Characterization of novel markers of senescence and their prognostic potential in cancer.

Cellular senescence is a terminal differentiation state that has been proposed to have a role in both tumour suppression and ageing. This view is supported by the fact that accumulation of senescent cells can be observed in response to oncogenic stress as well as a result of normal organismal ageing. Thus, identifying senescent cells in in vivo and in vitro has an important diagnostic and therapeutic potential. The molecular pathways involved in triggering and/or maintaining the senescent phenotype are not fully understood. As a consequence, the markers currently utilized to detect senescent cells are limited and lack specificity. In order to address this issue, we screened for plasma membrane-associated proteins that are preferentially expressed in senescent cells. We identified 107 proteins that could be potential markers of senescence and validated 10 of them (DEP1, NTAL, EBP50, STX4, VAMP3, ARMX3, B2MG, LANCL1, VPS26A and PLD3). We demonstrated that a combination of these proteins can be used to specifically recognize senescent cells in culture and in tissue samples and we developed a straightforward fluorescence-activated cell sorting-based detection approach using two of them (DEP1 and B2MG). Of note, we found that expression of several of these markers correlated with increased survival in different tumours, especially in breast cancer. Thus, our results could facilitate the study of senescence, define potential new effectors and modulators of this cellular mechanism and provide potential diagnostic and prognostic tools to be used clinically.

Mouse models for BRAF-induced cancers.

Oncogenic mutations in the BRAF gene are detected in approximately 7% of human cancer samples with a particularly high frequency of mutation in malignant melanomas. Over 40 different missense BRAF mutations have been found, but the vast majority (>90%) represent a single nucleotide change resulting in a valine-->glutamate mutation at residue 600 ((V600E)BRAF). In cells cultured in vitro, (V600E)BRAF is able to stimulate endogenous MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] and ERK phosphorylation leading to an increase in cell proliferation, cell survival, transformation, tumorigenicity, invasion and vascular development. Many of these hallmarks of cancer can be reversed by treatment of cells with siRNA (small interfering RNA) to BRAF or by inhibiting MEK, indicating that BRAF and MEK are attractive therapeutic targets in cancer samples with BRAF mutations. In order to fully understand the role of oncogenic BRAF in cancer development in vivo as well as to test the in vivo efficacy of anti-BRAF or anti-MEK therapies, GEMMs (genetically engineered mouse models) have been generated in which expression of oncogenic BRaf is conditionally dependent on the Cre recombinase. The delivery/activation of the Cre recombinase can be regulated in both a temporal and spatial manner and therefore these mouse models can be used to recapitulate the somatic mutation of BRAF that occurs in different tissues in the development of human cancer. The data so far obtained following Cre-mediated activation in haemopoietic tissue and the lung indicate that (V600E)BRAF mutation can drive tumour initiation and that its primary effect is to induce high levels of cyclin D1-mediated cell proliferation. However, hallmarks of OIS (oncogene-induced senescence) are evident that restrain further development of the tumour.

MEK kinase activity is not necessary for Raf-1 function.

Raf-1 protein kinase has been identified as an integral component of the Ras/Raf/MEK/ERK signalling pathway in mammals. Activation of Raf-1 is achieved by RAS:GTP binding and other events at the plasma membrane including tyrosine phosphorylation at residues 340/341. We have used gene targeting to generate a 'knockout' of the raf-1 gene in mice as well as a rafFF mutant version of endogenous Raf-1 with Y340FY341F mutations. Raf-1(-/-) mice die in embryogenesis and show vascular defects in the yolk sac and placenta as well as increased apoptosis of embryonic tissues. Cell proliferation is not affected. Raf-1 from cells derived from raf-1(FF/FF) mice has no detectable activity towards MEK in vitro, and yet raf-1(FF/FF) mice survive to adulthood, are fertile and have an apparently normal phenotype. In cells derived from both the raf-1(-/-) and raf-1(FF/FF) mice, ERK activation is normal. These results strongly argue that MEK kinase activity of Raf-1 is not essential for normal mouse development and that Raf-1 plays a key role in preventing apoptosis.

Expression and regulation of prostaglandin E receptor subtype mRNAs in rat sensory ganglia and spinal cord in response to peripheral inflammation.

Prostaglandins are known to act via seven transmembrane domain receptors to exert actions on both peripheral and central neurons resulting in changes in neuronal excitability. Prostaglandin E2, the prostaglandin most often associated with inflammation, itself acts on a family of closely related receptors, the EP receptors. Using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), we have shown that rat primary afferent neurons express the mRNA for all EP receptor subtypes, and that some, but not all EP receptor subtype mRNAs are down-regulated in sensory neurons in response to an acute peripheral inflammation. We also show for the first time that all EP receptor subtype mRNAs are expressed in rat lumbar spinal cord. Spinal cord EP receptor subtype mRNAs are also regulated in acute inflammation in a pattern distinct from the changes seen in sensory ganglia in response to the same inflammatory stimulus.

Disruption of the talin gene arrests mouse development at the gastrulation stage.

Studies on cultured cells show that the cytoskeletal protein talin plays a key role in cell spreading and the assembly of cell-extracellular matrix junctions. To examine the role of talin in vivo, we have generated mice with a targeted disruption of the talin gene. Heterozygotes are normal, but no surviving homozygous mutant animals were obtained, proving that talin is required for embryogenesis. Mutant embryos develop normally to the blastocyst stage and implant, but there is a gross disorganization of the embryos at gastrulation (6.5-7.5 days post coitum), and they die around 8.5-9.5 days post coitum. The embryonic ectoderm is reduced in size, with fewer cells, and is incompletely organised compared with wild-type embryos. The mutant embryos show disorganised extraembryonic tissues, and the ectoplacental and excocoelomic cavities are not formed. This seems to be because embryonic mesoderm accumulates as a mass on the posterior side of the embryos and fails to migrate to extraembryonic regions, although mesodermal cells are evident in the embryo proper. Spreading of trophoblast cells derived from cultured mutant blastocysts on fibronectin and laminin is also considerably reduced. Therefore, the fundamental deficit in these embryos seems to be a failure of cell migration at gastrulation.

Cyclooxygenase-1 is a marker for a subpopulation of putative nociceptive neurons in rat dorsal root ganglia.

Immunocytochemical and morphometric techniques were used to quantify the distribution of cyclooxygenase (cox)-containing neurons in rat L5 dorsal root ganglia (DRG). Cox-1 immunolabelling was almost exclusively restricted to small diameter DRG neurons (< 1000 microm2), and was extensively colocalized with calcitonin gene-related peptide (CGRP) and isolectin B4 (IB4). Cox-1 was present in 65% and 70% of CGRP- and IB4-labelled neurons, respectively. Cox-1 labelling was also found in neurons expressing the sensory neuron-specific (SNS) Na+ channel. Cox-2 labelling was absent in DRG from normal rats. In the Freund's adjuvant model of monoarthritis, the proportion of cox-1-positive DRG neurons was unchanged and no neurons were found to be labelled for cox-2. In primary tissue culture, cox-1 immunolabelling persisted in vitro for up to 9 days and was present in morphologically identical neurons. The selective expression of cox-1 in peripheral ganglia was confirmed by the small number of nodose ganglion neurons and superior cervical ganglion (SCG) neurons labelled for cox-1. These data suggest that cox-1 is a marker for a subpopulation of putative nociceptive neurons in vitro and in vivo, and suggests that the prostaglandins synthesized by these neurons may be important for nociceptor function. These data may have important implications for the mode and mechanism of action of non-steroidal anti-inflammatory drugs (NSAIDs).

Prostanoids synthesized by cyclo-oxygenase isoforms in rat spinal cord and their contribution to the development of neuronal hyperexcitability.

1. The responses of wide dynamic range spinal dorsal horn neurones to noxious mechanical stimulation of the ankle or knee joint were tested before and after spinal administration of the non-selective cyclooxygenase (COX) inhibitors, indomethacin and meclofenamic acid. Neither of these drugs altered the responses of these neurones to noxious mechanical stimulation. 2. Wind-up of a spinal nociceptive reflex evoked by electrical stimulation of the sural nerve at C-fibre strength was dose-dependently inhibited by intravenous administration of indomethacin, a non-selective COX inhibitor, and SC58125, a selective COX-2 inhibitor. Intrathecal administration of indomethacin also reduced the wind-up of this nociceptive reflex. 3. Western blot analysis of proteins extracted from normal rat spinal cord revealed the presence of both cyclo-oxygenase (COX)-1 and COX-2 proteins. 4. Immunocytochemistry of sections of normal rat spinal cord with specific COX-1 antiserum revealed little specific COX-1-like immunoreactivity in the grey matter. With the same antiserum, intense COX-1-like immunoreactivity was observed in the cytoplasm, nuclear membrane and axonal processes of small to medium sized (< 1000 microns2) dorsal root ganglion (DRG) cell bodies. 5. Immunocytochemistry of sections of normal rat spinal cord incubated with specific COX-2 antiserum showed intense COX-2-like immunoreactivity (COX-2-li) in the superficial dorsal horn of the spinal cord (laminae I and II) and around the central canal (lamina X). COX-2-li was also observed in some neurones in deep dorsal horn and in individual motor neurones in ventral horn. COX-2-li was not observed in the cell bodies of DRG. 6. Superfusion of the lumbar spinal cord of normal rats with artificial CSF and subsequent radioimmunoassay revealed the presence of prostaglandin D2 (PGD2) < PGE2, but not PGI2 (determined by measurement of the stable metabolite, 6-keto-PGF1 alpha) or PGF2 alpha. 7. These data suggest that eicosanoids synthesized by an active COX pathway in the spinal cord of normal animals may contribute to nociceptive processing, but only when the spinal cord neurones are rendered hyperexcitable following C-fibre stimulation. Selective inhibition of one or both of the COX isoforms in normal animals may represent a novel target for spinal analgesia.