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.     

Demonstration by light and ultrastructural immunoperoxidase study of alpha-fetoprotein-positive non-hepatoma cells and hepatoma cells during 3'-methyl-4-dimethylaminoazobenzene hepatocarcinogenesis

Immunoreaction of alpha-fetoprotein (AFP) has been described in cholangiolar "oval" cells in the early stage of 3'-methyl-4-dimethylaminoazobenzene hepatocarcinogenesis. The subcellular location of AFP in the oval cells was in the perinuclear space, rough endoplasmic reticulum and Golgi apparatus. In livers with hyperplastic nodules there were two different types of foci containing AFP-positive cells. One type had a normal nucleocytoplasmic ratio and was seen in well-preserved hepatic trabecular structures, and the other had less cytoplasm and occurred in trabecular structures in disarray. AFP-immunoreactivity in the former type was visible in the perinuclear space and rough endoplasmic reticulum but scarce in the Golgi apparatus, and in the latter type it was present in the proliferative smooth endoplasmic reticulum and in several parts of Golgi apparatus in the submembranous or pericanalicular areas. In livers with hepatocarcinoma, AFP immunoreaction was detected in well-differentiated hepatocellular carcinomas, and the subcellular location of AFP was in the perinuclear space, rough endoplasmic reticulum and many developed Golgi complexes. Therefore, AFP-positive cells in livers with hyperplastic nodules are a new cell population in hepatocarcinogenesis, and each type is morphologically different from the oval cell.     

Studies on the mechanism of different collagen glucosyltransferase reactions (Golgi apparatus, smooth endoplasmic reticulum, rough endoplasmic reticulum) in chick embryo liver.

1. The galactosylhydroxylysylglucosyltransferase (GGT) specific to collagen is located in the RER (rough endoplasmic reticulum), SER (smooth endoplasmic reticulum) and Golgi apparatus for the chick embryo liver. 2. The UDP-glucose collagen glucosyltransferase activities in chick embryo liver were solubilized by Nonidet P-40. 3. The mechanism of collagen glucosyltransferase reaction was studied with enzyme preparation of Golgi apparatus CF2, smooth endoplasmic reticulum CF4 and rough endoplasmic reticulum CF8. 4. For the three fractions, data obtained in experiments were consistent with a sequential ordered mechanism in which the substrates are bound to the enzyme in the following order: Mn2+, collagen and UDP-glucose substrate, with different values for Km and Vmax.     

Mechanisms of α-fetoprotein synthesis in hepatoma cells

Summary Immunoreaction of -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.     

B-type cytochromes in plasma membranes isolated from rat liver, in comparison with those of endomembranes

Fractions of plasma membranes, Golgi apparatus, endoplasmic reticulum (ER), and nuclear envelope were isolated from rat liver and were characterized by electron microsocpe and biochemical methods. The purity of the fractions was controlled by morphometry and by marker enzyme activities. Amounts of cytochromes b5, P-450, and P-420 were measured, as well as the NADPH- and NADPH-cytochrome c reductase activities. The pigments of the microsomal electron transport system were found in all membrane fractions in relatively high amounts, thus excluding an origin by microsomal contamination. Purified preparations of plasma membrane and Golgi apparatus contained approximately 30% of the cytochrome b5 and cytochrome P-450 + P-420 found in ER membranes. Plasma membranes were also characterized by a high ratio of P-420/450. Degradation of cytochromes P-450 and P-420 was relatively rapid in all fractions, except in the ER. Cytochrome b5 extracted from plasma membranes was spectrophotometrically and enzymatically indistinguishable from ER cytochrome b5. However, immunnlogical characterization with rabbit antibodies against the trypsin-resistant core of microsomal cytochrome b5 showed the presence of at least two types of cytochrome b5 in ER membranes, in contrast to the plasma membranes in which only one of these components was detected. This immunological differentiation also demonstrates that the plasma membrane-bound cytochrome b5 is endogenous to this membrane and does not reflect contamination by ER elements. We conclude that cytochromes b5, P-450, and P-420 are not confined only to ER and nuclear membranes but also occur in signficant amounts in Golgi apparatus and plasma membranes. The findings are discussed in relation to observations of similar redox components in Golgi apparatus, secretory vesicles, and plasma membranes of other cells.     

ISOLATION OF A GOLGI APPARATUS-RICH FRACTION FROM RAT LIVER : II. Enzymatic Characterization and Comparison with Other Cell Fractions

Enzymatic activities associated with Golgi apparatus-, endoplasmic reticulum-, plasma membrane-, mitochondria-, and microbody-rich cell fractions isolated from rat liver were determined and used as a basis for estimating fraction purity. Succinic dehydrogenase and cytochrome oxidase (mitochondria) activities were low in the Golgi apparatus-rich fraction. On the basis of glucose-6-phosphatase (endoplasmic reticulum) and 5'-nucleotidase (plasma membrane) activities, the Golgi apparatus-rich fraction obtained directly from sucrose gradients was estimated to contain no more than 10% endoplasmic reticulum- and 11% plasma membrane-derived material. Total protein contribution of endoplasmic reticulum, mitochondria, plasma membrane, microbodies (uric acid oxidase), and lysosomes (acid phosphatase) to the Golgi apparatus-rich fraction was estimated to be no more than 20–30% and decreased to less than 10% with further washing. The results show that purified Golgi apparatus fractions isolated routinely may exceed 80% Golgi apparatus-derived material. Nucleoside di- and triphosphatase activities were enriched 2–3-fold in the Golgi apparatus fraction relative to the total homogenate, and of a total of more than 25 enzyme-substrate combinations reported, only thiamine pyrophosphatase showed a significantly greater enrichment.     

Is cytochrome P-450 transported from the endoplasmic reticulum to the Golgi apparatus in rat hepatocytes?

The Golgi apparatus mediates intracellular transport of not only secretory and lysosomal proteins but also membrane proteins. As a typical marker membrane protein for endoplasmic reticulum (ER) of rat hepatocytes, we have selected phenobarbital (PB)-inducible cytochrome P- 450 (P-450[PB]) and investigated whether P-450(PB) is transported to the Golgi apparatus or not by combining biochemical and quantitative ferritin immunoelectron microscopic techniques. We found that P-450(PB) was not detectable on the membrane of Golgi cisternae either when P-450 was maximally induced by phenobarbital treatment or when P-450 content in the microsomes rapidly decreased after cessation of the treatment. The P-450 detected biochemically in the Golgi subcellular fraction can be explained by the contamination of the microsomal vesicles derived from fragmented ER membranes to the Golgi fraction. We conclude that when the transfer vesicles are formed by budding on the transitional elements of ER, P-450 is completely excluded from such regions and is not transported to the Golgi apparatus, and only the membrane proteins destined for the Golgi apparatus, plasma membranes, or lysosomes are selectively collected and transported.     

The distribution of the components of mixed-function oxidase between the rough and the smooth endoplasmic reticulum of liver cells

Mixed-function oxidase activity, when measured by the N-demethylation of ethylmorphine or the hydroxylation of aniline, is significantly higher in the smooth hepatic endoplasmic reticulum than in the rough. In the rabbit the smooth membrane/rough membrane activity ratios are significantly greater than 1 whether the activities are expressed per g. of liver (ratio 5), per mg. of protein (ratio 3-5), per mug. of phospholipid phosphorus (ratio 2), per unit of cytochrome P-450 (ratio 1.7) or per unit of NADPH-cytochrome c reductase activity (ratio 2). On the other hand, if the activities are normalized to the NADPH-cytochrome P-450 reductase, there is no significant difference between the rough and smooth membranes. These results suggest that, in the rabbit, the rate-limiting step is the reduction of cytochrome P-450. In contrast, in the rat the difference in activities can be explained by differences in the concentration of cytochrome P-450.     

The role of components of the endoplasmic reticulum in the biosynthesis of cytochrome P-450.

1. Antibodies have been prepared to rat hepatic cytochrome P-450 and their specificity demonstrated. These antibodies have been used to investigate the biosynthesis of cytochrome P-450 in vitro and in situ in various components of the endoplasmic reticulum. 2. A preparation of heavy rough endoplasmic reticulum translocates proteins newly biosynthesized in vitro vectorially into the luminal space and these are released by low concentrations of deoxycholate. A significant proportion of the radioactivity found in this released fraction is incorporated into cytochrome P-450. 3. Following incorporation of [14C]leucine by perfused rat liver, radioactively labelled cytochrome P-450 can be found in the intrascisternal content of heavy rough, light rough and smooth endopalsmic reticulum and also in a solublized Golgi preparation. 4. We suggest that at least part of the newly biosynthesized cytochrome P-450 is translocated into the intracisternal space of the rough endoplasmic and then passes through the other components of the endoplasmic reticulum before insertion at its ultimate membrane locus.     

Long-term survival of functional hepatocytes from adult rat in the presence of phenobarbital in primary culture

In the presence of phenobarbital (PB) at 3 mM, hepatocytes isolated from adult rats by a collagenase-perfusion technique survived well on plastic dishes for at least 49 days after initiation of primary culture. PB at concentrations less than 3 mM was ineffective for the maintenance of hepatocytes, and the maintenance of them was attained only in the continuous presence of 3 mM PB. The hepatocytes surviving in the presence of 3 mM PB were morphologically indistinguishable from the hepatocytes after 1-day attachment period, except for the presence of prominent nucleoli in the former. Although both the albumin secretion and tyrosine aminotransferase (TAT) activities of the cells decreased gradually up to day 7 with time in culture, both were thereafter maintained at relatively high levels at least up to day 35 of primary culture. The addition of 10 microM dexamethasone caused a 3-5-fold induction in TAT activity, and the cells were capable of responding to the hormone in this manner at least up to day 28 of primary culture. Furthermore, the cells also had glucose-6-phosphatase activity, even though the level of this enzyme activity was relatively low as compared with that of TAT activity. Survival of hepatocytes in the presence of 3 mM PB was further enhanced by simultaneous addition of dexamethasone (10 microM) and insulin (10 micrograms/ml). The sensitivity of hepatocytes to 3'-methyl-4-dimethylaminoazobenzene (0.24 mM) was remarkably reduced by treatment with PB at 3 mM. PB treatment decreased efficiently the falling rate of total cytochrome P-450 content, but did not induce P-450PB, which is the specific form of cytochrome P-450 induced by PB, in primary cultured hepatocytes. On the other hand, 3-methylcholanthrene (MC, 10 microM) caused an increase of both contents of total cytochrome P-450 and P-450MC, which is the specific form of cytochrome P-450 induced by MC, in primary cultured hepatocytes. However, MC was ineffective for the maintenance of hepatocytes in primary culture. The possible biological actions of PB on primary cultured hepatocytes are discussed on the basis of the experimental data obtained.     

Dose dependent suppression of hepatic cytochrome P-450 content by doxorubicin and Mitomycin-C: correlation with antipyrine biotransformation

Doxorubicin (DOX) and Mitomycin-C (MMC) are two anthraquinones which, when administered to rats, result in a decrease in the content of hepatic cytochrome P-450 and mixed function oxidase activities. DOX administration produced a dose-dependent immediate decrease in cytochrome P-450 content at all doses but a parallel dose-dependent decrease in the rate of antipyrine metabolite formation of the two higher doses. The lower dose of DOX produced an increase in metabolite formation and produced a less than 20% reduction in cytochrome P-450 content. MMC administration produced an immediate, modest (less than 10% of control levels) suppression of hepatic cytochrome P-450 content, and had no effect on antipyrine metabolite formation. These findings demonstrates that two drugs of the same class can produce similar suppressions of cytochrome P-450 content and that a threshold suppression of cytochrome P-450 content is needed to produce alterations in in vivo drug biotransformations.     

Distribution of NAD(P)H-dependent cytochrome P-450 mixed function oxidase system in the brush border membrane of rabbit kidney cortex.

Optical and magnetic studies were made on subfractions of rabbit kidney cortex. Cytochrome P-450 and cytochrome b5-dependent mixed function oxidase systems were localized mainly in the brush border membranes and microsomes. Cytochrome P-450-dependent mixed function oxidases in the membranes comprised both an NADPH-dependent system and an NADH-dependent system.     

Dietary orotic acid accentuates the hepatic response to phenobarbital in rats.

In rats treated with phenobarbital for 3 days and simultaneously fed a semisynthetic diet containing 1.0% orotic acid, the extent of the increases in liver microsomal phosphatidylcholine, phosphatidylethanolamine, total RNA, total protein, and cytochrome P-450 were significantly greater than they were in rats treated identically with phenobarbital but without dietary orotic acid. This is attributed primarily to the stimulation of hepatic phosphatidylcholine synthesis by dietary orotic acid. In the absence of phenobarbital, orotic acid was shown to cause some increase in liver smooth endoplasmic reticulum components, but not cytochrome P-450. Orotic acid also decreased the activity of microsomal phosphatidylethanolamine N-methyltransferase, which may have contributed to the increase in the microsomal content of phosphatidylethanolamine. The hypothesis is advanced that phospholipid availability is a limiting factor in the hepatic response to phenobarbital. When more phospholipid is available to provide the structural framework for biogenesis of endoplasmic reticulum, all of the hepatic actions of phenobarbital, including induction of cytochrome P-450, are amplified.     

Distribution of phosphatidylinositol biosynthetic activities among cell fractions from rat liver.

The distribution of activities for synthesis of phosphatidylinositol among cell fractions from rat liver was determined. Activity was concentrated in endoplasmic reticulum; rough and smooth fractions were nearly equal. Golgi apparatus exhibited a biosynthetic rate 44% that of endoplasmic reticulum. Plasma membranes and mitochondrial fractions were only 6% as active as endoplasmic reticulum. Thus, endoplasmic reticulum and Golgi apparatus fractions from rat liver catalyze the net synthesis of phosphatidylinositol in vitro, whereas plasma membrane and mitochondrial fractions do not.     

Transglutaminase activity during the differentiation of macrophages

For the examination of the participation of the microsomal electron transport system in mutagenic activation by 4-dimethylaminoazobenzene (DAB), 4-methylaminoazobenzene (MAB) and their 3′-methyl-derivatives (3′-methyl-DAB and 3′-methyl-MAB), monospecific antibodies to NADPH-cytochrome P-450 reductase, 3-methylcholanthrene-inducible major P-450 (MC-P-448) and phenobarbital-inducible major P-450 (PB-P-450) were used. In Ames' assay system, the antibody to NADPH-cytochrome P-450 reductase inhibited the mutagenicities of DAB, MAB, 3′-methyl-DAB and 3′-methyl-MAB by 94, 94, 90 and 87%, respectively. The antibody to MC-P-448 inhibited their mutagenicities by more than 90%, while the antibody to PB-P-450 inhibited the mutagenicities less than 20%. These results indicate that the microsomal electron transport system, especially MC-P-448, is involved in activation of these dyes.     

FINE STRUCTURAL ALTERATIONS IN CELL PARTICLES DURING CHEMICAL CARCINOGENESIS : III. Selective Action of Hepatic Carcinogens Other than 3'-Methyl-4-Dimethylaminoazobenzene on Different Types of Mitochondrial Swelling. Effect of Stimulated Liver Growth

The previous studies on the correlation between tumor incidence and changes in microsomal and mitochondrial swelling during feeding of 3'-methyl-4-dimethylaminoazobenzene to rats have been extended to other hepatic carcinogens. Administration of 4'-fluoro-4-dimethylaminoazobenzene, 4'-ethyl-2-methyl-4-dimethylaminoazobenzene, 2-acetylam-inofluorene, ethionine, and tannic acid were found to produce drastic alterations of the swelling of rat liver mitochondria. In contrast to these compounds, feeding of the non-carcinogenic azobenzene and 4-diethylaminoazobenzene produced only small changes in swelling. Significant modification in the over-all pattern of the swelling curve was observed when the usual concentration of 3'-methyl-4-dimethylaminoazobenzene was reduced, but not when the riboflavin level in the diet was increased tenfold. Feeding of high levels of this dye to the guinea pig did not affect mitochondrial swelling which is consistent with the resistance of this species to azo-dye carcinogenesis. Hypophysectomy provides protection against the alterations, produced by feeding 3'-methyl-4-dimethylaminoazobenzene, in the characteristics of thyroxine- or mercuric chloride-induced mitochondrial swelling. Studies with citric cycle substrates on mitochondrial swelling suggest block of the glutamate α-keto-glutarate pathway after feeding 3'-methyl-4-dimethylaminoazobenzene for 4 weeks. There is a considerable, but reversible, reduction of certain types of mitochondrial swelling in two situations associated with rapid liver growth: after partial hepatectomy and after intraperitoneal injection of 20-methylcholanthrene. Naphthacene, however, which also stimulates rapid liver growth, does not affect mitochondrial swelling.     

Effects of energy and/or protein restriction on hepatic microsomal mixed function oxidase in male broiler chicks

1. Effect of energy and/or protein intake on a mixed function oxidase system (MFO) in hepatic microsomes was studied in male broiler chicks. 2. Contents of cytochrome P-450 and b5 in 72 hr starvation were larger than those in 12 and 36 hr starvations. 3. Reduction of energy and protein intake did not change the MFO, except cytochrome P-450. 4. Reduction of energy intake under the same protein intake increased the cytochrome P-450 content and aminopyrine N-demethylase activity. An increase in protein intake under the same energy intake also increased the cytochrome P-450 content.     

Biosynthesis of rat liver cytochrome P-450 in mitochondria-associated rough endoplasmic reticulum and in rough microsomes in vivo

The hypothesis of a preferential biosynthesis of a major phenobarbital inducible form of hepatic cytochrome P-450 (P-450b) in mitochondria-associated rough endoplasmic reticulum (RERmito) was tested by measuring incorporation rates of [35S]methionine and delta-amino[3H]levulinate into the hemoprotein in adult rats. RERmito, rough microsomes (RM representing RER not associated with mitochondria) and smooth microsomes (SM) were quantitatively isolated from the same homogenate by rate zonal centrifugation and their content of P-450b determined by rocket immunoelectrophoresis. P-450b was isolated by immunoprecipitation from detergent-solubilized membrane fractions. The time course and rate of incorporation of [35S] methionine into immunoprecipitable P-450b of RERmito and of RM were similar at all time points studied (2-15 min) both under conditions of maximal induction (4 injections of phenobarbital in 4 days) and after a single injection of phenobarbital. The incorporation of [35S]methionine into P-450b of SM was slower at early time points (2-8 min) but similar to RERmito and RM after 15 min. In contrast, at short labeling periods (less than 8 min) more delta-amino[3H]levulinate was incorporated into P-450b of RERmito than into P-450b of RM and SM. No significant accumulation of free apocytochrome P-450b was found in either membrane fraction. These data indicate a close coordination of the biosynthesis and assembly of apocytochrome P-450b and its prosthetic heme but do not support the hypothesis of a major functional role of MITO X RER complexes in the synthesis of microsomal cytochrome P-450b.     

Solubilization of UDP-glucose collagen glucosyltransferase activities from chick embryo liver: Golgi apparatus, smooth and rough endoplasmic reticulum.

1. The choice of a suitable detergent for solubilization of UDP-glucose collagen glucosyltransferase (GGT) activities from chick embryo liver has been investigated. Several detergents were used (zwitterionic detergent as Chaps, and non-ionic detergents as Triton X-100, Nonidet P 40, Brij 35). 2. All the detergents with GGT activities were tested in Golgi apparatus, smooth and rough endoplasmic reticulum (SER, RER). 3. 80-100% GGT Golgi apparatus activity was easily solubilized at low concentrations in surfactant (0.5 mg/ml). 25-78% of SER and RER GGT activities were extracted at this concentration. 4. A higher level of detergent (5 mg/ml) was necessary to release all GGT activities of SER and RER. Protein extraction was identical to GGT activities.     

Biogenesis of very-low-density lipoproteins in rat liver. Intracellular distribution of apolipoprotein B

The hepatic subcellular distribution of apolipoprotein B (apo B) was studied quantitatively by using an enzyme immunoassay developed for apo B and by immunoadsorption-precipitation of [3H]leucine-labelled apo B. Over 50% (of 0.59 microgram/mg protein) of the apo B was located in the microsomal fraction. Further subfractionation of the microsomes revealed that 47% of the microsomal apo B was in the Golgi apparatus, while another 43% was associated with the rough endoplasmic reticulum. The smooth endoplasmic reticulum accounted for only 4% of the total. When rat livers were labelled with [3H]leucine for 10 min, the rough endoplasmic reticulum accounted for 80% of the total immunoadsorbed precipitable apo B radioactivity while the smooth accounted for 20%, with no contribution from the Golgi. However, only 8.7% of the total radioactive immunoadsorbed precipitable apo B was lipoprotein-associated, the remainder being membrane-bound. Lipoprotein-associated apo B radioactivity in the smooth endoplasmic reticulum accounted for 40%, with the rough contribution attributed at 50% and the Golgi at 9%. We concluded that (a) there are two major pools of apo B in rat liver microsomes; (b) although the apo B mass may be negligible in the smooth endoplasmic reticulum, the latter does play a role in lipoprotein biogenesis. The possible function of apo B associated with membranes of the microsomes is also discussed.