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.

Colorectal cancer cells with the BRAF(V600E) mutation are addicted to the ERK1/2 pathway for growth factor-independent survival and repression of BIM.

The RAF-mitogen-activated protein kinase kinase 1/2-extracellular signal-regulated kinase 1/2 (RAF-MEK1/2-ERK1/2) pathway is activated in many human tumours and can protect cells against growth factor deprivation; however, most such studies have relied upon overexpression of RAF or MEK constructs that are not found in tumours. Here we show that expression of the endogenous BRAF(V600E) allele in mouse embryonic fibroblasts from conditional knock-in transgenic mice activates ERK1/2, represses the BH3-only protein BIM and protects cells from growth factor withdrawal. Human colorectal cancer (CRC) cell lines harbouring BRAF(V600E) are growth factor independent for the activation of ERK1/2 and survival. However, treatment with the MEK1/2 inhibitors U0126, PD184352 or the novel clinical candidate AZD6244 (ARRY-142886) overcomes growth factor independence, causing CRC cell death. BIM is de-phosphorylated and upregulated following MEK1/2 inhibition in all CRC cell lines studied and knockdown of BIM reduces cell death, indicating that repression of BIM is a major part of the ability of BRAF(V600E) to confer growth factor-independent survival. We conclude that a single endogenous BRAF(V600E) allele is sufficient to repress BIM and prevent death arising from growth factor withdrawal, and CRC cells with BRAF(V600E) mutations are addicted to the ERK1/2 pathway for repression of BIM and growth factor-independent survival.

Analysis of the mammalian talin2 gene TLN2.

We have utilised genomic and EST databases to assemble the sequence of the human talin2 (TLN2) gene. Talin2 protein is similar in size and sequence to talin1 throughout its length (74% identity, 86% similarity). The major differences are in (i) the size of the genes, the TLN2 gene is >200 kb compared with approximately 30 kb for TLN1 due to a difference in intron size, although intron/exon boundaries, with the exception of two, are strictly conserved; (ii) the expression patterns, TLN1 gives rise to an approximately 8-kb mRNA which is observed in all tissues, whereas TLN2 gives rise to multiple transcripts with the highest levels in heart.

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.

Expression of the A-raf proto-oncogene in the normal adult and embryonic mouse.

We have determined the expression pattern of the A-raf proto-oncogene in the embryonic and adult mouse. Western blot analysis of protein lysates from tissues of adult mice show that p69A-raf is ubiquitously expressed, but that levels of expression vary among different tissues. To determine the cell-specific expression pattern of A-raf, we generated transgenic mice expressing the beta-galactosidase reporter gene from the A-raf promoter. We show that A-raf expression is highly specific within a given tissue, and we identify cell types expressing this gene in the adult testis, epididymis, vas deferens, seminal vesicle, ovary, oviduct, bladder, kidney, intestine, heart, spleen, thymus, and cerebellum. In the embryo, ubiquitous expression of the reporter gene is observed, but the highest levels of expression are specifically detected in the embryonic heart at stages 9.5-11.5 days post-coitum.

Reduced vas deferens contraction and male infertility in mice lacking P2X1 receptors.

P2X1 receptors for ATP are ligand-gated cation channels, present on many excitable cells including vas deferens smooth muscle cells. A substantial component of the contractile response of the vas deferens to sympathetic nerve stimulation, which propels sperm into the ejaculate, is mediated through P2X receptors. Here we show that male fertility is reduced by approximately 90% in mice with a targeted deletion of the P2X1 receptor gene. Male mice copulate normally--reduced fertility results from a reduction of sperm in the ejaculate and not from sperm dysfunction. Female mice and heterozygote mice are unaffected. In P2X1-receptor-deficient mice, contraction of the vas deferens to sympathetic nerve stimulation is reduced by up to 60% and responses to P2X receptor agonists are abolished. These results show that P2X1 receptors are essential for normal male reproductive function and suggest that the development of selective P2X1 receptor antagonists may provide an effective non-hormonal male contraceptive pill. Also, agents that potentiate the actions of ATP at P2X1 receptors may be useful in the treatment of male infertility.

Post-natal lethality and neurological and gastrointestinal defects in mice with targeted disruption of the A-Raf protein kinase gene.

The Ras/Raf/MEK/MAP kinase cascade transmits signals from activated cell-surface receptors to transcription factors in the nucleus and is an essential component of metazoan intracellular signaling pathways (see, for example, [1-6]). In the mouse, the Raf protein kinase family is comprised of three homologous genes, Raf-1, A-Raf and B-Raf [5] which are ubiquitously expressed in the developing embryo [7]. We have introduced into the mouse germ line a loss-of-function mutation in the X-chromosomal A-Raf gene, by homologous recombination in embryonic stem cells. On a predominantly C57 Bl/6 genetic background, A-Raf-deficient mice displayed neurological and intestinal abnormalities and died between 7 and 21 days post-partum. When the mutated allele was maintained on a predominantly 129/OLA background, by contrast, A-Raf-deficient animals survived to adulthood, did not display obvious intestinal abnormalities, were fertile, but did have a subset of the neurological defects.

Conditionally oncogenic forms of the A-Raf and B-Raf protein kinases display different biological and biochemical properties in NIH 3T3 cells.

The protein kinase domains of mouse A-Raf and B-Raf were expressed as fusion proteins with the hormone binding domain of the human estrogen receptor in mammalian cells. In the absence of estradiol, 3T3 and rat1a cells expressing delta A-Raf:ER and delta B-Raf:ER were nontransformed, but upon the addition of estradiol the cells became oncogenically transformed. Morphological oncogenic transformation was more rapid and distinctive in cells expressing delta B-Raf:ER compared with cells expressing delta A-Raf:ER. Biochemical analysis of cells transformed by delta A-Raf:ER and delta B-Raf:ER revealed several interesting differences. The activation of delta B-Raf:ER consistently led to the rapid and robust activation of both MEK and p42/p44 MAP kinases. By contrast, the activation of delta A-Raf:ER led to a weak activation of MEK and the p42/p44 MAP kinases. The extent of activation of MEK in cells correlated with the ability of the different Raf kinases to phosphorylate and activate MEK1 in vitro. delta B-Raf:ER phosphorylated MEK1 approximately 10 times more efficiently than delta Raf-1:ER and at least 500 times more efficiently than delta A-Raf:ER under the conditions of the immune-complex kinase assays. These results were confirmed with epitope-tagged versions of the Raf kinase domains expressed in insect cells. The activation of all three delta Raf:ER proteins in 3T3 cells led to the hyperphosphorylation of the resident p74raf-1 and mSOS1 proteins, suggesting the possibility of "cross-talk" between the different Raf kinases and feedback regulation of intracellular signaling pathways. The activation of either delta B-Raf:ER or delta Raf-1:ER in quiescent 3T3 cells was insufficient to promote the entry of the cells into DNA synthesis. By contrast, the activation of delta A-Raf:ER in quiescent 3T3 cells was sufficient to promote the entry of the cells into S phase after prolonged exposure to beta-estradiol. The delta Raf:ER system has allowed us to reveal significant differences between the biological and biochemical properties of oncogenic forms of the Raf family of protein kinases. We anticipate that cells expressing these proteins and other estradiol-regulated protein kinases will be useful tools in future attempts to unravel the complex web of interactions involved in intracellular signal transduction pathways.

Rapid induction of heparin-binding epidermal growth factor/diphtheria toxin receptor expression by Raf and Ras oncogenes.

We have used differential display PCR to search for mRNAs induced by delta Raf-1:ER, an estradiol-dependent form of Raf-1 kinase. Through this approach the gene encoding heparin-binding epidermal growth factor (HB-EGF) was identified as an immediate-early transcriptional target of oncogenic Raf kinases. Activation of delta Raf-1:ER and a conditional oncogenic form of B-Raf, delta B-RAF:ER, resulted in rapid and sustained induction of HB-EGF mRNA expression and secretion of mature HB-EGF from cells. Neutralizing anti-HB-EGF antisera prevented the delayed activation of the c-Jun amino-terminal kinases that is observed in cells transformed by delta Raf-1:ER. These results demonstrate that distinct signaling pathways can cross talk via the secretion of polypeptide growth factors. Furthermore, cells transformed by oncogenic Ras, which also induced HB-EGF expression, demonstrated a marked increase in sensitivity to the cytotoxic action of diphtheria toxin, for which the membrane anchored HB-EGF precursor acts as a cell-surface receptor.

Segregation of the Huntington disease region of human chromosome 4 in a somatic cell hybrid.

We have developed an X-irradiation:cell fusion procedure that segregates segments of human chromosomes lacking selectable markers and have used this approach to construct somatic cell hybrids retaining fragments of human chromosome 4 as the only human material. To identify hybrids retaining a small chromosomal fragment in the region of the Huntington disease (HD) gene, we used Southern blot analysis to screen 72 hybrid lines for the presence or absence of seven chromosome 4 single-copy loci. These data, combined with in situ hybridization experiments, identified three hybrids of interest. One of these cell lines, C25, stably retains a 10,000- to 20,000-kb fragment of distal 4p in the vicinity of the HD gene, translocated to a hamster chromosome. Field-inversion gel electrophoresis revealed no evidence of rearrangements in the human DNA present in C25. In combination with similar radiation hybrids, C25 is a valuable tool for isolating DNA probes near the HD gene.

Isolation and field-inversion gel electrophoresis analysis of DNA markers located close to the Huntington disease gene.

A radiation-induced hybrid cell line containing 10-20 million base pairs of DNA derived from the terminal part of human 4p16 in a background of hamster chromosomes has been used to construct a genomic library highly enriched for human sequences located close to the Huntington disease (HD) gene. Recombinant phage containing human inserts were isolated from this library and used as hybridization probes against two other radiation hybrids containing human fragments with chromosomal breaks in 4p16 and against a human-hamster somatic cell hybrid that retains only the 4p15-4pter part of chromosome 4. Of 121 human phage tested, 6 were mapped distal to the HD-linked D4S10 locus. Since the HD gene is located between D4S10 and the 4p telomere, all of these sequences are likely to be closer to HD than D4S10, and any one of them may be a distal flanking marker for the disease locus. Long-range restriction map analysis performed with a field-inversion gel system shows that the six new loci are distributed in different places within 4p16. Although it is not possible to establish an order for the six sequences with the FIGE data, the results demonstrate that the region detected by these probes must span at least 2000 kb of DNA.

Chromosome fragmentation by chromosome mediated gene transfer.

Chromosome mediated gene transfer (CMGT) can be used to construct human-mouse somatic cell hybrids containing fragments of human chromosomes. We have constructed two panels of hybrid cells by CMGT: the first panel contains fragments of the human X chromosome and the second panel contains fragments of the Y chromosome. The construction of the Y chromosome panel was facilitated by the cotransfer of chromosomes with a selectable plasmid; selection for the plasmid encoded marker enriched for cells which had incorporated chromosome fragments. This preselection was combined with antibody dependent enrichment using the monoclonal antibody 12E7, which reacts with a Y-encoded antigen. Detailed investigation of the two panels revealed extensive rearrangements of the transferred fragments and the preferential retention of sequences from the centromeric region of the donor chromosomes. These results suggest that CMGT will not be particularly useful in the construction of genetic maps, but nevertheless CMGT can be used for enriching for specific regions of the human genome in somatic cell hybrids.

Isolation of a sequence which maps close to the human sex determining gene.

A sequence mapping close to the human sex determining gene (TDF) has been isolated from a lambda library constructed with DNA derived from a chromosome transfectant hybrid cell line. This sequence is shown to be present in the DNA of X-Y interchange males at a very high frequency and, based on these studies, it is categorised with the sequence defined by the probe, GMGY3, as the closest known Y chromosome derived marker to TDF. In contrast to GMGY3, however, this locus shares no homology with any other human chromosome. Southern blot analysis also reveals specific hybridization to the Y chromosome of other primates. It therefore defines, for the first time, a conserved and Y chromosome unique locus that is near to TDF.

Mapping the limits of the human pseudoautosomal region and a candidate sequence for the male-determining gene.

The human Y chromosome is composed of two different parts: a pseudoautosomal region shared with the X chromosome which is responsible for sex chromosome pairing and a Y-specific part that encodes the sex determining gene. Previously we have shown that the pseudoautosomal gene MIC2 only rarely recombines between the sex chromosomes and, based on the elevated recombination rates in the pseudoautosomal region, we predicted that this gene would lie close to the Y-specific region. In this report we describe a test of this prediction using long-range restriction mapping techniques. We conclude that MIC2 is less than 200 kilobases (kb) away from Y-specific sequences. During these experiments we have identified an HTF island in a position consistent with the proposed location of the human sex determining gene.

Investigation of chromosome-mediated gene transfer using the HPRT region of the human X chromosome as a model.

A panel of over 50 hybrid cells containing varying portions of the long arm of the human X chromosome have been obtained by chromosome-mediated gene transfer (CMGT) of human chromosomes to mouse cells deficient in HPRT. This panel is used to investigate the size and integrity of transfected human chromosome fragments and also to examine the effect of including a selectable DNA plasmid in the transfection mix. Chromosomal rearrangements are found to be generated in the chromosome transfer process, and the human X centromeric region is detected in the transfected cells at an unusually high frequency. Extensive lengths of X chromosome DNA are transferred intact, suggesting potential uses of CMGT in cloning large genes and loci for which only the chromosomal map position is known.

Genetic analysis of the human Y chromosome by chromosome-mediated gene transfer.

Chromosome-mediated gene transfer (CMGT) can be used to segregate fragments of human chromosomes in human-rodent hybrid cells. As with all somatic cell genetics methods, a selection technique is needed to isolate the hybrid cell lines produced by CMGT. Expression of the MIC2 gene product on the cell surface (the 12E7 antigen) provides an endogenous selectable marker for the human Y chromosome. Using chromosome transfer followed by separation of 12E7 antigen-positive cells on the fluorescence-activated cell sorter, a series of cell lines containing segregated fragments of the Y chromosome have been derived. The possibility of using these fragments to derive fine structural mapping data for the Y chromosome is considered in this review.

Adaptation of museum specimens for use in anatomical teaching aids.

Colour transparencies are prepared of a re-colourized anatomical specimen after placing labels temporarily in position to indicate specific structures. The specimen is also radiographed to show skeletal and soft tissue structures and radio-opaque materials and letters may used to indicate certain parts. Having mounted the specimen it can then be placed in a teaching carrel together with the radiographs and a back-slide projector for viewing the colour transparencies. The student is guided through an examination of the specimen using a script or soundtape. Frequent cross-reference is made between the actual specimen, the colour transparencies on which certain parts of the specimen have been labelled, and also the radiographs. The examination is followed by self-assessment questions relevant to the specimen and these may include identification of structures on a photograph of the specimen and on duplicate radiographs. In the final section of the aid, the answers of the self-assessment questions are given.