Molecular genetics and mechanisms of apoptosis in carcinomas of the lung and pleura: therapeutic targets.

Cancers of the lung and pleura remain a major cause of cancer deaths, both in men and women, with strong causal relationships between cigarette smoking and asbestos fibres, and deaths from lung cancer and mesothelioma, respectively. The poor survival rates for small cell lung cancer and mesotheliomas argue powerfully for greater understanding of mechanisms of carcinogenesis, genetic abnormalities and the role of tumour suppressor genes and proteins in carcinomas of the lung and pleura. Despite progress in the development of newer cytotoxic drugs, lung cancer remains a lethal disease. Chemotherapy and radiotherapy produce only a modest improvement in survival of patients with advanced disease. Increased knowledge of molecular mechanisms of lung cancer and apoptosis are providing opportunities for treating lung cancer with new classes of molecularly targeted drugs. These novel therapies should target the abnormalities in lung cancer by maximizing the effects of anti-tumour molecules, with minimal side effects on normal tissues. Of the several molecular targets, those receiving attention are p53 gene replacement, Bcl-2 downregulation, apoptosis by induced by TNF, the FAS/CD95 receptor system and TRAIL, and inhibition of NF-kappaB. Although several studies have shown benefits, there is a need for well planned clinical trials of drugs that target the apoptotic cascade. Stem cell therapy and gene replacement offer the prospect of novel approaches that are likely in the near future to play a definitive role in the treatment of advanced lung cancer. Furthermore, with their apparent minimal toxicity to normal tissues, the newer molecular targets represent attractive investigational directions for innovative cancer therapies.

Activation of kinin B1 receptors induces chemotaxis of human neutrophils.

Kinins are biologically active peptides that are powerful mediators of cellular inflammation. They mimic the cardinal signs of inflammation by inducing vasodilatation and by increasing vascular permeability and pain. Neutrophils are chemoattracted to sites of inflammation by several stimuli. However, the evidence concerning the chemotactic effect of kinin peptides has been contradictory. We analyzed the chemotactic effect of kinin B(1) receptor agonists on neutrophils isolated from peripheral blood of human healthy subjects. Chemotaxis was performed using the migration under agarose technique. To test the effect of B(1) receptor agonists, each assay was carried out overnight at 37 degrees C in 5% CO(2)-95% air on neutrophils primed with 1 ng/ml interleukin-1beta. Simultaneous experiments were performed using unprimed cells or cells challenged with formyl-Met-Leu-Phe (fMLP). A clear chemotactic activity was observed when primed neutrophils were challenged with Lys-des[Arg(9)]-bradykinin (LDBK) or des[Arg(9)]-bradykinin at 10(-10) M but not when unprimed cells were used. A reduction in the chemotactic response was observed after priming of cells in the presence of 0.5 mM cycloheximide and 10 mug/ml brefeldin A, suggesting that some protein biosynthesis is required. Techniques such as reverse transcriptase-polymerase chain reaction and in situ hybridization confirmed the expression of the B(1) receptor mRNA, and immunocytochemistry and autoradiography demonstrated the expression of the B(1) receptor protein. In contrast to other chemoattractants such as fMLP, cytosolic intracellular calcium did not increase in response to the B(1) receptor agonist LDBK. A generation of kinin B(1) receptor agonists during the early phase of acute inflammation may favor the recruitment of neutrophils to the inflammatory site.

Excitation-secretion coupling in exocrine glands. Properties of cyclic AMP phosphodiesterase and adenylate cyclase from the submaxillary gland and pancreas.

1. The cyclic AMP phosphodiesterase in homogenates of the submaxillary gland and pancreas was found to be associated mainly with the 300,000 times g supernatant fraction. A Lineweaver-Burk plot showed a high-affinity (Km app. = 1.6 muM) and a low-affinity (Km app. greater than 100muM) component for the cyclic AMP substrate. The enzyme was magnesium dependent, and strongly inhibited by papaverine, theophylline and caffeine. Cyclic GMP inhibited cyclic AMP phosphodiesterase, but only in concentrations greatly exceeding that of the cyclic AMP. Calcium did not alter the activity of the enzyme. The activity of the submaxillary cyclic AMP phosphodiesterase was not influenced by noradrenaline, dopamine, histamine, 5-hydroxytryptamine or gamma-amino butyric acid, and that of the pancreatic enzyme by acetylcholine, pancreozymin or secretin. 2. Adenylate cyclases from guinea-pig submaxillary gland and cat pancreas are particulate enzymes. The highest specific activity was recovered from the 1500 times g pellet. Guineo-pig submaxillary adenylate cyclase was activated by fluoride, noradrenaline, isoprenaline and adrenaline. The noradrenaline activation was blocked by the beta-adrenoceptor blocker, propranolol, but not by the alphs-adrenoceptor blocker, phentolamine. Neither acetylcholine nor carbachol had any effect on the adenylate cyclase activity. The apparent Km value for the 10- minus 4 M noradrenaline activated adenylate cyclase activity was completely aboliched by 5 mM calcium. Cat pancreatic adenylate cyclase was clearly and consistently activated by secretin, but not by pancreozymin or carbachol.

Genealogy, expression, and cellular function of transforming growth factor-beta.

The transforming growth factor-beta (TGF-beta) gene superfamily expresses a large set of structurally and functionally related polypeptides. Three TGF-beta isoforms are regulated by specific genes and have been identified in mammals (TGF-beta1, -beta2, and -beta3). All three-protein isoforms are observed abundantly during development and display overlapping and distinct spatial and temporal patterns of expressions. Each isoform plays a distinct role, the nature of which depends on the cell type, its state of differentiation, and growth conditions, and on the other growth factors present. TGF-beta regulates many of the processes common to both tissue repair and disease, including angiogenesis, chemotoxins, fibroblast proliferation and the controlled synthesis, and degradation of matrix proteins, such as collagen and fibronectin. This review will examine the genealogy and mode of actions of TGF-beta on the cell types involved in inflammation and repair, as well as in carcinoma.

Endothelins: vasoactive modulators of renal function in health and disease.

Vasoactive autocoids with directly opposing actions on the renal vasculature, glomerular function, and in salt and water homeostasis have been demonstrated in the kidney. In the renal cortex, endothelin (ET)-1 and angiotensin-II cause vasoconstriction, decreasing renal blood flow, and glomerular filtration rate, whereas bradykinin and atrial natriuretic peptide cause vasodilation and increase glomerular capillary permeability. ET-1 causes vasoconstriction of the afferent and efferent arteries and outer medullary descending vasa recta, thereby decreasing vasa recta and papillary blood flow, while bradykinin has the opposite effect. ET-1 stimulates cell proliferation, increasing the expression of several genes, including collagenase, prostaglandin endoperoxidase synthase, and platelet-derived growth factor. ET-1 promotes natriuresis via the ET-B receptor, causing down-regulation of the epithelial Na(+) channel in the renal tubule. Thus, ETs affect three major aspects of renal physiology: vascular and mesangial tone, Na(+) and water excretion, and cell proliferation and matrix formation.

Pathophysiology of the kallikrein-kinin system in mammalian nervous tissue.

The nervous system and peripheral tissues in mammals contain a large number of biologically active peptides and proteases that function as neurotransmitters or neuromodulators in the nervous system, as hormones or cellular mediators in peripheral tissue, and play a role in human neurological diseases. The existence and possible functional relevance of bradykinin and kallidin (the peptides), kallikreins (the proteolytic enzymes), and kininases (the peptidases) in neurophysiology and neuropathological states are discussed in this review. Tissue kallikrein, the major cellular kinin-generating enzyme, has been localised in various areas of the mammalian brain. Functionally, it may assist also in the normal turnover of brain proteins and the processing of peptide-hormones, neurotransmitters, and some of the nerve growth factors that are essential for normal neuronal function and synaptic transmission. A specific class of kininases, peptidases responsible for the rapid degradation of kinins, is considered to be identical to enkephalinase A. Additionally, kinins are known to mediate inflammation, a cardinal feature of which is pain, and the clearest evidence for a primary neuronal role exists so far in the activation by kinins of peripherally located nociceptive receptors on C-fibre terminals that transmit and modulate pain perception. Kinins are also important in vascular homeostasis, the release of excitatory amino acid neurotransmitters, and the modulation of cerebral cellular immunity. The two kinin receptors, B2 and B1, that modulate the cellular actions of kinins have been demonstrated in animal neural tissue, neural cells in culture, and various areas of the human brain. Their localisation in glial tissue and neural centres, important in the regulation of cardiovascular homeostasis and nociception, suggests that the kinin system may play a functional role in the nervous system.

Kallikrein and kinin receptor genes.

Recent evidence increasingly supports the view that kinins exercise an important regulatory control in inflammation and in the growth and proliferation of cancer cells. The induction of tissue kallikrein (TK) gene results in either increased or new expression of this protease, resulting in an increased capacity to form kinins. The cellular actions of kinins are initiated and controlled by kinin B1 and B2 receptors. This review collates in detail current knowledge on the molecular profile and status of TK (hKLK1, hKLK2, and hKLK3) and the kinin B1 and B2 receptor genes. The development of TK inhibitors, as well as kinin receptor antagonists, for use in immune-modulated disorders and in tumours may provide a new generation of drugs of therapeutic value.

Natural products for cancer prevention: a global perspective.

The control of cancer, the second leading cause of death worldwide, may benefit from the potential that resides in alternative therapies. The primary carcinogens stem from a variety of agricultural, industrial, and dietary factors. Conventional therapies cause serious side effects and, at best, merely extend the patient's lifespan by a few years. There is thus the need to utilise alternative concepts or approaches to the prevention of cancer. This review focuses on the many natural products that have been implicated in cancer prevention and that promote human health without recognisable side effects. These molecules originate from vegetables, fruits, plant extracts, and herbs.

Upregulation of tissue kallikrein, kinin B1 receptor, and kinin B2 receptor in mast and giant cells infiltrating oesophageal squamous cell carcinoma.

BACKGROUND: The mitogenic kinin peptides formed by the serine protease, tissue kallikrein (TK1), stimulate the proliferation of tumour cells and, by increasing vascular permeability, enhance metastasis. Oesophageal mucosal epithelial cells are derived from the epithelial cell germ layer, which expresses the kallikrein-kinin cascade. AIM: To determine the cellular distribution of active TK1, prokallikrein, and the kinin B(1) and B(2) receptors in oesophageal carcinoma by immunocytochemistry and in situ hybridisation (ISH). METHODS: Fifty oesophageal specimens (33 biopsies and 17 resections) and 10 control specimens adjacent to tumour or normal oesophageal biopsies were studied. Specific antibodies were used to determine the cellular localisation of TK1, prokallikrein, and the kinin B(1) and B(2) receptors in normal and oesophageal specimens by standard immunohistochemical techniques. The intensity of immunolabelling was quantified by image analysis. Antisense probes for TK1 and the kinin B(1) and B(2) receptors were also used to localise mRNA. RESULTS: TK1 (active and prokallikrein) was expressed in the mucosa of normal and tumour oesophageal epithelium. In general, expression was highest in activated mast cells, followed by giant tumour cells. Immunolabelling results were confirmed by ISH experiments. CONCLUSIONS: This is the first demonstration that TK1 and kinin B(1) and B(2) receptors are expressed in oesophageal carcinoma. Because TK1 released from tumour cells enzymatically generates mitogenic kinins from its endogenous substrate, kininogen, it is possible that third generation kinin receptor antagonists, which have been shown to be cytotoxic to cancer cells, may be useful therapeutic agents in this disease.

Kinin receptors on human neurones.

Knowledge of the distribution of kinin receptors in the human brain will aid our understanding of the role of kinins in neurophysiology. Furthermore, induction of the kinin B1 receptor may be important in the pathogenesis of neural diseases. Using polyclonal antibodies directed to specific regions of the B1 and B2 kinin receptors and standard immunolabelling techniques, we report on the localisation of these receptors on neurones in specific areas of the human brain. B2 bradykinin receptors are present in neurones of the brain stem, basal nuclei, cerebral cortex, thalamus and hypothalamus. B2 immunolabelling was also observed in the endothelial lining of the superior sagittal dural sinus and ependyma of the lateral and third ventricles. B1 kinin receptors have been localised on neurones of the thalamus, spinal cord and hypothalamus. Although binding of labelled bradykinin to neuronal membranes has been demonstrated, this is the first conclusive evidence for the existence of immunoreactive B1, and further confirmation of B2 receptors on human neurones.

Opioid inhibition of adenylate cyclase in the striatum and vas deferens of the rat.

The activity of adenylate cyclase in striatal membrane-enriched fractions (25,000 g) was inhibited by morphine, beta-endorphin, [D-Ala2-D-Leu5] enkephalin (DADLenk), fentanyl and bremazocine. Whereas guanosine triphosphate (GTP) appeared essential for the expression of this effect, sodium chloride seemed to enhance the degree of inhibition. Dopamine stimulation and sodium fluoride activation of the enzyme was also suppressed by morphine, beta-endorphin and DADLenk. beta-Endorphin and DADLenk inhibited adenylate cyclase activity in vasa deferentia membrane-enriched fractions (25,000 g); both opioids required GTP and NaCl and were inhibited by a delta-opioid receptor antagonist and by naloxone. Morphine, bremazocine and tifluadom did not significantly alter the activity of the vas deferens enzyme. Basal cyclic AMP values of striatal slices were not significantly altered by morphine, beta-endorphin or DADLenk. However, dopamine-induced elevation of cyclic AMP was reduced by morphine and this effect of the opiate was suppressed by naloxone. Only beta-endorphin lowered the basal cyclic AMP values in the vas deferens. The physiological relevance of adenylate cyclase coupling to opioid receptor subtypes is considered.

Kallikrein, trypsin-like proteases and amylase in mammalian submaxillary glands.

1. The tissue concentration of kallikrein, trypsin-like proteases and amylase has been determined in the submaxillary glands of the cat, dog, rabbit, rat, hamster and guinea-pig.2. Submaxillary gland extracts of the six species showed a wide variation in kallikrein content. The esterase activity in the extracts, however, did not correlate with the kininogenase activity of kallikrein. Both activities in each of the six species were affected by proteinase inhibitors but not in the same way.3. Evidence is presented which indicates that trypsin-like proteases similar to those described in the rat, also occur in the hamster and cat submaxillary glands.4. An even greater spread of amylase activity was observed between the species.5. The assumption that mammalian submaxillary kallikreins are identical proteins is questioned.

Electrophoresis of isolated secretory granules from guinea-pig submaxillary gland.

1 The nature of the surface charge on secretory granules from the submaxillary gland of the guinea-pig has been studied by electrophoresis of the isolated granules. The granules suspended in buffered sucrose solutions move to the anode.2 Divalent ions reduce the anodal movements of the granules. Calcium and magnesium neutralize the net surface charge of the granules. This result supports the hypothesis that the electrostatic surface charge which can be reduced by calcium ions may be a major factor in controlling the fusion of secretory granules to the cell membrane, before release by exocytosis.3 The mechanisms which couple membrane events with enzyme or hormone release from secretory glands are considered.

Biosynthesis of noradrenaline in organ cultured hearts.

In hearts of newborn mice, maintained in organ culture in an in vitro environment, noradrenaline levels were reduced after 24 h but subsequently recovered, reaching at three days values significantly greater than those found at birth. The increased synthesis of noradrenaline was blocked by the inhibitor of tyrosine hydroxylase, alpha-methyl tyrosine methyl ester. The site and control of noradrenaline biosynthesis in cultured newborn hearts are discussed.

Modulation of cyclic AMP in isolated rat uterine tissue slices by porcine relaxin.

Porcine relaxin produced a rapid, dose-related rise of cyclic AMP values in rat uterine tissue incubated in vitro. In time-course experiments, peak cyclic AMP concentrations were observed in the uterine slices at 5 min; subsequently the values fell, at first rapidly and then more slowly with the tissue concentration remaining significantly raised at 15 min. Levels of cyclic GMP in the same tissue slices were not significantly altered by relaxin. Furthermore, no increase in basal cyclic AMP values was measured in control slices prepared from the rat heart or jejunum. An increase in cyclic AMP concentration comparable to that found in the rat uterus was observed in slices of porcine uterus and cervix but not of vagina when they were stimulated with porcine relaxin. Our results suggest that the hormonal action of relaxin on the uterus and cervix is mediated through receptors linked to the enzyme, adenylate cyclase.

Characterization of a tissue kallikrein in human prolactin-secreting adenomas.

Immunoreactive tissue kallikrein was co-localized with prolactin in all the eleven prolactin-secreting adenomas of the human anterior pituitary gland examined in this study. The intracellular distribution of immunoreactivity in the prolactin-secreting cells suggests that tissue kallikrein is located within the Golgi complex of these cells. Both the intracellular hormone-processing action and the kininogenase activity of tissue kallikrein may be of functional importance in human prolactinomas.

Tissue kallikrein is associated with prolactin-secreting cells within human growth hormone-secreting adenomas.

Tissue kallikrein is a serine protease which may be involved in the intracellular processing of prolactin in the anterior pituitary gland. The expression of tissue kallikrein, in the rat, is promoted by oestrogen and inhibited by dopamine. Human and rat prolactinomas contain markedly increased amounts of tissue kallikrein; this is comparatively reduced if patients are pretreated with the dopamine agonist, bromocriptine, before surgery. Some GH-secreting adenomas are mixed and also contain prolactin-secreting cells. We therefore investigated 27 GH-immunostaining human pituitary adenomas for the presence of immunoreactive tissue kallikrein. Sixteen of the adenomas had positive immunostaining for prolactin; eight of these patients had associated clinical hyperprolactinaemia before the tumour was removed. Tissue kallikrein immunoreactivity was found in ten adenomas, all of which also had prolactin immunopositivity. There was a close relationship between the percentage of cells staining for prolactin and tissue kallikrein but not for GH. A further eight adenomas had patchy positivity, i.e. less than 1% of cells immunostained for tissue kallikrein and six of these also had some prolactin-staining cells. Nine out of eleven purely GH-staining adenomas had no tissue kallikrein immunopositivity, the remaining two showing patchy staining. A review of bromocriptine responsiveness, as assessed by mean GH hormone levels during oral glucose tolerance tests before and after therapy was commenced, indicated that patients with adenomas which stained for prolactin and tissue kallikrein were more likely to respond to bromocriptine than those which failed to do so.

Development and characterization of a radioimmunoassay to measure human tissue kallikrein in biological fluids.

A direct radioimmunoassay has been developed to measure tissue kallikrein in human biological fluids. These fluids include serum, plasma, urine, pancreatic juice and saliva. Purified kallikreins from human urine and human saliva were used to raise rabbit antibody and each was labelled with Na125I for use in the radioimmunoassay. A comparison of the different antigen-antibody systems was then made. Bound and free enzyme were separated by a double-antibody technique. The usable range of the standard curve was from 2.5 to 100 micrograms kallikrein/l. The intra-assay coefficient of variation was 4.7%, the interassay coefficient of variation 8.9% and the recoveries of purified kallikrein added to the samples were 99.3, 96.0, 110.8 and 81.2% for urine, saliva, serum and plasma respectively. Parallel dilution curves were obtained for serum and plasma, as well as for urine, saliva and pancreatic juice. However, plasma anticoagulated with EDTA or heparin gave consistently lower values than serum, when measured in the radioimmunoassay. From eight different subjects plasma (EDTA) values were on average 50% lower than those of serum. Experiments designed to determine the cause of this difference revealed that treatment of blood with some anticoagulants, in particular heparin and EDTA, resulted in a marked reduction in the amount of measurable tissue kallikrein.

Comparison of the action of cholecystokinin, carbachol and vasoactive intestinal peptide on receptor-activated formation of cyclic GMP and cyclic AMP in the striatum and the pancreas.

Sulphated cholecystokinin octapeptide (CCK-8S) and sodium nitroprusside (SNP) increased the formation of cyclic GMP in rat striatal slices with no effect on cyclic AMP. CCK-8S, SNP and carbachol increased the formation of cyclic GMP in guinea-pig pancreatic lobules, but had no effect on levels of cyclic AMP. Vasoactive intestinal peptide (VIP) significantly stimulated the formation of cyclic AMP in both striatal and pancreatic tissue without effect on levels of cyclic GMP in these tissues. In rat striatal slices carbachol significantly inhibited the VIP-stimulated increase in cyclic AMP.

Modulation of guinea-pig lung adenylate cyclase by ovalbumin sensitization.

1. The regulation of lung adenylate cyclase was investigated in guinea pigs sensitized with high dose (300-500 micrograms kg-1) ovalbumin to raise IgG(I) and low dose (2.8-4.0 micrograms kg-1) to raise to IgG/IgE (II) antibodies. 2. Basal activity of sensitized II (IgG/IgE antibodies) lung adenylate cyclase was approximately double that of the control values. 3. Guanosine 5'-triphosphate (GTP) was a potent activator of adenylate cyclase from the sensitized II (IgG/IgE) lung membranes. 4. The sensitivity of lung membrane adenylate cyclase to stimulation by beta-adrenoceptor agonists or VIP was reduced whereas no significant difference was observed with histamine or bradykinin on the sensitized II membranes. 5. Possible mechanisms involved in the increase in basal activity and in the decreased sensitivity to beta-adrenoceptor- and VIP-mediated stimulation of adenylate cyclase by ovalbumin sensitization of guinea-pig lung membranes are discussed.