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Upper tract urothelial carcinoma (UTUC) may arise in the setting of hereditary non-polyposis colorectal cancer (Lynch syndrome [LS]) or sporadically. Variable frequencies of microsatellite instability (MSI) were found in UTUC. For advanced solid MSI tumors, targeted therapy with programmed death-ligand 1 inhibitors is available. Therefore, we aimed to determine the prevalence of mismatch repair (MMR) protein loss and MSI in UTUC using a tissue microarray approach and further molecular and correlation analysis.We studied the immunohistochemical expression of MLH1, MSH2, MSH6, and PMS2 on tissue microarrays containing formalin-fixed, paraffin-embedded samples of 128 patients with UTUC. MSI analysis was performed in 79 cases with deficient MMR protein expression, and/or in patients aged 60 years and below, and/or other tumors possibly related to LS.Loss of MMR protein expression was seen in 24 (18.8%) of 128 cases. MSI analysis revealed MSI-high in 29, MSI-low in 7 cases. The Fisher exact test demonstrated significant differences between MSI and loss of MMR protein expression, clinically possible LS, tumor growth pattern, inverted growth pattern, and death (P .001, P .001, P = .002, P = .003, and P = .033, respectively). MSI does not appear to influence survival (overall and progression-free), but there was a significant shorter progression-free survival in MSI-high versus MSS patients who had received chemotherapy.The frequency of MSI in UTUC was 36 (28.1%) of 128 patients with a good accuracy of immunohistochemistry. In daily practice, MSI screening especially is recommended in patients with advanced UTUC and inverted papillary tumor growth pattern with the aim of screening patients for possible targeted therapy.

AIMS This study was designed to test programmed cell death 1 (PD-1) expression of T cells, the hallmark of T cell exhaustion, in different 'immune-classes' of colorectal carcinoma microenvironments as delineated by unsupervised hierarchical cluster analysis. METHODS AND RESULTS A tissue microarray was made with punches from the invasive margins of 40 microsatellite-unstable and 34 microsatellite-stable colorectal carcinomas. Immune cells were phenotyped by CD8, granzyme B, CD4, FoxP3, CD68, S-100, PD-1 and programmed cell death ligand 1 (PD-L1) immunohistochemistry; tumour area per tissue spot was quantified by cytokeratin (CK)18 immunohistochemistry. For each tissue spot, intra-epithelial immune cells were counted and densities of the various immune cells were calculated. Unsupervised hierarchical cluster analysis with these data yielded a group of 'anergic/immune-naive' microenvironments (47.3%), a group of 'intermediates' (27.0%) and a group of 'immunoreactives' (25.7%) in which PD-1 expressing T cells were prominent. Sixteen of 19 tissue spots representing immunoreactive microenvironments derived from microsatellite-unstable tumours and three were from microsatellite-stable tumours. Further phenotyping of intra-epithelial T cells by sequential immunohistochemistry showed frequent granzyme B/CD8 co-expression, whereas PD-1/CD8 co-expression was more variable. Using receiver operating curve (ROC) analysis, assignment to immune classes was seen to be feasible with good sensitivity and specificity by CD8 counts only. CONCLUSION A subset of colorectal carcinoma microenvironments is distinguished from the rest by an immune cell composition suggestive of active host anti-tumour immune defence, but this appears to be antagonized by a brisk undercurrent of T cell exhaustion. This observation may have implications for selecting colorectal carcinoma patients for immune checkpoint therapy.

Hypermutator-type colorectal carcinomas are microsatellite-stable and have point mutations of the exonuclease domain of the DNA polymerase e or δ genes (POLE and POLD1, respectively), and an ultrahigh tumor mutational burden (TMB). These tumors may be associated with enhanced antitumor immunity and preferentially afflict younger patients, but this notion awaits validation by accrual of further cases for detailed correlative phenotypic and molecular study. We performed POLE and POLD1 exonuclease domain Sanger sequencing of 271 unselected colorectal carcinomas. We identified two microsatellite-stable tumors with somatic POLE p.P286R variants, both with ultrahigh TMBs as demonstrated by whole exome sequencing. A POLE p.V411L was found in another two microsatellite-stable tumors with ultrahigh TMBs. Two of these four tumors were from young patients (

// Claudia Maletzki 1, * , Franziska Beyrich 1, * , Maja Huhns 2 , Ernst Klar 3 , Michael Linnebacher 1 1 Molecular Oncology and Immunotherapy, Department of General Surgery, University of Rostock, 18057 Rostock, Germany 2 Institute of Pathology, University of Rostock, 18057 Rostock, Germany 3 Department of General Surgery, University of Rostock, 18057 Rostock, Germany * These authors have contributed equally to this work Correspondence to: Claudia Maletzki, email: claudia.maletzki@med.uni-rostock.de Keywords: murine tumor models, MMR deficiency, cell line establishment, tumor microenvironment, MSI target genes Received: February 29, 2016 Accepted: June 06, 2016 Published: July 18, 2016 ABSTRACT Mice lines homozygous negative for one of the four DNA mismatch repair (MMR) genes ( MLH1, MSH2, PMS2, MSH6 ) were generated as models for MMR deficient (MMR-D) diseases. Clinically, hereditary forms of MMR-D include Lynch syndrome (characterized by a germline MMR gene defect) and constitutional MMR-D, the biallelic form. MMR-D knockout mice may be representative for both diseases. Here, we aimed at characterizing the MLH1 -/- model focusing on tumor-immune microenvironment and identification of coding microsatellite mutations in lymphomas and gastrointestinal tumors (GIT). All tumors showed microsatellite instability (MSI) in non-coding mononucleotide markers. Mutational profiling of 26 coding loci in MSI + GIT and lymphomas revealed instability in half of the microsatellites, two of them ( Rfc3 and Rasal2 ) shared between both entities. MLH1 -/- tumors of both entities displayed a similar phenotype (high CD71, FasL, PD-L1 and CTLA-4 expression). Additional immunofluorescence verified the tumors’ natural immunosuppressive character (marked CD11b/CD200R infiltration). Vice versa , CD3 + T cells as well as immune checkpoints molecules were detectable, indicative for an active immune microenvironment. For functional analysis, a permanent cell line from an MLH1 -/- GIT was established. The newly developed MLH1 -/- A7450 cells exhibit stable in vitro growth, strong invasive potential and heterogeneous drug response. Moreover, four additional MSI target genes ( Nktr1 , C8a , Taf1b , and Lig4 ) not recognized in the primary were identified in this cell line. Summing up, molecular and immunological mechanisms of MLH1 -/- driven carcinogenesis correlate well with clinical features of MMR-D. MLH1 -/- knockout mice combine characteristics of Lynch syndrome and constitutional MMR-D, making them suitable models for preclinical research aiming at MMR-D related diseases.

Colorectal cancer (CRC) is rare in young patients without a confirmed family history of cancer. Reports of an increased prevalence of POLD1/POLE mutations in young patients with colorectal cancer have raised awareness and support routine genetic testing for patients with early-onset tumors. In cases of CRC without proven MMR-germline mutation, molecular analyses are warranted to confirm or rule out other familial CRC syndromes. This article describes the cases of two young male patients, who presented with locally advanced and metastatic CRC, and reports the results of the germline mutational analyses done for both patients. These cases demonstrate the importance of special care and molecular diagnostic procedures for young patients with CRC. Key Points Patients with colorectal cancer who are younger than 50 years at initial diagnosis (early onset) should routinely undergo genetic testing. Early- and very-early-onset patients (younger than 40 years) with absence of microsatellite instability should be considered for tumor mutation burden testing and/or DNA polymerase proofreading mutation. The mutational signature of HSP110 within mismatch repair deficiency-related tumors may help to identify patients likely to benefit from 5-fluorouracil-based chemotherapy. Intensified, maintained, and specific surveillance may help to reduce secondary tumor progression.

This study addressed if TP53 immunohistochemistry as a surrogate method for gene sequencing could be applied to colorectal carcinomas as successfully as recently reported for ovarian cancers. Sanger sequencing of the coding exons 2-11 of 87 tumors yielded a total of 65 mutations in 61 of the tumors. Immunohistochemistry was done with the Do-7 antibody. By a pattern recognition evaluation of immunohistochemistry, 44 cases were classified as "overexpressors" and 20 as having "wild-type" immunostaining; complete absence of or cytoplasmic immunostaining was seen in 9 and 4 cases, respectively. However, for 10 tumors, a confident distinction between overexpression and wild-type immunostaining was not possible ("indeterminates"). Quantitative analysis on digital images (i) using QuPath to determine the percentage of immunopositive cells and (ii) WEKA segmentation to obtain an index that quantified the intensities of tumor cells' nuclear immunostaining showed a continuous distribution of the data, explaining failure of assessment by pattern recognition in some cases. Quantitative data were then used to define cutoffs by receiver operator curve analysis, which allowed for predicting the mutational status of the TP53 gene with sensitivities of 0.89 and 0.95 for the 2 methods, respectively, and specificities of 0.81 for both. In conclusion, by a dedicated approach, TP53 immunohistochemistry works well as a surrogate method for molecular studies. Considering the potential predictive role of TP53 gene mutations in chemotherapy decisions, TP53 immunohistochemistry may be of value alongside with molecular gene studies, possibly even across different cancers.

anti-PD-L1 treatment effects tumour responses in a significant number of cases across various types of cancer and often is substantial (1). This has fostered considerable optimism in the field of cancer immunotherapy. Even at this juncture, surgical pathologists are requested to provide immunohistochemical assessment of PD-L1 expression in biopsy or surgical resection specimens, as for malignant melanoma, bladder cancer, and non-small cell lung cancer, at least, expression of PD-L1 appears to be predictive of therapeutic effects (1). In these types of cancer, PD-L1 immunohistochemistry decorates intra- and peritumoural immune cells as well as the tumour cells themselves to varying degrees, making it amenable to scoring; as would be expected for a cell surface receptor the immunostaining pattern is membranous. Effective anti-PD-L1 treatment of colorectal cancer patients in a phase 2 study was shown to be largely restricted to patients with high-degree microsatellite instable tumours (2) which by PD-L1 immunohistochemistry are reported in many cases as "peppered" with large numbers of PD-L1 positive immune cells, macrophages in particular (3), whereas the tumour cells themselves often are negative. This article is protected by copyright. All rights reserved.

Progression of solid cancers, colorectal carcinomas among them, from their primaries to metastatic lesions traditionally is thought to proceed by a stepwise acquisition of and selection for genomic aberrations. To test if patterns of genomic aberrations would be consistent with this model, we studied 10 colorectal carcinoma primary-metastasis pairs, 9 with 1 liver metastasis each and 1 with 2 metastases. Next-generation targeted sequencing (50-gene panel) with samples obtained from different regions of the primaries and their metastases demonstrated 1-11 gene mutations per lesion. But only in 2 tumors were there seen mutations in all samples from the metastasis and not any of the primaries (BRAFD594N and SMARCB1R377C mutation, respectively). However, allelotyping the multiregional samples with polymorphous microsatellite markers (17p13.1, D9S942, D9S1748, D5S346, D5S1385) and DNA methylation studies with a marker panel (MLH1, CDNK2A, NEUROG1, CRABP1, CACNA1G, IGF2, RUNX3, SOCS1) showed remarkably "insular" genomic aberrations in all cases for at least some of the analyses. The marked preponderance of mutations shared by the primaries and their metastases throughout the lesions over mutations private to metastases suggests that, at least in many cases, colorectal carcinomas might be endowed with a mutational load sufficient for fully fledged metastases even at a very early stage ("born bad"). But the very focal allelic imbalances and methylations observed here, hypothetically, could play a role in clinically metastasizing disease, a process of years rather than months and very much a matter of tumor-host interactions when tumor cells adapt to the host microenvironment.

Introduction C-kit overexpression has previously been described in chromophobe renal cell carcinoma (cpRCC) and renal oncocytoma (RO). However, so far no KIT mutations have been found. The objective of our study was to analyse c-kit in a large cohort of renal tumors and to perform KIT mutation analysis in a subset cpRCC and RO cases with overexpression of c-kit. Materials and methods We studied the immunohistochemical expression of c-kit on tissue microarrays containing formalin-fixed, paraffin-embedded samples of 948 patients with renal tumors. CpRCC and RO cases with c-kit overexpression ( n = 23) were analyzed for KIT mutations in exons 9, 11, 13, 14, 15, and 17. Results Expression of c-kit was found in 6/642 (0.9%) clear cell RCC, 3/154 (1.9%) papillary RCC, 54/69 (78.3%) cpRCC, 37/45 (82.2%) RO and 2/30 (6.7%) of other unclassified tumor types. In none of the RO and cpRCC cases analyzed, a KIT gene mutation was found. Conclusion C-kit expression is found in the majority of cpRCC and RO, but these tumors do not harbor the usual c-kit activating mutations. This may have implications for the use of tyrosine kinase inhibitors in patients with advanced cpRCC and c-kit expression.

Potatoes are a promising system for industrial production of the biopolymer cyanophycin as a second compound in addition to starch. To assess the efficiency in the field, we analysed the stability of the system, specifically its sensitivity to environmental factors. Field and greenhouse trials with transgenic potatoes (two independent events) were carried out for three years. The influence of environmental factors was measured and target compounds in the transgenic plants (cyanophycin, amino acids) were analysed for differences to control plants. Furthermore, non-target parameters (starch content, number, weight and size of tubers) were analysed for equivalence with control plants. The huge amount of data received was handled using modern statistical approaches to model the correlation between influencing environmental factors (year of cultivation, nitrogen fertilization, origin of plants, greenhouse or field cultivation) and key components (starch, amino acids, cyanophycin) and agronomic characteristics. General linear models were used for modelling, and standard effect sizes were applied to compare conventional and genetically modified plants. Altogether, the field trials prove that significant cyanophycin production is possible without reduction of starch content. Non-target compound composition seems to be equivalent under varying environmental conditions. Additionally, a quick test to measure cyanophycin content gives similar results compared to the extensive enzymatic test. This work facilitates the commercial cultivation of cyanophycin potatoes.

Cyanophycin (CP) is a proteinogenic polymer that can be substituted for petroleum in the production of plastic compounds and can also serve as a source of valuable dietary supplements. However, because there is no economically feasible system for large-scale industrial production, its application is limited. In order to develop a low-input system, CP-synthesis was established in the two commercial Nicotiana tabacum (N. tabacum) cultivars 'Badischer Geudertheimer' (BG) and 'Virginia Golta' (VG), by introducing the cyanophycin-synthetase gene from Thermosynecchococcus elongatus BP-1 (CphATe) either via crossbreeding with transgenic N. tabacum cv. Petit Havana SR1 (PH) T2 individual 51-3-2 or by agrobacterium-mediated transformation. Both in F1 hybrids (max. 9.4% CP/DW) and T0 transformants (max. 8.8% CP/DW), a substantial increase in CP content was achieved in leaf tissue, compared to a maximum of 1.7% CP/DW in PH T0 transformants of Huhns et al. (2008). In BG CP, yields were homogenous and there was no substantial difference in the variation of the CP content between primary transformants (T0), clones of T0 individuals, T1 siblings and F1 siblings of hybrids. Therefore, BG meets the requirements for establishing a master seed bank for continuous and reliable CP-production. In addition, it was shown that the polymer is not only stable in planta but also during silage, which simplifies storage of the harvest prior to isolation of CP.

A chimeric cyanophycin synthetase gene composed of the cph A Te coding region from the cyanobacterium Thermosynechococcus elongatus BP-1, the constitutive 35S promoter and the plastid targeting sequence of the integral photosystem II protein PsbY was transferred to the tobacco variety Petit Havanna SRI and the commercial potato starch production variety Albatros. The resulting constitutive expression of cyanophycin synthetase leads to polymer contents in potato leaf chloroplasts of up to 35 mg/g dry weight and in tuber amyloplasts of up to 9 mg/g dry weight. Both transgenic tobacco and potato were used for the development of isolation methods applicable for large-scale extraction of the polymer. Two different procedures were developed which yielded polymer samples of 80 and 90% purity, respectively

The production of biodegradable polymers that can be used to substitute petrochemical compounds in commercial products in transgenic plants is an important challenge for plant biotechnology. Nevertheless, it is often accompanied by reduced plant fitness. To decrease the phenotypic abnormalities of the sprout and to increase polymer production, we restricted cyanophycin accumulation to the potato tubers by using the cyanophycin synthetase gene (cphA(Te)) from Thermosynechococcus elongatus BP-1, which is under the control of the tuber-specific class 1 promoter (B33). Tuber-specific cytosolic (pB33-cphA(Te)) as well as tuber-specific plastidic (pB33-PsbY-cphA(Te)) expression resulted in significant polymer accumulation solely in the tubers. In plants transformed with pB33-cphA(Te), both cyanophycin synthetase and cyanophycin were detected in the cytoplasm leading to an increase up to 2.3% cyanophycin of dry weight and resulting in small and deformed tubers. In B33-PsbY-cphA(Te) tubers, cyanophycin synthetase and cyanophycin were exclusively found in amyloplasts leading to a cyanophycin accumulation up to 7.5% of dry weight. These tubers were normal in size, some clones showed reduced tuber yield and sometimes exhibited brown sunken staining starting at tubers navel. During a storage period over of 32 weeks of one selected clone, the cyanophycin content was stable in B33-PsbY-cphA(Te) tubers but the stress symptoms increased. However, all tubers were able to germinate. Nitrogen fertilization in the greenhouse led not to an increased cyanophycin yield, slightly reduced protein content, decreased starch content, and changes in the amounts of bound and free arginine and aspartate, as compared with control tubers were observed.

Summary The production of biodegradable polymers in transgenic plants is an important challenge in plant biotechnology; nevertheless, it is often accompanied by reduced plant fitness. In order to decrease the phenotypic abnormalities caused by cytosolic production of the biodegradable polymer cyanophycin, and to increase polymer accumulation, four translocation pathway signal sequences for import into chloroplasts were individually fused to the coding region of the cyanophycin synthetase gene (cphATe) of Thermosynechococcus elongatus BP-1, resulting in the constructs pRieske-cphATe, pCP24-cphATe, pFNR-cphATe and pPsbY-cphATe. These constructs were expressed in Nicotiana tabacum var. Petit Havana SRI under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter. Three of the four constructs led to polymer production. However, only the construct pPsbY-cphATe led to cyanophycin accumulation exclusively in chloroplasts. In plants transformed with the pCP24-cphATe and pFNR-cphATe constructs, water-soluble and water-insoluble forms of cyanophycin were only located in the cytoplasm, which resulted in phenotypic changes similar to those observed in plants transformed with constructs lacking a targeting sequence. The plants transformed with pPsbY-cphATe produced predominantly the water-insoluble form of cyanophycin. The polymer accumulated to up to 1.7% of dry matter in primary (T0) transformants. Specific T2 plants produced 6.8% of dry weight as cyanophycin, which is more than five-fold higher than the previously published value. Although all lines tested were fertile, the progeny of the highest cyanophycin-producing line showed reduced seed production compared with control plants.

Benign and malignant salivary gland tumours are clinically heterogeneous and show different histology. Little is known about the role of human herpes virus 8 (HHV-8), Epstein-Barr virus (EBV), and human papillomavirus (HPV) infection in salivary gland neoplasms. We investigated the presence of the three viruses in formalin-fixed, paraffin-embedded tissue samples in a cohort of 200 different salivary gland tumours. We performed EBV-LMP-1 and HHV-8 and p16 immunohistochemistry, a specific chip based hybridization assay for detection and typing of HPV and a chromogenic in situ hybridization for EBV analysis. Only one case, a polymorphic low-grade carcinoma, showed HHV-8 expression and one lymphoepithelial carcinoma was infected by EBV. In 17 cases (9%) moderate or strong nuclear and cytoplasmic p16 expression was detected. The HPV type was investigated in all of these cases and additionally in 8 Warthin’s tumours. In 19 cases HPV type 16 was detected, mostly in Warthin’s tumour, adenoid cystic carcinoma, and adenocarcinoma NOS. We concluded that HHV-8 infection and EBV infection are not associated with salivary gland cancer, but HPV infection may play a role in these tumour entities.

// Philippe Perot 1, 7, * , Christina Susanne Mullins 1, 2, * , Magali Naville 3 , Cedric Bressan 1 , Maja Huhns 4 , Michael Gock 5 , Florian Kuhn 5 , Jean-Nicolas Volff 3 , Veronique Trillet-Lenoir 2 , Michael Linnebacher 6, * , Francois Mallet 1, * 1 Cancer Biomarkers Research Group, Joint Unit Hospices Civils de Lyon, bioMerieux, Centre Hospitalier Lyon Sud, Pierre Benite, France 2 Centre d’Investigation des Therapeutiques en Oncologie et Hematologie, EMR 3738 Lyon Claude Bernard University, Institut de Cancerologie des Hospices Civils de Lyon, France 3 Institut de Genomique Fonctionnelle de Lyon, Ecole Normale Superieure de Lyon, CNRS/Universite Lyon I, Lyon, France 4 Institute of Pathology, University Medicine Rostock, Rostock, Germany 5 Department of General, Thoracic, Vascular and Transplantation Surgery, University Medicine Rostock, Rostock, Germany 6 Department of General Surgery, Molecular Oncology and Immunotherapy, University Medicine Rostock, Rostock, Germany 7 Current address: Institut Pasteur, Laboratory for Pathogen Discovery, Paris, France * These authors have contributed equally to this work Correspondence to: Michael Linnebacher, e-mail: michael.linnebacher@med.uni-rostock.de Francois Mallet, e-mail: francois.mallet@biomerieux.com Keywords: colorectal cancer, HERV-H, microsatellite instability, qRT-PCR, biomarker Received: June 28, 2015 Accepted: October 13, 2015 Published: October 23, 2015 ABSTRACT Background: Expression of the human endogenous retrovirus (HERV)-H family has been associated with colorectal carcinomas (CRC), yet no individual HERV-H locus expression has been thoroughly correlated with clinical data. Here, we characterized HERV-H reactivations in clinical CRC samples by integrating expression profiles, molecular patterns and clinical data. Expression of relevant HERV-H sequences was analyzed by qRT-PCR on two well-defined clinical cohorts ( n = 139 pairs of tumor and adjacent normal colon tissue) including samples from adenomas ( n = 21) and liver metastases ( n = 16). Correlations with clinical and molecular data were assessed. Results: CRC specific HERV-H sequences were validated and found expressed throughout CRC disease progression. Correlations between HERV-H expression and lymph node invasion of tumor cells ( p = 0.0006) as well as microsatellite instable tumors ( p < 0.0001) were established. No association with regard to age, tumor localization, grading or common mutations became apparent. Interestingly, CRC expressed elements belonged to specific young HERV-H subfamilies and their 5′ LTR often presented active histone marks. Conclusion: These results suggest a functional role of HERV-H sequences in colorectal carcinogenesis. The pronounced connection with microsatellite instability warrants a more detailed investigation. Thus, HERV-H sequences in addition to tumor specific mutations may represent clinically relevant, truly CRC specific markers for diagnostic, prognostic and therapeutic purposes.

Summary The production of biodegradable polymers in transgenic plants in order to replace petrochemical compounds is an important challenge for plant biotechnology. Polyaspartate, a biodegradable substitute for polycarboxylates, is the backbone of the cyanobacterial storage material cyanophycin. Cyanophycin, a copolymer of l-aspartic acid and l-arginine, is produced via non-ribosomal polypeptide biosynthesis by the enzyme cyanophycin synthetase. A gene from Thermosynechococcus elongatus BP-1 encoding cyanophycin synthetase has been expressed constitutively in tobacco and potato. The presence of the transgene-encoded messenger RNA (mRNA) correlated with changes in leaf morphology and decelerated growth. Such transgenic plants were found to produce up to 1.1% dry weight of a polymer with cyanophycin-like properties. Aggregated material, able to bind a specific cyanophycin antibody, was detected in the cytoplasm and the nucleus of the transgenic plants.

Low-passage cancer cell lines are versatile tools to study tumor cell biology. Here, we have employed four such cell lines, established from primary tumors of colorectal cancer (CRC) patients, to evaluate effects of the small molecule kinase inhibitors (SMI) vemurafenib, trametinib, perifosine, and regorafenib in anin vitrosetting. The mutantBRAF(V600E/V600K) inhibitor vemurafenib, but also the MEK1/2 inhibitor trametinib efficiently inhibited DNA synthesis, signaling through ERK1/2 and expression of genes downstream of ERK1/2 inBRAFmutant cells only. In case of the AKT inhibitor perifosine, three cell lines showed a high or intermediate responsiveness to the drug while one cell line was resistant. The multikinase inhibitor regorafenib inhibited proliferation of all CRC lines with similar efficiency and independent of the presence or absence ofKRAS, BRAF, PIK3CA, andTP53mutations. Regorafenib action was associated with broad-range inhibitory effects at the level of gene expression but not with a general inhibition of AKT or MEK/ERK signaling. In vemurafenib-sensitive cells, the antiproliferative effect of vemurafenib was enhanced by the other SMI. Together, our results provide insights into the determinants of SMI efficiencies in CRC cells and encourage the further use of low-passage CRC cell lines as preclinical models.

Composite potato plants offer an extremely fast, effective and reliable system for studies on gene functions in roots using antisense or inverted-repeat but not sense constructs for gene inactivation. Composite plants, with transgenic roots on a non-transgenic shoot, can be obtained by shoot explant transformation with Agrobacterium rhizogenes. The aim of this study was to generate composite potato plants (Solanum tuberosum) to be used as a model system in future studies on root-pathogen interactions and gene silencing in the roots. The proportion of transgenic roots among the roots induced was high (80–100 %) in the four potato cultivars tested (Albatros, Desiree, Sabina and Saturna). No wild-type adventitious roots were formed at mock inoculation site. All strains of A. rhizogenes tested induced phenotypically normal roots which, however, showed a reduced response to cytokinin as compared with non-transgenic roots. Nevertheless, both types of roots were infected to a similar high rate with the zoospores of Spongospora subterranea, a soilborne potato pathogen. The transgenic roots of composite potato plants expressed significantly higher amounts of β-glucuronidase (GUS) than the roots of a GUS-transgenic potato line event. Silencing of the uidA transgene (GUS) was tested by inducing roots on the GUS-transgenic cv. Albatros event with strains of A. rhizogenes over-expressing either the uidA sense or antisense transcripts, or inverted-repeat or hairpin uidA RNA. The three last mentioned constructs caused 2.5–4.0 fold reduction in the uidA mRNA expression. In contrast, over-expression of uidA resulted in over 3-fold increase in the uidA mRNA and GUS expression, indicating that sense-mediated silencing (co-suppression) was not functional in roots. The results suggest that composite plants offer a useful experimental system for potato research, which has gained little previous attention.

INTRODUCTION:A substantial part of the human genome originates from transposable elements, remnants of ancient retroviral infections. Roughly 8% of the human genome consists of about 400,000 LTR elements including human endogenous retrovirus (HERV) sequences. Mainly, the interplay between epigenetic and post-transcriptional mechanisms is thought to silence HERV expression in most physiological contexts. Interestingly, aberrant reactivation of several HERV-H loci appears specific to colorectal carcinoma (CRC). RESULTS:The expression of HERV-H Gag proteins (Gag-H) was assessed using novel monoclonal mouse anti Gag-H antibodies. In a flow cytometry screen four antibody clones were tested on a panel of primary CRC cell lines and the most well performing ones were subsequently validated in western blot analysis. Finally, Gag-H protein expression was analyzed by immune histology on cell line cytospins and on clinical samples. There, we found a heterogeneous staining pattern with no background staining of endothelial, stromal and infiltrating immune cells but diffuse staining of the cytoplasm for positive tumor and normal crypt cells of the colonic epithelium. CONCLUSION:Taken together, the Gag-H antibody clone(s) present a valuable tool for staining of cells with colonic origin and thus form the basis for future more detailed investigations. The observed Gag-H protein staining in colonic epithelium crypt cells demands profound analyses of a potential role for Gag-H in the normal physiology of the human gut.

The production of biodegradable polymers that can be used to substitute petrochemical compounds in commercial products in transgenic plants is an important challenge for plant biotechnology. Nevertheless, it is often accompanied by reduced plant fitness. To decrease the phenotypic abnormalities of the sprout and to increase polymer production, we restricted cyanophycin accumulation to the potato tubers by using the cyanophycin synthetase gene (cphA(Te)) from Thermosynechococcus elongatus BP-1, which is under the control of the tuber-specific class 1 promoter (B33). Tuber-specific cytosolic (pB33-cphA(Te)) as well as tuber-specific plastidic (pB33-PsbY-cphA(Te)) expression resulted in significant polymer accumulation solely in the tubers. In plants transformed with pB33-cphA(Te), both cyanophycin synthetase and cyanophycin were detected in the cytoplasm leading to an increase up to 2.3% cyanophycin of dry weight and resulting in small and deformed tubers. In B33-PsbY-cphA(Te) tubers, cyanophycin synthetase and cyanophycin were exclusively found in amyloplasts leading to a cyanophycin accumulation up to 7.5% of dry weight. These tubers were normal in size, some clones showed reduced tuber yield and sometimes exhibited brown sunken staining starting at tubers navel. During a storage period over of 32 weeks of one selected clone, the cyanophycin content was stable in B33-PsbY-cphA(Te) tubers but the stress symptoms increased. However, all tubers were able to germinate. Nitrogen fertilization in the greenhouse led not to an increased cyanophycin yield, slightly reduced protein content, decreased starch content, and changes in the amounts of bound and free arginine and aspartate, as compared with control tubers were observed.

The production of biodegradable polymers in transgenic plants is an important challenge in plant biotechnology; nevertheless, it is often accompanied by reduced plant fitness. In order to decrease the phenotypic abnormalities caused by cytosolic production of the biodegradable polymer cyanophycin, and to increase polymer accumulation, four translocation pathway signal sequences for import into chloroplasts were individually fused to the coding region of the cyanophycin synthetase gene (cphA(Te)) of Thermosynechococcus elongatus BP-1, resulting in the constructs pRieske-cphA(Te), pCP24-cphA(Te), pFNR-cphA(Te) and pPsbY-cphA(Te). These constructs were expressed in Nicotiana tabacum var. Petit Havana SRI under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter. Three of the four constructs led to polymer production. However, only the construct pPsbY-cphA(Te) led to cyanophycin accumulation exclusively in chloroplasts. In plants transformed with the pCP24-cphA(Te) and pFNR-cphA(Te) constructs, water-soluble and water-insoluble forms of cyanophycin were only located in the cytoplasm, which resulted in phenotypic changes similar to those observed in plants transformed with constructs lacking a targeting sequence. The plants transformed with pPsbY-cphA(Te) produced predominantly the water-insoluble form of cyanophycin. The polymer accumulated to up to 1.7% of dry matter in primary (T(0)) transformants. Specific T(2) plants produced 6.8% of dry weight as cyanophycin, which is more than five-fold higher than the previously published value. Although all lines tested were fertile, the progeny of the highest cyanophycin-producing line showed reduced seed production compared with control plants.

The production of biodegradable polymers in transgenic plants in order to replace petrochemical compounds is an important challenge for plant biotechnology. Polyaspartate, a biodegradable substitute for polycarboxylates, is the backbone of the cyanobacterial storage material cyanophycin. Cyanophycin, a copolymer of l-aspartic acid and l-arginine, is produced via non-ribosomal polypeptide biosynthesis by the enzyme cyanophycin synthetase. A gene from Thermosynechococcus elongatus BP-1 encoding cyanophycin synthetase has been expressed constitutively in tobacco and potato. The presence of the transgene-encoded messenger RNA (mRNA) correlated with changes in leaf morphology and decelerated growth. Such transgenic plants were found to produce up to 1.1% dry weight of a polymer with cyanophycin-like properties. Aggregated material, able to bind a specific cyanophycin antibody, was detected in the cytoplasm and the nucleus of the transgenic plants.