Underexpression of tumour suppressor LKB1 in clear cell renal cell carcinoma is common and confers growth advantage in vitro and in vivo.

BACKGROUND: Evidence suggests that dysregulation of energy-sensing pathways closely associates with renal cell carcinoma (RCC) development. The metabolic regulation is largely controlled by 5'-AMP activated protein kinase (AMPK) which is activated through phosphorylation by LKB1. METHODS: The expression of LKB1 was determined by reverse transcription-PCR using 10 clinical clear cell RCC (ccRCC) samples and their adjacent normal renal parenchyma, and by immunohistochemical staining of two tissue microarrays containing 201 ccRCC and 26 normal kidney samples. Expression of LKB1 was knocked down in human ccRCC 786-O cells (shLKB1) and compared with cells expressing scrambled control shRNA (shControl). AMPK signalling, proliferation, invasion, and VEGF secretion was measured. The cells were subcutaneously injected into mice to determine tumour growth in vivo. RESULTS: At the protein and transcript levels, a significant reduction in LKB1 expression in tumour compared with normal tissue was found. In vitro, knockdown of LKB1 resulted in reduced AMPK signalling and increased cellular proliferation, invasion, and VEGF secretion compared with shControl cells. In vivo, growth of shLKB1 ccRCC xenografts in nude mice was significantly increased compared with shControl xenografts. CONCLUSION: Collectively, our results suggest that LKB1 acts as a tumour suppressor in most sporadic cases of ccRCC and that underexpression of LKB1 is a common event in the disease.

Prenatal stress enhances severity of atherosclerosis in the adult apolipoprotein E-deficient mouse offspring via inflammatory pathways.

Atherosclerosis is the underlying cause of cardiovascular disease and stroke. Endothelial cell dysfunctions are early events in atherosclerosis, resulting in the recruitment of circulating monocytes. The immune system can elicit an inflammatory response toward the atherosclerotic lesion, thereby accelerating lesion growth. Risk factors for atherosclerosis include hypertension, smoking, stress perception or low birth weight. As prenatal stress challenge decreases the birth weight and affects the offspring's postnatal immune response, we aimed to investigate whether prenatal stress contributes to the development of atherosclerosis in mice. Syngenic pregnant apolipoprotein E-deficient (apoE-/-) dams were exposed to sound stress on gestation days 12.5 and 14.5. The presence and size of atherosclerotic plaques in the offspring at the age of 15 weeks was evaluated by histomorphology, accompanied by flow cytometric analysis of the frequency and phenotype of monocytes/macrophages and regulatory T (Treg) cells in the blood. Further, cytokine secretion of peripheral blood lymphocytes was analyzed. In response to prenatal stress challenge, an increased frequency of large atherosclerotic plaques was detectable in apoE-/- offspring, which was particularly profound in females. Prenatal stress also resulted in alterations of the offspring's immune response, such as a decreased frequency of Treg cells in blood, alterations of macrophage populations in blood and an increased secretion of inflammatory cytokines. We provide novel evidence that prenatally stressed adult offspring show an increased severity of atherosclerosis. As Treg cells are key players in dampening inflammation, the observed increase in atherosclerosis may be due to the lack of Treg cell frequency. Future interdisciplinary research is urgently required to understand the developmental origin of prenatal stress-induced atherosclerosis. The availability of our model may facilitate and foster such research endeavors.

Morphological, histomorphometric, and microstructural alterations in human bone metastasis from breast carcinoma.

Bone is one of the most common sites of breast cancer metastasis. Metastases are often associated with bone destruction and are a major cause of morbidity. We examined structural bone changes induced by metastatic tumor in bone biopsies from 33 patients with metastatic breast carcinoma (20 from patients with pathological femoral fracture and 13 with no fracture) and 20 normal controls. In all metastatic biopsies bone remodeling was shown to be tumor volume-dependent. Bone resorption and bone formation were biphasic with both increasing at earlier stages of metastatic bone disease and decreasing later on. A comparison of patients with fracture and no fracture did not reveal statistically significant differences in the extent of bone destruction or trabecular thinning. Bone histomorphometry showed limited ability to explain the higher bone volume loss in fracture patients (decreases of 42% and 25%, respectively, in fracture and nonfracture patients compared with controls). However, changes in bone quality, including increased disconnectivity and decreased connectivity, as evaluated by node-strut analysis, suggested that there were more structural changes in the fracture compared with the nonfracture group. The nonfracture group included six patients with no radiological evidence of bone metastasis (occult metastasis). They showed a higher tumor volume and a twofold lower eroded surface compared with the rest of the group. The decrease in bone volume (14% lower than controls) was below the limit of X-ray detection. Because we observed no increase in osteoclast-related parameters and no correlation between osteoclast surface and eroded surface, we believe that, in occult metastasis, osteoclastic bone resorption is not an important factor in overall bone resorption. Quantitatively, the eroded surface in direct contact with tumor cells was threefold higher than the osteoclast surface in occult metastasis, whereas the rest of the metastatic group (27 of 33) showed predominantly osteoclast-mediated eroded surface. Node-strut analysis on occult metastasis revealed a significant increase in disconnectivity without a concomitant significant decrease in bone volume and trabecular thinning. We conclude that, in occult metastasis, bone resorption may be more osteoclast-independent and other mechanisms involving the tumor cells may be more prevalent.

A mouse homologue of the Drosophila tumor suppressor l(2)tid gene defines a novel Ras GTPase-activating protein (RasGAP)-binding protein.

p120 GTPase-activating protein (GAP) down-regulates Ras by stimulating GTP hydrolysis of active Ras. In addition to its association with Ras, GAP has been shown to bind to several tyrosine-phosphorylated proteins in cells stimulated by growth factors or expressing transforming tyrosine kinase variants. Here we report the cloning and characterization of a novel GAP-binding protein, mTid-1, a DnaJ chaperone protein that represents the murine homolog of the Drosophila tumor suppressor l(2)tid gene. Three alternatively spliced variants of mTid-1 were isolated, two of which correspond to the recently identified hTid-1(L) and hTid-1(S) forms of the human TID1 gene that exhibit opposing effects on apoptosis. We demonstrate that both cytoplasmic precursor and mitochondrial mature forms of mTid-1 associate with GAP in vivo. Interestingly, although mTid-1 is found tyrosine-phosphorylated in v-src-transformed fibroblast cells, GAP selectively binds to the unphosphorylated form of mTid-1. In immunofluorescence experiments, GAP and Tid-1 were shown to colocalize at perinuclear mitochondrial membranes in response to epidermal growth factor stimulation. These findings raise the possibility that Tid chaperone proteins may play a role in governing the conformation, activity, and/or subcellular distribution of GAP, thereby influencing its biochemical and biological activity within cells.

Taxol (paclitaxel) involution of articular cartilage destruction in collagen induced arthritis: an ultrastructural demonstration of an increased superficial chondroprotective layer.

OBJECTIVE: To determine the ultastructural changes of Taxol (paclitaxel) involution of articular cartilage destruction in collagen induced arthritis (CIA) and to compare with articular cartilage from normal rats. METHODS: Forty-five Louvain rats were randomized to one of 3 protocols for structural analysis: (1) control group, (2) CIA group, and (3) Taxol treated CIA group. The latter group received 10 mg/kg body weight of Taxol at Days 10, 12, and 14 and 7.5 mg/kg body weight of Taxol on Days 16, 18, and 20 postimmunization with collagen type II. Eight days later, each group was examined by light microscopy and scanning and transmission electron microscopy. RESULTS: In Taxol treated rats, the morphology of the articular cartilage reverted to that observed in naive rats except for a striking increase in the thickness of the superficial amorphous layer covering the articular surface. CONCLUSION: The involution of CIA by Taxol suggests that this agent may be useful in the clinical treatment of RA.

Evidence that colitis is initiated by environmental stress and sustained by fecal factors in the cotton-top tamarin (Saguinus oedipus).

Parallel changes in spontaneously occurring inflammation in colonic Thiry-Vella loops and the in-line colon of cotton-top tamarins were studied in a colitis-inducing environment at 8 and 15 months following surgical preparation of the loops. Gross disease severity and numbers of inflammatory/immune cells per unit area of lamina propria in histological sections from endoscopic biopsies were analyzed. Cell counts and severity of colitis declined over time in the Thiry-Villa loops while the disease followed its characteristic course in the remaining large bowel and in the colons of controls. Perfusion of the loops with the animals' feces increased the density of the cellular infiltrate in the lamina propria in parallel with increased severity of inflammation. Electron micrographs of the colonic mucosa showed invasion by microorganisms. The predominant microorganism had characteristics of Helicobacter sp. The results implicate the fecal stream as a factor in the persistence of colitis in the tamarin model. Nevertheless, fecal factors appear not to be the primary trigger, as evidenced by findings that the disease is not expressed in wild-living tamarins and that it enters remission when affected animals are transferred to natural conditions from a colitis-inducing environment. Both an adverse environment and the fecal contents appear to be required for expression of the disease.

Immunolocalization of matrix metalloproteinases and their inhibitors in clinical specimens of bone metastasis from breast carcinoma.

Matrix metalloproteinases (MMPs) are essential in several stages of the metastatic process, and in normal bone development and remodeling. We explored whether the interaction between tumor cells and bone leads to changes in MMP and tissue inhibitor of MMP (TIMP) expression thus affecting osteolysis in metastatic bone disease. Using immunohistochemistry we have investigated the MMP/TIMP expression in tumor cells, fibroblasts, osteoblasts and osteoclasts. Thirty one specimens of bone metastasis from breast carcinoma were stained for MMP-1, -2, -9, MT1-MMP and TIMP-1, and -2 and compared with staining in normal breast tissue, primary breast carcinoma and normal bone. Specimens came from patients in three clinical scenarios: from open biopsies without or with pathological fracture, or bone marrow biopsies containing tumor from patients with pancytopenia but without clinical evidence of osteolysis. By bone histomorphometry the latter group showed a heavy tumor load not different from the open biopsy groups but displayed little active bone resorption and low numbers of osteoclasts. Cell type-specific MMP/TIMP expression was observed and the staining patterns were comparable between the three groups of patients. Though no major differences in the MMP/TIMP staining of tumor cells and fibroblasts were observed between bone metastasis and primary tumor, we showed that tumor cells do express MMPs capable of degrading bone matrix collagen. The number and activity of osteoclasts and osteoblasts was increased dramatically in bone metastases, their MMP/TIMP profiles, however, were not different from normal bone, suggesting that the mechanism of bone degradation by osteoclasts is not different from normal bone remodelling.

Taxol involution of collagen-induced arthritis: ultrastructural correlation with the inhibition of synovitis and neovascularization.

Collagen-induced arthritis (CIA) is an animal model of rheumatoid arthritis (RA) that can be regressed with Taxol (paclitaxel), a chemotherapeutic agent. To identify structural changes that occur with involution, the synovium from naive, untreated CIA, and Taxol-treated CIA rats were evaluated by light microscopy plus transmission and scanning electron microscopy. Analysis included detailed images of vascular networks using polymeric corrosion casts. The CIA synovium was morphologically similar to human RA synovium. In CIA, the integrity of the intimal lining is lost by Type-B synoviocytes becoming highly elongated and polarized toward the joint space, resulting in non-overlapping cellular processes and the elimination of the basal lamina. In addition, the lining expanded from a width of 6-10 microns in naives to 200-250 microns in CIA due primarily to increased numbers of both Type-A and -B synoviocytes and more interstitial matrix. Vascular corrosion casts of CIA synovium illustrated a marked increase in blood vessel volume and an extensive interconnecting vascular architecture; neovascular arrays were observed to project toward the synovial surface. In Taxol-treated CIA, the synoviocyte and neovascular components reverted to the naive synovium morphology, suggesting that this agent might be useful in the therapy of RA.

Effect of truncal vagotomy and capsaicin on mast cells and IgA-positive plasma cells in rat jejunal mucosa.

Immunocompetent cells, including mast cells and plasma cells (PC), in the intestinal mucosa are closely apposed to nerve fibres. Recent work has shown that vagal afferent nerves penetrate the jejunal mucosa and contact intestinal mucosal mast cells (IMMC); and that electrical stimulation of the vagus results in increased IMMC histamine content. To determine if the vagus nerve exerts a trophic effect on immunocompetent cells in the gut mucosa, the effects of truncal vagotomy and neonatal capsaicin treatment on IMMC and IgA containing PC in the lamina propria of rat jejunum were investigated. Three weeks after vagotomy, microdensitometric assessment of Alcian blue stained sections revealed 25% fewer IMMC in vagotomized animals than in controls (P < 0.05). Three months after neonatal capsaicin administration 28% fewer IMMC were found in treated rat jejunum, compared with littermate controls (P < 0.05). Three weeks post-surgery, IgA-PC densities were increased in both vagotomized animals (that also underwent pyloroplasty) and pyloroplasty controls, compared to animals subjected to laparotomy only. The proportion of lamina propria areas remained stable, indicating that the observations reflected real reductions in the numbers of IMMC. We also determined the densities of B-50 (a nerve growth-associated protein, also called GAP-43) immunoreactive nerve fibres in the lamina propria, as well as nerve profile areas, three weeks after vagotomy, and these parameters were unchanged. Taken together, these findings support the hypothesis that the vagus exerts a trophic effect on IMMC; and that capsaicin-sensitive nerves also affect the IMMC population. These data add to the growing body of evidence for a functional connection between IMMC and the nervous system.

Electrical stimulation of the vagus nerve modulates the histamine content of mast cells in the rat jejunal mucosa.

Mast cells are best known for their participation in allergic reactions. However, a number of recent studies suggest that mast cells are subject to nervous control. In the gut mucosa, mast cells are intimately associated with nerves, and the psychologically conditioned release of RMCP II (a mucosal mast cell-derived mediator) has been reported. These data suggest the potential for CNS regulation of intestinal mucosal mast cells. In this study, we stimulated the cervical vagi and found an increased histamine content in mucosal mast cells, without apparent degranulation. Furthermore, these changes could be prevented by subdiaphragmatic vagotomy. These data support the potential for intestinal mucosal mast cell regulation by the central nervous system and suggest modulation of mast cells without degranulation.

Ultrastructural examination of B-50(GAP-43) immunoreactivity in rat jejunal villi.

The lamina propria of rat jejunum is densely innervated with nerve fibres extending to the tips of the villi. A large number of these nerve fibres were previously shown to be B-50-immunoreactive at the light microscope level, whereas neurofilament immunoreactivity was found to be sparse in the mucosa. In this study we used immunoelectron microscopy to determine what proportion of nerve fibres in the lamina propria express B-50. Jejuna from male Lewis rats were immunolabelled for B-50 and neurofilament proteins. For electron microscopy, postembedding immunogold-silver techniques and LR White embedded tissues were used. Light microscopical immunostaining was performed by the streptavidin-biotin-peroxidase technique on deparaffinized tissue sections. We found that all ultrastructurally identifiable nerve profiles in jejunum were B-50 immunoreactive. Immunoelectron microscopy for neurofilament proteins failed to label fibres in the villi, whereas myelinated nerves in tongue sections processed in parallel (positive controls) were strongly neurofilament-protein-immunoreactive. The dominant B-50-positive and neurofilament-protein-negative phenotype supports the hypothesis of ongoing modelling or plasticity of intestinal mucosal nerves.

Endothelial injury causes degradation of adjacent basement membranes and promotes their invasion by A549 carcinoma cells.

Experiments in vivo have demonstrated that endothelial cell injury promotes the local arrest of circulating, intravascular cancer cells and the subsequent formation of metastatic tumors. The experiments described here were performed to test the hypothesis that injury of the endothelium also causes damage to the adjacent vascular basement membrane, which in turn facilitates the passage of cancer cells across the vessel wall. Confluent monolayers of bovine pulmonary artery endothelial cells were incubated with 3H-2-deoxyglucose or 3H-proline to label the endothelial cells or the basement membrane, respectively. After adding H2O2 to these cultures, damage of the endothelium and basement membrane was detected by release of the isotopes into the culture medium. The kinetics and magnitude of basement membrane degradation correlated with the damage to the endothelial cells. Evidence for involvement of endothelial proteases in basement membrane injury included identification of a 63-kD gelatinase in the culture medium, inhibition of injury by protease inhibitors and the inability of H2O2 to cause 3H-proline release when applied directly to basement membranes. Scanning electron microscopy demonstrated that a greater number of A549 lung adenocarcinoma cells attached to the basement membrane and endothelium at points of endothelial retraction. However, this was not due to an increase in the adhesive properties of the basement membrane. The media from injured endothelial cultures stimulated the motility of A549 cells in a Boyden chamber assay. Furthermore, in a 24-hour invasion assay, a greater number of A549 cells migrated through injured basement membranes than through control membranes. We conclude that endothelial cell injury can cause enzymatic damage to the underlying basement membrane and postulate that this can facilitate the transvascular passage of cancer cells in vivo.

Immunoelectron microscopic diagnosis in nephropathology.

An immunogold postembedding method for the ultrastructural localization of immunoglobulin G (IgG), IgA, IgM, C3, fibrinogen, and kappa and lambda immunoglobulin light chains was established. The method was performed on surplus tissue from renal biopsies fixed in 2% glutaraldehyde and embedded in acrylic LR White resin. Fifteen cases were studied and the localization of antigen in the dense deposits in glomeruli compared with the immunofluorescence (IF) findings on the same cases. The results obtained by IF and immunoelectron microscopy (IEM) were comparable for IgG and IgA. In most instances, IgM and C3 were more intensely labeled by IF than IEM. Some discrepancies were noted for fibrinogen. It is postulated that these differences are related to a partial loss of antigenicity due to fixation or chemical reaction with the embedding medium. The advantage of IEM resides in localizing antigens in very small deposits seen in early stages of glomerulonephritis. The method can supplement IF in diagnostically difficult cases or be substituted for it when material for IF is not available.