Fold Increase In Gene Expression Research Articles

Study the Gene Expression of PIK3CA in Tissue and Blood Samples of Iraqi Patients with Colorectal Cancer

Objective: The primary objective of this investigation is to explore the correlation between gene expression of PIKCA3 and colorectal cancer in both tissue and blood samples, while also performing a comparative analysis between these sample types.
 Methods: This research involved the collection of blood and tissue samples. The study was conducted at the Gastroenterology Hospital within the Medical City Department in Baghdad, Iraq, spanning from December 2021 to December 2022. A total of 80 volunteers, encompassing both genders and aged between 24 and 77, participated in the study. The Real Time qRT-PCR analysis method was employed to assess the expression of PIKCA3 genes within patient and control groups.
 Results: The findings indicate significant distinctions in Ct PIK3CA between blood and tissue samples across all study groups. Moreover, PIK3CA expression displayed a notable association with CRC patients, showing higher expression levels compared to the other groups. This disparity was particularly pronounced when comparing tissue and blood samples. Statistical analysis, specifically Receiver Operating Characteristic (ROC) Curve Analysis, affirmed the excellent predictive capacity of the AUC (Area Under the Curve) value. The study established that PIK3CA gene expression in tissue outperformed that in blood, demonstrating a considerably elevated gene expression in tissue (p<0.001). Notably, the fold increase in gene expression within CRC patients was significantly higher compared to the group of healthy participants, with values of 23.630 in blood samples and 124.810 in tissue samples, respectively. The present study underscores the prevalence of PIK3CA gene mutations in colorectal carcinomas.
 Conclusion: The frequent occurrence of PIK3CA gene mutations in colorectal carcinomas may hold promise for the development of targeted therapy combinations. These could involve inhibiting both the PI3K kinase itself and addressing associated pathway anomalies.

Abstract 2595: Role of VEGF/VEGFR autocrine in mediating EGFR TKI resistance in non-small cell lung cancer

Abstract The treatment paradigm for late stage non-small cell lung carcinoma (NSCLC) has shifted towards molecular targeted therapy like epidermal growth factor receptor (EGFR), one of the most studied targets. Tyrosine kinase inhibitors (TKIs) against EGFR have proven to be effective, however TKI resistance is common due to the acquisition of the T790M mutation in the EGFR kinase domain. There are currently very few studies suggesting the role of VEGFR in mediating erlotinib resistance. We suggest that over-expression of vascular endothelial growth factor receptors (VEGFRs) in tumors may create an autocrine loop that may mediate TKI resistance. The purpose of this study is to investigate the VEGF/VEGFR autocrine loop as a potential mechanism of TKI resistance via alternative signaling. H2170 NSCLC parental cells were obtained from ATCC. The resistant cells were obtained by treating parental cells with increasing concentrations of erlotinib. DNA sequencing was performed to exclude the presence of the T790M mutation. Quantitative real-time PCR was carried out for target genes coding for VEGF, VEGFR-2, and neuropilin-1 (NP-1). Fluorescence-activated cell sorting (FACS) analysis and immunofluorescence (IF) analysis were performed with antibodies against VEGFR-2 and NP-1. The viability of resistant cells was assessed after being treated with increasing concentrations of a VEGF inhibitor (VEGF neutralizing antibody) in medium with or without erlotinib using a cell proliferation assay (MTT). Thirty paraffin embedded tumor biopsies were immunostained (IHC) with an antibody against VEGFR-2. On the transcriptional level, real-time PCR revealed a 13.1, 3.4 and a 2.9 fold increase in gene expression for VEGF, VEGFR-2 and NP-1 respectively in the resistant cells compared to the parental cells. When looking at membrane bound receptors, FACS analysis showed a 2.1 and a 3.8 fold increase in the expression of VEGFR and NP-1 in resistant cells, respectively. IF studies correlated with the FACS results, showing significantly higher expression of both VEGFR-2 and NP-1 in the resistant cells. MTT assays demonstrated that the VEGF inhibitor was able to decrease cell death from 10 to 80% (0.1-10ug/ml) as evidenced by decreasing viability of resistant cells treated with increasing concentrations of VEGF inhibitor compared to the untreated control. Kaplan-Meier analysis revealed shorter median survival with high vs low expression of total VEGFR-2 expression (7mo vs 25mo, p=0.05) in NSCLC tumors in patients diagnosed with late stage NSCLC. Our results suggest that by up-regulating the VEGF/VEGFR autocrine system as an alternative signaling pathway to promote proliferation, tumor cells may bypass the inhibitory effect of TKI's on the EGFR pathway, leading to resistance. Furthermore, IHC demonstrated the presence of high expression of VEGFR-2 in tumors, suggesting its potential as a prognostic biomarker for lung cancer. Citation Format: Chike Osude, Leo Lin, Brad Foster, Neelu Puri. Role of VEGF/VEGFR autocrine in mediating EGFR TKI resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2595.

Profile of Gene Expression in the Kidneys of Mice with the insrr Gene Knockout

The insulin receptor-related receptor (IRR) is a receptor tyrosine kinase and a close homologue of the insulin receptor and the insulin-like growth factor receptor; however, peptide and protein ligands for this receptor have not been identified. IRR is encoded by the insrr gene and is found in some cell populations of kidneys, stomach, pancreas, as well as in some sympathetic and cholinergic neurons. In the present study, a comparative analysis of the transcriptomes of kidneys from wild-type mice and IRR-knockout mice has been performed by the method of deep sequencing and by the analysis of gene expression on microarrays and in real-time PCR. A significant change (a more than 1.5fold increase in gene expression in knockout animals) was found for the transcripts of two genes: hsd3b2 and igf2. The results suggest that the line of IRR-knockout mice can find application as an animal model in studies of the role of these genes in the kidney.

One-Step piggyBac Transposon-Based CRISPR/Cas9 Activation of Multiple Genes.

Neural cell fate is determined by a tightly controlled transcription regulatory network during development. The ability to manipulate the expression of multiple transcription factors simultaneously is required to delineate the complex picture of neural cell development. Because of the limited carrying capacity of the commonly used viral vectors, such as lentiviral or retroviral vectors, it is often challenging to perform perturbation experiments on multiple transcription factors. Here we have developed a piggyBac (PB) transposon-based CRISPR activation (CRISPRa) all-in-one system, which allows for simultaneous and stable endogenous transactivation of multiple transcription factors and long non-coding RNAs. As a proof of principle, we showed that the PB-CRISPRa system could accelerate the differentiation of human induced pluripotent stem cells into neurons and astrocytes by triggering endogenous expression of different sets of transcription factors. The PB-CRISPRa system has the potential to become a convenient and robust tool in neuroscience, which can meet the needs of a variety of in vitro and in vivo gain-of-function applications.

Porous polymer scaffold for on-site delivery of stem cells – Protects from oxidative stress and potentiates wound tissue repair

Wound healing by cell transplantation techniques often suffer setbacks due to oxidative stress encountered at injury sites. A porous polyethyleneglycol–polyurethane (PEG–PU) scaffold that facilitates cell delivery and boosts tissue repair was developed through semi-interpenetrating polymer network approach. The key physico-chemical properties assessed confirms these polymeric matrices are highly thermostable, barostable, degrade at an acidic pH (5.8), biodegradable, cytocompatible and possess excellent porosity. Mechanism of cellular penetration into porous polymer networks was evident by a ≥6 – fold increase in gene expression of MMP-13 and MMP-2 via activation of Akt and Erk. H2O2-induced apoptosis of mouse bone marrow stem cells (BMSCs) was abrogated in presence of polymer networks indicating a protective effect from oxidative stress. Transplantation of BMSC + PEG–PU at murine excisional splint wound site depicted significant increase in fibroblast proliferation, collagen deposition, anti-oxidant enzyme activities of catalase, SOD and GPx. Furthermore it significantly decreased expression of pro-inflammatory cytokines (IL-1β, TNF-α, IL-8, etc) with a concomitant increase in anti-inflammatory cytokines (IL-10, IL-13) at an early healing period of day 7. Finally, immunostaining revealed an enhanced engraftment and vascularity indicating an accelerated wound tissue closure. This pre-clinical study demonstrates the proof-of-concept and further necessitates their clinical evaluation as potential cell delivery vehicle scaffolds.

Influence of detoxified neem seed cake on diet digestibility, body weight change, hormonal profiles, immune response and testicular gene expression in male sheep

The present study was conducted to find out the effect of supplementing defatted and detoxified neem (Azadirachta indica) seed cake as a partial replacement of soybean meal (SBM) in the diet of growing lambs. Male lambs of uniform body weight (8.31±0.32kg) were randomly allotted to three groups such as control (diet containing SBM) and two test diets with defatted and detoxified neem cake (DNC) at two levels (34g and 68g per kg of diet) replacing 25% and 50% nitrogen of SBM (DNC-25 and DNC-50). The respective diets (concentrate mixtures along with finger millet (Eleucine corocana) straw) were fed individually to lambs for a period of 140 days. The repeated measure ANOVA indicated that lambs on DNC treatments grew on par with control across all the periods and no interaction was observed. The difference in the dry matter intake was not found to be significant (P>0.05) in any of the three study groups. Similarly apparent nutrient digestibility, nitrogen balance, rumen and hormonal profiles did not differ (P>0.05) across the groups. Humoral and cell mediated immune response measured in terms of antibody titre and lymphocyte proliferation assay was also the same (P>0.05) across the groups. The relative gene expression of IGF-I and LHR transcripts in testes tissue was positively influenced (P<0.05) in DNC-50 treatment (3–4 fold increase) as compared to control and DNC-25. No histopathological changes were observed in vital organs of sheep fed DNC diets compared to control. It can be concluded that based on the studied parameters, DNC can replace 50% of SBM nitrogen with 3–4 fold increase in gene expression in testes tissue.

Abstract 542: CEACAM6 is upregulated by the H Pylori CAG-A oncoprotein, and is a candidate biomarker for the diagnosis of early gastric cancer

Abstract Early detection of gastric cancers confers a significant mortality benefit, but remains a diagnostic challenge. We aim to identify and develop novel cell-surface biomarkers to improve the efficiency of gastric cancer detection by fluorescent endoscopy. Traditional approaches for surface biomarker discovery compare ‘normal’ and ‘malignant’ cell lines, but are prone to artifacts of cell line derivation and evolution. Our strategy exploits the dependence of gastric cancers on CAG-A, the Helicobacter Pylori oncoprotein. CAG-A incorporation is necessary and sufficient for gastric cancer pathogenesis, and is accordingly observed in early cancer specimens, but is intracellular and not expressed at high enough levels to be an applicable biomarker for cancer diagnosis. We therefore designed a strategy to screen for cell surface alterations induced by CAG-A overexpression, which may be retained in gastric cancers through oncogenic addiction. We used an inducible system for CAG-A expression in gastric MKN28 cells to compare the gene expression profile of cells with and without CAG-A overexpression, on the Affymetrix Gene 1.0 platform. We focused on putative membrane protein transcripts, to identify differentially upregulated candidates in CAG-A overexpressing cells. The top 10 hits from our screen were then analyzed in gene expression and RNAseq datasets from the Singapore Gastric Cancer Consortium, comparing the transcriptome of gastric cancer with adjacent normal tissue. Amongst our top hits in the in-vitro CAG-A screen, we noted a significant enrichment of the cell adhesion molecule CEACAM6 in gastric cancer samples when compared to normal tissue (∼8 fold increase in gene expression by microarray analysis (n = 100 normals, 200 cancers), and ∼200 fold increase in matched RNAseq analysis (n = 10 each)). We confirmed CEACAM6 to be upregulatd by CAG-A using RT-PCR on the inducible CAG-A system, and also in other gastric cell lines infected with H Pylori. Finally, we compared CEACAM6 protein expression on a gastric tissue microarray, using quantitative immunohistochemistry through automated spectral microscopy on the Vectra 2 platform. We confirm a 10-fold increase in CEACAM6 protein in gastric cancers as compared to normal tissue (n = 63 cancer, 52 normal). Fluorescently conjugated antibodies to these cell surface markers bind avidly to freshly resected xenografts (n = 3) of gastric cancer, without fixation. Together, these results suggest that CEACAM6 upregulation is a cell surface response to the introduction of H Pylori CAG-A in gastric epithelial cells, and is retained in a significant fraction of gastric cancers. It appears to be a promising candidate for a fluorescently conjugated biomarker to aid with the endoscopic diagnosis of early gastric cancer. Citation Format: Anand D. Jeyasekharan, Rony K. Roy, Michal M. Hoppe, Kar Tong Tan, Henry Yang, Patrick Tan, Phillip H. Koeffler, Khay Guan Yeoh, Singapore Gastric Cancer Consortium. CEACAM6 is upregulated by the H Pylori CAG-A oncoprotein, and is a candidate biomarker for the diagnosis of early gastric cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 542. doi:10.1158/1538-7445.AM2015-542

Novel mechanism linking noncanonical Wnt ligand induction to suppression of colorectal cancer cell proliferation.

648 Background: Malignant tissues are characterized by uncontrolled proliferation and dysfunctional differentiation. A central pathway in the control of proliferation and differentiation is the Wnt pathway. Wnt function is mediated by a canonical pathway (CP) which utilizes β-catenin and stimulates cell proliferation, and a non-canonical pathway (NCP) independent of β-catenin involved with differentiation. Studies suggest that dysfunctional Wnt signaling results from imbalance of CP components. The possibility that modulation of NCP components are involved in this process has not been studied comprehensively. Given their importance in constraining CP signaling and proliferation, the purpose of this study was to determine the capacity of NCP components to suppress human colorectal cancer (CRC) proliferation. Methods: Short term, primary CRC lines were established from resected tumors from patients with metastatic and/or recurrent disease. CRC were treated with LiCl, an activator of the CP, Dkk1, an inhibitor of the CP, and IWP-2, a pan inhibitor of CP and NCP Wnt ligand secretion. CRC proliferation and Wnt pathway gene expression were determined by MTS assays and quantitative gene expression respectively. Results: Unexpectedly, CRC proliferation was inhibited significantly (p&lt;0.01) by LiCl and stimulated modestly (ns) by Dkk1. This was associated with a 20 fold increase in gene expression for the NCP ligand, Wnt9A. LiCl treated cells incubated with IWP-2 reversed the unexpected LiCl-mediated suppression of CRC proliferation. Conclusions: The unexpected finding that LiCl suppresses proliferation and Dkk1 has the opposite effect indicate dysregulation of the Wnt pathway in CRC. Further support for this hypothesis is the fact that Wnt 9A, an NCP ligand is increased by LiCl in CRC, an effect that is not expected in non-malignant cells. That IWP-2, a pan Wnt ligand secretion inhibitor reverses these effects is consistent with these observations. This study is the first to demonstrate a correlation between NCP ligand induction and suppression of CRC proliferation and suggests that control of CRC proliferation may be achieved in patients by modalities that activate the NCP Wnt pathway.

Comparative Quantitative Gene Expression analysis Between Salinity Tolerant and Susceptible Varieties of Chickpea

Comparative Quantitative Gene Expression analysis Between Salinity Tolerant and Susceptible Varieties of Chickpea Salinity is one of the greatest limiting factors in agriculture. Therefore, the ability of plants to adapt saline conditions is crucial to sustain worldwide crop production. Present study is meant to identify gene specific markers for salt tolerance in Chickpea. Selected chickpea genotypes were already characterized for salt tolerance and susceptibility. Sixteen gene specific primers were designed from already identified abiotic stress resistant genes from database among which five were used for semiquantitative reverse transcription polymerase chain reaction. The expression levels of two of them allow discrimination between tolerant and susceptible genotypes. In tolerant genotypes (JG-62 and Pusa 1053) the expression level of these genes was higher as compared to susceptible genotypes (BG-256 and ICCV2). For primer 13 the relative fold increase in gene expression was 10 times as compared to 2 times in susceptible plants. For primer 6 the relative fold increase in gene expression was 5 folds in Pusa 1053 (tolerant) as compared to 4 folds in other genotypes. Gene expression was observed to be maximum at 200mM concentration in almost all genotypes for above two gene specific primers. So, primer 13 is a potent gene for gene pyramiding in susceptible varieties for salt stress.

Folate-targeted multifunctional amino acid-chitosan nanoparticles for improved cancer therapy.

Tumor targeting nanomaterials have potential for improving the efficiency of anti-tumoral therapeutics. However, the evaluation of their biological performance remains highly challenging. In this study we describe the synthesis of multifunctional nanoparticles decorated with folic acid-PEG and dual amino acid-modified chitosan (CM-PFA) complexed with DNA and their evaluation in organotypic 2D co-cultures of cancer-normal cells and also on 3D multicellular tumor spheroids models. The physicochemical characterization of CM-PFA multifunctional carriers was performed by FTIR, (1)H NMR and DLS. 2D co-culture models were established by using a 1:2 cancer-to-normal cell ratio. 3D organotypic tumor spheroids were assembled using micromolding technology for high throughput screening. Nanoparticle efficiency was evaluated by flow cytometry and confocal microscopy. The CM-PFA nanocarriers (126-176 nm) showed hemocompatibility and were internalized by target cells, achieving a 3.7 fold increase in gene expression. In vivo-mimicking 2D co-cultures confirmed a real affinity towards cancer cells and a negligible uptake in normal cells. The targeted nanoparticles penetrated into 3D spheroids to a higher extent than non-targeted nanocarriers. Also, CM-PFA-mediated delivery of p53 tumor suppressor promoted a decrease in tumor-spheroids volume. These findings corroborate the improved efficiency of this delivery system and demonstrate its potential for application in cancer therapy.

Application of increased temperature from an exogenous source to enhance gene electrotransfer

The presence of increased temperature for gene electrotransfer has largely been considered negative. Many reports have published on the lack of heat from electrotransfer conditions to demonstrate that their effects are from the electrical pulses and not from a rise in temperature. Our hypothesis was to use low levels of maintained heat from an exogenous source to aid in gene electrotransfer. The goal was to increase gene expression and/or reduce electric field. In our study we evaluated high and low electric field conditions from 90V to 45V which had been preheated to 40°C, 43°C, or 45°C. Control groups of non-heated as well as DNA only were included for comparison in all experiments. Luciferase gene expression, viability, and percent cell distribution were measured. Our results indicated a 2–4 fold increase in gene expression that is temperature and field dependent. In addition levels of gene expression can be increased without significant decreases in cell death and in the case of high electric fields no additional cell death. Finally, in all conditions percent cell distribution was increased from the application of heat. From these results, we conclude that various methods may be employed depending on the end user's desired goals. Electric field can be reduced 20–30% while maintaining or slightly increasing gene expression and increasing viability or overall gene expression and percent cell distribution can be increased with low viability.

The limited role of interferon-γ in systemic juvenile idiopathic arthritis cannot be explained by cellular hyporesponsiveness.

Systemic juvenile idiopathic arthritis (JIA) is an autoinflammatory syndrome in which the myelomonocytic lineage appears to play a pivotal role. Inflammatory macrophages are driven by interferon-γ (IFNγ), but studies have failed to demonstrate an IFN- induced gene signature in active systemic JIA. This study sought to characterize the status of an IFN-induced signature within affected tissue and to gauge the integrity of IFN signaling pathways within peripheral monocytes from patients with systemic JIA. Synovial tissue from 12 patients with active systemic JIA and 9 with active extended oligoarticular JIA was assessed by real-time polymerase chain reaction to quantify IFN-induced chemokine gene expression. Peripheral monocytes from 3 patients with inactive systemic JIA receiving anti-interleukin-1β (anti-IL-1β) therapy, 5 patients with active systemic JIA, and 8 healthy controls were incubated with or without IFNγ to gauge changes in gene expression and to measure phosphorylated STAT-1 (pSTAT-1) levels. IFN-induced chemokine gene expression in synovium was constrained in active systemic JIA compared to the known IFN-mediated extended oligoarticular subtype. In unstimulated peripheral monocytes, IFN-induced gene expression was similar between the groups, except that lower levels of STAT1, MIG, and PIAS were observed in patients with active disease, while higher levels of PIAS1 were observed in patients with inactive disease. Basal pSTAT-1 levels in monocytes tended to be higher in systemic JIA patients compared to healthy controls, with the highest levels seen in those with inactive disease. Upon stimulation of monocytes, the fold increase in gene expression was roughly equal between groups, except for a greater increase in STAT1 in patients with inactive systemic JIA compared to controls, and a greater increase in IRF1 in those with active compared to inactive disease. Upon stimulation, the fold increase in pSTAT-1 was highest in monocytes from patients with inactive systemic JIA. Monocytes in patients with active systemic JIA retain the ability to respond to IFNγ, suggesting that the lack of an IFN-induced gene signature in patients with active disease reflects a limited in vivo exposure to IFNγ. In patients with inactive systemic JIA who received treatment with anti-IL-1β, hyperresponsiveness to IFNγ was observed.

CCN3 suppresses mitogenic signalling and reinstates growth control mechanisms in Chronic Myeloid Leukaemia

CCN3, a tumour suppressor gene, is down-regulated as a result of BCR-ABL tyrosine kinase activity in Chronic Myeloid Leukaemia (CML). We have established a stable CCN3 expression model in the human K562 CML cell line and have further validated the role for CCN3 in the leukaemogenic process. K562 cells stably transfected with CCN3 (K562/CCN3; 2.25 × 10(6) copies per 50ng cDNA) demonstrated over 50% reduction in cell growth in comparison to cells stably transfected with empty vector (K562/control; p = 0.005). K562/CCN3 cells had reduced colony formation capacity (reduced by 29.7%, p = 0.03) and reduced mitogenic signalling in comparison to K562/control cells (reduced by 29.5% (p = 0.002) and 37.4% (p = 0.017) for phosphorylation levels of ERK and AKT respectively). K562/CCN3 cells showed an accumulation of events within the subG(0) phase of the cell cycle and increased apoptosis was confirmed by a three-fold increase in annexin V binding (p < 0.05). K562/CCN3 cells exposed to Imatinib (1μM and 5μM) showed an increase in events within the subG(0) phase of cell cycle over 96h and mirrored the enhanced cell kill demonstrated by Annexin staining. Wild type K562 cells treated with recombinant human Ccn3 (10nM) in combination with Imatinib (5μM) also displayed enhanced cell kill (p = 0.008). K562/CCN3 cells displayed increased adhesion to matrigel™ (2.92 ± 0.52 fold increase compared to K562/control) which was commensurate with increased expression of the alpha 6 and beta 4 integrins (6.53 ± 0.47 and 1.94 ± 0.07 fold increase in gene expression respectively (n = 3, p < 0.05)). CCN3 restores cellular growth regulatory properties that are absent in CML and sensitises CML cells to imatinib induced apoptosis. CCN3 may provide novel avenues for the development of alternate therapeutic strategies.

PHF23: A Novel Erythropoietin-Induced Gene Associated with AML and MDS.

Chromosomal translocations in a number of hematopoietic malignancies, including AML and MDS, result in the formation of NUP98 gene chimeras. Recently, fusion of NUP98 with a novel gene, PHF23 or plant homeodomain (PHD) finger 23, was reported in a cryptic translocation in a patient with AML (Reader et al., 2007). PHF23 is one of three known NUP98 fusion partners, associated with AML, which contain PHD finger motifs. In this study, we report a more detailed characterization of PHF23, which we originally identified by differential display PCR as an erythropoietin-induced gene in a murine model of terminal erythroid differentiation. In silico sequence analysis revealed a five exon gene containing a nuclear localization signal (NLS) as well as the C-terminal PHD motif. PHF23 mapped to chromosome 17p13.1, <450 kb from the TP53 gene, in a region frequently disrupted in cancer. Quantitative PCR analysis indicated Phf23 expression is not restricted to hematopoietic cells but demonstrates widespread tissue distribution, with increased expression levels in more proliferative tissues (∼6 fold increase in gene expression in the testis over mRNA levels in the liver). A ubiquitously expressed Phf23 splice variant (with mRNA concentrations ∼30–45% lower than full-length Phf23 gene expression levels) was generated using an alternative 3′ splice site within exon four, which exactly corresponded to the chr17 translocation breakpoint in the AML patient described. PHF23 is evolutionarily conserved, particularly within the PHD finger domain (>76% sequence identity between human and zebrafish PHD motifs), and is significantly related to another potentially oncogenic protein, PHF13/SPOC-1, associated with ovarian cancer. PHD motifs act as zinc fingers and the expanding family of PHD-containing proteins are implicated in chromatin regulation. Cellular localization studies using fluorescently tagged PHF23 indicated that both the full-length and spliced isoforms are located exclusively within the nucleus. We predicted that PHF23 has a similar structure to PHD motifs of known histone effector proteins, which influence transcription through recognition of specific epigenetic marks. This was demonstrated by immunoprecipitation and histone peptide binding assays showing that PHF23 associated with histone H3, primarily di- and tri-methylated H3K4, which are characteristic marks of active transcription. Microarray analysis of PHF23 mRNA expression levels in AML and MDS patient samples indicated no defined association with AML, however a positive correlation was demonstrated with a subset of MDS patients exhibiting erythroid dysplasia. This study suggests that PHF23, a novel histone effector protein, may play a role in aberrant transcription during defective hematopoiesis.

Contribution of Interferon-β to the Murine Macrophage Response to the Toll-like Receptor 4 Agonist, Lipopolysaccharide

Interferon-beta (IFN-beta) has been identified as the signature cytokine induced via the Toll-like receptor (TLR) 4, "MyD88-independent" signaling pathway in macrophages stimulated by Gram-negative bacterial lipopolysaccharide (LPS). In this study, we analyzed the responses of macrophages derived from wild-type (IFN-beta(+/+)) mice or mice with a targeted mutation in IFN-beta (IFN-beta(-/-)) to the prototype TLR4 agonist, Escherichia coli LPS. A comparison of basal and LPS-induced gene expression (by reverse transcription-PCR, real-time PCR, and Affymetrix microarray analyses) resulted in the identification of four distinct patterns of gene expression affected by IFN-beta deficiency. Analysis of a subset of each group of differentially regulated genes by computer-assisted promoter analysis revealed putative IFN-responsive elements in all genes examined. LPS-induced activation of intracellular signaling molecules, STAT1 Tyr-701, STAT1 Ser-727, and Akt, but not p38, JNK, and ERK MAPK proteins, was significantly diminished in IFN-beta(-/-) versus IFN-beta(+/+) macrophages. "Priming" of IFN-beta(-/-) macrophages with exogenous recombinant IFN-beta significantly increased levels of LPS-induced gene expression for induction of monocyte chemotactic protein 5, inducible nitric-oxide synthase, IP-10, and IL-12 p40 mRNA, whereas no increase or relatively small increases were observed for IL-1beta, IL-6, monocyte chemotactic protein 1, and MyD88 mRNA. Finally, IFN-beta(-/-) mice challenged in vivo with LPS exhibited increased survival when compared with wild-type IFN-beta(+/+) controls, indicating that IFN-beta contributes to LPS-induced lethality; however, not to the extent that one observes in mice with more complete pathway deficiencies (e.g. TLR4(-/-) or TRIF(-/-) mice). Collectively, these findings reveal unanticipated regulatory roles for IFN-beta in response to LPS in vitro and in vivo.

Global Gene Expression Changes during Arsenic Trioxide -Induced Apoptosis in Myeloma Cell Lines Expressing Mutant or w.t p53.

Arsenic trioxide (ATO) is an effective chemotherapeutic agent for the treatment of acute promyelocytic leukemia and is being tested in phase II studies in various types of hematological malignancies and solid tumors. We have previously shown that ATO is a potent inducer of apoptosis in multiple myeloma cells, engaging primarily the intrinsic apoptotic pathway in cells expressing w.t. p53. In contrast, in cells expressing mutant p53, both the intrinsic and extrinsic apoptotic pathways are engaged. These findings were further supported by a recent study using a p53 temperature sensitive (p53Ts) mutant cell line, BRK, expressing w.t. p53 at 32°C and mutant p53 phenotype at 37°C (Akay et al. Akay et al., AACR ; Abstract #5344, 2005). Furthermore, myeloma cells expressing w.t. p53 transfected with SiRNA for p53 or p21 behaved like cells with mutant p53 (Kircelli et al. ASH presentation,2005). In order to identify new genes affected by ATO we used the Affymetrix Microarray technology to compare global gene expression in IM9 myeloma cells (w.t. p53) and U266 myeloma cells (mutant p53) following 0, 1,5 and 10 hours of treatment with ATO. We found ≥2 fold increase in gene expression by Affymetrix Microarray Suite Software (MAS) in 94 genes at 5 h and 455 genes at 10 h with an increase in 134, at both 5 and 10 h of treatment. By similar analysis, 263 genes were decreased at 5 h and 679 genes were decreased at 10 h with 204 genes decreased at both time points. Similar analysis with GeneSpring (GS) software revealed an increase in 202 genes and a decrease in 233 genes at both 5 and 10 h. Combination of the 2 analysis methods yielded 90 consistent increasers and 64 consistent decreasers. A great number of these genes in whom we detected changes in this study are genes that were previously identified by us and by others using Western immunoblotting. In addition, we observed differential effect of ATO in IM9 and U266 myeloma cells in apoptosis-related genes (HRK, BID, MCL1); cell cycle-related genes (GADD45, Cyclin D1, cyclin D2 and cyclin D3); signal transduction proteins (ERK, NFkB, ATM, ATR, CHK2, TRAILR2, TNF-R5/6 and VEGF); in chaperon proteins; cyclophyllin B; SAT, ToPoIIA and others. Systematic validation of these changes on the protein levels is ongoing.