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Biomedical subjects

R B Bankert

Publications and source records attributed to R B Bankert.

At least 55 records · Page 3Linked to original sources
PubMed1989

Cell surface glycoprotein associated with human lung tumors that is similar to but distinct from the epidermal growth factor receptor.

A cell surface glycoprotein (gp160) present on the surface of non-small cell human lung tumors is characterized and compared with the epidermal growth factor receptor (EGFR). The epitope on gp160 recognized by monoclonal antibody 5E8 is shown to be part of the protein moiety of the molecule and is found to be relatively stable. The epitope is stable over a wide pH range and after treatment with urea as well as most ionic and non-ionic detergents. We have observed that gp160 is similar in several respects to the EGFR. However, despite the similarities, several independent lines of experimental evidence presented here suggest that gp160 and the EGFR are distinct molecules. The first evidence suggesting that these two molecules are different is that the EGFR, but not gp160, is constitutively detectable in the A431 cell tissue culture supernatant, and that a pulse of these cells with epidermal growth factor (under conditions which permit the internalization of the receptor-ligand complexes) significantly reduces the expression of the EGFR without noticeably affecting the level of gp160 on the cell surface. Two very different immunofluorescent patterns marking the position of gp160 and EGFR are observed using monoclonal antibodies specific for each molecule. Using an enzyme-linked immunosorbent assay, it was determined that these same monoclonal antibodies do not cross-inhibit one another, and it was established that gp160, but not EGFR, was retained on an affinity column containing anti-gp160 antibodies immobilized to the solid matrix. An additional finding that supports the notion that gp160 and the EGFR are distinct molecules is that one human lung tumor cell line (Calu-3) has been identified which expresses gp160 but not the EGFR on its surface. These results indicate that there are characteristics which distinguish gp160 from the EFGR, and we establish here that these distinguishing features reflect differences at the protein moiety and not simply differential glycosylation. We conclude from these studies that we have identified and characterized a cell surface molecule that resembles in several respects the epidermal growth factor receptor. This cell surface molecule represents a potentially useful target for the immunotherapy and diagnosis of human non-small cell lung cancer.

Antibodies, Monoclonal↗
PubMed1989

Generation of somatic variants of a B cell hybrid mediated by a non-cytolytic L3T4+ idiotype-specific T cell.

We previously reported that idiotype (Id)-loss, stable somatic variants of a B cell hybrid, 2C3E1, are generated both in vitro and in vivo, after interaction of the Id-positive tumor cells with autologous Id-specific effector T cells. The present investigation was undertaken to elucidate further the nature and functional characteristics of the effector T cells. We report here that the idiotype-specific cells mediating the generation of Id- tumor variants are Thy1+ L3T4+ Lyt-2- cells, which respond to specific idiotypic stimulation by secreting IL-2 in vitro. No IL-2 is secreted in response to unrelated Ig or an Id/Ig-2C3E1 tumor variant. Furthermore, the Id-specific T cells exert strong suppressive effects on the expression of 2C3E1 Ig and the effects can be reversed by blocking the L3T4+ T cells with monoclonal anti-L3T4 antibody in vitro during the initial 3 days of co-culture. After 4 days, the T cell-mediated suppression of the 2C3E1-Id is irreversible. In addition to the in vitro studies we have determined that the administration of anti-L3T4 mAb to mice just before priming with idiotype-bearing tumor cells also abrogates the suppressive effects of the idiotype primed spleen cells on Ig expression of 2C3E1. To study the Id-specific effector T cells in more detail we have generated functional Id-specific L3T4+ T cell lines. These T cell lines have been shown to recapitulate the generation of Id- tumor variants that we observed with Id-primed spleen cells. It is concluded that L3T4+, Id-specific Ts cells are responsible for the generation of somatic variants of the B cell hybrid 2C3E1 and that the induction or selection of these variants progresses from a reversible phase to an irreversible phase.

Animals↗
PubMed1989

Immunospecific targeting of cytosine arabinonucleoside-containing liposomes to the idiotype on the surface of a murine B-cell tumor in vitro and in vivo.

A new tumor model is described that is suitable for the evaluation of antibody-directed drug-delivery protocols and a modification in the procedure for covalently coupling antibody to the surface of drug-containing liposomes is presented. These immunospecific liposomes containing cytosine arabinonucleoside (Ara-C) have been tested in vitro and in vivo for their ability to kill a B-cell tumor. The target of the immunospecific-Ara-C liposomes is the idiotype associated with an antigen-specific immunoglobulin receptor on the cell surface of a murine B-cell hybrid (2C3). Affinity-purified antibodies specific for the idiotype were covalently coupled to modified lipid on the surface of the large unilamelar liposomes containing drug. These liposomes were shown to kill idiotype-positive 2C3 cells in vitro, but not idiotype-negative variants of this same cell line. It was also established in vitro that the drug-containing liposomes were at least 40 times more efficient than free Ara-C in the killing of the tumor cells. The 2C3 tumor was also propagated in vivo following the i.p. administration of tumor cells. The tumor grew initially as multiple foci within the peritoneum and subsequently spread to the spleen. Tumor-bearing mice were treated either with free Ara-C or with immunospecific liposomes containing Ara-C. Tumor growth in the primary tumor nodules and in the spleen was monitored by the administration of bromodeoxyuridine to the tumor-bearing animals followed by the immunofluorescent staining of cells with a monoclonal anti-bromodeoxyuridine antibody to estimate the proportion of cells in S phase. Our data from five out of seven animal experiments shows that the immunospecific-Ara-C liposomes, but not free drug, reduced tumor growth in the spleen. However, neither the liposomes containing drug nor the free drug were able to alter the growth of the primary tumor nodules growing in the peritoneal cavity. These results suggest that immunospecific-Ara-C containing liposomes may be useful in conjunction with other cytoreductive protocols in controlling tumor growth or preventing the spread of the tumor to other sites, but that immunospecific-Ara-C containing liposomes by themselves are not likely to eliminate an established tumor in vivo. We also demonstrate here that the administration of immunospecific-Ara-C containing liposomes in an animal having high levels of circulating tumor-associated antigen (i.e., IgG containing the idiotype) represents a potential clinically relevant hazard which must be considered when designing antibody-directed drug-delivery protocols.

Animals↗
PubMed1988

Activation induces a rapid reorganization of spectrin in lymphocytes.

Lymphocyte activation results in a rapid reorganization of the cytoskeletal protein spectrin. Immediately following an activation signal, there is fragmentation of a spectrin-rich cytoplasmic structure and subsequent translocation of the fragments to defined areas of the plasma membrane in both antigen-specific T cell hybridomas and lymph node T cells. These dramatic changes have been documented by light and electron microscopic immunolocalization and by immunoblot analysis of plasma membrane-enriched preparations. A T cell hybridoma variant lacking the spectrin-rich cytoplasmic structure of the parental line does not redistribute spectrin and produces little or no IL-2 in response to antigen-dependent activation. This suggests a functional link between spectrin distribution and activation potential. We propose that the cytoplasmic structure functions as an organizing center or reservoir for spectrin that is sensitive to signaling at the cell surface.

Animals↗
PubMed1988

Selective growth inhibition of human lung cancer cell lines bearing a surface glycoprotein gp160 by 125I-labeled anti-gp160 monoclonal antibody.

Monoclonal antibody 5E8 which is specific for a Mr 160,000 glycoprotein (gp160) on the surface of human lung cancer was radiolabeled with 125I. Radiolabeled 5E8 antibody is shown here to suppress the growth of gp160 positive human lung tumor cell lines in a dose-dependent fashion, but this same radiolabeled antibody does not alter the growth of gp160 negative lung tumor cell lines. Neither the unlabeled 5E8 nor a control radiolabeled monoclonal antibody has any effect upon the growth of gp160 positive tumors. The specificity of radiolabeled antibody mediated tumor killing is further demonstrated by the ability of unlabeled 5E8 to inhibit tumor killing by 125I-5E8. The efficiency with which the labeled tumor specific antibody suppressed tumor colony formation is enhanced by increasing the molar ratio of 125I to 5E8. This ratio could be increased to a level of two without affecting the capacity of the antibody to bind to the cell surface antigen. An attempt to increase the efficiency of tumor killing by the addition of a second antibody subsequent to incubation with 125I-5E8 was unsuccessful. These results indicate that 125I is a viable isotope and gp160 represents an appropriate target for radioimmunotherapy of human lung cancer.

Animals↗
PubMed1988

Selective elimination of idiotype-binding cells in vivo by a drug-idiotype conjugate demonstrates the functional significance of these cells in immune regulation.

A receptor-specific cytotoxic drug delivery system has been used to eliminate idiotype-binding cells in vivo to ascertain the possible functional significance of these cells in regulating the humoral immune response to dextran. Protein M104E, a mouse myeloma protein that binds dextran, expresses a private idiotope that is present on a significant proportion of the normal dextran-specific antibody repertoire. Immunocompetent cells that bind and internalize M104E idiotype-bearing molecules were eliminated by the intravenous administration of a single dose of cytosine arabinonucleoside conjugated to purified M104E protein. The administration of this cytotoxic drug-idiotype conjugate had a profound effect upon the expression of the M104E idiotype in euthymic but not in athymic BALB/c mice following immunization with dextran. In euthymic mice, the depletion of the idiotype-binding cells resulted in a marked elevation in the level of M104E idiotype present in the immune sera. Moreover, treated but not control mice developed idiotype-positive molecules that did not bind dextran. These results demonstrate the functional significance of idiotype-binding cells in the regulation of individual clonotypes during an immune response.

Animals↗
PubMed1988

Heterogeneity in lymphocyte spectrin distribution: ultrastructural identification of a new spectrin-rich cytoplasmic structure.

Spectrin-like proteins are found in a wide variety of non-erythroid cells where they generally occur in the cell cortex near the plasma membrane. To determine the intracellular distribution of alpha-spectrin (alpha-fodrin) in lymphocytes, we have developed an immunoperoxidase method to localize this protein at the ultrastructural level. Of considerable interest, particularly with regard to our efforts to determine the function of spectrin in this cell type, was the finding that its subcellular localization and its relationship with the plasma membrane can vary dramatically. Based on its position in the cell, alpha-spectrin can occur in two forms in lymphocytes: one that associates closely with the plasma membrane and another that occurs at some distance from the cell periphery, either as a single large aggregate or as several smaller ones. The single large aggregate of spectrin is a stable feature in a number of lymphocyte cell lines and hybrids which were used to examine its ultrastructural characteristics. A previously undescribed cellular structure, consisting of a meshwork of spectrin filaments and membranous vesicles, was identified in these cells. This structure could be induced to dissipate in response to membrane perturbants (e.g., hyperthermia and phorbol esters, known effectors of lymphocyte function and differentiation) and the patterns resulting from the redistribution of spectrin were a reflection of those observed routinely in lymphocytes in situ. The correlation between naturally occurring spectrin localization patterns and those seen after membrane perturbation suggested the possibility that spectrin distribution is indicative of particular maturation stages or functional states in lymphocytes. The implications of these findings with regard to the role of spectrin in lymphocyte function are discussed.

Carrier Proteins↗
PubMed1987

Human lung tumor growth established in the lung and subcutaneous tissue of mice with severe combined immunodeficiency.

We report here that a mouse mutant (C.B-17 scid) which lacks functional B- and T-lymphocytes can be used to propagate a human lung tumor. The heterotransplanted tumor cells generated palpable s.c. tumors by 18 days in 100% of the mice inoculated s.c. with greater than 4 X 10(6) cells. All tumors grew progressively with no sign of regression. A portion of the scid mice given injections i.v. of the human lung tumor cells developed multiple tumor nodules in the lung by 15 weeks after the inoculation of tumor cells. The tumor nodules were shown by karyotype analysis to be human cells, and the histopathology of the tumor nodules revealed a pattern of growth that was consistent with that of the original tumor. The human lung tumor used in the study expresses an Mr 160,000 cell surface glycoprotein that has been shown to occur on a large proportion of human lung tumors and tumor cell lines. A monoclonal antibody specific for Mr 160,000 glycoprotein was used to demonstrate that this tumor-associated antigen is stably expressed by the s.c. tumors and the lung tumor nodules in the scid mice. The mutant mice with this severe combined immunodeficiency represent a new and viable model for propagating human tumors and for evaluating the efficacy of novel drug delivery protocols in the treatment of human cancer.

Animals↗
PubMed1987

Idiotype-specific T lymphocytes responsible for the selection of somatic variants of a B cell hybrid.

A B cell hybrid (2C3E1), which when cultivated in vitro stably expresses a serologically defined private idiotype on the cell surface, undergoes a somatic variation which culminates in the generation of idiotype-negative variants when propagated as a tumor in syngeneic BALB/c mice. Lyt-1+ and Lyt-2- BALB/c T cells derived from appropriately primed spleen cells when co-cultured with 2C3E1 tumor cells in vitro are responsible for the generation or selection of the idiotype-negative tumor variants. The idiotype-specific effector T cells responsible for the variant production in vitro and in vivo are triggered either by viable or irradiated 2C3E1 tumor cells, but not by soluble idiotype-positive monoclonal antibody secreted by the tumor. The idiotype-positive antibody did stimulate the production of the effector T cells when the soluble protein was covalently linked to the plasma membrane of BALB/c spleen cells, but not when the idiotype-positive protein was presented on allogeneic C57BL/6 spleen cells. A single exposure of spleen cells to 2C3E1 cells in vivo was sufficient to prime the idiotype-specific T cells, but the frequency and reproducibility of variant selection increased when the in vivo primed effector T cells were restimulated by irradiated 2C3E1 cells in vitro prior to co-cultivation with the wild-type 2C3E1 cells. The initial variant B cell lines could be segregated into several different phenotypes. However, after extensive cultivation either in vitro or after repeated transfer in vivo, all of the variant cell lines acquired a single stable phenotype that was characterized by the loss of both the surface and secreted idiotype marker and antigen-binding activity associated with the wild-type 2C3E1 cells.

Animals↗
PubMed1987

A new cross-reactive idiotype-defined family in the phthalate humoral immune response of mice. I. Linkage of VH-Xmp to IgCH allotype locus and mapping with respect to other known VH genes.

A cross-reactive idiotype family was previously identified from a very large library of phthalate-specific hybridoma clones. The prototype of this idiotype family is the hybridoma, 2E9, secreting an IgM antibody with phthalate specificity. A portion of both primary and secondary anti-phthalate antibodies elicited in all BALB/c mice tested expresses the 2E9 cross-reactive idiotype. This idiotype has now been found in the anti-phthalate antibodies of several other inbred strains of mice (A/HeHa, DBA/2, and C3Hf/HeHa) tested but not in C57BL/6 mice. Anti-phthalate antibodies elicited from congenic mice BC.8, which express the same IgCH allotype as BALB/c mice but possess C57BL/6 genetic background, contain the 2E9 cross-reactive idiotype, whereas this idiotype is not expressed on the anti-phthalate antibodies derived from another congenic mouse CB.20, which expresses a C57BL/6 IgCH allotype and a genetic background of the BALB/c strain. These results indicate that the gene controlling the 2E9 idiotype is closely linked to the IgCH allotype locus. The 2E9 cross-reactive idiotype was also found in all of the F1 mice (BALB/c X C57BL/6) tested, and the level of expression of this idiotype in the F1 mice was quantitatively equivalent to the allotype/idiotype homozygous mice. The expression of the 2E9 idiotype in the phthalate repertoire has been followed in 12 different wild mouse populations. As expected, the 2E9 idiotype was observed in a large proportion of the wild mouse strains. Surprisingly, several examples of nonconcordance in the expression of idiotype and allotype were observed in these mice. One likely explanation for the linkage breakdown is a crossing over of the heavy chain constant and variable region gene complexes. In the SM/J inbred strain of mice, where such a crossover has occurred, nonconcordance between allotype and 2E9 idiotype expression was demonstrated. By using the recombinant inbred BXD strains of mice, the VH gene encoding the 2E9 idiotype has been mapped with respect to other known VH gene families. Relative to other VH genes the VH-Xmp is situated very close to the IgCH gene region.

Chromosome Mapping↗
PubMed1987

Expression of mu and gamma 1 membrane forms of immunoglobulin segregate in somatic cell hybrids.

In the present investigation, we have utilized the somatic cell hybridization technique to generate an experimental model for studying the differential expression of membrane (mIg) and secreted (sIg) forms of immunoglobulin that characterize different stages of B cell development. We describe here that fusion of the dextran-binding myeloma, MOPC 104E (mu, lambda 1) and the phthalate-binding B cell hybridoma, 2C3E1 (gamma 1, kappa) results in the formation of antigen-specific, double hybrids (tribrids) that coexpress both parental secreted forms of Ig but express only one of the two possible membrane forms of immunoglobulin (Ig). This segregated expression of membrane Ig is a new and unexpected finding that has been substantiated here by both immunological and biochemical methods. Analysis by SDS-containing polyacrylamide gels (SDS-PAGE) reveals distinct and characteristic migration patterns for each of the four Ig heavy chains in the tribrids (mu membrane, mu secreted, gamma 1 membrane and gamma 1 secreted). Immunochemical analysis of the immunoglobulin from the tribrids confirms the coexpression of both secreted forms of immunoglobulin in most of the tribrid lines tested and indicates that about 30% of the tribrids express only phthalate-specific gamma 1 membrane Ig, while 38% express only dextran-binding mu membrane Ig. About 30% of the tribrids secrete both antibodies but express no membrane form and less than 1% are non-secretors. Approximately 2% initially express both membrane forms of Ig, as determined by immunocytoadherence assay using appropriate target cells but subsequently express only one membrane form during propagation in vitro. SDS-PAGE analysis of surface labeled tribrids confirms that in tribrids expressing membrane Ig, only a single mIg is synthesized. These results suggest that the expression of the secreted and membrane forms of immunoglobulin are separately regulated and the tribrids represent a model with which to study the mechanisms involved in the regulation of each structurally distinct immunoglobulin form.

Animals↗
PubMed1987

Antigen-specific drug-targeting used to manipulate an immune response in vivo.

The administration of dextran-conjugated cytosine arabinonucleoside (araC) to BALB/c mice at various times prior to but not subsequent to immunization with native dextran renders mice unresponsive to this thymic-independent antigen. These results demonstrate that the primary immune response to an antigen can be selectively and efficiently suppressed or eliminated in vivo by the delivery of a single dose of an appropriate antigen-cytotoxic drug conjugate. Evidence presented here indicates that the dextran-araC conjugate (toxogen) acts directly and selectively upon unprimed dextran-specific antibody-forming cell precursors, presumably by binding to their receptors and subsequent internalization of the resultant receptor-toxogen complexes. The resistance of antigen-primed mice to the cytotoxic effect of the toxogen could result from the failure of dextran-primed cells to reexpress antigen-specific receptors, from an alternative processing of the toxogen, or from the inability of the antigen-primed cells to internalize a second round of receptor-ligand complexes. We also determined that B cells responding to thymic-dependent antigens were not affected by the prior exposure to a toxogen. The inability to eliminate or suppress the primary response to a thymic-dependent antigen via the administration of a cytotoxic drug-antigen conjugate distinguishes the thymic-independent set of B cells from the thymic-dependent B-cell repertoire. The difference between these two B-cell compartments could be due either to differences in the amount of ligand bound to receptors or to differences in the trafficking patterns of receptor-ligand complexes within each cell type.

Animals↗
PubMed1987

Generation and use of an antigen-specific hybrid to study B-cell function.

Several important events are known to occur following the crosslinking of membrane-associated immunoglobulin (mIg) on B-lymphocytes. Among these include the internalization and re-expression of mIg. We have addressed two questions regarding the re-expression of mIg following an antigen-induced clearance of this B-cell receptor. 1) How long does it take for a cell to re-express its receptors after the first or second exposure to antigen, and 2) is this re-expression dependent upon the synthesis of new mIg? These questions, which in the past have been addressed primarily with heterogeneous populations of cells and using anti-immunoglobulin instead of antigen, are vital to understanding B-cell activation as well as antigen processing events. In order to address these questions, we have produced and characterized an antigen-specific B-cell hybrid 2C3E1 that expresses a membrane-associated form of immunoglobulin. A hybrid specific for a charged hapten (phthalate) was selected to avoid the possibility of nonspecific hydrophobic interactions of the binding ligand with the plasma membrane. After establishing the presence of mIg biochemically and demonstrating the ability of phthalate-keyhole limpet hemocyanin to induce the clearance of the phthalate-specific receptor from the plasma membrane, it was determined that re-expression of antigen-specific receptors was detected within one hour and completed by six hours after the first or second pulse with antigen and that this re-expression did not require the synthesis of new receptors. This later finding is novel and suggests that B-cell receptors are re-utilized or that a presynthesized pool of mIg exists within the cytoplasm of these cells. During the characterization of the 2C3E1 cell line, it was determined that this hybrid also produced a secreted form of the phthalate-specific immunoglobulin (sIg). An unexpected subsequent observation was that sIg was found associated with the cell surface in addition to membrane Ig. A likely source of this unexpected surface-associated sIg was observed as a vesicle on the surface of many cells that is associated with a high concentration of sIg. This finding and its possible relevance to Ig secretion per se are considered further in the paper which follows this one.

Animals↗
PubMed1987

Biochemical and ultrastructural characterization of a novel cell structure associated with immunoglobulin secretion in B-lymphocytes.

In the companion paper, it was established that a secretory form of immunoglobulin, sIg, is present at or near the cell surface. This unexpected occurrence of sIg was postulated to be due to the labelling of sIg which remains temporarily associated with the cell packaged in a vesicle which appears to bud from the plasma membrane at a single pole of the cell. The question that is addressed in this report is whether or not this polar accumulation of sIg represents a common pathway for the exit of this protein which is used by antibody-producing cells. This question is important since, in spite of the fact that the intracellular events associated with immunoglobulin synthesis (processing and movement between subcellular compartments) have been defined, very little data exists on how or where immunoglobulin finally leaves the plasma cell. This question was approached here by first demonstrating that the polar immunoglobulin secretory vesicles (ISV) are associated with several sIg-producing cells including other hybridomas, B-cell lines, and mitogen-activated spleen cells. The second approach was to characterize the ISV on the cell ultrastructurally and to establish that these vesicles are released from the cell carrying with them sIg. Isolated vesicles released from biosynthetically labeled Ig-producing cells were analyzed by SDS-PAGE in order to confirm the presence of sIg and to determine the number of other proteins associated with the ISV, their molecular weights, and the degree of disulfide crosslinking of the molecules comprising this structure. Finally, the kinetics of sIg release was established by a pulse chase protocol for biosynthetically labeled cells, and by monitoring the release of radioactive Ig from surface iodinated cells. As was predicted from our biochemical studies of the ISV, we observed a very slow phase of sIg release as well as a rapid release phase. Our studies have established that at least one of the pathways for the release of Ig from hybridomas, B-cell lines, and normal splenic B-cells is via a polar multivesiculated structure that we have termed ISV, and that the sIg can be released either as a free form of the protein or packaged within a satellite vesicle which may release the sIg later and perhaps at considerable distance from the cell that produced it.

Animals↗
PubMed1987

Effects of hyperthermia on spectrin expression patterns of murine lymphocytes.

In this study the influence of whole-body hyperthermia on the distribution of spectrin in murine lymphocytes isolated from various lymphoid tissues is examined. Lymphocytes normally vary in terms of the pattern of spectrin distribution within the cell. In certain populations of lymphocytes, spectrin is distributed into a dense submembranous aggregate that can be easily identified by immunofluorescence microscopy. In these lymphocytes, little or no spectrin is seen at the plasma membrane region in the rest of the cell. Other lymphocytes have no such cytoplasmic aggregates, and the protein is seen at the region of the plasma membrane. Following whole-body hyperthermia (40.5 degrees C for 90 min) there is a 100% increase in cells exhibiting polar spectrin aggregates in the spleen, while lymphocytes from the thymus show no alteration in the number of cells showing such aggregates. The increase in the percentage of splenic cells that express aggregated spectrin is a result of increases occurring in both T- and B-cell subsets. This increase gradually returns to control levels by 48 h post-heating. During recovery to control levels this phenomenon is resistant to additional changes when a second heat treatment is applied. The effects described above are not observed when the experiments are performed in vitro; therefore, it is likely that the in vivo heat-induced alteration in the splenic lymphocyte population reflects the physiological response of lymphocytes to stimuli during a natural fever. The role that spectrin may play in the modulation of lymphocyte membrane properties is discussed.

Animals↗
PubMed1986

Immunofluorescent patterns of spectrin in lymphocyte cell lines.

Spectrin, a membrane-associated cytoskeletal protein, has been observed in all of 45 lymphoid and myeloid cell lines examined. For these experiments, formalin-fixed cells from randomly selected lines propagated by using conventional tissue culture procedures were examined by immunofluorescence, using an antibody directed against chicken erythrocyte alpha-spectrin. Two distinct immunofluorescent patterns of spectrin distribution were identified. In most lines examined (16 mouse and 18 human lymphoid or myeloid lines), spectrin was symmetrically distributed near the submembranous region of the plasma membrane. In the remainder of the cell lines examined, a second pattern was observed; in these cultures, the cells contain a polar submembranous aggregate of spectrin with little staining at the rest of the plasma membrane. Long-term T lymphocyte cell lines in which greater than 60% of the cells expressed a polar submembranous aggregate of spectrin (PSA-S) include mouse cell lines EL-4, LBRM-33, CT-6X, NIXT, 22CM-37, and 7ON-2 and human lines JM and PEER. Other established cultures in which PSA-S were observed included the human macrophage-like line U-937 and gibbon T cell line MLA-144. Phorbol myristate acetate or mezerin caused a reversible alteration in the distribution of spectrin in these cell lines. These drugs, which increase membrane fluidity, caused a complete but temporary symmetrical redistribution of the spectrin aggregate. Our results indicate that the pattern of spectrin distribution, either aggregated or evenly dispersed, is a stable characteristic (but one that can be altered) in various cell lines, and that because similar variations in pattern have been noted in situ, it is likely that the pattern present in any given cell line reflects a characteristic associated with a particular stage of a cell's maturation. It is anticipated that these cell lines, positive and negative for the expression of natural polarity of spectrin distribution, will provide useful models for future studies to define further the role of spectrin in lymphocyte plasma membrane functions.

Animals↗
PubMed1986

Membrane-associated glycoprotein (gp 160) identified on human lung tumors by a monoclonal antibody.

A monoclonal antibody (5E8) has been used to identify and structurally characterize a previously unreported macromolecule present on the surface of human lung tumors. This antibody was derived from a hybrid clone that was produced using spleen cells of mice immunized with a surgically excised squamous cell carcinoma. Using immunofluorescence, the 5E8 antibody was observed to stain many different human lung tumor cell lines and surgically excised human lung tumors including squamous cell carcinomas, adenocarcinomas, alveolar carcinomas, and a portion of the large cell tumors tested. With few exceptions, notably the basal layer of the skin, little or no detectable staining of 5E8 to normal human tissues (lung, brain, kidney, heart, stomach, breast, erythrocytes, or lymphocytes) was observed. The 5E8 antibody was used to immunoprecipitate detergent lysates of biosynthetically labeled or surface radioiodinated lung tumors. Analysis of the immunoprecipitates by sodium dodecyl sulfate gel electrophoresis revealed a major band and a faster migrating second minor band. The molecular weights of these two proteins were estimated to be 160,000 and 120,000, respectively. The addition of a reducing agent to the gels did not alter the migration pattern of the immunoprecipitated macromolecules. The removal of a terminal carbohydrate, sialic acid, did not restrict the binding of 5E8 to the tumor-associated antigen. However, labeling studies using galactose oxidase and tritiated borohydride revealed the presence of galactose on the immunoprecipitated protein. This major Mr 160,000 glycoprotein that was identified on two different human lung tumor cell lines was also found on a human large cell tumor tissue obtained by surgical biopsy. The 5E8 antibody and the Mr 160,000 glycoprotein that it recognizes represent two very useful components with which to test several new antibody-mediated drug delivery systems in the treatment of human lung tumors. The tumor-associated glycoprotein also represents a potential analyte for a diagnostic or prognostic immunoassay for lung cancer.

Antibodies, Monoclonal↗