Low-expression of E-cadherin in leukaemia cells causes loss of homophilic adhesion and promotes cell growth

E-cadherin (epithelial cadherin) belongs to the calcium-dependent adhesion molecule superfamily and is implicated in the interactions of haematopoietic progenitors and bone marrow stromal cells. Adhesion capacity to bone marrow stroma was impaired for leukaemia cells, suggesting that a breakdown of adhesive mechanisms governed by an adhesion molecule may exist in leukaemic microenvironment. We previously found that E-cadherin was low expressed in primary acute leukaemia cells compared with normal bone marrow mononuclear cells. In this study, we investigate the functional importance of low E-cadherin expression in leukaemia cell behaviours and investigate its effects in the abnormal interaction of leukaemic cells with stromal cells. After expression of E-cadherin was restored by a demethylating agent in leukaemia cells, E-cadherin-specific adhesion was enhanced. Additionally, siRNA (small interfering RNA)-mediated silencing of E-cadherin in Raji cells resulted in a reduction of cell homophilic adhesion and enhancement of cell proliferation and colony formation. These results suggest that low expression of E-cadherin contributes to the vigorous growth and transforming ability of leukaemic cells.     

Relationship between growth arrest and autophagy in midgut programmed cell death in Drosophila

Autophagy has been implicated in both cell survival and programmed cell death (PCD), and this may explain the apparently complex role of this catabolic process in tumourigenesis. Our previous studies have shown that caspases have little influence on Drosophila larval midgut PCD, whereas inhibition of autophagy severely delays midgut removal. To assess upstream signals that regulate autophagy and larval midgut degradation, we have examined the requirement of growth signalling pathways. Inhibition of the class I phosphoinositide-3-kinase (PI3K) pathway prevents midgut growth, whereas ectopic PI3K and Ras signalling results in larger cells with decreased autophagy and delayed midgut degradation. Furthermore, premature induction of autophagy is sufficient to induce early midgut degradation. These data indicate that autophagy and the growth regulatory pathways have an important relationship during midgut PCD. Despite the roles of autophagy in both survival and death, our findings suggest that autophagy induction occurs in response to similar signals in both scenarios.     

The Arabidopsis aberrant growth and death2 mutant shows resistance to Pseudomonas syringae and reveals a role for NPR1 in suppressing hypersensitive cell death

A novel Arabidopsis mutant has been identified with constitutive expression of GST1-GUS using plants with a pathogen-responsive reporter transgene containing the beta-glucuronidase (GUS) coding region driven by the GST1 promoter. The recessive mutant, called agd2 (aberrant growth and death2), has salicylic acid (SA)-dependent increased resistance to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae, elevated SA levels, a low level of spontaneous cell death, callose deposition, and enlarged cells in leaves. The enhanced resistance of agd2 to virulent P. syringae requires the SA signaling component NONEXPRESSOR OF PR1 (NPR1). However, agd2 renders the resistance response to P. syringae carrying avrRpt2 NPR1-independent. Thus agd2 affects both an SA- and NPR1-dependent general defense pathway and an SA-dependent, NPR1-independent pathway that is active during the recognition of avirulent P. syringae. agd2 plants also fail to show a hypersensitive cell death response (HR) unless NPR1 is removed. This novel function for NPR1 is also apparent in otherwise wild-type plants: npr1 mutants show a stronger HR, while NPR1-overproducing plants show a weaker HR when infected with P. syringae carrying the avrRpm1 gene. Spontaneous cell death in agd2 is partially suppressed by npr1, indicating that NPR1 can suppress or enhance cell death depending on the cellular context. agd2 plants depleted of SA show a dramatic exacerbation of the cell-growth phenotype and increased callose deposition, suggesting a role for SA in regulating growth and this cell-wall modification. AGD2 may function in cell death and/or growth control as well as the defense response, similarly to what has been described in animals for the functions of NFkappaB.     

Partial loss of Ascl2 function affects all three layers of the mature placenta and causes intrauterine growth restriction

Several imprinted genes have been implicated in the regulation of placental function and embryonic growth. On distal mouse chromosome 7, two clusters of imprinted genes, each regulated by its own imprinting center (IC), are separated by a poorly characterized region of 280 kb (the IC1–IC2 interval). We previously generated a mouse line in which this IC1–IC2 interval has been deleted (Del^7AI allele) and found that maternal inheritance of this allele results in low birth weights in newborns. Here we report that Del^7AI causes a partial loss of Ascl2, a maternally expressed gene in the IC2 cluster, which when knocked out leads to embryonic lethality at midgestation due to a lack of spongiotrophoblast formation. The hypomorphic Ascl2 allele causes embryonic growth restriction and an associated placental phenotype characterized by a reduction in placental weight, reduced spongiotrophoblast population, absence of glycogen cells, and an expanded trophoblast giant cell layer. We also uncovered severe defects in the labyrinth layer of maternal mutants including increased production of the trilaminar labyrinth trophoblast cell types and a disorganized labyrinthine vasculature. Our results have important implications for our understanding of the role played by the spongiotrophoblast layer during placentation and show that regulation of the dosage of the imprinted gene Ascl2 can affect all three layers of the chorio-allantoic placenta.     

Bcl-2 inhibits cell death of serum-free mouse embryo cells caused by epidermal growth factor deprivation

SFME cells are brain-derived neural precursor cells that are acutely dependent on epidermal growth factor (EGF) for survival, undergoing apoptosis within 24 h after EGF withdrawal. Because the expression of the protooncogene bcl-2 inhibits apoptosis induced by the withdrawal of interleukins or nerve growth factor in some growth factor-dependent haematopoietic or neuronal cell cultures, we examined the effect of Bcl-2 expression on cell death of SFME cells in the absence of EGF. SFME cells expressing human Bcl-2 showed prolonged survival when deprived of EGF compared to control cells not expressing Bcl-2. A significant fraction of Bcl-2-expressing cells remained viable for 4 days in the absence of EGF and resumed proliferation upon readdition of EGF to the cultures. These results suggest that apoptosis induced by EGF withdrawal in SFME cells may share common mechanisms with other growth factor-related apoptotic systems.     

CASK inhibits ECV304 cell growth and interacts with Id1

Calcium/calmodulin-dependent serine protein kinase (CASK) is generally known as a scaffold protein. Here we show that overexpression of CASK resulted in a reduced rate of cell growth, while inhibition of expression of endogenous CASK via RNA-mediated interference resulted in an increased rate of cell growth in ECV304 cells. To explore the molecular mechanism, we identified a novel CASK-interacting protein, inhibitor of differentiation 1 (Id1) with a yeast two-hybrid screening. Furthermore, endogenous CASK and Id1 proteins were co-precipitated from the lysates of ECV304 cells by immunoprecipitation. Mammalian two-hybrid protein-protein interaction assays indicated that CASK possessed a different binding activity for Id1 and its alternative splicing variant. It is known that Id proteins play important roles in regulation of cell proliferation and differentiation. Thus, we speculate that the regulation of cell growth mediated by CASK may be involved in Id1. Our findings indicate a novel function of CASK, the mechanism that remains to be further investigated.     

Overexpression of Bcl-2 or Bcl-xL prevents spiral ganglion neuron death and inhibits neurite growth

Spiral ganglion neurons (SGNs) provide afferent innervation to the cochlea and rely on contact with hair cells (HCs) for their survival. Following deafferentation due to hair cell loss, SGNs gradually die. In a rat culture model, we explored the ability of prosurvival members of the Bcl-2 family of proteins to support the survival and neurite outgrowth of SGNs. We found that overexpression of either Bcl-2 or Bcl-xL significantly increases SGN survival in the absence of neurotrophic factors, establishing that the Bcl-2 pathway is sufficient for SGN cell survival and that SGN deprived of trophic support die by an apoptotic mechanism. However, in contrast to observations in central neurons and PC12 cells where Bcl-2 appears to promote neurite growth, both Bcl-2 and Bcl-xL overexpression dramatically inhibit neurite outgrowth in SGNs. This inhibition of neurite growth by Bcl-2 occurs in nearly all SGNs even in the presence of multiple neurotrophic factors implying that Bcl-2 directly inhibits neurite growth rather than simply rescuing a subpopulation of neurons incapable of extending neurites without additional stimuli. Thus, although overexpression of prosurvival members of the Bcl-2 family prevents SGN loss following trophic factor deprivation, the inhibition of neurite growth by these molecules may limit their efficacy for support of auditory nerve maintenance or regeneration following hair cell loss.     

Calmodulin-dependent and -independent apoptosis in cells of a Drosophila neuronal cell line

This study was undertaken to reveal apoptotic pathways in neurons using a Drosophila neuronal cell line derived from larval central nervous system. We could induce apoptotic cell death in the cells by a Ca²⁺ ionophore (A23187), a protein kinase inhibitor (H-7), an RNA synthesis inhibitor (actinomycin D) and a protein synthesis inhibitor (cycloheximide). All the apoptosis induced by each chemical required Ca²⁺ ions, although the origin of Ca²⁺ ions were different: apoptosis induced by A23187 was dependent on extracellular Ca²⁺ ions whereas those by the other three chemicals utilized intracellular Ca²⁺ ions. Furthermore, different reactions to W-7, a calmodulin inhibitor, were found: W-7 prevented the cell death by each of the three chemicals but not by A23187. Based on the results, we proposed that the apoptotic pathways are classified into two types in individual cells. One pathway induced by H-7, actinomycin D or cycloheximide is calmodulin-dependent (pathway H), and another induced by A23187 is calmodulin-independent (pathway A).     

Upf1 stimulates degradation of the product derived from aberrant messenger RNA containing a specific nonsense mutation by the proteasome

Aberrant messenger RNAs containing a premature termination codon (PTC) are eliminated by the nonsense‐mediated mRNA decay (NMD) pathway. Here, we show that a crucial NMD factor, up frameshift 1 protein (Upf1), is required for rapid proteasome‐mediated degradation of an aberrant protein (PTC product) derived from a PTC‐containing mRNA. Western blot and pulse–chase analyses revealed that Upf1 stimulates the degradation of specific PTC products by the proteasome. Moreover, the Upf1‐dependent, proteasome‐mediated degradation of the PTC product was also stimulated by mRNAs harbouring a faux 3′ untranslated region (3′‐UTR). These results indicate that protein stability might be regulated by an aberrant mRNA 3′‐UTR.     

Suppression of PTP1B in gastric cancer cells in vitro induces a change in the genome‐wide expression profile and inhibits gastric cancer cell growth

PTP1B (protein tyrosine phosphatase 1B) is a member of the superfamily of PTPs (protein tyrosine phosphatases) and has been implicated in cancer pathogenesis. However, the role of PTP1B in gastric cancer is still unknown. Here, we first detected the PTP1B expression in six gastric cancer cell lines and in the immortalized gastric mucosal epithelial cell line GES‐1 by RT‐PCR and Western blot. Then, we measured the change of the genome‐wide expression profile in MKN28 gastric cancer cells transfected with a plasmid expressing PTP1B‐specific small interfering RNA by microarray analysis. Our results showed that PTP1B was overexpressed in gastric cancer cells, and inhibition of PTP1B expression dramatically inhibited gastric cancer cell growth in vitro and in vivo. In addition, microarray analysis revealed that inhibition of PTP1B induced changes in the genome‐wide expression profile. These changes may be related to cell growth. Taken together, our data suggested that PTP1B may be a candidate oncogene in gastric cancer.     

Caveolin-3 inhibits growth signal in cardiac myoblasts in a Ca2+-dependent manner

Caveolin, a major protein component of caveolae, directly interacts with multiple signaling molecules, such as Ras and growth factor receptors, and inhibits their function. However, the role of the second messenger system in mediating this inhibition by caveolin remains poorly understood. We examined the role of Ca2+-dependent signal in caveolin- mediated growth inhibition using a rat cardiac myoblast cell line (H9C2), in which the expression of caveolin- 3, the muscle specific subtype, can be induced using the LacSwitch system. Upon induction with IPTG and serum-starvation, the expression of caveolin-3 was increased by 3.3-fold relative to that of mock-induced cells. The recombinant caveolin-3 was localized to the same subcellular fraction as endogenous caveolin-3 after sucrose gradient purification. Angiotensin II enhanced ERK phosphorylation, but this enhancement was significantly decreased in caveolin-3-induced cells in comparison to that in mock-induced cells. Similarly, when cells were stimulated with fetal calf serum, DNA synthesis, as determined by [3H]-thymidine incorporation, was significantly decreased in caveolin- 3-induced cells. When cells were treated with Ca2+ chelator (BAPTA and EGTA), however, this attenuation was blunted. Calphostin (PKC inhibitor), but not cyclosporine A treatment (calcineurin inhibitor), blunted this attenuation in caveolin-3 induced cells. Our findings suggest that caveolin exhibits growth inhibition in a Ca2+-dependent manner, most likely through PKC, in cardiac myoblasts.     

NUSAP1 knockdown inhibits cell growth and metastasis of non-small-cell lung cancer through regulating BTG2/PI3K/Akt signaling

Non-small-cell lung cancer (NSCLC) is the most common malignancy along with high mortality rate worldwide. Recently, nucleolar and spindle-associated protein 1 (NUSAP1) has been reported to be involved in the malignant progression of several cancers. However, in NSCLC, the biological function of NUSAP1 and its molecular mechanism have not been reported. Here, our findings indicated that the NUSAP1 messenger RNA expression level was remarkably upregulated in NSCLC tissues compared with that of adjacent normal tissues. We also found that NUSAP1 gene expression was notably upregulated in NSCLC cell lines (A549, 95-D, H358, and H1299) compared with that of normal human bronchial epithelial cell line (16HBE). Subsequently, the biological function of NUSAP1 was investigated in A549 and H358 cells transfected with NUSAP1 small interfering RNA (siRNA), respectively. Results showed that NUSAP1 knockdown inhibited NSCLC cell proliferation, and promoted cell apoptosis. Furthermore, the number of cell migration and invasion was significantly suppressed by NUSAP1 knockdown. In addition, our results indicated that NUSAP1 knockdown increased the gene expression of B-cell translocation gene 2 (BTG2), but decreased the expression levels of phosphoinositide 3-kinase (PI3K) and phosphorylated serine/threonine kinase (p-AKT). BTG2 siRNA partly abrogates the effect of NUSAP1 knockdown on BTG2 gene expression. Fumonisin B1 (FB1), a AKT activator, reversed the effect of NUSAP1 knockdown on the biological function in NSCLC. Taken together, NUSAP1 knockdown promotes NSCLC cell apoptosis, and inhibits cell proliferation, cell migration, and invasion, which is associated with regulating BTG2/PI3K/Akt signal pathway. Our findings suggest that NUSAP1 is a promising molecular target for NSCLC treatment.     

Cell surface expression of 4β-galactosyltransferase accompanies rat parotid gland acinar cell transition to growth

Rat parotid gland acinar cells stimulated to divide by a chronic regimen of isoproterenol demonstrate a dramatic increase in the synthesis of the glycosyltransferase 4β-galactosyltransferase. A plasma membrane localization for much of the increase in 4β-galactosyltransferase was determined by density gradient membrane fractionation. Golgi-enriched fractions showed no increase in specific activity, while plasma membrane activity increased 40-fold. This selective increase at the cell surface was confirmed by immunofluorescence of intact, nonpermeabilized cells from treated glands, using a monospecific antibody prepared against the purified bovine milk transferase. In detergent-permeabilized cells staining of nontreated cells was seen only as groups of perinuclear vesicles, presumed to be Golgi apparatus. In isoproterenol-treated and permcabilized cells both presumptive Golgi and cell surface staining was apparent. Enzyme assays performed on intact cells established that the enzyme's active site was oriented to the exterior of the cells. The transferase could be detected as early as 3 hr after the primary challenge with isoproterenol. Pretrcatment of rats with cycloheximide prevented its appearance.     

Cell-cell contact and growth regulation of pinocytosis in 3T3 cells

In sub-confluent cultures of Balb/c-3T3 cells, pinocytosis rates were increased after exposure to specific growth factors (serum; platelet-derived growth factor, PDGF; epidermal growth factor, EGF). Conversely, as cells became growth-inhibited with increasing culture density, there was a corresponding decline in pinocytosis rate per cell. In order to test whether density-inhibition of pinocytosis was influenced either by the growth cycle or by cell contact independently of growth, cells were induced into a quiescent state at a range of subconfluent and confluent densities. Under such conditions, cell density did not significantly inhibit pinocytosis rate. When confluent quiescent cultures in 2.5% serum were exposed to 10% serum, the resulting round of DNA synthesis was accompanied by enhanced pinocytosis per cell, even though the cells were incontact with one another. Furthermore, in a SV40-viral transformed 3T3 cell line, both the growth fraction and the pinocytosis rate per cell remained unchanged over a wide range of culture densities. These studies indicate that density-dependent inhibition of pinocytosis in 3T3 cells appears to be secondary to growth-inhibition rather than to any direct physical effects of cell–cell contact.