Murine thioglycollate-elicited peritoneal macrophages were cultured in the presence of a variety of fatty acids added as complexes with bovine serum albumin. All fatty acids tested were taken up readily by the cells and both neutral and phospholipid fractions were enriched with the fatty acid provided in the medium. This generated a range of cells enriched in saturated, monounsaturated or polyunsaturated fatty acids, including n-3 acids of fish oil origin. Saturated fatty acid enrichment enhanced macrophage adhesion to both tissue culture plastic and bacterial plastic compared with enrichment with polyunsaturated fatty acids. Macrophages enriched with the saturated fatty acids myristate or palmitate showed decreases of 28% and 21% respectively in their ability to phagocytose unopsonized zymosan particles. Those enriched with polyunsaturated fatty acids showed 25-55% enhancement of phagocytic capacity. The greatest rate of uptake was with arachidonate-enriched cells. Phagocytic rate was highly correlated with the saturated/unsaturated fatty acid ratio, percentage of polyunsaturated fatty acid and index of unsaturation, except for macrophages enriched with fish-oil-derived fatty acids; they showed lower phagocytic activity than expected on the basis of their degree of unsaturation. These results suggest that membrane fluidity is important in determining macrophage adhesion and phagocytic activity. However, in the case of phagocytosis, this effect may be partially overcome if the cells are enriched with fish-oil-derived fatty acids. Thus it may be possible to modulate the activity of cells of the immune system, and so an immune response, by dietary lipid manipulation.

Normal senescence in human melanocytes requires p16 activity. p53 contributes to a delayed form of senescence that requires telomere shortening, in p16-deficient melanocytes. These findings provide some basis for the role of p16 in melanoma susceptibility.

Replicative senescence in human fibroblasts is absolutely dependent on the function of the phosphoprotein p53 and correlates with activation of p53-dependent transcription. However, no evidence for posttranslational modification of p53 in senescence has been presented, raising the possibility that changes in transcriptional activity result from upregulation of a coactivator. Using a series of antibodies with phosphorylation-sensitive epitopes, we now show that senescence is associated with major changes at putative regulatory sites in the N and C termini of p53 consistent with increased phosphorylation at serine-15, threonine-18, and serine-376 and decreased phosphorylation at serine-392. Ionizing and UV radiation generated overlapping but distinct profiles of response, with increased serine-15 phosphorylation being the only common change. These results support a direct role for p53 in signaling replicative senescence and are consistent with the generation by telomere erosion of a signal which shares some but not all of the features of DNA double-strand breaks.Normal human somatic cells (with the possible exception of stem cells) are capable of only a finite number of cell divisions, after which they enter a nondividing though viable state termed replicative senescence (22,55). The significance of this phenomenon for human health is two-edged. On the one hand, it imposes a natural obstacle to clonal expansion, which probably plays a vital part in limiting tumor development (2, 38, 59). On the other hand, in some tissues, notably skin and blood vessels, it may account for progressive functional abnormality with advancing age. This may result directly from loss of regenerative capacity but also indirectly through senescence-associated biochemical changes, a good example being the increased collagenase secretion by ageing dermal fibroblasts, which may be significant even when only a minority of cells are overtly senescent (11,34). Knowledge of the underlying mechanisms of cellular senescence is therefore central to both cancer and aging research.We and others have demonstrated that one key signal pathway mediating replicative senescence involves the phosphoprotein p53, more widely recognized for its role as a tumor suppressor, which is known to mediate growth arrest in response to a wide variety of cellular stress signals including DNA damage (31, 40). Experimental abrogation of p53 function prevents fibroblasts from entering senescence normally and indeed can reverse established senescence, demonstrating that p53, if not sufficient, is certainly necessary for this process (5,6,20). Furthermore, growth arrest in senescence is tightly correlated with switching on of the transcriptional transactivation function of p53, as revealed by the use of reporter constructs and by DNA binding assays (1,7,50).Nevertheless, senescence has not thus far been shown to lead to any of the range of posttranslational modifications of the p53 protein which bring about its activation in response to other signals such as DNA damage (19). ...

The accumulation of genetic abnormalities in a developing tumor is driven, at least in part, by the need to overcome inherent restraints on the replicative life span of human cells, two of which-senescence (M1) and crisis (M2)-have been well characterized. Here we describe additional barriers to clonal expansion (M int ) intermediate between M1 and M2, revealed by abrogation of tumor-suppressor gene (TSG) pathways by individual human papillomavirus type 16 (HPV16) proteins. In human fibroblasts, abrogation of p53 function by HPVE6 allowed escape from M1, followed up to 20 population doublings (PD) later by a second viable proliferation arrest state, M int E6, closely resembling M1. This occurred despite abrogation of p21 WAF1 induction but was associated with and potentially mediated by a further ϳ3-fold increase in p16 INK4a expression compared to its level at M1. Expression of HPVE7, which targets pRb (and p21 WAF1 ), also permitted clonal expansion, but this was limited predominantly by increasing cell death, resulting in a M int E7 phenotype similar to M2 but occurring after fewer PD. This was associated with, and at least partly due to, an increase in nuclear p53 content and activity, not seen in younger cells expressing E7. In a different cell type, thyroid epithelium, E7 also allowed clonal expansion terminating in a similar state to M int E7 in fibroblasts. In contrast, however, there was no evidence for a p53-regulated pathway; E6 was without effect, and the increases in p21 WAF1 expression at M1 and M int E7 were p53 independent. These data provide a model for clonal evolution by successive TSG inactivation and suggest that cell type diversity in life span regulation may determine the pattern of gene mutation in the corresponding tumors.Human tumors develop by a process of clonal evolution mediated by the acquisition of successive molecular abnormalities and driven, at least in part, by the need to overcome the inherent controls which limit the proliferative life span of normal human cells (51).Two of these proliferative life span barriers (PLBs)-senescence and crisis-have been well characterized, particularly with respect to human fibroblast models. These cells normally undergo around 40 to 70 population doublings (PD) (depending on age of donor) after which, even in ideal culture conditions, they enter a stable proliferative arrest in which they remain viable for many months (27). Escape from this state of replicative senescence, or mortality stage 1 (M1) (48), can be conferred by expression of a variety of DNA tumor virus genes, including simian virus 40 (SV40) T and human papillomavirus type 16 (HPV16) E6 plus E7, which target a common set of cell cycle regulatory tumor suppressor gene products, notably p53 and pRb (14, 41). The resulting clones are capable of at least an additional 30 PD, after which further expansion is limited by a second PLB termed crisis (or M2), which is due not so much to decreasing proliferation as to increasing cell death. Escape from this state is associated with stabiliz...

Hepatocyte Growth Factor (HGF) receptor, encoded by the protooncogene c-met, is overexpressed in many human tumours, including those of thyroid epithelium. The absence in most cases of any primary structural abnormality of the met gene suggests that overexpression is secondary to mutation of other gene(s). To test this hypothesis we investigated the e ect on met expression of two activated oncogenes known to play a major role in thyroid oncogenesis, ras and ret. To minimize the possibility of unknown co-operating events, we introduced these genes directly into normal human thyrocytes in primary culture using amphotropic retroviral vectors and assessed met expression as early as possible in the resulting epithelial colonies. Double immuno¯uorescence revealed expression of met protein, strictly localized to cells expressing the mutant ras and ret vectors, expression in background normal cells being barely detectable. In contrast, colonies induced to proliferate at a comparable rate by a vector expressing SV40 T showed no increase in met expression. To permit quantitation by Western blotting we also extended these ®ndings to a thyroid cell line (R18) containing a zincinducible mutant ras gene. Induction of the oncogene led to a fourfold increase in met protein expression. We conclude that overexpression of met is induced by activation of the ras or ret signalling pathway and not simply by deregulation of the cell cycle per se. The data suggest that the proliferative advantage conferred by these oncogenes may be in part due to the resulting sensitization of tumour epithelium to paracrine HGF secreted by stromal cells.

The tumour suppressor gene p53 plays a major role in the cellular response to DNA damage, mediating growth arrest and/or apoptosis. Phosphorylation of the protein occurs at numerous sites in vivo and is likely to be a major mechanism for modulation of its activity as a transcriptional transactivator. Not surprisingly, therefore, p53 has been intensively studied by 32 P metabolic labelling. Here we show however, using normal human ®broblasts, that typical labelling conditions induce (i) a p53-dependent inhibition of DNA synthesis and (ii) an increase in the cellular content of p53 protein detectable by the phosphorylation-sensitive antibody DO-1 but not by antibody DO-12. These data demonstrate for the ®rst time that 32 P labelling is sucient to induce a biologically-signi®cant, p53-mediated cellular response and strongly suggest that it perturbs the phosphorylation state of p53 which it is being used to measure. This highlights the need to re-evaluate earlier data by nonradioactive approaches using phospho-speci®c antibodies.

Current evidence suggests the papillary thyroid carcinoma oncogene (RET/PTC) generates papillary thyroid carcinomas in one genetic step. We tested a resulting prediction that RET/PTC expression in thyroid epithelium should be sufficient to cause the changes in nuclear morphology diagnostic of this tumor. Primary cultures of human thyroid epithelial cells were infected with a RET/PTC retroviral construct. Morphological scoring by two independent cytopathologists shows RET/PTC expression by immunohistochemistry to be highly associated (p << 0.0001) with an irregular nuclear contour and a euchromatic appearance compared with non-expressing cells in the same cultures. The altered nuclear morphology is not due to gene transfer or transformation per se as primary thyroid cell cultures infected with a retroviral H-RAS construct differ from RET/PTC-infected cells by showing round nuclear envelopes and coarser chromatin, as determined by the independent scoring of two cytopathologists (p << 0.0001). In addition, RET/ PTC-transfected cells appear to disperse, whereas RAS-transfected cells grow as discrete colonies. The results provide additional support for the hypothesis that RET/PTC is sufficient to cause papillary thyroid carcinomas. A signaling pathway downstream of RET/ PTC leads to restructuring of the nuclear envelope and chromatin, and the signal does not depend entirely, if at all, on a RAS pathway.

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