Growth control in cultured 3T3 fibroblasts II. Molecular properties of a fraction enriched in growth inhibitory activity

Treatment of sparse, proliferating cultures of 3T3 cells with medium conditioned by exposure to density-inhibited 3T3 cultures resulted in an inhibition of growth and division in the target cells when compared to similar treatment with unconditioned medium. This growth inhibitory activity was fractionated by ammonium sulfate precipitation and gel filtration, yielding one fraction that was 35-fold enriched in specific activity. Analysis of the chemical and biological properties of this highly active fraction indicated that: (a) it is an endogenous cell product, synthesized by the 3T3 cells and shed into the medium; (b) it is a protein and its activity is sensitive to treatment with pronase; (c) the constituent polypeptide chains have molecular weights of 10,000 and 13,000; and (d) it is not cytotoxic and its effect on target cells are reversible. These results suggest that we have partially purified from conditioned medium an endogenous growth regulatory factor that may play a role in density-dependent inhibition of growth in cultured fibroblasts. We propose the term Fibroblast Growth Regulator to describe this class of molecules.     

Epidermal growth factor receptor. Characterization of a monoclonal antibody specific for the receptor of A431 cells

A monoclonal antibody to the epidermal growth factor (EGF) receptor of A431 cells was obtained after fusion of immunized BALB/c mouse spleen cells with NS-1 myeloma cells. Specific binding of the antibody to the plasma membrane of A431 cells was demonstrated by indirect immunofluorescence and electron microscopy. The antibody did not react with human KB cells, normal rat kidney cells, or Swiss 3T3 cells. The antibody is an IgG3K; it specifically immunoprecipitated a Mr approximately 170,000 protein from radiolabeled A431 cell extracts. This protein is phosphorylated in a EGF-dependent manner in intact A431 cells and in Triton X-100-solubilized plasma membranes. The specificity of the interaction of the antibody with the Mr = 170,000 protein was confirmed by electrophoretic transfer of A431 cell proteins to nitrocellulose followed by incubation with the antibody and 125I-protein A. When 125I-EGF was covalently cross-linked to its receptor, the 125I-EGF-receptor complex was specifically precipitated by the antibody. The monoclonal antibody did not inhibit the binding of 125I-EGF to its receptor in intact A431 cells and also failed to stimulate the phosphorylation of the Triton X-100-solubilized EGF receptor. The results indicate that the antibody and EGF bind to different sites on the EGF receptor. The antibody will be useful for isolating the EGF receptor in an unactivated form.     

The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis.

Mouse anti-Fas monoclonal antibody has a cytolytic activity on human cells that express the antigen. Complementary DNAs encoding the cell surface antigen Fas were isolated from a cDNA library of human T cell lymphoma KT-3 cells. The nucleotide sequence of the cDNAs revealed that the molecule coding for the Fas antigen determinant is a 319 amino acid polypeptide (Mr 36,000) with a single transmembrane domain. The extracellular domain is rich in cysteine residue, and shows a similarity to that of human tumor necrosis factor receptors, human nerve growth factor receptor, and human B cell antigen CD40. Murine WR19L cells or L929 cells transformed with the human Fas antigen cDNA were killed by the anti-Fas antibody in the process known as apoptosis.     

Neurotrophic action of gliostatin on cortical neurons. Identity of gliostatin and platelet-derived endothelial cell growth factor.

Gliostatin is a polypeptide growth inhibitor of apparent M(r) = 100,000 with a homodimeric structure comprising two 50-kDa subunits, acting on astrocyte as well as astrocytoma cells (Asai, K., Hirano, T., Kaneko, S., Moriyama, A., Nakanishi, K., Isobe, I., Eksioglu, Y.Z., and Kato, T. (1992) J. Neurochem., 59, 307-317). The amino acid sequences of 13 tryptic peptides including the amino terminus were completely identical to those of platelet-derived endothelial cell growth factor (PD-ECGF) (Ishikawa, F., Miyazono, K., Hellman, U., Drexler, H., Wernstedt, C., Hagiwara, K., Usuki, K., Takaku, F., Risau, W., and Heldin, C.-H. (1989) Nature 338, 557-562). Gliostatin and PD-ECGF, purified from human placenta, shared growth inhibition on glial cells and growth promotion on endothelial cells, and exhibited similar values for half-maximal dose of glial growth inhibition (ID50 = 1.3 nM) and the half-maximal concentration of endothelial growth promotion (EC50 = 1.0 nM), suggesting that both factors evoke the biological actions through an identical receptor on each cell surface. We have further demonstrated evidence of a novel neurotrophic action of gliostatin/PD-ECGF toward embryonic rat cortical neurons in culture. The half-maximal concentration of gliostatin/PD-ECGF for neurotrophic action was 0.3 nM. All actions on glial, endothelial, and neuronal cells, were abolished by a monoclonal antibody against gliostatin. These data indicate that gliostatin/PD-ECGF may play important roles on development and regeneration of the central nervous system and may also involve the induction of angiogenesis for the formation of blood brain barrier.     

Purification and characterization of a growth factor from guinea pig harderian gland

A polypeptide growth factor, Harderian gland-derived growth factor (HGDGF), has been purified approximately 43,000-fold from guinea pig Harderian gland by column chromatography on TSK gel DEAE-5PW, blue-Sepharose CL-6B, and Superose 12. The yield was approximately 10%. The Superose 12 fraction was further purified by Aquapore BU-300 reversed-phase chromatography to apparent homogeneity. HGDGF was eluted from TSK gel DEAE-5PW at 0.20-0.35 M NaCl, with a linear gradient of 0.15-0.80 M NaCl and at 2.20 M NaCl from blue-Sepharose CL-6B. The activity of HGDGF toward human embryonic cells (TIG-3) was quantitated, [3H]thymidine incorporation for 48 h being stimulated in a linear and dose-dependent manner. Purified HGDGF has a molecular weight of approximately 13,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular sieve column chromatography. HGDGF is labile to treatment with SH reagents or acetic acid. Both trypsin digestion and boiling decrease the activity of HGDGF. Its pI is 5.1. HGDGF stimulates the multiplication of TIG-3 cells but has no effect on human endothelial cells K2T1 or A2T2 which require fibroblast growth factor for growth. HGDGF appears to differ from other growth factors, suggesting that it is a previously undescribed growth factor.     

Partial purification and characterization of a growth factor present in goat's colostrum. Similarities with platelet-derived growth factor.

A factor in goat's colostrum which stimulates DNA synthesis and cell proliferation in Swiss 3T3 fibroblasts has been purified approx. 350-fold by a sequence of acid precipitation, cation-exchange chromatography and gel filtration. The growth factor is a highly basic, heat stable (100 degrees C for 5 min) polypeptide with Mr approx. 35000. The polypeptide resists denaturation by guanidinium chloride or urea but is totally inactivated by treatment with reducing agents. The factor, which we have termed colostric basic growth factor ( CBGF ), inhibits the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 fibroblasts but does not inhibit 125I-EGF binding to epidermoid A431 cells. CBGF interacts synergistically with plasma in stimulating DNA synthesis in quiescent Swiss 3T3 cells. The chemical and biological properties of CBGF are thus very similar to the properties reported for the human platelet-derived growth factor. Although high concentrations of CBGF are present in the colostrum of goats, cows, and sheep, the milk of these species contains little or no factor. The origin and possible functions of CBGF are unknown.     

K562 erythroleukemia cells express cytokeratins 8, 18, and 19 and epithelial membrane antigen that disappear after induced differentiation.

The effects of differentiation-modulating drugs were studied on the expression of intermediate filaments (IFs) in the human K562 erythroleukemic cell line. The untreated cells contained typical cytoplasmic coiling bundles, positive for both vimentin and cytokeratin as judged by indirect immunofluorescence microscopy with monoclonal antibodies (Mabs). Some of the cells also showed bright immunoreactivity for epithelial membrane antigen (EMA), as revealed with a Mab and polyclonal antiserum. When exposed to hemin or to sodium butyrate, most of the cells became cytokeratin negative within 3 days and showed dispersion of vimentin fibrils. Upon exposure to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), the amount of both vimentin and cytokeratin appeared to be greatly increased within 3 days and was found both in dispersed cytoplasmic fibrils, in large spherical, eccentric aggregates, as well as in cytoplasmic fibrils in cells spreading on fibronectin. TPA induced a complete loss of proliferation, as judged by immunostaining with the Mab Ki-67. The effects of TPA were found to be irreversible and could be induced by only a short exposure to the drug. Western blotting analysis and monoclonal antibodies to individual cytokeratins revealed that untreated K562 cells expressed Mr 52,000 (No. 8), 46,000 (No. 18), and 40,000 (No. 19) cytokeratin polypeptides, which disappeared when the cells were exposed to hemin or to sodium butyrate to induce erythroid differentiation but were greatly enhanced when exposed to TPA. The monoclonal anti EMA antibody reacted in K562 cells with a single Mr 320,000 polypeptide that was also revealed in MCF-7 breast carcinoma cells. Human bone marrow cells or other leukemic cell lines with erythroid differentiation capacity (HEL and KG-1) did not contain cytokeratin- or EMA-immunoreactive cells, suggesting that in K562 cells these properties may rather represent abnormal cytodifferentiation or retrodifferentiation toward early embryonic mesenchymal cells, than a more general expression of epithelial features in human leukemic cells.     

Expression and characterization of transforming growth factor alpha precursor protein in transfected mammalian cells.

Analysis of a cDNA clone derived from retrovirus-transformed rat fibroblasts has recently suggested that the mature 50-amino-acid form of transforming growth factor alpha (TGF alpha) is derived from a 159-amino-acid transmembrane precursor by proteolytic cleavage. To understand the processing of the TGF alpha precursor molecule in more detail, we have expressed this protein in baby hamster kidney (BHK) fibroblasts under control of the metal-ion-inducible metallothionein promoter and characterized the expressed precursor with site-specific antipeptide antibodies. One of the BHK transfectants, termed 5:2, expressed the TGF alpha mRNA in a cadmium- and zinc-inducible manner. The TGF alpha precursor protein was detected by immunoprecipitation analysis of radiolabeled cell cultures. In the induced 5:2 cells, a polypeptide of Mr 13,000 to 17,000 was readily identified by peptide antisera made to three different regions of the TGF alpha precursor protein. No such protein species were observed in BHK cells treated with cadmium and zinc or in uninduced 5:2 cells. However, two cell lines known to produce TGF alpha naturally, Leydig testicular tumor cells and Snyder-Theilan feline sarcoma virus-transformed Fisher rat embryo fibroblasts, possessed detectable levels of immunologically related Mr 13,000 to 17,000 proteins. Cell fractionation studies indicate that the Mr 13,000 to 17,000 species expressed in induced 5:2 cells is membrane associated, consistent with predictions based on the cDNA sequence of the TGF alpha precursor. Media conditioned by induced 5:2 cells contained epidermal growth factor receptor-competing activity, which, upon size fractionation, was similar in size to the mature processed form of TGF alpha. These data show that these nontransformed BHK cells possess the ability to process the TGF alpha precursor molecule into its native form.     

Growth factor-stimulated protein phosphorylation in 3T3-L1 cells. Evidence for protein kinase C-dependent and -independent pathways

Exposure of serum-deprived 3T3-L1 fibroblasts to phorbol 12-myristate 13-acetate (PMA), synthetic diacylglycerols, platelet-derived growth factor (PDGF), or pituitary fibroblast growth factor (FGF) resulted in stimulated phosphorylation of an acidic, multicomponent, soluble protein of Mr 80,000. Phosphorylation of this protein was promoted to a lesser extent by epidermal growth factor; however, neither insulin nor dibutyryl cAMP was effective. Phosphoamino acid analysis and peptide mapping of the Mr 80,000 32P-protein after exposure of fibroblasts to PDGF revealed identical patterns to those obtained with PMA or diacylglycerols. In contrast to the Mr 80,000 protein, proteins of Mr 22,000 (and pI 4.4) and Mr 31,000 were also phosphorylated in response to insulin as well as to PMA, diacylglycerols, epidermal growth factor, PDGF, and FGF in these cells. Similar findings were noted in fully differentiated 3T3-L1 adipocytes. Preincubation of the cells with high concentrations of active phorbol esters abolished specific [3H]phorbol 12,13-dibutyrate binding, protein kinase C activity, and immunoreactivity and also prevented stimulated phosphorylation of the Mr 80,000 protein by PMA, diacylglycerols, PDGF, or FGF, supporting the contention that this effect was mediated through protein kinase C. The stimulated phosphorylation of the Mr 22,000 and 31,000 proteins in response to PMA was also abolished by such pretreatment. In contrast, the ability of insulin, PDGF, and FGF to promote phosphorylation of the Mr 22,000 and 31,000 proteins was unaffected in the protein kinase C-deficient cells. We conclude that PDGF and FGF may exert some of their effects on these cells through at least two distinct pathways of protein phosphorylation, phorbol ester-like (P) activation of protein kinase C, and an insulin-like (I) pathway exemplified by phosphorylation of the Mr 22,000 and 31,000 proteins.     

Characterization of transforming growth factors produced by the insulin-independent teratoma-derived cell line 1246-3A

The 1246-3A cell line is an insulin-independent variant derived from the adipogenic cell line 1246. Data presented in this paper indicate that the 1246-3A cell line releases in its culture medium two types of transforming growth factors, TGF-alpha- and TGF-beta-like polypeptides, and a growth inhibitor. TGF-alpha like polypeptide eluted from Biogel P60 column into two fractions with an apparent molecular weight of 50 kDa and 13 kDa. These high-molecular-weight TGF-alpha-like factors competed with 125I-EGF for binding to epidermal growth factor (EGF) receptors and were specifically immunoprecipitated by incubation with antirat TGF-alpha antibody, not by incubation with anti-EGF antibody. Both fractions promoted anchorage-independent growth of normal rat kidney NRK cells in the absence of EGF and stimulated DNA synthesis in quiescent Balb/c-3T3 cells in a fashion similar to EGF and synthetic TGF-alpha. In addition to secreting TGF-alpha-like polypeptides, 1246-3A cells produce TGF-beta. This polypeptide, eluted from Biogel P60 chromatography with an apparent molecular weight of 25 kDa, promoted anchorage-independent growth of NRK cells in the presence of EGF and was growth inhibitory for Chinese hamster lung fibroblasts CCL 39 cells. Interestingly, another growth inhibitory activity was detected in Biogel P60 fractions and eluted with an apparent molecular weight of between 9.5-11 kDa. This fraction was different from TGF-beta and TGF-alpha as determined by specific radioreceptor competition assays. TGF-alpha and TGF-beta-like polypeptides could represent autocrine growth stimulators for the insulin-independent 1246-3A cells and act in synergy with insulin-related factor (IRF) for an optimal stimulation of 1246-3A cell proliferation in serum-free medium.     

Oncogenic ras causes resistance to the growth inhibitor insulin-like growth factor binding protein-3 (IGFBP-3) in breast cancer cells.

Insulin-like growth factor binding protein-3 (IGFBP-3) inhibits proliferation and promotes apoptosis in normal and malignant cells. In MCF-10A human mammary epithelial cells, 30 ng/ml human plasma-derived IGFBP-3 inhibited DNA synthesis to 70% of control. This inhibition appeared IGF-independent, since neither an IGF-receptor antibody nor IGFBP-6 inhibited DNA synthesis. Malignant transformation of MCF-10A cells by transfection with Ha-ras oncogene abolished the inhibitory effect of IGFBP-3, concomitant with an increase in IGFBP-3 secretion and cell association of approximately 60 and 300%, respectively. When mitogen-activated protein (MAP) kinase activation was partially inhibited using PD 98059, IGFBP-3 sensitivity in ras-transfected cells was restored, with a significant inhibitory effect at 10 ng/ml IGFBP-3. PD 98059 had no effect on IGFBP-3 secretion or cell association by ras-transfected or parent MCF-10A cells. Hs578T, a tumor-derived breast cancer cell line that expresses activated Ha-ras, similarly has a high level of secreted and cell-associated IGFBP-3. In the absence of PD 98059, DNA synthesis by Hs578T cells was reduced to 70% of control by 1000 ng/ml IGFBP-3. PD 98059 increased sensitivity to IGFBP-3, so that this level of inhibition was achieved with 100 ng/ml IGFBP-3. These results suggest that MAP kinase activation by oncogenic ras expression causes IGFBP-3 resistance, a possible factor in the dysregulation of breast cancer cell growth.     

Identification of the avian homologues of mammalian CD4 and CD8 antigens

Two mAb were produced against chicken T cells. The CT4 antibody precipitated a polypeptide of Mr 64,000 under both reducing and non-reducing conditions. The CT8 antibody precipitated a molecule of Mr 63,000 under non-reducing conditions and polypeptide chains of Mr 34,000 under reducing conditions, suggesting that the CT8 molecule is a disulfide-linked homodimer. Tissue distribution studies by immunofluorescence revealed that the CT4 and CT8 Ag were expressed by the majority of thymocytes and by subpopulations of CT3+ cells in peripheral tissues. The CT4 reactive molecule was found on approximately 70% of thymocytes, 10% splenocytes, and 45% of lymphoid cells in blood. The CT8 reactive molecule was expressed on approximately 80% of thymocytes, 50% of spleen cells, and 15% of blood lymphocytes. Two-color immunofluorescence indicated that the CT4 and CT8 Ag were expressed together on most thymocytes and on mutually exclusive subsets of cells in the spleen and blood. Ontogenic studies revealed a sharp increase in the frequencies of CT4+ and CT8+ cells in the thymus between days 13 and 16 embryonic life. Both CT4 and CT8 antibodies inhibited PHA- and Con A-induced proliferative responses of splenocytes, and the degree of inhibition correlated with the frequencies of CT4+ and CT8+ lymphoblasts. Treatment of spleen cells with CT4 antibody and inhibited PWM-induced IL-2 production, and removal of CT8+ cells inhibited the cytolytic activity induced by allogeneic lymphocyte stimulation. Macrophages did not express detectable CT4 reactivity. These results suggest that the CD4 and CD8 molecules and their tissue-restricted patterns of expression are highly conserved in birds and mammals.     

Cross-linking of human T cell receptor proteins: association between the T cell idiotype beta subunit and the T3 glycoprotein heavy subunit

Bifunctional cross-linking reagents DSP, DSS, and BSOCOES were used to cross-link 125I-surface-labeled viable T lymphocytes. The cross-linked cells were solubilized in Nonidet-P40, immunoprecipitated with anti-Ti (monoclonal antibody T40/25) or anti-T3 (monoclonal antibodies UCHT-1 or OKT3), and analyzed by SDS-PAGE. With all three cross-linkers, the intact cross-linked products obtained with monoclonal antibody T40/25 from HPB-ALL cells were 20-30 kd heavier than the Ti dimer (Mr 80,000). When the DSP cross-linked product was isolated using either anti-Ti or anti-T3 monoclonal antibodies and then cleaved, bands having molecular weights identical with both the Ti and T3 subunits were obtained. The two-dimensional SDS-PAGE analysis (nonreducing followed by reducing conditions) of the DSS and BSOCOES cross-linked products revealed the specifically cross-linked bands to have Mr 40,000 and Mr 28,000. These data indicate that the Ti molecule and the T3 molecule are spatially associated on the cell surface and suggest the predominant association is between the Ti beta subunit (Mr 40,000) and the T3 heavy subunit (Mr 28,000).     

Interleukin 2-driven T lymphocyte proliferation is dependent upon a surface antigen distinct from the interleukin 2 receptor: requirements for inhibition of T-cell proliferation by monoclonal antibody 5C3

A monoclonal antibody (5C3) to an antigen expressed on activated guinea pig T lymphocytes that did not react with the interleukin 2 (IL-2) receptor, but inhibited IL-2-driven proliferative responses has been previously characterized. The present study provides further analysis of the inhibitory capacity of 5C3 for T-cell proliferation and of the relationship between the expression of the antigen defined by 5C3 and the capacity of cells to respond to IL-2. 5C3 inhibited proliferation of T-cell blasts to IL-2-containing fluids when added as late as 8 hr prior to termination of a 26-hr culture. 5C3 pretreatment of the IL-2-responsive blast cells was also sufficient to detect significant inhibition of proliferation. FACS analysis of these blasts indicated that maximal 5C3 binding was required for pretreatment to result in inhibition of IL-2-driven proliferation. Delayed addition of 5C3 to culture or pretreatment with 5C3 of responding cells also resulted in inhibition of proliferation of immune T lymphocytes to antigen-pulsed-presenting cells. Lastly, although modulated 5C3- blasts failed to proliferate to IL-2, induction of the 5C3-bearing molecule on these 5C3- blasts correlated with restoration of the ability of these cells to proliferate to IL-2. Collectively, these results further support the hypothesis that monoclonal antibody 5C3 interferes with a critical signal in the IL-2 growth pathway.     

Antibodies to the common leukocyte antigen (T200) inhibit an early phase in the activation of resting human B cells

T191, a monoclonal antibody reactive with the T200 common leukocyte antigen, profoundly inhibits an early event(s) associated with alpha-immunoglobulin M (alpha IgM)/T cell replacing factor (TRF) or alpha IgM/recombinant interleukin 1 and 2 (rIL 1 and rIL 2)-induced tonsillar B cell proliferation. Kinetic analysis of T191-mediated inhibition indicated that the antibody exerts its effect within 12 to 24 hr of the initiation of cultures and rapidly loses its activity thereafter. Small resting B cells are most sensitive to T191 inhibition, whereas B cells with increasing buoyant density (presumably reflecting stages of increased activation) become progressively T191 insensitive. Analysis of RNA synthesis subsequent to alpha IgM crosslinking of surface immunoglobulin demonstrated that T191 reduced [3H]uridine incorporation by up to 38% during the first 20 hr of culture. In contrast to the effects seen with alpha IgM stimulated B cells, T191 had no inhibitory effect upon phorbol myristate acetate-induced B cell proliferation. The inhibitory effect upon B cell proliferation observed with T191 is not unique among other alpha-T200 antibodies. Four of five previously described alpha-T200 monoclonal antibodies had similar inhibitory effects (82 to 57% maximum inhibition of [3H]thymidine incorporation). However, 13.3, an alpha-T200 monoclonal antibody previously shown to block natural killer (NK) cell-mediated killing was without effect. Likewise, those antibodies capable of inhibiting B cell proliferation failed to block NK-mediated cytolysis. Antibody binding experiments together with proliferation inhibition studies suggest that all of the monoclonal antibodies tested recognized distinct epitopes on the T200 antigen. Both observations are of significance because they demonstrate that the effects seen with anti-T200 antibodies represent an interference with highly specific functional regions on the T200 molecules.     

Influence of sex hormones on Coxsackie B-3 virus infection in Balb/c mice

Background and “spontaneous” proliferation are terms often used for the proliferative activity normally exhibited by peripheral blood mononuclear cells (MNC) in vitro. In this report, we show that Interleukin-2 (IL-2) added to unfractionated MNC but not to isolated T or non-T cells significantly increased their proliferative activity. The cells responding to IL-2 stimulation from MNC were OKT3 positive lymphocytes. In addition, treatment of MNC with either a monoclonal anti-HLA-DR antibody (in the absence of C′) or Cyclosporin-A strongly suppressed the “background” whereas treatment of MNC with the 3A1 monoclonal anti-human T cell antibody did not modify “spontaneous” proliferation of these cells. IL-2 could not restore or increase the proliferative activity of MNC exposed to the anti-HLA-DR antibody or Cyclosporin-A while the T cell growth factor significantly enhanced proliferation of MNC cultured in the presence of the OKT4 antibody. Taken together these results strongly suggest that IL-2 responding T cells from MNC become sensitive to IL-2 by interacting with HLA-DR antigens on B lymphocytes and/or monocytes contained in MNC (resting T cells are Dr^?). By a similar mechanism we have previously shown that T cells acquire responsiveness to IL-2 in the autologous mixed lymphocyte reaction (AMLR). Since all the cells that participate in AMLR are present in MNC, we postulate that a “mini” AMLR taking place within MNC may explain the “spontaneous” proliferation of peripheral blood mononuclear cells.     

A T helper cell hybridoma produces an antigen-specific regulatory activity. Relationship to the T cell receptor by serology and antigenic fine specificity.

We have previously shown that a T cell hybridoma, A1.1, constitutively produces an Ag-specific regulatory factor with specificity for poly-18, a synthetic polypeptide. This cell also responds to poly-18 plus I-Ad by producing lymphokines. The antigenic specificity of the factor and the T cell appeared to be the same. This suggested the possibility that some part of the TCR, responsible for antigenic specificity of the cell, also imparts specificity to the A1.1-derived factor. This was supported by the observation that the factor was bound and eluted from a monospecific anti-TCR antiserum. Further, we demonstrated that antisense oligodeoxynucleotides corresponding to the TCR V alpha of A1.1 (but not TCR V beta) block production of the Ag-specific factor. Herein, we report recent findings that strengthen the proposed relationship between the TCR and the A1.1-derived factor. The factor was bound and eluted from a monoclonal anti-TCR C alpha antibody, but not from anti-TCR beta, anti-V beta 6, nor anti-CD3 epsilon. The anti-TCR C alpha antibody bound a Mr 46-kDa protein from A1.1 supernatants, which is the same apparent size at which activity could be eluted from an SDS-PAGE gel separation of concentrated factor. Antigenic fine-specificity analysis revealed that two amino acids in poly-18 are critical for the recognition of the antigen by the Ag-specific factor. These two amino acids appear to be those recognized by the TCR. The factor that was bound and eluted from the monoclonal anti-TCR C alpha showed this fine-specificity as well. This, combined with our earlier studies, supports the view that the A1.1-derived factor is encoded, at least in part, by TCR-alpha.     

Expression of platelet glycoprotein Ib alpha in HEL cells

We have previously shown that platelet glycoprotein Ib is expressed in a minority of cells of the human leukemic cell line HEL (Tabilio, A., Rosa, J. P., Testa, U., Kieffer, N., Nurden, A. T., Del Canizo, M. C., Breton-Gorius, J., and Vainchenker, W. (1984) EMBO J. 3, 453-459). In this report, we have selected a stable HEL subclone with increased expression of glycoprotein (GP) Ib as assessed by 6 different monoclonal antibodies in order to investigate the biochemical characteristics of this glycoprotein. A single polypeptide chain of apparent Mr = 60,000 was precipitated under reducing and nonreducing conditions by a specific polyclonal anti-platelet glycocalicin antibody and two anti-GPIb alpha monoclonal antibodies (AN51 and AP1), both from surface-labeled and metabolically labeled HEL cells. We were unable to demonstrate the presence of a polypeptide corresponding to the beta subunit of GPIb or GPIX which is closely associated with GPIb. Competitive immunoprecipitation performed in the presence of an excess amount of cold platelet glycocalicin completely displaced the Mr = 60,000 polypeptide. Synthesis of N-linked oligosaccharide chains on this Mr = 60,000 polypeptide was inhibited by the antibiotic tunicamycin, and a shift of the apparent Mr from 60,000 to 48,000 was observed. O-Linked oligosaccharide chains identical to platelet GPIb hexasaccharides were deficient or incomplete since no peanut agglutinin binding to the Mr = 60,000 polypeptide was observed after neuraminidase treatment of HEL cells. Thus, our results provide evidence that the Mr = 60,000 polypeptide expressed on the surface membrane of HEL cells is closely related to platelet GPIb and corresponds to an incompletely or abnormally O-glycosylated GPIb alpha subunit.     

Effect of T3 modulation on pokeweed mitogen-induced T cell activation: evidence for an alternative pathway of T cell activation

Modulation of the T3 molecule on human T cells with monoclonal anti-T3 antibodies has been shown to result in the disappearance of the T3-Ti complex from the membrane and to preclude subsequent T cell activation by various mitogenic and antigenic stimuli. We have examined the effect of T3 modulation on pokeweed mitogen (PWM)-induced T cell activation. T3 modulation was accomplished by incubating peripheral blood mononuclear cells (PBMC) or mixtures of T cells and non-T cells at 37 degrees C for 18 hr in the presence of UCHT-1, a mouse IgG1 anti-T3 monoclonal antibody. Only donors whose PBMC were unresponsive to the mitogenic activity of this antibody were selected. Although T3 modulation resulted in complete to substantial inhibition of T cell proliferation induced by low PWM concentrations of 5 or 50 ng/ml, it had no effect on T cell proliferation when PWM was added at a concentration of 0.5 and 5 micrograms/ml. The results demonstrate that the higher doses of PWM can induce T cell proliferation via an alternative pathway that does not involve participation of the T3-Ti complex. In contrast, irrespective of the PWM dose added, T3 modulation almost totally inhibited PWM-induced interleukin 2 (IL 2) production. The differential effect of T3 modulation on IL 2 production and on T cell proliferation induced by high doses of PWM suggests that this alternative pathway of T cell proliferation is IL 2 independent. This suggestion was additionally substantiated by the lack of effect of anti-Tac, and anti-IL 2 receptor antibody, on PWM-induced proliferation of T3-modulated T cells. In conclusion our data demonstrate that high doses of PWM can induce T cells to proliferate via an alternative pathway that does not involve perturbation of the T3-Ti complex.     

Membrane topology and quaternary structure of cardiac gap junction ion channels.

The membrane topology and quaternary structure of rat cardiac gap junction ion channels containing alpha 1 connexin (i.e. Cx43) have been examined using anti-peptide antibodies directed to seven different sites in the protein sequence, cleavage by an endogenous protease in heart tissue and electron microscopic image analysis of native and protease-cleaved two-dimensional membrane crystals of isolated cardiac gap junctions. Specificity of the peptide antibodies was established using dot immunoblotting, Western immunoblotting, immunofluorescence and immunoelectron microscopy. Based on the folding predicted by hydropathy analysis, five antibodies were directed to sites in cytoplasmic domains and two antibodies were directed to the two extracellular loop domains. Isolated gap junctions could not be labeled by the two extracellular loop antibodies using thin-section immunogold electron microscopy. This is consistent with the known narrowness of the extracellular gap region that presumably precludes penetration of antibody probes. However, cryo-sectioning rendered the extracellular domains accessible for immunolabeling. A cytoplasmic "loop" domain of at least Mr = 5100 (residues (101 to 142) is readily accessible to peptide antibody labeling. The native Mr = 43,000 protein can be protease-cleaved on the cytoplasmic side of the membrane, resulting in an Mr approximately 30,000 membrane-bound fragment. Western immunoblots showed that protease cleavage occurs at the carboxy tail of the protein, and the cleavage site resides between amino acid residues 252-271. Immunoelectron microscopy demonstrated that the Mr approximately 13,000 carboxy-terminal peptide(s) is released after protease cleavage and does not remain attached to the Mr approximately 30,000 membrane-bound fragment via non-covalent interactions. Electron microscopic image analysis of two-dimensional membrane crystals of cardiac gap junctions revealed that the ion channels are formed by a hexagonal arrangement of protein subunits. This quaternary arrangement is not detectably altered by protease cleavage of the alpha 1 polypeptide. Therefore, the Mr approximately 13,000 carboxyterminal domain is not involved in forming the transmembrane ion channel. The similar hexameric architecture of cardiac and liver gap junction connexins indicates conservation in the molecular design of the gap junction channels formed by alpha or beta connexins.