Regulation of cell adhesion and anchorage-dependent growth by a new beta 1-integrin-linked protein kinase.

The interaction of cells with the extracellular matrix regulates cell shape, motility, growth, survival, differentiation and gene expression, through integrin-mediated signal transduction. We used a two-hybrid screen to isolate genes encoding proteins that interact with the beta 1-integrin cytoplasmic domain. The most frequently isolated complementary DNA encoded a new, 59K serine/threonine protein kinase, containing four ankyrin-like repeats. We report here that this integrin-linked kinase (ILK) phosphorylated a beta 1-integrin cytoplasmic domain peptide in vitro and coimmunoprecipitated with beta 1 in lysates of mammalian cells. Endogenous ILK kinase activity was reduced in response to fibronectin. Overexpression of p59ILK disrupted epithelial cell architecture and inhibited adhesion to integrin substrates, while inducing anchorage-independent growth. We propose that ILK is a receptor-proximal protein kinase regulating integrin-mediated signal transduction.

Stimulation of tyrosine phosphorylation and accumulation of GTP-bound p21ras upon antibody-mediated alpha 2 beta 1 integrin activation in T-lymphoblastic cells.

Integrins are a family of heterodimeric integral plasma membrane proteins that behave as receptors for components of the extracellular matrix and also mediate cell to cell adhesion. Occupation of integrins can result in the transduction of intracellular signals, leading to cytoskeletal reorganization, tyrosine phosphorylation, and induction of gene expression. We report here that the ligation of alpha 2 beta 1 integrin by collagen-adhesion stimulatory anti-alpha 2 and anti-beta 1 antibodies resulted in the accumulation of p21ras in the active GTP-bound state in Jurkat T-lymphoblastoid cells. The activation was accompanied by the tyrosine phosphorylation of proteins of 47-52 kDa. This stimulation of tyrosine phosphorylation and p21ras activation was specific for the activating antibodies and occurred within 2 min of the addition of these antibodies. Although treatment of the cells with the protein kinase C activator, phorbol 12-myristate 13-acetate also resulted in an induction of both cell attachment to collagen and of p21ras activation, tyrosine phosphorylation was not observed. These results demonstrate that alpha 2 beta 1 integrin activation can result in the specific stimulation of tyrosine phosphorylation of 47-52-kDa proteins, as well as activation of a signaling pathway involving p21ras.

Subtypes of betaglycan and of type I and type II transforming growth factor-beta (TGF-beta) receptors with different affinities for TGF-beta 1 and TGF-beta 2 are exhibited by human placental trophoblast cells.

Transforming growth factor-beta is likely to be an important factor controlling placental activities, including growth, differentiation, invasiveness, hormone production, and immunosuppression. We have used a chemical cross-linking technique with either 125I-TGF-beta 1 or 125I-TGF-beta 2 and bis(sulfosuccinimidyl) suberate (BS3) to characterize TGF-beta binding components on human placental cells in primary culture. Trophoblast-enriched primary cultures exhibited a predominant affinity-labelled complex characteristic of membrane-anchored betaglycan (formerly termed the Type III TGF-beta receptor) and relatively low levels of the Type I and Type II TGF-beta receptor complexes. The results from affinity labelling saturation and competition experiments with TGF-beta 1 and TGF-beta 2 suggest the existence of two distinct subtypes of betaglycan: one subtype has a lower capacity and higher affinity, binds both TGF-beta 1 and TGF-beta 2, yet has a preferential affinity for TGF-beta 2; the second subtype has a higher capacity and lower affinity and binds TGF-beta 1 exclusively. In contrast, mesenchymal cell-enriched placental primary cultures possessed only one subtype of the betaglycan component that binds the two TGF-beta isoforms with similar affinities and capacities as observed on most cell lines. These experiments demonstrate that the betaglycan component which exhibits a higher affinity for TGF-beta 2 than for TGF-beta 1, that we had observed previously on term placental membranes, is actually present on trophoblast cells. In addition to the two distinctive betaglycan subtypes, subtypes of the Type I and II TGF-beta receptors were detected on the trophoblast-enriched cultures. In competition experiments, when 125I-TGF-beta 1 was used as the radiotracer, the Type I and II TGF-beta receptors show a much higher affinity for TGF-beta 1 than for TGF-beta 2, as observed with other cell types. However, when 125I-TGF-beta 2 was used, low abundance subtypes of both the Type I and II receptors that show similar affinities for TGF-beta 1 and TGF-beta 2 were also revealed.

The effect of cyclosporine A on infection of susceptible cells by human immunodeficiency virus type 1.

The effect of cyclosporine A (CyA) on the ability of the human immunodeficiency virus type 1 (HIV-1) to infect the H-9 T-cell leukemic line, as well as interleukin-2 (IL-2)-grown human peripheral blood-derived lymphocytes, has been studied. Pretreatment of H-9 cells and human lymphocytes with CyA over 24 hours completely prevented viral infection over a 21-day period, whereas the addition of drug at two hours postinfection with HIV-1 had a significant inhibitory effect on viral replication and expression of the virus-specific antigens p17 and p24. However, if CyA was added at later times to these lymphocytic cells, this inhibitory effect was lost. Indeed, the removal of CyA from cultures that had been treated from two hours after infection led to the rapid production of progeny virus. HIV-1 was able to infect peripheral blood lymphocytes obtained from each of four kidney allograft recipients on long-term CyA antirejection therapy, as long as drug was not included in the culture medium. In addition, we asked what effect pretreatment with CyA of cells of the U-937 monocytic line and primary cultures of human monocytes/macrophages might have on infection by HIV-1. CyA had no demonstrable effect on the ability of HIV-1 to infect cells of either type.

Heterogeneity of HIV-1 replication and antigen expression in EBV-transformed B cell lines.

Over a period of six months, we have followed a total of six different EBV-transformed B cell lines, each of which has been infected by the human immunodeficiency virus (HIV-1). The results indicate that all of these lines were initially able to produce progeny HIV-1, but that over time three of them ceased to produce infectious virus and two lines failed to elaborate significant levels of reverse transcriptase activity into the medium. We further found that two of the latter cell lines continued to express the viral antigens p17, p24 and/or gp41, as determined by indirect immunofluorescence assay, at times after progeny HIV-1 could no longer be detected. Those cell lines which continued to secrete progeny HIV-1 did so intermittently with large amounts of virus production on some days but not others. We further found that treatment of such cells with 3' azido-3'-deoxythymidine (AZT) completely inhibited production of progeny HIV-1.

Infection of human monocytes/macrophages by HIV-1: effect on secretion of IL-1 activity.

We have infected peripheral blood-derived monocyte/macrophage cultures with HIV-1 in order to determine the effect of such infection on cellular immunoregulatory function. We have confirmed that monocytes/macrophages are susceptible to infection by HIV-1, as determined by in situ hybridization using a HIV-1-specific RNA probe and by the presence of reverse transcriptase activity in culture supernatants. The cells employed efficiently supported viral replication in the absence of significant cytopathic effect, and secreted as little as 20% of the amount of IL-1 activity of uninfected controls in response to stimulation with either latex beads or lipopolysaccharide. This effect was not observed when UV-inactivated HIV-1 was used to infect the cells.

Differential susceptibility of human lymphocyte cultures to infection by HIV.

We have assessed the ability of HIV to infect cultures of peripheral blood lymphocytes derived from different healthy donors, varying in age between 20 and 76. The results indicate that cells from all of these people can be infected, although the percentage of infected cells varied from case to case. A similar variation was observed when attempts were made to infect cells from the same donor on more than one occasion. In most cases, infection by HIV led to persistence of an activated cell state, as indicated by the presence of Tac Ag or IL-2 receptor, at the cell surface. Co-incubation of HIV-infected lymphocytes with uninfected cells did not affect the ability of the latter either to respond to phytohaemagglutinin (PHA) or to express Tac Ag.

Deficiency of myeloperoxidase and abnormal chromosome 1 occurs in variant (HL60) promyelocytes.

Maturation of normal polymorphonuclear neutrophils is characterized by successive periods of granule synthesis, a process which frequently is abnormal in leukemia. Recently, the human leukemic cell line HL60, displaying a promyelocytic phenotype, has been used to study granulocyte maturation. We describe a variant line of HL60, called HL60-A7, resulting from growth in actinomycin D, which contains atypical large azurophilic granules deficient in myeloperoxidase. The products of in-vitro translation of A7 RNA contained less than 5% of the immunoreactive MPO found in the parent line. Electrophoresis of plasma membrane polypeptides radioiodinated by the lactoperoxidase technique revealed several differences. Karyotypic analysis identified a consistent chromosome 1q+ abnormality which was not found in any of the parental cells examined. This constellation of differences between HL60 and HL60-A7, i.e. MPO deficiency, abnormal granule morphology, cell surface changes, and further cytogenetic abnormalities, may point to a common site sensitive to altered regulation in some leukemic promyelocytes.

Antigens differentially expressed on surface and cytoplasmic structures of human myeloid cells.

Specialized internal granules are a major feature of myeloid differentiation and are deficient in most acute myeloid leukemia cells. Although they arise from the same synthetic apparatus as does the plasma membrane, their relationship to it is not well characterized in human tissues. Using murine monoclonal antibodies, we have identified myeloid-related structures that illustrate three possible modes of antigen expression in these organelles. Immunocytochemical studies with the light microscope have shown that the first (D51) was restricted to the surface of neutrophils, monocytes, megakaryocytes and platelets; a second (D46) was found on the surface of blastic cell lines but on only internal components of mature granulocytes; the third (H36/71) appeared on both the surface and internal particles of promyelocytes, myelocytes and polymorphs. These model antigens may be used to study the control of granule synthesis in normal and leukemic cells.

Deficiency of a major myeloid antigen in neutrophils of patients with refractory anemia and excess myeloblasts.

The expression of a major granulocyte-related antigen was assessed on the peripheral blood neutrophils of patients with the syndrome of refractory anemia with excess blasts. Of thirteen patients studied six showed a marked decrease in the number of cells with detectable staining. These results indicate that, although the neutrophils may appear normal by standard morphological criteria in this disease, they may be defective in a major component associated with myeloid maturation.

Loss of promyelocytic maturation in HL60 sublines: a potential model for leukaemia progression.

The majority of cells of the human leukaemic line HL60 appear to be promyelocytes which can be stimulated to mature into functioning neutrophils. We report here the derivation of stable subclones from the parent line which differ. Two forms of variant were obtained: (1) those with a defined lesion, selected by resistance to kill by 6-thioguanine, which matured in the presence of DMSO and retinoic acid but not in hypoxanthine. They also continued to express peroxidase and a major granulocyte antigen, and (2) those obtained by clonal growth in DMSO which did not mature in the presence of any compound tested including hypoxanthine, DMSO, retinoic acid and actinomycin D and lacked granules, peroxidase and the granulocyte antigen. The concurrent loss of the characteristic of maturability and the phenotype of myeloid commitment suggests that the control of the two phenomena may be related. These variant cell lines provide a useful model in which to study how human leukaemic cells may become arrested in less differentiated stages of development.