Epithelial cell biology最新文献

The contribution of adhesion to an extracellular matrix in the polarized spatial organization of keratinocytes was studied in dispase-detached cultures stored as floating sheets. Proliferating and terminally differentiating cell populations were, therefore, localized on tissue sections by their DNA-synthesizing ability and involucrin immunostaining, respectively. A progressive reorganization was induced from superposed proliferating and differentiating layers into clusters exhibiting differentiating cells on the outside. Measurements of proliferation and terminal differentiation in detached cultures revealed the progressive disappearance of proliferating cells, followed by an increase in involucrin-positive cells. Attempts to block the spatial reorganization by the addition of components of the extracellular matrix remained unsuccessful. These results suggest that basal anchorage is responsible for the superposition of proliferating and differentiating cells in keratinocyte cultures. They afford new arguments for the induction of terminal differentiation in non-adhesive keratinocytes which exhibit a concomitant modification of cell shape.

Eicosanoids are thought to play an important role in the pathogenesis of inflammatory skin diseases. The object of the present study was the detection and characterization of putative 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) binding sites in normal human keratinocytes. Keratinocytes were obtained from foreskin and dermatome-shaved normal human skin. Radioligand binding assays were performed with 12(S)-[3H]HETE on cultured cells. Analysis of saturation curves suggested a one-site model for 12(S)-HETE binding with a KD of 3.84 +/- 0.18 nM and receptor number Bmax of 2.32 +/- 0.12 x 10(5) per cell. Ligand binding was reversible. The rank order of potency in competition for 12(S)-[3H]HETE was 12(S)-HETE > 12(R)- HETE > or = leukotriene B4. Preincubation of cells with 12(S)-HETE (2 x 10(-6) M) resulted in down-regulation of the binding site by approximately 50%. The identification and characterization of specific 12(S)-HETE binding sites on normal human keratinocytes should enable further elucidation of the role of 12-HETE in cutaneous biology and in the pathophysiology of psoriasis and other inflammatory and hyperproliferative dermatoses.

Epithelia differ regionally in their patterns of phenotypic expression. The junctional epithelium (JE) that attaches the oral mucosa to the teeth is a unique tissue that shows a pattern of differentiation unlike other oral epithelia and forms basal lamina against the non-vital tooth surface. The mechanisms that establish this unusual phenotype and the developmental origin of this epithelium are both uncertain. The formation of JE by downgrowth of the oral gingival epithelium (OGE) during tooth eruption has been suggested but morphological studies indicate that it may be derived from the reduced enamel epithelium (REE) that covers the crown of the unerupted tooth. These epithelia of potential origin differ in their developmental histories: intrinsic differences between them could thus significantly influence the phenotype of an epithelium formed from them. The patterns of phenotypic expression of specimens of dissected JE, OGE and REE, and of cell cultures of these epithelia grown under standardized conditions, were examined (1) by immunocytochemistry using monoclonal antibodies with specificity for individual cytokeratins, vimentin and ICAM-1, and (2) by two-dimensional SDS-PAGE and immunoblotting. The results indicated that, in vivo, OGE expressed keratin markers typical of differentiating mucosal epithelium; JE and REE, in contrast, lacked expression of most such markers but expressed keratins typical of simple epithelia together with some undefined keratin peptides. All epithelia showed changes in vitro but OGE remained different from JE and REE. OGE lost expression of the differentiation markers K1, K10 and K13; it acquired some expression of K19, but less than JE and REE. Cultures of JE and REE retained some expression of ICAM-1 and K8 and K18, and consistently acquired high levels of vimentin expression. These findings indicate that differences persist in standardized culture conditions and that these are apparently of an intrinsic nature. They support a concept of the origins of JE from REE and suggest that the unusual in vivo phenotype of JE results partly from intrinsic differences acquired during its development.

We examined the fibronectin-adhesive properties of clones from a rat colonic cell line exhibiting distinct tumorigenicity in a syngeneic host. These cells were originally selected on the basis of differential adhesion to plastic surfaces. The TR cell line, when injected subcutaneously, forms a tumour which grows progressively and gives off metastases, whereas the TS cell line forms a small tumour which regresses within a few weeks. The regression is largely mediated by immunological factors and involves a fibroblastic reaction. REGb, a clone from the TS subline, adhered better to fibronectin or RGDS tetrapeptide than did PROb, a clone from the TR subline. However, there was little binding to the RGD tripeptide with either clone. The degree of adhesion was dependent on time and substrate concentration. After 6 h of incubation, 38% and 55% respectively of PROb and REGb cells bound to plates coated with 10 micrograms/ml fibronectin. Adhesion of both clones to fibronectin was inhibited to various degrees when cells were preincubated with RGDS, GRGDS or GRADSPK peptides, whereas other synthetic peptides such as RGD, GRGD or GRGFSPK were ineffective. Binding experiments using 125I-labelled fibronectin showed 39,000 fibronectin receptor sites on REGb cells but only 17,000 on PROb cells. Flow cytometry analysis using both anti-alpha 5 and anti-beta 1 integrins showed more fibronectin receptor sites on REGb than on PROb cells. Both approaches were in accordance with the higher adhesiveness of the REGb clone to fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)

Using tritiated thymidine (3HTdR) labelling in vivo in the mouse we have determined the labelling index (LI%) at each cell position along the sides of sections of crypts in the small and large bowel. We have compared LI versus cell position frequency plots obtained in this way with those obtained using bromodeoxyuridine (BrdUrd) in vivo in the small intestine. Both thymidine analogues give identical patterns and similar levels of labelling: for example, the overall LI is 29.1% after 3HTdR and 34.7% after BrdUrd in the mouse ileum. Similar data have been obtained following in vivo labelling in humans with BrdUrd prior to gastrointestinal surgery for cancer and in mouse colon following 3HTdR labelling. Comparisons between the mouse and human data show that the spatial distribution of label within the crypts occurs at the same relative positions in the two species. However, the intestinal crypts are between 2-fold and 4-fold larger, particularly in their length, in the human: for example, 250 and 450 cells per crypt for ileum and 590 and 2000 cells per crypt for the colon in mouse and human respectively. The absolute value of the maximum LI in the mouse small intestine (56.5%) is higher than it is in the human (26.3%). However, the patterns of proliferation are similar in the two species under steady-state conditions.

The association between sexual activity and cancer, first described in carcinoma of the cervix, has been expanded to include the majority of anogenital squamous epithelial carcinomas. Current evidence suggests that human papillomavirus (HPV) may be of great importance in the development of these tumours, whilst herpes simplex type 2 virus (HSV-2) and human immunodeficiency virus (HIV) may play minor roles. Certain types of HPV DNA, including types 16, 18, 31, 33 and 39 are found in most but not all anogenital cancers and pre-invasive neoplastic conditions. Viral genes E6 and E7 of HPV 16 and 18 are regularly expressed in HPV-positive tumours. In vitro, E6 and E7 genes have transforming properties which correlate with their ability to bind naturally occurring growth regulation proteins p53 and pRB. It has, however, become apparent that HPV alone does not provide the full aetiological explanation of sexually related carcinomas. The finding of latent, non-sexually-acquired HPV in a sizable proportion of the community, including children, has confounded simple theories of HPV transmission and cancer. Furthermore, in vitro experiments suggest that other factors may potentiate the effects of HPV. HSV-2 may possibly function as cofactor as it can synergize with HPV to cause transformation in vitro, and can transactivate HPV gene expression. HIV is associated with an increased rate of anogenital malignancies, particularly of the anus. Tumours in HIV-positive patients appear to have a worse prognosis, even before the onset of AIDS.

Transglutaminases stabilize a variety of biological structures by cross-linking constituent proteins. This action appears physiologically important in stabilizing (1) keratinocyte cornified envelopes, (2) fibrin clots, (3) the copulation plug in rodents, and (4) the fertilized egg surface in aquatic species. Several transglutaminases that participate in such processes have been well characterized and found, though highly divergent, to differ in sequence primarily at the amino terminus. Comparison of their gene structures suggests a likely mechanism by which new members may arise that assume a diversity of functions. The functions of some members of this family are presently unknown, including the tissue transglutaminase found in many mammalian cell types, and those found in plants. Most of the transglutaminases identified are soluble enzymes, but several that are membrane-bound have gained recognition recently. The best characterized of the latter is keratinocyte transglutaminase, which is anchored in the membrane by acylated fatty acid. Important for proper epidermal cell maturation, expression of this enzyme is greatly altered by physiological effectors and toxic agents. In addition, it is induced by cultivation of cells from non-squamous epithelia. Thus, it is a promising marker for helping to elucidate the molecular basis by which keratinocyte differentiation is elicited or altered.

Mouse secondary palate morphogenesis is accompanied by distinctive patterns of proliferation in the palatal epithelium which latterly reflect its region-specific differentiation into oral, nasal and medial edge phenotypes. Isolated intact embryonic palatal epithelial sheets were cultured prior to, and during, the critical period of epithelial differentiation in chemically defined culture medium with, and without, 10% donor calf serum. The spatial and temporal patterns of proliferative activity were investigated by immunocytochemistry in 'pulse' and 'continuous' labelling experiments using bromodeoxyuridine (BrdUrd). Continuously labelled cultures exhibited extensive proliferation throughout the oral, nasal and medial edge regions. Pulse labelled cultures demonstrated a shift in mitotic activity from nasal to oral epithelial cells probably representing the cell turnover associated with the respective differentiated phenotypes. Medial edge cells became post-mitotic within the first 19 h of culture. Our defined culture system coupled with the immunocytochemical detection of cell proliferation using BrdUrd offers a rapid and precise method for the further investigation of palatal epithelial proliferation and its regulation by extrinsic factors.

Several studies have linked abnormal actin cytoskeletal organization and mutant actin genes with neoplastic transformation. Using in situ hybridization techniques we looked at expression of beta actin mRNA at a cellular level in normal, benign and neoplastic human colorectal tissues and correlated the level of expression with the extent of actin cytoskeletal organization in sequential sections. Normal tissues showed light labelling with the actin riboprobe, but non-neoplastic crypts, with evidence of regeneration and repair, showed greater levels. Higher levels of actin mRNA expression were found in adenomas and metaplastic polyps, but the highest levels were found in carcinomas, particularly those that were poorly differentiated. Cytoskeletal actin organization was, however, reduced in colorectal neoplasia. Normal mucosa showed the highest level of cytoskeletal actin organization, as assessed by phalloidin binding, and adenomas and metaplastic polyps also stained strongly. In contrast, phalloidin binding to poorly differentiated carcinomas was absent or very weak. The inverse relationship between actin mRNA expression and actin filament organization was confined to the carcinomas studied and may indicate defects in actin binding proteins or in post-transcriptional or translational events.