Detecting plant silica fibres in animal tissue by confocal fluorescence microscopy.

Silica fibres from the inflorescence bracts of the grass Phalaris canariensis L. cause dermatitis, and have been implicated in the aetiology of oesophageal cancer in northeastern Iran. Here we describe a method for labelling these fibres so that they can be located in mammalian tissue. Fluorescein was covalently linked to isolated, purified fibres with the silane coupling agent 3-aminopropyl triethoxysilane. The labelled hairs were then rubbed into the backs of mice. These were later killed and their skin fixed, stained and sliced at a thickness of 250 microns. A confocal laser scanning microscope gave brilliant images of the fibres at any depth up to 100 microns or more beneath the surface of the slice. Fibres penetrated deeply into the dermis. Several cubic millimetres of tissue could be surveyed in 1 h. The number of fibres present was approximately 2 mm-3 initially, falling to 0.1 mm-3 after 7 days.

Actin cytoskeleton and motility in rat sarcoma cell populations with different metastatic potential.

We have studied the organisation of the actin cytoskeleton in three related rat sarcoma cell populations of differing malignancy. They were derived by neoplastic progression from a population which had transformed spontaneously in vitro, and were distinguished by their ability to give rise to reproducibly different numbers of metastases, ranging from 10% to 80% of the animals inoculated. We found characteristic differences in the arrangement of the actin cytoskeleton. Confocal three-dimensional microscopy showed that nearly all of the least malignant population contained conspicuous actin stress fibres lying in the lower part of the cell parallel to the substratum and no other actin structures. Actin in the intermediate population was typically situated in a diffuse layer underlying the whole plasma membrane, in which no fibres could be seen. Two thirds of the most malignant population consisted of more rounded cells filled with a three-dimensional network of fine oblique actin fibres. There were focal contacts in all these cells; their area showed a regular decrease from 1.3 microns 2 to 0.4 microns 2. The differences in actin distribution were accompanied by differences in motility, which increased as malignancy increased. When individual cells were fixed after they had been tracked by time-lapse, their cytoskeleton type correlated with the speed at which they had moved. All these differences were enhanced at low pH. These findings point to the possibility that the three-dimensional network of fine actin fibres in acid culture could be a measure of the malignant potential of transformed cells in vitro.

Changes in the abundance and distribution of actin and associated proteins during terminal differentiation of human epidermal keratinocytes.

We have examined the abundance and distribution of actin and several actin-associated proteins in human epidermal keratinocytes before and after initiation of terminal differentiation. Keratinocytes were placed in suspension in methylcellulose for 1 h or 24 h and then extracted for immunoblotting. At 24 h, when the proportion of cells expressing the terminal differentiation marker, involucrin, had increased approximately 3-fold, there were marked decreases in the levels of vinculin, talin, filamin and gelsolin. The level of actin was unchanged and the level of alpha-actinin decreased only slightly. To complement the immunoblot analysis, we also examined the distribution of each protein in basal (involucrin-negative) and suprabasal (involucrin-positive) cells in stratified colonies, using confocal microscopy. Gelsolin, filamin, vinculin, talin, alpha-actinin and filamentous actin were all less abundant in suprabasal cells than in basal cells. There were also differences in the distribution of all the proteins in the basal compared to the suprabasal layers. In addition to the changes associated with terminal differentiation, there was variation in the distribution of focal contacts and stress fibres and in gelsolin levels between basal cells at the periphery of colonies and those in the centre. These results are discussed in the context of the known association of the actin cytoskeleton with receptors of the integrin family, the loss of integrins that occurs during keratinocyte terminal differentiation, and the possible role of the cytoskeleton in signalling between integrins and the nucleus.

Limitation of substratum size alters cytoskeletal organization and behaviour of Swiss 3T3 fibroblasts.

Cell spreading and adhesion formation in Swiss 3T3 cells was studied on circular adhesive islands of size 400-500 microns 2 made by evaporating palladium through a mask onto an underlying non-adhesive surface. Cell spreading was limited since focal contacts were restricted to the palladium. On islands less than 2000 microns 2, focal contacts and actin bundles were arranged at the cell periphery. On islands less than 1000 microns 2, the size and number of focal contacts were reduced. Focal contacts may be important regulators of proliferation, but they do not seem to form a deterministic link between substratum contact and proliferative stimulus.

Cell shape controls terminal differentiation of human epidermal keratinocytes.

Cultures of human epidermal keratinocytes provide a useful experimental model with which to study the factors that regulate cell proliferation and terminal differentiation. One situation that is known to trigger premature terminal differentiation is suspension culture, when keratinocytes are deprived of substratum and intercellular contact. We have now investigated whether area of substratum contact, and hence cell shape, can regulate terminal differentiation. Keratinocytes were grown on circular adhesive islands that prevented cell-cell contact. By varying island area we could vary cell shape from fully spread to almost spherical. We found that when substratum contact was restricted, DNA synthesis was inhibited and expression of involucrin, a marker of terminal differentiation, was stimulated. Inhibition of proliferation was not a sufficient stimulus for involucrin synthesis in fully spread cells. When DNA synthesis and involucrin expression were plotted against contact area, classic dose-response curves were obtained. Thus cell shape acts as a signal for the terminal differentiation of keratinocytes in culture.

A fine fibrous silica contaminant of flour in the high oesophageal cancer area of north-east Iran.

We report here the discovery and characterization of a fibrous mineral contaminant of the diet in that area of north-east Iran where oesophageal cancer has a very high incidence. This contaminant has a smoothly tapering shape and is between 50 and 150 micrometers long. The greatest diameter is between 1 and 10 micrometers and this decreases to a sharply pointed tip with a radius of curvature of between 0.25 and 0.60 micrometers. Electron microscope X-ray analysis shows that this fibre consists almost entirely of silica. It is free from alkali metals, aluminium and iron, and therefore differs from other known natural or manmade mineral fibres. Examination of the seeds of more than sixty different species of weed know to contaminate the wheat in this area of the Middle East shows that the fibre originates from the seeds of the common Mediterranean grass Phalaris minor. This seed bears fibres of the same dimensions, composition and birefringence, borne upon the inflorescence bracts which envelop the pericarp of the seeds of this and other members of the phalaris genus. They are broken off from the seed when the wheat is milled but persist in the flour, where up to 3,000 are found in each gram. Similar fibres can be isolated in quantity from the seeds of related species which are grown commercially, and they have a similar size and composition. When cells of the 3T3 mouse fibroblast line are exposed to these fibres in semi-solid suspension culture, their proliferation is stimulated more than 100-fold. We present an hypothesis for the involvement of these plant mineral fibres in the aetiology of oesophageal cancer in Iran and in other areas of high incidence.