Robert E. Priest

Differentiation in human amniotic fluid cell cultures: I: Collagen production.

The collagen produced by differentiated cells cultured from human amniotic fluid was characterized in two ways. By chain composition and by 4-hydroxyproline:3-hydroxyproline isomer ratio, the collagen synthesized by F-type (fibroblast) cells was indistinguishable from that made by cultured fetal dermal fibroblasts. The predominant cells in young amniotic fluid cultures, termed AF-type, produced collagen with a lower isomer ratio, resembling that of basement membrane collage. The chain composition, as determined by chromatography on carboxymethyl cellulose, varied for different cultures of the AF-type, but the major pattern was consistent with that of basement membrane collagen. On the basis of these characteristics, F cells are of fibroblast origin, whereas most AF cells are of a different origin either endothelial or epithelial. Other evidence (Megaw et al., 1977) suggests an epithelial origin for AF cells.

Differentiation in human amniotic fluid cell cultures: Chorionic gonadotropin production

SummaryTwo of the distinguishable cell classes subcultured from human amniotic fluid were examined for their capability to produce human chorionic gonadotropin (hCG) as determined by radioimmunoassay. The class that predominates in most cultures used for prenatal genetic diagnosis, previously termed AF (for amniotic fluid), secretes hCG into the culture medium. Dermal fibroblasts do not, nor does another type of cultured cell from amniotic fluid, previously termed F because of a resemblance to fibroblasts. Primary AF cultures produce more hCG than do subcultures. Evidence that this hormone is intact hCG is provided by its immunoreactivity with antisera raised against the β-subunit and against the intact molecule of hCG. Furthermore, a dose-response curve for hormone in culture medium is parallel to that of highly purified intact hCG. It is postulated that AF cultures are derived from fetal membranes and retain properties of trophoblast.

Differentiation in human amniotic fluid cell cultures: II: Secretion of an epithelial basement membrane glycoprotein.

Cells obtained by amniocentesis for prenatal diagnosis were grown in vitro and examined for the presence of a glycoprotein component epithelial basement membrane. Isolated colonies or clones of amniotic fluid-type cells secrete the glycoprotein, which was identified in association with the cells using indirect immunofluorescent antibody techniques. In addition, the glycoprotein was isolated from tissue culture medium and identified as a component of epithelial basement membranes by passive haemagglutination (PHA) and immunodiffusion assays. Fibroblast-type cells do not secrete the glycoprotein. These results correlate well with the synthesis of type IV collagen by amniotic fluid cells reported in the accompanying paper (Priest et al., 1977) and indicate that amniotic fluid cells are epithelial in origin.

Oxygen dependence of oestrogen production by human placental microsomes and cultured choriocarcinoma cells.

The oxygen dependence of oestrogen (oestrone and 17 beta-oestradiol) formation from androstenedione and testosterone was studied in term human placental microsomes and in cultured human choriocarcinoma cells (BeWo line). Incubations were performed under various steady-state oxygen concentrations and the production of oestrone and 17 beta-oestradiol quantitated by specific radioimmunoassays. The aromatization of C19-steroids by both placental microsomes and choriocarcinoma cells was shown to be oxygen dependent over a wide range of O2 concentrations. The results indicate that placental oxygenation may be a critical factor in determining oestrogen production in vivo. Therefore, impaired oestrogen biosynthesis due to hypoxia could be an important factor in a variety of physiological and pathological conditions.

Metabolism of [4-14C]androstenedione by cells cultured from human amniotic fluid

The conversion of [14C]androstenedione by human fibroblast (F) and amniotic fluid (AF) cells obtained by amniocentesis was investigated using human dermal fibroblasts (DF) as controls. Cell suspensions were incubated with [14C]androstenedione in the presence of a NADPH generating system. Steroid reaction products were separated from unreacted substrate by chromatography on micro-columns of magnesium oxide, partially resolved by partition chromatography on celite and further characterized by thin-layer chromatography and recrystallization to constant specific activity. In the case of F cells the pattern of metabolism was qualitatively similar to that of DF cells; the predominant metabolite was testosterone and several uncharacterized metabolites were detectable. However testosterone was the only metabolite isolated from incubations with AF cells. The results demonstrate distinct differences in the capacity of AF and F type cells to metabolize [14C]androstenedione and support the view that F cells resemble typical fibroblasts from dermis or other connective tissues.

Isolation and characterization of hydroxyproline-containing proteins secreted by a murine carcinoma cell culture

A collagenous protein was isolated from a murine carcinoma cell culture, which has been shown to synthesize basement membrane. The molecular weight of this protein was estimated to be 155 000. It eluted from carboxymethyl-cellulose in the region near the alpha 1 and beta 11 components of calf skin collagen. 63--69% of the peptide-bound prolines were hydroxylated, and the 4-/3-hydroxyproline ratios ranged from 12 : 1 to 14 : 1. About 95% of the hydroxylysines in the peptide were glycosylated, and almost all of them were in the glucosylgalactosyl dissacharide form. Judging from the posttranslational characteristics, this collagenous protein is probably of basement membrane type.

CHARACTERIZATION OF hCG REGULATION IN CULTURED HUMAN AMNIOTIC FLUID CELLS: II. Mechanisms for Stimulation

SummaryWe showed previously that sodium butyrate stimulated human chorionic gonadotropin (hCG) measured by radioimmunoassay of medium from human second trimester amniotic fluid cell cultures, termed AF cells. We now find that stimulation of hCG in the presence of sodium butyrate takes as long as 20 h. When AF cells are preincubated with sodium butyrate, hCG levels increase in direct relation to length of the preincubation period. These findings suggest that elevation of hCG is not due merely to a release of hormone from the cells. Addition of cycloheximide or Actinomycin D inhibited protein synthesis and RNA synthesis, respectively, and prevented the stimulation of hCG by sodium butyrate. These results lend support for a mechanism of regulation involving protein and RNA synthesis, the increase in hCG levels being due to new synthesis of the hormone.Other agents reported to influence hCG production by different types of cell cultures include dibutyryl cyclic AMP, epidermal growth factor (EGF), methotrexate, and hydroxyurea. Dibutyryl cyclic AMP and EGF have no effect on hCG production in our AF cells: methotrexate causes a minimal increase, hydroxyurea causes a further increase, but sodium butyrate has the strongest stimulatory effect.We conclude that amniotic fluid cells in culture are susceptible to environmental agents capable of modulating synthesis of hCG by mechanisms involving synthesis of RNA and protein.

Purification and characterization of a collagenous protein secreted by a murine teratocarcinoma-derived cell line

A collagenous protein could be precipitated by (NH4)2SO4 from the culture medium of a murine teratocarcinoma-derived cell line (Ko, C.Y., Johnson, L.D. and Priest, R.E. (1979) Biochim. Biophys. Acta 581, 252-259). Further purification of this protein was achieved by combining DEAE-cellulose chromatography with either CM-cellulose or molecular sieve chromatography. The collagenous polypeptides had subunit molecular weights of 160 000, if determined by molecular sieve chromatography, or 190 000, if determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and they are not linked by disulphide bridges. Amino acid composition of this collagen is similar to that of a murine type IV collagen isolated from EHS sarcoma (Timpl et al. (1978) Eur. J. Biochem. 84, 43-52). The most prominent peptides resulting from cleavage of the protein by CNBr had estimated molecular weights of 25 000, 23 000, 11 700 and 9400. Pepsin treatment of this collagen under non-denaturing conditions produced three major fragments having molecular weights of 70 000, 45 000 and 43 000. We conclude that the collagen secreted by the murine teratocarcinoma-derived cell culture is a type IV basement membrane collagen. Therefore, this culture system should provide a continuous source of type IV collagen, which may be used to study the interaction of this collagen with other basement membrane components.