Serum-deprived Medium Research Articles

Protein Kinase Cα-RhoA Cross-talk in CCL2-induced Alterations in Brain Endothelial Permeability

Monocyte chemoattractant protein-1 (MCP-1 or CCL2) regulates blood-brain barrier permeability by inducing morphological and biochemical alterations in the tight junction (TJ) complex between brain endothelial cells. The present study used cultured brain endothelial cells to examine the signaling networks involved in the redistribution of TJ proteins (occludin, ZO-1, ZO-2, claudin-5) by CCL2. The CCL2-induced alterations in the brain endothelial barrier were associated with de novo Ser/Thr phosphorylation of occludin, ZO-1, ZO-2, and claudin-5. The phosphorylated TJ proteins were redistributed/localized in Triton X-100-soluble as well as Triton X-100-insoluble cell fractions. Two protein kinase C (PKC) isoforms, PKCalpha and PKCzeta, had a significant impact on this event. Inhibition of their activity using dominant negative mutants PKCalpha-DN and PKCzeta-DN diminished CCL2 effects on brain endothelial permeability. Previous data indicate that Rho/Rho kinase signaling is involved in CCL2 regulation of brain endothelial permeability. The interactions between the PKC and Rho/Rho kinase pathways were therefore examined. Rho, PKCalpha, and PKCzeta activities were knocked down using dominant negative mutants (T17Rho, PKCalpha-DN, and PKCzeta-DN, respectively). PKCalpha and Rho, but not PKCzeta and Rho, interacted at the level of Rho, with PKCalpha being a downstream target for Rho. Double transfection experiments using dominant negative mutants confirmed that this interaction is critical for CCL2-induced redistribution of TJ proteins. Collectively these data suggest for the first time that CCL2 induces brain endothelial hyperpermeability via Rho/PKCalpha signal pathway interactions.

Production of GM-CSF by CML Cells Can Modulate the Anti-Proliferative and Pro-Apoptotic Effects of Imatinib on CML CD34+ Cells.

CML cells gain a survival advantage over normal cells due to dysregulated tyrosine kinase activity of BCR/ABL protein, which phosphorylates a number of proteins, potentially activating multiple signal transduction pathways. Imatinib specifically targets BCR/ABL protein and results in anti-proliferation and apoptosis. Even in the absence of mutation, the leukaemic clone may not be eradicated by imatinib, suggesting that leukaemic stem cells may not be absolutely reliant on BCR/ABL activity for survival. Reports on GM-CSF production by CML cells suggest a possible autocrine role, therefore we examined if GM-CSF could protect CML CD34+ cells from imatinib. CD34+ cells from peripheral blood of patients with CML in chronic phase (n=9) and from normal bone marrow donors (n=5), were labeled with CFSE (Carboxy-Fluorescein diacetate Succinimidyl Ester) to enable tracking of cell division. Normal and CML samples were cultured with and without GM-CSF (300pg/mL) to assess response to this cytokine as a single agent. CML CD34+ cells were cultured for 3 days in serum deprived medium with imatinib only, GM-CSF (300 pg/mL) + imatinib, GM-CSF (300pg/mL) + E21R (a GM-CSF analogue able to block cytokine binding) (10mg/mL), and GM-CSF (300pg/mL) + E21R (10mg/mL) + imatinib. In each condition, imatinib was titrated over the range of 0 to 10 microM. Cultures were analysed by flow cytometry to evaluate the proliferation index (PI), a ratio of final cultured cell to precursor cell number, where a PI of 1 represents no proliferation, and a PI of 2 corresponds to approximately three division cycles on average. Data are summarised in Table 1.Effect of GM-CSF and imatinib on proliferationCultureProliferation IndexNormal Control1.07+/−0.06* NSNormal+GM-CSF1.11+/−0.03*# NSCML Control1.43+/−0.18** p>0.01#, & NSCML+GM-CSF2.30+/−0.40**CML+10μM imatinib1.18+/−0.09*** NSCML+10μM imatinib+GM-CSF1.54+/−0.19@, &CML+10μM imatinib+GM-CSF+E21R1.17+/−0.09***@ p>0.01Statistics using Student t test, symbols denote comparisons between culture conditionsGM-CSF induced strong proliferation in all CML, but not normal samples. Imatinib reduced proliferation of CML CD34+ cells at 1 microM and above, and the addition of GM-CSF reduced this proliferative effect at concentrations of imatinib up to 10 microM. The protective effect of GM-CSF was clearly blocked using E21R. When the proliferation of CML CD34+ cells cultured within the total mononuclear fraction was compared to purified CD34+ cells, it was found that there was an approximate 40% enhancement of PI at each concentration of imatinib. This enhanced proliferation was inhibited by the addition of E21R, indicating GM-CSF may be produced by non-CD 34+ cells. ELISpot assay confirmed the production of GM-CSF by non-CD34+ CML cells (34±23%), but not CD34+ cells (0.06±0.02%). Fluorescent inhibitor of apoptosis (FLICA) assay showed that GM-CSF could reduce the proportion of cells with activated caspases induced by imatinib by over 40% during a 3 day in vitro culture, promoting increased cell survival. As the GM-CSF receptor and BCR/ABL may share common signaling pathways, during blockade of BCR/ABL activity by imatinib, GM-CSF can compensate to maintain cell viability and proliferation. These findings have implications for optimizing imatinib therapy by manipulating cytokine signaling.

Neutrophil Elastase Modulates Granulopoiesis through the Interaction of G-CSF and G-CSF Receptor.

Primary granule proteases provide negative feedback on granulopoiesis. Recent study showed that neutrophil elastase potentially inhibits granulopoiesis by enzymatic digestion of G-CSF. We studied the effects of neutrophil elastase on granulopoiesis in the interaction of G-CSF and G-CSF receptor (G-CSFR). Primitive myeloid cells of the bone marrow in normal subjects were purified based on the expression of CD34 and G-CSFR by FACS Vantage. The cells were incubated with or without human neutrophil elastase for 2 hours in serum-deprived media. Cells were washed once and then cultured in various serum-deprived conditions. The proliferation and colony formation of CD34+/G-CSFR+ cells in the presence of G-CSF were significantly reduced by the pretreatment of elastase. Furthermore, those of CD34+/G-CSFR+ cells in response to hematopoietic factors without G-CSF were similarly inhibited by elastase. On the other hand, the responses of CD34+/G-CSFR− cells were not affected by the treatment of elastase in the stimulation of any hematopoietic factors. When CD34+ cells were incubated with or without elastase, further G-CSFR expression was analysed by flow cytometry. The treatment of elastase did not affect the expression of surface expression of G-CSFR on CD34+ cells. Next, the binding of G-CSF to G-CSFR was examined by flow cytometry using antibody to G-CSF. The mean channel of fluorescence of G-CSF binding to G-CSFR-positive cells was reduced by the treatment of elastase, suggesting the loss of the binding of G-CSF to G-CSFR. When CD34+/G-CSFR+ cells were treated with elastase, the observation by confocal immunofluorescence microscopy revealed neither accumulation of elastase on the surface of cell membrane nor in intracellular compartment. No evidences that cells treated with elastase directly cleave the G-CSF leading to inactivation of G-CSF. Collectively, these results suggest that G-CSFR-positive cells are selectively affected by neutrophil elastase, and that neutrophil elastase as negative regulator may play a role in modulating granulopoiesis through G-CSF-G-CSFR interaction.

Preadipocytes in the human subcutaneous adipose tissue display distinct features from the adult mesenchymal and hematopoietic stem cells

The stroma-vascular fraction (SVF) of human adipose tissue has recently been described to be composed of endothelial cells identified as CD34+/CD31+ cells, infiltrated/resident macrophages defined as CD14+/CD31+ cells, and a new cell population characterized as CD34+/CD31- cells. To elucidate the cell identity of the adipocyte precursor cells, fluorescent activating cell sorter (FACS) analyses were performed on crude SVF cultured under adipogenic conditions, i.e., serum-deprived medium containing insulin, cortisol, triiodothyronine, and supplemented with a PPARgamma agonist for the first 3 days. The progressive accumulation of lipid droplets was associated with a selective enrichment of the CD34+/CD31- cell population whereas control experiments performed in medium supplemented with 10% serum showed an overall downregulation of the three cell markers without adipogenesis. Among the different cell subsets, the CD34+/CD31- subset was the unique cell fraction able to answer to adipogenic culture conditions. Indeed, a time-dependent expression of adipocyte markers as well as acquisition of adipocyte-typical metabolic activities were observed. In parallel, the gene expression of lipogenic and lipolytic enzymes increased. The ability to differentiate into adipocytes was restricted to cells that did not express the mesenchymal stem cell marker CD105. Furthermore, the CD34+/CD31- cells did not respond to culture conditions used for hematopoietic colony assays. Taken together, the present study demonstrates that adipocyte progenitor cells, i.e., the preadipocytes, are included in the CD34+/CD31- cell fraction, which displays distinct features from the adult mesenchymal and hematopoietic stem cells.

Matrix Metalloproteinase-3: A Novel Signaling Proteinase from Apoptotic Neuronal Cells That Activates Microglia

Microglial activation and inflammation are associated with progressive neuronal apoptosis in neurodegenerative human brain disorders. We sought to investigate molecular signaling mechanisms that govern activation of microglia in apoptotic neuronal degeneration. We report here that the active form of matrix metalloproteinase-3 (MMP-3) was released into the serum-deprived media (SDM) of PC12 cells and other media of apoptotic neuronal cells within 2-6 h of treatment of the cells, and SDM and catalytic domain of recombinant MMP-3 (cMMP-3) activated microglia in primary microglia cultures as well as BV2 cells, a mouse microglia cell line. Both SDM and cMMP-3 induced generation of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), IL-1beta, and interleukin-1 receptor antagonist but not IL-12 and inducible nitric oxide synthase, which are readily induced by lipopolysaccharide, in microglia, suggesting that there is a characteristic pattern of microglial cytokine induction by apoptotic neurons. Neither glial cell line-derived neurotrophic factor nor anti-inflammatory cytokines, such as IL-10 and transforming growth factor-beta1, were induced. SDM and cMMP-3 extensively released TNF-alpha from microglia and activated the nuclear factor-kappaB pathway, and these microglial responses were totally abolished by preincubation with an MMP-3 inhibitor, NNGH [N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid]. MMP-3-mediated microglial activation mostly depended on ERK (extracellular signal-regulated kinase) phosphorylation but not much on either JNK (c-Jun N-terminal protein kinase) or p38 activation. Conditioned medium of SDM- or cMMP-3-activated BV2 cells caused apoptosis of PC12 cells. These results strongly suggest that the distinctive signal of neuronal apoptosis is the release of active form of MMP-3 that activates microglia and subsequently exacerbates neuronal degeneration. Therefore, the release of MMP-3 from apoptotic neurons may play a major role in degenerative human brain disorders, such as Parkinson's disease.

Isolation and Expansion of Functional Differentiating Mesenchymal Stem Cells with a Serum Deprived Medium.

The infusion of mesenchymal stem cells (MSC) is an increasingly utilized therapeutic approach for congenital and acquired diseases in regenerative medicine. Recently, MSC have been used for the treatment of graft-versus-host disease after allogeneic stem cell transplantation[1]. These strategies require ex vivo MSC expansion with media containing fetal bovine serum (FBS). Since the use of FBS may be associated with immunological responses against the cultured MSC[2] and may unknowingly transmit prions[4], the development of a serum free medium (SFM) for MSC isolation and expansion is essential for the widely applicable MSC therapy.We evaluated a novel serum deprived medium (Quantum 333R) for the isolation and expansion of MSC as compared to the standard medium (SM), DMEM + 10% FBS. Fibroblastoid colony forming unit (CFU-F), as MSC progenitors, and MSC expansion potential were considered as measurable parameters. The number of CFU-F found at 14 days of culture in the SFM (average: 1 CFU-F/ 1.78 x 105 bone marrow mononuclear cells, BMMNC) and the SM (1 CFU-F/ 2.3 x 105 BMMNC) were similar (p=0.18 t-test), but the trend suggested that the SFM may be superior. Passage 5 MSC in both culture conditions showed similar percentages of proliferating, i. e. BrdU+ cells: 7,5% (+/− 1,3%) and 10% (+/− 2,1%) in MSF and in SM, respectively. Flow cytometric analysis of MSC for side and forward scatter properties showed that MSCSFM were smaller in the side scatter than MSC SM. Further immunophenotypical characterization showed, in the MSCSFM, the absence of CD45, CD34, CD31 and the presence of CD90, CD105, CD73 confirming the phenotype of the “classical” MSCSM. CD73 and CD105 were upregulated in MSCSFM in comparison to MSCSM. Still, MSCSFM retained the capacity to inhibit the cytokine-induced proliferation of PBMC as reported for MSCSM. In addition, to test the differentiation potential of MSCSFM, the cells were successfully driven into osteogenic, adipogenic, myogenic and chondrogenic phenotypes. Our data support that Quantum 333R is able to isolate and expand MSC clonogenic precursors without affecting their proliferation, immunophenotype, differentiation and immunomodulatory potentials, thus suggesting a superior technique to isolate clinical grade cell population for safer clinical applications.

Eosinophil Adhesion to Cholinergic IMR-32 Cells Protects against Induced Neuronal Apoptosis

Eosinophils release a number of mediators that are potentially toxic to nerve cells. However, in a number of inflammatory conditions, such as asthma and inflammatory bowel disease, it has been shown that eosinophils localize to nerves, and this is associated with enhanced nerve activity. In in vitro studies, we have shown that eosinophil adhesion via neuronal ICAM-1 leads to activation of neuronal NF-kappaB via an ERK1/2-dependent pathway. In this study, we tested the hypothesis that eosinophil adhesion to nerves promotes neural survival by protection from inflammation-associated apoptosis. Exposure of differentiated IMR-32 cholinergic nerve cells to IL-1beta, TNF-alpha, and IFN-gamma, or culture in serum-deprived medium, induced neuronal apoptosis, as detected by annexin V staining, caspase-3 activation, and DNA laddering. Addition of human eosinophils to IMR-32 nerve cells completely prevented all these features of apoptosis. The mechanism of protection by eosinophils was by an adhesion-dependent activation of ERK1/2, which led to the induced expression of the antiapoptotic gene bfl-1. Adhesion to nerve cells did not influence the expression of the related genes bax and bad. Thus, prevention of apoptosis by eosinophils may be a mechanism by which these cells regulate neural plasticity in the peripheral nervous system.

Krüppel-like factor 5 mediates the transforming activity of oncogenic H-Ras.

Previous studies indicate that Krüppel-like factor 5 (KLF5), also known as intestinal-enriched Krüppel-like factor (IKLF), is a positive regulator of cell proliferation and gives rise to a transformed phenotype when overexpressed. Here we demonstrate that levels of KLF5 transcript and protein are significantly elevated in oncogenic H-Ras-transformed NIH3T3 cells. These cells display an accelerated rate of proliferation in both serum-containing and serum-deprived media and form anchorage-independent colonies in soft agar assays. H-Ras-transformed cells also contain elevated mitogen-activated protein kinase (MAPK) activity. When treated with inhibitors of MEK (MAPK kinase), H-Ras-transformed cells lose their growth advantage and no longer form colonies. Significantly, levels of KLF5 transcript and protein are substantially reduced in H-Ras-transformed cells treated with MEK inhibitors. Moreover, inhibition of KLF5 expression in H-Ras-transformed cells with KLF5-specific small interfering RNA (siRNA) leads to a decreased rate of proliferation and a significant reduction in colony formation. H-Ras-transformed cells also contain elevated levels of Egr1 that are diminished by MEK inhibitors. Inhibition of Egr1 by siRNA results in a reduced level of KLF5, indicating that Egr1 mediates the inductive action of MAPK on KLF5. Lastly, KLF5 activates expression of cyclin D1. These findings indicate that the increased expression of KLF5 in H-Ras-transformed cells is secondary to increased MAPK activity from H-Ras overexpression and that the elevated level of KLF5 is in part responsible for the proproliferative and transforming activities of oncogenic H-Ras.

Induction of the early response protein EGR-1 in human tumour cells after ionizing radiation is correlated with a reduction of repair of lethal lesions and an increase of repair of sublethal lesions

The role of EGR-1 in potentially lethal damage repair (PLDR) was studied. Induction of the early response protein EGR-1 and survival after ionizing radiation of two human tumour cell lines after culturing for 48 h in serum-deprived medium was investigated. The glioblastoma cell line (Gli-6) and a lung carcinoma cell line (SW-1573) were selected as these cell lines differ considerably in the degree of PLD repair after radiation. In both cell lines induction of EGR-1 protein was observed between 30-120 min after treatment with 10 Gy in serum-deprived cultures. In cells growing in medium with normal serum no induction of EGR-1 was observed. No difference in EGR-1 expression levels between the two cell lines was detected. Linear-Quadratic analysis of the survival curves showed a much larger difference between the values of alpha after immediate and delayed plated cells of the cultures in normal serum as compared to cells cultured in serum-deprived medium. The cells cultured in serum-deprived medium showed much larger difference between the values of beta. This indicates that induction of EGR-1 is correlated with a reduction of repair of lethal lesions (PLDRalpha) and with an increase of repair of sublethal lesions (PLDRbeta).

Apoptosis in serum-deprived vascular smooth muscle cells: evidence for cell volume-independent mechanism.

Shrinkage is the earliest hallmark of cells undergoing apoptosis. This study examines the role of this phenomenon in the onset of vascular smooth muscle cell (VSMC) apoptosis triggered by growth factor withdrawal. In hyperosmotic media, VSMC showed the same amplitude of shrinkage but were more resistant to apoptosis than endothelial, epithelial and immune system cells. As with growth factor withdrawal, apoptosis in hyperosmotically-shrunken VSMC was sharply potentiated by transfection with E1A-adenoviral protein and was suppressed by activation of cAMP signaling as well as by the pan-caspase inhibitor z-VAD.fmk. Both cell shrinkage and apoptosis in VSMC-E1A treated with hyperosmotic medium were potentiated under sustained Na+, K+ pump inhibition with ouabain that was in contrast to inhibition of apoptosis documented in ouabain-treated, serum-deprived cells. After 1-hr incubation in serum-deprived medium, VSMC-E1A volume declined by approximately 15%. Transfer from hypotonic to control medium decreased VSMC-E1A volume by approximately 25% without any induction of apoptosis. Neither swelling in hyposmotic medium nor dissipation of the transmembrane gradient of K+ and major organic osmolytes protected serum-deprived VSMC-E1A from apoptosis. Thus, our results show that similarly to immune system, endothelial and epithelial cells, extensive VSMC shrinkage in hyperosmotic medium leads to the development of apoptosis. In contrast to hyperosmotic medium, the modest cell volume decrease occurring in serum-deprived VSMC does not contribute to triggering of the apoptotic machinery.

Insulin-like growth factor binding protein-3 mediates serum starvation- and doxorubicin-induced apoptosis in H9c2 cardiac cells.

Insulin-like growth factor binding protein 3 (IGFBP-3) modulates the activity of IGF-I, which exerts antiapoptotic action upon the myocardiocyte. IGFBP-3 also exerts IGF-independent actions to inhibit cell growth and induce apoptosis, mediating the effects of several antiproliferative agents. We hypothesized that IGFBP-3 mediates cardiomyocyte apoptosis. IGFBP-3 expression was studied in H9c2 rat cardiac cells cultured in serum-deprived medium in the absence or presence of 1 microM doxorubicin during a 72 h time-span. To a greater degree than serum withdrawal, doxorubicin induced IGFBP-3 up-regulation that was time-dependent. IGFBP-3 mRNA levels positively correlated with the degree of apoptosis. Exogenous IGFBP-3 decreased cell viability and induced apoptosis in serum-starved cells exposed to doxorubicin. IGFBP-3 antisense oligonucleotides markedly decreased apoptosis induced by either serum withdrawal or doxorubicin. Binding studies revealed specific high-affinity sites for IGFBP-3 in H9c2 cardiomyocytes, with binding characteristics typical of receptor-ligand interactions. These findings indicate that IGFBP-3 could play proapoptotic action at the myocardial level and suggest a novel role for this protein in cardiovascular dysfunction.

Inhibitory Role of the Somatostatin Receptor SST2 on the Intracrine-regulated Cell Proliferation Induced by the 210-Amino Acid Fibroblast Growth Factor-2 Isoform: IMPLICATION OF JAK2

The fibroblast growth factor (FGF)-2 isoform of 210 amino acids (HMW FGF-2) contains a nuclear localization sequence (NLS) and is targeted to the nucleus. This FGF-2 isoform allows cells to grow in low serum concentrations through still unknown mechanisms called intracrine regulations. Different peptide hormones and cytokines have been found to be nuclearized through NLS and to induce cell proliferation. The existence of molecules acting as negative regulators of the intracrine-induced cell growth has not been explored. Pancreatic cells AR4-2J were stably transfected to express selectively the HMW FGF-2. We demonstrated that activation of the somatostatin receptor subtype SST2 by the somatostatin analogue RC-160 in serum-deprived medium inhibits the mitogenic effect of the HMW FGF-2, without affecting growth of control cells. The signaling pathway implicates Galphai/JAK2/SHP-1. The Galphai inhibitor pertussis toxin and the JAK2 inhibitor AG490 abrogate the inhibitory effect of RC-160 on HMW FGF-2-induced cell growth. Co-immunoprecipitation studies demonstrate the constitutive association of JAK2 and SHP-1, and RC-160 induces a rapid activation of both proteins followed by the dissociation of the complex. AG490 prevents the RC-160 induced SHP-1 activation indicating the implication of JAK2 in this process. JAK2 and SHP-1 are immunoprecipitated with SST2 in basal conditions indicating the existence of a functional signaling complex at the receptor level. In summary, these data provide the following evidence: 1) the intracrine-induced proliferation can be reversed by extracellular acting polypeptides; 2) SST2 inhibitory signaling may involve the JAK2/SHP-1 pathway.

Cell cycle synchronization of porcine granulosa cells in G1 stage with mimosine

The success of somatic cell nuclear transfer depends critically on the cell cycle stage of the donor nucleus and the recipient cytoplast. Karyoplasts in the G0 or G1 stages are considered to be the most suitable for nuclear transfer. In the present study, we used a reversible cell cycle inhibitor, mimosine, to synchronize porcine granulosa cells (GCs) in G1 phase of the cell cycle. Porcine GCs were obtained from 3 to 5 mm ovarian follicles of slaughtered gilts. The effect of mimosine on the proliferation, DNA synthesis and cell cycle stage of cultured cells was examined by incorporation of radiochemical 3 H -thymidine, immunocytochemical detection of incorporated thymidine analogue 5-bromo-2-deoxyuridine (BrdU) and flow cytometry analyses. Mimosine treatment of pig GCs for 24 h resulted in proliferation arrest in vitro. Treatment with 0.5 mM mimosine significantly ( P<0.05) inhibited 3 H -thymidine incorporation after 24 h of culture (4.6% ± 0.1) and after 24 h of culture in serum deprived medium (41.3% ± 3.8), in comparison to controls (100%). Inhibition of DNA synthesis was further confirmed by immunocytochemical and flow cytometry analyses. Compared with controls (78.2%), mimosine treatment for 24 h increased the proportion of G0/G1 cells in the culture (85.7%) more effectively than serum starvation (SS; 81.2%). Mimosine-caused G1 arrest of porcine GCs was fully reversible and cells continued to proliferate after removing the drug, especially when they were stimulated by EGF.

Contribution of the Helix-Loop-Helix Factor Id2 to Regulation of Vascular Smooth Muscle Cell Proliferation

Smooth muscle cell (SMC) proliferation plays a key role in vascular proliferative disorders. The molecular mechanisms that control cell cycle entry of SMCs in response to vascular injury are not well understood. Id2 (inhibitor of DNA binding) is a member of the helix-loop-helix (HLH) family of transcription regulators that are known to promote cell cycle progression. Thus, we investigated the role of Id2 in SMC growth and cell cycle regulation. The results demonstrated that overexpression of Id2 resulted in a significant enhancement of SMC growth via increased S-phase entry. A possible mechanism of Id2-enhanced SMC growth is via regulation of p21 expression, as overexpression of Id2-inhibited transcriptional activity of a 2.3-kb p21 promoter/luciferase reporter construct as well as p21 protein levels. Id2 enhancement of SMC growth and inhibition of p21 expression were dependent on phosphorylation of Id2 by cyclin E/cdk2, as an Id2 cDNA containing a mutation in the cdk2 phosphorylation site (serine 5) failed to regulate SMC cell cycle progression or p21 promoter activity. The mechanism of cyclin E/cdk2 control of the Id2 effect may in part involve regulation of nuclear transport; unlike wild-type Id2, the Id2 mutant was not transported to the nucleus. Finally, in a rat carotid model of arterial injury, Id2 was expressed in a temporal pattern that parallels the kinetics of cellular proliferation. In summary, these results provide evidence that the Id2 protein is integrated into the cell cycle regulatory cascade that results in SMC proliferation following vascular injury and suggest that this effect is at least in part via a cdk2-dependent inhibition of p21 gene expression.

Generation of cloned goats (Capra hircus) from transfected foetal fibroblast cells, the effect of donor cell cycle.

The neomycin-resistant gene (neo(r)) is probably the most commonly used selectable marker gene in gene targeting and gene transfection research. In this study, the neo(r) gene construct was introduced into in vitro cultured goat foetal fibroblast cells (IV-5), and the cells were selected with 900 microg/ml G418. The G418-resistant colonies were analysed by neo-specific PCR, karyotyping and anti-intermediate filament proteins antibody (anti-vimentin) staining. Cell cycle analysis of the neo(r) positive foetal fibroblast cell colony (IV-5.1) cultured in a variety of cell cycle-arresting medium indicated that 74.2% of cells cultured in serum-deprived medium for 3 days and 71.7% of cells grown to confluence were at G0/G1 stage of cell cycle, respectively, in comparison to 61.6% of cells in normal culture (cycling) medium. Nocodazole treatment for 17 hr in vitro culture could increase the number of cells at G2/M stage of cell cycle from 20.3% (in cycling medium) to 39.7%. In total, one early pregnancy was observed by B ultra-sound scanning in a surrogate transferred with cloned embryos from IV-5.1 cells at M stage (cells were cultured in nocodazole medium). Seven cloned goats, including two that miscarried at a late stage, were derived from the IV-5.1 cell clone cultured in starved medium (G0). Indeed, one surrogate receiving three blastocysts reconstituted from the starved donor cells, gave birth to three live cloned goats, all of which are healthy and doing well. PCR, Southern blot and G418 resistance in vitro of fibroblast cells from cloned goats confirmed that all cloned goats are positive for neo(r) transgene. This study demonstrates that a foreign gene, such as the neo-resistant gene, can be introduced into goat foetal fibroblast cells, and that the resulting transgenic cells are capable of being cloned to produce 100% transgenic animals.

Broad distribution of colony-forming cells with erythroid, myeloid, dendritic cell, and NK cell potential among CD34(++) fetal liver cells.

The generation of erythroid, myeloid, and lymphoid cells from human fetal liver progenitors was studied in colony-forming cell (CFC) assays. CD38(-) and CD38(+) progenitors that expressed high levels of CD34 were grown in serum-deprived medium supplemented with kit ligand, flk2/flt3 ligand, GM-CSF, c-mpl ligand, erythropoietin, and IL-15. The resulting colonies were individually analyzed by flow cytometry. CD56(+) NK cells were detected in 21.9 and 9.9% of colonies grown from CD38(-) and CD38(+) progenitors, respectively. NK cells were detected in mostly large CD14(+)/CD15(+) myeloid colonies that also, in some cases, contained red cells. NK cells were rarely detected in erythroid colonies, suggesting an early split between the erythroid and the NK cell lineages. CD1a(+) dendritic cells were also present in three-quarters of the colonies grown from CD38(-) and CD38(+) progenitors. Multilineage colonies containing erythrocytes, myeloid cells, and NK cells were present in 13.7 and 2.7% of colonies grown from CD38(-) and CD38(+) progenitors, respectively. High proliferative-potential CFCs that generated multilineage colonies were also detected among both populations of progenitors. The total number of high proliferative-potential CFCs with erythroid, myeloid, and NK cell potential was estimated to be 2-fold higher in the CD38(+) fraction compared with the CD38(-) fraction because of the higher frequency of CD38(+) cells among CD34(++) cells. The broad distribution of multipotent CFCs among CD38(-) and CD38(+) progenitors suggests that the segregation of the erythroid, myeloid, and lymphoid lineages may not always be an early event in hemopoiesis. Alternatively, some stem cells may be present among CD38(+) cells.

GROWTH INHIBITION, MORPHOLOGICAL DIFFERENTIATION AND STIMULATION OF SURVIVAL IN NEURONAL CELL TYPE (Neuro-2a) TREATED WITH TROPHIC MOLECULES

Trophic molecules are key regulators of survival, growth and differentiation of neural cells. Neuronal cell type Neuro-2a is a good model to study development and molecules modulating this process, and retinoic acid (RA) and neurotrophins (NGF, BDNF, NT-3 and NT-4) have been shown to be active in this modulation. The purpose of the present study was the functional analysis of these trophic molecules in our short-term bioassay of Neuro-2a cells, an immortalised murine neuroblastoma cell line. Through cell counting, image process and arithmetic combination of digital parameters of treated and untreated cultures, we show that RA inhibits growth and induces morphological neuronal phenotype of treated cells. Through DNA labelling with BrdU we also show that NGF, BDNF, and NT-3 increase survival and proliferation of cells, grown in serum-deprived media. From these results we conclude that neurotrophins have manifest trophic effects on cells improving survival, growth and proliferation and we also confirm the growth arrest and differentiation properties of RA on Neuro-2a cells.

High-Efficiency Retroviral Transduction of Fetal Liver CD38–CD34++ Cells: Implications for in utero and ex utero Gene Therapy

Objectives: Defining methods for the efficient transduction of fetal stem cells could lead to novel fetal therapies for blood cell disorders and other birth defects. In this study, we analyzed the effects of various parameters on the retroviral transduction of primitive hematopoietic progenitors/stem cells isolated from fetal liver. Methods: Candidate stem cells were isolated by fluorescence-activated cell sorting from midtrimester human livers based on the phenotype CD38–CD34++lineage– (lineage = glycophorin A, CD3, CD14, CD19, CD20 and CD56). A murine retroviral vector with a truncated human low-affinity nerve growth factor receptor (ΔNGFR) gene was used to transduce the candidate stem cells. Marker gene expression was monitored by flow cytometry using an anti-NGFR mAb. Candidate stem cells were transduced immediately after isolation or after up to 4 days of culture in serum-deprived medium containing the growth factors kit ligand and granulocyte-macrophage colony-stimulating factor. The effects on transduction efficiency of the addition of 4 µg/ml protamine sulfate and/or centrifugation to concentrate the candidate stem cells and virus were tested. After transduction, the cells were expanded for 10–21 days before determining the frequency of NGFR+ cells among the different hematopoietic progeny. Results: Efficient transduction of candidate stem cells, at an average rate of 46%, was achieved after 3 days of culture with a single exposure to virus. Longer than 3 days of culture or repeated exposure to viral supernatant did not significantly improve the rate of transduction. The use of centrifugation at 1,200 g for 1 h and the addition of protamine sulfate during the transduction procedure were critical to achieving a high rate of transduction. Marker gene expression was observed on the progeny of the transduced cells in conjunction with CD34 (progenitors), glycophorin A (erythrocytes), CD14 (monocytes), CD15 (granulocytes) and CD41 (megakaryocytes). Conclusions: This study demonstrates that the efficient transduction of fetal candidate stem cells can be achieved under defined culture conditions using a retroviral vector. These results encourage further examination of in utero and ex utero gene therapy as a means of treating birth defects.

Glial cell line-derived neurotrophic factor promotes survival and induces differentiation through the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathway respectively in PC12 cells.

PC12-GFRalpha1 cells, a clonal cell line engineered to express glial cell line-derived neurotrophic factor receptor alpha1 were constructed. Given glial cell line-derived neurotrophic factor could induce the differentiation and promote the survival of PC12-GFRalpha1 cells at low concentrations, the cells provide an unlimited source of monoclonal cells for studies on the signal transduction pathway of glial cell line-derived neurotrophic factor. To characterize the involvement of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways in the biological effect of glial cell line-derived neurotrophic factor, we used the mitogen-activated protein kinase kinase inhibitor PD98059 and the phosphatidylinositol 3-kinase inhibitor LY294002. PD98059 blocked glial cell line-derived neurotrophic factor-induced PC12-GFRalpha1 cells neurite formation in a dose-dependent manner, without significantly altering cell viability. LY294002 reversed the survival-promoting effect of glial cell line-derived neurotrophic factor on the PC12-GFRalpha1 cells in serum-deprived medium. The present study demonstrates that phosphatidylinositol 3-kinase pathway seems to mediate the survival-promoting effect of glial cell line-derived neurotrophic factor on PC12-GFRalpha1 cells, while the activation of mitogen-activated protein kinase pathway could be an important step in mediating PC12-GFRalpha1 cells differentiation induced by glial cell line-derived neurotrophic factor. Therefore, it is inferred that similar intracellular signaling components are used by distinct growth factors toward a common biological effect.

Flow-Induced DNA Synthesis Requires Signaling to a Translational Control Pathway

Background. The mTOR translational control pathway that signals to the P70/P85 S6 kinase (pp70S6k) is essential for mitogenesis. We have previously shown that pp70S6k is activated by fluid flow. We hypothesized that oscillatory fluid flow in the absence of exogenous mitogens would induce endothelial cells to synthesize DNA via activation of the mTOR pathway. For comparison, we also studied the ERK1/2 transcriptional signaling pathway.Methods. Confluent human umbilical vein endothelial cells (HUVECs) were exposed to oscillatory flow (12 dyn/cm2 peak shear stress; 3.3 Hz) or kept static in serum-deprived culture medium. Rapamycin or PD98059 was used to inhibit pp70S6k or ERK1/2 activation, respectively.Results. Oscillatory flow activated both the pp70S6k and ERK1/2 signaling pathways. Rapamycin blocked activation of pp70S6k but not ERK1/2, while PD98059 blocked ERK1/2 but not pp70S6k. DNA synthesis, as measured by [3H]thymidine uptake, increased by approximately twofold (P < 0.01) in HUVEC cultures exposed to oscillatory flow compared with those kept static. Rapamycin completely abolished the flow-induced increase in DNA synthesis while PD98059 did not. Oscillatory flow upregulated expression of cyclin-dependent kinases 1 and 4 mRNA in a temporal pattern consistent with cell cycle entry; rapamycin also inhibited these changes.Conclusions. Oscillatory flow activates both the ERK 1/2 and pp70S6k signaling pathways in HUVECs and induces DNA synthesis in the absence of other exogenous mitogens. Complete blockade of [3H]thymidine uptake by the mTOR pathway inhibitor rapamycin indicates that separate and distinct signaling to a translational control pathway is necessary to mediate flow-induced DNA synthesis by endothelial cells. Oscillatory flow-induced endothelial proliferation may contribute to atherogenesis.