alpha-Fetoprotein production and erythropoiesis in cell cultures of human fetal liver.

alpha-Fetoprotein and the synthesis of heme associated with hemoglobin were measured simultaneously in short-term cultures of human fetal liver cells to correlate the relationship of alpha-fetoprotein to erythroid cell function. Both synthetic processes decreased exponentially during the first 5 days of culture. The use of media supplemented with different batches of fetal calf serum and porcine portal vein serum indicated that the optimal conditions for the production of alpha-fetoprotein were different from those required for the synthesis of heme associated with hemoglobin. Moreover, the alpha-fetoprotein-producing cells could be separated from erythroid cells after velocity sedimentation in Ficoll gradients. Although it is well known that erythropoiesis and alpha-fetoprotein production occur simultaneously during ontogenesis, alpha-fetoprotein itself (0.01-100 micron g/ml) did not stimulate heme synthesis in liver erythroid cells. Erythropoietin did not stimulate alpha-fetoprotein production. It is concluded that there is no cause-effect relationship between alpha-fetoprotein production and erythroid cell fuction in human fetal liver cells and that the two processes occur independently in different cell types.     

Proliferation and morphology of chick embryo cells cultured in the presence of horse serum and hemoglobin

Summary We have shown previously that hemoglobin greatly stimulates chick embryo cell proliferation in Eagle's minimal essential medium supplemented with horse serum. In the present study we compared the effects of horse serum plus 10 μM hemoglobin to those of fetal bovine serum on subcultures of chick embryo cells serially propagated at high cell densities. The cells became elongated in the presence of fetal bovine serum and their rate of proliferation progressively decreased, whereas they became polygonal in the presence of horse serum plus hemoglobin and proliferated well in successive cell passages. The polygonal cell obtained in the presence of horse serum plus hemoglobin rapidly elongated if cultured at low cell densities in the presence of fetal bovine serum, but, in contrast, elongated cells did not yield polygonal cells if cultured at low densities in the presence of horse serum plus hemoglobin. It is possible that the polygonal and elongated cells are undifferentiated cells and differentiating myogenic cells, respectively.     

Prostaglandin E2 mediated effects on the synthesis of fetal and adult hemoglobin in blood erythroid bursts

The effects of prostaglandin E2 (PGE2) in association with erythropoietin on the synthesis of fetal and adult hemoglobin in peripheral blood-derived erythroid burst colonies from normal adults and from patients with sickle cell anemia were investigated. The synthesized hemoglobin at the end of 8, 14 or 18 days in culture was separated by DEAE-cellulose chromatography of 35S-methionine labelled hemoglobin. Quantitative estimation of the synthesized hemoglobin phenotypes, for the three indicated culture periods, showed preferential synthesis of Hb F in addition to an overall increase in hemoglobin synthesis in PGE2 treated colonies. Furthermore, the reactivation of fetal hemoglobin production by PGE2 was more pronounced when the adherent cells were included in the culture dishes. These results indicate that the addition of PGE2 to culture dishes presumably constitutes an environmental change to promote the functional changes seen in the blood erythroid bursts in terms of Hb synthesis and switching.     

Prostaglandin E2 mediated effects on the synthesis of fetal and adult hemoglobin in blood erythroid bursts

The effects of prostaglandin E₂ (PGE₂) in association with erythropoietin on the synthesis of fetal and adult hemoglobin in peripheral blood-derived erythroid burst colonies from normal adults and from patients with sickle cell anemia were investigated. The synthesized hemoglobin at the end of 8, 14 or 18 days in culture was separated by DEAE-cellulose chromatography of ³⁵S-methione labelled hemoglobin. Quantitative estimation of the synthesized hemoglobin phenotypes, for the three indicated culture periods, showed preferential synthesis of Hb F in addition to an overall increase in hemoglobin synthesis in PGE₂ treated colonies. Furthermore, the reactivation of fetal hemoglobin production by PGE₂ was more pronounced when the adherent cells were included in the culture dishes. These results indicate that the addition of PGE₂ to culture dishes presumably constitutes an environmental change to promote the functional seen in the blood erythroid bursts in terms of Hb synthesis and switching.     

The effect of progesterone on hemoglobin synthesis in suspension cultures of fetal erythroid cells from calf liver

When fetal calf liver erythroid cells were incubated in the presence of small amounts of progesterone (10(-7)-10(-8) M), the hemoglobin synthesis in these cells was significantly increased. The increase in the amount of radioactivity in de novo synthesized hemoglobins could be demonstrated when techniques such as isoelectric focusing, chromatography on DEAE-cellulose and gel chromatography on Sephadex G-100 were used to isolate the hemoglobin fraction. Using the latter technique, it was shown that the synthesis of cytoplasmic non-hemoglobin proteins in erythroid-cell lysates was also stimulated by progesterone. The presence of hepatocytes in culture nullified the hormone action. It was necessary that progesterone was present during the first hours of culture. Delayed addition of the steroid to the cells had no effect on hemoglobin synthesis. Erythropoietin was necessary to obtain stimulation by progesterone. These results suggest that the target cell of the hormone is an erythropoietin-sensitive cell. High concentrations of progesterone (10(-4) M) strongly inhibited hemoglobin synthesis in fetal calf erythroid cells. Culture of cells under this condition, however, gives rise to a cell population that preferentially synthesizes adult hemoglobin. Our results suggest that in the erythropoietic calf liver, high concentrations of progesterone may preferentially stimulate adult hemoglobin synthesis, or that those cells which have a high capacity to synthesize adult hemoglobins are less sensitive to toxic concentrations of the hormone. The effects of stimulation of hemoglobin synthesis in fetal calf erythroid cells occur at hormone concentrations that suggest a possible physiological role of progesterone in fetal, and eventually also in maternal, erythropoiesis.     

Fetal hemoglobin: in vivo stimulation by D-thyroxine in adult marmosets.

Reactivation of fetal hemoglobin gene expressions was studied in new-world monkeys (marmosets). After a series of intraperitoneal injections of D-thyroxine, fetal hemoglobin production was increased in three experimental animals. Control animals were unaffected by the injection of the solvent. Because the percentage of cells containing fetal hemoglobin exceeded the total percentage of fetal hemoglobin, it was concluded that thyroid hormone influenced the synthesis of fetal hemoglobin rather than the development of a population of fetal cells.     

Effects of acidified fetal bovine serum on the fibrinolytic activity and growth of cells in culture.

The fibrinolytic activity of cells in culture varied with the type of serum employed in the growth medium. Degradation of iodinated fibrin occurred slowly when Rous sarcoma virus-transformed chick embryo fibroblasts were grown in medium containing fetal bovine serum (FBS), and rapidly when chicken serum was employed. This difference reflected the low plasminogen and high inhibitor content of FBS. The inhibitors were found to be serum macromolecules that were precipitated with ammonium sulfate or polyethylene glycol, and were inactivated by boiling or upon exposure to acidic conditions. No inhibitor activity was detected in fetuin, one of the major proteins present in FBS. Acidified FBS was similar to chicken serum in that both supported high rates of cell-mediated fibrinolytic activity. Although virally transformed hamster, mouse and chicken cells grew well in acid-treated FBS, their normal counterparts did not. Apparently, acifification resulted in the formation of materials that were toxic to normal cells. These agents rapidly blocked cellular DNA synthesis.     

Effect of phase of growth and hyperlipidemic serum on the synthesis of collagen in rabbit aortic smooth muscle cells in culture

The aim of this study was to elucidate wheter long-term cultivation in the presence of hyperlipidemic serum is able to induce changes in the rate of synthesis of collagen and other proteins by arterial smooth muscle cells. Rabbit aortic medial cells were grown in 10% sera and their collagen and total protein synthesis were studied by incubation of the cells with radioactive proline. When the cells were grown in fetal calf serum, their collagen synthesis was low after trypsinization but reached a constant level in one week, whereafter it remained within 4--5% of total protein synthesis for up to 30 days. Cultivation in hyperlipidemic rabbit serum for up to 14 days caused an accumulation of lipid droplets in the cells, but there were no detectable changes in the rate of collagen of total protein synthesis when compared with cells grown in normal rabbit serum.     

Embryonic and fetal hemoglobin synthesis in K562 cell line

K562 cell line was grown in liquid suspension and in plasma clot cultures. Morphological studies revealed the presence of a minority of cells, which were identified as erythroblasts. However, the majority of the cells remained unidentified. Biochemical studies confirmed the synthesis of hemoglobin by K562 cells. The pattern of hemoglobin (Hb) production was of the embryonic type, with the presence of small amount of fetal Hb. The addition of several inducers, like Epo and butyrate, was unable to modify the pattern of Hb production of K562. In contrast, the addition of hemin increased the synthesis of Hb and stimulated the synthesis of fetal Hb and probably adult Hb.     

Density distribution and cation composition of red blood cells in newborn puppies

The density distribution and cation composition of red blood cells from newborn puppies have been studied. The density distribution of red cells from a newborn puppy in a bovine serum albumin density gradient resembles a normal distribution with a peak density at a region less than that found for adult dog red cells. In two weeks the whole distribution shifts toward a more dense region, and a second cell peak appears so that the distribution becomes bimodal. This second cell peak is smaller than the original peak, and it appears at a region of lower density. In nine weeks the distribution becomes a normal one again, but the peak density corresponds to the peak density of the second cell peak which first appeared at two weeks. Evidence has been obtained to show that fetal red cells are located in the more dense cell peak and neonatal cells are in the less dense second peak. These results were obtained by labeling fetal cells with Cr⁵¹ and neonatal cells with Fe⁵⁹. The analysis of the cation content of these cells shows that fetal cells contain more K and Na and have a higher K/Na ratio than adult red cells. Furthermore, neonatal cells contain considerably less cation and hemoglobin than do fetal cells. From a study of the cation and hemoglobin content of red cells appearing in various density fractions it is concluded that fetal cells lose K and Na during the first two weeks after birth. Thus, the change in the density disribution of the erythrocytes is thought to be due to two factors: (1) An increase in the density of fetal cells due to the loss of K and Na and, hence, water during the first two weeks after birth, and (2) the entry of less dense neonatal cells into the circulation.     

Limited synthesis of labile hemoglobin A1 in vivo and in vitro by the preexisting hemoglobin A1 in diabetic subjects

The synthesis of labile hemoglobin A1 in vivo was studied in subjects with non-insulin dependent diabetes mellitus, impaired and normal glucose tolerance. The labile hemoglobin A1 index defined as delta labile hemoglobin A1 divided by delta plasma glucose at 30 min after oral glucose load, representing the rate of labile hemoglobin A1 synthesis in vivo, was low in diabetic subjects and high in normal subjects, showing an inverse correlation with the amount of preexisting hemoglobin A1. The study on the synthesis of labile hemoglobin A1 in vitro showed a lower initial rate of synthesis and a smaller increase in labile hemoglobin A1 at saturation in red blood cells from diabetic subjects with a relatively large amount of preexisting hemoglobin A1, as opposed to red blood cells from normal subjects. Although the further study is necessary in which delta plasma glucose levels are kept relatively constant in each of 3 groups by glucose-clamp methods, our data suggest that the synthesis of labile hemoglobin A1 is limited in vivo and in vitro in diabetic subjects by the preexisting hemoglobin A1 due to the saturability of its synthesis.     

On the kinetics of erythroid cell differentiation in fetal mice. I. Microspectrophotometric determination of the hemoglobin content in erythroid cells during gestation

In a microspectrophotometric study, photographic emulsions and a computer are used for measuring the hemoglobin content of a large number (about 50,000) of erythroid cells in fetal mice. Histograms of the hemoglobin content in erythroid cells illustrate the kinetics of erythropoiesis in yolk sac derived nucleated cells in the fetal peripheral blood, in fetal liver, and in fetal spleen. After the occasional extrusion of their nucleus, yolk sac derived erythrocytes remain as “macrocytes” in fetal circulation two or three days longer than the nucleated yolk sac derived erythrocytes do. Erythrocytes in fetal liver have a constant hemoglobin content of 28 pg ² until day 17 of gestation. During further erythropoiesis in liver and then in the spleen, this amount is gradually adapted to the normal hemoglobin content in red blood cells of 16 pg.     

Similar content of phospholipids and gangliosides in normal and homozygous familial hypercholesterolemia fibroblasts.

The cellular content of total and individual phospholipids and gangliosides was measured in fibroblasts cultured from four normal subjects, three patients with lysosomal lipid storage diseases, and two subjects with homozygous familial hypercholesterolemia. Measurements were made on cells grown in medium containing fetal calf serum under conditions in which normal cells derive cholesterol for cell growth from low density lipoprotein present in the fetal calf serum, whereas familial hypercholesterolemia homozygote cells, which lack cell surface low density lipoprotein receptors, derive cholesterol from endogenous synthesis. No difference was observed in the cellular content of total or individual phospholipids and gangliosides in the normal and familial hypercholesterolemia homozygote cells. In contrast, cells from a patient with Niemann-Pick disease and a patient with Sandhoff disease showed elevations in the content of sphingomyelin and complex gangliosides, respectively.     

Lack of a correlation between polyamine synthesis and DNA synthesis by cultured rat liver cells and fibroblasts

Growth stimulation of either fetal rat liver cells or rat embryo fibroblasts in culture results in considerable increases in intracellular polyamine levels as cells proceed through the cell cycle. Treatment of such cell cultures with appropriate levels of two inhibitors of polyamine synthesis, namely α-hydrazino ornithine and methylglyoxal bis(guanylhydrazone), can essentially completely block these increases in cellular polyamine content. Under such conditions, where the elevation in intracellular polyamine content is prevented, cell cultures are nevertheless able to initiate DNA synthesis and subsequently synthesize DNA at rates comparable to untreated control cultures that have been growth-stimulated. These two cell types therefore contain sufficient polyamines when in a resting state (G₁) to enable them to enter from G₁ into S phase and traverse S phase at normal rates in the absence of further polyamine synthesis. The recruitment of cells into the first cell cycle, through serum stimulation of growth, therefore appears not to be mediated or regulated by the increases in intracellular levels of polyamines that occurs under these conditions. Conversely, the arrest of growth of these cell types resulting from serum deprivation is not mediated by a limitation of intracellular polyamine content.     

Differential control of the synthesis of two hemoglobin beta chains in normal mice

A fetal-to-adult switch in the proportion of the mouse minor hemoglobin is described. Although mice have no fetal hemoglobin per se, the timing of this switch in the mouse suggests that the mechanism of its control may directly parallel that of the human switch from fetal to adult hemoglobin expression. The mouse minor hemoglobin is expressed only in strains with the "diffuse" allele for the beta chain complex locus. Fetal liver cells of these mice synthesize a much greater proportion of the betaminor globin chain that do adult hematopoietic cells. Consequently, circulating fetal erythrocytes carry a high level of the minor hemoglobin containing it. By the time of birth, a lowered proportion of betaminor is synthesized in the liver. This low proportion continues to be expressed during early erythroid maturation in the adult. The fetal-to-adult switch is the first indication that in normal mice, the two beta chain loci can be expressed noncoordinately. The similarity between the patterns of the decline of the minor hemoglobin in mice and of the disappearance of fetal hemoglobin in humans suggests that the minor hemoglobin in the "diffuse" mouse may function to some degree as a fetal hemoglobin in the period between the disappearance of the embryonic hemoglobins and the time of birth.     

Altered growth factor requirements and cell cycle control in rat hepatoma cells versus adult rat hepatocytes in culture

Adult rat hepatocytes multiply in primary cultures when incubated in arginine-free MX-83 medium supplemented with dialyzed fetal calf serum, insulin, glucagon, hydrocortisone, epidermal growth factor, and transferrin. In the absence of mitogens, the fraction of the cells engaged in DNA synthesis dropped sharply. However, cells initiated DNA synthesis in response to the mitogenic mixture indicating that hepatocyte proliferation is controlled by G1----S transition rates. In contrast, rat hepatoma line DTH-3, derived from Morris 7777 "minimal deviation" hepatoma, required only insulin for proliferation in chemically defined MX-83 medium. The lengths of their cell cycle phases varied with the growth rate. The phases of the growth cycle were proportionately shortened (expanded) when the growth rate was increased (decreased). It is concluded that DTH-3 hepatoma cells, which display a decreased growth factor requirement as compared with adult rat hepatocytes differ from normal hepatocytes by fundamental alterations in the mechanisms controlling the progression of the cell cycle.     

Growth control in primary fetal rat liver cells in culture.

Primary fetal rat liver cells cultured in medium deficient in, but not free of, arginine in the presence of dialyzed fetal calf serum grow until the final cell density is attained and cells become quiescent in the Go phase of the cell cycle. When growing cells are transferred into arginine free medium, cells become reversibly arrested in Go. Fetal rat liver cells can be induced to synthesize DNA by addition of high levels of arginine to serum free medium. Low arginine levels in the culture medium do not induce cell growth unless serum is present. Serum stimulates arginine uptake in fetal rat liver cells suggesting that serum growth factor(s) act by increasing intracellular arginine levels high enough to initiate the growth cycle. Fractionation of fetal calf serum by gel filtration on G-200 Sephadex yields a partially purified arginine uptake stimulating activity which is eluted from the column in the same fractions that contain fetal rat liver cell growth promoting activity. Insulin induces DNA synthesis in quiescent fetal rat liver cells. Glucagon reverses the stimulatory effects of insulin. N-6,O-2-Dibutyryl adenosine 3:5-cyclic monophosphoric acid (But2c-AMP) (10-minus4 M) and theophilline (10-minus3 M) inhibit arginine uptake and the initiation of DNA synthesis by serum. The role of arginine in the control of DNA synthesis in fetal rat liver cells and the mechanism of action of serum growth factors are discussed.     

Effects of serum and conditioned medium on protein degradation, migration of nonhistone proteins to the nucleus, and DNA synthesis in transformed cells

Stimulation of resting transformed cells (Chang liver cells), prelabeled with [3H] leucine, with fetal calf serum, caused increased nuclear translocation of [3H] nonhistone proteins ([3H] NHP) and DNA synthesis and a parallel inhibition of proteolysis of cellular proteins. [3H] NHP migration was independent of protein synthesis. Fractionation of the nuclear proteins in a pH gradient of 2.5-6.5, showed that [3H] NHP fractions with high degradation rates in resting cells corresponded to the [3H] NHP fractions with high migration rates in stimulated cells, suggesting that degradation and migration of [3H] NHP are linked. Conditioned medium (COM) produced by Chang cells had similar effects as serum, suggesting that factors produced by these transformed cells, control cell growth by a mechanism that is similar to serum. The lysosomotropic amine eserine had similar effects as serum and COM. Based on the similarity of the effects, it would appear that serum and COM inhibit lysosomal proteolysis. It is proposed that serum and COM induce NHP migration to the nucleus by inhibiting lysosomal degradation of these proteins. Serum and COM caused also migration of [3H] histones to the nucleus, however the mechanism is not clear.     

Induced changes in the rates of uridine- 3 H uptake and incorporation during the G 1 and S periods of synchronized Chinese hamster cells

The rates of uridine-5-³H incorporation into RNA and the rates of uridine uptake into the acid-soluble pool during the cell cycle of V79 Chinese hamster cells were examined. Cells cultured on Eagle''s minimal essential medium supplemented with fetal calf serum, lactalbumin hydrolysate, glutamine, and trypsin displayed rates of incorporation and uptake which increased only slightly during G₁ and accelerated sharply as DNA synthesis commenced. In contrast, cells cultured on minimal essential medium supplemented only with calf serum exhibited rates of incorporation and uptake which increased linearly through both G₁ and S. The transition from one pattern to the other can be induced within 24 hr and is completely reversible. The nonlinear pattern exhibited by cells grown on the supplemented fetal calf serum medium can also be overcome with high exogenous uridine concentrations. In the presence of 200 µM uridine, these cells display a linear pattern of increase in rates of uridine incorporation and uptake. It is concluded that at lower uridine concentrations the pattern of increase in the rate of uridine incorporation into RNA during the cell cycle for a given population of cells is dependent upon the rate of uridine entry into the cell, and that this pattern is not rigidly determined but can be modified by culture conditions.     

The control of δ-crystallin gene expression during lens cell development: Dissociation of cell elongation,cell division, δ-crystallin synthesis,and δ-crystallin mRNA accumulation

Previous studies have shown that freshly explanted 6-day-old embryonic chick lens epithelial cells elongate, differentially increase their synthesis of δ-crystallin, and accumulate δ-crystallin mRNA when cultured with fetal calf serum; in contrast, precultured serum-starved 6-day-old and freshly explanted 19-day-old embryonic epithelial cells divide when treated with fetal calf serum. We have explored whether the stimulation of δ-crystallin gene expression (as measured by δ-crystallin synthesis and δ-crystallin mRNA accumulation) is affected by inhibiting lens cell elongation with colchicine, and whether δ-crystallin gene expression is increased in lens epithelial cells stimulated to divide by treatment with fetal calf serum, as it is in those stimulated to elongate by treatment with serum. Three new findings were made in this study. First, the stimulation of δ-crystallin gene expression does not require elongation of the cultured lens cells. Second, a decreased proportion of δ-crystallin synthesis is observed in lens epithelial cells during normal development and during serum starvation; in neither case is this decrease associated with a reduction in the number of δ-crystallin mRNA sequences per cell. Finally, serum stimulation of lens cell division does not increase the proportion of δ-crystallin synthesis, but can promote the accumulation of δ-crystallin mRNA. Thus, the relative proportion of δ-crystallin synthesized during chick lens development is not solely a function of the number of δ-crystallin mRNA sequences in the lens cells.