An Increase in HSF1 Expression Directs Human Mammary Epithelial Cells toward a Mesenchymal Phenotype.

HSF1 is a well-known heat shock protein expression regulator in response to stress. It also regulates processes important for growth, development or tumorigenesis. We studied the HSF1 influence on the phenotype of non-tumorigenic human mammary epithelial (MCF10A and MCF12A) and several triple-negative breast cancer cell lines. MCF10A and MCF12A differ in terms of HSF1 levels, morphology, growth in Matrigel, expression of epithelial (CDH1) and mesenchymal (VIM) markers (MCF10A are epithelial cells; MCF12A resemble mesenchymal cells). HSF1 down-regulation led to a reduced proliferation rate and spheroid formation in Matrigel by MCF10A cells. However, it did not affect MCF12A proliferation but led to CDH1 up-regulation and the formation of better organized spheroids. HSF1 overexpression in MCF10A resulted in reduced CDH1 and increased VIM expression and the acquisition of elongated fibroblast-like morphology. The above-mentioned results suggest that elevated levels of HSF1 may direct mammary epithelial cells toward a mesenchymal phenotype, while a lowering of HSF1 could reverse the mesenchymal phenotype to an epithelial one. Therefore, HSF1 may be involved in the remodeling of mammary gland architecture over the female lifetime. Moreover, HSF1 levels positively correlated with the invasive phenotype of triple-negative breast cancer cells, and their growth was inhibited by the HSF1 inhibitor DTHIB.

Potential Biomarkers of Skin Melanoma Resistance to Targeted Therapy-Present State and Perspectives.

Melanoma is the most aggressive skin cancer, the number of which is increasing worldwide every year. It is completely curable in its early stage and fatal when spread to distant organs. In addition to new therapeutic strategies, biomarkers are an important element in the successful fight against this cancer. At present, biomarkers are mainly used in diagnostics. Some biological indicators also allow the estimation of the patient's prognosis. Still, predictive markers are underrepresented in clinics. Currently, the only such indicator is the presence of the V600E mutation in the BRAF gene in cancer cells, which qualifies the patient for therapy with inhibitors of the MAPK pathway. The identification of response markers is particularly important given primary and acquired resistance to targeted therapies. Reliable predictive tests would enable the selection of patients who would have the best chance of benefiting from treatment. Here, up-to-date knowledge about the most promising genetic and non-genetic resistance-related factors is described. These are alterations in MAPK, PI3K/AKT, and RB signaling pathways, e.g., due to mutations in NRAS, RAC1, MAP2K1, MAP2K2, and NF1, but also other changes activating these pathways, such as the overexpression of HGF or EGFR. Most of them are also potential therapeutic targets and this issue is also addressed here.

NGS Analysis of Liquid Biopsy (LB) and Formalin-Fixed Paraffin-Embedded (FFPE) Melanoma Samples Using Oncomine™ Pan-Cancer Cell-Free Assay.

Next-generation sequencing (NGS) in liquid biopsies may contribute to the diagnosis, monitoring, and personalized therapy of cancer through the real-time detection of a tumor's genetic profile. There are a few NGS platforms offering high-sensitivity sequencing of cell-free DNA (cfDNA) samples. The aim of this study was to evaluate the Ion AmpliSeq HD Technology for targeted sequencing of tumor and liquid biopsy samples from patients with fourth-stage melanoma. Sequencing of 30 samples (FFPE tumor and liquid biopsy) derived from 14 patients using the Oncomine™ Pan-Cancer Cell-Free Assay was performed. The analysis revealed high concordance between the qPCR and NGS results of the BRAF mutation in FFPE samples (91%), as well as between the FFPE and liquid biopsy samples (91%). The plasma-tumor concordance of the non-BRAF mutations was 28%. A total of 17 pathogenic variants in 14 genes (from 52-gene panel), including TP53, CTNNB1, CCND1, MET, MAP2K1, and GNAS, were identified, with the CTNNB1S45F variant being the most frequent. A positive correlation between the LDH level and cfDNA concentration as well as negative correlation between the LDH level and time to progression was confirmed in a 22-patient cohort. The analysis showed both the potential and limitations of liquid biopsy genetic profiling using HD technology and the Ion Torrent platform.

Evaluation of the Role of ITGBL1 in Ovarian Cancer.

In our previous microarray study we identified two subgroups of high-grade serous ovarian cancers with distinct gene expression and survival. Among differentially expressed genes was an Integrin beta-like 1 (ITGBL1), coding for a poorly characterized protein comprised of ten EGF-like repeats. Here, we have analyzed the influence of ITGBL1 on the phenotype of ovarian cancer (OC) cells. We analyzed expression of four putative ITGBL1 mRNA isoforms in five OC cell lines. OAW42 and SKOV3, having the lowest level of any ITGBL1 mRNA, were chosen to produce ITGBL1-overexpressing variants. In these cells, abundant ITGBL1 mRNA expression could be detected by RT-PCR. Immunodetection was successful only in the culture media, suggesting that ITGBL1 is efficiently secreted. We found that ITGBL1 overexpression affected cellular adhesion, migration and invasiveness, while it had no effect on proliferation rate and the cell cycle. ITGBL1-overexpressing cells were significantly more resistant to cisplatin and paclitaxel, major drugs used in OC treatment. Global gene expression analysis revealed that signaling pathways affected by ITGBL1 overexpression were mostly those related to extracellular matrix organization and function, integrin signaling, focal adhesion, cellular communication and motility; these results were consistent with the findings of our functional studies. Overall, our results indicate that higher expression of ITGBL1 in OC is associated with features that may worsen clinical course of the disease.

Biological Factors behind Melanoma Response to Immune Checkpoint Inhibitors.

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.

Genetic Profiling of Advanced Melanoma: Candidate Mutations for Predicting Sensitivity and Resistance to Targeted Therapy.

BACKGROUND: Molecularly targeted therapy has revolutionized the treatment of advanced melanoma. However, despite its high efficiency, a majority of patients experience relapse within 1 year of treatment because of acquired resistance, and approximately 10-25% patients gain no benefit from these agents owing to intrinsic resistance. This is mainly caused by the genetic heterogeneity of melanoma cells. OBJECTIVE: We aimed to validate the predictive significance of selected genes in advanced melanoma patients before treatment with BRAF/MEK inhibitors. PATIENTS AND METHODS: Archival DNA derived from 37 formalin-fixed paraffin-embedded pre-treatment advanced melanoma samples of patients treated with targeted therapy was used for next-generation sequencing analysis using the Ion Torrent platform. The AmpliSeq Custom Panel comprised coding sequences or hot spots of 23 melanoma genes: ATM, BRAF, CDK4, CDKN2A, CTNNB1, EGFR, HOXD8, HRAS, IDH1, KIT, KRAS, MAP3K8, MAP2K1, MAP2K2, MITF, MYC, NF1, NRAS, PAX5, PIK3R1, PTEN, RAC1, and RB1. The sequences were evaluated for genomic alterations and further validated using Sanger sequencing. RESULTS: Our analysis revealed non-BRAF genetic alterations in 28 out of 37 samples (75.7%). Genetic changes were identified in PTEN, CDK4, CDKN2A, CTNNB1, EGFR, HOXD8, HRAS, KIT, MAP2K1, MAP2K2, MITF, MYC, NF1, PAX5, RAC1, and RB1. Fifteen known pathogenic mutations (single nucleotide variants or indels) and 11 variants of unknown significance were detected. Statistical analysis revealed an association between the presence of pathogenic mutations and time to progression during treatment with combination therapy. CONCLUSIONS: Pathogenic mutations identified by gene panel sequencing have potential predictive value for targeted therapy of melanoma and are worth further validation in a larger series of cases. The role of some known mutations (e.g. CDK4R24, PTEN c.801 + 1G > A, CTNNB1S45F) as well as variants of unknown significance identified in this study (e.g. MITFR316K, KITG498S) in the generation of resistance to BRAF/MEK inhibitors should be further investigated.

Prognostic significance of Epstein-Barr virus viral load in patients with T1-T2 nasopharyngeal cancer.

Nasopharyngeal cancer (NPC) is highly prevalent in southern Chinese populations but it is rare in most parts of the world. A few studies were performed in nonendemic regions of the world, and suggested the prognostic value of Epstein-Barr virus (EBV) DNA load in blood. In this study, EBV DNA presence and viral load (VL) level in the blood of patients with NPC in Polish population were presented. In addition, its prognostic value for locoregional control among other clinicopathological features was evaluated. Patients with carcinoma of the nasopharynx treated definitively with radiotherapy or radiochemotherapy were included in the study. Real-time polymerase chain reaction was performed for quantitating of EBV DNA in plasma. Among patients with NPC, 51% (22 of 43) were classified as EBV-positive with the mean of the VL of 4934 ± 8693 copies/mL. Multiple regression analysis between log EBV DNA VL and clinical parameters revealed that the most important factors increasing the VLs were advanced N disease together with no-smoking status and advanced T tumors. Multivariate Cox regression analysis revealed that T3-T4 tumors were an independent prognostic factor for poor locoregional control. Analysis for the subgroup of patients with T1-T2 tumors showed that T1-T2 EBV-negative patients had better locoregional control compared with T1-T2 EBV-positive, though without statistical significance. In conclusion, it seems that EBV DNA determination may have an important role in diagnostics of patients with NPC with T1-T2 tumors indicating a subgroup with poorer prognosis, though it needs to be proven on a larger cohort.

Molecular background of skin melanoma development and progression: therapeutic implications.

Melanoma is the most aggressive skin cancer with an increasing number of cases worldwide and curable mostly in its early stage. The improvement in patients' survival in advanced melanoma has been achieved only recently, due to development of new biological drugs for targeted therapies and immunotherapy. Further progress in the treatment of melanoma is clearly dependent on the better understanding of its complex biology. This review describes the most important molecular mechanisms and genetic events underlying skin melanoma development and progression, depicts the way of action of newly developed drugs and indicates new potential therapeutic targets.

Establishment and Characterization of the Novel High-Grade Serous Ovarian Cancer Cell Line OVPA8.

High-grade serous ovarian carcinoma (HGSOC) is the most frequent histological type of ovarian cancer and the one with worst prognosis. Unfortunately, the majority of established ovarian cancer cell lines which are used in the research have unclear histological origin and probably do not represent HGSOC. Thus, new and reliable models of HGSOC are needed. Ascitic fluid from a patient with recurrent HGSOC was used to establish a stable cancer cell line. Cells were characterized by cytogenetic karyotyping and short tandem repeat (STR) profiling. New generation sequencing was applied to test for hot-spot mutations in 50 cancer-associated genes and fluorescence in situ hybridization (FISH) analysis was used to check for TP53 status. Cells were analyzed for expression of several marker genes/proteins by reverse-transcription polymerase chain reaction (RT-PCR), fluorescence-activated cell sorting (FACS), and immunocytochemistry (ICC). Functional tests were performed to compare OVPA8 cells with five commercially available and frequently used ovarian cancer cell lines: SKOV3, A2780, OVCAR3, ES2, and OAW42. Our newly-established OVPA8 cell line shows morphologic and genetic features consistent with HGSOC, such as epithelial morphology, multiple chromosomal aberrations, TP53 mutation, BRCA1 mutation, and loss of one copy of BRCA2. The OVPA8 line has a stable STR profile. Cells are positive for EpCAM, CK19, and CD44; they have relatively low plating efficiency/ability to form spheroids, a low migration rate, and intermediate invasiveness in matrigel, as compared to other ovarian cancer lines. OVPA8 is sensitive to paclitaxel and resistant to cisplatin. We also tested two FGFR inhibitors; OVPA8 cells were resistant to AZD4547 (AstraZeneca, London, UK), but sensitive to the new inhibitor CPL304-110-01 (Celon Pharma, Lomianki/Kielpin, Poland). We have established and characterized a novel cell line, OVPA8, which can be a valuable preclinical model for studies on high-grade serous ovarian cancer.

Bis-anthracycline WP760 abrogates melanoma cell growth by transcription inhibition, p53 activation and IGF1R downregulation.

Anthracycline chemotherapeutics, e.g. doxorubicin and daunorubicin, are active against a broad spectrum of cancers. Their cytotoxicity is mainly attributed to DNA intercalation, interference with topoisomerase activity, and induction of double-stranded DNA breaks. Since modification of anthracyclines can profoundly affect their pharmacological properties we attempted to elucidate the mechanism of action, and identify possible molecular targets, of bis-anthracycline WP760 which previously demonstrated anti-melanoma activity at low nanomolar concentrations. We studied the effect of WP760 on several human melanoma cell lines derived from tumors in various development stages and having different genetic backgrounds. WP760 inhibited cell proliferation (IC50 = 1-99 nM), impaired clonogenic cell survival (100 nM), and inhibited spheroid growth (≥300 nM). WP760 did not induce double-stranded DNA breaks but strongly inhibited global transcription. Moreover, WP760 caused nucleolar stress and led to activation of the p53 pathway. PCR array analysis showed that WP760 suppressed transcription of ten genes (ABCC1, MTOR, IGF1R, EGFR, GRB2, PRKCA, PRKCE, HDAC4, TXNRD1, AKT1) associated with, inter alia, cytoprotective mechanisms initiated in cancer cells during chemotherapy. Furthermore, WP760 downregulated IGF1R and upregulated PLK2 expression in most of the tested melanoma cell lines. These results suggest that WP760 exerts anti-melanoma activity by targeting global transcription and activation of the p53 pathway and could become suitable as an effective therapeutic agent.

Unsupervised analysis reveals two molecular subgroups of serous ovarian cancer with distinct gene expression profiles and survival.

PURPOSE: Ovarian cancer is typically diagnosed at late stages, and thus, patients' prognosis is poor. Improvement in treatment outcomes depends, at least partly, on better understanding of ovarian cancer biology and finding new molecular markers and therapeutic targets. METHODS: An unsupervised method of data analysis, singular value decomposition, was applied to analyze microarray data from 101 ovarian cancer samples; then, selected genes were validated by quantitative PCR. RESULTS: We found that the major factor influencing gene expression in ovarian cancer was tumor histological type. The next major source of variability was traced to a set of genes mainly associated with extracellular matrix, cell motility, adhesion, and immunological response. Hierarchical clustering based on the expression of these genes revealed two clusters of ovarian cancers with different molecular profiles and distinct overall survival (OS). Patients with higher expression of these genes had shorter OS than those with lower expression. The two clusters 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.

The putative oncogene, CRNDE, is a negative prognostic factor in ovarian cancer patients.

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.

Global gene expression profiling in three tumor cell lines subjected to experimental cycling and chronic hypoxia.

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.

Gene expression analysis in ovarian cancer - faults and hints from DNA microarray study.

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.

Impact of heat shock transcription factor 1 on global gene expression profiles in cells which induce either cytoprotective or pro-apoptotic response following hyperthermia.

BACKGROUND: Elevated 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. RESULTS: To 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. CONCLUSIONS: Observed 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.

[Role of tumor microenvironment in the formation and progression of skin melanoma]. / Rola mikrosrodowiska nowotworowego w powstaniu i progresji czerniaka skóry.

Melanoma develops from melanocytes localized mainly in skin, to a lesser extent in uvea and mucosal tissue. It is one of the most aggressive tumors, resistant to standard therapies. It is caused mainly by UV radiation. Approximately 10% of melanomas have a familial background. Transformation of melanocytes is driven mainly by mutations in genes coding for proteins regulating proliferation, cell growth, cell cycle, and death. Although genetic changes are indisputable causes of melanoma formation, the key role of the skin microenvironment is becoming more and more acknowledged. Normal cells of the skin, such as keratinocytes, fibroblasts, endothelial, and immune cells, alongside melanocytes constitute a special microenvironment in which disturbed homeostasis may facilitate melanoma development. The process of transformation is initiated by aberrant interaction of keratinocytes and melanocytes. Proliferation and invasive growth of the developing neoplasm may be further facilitated by myofibroblasts as well as macrophages residing within the tumor. They are able to stimulate angiogenesis and suppress the immune response. Macrophages may, additionally, create aggressive hybrid cells through fusion with melanoma cells. Indispensable in the process of metastasis are endothelial cells which co-form blood vessels, the main routes of tumor cells' seeding. All of the aforementioned processes take place in a tumor growth-promoting atmosphere of low oxygen tension in the skin, further decreasing during tumor growth. Probably the tumor growth-promoting microenvironment is one of the main factors responsible for melanoma aggressiveness.

Melanoma-associated genes, MXI1, FN1, and NME1, are hypoxia responsive in murine and human melanoma cells.

Hypoxia can influence aggressiveness of melanoma by inducing specific gene expression profiles. In our previous microarray study, we identified more than 430 hypoxia-responsive genes in the B16-F10 murine melanoma cell line in vitro. Of the genes identified, seven genes: galectin 3 (Lgals3), melanoma cell adhesion molecule (Mcam), fibronectin 1 (Fn1), signal transducer and activator of transcription 3 (Stat3), microphthalmia-associated transcription factor (Mitf), max interacting protein 1 (Max1), and non-metastatic cells 1, protein (NM23A) expressed in (Nme1) are known to be associated with melanoma, but have not yet been reported as being regulated by hypoxia in human melanoma cells. In this study, we investigated whether the expression of these genes is modulated by hypoxia in microdissected areas of experimental B16-F10 tumors in vivo, as well as in commercially available human melanoma cell lines (WM35, WM1552C, WM793B, WM278, 1205Lu, and 451Lu) exposed to hypoxic conditions in vitro. Our analysis revealed significant agreement between the in-vitro and in-vivo results showing that all genes except Mitf were hypoxia regulated in the oxygen-deprived tumor regions (P<0.05). In contrast, three genes (NME1, MXI1 and FN1) proved to be hypoxia regulated in both human and mouse melanoma cells (P<0.05). Our results link these genes, for the first time, with hypoxic microenvironment of melanoma and imply that the widely used B16-F10 melanoma experimental tumor model could be a convenient research tool for further investigation of their role in the development and course of this malignancy.

Gene expression profile of B 16(F10) murine melanoma cells exposed to hypoxic conditions in vitro.

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.

A receptor kinase gene of the LysM type is involved in legume perception of rhizobial signals.

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.

A genetic linkage map of the model legume Lotus japonicus and strategies for fast mapping of new loci.

A genetic map for the model legume Lotus japonicus has been developed. The F(2) mapping population was established from an interspecific cross between L. japonicus and L. filicaulis. A high level of DNA polymorphism between these parents was the source of markers for linkage analysis and the map is based on a framework of amplified fragment length polymorphism (AFLP) markers. Additional markers were generated by restriction fragment length polymorphism (RFLP) and sequence-specific PCR. A total of 524 AFLP markers, 3 RAPD markers, 39 gene-specific markers, 33 microsatellite markers, and six recessive symbiotic mutant loci were mapped. This genetic map consists of six linkage groups corresponding to the six chromosomes in L. japonicus. Fluorescent in situ hybridization (FISH) with selected markers aligned the linkage groups to chromosomes as described in the accompanying article by Pedrosa et al. 2002(this issue). The length of the linkage map is 367 cM and the average marker distance is 0.6 cM. Distorted segregation of markers was found in certain sections of the map and linkage group I could be assembled only by combining colormapping and cytogenetics (FISH). A fast method to position genetic loci employing three AFLP primer combinations yielding 89 markers was developed and evaluated by mapping three symbiotic loci, Ljsym1, Ljsym5, and Ljhar1-3.