20 research outputs found
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 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 mechanis
Increased <i>FKH1</i> or <i>FKH2</i> expression improves stress resistance and extends CLS and RLS.
<p>(A) Schematic representation of scheme used to integrate the <i>GAL1</i>/<i>10</i> promoter upstream of <i>FKH1</i> and <i>FKH2</i>. The <i>LEU2</i> PCR product, containing 300 basepairs of <i>LEU2</i> promoter (incorporated for selection purposes), was flanked by 60 basepairs of homology to the <i>FKH</i> promoter and to the <i>GAL1</i>/<i>10</i> promoter. The <i>GAL1</i>/<i>10</i> promoter PCR fragment was flanked by 60 basepairs of homology to the 3β² end of <i>LEU2</i> and to the 5β² end of the <i>FKH</i> gene. Cells were cotransformed with both products and selected on leu<sup>β</sup> plates. Cells harboring <i>FKH1</i> and <i>FKH2</i> under the control of the <i>GAL1</i>/<i>10</i> promoter were generated by crossing the single integrated strains. (B) Cells overexpressing (OE) <i>FKH1</i> and/or <i>FKH2</i> from the <i>GAL1</i>/<i>10</i> promoter were grown overnight in 2% YPD, then spot diluted onto the plates shown. The plates were incubated at 30Β°C for 2 to 5 days. (C) <i>FKH1-TAP</i> OE cells were grown overnight in 2% glucose. The next day, the cells were washed and resuspended in media containing 2% galactose. Samples were taken every two hours for 10 hours for Western analysis using antibodies against TAP and GAPDH. Cells expressing <i>FKH1-TAP</i> and <i>FKH1</i> under their own promoter were used as controls. (D) <i>FKH2-TAP</i> OE cells were analyzed as described above for <i>FKH1-TAP</i> OE cells.</p
The Fkh proteins and the APC work together to promote extended CLS and stress response.
<p>(A) The <i>FKH</i> genes were deleted in <i>apc5<sup>CA</sup></i> cells by multiple rounds of genetic crosses. The strains shown were grown overnight at 30Β°C in YPD, then spot diluted onto YPD plates and incubated at 30 or 37Β°C. (B) WT and <i>apc5<sup>CA</sup></i> cells were transformed with plasmids expressing <i>FKH1</i> or <i>FKH2</i> from a galactose inducible promoter, or an empty vector control plasmid. Individual transformed colonies were grown overnight and then spot diluted onto SD ura- plates supplemented with either 2% glucose or 2% galactose. The plates were incubated at 30 or 37Β°C. (C) The mutants used above were grown to stationary phase and maintained in depleted media (DM) for the remainder of the experiment. Colony forming units were determined every other day and a survival curve was plotted. Standard error is shown for at least 3 repeats. (D) CLS was determined for the strains used above. Rather than maintenance in DM, the cells were washed once they reached stationary phase and maintained in H<sub>2</sub>O for the remainder of the experiment. Standard error is shown for at least 3 repeats. The experiments in (C) and (D) were started from the same cultures. (E) The panel of strains shown were grown overnight at 30Β°C to early log phase growth. Proteins were extracted and analyzed by Westerns to assess total histone H2B, H3 and H4 protein levels. A Ponceau S stained gel is included to shown equivalence of protein load. (F) WT, <i>apc5<sup>CA</sup></i> and <i>fkh1Ξ fkh2Ξ</i> cells encoding <i>BAR1</i> were grown to early log phase, then arrested with 2 Β΅g/ml Ξ± factor for 1.5 hours in pH 3.5 YPD media. Another 2 Β΅g/ml Ξ± factor was added followed by another hour of incubation. Cycloheximide was then added with continued incubation. Samples were removed at the times shown and Clb2 protein levels were determined by Western blotting. Antibodies against GAPDH were included as a load control. The same blot was divided and used for the Clb2 and GAPDH Westerns. Samples were taken before and after G1 arrest for FACS analysis.</p
The APC and the Fkh proteins provide overlapping function to respond to oxidative stress.
<p>(A) CLS was performed using the strains shown in the presence of 25 mM H<sub>2</sub>O<sub>2</sub>. The cells were grown to stationary phase, and then H<sub>2</sub>O<sub>2</sub> was added to the cultures. Colony forming units were determined every other day for the remainder of the experiment. Survival curves are shown. (B) The strains described above were used to determine resistance to oxidative stress. Day 5 stationary phase cells were exposed to 100 mM H<sub>2</sub>O<sub>2</sub> for 1 hour at 30Β°C. Controls were not treated with H<sub>2</sub>O<sub>2</sub>. Diluted cells were then plated onto YPD plates until colonies formed. The percent survival was determined, with standard error shown for at least 3 replicates.</p
<i>Saccharomyces cerevisiae</i> strains used in this study.
<p><i>Saccharomyces cerevisiae</i> strains used in this study.</p
The Fkh proteins are present in the nucleus of stationary phase cells.
<p>(A) Cells expressing endogenously TAP-tagged <i>FKH1</i> were grown to stationary phase and either left in DM or transferred to H<sub>2</sub>O for the remainder of the experiment. Samples were removed on days 1 and 5 for Western analysis using antibodies against the TAP epitope, or GAPDH as a load control. Fkh2-TAP was also observed in stationary phase cells (data not shown). (B) Cells expressing endogenously tagged <i>FKH1</i>- or <i>FKH2</i>-GFP were grown to day 5 stationary phase while maintained in DM. Cells were observed to harbor both Fkh1 and Fkh2 nuclear staining. (C) Day 13 stationary phase cells expressing Fkh2-GFP were imaged, showing reduced nuclear staining in cells maintained in DM. (D) The percentage of nuclear localized Fkh1-GFP or Fkh2-GFP was determined as cells aged in either DM or H<sub>2</sub>O.</p
<i>FKH1</i> and <i>FKH2</i> encode redundant determinants of lifespan and stress response.
<p>(A) and (B) The cells shown were grown in CM (2% glucose) to stationary phase. The cells were either left in (A) depleted CM (DM), or (B) washed and resuspended in H<sub>2</sub>0 for the remainder of the experiment. Colony counts were performed every other day throughout the experiment. The day when the colony count peaked was considered Day 1. Standard error is shown for at least 3 replicates. (C) Acute oxidative stress in stationary phase cells. WT and <i>fkh1Ξ fkh2Ξ</i> cells were grown to day 5 of stationary phase with maintenance in either DM or H<sub>2</sub>O. 100 mM H<sub>2</sub>O<sub>2</sub> was then added to one half of each sample and incubated for 60 minutes at 30Β°C. Diluted cells were then plated on YPD media and the colony forming units were counted. Survival was determined by dividing the colony forming units following H<sub>2</sub>O<sub>2</sub> treated by untreated samples. Standard error of at least 3 replicates is shown. (D) Chronic oxidative stress in mitotically active and stationary phase cells. WT and <i>fkh1Ξ fkh2Ξ</i> cells from overnight log phase cultures or day 5 stationary phase cultures were treated with 100 mM H<sub>2</sub>O<sub>2</sub> at 30Β° for 1 hour, as above, then spot diluted onto YPD plates in the absence of stress. The plates were grown at 30Β°C for 3 days.</p
Increased expression of the <i>FKH</i> genes increases lifespan and stress response.
<p>(A) The <i>FKH</i> OE cells were grown to stationary phase, then either maintained in DM, or 0.05% galactose was added. The cells were incubated for an additional 5 days, then split, with one half treated with 100 mM H<sub>2</sub>O<sub>2</sub> for 1 hour. The other half served as the untreated control. Following the 1 hour incubation, the cells were diluted and plated onto YPD until colony forming units formed. Survival was determined by dividing the treated cells by the untreated cells. Standard error is shown for at least 3 replicates. (B) CLS was determined for the OE strains when maintained solely in DM (left panel) or in DM supplemented with 0.05% galactose (right panel). Standard error is shown for at least 3 replicates. (C) RLS was determined for the OE strains on 2% sucrose plates or sucrose plates supplemented with 0.1% galactose. Typical results are shown.</p
Model depicting how the APC and the Fkh proteins may interact under normal and stress conditions.
<p>Under normal conditions, the Fkh proteins play a role in activating the APC. This in turn mediates progression through mitosis and maintenance of G1. This interaction is depicted by bold arrows. Under stress conditions, both the APC and the Fkh proteins respond via different mechanisms. The Fkh proteins likely promote the expression of stress response proteins. The APC acts according to a mechanism that remains uncharacterized, but likely requires ubiquitin-dependent processes.</p
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 HIF1Ξ± protein in DOX resistant cells, and in cells with forced expression of TFPI1, suggesting TFPI1 induces HIF1Ξ±. 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 HIF1Ξ±, 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 HIF1Ξ±. This in turn activates a pathway leading to drug resistance