Number Of Cycling Cells Research Articles

Tissue homeostasis in sponges: Quantitative analysis of cell proliferation and apoptosis.

Tissues of multicellular animals are maintained due to a tight balance between cell proliferation and programmed cell death. Sponges are early branching metazoans essential to understanding the key mechanisms of tissue homeostasis. This article is dedicated to the comparative analysis of proliferation and apoptosis in intact tissues of two sponges, Halisarca dujardinii (class Demospongiae) and Leucosolenia variabilis (class Calcarea). Labeled nucleotides EdU and anti-phosphorylated histone 3 antibodies reveal a considerable number of cycling cells in intact tissues of both species. Quantitative DNA staining reveals the classic cell cycle distribution curve. The main type of cycling cells are choanocytes - flagellated cells of the aquiferous system. The rate of proliferation remains constant throughout various areas of sponge bodies that contain choanocytes. The EdU tracking experiments conducted in H. dujardinii indicate that choanocytes may give rise to mesohyl cells through migration. The number of apoptotic cells in tissues of both species is insignificant, although being comparable to the renewing tissues of other animals. In vivo studies with tetramethylrhodamine ethyl ester and CellEvent Caspase-3/7 indicate that apoptosis might be independent of mitochondrial outer membrane permeabilization. Altogether, a combination of confocal laser scanning microscopy and flow cytometry provides a quantitative description of cell proliferation and apoptosis in sponges displaying either rapid growth or cell turnover.

276 SCRNA SEQUENCING OF SINGLE EAC ORGANOID REVEALS HETEROGENEITY OF STEM CELL LIKE POPULATIONS

Abstract Tumor heterogeneity is one of the main contributor of poor response to treatment and metastasis. Esophageal adenocarcinoma (EAC) is known to be second only to melanoma in terms of heterogeneity. Robust in vitro models of EAC are lacking. In our patient derived organoid (PDO) culture, we identified different organoid morphologies from the same patient. We decided to investigate the transcriptional programs of the different organoid morphologies. Methods EAC organoids were established from endoscopic biopsies from 4 different patients. Based on their morphology, individual organoids were isolated and dissociated into single cell suspensions. The 10X Genomics platform was used for single cell RNA sequencing. Results The two morphologies identified by histology were cystic and intestinal. For the two intestinal populations the samples could be divided into stem-like (905/1646 and 1472/1926) and differentiated populations. Interestingly, we observed differences in the number of cycling cells in these Lgr5+ stem-like populations (905/905 and 337/1926). For the cystic samples the two populations could also be divided into stem-like populations (758/923 and 284/784) and differentiated populations. We also observed cell cycle differences in the Lgr5+ stem-like populations in these samples. RNA velocity analyses on each sample verified a differentiation trajectory from the stem-like to the differentiated cells. Conclusion Studying EAC organoids provides us with the ability to profile the transcriptional programs of progenitor-like cells, which are difficult to isolate from gross tumor. We demonstrate that we can isolate individual organoids and study them at single-cell resolution to evaluate the different types of organoid generating cells based on their morphology.

GDNF/GFRα1 Complex Abrogates Self-Renewing Activity of Cortical Neural Precursors Inducing Their Differentiation.

SummaryThe balance between factors leading to proliferation and differentiation of cortical neural precursors (CNPs) determines the correct cortical development. In this work, we show that GDNF and its receptor GFRα1 are expressed in the neocortex during the period of cortical neurogenesis. We show that the GDNF/GFRα1 complex inhibits the self-renewal capacity of mouse CNP cells induced by fibroblast growth factor 2 (FGF2), promoting neuronal differentiation. While GDNF leads to decreased proliferation of cultured cortical precursor cells, ablation of GFRα1 in glutamatergic cortical precursors enhances its proliferation. We show that GDNF treatment of CNPs promoted morphological differentiation even in the presence of the self-renewal-promoting factor, FGF2. Analysis of GFRα1-deficient mice shows an increase in the number of cycling cells during cortical development and a reduction in dendrite development of cortical GFRα1-expressing neurons. Together, these results indicate that GDNF/GFRα1 signaling plays an essential role in regulating the proliferative condition and the differentiation of cortical progenitors.

A Versatile Bioreactor for Dynamic Suspension Cell Culture. Application to the Culture of Cancer Cell Spheroids.

A versatile bioreactor suitable for dynamic suspension cell culture under tunable shear stress conditions has been developed and preliminarily tested culturing cancer cell spheroids. By adopting simple technological solutions and avoiding rotating components, the bioreactor exploits the laminar hydrodynamics establishing within the culture chamber enabling dynamic cell suspension in an environment favourable to mass transport, under a wide range of tunable shear stress conditions. The design phase of the device has been supported by multiphysics modelling and has provided a comprehensive analysis of the operating principles of the bioreactor. Moreover, an explanatory example is herein presented with multiphysics simulations used to set the proper bioreactor operating conditions for preliminary in vitro biological tests on a human lung carcinoma cell line. The biological results demonstrate that the ultralow shear dynamic suspension provided by the device is beneficial for culturing cancer cell spheroids. In comparison to the static suspension control, dynamic cell suspension preserves morphological features, promotes intercellular connection, increases spheroid size (2.4-fold increase) and number of cycling cells (1.58-fold increase), and reduces double strand DNA damage (1.5-fold reduction). It is envisioned that the versatility of this bioreactor could allow investigation and expansion of different cell types in the future.

IFN gamma regulates proliferation and neuronal differentiation by STAT1 in adult SVZ niche.

The adult subventricular zone (SVZ) is the main neurogenic niche in normal adult brains of mice and rats. Interferon gamma (IFNγ) has somewhat controversially been associated with SVZ progenitor proliferation and neurogenesis. The in vivo involvement of IFNγ in the physiology of the adult SVZ niche is not fully understood and its intracellular mediators are unknown. Here we show that IFNγ, through activation of its canonical signal transducer and activator of transcription 1 (STAT1) pathway, acts specifically on Nestin+ progenitors by decreasing both progenitor proliferation and the number of cycling cells. In addition, IFNγ increases the number of neuroblasts generated without shifting glial fate determination. The final result is deficient recruitment of newborn neurons to the olfactory bulb (OB), indicating that IFNγ-induced stimulation of neuronal differentiation does not compensate for its antiproliferative effect. We conclude that IFNγ signaling via STAT1 in the SVZ acts dually as an antiproliferative and proneurogenic factor, and thereby regulates neurogenesis in normal adult brains.

Acute effects of wheel running on adult hippocampal precursor cells in mice are not caused by changes in cell cycle length or S phase length.

Exercise stimulates cellular brain plasticity by extending the pool of proliferating neural precursor cells in the adult hippocampus. This effect has been investigated extensively, but the most immediate cellular effect induced by exercise that results in this acute increase in the number of cycling cells remained unclear. In the developing brain as well as adult pathological models, cell cycle alterations have a major influence on the balance between proliferative and neurogenic divisions. In this study we investigated whether this might also apply to the acute physiological pro-neurogenic stimulus of physical exercise in adulthood. Do changes in cell cycle precede the measurable increase in proliferation? After 5 days of voluntary wheel running, however, we measured only a very small, statistically not significant acceleration in cell cycle, which could not quantitatively explain the observed increase in proliferating cells after exercise. Thus, at this acute stage, changes at the level of cell cycle control is not the primary causal mechanism for the expansion of the precursor cell population, although with time after the stimulus changes in cell cycle of the entire population of labeled cells might be the result of the expanded pool of cells that have progressed to the advanced neurogenic stages with shorter cell cycle length.

Abstract 3735: Preclinical studies of salinomycin as a therapeutic agent for pediatric high-grade glioma cancer stem cells.

Abstract Brain tumors are the most prevalent childhood solid tumors. In the low-grade astrocytomas complete surgical resection has achieved over 90% five-year survival rate. In contrast, the aggressive anaplastic astrocytoma high-grade 3 and the fast growing glioblastoma multiforme high-grade 4 are often refractory to standard therapies; the majority of these patients at presentation are beyond cure by surgery and or radiotherapy. Emerging data substantiate the idea that a small pool of post-therapy residual cells within a tumor displaying stem-like properties is responsible for relapse. Therefore, selectively targeting these cancer stem-like cells (CSCs) may lead to advances in the treatment of these tumors. Salinomycin, an antibiotic potassium ionophore, has been shown to be highly effective against breast cancer stem cells and other malignancies; however, its potential use has not been studied in childhood astrocytomas. Recent studies indicate that salinomycin exerts its effect on components of the Wnt signaling cascade. In this study, we undertook in vitro and in vivo analyses on the effect of salinomycin on four pediatric high-grade astrocytoma cell lines, UW479 (provided by Dr. John Silber, University of Washington), SF188 (from Dr. Daphne Haas-Kogan, UCSF), SJ-GBM2, and CHLA-200 (COG Cell Culture and Xenograft Repository). Our results indicated that salinomycin was able to inhibit cellular viability in a dose-dependent manner. The soft-agar anchorage independent growth assay showed salinomycin inhibition of colony formation in 3 of the 4 cell lines tested. A decrease in the number of cycling cells (S-phase) was observed in cells treated with salinomycin. Notably, a 95% reduction in the glioma stem cells, read-out as CD133+, and impairment in the ability to form spheres (read-out as self-renewal) was observed. A xenograft mouse model was used to determine the effect of salinomycin on tumor growth in vivo and is ongoing. Salinomycin's modulation of Wnt targets measured by qRT-PCR, western blot analysis, and Bio-Plex protein assays, is in progress. Our data suggest that salinomycin could be exploited for the treatment of the aggressive form of pediatric gliomas. Citation Format: Emily S. Moses, Judy C. Chang, Jaclyn Y. Hung. Preclinical studies of salinomycin as a therapeutic agent for pediatric high-grade glioma cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3735. doi:10.1158/1538-7445.AM2013-3735

Role of neurotrophins on postnatal neurogenesis in the thalamus: prenatal exposure to ethanol

A second wave of neuronal generation occurs in the ventrobasal nucleus of the rat thalamus (VB) during the first three postnatal weeks. The present study tested the hypotheses (1) that postnatal neurogenesis in the VB is neurotrophin-regulated and (2) that ethanol-induced changes in this proliferation are mediated by neurotrophins. The first studies examined the effects of neurotrophins on the numbers of cycling cells in ex vivo preparations of the VB from 3-day-old rats. The proportion of cycling (Ki-67-positive) VB cells was higher in cultured thalamic slices treated with neurotrophins than in controls. Interestingly, this increase occurred with nerve growth factor (NGF) alone or with a combination of NGF and brain-derived neurotrophic factor (BDNF), but not with BDNF alone. Based on these data, the VBs from young offspring of pregnant rats fed an ethanol-containing or an isocaloric non-alcoholic liquid diet were examined between postnatal day (P) 1 and P31. Studies used enzyme-linked immunosorbent assays and immunoblots to explore the effects of ethanol on the expression of neurotrophins, their receptors, and representative signaling proteins. Ethanol altered the expression of neurotrophins and receptors throughout the first postnatal month. Expression of NGF increased, but there was no change in the expression of BDNF. The high affinity receptors (TrkA and TrkB) were unchanged but ethanol decreased expression of the low affinity receptor, p75. One downstream signaling protein, extracellular signal-regulated kinase (ERK), decreased but Akt expression was unchanged. Thus, postnatal cell proliferation in the VB of young rat pups is neurotrophin-responsive and is affected by ethanol.

Dissociation of EphB2 Signaling Pathways Mediating Progenitor Cell Proliferation and Tumor Suppression

Signaling proteins driving the proliferation of stem and progenitor cells are often encoded by proto-oncogenes. EphB receptors represent a rare exception; they promote cell proliferation in the intestinal epithelium and function as tumor suppressors by controlling cell migration and inhibiting invasive growth. We show that cell migration and proliferation are controlled independently by the receptor EphB2. EphB2 regulated cell positioning is kinase-independent and mediated via phosphatidylinositol 3-kinase, whereas EphB2 tyrosine kinase activity regulates cell proliferation through an Abl-cyclin D1 pathway. Cyclin D1 regulation becomes uncoupled from EphB signaling during the progression from adenoma to colon carcinoma in humans, allowing continued proliferation with invasive growth. The dissociation of EphB2 signaling pathways enables the selective inhibition of the mitogenic effect without affecting the tumor suppressor function and identifies a pharmacological strategy to suppress adenoma growth.

Ionizing Radiation Induces Ataxia Telangiectasia Mutated-Dependent Checkpoint Signaling and G2 But Not G1 Cell Cycle Arrest in Pluripotent Human Embryonic Stem Cells

Human embryonic stem (ES) cells are highly sensitive to environmental insults including DNA damaging agents, responding with high levels of apoptosis. To understand the response of human ES cells to DNA damage, we investigated the function of the ataxia telangiectasia mutated (ATM) DNA damage signaling pathway in response to gamma-irradiation. Here, we demonstrate for the first time in human ES cells that ATM kinase is phosphorylated and properly localized to the sites of DNA double-strand breaks within 15 minutes of irradiation. Activation of ATM kinase resulted in phosphorylation of its downstream targets: Chk2, p53, and Nbs1. In contrast to murine ES cells, Chk2 and p53 were localized to the nucleus of irradiated human ES cells. We further show that irradiation resulted in a temporary arrest of the cell cycle at the G(2), but not G(1), phase. Human ES cells resumed cycling approximately 16 hours after irradiation, but had a fourfold higher incidence of aberrant mitotic figures compared to nonirradiated cells. Finally, we demonstrate an essential role of ATM in establishing G(2) arrest since inhibition with the ATM-specific inhibitor KU55933 resulted in abolishment of G(2) arrest, evidenced by an increase in the number of cycling cells 2 hours after irradiation. In summary, these results indicate that human ES cells activate the DNA damage checkpoint, resulting in an ATM-dependent G(2) arrest. However, these cells re-enter the cell cycle with prominent mitotic spindle defects.

Deciphering JunB Function in Regulating Hematopoietic Stem Cell Functions.

The AP-1 transcription factor JunB plays a key role in controlling normal homeostasis of the hematopoietic stem cell (HSC) compartment and acts as a tumor suppressor in mice. We have previously shown that inactivating JunB expression in HSC causes an aberrant stem cell expansion leading to myeloproliferative disorder (MPD) development and leukemia progression. JunB-deficient HSC are the leukemia-initiating (leukemic) stem cells (LSC) responsible for the initiation and maintenance of this disease. We have now investigated the mechanisms by which loss of JunB alters HSC properties. We found that loss of JunB severely impaired HSC transplantability and cell cycle regulation. Limit dilution transplantation experiments with purified HSC (Lin−/c-Kit+/Sca-1+/Flk2- cells) revealed that junB-deficient HSC are extremely poor at providing engraftment on a cell-by-cell basis. Competitive bone marrow transplantation experiments confirmed that junB-deficient cells are on average 50% less efficient than normal cells in providing engraftment, although in every case where they did engraft, they mediated multilineage reconstitution followed by myeloid expansion and MPD development. JunB-deficient HSC also displayed rapid exhaustion of their self-renewal activity following serial transplantation. At the cellular level, the absence of JunB profoundly deregulated HSC proliferation and cell cycle distribution. Direct analysis of junB-deficient HSC revealed a striking decrease in the number of quiescent G0 cells (30% vs. 70% in normal HSC) and a correlative increase in the number of cycling cells. Quantitative RT-PCR analysis of junB-deficient HSC indicated a global decrease in the expression level of early G1 cyclins and almost all cyclin-dependant kinase inhibitors, associated with an increase in the expression level of late G1 and S-G2/M cyclins. These results provide a molecular understanding of junB-deficient HSC proliferation and strongly suggests that faster trafficking through the cell cycle is a central feature of their leukemic behavior. We also found that loss of JunB severely impaired HSC migratory response, with junB-deficient HSC displaying increased random diffusion and severely blunted response to the chemoattractant SDF1α. In fact, flow cytometry, qRT-PCR and microarray analyses revealed that several molecules involved in cell adhesion/migration (including CXCR4, LFA-1 and VLA-4) are deregulated in junB-deficient HSC. Finally, using short-term in vivo homing assays and intrafemoral injections we established that the engraftment defect exhibited by junB-deficient HSC is not due to a defect in their ability to home to the bone marrow (BM) cavity but to their defective ability to respond and to be maintained in this microenvironment. We are now studying how junB-deficient HSC localize/interact with cellular and molecular components of the BM niches contributes to their loss of quiescence and leukemic expansion. Taken together, these results provide a cellular and molecular understanding of how JunB controls the homeostasis of the stem cell compartment by coordinating stem cell maintenance with stem cell migration. They raise the exciting possibility that interfering with the deregulated cell cycle and altered maintenance of LSC in the BM microenvironment could lead to their specific eradication without impacting on normal HSC function.

FLT-PET in Haematological Disorders: A Novel Technique To Analyze the Proliferative Activity of the Bone Marrow Compartment.

Few diagnostic procedures are available to determine the degree of bone marrow cellularity and the numbers of cycling cells in patients with hematopoietic disorders. Therefore non-invasive imaging of the bone marrow compartment may be helpful. Recently the PET tracer [18F]-3′-fluoro-3′-deoxy-L-thymidine (FLT) has been developed. FLT uptake is related to the DNA synthesis rate and increases with higher proliferation rates and may therefore visualise the high cycling activity of haematopoietic cells in the bone marrow compartment. In the present study the feasibility of visualisation and quantification of the bone marrow compartment activity with FLT-PET was used to distinguish different haematological disorders.Clinical and laboratory data of 18 patients with low-risk myelodysplasia (MDS), chronic myeloproliferative disorders, myelofibrosis, aplastic anemia or multiple myeloma were correlated with the results of FLT-PET imaging using visual analysis and SUV. Findings were compared with those in normal controls (n=14).With SUV analysis of the femura, thoracic and lumbar vertebral body in conjunction with visual analysis a distinction could be made between MDS (n=9), chronic myeloproliferative disorders (n=3) and myelofibrosis (n=3) compared to normal controls (p<0.001). A significant increase in FLT uptake in the defined areas was observed in all the studied patients with MDS and myeloproliferative disorders. In addition a significant expansion of the bone marrow compartment was shown (p<0.001). No correlation was found between Ki-67 staining of bone marrow biopsies and FLT uptake. In contrast, patients with myelofibrosis demonstrated a decline in bone marrow FLT uptake compared to normal controls but a significant increase in liver and spleen uptake. Osteolytic lesions of multiple myeloma patients and patients with aplastic anemia showed a strong reduction in bone marrow FLT uptake.The results indicate that FLT-PET can be used to visualize the proliferative activity of the bone marrow compartment and may be helpful to distinguish separate haematological disorders.

Effects of rhIL-7 administration in humans on in vivo expansion of naïve, memory and effector subsets of CD4+ &amp; CD8+ T-cells

2504 Background: IL-7 has a critical and non-redundant role in T-cell lymphopoiesis and peripheral T-cell homeostasis. IL-7 administration may prove clinically valuable in conditions of disease induced (HIV) or iatrogenic T-cell depletion and for modulation of vaccine immune responses. In the first phase I study in humans, recombinant human interleukin-7 (“CYT 99–007”, Cytheris Inc., Rockville, MD) was administered subcutaneously every other day for two weeks in adults with refractory malignancies at 3, 10, 30 and 60 μg/kg/dose. Biologic activity, defined as a 50% increase over baseline of peripheral blood CD3+ T-cells, was seen at and above the 10μg/kg/dose in all patients. The kinetics of proliferation and expansion of peripheral blood T-cell subsets were analyzed. Methods: Multicolor flow cytometry was performed at baseline, 1, 2 and 3 weeks. Among CD4+ cells, the most naïve were defined as CD45RA+ /CD31+. Among CD4+ &amp; CD8+ cells, the main naïve, memory and effector populations were defined respectively as CD45RA+/CD27+, CD45RA-/CD27+ and CD45RA-/CD27-. Within each subset, the number of cells in cycle was defined by Ki67 staining. Results: Following IL-7 therapy, there was marked proliferation of all T-cells subsets, peaking at week 1, most striking for the naive subsets with 30–70% of circulating cells induced to cycle. Proliferation rates were halved by week 2 despite continuation of treatment, coincident with the observed down-regulation of the IL-7 receptor. Cycling returned to baseline by week 3. Significant proliferation was also induced in effector and memory CD4+ and CD8+ T-cells but to a lesser magnitude, resulting in a greater net expansion of the naïve subsets, still ongoing one week after the end of treatment. Conclusions: IL-7 administration induces marked expansion of naïve, memory and effector CD4+ &amp; CD8+ T-cells in humans. Consistent with the known down-regulation of the IL-7 receptor upon IL-7 exposure, proliferation rates decrease during the second week of treatment. rhIL-7 induced T-cell expansion may prove clinically valuable in adoptive immunotherapy as an adjunct to tumor vaccination and / or immunorestorative agent. [Table: see text]

Differential alteration of stem and other cell populations in ducts and lobules of TGFα and c-Myc transgenic mouse mammary epithelium

Genes associated with proliferation are active in stem and progenitor cells, and their over-expression can promote cancer. Two such genes, c-Myc and TGFα, promote morphologically dissimilar mammary tumors in transgenic mice. We investigated whether their over-expression affects population size and cell cycle activity in stem and other cell populations in non-neoplastic mammary epithelia. Results indicated that both cell population and cell cycle regulation are cell type- and microenvironment-specific. To create a tool for identifying and categorizing the five cellular phenotypes by light microscopy, we adapted previously established ultrastructural criteria. Using nulliparous MMTV- c-myc or MT- tgfα mice, we determined and compared the relative sizes the putative stem, progenitor and differentiated cell populations. PCNA staining was used to compare the portion of each cell population in the cell cycle. Cell population sizes were analyzed relative to: (1) their location in ducts versus lobules (microenvironment), (2) genotype, and (3) cell type. Population sizes differed significantly by genotype, depending on microenvironment ( p = 0.0008), by genotype, depending on cell type ( p < 0.0001), and by microenvironment, depending on cell type ( p = 0.03). The number of cycling cells was also affected by all three factors, confirming that the interplay of cell type, gene expression and three-dimensional organization are very important in tissue morphogenesis and function. We describe a structure in mammary epithelium consistent with that of a stem cell niche, and show that it is altered in MMTV- c-myc and likely altered in MT TGFα transgenic epithelia.

Cell Proliferation and Granule Cell Dispersion in Human Hippocampal Sclerosis

Granule cell dispersion (GCD) is observed in approximately 40% of cases of hippocampal sclerosis (HS) in patients with epilepsy. Studies in animal models suggest that GCD may be a consequence of enhanced proliferation of granule cell precursors as a result of seizures. We quantified the number of cells in cycle in subfields of the hippocampus with immunohistochemistry for Mcm2 in 14 HS cases with or without severe GCD compared to 6 epilepsy patients without classical HS or GCD as well as 5 postmortem controls. Higher numbers of Mcm2-positive cells were seen in the region of the granule cell layer in patients with severe GCD, and immunolabeling with Geminin and Ki-67 confirmed a proportion were progressing through cycle. Double labeling with Mcm2 and GFAP confirmed the majority of these cycling cells were GFAP-negative and occasional cells stained colocalized with stem cell marker nestin. These findings support the view that GCD may be a phenomenon related to increased progenitor cell proliferation in patients with hippocampal damage and chronic epilepsy.

Transgenic mice expressing F3/contactin from the TAG-1 promoter exhibit developmentally regulated changes in the differentiation of cerebellar neurons.

F3/contactin (CNTN1) and TAG-1 (CNTN2) are closely related axonal glycoproteins that are differentially regulated during development. In the cerebellar cortex TAG-1 is expressed first as granule cell progenitors differentiate in the premigratory zone of the external germinal layer. However, as these cells begin radial migration, TAG-1 is replaced by F3/contactin. To address the significance of this differential regulation, we have generated transgenic mice in which F3/contactin expression is driven by TAG-1 gene regulatory sequences, which results in premature expression of F3/contactin in granule cells. These animals (TAG/F3 mice) display a developmentally regulated cerebellar phenotype in which the size of the cerebellum is markedly reduced during the first two postnatal weeks but subsequently recovers. This is due in part to a reduction in the number of granule cells, most evident in the external germinal layer at postnatal day 3 and in the inner granular layer between postnatal days 8 and 11. The reduction in granule cell number is accompanied by a decrease in precursor granule cell proliferation at postnatal day 3, followed by an increase in the number of cycling cells at postnatal day 8. In the same developmental window the size of the molecular layer is markedly reduced and Purkinje cell dendrites fail to elaborate normally. These data are consistent with a model in which deployment of F3/contactin on granule cells affects proliferation and differentiation of these neurons as well as the differentiation of their synaptic partners, the Purkinje cells. Together, these findings indicate that precise spatio-temporal regulation of TAG-1 and F3/contactin expression is critical for normal cerebellar morphogenesis.

Thymidylate synthase protein expression and activity are related to the cell proliferation rate in human cancer cell lines

Aims: To ascertain whether the expression and enzyme activity of thymidylate synthase (TS) are related to the rapidity of cell proliferation in human cancer cell lines. Methods: Ten asynchronously growing...

Characterization of autonomous thyroid adenoma: metabolism, gene expression, and pathology.

Fifty-one in vivo characterized autonomous single adenomas have been studied for functional parameters in vitro, for gene and protein expression and for pathology, and have been systematically compared to the corresponding extratumoral quiescent tissue. The adenomas were characterized by a high level of iodide trapping that corresponds to a high level of Na+ /iodide symporter gene expression, a high thyroperoxidase mRNA and protein content, and a low H2O2 generation. This explains the iodide metabolism characteristics demonstrated before, ie, the main cause of the "hot" character of the adenomas is their increased iodide transport. The adenomas spontaneously secreted higher amounts of thyroid hormone than the quiescent tissue and in agreement with previous in vivo data, this secretion could be further enhanced by thyrotropin (TSH). Inositol uptake was also increased but there was no spontaneous increase of the generation of inositol phosphates and this metabolism could be further activated by TSH. These positive responses to TSH are in agreement with the properties of TSH-stimulated thyroid cells in vitro and in vivo. They are compatible with the characteristics of mutated TSH receptors whose constitutive activation accounts for the majority of autonomous thyroid adenomas in Europe. The number of cycling cells, as evaluated by MIB-1 immunolabeling was low but increased in comparison with the corresponding quiescent tissue or normal tissue. The cycling cells are observed mainly at the periphery; there was very little apoptosis. Both findings account for the slow growth of these established adenomas. On the other hand, by thyroperoxidase immunohistochemistry, the whole lesion appeared hyperfunctional, which demonstrates a dissociation of mitogenic and functional stimulations. Thyroglobulin, TSH receptor, and E-cadherin mRNA accumulations were not modified in a consistent way, which confirms the near-constitutive expression of the corresponding genes in normal differentiated tissue. On the contrary, early immediate genes expressions (c-myc, NGF1B, egr 1, genes of the fos and jun families) were decreased. This may be explained by the proliferative heterogeneity of the lesion and the previously described short, biphasic expression of these genes when induced by mitogenic agents. All the characteristics of the autonomous adenomas can therefore be explained by the effect of the known activating mutations of genes coding for proteins of the TSH cyclic adenosine monophosphate (cAMP) cascade, all cells being functionally activated while only those at the periphery multiply. The reason of this heterogeneity is unknown.

Two-Month Glucocorticoid Treatment Increases Proliferation in the Stomach and Large Intestine of Rats

Background/Aims: It has been shown that acute or short-term treatments with glucocorticoids lead to a marked decrease in proliferation in the stomach and large intestine. The effects of more prolonged glucocorticoid treatment on cell renewal in these organs are not known. The present work was therefore undertaken to examine the proliferative activity in the stomach and colon during 2 months of glucocorticoid treatment in comparison with shorter treatments. Methods: Rats were treated with either the glucocorticoid triamcinolone acetonide or vehicle for 63, 33 or 3 days. Proliferation was assessed in the glandular epithelium of the fundal part of the stomach and in the epithelium of the colonic crypts using three criteria: the mitotic index; the bromodeoxyuridinelabelling index, and the proliferating cell nuclear antigen-labelling index (percentage of mitotic or labelled cells). Results: Treatment with glucocorticoid for 63 days resulted in a very significant increase in all proliferative parameters tested in the gastric mucosa and the colonic crypts. On the contrary, treatments with glucocorticoid for 3 or 33 days had a marked inhibitory influence on proliferation in these tissues. Conclusion: As opposed to treatments for 3 or 33 days, glucocorticoid treatment for 2 months leads to an increase in the number of cycling cells in the gastric and colonic mucosae.

Elevations of DNA topoisomerase I in transitional cell carcinoma of the urinary bladder: Correlation with DNA topoisomerase II-alpha and p53 expression

DNA topoisomerase I (topo I) is the molecular target of the camptothecin group of antitumor drugs. Laboratory studies have indicated that cells sensitive to these drugs contain elevated levels of topo I. In this study, we immunostained 49 cases of transitional cell carcinoma from the urinary bladder with a monoclonal antibody directed against human topo I. We found elevated expression of the enzyme in 77% (38 of 49). This included three of six grade 1 tumors (50%), 9 of 15 grade II tumors (60%), 14 of 15 grade III tumors (93%) and 12 of 13 grade IV tumors (92%). Because the number of cycling cells in a tumor also may be an important determinant of topo I drug response, a proliferation index (topo II-alpha) also was performed for each case. The average topo II-alpha index of grade I tumors was 7.5 × 3.8; for grade II tumors, 20.1 ± 10.5; for grade III tumors, 40.3 × 8.2; and for grade IV tumors, 50.5 ± 13.0. Because a functional p53 tumor suppressor gene may be necessary for anticancer drug response, we also evaluated our cases for alteration in p53 function. Mutations in the p53 tumor suppressor gene, estimated by immunohistochemical staining, were common, occurring in 23 of 49 cases (47%). The number of cases with elevated topo I, a large growth fraction, and a functional p53 tumor suppressor gene was 4 of 49 (8%). Our results suggest that a small population of patients with transitional cell carcinoma of the urinary bladder may have tumors with molecular features suggesting responsiveness to the new anticancer drugs targeting topo I.