Activation of the Anaphase Promoting Complex Reverses Multiple Drug Resistant Cancer in a Canine Model of Multiple Drug Resistant Lymphoma.

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 went into remission showed that APC substrate mRNAs were decreased, indicating that the APC was activated during remission. In vitro validation using canine lymphoma cells selected for resistance to chemotherapeutic drugs confirmed that APC activation restored MDR chemosensitivity, and that APC activity was reduced in MDR cells. This supports the idea that rapidly pushing MDR cells that harbor high loads of chromosome instability through mitosis, by activating the APC, contributes to improved survival and disease-free duration.

Smc5/6 in the rDNA modulates lifespan independently of Fob1​.

The ribosomal DNA (rDNA) in Saccharomyces cerevisiae is in one tandem repeat array on Chromosome XII. Two regions within each repetitive element, called intergenic spacer 1 (IGS1) and IGS2, are important for organizing the rDNA within the nucleolus. The Smc5/6 complex localizes to IGS1 and IGS2. We show that Smc5/6 has a function in the rDNA beyond its role in homologous recombination (HR) at the replication fork barrier (RFB) located in IGS1. Fob1 is required for optimal binding of Smc5/6 at IGS1 whereas the canonical silencing factor Sir2 is required for its optimal binding at IGS2, independently of Fob1. Through interdependent interactions, Smc5/6 stabilizes Sir2 and Cohibin at both IGS and its recovery at IGS2 is important for nucleolar compaction and transcriptional silencing, which in turn supports rDNA stability and lifespan.

High-Throughput Rapid Yeast Chronological Lifespan Assay.

The budding yeast is a valuable model system for discovering molecular mechanisms underlying cellular aging. This is due to the ease of performing genetic manipulations in yeast and the vast number of evolutionarily conserved genes that have been found to regulate cellular health and lifespan from yeast to humans. Lifespan assays are an essential tool for examining the effects of these genes on longevity. There are two ways lifespan is measured in yeast: replicative lifespan (RLS) and chronological lifespan (CLS). RLS is a measure of how many divisions an individual mother cell will undergo. CLS measures the length of time nondividing cells survive. Previously described CLS assays involved diluting and plating cells of a culture and counting the colonies that arose. While effective, this method is both time and labor intensive. Here, we describe a method for a high-throughput rapid CLS assay that is both time- and cost-efficient.

The Nuclear Lamina: Protein Accumulation and Disease.

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.

Activating the Anaphase Promoting Complex to Enhance Genomic Stability and Prolong Lifespan.

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.

Mitotic degradation of yeast Fkh1 by the Anaphase Promoting Complex is required for normal longevity, genomic stability and stress resistance.

The Saccharomyces cerevisiae Forkhead Box (Fox) orthologs, Forkheads (Fkh) 1 and 2, are conserved transcription factors required for stress response, cell cycle progression and longevity. These yeast proteins play a key role in mitotic progression through activation of the ubiquitin E3 ligase Anaphase Promoting Complex (APC) via transcriptional control. Here, we used genetic and molecular analyses to demonstrate that the APC E3 activity is necessary for mitotic Fkh1 protein degradation and subsequent cell cycle progression. We report that Fkh1 protein degradation occurs specifically during mitosis, requires APCCdc20 and proteasome activity, and that a stable Fkh1 mutant reduces normal chronological lifespan, increases genomic instability, and increases sensitivity to stress. Our data supports a model whereby cell cycle progression through mitosis and G1 requires the targeted degradation of Fkh1 by the APC. This is significant to many fields as these results impact our understanding of the mechanisms underpinning the control of aging and cancer.

A Genome Scale Screen for Mutants with Delayed Exit from Mitosis: Ire1-Independent Induction of Autophagy Integrates ER Homeostasis into Mitotic Lifespan.

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 aggregates from the cytoplasm to the vacuoles, and leads to a profound enhancement of daughter cell production. We demonstrate that this catabolic network is evolutionarily conserved, as it also extends reproductive lifespan in the nematode Caenorhabditis elegans. Our data provide evidence that catabolism of protein aggregates, a natural byproduct of high protein synthesis and turn over in dividing cells, is among the drivers of mitotic longevity in eukaryotes.

Replicative and chronological life-span assays.

Life-span assays in yeast are invaluable in characterizing the functions of gene products on cellular aging. Replicative life-span (RLS) is a measure of the number of divisions an individual cell can undergo. In this assay daughter cells are removed using a tetrad dissection microscope with a micromanipulator and scored. Chronological life-span (CLS) measures the length of time nondividing cells survive. A culture is grown to stationary phase with samples removed over time to assess the survival within the population. The strength of the yeast system lies in the ease of genetically manipulating genes of interest and the evolutionarily conserved nature of the genes found to influence longevity. Here, we describe methods used to measure yeast RLS and CLS.

TFPI1 mediates resistance to doxorubicin in breast cancer cells by inducing a hypoxic-like response.

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 resistant cells lines, from humans and rats, indicating evolutionarily conservation of the effect. Elevated TFPI1 in DOX resistant cells was active, as thrombin protein levels were coincidentally low. We observed elevated HIF1α protein in DOX resistant cells, and in cells with forced expression of TFPI1, suggesting TFPI1 induces HIF1α. TFPI1 also induced c-MYC, c-SRC, and HDAC2 protein, as well as DOX resistance in parental cells. Growth of cells in 1% O2 induced elevated HIF1α, BCRP and MDR-1 protein, and these cells were resistant to DOX. Our in vitro results were consistent with in vivo patient datasets, as tumors harboring increased BCRP and MDR-1 expression also had increased TFPI1 expression. Our observations are clinically relevant indicating that DOX treatment induces an anticoagulation cascade, leading to inhibition of thrombin and the expression of HIF1α. This in turn activates a pathway leading to drug resistance.

The anaphase promoting complex regulates yeast lifespan and rDNA stability by targeting Fob1 for degradation.

Genomic stability, stress response, and nutrient signaling all play critical, evolutionarily conserved roles in lifespan determination. However, the molecular mechanisms coordinating these processes with longevity remain unresolved. Here we investigate the involvement of the yeast anaphase promoting complex (APC) in longevity. The APC governs passage through M and G1 via ubiquitin-dependent targeting of substrate proteins and is associated with cancer and premature aging when defective. Our two-hybrid screen utilizing Apc5 as bait recovered the lifespan determinant Fob1 as prey. Fob1 is unstable specifically in G1, cycles throughout the cell cycle in a manner similar to Clb2 (an APC target), and is stabilized in APC (apc5(CA)) and proteasome (rpn10) mutants. Deletion of FOB1 increased replicative lifespan (RLS) in wild type (WT), apc5(CA), and apc10 cells, and suppressed apc5(CA) cell cycle progression and rDNA recombination defects. Alternatively, increased FOB1 expression decreased RLS in WT cells, but did not reduce the already short apc5(CA) RLS, suggesting an epistatic interaction between apc5(CA) and fob1. Mutation to a putative L-Box (Fob1(E420V)), a Destruction Box-like motif, abolished Fob1 modifications, stabilized the protein, and increased rDNA recombination. Our work provides a mechanistic role played by the APC to promote replicative longevity and genomic stability in yeast.

The yeast forkhead transcription factors fkh1 and fkh2 regulate lifespan and stress response together with the anaphase-promoting complex.

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 fkh1Δ fkh2Δ 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) fkh1Δ fkh2Δ, 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.

Lithocholic bile acid selectively kills neuroblastoma cells, while sparing normal neuronal cells.

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 driven by mitochondrial outer membrane permeabilization and initiator caspase-9 activation, but also the extrinsic (death receptor) pathway of apoptosis involving activation of the initiator caspase-8. Based on these data, we propose a mechanism underlying a potent and selective anti-tumor effect of LCA in cultured human NB cells. Moreover, our finding that LCA kills cultured human breast cancer and rat glioma cells implies that it has a broad anti-tumor effect on cancer cells derived from different tissues and organisms.

A role for the anaphase promoting complex in hormone regulation.

To increase our knowledge of anaphase promoting complex (APC/C) function during plant development, we characterized an Arabidopsis thaliana T-DNA-insertion line where the T-DNA fell within the 5' regulatory region of the APC10 gene. The insert disrupted endogenous expression, resulting in overexpression of APC10 mRNA from the T-DNA- internal CaMV 35S promoter, and increased APC10 protein. Overexpression of APC10 produced phenotypes resembling those of known auxin and ethylene mutants, and increased expression of two tested auxin-regulated genes, small auxin up RNA (SAUR) 15 and SAUR24. Taken together, our data suggests that elevated APC10 likely mimics auxin and ethylene sensitive phenotypes, expanding our understanding of proteolytic processes in hormone regulation of plant development.

The Saccharomyces cerevisiae anaphase-promoting complex interacts with multiple histone-modifying enzymes to regulate cell cycle progression.

The anaphase-promoting complex (APC), a large evolutionarily conserved ubiquitin ligase complex, regulates cell cycle progression through mitosis and G(1). Here, we present data suggesting that APC-dependent cell cycle progression relies on a specific set of posttranslational histone-modifying enzymes. Multiple APC subunit mutants were impaired in total and modified histone H3 protein content. Acetylated H3K56 (H3K56(Ac)) levels were as reduced as those of total H3, indicating that loading histones with H3K56(Ac) is unaffected in APC mutants. However, under restrictive conditions, H3K9(Ac) and dimethylated H3K79 (H3K79(me2)) levels were more greatly reduced than those of total H3. In a screen for histone acetyltransferase (HAT) and histone deacetylase (HDAC) mutants that genetically interact with the apc5(CA) (chromatin assembly) mutant, we found that deletion of GCN5 or ELP3 severely hampered apc5(CA) temperature-sensitive (ts) growth. Further analyses showed that (i) the elp3Δ gcn5Δ double mutant ts defect was epistatic to that observed in apc5(CA) cells; (ii) gcn5Δ and elp3Δ mutants accumulate in mitosis; and (iii) turnover of the APC substrate Clb2 is not impaired in elp3Δ gcn5Δ cells. Increased expression of ELP3 and GCN5, as well as genes encoding the HAT Rtt109 and the chromatin assembly factors Msi1 and Asf1, suppressed apc5(CA) defects, while increased APC5 expression partially suppressed elp3Δ gcn5Δ growth defects. Finally, we demonstrate that Gcn5 is unstable during G(1) and following G(1) arrest and is stabilized in APC mutants. We present our working model in which Elp3/Gcn5 and the APC work together to facilitate passage through mitosis and G(1). To progress into S, we propose that at least Gcn5 must then be targeted for degradation in an APC-dependent fashion.

Troglitazone reverses the multiple drug resistance phenotype in cancer cells.

A major problem in treating cancer is the development of drug resistance. We previously demonstrated doxorubicin (DOX) resistance in K562 human leukemia cells that was associated with upregulation of glyoxalase 1 (GLO-1) and histone H3 expression. The thiazolidinedione troglitazone (TRG) downregulated GLO-1 expression and further upregulated histone H3 expression and post-translational modifications in these cells, leading to a regained sensitivity to DOX. Given the pleiotropic effects of epigenetic changes in cancer development, we hypothesized that TRG may downregulate the multiple drug resistance (MDR) phenotype in a variety of cancer cells. To test this, MCF7 human breast cancer cells and K562 cells were cultured in the presence of low-dose DOX to establish DOX-resistant cell lines (K562/DOX and MCF7/DOX). The MDR phenotype was confirmed by Western blot analysis of the 170 kDa P-glycoprotein (Pgp) drug efflux pump multiple drug resistance protein 1 (MDR-1), and the breast cancer resistance protein (BCRP). TRG markedly decreased expression of both MDR-1 and BCRP in these cells, resulting in sensitivity to DOX. Silencing of MDR-1 expression also sensitized MCF7/DOX cells to DOX. Use of the specific and irreversible peroxisome proliferator-activated receptor gamma (PPARgamma) inhibitor GW9662 in the nanomolar range not only demonstrated that the action of TRG on MCF/DOX was PPARgamma-independent, but indicated that PPARgamma may play a role in the MDR phenotype, which is antagonized by TRG. We conclude that TRG is potentially a useful adjunct therapy in chemoresistant cancers.

Phase 2 protein inducers in the diet promote healthier aging.

Oxidative stress drives many aging-associated problems. Because oxidative stress can be decreased by induction of phase 2 proteins, we hypothesized that incorporating the phase 2 protein inducer 2(3)-tert-butyl-4-hydroxyanisole (tBHA) into the diet would result in healthier aging. C57BL/6 mice were placed either on control mouse chow diet or on chow containing tBHA and were examined at 6, 12, and 18 months. Dietary tBHA resulted in the antioxidant response activation, decreased both oxidative stress and pro-inflammatory gene expression in tissues examined, counteracted the decrease in the transcription factors peroxisome proliferator-activated receptor-gamma and increase in CCAAT/enhancer binding protein-alpha levels seen in liver with aging, and was associated with mice having less weight gain, despite having no differences in food consumption, and better locomotor function. We conclude that simple changes in the diet such as incorporation of phase 2 protein inducers can have a profound influence on health and, thereby, the aging process.

Decondensation of Xenopus sperm chromatin using Saccharomyces cerevisiae whole-cell extracts.

Biochemical studies using highly condensed Xenopus sperm chromatin and protein extracts prepared from multiple systems have lead to the identification of conserved proteins involved in chromosome decondensation. However, mutations to these proteins are unavailable as the systems used are not amenable to genetic studies. We took a genetic approach to isolating chromosome decondensation mutants by incubating Xenopus sperm chromatin with whole-cell extracts prepared from the Hartwell library of random temperature sensitive (ts) yeast cells. We show that decondensation of Xenopus sperm chromatin using wild type yeast extracts was rapid, ATP- and extract-dependent, and resistant to heat, N-ethylmaleimide, protease K, RNase A, and micrococcal nuclease. From 100 mutant extracts screened, we obtained one strain, referred to as rmc4, that was chromosome decondensation defective. The mutant was slow growing and exhibited germination defects. Low concentrations of rmc4 extract would eventually decondense sperm heads, and fractionation of the mutant extract produced a decondensation competent fraction, suggesting the presence of an overactive inhibitor in rmc4 cells. We performed a multicopy suppressor screen that identified PDE2, a gene encoding a protein that inhibits protein kinase A (PKA) activity. As PKA was previously shown in human cells to maintain condensed chromatin, our results suggest that PKA activity is elevated in rmc4 cells, causing a decondensation defect. Thus, our experiments reveal that yeast encodes an evolutionarily conserved chromosome decondensation activity that can be genetically manipulated.

Troglitazone overcomes doxorubicin-resistance in resistant K562 leukemia cells.

Human myeloid leukemia cells become resistant to doxorubicin (DOX) treatment and this resistance is correlated with an increased glyoxalase 1 (GLO1) expression. Troglitazone (TRG) is an anti-diabetic thiazolidinedione drug previously used to treat insulin-resistance in Type 2 diabetes. We previously showed that TRG down regulates GLO1 gene expression in a number of cell types and reasoned that TRG might be a useful adjunct therapy to overcome DOX resistance. Here we show that TRG treatment overcomes the resistance to DOX in the DOX-resistant K562 human leukemia cells. Higher doses of TRG were found to alter histone H3:H2B ratios with a decreased ratio in DOX-sensitive and increased ratio in DOX-resistant lines. Furthermore, phosphorylated H3 was seen in DOX-resistant but not in DOX-sensitive cells. We conclude that the downstream effect of TRG in DOX-resistant cells may be interference with normal cell cycle events leading to genomic instability. Our data suggest that TRG may be a useful adjunct therapy in circumventing drug resistance in K562 leukemia cells.