Katharina Anna Sterenczak

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, Alzheimers 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. Alzheimers 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.

HMGA1 and HMGA2 expression and comparative analyses of HMGA2, Lin28 and let-7 miRNAs in oral squamous cell carcinoma

BackgroundHumans and dogs are affected by squamous cell carcinomas of the oral cavity (OSCC) in a considerably high frequency. The high mobility group A2 (HMGA2) protein was found to be highly expressed in human OSCC and its expression was suggested to act as a useful predictive and prognostic tool in clinical management of oral carcinomas. Herein the expression of HMGA2 and its sister gene HMGA1 were analysed within human and canine OSCC samples. Additionally, the HMGA negatively regulating miRNAs of the let-7 family as well as the let-7 regulating gene Lin28 were also comparatively analysed. Deregulations of either one of these members could affect the progression of human and canine OSCC.MethodsExpression levels of HMGA1, HMGA2, Lin28, let-7a and mir-98 were analysed via relative qPCR in primary human and canine OSCC, thereof derived cell lines and non-neoplastic samples. Additionally, comparative HMGA2 protein expression was analysed by immunohistochemistry.ResultsIn both species, a significant up-regulation of the HMGA2 gene was found within the neoplastic samples while HMGA1 expression did not show significant deregulations. Comparative analyses showed down-regulation of mir-98 in human samples and up-regulation of let-7a and mir-98 in canine neoplastic samples. HMGA2 immunostainings showed higher intensities within the invasive front of the tumours than in the centre of the tumour in both species.ConclusionsHMGA2 could potentially serve as tumour marker in both species while HMGA1 might play a minor role in OSCC progression. Comparative studies indicate an inverse correlation of HMGA2 and mir-98 expression in human samples whereas in dogs no such characteristic could be found.

RAGE splicing variants in mammals.

The receptor for advanced glycation end products (RAGE) is a multiligand receptor of environmental stressors which plays key roles in pathophysiological processes, including immune/inflammatory disorders, Alzheimers disease, diabetic arteriosclerosis, tumorigenesis, and metastasis. Besides the full-length RAGE protein in humans nearly 20 natural occurring RAGE splicing variants were described on mRNA and protein level. These naturally occurring isoforms are characterized by either N-terminally or C-terminally truncations and are discussed as possible regulators of the full-length RAGE receptor either by competitive ligand binding or by displacing the full-length protein in the membrane. Accordingly, expression deregulations of the naturally occurring isoforms were supposed to have significant effect on RAGE-mediated disorders. Thereby the soluble C-truncated RAGE isoforms present in plasma and tissues are the mostly focused isoforms in research and clinics. Deregulations of the circulating levels of soluble RAGE forms were reported in several RAGE-associated pathological disorders including for example atherosclerosis, diabetes, renal failure, Alzheimers disease, and several cancer types. Regarding other mammalian species, the canine RAGE gene showed high similarities to the corresponding human structures indicating RAGE to be evolutionary highly conserved between both species. Similar to humans the canine RAGE showed a complex and extensive splicing activity leading to a manifold pattern of RAGE isoforms. Due to the similarities seen in several canine and human diseases-including cancer-comparative structural and functional analyses allow the development of RAGE and ligand-specific therapeutic approaches beneficial for human and veterinary medicine.

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

BackgroundProstate 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.MethodsFor 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.ResultsThe 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.ConclusionTumours 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.

Longitudinal MRI contrast enhanced monitoring of early tumour development with manganese chloride (MnCl2) and superparamagnetic iron oxide nanoparticles (SPIOs) in a CT1258 based in vivo model of prostate cancer

BackgroundCell lines represent a key tool in cancer research allowing the generation of neoplasias which resemble initial tumours in in-vivo animal models. The characterisation of early tumour development is of major interest in order to evaluate the efficacy of therapeutic agents. Magnetic resonance imaging (MRI) based in-vivo characterisation allows visualisation and characterisation of tumour development in early stages prior to manual palpation. Contrast agents for MRI such as superparamagnetic iron oxide nanoparticles (SPIOs) and manganese chloride (MnCl2) represent powerful tools for the in-vivo characterisation of early stage tumours. In this experimental study, we labelled prostate cancer cells with MnCl2 or SPIOs in vitro and used 1 T MRI for tracing labelled cells in-vitro and 7 T MRI for tracking in an in-vivo animal model.MethodsLabelling of prostate cancer cells CT1258 was established in-vitro with MnCl2 and SPIOs. In-vitro detection of labelled cells in an agar phantom was carried out through 1 T MRI while in-vivo detection was performed using 7 T MRI after subcutaneous (s.c.) injection of labelled cells into NOD-Scid mice (n = 20). The animals were scanned in regular intervals until euthanization. The respective tumour volumes were analysed and corresponding tumour masses were subjected to histologic examination.ResultsMnCl2in-vitro labelling resulted in no significant metabolic effects on proliferation and cell vitality. In-vitro detection-limit accounted 105 cells for MnCl2 as well as for SPIOs labelling. In-vivo 7 T MRI scans allowed detection of 103 and 104 cells. In-vivo MnCl2 labelled cells were detectable from days 4–16 while SPIO labelling allowed detection until 4 days after s.c. injection. MnCl2 labelled cells were highly tumourigenic in NOD-Scid mice and the tumour volume development was characterised in a time dependent manner. The amount of injected cells correlated with tumour size development and disease progression. Histological analysis of the induced tumour masses demonstrated characteristic morphologies of prostate adenocarcinoma.ConclusionsTo the best of our knowledge, this is the first study reporting direct in-vitro MnCl2 labelling and 7 T based in-vivo MRI tracing of cancer cells in a model of prostate cancer. MnCl2 labelling was found to be suitable for in-vivo tracing allowing long detection periods. The labelled cells kept their highly tumourigenic potential in-vivo. Tumour volume development was visualised prior to manual palpation allowing tumour characterisation in early stages of the disease.

Influence of P-glycoprotein modulation on plasma concentrations and pharmacokinetics of orally administered prednisolone in dogs

OBJECTIVE To evaluate the impact of modulation of the membrane-bound efflux pump P-glycoprotein (P-gp) on plasma concentrations of orally administered prednisolone in dogs. ANIMALS 7 healthy adult Beagles. PROCEDURES Each dog received 3 treatments (control [no treatment], rifampicin [100 mg/d, PO, for 21 days, as an inducer of P-gp], and ketoconazole [100 mg/d, PO, for 21 days, as an inhibitor of P-gp]). A single dose of prednisolone (1 mg/kg, PO) was administered on day 8 of each treatment period. There was a 7-day washout period between subsequent treatments. Plasma concentrations of prednisolone were determined by use of a validated liquid chromatography-tandem mass spectrometry method. Duodenum and colon biopsy specimens were obtained endoscopically from anesthetized dogs and assessed for P-gp protein labeling via immunohistochemical analysis and mRNA quantification via real-time PCR assay. Total fecal collection was performed for evaluation of effects of P-gp modulation on digestion of nutrients. RESULTS Rifampicin treatment upregulated duodenal P-gp in dogs and significantly reduced the area under the plasma concentration-time curve of prednisolone. Ketoconazole typically downregulated expression of duodenal P-gp, with a subsequent increase in the area under the plasma concentration-time curve of prednisolone. There was a noticeable interindividual difference in response. Digestion of nutrients was not affected. CONCLUSIONS AND CLINICAL RELEVANCE Modulation of P-gp expression influenced plasma concentrations of prednisolone after oral administration in dogs. Thus, treatment response to prednisolone may be influenced by coadministration of P-gp-modulating medications or feed ingredients.

Expression of the high mobility group A1 (HMGA1) and A2 (HMGA2) genes in canine lymphoma: analysis of 23 cases and comparison to control cases.

Overexpression of high mobility group A (HMGA) genes was described as a prognostic marker in different human malignancies, but its role in canine haematopoietic malignancies was unknown so far. The objective of this study was to analyse HMGA1 and HMGA2 gene expression in lymph nodes of canine lymphoma patients. The expression of HMGA1 and HMGA2 was analysed in lymph node samples of 23 dogs with lymphoma and three control dogs using relative quantitative real-time RT-PCR. Relative quantity of HMGA1 was significantly higher in dogs with lymphoma compared with reference samples. HMGA2 expression did not differ between lymphoma and control dogs. With the exception of immunophenotype, comparison of disease parameters did not display any differences in HMGA1 and HMGA2 expression. The present findings indicate a role of HMGA genes in canine lymphoma. This study represents the basis for future veterinary and comparative studies dealing with their diagnostic, prognostic and therapeutic values.

Effects of high-mobility group a protein application on canine adipose-derived mesenchymal stem cells in vitro.

Multipotency and self-renewal are considered as most important features of stem cells to persist throughout life in tissues. In this context, the role of HMGA proteins to influence proliferation of adipose-derived mesenchymal stem cell (ASCs) while maintaining their multipotent and self-renewal capacities has not yet been investigated. Therefore, extracellular HMGA1 and HMGA2 application alone (10–200 ng/mL) and in combination with each other (100, 200 ng/mL each) was investigated with regard to proliferative effects on canine ASCs (cASCs) after 48 hours of cultivation. Furthermore, mRNA expression of multipotency marker genes in unstimulated and HMGA2-stimulated cASCs (50, 100 ng/mL) was analyzed by RT-qPCR. HMGA1 significantly reduced cASCs proliferation in concentrations of 10–200 ng/mL culture medium. A combination of HMGA1 and HMGA2 protein (100 and 200 ng/mL each) caused the same effects, whereas no significant effect on cASCs proliferation was shown after HMGA2 protein application alone. RT-qPCR results showed that expression levels of marker genes including KLF4, SOX2, OCT4, HMGA2, and cMYC mRNAs were on the same level in both HMGA2-protein-stimulated and -unstimulated cASCs. Extracellular HMGA protein application might be valuable to control proliferation of cASCs in context with their employment in regenerative approaches without affecting their self-renewal and multipotency abilities.