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J F Lesley

Publications and source records attributed to J F Lesley.

8 recordsLinked to original sources
PubMed1998

Site-specific de-N-glycosylation of CD44 can activate hyaluronan binding, and CD44 activation states show distinct threshold densities for hyaluronan binding.

CD44 is a cell surface receptor for the glycosaminoglycan hyaluronan (HA). Not all CD44-positive cells bind HA, and binding ability is strictly regulated. Three different HA binding states have been defined: inactive, inducible (by certain CD44-specific monoclonal antibodies), and constitutively active. The observation that sets of genetically related cell lines representing different HA binding states showed correlated differences in N-glycosylation of CD44, and that inhibition of N-glycosylation enhanced HA binding (Lesley et al., J. Exp. Med., 182: 431-437, 1995) led us to examine directly whether specific N-glycosylation site modifications were involved in regulating the HA binding function. CD44-negative, -active, and inducible cell lines were stably transfected with mutant constructs in which each of the five N-glycosylation sites of murine CD44 had been separately inactivated. Ability to bind soluble HA was examined over a range of CD44 expression levels. For the active cell line, AKR1, transfectants for all N-glycosylation mutants bound HA as well as did transfectants for wild type CD44. No inhibitory effects of inactivating specific N-glycosylation sites were observed. HA binding was activated when two of the mutant constructs were transfected into a novel CD44-negative inducible cell line. Inactivation of N-glycosylation sites at residues 25 or 120 converted the inducible cell line to constitutively active, whereas inactivation of other sites had little or no effect. Fusion proteins secreted from inactive, inducible, or active cell lines were purified, bound to beads, and assayed for HA binding activity by flow cytometric analysis. Fusion proteins derived from inactive, inducible, and constitutively active cells exhibited three distinguishable "threshold" densities required for HA binding ability. The results imply that the CD44 molecules produced in cells in these three activation states have intrinsic differences in HA binding function. Treatment of the fusion proteins with neuraminidase altered the HA binding state, and glycosylation mutations that affected the phenotype of the inducible cell line lowered the threshold required for HA binding of CD44-immunoglobulin fusion proteins derived from the inducible cell line. Thus, alterations of glycosylation of CD44 itself can affect HA binding ability as manifested by a change in HA binding state.

Antibodies, Monoclonal↗
PubMed1987

Effects of monoclonal antibodies that block transferrin receptor function on the in vivo growth of a syngeneic murine leukemia.

The ability of monoclonal antibodies (MAbs) against the murine transferrin receptor to inhibit the growth of transplanted syngeneic AKR/J SL-2 leukemic cells has been investigated. Two rat IgM antibodies, RI7 208 and REM 17.2, which both block transferrin receptor function, inhibited the growth of SL-2 leukemic cells in vitro at concentrations of 5-10 micrograms per ml. However, RI7 208 was more effective than REM 17.2 in prolonging survival of tumor-bearing mice. The antitumor effects of RI7 208 MAb were dependent on both the antibody dose and number of leukemic cells inoculated. The serum clearance of [75Se]methionine-labeled RI7 208 and REM 17.2 antibodies was similar and consisted of an initial rapid phase over the first 2 days followed by a slower phase. A single dose of 2 mg of antibody maintained a serum MAb concentration (greater than 10 micrograms/ml) sufficient to inhibit SL-2 leukemic cell growth in vitro for 2-3 days. The liver, kidney, and spleen were the major sites at which each of the antibodies accumulated regardless of whether trace or saturating amounts of antibody were administered. The specific activity of antibody found in s.c. SL-2 tumors was about 2-fold less than that of liver. It was shown that multiple doses of R17 208 MAb administered on a schedule aimed at maintaining a therapeutic serum level of MAb for 1-3 weeks were more effective than a single dose. Further, administration of RI7 208 MAb, in combination with the anti-Thy-1.1 MAb 19E12, was more effective than either antibody alone. SL-2 mutant cells were selected that were resistant to growth inhibitory effects of RI7 208 in vitro. The effects of RI7 208 MAb on the growth of these mutant cells in vivo suggests the major mechanism by which the MAb inhibits SL-2 tumor growth is by directly blocking receptor function. Acute toxicity associated with administration of the MAb was minimal. However, assays of myeloid and erythroid colony-forming units in bone marrow and spleen of mice given multiple doses of RI7 208 showed a depression of stem cell activity in bone marrow and elevated numbers of erythroid and cellular colony-forming units in the spleen.

Animals↗
PubMed1985

Inhibition of cell growth by monoclonal anti-transferrin receptor antibodies.

Five anti-murine transferrin receptor monoclonal antibodies have been characterized with respect to immunoglobulin class, effects on binding of transferrin, and effects on AKR1 lymphoma cell growth in vitro. The immunoglobulin M (IgM) antibodies, but not the IgG antibodies, prevent cell growth. We suggest that the profound effects of the IgM antibodies on cell growth are probably due to extensive cross-linking of cell surface receptors. In support of this, we are able to mimic the growth-inhibiting effects of the IgM antibodies by adding antiimmunoglobulin to an IgG antibody. By flow microfluorimetry, we show that an IgG antibody by itself induces up to a 10-fold downward regulation in the cell surface transferrin receptor, which is accompanied by accelerated receptor degradation. A similar downward regulation is seen in mutant cells resistant to growth inhibition by an IgM antibody, when grown in the selecting antibody. Wild-type cells grown in the presence of IgM antibody do not show receptor downward regulation. Inhibitory effects of antibody plus antiimmuoglobulin on mutant cells are also consistent with extensive cross-linking causing inhibition of growth.

Animals↗
PubMed1984

Selection of cell lines resistant to anti-transferrin receptor antibody: evidence for a mutation in transferrin receptor.

Some anti-murine transferrin receptor monoclonal antibodies block iron uptake in mouse cell lines and inhibit cell growth. We report here the selection and characterization of mutant murine lymphoma cell lines which escape this growth inhibition by anti-transferrin receptor antibody. Growth assays and immunoprecipitation of transferrin receptor in hybrids between independently derived mutants or between mutants and antibody-susceptible parental cell lines indicate that all of the selected lines have a similar genetic alteration that is codominantly expressed in hybrids. Anti-transferrin receptor antibodies and transferrin itself still bind to the mutant lines with saturating levels and Kd values very similar to those of the parental lines. However, reciprocal clearing experiments by immunoprecipitation and reciprocal blocking of binding to the cell surface with two anti-transferrin receptor antibodies indicate that the mutant lines have altered a fraction of their transferrin receptors such that the growth-inhibiting antibody no longer binds, whereas another portion of their transferrin receptors is similar to those of the parental lines and binds both antibodies. These results argue that the antibody-selected mutant cell lines are heterozygous in transferrin receptor expression, probably with a mutation in one of the transferrin receptor structural genes.

Animals↗
PubMed1978

Thy-L antigen expression on rat brain cell lines.

Cell lines derived from the central nervous system of rats were screened serologically for the presence of Thy-1 (theta), a cell surface differentiation antigen shared by brain and thymus of rats and mice. Both cytotoxicity absorption and indirect immunofluorescence assays were performed using a rabbit anti-rat thymocyte serum (ATS) with Thy-1 specificity. The complement-dependent cytotoxicity of ATS detected a mouse-rat cross-reacting determinant of the molecule bearing the Thy-1 antigen. Of 20 lines tested, 1 of 6 neuronal and 7 of 14 non-neuronal lines expressed Thy-1, as judged by their capacity to absorb ATS cytotoxicity for a Thy-1 positive thymoma line. Similar results were obtained in quantitative absorption assays of these lines employing a mouse anti-Thy 1.1 alloantiserum, but the xenoantiserum (ATS) was more sensitive for detecting the rat molecule bearing Thy-1. Indirect immunofluorescence, which was performed on several of the lines, yielded results in complete agreement with the cytotoxicity absorption assays, and revealed a generalized distribution of antigen in a speckled or patchy pattern over the membrane of cell bodies and processes.

Animals↗
PubMed1971

Immunoglobulins on the surface of thymus-derived cells engaged in the initiation of a humoral immune response.

Preculture treatment of normal spleen cells with antiserum against mouse kappa light chains and complement was found to inhibit in vitro responses of these cells to TNP and erythrocyte (carrier) antigens, primarily by elimination of a thymus-derived helper component required for the response. Spleen populations inactivated in this way could be reconstituted with irradiated, carrier-immune spleen cells or with carrier-educated thymus-derived spleen cells. The ability of helper populations (i.e. irradiated, carrier-immune spleen cells or carrier-educated thymus-derived spleen cells) to enhance the response of normal spleen cells to hapten was eliminated by pretreatment of the helper cells with anti-kappa serum and complement. No significant effect of anti-kappa and complement treatment on precursor cell populations in normal spleen or bone-marrow-derived spleen could be demonstrated. The data are interpreted as evidence for the presence of immunoglobulin components. The function of these molecules is not established but it would be reasonable to assume that they are involved in antigen recognition, on the surface of thymus-derived cells.

Animals↗