Endogenous estrogens and estrogen metabolites play an important role in the pathogenesis and development of human breast, endometrial, and ovarian cancers. Increasing evidence also supports their involvement in the development of certain lung, colon and prostate cancers.In this study we systemically surveyed endogenous estrogen and estrogen metabolite levels in each of the NCI-60 human tumor cell lines, which include human breast, central nerve system, colon, ovarian, prostate, kidney and non-small cell lung cancers, as well as melanomas and leukemia. The absolute abundances of these metabolites were measured using a liquid chromatography-tandem mass spectrometry method that has been previously utilized for biological fluids such as serum and urine.6 cells) and 17β-estradiol (753.45 pg/106 cells). In comparison, the ovarian cancer cell line with the highest levels of these estrogens contained only 19.79 and 139.32 pg/106 cells of estrone and 17β-estradiol, respectively. The highest levels of estrone and 17β-estradiol in breast cancer cell lines were only 8.45 and 87.37 pg/106 cells in BT-549 and T-47D cells, respectively.Endogenous estrogens and estrogen metabolites were found in all NCI-60 human tumor cell lines and some were substantially elevated and exceeded the levels found in well known estrogen-dependent and estrogen receptor-positive tumor cells such as MCF-7 and T-47D. While estrogens were expected to be present at high levels in cell lines representing the female reproductive system , other cell lines, such as leukemia and colon, also contained very high levels of these steroid hormones. The leukemia cell line RMPI-8226 contained the highest levels of estrone (182.06 pg/10The data provided evidence for the presence of significant amounts of endogenous estrogens and estrogen metabolites in cell lines not commonly associated with these steroid hormones. This broad discovery of endogenous estrogens and estrogen metabolites in these cell lines suggest that several human tumors may be beneficially treated using endocrine therapy aimed at estrogen biosynthesis and estrogen-related signaling pathways. Today it is recognized that E2 exerts some effect on almost every organ in the body + of EM-Dansyl and SI-EM-Dansyl were similar to those previously described [LC-MSescribed ,10.13C6-E2 was used as the internal standard for E2; 13C6-E1 for E1; d3-E3 for E3, 16-ketoE2, and 16α-OHE1; d3-16-epiE3 for 16-epiE3 and 17-epiE3; d5-2-MeOE2 for 2-MeOE2, 4-MeOE2, 2-MeOE1, 4-MeOE1, and 3-MeOE1; d5-2-OHE2 for 2-OHE2, 2-OHE1, and 4-OHE1.Quantification of EMs was carried out using Xcalibur™ Quan Browser (Thermo Electron) as previously described ,21. Brie1, E2, 16-ketoE2, 16α-OHE1, E3, 2-MeOHE1, 2-MeOHE2, and 2-OHE1. The chromatograms showing the eight quantified endogenous EMs for an ovarian (SK-OV-3) and colon cancer cell line (HCC-2998) are shown in Figure 1 was found in the non-small cell lung cancer cell line NCI-H460.The levels of endogenous estrogens and EMs were measured in the NCI-60 cell lines, which comprise breast (n = 5), CNS (n = 6), colon (n = 7), leukemia (n = 6), melanoma (n = 9), non-small cell lung (n = 9), ovarian (n = 9), prostate (n = 2), and renal (n = 8) cancers. This study focused on measuring only the unconjugated, active forms of the EMs. Glucoronidated and sulfated forms of the EMs were not included in the analysis. All NCI-60 human tumor cell lines showed significant levels of EWithin the same type of cancer, different tumor cell lines had substantially different levels of EMs Table . For exa2 was by far the most abundant unconjugated estrogen followed by E1 and 2-OHE2  were amongst those with the highest E2 levels. OVCAR-4 and -5 are both ERα-negative, ERβ-positive ovarian cell lines who's growth is insensitive to E2 treatment [2 [2 (753 pg/106 cells). In fact, its E2 levels were more than 3.5-fold higher than the colon cell line HCC-2998, which contained the next highest level of E2 (209 pg/106 cells). This result correlates with a previous study showing that RMPI-8226 cells possess the highest ER levels compared to other leukemia and myeloid cell lines tested [2 treatment. When the cells are maintained in a medium containing physiological concentrations  of E2, cell growth is stimulated; however, pharmacological concentrations (10-6 M) of E2 inhibit their growth [The cell lines with the highest Ereatment . While Stment [2 . The leus tested . Previour growth . Adding 2 levels are shown in Figure 2 levels ranged from 1.31 to 12.25 pg/106 cells. To put into perspective the range of E2 values found in all the cell lines, SW-620 colon cells contained almost 575-fold less E2 than RMPI-8226 cells. The finding that colon cell lines generally contain low levels of E2 is consistent with a previous study that found ERs are present in colorectal tumors and human colonic cancer cell lines at very low levels [Cell lines with the lowest E2, the leukemic cell line RPMI-8226 contained the highest levels of E1 of the NCI-60 cell lines  was sixth on the list of cell lines containing the most E1. Two non-small cell lung carcinoma cell lines (EKVX and NCI-H226) were amongst the top ten in both E1 and E2 levels. This result is interesting considering that females that never smoked are far more likely to develop lung carcinoma than never-smoked males, suggesting a gender difference exists in the clinical and pathophysiology of lung cancer [in situ in male and female lung cancers suggest that estrogens may contribute to the manifestation and progression of lung carcinoma [As with Es Figure . The amog cancer . Recent arcinoma .1 levels are shown in Figure 1 levels . Their E1 levels ranged from 0.14 to 1.12 pg/106 cells. The levels of E1 found in KM12 colon cancer cells was approximately 1,300-fold less than that found in RPMI-8226 leukemia cells. Two leukemia cell lines, CCRF-CEM and K562, which were amongst those possessing the lowest E2 levels, also contained low E1 levels.Cell lines with the lowest E6 cells). This value was almost twice as high as the cell types with the next highest total EM levels. Ovarian (96.33 pg/106 cells) and breast (83.18 pg/106 cells) cancer cells, which are commonly associated with estrogens, contained the second and sixth highest levels of total EMs. In fact, five of the cancer cell lines  had total EM levels between 80 and 100 pg/106 cells. A noticeable feature of Table 6 cells with a SD of 28.68; dropping their overall rank from first to sixth. The cell types having the three highest EM levels after eliminating the cell lines with the highest and lowest concentrations were (in order) ovarian , melanoma , and breast .To identify general trends within the various cell lines tested, the means and standard deviations (SDs) of the total EM levels found in the cell types analyzed. As shown in Table 1  and E2 . Only four other cell lines, SF-539 (CNS), NCI-H226 (NSCL), RMPI-8226 (leukemia) and HCC-2998 (colon), contained higher levels of total estrogens. High-affinity E2 receptors have been reported for primary human melanomas [2 may inhibit the growth of these melanoma tumors [2 has been shown to inhibit their growth, while pre-treating the cells with tamoxifen (an anti-estrogen) blocks the effects of E2 [2 within the melanoma cell lines may prevent uncontrolled cell proliferation by acting back upon the cells and binding to the type II ER.The finding that the melanoma cell lines contained relatively high levels of endogenous estrogen and EMs is interesting. Two melanoma cell lines in particular, SK-MEL-28 and UACC-257, were among those containing the highest levels of Ea tumors . This rets of E2 . The pre3, 16αOHE1, and 16-epiE3 were less abundant except for in HCT-15 colon tumor cells, which had a greater amount of E3 than E2. The catechol estrogen 2-OHE2 was the only catechol estrogen detected in the tumor cell lines, except for the NSCL cancer cell line NCI-H460, which also contains a relatively high level of 2-OHE1  in the conjugated forms in the circulation [1, E2, E3, 2-MeOHE1 and 2-MeOHE2 are detected in their unconjugated forms [1, 16-epiE3, and 2-OHE2. Unfortunately, it is difficult to directly compare the estrogen and EM levels as the cell line concentrations are recorded in pg/106 cells, while those in serum are measured as pg/ml. The fact that more compounds are detected in their unconjugated forms in the cell lines, however, suggests that, in general, the concentrations of estrogens and EMs are higher in cells than in the circulation.This study measured the unconjugated levels of endogenous estrogens and EMs in the NCI-60 cell panel. From our previous experience, if we had measured the conjugated levels by adding a sulfatase/glucoronidase enzyme to deconjugate sulfated and glucoronidated molecules prior to LC-MSculation . While ted forms . In the et al. [2 levels were measured in this study. Of these, detectable ERα levels were found for SKOV-3, OVCAR-4, UACC-257, SK-MEL-28, and SF-539 cell lines. No ERα mRNA was detected for HCC-2998, NCI-H226, EKVX, and OVCAR-5 cell lines. The cell lines with the highest levels of ERα mRNA were SK-OV-3, and two breast cancer cell lines, MCF-7 and T-47D. Of these, only SK-OV-3 was among the cell lines containing the highest amounts of E2. We also compared the ERα and E2 levels within the nine melanoma cell lines analyzed in both studies. In this case, the six melanoma cell lines with detectable levels of ERα mRNA  contained the highest amounts of E2 within that group. The melanoma cell lines containing the lowest E2 concentrations  did not show detectable levels of ERα. Overall, there is no obvious correlation between ERα and E2 levels; however, only about 25% of the cell lines had detectable levels of ERα whereas E2 could be measured in every one.To determine if ER status correlates with the levels of estrogens and EMs identified in the various cell lines, we compare our data to that published by Holbeck et al. , who mea2 approach with the capability of measuring up to 15 different EMs to measure the levels of endogenous estrogens within the NCI-60 cell lines. Eight of the measured endogenous estrogens were consistently observed in all of the NCI-60 cell lines, providing an unprecedented view of these metabolites within these cancer cell lines. What is particularly striking is that the levels of EMs in well-known estrogen-dependent cancers such as ovarian and breast were not substantially greater than those found in other types of cancer cell lines. In fact, none of the breast cancer cell lines were amongst the top ten that contained the highest levels of E1 or E2. Cell lines not generally associated with estrogens, such as leukemia, colon, CNS, and NSCL, were found to have appreciable levels of these metabolites. The broad presence of EMs within the NCI-60 cell lines suggests that many cancers outside of the reproductive system may respond to treatments with anti-estrogens such as tamoxifen, toremifene, and fulvestrant. Considering technologies for measuring estrogen levels in biological samples is considerably improved, it is now worth the effort to test various tumors for the levels of these metabolites.This study utilized an LC-MS3: 16-epiestriol; 16-ketoE2: 16-ketoestradiol; 16α-OHE1: 16α-hydroxyestrone; 17-epiE3: 17-epiestriol; 2-MeOE1: 2-methoxyestrone; 2-MeOE2: 2-methoxyestradiol; 2-OHE1: 2-hydroxyestrone; 2-OHE2: 2-hydroxyestradiol; 3-MeOE1: 2-hydroxyestrone-3-methyl ether; 4-MeOE1: 4-methoxyestrone; 4-MeOE2: 4-methoxyestradiol; 4-OHE1: 4-hydroxyestrone; CNS: central nervous system; E1: estrone; E2: estradiol; E3: estriol; EM: estrogen metabolite; ER: estrogen receptor; LC: liquid chromatography; MS2: tandem mass spectrometry; NSCL: non-small cell lung; SD: standard deviation; SI-EM: stable isotope-labeled estrogen. Bre: breast; Col: colon; Leu: leukemia; Mel: melanoma; Ovc: ovarian; Pros: prostate; Ren: renal.16-epiEThe authors declare that they have no competing interests.TV participated in the conception, design, and implementation of the experiments, the analysis and interpretation of the data and drafting of the manuscript. XX participated in the conception, design, and implementation of the experiments, conducted the sample preparation and data acquisition and participated in the analysis and interpretation of the data and drafting of the manuscript. All authors have read and approved the final version of the manuscript for publication.


Lung cancer is the leading cause of cancer deaths worldwide. In the United States, only one in six lung cancer patients survives five years after diagnosis. These statistics may improve if new therapeutic targets are identified. We previously reported that an enzyme of fatty acid metabolism, very long-chain acyl-CoA synthetase 3 (ACSVL3), is overexpressed in malignant glioma, and that depleting glioblastoma cells of ACSVL3 diminishes their malignant properties. To determine whether ACSVL3 expression was also increased in lung cancer, we studied tumor histologic sections and lung cancer cell lines. Immunohistochemical analysis of normal human lung showed moderate ACSVL3 expression only in bronchial epithelial cells. In contrast, all of 69 different lung tumors tested, including adeno-, squamous cell, large cell, and small cell carcinomas, had robustly elevated ACSVL3 levels. Western blot analysis of lung cancer cell lines derived from these tumor types also had significantly increased ACSVL3 protein compared to normal bronchial epithelial cells. Decreasing the growth rate of lung cancer cell lines did not change ACSVL3 expression. However, knocking down ACSVL3 expression by RNA interference reduced cell growth rates in culture by 65–76%, and the ability of tumor cells to form colonies in soft agar suspension by 65–80%. We also conducted studies to gain a better understanding of the biochemical properties of human ACSVL3. ACSVL3 mRNA was detected in many human tissues, but the expression pattern differed somewhat from that of the mouse. The enzyme activated long- and very long-chain saturated fatty acid substrates, as well as long-chain mono- and polyunsaturated fatty acids to their respective coenzyme A derivatives. Endogenous human ACSVL3 protein was found in a punctate subcellular compartment that partially colocalized with mitochondria as determined by immunofluorescence microscopy and subcellular fractionation. From these studies, we conclude that ACSVL3 is a promising new therapeutic target in lung cancer. SLC27A1-6.Acyl-CoA synthetases (ACS) catalyze the ATP-dependent thioesterification of fatty acids (FA) to coenzyme A (CoA) We previously described properties of mouse ACSVL3  Adult mouse brain sections showed weak ACSVL3 immunostaining in cortical neurons, hippocampal neurons, and cerebellar Purkinje cells, but the protein was not detected in glial cells 14C]palmitic acid (C16∶0), [1-14C]oleic acid (C18∶1), [1-14C]arachidonic acid (C20∶4), [1-14C]docosahexaenoic acid (C22∶6) and [1-14C]lignoceric acid (C24∶0) were obtained from Moravek Biochemicals Inc. . [1-14C]linoleic acid (C18∶2) was from American Radiolabeled Chemicals . Polyclonal sheep antiserum to 70 kDa peroxisomal membrane protein (PMP70) was a gift from Dr. S. Gould (Johns Hopkins Univ. Sch. Med.). Mouse monoclonal antibody to protein disulfide isomerase and polyclonal rabbit antibody to manganese-superoxide dismutase (MnSOD), were from Stressgen . Polyclonal rabbit antibody to ATP synthase was from Chemicon . Polyclonal rabbit antibody to phosphatidylethanolamine N-methyltransferase 2 (PEMT), was a gift from Dr. D. Vance . Affinity-purified polyclonal rabbit anti-ACSVL3 antibody was previously described -labeled FA  to their CoA thioesters was performed as described previously COS-1 cells were transfected with the ACSVL3 expression plasmid p435 or empty vector via electroporation as described previously H460 and EKVX cells were grown to near confluence, harvested by gentle trypsinization, and washed with phosphate-buffered saline. Cell pellets containing 1.0 mg protein were extracted, derivatized with pentafluorobenzyl bromide, and quantitated by capillary gas chromatography-electron-capture negative-ion mass spectrometry (GC/MS) as described by Lagerstedt et al. ACSVL3 mRNA expression was examined by RNA dot blot analysis using a multiple tissue expression (MTE™) array containing 61 adult human tissue samples . The sigTo establish the size of ACSVL3 mRNA, a human multiple tissue Northern blot was hybridized with the probe used for the MTE blot. This probe detected a 2.7 kb ACSVL3 transcript in all tissues except brain , top. ThWe analyzed the functional capability of human ACSVL3 protein to activate FA to their CoA thioesters following overexpression in COS-1 cells. Compared to COS-1 cells transfected with empty vector, cells expressing ACSVL3 showed increased ACS activity when assayed with several FA substrates. Statistically significant increases in activation of palmitic acid (C16∶0), oleic acid (C18∶1ω9), α-linoleic acid (C18∶2ω6), and arachidonic acid (C20∶4ω6) were observed . AlthougIndirect immunofluorescence was used to localize endogenous ACSVL3 in the human hepatoma cell line HepG2. ACSVL3 exhibited a punctate fluorescence pattern when observed by confocal microscopy . ACSVL3 To characterize further the subcellular localization of ACSVL3, HepG2 cells were fractionated by differential centrifugation. Homogenates were separated into fractions enriched in nuclei (N), mitochondria (M), peroxisomes (L), endoplasmic reticulum (P), and the membrane-free cytosol (S), and analyzed by Western blotting. ACSVL3 was found primarily in the M-fraction , as indiBecause colocalization of ACSVL3 and mitochondria by immunofluorescence was not complete, we speculated that ACSVL3 might be found in the mitochondria-associated membrane (MAM) fraction, an endoplasmic reticulum-derived compartment that sediments with mitochondria during differential centrifugation We previously reported that, despite weak expression in brain and essentially no detectable protein in glia, ACSVL3 levels were robustly elevated in malignant glioma and in human glioblastoma cell lines To confirm and extend these observations, we examined ACSVL3 expression in normal human bronchial epithelial cells (HBEC), and in several lung cancer cell lines. ACSVL3 expression was barely detectable in HBEC . Cell liHBEC grow at a slow rate as they are cultured in serum-free synthetic medium (BEGM) to prevent squamous differentiation Stable knockdown of ACSVL3 expression in glioblastoma cells decreased their malignant phenotype in culture in vitro growth phenotype.We then measured the effect of ACSVL3 knockdown on proliferation of cultured H460 and H82 lung cancer cell lines. As shown in To begin to understand the role of ACSVL3 on tumor cell lipid metabolism, we analyzed the FA composition of control and ACSVL3 knockdown H460 and EKVX cells. No substantive differences in long-chain saturated FA containing 14–18 carbons between control and ACSVL3 knockdown were observed for either cell line . SaturatAlthough ACSs catalyze a fundamental reaction in cellular metabolism, the specific physiological function(s) of each of the 26 human ACS enzymes are only beginning to be elucidated. Based on tissue and cell expression pattern, subcellular location, substrate specificity, and other properties, we and others have suggested that each ACS likely plays a relatively specific role by channeling the acyl-CoA reaction product toward a particular metabolic fate While physiological effects of gene manipulation in mice cannot always be extrapolated to humans, studies of knockout (KO) and transgenic mice have begun to address the functions of several members of the very long-chain ACS subfamily that includes ACSVL3. KO of ACSVL1  was associated with decreased peroxisomal β-oxidation of very long-chain FA in liver and kidney To date, ACSVL3 is the only member of the very long-chain ACS family that has been associated with cancer in humans Both overexpression and knockdown studies have shown that ACSVL3 catalyzes the formation of fatty acyl-CoAs In this report, we show that several biological properties of human ACSVL3 are similar to those previously reported for the mouse enzyme Results reported here demonstrate that ACSVL3 is highly over-produced in all types of human lung tumors, including adeno-, squamous cell, non-small cell, large cell, and small cell carcinomas. All of 69 tumors examined  exhibited rather robust immunostaining. This observation is similar to that made previously with a human glioma tissue array Cell lines representing the major types of lung tumors present on the tissue microarray were investigated, and ACSVL3 was found to be highly overexpressed in all cell lines tested. All cell lines are well-characterized and exhibit in vitro malignant features such as rapid growth and the ability to form colonies in soft-agar suspension. Slowing the growth rate of these cancer cells by maintaining them in a serum-free medium was not sufficient to decrease ACSVL3 levels. In contrast, depleting lung cancer cells of ACSVL3 significantly decreased their rapid growth rates, and impaired their ability to grow in suspension culture. These observations, along with similar findings in human glioblastoma cell lines The role of ACSVL3 in lipid metabolism has not been fully elucidated. To begin to address this, we examined the fatty acid composition of control and ACSVL3 knockdown H460 and EKVX cells. We found decreased levels of C26∶0, consistent with the proposed role for ACSVL family enzymes in saturated very long-chain FA metabolism. Interestingly, ACSVL3 knockdown affected the cellular levels of n-7 monounsaturated FA. Surprisingly little is known about the metabolic functions of n-7 FA in either normal or cancer cells; our results suggest that further investigation is warranted. Decreased ACSVL3 expression had somewhat different effects on levels of other FA in the two cell lines, indicating that this enzyme may play cell-specific roles in cancer cell lipid metabolism.Lung cancer remains the leading cause of cancer deaths in the U.S.


This work was conducted by investigators at submitting institutions and scientists at Coriell Institute for Medical Research, where the NIGMS Repository is hosted. Recently, approximately 900 cell lines, mostly chosen from the Chromosomal Aberrations and Heritable Diseases collections, have been further characterized in detail at the Coriell Institute using the Affymetrix Genome-Wide Human SNP Array 6.0 to detect copy number variations and copy number neutral loss of heterozygosity. A database containing detailed cytogenetic and genomic information for these cell lines has been constructed and is freely available through several sources, such as the NIGMS Repository website and the University of California at Santa Cruz Genome Browser. As additional cell lines are analyzed and subsequently added into it, the database will be maintained dynamically.The Human Genetic Cell Repository sponsored by the National Institute of General Medical Sciences (NIGMS) contains more than 11,000 cell lines and DNA samples collected from numerous individuals. All of these cell lines and DNA samples are categorized into several collections representing a variety of disease states, chromosomal abnormalities, heritable diseases, distinct human populations, and apparently healthy individuals. Many of these cell lines have previously been studied with detailed conventional cytogenetic analyses, including G-banded karyotyping and fluorescence  The National Institute of General Medical Sciences (NIGMS) established the Human Genetic Cell Repository (NIGMS Repository) 40 years ago. The NIGMS Repository is maintained by the Coriell Institute for Medical Research (Coriell Institute) in Camden, New Jersey. To date, more than 11,000 unique samples have been donated by different individuals for the production of cell lines and genomic DNA. These samples are distributed among several collections and represent a variety of chromosomal abnormalities, heritable diseases, numerous distinct human populations, and apparently healthy individuals. All of the samples distributed through the NIGMS Repository have been intensively analyzed by the investigators who submitted the samples originally and/or scientists at the Coriell Institute. As part of its mission to update/supplement the phenotypic information associated with the NIGMS Repository samples, the Coriell Institute utilizes the latest cytogenetic and genomic techniques to analyze samples.de novo analysis without previous phenotypic information. In 2007, the annual volume of CMA tests performed clinically was more than 10,000 in the United States alone , has become a widely performed genomic diagnostic test . CMA hases alone . Up to de.g., gender, age at time of sample collection, and name of diagnosed disease or category of aberration), were deposited in the Database of Genotypes and Phenotypes (dbGaP) under the project name “Genotyping NIGMS Chromosomal Aberration and Inherited Disorder Samples” (Study Accession ID: phs000269.v1.p1). Access to the data is available through dbGaP’s Authorized Access mechanism (https://dbgap.ncbi.nlm.nih.gov/aa/wga.cgi?page=login). Recently, more than 200 additional NIGMS Repository cell lines have been analyzed with the SNP Array 6.0. A large fraction of these additional lines are newly recruited samples from submitters around the world and represent new collections for diseases or disease spectrums not previously held by the Repository, e.g., monosomy 1p36 syndrome, congenital muscular dystrophies, and chromosome 15q11-q13 duplication syndrome, among others. The new CEL files generated from these new cell lines will be available in the dbGaP as well.In 2010, 716 samples derived from 697 individuals from NIGMS Repository collections were analyzed for CNVs using the SNP Array 6.0. The majority of these samples were chosen from the Chromosomal Aberrations and Heritable Diseases collections. The resulting data, intensity data files (CEL files), and genotype data files (CHP files), along with a limited set of phenotypic information (All data files (CEL files) were analyzed in Genotyping Console software (Affymetrix). The Contrast QC and MAPD metrics generated by the software were used for quality control purposes. Values of 0.4 and 0.35, respectively, were used as cutoffs for high-quality data. For reporting purposes, the copy number and AOH files (CNCHP files) produced by the Genotyping Console software were subsequently analyzed using Chromosome Analysis Suite (ChAS) software (Affymetrix). All CEL files have been re-analyzed with Nexus 6.0 software  with the same parameters described below to confirm all the CNV calls included in this dataset.e.g., polymerase chain reaction, multiplex ligation-dependent probe amplification, DNA sequencing) were reanalyzed with new parameters  that would permit detection of segments of <100 kb. To minimize the possibility of missing a potential pathogenic abnormality, all CNVs (≥25 kb) involving one or more of the genes described in the Online Mendelian Inheritance in Man (OMIM) Morbid Map were reported, even when, according to the clinical information, they were detected in apparently healthy individuals. This is necessary to point out that some of the CNVs, many of which have a size of 25 −100 kb but involve one or more OMIM Morbid Map genes, may not have been further confirmed by other methods at Coriell Institute. The primary goal of this analysis was to extract as much copy number information as possible from each cell line, regardless of clinical pathogenicity. Therefore, the parameters used to generate the database would not necessarily be suitable for clinical diagnostic purposes. Clinical scientists who wish to reanalyze the CEL files should refer to the guidelines issued by the ACMG for the appropriate parameters to be used  in the majority of the cell lines were a segment size ≥100 kb consisting of at least 10 contiguous markers (CNV-only probes) and a confidence score of 95. The minimum parameters used to call a region of AOH were a segment size ≥5 Mb consisting of at least 10 contiguous markers (SNP-only probes) and a confidence score of 95. In addition to analysis using the above default parameters, some samples with a known small pathogenic deletion or duplication previously detected by other methods  (in situ hybridization (FISH) analyses, as well as those subtle CNVs (≥25 kb) which have not been detected by conventional methods, but involve one or more OMIM genes as outlined previously. AOH has been detected in many cell lines in this dataset. Examples of AOH in the dataset include AOH affecting one or more segments of a chromosome; a whole chromosome or even the whole genome of a cell line. In general, the clinical significance of the majority of AOH, especially the AOH only affecting chromosomal segment(s) detected in this study, is unknown. The detected AOH regions have not been further confirmed by other methods, such as methylation testing. Therefore, the majority of AOH information has not been integrated into the ISCN reports except in special cases, e.g., GM01225, in which AOH was detected on 14 segments involving 9 different chromosomes (no including the X chromosome) . The term “critical CNVs” refers to all chromosomal abnormalities detected or confirmed by G-banded karyotype and/or fluorescence omosome) ; GM11496Through the aforementioned NIGMS Repository online catalog, researchers can find all relevant genomic analyses for each cell line. Within the ISCN entry on the Overview tab, all results from G-banded karyotype, FISH, and/or CMA analyses have been presented according to the ISCN 2013 guidelines . Users of the data, especially those comparing it with results obtained by using different platforms than the SNP Array 6.0, are reminded that many factors, including the probe density, analysis parameters, and genome build/annotation files used, will impact the final results.Second, a spreadsheet including sample ID and short and long ISCN nomenclature describing the chromosomal abnormalities of all cell lines in this database is available both online and as a supplement to this report  from 602 cell lines has been integrated into a custom track within the University of California at Santa Cruz Genome Browser so far, and they are categorized in the group “Phenotype and Disease Associations” under the name “Coriell CNVs.” This information is readily accessible and searchable by all research scientists.The samples in the NIGMS Repository have been used extensively by scientists around the world for a wide variety of research purposes. Publications that use NIGMS Repository samples continually appear in the scientific literature. A list of recent publications can be found on the NIGMS Repository website. Although many investigators may have performed copy number analysis on individual samples of the NIGMS Repository and shared their findings with the Coriell Institute, this database represents the first concerted effort to generate and curate genome-wide copy number data for a large number of samples. Scientists may find numerous mutations in this database that have not been revealed before. For example, a preliminary analysis of the CMA data from approximate 700 cell lines in this database  has resulted in the annotation of 1120 clinically pathogenic and 4470 most likely benign or uncertain clinical significance CNVs (gain or loss) involving all autosomal and sex chromosomes, according to the ACMG guidelines for interpretation and reporting of the CMA results , and (3) methods used to validate the CMA assay . As a result, the development of reference materials that can be widely used for CMA validation has become necessary. The Genetic Testing Reference Materials Coordination Program, led by the Center for Disease Control and Prevention, has been focusing on creating a genomic DNA reference material panel of at least 96 extensively analyzed cell lines containing a broad range of chromosomal abnormalities, e.g., microdeletions and microduplications, subtelomeric abnormalities, UPD, as well as other genetic variations. To date, as part of the Genetic Testing Reference Materials Coordination Program, a panel of 45 cell lines, all chosen from the NIGMS Repository, has been tested with several commercial CMA platforms by their producers . Development of characterized genomic DNA reference material panels for clinical chromosomal microarrays. Presented at the 12th International Congress of Human Genetics/61st Annual Meeting of The American Society of Human Genetics, October 12, 2011, Montreal, Canada). The resulting data have been analyzed by several Cytogenetics experts from various institutes. Currently, this panel of 45 cell lines (“CNVPANEL01”) (Table S2) has been recommended to be applied for developing and validating a CMA assay. It is specifically listed and accessible on the NIGMS Repository website. All the other cell lines documented in this database serve as the pool from which the rest of the specimens for the complete CNV panel will be chosen. This database covers the majority of the most commonly encountered chromosomal conditions in clinical practice, as well as many rarely seen chromosomal abnormalities. Scientists are encouraged to use samples from the CNVPANEL01 or any other cell lines in the database to validate their own CMA assays.Although the CMA assay has been recommended as the first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies, the results can vary between individual laboratories . These cell lines are good resources to test the ability of a platform to detect mosaic karyotypes.Many laboratories focus their testing on a certain disease or disease spectrum. Under these circumstances, a CMA assay is expected to be capable of detecting as many chromosomal conditions related to the disease or disease spectrum as possible. This database can assist laboratories in identifying relevant samples for several diseases and disease spectrums. For example, there are currently 23 cell lines from individuals with PWS and their family members included in this database. The spectrum of variants contained in this subset of cell lines includes microdeletions of various sizes within the 15q11.2-q13 region, as well as examples of both isodisomic and heterodisomic (previously confirmed with methylation tests by the submitters and investigators who used the cell lines) maternal UPD (matUPD) . Many other cell lines in the database also have one or more chromosomal abnormalities involving chromosome 15. Therefore, these cell lines can also be used to build a reference material panel to validate a CMA assay with emphasis on PWS and/or abnormalities on chromosome 15. Other examples of large subsets of samples in the database include, but are not limited to, monosomy 1p36 syndrome, Duchenne muscular dystrophy, Cri-Du-Chat syndrome, and Williams-Beuren syndrome. Many cell lines also have exhibited mosaic karyotypes of different degrees during G-banded karyotype analysis. The mosaic status has also detected by the SNP array 6.0 in some cell lines (Z. Tang, N. Gerry, D. Berlin, A. MacMillan, M. Wineburg, B. A. Frederick, L. Toji, G. A. Toruner, C. Beiswanger. Analysis of 24 Cell Lines of Mosaicism by the Affymetrix Genome-Wide Human SNP Array 6.0. Presented at the 12All investigators are encouraged to provide feedback regarding findings they obtained from analyzing any of the cell lines included in the database, regardless of the assay or platforms used, so that more information about each cell line can be added to the database and shared with the research community. Feedback can be relayed either through the web-based customer services forms or through personal communication with scientists at Coriell Institute by sending an e-mail to NIGMS@coriell.org.In summary, a database containing genomic information for approximately 900 cell lines, which are mostly chosen from the Chromosomal Aberrations Collection and Heritable Diseases Collection of the NIGMS repository and intensively analyzed by karyotype, FISH, and CMA assays at the Coriell Institute, has been constructed. We believe that this database will serve the clinical and research communities with cell lines and/or DNA samples containing detailed genomic information. This database will be maintained dynamically as additional cell lines are analyzed and subsequently added.


Adult T-cell leukemia-lymphoma (ATL) is an aggressive peripheral T-cell neoplasm that develops after long-term infection with human T-cell leukemia virus (HTLV-1). SIRT1, a nicotinamide adenine dinucleotide (NAD+)-dependent histone/protein deacetylase, plays a crucial role in various physiological processes, such as aging, metabolism, neurogenesis and apoptosis, due to its ability to deacetylate numerous substrates, such as histone and NF-κB, which is implicated as an exacerbation factor in ATL. Here, we assessed how SIRT1 is regulated in primary ATL cells and leukemic cell lines. SIRT1 expression in ATL patients was significantly higher than that in healthy controls, especially in the acute type. Sirtinol, a SIRT1 inhibitor, induced significant growth inhibition or apoptosis in cells from ATL patients and leukemic cell lines, especially HTLV-1-related cell lines. Sirtinol-induced apoptosis was mediated by activation of the caspase family, and degradation of SIRT1 in the nucleus. Interestingly, NAD+ augmented sirtinol-induced apoptosis in leukemic cell lines. Thus, the SIRT1 inhibitor acted as a tumor suppressor, where NAD+ accelerated the SIRT1 inhibitor-induced apoptosis. These results suggest that SIRT1 is a crucial anti-apoptotic molecule in ATL cells, and that SIRT1 inhibitors may be useful therapeutic agents for leukemia, especially in patients with ATL.


Malignant mesothelioma is an aggressive tumour of serosal surfaces most commonly pleura. Characterised cell lines represent a valuable tool to study the biology of mesothelioma. The aim of this study was to develop and biologically characterise six malignant mesothelioma cell lines to evaluate their potential as models of human malignant mesothelioma.Five lines were initiated from pleural biopsies, and one from pleural effusion of patients with histologically proven malignant mesothelioma. Mesothelial origin was assessed by standard morphology, Transmission Electron Microscopy (TEM) and immunocytochemistry. Growth characteristics were assayed using population doubling times. Spectral karyotyping was performed to assess chromosomal abnormalities. Authentication of donor specific derivation was undertaken by DNA fingerprinting using a panel of SNPs.Most of cell lines exhibited spindle cell shape, with some retaining stellate shapes. At passage 2 to 6 all lines stained positively for calretinin and cytokeratin 19, and demonstrated capacity for anchorage-independent growth. At passage 4 to 16, doubling times ranged from 30–72 hours, and on spectral karyotyping all lines exhibited numerical chromosomal abnormalities ranging from 41 to 113. Monosomy of chromosomes 8, 14, 22 or 17 was observed in three lines. One line displayed four different karyotypes at passage 8, but only one karyotype at passage 42, and another displayed polyploidy at passage 40 which was not present at early passages. At passages 5–17, TEM showed characteristic features of mesothelioma ultrastructure in all lines including microvilli and tight intercellular junctions.in vitro model systems for studying cellular, molecular and genetic aspects of mesothelioma.These six cell lines exhibit varying cell morphology, a range of doubling times, and show diverse passage-dependent structural chromosomal changes observed in malignant tumours. However they retain characteristic immunocytochemical protein expression profiles of mesothelioma during maintenance in artificial culture systems. These characteristics support their potential as  The incidence is variable worldwide but is highest in Australia: 47 new cases per 100,000 population Advantages of cell line models include that they are relatively inexpensive compared with animal models, limitlessly renewable, able to be manipulated by transfections or knockdowns for investigation of gene function, and are practical for high-throughput screening studies. Furthermore, lacking stromal and inflammatory cell content, they provide a relatively pure source of tumour which is superior for estimation of gene copy number. Cultured cells derived from many different tumour types have been shown to retain hallmarks of cancer with the exception of angiogenesis In vitro screening assays in cell line models can be useful to predict therapeutic responses and mesothelioma cell lines have proved useful models for testing conventional, targeted, and gene-based therapies Mesothelioma cell lines were useful tools to demonstrate that the NF2 tumour suppressor gene product, merlin, exerts its anti-proliferative effect in mesothelioma by suppression of p21 activated kinase-induced cyclin D1 expression in vitro models of human disease, cell lines must reflect biological and genetic changes of human disease closely and stably.Although characterised mesothelioma cell lines exist This study aimed 1) to establish a bio-tool of annotated human malignant mesothelioma tumour cell lines characterised for morphology, doubling times, immunocytochemical properties, and chromosomal alterations, and 2) to define passage related changes in cytogenetic profiles of human mesothelioma cell lines in long term culture.Patients with malignant mesothelioma attending clinics at The Prince Charles Hospital (TPCH) gave written informed consent to donate pleural biopsy tissue and/or pleural fluid after sufficient diagnostic samples were obtained. The study was approved by the Ethics Committee at The Prince Charles Hospital (EC27121). Demographic, clinical and pathology data for the six donors are detailed in Methods to generate cell lines were as follows:Tissue samples from video assisted thoracoscopic (VAT) biopsies were cut into small pieces (approximately 1–3 mm) and plated in sterile petri dishes containing 4 ml medium. Fresh medium (R-10) was added periodically until adherent cell outgrowth was observed. To prepare cell lines, tissue pieces were removed and cells were harvested to fresh flasks and allowed to grow to confluence. Medium was changed every three days and subculturing was performed when the cells became 70–80% confluent.2 flasks . Medium (R-10) was changed every three days to remove non-adherent cells. Subculturing was performed by treating cells with 0.25% trypsin-EDTA  for two min at 37°C at confluence.Pleural fluid samples were centrifuged (1766 rpm for 7 min) and the pellets were washed in phosphate buffered saline  and distributed into 75 cm2. Early passage cells were stored in liquid nitrogen in freezing medium (90% FCS and 10% DMSO).All cells were cultured in RPMI 1640 (Gibco Cat No 21870-076) with 10% FCS  supplemented with 1% Penicillin/Streptomycin/Glutamine (Gibco Cat No 10378-016) and 0.2% fungizone (Invitrogen Cat No 04195780D) (R-10) and maintained at 37°C in a humidified atmosphere at 5% COAll cell lines tested negative for mycoplasma infection using a conventional PCR based Mycoplasma detection kit, Venor GEM (Minerva biolabs) (data not shown).Six cell lines were selected for characterisation. Clinical and demographic details for the six donors and clinical pathology of the primary mesothelioma tumours are shown in en bloc with uranyl acetate and embedded in Procure 812 Epoxy Resin. Ultrathin sections were cut at 70–90 nm, stained with lead citrate, and examined by transmission electron microscope .Cells were grown over cover slips to 70% confluence, washed and fixed in gluteraldehyde buffer at 4°C for 1 hour and then examined by light microscopy. Transmission electron microscopy (TEM) was used for ultra-structural characterisation. Cells at 70–80% confluency were washed with 1×PBS and fixed with 2 ml 3% gluteraldehyde buffer at 4°C for 1 hr. Cells were harvested with a rubber scraper and spun at 1200 rpm for 5 min. Supernatant was discarded and fresh gluteraldehyde buffer was added. Cell pellets were secondarily fixed with osmium tetroxide and stained To verify derivation, cell line profiles for 45 SNPs generated by Sequenom using the iPLEX Sample ID Plus Panel were compared to peripheral constitutional DNA SNP profiles from each donor.2) and grown in medium (R-10). Cells were counted after 24 hr, 48 hr, 72 hr, 96 hrs, 7 days, 9 days and 11 days. Cell viability was assessed by trypan blue exclusion. Doubling time was calculated by the algorithm provided by http://www.doubling-time.com. 5000 cells of each line at passage number 8–16 were seeded into triplicate wells of a 24 well plate  in antifade medium. Selected metaphases were captured and analysed using the SD 300 bioimaging system (ASI Ltd), using manufacturer’s software for acquisition and analysis of colour chromosomes (HiSKY). DAPI images were captured separately and chromosomes were then sorted automatically into a karyotype table and analysed for structural and/or numerical aberrations. From each sample 7–10 metaphases were randomly chosen for full analysis from different areas of the slides.Chromosome preparations of the cells were made as follows: Colcemid (Demecolcine Solution D1925 Sigma 10 ug/ml in HBSS)  was added to semi- confluent cultures (70%) 16–17 hours before harvesting. Metaphase cells were harvested by trypsinizing, treated with a hypotonic solution (0.56% KCl) for 15 min at 37°C, and fixed twice in acetic acid (one part)/methanol (three parts). Cells were dropped onto clean slides and sent to Applied Spectral Imaging (Israel) for Spectral Karyotyping (SKY). A SKY probe containing all 24 labelled chromosome libraries was hybridized simultaneously to the metaphases. After washingStaining indicative of mesothelial origin was performed using the following antibodies: anti-calretinin  at a dilution of 1∶50, and anti-cytokeratin-19  at a dilution of 1∶100 for staining cells. Cells were grown on cover slips to 80% confluency. Staining with immunocytochemical markers was performed using streptavidin–biotin horseradish peroxidase (SAB/HRP) kit  with an incubation time of 1 hour at room temperature for each primary antibody. Intensity of immunocytochemical reactions was evaluated by a semi-quantitative method using a scale defined as follows: negative: no positive cells, weak: <10%, medium: 10–60%, high: >60%. Cell line JU77, a previously well-characterised malignant mesothelioma cell line Anchorage-independent growth strongly correlates with tumourogenicity Viable cells from all lines reported here were retrievable from cryopreservation, subsequently grew to confluency, and were propagated by passaging. DNA fingerprinting was undertaken on four of the cell lines showing matching SNP profiles between blood and cell line DNA  (data not shown) verifying derivation of each line from the donor thus authenticating the annotation of these lines against clinicopathologic characteristics of the donor patients and their tumours.All cell lines  formed monolayers with various morphological cell types , Table 2Growth characteristics were studied at the following passages (p) - MM04 (p11) MM05 (p16), MM12 (p5), MM13 (p9), MM1081 (p6) and PF1038 (p6). Doubling times ranged between 30 hr and 72 hrs . No relaChromosomal abnormalities are listed in Two cell lines (MM04 and MM05) studied at both early (p8 and p5 respectively) and late (p40 and p42 respectively) passages displayed chromosomal abnormalities. MM05 developed polyploidy at late passage while continuing to show similar abnormalities as observed in early passages. Late passages showed similar translocations in chromosome 1, 8, 11 and 17 as in early passages . Early pEstablishment of primary cell lines as a disease model is potentially of considerable importance in the study of mesothelioma at cellular, molecular and genetic levels. Mesothelioma is readily propagated in cell culture from human tumour biopsies and pleural effusions. Over a period of four years (2008–2011), we established twenty-one cell lines , HBME-1, thrombomodulin, WT-1, mesothelin, and podoplanin as mesothelial markers and pCEA, Ber-Ep4, TTF-2, B72.3 as epithelial markers Mesothelioma cell lines exhibit complex structural and numerical chromosomal abnormalities on spectral karyotypic analysis - a molecular cytogenetic technique by which even complex chromosomal abnormalities can be detected, which may not be revealed by routine G banding or FISH techniques. Monosomy 17, found in three of five cell lines, has been reported previously in mesothelioma Genomic instability is recognized to occur during long term culturing of tumour cell lines in vitro model systems of human disease. Characterization of these six cell lines adds to the bioresources available for future mesothelioma research.Mesothelioma lines available from various cell banks may be affected by culture related cytogenetic and other alterations affecting their suitability for various research purposes. Annotation of repository cell lines with evidence of authenticated derivation, passage number, and comprehensive characterisation is essential to underpin their utility as


Moreover, a non-biased screen of ∼3400 compounds, including FDA-approved drugs, revealed similar sensitivity of RB-proficient and -deficient TNBC cells. Finally, ESA+/CD24−/low/CD44+ cancer stem cells from RB-negative TNBC lines were consistently more sensitive to gamma-irradiation than RB-positive lines, whereas the effect of chemotherapy on the cancer stem cell fraction varied irrespective of RB1 expression. Our results suggest that patients carrying RB-deficient TNBCs would benefit from gamma-irradiation as well as doxorubicin and methotrexate therapy, but not necessarily from many other anti-neoplastic drugs.Triple negative breast cancer (TNBC) includes basal-like and claudin-low subtypes for which only chemotherapy and radiation therapy are currently available. The retinoblastoma (RB1) tumor suppressor is frequently lost in human TNBC. Knockdown of RB1 in luminal BC cells was shown to affect response to endocrine, radiation and several antineoplastic drugs. However, the effect of RB1 status on radiation and chemo-sensitivity in TNBC cells and whether RB1 status affects response to divergent or specific treatment are unknown. Using multiple basal-like and claudin-low cell lines, we hereby demonstrate that RB-negative TNBC cell lines are highly sensitive to gamma-irradiation, and moderately more sensitive to doxorubicin and methotrexate compared to RB-positive TNBC cell lines. In contrast, RB1 status did not affect sensitivity of TNBC cells to multiple other drugs including cisplatin (CDDP), 5-fluorouracil, idarubicin, epirubicin, PRIMA-1 Triple negative breast cancer (TNBC) represents a collection of tumors that lack expression of estrogen (ER) and progesterone (PR) receptors as well as the receptor tyrosine kinase HER2 TNBC makes up 10–30% of all breast cancer cases. Compared to other subtypes, TN tumors are associated with poor prognosis, in part due to a lack of targeted treatment. Clinically, TNBCs respond more favorably to chemotherapy than other types, however prognosis still remains poor due to a greater risk of distal recurrence, with a rapid rise in relapse in the first 3 years post diagnosis The tumor suppressor RB1 is often lost by mutation, deletion or transcriptional silencing as well as by hyper-phosphorylation of its gene product, pRb, in many human malignancies + breast cancer. However, RB1 is most commonly lost in TNBC, not in ER+ luminal tumors Acute inactivation of RB1 in hormone-dependent luminal breast and colon cancer cells increases response to several antineoplastic drugs, suggesting that RB-deficiency affects therapeutic outcome in certain tumor types including ERBC cell lines were shown to maintain many genomic and transcriptional characteristics of primary breast tumors from which they were derived and are therefore useful as surrogates for breast cancer The aforementioned Western blot analysis allowed us to compare pRb protein expression to RB1 transcript levels using publicly available microarray data sets for over 20 TNBC cell lines. We used the following data sets: Neve/Gray + breast cancer). Consistent with a previous observation that cyclin E1 is elevated in basal-like breast cancer + vs. RB− TNBC lines.We next investigated the expression RB1 relative to components of the RB1 pathway including the cyclins  and cyclin-dependent kinases , using the same four published microarray data sets  treated with 5–10 Gy. The RB-null lines analyzed were MDA-MB-468, Du4475, MDA436 and Bt549; RB-proficient lines were MDA-MB-231, HCC38, HCC70 and Hs578t. Interestingly, although radiation treatment was effective against all TNBC lines, the RB-null lines were significantly more sensitive to radiation than RB-positive lines at a dose of 10 Gy (p = 0.0024)  and 5-fluorouracil (5-FU), which are commonly used to treat TNBC −  and 2 RB+ TNBC lines . The majority of strong hits were known chemotherapeutics such as doxorubicin and idarubicin . In another study we screened 3185 compounds against the same 4 TNBC lines ;  Thus, loss of RB1 does not lead to a general increase in chemo-sensitivity to a multitude of compounds, including antineoplastic drugs. Notably, one drug, phenylmercuric acetate, diminished growth of the RB− lines to 1% viability , therefore leading to an average of 48% inhibition. Given these results, and the fact that phenylmercuric acetate is a mercury-containing compound To determine whether RB1 status had a global effect on chemo-sensitivity, we performed a screen of 268 FDA-approved drugs using two RBiability , arrow. + and 5 RB− lines, representing basal-like and claudin-low subtypes. Drugs tested included idarubicin, which scored highly in our screen of TNBC lines . However, there was no significant difference between the average dose response curves for RB+ and RB− lines . pRb phosphorylation state did not affect response to treatment with these drugs, with the exception of idarubicin, which contrary to our expectations killed hypo-phosphorylated pRb lines slightly better than hyper-phosphorylated pRb cells . There was also no difference in response to these chemotherapeutics between basal-like  and claudin-low  cell lines . We note that two of the TNBC lines we analyzed  carry both RB1 and BRCA1 mutations. Drug sensitivity of these tumor cells was indistinguishable from sensitivity of other RB− (but Brca1 proficient) or RB+ TNBC cell lines (data not shown).Next, we analyzed the effect of RB1 status on selected anti-neoplastic drugs used to treat different types and grades of breast cancer. For this, we used our panel of 9 RB−/ESA+/CD24−/low/CD44++ and RB− lines with CDDP, doxorubicin or gamma-irradiation , capable of sustaining tumorigenesis, at their apex We found that pRb expression is lost in 33% of human basal-like cell lines, in agreement with recent genomic analysis of primary basal-like tumors Inactivation of RB1 is thought to increase sensitivity to DNA damaging agents, as cells are unable to halt cell cycle progression and repair damaged DNA. This notion is supported by studies on BC patient cohorts showing that women with triple negative disease have a better prognosis and longer disease-free-survival if they are pRb-deficient and treated with chemotherapy Previous studies using hormone-sensitive RB1 proficient BC lines  demonstrated that silencing of RB1 significantly increased sensitivity to 5-FU, methotrexate and CDDP + and basal-like breast cancer patients to therapy + cohorts. It would be important to determine whether TNBC cells with RB-pathway disruption are as sensitive to irradiation and doxorubicin as bona fide RB-deficient TNBC cells, and whether they share a similar spectrum of drug sensitivity.Another recent study shows that an RB-pathway-loss signature predicts response of ER− lines by 10–20% more than RB+ lines along the entire dose-response curve (p = 0.0043). Methotrexate exerts its effect by inhibiting purine/DNA synthesis. Therefore, one would expect that other drugs affecting DNA synthesis would also be more effective against RB-deficient TNBC cells. However, this is clearly not the case since treatment with 5-FU, which inhibits thymidine synthesis, shows a reversed sensitivity profile with RB− cells being less sensitive than RB+ lines (− lines more efficiently than RB+ cells (p = 0.0140). Finally, CDDP exerts its effect by cross-linking DNA, where RB-independent pathways regulate response to this stress − cells relative to RB+ TNBC lines. Together, these results suggest that RB status affects response to specific drugs, and this may be important for development of drugs that selectively target RB-deficient tumor cells.Our study demonstrates no difference in sensitivity between RB-deficient and RB-proficient cell lines towards a large number of anti-neoplastic drugs and other compounds. However, we identified selective response to specific compounds. For example, methotrexate inhibited RBB+ lines . Further+/CD24−/low/CD44+ CSC fraction in RB-null TNBC lines was significantly more susceptible to as low doses of irradiation (5 Gy) compared to CSCs from RB-proficient lines (p = 0.038). In the clinic, TNBC patients may receive a biological equivalence of >60 Gy Intriguingly, our findings on the role of RB1 status suggest that radiation therapy is most effective against TNBC lacking pRb protein. We observed a decrease in cell viability for RB-null lines when treated with 5 Gy of radiation, and a further decrease at 10 Gy (p = 0.0024). Moreover, the ESA2 in attachment plates (Du4475 grew in attachment plates as suspended aggregates). Cell lines Du4475, Bt549, MDA-MB-436, MDA-MB-468, MDA-MB-231, and HCC1569 were kind gifts from Dr. Mona Gauthier. HCC3153 was a gift from Dr. Tak Wah Mak lab. The remaining breast cancer cell lines were purchased from the America Type Culture Collection (ATCC) . All cell lines are available from the ATCC.MDA-MB-231, MDA-MB-468, MDA-MB-157, Hs578T, and MCF7 were maintained in DMEM containing 10% FBS and 1% PEST. MDA-MB-436 was maintained in DMEM containing 10% FBS, 1% PEST, and supplemented with 10 µg/ml insulin. HCC70, HCC1937, HCC38, HCC1954, HCC1569, HCC1187, HCC3153, HCC1500, Du4475, and Bt549 were maintained in RPMI containing 10% FBS and 1% PEST. All cell lines were grown at 37°C with 5% CO3VO4, and 1∶100 protease inhibitor cocktail ). Protein concentration was determined by Bio-Rad dye-binding assay . Proteins in total cell lysates were fractionated by SDS-PAGE and transferred onto nitrocellulose membranes using electrophoresis for subsequent immunoblotting. Membranes were blocked with 5% nonfat dried milk in phosphate-buffered saline containing 0.05% Tween 20 (PBST) at R.T. for 1 h and incubated at 4°C overnight with mouse anti-pRb primary antibody , rabbit anti-ppRb-ser795 primary antibody , or rabbit anti-tubulin primary antibody . Membranes were washed with PBST buffer 3 times, 5 min each and incubated with HRP-conjugated anti-mouse, or anti-rabbit, IgG secondary antibody  for 1 h. After further washing, the membranes were allowed to react with ECL , the signal was detected using autoradiography film and developed using a Konica SRX-101A developer. Primary antibodies were diluted 1∶1000 in PBS with 5% BSA; secondary antibodies were diluted 1∶2000 in PBS with 5% nonfat dried milk.Each TNBC cell line was cultured in 10 cm dishes with their corresponding media, treated with 0.25% trypsin (Sigma-Aldrich), washed with PBS, pelleted and lysed with lysis buffer , which includes marketed drugs, natural products with unknown biological properties, and other non-drug molecular entities. The Prestwick library is composed of 1185 drugs from Prestwick Chemical  , and contains only marketed drugs approved by the FDA, EMEA and other agencies. Both libraries have been successfully implemented in drug reposition and combination strategies Screens were performed in the S.M.A.R.T. Facility of the Samuel Lunenfeld Research Institute. All libraries were prepared in 100% DMSO to facilitate pinning. The final concentration of DMSO in each screen was 0.4%. The breast cancer lines MDA-MB-231, MDA-MB-436, Bt549, and HCC70 were seeded with their corresponding media in 384-well plates at a density of 900 cells/well in a total volume of 50 µL/well. The following day, plates were pinned with a drug library to reach a final concentration of 1 µM (Sequoia and Spectrum libraries), or 0.8 µM (Prestwick library). Alamar blue (Invitrogen) was added three days post drug pinning at 10% of the volume (5 µL/well), and cell viability was read 4–6 h later using a Pherastar plate reader. The Spectrum library consists of 2000 drugs from MicroSource Discovery Systems (USA) , and PD-0332991 . The source of radiation was from a Cesium-137 Gammacell Irradiator.Unless otherwise stated, drugs used in this study were obtained from Sigma-Aldrich . Compounds used to establish dose-response curves include cisplatin/CDDP, doxorubicin, idarubicin, epirubicin, 5-fluorouracil, methotrexate, fludarabine, PRIMA-13 cells/well) and treated the following day, leaving 100 µL final volume of media. Three days (72 h) post treatment 30 µL of 2 mg/mL MTT -2, 5-diphenyl tetrazolium bromide, Sigma-Aldrich) was added to each well and incubated for 2–4 h, depending on cell type. MTT/media solution was aspirated and replaced with 100 µL DMSO and left at R.T. for 15–20 min to dissolve the formazan dye. After gentle agitation to ensure even mixture of the dye, a 96-well microplate reader (Molecular Devices) was used to determine the optical density (OD) of each well at 570 nm. Viability (%) was determined by (treatment group OD/untreated control group OD) ×100%, using DMSO as a blank. Each assay was performed in triplicate, and repeated at least 3 times.Cells were seeded in 96-well plates at their optimal density (2–5×106 cells were incubated with 5 µL mouse anti-human ESA/Ep-CAM/CD326-PE (BioLegend), CD24-FITC (BD Biosciences), and CD44-APC (BD Biosciences) antibody at R.T., in the dark, for 15–20 min with occasional pulse vortexing. Cells were then washed and strained to single cells into 5 mL polystyrene round-bottom FACS tubes  to a total volume of 500 µL (1×106 cells/mL). Finally, 5 µL 7-AAD (BD Biosciences) was added to each tube as a viability marker, and cells were processed on a FACSCaliber (Becton Dickinson) no longer than 1 h post-staining. In all experiments, 7-AAD exclusion, side scatter and forward scatter profiles were used to eliminate dead cells and debris.Cell lines were plated at optimal densities and treated the following day with their respective IC50 of cisplatin/CDDP or doxorubicin, or with exposure to cesium-137 radiation to a dose of 5 Gy. After 72 h, the supernatant from each treatment group was collected before cells were trypsinized to single cell suspension. Trypsinized cells were added to their respective supernatants, washed with PBS, pelleted, resuspended in serum-free PBS, and counted. In a volume of 100 µL serum-free PBS, 0.5–1.0×10Statistical analyses for curve comparisons were performed on Prism 6 GraphPad Software using nonlinear regression analysis with a significance cut-off of p = 0.05. The p-values for gamma-irradiation and CSC analyses were calculated using a two-tailed t-test method. Statistical analysis for cyclin/Cdk expression was determined by ANOVA.Figure S1Drugs ranked by RB status and superimposed for comparison. (A) Sequoia library , (B) Spectrum library , and (C) Prestwick library .(TIF)Click here for additional data file.Figure S2met or fludarabine.Effect of RB1 status on dose-response curves for 14 human derived TNBC lines treated with PRIMA-1 (A) Response for each individual line. Values represent the average of 3–5 assays, each performed in triplicate. (B) Average response based on RB1 status. RB+ lines: MDA-MB-231, HCC38, Hs578t, MDA-MB-157, HCC1954, HCC1569, HCC3153, SUM149 and HCC70. RB− lines: MDA-MB-436, MDA-MB-468, Bt549, Du4475 and HCC1937. PRIMA-1met, p = 0.9347. fludarabine, p = 0.6875. (C) Average response for hyper- and hypo-phosphorylated pRb states. fludarabine, p = 0.2484. PRIMA-1met, p = 0.9884. (D) Average response for BaA and BaB subtypes. fludarabine, p = 0.1748. PRIMA-1met, p = 0.8237. p-values calculated using nonlinear regression analysis.(TIF)Click here for additional data file.


In vitro cell culture experiments with primary cells have reported that cell proliferation is retarded in the presence of ambient compared to physiological O2 levels. Cancer is primarily a disease of aberrant cell proliferation, therefore, studying cancer cells grown under ambient O2 may be undesirable. To understand better the impact of O2 on the propagation of cancer cells in vitro, we compared the growth potential of a panel of ovarian cancer cell lines under ambient (21%) or physiological (3%) O2.2, but some display insensitivity to changes in O2 concentration. Further analysis revealed an association between defective G2/M cell cycle transition regulation and O2 insensitivity resultant from overexpression of 14-3-3 σ. Targeting 14-3-3 σ overexpression with RNAi restored O2 sensitivity in these cell lines. Additionally, we found that metastatic ovarian tumors frequently overexpress 14-3-3 σ, which in conjunction with phosphorylated RB, results in poor prognosis.Our observations demonstrate that similar to primary cells, many cancer cells maintain an inherent sensitivity to O2 concentration. Although a direct link between O2 insensitivity and metastasis was not determined, this investigation showed that an O2 insensitive phenotype in cancer cells to correlate with metastatic tumor progression.Cancer cells show differential proliferative sensitivity to changes in O However, it has been reported that altered concentrations of O2 results in clear differences in cell proliferation and response to drug treatment in the cancer cells Cell lines derived from cancer patients provide an experimentally manipulable model system that facilitates investigations into cancer biology and its therapy. The unlimited proliferation potential of cancer cells is a major hallmark of malignancy, however the use of standard tissue culture protocols often restricts cell proliferation, as observed with primary cell lines in vitro investigations have demonstrated the advantages of physiological O2 for tissue culture. For example, the biological behavior of primary cell cultures with a physiological concentration of O2 (2.7–5.3%) is far superior compared to the standard practice of growing cells under atmospheric or “ambient” O2 concentration (21% O2) Oxygen, in addition to nutrients and growth factors, is vital for proper cell growth and its availability has a direct impact on cellular metabolism, signaling pathways, proliferation, differentiation and survival 2 tension in cancer biology is well established. For example, the fact that many cancers exist in a ‘hypoxic’ state has led to the development of hypoxia-targeted therapy 2 is <1% for most solid tumors, however the hypoxic concentration could vary based on the cell types and the normal perfusion status 2 tension varies from 2.7–5.3% in the interstitial space 2 may miss pertinent biological observations. This may be particularly important when attempting to study the progression of cancer to metastatic disease, which is a significant event in cancer etiology and is associated with poor prognosis 2 tension in different compartments of the body, an understanding of the effect of O2 concentration on cancer cell proliferation could provide useful insights into the mechanisms involved in the pathological progression of cancer.The importance of considering O2) 2) 2 conditions is both important and revealing, it must be noted that these previous studies did not investigate the response of p53 at physiological (3%) O2 and ambient (21%) O2. p21 and 14-3-3 σ are transcriptional targets of p53 that are involved in regulating G1/S and G2/M transitions of the cell cycle by targeting CDK2 and CDC2 , respectively 2 concentration result in altered cell cycle progression in primary cells but cancer cells frequently display cell cycle control defects, there is clearly the potential that these defects may impact how cancer cells respond to altered O2 levels in a manner that could have a profound influence on cancer progression.Cancer cells that have acquired mutations in either oncogenes or tumor suppressor genes display a characteristic uncontrolled proliferation phenotype 2. Interestingly, some of the ovarian cancer cell lines had a normal response to O2 concentration, (i.e. reduced cell proliferation with increased O2 concentration) while the proliferation of other ovarian cancer cell lines was unaffected by this O2 increase. Further, our investigations revealed that 14-3-3 σ and its role in the cell cycle influence the proliferative response to altered O2 levels. Considering the variation in partial pressure of oxygen throughout the body and the potential importance that this context may have on cancer progression, it is crucial to understand the affect of O2 concentration on cancer cell proliferation and cancer progression. We provide evidence that acquisition of O2 insensitivity may be a component in cancer progression and a hallmark of successful metastatic disease.Here we examined the biological behavior of ovarian cancer cells under physiological and ambient O2) and ambient (21% O2) oxygen concentration using A2780 ovarian cancer cells and observed that 12 days of cell culture under these conditions resulted in a 2.6 fold growth suppression under 21% O2  and ambient (21% O2) oxygen concentrations. Since the serum present in cell culture medium can also have a dominant influence on growth, we also tested the affect of various concentrations of serum. Regardless of the amount of serum present in the growth medium, culturing in 21% O2 generally resulted in a significant decrease in cell proliferation for four of the ovarian cancer cell lines  compared to 3% O2 . In contrast to the effect of O2 levels, increasing the concentration of serum resulted in a proportional growth increase in the ovarian cancer cell lines A2780, OVCAR5 and OVCAR8  in SKOV3, while HeyA8 serum concentration between 2 and 10% serum had the greatest effect at 3% O2 (p<10−5)  O2 levels. We therefore categorized the ovarian cancer cell lines based on these differences, being either O2 sensitive  or insensitive (SKOV3 and HeyA8)  . Increas(p<10−5) . Togethed HeyA8) . Overall2 insensitivity and differences in proliferation resulted from differences in the doubling time of each cell line. For example, if SKOV3 and HeyA8 (the O2 insensitive cell lines) proliferate more slowly, O2 dependent proliferation changes may be too trivial to measure. Therefore, we measured the cell doubling time for all ovarian cancer cell lines. Our results showed that under standard tissue culture conditions (10% serum and 21% O2) the doubling time for all ovarian cancer cell lines were somewhat similar (<24 hours) except for HOC8, which had an extended doubling time of about 45.5±4.9 hours  the percentage of the cell population in the G2 phase was increased significantly in 21% O2. However a significant increase in G2 was not observed in the fourth O2 sensitive cell line, HOC8 , we did not observe a significant increase in the percentage of cells in G2 when the O2 levels were increased. These results suggest that although the G1 and S phases of the cell cycle are responding similarly to changes in O2 concentration in both O2 sensitive and insensitive cell lines, it is the G2 phase of the cell cycle that is not responsive to O2 concentration in the O2 insensitive cell lines. Therefore, the difference in cell cycle response observed with these ovarian cancer cell lines might be at the level of regulation during the cell cycle progression from G2 to M phase. It is also possible that the changes observed with G2 and O2 sensitivity in these cancer cell lines is reflected in the mitotic component of the cell cycle. Our observation of the mitotic cells present in the O2 sensitive and insensitive cell lines grown under 3% and 21% O2 supports this conclusion; the O2 sensitive cell lines show a proportionate decrease in the mitotic cell population observed at 21% O2 compared to 3% O2, (2 (2 insensitive cell lines (HeyA8 and SKOV3) the proportion of mitotic cells remained unaltered regardless of O2 concentrations , resulting in a distinct phenotype.Considering the differences in proliferation observed for ovarian cancer cell lines grown under either 3% or 21% O S phase , which wne, HOC8 . Similarr 21% O2 . Consideo 3% O2, , corresp% O2,  compared to the O2-sensitive cell lines   compared to O2 insensitive cell lines (HeyA8 and SKOV3)  were considerably lower in O2 insensitive cell lines (HeyA8 and SKOV3) compared to O2 sensitive cell lines  (2 insensitive cell lines.Thus far we have demonstrated that Oll lines . Althoug OVCAR8) , we obsem levels . This com levels . Howeverd SKOV3) . Therefond HOC8) . These o2 insensitive cell lines (HeyA8 and SKOV3), we explored the possibility that impairment was due to a defect in any of these molecular regulators. Western blot analysis found p53 and p21 to be overexpressed in one O2-insensitive cell line (HeyA8). However, both were absent in the other O2-insensitive cell line (SKOV3), and the expression pattern for these proteins remained unaltered regardless of changes in O2 or serum concentration  compared to the O2-sensitive cell lines. Although, the level of 14-3-3 σ expression was considerably lower in all O2-sensitive cell lines compared to HeyA8 and SKOV3, we did observe an increase in the expression of 14-3-3 σ at 21% O2 with A2780 , but not in HOC8 (2-insensitive cell lines (HeyA8 and SKOV3) , barely detectable levels in HOC8,  , with th pattern  and cell pattern  revealedin HOC8,  and a co2-sensitive cell lines  showed increased expression of CDC2 and cyclin B combined with low level of 14-3-3 σ expression. This suggests that the cell cycle components required for a dynamic proliferative response to differences in the O2 concentration is present in these cell lines. However in the O2-insensitive cell lines that express high levels of 14-3-3 σ and low levels of CDC2 and CDC25C such a dynamic cell cycle response to changes in O2 concentration could be impaired. We therefore pursued the possibility that this inverse correlation between 14-3-3 σ and CDC2 might be important for the O2-sensitive regulation of G2/M transition.In summary, the O2-insensitivity that needs to be confirmed. Therefore, we wanted to confirm that 14-3-3 σ does indeed affect O2-dependent proliferation. For this part of the study, we restricted our analysis to two cell lines with wild type p53: the O2-sensitive A2780 2-insensitive HeyA8 cell lines 2 and 21% O2. In the O2-sensitive A2780 cell line, the localization of 14-3-3 σ was restricted to the cytoplasm under 3% O2 , compared to 3% O2  and screened 57 different ovarian cancer cell lines for the levels of 14-3-3 σ and CDC2, as well as phospho-RB as an indicator of CDC2 activity. Cell lines with the same name but from different labs or different passages were considered to be different. We therefore set the analysis criteria on the RPPA array to detect high phospho-RB (P-RB) and either high 14-3-3 σ or high CDC2. In the context of high levels of P-RB, this criteria should indicate that either 14-3-3 σis dysfunctional or that active CDC2 is uninhibited, perhaps due to methylated 14-3-3 σ or inhibition of CDC2 degradation 2 insensitivity pattern we have described. Amongst these 16 cell lines, 6 also have increased levels of CDC2 while the remainder had decreased levels of CDC2. This suggests that this protein profile is not exclusive to the cell lines we originally identified and might be representative of a relatively common phenomenon. We therefore determined whether this O2-insensitive associated 14-3-3 σ/CDC2/P-RB protein profile is also observed in ovarian tumor samples. Using the same criteria as with the cell line RPPA, we examined 205 ovarian tumor specimens using RPPA. This analysis revealed that 27% of ovarian tumors (56) had elevated levels of both 14-3-3 σ and P-RB, and amongst these, 34 also had elevated levels of CDC2 expression  we identified 47 of 158 tumors with associated clinical data. A Kaplan-Meier survival estimate shows that patients with the O2-insensitive associated protein profile have a poor survival outcome  is both relatively common in ovarian cancer and associated with poor prognosis, we went on to determine directly whether metastatic ovarian tumors exhibit an overt 14-3-3 σ signature. Of note, the ovarian tumors represented in the ovarian tumor RPPA are from primary sites and thus do not necessarily provide an accurate representation of the protein profile in the metastatic cancer. We therefore expect that metastatic tumors or primary tumors that give rise to metastatic tumors will exhibit a more overt 14-3-3 σ signature than primary tumors. In fact, an increased expression of 14-3-3 σ has been previously reported with other tumors a–c). Increased expression of 14-3-3 σ in the metastatic primary tumors compared to normal tissue or malignant tumors without metastasis were observed to be statistically significant by the Fisher’s exact test  and ambient (21%) oxygen, show that 21% O2 causes increased oxidative stress and induces senescence 2- ES cells otherwise differentiate under ambient O2 as reviewed in 2, a 20% growth suppression was observed with 21% O2 by three days   cell lines 2), but a reduced proportion of cells in G2 and increased proliferation when cultured at 5% O22 has also been observed to be accompanied by a reduction in the G2 cell population 2-sensitive phase of the cell cycle. Exploring the possible molecular mechanisms that render ovarian cancer cells either sensitive or insensitive to oxygen has clearly demonstrated that it is 14-3-3 σ and its inability to control CDC2 dependent G2/M transition in response to O2 levels that results in oxygen-insensitive cell lines. Although expression of 14-3-3 σ is regulated by p53 2-sensitivity is independent of p53. If the decrease in 14-3-3 σ is associated with oxygen-sensitive increase in proliferation, then silencing the expression of 14-3-3 σ in oxygen-insensitive cell lines should restore proliferative sensitivity to oxygen. In fact, our experiments show that RNAi mediated silencing of 14-3-3 σ in HeyA8 cells restored oxygen sensitivity (2-insenstive HeyA8 cells provides the first indication for the possible mechanistic basis of this dysregulation (2 insensitive p53 null cell line SKOV3).There is an increasing interest to study cell biology under the context of physiological Oree days  and althree days . The acctrations . This obd SKOV3) . These pto 3% O2 , suggestsitivity  and in asitivity . This sugulation . Other r2-sensitivity and, consequently, cell proliferation is most important when trying to recapitulate in vivo responses where physiological O2 tensions vary from 2.7–5% in the interstitial space (where many cancer cells reside) to 14.7% in the arterial circulation and lung 2-sensitive cancer cells were to dislodge from a primary interstitial space and migrate to the lungs via blood circulation, the increased O2 concentration would restrict proliferation. In contrast, we speculate that oxygen insensitive cancer cells would have a selective advantage compared to sensitive ones, being better able to thrive in the conditions of increased oxygen concentration. In fact, 14-3-3 σ is frequently over-expressed in many thyroid in vitro growth conditions because transient over-expression of 14-3-3 σ renders O2-sensitive A2780 cell line insensitive to increased levels of O2 , compared with ambient (21%) O2 is a prudent approach to identify and understand some of the behavioral diversity observed in cancer.In conclusion, there are many advantages to studying cancer biology under physiological O2 and O2 monitors and controllers. To maintain 3% O2, the incubator received an additional supply of nitrogen gas.Ovarian cancer cells were grown in RPMI 1640  or DMEM (HeyA8 and HOC8) supplemented with 10% heat inactivated Fetal Bovine Serum  and 200 units of penicillin/streptomycin and 0.5 µg amphotericin-B. Transfection was performed using Amaxa Nucleofector technology (Lonza) as described previously Cell proliferation was determined using Celltitre-Glo  per manufacturer instructions, as described previously 2. Finally, cells were washed, resuspended in phosphate buffered saline and stained with DAPI, as described The number of mitotic cells were quantified based the method as described Protein lysates and western blot analysis were preformed as previously described 6 cells per well in a 10 cm dish containing growth medium, antibiotics and appropriate concentrations of FBS. Dishes were then returned to the incubators set for the different oxygen conditions. Following three days of incubation, cells were harvested and prepared for FACS analysis as described previously Cells were trypsinized and seeded to a final density of 1×102. Control wells were treated with 1.5 µg/ml nocodazole (Sigma Aldrich) for 16 hours, fixed with 16% formaldehyde, permeabilized, blocked and stained with reagents consisting anti-phospho-histone H3 primary antibody, as per instructions provided in the kit. Stained cells were analyzed with a Zeiss Axiovert 200M inverted fluorescent microscope using 10X magnification and Openlab (PerkinElmer) image acquisition software. 100–250 cells per replicate were counted for phospho-histone H3 positive cells.The number of cells in M phase were quantified based on mitosis-specific histone H3 phosphorylation in the ovarian cancer cell lines using the Cellomics® Cell Cycle Kit I (Thermo Scientific) as per the manufacturer’s recommended protocol. Briefly, 96 well collagen coated plates were used to seed cells at a final concentration of 1000 cells/well in their respective media. Cells were then incubated for three days at 37°C in 3% or 21% O5 cells per fibronectin (Sigma) coated 12.5 mm2 glass coverslip mounted in each well of a 12-well plate. Cells were maintained in complete growth medium supplemented with 10% fetal bovine serum and allowed to grow for three days in the presence of 21% or 3% oxygen. For the detection of 14-3-3 σ or CDC2 by immunofluorescence, cells were processed as described previously A2780 cells transfected with a 14-3-3 σ cDNA expression construct or HeyA8 cells transfected with 14-3-3 σ siRNA were seeded at a final density of 10Protein lysates from 57 cancer cell lines or 205 primary ovarian cancer tumors were spotted in RPPA slides and processed for expression analysis, as described previously log-transformed RPPA data was first examined to remove non ovarian cancer cell lines. We then examined all replicated representations from the same source as annotated to reduce down to 57 ovarian cancer cell lines or 205 patient samples (from each source) by taking the median protein expression level of all replicates. An additional cell-line specific normalization step was performed in which median expression levels for each protein was first determined and then subtracted from individual RPPA experiments. The anchored heatmap (termed after anchored over/under-expression orientation) was generated by requiring RB, 14-3-3 σ and CDC2 to be arranged from over-expressed to under-expressed recursively from the given cell-line order, but exact positions of each protein was determined by hierarchical clustering algorithm with Euclidean distance as similarity measure and average lineage from all cell-lines, as shown in Tissue arrays (OV951-1) consisting of normal and malignant tissues from primary or metastatic sites were purchased from US Biomax Inc. Slides were processed for immunohistochemistry and analyzed, as described previously 2, a Student t-test was performed, and ANOVA was performed to compare the different cell cycle profiles with the panel of ovarian cancer cell lines. Kaplan-Meier survival analysis with p-value determined with log-rank test was performed using MATLAB  for RPPA data consisting patient specimens. For Kaplan-Meier survival analysis the data was censored based on patient's vital status. Statistical analysis for the correlation of 14-3-3 σ expression with the various pathological grades of ovarian tumors determined based on immunohistochemistry was analyzed by a Fisher’s exact test using R.To determine significant differences to proliferation under 3% or 21% OFigure S12 in ovarian cancer cells.Western blot analysis of phospho and total p53, and p21, which are major upstream regulators of G2/M cell cycle progression and the relevance to 21% or 3% O O2 insensitive cell lines are indicated by asterisk and italics.(EPS)Click here for additional data file.Table S1In vitro cell doubling time for the ovarian cancer cell lines.(XLS)Click here for additional data file.Table S2Raw data from cell line RPPA for Phospho-Rb, p53, CDC2 and 14-3-3 σ expression. The table contains following columns: 1) Unique ID, 2) Original cell-line or with treatment, 3) Cell-line's contributing Lab/source; 4) Cell type, 5) Cell-line name, 6–9) log2 transformed protein expression ratios  as the raw measurement provided by the MD Anders Cancer Center RPPA core facility.(XLS)Click here for additional data file.Table S3Raw data from OVSS2 RPPA for Phospho Rb, CDC2 and 14-3-3 σ expression. The table contains following columns: 1) Unique ID, 2) Tumor ID in various databases (DBs), 3) Tumor source institution; 4) patient age at diagnosis (in months), 5) tumor stage, 6) grade , 7) Overall survival (in months), 8) Vital Status , 9–12) log2 transformed protein expression ratios  as the raw measurement provided by the MD Anderson Cancer Center RPPA core facility.(XLS)Click here for additional data file.


PSI+] and [RNQ+] prions, which indicates that some prion variants are more sensitive to changes in the M-domain mobility than others. Thus, we provide evidence that regulation of the M-domain of Hsp104 is critical for efficient prion propagation. This shows the importance of elucidating the function of the M-domain in order to understand the role of Hsp104 in the propagation of different prions and prion variants.Molecular chaperones play a significant role in preventing protein misfolding and aggregation. Indeed, some protein conformational disorders have been linked to changes in the chaperone network. Curiously, in yeast, chaperones also play a role in promoting prion maintenance and propagation. While many amyloidogenic proteins are associated with disease in mammals, yeast prion proteins, and their ability to undergo conformational conversion into a prion state, are proposed to play a functional role in yeast biology. The chaperone Hsp104, a AAA+ ATPase, is essential for yeast prion propagation. Hsp104 fragments large prion aggregates to generate a population of smaller oligomers that can more readily convert soluble monomer and be transmitted to daughter cells. Here, we show that the middle (M) domain of Hsp104, and its mobility, plays an integral part in prion propagation. We generated and characterized mutations in the M-domain of Hsp104 that are predicted to stabilize either a repressed or de-repressed conformation of the M-domain . We show that the predicted stabilization of the repressed conformation inhibits general chaperone activity. Mutation to the de-repressed conformation, however, has differential effects on ATP hydrolysis and disaggregation, suggesting that the M-domain is involved in coupling these two activities. Interestingly, we show that changes in the M-domain differentially affect the propagation of different variants of the [ Saccharomyces cerevisiaeProtein aggregates pose a considerable challenge to cellular homeostasis and contribute to the pathogenesis of numerous neurodegenerative diseases. As such, protein misfolding and aggregation are guarded against by molecular chaperones, which act as the cell's first line of defense by maintaining proteostasis. In bacteria, fungi, and plants, the Hsp100 chaperones, together with Hsp70 and Hsp40 co-chaperones, are responsible for disaggregating protein aggregates and promoting cell survival and recovery from cell stress In addition to its role in protein disaggregation, Hsp104 is required for yeast prion propagation PSI+], has been proposed to play a role in creating genetic diversity by promoting translation of normally silent regions of the genome de novo formation of [PSI+] is regulated by another yeast prion, [RNQ+], resulting from the aggregation of the Rnq1 protein PSI+] has been shown to increase cell viability under various stresses, but can be detrimental in others, suggesting that the formation and propagation of [PSI+] is an important biological process that must be strictly regulated, in part by Hsp104 One of the best-characterized yeast prions is formed from the reversible aggregation of the translation termination factor, Sup35 Hsp104 can be divided into five functionally distinct, yet cooperative, domains. The N-terminal domain is not required for either prion propagation or thermotolerance, but has been proposed to be a site for substrate binding, as well as an interaction site for the Hsp70 and Hsp40 co-chaperones The M-domain is a coiled-coil insertion between NBD1 and NBD2 and is characteristic of Hsp100 chaperones that function as disaggregases, including the bacterial homolog, ClpB In the present study, we generated mutations in the M-domain of Hsp104 analogous to the previously characterized repressed and de-repressed mutations in ClpB S. cerevisiae strains were derivatives of 74-D694 and were grown using standard culture techniques. Strains were grown in YPD  or synthetic defined media  lacking amino acids that correlated with plasmid auxotrophic markers.All HSP104 were generated by bridge PCR using as the template, pRS313-phs-HSP104HSP104 from the HSP104 promoter (phs). Bridge PCR products and pRS313-phs-HSP104 were digested with EcoRI and Bsu36I, which are endogenous restriction sites in the HSP104 open-reading frame, and ligated together. Hsp104 mutants were also cloned into pProEx-HTb-HSP104hsp104-V426I plasmid was generated by PCR amplifying genomic DNA from the EMS mutagenized strain, then digesting and ligating as described.For expression of the Hsp104 mutants in vivo, point mutations in PSI+] in 74-D694 were previously characterized and kindly provided by Y. Chernoff and S. Liebman PSI+] variants and harboring the Hsp104 mutants, cells propagating each variant were mated to an hsp104Δ (hsp104::leu2) strain and diploids were selected. The mutant pRS313-phs-HSP104 plasmids were transformed into the heterozygous diploids, the diploids were sporulated, and haploids were selected on media lacking histidine and leucine. Colonies were verified as haploids by mating-type testing.The strong and weak variants of [RNQ+] variant yeast strains RNQ+] variants, we created HSP104 plasmid shuffle strains. First, pRS316-phs-HSP104RNQ+] variants. HSP104 on the chromosome was deleted by transforming the hphMX4 cassette amplified from pAG32 using oligonucleotides 5′GAAAAAAGAAATCAACTACACGTACCATAAAATATACAGAATATCAGCTGAAGCTTCGTACGC and 5′GATTCTTGTTCGAAAGTTTTTAAAAATCACACTATATTAAAGCATAGGCCACTAGTGGATCTG, containing flanking homology to the HSP104 promoter and terminator. Deletion of HSP104 was confirmed by PCR in Ura+ HygBR colonies. These strains were then transformed with each of the mutant pRS313-phs-hsp104 plasmids, selected on media lacking histidine and uracil, grown overnight in liquid media lacking just histidine, and then plated on media lacking histidine and containing 5-fluoroorotic acid  to select for cells that had lost the pRS316-phs-HSP104 plasmid. Colonies that were His+ ura− were used for further analysis.The [PSI+] yeast strain was subjected to EMS mutagenesis as previously described HSP104 by back-crossing to an hsp104Δ strain and analyzing the progeny for segregation of the prion-dependent nonsense suppression phenotype. Genomic DNA was PCR amplified and sequenced to identify the point mutations in HSP104.The strong  cells), translation termination is efficient, and the ade1-14 colonies appear red in color and do not grow on media lacking adenine. Conversely, when Sup35 is in a prion state, it is aggregated and less functional, and the [PSI+] colonies are Ade+ (light pink in color on YPD and able to grow on media lacking adenine). From our screen, we identified a candidate that caused the [PSI+] cells to change from a light pink phenotype to a sectoring colony color phenotype  as compared to wild type HSP104 cells in which loss of [PSI+] is rarely observed  from its native promoter and as the only copy of Hsp104  tolerant . Conversolerance . Interes strains . This lo7D cells .hsp104-D434A cells resembled the hsp104Δ strain harboring a vector only, in that there appeared to be no increase in the amount of resolubilized luciferase over time . We first noticed that hsp104-D434A had a dominant curing effect and resulted in red [psi−] diploids  with the haploids. We found that hsp104-V426I, hsp104-V426C, and hsp104-K480C cells still maintained aggregates of Sup35, while hsp104-D434A cells did not  or multiple-dot (m.d.) pattern of fluorescence that describes the appearance of Rnq1-GFP aggregates in [RNQ+] cells RNQ+] variants that harbor the s.d. fluorescence pattern can facilitate the induction of [PSI+] at low, medium, high, and very high levels upon Sup35 over-expression. Only one established m.d. variant of [RNQ+] has been characterized and it exhibits a high rate of [PSI+] induction. We utilized these five [RNQ+] variants to ascertain the effect of the M-domain mutants on conformational variants of [RNQ+].We next examined the ability of the M-domain mutants to propagate several different variants of the [hsp104Δ cells that were complemented by wild type HSP104 from a plasmid and propagated one of the [RNQ+] variants as a starting point to replace HSP104 with the M-domain mutants. We transformed the plasmids containing the Hsp104 M-domain mutants into these strains and subsequently selected for cells that eliminated wild type HSP104 by growing them on media that counter-selected against cells containing the URA3-marked HSP104 plasmid. Interestingly, we observed a differential effect of the mutants on the propagation of the [RNQ+] variants by both SDD-AGE analysis , hsp104-V426I, hsp104-V426C, hsp104-D434A, hsp104-K480C, or hsp104-Y507D from the native HSP104 promoter, or an empty vector control (EV), were grown at 30°C to an OD600 ∼1.0, lysed, and subjected to SDS-PAGE and western blot using anti-Hsp104 and anti-Pgk1 antibodies.(TIF)Click here for additional data file.