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Biomedical subjects

F M Watt

Publications and source records attributed to F M Watt.

At least 109 records · Page 6Linked to original sources
PubMed1991

Cell culture models of differentiation.

It is now possible to culture cells from most organs of the body under conditions in which they continue to express at least some of their differentiated traits, and to model some of the differentiation processes that occur during embryonic and adult life. How much can these cultures tell us about the acquisition and maintenance of the differentiated state? To answer this question I shall outline the features of several cell culture models, dividing them into categories according to whether they mimic differentiation during development, differentiation of adult stem cell progeny, or the transition from one differentiated phenotype to another. In spite of the diversity of cell types under consideration, it is possible to detect some common themes: the stability of the differentiated state; the relationship between proliferation and differentiation; the relative importance of intrinsic cellular programming and environmental regulation; and possible mechanisms for transcriptional control of the genes that are activated during differentiation. In recent years cell culture models have yielded a great deal of information about differentiation and the way is now clear for even more exciting discoveries.

Animals↗
PubMed1991

Expression of beta 1, beta 3, beta 4, and beta 5 integrins by human epidermal keratinocytes and non-differentiating keratinocytes.

We have compared the adhesive properties and integrin expression profiles of cultured human epidermal keratinocytes and a strain of nondifferentiating keratinocytes (ndk). Both cell types adhered to fibronectin, laminin, and collagen types I and IV, but ndk adhered more rapidly and at lower coating concentrations of the proteins. Antibody blocking experiments showed that adhesion of both cell types to fibronectin was mediated by the alpha 5 beta 1 integrin and to laminin by alpha 3 beta 1 in synergy with alpha 2 beta 1. Keratinocytes adhered to collagen with alpha 2 beta 1, but an antibody to alpha 2 did not inhibit adhesion of ndk to collagen. Both cell types adhered to vitronectin by alpha v-containing integrins. Immunoprecipitation of surface-iodinated and metabolically labeled cells showed that in addition to alpha 2 beta 1, alpha 3 beta 1, and alpha 5 beta 1, both keratinocytes and ndk expressed alpha 6 beta 4 and alpha v beta 5. ndk expressed all these integrins at higher levels than normal keratinocytes. ndk, but not normal keratinocytes, expressed alpha v beta 1 and alpha v beta 3; they also expressed alpha 1 beta 1, an integrin that was not consistently detected on normal keratinocytes. Immunofluorescence experiments showed that in stratified cultures of normal keratinocytes integrin expression was confined to cells in the basal layer; terminally differentiating cells were unstained. In contrast, all cells in the ndk population were integrin positive. Our observations showed that the adhesive properties of ndk differ from normal keratinocytes and reflect differences in the type of integrins expressed, the level of expression and the distribution of integrins on the cell surface. ndk thus have a number of characteristics that distinguish them from normal basal keratinocytes.

Antibodies↗
PubMed1991

Two strains of human keratinocytes transfected with HPV16 DNA: comparison with the normal parental cells.

We have transfected two strains of normal human epidermal keratinocytes, v and u, with a plasmid containing the HPV16 genome, and compared the growth and terminal differentiation capacity of the transfectants, vp and up, with the parental cells. vp and up contained integrated HPV16 DNA and expressed a number of viral mRNAs; unlike v and u, vp and up were aneuploid and had chromosomal alterations. Their growth rate was similar to the parental cells, although they reached a higher confluent density. In contrast to v and u, vp and up grew well in the absence of 3T3 feeders and after 14 months in culture have reached passage 60. vp and up contained desmosomes and keratin filaments, but their capacity for stratification was reduced, as were the proportions of cells expressing the terminal differentiation marker, involucrin. Involucrin mRNA levels in vp and up were lower than in the parental cells but increased after 24 h in suspension, as observed in the normal cells. u and v did not form colonies in soft agar, whereas up had a colony-forming efficiency of approximately 0.1% and vp of less than 0.1%. vp and up did not form tumours in nude mice; like the parental cells they formed cysts a week after injection, but the cysts were less well organized than those formed by v and u. In conclusion, we have obtained two lines of immortalized human keratinocytes that are not tumorigenic, but have a reduced capacity for terminal differentiation. The ability to compare matched parental and transfected cells offers considerable potential for further studies of the role of human papilloma viruses in carcinogenesis.

Animals↗
PubMed1991

Expression of E-cadherin, P-cadherin and involucrin by normal and neoplastic keratinocytes in culture.

We have compared expression of involucrin, E-cadherin and P-cadherin in cultures of normal keratinocytes and in five different lines derived from squamous cell carcinomas (SCCs), using Northern analysis and immunofluorescence. In normal keratinocytes there was an inverse correlation between P-cadherin and involucrin expression, whereas E-cadherin was expressed by both basal and terminally differentiating cells. In SCC lines involucrin expression was lower than in normal keratinocytes, and there was variable expression of P- and E-cadherin: E-cadherin mRNA levels tended to be lower in SCC lines than in normal keratinocytes, whereas P-cadherin levels were similar. Our results are consistent with observations of cadherin expression in vivo and suggest that the cultures provide a useful experimental model for investigating the role of cadherins in determining the spatial organization of normal and neoplastic keratinocytes.

Cadherins↗
PubMed1991

Integrin expression during human epidermal development in vivo and in vitro.

In order to investigate the role of extracellular matrix receptors of the integrin family in establishing the spatial organization of epidermal kerotinocytes, we used immunofluorescence microscopy to examine the expression of a range of integrin subunits during development of human palm and sole skin. All of the integrins expressed during development were also present in mature epidermis and were largely confined to the basal layer of keratinocytes in a pericellular distribution. The alpha 3 and beta 1 subunits were expressed prior to the initiation of stratification and did not change in abundance or distribution during subsequent development. alpha 4 and beta 3 were not detected at any time in the epidermis. Every other subunit examined showed spatial or temporal changes in expression. Staining for alpha 1 was strong before stratification and until mid-development, but was greatly decreased in neonatal epidermis. alpha 2 was first detected in small patches of basal cells prior to stratification, and thereafter was found in the entire basal layer, with greater staining in developing sweat glands. alpha 5 was not expressed until mid-development, and then primarily in developing sweat glands, with faint expression in neonatal epidermis. alpha v was detected following stratification, in developing sweat glands, and occasionally in neonatal epidermis. alpha 6 and beta 4 were peribasally expressed before stratification, but thereafter became concentrated at the basal cell surface in contact with the basement membrane, co-localizing with hemidesmosomes as determined by staining with bullous pemphigoid antiserum. We also examined the distribution of three known ligands for keratinocyte integrins: laminin and collagen type IV were present in the basement membrane zone at all stages of development, whereas fibronectin was only evident there until about 13 weeks estimated gestational age. Finally, we found that the changes in integrin expression that occur on initiation of stratification in vivo could be reproduced in organ cultures of developing skin; such cultures therefore provided a useful experimental model for further studies of the role of integrins in epidermal stratification.

Embryonic and Fetal Development↗
PubMed1991

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.

3T3 Cells↗
PubMed1991

Decreased expression of fibronectin and the alpha 5 beta 1 integrin during terminal differentiation of human keratinocytes.

We have examined the expression of fibronectin and the alpha 5 beta 1 fibronectin receptor during terminal differentiation of human epidermal keratinocytes, using involucrin as a terminal differentiation marker. The levels of mRNAs encoding fibronectin and the alpha 5 and beta 1 integrin subunits were measured in keratinocyte populations that had been enriched for involucrin-negative or -positive cells by unit gravity sedimentation or suspension-induced terminal differentiation. All three mRNAs decreased in abundance during terminal differentiation, and the corresponding proteins were localised by immunofluorescence to the basal layer in stratified colonies. We also examined expression in ndk, a strain of epidermal cells with a complete block in terminal differentiation, which, as a result, do not express involucrin. Messenger RNA levels for fibronectin and the alpha 5 and beta 1 subunits were higher in ndk, than in unfractionated keratinocytes and the corresponding proteins were expressed by all ndk, consistent with a basal keratinocyte phenotype. We conclude that expression of fibronectin and the alpha 5 beta 1 fibronectin receptor decreases during terminal differentiation and that such changes are likely to play a role in the selective migration of terminally differentiating cells from the basal epidermal layer.

Antibodies, Monoclonal↗
PubMed1991

Production of scatter factor by ndk, a strain of epithelial cells, and inhibition of scatter factor activity by suramin.

ndk are a strain of human epidermal keratinocytes that do not undergo terminal differentiation and which grow as single cells rather than compact colonies. We show that ndk are motile and secrete an epithelial scatter factor that has the same biochemical and immunological properties as the scatter factor previously purified from ras-transformed 3T3 fibroblasts. We have found that suramin, a polyanionic detergent, will reverse the activity of scatter factor from either cell type in the standard MDCK activity assay. When added to ndk cultures, suramin causes the cells to grow in coherent patches. This morphological change is accompanied by alterations in the distribution of actin and integrins, but not by stratification or terminal differentiation. The effect is reversed upon removal of suramin. We propose that the motile phenotype of ndk is due, at least in part, to autocrine production of scatter factor and that suramin may be useful for further studies of scatter factor binding to the cell surface.

Actins↗
PubMed1991

Mitogens induce calcium transients in both dividing and terminally differentiating keratinocytes.

During terminal differentiation, keratinocytes lose the ability to divide. One indicator of responsiveness to certain growth factors is a transient rise in the intracellular concentration of free calcium ions ([Ca2+]i). The aim of our experiments was to discover whether or not terminally differentiating keratinocytes have lost the ability to exhibit an increase in [Ca2+]i in response to factors that stimulate [3H]thymidine incorporation and increase [Ca2+]i in undifferentiated keratinocytes. [Ca2+]i was measured with the calcium indicator dye FURA-2 and by a ratio imaging method. Expression of involucrin, a precursor of the keratinocyte cornified envelope, was used as a marker of terminal differentiation. Measurements were made on stratified colonies of cells grown in standard medium (containing 1.8 mM calcium ions) and on cell monolayers in low calcium medium (0.1 mM). Treatment of serum-starved monolayers with substance P, bombesin or complete growth medium containing 10% fetal calf serum resulted in increased [3H]thymidine incorporation. A switch from low calcium to standard medium also stimulated [3H]thymidine incorporation whether or not the cells had been serum-starved. In each experiment some cells showed an increase in [Ca2+]i while others did not. However, the heterogeneity in the [Ca2+]i response did not reflect the terminal differentiation status of individual cells: both involucrin-positive and -negative cells were found in the responding and nonresponding populations. Involucrin-positive and -negative areas of stratified cultures also underwent a transient increase in [Ca2+]i in response to serum-containing medium. Our data therefore indicate that both proliferating (involucrin-negative) and post-mitotic, terminally differentiating (involucrin-positive) keratinocytes can respond to mitogenic stimuli by an increase in [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood↗
PubMed1990

Changes in keratinocyte adhesion during terminal differentiation: reduction in fibronectin binding precedes alpha 5 beta 1 integrin loss from the cell surface.

During terminal differentiation keratinocytes move out of the basal layer of the epidermis and thereby lose contact with the basement membrane. We show that terminal differentiation in culture involves loss of adhesiveness to fibronectin, laminin, and collagen types I and IV. The adhesive changes precede, by several hours, loss of the alpha 2 beta 1, alpha 3 beta 1, and alpha 5 beta 1 integrins from the cell surface. Keratinocyte adhesion to fibronectin is mediated by the alpha 5 beta 1 integrin, and the decrease in adhesion of intact cells to fibronectin is correlated with a decrease in the ability of alpha 5 beta 1 receptors to bind fibronectin. Thus modulation of integrin function early in terminal differentiation may be an early event determining cell migration out of the basal layer.

Cell Adhesion↗
PubMed1990

DNA amplification is rare in normal human cells.

Three types of normal human cells were selected in tissue culture with three drugs without observing a single amplification event from a total of 5 x 10(8) cells. No drug-resistant colonies were observed when normal foreskin keratinocytes were selected with N-(phosphonacetyl)-L-aspartate or with hydroxyurea or when normal mammary epithelial cells were selected with methotrexate. Some slightly resistant colonies with limited potential for growth were obtained when normal diploid fibroblast cells derived from fetal lung were selected with methotrexate or hydroxyurea but careful copy-number analysis of the dihydrofolate reductase and ribonucleotide reductase genes revealed no evidence of amplification. The rarity of DNA amplification in normal human cells contrasts strongly with the situation in tumors and in established cell lines, where amplification of oncogenes and of genes mediating drug resistance is frequent. The results suggest that tumors and cell lines have acquired the abnormal ability to amplify DNA with high frequency.

Breast↗
PubMed1990

Characterisation of the peanut lectin-binding glycoproteins of human epidermal keratinocytes.

We have previously shown that peanut lectin (PNA) binding is a useful marker of keratinocyte terminal differentiation and have identified two PNA-binding glycoproteins with electrophoretic mobilities of approximately 250 kDa and 110 kDa [11]. We now report that in epidermis and stratified cultures of keratinocytes the binding patterns of PNA and the sialic acid-specific lectin Limax flavus agglutinin (LFA) are complementary, with LFA showing specificity for cells in the basal layer. LFA bound to the 250-kDa glycoprotein immunoprecipitated with an antiserum raised against the PNA-binding glycoproteins (anti PNA-gp), but not to the 110-kDa glycoprotein; it also bound additional high-molecular-weight material. These data suggest that the 250-kDa glycoprotein is expressed in the basal layer in a form with terminal sialic acid residues and suprabasally in a form with terminal galactose. LFA and anti-PNA-gp stained all cells in a range of cultured epithelial lines tested, whereas PNA stained only cells that had lost contact with the culture substratum, raising the possibility that loss of sialic acid residues is associated with stratification. Anti PNA-gp recognized glycoproteins of differing mobilities in these lines. Anti PNA-gp also stained epithelial cells in all tissues tested. In keratinocytes the PNA-binding glycoproteins were localised to the cell surface by immunoelectron microscopy; they were abundant on the microvilli and absent from desmosomal junctions. In conclusion, we have obtained further information about the nature of the PNA-binding glycoproteins in keratinocytes and related glycoproteins in other epithelial cell types.

Cells, Cultured↗
PubMed1990

Subpopulations of chondrocytes from different zones of pig articular cartilage. Isolation, growth and proteoglycan synthesis in culture.

Articular cartilage varies in ultrastructure and composition with distance from the articular surface. We have cultured chondrocytes from different zones of pig articular cartilage and investigated whether there are intrinsic differences in their behaviour that might account for the variation observed in intact tissue. On isolation, cells from the upper third of the cartilage were smaller than those of the lower third, but this difference was not maintained in culture. Upper zone cells attached and spread more slowly than lower zone cells; morphological differences between the two populations could be seen for several weeks. The growth rates of the two populations were similar, but upper zone cells reached a lower confluent density. Levels of protein synthesis were similar for both populations, but upper zone cells deposited less proteoglycan in the cell layer. On isolation, the percentage of upper zone cells that stained positive with MZ15, a monoclonal antibody to keratan sulphate, was smaller than the percentage of lower zone cells, but this difference was lost after several days in culture. Nevertheless, the keratan sulphate content of proteoglycan synthesised by lower zone chondrocytes at high density was greater than of that synthesised by upper zone cells. The proportion of nonaggregating proteoglycan was greater in upper than lower zone cartilage and this difference was also observed in long-term cultures. proteoglycans were further characterised by composite and polyacrylamide gel electrophoresis and by immunoblotting; differences detected in cartilage extracts were not, however, maintained in culture; instead, the small proteoglycans synthesised by both upper and lower zone cells varied with plating density. Finally, alkaline phosphatase, a marker of hypertrophic, calcifying cartilage, was only expressed in lower zone cultures. We conclude that some of the observed heterogeneity of articular cartilage reflects intrinsic differences between the cells of different zones, whereas some may reflect the response of chondrocytes to different environmental conditions.

Alkaline Phosphatase↗
PubMed1989

Fibronectin inhibits the terminal differentiation of human keratinocytes.

In the epidermis proliferation of keratinocytes is restricted to the basal layer, which is in contact with the basement membrane, and cells undergo terminal differentiation as they move upwards through the suprabasal layers. In stratified cultures of human keratinocytes, upward migration is a consequence, not a cause, of terminal differentiation and occurs because keratinocytes become less adhesive to their substratum and to one another. Most keratinocytes can be induced to differentiate to completion by placing them in suspension in methylcellulose: within 12 h DNA synthesis is irreversibly inhibited and by 24 h most cells express involucrin (ref 4; P. A. Hall, J.C.A. and F.M.W., unpublished observations). Here we report that when fibronectin is added to the methylcellulose, keratinocytes still withdraw from the cell cycle, but induction of involucrin expression is largely inhibited. The effect of fibronectin is concentration- and time-dependent and is mediated by a receptor of the integrin family. These results provide an explanation for why overt terminal differentiation is normally restricted to suprabasal cells, whereas cell-cycle withdrawal occurs within the basal layer; they also have important implications for the mechanism of epidermal wound healing. Furthermore, our data show that the binding of an extracellular matrix protein to its receptor can regulate differentiated gene expression in the absence of changes in cell shape.

Cell Differentiation↗
PubMed1989

Heterogeneous expression of keratin, involucrin, and extracellular matrix among subpopulations of a poorly differentiated human cervical carcinoma: possible relationships to patterns of invasion.

Undifferentiated cervical carcinomas vary considerably in their intercellular organization and patterns of invasion. In spite of its clinical significance, the basis for such variation is poorly understood. We investigated the cellular properties that may be responsible for this diversity, using as a model two human cervical carcinoma cell lines that were derived from the same tumor specimen and the same clone. It was shown previously that, in spite of their common origin, each line forms a histologically distinct type of undifferentiated carcinoma when heterotransplanted in vivo: cells of line C-4I grow as compact expanding masses with central necrosis, while tumors of line C-4II infiltrate host tissues as small, well-vascularized, dispersed cell groups. The characteristic behavior of each line was retained in culture, where C-4I cells formed highly multilayered cohesive colonies, while C-4II cells formed diffuse, monolayered colonies and shed into the culture medium. These observations as well as ultrastructural data suggested that each line may be arrested at a different stage of stratified squamous differentiation. In the present study, this hypothesis was tested by examining specific differentiation markers. An analysis of the cultures by immunofluorescence microscopy and immunoblotting revealed that keratin was more abundant in the compact C-4I line than in the dispersed C-4II line. C-4I cells expressed keratins 5, 6, 8, 16, 18, and 19, while C-4II expressed only keratins 8, 16, 18, and 19. In the multilayered C-4I colonies, involucrin-positive cells occurred in the apical cell layers only. In C-4II, involucrin-positive cells occurred in monolayers and domes, and they were most consistently located apically in crowded cultures. Laminin was secreted by both lines, but only C-4II cells deposited a fibronectin matrix. The results suggest that C-4I cells resemble normal cervical cells at the spinous stage of stratified squamous differentiation, while C-4II cells resemble basal/suprabasal cells. The different growth patterns of the tumors, formed by the lines in vivo, therefore likely reflect functional and behavioral differences that normally exist between spinous and basal cervical epithelial cells. The results suggest that differentiation-related functional properties may lead to histological diversity among cervical carcinomas that are categorized as undifferentiated by histopathological criteria.

Carcinoma↗
PubMed1989

Pentapeptide inhibitor of epidermal mitosis: production and responsiveness in cultures of normal, transformed and neoplastic human keratinocytes.

A pentapeptide, pyroGlu-Glu-Asp-Ser-GlyOH, was previously isolated from mouse skin and shown to inhibit reversibly proliferation of murine keratinocytes, both in intact skin and in culture. In the present report we have shown that proliferation of normal human keratinocytes in culture is also inhibited by the pentapeptide, whether the cells are grown under standard conditions or prevented from stratifying in medium containing a low concentration of calcium ions. An SV40-transformed line of human keratinocytes, SVK14, was completely insensitive to the pentapeptide and a line derived from a squamous cell carcinoma of the oral cavity, SCC-9, was less sensitive than the normal cells. The effect of the pentapeptide on terminal differentiation was determined by measuring the proportion of cells expressing involucrin after a single dose or repeated doses of the pentapeptide: no consistent change in the number of terminally differentiating cells was observed. Cell extracts and conditioned medium from all three cell types contained the epidermal pentapeptide, suggesting a role for the peptide in both autocrine (normal keratinocytes) and paracine (SVK14, SCC-9) growth control. A proportion of the pentapeptide isolated from conditioned medium was phosphorylated; since it was as active as the non-phosphorylated form, when assayed on mouse epidermis, the role of phosphorylation remains to be determined.

Amino Acid Sequence↗