The purpose of this study was to identify novel marbling-related genes by comparison of the global gene expression in semitendinosus muscle of 15-month-old Limousin (LIM), Holstein-Friesian (HF) and ...Hereford (HER) bulls. Muscle of LIM was lean with low intramuscular fat (IMF) content (0.53%) unlike the marbled muscles of HER and HF characterized by higher amounts of IMF (1.10 and 0.81%, respectively). The comparison of muscle transcriptional profile between marbled and lean beef revealed significant differences in expression of 144 genes, presumably involved in consecutive stages of adipose tissue development, such as preadipocyte proliferation and differentiation, adipocyte maturation, lipid filling and lipid metabolism leading to increased IMF deposition and marbling development. Correlation coefficients and regression analysis for nine of them (gadd45a, pias3, ccrn4l, diras3, pou5f1, hoxa9, atp2a2 and pim1) validated by real-time qPCR confirmed their moderate-high correlation with IMF% and explained up to 70.5% of the total variability in IMF deposition in the bulls.
MicroRNAs (miRNAs) are small non‐coding RNAs that participate in the regulation of gene expression. Their role during mammary gland development is still largely unknown. In this study, we performed a ...microarray analysis to identify miRNAs associated with high mammogenic potential of the bovine mammary gland. We identified 54 significantly differentially expressed miRNAs between the mammary tissue of dairy (Holstein‐Friesian, HF) and beef (Limousin, LM) postpubertal heifers. Fifty‐two miRNAs had higher expression in the mammary tissue of LM heifers. The expression of the top candidate miRNAs (bta‐miR‐10b, bta‐miR‐29b, bta‐miR‐101, bta‐miR‐375, bta‐miR‐2285t, bta‐miR‐146b, bta‐let7b, bta‐miR‐107, bta‐miR‐1434‐3p) identified in the microarray experiment was additionally evaluated by qPCR. Enrichment analyses for targeted genes revealed that the major differences between miRNA expression in the mammary gland of HF versus LM were associated with the regulation of signalling pathways that are crucial for mammary gland development, such as TGF‐beta, insulin, WNT and inflammatory pathways. Moreover, a number of genes potentially targeted by significantly differentially expressed miRNAs were associated with the activity of mammary stem cells. These data indicate that the high developmental potential of the mammary gland in dairy cattle, leading to high milk productivity, depends also on a specific miRNA expression pattern.
Cancer stem cells (CSCs) display both unique self-renewal ability as well as the ability to differentiate into many kinds of cancer cells. They are supposed to be responsible for cancer initiation, ...recurrence and drug resistance. Despite the fact that a variety of methods are currently employed in order to target CSCs, little is known about the regulation of their phenotype and biology by miRNAs. The aim of our study was to assess miRNA expression in canine mammary cancer stem-like cells (expressing stem cell antigen 1, Sca-1; CD44 and EpCAM) sorted from canine mammary tumour cell lines (CMT-U27, CMT-309 and P114). In order to prove their stem-like phenotype, we conducted a colony formation assay that confirmed their ability to form colonies from a single cell. Profiles of miRNA expression were investigated using Agilent custom-designed microarrays. The results were further validated by real-time rt-PCR analysis of expression of randomly selected miRNAs. Target genes were indicated and analysed using Kioto Encyclopedia of Genes and Genomes (KEGG) and BioCarta databases. The results revealed 24 down-regulated and nine up-regulated miRNAs in cancer stem-like cells compared to differentiated tumour cells. According to KEGG and BioCarta databases, target genes (n=240) of significantly down-regulated miRNAs were involved in transforming growth factor-beta signaling, mitogen-activated protein kinases (MAPK) signaling pathway, anaplastic lymphoma receptor tyrosine kinase (ALK) and peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC1A) pathways. The analysis of single-gene overlapping with different pathways showed that the most important genes were: TGFBR1, TGFBR2, SOS1, CHUK, PDGFRA, SMAD2, MEF2A, MEF2C and MEF2D. All of them are involved in tumor necrosis factor-beta signaling and may indicate its important role in cancer stem cell biology. Increased expression of TGFBR2, SMAD2, MEF2A and MEF2D in canine mammary cancer stem-like cells was further confirmed by real-time-qPCR. The results of our study point at epigenetic differences between cancer stem-like cells and differentiated tumour cells, which may be important not only for veterinary medicine but also for comparative oncology.
Successful lactation depends on a controlled process of cell proliferation and mammary gland growth during pregnancy and cell death during involution. The main type of cell death responsible for ...involution of bovine mammary gland is apoptosis, but programmed cell death type II (PCD II)--autophagic cell death is also observed. The regulation of mammary epithelial cells (MEC) apoptosis occurs at three levels, the molecular regulators of autophagy have not been identified yet. There are possible correlations between both processes, because cells sharing morphological features of apoptosis and autophagy were found in involuting mammary gland. Autophagy seems to be cellular defense against starvation, when it fails, a secondary response of apoptotic cell death is triggered.
Skeletal myoblasts (SMs) transplantations are the focus of intense investigation due to their therapeutic potential for cardiac repair. Heart diseases are the main problem in humans worldwide, ...resulting very often in death of patients. Consequently, investigators sought methods which could improve myocardial function in patients with cardiac failure. Stem cells therapy belongs to one of the highly promising and the most realistic methods of treating myocardial infarction. Despite the fact that many cell types were transplanted into damaged myocardium, autologous skeletal myoblasts seem to be the most encouraging cell source for myocardial repair due to their myogenic and contractile phenotype, biochemical and functional similarities to cardiac cells, high proliferative capacity in vitro and resistance to ischemia. Moreover, engrafted myoblasts could repopulate post-infarction scar and improve cardiac function. Unfortunately, transplanted SMs do not functionally or electrically integrate with the host myocardium, which could also cause ventricular arrhythmias, therefore, the use of these cells still remains a subject of intensive studies aiming at improvement of this therapeutic method. In this paper we recapitulate the current state of knowledge concerning the use of skeletal myoblasts in the treatment of post-infarction scar tissue, and discuss the problems resulting from their applications.
Insulin-like growth factor-I is involved in mammary gland development, promoting proliferation and inhibiting apoptosis of mammary epithelial cells (MECs). Mitogenic actions of IGF-I are mainly ...mediated by the phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway. We have found that in the presence of IGF-I bovine BME-UV1 MECs cultured on reconstituted basement membrane form large spheroids with disrupted polarity and no cavity in the center. These cells showed enhanced phosphorylation of Akt, decreased level of cleaved caspase-3, and sustained proliferative activity throughout the 16-d period of 3-dimensional culture. Inhibition of the PI3K/Akt pathway by a specific inhibitor of PI3K, LY294002, resulted in the restoration of the normal acinar phenotype. However, this effect was noted only when LY294002 was added in the second week of 3-dimensional culture, which corresponded with the time of cell cycle arrest and polarity formation under control conditions. Normal development of acini was also obtained when BME-UV1 cells were treated simultaneously with IGF-I and 17β-estradiol. The addition of 17β-estradiol regulated Akt activation, enabling the subsequent initiation of polarization processes. 17β-Estradiol also increased the level of IGFBP-3 protein in MECs cultured on Matrigel in the presence of IGF-I. The presented results indicate important interactions between signaling pathways activated by estrogen and IGF-I, which regulate alveologenesis in bovine mammary gland.
Apoptosis - programmed cell death (PCD) type I is physiological process responsible for cell loss during mammary gland involution after natural weaning or litter removal in rodents, after weaning in ...sow and during drying off in goat and cow. The regulation of mammary epithelial cell (MEC) apoptosis in bovine mammary gland occurs at three levels. The first level comprises intracellular regulatory proteins, e.g. Bcl-2 family death promoters and inhibitors. The second level is represented by intramammary inductors of apoptosis, e.g. FIL, IGFBPs, Fas ligand, TGF-betas. The expression and activity of these auto/paracrine inductors of apoptosis is controlled and modulated by the third level factors, e.g. systemic galactopoetic hormones, nutrition, reproductive status and milking management. Our recent study proved that apoptosis in involuting bovine mammary gland is accompanied by increased intensity of autophagy, regarded as a cytoprotective process but in advanced stage as a PCD type II. Moreover, we have reported for the first time the ability of TGF-beta(1) to induce both apoptosis and autophagy in bovine BME-UV1 MEC. Much more pronounced heterogeneity of PCD was observed when breast cancer cells were exposed to anticancer drugs. The primary responses of breast cancer MCF-7 cells to camptothecin (CPT) are apoptosis and autophagy (as a cytoprotective process). In this case autophagy occurs in cells which are resistant to apoptosis as a tool of cancer cell survival. The fail-safe responses of breast cancer cells to persisting CPT-induced stress are apoptosis accompanied by morphological and biochemical features of autophagy or type II PCD with advanced subcellular degradation. The threshold between autophagy as a cytoprotective process (reversible) or PCD (irreversible) is difficult to establish and probably depends on the extent of degradation of cellular components. Proapoptotic protein Bid may serve as a molecular switch between apoptosis and autophagy. Bid knock down in MCF-7 cells exposed to CPT leads to a shift of cell death from apoptosis to autophagy. Since bid and other proapoptotic genes undergo mutations in malignant cells, the ability of cancer cells commitment to autophagy may have important therapeutic implications.
Mammary epithelial cells (MECs) are characterized by specific spatial architecture with several distinguishing features such as: polarized morphology, specialized cell-cell contacts, and attachment ...to an underlying basement membrane. Three dimensional (3D) basement membrane cultures provide a unique opportunity to model the architecture of epithelium in vitro. The aim of this study was to characterize the growth of bovine mammary epithelial cell line BME-UV1 in 3D culture on Matrigel and identification of differently expressed genes in bovine MECs forming polarized structures in comparison to conventional monolayer (2D) cell culture. We demonstrate that BME-UV1 cells grown on Matrigel form polarized acinar structures during 16 days of culture. A microarray study has proven that the difference in spatial architecture between MECs cultured in monolayer and 3D system is reflected by differences in transcriptomic profile. Microarray data analysis showed 40 differentially expressed genes with statistical significance (p<0.05) and characterized biological functions. Identified genes comprised of cytoskeletal proteins, extracellular matrix components, kinases such as: Rac serine/threonine kinase, SRPK, protooncogene tyrosine-protein kinase ABL1, uridine cytidine kinase and proteins with nucleic acid binding / transcription factor activity. Products of those genes are involved in processes which are known to participate in regulating mammary gland polarization and function.
Role of autophagy in mammary gland development Gajewska, M; Sobolewska, A; Kozlowski, M ...
Journal of physiology and pharmacology : an official journal of the Polish Physiological Society
59 Suppl 9
Journal Article
Recenzirano
Autophagy is a highly conserved catabolic process responsible for degradation and recycling of long-lived proteins and organelles by lysosomes. This degradative pathway, together with proteasome ...system is particularly important during development and under certain environmental stress conditions. This review summarizes the latest achievements of studies aiming to explore the role of autophagy in development and differentiation of eukaryotic cells. It shows the importance of this process in the development of lower eukaryotic organisms such as Dicyostelium discoideum, and Caenorhabditis elegans, as well as functions of autophagy and autophagy related genes (Atg) in development and differentiation of higher eukaryotic organisms. The review is focused on the results of studies conducted on mammary gland, as it is a good model for studying the mechanisms controlling higher eukaryotic organisms' development. Studies have shown that autophagy is involved in the removal of epithelial cells during formation of alveolar structures, indicating its role in mammogenesis. There are also evidences of involvement of Atg's in epithelial tumors development. Context dependent manipulations of autophagic pathways may create more effective anticancer therapies in the future.
Matrix Metalloproteinase-2 (MMP-2) is an enzyme that degrades components of the extracellular matrix and thus plays a pivotal role in cell migration during physiological and pathological processes ...(e.g. gastric, pancrcreatic, prostate, and breast cancer). MMP-2 expression is dependent on extracellular matrix metalloproteinase inducer (EMMPRIN), Her2/neu, growth factors, cytokines, and hormones. Pro-MMP-2 activation needs MT1-MMP and TIMP-2 contribution. The active forms of MMPs subsequently release a cascade of activation of the remaining pro-MMPs. Inactivation of the physiological function of MMPs, or even pro-MMPs, is accomplished by non-covalent TIMP binding. The detection of active MMP-2 alone or the rate of pro-MMP-2 and active MMP-2 is considered a very sensitive indicator of cancer metastasis. Modulation of MMP-2 expression and activation through specific inhibitors and activators may thus provide a new mechanism for breast cancer treatment. Degradation of the cellular network established by adhesion molecules such as E-cadherin or ALCAM/CD166 causes tumor tissue relaxation, increases metastasis, and correlates with shortened survival in patients with primary breast carcinoma. A low level of MMP-2 is linked to favorable prognosis in patients with a hormone receptor-negative tumor, usually associated with high risk. Blocking MMP-2 secretion and activation during breast carcinoma development may decrease metastasis. Besides zoledronic acid and bisphosphonates, the new synthetic metalloproteinase blockers (MMPIs) batimastat, marimastat, and tetracycline derivates have been investigated in anticancer therapy. Recent research shows that modified synthetic siRNA targeting TIMP-2 may also regulate the balance between MMPs and TIMP-2 and thus decrease the degradation of extracellular matrix and prevent distant metastasis.
