Transcriptomic profile of semitendinosus muscle of bulls of different breed and performance.

The aim of the study was to compare the transcriptomic profiles of fully differentiated skeletal muscle derived from bulls belonging to different breeds of varying performance. Microarray analyses were performed to determine the differences in the expression profiles of genes between semitendinosus muscles of 15-month-old beef-breed bulls (Limousin-LIM and Hereford-HER) and dairy-breed bulls (Holstein Friesian-HF). These analyses allowed for the identification of those genes the expression of which is similar and characteristic of fully differentiated muscle in beef breeds, but differs in skeletal muscle of a typical dairy breed. The analysis revealed 463 transcripts showing similar expression in the semitendinosus muscle of beef breeds (LIM/HER), in comparison with the dairy breed (HF). Among the identified genes, 227 were upregulated and 236 were downregulated in beef breeds. The ontological analyses revealed that the largest group of genes similarly expressed in LIM and HER was involved in the processes of protein metabolism and development of muscle organ. In beef breeds, some genes involved in protein synthesis and proteolysis showed an upregulation, including ctsd, ctsf, fhl2, fhl3, fst, sirt1, and trim63, whereas some were downregulated, including bmpr1a, bmpr2, mstn, smad2, hspa8, gsk3ß, and tgfß2. The expression of the chosen genes was confirmed by RT-qPCR technique. Thus, it can be assumed that the identified genes involved in the regulation of growth and development of muscle tissue and the processes of protein metabolism in the examined cattle breeds may be responsible for the greater gain of muscle mass in beef-breed bulls.

Transcriptomic Profile of Primary Culture of Skeletal Muscle Cells Isolated from Semitendinosus Muscle of Beef and Dairy Bulls.

The aim of the study was to identify differences in the transcriptomic profiles of primary muscle cell cultures derived from the semitendinosus muscle of bulls of beef breeds (Limousin (LIM) and Hereford (HER)) and a dairy breed (Holstein-Friesian (HF)) (n = 4 for each breed). Finding a common expression pattern for proliferating cells may point to such an early orientation of the cattle beef phenotype at the transcriptome level of unfused myogenic cells. To check this hypothesis, microarray analyses were performed. The analysis revealed 825 upregulated and 1300 downregulated transcripts similar in both beef breeds (LIM and HER) and significantly different when compared with the dairy breed (HF) used as a reference. Ontological analyses showed that the largest group of genes were involved in muscle organ development. Muscle cells of beef breeds showed higher expression of genes involved in myogenesis (including erbb-3, myf5, myog, des, igf-1, tgfb2) and those encoding proteins comprising the contractile apparatus (acta1, actc1, myh3, myh11, myl1, myl2, myl4, tpm1, tnnt2, tnnc1). The obtained results confirmed our hypothesis that the expression profile of several groups of genes is common in beef breeds at the level of proliferating satellite cells but differs from that observed in typical dairy breeds.

Bridging the Gap between Alzheimer's Disease and Alzheimer's-like Diseases in Animals.

The average life span steadily grows in humans and in animals kept as pets or left in sanctuaries making the issue of elderly-associated cognitive impairment a hot-spot for scientists. Alzheimer's disease (AD) is the most prevalent cause of progressive mental deterioration in aging humans, and there is a growing body of evidence that similar disorders (Alzheimer's-like diseases, ALD) are observed in animals, more than ever found in senescent individuals. This review reveals up to date knowledge in pathogenesis, hallmarks, diagnostic approaches and modalities in AD faced up with ALD related to different animal species. If found at necropsy, there are striking similarities between senile plaques (SP) and neurofibrillary tangles (NFT) in human and animal brains. Also, the set of clinical symptoms in ALD resembles that observed in AD. At molecular and microscopic levels, the human and animal brain histopathology in AD and ALD shows a great resemblance. AD is fatal, and the etiology is still unknown, although the myriad of efforts and techniques were employed in order to decipher the molecular mechanisms of disease onset and its progression. Nowadays, according to an increasing number of cases reported in animals, apparently, biochemistry of AD and ALD has a lot in common. Described observations point to the importance of extensive in vivo models and extensive pre-clinical studies on aging animals as a suitable model for AD disease.

[Molecular actions of 17ß-estradiol and progesterone and their relationship with cellular signaling pathways]. / Molekularne aspekty dzialania 17ß-estradiolu i progesteronu w komókowych szlakach sygnalwych.

SEX STEROIDS: 17ß-estradiol and progesterone play a major role in modulation of reproductive functions of the organism and participate in regulation of a broad spectrum of cellular processes in target cells via their specific receptors. Our understanding of molecular mechanisms of sex steroid action has significantly developed over the last years. Apart from the well-established effect of sex steroids on regulation of gene expression, some rapid nongenomic mechanisms have been identified, which are involved in modulation of the activity of several cellular, membrane-bound and cytoplasmic regulatory proteins. 17ß-estradiol and progesterone regulate several signal transduction pathways, which involve activation of enzymes such as mitogen-activated protein kinases (MAPK), phosphatidylinositol 3-kinase and tyrosine kinases. Biological effects of sex steroids action constitute a complex interplay of genomic and nongenomic mechanisms, and depend on the physiological and genetic context of the target cell. Understanding the molecular mechanisms of sex steroids action is therefore important and may broaden our knowledge about their role in both physiological and pathological processes. This review provides a comprehensive insight into the molecular actions of 17ß-estradiol and progesterone, aiming to present the role of these sex steroids in regulation of cellular signaling pathways.

Functional interactions between 17 ß -estradiol and progesterone regulate autophagy during acini formation by bovine mammary epithelial cells in 3D cultures.

Mammary gland epithelium forms a network of ducts and alveolar units under control of ovarian hormones: 17-beta-estradiol (E2) and progesterone (P4). Mammary epithelial cells (MECs) cultured on reconstituted basement membrane (rBM) form three-dimensional (3D) acini composed of polarized monolayers surrounding a lumen. Using the 3D culture of BME-UV1 bovine MECs we previously demonstrated that autophagy was induced in the centrally located cells of developing spheroids, and sex steroids increased this process. In the present study we showed that E2 and P4 enhanced the expression of ATG3, ATG5, and BECN1 genes during acini formation, and this effect was accelerated in the presence of both hormones together. The stimulatory action of E2 and P4 was also reflected by increased levels of Atg5, Atg3, and LC3-II proteins. Additionally, the activity of kinases involved in autophagy regulation, Akt, ERK, AMPK, and mTOR, was examined. E2 + P4 slightly increased the level of phosphorylated AMPK but diminished phosphorylated Akt and mTOR on day 9 of 3D culture. Thus, the synergistic actions of E2 and P4 accelerate the development of bovine mammary acini, which may be connected with stimulation of ATGs expression, as well as regulation of signaling pathways (PI3K/Akt/mTOR; AMPK/mTOR) involved in autophagy induction.

Comparison of stem/progenitor cell number and transcriptomic profile in the mammary tissue of dairy and beef breed heifers.

Bovine mammary stem cells (MaSC) are a source of ductal and lobulo-alveolar tissue during the development of the mammary gland and its remodeling in repeating lactation cycles. We hypothesize that the number of MaSC, their molecular properties, and interactions with their niche may be essential in order to determine the mammogenic potential in heifers. To verify this hypothesis, we compared the number of MaSC and the transcriptomic profile in the mammary tissue of 20-month-old, non-pregnant dairy (Holstein-Friesian, HF) and beef (Limousin, LM) heifers. For the identification and quantification of putative stem/progenitor cells in mammary tissue sections, scanning cytometry was used with a combination of MaSC molecular markers: stem cell antigen-1 (Sca-1) and fibronectin type III domain containing 3B (FNDC3B) protein. Cytometric analysis revealed a significantly higher number of Sca-1(pos)FNDC3B(pos) cells in HF (2.94 ± 0.35%) than in LM (1.72 ± 0.20%) heifers. In HF heifers, a higher expression of intramammary hormones, growth factors, cytokines, chemokines, and transcription regulators was observed. The model of mammary microenvironment favorable for MaSC was associated with the regulation of genes involved in MaSC maintenance, self-renewal, proliferation, migration, differentiation, mammary tissue remodeling, angiogenesis, regulation of adipocyte differentiation, lipid metabolism, and steroid and insulin signaling. In conclusion, the mammogenic potential in postpubertal dairy heifers is facilitated by a higher number of MaSC and up-regulation of mammary auto- and paracrine factors representing the MaSC niche.

Macrophages mediate a switch between canonical and non-canonical Wnt pathways in canine mammary tumors.

OBJECTIVE: According to the current hypothesis, tumor-associated macrophages (TAMs) are "corrupted" by cancer cells and subsequently facilitate, rather than inhibit, tumor metastasis. Because the molecular mechanisms of cancer cell-TAM interactions are complicated and controversial we aimed to better define this phenomenon. METHODS AND RESULTS: Using microRNA microarrays, Real-time qPCR and Western blot we showed that co-culture of canine mammary tumor cells with TAMs or treatment with macrophage-conditioned medium inhibited the canonical Wnt pathway and activated the non-canonical Wnt pathway in tumor cells. We also showed that co-culture of TAMs with tumor cells increased expression of canonical Wnt inhibitors in TAMs. Subsequently, we demonstrated macrophage-induced invasive growth patterns and epithelial-mesenchymal transition of tumor cells. Validation of these results in canine mammary carcinoma tissues (n = 50) and xenograft tumors indicated the activation of non-canonical and canonical Wnt pathways in metastatic tumors and non-metastatic malignancies, respectively. Activation of non-canonical Wnt pathway correlated with number of TAMs. CONCLUSIONS: We demonstrated that TAMs mediate a "switch" between canonical and non-canonical Wnt signaling pathways in canine mammary tumors, leading to increased tumor invasion and metastasis. Interestingly, similar changes in neoplastic cells were observed in the presence of macrophage-conditioned medium or live macrophages. These observations indicate that rather than being "corrupted" by cancer cells, TAMs constitutively secrete canonical Wnt inhibitors that decrease tumor proliferation and development, but as a side effect, they induce the non-canonical Wnt pathway, which leads to tumor metastasis. These data challenge the conventional understanding of TAM-cancer cell interactions.

Migrastatin analogues inhibit canine mammary cancer cell migration and invasion.

BACKGROUND: Cancer spread to other organs is the main cause of death of oncological patients. Migration of cancer cells from a primary tumour is the crucial step in the complex process of metastasis, therefore blocking this process is currently the main treatment strategy. Metastasis inhibitors derived from natural products, such as, migrastatin, are very promising anticancer agents. Thus, the aim of our study was to investigate the effect of six migrastatin analogues (MGSTA-1 to 6) on migration and invasion of canine mammary adenocarcinoma cell lines isolated from primary tumours and their metastases to the lungs. Canine mammary tumours constitute a valuable tool for studying multiple aspect of human cancer. RESULTS: OUR RESULTS SHOWED THAT TWO OF SIX FULLY SYNTHETIC ANALOGUES OF MIGRASTATIN: MGSTA-5 and MGSTA-6 were potent inhibitors of canine mammary cancer cells migration and invasion. These data were obtained using the wound healing test, as well as trans-well migration and invasion assays. Furthermore, the treatment of cancer cells with the most effective compound (MGSTA-6) disturbed binding between filamentous F-actin and fascin1. Confocal microscopy analyses revealed that treatment with MGSTA-6 increased the presence of unbound fascin1 and reduced co-localization of F-actin and fascin1 in canine cancer cells. Most likely, actin filaments were not cross-linked by fascin1 and did not generate the typical filopodial architecture of actin filaments in response to the activity of MGSTA-6. Thus, administration of MGSTA-6 results in decreased formation of filopodia protrusions and stress fibres in canine mammary cancer cells, causing inhibition of cancer migration and invasion. CONCLUSION: Two synthetic migrastatin analogues (MGSTA-5 and MGSTA-6) were shown to be promising compounds for inhibition of cancer metastasis. They may have beneficial therapeutic effects in cancer therapy in dogs, especially in combination with other anticancer drugs. However, further in vivo studies are required to verify this hypothesis.

Relationship between clinical data and gene expression in the HER2/ErbB2-dependent signaling pathway in patients with acute heart failure.

Anticancer treatment with the human epidermal growth factor receptor (HER) 2 inhibitors can lead to significant myocardial dysfunction. The primary aim of the study was to estimate the possible association between gene expression in the ErbB signaling pathway and selected clinical event data in patients with acute heart failure. Twenty-four patients (19 males), aged 68.6 ± 12.3 years, were diagnosed and treated due to acute heart failure. The globaltest method was used for the correlation between blood nuclear cells' gene expression in the ErbB pathway (KEGG pathway id 04012) and important clinical data. Decreased expression of ErbB2/HER2 was found to be associated with the release of troponin and the need for inotropic support, whereas decreased neuregulin 1 (NRG1) expression was found to be associated with a decrease of ejection fraction below 40 % (globaltest p-value < 0.05). In summary, the ErbB signaling pathway and, especially, HER2/ErbB2 receptor expression are significantly associated with some of the recognized, clinically significant parameters of patients with acute heart failure. Evaluation of the molecular function of the HER2 receptor may be essential for the prognosis and targeted therapy of heart diseases.

Five markers useful for the distinction of canine mammary malignancy.

BACKGROUND: Spontaneous canine mammary tumors constitute a serious clinical problem. There are significant differences in survival between cases with different tumor grades. Unfortunately, the distinction between various grades is not clear. A major problem in evaluating canine mammary cancer is identifying those, that are "truly" malignant. That is why the aim of our study was to find the new markers of canine malignancy, which could help to diagnose the most malignant tumors. RESULTS: Analysis of gene expression profiles of canine mammary carcinoma of various grade of malignancy followed by the boosted tree analysis distinguished a `gene set`. The expression of this gene set (sehrl, zfp37, mipep, relaxin, and magi3) differs significantly in the most malignant tumors at mRNA level as well as at protein level. Despite this `gene set` is very interesting as an additional tool to estimate canine mammary malignancy, it should be validated using higher number of samples. CONCLUSIONS: The proposed gene set can constitute a `malignancy marker` that could help to distinguish the most malignant canine mammary carcinomas. These genes are also interesting as targets for further investigations and therapy. So far, only two of them were linked with the cancer development.

Expression and role of PGP, BCRP, MRP1 and MRP3 in multidrug resistance of canine mammary cancer cells.

BACKGROUND: In both women and female dogs, the most prevalent type of malignant neoplasm is the spontaneous mammary tumor. In dogs, half of these are malignant. The treatment of choice for the canine patients is surgical mastectomy. Unfortunately, it often fails in high-risk, locally invasive mammary tumors as of during the time of the surgery the micro-metastases are present. Moreover, there are neither large studies conducting to prove of the benefit from the chemotherapy in dogs nor established chemotherapy treatment protocols available. Additionally, the effectiveness of each individual chemotherapeutic agent and drug resistance of canine mammary cancer have not yet been characterized. That has become the aim of our study, to assess the expression of PGP, BCRP, MRP1 and MRP3 in canine mammary cancer cell lines and to investigate their role in cancer resistance to vinblastine, cisplatin and cyclophosphamide with using RNAi approach. RESULTS: The results suggested that in canine mammary cancer, the vinblastine efflux was mediated by PGP and MRP1 proteins, cisplatin efflux was mediated by all four examined efflux pumps (PGP, BCRP, MRP1 and MRP3), whereas cyclophosphamide resistance was related to BCRP activity. RNAi silencing of these efflux pumps significantly decreased IC50 doses of the examined drugs in canine mammary carcinoma cells. CONCLUSIONS: Our results have indicated the treatment of cells involving use of the siRNA targeting efflux pumps could be a beneficial approach in the future.

CSF-1R as an inhibitor of apoptosis and promoter of proliferation, migration and invasion of canine mammary cancer cells.

BACKGROUND: Tumor-associated macrophages (TAMs) have high impact on the cancer development because they can facilitate matrix invasion, angiogenesis, and tumor cell motility. It gives cancer cells the capacity to invade normal tissues and metastasize. The signaling of colony-stimulating factor-1 receptor (CSF-1R) which is an important regulator of proliferation and differentiation of monocytes and macrophages regulates most of the tissue macrophages. However, CSF-1R is expressed also in breast epithelial tissue during some physiological stages i.g.: pregnancy and lactation. Its expression has been also detected in various cancers. Our previous study has showed the expression of CSF-1R in all examined canine mammary tumors. Moreover, it strongly correlated with grade of malignancy and ability to metastasis. This study was therefore designed to characterize the role of CSF-1R in canine mammary cancer cells proliferation, apoptosis, migration, and invasion. As far as we know, the study presented hereby is a pioneering experiment in this field of veterinary medicine. RESULTS: We showed that csf-1r silencing significantly increased apoptosis (Annexin V test), decreased proliferation (measured as Ki67 expression) and decreased migration ("wound healing" assay) of canine mammary cancer cells. Treatment of these cells with CSF-1 caused opposite effect. Moreover, csf-1r knock-down changed growth characteristics of highly invasive cell lines on Matrigel matrix, and significantly decreased the ability of these cells to invade matrix. CSF-1 treatment increased invasion of cancer cells. CONCLUSION: The evidence of the expression and functional role of the CSF-1R in canine mammary cancer cells indicate that CSF-1R targeting may be a good therapeutic approach.

Gene expression profiles in canine mammary carcinomas of various grades of malignancy.

BACKGROUND: The frequency of mammary malignancies in canine patients is even three times over than in human. In various types of cancer different intracellular signalling pathways are perturbed, thus the patients with pathologically the same type of cancer often have dissimilar genetic defects in their tumours and respond in a heterogeneous manner to anticancer treatment. That is why the objective of the hereby study was to assess the gene expression profiles in canine mammary carcinomas (in unsupervised manner) classified by pathologists as grade 1 (well differentiated), grade 2 (moderately differentiated) and grade 3 (poorly differentiated) and compare their molecular and pathological classifications. RESULTS: Our unsupervised analysis classified the examined tissues into three groups. The first one significantly differed from the others and consisted of four carcinomas of grade 3 and one carcinoma of grade 2. The second group consisted of four grade 1 carcinomas. The very heterogeneous (based on their pathological parameters) group was the last one which consisted of two grade 1 carcinomas, two grade 3 carcinomas and five grade 2 carcinomas. Hierarchical dendrogram showed that the most malignant tumour group had significantly distinct gene expression. CONCLUSIONS: Molecular classification of canine mammary tumours is not identical with pathological classification. In our opinion molecular and pathological characterization of canine mammary malignancy can complement one another. However, furthers studies in this field are required.

A role of ghrelin in canine mammary carcinoma cells proliferation, apoptosis and migration.

BACKGROUND: Ghrelin is a natural ligand of the growth hormone secretagogue receptor (GHS-R). They are often co-expressed in multiple human tumors and related cancer cell lines what can indicate that the ghrelin/GHS-R axis may have an important role in tumor growth and progression. However, a role of ghrelin in canine tumors remains unknown. Thus, the aim of our study was two-fold: (1) to assess expression of ghrelin and its receptor in canine mammary cancer and (2) to examine the effect of ghrelin on carcinoma cells proliferation, apoptosis, migration and invasion. The expression of ghrelin and its receptor in canine mammary cancer tissues and cell lines (isolated from primary tumors and their metastases) was examined using Real-time qPCR and immunohistochemistry. For apoptosis analysis the Annexin V and propidium iodide dual staining was applied whereas cell proliferation was evaluated by MTT assay and BrdU incorporation test. The influence of ghrelin on cancer cells migration and invasion was assessed using Boyden chamber assays and wound healing assay. RESULTS: The highest expression of ghrelin was observed in metastatic cancers whereas the lowest expression of ghrelin receptor was detected in tumors of the 3rd grade of malignancy. Higher expression of ghrelin and its receptor was detected in cancer cell lines isolated from metastases than in cell lines isolated from primary tumors. In vitro experiments demonstrated that exposure to low doses of ghrelin stimulates cellular proliferation, inhibits apoptosis and promotes motility and invasion of canine mammary cancer cells. Growth hormone secretagogue receptor inhibitor ([D-Lys3]-GHRP6) as well as RNA interference enhances early apoptosis. CONCLUSION: The presence of ghrelin and GHS-R in all of the examined canine mammary tumors may indicate their biological role in cancer growth and development. Our experiments conducted in vitro confirmed that ghrelin promotes cancer development and metastasis.

White blood cell transcriptome correlates with renal function in acute heart failure.

It is notoriously difficult to classify patients with acute heart failure (AHF) because of variations in clinical presentation, different etiologies, the impact of comorbidities, and variable prognoses. In this study, we used DNA whole-genome microarrays to classify 24 patients with AHF based on the transcriptome of their peripheral blood nuclear cells. The main purpose was to verify whether any transcriptomic sub-clusters had clinical correlations. We identified two distinct groups of transcriptomic profiles that correlated with normal (1.125 mg/dL) and increased (1.783 mg/dL) mean blood creatinine concentrations. These two subgroups of patients (n = 12) differed in the expression of more than 6000 genes and 108 signaling pathways. The most significant regulated signaling pathway was the aldosterone-regulated sodium reabsorption pathway and the most significant regulated genes included the angiotensin-converting enzyme gene. This suggests that kidney impairment in patients with AHF is related to dysregulation of the renin-angiotensin-aldosterone system. The interesting findings of our study were the significant differences in expression of genes belonging to the aldosterone-regulated signaling pathway: Na+/K+ transporting ATPase and NEDD4L (neuronal precursor cell expressed developmentally down-regulated 4-like) between patients with and without renal dysfunction. Future studies of blood-cell transcriptomic profiles in patients with AHF will provide further insights into the molecular pathogenesis of this cardiorenal disorder.

The gene expression profiles of canine mammary cancer cells grown with carcinoma-associated fibroblasts (CAFs) as a co-culture in vitro.

BACKGROUND: It is supposed that fibroblasts present in tumour microenvironment increase cancer invasiveness and its ability to metastasize but the mechanisms have not been clearly defined yet. Thus, the current study was designed to assess changes in gene expression in five various cancer cell lines grown as a co-culture with the carcinoma-associated fibroblasts (CAFs) in vitro. RESULTS: A carcinoma-associated fibroblast cell line was isolated from a canine mammary cancer. Then, a co-culture of cancer cells with the CAFs was established and maintained for 72 hrs. Having sorted the cells, a global gene expression in cancer cells using DNA microarrays was examined. The analysis revealed an up-regulation of 100 genes and a down-regulation of 106 genes in the cancer cells grown as a co-culture with the CAFs in comparison to control conditions. The PANTHER binomial statistics tool was applied to determine statistically over-manifested pathways (p < 0.05). Bulk of the up-regulated genes are involved in the adhesion, the angiogenesis, the epithelial-mesenchymal transition (EMT) and generally take part in the developmental processes. These results were further confirmed using real-time qPCR. Moreover, a wound-healing assay and growth characteristics on Matrigel matrix showed that CAFs increase cancer cell migration and matrix invasion. CONCLUSION: The results of the current study showed that the co-culturing of cancer cells and the CAFs caused significant changes to the cancer gene expression. The presence of the CAFs in a microenvironment of cancer cells promotes adhesion, angiogenesis and EMT.

Global gene expression profiles of canine macrophages and canine mammary cancer cells grown as a co-culture in vitro.

BACKGROUND: Solid tumours comprise various cells, including cancer cells, resident stromal cells, migratory haemopoietic cells and other. These cells regulate tumour growth and metastasis. Macrophages constitute probably the most important element of all interactions within the tumour microenvironment. However, the molecular mechanism, that guides tumour environment, still remains unknown. Exploring the underlying molecular mechanisms that orchestrate these phenomena has been the aim of our study.A co-culture of canine mammary cancer cells and macrophages was established and maintained for 72 hrs. Having sorted the cells, gene expression in cancer cells and macrophages, using DNA microarrays, was examined. The results were confirmed using real-time qPCR and confocal microscopy. Moreover, their ability for migration and invasion has been assessed. RESULTS: Microarray analysis showed that the up-regulated genes in the cancer cell lines are involved in 15 highly over-manifested pathways. The pathways that drew our diligent attention included: the inflammation pathway mediated by chemokine and cytokine, the Toll receptor signalling pathway and the B cell activation. The up-regulated genes in the macrophages were involved in only 18 significantly over-manifested pathways: the angiogenesis, the p53 pathway feedback loops2 and the Wnt signalling pathway. The microarray analysis revealed that co-culturing of cancer cells with macrophages initiated the myeloid-specific antigen expression in cancer cells, as well as cytokine/chemokine genes expression. This finding was confirmed at mRNA and protein level. Moreover, we showed that macrophages increase cancer migration and invasion. CONCLUSIONS: The presence of macrophages in the cancer environment induces acquisition of the macrophage phenotype (specific antigens and chemokines/cytokines expression) in cancer cells. We presumed that cancer cells also acquire other myeloid features, such as: capabilities of cell rolling, spreading, migration and matrix invasion (what has also been confirmed by our results). It may, perhaps, be the result of myeloid-cancer cell hybrid formation, or cancer cells mimicking macrophages phenotype, owing to various proteins secreted by macrophages.

Role and regulation of autophagy in the development of acinar structures formed by bovine BME-UV1 mammary epithelial cells.

Autophagy is a catabolic process providing an alternative energy source for cells under stressful conditions such as starvation, growth factor deprivation or hypoxia. During involution of the bovine mammary gland autophagy is induced in mammary epithelial cells (MECs) as a survival mechanism, and is tightly regulated by hormones and growth factors necessary for gland development. In the present study we adapted the three-dimensional culture model to investigate the role of autophagy during formation of alveoli-like structures by bovine BME-UV1 MECs grown on extracellular matrix (ECM) components. Using confocal microscopy and Western-blot analyses of autophagic and apoptotic markers: LC3, and cleaved caspase-3, we showed that autophagy was induced in centrally localized cells within the developing acini. These cells lacked a direct contact with ECM, and formed a distinct population from the outer layer of cells. Induction of autophagy preceded apoptosis, but did not inhibit the formation of a hollow lumen. In the presence of steroid hormones: 17ß-estradiol and progesterone, although autophagy was augmented, acini formation proceeded normally. In contrast, the major lactogenic hormone: prolactin, which supports functional differentiation of alveoli, did not alter induction of autophagy within the spheroids. BME-UV1 cells cultured on Matrigel in the presence of growth factors IGF-I and EGF formed larger, underdeveloped acini without lumens due to caspase-3 inhibition, and sustained autophagy in the centre of the spheroids, while TGF-ß1 accelerated apoptosis, and increased autophagy significantly. Our observations suggest that sex steroids 17ß-estradiol and progesterone, as well as growth factor TGF-ß1 may regulate the development of the bovine mammary gland by inducing autophagy in addition to regulating proliferation and apoptosis of MECs. These data indicate that autophagy may play an important role during alveolargenesis.

Density of Gr1-positive myeloid precursor cells, p-STAT3 expression and gene expression pattern in canine mammary cancer metastasis.

The very recent studies on human and mice models have indicated an important role of myeloid precursor cells (progenitors or not fully differentiated cells that express the Gr1 antigen also called Gr1-positive myeloid suppressor cells) in the tumor progression and metastasis. They are thought to suppress the immune system and promote angiogenesis via Signal transducer and activator of transcription 3 (STAT3) activation. As of now there is no data available on the correlation of Gr1-positive cell number, phosphorylated STAT3 (p-STAT3) expression and cancer ability to metastasis. Thus, we counted the myeloid precursor cell number and analyzed p-STAT3 expression in 50 canine mammary tumors that gave local/distant metastases and did not metastasize. We showed that the number of Gr1-positive cells and p-STAT3 expression are significantly higher (p < 0.001) in the metastatic tumors than in the non-metastatic ones. We also observed higher expression of p-STAT3 in the canine mammary cancer cell lines with metastatic potential than in other cell lines (p < 0.001). Moreover, the number of myeloid precursors and p-STAT3 expression in metastatic tumors correlate strongly. The tumor infiltrating myeloid precursor cells may invigorate the STAT3 activity (probably via vascular endothelial growth factor - VEGF) that contributes to the tumor angiogenesis and furthermore tumor`s ability to metastasize. The analysis of gene expression in canine mammary cancer cell lines with metastatic potential indicated that semaphorin 3B (SEMA3B) and neuropilin receptors (NRP) may also be important elements in this process. Thus, we discuss the possible interactions within the tumor that may be required for cancer metastatis.