Surface molecules regulating rolling and adhesion to endothelium of neutrophil granulocytes and MDA-MB-468 breast carcinoma cells and their interaction

The extravasation of leukocytes and tumor cells is a multi-step process with the involvement of various adhesion molecules depending on the three steps rolling, adhesion, and diapedesis. We have developed an in vitro model, by which we investigated the rolling and adhesion of neutrophil granulocytes and MDA-MB-468 human breast carcinoma cells to lung endothelial cells under physiological flow-conditions. We found that norepinephrine had an inhibitory function on the fMLP-promoted adhesion of neutrophil granulocytes due to a down-regulation of β2-integrin. Furthermore, neutrophil granulocytes serve as linking cells for the interaction of the MDA-MB-468 cells with the endothelium, which are both β2-integrin negative, but express the β2-integrin ligand ICAM-1. In addition, we show here that N-cadherin is up-regulated on the endothelial cells and on neutrophil granulocytes in response to fMLP. This up-regulation resulted in a significant increase of adherent MDA-MB-468 cells, which are also N-cadherin positive. Received 3 September 2007; received after revision 17 October 2007; accepted 22 October 2007     

UDP-glucose ceramide glucosyltransferase activates AKT,promoted proliferation,and doxorubicin resistance in breast cancer cells

The UDP-glucose ceramide glucosyltransferase (UGCG) is a key enzyme in the synthesis of glycosylated sphingolipids, since this enzyme generates the precursor for all complex glycosphingolipids (GSL), the GlcCer. The UGCG has been associated with several cancer-related processes such as maintaining cancer stem cell properties or multidrug resistance induction. The precise mechanisms underlying these processes are unknown. Here, we investigated the molecular mechanisms occurring after UGCG overexpression in breast cancer cells. We observed alterations of several cellular properties such as morphological changes, which enhanced proliferation and doxorubicin resistance in UGCG overexpressing MCF-7 cells. These cellular effects seem to be mediated by an altered composition of glycosphingolipid-enriched microdomains (GEMs), especially an accumulation of globotriaosylceramide (Gb3) and glucosylceramide (GlcCer), which leads to an activation of Akt and ERK1/2. The induction of the Akt and ERK1/2 signaling pathway results in an increased gene expression of multidrug resistance protein 1 (MDR1) and anti-apoptotic genes and a decrease of pro-apoptotic gene expression. Inhibition of the protein kinase C (PKC) and phosphoinositide 3 kinase (PI3K) reduced MDR1 gene expression. This study discloses how changes in UGCG expression impact several cellular signaling pathways in breast cancer cells resulting in enhanced proliferation and multidrug resistance.     

Myosin II in mechanotransduction: master and commander of cell migration, morphogenesis, and cancer

Mechanotransduction encompasses the role of mechanical forces in controlling cell behavior by activating signal transduction pathways. Most forces at a cellular level are caused by myosin II, which contracts and cross-links actin. Myosin II-dependent forces are transmitted through the actin cytoskeleton to molecular endpoints that promote specific cellular outcomes, e.g., cell proliferation, adhesion, or migration. For example, most adhesive and migratory phenomena are mechanically linked by a molecular clutch comprised of mechanosensitive scaffolds. Myosin II activation and mechanosensitive molecular mechanisms are finely tuned and spatiotemporally integrated to coordinate morphogenetic events during development. Mechanical events dependent on myosin II also participate in tumor cell proliferation, invasion, and metastatic dissemination. Specifically, tumor cells alter the mechanical properties of the microenvironment to create favorable conditions for proliferation and/or dissemination. These observations position myosin II-dependent force generation and mechanotransduction at the crossroads between normal development and cancer.     

TRAIL promotes the survival,migration and proliferation of vascular smooth muscle cells

Human and rat primary sub-cultured vascular smooth muscle cells (VSMCs) showed clear expression of the death receptors TRAIL-R1 and TRAIL-R2; however, recombinant soluble TRAIL did not induce cell death when added to these cells. TRAIL tended to protect rat VSMCs from apoptosis induced either by inflammatory cytokines tumor necrosis factor- + interleukin-1 + interferon- or by prolonged serum withdrawal, and promoted a significant increase in VSMC proliferation and migration. Of note, all the biological effects induced by TRAIL were significantly inhibited by pharmacological inhibitors of the ERK pathway. Western blot analysis consistently showed that TRAIL induced a significant activation of ERK1/2, and a much weaker phosphorylation of Akt, while it did not affect the p38/MAPK pathway. Taken together, these data strengthen the notion that the TRAIL/TRAIL-R system likely plays a role in the biology of the vascular system by affecting the survival, migration and proliferation of VSMCs.Received 6 May 2004; received after revision 7 June 2004; accepted 8 June 2004     

Influence of ethanol on calcium, inositol phospholipids and intracellular signalling mechanisms

Studies have implicated Ca++ in the actions of ethanol at many biochemical levels. Calcium as a major intracellular messenger in the central nervous system is involved in many processes, including protein phosphorylation enzyme activation and secretion of hormones and neurotransmitters. The control of intracellular calcium, therefore, represents a major step by which neuronal cells regulate their activities. The present review focuses on three primary areas which influence intracellular calcium levels; voltage-dependent Ca++ channels, receptor-mediated inositol phospholipid hydrolysis, and Ca++/Mg++-ATPase, the high affinity membrane Ca++ pump. Current research suggests that a subtype of the voltage-dependent Ca++ channel, the dihydropyridine-sensitive Ca++ channel, is uniquely sensitive to acute and chronic ethanol treatment. Acute exposure inhibits, while chronic ethanol exposure increases 45Ca++-influx and [3H]dihydropyridine receptor binding sites. In addition, acute and chronic exposure to ethanol inhibits, then increases Ca++/Mg++-ATPase activity in neuronal membranes. Changes in Ca++ channel and Ca++/Mg++-ATPase activity following chronic ethanol may occur as an adaptation process to increase Ca++ availability for intracellular processes. Since receptor-dependent inositol phospholipid hydrolysis is enhanced after chronic ethanol treatment, subsequent activation of protein kinase-C may also be involved in the adaptation process and may indicate increased coupling for receptor-dependent changes in Ca++/Mg++-ATPase activity. The increased sensitivity of three Ca++-dependent processes suggest that adaptation to chronic ethanol exposure may involve coupling of one or more of these processes to receptor-mediated events.     

Influence of ethanol on calcium,inositol phospholipids and intracellular signalling mechanisms

Summary Studies have implicated Ca⁺⁺ in the actions of ethanol at many biochemical levels. Calcium as a major intracellular messenger in the central nervous system is involved in many processes, including protein phosphorylation enzyme activation and secretion of hormones and neurotransmitters. The control of intracellular calcium, therefore, represents a major step by which neuronal cells regulate their activities. The present review focuses on three primary areas which influence intracellular calcium levels; voltage-dependent Ca⁺⁺ channels, receptor-mediated inositol phospholipid hydrolysis, and Ca⁺⁺/Mg⁺⁺-ATPase, the high affinity membrane Ca⁺⁺ pump.Current research suggests that a subtype of the voltage-dependent Ca⁺⁺ channel, the dihydropyridine-sensitive Ca⁺⁺ channel, is uniquely sensitive to acute and chronic ethanol treatment. Acute exposure inhibits, while chronic ethanol exposure increases⁴⁵Ca⁺⁺-influx and [³H]dihydropyridine receptor binding sites. In addition, acute and chronic exposure to ethanol inhibits, then increases Ca⁺⁺/Mg⁺⁺-ATPase activity in neuronal membranes. Changes in Ca⁺⁺ channel and Ca⁺⁺/Mg⁺⁺-ATPase activity following chronic ethanol may occur as an adaptation process to increase Ca⁺⁺ availability for intracellular processes. Since receptor-dependent inositol phospholipid hydrolysis is enhanced after chronic ethanol treatment, subsequent activation of protein kinase-C may also be involved in the adaptation process and may indicate increased coupling for receptor-dependent changes in Ca⁺⁺/Mg⁺⁺-ATPase activity.The increased sensitivity of three Ca⁺⁺-dependent processes suggest that adaptation to chronic ethanol exposure may involve coupling of one or more of these processes to receptor-mediated events.     

Emerging mechanisms and consequences of calcium regulation of alternative splicing in neurons and endocrine cells

Alternative splicing contributes greatly to proteomic complexity. How it is regulated by external stimuli to sculpt cellular properties, particularly the highly diverse and malleable neuronal properties, is an underdeveloped area of emerging interest. A number of recent studies in neurons and endocrine cells have begun to shed light on its regulation by calcium signals. Some mechanisms include changes in the trans-acting splicing factors by phosphorylation, protein level, alternative pre-mRNA splicing, and nucleocytoplasmic redistribution of proteins to alter protein–RNA or protein–protein interactions, as well as modulation of chromatin states. Importantly, functional analyses of the control of specific exons/splicing factors in the brain point to a crucial role of this regulation in synaptic maturation, maintenance, and transmission. Furthermore, its deregulation has been implicated in the pathogenesis of neurological disorders, particularly epilepsy/seizure. Together, these studies have not only provided mechanistic insights into the regulation of alternative splicing by calcium signaling but also demonstrated its impact on neuron differentiation, function, and disease. This may also help our understanding of similar regulations in other types of cells.     

Novel actions of tyrphostin AG 879: inhibition of RAF-1 and HER-2 expression combined with strong antitumoral effects on breast cancer cells

Binding of growth factors to cell surface receptors activates protein tyrosine kinases (PTKs) that initiate cascades of downstream signaling events including the mitogen-activated protein (MAP) kinase cascade. This study reports that the PTK inhibitor AG 879 inhibits proliferation of human breast cancer cells through an effect involving inhibition of MAP kinase activation, but which cannot be explained by effects of AG 879 on its known PTK targets. Instead, AG 879 markedly inhibits expression of the RAF-1 gene, which encodes an upstream MAP kinase kinase kinase. Additionally, expression of HER-2, but not of other genes tested, is inhibited by this compound. These novel effects have to be considered when using AG 879 as a TRK-A and HER-2 inhibitor but may have useful therapeutic implications.     

Mitochondria dynamism: of shape,transport and cell migration

Mitochondria are highly dynamic and functionally versatile organelles that continuously fragment and fuse in response to different physiological needs of the cell. The list of proteins that strictly regulate the morphology of these organelles is constantly growing, adding new players every day and new pieces to the comprehension and elucidation of this complex machinery. The structural complexity of mitochondria is only paralled by their functional versatility. Indeed, changes in mitochondria shape play critical roles in vertebrate development programmed cell death and in various processes of normal cell physiology, such as calcium signaling, reactive oxygen species production, and lifespan. Here, we present the latest findings on the regulation of mitochondrial dynamics and some of their physiological roles, focusing on cell migration. In cells where migration represents a crucial function in their physiology, such as T and tumoral metastatic cells, mitochondria need to be fragmented and recruited to specific subcellular regions to make movement possible. In depth analysis of this role of mitochondrial dynamics should help in identifying potential targeted therapy against cancer or in improving the immune system’s efficiency.     

Melatonin and cell death: differential actions on apoptosis in normal and cancer cells

Melatonin is a natural compound synthesized by a variety of organs. It has been shown to function as a cell-protective agent. Since 1994, when the first paper was published documenting the role of melatonin in apoptosis, the number of reports in this area has increased rapidly. Much of the research conducted falls into three major categories: first, the role of melatonin in inhibiting apoptosis in immune cells; second, the role of melatonin in preventing neuronal apoptosis and finally, the role of melatonin in increasing apoptotic cell death in cancer cells. The mechanisms whereby melatonin influences apoptosis have not clarified, although a number of mechanistic options have been suggested. Apoptotic cell death is a physiological phenomenon related to homeostasis and proper functioning of tissues and organs; however, a failure in the apoptotic program is related to a number of diseases. The participation of melatonin in apoptosis in numerous cell types and its potential importance in a variety of diseases such as immunodeficiency, neurodegeneration and cancer is summarized in this review.Received 14 November 2002; received after revision 16 January 2003; accepted 10 February 2003     

Lack of relationship between Langerhans cells,epidermal cell proliferation and epidermal G1 chalone

Summary Mouse tail interscale epidermis contains numerous Langerhans cells, whereas the adjacent scale regions are devoid of these cells. No difference in a) proliferative activity and b) inhibitory effect of the epidermal G1 chalone can be demonstrated in both regions. A direct relationship between Langerhans cells and growth control may be excluded.     

Tightrope act: autophagy in stem cell renewal, differentiation, proliferation, and aging

Autophagy is a constitutive lysosomal catabolic pathway that degrades damaged organelles and protein aggregates. Stem cells are characterized by self-renewal, pluripotency, and quiescence; their long life span, limited capacity to dilute cellular waste and spent organelles due to quiescence, along with their requirement for remodeling in order to differentiate, all suggest that they require autophagy more than other cell types. Here, we review the current literature on the role of autophagy in embryonic and adult stem cells, including hematopoietic, mesenchymal, and neuronal stem cells, highlighting the diverse and contrasting roles autophagy plays in their biology. Furthermore, we review the few studies on stem cells, lysosomal activity, and autophagy. Novel techniques to detect autophagy in primary cells are required to study autophagy in different stem cell types. These will help to elucidate the importance of autophagy in stem cells during transplantation, a promising therapeutic approach for many diseases.     

Lack of relationship between Langerhans cells, epidermal cell proliferation and epidermal G1 chalone.

Mouse tail interscale epidermis contains numerous Langerhans cells, whereas the adjacent scale regions are devoid of these cells. No difference in a) proliferative activity and b) inhibitory effect of the epidermal G1 chalone can be demonstrated in both regions. A direct relationship between Langerhans cells and growth control may be excluded.     

Differentiation antigens in keratinocytes II: cytoplasmic specificity expressed in the basal cell of the external root sheath of hairs but not in the basal cells of epidermis]

By means of indirect immunofluorescence with sera obtained from patients after bone marrow transplantation it has been demonstrated that antibodies were reacting with the cytoplasm of basal cells ((C.B.C.) in the external root sheath of hairs (E.R.S.). Some sera were found to react with C.B.C. of the E.R.S. but not with the C.B.C of the epidermis. The change in the antigenic properties of the basal cells was found to be situated at the orifice of the sebaceous gland duct.     

Killing cancer by targeting genes that cancer cells have lost: allele-specific inhibition,a novel approach to the treatment of genetic disorders

Oligonucleotide-based drugs are now rapidly establishing themselves as an important tool in both research and treatment of genetic disorders. In the past many problems were encountered in using antisense oligonucleotides. Our expanding knowledge and new oligonucleotide chemistries are giving us the chance to treat serious genetic disorders such as cancer in novel, elegant and effective ways not previously possible. In addition, recent knowledge about RNA interference may add to these new approaches for disease treatment with oligonucleotide-based drugs. In this review we discuss one such novel therapeutic strategy against cancer called allele-specific inhibition (ASI). ASI is an approach where cancer cells are attacked at one of the few widely occurring and consistently weak points: the loss of large segments of DNA. Oligonucleotide-based drugs may provide the required selectivity for this therapeutic approach.     

Pharmacological studies on a plant lectin,Aloctin A. I. Growth inhibition of mouse methylcholanthrene-induced fibrosarcoma (Meth A) in ascites form by Aloctin A

Summary A glycoprotein isolated fromAloe arborescens Mill markedly inhibited the growth of a syngeneic transplantable fibrosarcoma of mice, Meth A, in ascites form. There is evidence that the inhibition mechanism is host-mediated and not a direct toxic effect on the tumor cell.Acknowledgment. We thank Miss M. Hayama for her technical help.To whom all correspondence should be addressed.