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.     

Comparison of polypeptides from cultured human fibroblasts and sarcoma cells.

The proteins in cell layers of cultured normal diploid human skin (ES, ER) and lung (WI-38) fibroblasts were compared to those of SV40-transformed human fibroblasts (WI-38/VA-13), human rhabdomyosarcoma (RD) and fibrosarcoma (HT-1080) cells using metabolic amino acid and sugar labeling and surface labeling with tritiated sodium borohydride after oxidation with galactose oxidase. The labeled proteins were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography (fluorography). A transformation-associated decrease in the pericellular glycoprotein fibronectin (subunit molecular weight, 220 000) and in the synthesis of a set of polypeptides in the 130 000--180 000 dalton region was seen. Synthesis of a glycosylated 160 000 dalton polypeptide was markedly reduced. In transformed cells distinct increases of several specific polypeptides was detected in both [35S]methionine and [3H] mannose incorporation experiments but not using the surface labeling method.     

Role of nonhistone chromosomal proteins in determining circular dichroism spectra of chromatin.

Complexing of histone proteins, from WI-38 cells with pure DNA from WI-38 cells, causes a marked decrease in the amplitude of the positive ellipticity band and a red shift in circular dichroism spectra in the 250–300 nm region. Total nonhistone chromosomal proteins from WI-38 cells (without histones) cause an analogous effect, but of significantly reduced magnitude. However, the two effects are not additive, because, when DNA is complexed with both histones and nonhistones, the amplitude of the positive ellipticity band has an intermediate value, between the histone-DNA complex and the nonhistone-DNA complex. Removal of certain nonhistone proteins from chromatin of WI-38 cells, by extraction with 0.25–0.35 m NaCl, causes a decrease in the positive circular dichroism band in the 250–300 nm region. Removal of histones and other nonhistone proteins from chromatin by extraction with 0.75 and 1.5 m NaCl causes a strong increase in positive ellipticity. This suggests the existence of modest but definite effects of nonhistone proteins in determining DNA conformation in native chromatin. Taken as a whole, nonhistone chromosomal proteins have a weaker but analogous effect to that of histones, while the nonhistone proteins extractable with 0.25–0.35 m NaCl have an opposite effect.     

Degradation of proteins synthesized in the presence of cycloheximide in normal and transformed fibroblasts

Monolayer cultures of 3T3 and WI-38 fibroblasts were pulse labeled with radioactive leucine in the presence of cycloheximide. The rate of protein degradation was measured and compared with that of SV-40 virus transformed cells. The results clearly show that normal and transformed cells have essentially identical rates of degradation of proteins synthesized in the presence of cycloheximide. These findings indicate that the lower rates of protein degradation observed in some transformed cells is not a general rule.     

Changes in template activity and structure of nuclei from WI-38 cells in the prereplicative phase.

Quiescent confluent monolayers of WI-38 fibroblasts were stimulated to proliferate by either adding 10% fetal calf serum or by trypsinization and replating at lower density. The length of the prereplicative phase was 12 hr after serum stimulation and 18 hr after trypsinization and replating at lower density. Nuclei were isolated from WI-38 cells at different time intervals after either type of stimulation and their template activity, circular dichroism spectra, and ability to bind ethidium bromide were investigated. All these parameters were similarly increased after either type of stimulation. However, these changes, like the onset of DNA synthesis, were delayed 6 hr in cells trypsinized and replated at lower density. While there were no detectable changes in nuclear protein content after serum stimulation, at least 40% of nuclear protein, mostly nonhistone chromosomal proteins, were lost after trypsinization. The amount of nuclear proteins returned to prestimulation levels only 6-8 hr after replating. These data seem to suggest that nonhistone chromosomal proteins lost by trypsinization are essential for the entrance of WI-38 cells into the "prereplicative phase".     

Early effects of serum on phospholipid metabolism in untransformed and oncogenic virus-transformed cultured fibroblasts.

The addition of serum to previously serum-deprived 3T3 fibroblasts in culture caused a pronounced, rapid and selective stimulation of the incorporation of [³²P]phosphate into phosphatidyl inositol. Comparison of the content of radioactivity in phosphatidyl inositol after a short pulse with that obtained following a prolonged labeling period showed that serum accelerated the rate of the turnover (and not the net accumulation) of this substance. In cells transformed by SV-40 virus, the rate of labeling of phosphatidyl inositol was relatively high, and was not influenced significantly by the deprivation of serum or its resupplementation. It is suggested that the rate of phosphatidyl inositol turnover may be related to the state of the mobility of membrane constituents, and that this process escapes the control of serum factors in malignantly transformed cells.     

Effect of low extracellular Ca2+ on growth spreading area,cytoplasmic Ca2+ concentration,and intracellular pH in normal and transformed human fibroblasts

The transformation of certain cells reduces the requirement of extracellular Ca²⁺ for growth. The SV-40 transformed human lung fibroblasts, WI-38 VA13, require less Ca²⁺ than normal WI-38 cells. Spreading area of normal cells decreases when cultured in 10 μM Ca²⁺ medium. Intracellular calcium concentration ([Ca²⁺]_i), of the normal and transformed cells cultured in 10μM and 2 mM Ca²⁺ media was measured by the fluorescence microscope technique using fura-2 as a probe. The [Ca²⁺], is measured in the resting state and during mobilization by serum or bradykinin stimulation. The lowering of extracellular calcium concentration results in a decrease in the resting state [Ca²⁺],_i of both normal and transformed cells. Although the total decrease in [Ca²⁺]_i is the same for both cell, the rate of decrease is much faster in normal cells than in transformed cells. Low extracellular Ca²⁺ reduces the number of cells responsive to the serum or bradykinin stimulation and decreases the peak [Ca²⁺]_i value in both cells. In addition, we investigated, using BCECF as a fluorecent probe, the intracellular pH (pH_i) of normal and transformed cells maintained at low and normal Ca²⁺. The low Ca²⁺ condition makes pH_i acidic in normal cells but not in transformed cells. The acidification of the normal cell is accompanied by a decrease in the spreading area of the cells. The decrease of the cell attacment, followed by the reduced spreading area, induced the acidic pH_i. These results suggest that the reduced Ca²⁺ requirement of transformed cells for growth is related to the mechanism of pH_i regulation rather than Ca²⁺ homeostasis and, possibly, to the anchorage-independent growth, which is a unique feature of transformed cells. © 1993 Wiley-Liss, Inc.     

Isolation and characterization of the plasma membrane of L-1210 cells. Iodination as a marker for the plasma membrane

Mouse leukemia L-1210 cells were iodinated with ¹²⁵I; this permitted the development of a method for the isolation of the plasma membranes. These show a 10- to 16-fold increase in the specific activity of ¹²⁵I over that of the cell homogenate and a 20-fold increase in the specific activities of 5′-nucleotidase and alkaline phosphate; no mitochondrial or microsomal marker enzyme activities were detected. Sodium dodecyl sulfate gel electrophoresis of the plasma membranes shows approx. 40 peptides with molecular weights ranging from 10 000 to over 200 000; a polypeptide ($\text{M}{}_{\mathrm{<mtext>m</mtext>}}$ 50 000) predominates. Of 13 iodinated surface membrane proteins, the major radioactive peptide has a molecular weight of 85 000. The importance of the selection of the appropriate gel sytem for the analysis of membrane proteins is emphasized.     

Simian Virus 40 Gene A Regulates the Association Between a Highly Phosphorylated Protein and Chromatin and Ribosomes in Simian Virus 40-Transformed Cells

The species of proteins associated with chromatin and ribosomes of simian virus 40 (SV40)-transformed and untransformed monkey, mouse, and rat cells have been compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after phosphorylation of these proteins either in vivo or in vitro. In vitro phosphorylation was carried out by protein kinase associated with these organelles and [gamma-(32) P]ATP as the phosphoryl donor. The reaction products contained both phosphoserine and phosphothreonine in approximately equal amounts. The electrophoretic analysis of the phosphorylated proteins revealed that the highly phosphorylated protein with a molecular weight of approximately 90,000 (90K protein) was associated with chromatin and ribosomes from transformed cells but not from untransformed cells. The 90K protein could be extracted from chromatin and ribosomes with 0.5 to 1.0 M NaCl or KCl. The 90K protein was still associated with the runoff ribosomes prepared by the puromycin reaction of the post-mitochondrial supernatant in the protein-synthesizing system. In vitro phosphorylation of chromatin and ribosomes from SV40 tsA-transformed mouse and rat cells indicated that the amounts of 90K protein associated with these organelles decreased greatly when the cells were cultivated at the restrictive temperature. A similar temperature-dependent decrease in the amount of (32)P-labeled 90K protein was observed in nonhistone chromosomal and ribosome-associated protein fractions prepared from SV40 tsA-transformed cells labeled with [(3)H]leucine and [(32)P]orthophosphate in vivo. In vitro phosphorylated 90K protein in nonhistone chromosomal and ribosome-associated proteins extracted with high salt was not immunoprecipitated with anti-SV40 T sera.     

Regulation of protein degradation in normal and transformed human cells. Effects of growth state, medium composition, and viral transformation.

In WI-38, a normal human fibroblast, the rates of degradation of short lived and long lived proteins are identical whether the cultures are growing exponentially or are density-inhibited. Replacement of the growth medium with fresh medium does not alter these rates. In VA-13, an SV-40 transformed derivative of WI-38, the rates of protein degradation are also independent of growth rate and fresh medium. However, in both WI-38 and VA-13 the rate of long lived protein degradation increases as the serum concentration is reduced below 5%. After complete serum withdrawal, the rate increases by 60 to 100% in both cell types. Withdrawal of arginine and phenylalanine triples the rate of long lived protein degradation, while addition of 10% dialyzed serum to this amino acid-deficient medium reduces the effect to twice that of the controls. Incubation of both types of cells in phosphate-buffered saline also increases protein degradation. This effect is reduced by glucose, albumin, and dialyzed serum. Therefore, the rate of protein degradation is independent of growth rate in normal and transformed human cells. However, the rate of degradation is closely coupled to certain medium alterations.     

Methionine biosynthesis in normal and transformed fibroblasts.

SV-40 transformed human fibroblasts show a growth requirement for methionine, whereas normal fibroblasts do not. Activities of the N⁵-methyltetrahydrofolate-homocysteine transmethylase and N^5–10-methylenetetrahydrofolate reductase in extracts of both cell lines are similar. These observations indicate that the absolute growth requirement for methionine observed in these transformed cells does not necessarily involve a deficiency in enzymes related to methionine synthesis.     

DNA-cellulose column chromatography of phosphorylated nucleolar nonhistone proteins

Summary A salt-extraction procedure was used to isolate a nucleolar nonhistone protein fraction, containing [³²P]phosphoserine, from the nucleoli of Novikoff hepatoma ascites cells. These proteins are similar in amino-acid composition to whole nuclear (chromosomal) nonhistone proteins. DNA-cellulose column chromatography showed that this fraction contains DNA-binding phosphoproteins, some of which will bind only to homologous (Novikoff) nucleolar or nuclear DNA.     

Inducers of DNA synthesis: levels higher in transformed cells than in normal cells

The objective of this study was to determine whether transformed cells have greater DNA synthesis-inducing ability (DSIA) than normal cells when fused with G1 phase cells. HeLa cells synchronized in G1 phase, prelabeled with large latex beads, were fused separately with (a) quiescent human diploid fibroblasts (HDF), (b) HDF partially synchronized in late G1, and random populations of (c) HeLa, (d) WI-38, (e) SV-40 transformed WI-38, (f) CHO, (g) chemically transformed mouse cells (AKR-MCA), and (h) T98G human glioblastoma cells (all prelabeled with small latex beads) using UV-inactivated Sendai virus. The fusion mixture was incubated with [3H] thymidine, sampled at regular intervals, and processed for radioautography. Among the heterodikaryons, the frequency of those with a labeled and an unlabeled nuclei (L/U) were scored as a function of time after fusion. The faster the induction of DNA synthesis in HeLa G1, the steeper the drop in the L/U class and hence the higher DSIA in the S phase cells. The DSIA, which is indicative of the intracellular levels of the inducers of DNA synthesis, was the highest in HeLa and virally transformed WI-38 cells and the lowest in normal human diploid fibroblasts (HDF) while those of chemically and spontaneously transformed cells are intermediate between these two extremes. Higher level of DNA synthesis inducers appears to be one of the pleotropic effects of transformation by DNA tumor viruses. These studies also revealed that initiation of DNA synthesis per se is regulated by the presence of inducers and not by inhibitors.     

Isolation and characterization of a membrane-bound proteinase from rat liver.

The proteinase previously found in chromatin prepared from a total rat liver homogenate was purified from the rat liver mitochondrial fraction. The membrane-bound enzyme is solubilized in either 0.6% digitonin or 0.5 m phosphate buffer. After a 1330-fold purification, the enzyme appears homogeneous by acrylamide-gel electrophoresis. Sucrose density gradient centrifugation indicated a molecular weight of 22,500, a molecular weight of 23,500 ± 10% has been estimated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme showed a high substrate specificity. Among several proteins tested, only glucagon, nonhistone chromosomal proteins, and histones are good substrates. A limited proteolysis was found for the very-lysine-rich histone H1, which was split into a high molecular weight fragment (M_r 13,000). The highly phosphorylated histone H1 isolated from regenerating rat liver 24 h after partial hepatectomy exhibited the same susceptibility to the proteinase as H1 from normal liver. Large polypeptides of a nonhistone chromosomal protein fraction were degraded more rapidly than the small ones. N-Acetyl-l-tyrosine ethyl ester was used with alcohol dehydrogenase and NAD in a coupled enzyme assay for the proteinase. The apparent Michaelis constant for the hydrolysis of N-acetyl-l-tyrosine ethyl ester is 5.0 × 10^?3m. The proteinase has catalytic properties simlar to trypsin and chymotrypsin. The pH optimum was around 8, soybean trypsin inhibitor depressed the enzymatic activity, and the serine modifying reagents diisopropyl phosphofluoridate and phenylmethanesulfonyl fluoride inactivated the enzyme. The affinity reagent for chymotrypsin-like active sites, l-1-tosylamido-2-phenylethyl chloromethyl ketone, inactivated the proteinase.     

ANALYSIS OF POLYPEPTIDES ASSOCIATED WITH SHOOT FORMATION IN TOBACCO CALLUS CULTURES

Callus lines of Nicotiana tabacum were selected for competence and lack of competence in shoot formation. Changes in total and chromosomal polypeptides in these shoot-forming and nonshoot-forming tobacco cultures were examined by twodimensional polyacrylamide gel electrophoresis. Qualitative and quantitative differences in total, nonhistone chromosomal, and basic chromosomal polypeptides were evident throughout the 7-d test period. The analysis of total proteins identified polypeptides specific to shoot-forming and nonshoot-forming tissue during the 7-d sampling period. A small number of basic chromosomal proteins were found solely in shoot-forming or nonshoot-forming tissue. One basic chromosomal protein was detected in only nonshoot-forming tissue at all sampling times. Two proteins, although present in shoot-forming tissue, were present at elevated levels in the nonshoot-forming cultures. No temporal changes in basic proteins over the 7-d incubation period were observed. Qualitative differences in total nonhistone chromosomal polypeptides in the shoot-forming and nonshoot-forming tissue were also observed. Differences in chromosomal polypeptides were observed. In contrast to the basic chromosomal proteins, temporal variation in the nonhistone chromosomal polypeptides was demonstrated. Throughout the 7-d sampling period, 29 and 12 nonhistone chromosomal polypeptides varied qualitatively in shoot-forming and nonshoot-forming callus cultures, respectively. In vitro labeling with ³²P-orthophosphate indicated that approximately 1.0% and 0.3% of the nonhistone chromosomal proteins were phosphorylated in the shoot-forming and nonshoot-forming cultures. Of these phosphorylated polypeptides, one was present in nonshoot-forming tissue and three were detected only in the shoot-forming tissue. Phosphorylation occurred at serine or threonine residues.     

Changes in membrane transport function in G0 and G1 cells

Confluent quiescent monolayers of aneuploid and euploid cells in culture can be stimulated to proliferate by appropriate nutritional changes. In confluent monolayers of WI-38 human diploid fibroblasts the uptake of cycloleucine is increased three hours after these cells are stimulated to proliferate by a change of medium plus 10% serum. No changes in the uptake of cycloleucine are observed in logarithmically-growing WI-38 cells exposed to fresh medium plus 10% serum, or in WI-38 confluent monolayers in which the conditioned medium has been replaced by fresh medium with 0.3% serum (a change that does not cause stimulation of cellular proliferation in WI-38 cells). In 3T6 cells in the stationary phase stimulated to proliferate by nutritional changes, there is a prompt increase in the uptake of cycloleucine, within one hour after stimulation of cell proliferation. Similar results were obtained with stationary 2RA cells which are SV-40 transformed WI-38 fibroblasts. In addition, chromatin template activity which is known to increase in the early stages after stimulation of confluent WI-38 cells, was unchanged in confluent 3T6 or 2RA cells stimulated to proliferate. These results show that at least two of the very early biochemical events occurring in response to stimulation of cell proliferation are different in WI-38 diploid cells and in aneuploid 2RA or 3T6 cells. It is proposed that WI-38 cells in the stationary phase are arrested in the G₀ phase of the cell cycle, while 2RA and 3T6 cells are arrested in the G₁ phase.     

In vivo and in vitro phosphoarylation of nuclear proteins in rat liver.

The incorporation of 32P into nuclear nonhistone proteins was compared in rat liver in vivo, in liver slices incubated in vitro, and in isolated nuclei incubated with gamma-[32P]ATP. The highest specific activities of nuclear phosphorproteins were obtained by incubating isolated nuclei. However, the Radioactivity profiles of polyacrylamide gel electrophoretograms of these proteins differed from those obtained in vivo or in liver slice experiments. A group of low molecular weight nonhistone proteins exhibited a very high incporation of labelled phosphate. These proteins could be obtained from the interface when the phosphoproteins were isolated by the buffered phenol extraction procedure. Phosphorylated proteins were also obtained from three cytoplasmic fractions (mitochondria, microsomes, and cytosol). The specific activities of these proteins were much lower than of the nuclear phosphoproteins.