In vitro growth properties and PCR ras gene analysis of a murine embryonal carcinoma cell line harboring an amplified c-Ki-ras gene

Ras proto-oncogenes are thought to be involved in both proliferation and malignant processes. We examined some growth properties of a murine embryonal carcinoma cell line (ECC), PCC4, that have been shown previously to be amplified for the c-KI-ras gene. Our results show that doubling time and plating efficiency are not significantly affected by such amplification. To examine the possible link between malignant behavior and c-Ki-ras alteration, we investigated activating mutations in this PCC4 cell line as well as in other ECC. Analysis of the in vitro amplified Ki-ras gene by PCR technology has not revealed point mutations in any of the ECC examined.     

Mutation analysis of BRAF, MEK1 and MEK2 in 15 ovarian cancer cell lines: implications for therapy

Background

Among gynecologic cancers, ovarian cancer is the second most common and has the highest death rate. Cancer is a genetic disorder and arises due to the accumulation of somatic mutations in critical genes. An understanding of the genetic basis of ovarian cancer has implications both for early detection and for therapeutic intervention in this population of patients.

Methodology/Principal Findings

Fifteen ovarian cancer cell lines, commonly used for in vitro experiments, were screened for mutations using bidirectional direct sequencing in all coding regions of BRAF, MEK1 and MEK2. BRAF mutations were identified in four of the fifteen ovarian cancer cell lines studied. Together, these four cell lines contained four different BRAF mutations, two of which were novel. ES-2 had the common B-Raf p.V600E mutation in exon 15 and Hey contained an exon 11 missense mutation, p.G464E. The two novel B-Raf mutants identified were a 5 amino acid heterozygous deletion p.N486-P490del in OV90, and an exon 4 missense substitution p.Q201H in OVCAR 10. One of the cell lines, ES-2, contained a mutation in MEK1, specifically, a novel heterozygous missense substitution, p.D67N which resulted from a nt 199 G→A transition. None of the cell lines contained coding region mutations in MEK2. Functional characterization of the MEK1 mutant p.D67N by transient transfection with subsequent Western blot analysis demonstrated increased ERK phosphorylation as compared to controls.

Conclusions/Significance

In this study, we report novel BRAF mutations in exon 4 and exon 12 and also report the first mutation in MEK1 associated with human cancer. Functional data indicate the MEK1 mutation may confer alteration of activation through the MAPK pathway. The significance of these findings is that BRAF and MEK1/2 mutations may be more common than anticipated in ovarian cancer which could have important implications for treatment of patients with this disease and suggests potential new therapeutic avenues.     

A murine cell line (2E10.4.13) produces five hemopoietic stimulators, and interleukin-2 and interleukin-3

An interleukin-2 (IL-2)-independent murine lymphocyte clone (2E10.4.13) with the Thy1+Lyt1+2-T200+ phenotype was separated from the original IL-2-dependent natural killer (NK) cell line (PEC-1). Erythroid burst-promoting activity (BPA), erythropoietin (Ep), granulocyte/macrophage, megakaryocyte and eosinophil colony-stimulating factors (GM-, MK- and Eo-CSF), IL-2 and Interleukin-3 (IL-3) were produced when these cells were stimulated with phorbol myristate acetate (PMA). When the conditioned medium was run through ion-exchange high-performance liquid chromatography, BPA, Ep, GM-CSF, MK-CSF and Eo-CSF were eluted in the same region as IL-3. In contrast, MK-CSF, much of the GM-CSF and half of the Eo-CSF were eluted in a distinct region where no IL-3 was detected. Chemical analyses of the hemopoietic factors derived from a single T inducer clone indicated that all the hemopoietic activities were associated with IL-3 activity. Some CSF activities (GM-, MK- and Eo-CSF) also could be mediated by the distinct molecules from IL-3, evidence that heterogeneous molecules are responsible for CSF activity.     

MAGE-A1,A2,A3,A4在膀胱移行细胞癌组织及细胞株中基因表达的研究

目的：研究膀胱移行细胞癌（TCC）中黑色素瘤抗原（MAGE）基因表达.方法：逆转录聚合酶链反应（RT-PCR）技术检测20例膀胱TCC患者癌组织和3株膀胱TCC细胞株T24、EJ、BIU87中MAGE-A1、A2、A3、A4基因mRNA表达.结果：20例膀胱TCC癌组织中19例（95%）至少表达一种MAGE-A基因,12例MAGE-A1阳性（60%）,16例MAGE-A2阳性（80%）,11例MAGE-A3阳性（55%）,18例MAGE-A4阳性（90%）,MAGE-A1-4均阳性8例（40%）.膀胱TCC细胞株T24中MAGE-A1-4基因均表达,EJ中MAGE-A3、A4基因表达,BIU87中MAGE-A2、A3、A4基因表达.结论：MAGE基因在膀胱TCC中有较高表达,可望成为膀胱TCC免疫治疗的靶基因.     

1 alpha, 25-Dihydroxyvitamin D3 directly induces fusion of alveolar macrophages by a mechanism involving RNA and protein synthesis, but not DNA synthesis

The results of our present study indicate that 1 alpha, 25-dihydroxyvitamin D3[1 alpha, 25(OH)2D3] directly induces fusion of mouse alveolar macrophages without any participation of T-lymphocytes by a mechanism involving RNA and protein synthesis but not DNA synthesis. We have reported that 1 alpha, 25(OH)2D3 induces fusion of alveolar macrophages by a direct mechanism and by a spleen cell-mediated indirect mechanism [(1983) Proc. Natl. Acad. Sci. USA 80, 5583-5587]. Alveolar macrophages pretreated with or without anti-Thy 1.2 antibody and complement fused similarly when they were incubated with 1 alpha, 25(OH)2D3. The vitamin suppressed DNA synthesis, but it significantly enhanced RNA and protein synthesis. The 1 alpha, 25(OH)2D3-induced fusion was blocked by adding actinomycin D or cycloheximide, but not by hydroxyurea.     

The human rhabdomyosarcoma cell line A204 lays down a highly insoluble matrix composed mainly of alpha 1 type-XI and alpha 2 type-V collagen chains.

The biosynthesis of collagen by the A204 cell line was examined using polyclonal antibodies raised against collagen type V and type XI. The study of the pepsin-digested collagen showed that it is composed mainly of alpha 1(XI) and alpha 2(V) collagen chains in an apparent 2:1 ratio, suggesting the formation of heterotypic molecules [alpha 1(XI)]2 alpha 2(V). The existence of this chain stoichiometry was further demonstrated by immunoprecipitation of the molecule with an antibody recognizing alpha 2(V) but not alpha 1(XI) collagen chains. Electron microscopy analyses of 24-h cultures showed that this matrix is composed of thin fibrils, that can be decorated with immunogold-labelled anti-(type-V collagen) IgG, but not with anti-(type-XI collagen) IgG. The collagen matrix laid down by A204 cells is highly insoluble. In the presence of beta-aminopropionitrile, an inhibitor of lysyl oxidase, only a small proportion of intact collagen could be extracted without proteolytic treatment. Immunoblotting of intact medium collagen from cultures performed in the presence of beta-aminopropionitrile showed four distinct bands with each antibody. The migration of the bands, stained with anti-(type-V collagen) IgG, had apparent molecular masses of 127, 149, 161 and 198 kDa (compared to globular standards) while the bands stained with anti-(type-XI collagen) IgG had apparent masses of 145, 182, 207 and 225 kDa. These data indicate that type-V and type-XI collagen chains can assemble in heterotypic isoforms. In this system, the synthesized isoforms are able to aggregate into a highly cohesive matrix and they undergo a proteolytic processing closely similar to that of other fibrillar collagens.     

Effects of propentofylline on adenosine receptor activity in Chinese hamster ovary cell lines transfected with human A1, A2A, or A2B receptors and a luciferase reporter gene.

Propentofylline is neuroprotective in vivo, but its mechanism of action is not completely understood. Previously, propentofylline was shown to block adenosine transport processes, to inhibit three adenosine receptor subtypes, and to inhibit cAMP phosphodiesterase. We tested the effect of propentofylline on adenosine receptor function in Chinese hamster ovary (CHO) cells transfected with human adenosine A1, A2A, or A2B receptors and a luciferase reporter gene under control of a promoter sequence containing several copies of the cAMP response element. We investigated the concentration-dependent inhibitory effects of propentofylline on cAMP phosphodiesterase, adenosine transport processes, and adenosine A1, A2A, and A2B receptors. At concentrations > or = 1 mM, propentofylline increased luciferase activity probably as a result of inhibition of cAMP phosphodiesterase. Inhibition of [3H]adenosine uptake by propentofylline was concentration dependent, with IC50 values of 37-39 microM for the three cell types. Agonist-activated adenosine A1 receptors were antagonized by 100 microM propentofylline, but inhibition of agonist-stimulated A2A or A2B receptors was not observed. In contrast, A1 and A2A receptor mediated effects of adenosine were enhanced by propentofylline at concentrations of 1 and 100 microM, respectively. These data indicate that the net effects of propentofylline in vivo will be dependent on the concentrations of propentofylline and adenosine available and on the subtypes of adenosine receptors, phosphodiesterases, and nucleoside transporters present.     

Establishment and characterization of a novel ovarian clear cell carcinoma cell line,TU-OC-2, with loss of ARID1A expression

A new cell line of human ovarian clear cell carcinoma (CCC), TU-OC-2, was established and characterized. The cells were polygonal in shape, grew in monolayers without contact inhibition and were arranged in islands like pieces of a jigsaw puzzle. The chromosome numbers ranged from 41 to 96. A low rate of proliferation was observed and the doubling time was 37.5 h. The IC₅₀ values of cisplatin, 7-ethyl-10-hydroxycamptothecin (SN38), which is an active metabolite of camptothecin, and paclitaxel were 7.7 μM, 17.7 nM and 301 nM, respectively. The drug sensitivity assay indicated that TU-OC-2 was sensitive to SN38, but resistant to cisplatin and paclitaxel. Mutational analysis revealed that TU-OC-2 cells have no mutations of PIK3CA in exons 9 and 20 and of TP53 in exons 4–9. We observed the loss of ARID1A protein expression in TU-OC-2 cells by western blot analysis and in the original tumor tissue by immunohistochemistry. This cell line may be useful for studying the chemoresistant mechanisms of CCC and exploring novel therapeutic targets such as the ARID1A-related signaling pathway.     

The HepaRG cell line,a superior in vitro model to L-02, HepG2 and hiHeps cell lines for assessing drug-induced liver injury

Drug-induced liver injury (DILI) is a leading cause of discontinuation of new drug approval or withdrawal of marketed medicine based on safety due to organ vulnerability. The aim of this research is to investigate the potential abilities of four different in vitro cell models (L-02, HepG2, HepaRG, and hiHeps cell lines) in assessing marketed drugs labeled with apparently different types of liver injury. A total of 17 drugs with versatile pharmacological profiles were chosen, of which, 14 drugs are recognized as DILI agents and 3 drugs are DILI irrelevant. Preliminary cellular screening assays indicated that the HepaRG cell line had an advantage over other cell lines in predicting drugs associated with DILI in vitro as it had the highest Youden’s index (71.4 %). A multi-parametric screening assay showed that oxidative stress, mitochondrial damage, and disorders of neutral lipid metabolism were changed notably in the HepaRG cell line after DILI-related drugs exposure, accounting for its high sensitivity in comparison with other three cell lines. In addition, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) all correlated with the cytotoxic effects of diclofenac sodium (p?<?0.05), buspirone hydrochloride (p?<?0.01), and danazol (p?<?0.01) in the HepaRG cell line. We conclude that the HepaRG cell line is a superior in vitro cell model to other three cell lines for evaluating drugs with DILI potential.     

Establishment and proteomic characterization of a novel synovial sarcoma cell line,NCC-SS2-C1

Synovial sarcoma is an aggressive mesenchymal tumor, characterized by the presence of unique transfusion gene, SS18–SSX. Cell lines enable researchers to investigate the molecular backgrounds of disease and the significance of SS18–SSX in relevant cellular contexts. We report the establishment and proteomic characterization of a novel synovial sarcoma cell line. Primary tissue culture was performed using tumor tissue of synovial sarcoma. The established cell line was authenticated by assessing its DNA microsatellite short tandem repeat analysis and characterized by in vitro assay. Proteomic study was achieved by mass spectrometry, and the results were analyzed by treemap. The cell line NCC-SS2-C1 was established from a primary tumor tissue of a synovial sarcoma patient. The cell line has grown well for 11 mo and has been subcultured more than 15 times. The established cells were authenticated by assessing their short tandem repeat pattern comparing with that of original tumor tissue. The cells showed polygonal in shape and formed spheroid when seeded on the low-attachment dish. Proteomic analysis revealed the molecular pathways which are unique to the original tumor tissue or the established cell line. In conclusion, a novel synovial sarcoma cell line NCC-SS2-C1 was successfully established from the primary tumor tissue. The cell line has characteristic transfusion SS18–SSX and poses aggressive in vitro growth and capability of spheroid formation. Thus, NCC-SS2-C1 cell line will be a useful tool for investigation of the mechanisms of disease and the biological role of fusion gene.     

A colony-stimulating factor 1 (CSF-1) receptor/platelet-derived growth factor-beta receptor gene fusion confers CSF-1 independence and tumorigenicity on a c-myc-immortalized monocyte cell line.

Monocytes and macrophages express the receptor for the hematopoietic growth factor colony-stimulating factor 1 (CSF-1) and require this factor for growth in culture. A murine monocyte tumor cell line that lacks the usual requirement for CSF-1 was isolated. On the basis of the similarity of the structures of the CSF-1 and platelet-derived growth factor (PDGF) receptors and because monocytes normally secrete PDGF, we analyzed the tumor cell line for anomalous expression of the PDGF-R beta gene. Two different cDNAs that each contain sequences corresponding to the complete coding sequence of PDGF-R beta fused (in frame) to the amino-terminal half of the CSF-1 receptor were isolated. Introduction of these PDGF-R beta-related cDNAs into two partially transformed, CSF-1-dependent monocyte cell lines resulted in autonomous growth and cell transformation. These monocyte cell lines exhibit a novel form of growth factor receptor activation that can lead to oncogenic growth in collaboration with the c-myc oncogene.