Receptor Protein Expression Levels Research Articles

Epidermal growth factor receptor (EGFR) mRNA levels and protein expression levels in primary colorectal cancer and corresponding liver metastases

High expression levels of EGFR mRNA are reported to be associated with a higher response probability in epidermal growth factor receptor (EGFR) targeted drugs. Our aim was to determine how EGFR gene expression levels in primary colorectal cancer (CRC) were related to those in liver metastases. 31 pairs of primary CRC and corresponding liver metastases were analyzed. Gene expression level was measured using real-time RT-PCR. No significant difference was observed between median mRNA expression levels of EGFR in primary cancer and those in corresponding liver metastases (P = 0.99). When matched tissue sets were compared on an individual basis, there was a significant correlation for EGFR mRNA expression between primary cancer and corresponding liver metastases (rs = 0.78, P < 0.0001). A good prediction of EGFR mRNA levels in liver metastases can be obtained by measuring those in the primary CRC.

Glucocorticoid pathway mediated the inhibition of testosterone in rats exposed to dibutyl phthalate

To investigate the inhibitory mechanisms of testosterone (T) biosynthesis in rats exposed to dibutyl phthalate (DBP). Male Wistar rats were randomly divided into five groups by weight, including 0.25, 0.50, 1.00, 2.00 g/kg DBP groups and corn oil control group, with 16 rats in each group. DBP was administered by gavage once a day. After 30 days exposure, eight rats in each group were killed and the others were killed after 15 days without DBP administration. The levels of T and glucocorticoid (GC) in serum were determined by radioimmunoassay. The expression levels of 11 beta-dedroxysteriod dehydrogenase (11 beta-HSD) mRNA and steroidogenesis acute regulatory protein (StAR) mRNA were determined by RT-PCR. The protein expression level of glucocorticoid receptor (GR) was investigated by Western blotting. During exposure period, in 1.00 and 2.00 g/kg DBP groups, the levels of T were (0.260 +/- 0.218) ng/ml and (0.260 +/- 0.342) ng/ml, the levels of GC were (13.470 +/- 5.661) ng/ml and (13.740 +/- 3.977) ng/ml, the levels of T and GC in control group were (1.045 +/- 1.222) ng/ml and (9.224 +/- 3.496) ng/ml. There were statistic differences between 1.00 and 2.00 g/kg DBP groups and control group (t(T) values were -2.295 and -2.295, t(GC) values were 2.159 and 2.296, respectively, P < 0.05). The expression level of StAR mRNA was significantly down-regulated in 1.00 and 2.00 g/kg DBP groups, while StAR/beta-Actin values were 0.657 +/- 0.060 and 0.407 +/- 0.033, and compared to control group (0.871 +/- 0.081), there was statistic difference (t values were -3.707 and -8.037, P < 0.05). In 1.00 and 2.00 g/kg DBP groups, the expression of 11 beta-HSD mRNA and the expression of GR protein were increased in DBP dose-dependent manner, while 11 beta-HSD/beta-Actin values were 0.538 +/- 0.138 and 0.988 +/- 0.133, and GR/beta-Actin were 0.785 +/- 0.106 and 0.956 +/- 0.076, respectively. There were statistic difference, as compared to the controls (0.285 +/- 0.106 and 0.275 +/- 0.035) (t(11 beta-HSD/beta-Actin) values were 2.829 and 7.860, t(GR/beta-Actin) values were 8.064 and 10.77, respectively, P < 0.05).Linear correlation and regression revealed that there were positive correlation between DBP dose and the expression levels of 11 beta-HSD mRNA and GR protein, with r values of 0.766 and 0.790, respectively. In post-exposure period, there were no statistic differences of all above index among DBP groups and control group. DBP might inhibit T production in rats through GR mediation.

African lungfish,Protopterus annectens, possess an arginine vasotocin receptor homologous to the tetrapod V2-type receptor

In tetrapods, arginine vasopressin and its counterpart, arginine vasotocin (AVT), are involved in renal water conservation through vascular V1a-type and tubular V2-type receptors, and only the former has thus far been cloned in fish. We successfully cloned the V1a-type and V2-type AVT receptor from the kidney of the African lungfish, Protopterus annectens, and the deduced amino acid sequences exhibited high homology with amphibian V1a- and V2-type receptors, respectively. Functional analysis showed that AVT addition to CHO cells transfected with lungfish V1a-type receptor increased [Ca2+]i in a concentration-dependent manner, whereas CHO cells transfected with lungfish V2-type receptor responded with cAMP accumulation after AVT stimulation. Lungfish V2-type receptor mRNA was strongly expressed in the heart and kidney, while V1a-type receptor mRNA was ubiquitously expressed in all the tissues examined. In the kidney, immunohistochemistry using a specific antibody to lungfish V2-type receptor showed localization in the basolateral area of the cells in the late part of the distal tubules. Artificial estivation (EST) for 90 days significantly increased plasma osmolality and sodium and urea concentrations. There was no significant difference in the V2-type receptor mRNA and protein expression levels in the kidney between the freshwater and EST lungfish, while the AVT precursor mRNA level in the hypothalamus was remarkably higher in the EST lungfish. Our results indicate that African lungfish possess a functional V2-type receptor similar to that in tetrapods, suggesting that elevated plasma AVT during estivation exerts a renal tubular antidiuretic effect through the V2-type receptor expressed in the distal segments of lungfish kidney.

Human Placental Adenosine Receptor Expression is Elevated in Preeclampsia and Hypoxia Increases Expression of the A 2A Receptor

Placental hypoxia as a result of impaired trophoblast invasion is suggested to be involved in the pathophysiology of preeclampsia. Hypoxia is a potent stimulus for the release of adenosine, and the actions of adenosine are mediated through four adenosine receptors, A 1, A 2A, A 2B and A 3. We investigated the presence, distribution and expression of adenosine receptor subtypes in the human placenta, the expression of the adenosine receptors in placentas from pregnancies complicated by preeclampsia, small for gestational age (SGA) infants and uncomplicated pregnancies, and the effect of hypoxia on placental adenosine receptor expression. Immunofluorescent microscopy localized A 1, A 2A, A 2B and A 3 adenosine receptors to the syncytiotrophoblast, endothelial cells and myofibroblasts within the human placenta. Adenosine receptor protein and message expression levels were significantly higher in placentas from preeclamptic pregnancies with or without SGA infants, but not different in pregnancies with SGA infants alone. In vitro exposure of placental villous explants to hypoxia (2% oxygen) increased the expression of A 2A adenosine receptor 50%. These data indicate that all four known adenosine receptors are expressed in the human placenta and adenosine receptor expression is significantly higher in pregnancies complicated by preeclampsia. These data are consistent with the hypothesis that differences in placental adenosine receptors may contribute to alterations in placental function in preeclampsia.

The expression of ERα, ERβ and PR in lobular carcinoma in situ of the breast determined using laser microdissection and real‐time PCR

To determine the status of oestrogen receptor (ER) subtypes (ERalpha and ERbeta) in lobular carcinoma in situ (LCIS) of the breast. Forty-seven cases of LCIS and six cases with normal breast lobules were subjected to immunohistochemistry and evaluated for ERalpha, ERbeta and progesterone receptor (PR) expression. mRNA for ERalpha, ERbeta1 and ERbeta2 were quantified in LCIS and normal lobules using laser microdissection coupled with real-time polymerase chain reaction. LCIS showed a higher level of steroid receptor protein expression than normal lobules. There was no difference in ERbeta1 gene or ERbeta protein expression between normal lobules, pure LCIS, or LCIS associated with invasive breast cancer. No significant difference in expression of either ERalpha or ERbeta was found between pure LCIS and LCIS associated with invasive cancer. However, PR was significantly lower in those cases of LCIS with associated invasive than in those without synchronous invasive disease. Increased expression of steroid receptors in LCIS suggests their possible role in the biology of LCIS and, for PR, could influence the predisposition of women diagnosed with LCIS to develop invasive breast carcinoma.

Long-term administration of tempol attenuates postinfarct ventricular dysfunction and sympathetic activity in rats

The purpose of this study was to determine whether the long-term administration of tempol attenuates postinfarct ventricular dysfunction and sympathetic activity in rats. Myocardial infarction (MI) was induced by left descending coronary artery ligation. Tempol was orally administered in drinking water (2 mmol/L), which was initiated 4 h after infarction and continued for 6 weeks. Tempol prevented not only the increases in left ventricular end-diastolic pressure and volume but also the decreases in ejection fraction and peak velocities of contraction in MI rats. The treatment normalized the increased renal sympathetic nerve activity (RSNA) and plasma norepinephrine level, as well as the enhanced cardiac sympathetic afferent reflex (CSAR; an excitatory cardiovascular reflex partially contributing to the sympathetic activation in chronic heart failure) and the RSNA responses to microinjection of angiotensin II into paraventricular nucleus in MI rats. Furthermore, tempol prevented the increased AT(1) receptor protein expression and superoxide anion level in both paraventricular nucleus and rostral ventrolateral medulla in MI rats. In conclusion, long-term administration of tempol attenuates ventricular dysfunction and normalizes sympathetic neural control in MI rats. The normalization of the CSAR, levels of superoxide anions and AT(1) receptor expression, and the response to angiotensin II in the paraventricular nucleus and rostral ventrolateral medulla may partially contribute to the beneficial effects of tempol on central sympathetic control.

Effects of hypergravity on histamine H1 receptor mRNA expression in hypothalamus and brainstem of rats: implications for development of motion sickness

Conclusion: The study findings suggest that histamine was released from the axon terminals in the hypothalamus and brainstem and the released histamine activated post-synaptic H1 receptors there, resulting in the development of motion sickness. Objectives: We first examined which subtype of post-synaptic histaminergic receptor was responsible for the development of motion sickness. We then examined whether H1 receptors were up-regulated in various areas of the rat brain after 2 G hypergravity load, because the stimulation of H1 receptor was reported to up-regulate the level of H1 receptor protein expression through augmentation of H1 receptor mRNA expression. Materials and methods: For this purpose, we used an animal model of motion sickness, using pica (eating non-nutritive substances such as kaolin), as a behavioral index in rats. Results: After 2 G hypergravity load, rats ate a significant amount of kaolin, indicating that they suffered from motion sickness. The hypergravity-induced kaolin intake was suppressed by mepyramine, but not by terfinadine or zolantizine. This finding indicates that cerebral post-synaptic H1 but not H2 or peripheral H1 receptors play an important role in the development of motion sickness. The expression of H1 receptor mRNA was up-regulated in the hypothalamus and brainstem, but not in the cerebral cortex after 2 G hypergravity load in rats.

Up-regulation of endothelin type B receptors in the human internal mammary artery in culture is dependent on protein kinase C and mitogen-activated kinase signaling pathways

BackgroundUp-regulation of vascular endothelin type B (ETB) receptors is implicated in the pathogenesis of cardiovascular disease. Culture of intact arteries has been shown to induce similar receptor alterations and has therefore been suggested as a suitable method for, ex vivo, in detail delineation of the regulation of endothelin receptors. We hypothesize that mitogen-activated kinases (MAPK) and protein kinase C (PKC) are involved in the regulation of endothelin ETB receptors in human internal mammary arteries.MethodsHuman internal mammary arteries were obtained during coronary artery bypass graft surgery and were studied before and after 24 hours of organ culture, using in vitro pharmacology, real time PCR and Western blot techniques. Sarafotoxin 6c and endothelin-1 were used to examine the endothelin ETA and ETB receptor effects, respectively. The involvement of PKC and MAPK in the endothelin receptor regulation was examined by culture in the presence of antagonists.ResultsThe endohtelin-1-induced contraction (after endothelin ETB receptor desensitization) and the endothelin ETA receptor mRNA expression levels were not altered by culture. The sarafotoxin 6c contraction, endothelin ETB receptor protein and mRNA expression levels were increased after organ culture. This increase was antagonized by; (1) PKC inhibitors (10 μM bisindolylmaleimide I and 10 μM Ro-32-0432), and (2) inhibitors of the p38, extracellular signal related kinases 1 and 2 (ERK1/2) and C-jun terminal kinase (JNK) MAPK pathways (10 μM SB203580, 10 μM PD98059 and 10 μM SP600125, respectively).ConclusionIn conclusion, PKC and MAPK seem to be involved in the up-regulation of endothelin ETB receptor expression in human internal mammary arteries. Inhibiting these intracellular signal transduction pathways may provide a future therapeutic target for hindering the development of vascular endothelin ETB receptor changes in cardiovascular disease.

Relationship between antidepressants and IGF-1 system in the brain: Possible role in cognition

Antidepressants facilitate neuroplasticity by stimulating trophic factors. This study evaluated the effect of fluoxetine (FLX) treatment on insulin-like growth factor-1 (IGF-1) in the rat brain and its role in the effect of FLX on cognition. IGF-1 receptor (IGF-1R) protein expression and IGF-1 mRNA levels were assessed in rat frontal cortex (FC) and hippocampus, in FLX-treated [15 mg/kg, orally; 3 (acute) or 21 (repeated) days] male Wistar rats. Rats were subjected to the Morris Water Maze test. Acute FLX administration decreased IGF-1 mRNA levels in the FC and hippocampus and increased IGF-1R levels in the FC. Repeated FLX increased both mRNA and IGF-1R levels in the FC. Repeated, but not acute, FLX treatment decreased IGF-1 mRNA in the hippocampus. FLX did not affect cognitive performance. Thus, repeated FLX treatment leads to upregulation of IGF-1 system is FC. It is possible that FLX affect FC neuroplasticity through activation of the IGF-1 system.

The Anti-Cancer Effect of A3 Adenosine Receptor Agonists: A Novel, Targeted Therapy.

The A3 adenosine receptor (A3AR) is highly expressed in various human solid tumor cells whereas low expression is found in the adjacent normal tissues. Activation of the A3AR with synthetic highly selective agonists, such as IB-MECA, Cl-IB-MECA or LJ529, induces tumor growth inhibition of melanoma, lymphoma, breast, hepatoma, prostate and colon carcinoma cells both in vitro and in vivo. Two molecular events take place upon receptor activation and include: a. receptor internalization and subsequent degradation, followed by decreased receptor mRNA and protein expression level. b. modulation of down-stream signal transduction pathways, including those related to Wnt and NF-κB. Subsequently, the levels of cyclin D1 and c-Myc are decreased leading to tumor growth inhibition. IB-MECA synergizes with chemotherapeutic agents to yield an additive anti-tumor effect and protects against myelotoxicity induced by chemotherapy. Taken together, A3AR agonists may be suggested as a new family of orally bioavailable compounds to be developed as potent inhibitors of malignant diseases.

Neurotoxicity of HIV-1 Tat protein: Involvement of D1 dopamine receptor

Neurotoxic viral proteins released from HIV-infected cells are believed to play a major role in the pathogenesis of the dementia displayed in a significant number of AIDS patients. HIV-1 associated neuropathology severely affects dopaminergic regions of the brain. Growing evidence indicates that HIV-1 neurotoxic proteins, such as Tat may affect the function of the dopamine transmission system. In turn, molecular components of dopamine neurotransmission may participate in a complex network of Tat-induced cell responses which result in neurodegeneration. In this study we investigated whether D1 dopamine receptors are involved in the mechanism of Tat neurotoxicity in primary rat neuronal cell cultures. We found that in rat midbrain cell cultures, which express significant levels of D1 dopamine receptors, the specific D1 antagonist SCH 23390 attenuates the cell death caused by HIV-1 Tat. In rat hippocampal cell cultures, where the expression of D1 receptors is low, SCH 23390 did not change the toxicity of Tat. Thus, the protective effect of SCH 23390 in rat primary neuronal cell cultures is a function of the level of D1 receptor protein expression. Our results provide further evidence for the involvement of the dopaminergic transmission system in the mechanism of HIV-1 Tat neurotoxicity.

Assessment of epidermal growth factor receptor (EGFR, ErbB1) and HER2 (ErbB2) protein expression levels and response to lapatinib (Tykerb, GW572016) in an expanded panel of human normal and tumour cell lines.

Lapatinib (Tykerb, GW572016), a potent inhibitor of the catalytic activities of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) (ErbB2), inhibits population growth of selected EGFR and HER2 overexpressing cell lines. Previous studies with a small number of cell lines suggest a correlation between overexpression of EGFR and/or HER2 and sensitivity to growth inhibition by lapatinib; however, the precise determinants of lapatinib selectivity for tumour and/or other cells remain unclear. To clarify the determinants of its selectivity in cultured cells, lapatinib-induced cell population growth inhibition and relative EGFR and HER2 protein expression were quantified in 61 different human tumour cell lines from 12 tumour types, two oncogene transformed human cell lines and two normal human cell cultures. Using statistical tools to analyse the data, a model describing the relationship between lapatinib IC(50) (the response variable) and EGFR and HER2 expression and tissue type (explanatory variables) was derived. The results suggest that simultaneous consideration of EGFR and HER2 expression, as well as tissue type yields the best determinant of lapatinib selectivity in cultured cells.

The epidermal growth factor receptor intron1 (CA) n microsatellite polymorphism is a potential predictor of treatment outcome in patients with advanced lung cancer treated with Gefitinib

The goal of our study was to assess the association between R497K and intron1 (CA) n repeat genetic polymorphisms of the EGF (epidermal growth factor) receptor and the clinical outcome of patients with advanced non-small cell lung cancer treated with EGF receptor tyrosine kinase inhibitor. We determined the genotypes for R497K and intron1 (CA) n repeat genetic polymorphisms of 70 Chinese patients with advanced non-small cell lung cancer. Genetic polymorphisms were correlated with the clinical outcome of treatment with EGF receptor tyrosine kinase inhibitor. In a subgroup of patients whose tumor tissues were available for mutation analysis and IHC (immunohistochemistry) assay, the associations between the EGF receptor mutations, the EGF receptor protein expression levels and EGF receptor polymorphisms were analyzed. The results indicated that patients with a lower number of EGF receptor CA repeats (any allele ≤ 16 CA) were more likely to have higher EGF receptor protein expression levels, better response, and longer survival time than were patients with a higher number of CA repeats (both alleles > 16 CA) after therapy targeted at the EGF receptor ( P = 0.021; P = 0.014; P = 0.0392, respectively). In contrast, the R497K polymorphism had no relationship with EGF receptor protein expression levels or the clinical outcome of the patients treated with EGF receptor tyrosine kinase inhibitor ( P = 0.49; P = 0.452, respectively), and there were no associations between the two polymorphisms and somatic mutations ( P = 0.916; P = 0.562, respectively). Overall, our data suggest that the intron1 (CA) n polymorphism of the EGF receptor gene may be associated with the sensitivity to and the prognosis of non-small cell lung cancer after EGF receptor targeted inhibitor treatment.

Abnormalities of Prostaglandins and Cyclooxygenase Enzymes in Female Patients With Slow-Transit Constipation

Chronic constipation due to slow transit (STC) is more common in female than in male patients. We have previously shown that these gender differences may be due to over expression of progesterone (PG) receptors that alter G protein patterns. We sought to elucidate the mechanisms responsible for the impaired basal colonic motility in female patients with STC. Muscle tissues from females with STC and controls with adeno-carcinoma of the colon were studied. Prostaglandins were determined by immunoassay, COX enzymes by Western blot and COX enzymes and progesterone receptors mRNA by RT-PCR. STC patients had impaired colonic motility index, lower TxA(2) and PGF(2) and higher PGE(2) levels than controls. STC had lower COX-1 protein and mRNA levels and higher COX-2 protein and mRNA levels than controls. These abnormalities were reproduced in normal colonic muscle cells treated with PG for 6 h. STC patients had higher PG receptor protein expression and mRNA levels than controls suggesting over expression of these receptors. These findings suggest that the impaired motility index of STC is due to abnormal levels of prostaglandin and COX enzymes, probably caused by an over expression of PG receptors that make muscle cells more sensitive to circulating levels of PG.

Substance P Is Responsible for Physiological Alterations Such as Increased Chloride Ion Secretion and Glucose Malabsorption in Cryptosporidiosis

Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium, causes self-limited diarrhea in immunocompetent hosts and severe life-threatening diarrhea in AIDS patients. Highly active antiretroviral therapy has been used to effectively treat cryptosporiosis in some but not all AIDS patients. Therefore, there is an urgent need for innovative drugs to treat this disease. Cryptosporidium infection results in intestinal pathophysiological changes such as glucose malabsorption, increased chloride ion (Cl(-)) secretion, and epithelial barrier disruption, leading to disease pathogenesis. In order to develop tools to combat this opportunistic pathogen, it is vital to understand mediators involved in disease pathogenesis. Substance P (SP), a neuropeptide and pain transmitter, is located in the gastrointestinal tract. SP can cause Cl(-) secretion in human gastrointestinal explants. However, its role in cryptosporidiosis has not been fully studied. Jejunal samples from macaques before and after Cryptosporidium parvum infection were assayed for SP and SP receptor mRNA and protein levels by reverse transcription-PCR and by immunohistochemistry and enzyme-linked immunosorbent assay, respectively. The role of SP in pathophysiological alterations, such as Cl(-) secretion and glucose malabsorption, was studied using tissues derived from macaques infected with C. parvum by the Ussing chamber technique. SP and SP receptor mRNA and protein expression levels were increased in jejunal samples following C. parvum infection and were accompanied by increased basal ion secretion and glucose malabsorption. In vitro treatment of samples obtained from infected macaques with the SP receptor antagonist aprepitant (Emend; Merck, Whitehouse Station, NJ) completely reversed the increase in basal ion secretion and corrected the glucose malabsorption. Our findings raise the possibility of using SP receptor antagonists for the treatment of symptoms associated with cryptosporidiosis.

Expression of the estrogen receptor in blood neutrophils of dairy cows during the periparturient period

During the period around parturition, cows experience an increased susceptibility to inflammatory disorders in the mammary gland and uterus. This increased susceptibility has been correlated with a decreased functionality of neutrophils, major components in the innate immune defence. As sex steroid levels vary extensively in the period around parturition, an influence of these changes on the functionality of neutrophils has been suggested. Indeed, it has been shown that 17β-estradiol affects some functions of bovine neutrophils. In spite of these observations, receptors for 17β-estradiol have not yet been demonstrated in these cells. The investigation of the presence of estrogen receptors in bovine neutrophils was therefore the main objective of this study. The expression of estrogen receptors was evaluated at the protein level by flow cytometry, and at the mRNA level by polymerase chain reaction. A clear positive signal was obtained using flow cytometry for the estrogen receptor protein in bovine neutrophils. Further discrimination between the estrogen receptor subtypes α and β revealed the expression of the estrogen receptor β, whereas for the estrogen receptor α no reproducible positive signal could be obtained with the available antibodies. Both subtypes were found at the mRNA level. Subsequently, the estrogen receptor protein expression level in neutrophils obtained from cows in early lactation was compared with those from cows in late pregnancy. Additionally, the influence of endogenous 17β-estradiol and progesterone levels was assessed. No difference was found for the estrogen receptor protein expression in neutrophils from cows in early lactation compared with late gestation neither were the endogenous 17β-estradiol and progesterone levels correlated with the protein expression.

Stimulation of Vascular Smooth Muscle Cell Migration by Macrophage Migration Inhibitory Factor

Macrophage migration inhibitory factor (MIF) is a well known proinflammatory factor that influences the migration and proliferation of various cell types, predominantly monocytes and macrophages. Recent evidence suggests an important role for MIF in the progression of atherosclerosis and restenosis. For this reason, we studied the effect of MIF on platelet-derived growth factor-BB (PDGF-BB)-induced migration and PDGF receptor protein expression in vascular smooth muscle cells (VSMCs). Furthermore, the possibility of MIF influencing the migration of VSMCs was investigated. Our results show that short-term incubation of MIF is able to enhance PDGF-BB-induced migration. Long-term incubation decreases PDGF-BB-induced migration, but preserves a short-term stimulatory effect. These effects are not regulated at the level of PDGF receptor protein expression. MIF also acts as a chemoattractant for VSMCs, with a maximum response at 15 ng/ml. In contrast, the proliferation of VSMCs was unaffected by MIF. We conclude that MIF has a biphasic effect on VSMC migration. It remains unclear whether this effect is direct or involves the secretion of unidentified promigratory factors. Exogenous MIF does not stimulate VSMC proliferation; however, a role for MIF in proliferation cannot be fully ruled out. In view of the known key contributions of macrophage-derived MIF and VSMCs, the observed effects may well play a role in the progression of atherosclerosis and restenosis.

Impaired proliferation of non-parenchymal cells participates in an impairment of liver regeneration in db/db mice

In this study, we examined the possibility that impaired proliferation of non-parenchymal cells affects in an impairment of liver regeneration in db/db mice, which are congenitally deficient in receptors for leptin. Liver regeneration after a two thirds partial hepatectomy (2/3 PH) was impaired in 10-week-old female db/db mice. The proliferation of both hepatocytes and non-parenchymal cells estimated from a bromodeoxyuridine (BrdU) labeling index was suppressed, and the protein expression of vascular endothelial growth factor was blocked in db/db mice. Although the extent of fatty change and the level of epidermal growth factor receptor protein expression in the liver were improved in 5-week-old db/db mice, the regeneration of liver was impaired after 2/3 PH in both 5- and 10-week-old db/db mice. These results suggested that suppressed proliferation of non-parenchymal cells contributes to the impairment of liver regeneration in db/db mice. As leptin has also the angiogenic effect, the angiogenic inhibitor FR-118487 was administered to ICR mice to examine liver regeneration after 2/3 PH, and the rate of regeneration was affected. In conclusion, it is suggested that the suppressed proliferation of non-parenchymal cells contributes to the impairment of liver regeneration probably through a disrupted angiogenesis in db/db mice.

Immunohistochemically determined total epidermal growth factor receptor levels not of prognostic value in newly diagnosed glioblastoma multiforme: Report from the Radiation Therapy Oncology Group

The Radiation Therapy Oncology Group (RTOG) performed an analysis of patterns of immunohistochemically detected total epidermal growth factor receptor (EGFR) protein expression levels and their prognostic significance on archival tissue in newly diagnosed glioblastoma multiforme (GBM) patients from prior prospective RTOG clinical trials. Patients in this study had been treated on previous RTOG GBM trials (RTOG 7401, 7918, 8302, 8409, 9006, 9305, 9602, and 9806). Tissue microarrays were prepared from 155 patients enrolled in these trials. These specimens were stained using a mouse monoclonal antibody specific for the extracellular binding domain of EGFR to detect total EGFR (including both wild-type phosphorylated and wild-type unphosphorylated isoforms with some cross-reactivity with EGFRvIII). The intensity of total EGFR protein expression was measured by computerized quantitative image analysis using the SAMBA 4000 Cell Image Analysis System. The parameters measured were the mean optical densities over the labeled areas and the staining index, which represents the proportion of stained area relative to the mean stain concentration. Both parameters were correlated with the clinical outcome. No differences in either overall or progression-free survival could be demonstrated by the mean optical density class or mean optical density quartile or the staining index of total EGFR immunostaining among the representative RTOG GBM cases. Total EGFR protein expression levels, as measured immunohistochemically, do not appear to be of prognostic value in newly diagnosed GBM patients. Given the accumulating clinical evidence of the activity of anti-EGFR agents in GBM and the preclinical data suggesting the important role of downstream mediators as effectors of EGFR signaling, the RTOG is conducting additional investigations into the prognostic value of activation patterns of EGFR signaling, both at the level of the receptor (e.g., EGFRvIII, phospho-EGFR) and at the level of downstream signal transduction pathways (e.g., PI3K, Ras/MAPK pathways).

Role of extracellular cysteine residues in dimerization/oligomerization of the human prostacyclin receptor

Prostacyclin activation of prostanoid IP receptors may result in pain sensation, inflammatory responses, inhibition of platelet aggregation, and vasodilation in vascular tissue. The prostanoid IP receptor is a G-protein-coupled receptor. In the present study, we investigated the determinants responsible, at least in part, for the prostacyclin receptor (IP) dimerization/oligomerization. Using co-immunoprecipitation of differentially tagged IP expressed in COS-7 cells, we demonstrate that IP can form dimers and oligomers. Treatment of IP-expressing cells with the stable agonist carbaprostacyclin failed to alter the ratios of oligomeric/dimeric/monomeric forms of the receptor, suggesting that IP dimerization/oligomerization is an agonist-independent process. The reducing agents dithiothreitol and 2-mercaptoethanol were highly efficient in converting the receptor from its oligomeric form to the monomeric state, indicating the involvement of disulfide bonds in IP oligomerization. Immunoblotting of the osteoblastic MG-63 cell line lysates with an anti-IP specific antibody revealed the presence of endogenous IP oligomers which were converted to dimers and monomers upon treatment with dithiothreitol. Individual substitutions of the four extracellular IP Cys residues (Cys 5, Cys 92, Cys 165 and Cys 170) for Ser resulted in greatly decreased receptor protein expression in COS-7 cells. The C92–170S double mutant showed receptor protein expression level similar to the individual mutants. However, expression of the C92–165S and C165–170S mutants was drastically reduced, suggesting that there was formation of disulfide bonds between Cys 5 and Cys 165, and between Cys 92 and Cys 170. The Cys receptor mutants showed altered oligomer/dimer/monomer ratios. Dimerization/oligomerization likely occurs intracellularly since these Cys receptor mutants could still form dimers/oligomers despite their lack of expression at the cell surface.