WI-38 Human Diploid Fibroblasts Research Articles

Synthesis of nuclear proteins during DNA repair synthesis in human diploid fibroblasts damaged with ultraviolet radiation of N-acetoxy-2-acetylaminofluroene.

We have examined the accumulation of newly synthesized nuclear proteins into nuclei during DNA repair synthesis in confluent WI-38 human diploid fibroblasts damaged with ultraviolet radiation or N-acetoxy-2-acetylaminofluroene. In contrast to a marked stimulation of DNA repair synthesis, stimulation of amino acid incorporation into histone polypeptides or into the various molecular weight classes of nonhistone nuclear proteins was not observed. These results suggest that detectable stimulation of newly synthesized nuclear protein incorporation into nuclei does not accompany DNA repair synthesis induced by ultraviolet radiation or a direct acting chemical carcinogen. At least for the special case of repair, DNA synthesis may be uncoupled from histone synthesis.

Phosphorylation of nonhistone chromosomal proteins early during the prereplicative phase of the cell cycle of WI-38 human diploid fibroblasts

Stimulation of confluent monolayers of nondividing WI-38 cells to proliferate is accompanied by a doubling in the rate of phosphorylation of nonhistone chromosomal proteins during the first hour. The use of cycloheximide to inhibit protein synthesis does not appear to affect phosphorylation of the nonhistones. Nondividing and serum-stimulated cells exhibit qualitative differences in the phosphorylation and dephosphorylation of individual polypeptide peaks resolved by polyacrylamide gel electrophoresis. These results are consistent with other evidence implicating nonhistone protein phosphorylation as an early event involved in the activation of transcription occurring during cell proliferation.

Translational control of protein synthesis in stimulated WI-38 fibroblasts

A cell-free protein synthesis system employing ribosomes from WI-38 human diploid fibroblasts was developed and its optimum MgCl 2 and KCl levels and pH value found. The rate at which ribosomes are able to incorporate radioactive leucine into proteins ([ 14C]leucine incorporation/10 min/100 μg rRNA) and the number of growing peptide chains [ 3H]puromycinpeptides formed/100 μg rRNA) was determined. When confluent monolayers of WI-38 cells were stimulated to proliferate by serum, a transient increase in the rate of peptide elongation by ribosomes was observed at 60 min after stimulation. This increase was not affected by the presence of actinomycin D (10 μg/ml) in the stimulating medium. A change in the relative amount of certain ribosome-associated proteins accompanied the increased elongation rate of peptide growth. The alteration in associated proteins could not be accounted for by an increased synthesis of protein. Finally, the early activation of ribosomes in stimulated WI-38 cells appears to result from the removal of an inhibitor(s) of ribosome function.

Structural alterations of chromatin in phase III WI38 human diploid fibroblasts

Circular dichroism spectra of chromatin from phase II (middle-aged) and phase III (old) WI38 human diploid fibroblasts are different. These differences were evident in populations of confluent WI38 cells and were associated with different kinetics when the confluent monolayers were stimulated to proliferate by a nutritional change. The kinetic differences consisted of an increased length of the prereplicative phase and a decreased percentage of cells entering DNA synthesis in the older cell population. The structural differences between the chromatins of middle- and late-passage cells were abolished when both chromatins were extracted with 0.25 M NaCl. Finally, analysis of the 0.25 M NaCl extract showed differences in the gel electrophoretic profiles of the extracted proteins.

Stimulated phosphorylation of non-histone phosphoproteins in SV-40 transformed WI-38 human diploid fibroblasts

Because the phosphorylation of non-histone proteins has been suggested to play a role in the regulation of eukaryotic gene transcription, we have compared the phosphorylation of these proteins in normal and SV-40 transformed WI-38 human diploid fibroblasts. The rate of phosphorylation was found to be roughly ten-fold higher in the transformed cells, and this striking difference could not be accounted for by either an increased rate of phosphate transport or by the synthesis of new species of non-histone proteins which subsequently become phosphorylated. To our knowledge this is the most dramatic alteration in non-histone protein phosphorylation ever described, and therefore may have important implications for our understanding of malignant transformation.

Conformational changes in chromatin from density inhibited WI-38 fibroblasts stimulated to proliferate.

Quiescent confluent monolayers of WI-38 human diploid fibroblasts can be stimulated to proliferate by replacing the old medium with fresh medium plus 10% serum. Circular dichroism spectra of chromatin from stimulated cells between 2 and 10 hrs after stimulation show an increase in positive ellipticity maxima and a blue shift in the 250-300 nm region. These changes are reversed when the stimulated cells enter DNA synthesis (which, in the present conditions, begins to increase at 12-15 hrs and reaches a peak at 20 hrs). The circular dichroism changes occurring 3 hrs after stimulation have been studied in greater detail. They consist in a 35% (average) increase in positive ellipticity and a blue shift in the 250-300 nm region. Changes in the gamma less than 244 nm region are less consistent. The differences between chromatins of stimulated and unstimulated cells are abolished when both chromatins are washed with 0.25 M NaC1. This procedure removes 10-12% of chromosomal proteins, which chromatograph with non-histone proteins. DNA, RNA and histones could not be detected in the 0.25 M NaC1 extract. In gel electrophoretic profiles of radioactively labelled chromosomal proteins from stimulated and unstimulated WI-38 cells there were no detectable differences between histones. The non-histone proteins of stimulated cells showed one radioactive peak which was increased above the level of non-histone proteins from control cells. These results show that structural changes occur in the chromatin of WI-38 cells stimulated to proliferate several hrs before the onset of DNA synthesis. The fact that differences in the chromatins can be abolished by washing with 0.25 M NaC1 could give a clue as to the mechanisms responsible for these structural changes.

Chicken antichromatin antibodies: specificity to different chromatin fractions.

Specific complement-fixing and precipitin antibodies were induced in chicken to chromatin from WI-38 human diploid fibroblast. Chicken antibodies were chosen because they present two major advantages with respect to rabbit antibodies: (a) the optimal NaCl concentration for chicken precipitin is 1.5 M, which is also an optimal solvent for chromatin proteins that are insoluble at the lower salt concentration required by rabbit precipitin; (b) chicken antichromatin antibodies require an antigen concentration much lower with respect to rabbit antibodies for saturation at the complement-fixation reaction. These antibodies are species specific and they react at the complement-fixation and precipitin assay not only with the whole chromatin but also with dissociated proteins, albeit at a higher concentration of both antigen and antibodies. The specificity of these antibodies to different fractions of chromosomal proteins and of the chromatin after washing with a solution at a different sodium chloride concentration has been investigated.

Effect of 5-bromodeoxyuridine on the transcriptional properties of the genome in WI-38 human diploid fibroblasts

Growth of WI-38 diploid fibroblasts in a medium containing 5-bromodeoxyuridine (BrdU) resulted in an increased GMP and a decreased AMP incorporation into the RNA synthesised in vitro on a chromatin template. This effect was similar to that previously reported using 3T6 mouse fibroblasts-1. Substitution of thymidine by BrdU in DNA, also altered the characteristics of the DNA template itself, since the increased incorporation of guanine and decreased incorporation of adenine into RNA were evident also when purified, isolated DNA was used as template. The extent of replacement of AMP by GMP was proportional to the extent of replacement of thymidine by BrdU. Although there are variations in the base composition of RNA transcribed from BrdU-containing DNA templates, there are no significant difference in overall template activity or in the number of available chromatin binding sites for E. coli RNA polymerase. Confluent monolayers of BrdU-treated WI-38 fibroblasts are still able to respond with cell proliferation to a change of medium, as evidenced by an increased incorporation of (-3H)thymidine and an increase in chromatin template activity. The length of the prereplicative phase is similar in both BrdU-treated and untreated cells, although the magnitude of the increase of (-3H)thymidine incorporation is reduced by approximately 30% after BrdU treatment. The increase in chromatin template activity is associated with an increase in the number of chromatin binding sites for E. COLI RNA polymerase, suggesting that the presence of BrdU does not interfere with the availability of initiation sites or alter the actual rate of transcription.

Properties of the genome in normal and SV40 transformed WI38 human diploid fibroblasts: II. Metabolism and binding of histones

Composition, metabolism and extractability of histone fractions from WI38 human diploid fibroblasts and SV40 transformed WI38 fibroblasts are compared. Two alternate procedures were used for isolation of nuclei which allow for either optimal recovery of arginine-rich histones F3 (III) and F2a1 (IV) or for optimal retention of lysine-rich F1 (I) and slightly lysine rich F2b (II b2). While the relative amount of each histone fraction was found to be similar in normal and SV40 transformed cells, substantial increases in the levels of F 3 acetylation and F1 and F2a2 phosphorylation are reported for the histones of SV40 transformed cells. Differences in extractability of arginine-rich histones with 0.25 M HCl are also reported. While F 3 is extracted more rapidly than F 2a1 from nuclei of normal WI38 fibroblasts, the reverse is true in SV40 transformed WI38 cells. These differences are discussed in relation to modification reactions, binding of histones to DNA and SV40-induced alterations in gene readout.

Properties of the genome in normal and SV40 transformed WI38 human diploid fibroblasts: I. Composition and metabolism of nonhistone chromosomal proteins

The composition and metabolism of nonhistone chromosomal proteins associated with the genome of normal and SV40 transformed WI38 human diploid fibroblasts were examined. Evidence is presented which indicates variations in the relative protein content, specific activity and phosphorylation of several defined molecular weight classes of these chromosomal polypeptides. In addition, it is shown that blocking DNA replication with cytosine arabinoside does not inhibit the incorporation of 3H-leucine into nonhistone chromosomal proteins of SV40 transformed cells. The possibility that the nonhistone chromosomal proteins of human diploid fibroblasts transformed by SV40 virus may be involved in the regulation of viral induced alterations in gene expression is discussed.

Properties of the genome in normal and SV-40 transformed WI-38 human diploid fibroblasts. III. Turnover of nonhistone chromosomal proteins and their phosphate groups

The turnover of nonhistone chromosomal proteins and their phosphate groups was compared in normal and in SV-40 virus transformed WI-38 human diploid fibroblasts. Cells were pulse labelled with tryptophan- 3H and 32P for 30 minutes and the specific activities of tryptophan- 3H and 32P in the various molecular weight classes of nonhistone chromosomal proteins were determined during the first four hours following termination of labelling. While a rapid turnover of high molecular weight nonhistone polypeptides (142, 000 to 200, 000 Daltons) is evident after one hour in SV_40 transformed cells, the specific activities of these nonhistone chromosomal polypeptides are not significantly decreased in normal cells. In contrast, a rapid turnover of low molecular weight (30, 000 to 51, 000 Daltons) nonhistone chromosomal proteins occurs during the first hour in normal WI-38 cells with no corresponding decrease in the specific activities of these proteins in SV-40 transformed cells. There is no apparent net turnover of phosphate groups on nonhistone chromosomal proteins in either normal or SV-40 transformed cells four hours following pulse labelling. Rather, during the first four hours significant fluctuations are observed in the 32P specific activities of defined molecular weight fractions. Taken together with previous reports of differences in the composition, synthesis and phosphorylation of nonhistone chromosomal proteins in normal and SV-40 transformed human diploid cells the present results further indicate the complex nature of the alterations in these proteins which accompany viral transformation.

Gene activation in human diploid cells: Age-dependent modifications in the stability of messenger RNAs for nonhistone chromosomal proteins

Serum stimulation of early as well as late passages of nondividing WI-38 human diploid fibroblasts to proliferate, results in DNA synthesis beginning at 12 hours and mitosis at 20 hours. A 2-fold increase in the transcriptional activity of chromatin isolated from early and late passage WI-38 cells is evident one hour following serum stimulation. An increased synthesis and association with the genome of two defined molecular weight classes of nonhistone chromosomal proteins one hour following serum stimulation of early and late passage cells is also observed. The increased chromatin template activity and nonhistone chromosomal protein synthesis occur in early passage cells stimulated to proliferate in the presence of actinomycin D. However, when late passage WI-38 cells are stimulated in the presence of actinomycin D, increases in chromatin template activity and nonhistone chromosomal protein synthesis are not observed one hour following serum stimulation. The possibility that nonhistone chromosomal protein synthesis and activation of transcription early during the prereplicative phase of the cell cycle in early passage human diploid fibroblasts may be regulated at the translational level is discussed. Consideration is also given to the possibility that there may be an age-dependent modification in such a regulatory mechanism.

Changes in polyamine metabolism in WI38 cells stimulated to proliferate

Quiescent confluent monolayers of WI38 human diploid fibroblasts were stimulated to proliferate by replacement of the exhausted medium with fresh medium containing 10% fetal calf serum. The cellular content of the polyamines, putrescine, spermidine, and spermine was studied at various intervals after the nutritional change. The putrescine content increased during the pre-replicative phase of the cell cycle, whereas the content of spermidine and spermine did not increase until after the initiation of DNA synthesis. By varying the composition of the stimulating medium it was possible to alter the percentage of cells that were stimulated to proliferate. Measurement of the cellular polyamine content and 3H-thymidine ( 3H-TdR) incorporation into DNA at the time of the maximal rate of DNA synthesis showed that the magnitude of putrescine accumulation depended on the percentage of cells that were stimulated to proliferate. These results indicate that there may be a connection between polyamine synthesis and subsequent DNA replication.

Changes in the G0 state of WI-38 fibroblasts at different times after confluence

Some events in the prereplicative phase of WI-38 human diploid fibroblasts stimulated to proliferate are found to be a function of the length of time the cells have been quiescent. At 5 days after plating, when the cells first become confluent, the prereplicative phase upon stimulation by a nutritional change is relatively short, DNA synthesis begins at 8 h after stimulation, and there is no increase in chromatin template activity. At 9 days after plating the prereplicative phase of stimulated cells is lengthened to 14 h and there is an increase in chromatin template activity within 1 h of stimulation. Finally, in 18-day cells, the prereplicative phase is lengthened even further to 20 h, and there is a lag after stimulation before the increase in chromatin template activity. It is proposed that confluent WI-38 cells initially arrest in G 1, subsequently pass into G 0, and continue to go deeper into G 0 as they remain quiescent.

Utilization of pre-existing messenger RNAs for nonhistone chromosomal protein synthesis in WI-38 human diploid fibroblasts

Contact-inhibited monolayers of WI-38 human diploid fibroblasts show an increase in chromatin template activity within 1 h following stimulation to proliferate. Direct evidence that nonhistone chromosomal proteins are responsible for this increased transcriptional activity of the genome comes from previous studies which demonstrate that the template activity of chromatin reconstituted with nonhistone chromosomal proteins from WI-38 cells 1 h following stimulation is higher than the template activity of chromatin reconstituted with nonhistone chromosomal proteins from contact-inhibited cells. The present studies indicate that there is an increase in the amount of two specific newly synthesized molecular weight classes of nonhistone chromosomal proteins in chromatin 1 h following the stimulation of contact-inhibited WI-38 cells to proliferate and that the synthesis of these nonhistone chromosomal proteins occurs in the absence of new RNA synthesis. The possibility that such nonhistone chromosomal protein synthesis early during the prereplicative phase of the cell cycle may be regulated at the translational level is discussed.

Presence of two active X chromosomes in hybrids between normal human and SV40-transformed fibroblasts from patients with the Lesch-Nyhan syndrome

Skin fibroblasts (LNSV) derived from a hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient patient with the Lesch-Nyhan syndrome, who has glucose-6-phosphate dehydrogenase (G6PD) type A, were transformed with SV40 and hybridized with WI38 human diploid fibroblasts derived from a female embryo which have normal HGPRT and G6PD type B activities. The hybrid clones selected in hypoxanthine, aminopterin and thymidine (HAT) medium, were essentially tetraploid and contained three X and one Y chromosomes. These hybrids contained HGPRT, types A and B and the AB heteropolymeric form of G6PD enzymes which were indicative that in these cells X linked genes of both parental cells were fully active. Hybrids back-selected in medium containing 8-azaguanine (8-AG) contained only two X chromosomes. They had no HGPRT activity and they contained only G6PD type A enzyme. It is concluded that the hybrid cells which grew in the presence of 8-AG retained the X chromosome of the LNSV parental cell and apparently the inactive X of the WI 38 cell.

Modifications in the chromosomal proteins of SV-40 transformed WI-38 human diploid fibroblasts

Summary The composition and metabolism of chromosomal proteins associated with the genome of WI-38 human diploid fibroblasts and SV-40 transformed WI-38 fibroblasts were compared. While the five major histone fractions are present in normal and SV-40 transformed cells, variations in the relative contents and rates of synthesis of specific histone fractions are described. Differences in the protein contents and rates of synthesis of several molecular weight classes of nonhistone chromosomal proteins in normal and SV-40 transformed WI-38 cells are also reported.

Changes in the number of binding sites for ribonucleic acid polymerase in chromatin of WI-38 fibroblasts stimulated to proliferate.

1. When WI-38 human diploid fibroblasts form confluent monolayers, DNA synthesis and cell division almost completely cease. A change of medium causes these density-inhibited cells to proliferate and within 1h after the application of the stimulus there is an increase in template activity of the chromatin isolated from stimulated cells. 2. The number of binding sites for Escherichia coli RNA polymerase was determined on chromatin from WI-38 cells by two different methods, i.e. incorporation of [(3)H]UTP into RNA in the absence of reinitiation, and incorporation of [gamma-(32)P]GTP into chain termini. 3. Both methods indicate that the capacity of chromatin to bind E. coli RNA polymerase is increased in WI-38 cells stimulated to proliferate. 4. The increase in the number of binding sites for E. coli RNA polymerase parallels the increase in chromatin template activity and suggests that the latter reflects an increase in the number of initiation sites, rather than an increase in the rate of transcription.

Immunofluorescent study of chromatin proteins in cultured fibroblasts

Antibodies against chromatin from 3T6 mouse fibroblasts and WI-38 human diploid fibroblasts were prepared by immunization of rabbits. Immunofluorescent studies showed species-specificity of these antichromatin antibodies. Furthermore, using anti-3T6 chromatin antibodies against 3T6 cells and anti-WI-38 chromatin antibodies against WI-38 cells, we observed, by immuno-fluorescent techniques, granular fluorescence in the diffusely stained nucleus and diffuse fluorescence in the cytoplasm. These results raise the possibility of the presence of a cytoplasmic pool of chromatin proteins.

The Effects of Epinephrine and Prostaglandin E1 on Cyclic Adenosine 3′ : 5′-Monophosphate Levels in WI-38 Fibroblasts

Abstract 1. Intact WI-38 human diploid lung fibroblasts were shown to respond to epinephrine (3 x 10-7 m) and prostaglandin E1 (PGE1) (0.57 x 10-7 m) with increased cyclic adenosine 3' : 5'-monophosphate (cyclic AMP) levels. Corticotropin (1.25 units per ml), insulin (0.02 to 200 milliunits per ml), and glucagon (5 x 10-7 m) were ineffective. Although the response to PGE1 was not clearly maximal at concentrations up to 2.8 x 10-5 m, the response to 5.7 x 10-6 m PGE1 was greater than the maximal response to epinephrine (10 x 10-6 m) under all conditions studied. 2. Theophylline (2 mm) potentiated the action of PGE1 on cyclic AMP levels by at least 2-fold in all experiments, but was less effective with epinephrine; in several experiments theophylline did not potentiate the effects of the catecholamine. 3. Propranolol (0.1 mm) completely inhibited the effect of epinephrine but only slightly diminished that of PGE1. 4. Cells grown to high population density responded poorly or not at all to epinephrine while cells at low population densities responded very well. A converse relationship was observed between PGE1 action and population density, although it was not as dramatic. The effect of population density on the epinephrine stimulation did not appear to be due to a difference in sensitivity of the populations to epinephrine. 5. Cyclic AMP appeared in the incubation medium of WI-38 cells treated with epinephrine or PGE1. This appearance reached maximal levels more slowly than did intracellular cyclic AMP but represented a considerable amount of cyclic nucleotide.