Biomedical subjects

M A Loo

Publications and source records attributed to M A Loo.

7 recordsLinked to original sources

PubMed1998

Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome.

Maturation of wild-type CFTR nascent chains at the endoplasmic reticulum (ER) occurs inefficiently; many disease-associated mutant forms do not mature but instead are eliminated by proteolysis involving the cytosolic proteasome. Although calnexin binds nascent CFTR via its oligosaccharide chains in the ER lumen and Hsp70 binds CFTR cytoplasmic domains, perturbation of these interactions alone is without major influence on maturation or degradation. We show that the ansamysin drugs, geldanamycin and herbimycin A, which inhibit the assembly of some signaling molecules by binding to specific sites on Hsp90 in the cytosol or Grp94 in the ER lumen, block the maturation of nascent CFTR and accelerate its degradation. The immature CFTR molecule was detected in association with Hsp90 but not with Grp94, and geldanamycin prevented the Hsp90 association. The drug-enhanced degradation was decreased by lactacystin and other proteasome inhibitors. Therefore, consistent with other examples of countervailing effects of Hsp90 and the proteasome, it would seem that this chaperone may normally contribute to CFTR folding and, when this function is interfered with by an ansamycin, there is a further shift to proteolytic degradation. This is the first direct evidence of a role for Hsp90 in the maturation of a newly synthesized integral membrane protein by interaction with its cytoplasmic domains on the ER surface.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed1996

Dichotomy of astrocytoma migration and proliferation.

Astrocytomas often show high rates of local invasion that lead to local recurrence of the disease. Histologically, the most highly invasive astrocytoma cells are detected in isolation rather than as nests of tumor. Our study attempted to determine whether the migratory response to extracellular substrates influences the proliferative behavior of these highly invasive cells. The preferential and specific migratory response of human astrocytoma cells to extracellular matrix proteins was assessed by a microliter scale migration assay. Growth curve studies on protein ligands permissive (merosin) for cell migration indicated that the lag phase was protracted compared with cells seeded on non-permissive proteins (vitronectin). Once a certain cell density was reached, logarithmic proliferation was indistinguishable on the different proteins. The proliferation index of populations of cells migrating on merosin and vitronectin was measured by both BrdU incorporation and MIB-1 immunocytochemistry labeling. Cells seeded on vitronectin showed higher proliferation throughout the population than cells seeded on merosin. On merosin, the more migratory cells at the periphery were less proliferative than non-migratory cells in the central region of that population. The integrin-associated signal transduction protein, p125FAK, was heavily localized in the membrane of non-migrating cells and largely absent in migrating astrocytoma cells. We conclude that temporally, proliferation and migration are mutually exclusive behaviors. Cell density or non-permissive substrates that inhibit cell motility favor a more proliferative phenotype. Conversely, active migration suppresses cell proliferation.

Astrocytoma↗

PubMed1996

Contrasting migratory response of astrocytoma cells to tenascin mediated by different integrins.

Tenascin, an extracellular matrix protein, is expressed in human gliomas in vitro and in vivo. The distribution of tenascin at the invasive edge of these tumors, even surrounding solitary invading cells, suggests a role for this protein as a regulator of glioma cell migration. We tested whether purified tenascin, passively deposited on surfaces, influenced the adhesion or migration of a human gliomaderived cell line, SF-767. Adhesion of glioma cells to tenascin increased in a dose-dependent fashion up to a coating concentration of 10 micrograms/ml. Higher coating concentrations resulted in progressively fewer cells attaching. Cell adhesion could be blocked to basal levels using anti-beta 1 integrin antibodies. In contrast, when anti-alpha v antibodies were added to the medium of cells on tenascin, cell adhesion was enhanced slightly. Using a microliter scale migration assay, we found that cell motility on tenascin was dose dependently stimulated at coating concentrations of 1 and 3 micrograms/ml, but migration was inhibited below levels of non-specific motility when tested at coating concentrations of 30 and 100 micrograms/ml. Migration on permissive concentrations of tenascin could be reversibly inhibited with anti-beta 1, while treatment with anti-alpha v antibodies increased migration rates. We conclude that SF-767 glioma cells express two separate integrin receptors that mediate contrasting adhesive and migratory responses to tenascin.

Astrocytoma↗

PubMed1995

Multiple proteolytic systems, including the proteasome, contribute to CFTR processing.

The molecular components of the quality control system that rapidly degrades abnormal membrane and secretory proteins have not been identified. The cystic fibrosis transmembrane conductance regulator (CFTR) is an integral membrane protein to which this quality control is stringently applied; approximately 75% of the wild-type precursor and 100% of the delta F508 CFTR variant found in most CF patients are rapidly degraded before exiting from the ER. We now show that this ER degradation is sensitive to inhibitors of the cytosolic proteasome, including lactacystin and certain peptide aldehydes. One of the latter compounds, MG-132, also completely blocks the ATP-dependent conversion of the wild-type precursor to the native folded form that enables escape from degradation. Hence, CFTR and presumably other intrinsic membrane proteins are substrates for proteasomal degradation during their maturation within the ER.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Substrates for astrocytoma invasion.

A better understanding of the influences of specific extracellular substrates, including proteins, glycosaminoglycans, and parenchymal cells, on the invasive behavior of glioma cells would potentially lead to novel forms of treatment aimed at confining the tumor. A monolayer, microliter scale assay was used to investigate how different substrates influenced glioma migration. Basal or unspecific movement (range, 10-260 microns/d) was determined by observing a panel of seven established human glioma cell lines. Migration rates two to five times higher than this basal activity were referred to as preferential and specific glioma migration; these rates generally occurred on merosin and tenascin. Collagen, fibronectin, or vitronectin were less supportive of migration. The glioma cells migrated on hyaluronic acid, but they did not migrate to the extent generally found on the extracellular matrix proteins. Glioma-derived extracellular matrix also served to promote cell migration. This finding implicates a role for either glioma remodeling or synthesis of a permissive environment for local dissemination that may be independent of the constitutive matrix proteins normally found in the brain. Although the glioma cells were able to migrate over monolayers of other glioma cells, they were unable to migrate over astrocytes and fibroblasts. Our findings indicate that the invasive behavior of glioma cells in situ is most likely a consequence of the interplay between the cells' manipulation of the environment and the constitutive ligands associated with specific regions or structures of the brain.

Astrocytoma↗

PubMed1994

Determinants of human astrocytoma migration.

A unique characteristic of astrocytic malignancies is their frequent dissemination through the brain. Cellular determinants of migration include adhesion to the substratum, restructuring of the actin cytoskeleton to generate motion, and (in the setting of invasion into tissue) secretion of enzymes for remodeling interstitial space to accommodate forward motion of the migrating cell. In order to better understand these features in the context of local brain invasion by astrocytoma cells, the adhesion and migratory properties of these cells have been investigated in an in vitro monolayer system. Adhesion of 8 different astrocytoma cell lines to different purified human extracellular matrix (ECM) proteins (collagen type IV, cellular fibronectin, laminin, and vitronectin) revealed that there is no "astrocytoma-specific" ECM protein that consistently leads to high cell binding. Similarly, migration of astrocytoma cells was found to be variable and dependent on different ECM proteins. Laminin was frequently the most permissive for adhesion and migration. Adhesion to collagen, fibronectin, and vitronectin was integrin dependent and could be blocked using anti-beta 1 integrin antibodies; in contrast, attachment to laminin could not be blocked using these antibodies. A comparison of adhesion with migration for each of the cell lines on each of the 4 ECM proteins revealed that poor adhesion was associated with minimal migration and that frequently, high adhesion was correlated with rapid migration. When tested for migration on autologous, cell-derived ECM, none of the cell lines were as migratory as they were on one of the purified ECM proteins, with the exception of SF767 cells. Furthermore, it was found that ECM from SF767 cells promoted the migration of other astrocytoma cells. The results from this study indicate that migration is a constitutive behavior of glioma cells which is dependent on, or modified by, the presence or absence of permissive ligands in the environment.

Astrocytoma↗

PubMed1994

The role of extracellular matrix in human astrocytoma migration and proliferation studied in a microliter scale assay.

Ligands in the extracellular matrix (ECM) are known to mediate migration of normal as well as tumor cells via adhesion molecules such as the integrin receptor family. We develop a microliter scale (15-20 microliters total volume) monolayer migration assay to investigate the ability of astrocytoma cells to disperse on surfaces coated with purified human ECM protein ligands. In this system the rate of radial migration of the cell population was constant over time. For human astrocytoma cell lines U-251 and SF-767, laminin and collagen type IV supported a migratory phenotype; fibronectin and vitronectin only minimally supported migration. The different ECM proteins also influenced growth rate: cells on laminin and collagen had a protracted lag phase. Furthermore, migrating cells seeded on laminin or collagen showed a lower labeling index than did stationary cells in the central, crowded region on the same substrate. This micro-scale migration assay should enable detailed molecular and biochemical studies of the determinants of migration.

Astrocytoma↗