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Biomedical subjects

M K Cathcart

Publications and source records attributed to M K Cathcart.

At least 19 recordsLinked to original sources
PubMed1992

Oxygen free radical generation and regulation of proliferative activity of human mononuclear cells responding to different mitogens.

We have compared various mitogenic stimuli for their ability to induce hydrogen peroxide (H2O2) and superoxide anion (O2-) production by PBMC and the effect of these reactive oxygen species and hydroxyl radical (OH.) has been assessed on proliferation. Our results show that pokeweed mitogen (PWM) stimulated PBMC to release H2O2 which interfered with proliferation since inclusion of catalase enhanced PBMC thymidine uptake. In contrast, phytohemagglutinin (PHA) and monoclonal antibody to CD3 (alpha CD3) did not induce PBMC to generate H2O2. O2- release by PBMC, which is readily induced by phorbol myristate acetate (PMA), did not occur when the cells were stimulated with PWM, PHA, or alpha CD3. In correlation, the O2- scavenger enzyme superoxide dismutase (SOD) had no effect on the proliferative response of the cells to the same mitogens, whereas it impaired the thymidine uptake of PMA-stimulated PBMC. A regulatory role for OH. was implied by studies using a battery of OH scavengers known to inhibit PMA-stimulated PBMC proliferation. OH. scavengers markedly inhibited the lymphoblastic transformation of alpha CD3-stimulated cells but had little or no effect on PHA- and PWM-stimulated PBMC. Thus, one manner by which PBMC proliferation is regulated is through oxygen free radical production which varies depending on the type of mitogenic stimulus.

Catalase↗
PubMed1991

Lipoxygenase-mediated transformation of human low density lipoprotein to an oxidized and cytotoxic complex.

We have been studying the mechanisms involved in the oxidative modification of low density lipoprotein (LDL) that lead to its transformation to a cytotoxic complex. Here we examine the direct effect-of soybean lipoxygenase (SLO), a 15-lipoxygenase, on normal human LDL. SLO oxidized LDL and rendered it cytotoxic; agents known to interfere with lipoxygenase activity inhibited this reaction. Enhancement of both the SLO-mediated LDL oxidation and the conversion of LDL to a cytotoxin was observed when either superoxide dismutase or copper (II) (3,5,-diisopropylsalicylic acid)2, both of which dismute superoxide anion, were included during the incubation of SLO with LDL. In contrast, catalase inhibited this reaction in the presence or absence of agents that dismute superoxide anion. Thus, purified lipoxygenase can mediate LDL modification and superoxide anion inhibits this reaction, Furthermore, H2O2 is essential for SLO-mediated LDL oxidation and conversion of LDL to a cytotoxin.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1990

Activated human monocytes oxidize low-density lipoprotein by a lipoxygenase-dependent pathway.

Monocyte-mediated oxidation of low-density lipoprotein (LDL) converts the lipoprotein to a potent cytotoxin. The oxidation process requires monocyte activation and requires superoxide anion since it can be blocked by superoxide dismutase. In this study, the requirement for lipoxygenase activity is shown, in that 1) inhibitors of lipoxygenase prevent the alteration of LDL, 2) copper (II) (3,5-diisopropylsalicylic acid), an agent shown to enhance lipoxygenase activity in a cell-free system, similarly enhances monocyte-mediated LDL alteration, and 3) the (3,5-diisopropylsalicylic acid)-enhanced monocyte-mediated modification of LDL can be completely blocked by inhibitors of lipoxygenase or by superoxide dismutase. These data suggest an integral role for monocyte lipoxygenase in the generation by activated monocytes of the extracellular superoxide anion that participates in the oxidation of LDL and the conversion of LDL to a cytotoxin. Monocyte-modified LDL may be a mediator in tissue damage that accompanies atherosclerosis or occurs at sites of inflammation.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1990

Selective suppression of lymphokine production by human hybridoma suppressor factor (HSF).

We have made a human thymus cell hybridoma that secretes an immunosuppressive monoclonal lymphokine, referred to as hybridoma suppressor factor (HSF). This factor modulates the function of CD4+ cells suppressing their IL-2 production and suppressing PWM-induced B cell differentiation into Ig producing cells. Here we have examined the effect of HSF on the generation of T cell-derived lymphokines that regulate B cell growth and differentiation as well as the expression of other proteins involved in the control of T cell growth i.e., the p55 chain of the IL-2R and the transferrin receptor (TFR). HSF suppressed IFN-gamma activity produced by mitogen-stimulated PBMC without affecting the generation of lymphokines responsible for BCGF and BCDF activities. Additionally, HSF did not inhibit the expression of either IL-2R (p55) or TFR by activated T cells in spite of causing the suppression of IL-2 production. This evidence was further supported by experiments in which HSF selectively suppressed the accumulation of IL-2 mRNA without affecting IL-2R (p55) mRNA expression in mitogen-stimulated PBMC. The selective action of HSF may help to clarify the regulatory mechanisms involved in lymphokine gene expression as well as provide a way by which immune responses involved in autoimmunity and transplant rejection may be interrupted.

Antibody Formation↗
PubMed1989

Generation of a lipid-like cytotoxin from human CD16+ natural killer cells.

Supernatants from unstimulated CD16+ natural killer (NK) cells or from CD16+ NK cells cocultured with K562 tumor cells (to generate NK cytotoxic factor) were both cytotoxic to target cells. Interleukin 2 stimulation of the CD16+ NK cells in the absence of tumor cell stimulation resulted in supernatants which mediated an increased cytotoxicity as compared to the unstimulated supernatants. The cytotoxic activity was recovered in the chloroform fraction of a Bligh-Dyer lipid extraction suggesting that the toxic moiety in the CD16+ NK cell-derived supernatants might be a lipid. Separation of the cytotoxic supernatants into Mr less than 10,000 and Mr greater than 10,000 fractions revealed that the Mr less than 10,000 fraction of both supernatants had no detectable protein but retained cytotoxicity equal to that of the matched unfractionated supernatant. For convenience, we refer to this lipid-like cytotoxin in the Mr less than 10,000 fraction of the supernatants from unstimulated CD16+ NK cells as lipotoxin (LTX) and the cytotoxin in the Mr less than 10,000 fraction of supernatant from interleukin 2 stimulated CD16+ NK cells as LTX*. Increasing concentrations of LTX and LTX* caused a dose related increase in cytotoxicity. Both LTX and LTX* mediated killing as early as 18 h and their cytotoxicity was not significantly affected by heating at 56 degrees for 2 h or by freezing and thawing. Heating at 63 degrees C resulted in a decrease in cytotoxic activity of 10 to 20%. The less than 10,000 dalton fraction of supernatants from both unstimulated and interleukin 2 stimulated CD3- cells (a crude NK cell population) mediated greater cytotoxicity than the CD3+ cell supernatants, and the majority of cytotoxicity from the CD3- cell supernatants was recovered in this fraction. Thus, NK cells were more efficient producers of the lipid-like cytotoxin than T-cells but whether LTX made by NK cells can also be made by T-cells remains to be determined. We propose that lipotoxin: (a) coexists with protein cytotoxins in NK cell supernatant preparations; (b) mediates significant cytotoxicity when separated from proteinaceous cytotoxins; (c) is responsible for the spontaneously secreted cytotoxic activity observed by others; (d) is distinct from previously reported proteinaceous cytotoxins, e.g., NK cytotoxic factor, tumor necrosis factor alpha, and cytolysin/perforin; (e) accounts for the lipophilic nature of cytotoxic factor activity in NK cell supernatants; and (f) causes the cytotoxic activity observed in a small molecular weight fraction of stimulated NK cell supernatants.

Antigens, Differentiation↗
PubMed1989

Superoxide anion participation in human monocyte-mediated oxidation of low-density lipoprotein and conversion of low-density lipoprotein to a cytotoxin.

Human monocytes, upon activation with opsonized zymosan, altered low-density lipoprotein (LDL) during a 24-h co-incubation, resulting in its oxidation and acquisition of cytotoxic activity against target fibroblast cell lines. Both the oxidation of LDL and its conversion to a cytotoxin were enhanced with time of incubation, with the most substantial changes occurring after 6 h of culture of LDL with activated monocytes. Unactivated monocytes did not mediate either alteration. Superoxide anion (O2-) participated in both the oxidation of LDL and its conversion to a cytotoxin since addition of superoxide dismutase (SOD) at the beginning of the co-incubation inhibited, in a concentration dependent fashion, both the monocyte-mediated oxidation and the monocyte-mediated conversion of LDL to a cytotoxin. As expected, the rate of superoxide anion release was greatest during the respiratory burst, very early in the 24-h incubation (0 to 2 h); however, exposure of LDL to monocytes during the respiratory burst was not required for LDL oxidation. The lower levels of O2- released by the cells hours after the respiratory burst had subsided were sufficient to lead to the initiation of LDL oxidation. Three results indicated that the oxidative modification of LDL into a cytotoxin required O2(-)-independent free radical propagation after O2(-)-dependent initiation. First, oxidation of LDL exposed to the activated, superoxide anion-releasing monocytes for 6 h could be almost completely blocked by the addition at 6 h of the general free radical scavenger butylated hydroxytoluene, but not by SOD. Second, LDL oxidation proceeded even after removal of LDL from the superoxide anion-producing, activated cells after various durations of exposure. Third, the development of substantial levels of lipid peroxidation products and the development of greater cytotoxicity occurred after 6 h of exposure of LDL to activated cells, long after peak O2- release had subsided. These results lead us to conclude that monocyte-mediated oxidation of LDL, leading to its transformation into a cytotoxin, requires release of O2- occurring as a result of activation but not necessarily during the respiratory burst, and also requires O2(-)-independent free radical propagation. The modification of LDL into a potent toxin by activated monocytes may explain the tissue damage in atherosclerotic lesions and other pathologic sites in which inflammatory cells congregate.

Cytotoxins↗
PubMed1988

Oxidative modification of low density lipoprotein (LDL) by activated human monocytes and the cell lines U937 and HL60.

Human peripheral blood monocytes, upon activation, have the capacity to oxidize low density lipoprotein (LDL) and render the LDL toxic to cultured cells. Previous studies by our laboratory indicate that this process is mediated by free radicals in that it can be prevented by addition of free radical scavengers and antioxidants during the incubation of monocytes with LDL. Here we report that optimal modification of LDL by monocytes was influenced by media composition. In the absence of added metal ions, oxidation was distinctly dependent on the concentration of monocytes as well as LDL concentration. Exposure of monocytes to lipopolysaccharide or stimulation of phagocytosis by opsonized zymosan resulted in marked enhancement of LDL oxidation compared to other activating agents. After exposure to activated monocytes, lipid oxidation products in the supernatant were found both in a high molecular weight fraction containing LDL (greater than 30,000 Daltons) and in a lipoprotein-free, low molecular weight fraction (less than 30,000 Daltons), yet only the high molecular weight, LDL-containing fraction was toxic to target cells. In addition, human myelomonocytic cell lines U937 and HL60 were shown to mediate oxidation of LDL. As with monocytes, exposing these cells to opsonized zymosan caused the level of LDL oxidation to be significantly enhanced. These findings offer further insight into the mechanisms of monocyte-mediated oxidation of lipoproteins and will facilitate studies investigating the role of monocyte-modified LDL in tissue injury.

Culture Media↗
PubMed1988

Two distinct subsets of patients with systemic lupus erythematosus.

This study was undertaken to examine the levels and function of peripheral blood immunoregulatory T cell subpopulations in systemic lupus erythematosus (SLE). T cell subpopulations can be distinguished by the T cell differentiation antigens CD4 (recognized by the monoclonal antibodies OKT4 or Leu3) and CD8 (recognized by the monoclonal antibodies OKT8 or Leu2). All SLE patients tested had normal percentages of CD8 cells in their peripheral blood. The SLE patients, however, fell into two groups based on their CD4 cell numbers. Fifty-five percent of the SLE patients had normal levels of CD4 cells (Group A) and therefore normal CD4/CD8 cell ratios, whereas 45% of the SLE patient population had markedly depressed CD4 cell levels (Group B) and significantly low CD4/CD8 cell ratios. T cells from normal donors and SLE patients were further examined for their ability to stimulate allogeneic normal B/M phi cells to secrete IgM in the presence of pokeweed mitogen (PWM). Utilizing this assay system two forms of immunosuppression were observed: (1) that mediated by high concentrations of purified CD4 cells and (2) that mediated by CD8 cells. High concentrations of purified CD4 cells, added to a constant number of allogeneic normal B/M phi cells, suppressed PWM-stimulated IgM synthesis. Group B SLE patients, with significantly low CD4 cell numbers, had defective CD4 cell-mediated suppression which was concentration dependent. This result was confirmed in a study using identical twins discordant for SLE. In this case CD4 cells from the SLE twin did not induce immunosuppression at a high concentration of CD4 cells whereas similar concentrations of CD4 cells from the normal twin resulted in suppression. SLE patients (Group A) with normal levels of CD4 cells had normally immunosuppressive CD4 cells. Suppression mediated by CD8 cells was demonstrated by the fact that removal of CD8 cells resulted in enhanced IgM synthesis induced by the remaining CD4 cells. Although all the SLE patients in this study had normal peripheral blood levels of CD8 cells, SLE Group A patients had defective CD8 cell suppression whereas CD8 function appeared to be normal in Group B patients. These results suggest that in SLE patients with depressed CD4 cell numbers (Group B) there is a corresponding defect in CD4 cell function. We demonstrate that in SLE Group B patients, defective suppression is due to a subset of T cells that bear the CD4 antigen. The SLE patient population (Group A) with normal CD4/CD8 ratios and normally functioning CD4 cells, however, appear to have normal CD4 cell-mediated suppression but defective CD8 suppressor cell function.

Antibody Formation↗
PubMed1988

Human hybridoma suppressor factor (HSF) inhibits IL2 production in addition to suppressing immunoglobulin production.

The human thymus cell hybridoma, 8E-24, secretes a potent immunosuppressive factor(s), HSF, which inhibits polyclonal immunoglobulin (Ig) production. Our current studies reveal that this suppression is monocyte dependent in that its suppressive activity for Ig production was not observed in monocyte-depleted lymphocyte cultures but was restored by addition of monocytes. The requirement for monocytes was equally satisfied by autologous or allogeneic monocytes or monocyte conditioned media. Although the suppression mediated by HSF required the presence of monocytes, the mechanism for monocyte participation appears to be different from that reported for suppression mediated by soluble immune response suppressor (SIRS) in that general oxygen free radical scavengers did not inhibit this activity. Further information on the mechanism of action of HSF was obtained from studies on its suppressive effect on the phytohemagglutinin(PHA)-induced proliferative response. In this system HSF significantly suppressed PHA induced interleukin 2(IL2) production of peripheral blood mononuclear cells (PBMC) in a dose dependent fashion without inhibiting the proliferative response of CTLL-20 target cells to IL2. Interleukin 1 (IL1) production by monocyte cultures was also not suppressed by HSF. These results indicate that HSF interferes with IL2 production and not its induction by IL1 or its interaction with the IL2 receptor. To investigate the role of HSF-induced IL2 suppression in the pokeweed mitogen (PWM) antibody synthesis assay, the time course of IgG, IgM, IL1, and IL2 production of PWM stimulated PBMC cultures was examined. Results showed that the peak of IL2 production occurred on the second day of culture and was significantly suppressed by HSF while IL1 production was not affected during the seven day culture period. Similar suppression of IL2 and IgM production was observed in cultures of B cells and T4+ cells. Reconstitution of the IL2 levels in these cultures with recombinant IL2 completely restored antibody production. These results suggest that HSF in the presence of monocytes modulates the function of T4+ cells by inhibiting IL2 production. The inhibition of IL2 production by HSF appears to be responsible for the suppression of antibody production.

Biological Factors↗
PubMed1987

A multi-step isolation scheme for obtaining CD16+ human natural killer cells.

A multi-step isolation scheme capitalizing on negative selection protocols is described for obtaining an enriched population of CD16+ human natural killer (NK) cells. The isolation scheme consists of incubating peripheral blood mononuclear cells (MNC) on nylon wool, rosetting the nylon wool non-adherent cells with sheep red blood cells (SRBCs) for 1 h at 29 degrees C and then utilizing a 'panning' technique to remove CD3+, non-rosetting cells. The final working cell population contained 70-80% CD16+ cells, 15% CD2+ cells, 1-3% CD3+ cells, 5-7% SIg+ cells and no detectable MO2+ cells. In comparing the final NK cell population from the multi-step isolation protocol to NK cells obtained by the Percoll density gradient centrifugation technique, the multistep method: (1) yielded a higher percentage of CD16+ cells, (2) mediated a greater degree of cytotoxicity at a 25:1 E:T ratio, and (3) contained fewer contaminating monocytes/macrophages (none were detectable). In addition, the multi-step scheme allowed recovery of 30% of the total CD16+ cells present compared to only 7% recovered by the Percoll density gradient technique. Pretreatment of the enriched NK cells, obtained from the multi-step scheme, with interleukin-2 (3.5 and 7.0 U/ml of activity) resulted in an increase in NK cell-mediated cytotoxicity. In addition, these cells were as effective at synthesizing the cytotoxin, NKCF, at a 25:1 E:T ratio as at 50:1 and 100:1 E:T ratios. This multi-step isolation scheme consistently yields a high percentage of CD16+ NK cells and thus may greatly facilitate studies on the mechanism(s) involved in NK cell-mediated cytotoxicity and may further the study of the cytotoxins involved.

Antigens, Surface↗
PubMed1987

Abnormal lymphocyte function in scleroderma: a study on identical twins.

This paper describes immunologic studies on a set of identical twins discordant for the presence of scleroderma. The affected twin had a low absolute T-cell count, low numbers of T4 helper/inducer cells, and an increase in the T8 suppressor/cytotoxic cell count. The T cells of the patient responded poorly to mitogens and to allogeneic and autologous stimuli. By contrast, T-cell-helper activity for pokeweed mitogen-induced IgM synthesis was markedly enhanced in the patient. Furthermore, activated mononuclear cell supernatants from the patient markedly enhanced the synthesis of collagen by normal cultured fibroblasts. The unaffected twin by contrast displayed normal responses in these assays. The results suggest that the immunologic defects in scleroderma are not entirely genetically determined.

Diseases in Twins↗
PubMed1987

Excessive helper T-cell function in patients with idiopathic pulmonary fibrosis: correlation with disease activity.

Peripheral blood T lymphocytes from patients with idiopathic pulmonary fibrosis and matched normal controls were examined for their helper function in an in vitro antibody synthesis assay. This assay measures the dose-related T-cell regulation of antibody production by B cells in the presence of pokeweed mitogen. Eight patients of 14 expressed significantly increased helper T-cell activity, three exhibited no change, and three had depressed helper T-cell function. All of the patients with excessive helper T-cell function had an active neutrophilic alveolitis as determined by bronchoalveolar lavage on the day of study. Five of the 14 patients studied were determined to have a low percentage of neutrophils (less than 10%) in their BAL fluid. None of these were found to express excessive helper T-cell function; in fact three of the five had depressed helper T-cell function. No correlation between steroid therapy or smoking history and the expression of excessive helper function was observed. None of the peripheral blood T-cells from IPF patients were actively producing IL-2 in vitro without further stimulation, providing evidence against constitutive production in vivo. T cells were also examined for their ability to produce lymphokines promoting fibroblast proliferation. Enhanced stimulation of fibroblast proliferation was shown to positively correlate with disease activity as determined by the degree of neutrophilic alveolitis (r = 0.68). The significant correlation between neutrophilic alveolitis and excessive helper T-cell function observed here suggests that altered systemic immunoregulation accompanies local inflammation. The further participation of patient T cells in promoting fibroblast proliferation may contribute to the development of fibrosis, or to the contrary may be an attempt to limit the fibrotic process.

Aged↗
PubMed1986

Suppression of polyclonal immunoglobulin production by a soluble factor produced by a human thymus hybridoma.

A human thymus cell hybridoma was established using thymus cells obtained from a patient with common variable hypogammaglobulinemia and associated thymoma. This hybridoma secreted a suppressor factor for polyclonal antibody synthesis. Supernatants of this hybrid showed 40-80% suppression of both IgM and IgG synthesis by pokeweed mitogen-stimulated human peripheral blood lymphocytes. Hybridoma supernatants were suppressive for immunoglobulin production only if added within the initial 48 h of the seven-day culture period. Suppression of antibody production by the hybridoma supernatant was prevented by preabsorption with T lymphocytes. Further, the suppressor factor was shown to inhibit antibody production in reconstructed cultures containing T4+ cells and B cells, yet the suppression could be abrogated by increasing the number of T4+ cells. The hybrid supernatant had no affect on the proliferation of human mononuclear cells in response to pokeweed mitogen, lipopolysaccharide, concanavalin A or alloantigen but inhibited phytohemagglutinin-induced proliferation. The target cell population for the inhibition of phytohemagglutinin responsiveness was shown to be a T4+ lymphocyte (helper inducer T cell). These results suggest that thymus hybridoma cells can produce immunoregulatory products that act through the modulation of T4+ lymphocyte function. To our knowledge this is the first human thymus cell hybridoma to be reported. Studies on such cell lines may provide important information on immunoregulatory thymic factors.

Antibody Formation↗
PubMed1985

Monocytes and neutrophils oxidize low density lipoprotein making it cytotoxic.

Free radicals are believed to be involved in leukocyte induced tissue injury. The present studies were performed to determine whether low density lipoprotein (LDL) might serve as a mediator of tissue injury after leukocyte induced free radical oxidation of LDL. Our results show that incubation of LDL with monocytes or polymorphonuclear leukocytes (PMN) leads to oxidation of the lipoprotein rendering it toxic to proliferating fibroblasts. Monocyte activation enhances these effects. Butylated hydroxytoluene (BHT), vitamin E (vit E) and glutathione (GSH) virtually prevent the oxidation of LDL and the formation of cytotoxic LDL, indicating that these alterations are mediated by leukocyte-derived free radicals. This is the first demonstration that short-lived free radicals emanating from phagocytic cells could mediate cell injury through the action of a stable cytotoxin formed by the oxidation of LDL. The fact that lipoproteins can transfer a cytotoxic effect from leukocytes to proliferating cells reveals a pathway for cell destruction which may have implications in atherosclerotic plaque progression, macrophage mediated toxicity to tumor cells and tissue injury by inflammatory processes.

Cells, Cultured↗
PubMed1985

Partial purification and characterization of B cell growth factor constitutively secreted by human T-cell hybridoma.

Human T cell hybridomas were established by fusion of PHA-activated PBL with the 8-azaguanine resistant human T-leukemic cell line CEM-CM3. High levels of B cell growth factor (BCGF) activity were detected in the supernatants of hybridoma C8-2B2 and its subclones. Hybridoma C8-2B2, in addition to the Leu 3a, also expressed the OKT11 surface marker which was not detectable on the parent CEM-CM3 cells. BCGF from the culture supernatant was purified by combined use of salt fractionation and gel filtration to 36.6 fold with 23.9% recovery of activity. The BCGF produced by hybridoma C8-2B2 has a molecular weight range of 16,000-20,000 in two major electrophoretically different forms with pI values of 6.4 and 7.4.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1979

Initial studies of the molecular organization of the cell-substrate adhesion site.

Using selective extraction reagents and non-penetrating probes, studies have been initiated on the molecular organization of substrate-attached material, adhesion sites which pinch off from the cell surface of normal Balb/c 3T3 or SV40-transformed Balb/c 3T3 (SVT2) cells and which remain bound to the serum-coated substrate during EGTA-mediated detachment of cells. Extraction of SVT2 adhesion sites with non-ionic detergents resulted in (a) only small amounts of leucine-radiolabeled protein and glucosamine-radiolabeled polysaccharide being solubilized; (b) selective solubilization of 80% of the adhesion site actin, and (c) solubilization of 95% of the phospholipid from these membranous pools. ATP in combination with potassium chloride extracted 60% of the actin. The 3T3 and SVT2 adhesion site proteins which are accessible to lactoperoxidase-catalyzed iodination were also determined. Many of the serum-derived proteins, bound to the substrate, were iodinated during iodination treatment of serum-coated or substrate-attached material-coated substrates, whereas the cellular proteins in the adhesion sites were not iodinated even though they were present in larger quantity as revealed by Coomassie blue staining. Iodination of cells, followed by their EGTA-mediated detachment and reattachment to fresh serum-coated substrates, indicated that the principal iodinated cell surface component deposited in new adhesion sites is the large external transformation-sensitive glycoprotein (even though large external transformation-sensitive glycoprotein is not the only principal iodinated cell surface component of these cells). These studies further establish the selective enrichment in this adhesive material of specific cell surface components and indicate that they are tenaciously bound at the interface between the serum coating and the undersurface of the adhesion site membranous pools.

Actins↗
PubMed1979

Phospholipid composition of substrate adhesion sites of normal, virus-transformed, and revertant murine cells.

The phospholipid composition of cell-substratum adhesion sites, obtained after EGTA-mediated detachment of cells from the tissue-culture substratum, was determined for [32P]orthophosphate radiolabeled Balb/c 3T3, SV40-transformed (SVT2), and concanavalin A selected revertant variant cell lines. All of the major phospholipid classes were found in the substrate-attached material, but there was an enrichment for specific phospholipid species in this adhesive material as compared to whole-cell and surface-enriched membranes. The phospholipid composition was remarkable similar for the whole-cell and surface-enriched membrane fractions from the three cell lines. However, pronounced differences in the phospholipid composition of the adhesion sites were observed as a result of viral transformation--SVT2 sites were clearly enriched in phosphatidylethanolamine and depleted in phosphatidylcholine when compared to 3T3 sites. This alteration in adhesion site phospholipids of transformed cells reverted to 3T3-like values in the adhesive material of revertant cells. The composition of adhesive material of newly attaching cells was also examined to differentiate compositional differences between "footpad" adhesion sites and "footprints", adhesive material pinched off from the posterior of cells as they move across the substratum. Pulse and pulse-chase analyses of the [32P]phospholipids revealed some differences in synthesis and turnover rates in the three cell lines; in addition, altered rates of deposition of newly synthesized material into adhesion sites of transformed cells were observed. These data afford further evidence that the cell-substratum adhesion sites are highly specialized areas of the cell surface enriched in components which are intricately involved in the adhesive process. The transformation-dependent changes in adhesion site phospholipids may help to determine the basis for the altered adhesive properties of transformed cells.

Animals↗