Shc is a substrate of the rat intestinal epidermal growth factor receptor tyrosine kinase

Epidermal growth factor (EGF) has been shown to induce intestinal proliferation and maturation; however, little information is available regarding substrates of the intestinal EGF receptor tyrosine kinase. The purpose of this study was to determine if src homologous collagen-like protein (Shc) was an in vivo substrate of the intestinal EGF receptor. Ten-day-old rats were treated with EGF or were breast-fed. In some experiments, IEC-6 cells were treated with EGF. Intestinal tissue and cell fractions were studied by immunodetection to compare the tyrosine phosphorylation state and the subcellular localization of intestinal proteins. The total tyrosine phosphorylation state of intestinal proteins was increased threefold by EGF. Tyrosine phosphorylation of the EGF receptor and Shc were rapidly increased by EGF. The association of Grb2 with Shc increased fourfold and fivefold. Plasma membrane translocation of Shc and associated phosphotyrosyl proteins was increased within 30 seconds of EGF treatment. Shc is a substrate of the intestinal EGF receptor in vivo. EGF-induced association of Shc with the adapter protein Grb2 may have implications for activation of the p21^ras signaling pathway in the intestine. The EGF-induced membrane association of Shc with two other phosphotyrosyl proteins suggests involvement of Shc in additional aspects of EGF-receptor signaling in the intestine.     

Changes in the site- and histology-specific incidence of gastric cancer during a 50-year period

In contrast to the dramatic decrease in the overall incidence of gastric cancer, there has been a reported increase in the incidence of cases located in the gastric cardia. The aim of this study was to identify changes in site- and histology-specific incidence rates of gastric adenocarcinoma during a 50-year period. The Rochester Epidemiology Project medical records linkage system was used to identify all cases of gastric adenocarcinoma among residents of Olmsted County, Minnesota, between 1941 and 1990 (n = 342). Each patient's complete (inpatient and outpatient) medical records were reviewed and tumor location determined from pathological, surgical, endoscopic, and radiological reports. All available histological specimens (n = 246) were reviewed independently. The overall incidence of gastric cancer decreased from 48.8 per 100,000 person-years in the 1940s to 11.6 per 100,000 in the 1980s, whereas the incidence of adenocarcinoma of the cardia did not change significantly during the 50-year period. The incidence of adenocarcinoma of the esophagogastric junction increased from 0.0 to 1.9 per 100,000 person-years, but the number of cases was small. The incidence of adenocarcinoma of the gastric cardia has not increased in this population. The reported increase in cardia cancer in other populations may be due to an increasing incidence of adenocarcinoma of the esophagogastric junction.     

Regulation of protein secretion into bile: Studies in mice with a disrupted mdr2 p-glycoprotein gene

Protein is secreted into bile via several independent pathways. The aim of this study was to investigate whether these pathways are influenced by secretion of biliary lipid. Protein secretion and biliary lipid output were studied in wildtype mice (+/+), heterozygotes (+/−), and homozygotes (−/−) for mdr2 gene disruption. Biliary lipid and protein output were varied by infusion with taurocholate (TC) and tauroursodeoxycholate (TUDC). Exocytosis and transcytosis were unaltered in (−/−) mice. Infusion with TC strongly induced secretion of alkaline phosphatase in (−/−) mice but had little effect in (+/−) and (+/+) mice. Infusion with TUDC had little effect on alkaline phosphatase output. In contrast, both TUDC and TC strongly stimulated secretion of aminopeptidase N and lysosomal enzymes in (+/+) mice but had no effect in (−/−) animals. Aminopeptidase N secretion correlated with phospholipid output, but only at high flux. At low flux, aminopeptidase N was secreted independently from both phospholipid and bile salts. The canalicular membrane enzymes alkaline phosphatase and aminopeptidase N are secreted via separate pathways. Part of alkaline phosphatase output is controlled by bile salt hydrophobicity, whereas at high lipid flux, aminopeptidase N secretion seems to be coupled to phospholipid output. Lysosomal enzymes follow the latter pathway.     

Microsatellite instability in human colonic cancer is not a useful clinical indicator of familial colorectal cancer

Microsatellite instability is a property of most tumors occurring in the context of hereditary nonpolyposis colon cancer. Instability also occurs in 10%–15% of apparently sporadic colorectal cancers, and it has been hypothesized that this instability may indicate a genetic predisposition to colonic cancer. This study evaluated whether there is a clinically useful association between colon cancer instability and a family history of cancer. Colon cancer cases (n = 188) from a population-based study were evaluated for microsatellite instability with 10 polymerase chain reaction primer sets. Instability results were compared with family history and other clinical and biological characteristics. Microsatellite instability was found in 16.5% of tumors. It was predominantly a feature of right-sided tumors (P = 0.003) and was associated with the youngest and oldest ages at diagnosis (P = 0.01). Instability was not associated with family history of cancer, sex of the individual, or the glutathione-S-transferase mu 1 null genotype. Although some very small, and as yet undefined, proportion of colon cancer may be caused by inherited mutations leading to microsatellite instability, tumoral instability by itself is not a marker for familiality and should not be considered as evidence for an inherited syndrome.     

Incidence of occult cancer in children presenting with musculoskeletal symptoms: a 10-year survey in a pediatric rheumatology unit

OBJECTIVES: To assess the frequency and types of cancer found in children presenting to our Unit with musculoskeletal symptoms over a 10-year period. METHODS: The medical records of patients with musculoskeletal symptoms and a final diagnosis of cancer were reviewed. In each case age, gender, presenting symptoms, laboratory data, diagnostic procedures, provisional and final diagnoses, and time between clinical onset and correct diagnosis were reviewed. RESULTS: An underlying neoplasia was found in 10 of 1,254 patients (<1%) complaining of musculoskeletal symptoms. The types of malignancies found included acute lymphocytic leukemia (ALL) (6 cases), lymphoma (2 cases), neuroblastoma (1 case), and Ewing's sarcoma (1 case). The mean time between disease onset and final diagnosis was 3.2 months. The most common presenting feature was monoarthritis, involving the larger joints such as the elbows, knees or ankles. Juvenile idiopathic arthritis (JIA) was the most frequent provisional diagnosis. In the preliminary hematologic evaluation, eight patients had an increased erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP) value. White blood cell (WBC) count was normal in almost all children, with a normal differential count. Lactic dehydrogenase (LDH) was raised in all children. Bone marrow aspirates and lymph node or bone biopsies were necessary to reach the final diagnosis. CONCLUSIONS: A malignancy should always be excluded in children with musculoskeletal symptoms, especially when the clinical pattern is not characteristic of a specific rheumatic disease. Routine laboratory tests may be misleading. The simultaneous presence of high LDH or alpha-hydroxybutyric dehydrogenase (alpha-HBDH) levels and raised ESR or CRP, even with normal blood cell counts, should lead to additional investigations. RELEVANCE: All patients presenting with arthritis or other musculoskeletal symptoms should have a thorough clinical examination. Disproportionate pain levels and an atypical pattern of "arthritis," especially in the presence of systemic manifestations, suggest a possible underlying malignancy.     

Depolarization induces insulin-like growth factor binding protein-2 expression in vivo via NMDA receptor stimulation

The effect of depolarization and N-methyl-D-aspartate (NMDA) receptor blockade on insulin-like growth factor-I (IGF-I), IGF binding protein-2 (IGFBP-2) and IGFBP-4 expression was analysed in vivo. Depolarization was induced in adult rat brains by applying 3 M KCl to the exposed cortex for 10 min. A subgroup of animals also received daily injections of MK-801. Four days after KCl exposure, the brains were analysed by in situ hybridization, immunohistochemistry and TUNEL. A significant upregulation of IGFBP-2 mRNA and protein was detected in astrocytes after KCl exposure This upregulation was reduced by MK-801 treatment. No alterations in IGF-I or IGFBP-4 mRNA levels were noted. We did not detect TUNEL positive cells, morphological signs of necrosis or apoptosis, or neuronal loss in the depolarized zone. Taken together, these findings indicate that upregulation of IGFBP-2 by depolarization is mediated by NMDA receptors, and, as no neuronal damage was detected, astrocytic NMDA receptors may be responsible for this upregulation.     

Total and free insulin-like growth factor I, insulin-like growth factor binding protein 3 and acid-labile subunit reflect clinical activity in acromegaly

The aim was to evaluate, markers of disease activity in acromegaly in relation to perceived disease activity. Thirty-seven consecutively treated, acromegalic patients, classified by clinical symptoms as inactive (n=16), slightly active (n=10) and active (n=11), entered the study. When evaluating the inactive and the active groups, we found that positive and negative predictive values (PV(pos), PV(neg)) for clinical disease activity of total and free insulin-like growth factor-I (IGF-I) were 0.59, 0.90 and 1.00, 0.82 respectively. Acid-labile subunit (ALS) showed diagnostic merit similar to insulin-like growth factor binding protein-3 (IGFBP-3) with PV(pos) of 0.69 and 0.71 and PV(neg) of 0.91 and 0.92 respectively. We conclude that free IGF-I is more closely related than total IGF-I to perceived disease activity and is as such useful when evaluating previously treated acromegaly for disease activity. Total IGF-I, IGFBP-3 and ALS possess a higher PV(neg) for the clinical disease activity. None of the parameters can at present be claimed to be superior to the others and thus all the measured parameters are recommended to be part of the evaluation of acromegalic patients.     

Roles of insulin-like growth factor-I (IGF-I) and IGF-I binding protein-2 (IGFBP2) and -5 (IGFBP5) in developing chick limbs

Insulin-like growth factor-I (IGF-I) and the IGF-I binding proteins (IGFBPs) which modulate IGF-I action have been implicated in the development of the vertebrate limbs and skeleton. We have examined the distribution of IGF-I, IGFBP2 and IGFBP5 in developing chick limb buds and have investigated their functional roles and relationships during chick limb development. IGF-I and IGFBP2 are co-expressed throughout the lateral plate from which limbs form, although IGFBP2, unlike IGF-I, does not promote formation of rudimentary limb buds from non-limb-forming flank regions in vitro. During limb outgrowth, IGF-I is present in non-AER limb ectoderm, but little IGF-I is present in the AER itself, suggesting that restriction of endogenous IGF-I activity may be required for proper AER function. Consistent with this possibility, the ectoderm of mutant limbless and wingless wing buds, which fail to form an AER, continues to express IGF-I. We also found that the AER contains abundant IGFBP2 but that IGFBP2 is not present in limb subridge mesoderm. In contrast, IGFBP2 is present in the distal mesoderm of mutant limbless or wingless limb buds, which fail to grow out. This suggests that attenuation of IGFBP2 expression is controlled by the AER and that cessation of IGFBP2 expression may be necessary for the proliferation and suppression of differentiation of subridge mesoderm that is required for limb outgrowth to occur. Consistent with this possibility, we found that exogenous IGFBP2 inhibits the anti-differentiative activity of the AER in vitro. We also found that regions of cell death in the limb contain abundant IGF-I-immunoreactive cells, consistent with a role for IGF-I in apoptosis. During skeletogenesis, IGF-I and IGFBP2 are co-localized to the condensing central core of the limb, implicating these factors as potential regulators of the onset of chondrogenic differentiation. Intriguingly, we found that IGF-I and IGFBP2 have opposing effects on chondrogenesis, as IGF-I stimulates but IGFBP2 inhibits accumulation of cartilage matrix by micromass cultures in vitro. Long [R(3)] IGF-I, an analog of IGF-I that cannot bind IGFBPs, is more effective than IGF-I in stimulating matrix accumulation, consistent with a negative role for IGFBP2 in chondrogenesis. As the chondrocytes of the limb mature, IGF-I is present only in terminal hypertrophic chondrocytes, which undergo programmed cell death, while IGFBP2 becomes localized to prehypertrophic and hypertrophic chondrocytes, suggesting involvement in chondrocyte maturation. Consistent with this possibility, we found that exogenous IGFBP2 induces precocious expression of Indian hedgehog, a marker of prehypertrophy, in maturing chondrocytes in vitro. IGF-I and IGFBP2 are also present in the osteoblasts, clasts and nascent matrix of the long bones, consistent with roles in endochondral bone formation. Unlike in rodent limbs, IGFBP5 is not expressed by chick limb ectoderm or AER. IGFBP5 expression is highly localized to developing limb musculature and, later, to the developing skeletal elements where it is expressed by osteoblast precursers and osteoblasts. The results of this study suggest potential novel roles for IGF-I and IGFBP2 in several aspects of limb development including limb outgrowth and AER activity, programmed cell death, chondrogenesis and chondrocyte maturation.     

Maternal nutrition affects the ability of treatment with IGF-I and IGF-II to increase growth of the placenta and fetus, in guinea pigs

The aim of this study was to investigate how administration of IGF-I and IGF-II, during early to mid pregnancy, affects maternal growth and body composition as well as fetal and placental growth, in ad libitum fed, and in moderately, chronically food restricted guinea pigs. From day 20 of gestation, mothers (3-4 months old) were infused with IGF-I, IGF-II (565 microg/day) or vehicle for 17 days and then killed on day 40 of gestation. Maternal organ weights, fetal and placental weights were assessed. Treatment with IGFs did not alter body weight gain and had small effects on body composition in the mothers. Both IGF-I and IGF-II increased fetal and placental weights in ad libitum fed dams and IGF-I increased placental weight in food restricted dams. In conclusion, treatment with IGF-I during the first half of pregnancy stimulates placental growth in both ad libitum fed and food restricted guinea pigs without affecting maternal growth while fetal growth is stimulated by IGF treatment only in ad libitum fed animals.     

The changes of IGF binding proteins after rhGH administration to patients totally dependent on parenteral nutrition

The objective was to study the effect of recombinant human growth hormone (rhGH) administration to patients with chronic malnutrition maintained on total parenteral nutrition (TPN) on the levels of insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) during a double-blind trial. After 1 week of TPN the patients were randomized into group I (placebo) or group II (rhGH). Samples were collected on the first day (start of the TPN) to measure basal values, the seventh day to study the effect of TPN and the 10th, 14th and 21st days to evaluate the rhGH effect. Basal laboratory evaluation, nutritional status and glucose tolerance were assessed using standard laboratory techniques. Radioimmunoassays were used to analyse IGF-I, free IGF-I (fIGF-I) and IGFBP1-3. Electrophoresis with Western ligand blotting and Western immunoblotting was applied to find the pattern of IGFBPs. TPN had no effect on the circulating IGF-I concentration and the pattern of IGFBPs present in the studied groups of patients. The rhGH administration led to significant increases of IGF-I, total IGFBP-3, glycosylated IGFBP-3 (39, 42 kDa) and the 29 kDa fragment of IGFBP-3 and the decrease of IGFBP-2 during the trial (P<0.05). The mean levels of IGFBP-1, fIGF-I and the parameters of nutritional status in group II during the trial were not significantly influenced by rhGH. However, it has been found that IGFBP-1 and fIGF-I levels were correlated with the levels of the weekly nitrogen balance of each patient in group II at the end of the trial. In spite of the significant changes of IGF-I, IGFBP-2, total IGFBP-3 and IGFBP-3 (29 kDa proteolytic fragment) after rhGH administration to patients with malnutrition, maintained on parenteral nutrition, the increase of nitrogen balance was seen only in patients who decreased their IGFBP-1 and increased bioavailable IGF-I as reflected by measurement of fIGF-I. The levels of IGFBP-1 may provide a useful marker of IGF-I bioavailability in monitoring the efficiency of the rhGH therapy in malnourished patients.     

Inhibition of human osteoblast marker gene expression by retinoids is mediated in part by insulin-like growth factor binding protein-6

All-trans -retinoic acid (atRA) inhibits osteoblast marker gene expression and markedly increases expression of insulin-like growth factor binding protein-6 (IGFBP-6) in human osteoblasts. The possibility that IGFBP-6 inhibits the osteoblast phenotype and also mediates the inhibitory effect of atRA on osteoblast marker gene expression was explored using an antisense approach. Stable human osteoblast-like osteosarcoma SaOS-2 cells were prepared that expressed antisense IGFBP-6 RNA under basal and atRA-stimulated conditions. The functional expression of IGFBP-6 antisense RNA was confirmed by measuring IGFBP-6 mRNA by Northern analysis or by measuring IGFBP-6 protein in the conditioned media (CM) by radioimmunoassay. Antisense clones produced less mRNA and had less IGFBP-6 protein in the CM than controls. IGFBP-6 protein levels in the CM were inversely correlated with alkaline phosphatase (ALP) activity, whereas IGFBP-3 and IGFBP-4 protein levels were not. We reasoned that atRA would have little or no effect on ALP activity in IGFBP-6 antisense clones if atRA mediated its inhibitory effects by recruiting IGFBP-6. In the majority of IGFBP-6 antisense clones with the lowest IGFBP-6 mRNA and CM protein levels and only modest changes in other IGF system components, atRA did not significantly decrease ALP activity. These findings provide evidence that atRA recruits IGFBP-6 to inhibit the human osteoblast phenotype.