Proliferation Of Cell Lineages Research Articles

Recombinant Erythropoietin Induces Oligodendrocyte Progenitor Cell Proliferation After Traumatic Brain Injury and Delayed Hypoxemia

Traumatic brain injury (TBI) can result in axonal loss and demyelination, leading to persistent damage in the white matter. Demyelinated axons are vulnerable to pathologies related to an abnormal myelin structure that expose neurons to further damage. Oligodendrocyte progenitor cells (OPCs) mediate remyelination after recruitment to the injury site. Often this process is inefficient due to inadequate OPC proliferation. To date, no effective treatments are currently available to stimulate OPC proliferation in TBI. Recombinant human erythropoietin (rhEPO) is a pleiotropic neuroprotective cytokine, and its receptor is present in all stages of oligodendroglial lineage cell differentiation. Therefore, we hypothesized that rhEPO administration would enhance remyelination after TBI through the modulation of OPC response. Utilizing a murine model of controlled cortical impact and a primary OPC culture in vitro model, we characterized the impact of rhEPO on remyelination and proliferation of oligodendrocyte lineage cells. Myelin black gold II staining of the peri-contusional corpus callosum revealed an increase in myelinated area in association with an increase in BrdU-positive oligodendrocytes in injured mice treated with rhEPO. Furthermore, morphological analysis of OPCs showed a decrease in process length in rhEPO-treated animals. RhEPO treatment increased OPC proliferation after in vitro CSPG exposure. Erythropoietin receptor (EPOr) gene knockdown using siRNA prevented rhEPO-induced OPC proliferation, demonstrating that the rhEPO effect on OPC response is EPOr activation dependent. Together, our findings demonstrate that rhEPO administration may promote myelination by increasing oligodendrocyte lineage cell proliferation after TBI.

The suppressive functions of Rora in B lineage cell proliferation and BCR/ABL1-induced B-ALL pathogenesis.

RORA plays an important role in regulating circadian rhythms, inflammation, metabolism and cellular development. Herein, we explore the roles of Rora in B cell proliferation and differentiation, as well as in Ph+ B-ALL. By using Roraloxp/loxp Mx-1-Cre mice, Rora was deleted in hematopoietic cells post Pipc induction. Rora deficiency mice were associated with an obvious accumulation of B cells in the peripheral blood, bone marrow, and spleen. On the other hand, activation of Rora with Cholesterol sulfate (CS) was associated with decreased B cell numbers. RNA-seq analysis revealed that the transcription level of Lmo1 was decreased in Rora deficient B cells. Moreover, the expression of RORA was shown to be decreased in Ph+ B-ALL cells compared to peripheral blood derived B cells from healthy donors. The overexpression of Rora in BaF3 cells with BCR/ABL1 was also associated with impeded the cell growth and an increased apoptotic rate compared to cells transduced with BCR/ABL1 alone. The co-expression of BCR/ABL1 and Rora induced B-ALL mouse model was associated with the significant inhibition of BCR/ABL1-transformed cell growth and prolonged the survival of the diseased mice. These results suggest a novel role for Rora in B cell development and Ph+ leukemogenesis.

Environmental and sex-specific molecular signatures of glioma causation.

BackgroundThe relative importance of genetic and environmental risk factors in gliomagenesis remains uncertain.MethodsUsing whole-exome sequencing data from 1105 adult gliomas, we evaluate the relative contribution to cancer cell lineage proliferation and survival of single-nucleotide mutations in tumors by IDH mutation subtype and sex. We also quantify the contributions of COSMIC cancer mutational signatures to these tumors, identifying possible risk exposures.Results IDH-mutant tumors exhibited few unique recurrent substitutions—all in coding regions, while IDH wild-type tumors exhibited many substitutions in non-coding regions. The importance of previously reported mutations in IDH1/2, TP53, EGFR, PTEN, PIK3CA, and PIK3R1 was confirmed; however, the largest cancer effect in IDH wild-type tumors was associated with mutations in the low-prevalence BRAF V600E. Males and females exhibited mutations in a similar set of significantly overburdened genes, with some differences in variant sites—notably in the phosphoinositide 3-kinase (PI3K) pathway. In IDH-mutant tumors, PIK3CA mutations were located in the helical domain for females and the kinase domain for males; variants of import also differed by sex for PIK3R1. Endogenous age-related mutagenesis was the primary molecular signature identified; a signature associated with exogenous exposure to haloalkanes was identified and noted more frequently in males.ConclusionsCancer-causing mutations in glioma primarily originated as a consequence of endogenous rather than exogenous factors. Mutations in helical vs kinase domains of genes in the phosphoinositide 3-kinase (PI3K) pathway are differentially selected in males and females. Additionally, a rare environmental risk factor is suggested for some cases of glioma—particularly in males.

Live Visualization of ERK Activity in the Mouse Blastocyst Reveals Lineage-Specific Signaling Dynamics.

FGF/ERK signaling is crucial for the patterning and proliferation of cell lineages that comprise the mouse blastocyst. However, ERK signaling dynamics have never been directly visualized in live embryos. To address whether differential signaling is associated with particular cell fates and states, we generated a targeted mouse line expressing an ERK-kinase translocation reporter (KTR) that enables live quantification of ERK activity at single-cell resolution. 3D time-lapse imaging of this biosensor in embryos revealed spatially graded ERK activity in the trophectoderm prior to overt polar versus mural differentiation. Within the inner cell mass (ICM), all cells relayed FGF/ERK signals with varying durations and magnitude. Primitive endoderm cells displayed higher overall levels of ERK activity, while pluripotent epiblast cells exhibited lower basal activity with sporadic pulses. These results constitute a direct visualization of signaling events during mammalian pre-implantation development and reveal the existence of spatial and temporal lineage-specific dynamics.

Combination therapy of apo-transferrin and thyroid hormones enhances remyelination.

The current study presents two different approaches with a view to elucidating the interaction between thyroid hormones (TH) and apo-transferrin (aTf) and their role in myelination and remyelination. First, in vitro assays were conducted to determine the single and combined effects of aTf and triiodothyronine (T3) on oligodendroglial cell lineage proliferation and oligodendrocyte (OLG) maturation in primary cultures. Results revealed higher proliferation rates upon single aTf treatment but Control values upon T3 and aTf + T3 treatments. In addition, both aTf and T3 accelerated OLG maturation, with the greatest effects being exerted by combined aTf + T3 administration in terms of both myelin basic protein (MBP) expression and morphological complexity. Second, in vivo assays were carried out to establish single and combined effects of aTf and T3, as well as TH receptor (THR) inhibitor I-850, on remyelination following a CPZ-induced demyelination protocol. Results showed an increase in myelin deposition and the number of mature remyelinating OLG upon single treatments, but a synergic effect upon combined aTf + T3 treatment which was prevented by THR inhibition. It may be thus concluded that combined treatment yielded the most beneficial effects on OLG maturation parameters in vitro and remyelinating capacity in vivo when compared to single treatments. These findings may help explore the development of new target molecules in the treatment of demyelinating diseases.

ERK5 negatively regulates Kruppel-like factor 4 and promotes osteogenic lineage cell proliferation in response to MEK5 overexpression or fluid shear stress

ABSTRACT Aim of the study: Fluid shear stress (FSS) plays a critical role in osteoblast proliferation via extracellular signal-regulated kinase 5 (ERK5). Kruppel-like factor 4 (KLF4) knockout robustly enhances bone formation due to increased osteoblast differentiation and mineralization. However, the effect of KLF4 on osteoblast proliferation is unresolved. Therefore, the aim of our study was to investigate the effect of KLF4 on osteogenic lineage cell proliferation and the relationship between KLF4 and ERK5. Materials and methods: MC3T3-E1 cells were treated with FSS and/or KLF4 siRNA, cell viability was accessed by Edu labeling and CCK-8 assay, and proliferative gene expression were assessed by PCR array. Bone marrow stromal cells (BMSCs) were infected with adenovirus expressing KLF4 and/or constitutively active MEK5, cell viability was evaluated using crystal violet staining, colony formation assay, and cell WST1 assay. The levels of KLF4 and ERK5 phosphorylation were identified through qRT-PCR and western blot, respectively. Results: KLF4 expression was significantly down-regulated by FSS exposure, however, this was reversed by ERK5 siRNA. KLF4 overexpression inhibited colony formation efficiency and cell viability in BMSCs. Adenoviruses expressing constitutively active MEK5 increased ERK5 phosphorylation, which inhibited KLF4 expression, and promoted BMSC proliferation. FSS-induced osteoblast proliferation also involved elevation of Cyclin B2 and Cdc14b as well as repressed expression of P27. Conclusions: KLF4 negatively regulates osteogenic lineage cell proliferation, and ERK5 negatively regulates KLF4 expression and promotes osteogenic lineage cell proliferation.

JAK-2-Positive Latent Myeloproliferative Neoplasms with Splanchnic Vein Thrombosis- from the Hematopathology Perspective

Introduction: Philadephia-negative Myeloproliferative neoplasms (MPNs) include polycythemia Vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The diagnosis of MPNs and further sub classification are based mainly on WHO diagnostic criteria which rely on the presence of an evidence of single or multiple cell lineage proliferation reflected by elevated hemoglobin, and / leukocytosis and /thrombocytosis. In addition, to presence of certain pathologic features in the bone marrow (BM) including hypercellularity , panmyelosis, proliferation of megakaryocytes, the presence of atypical megakaryocytes and or increased marrow fibrosis. This is in addition to the presence of molecular genetic marker specific for MPN including JAK-2, MPL and CALR. Making a diagnosis of MPN in absence of the previously mentioned features is always challenging. Herein, we analyze the clinical, pathologic and molecular genetics features for five cases presented with splanchnic vein thrombosis and found to be positive for JAK-2 V617F. However, from the hematpathologic point of view, apart from JAK-2 positivity, none of these cases had fulfilled the WHO diagnostic criteria for MPNs. Although the BM specimens were reviewed by at least two experienced hematopathologists, it was so difficult to conclude the diagnosis and or do further sub classification into a specific MPN category. Objective: The aim of this report is to highpoint the difficulty in establishing the diagnosis within this group of patients based on the current WHO diagnostic criteria, to focus on BM pathologic features of this group of patients. Patients and Methods: The patients reported here were referred to our center presenting with portal vein, superior mesenteric or splenic vein thrombosis. Five adult female patients were recognized (2015 and 2019). The mean age at diagnosis is 40.8 years (27-56). None of these cases had an increase in peripheral blood counts except for patient (1) which had mild leukocytosis and thrombocytosis. All patients showed normocellular or slightly hypercellular BM. Slight erythroid predominance was reported in patient (2) and (4). Megakaryocytes were increased in all patients except for patient (2). Interestingly, megakaryocytes morphology was similar in all five patients as they showed anisocytosis with some degree of pleomorphism with predominant of large forms, no clustering and no abnormal topography was noted (figure 1 and 2). All patients showed no significant increase in reticulin fibrosis. Sub-classification into a specific MPN category cannot be performed in all patients. Next generation sequence had been used to analyze targeted regions in recurrently mutated genes in myeloid neoplasia. JAK-2V617F mutation was detected in all studied patients. In patient number (2) JAK-2 mutation was detected at a low level (4%), however, on follow-up, the mutation level increased in addition to detection of 3bp insertion/deletion in CALR gene resulting in a mutation other than type I or type II. table (1). Discussion: Splanchnic veins thrombosis (SVT) includes the portal vein thrombosis (PVT), mesenteric (MVT) splenic vein thrombosis, and the Budd Chiari syndrome (BCS). SVTs are rare events, estimated by 0.7 per 100,000 patients per year for PVT [Rajani et al. 2010]. In a laboratory setting, the diagnosis of latent MPN in patients with SVT is challenging due to absence of elevated blood counts, probably caused by portal hypertension and splenomegaly that result from SVT and MPN. However, the reason why this group of patients does not have overt features of myeloproliferation (particularly) the hypercellularity in the BM is not yet clear. It is possible that JAK-2 positivity induced venous thrombosis longer time before development of the full blown picture of MPN. However, follow-up BM studies for this group of patients might help in better characterization of the pathologic features of these patients. These patients are included in the MPN, unclassifiable subcategory in the current WHO classification. However, based on our findings and the previously published data it seems that these patients have distinctive clinical and pathological features that necessitate having them in a separate MPN entity that might have less strict diagnostic criteria that might include clinical symptoms especially venous thrombosis in large vessels. Separating this group of patients will help to study them thoroughly. Disclosures No relevant conflicts of interest to declare.

Selective Pressures on Human Cancer Genes along the Evolution of Mammals.

Cancer is a disease driven by both somatic mutations that increase survival and proliferation of cell lineages and the evolution of genes associated with cancer risk in populations. Several genes associated with cancer in humans, hereafter cancer genes, show evidence of germline positive selection among species. Taking advantage of a large collection of mammalian genomes, we systematically looked for signatures of germline positive selection in 430 cancer genes available in COSMIC. We identified 40 cancer genes with a robust signal of positive selection in mammals. We found evidence for fewer selective constraints—higher number of non-synonymous substitutions per non-synonymous site to the number of synonymous substitutions per synonymous site (dN/dS)—and higher incidence of positive selection—more positively selected sites—in cancer genes bearing germline and recessive mutations that predispose to cancer. This finding suggests a potential association between relaxed selection, positive selection, and risk of hereditary cancer. On the other hand, we did not find significant differences in terms of tissue or gene type. Human cancer genes under germline positive selection in mammals are significantly enriched in the processes of DNA repair, with high presence of Fanconi anaemia/Breast Cancer A (FA/BRCA) pathway components and T cell proliferation genes. We also show that the inferred positively selected sites in the two genes with the strongest signal of positive selection, i.e., BRCA2 and PTPRC, are in regions of functional relevance, which could be relevant to cancer susceptibility.

Life history, immunity, Peto's paradox and tumours in birds.

Cancer and tumours may evolve in response to life-history trade-offs between growth and duration of development on one hand, and between growth and maintenance of immune function on the other. Here, we tested whether (i) bird species with slow developmental rates for their body size experience low incidence of tumours because slow development allows for detection of rapid proliferation of cell lineages. We also test whether (ii) species with stronger immune response during development are more efficient at detecting tumour cells and hence suffer lower incidence of tumours. Finally, we tested Peto's paradox, that there is a positive relationship between tumour incidence and body mass. We used information on developmental rates and body mass from the literature and of tumour incidence (8468 birds) and size of the bursa of Fabricius for 7659 birds brought to a taxidermist in Denmark. We found evidence of the expected negative relationship between incidence of tumours and developmental rates and immunity after controlling for the positive association between tumour incidence and body size. These results suggest that evolution has modified the incidence of tumours in response to life history and that Peto's paradox may be explained by covariation between body mass, developmental rates and immunity.

SDF-1 and CXCR4 play an important role in adult SVZ lineage cell proliferation and differentiation

Evidence has shown that stromal cell-derived factor (SDF-1/CXCL12) plays an important role in maintaining adult neural progenitor cells (NPCs). SDF-1 is also known to enhance recovery by recruiting NPCs to damaged regions and recent studies have revealed that SDF-1α exhibits pleiotropism, thereby differentially affecting NPC subpopulations. In this study, we investigated the role of SDF-1 in in vitro NPC self-renewal, proliferation and differentiation, following treatment with different concentrations of SDF-1 or a CXCR4 antagonist, AMD3100. We observed that AMD3100 inhibited the formation of primary neurospheres. However, SDF-1 and AMD3100 exhibited no effect on proliferation upon inclusion of growth factors in the media. Following growth factor withdrawal, AMD3100 and SDF-1 treatment resulted in differential effects on NPC proliferation. SDF-1, at a concentration of 500ng/ml, resulted in an increase in the relative proportion of oligodendrocytes following growth factor withdrawal-induced differentiation. Using CXCR4 knockout mice, we observed that SDF-1 affected NPC proliferation in the sub-ventricular zone (SVZ). We also investigated the occurrence of differential CXCR4 expression at different stages during lineage progression. These results clearly indicate that signaling interactions between SDF-1 and CXCR4 play an important role in adult SVZ lineage cell proliferation and differentiation.

Abstract 5113: Synergistic effects of Deferasirox and Imatinib in chronic myeloid leukemia cells

Abstract Chronic myelogenous leukemia (CML) is diagnosed by abnormal myeloid lineage cell proliferation and the presence of the Bcr-Abl fusion gene encoding abnormal tyrosine kinase. Imatinib mesylate (IM) is the first developed Bcr/Abl tyrosine-kinase inhibitor for the treatment of CML. Despite of high response rates, significant numbers of CML patients develop the resistance against these kinase inhibitors. Deferasirox (DFO) is an oral iron chelating agent that has been used to treat acute iron detoxification and chronically iron overloaded. Recent reports have been shown that iron chelators induce anti-proliferative effect in multiple leukemia cells. However, their roles and underlying mechanism in CML cell lines are not yet fully understood. In this study, we evaluated the combination effects of Deferasirox and Imatinib in CML cell line, and investigated their apoptosis -inducing effects as the potential mechanism in this process. Two CML cell lines (K562, KU812) were treated with various concentrations of Deferasirox, Imatinib and combination of two drugs. The viability and the level of apoptosis of the treated cells will be measured with MTT assay, FACS analysis and western blot. The results showed that both of them induce the growth inhibitory effects in dose and Time dependent fashion in both CML cell lines. Cell cycle analysis by FACS indicated that cell lines treated with combination therapy showed an increase in the proportion of cells in sub-G1 phase, compared to single treatment cell lines. Exposure of CML cells to IM/DFO also resulted in the increase in the percentage of annexin V-positive and PI-negative cells. The results by multiple in vitro experimental model, showed that IM/DFO induce CML cells to undergo apoptosis with the activation of caspase9 and 3, the cleavage of PARP and reduced significantly in parallel with BCR/ABL phosphorylation compared with treatment with IM or DFO alone. Importantly, inhibition of NF-kB activity demonstrated that IM/DFO-induced apoptotic pathway was regulated by the NF-kB signaling cascade. To determine the effect of IM/DFO on NF-κB transcriptional activity, a plasmid containing the luciferase reporter regulated by NF-κB cis-acting elements was used. The results showed that IM/DFO decreased NF-κB transcriptional activity in K562 and KU812 cells, respectively, compared to single treated cells. IM/DFO regulated the nuclear activation of NF-κB. In conclusion, this finding indicates that combination of Imatinib and Deferasirox, promotes apoptosis of CML cells by sequential regulation of the BCR/ABL expression followed by the inhibition of NF-kB signaling pathway. Citation Format: Dae Sik Kim, Myoung Hee Kang, Yoo Jin Na, Jung Lim Kim, Bo Ram Kim, Kyong Hwa Park, Sang Cheul Oh, Jae Hong Seo, Chul Won Choi, Jun Suk Kim. Synergistic effects of Deferasirox and Imatinib in chronic myeloid leukemia cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5113. doi:10.1158/1538-7445.AM2014-5113

Dense‐map genome scan for dyslexia supports loci at 4q13, 16p12, 17q22; suggests novel locus at 7q36

Analysis of genetic linkage to dyslexia was performed using 133,165 array-based SNPs genotyped in 718 persons from 101 dyslexia-affected families. Results showed five linkage peaks with lod scores >2.3 (4q13.1, 7q36.1-q36.2, 7q36.3, 16p12.1, and 17q22). Of these five regions, three have been previously implicated in dyslexia (4q13.1, 16p12.1, and 17q22), three have been implicated in attention-deficit hyperactivity disorder (ADHD, which highly co-occurs with dyslexia; 4q13.1, 7q36.3, 16p12.1) and four have been implicated in autism (a condition characterized by language deficits; 7q36.1-q36.2, 7q36.3, 16p12.1, and 17q22). These results highlight the reproducibility of dyslexia linkage signals, even without formally significant lod scores, and suggest dyslexia predisposing genes with relatively major effects and locus heterogeneity. The largest lod score (2.80) occurred at 17q22 within the MSI2 gene, involved in neuronal stem cell lineage proliferation. Interestingly, the 4q13.1 linkage peak (lod 2.34) occurred immediately upstream of the LPHN3 gene, recently reported both linked and associated with ADHD. Separate analyses of larger pedigrees revealed lods >2.3 at 1-3 regions per family; one family showed strong linkage (lod 2.9) to a known dyslexia locus (18p11) not detected in our overall data, demonstrating the value of analyzing single large pedigrees. Association analysis identified no SNPs with genome-wide significance, although a borderline significant SNP (P = 6 × 10(-7)) occurred at 5q35.1 near FGF18, involved in laminar positioning of cortical neurons during development. We conclude that dyslexia genes with relatively major effects exist, are detectable by linkage analysis despite genetic heterogeneity, and show substantial overlapping predisposition with ADHD and autism.

Parathyroid hormone affects the fibroblast growth factor–proteoglycan signaling axis to regulate osteosarcoma cell migration

Parathyroid hormone (PTH)(1-34), which has been established to have a dual effect on bone metabolism, was recently found to regulate osteosarcoma cell migration. A significant part of the bone anabolic action of PTH(1-34) is attributed to fibroblast growth factor (FGF)-2 stimulation. Furthermore, it was recently suggested that the FGF-proteoglycan axis may form an extracellular matrix-related regulatory feedback loop that controls osteoblastic lineage cell proliferation and execution of the osteogenic program. In this study, we investigated the possible participation of FGF-2 signaling in PTH(1-34)-dependent osteosarcoma cell migration. FGF-2 treatment of osteosarcoma cells resulted in a significant increase (P ≤ 0.01) in MG63 cell migration, similar to that caused by PTH(1-34). mRNA expression analysis of cells treated with PTH(1-34) showed a strong increase in FGF-2 transcript levels (P = 0.0015). Interestingly, the addition of FGF-2 to MG63 cells led to significant downregulation of small leucine-rich proteoglycan biglycan expression at both the mRNA (P ≤ 0.0001) and protein (60%) levels. In order to examine the significance of biglycan on MG63 cell migration, transfection with short interfering RNA specific for biglycan was performed, resulting in a significant increase (P ≤ 0.01) in the migration capacity of biglycan-deficient MG63 cells. In contrast, exogenous human recombinant biglycan strongly inhibited the migration of these cells (P ≤ 0.01). Finally, a direct correlation between PTH(1-34) action and biglycan expression was established by the finding of a significant decrease (P ≤ 0.01) in biglycan transcript levels in PTH(1-34)-treated cells. To summarize, the present study demonstrates a novel cooperative mechanism of PTH(1-34) and FGF-2 action that results in specific alteration of the biglycan extracellular matrix content to regulate osteosarcoma cell migration.

RNA Interference Reveals a Requirement for Both p18INK4c and p27Kip1 in B Lymphopoiesis

Cyclin-dependent kinase inhibitors (CKIs) p18(INK4c) (p18) and p27(Kip1) (p27) were reported to be able to modulate self-renewal and differentiation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells, and regulate the lineage cell proliferation and maturation into the terminal elements; however, whether p18 and p27 in HSCs affect the development of lineage cells, especially B lymphocytes, in the reconstituted blood is unknown. Here we employed small-interference RNA (siRNA) technique, which provides a powerful tool for tissue-targeted knockdown of genes, to evaluate the biological functions of the p18 and p27 in the hematopoiesis process. We knocked down the expression of p18, p27 alone or both in the isolated sca-1(+) bone marrow cells by lentiviral vector-based siRNA system, and transplanted these cells into lethally irradiated C57BL/6J mice to evaluate the effect of these two genes on reconstituted lymphocyte development. The knockdown of p18 or p27 alone or both was proved to be effective as verified by western blotting. FACS analysis results showed that compared with the control group, the B lymphocytes were both significantly lower in p18, p27 alone and especially in both p18 and p27 knockdown group in reconstituted peripheral blood; and the B lymphocytes showed similar trend in bone marrow. Interestingly, the differentiation to T cells was not greatly changed, only with the dramatic decrease of the CD4/CD8 ratio. Overexpression of the antiapoptotic protein Bcl2 could not rescue the B lymphopoiesis. All these results demonstrate that p18 and p27 are collaboratively involved in B lymphopoiesis, and simultaneous knockdown of p18 and p27 probably blocks the differentiation from HSCs to B lymphocytes, but not triggers apoptosis of B cell precursors.

Calcium‐mediated differentiation of ameloblast lineage cells in vitro

Calcium is a key component of the mineralized enamel matrix, but may also have a role in ameloblast cell differentiation. In this study we used human ameloblast lineage cells to determine the effect of calcium on cell function. Primary human ameloblast lineage cells were isolated from human fetal tooth buds. Cells were treated with calcium ranging from 0.05 to -1.8 mM. Cell morphology was imaged by phase contrast microscopy, and amelogenin was immunolocalized. Proliferation of cells treated with calcium was measured by BrdU immunoassay. The effect of calcium on mRNA expression of amelogenin, Type 1 collagen, DSPP, amelotin, and KLK-4 was compared by PCR analysis. Von Kossa staining was used to detect mineral formation after cells were pretreated with calcium. Calcium induced cell organization and clustering at 0.1 and 0.3 mM concentrations. Increasing concentrations of calcium significantly reduced ameloblast lineage cell proliferation. The addition of 0.1 mM calcium to the cultures upregulated expression of amelogenin, Type I collagen, and amelotin. After pretreatment with 0.3 mM calcium, the cells could form a mineralized matrix. These studies, which utilized human ameloblast lineage cells grown in vitro, showed that the addition of calcium at 0.1 and 0.3 mM, induced cell differentiation and upregulation of amelogenin Type I collagen and amelotin.

Thymocyte costimulating antigen is CD26 (dipeptidyl-peptidase IV). Costimulation of granulocyte, macrophage, and T lineage cell proliferation via CD26.

The rat thymocyte costimulating protein appears to be involved in the regulation of CD4-/CD8- thymocyte proliferation in vitro. We show that thymocyte costimulating protein is dipeptidyl peptidase IV (E.C.N.3.4.14.5) also known as CD26. Some bone marrow cells as well as CD4-/CD8- and, and to a lesser extent, more differentiated T cells respond by proliferating to a CD26 specific mAb-mediated costimulus that does not influence dipeptidyl dipeptidase IV enzyme activity. This suggests the existence of a CD26-linked regulatory mechanism of proliferation that is operational on granulocyte- and macrophage-lineage cells and throughout T cell development.

Autocrine inhibition of mitotic activity in cultured oligodendrocyte-type-2 astrocyte (O-2A) precursor cells.

During development, oligodendrocytes are generated from a bipotential glial stem cell, the oligodendrocyte-type-2 astrocyte precursor (O-2A). O-2A cells are under the mitogenic influence of the platelet-derived growth factor (PDGF) released from type-1 astrocytes. In vitro experiments have shown that O-2A cells stimulated by PDGF are limited to a set number of divisions and then differentiate to oligodendrocytes by becoming unresponsive to the growth factor. In the healthy adult central nervous system, oligodendrocyte proliferation remains generally quiescent and is possibly under negative growth control. The view that O-2A lineage cells are capable of negatively regulating their own proliferation is supported by the demonstration that conditioned medium obtained from O-2A cultures inhibits their DNA synthesis. In addition to O-2A cells, the newly established CG4 cell line, a derivative of O-2A cells, was found to inhibit O-2A lineage cell proliferation. The antiproliferative activity was present in the media conditioned by CG4 cells that were expanded as undifferentiated O-2A precursors, as well as by CG4 cells induced to differentiate to nonproliferating oligodendrocytes. Moreover, the inhibitory activity was produced by CG4 cells (source cells) propagated by various mitogens. The inhibition of mitotic activity was nearly complete, dose-dependent, fully reversible, and exhibited when CG4 cells (test cells) were stimulated to divide by various mitogens, such as PDGF, basic fibroblast growth factor, or medium conditioned by the neuronal B104 cell line. The inhibition of proliferation was accompanied by the conversion of the phenotype of CG4 cells, from A2B5+/O4- precursors to A2B5-/O4+ pro-oligodendrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)