K-252a-induced polyploidization and differentiation of a human megakaryocytic cell line, Meg-J: transient elevation and subsequent suppression of cyclin B1 and cdc2 expression in the process of polyploidization

Megakaryocytes are unique haemopoietic cells which undergo DNA replication, giving rise to polyploid cells. However, little is known about the mechanism of megakaryocytic polyploidization. To address this issue, we used the human megakaryocytic cell line Meg-J. In the presence of K-252a (an indolocarbasole derivative), Meg-J cells stopped proliferation and exhibited additional megakaryocytic features, including morphological changes, polyploidization, and increases in the levels of surface expression of platelet glycoprotein (GP) IIb/IIa and GPIb. Thrombopoietin (TPO) promoted the K-2 52a-induced polyploidization and megakaryocytic differentiation. In the process of K-252a-induced polyploidization, levels of expression of both cdc2 and cyclin B1 were elevated transiently and subsequently decreased. This suggested that the polyploidization process in Meg-J cells was at least in part associated with a transient elevation and subsequent decrease in the expression of cdc2/cyclin B1 complex, a critical kinase involved in G2/M cell cycle transition.     

Loss of heterozygosity analysis in a human fibrosarcoma cell line

Loss of heterozygosity (LOH) is an important event in tumor formation. We have used polymorphic microsatellite repeat markers to identify and characterize LOH in spontaneous mutants of a human cell line, MR12-1, that is heterozygous for the adenine phosphoribosyltransferase gene (APRT+/-) located on chromosome 16q24.3. Initially, clones without extensive LOH (which are likely derived as a consequence of intragenic point mutations) and clones with multilocus LOH (which are likely due to major chromosome alterations) were identified. Clones with major regions of LOH were further characterized by assaying additional informative microsatellite markers. Analysis of 20 spontaneously-arising, independent APRT-/- clones from MR12-1 demonstrated that nine of the mutants retained both copies of APRT and 11 had undergone multilocus genetic alterations. The nature of LOH in four of the latter clones has been examined in detail by karyotype and fluorescence in situ hybridization analysis (Shao et al., 1996). These data demonstrate that LOH of chromosome 16 may be due to mitotic recombination, interstitial or partial deletion, or to more complex mechanisms. LOH in these clones may be a consequence of events similar to those observed in many tumors.     

cAMP mediates transepithelial K+ and Na+ transport in a strial marginal cell line

The recently described gastrointestinal glutathione peroxidase (GI-GPx) is the fourth member of the family of the selenoenzymes glutathione peroxidases (GPx). In contrast to the more uniform distribution of, for example, the classical glutathione peroxidase (cGPx), it is expressed exclusively in the gastrointestinal tract and has, therefore, been suggested to function as a primary barrier against alimentary hydroperoxides. In order to get an idea of its relative importance we investigated its position in the hierarchy of selenoprotein expression. The selenium-dependent expression of GI-GPx was analyzed in comparison with that of other GPx types at the level of mRNA and protein in HepG2 and CaCo-2 cells. Furthermore, the selenocysteine insertion sequence (SECIS) efficiencies of GI-GPx, phospholipid hydroperoxide glutathione peroxidase (PHGPx) and cGPx in response to selenium were determined by a reporter-gene assay in human hepatoma cells and baby hamster kidney cells. GI-GPx mRNA levels increased during selenium deficiency, whereas cGPx mRNA levels decreased and PHGPx mRNA levels remained almost unaffected. In cells grown in selenium-poor media, all GPx-types were low in both activity and immunochemical reactivity. Upon selenium repletion immunoreactive GI-GPx protein reached a plateau after 10 h, whereas cGPx started to be expressed at 24 h and did not reach its maximum level before 3 days. SECIS efficiencies decreased in the order PHGPx > cGPx > GI-GPx. The augmentation of SECIS efficiencies by selenium was highest for cGPx and intermediate for PHGPx, whereas it was marginal for GI-GPx. The high mRNA stability under selenium restriction, the speed of biosynthesis upon selenium repletion and the marginal effect of selenium on the SECIS efficiency indicate that of the GPx isotypes, GI-GPx ranks highest in the hierarchy of selenoproteins and point to a vital role of GI-GPx in the gastrointestinal tract.     

Expression and activation of protein kinase C isoforms in a human megakaryocytic cell line

Megakaryocytes undergo a unique differentiation program, becoming polyploid through repeated cycles of DNA synthesis without concomitant cell division. We have shown previously that phorbol 12-myristate 13-acetate (PMA) induces the Dami human megakaryocytic cell line to become polyploid and to express platelet-specific proteins, including von Willebrand factor (vWF) and glycoprotein Ib (GpIb). Phorbol esters are thought to regulate gene expression principally through the activation of protein kinase C (PKC), a family of structurally related kinases with potentially unique activation requirements and substrate specificities. A survey of PKC isoforms in Dami cells revealed that, by both Western and Northern analyses, PKC isoforms alpha, beta, delta, epsilon, eta, theta, and zeta were reproducibly detected. PKC-gamma was not detected. In order to define the role of individual PKC isoforms in megakaryocytic maturation, PMA and 2-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), a putative selective activator of the PKC-beta 1 isotype, were compared for their effects on Dami cell maturation. Treatment with either dPPA or PMA caused Dami cells to cease proliferating, to become polyploid, and to express vWF. We also examined dPPA and PMA for their ability to activate and to downregulate expression of different PKC isoforms. Fifteen-minute treatment with PMA resulted in the translocation of PKC isoforms alpha, epsilon, and theta from the cytosolic to the membrane fraction; twenty-four hour treatment resulted in the downregulation of these isoforms. In contrast, dPPA was found to be a potent activator of PKC-epsilon alone and exhibited weaker effects on alpha and theta. These data suggest that PKC isoforms beta, delta, eta, and zeta, which appear not to be activated by either phorbol ester, are unlikely to be primarily involved in megakaryocytic maturation in response to these agents. The isoforms that are translocated by both phorbol esters-PKC isoforms alpha and theta, and particularly epsilon-are more likely to transduce the signals that stimulate Dami cell differentiation.     

Involvement of protein kinase C-delta in CD28-triggered cytotoxicity mediated by a human leukaemic cell line YT

Is shown, that the process of red blood sedimentation under the conditions of standard measurement of a clinical parameter ESR (erythrocyte sedimentation rate) is multistage and non-monotonous. Red blood sedimentation is preceded by the "latent" period lasting up to tens of minutes. When sedimentation begins, multiple accelerations and decelerations in the movement of the boundary between red blood and clean plasma are observed. The differences in dynamics of red blood sedimentation (DRBS) of healthy donors and patients with sharp myocardial infarction are revealed. The form of DRBS diagrams depends upon metabolic activity of blood. We suggest that DRBS represents the active reaction of blood, as of an integral surviving tissue, on stress factors acting on it under the conditions of ESR determination.     

Biological consequences of p160v-abl protein tyrosine kinase activity in a primitive, multipotent haemopoietic cell line

A temperature sensitive abl protein tyrosine kinase gene was transferred into a multipotent haemopoietic stem cell line, and the primary biological effects of expression of the gene were examined at the permissive and non-permissive temperatures. Unlike previous studies in factor-dependent cell lines, we found that expression of the functional abl protein tyrosine kinase did not lead to growth autonomy. Furthermore, the cells were still able to undergo terminal myeloid differentiation. However, expression of the functional gene did lead to a delay in maturation with a concomitant increase in cell production, had a modest effect in terms of delayed apoptosis particularly when the cells were maintained at a high cell density, and slightly increased the response to sub-optimal concentrations of IL-3. In many respects, therefore, the effects of abl protein tyrosine kinase in these cells mimics the effect of bcr/abl in primary haemopoietic cells where growth factor independence and an aberrant differentiation profile are relatively late events in clonal evolution and are not intermediate consequences of activation of the abl gene.     

Activation of protein kinase C by intracellular free calcium in the motoneuron cell line NSC-19

Clotrimazole (CLT), a member of the antifungal imidazole family of compounds, has been found to inhibit both calcium (Ca2+)-activated 86Rb and potassium (K) fluxes of human red cells and to inhibit red cell binding of 125I-charybdotoxin (ChTX) [11]. We have now used patch-clamp techniques to demonstrate reversible inhibition of whole cell KCa2+ currents in murine erythroleukemia (MEL) cells by submicromolar concentrations of CLT. Inhibition was equivalent whether currents were elicited by bath application of the Ca2+ ionophore A23187 or by dialyzing cells with a pipette solution containing micromolar concentrations of free Ca2+. The extent of inhibition of whole cell MEL KCa2+ currents was voltage-dependent, decreasing with increasing test potential. We also determined the single channel basis of the CLT inhibition in MEL cells by demonstrating the inhibition of a calcium-activated, ChTX-sensitive K channel by CLT in outside-out patches. The channel was also blocked by the des-imidazolyl metabolite of CLT, 2-chlorophenyl-bisphenyl-methanol (MET II) [15], thus demonstrating that the imidazole ring is not required for the inhibitory action of CLT. Single KCa2+ channels were also evident in inside-out patches of MEL cells. Block of K current by CLT was not unique to MEL cells. CLT also inhibited a component of the whole cell K current in PC12 cells. Channel specificity of block by CLT was determined by examining its effects on other types of voltage-sensitive currents. CLT block showed the following rank order of potency: K currents in PC12 cells > Ca2+ currents in PC12 cells > Na currents in sympathetic neurons. These results demonstrate that direct inhibition of single KCa2+ by CLT can be dissociated from inhibition of cytochrome P-450 in MEL cells.     

Theoretical analysis of a morphine withdrawal phenotype in a cultured cell line

We have previously described a delta-opioid receptor-expressing cultured cell line that proliferates in a defined medium and responds to chronic morphine treatment with an inhibition of its rate of proliferation. To help provide an explanation for this behavior, we have used computer simulation of cell cycle kinetics to analyze the observed rates of proliferation of these cells in the presence and absence of morphine, and after withdrawal of morphine treatment. We questioned whether the difference in cell kinetics observed for the cell populations under the different treatments could be due to changes in the length of the cell cycle, withdrawal of cells from the cycle into a quiescent state, or differences in cell renewal. This was investigated by comparing observed cell numbers as a function of time with the results of different computer simulations using different values for these parameters. We found that we can provide a satisfactory explanation of the experimental observations on the basis of changes in a small set of parameters: Untreated cells experience a slowdown of cell proliferation at about the culture density where multiple cell-cell contacts are made and, beginning then, a large fraction are shunted from G1 into a quiescent state. Chronic morphine treatment inhibits proliferation by slowing passage through G1, but the cells remain as sensitive to cell-cell contacts as the untreated cells. After drug withdrawal following a 6 day treatment with morphine, the cells exhibit a large temporary increase in their rate of proliferation compared with control or chronically treated cells but about 48 hours after withdrawal, when cell-cell contacts just begin to be made, the cells return to almost their pre-treatment total cell cycle time and, as before, a large fraction are shunted into a quiescent state. Taken in conjunction with previously published results, the present ones indicate a possible interaction between morphine-induced and insulin-induced nuclear signaling pathways to the nucleus.     

The role of 80K/MARCKS, a specific substrate of protein kinase C, in cell growth and tumour progression

Since its discovery more than a decade ago [Wu et al., 1982; Rozengurt et al., 1983], the 80-87 kDa myristoylated alpha lanine-rich C-kinase substrate (80K/MARCKS) protein has attracted a great deal of attention from researchers interested in cell growth and tumour progression. However, despite its ubiquitous distribution, a definitive functional role for 80K/MARCKS has not been found. The purpose of this review is to describe the properties, distribution and regulation of 80K/MARCKS and to discuss some of the most recent findings, both from our laboratory and from others, that have suggested a functional role for this protein in modulating cell growth and tumour progression. Furthermore, I will present data from our laboratory that implicates 80K/MARCKS as a novel tumour suppressor in cells of melanocyte origin.     

The mitogen-activated protein kinase pathway inhibits ceramide-induced terminal differentiation of a human monoblastic leukemia cell line, U937

This communication describes an extracellular signal-regulated kinase kinase (MEK)-dependent signal transduction pathway that prevents the terminal differentiation of a hemopoietic cell line. Both PMA and the cell-permeable ceramide, C2-ceramide, caused differentiation of U937 cells, but with distinct cell morphology and CD11b/CD14 surface expression. While PMA activated extracellular signal-regulated kinase (ERK), a downstream kinase of Raf-MEK signaling, C2-ceramide activated c-Jun NH2-terminal kinase (JNK), an anchor kinase of stress-induced signaling. Furthermore, only C2-ceramide stimulated an induction of cell cycle arrest that was associated with stable expression of p21CIP1 and retinoblastoma nuclear phosphoprotein dephosphorylation. Expression of p21CIP1 and JNK activation were also observed in sphingosine-treated cells, whereas sphingosine did not induce detectable differentiation. Concomitant stimulation with C2-ceramide and PMA resulted in the PMA phenotype, and cell cycle arrest was absent. ERK activation was enhanced by C2-ceramide plus PMA stimulation, whereas the activation of JNK was aborted. Strikingly, the inhibition of MEK with PD98059 altered the phenotype of C2-ceramide- and PMA-stimulated U937 cells to that of cells treated with C2-ceramide alone. Thus, ERK and JNK pathways deliver distinct signals, and the ERK pathway is dominant to the JNK cascade. Furthermore, differentiation and cell cycle arrest caused by C2-ceramide rely on independent signaling pathways, and JNK is an unlikely signaling element for this differentiation. Importantly, during C2-ceramide and PMA costimulation, the JNK pathway is not simply blocked by ERK activation; rather, cross-talk between these MAP kinase pathways acts to simultaneously augment ERK activity and down-regulate JNK activity.     

Reoxygenation injury in a cultured corneal epithelial cell line protected by the uptake of lactoferrin

PURPOSE: To investigate whether reoxygenation after extended hypoxia causes cellular damage in cultured corneal epithelial cells and to demonstrate the protective effects of lactoferrin. METHODS: Immortalized human corneal epithelial cells (T-HCECs) were cultured to confluence in 96-well culture plates, subjected to stringent hypoxia (1% O2, 5% CO2, 94% N2 at 37 degrees C) for 24 hours, and returned to normoxic conditions (5% CO2, 95% air at 37 degrees C). Cell viability was observed by 1 microM propidium iodide staining 0, 2, 4, and 6 hours after reoxygenation. Inhibition studies were performed after 2 hours' reoxygenation, using 2 mM iron chelator desferrioxamine and 0.2 mg/ml lactoferrin. Confocal immunocytochemistry for human lactoferrin and western blot analysis for lactoferrin-induced ferritin were performed in cultured T-HCECs to demonstrate the internalization of lactoferrin after application. RESULTS: After 2 hours, reoxygenation of T-HCECs after hypoxia produced an increase in cell death that was significantly greater than that observed in normoxic control cells or in cells subjected to hypoxia for the same time span without reoxygenation. The addition of desferrioxamine and lactoferrin at the time of reoxygenation significantly attenuated cellular damage. Confocal immunocytochemistry revealed that lactoferrin is taken into the cytoplasm of T-HCECs as early as 30 minutes after application. This was also demonstrated in western blot analysis by the upregulation of intracellular ferritin at 18 hours by the addition of iron-bound lactoferrin but not by iron-free lactoferrin. CONCLUSION: Reoxygenation is responsible for increased cellular damage after extensive hypoxia, which is attenuated by chelators of free iron in the cytosol, including the major tear protein lactoferrin.     

Effects of protein kinase inhibitors on heat-induced hsp72 gene expression in a human glioblastoma cell line

This study aims to evaluate the performance of a new diagnostic method (LCx Tuberculosis Assay, Abbott Laboratories) based on Ligase Chain Reaction (LCR) technology, for the detection of Mycobacterium tuberculosis in respiratory and non-respiratory specimens and compare it with standard microbiological data and the clinical diagnosis of tuberculosis. Nine hundred specimens were collected from patients with a high suspicion of tuberculosis (740 respiratory samples and 160 non-respiratory specimens). The study was divided into two separate groups: samples washed and distilled water (207 samples) and unwashed samples that were directly resuspended in phosphate buffer (693 samples). The overall sensitivity, specificity, positive and negative predictive values of samples washed with distilled water after decontamination with SDS-NaOH were: 54%, 100%, 100%, and 94%, respectively. If these results were divided according to origin of specimens, the sensitivity, specificity, positive and negative predictive values in respiratory and non-respiratory samples were 54.5%, 100%, 100%, 94% and 50 100%, 100%, 93%, respectively. In contrast, for the non-washed samples, values were 85%, 95%, 80% and 98%, respectively. Respiratory and non-respiratory samples gave values of 84%, 96%, 77%, and 97.5% versus 89%, 99%, 94%, and 98%. The LCx M. tuberculosis assay is a novel, semi-automated assay and a rapid and highly specific technique for screening all forms of tuberculosis, including non-respiratory forms.     

cAMP, an activator of protein kinase A, suppresses the expression of sonic hedgehog

In Drosophila, it has been shown that protein kinase A and hedgehog have antagonistic actions during the formation of imaginal disks. In vertebrate skin, sonic hedgehog is expressed specifically in the feather bud epithelia. using an in vitro explant culture model we showed that dibutyryl cAMP, a protein kinase A (PKA) activator, suppresses the expression of Sonic hedgehog, (Shh) and continuous feather growth. The results suggest that Shh and PKA also have antagonistic action during vertebrate skin morphogenesis.     

Host TIMP-1 overexpression confers resistance to experimental brain metastasis of a fibrosarcoma cell line

Within the tumor-stromal microenvironment a disrupted balance between matrix metalloproteinases (MMPs) and their inhibitors compromises the integrity of the extracellular matrix and promotes malignancy. Tissue inhibitors of metalloproteinases (TIMPs) have been linked to tumor suppression in studies of genetically altered tissue culture cells and in analyses of clinical specimens in situ. We generated transgenic mice as a model system to test the relationship between TIMP-1 levels in a host organ and susceptibility to experimentally targeted metastasis. Ectopically overexpressed TIMP-1 in the brain resulted in a tissue microenvironment with elevated protein levels of this natural MMP inhibitor. Metastatic challenge provided by lacZ-tagged fibrosarcoma cells permitted high-resolution analysis of metastatic load and pattern. We found that elevated host TIMP-1 imposed resistance to experimental metastasis of fibrosarcoma: In TIMP-1 overexpressing mice, brain metastases were significantly reduced by 75% compared to wild-type littermates. Our findings demonstrate that ectopic TIMP-1 expression efficiently exerts a suppressive effect on metastasizing tumor cells.     

Regulation of cell motility by mitogen-activated protein kinase

Cell interaction with adhesive proteins or growth factors in the extracellular matrix initiates Ras/mitogen-activated protein (MAP) kinase signaling. Evidence is provided that MAP kinase (ERK1 and ERK2) influences the cells' motility machinery by phosphorylating and, thereby, enhancing myosin light chain kinase (MLCK) activity leading to phosphorylation of myosin light chains (MLC). Inhibition of MAP kinase activity causes decreased MLCK function, MLC phosphorylation, and cell migration on extracellular matrix proteins. In contrast, expression of mutationally active MAP kinase kinase causes activation of MAP kinase leading to phosphorylation of MLCK and MLC and enhanced cell migration. In vitro results support these findings since ERK-phosphorylated MLCK has an increased capacity to phosphorylate MLC and shows increased sensitivity to calmodulin. Thus, we define a signaling pathway directly downstream of MAP kinase, influencing cell migration on the extracellular matrix.     

alpha-tocopherol transfer protein stimulates the secretion of alpha-tocopherol from a cultured liver cell line through a brefeldin A-insensitive pathway

Vitamin E (alpha-tocopherol) is a fat-soluble antioxidant that is transported by plasma lipoproteins in the body. alpha-Tocopherol taken up by the liver with lipoprotein is thought to be resecreted into the plasma in very low density lipoprotein (VLDL). alpha-Tocopherol transfer protein (alphaTTP), which was recently identified as a product of the causative gene for familial isolated vitamin E deficiency, is a cytosolic liver protein and plays an important role in the efficient recycling of plasma vitamin E. To throw light on the mechanism of alphaTTP-mediated alpha-tocopherol transfer in the liver cell, we devised an assay system using the hepatoma cell line McARH7777. Using this system, we found that the secretion of alpha-tocopherol was more efficient in cells expressing alphaTTP than in matched cells lacking alphaTTP. Brefeldin A, which effectively inhibits VLDL secretion by disrupting the Golgi apparatus, had no effect on alpha-tocopherol secretion, indicating that alphaTTP-mediated alpha-tocopherol secretion is not coupled to VLDL secretion. Among other agents tested, only 25-hydroxycholesterol, a modulator of cholesterol metabolism, inhibited alpha-tocopherol secretion. This inhibition is most likely mediated by oxysterol-binding protein. These results suggest that alphaTTP present in the liver cytosol functions to stimulate secretion of cellular alpha-tocopherol into the extracellular medium and that the reaction utilizes a novel non-Golgi-mediated pathway that may be linked to cellular cholesterol metabolism and/or transport.     

Stimulation of Jun N-terminal kinase (JNK) by gonadotropin-releasing hormone in pituitary alpha T3-1 cell line is mediated by protein kinase C, c-Src, and CDC42

The signaling of ligands operating via heterotrimeric G proteins is mediated by a complex network that involves sequential phosphorylation events. Signaling by the G protein-coupled receptor GnRH was shown to include elevation of Ca2+ and activation of phospholipases, protein kinase C (PKC) and extra-cellular signal-regulated kinase (ERK). In this study, GnRH was shown to activate Jun N-Terminal Kinase (JNK)/SAPK in alpha T3-1 cells in a PKC- and tyrosine kinase-dependent manner. GnRH as well as tumor-promoting agent (TPA) also increased c-Src activity, which peaked at 2 min after GnRH stimulation and was sensitive both to PKC and to tyrosine kinase inhibitors. Coexpression of Csk, which serves as a Src-dominant interfering kinase, and constitutively active forms of Src, together with JNK, confirmed the involvement of c-Src downstream of PKC in the GnRH-JNK pathway. Coexpression of dominant negative and constitutively active forms of CDC42, Rac1, Ras, MEKK1, and MEK1 with JNK indicated that JNK activation by GnRH and TPA is mediated by CDC42 and MEKK1. Ras and MEK1, which are involved in a related mitogen-activated protein kinase (MAPK) pathway, did not affect JNK activation in alpha T3-1 cells. Taken together, our results suggest that GnRH stimulation of JNK activity is mediated by a unique pathway that includes sequential activation of PKC, c-Src, CDC42, and probably also MEKK1.     

Dolichol-dependent protein glycosylation in a deoxy-glucose-resistant baby-hamster-kidney cell line

The formation in vivo of lipid-linked oligosaccharides is inhibited by deoxy-glucose in wild-type BHK cells but not in a cell-line (dGR) selected for resistance towards deoxyglucose. On the other hand, the formation in vitro of lipid-linked oligosaccharides by membranes from dGR (and wild-type) cells is inhibited by GDPdeoxyglucose, the main metabolite responsible for inhibition of protein glycosylation by deoxyglucose. Our results suggest increased pools of GDP mannose and decreased amounts of GDPdeoxyglucose in the mutant cell line. The enlarged ratio of GDPmannose to GDPdeoxyglucose in the dGR cells treated with deoxyglucose is shown to moderate the inhibition of formation of lipid-linked oligosaccharides, and this explains the capacity of the dGR-cells to grow in the presence of deoxyglucose.