In-house abbreviated qualification of a real-time polymerase chain reaction method and strategies to amplify mycoplasma detection in human mesenchymal stromal cells.

BACKGROUND AIMS: In this study, the authors performed an in-house abbreviated qualification of a commercially available real-time polymerase chain reaction (PCR) kit for limit of detection (LOD), matrix interference and ruggedness of mycoplasma detection in a human bone marrow-derived mesenchymal stromal cell (MSC(M)) investigational cell product (NCT02351011). The approach used was similar to an abbreviated qualification the authors previously conducted for endpoint PCR, which was accepted by Canadian regulators for final product release of the same MSC(M) investigational cell product for treatment of osteoarthritis patients (NCT02351011). With patient consent, biobanked MSCs(M) were re-analyzed by real-time PCR for mycoplasma detection to conduct in-house qualification of the kit. METHODS: LOD was determined by spiking MSCs(M) with a series of 10-fold dilutions of two commercially available genomic DNA (gDNA) reference standards for Mycoplasma arginini (M. arginini) and Mycoplasma hominis (M. hominis). Matrix interference was tested by using 10-fold dilutions of MSC(M)s down to 4500 cells/mL. Polyadenylic acid (poly[A]) was used to improve DNA recovery in samples with 4500-45 000 MSCs(M)/mL. Real-time PCR tests performed on different days were compared to evaluate ruggedness. RESULTS: Real-time PCR analysis showed a conservative LOD of 40 genome copies (GCs)/mL and 240 GCs/mL, which are equivalent to 10 colony-forming units (CFUs)/mL, for M. arginini and M. hominis, respectively. According to a less conservative manufacturer-based criterion for positivity, the kit detected 0.4 GC/mL (0.1 CFU/mL) and 24 GCs/mL (1 CFU/mL) M. arginini and M. hominis, respectively. Real-time PCR with different MSC(M) dilutions did not show matrix interference. However, DNA recovery was compromised at MSC(M) concentrations at or below 45 000 cells/mL. The addition of poly(A) as a DNA carrier improved DNA recovery and allowed an LOD, considered here to be equivalent to 10 CFUs/mL, to be achieved, which was not possible in diluted MSC(M) samples (≤45 000 cells/mL) in the absence of poly(A). Ruggedness was demonstrated with tests (n = 18) performed on different days, with an average overall inter-assay percent coefficient of variation of less than 4 for M. arginini (3.62 [400 GCs/mL], 3.61 [40 GCs/mL]) and less than 3 for M. hominis (2.83 [2400 GCs/mL], 1.95 [240 GCs/mL]). CONCLUSIONS: A commercially available real-time PCR mycoplasma detection kit was qualified for evaluating mycoplasma contamination in investigational MSC(M) products and met the criteria used previously (and accepted by Canadian regulators) for in-house qualification of an endpoint PCR mycoplasma detection kit, and the addition of poly(A) addressed the poor recovery of mycoplasma gDNA in samples with low cell numbers.

Leptin enhances hypothalamic lactate dehydrogenase A (LDHA)-dependent glucose sensing to lower glucose production in high-fat-fed rats.

The responsiveness of glucose sensing per se to regulate whole-body glucose homeostasis is dependent on the ability of a rise in glucose to lower hepatic glucose production and increase peripheral glucose uptake in vivo In both rodents and humans, glucose sensing is lost in diabetes and obesity, but the site(s) of impairment remains elusive. Here, we first report that short-term high-fat feeding disrupts hypothalamic glucose sensing to lower glucose production in rats. Second, leptin administration into the hypothalamus of high-fat-fed rats restored hypothalamic glucose sensing to lower glucose production during a pancreatic (basal insulin)-euglycemic clamp and increased whole-body glucose tolerance during an intravenous glucose tolerance test. Finally, both chemical inhibition of hypothalamic lactate dehydrogenase (LDH) (achieved via hypothalamic LDH inhibitor oxamate infusion) and molecular knockdown of LDHA (achieved via hypothalamic lentiviral LDHA shRNA injection) negated the ability of hypothalamic leptin infusion to enhance glucose sensing to lower glucose production in high fat-fed rats. In summary, our findings illustrate that leptin enhances LDHA-dependent glucose sensing in the hypothalamus to lower glucose production in high-fat-fed rodents in vivo.

Expression Levels of the Tyrosine Hydroxylase Gene and Histone Modifications Around its Promoter in the Locus Coeruleus and Ventral Tegmental Area of Rats during Forced Abstinence from Morphine.

BACKGROUND: Epigenetic mechanisms such as histone modifications may be involved in the structural and behavioral changes associated with addiction. We studied whether morphine-induced changes in mRNA levels of the catecholamine biosynthesis enzyme, tyrosine hydroxylase (TH), are associated with histone modifications around the promoter of this gene in the locus coeruleus (LC) and ventral tegmental area (VTA) of rats. METHODS: Dependence was induced in rats by intraperitoneal injections of morphine for 11 days. The animals were killed 2 h (chronic morphine), 24 h and 7 days (spontaneous withdrawal) after the last injection of morphine. RESULTS: Analysis of our real-time quantitative reverse transcription PCR results by 1-way ANOVA showed significant upregulation (5.13 ± 0.39 folds) of LC levels of the TH transcript 24 h after the last injection of morphine to rats, when compared with 2 h and 7 days time points. Chronic morphine and morphine abstinence failed to cause any significant changes in the levels of TH mRNA in the VTA after cessation of morphine. Consistently, chromatin immunoprecipitation real-time quantitative PCR assays revealed that 24 h after the last injection of morphine, levels of H3 acetylation were significantly increased (4.12 ± 0.38 folds) at the promoter of the TH gene in the LC but not in the VTA. Our data also showed that histone H3 trimethylation failed to change around the TH gene promoter either in the VTA or in the LC after morphine abstinence. CONCLUSIONS: Results of the present study, for the first time, demonstrate the involvement of histone H3 acetylation in the regulation of TH gene expression in the LC of rats during forced abstinence from morphine.

The Study of DNA Methyltransferase-3B Promoter Variant Genotype among Iranian Sporadic Breast Cancer Patients.

BACKGROUND: DNA methyltransferase-3B (DNMT3B) is an important enzyme responsible for maintaining the DNA methylation pattern in eukaryotic cells. In this study we have investigated the correlation between the 46359C→T polymorphism in the DNMT3B gene and the risk of breast cancer incidence among sporadic breast cancer patients in Fars Province, Southern Iran. METHODS: In this case-control study, 100 breast cancer patients and 138 healthy control subjects were genotyped for the DNMT3B gene by the polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: The genotype frequency in the case (CC 27%, CT 47%, TT 26%) group significantly (P=0.008) differed from the control (CC 19.56%, CT 67.3%, TT 13%) group. We observed a decreased association between the CT genotype and lymph node involvement in breast cancer patients. Our results have shown that in comparison to the homozygous CC genotype carriers the DNMT3B-CT genotype has a significantly lower risk for breast cancer (OR=0.515, 95% CI=0.267-0.994, P=0.048). CONCLUSION: Our case-control study showed that the CT genotype was significantly associated with decreased breast cancer risk. Consistent with these results, a significant decrease of CT genotype among lymph node positive breast cancer patients was observed. However, a larger study population with more clinical data is needed to confirm these results.

Investigating protease-mediated peptides of inflammation and tissue remodeling as biomarkers associated with flares in psoriatic arthritis.

BACKGROUND: Psoriatic arthritis (PsA) is an inflammatory arthritis associated with psoriasis. PsA disease involves flares, which are associated with increased joint inflammation and tissue remodeling. There is a need for identifying biomarkers related to PsA disease activity and flares to improve the management of PsA patients and decrease flares. The tissue turnover imbalance that occurs during the inflammatory and fibro-proliferative processes during flares leads to an increased degradation and/or reorganization of the extracellular matrix (ECM), where increased proteolysis plays a key role. Hence, protease-mediated fragments of inflammatory and tissue-remodeling components could be used as markers reflecting flares in PsA patients. METHODS: A broad panel of protease-mediated biomarkers reflecting inflammation and tissue remodeling was measured in serum and synovial fluid (SF) obtained from PsA patients experiencing flares (acutely swollen joint[s], PsA-flare). In serum, biomarker levels assessed in PsA-flare patients were compared to controls and in early-diagnosed PsA patients not experiencing flares (referred to as PsA without flare). Furthermore, the biomarker levels assessed in SF from PsA-flare patients were compared to the levels in SF of osteoarthritis (OA) patients. RESULTS: In serum, levels of the PRO-C3 and C3M, reflecting formation and degradation of the interstitial matrix, were found significantly elevated in PsA-flare compared to controls and PsA without flare. The remodeling marker of the basement membrane, PRO-C4, was significantly elevated in PsA-flare compared to PsA without flare. The inflammation and immune cell activity related markers, CRPM, VICM, and CPa9-HNE were significantly elevated in PsA-flare patients compared to controls and PsA without flare. In addition, VICM (AUC = 0.71), CPa9-HNE (AUC = 0.89), CRPM (AUC = 0.76), and PRO-C3 (AUC = 0.86) showed good discriminatory performance for separating PsA-flare from PsA without flare. In SF, the macrophage activity marker, VICM, was significantly elevated whereas the type II collagen formation marker, PRO-C2, was significantly reduced in the PsA-flare compared to OA. The combination of five serum markers reflecting type III and IV collagen degradation (C3M and C4M, respectively), type III and VI collagen formation (PRO-C3 and PRO-C6, respectively), and neutrophil activity (CPa9-HNE) showed an excellent discriminatory performance (AUC = 0.98) for separating PsA-flare from PsA without flares. CONCLUSIONS: The serum biomarker panel of C3M, C4M, PRO-C3, PRO-C6, and CPa9-HNE reflecting synovitis, enthesitis, and neutrophil activity may serve as novel tool for quantitatively monitoring flares in PsA patients.

Mutant p53 binds to estrogen receptor negative promoter via DNMT1 and HDAC1 in MDA-MB-468 breast cancer cells.

DNA methylation and histone deacetylation are two epigenetic mechanisms involved in the lack of estrogen receptor (ER) expression. Our previous studies demonstrated that mutant p53 along with repression complex proteins including DNMT1, HDAC1 and MeCP2 is associated with ER-negative promoter in MDA-MB-468 cells. To elucidate the molecular mechanism of estrogen receptor 1 (ESR1) gene silencing in these cells, we down-regulated DNMT1 and HDAC1 expression using siRNAs and studied the ability of DNMT1, HDAC1, MeCP2 and p53 in binding to ESR1 promoter CpG island. Our results showed that DNMT1 or HDAC1 down-regulation disassembled the repression complex proteins and mutant p53 from ER-negative promoter. The partial demethylation of ESR1 promoter and ER re-expression in down-regulated cells supports these findings. In vivo binding studies demonstrated that mutation of p53 protein in this cell line did not affect its binding capacity to DNMT1, HDAC1 and MeCP2 proteins. Our observations suggest that not only histone deacetylase activity of HDAC1 contributes to inactivation of methylated ESR1 gene but also HDAC1 presence on ESR1 promoter is important for assembly of DNMT1 in repression complex. In addition, our data revealed that mutant p53 protein binds to the promoter of ESR1 through direct interaction with HDAC1 and indirect interaction with DNMT1, MeCP2 proteins in the ER-negative MDA-MB-468 cells.

Expression levels of the BDNF gene and histone modifications around its promoters in the ventral tegmental area and locus ceruleus of rats during forced abstinence from morphine.

Brain-derived neurotrophic factor (BDNF) plays a role in mediating molecular, cellular, and behavioral adaptations underlying drug addiction. Here, we examined the influence of withdrawal from repeated morphine treatment on the expression of BDNF mRNA in the ventral tegmental area (VTA) and locus coeruleus (LC) of the rat brain. We also studied whether alternations in mRNA levels of BDNF in these tissues are associated with histone modifications around promoters II and III of the BDNF gene. Thus, chromatin immunoprecipitation (CHIP) and quantitative (q)-PCR were employed to assess acetylation of histone H3 at K9/K14 and trimethylation of histone H3 at K9. Results of qRT-PCR showed that levels of BDNF mRNA in both VTA and LC were significantly increased 7 days rather than 2 h or 24 h following the last injection of morphine. Consistently, CHIP and qPCR analysis revealed that on day 7 of morphine abstinence, both VTA and LC levels of histone methylation at BDNF promoters II and III of morphine treated rats were significantly lower than control animals. Morphine withdrawal caused only a significant increase in H3 acetylation at the promoter II in the LC. These data demonstrate the involvement of histone H3 methylation in the regulation of gene expression in the VTA and LC of rats during forced abstinence of morphine.

Epigenetic and genetic analysis of WNT signaling pathway in sporadic colorectal cancer patients from Iran.

The WNT signaling is deregulated in most human colorectal cancers (CRC). Promoter methylation has been proposed as an alternative mechanism to inactivate genes in tumors. To gain insight into the methylation silencing of the WNT pathway during colorectal carcinogenesis, we examined the aberrant methylation profile of four genes, APC, Axin1, Axin2, and GSK3ß in an unselected series of 112 sporadic colorectal tumors by methylation specific PCR. It has been suggested that the Axin2 C148T SNP is associated with the risk of developing certain types of cancers. To assess the contribution of Axin2 SNP to CRC susceptibility, we examined the Axin2 C148T genotype in CRC patients and 170 healthy controls by PCR-RFLP. The frequency of CRCs with at least one gene methylated was 18.75%. Promoter methylation of Axin2 and APC genes was detected in 7.1 and 11.9% of tumors, respectively. No aberrant methylation was found in Gsk3ß and Axin1 gene in these tumor series. The methylation status of APC had no significant association with clinical parameters. But, promoter methylation of Axin2 was sex-related, occurring more frequently in females (P = 0.002). The frequency of Axin2 C148T genotypes were similar in patients and controls. Moreover, we observed no association between the Axin2 SNP and risk of CRC in patients stratified by age, sex, and smoking status. However, the heterozygote CT genotype was associated with a reduced CRC risk in distal patients compared with proximal patients (OR = 0.3; 95% CI 0.1-0.9, P = 0.04). Our findings indicate that Axin1 and GSK3ß methylation play a minor role in colorectal carcinogenesis.

Effect of oral resveratrol on the BDNF gene expression in the hippocampus of the rat brain.

Resveratrol is a plant polyphenolic compound. Evidence indicates that resveratrol has beneficial effects against aging and neurodegenerative diseases. The goal of our study was in vivo examination of the effects of resveratrol on the abundance of mRNA encoding Brain Derived Neurotrophic Factor (BDNF) in the hippocampus of rat brain. Rats were administrated orally by different doses (2.5-20 mg/kg bwt) of resveratrol for 3, 10 and 30 days. Saline was used as control and 10% ethanol in saline was used as vehicle for resveratrol. Measurement of BDNF mRNA by Real-time RT-PCR showed that levels of the mRNA for BDNF were significantly and dose dependently elevated in the hippocampal tissues of rats. The findings suggest that the neuroprotective effects of resveratrol may be at least partly due to its inducing effects on the expression levels of the BDNF mRNA.

Corrigendum: Proteinase-Mediated Macrophage Signaling in Psoriatic Arthritis.

[This corrects the article DOI: 10.3389/fimmu.2020.629726.].

Proteinase-Mediated Macrophage Signaling in Psoriatic Arthritis.

Objective: Multiple proteinases are present in the synovial fluid (SF) of an arthritic joint. We aimed to identify inflammatory cell populations present in psoriatic arthritis (PsA) SF compared to osteoarthritis (OA) and rheumatoid arthritis (RA), identify their proteinase-activated receptor 2 (PAR2) signaling function and characterize potentially active SF serine proteinases that may be PAR2 activators. Methods: Flow cytometry was used to characterize SF cells from PsA, RA, OA patients; PsA SF cells were further characterized by single cell 3'-RNA-sequencing. Active serine proteinases were identified through cleavage of fluorogenic trypsin- and chymotrypsin-like substrates, activity-based probe analysis and proteomics. Fluo-4 AM was used to monitor intracellular calcium cell signaling. Cytokine expression was evaluated using a multiplex Luminex panel. Results: PsA SF cells were dominated by monocytes/macrophages, which consisted of three populations representing classical, non-classical and intermediate cells. The classical monocytes/macrophages were reduced in PsA compared to OA/RA, whilst the intermediate population was increased. PAR2 was elevated in OA vs. PsA/RA SF monocytes/macrophages, particularly in the intermediate population. PAR2 expression and signaling in primary PsA monocytes/macrophages significantly impacted the production of monocyte chemoattractant protein-1 (MCP-1). Trypsin-like serine proteinase activity was elevated in PsA and RA SF compared to OA, while chymotrypsin-like activity was elevated in RA compared to PsA. Tryptase-6 was identified as an active serine proteinase in SF that could trigger calcium signaling partially via PAR2. Conclusion: PAR2 and its activating proteinases, including tryptase-6, can be important mediators of inflammation in PsA. Components within this proteinase-receptor axis may represent novel therapeutic targets.

Association between HIC1 and RASSF1A promoter hypermethylation with MTHFD1 G1958A polymorphism and clinicopathological features of breast cancer in Iranian patients.

BACKGROUND: Ras-associated domain family 1 (RASSF1A) and hypermethylated in cancer (HIC1) genes are methylated more frequently in breast cancer. Genetic factors that alter the DNA methylation levels in normal and tumor tissues could therefore influence the susceptibility to this tumor phenotype. We determined the frequency of aberrant methylation of HIC1 and RASSF1A gene promoters and their association with methylene tetrahydrofolate dehydrogenase (MTHFD1) G1958A polymorphism and major clinical and pathological features of breast cancer in Iranian women. METHODS: DNA was extracted from 81 primary breast tumors and 100 control blood samples. Gene promoter methylation was analyzed by methylation-specific polymerase chain reaction. RESULTS: Eighty four percent of the breast cancer samples showed total methylation in at least one of two tested loci. We detected HIC1 hypermethylation in 79% of invasive and metastasis tumors and RASSF1A gene hypermethylation in 51% of them. We found no association between HIC1 and RASSF1A gene hypermethylation and MTHFD1 G1958A polymorphism, but a significant correlation between methylation of HIC1 and RASSF1A promoters was indicated (r = 0.24, P = 0.02). There was a combination between hypermethylation of HIC1 locus and nodal involvement in the studied population (p=0.03). We found a significant association between total methylation and nodal involvement (P = 0.01) as well as tumor size more than 2 cm in all cases (P = 0.02). CONCLUSION: Methylation of HIC1 and RASSF1A promoters can be used as epigenetic markers to detect the malignant progression of breast carcinoma in Iranian women patients.

Expression and Activity of Platelet Endothelial Nitric Oxide Synthase Are Decreased in Patients with Coronary Thrombosis and Stenosis.

BACKGROUND: Nitric Oxide (NO) which is synthesized by endothelial Nitric Oxide Synthase (eNOS) in both vascular tissues and platelets plays an important role as a protective mediator in the cardiovascular system. It modulates blood pressure, vasodilation and thrombosis. In this regard, eNOS activity and gene expression in platelets and NO levels in patients' plasmas with Coronary Thrombosis (CT) and stenosis diseases were determined. METHODS: Blood samples were collected from 60 subjects that where divided into three equal groups [without coronary artery disease, with CT and less than 70% Coronary Stenosis (CS)]. NO concentration in plasma was measured by the Griess reagent system. The eNOS activity was assessed based on a fluorimetric detection system in platelets and the gene expression was quantified by the real time-reverse transcription-polymerase chain reaction. RESULTS: There was a significant decrease in the amount of NO concentration in the plasma of subjects with CT (0.53±0.09 µM, p<0.01) and CS (1.31±0.11 µM, p<0.01) compared to the control group (2.6±0.10 µM). The activity levels of eNOS enzyme were significantly lower in patients' platelets with CT (0.68±0.013 UF/mn, p<0.01) and CS (0.85±0.017 UF/mn, p<0.01) than the control cases (1.29±0.019 UF/mn). These data were consistent with the reduction of the expression levels of eNOS in patients with CT (75 folds) and CS (4 folds) lower than the control cases. CONCLUSION: Patients with CT and CS possessed reduced eNOS activity and gene expression in their platelets. Decreased plasma concentration of NO in these patients confirmed the potential significance of the diagnostic and prognostic value of NO in the subjects' plasma with vascular disease risk.

Inhibition of upper small intestinal mTOR lowers plasma glucose levels by inhibiting glucose production.

Glucose homeostasis is partly controlled by the energy sensor mechanistic target of rapamycin (mTOR) in the muscle and liver. However, whether mTOR in the small intestine affects glucose homeostasis in vivo remains unknown. Here, we first report that delivery of rapamycin or an adenovirus encoding the dominant negative acting mTOR-mutated protein into the upper small intestine is sufficient to inhibit small intestinal mTOR signaling and lower glucose production in rodents with high fat diet-induced insulin resistance. Second, we found that molecular activation of small intestinal mTOR blunts the glucose-lowering effect of the oral anti-diabetic agent metformin, while inhibiting small intestinal mTOR alone lowers plasma glucose levels by inhibiting glucose production in rodents with diabetes as well. Thus, these findings illustrate that inhibiting upper small intestinal mTOR is sufficient and necessary to lower glucose production and enhance glucose homeostasis, and thereby unveil a previously unappreciated glucose-lowering effect of small intestinal mTOR.

Lactobacillus gasseri in the Upper Small Intestine Impacts an ACSL3-Dependent Fatty Acid-Sensing Pathway Regulating Whole-Body Glucose Homeostasis.

Long-chain acyl-CoA synthetase (ACSL)-dependent upper small intestinal lipid metabolism activates pre-absorptive pathways to regulate metabolic homeostasis, but whether changes in the upper small intestinal microbiota alter specific fatty acid-dependent pathways to impact glucose homeostasis remains unknown. We here first find that upper small intestinal infusion of Intralipid, oleic acid, or linoleic acid pre-absorptively increases glucose tolerance and lowers glucose production in rodents. High-fat feeding impairs pre-absorptive fatty acid sensing and reduces upper small intestinal Lactobacillus gasseri levels and ACSL3 expression. Transplantation of healthy upper small intestinal microbiota to high-fat-fed rodents restores L. gasseri levels and fatty acid sensing via increased ACSL3 expression, while L. gasseri probiotic administration to non-transplanted high-fat-fed rodents is sufficient to restore upper small intestinal ACSL3 expression and fatty acid sensing. In summary, we unveil a glucoregulatory role of upper small intestinal L. gasseri that impacts an ACSL3-dependent glucoregulatory fatty acid-sensing pathway.

Dynamin-Related Protein 1-Dependent Mitochondrial Fission Changes in the Dorsal Vagal Complex Regulate Insulin Action.

Mitochondria undergo dynamic changes to maintain function in eukaryotic cells. Insulin action in parallel regulates glucose homeostasis, but whether specific changes in mitochondrial dynamics alter insulin action and glucose homeostasis remains elusive. Here, we report that high-fat feeding in rodents incurred adaptive dynamic changes in mitochondria through an increase in mitochondrial fission in parallel to an activation of dynamin-related protein 1 (Drp1) in the dorsal vagal complex (DVC) of the brain. Direct inhibition of Drp1 negated high-fat-feeding-induced mitochondrial fission, endoplasmic reticulum (ER) stress, and insulin resistance in the DVC and subsequently restored hepatic glucose production regulation. Conversely, molecular activation of DVC Drp1 in healthy rodents was sufficient to induce DVC mitochondrial fission, ER stress, and insulin resistance. Together, these data illustrate that Drp1-dependent mitochondrial fission changes in the DVC regulate insulin action and suggest that targeting the Drp1-mitochondrial-dependent pathway in the brain may have therapeutic potential in insulin resistance.

Additive effect of recombinant Mycobacterium tuberculosis ESAT-6 protein and ESAT-6/CFP-10 fusion protein in adhesion of macrophages through fibronectin receptors.

BACKGROUND/PURPOSE: Tuberculous granulomas are the sites of interaction between the T cells, macrophages, and extracellular matrix (ECM) to control the infection caused by Mycobacterium tuberculosis (M. tuberculosis). A predominant role of RD-1-encoded secretory proteins, early secreted antigenic target-6 (ESAT-6), and culture filtrate protein-10 (CFP-10) in the formation of granulomas has recently been emphasized. However, the precise molecular events that induce the formation of these granulomatous structures are yet to be elucidated. Macrophages use integrins to adhere to fibronectin (FN) as a major component of the ECM. The major goal of this study was to investigate whether recombinant M. tuberculosis antigens can modulate integrin-mediated macrophage adhesion. METHODS: Differentiated THP-1 cell line was stimulated with recombinant ESAT-6, CFP-10, and ESAT-6/CFP-10 proteins and evaluated for alterations in the expression levels of α5ß1 and α4ß1 by semiquantitative real-time polymerase chain reaction. The role of these recombinant antigens in the cytoskeleton rearrangement was determined by adhesion assay and immunofluorescent microscopy. RESULTS: Our data showed that ESAT-6 and ESAT-6/CFP-10 fusion proteins could induce adhesion of macrophages to FN through α4ß1 integrin. An increased expression level of α4ß1 integrin in comparison with α5ß1 integrin in differentiated THP-1 cells was also observed. Results of immunofluorescence studies showed that recombinant proteins-treated THP-1 cells form well-organized stress fibers and focal contacts containing vinculin compared with untreated THP-1 cells. CONCLUSION: Increased expression level of α4ß1 in differentiated THP-1 cells could suggest the important role of α4ß1 integrin in adhesion and focal contact formation of macrophages exposed to M. tuberculosis antigens.

TP53 Binding to BRCA1 and RAD51 in MCF7 and MDA-MB-468 Breast Cancer Cell Lines In vivo and In vitro.

BACKGROUND: Tumour suppressor genes such as TP53, BRCA1 and RAD51 are involved in DNA repair and their malfunctions result in genomic instability and cancer. Wild type (WT) TP53 binds to BRCA1and RAD51 in vivo and in vitro. However, mutated TP53 in tumours can interfere with WT TP53 function. We studied how mutation of TP53 in MDA-MB-468 cell line could affect its binding capacity and interfere with WT TP53 interaction with these DNA repair proteins. METHODS: Binding capacity of mutated TP53 in MDA-MB-468 breast cancer cell line to BRCA1 and RAD51 proteins in comparison to WT TP53 in MCF7 cell line was studied by Immunoprecipitation. In vitro studies were performed by GST-WT p53 pull-down assays in these cell lines to assess the interaction of GST-WT p53 with BRCA1 and RAD51 proteins. RESULTS: The results showed that mutated TP53 in MDA-MB-468 cells interacted with BRCA1 protein in vivo and did not effect WT TP53 binding to this protein in vitro. The Immunoprecipitation assays revealed that the mutated TP53 did not bind to RAD51 in comparison to WT TP53. However, this mutated protein could not interfere with binding of RAD51 to GST-WT p53 in MDA-MB-468 cell line by in vitro experiment. CONCLUSION: It was found that WT TP53 interactions with BRCA1 and RAD51 did not interfere with mutated TP53 in MDA-MB-468 cell line. In addition, RAD51 did not bind to TP53 with R273C mutation in vivo.

UBE2Q1 in a Human Breast Carcinoma Cell Line: Overexpression and Interaction with p53.

The p53 tumor suppressor protein is a principal mediator of growth arrest, senescence, and apoptosis in response to a broad array of cellular damage. p53 is a substrate for the ubiquitin-proteasome system, however, the ubiquitin-conjugating enzymes (E2s) involved in p53 ubiquitination have not been well studied. UBE2Q1 is a novel E2 ubiquitin conjugating enzyme gene. Here, we investigated the effect of UBE2Q1 overexpression on the level of p53 in the MDA-MB-468 breast cancer cell line as well as the interaction between UBE2Q1 and p53. By using a lipofection method, the p53 mutated breast cancer cell line, MDA-MB-468, was transfected with the vector pCMV6-AN-GFP, containing UBE2Q1 ORF. Western blot analysis was employed to verify the overexpression of UBE2Q1 in MDA-MB-468 cells and to evaluate the expression level of p53 before and after cell transfection. Immunoprecipitation and GST pull-down protocols were used to investigate the binding of UBE2Q1 to p53. We established MDA-MB-468 cells that transiently expressed a GFP fusion proteins containing UBE2Q1 (GFP-UBE2Q1). Western blot analysis revealed that levels of p53 were markedly lower in UBE2Q1 transfected MDA-MB-468 cells as compared with control MDA-MB-468 cells. Both in vivo and in vitro data showed that UBE2Q1 co-precipitated with p53 protein. Our data for the first time showed that overexpression of UBE2Q1can lead to the repression of p53 in MDA-MB-468 cells. This repression of p53 may be due to its UBE2Q1 mediated ubiquitination and subsequent proteasome degradation, a process that may involve direct interaction of UBE2Q1with p53.

Quantitative Analysis of ErbB1 and ErbB2 Genes Amplification by a High Performance Liquid Chromatography.

BACKGROUND: Genes for human epidermal growth factor receptors B1 (ErbB1) and B2 (ErbB2) were amplified in breast and ovarian cancers. Both of them were associated with aggressive disease and worse prognosis. The ErbB1 or ErbB2 status of a tumor may provide an indication of the response to ErbB1 and ErbB2 -targeted therapies. For accurate and rapid assessment of amplification of ErbB1 and ErbB2 oncogenes, a High Performance Liquid Chromatography (HPLC) method was developed in this study. METHODS: DNA was extracted from 30 primary breast tumors and 20 blood samples of healthy donors. ErbB1 and ErbB2 genes along with a reference gene were co-amplificated by Polymerase Chain Reaction (PCR). The PCR products were separated and quantified using an anion-exchange column within 30 min and in a single step. Optimum resolution was obtained when a sodium chloride gradient and a column temperature of 35°C were used. The results of HPLC analysis of ErbB1 and ErbB2 PCR products were compared with real time PCR method as a gold standard test for 7 tumor samples. RESULTS: The proposed HPLC method was confirmed by real time PCR method. Twenty two and ten of the specimens in our breast cancer cohort showed more than a two-fold amplification of ErbB2 and ErbB1 oncogenes, respectively. CONCLUSION: Our results were confirmed by real time PCR and showed that HPLC method is a specific, cheap and clinically applicable analytical approach for assessment of ErbB1 and ErbB2 statuses in breast tumors.