Effects of serum,cycloheximide and actinomycin D on protein secretion by quiescent mouse embryo fibroblasts

Quiescent secondary cultures of Swiss mouse embryo fibroblasts secrete several proteins in response to the addition of 20% fetal calf serum (FCS). Of these proteins, a polypeptide of molecular weigth (Mr) 48 000 (48 K) was identified in the medium within an hour of mitogenic stimuli. In the next hour an additional protein of Mr 26000 (26 K) appeared in the medium. These two proteins were absent in the conditioned medium of quiescent cells. A third protein of molecular weight 45,000 (45 K) was found in small quantities in the conditioned medium of quiescent cells but a 2–3 fold increase in the level of this protein was observed in the medium of stimulated cells. The level of the serum-induced 45 K protein was much higher in the medium of cells that were treated with cycloheximide (CH) and FCS than that found in the medium of cells treated with FCS alone. A 40000 dalton protein was found to be a quiescence specific protein which was observed in large amounts in the medium of quiescent cells; the level of this protein gradually declined in the conditioned medium as the cells entered into the proliferative phase. Actinomycin D specifically inhibited the level of the 45 K secreted protein and a 29 K intracellular protein when added along with CH. In contrast to the inhibition of the synthesis of mitogen induced proteins, actinomycin D super-induced the intracellular and extracellular levels of the matrix proteins fibronectin and procollagens.     

Purification and characterization of a DNA synthesis inhibitor protein from mouse embryo fibroblasts

A DNA synthesis inhibitor protein was purified from the conditioned medium of cycloheximide treated mouse embryo fibroblasts. This protein has a molecular weight of 45,000 as determined by gel filtration and Polyacrylamide gel electrophoresis. The levels of the [³⁵S] methionine la belled 45 kDa protein in the medium and matrix were monitored across two cell cycles in synchronized cultures. The 45 kDa protein was present in higher levels in the medium of non-S-phase cells depicting a peak between the two S-phases. The DNA synthesis inhibitor protein was immunologically related to a chicken DNA-binding protein which showed similar cell cycle specific variations at the intracellular level. The purified 45 kDa protein inhibited DNA synthesis in murine and human cells. In mouse embryo fibroblasts, the DNA synthesis was inhibited to an extent of 86% by 0.25 μg/ml of the inhibitor, while higher amounts of the inhibitor were required to arrest DNA synthesis in human skin fibroblasts: in these cells, 4 μg/ml of the inhibitor inhibited DNA synthesis to an extent of 50%. The high levels of the 45 kDa protein in the medium of non-S phase cells and its DNA synthesis inhibitory potential suggest that this protein may be involved in the regulation of DNA synthesis during the cell cycle.     

Changes induced by expression of the v-src gene in the regulation of cell proliferation. Hypothesis and preliminary results

Similarities between the mode of action of growth factors and the oncogene product (pp 60 src protein) of Rous Sarcoma virus have been described. However, a major difference is that addition of growth factors does not induce a malignant transformation of cells. The present work proposes a hypothesis concerning this difference. Various data suggest that density-dependent inhibition (DDI) of growth in non-transformed cells is due to the diffusion of growth inhibitory molecules. Inhibitory factors of 45 K (IDF 45) and 12 K have been fractionated. We assume that the stimulation of DNA synthesis induced by growth factor addition to dense quiescent cultures of non-transformed cells leads to an increase in the activity of autocrine inhibitory molecules in such a manner that the growth factor stimulatory effect is only transient, and cells re-enter the Go phase. On the contrary, the stimulation of DNA synthesis by v-src transformation would not be counterbalanced by inhibitory diffusing factors and cells would not enter Go phase. We present preliminary results which support this assumption. Dense quiescent cultures of chick embryo fibroblasts infected by Ny 68 virus (ts mutant for transformation of Rous Sarcoma virus) were stimulated to proliferate either by addition of growth factors in cultures maintained at 41 degrees C or by expression of transformation (by the cell transfer from 41 to 37 degrees C, the permissive temperature for expression of transformation). Stimulation of DNA synthesis by growth factors was totally inhibited by the inhibitory diffusing factors of 45 K (IDF45) whereas the stimulation of DNA synthesis produced by transformation was reproducibly not decreased by IDF45.     

Synthesis of DNA-binding proteins during the cell cycle of WI-38 cells

Synthesis of DNA-binding proteins was investigated in WI-38 human diploid fibroblast cultures after stimulation with serum containing medium. Density-inhibited confluent monolayers of young (phase II) and aging (phase III) WI-38 cells can be stimulated to synthesize DNA by replacing the medium with fresh medium containing 10% fetal calf serum. Of the phase II cells, 35–50% showed a partially synchronized burst of DNA-synthesizing activity between 15 and 24 h whereas only 4–6% of phase III cells showed DNA-synthesizing activity at 20 h, and that cell fraction was increasing even at 38 h. This suggests either an extremely prolonged G 1 in stimulated phase III cells, or a heterogeneity of the population (e.g., a mixed population of pre- and postmitotic cells) for phase III cells. At various times after the change of medium, DNA-binding protein synthesis was examined in these stimulated cultures. Protein of mol. wt 20 000–25 000 D accumulated rapidly during early G 1 and declined thereafter, whereas larger protein (40 000 and 68 000 D) accumulated during the late G 1 or G 1-S transition period indicating that accumulation of these proteins is associated with the onset of DNA synthesis in the serum-stimulated cells. In cultures where the DNA synthesis has been reduced or inhibited by an excess of thymidine, hydroxyurea or dibutyryl cAMP, the accumulation of the larger proteins (40 000 and 68 000 D) was neglible as compared with non-stimulated cultures. Hydrocortisone did not exert any effect on the DNA-binding protein synthesis in phase II cells. However, it seems to increase the cell fraction which can respond to the serum factor in phase III cells as evidenced from the pattern of DNA-binding proteins synthesis.     

Influence of external diffusible factors on myogenesis of the cells of the line L6

The influence of external diffusible factors on the terminal differentiation of cells of the myogenic line L₆ has been studied. The cultures were fed either with medium which had been depleted of mitogenic factors by previous incubation in the presence of myogenic cells, or with standard medium to which proteins secreted by myoblasts had been added. We present evidence that the length of the proliferative phase of the cultures is largely dependent upon environmental cues. However, by inhibiting DNA replication by a variety of means during this phase, we show that in order to differentiate, DNA synthesis is needed for myogenic cells of this line.Once the myoblasts have initiated their last presumptive round of DNA synthesis, they cannot be induced to undergo further DNA replication by environmental factors. Cloning experiments showed that, at this time, the cells lose their proliferative capacity. Our data strongly suggest that, at this stage, cells of line L₆ become irreversibly committed for differentiation. The fusion rate of the committed myoblasts could be significantly increased by proteins secreted by proliferating myogenic cells, but not by those secreted by myotubes.     

Inhibitory diffusible factor IDF45, a G1 phase inhibitor

An inhibitory diffusible factor of 45 kDa (IDF45) was isolated from medium conditioned by dense cultures of 3T3 cells. The procedure involved Bio-Gel P150 chromatography and 2 reverse-phase FPLC. After the final step of purification, 60 ng/ml of IDF45 inhibited 50% of alpha-globulin-stimulated DNA synthesis. It was shown that IDF45 acted in the G1 phase of the cell cycle. When added for 8 h in the G1 phase of the cell cycle, it was able to inhibit DNA synthesis in the S phase which followed this G1 phase. Furthermore, IDF45 inhibited the early stimulation of RNA synthesis induced by alpha-globulin.     

Density-dependent growth control of adult rat hepatocytes in primary culture

Adult rat hepatocytes in primary culture, which show various liver functions, did not show any mitosis at confluent cell density, although they entered the S phase and remained in the G2 phase, judging by cytofluorometry, when insulin and epidermal growth factor (EGF) were added to 2-day cultures (Tomita, Y., Nakamura, T., & Ichihara, A. (1981) Exp. Cell Res. 135, 363-371). However, when the cell density was decreased by half or one third, the number of nuclei and cell number increased to 1.5-2.0 times that after culture for 35 h with insulin and EGF. Moreover, at these lower densities, DNA synthesis started much earlier, although at the usual high density DNA synthesis with these two hormones did not start until the hepatocytes had been cultured for over 40 h. These results suggest that proliferation of mature rat hepatocytes is regulated by the cell density. First, cells in G0 enter the G1 phase density-dependently; then cells in the G1 phase seem to be stimulated to enter the S phase by insulin and EGF, and a low cell density may permit cells after DNA synthesis to enter the M phase. DNA synthesis of rat hepatocyte cultures at low cell density was strongly inhibited by co-culture with a dense culture. Therefore, the density-dependent mechanism of hepatocyte proliferation seems to involve regulation by a soluble inhibitor(s) secreted by the hepatocytes into the culture medium.     

Dolichol-Linked Glycoprotein Synthesis in G1 Is Necessary for DNA Synthesis in Synchronized Primary Cultures of Cerebral Glia

Primary cultures of newborn rat cerebrum, which are composed of glial cells (principally astroglia), were used for examining the relationship between dolichol-linked glycoprotein synthesis and DNA synthesis in developing cerebral glia. The cells were synchronized by reducing the content of fetal calf serum in the culture medium from 10 to 0.1% (vol/vol) for 48 h between days 4 and 6 in culture. Reversal of the quiescent state by return of the cultures to 10% serum causes a marked increase in DNA synthesis 12-24 h later. A sharp increase in glycoprotein synthesis (incorporation of [3H]mannose) occurred in the first 12 h after serum repletion, preceding the increase in DNA synthesis. Tunicamycin, an inhibitor of the dolichol-linked pathway to glycoprotein synthesis at the first committed step in oligosaccharide formation, promptly and completely prevented the increase in glycoprotein synthesis and, in addition, the subsequent increase in DNA synthesis. The effects of tunicamycin on glycoprotein and DNA syntheses were reversible, and no comparable effect on total protein synthesis was observed. When tunicamycin was added only during a temporally circumscribed period in G1, i.e., from 3 to 9 h after serum repletion, the increase in DNA synthesis between 12 and 24 h after repletion was still markedly inhibited, i.e., to approximately 45% of the value in untreated cultures. The data thus show that there is a requirement for dolichol-linked glycoprotein synthesis for the subsequent occurrence of DNA synthesis and that this requirement is expressed late in the G1 phase of the cell cycle.     

Mechanism of α Factor Biosynthesis in Saccharomyces cerevisiae

The biosynthesis of alpha factor, a mating-type-specific regulatory oligopeptide which is secreted by Saccharomyces cerevisiae cells of alpha mating type, was studied. In batch cultures only small amounts of the peptide were synthesized during the exponential growth phase. During the stationary phase, alpha factor was produced at a constant rate and accumulated in the culture medium. Inhibition of translation in wild-type cells by cycloheximide, or in mutant strains under conditions which blocked protein or ribonucleic acid (RNA) synthesis completely inhibited the production of alpha factor. These results indicate that the factor is produced by ribosomal translation of a specific messenger RNA and not by an extraribosomal mechanism of peptide synthesis.     

Effect of cell physiological state on infection by rat virus

Infection by rat virus has been studied in cultures of rat embryo cells to evaluate the Margolis-Kilham hypothesis that the virus preferentially infects tissues with actively dividing cells. An enhancement of infection was seen in cultures infected 10 hr after fresh medium was added as compared to infection of stationary cultures (infected before addition of fresh medium). Since addition of fresh medium stimulates deoxyribonucleic acid (DNA) synthesis, the number of cells per culture synthesizing DNA at the time of infection was compared with the proportion of cells which synthesized viral protein. Cells were infected before the medium change and 10 or 24 hr after the medium change and were pulse-labeled with ³H-thymidine at the time virus was added. The cells were allowed to initiate viral protein synthesis before they were fixed and stained with fluorescein-conjugated anti-rat virus serum. Fluorescence microscopy permitted both labels to be counted simultaneouly and showed that the greatest proportion of cells synthesizing viral protein were those which had incorporated ³H-thymidine at the time of infection.     

Studies of fibronectin synthesized by cultured chick hepatocytes

We have adapted a chick embryo liver cell system for studying the synthesis of proteins secreted by hepatocytes. In primary liver cell cultures maintained for several days in arginine-deficient medium containing ornithine (0.7 mM) and carbamyl phosphate (1 mM), only hepatocytes demonstrated normal morphological and biosynthetic characteristics, indicating that they possessed a functional ornithine cycle as a source of arginine production. Non-parenchymal liver cells, such as fibroblasts, which lack the ornithine cycle were excluded. Hepatocytes in arginine-deficient or arginine-containing medium synthesized fibronectin (Fn) over several days at a constant rate of 3 micrograms +/- 1 microgram/mg cell protein per day, with fibronectin representing approximately 3% of the total secreted hepatocyte proteins during any culture period after the first 24 h. Pulse-chase experiments indicated that Fn synthesis and secretion was relatively rapid (t1/2 = 45 min) and represented approximately 95% of the intracellularly labelled Fn. This Fn is secreted predominantly as a 450 kD dimer with a subunit size that is indistinguishable from the plasma form as assessed by one-dimensional electrophoretic analysis. Continuous exposure of hepatocytes to insulin caused a moderate decrease (26%) in Fn synthesis, whereas there was no effect of short-term exposure. In contrast, dexamethasone stimulated Fn production 2-3-fold, consistent with its known ability to stimulate hepatocyte production of acute phase proteins. Under these conditions, electrophoretic analyses showed that an increased quantity of intact hepatocyte Fn was produced having the same molecular size of plasma Fn.     

Immediate induction of a 45 K secreted glycoprotein by serum and growth factors in quiescent mouse 3T3 cells : Two-dimensional gel analysis

Stimulation of quiescent 3T3 cells by serum dramatically induces the synthesis of a group of secreted polypeptides with a molecular weight (MW) of 45 K (p45 A, B, C, D). The synthesis of these polypeptides increases 10-fold during the first 2 h. Cycloheximide superinduces the 45 K polypeptides and actinomycin D (actD) blocks completely their induction by serum. Peptide mapping analysis and pulse-chase experiments revealed that p45 A is a precursor of polypeptides p45 B, C, D. Tunicamycin treatment inhibits the synthesis of all four polypeptides but a new related protein appears, p-p45, the unglycosylated precursor. In the presence of tunicamycin, p-p45 is also found in the medium, demonstrating that glycosylation is not essential for the secretion. In vitro translation experiments show that the levels of p45 mRNA present in stimulated cells are severalfold higher than that of non-stimulated cells. Purified growth factors, such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and epidermal growth factor (EGF) induce the synthesis of p45 in quiescent cells.     

Regulation of cell proliferation inhibitory and stimulatory factors diffused by 3T3 cultured cells

The growth rate of normal cells multiplied in vitro decreases as the cell density of the culture increases. Previous results suggested that this density-dependent inhibition of growth in nontransformed cells was due to the diffusion of growth inhibitory substances in the medium of dense cultures. In this paper, we demonstrate that dense cultures of 3T3 cells secrete inhibitory and stimulatory factors. Macromolecules of conditioned medium were fractionated on Biogel P150 and the different fractions were tested on quiescent cultures of 3T3 cells stimulated or not to proliferate by addition of alpha globulin. When target cells were not stimulated to proliferate by addition of exocrine growth factors, we observed the inhibitory activity of a large molecular weight inhibitor (IDF45) and the stimulatory activity of autocrine growth factors (fraction about 35 and 10 K molecular weight), on the incorporation of 14C inosine into nucleotide pool and RNA. However, DNA synthesis was significantly stimulated with fraction 10 K only. This discrepancy between the stimulation of RNA and DNA synthesis may be explained by the presence, simultaneously, of inhibitory and stimulatory factors in fraction 35 and 10 K molecular weight. The presence of inhibitory factor was demonstrated when the fractions were tested on target cells stimulated to proliferate by alpha globulin addition and labeled with 14C thymidine. In these conditions, the stimulatory activity of autocrine growth factors was not observable, and only the inhibitory activity on DNA synthesis of fractions 35 and 10 K appeared. It is tempting to assume that the regulation of in vitro cell proliferation is determined by the balance between these antagonist stimulatory and inhibitory autocrine growth factors.     

In vitro culture of cryopreserved bovine mammary cells on collagen gels: synthesis and secretion of casein and lactoferrin

The preparation, cryopreservation, and culture on type I collagen gels of lactating bovine mammary cells with prolonged milk protein synthesis and secretion in vitro is described. Cryopreserved cells prepared as acinar fragments from either lactating or developing mammary glands attached to the collagen substratum within 24-48 hr after plating in serum and hormone supplemented medium. During continued culture in hormone-supplemented (insulin, cortisol, and prolactin) serum-free medium outgrowth of cells from the attached acinar fragments was observed beginning on day 2, with continued outgrowth to near confluence by day 6. Two morphologically distinct cell types were evident; initial outgrowth was by large polygonal cells that were subsequently overlain by spindle-shaped cells. Cells from both lactating and developing mammary glands sustained substantial milk protein secretion for at least 14 days in culture. Alpha S1-casein synthesis and secretion in cultures of lactating mammary cells was dependent on a critical minimum cell population density, below which alpha S1-casein was not secreted. In contrast, lactoferrin (LF) secretion into the medium increased linearly with the increase in cell population density. Cells cryopreserved up to 16 months secreted LF at levels comparable to fresh cultures of the same cells.     

INHIBITION OF CELL GROWTH IN THE G1 PHASE BY ADENOSINE 3'',5''-CYCLIC MONOPHOSPHATE

Incorporation of tritiated thymidine into acid-precipitable material was used to measure the rate of DNA synthesis in secondary cultures of human diploid fibroblasts. Confluent cultures of human diploid fibroblasts, which are synchronized in the G₁ phase due to contact inhibition, were released from growth inhibition either by the addition of fresh medium to the cultures or by trypsinization and replating at nonconfluent densities. Either treatment resulted in a synchronous wave of DNA synthesis beginning 10–15 h after treatment and peaking at 20–25 h. In confluent cultures stimulated by fresh medium, either the addition of 0.25 mM N⁶, O²-dibutyryl-adenosine 3',5'-cyclic monophosphate (db-cAMP) to the medium in the interval 4–8 h after stimulation or the replacement of the fresh medium in that same 4 h interval with the depleted medium present on the cells for the 2 day period before stimulation delayed the synchronous onset of DNA synthesis in the cultures by about 4 h. In nonconfluent cultures freshly seeded from trypsinized confluent cultures, this same depleted medium obtained after a 2 day incubation of fresh medium on confluent cultures is shown to support the progress of the cells into S phase; however, the addition of 0.25 mM db-cAMP to the medium 3½ h after replating still partially prevented the initiation of DNA synthesis in the cultures. The results are discussed in terms of the role of serum and cAMP in the control of cell growth in fibroblast cultures.     

In vitro primary satellite cell growth and differentiation within litters of pigs

Postnatal muscle growth is dependent on satellite cell (SC) proliferation, differentiation and fusion to increase the DNA content of existing muscle fibres and thereby the capacity to synthesize protein. The purpose of the present study was to examine the ability of isolated SCs from low, medium and high weaning weight litter mates of pigs to proliferate and differentiate, and to affect protein synthesis and degradation after fusion into myotubes. At 6 weeks of age, SCs from the lowest weight (LW), medium weight (MW) and highest weight (HW) female pigs within eight litters were isolated. Thereby, eight cultures of SCs were established for each of the three weight groups within litter, representing three groups of SCs from pigs exhibiting differences in postnatal muscle growth performance. Proliferation was estimated as the number of viable cells at different time points after seeding. SC differentiation was evaluated by measuring the activity of the muscle-specific enzyme, creatine phosphokinase, and protein synthesis and degradation were measured by incorporation and release of 3H-tyrosine, respectively. A tendency towards a difference in proliferation between SC cultures was found (P = 0.09). This was evident as the number of viable cells at day 3 was lower in cultures from LW pigs than from HW (P < 0.05) and MW (P < 0.01) pigs. Differentiation was significantly different between cultures (P < 0.05). There was a significant difference between LW and MW cultures at 72 h (P < 0.05), and a tendency towards a difference between LW and HW cultures at 45 h (P = 0.07). Protein synthesis per μg protein or per μg DNA did not differ among SC cultures from LW, MW and HW pigs. Neither did protein degradation rate differ significantly among SC cultures from LW, MW and HW pigs. Overall, the results show that SCs from LW pigs seem to proliferate and differentiate at a slower rate than SCs from MW and HW pigs. The results found in this study show no difference in the ability of SCs to affect protein synthesis or degradation between SCs from litter mates exhibiting different growth rates in vivo.     

Improved purification and some properties of megacin Cx, a bacteriocin produced by Bacillus megaterium

Megacin Cx is a bacteriocidal protein, previously described by Durner and Mach (Durner, K. (1970) Z. Allg. Mikrobiol. 10, 93-102; Durner, K. (1970) Z. Allg. Mikrobiol. 10, 373-382; Durner, K., and Mach, F. (1966) Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. I Orig. 2, 120, 565-575), which is released into the medium during growth by Bacillus megaterium strain 337. We have optimized the culturing conditions to achieve reproducibly high yields of this protein; under these conditions, megacin Cx represents about one-half of the protein secreted into the medium. We have developed improved methods of purifying the protein to homogeneity. The active form of megacin Cx is a monomeric protein, a single polypeptide of Mr = 210,000, as determined by analytical ultracentrifugation and gel electrophoresis in sodium dodecyl sulfate. Like many other proteins secreted by Gram-positive organisms, megacin Cx contains remarkably little methionine and cysteine. The purified protein blocks protein synthesis in sensitive cells, but has little immediate effect upon nucleic acid synthesis. Our purification procedure separates megacin Cx from another activity in the culture medium which blocks the synthesis of RNA and DNA, as well as synthesis of protein, in sensitive cells.     

Stimulation of growth of primary cultured adult rat hepatocytes without growth factors by coculture with nonparenchymal liver cells

DNA synthesis of adult rat parenchymal hepatocytes alone in primary culture can be stimulated only by the addition of humoral growth factors to the culture medium. However, when parenchymal hepatocytes were cocultured with nonparenchymal liver cells from adult rats, their DNA synthesis was markedly stimulated in the absence of added growth factors or calf serum. DNA synthesis of parenchymal hepatocytes was not stimulated by conditioned medium from nonparenchymal liver cells and was greatest when the parenchymal cells were plated on 24-h cultures of nonparenchymal liver cells. A dead feeder layer of nonparenchymal cells was almost as effective as a feeder layer of viable nonparenchymal cells. These results suggest that the stimulation of DNA synthesis in parenchymal hepatocytes was not due to some soluble factors secreted by nonparenchymal liver cells but to an insoluble material(s) produced by the nonparenchymal liver cells. This insoluble material(s) was collagenase- and acid-sensitive, suggesting that it was a protein containing collagen. The effect of nonparenchymal liver cells was specific: coculture with hepatoma cells, liver epithelial cells, or Swiss 3T3 cells did not stimulate DNA synthesis in parenchymal hepatocytes. Added insulin and epidermal growth factor showed additive effects with nonparenchymal cells in the cocultures. These results suggest that DNA synthesis in parenchymal hepatocytes is stimulated not only by various humoral growth factors but also by cell-cell interaction between parenchymal and nonparenchymal hepatocytes, possibly endothelial cells. This cell-cell interaction may be important in repair of liver damage and liver regeneration.     

Effect of dimethyl sulfoxide on mouse embryo fibroblasts: inhibition of plasminogen activator inhibitor deposition and interference with early events of serum-stimulated growth

Quiescent and serum-stimulated cultures of Swiss mouse embryo fibroblasts (MEF) showed alterations in cell morphology including an enlargement in size upon treatment with 2% dimethyl sulfoxide (DMSO). Treatment of MEF and monkey kidney epithelial cells (MK2) with 2% DMSO at the early periods of serum-stimulated growth inhibited RNA, protein and DNA synthesis. DMSO treatment of cells at late stages of serum-stimulated growth (MEF after 1 hr and MK2 cells after 3 hr of stimulation) had little effect on DNA and protein synthesis although cell enlargement occurred in these cells. When the [35S]methionine labelled proteins of the control and the DMSO treated cells were analysed by high resolution polyacrylamide gel electrophoresis, no apparent difference was observed in the pattern of intracellular proteins of these cells. In contrast, the extracellular levels of two serum-induced secreted proteins of MEF (Mr 48,000 and 26,000) were dramatically reduced by DMSO treatment. The DMSO sensitive 48 kDa protein was found to be the major component of the extracellular matrix, while the 26 kDa protein was not. The 48 kDa protein was identified as plasminogen activator inhibitor (PAI-1). Densitometric quantitation showed a gradual accumulation of this protein in the matrix of serum-stimulated cells. The deposition of this protein in the matrix was inhibited by DMSO. Flow-cytometric quantitation of indirect immunofluorescence indicated higher intracellular levels of the 48 kDa protein in fetal calf serum (FCS) + DMSO treated cells, suggesting that the low level of this protein in the medium of DMSO treated cells is probably due to lack of transport of this protein from the cells into the medium.