G protein-coupled receptors (GPCR) can participate in a number of signaling pathways, and this property led to the concept of biased GPCR agonism. Agonists, antagonists and allosteric modulators can bind to GPCRs in different ways, creating unique conformations that differentially modulate signaling through one or more G proteins. A unique neuromedin B (NMBR) GPCR-signaling platform controlling mammalian neuraminidase-1 (Neu1) and matrix metalloproteinase-9 (MMP9) crosstalk has been reported in the activation of the insulin receptor (IR) through the modification of the IR glycosylation. Here, we propose that there exists a biased GPCR agonism as small diffusible molecules in the activation of Neu1-mediated insulin receptor signaling. GPCR agonists bombesin, bradykinin, angiotensin I and angiotensin II significantly and dose-dependently induce Neu1 sialidase activity and IR activation in human IR-expressing rat hepatoma cell lines (HTC-IR), in the absence of insulin. Furthermore, the GPCR agonist-induced Neu1 sialidase activity could be specifically blocked by the NMBR inhibitor, BIM-23127. Protein expression analyses showed that these GPCR agonists significantly induced phosphorylation of IR? and insulin receptor substrate-1 (IRS1). Among these, angiotensin II was the most potent GPCR agonist capable of promoting IR? phosphorylation in HTC-IR cells. Interestingly, treatment with BIM-23127 and Neu1 inhibitor oseltamivir phosphate were able to block GPCR agonist-induced IR activation in HTC cells in vitro. Additionally, we found that angiotensin II receptor (type I) exists in a multimeric receptor complex with Neu1, IR? and NMBR in na�ve (unstimulated) and stimulated HTC- IR cells with insulin, bradykinin, angiotensin I and angiotensin II. This complex suggests a molecular link regulating the interaction and signaling mechanism between these molecules on the cell surface. These findings uncover a biased GPCR agonist-induced IR transactivation signaling axis, mediated by Neu1 sialidase and the modification of insulin receptor glycosylation.

Aberrant cell surface glycan alterations such as glycosylation have been linked to a variety of cancers. These findings have warranted a deeper investigation into whether fucosylation may play a role in prostate cancer, which affects roughly 1.1 million people worldwide. Fucosylation is a type of cell surface glycosylation that involves the transfer of an L-fucose from a GDP-fucose to an acceptor substrate. Additionally, Asapargine N-linked glycans are typically the target of core fucosylation. Overexpression of Fucosyltransferase-8 (Fut-8), which catalyzes an ?-1,6 fucose transfer and is responsible for ???core fucosylation???, has been detected in aggressive prostate cancers with high Gleason scores. With the growing use of multicellular tumor spheroids (MTS) to study complex interactions in the tumor microenvironment, prostate spheroids (or prostaspheres) constitute a good three-dimensional model for studying the role of fucosylated proteins in tumor formation. Prostaspheres can be generated from DU145 and PC3 prostate cancer cell lines using the cyclo-RGDfK(TPP) peptide, a novel method that enables rapid, inexpensive and true spheroid formation. I have shown that DU145 prostate cancer cells express core fucose with immunochemistry (ICC) using the Aspergillus oryzae lectin (AOL), which binds core fucose residues. Using a lectin inhibition assay, I have also shown that pre-treating prostaspheres generated from DU145 prostate cancer cells with AOL result in the formation of spheroids with a reduced volume. Therefore, in addition to the previously established role of sialylation in MTS formation, fucosylation is also likely contributing to prostate tumor formation. The role of glycosylation in tumor spheroid formation is an emerging yet poorly understood area of research that can enhance our understanding of the mechanisms behind tumor growth in humans. Novel applications of glycobiology to the recent advancements in MTS with the advent of the cyclo-RGDfK(TPP) peptide method give rise to the possibility of optimizing current MTS models.

Statement of the Problem: During pathogen-mediated inflammation, host peripheral blood mononuclear cells/monocytes/ macrophages (PBMC/MO/M??) play a critical role in exacerbating or resolving disease by (a) priming of na�ve and resident cells for selective host responsiveness; (b) extensive release of cytokines, reactive oxygen species, and nitric oxide; and, (c) development of hyporesponsiveness (tolerance/refractoriness) where selective host responses are repressed. Mechanisms of bacteria-triggered development of diseases in patients provide a prototype example of each of the responses elicited to various extents during disease progression/resolution. It is well-established that the human host innate immune system has developed the capacity to recognize and respond immunologically to multiple bacterial structures, such as lipopolysaccharides (LPS), peptidoglycan, and CpG DNA. However, all cells behave differentially with respect to agonists. The mechanisms underlying the modulation of these immune cells with respect to LPS are not well defined and conflicting results are reported. The purpose of this study is to understand the role of LPS in innate immune responses, such as induction of growth factors, priming, proliferation, differentiation, cytokines, nitric oxide, autophagy and death in PBMC/MO/M?? based on the proteasome???s proteases (a complex that degrades key regulatory proteins). A novel model based on the function of subunits of the proteasome was developed in both human and murine cells, based on genomics, proteomics, and signaling pathways. Several hormones, dietary nutrients, and vitamins were also tested in PBMC/MO/M?? from normal and diseased subjects with surprising results. Conclusion & Significance: The innate immune response induced by LPS in human monocytes (huMO) and mouse M??, as described above is largely dependent on the change in composition/function of cellular proteasomes in cells. This novel information will lead to the development of drugs for sepsis, diabetes, asthma, cancer, AIDS, and neurological diseases, based on solid findings on the modulation of proteasomes in immune cells.

One of the hallmarks of cancer is dysregulated metabolism. A subset of many cancers possess unique mitochondrial characteristics such as increased mitochondrial mass and mitochondrial DNA, a higher reliance on oxidative phosphorylation (OXPHOS) and a lower spare reserve capacity compared to their normal counterparts. We focused on a novel non-mutant therapeutic target, the mitochondrial RNA polymerase (POLRMT) which indirectly controls oxidative phosphorylation (OXPHOS) by transcribing 13 essential subunits of the mitochondrial electron transport chain. While mitochondria are ubiquitous in all cells, their unique dysregulation in some cancer cells and heightened response to POLRMT depletion indicates a real possibility for anti-cancer therapy. This study focused on acute myeloid leukemia as one type of cancer with a high reliance on OXPHOS. Using the ribonucleoside analogue 4???-azidocytidine, a chain terminator of mitochondrial transcription, we observed that 4???-azidocytidine decreased growth and viability of leukemia and lymphoma cell lines. An unanticipated finding was that, paradoxically, 4???-azidocytidine increases nascent mitochondrial gene expression at short timepoints. Since POLRMT also acts as a primase for mitochondrial DNA replication, we asked whether mitochondrial DNA levels were affected by 4'-azidocytidine, but it did not alter mitochondrial DNA abundance. Our interpretation of the elevation in nascent mitochondrial RNA was that cancer cells stabilize mitochondrial RNA to prevent its degradation. Using genome-wide RNA-sequencing, we observed that 4???-azidocytidine decreased total mitochondrial gene expression while the expression of nuclear genes was generally unaffected, with the exception of a specific increase in the expression of nuclear genes encoding mitochondrial electron transport chain subunits, likely as a compensatory response. 4'-azidocytidine decreased the levels of mitochondrial DNA-encoded protein subunits, and inhibited mitochondrial respiration in leukemia cell lines. We treated primary AML patient samples and observed a heterogeneous mitochondrial gene expression response to 4'-azidocytidine.

Statement of the Problem: It has long been accepted that any hereditary factor in any organism is represented by DNA sequences. This idea became fundamental in molecular genetics and was implicitly transformed into the DNA theory of inheritance. All basic genetic terms (genotype, gene, allele, mutation, recombination, etc.) were considered as specific DNA sequences or their alterations. However, multiple examples of stable epigenetic inheritance lacking any distinctions in DNA sequences were recently discovered, and the most exciting among them is protein inheritance. Amyloid hereditary prions in fungi were considered as ???protein-only??? hereditary factors, which features were determined entirely by protein conformation. As a result, the principal question arises whether the DNA theory of inheritance is wrong or not. Methodology & Theoretical Orientation: Considering different variants of the same hereditary prion as prion alleles, we examined the molecular nature of such variety. Findings: To perpetuate stably in cell generations a certain prion allele requires two entities: a specific state of the prion protein, and the corresponding DNA sequence to provide reproduction of the prion particles. We name these entities as the DNA determinant and the epigenetic determinant, respectively. Thus, a certain prion allele is a bimodular hereditary system depending on both the DNA determinant and the epigenetic determinant. Alteration of any of these two determinants may result in the establishment of a novel prion allele. Moreover, similar regularities are characteristic to all other cases of epigenetic inheritance, irrespective to the underlying mechanisms. Conclusion & Significance: The hereditary role of DNA is fundamental for any known mechanisms of inheritance, including epigenetic. However, it becomes an element of a more complicated concept: in addition to ???DNA-only??? hereditary factors, various bimodular hereditary factors also exist.

Statement of the Problem: The Section on DNA Helicases, NIA-NIH investigates the roles of human DNA helicases in genomic stability, prompted by the findings of hereditary helicase disorders characterized by accelerated aging and cancer. Methodology & Theoretical Orientation: Building on our discovery of small molecule inhibitors of the WRN helicase defective in Werner syndrome, we are conducting studies with compounds to target human helicases for inhibition (or activation) in cell-based model systems as a novel approach to assign specific helicase functions in vivo that may pave the way for development of therapeutic strategies. Of particular interest are helicase-interacting compounds that accentuate the effects of anti-cancer drugs based on a DNA damaging paradigm in genetically defined mutant backgrounds. Development of preclinical models is underway to evaluate the efficacy of helicase-targeted drugs for personalized medicine. In a parallel effort, we are conducting RNA interference screens with isogenic helicase-deficient and helicase-proficient cell lines to identify novel interactions between human helicases and target genes. Conclusion & Significance: These studies are built upon the premise that DNA helicases exhibit a network of synthetic lethal interactions that may be exploited to overcome resistance to existing anti-cancer therapies.

Temozolomide is a DNA damaging agent that is a frontline therapeutic agent against brain tumors including glioblastoma (GBM). Unfortunately, resistance to temozolomide occurs frequently, thus limiting the overall utility of the agent. Resistance can be caused by a variety of different cellular defects. However, an emerging mechanism of resistance reflects the ability to DNA polymerases to misreplicate DNA lesions that are left unrepaired. In this study, we provide evidence that the therapeutic efficacy of temozolomide can be significantly increased by co-administration of an artificial nucleoside, denoted as 5-nitroindolyl-2???-deoxyriboside (5-NIdR), that efficiently and selectively inhibits the replication of DNA lesions generated by temozolomide. Conversion of 5-NIdR to the corresponding nucleoside triphosphate 5-NITP in vivo creates a potent inhibitor of several human DNA polymerases that can replicate damaged DNA. This inhibition accounts for the ability of 5-NIdR to synergize with temozolomide to increase apoptosis of tumor cells. In a murine xenograft model of GBM, treatment with temozolomide only delayed tumor growth while co-administering 5-NIdR with temozolomide caused complete tumor regression. Preliminary toxicology studies demonstrate that high doses of 5-NIdR do not produce the side effects typically observed with conventional nucleoside analogs such as fludarabine and gemcitabine. Collectively, these preclinical results demonstrate pharmacological proof of concept for the coordinate inhibition of translesion DNA synthesis as a strategy to improve chemotherapeutic responses in aggressive brain tumors.