Plants belonging to the legume family develop nitrogen-fixing root nodules in symbiosis with bacteria commonly known as rhizobia. The legume host encodes all of the functions necessary to build the specialized symbiotic organ, the nodule, but the process is elicited by the bacteria. Molecular communication initiates the interaction, and signals, usually flavones, secreted by the legume root induce the bacteria to produce a lipochitin-oligosaccharide signal molecule (Nod-factor), which in turn triggers the plant organogenic process. An important determinant of bacterial host specificity is the structure of the Nod-factor, suggesting that a plant receptor is involved in signal perception and signal transduction initiating the plant developmental response. Here we describe the cloning of a putative Nod-factor receptor kinase gene (NFR5) from Lotus japonicus. NFR5 is essential for Nod-factor perception and encodes an unusual transmembrane serine/threonine receptor-like kinase required for the earliest detectable plant responses to bacteria and Nod-factor. The extracellular domain of the putative receptor has three modules with similarity to LysM domains known from peptidoglycan-binding proteins and chitinases. Together with an atypical kinase domain structure this characterizes an unusual receptor-like kinase.

The introduction of microarray techniques to cancer research brought great expectations for finding biomarkers that would improve patients’ treatment; however, the results of such studies are poorly reproducible and critical analyses of these methods are rare. In this study, we examined global gene expression in 97 ovarian cancer samples. Also, validation of results by quantitative RT-PCR was performed on 30 additional ovarian cancer samples. We carried out a number of systematic analyses in relation to several defined clinicopathological features. The main goal of our study was to delineate the molecular background of ovarian cancer chemoresistance and find biomarkers suitable for prediction of patients’ prognosis. We found that histological tumor type was the major source of variability in genes expression, except for serous and undifferentiated tumors that showed nearly identical profiles. Analysis of clinical endpoints [tumor response to chemotherapy, overall survival, disease-free survival (DFS)] brought results that were not confirmed by validation either on the same group or on the independent group of patients. CLASP1 was the only gene that was found to be important for DFS in the independent group, whereas in the preceding experiments it showed associations with other clinical endpoints and with BRCA1 gene mutation; thus, it may be worthy of further testing. Our results confirm that histological tumor type may be a strong confounding factor and we conclude that gene expression studies of ovarian carcinomas should be performed on histologically homogeneous groups. Among the reasons of poor reproducibility of statistical results may be the fact that despite relatively large patients’ group, in some analyses one has to compare small and unequal classes of samples. In addition, arbitrarily performed division of samples into classes compared may not always reflect their true biological diversity. And finally, we think that clinical endpoints of the tumor probably depend on subtle changes in many and, possibly, alternative molecular pathways, and such changes may be difficult to demonstrate.

Hypoxia is one of the most important features of the tumor microenvironment, exerting an adverse effect on tumor aggressiveness and patient prognosis. Two types of hypoxia may occur within the tumor mass, chronic (prolonged) and cycling (transient, intermittent) hypoxia. Cycling hypoxia has been shown to induce aggressive tumor cell phenotype and radioresistance more significantly than chronic hypoxia, though little is known about the molecular mechanisms underlying this phenomenon. The aim of this study was to delineate the molecular response to both types of hypoxia induced experimentally in tumor cells, with a focus on cycling hypoxia. We analyzed in vitro gene expression profile in three human cancer cell lines (melanoma, ovarian cancer, and prostate cancer) exposed to experimental chronic or transient hypoxia conditions. As expected, the cell-type specific variability in response to hypoxia was significant. However, the expression of 240 probe sets was altered in all 3 cell lines. We found that gene expression profiles induced by both types of hypoxia were qualitatively similar and strongly depend on the cell type. Cycling hypoxia altered the expression of fewer genes than chronic hypoxia (6,132 vs. 8,635 probe sets, FDR adjusted p<0.05), and with lower fold changes. However, the expression of some of these genes was significantly more affected by cycling hypoxia than by prolonged hypoxia, such as IL8, PLAU, and epidermal growth factor (EGF) pathway-related genes (AREG, HBEGF, and EPHA2). These transcripts were, in most cases, validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Our results indicate that experimental cycling hypoxia exerts similar, although less intense effects, on the examined cancer cell lines than its chronic counterpart. Nonetheless, we identified genes and molecular pathways that seem to be preferentially regulated by cyclic hypoxia.

The CRNDE gene seems to play an oncogenic role in cancers, though its exact function remains unknown. Here, we tried to assess its usefulness as a molecular prognostic marker in ovarian cancer. Based on results of our microarray studies, CRNDE transcripts were further analyzed by Real-Time qPCR-based profiling of their expression. The qPCR study was conducted with the use of personally designed TaqMan assays on 135 frozen tissue sections of ovarian carcinomas from patients treated with platinum compounds and either cyclophosphamide (PC, N = 32) or taxanes (TP, N = 103). Elevated levels of two different CRNDE transcripts were a negative prognostic factor; they increased the risk of death and recurrence in the group of patients treated with TP, but not PC (DNA-damaging agents only). Higher associations were found for overexpression of the short CRNDE splice variant (FJ466686): HR 6.072, 95% CI 1.814–20.32, p = 0.003 (the risk of death); HR 15.53, 95% CI 3.812–63.28, p < 0.001 (the risk of recurrence). Additionally, accumulation of the TP53 protein correlated with decreased expression of both CRNDE transcripts in tumor cells. Our results depict CRNDE as a potential marker of poor prognosis in women with ovarian carcinomas, and suggest that its significance depends on the therapeutic regimen used.

BackgroundElevated temperatures induce activation of the heat shock transcription factor 1 (HSF1) which in somatic cells leads to heat shock proteins synthesis and cytoprotection. However, in the male germ cells (spermatocytes) caspase-3 dependent apoptosis is induced upon HSF1 activation and spermatogenic cells are actively eliminated.ResultsTo elucidate a mechanism of such diverse HSF1 activity we carried out genome-wide transcriptional analysis in control and heat-shocked cells, either spermatocytes or hepatocytes. Additionally, to identify direct molecular targets of active HSF1 we used chromatin immunoprecipitation assay (ChIP) combined with promoter microarrays (ChIP on chip). Genes that are differently regulated after HSF1 binding during hyperthermia in both types of cells have been identified. Despite HSF1 binding to promoter sequences in both types of cells, strong up-regulation of Hsps and other genes typically activated by the heat shock was observed only in hepatocytes. In spermatocytes HSF1 binding correlates with transcriptional repression on a large scale. HSF1-bound and negatively regulated genes encode mainly for proteins required for cell division, involved in RNA processing and piRNA biogenesis.ConclusionsObserved suppression of the transcription could lead to genomic instability caused by meiotic recombination disturbances, which in turn might induce apoptosis of spermatogenic cells. We propose that HSF1-dependent induction of cell death is caused by the simultaneous repression of many genes required for spermatogenesis, which guarantees the elimination of cells damaged during heat shock. Such activity of HSF1 prevents transmission of damaged genetic material to the next generation.

Hypoxia is an important feature of tumor microenvironment, exerting far-reaching effects on cells and contributing to cancer progression. Previous studies have established substantial differences in hypoxia response between various cell lines. Investigating this phenomenon in melanoma cells contributes to a better understanding of cell lineage-specific hypoxia response and could point out novel hypoxia-regulated genes. We investigated transcriptional activity of B 16(F10) murine melanoma cells cultured for 24 h under hypoxic (nominal 1% O2, 15 samples including controls) and hypoxia-mimicking conditions (cobalt chloride, 100 or 200 microM, 6 samples including controls). Gene expression profiles were analyzed using MG-U74Av2 oligonucleotide microarrays. Data analysis revealed 2541 probesets (FDR <5%) for 1% oxygen experiment and 364 probesets (FDR <5%) for cobalt chloride, which showed differences in expression levels. Analysis of hypoxia-regulated genes (true hypoxia, 1% O2) by stringent Family-Wise Error Rate estimation indicated 454 significantly changed transcripts (p < 0.05). The most upregulated genes were Lgals3, Selenbpl, Nppb (more than ten-fold increase). We observed significant differences in expression levels of genes regulating glycolysis (Pfkp, Hk2, Aldo3, Eno2), apoptosis (Bnip3, Bnip31, Cdknla), transcription (Bhlhb2, Sap30, Atf3, Mxil), angiogenesis (Vegfa, Adm, Anxa2, Ctgf), adhesion (Pkp2, Itga4, Mcam), migration (Cnn2, Tmsb4x), and other processes. Both true hypoxia and hypoxia mimicry induced HIF-1-regulated genes. However, unsupervised analysis (Singular Value Decomposition) revealed distinct differences in gene expression between these two experimental conditions. Contrary to hypoxia, cobalt chloride caused suppression of gene expression rather than stimulation, especially concerning transcripts related to proliferation, immune response, DNA repair, and melanin biosynthesis.

Modern immunotherapy together with targeted therapy has revolutionized the treatment of advanced melanoma. Inhibition of immune checkpoints significantly improved the median overall survival and gave hope to many melanoma patients. However, this treatment has three serious drawbacks: high cost, serious side effects, and an effectiveness limited only to approximately 50% of patients. Some patients do not derive any or short-term benefit from this treatment due to primary or secondary resistance. The response to immunotherapy depends on many factors that fall into three main categories: those associated with melanoma cells, those linked to a tumor and its microenvironment, and those classified as individual ontogenic and physiological features of the patient. The first category comprises expression of PD-L1 and HLA proteins on melanoma cells as well as genetic/genomic metrics such as mutational load, (de)activation of specific signaling pathways and epigenetic factors. The second category is the inflammatory status of the tumor: “hot” versus “cold” (i.e., high versus low infiltration of immune cells). The third category comprises metabolome and single nucleotide polymorphisms of specific genes. Here we present up-to-date data on those biological factors influencing melanoma response to immunotherapy with a special focus on signaling pathways regulating the complex process of anti-tumor immune response. We also discuss their potential predictive capacity.

did not derive from high-versus low-grade serous carcinomas and were unrelated to histological (ovarian vs. fallopian) origin. Interestingly, there was considerable overlap between identified prognostic signature and a recently described invasion-associated signature related to stromal desmoplastic reaction. Several genes from this signature were validated by quantitative PCR; two of them-DSPG3 and LOX-were validated both in the initial and independent sets of samples and were significantly associated with OS and disease-free survival. Conclusions We distinguished two molecular subgroups of serous ovarian cancers characterized by distinct OS. Among differentially expressed genes, some may potentially be used as prognostic markers. In our opinion, unsupervised methods of microarray data analysis are more effective than supervised methods in identifying intrinsic, biologically sound sources of variability. Moreover, as histological type of the tumor is the greatest source of variability in ovarian cancer and may interfere with analyses of other features, it seems reasonable to use histologically homogeneous groups of tumors in microarray experiments.