Cell lineage relationship in the stomach of normal and genetically manipulated mice

The oxyntic mucosa of the mouse stomach is lined with a heterogeneous population of cells that form numerous short pits continuous with long tubular glands. Tritiated thymidine radioautography has made it possible to pinpoint the origin of all cell types and to follow the differentiation/migration of different cell lineages along the pit-gland unit. The proliferating multipotent stem cells functionally anchored in the upper glandular region, the isthmus, give rise to three main lineage precursors: 1) pre-pit cells, which migrate upward to the pit while differentiating into mucus-producing pit cells; 2) pre-neck cells, which migrate downward to the glandular neck while differentiating into mucus-producing neck cells that, by approaching the glandular base, gradually change their phenotype into pepsinogen- and intrinsic factor-producing zymogenic cells; 3) pre-parietal cells, which differentiate into acid-producing parietal cells in the isthmus and then undergo bipolar migration towards the pit and the glandular base. Thus, parietal cells are the only cells that complete their differentiation in the isthmus and then migrate to be scattered throughout the pit-gland unit. To determine whether parietal cells play a role in controlling decisions about cell fate within the pit-gland unit, the gastric epithelium has been examined in transgenic mice expressing the H,K-ATPase ß-subunit-1035 to +24/simian virus 40 large T antigen fusion gene. The blockade in parietal cell differentiation in these mice produces an amplification of lineage precursors, a marked depletion of zymogenic cells and an increase in pit cell census. Ablation of parietal cells in another transgenic mouse model expressing the H,K-ATPase ß-subunit-1035 to +24/diphtheria toxin fragment A fusion gene also produces amplification of lineage precursors, and similar effects on zymogenic and pit cell census. These findings strongly suggest that parietal cells produce regulatory signals that control the cellular differentiation program of both pit and zymogenic cell lineages, and would hopefully improve our ability to identify the cellular pathways leading to malignant transformation

Regulation of T cell response to leishmania antigens by determinants of histocompatibility leukocyte class I and II molecules

It has been shown that HLA class I molecules play a significant role in the regulation of the proliferation of T cells activated by mitogens and antigens. We evaluated the ability of mAb to a framework determinant of HLA class I molecules to regulate T cell proliferation and interferon gamma (IFN-g) production against leishmania, PPD, C. albicans and tetanus toxoid antigens in patients with tegumentary leishmaniasis and healthy subjects. The anti-major histocompatibility complex (MHC) mAb (W6/32) suppressed lymphocyte proliferation by 90% in cultures stimulated with aCD3, but the suppression was variable in cultures stimulated with leishmania antigen. This suppression ranged from 30-67% and was observed only in 5 of 11 patients. IFN-g production against leishmania antigen was also suppressed by anti-HLA class I mAb. In 3 patients IFN-g levels were suppressed by more than 60%, while in the other 2 cultures IFN-g levels were 36 and 10% lower than controls. The suppression by HLA class I mAb to the proliferative response in leishmaniasis patients and in healthy controls varied with the antigens and the patients or donors tested. To determine whether the suppression is directed at antigen presenting cells (APCs) or at the responding T cells, experiments with antigen-primed non-adherent cells, separately incubated with W6/32, were performed. Suppression of proliferation was only observed when the W6/32 mAb was added in the presence of T cells. These data provide evidence that a mAb directed at HLA class I framework determinants can suppress proliferation and cytokine secretion in response to several antigens.

Genetic alterations in head and neck squamous cell carcinomas

The genetic alterations observed in head and neck cancer are mainly due to oncogene activation (gain of function mutations) and tumor suppressor gene inactivation (loss of function mutations), leading to deregulation of cell proliferation and death. These genetic alterations include gene amplification and overexpression of oncogenes such as myc, erbB-2, EGFR and cyclinD1 and mutations, deletions and hypermethylation leading to p16 and TP53 tumor suppressor gene inactivation. In addition, loss of heterozygosity in several chromosomal regions is frequently observed, suggesting that other tumor suppressor genes not yet identified could be involved in the tumorigenic process of head and neck cancers. The exact temporal sequence of the genetic alterations during head and neck squamous cell carcinoma (HNSCC) development and progression has not yet been defined and their diagnostic or prognostic significance is controversial. Advances in the understanding of the molecular basis of head and neck cancer should help in the identification of new markers that could be used for the diagnosis, prognosis and treatment of the disease.

Differential regulation of vitamin D receptor expression in distinct leukemic cell lines upon phorbol ester-induced growth arrest

A close correlation between vitamin D receptor (VDR) abundance and cell proliferation rate has been shown in NIH-3T3 fibroblasts, MCF-7 breast cancer and in HL-60 myeloblastic cells. We have now determined if this association occurs in other leukemic cell lines, U937 and K562, and if VDR content is related to c-myc expression, which is also linked to cell growth state. Upon phorbol myristate acetate (PMA) treatment, cells from the three lineages (HL-60, U937 and K562) differentiated and expressed specific surface antigens. All cell lines analyzed were growth inhibited by PMA and the doubling time was increased, mainly due to an increased fraction of cells in the G0/G1 phase, as determined by flow cytometry measurements of incorporated bromodeoxyuridine and cell DNA content. C-myc mRNA expression was down-regulated and closely correlated to cell growth arrest. However, VDR expression in leukemic cell lines, as determined by immunofluorescence and Northern blot assays, was not consistently changed upon inhibition of cell proliferation since VDR levels were down-regulated only in HL-60 cells. Our data suggest that VDR expression cannot be explained simply as a reflection of the leukemic cell growth state.

The pathogenesis of tropical spastic paraparesis/human T-cell leukemia type I-associated myelopathy

Tropical spastic paraparesis/human T-cell leukemia type I-associated myelopathy (TSP/HAM) is caused by a human T-cell leukemia virus type I (HTLV-I) after a long incubation period. TSP/HAM is characterized by a chronic progressive paraparesis with sphincter disturbances, no/mild sensory loss, the absence of spinal cord compression and seropositivity for HTLV-I antibodies. The pathogenesis of this entity is not completely known and involves a multivariable phenomenon of immune system activation against the presence of HTLV-I antigens, leading to an inflammatory process and demyelination, mainly in the thoracic spinal cord. The current hypothesis about the pathogenesis of TSP/HAM is: 1) presence of HTLV-I antigens in the lumbar spinal cord, noted by an increased DNA HTLV-I load; 2) CTL either with their lytic functions or release/production of soluble factors, such as CC-chemokines, cytokines, and adhesion molecules; 3) the presence of Tax gene expression that activates T-cell proliferation or induces an inflammatory process in the spinal cord; 4) the presence of B cells with neutralizing antibody production, or complement activation by an immune complex phenomenon, and 5) lower IL-2 and IFN-gamma production and increased IL-10, indicating drive to a cytokine type 2 pattern in the TSP/HAM subjects and the existence of a genetic background such as some HLA haplotypes. All of these factors should be implicated in TSP/HAM and further studies are necessary to investigate their role in the development of TSP/HAM.

RNA and DNA aptamers as potential tools to prevent cell adhesion in disease

Recent research has shown that receptor-ligand interactions between surfaces of communicating cells are necessary prerequisites for cell proliferation, cell differentiation and immune defense. Cell-adhesion events have also been proposed for pathological conditions such as cancer growth, metastasis, and host-cell invasion by parasites such as Trypanosoma cruzi. RNA and DNA aptamers (aptus = Latin, fit) that have been selected from combinatorial nucleic acid libraries are capable of binding to cell-adhesion receptors leading to a halt in cellular processes induced by outside signals as a consequence of blockage of receptor-ligand interactions. We outline here a novel approach using RNA aptamers that bind to T. cruzi receptors and interrupt host-cell invasion in analogy to existing procedures of blocking selectin adhesion and function in vitro and in vivo.

A fucan from the brown seaweed Spatoglossum schröederi inhibits Chinese hamster ovary cell adhesion to several extracellular matrix proteins

Fucans, a family of sulfated polysaccharides present in brown seaweed, have several biological activities. Their use as drugs would offer the advantage of no potential risk of contamination with viruses or particles such as prions. A fucan prepared from Spatoglossum schröederi was tested as a possible inhibitor of cell-matrix interactions using wild-type Chinese hamster ovary cells (CHO-K1) and the mutant type deficient in xylosyltransferase (CHO-745). The effect of this polymer on adhesion properties with specific extracellular matrix components was studied using several matrix proteins as substrates for cell attachment. Treatment with the polymer inhibited the adhesion of fibronectin to both CHO-K1 (2 x 10(5))()and CHO-745 (2 x 10(5) and 5 x 10(5)) cells. No effect was detected with laminin, using the two cell types. On the other hand, adhesion to vitronectin was inhibited in CHO-K1 cells and adhesion to type I collagen was inhibited in CHO-745 cells. In spite of this inhibition, the fucan did not affect either cell proliferation or cell cycle. These results demonstrate that this polymer is a new anti-adhesive compound with potential pharmacological applications.

Cytogenetic description of breast fibroadenomas: alterations related solely to proliferation?

Twelve breast fibroadenomas were analyzed cytogenetically and only four were found to have clonal alterations. The presence of chromosomal alterations in fibroadenomas must be the consequence of the proliferating process and must not be related to the etiology of this type of lesion. In contrast, the few fibroadenomas that exhibit chromosomal alterations are likely to be those presenting a risk of neoplastic transformation. Clonal numerical alterations involved chromosomes 8, 18, 19, and 21. Of the chromosomal alterations found in the present study, only monosomy of chromosomes 19 and 21 has been reported in breast fibroadenomas. The loss of chromosome 21 was the most frequent alteration found in our sample. The study of benign proliferations and their comparison with chromosome alterations in their malignant counterparts ought to result in a better understanding of the genes acting on cell proliferation alone, and of the genes that cause these cells to exhibit varied behaviors such as recurrences, spontaneous regression and fast growth.

Cyclic AMP increases the survival of ganglion cells in mixed retinal cell cultures in the absence of exogenous neurotrophic molecules, an effect that involves cholinergic activity

Natural cell death is a well-known degenerative phenomenon occurring during development of the nervous system. The role of trophic molecules produced by target and afferent cells as well as by glial cells has been extensively demonstrated. Literature data demonstrate that cAMP can modulate the survival of neuronal cells. Cultures of mixed retinal cells were treated with forskolin (an activator of the enzyme adenylyl cyclase) for 48 h. The results show that 50 µM forskolin induced a two-fold increase in the survival of retinal ganglion cells (RGCs) in the absence of exogenous trophic factors. This effect was dose dependent and abolished by 1 µM H89 (an inhibitor of protein kinase A), 1.25 µM chelerythrine chloride (an inhibitor of protein kinase C), 50 µM PD 98059 (an inhibitor of MEK), 25 µM Ly 294002 (an inhibitor of phosphatidylinositol-3 kinase), 30 nM brefeldin A (an inhibitor of polypeptide release), and 10 µM genistein or 1 ng/ml herbimycin (inhibitors of tyrosine kinase enzymes). The inhibition of muscarinic receptors by 10 µM atropine or 1 µM telenzepine also blocked the effect of forskolin. When we used 25 µM BAPTA, an intracellular calcium chelator, as well as 20 µM 5-fluoro-2'-deoxyuridine, an inhibitor of cell proliferation, we also abolished the effect. Our results indicate that cAMP plays an important role controlling the survival of RGCs. This effect is directly dependent on M1 receptor activation indicating that cholinergic activity mediates the increase in RGC survival. We propose a model which involves cholinergic amacrine cells and glial cells in the increase of RGC survival elicited by forskolin treatment.

Multicellular spheroids of bone marrow stromal cells: a three-dimensional in vitro culture system for the study of hematopoietic cell migration

Cell fate decisions are governed by a complex interplay between cell-autonomous signals and stimuli from the surrounding tissue. In vivo cells are connected to their neighbors and to the extracellular matrix forming a complex three-dimensional (3-D) microenvironment that is not reproduced in conventional in vitro systems. A large body of evidence indicates that mechanical tension applied to the cytoskeleton controls cell proliferation, differentiation and migration, suggesting that 3-D in vitro culture systems that mimic the in vivo situation would reveal biological subtleties. In hematopoietic tissues, the microenvironment plays a crucial role in stem and progenitor cell survival, differentiation, proliferation, and migration. In adults, hematopoiesis takes place inside the bone marrow cavity where hematopoietic cells are intimately associated with a specialized three 3-D scaffold of stromal cell surfaces and extracellular matrix that comprise specific niches. The relationship between hematopoietic cells and their niches is highly dynamic. Under steady-state conditions, hematopoietic cells migrate within the marrow cavity and circulate in the bloodstream. The mechanisms underlying hematopoietic stem/progenitor cell homing and mobilization have been studied in animal models, since conventional two-dimensional (2-D) bone marrow cell cultures do not reproduce the complex 3-D environment. In this review, we will highlight some of the mechanisms controlling hematopoietic cell migration and 3-D culture systems.

Effects of gamma-radiation on cell growth, cycle arrest, death, and superoxide dismutase expression by DU 145 human prostate cancer cells

Gamma-irradiation (gamma-IR) is extensively used in the treatment of hormone-resistant prostate carcinoma. The objective of the present study was to investigate the effects of 60Co gamma-IR on the growth, cell cycle arrest and cell death of the human prostate cancer cell line DU 145. The viability of DU 145 cells was measured by the Trypan blue exclusion assay and the 3(4,5-dimethylthiazol-2-yl)-2,5,diphenyltetrazolium bromide test. Bromodeoxyuridine incorporation was used for the determination of cell proliferation. Cell cycle arrest and cell death were analyzed by flow cytometry. Superoxide dismutase (SOD), specifically CuZnSOD and MnSOD protein expression, after 10 Gy gamma-IR, was determined by Western immunoblotting analysis. gamma-IR treatment had a significant (P < 0.001) antiproliferative and cytotoxic effect on DU 145 cells. Both effects were time and dose dependent. Also, the dose of gamma-IR which inhibited DNA synthesis and cell proliferation by 50% was 9.7 Gy. Furthermore, gamma-IR induced cell cycle arrest in the G2/M phase and the percentage of cells in the G2/M phase was increased from 15% (control) to 49% (IR cells), with a nonsignificant induction of apoptosis. Treatment with 10 Gy gamma-IR for 24, 48, and 72 h stimulated CuZnSOD and MnSOD protein expression in a time-dependent manner, approximately by 3- to 3.5-fold. These data suggest that CuZnSOD and MnSOD enzymes may play an important role in the gamma-IR-induced changes in DU 145 cell growth, cell cycle arrest and cell death.

Nuclear calcium signaling: a cell within a cell

Calcium (Ca2+) is a versatile second messenger that regulates a wide range of cellular functions. Although it is not established how a single second messenger coordinates diverse effects within a cell, there is increasing evidence that the spatial patterns of Ca2+ signals may determine their specificity. Ca2+ signaling patterns can vary in different regions of the cell and Ca2+ signals in nuclear and cytoplasmic compartments have been reported to occur independently. No general paradigm has been established yet to explain whether, how, or when Ca2+ signals are initiated within the nucleus or their function. Here we highlight that receptor tyrosine kinases rapidly translocate to the nucleus. Ca2+ signals that are induced by growth factors result from phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate formation within the nucleus rather than within the cytoplasm. This novel signaling mechanism may be responsible for growth factor effects on cell proliferation.

Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system

Future clinical applications of human embryonic stem (hES) cells will require high-yield culture protocols. Currently, hES cells are mainly cultured in static tissue plates, which offer a limited surface and require repeated sub-culturing. Here we describe a stirred system with commercial dextran-based microcarriers coated with denatured collagen to scale-up hES cell production. Maintenance of pluripotency in the microcarrier-based stirred system was shown by immunocytochemical and flow cytometry analyses for pluripotency-associated markers. The formation of cavitated embryoid bodies expressing markers of endoderm, ectoderm and mesoderm was further evidence of maintenance of differentiation capability. Cell yield per volume of medium spent was more than 2-fold higher than in static plates, resulting in a significant decrease in cultivation costs. A total of 10(8) karyotypically stable hES cells were obtained from a unitary small vessel that needed virtually no manipulation during cell proliferation, decreasing risks of contamination. Spinner flasks are available up to working volumes in the range of several liters. If desired, samples from the homogenous suspension can be withdrawn to allow process validation needed in the last expansion steps prior to transplantation. Especially when thinking about clinical trials involving from dozens to hundreds of patients, the use of a small number of larger spinners instead of hundreds of plates or flasks will be beneficial. To our knowledge, this is the first description of successful scale-up of feeder- and Matrigel™-free production of undifferentiated hES cells under continuous agitation, which makes this system a promising alternative for both therapy and research needs.

Effect of aging and oral tolerance on dendritic cell function

Oral tolerance can be induced in some mouse strains by gavage or spontaneous ingestion of dietary antigens. In the present study, we determined the influence of aging and oral tolerance on the secretion of co-stimulatory molecules by dendritic cells (DC), and on the ability of DC to induce proliferation and cytokine secretion by naive T cells from BALB/c and OVA transgenic (DO11.10) mice. We observed that oral tolerance could be induced in BALB/c mice (N = 5 in each group) of all ages (8, 20, 40, 60, and 80 weeks old), although a decline in specific antibody levels was observed in the sera of both tolerized and immunized mice with advancing age (40 to 80 weeks old). DC obtained from young, adult and middle-aged (8, 20, and 40 weeks old) tolerized mice were less efficient (65, 17 and 20%, respectively) than DC from immunized mice (P < 0.05) in inducing antigen-specific proliferation of naive T cells from both BALB/c and DO11.10 young mice, or in stimulating IFN-g, IL-4 and IL-10 production. However, TGF-β levels were significantly elevated in co-cultures carried out with DC from tolerant mice (P < 0.05). DC from both immunized and tolerized old and very old (60 and 80 weeks old) mice were equally ineffective in inducing T cell proliferation and cytokine production (P < 0.05). A marked reduction in CD86+ marker expression was observed in DC isolated from both old and tolerized mice (75 and 50%, respectively). The results indicate that the aging process does not interfere with the establishment of oral tolerance in BALB/c mice, but reduces DC functions, probably due to the decline of the expression of the CD86 surface marker.

The miR-17-92 cluster regulates FOG-2 expression and inhibits proliferation of mouse embryonic cardiomyocytes

MicroRNAs (miRNAs) have gradually been recognized as regulators of embryonic development; however, relatively few miRNAs have been identified that regulate cardiac development. A series of recent papers have established an essential role for the miRNA-17-92 (miR-17-92) cluster of miRNAs in the development of the heart. Previous research has shown that the Friend of Gata-2 (FOG-2) is critical for cardiac development. To investigate the possibility that the miR-17-92 cluster regulates FOG-2 expression and inhibits proliferation in mouse embryonic cardiomyocytes we initially used bioinformatics to analyze 3’ untranslated regions (3’UTR) of FOG-2 to predict the potential of miR-17-92 to target it. We used luciferase assays to demonstrate that miR-17-5p and miR-20a of miR-17-92 interact with the predicted target sites in the 3’UTR of FOG-2. Furthermore, RT-PCR and Western blot were used to demonstrate the post-transcriptional regulation of FOG-2 by miR-17-92 in embryonic cardiomyocytes from E12.5-day pregnant C57BL/6J mice. Finally, EdU cell assays together with the FOG-2 rescue strategy were employed to evaluate the effect of proliferation on embryonic cardiomyocytes. We first found that the miR-17-5p and miR-20a of miR-17-92 directly target the 3’UTR of FOG-2 and post-transcriptionally repress the expression of FOG-2. Moreover, our findings demonstrated that over-expression of miR-17-92 may inhibit cell proliferation via post-transcriptional repression of FOG-2 in embryonic cardiomyocytes. These results indicate that the miR-17-92 cluster regulates the expression of FOG-2 protein and suggest that the miR-17-92 cluster might play an important role in heart development.