A review of in vitro modelling approaches to the identification and modulation of squamous metaplasia in the human tracheobronchial epithelium

Squamous metaplasia in the tracheobronchial epithelium (TBE) involves the replacement of the normal pseudostratified mucociliary epithelium with a stratified squamous epithelium. Squamous metaplasia is considered to be an adaptive response that protects the lumen from the effects of inhaled airborne pollutants, but which might also feature as a pre-neoplastic lesion preceding squamous cell carcinoma. With the exception of transglutaminase I, involucrin, and cytokeratins 5, 6 and 13, few markers that contribute to the squamous phenotype have been identified in human TBE that can be used in diagnosis or to monitor its development in laboratory investigations, and current models are inadequate to provide statistically meaningful data. Therefore, new predictive markers have been identified, and new techniques established, in epithelial in vitro models capable of expressing squamous characteristics, which will be used to identify hazardous exposures and elucidate the mechanisms by which they induce their effects. A protocol for the quantitative detection of transglutaminase activity has been standardised in keratinocytes, based on the enzymatic incorporation of fluorescein-cadaverine (FC) into bis(gamma-glutamyl) polyamine cross-links. The specificity of this compound as a transglutaminase substrate was demonstrated by using a range of competitive transglutaminase inhibitors, and by modulation of the squamous pathway. FC incorporation was localised to the cell membrane of terminally differentiating cells, and was not visible in basal, proliferating cells. High calcium-containing medium, nicotine and cigarette smoke condensates (CSC) induced an increase in FC incorporation, providing evidence of their role in enhancing the squamous pathway. Analysis by flow cytometry was used to provide a quantitative assessment of a range of optimised squamous differentiation markers, identified in normal human bronchial epithelia and in a bronchial cell line. Transglutaminase I was induced in a time-dependent manner, in post-confluent cells induced to differentiate down the squamous pathway, whereas involucrin was ubiquitously expressed and the levels of cytokeratins 5, 6 and 18 were reduced. The response of these and other differentiation markers to squamous-inducing conditions is being explored.     

Role of protein kinase C alpha in calcium induced keratinocyte differentiation: defective regulation in squamous cell carcinoma

Calcium induces both involucrin and transglutaminase-K in normal keratinocytes (NHK) but not in squamous carcinoma cell lines (SCC). The protein kinase C (PKC) agonist phorbol myristoyl acetate potentiates and the PKC antagonist Ro31-8220 blocks the ability of calcium to stimulate the involucrin promoter in normal human keratinocytes but not in SCC4. We thus examined the ability of calcium to regulate the levels of five PKC isozymes in NHK and two SCC. In the normal keratinocytes, the levels of PKC [alpha], PKC [delta], PKC [eta], and PKC [zeta] increased over the first one to two weeks in a calcium-and time-dependent manner. PKC [epsilon] decreased in a time-and calcium-dependent fashion over the three-week period. All five isozymes showed little change during culture in SCC4 at any calcium concentration. Calcium and time of culture had partial effects on SCC12B2, a carcinoma that shows partial differentiation characteristics. Since PKC [alpha] is the only calcium responsive PKC isozyme in keratinocytes and most likely to be directly involved in calcium induced differentiation, we evaluated the effect of inhibiting its production with antisense oligonucleotides on calcium-regulated markers of differentiation. We found that the PKC [alpha] specific antisense oligonucleotide blocked calcium stimulated involucrin promoter activity as well as PKC [alpha], involucrin, and transglutaminase protein production, whereas the sense oligonucleotide control did not. We conclude that although a number of PKC isozymes are regulated during calcium-induced differentiation, PKC [alpha] plays a necessary role in mediating calcium-induced differentiation. Failure to regulate PKC [alpha] in SCC4 may underlie at least part of the failure of calcium to promote differentiation in these cells.     

Differentiation of normal and tumoral human keratinocytes cultured on dermis: Reconstruction of either normal or tumoral architecture

Summary Normal human keratinocytes isolated from skin and squamous carcinoma cells established from a human tumor (TR146 cell line) both exhibit limited morphologic differentiation when they are grown on conventional plastic dishes. However, when they are seeded on human de-epidermized dermis and cultured at the air-liquid interface, they are able to reform an epithelium having the morphology of the tissue of origin (i.e. skin or squamous carcinoma). The distribution in such reconstructed tissues of differentiation markers such as bullous pemphigoid antigen, 67K keratin, involucrin, membrane-bound transglutaminase, and filaggrin was very similar to their distribution in normal skin and squamous carcinoma specimens, respectively. The degree of differentiation is for both cell types extremely sensitive to culture conditions such as retinoic acid concentration, emersion of the cultures, etc. These results show that subcultured normal or tumoral keratinocytes are able to recover their specific morphogenetic potential when cultured in an environment close to their in vivo situation.     

Induction of Differentiation in Normal Human Keratinocytes by Adenovirus-Mediated Introduction of the η and δ Isoforms of Protein Kinase C

Protein kinase C (PKC) plays a crucial role(s) in regulation of growth and differentiation of cells. In the present study, we examined possible roles of the α, δ, η, and ζ isoforms of PKC in squamous differentiation by overexpressing these genes in normal human keratinocytes. Because of the difficulty of introducing foreign genes into keratinocytes, we used an adenovirus vector system, Ax, which allows expression of these genes at a high level in almost all the cells infected for at least 72 h. Increased kinase activity was demonstrated in the cells overexpressing the α, δ, and η isoforms. Overexpression of the η isoform inhibited the growth of keratinocytes of humans and mice in a dose (multiplicity of infection [MOI])-dependent manner, leading to G₁ arrest. The η-overexpressing cells became enlarged and flattened, showing squamous cell phenotypes. Expression and activity of transglutaminase 1, a key enzyme of squamous cell differentiation, were induced in the η-overexpressing cells in dose (MOI)- and time-dependent manners. The inhibition of growth and the induction of transglutaminase 1 activity were found only in the cells that express the η isoform endogenously, i.e., in human and mouse keratinocytes but not in human and mouse fibroblasts or COS1 cells. A dominant-negative η isoform counteracted the induction of transglutaminase 1 by differentiation inducers such as a phorbol ester, 1α,25-dihydroxyvitamin D₃, and a high concentration of Ca²⁺. Among the isoforms examined, the δ isoform also inhibited the growth of keratinocytes and induced transglutaminase 1, but the α and ζ isoforms did not. These findings indicate that the η and δ isoforms of PKC are involved crucially in squamous cell differentiation.     

Coordination of keratinocyte programming in human SCC-13 squamous carcinoma and normal epidermal cells

Exploiting the sensitivity of neoplastic keratinocytes to physiological effectors, this work analyzes the degree of coordination among differentiation markers in the established human epidermal squamous carcinoma cell line SCC-13 in comparison to normal human epidermal cells. This analysis showed that overall keratin content was modulated substantially and in parallel with particulate transglutaminase activity in response to variation of calcium, retinoic acid, and hydrocortisone concentrations in the medium. The changes in keratin expression were evident primarily in the striking stimulation by hydrocortisone or calcium and the virtual suppression by retinoic acid of species in the 56-58 kd region, which have not previously been reported subject to such physiological modulation. In contrast, involucrin levels were coordinated only to a limited degree with particulate transglutaminase activity and keratin content. The very low involucrin levels observed in low calcium medium were increased 5- to 10-fold in high calcium medium. However, they were also increased 5- to 30-fold in low calcium medium by retinoic acid, a clear example of uncoupling. Activities of the tissue transglutaminase were altered considerably by the various culture conditions but were not obviously coordinated to keratinocyte markers. In normal epidermal cells, the suppressive effect of retinoic acid was much more evident with particulate transglutaminase than involucrin levels. While calcium had a large stimulatory effect on both markers, hydrocortisone had little or no influence. These results emphasize the potential importance of quantitative analysis of differentiation markers for resolving the contribution of physiological elements in coordination of cellular programming.     

Participation of membrane-associated proteins in the formation of the cross-linked envelope of the keratinocyte

Cultured keratinocytes, like those in natural squamous epithelia, form submembranous protein envelopes cross-linked by cellular transglutaminase. During the cross-linking, the cytosolic protein involucrin becomes incorporated into the envelope and can no longer be extracted by detergents. We show here that when transglutaminase is activated in cultured keratinocytes, at least six other proteins also become nonextractable. In contrast to involucrin, these proteins are associated with membranes. Two of the proteins (210 and 195 kd) are differentiated products specific to the keratinocyte; like involucrin, they are absent from small keratinocytes and fibroblasts, but appear in larger keratinocytes during the course of their terminal differentiation. The other proteins that become nonextractable cannot be destined exclusively for envelope formation since they are also present in fibroblasts. Transglutaminase is used by the mature (large) keratinocyte to make a detergent-resistant envelope from what appears to be a mixture of differentiation-specific and nonspecific proteins, both membrane-bound and cytosolic.     

Functional characterization of cultured cells derived from an intraepidermal carcinoma of the skin (IEC-1)

We have successfully isolated a cell line (IEC-1) from an intraepidermal carcinoma of the skin of a patient and compared its behavior, in vitro, to normal human epidermal keratinocytes (HEK) and squamous cell carcinoma cell lines (SCCs). HEK differentiation comprises an initial growth arrest followed by an induction of squamous differentiation-specific genes such as transglutaminase type 1 (TG-1). Using thymidine uptake and TG-1 induction as markers of proliferation and differentiation, respectively, we were able to show that HEKs and the IEC-1 cells undergo growth arrest and induce TG-1 mRNA expression in response to various differentiation-inducing stimuli, while neoplastic SCC cell lines did not. However, differentiation in HEKs was an irreversible process whereas differentiation of the IEC-1 cells was reversible. Furthermore, growth of IEC-1 cells in organotypic raft cultures revealed differences in their ability to complete a squamous differentiation program compared with that of normal HEKs. The IEC-1 cells also exhibited a transitional phenotype with respect to replicative lifespan; HEKs had a lifespan of 4-6 passages, IEC-1 cells of 15-17 passages, and SCC cells were immortal. These alterations in IEC-1 cell behavior were not associated with functional inactivation or mutations of the p53 gene. These data indicate that the IEC-1 cells, derived from a preneoplastic skin tumor, exhibit differences in their ability to undergo terminal differentiation and have an extended replicative lifespan.     

Induction of normal and psoriatic phenotypes in submerged keratinocyte cultures

Lesional psoriatic epidermis displays a number of phenotypic changes that are distinct from the differentiation program found in normal interfollicular epidermis. In psoriatic epidermis, keratinocytes are hyperproliferative and several differentiation-associated molecules are expressed that are absent in normal skin (e.g., cytokeratins (CK) 6, 16, and 17, and the epidermal proteinase inhibitor SKALP/elafin). In addition, several molecules which are normally restricted to the stratum granulosum are strongly upregulated in the stratum spinosum (e.g., psoriasis-associated fatty acid binding protein (PA-FABP), psoriasin, involucrin, and transglutaminase). The aim of this study was to develop in vitro culture systems which (a) would allow to study the induction of normal and psoriatic differentiation pathways, and (b) would be amenable for screening of antipsoriatic drugs. Here we have investigated several models for induction of differentiation with respect to the expression of markers for the normal and psoriatic phenotype. Cell cycle parameters and expression levels of CK1, CK10, CK16, SKALP/elafin, transglutaminase, involucrin, psoriasin, and PA-FABP were assessed in these models using flow cytometry, immunocytochemistry, and Northern blot analysis. We observed that induction of differentiation with fetal calf serum resembled the psoriatic phenotype (sustained hyperproliferation; high levels of CK16, SKALP/elafin, transglutaminase, and involucrin; moderate psoriasin expression), whereas differentiation induced by growth factor depletion in a confluent culture resembled the normal differentiation phenotype (low proliferative rate; high expression levels of CK1 and CK10; moderate expression of involucrin and transglutaminase; low expression levels of SKALP/elafin and CK16; absence of psoriasin). We propose that these models can be used to study expression and pharmacological modulation of selected differentiation genes and the coordinated expression of sets of genes associated with epidermal differentiation programs. © 1996 Wiley-Liss, Inc.     

Induction of keratinocyte type-I transglutaminase in epithelial cells of the rat

Using immunogold-silver techniques, we have demonstrated that, in rats, type-I (keratinocyte) transglutaminase is expressed primarily in stratified squamous epithelia of the integument, the upper digestive tract, and the lower female genital tract. In these epithelia, the enzyme was found to be present predominantly in the granular layer, but was evident at low levels even in the basal layer, especially in the genital tract. No immunoreactivity was detected in glandular, columnar, or transitional epithelia or in soft tissues. However, considerable enzyme antigenicity was observed in the endometrium and in major ducts of the pancreas and mammary glands of near-term pregnant and early postpartum females. In cultures, substantial immunoreactivity was readily identifiable not only in epidermal, vaginal, and esophageal epithelial cells (immunopositive in vivo), but also in urinary bladder, seminal vesicle, and tracheal epithelial cells (immunonegative in vivo). Primary epithelial outgrowths from bladder and seminal vesicle tissue explants were immunopositive, demonstrating rapid adaptation to the culture environment. These results reveal three distinct levels of regulation of transglutaminase expression in various cell types: during the differentiation of keratinocytes, during pregnancy, being evident principally in the endometrium but detectable elsewhere as well, and during the cultivation of certain epithelia which do not normally express the enzyme in vivo. We conclude that type-I transglutaminase may be a valuable marker for elucidating the regulation of normal epithelial differentiation and squamous metaplasia.     

The role of phospholipase C-gamma1 in 1alpha,25-dihydroxyvitamin D(3) regulated keratinocyte differentiation

Phospholipase C-gamma1 (PLC-gamma1) is the most abundant member of the phospholipase C family expressed in human keratinocytes. PLC-gamma1 is induced by 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) in normal keratinocytes via a DR6-type vitamin D responsive element. This regulation is not observed in transformed keratinocytes. The role of PLC-gamma1 in mediating 1alpha,25(OH)(2)D(3) and calcium-regulated differentiation was then tested. Both specific PLC inhibitors and antisense constructs which selectively block PLC-gamma1 production prevented 1alpha,25(OH)(2)D(3) and calcium from inducing markers of differentiation such as involucrin and transglutaminase. These studies demonstrate that PLC-gamma1 induction by 1alpha,25(OH)(2)D(3) is critical to the ability of this hormone to regulate keratinocyte differentiation.     

Targeted ablation of the murine involucrin gene

Involucrin is synthesized in abundance during terminal differentiation of keratinocytes. Involucrin is a substrate for transglutaminase and one of the precursors of the cross-linked envelopes present in the corneocytes of the epidermis and other stratified squamous epithelia. These envelopes make an important contribution to the physical resistance of the epidermis. We have generated mice lacking involucrin from embryonic stem cells whose involucrin gene had been ablated by homologous recombination. These mice developed normally, possessed apparently normal epidermis and hair follicles, and made cornified envelopes that could not be distinguished from those of wild-type mice. No compensatory increase of mRNA for other envelope precursors was observed.     

Differentiation of cultured epidermal keratinocytes related to sterol metabolism and its retardation by chemical carcinogens

The amount of 5 beta-cholest-7-en-3 beta-ol of mouse dorsal skin was increased after parturition until 10 days of age, reaching a maximum 5 beta-cholest-7-en-3 beta-ol/5-cholesten-3 beta-ol ratio of 0.43. [2-14C]Acetate was incorporated almost exclusively into 5-cholesten-3 beta-ol in the basal cell culture of epidermal keratinocytes. However, when the concentration of calcium was changed from 0.07 to 1.9 mM to induce terminal differentiation of keratinocytes, a definite amount of radioactive acetate was incorporated into 5 beta-cholest-7-en-3 beta-ol. The extent of the incorporation was increased at least until 72 h after changing medium, and the ratio of radioactive 5 beta-cholest-7-en-3 beta-ol/radioactive 5-cholesten-3 beta-ol was constantly increased in this period, indicating that the accumulation of 5 beta-cholest-7-en-3 beta-ol in the cell is concomitant with the differentiation of the cell. Pretreatment with chemical carcinogens such as 7,12-dimethylbenz[a]anthracene and 20-methylcholanthrene inhibited the incorporation of radioactive acetate into 5 beta-cholest-7-en-3 beta-ol in the high calcium medium at least for the initial 24 h. After that, the incorporation was gradually restored to the normal level. Pretreatment with a tumor promoter, such as 12-O-tetradecanoyl-phorbol 13-acetate, however, did not inhibit the incorporation. Thus, sterol metabolism is suggested to be a useful indicator for differentiation of epidermal keratinocytes.     

Regulation of transglutaminase type II by transforming growth factor-beta 1 in normal and transformed human epidermal keratinocytes

This study examines the effect of transforming growth factor-beta 1 (TGF-beta 1) on the expression of Type I and II transglutaminase in normal human epidermal keratinocytes (NHEK cells). Treatment of undifferentiated NHEK cells with 100 pM TGF-beta 1 caused a 10- to 15-fold increase in the activity of a soluble transglutaminase. Based on its cellular distribution and immunoreactivity this transglutaminase was identified as Type II (tissue) transglutaminase. TGF-beta 1 did not enhance the levels of the membrane-bound Type I (epidermal) transglutaminase activity which is induced during squamous cell differentiation and did not increase Type II transglutaminase activity in differentiated NHEK cells. Several SV40 large T antigen-immortalized NHEK cell lines also exhibited a dramatic increase in transglutaminase Type II activity after TGF-beta 1 treatment; however, TGF-beta 1 did not induce any significant change in transglutaminase activity in the carcinoma-derived cell lines SCC-13, SCC-15, and SQCC/Y1. Half-maximal stimulation of transglutaminase Type II activity in NHEK cells occurred at a dose of 15 pM TGF-beta 1. TGF-beta 2 was about equally effective. This enhancement in transglutaminase activity was related to an increase in the amount of transglutaminase Type II protein as indicated by immunoblot analysis. Northern blot analyses using a specific cDNA probe for Type II transglutaminase showed that exposure of NHEK cells to TGF-beta 1 caused a marked increase in the mRNA levels of this enzyme which could be observed as early as 4 h after the addition of TGF-beta 1. Maximal induction of transglutaminase Type II mRNA occurred between 18 and 24 h. The increase in Type II transglutaminase mRNA levels was blocked by the presence of cycloheximide, suggesting that this increase in mRNA by TGF-beta 1 is dependent on protein synthesis.     

The extracellular calcium-sensing receptor is required for calcium-induced differentiation in human keratinocytes

In cultured keratinocytes, the acute increase of the extracellular calcium concentration above 0.03 mM leads to a rapid increase in intracellular calcium concentration ([Ca(2+)]i) and inositol trisphosphate production and, subsequently, to the expression of differentiation-related genes. Previous studies demonstrated that human keratinocytes express the full-length extracellular calcium-sensing receptor (CaR) and an alternatively spliced variant lacking exon 5 and suggested their involvement in calcium regulation of keratinocyte differentiation. To understand the role of the CaR, we transfected keratinocytes with an antisense human CaR cDNA construct and examined its impact on calcium signaling and calcium-induced differentiation. The antisense CaR cDNA significantly reduced the protein level of endogenous CaRs. These cells displayed a marked reduction in the rise in [Ca(2+)]i in response to extracellular calcium or to NPS R-467, a CaR activator, whereas the ATP-evoked rise in [Ca(2+)]i was not affected. Calcium-induced inhibition of cell proliferation and calcium-stimulated expression of the differentiation markers involucrin and transglutaminase were also blocked by the antisense CaR cDNA. When cotransfected with luciferase reporter vectors containing either the involucrin or transglutaminase promoter, the antisense CaR cDNA suppressed the calcium-stimulated promoter activities. These results indicate that CaR is required for mediating calcium signaling and calcium-induced differentiation in keratinocytes.     

Characterization of transglutaminase activity in rabbit tracheal epithelial cells. Regulation by retinoids

Rabbit tracheal epithelial cells undergo terminal cell division, start to express a squamous phenotype, and form cross-linked envelopes when reaching the plateau phase of the growth curve. This terminal differentiation is accompanied by a 20-30-fold increase in the activity of the cross-linking enzyme transglutaminase. This activity is found almost solely in the particulate fraction of homogenized cells and can be solubilized by nonionic detergents. This transglutaminase crossreacts with a monoclonal antibody raised against type I transglutaminase, but does not react with an antiserum against type II transglutaminase. The tracheal transglutaminase contains a protein subunit of approximately 92 kDa. The omission of epidermal growth factor from the medium or the addition of fetal bovine serum, conditions that induce terminal cell division and expression of a squamous phenotype, enhance transglutaminase activity. High calcium concentrations only stimulate transglutaminase activity after the cells become committed to terminal cell division. Retinoids, which inhibit the expression of the squamous phenotype but not terminal cell division, inhibit the enhancement in transglutaminase activity induced by either confluency or serum, indicating that this enzyme activity is under the control of retinoids. Some retinoids are active at concentrations as low as 10(-12) M. The ability of retinoids to inhibit transglutaminase activity correlates well with their capacity to bind to the retinoic acid-binding protein. Our results show that the increase in transglutaminase activity correlates with the induction of the terminal differentiated phenotype and suggest that this enzyme can function as a marker for this program of differentiation of rabbit tracheal epithelial cells in culture. Our results identify the transglutaminase as type I transglutaminase and are in agreement with the concept that this transglutaminase is involved in the formation of cross-linked envelopes.     

A miR‐34a‐SIRT6 axis in the squamous cell differentiation network

Squamous cell carcinomas (SCCs) are highly heterogeneous tumours, resulting from deranged expression of genes involved in squamous cell differentiation. Here we report that microRNA‐34a (miR‐34a) functions as a novel node in the squamous cell differentiation network, with SIRT6 as a critical target. miR‐34a expression increases with keratinocyte differentiation, while it is suppressed in skin and oral SCCs, SCC cell lines, and aberrantly differentiating primary human keratinocytes (HKCs). Expression of this miRNA is restored in SCC cells, in parallel with differentiation, by reversion of genomic DNA methylation or wild‐type p53 expression. In normal HKCs, the pro‐differentiation effects of increased p53 activity or UVB exposure are miR‐34a‐dependent, and increased miR‐34a levels are sufficient to induce differentiation of these cells both in vitro and in vivo. SIRT6, a sirtuin family member not previously connected with miR‐34a function, is a direct target of this miRNA in HKCs, and SIRT6 down‐modulation is sufficient to reproduce the miR‐34a pro‐differentiation effects. The findings are of likely biological significance, as SIRT6 is oppositely expressed to miR‐34a in normal keratinocytes and keratinocyte‐derived tumours.     

Regulation of lipid synthesis in relation to keratinocyte differentiation capacity

Cultured keratinocytes and squamous carcinoma cells provide a useful model system for studying the processes involved in the regulation of differentiation, as the differentiation capacity of the cells can be modulated experimentally by changing the extracellular calcium concentration. Furthermore, the squamous carcinoma cell lines exhibit a defect in their differentiation capacity which they express to different extents. In this paper, the effect of external lipoproteins has been studied on lipid synthesis in normal keratinocytes and three squamous carcinoma cell (SCC) lines which showed a decreasing capacity to differentiate in the order of normal keratinocytes greater than SCC-12F2 greater than SCC-15 greater than SCC-4. The ability of the cells to form cornified envelopes was taken as a measure of differentiation capacity. The rate of total lipid synthesis as well as the phospholipid-neutral lipid ratio decreased in the order SCC-4 greater than SCC-15 greater than SCC-12F2 greater than or equal to normal keratinocytes, clearly correlating with the differentiation capacity of the cells. Because of the high rate of phospholipid synthesis and the low rate of ceramide synthesis, it is concluded that, under these in vitro conditions used, the maturation of keratinocytes proceeds to a lesser extent than that seen under in vivo conditions. In proliferating cells, in which the low-density lipoprotein (LDL) receptor is operative to a high extent, the rate of lipogenesis, especially that of neutral lipids, responded dramatically to changes of extracellular lipoprotein concentration. In the presence of lipoproteins a marked decrease of cholesterol and triacylglycerol synthesis and an increase of cholesterol ester synthesis has been observed. On the other hand, in differentiating cells lipogenesis appeared to be independent of extracellular lipoproteins, due to the absence of the LDL uptake mechanism, the only exception being the synthesis of triacylglycerols, the rate of which could be modulated to a certain extent by extracellular lipoproteins. The results presented here demonstrate a close inverse relationship between the regulation of lipogenesis by extracellular lipoproteins and the ability of the cells to differentiate.     

Absence of differentiation-related expression of keratin 10 in earlystages of vulvar squamous carcinoma

Using specific monoclonal antibodies (DE-K10 and DE-SCK respectively), the expression of some differentiation-related epidermal keratins was studied in 38 human vulvar squamous carcinomas. In the epidermis, expression of keratin 10 (K10) strictly paralleled the extent of differentiation; it was absent in the basal layer, appeared in the first suprabasal layers and increased in concentration towards the granular layer. However, K10 was rarely detected (1 case out of 12) in early stages of vulvar squamous carcinomas (tumours less than 2 cm, clinical stage I) regardless of the tumour grade. In larger and more advanced tumours (greater than 2 cm, clinical stages II and III), K10 was detected in 21 out of 26 cases. Its expression appeared to be related to maturation of malignant keratinocytes, being preferentially detected in more-differentiated parts. Occasionally however, cells that did not show histological signs of keratinisation were also K10-positive. Modified stratum corneum keratins (recognized specifically by monoclonal antibody DE-SCK) were detected in the most keratinized areas (horn pearls and their close vicinity) of some K10-positive tumours, i.e., in a pattern close to their normal expression in terminally differentiated epidermal cells. These data suggest differences in the regulation of K10 expression during the differentiation processes in the normal keratinising squamous epithelium and in squamous carcinomas. While the normal pattern of vulvar epithelial differentiation is accompanied by an increasing expression of K10, malignant keratinocytes, also when these are histologically moderately or well differentiated, cease expressing this keratin in the early stages of tumour development.     

Induction of keratinocyte type-I transglutaminase in epithelial cells of the rat

Abstract. Using immunogold-silver techniques, we have demonstrated that, in rats, type-I (keratinocyte) transglutaminase is expressed primarily in stratified squamous epithelia of the integument, the upper digestive tract, and the lower female genital tract. In these epithelia, the enzyme was found to be present predominantly in the granular layer, but was evident at low levels even in the basal layer, especially in the genital tract. No immunoreactivity was detected in glandular, columnar, or transitional epithelia or in soft tissues. However, considerable enzyme antigenicity was observed in the endometrium and in major ducts of the pancreas and mammary glands of near-term pregnant and early postpartum females. In cultures, substantial immunoreactivity was readily identifiable not only in epidermal, vaginal, and esophageal epithelial cells (immunopositive in vivo), but also in urinary bladder, seminal vesicle, and tracheal epithelial cells (immunonegative in vivo). Primary epithelial outgrowths from bladder and seminal vesicle tissue explants were immunopositive, demonstrating rapid adaptation to the culture environment. These results reveal three distinct levels of regulation of transglutaminase expression in various cell types: (1) during the differentiation of keratinocytes, (2) during pregnancy. being evident principally in the endometrium but detectable elsewhere as well, and (3) during the cultivation of certain epithelia which do not normally express the enzyme in vivo. We conclude that type-I transglutaminase may be a valuable marker for elucidating the regulation of normal epithelial differentiation and squamous metaplasia.