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M K Collins

Publications and source records attributed to M K Collins.

90 records · Page 5Linked to original sources
PubMed1989

Species specificity of interleukin 2 binding to individual receptor components.

The affinity of recombinant human and murine interleukin 2 (IL 2), which differ significantly in structure in the amino terminal alpha helix, for the human or murine interleukin 2 receptors was compared. Murine IL 2 showed a 10-fold higher affinity than human IL 2 for the murine high-affinity receptor expressed on T lymphocytes, whereas human IL 2 bound 100-fold more strongly than murine IL 2 to the human high-affinity receptor. As the high-affinity IL 2 receptor on T lymphocytes has been demonstrated to be composed of two IL 2 binding components, p55 and p75, the role of the individual chains in the determination of species specificity was then investigated. Recombinant retroviral vectors were used to generate populations of 3T3 fibroblasts expressing either human or murine p55 in isolation, and their binding characteristics were determined. While the murine p55 showed an identical species preference to the murine p55/p75 high-affinity complex, the human p55 had an equal affinity for human and murine IL 2. This suggested that the human p75 chain was responsible for the preference for human IL 2 shown by the human high-affinity receptor, which was confirmed by performing a binding experiment using gibbon MLA 144 cells, expressing only p75. These data suggest that the amino terminal region of the IL 2 molecule interacts with the p75 chain of the IL 2 receptor.

Animals↗
PubMed1988

Transfer of functional EGF receptors to an IL3-dependent cell line.

Epidermal growth factor (EGF) is a small protein that acts as a mitogen for various epidermal, epithelial, and fibroblastic cells that bear specific EGF receptors. The molecule that binds EGF is a 175-kD transmembrane protein, with an extracellular ligand binding domain and an intracellular domain that possesses tyrosine kinase activity, thought to be involved in the mitogenic signalling process. Here we have constructed a recombinant murine retrovirus that transduces a human cDNA encoding the 175-kD protein and used this retrovirus to infect BAF3, a murine, bone marrow-derived cell line, which is dependent on the haematopoietic factor interleukin-3 (IL3) for its growth in culture. The EGF receptors expressed in the infected cells exhibit two affinity states, as well as EGF-stimulated autophosphorylation. Furthermore, EGF can replace IL3 in supporting short-term proliferation of these cells. These data identify functional properties of the EGF receptor upon expression of the 175-kD EGF binding protein in a haemotopoietic cell that does not express endogenous receptors. They also suggest that gene transfer of growth factor receptors to heterologous cells may allow novel growth stimuli to be exploited.

Animals↗
PubMed1988

The local use of vitamin D to increase the rate of orthodontic tooth movement.

The purpose of the study was to determine if the rate and amount of orthodontic tooth movement in a sample of cats could be enhanced by the injection of a vitamin D metabolite 1,25-dihydroxycholecalciferol (1,25D) into the periodontal ligament. After 21 days of canine retraction with a light-wire retraction spring, the teeth that had received weekly intraligamentous injections of a solution of 1,25D in dimethylsulfoxide (DMSO) had moved 60% further than matched control teeth (P less than 0.05). At the histologic level, increased numbers of mononuclear osteoclasts were recruited and activated, resulting in greater amounts of alveolar bone resorption on the pressure side of the periodontal ligament. No obvious clinical, microscopic, or biochemical side effects were noted.

Animals↗
PubMed1988

Autocrine regulation of T-lymphocyte proliferation: differential induction of IL-2 and IL-2 receptor.

Three stimuli were used to compare the signals necessary for interleukin (IL-2) receptor expression and IL-2 production: triggering of the T-cell antigen receptor/CD3 complex (Ti/CD3) by CD3 antibodies, activation of protein kinase C (PKC) by phorbol esters, and elevation of intracellular calcium levels by calcium ionophore. The salient observations were that IL-2 responsiveness, which reflects IL-2 receptor expression, and T-cell proliferation which requires both IL-2 production and IL-2 receptor expression, are not co-ordinately regulated. Firstly, a low threshold of CD3 activation or a brief (1 hr) exposure of T cells to maximal CD3 stimulation is sufficient to induce IL-2 responsiveness, but higher levels of activation for a prolonged period are necessary to ensure a T-cell proliferative response. Secondly, in response to optimal T-cell stimulation there is a short (2-4 day) period of T-cell proliferation followed by a prolonged phase of IL-2 responsiveness (10-14 days). Differences in the kinetics and signalling requirements for IL-2 receptor expression and IL-2 production, regulated at the level of mRNA expression, provide a molecular basis for these observations. A major difference between induction of IL-2 production and IL-2 receptor expression is that the dual signals of calcium and PKC are necessary for IL-2 production, but a sole stimulus of PKC is sufficient for IL-2 receptor expression. Also, a low level stimulation of PKC will induce IL-2 receptor expression but higher levels of PKC stimulation are required for IL-2 production. As a consequence, triggering of a single receptor, namely the Ti/CD3 complex, results in IL-2 responsiveness, but an additional signal that activates PKC is necessary for IL-2 production. These observations suggest that a Ca2+/PKC dual signal model does not explain completely the signal transduction pathways that regulate T-cell growth. Moreover, precise regulatory mechanisms have evolved to control the homeostasis of the autocrine proliferative response of a T-cell population.

Antibodies, Monoclonal↗
PubMed1985

Transcripts from an aberrantly re-arranged human T-cell receptor beta-chain gene.

A number of cDNA clones homologous to the human T-cell receptor beta-chain gene have been isolated from a library constructed from the human leukaemic cell line Jurkat. The nucleotide sequences of two of these clones demonstrate that Jurkat synthesizes an RNA, probably derived from an aberrantly rearranged C beta 1 gene, which cannot encode a functional beta-chain. This transcript contains a novel joining region and similar transcripts appear to be relatively abundant in several T-cell lines.

Base Sequence↗
PubMed1985

Regulation of T-cell receptor gene expression in human T-cell development.

A cDNA clone encoding the alpha chain of the human T-cell antigen receptor was isolated by screening a library from the human T-cell line Jurkat with a mouse alpha-chain cDNA clone. This human alpha-chain clone, together with a human antigen receptor beta-chain cDNA clone, was used to determine the stage of T-cell development at which antigen receptor mRNAs first appear. Blot-hybridization analysis of mRNA isolated from a panel of human thymic tumor lines clearly demonstrated that beta-chain transcripts could be detected in all T-lineage cells. However, alpha-chain transcripts were only found in the most phenotypically mature lines, which express the antigen receptor-associated molecule T3. Furthermore, beta-chain transcripts were abundant in RNA prepared from purified T3-negative thymocytes, whereas alpha-chain transcripts were virtually absent. From these results we conclude that alpha-chain expression occurs later in thymic ontogeny than that of the beta chain and propose that it controls surface expression of the antigen receptor-T3 complex.

Amino Acid Sequence↗
PubMed1984

A human T-cell antigen receptor beta chain gene maps to chromosome 7.

cDNA clones which encode the human and mouse T cell antigen receptor beta chain gene have previously been isolated. We have used a mouse cDNA clone to map the chromosomal position of a human beta chain gene. Southern blot analysis of DNA prepared from somatic cell hybrids has assigned this gene to chromosome 7. The use of a hybrid containing a chromosome 7 translocation has further localised this gene to the region 7q22-qter.

Animals↗
PubMed1984

Homologous and heterologous mitogenic desensitization of Swiss 3T3 cells to phorbol esters and vasopressin: role of receptor and postreceptor steps.

Prolonged treatment of Swiss 3T3 cells with phorbol 12,13 dibutyrate (PDB) rendered the cells refractory to subsequent mitogenic stimulation by both PDB and vasopressin. In contrast, the cells retained full responsiveness to a wide variety of other mitogens. An early response to vasopressin and phorbol esters, inhibition of (125I)-labeled epidermal growth factor [(125I)-EGF] binding, was also substantially decreased in PDB pretreated cells. The cross desensitization was not produced by vasopressin; this ligand induced homologous but not heterologous desensitization. Exposure of Swiss 3T3 cells to PDB caused a down regulation of (3H)-PDB receptors but did not reduce the binding of vasopressin to refractory cells. The time-course (t1/2 = 7 h) and dependence on PDB concentration (half maximal at 20 nM) for this phorbol ester receptor loss paralleled the induction of the mitogenic desensitizations to both PDB and vasopressin. However, the time-course of recovery revealed an important dissociation between receptor presence and mitogenic response. When Swiss 3T3 cultures, which had been pretreated with PDB, were washed to remove this ligand and incubated in its absence for 24 h, both (3H)-PDB receptors and PDB or vasopressin inhibition of (125I)-EGF binding were almost completely restored to control levels. However the homologous and heterologous mitogenic desensitizations showed a very different reversal time. After a 24-h recovery period PDB-treated refractory cells were still unable to synthesize DNA in response to PDB or vasopressin. The mitogenic desensitizations were however completely reversible; after a 48-h incubation in the absence of PDB the cells responded fully to the mitogenic actions of PDB or vasopressin. This finding suggests that a further postreceptor step was also desensitized by prolonged PDB treatment. The presence of a low level of cycloheximide during the PDB pretreatment blocked induction of this postreceptor refractoriness. We propose that this refractory postreceptor step selectively blocks both PDB and vasopressin stimulation of DNA synthesis and may represent the point at which the mitogenic pathways of phorbol esters and vasopressin converge.

Animals↗
PubMed1984

Insulin induces a selective heterologous desensitization of the mitogenic response of Swiss 3T3 cells to vasopressin.

Prior incubation of confluent, quiescent cultures of Swiss 3T3 cells with insulin leads to a selective loss of mitogenic stimulation on re-addition of the combination of vasopressin and insulin in serum-free medium. The desensitization is specific for the action of vasopressin as insulin is fully active in the refractory cells when added in combination with other mitogens, whereas vasopressin is not. A prolonged treatment with insulin is required for induction of the refractoriness, half-maximal loss of response occurs after about 7 h and desensitization is complete after 12 h treatment. The refractory cells recover their response to vasopressin after more than 24 h incubation in the absence of insulin. A rapid response of the cells to vasopressin, inhibition of 125I-epidermal growth factor (125I-EGF) binding, is also desensitized by insulin. Desensitization is induced by insulin-like growth factor I (IGF-I), and partially by desoctapeptide insulin, but not by insulin B chain. Although the characteristics of insulin-induced desensitization are very similar to those of the homologous desensitization induced by vasopressin treatment, insulin does not bind to vasopressin antiserum or the [3H]vasopressin receptors of Swiss 3T3 cells. Insulin treatment also does not lead to any down-regulation of [3H]vasopressin receptors, and the refractoriness of the cells must therefore lie at a post-receptor step. Both insulin- and vasopressin-induced refractoriness to the mitogenic action of vasopressin can be blocked by a low level of cycloheximide. Both these agents therefore seem to induce the synthesis of specific protein(s) which selectively inhibit the mitogenic response of the cell to vasopressin.

Animals↗
PubMed1983

Platelet-derived growth factor treatment decreases the affinity of the epidermal growth factor receptors of Swiss 3T3 cells.

Platelet-derived growth factor (PDGF) inhibits markedly (90%) the binding of a low concentration (0.5 ng/ml) of 125I-labeled epidermal growth factor (125I-EGF) to Swiss 3T3 cells in a dose, time, and temperature-dependent manner. In contrast, PDGF inhibits the binding of a high concentration (20 ng/ml) of 125I-EGF by only 20%. Detailed Scatchard analysis of 125I-EGF binding to Swiss 3T3 cells at 4 degrees C, after a 2-h incubation with a saturating level of PDGF at 37 degrees C, demonstrates that this PDGF pretreatment does not change the number of EGF receptors (70,000 sites/cell), but causes a large decrease in the affinity of the receptors, from a mixed population of 0.5-12 ng/ml, to an affinity of 13 ng/ml. A parallel treatment at 37 degrees C with EGF itself causes a 70% decrease in the number of EGF receptors. Therefore, we conclude, in contrast to previous reports, that PDGF (unlike EGF) does not cause down-regulation of EGF receptors. Rather PDGF induces a potent, temperature-dependent change in the affinity of the EGF receptor population ("transmodulation").

Animals↗
PubMed1983

Mitogenic effect of prostaglandin E1 in Swiss 3T3 cells: role of cyclic AMP.

Addition of prostaglandin E1 (PGE1) to quiescent cultures of Swiss 3T3 cells rapidly elevates the intracellular levels of cAMP and increases the activity of adenylate cyclase in particulate fractions of these cells. In the presence of insulin, PGE1 stimulates the reinitiation of DNA synthesis. Both effects (increase in cellular cAMP and stimulation of DNA synthesis) are markedly potentiated by 1-methyl-3-isobutyl xanthine (IBMX) or by 4-(3-butoxy-4 methoxy benzyl)-2-imidazolidine (Ro 20-1724), both of which are potent inhibitors of cyclic nucleotide phosphodiesterase activity. In the presence of 50 microM IBMX, PGE1 caused a dose-dependent increase in cAMP levels and in [3H]thymidine incorporation into acid-insoluble material at concentrations (5-50 ng/ml) that are orders of magnitude lower than those used in previous studies (50 micrograms/ml) to demonstrate growth-inhibitory effects. Thus, the inhibitory effects produced by adding high concentrations of PGE1 on the initiation of DNA synthesis in Swiss 3T3 cells are not mediated by cAMP and should be regarded as nonspecific. In contrast, the mitogenic activity of PGE1 parallels its ability to increase the intracellular levels of cAMP. The findings support the proposition that a sustained increase in the level of this cyclic nucleotide acts as a mitogenic signal for confluent and quiescent Swiss 3T3 cells.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Vasopressin induces selective desensitization of its mitogenic response in Swiss 3T3 cells.

Prior incubation of quiescent cultures of Swiss 3T3 cells with vasopressin leads to loss of mitogenic stimulation on its subsequent addition in the presence of a synergistic growth factor. This desensitization is selective for vasopressin, requires prolonged incubation (half-maximal desensitization after 12 hr of treatment) for its induction, and is reversed after a 48-hr incubation in the absence of vasopressin. It is elicited by concentrations of vasopressin, and several analogues, similar to those required for stimulation of DNA synthesis. Inhibition of 125I-labeled epidermal growth factor binding and stimulation of 86Rb+ uptake by vasopressin are also selectively decreased in the refractory cells. The vasopressin receptors that mediate mitogenesis in Swiss 3T3 cells are of the pressor type, not coupled to adenylate cyclase. These cells bind [3H]vasopressin in a specific and saturable (Kd = 1 X 10(-8) M) manner. The receptors are down-regulated after prolonged vasopressin treatment; however, this cannot provide a complete explanation of desensitization because cells that are completely refractory to vasopressin retain 60% of their [3H]vasopressin binding sites. Vasopressin refractoriness must therefore occur partly at a post-receptor locus.

Animals↗
PubMed1982

Binding of phorbol esters to high-affinity sites on murine fibroblastic cells elicits a mitogenic response.

The level of occupancy of a single class of high-affinity (3H)-phorbol 12, 13-dibutyrate (PDBu) binding sites (Kd = 26 nM) in quiescent Swiss 3T3 cells was correlated with the dose of PDBu required to stimulate rapid (increase in 86Rb uptake, decrease in epidermal growth factor receptor affinity) and long-term (induction of 2-deoxyglucose uptake, initiation of DNA synthesis) events of the proliferative response. Further, structural analogues of PDBu showed identical relative potencies in the inhibition of (3H)-PDBu binding and stimulation of DNA synthesis. Finally, prolonged (24-hour) incubation of Swiss 3T3 or whole mouse embryo fibroblasts with 400 nM PDBu led to a decrease in the number of (3H)-PDBu binding sites (down-regulation) with a parallel loss of rapid and long-term responses of the cells to PDBu (desensitization). These findings indicate that the high-affinity (3H)-PDBu binding sites mediate the mitogenic effects of phorbol esters in fibroblastic cells.

Animals↗
PubMedDate not supplied

Monoclonal antibodies for the treatment of metastases. Evaluation of strategies using a syngeneic rat model.

To investigate critical factors influencing the localization and antitumor effects of monoclonal antibodies (MAb) or toxic conjugates, we have adapted a single rat sarcoma, HSN, for preferential growth in the lungs, liver, and lymph nodes (the major sites of metastasis in humans) and have raised a panel of syngeneic rat MAbs to a stably-expressed cell surface antigen. Using this model we have shown that localization in tumors is significantly influenced by their anatomical location and vascularization, and the degree of MAb interaction with host cells. Uptake in small hepatic tumors was excellent, but access to lung tumors was limited by the poor permeability of pulmonary vessels. HSN cells transfected with th human IL-2 gene and coinjected in low numbers with parental tumors secreted sufficient cytokine to enhance the local permeability of vessels and doubled MAb localization in tumors without any systemic toxicity, suggesting that regional delivery of IL-2 may be used to enhance MAb localization in this situation. In order to extent the applicability of the model to studies of MAbs raised against human tumor targets, we have transfected the human c-erb B-2 gene (homolog of the rat neu) into the highly metastatic HSN.LV subline. MAbs raised against the external domain of the p185 product can now be screened for their ability to localize in metastases, and for various conjugates to inhibit tumor growth either independently of, or in association with, a fully functional immune system.

Animals↗
PubMedDate not supplied

The human T-cell receptor alpha-chain gene maps to chromosome 14.

The T-cell receptor for antigen has been identified as a disulphide-linked heterodimeric glycoprotein of relative molecular mass (Mr) 90,000 comprising an alpha- and a beta-chain. The availability of complementary DNA clones encoding mouse and human beta-chains has allowed a detailed characterization of the genomic organization of the beta-chain gene family and has revealed that functional beta-chain genes in T cells are generated from recombination events involving variable (V), diversity (D), joining (J) and constant (C) gene segments. Recently, cDNA clones encoding mouse and human alpha-chains have been described; the sequences of these clones have indicated that functional alpha-chain genes are also generated from multiple gene segments. It is possible that chromosomal translocations involving T-cell receptor alpha- and beta-chain genes have a role in T-cell neoplasms in much the same way as translocations involving immunoglobulin genes are associated with oncogenic transformation in B cells. In the latter case, the chromosomal localization of the immunoglobulin genes provided one of the first indications of the involvement of such translocations in oncogenic transformation. The chromosomal assignment of the alpha- and beta-chain genes may, therefore, provide equally important clues for T-cell neoplastic transformation. The chromosomal location of the mouse and human beta-chain gene family has been determined: the murine gene lies on chromosome 6 (refs 12, 13) whereas the human gene is located on chromosome 7 (refs 13, 14). Here we use a cDNA clone encoding the human alph-chain to map the corresponding gene to chromosome 14.

Animals↗