Cell Line-conditioned Medium Research Articles

Exosomal lncAY927529 enhances prostate cancer cell proliferation and invasion through regulating bone microenvironment

ABSTRACT Exosomes mediate the interaction between cancer cells and their microenvironment, and play a key role in tumor development. Although exosomes can package lncRNAs to mediate extracellular communication, the role of exosomal lncRNA AY927529 in prostate cancer (PCa) remains unclear. Exosomes were extracted from normal human prostatic epithelial cell lines (BPH-1 and RWPE-1) and PCa cell lines (VCaP and LNCaP, DU145, PC3) by ultrahigh speed centrifugation. Results of Western blot indicated that Alix, HSC70 and TSGl01 protein levels were upregulated in exosomes derived from PCa cells. LncAY927529 level was upregulated in PCa cells and exosomes derived from PCa patient serum and human PCa cells. CCK-8, Transwell and Flow cytometry assays demonstrated that bone marrow stromal cell line (ST2) conditioned medium (ST2-CM), treated with exosomes derived from PCa cells with high lncAY927529 level, promoted proliferation and invasion of PC3 and DU145 cells, and inhibited cell apoptosis. RT-qPCR assay indicated that lncAY927529 level was downregulated in PC3 and DU145 cells, exosomes derived from PCa cells (PCa-Exo) and ST2-CM treated with PCa-Exo with low expression of lncAY927529, and overexpression of lncAY927529 had the opposite results. In addition, Western blot assay showed that the autophagy related protein LC3II level was increased in ST2 cells treated with exosomes derived from DU145 cells with high expression of lncAY927529, and LC3I protein level was decreased. CXCL14 acted as a RNA-binding protein of lncAY927529, and exosome-mediated lncAY927529 positively regulated CXCL14 levels in ST2 cells. In general, exosome-mediated lncAY927529 could promote PCa cell proliferation and invasion by regulating bone microenvironment, suggesting that exosomal lncAY927529 may be a potential molecular diagnostic marker of PCa.

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma.

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroidmodel which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 µL media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 µL media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Secreted cellular prion protein binds doxorubicin and correlates with anthracycline resistance in breast cancer.

Anthracyclines are amongst the most effective chemotherapeutics ever developed, but they produce grueling side-effects, serious adverse events and resistance often develops over time. We found that these compounds can be sequestered by secreted cellular Prion protein (PrPC), blocking their cytotoxic activity. This effect was dose-dependent using either cell line-conditioned medium or human serum as a source of PrPC. Genetic depletion of PrPC or inhibition of binding via chelation of ionic copper prevented the interaction and restored cytotoxic activity. This was more pronounced for doxorubicin than its epimer, epirubicin. Investigating the relevance to breast cancer management, we found that the levels of PRNP transcript in pre-treatment tumor biopsies stratified relapse-free survival after neoadjuvant treatment with anthracyclines, particularly amongst doxorubicin-treated patients with residual disease at surgery (p=2.8E-08). These data suggest that local sequestration could mediate treatment resistance. Consistent with this, tumor cell expression of PrPC protein correlated with poorer response to doxorubicin but not epirubicin in an independent cohort analyzed by immunohistochemistry, particularly soluble isoforms released into the extracellular environment by shedding (p=0.015). These findings have important potential clinical implications for frontline regimen decision-making. We suggest there is warranted utility for prognostic PrPC/PRNP assays to guide chemo-sensitization strategies that exploit an understanding of PrPC-anthracycline-copper ion complexes.

Abstract P2-01-07: M2-like tumor-associated macrophages require Tartrate-resistant acid phosphatase as overall function to promote breast cancer metastasis

Abstract Tartrate-resistant acid phosphatase (TRAP) is a member of the purple acid phosphatase family and is also known as type 5 acid phosphatase. It is mainly expressed in cells of the monocyte lineage, including osteoclasts, macrophages, and dendritic cells (DCs). There are two isoforms of TRAP enzyme: TRAP-5a and TRAP5b, and TRAP-5b is produced by post-translational modification of TRAP-5a. TRAP-5b is the major isoform of TRAP secreted by osteoclasts and it has been shown that TRAP-5b activity correlates with the number of osteoclasts and serum activity in rat and human studies. In contrast, macrophages and DCs are thought to secrete TRAP-5a as the major subtype, whereas TRAP-5a is a non-specific marker of macrophage activation. The recent studies show that higher-grade proliferative tumors with much necrosis were associated with abundant macrophage expressing TRAP. Here we generate TRAP-/- knockout mice to study the function of TRAP in tumorigenesis. In vitro experiments have found that the expression of TRAP in macrophages is essential for the full promoting tumor function of M2-like tumor-associated macrophages (TAM) including tumor growth and metastasis. Marrow-derived macrophages (BMDM) extracted from the TRAP-/- mice lost promote tumor migration activity. The 4T1 tumor (murine breast cancer cell line) conditioned medium was used to induce TAM formation found M2-like TAM formation in wild type BMDM was promoted higher than that of TRAP-/- BMDM. In contrast, the TRAP-/- is much higher M1-like TAM formation than the wild type. Xenograft tumor formation assays were used to monitor tumor formation by combining 4T1 and different M2 polarized macrophages into BALB/c mice. M2-polarized macrophages with trap-defective BMDM showed reduced promoted neoplastic activity compared to wild type. In MMTV-PyMT transgenic mice, spontaneous tumor formation in knockout trap-/- significantly repressed tumor formation compared to TRAP+/+ wild type mice. Finally, we hypothesized that TRAP is essential for the full function of M2-like tumor-associated macrophages and promotes tumor metastasis. Citation Format: Dai M-S, Lo H-C, Chen L-C, Janckilla A, Tseng S-F. M2-like tumor-associated macrophages require Tartrate-resistant acid phosphatase as overall function to promote breast cancer metastasis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-01-07.

Renal Differentiation of Mesenchymal Stem Cells Seeded on Nanofibrous Scaffolds Improved by Human Renal Tubular Cell Lines-Conditioned Medium.

Kidney injuries and renal dysfunctions are one of the most important clinical problems, and tissue engineering could be a valuable method for solving it. The objective of this study was to investigate the synergistic effect of renal cell line-conditioned medium and Polycaprolactone (PCL) nanofibers on renal differentiation of human mesenchymal stem cells (MSCs). In the current study, after stem cells isolation and characterization, PCL nanofibrous scaffold was fabricated using electrospinning methods and characterized morphologically, mechanically, and for biocompatibility. The renal differentiation of seeded MSCs on the surface of PCL nanofibers with and without human renal tubular cell lines-conditioned medium was investigated by evaluation of eight important renal-related genes expression by real-time reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry. Fabricated nanofibrous scaffolds were good in all characterized items. Almost highest expression of all genes was detected in stem cells seeded on PCL under conditioned media in comparison with the stem cells seeded on PCL, tissue culture polystyrene (TCPS) under renal induction medium, and TCPS under conditioned medium. According to the results, PCL nanofibers in contribution with conditioned medium can provide the optimal conditions for renal differentiation of MSCs and could be a promising candidate for renal tissue engineering application.

Infusion of human embryonic kidney cell line conditioned medium reverses kainic acid induced hippocampal damage in mice

Background aimsHippocampal neurodegeneration is one of the hallmarks in neurological and neurodegenerative diseases such as temporal lobe epilepsy and Alzheimer disease. Human embryonic kidney (HEK) cells are a mixed population of cells, including neurons, and their conditioned medium is enriched with erythropoietin (EPO). Because EPO is a known neuroprotectant, we hypothesized that infusion of HEK cells or HEK-conditioned medium (HEK-CM) may provide neuroprotection against kainic acid (KA)-induced hippocampal damage in mice. MethodsAdult CF1 mice were treated with KA to induce hippocampal damage. On 3rd and 5th days after KA treatment, HEK cells or HEK-CM was infused intravenously through the tail vein. On the 7th and 8th days after KA treatment, all groups of mice were subjected to cognitive and depression assessment by use of a novel object recognition test and a forced swim test, respectively. Subsequent to this assessment, mice were killed and the brain samples were used to assess the histopathology and messenger RNA expression for EPO and B-cell lymphoma-2 (Bcl-2). ResultsWe found that infusion of HEK cells/HEK-CM improves cognitive function and alleviates symptoms of depression. Histological assessment demonstrates complete neuroprotection against KA-mediated excitotoxicity, and the hippocampal cytoarchitecture of HEK cells/HEK-CM treated mice was comparable to normal control mice. HEK cells/HEK-CM treatment could provide neuroprotection by upregulating the endogenous EPO and Bcl-2 in KA-treated mice. ConclusionsOur present data demonstrate for the first time that infusion of HEK cells/HEK-CM can prevent excitotoxic hippocampal damage and alleviate consequent behavioral abnormalities.

Transfer of malignant trait to immortalized human cells following exposure to human cancer serum.

BackgroundHuman cancer cells can transfer signaling molecules to neighboring and distant cells predisposing them to malignant transformation. This process might contribute to tumor progression and invasion through delivery of oncogenes or inhibitors of tumor suppressor genes, derived from the primary tumor cells, to susceptible target cells. The oncogenic potential of human cancer serum has been described in immortalized mouse fibroblasts but has not been shown yet in human cells. The objective of this study was to determine whether metastatic cancer patient sera have the ability to induce neoplastic transformation in immortalized human embryonic kidney (HEK293) cells, human embryonic stem cells (hESCs), human mesenchymal stem cells (hMSCs) and human adult liver fibroblasts (hALFs).MethodsEarly passage HEK293 cells, hESCs, hMSCs and hALFs were exposed to cancer patient serum, or cancer cells-derived condition medium for 3 weeks. Treated cells were analyzed for cell proliferation and transformation both in vitro and in vivo.ResultsHEK293 cells exposed to cancer serum increased their proliferative capability and displayed characteristics of transformed cells, as evaluated by in vitro anchorage-independent growth assay and in vivo tumorigenesis in immunodeficient mice. The same phenotypes were acquired when these cells were cultured in cancer cell line conditioned medium suggesting that the putative oncogenic factors present in the serum might derive directly from the primary tumor. Histopathological analyses revealed that the tumors arising from cancer patient serum and conditioned medium-treated HEK293 cells were poorly differentiated and displayed a high proliferative index. In contrast, neither of these phenomena was observed in treated hMSCs and hALFs. Intriguingly enough, hESC-treated cells maintained their self-renewal and differentiation potentials, as shown by in vitro sphere formation assay and in vivo development of teratomas in immunodeficient mice.ConclusionOur results indicate that cancer patients serum is able to induce oncogenic transformation of HEK293 cells and maintain the self-renewal of hESCs. To our knowledge, this is the first study that demonstrates the oncogenic transformation potential of cancer patient serum on human cells. In depth characterization of this process and the molecular pathways involved are needed to confirm its validity and determine its potential use in cancer therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-014-0086-5) contains supplementary material, which is available to authorized users.

Primary Effusion Lymphoma: Secretome Analysis Reveals Novel Candidate Biomarkers with Potential Pathogenetic Significance

Primary Effusion Lymphoma: Secretome Analysis Reveals Novel Candidate Biomarkers with Potential Pathogenetic Significance

Circulating CD14+CD204+ Cells Predict Postoperative Recurrence in Non–Small-Cell Lung Cancer Patients

The expression of CD204 on macrophages in the stroma of the primary tumor is reportedly correlated with an unfavorable prognosis for lung cancer. The purpose of this study is to investigate the correlation among the number of CD204 tumor-associated macrophages infiltrating the stroma of the primary tumor, the number of circulating CD14CD204 cells from the pulmonary vein (PV), and recurrence-free probability in non-small-cell lung cancer patients. Human mononuclear cells were isolated from the PV of resected lungs. We examined the expressions of CD14 and CD204 on these cells by flow cytometry. Immunohistochemical staining for CD204 was performed in the resected specimens. The number of CD14CD204 cells from the PV was found to be correlated with the number of CD204 tumor-associated macrophages identified in the stroma of the tumor. Significantly more cases with high levels of CD14CD204 cells from the PV were found to have developed early recurrences. CD14CD204 cells, which were polarized to the tumor-promoting phenotype cultured in lung cancer cell line-conditioned medium, facilitated the lung metastasis of cancer cells more effectively than CD14CD204 cells in our in vivo mouse model. In multivariate analysis, only the high number of CD14CD204 cells from the PV was found to be a statistically significant independent risk factor for early recurrence. Our results showed the possibility that circulating CD14CD204 cells contribute to the metastasis of cancer cells. The blockage of circulating CD14CD204 cells activity may prevent postoperative recurrence in resected non-small-cell lung cancer patients.

Transfer of malignant trait to immortalized human cells following exposure to human cancer serum

BackgroundHuman cancer cells can transfer signaling molecules to neighboring and distant cells predisposing them to malignant transformation. This process might contribute to tumor progression and invasion through delivery of oncogenes or inhibitors of tumor suppressor genes, derived from the primary tumor cells, to susceptible target cells. The oncogenic potential of human cancer serum has been described in immortalized mouse fibroblasts but has not been shown yet in human cells. The objective of this study was to determine whether metastatic cancer patient sera have the ability to induce neoplastic transformation in immortalized human embryonic kidney (HEK293) cells, human embryonic stem cells (hESCs), human mesenchymal stem cells (hMSCs) and human adult liver fibroblasts (hALFs).MethodsEarly passage HEK293 cells, hESCs, hMSCs and hALFs were exposed to cancer patient serum, or cancer cells-derived condition medium for 3 weeks. Treated cells were analyzed for cell proliferation and transformation both in vitro and in vivo.ResultsHEK293 cells exposed to cancer serum increased their proliferative capability and displayed characteristics of transformed cells, as evaluated by in vitro anchorage-independent growth assay and in vivo tumorigenesis in immunodeficient mice. The same phenotypes were acquired when these cells were cultured in cancer cell line conditioned medium suggesting that the putative oncogenic factors present in the serum might derive directly from the primary tumor. Histopathological analyses revealed that the tumors arising from cancer patient serum and conditioned medium-treated HEK293 cells were poorly differentiated and displayed a high proliferative index. In contrast, neither of these phenomena was observed in treated hMSCs and hALFs. Intriguingly enough, hESC-treated cells maintained their self-renewal and differentiation potentials, as shown by in vitro sphere formation assay and in vivo development of teratomas in immunodeficient mice.ConclusionOur results indicate that cancer patients serum is able to induce oncogenic transformation of HEK293 cells and maintain the self-renewal of hESCs. To our knowledge, this is the first study that demonstrates the oncogenic transformation potential of cancer patient serum on human cells. In depth characterization of this process and the molecular pathways involved are needed to confirm its validity and determine its potential use in cancer therapy.

Induced pluripotent stem cells’ self-renewal and pluripotency is maintained by a bovine granulosa cell line-conditioned medium

Induced pluripotent stem cells (iPSCs) are a promising type of stem cells, comparable to embryonic stem cells (ESCs) in terms of self-renew and pluripotency, generated by reprogramming somatic cells. These cells are an attractive approach to supply patient-specific pluripotent cells, for producing in vitro models of disease, drug discovery, toxicology and potentially treating degenerative disease circumventing immune rejection. In spite of the great advance since iPSCs’ establishment, their obtention and propagation is an increasing area of great interest.In a recent work, we have shown that the conditioned medium from a bovine granulosa cell line (BGC-CM) is able to preserve the basic properties of mESCs. Therefore, based on our previous results and the reported resemblance between iPSCs and ESCs, we hypothesized that BGC-CM could provide a favorable context to culturing iPSCs. In this work, we have reprogrammed mouse embryonic fibroblasts obtaining iPSC lines, and showed that they can be propagated in BGC-CM while maintaining self-renewal and pluripotency, evidenced by expression of specific gene markers and capability of in vitro and in vivo differentiation to cell types from the three germ layers. We believe that these findings may provide a novel context to propagate iPSCs to study the molecular mechanisms involved in self-renewal and pluripotency.

Maintenance of Murine Embryonic Stem Cells' Self-Renewal and Pluripotency with Increase in Proliferation Rate by a Bovine Granulosa Cell Line-Conditioned Medium

Murine embryonic stem cells (mESCs) are pluripotent cells that can be propagated in an undifferentiated state in continuous culture on a feeder layer or without feeders in the presence of leukemia inhibitory factor (LIF). Although there has been a great advance since their establishment, ESC culture is still complex and expensive. Therefore, finding culture conditions that maintain the self-renewal of ESCs, preventing their differentiation and promoting their proliferation, is still an area of great interest. In this work, we studied the effects of the conditioned medium from a bovine granulosa cell line (BGC-CM) on the maintenance of self-renewal and pluripotency of mESCs. We found that this medium is able to maintain mESCs' self-renewal while preserving its critical properties without LIF addition. mESCs cultured in BGC-CM expressed the stem cell markers Oct4, Sox2, Nanog, SSEA-1, Klf4, Rex1, and ECAT1. Moreover, mESCs cultured in BGC-CM gave rise to embryoid bodies and teratomas that differentiated effectively to diverse cell populations from endoderm, mesoderm, and ectoderm. Further, we found that mESCs cultured in BGC-CM have an increased proliferation rate compared with cells grown in the mESC standard culture medium supplemented with LIF. These findings may provide a powerful tool to culture mESCs for long periods of time with high proliferation rate while preserving its basic characteristics, contributing to the application of these cells to assess potential tissue engineering and cellular therapy applications.

Short-term culture for acute myeloid leukemia blast cells

This study was carried out to separate acute myeloid leukemia (AML) blast cells and studies their proliferation in short-term culture. The separation procedure include three steps; Ficoll gradient separation, depletion of macrophages and depletion of (lymphocytes and of monocytes) for preparation of highly pure native AML blast cells from blood samples collected from patients with moderate blast percentage. Results showed that this procedure is an inefficient due to a decrease in total cell number and contamination with other cells after each separation step. Proliferation of native AML blast cells in short term-culture by cultivating isolated AML blast cells in RPMI medium supplemented with 20% human plasma at a concentration 1×106/ml in the presence and absence of colony -stimulating factor which was provided by conditioned media (PHA-leucocytes-, plasmacytoma cell line- and Hep-2 conditioned medium. The effect of each conditioned medium on proliferation of AML blast cells was studied separately. Results showed that plasmacytoma cell line conditioned medium didnot stimulate the proliferation of native AML blast cells, while cells seeded on media containing 10%PHA-LCM showed an increase in cell number and growth of the cells was observed for approximately 3 days and then decreased.

Role of NF-κB in hematopoietic niche function of osteoblasts after radiation injury

Hematopoietic tissue is very sensitive to ionizing radiation (IR). In adult mammalian bone marrow, hematopoietic stem and progenitor cells (HSPC) reside next to the endosteal bone surface, which is lined primarily by osteoblastic cells. In the present study, we proposed to investigate the mechanisms by which osteoblasts in the hematopoietic niche regulate survival, proliferation, and differentiation of HSPC after radiation injury. Human primary CD34+ HSPC were cultured with human fetal osteoblast (hFOB) cell line cells or conditioned medium (CM) from hFOB cells with or without irradiation. Survival, apoptosis, and cell cycle were analyzed using clonogenic and flow cytometric assays. Cytokine and chemokine expression were measured by cytokine array and enzyme-linked immunosorbent assay. Their regulatory activities were assessed by quantitative real-time polymerase chain reaction, small interfering (si)RNA transfection, immunoblotting, and transbinding assays. Survival of gamma-irradiated CD34+ HSPC was significantly enhanced by coculture with hFOB cells or by CM from hFOB cells. There were six factors in hFOB cell lysates and five factors released into hFOB CM enhanced by IR. IR induced phosphorylation of p53, c-Jun, and p38 and downstream p21 expression, as well as cell cycle arrest and apoptosis in hFOB cells. However, IR also induced phosphorylation of nuclear factor (NF)-kappaBp65 (ser536) and NF-kappaB activation in hFOB cells. Inhibition of NF-kappaB expression with siRNA upregulated p21, inhibited release of cytokines and chemokines, and induced hFOB and CD34+ cell apoptosis. NF-kappaB is a radiation-induced prosurvival factor in human osteoblastic cells. NF-kappaB gene knockdown abrogated the hematopoietic niche function of hFOB cells in supporting survival of CD34+ cells after IR.

Characterisation of 6 Factor XI Missense Mutations.

Factor XI deficiency (MIM 264900 ) is an autosomal bleeding disorder of variable clinical severity. In contrast to haemophilia A or B the clinical symptoms do not correlate well with plasma levels of factor XI; it is therefore difficult to predict the bleeding tendency from either the factor level or the molecular defect. Causative mutations, outside of the Ashkenazi Jewish population, are highly heterogeneous, with 79 different mutations currently recorded on the Factor XI Deficiency Mutation Database (http://www.factorxi.com/ ), and this number being added to at a rapid rate. The identification of novel missense mutations leaves the dilemma as to whether these are causative or not. The in vitro expression of novel missense mutations is currently the preferred method for assigning causality.We have utilised site-directed mutagenesis and lipofectamine transfection to express novel factor XI missense mutations in a Baby Hamster Kidney cell line (BHK 570). Three novel /previously uncharacterised missense mutations, Met-18Ile, Met102Thr and Thr575Met, and a change previously reported as a polymorphism, Arg378Cys, were expressed in vitro. Two further missense mutations, on which expression data had been reported, were also expressed as expression negative (Tyr133Ser) and expression positive (Pro520Leu) controls, along with the wild-type FXI cDNA vector. A sandwich ELISA assay failed to detect factor XI antigen in the conditioned media of cell lines expressing the Met-18Ile, Met102Thr or the Tyr133Ser mutant FXI cDNAs but detected good FXI expression from the wild-type FXI cDNA. FXI antigen was detected in the cell lysates of all three cell lines, at similar levels to the wild-type cell line, demonstrating that factor XI was being synthesised in but not being secreted by these cell lines. Preliminary data for the Thr575Met, Arg378Cys and Pro520Leu cell lines shows that a significant amount of FXI antigen is being secreted into the conditioned media, with levels broadly similar for all three cell lines. However, levels in the wild-type cell line are approximately 6-fold higher. Further work is on-going to see whether this is a true reflection of secretion efficiency or the effect of differences in cell growth, plasmid incorporation etc. Levels of FXI antigen in cell lysates from these cell lines were broadly similar. Functional studies on these proteins is on-going. The Arg378Cys ‘mutation', although previously reported as a polymorphism, was detected in a factor XI deficient father and daughter, with no other genetic variation detectable. Secretion of the Arg378Cys protein was surprising as the creation of a free Cysteine residue was expected to cause structural disruption resulting in a CRM- phenotype, as seen in the Arg308Cys and Trp501Cys mutants. We await the results of the function studies with interest.The Met-18Ile mutation was of particular interest as the mutation results in the loss of the initiator Methionine. Western blot analysis failed to detect any size difference between factor XI antigen recovered from cell lysates from the Met-18Ile and wild-type cell lines. We intend to purify the M-18I FXI protein and subject it to tandem MS analysis to try and determine the amnio acid sequence of this mutant protein.

Secretion of interleukin-6 and prostaglandin E 2 during uveal melanoma-monocyte in vitro interactions

Host–tumor interactions in uveal melanoma are not well understood. It is believed that the cytokine interleukin-6 and the lipid mediator autacoid prostaglandin E 2 are involved in tumor growth, proliferation, tumor cell survival, and angiogenesis. These cytokines have been shown to be poor prognostic markers in uveal and cutaneous melanoma. In this study, we investigated the levels of interleukin-6 and prostaglandin E 2 in monocyte and uveal melanoma conditioned medium. Five human uveal melanoma cell lines (92·1, MKT-BR, OCM-1, SP6·5 and UW-1), and one monocyte cell line (28SC) were seeded in 6 well plates at a concentration of 1×10 6 cells ml −1. After 18 hr melanoma conditioned medium was placed on the monocyte cell line and monocyte conditioned medium was placed on each uveal melanoma cell line. Tumor cells and monocytes incubated in fresh medium after 18 hr were used as controls. Interleukin-6 and prostaglandin E 2 levels were determined by immunoassays prior to media transfer and 6, 12, 24, and 36 hr thereafter. In the absence of conditioned medium, neither product showed baseline levels of expression. Interleukin-6 but not prostaglandin E 2, which remained undetectable for the duration of the study, showed up-regulation of expression after incubation in conditioned medium. 28SC incubated in melanoma conditioned medium expressed higher levels of interleukin-6 than did uveal melanoma cells incubated in monocyte conditioned medium. In addition each cell line exhibited a distinct pattern of expression with individual cell lines exhibiting peak levels of cytokine production at different time points. The results of this study offer insight into the mechanism by which interleukin 6 may be involved in tumor–host interactions potentially favoring tumor growth, survival, and proliferation.

Heparin-binding EGF-like growth factor mediates the biological effects of P450 arachidonate epoxygenase metabolites in epithelial cells.

In addition to its important functions in detoxification of foreign chemicals and biosynthesis of steroid hormones, the cytochrome P450 enzyme system metabolizes arachidonate to 14,15-epoxyeicosatrienoic acid (14,15-EET). This study demonstrates that a P450 arachidonate epoxygenase metabolite can activate cleavage of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and delineates an essential role for HB-EGF in the mitogenic effects of this lipid mediator. Blockade of HB-EGF processing or EGF receptor (EGFR) inhibited 14,15-EET-stimulated early mitogenic signals and DNA synthesis. 14,15-EET failed to induce mitogenesis in cell lines expressing minimal HB-EGF, whereas 14,15-EET induced soluble HB-EGF release into the conditioned media of cell lines that both express high levels of HB-EGF and display mitogenic response to this lipid mediator. Moreover, transfection of a bacterial 14,15-epoxygenase established intracellular endogenous 14,15-EET biosynthesis in cultured cell systems, which allowed direct confirmation of involvement of EGFR transactivation in the endogenous 14,15-EET-mediated mitogenic signaling pathway. This mechanism involves EET-dependent activation of metalloproteinases and release of the potent mitogenic EGFR ligand, HB-EGF.

Trophic factor secreting kidney cell lines: in vitro characterization and functional effects following transplantation in ischemic rats

Several kidney cell lines were investigated for their ability to produce glial cell line-derived neurotrophic factor (GDNF). Cell line-conditioned medium was analyzed using ELISA and two cell lines were identified which produce GDNF in physiologically active concentrations. ELISA analyses revealed that conditioned medium from these two cell lines also contained PDGF, bFGF, TGFβ1 and TGFβ2. Both of these cell lines were then transplanted into the striatal penumbra of rats, 1 h following middle cerebral artery occlusion. Behavioral testing revealed that both cell lines reduced the deficit associated with cerebral ischemia and reduced the infarct volume relative to controls. Reduction of infarct volume was likely achieved by the action of GDNF and/or other growth factors produced by the cells.

In vitro rearing of Edovum puttleri, an egg parasitoid of the Colorado potato beetle, from egg to pupal stage in artificial diets devoid of insect sources: Effects of dietary amino acid and carbohydrate levels

Previous studies have demonstrated that Edovum puttleri, an egg parasitoid of the Colorado potato beetle, can be reared through the pupal stage in an artificial diet containing either hemolymph from Manduca sexta or CPB embryonic cell line-conditioned medium. In order to improve the diet and reduce production costs, i.e., eliminate the insect-derived components, the effects of 13 amino acids and 20 carbohydrates on the growth and development of the parasitoid were determined. In the presence of any one of five of the amino acids (arginine, glutamine, lysine, threonine and valine) at a concentration of 1%, or eight of the carbohydrates (cellobiose, fructose, gentiobiose, glucose, lactose, melinbiose, sorbitol and threhalose), also at a concentration of 1%, more than 75% of the larvae molted to the second instar. At higher concentrations (2 and 3%), all five of the amino acids and most of the eight sugars promoted third instar formation. Prepupal formation was also observed. In the presence of threonine (at 2 and 3%) or glutamine (at 3%), between 22 and 29% of the parasitoids formed prepupae. Of the six sugars (at 3%) that promoted prepupal formation, lactose and sorbitol which promoted 18 and 20% prepupation, respectively, were the most effective. In the presence of all 13 amino acids (each at 3%) or all 20 carbohydrates (each at 3%), 36 and 39%, respectively, of the parasitoids formed prepupae. At lower concentrations, reduced percentages of prepupal formation were observed. Since the amino acids, glutamine and threonine, and the sugars, fructose, gentiobiose, glucose, lactose, sorbitol and trehalose, when added individually to the diet (at a concentration of 3%) were most effective in promoting prepupal formation, the effect of one of these two amino acids in combination with each of the six sugars was tested. Glutamine in combination with lactose or sorbitol was able to promote pupation (≤6.3%), and threonine in combination with any one of five of the sugars (lactose and sorbitol were most effective) supported pupal formation (≤6.7%). Our study demonstrated that E. puttleri can be successfully reared from the egg through the pupal stage in an artificial diet devoid of insect materials.