Janice Yang Chou

Human pregnancy-specific β1-glycoprotein: A new member of the carcinoembryonic antigen gene family

The amino acid sequence of human pregnancy-specific beta 1-glycoprotein (PS beta G) as deduced from the cDNA sequence contains two repeated protein domains (1a and 2a) of 93 amino acids each. PS beta G shows strong homology to human carcinoembryonic antigen (CEA) at both nucleotide and amino acid levels. CEA contains eight domains including a N-terminal domain, three repeated domains (I, II, and III), each containing two subdomains (A and B), and a hydrophobic carboxyl-terminal domain. PS beta G contains a CEA-like N-terminal domain, two repeated domains similar to the A subdomains of CEA which is followed by a domain (1b) similar to the B subdomains of CEA, but lacks a hydrophobic carboxyl-terminal domain. The positions of the cysteine residues in each domain also are conserved, indicating that PS beta G and CEA are two members of the same gene family.

Regulators of fetal liver differentiation in vitro

Seventeen-day-old fetal rat hepatocytes were employed to examine factors required to promote differentiation in vitro. In the absence of effectors, primary fetal hepatocytes dedifferentiated, as characterized by the rapid decline in synthesis of fetal alpha-fetoprotein (AFP), albumin, and transferrin. On the other hand, cells maintained in the presence of glucocorticoid hormone produced high levels of albumin and transferrin. Glucocorticoid could not prevent the decline in fetal AFP synthesis, but induced synthesis of the 65K variant AFP--the major AFP species produced by adult rat liver. Fetal hepatocytes maintained in the presence of 8-bromo-cAMP (8-BrcAMP), or methyl isobutyl xanthine (MIX), an agent that increases intracellular cAMP levels, synthesized high levels of fetal AFP and albumin but reduced levels of transferrin. Both glucocorticoid and 8-BrcAMP or MIX induced expression of adult liver-specific genes such as tyrosine aminotransferase (TAT) and phosphoenolpyruvate carboxykinase (PEPCK), suggesting that these fetal hepatocytes have matured. Cells maintained in the presence of glucocorticoid hormone and MIX (or 8-BrcAMP) contained more albumin, TAT, and PEPCK mRNAs and synthesized increased amounts of the 65K variant AFP than those with either agent alone. However, the glucocorticoid/MIX cells produced intermediate levels of the fetal AFP and transferrin. Our data indicate that both glucocorticoid hormone and cAMP are necessary for optimal differentiation of fetal hepatocytes in vitro.

Regulation of rat liver maturation in vitro by glucocorticoids.

The biochemistry of liver maturation was studied by using the RLA209-15 fetal rat hepatocyte line that is temperature sensitive for maintenance of the differentiated fetal liver phenotype. At 33 degrees C these cells were dedifferentiated; but at 40 degrees C they were phenotypically differentiated and, like normal fetal hepatocytes, synthesized moderate levels of albumin and transferrin, high levels of authentic (69,000 and 73,000 molecular weight) rat fetal alpha-fetoprotein (AFP), and low levels of a 65,000-molecular-weight variant AFP. Our results indicated that administration of glucocorticoid hormones to RLA209-15 cells at 40 degrees C induced a series of events associated with normal hepatocyte maturation; synthesis of fetal AFP was inhibited, whereas the synthesis of variant AFP, albumin, transferrin, tyrosine aminotransferase, and alpha 1-acid glycoprotein was induced. The variant AFP was produced by RLA209-15 cells at both temperatures and was encoded by an mRNA of 1.7 kilobases (kb). The fetal AFP was encoded by an mRNA of 2.2 kb. Normal adult rat liver contained three AFP mRNAs of 2.2 (minor), 1.7, and 1.5 kb. The 1.7-kb adult liver AFP mRNA comigrated with the RNA found in RLA209-15 cells, and both directed the synthesis of a 50,000-molecular-weight precursor polypeptide of the variant AFP. Administration of glucocorticoids to RLA209-15 cells grown at 33 degrees C stimulated synthesis of both the fetal and variant AFPs, but the levels of the 2.2-kb AFP mRNA were preferentially increased. RLA209-15 cells contained two glucocorticoid receptor mRNAs of 6.8 and 4.5 kb. The glucocorticoid-mediated maturation described above was blocked by the antiglucocorticoid RU486.

Role of retinoic acid in maturation of fetal liver cells inmvitro

Regulation of the biosynthesis of alpha-fetoprotein and albumin was studied in a temperature-sensitive fetal rat hepatocyte line (RLA209-15) which exhibits a differentiated phenotype when grown at 40 degrees C. Retinoic acid inhibited alpha-fetoprotein production but increased albumin production. This retinoid also changed the proportion of three forms of alpha-fetoprotein; the biosynthesis of the 73,000- and 69,000-dalton variants, which are indistinguishable from authentic rat alpha-fetoprotein, was inhibited and an additional 65,000-dalton variant was induced. It has previously been shown that alpha-fetoprotein production decreases during maturation whereas albumin production increases. Our data suggest that retinoic acid induces maturation of fetal liver cells in vitro. Further, the 65,000-dalton alpha-fetoprotein variant may be characteristic of liver maturation.

Alpha-fetoprotein synthesis in transformed fetal rat liver cells

We have reported that transformed fetal liver cells produced a variant alpha-fetoprotein of 65K that differed from the mature alpha-fetoprotein of 69K and 73K in the polypeptide backbone. In the present study, we demonstrated that the biosynthetic pathway of the variant alpha-fetoprotein differed from that of the mature alpha-fetoprotein. The 65K variant was synthesized first as a preprotein of 49.5K which was processed to a polypeptide of 59K in the presence of microsomal membranes. The latter was the precursor of the variant alpha-fetoprotein found in cells and medium of transformed fetal liver cells. The 65K alpha-fetoprotein was encoded by a mRNA of 16S while mature AFP was encoded by a mRNA of 20S.

Expression of the α-fetoprotein gene in adult rat liver

Abstract The adult rat liver contains three α-fetoprotein (AFP) mRNAs of 2.2 (minor), 1.7, and 1.5 kb. These transcripts share a common 3′ sequence, but the 1.7- and 1.5-kb AFP mRNAs lack sequences present in the first seven 5′ exons of the 2.2-kb AFP mRNA. S1 nuclease analysis maps the 1.7-kb mRNA at the 5′ boundary of the eighth exon of the 2.2-kb AFP mRNA and the 1.5-kb mRNA in the middle of the eight exon. In a transformed fetal rat liver cell line, we have previously identified a 1.7-kb AFP mRNA which is encoded by an AFP cDNA (ARFP5) isolated from an adult rat liver cDNA library. The 90-bp 5′ sequence of ARFP5, which is located in the seventh intron of the rat AFP gene, is not present in the 2.2-kb fetal AFP mRNA, although ARFP5 does contain nucleotide sequence present in the 2.2-kb AFP mRNA extending from the beginning of its eighth exon (nucleotide 873) to the 3′ end. The 1.7-kb RNA in adult liver also hybridizes with the 90-bp 5′ sequence of ARFP5, suggesting that the two 1.7-kb AFP mRNAs are similar. The developmental profile of these AFP transcripts shows that fetal rat liver contains mainly the 2.2-kb mRNA which decreases to a very low level around the fifth week after birth. The 1.7- and 1.5-kb AFP mRNAs can be visualized about the third and fourth weeks after birth, respectively, and are the major AFP mRNAs in the liver of rats that are older than 4 weeks. The levels of the AFP mRNAs in adult liver are approximately 0.4% of the AFP mRNA level in 18-day-old fetal liver. Both the 1.7- and 1.5-kb AFP mRNAs are actively translated; they direct the cell-free synthesis of two polypeptides of 50K and 44K. A 50K polypeptide is also the encoded product of the 1.7-kb mRNA in the transformed fetal rat liver cell line, suggesting that the 44K polypeptide may be encoded by the 1.5-kb AFP mRNA.

Multiple forms of alkaline phosphatase in untreated and 5-bromo-2'-deoxyuridine-treated choriocarcinoma cells.

Abstract The alkaline phosphatases present in choriocarcinoma cells, either untreated or treated with 5-bromo-2′-deoxyuridine (BrdUrd), were purified and characterized. Three forms of phosphatase [I, IIa (or IIIa), and IIb (or IIIb)]were isolated from both the untreated and BrdUrd-treated cells. Although BrdUrd induced the synthesis of all three forms of alkaline phosphatase in these cells, the synthesis of forms IIa and IIb was, however, preferentially stimulated. The forms of phosphatase in choriocarcinoma cells resembled each other in their kinetic properties and thermal lability, but differed in their molecular weights and in their electrophoretic mobilities in nondenaturing polyacrylamide gels. All three phosphatases were inactivated by antiserum to term-placental alkaline phosphatase. The alkaline phosphatases from choriocarcinoma cells differed, however, from the enzyme from term placentas in several physicochemical properties. The phosphatases from choriocarcinoma cells had a lower K m value for p -nitrophenyl phosphate, were more sensitive to inhibition by l -leucine, levamisole, l-p -bromotetramisole, and EDTA, and were more heat-labile. Phosphatase I comigrated with term-placental alkaline phosphatase on nondenaturing polyacrylamide electrophoretic gels, but phosphatases IIa and IIb migrated more slowly. The apparent molecular weights of phosphatase forms I, IIa, and IIb were estimated by gel filtration and polyacrylamide gel electrophoresis to be 115,000, 240,000, and 510,000, respectively. Although three molecular forms of alkaline phosphatase occurred in choriocarcinoma cells, the subunit molecular weight of these phosphatases appeared to be identical to each other and to the subunit of term-placental alkaline phosphatase (63,000 MW). The alkaline phosphatase in choriocarcinoma cells therefore exists in the dimeric, tetrameric, and octameric forms.

Cloning and expression of a new pregnancy-specific β1-glycoprotein member

Abstract Human pregnancy-specific β1-glycoproteins (PSGs) are a group of closely related placental glycoproteins and members of the immunoglobulin superfamily. To study possible functional roles of PSG, three type I cDNA ciones which encode PSG in a N-A1-A2-B2-C domain arrangement have been characterized in this laboratory. Now, we report the cloning of a type II cDNA (PSG9) which is 1512 bp and encodes a PSG of 332 amino acids in a N-A1-B2-C domain arrangement. In vitro expression experiments demonstrate that PSG9 encodes a 36 kDa nonglycosylated protein which is processed to a glycosylated protein of 46 kDa. Ribonuclease protection analyses show that PSG mRNAs that share sequence similarity with the PSG9 transcript are expressed in both human placenta and placental fibroblasts.