Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets

Background: Melanoma progression occurs through three major stages: radial growth phase (RGP), confined to the epidermis; vertical growth phase (VGP), when the tumor has invaded into the dermis; and metastasis. In this work, we used suppression subtractive hybridization (SSH) to investigate the molecular signature of melanoma progression, by comparing a group of metastatic cell lines with an RGP-like cell line showing characteristics of early neoplastic lesions including expression of the metastasis suppressor KISS1, lack of alpha v beta 3-integrin and low levels of RHOC. Methods: Two subtracted cDNA collections were obtained, one (RGP library) by subtracting the RGP cell line (WM1552C) cDNA from a cDNA pool from four metastatic cell lines (WM9, WM852, 1205Lu and WM1617), and the other (Met library) by the reverse subtraction. Clones were sequenced and annotated, and expression validation was done by Northern blot and RT-PCR. Gene Ontology annotation and searches in large-scale melanoma expression studies were done for the genes identified. Results: We identified 367 clones from the RGP library and 386 from the Met library, of which 351 and 368, respectively, match human mRNA sequences, representing 288 and 217 annotated genes. We confirmed the differential expression of all genes selected for validation. In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking. Interestingly, 19% of the genes from the RGP library map to chromosome 1 against 4% of the ones from Met library. Conclusion: This study identifies two populations of genes differentially expressed between melanoma cell lines from two tumor stages and suggests that these sets of genes represent profiles of less aggressive versus metastatic melanomas. A search for expression profiles of melanoma in available expression study databases allowed us to point to a great potential of involvement in tumor progression for several of the genes identified here. A few sequences obtained here may also contribute to extend annotated mRNAs or to the identification of novel transcripts.

Suppression subtractive hybridization analysis reveals expression of conserved and novel genes in male accessory glands of the ant Leptothorax gredleri

Background: During mating, insect males eject accessory gland proteins (Acps) into the female genital tract. These substances are known to affect female post-mating behavior and physiology. In addition, they may harm the female, e. g., in reducing its lifespan. This is interpreted as a consequence of sexual antagonistic co-evolution. Whereas sexual conflict abounds in non-social species, the peculiar life history of social insects (ants, bees, wasps) with lifelong pair-bonding and no re-mating aligns the reproductive interests of the sexes. Harming the female during mating would negatively affect male fitness and sexual antagonism is therefore not expected. Indeed, mating appears to increase female longevity in at least one ant species. Acps are presumed to play a role in this phenomenon, but the underlying mechanisms are unknown. In this study, we investigated genes, which are preferentially expressed in male accessory glands of the ant Leptothorax gredleri, to determine which proteins might be transferred in the seminal fluid. Results: By a suppression subtractive hybridization protocol we obtained 20 unique sequences (USs). Twelve had mutual best matches with genes predicted for Apis mellifera and Nasonia vitripennis. Functional information (Gene Ontology) was available only for seven of these, including intracellular signaling, energy-dependent transport and metabolic enzyme activities. The remaining eight USs did not match sequences from other species. Six genes were further analyzed by quantitative RT-PCR in three life cycle stages of male ants. A gene with carboxy-lyase activity and one of unpredicted function were significantly overexpressed in accessory glands of sexually mature males. Conclusions: Our study is the first one to investigate differential gene expression in ants in a context related to mating. Our findings indicate that male accessory glands of L. gredleri express a series of genes that are unique to this species, possibly representing novel genes, in addition to conserved ones for which functions can be predicted. Identifying differentially expressed genes might help to better understand molecular mechanisms involved in reproductive processes in eusocial Hymenoptera. While the novel genes could account for rapidly evolving ones driven by intra-sexual conflict between males, conserved genes imply that rather beneficial traits might get fixed by a process described as inter-sexual cooperation between males and females.

Differential display and suppression subtractive hybridization analysis of the pulp of ripening banana

The fruit of banana undergoes several important physico-chemical changes during ripening. Analysis of gene expression would permit identification of important genes and regulatory elements involved in this process. Therefore, transcript profiling of preclimacteric and climacteric fruit was performed using differential display and Suppression subtractive hybridization. Our analyses resulted in the isolation of 12 differentially expressed cDNAs, which were confirmed by dot-blots and northern blots. Among the sequences identified were sequences homologous to plant aquaporins, adenine nucleotide translocator, immunophilin, legumin-like proteins, deoxyguanosine kinase and omega-3 fatty acid desaturase. Some of these cDNAs correspond to newly isolated genes involved in changes related to the respiratory climacteric, or stress-defense responses. Functional characterization of ripening-associated genes could provide information useful in controlling biochemical pathways that would have an impact on banana quality and shelf life. (C) 2009 Elsevier B.V. All rights reserved.

PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori

Genes that are characteristic of only certain strains of a bacterial species can be of great biologic interest. Here we describe a PCR-based subtractive hybridization method for efficiently detecting such DNAs and apply it to the gastric pathogen Helicobacter pylori. Eighteen DNAs specific to a monkey-colonizing strain (J166) were obtained by subtractive hybridization against an unrelated strain whose genome has been fully sequenced (26695). Seven J166-specific clones had no DNA sequence match to the 26695 genome, and 11 other clones were mixed, with adjacent patches that did and did not match any sequences in 26695. At the protein level, seven clones had homology to putative DNA restriction-modification enzymes, and two had homology to putative metabolic enzymes. Nine others had no database match with proteins of assigned function. PCR tests of 13 unrelated H. pylori strains by using primers specific for 12 subtracted clones and complementary Southern blot hybridizations indicated that these DNAs are highly polymorphic in the H. pylori population, with each strain yielding a different pattern of gene-specific PCR amplification. The search for polymorphic DNAs, as described here, should help identify previously unknown virulence genes in pathogens and provide new insights into microbial genetic diversity and evolution.

Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries.

A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model system, the SSH technique enriched for rare sequences over 1,000-fold in one round of subtractive hybridization. We demonstrate its usefulness by generating a testis-specific cDNA library and by using the subtracted cDNA mixture as a hybridization probe to identify homologous sequences in a human Y chromosome cosmid library. The human DNA inserts in the isolated cosmids were further confirmed to be expressed in a testis-specific manner. These results suggest that the SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes.

Identification Of Target Genes Using Gene Expression Profile Of Granulocytes From Patients With Chronic Myeloid Leukemia Treated With Tyrosine Kinase Inhibitors.

Differential gene expression analysis by suppression subtractive hybridization with correlation to the metabolic pathways involved in chronic myeloid leukemia (CML) may provide a new insight into the pathogenesis of CML. Among the overexpressed genes found in CML at diagnosis are SEPT5, RUNX1, MIER1, KPNA6 and FLT3, while PAN3, TOB1 and ITCH were decreased when compared to healthy volunteers. Some genes were identified and involved in CML for the first time, including TOB1, which showed a low expression in patients with CML during tyrosine kinase inhibitor treatment with no complete cytogenetic response. In agreement, reduced expression of TOB1 was also observed in resistant patients with CML compared to responsive patients. This might be related to the deregulation of apoptosis and the signaling pathway leading to resistance. Most of the identified genes were related to the regulation of nuclear factor κB (NF-κB), AKT, interferon and interleukin-4 (IL-4) in healthy cells. The results of this study combined with literature data show specific gene pathways that might be explored as markers to assess the evolution and prognosis of CML as well as identify new therapeutic targets.

Transcriptional profiling reveals the expression of novel genes in response to various stimuli in the human dermatophyte Trichophyton rubrum

Background: Cutaneous mycoses are common human infections among healthy and immunocompromised hosts, and the anthropophilic fungus Trichophyton rubrum is the most prevalent microorganism isolated from such clinical cases worldwide. The aim of this study was to determine the transcriptional profile of T. rubrum exposed to various stimuli in order to obtain insights into the responses of this pathogen to different environmental challenges. Therefore, we generated an expressed sequence tag (EST) collection by constructing one cDNA library and nine suppression subtractive hybridization libraries. Results: The 1388 unigenes identified in this study were functionally classified based on the Munich Information Center for Protein Sequences (MIPS) categories. The identified proteins were involved in transcriptional regulation, cellular defense and stress, protein degradation, signaling, transport, and secretion, among other functions. Analysis of these unigenes revealed 575 T. rubrum sequences that had not been previously deposited in public databases. Conclusion: In this study, we identified novel T. rubrum genes that will be useful for ORF prediction in genome sequencing and facilitating functional genome analysis. Annotation of these expressed genes revealed metabolic adaptations of T. rubrum to carbon sources, ambient pH shifts, and various antifungal drugs used in medical practice. Furthermore, challenging T. rubrum with cytotoxic drugs and ambient pH shifts extended our understanding of the molecular events possibly involved in the infectious process and resistance to antifungal drugs.

Epigenetic Silencing of CRABP2 and MX1 in Head and Neck Tumors

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease affecting the epithelium of the oral cavity, pharynx and larynx. Conditions of most patients are diagnosed at late stages of the disease, and no sensitive and specific predictors of aggressive behavior have been identified yet. Therefore, early detection and prognostic biomarkers are highly desirable for a more rational management of the disease. Hypermethylation of CpG islands is one of the most important epigenetic mechanisms that leads to gene silencing in tumors and has been extensively used for the identification of biomarkers. In this study, we combined rapid subtractive hybridization and microarray analysis in a hierarchical manner to select genes that are putatively reactivated by the demethylating agent 5-aza-2'-deoxycytidine (5Aza-dC) in HNSCC cell lines (FaDu, UM-SCC-14A, UM-SCC-17A, UM-SCC-38A). This combined analysis identified 78 genes, 35 of which were reactivated in at least 2 cell lines and harbored a CpG island at their 5' region. Reactivation of 3 of these 35 genes (CRABP2, MX1, and SLC15A3) was confirmed by quantitative real-time polymerase chain reaction (PCR; fold change, >= 3). Bisulfite sequencing of their CpG islands revealed that they are indeed differentially methylated in the HNSCC cell lines. Using methylation-specific PCR, we detected a higher frequency of CRABP2 (58.1% for region 1) and MX1 (46.3%) hypermethylation in primary HNSCC when compared with lymphocytes from healthy individuals. Finally, absence of the CRABP2 protein was associated with decreased disease-free survival rates, supporting a potential use of CRABP2 expression as a prognostic biomarker for HNSCC patients.

Differential expression of genes in salivary glands of male Rhipicephalus (Boophilus)microplus in response to infection with Anaplasma marginale

Background: Bovine anaplasmosis, caused by the rickettsial tick-borne pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), is vectored by Rhipicephalus (Boophilus) microplus in many tropical and subtropical regions of the world. A. marginale undergoes a complex developmental cycle in ticks which results in infection of salivary glands from where the pathogen is transmitted to cattle. In previous studies, we reported modification of gene expression in Dermacentor variabilis and cultured Ixodes scapularis tick cells in response to infection with A. marginale. In these studies, we extended these findings by use of a functional genomics approach to identify genes differentially expressed in R. microplus male salivary glands in response to A. marginale infection. Additionally, a R. microplus-derived cell line, BME26, was used for the first time to also study tick cell gene expression in response to A. marginale infection. Results: Suppression subtractive hybridization libraries were constructed from infected and uninfected ticks and used to identify genes differentially expressed in male R. microplus salivary glands infected with A. marginale. A total of 279 ESTs were identified as candidate differentially expressed genes. Of these, five genes encoding for putative histamine-binding protein (22Hbp), von Willebrand factor (94Will), flagelliform silk protein (100Silk), Kunitz-like protease inhibitor precursor (108Kunz) and proline-rich protein BstNI subfamily 3 precursor (7BstNI3) were confirmed by real-time RT-PCR to be down-regulated in tick salivary glands infected with A. marginale. The impact of selected tick genes on A. marginale infections in tick salivary glands and BME26 cells was characterized by RNA interference. Silencing of the gene encoding for putative flagelliform silk protein (100Silk) resulted in reduced A. marginale infection in both tick salivary glands and cultured BME26 cells, while silencing of the gene encoding for subolesin (4D8) significantly reduced infection only in cultured BME26 cells. The knockdown of the gene encoding for putative metallothionein (93 Meth), significantly up-regulated in infected cultured BME26 cells, resulted in higher A. marginale infection levels in tick cells. Conclusions: Characterization of differential gene expression in salivary glands of R. microplus in response to A. marginale infection expands our understanding of the molecular mechanisms at the tick-pathogen interface. Functional studies suggested that differentially expressed genes encoding for subolesin, putative von Willebrand factor and flagelliform silk protein could play a role in A. marginale infection and multiplication in ticks. These tick genes found to be functionally relevant for tick-pathogen interactions will likely be candidates for development of vaccines designed for control of both ticks and tick-borne pathogens.

Identification of COL6A1 as a differentially expressed gene in human astrocytomas

Diffuse infiltrating gliomas are the most common tumors of the central nervous system. Gliomas are classified by the WHO according to their histopathological and clinical characteristics into four classes: grade I (pilocytic astrocytoma), grade II (diffuse astrocytoma), grade III (anaplastic astrocytoma), and grade IV (glioblastoma multiforme). Several genes have already been correlated with astrocytomas, but many others are yet to be uncovered. By analyzing the public SAGE data from 21 patients, comprising low malignant grade astrocytomas and glioblastomas, we found COL6A1 to be differentially expressed, confirming this finding by real time RT-PCR in 66 surgical samples. To the best of our knowledge, COL6A1 has never been described in gliomas. The expression of this gene has significantly different means when normal glia is compared with low-grade astrocytomas (grades I and II) and high-grade astrocytomas (grades III and IV), with a tendency to be greater in higher grade samples, thus rendering it a powerful tumor marker.

Searching for Moniliophthora perniciosa pathogenicity genes

The basidiomycete Moniliophthora perniciosa is the causal agent of witches` broom disease of Theobroma cacao (cacao). Pathogenesis mechanisms of this hemibiotrophic fungus are largely unknown. An approach to identify putative pathogenicity genes is searching for sequences induced in mycelia grown under in vitro conditions. Using this approach, genes from M. perniciosa induced under limiting nitrogen and light were identified from a cDNA library enriched by suppression subtractive hybridization as potential putative pathogenicity genes. From the 159 identified unique sequences, 59 were annotated and classified by gene ontology. Two sequences were categorized as ""Defence genes, Virulence, and Cell response"" presumably coding for allergenic proteins, whose homologues from other fungi are inducers of animal or plant defences. Differential gene expression was evaluated by quantitative amplification of reversed transcripts (RT-qPCR) of the putative identified genes coding for the two allergenic proteins (Aspf13 and 88KD), and for the enzymes Arylsulfatase (AS); Aryl-Alcohol Oxidase; Aldo-Keto Reductase (AK); Cytochrome P450 (P450); Phenylalanine Ammonia-Lyase; and Peroxidase from mycelia grown under contrasting N concentrations. All genes were validated for differential expression, except for the putative Peroxidase. The same eight genes were analysed for expression in susceptible plants inoculated with M. perniciosa, and six were induced during the early asymptomatic stage of the disease. In infected host tissues, transcripts of 88KD and AS were found more abundant at the biotrophic phase, while those from Aspf13, AK, PAL, and P450 accumulated at the necrotrophic phase, enabling to suggest that mycelia transition from biotrophic to necrotrophic might occur earlier than currently considered. These sequences appeared to be virulence life-style genes, which encode factors or enzymes that enable invasion, colonization or intracellular survival, or manipulate host factors to benefit the pathogen`s own survival in the hostile environment. (C) 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Identification and validation of candidate Myb target genes

While a considerable number of candidate Myb target genes have been reported to date, most of these are likely to play little or no role in transformation by myb oncogenes. Here we have used a conditionally myb-transformed myeloid cell line (ERMYB) to further examine Myb regulation of one candidate target gene-c-myc-that has the potential to affect cell proliferation. It was found that the major influence on c-myc expression was the presence of cytokine (GM-CSF) rather than Myb activity. We also describe the application of PCR-based subtractive hybridization and low-density cDNA array screening, in conjunction with the ERMYB line, to the identification of additional Myb target genes. Preliminary identification of a number of candidates is reported; these include myeloperoxidase, which is known to have essential Myb-binding sites in its regulatory region. (C) 2001 Academic Press.

Regulation of the gene encoding glutathione S-transferase M1 (GSTM1) by the Myb oncoprotein

The identification of Myb 'target' genes will not only aid in the understanding of how overexpression of Myb, or expression of activated forms of Myb, leads to cellular transformation but will also shed light on its role in normal cells. Using a combination of an estrogen-regulated Myb-transformed cell line (ERMYB) and PCR-based subtractive hybridization, we have identified the gene (GSTM1) encoding the detoxification enzyme glutathione S-transferase M1 as being transcriptionally upregulated by Myb. Functional analysis of the GSTM1 promoter using reporter assays indicated that both the DNA binding and transactivation domains of Myb were required for transcriptional activation. Mutational analysis of consensus Myb-binding sites (MBS) in the promoter and electrophoretic mobility gel shift analysis indicated that one of the three potential MBS can bind Myb protein, and is the primary site involved in the regulation of this promoter by Myb.

Representational Difference Analysis (RDA) reveals differential expression of conserved as well as novel genes during caste-specific development of the honey bee (Apis mellifera L.) ovary

In highly eusocial insects, such as the honey bee, Apis mellifera, the reproductive bias has become embedded in morphological caste differences. These are most expressively denoted in ovary size, with adult queens having large ovaries consisting of 150-200 ovarioles each, while workers typically have only 1-20 ovarioles per ovary. This morphological differentiation is a result of hormonal signals triggered by the diet change in the third larval instar, which eventually generate caste-specific gene expression patterns. To reveal these we produced differential gene expression libraries by Representational Difference Analysis (RDA) for queen and worker ovaries in a developmental stage when cell death is a prominent feature in the ovarioles of workers, whereas all ovarioles are maintained and extend in length in queens. In the queen library, 48% of the gene set represented homologs of known Drosophila genes, whereas in the worker ovary, the largest set (59%) were ESTs evidencing novel genes, not even computationally predicted in the honey bee genome. Differential expression was confirmed by quantitative RT-PCR for a selected gene set, denoting major differences for two queen and two worker library genes. These included two unpredicted genes located in chromosome 11 (Group11.35 and Group11.31, respectively) possibly representing long non-coding RNAs. Being candidates as modulators of ovary development, their expression and functional analysis should be a focal point for future studies. (C) 2011 Elsevier Ltd. All rights reserved.

Differentially expressed genes in eutopic and ectopic endometrium of women with endometriosis

Objective: To elucidate the potential mechanisms involved in the physiopathology of endometriosis. We analyzed the differential gene expression profiles of eutopic and ectopic tissues from women with endometriosis. Design: Prospective laboratory study. Setting: University hospital. Patient(s): Seventeen patients in whom endometriosis was diagnosed and 11 healthy fertile women. Intervention(s): Endometrial biopsy specimens from the endometrium of healthy women without endometriosis and from the eutopic and ectopic endometrium tissues of patients with endometriosis were obtained in the early proliferative phase of the menstrual cycle. Main Outcome Measure(s): Six paired samples of eutopic and ectopic tissue were analyzed by subtractive hybridization. To evaluate the expression of genes found by rapid subtraction hybridization methods, we measured CTGF, SPARC, MYC, MMP and IGFBP1 genes by real-time polymerase chain reaction in all samples. Result(s): This study identified 291 deregulated genes in the endometriotic lesions. Significant expression differences were obtained for SPARC, MYC, and IGFBP1 in the peritoneal lesions and for MMP3 in the ovarian endometriomas. Additionally, significant differences were obtained for SPARC and IGFBP1 between the peritoneal and ovarian lesions. No significant differences were found for the studied genes between the control and the eutopic endometrium. Conclusion(s): This study identified 291 genes with differential expression in endometriotic lesions. The deregulation of the SPARC, MYC, MMP3, and IGFBP1 genes may be responsible for the loss of cellular homeostasis in endometriotic lesions. (Fertil Steril(R) 2010;93:1750-73. (C) 2010 by American Society for Reproductive Medicine.)