Leprosy, caused by Mycobacterium leprae, rarely affects children younger than 5 years. Here, we s... more Leprosy, caused by Mycobacterium leprae, rarely affects children younger than 5 years. Here, we studied a multiplex leprosy family that included monozygotic twins aged 22 months suffering from paucibacillary leprosy. Whole genome sequencing identified three amino acid mutations previously associated with Crohn’s disease and Parkinson’s disease as candidate variants for early onset leprosy: LRRK2 N551K, R1398H and NOD2 R702W. In genome-edited macrophages, we demonstrated that cells expressing the LRRK2 mutations displayed reduced apoptosis activity following mycobacterial challenge independently of NOD2. However, employing co-immunoprecipitation and confocal microscopy we showed that LRRK2 and NOD2 proteins interacted in RAW cells and monocyte-derived macrophages, and that this interaction was substantially reduced for the NOD2 R702W mutation. Moreover, we observed a joint effect of LRRK2 and NOD2 variants on Bacillus Calmette-Guérin (BCG)-induced respiratory burst, NF-κB activation ...
Introduction Globally, tuberculosis (TB) is the leading cause of death due to a single bacterial ... more Introduction Globally, tuberculosis (TB) is the leading cause of death due to a single bacterial pathogen. In 2019, there were an estimated 1.4 million deaths caused by TB, which included 208,000 people living with the human immunodeficiency virus (HIV) (1). Of the estimated 10 million people who fell ill with TB, 820,000 were persons living with HIV (PLWH), consistent with a 2.0-fold higher mortality due to TB in HIV-positive relative to HIV-negative persons. Globally, the proportion of notified HIV-positive TB cases on antiretroviral therapy was 88% with large gaps of antiretrovi-ral therapy (ART) coverage among countries (1). However, even people on long-term ART still have substantially increased risk of developing TB (2), which is usually the result of new infection rather than reactivation of latent TB (3-6). Transmission of Mycobacterium tuberculosis (M. tuberculosis) occurs by aerosols that are inhaled by an exposed person. Results from the mouse model show that after reaching the lung alveoli, M. tuberculosis bacilli are being rapidly taken up by alveolar macrophages (AMs). Following a delay of approximately 10 days, successful establishment of infection occurs once M. tuberculosisinfected AMs traverse the airway epithelium and establish themselves in the lung interstitium. There, transfer of M. tuberculosis to permissive inflammatory macrophages occurs and T cell priming is initiated in the draining lymph nodes (7). However, much of the AM-M. tuberculosis interaction in the alveoli remains unknown. In humans, not each exposure results in successful infection, and a subset of highly exposed persons entirely resist infection, as inferred from the absence of CD4 + T cell anti-M. tuberculosis immunity (8, 9). Hence it seems possible that human AMs are capable of limiting transfer of inhaled M. tuberculosis bacilli to the Persons living with HIV (PLWH) are at increased risk of tuberculosis (TB). HIV-associated TB is often the result of recent infection with Mycobacterium tuberculosis (M. tuberculosis) followed by rapid progression to disease. Alveolar macrophages (AMs) are the first cells of the innate immune system that engage M. tuberculosis, but how HIV and antiretroviral therapy (ART) affect the anti-mycobacterial response of AMs is not known. To investigate the impact of HIV and ART on the transcriptomic and epigenetic response of AMs to M. tuberculosis, we obtained AMs by bronchoalveolar lavage from 20 PLWH receiving ART, 16 control subjects who were HIV-free (HC), and 14 subjects who received ART as preexposure prophylaxis (PrEP) to prevent HIV infection. Following in vitro challenge with M. tuberculosis, AMs from each group displayed overlapping but distinct profiles of significantly up-and downregulated genes in response to M. tuberculosis. Comparatively, AMs isolated from both PLWH and PrEP subjects presented a substantially weaker transcriptional response. In addition, AMs from HC subjects challenged with M. tuberculosis responded with pronounced chromatin accessibility changes while AMs obtained from PLWH and PrEP subjects displayed no significant changes in their chromatin state. Collectively, these results revealed a stronger adverse effect of ART than HIV on the epigenetic landscape and transcriptional responsiveness of AMs.
Leprosy, caused by Mycobacterium leprae, has a long incubation period and cases with age-of-onset... more Leprosy, caused by Mycobacterium leprae, has a long incubation period and cases with age-of-onset <5 years are rare. Here, we studied a three-generational multiplex leprosy family which included monozygotic twins age <24 months suffering from paucibacillary leprosy. Whole genome sequencing identified a homozygous double mutation in the LRRK2 gene (N551K, R1398H) and a heterozygous mutation in NOD2 (R702W) as candidate variants underlying the early onset phenotype in the twins. The same amino acid substitutions had previously been identified as shared risk-modulating factors for Crohn’s disease and Parkinson’s disease. To evaluate the functional impact of the LRRK2 mutations, we employed genome editing in RAW264.7 cells. Cells expressing the LRRK2 variants displayed reduced respiratory burst and apoptosis following mycobacterial challenge. Moreover, the BCG-induced respiratory burst was significantly lower in LRRK2 wild-type-expressing cells transfected with NOD2 R702W compared...
Type-1 reactions (T1Rs) are pathological inflammatory episodes and main contributors to nerve dam... more Type-1 reactions (T1Rs) are pathological inflammatory episodes and main contributors to nerve damage in leprosy. Here, we evaluate the gene-wise enrichment of rare protein altering variants in seven genes where common variants were previously associated with T1R. We selected 474 Vietnamese leprosy-patients of which 237 were T1R-affected and 237 were T1R-free matched controls. Gene-wise enrichment of nonsynonymous variants was tested with both kernel based (SKAT) and burden methods. Of the seven genes tested two showed statistical evidence of association with T1R. For the LRRK2 gene an enrichment of nonsynonymous variants was observed in T1R-free controls (p SKAT-O= 1.6x10-4). This gene-wise association was driven almost entirely by the gain of function variant R1628P (p = 0.004; OR = 0.29). The second gene-wise association was found for the Parkin coding gene PRKN (formerly PARK2) where seven rare variants were enriched in T1R-affected cases (p SKAT-O = 7.4x10-5). Mutations in both ...
Cell death plays an important role in tumorigenesis, growth, and progression and affects the effi... more Cell death plays an important role in tumorigenesis, growth, and progression and affects the efficiency of chemotherapy to a great extent. Apoptosis is usually regarded as the principal mechanism of chemotherapy-induced cell death. However, the dysregulation of apoptosis occurs commonly in many cancers, which lowers the effectiveness of therapy and allows cells to survive. The mechanisms by which cells acquire this resistance to chemotherapy are not fully understood. Several studies uncovered alternative cell death pathways that are mechanistically distinct from apoptosis. These pathways, including autophagy and necrosis, represent potential targets for novel cancer treatment. By modulating the key regulatory molecules involved in the different types of cell death, more effective and less toxic chemotherapy might be developed. In this chapter, we describe the signaling pathways and the molecular events that are involved in these three major forms of programmed cell death. Additionally, we also discuss the emerging therapies targeting these cell death pathways as new strategies against cancer.
Zhonghua jie he he hu xi za zhi = Zhonghua jiehe he huxi zazhi = Chinese journal of tuberculosis and respiratory diseases, 2003
To investigate the effect of M. tuberculosis infection on actin in host-cells. The form and distr... more To investigate the effect of M. tuberculosis infection on actin in host-cells. The form and distribution of fibrous actin and changes of actin expression were observed by confocal microscopy and Western blot analysis in macrophages infected with either M. tuberculosis H(37)R(a) or H(37)R(v) at 6 h, 12 h and 24 h after infection. Non-infected macrophages or macrophages treated with dead M. tuberculosis H(37)R(v) served as controls. F-actin aggregated and the actin expression was suppressed in macrophages infected with H(37)R(a) or H(37)R(v), and the effect appeared earlier in cells infected by the virulent H(37)R(v) strain than in cells infected by the avirulent H(37)R(a) strain. In macrophages treated with the dead H(37)R(v), actin was not affected. The results suggest that in the process of infection, M. tuberculosis evades the bactericidal mechanisms possibly by secretion of certain proteins or factors which affect the host-cell actin.
Promoter deletion analysis is a useful tool for identifying important regulatory regions involved... more Promoter deletion analysis is a useful tool for identifying important regulatory regions involved in transcriptional control of gene expression. In this approach, a series of promoter deletion fragments are fused to a reporter gene, such as chloramphenicol acetyltransferase or luciferase gene in a vector, and then transfected into cells for induction. Screening the expression level of the reporter gene using either a qualitative or a quantitative assay, allows to identify the regulatory regions of interest (e.g., cis-acting elements or enhancer) in the promoter.Luciferase genes have been widely used as reporter genes for their sensitivity and efficiency. Firefly and Renilla luciferases are two commonly used reporters, which oxidize different substrates to generate quantifiable luminescence. Therefore, the enzymatic activities of firefly and Renilla luciferases can be sequentially measured in a single sample by controlling reaction conditions. Here, we describe a dual-luciferase reporter assay, where the promoter of interest is fused to a firefly luciferase reporter and is co-transfected into cells with an internal control vector (pRL-CMV) to express Renilla luciferase. Both the Firefly and Renilla luciferases are measured using a dual-luciferase reporter assay system which improves experimental accuracy.
Sheng li xue bao : [Acta physiologica Sinica], Jan 25, 2003
Expression microarray was employed in this study to investigate whether the ion channels and thei... more Expression microarray was employed in this study to investigate whether the ion channels and their regulatory elements encoding genes participate in the immune response to Mycobacterium tuberculosis infection. The results of a virulent strain were compared with those of the clinically isolated strains. The data demonstrate that K(+), Na(+), Ca(2+) and Cl(-) channels and their regulatory elements, such as the G protein, receptor and second messenger, protein kinase and protein phosphatase were involved in the immune reaction. The clinical strain affected more types of ion channels and respective regulatory elements. The data provides clues for further scrutiny into the role of ion channels and related elements in the interaction between Mycobacterium tuberculosis and host macrophage.
Toll-like receptor ligands (TLRLs) produced by various pathogens activate mitogen-activated prote... more Toll-like receptor ligands (TLRLs) produced by various pathogens activate mitogen-activated protein kinases (MAPKs). While the dependence on p38 MAPK activation for the induction of inflammatory genes by the TLR4L, lipopolysaccharide (LPS), has been well documented, the importance of the p38 pathway in gene regulation by other TLRLs is less well understood. We have focused our analysis on two TLRLs with therapeutic potential, imidazoquinoline S28463 (TLR7L) and CpG DNA (TLR9L), to explore in detail their effects on the regulation of gene expression in macrophages. Here we report that activation of the p38 MAPK/MK2 pathway is crucial for both S28463- and CpG-induced cytokine and chemokine production. We show that the stability of TNF mRNA induced by CpG DNA and S28463 is not dependent on the p38 MAPK/MK2 pathway, in contrast to LPS-induced TNF mRNA. Using a GFP reporter construct under the control of the 3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; untranslated region of the TNF gene, we demonstrate that S28463 and CpG DNA-induced MK2 signalling regulates TNF mRNA primarily at the translational level, whereas LPS-induced MK2 signalling regulates both the stability and translational efficiency of TNF mRNA. Overall, these data provide insight into distinct molecular mechanisms of gene expression regulation by different Toll-like receptor ligands.
BackgroundSecreted protein, acidic and rich in cysteine (SPARC) is a matricellular protein that m... more BackgroundSecreted protein, acidic and rich in cysteine (SPARC) is a matricellular protein that mediates cell-matrix interactions. It has been shown, depending on the type of cancer, to possess either pro- or anti-tumorigenic properties. The transcriptional regulation of the SPARC gene expression has not been fully elucidated and the effects of anti-cancer drugs on this process have not been explored.ResultsIn the present study, we demonstrated that chromatin remodeling factor Brg-1 is recruited to the proximal SPARC promoter region (-130/-56) through an interaction with transcription factor Sp1. We identified Brg-1 as a critical regulator for the constitutive expression levels of SPARC mRNA and protein in mammary carcinoma cell lines and for SPARC secretion into culture media. Furthermore, we found that Brg-1 cooperates with Sp1 to enhance SPARC promoter activity. Interestingly, fenretinide [N-4(hydroxyphenyl) retinamide, 4-HPR], a synthetic retinoid with anti-cancer properties, wa...
Studies have shown that nuclear translocation of actin occurs under certain conditions of cellula... more Studies have shown that nuclear translocation of actin occurs under certain conditions of cellular stress; however, the functional significance of actin import remains unclear. Here, we demonstrate that during the phorbol 12-myristate 13-acetate (PMA)-induced differentiation of HL-60 cells toward macrophages, β-actin translocates from the cytoplasm to the nucleus and that this process is dramatically inhibited by pretreatment with p38 mitogen-activated protein kinase inhibitors. Using chromatin immunoprecipitation-on-chip assays, the genome-wide maps of β-actin binding to gene promoters in response to PMA treatment is analyzed in HL-60 cells. A gene ontology-based analysis shows that the identified genes belong to a broad spectrum of functional categories such as cell growth and differentiation, signal transduction, response to external stimulus, ion channel activity, and immune response. We also demonstrate a correlation between β-actin occupancy and the recruitment of RNA polymera...
The solute carrier family 11 member 1 ( SLC11A1 , formerly NRAMP1 ) gene is associated with infec... more The solute carrier family 11 member 1 ( SLC11A1 , formerly NRAMP1 ) gene is associated with infectious and autoimmune diseases and plays an important role in macrophage activation. Human SLC11A1 mRNA contains an AU-rich element (ARE) within the 3′ untranslated region; however, its role in the regulation of SLC11A1 gene expression has not been elucidated. Here we analyze the expression of SLC11A1 in human monocytes and HL-60 cells and then use HL-60 cells as a model to determine whether RNA-binding protein HuR is associated with the ARE and involved in SLC11A1 mRNA turnover. Our results demonstrate a binding of HuR to the SLC11A1 ARE in phorbol myristate acetate (PMA)-differentiated cells dramatically increased compared to that in undifferentiated cells. Interestingly, PMA-induced accumulation of cytoplasmic HuR occurs in parallel with an increase in the binding of HuR to SLC11A1 ARE and with an increase in the SLC11A1 mRNA level. This suggests that HuR's cytoplasmic localization...
Secretory leukocyte protease inhibitor (SLPI) is an anti-inflammatory protein that is observed at... more Secretory leukocyte protease inhibitor (SLPI) is an anti-inflammatory protein that is observed at high levels in asthma patients. Resiquimod, a TLR7/8 ligand, is protective against acute and chronic asthma, and it increases SLPI expression of macrophages in vitro. However, the protective role played by SLPI and the interactions between the SLPI and resiquimod pathways in the immune response occurring in allergic asthma have not been fully elucidated. To evaluate the role of SLPI in the development of asthma phenotypes and the effect of resiquimod treatment on SLPI, we assessed airway resistance and inflammatory parameters in the lungs of OVA-induced asthmatic SLPI transgenic and knockout mice and in mice treated with resiquimod. Compared with wild-type mice, allergic SLPI transgenic mice showed a decrease in lung resistance (p < 0.001), airway eosinophilia (p < 0.001), goblet cell hyperplasia (p < 0.001), and plasma IgE levels (p < 0.001). Allergic SLPI knockout mice dis...
Journal of Experimental & Clinical Cancer Research, 2012
Background It is mandatory to confirm the absence of mutations in the KRAS gene before treating m... more Background It is mandatory to confirm the absence of mutations in the KRAS gene before treating metastatic colorectal cancers with epidermal growth factor receptor inhibitors, and similar regulations are being considered for non-small cell lung carcinomas (NSCLC) and other tumor types. Routine diagnosis of KRAS mutations in NSCLC is challenging because of compromised quantity and quality of biological material. Although there are several methods available for detecting mutations in KRAS, there is little comparative data regarding their analytical performance, economic merits, and workflow parameters. Methods We compared the specificity, sensitivity, cost, and working time of five methods using 131 frozen NSCLC tissue samples. We extracted genomic DNA from the samples and compared the performance of Sanger cycle sequencing, Pyrosequencing, High-resolution melting analysis (HRM), and the Conformité Européenne (CE)-marked TheraScreen DxS and K-ras StripAssay kits. Results and conclusio...
The solute carrier family 11 member 1 (SLC11A1) gene is strictly regulated and exclusively expres... more The solute carrier family 11 member 1 (SLC11A1) gene is strictly regulated and exclusively expressed in myeloid lineage cells. However, little is known about the transcriptional regulation of the SLC11A1 gene during myeloid development. In this study, we used HL-60 cells as a model to investigate the regulatory elements/factors involved in the transactivation of the SLC11A1 gene during phorbol 12-myristate 13-acetate (PMA)induced macrophage differentiation of HL-60 cells. Promoter deletion analysis showed that a 7-base AP-1-like element (TGACTCT) was critical for the responsiveness of the SLC11A1 promoter to PMA. Stimulation by PMA induced the binding of ATF-3 and the recruitment of two components of the SWI/SNF complex, BRG1 and -actin, to this element in an ATF-3-dependent manner. RNAi-mediated depletion of ATF-3 or BRG1 markedly decreased SLC11A1 gene expression and its promoter activity induced by PMA. Luciferase reporter experiments demonstrated that ATF-3 cooperated with BRG1 and -actin to activate the SLC11A1 promoter. Furthermore, we showed that PMA can induce the proximal (GT/AC) n repeat sequence to convert to the Z-DNA structure in the SLC11A1 gene promoter, and depletion of BRG1 resulted in a significant decrease of Z-DNA formation. Our results demonstrated that recruitment of the SWI/SNF complex initiated Z-DNA formation and subsequently helped to transactivate the SLC11A1 gene.
Leprosy, caused by Mycobacterium leprae, rarely affects children younger than 5 years. Here, we s... more Leprosy, caused by Mycobacterium leprae, rarely affects children younger than 5 years. Here, we studied a multiplex leprosy family that included monozygotic twins aged 22 months suffering from paucibacillary leprosy. Whole genome sequencing identified three amino acid mutations previously associated with Crohn’s disease and Parkinson’s disease as candidate variants for early onset leprosy: LRRK2 N551K, R1398H and NOD2 R702W. In genome-edited macrophages, we demonstrated that cells expressing the LRRK2 mutations displayed reduced apoptosis activity following mycobacterial challenge independently of NOD2. However, employing co-immunoprecipitation and confocal microscopy we showed that LRRK2 and NOD2 proteins interacted in RAW cells and monocyte-derived macrophages, and that this interaction was substantially reduced for the NOD2 R702W mutation. Moreover, we observed a joint effect of LRRK2 and NOD2 variants on Bacillus Calmette-Guérin (BCG)-induced respiratory burst, NF-κB activation ...
Introduction Globally, tuberculosis (TB) is the leading cause of death due to a single bacterial ... more Introduction Globally, tuberculosis (TB) is the leading cause of death due to a single bacterial pathogen. In 2019, there were an estimated 1.4 million deaths caused by TB, which included 208,000 people living with the human immunodeficiency virus (HIV) (1). Of the estimated 10 million people who fell ill with TB, 820,000 were persons living with HIV (PLWH), consistent with a 2.0-fold higher mortality due to TB in HIV-positive relative to HIV-negative persons. Globally, the proportion of notified HIV-positive TB cases on antiretroviral therapy was 88% with large gaps of antiretrovi-ral therapy (ART) coverage among countries (1). However, even people on long-term ART still have substantially increased risk of developing TB (2), which is usually the result of new infection rather than reactivation of latent TB (3-6). Transmission of Mycobacterium tuberculosis (M. tuberculosis) occurs by aerosols that are inhaled by an exposed person. Results from the mouse model show that after reaching the lung alveoli, M. tuberculosis bacilli are being rapidly taken up by alveolar macrophages (AMs). Following a delay of approximately 10 days, successful establishment of infection occurs once M. tuberculosisinfected AMs traverse the airway epithelium and establish themselves in the lung interstitium. There, transfer of M. tuberculosis to permissive inflammatory macrophages occurs and T cell priming is initiated in the draining lymph nodes (7). However, much of the AM-M. tuberculosis interaction in the alveoli remains unknown. In humans, not each exposure results in successful infection, and a subset of highly exposed persons entirely resist infection, as inferred from the absence of CD4 + T cell anti-M. tuberculosis immunity (8, 9). Hence it seems possible that human AMs are capable of limiting transfer of inhaled M. tuberculosis bacilli to the Persons living with HIV (PLWH) are at increased risk of tuberculosis (TB). HIV-associated TB is often the result of recent infection with Mycobacterium tuberculosis (M. tuberculosis) followed by rapid progression to disease. Alveolar macrophages (AMs) are the first cells of the innate immune system that engage M. tuberculosis, but how HIV and antiretroviral therapy (ART) affect the anti-mycobacterial response of AMs is not known. To investigate the impact of HIV and ART on the transcriptomic and epigenetic response of AMs to M. tuberculosis, we obtained AMs by bronchoalveolar lavage from 20 PLWH receiving ART, 16 control subjects who were HIV-free (HC), and 14 subjects who received ART as preexposure prophylaxis (PrEP) to prevent HIV infection. Following in vitro challenge with M. tuberculosis, AMs from each group displayed overlapping but distinct profiles of significantly up-and downregulated genes in response to M. tuberculosis. Comparatively, AMs isolated from both PLWH and PrEP subjects presented a substantially weaker transcriptional response. In addition, AMs from HC subjects challenged with M. tuberculosis responded with pronounced chromatin accessibility changes while AMs obtained from PLWH and PrEP subjects displayed no significant changes in their chromatin state. Collectively, these results revealed a stronger adverse effect of ART than HIV on the epigenetic landscape and transcriptional responsiveness of AMs.
Leprosy, caused by Mycobacterium leprae, has a long incubation period and cases with age-of-onset... more Leprosy, caused by Mycobacterium leprae, has a long incubation period and cases with age-of-onset <5 years are rare. Here, we studied a three-generational multiplex leprosy family which included monozygotic twins age <24 months suffering from paucibacillary leprosy. Whole genome sequencing identified a homozygous double mutation in the LRRK2 gene (N551K, R1398H) and a heterozygous mutation in NOD2 (R702W) as candidate variants underlying the early onset phenotype in the twins. The same amino acid substitutions had previously been identified as shared risk-modulating factors for Crohn’s disease and Parkinson’s disease. To evaluate the functional impact of the LRRK2 mutations, we employed genome editing in RAW264.7 cells. Cells expressing the LRRK2 variants displayed reduced respiratory burst and apoptosis following mycobacterial challenge. Moreover, the BCG-induced respiratory burst was significantly lower in LRRK2 wild-type-expressing cells transfected with NOD2 R702W compared...
Type-1 reactions (T1Rs) are pathological inflammatory episodes and main contributors to nerve dam... more Type-1 reactions (T1Rs) are pathological inflammatory episodes and main contributors to nerve damage in leprosy. Here, we evaluate the gene-wise enrichment of rare protein altering variants in seven genes where common variants were previously associated with T1R. We selected 474 Vietnamese leprosy-patients of which 237 were T1R-affected and 237 were T1R-free matched controls. Gene-wise enrichment of nonsynonymous variants was tested with both kernel based (SKAT) and burden methods. Of the seven genes tested two showed statistical evidence of association with T1R. For the LRRK2 gene an enrichment of nonsynonymous variants was observed in T1R-free controls (p SKAT-O= 1.6x10-4). This gene-wise association was driven almost entirely by the gain of function variant R1628P (p = 0.004; OR = 0.29). The second gene-wise association was found for the Parkin coding gene PRKN (formerly PARK2) where seven rare variants were enriched in T1R-affected cases (p SKAT-O = 7.4x10-5). Mutations in both ...
Cell death plays an important role in tumorigenesis, growth, and progression and affects the effi... more Cell death plays an important role in tumorigenesis, growth, and progression and affects the efficiency of chemotherapy to a great extent. Apoptosis is usually regarded as the principal mechanism of chemotherapy-induced cell death. However, the dysregulation of apoptosis occurs commonly in many cancers, which lowers the effectiveness of therapy and allows cells to survive. The mechanisms by which cells acquire this resistance to chemotherapy are not fully understood. Several studies uncovered alternative cell death pathways that are mechanistically distinct from apoptosis. These pathways, including autophagy and necrosis, represent potential targets for novel cancer treatment. By modulating the key regulatory molecules involved in the different types of cell death, more effective and less toxic chemotherapy might be developed. In this chapter, we describe the signaling pathways and the molecular events that are involved in these three major forms of programmed cell death. Additionally, we also discuss the emerging therapies targeting these cell death pathways as new strategies against cancer.
Zhonghua jie he he hu xi za zhi = Zhonghua jiehe he huxi zazhi = Chinese journal of tuberculosis and respiratory diseases, 2003
To investigate the effect of M. tuberculosis infection on actin in host-cells. The form and distr... more To investigate the effect of M. tuberculosis infection on actin in host-cells. The form and distribution of fibrous actin and changes of actin expression were observed by confocal microscopy and Western blot analysis in macrophages infected with either M. tuberculosis H(37)R(a) or H(37)R(v) at 6 h, 12 h and 24 h after infection. Non-infected macrophages or macrophages treated with dead M. tuberculosis H(37)R(v) served as controls. F-actin aggregated and the actin expression was suppressed in macrophages infected with H(37)R(a) or H(37)R(v), and the effect appeared earlier in cells infected by the virulent H(37)R(v) strain than in cells infected by the avirulent H(37)R(a) strain. In macrophages treated with the dead H(37)R(v), actin was not affected. The results suggest that in the process of infection, M. tuberculosis evades the bactericidal mechanisms possibly by secretion of certain proteins or factors which affect the host-cell actin.
Promoter deletion analysis is a useful tool for identifying important regulatory regions involved... more Promoter deletion analysis is a useful tool for identifying important regulatory regions involved in transcriptional control of gene expression. In this approach, a series of promoter deletion fragments are fused to a reporter gene, such as chloramphenicol acetyltransferase or luciferase gene in a vector, and then transfected into cells for induction. Screening the expression level of the reporter gene using either a qualitative or a quantitative assay, allows to identify the regulatory regions of interest (e.g., cis-acting elements or enhancer) in the promoter.Luciferase genes have been widely used as reporter genes for their sensitivity and efficiency. Firefly and Renilla luciferases are two commonly used reporters, which oxidize different substrates to generate quantifiable luminescence. Therefore, the enzymatic activities of firefly and Renilla luciferases can be sequentially measured in a single sample by controlling reaction conditions. Here, we describe a dual-luciferase reporter assay, where the promoter of interest is fused to a firefly luciferase reporter and is co-transfected into cells with an internal control vector (pRL-CMV) to express Renilla luciferase. Both the Firefly and Renilla luciferases are measured using a dual-luciferase reporter assay system which improves experimental accuracy.
Sheng li xue bao : [Acta physiologica Sinica], Jan 25, 2003
Expression microarray was employed in this study to investigate whether the ion channels and thei... more Expression microarray was employed in this study to investigate whether the ion channels and their regulatory elements encoding genes participate in the immune response to Mycobacterium tuberculosis infection. The results of a virulent strain were compared with those of the clinically isolated strains. The data demonstrate that K(+), Na(+), Ca(2+) and Cl(-) channels and their regulatory elements, such as the G protein, receptor and second messenger, protein kinase and protein phosphatase were involved in the immune reaction. The clinical strain affected more types of ion channels and respective regulatory elements. The data provides clues for further scrutiny into the role of ion channels and related elements in the interaction between Mycobacterium tuberculosis and host macrophage.
Toll-like receptor ligands (TLRLs) produced by various pathogens activate mitogen-activated prote... more Toll-like receptor ligands (TLRLs) produced by various pathogens activate mitogen-activated protein kinases (MAPKs). While the dependence on p38 MAPK activation for the induction of inflammatory genes by the TLR4L, lipopolysaccharide (LPS), has been well documented, the importance of the p38 pathway in gene regulation by other TLRLs is less well understood. We have focused our analysis on two TLRLs with therapeutic potential, imidazoquinoline S28463 (TLR7L) and CpG DNA (TLR9L), to explore in detail their effects on the regulation of gene expression in macrophages. Here we report that activation of the p38 MAPK/MK2 pathway is crucial for both S28463- and CpG-induced cytokine and chemokine production. We show that the stability of TNF mRNA induced by CpG DNA and S28463 is not dependent on the p38 MAPK/MK2 pathway, in contrast to LPS-induced TNF mRNA. Using a GFP reporter construct under the control of the 3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; untranslated region of the TNF gene, we demonstrate that S28463 and CpG DNA-induced MK2 signalling regulates TNF mRNA primarily at the translational level, whereas LPS-induced MK2 signalling regulates both the stability and translational efficiency of TNF mRNA. Overall, these data provide insight into distinct molecular mechanisms of gene expression regulation by different Toll-like receptor ligands.
BackgroundSecreted protein, acidic and rich in cysteine (SPARC) is a matricellular protein that m... more BackgroundSecreted protein, acidic and rich in cysteine (SPARC) is a matricellular protein that mediates cell-matrix interactions. It has been shown, depending on the type of cancer, to possess either pro- or anti-tumorigenic properties. The transcriptional regulation of the SPARC gene expression has not been fully elucidated and the effects of anti-cancer drugs on this process have not been explored.ResultsIn the present study, we demonstrated that chromatin remodeling factor Brg-1 is recruited to the proximal SPARC promoter region (-130/-56) through an interaction with transcription factor Sp1. We identified Brg-1 as a critical regulator for the constitutive expression levels of SPARC mRNA and protein in mammary carcinoma cell lines and for SPARC secretion into culture media. Furthermore, we found that Brg-1 cooperates with Sp1 to enhance SPARC promoter activity. Interestingly, fenretinide [N-4(hydroxyphenyl) retinamide, 4-HPR], a synthetic retinoid with anti-cancer properties, wa...
Studies have shown that nuclear translocation of actin occurs under certain conditions of cellula... more Studies have shown that nuclear translocation of actin occurs under certain conditions of cellular stress; however, the functional significance of actin import remains unclear. Here, we demonstrate that during the phorbol 12-myristate 13-acetate (PMA)-induced differentiation of HL-60 cells toward macrophages, β-actin translocates from the cytoplasm to the nucleus and that this process is dramatically inhibited by pretreatment with p38 mitogen-activated protein kinase inhibitors. Using chromatin immunoprecipitation-on-chip assays, the genome-wide maps of β-actin binding to gene promoters in response to PMA treatment is analyzed in HL-60 cells. A gene ontology-based analysis shows that the identified genes belong to a broad spectrum of functional categories such as cell growth and differentiation, signal transduction, response to external stimulus, ion channel activity, and immune response. We also demonstrate a correlation between β-actin occupancy and the recruitment of RNA polymera...
The solute carrier family 11 member 1 ( SLC11A1 , formerly NRAMP1 ) gene is associated with infec... more The solute carrier family 11 member 1 ( SLC11A1 , formerly NRAMP1 ) gene is associated with infectious and autoimmune diseases and plays an important role in macrophage activation. Human SLC11A1 mRNA contains an AU-rich element (ARE) within the 3′ untranslated region; however, its role in the regulation of SLC11A1 gene expression has not been elucidated. Here we analyze the expression of SLC11A1 in human monocytes and HL-60 cells and then use HL-60 cells as a model to determine whether RNA-binding protein HuR is associated with the ARE and involved in SLC11A1 mRNA turnover. Our results demonstrate a binding of HuR to the SLC11A1 ARE in phorbol myristate acetate (PMA)-differentiated cells dramatically increased compared to that in undifferentiated cells. Interestingly, PMA-induced accumulation of cytoplasmic HuR occurs in parallel with an increase in the binding of HuR to SLC11A1 ARE and with an increase in the SLC11A1 mRNA level. This suggests that HuR's cytoplasmic localization...
Secretory leukocyte protease inhibitor (SLPI) is an anti-inflammatory protein that is observed at... more Secretory leukocyte protease inhibitor (SLPI) is an anti-inflammatory protein that is observed at high levels in asthma patients. Resiquimod, a TLR7/8 ligand, is protective against acute and chronic asthma, and it increases SLPI expression of macrophages in vitro. However, the protective role played by SLPI and the interactions between the SLPI and resiquimod pathways in the immune response occurring in allergic asthma have not been fully elucidated. To evaluate the role of SLPI in the development of asthma phenotypes and the effect of resiquimod treatment on SLPI, we assessed airway resistance and inflammatory parameters in the lungs of OVA-induced asthmatic SLPI transgenic and knockout mice and in mice treated with resiquimod. Compared with wild-type mice, allergic SLPI transgenic mice showed a decrease in lung resistance (p < 0.001), airway eosinophilia (p < 0.001), goblet cell hyperplasia (p < 0.001), and plasma IgE levels (p < 0.001). Allergic SLPI knockout mice dis...
Journal of Experimental & Clinical Cancer Research, 2012
Background It is mandatory to confirm the absence of mutations in the KRAS gene before treating m... more Background It is mandatory to confirm the absence of mutations in the KRAS gene before treating metastatic colorectal cancers with epidermal growth factor receptor inhibitors, and similar regulations are being considered for non-small cell lung carcinomas (NSCLC) and other tumor types. Routine diagnosis of KRAS mutations in NSCLC is challenging because of compromised quantity and quality of biological material. Although there are several methods available for detecting mutations in KRAS, there is little comparative data regarding their analytical performance, economic merits, and workflow parameters. Methods We compared the specificity, sensitivity, cost, and working time of five methods using 131 frozen NSCLC tissue samples. We extracted genomic DNA from the samples and compared the performance of Sanger cycle sequencing, Pyrosequencing, High-resolution melting analysis (HRM), and the Conformité Européenne (CE)-marked TheraScreen DxS and K-ras StripAssay kits. Results and conclusio...
The solute carrier family 11 member 1 (SLC11A1) gene is strictly regulated and exclusively expres... more The solute carrier family 11 member 1 (SLC11A1) gene is strictly regulated and exclusively expressed in myeloid lineage cells. However, little is known about the transcriptional regulation of the SLC11A1 gene during myeloid development. In this study, we used HL-60 cells as a model to investigate the regulatory elements/factors involved in the transactivation of the SLC11A1 gene during phorbol 12-myristate 13-acetate (PMA)induced macrophage differentiation of HL-60 cells. Promoter deletion analysis showed that a 7-base AP-1-like element (TGACTCT) was critical for the responsiveness of the SLC11A1 promoter to PMA. Stimulation by PMA induced the binding of ATF-3 and the recruitment of two components of the SWI/SNF complex, BRG1 and -actin, to this element in an ATF-3-dependent manner. RNAi-mediated depletion of ATF-3 or BRG1 markedly decreased SLC11A1 gene expression and its promoter activity induced by PMA. Luciferase reporter experiments demonstrated that ATF-3 cooperated with BRG1 and -actin to activate the SLC11A1 promoter. Furthermore, we showed that PMA can induce the proximal (GT/AC) n repeat sequence to convert to the Z-DNA structure in the SLC11A1 gene promoter, and depletion of BRG1 resulted in a significant decrease of Z-DNA formation. Our results demonstrated that recruitment of the SWI/SNF complex initiated Z-DNA formation and subsequently helped to transactivate the SLC11A1 gene.
Uploads
Papers by Yong Zhong Xu