Epithelial cell biology最新文献

We have investigated expression of pemphigus vulgaris (PV) antigen, desmoglein 3 (Dsg3), and pemphigus foliaceus (PF) antigen, desmoglein 1 (Dsg1), in various tissues. Immunofluorescence studies suggested that Dsg1 and Dsg3 are preferentially expressed in the upper and lower epidermis, respectively. With immunoblotting of human epidermal extracts, all PV sera reacted with Dsg3 but not with Dsg1. By contrast, only half of PF sera reacted with Dsg1 but none reacted with Dsg3. These results confirm the distinct antibody specificity between PV and PF sera. They also suggest that PV sera contain antibodies against linear epitopes present even on the denatured antigen, while some PF sera contain antibodies only against conformational epitopes present on the native antigen. With immunoblotting of bovine desmosome preparations, certain PV sera and an anti-Dsg monoclonal antibody reacted with both Dsg1 and Dsg3. Affinity-purification of these PV antibodies suggested that the simultaneous reactivity with Dsg1 and Dsg3 was produced by two different subsets of antibodies and not by cross-reactivity of single antibodies. This study indicates that pemphigus serum is a useful probe with which we learn various aspects of keratinocyte biology.

We have previously shown that the 180-kD bullous pemphigoid antigen (BPAII), which is a transmembrane collagenous protein of hemidesmosomes, is distributed at adhesion sites on glass coverslips on the basal membrane forming a concentric ring, or arch pattern, in a human squamous cell carcinoma cell line (DJM-1), when studied by immunofluorescence microscopy using monoclonal antibodies to BPA II. This concentric ring/arch pattern of "footsteps" of BPA II has been shown to be collapsed in association with a transient activation of protein kinase C by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In the present study, therefore, the effects of TPA on the phosphorylation of BPA II was examined. DJM-1 cells, which were metabolically labelled with [32Pi], were lysed and the extracts were subjected to immunoprecipitation with anti-BPAII and anti-230 kDa bullous pemphigoid antigen (BPAI) monoclonal antibodies. The results showed that only BPA II, but not BPA I, was phosphorylated at serine residues before TPA treatment. After TPA treatment phosphorylation was prominently increased so as to generate a 190 kDa-phosphorylated peptide. This 190-kDa peptide was reacted with anti-BPA II monoclonal antibodies by immunoblotting, and it was not detected when cells were pretreated with a specific protein kinase C inhibitor (H7) before TPA treatment, suggesting that the 190 kDa peptide is phosphorylated BPAII with TPA. Prolonged treatment with TPA abolished both of 180- and 190-kDa BPA II from Triton X-100-soluble fractions. These findings suggest that the BPA II, but not BPA I, is a substrate of protein kinase C, and the generation of 190-kDa-phosphorylated BPA II has a key role in the TPA-induced collapse of the assembly of BPA II on the basal plasma membrane, probably, at hemidesmosomes.

The present study was aimed at re-evaluating the mechanism by which partial hepatectomy (PH) and the resulting mitogenic stimulation act to positively modulate the action of a chemical hepatocarcinogen applied 24 hours after surgery. Using as a marker the immunocytochemical expression of glutathione S-transferase placental form (GST-P), we compared the early incidence of single altered hepatocytes and the time-course of appearance and growth of subsequently developed liver neoplasia in diethylnitrosamine (DENA) treated sham-operated or partially hepatectomized animals. The data showed that, compared to sham-operation, partial hepatectomy increases the frequency of early GST-P positive single hepatocytes appearing 72 h after treatment with DENA (50 mg/kg b.w.), indicating a positive (co-carcinogenic) effect of PH on the frequency of the initiation event. At 2 months after this treatment, foci of altered hepatocytes were about 5 times more numerous in PH-pretreated than in sham-operated animals and their average volume about four times greater. The latter difference persisted for up to 10 months after carcinogen treatment, indicating that the lesions had grown at similar rates in the meantime. The simplest hypothesis to account for this difference in size of the lesions is that it is due to the 2-3 waves of normal-like cell division the altered hepatocytes underwent during the very first days after removal of part of the liver mass, rather than to true promotion or to an intrinsic difference in proliferative phenotype of the initiated cells. The increased size and higher frequency of the lesions in PH-pretreated rats concur to increase the population of cells at risk towards subsequent events implied in multistep progression and, by this, to positively modulate the latter. Accordingly, only PH treated rats had developed neoplastic nodules at 10 months and tumours at 15 months. This can therefore be explained without resorting to the currently held view that cell divisions in PH would increase the carcinogenic efficiency of DENA, due to the fact that resulting cell divisions would intervene before full repair of DENA-induced DNA damages.

We previously demonstrated that spreading and clustering of in vitro cultured human keratinocytes are autocrine-induced phenomena, mediated by keratinocyte-secreted soluble factor. In this paper, the effects of this factor on spreading, number of dendrites and cell-cell contacts of the two cellular components of skin, melanocytes and fibroblasts have been studied 24 h after plating cells on uncoated plastic surfaces in MCDB153 serum-free medium or in the same medium conditioned by keratinocytes (KCM). Spreading of melanocytes present in the epidermal cell population remained constant at increasing cell density, while that of keratinocytes showed a statistically significant increase. Moreover, time-course experiments showed that the rate of spreading was faster for melanocytes. At increasing epidermal cell density, a statistically significant increase in number of dendrites and cell-cell contacts of melanocytes was observed. Similar results were obtained when melanocytes were plated both in coculture with keratinocytes (as epidermal cell cultures) or as a pure cell population in keratinocyte conditioned medium (KCM), suggesting that the observed phenomena are due to keratinocyte-secreted soluble factors and not to direct keratinocyte-melanocyte interactions. The addition of nerve growth factor (NGF) to fresh medium or addition of an inactivating anti-NGF monoclonal antibody (alpha D11) to KCM did not affect the number of dendrites or cell-cell contacts of melanocytes. Keratinocyte-secreted soluble factor(s) present in KCM also dramatically influenced morphology and cell-cell contacts of human dermal fibroblasts.

The excretory/secretory (ES) products of the nematode parasite Trichostrongylus colubriformis have been found to increase the in vitro proliferation of the epithelial cell line HT29-D4. To assess the specificity of this effect, ES products from other trichostrongyle species were tested on colonic (HT29-D4) and gastric (HGT-1) tumour cell lines. Adult worms of six different nematode species, parasites of the stomach or the small intestine of ruminants, were incubated in vitro in Dulbecco's Modified Eagle's Medium for 24 h. The conditioned media were then added at different concentrations to the culture medium of the two cell lines. A stimulation of the HT29-D4 cell growth occurred with the ES products of two parasite species of the small intestine, at the concentrations of 0.1 microgram protein/ml (Trichostrongylus vitrinus) and 1.0-5.0 micrograms/ml (Cooperia curticei). Inversely, a decrease in cell number was observed with the ES products of another intestinal species, Nematodirus battus at concentrations of 1.0-5.0 micrograms/ml. With the ES products of the abomasal nematodes, a proliferation of HT29-D4 cells was obtained at 0.25-5.0 micrograms/ml with ES products of Teladorsagia circumcincta but no significant effect was observed for Haemonchus contortus. On the tumoral gastric cell line HGT-1, the ES products from the 6 nematode species gave a similar stimulative effect. These in vitro results suggest that nematode parasite species secrete or excrete component(s) which could affect the epithelial regeneration of the host digestive tract.

Topical application of retinoic acid (RA) induces skin irritation, increased epidermal DNA synthesis and hyperplasia by an unknown mechanism, possibly in common with other hyperplasiogens. To further characterize this regeneration-like effect of RA, the cell cycle traverse in hairless mouse epidermis in vivo after application of a single dose of 100 nmol RA was investigated. Two BrdUrd labelled basal cell cohorts, one exposed to RA during the S phase, the other stimulated by RA into S phase 16 h earlier, were followed for 30 h in their progression through the cell cycle. The basal cells were isolated and prepared for bivariate BrdUrd/DNA flow cytometry analysis (FCM). Both cohorts showed induced proliferation of cells stimulated from G0/G1 into S phase as shown by increase in the S phase fraction, but without reduction in cell cycle time as expected for regenerative growth. This implies that RA stimulates proliferation differently than other hyperplasiogens, and hence may induce irritation and growth stimulation through another mechanism.

In this morphological study the (ultra)structural changes that lead to contraction of detached cultured epithelium were investigated. Keratinocytes, isolated from human skin and oral mucosa, were grown to form stratified cell sheets. The multilayers were examined with light and electron microscopy before, during and after detachment from the culture vessel. Attached epithelium had a stretched morphology with flattened cells and nuclei. Evidence is provided that after enzymatical detachment with dispase (1) basal cells became columnar by contraction of actin bundles in the basal cortex, which was accompanied by blebbing of the basal cell membrane; (2) in all cell layers cytokeratin bundles contracted resulting in displacement of desmosomes and a spherical shape of the cells and nuclei. By slow dispase-detachment at 4 degrees C or by quick mechanical detachment, shrinkage of the sheet was partly suppressed but contraction of cytokeratin and related events occurred indicating that these were the result of the spontaneous reassembly of the intermediate filament system. The results suggested that the shape and ultrastructure of all cells in an epithelial multilayer are dependent on the interaction of the basal cells with the underlying extracellular matrix.

The presence of the nematode parasite Trichostrongylus colubriformis in the small intestine is associated with an increase in epithelial renewal. To assess the possible role of excretory/secretory products from the worm on cell proliferation, adult Trichostrongylus colubriformis were incubated in vitro in Dulbecco's Modified Eagle's Medium for 24 h and the conditioned medium was added to the culture medium of the transformed epithelial cell line HT29-D4. A stimulation of the HT29-D4 cell growth was ascertained at concentrations of 0.25-1.0 micrograms protein/ml using counts of cell numbers, the MTT method and incorporation of tritiated thymidine. An increased incorporation of tritiated thymidine was also observed with the excretory/secretory products from T. colubriformis fourth stage larvae at 1.0 microgram/ml. Dialysis of the medium conditioned by the worms indicated that the molecular weight of the factor is greater than 8000 Daltons in size. Heat treatment, acid hydrolysis and precipitation by trichloracetic acid of the conditioned medium resulted in the disappearance of the proliferative effect while treatment with trypsin partially depleted the stimulative activity. These results suggest that T. colubriformis produce some protein factor which could increase the epithelial regeneration in the host small intestine.

The present study investigates the spatial organisation of epithelial cell proliferation in human small intestinal xenografts, in order that direct comparisons can be made with paediatric small bowel. For this purpose we employed the MIB-1 (Ki-67) monoclonal antibody and [3H]thymidine to analyse the crypt growth fraction and DNA synthesising (S-phase) cells, respectively. The spatial distribution of cycling (MIB-1+) cells was appropriately confined to the xenograft crypts where it closely resembled that of paediatric intestine, both in terms of the labelling index and an ability to form runs of labelled cells, thereby demonstrating synchronous patterns of cell division. In addition, the S-phase representation in xenograft intestine was uniform throughout the crypt proliferation compartment thereby indicating cell-cycle homogeneity. This chimeric model system now provides a new approach to investigate altered proliferative responses of human gut to a number of potentially harmful substances e.g. carcinogens, the assessment of which is not feasible in patients or volunteers.

DMH-induced (25mg/kg/wk) rodent intestinal carcinogenesis was re-examined using histometry under various conditions including modulation of host natural killer (NK) cell activity and mutagen administration. Early lesion was enterocyte hyperplasia all along the intestinal tract. Cell kinetic analysis showed that it was caused by a slightly altered but still functional "initiated" enterocyte population added to the normal population which remained unchanged. Primarily, initiated stem (IS) cells were added which then produced their initiated progeny which still renewed normally. NK cells countered the initiated cells selectively, either inhibiting IS cell proliferation or killing them when activated by lymphokines. Evidence was obtained that the IS cells could further transform into preneoplastic stem (PS) and then neoplastic stem (NS) cells under the influence of promoters and mutagens, respectively. Subsequent transformation of normal stem cells into IS, PS, and NS cells apparently is the basis of carcinogenesis. These produce lesions, hyperplastic, preneoplastic, and neoplastic, respectively, only when and where NK activity is inhibited. Under the influence of normal NK cells, they remain dormant (nonproliferative), compatible with normal life.