Troy Harkness - Academia.edu (original) (raw)

Papers by Troy Harkness

Cancers

Like humans, canine lymphomas are treated by chemotherapy cocktails and frequently develop multip... more Like humans, canine lymphomas are treated by chemotherapy cocktails and frequently develop multiple drug resistance (MDR). Their shortened clinical timelines and tumor accessibility make canines excellent models to study MDR mechanisms. Insulin-sensitizers have been shown to reduce the incidence of cancer in humans prescribed them, and we previously demonstrated that they also reverse and delay MDR development in vitro. Here, we treated canines with MDR lymphoma with metformin to assess clinical and tumoral responses, including changes in MDR biomarkers, and used mRNA microarrays to determine differential gene expression. Metformin reduced MDR protein markers in all canines in the study. Microarrays performed on mRNAs gathered through longitudinal tumor sampling identified a 290 gene set that was enriched in Anaphase Promoting Complex (APC) substrates and additional mRNAs associated with slowed mitotic progression in MDR samples compared to skin controls. mRNAs from a canine that we...

Biomedicines, 2020

Cellular health is reliant on proteostasis—the maintenance of protein levels regulated through mu... more Cellular health is reliant on proteostasis—the maintenance of protein levels regulated through multiple pathways modulating protein synthesis, degradation and clearance. Loss of proteostasis results in serious disease and is associated with aging. One proteinaceous structure underlying the nuclear envelope—the nuclear lamina—coordinates essential processes including DNA repair, genome organization and epigenetic and transcriptional regulation. Loss of proteostasis within the nuclear lamina results in the accumulation of proteins, disrupting these essential functions, either via direct interactions of protein aggregates within the lamina or by altering systems that maintain lamina structure. Here we discuss the links between proteostasis and disease of the nuclear lamina, as well as how manipulating specific proteostatic pathways involved in protein clearance could improve cellular health and prevent/reverse disease.

ABSTRACTLike humans, canines spontaneously develop lymphomas that are treated by chemotherapy coc... more ABSTRACTLike humans, canines spontaneously develop lymphomas that are treated by chemotherapy cocktails and frequently develop multiple drug resistance (MDR). Their shortened clinical timelines and tumor accessibility make them excellent models to study MDR mechanisms. We previously demonstrated that adjunct treatment ofin vitroMDR cell lines with insulin-sensitizers effectively restored MDR chemosensitivity and prevented MDR development. This study extends the use of an insulin-sensitizer to clinical and tumor responsesin vivoin volunteer canines with MDR lymphoma, including assessing changes in MDR protein biomarkers and global gene expression. Longitudinal tumor sampling and analysis of MDR cases throughout treatment allowed a correlation betweenin vivomolecular mechanisms and clinical responsiveness. We found reduced MDR biomarkers within all tumors, yet only one canine entered clinical remission. Analysis of tumor samples during remission and relapse allowed comparison of gene ...

Journal of Biological Methods, 2018

The budding yeast Saccharomyces cerevisiae is a major model system in the study of aging. Like me... more The budding yeast Saccharomyces cerevisiae is a major model system in the study of aging. Like metazoans, yeast lifespan is extended by caloric restriction and treatment with pharmacological agents which extend lifespan. A major workhorse of aging research in budding yeast is the chronological lifespan assay. Traditionally, chronological lifespan assays consist of taking regular samples of aging yeast cultures, plating out aliquots on agar, and counting the resulting colonies. This method, while highly reliable, is labor-intensive and expensive in terms of materials consumed. Here, we report a novel MTT-based method for assessing chronological lifespan in yeast. We show that this method is equal to the colony counting method in its rigorous and reliable measurement of lifespan extension in yeast as a result of caloric restriction, and is able to distinguish known long-lived and short-lived yeast strains. We have further developed this method into a high-throughput assay that allows ...

International Journal of Molecular Sciences, 2018

In aging cells, genomic instability is now recognized as a hallmark event. Throughout life, cells... more In aging cells, genomic instability is now recognized as a hallmark event. Throughout life, cells encounter multiple endogenous and exogenous DNA damaging events that are mostly repaired, but inevitably DNA mutations, chromosome rearrangements, and epigenetic deregulation begins to mount. Now that people are living longer, more and more late life time is spent suffering from age-related disease, in which genomic instability plays a critical role. However, several major questions remain heavily debated, such as the following: When does aging start? How long can we live? In order to minimize the impact of genomic instability on longevity, it is important to understand when aging starts, and to ensure repair mechanisms remain optimal from the very start to the very end. In this review, the interplay between the stress and nutrient response networks, and the regulation of homeostasis and genomic stability, is discussed. Mechanisms that link these two networks are predicted to be key lifespan determinants. The Anaphase Promoting Complex (APC), a large evolutionarily conserved ubiquitin ligase, can potentially serve this need. Recent work demonstrates that the APC maintains genomic stability, mounts a stress response, and increases longevity in yeast. Furthermore, inhibition of APC activity by glucose and nutrient response factors indicates a tight link between the APC and the stress/nutrient response networks.

eLife, Aug 17, 2018

Translational efficiency correlates with longevity, yet its role in lifespan determination remain... more Translational efficiency correlates with longevity, yet its role in lifespan determination remains unclear. Using ribosome profiling, translation efficiency is globally reduced during replicative aging in budding yeast by at least two mechanisms: Firstly, Ssd1 is induced during aging, sequestering mRNAs to P-bodies. Furthermore, Ssd1 overexpression in young cells reduced translation and extended lifespan, while loss of Ssd1 reduced the translational deficit of old cells and shortened lifespan. Secondly, phosphorylation of eIF2α, mediated by the stress kinase Gcn2, was elevated in old cells, contributing to the global reduction in translation without detectable induction of the downstream Gcn4 transcriptional activator. tRNA overexpression activated Gcn2 in young cells and extended lifespan in a manner dependent on Gcn4. Moreover, overexpression of Gcn4 sufficed to extend lifespan in an autophagy-dependent manner in the absence of changes in global translation, indicating that Gcn4-m...

PloS one, 2017

Multiple drug resistant (MDR) malignancy remains a predictable and often terminal event in cancer... more Multiple drug resistant (MDR) malignancy remains a predictable and often terminal event in cancer therapy, and affects individuals with many cancer types, regardless of the stage at which they were originally diagnosed or the interval from last treatment. Protein biomarkers of MDR are not globally used for clinical decision-making, but include the overexpression of drug-efflux pumps (ABC transporter family) such as MDR-1 and BCRP, as well as HIF1α, a stress responsive transcription factor found elevated within many MDR tumors. Here, we present the important in vitro discovery that the development of MDR (in breast cancer cells) can be prevented, and that established MDR could be resensitized to therapy, by adjunct treatment with metformin. Metformin is prescribed globally to improve insulin sensitivity, including in those individuals with Type 2 Diabetes Mellitus (DM2). We demonstrate the effectiveness of metformin in resensitizing MDR breast cancer cell lines to their original trea...

Genetics, Sep 10, 2017

Aging in eukaryotes is accompanied by widespread deterioration of the somatic tissue. Yet, abolis... more Aging in eukaryotes is accompanied by widespread deterioration of the somatic tissue. Yet, abolishing germ cells delays the age-dependent somatic decline in Caenorhabditis elegans In adult worms lacking germ cells, the activation of the DAF-9/DAF-12 steroid signalling pathway in the gonad recruits DAF-16 activity in the intestine to promote longevity-associated phenotypes. However, the impact of this pathway on the fitness of normally reproducing animals is less clear. Here, we explore the link between progeny production and somatic aging and identify the loss of lysosomal acidity, a critical regulator of the proteolytic output of these organelles, as a novel biomarker of aging in C. elegans The increase in lysosomal pH in older worms is not a passive consequence of aging, but instead is timed with the cessation of reproduction and correlates with the reduction in proteostasis in early adult life. Our results further implicate the steroid signalling pathway and DAF-16 in dynamically...

Aging, 2016

The molecular genetics governing eukaryotic longevity are strongly conserved from yeast to mammal... more The molecular genetics governing eukaryotic longevity are strongly conserved from yeast to mammals [1]. The genetic malleability of yeast has facilitated discoveries that have determined factors involved in both replicative (divisions of a single cell, mitotic) and chronological (population survival time, post-mitotic) lifespan, with corresponding orthologs in higher eukaryotes subsequently proven to carry out similar roles. Specifically, the technique of yeast replicative lifespan (RLS) follows how many mitotic divisions a yeast cell will go through prior to senescence, whereas chronological lifespan (CLS) measures how long a postmitotic population of cells will remain metabolically active [2, 3]. These are two very different measures of the yeast life cycle, and are dictated by similar, yet distinct, sets of genes [3]. Intertwined with the genes affecting aging are those related to stress response and Research Paper adaptive survival. The Anaphase Promoting Complex (APC) and the yeast Forkhead (Fkh) proteins (Fkh1 and Fkh2) interact to play important roles in both lifespan and stress response [4-6]. Stress response genes play a significant role in lifespan in multiple model systems [7-9]. The evolutionarily conserved Fox family of proteins are stress response transcription factors that are critical for properly controlling apoptosis, autophagy, metabolism and cell proliferation [10-12]. This, in turn, improves cell health, survival, and extension of lifespan. In higher eukaryotes including flies, mice, and worms, mutations of various Fox family members results in significant elevations of cancer incidence, supporting their role in apoptosis and proliferation [13, 14]. Higher eukaryotic Fox proteins undergo numerous post-translational modifications including phosphorylation, and ubiquitin-dependent degradation by the proteasome, leading to a variety of

Cancers, 2015

Early detection and improved therapies for many cancers are enhancing survival rates. Although ma... more Early detection and improved therapies for many cancers are enhancing survival rates. Although many cytotoxic therapies are approved for aggressive or metastatic cancer; response rates are low and acquisition of de novo resistance is virtually universal. For decades; chemotherapeutic treatments for cancer have included anthracyclines such as Doxorubicin (DOX); and its use in aggressive tumors appears to remain a viable option; but drug resistance arises against DOX; as for all other classes of compounds. Our recent work suggests the anticoagulant protein Tissue Factor Pathway Inhibitor 1α (TFPI1α) plays a role in driving the development of multiple drug resistance (MDR); but not maintenance; of the MDR state. Other factors; such as the ABC transporter drug efflux pumps MDR-1/P-gp (ABCB1) and BCRP (ABCG2); are required for MDR maintenance; as well as development. The patient population struggling with therapeutic resistance specifically requires novel treatment options to resensitize...

PLoS genetics, 2015

Proliferating eukaryotic cells undergo a finite number of cell divisions before irreversibly exit... more Proliferating eukaryotic cells undergo a finite number of cell divisions before irreversibly exiting mitosis. Yet pathways that normally limit the number of cell divisions remain poorly characterized. Here we describe a screen of a collection of 3762 single gene mutants in the yeast Saccharomyces cerevisiae, accounting for 2/3 of annotated yeast ORFs, to search for mutants that undergo an atypically high number of cell divisions. Many of the potential longevity genes map to cellular processes not previously implicated in mitotic senescence, suggesting that regulatory mechanisms governing mitotic exit may be broader than currently anticipated. We focused on an ER-Golgi gene cluster isolated in this screen to determine how these ubiquitous organelles integrate into mitotic longevity. We report that a chronic increase in ER protein load signals an expansion in the assembly of autophagosomes in an Ire1-independent manner, accelerates trafficking of high molecular weight protein aggregat...

The Journal of biological chemistry, Jan 13, 2015

The enzyme family of heterotrimeric AMP-dependent protein kinases (AMPKs) is activated upon low e... more The enzyme family of heterotrimeric AMP-dependent protein kinases (AMPKs) is activated upon low energy states, conferring a switch towards energy-conserving metabolic pathways through immediate kinase actions on enzyme targets, and delayed alterations in gene expression through its nuclear relocalization. This family is evolutionarily conserved, including the presence of an Ubiquitin-associated (UBA) motif in most catalytic subunits. The potential for the UBA domain to promote protein-associations or direct subcellular location, as seen in other UBA-containing proteins, led us to query if the UBA domain within the yeast AMPK ortholog, SNF1 kinase, was important in these aspects of its regulation. Here, we demonstrate that conserved UBA motif mutations significantly alter SNF1 kinase activation and biological activity including enhanced allosteric subunit associations, and increased oxidative stress resistance and lifespan. Significantly, the enhanced UBA-dependent longevity and oxid...

Frontiers in physiology, 2012

The spatiotemporal dynamics of longevity-defining cellular processes and its modulation by geneti... more The spatiotemporal dynamics of longevity-defining cellular processes and its modulation by genetic, dietary, and pharmacological anti-aging interventions. Front. Physio. 3:419.

Frontiers in physiology, 2012

The longevity of an organism depends on the health of its cells. Throughout life cells are expose... more The longevity of an organism depends on the health of its cells. Throughout life cells are exposed to numerous intrinsic and extrinsic stresses, such as free radicals, generated through mitochondrial electron transport, and ultraviolet irradiation. The cell has evolved numerous mechanisms to scavenge free radicals and repair damage induced by these insults. One mechanism employed by the yeast Saccharomycescerevisiae to combat stress utilizes the Anaphase Promoting Complex (APC), an essential multi-subunit ubiquitin-protein ligase structurally and functionally conserved from yeast to humans that controls progression through mitosis and G1. We have observed that yeast cells expressing compromised APC subunits are sensitive to multiple stresses and have shorter replicative and chronological lifespans. In a pathway that runs parallel to that regulated by the APC, members of the Forkhead box (Fox) transcription factor family also regulate stress responses. The yeast Fox orthologs Fkh1 an...

Oncotarget, 2011

Aging is one of the major risk factors of cancer. The onset of cancer can be postponed by pharmac... more Aging is one of the major risk factors of cancer. The onset of cancer can be postponed by pharmacological and dietary anti-aging interventions. We recently found in yeast cellular models of aging that lithocholic acid (LCA) extends longevity. Here we show that, at concentrations that are not cytotoxic to primary cultures of human neurons, LCA kills the neuroblastoma (NB) cell lines BE(2)-m17, SK-n-SH, SK-n-MCIXC and Lan-1. In BE(2)-m17, SK-n-SH and SK-n-MCIXC cells, the LCA anti-tumor effect is due to apoptotic cell death. In contrast, the LCA-triggered death of Lan-1 cells is not caused by apoptosis. While low concentrations of LCA sensitize BE(2)-m17 and SK-n-MCIXC cells to hydrogen peroxide-induced apoptotic cell death controlled by mitochondria, these LCA concentrations make primary cultures of human neurons resistant to such a form of cell death. LCA kills BE(2)-m17 and SK-n-MCIXC cell lines by triggering not only the intrinsic (mitochondrial) apoptotic cell death pathway drive...

PLoS Genetics, 2012

Forkhead box O (FOXO) transcription factors have a conserved function in regulating metazoan life... more Forkhead box O (FOXO) transcription factors have a conserved function in regulating metazoan lifespan. A key function in this process involves the regulation of the cell cycle and stress responses including free radical scavenging. We employed yeast chronological and replicative lifespan assays, as well as oxidative stress assays, to explore the potential evolutionary conservation of function between the FOXOs and the yeast forkhead box transcription factors FKH1 and FKH2. We report that the deletion of both FKH genes impedes normal lifespan and stress resistance, particularly in stationary phase cells, which are non-responsive to caloric restriction. Conversely, increased expression of the FKHs leads to extended lifespan and improved stress response. Here we show the Anaphase-Promoting Complex (APC) genetically interacts with the Fkh pathway, likely working in a linear pathway under normal conditions, as fkh1D fkh2D post-mitotic survival is epistatic to that observed in apc5 CA mutants. However, under stress conditions, post-mitotic survival is dramatically impaired in apc5 CA fkh1D fkh2D, while increased expression of either FKH rescues APC mutant growth defects. This study establishes the FKHs role as evolutionarily conserved regulators of lifespan in yeast and identifies the APC as a novel component of this mechanism under certain conditions, likely through combined regulation of stress response, genomic stability, and cell cycle regulation.

PloS one, 2014

Thrombin and hypoxia are important players in breast cancer progression. Breast cancers often dev... more Thrombin and hypoxia are important players in breast cancer progression. Breast cancers often develop drug resistance, but mechanisms linking thrombin and hypoxia to drug resistance remain unresolved. Our studies using Doxorubicin (DOX) resistant MCF7 breast cancer cells reveals a mechanism linking DOX exposure with hypoxic induction of DOX resistance. Global expression changes between parental and DOX resistant MCF7 cells were examined. Westerns, Northerns and immunocytochemistry were used to validate drug resistance and differentially expressed genes. A cluster of genes involved in the anticoagulation pathway, with Tissue Factor Pathway Inhibitor 1 (TFPI1) the top hit, was identified. Plasmids overexpressing TFPI1 were utilized, and 1% O2 was used to test the effects of hypoxia on drug resistance. Lastly, microarray datasets from patients with drug resistant breast tumors were interrogated for TFPI1 expression levels. TFPI1 protein levels were found elevated in 3 additional DOX re...

Journal of Cell Biology, 1994

The novel genetic method of "sheltered RIP" (repeat induced point mutation) was used to... more The novel genetic method of "sheltered RIP" (repeat induced point mutation) was used to generate a Neurospora crassa mutant in which MOM19, a component of the protein import machinery of the mitochondrial outer membrane, can be depleted. Deficiency in MOM19 resulted in a severe growth defect, but the cells remained viable. The number of mitochondrial profiles was not grossly changed, but mutant mitochondria were highly deficient in cristae membranes, cytochromes, and protein synthesis activity. Protein import into isolated mutant mitochondria was decreased by factors of 6 to 30 for most proteins from all suborganellar compartments. Proteins like the ADP/ATP carrier, MOM19, and cytochrome c, whose import into wild-type mitochondria occurs independently of MOM19 became imported normally showing that the reduced import activities are solely caused by a lack of MOM19. Depletion of MOM19 reveals a close functional relationship between MOM19 and MOM22, since loss of MOM19 led to...

Molecular Cell, 2010

Changes to the chromatin structure accompany aging, but the molecular mechanisms underlying aging... more Changes to the chromatin structure accompany aging, but the molecular mechanisms underlying aging and the accompanying changes to the chromatin are unclear. Here, we report a mechanism whereby altering chromatin structure regulates life span. We show that normal aging is accompanied by a profound loss of histone proteins from the genome. Indeed, yeast lacking the histone chaperone Asf1 or acetylation of histone H3 on lysine 56 are short lived, and this appears to be at least partly due to their having decreased histone levels. Conversely, increasing the histone supply by inactivation of the histone information regulator (Hir) complex or overexpression of histones dramatically extends life span via a pathway that is distinct from previously known pathways of life span extension. This study indicates that maintenance of the fundamental chromatin structure is critical for slowing down the aging process and reveals that increasing the histone supply extends life span.

Cancers

Like humans, canine lymphomas are treated by chemotherapy cocktails and frequently develop multip... more Like humans, canine lymphomas are treated by chemotherapy cocktails and frequently develop multiple drug resistance (MDR). Their shortened clinical timelines and tumor accessibility make canines excellent models to study MDR mechanisms. Insulin-sensitizers have been shown to reduce the incidence of cancer in humans prescribed them, and we previously demonstrated that they also reverse and delay MDR development in vitro. Here, we treated canines with MDR lymphoma with metformin to assess clinical and tumoral responses, including changes in MDR biomarkers, and used mRNA microarrays to determine differential gene expression. Metformin reduced MDR protein markers in all canines in the study. Microarrays performed on mRNAs gathered through longitudinal tumor sampling identified a 290 gene set that was enriched in Anaphase Promoting Complex (APC) substrates and additional mRNAs associated with slowed mitotic progression in MDR samples compared to skin controls. mRNAs from a canine that we...

Biomedicines, 2020

Cellular health is reliant on proteostasis—the maintenance of protein levels regulated through mu... more Cellular health is reliant on proteostasis—the maintenance of protein levels regulated through multiple pathways modulating protein synthesis, degradation and clearance. Loss of proteostasis results in serious disease and is associated with aging. One proteinaceous structure underlying the nuclear envelope—the nuclear lamina—coordinates essential processes including DNA repair, genome organization and epigenetic and transcriptional regulation. Loss of proteostasis within the nuclear lamina results in the accumulation of proteins, disrupting these essential functions, either via direct interactions of protein aggregates within the lamina or by altering systems that maintain lamina structure. Here we discuss the links between proteostasis and disease of the nuclear lamina, as well as how manipulating specific proteostatic pathways involved in protein clearance could improve cellular health and prevent/reverse disease.

ABSTRACTLike humans, canines spontaneously develop lymphomas that are treated by chemotherapy coc... more ABSTRACTLike humans, canines spontaneously develop lymphomas that are treated by chemotherapy cocktails and frequently develop multiple drug resistance (MDR). Their shortened clinical timelines and tumor accessibility make them excellent models to study MDR mechanisms. We previously demonstrated that adjunct treatment ofin vitroMDR cell lines with insulin-sensitizers effectively restored MDR chemosensitivity and prevented MDR development. This study extends the use of an insulin-sensitizer to clinical and tumor responsesin vivoin volunteer canines with MDR lymphoma, including assessing changes in MDR protein biomarkers and global gene expression. Longitudinal tumor sampling and analysis of MDR cases throughout treatment allowed a correlation betweenin vivomolecular mechanisms and clinical responsiveness. We found reduced MDR biomarkers within all tumors, yet only one canine entered clinical remission. Analysis of tumor samples during remission and relapse allowed comparison of gene ...

Journal of Biological Methods, 2018

The budding yeast Saccharomyces cerevisiae is a major model system in the study of aging. Like me... more The budding yeast Saccharomyces cerevisiae is a major model system in the study of aging. Like metazoans, yeast lifespan is extended by caloric restriction and treatment with pharmacological agents which extend lifespan. A major workhorse of aging research in budding yeast is the chronological lifespan assay. Traditionally, chronological lifespan assays consist of taking regular samples of aging yeast cultures, plating out aliquots on agar, and counting the resulting colonies. This method, while highly reliable, is labor-intensive and expensive in terms of materials consumed. Here, we report a novel MTT-based method for assessing chronological lifespan in yeast. We show that this method is equal to the colony counting method in its rigorous and reliable measurement of lifespan extension in yeast as a result of caloric restriction, and is able to distinguish known long-lived and short-lived yeast strains. We have further developed this method into a high-throughput assay that allows ...

International Journal of Molecular Sciences, 2018

In aging cells, genomic instability is now recognized as a hallmark event. Throughout life, cells... more In aging cells, genomic instability is now recognized as a hallmark event. Throughout life, cells encounter multiple endogenous and exogenous DNA damaging events that are mostly repaired, but inevitably DNA mutations, chromosome rearrangements, and epigenetic deregulation begins to mount. Now that people are living longer, more and more late life time is spent suffering from age-related disease, in which genomic instability plays a critical role. However, several major questions remain heavily debated, such as the following: When does aging start? How long can we live? In order to minimize the impact of genomic instability on longevity, it is important to understand when aging starts, and to ensure repair mechanisms remain optimal from the very start to the very end. In this review, the interplay between the stress and nutrient response networks, and the regulation of homeostasis and genomic stability, is discussed. Mechanisms that link these two networks are predicted to be key lifespan determinants. The Anaphase Promoting Complex (APC), a large evolutionarily conserved ubiquitin ligase, can potentially serve this need. Recent work demonstrates that the APC maintains genomic stability, mounts a stress response, and increases longevity in yeast. Furthermore, inhibition of APC activity by glucose and nutrient response factors indicates a tight link between the APC and the stress/nutrient response networks.

eLife, Aug 17, 2018

Translational efficiency correlates with longevity, yet its role in lifespan determination remain... more Translational efficiency correlates with longevity, yet its role in lifespan determination remains unclear. Using ribosome profiling, translation efficiency is globally reduced during replicative aging in budding yeast by at least two mechanisms: Firstly, Ssd1 is induced during aging, sequestering mRNAs to P-bodies. Furthermore, Ssd1 overexpression in young cells reduced translation and extended lifespan, while loss of Ssd1 reduced the translational deficit of old cells and shortened lifespan. Secondly, phosphorylation of eIF2α, mediated by the stress kinase Gcn2, was elevated in old cells, contributing to the global reduction in translation without detectable induction of the downstream Gcn4 transcriptional activator. tRNA overexpression activated Gcn2 in young cells and extended lifespan in a manner dependent on Gcn4. Moreover, overexpression of Gcn4 sufficed to extend lifespan in an autophagy-dependent manner in the absence of changes in global translation, indicating that Gcn4-m...

PloS one, 2017

Multiple drug resistant (MDR) malignancy remains a predictable and often terminal event in cancer... more Multiple drug resistant (MDR) malignancy remains a predictable and often terminal event in cancer therapy, and affects individuals with many cancer types, regardless of the stage at which they were originally diagnosed or the interval from last treatment. Protein biomarkers of MDR are not globally used for clinical decision-making, but include the overexpression of drug-efflux pumps (ABC transporter family) such as MDR-1 and BCRP, as well as HIF1α, a stress responsive transcription factor found elevated within many MDR tumors. Here, we present the important in vitro discovery that the development of MDR (in breast cancer cells) can be prevented, and that established MDR could be resensitized to therapy, by adjunct treatment with metformin. Metformin is prescribed globally to improve insulin sensitivity, including in those individuals with Type 2 Diabetes Mellitus (DM2). We demonstrate the effectiveness of metformin in resensitizing MDR breast cancer cell lines to their original trea...

Genetics, Sep 10, 2017

Aging in eukaryotes is accompanied by widespread deterioration of the somatic tissue. Yet, abolis... more Aging in eukaryotes is accompanied by widespread deterioration of the somatic tissue. Yet, abolishing germ cells delays the age-dependent somatic decline in Caenorhabditis elegans In adult worms lacking germ cells, the activation of the DAF-9/DAF-12 steroid signalling pathway in the gonad recruits DAF-16 activity in the intestine to promote longevity-associated phenotypes. However, the impact of this pathway on the fitness of normally reproducing animals is less clear. Here, we explore the link between progeny production and somatic aging and identify the loss of lysosomal acidity, a critical regulator of the proteolytic output of these organelles, as a novel biomarker of aging in C. elegans The increase in lysosomal pH in older worms is not a passive consequence of aging, but instead is timed with the cessation of reproduction and correlates with the reduction in proteostasis in early adult life. Our results further implicate the steroid signalling pathway and DAF-16 in dynamically...

Aging, 2016

The molecular genetics governing eukaryotic longevity are strongly conserved from yeast to mammal... more The molecular genetics governing eukaryotic longevity are strongly conserved from yeast to mammals [1]. The genetic malleability of yeast has facilitated discoveries that have determined factors involved in both replicative (divisions of a single cell, mitotic) and chronological (population survival time, post-mitotic) lifespan, with corresponding orthologs in higher eukaryotes subsequently proven to carry out similar roles. Specifically, the technique of yeast replicative lifespan (RLS) follows how many mitotic divisions a yeast cell will go through prior to senescence, whereas chronological lifespan (CLS) measures how long a postmitotic population of cells will remain metabolically active [2, 3]. These are two very different measures of the yeast life cycle, and are dictated by similar, yet distinct, sets of genes [3]. Intertwined with the genes affecting aging are those related to stress response and Research Paper adaptive survival. The Anaphase Promoting Complex (APC) and the yeast Forkhead (Fkh) proteins (Fkh1 and Fkh2) interact to play important roles in both lifespan and stress response [4-6]. Stress response genes play a significant role in lifespan in multiple model systems [7-9]. The evolutionarily conserved Fox family of proteins are stress response transcription factors that are critical for properly controlling apoptosis, autophagy, metabolism and cell proliferation [10-12]. This, in turn, improves cell health, survival, and extension of lifespan. In higher eukaryotes including flies, mice, and worms, mutations of various Fox family members results in significant elevations of cancer incidence, supporting their role in apoptosis and proliferation [13, 14]. Higher eukaryotic Fox proteins undergo numerous post-translational modifications including phosphorylation, and ubiquitin-dependent degradation by the proteasome, leading to a variety of

Cancers, 2015

Early detection and improved therapies for many cancers are enhancing survival rates. Although ma... more Early detection and improved therapies for many cancers are enhancing survival rates. Although many cytotoxic therapies are approved for aggressive or metastatic cancer; response rates are low and acquisition of de novo resistance is virtually universal. For decades; chemotherapeutic treatments for cancer have included anthracyclines such as Doxorubicin (DOX); and its use in aggressive tumors appears to remain a viable option; but drug resistance arises against DOX; as for all other classes of compounds. Our recent work suggests the anticoagulant protein Tissue Factor Pathway Inhibitor 1α (TFPI1α) plays a role in driving the development of multiple drug resistance (MDR); but not maintenance; of the MDR state. Other factors; such as the ABC transporter drug efflux pumps MDR-1/P-gp (ABCB1) and BCRP (ABCG2); are required for MDR maintenance; as well as development. The patient population struggling with therapeutic resistance specifically requires novel treatment options to resensitize...

PLoS genetics, 2015

Proliferating eukaryotic cells undergo a finite number of cell divisions before irreversibly exit... more Proliferating eukaryotic cells undergo a finite number of cell divisions before irreversibly exiting mitosis. Yet pathways that normally limit the number of cell divisions remain poorly characterized. Here we describe a screen of a collection of 3762 single gene mutants in the yeast Saccharomyces cerevisiae, accounting for 2/3 of annotated yeast ORFs, to search for mutants that undergo an atypically high number of cell divisions. Many of the potential longevity genes map to cellular processes not previously implicated in mitotic senescence, suggesting that regulatory mechanisms governing mitotic exit may be broader than currently anticipated. We focused on an ER-Golgi gene cluster isolated in this screen to determine how these ubiquitous organelles integrate into mitotic longevity. We report that a chronic increase in ER protein load signals an expansion in the assembly of autophagosomes in an Ire1-independent manner, accelerates trafficking of high molecular weight protein aggregat...

The Journal of biological chemistry, Jan 13, 2015

The enzyme family of heterotrimeric AMP-dependent protein kinases (AMPKs) is activated upon low e... more The enzyme family of heterotrimeric AMP-dependent protein kinases (AMPKs) is activated upon low energy states, conferring a switch towards energy-conserving metabolic pathways through immediate kinase actions on enzyme targets, and delayed alterations in gene expression through its nuclear relocalization. This family is evolutionarily conserved, including the presence of an Ubiquitin-associated (UBA) motif in most catalytic subunits. The potential for the UBA domain to promote protein-associations or direct subcellular location, as seen in other UBA-containing proteins, led us to query if the UBA domain within the yeast AMPK ortholog, SNF1 kinase, was important in these aspects of its regulation. Here, we demonstrate that conserved UBA motif mutations significantly alter SNF1 kinase activation and biological activity including enhanced allosteric subunit associations, and increased oxidative stress resistance and lifespan. Significantly, the enhanced UBA-dependent longevity and oxid...

Frontiers in physiology, 2012

The spatiotemporal dynamics of longevity-defining cellular processes and its modulation by geneti... more The spatiotemporal dynamics of longevity-defining cellular processes and its modulation by genetic, dietary, and pharmacological anti-aging interventions. Front. Physio. 3:419.

Frontiers in physiology, 2012

The longevity of an organism depends on the health of its cells. Throughout life cells are expose... more The longevity of an organism depends on the health of its cells. Throughout life cells are exposed to numerous intrinsic and extrinsic stresses, such as free radicals, generated through mitochondrial electron transport, and ultraviolet irradiation. The cell has evolved numerous mechanisms to scavenge free radicals and repair damage induced by these insults. One mechanism employed by the yeast Saccharomycescerevisiae to combat stress utilizes the Anaphase Promoting Complex (APC), an essential multi-subunit ubiquitin-protein ligase structurally and functionally conserved from yeast to humans that controls progression through mitosis and G1. We have observed that yeast cells expressing compromised APC subunits are sensitive to multiple stresses and have shorter replicative and chronological lifespans. In a pathway that runs parallel to that regulated by the APC, members of the Forkhead box (Fox) transcription factor family also regulate stress responses. The yeast Fox orthologs Fkh1 an...

Oncotarget, 2011

Aging is one of the major risk factors of cancer. The onset of cancer can be postponed by pharmac... more Aging is one of the major risk factors of cancer. The onset of cancer can be postponed by pharmacological and dietary anti-aging interventions. We recently found in yeast cellular models of aging that lithocholic acid (LCA) extends longevity. Here we show that, at concentrations that are not cytotoxic to primary cultures of human neurons, LCA kills the neuroblastoma (NB) cell lines BE(2)-m17, SK-n-SH, SK-n-MCIXC and Lan-1. In BE(2)-m17, SK-n-SH and SK-n-MCIXC cells, the LCA anti-tumor effect is due to apoptotic cell death. In contrast, the LCA-triggered death of Lan-1 cells is not caused by apoptosis. While low concentrations of LCA sensitize BE(2)-m17 and SK-n-MCIXC cells to hydrogen peroxide-induced apoptotic cell death controlled by mitochondria, these LCA concentrations make primary cultures of human neurons resistant to such a form of cell death. LCA kills BE(2)-m17 and SK-n-MCIXC cell lines by triggering not only the intrinsic (mitochondrial) apoptotic cell death pathway drive...

PLoS Genetics, 2012

Forkhead box O (FOXO) transcription factors have a conserved function in regulating metazoan life... more Forkhead box O (FOXO) transcription factors have a conserved function in regulating metazoan lifespan. A key function in this process involves the regulation of the cell cycle and stress responses including free radical scavenging. We employed yeast chronological and replicative lifespan assays, as well as oxidative stress assays, to explore the potential evolutionary conservation of function between the FOXOs and the yeast forkhead box transcription factors FKH1 and FKH2. We report that the deletion of both FKH genes impedes normal lifespan and stress resistance, particularly in stationary phase cells, which are non-responsive to caloric restriction. Conversely, increased expression of the FKHs leads to extended lifespan and improved stress response. Here we show the Anaphase-Promoting Complex (APC) genetically interacts with the Fkh pathway, likely working in a linear pathway under normal conditions, as fkh1D fkh2D post-mitotic survival is epistatic to that observed in apc5 CA mutants. However, under stress conditions, post-mitotic survival is dramatically impaired in apc5 CA fkh1D fkh2D, while increased expression of either FKH rescues APC mutant growth defects. This study establishes the FKHs role as evolutionarily conserved regulators of lifespan in yeast and identifies the APC as a novel component of this mechanism under certain conditions, likely through combined regulation of stress response, genomic stability, and cell cycle regulation.

PloS one, 2014

Thrombin and hypoxia are important players in breast cancer progression. Breast cancers often dev... more Thrombin and hypoxia are important players in breast cancer progression. Breast cancers often develop drug resistance, but mechanisms linking thrombin and hypoxia to drug resistance remain unresolved. Our studies using Doxorubicin (DOX) resistant MCF7 breast cancer cells reveals a mechanism linking DOX exposure with hypoxic induction of DOX resistance. Global expression changes between parental and DOX resistant MCF7 cells were examined. Westerns, Northerns and immunocytochemistry were used to validate drug resistance and differentially expressed genes. A cluster of genes involved in the anticoagulation pathway, with Tissue Factor Pathway Inhibitor 1 (TFPI1) the top hit, was identified. Plasmids overexpressing TFPI1 were utilized, and 1% O2 was used to test the effects of hypoxia on drug resistance. Lastly, microarray datasets from patients with drug resistant breast tumors were interrogated for TFPI1 expression levels. TFPI1 protein levels were found elevated in 3 additional DOX re...

Journal of Cell Biology, 1994

The novel genetic method of "sheltered RIP" (repeat induced point mutation) was used to... more The novel genetic method of "sheltered RIP" (repeat induced point mutation) was used to generate a Neurospora crassa mutant in which MOM19, a component of the protein import machinery of the mitochondrial outer membrane, can be depleted. Deficiency in MOM19 resulted in a severe growth defect, but the cells remained viable. The number of mitochondrial profiles was not grossly changed, but mutant mitochondria were highly deficient in cristae membranes, cytochromes, and protein synthesis activity. Protein import into isolated mutant mitochondria was decreased by factors of 6 to 30 for most proteins from all suborganellar compartments. Proteins like the ADP/ATP carrier, MOM19, and cytochrome c, whose import into wild-type mitochondria occurs independently of MOM19 became imported normally showing that the reduced import activities are solely caused by a lack of MOM19. Depletion of MOM19 reveals a close functional relationship between MOM19 and MOM22, since loss of MOM19 led to...

Molecular Cell, 2010

Changes to the chromatin structure accompany aging, but the molecular mechanisms underlying aging... more Changes to the chromatin structure accompany aging, but the molecular mechanisms underlying aging and the accompanying changes to the chromatin are unclear. Here, we report a mechanism whereby altering chromatin structure regulates life span. We show that normal aging is accompanied by a profound loss of histone proteins from the genome. Indeed, yeast lacking the histone chaperone Asf1 or acetylation of histone H3 on lysine 56 are short lived, and this appears to be at least partly due to their having decreased histone levels. Conversely, increasing the histone supply by inactivation of the histone information regulator (Hir) complex or overexpression of histones dramatically extends life span via a pathway that is distinct from previously known pathways of life span extension. This study indicates that maintenance of the fundamental chromatin structure is critical for slowing down the aging process and reveals that increasing the histone supply extends life span.