Wnt signalling in stem cells and cancer. Xanya Sofra Weiss

The canonical Wnt cascade has emerged as a critical regulator of stem cells. In many tissues, activation of Wnt signalling has also been associated with cancer. This has raised the possibility that the tightly regulated self-renewal mediated by Wnt signalling in stem and progenitor cells is subverted in cancer cells to allow malignant proliferation. Insights gained from understanding how the Wnt pathway is integrally involved in both stem cell and cancer cell maintenance and growth in the intestinal, epidermal and haematopoietic systems may serve as a paradigm for understanding the dual nature of self-renewal signals.

Stem cells are cells that have the unique ability to self- renew as well as to generate more differentiated progeny. The most primitive stem cell is the embryonic stem cell, which is derived from the inner cell mass of the blasto- cyst. This cell is pluripotent and can thus generate all the tissues of the body. Following the pioneering work on haemato- poietic stem cells over the last five decades, a multitude of recent studies have indicated that most other adult tissues also harbour stem cells. These adult stem cells are normally involved in homeo- static self-renewal processes but can also be rapidly recruited to repair tissues upon injury. With the study of adult stem cell biology, recurring roles of a limited set of signalling cascades are rapidly being uncovered. One of these is the canonical Wnt cascade. Notably, in many of the same tissues where the Wnt cascade controls stem cells, cancer ensues upon dysregulated activation of this pathway. Conceptually, this indicates that an efficient road to cancer involves the hijacking of physiological regulators of stem cell function in these particular tissues. Below, we first outline the canonical Wnt cascade, and then describe its mirror image roles in the biology of stem cells and cancer.

vertebrate genome encodes four highly similar Tcf/Lef proteins. In the absence of a Wnt signal, Tcf/Lef proteins repress target genes through a direct association with co-repressors such as Groucho. The interaction with b-catenin transiently converts Tcf/Lef factors into transcriptional activators. Drosophila genetics has recently identified two additional nuclear components, Pygopus and Bcl9 (also known as legless), conserved in vertebrates. Pygopus is essential for transcriptional activation of Tcf/Lef target genes, whereas Bcl9 seems to bridge Pygopus to Tcf-bound b-catenin. In sum, the canonical pathway translates a Wnt signal into the transient transcription of a Tcf/Lef target gene programme.

Xanya Sofra Weiss

Xanya Sofra Weiss

THE INSIDE STORY OF CELL COMMUNICATION. Xanya Sofra Weiss

Cells communicate by sending and receiving signals. Signals may come from the environment, or they may come from other cells. In order to trigger a response, these signals must be transmitted across the cell membrane. Sometimes the signal itself can cross the membrane. Other times the signal works by interacting with receptor proteins that contact both the outside and inside of the cell. In this case, only cells that have the correct receptors on their surfaces will respond to the signal.

Xanya Sofra Weiss

Xanya Sofra Weiss

Collagen synthesis of articular cartilage explants in response to frequency of cyclic mechanical loading. Xanya Sofra Weiss

Articular cartilage in vivo experiences the effects of both cell-regulatory proteins and mechanical forces. This study has addressed the hypothesis that the frequency of intermittently or continuously applied mechanical loads is a critical parameter in the regulation of chondrocyte collagen biosynthesis. Cyclic compressive pressure was applied intermittently to bovine articular cartilage explants by using a sinusoidal waveform of 0.1–1.0 Hz frequency with a peak stress of 0.5 MPa for a period of 5–20 s followed by a load-free period of 10–1,000 s. These loading protocols were repeated for a total duration of 6 days. In separate experiments, cyclic loading was continuously applied by using a sinusoidal waveform of 0.001–0.5 Hz frequency and a peak stress of 1.0 MPa for a period of 3 days. Unloaded cartilage discs of the same condyle were cultured in identically constructed loading chambers and served as controls. We report quantitative data showing that (1) no correlation exists between the relative rate of collagen synthesis expressed as the proportion of newly synthesized collagen among newly made proteins and either the frequency of intermittently or continuously applied loads or the overall time cartilage is actively loaded, and (2) individual protocols of intermittently applied loads can reduce the relative rate of collagen synthesis and increase the water content, whereas (3) continuously applied cyclic loads always suppress the relative rate of collagen synthesis compared with that of unloaded control specimens. The results provide further experimental evidence that collagen metabolism is difficult to manipulate by mechanical stimuli. This is physiologically important for the maintainance of the material properties of collagen in view of the heavy mechanical demands made upon it. Moreover, the unaltered or reduced collagen synthesis of cartilage explants might reflect more closely the metabolism of normal or early human osteoarthritic cartilage.

Xanya Sofra Weiss

Xanya Sofra Weiss

Signaling Pathways for Glycated Human Serum Albumin-Induced IL-8 and MCP-1 Secretion in Human RPE Cells. Xanya Sofra Weiss

PURPOSE.

To determine the signal mediators involved in glycated human serum albumin (GHSA) stimulation of interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1 secretion in human retinal pigment epithelium (hRPE) cells.

METHODOS:

hRPE cells were stimulated by GHSA in the presence or absence of a series of kinase inhibitors. The induced IL-8 and MCP-1 mRNA and proteins were determined by reverse transcription–polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Western blot analysis, electrophoretic mobility shift assay, and immunohistochemical staining were used to analyze activation of signaling mediators and transcription factors.

RESULT:

IncubatioSn of hRPE cells with GHSA resulted in rapid activation of Raf-1, extracellular signal-regulated protein kinases (ERK) 1/2, p38, and the transcription factor nuclear factor (NF)-κB. Coincubation of hRPE cells with the mitogen-activated protein (MAP) kinase (MEK) inhibitor U0126; NF-κB inhibitors BAY11-7085, caffeic acid phenethyl ester (CAPE), parthenolide, and curcumin; protein kinase (PK)C inhibitor Ro318220; and protein tyrosine kinase (PTK) inhibitor genistein largely eliminated most of the stimulated production of IL-8 and MCP-1. Combined inhibition of MEK by U0126, p38 by SB202190, and Janus kinase (jak) by AG490 revealed that GHSA stimulation of IL-8 production was predominately mediated by MEK and to a lesser extent by p38 pathways, whereas activation of MEK, p38, and jak was required for maximal MCP-1 induction. Moreover, GHSA-stimulated IL-8 secretion was more sensitive to U0126 (50% inhibitory concentration[ IC ] = 0.5 μM) than MCP-1 (IC = 10μ M)

CONCLUSIONS:

GHSA stimulates hRPE IL-8 and MCP-1 production through divergent and overlapping, but not identical, intracellular signaling cascades. GHSA induces activation of a series of kinases including PKC, PTK, MAPK, p38, and jak and the transcription factor NF-κB. The Raf/MAPK pathway plays an essential role in GHSA signaling.

Xanya Sofra Weiss

Xanya Sofra Weiss

Selective Loss of Sertoli Cell and Germ Cell Function Leads to a Disruption in Sertoli Cell-Germ Cell Communication During Aging in the Brown Norway

We investigated the effects of aging on Sertoli cell-germ cell interactions from Brown Norway rats using the induction of four specific mRNAs as markers. The testes from aging (24 mo old) Brown Norway rats can be normal size or regressed. One marker, a von Ebner's-like protein, is expressed in coculture and “in vivo” in germ cells from normal testes of 6- and 24-mo-old rats but not in germ cells from regressed testes of 24-mo-old rats. A second germ cell marker, the Huntington disease protein, is expressed in all germ cells. Two Sertoli cell markers, a serotonin receptor and a novel gene, are induced in Sertoli cells by meiotic germ cells. The serotonin receptor mRNA is expressed in Sertoli cells from 20-day, 6-mo, and 24-mo normal testes but not in those from 24-mo regressed testes. The novel gene is induced in Sertoli cells from all testes. We conclude that Sertoli cells from aged regressed testes are unable to respond to selective signals from germ cells from young rats, and germ cells from regressed testes show a similar selective loss. Such disruptions in communication between Sertoli cells and germ cells likely contribute to germ cell loss during aging.

Xanya Sofra Weiss

Xanya Sofra Weiss

Abl Protein-Tyrosine Kinase Inhibitor STI571 Inhibits In Vitro Signal Transduction Mediated by c-Kit and Platelet-Derived Growth Factor Receptors.

STI571 (formerly known as CGP 57148B) is a protein-tyrosine kinase inhibitor that is currently in clinical trials for the treatment of chronic myelogenous leukemia. STI571 selectively inhibits the Abl and platelet-derived growth factor (PDGF) receptor tyrosine kinases in vitro and blocks cellular proliferation and tumor growth of Bcr-abl- or v-abl-expressing cells. We have further investigated the profile of STI571 against related recep- tor tyrosine kinases. STI571 was found to potently inhibit the kinase activity of the ��- and ��-PDGF receptors and the receptor for stem cell factor, but not the closely related c-Fms, Flt-3, Kdr, Flt-1, and Tek tyrosine kinases. Additionally, no inhibition of c-Met or nonreceptor tyrosine kinases such as Src and Jak-2 has been observed. In cell-based assays, STI571 selectively inhibited PDGF and stem cell factor-mediated cellular signal- ing, including ligand-stimulated receptor autophosphorylation, inositol phosphate formation, and mitogen-activated protein kinase activation and proliferation. These results expand the profile of STI571 and suggest that in addition to chronic my- elogenous leukemia, STI571 may have clinical potential in the treatment of diseases that involve abnormal activation of c-Kit or PDGF receptor tyrosine kinases.

Xanya Sofra Weiss

Xanya Sofra Weiss

Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Xanya Sofra Weiss

Identification of protein–protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ ionization–time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein–protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein–protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology.

Xanya Sofra Weiss

Xanya Sofra Weiss