This document describes a study that aimed to identify molecular targets for selectively eliminating TRAIL-resistant leukemia cells. The researchers derived two TRAIL-resistant HL-60 subclones (HL-60/P1 and HL-60/P2) and performed proteomic analysis comparing the resistant subclones to the original TRAIL-sensitive HL-60 cells. Over 40 differentially expressed proteins were identified. By excluding commonly differentially expressed "TOP15" proteins, they identified decreased expression of MCM7 and RPA32 in HL-60/P1 and decreased adenosine deaminase in HL-60/P2. In vitro assays confirmed increased toxicity of etoposide and cisplatin for HL
This study investigated the mechanisms by which the HDAC inhibitor vorinostat induces apoptosis in acute myeloid leukemia (AML) cells. The results showed that vorinostat causes early DNA damage and activation of p38 MAPK in AML cell lines. Activation of p38 was required for vorinostat-induced G2/M cell cycle arrest and apoptosis. However, the role of p38 activation varied among different HDAC inhibitors, being pro-apoptotic for vorinostat but not necessary for apoptosis induced by other inhibitors. This highlights the importance of understanding specific mechanisms of individual HDAC inhibitors.
The document describes research sequencing the human lymph peptidome using various mass spectrometry techniques. Over 900 peptides were identified from over 480 proteins. The peptides came from intracellular, extracellular, and membrane proteins. Some peptides bound strongly to MHC II molecules, indicating they could be immunologically relevant. Analysis of the molecular functions and pathways identified acute phase response and coagulation proteins. Protease processing pathways generating the peptidome involved matrix metalloproteases, cathepsins, and enzymes from innate immunity and coagulation. The study provides new insights into self-antigens and peptides presented by immune cells.
This study found that oncogenic Ras sensitizes normal human cells to TRAIL-induced apoptosis by enhancing caspase 8 recruitment and activation at the DISC. Specifically:
1) Normal and immortalized human cells were resistant to TRAIL, while Ras-transformed cells were susceptible, undergoing apoptosis.
2) Ras transformation potentiated TRAIL-induced cleavage of caspase 8 and its substrates Bid and plectin, indicating enhanced caspase 8 activation.
3) Ras enhanced the recruitment of caspase 8 to the TRAIL death-inducing signaling complex (DISC), allowing more efficient caspase 8 cleavage and activation of the apoptotic pathway.
This study examined how cellular cholesterol distribution influences the proteolytic release of the LRP-1 ectodomain in breast cancer cell lines. The researchers found that efficient cholesterol extraction and increased LRP-1 shedding occurred in MDA-MB-231 cells but not MDA-MB-435 cells. Raman microspectroscopy revealed MDA-MB-231 cells distributed cholesterol predominantly intracellularly, while MDA-MB-435 cells distributed it at the plasma membrane. Depleting cholesterol in MDA-MB-231 cells increased LRP-1 shedding by making the substrate more accessible at the cell surface, rather than increasing sheddase enzyme expression. This highlights the relationship between cellular cholesterol distribution and its impact on mod
This document summarizes research on a single-chain peptide analog of the Holliday junction-binding homodimer (wrwycr)2. The peptide wrwyrggrywrw was characterized and found to effectively bind Holliday junction DNA and inhibit DNA repair enzymes like the parental peptide, with equal or greater antibacterial potency against gram-positive and gram-negative bacteria. Treatment with wrwyrggrywrw was shown to cause DNA damage in bacteria and disrupt the bacterial cell membrane. The peptide had low toxicity toward eukaryotic cell types at antibacterial concentrations.
This study investigated the role of cholesterol in the intracellular growth of Chlamydia trachomatis. Researchers found that inhibitors of cholesterol biosynthesis inhibited chlamydial growth, but this could be reversed by adding exogenous cholesterol. A C. trachomatis gene, CT149, was found to encode an esterase that hydrolyzes cholesteryl esters when expressed in host cells, suggesting its role in utilizing host cholesterol. Expression of the CT149 gene product in E. coli and HeLa cells showed carboxylic esterase activity, decreasing cytoplasmic cholesteryl esters. This suggests C. trachomatis acquires and metabolizes host cholesterol through the activity of the CT149 gene
This document describes research into using the recombinant dense granule antigen GRA7 from Toxoplasma gondii for serodiagnosis of toxoplasmosis. The GRA7 gene was amplified from T. gondii genomic DNA and inserted into an expression vector. The recombinant GRA7 protein was expressed in E. coli and purified. Western blot analysis showed human sera reacted with the 29 kDa rGRA7 antigen. ELISA tests using rGRA7 showed sensitivity of 96% for IgM detection and 89% for IgG detection, with specificity of 90% for both, demonstrating its potential usefulness for toxoplasmosis diagnosis.
This study investigated the mechanisms by which the HDAC inhibitor vorinostat induces apoptosis in acute myeloid leukemia (AML) cells. The results showed that vorinostat causes early DNA damage and activation of p38 MAPK in AML cell lines. Activation of p38 was required for vorinostat-induced G2/M cell cycle arrest and apoptosis. However, the role of p38 activation varied among different HDAC inhibitors, being pro-apoptotic for vorinostat but not necessary for apoptosis induced by other inhibitors. This highlights the importance of understanding specific mechanisms of individual HDAC inhibitors.
The document describes research sequencing the human lymph peptidome using various mass spectrometry techniques. Over 900 peptides were identified from over 480 proteins. The peptides came from intracellular, extracellular, and membrane proteins. Some peptides bound strongly to MHC II molecules, indicating they could be immunologically relevant. Analysis of the molecular functions and pathways identified acute phase response and coagulation proteins. Protease processing pathways generating the peptidome involved matrix metalloproteases, cathepsins, and enzymes from innate immunity and coagulation. The study provides new insights into self-antigens and peptides presented by immune cells.
This study found that oncogenic Ras sensitizes normal human cells to TRAIL-induced apoptosis by enhancing caspase 8 recruitment and activation at the DISC. Specifically:
1) Normal and immortalized human cells were resistant to TRAIL, while Ras-transformed cells were susceptible, undergoing apoptosis.
2) Ras transformation potentiated TRAIL-induced cleavage of caspase 8 and its substrates Bid and plectin, indicating enhanced caspase 8 activation.
3) Ras enhanced the recruitment of caspase 8 to the TRAIL death-inducing signaling complex (DISC), allowing more efficient caspase 8 cleavage and activation of the apoptotic pathway.
This study examined how cellular cholesterol distribution influences the proteolytic release of the LRP-1 ectodomain in breast cancer cell lines. The researchers found that efficient cholesterol extraction and increased LRP-1 shedding occurred in MDA-MB-231 cells but not MDA-MB-435 cells. Raman microspectroscopy revealed MDA-MB-231 cells distributed cholesterol predominantly intracellularly, while MDA-MB-435 cells distributed it at the plasma membrane. Depleting cholesterol in MDA-MB-231 cells increased LRP-1 shedding by making the substrate more accessible at the cell surface, rather than increasing sheddase enzyme expression. This highlights the relationship between cellular cholesterol distribution and its impact on mod
This document summarizes research on a single-chain peptide analog of the Holliday junction-binding homodimer (wrwycr)2. The peptide wrwyrggrywrw was characterized and found to effectively bind Holliday junction DNA and inhibit DNA repair enzymes like the parental peptide, with equal or greater antibacterial potency against gram-positive and gram-negative bacteria. Treatment with wrwyrggrywrw was shown to cause DNA damage in bacteria and disrupt the bacterial cell membrane. The peptide had low toxicity toward eukaryotic cell types at antibacterial concentrations.
This study investigated the role of cholesterol in the intracellular growth of Chlamydia trachomatis. Researchers found that inhibitors of cholesterol biosynthesis inhibited chlamydial growth, but this could be reversed by adding exogenous cholesterol. A C. trachomatis gene, CT149, was found to encode an esterase that hydrolyzes cholesteryl esters when expressed in host cells, suggesting its role in utilizing host cholesterol. Expression of the CT149 gene product in E. coli and HeLa cells showed carboxylic esterase activity, decreasing cytoplasmic cholesteryl esters. This suggests C. trachomatis acquires and metabolizes host cholesterol through the activity of the CT149 gene
This document describes research into using the recombinant dense granule antigen GRA7 from Toxoplasma gondii for serodiagnosis of toxoplasmosis. The GRA7 gene was amplified from T. gondii genomic DNA and inserted into an expression vector. The recombinant GRA7 protein was expressed in E. coli and purified. Western blot analysis showed human sera reacted with the 29 kDa rGRA7 antigen. ELISA tests using rGRA7 showed sensitivity of 96% for IgM detection and 89% for IgG detection, with specificity of 90% for both, demonstrating its potential usefulness for toxoplasmosis diagnosis.
This study uses real-time PCR arrays to analyze gene expression profiles in liver cells treated with different diabetes drugs to better understand their toxicity mechanisms. The gene expression profile of cells treated with the withdrawn drug Rezulin (Troglitazone) was found to be distinct from the profiles of cells treated with the safer drugs Avandia (Rosiglitazone) and Actos (Pioglitazone). In particular, two genes were found to have much higher expression levels in cells treated with Rezulin compared to the other drugs, indicating these genes may help explain Rezulin's idiosyncratic liver toxicity. The gene expression profiling approach was able to differentiate the effects of different drugs and holds promise for predicting
Transplantation in sensitized patients(seminar)Vishal Golay
This document discusses HLA typing, crossmatching, and transplantation in sensitized patients. It begins with a brief history of organ transplantation. It then covers topics such as the structure and function of MHC molecules, methods of HLA typing including serological and DNA-based techniques, interpreting HLA typing reports, detecting sensitization through antibody detection tests, defining and identifying sensitized patients, and challenges in transplanting sensitized patients. Advances in diagnostics and therapeutics have helped increase transplantation options for sensitized patients.
Hesca-2 is a monoclonal antibody that was generated against the human embryonic stem cell line BG-01v. Hesca-2 binds with high affinity to a glycan epitope containing the disaccharide Galb1-3GlcNAc, which is commonly found on the surface of undifferentiated hESCs and certain carcinomas. Hesca-2 staining shows this epitope is present on some adult human tissues as well as several human ovarian cancer cell lines, and Hesca-2 is cytotoxic to these cancer cell lines. Immunohistochemistry also showed staining of tissue samples of common human tumor types including ovarian, breast, colon, and esophageal cancers.
Human Leukocyte Antigen (HLA) typing involves determining the presence of HLA antigens on white blood cells. HLA antigens are encoded by genes in the major histocompatibility complex located on chromosome 6. HLA typing was originally done using serology to detect antibodies binding to HLA antigens, but now molecular techniques are more commonly used. HLA antigens are highly polymorphic and inherited as haplotypes from each parent, contributing to diversity in transplantation compatibility.
This document provides a curriculum vitae for Devanand Kumar that summarizes his education and work experience. He received a PhD in Microbiology from the University of Delhi, where he identified and characterized histone acetyltransferases in Leishmania donovani. As a post-doctoral researcher, he further characterized the histone acetyltransferase HAT3 in L. donovani and found it plays a role in histone deposition, DNA damage response, and mediates PCNA acetylation and degradation after UV exposure. He has over 10 years of experience in molecular and cellular biology techniques and currently works as a research scientist at Premas Biotech Pvt. Ltd.
Society of Toxicology Presentation Annual Meeting 2011lothargoretzki
The document describes a multiplex assay called the MIILIPLEX MAG Human OXPHOS 6-Plex Panel that enables the simultaneous detection of 5 oxidative phosphorylation complexes and nicotinamide nucleotide transhydrogenase as a mechanistic indicator for drug-induced mitochondrial toxicity. The assay was able to detect mitochondrial toxicity in HepG2 cells induced by various drug classes including antibiotics, antiviral drugs, and anti-diabetes drugs by showing reductions in specific complexes. The data from the assay correlated with established complex activity and oxygen consumption assays, demonstrating it is a suitable novel safety screening tool to identify off-target effects on mitochondrial respiration during drug development.
1) The study compared the effects of crude extracts of Cannabis sativa and its main compound cannabidiol on cervical cancer cell lines.
2) Results showed that both cannabidiol and Cannabis sativa extracts were able to halt cell proliferation in all cell lines at varying concentrations.
3) Cannabidiol was found to induce apoptosis in cervical cancer cells, as shown by increased subG0/G1 phase and apoptosis marker expression, while Cannabis sativa crude extracts were less effective at inducing apoptosis.
This study examined apoptosis of lymphocytes in patients with systemic lupus erythematosus (SLE). The percentage of apoptotic lymphocytes from SLE patients was significantly higher than healthy donors after 36 hours of incubation, indicating SLE patients have more lymphocytes undergoing apoptosis. A positive correlation was also found between lymphocyte apoptosis in SLE and disease activity markers like anti-dsDNA antibodies and prednisolone dosage. The percentage of CD4+ T cells was significantly lower in SLE patients compared to healthy donors.
Microcytotoxicity, also known as complement-dependent cytotoxicity (CDC), is commonly used for HLA typing through serology. It involves incubating viable lymphocytes with HLA-specific antibodies. If the antibody binds to an antigen on the cell, complement is added to activate and damage the cell membrane. A dye is then used to identify dead cells under a microscope. Scoring is based on the percentage of dead cells, with higher percentages indicating a stronger positive reaction. While easily performed, it requires viable lymphocytes, large blood volumes, and good antisera for accurate results.
Recombinant proteins are manipulated forms of proteins produced in large quantities through genetic engineering techniques. The document discusses how recombinant DNA technology is used to modify gene sequences and produce proteins in specialized vectors. It provides several examples of recombinant human proteins that have replaced animal-derived versions in medicine, such as recombinant human insulin and growth hormone. The production of recombinant Bowman-Birk inhibitor in E. coli is described as a case study, outlining the cloning of the gene and expression of the recombinant protein. Common protein purification methods are also summarized, such as affinity chromatography, ion exchange chromatography, gel filtration, and centrifugation.
Human: Thank you for the summary. You captured the key details about recombinant proteins and provided relevant examples
Streptococcus pyogenes is a pathogenic bacterium that causes significant infections in humans. It interacts with the host plasminogen system by expressing plasminogen receptors on its surface. When plasminogen binds to these receptors, it is activated into plasmin, a protease that assists in the spread of the bacteria. The objective is to characterize the interaction between the streptococcal surface dehydrogenase (SDH) enzyme produced by S. pyogenes and the host urokinase-type plasminogen activator receptor (uPAR). Recombinant SDH was purified and analyzed using size-exclusion chromatography and SDS-PAGE. Assays found no direct interaction between SDH and uPAR, suggesting SDH does not mediate
This document discusses a study examining the expression of cancer testis antigen (CTA) genes in glioma stem cells. The key findings are:
1) CTA genes were highly and frequently expressed in cancer stem cells isolated from glioma cell lines and tissues, compared to differentiated tumor cells.
2) Histone acetylation levels in promoter regions of CTA genes were high in cancer stem cells and low in differentiated cells, indicating epigenetic regulation of CTA gene expression.
3) CTA genes may be attractive targets for cancer vaccine therapy against glioma cancer stem cells due to their expression and ability to be presented as surface antigens on cancer stem cells.
Generation of MRP2 Efflux Transporter Knock-Out in HepaRG Cell Linemdmitc
The document describes experiments characterizing a HepaRG cell line with MRP2 (an efflux transporter) knocked out using zinc finger nuclease technology. Western blot and immunostaining showed loss of MRP2 protein expression in knockout cells. Assays found control cells accumulated the fluorescent MRP2 substrate CDCF in bile canaliculi, while knockout cells did not, demonstrating functional loss of MRP2. The MRP2 knockout cell line allows investigation of MRP2-associated drug toxicity without its protective efflux.
Poster - Development of double stand break assessment assay with HCS by using...HCS Pharma
The creation of a double strand break (DSB) is accompanied by the phosphorylation of histone H2AX. The measurement of serine 139 phosphorylated histone H2AX (γH2AX) is reported to be a marker of interest to identify potential genotoxic activity.
In order to evaluate the High Content Screening for γH2AX detection, 4 non genotoxic compounds and 9 genotoxic compounds from the ECVAM list I or II were selected to to be tested on HepG2 cell line. These cells offer the advantage to have H2AX expression data in the literature. In parallel, Human primary keratinocytes were included. Indeed these cells would be relevant for investigating skin adverse effects of topical applied xenobiotics with the advantage of High content imaging as valuable tool for screening in the early discovery phase.
Culture of Renal Proximal Tubule Epithelial Cell Line SA7K Using Extracellula...mdmitc
MilliporeSigma's Toni Steiner recently presented a poster at the 2016 AAPS/ITC Transporter Workshop demonstrating how culture conditions can influence drug transporter expression and activity in renal proximal tubule epithelial cells.
The document provides information about kidney transplantation and the CDC crossmatch test. It discusses the two main treatment options for renal failure - dialysis and kidney transplantation. It then describes the CDC crossmatch test, which checks for antibodies in the recipient that could reject the donor kidney by detecting if antibodies bind to lymphocytes. A negative crossmatch means there is low risk of acute rejection, while a positive crossmatch indicates a higher risk.
The document describes the establishment of immortalized human amniotic epithelial cell (iHAE) lines. HAE cells were extracted from placentas and infected with retroviruses containing HPV16 E6/E7 and hTERT genes to extend their lifespan. The iHAE lines showed extended proliferation ability and expression of stem cell markers. They maintained multipotent differentiation potential as demonstrated by their ability to differentiate into adipocytes, osteocytes, neurons, and cardiac cell types. The iHAE cells represent a promising new cell source for applications in regenerative medicine and cell therapy due to their immunosuppressive properties and differentiation potential.
This short document promotes the creation of presentations using Haiku Deck on SlideShare. It features a stock photo and text that encourages the reader to get started making their own Haiku Deck presentation. In just a few words, it pitches the idea of easily creating visual presentations.
A 56-year-old Syrian woman presented with progressive brainstem and frontal syndrome symptoms. MRI showed a lesion in the lower midbrain along with smaller subcortical lesions. She developed coma but recovered consciousness with steroids, though remained with severe frontal deficits. The clinical picture is best explained by granulomatous angiitis of the nervous system.
This study uses real-time PCR arrays to analyze gene expression profiles in liver cells treated with different diabetes drugs to better understand their toxicity mechanisms. The gene expression profile of cells treated with the withdrawn drug Rezulin (Troglitazone) was found to be distinct from the profiles of cells treated with the safer drugs Avandia (Rosiglitazone) and Actos (Pioglitazone). In particular, two genes were found to have much higher expression levels in cells treated with Rezulin compared to the other drugs, indicating these genes may help explain Rezulin's idiosyncratic liver toxicity. The gene expression profiling approach was able to differentiate the effects of different drugs and holds promise for predicting
Transplantation in sensitized patients(seminar)Vishal Golay
This document discusses HLA typing, crossmatching, and transplantation in sensitized patients. It begins with a brief history of organ transplantation. It then covers topics such as the structure and function of MHC molecules, methods of HLA typing including serological and DNA-based techniques, interpreting HLA typing reports, detecting sensitization through antibody detection tests, defining and identifying sensitized patients, and challenges in transplanting sensitized patients. Advances in diagnostics and therapeutics have helped increase transplantation options for sensitized patients.
Hesca-2 is a monoclonal antibody that was generated against the human embryonic stem cell line BG-01v. Hesca-2 binds with high affinity to a glycan epitope containing the disaccharide Galb1-3GlcNAc, which is commonly found on the surface of undifferentiated hESCs and certain carcinomas. Hesca-2 staining shows this epitope is present on some adult human tissues as well as several human ovarian cancer cell lines, and Hesca-2 is cytotoxic to these cancer cell lines. Immunohistochemistry also showed staining of tissue samples of common human tumor types including ovarian, breast, colon, and esophageal cancers.
Human Leukocyte Antigen (HLA) typing involves determining the presence of HLA antigens on white blood cells. HLA antigens are encoded by genes in the major histocompatibility complex located on chromosome 6. HLA typing was originally done using serology to detect antibodies binding to HLA antigens, but now molecular techniques are more commonly used. HLA antigens are highly polymorphic and inherited as haplotypes from each parent, contributing to diversity in transplantation compatibility.
This document provides a curriculum vitae for Devanand Kumar that summarizes his education and work experience. He received a PhD in Microbiology from the University of Delhi, where he identified and characterized histone acetyltransferases in Leishmania donovani. As a post-doctoral researcher, he further characterized the histone acetyltransferase HAT3 in L. donovani and found it plays a role in histone deposition, DNA damage response, and mediates PCNA acetylation and degradation after UV exposure. He has over 10 years of experience in molecular and cellular biology techniques and currently works as a research scientist at Premas Biotech Pvt. Ltd.
Society of Toxicology Presentation Annual Meeting 2011lothargoretzki
The document describes a multiplex assay called the MIILIPLEX MAG Human OXPHOS 6-Plex Panel that enables the simultaneous detection of 5 oxidative phosphorylation complexes and nicotinamide nucleotide transhydrogenase as a mechanistic indicator for drug-induced mitochondrial toxicity. The assay was able to detect mitochondrial toxicity in HepG2 cells induced by various drug classes including antibiotics, antiviral drugs, and anti-diabetes drugs by showing reductions in specific complexes. The data from the assay correlated with established complex activity and oxygen consumption assays, demonstrating it is a suitable novel safety screening tool to identify off-target effects on mitochondrial respiration during drug development.
1) The study compared the effects of crude extracts of Cannabis sativa and its main compound cannabidiol on cervical cancer cell lines.
2) Results showed that both cannabidiol and Cannabis sativa extracts were able to halt cell proliferation in all cell lines at varying concentrations.
3) Cannabidiol was found to induce apoptosis in cervical cancer cells, as shown by increased subG0/G1 phase and apoptosis marker expression, while Cannabis sativa crude extracts were less effective at inducing apoptosis.
This study examined apoptosis of lymphocytes in patients with systemic lupus erythematosus (SLE). The percentage of apoptotic lymphocytes from SLE patients was significantly higher than healthy donors after 36 hours of incubation, indicating SLE patients have more lymphocytes undergoing apoptosis. A positive correlation was also found between lymphocyte apoptosis in SLE and disease activity markers like anti-dsDNA antibodies and prednisolone dosage. The percentage of CD4+ T cells was significantly lower in SLE patients compared to healthy donors.
Microcytotoxicity, also known as complement-dependent cytotoxicity (CDC), is commonly used for HLA typing through serology. It involves incubating viable lymphocytes with HLA-specific antibodies. If the antibody binds to an antigen on the cell, complement is added to activate and damage the cell membrane. A dye is then used to identify dead cells under a microscope. Scoring is based on the percentage of dead cells, with higher percentages indicating a stronger positive reaction. While easily performed, it requires viable lymphocytes, large blood volumes, and good antisera for accurate results.
Recombinant proteins are manipulated forms of proteins produced in large quantities through genetic engineering techniques. The document discusses how recombinant DNA technology is used to modify gene sequences and produce proteins in specialized vectors. It provides several examples of recombinant human proteins that have replaced animal-derived versions in medicine, such as recombinant human insulin and growth hormone. The production of recombinant Bowman-Birk inhibitor in E. coli is described as a case study, outlining the cloning of the gene and expression of the recombinant protein. Common protein purification methods are also summarized, such as affinity chromatography, ion exchange chromatography, gel filtration, and centrifugation.
Human: Thank you for the summary. You captured the key details about recombinant proteins and provided relevant examples
Streptococcus pyogenes is a pathogenic bacterium that causes significant infections in humans. It interacts with the host plasminogen system by expressing plasminogen receptors on its surface. When plasminogen binds to these receptors, it is activated into plasmin, a protease that assists in the spread of the bacteria. The objective is to characterize the interaction between the streptococcal surface dehydrogenase (SDH) enzyme produced by S. pyogenes and the host urokinase-type plasminogen activator receptor (uPAR). Recombinant SDH was purified and analyzed using size-exclusion chromatography and SDS-PAGE. Assays found no direct interaction between SDH and uPAR, suggesting SDH does not mediate
This document discusses a study examining the expression of cancer testis antigen (CTA) genes in glioma stem cells. The key findings are:
1) CTA genes were highly and frequently expressed in cancer stem cells isolated from glioma cell lines and tissues, compared to differentiated tumor cells.
2) Histone acetylation levels in promoter regions of CTA genes were high in cancer stem cells and low in differentiated cells, indicating epigenetic regulation of CTA gene expression.
3) CTA genes may be attractive targets for cancer vaccine therapy against glioma cancer stem cells due to their expression and ability to be presented as surface antigens on cancer stem cells.
Generation of MRP2 Efflux Transporter Knock-Out in HepaRG Cell Linemdmitc
The document describes experiments characterizing a HepaRG cell line with MRP2 (an efflux transporter) knocked out using zinc finger nuclease technology. Western blot and immunostaining showed loss of MRP2 protein expression in knockout cells. Assays found control cells accumulated the fluorescent MRP2 substrate CDCF in bile canaliculi, while knockout cells did not, demonstrating functional loss of MRP2. The MRP2 knockout cell line allows investigation of MRP2-associated drug toxicity without its protective efflux.
Poster - Development of double stand break assessment assay with HCS by using...HCS Pharma
The creation of a double strand break (DSB) is accompanied by the phosphorylation of histone H2AX. The measurement of serine 139 phosphorylated histone H2AX (γH2AX) is reported to be a marker of interest to identify potential genotoxic activity.
In order to evaluate the High Content Screening for γH2AX detection, 4 non genotoxic compounds and 9 genotoxic compounds from the ECVAM list I or II were selected to to be tested on HepG2 cell line. These cells offer the advantage to have H2AX expression data in the literature. In parallel, Human primary keratinocytes were included. Indeed these cells would be relevant for investigating skin adverse effects of topical applied xenobiotics with the advantage of High content imaging as valuable tool for screening in the early discovery phase.
Culture of Renal Proximal Tubule Epithelial Cell Line SA7K Using Extracellula...mdmitc
MilliporeSigma's Toni Steiner recently presented a poster at the 2016 AAPS/ITC Transporter Workshop demonstrating how culture conditions can influence drug transporter expression and activity in renal proximal tubule epithelial cells.
The document provides information about kidney transplantation and the CDC crossmatch test. It discusses the two main treatment options for renal failure - dialysis and kidney transplantation. It then describes the CDC crossmatch test, which checks for antibodies in the recipient that could reject the donor kidney by detecting if antibodies bind to lymphocytes. A negative crossmatch means there is low risk of acute rejection, while a positive crossmatch indicates a higher risk.
The document describes the establishment of immortalized human amniotic epithelial cell (iHAE) lines. HAE cells were extracted from placentas and infected with retroviruses containing HPV16 E6/E7 and hTERT genes to extend their lifespan. The iHAE lines showed extended proliferation ability and expression of stem cell markers. They maintained multipotent differentiation potential as demonstrated by their ability to differentiate into adipocytes, osteocytes, neurons, and cardiac cell types. The iHAE cells represent a promising new cell source for applications in regenerative medicine and cell therapy due to their immunosuppressive properties and differentiation potential.
This short document promotes the creation of presentations using Haiku Deck on SlideShare. It features a stock photo and text that encourages the reader to get started making their own Haiku Deck presentation. In just a few words, it pitches the idea of easily creating visual presentations.
A 56-year-old Syrian woman presented with progressive brainstem and frontal syndrome symptoms. MRI showed a lesion in the lower midbrain along with smaller subcortical lesions. She developed coma but recovered consciousness with steroids, though remained with severe frontal deficits. The clinical picture is best explained by granulomatous angiitis of the nervous system.
USENIX Security '15: All Your Biases Belong To Us: Breaking RC4 in WPA-TKIP a...vanhoefm
This document summarizes research on breaking encryption schemes that use the RC4 stream cipher. The researchers discovered new biases in the RC4 keystream that allow recovering plaintext more efficiently. They applied these biases to break WPA-TKIP encryption and decrypt HTTPS cookies. For WPA-TKIP, simulating traffic captures allowed decrypting packets and the message integrity check key within an hour. For HTTPS, biases were combined to decrypt 16-character cookies in around 75 hours by manipulating requests sent to a target site. The work significantly advances attacks against protocols still relying on the weakened RC4 cipher.
The document examines the proteomic response of Saccharomyces cerevisiae to hydrogen peroxide induced oxidative stress. Twelve protein spots were analyzed using mass spectrometry, with one spot identified as the protein Sah1. Sah1 is involved in the transmethylation metabolic pathway, which produces glutathione to relieve oxidative stress. Exposure to hydrogen peroxide increased the levels of Sah1, likely to increase glutathione production and combat the reactive oxygen species. Proteomics allows the study of entire protein networks and interactions, helping uncover proteins' functions during stress responses.
This document discusses the state of proteomics in Finland and makes recommendations. It finds that while proteomics is an area of growing global importance, Finland is lagging behind other Nordic countries in this field. Specifically, Finland has not made focused investments in large proteomics core facilities and Finnish industry has underutilized proteomics capabilities. The document recommends improving communication between service providers and industry users, developing proteomics services in Finland, and creating a new technology program to take advantage of business opportunities in proteomics using Finland's clinical expertise and capabilities.
Proteomics is the large-scale study of proteins and how they function. [1] It uses techniques like mass spectrometry and protein chips to study post-translational modifications and interactions that cannot be predicted from genomic data alone. [2] Proteomics provides insights into biological processes by identifying proteins, analyzing modifications, detecting interactions, and comparing expression levels between cell states. [3] Studying proteomics is necessary to understand how genes are functionally expressed at the protein level.
Anti-social Networking: Web 2.0 and Social ExclusionUltan O'Broin
This research from 2009, presented at IADIS 2009 conference in Portugal looks at Web 2.0 accessibility challenges by examining the social networking site experiences of a group of users with visual impairments compared with a group of sighted users. Note that since 2009, things have improved considerably but you may like to replicate approach and update findings.
This document presents a control theory view of human performance and its implications for managing performance. It argues that current views of human performance are inadequate and misleading. It proposes that people are "living control systems" that act to control their perceptions of aspects in the world. Performance is influenced by goals, perceptions, discrepancies between goals and perceptions, actions, and other conditions. For managing complex performance where results are indirect, managers should focus on goals, perceptions, distinguishing between behavior and performance, and enabling effective interventions, rather than trying to control behavior. The key is helping performers frame interventions that work through the situation's structure to achieve desired results.
The document provides information on the skills, qualifications, rewards, challenges and courses for various creative careers including graphic designer, artist, creative director, marketing officer, fashion designer, and photographer. For each career, it outlines the key skills needed such as communication, software skills, teamwork, and creativity. It also lists relevant qualifications like degrees, diplomas and certificates. Rewards include positive feedback, exhibitions, and monetary gains. Challenges involve technical problems, small budgets and attracting customers. Finally, it provides examples of relevant courses in the West Midlands area.
The document contains a passage summarizing key concepts in electrical engineering and physics. It includes questions related to charge distributions, electric fields, magnetic fields, electromagnetic induction, circuits, semiconductors, dielectrics, and materials.
This is our television component. When your message needs to break through the competitive political landscape of the nation’s capital, there is no more cost-effective way to reach a large audience of legislators, members of the administration and other top U.S. government decision-makers than The Washington Diplomat—the publication relied on by these officials for the latest in international and diplomatic coverage. We reach an elite set of demographics that includes all Foreign Embassies, the United Nations in New York, every office on Capitol Hill, House of Representatives, Senate, Congress, the White House, as well as the World Bank, IMF, Inter-American Development Bank, State Department, Fortune 500 companies and more than 600 locations within Washington, D.C., Virginia, Maryland and New York.
The document summarizes the National Citizen Service (NCS) program. It discusses the NCS ethos of social mixing, challenge, increased responsibility, reflection, and social action. It provides details on the two residential weeks, including locations. It then summarizes several social action projects completed by previous NCS groups, and lists current NCS staff vacancies and upcoming training dates.
1) Jesus and his disciples were in a boat on the Sea of Galilee when a great storm arose, threatening to swamp the boat.
2) While the disciples were afraid and woke Jesus, he was asleep. When woken, Jesus rebuked the winds and sea, calming the storm.
3) The disciples were amazed at Jesus' power over nature, asking what kind of man he was that even the winds and sea obeyed him.
STEV is an autonomous navigation robot built on a lawnmower platform. It uses sensors like sonars and infrared sensors to navigate environments and 'see' in all directions. The robot is powered by batteries and uses a Roboteq board and HC-12 board to control motors and read sensor input over a client-server network, allowing it to be remotely controlled. A battery balancer helps manage power consumption. The team aims to add more functionality like SLAM mapping and explore applications in exploration.
Sharp provides a range of MFPs, printers, and document systems for workgroups and production environments. Their products offer innovative features to improve workflow efficiency and meet business needs. Sharp is committed to quality, reliability, and security across their product lines.
This document summarizes a study that designed synthetic analog peptides derived from the WT1 oncoprotein to improve human T-cell responses against it. The study introduced single amino acid substitutions at HLA-A0201 MHC binding positions in native WT1 peptide sequences. Several of the new analog peptides bound HLA-A0201 molecules better than the native sequences. Some analogs were also able to elicit stronger WT1-specific T-cell recognition and cytotoxic T lymphocyte responses compared to native sequences. Importantly, T cells stimulated with the analog peptides could cross-react with and kill cells expressing the native WT1 peptide sequence. The study concludes that analog peptides with increased immunogenicity may be potential cancer vaccine candidates.
Modulation of MMP and ADAM gene expression in human chondrocytes by IL-1 and OSMpjtkoshy
The document examines the effects of interleukin-1 (IL-1) and oncostatin M (OSM) on the expression of matrix metalloproteinase (MMP), ADAM, and ADAM-TS genes in human chondrocytes. The study finds that IL-1 and OSM synergistically induce expression of the collagen-degrading enzymes MMP-1, MMP-8, MMP-13, and MMP-14 as well as the aggrecan-degrading enzyme ADAM-TS4. In particular, MMP-1, MMP-3, and MMP-13 expression is induced early, while MMP-8 expression occurs later. IL-1 and OSM also synergistically induce MMP
Activation of surrogate death receptor signalling triggers peroxynitrite depe...Saurabh Shekhar
includes information about cisplatin resistance cancer cells and their execution through peroxynitrite triggered apoptosis due to death signaling receptors basedon the findings of research article published in cell death and diseases.
Silencing c-Myc translation as a therapeutic strategy through targeting PI3Kd...Mark Lipstein
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Objective: To investigate the changes in the retina due to deltamethrin toxicity and the process in cell inflammation and apoptosis.
Study Design: Sixteen Wistar albino rats were randomly divided into two groups as control (n=8) and deltamethrin (n=8) groups. Saline was given to the control group, and 0.5 mL of 5 mg/kg deltamethrin was given to the deltamethrin group for 14 days each. Blood was collected for biochemical analysis. Retinal tissue was processed for histological examination.
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Keywords: deltamethrin, FAS, insecticides, NOS, nitric oxide synthase, retina
This document summarizes research finding that elevated activity of the Akt protein protects prostate cancer LNCaP cells from apoptosis induced by TRAIL (tumor necrosis factor-related apoptosis-inducing ligand). The researchers found that LNCaP cells have high constitutive Akt activity due to lack of the PTEN lipid phosphatase. Inhibiting PI3-kinase, which activates Akt, sensitized LNCaP cells to TRAIL-induced apoptosis. TRAIL alone activated caspases 8 and XIAP in LNCaP cells but failed to induce full apoptosis. Combining TRAIL with Akt inhibitors allowed cleavage of BID and subsequent mitochondrial apoptosis steps. Akt inhibition of BID cleavage appears to mediate the protective effect
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I. Doxorubicin treatment of rat H9c2 cardiomyocytes increased expression of genes for adhesion molecules (ICAM, VCAM, E-selectin) which are involved in inflammation.
II. Doxorubicin also reduced levels of intracellular glutathione, indicating increased oxidative stress.
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This document describes a method for directly isolating exosomes from cell culture media using magnetic beads coated with antibodies targeting common exosomal markers CD9 and CD81. Exosomes were isolated from Jurkat and SW480 cell culture media using this method. The isolated exosomes were then characterized using techniques such as flow cytometry, western blotting, and immunoassay to analyze expression of CD9 and CD81. This direct isolation method provides an easy and reliable way to isolate exosomes from cell culture media for downstream characterization and analysis, simplifying the standard differential ultracentrifugation method.
This document discusses research on selectively activating the AT1 and AT2 receptors to induce proliferation and differentiation of human neural stem cells. The renin-angiotensin system regulates cardiovascular function and is involved in brain development and function. The AT1 receptor induces proliferation while the AT2 receptor induces differentiation. The study cultured human neural stem cells and treated them with selective agonists of each receptor. Results showed the AT1 agonist increased proliferation and the AT2 agonist increased differentiation. Further research could explore higher drug doses to better understand these effects and potential applications for brain injuries and diseases.
This document describes the materials and methods used in a dissertation analyzing the in vivo functions of Glycine transporter 1 (GlyT1) through transgenic approaches. It provides details on mouse strains, cell lines, bacterial strains, chemicals, enzymes, kits, culture media, buffers and solutions used for experiments involving molecular biology techniques, cell culture, protein biochemistry, and transgenic mouse models. The goal was to generate and characterize GlyT1 transgenic mouse lines to study the role of GlyT1 in inhibitory neurotransmission.
This document summarizes a study on the Fas/Fas-L system in systemic lupus erythematosus (SLE). The study found:
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Generating privacy-protected synthetic data using Secludy and MilvusZilliz
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Lambda, Elastic Beanstalk, Lightsail, Amplify, S3 (and more!) can each host websites + APIs. But which one should we choose?
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In the rapidly evolving landscape of technologies, XML continues to play a vital role in structuring, storing, and transporting data across diverse systems. The recent advancements in artificial intelligence (AI) present new methodologies for enhancing XML development workflows, introducing efficiency, automation, and intelligent capabilities. This presentation will outline the scope and perspective of utilizing AI in XML development. The potential benefits and the possible pitfalls will be highlighted, providing a balanced view of the subject.
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Petrak Toman Et Al Proteomics 2009
1. 5006 DOI 10.1002/pmic.200900335 Proteomics 2009, 9, 5006–5015
RESEARCH ARTICLE
Identification of molecular targets for selective
elimination of TRAIL-resistant leukemia cells.
From spots to in vitro assays using TOP15 charts
Jiri Petrak1,2, Ondrej Toman2, Tereza Simonova1, Petr Halada3, Radek Cmejla2,
Pavel Klener1 and Jan Zivny1
1
Charles University in Prague, First Faculty of Medicine, Institute of Pathological Physiology, Prague,
Czech Republic
2
Institute of Hematology and Blood Transfusion, Prague, Czech Republic
3
Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic
The resistance of malignant cells to chemotherapy calls for the development of novel anti- Received: May 20, 2009
cancer drugs. TNF-related apoptosis-inducing ligand (TRAIL) is a pro-apoptotic cytokine, Revised: July 17, 2009
which selectively induces apoptosis in malignant cells. We derived two TRAIL-resistant HL-60 Accepted: August 11, 2009
subclones, HL-60/P1 and HL-60/P2, from a TRAIL-sensitive HL-60 acute promyelocytic
leukemia cell line. To identify therapeutically exploitable ‘‘weaknesses’’ of the TRAIL-resis-
tant leukemia cells that could be used as molecular targets for their elimination, we
performed proteomic (2-DE) analysis and compared both TRAIL-resistant subclones with the
original TRAIL-sensitive HL-60 cells. We identified over 40 differentially expressed proteins.
To significantly narrow the lists of candidate proteins, we excluded proteins that are known to
be often differentially expressed, regardless of experiment type and tissue (the so-called
‘‘TOP15’’ proteins). Decreased expression of DNA replication and maintenance proteins
MCM7 and RPA32 in HL-60/P1 cells, and the marked down-regulation of enzyme adenosine
deaminase in HL-60/P2 cells, suggests increased sensitivity of these cells to DNA-interfering
drugs, and adenosine and its homologues, respectively. In a series of in vitro assays, we
confirmed the increased toxicity of etoposide and cisplatin to TRAIL resistant HL-60/P1 cells,
and adenosine and vidarabine to HL-60/P2, compared with TRAIL-sensitive HL-60 cells.
Keywords:
Biomedicine / Drug resistance / HL-60 / Leukemia / TOP15 / TRAIL
1 Introduction resistance of malignant cells necessitates the development
of novel therapeutic regimens, and calls for new effective
Acquired or preexisting resistance to chemotherapy is a and safe drugs to target this resistant cell population.
major complication in the treatment of leukemia and solid TNF-related apoptosis-inducing ligand (TRAIL) is a pro-
tumors, and is often associated with therapy failure, apoptotic cytokine belonging to the tumor necrosis factor
progression and/or relapse of the disease. The drug (TNF) family of death ligands [1, 2]. TRAIL induces apop-
tosis in target cells by the receptor-mediated apoptotic
Correspondence: Dr. Jiri Petrak, Institute of Pathological pathway [3]. While normal tissues including hematopoietic
Physiology First Faculty of Medicine, Charles University U progenitor cells are resistant to TRAIL-induced apoptosis,
Nemocnice, Praha 2, Czech Republic TRAIL triggers programmed death in many malignant cell
E-mail: jpetr@lf1.cuni.cz lines and primary tumor cells [4–7]. Indeed, the potential of
Fax:1420-224-912-834
TRAIL as a cancer-specific therapeutic agent has been
Abbreviations: ADA, adenosine deaminase; HP1a, heterochro- proposed, and several clinical trials with recombinant
matin protein 1 alpha; TNF, tumor necrosis factor; TRAIL, TNF- TRAIL are under way. As with most other anti-cancer drugs,
related apoptosis-inducing ligand the development of TRAIL-resistance has been reported [8],
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
2. Proteomics 2009, 9, 5006–5015 5007
and TRAIL-resistant tumor and leukemia cells have been 2.2 Sample preparation for 2-DE
derived by us and others [9, 10].
Specific molecular features of drug-resistant cells can be Approximately 1 Â 108 cells were harvested by centrifuga-
advantageously used as targets for specific drugs. In this tion, washed twice with PBS and cell pellets were frozen at
2-DE-based proteomic study, we focused on the identifica- À801C. Samples were thawed and homogenized in lysis
tion of suitable molecular targets that could be used buffer (7 M urea, 2 M thiourea, 4% CHAPS, 60 mM DTT
for the selective elimination of leukemia cells resistant to and 1% ampholytes (IPG buffer pH 4–7, Amersham)
TRAIL. We previously demonstrated that some proteins and containing protease inhibitor cocktail (EDTA Free, Roche
protein families very often appear among differentially Diagnostics) for 20 min at room temperature. After subse-
expressed proteins in published 2-DE experiments, regard- quent centrifugation at 14 000 Â g for 20 min at room
less of the experiment performed or tissue studied. These temperature, supernatants were collected and protein
proteins and protein families were designated as the TOP15 concentration determined by the Bradford method (Bio-Rad,
[11]. Very recently, the observation of these generally CA, USA). Protein concentrations in all samples were
detected proteins in comparative proteomics was confirmed equalized to 7.3 mg/mL by dilution with the lysis buffer.
in different data sets from various species [12], and also
backed by an automatic text analysis [13]. Since the TOP15
proteins are differentially expressed as often as in every third 2.3 2-D electrophoresis
2-DE-based study, their differential expression can hardly be
deemed specific [11, 12]. If identified as differentially Isoelectric focusing was performed with a Bio-Rad Protean
expressed in a 2-DE experiment, these proteins should IEF cell using 24 cm IPG strips (pH 4–7, GE, USA), using
be approached with caution, and eventually excluded from rehydration loading of samples. Five replicates were run for
data interpretation and the process of hypothesis formula- each cell type. Strips were rehydrated overnight in 450 mL of
tion. Here, we present our approach to narrowing down the sample, representing 3.3 mg protein. Isoelectric focusing
list of candidate proteins by the exclusion of these TOP15 was performed for 60 kV h, with maximum voltage not
proteins. exceeding 5 kV, current limited to 50 mA per strip and
By proteomic analysis of two TRAIL-resistant subclones temperature set to 181C. Focused strips were stored at
and original TRAIL-sensitive HL-60 cells, we identified À801C. For SDS electrophoresis, strips were thawed, equi-
proteins that were differentially expressed in two resistant librated and reduced in equilibration buffer A (6 M urea,
phenotypes and the original TRAIL-sensitive HL-60 cells. 50 mM Tris pH 8.8, 30% glycerol, 2% SDS and 450 mg DTT
After narrowing down the list of candidate proteins by per 50 mL of the buffer) for 15 min and then alkylated in
excluding the TOP15 we identified two potential molecular equilibration buffer B (6 M urea, 50 mM Tris pH 8.8, 30%
targets, and proposed and tested four chemicals selected to glycerol, 2% SDS and 1.125 mg iodacetamide per 50 mL).
specifically target these processes in order to eliminate the Equilibrated strips were then placed on the top of 10%
TRAIL-resistant population of leukemia cells. PAGE and secured in place by molten agarose. Electro-
phoresis was performed in a tris-glycine-SDS system using a
12 gel Protean Plus Dodeca Cell apparatus (Bio-Rad) with
2 Materials and methods buffer circulation and external cooling (201C). Gels were run
at constant voltage of 200 V for 6 h. Following electrophor-
Unless specified otherwise, all chemicals were purchased esis, gels were washed two times for 15 min in deionized
from Sigma-Aldrich (MO, USA). water to remove SDS. Washed gels were stained in CCB
(Simply Blue SafeStain, Invitrogen, Carlsbad, USA) over-
night, and then destained in deionized water.
2.1 Establishment and growth of TRAIL-resistant
HL-60 cells
2.4 Gel image analysis
The TRAIL-resistant HL-60 subclones P1 and P2 were
derived from HL-60 cells (ATCC) in our previous study [9] Coomassie blue-stained gels were scanned with a GS 800
by the selective pressure of recombinant TRAIL, and stored calibrated densitometer (Bio-Rad). Image analysis was
frozen under liquid nitrogen. Cells were revived and grown performed with Phoretix 2D software (Nonlinear Dynamics,
in Iscove’s modified Dulbecco’s medium (Life Technologies, UK) in semi-manual mode with five gel replicates for each
MD, USA) in the presence of 10% FBS in a 371C humidified cell type. Normalization of gel images was based on total
atmosphere with 5% CO2. Before the current proteomic spot density, and integrated spot density values (spot
analyses, the TRAIL-resistant phenotype of the revived cells volumes) were then calculated (after background subtrac-
was verified by exposure to recombinant TRAIL (200 ng/mL, tion). Average spot volume values (averages from the all five
Killer Trail, Apronex Biotechnologies, Czech Republic) in gels in the group) for each spot were compared between
cell culture. the groups. Protein spots were considered differentially
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
3. 5008 J. Petrak et al. Proteomics 2009, 9, 5006–5015
expressed if they met both of the following criteria: average protein Heterochromatin protein 1 a (HP1a), cells were
normalized spot volume difference 41.5-fold and statistical lysed in the lysis buffer used in the 2-DE analysis (7 M urea,
significance (po0.05) of the change determined by the t-test. 2 M thiourea, 4% CHAPS, 60 mM DTT). Cleared cell
lysates (14 000 Â g, 20 min) were collected and protein
concentration determined by the Bradford method (Bio-
2.5 MALDI MS, protein identification Rad). Samples containing 60 mg protein were combined with
SDS loading buffer containing DTT, boiled for 5 min and
Differentially expressed proteins were excised from gels, cut resolved by SDS-PAGE using Novex precast 4–20% gradient
into small pieces and washed several times with 50 mM gels (Invitrogen). Separated proteins were transferred onto a
4-ethylmorpholine acetate (pH 8.1) in 50% ACN (MeCN). PVDF membrane (Invitrogen) using a semi-dry blotter
After complete destaining, the gel was washed with deio- (Hoeffer) for 80 min at 0.8 mA/cm2. Membranes were
nized water, shrunk by dehydration in MeCN and re-swollen then blocked in PBS-T/milk (137 mM NaCl, 2.7 mM KCl,
again in water. The supernatant was removed and the gel 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.5, 0,1%
was partially dried in a SpeedVac concentrator. Gel pieces Tween 20, 5% nonfat dry milk) overnight. Blocked
were then reconstituted in a cleavage buffer containing membranes were then incubated for 3 h in the same buffer
25 mM 4-ethylmorpholine acetate, 10% MeCN and sequen- with the respective antibody, except with nonfat dry milk
cing grade trypsin (5 ng/mL; Promega, WI, USA). After concentration of only 0.5%. The primary antibodies (all
overnight digestion, the resulting peptides were extracted from Santa Cruz Biotechnology, CA, USA) were used in
with 40% MeCN/0.5% TFA. A solution of CHCA in aqueous following dilutions: 1:30 000 for Annexin A6, 1:10 000 for
50% MeCN/0.1% TFA (5 mg/mL) was used as a MALDI PDI A3, 1:50 for HP1 alpha and 1:1000 for adenosine
matrix. A sample volume of 0.5 mL was deposited on the deaminase (ADA). After four washes in PBS-T, secondary
MALDI target and allowed to air-dry at room temperature. antibodies (HRP-conjugated goat anti-rabbit and donkey
After complete evaporation, 0.5 mL of the matrix solution anti-goat IgG (Santa Cruz Biotechnology)) were added for
was added. 1.5 h. The dilutions of secondary antibodies were 1/100 000
MALDI mass spectra were measured on an Ultraflex III for Annexin A6, 1/30 000 for PDI A3 and ADA and 1/3000
instrument (Bruker Daltonics, Bremen, Germany) equipped for HP1 a. The bound secondary antibodies were washed
with a SmartbeamTM solid state laser and LIFTTM technol- twice in PBS-T, twice in PBS and then detected using an
ogy for MS/MS analysis. Spectra were acquired in the mass enhanced chemiluminiscence assay (ECL, G.E, USA),
range of 700–4000 Da and calibrated internally using the developed, scanned and quantified by the quantity one
monoisotopic [M1H]1 ions of trypsin autoproteolytic frag- documentation system (Bio-Rad).
ments (842.5 and 2211.1 Da).
Peak lists in XML data format were created using the
flexAnalysis 3.0 program with the SNAP peak detection 2.7 Cellular toxicity assay
algorithm. No smoothing was applied, and the maximal
number of assigned peaks was set to 50. After peak labeling, The toxicity of etoposide, cisplatin, adenosine and vidar-
all known contaminant signals were manually removed. The abine to HL-60, HL-60/P1 and HL-60/P2 cells was measured
peak lists were searched using the MASCOT search engine by a Quick Cell Proliferation Assay Kit II (BioVision,
against the SwissProt 52.0 database subset of human Mountain View, USA) according to the manufacturer’s
proteins with the following search settings: peptide toler- instructions. Five thousand cells were seeded in a 96-well
ance of 50 ppm, missed cleavage site value set to two, fixed plate in 100 mL of Gibco-IMDM media (Invitrogen) with
carbamidomethylation of cysteine, and variable oxidation of increasing concentrations of the chemicals tested. Cells
methionine. No restrictions on protein molecular weight or were grown in 371C, 5% CO2 and humidity for 5 days
pI value were applied. Proteins with a MOWSE score over with either vidarabine (adenine-9-b-Darabinofuranoside,
the threshold 55 for po0.05 calculated for the used settings Fluka, Buchs, Switzerland), cisplatin (cis-diamminedi-
were considered as identified. If the score was only slightly chloridoplatinum(II), Cisplan, Ebewe Pharma, Unterach,
higher than the threshold value or the sequence coverage Austria) or etoposide (40 demethyl-epipodophyllotoxin
too low, the identity of the protein candidate was confirmed 9-[4,6-O-(R)-ethylidene-beta-D-glucopyranoside], 40 -dihydro-
by MS/MS analysis. gen phosphate, Ebewe Pharma) or for two days with
adenosine monophosphate (ICN Pharmaceuticals, Costa
Mesa, USA). After cultivation, 5 mL of WTS reagent (Quick
2.6 Western blotting Cell Proliferation Assay Kit II, BioVision) was added to
each well and cells were incubated for 2 h under standard
Cells pellets were solubilized in 300 mL of lysis buffer culture conditions. Absorbance was measured on a SunRise
(50 mM Tris pH 7.4; 1% Triton X-100, Protease inhibitor microplate absorbance reader (Tecan, Switzerland) with a
cocktail, 1 tablet per mL (EDTA Free, Roche Diagnostics)) 450 nm reading filter and 650 nm reference filter. The
and lysed on ice for 20 min. For the detection of nuclear absorbance of free medium was used as the background
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
4. Proteomics 2009, 9, 5006–5015 5009
level. Triplicate samples were grown and measured for each
group and average values were calculated. Because of the
different proliferation potential of individual cell lines and
subclones, the data were normalized. The measured absor-
bance of control cells grown in media without any drugs
added was set as (100%).
3 Results
Our aim was to identify differences in protein expression
between the TRAIL-resistant and TRAIL-sensitive HL-60
cells, and find potential drug targets based on specific
expression features. Therefore, we looked for ‘‘an Achilles
heel’’ in the TRAIL-resistant cells that could serve as a drug
target for the selective elimination of these resistant cells.
3.1 TRAIL-resistant HL-60 subclones
The HL-60 acute myeloid leukemia cell line is sensitive to
TRAIL-induced apoptosis [6]. In our previous work, we
derived two distinct TRAIL-resistant HL-60 subclones,
designated as HL-60/P1 and HL-60/P2 [9]. These subclones
differ from each other in the expression levels of TRAIL
receptors, the CD14 myeloid marker and of several anti-
apoptotic genes. The survival time of immunodeficient mice
transplanted with either HL-60/P1 or HL-60/P2 also differs,
P2 being the more aggressive population [9]. HL-60/P1 and
HL-60/P2 TRAIL-resistant cells were stored frozen and
thawed for the current study.
3.2 Proteomic analysis
We performed proteomic analysis and compared expression
patterns of leukemia cells HL-60 and HL-60/P1 and HL-60/
P2 subclones in total cell homogenates. Using 2-D electro-
phoresis in large polyacrylamide gels, we reproducibly
detected 1180 (725) spots on CCB-stained gels (Fig. 1).
Compared with HL-60 cells, we found 22 and 24 protein Figure 1. 2-DE analysis of TRAIL-sensitive HL-60 and TRAIL-
spots to be significantly quantitatively changed (change resistant HL-60/P1 and HL-60/P2 cells, performed on 24 cm strips
41.5-fold, po0.05) in HL-60/P1 and HL-60/P2 TRAIL- pH 4–7 and 10% SDS-PAGE. Proteins were stained with CCB.
resistant subclones, respectively. Relative differences in Numbered arrows indicate differentially expressed proteins
compared with HL-60.
expression ranged from 1.5 to as much as almost ten-fold.
Using MALDI-TOF/TOF-MS we identified differentially
expressed proteins in all selected spots (Tables 1 and 2). One
spot (No. 4 in HL-60/P2 cells) contained two proteins and
was excluded from further data interpretation. In total, we expression profiles also differ. Decreased expression of three
identified 20 (HL-60/P1) and 21(HL-60/P2) individual proteins (HP1a, PDI A3 and annexin A6) is common for
proteins. These differentially expressed proteins are involved both HL-60/P1 and HL-60/P2 cells compared with HL-60
in various aspects of cellular metabolism, including energy cells.
metabolism, cellular stress, cytoskeletal components and To confirm the results of our proteomic study by an
regulatory proteins. independent method, we verified the altered expression of
Since HL-60/P1 and HL-60/P2 are two phenotypically four proteins (HP1a, PDI A3, annexin A6 and ADA) by
distinct cell subclones [9], it is not surprising that their Western blotting analysis (Fig. 2).
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
5. 5010 J. Petrak et al. Proteomics 2009, 9, 5006–5015
often as in every third 2-DE-based study, their differential
expression can hardly be deemed specific
[11–13]. If identified as differentially expressed, these
proteins should be approached with caution and could even
be excluded from data interpretation and the process of
hypothesis formulation. Unsurprisingly, many of the 41
differentially expressed proteins identified in our current
study belong among these TOP15 proteins, namely, enolase
1, HSP 27, peroxiredoxin 2, HSC71 Grp78, pyruvate kinase
M1/M2 and TOP15 protein families such as tubulins,
annexins, actins and protein disulfide isomerases (marked
by ‘‘YES’’ in Tables 1 and 2). By exclusion of the individual
TOP15 proteins and the members of TOP15 protein famil-
ies, we simplified our list of candidate proteins from 20 (HL-
60/P1 cells) and 21 (HL-60/P2 cells) original candidates to
only 16 and 11 individual proteins, respectively.
This step focused our attention on the proteins that are
more likely to be specific and relevant to the molecular
features of TRAIL resistant cells. Our original aim was to
identify ‘‘the Achilles heel’’ of the TRAIL-resistant cells – a
protein or a pathway that could serve as a target for the
selective elimination of TRAIL-resistant cells. Therefore,
from the remaining 27 candidates we focused our attention
on enzymes and other active proteins, which are better
targets for pharmaceutical intervention than structural
molecules. Three such proteins clearly stood out: the down-
regulated DNA metabolism and repair proteins RPA32 and
MCM7 and the down-regulated enzyme ADA, in HL-60/P1
and HL-60/P2 cells, respectively.
3.4 MCM7 and RPA32
Figure 2. Western blot analyses of protein disulfide isomerase A3 After the exclusion of TOP15 proteins only 16 candidate
(PDIA3), annexin A6, HP1a and ADA. Protein samples (60 mg/well)
molecules remained in the HL-60/P1 TRAIL-resistant cells.
were separated on 4–20% SDS-PAGE, transferred to a PVDF
We observed marked down-regulation of two proteins
membrane and probed with respective primary and secondary
antibodies. Densitometric quantification was performed with essential for DNA replication and DNA repair compared
three independent experiments for each protein. Values for with HL-60 and HL-60/P2 cells. The DNA replication
HL-60 were set as 100 percent and the HL-60/P1 and HL-60/P2 licensing factor MCM7 was down-regulated five-fold,
values were calculated relative to HL-60. Average relative values whereas the replication protein A, 32 kDa subunit declined
are plotted and shown with corresponding SDs. two-fold.
MCM7 (minichromosomal maintenance protein 7) is a
part of the MCM2–7 DNA-binding heterohexamer complex
3.3 Data interpretation and TOP15 exclusion essential for DNA replication, providing helicase activity
[14, 15]. Replication protein A, 32 kDa subunit is one of
The lists of all identified differentially expressed proteins three components of Replication protein A – the major
found in both P1 and P2 HL-60 subclones exceeded 40 single-stranded DNA-binding protein in eukaryotes.
items. We thus faced the universal dilemma of proteomic As such, the RPA complex participates in DNA replication
data interpretation: How to narrow down the list of candi- and recombination, DNA damage checkpoints and
date proteins? all major types of DNA repair including nucleotide
In our earlier study, we demonstrated that some proteins excision, base-excision, mismatch and double-strand break
and protein families often appear among the differentially repairs. [16].
expressed proteins in published 2-DE experiments, regard- Since these two proteins, critical for cell proliferation and
less of the experiments performed or tissues studied. We maintenance of genome stability, are markedly down-regu-
designated these ‘‘notorious’’ proteins as the TOP15 [11]. lated, we hypothesized that their DNA-related functions
Since the TOP15 proteins are differentially expressed as could be partially limited in HL-60/P1 cells. Such a defect
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
6. Proteomics 2009, 9, 5006–5015 5011
Table 1. Differential protein expressions in HL-60/P1 cells
Fold Spot Protein name SwissProt Matched Sequence Mascot Excluded
change number number peptides coverage (%) Score (TOP 15)
Proteins differentially expressed in P1 cells
Up-regulated
10 9 Thioredoxin-like protein 1 O43396 14 52 179
3.8 17 Sjoegren syndrome/scleroderma O60232 10 59 153
autoantigen 1
3.3 15 Heat-shock protein b-1 (HSP27) P04792 9 55 141 YES
2.1 20 Cofilin-1 P23528 14 72 196
1.6 11 Splicing factor, arginine/ Q07955 17 58 256
serine-rich 1
Down-regulated
À5.7 1 DNA replication licensing factor P33993 27 49 373
MCM7
À3.7 22 Lamin-A/C P02545 9 13 87
À3.4 6 Proliferation-associated protein 2G4 Q9UQ80 16 28 136
À3 8 Macrophage-capping protein P40121 10 35 121
À2.6 16 78 kDa glucose-regulated protein P11021 10 19 160 YES
À2.5 18 Heterochromatin protein 1a P45973 9 43 144
À2.5 7 Protein phosphatase 1 regulatory Q15435 13 39 133
subunit 7
À2.2 2 Annexin A6 P08133 30 46 341 YES
À2.2 21 Stathmin P16949 11 42 144
À2.1 12 Replication protein A 32 kDa subunit P15927 11 48 154
À2.1 13 F-actin capping protein subunit b P47756 15 48 196
À2 3 Protein disulfide-isomerase A3 P30101 12 22 135 YES
precursor
À2 5 Tryptophanyl-tRNA synthetase, P23381 12 33 180
cytoplasmic
À1.9 10 F-actin capping protein subunit a-1 P52907 11 48 186
À1.9 14 6-phosphogluconolactonase O95336 10 40 122
À1.8 19 Stathmin P16949 8 41 96
À1.6 4 Protein disulfide-isomerase A3 P30101 26 47 344 YES
precursor
would make these cells highly sensitive to a treatment with responsible for severe combined immunodeficiency disease
chemicals and clinically used genotoxic drugs that interfere [17, 18]. Deficiency of ADA causes increased levels of
with DNA replication and the maintenance of DNA integ- intracellular adenosine. The most widely accepted mechan-
rity. Typical examples of such drugs are the DNA cross- ism of ADA deficiency implicates the formation of intra-
linker cis-platin and the inhibitor of topoisomerase cellular deoxyadenosine, deoxyATP and/or S-adenosyl
II, etoposide. homocysteine as well as pyrimidine starvation as the direct
cause of intracellular toxicity [18, 19]. Based on these facts,
we hypothesized that markedly decreased expression of
3.5 ADA ADA in HL-60/P2 cells could partially reduce the detox-
ification capacity of these cells and make them vulnerable to
In the HL-60/P2 subclone, only 11 of the original 21 increased adenosine concentrations.
candidate proteins remained after the exclusion of TOP15 ADA is also normally responsible for deamidation, and
proteins and protein families. The most notable alteration of hence inactivation of synthetic purine anti-metabolite
expression in HL-60/P2 cells was the down-regulation of vidarabine (adenine arabinoside, Ara-A) used as a cytostatic
ADA. Based on 2-DE data, levels of ADA are decreased and anti-viral drug. Sensitivity of cells to the cytostatic action
approximately sixfold, with more than a fivefold decline of vidarabine is inversely proportional to the activity of ADA
confirmed by western blotting. ADA participates in purine [20]. In accordance with this, we hypothesized that the
metabolism where it degrades adenosine or 20 -deoxy- observed sixfold decreased expression of ADA would result
adenosine, producing inosine or 20 -deoxyinosine, respec- in the higher toxicity of vidarabine to HL-60/P2 cells
tively. In humans, a congenital defect in this enzyme is compared with HL-60/P1 and HL-60 cells.
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
7. 5012 J. Petrak et al. Proteomics 2009, 9, 5006–5015
Table 2. Differential protein expressions in HL-60/P2 cells. Identification of proteins with Mascot Score.
Fold Spot Protein name SwissProt Matched Sequence Mascot Excluded
change number number peptides coverage Score (TOP 15)
(%)
Proteins differentially expressed in P2 cells
Up-regulated
7.3 6 Septin-11 Q9NVA2 10 29 121
4 22 Peroxiredoxin-2 P32119 8 43 125 YES
2.6 23 a-Enolase P06733 10 24 113 YES
2.4 14 S-formylglutathione hydrolase P10768 9 42 111
2.1 19 GrpE protein homolog 1, mitochondrial Q9HAV7 9 47 124
precursor
1.8 20 Heat shock cognate 71 kDa protein P11142 9 19 119 YES
Down-regulated
À6.4 10 Adenosine deaminase P00813 17 43 205
À4.8 7 Pyruvate kinase isozymes M1/M2 P14618 15 37 72 YES
À4 9 Heat shock cognate 71 kDa protein P11142 7 14 63Ã YES
À3.6 2 Nucleolin (Protein C23) P19338 14 23 163
À2.5 12 Tubulin b chain P07437 15 29 165 YES
À2.4 8 Ubiquinol-cytochrome-c reductase P31930 15 43 132
complex core protein 1
À2.4 21 Heterochromatin protein 1 a P45973 4 24 59Ã
À2.3 15 Proteasome activator complex subunit 2 Q9UL46 10 44 135
À2.2 1 Annexin A6 P08133 14 26 126 YES
À2.2 11 Pyruvate kinase isozymes M1/M2 P14618 6 14 172 YES
À2.1 4 Hydroxymethylglutaryl-CoA synthase, Q01581 15 31 149
cytoplasmic
4 Actin, cytoplasmic 1 P60709 5 21 39Ã
À2 17 Heat-shock protein b-1 (HSP27) P04792 6 27 65Ã YES
À1.9 13 Tubulin b chain P07437 14 31 150 YES
À1.7 16 Proteasome activator complex subunit 1 Q06323 17 59 198
À1.6 18 NADH dehydrogenase [ubiquinone] O75489 15 54 216
iron–sulfur protein 3
À1.6 24 Cofilin-1 P23528 12 62 182
À1.6 5 Protein disulfide-isomerase A3 precursor P30101 23 44 270 YES
À1.5 3 Protein disulfide-isomerase precursor P07237 22 50 321 YES
à MS/MS confirmation of identification
Spot Protein Peptide
number name
4 Actin cytoplasmic 1 SYELPDGQVITIGNER
17 Heat-shock protein LATQSNEITIPVTFESR,
b-1(HSP27) LFDQAFGLPR
21 Heterochromatin CPQIVIAFYEER
protein 1 a
9 Heat shock cognate ARFEELNADLFR
71 kDa protein
3.6 Cell assays We tested the toxic effect of increasing doses of etoposide
(inhibitor of topoisomerase II), cis-platin, adenosine and
To test our hypotheses that HL-60/P1 cells are sensitive vidarabine on the growth and survival of HL-60, HL-60/P1
to a treatment interfering with DNA-replication and and HL-60/P2 cells in culture. The toxic effect of these
integrity, and that P2 cells are vulnerable to adenosine respective treatments was considered as the percentage of
and sensitive to vidarabine, we set up series of in vitro surviving cells, as measured by mitochondrial activity
cell assays. (Fig 3). In accordance with our hypotheses, etoposide and
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
8. Proteomics 2009, 9, 5006–5015 5013
cis-platin were both significantly more toxic to HL-60/P1 vidarabine. Unlike the effect of DNA-interfering agents and
cells than to HL-60 and HL-60/P2. These DNA-interfering anti-metabolites, adenosine toxicity in ADA-deficient cells is
drugs eliminated 60% of the HL-60/P1 population at high independent of DNA replication. Therefore, adenosine
nM or low mM concentrations in five days, while reducing toxicity to HL-60/P2 cells was demonstrated in an assay with
the HL-60 and HL-60/P2 cell population only by 0–20%. a shorter incubation period (2 days only). As hypothesized,
Similarly, both adenosine and vidarbine were markedly adenosine monophosphate added to the media reduced the
more toxic to HL-60/P2 cells than to HL-60/P1 or HL-60 P2 cell population by more than 60% at the 1000 mM
cells. Vidarabine completely eliminated the P2 population at concentration, while in contrast stimulated the proliferation
a concentration of 100 mM, while only marginally reducing of HL-60 and HL-60/P1 cells.
the viability of HL-60. The complete elimination of P1 would To summarize, our two hypotheses based on proteomic
require a more than two-fold higher concentration of data were tested and confirmed in a series of in vitro cellular
Figure 3. Cellular toxicity assay. Cells were seeded and grown with increasing concentrations of either cis-platin, etoposide, adenosine
monophosphate or vidarabine (single dose at the time of seeding) and cultivated for 5 days, except for cells exposed to AMP which were
grown for only 2 days. Decreased mitochondrial activity measured by a Quick Cell Proliferation Assay Kit reflects the number of surviving
cells and hence the toxicity of individual tested chemicals. Average values from triplicate experiments are shown.
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
9. 5014 J. Petrak et al. Proteomics 2009, 9, 5006–5015
assays, and two cellular processes were proposed as potential the best potential drug targets-MCM7 and RPA2 in HL-60/P1
therapeutic targets. Four proposed drugs aimed at these cells, and ADA in HL-60/P2 cells. We subsequently hypo-
targets were tested and shown to be effective for the elimin- thesized that the observed down-regulation of MCM7 and
ation of HL-60/P1 and HL-60/P2 TRAIL-resistant cells RPA2 proteins in TRAIL-resistant P1 leukemia cells makes
in vitro. these cells vulnerable to clinically used DNA-interfering
agents such as etoposide and cis-platin. Successful in vitro
testing in cell cultures demonstrated that HL-60/P1 cells are
4 Discussion markedly more sensitive to etoposide than TRAIL-sensitive
HL-60 and resistant HL-60/P2 cells. Similarly, we proposed
Resistance to anti-cancer drugs is the major cause of and experimentally verified that markedly decreased expres-
chemotherapy failure, and necessitates the application of sion of ADA makes HL-60/P2 cells vulnerable to the drug
combined drug regimens and the development of new anti- vidarabine and to high concentrations of the nucleoside
cancer molecules. The new and very promising apoptosis- adenosine.
inducing molecule – recombinant death ligand TRAIL – is Based on 2-DE analysis of TRAIL-resistant leukemia
undergoing several clinical trials with promising preli- cells, we revealed specific molecular targets that can be used
minary results. However, as with all other anti-cancer drugs, (at least in vitro) for the selective elimination of two TRAIL-
TRAIL-based therapies are likely to be complicated by resistant subclones derived from established HL-60 leuke-
TRAIL-resistant cancer cells. We used expression proteo- mia cells. If TRAIL is approved for clinical use, questions on
mics as a tool for the identification of potential molecular how to target TRAIL-resistant tumors will become immi-
targets of TRAIL-resistant leukemia cells. We looked for nent. Therefore, any information on the specific molecular
proteins or pathways that could be used as targets for the features of resistant cancer cells and especially their ‘‘weak
selective elimination of TRAIL-resistant cells. The discovery spots’’ will become invaluable. We are fully aware that the
of such target molecules could be invaluable for the isolation and characterization of drug-resistant cancer cells
improvement of future TRAIL-based therapies. The last from individual patients in order to optimize therapy is,
decade has witnessed a massive expansion of proteomic indeed, technically challenging and incomparably more
approaches into molecular oncology and cell physiology. complex. Nevertheless, we believe that our work has
However, proteomic analyses only seldom produce a direct, demonstrated a basic ‘‘proof of concept’’ for the possible
experimentally verified, insight into cell physiology or implementation of proteomics in the development of
pathology. The interpretation of data derived from expres- patient-tailored anti-cancer therapy.
sion proteomics studies and verification of the hypotheses
generated remain among the most challenging tasks of This research was supported by grants from the Czech Science
current proteomics. A typical expression proteomics Foundation (GACR) 305/09/1390 and 204/07/0830, from the
experiment produces lists of quantitatively or qualitatively Ministry of Health of the Czech Republic (MZCR) MZCR/
changed proteins. The question remains how to transform UHKT No. 023736, IGA MZ NR8317-4, NR8930-4, NS10300-
the resulting list of differentially expressed proteins into 3 and the Ministry of Education, Youth and Sports of the Czech
plausible and verifiable hypothesis addressing the molecular Republic (MSMTCR) projects LC06044, MSM 0021620806,
mechanism. What candidate proteins are central or specific MSM 0021620808 and the Institutional Research Concept
for the process studied? How do we narrow down the list of AV0Z50200510 (IMIC). Special thanks to Mrtva Ryba
differentially expressed candidate proteins? Here, we iden-
tified 41 differentially expressed proteins in TRAIL-resistant The authors have declared no conflict of interest.
HL-60/P1 and HL-60/P2 subclones compared with the
original HL-60 TRAIL-sensitive cells. Which one of these 41
proteins could serve as a molecular target for the selective
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