Pleiotropic Actions of Forskolin Result in Phosphatidylserine Exposure in Primary Trophoblasts

Forskolin is an extract of the Coleus forskholii plant that is widely used in cell physiology to raise intracellular cAMP levels. In the field of trophoblast biology, forskolin is one of the primary treatments used to induce trophoblastic cellular fusion. The syncytiotrophoblast (ST) is a continuous multinucleated cell in the human placenta that separates maternal from fetal circulations and can only expand by fusion with its stem cell, the cytotrophoblast (CT). Functional investigation of any aspect of ST physiology requires in vitro differentiation of CT and de novo ST formation, thus selecting the most appropriate differentiation agent for the hypothesis being investigated is necessary as well as addressing potential off-target effects. Previous studies, using forskolin to induce fusion in trophoblastic cell lines, identified phosphatidylserine (PS) externalization to be essential for trophoblast fusion and showed that widespread PS externalization is present even after fusion has been achieved. PS is a membrane phospholipid that is primarily localized to the inner-membrane leaflet. Externalization of PS is a hallmark of early apoptosis and is involved in cellular fusion of myocytes and macrophages. We were interested to examine whether PS externalization was also involved in primary trophoblast fusion. We show widespread PS externalization occurs after 72 hours when fusion was stimulated with forskolin, but not when stimulated with the cell permeant cAMP analog Br-cAMP. Using a forskolin analog, 1,9-dideoxyforskolin, which stimulates membrane transporters but not adenylate cyclase, we found that widespread PS externalization required both increased intracellular cAMP levels and stimulation of membrane transporters. Treatment of primary trophoblasts with Br-cAMP alone did not result in widespread PS externalization despite high levels of cellular fusion. Thus, we concluded that widespread PS externalization is independent of trophoblast fusion and, importantly, provide evidence that the common differentiation agent forskolin has previously unappreciated pleiotropic effects on trophoblastic cells.     

Arsenic Trioxide Overcomes Rapamycin-Induced Feedback Activation of AKT and ERK Signaling to Enhance the Anti-Tumor Effects in Breast Cancer

Inhibitors of the mammalian target of rapamycin (mTORi) have clinical activity; however, the benefits of mTOR inhibition by rapamycin and rapamycin-derivatives (rapalogs) may be limited by a feedback mechanism that results in AKT activation. Increased AKT activity resulting from mTOR inhibition can be a result of increased signaling via the mTOR complex, TORC2. Previously, we published that arsenic trioxide (ATO) inhibits AKT activity and in some cases, decreases AKT protein expression. Therefore, we propose that combining ATO and rapamycin may circumvent the AKT feedback loop and increase the anti-tumor effects. Using a panel of breast cancer cell lines, we find that ATO, at clinically-achievable doses, can enhance the inhibitory activity of the mTORi temsirolimus. In all cell lines, temsirolimus treatment resulted in AKT activation, which was decreased by concomitant ATO treatment only in those cell lines where ATO enhanced growth inhibition. Treatment with rapalog also results in activated ERK signaling, which is decreased with ATO co-treatment in all cell lines tested. We next tested the toxicity and efficacy of rapamycin plus ATO combination therapy in a MDA-MB-468 breast cancer xenograft model. The drug combination was well-tolerated, and rapamycin did not increase ATO-induced liver enzyme levels. In addition, combination of these drugs was significantly more effective at inhibiting tumor growth compared to individual drug treatments, which corresponded with diminished phospho-Akt and phospho-ERK levels when compared with rapamycin-treated tumors. Therefore, we propose that combining ATO and mTORi may overcome the feedback loop by decreasing activation of the MAPK and AKT signaling pathways.     

The immunostimulatory effect of T cells and T cell lymphokines on murine fetally derived placental cells

Evidence for maternal immune recognition of the fetus can be found during pregnancy, yet the conceptus remains unharmed. Indeed, in some cases immunizing the mother with cells sharing histocompatibility antigens with the fetus is beneficial to fetal survival. This could be due to the effect of maternally derived lymphokines on placental growth and function, according to the immunostimulation hypothesis. We demonstrate here that placental cells in culture proliferate upon the addition of T cell-derived lymphokines. The lymphokine activity has been separated from IL 2 and B cell growth factor, and copurified with IL 3 and granulocyte-macrophage colony-stimulating factor (CSF-GM). Recombinant CSF-GM and recombinant IL 3 showed a similar effect. The placental cells that proliferate in culture are of fetal origin and are characterized by strong adherence, phagocytosis, nonspecific esterase staining, and response to the macrophage-specific colony-stimulating factor CSF-1. In addition, treatment of pregnant females with anti-thymocyte serum as well as anti-Ly-2.1 monoclonal antibody, at gestational times before Ly-2 antigen appearance in the fetus, leads to a reduction of the proliferative and phagocytic capacity of day 12 placentae. These results clearly demonstrate that maternal T cells act upon fetally derived placental cells to improve their proliferative and phagocytic potential, and thus provide evidence for the immunostimulatory role of these cells during pregnancy.     

Confirmation of quantitative trait loci affecting fatness in chickens

In this report we describe the analysis of an advanced intercross line (AIL) to confirm the quantitative trait locus (QTL) regions found for fatness traits in a previous study. QTL analysis was performed on chromosomes 1, 3, 4, 15, 18, and 27. The AIL was created by random intercrossing in each generation from generation 2 (G₂) onwards until generation 9 (G₉) was reached. QTL for abdominal fat weight (AFW) and/or percentage abdominal fat (AF%) on chromosomes 1, 3 and 27 were confirmed in the G₉ population. In addition, evidence for QTL for body weight at the age of 5 (BW5) and 7 (BW7) weeks and for the percentage of intramuscular fat (IF%) were found on chromosomes 1, 3, 15, and 27. Significant evidence for QTL was detected on chromosome 1 for BW5 and BW7. Suggestive evidence was found on chromosome 1 for AFW, AF% and IF%, on chromosome 15 for BW5, and on chromosome 27 for AF% and IF%. Furthermore, evidence on the chromosome-wise level was found on chromosome 3 for AFW, AF%, and BW7 and on chromosome 27 for BW5. For chromosomes 4 and 18, test statistics did not exceed the significance threshold.     

Evidence of homing following translocation of long‐tailed bats (Chalinolobus tuberculatus) at Grand Canyon Cave,New Zealand

Abstract

Homing behaviour in the New Zealand long‐tailed bat (Chalinolobus tuberculatus), a temperate insectivorous species, was investigated at Grand Canyon Cave, central North Island. A pilot study of nine adult male bats was conducted to determine whether use of the cave was regular enough for a homing study. Eight bats returned to the cave over the 3‐week monitoring period, six on the night following release. Nine additional bats carrying radio transmitters were then released at three sites (three at each site) c. 5, 10 and 20 km due east of the cave; sites estimated to be located within, on the border of, and outside the population's known familiar area respectively. All but one of these nine bats were subsequently detected at the cave. Results suggest that adult long‐tailed bats are able to return home following displacement both inside and outside their familiar area. Implications of these findings for translocations of bats and the possession of a potential long‐distance navigation system by this species are discussed.     

Identification of methylation markers for the prediction of nodal metastasis in oral and oropharyngeal squamous cell carcinoma

Hypermethylation is an important mechanism for the dynamic regulation of gene expression, necessary for metastasizing tumour cells. Our aim is to identify methylation tumour markers that have a predictive value for the presence of regional lymph node metastases in patients with oral and oropharyngeal squamous cell carcinoma (OOSCC). Significantly differentially expressed genes were retrieved from four reported microarray expression profiles comparing pN0 and pN+ head-neck tumours, and one expression array identifying functionally hypermethylated genes. Additional metastasis-associated genes were included from the literature. Thus genes were selected that influence the development of nodal metastases and might be regulated by methylation. Methylation-specific PCR (MSP) primers were designed and tested on 8 head-neck squamous cell carcinoma cell lines and technically validated on 10 formalin-fixed paraffin-embedded (FFPE) OOSCC cases. Predictive value was assessed in a clinical series of 70 FFPE OOSCC with pathologically determined nodal status. Five out of 28 methylation markers (OCLN, CDKN2A, MGMT, MLH1 and DAPK1) were frequently differentially methylated in OOSCC. Of these, MGMT methylation was associated with pN0 status (P = 0.02) and with lower immunoexpression (P = 0.02). DAPK1 methylation was associated with pN+ status (P = 0.008) but did not associate with protein expression. In conclusion, out of 28 candidate genes, two (7%) showed a predictive value for the pN status. Both genes, DAPK1 and MGMT, have predictive value for nodal metastasis in a clinical group of OOSCC. Therefore DNA methylation markers are capable of contributing to diagnosis and treatment selection in OOSCC. To efficiently identify additional new methylation markers, genome-wide methods are needed.