Enhancement of cell proliferation in various mammalian cell lines by gene insertion of a cyclin-dependent kinase homolog

<p>Abstract</p> <p>Background</p> <p>Genomics tools, particularly DNA microarrays, have found application in a number of areas including gene discovery and disease characterization. Despite the vast utility of these tools, little work has been done to explore the basis of distinct cellular properties, especially those important to biotechnology such as growth. And so, with the intent of engineering cell lines by manipulating the expression of these genes, anchorage-independent and anchorage-dependent HeLa cells, displaying markedly different growth characteristics, were analyzed using DNA microarrays.</p> <p>Results</p> <p>Two genes, cyclin-dependent kinase like 3 (<it>cdkl3</it>) and cytochrome c oxidase subunit (<it>cox15</it>), were up-regulated in the faster growing, anchorage-independent (suspension) HeLa cells relative to the slower growing, anchorage-dependent (attached) HeLa cells. Enhanced expression of either gene in the attached HeLa cells resulted in elevated cell proliferation, though insertion of <it>cdkl3 </it>had a greater impact than that of <it>cox15</it>. Moreover, flow cytometric analysis indicated that cells with an insert of <it>cdkl3 </it>were able to transition from the G0/G1 phases to the S phase faster than control cells. In turn, expression of <it>cox15 </it>was seen to increase the maximum viable cell numbers achieved relative to the control, and to a greater extent than <it>cdkl3</it>. Quantitatively similar results were obtained with two Human Embryonic Kidney-293 (HEK-293) cell lines and a Chinese Hamster Ovary (CHO) cell line. Additionally, HEK-293 cells secreting adipocyte complement-related protein of 30 kDa (acrp30) exhibited a slight increase in specific protein production and higher total protein production in response to the insertion of either <it>cdkl3 </it>or <it>cox15</it>.</p> <p>Conclusion</p> <p>These results are consistent with previous studies on the functionalities of <it>cdkl3 </it>and <it>cox15</it>. For instance, the effect of <it>cdkl3 </it>on cell growth is consistent with its homology to the <it>cdk3 </it>gene which is involved in G1 to S phase transition. Likewise, the increase in cell viability due to <it>cox15 </it>expression is consistent with its role in oxidative phosphorylation as an assembly factor for cytochrome c oxidase and its involvement removing apoptosis-inducing oxygen radicals. Collectively, the present study illustrates the potential of using microarray technology to identify genes influential to specific cellular processes with the possibility of engineering cell lines as desired to meet production needs.</p

Interleukin-6 Synthesis in Human Chondrocytes Is Regulated via the Antagonistic Actions of Prostaglandin (PG)E2 and 15-deoxy-Δ12,14-PGJ2

BACKGROUND: Elevated levels of interleukin-6 (IL-6), prostaglandin (PG)E(2), PGD(2) and its dehydration end product 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)) have been detected in joint synovial fluids from patients with rheumatoid arthritis (RA). PGE(2) directly stimulates IL-6 production in human articular chondrocytes. However, the effects of PGD(2) and 15d-PGJ(2) in the absence or presence of PGE(2) on IL-6 synthesis in human chondrocytes have yet to be determined. It is believed that dysregulated overproduction of IL-6 is responsible for the systemic inflammatory manifestations and abnormal laboratory findings in RA patients. METHODOLOGY/PRINCIPAL FINDINGS: Using the T/C-28a2 chondrocyte cell line as a model system, we report that exogenous PGE(2) and PGD(2)/15d-PGJ(2) exert antagonistic effects on IL-6 synthesis in human T/C-28a2 chondrocytes. Using a synthesis of sophisticated molecular biology techniques, we determined that PGE(2) stimulates Toll-like receptor 4 (TLR4) synthesis, which is in turn responsible for the activation of the ERK1/2, PI3K/Akt and PKA/CREB pathways that phosphorylate the NF-κB p65 subunit leading to NF-κB activation. Binding of the activated NF-κB p65 subunit to IL-6 promoter induces IL-6 synthesis in human T/C28a2 chondrocytes. PGD(2) or 15d-PGJ(2) concurrently downregulates TLR4 and upregulates caveolin-1, which in turn inhibit the PGE(2)-dependent ERK1/2, PI3-K and PKA activation, and ultimately with NF-κB-dependent IL-6 synthesis in chondrocytes. CONCLUSIONS/SIGNIFICANCE: We have delineated the signaling cascade by which PGE(2) and PGD(2)/15d-PGJ(2) exert opposing effects on IL-6 synthesis in human chondrocytes. Elucidation of the molecular pathway of IL-6 synthesis and secretion by chondrocytes will provide insights for developing strategies to reduce inflammation and pain in RA patients

Aseptic Meningitis with Urinary Retention: A Case Report

Introduction. Aseptic meningitis is serious inflammation of the meninges caused by agents including viruses, non-viral pathogens, non-infectious conditions and chemicals. Case Presentation. This study concerns the case of a 16-year-old healthy Greek female with persistent fever, mild headache and acute urinary retention, secondary to aseptic meningitis. Physical examination revealed no distinct signs of meningeal irritation. The urinary bladder was palpable, painless and over-distended. Serology carried out for common viruses was as follows: CMV IgG (−), CMV IgM (−), HSV IgG (−), HSV IgM (+), VZ IgG (+), VZ IgM (−), EBV IgG (−) and EBV IgM (+). During recovery in hospital, three trials of removing a urinary catheter were carried out; during the first two attempts the patient was unable to urinate and had a loss of bladder sensation. On the third attempt the patient had modest bladder perception but she left a post-voiding residual, and was instructed to perform bladder self-catheterization. Seven days after being discharged the patient underwent a full recovery. Conclusion. There are few reports concerning aseptic meningitis together with acute urinary retention. A number of these cases concern so-called “meningitis-retention syndrome,” which implies an underlying CNS mechanism, while others concerned an underlying peripheral nervous system mechanism

Piezo2 channel regulates RhoA and actin cytoskeleton to promote cell mechanobiological responses

International audienceActin polymerization and assembly into stress fibers (SFs) is central to many cellular processes. However, how SFs form in response to the mechanical interaction of cells with their environment is not fully understood. Here we have identified Piezo2 mechanosensi-tive cationic channel as a transducer of environmental physical cues into mechanobiological responses. Piezo2 is needed by brain metastatic cells from breast cancer (MDA-MB-231-BrM2) to probe their physical environment as they anchor and pull on their surroundings or when confronted with confined migration through narrow pores. Piezo2-mediated Ca 2+ influx activates RhoA to control the formation and orientation of SFs and focal adhesions (FAs). A possible mechanism for the Piezo2-mediated activation of RhoA involves the recruitment of the Fyn kinase to the cell leading edge as well as calpain activation. Knockdown of Piezo2 in BrM2 cells alters SFs, FAs, and nuclear translocation of YAP; a phenotype rescued by overexpression of dominant-positive RhoA or its downstream effector, mDia1. Consequently, hallmarks of cancer invasion and metastasis related to RhoA, actin cytoskeleton, and/or force transmission, such as migration, extracellular matrix degradation, and Serpin B2 secretion, were reduced in cells lacking Piezo2. mechanotransduction | calcium signaling | RhoA | actin stress fibers | cance

Piezo2 channel regulates RhoA and actin cytoskeleton to promote cell mechanobiological responses

Actin polymerization and assembly into stress fibers (SFs) is central to many cellular processes. However, how SFs form in response to the mechanical interaction of cells with their environment is not fully understood. Here we have identified Piezo2 mechanosensitive cationic channel as a transducer of environmental physical cues into mechanobiological responses. Piezo2 is needed by brain metastatic cells from breast cancer (MDA-MB-231-BrM2) to probe their physical environment as they anchor and pull on their surroundings or when confronted with confined migration through narrow pores. Piezo2-mediated Ca2+ influx activates RhoA to control the formation and orientation of SFs and focal adhesions (FAs). A possible mechanism for the Piezo2-mediated activation of RhoA involves the recruitment of the Fyn kinase to the cell leading edge as well as calpain activation. Knockdown of Piezo2 in BrM2 cells alters SFs, FAs, and nuclear translocation of YAP; a phenotype rescued by overexpression of dominant-positive RhoA or its downstream effector, mDia1. Consequently, hallmarks of cancer invasion and metastasis related to RhoA, actin cytoskeleton, and/or force transmission, such as migration, extracellular matrix degradation, and Serpin B2 secretion, were reduced in cells lacking Piezo2

Targeting hypersialylation in pancreatic ductal adenocarcinoma to reverts their malignant phenotype

Trabajo presentado en el 15th Jenner Glycobiology and Medicine Symposium, celebrado en Oporto (Portugal), del 14 al 16 de junio de 202

DETECTION OF CALR MUTATIONS USING HIGH RESOLUTION MELTING CURVE ANALYSIS (HRM-A); APPLICATION ON A LARGE COHORT OF GREEK ET AND MF PATIENTS

Background and Objectives Somatic mutations in the calreticulin gene (CALR) are detected in approximately 70% of patients with essential thrombocythemia (ET) and primary or secondary myelofibosis (MF), lacking the JAK2and MPLmutations. To determine the prevalence of CALRframeshift mutations in a population of MPN patients of Greek origin, we developed a rapid low-budget PCR-based assay and screened samples from 5 tertiary Haematology units. This is a first of its kind report of the Greek patient population that also disclosed novel CALRmutants.   Methods MPN patient samples were collected from different clinical units and screened for JAK2and MPLmutations after informed consent was obtained. Negative samples were analyzed for the presence of CALRmutations. To this end, we developed a modified post Real Time PCR High Resolution Melting Curve analysis (HRM-A) protocol. Samples were subsequently confirmed by Sanger sequencing.   Results Using this protocol we screened 173 MPN, JAK2and MPLmutation negative, patients of Greek origin, of whom 117 (67.63%) displayed a CALRexon 9 mutation. More specifically, mutations were detected in 90 out of 130 (69.23%) essential thrombocythaemia cases (ET), in 18 out of 33 (54.55%) primary myelofibrosis patients (pMF) and in 9 out of 10 (90%) cases of myelofibrosis secondary to ET (post-ET sMF). False positive results were not detected. The limit of detection (LoD) of our protocol was 2%. Furthermore, our study reavealed 6 rare novel mutations which are to be added in the COSMIC database.    Conclusions Overall, our method could rapidly and cost-effectively detect the mutation status in a representative cohort of Greek patients; the mutation make-up in our group was not different from what has been published for other national groups

Evaluation of the Revised International Staging System in an independent cohort of unselected patients with multiple myeloma

The Revised International Staging System (R-ISS) was recently introduced in order to improve risk stratification over that provided by the widely used standard International Staging System. In addition to the parameters of the standard system, the R-ISS incorporates the presence of chromosomal abnormalities detected by interphase fluorescence in situ hybridization [t(4;14), t(14;16) and del17p] and elevated serum lactate dehydrogenase. The R-ISS was formulated on the basis of a large dataset of selected patients who had participated in clinical trials and has not been validated in an independent cohort of unselected patients. Thus, we evaluated the R-ISS in 475 consecutive, unselected patients, treated in a single center. Our patients were older and more often had severe renal dysfunction than those in the original publication on the R-ISS. As regards distribution by group, 18% had R-ISS-1, 64.5% R-ISS-2 and 18% R-ISS-3. According to R-ISS group, the 5-year survival rate was 77%, 53% and 19% for R-ISS-1, -2 and -3, respectively (P75 years. However, in patients with severe renal dysfunction the distinction between groups was less clear. In conclusion, our data in consecutive, unselected patients, with differences in the characteristics and treatment approaches compared to the original International Myeloma Working Group cohort, verified that R-ISS is a robust tool for risk stratification of newly diagnosed patients with symptomatic myeloma