Prolonged insulin treatment sensitizes apoptosis pathways in pancreatic ß cells.

Insulin resistance results from impaired insulin signaling in target tissues that leads to increased levels of insulin required to control plasma glucose levels. The cycle of hyperglycemia and hyperinsulinemia eventually leads to pancreatic cell deterioration and death by a mechanism that is yet unclear. Insulin induces ROS formation in several cell types. Furthermore, death of pancreatic cells induced by oxidative stress could be potentiated by insulin. Here, we investigated the mechanism underlying this phenomenon. Experiments were done on pancreatic cell lines (Min-6, RINm, INS-1), isolated mouse and human islets, and on cell lines derived from nonpancreatic sources. Insulin (100nM) for 24h selectively increased the production of ROS in pancreatic cells and isolated pancreatic islets, but only slightly affected the expression of antioxidant enzymes. This was accompanied by a time- and dose-dependent decrease in cellular reducing power of pancreatic cells induced by insulin and altered expression of several ER stress response elements including a significant increase in Trb3 and a slight increase in iNos The effect on iNos did not increase NO levels. Insulin also potentiated the decrease in cellular reducing power induced by H2O2 but not cytokines. Insulin decreased the expression of MCL-1, an antiapoptotic protein of the BCL family, and induced a modest yet significant increase in caspase 3/7 activity. In accord with these findings, inhibition of caspase activity eliminated the ability of insulin to increase cell death. We conclude that prolonged elevated levels of insulin may prime apoptosis and cell death-inducing mechanisms as a result of oxidative stress in pancreatic cells.

Periparturient dairy cows do not exhibit hepatic insulin resistance, yet adipose-specific insulin resistance occurs in cows prone to high weight loss.

The periparturient period in dairy cows is associated with alterations in insulin action in peripheral tissues; however, the molecular mechanism underlying this process is not completely understood. The objective was to examine the response to a glucose tolerance test (GTT) and to analyze insulin signaling in liver and adipose tissues in pre- and postpartum dairy cows. Liver and adipose tissue biopsies were taken before and after GTT, at 17d prepartum and again at 3 to 5d postpartum from 8 high-yielding Israeli Holstein dairy cows. Glucose clearance rate after GTT was similar pre- and postpartum. Basal insulin concentrations and the insulin response to GTT were approximately 4-fold higher prepartum than postpartum. In accordance, phosphorylation of the hepatic insulin receptor after GTT was higher prepartum than postpartum. Across periods, a positive correlation was observed between the basal and peak plasma insulin and phosphorylated insulin receptor after GTT in the liver. Hepatic phosphorylation of protein kinase B after GTT was elevated pre- and postpartum. Conversely, in adipose tissue, phosphorylation of protein kinase B after GTT pre- and postpartum was increased only in 4 out of 8 cows that lost less body weight postpartum. Our results demonstrate that hepatic insulin signaling is regulated by plasma insulin concentrations as part of the homeorhetic adjustments toward calving, and do not support a model of hepatic insulin resistance in periparturient cows. Nevertheless, we suggest that specific insulin resistance in adipose tissue occurs pre- and postpartum only in cows prone to high weight loss. The different responses among these cows imply that genetic background may affect insulin responsiveness in adipose tissue pre- and postpartum.

Otubain 2 is a novel promoter of beta cell survival as revealed by siRNA high-throughput screens of human pancreatic islets.

AIMS/HYPOTHESIS: Pro-inflammatory cytokines induce death of beta cells and hamper engraftment of transplanted islet mass. Our aim was to reveal novel genes involved in this process, as a platform for innovative therapeutic approaches. METHODS: Small interfering RNA (siRNA) high-throughput screening (HTS) of primary human islets was employed to identify novel genes involved in cytokine-induced beta cell apoptosis. Dispersed human islets from nine human donors, treated with a combination of TNF-α, IL-1ß and IFN-γ were transfected with ∼730 different siRNAs. Caspase-3/7 activity was measured, results were analysed and potential anti- and pro-apoptotic genes were identified. RESULTS: Dispersed human pancreatic islets appeared to be suitable targets for performance of siRNA HTS. Using this methodology we found a number of potential pro- and anti-apoptotic target hits that have not been previously associated with pancreatic beta cell death. One such hit was the de-ubiquitinating enzyme otubain 2 (OTUB2). OTUB2 knockdown increased caspase-3/7 activity in MIN6 cells and primary human islets and inhibited insulin secretion and increased nuclear factor-κB (NF-κB) activity both under basal conditions and following cytokine treatment. CONCLUSIONS: Use of dispersed human islets provides a new platform for functional HTS in a highly physiological system. Employing this technique enabled the identification of OTUB2 as a novel promoter of viability and insulin secretion in human beta cells. OTUB2 acts through the inhibition of NF-κB signalling, which is deleterious to beta cell survival. siRNA screens of human islets may therefore identify new targets, such as OTUB2, for therapeutic intervention in type 1 diabetes and islet transplantation.

Hepatitis C core protein impairs insulin downstream signalling and regulatory role of IGFBP-1 expression.

Chronic infection with hepatitis C virus (HCV), mainly genotype 1, has been shown to be associated with insulin resistance and type 2 diabetes. The mechanisms underlying this association are partly understood. Increased levels of tumor necrosis factor (TNF)-α occurring in HCV infection have an important role in HCV-mediated insulin resistance; however, other direct effects of HCV core protein on disrupting insulin signalling have been suggested. The insulin receptor substrate (IRS) proteins are key players in insulin signal transduction and are the major substrates of the insulin receptor. To further elucidate the direct effect of HCV core protein on insulin signalling. We studied the direct effects of HCV core protein in two cell lines transfected with HCV core protein. We found several impairments in the insulin signalling cascade which could be attributed to a significant proteasomal degradation of IRS-1 protein, in a dose-dependent way. In addition, our data show that liver cells transfected by HCV core protein show a marked attenuation of the regulatory inhibitory role of insulin on insulin growth factor binding protein-1 (IGFBP-1) expression. Since IGFBP-1 may have a role in glucose regulation and hepatic insulin sensitivity, this effect of HCV core protein can contribute to insulin resistance in chronic HCV infection. Our data suggest that the degradation of IRS-1 by HCV core protein translates to impaired ability of insulin to inhibit the expression of the target gene IGFBP-1 in the liver and may serve as a novel mechanism for insulin resistance and hyperglycaemia.

An siRNA screen identifies transmembrane 7 superfamily member 3 (TM7SF3), a seven transmembrane orphan receptor, as an inhibitor of cytokine-induced death of pancreatic beta cells.

AIMS/HYPOTHESIS: Pro-inflammatory cytokines induce death of pancreatic beta cells, leading to the development of type 1 diabetes. We sought to identify novel players and the underlying mechanisms involved in this process. METHODS: A high-throughput screen of 3,850 mouse small interfering RNAs (siRNAs) was performed in cytokine-treated MIN6 beta cells. Cells were transfected with the different siRNAs and then treated with a combination of TNFα, IL-1ß and IFNγ. Cellular apoptosis (caspase-3/7 activity), and changes in cellular reducing power and cell morphology were monitored. The resulting data were analysed and the corresponding z scores calculated. RESULTS: Several gene families were identified as promoting cytokine-induced beta cell apoptosis, the most prominent being those encoding ubiquitin ligases and serine/threonine kinases. Conversely, deubiquitinating enzymes appeared to reduce apoptosis, while protein phosphatases were mainly associated with lowering cellular reducing power. The screen suggested with high confidence the involvement of several novel genes in cytokine-induced beta cell death, including Camkk2, Epn3, Foxp3 and Tm7sf3, which encodes an orphan seven transmembrane receptor. siRNAs to Tm7sf3 promoted cytokine-induced death of MIN6 cells and human pancreatic islets, and abrogated insulin secretion in these cells. These findings implicate transmembrane 7 superfamily member 3 as a potential new player in the inhibition of cytokine-induced death and in the promotion of insulin secretion from pancreatic beta cells. CONCLUSIONS/INTERPRETATION: The signalling pathways and novel genes that we identified in this screen and that mediate beta cell death offer new possible targets for therapeutic intervention in diabetes and its adverse complications.

Uncoupling insulin signalling by serine/threonine phosphorylation: a molecular basis for insulin resistance.

Insulin resistance refers to a decreased capacity of circulating insulin to regulate nutrient metabolism. Recent studies reveal that agents that induce insulin resistance exploit phosphorylation-based negative feedback control mechanisms otherwise utilized by insulin itself to uncouple the insulin receptor from its downstream effectors and thereby terminate insulin signal transduction. This article focuses on the Ser/Thr protein kinases which phosphorylate insulin receptor substrates and the major Ser sites that are phosphorylated, as key elements in the uncoupling of insulin signalling and the induction of an insulin resistance state.

Combined analysis of tumor growth pattern and expression of endogenous lectins as a prognostic tool in primary testicular cancer and its lung metastases.

The aim of this study was to glyco- and immunohistochemically analyze expression of distinct growth/adhesion-related markers of primary testicular carcinomas and their lung metastases in relation to the risk of developing lung metastases and survival of patients, and to correlate immunohistochemical staining profile and syntactic structure analysis in order to delineate new prognostic parameters for this tumor type. Clinical features of 50 patients with primary testicular carcinomas and their corresponding lung metastases were evaluated and compared to those of a control cohort of 25 cases. The set of eight probes including labeled galectins-1 and -3, specific non-cross-reactive antibodies against galectins-1, -3, and -8 as well as anti-Ki-67, anti-bcl-2, and anti-p53 was applied to formalin-fixed, paraffin-embedded tumor sections of both primary and metastatic lesions. Syntactic structure analysis computed staining intensities and structural features of the tumor cells. These parameters were set into relation separately and in combination to clinical data including tumor stages, smoking habits, applied cytostatic therapy, disease-free interval, and survival. The risk of testis cancer patients to develop lung metastases depends in descending order on the tumor cell type (non-seminoma versus seminoma), tumor cell heterogeneity (mixed versus monomorphous cell type), age of patients, and pT stage. The extent of differential expression of galectin-related features between primary and secondary lesions was pronounced. Prognostic correlations for distinct galectin-related features were delineated in combination with data from syntactic structure analysis, for example cluster radius of galectin-3-positive tumor cells and post-surgical and total survival. Lengths of disease-free interval and total survival of patients were also correlated to characteristics obtained by syntactic structure analysis and their combination with galectin data in the first place, then to smoking habits, percentage of proliferating cells in the primary and secondary tumors, and finally to expression of certain galectins and of p53. Patients with non-seminoma testicular cancer should be thoroughly controlled for lung metastases. Regarding marker selection, our study underscores that further investigation of the growth-regulatory network of galectins is clearly warranted.

Role of Ser/Thr kinases in the uncoupling of insulin signaling.

Insulin resistance refers to a decreased capacity of circulating insulin to regulate nutrient metabolism. It is associated with the development of type II diabetes, a 21st century epidemic. Recent studies reveal that agents that induce insulin resistance exploit phosphorylation-based negative feedback control mechanisms otherwise utilized by insulin itself, to uncouple the insulin receptor from its downstream effectors and thereby terminate insulin signal transduction. This article focuses on the cardinal role of Ser/Thr protein kinases, which phosphorylate insulin receptor substrates, as key players in the uncoupling of insulin signaling and the induction of an insulin resistance state.

Galectin fingerprinting by immuno- and lectin histochemistry in cutaneous lymphoma.

Owing to their relevance for growth regulation and cell adhesion monitoring the expression of galectins (endogenous beta-galactoside-binding lectins) and their binding sites has relevance for tumor biology. Using galectin-type-specific reagents (non-crossreactive antibodies for proto-type galectin-1, chimera-type galectin-3 and tandem-repeat-type galectins-4 and -8, and labeled galectins-1, -3, and -4) we determined galectin expression in cutaneous T cell lymphomas (CTCL) and controls. In addition to commonly studied galectins-1 and -3, tandem-repeat-type galectins could be detected, i.e., galectin-8 in six from 15 cases and galectin-4 in one of 15 cases. In view of relevant ligands such as bcl-2 or integrins the presence of galectins-3 and -8 seems to be possibly related to loss of proliferation control and change in cell adhesion properties that are involved in clonal expansion and epidermal spread of malignant T cell clones. Successful chemotherapy of CTCL alters galectin expression selectively as shown for liposomal doxorubicin.

Galectin-8 expression decreases in cancer compared with normal and dysplastic human colon tissue and acts significantly on human colon cancer cell migration as a suppressor.

BACKGROUND AND AIMS: Galectins are beta-galactoside binding proteins. This ability may have a bearing on cell adhesion and migration/proliferation in human colon cancer cells. In addition to galectins-1 and -3 studied to date, other members of this family not investigated in detail may contribute to modulation of tumour cell features. This evident gap has prompted us to extend galectin analysis beyond the two prototypes. The present study deals with the quantitative determination of immunohistochemical expression of galectin-8 in normal, benign, and malignant human colon tissue samples and in four human colon cancer models (HCT-15, LoVo, CoLo201, and DLD-1) maintained both in vitro as permanent cell lines and in vivo as nude mice xenografts. The role of galectin-8 (and its neutralising antibody) in cell migration was investigated in HCT-15, LoVo, CoLo201, and DLD-1 cell lines. METHODS: Immunohistochemical expression of galectin-8 and its overall ability to bind to sugar ligands (revealed glycohistochemically by means of biotinylated histochemically inert carrier bovine serum albumin with alpha- and beta-D-galactose, alpha-D-glucose, and lactose derivatives as ligands) were quantitatively determined using computer assisted microscopy. The presence of galectin-8 mRNA in the four human colon cancer cell lines was examined by reverse transcriptase-polymerase chain reaction. In vitro, cellular localisation of exogenously added galectin-8 in the culture media of these colon cancer cells was visualised by fluorescence microscopy. In vitro galectin-8 mediated effects (and the influence of its neutralising antibody) on migration levels of living HCT-15, LoVo, CoLo201, and DLD-1 cells were quantitatively determined by computer assisted phase contrast microscopy. RESULTS: A marked decrease in immunohistochemical expression of galectin-8 occurred with malignancy development in human colon tissue. Malignant colon tissue exhibited a significantly lower galectin-8 level than normal or benign tissue colon cancers; those with extensive invasion capacities (T3-4/N+/M+) harboured significantly less galectin-8 than colon cancers with localised invasion capacities (T1-2/N0/M0). The four experimental models (HCT-15, LoVo, CoLo201, and DLD-1) had more intense galectin-8 dependent staining in vitro than in vivo. Grafting the four experimental human colon cancer models onto nude mice enabled us to show that the immunohistochemical expression of galectin-8 was inversely related to tumour growth rate. In vitro, galectin-8 reduced the migration rate of only those human experimental models (HCT-15 and CoLo201) that exhibited the lowest growth rate in vivo. CONCLUSIONS: Expression of galectin-8 correlated with malignancy development, with suppressor activity, as shown by analysis of clinical samples and xenografts. In vitro, only the two models with low growth rates were sensitive to the inhibitory potential of this galectin. Future investigations in this field should involve fingerprinting of these newly detected galectins, transcending the common focus on galectins-1 and -3.

Insulin resistance: a phosphorylation-based uncoupling of insulin signaling.

Insulin resistance refers to a decreased capacity of circulating insulin to regulate nutrient metabolism. It is associated with the development of type 2 diabetes--an ever-increasing epidemic of the 21st century. Recent studies reveal that agents that induce insulin resistance exploit phosphorylation-based negative-feedback control mechanisms, otherwise utilized by insulin itself, to uncouple the insulin receptor from its downstream effectors and thereby terminate insulin signal transduction. This article describes recent findings that present novel viewpoints of the molecular basis of insulin resistance, focusing on the cardinal role of Ser/Thr protein kinases as emerging key players in this arena.

Detection of macrophage migration inhibitory factor (MIF) in human cholesteatomas and functional implications of correlations to recurrence status and to expression of matrix metalloproteinases-3/9, retinoic acid receptor-beta, and anti-apoptotic galectin-3.

OBJECTIVES: To investigate whether the expression of the macrophage migration inhibitory factor (MIF) 1) is detectable, 2) changes in relation to recurrence and infection status, and 3) relates to the levels of expression of growth regulators/differentiation markers, including galectin-1, -3, and -8, retinoid acid receptors (RAR)]-alpha, -beta, and -gamma, binding sites for sarcolectin, and invasion markers (cathepsins -B and -D, and matrix metalloproteinases [MMP]-2, -3, and -9) in human cholesteatomas. STUDY DESIGN: An analysis of 56 cholesteatomas resected by the same surgeon using canal wall up and canal wall down surgical procedures. METHODS: The immunohistochemical levels of expression of MIF and the proteases were quantitatively determined (using computer-assisted microscopy) on routine histologic slides by specific antibodies, and statistically correlated to parameters of the other markers determined previously in conjunction with data on apoptosis/proliferation. RESULTS: MIF expression was detected. It was significantly higher in the epithelium (P =.002) and vessels (P =.04) of the connective tissues (but not in the connective tissue itself) of recurrent as opposed to non-recurrent cholesteatomas. The MIF expression is significantly correlated (P =.006) to the RAR beta expression in non-infected cholesteatomas, and to MMP-3 (P <.01) and anti-apoptotic galectin-3 (P =.01) in infected cholesteatomas. The level of MIF expression was also correlated significantly to MMP-9 (P = 0.003), RAR beta (P <.001), and galectin-8 (P =.003) expression in the cholesteatomas regardless of their infection status. CONCLUSIONS: MIF expression in human cholesteatomas is related to the levels of biologic aggressiveness reflected in their recurrence status and MMP expression, and to the differentiation status reflected in their galactin and RAR beta expressions. Together with galectin-3, it could cooperate to form an anti-apoptotic feedback loop.

The levels of expression of galectin-3, but not of galectin-1 and galectin-8, correlate with apoptosis in human cholesteatomas.

OBJECTIVES: To investigate whether galectins 1, 3, and 8 are expressed in human cholesteatomas and whether any such expression does correlate with the level of apoptosis, which is, as we have previously shown, predictive of recurrence.7 STUDY DESIGN: The analysis of 52 cholesteatomas resected by the same surgeon by means of canal wall up and canal wall down procedures. METHODS: The immunohistochemical levels of expression of galectins 1, 3, and 8 were quantitatively determined (using computer-assisted microscopy) on conventional histological slides by means of specific anti-galectin-1, anti-galectin-3, and anti-galectin-8 antibodies. The level of apoptosis in each cholesteatoma under study had already been determined 7 by means of the in situ labeling of nuclear DNA fragmentation (Tolt-mediated dUTP nick end labeling [TUNEL] staining). RESULTS: Galectin-1 was expressed markedly in both the epithelial and the connective tissue areas of all the cholesteatomas under study. The levels of expression of galectin-3 and galectin-8 were considerably lower than that of galectin-1. The level of expression of galectin-3 correlated both highly and positively with the level of apoptosis. CONCLUSIONS: An upregulation of galectin-3 (known to have an antiapoptotic and antianoikis effect in certain model systems) expression, which is associated with pronounced apoptotic activity, could have a physiologically protective effect against the characteristically substantial apoptotic features occurring in recurrent cholesteatomas.

Galectin-8 functions as a matricellular modulator of cell adhesion.

The interaction of cells with the extracellular matrix regulates cell adhesion and motility. Here we demonstrate that different cell types adhere and spread when cultured in serum-free medium on immobilized galectin-8, a mammalian beta-galactoside-binding protein. At maximal doses, galectin-8 is equipotent to fibronectin in promoting cell adhesion and spreading. Cell adhesion to immobilized galectin-8 is mediated by sugar-protein interactions with integrins, and galectin-8 triggers integrin-mediated signaling cascades including Tyr phosphorylation of focal adhesion kinase and paxillin. Cell adhesion is potentiated in the presence of Mn(2+), whereas it is interrupted in the presence of soluble galectin-8, integrin beta(1) inhibitory antibodies, EDTA, or thiodigalactoside but not by RGD peptides. Furthermore, cells readily adhere onto immobilized monoclonal galectin-8 antibodies, which are equipotent to integrin antibodies in promoting cell adhesion. Cell adhesion to immobilized galectin-8 is partially inhibited by serum proteins, suggesting that complex formation between immobilized galectin-8 and serum components generates a matrix that is less supportive of cell adhesion. Accordingly, cell motility on immobilized galectin-8 readily takes place in the presence of serum. Truncation of the C-terminal half of galectin-8, including one of its two carbohydrate recognition domains, largely abolishes its ability to modulate cell adhesion, indicating that both carbohydrate recognition domains are required to maintain a functional form of galectin-8. Collectively, our findings implicate galectin-8 as a physiological modulator of cell adhesion. When immobilized, it functions as a matrix protein equipotent to fibronectin in promoting cell adhesion by ligation and clustering of cell surface integrin receptors. In contrast, when present in excess as a soluble ligand, galectin-8 (like fibronectin) forms a complex with integrins that negatively regulates cell adhesion. Because of its dual effects on the adhesive properties of the cells and its association with fibronectin, galectin-8 might be considered a novel type of matricellular protein.

The levels of retinoid RARbeta receptors correlate with galectin-1, -3 and -8 expression in human cholesteatomas.

Cholesteatoma is a benign disease characterized by the presence of an unrestrained growth and the accumulation of keratin debris in the middle ear cavity. This often recurs, even when surgical resection is thought to be complete. In a previous study we showed that cholesteatomas with the highest apoptotic indices recurred more rapidly and also exhibited a high level of p53 immunopositive cells. In view of their relevance to the characterization of the cell differentiation status, the present study focuses on the expression of retinoid acid receptors (RARs) and galectins in human cholesteatomas. Retinoids control the differentiation processes in keratinocytes while galectins play strikingly modulatory roles at apoptosis and cell adhesion levels in a wide variety of tissue (embryonic, normal and neoplastic). To clarify the expression of these two protein families in human cholesteatomas we examined and quantified the levels of immunohistochemical expression of RARalpha, beta and gamma, and also galectin-1, -3 and -8 in a series of 70 human cholesteatomas. Our data show clearly that predominantly RARbeta and galectin-1 were expressed. The RARgamma concentration was significantly lower than that of the RARalpha; this was also observed for the galectin-8 concentration in comparison with the galectin-3 one. Furthermore, the level of RARbeta expression correlated highly (P=0.00001) with the level of galectin-8 expression, which also correlated significantly with the level of RARalpha and RARgamma expression. In addition, this parameter also correlated with the level of galectin-1 and galectin-3 expression. These data suggest that cholesteatomas may originate in an undifferentiated population of keratinocytes, and that a relation may exist between retinoid activity and galectins.

The insulin-like growth factor I receptor-induced interaction of insulin receptor substrate-4 and Crk-II.

Stimulation of the insulin or insulin-like growth factor (IGF)-I receptor results in activation of several signaling pathways. Proteins of the insulin receptor substrate (IRS) family play important roles in mediating these signaling cascades. To date, four members of the IRS family of docking proteins have been characterized. Recently, we have reported that stimulation of the IGF-I receptor in 293 HEK cells regulates interaction of the newly discovered IRS-4 molecule with the Crk family of proteins. In the present study, we characterize the molecular basis of these interactions. C- and N termini truncation analysis of IRS-4 demonstrated that the region between amino acids 678 and 800 of the IRS-4 molecule is involved in this interaction. This region contains a cluster of four tyrosines (Y(700), Y(717), Y(743), and Y(779)). We hypothesize that one or more of these tyrosines are involved in the interaction between the SH2 domain of the Crk-II molecule when IRS-4 is phosphorylated upon IGF-I receptor activation. Additional mutational analyses confirmed this hypothesis. Interestingly, none of these four tyrosines was individually critical for the interaction between Crk-II and IRS-4, but when all four tyrosines were simultaneously mutated to phenylalanine, the IGF-I induced interaction between these molecules was abolished. Taken together, these results suggest a novel mechanism of Crk-II binding to tyrosine phosphorylated proteins.

Insulin stimulates PKCzeta -mediated phosphorylation of insulin receptor substrate-1 (IRS-1). A self-attenuated mechanism to negatively regulate the function of IRS proteins.

Incubation of rat hepatoma Fao cells with insulin leads to a transient rise in Tyr phosphorylation of insulin receptor substrate (IRS) proteins. This is followed by elevation in their P-Ser/Thr content, and their dissociation from the insulin receptor (IR). Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, abolished the increase in the P-Ser/Thr content of IRS-1, its dissociation from the IR, and the decrease in its P-Tyr content following 60 min of insulin treatment, indicating that the Ser kinases that negatively regulate IRS-1 function are downstream effectors of PI3K. PKCzeta fulfills this criterion, being an insulin-activated downstream effector of PI3K. Overexpression of PKCzeta in Fao cells, by infection of the cells with adenovirus-based PKCzeta construct, had no effect on its own, but it accelerated the rate of insulin-stimulated dissociation of IR.IRS-1 complexes and the rate of Tyr dephosphorylation of IRS-1. The insulin-stimulated negative regulatory role of PKCzeta was specific and could not be mimic by infecting Fao cells with adenoviral constructs encoding for PKC alpha, delta, or eta. Because the reduction in P-Tyr content of IRS-1 was accompanied by a reduced association of IRS-1 with p85, the regulatory subunit of PI3K, it suggests that this negative regulatory process induced by PKCzeta, has a built-in attenuation signal. Hence, insulin triggers a sequential cascade in which PI3K-mediated activation of PKCzeta inhibits IRS-1 functions, reduces complex formation between IRS-1 and PI3K, and inhibits further activation of PKCzeta itself. These findings implicate PKCzeta as a key element in a multistep negative feedback control mechanism of IRS-1 functions.

Galectins are differentially expressed in supratentorial pilocytic astrocytomas, astrocytomas, anaplastic astrocytomas and glioblastomas, and significantly modulate tumor astrocyte migration.

Galectins, a family of mammalian lectins with specificity to beta-galactosides, are involved in growth-regulatory mechanisms and cell adhesion. A relationship is assumed to exist between the levels of expression of galectins and the level of malignancy in human gliomas. A comparative study of this aspect in the same series of clinical samples is required to prove this hypothesis. Using computer-assisted microscopy, we quantitatively characterized by immunohistochemistry the levels of expression of galectins-1, -3 and -8 in 116 human astrocytic tumors of grades I to IV. Extent of transcription of galectins-1, -3, and -8 genes was investigated in 8 human glioblastoma cell lines by means of RT-PCR techniques. Three of these cell lines were grafted into the brains of nude mice in order to characterize in vivo the galectins-1, -3 and -8 expression in relation to the patterns of the tumor invasion of the brain. The role of galectin-1, -3 and -8 in tumor astrocyte migration was quantitatively determined in vitro by means of computer-assisted phase-contrast videomicroscopy. The data indicate that the levels of galectin-1 and galectin-3 expression significantly change during the progression of malignancy in human astrocytic tumors, while that of galectin-8 remains unchanged. These three galectins are involved in tumor astrocyte invasion of the brain parenchyma since their levels of expression are higher in the invasive parts of xenografted glioblastomas than in their less invasive parts. Galectin-3, galectin-1, and to a lesser extent galectin-8, markedly stimulate glioblastoma cell migration in vitro. Since bands for the transcripts of human galectins-2, -4 and -9 were apparently less frequent and intense in the 8 human glioblastoma cell lines, this system provides an excellent model to assign defined roles to individual galectins and delineate overlapping and distinct functional aspects.

Benign metastasizing leiomyoma of the uterus: documentation of clinical, immunohistochemical and lectin-histochemical data of ten cases.

The clinical histories of 10 women suffering from benign metastasizing leiomyoma (BML) after hysterectomy and information on lung lesions detected in these women are presented, together with corresponding data for 2 women with metastasizing leiomyosarcoma of the uterus for comparison: gross appearance, survival, and light microscopical, immunohistochemical and lectin-histochemical findings are reported. All patients with BML had undergone hysterectomy for uterus leiomyomatosus without any detection of sarcomatous lesions in the uterus wall. After a median period of 14.9 years intrapulmonary masses were detected by imaging techniques. On average, six nodules with a mean diameter of 1.8 cm were seen. Resection of the lesions was performed in all cases. The immunohistochemical and lectin-histochemical examination of the tumors included analysis of the proliferation-associated protein Ki-67, the p53 protein, estrogen and progesterone receptor, sarcolectin as an indicator of the presence of lymphokine macrophage migration inhibitory factor, antibodies and the labeled protein to assess galectin (galactoside-binding animal lectin)-dependent parameters, analysis of tumor vascularization (CD-34), and expression of bcl-2, vimentin, smooth muscle actin, desmin, and keratin. The lesions were characterized by low proliferation activity of 2.9% (measured with Ki-67), frequent hormone receptor expression (8 of the 10 cases presented hormone-specific receptors), low to moderate vascularization compared with metastases from the two uterine sarcomas, remarkable p53 overexpression and frequent expression of the lymphokine, the galectins and accessible binding sites. The median survival of the BML patients was 94 months after excision of the intrapulmonary lesions, and the maximum survival of the two sarcoma patients was 22 months. The results recorded in this patient sample with the methodology applied suggest that benign metastasizing leiomyomas are a slow-growing variant of leiomyosarcoma of the uterus, which becomes clinically apparent at a young age and progresses with low velocity.