D1 Cells Research Articles

Identification of USF2 as a key regulator of Runx2 expression in mouse pluripotent mesenchymal D1 cells

Runx2 is one of the most important transcription factors directing the osteogenesis of mesenchymal stem cells and osteoblastic functions. It is likely that the factors controlling Runx2 expression would trigger the early steps of osteoblast differentiation. By using a reporter gene assay for 4.5 kb Runx2 promoter, it was found that the first 305 bp of Runx2 promoter are active in D1 cells. Within this region, electromobility shift assays (EMSAs) delineated a 6 bp of CACATG bound specifically by the proteins from D1 cell nuclear extract. Antibody super-shift and DNA-coupling magnetic bead pull-down assay indicated that the protein bound to this sequence is USF2. Site-specific mutagenesis revealed that this sequence contributed to the activity of 305 bp Runx2 promoter. Thus, we suggest that USF2 might be one of the regulators for the expression of the Runx2 gene in D1 cells.

Interference of EGFP RNA in human NT-2/D1 cell lines using human U6 promoter-based siRNA PCR products

RNA interference (RNAi), a process of sequence-specific gene suppression, has been known as a natural gene regulatory mechanism in a wide range of lower organisms. Recently, we have reported that a transfection of human U6 promoter (hU6) driven hairpin small-interference RNA (siRNA) plasmid specifically knocks down the target gene by post-transcriptional gene silencing in mammalian cells. Here we report that transfection of polymerase chain reaction (PCR) products, containing human U6 promoter with hairpin siRNA, knocks down the target gene expression in human teratocarcinoma NT-2/D1 cells. Moreover, we showed 3′ end termination sequence, 5 Ts, is not critical elements for knocking down in PCR-based siRNA system. Therefore, the PCR-based siRNA system is a promising tool not only for the screening but also to temporally regulate gene expression in the human progenitor cells.

Contribution of guanine exchange factor H1 in phorbol ester-induced apoptosis

Phorbol-12-myristate-13-acetate (PMA) treatment induces erythroblastoma D2 cells kept in suspension to undergo RhoA-dependent contraction and to become proapoptotic, while attached cells are induced to differentiate accompanied by the reduction of RhoA activity. In this study, we found that guanine exchange factor H1 (GEF-H1) is highly expressed in D2 cells. Depletion of GEF-H1 expression in D2 cells decreased RhoA activity and prevented PMA-induced contraction and apoptosis. Upon PMA stimulation, GEF-H1 became associated with microtubules in cells that were induced to differentiate. As a contrast, in the proapoptotic population of cells GEF-H1 stayed in the cytoplasm without showing PMA-responsive microtubule translocation. Given that GEF-H1 is inactivated when associated with microtubules and its release into cytosol due to depolymerization of microtubules activates RhoA, our results demonstrated that nonmicrotubule-associated GEF-H1 in D2 cells contributes to the sustained activation of RhoA/ROCK signaling in suspension cells, making cells susceptible to PMA-induced apoptosis.

Evidence for the Role of Adenosine 5′-Triphosphate-Binding Cassette (ABC)-A1 in the Externalization of Annexin 1 from Pituitary Folliculostellate Cells and ABCA1-Transfected Cell Models

Annexin 1 (ANXA1), a 37-kDa protein, is a member of the superfamily of Ca(2+)- and phospholipid-binding annexin proteins. In the anterior pituitary, ANXA1 is expressed mainly by folliculostellate (FS) cells and mediates the early delayed feedback inhibition exerted by glucocorticoids on the release of ACTH and other pituitary hormones. It has been previously demonstrated that TtT/GF cells (a FS cell line) express and externalize ANXA1 in response to glucocorticoid treatment. However, ANXA1 lacks a cleavable signal sequence and externalization is not affected by inhibitors of the secretory pathway. We have previously shown that glyburide, an ATP-binding cassette (ABC) transporter inhibitor, inhibits the externalization of ANXA1 from TtT/GF cells and pituitary tissue. Here we investigated whether ABCA1 is involved in ANXA1 externalization. The use of the ABCA1-transporter inhibitors geranyl-geranyl pyrophosphate and sulfobromophthalein significantly inhibited ANXA1 externalization. Partial silencing of ABCA1 expression in TtT/GF cells by siRNA also significantly decreased the amount of cell surface ANXA1. However, anterior pituitary tissue from ABCA1-null mice was found to externalize ANXA1 normally. Because compensation by other ABC family members may occur in vivo, ANXA1 externalization was studied in two transfection models: Xenopus oocytes injected with ABCA1 mRNA and AtT20 D1 corticoctroph cells cotransfected with ABCA1-green fluorescent protein and ANXA1. ABCA1-expressing oocytes, but not water-injected controls, were found to externalize ANXA1. Expression of ABCA1 in AtT20 D1 cells significantly increased the amount of cell surface ANXA1, compared with mock-transfected and ANXA1-only transfected controls. Together these data provide evidence for a role of ABCA1 in ANXA1 export.

Amyloid precursor protein is involved in staurosporine induced glial differentiation of neural progenitor cells

Staurosporine (STS) has been reported as not only a pro-apoptotic agent, but also a terminal differentiation inducer in several neuroblastoma cell lines. Here, we report involvement of amyloid precursor protein (APP) in a STS induced astrocytic differentiation of human neural progenitor cells (NT-2/D1). We found that STS-treated NT-2/D1 cells expressed astrocyte-specific glial fibrillary acidic protein (GFAP), aspartate transporter, and glutamate transporter-1 with a distinctive astrocytic morphology. STS treatment increased GFAP promoter activity and increased expression and secretion of APP in NT-2/D1 cell culture. Overexpressed APP enhanced GFAP promoter activity and expression of GFAP, while gene silencing of APP by RNA interference decreased GFAP expression. These results indicate involvement of APP in STS induced astrocytic differentiation of NT-2/D1 cells. Furthermore, suppression of ERK1/2 phosphorylation, which is known to regulate APP expression by a MEK1 inhibitor, PD098059, reduced both APP and GFAP expression in STS treated NT-2/D1 cells. Thus, STS may induce astrocytic differentiation of NT-2/D1 by increasing APP levels associate with activation of ERK pathway.

Celecoxib leads to G2/M arrest by induction of p21 and down-regulation of cyclin B1 expression in a p53-independent manner

A unique in vitro system has been developed in our lab that consists of normal enterocytes derived from the rat ileum (IEC-18 cells) and their transformed derivatives with c-K-ras (R1 cells), anti-sense bak (B3 cells) and cyclin D1 (D1 cells). R1 and B3 cells express high level of COX-2 protein and PGE2. IEC 18 and D1 cells express negligible amount of COX-2, and produce very low level of PGE2. A relatively low dose of celecoxib (5-10 microM) induced G2/M arrest, followed by induction of apoptosis in the transformed but not in the normal cells. Down-regulation of cyclin B1 and up-regulation of p21 expressions independent of p53 might have cause this cell cycle block. Growth inhibition was related to COX-2 function with 90-95% reduction in PGE2 production. These findings may be of clinical importance, since low concentration of celecoxib can be achieved in human serum following standard anti-inflammatory (100-200 mg bid) regime.

Forebrain dopamine receptors in cognitive, memory and learning processes

Neurons that utilize dopamine (DA) as a neurotransmitter have attracted great interest because of their involvement in the behavioral, endocrine and descending control of major brain functions. DA is known to exert its physiological effects on target neurons through D1-like (D1 and D5) and D2-like (D2, D3 and D4) receptor subtypes. To better understand the DA modulation of brain functions, the distribution and cellular localization of D1 and D2 DA receptors in the rat forebrain is reviewed, and their possible role in cognitive aspects of behavior is discussed. In general, DA receptors are differentially expressed and mostly distributed in different target fields of DA neurons. Both D1 and D2 receptor gene products are found in the cerebral cortex, basal ganglia and hippocampal formation, albeit in different cell groups or neuronal subpopulations, whereas D3 receptors are mainly located in limbic regions. In the cortex, mRNA signals are seen in all the areas and cortical layers except layer I. In the striatum, the most intense signal is found in the caudate-putamen, nucleus accumbens and olfactory tubercle where a large number of cells are strongly labeled for D1 and D2. In the globus pallidus only scattered D2 mRNA-containing cells are present. In contrast, no D1 or D2 messages can be seen in the ventral pallidum. In the basal forebrain, mRNA encoding the D1 receptor is detected in the islands of Calleja. The medial and lateral septal nuclei show a low D2 signal. In the amygdaloid nuclear complex, the strongest D1 receptor message is observed in the basomedial and basolateral nuclei. Conversely, the highest density of D2 mRNA-expressing cells is revealed in the central nucleus. Moderately labeled for D1 and D2 cells are scattered throughout the anterior and posterior subdivisions of the bed nucleus of stria terminalis, and within all subfields of the hippocampal formation and dentate granule cell layer. Differential regional and cellular distribution of DA receptors in the forebrain provides anatomical evidence for an area-specific regulation of the DAergic neurotransmission. It can be inferred that DA facilitates learning, memory and cognition processes via activation of both the D1 and D2 receptors. Biomedical Reviews 2005; 16: 59-75.

A useful new classification of dysmorphic urinary erythrocytes

Among dysmorphic urinary erythrocytes (D cells), G1 cells or doughnut-shaped erythrocytes with one or more blebs are considered to be reliable markers for glomerular diseases. However, although there are many D cells with cytoplasmic color loss and without blebs in the urinary sediment, the significance of these cells is not clear. In this study, we devised a classification system for D cells and examined the relation between these cell types and urinalysis data. We classified D cells into three types (D1, D2, and D3 cells): D1 cells showed a ring-like shape and severe loss of cytoplasmic color with protrusions or blebs; D2 cells showed a doughnut-like shape and moderate cytoplasmic color loss with protrusions or blebs; and D3 cells showed a doughnut-like shape and mild cytoplasmic color loss without protrusions or blebs. We calculated the numbers of D cells of each type in 45 patients with glomerular diseases and in 303 general outpatients. This was done by bright-field microscopy modified for the analysis of urinary sediment, and we also examined the significance of these cell types. In the 45 patients with glomerular diseases, the numbers of D1, D2, and D3 cells correlated with urine levels of proteinuria and hematuria and numbers of cellular and fatty casts. Numbers of D1 and D2 cells correlated with urine concentrations of albumin and N-acetyl-beta-D-glucosaminidase, and the proportions of D1 and D2 cells in D cells increased with the activity of glomerular diseases classified by urinalysis data. Only the number of D1 cells correlated with the urine concentration of potassium, which may increase in hemolysis. In the 303 outpatients, the sensitivity of D3 cells and D1 and/or D2 cells (G1 cells) was 73% and 46%, respectively, for the detection of glomerular diseases and the specificity was 93% and 99%, respectively. These data indicate that the D3 cell is a sensitive marker for glomerular diseases, and that D1 and/or D2 cells are markers for severe glomerular diseases.

Green tea catechin enhances osteogenesis in a bone marrow mesenchymal stem cell line

Green tea has been reported to possess antioxidant, antitumorigenic, and antibacterial qualities that regulate the endocrine system. Previous epidemiological studies found that the bone mineral density (BMD) of postmenopausal women with a habit of tea drinking was higher than that of women without habitual tea consumption. However, the effects of green tea catechins on osteogenic function have rarely been investigated. In this study, we tested (-)-epigallocatechin-3-gallate (EGCG), one of the green tea catechins, on cell proliferation, the mRNA expressions of relevant osteogenic markers, alkaline phosphatase (ALP) activity and mineralization. In a murine bone marrow mesenchymal stem cell line, D1, the mRNA expressions of core binding factors a1 (Cbfa1/Runx2), osterix, osteocalcin, ALP increased after 48 h of EGCG treatment. ALP activity was also significantly augmented upon EGCG treatment for 4 days, 7 days and 14 days. Furthermore, mineralizations assayed by Alizarin Red S and von Kossa stain were enhanced after EGCG treatment for 2-4 weeks in D1 cell cultures. However, a 24-h treatment of EGCG inhibited thymidine incorporation of D1 cells. These results demonstrated that long-term treatment of EGCG increases the expressions of osteogenic genes, elevates ALP activity and eventually stimulates mineralization, in spite of its inhibitory effect on proliferation. This finding suggests that the stimulatory effects of EGCG on osteogenesis of mesenchymal stem cells may be one of the mechanisms that allow tea drinkers to possess higher BMD.

Post-irradiation phosphorylation of structural maintenance chromosome 1 (SMC1) is independent of the Fanconi protein pathway

To confirm the sensitivity of cells from patients with Fanconi anemia (FA) to ionizing radiation, and to determine whether the phosphorylation of structural maintenance chromosome 1 (SMC1) was associated with radiosensitivity, as it is in other DNA repair disorders. Using lymphoblastoid cell lines from FA patients before and after exposure to ionizing radiation, the colony survival assay, radioresistant DNA synthesis, and SMC1 phosphorylation were measured. FA lymphoblastoid cell lines that had been transfected with the wild-type FANC gene were used as controls. Cells from FA patients of six complementation groups were radiosensitive. Despite this, SMC1 phosphorylation was normal in each case; radioresistant DNA synthesis, a measure of S phase checkpoint integrity, was defective in FANCD2 lymphoblastoid cell lines and was corrected in FANCD2 + D2 cells. The data indicate that the FANC pathway proteins play a major role in the cellular responses to ionizing radiation, but not in SMC1 phosphorylation or in the S phase checkpoint of FANCD2-deficient cells. Thus, SMC1 activation is not a common denominator of radiosensitivity, as has been suggested by radiation responses of cells from ataxia-telangiectasia, Nijmegen breakage syndrome, or Mre11 deficiency patients.

Hsp90 restrains ErbB‐2/HER2 signalling by limiting heterodimer formation

ErbB-2/HER2 is an oncogenic tyrosine kinase that regulates a signalling network by forming ligand-induced heterodimers with several growth factor receptors of the ErbB family. Hsp90 and co-chaperones regulate degradation of ErbB-2 but not other ErbB members. Here, we report that the role of Hsp90 in modulating the ErbB network extends beyond regulation of protein stability. The capacity of ErbB-2 to recruit ligand-bound receptors into active heterodimers is limited by Hsp90, which is dissociated from ErbB-2 following ligand-induced heterodimerization. We show that Hsp90 binds a specific loop within the kinase domain of ErbB-2, thereby restraining heterodimer formation and catalytic function. These results define a role for Hsp90 as a molecular switch regulating the ErbB signalling network by limiting formation of ErbB-2-centred receptor complexes.

Interleukin-7 Inactivates the Pro-apoptotic Protein Bad Promoting T Cell Survival

Interleukin-7 (IL-7) is a cytokine that is required for T cell development and survival. The anti-apoptotic function of IL-7 is partly through induction of Bcl-2 synthesis and cytosolic retention of Bax. Here we show that the Bcl-2 homology 3 domain-only protein, Bad, is involved in cell death following IL-7 withdrawal from D1 cells, an IL-7-dependent murine thymocyte cell line. IL-7 stimulation resulted in the inactivation of Bad by phosphorylation at Ser-112, -136, and -155. The phosphoinositide 3-kinase (PI3K)/Akt pathway has been implicated previously in Bad phosphorylation. In response to IL-7, the PI3K/Akt pathway induced phosphorylation at Ser-136 and -155, but Ser-112 was partly independent of the PI3K/Akt pathway, indicating an as yet unknown pathway in this response. Following IL-7 withdrawal, dephosphorylated Bad translocated from cytosol to mitochondria, bound to Bcl-2, and accelerated cell death. Thus, the inactivation of Bad contributes to the survival function of IL-7.

Cellular interactions in the tropism of prostate cancer to bone.

At autopsy >or=80% of prostate cancers have established macrometastases in marrow containing bone. The mechanism(s) to explain this remarkable level of bone involvement remain to be elucidated. We examined the adhesive and invasive behavior of prostate cancer cells to osteoblastic and human bone marrow endothelial cells (HBME-1) in an attempt to explain the tropism of prostate cells for bone. We found an inverse relationship between adhesion and prostate cell tumorigenicity and metastatic potential. Relative cell adhesion of P69 between cell lines was 1.74-fold (95% confidence interval [CI] = 1.15-2.64) and 1.58-fold (95% CI = 0.94-2.68) greater at 1 hr and 2 hr, respectively, than LNCaP that was essentially equivalent to C4-2 cells when using an osteoblastic cell line, D1 as the substrate. Similar results were acquired when HBME-1 were used as substratum. There was a marked increase in adhesion of the poorly tumorigenic cell line P69 as compared to the cancer cells to HBME-1. P69 adhesion was 2.78-fold (95% CI = 1.87-4.84) and 2.0-fold (95% CI = 1.43-2.80) greater at 1 hr and 2 hr, respectively when compared to LNCaP or C4-2 cells. D1 cells, a bone homing osteoblastic precursor, behaved contrary to the metastatic, bone-colonizing C4-2 cell line and bound best to other bone cells but not as well as a non-homing fetal bone marrow-derived cell line, D2. Invasion of prostate cancer cells through HBME-1 lawns was examined at 8 hr and 16 hr. In contrast to the adhesion studies, the invasion of the more aggressive C4-2 cells was 3.46-fold (95% CI = 1.18-10.17) and 2.65-fold (95% CI = 1.26-5.56) greater at 8 hr and 16 hr, respectively than LNCaP cells. Similarly, LNCaP cell invasion was 1.73-fold (95% CI = 0.69-4.37) and 2.35-fold (95% CI = 1.41-3.93) greater at 8 hr and 16 hr, respectively than P69 cells at the invasion of HBME-1 monolayers. At 8 hr, C4-2 cells had 6.0-fold (95% CI = 2.63,13.65) higher invasive potential than P69 cells. Phage display biopanning of LNCaP cells versus C4-2 cells in vitro using 4 separate techniques repeatedly identified the same peptide in support of minimal cell surface changes associated with the ability of C4-2 cells to metastasize to bone. As integrins are vital to cell adhesion and migration, we examined the integrin subunit expression in the prostate cell lines. The expression of integrin subunits is much higher in the nontumorigenic cell line, P69, whereas the differences in integrin expression between LNCaP and C4-2 are negligible. Only alpha(2) and beta(5) integrin subunits increase from LNCaP to C4-2. Given that C4-2 cells spontaneously metastasize to bone in vivo and LNCaP cells do not, these studies imply that the ability of a metastatic prostate cancer cell to colonize the bone is not completely dependent upon the ability of the cancer cell to adhere to either osteoblastic cells or to the bone marrow endothelial cell lining. Therefore, the initial interaction between the bone endothelium or stroma and prostate cells is not accurately referred to as a tropic or homing response. The invasion assay results indicate that the invasive potential of the cell more accurately reflects the bone colonizing potential of a prostate cancer cell. It is likely that bidirectional paracrine interactions, subsequent to marrow adhesion, between prostate cancer cells and the bone microenvironment are what determine the successful colonization of the bone by prostate cancer cells. Further, functional changes in surface proteins that are involved in invasion are likely to occur without major changes in levels of cell surface protein expression. Functional integrin association, substratum usage and outside in signaling are more likely to predict metastatic behavior.

Nanoscale RGD Peptide Organization Regulates Cell Proliferation and Differentiation

ABSTRACTRGD (arginine-glycine-aspartic acid) containing peptide sequences, common cell attachment sites present in many extracellular matrix (ECM) proteins, mediate many important cellular processes. The role of nanoscale organization of RGD peptides in the regulation of the adhesion, proliferation and differentiation of both preosteoblasts (MC3T3-E1) and multipotential (D1) cell lines in vitro was investigated in this study. Alginate polymer chains with varying RGD peptide degree of substitution were mixed with unmodified polymer chains at different ratios to allow variation of RGD peptide spacing in the nanometer scale, independently of the overall bulk density of peptides presented from the material. Proliferation of both cell types was observed to be closely correlated to RGD island (defined as a cluster of RGD peptides) spacing, independently of overall bulk ligand density, following cell adhesion to alginate hydrogels. Increased RGD island spacing was observed to promote spreading of MC3T3-E1 cells while simultaneously suppressing their proliferation. However, increased RGD island spacing decreased spreading of D1 cells while also decreasing proliferation. Moreover, differentiation of preosteoblasts was significantly upregulated in response to decreased RGD island spacing, whereas differentiation of multipotential cells was modestly regulated by this variable. These results demonstrate that the nanoscale organization of adhesion ligands may be an important variable in controlling cell phenotype and function. In addition, cellular responses to nanoscale ligand organization differ depending on the cell type, and this may be related to the differentiation stage of the cells.

Addicting drugs utilize a synergistic molecular mechanism in common requiring adenosine and Gi-beta gamma dimers.

The mesolimbic dopamine system and cAMP-dependent/protein kinase A (PKA) pathways are strongly implicated in addictive behaviors. Here we determine the role of dopamine D2 receptors (D2) in PKA signaling responses to delta-opioid (DOR) and cannabinoid (CB1) receptors. We find in NG108-15/D2 cells and in cultured primary neurons that a brief exposure to saturating concentrations of DOR and CB1 agonists increases cAMP, promotes PKA C alpha translocation and increases cAMP-dependent gene expression. Activation of PKA signaling is mediated by Gi-beta gamma dimers. Importantly, subthreshold concentrations of DOR or CB1 agonists with D2 agonists, which are without effect when added separately, together activate cAMP/PKA signaling synergistically. There is also synergy between DOR or CB1 with ethanol, another addicting agent. In all instances, synergy requires adenosine activation of adenosine A2 receptors and is mediated by beta gamma dimers. Synergy by this molecular mechanism appears to confer hypersensitivity to opioids and cannabinoids while simultaneously increasing the sensitivity of D2 signaling when receptors are expressed on the same cells. This mechanism may account, in part, for drug-induced activation of medium spiny neurons in the nucleus accumbens.

Evidence of increased dopamine receptor signaling in food-restricted rats

It is well established that chronic food restriction enhances sensitivity to the rewarding and motor-activating effects of abused drugs. However, neuroadaptations underlying these behavioral effects have not been characterized. The purpose of the present study was to explore the possibility that food restriction produces increased dopamine (DA) receptor function that is evident in behavior, signal transduction, and immediate early gene expression. In the first two experiments, rats received intracerebroventricular (i.c.v.) injections of the D1 DA receptor agonist SKF-82958, and the D2/3 DA receptor agonist quinpirole. Both agonists produced greater motor-activating effects in food-restricted than ad libitum-fed rats. In addition, Fos-immunostaining induced by SKF-82958 in caudate-putamen (CPu) and nucleus accumbens (Nac) was greater in food-restricted than ad libitum-fed rats, as was staining induced by quinpirole in globus pallidus and ventral pallidum. In the next two experiments, neuronal membranes prepared from CPu and Nac were exposed to SKF-82958 and quinpirole. Despite the documented involvement of cyclic AMP (cAMP) signaling in D1 DA receptor-mediated c-fos induction, stimulation of adenylyl cyclase (AC) activity by SKF-82958 in CPu and Nac did not differ between groups. Food restriction did, however, decrease AC stimulation by the direct enzyme stimulant, forskolin, but not NaF or MnCl 2, suggesting a shift in AC expression to a less catalytically efficient isoform. Finally, food restriction increased quinpirole-stimulated [ 35S]guanosine triphosphate-γS binding in CPu, suggesting that increased functional coupling between D2 DA receptors and G i may account for the augmented behavioral and pallidal c-Fos responses to quinpirole. Results of this study support the hypothesis that food restriction leads to neuroadaptations at the level of postsynaptic D1 and D2 receptor-bearing cells which, in turn, mediate augmented behavioral and transcriptional responses to DA. The signaling pathways mediating these augmented responses remain to be fully elucidated.

IKKalpha regulates mitogenic signaling through transcriptional induction of cyclin D1 via Tcf.

The Wnt/beta-catenin/Tcf and IkappaB/NF-kappaB cascades are independent pathways involved in cell cycle control, cellular differentiation, and inflammation. Constitutive Wnt/beta-catenin signaling occurs in certain cancers from mutation of components of the pathway and from activating growth factor receptors, including RON and MET. The resulting accumulation of cytoplasmic and nuclear beta-catenin interacts with the Tcf/LEF transcription factors to induce target genes. The IkappaB kinase complex (IKK) that phosphorylates IkappaB contains IKKalpha, IKKbeta, and IKKgamma. Here we show that the cyclin D1 gene functions as a point of convergence between the Wnt/beta-catenin and IkappaB pathways in mitogenic signaling. Mitogenic induction of G(1)-S phase progression and cyclin D1 expression was PI3K dependent, and cyclin D1(-/-) cells showed reduced PI3K-dependent S-phase entry. PI3K-dependent induction of cyclin D1 was blocked by inhibitors of PI3K/Akt/IkappaB/IKKalpha or beta-catenin signaling. A single Tcf site in the cyclin D1 promoter was required for induction by PI3K or IKKalpha. In IKKalpha(-/-) cells, mitogen-induced DNA synthesis, and expression of Tcf-responsive genes was reduced. Reintroduction of IKKalpha restored normal mitogen induction of cyclin D1 through a Tcf site. In IKKalpha(-/-) cells, beta-catenin phosphorylation was decreased and purified IKKalpha was sufficient for phosphorylation of beta-catenin through its N-terminus in vitro. Because IKKalpha but not IKKbeta induced cyclin D1 expression through Tcf activity, these studies indicate that the relative levels of IKKalpha and IKKbeta may alter their substrate and signaling specificities to regulate mitogen-induced DNA synthesis through distinct mechanisms.

Ultrastructural immunohistochemical localization of gastrin, somatostatin and serotonin in endocrine cells of human antral gastric mucosa

Five types of endocrine cells are found in the human antral gastric mucosa: gastrin (G) cells, somatostatin (D) cells, enterochromaffin (EC) cells and cells with an unknown secretory product (D1 cells and P cells). The content of secretory granules, gastrin, somatostatin and serotonin, was evaluated using electron microscopic immunohistochemistry and was compared with the granular content in G cells, D cells and EC cells as determined by routine electron microscopy. Semi-quantitative scoring of the granular content was performed on a scale 1-4 (empty-full). The content of gastrin (2.5 +/- 0.2) and somatostatin (3.3 +/- 0.2) in the granules was not different from the granular content in G cells (2.5 +/- 0.3; p > 0.05) and D cells (3.5 +/- 0.2; p > 0.05). Gastrin was also found in G cells in a nongranular form. The content of serotonin in granules (2.8 +/- 0.3) was smaller than the granular content in EC cells (3.7 +/- 0.2; p < 0.05). In intermediate-full and intermediate-empty granules, serotonin was localized in the periphery of granules whereas the granular content in EC cells was localized in an eccentric or central pattern. The granular content of D1 cells and P cells was 3.8 +/- 0.2, and 3.4 +/- 0.2, respectively. It is concluded that gastrin and somatostatin immunostaining in granules of G cells and D cells reflects the granular content in G cells and D cells, respectively, whereas serotonin immunostaining does not agree with the granular content of EC cells.

Characterization of an interleukin-7-dependent thymic cell line derived from a p53 −/− mouse

In order to study the response of T cells to IL-7, we aimed to generate an IL-7-dependent thymocyte line. CD4 −CD8 − thymocytes from a p53 −/ − mouse were continuously propagated in interleukin-7 (IL-7), and after 2 months there developed an immortal line termed “D1.” The D1 line has retained a stable dependency on IL-7. Withdrawal of IL-7 from D1 cells induced arrest in G1 phase of the cell cycle, followed by apoptosis. In addition to IL-7, several other cytokines that employ γ c as part of their receptor were also capable of stimulating D1 cell survival and proliferation. Gene induction by IL-7 was analyzed in D1 cells using RNase protection and array analysis and revealed a number of transcripts potentially involved in cell cycle, apoptosis and signaling.

Cytosolic retention of phosphorylated extracellular signal-regulated kinase and a Rho-associated kinase-mediated signal impair expression of p21(Cip1/Waf1) in phorbol 12-myristate-13- acetate-induced apoptotic cells.

In response to treatment with phorbol-12-myristate-13-acetate (PMA), the half-population of erythromyeloblast D2 cells, a cytokine-independent variant of TF-1 cells, displayed adhesion and differentiated into a monocyte/macrophage-like morphology, while the other half-population remained in suspension and underwent apoptosis. Expression of the cell cycle inhibitor p21(Cip1/Waf1) was induced after PMA treatment in the adherent cells but not in the proapoptotic cells. We investigated the mechanism responsible for the impairment of p21(Cip1/Waf1) induction in PMA-induced proapoptotic cells. We demonstrated that in PMA-induced adherent cells, upregulation of p21(Cip1/Waf1) requires the activation and nuclear translocation of phosphorylated extracellular signal-regulated kinase (phospho-ERK). Although ERK was phosphorylated to comparable levels in PMA-induced proapoptotic and adherent cells, nuclear distribution of phospho-ERK was seen only in the adherent, not in the proapoptotic cells. We also found that only PMA-induced proapoptotic cells contained the phosphorylated form of myosin light chain, which is dependent on Rho-associated kinase (ROCK) activation, and that expression of a dominant-active form of ROCK suppressed activation of the p21(Cip1/Waf1) promoter during PMA induction. Finally, we demonstrated that inhibition of ROCK restores nuclear distribution of phospho-ERK and activation of p21(Cip1/Waf1) expression. Based on these findings, we propose that a ROCK-mediated signal is involved in interfering with the process of ERK-mediated p21(Cip1/Waf1) induction in PMA-induced proapoptotic TF-1 and D2 cells.