No abstract

The expression of phenotypic markers was examined during fetal and neonatal differentiation of rat tracheal epithelial (RTE) cells. The rat counterpart of human keratin 18 was predominantly found in columnar cells in the adult trachea. It was detected in the primordial tracheal epithelium first seen on gestational day (GD) 12 (term = 21.5 days). Staining intensity gradually increased, and by GD 17 it was principally localized to the apical portion of the epithelium. The rat counterpart of human keratin 19 was barely detectable in the trachea on GD 13 but became abundant in almost all RTE cells on and after GD 19. Morphologically and immunocytochemically identifiable secretory and ciliated cells appeared on GD 18. Ciliated cell number slowly rose while secretory cells increased dramatically on GD 19 through postnatal day 1. The secretory granule antigens detected by monoclonal antibodies RTE 9 and 11 were rare in the adult trachea but were highly expressed in virtually all of the perinatal secretory cells. In contrast, the epitope detected by monoclonal antibody RTE 12, which was present in all adult tracheal surface secretory cells, did not appear until postnatal day 1 and slowly increased. These results demonstrate marked shifts in the biochemical composition of secretory cells during development and postnatal maturation. For the above-mentioned molecules, a similar expression pattern was observed during epithelial regeneration in tracheal grafts (Am. J. Respir. Cell Mol. Biol. 1992; 7:30-41). Pseudo-stratification of the epithelium and basal cells was first observed on GD 20. Keratin 14, which is confined to basal cells in the normal adult trachea, was not present in the nascent basal cells but appeared after postnatal day 1. In contrast to the present results, during epithelial regeneration in tracheal grafts keratin 14 appeared before markers of highly differentiated secretory or ciliated cells. Thus, the biochemical sequence of cellular differentiation during regeneration did not precisely recapitulate development.

The synthesis of the major surfactant protein, SP-A, was studied in activated alveolar type II cells isolated from the lungs of rats exposed to silica by intratracheal instillation. Exposure of rats to silica resulted in large increases in the levels of disaturated phosphatidylcholine and SP-A in the extracellular and intracellular surfactant compartments. Isolated type II cells were used to determine if the observed increases in SP-A were associated with increased SP-A synthesis. Type II cells were isolated by a combination of elastase digestion, centrifugal elutriation, and differential adherence on IgG-coated petri dishes. Type II cells from silica-treated lungs were separated into two populations, designated type IIA and type IIB. The type IIB, or activated population, consisted of type II cells that were larger than normal type II cells and, in addition, contained larger and more numerous lamellar bodies than normal type II cells. Type IIB cells contained 4.3-fold higher levels of SP-A compared to normal type II cells. SP-A synthesis was measured by incubating freshly isolated cells with [35S]Translabel (70% [35S]methionine, 15% [35S]cysteine) for up to 4 h in methionine-free medium, followed by immunoprecipitation of newly synthesized protein. The rate of SP-A synthesis was increased approximately 6.7-fold in the activated type II cells. Analysis of the newly synthesized protein by one-dimensional SDS-PAGE indicated three intracellular forms of SP-A with molecular weights of approximately 26,000, 30,000, and 34,000. In type II cells from control rats, the 34-kD protein accounted for approximately 93% of the newly synthesized SP-A after 4 h of incubation; only a small amount of radioactivity was associated with the lower molecular weight species. The increased biosynthesis of SP-A in the activated type II cells was associated with a 7.3-fold increase in the level of SP-A mRNA. These results indicate that the content and synthesis of SP-A are both highly elevated in activated type II cells and that these increases may be due to increased levels of SP-A mRNA.

The more differentiated components of a primary tumor may produce substances that reduce the growth rate and metastatic potential of more aggressive components. In the Dunning R-3327 prostatic adenocarcinoma model, cancer cell motility is required for metastatic potential. Medium conditioned by the non-motile, non-metastatic G subline contains proteins of molecular weight 50-100 kDa that inhibited the motility of the highly motile, highly metastatic MAT-LyLu subline. G subline-conditioned medium was separated by DEAE-cellulose chromatography using a linear gradient of 0-0.5 M NaCl in 100 mM Tris at pH 8.3. The motility inhibitory activity of G-conditioned medium was localized to column fractions 51-70 that contained 18% of the applied protein and only 6.5% of the proteins secreted by the G cells. Analysis of pooled fractions 51-60 and 61-70 by two-dimensional gel electrophoresis identified five protein families, with a total of 12 charged proteins of molecular weights approximating 66, 54, 50, 41 and 34 kDa, that were not present or present in reduced quantities in column fractions that did not inhibit motility. Isolation and identification of motility inhibitory protein may prove it the first substance discovered that is produced by a more differentiated component of a neoplasm that directly inhibits a metastasis-associated property.