Effect of long-term culture of mouse embryonic stem cells under low oxygen concentration as well as on glycosaminoglycan hyaluronan on cell proliferation and differentiation

Objectives: Maintaining undifferentiated stem cells in defined conditions is of critical importance to improve their in vitro culture. We have evaluated the effects of culturing mouse stem (mES) cells under physiological oxygen concentration as well as by replacing fibroblast feeder layer (mEF) with gelatin or glycosaminoglycan hyaluronan (HA), on cell proliferation and differentiation. Materials and methods: After 3 days culture or after long‐term cell culture under different conditions, levels of apoptotic cell death were determined by cell cycle and TUNEL (TdT‐mediated dUTP nick end labelling) assays and levels of cell proliferation by CFSE (5‐(and‐6)‐carboxyfluorescein diacetate succinimidyl ester) labelling. We assessed spontaneous differentiation into cardiomyocytes and mRNA expression of pluripotency and differentiation biomarkers. Results: After 3 days culture under hypoxic conditions, levels of proliferation and apoptosis of mES cells were higher, in correlation with increase in intracellular reactive oxygen species. However, when cells were continuously grown for 1 month under those conditions, the level of apoptosis was, in all cases, under 4%. Hypoxia reduced spontaneous differentiation of mES into cardiomyocytes. Long‐term culture on HA was more effective in maintaining the pluripotent state of the mES cells when compared to that on gelatin. Level of terminal differentiation was highest on mEF, intermediate on HA and lowest on gelatin. Conclusions: Our data suggest that hypoxia is not necessary for maintaining pluripotency of mES cells and appeared to be detrimental during ES differentiation. Moreover, HA may offer a valuable alternative for long‐term culture of mES cells in vitro.     

Spontaneously immortalized mouse mesothelial cells display characteristics of malignant transformation

Abstract. Objectives: Mesotheliomas occur in occult serous cavities after chronic exposure of mesothelial cells to asbestos fibres. Molecular events that contribute to the development of this cancer are therefore not readily accessible for study. We have used in vitro culture systems to study and compare induced and spontaneous transformation events in primary mouse mesothelial cells. Materials and methods: Mouse mesothelial cells were cultivated until small populations of proliferating cells emerged from senescing cultures. Spontaneously transformed cultures of cells were characterized and compared to malignantly transformed cells. Results: Human mesothelial cells had a finite lifespan of 10–15 population doublings when cultured in vitro; mouse mesothelial cells typically exhibit this same pattern. Here, we show that mouse mesothelial cells can be cultured for extended periods and that these cells can transform spontaneously. Lines of spontaneously transformed cells generated in this study are immortal and growth factor‐independent. They display the salient characteristic features of transformation, including increased proliferation rate, lack of contact inhibition, aneuploidy and ability to grow in anchorage‐independent conditions. A subset of these cell lines developed into tumours in syngeneic mice. Comparative gene expression analysis demonstrated that spontaneously transformed cell lines were more closely related to neoplastic cells than to primary cells. Conclusion: These findings have implications for interpretation of in vitro transformation studies, demonstrating broad similarity between spontaneous and induced genetic changes.     

Spontaneous transformation of adult mesenchymal stem cells from cynomolgus macaques in vitro

Mesenchymal stem cells (MSCs) have shown potential clinical utility in cell therapy and tissue engineering, due to their ability to proliferate as well as to differentiate into multiple lineages, including osteogenic, adipogenic, and chondrogenic specifications. Therefore, it is crucial to assess the safety of MSCs while extensive expansion ex vivo is a prerequisite to obtain the cell numbers for cell transplantation. Here we show that MSCs derived from adult cynomolgus monkey can undergo spontaneous transformation following in vitro culture. In comparison with MSCs, the spontaneously transformed mesenchymal cells (TMCs) display significantly different growth pattern and morphology, reminiscent of the characteristics of tumor cells. Importantly, TMCs are highly tumorigenic, causing subcutaneous tumors when injected into NOD/SCID mice. Moreover, no multiple differentiation potential of TMCs is observed in vitro or in vivo, suggesting that spontaneously transformed adult stem cells may not necessarily turn into cancer stem cells. These data indicate a direct transformation of cynomolgus monkey MSCs into tumor cells following long-term expansion in vitro. The spontaneous transformation of the cultured cynomolgus monkey MSCs may have important implications for ongoing clinical trials and for models of oncogenesis, thus warranting a more strict assessment of MSCs prior to cell therapy.     

Steroid production,cell proliferation,and apoptosis in cultured bovine antral and mural granulosa cells: Development of an in vitro model to study estradiol production

This study was undertaken to characterize the relationship between changes in steroid production, cell cycle activity (ie, cell proliferation) and apoptosis in antral and mural bovine granulosa cells cultured in vitro. This was done to select conditions promoting optimal estradiol production by bovine granulosa cells cultured in completely defined conditions. In the first experiment, antral granulosa cells were cultured over the entire 4 days of the culture period in the presence of either 0, 2, or 10 ng/ml of FSH (chronic conditions) or were maintained under minimal FSH support (0.5 ng/ml FSH) for the first 3 days of culture and then were challenged over the fourth day of culture with either 0, 2, or 10 ng/ml FSH (challenged conditions). Compared with cells exposed to constant FSH levels (chronic conditions), the FSH-induced production of estradiol was higher (P < 0.006) and that of progesterone was lower (P < 0.02) over the last 24 h of culture, when antral granulosa cells were maintained under minimal FSH support during the first 3 days of culture (challenged conditions). In the second experiment, dynamics of estradiol and progesterone productions, conversion of [¹⁴C]androstenedione into subsequent steroid metabolites, DNA content, cell cycle activity, and apoptosis (as assessed by flow cytometry) of antral and mural granulosa cells over the first 3 days of culture under minimal FSH support and in response to a challenge with FSH during the last 24 h of culture were evaluated. Estradiol production as well as the conversion of androstenedione into testosterone and estradiol were greater (P< 0.01) in antral than in mural granulosa cells cultured under challenged conditions. A higher proportion of mural than antral granulosa cells were in the proliferative state at the end of culture (P < 0.03). This may be related to the decreased ability of mural cells to produce estradiol. FSH suppressed (P < 0.05) the spontaneous onset of apoptosis in both cell types. These results suggest that functional differences between these two cell compartments need to be considered in studying bovine granulosa cells in vitro. Because of their large (400 to 600%) FSH-induced estradiol production, antral granulosa cells cultured under challenged conditions provide a model that can be used to examine substances for their ability to alter estradiol production and apoptosis in bovine granulosa cells. Mol. Reprod. Dev. 50:170–177, 1998. © 1998 Wiley-Liss, Inc.     

A Combination of Cytokines Rescues Highly Purified Leukemic CLL B-Cells from Spontaneous Apoptosis In Vitro

B-chronic lymphocytic leukemia (B-CLL), the most common human leukemia, is characterized by predominantly non-dividing malignant mature CD5+ B lymphocytes with an apoptosis defect. Various microenvironmental stimuli confer a growth advantage on these leukemic cells and extend their survival in vivo. Nevertheless, when cultured in vitro, CLL B-cells rapidly die from apoptosis. Certain cytokines may extend the survival capacity of CLL B-cells in vitro and individual anti-apoptotic effects of several cytokines have been reported. The potential cumulative effect of such cytokines has not been studied. We therefore investigated the effects on CLL B-cells survival in vitro of humoral factors, polyclonal lymphocyte activators and a combination of cytokines known for their anti-apoptotic effects. Purified CLL B-cells were cultured in the presence or absence of various soluble molecules and the leukemic cell response was assessed in terms of viability. Apoptotic cell death was detected by flow cytometry using annexinV and 7-amino-actinomycin. The survival of CLL B-cells in vitro was highly variable. When tested separately, cytokines (IL-2, -6, -10, -12, -15, -21, BAFF and APRIL) improved CLL B cell survival moderately; in combination, they significantly enhanced survival of these cells, even up to 7 days of culture. We also report that humoral factors from autologous serum are important for survival of these malignant cells. Our findings support the concept that the CLL microenvironment is critical and suggest that soluble factors may contribute directly to the prolonged survival of CLL B-cells. Therefore, the combination of cytokines we describe as providing strong resistance to apoptosis in vitro might be used to improve the treatment of CLL.     

Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum-free medium: Clonal analyses,growth kinetics,and cell cycle studies

The effects of growth factors, hormones, and calcium on the growth and differentiation of secondary cultures of normal human prokeratinocytes, i.e., proliferative keratinocytes, derived from adult or neonatal skin were determined by culture in serum-free basal medium, MCDB 153. Clonal growth was achieved when MCDB 153 was supplemented with either epidermal growth factor (EGF) or bovine pituitary extract (BPE), provided insulin was present. In the absence of insulin, however, both EGF and BPE were required for clonal growth. Using this assay, it was established that colony-forming efficiency is independent of calcium concentrations above 0.03 mM and averages 56%; colony size, however, was influenced by calcium and EGF concentrations. Optimal clonal growth occurred in medium containing 10 ng/ml EGF and 0.3 mM calcium. By contrast, differentiation was enhanced by the combination of low EGF (0.1 ng/ml) and high calcium (2 mM). This suggests that an inverse relationship exists between the growth response (extent of clonal growth) and the differentiation response (extent of differentiation). These results suggest that proliferation and differentiation are regulated in an integrated manner. Detailed kinetic studies and cytofluorimetric and autoradiographic analyses also showed that exponentially growing secondary cultures of adult and neonatal prokeratinocytes have a 24-hour cell generation time with G₁, S, G₂, and M phases of 12, 8, 3, and 1 hours, respectively. In addition, the data show that such cells can be growth arrested in medium that does not induce differentiation and that such a procedure significantly limits the cell's subsequent proliferative potential. Furthermore, prolonged culture of adult (> 30 population doublings) and neonatal prokeratinocytes (> 50 population doublings) is associated with senescence and the G₁ arrest of noncycling cells.     

Reciprocal control of apoptosis and proliferation in cultured rat hepatoma arl-6 cells: Roles of nutrient supply, serum, and oxidative stress

Summary In order to understand how cancer cells accumulate, rat hepatoma ARL-6 cells were cultured for 8 d to identify factors involved in spontaneous cell proliferation and apoptosis. With increasing time in culture, the proportion of cells in the proliferative phases of the cell cycle and the rate of deoxyribonucleic acid (DNA) synthesis decreased. The waning of proliferation was associated with a gradual reduction of cell viability, and this was temporally related to the appearance of typical apoptotic morphology and DNA laddering. Medium replacement or supplementation with fetal calf serum (FCS) suppressed apoptosis, while medium change, but not fetal calf serum alone, enhanced cell proliferation. Apoptosis was also suppressed by dimethyl sulfoxide (DMSO), but supplementary glutathione was without effect. Expression of poly(adenosine diphosphate[ADP]-ribose)polymerase peaked on days 3–4 of culture, and was followed by a progressive decrease thereafter, consistent with proteolytic cleavage. This decrease was prevented to varying extents by complete medium replacement, FCS and DMSO, indicating a close temporal relationship between poly(ADP-ribose)polymerase activation and apoptosis. Expression of Fas and Bcl-2 did not change appreciably over the 8-d culture, but there was a gradual increase in Bax expression; medium change, FCS and DMSO all partly inhibited Bax expression. These data indicate that spontaneous apoptosis in cultured ARL-6 cells is inversely related to cell proliferation, and that nutrient supply, and to a lesser extent, serum-derived factors and oxidative stress modulate apoptosis in this system. Proteolytic cleavage of poly(ADP-ribose)polymerase and expression of Bax are likely to be mechanistically involved with the control of spontaneous apoptosis in ARL-6 cells, whereas changes in the levels of Fas and Bcl-2 do not play a role.     

Serum‐specific stimulation of proliferation and mineralization of fish bone‐derived cells

Teleost fish have recently been implemented as suitable model organisms to study vertebrate development, in particular skeletogenesis. In vitro cell systems derived from fish bone have been successfully established, although their development has been hampered by the limited availability of fish serum to supplement culture medium. Commercially available sera are mostly of mammalian origin and thus not necessarily adequate to fish cell growth. The main objective of this work was to compare proliferative and mineralogenic potential of bovine and fish sera using fish bone‐derived cell lines VSa13 and VSa16. Fish serum was shown to (i) strongly stimulate cell proliferation in an apparent dose‐dependent and cell type‐specific manner, (ii) induce morphological changes, and (iii) enhance extracellular matrix mineralization of bone cells, although cytotoxic for fish osteoblast‐like cells at the concentration tested. To better understand mechanisms underlying mineralogenic effect of fish serum in fish chondrocytes, expression of several mineralization‐related genes was evaluated by qPCR. Regulation of matrix Gla protein (MGP) and bone morphogenetic protein 2 (BMP2) gene expression was modified upon culture with fish serum in a way compatible with an early onset and an increase in mineralization. In conclusion, fish serum was shown to be more adequate to proliferation and differentiation/mineralization of fish bone‐derived cells.     

An improved method of cell culture system from eye stalk,hepatopancreas, muscle,ovary, and hemocytes of <Emphasis Type="Italic">Penaeus vannamei</Emphasis>

Improved methods of cell culture from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of shrimp (Penaeus vannamei) were established using synthetic media and shrimp muscle extract (SME). For hemocytes and ovarian cell cultures, Grace’s insect medium supplemented with 10% (v/v) fetal bovine serum and 10% SME (v/v) showed enhanced attachment and proliferation of the cells. The hemocyte and ovarian cell cultures could be maintained for 48 and 66 days, respectively, and have been sub-cultured four and six times, respectively. Both ovary and hemocyte cell cultures contained primarily epithelial-like cells. Cells derived from ovary tissue grew preferably between 26°C and 28°C with 5% CO₂. Although the temperature preference of hemocyte cells was the same as ovarian cells, CO₂ supplementation did not show any difference in the growth of hemocyte cells. When the shrimp were injected with lipopolysaccharide (8 μg/g of shrimp) and hemolymph was drawn 24 h post-injection, the in vitro multiplicity of hemocytes dramatically improved. The growth of eye stalk, hepatopancreas, and muscle-derived cells was much less compared to ovarian cells and hemocytes under the conditions described above. The optimal culture conditions for ovarian cells and hemocytes were also different from that for eye stalk, hepatopancreas, and muscle cell culture. The proliferation efficiencies of primary cultures of hepatopancreas, eyestalk, and muscle cells were about 30, 12, and <7 d, respectively. The improved culture conditions described here, particularly for hemocytes and ovary, will be very useful for in vitro studies involving viruses infecting shrimp and in shrimp genomic studies.     

Recombinant human midkine stimulates proliferation of articular chondrocytes

Objectives: Midkine, a heparin‐binding growth factor, promotes population growth, survival and migration of several cell types, but its effect on articular chondrocytes remains unknown. The aim of this study was to investigate its role on proliferation of articular chondrocytes in vitro and in vivo. Materials and methods: Bromodeoxyuridine incorporation and MTT assays were performed to examine the proliferative effect of recombinant human midkine (rhMK) on primary articular chondrocytes. Activation of extracellular signal‐regulated kinase (ERK) and phosphatidylinositol 3‐kinase (PI3K) was analysed using western blot analysis. Systemic and local delivery of rhMK into mice and rats was preformed to investigate the proliferative effect of rhMK in vivo, respectively. Histological evaluation, including measurement of articular cartilage thickness, cell density, matrix staining and immunostaining of proliferating cell nuclear antigen was carried out. Results: rhMK promoted proliferation of articular chondrocytes cultured in a monolayer, which was mediated by activation of ERK and PI3K. The proliferative role of rhMK was not coupled to dedifferentiation of culture‐expanded cells. Consistent with its action in vitro, rhMK stimulated proliferation of articular chondrocytes in vivo when it was administered subcutaneously and intra‐articularly in mice and rats, respectively. Conclusion: Our results demonstrate that rhMK stimulates proliferation of primary articular chondrocytes in vitro and in vivo. The results of this study warrant further examination of rhMK for treatment of animal models of articular cartilage defects.     

TGFβ1 induces growth arrest and apoptosis but not ciliated cell differentiation in rat tracheal epithelial cell cultures

Summary We are studying the regulation of ciliated cell differentiation using an in vitro model of tracheal regeneration. Previously, we reported that removal of growth stimulating compounds such as epidermal growth factor (EGF) and cholera toxin reduced DNA synthesis and cell number while increasing ciliated cell differentiation (Clark et al., 1995). This result suggested that the induction of growth arrest may stimulate terminal differentiation of airway epithelial cells into ciliated cells. Transforming growth factor βs (TGFβs) inhibit epithelial cell proliferation and have also been shown to stimulate epithelial cell differentiation. In this study, the effect of TGFβ₁ on growth and ciliated cell differentiation of rat tracheal epithelial (RTE) cells was examined. TGFβ₁ inhibited [³H]thymidine incorporation by RTE cells in a dose-dependent manner. A 40% inhibition was observed after a 24-h incubation with 10 pM TGFβ₁. Continuous treatment with TGFβ₁ (1–50 pM) also reduced cell number during the time when ciliogenesis occurs. This reduction resulted in part from a loss of cells through exfoliation, in addition to the inhibition of proliferation. The exfoliated cells exhibited several morphological features characteristic of apoptosis, including shrunken cells, condensed and fragmented nuclei, and intact organelles. In addition, electrophoretic analysis of genomic DNA analysis isolated from exfoliated cells demonstrated the presence of a nucleosomal ladder. However, in contrast to the removal of EGF, treatment with TGFβ₁ for 7 d did not increase ciliated cell differentiation. TGFβ1 is, therefore, capable of inhibiting proliferation and increasing apoptosis in RTE cells without stimulating ciliated cell differentiation.     

Effects of Citalopram on Sutural and Calvarial Cell Processes

The use of selective serotonin reuptake inhibitors (SSRIs) for the treatment of depression during pregnancy is suggested to increase the incidence of craniofacial abnormalities including craniosynostosis. Little is known about this mechanism, however based on previous data we propose a mechanism that affects cell cycle. Excessive proliferation, and reduction in apoptosis may lead to hyperplasia within the suture that may allow for differentiation, bony infiltration, and fusion. Here we utilized in vivo and in vitro analysis to investigate this proposed phenomenon. For in vivo analysis we used C57BL–6 wild-type breeders treated with a clinical dose of citalopram during the third trimester of pregnancy to produce litters exposed to the SSRI citalopram in utero. At post-natal day 15 sutures were harvested from resulting pups and subjected to histomorphometric analysis for proliferation (PCNA) and apoptosis (TUNEL). For in vitro studies, we used mouse calvarial pre-osteoblast cells (MC3T3-E1) to assess proliferation (MTS), apoptosis (Caspase 3/7-activity), and gene expression after exposure to titrated doses of citalopram. In vivo analysis for PCNA suggested segregation of effect by location, with the sagittal suture, showing a statistically significant increase in proliferative response. The coronal suture was not similarly affected, however there was a decrease in apoptotic activity at the dural edge as compared to the periosteal edge. No differences in apoptosis by suture or area due to SSRI exposure were observed. In vitro results suggest citalopram exposure increased proliferation and proliferative gene expression, and decreased apoptosis of the MC3T3-E1 cells. Decreased apoptosis was not confirmed in vivo however, an increase in proliferation without a concomitant increase in apoptosis is still defined as hyperplasia. Thus prenatal SSRI exposure may exert a negative effect on post-natal growth through a hyperplasia effect at the cranial growth sites perhaps leading to clinically significant craniofacial abnormalities.     

Evaluating the mechanism underlying antitumor effect of interleukin 27 on B cells of chronic lymphocytic leukemia patients

Chronic lymphocyte leukemia (CLL) is a B-cell malignancy resisted to apoptosis. Recently, some studies indicated that cytokines such as interleukin 27 (IL-27) can reduce B-cell proliferation. The aim of this study is to evaluate the mechanism underlying the proapoptotic effect of IL-27 on B cells of patients with CLL in comparison with B cells of normal subjects. The effect of IL-27 on the antitumor activity of natural killer (NK) and T cells was also evaluated. Peripheral blood mononuclear cells (PBMCs) were isolated from 35 patients with CLL and 15 normal subjects. B cells and PBMCs were cocultured with IL-27 and B cells apoptosis to evaluate proliferation. Both messenger RNA and protein expression of IL-27 and IL-27 receptor were determined using flow cytometry and real-time polymerase chain reaction analysis. To evaluate the apoptotic effect of IL-27 on B cells of patients with CLL, Annexin V-FITC and 7-AAD (BioLegend) fluorescent dyes were used. In addition, the IL-27 effect on activation of T cell and NK cell was determined by determining CD96 molecule expression. IL-27 and IL-27 receptor expression in patients with CLL was significantly lower than that of normal subjects (p < .05). IL-27 enhanced apoptosis of B cells in patients with CLL (p < .05) but this effect was not significantly observed in B cells of normal subjects (p > .05). Consequently, IL-27 reduced the proliferation of B cells and enhanced NK cell activity (p < .05). IL-27, through inducing apoptosis, can exert an inhibitory effect on cancer B cells of CLL patients with minimal effect on normal B cells.     

Increased Spontaneous Apoptosis of Rat Primary Neurospheres In Vitro After Experimental Autoimmune Encephalomyelitis

Survival of neuronal progenitors (NPCs) is a critical determinant of the regenerative capacity of brain following cellular loss. Herein, we report for the first time, the increased spontaneous apoptosis of the first acute phase of Experimental Autoimmune Encephalomyelitis (EAE) derived neurospheres in vitro. Neuronal as well as oligodendroglial loss occurs during experimental autoimmune encephalomyelitis (EAE). This loss is replenished spontaneously by the concomitant increase in the NPC proliferation evidenced by the presence of thin myelin sheaths in the remodeled lesions. However, remyelination depends upon the survival of NPCs and their lineage specific differentiation. We observed significant increase (P < 0.001) in number of BrdU (+) cells in ependymal subventricular zone (SVZ) in EAE rats. EAE derived NPCs showed remarkable increase in S-phase population which was indeed due to the decrease in G-phase progeny suggesting activation of neuronal progenitor cells (NPCs) from quiescence. However, EAE derived neurospheres showed limited survival in vitro which was mediated by the significantly (P < 0.01) depolarized mitochondria, elevated Caspase-3 (P < 0.001) and fragmentation of nuclear DNA evidenced by single cell gel electrophoresis. Our results suggest EAE induced spontaneous apoptosis of NPCs in vitro which may increase the possibility of early stage cell death in the negative regulation of the proliferative cell number and may explain the failure of regeneration in human multiple sclerosis.     

Cell lines derived from mouse neural crest are representative of cells at various stages of differentiation

In order to study mammalian neural crest differentiation in vitro, a series of clonal neural crest (NC) cell lines have been generated by infection of migrating mouse neural crest cells with two recombinant retroviruses containing either the c-myc or N-myc proto-oncogenes. Many cell lines were generated which could be subdivided into three groups based on their appearance in culture. Eleven of these cell lines representative of each of the morphological groups were characterized for the expression of six antigenic markers expressed by neural cells. In addition, mRNA was prepared from these cell lines and analyzed for the expression of a number of neural specific genes. These analyses show that the cell lines are representative of the following cell types: (1) neural crest-like cell lines that do not differentiate in 10% serum; (2) progenitor cell lines, some of which can partially differentiate in culture; and (3) mature neuronal cell lines or bipotential cell lines. Southern blot analysis of DNA from these lines indicated that they have multiple integration sites for the provirus and suggest that phenotypically different cell types have arisen from a single cell. None of the cell lines showed any proliferative or morphological response to nerve growth factor (NGF), whereas over two-thirds of the lines showed both marked proliferative and morphological responses to fibroblast growth factor (FGF). These data indicate that we have generated a range of cell lines representative of a spectrum of mouse neural crest derivatives.     

The MEC1 and MEC2 Lines Represent Two CLL Subclones in Different Stages of Progression towards Prolymphocytic Leukemia

The EBV carrying lines MEC1 and MEC2 were established earlier from explants of blood derived cells of a chronic lymphocytic leukemia (CLL) patient at different stages of progression to prolymphocytoid transformation (PLL). This pair of lines is unique in several respects. Their common clonal origin was proven by the rearrangement of the immunoglobulin genes. The cells were driven to proliferation in vitro by the same indigenous EBV strain. They are phenotypically different and represent subsequent subclones emerging in the CLL population. Furthermore they reflect the clinical progression of the disease. We emphasize that the support for the expression of the EBV encoded growth program is an important differentiation marker of the CLL cells of origin that was shared by the two subclones. It can be surmised that proliferation of EBV carrying cells in vitro, but not in vivo, reflects the efficient surveillance that functions even in the severe leukemic condition. The MEC1 line arose before the aggressive clinical stage from an EBV carrying cell within the subclone that was in the early prolymphocytic transformation stage while the MEC2 line originated one year later, from the subsequent subclone with overt PLL characteristics. At this time the disease was disseminated and the blood lymphocyte count was considerably elevated. The EBV induced proliferation of the MEC cells belonging to the subclones with markers of PLL agrees with earlier reports in which cells of PLL disease were infected in vitro and immortalized to LCL. They prove also that the expression of EBV encoded set of proteins can be determined at the event of infection. This pair of lines is particularly important as they provide in vitro cells that represent the subclonal evolution of the CLL disease. Furthermore, the phenotype of the MEC1 cells shares several characteristics of ex vivo CLL cells.     

Effect of retinoic acid on the proliferation and tumorigenicity of mouse mastocytoma cells

Retinoic acid (RA) inhibited the in vitro growth of the mouse mast cell tumor line P₈₁₅ in a dose- and time-dependent manner. The inhibition was accompanied by an increase in the amount of neutral intracellular mucopolysaccharides. Study of cell cycle kinetics showed that exposure to retinoic acid led to a slowing-down of the cell-cycle progression possibly related to a more differentiated cell population disclosed by microscopy with a lower proliferative capacity. In vivo, delays in both tumor appearance and mouse mortality were observed after injecting RA into mice bearing mastocytomas. These results suggest that RA could be of interest in the treatment of human malignant systemic mastocytosis with proliferation of immature mast cells.     

miR-483 inhibits bovine myoblast cell proliferation and differentiation via IGF1/PI3K/AKT signal pathway

MicroRNAs (miRNAs) have been established to regulate skeletal muscle development in mammals. However, few studies have been conducted on the regulation of proliferation and differentiation of bovine myoblast cells by miRNAs. The aim of our study was to explore the function of miR-483 in cell proliferation and differentiation of bovine myoblast. Here, we found that miR-483 declined in both proliferation and differentiation stages of bovine myoblast cells. During the proliferation phase, the overexpression of miR-483 downregulated the cell cycle–associated genes cyclin-dependent kinase 2 (CDK2), proliferating cell nuclear antigen (PCNA) messenger RNA (mRNA), and the protein levels. At the cellular level, cell cycle, cell counting kit-8, and 5-ethynyl-2´-deoxyuridine results indicated that the overexpression of miR-483 block cell proliferation. During differentiation, the overexpression of miR-483 led to a decrease in the levels of the myogenic marker genes MyoD1 and MyoG mRNA and protein. Furthermore, the immunofluorescence analysis results showed that the number of MyHC-positive myotubes was reduced. In contrast, the opposite experimental results were obtained concerning both proliferation and differentiation after the inhibition of miR-483. Mechanistically, we demonstrated that miR-483 target insulin-like growth factor 1 (IGF1) and downregulated the expression of key proteins in the PI3K/AKT signaling pathway. Altogether, our findings indicate that miR-483 acts as a negative regulator of bovine myoblast cell proliferation and differentiation.