Expression and localization of thymidine phosphorylase/platelet-derived endothelial cell growth factor in skin and cutaneous tumors.

Thymidine phosphorylase/platelet-derived endothelial cell growth factor (TPase/PD-ECGF) is a catabolic enzyme that has been shown to be chemotactic for endothelial cells in vitro and angiogenic in vivo. TPase/PD-ECGF expression is increased in a variety of tumors. In the skin, TPase is active in normal keratinocytes in vitro and in vivo. Our objective was to study the expression and localization of TPase/PD-ECGF by immunohistochemical analysis in normal skin and cutaneous tumors and to correlate this information with enzymatic activity of TPase. TPase/PD-ECGF expression was observed in keratinocytes with intense staining of the infundibulum of hair follicles but no staining of hair bulbs. Expression localized primarily to the nucleus of keratinocytes in the basal layer but was more intense and cytoplasrmic in suprabasal keratinocytes. Increased expression of TPase/PD-ECGF in differentiated cells was confirmed by in vitro studies of TPase activity. In cutaneous tumors, there was positive staining for TPase/ PD-ECGF in squamous cell carcinomas (10/10), eccrine poromas (3/4), eccrine syringomas (4/4), trichoepitheliomas (1/3), and tumors of the follicular infundibulum (2/3) and melanomas (5/8). There was no staining of any intradermal nevi (0/2), basal cell carcinomas (0/10) or Merkel cell carcinoma (0/1). We conclude TPase/PD-ECGF is found throughout the epidermis and its expression increases with differentiation of keratinocytes. In cutaneous tumors, expression of TPase/PD-ECGF may be linked to the cell of origin of the tumor as well as the tumor's degree of differentiation.

Beta-L-1,3-dioxolane-cytidine: a novel nucleoside that inhibits proliferation and induces differentiation of keratinocytes in vitro.

beta-L-1,3-Dioxolane-cytidine (L-(-)-OddC) is a novel L-nucleoside, and its antitumor activity is under investigation in clinical trials. To evaluate the potential of L-(-)-OddC for treating hyperproliferative diseases of the skin, we examined its activity in human keratinocytes in vitro. The dose of L-(-)-OddC that inhibited the rate of proliferation of keratinocytes by 50% was 50 nM. L-(-)-OddC was about as cytotoxic as 9-beta-D-arabinofuranosylcytosine but was about 1,000 time more potent than 3'-azidothymidine. L-(-)-OddC caused irreversible growth arrest and induced differentiation of keratinocytes. L-(-)-OddC altered morphology, increased the cell size of keratinocytes and increased the expression of involucrin. These data suggest that L-(-)-OddC may have potential as a therapeutic agent against hyperproliferative skin diseases.

CD44 expression on epidermal melanocytes.

We examined CD44 expression on melanocytes to begin to understand what role CD44 might have in the normal behavior of melanocytes and to provide a basis for comparing CD44 expression in melanoma cells. CD44 was expressed on the entire surface of melanocytes and accentuated at the tips of dendritic processes. Two predominant forms of CD44 are expressed on cultured human foreskin melanocytes. One form has the covalent addition of chondroitin sulfate, whereas the other form has no chondroitin sulfate. Both use the hematopoietic, or CD44H, core protein. Using polymerase chain reaction primers that span the site where alternative splicing of CD44 occurs, we found only the cDNA coding CD44H. 12-O-Tetradecanoylphorbol 13-acetate increases the size of the chondroitin sulfate chain(s) attached to CD44 but not the proportion of CD44 molecules that carry chondroitin sulfate. Ninety percent of proteoglycans on melanocytes are chondroitin sulfate proteoglycans, and the CD44 chondroitin sulfate proteoglycan represented 10% of that total. These data show that CD44H is expressed as a "part-time" chondroitin sulfate proteoglycan on normal cultured melanocytes.

Increased Na(+)-H+ antiporter activity in apical membrane vesicles from mutant LLC-PK1 cells.

In whole cell experiments, the PKE20 mutant of the renal epithelial cell line LLC-PK1 displays a severalfold elevation of Na(+)-H+ antiporter activity at the apical surface (J.G. Haggerty, N. Agarwal, R.F. Reilly, E. A. Adelberg, and C.W. Slayman. Proc. Natl. Acad. Sci. USA 85: 6797-6801, 1988). The present study was undertaken to explore the properties of the mutant at the membrane level. Apical membrane vesicles were prepared by the magnesium-aggregation technique, with a similar enrichment (ca. 10-fold) of the marker enzyme gamma-glutamyltranspeptidase in vesicles from parent and mutant cell lines. In both cases, 22Na influx was stimulated by an inside-acid pH gradient, inhibited by ethylisopropylamiloride (EIPA), and unaffected by valinomycin, indicating that it was mediated by Na(+)-H+ antiport. Quantitatively, PKE20 vesicles showed a 4.2-fold increase in the maximal velocity of Na(+)-H+ antiporter activity compared with the parent, with only minor increases in the activity of two other Na(+)-dependent transporters (14-56% for alpha-methylglucoside and L-glutamate). Dose-response curves for EIPA indicated that the increased Na(+)-H+ antiport activity in PKE20 vesicles was due to an increased activity of the relatively amiloride-resistant form of the Na(+)-H+ antiporter with little or no change in the amiloride-sensitive form. No differences in polypeptide composition of the two vesicle preparations could be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Taken together, the results indicate that the mutation in PKE20 is expressed at the membrane level and is specific for the relatively amiloride-resistant Na(+)-H+ antiporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacologically different Na/H antiporters on the apical and basolateral surfaces of cultured porcine kidney cells (LLC-PK1).

Proximal tubule cells of the kidney contain, on their apical surface, an amiloride-sensitive Na/H antiporter that functions in Na reabsorption and proton secretion. We have investigated the localization of the antiporter in a cloned cell line of porcine renal origin, LLC-PK1/Cl4, which is often considered to be a useful model of the proximal tubule. Transport measurements were performed with differentiated monolayers grown on Nuclepore filters, permitting independent access to the apical and basolateral cell surfaces. In control experiments with LLC-PK1/Cl4 monolayers, three marker transport systems showed the expected polarity: 87% of ouabain-sensitive Rb uptake was at the basolateral surface, and 99% of Na-dependent alpha-methylglucoside transport and 93% of Na-dependent D-aspartate (L-glutamate) transport were at the apical surface. By contrast, the monolayers displayed significant Na/H antiporter activity (assayed as ethylisopropylamiloride-sensitive 22Na uptake) at both cell surfaces, with an apical uptake rate amounting to 44% and a basolateral rate amounting to 56% of the total. Significantly, the apical and basolateral antiporters could readily be distinguished from one another on the basis of ethylispropylamiloride sensitivity. The apical system had an IC50 of 13 microM, close to that reported for kidney brush border vesicle preparations, whereas the basolateral system had an IC50 of 44 nM, similar to values seen in undifferentiated LLC-PK1 cells and other cultured cell lines. The PKE20 mutant, previously selected from LLC-PK1/Cl4 on the basis of resistance to ethylisopropylamiloride, was found to overexpress the more resistant antiporter both during rapid growth and on its apical cell surface at confluence; normal amounts of the more sensitive antiporter were seen on the basolateral surface of confluent PKE20 cells. Taken together, these results suggest that there are two distinct forms of the Na/H antiporter, which are under separate genetic control.

Restoration of 125I-alpha-bungarotoxin binding activity to the alpha subunit of Torpedo acetylcholine receptor isolated by gel electrophoresis in sodium dodecyl sulfate.

The four subunits (alpha, beta, gamma, delta) of the acetylcholine receptor from Torpedo californica have been isolated by preparative gel electrophoresis in sodium dodecyl sulfate. After removal of the sodium dodecyl sulfate by dialysis of the polypeptides against a cholate-containing buffer, the alpha subunit, but not the other chains, binds 125I-alpha-bungarotoxin in a saturable manner. The binding affinity, 0.1-0.2 microM, is approximately 10(4)-fold lower than that observed for native acetylcholine receptor. For three preparations of alpha subunit, 1 mol of subunit bound 0.87, 0.38, and 0.33 mol of 125I-alpha-bungarotoxin at saturation. The binding was inhibited by cholinergic ligands, although the apparent affinities of these ligands for alpha were 50-100-fold lower than that found for the native receptor. These results indicate that at least part of the alpha-bungarotoxin binding site resides on the alpha subunit.