Epstein-Barr virus-associated gastric cancer reveals intratumoral heterogeneity of PIK3CA mutations.

Background: Recent whole-genome sequencing identified four molecular subtypes of gastric cancer (GC), of which the subgroup of Epstein-Barr virus-associated GC (EBVaGC) showed a significant enrichment of PIK3CA mutations. We here aimed to validate independently the enrichment of PIK3CA mutations in EBVaGC of a Central European GC cohort, to correlate EBV status with clinico-pathological patient characteristics and to test for a major issue of GC, intratumoral heterogeneity. Patients and methods: In a first step, 484 GCs were screened for EBV and PIK3CA hot spot mutations of exon 9/20 using EBER in situ hybridization and pyrosequencing, respectively. Secondly, an extended sequencing of PIK3CA also utilizing next generation sequencing was carried out in all EBVaGCs and 96 corresponding lymph node metastases. Results: Twenty-two GCs were EBER-positive, all being of latency type I. Intratumoral heterogeneity of EBER-positivity was found in 18% of EBVaGCs. Twenty-three GCs held PIK3CA mutations in hot spot regions of exon 9 or 20, being significantly more common in EBVaGCs (P < 0.001). Subsequent extended sequencing of PIK3CA of EBVaGCs showed that 14% harvested three to five different PIK3CA genotypes (including wildtype) in the same primary tumor, albeit in histologically and spatially distinct tumor areas, and that intratumoral heterogeneity of PIK3CA was also present in the corresponding lymph node metastases. Conclusions: Our findings unravel issues of tumor heterogeneity and illustrate that the assessment of the EBV status in tissue biopsies might carry the risk of sampling errors, which may significantly hamper adequate molecular tumor classification in a more clinical setting. Moreover, this is the first report of intratumoral heterogeneity of PIK3CA mutations in GC, and our findings lead to the conclusion that PIK3CA mutant and -wildtype tumor subclones are skilled to metastasize independently to different regional lymph nodes.

HER2/neu testing in primary colorectal carcinoma.

BACKGROUND: Anti-HER2/neu therapy is well-established in breast and gastric carcinoma. The increased understanding of this pathway led to the identification of new promising drugs in addition to trastuzumab, offering further perspectives. The role of HER2/neu in colorectal carcinoma is controversially discussed, as discrepant data has been reported. METHODS: Here, we retrospectively assessed the prevalence of HER2/neu positivity in a large series of colorectal carcinoma, testing HER2/neu status according to current recommendations. We correlated the results to clinico-pathological data and patient survival. RESULTS: Overall, in 1645 primary colorectal carcinoma cases, 1.6% of the cases were HER2/neu positive. HER2/neu positivity significantly correlated with higher UICC stages (P=0.017) and lymph node metastases (P=0.029). In the subgroup of sigmoideal and rectal carcinomas, positive HER2/neu status was associated with T-category (P=0.041) and higher UICC stages (P=0.022). Although statistically not significant, HER2/neu-positive colorectal carcinomas displayed a tendency to poorer overall survival. CONCLUSIONS: These results illustrate the importance of testing HER2/neu by approved diagnostic techniques and scoring systems. We assume that although the prevalence of HER2/neu positivity in colorectal carcinoma is low, HER2/neu testing in advanced, nodal-positive colorectal carcinoma is reasonable, offering a potential target in high risk colorectal carcinoma.

Metastatic melanoma of unknown primary resembles the genotype of cutaneous melanomas.

BACKGROUND: Although 90% of all melanomas are of cutaneous origin, some patients present with melanoma metastases of unknown origin (MUP). Commonly, in these patients an extensive search for the primary tumor is carried out. In the past, genetic analyses have shown substantial differences in pathogenetic mutations among cutaneous, acral and mucosal melanomas. The aim of this study was to assess the mutational status of MUP in order to better characterize the putative origin of the primary tumor and to evaluate potential prognostic factors. PATIENTS AND METHODS: The medical records of 44 patients with MUP were analyzed and a survival analysis was conducted. In total, 66 paraffin samples of 44 patients were analyzed, and in 15 patients multiple metastases were tested. Mutational analysis of the BRAF, NRAS and KIT genes was carried out. RESULTS: Twenty-three patients (52.3%) had a mutation in the BRAF gene and 12 patients (23.8%) had a mutation in the NRAS gene. There were neither mutations in the KIT gene. In patients with multiple samples, there was 100% consistency regarding mutational status among the different metastases. The median overall survival (OS) was 86.4 months (39-134). The American Joint Committee on Cancer stage at first diagnosis of metastatic melanoma (stage III versus IV) was significantly associated with OS (P < 0.001), BRAF or NRAS mutation status had no significant prognostic impact on clinical outcomes. CONCLUSIONS: MUP resembles the genotype of cutaneous melanoma and not that of mucosal melanomas.

Members of the EpCAM signalling pathway are expressed in gastric cancer tissue and are correlated with patient prognosis.

BACKGROUND: We investigated the expression of members of the epithelial cell adhesion molecule (EpCAM) signalling pathway in gastric cancer (GC) testing the following hypotheses: are these molecules expressed in GC and are they putatively involved in GC biology. METHODS: The study cohort consisted of 482 patients. The following members of the EpCAM signalling pathway were analysed by immunohistochemistry and were correlated with various clinico-pathological patient characteristics: extracellular domain of EpCAM (EpEX), intracellular domain of EpCAM (EpICD), E-cadherin, ß-catenin, presenilin-2 (PSEN2), and ADAM17. RESULTS: All members of the EpCAM signalling pathway were differentially expressed in GC. The expression correlated significantly with tumour type (EpEX, EpICD, E-cadherin, ß-catenin, and PSEN2), mucin phenotype (EpEX, EpICD, ß-catenin, and ADAM17), T-category (EpEX, E-cadherin, and ß-catenin), N-category (EpEX and ß-catenin), UICC tumour stage (EpEX, EpICD, ß-catenin, and PSEN2), tumour grade (EpEX, EpICD, E-cadherin, ß-catenin, and PSEN2), and patients' survival (EpEX, EpICD, and PSEN2). A significant coincidental expression in GC was found for EpEX, EpICD, E-cadherin, ß-catenin, PSEN2, and ADAM17. Decreased immunodetection of EpEX in locally advanced GC was not associated with decreased EpCAM mRNA levels. CONCLUSION: All members of the EpCAM signalling pathway are expressed in GC. The expression correlated significantly with each other and with various clinico-pathological patient characteristics, including patients' survival. Thus, the EpCAM signalling pathway is a highly interesting putative therapeutic target in GC.

Investigation of interfacial strength in nacre-mimicking tungsten heavy alloys for nuclear fusion applications.

Tungsten heavy alloys have been proposed as plasma facing material components in nuclear fusion reactors and require experimental investigation in their confirmation. For this purpose, a 90W-7Ni-3Fe alloy has been selected and microstructurally manipulated to present a multiphase brick-and-mortar structure of W-phase 'bricks' surrounded by a ductile 'mortar'. This work draws inspiration from nature to artificially imitate the extraordinary combination of strength and stiffness exhibited by mollusks and produce a nacre-mimicking metal matrix composite capable of withstanding the extremely hostile environment of the reactor interior and maintaining structural integrity. The underlying mechanisms behind this integrity have been probed through high-resolution structural and chemical characterization techniques and have revealed chemically diffuse phase boundaries exhibiting unexpected lattice coherency. These features have been attributed to an increase in the energy required for interfacial decohesion in these systems and the simultaneous expression of high strength and toughness in tungsten heavy alloys.

High-efficient nonviral transfection of the rat thyroid cell line FRTL-5.

FRTL-5 cells are used in many laboratories as an in vitro system of thyroid follicular cells since they share many properties of human thyrocytes. However, the use of FRTL-5 cells for experimental modifications is limited by low transfection efficiencies of lipid-based transfections and the need for cumbersome viral transduction protocols. A new technology - nucleofection - has become available for cell lines that are difficult to transfect. Here, we report the application and optimization of this method in FRTL-5 cells. Using the green fluorescent protein (GFP) as a reporter gene, FRTL-5 cells were easily transfectable with efficiencies over 60%. In addition, the simultaneous transfer of siRNA against GFP was feasible and allowed suppression of GFP over at least 4 days. Furthermore nucleofection was successful for establishing stable FRTL-5 cell clones. In conclusion, this optimized fast and efficient nucleofection protocol offers new properties for the experimental use of FRTL-5 cells.

Efficient non-viral transfection of primary human adult chondrocytes in a high-throughput format.

OBJECTIVE: The development of a reliable high-throughput transfection protocol for primary human articular chondrocytes. METHODS: Primary human chondrocytes were isolated from adult knee cartilage by an optimized enzymatic digestion protocol and cultivated in high-density monolayer culture for 3-5 days. Isolated chondrocytes were transfected with a green fluorescent protein (GFP)-expressing reporter construct using amaxa's Nucleofector 96-well Shuttle System. Transfection efficiencies were measured by fluorescence activated cell sorting and cell viability was determined by an adenosine-5'-triphosphate (ATP) assay. siRNA oligonucleotides (against glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) were transfected into the cells using the optimized nucleofection protocol and mRNA knockdown values were determined by a branched-DNA assay. RESULTS: Transfection efficiencies of more than 70% of surviving cells were achieved routinely with the nucleofection protocol presented in this article. Cell viability 24h post transfection was around 80%. The cell number used per transfection was reduced to 2x10(5) per sample. In addition, the protocol proved to be well suited for the transfer of siRNA molecules into primary human chondrocytes with suppression rates on the mRNA level of more than 95% (for GAPDH). CONCLUSIONS: We present the successful use of nucleofection on primary human chondrocytes using a microtiter plate compatible format that for the first time allows the efficient transfection of up to 96 samples in parallel. The optimized nucleofection protocol is offering maximum substrate flexibility by allowing transfer of DNA and siRNA oligonucleotides with the same set of parameters. Moreover, the transfection procedure requires substantially lower cell numbers than single cuvette protocols and is therefore perfectly suited for applications requiring multiple experimental replicates.

DNA methylation is not responsible for p21WAF1/CIP1 down-regulation in osteoarthritic chondrocytes.

OBJECTIVE: In this study, we were interested in the overall methylation level in aged and degenerated cartilage. Also, we looked at one gene which might be involved in the re-initiation of replicative activity in osteoarthritis (OA) chondrocytes, p21(WAF1/CIP1). p21(WAF1/CIP1) was previously suggested to be down-regulated in OA chondrocytes and is known to be regulated by epigenetic modulation. METHODS: Total methylation levels were analyzed by high pressure liquid chromatography (HPLC), mRNA expression of p21(WAF1/CIP1) and DNMT enzymes by real-time polymerase chain reaction. The methylation status of the p21(WAF1/CIP1)- promotor using bisulfite genomic sequencing was evaluated. RESULTS: General methylation analysis of genomic DNA showed no difference in between normal and aged/OA chondrocytes. Also no difference in methylation of the promotor of the p21(WAF1/CIP1) gene was detectable, which was significantly down-regulated in OA chondrocytes. DNMT1 and DNMT3a were expressed with no significant changes of expression levels found in OA chondrocytes. CONCLUSION: Cell cycle progression inhibitor p21(WAF1/CIP1) is expressed in normal and significantly down-regulated in OA articular chondrocytes, which may mediate the re-initiation of cell proliferation in OA cartilage. However, the suppression of p21(WAF1/CIP1) mRNA expression is not due to hypermethylation of its promotor. No overall changes in genome methylation levels were found in aged or OA cartilage. Interestingly, significant expression of DNA methyltransferases was found in articular chondrocytes, which supports that DNA methylation could still be a relevant mechanism of gene regulation in (osteoarthritic) chondrocytes, though not on an overall genomic level nor specifically for the regulation of the p21(WAF1/CIP1) gene.

Characterizing a rat Brca2 knockout model.

Evidence exists that BRCA2 carriers may have an elevated risk of breast, ovarian, colon, prostate, and pancreatic cancer. In general, carriers are defined as individuals with protein truncating mutations within the BRCA2 gene. Many Brca2 knockout lines have been produced and characterized in the mouse. We previously produced a rat Brca2 knockout strain in which there is a nonsense mutation in exon 11 between BRC repeats 2 and 3, and a truncated protein is produced. Interestingly, while such a mutation in homozygous mice would lead to limited survival of approximately 3 months, the Brca2-/- rats are 100% viable and the vast majority live to over 1 year of age. Brca2-/- rats show a phenotype of growth inhibition and sterility in both sexes. Aspermatogenesis in the Brca2-/- rats is due to a failure of homologous chromosome synapsis. Long-term phenotypes include underdeveloped mammary glands, cataract formation and lifespan shortening due to the development of tumors and cancers in multiple organs. The establishment of the rat Brca2 knockout model provides a means to study the role of Brca2 in increasing cancer susceptibility and inducing a novel ocular phenotype not previously associated with this gene.

[Supplying surgery blocs in red cell concentrates: a collective approach to improve practices]. / Approvisionnement des blocs opératoires en concentrés érythrocytaires: une démarche collective d'amélioration des pratiques.

The need to adapt red blood cells concentrates management in surgery blocs and resuscitation to the changes of the legal framework has lead to a collective approach to improve practices. Gathered by the regional hemovigilance coordinators of the Drass Ile-de-France (regional office of health and social actions), representatives of doctors' ordering transfusions and hemovigilance correspondents of the Assistance publique-Hôpitaux de Paris and representatives of the EFS (French blood establishment) Ile-de-France, together with representatives of the Afssaps (French health products safety agency), have coordinated an assessment of local transfusion practices in surgery blocs and resuscitation that have to be compliant. Each hospital then offered local improvement actions, approved by regional and national instances. We present this original and collective approach of assessing practices leading to offers that both respond to a legal framework and improve blood products flows without damaging transfusion security.

The Mcs7 quantitative trait locus is associated with an increased susceptibility to mammary cancer in congenic rats and an allele-specific imbalance.

Identification of high-penetrance breast cancer genes such as Brca1 has been accomplished by analysing familial cases. However, these genes occur at low frequency and do not account for the majority of genetic risk. Identification of low-penetrance alleles that occur commonly in populations may benefit from unbiased genome-wide screening. One such approach uses linkage studies in rodent models to identify homologous human candidates. The Wistar Kyoto (WKy) rat is resistant to mammary carcinomas induced with 7,12-dimethybenz[a]anthracene (DMBA), whereas the Wistar Furth (WF) strain is susceptible. Previous genome-wide linkage studies in crosses of these strains identified three WKy resistance quantitative trait loci, Mcs5, Mcs6 and Mcs8, and one predicted to increase susceptibility, Mcs7. The Mcs7 region on rat chromosome 10 (RNO10) is orthologous to human 17q, a common site of genetic aberrations in breast cancer. Here, we establish the independent phenotype conferred by Mcs7 using congenic rats carrying the WKy Mcs7 locus on a WF background. Tumor multiplicity was significantly higher ( approximately 50%) in DMBA-treated congenics homozygous and heterozygous for the WKy allele at the Mcs7 locus, compared to controls. We also investigated allelic imbalance (AI) in mammary carcinomas from (WKy x WF)F1 rats and Mcs7 heterozygous congenics. Of the four known WKy Mcs loci tested, only Mcs7 displayed AI. The pattern of AI in carcinomas from both F1 and Mcs7 congenic rats was similar, suggesting a WF allelic loss. Together, these data suggest that one or more breast cancer-related genes are located within the dominantly acting WKy allele at the Mcs7 locus.

IL-1beta and BMPs--interactive players of cartilage matrix degradation and regeneration.

Intact human adult articular cartilage is central for the functioning of the articulating joints. This largely depends on the integrity of its extracellular matrix, given the high loading forces during movements in particular in the weight-bearing joints. Unlike the first impression of a more or less static tissue, articular cartilage shows - albeit in the adult organism a slow--tissue turnover. Thus, one of the most important questions in osteoarthritis research is to understand the balance of catabolic and anabolic factors in articular cartilage as this is the key to understand the biology of cartilage maintenance and degeneration. Anabolic and catabolic pathways are very much intermingled in articular cartilage. The balance between anabolism and catabolism is titrated on numerous levels, starting from the mediator-synthesizing cells which express either catabolic or anabolic factors. Also, on the level of the effector cells (i.e. chondrocytes) anabolic and catabolic gene expression compete for a balance of matrix homeostasis, namely the synthesis of matrix components and the expression and activation of matrix-degrading proteases. Also, there are multiple layers of intracellular cross-talks in between the anabolic and catabolic signalling pathways. Maybe the most important lesson from this overview is the notion that the anabolic-catabolic balance as such counts and not so much sufficient net anabolism or limited catabolism alone. Thus, it might be neither the aim of osteoarthritis therapy to foster anabolism nor to knock down catabolism, but the balance of anabolic-catabolic activities as a total might need proper titration and balancing.

MMP-9/gelatinase B is a gene product of human adult articular chondrocytes and increased in osteoarthritic cartilage.

OBJECTIVE: Collagen fibril degeneration involves initially the cleavage within the triple helix by the collagenases 1 (MMP-1) and 3 (MMP-13), but then mainly involves also the gelatinases A (MMP-2) and B (MMP-9). The objective of this study was to determine the quantitative expression levels as well as the distribution in normal and osteoarthritic cartilage of gelatinase B and in cultured articular chondrocytes with and without stimulation by Il-1Beta. METHODS: Conventional and real-time quantitative PCR technology and immunohistochemistry were used to determine gelatinase B expression on the mRNA and protein level. RESULTS: Conventional PCR analysis could demonstrate the presence of gelatinase B mRNA only in osteoarthritic chondrocytes. Real-time quantitative PCR confirmed the increased expression of gelatinase B mRNA expression in osteoarthritic chondrocytes. No significant up-regulation of gelatinase B was observed by Il-1Beta. Immunostaining for gelatinase B showed the presence of gelatinase B in a subset of normal and in a large portion of osteoarthritic chondrocytes with a more extended distribution in the latter. CONCLUSION: In osteoarthritic cartilage destruction, gelatinase B is involved in collagen destruction though still at a very much lower level than gelatinase A. Only a very small subset of normal adult articular chondrocytes express gelatinase B in vivo suggesting that gelatinase B unlike gelatinase A is hardly or only very focally involved in physiological collagen turnover.

Reduction in the frequency of activated ras oncogenes in rat mammary carcinomas with increasing N-methyl-N-nitrosourea doses or increasing prolactin levels.

The role of c-Ha-ras-1 oncogene activation in the multistage biological process of N-methyl-N-nitrosourea (NMU)-induced mammary carcinogenesis was investigated. The average yield of NMU-induced mammary tumors in Wistar-Furth rats was altered by modification of either the initiation or promotion/progression stage of carcinogenesis. Initiation was varied by the use of different doses of NMU from 20 to 50 mg/kg. Tumor yield was increased with increasing NMU doses. However, the frequency of mammary tumors with activated c-Ha-ras-1 decreased in a linear fashion with increasing NMU doses. Promotion/progression was varied by increasing prolactin levels starting approximately 2 weeks after NMU administration. This hormonal manipulation increased tumor yield, while reducing the frequency of tumors with activated ras. It is postulated that ras activation represents one of several possible mechanisms by which NMU initiates mammary carcinogenesis. Furthermore, initiated cells without activated ras are more dependent on epigenetic promotional events provided by either prolactin or NMU than are ras-initiated cells.

Limonene-induced regression of mammary carcinomas.

Dietary administration of the monocyclic monoterpenoid d-limonene causes complete regression of both dimethylbenz[alpha]anthracene- and N-nitroso-N-methylurea-induced rat mammary carcinomas. Carcinomas regress when limonene is added to the diet either when the tumor is small and still capable of spontaneously regressing or when it is large and progressed beyond the stage when it is susceptible to spontaneous regression. The limonene dose-tumor regression response relationship is steep. Significant regressions are not observed at 5% dietary levels, while a majority of tumors completely regress above a 7.5% dietary level. Limonene appears to act in a cytostatic fashion. Its removal from the diet results in a significant number of tumor recurrences. Regressing tumors have a unique histopathological appearance that is not associated with gross cytotoxicity, immune cell involvement, or apoptosis. Preliminary analysis suggests a remodeling/redifferentiation event underlying regression. The underlying mechanism of action of limonene in causing tumor regression is unknown. However, it should be noted that limonene can selectively inhibit the isoprenylation of small G proteins. Monoterpenoids such as limonene represent a novel class of anticancer drugs with the potential to cause tumor regressions with limited toxicity.

Increased mannose 6-phosphate/insulin-like growth factor II receptor and transforming growth factor beta 1 levels during monoterpene-induced regression of mammary tumors.

The monoterpenes represent a potentially new class of breast cancer therapeutic agents. We have shown that d-limonene induces the regression of advanced rat mammary adenocarcinomas. These regressing tumors have an increased cellular concentration of both the mannose-6-phosphate/insulin-like growth factor II receptors and transforming growth factor beta 1. The terpene-induced regression of mammary tumors may result in part from the mitoinhibitory and differentiation properties of active transforming growth factor beta 1. Furthermore, the activation of transforming growth factor beta 1 in these tumors is likely to be facilitated by the increased concentration of the mannose-6-phosphate/insulin-like growth factor II receptors in the mammary tumor cells. Tumors not responding to terpene therapy lacked a rise in the mannose-6-phosphate/insulin-like growth factor II receptor level which may relate to the fact that this gene is hemizygous due to maternal imprinting.

Limonene chemoprevention of mammary carcinoma induction following direct in situ transfer of v-Ha-ras.

Monoterpenes, including limonene and its in vivo rat plasma metabolites, have been shown to be inhibitors of protein isoprenylation of small G proteins, including p21 ras. In addition, dietary limonene has been shown to be capable of preventing the development and causing the regression of chemically induced mammary carcinomas, many of which contain activated ras oncogenes. On the basis of these observations, it was hypothesized that a possible mechanism by which limonene exerts its effects on the chemoprevention and regression of mammary tumors involves the inhibition of protein isoprenylation of the small G protein p21. In the first study, we asked whether dietary limonene was able to prevent the development of mammary carcinomas which were induced using direct retroviral gene transfer of v-Ha-ras into the mammary parenchyma in situ. Limonene modified neither the rate of gene transfer nor the stability of gene expression. However, limonene did greatly inhibit the formation of mammary carcinomas induced by the insertion of activated ras. In a follow-up study, we asked whether chemoprevention by limonene was preferentially effective against a subset of chemically induced mammary carcinomas with activated ras. Rats were fed limonene to prevent the development of N-nitroso-N-methylurea-induced mammary tumors, a majority of which contain the activated Ha-ras oncogene. As expected, limonene administration increased the latency period and lowered the frequency of mammary carcinoma development as compared to controls. However, tumor characterization revealed that limonene treatment did not alter the percentage of carcinomas with activated ras. These studies are consistent with the above studies in that limonene is effective in preventing mammary carcinomas with activated ras. Interestingly, carcinomas without activated ras were prevented to the same extent as those with the activated oncogene.

Activation of the transforming growth factor beta signaling pathway and induction of cytostasis and apoptosis in mammary carcinomas treated with the anticancer agent perillyl alcohol.

The mechanisms of action of the anticancer agent perillyl alcohol (POH), presently in Phase II clinical trials, were investigated in advanced rat mammary carcinomas. Gross and ultrastructural morphology of POH-mediated tumor regression indicated that apoptosis accounted for the marked reduction in the epithelial compartment. Characterization of cell growth and death indices revealed that apoptosis was induced within 48 h of chemotherapy, before the induction of cytostasis. RNA expression studies, based on a multiplexed-nuclease protection assay, demonstrated that cell cycle- and apoptosis-related genes were differentially expressed within 48 h of POH treatment; p21(Cip1/WAF1), bax, bad, and annexin I were induced; cyclin E and cyclin-dependent kinase 2 were repressed; and bcl-2 and p53 were unchanged. Next, a potential role for transforming growth factor beta (TGF-beta) signaling in POH-mediated carcinoma regression was explored. RNA expression studies, again based on a multiplexed-nuclease protection assay, showed that TGF-beta-related genes were induced and temporally regulated during POH treatment: (a) c-jun and c-fos were transiently induced within 12 h of chemotherapy; (b) TGF-beta1 was induced within 24 h of chemotherapy; (c) the mannose 6-phosphate/insulin-like growth factor II receptor and the TGF-beta type I and II receptors were induced within 48 h of chemotherapy; and (d) smad3 was induced during active carcinoma regression. In situ protein expression studies, based on fluorescence-immunohistochemistry in concert with confocal microscopy, confirmed up-regulation and demonstrated colocalization of TGF-beta1, the mannose 6-phosphate/insulin-like growth factor II receptor, the TGF-beta type I and II receptors, and Smad2/Smad3 in epithelial cells. Nuclear localization of Smad2/Smad3 indicated that the TGF-beta signaling pathway was activated in regressing carcinomas. Subpopulations of Smad2/Smad3-positive and apoptotic nuclei colocalized, indicating a role for Smads in apoptosis. Thus, Smads may serve as a potential biomarker for anticancer activity. Importantly, none of the POH-mediated anticancer activities were observed in normal mammary gland.

Recurrent network interactions underlying flow-field selectivity of visual interneurons.

Motion-sensitive large-field neurons found at higher processing stages in many species often exhibit a remarkable selectivity for particular flow fields. However, the underlying neural mechanisms are not yet understood. We studied this problem in the so-called lobula plate tangential cells (LPTCs) of the fly. Investigating the connectivity between LPTCs by means of dual recordings, we find two types of connections: (1) heterolateral connections between LPTCs of both hemispheres and (2) ipsilateral connections between LPTCs within one lobula plate. The circuit is suitable to amplify incoming, dendritic signals in the case of rotatory flow fields and to reduce them in the case of other flow-field structures. In addition to feedforward connectivity, thus, the flow-field selectivity of LPTCs may be significantly attributable to recurrent excitation involving the network of large-field neurons in both brain hemispheres.

Genetic loci controlling breast cancer susceptibility in the Wistar-Kyoto rat.

In this study, the Wistar-Kyoto (WKy) rat was genetically characterized for loci that modify susceptibility to mammary carcinogenesis. We used a genetic backcross between resistant WKy and susceptible Wistar-Furth (WF) rats as a panel for linkage mapping to genetically identify mammary carcinoma susceptibility (Mcs) loci underlying the resistance of the WKy rat. Rats were phenotyped for DMBA-induced mammary carcinomas and genotyped using microsatellite markers. To detect quantitative trait loci (QTL), we analyzed the genome scan data under both parametric and nonparametric distributional assumptions and used permutation tests to calculate significance thresholds. A generalized linear model analysis was also performed to test for interactions between significant QTL. This methodology was extended to identify interactions between the significant QTL and other genome locations. Chromosomes 5, 7, 10, and 14 were found to contain significant QTL, termed Mcs5, Mcs6, Mcs7, and Mcs8, respectively. The WKy alleles of Mcs5, -6, and -8 are associated with mammary carcinoma resistance; the WKy allele of Mcs7 is associated with an increased incidence of mammary cancer. In addition, we identified an interaction between Mcs8 and a region on chromosome 6 termed Mcsm1 (modifier of Mcs), which had no significant main effect on mammary cancer susceptibility in this genetic analysis.