Microsatellite instability analysis in tumors with different mechanisms for total loss of HLA expression

Down-regulation of the expression of major histocompatibility complex molecules is a frequent event that is associated with the poor immunogenicity of tumor cells. Acquired resistence to T-cell-based immunotherapy has been associated with loss of functional β2-microglobulin expression. This anomaly appears to be particularly relevant in tumors exhibiting a defect in DNA-mismatch repair, and induces structural abnormalities in HLA cell-surface expression that are not reversible by cytokine treatment. We examined HLA expression in 118 melanoma, colon or larynx tumors to identify total loss of HLA class I expression with or without somatic β2-microglobulin gene mutation. Microsatellite instability was investigated in these tumors to determine whether a replication error phenotype (RER⁺) implied a particular alteration in HLA phenotype. A total of 7.6% of the tumors showed the RER⁺ phenotype, and 12.7% were HLA-ABC-negative. In the RER⁺ group, only one tumor was HLA-ABC-negative and no β2-microglobulin mutation was identified. In contrast, in the HLA-ABC-negative group, only one tumor showed microsatellite instability. None of the three melanomas that contained β2-microglobulin mutation exhibited the mutator phenotype. These findings suggest that β2-microglobulin mutation in human melanoma tumors may arise through a mechanism that does not necessarily involve microsatellite instability. Our results also indicate that somatic mutations of the β2-microglobulin gene are not the main mechanism of total loss of HLA expression. Received: 14 June 1999 / Accepted: 16 September 1999     

Human cancer, carcinogenic exposures and mutation spectra

Exposure of mammalian cells to alkylating agents causes transfer of alkyl groups to N- as well as O-atoms of DNA bases. Especially the O-alkylated G and T bases have strong mutagenic properties, since they are capable of mispairing during replication. The mutagenic potential of N-alkylbases is less clear although specific base excision repair (BER) pathways exist which remove those lesions from the DNA. We investigated the relative contribution of N-alkylations to mutation induction at the Hprt gene in cultured Chinese hamster ovary cells (CHO). To this end BER activity in CHO cells was modulated by introduction of an expression vector carrying the rat N-alkylpurine-DNA glycosylase (APDG) gene, which codes for a glycosylase that is able to remove 3-methyladenine and 7-methylguanine from DNA thereby generating apurinic sites. Upon selection of a CHO clone which 10 times overproduced APDG compared to control CHO cells, mutation induction, the mutational spectrum, and cell survival were determined in both cell lines following treatment with methyl methanesulfonate (MMS). The results show that over-expression of APDG renders CHO cells more sensitive for mutation induction as well as cytotoxicity induced by MMS. The involvement of apurinic sites in induction of base pair changes at positions where 3-methyladenine was induced is inferred from the observation that the mutational spectrum of MMS-induced mutations in APDG-CHO cells showed twice as much base pair changes at AT base pairs (33.3%) compared to the spectrum of MMS-induced mutations in CHO-control cells (15.8%).     

Notch inhibits Ptf1 function and acinar cell differentiation in developing mouse and zebrafish pancreas

Notch signaling regulates cell fate decisions in a variety of adult and embryonic tissues, and represents a characteristic feature of exocrine pancreatic cancer. In developing mouse pancreas, targeted inactivation of Notch pathway components has defined a role for Notch in regulating early endocrine differentiation, but has been less informative with respect to a possible role for Notch in regulating subsequent exocrine differentiation events. Here, we show that activated Notch and Notch target genes actively repress completion of an acinar cell differentiation program in developing mouse and zebrafish pancreas. In developing mouse pancreas, the Notch target gene Hes1 is co-expressed with Ptf1-P48 in exocrine precursor cells, but not in differentiated amylase-positive acinar cells. Using lentiviral delivery systems to induce ectopic Notch pathway activation in explant cultures of E10.5 mouse dorsal pancreatic buds, we found that both Hes1 and Notch1-IC repress acinar cell differentiation, but not Ptf1-P48 expression, in a cell-autonomous manner. Ectopic Notch activation also delays acinar cell differentiation in developing zebrafish pancreas. Further evidence of a role for endogenous Notch in regulating exocrine pancreatic differentiation was provided by examination of zebrafish embryos with homozygous mindbomb mutations, in which Notch signaling is disrupted. mindbomb-deficient embryos display accelerated differentiation of exocrine pancreas relative to wild-type clutchmate controls. A similar phenotype was induced by expression of a dominant-negative Suppressor of Hairless [Su(H)] construct, confirming that Notch actively represses acinar cell differentiation during zebrafish pancreatic development. Using transient transfection assays involving a Ptf1-responsive reporter gene, we further demonstrate that Notch and Notch/Su(H) target genes directly inhibit Ptf1 activity, independent of changes in expression of Ptf1 component proteins. These results define a normal inhibitory role for Notch in the regulation of exocrine pancreatic differentiation.     

Delimiting the geographical background in species distribution modelling

Aim The extent of the study area (geographical background, GB) can strongly affect the results of species distribution models (SDMs), but as yet we lack objective and practicable criteria for delimiting the appropriate GB. We propose an approach to this problem using trend surface analysis (TSA) and provide an assessment of the effects of varying GB extent on the performance of SDMs for four species. Location Mainland Spain. Methods Using data for four well known wild ungulate species and different GBs delimited with a TSA, we assessed the effects of GB extent on the predictive performance of SDMs: specifically on model calibration (Miller’s statistic) and discrimination (area under the curve of the receiver operating characteristic plot, AUC; sensitivity and specificity), and on the tendency of the models to predict environmental potential when they are projected beyond their training area. Results In the training area, discrimination significantly increased and calibration decreased as the GB was enlarged. In contrast, as GB was enlarged, both discriminatory power and calibration decreased when assessed in the core area of the species distributions. When models trained using small GBs were projected beyond their training area, they showed a tendency to predict higher environmental potential for the species than those models trained using large GBs. Main conclusions By restricting GB extent using a geographical criterion, model performance in the core area of the species distribution can be significantly improved. Large GBs make models demonstrate high discriminatory power but are barely informative. By delimiting GB using a geographical criterion, the effect of historical events on model parameterization may be reduced. Thus purely environmental models are obtained that, when projected onto a new scenario, depict the potential distribution of the species. We therefore recommend the use of TSA in geographically delimiting the GB for use in SDMs.     

Differential distribution of sialic acid in alpha2,3 and alpha2,6 linkages in the apical membrane of cultured epithelial cells and tissues.

We used lectin cytochemistry and confocal microscopy to examine the distribution of sialic acid in epithelial cells. Maackia amurensis lectin and Sambuccus nigra agglutinin were used to detect alpha2,3 and alpha2,6 sialic acid, respectively. In Caco-2, HT-29 5M12, and MCF-7 cells, which express sialic acid mainly in one type of linkage, the majority of the signal was observed in the apical membrane. In cells that bound both lectins, alpha2,3 sialic acid was distributed apically, whereas alpha2,6 sialic acid showed a broader distribution. In IMIM-PC-1 cultures, alpha2,3 sialic acid was detected mainly in the apical membrane, whereas alpha2,6 sialic acid was more abundant in the basolateral domain of polarized cells. In these cells, treatment with GalNAc-O-benzyl led to reduced alpha2,3 levels and to an increase and redistribution of alpha2,6 to the apical domain. Similarly, sialic acid was predominantly expressed apically in all epithelial tissues examined. In conclusion, (a) sialic acid is mainly distributed to the apical membrane of epithelial cells, (b) there is a hierarchy in the distribution of sialic acids in polarized epithelial cells, i.e., alpha2,3 is preferred to alpha2,6 in the apical membrane, and (c) IMIM-PC-1 cells are a good model in which to study the regulation of the levels and distribution of sialic acids.     

Calcium-dependent modulation of human glycine receptors expressed in cultivated cell lines

Glycine receptors (GlyRs) provide the main inhibitory neurotransmission in spinal cord and brain-stem synapses of vertebrates. Fucile et al. (2000) discovered that elevation of intracellular Ca²⁺ caused rapid potentiation of GlyRs. This modulation develops in less than 100 ms. It is characterized by an increase in GlyR apparent affinity for glycine. It has been suggested that the phenomenon of Ca-induced potentiation involves an unknown Ca²⁺-binding protein (CaBP). Using the yeast two-hybrid system, screening of a human brain cDNA library against the cytoplasmic loop of human alpha 1 subunit (GlyRh1) allowed us to identify five new interactors. One of them belongs to the family of Ca-binding proteins. We analyzed the effect of “short” forms of this protein (CaBP-1) on functional properties of GlyRh1 expressed in HEK-293 and CHO cells. Using whole-cell recordings and rapid agonist application, we constructed concentration dependences of glycine-induced currents. This analysis revealed statistically significant differences in EC₅₀s between control cells (expressing only GlyRh1) and those expressing CaBP-1. In HEK-293 cells recorded under conditions of low intracellular Ca concentration (BAPTA 20 mM in the recording pipette), EC₅₀ for glycine in control cells and expressing GlyRh1+CaBP-1 were, correspondingly, 68 ± 49 μM (n = 29) and 409 ± 421 μM (n = 60). In CHO cells, EC₅₀ were 54 ± 43 μM (n = 25) and 123 ± 104 μM (n = 28). The differences were not statistically significant for recordings made with an intracellular solution containing high Ca concentration (50 μM). Under these conditions, EC₅₀ values were correspondingly 35 ± 28 μM (n = 7) and 64 ± 38 μM (n = 7). These results suggest that CaBP-1 causes a decrease of GlyR sensitivity to agonist interacting with the cytoplasmic domain of GlyR.     

Separation by FPLC chromatofocusing of UDP-glucosyltransferases from three developmental stages of Drosophila melanogaster

Variation of UDP-glucosyltransferase activity, during Drosophila melanogaster development, was analyzed. The endogenous metabolite xanthurenic acid and the xenobiotic compounds 1-naphthol and 2-naphthol were used as substrates. Developmentally regulated differences were observed for the three substrates, suggesting the presence of UDP-glucosyltransferase isoenzymes. This was further confirmed by FPLC chromatofocusing on a Mono P column: seven peaks of UDP-glucosyltransferase activity (pHs: ≥6.3, 5.8, 5.5, 4.9, 4.5, 4.2, ≤4.0) with either single or overlapping substrate specificity were detected. A single xanthurenic acid:UDP-glucosyltransferase activity (pl 5.8) was found throughout development. In contrast, a gradual increase in the number of 2-napthol:UDP-glucosyltransferase isoenzymes (pl from 6.3 to 4.0) was observed during development, whereas no isoenzymes specific for 1-naphthol were resolved. Based on the distribution and substrate specificity of the eluted peaks in the three developmental stages analyzed, the presence of seven or possibly eight UDP-glucosyltransferase isoenzymes is proposed. Arch. Insect Biochem. Physiol. 34:347–358, 1997. © 1997 Wiley-Liss, Inc.