Remko R. Bosch

Expression of the transcription factor Ets-1 is an independent prognostic marker for relapse-free survival in breast cancer.

The transcription factor Ets-1 regulates the expression of several angiogenic and extracellular matrix remodeling factors, and might be implicated in disease progression of breast cancer. In the present study, the prognostic value of Ets-1 expression was assessed by quantitative real-time fluorescence RT–PCR in 123 sporadic primary breast cancer samples of patients with a median follow-up time of 62 months. Ets-1 expression levels correlated significantly with VEGF and PAI-1 in the same tissue. In univariate (P=0.0011) and multivariate (P=0.005) analyses, Ets-1 expression showed significant prognostic value for relapse-free survival. Ets-1 is a strong, independent predictor of poor prognosis in breast cancer. This seems – at least in part – to be attributable to its role in transcriptional regulation of factors involved in angiogenesis (VEGF), and extracellular matrix remodeling (PAI-1).

Molecular Beacon Reverse Transcription-PCR of Human Chorionic Gonadotropin-β-3, -5, and -8 mRNAs Has Prognostic Value in Breast Cancer

BACKGROUND The beta-subunit of human chorionic gonadotropin (hCG) is encoded by four genes, of which expression of the hCGbeta-3, -5, and -8 genes could have prognostic value in breast cancer. METHODS Applying a new, modified Molecular Beacon reverse transcription-PCR assay, we investigated the prognostic value of the hCGbeta-3, -5, and -8 gene transcripts in 129 sporadic unilateral breast cancer samples from patients with a median follow-up of 62.3 months. RESULTS Expression of hCGbeta-3, -5, -8 was significantly (P = 0.020) associated with relapse-free survival (RFS). In multivariate survival analysis, hCGbeta-3, -5, and -8 maintained prognostic value for RFS, with high expression predicting shorter RFS (P = 0.015; hazard ratio, 2.25; 95% confidence interval, 1.17-4.34). Only 1 of 24 (4%) node-negative patients with low hCGbeta-3, -5, -8 expression relapsed, in contrast to 7 of 26 (27%) patients with high expression (P = 0.046). CONCLUSIONS Expression of hCGbeta-3, -5, -8, which differ by only one nucleotide from other hCGbeta genes, can be assessed by our modified Molecular Beacon assay in breast cancer tissues. Expression of hCGbeta-3, -5, -8 has independent, prognostic value for RFS in breast cancer and may help identify node-negative patients with poor prognosis.

Hexosamines are unlikely to function as a nutrient-sensor in 3T3-L1 adipocytes: a comparison of UDP-hexosamine levels after increased glucose flux and glucosamine treatment.

Whether the hexosamine biosynthesis pathway acts as a nutrient-sensing pathway is still unclear. Glucose is directed into this pathway by GFAT. Because the activity of GFAT is tightly regulated, we examined whether UDP-hexosamine levels can increase significantly and dose-dependently in response to elevated glucose concentrations. In glucosamine-treated 3T3-L1 adipocytes, inhibition of insulin-stimulated glucose uptake was highly correlated with UDP-hexosamine levels (r=−0.992; p<0.0001 for UDP-GlcNAc and r=−0.996; p<0.0001 for UDP-GalNAc). Incubation of 3T3-L1 adipocytes with 0.1 µM insulin for 24 h in medium containing 1 and 5 mM glucose increased the rate of glucose uptake by 365% and 175% compared to untreated cells, respectively. This increase was not observed when the cells were incubated for 24 h with insulin in medium containing 10 or 25 mM glucose. However, treatment of cells with insulin and 1, 5, 10, or 25 mM glucose resulted in similar increases in levels of UDP-GlcNAc and UDP-GalNAc that always amounted to approx 30–40% above baseline values. This led us to conclude that despite exposure of adipocytes to conditions of extreme and prolonged glucose disposal, the increases in cellular UDP-hexosamines were minimal and not dependent on the extracellular glucose concentration. Taken together, our results are in line with the hypothesis that in glucosamine-treated adipocytes UDP-hexosamines influence insulin-stimulated glucose uptake. However, our observations in glucose-treated adipocytes argue against the possibility that UDP-hexosamines function as a nutrient-sensor, and question the role of the hexosamine biosynthesis pathway in the pathogenesis of insulin resistance.

Regulation of GLUT1-mediated glucose uptake by PKCλ-PKCβII interactions in 3T3-L1 adipocytes

Members of the PKC (protein kinase C) superfamily play key regulatory roles in glucose transport. How the different PKC isotypes are involved in the regulation of glucose transport is still poorly defined. PMA is a potent activator of conventional and novel PKCs and PMA increases the rate of glucose uptake in many different cell systems. In the present study, we show that PMA treatment increases glucose uptake in 3T3-L1 adipocytes by two mechanisms: a mitogen-activated protein kinase kinase-dependent increase in GLUT1 (glucose transporter 1) expression levels and a PKClambda-dependent translocation of GLUT1 towards the plasma membrane. Intriguingly, PKClambda co-immunoprecipitated with PKCbeta(II) and did not with PKCbeta(I). Previously, we have described that down-regulation of PKCbeta(II) protein levels or inhibiting PKCbeta(II) by means of the myristoylated PKCbetaC2-4 peptide inhibitor induced GLUT1 translocation towards the plasma membrane in 3T3-L1 adipocytes. Combined with the present findings, these results suggest that the liberation of PKClambda from PKCbeta(II) is an important factor in the regulation of GLUT1 distribution in 3T3-L1 adipocytes.

Exploring levels of hexosamine biosynthesis pathway intermediates and protein kinase C isoforms in muscle and fat tissue of zucker diabetic fatty rats

Many studies suggest that insulin resistance develops and/or is maintained by an increased flux of glucose through the hexosamine biosynthesis pathway. This pathway may attenuate insulin-stimulated glucose uptake by activating protein kinase C (PKC). Therefore, we investigated whether the concentrations of the major hexosamine metabolites, uridine diphosphate-N-acetyl-glucosamine (UDP-GlcNAc) and uridine diphosphate-N-acetyl-galactosamine (UDP-GalNAc), and the expression levels of PKC isoforms were affected in Zucker Diabetic Fatty (ZDF) rats, an animal model widely used to study type 2 diabetes mellitus. At the age of 6 wk, control and ZDF rats were normoglycemic. Whereas control rats remained normoglycemic, the ZDF rats became hyperglycemic. The amount of UDP-GlcNAc and UDP-GalNAc in muscle tissue of ZDF rats was similar at 6, 12, 18, and 24 wk of age. Moreover, the concentration of both hexosamines did not differ among ZDF, phlorizin-treated ZDF, and control rats. Western blot analysis revealed that PKCα, δ, ɛ, and ζ, but not PKCβ and γ, were expressed in muscle and fat tissues from 6- and 24-wk-old control and ZDF rats. In addition, we did not observe changes in the expression levels of the PKC isoforms following prolonged hyperglycemia. Taken together, these findings indicate that the amounts of several metabolites from the hexosamine biosynthesis pathway and PKC isoforms, both hypothesized to be important in the development and/or maintenance of the insulin-resistant state of muscle and fat tissue, are not different in ZDF compared with nondiabetic rats.

Rat pancreatic acinar cells express a cytosolic phospholipase D1b isoform that is not regulated by cholecystokinin.

Abstract. Evidence for the presence of a regulated phospholipase D (PLD) activity in pancreatic acinar cells is conflicting. Such knowledge is important because signal-activated PLD has been implicated in, amongst other things, regulated exocytosis. In this study, freshly isolated rat pancreatic acini were used to identify PLD transcripts by RT-PCR, to assess the presence and subcellular localization of PLD protein by Western blotting and to evaluate the presence of secretagogue-regulated PLD activity by means of the PLD-catalysed transphosphatidylation reaction. Transcripts of PLD1b and PLD2, but not PLD1a, were present in acinar cells. Moreover, a specific anti-human PLD1 antibody demonstrated the expression of substantial amounts of PLD1 protein. Intriguingly, however, the distribution pattern of acinar PLD1 seen following subcellular fractionation was clearly atypical in that immunoreactivity occurred predominantly in the acinar cytosol. Pretreatment of intact acini with a phorbol ester (4β-phorbol 12-myristate 13-acetate, PMA) to activate PLD1 protein kinase C (PKC) dependently did not change the subcellular distribution of PLD1. Similarly, pretreatment of a broken cell preparation of acini with guanosine 5′-O-(3-thiotriphosphate) (GTPγS) to activate PLD via small GTPases and PMA also did not influence this distribution. In the presence of ethanol, cholecystokinin-(26–33)-peptide amide (CCK8) did not increase the amount of radiolabelled phosphatidylethanol (PtdEth) in intact acini prelabelled with either o-[32P]phosphate or [3H]myristic acid. Similarly, an increased cytosolic Ca2+ concentration evoked by the specific inhibitor of the endoplasmic reticulum Ca2+-ATPase, thapsigargin, did not stimulate acinar PLD activity whereas high-level PKC activation with PMA elicited slight stimulation. In contrast, all three stimuli are known to increase PLD activity readily in Chinese hamster ovary (CHO) cells expressing the rat pancreatic acinar cell CCKA receptor. Finally, the combination of PMA and GTPγS did not increase PLD activity following homologous reconstitution of acinar cytosol and membranes, whereas the same manoeuvre resulted in marked stimulation of PLD activity in CHO cells. Heterologous reconstitution experiments revealed that PLD activity in CHO membranes was stimulated readily in the presence of acinar cytosol, indicating that the acinar cytosol contains the necessary factors for PMA/GTPγS-induced stimulation of membrane PLD activity. In contrast, CHO cell cytosol did not confer PMA/GTPγS-stimulation of PLD activity on acinar membranes, in agreement with the predominantly cytosolic localization of acinar PLD. The present findings show that rat pancreatic acinar cells express a cytosolic PLD1 isoform that is not regulated by the physiologically important secretagogue CCK.

Hormonal regulation of phospholipase D activity in Ca(2+) transporting cells of rabbit connecting tubule and cortical collecting duct.

Phospholipase D (PLD) is distributed widely in mammalian tissues where it is believed to play an important role in the regulation of cell functions and cell fate by a variety of extracellular signals. In this study, we used primary cultures of rabbit connecting tubule (CNT) and cortical collecting duct (CCD) cells, grown to confluence on a permeable support, to investigate the possible involvement of PLD in the mechanism of action of hormones that regulate Ca(2+) reabsorption. RT-PCR revealed the presence of transcripts of PLD1b and PLD2, but not PLD1a, in these cultures. Moreover, the expression of substantial amounts of PLD1 protein was demonstrated by Western blotting. To measure PLD activity, cells were labelled with [(3)H]myristic acid after which the PLD-catalysed formation of radiolabelled phosphatidylethanol ([(3)H]PtdEth) was measured in the presence of 1% (v/v) ethanol. Deamino-Cys,D-Arg(8)-vasopressin (dDAVP) and N(6)-cyclopentyladenosine (CPA), two potent stimulators of Ca(2+) transport across these monolayers, stimulated PLD activity as was indicated by a marked increase in [(3)H]PtdEth. Similarly, ATP, a potent inhibitor of dDAVP- and CPA-stimulated Ca(2+) transport, increased the formation of [(3)H]PtdEth. PLD activity was furthermore increased by 8Br-cAMP and following acute (30 min) stimulation of protein kinase C (PKC) with a phorbol ester (PMA). Chronic PMA treatment (120 h) to downregulate phorbol ester-sensitive PKC isoforms did not affect PLD activation by dDAVP, CPA and 8Br-cAMP, while markedly decreasing the effect of ATP and abolishing the effect of PMA. The PKC inhibitor chelerythrine significantly reduced PLD activation by dDAVP, CPA and 8Br-cAMP, without changing the effect of ATP. The inhibitor only partially reduced the effect of PMA. This study shows that Ca(2+) transporting cells of CNT and CCD contain a regulated PLD activity. The physiological relevance of this activity, which is not involved in Ca(2+) reabsorption, remains to be established.

Analysis of Agonist-Induced Cell Recruitment in Terms of Intracellular Calcium Mobilization in a Population of Enzymatically Dispersed Pancreatic Acinar Cells

The term cell recruitment is often used to describe the accumulation of motile cells such as neutrophils, lymphocytes, macrophages and mast cells at the site of release of some chemotactic factor. In the broadest sense of the word, however, this term applies to every situation in which a signal mobilizes cells to perform some kind of cellular activity. For instance, in a population of enzymatically dispersed pancreatic acinar cells the peptide hormone cholecystokinin (CCK) both time- and dose-dependently increases the number of cells displaying repetitive changes in free cytosolic calcium concentration (Fig. 29.1). This particular experiment clearly demonstrates that only part of the cells respond to stimulation with a submaximal concentration of 10 pM of the COOH-terminal octapeptide of CCK (CCK8), with a delay greatly differing between individual cells. As a result, maximal recruitment is reached only at 10 min following the onset of stimulation. By contrast, the vast majority of cells respond within the first minute of stimulation with a close to maximal hormone concentration of 1 nM. Such detection of differences in sensitivity among individual cells was enabled by the development of digital imaging techniques allowing simultaneous monitoring of agonist-induced changes in [Ca2+]i in large numbers of individual cells loaded with Ca2+-sensitive fluorescent dye. This chapter aims to give a detailed description of the method used in our laboratory to analyze agonist-induced cell recruitment in terms of intracellular calcium mobilization.