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.     

Differential effects of dimethyl sulfoxide and sodium butyrate on alpha-fetoprotein, albumin, and transferrin production by rat hepatomas in culture

Radioimmunoassay was used to determine alpha-fetoprotein (AFP), albumin, and transferrin production (ng/10(5) cells/24 h) by two cell lines (7777 and 8994) derived from chemically induced rat hepatomas. alpha-Fetoprotein production was high (2000 to 4400) in 7777, but was very low (0.2 to 0.4) in 8994. Albumin production varied from 0.4-0.8 (7777) to 14-26 (8994). Both lines produced substantial amounts of transferrin (180 to 240 by 7777 and 29 to 42 by 8994). Addition of dimethyl sulfoxide (DMSO, 1 to 4%) or sodium butyrate (BA, 0.5 to 2.0 mM) to the medium inhibited growth in both lines, but 8994 was more sensitive to these agents than 7777. Dimethyl sulfoxide treatment (2 to 4%) resulted in a dose-related decrease (less than 10% of control at 4% DMSO) in AFP, albumin, and transferrin production by 7777, but in 8994, DMSO (1 to 2%) resulted in an increase (up to sixfold) in albumin and transferrin production, without affecting AFP production. By contrast, BA (2 to 4 mM) stimulated the production of all three proteins in both lines, most notably that of albumin (up to sixfold) by 7777 and that of AFP (up to 20-fold) by 8994. It is concluded that both DMSO and BA can enhance the expression of differentiated functions of the hepatoma cell, and that DMSO at the same time can suppress the expression of an oncofetal function. However, neither DMSO nor BA is selective in its effects on specific genes (i.e., normal, adult vs. oncofetal genes), and it appears that their effects may be the result of a more general phenomenon, the expression of which may be related to the stage of differentiation of the cell.     

Effects of hexamethylene bisacetamide onα-fetoprotein,albumin, and transferrin production by two rat hepatoma cell lines

Summary α-Fetoprotein (AFP), albumin, and transferrin production by two rat hepatoma cell lines, McA-RH 7777 (7777) and McA-RH 8994 (8994), was determined after treatment with hexamethylene bisacetamide (HMBA, 2 to 6 mM). Radioimmunoassays were used to determine the levels of both secreted and intracellular AFP, albumin, and transferrin. Line 7777 normally produces large quantities of AFP and small quantities of albumin, thus resembling the less differentiated fetal liver with respect to the synthesis of these two proteins. Line 8994 normally produces small quantities of AFP and relatively larger amounts of albumin, thus resembling hepatic functions characteristic of a more differentiated state. After treatment with HMBA for a period of 28 to 96 h a threefold increase in AFP secretion by 7777 and a dose related increase in AFP, albumin, and transferrin secretion by 8994 were observed. In contrast, the secretion of albumin and transferrin in 7777 was inhibited by 60 and 40%, respectively, following treatment with HMBA. The intracellular concentrations of AFP in 7777 and AFP, albumin, and transferrin in 8994 were increased by treatment with HMBA indicating that HMBA is able to stimulate the synthesis of these proteins. The intracellular concentration of AFP, albumin, and transferrin in 7777, when expressed as a percentage of the extracellular concentration of these proteins, did not change significantly during HMBA treatment, indicating that the observed decrease in secreted albumin and transferrin by 7777 is due to decreased synthesis. Similarly, in Line 8994, when the intracellular concentration of the three proteins was expressed as percentage of the extracellular concentration, the only significant change observed was an increase in AFP after 72 h of HMBA (5 mM) treatment. The observed changes in the synthesis of AFP, albumin, and transferrin in both 7777 and 8994 after HMBA treatment were reversible, as judged by the return to control values upon removal of HMBA from the culture medium. Thus, HMBA stimulates synthesis of the oncofetal protein AFP, a result that appears to be independent of the stage of differentiation of the cell. However, its effect on the synthesis of albumin and transferrin are opposite in the two cell lines, suggesting that the regulation of the synthesis of these two proteins is controlled by factors or conditions that are dependent upon the stage of differentiation of the hepatoma cell lines.     

Regulation of rat liver phosphofructokinase levels in Morris hepatomas.

The levels of the major liver phosphofructokinase isozyme (PFK-L₂) and the PFK regulatory factor were measured in adult and fetal liver as well as Morris hepatomas of different differentiation states. The results indicate that both the PFK-L₂ activity and the PFK regulatory factor levels in well and highly differentiated hepatomas are not statistically different from the amounts found in adult liver. In the poorly differentiated hepatomas and fetal liver, the levels of both PFK-L₂ and PFK regulatory factor are approximately 2 and 3 fold greater, respectively, than what was found in adult liver.     

Interaction of trans-acting factors with the proximal promoter of the mouse alpha-fetoprotein gene.

1. The alpha-Fetoprotein (AFP) gene is expressed during fetal life, but not in adult cells. Also, the AFP gene is expressed in most hepatomas. 2. Using gel retardation (band-shift) assays under very stringent conditions we have compared the binding of trans-acting factors to the proximal enhancer (-202, +34) region of the AFP gene. 3. We have detected the presence of two retarded bands in experiments performed with adult rat hepatocytes and the Fa32 cell line (which does not produce AFP) but only one band is observed with the HepG2 cell line (which produces AFP) and fetal liver. 4. We relate the two retarded bands to a glucocorticoid response element and, tentatively, to the C/EBP trans-acting fractor.     

Differential effects of dimethyl sulfoxide and sodium butyrate on α-fetoprotein,albumin, and transferrin production by rat hepatomas in culture

Summary Radioimmunoassay was used to determine α-fetoprotein (AFP), albumin, and transferrin production (ng/10⁵ cells/24 h) by two cell lines (7777 and 8994) derived from chemically induced rat hepatomas. α-Fetoprotein production was high (2000 to 4400) in 7777, but was very low (0.2 to 0.4) in 8994. Albumin production varied from 0.4–0.8 (7777) to 14–26 (8994). Both lines produced substantial amounts of transferrin (180 to 240 by 7777 and 29 to 42 by 8994). Addition of dimethyl sulfoxide (DMSO, 1 to 4%) or sodium butyrate (BA, 0.5 to 2.0 mM) to the medium inhibited growth in both lines, but 8994 was more sensitive to these agents than 7777. Dimethyl sulfoxide treatment (2 to 4%) resulted in a dose-related decrease (<10% of control at 4% DMSO) in AFP, albumin, and transferrin production by 7777, but in 8994, DMSO (1 to 2%) resulted in an increase, (up to sixfold) in albumin and transferrin production, without affecting AFP production. By contrast, BA (2 to 4 mM) stimulated the production of all three proteins in both lines, most notably that of albumin (up to sixfold) by 7777 and that of AFP (up to 20-fold) by 8994. It is concluded that both DMSO and BA can enhance the expression of differentiated functions of the hepatoma cell, and that DMSO at the same time can suppress the expression of an oncofetal function. However, neither DMSO nor BA is selective in its effects on specific genes (i.e., normal, adult vs. oncofetal genes), and it appears that their effects may be the result of a more general phenomenon, the expression of which may be related to the stage of differentiation of the cell.     

Nuclear topoisomerase I and DNase activities in rat diethylnitrosamine-induced hepatoma, in regenerating and fetal liver.

DNase activity in the presence of Ca2+ + Mg2+, Mg2+ alone, Mn2+ alone, or EDTA, and topoisomerase I activity were measured in nuclear extracts of diethylnitrosamine (DEN)-induced hepatomas, regenerating, fetal, and normal rat livers. In hepatoma tissue, the Ca/Mg-dependent DNase activity was lower than in normal tissue and nearly the same as in fetal liver. In the poorly differentiated hepatomas, Mn-dependent DNase activity was higher than in both moderately and well differentiated ones and than in normal liver tissue. The activity of topoisomerase I in hepatomas and in regenerating liver was lower than in normal liver tissue.     

DNA content in cells of spontaneous hepatomas in CBA mice

The DNA content was measured in the cells of 37 well-differentiated spontaneous hepatomas from 20 CBA mice aged 18 to 22 months. In all the cases, the DNA distribution pattern was polymodal, with the class of tetraploid cells being predominant. It was independent of the tumor ability to produce AFP. No signs of aneuploidy were noted. The cells of spontaneous hepatomas were characterized by a high level of polyploidy and by a dramatic reduction in the number of binuclear cells as compared with liver cells. A tendency was recorded toward positive correlation between the degree of cellular polyploidy and tumor size. The high level of polyploidy seen in spontaneous hepatomas which is also characteristic of the normal liver is considered to be a sign of tumor maturity.     

Alpha-fetoprotein (AFP)-derived peptides as epitopes for hepatoma immunotherapy: a commentary

The various immunological roles of human alpha-fetoprotein (HAFP), and its correlation with hepatomas, that is, hepatocellular carcinomas (HCCs), are not often addressed together in biomedical reports considering that HAFP is an established biomarker for hepatomas. Studies reporting measurement of HAFP serum levels in hepatoma patients in basic/clinical research settings has greatly increased over the years. Recent reports have now expanded our base knowledge in the mounting of an immune response against AFP, a self antigen, during hepatoma tumorigenesis. Advances in the detection and identification of AFP-derived peptide epitopes are opening new vistas of knowledge regarding the immunological role of AFP-peptides as T cell stimulating antigens in the course of hepatoma growth and progression. The present commentary addresses HAFP-derived peptides as immunologic responders in HCC and their use in the study and generation of AFP-peptide sensitized T cells directed against hepatoma cells. Attempts were further made to relate the AFP-derived peptide epitopes to T cell activities during the course of hepatoma immunotherapies and to profile the traits and properties of the peptides themselves. Hence, the present commentary was divided into two sections; (1) the characterization, properties, and traits of AFP peptide epitopes, and (2) the use of AFP-derived peptides in the therapeutic induction of T cells primed against hepatoma cells using both in vivo and in vitro models.     

Cloning of rat alpha-fetoprotein 3'-terminal complementary deoxyribonucleic acid sequences and preparation of radioactively labeled hybridization probes from cloned deoxyribonucleic acid inserts

Double-stranded complementary deoxyribonucleic acid (cDNA) was synthesized from rat yolk sac alpha-fetoprotein (AFP) mRNA, inserted into the PstI site of plasmid pBR322 by an oligo(deoxyguanylic acid).oligo(deoxycytidylic acid) joining technique, and cloned in Escherichia coli chi 1776. A plasmid containing an inserted AFP double-stranded cDNA with a contiguous poly(adenylic acid) [poly(A)] segment was identified and subsequently employed in a new method for preparing AFP-specific hybridization probe. Following an initial digestion of the AFP plasmid with HindIII to create an open, recessed 3' end, lambda exonuclease III was employed to remove the DNA strand opposite the coding strand of the cDNA insert. Oligo(thymidylic acid) was then annealed to the poly(A) segment and employed as primer for E. coli DNA polymerase I to synthesize a 32P-labeled cDNA copy of the AFP coding strand. The single-stranded cDNA product was easily isolated by sedimentation through isokinetic alkaline sucrose gradients. Hybridization with this AFP-specific cDNA probe showed that the yolk sac contained a 6-fold greater concentration of AFP mRNA than that of the fetal liver. AFP mRNA was also found in the normal adult liver, but at a much lower level than in the fetal liver. The concentrations of AFP mRNA in Morris hepatomas 7777 and 8994, however, were significantly elevated to a 2- to 3-fold higher concentration that in the fetal liver.     

Mechanisms of alpha-fetoprotein synthesis in hepatoma cells. Combined electron microscopic immunocytochemistry and autoradiography

Immunoreaction of alpha-fetoprotein (AFP) was detected not only in well-differentiated hepatocellular carcinoma but also in hepatocytes forming foci in livers with hyperplastic nodules during 3'-methyl-4-dimethylaminoazobenzene hepatocarcinogenesis. The subcellular location of AFP in hepatoma cells was in the rough endoplasmic reticulum, perinuclear space and well-developed Golgi apparatus around the nucleus. In livers with hyperplastic nodules it was also in some parts of the smooth endoplasmic reticulum and Golgi regions in hepatocytes in the vicinity of submembranous areas or bile canaliculi. These findings suggest that the Golgi apparatus in hepatoma cells acts mainly as an organelle for glycosylation of AFP and that the Golgi complexes in the hepatocytes in livers with hyperplastic nodules are organelles for secretion of AFP. Combined light microscopic immunoperoxidase study and autoradiography with 3H-thymidine revealed a higher cumulative labeling index in AFP-positive hepatoma cells than in non-tumorous areas. Combined electron microscopic immunoperoxidase study and autoradiography showed that hepatoma cells with AFP immunoreactivity only in the rough endoplasmic reticulum had a significantly higher labeling index than did cells with AFP immunoreactivity in both rough endoplasmic reticulum and Golgi apparatus. These findings suggest that AFP is synthesized in hepatoma cells before or during the stage of their DNA synthesis and is then transported to the Golgi apparatus.     

"Contact inhibition" of alpha-fetoprotein synthesis and junctional communication in adult mouse hepatocyte culture

Synthesis of oncofetal serum protein alpha-fetoprotein (AFP) may be reexpressed in adult differentiated mouse hepatocytes both in regenerating liver and in primary monolayer culture of intact adult liver. We have found that appearance of AFP in these cultures was strongly correlated with the loss of junctional communication between hepatocytes as tested by the dye transfer method. When in hepatocyte culture the gradient of cell density was formed, and the cells in the center of the dense monolayer retained an epithelial morphology and junctional communication and were AFP-negative during 5 days of culture. At the periphery of the monolayer hepatocytes lost junctional communication by the third day of cultivation. They acquired fibroblast-like morphology, formed multilayered sheets, and started to produce AFP. These findings suggest that reexpression of AFP synthesis may be regulated through a process related to "contact inhibition" and junctional communication might play an important role in the phenomenon.     

Messenger RNA for manganese and copper-zinc superoxide dismutases in hepatomas: correlation with degree of differentiation

Total and polyadenylylated RNA have been isolated from two Morris hepatomas with different degree of differentiation and from the normal liver of the corresponding tumor-bearing inbred rats. The analysis of mRNA has been performed by Northern hybridization using 32P-dA-tailed synthetic deoxyoligonucleotide probes, 33-mer for Mn superoxide dismutase (SOD) and 36-mer for CuZnSOD, derived from the nucleotide sequences of the rat enzyme cDNAs. Two distinct mRNA species (about 850 and 1080 nucleotides) have been identified by using the MnSOD probe. CuZnSOD is translated from a single message of about 720 nucleotides. The total MnSOD mRNA concentration is decreased by 43% and 57% in the hepatomas 9618A (highly differentiated) and 3924A (poorly differentiated), respectively. CuZnSOD mRNA is practically unchanged in the hepatoma 9618A whereas it is reduced by 80% in the hepatoma 3924A. Comparison of the enzyme activities and mRNA levels indicates a good correlation only for hepatoma 3924A, suggesting that the changes of both SODs are regulated pretranslationally. From the data obtained it is also inferred that the mRNA levels of MnSOD respond more readily than those of CuZnSOD to changes in differentiation.     

The synthesis of sphingomyelin in the Morris hepatomas 7777 and 5123D is restricted to the plasma membrane

The site of sphingomyelin synthesis in the rapidly growing Morris hepatomas 7777 and 5123D was determined by incubating plasma membrane, mitochondrial and microsomal membrane fractions with vesicles of phosphatidyl[methyl-14C]choline in the presence of phosphatidylcholine transfer protein. In agreement with a previous study on rat liver (Voelker, D.R. and Kennedy, E.P., 1982, Biochemistry 21, 2753-2759) we have demonstrated that sphingomyelin synthesis in these hepatomas is restricted to the plasma membrane. The greatly elevated sphingomyelin content of mitochondria and microsomes (Hostetler, K.Y., Zenner, B.D. and Morris, H.P., 1979, Cancer Res. 39, 2978-2983) suggests that rapidly growing hepatomas, in contrast to liver, have an effective mechanism of intracellular sphingomyelin transfer.     

Synthesis of alpha-fetoprotein by the pre-implantation and post-implantation bovine embryo

The synthesis of alpha 1-fetoprotein (AFP) was measured by radioimmunoassay in tissues and fluids of 19 bovine embryos (14-46 days of gestation) and in tissue cultures of 4 pre-implantation embryos (17-27 days) by incorporation of radioactive methionine. AFP was first detected in Day-14 trophoblasts and secretion of AFP into allantoic fluid occurred by Day 16. Embryonic tissues and fluids in pre-implantation and post-implantation embryos contained levels of AFP that were 550 to 1 500 000 times higher than those found in maternal serum (3.9-298 000 compared with 0.07-0.25 ng/mg protein). High levels of AFP were also found in uterine fluid which suggested significant transfer of this protein from the early post-implantation conceptus. The major sites of AFP synthesis were yolk sac and fetal liver. It is concluded that the synthesis of bovine AFP is not initiated by events associated with implantation.     

25 years of the study of alpha-fetoprotein

alpha-Fetoprotein (AFP) is an embryonic serum protein. Only traces of AFP are present in blood of adult man or animal, but its level substantially increases during liver regeneration and in cases of primary liver carcinoma or embryonic carcinomas. Different aspects of AFP studies (localization of its synthesis, structure and functions, genetic control of its synthesis and its use for cancer diagnostics) are reviewed. The main consideration is given to the cellular mechanisms of regulation of AFP synthesis. Hypothesis of "structural repression of AFP" is substantiated. According to it formation of liver trabeculae leads to suppression of AFP synthesis in hepatocytes, whereas escape of hepatocyte from trabecula results in activation of AFP synthesis. A new experimental model is described: AFP synthesis is activated in primary hepatocyte culture and is suppressed during three-dimensional growth of hepatocytes in collagen gel or mixed culture with nonparenchymal liver cells.     

The expressions of oncogenes and liver-specific genes in Morris hepatomas

The expression of three liver-specific genes and four oncogenes was studied in the Morris hepatomas 8994, 7288c, 7777, 5123tc, and 7800. Total RNA isolated from these tumors was probed with cDNA's for alpha-fetoprotein (AFP), albumin, tyrosine aminotransferase (TAT), and the oncogenes Ha-ras, Ki-ras, myc and src. When compared to mRNA's levels expressed in normal adult liver, we found AFP levels elevated in AFP-producing tumors, albumin and TAT mRNA levels depressed in all tumors, except TAT is elevated in 5123tc and the oncogenes with the exception of src elevated in all tumors. These results argue against a coordinated expression of these genes as a result of transformation, but suggest that oncogene expression is related to tumorigenesis or proliferation.