Comparative High-Resolution Transcriptome Sequencing of Lymphoma Cell Lines and de novo Lymphomas Reveals Cell-Line-Specific Pathway Dysregulation.

In dogs as well as humans, lymphoma is one of the most common hematopoietic malignancies. Furthermore, due to its characteristics, canine lymphoma is recognized as a clinically relevant in vivo model to study the corresponding human disease. Immortalized cell lines are widely used as in vitro models to evaluate novel therapeutic agents and characterize their molecular mechanisms. However, it is known that long-term cultivation leads to clonal selection, genetic instability, and loss of the initial heterogenic character, limiting the usefulness of cell lines as preclinical models. Herein, we present a systematic characterization and comparison of the transcriptomic landscape of canine primary B- and T-cell lymphomas, five lymphoid cell lines (CLBL-1, CLBL-1M, GL-1, CL-1, and OSW) and four non-neoplastic control samples. We found that lymphomas and cell lines exhibit a common "differentiation and proliferation signature". However, our analysis also showed that, independently of the cell of origin, the transcriptional signatures of lymphomas are more similar to each other than they are to those of cell lines. In particular, we observed that not all common therapeutic targets are similarly expressed between lymphomas and lymphoid cell lines, and provide evidence that different lymphoid cell-lines should be used to model distinct aspects of lymphoma dysregulation.

Analysis of circulating DNA distribution in pregnant and nonpregnant dairy cows.

Circulating nucleic acids (CNAs) are free-floating, cell-free DNA and RNA molecules in the circulation of healthy and diseased humans and animals. The aim of this study was to identify differences in CNA distribution in serum samples from multiparous pregnant (n = 24) and nonpregnant (n = 16) dairy cows at different days of gestation (Days 0, 20, and 40). A modified serial analysis of gene expression procedure was used to generate concatemerized short sequence tags from isolated serum DNA. A total of 6.1 × 10(6) tags were recovered from analyzed samples (n = 40). Significant differences between the pregnant and nonpregnant groups were detected in chromosomal regions, protein-coding sequences, and single genes (P < 0.05). Approximately 23% (1.4 × 10(6) tags) of the total sequence pool were present exclusively in the analyzed serum samples of pregnant cows. Of these tag sequences, seven originated from genomic regions and 13 from repetitive elements. Comparative BLAST analysis identified the repetitive tags as BovB (non-long terminal repeat retrotransposons/long interspersed nuclear elements), Art2A, BovA2, and Bov-tA2 (short interspersed nuclear elements). To our knowledge, this is the first study to comprehensively characterize the circulating, cell-free DNA profile in sera from pregnant and nonpregnant cows across early gestation.

Early pregnancy diagnosis in dairy cows using circulating nucleic acids.

Early and accurate pregnancy diagnosis in dairy cattle is a prerequisite for successful herd management. However, most of the currently available methods allow an early diagnosis only approximately 30 days after insemination. Recently, circulating nucleic acids (CNAs) have been used successfully as biomarkers in prenatal diagnosis at different gestational stages in human and animals. Here we show that CNAs can also be used as markers for the detection of early pregnancy in cattle. Serum samples were collected from multiparous pregnant (N = 24) and nonpregnant (N = 16) dairy cows at different days after insemination (Days 0, 20, and 40). Isolated serum DNA was preprocessed using a modified serial analysis of gene expression technique, which generated concatemerized short sequence tags for downstream next generation sequencing. Bioinformatic analysis identified sequence tags specific for pregnant dairy cows at Day 20 after insemination. The identified CNA-tags originated from repetitive regions of the bovine genome. Tag sequences that showed increased hit counts per animal were used to design quantitative real-time polymerase chain reaction assays. These quantitative polymerase chain reaction assays were applied to CNA samples from matched pregnant (N = 12) and nonpregnant cows (N = 16) at different times after insemination (Day 0, 20, and 40). At Day 20 after insemination the quantities of the interspersed repeats Art2A and BovB were increased in the pregnant cows compared with the nonpregnant control cows (P < 0.05). The best performing CNA biomarker BovB yielded an area under the receiver operating characteristic curve of 0.76. At a defined cutoff value, the pregnant and the control groups can be distinguished with a sensitivity of 83% and specificity of 75%. These results suggest that CNAs can be used as biomarkers for the detection of early pregnancy in cattle.

Comparison of nanoparticle-mediated transfection methods for DNA expression plasmids: efficiency and cytotoxicity.

BACKGROUND: Reproducibly high transfection rates with low methodology-induced cytotoxic side effects are essential to attain the required effect on targeted cells when exogenous DNA is transfected. Different approaches and modifications such as the use of nanoparticles (NPs) are being evaluated to increase transfection efficiencies. Several studies have focused on the attained transfection efficiency after NP-mediated approaches. However, data comparing toxicity of these novel approaches with conventional methods is still rare.Transfection efficiency and methodology-induced cytotoxicity were analysed after transfection with different NP-mediated and conventional approaches. Two eukaryotic DNA-expression-plasmids were used to transfect the mammalian cell line MTH53A applying six different transfection protocols: conventional transfection reagent (FuGENE HD, FHD), FHD in combination with two different sizes of stabilizer-free laser-generated AuNPs (PLAL-AuNPs_S1,_S2), FHD and commercially available AuNPs (Plano-AuNP), and two magnetic transfection protocols. 24 h post transfection efficiency of each protocol was analysed using fluorescence microscopy and GFP-based flow cytometry. Toxicity was assessed measuring cell proliferation and percentage of propidium iodide (PI%) positive cells. Expression of the respective recombinant proteins was evaluated by immunofluorescence. RESULTS: The addition of AuNPs to the transfection protocols significantly increased transfection efficiency in the pIRES-hrGFPII-eIL-12 transfections (FHD: 16%; AuNPs mean: 28%), whereas the magnet-assisted protocols did not increase efficiency. Ligand-free PLAL-AuNPs had no significant cytotoxic effect, while the ligand-stabilized Plano-AuNPs induced a significant increase in the PI% and lower cell proliferation. For pIRES-hrGFPII-rHMGB1 transfections significantly higher transfection efficiency was observed with PLAL-AuNPs (FHD: 31%; PLAL-AuNPs_S1: 46%; PLAL-AuNPs_S2: 50%), while the magnet-assisted transfection led to significantly lower efficiencies than the FHD protocol. With PLAL-AuNPs_S1 and _S2 the PI% was significantly higher, yet no consistent effect of these NPs on cell proliferation was observed. The magnet-assisted protocols were least effective, but did result in the lowest cytotoxic effect. CONCLUSIONS: This study demonstrated that transfection efficiency of DNA-expression-plasmids was significantly improved by the addition of AuNPs. In some combinations the respective cytotoxicity was increased depending on the type of the applied AuNPs and the transfected DNA construct. Consequently, our results indicate that for routine use of these AuNPs the specific nanoparticle formulation and DNA construct combination has to be considered.

Co-transfection of plasmid DNA and laser-generated gold nanoparticles does not disturb the bioactivity of GFP-HMGB1 fusion protein.

Ultrashort pulsed laser ablation in liquids represents a powerful tool for the generation of pure gold nanoparticles (AuNPs) avoiding chemical precursors and thereby making them especially interesting for biomedical applications. However, because of their electron accepting properties, laser-generated AuNPs might affect biochemical properties of biomolecules, which often adsorb onto the nanoparticles. We investigated possible effects of such laser-generated AuNPs on biological functionality of DNA molecules. We tested four differently sized and positively charged AuNPs by incubating them with recombinant eGFP-C1-HMGB1 DNA expression plasmids that code for eGFP fusion proteins and contain the canine architectural transcription factor HMGB1. We were able to show that successfully transfected mammalian cells are still able to synthesize and process the fusion proteins. Our observations revealed that incubation of AuNP with the plasmid DNA encoding the recombinant canine HMGB1 neither prevented the mediated uptake of the vector through the plasma membrane in presence of a transfection reagent nor had any effect on the transport of the synthesized fusion proteins to the nuclei. Biological activity of the recombinant GFP-HMGB1 fusion protein appears to have not been affected either, as a strong characteristic protein accumulation in the nucleus could be observed. We also discovered that transfection efficiencies depend on the size of AuNP. In conclusion, our data indicate that laser-generated AuNPs present a good alternative to chemically synthesized nanoparticles for use in biomedical applications.

Cloning, characterisation, and comparative quantitative expression analyses of receptor for advanced glycation end products (RAGE) transcript forms.

RAGE is a member of the immunoglobulin superfamily of cell surface molecules playing key roles in pathophysiological processes, e.g. immune/inflammatory disorders, Alzheimer's disease, diabetic arteriosclerosis and tumourigenesis. In humans 19 naturally occurring RAGE splicing variants resulting in either N-terminally or C-terminally truncated proteins were identified and are lately discussed as mechanisms for receptor regulation. Accordingly, deregulation of sRAGE levels has been associated with several diseases e.g. Alzheimer's disease, Type 1 diabetes, and rheumatoid arthritis. Administration of recombinant sRAGE to animal models of cancer blocked tumour growth successfully. In spite of its obvious relationship to cancer and metastasis data focusing sRAGE deregulation and tumours is rare. In this study we screened a set of tumours, healthy tissues and various cancer cell lines for RAGE splicing variants and analysed their structure. Additionally, we analysed the ratio of the mainly found transcript variants using quantitative Real-Time PCR. In total we characterised 24 previously not described canine and 4 human RAGE splicing variants, analysed their structure, classified their characteristics, and derived their respective protein forms. Interestingly, the healthy and the neoplastic tissue samples showed in majority RAGE transcripts coding for the complete receptor and transcripts showing insertions of intron 1.

Establishing an in vivo model of canine prostate carcinoma using the new cell line CT1258.

BACKGROUND: Prostate cancer is a frequent finding in man. In dogs, malignant disease of the prostate is also of clinical relevance, although it is a less common diagnosis. Even though there are numerous differences in origin and development of the disease, man and dog share many similarities in the pathological presentation. For this reason, the dog might be a useful animal model for prostate malignancies in man.Although prostate cancer is of great importance in veterinary medicine as well as in comparative medicine, there are only few cell lines available. Thus, it was the aim of the present study to determine whether the formerly established prostate carcinoma cell line CT1258 is a suitable tool for in vivo testing, and to distinguish the growth pattern of the induced tumours. METHODS: For characterisation of the in vivo behaviour of the in vitro established canine prostate carcinoma cell line CT1258, cells were inoculated in 19 NOD.CB17-PrkdcScid/J (in the following: NOD-Scid) mice, either subcutaneously or intraperitoneally. After sacrifice, the obtained specimens were examined histologically and compared to the pattern of the original tumour in the donor. Cytogenetic investigation was performed. RESULTS: The cell line CT 1258 not only showed to be highly tumourigenic after subcutaneous as well as intraperitoneal inoculation, but also mimicked the behaviour of the original tumour. CONCLUSION: Tumours induced by inoculation of the cell line CT1258 resemble the situation in naturally occurring prostate carcinoma in the dog, and thus could be used as in vivo model for future studies.

Genomic characterisation, chromosomal assignment and in vivo localisation of the canine high mobility group A1 (HMGA1) gene.

BACKGROUND: The high mobility group A1 proteins (HMGA1a/HMGA1b) are highly conserved between mammalian species and widely described as participating in various cellular processes. By inducing DNA conformation changes the HMGA1 proteins indirectly influence the binding of various transcription factors and therefore effect the transcription regulation. In humans chromosomal aberrations affecting the HMGA1 gene locus on HSA 6p21 were described to be the cause for various benign mesenchymal tumours while high titres of HMGA1 proteins were shown to be associated with the neoplastic potential of various types of cancer. Interestingly, the absence of HMGA1 proteins was shown to cause insulin resistance and diabetes in humans and mice. Due to the various similarities in biology and presentation of human and canine cancers the dog has joined the common rodent animal model for therapeutic and preclinical studies. Accordingly, the canine genome was sequenced completely twice but unfortunately this could not solve the structure of canine HMGA1 gene. RESULTS: Herein we report the characterisation of the genomic structure of the canine HMGA1 gene consisting of 7 exons and 6 introns spanning in total 9524 bp, the in vivo localisation of the HMGA1 protein to the nucleus, and a chromosomal assignment of the gene by FISH to CFA12q11. Additionally, we evaluated a described canine HMGA1 exon 6 SNP in 55 Dachshunds. CONCLUSION: The performed characterisations will make comparative analyses of aberrations affecting the human and canine gene and proteins possible, thereby providing a basis for revealing mechanisms involved in HMGA1 related pathogenesis in both species.

Chromosomal assignment of canine THADA gene to CFA 10q25.

BACKGROUND: Chromosomal translocations affecting the chromosome 2p21 cluster in a 450 kb breakpoint region are frequently observed in human benign thyroid adenomas. THADA (thyroid adenoma associated) was identified as the affected gene within this breakpoint region. In contrast to man tumours of the thyroid gland of dogs (Canis lupus familiaris) constitute mainly as follicular cell carcinomas, with malignant thyroid tumours being more frequent than benign thyroid adenomas. In order to elucidate if the THADA gene is also a target of chromosomal rearrangements in thyroid adenomas of the dog we have physically mapped the canine THADA gene to canine chromosome 10.A PCR was established to screen a canine genome library for a BAC clone containing the gene sequence of canine THADA. Further PCR reactions were done using the identified BAC clone as a template in order to verify the corresponding PCR product by sequencing.Canine whole blood was incubated with colcemid in order to arrest the cultured cells in metaphases. The verified BAC DNA was digoxigenin labeled and used as a probe in fluorescence in situ hybridization (FISH). Ten well spread metaphases were examined indicating a signal on canine chromosome 10 on both chromatids. A detailed fine mapping was performed indicating the canine THADA gene locus on the q-arm of chromosome 10. RESULTS: The canine THADA gene locus was mapped on chromosome 10q25. Our mapping results obtained in this study following the previously described nomenclature for the canine karyotype. CONCLUSION: We analysed whether the THADA gene locus is a hotspot of canine chromosomal rearrangements in canine neoplastic lesions of the thyroid and in addition might play a role as a candidate gene for a possible malignant transformation of canine thyroid adenomas. Although the available cytogenetic data of canine thyroid adenomas are still insufficient the chromosomal region to which the canine THADA has been mapped seems to be no hotspot of chromosomal aberrations seen in canine thyroid adenomas.

A domain of the thyroid adenoma associated gene (THADA) conserved in vertebrates becomes destroyed by chromosomal rearrangements observed in thyroid adenomas.

THADA, mapping to chromosomal band 2p21 is target gene of specific chromosomal rearrangements observed in thyroid benign tumors. Thus, it is one of the most common gene targets in chromosomal rearrangements in benign epithelial tumors. Nevertheless, nothing is known about the function of its protein. Therefore, we have analyzed the genetic structure of THADA homologous genes in selected vertebrates (Canis familiaris, Chlorocebus aethiops, Gallus gallus, and Mus musculus), which are not characterized up to now. The coding sequences of the mRNA of these species have been sequenced and analyzed revealing similarities to ARM repeat structures which indicates an involvement in protein-protein interactions. Using multiple alignments we identified the most conserved part of the protein (aa 1033-1415 Homo sapiens) with an identity of 70.5% between the most different organisms implying a putative important functional domain. The truncations observed in human thyroid adenomas disrupt this conserved domain of the protein indicating a loss of function of THADA contributing to the development of the follicular neoplasias of the thyroid.

Comparison of the human and canine cytokines IL-1(alpha/beta) and TNF-alpha to orthologous other mammalians.

The cytokines interleukin-1 (IL-1alpha and IL-1beta) and the tumor necrosis factor-alpha (TNF-alpha) both play a major role in the initiation and regulation of inflammation and immunity responses. Polymorphisms within the gene sequences of these cytokines IL-1 and TNF-alpha have been proposed to play an important role in the pathogenesis of certain diseases. Affecting nearly every organ, various diseases, including some cancers, are described to be associated with an increased level of IL-1 and TNF-alpha proteins, for example, solid tumors, hematologic malignancies, malignant histiocytosis, autoimmune disorders, Alzheimer's disease, Parkinson's disease, sepsis, and rheumatoid arthritis. Regarding genetic backgrounds and pathways, numerous canine diseases show close similarities to their human counterparts. As a genetic model, the dog could be used to unravel the genetic mechanisms, for example, in particular the predispositions, the development, and progression of cancer and metabolic diseases. The identity comparison of gene and protein sequences of different species could be used to elucidate the structure and function of the genes and proteins by identifying the evolutionary conserved regions and domains. Herein we analyzed in detail the mRNA and protein structures and identities of the present known mammalian (human, canine, murine, rat, ovine, equine, feline, porcine, and bovine) TNF-alpha, IL-1alpha, and IL-1beta mRNAs and proteins. Additionally, based on the canine genome sequence, we derived in silico the complete mRNA structures of the IL-1alpha and IL-1beta mRNAs.

Cloning and characterization of the canine receptor for advanced glycation end products.

Metastasis is one of the major problems when dealing with malignant neoplasias. Accordingly, the finding of molecular targets, which can be addressed to reduce tumour metastasising, will have significant impact on the development of new therapeutic approaches. Recently, the receptor for advanced glycation end products (RAGE)-high mobility group B1 (HMGB1) protein complex has been shown to have significant influence on invasiveness, growth and motility of tumour cells, which are essential characteristics required for metastatic behaviour. A set of in vitro and in vivo approaches showed that blocking of this complex resulted in drastic suppression of tumour cell growth. Due to the similarities of human and canine cancer the dog has joined the common rodent animal model for therapeutic and preclinical studies. However, complete characterisation of the protein complex is a precondition to a therapeutic approach based on the blocking of the RAGE-HMGB1 complex to spontaneously occurring tumours in dogs. We recently characterised the canine HMGB1 gene and protein completely. Here we present the complete characterisation of the canine RAGE gene including its 1384 bp mRNA, the 1215 bp protein coding sequence, the 2835 bp genomic structure, chromosomal localisation, gene expression pattern, and its 404 amino acid protein. Furthermore we compared the CDS of six different canine breeds and screened them for single nucleotide polymorphisms.

Absence of ras-gene hot-spot mutations in canine fibrosarcomas and melanomas.

Point mutations within ras proto-oncogenes, particularly within the mutational hot-spot codons 12, 13 and 61, are frequently detected in human malignancies and in different types of experimentally-induced tumours in animals. So far little is known about ras mutations in naturally occurring canine fibrosarcomas or K-ras mutations in canine melanomas. To elucidate whether ras mutations exist in these naturally occurring tumours in dogs, in the present study we screened 13 canine fibrosarcomas, 2 feline fibrosarcomas and 11 canine melanomas for point mutations, particularly within the mutational hot-spots, making this the first study to investigate a large number of canine fibrosarcomas. None of the samples showed a K- or N-ras hot spot mutation. Thus, our data strongly suggest that ras mutations at the hot-spot loci are very rare and do not play a major role in the pathogenesis of the spontaneously occurring canine tumours investigated.

The canine HMGA1.

Due to the emerging advantages of numerous canine diseases as a genetic model for their human orthologs, the dog could join the mouse as the species of choice to unravel genetic mechanisms, e.g. of cancer predisposition, development and progression. However, precondition for such studies is the characterisation of the corresponding canine genes. Human and murine HMGA1 non-histone proteins participate in a wide variety of cellular processes including regulation of inducible gene transcription, integration of retroviruses into chromosomes, and the induction of neoplastic transformation and promotion of metastatic progression of cancer cells. Chromosomal aberrations affecting the human HMGA1 gene at 6p21 were described in several tumours like pulmonary chondroid hamartomas, uterine leiomyomas, follicular thyroid adenomas and others. Over-expression of the proteins of HMGA1 is characteristic for various malignant tumours suggesting a relation between high titer of the protein and the neoplastic phenotype. In this study, we characterised the molecular structure of the canine HMGA1 cDNA, its splice variants and predicted proteins HMGA1a and HMGA1b. Furthermore, we compared the coding sequence(s) (CDS) of both splice variants for 12 different breeds, screened them for single nucleotide polymorphisms (SNPs) and characterised a basic expression pattern.