors activated the UPR in some cells but not in others. My observation may have implications for the design of combination regimens that are based on exploiting proteasome inhibitor-induced ER stress.^ Although proteasome inhibitors have shown modest activity on prostate cancer, there is general consensus that no single agent is likely to have significant activity in prostate cancer. In the second part of this dissertation I attempted to exploit the effects of proteasome inhibition on the UPR to design a combination therapy that would enhance cancer cell death. Autophagy is a lysosome dependent degradation pathway that functions to eliminate long-lived protein and subcellular structures. Targeting autophagy has been shown to inhibit tumors in preclinical studies. I found that inhibition of autophagy with chloroquine or 3-methyladenine enhanced proteasome inhibitor induced cell death and the effects were associated with increased intracellular stress as marked by aggresome formation. Multiple cancers appear to be resistant to proteasome inhibition treatment alone. The implications of synergy for the combined inhibition of autophagy and the proteasome would likely apply to other cancers aside from prostate cancer. ^