Intrinsic FAK activity and Y925 phosphorylation facilitate an angiogenic switch in tumors.

Elevated focal adhesion kinase (FAK) expression occurs in advanced cancers, yet a signaling role for FAK in tumor progression remains undefined. Here, we suppressed FAK activity in 4T1 breast carcinoma cells resulting in reduced FAK Y925 phosphorylation, Grb2 adaptor protein binding to FAK, and signaling to mitogen-activated protein (MAP) kinase (MAPK). Loss of a FAK-Grb2-MAPK linkage did not affect 4T1 cell proliferation or survival in culture, yet FAK inhibition reduced vascular endothelial growth factor (VEGF) expression and resulted in small avascular tumors in mice. This FAK-Grb2-MAPK linkage was essential in promoting angiogenesis as reconstitution experiments using Src-transformed FAK-null fibroblasts revealed that point mutations affecting FAK catalytic activity (R454) or Y925 phosphorylation (F925) disrupted the ability of FAK to promote MAPK- and VEGF-associated tumor growth. Notably, in both FAK-inhibited 4T1 and Src-transformed FAK-null cells, constitutively activated (CA) mitogen-activated protein kinase kinase 1 (MEK1) restored VEGF production and CA-MEK1 or added VEGF rescued tumor growth and angiogenesis. These studies provide the first biological support for Y925 FAK phosphorylation and define a novel role for FAK activity in promoting a MAPK-associated angiogenic switch during tumor progression.

A distinct pathway of cell-mediated apoptosis initiated by granulysin.

Granulysin is an antimicrobial and tumoricidal molecule expressed in granules of CTL and NK cells. In this study, we show that granulysin damages cell membranes based upon negative charge, disrupts the transmembrane potential (Deltapsi) in mitochondria, and causes release of cytochrome c. Granulysin-induced apoptosis is blocked in cells overexpressing Bcl-2. Despite the release of cytochrome c, procaspase 9 is not processed. Nevertheless, activation of caspase 3 is observed in granulysin-treated cells, suggesting that granulysin activates a novel pathway of CTL- and NK cell-mediated death distinct from granzyme- and death receptor-induced apoptosis.

Granulysin, a T cell product, kills bacteria by altering membrane permeability.

Granulysin, a protein located in the acidic granules of human NK cells and cytotoxic T cells, has antimicrobial activity against a broad spectrum of microbial pathogens. A predicted model generated from the nuclear magnetic resonance structure of a related protein, NK lysin, suggested that granulysin contains a four alpha helical bundle motif, with the alpha helices enriched for positively charged amino acids, including arginine and lysine residues. Denaturation of the polypeptide reduced the alpha helical content from 49 to 18% resulted in complete inhibition of antimicrobial activity. Chemical modification of the arginine, but not the lysine, residues also blocked the antimicrobial activity and interfered with the ability of granulysin to adhere to Escherichia coli and Mycobacterium tuberculosis. Granulysin increased the permeability of bacterial membranes, as judged by its ability to allow access of cytosolic ss-galactosidase to its impermeant substrate. By electron microscopy, granulysin triggered fluid accumulation in the periplasm of M. tuberculosis, consistent with osmotic perturbation. These data suggest that the ability of granulysin to kill microbial pathogens is dependent on direct interaction with the microbial cell wall and/or membrane, leading to increased permeability and lysis.

Self-recognition of CD1 by gamma/delta T cells: implications for innate immunity.

The specificity of immunoglobulins and alpha/beta T cell receptors (TCRs) provides a framework for the molecular basis of antigen recognition. Yet, evolution has preserved a separate lineage of gamma/delta antigen receptors that share characteristics of both immunoglobulins and alpha/beta TCRs but whose antigens remain poorly understood. We now show that T cells of the major tissue gamma/delta T cell subset recognize nonpolymorphic CD1c molecules. These T cells proliferated in response to CD1+ presenter cells, lysed CD1c+ targets, and released T helper type 1 (Th1) cytokines. The CD1c-reactive gamma/delta T cells were cytotoxic and used both perforin- and Fas-mediated cytotoxicity. Moreover, they produced granulysin, an important antimicrobial protein. Recognition of CD1c was TCR mediated, as recognition was transferred by transfection of the gamma/delta TCR. Importantly, all CD1c-reactive gamma/delta T cells express V delta 1 TCRs, the TCR expressed by most tissue gamma/delta T cells. Recognition by this tissue pool of gamma/delta T cells provides the human immune system with the capacity to respond rapidly to nonpolymorphic molecules on professional antigen presenting cells (APCs) in the absence of foreign antigens that may activate or eliminate the APCs. The presence of bactericidal granulysin suggests these cells may directly mediate host defense even before foreign antigen-specific T cells have differentiated.

Biosynthesis of granulysin, a novel cytolytic molecule.

Granulysin is a newly described lytic molecule expressed by CTL and NK cells. Three mRNA (519, 520, and 522) and two protein products of 15 and 9 kDa are encoded by the granulysin gene. Stable transfectants overexpressing the predominate 520 mRNA were generated to determine the protein products originating from the translation of this message. A transfectant of the NK cell tumor YT overexpressed both 15 and 9 kDa proteins while a transfectant of the T cell tumor HuT78 produced mainly 15 kDa granulysin. Thus the 520 mRNA is sufficient for production of both 15 and 9 kDa granulysin. 9 kDa granulysin accumulated via post-translational processing of 15 kDa protein and was present intracellularly but not in the cell culture supernatant, indicating specific retention of the 9 kDa protein. An inhibitor of granule acidification, concanamycin A, blocked the processing of 15 kDa granulysin to the 9 kDa form. A deduced structural difference between the two forms of the protein and a decrease in lytic activity of 9 kDa granulysin at granule pH suggest two mechanisms by which a granulysin expressing cell is protected from autolysis during the biosynthesis of this potentially harmful molecule.

An antimicrobial activity of cytolytic T cells mediated by granulysin.

Cytolytic T lymphocytes (CTLs) kill intracellular pathogens by a granule-dependent mechanism. Granulysin, a protein found in granules of CTLs, reduced the viability of a broad spectrum of pathogenic bacteria, fungi, and parasites in vitro. Granulysin directly killed extracellular Mycobacterium tuberculosis, altering the membrane integrity of the bacillus, and, in combination with perforin, decreased the viability of intracellular M. tuberculosis. The ability of CTLs to kill intracellular M. tuberculosis was dependent on the presence of granulysin in cytotoxic granules, defining a mechanism by which T cells directly contribute to immunity against intracellular pathogens.

Granulysin-induced apoptosis. I. Involvement of at least two distinct pathways.

Granulysin is a newly described cytolytic molecule released by CTL and NK cells via granule-mediated exocytosis. It shares homology with saposin-like proteins, including NK-lysin and amoebapores, and has been implicated in the lysis of tumor cells and microbes. In the present study we show that recombinant granulysin alone induces apoptosis of Jurkat cells. This apoptosis is associated with a sixfold increase in the ceramide/sphingomyelin ratio, implicating the activation of sphingomyelinases. Granulysin- and ceramide-induced apoptosis are similar in that they both are only minimally inhibited by the more selective cysteine protease p32 (caspase 3)-like caspase inhibitor N-acetyl-Asp-Glu-Val-Asp aldehyde, while they are significantly inhibited by the more general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk). Nevertheless, while Z-VAD-fmk almost completely inhibits ceramide-induced apoptosis, a Z-VAD-fmk-resistant component was observed using granulysin. Granulysin also causes apoptosis in cells depleted of sphingomyelin by prolonged treatment with the ceramide synthase inhibitor fumonisin B1. These data indicate that granulysin induces target cell death by both ceramide- and caspase-dependent and -independent pathways.

Processing, subcellular localization, and function of 519 (granulysin), a human late T cell activation molecule with homology to small, lytic, granule proteins.

CTL and NK cells share a common cytolytic mechanism that involves regulated exocytosis of lytic molecules stored within cytoplasmic granules. Here we describe the processing, subcellular localization, and function of a T and NK cell-specific granule protein that shares homology with small, lytic granule-associated molecules. The gene coding for this protein, 519, is expressed late after T cell activation. Antisera raised against a 519/glutathione-S-transferase fusion protein and a series of peptides derived from the 519 protein sequence permitted the identification of two small CTL protein products of 15 and 9 kDa that are exocytosed after stimulation through the TCR. The 9-kDa product is a processed form of 519 and differs from the 15-kDa product in both its amino and carboxyl terminus. While both 519 proteins are found in cytoplasmic granules, the 9-kDa form is also present in dense, highly cytolytic granules. Functional studies indicate that this protein is lytic against tumor cell targets. The cell type- and stage-specific expression pattern of 519 along with its subcellular localization are reminiscent of molecules that play a vital role in granule-mediated cytolysis by CTL and NK cells. Its lytic activity suggests the involvement of 519 in CTL effector function.

Molecular site specificity of pyridinoline and pyrrole cross-links in type I collagen of human bone.

Compared with soft tissue collagens, bone type I collagen displays a distinctive pattern of covalent cross-linking, with evidence of preferred sites of molecular interaction and a prominence of both immature, divalent cross-links and mature, trivalent cross-links in the adult tissue. In this study the site-specificity of the mature cross-links in human bone collagen was examined. Peptides containing fluorescent pyridinoline cross-links and Ehrlich's-reactive pyrrole cross-links were isolated from a bacterial collagenase digest of demineralized bone matrix. The digest was fractionated by molecular sieve chromatography, monitoring for peptide absorbance, pyridinoline fluorescence, pyrroles by Ehrlich's reagent, and immunoassay for cross-linked N-telopeptides. Individual cross-linked peptides were resolved by ion-exchange and reverse-phase HPLC. Structures were established by NH2-terminal microsequencing, cross-link analysis, electrospray mass spectrometry, and immunoassay. Two, about equally occupied, sites of pyridinoline cross-linking were identified, N-telopeptide to helix and C-telopeptide to helix. Pyrroles were alternative cross-linking products at the same sites, but concentrated (85%) at the N-telopeptide end. Only one combination of chains was cross-linked by pyridinolines at the C-telopeptide to helix site, [alpha1(I)C]2alpha1(I)helix. Several peptide combinations arose from the N-telopeptide to helix site, but the main source of pyridinolines was from the locus, alpha1(I)Nalpha2(I)Nalpha1(I)helix. Pyridinolines linking two alpha1(N) telopeptides were a minor component. Pyrroles were concentrated at the locus, alpha1(I)Nalpha2(I)Nalpha2(I)helix. The cross-link ratio of hydroxylysylpyridinoline to lysylpyridinoline differed between N-telopeptide and C-telopeptide sites, and between the individual interchain combinations. Cross-linked N-telopeptides accounted for two-thirds of the total lysylpyridinoline in bone. N-telopeptide pyridinoline fluorescence was quenched on chromatography, so that reliance on peptide fluorescence alone can underestimate the level of N-telopeptide pyridinoline cross-linking.

Monitoring bone resorption in early postmenopausal women by an immunoassay for cross-linked collagen peptides in urine.

A new immunoassay using an ELISA approach for measuring urinary excretion of cross-linked N-telopeptides of type 1 collagen was evaluated as a specific measure of bone resorption. The assay was applied to 65 early postmenopausal women who participated in a placebo-controlled trial of the aminobisphosphonate, alendronate sodium. Eight blood and urine samples were collected over a 9 month interval. Baseline cross-linked peptide excretion varied from 26 to 216 pmol BCE (bone collagen/mumol Cr. Within-subject variability (CV) for cross-linked peptide excretion was 20.2% over the 9 months in placebo-treated subjects, substantially less than that observed for other biochemical markers of bone resorption: 45, 53, and 63% for fasting urinary calcium and hydroxyproline and 24 h urinary lysylpyridinoline (HPLC assay), respectively. Baseline cross-linked peptide excretion correlated significantly (p < 0.001) with baseline total urine lysylpyridinoline and serum osteocalcin, but not with the other biochemical markers. Initial peptide excretion also correlated inversely with lumbar spine bone mineral density at entry (r = -0.26, p < 0.05). Treatment for 6 weeks with alendronate produced a dose-dependent suppression of cross-linked peptide excretion (0 +/- 8, 29 +/- 6, 56 +/- 5, and 64 +/- 3% for 0, 5, 20, and 40 mg, respectively, p < 0.01 versus placebo for treatment effect), with a return toward pretreatment values during follow-up. Measurement of the urinary cross-linked N-telopeptides of type I collagen by this new ELISA approach appears promising as a simple and reliable method to assess overall bone resorption.(ABSTRACT TRUNCATED AT 250 WORDS)

Nurses' care during labor: its effect on the cesarean birth rate of healthy, nulliparous women.

This retrospective study was designed to determine the influence of nurses' care during labor and delivery on the cesarean birth rate of healthy, nulliparous women. Labor and delivery nurses in a large, nonprofit hospital were grouped according to the cesarean birth rates of their healthy, nulliparous patients in spontaneous labor. Large differences in cesarean birth rates between nurses in the lowest quintile (near 4.9%) and the highest quintile (near 19%) were not explained by differences in maternal age and gravidity, attendance of mother at childbirth class, insurance status, reliance on public assistance, physician who attended labor, use of epidural anesthesia, augmentation of labor, dilation when the nurse assumed care, infant weight, or gestational age. In multivariate analysis, the elapsed time between when the nurse assumed care and birth was significantly shorter for patients of nurses in the lowest quintile of cesarean birth rate (4.4 hrs) compared with patients of nurses in the highest quintile (5.6 hrs). The former were also less likely to have forceps used to assist vaginal delivery than the latter (13% vs 26%). Nurses in the lowest quintile of cesarean birth rates were more likely to use a form to record psychosocial data than nurses in the highest quintile (35% vs 15%). The study suggests that nurses' care during labor is an important factor influencing cesarean birth rates.

A specific immunoassay for monitoring human bone resorption: quantitation of type I collagen cross-linked N-telopeptides in urine.

Peptides of low molecular weight that contain pyridinoline cross-links were isolated from adolescent human urine. A fraction was selected that was enriched in the N-telopeptide-to-helix intermolecular cross-linking domain of bone type I collagen. Mouse monoclonal antibodies were generated against these urinary peptides conjugated to a carrier protein as immunogen. A high-affinity antibody was identified that specifically bound to the trivalent peptides derived from the N-telopeptide-to-helix pyridinoline cross-linking site in type I collagen of human bone. This was confirmed by the direct isolation from human bone collagen of similar fragments recognized selectively by the antibody. A sensitive inhibition ELISA was established on microtiter plates that could quantify the bone-derived peptides in human urine. The assay, which can be run directly on untreated urine, was thoroughly tested against samples from normal subjects and from patients with metabolic bone disease. For example, strong correlations with urinary hydroxyproline and total pyridinoline cross-links were found in patients with Paget's disease of bone. The method shows considerable promise as a rapid and specific index of human bone resorption rates, with greatly improved specificity and convenience over total pyridinoline analysis. Potential applications include the study of normal metabolism, the diagnosis and monitoring of bone disease, and evaluating the effectiveness of antiresorption therapies.