Murine amniotic fluid stem cells contribute mesenchymal but not epithelial components to reconstituted mammary ducts

Introduction: Amniotic fluid harbors cells indicative of all three germ layers, and pluripotent fetal amniotic fluid stem cells (AFSs) are considered potentially valuable for applications in cellular therapy and tissue engineering. We investigated whether it is possible to direct the cell fate of AFSs in vivo by transplantation experiments into a particular microenvironment, the mammary fat pad. This microenvironment provides the prerequisites to study stem cell function and the communication between mesenchymal and epithelial cells. On clearance of the endogenous epithelium, the ductal tree can be reconstituted by the transfer of exogenously provided mammary stem cells. Analogously, exogenously provided stem cells from other tissues can be investigated for their potential to contribute to mammary gland regeneration. Methods: We derived pluripotent murine AFSs, measured the expression of stem cell markers, and confirmed their in vitro differentiation potential. AFSs were transplanted into cleared and non cleared fat pads of immunocompromised mice to evaluate their ability to assume particular cell fates under the instructive conditions of the fat-pad microenvironment and the hormonal stimulation during pregnancy. Results: Transplantation of AFSs into cleared fat pads alone or in the presence of exogenous mammary epithelial cells caused their differentiation into stroma and adipocytes and replaced endogenous mesenchymal components surrounding the ducts in co-transplantation experiments. Similarly, transplantation of AFSs into fat pads that had not been previously cleared led to AFS-derived stromal cells surrounding the elongating endogenous ducts. AFSs expressed the marker protein α-SMA, but did not integrate into the myoepithelial cell layer of the ducts in virgin mice. With pregnancy, a small number of AFS-derived cells were present in acinar structures. Conclusions: Our data demonstrate that the microenvironmental cues of the mammary fat pad cause AFSs to participate in mammary gland regeneration by providing mesenchymal components to emerging glandular structures, but do not incorporate or differentiate into ductal epithelial cells

The inhibition of Stat5 by a peptide aptamer ligand specific for the DNA binding domain prevents target gene transactivation and the growth of breast and prostate tumor cells

The signal transducer and activator of transcription Stat5 is transiently activated by growth factor and cytokine signals in normal cells, but its persistent activation has been observed in a wide range of human tumors. Aberrant Stat5 activity was initially observed in leukemias, but subsequently also found in carcinomas. We investigated the importance of Stat5 in human tumor cell lines. shRNA mediated downregulation of Stat5 revealed the dependence of prostate and breast cancer cells on the expression of this transcription factor. We extended these inhibition studies and derived a peptide aptamer (PA) ligand, which directly interacts with the DNA-binding domain of Stat5 in a yeast-two-hybrid screen. The Stat5 specific PA sequence is embedded in a thioredoxin (hTRX) scaffold protein. The resulting recombinant protein S5-DBD-PA was expressed in bacteria, purified and introduced into tumor cells by protein transduction. Alternatively, S5-DBD-PA was expressed in the tumor cells after infection with a S5-DBD-PA encoding gene transfer vector. Both strategies impaired the DNA-binding ability of Stat5, suppressed Stat5 dependent transactivation and caused its intracellular degradation. Our experiments describe a peptide based inhibitor of Stat5 protein activity which can serve as a lead for the development of a clinically useful compound for cancer treatment

Localization of the ~12 kDa Mr discrepancy in gel migration of the mouse glucocorticoid receptor to the major phosphorylated cyanogen bromide fragment in the transactivating domain

The intact wild-type mouse glucocorticoid receptor has a theoretical molecular weight of ~86 kDa based on amino acid sequence, but on SDS-polyacrylamide gel electrophoresis it migrates as a protein of ~98 kDa. It is not known where the unusual primary structure or covalent modification responsible for this anomalous migration is located within the amino acid chain. In the course of examining the pattern of fragmentation of 32P-labeled glucocorticoid receptors from Chinese hamster ovary (CHO) cells containing amplified mouse receptor cDNA, we have found a localized region in the amino-terminal half of the receptor that accounts for this anomalous behavior. Cyanogen bromide treatment of the intact receptor produces a 23.4 kDa (theoretical) fragment consisting of residues 108-324 and containing all of the identified phosphorylated serines within the receptor. We find that the only large resolvable 32P-labeled receptor fragment produced after complete cyanogen bromide cleavage of intact receptors migrates with an apparent molecular weight of ~35 kDa. Because the apparent difference between the theoretical and the experimentally observed molecular weights of this cyanogen bromide fragment is essentially the same as the difference between the theoretical and experimental molecular weights of the intact mouse glucocorticoid receptor, we propose that some feature lying within this fragment accounts for slower migration. Although the existence of an additional phosphorylation site lying within the 15 kDa tryptic receptor fragment containing the DNA-binding domain has been contested, we also demonstrate that this fragment of the mouse glucocorticoid receptor is phosphorylated in vivo upon incubation of CHO cells in growth medium containing [32P]orthophosphate.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30424/1/0000045.pd

STAT3 Induction of miR-146b Forms a Feedback Loop to Inhibit the NF-kB to IL-6 Signaling Axis and STAT3-Driven Cancer Phenotypes

Interleukin-6 (IL-6)–mediated activation of signal transducer and activator of transcription 3 (STAT3) is a mechanism by which chronic inflammation can contribute to cancer and is a common oncogenic event. We discovered a pathway, the loss of which is associated with persistent STAT3 activation in human cancer. We found that the gene encoding the tumor suppressor microRNA miR-146b is a direct STAT3 target gene, and its expression was increased in normal breast epithelial cells but decreased in tumor cells. Methylation of the miR-146b promoter, which inhibited STAT3-mediated induction of expression, was increased in primary breast cancers. Moreover, we found that miR-146b inhibited nuclear factor kB (NF-kB)–dependent production of IL-6, subsequent STAT3 activation, and IL-6/STAT3–driven migration and invasion in breast cancer cells, thereby establishing a negative feedback loop. In addition, higher expression of miR-146b was positively correlated with patient survival in breast cancer subtypes with increased IL6 expression and STAT3 phosphorylation. Our results identify an epigenetic mechanism of crosstalk between STAT3 and NF-kB relevant to constitutive STAT3 activation in malignancy and the role of inflammation in oncogenesis

Linear and cooperative signaling: roles for Stat proteins in the regulation of cell survival and apoptosis in the mammary epithelium

The mammary epithelium undergoes cyclical periods of cellular proliferation, differentiation and regression. These processes are under the control of the hormones secreted during pregnancy, lactation and involution. Signaling pathways have been identified that connect the hormonal stimuli with the transcription of genes responsible for the determination of the cellular fate. The kinetics of induction and deinduction have suggested that cytokine-activated Stat proteins play a crucial role. Stat5 is strongly activated towards the end of pregnancy, persists in an activated state during pregnancy and is rapidly inactivated after cessation of suckling. Stat3 activation is hardly detectable during lactation, but is strongly induced at the onset of involution. The phenotypes of mice in which these genes have been inactivated through homologous recombination corroborate some of the functional assignments deducted from the activation pattern. Stat3 activation seems to be a driving force in the induction of apoptosis early in the involution period

Monitoring of the Sublingual Microcirculation During Cardiac Surgery:Current Knowledge and Future Directions

Handheld vital microscopes allow for direct observation of the sublingual microcirculatory perfusion during cardiac surgery. Through the use of handheld vital microscopes, it has been shown that cardiac surgery with cardiopulmonary bypass is associated with reduced and heterogenous microcirculatory perfusion. Microcirculatory impairment can result in inadequate tissue perfusion, leading to perioperative complications and poor outcome. Because microcirculatory impairment can occur despite stable or improved global hemodynamics, there is a yet unmet need for specific monitoring of the microcirculation. Technological advancements may facilitate point-of-care monitoring of microcirculatory perfusion using automated real-time analysis of microcirculatory measurements. Thus, microcirculatory monitoring may create new opportunities for specific microcirculatory treatment as part of hemodynamic management. The implementation of microcirculatory variables into personalized treatment concepts has the potential to improve hemodynamic management during cardiac surgery and thereby improve patient outcomes. Therefore, specific treatment strategies need to be developed to prevent or treat alterations of the microcirculatory perfusion. In the future, the use of handheld vital microscopes for microcirculatory monitoring may help to improve hemodynamic management and outcomes for patients undergoing cardiac surgical procedures

Heat shock protein-90-alpha, a prolactin-STAT5 target gene identified in breast cancer cells, is involved in apoptosis regulation

Introduction The prolactin-Janus-kinase-2-signal transducer and activator of transcription-5 (JAK2-STAT5) pathway is essential for the development and functional differentiation of the mammary gland. The pathway also has important roles in mammary tumourigenesis. Prolactin regulated target genes are not yet well defined in tumour cells, and we undertook, to the best of our knowledge, the first large genetic screen of breast cancer cells treated with or without exogenous prolactin. We hypothesise that the identification of these genes should yield insights into the mechanisms by which prolactin participates in cancer formation or progression, and possibly how it regulates normal mammary gland development. Methods We used subtractive hybridisation to identify a number of prolactin-regulated genes in the human mammary carcinoma cell line SKBR3. Northern blotting analysis and luciferase assays identified the gene encoding heat shock protein 90-alpha (HSP90A) as a prolactin-JAK2-STAT5 target gene, whose function was characterised using apoptosis assays. Results We identified a number of new prolactin-regulated genes in breast cancer cells. Focusing on HSP90A, we determined that prolactin increased HSP90A mRNA in cancerous human breast SKBR3 cells and that STAT5B preferentially activated the HSP90A promoter in reporter gene assays. Both prolactin and its downstream protein effector, HSP90α, promote survival, as shown by apoptosis assays and by the addition of the HSP90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), in both untransformed HC11 mammary epithelial cells and SKBR3 breast cancer cells. The constitutive expression of HSP90A, however, sensitised differentiated HC11 cells to starvation-induced wild-type p53-independent apoptosis. Interestingly, in SKBR3 breast cancer cells, HSP90α promoted survival in the presence of serum but appeared to have little effect during starvation. Conclusions In addition to identifying new prolactin-regulated genes in breast cancer cells, we found that prolactin-JAK2-STAT5 induces expression of the HSP90A gene, which encodes the master chaperone of cancer. This identifies one mechanism by which prolactin contributes to breast cancer. Increased expression of HSP90A in breast cancer is correlated with increased cell survival and poor prognosis and HSP90α inhibitors are being tested in clinical trials as a breast cancer treatment. Our results also indicate that HSP90α promotes survival depending on the cellular conditions and state of cellular transformation

Prolactin-mediated gene activation in mammary epithelial cells

International audienceMammary epithelial cells grow and develop with the onset of sexual maturity. In addition, lobular alveolar structures are formed during pregnancy, and quiescent differentiated cells secrete high levels of milk proteins after parturition. These events are governed by multiple hormones and growth factors and involve the sequential and synergistic action of functionally distinct signal transduction pathways. Milk protein genes have been analyzed and composite response elements have been identified in the promoter sequences. Transcription factors, which relay the hormonal signals, bind to these sequences. The factor that confers prolactin simulation to milk protein gene transcription has recently been identified. MGF/Stat5 is a latent transcription factor that becomes activated by a tyrosine-specific protein kinase, Jak2, associated with the prolactin receptor. Tyrosine phosphorylation converts the latent factor into one with DNA-binding and transcriptional activation potential. The regulation of MGF/Stat5 in vitro and in vivo indicates that it is a central component of the lactogenic hormone signaling pathway. Involvement of MGF/Stat5 in the signaling by other cytokines indicates that the same factor might be involved in regulation of growth-promoting genes, primarily in hematopoietic cells