Changes in the organization of membrane lipids during human platelet activation. Study by fluorescent and freeze-fracture cytochemistry

Modifications in the membrane lipid organization of human platelets activated with different agents (adenosine 5'-diphosphate, thrombin, collagen type I, and monosaccharides such as fucose, mannose, and galactose) were analyzed in vitro by using three lipid markers. Cholesterol was detected upon interaction with filipin, the anionic phospholipids were reacted with polymyxin B, and alterations in the degree of lipid packing were evaluated with the lipophilic fluorescent probe merocyanine 540, which reportedly inserts into bilayer domains whose lipids are more disordered. Filipin-sterol complexes and polymyxin B-anionic phospholipid complexes form characteristic membrane deformations which were examined in freeze-fracture preparations, whereas the merocyanine 540 binding to platelet membrane was recorded by fluorescent microscopy. In contrast to the resting cells, thrombin-stimulated platelets displayed an uneven distribution of filipin-sterol complexes which occurred in much higher density on the cell body than on pseudopods: on the latter, apparently cholesterol-free domains were very common. Unlike the non-stimulated cells, the platelets aggregated with the various agents employed showed characteristic polymixin B-anionic phospholipid complexes deformations of plasmalemma suggesting the appearance in uneven concentration of anionic phospholipids in the outer membrane leaflet. Incubation with merocyanine 540 did not result in staining of resting platelets when these were maintained in plasma, but a slight fluorescence was observed when platelets were kept in Tyrode buffer. However, platelets stimulated with thrombin, collagen type I, and monosaccharides bound very heavily the fluorescent dye; platelets aggregated with adenosine-5'-diphosphate bound only small amounts of merocyanine 540. The results showed that, during activation by different agents, modifications in lipid membrane organization include alterations in cholesterol and anionic phospholipid distribution, transbilayer movement of anionic phospholipids accompanied by more disordered membrane.     

The significance ofheregulin in breast cancer tumor progression and drug resistance

Summary TheerbB-2 receptor plays an important role in the prognosis of breast cancer and is expressed at high levels in nearly 30% of tumors in breast cancer patients. While evidence accumulates to support the relationship betweenerbB-2 overexpression and poor overall survival in human breast cancer, understanding of the biological consequence(s) oferbB-2 overexpression remains elusive. The discovery ofheregulin has allowed us to identify a number of related but distinct biological endpoints which appear responsive to signal transduction through theerbB-2/4 receptor. These endpoints of growth, invasiveness, and differentiation have clear implications for the emergence, maintenance, and/or control of malignancy, and represent established endpoints in the assessment of malignant progression in human breast cancer. Preliminary studiesin vitro have shown thatheregulin induces a biphasic growth effect on cells witherbB-2 overexpression. Interestingly, we observed that expression ofheregulin correlates with a more aggressive/invasive, vimentin-positive phenotype in breast cancer cells lines. Therefore, we have postulated thatheregulin is involved in breast cancer tumor progression. We have shown thatheregulin inducesin vitro chemoinvasion and chemotaxis of breast cancer cells as well as growth in an anchorage dependent and independent manner. Interestingly, aheregulin neutralizing antibody inhibits chemotaxis and results in cell growth inhibition and blockade of the invasive phenotype. Strikingly, genetically engineered cells which constitutively expressheregulin demonstrate critical phenotypic changes that are associated with a more aggressive phenotype. Specifically, these cells are no longer dependent on estrogen for growth and are resistant to tamoxifenin vitro andin vivo, and moreover these cells metastasize to lymph nodes in athymic nude mice. These tumors appear to have lostbcl-2 expression as compared with the control tumors. In addition, presumably by activation/regulation of topoisomerase II, theheregulin-transfected cells become exquisitely sensitive to doxorubicin and VP-16. Clearly, mechanistic aspects of theerbB-2/4 andheregulin interaction need to be understood from a therapeutic standpoint which could provide additional insights into synergistic treatments for certain patients, or improve treatment regimens for a large number of women. The study ofheregulin and its co-expression witherbB-2/4 receptor and the assessment of its involvement in the progression from the in situ stage of breast tumors to the invasive one will additionally increase the relevance ofheregulin as a prognostic/diagnostic factor. We believe that our studies provide new insights into breast cancer diagnosis, prognosis, and treatment.Presented at the symposium "New Approaches in the Therapy of Breast Cancer", Georgetown University Medical Center, Washington DC, October 1994, generously supported by an education grant from Bristol-Myers Squibb.     

Heregulin,a new regulator of telomere length in human cells

The growth factor heregulin (HRG) promotes breast cancer (BC) tumorigenesis and metastasis and differentially modulates BC cell responses to DNA-damaging agents via its dual extracellular and nuclear localization. Given the central role of telomere dysfunction to drive carcinogenesis and to alter the chemotherapeutic profile of transformed cells, we hypothesized that an unanticipated nuclear function of HRG might be to regulate telomere length. Engineered overexpression of the HRGβ2 isoform in non-aggressive, HRG-negative MCF-7 BC cells resulted in a significant shortening of telomeres (up to 1.3 kb) as measured by Southern blotting of telomere terminal restriction fragments. Conversely, antisense-mediated suppression of HRGβ2 in highly aggressive, HRG-overexpressing MDA-MB-231 and Hs578T cells increased telomere length up to 3.0 kb. HRGβ2 overexpression promoted a marked upregulation of telomere-binding protein 2 (TRF2) protein expression, whereas its knockdown profoundly decreased TRF2 expression. Double staining of endogenous HRGβ2 with telomere-specific peptide nucleic acid probe/fluorescence in situ hybridization (PNA/FISH) revealed the partial localization of HRG at the chromosome ends. Moreover, a predominantly nucleoplasmic staining pattern of endogenous HRGβ2 appeared to co-localize with TRF2 and, concomitantly with RAP1, a telomere regulator that specifically interacts with TRF2. Small interfering RNA-mediated knockdown of HRG decreased the expression of TRF2 and RAP1, decreased their presence at chromosome ends, and coincidentally resulted in the formation of longer telomeres. This study uncovers a new function for HRGβ2 in controlling telomere length, in part due to its ability to regulate and interact with the telomere-associated proteins TRF2 and RAP1.     

The Growing Demand for Hospice and Palliative Medicine Physicians: Will the Supply Keep Up?

Context

The need for hospice and palliative care is growing rapidly as the population increases and ages and as both hospice and palliative care become more accepted. Hospice and palliative medicine (HPM) is a relatively new physician specialty, currently training 325 new fellows annually. Given the time needed to increase the supply of specialty-trained physicians, it is important to assess future needs to guide planning for future training capacity.

The angiogenic factor CYR61 in breast cancer: molecular pathology and therapeutic perspectives

CYR61 (CNN1), a member of the cysteine rich 61/connective tissue growth factor/nephroblastoma overexpressed (CYR61/CTFG/NOV) family of growth regulators (CNN), is a pro-angiogenic factor that mediates diverse roles in development, cell proliferation, and tumorigenesis. We have recently shown that CYR61 is overexpressed in invasive and metastatic human breast cancer cells. Accordingly, elevated levels of CYR61 in breast cancer are associated with more advanced disease. Unfortunately, the exact mechanisms by which CYR61 promotes an aggressive breast cancer phenotype are still largely unknown. This review examines the functional role of CYR61 in breast cancer disease, presenting evidence that CYR61 signaling may play a major role in estrogen- as well as growth factor-dependent breast cancer progression. We also emphasize the functional significance of the molecular connection of CYR61 and its integrin receptor alpha(v)beta(3) enhancing breast cancer aggressiveness. Moreover, we describe experimental evidence that establishes a novel role for CYR61 determining the protection of human breast cancer cells against chemotherapy-induced apoptosis through its interactions with the integrin receptor alpha(v)beta(3). All these findings delineate a new noteworthy function of a CYR61/alpha(v)beta(3) autocrine-paracrine signaling pathway within both angiogenesis and breast cancer progression, which would allow a dual anti-angiogenic and anti-tumor benefit with a single drug.     

Family involvement moderates the relationship between perceived recovery orientation of services and personal narratives among Chinese with schizophrenia in Hong Kong: a 1-year longitudinal investigation

Social Psychiatry and Psychiatric Epidemiology - Family has been found to have an influential role on clinical and recovery outcomes of people with schizophrenia. While recovery-oriented services...     

Factors inducing synthesis of metallothioneins

Metallothioneins (MT) are the widespread proteins in animal world. These proteins are characterized by the great invariability of their structure. While isolated from the different organs of different animals they only slightly differ in the aminoacid composition from one another. Studies on the structure and biological functions of MT have identified four major isoforms (MT-I, -II, -III and -IV) in mammals. Both MT-I and MT-II genes are expressed in various organs and they are inducible by a variety of metals, hormones, cytokines and xenobiotics. MT-III is expressed only in brain and no specific environmental regulators have been identified. MT-IV is only expressed in differentiating cells of stratified squamous epithelia.     

Unfractionated and low molecular weight heparin affect fibrin structure and angiogenesis in vitro

Cancer patients treated for venous thromboembolism with low molecular weight heparin (LMWH) have a better survival rate than patients treated with unfractionated heparin (UFH). Because fibrin-associated angiogenesis is an important determinant in the progression and metastasis of many solid tumors, the effects of heparins on in vitro angiogenesis were investigated. Both UFH and LMWH inhibited bFGF-induced proliferation of human microvascular endothelial cells (hMVECs) to the same the extent (36-60%). VEGF165-induced proliferation was inhibited to a to a lesser extent (19-33%). Turbidity measurements and electron microscopy showed that the presence of LMWH during polymerization of the fibrin matrix led to a more transparent rigid network with thin fibrin bundles, whereas the presence of UFH resulted in a more opaque more porous network with thick fibrin fibers. We used a human in vitro angiogenesis model, which consisted of hMVECs seeded on top of a fibrin matrix, and stimulated the cells with basic fibroblast growth factor plus tumor necrosis factor a to induce capillary-like tubular structures. The formation of capillary-like tubular structures was retarded with matrices polymerized in the presence of LMWH (46% inhibition compared with a control matrix for both 1.5 and 10 units/ml LMWH), whereas matrices polymerized in the presence of UFH facilitated tubular structure formation (72 and 36% stimulation compared with a control matrix for 1.5 and 10 units/ml UFH, respectively). Similar results were obtained for cells stimulated with vascular endothelial growth factor plus tumor necrosis factor alpha. These data demonstrate the inhibitory effect of heparins on proliferation of hMVECs and provide a novel mechanism by which LMWH may affect tumor progression, namely reduced ingrowth of microvascular structures in a fibrinous stroma matrix by rendering it less permissive for invasion.