Characteristics of intracellular metabolism of procollagen and other proteins in human embryo fibroblasts in trisomy for chromosome 7

A comparative study of the relative rates of intracellular total protein metabolism in diploid and aneuploid (with trisomy for chromosome 7) human embryo fibroblasts in the logarithmic and stationary growth phases was carried out. Using double labeling with [14C]proline (24 hrs) and [3H]proline (3 hrs), it was found that: the rates of intracellular protein metabolism during transition to the stationary phase of growth are increased in diploid cells and decreased in cells with trisomy for chromosome 7; the relative rate of protein metabolism in the logarithmic phase is higher in trisomic cells than in diploid ones. The intracellular degradation of procollagen in trisomic cells is increased approximately by 17% as compared to normal fibroblasts. Treatment of cell lysates with bacterial collagenase revealed the presence of procollagen incomplete degradation products in anomalous fibroblasts. The observed differences in the rates and mode of protein metabolism during transition of diploid and trisomic fibroblasts to the stationary phase of growth suggest that the odd autosome interferes with the normal coordinated activity of genes in chromosomes.     

Changes in the level of sister chromatid exchanges in cultured Chinese hamster cells undergoing limited proliferation

"Stationary phase ageing" of cultured Chinese hamster cells (when proliferation rate decreases and in the stationary growth phase) produces an increase in the frequency of spontaneous sister chromatid exchanges (SCE). Thiophosphamide-induced (24 h) frequency of SCE increases from 2-day to 5-day "age" and later (in the stationary phase) is practically the same. The "stationary ageing" cultured cells are suggested to be used as a model system for studying molecular-genetic age changes.     

Kinetics of protein degradation in diploid and trisomic human fibroblasts

The degradation rate of long-lived and short-lived proteins was determined in diploid fibroblasts and fibroblasts with trisomy 7 derived from human embryos. Two fractions of proteins were detected in the exponentially growing diploid fibroblasts with half-lives (T 1/2) 37 and 19 hours. The rate of protein degradation increases in diploid fibroblasts as they approach confluence and protein fractions with T 1/2 30, 18 and 12 hours appear. The rate of protein degradation in trisomic fibroblasts does not change for the long-lived and short-lived proteins and is the same in both exponential (T 1/2 31 and 14 hours) and stationary phase (T 1/2 33 and 17 hours). The relative amount of the short-lived proteins in trisomic fibroblasts in the stationary phase decreased as compared with the one in diploid fibroblasts. It is apparent that a mechanism of regulation of protein catabolism in trisomic fibroblasts is impaired.     

Characteristics of DNA replication in the long-term culture of human cells at the stationary phase

Peculiarities of DNA replication in cultured human diploid fibroblasts in logarithmic and stationary phases were studied using DNA autoradiography. The rate of DNA replication fall from 30-36 mu/hour at active proliferative phase to 18-20 mu/hour at late stationary phase. This phenomenon is characteristic of stationary cultures after stimulation to proliferate by changing medium as well as by culturing without stimulation. Possible mechanisms of DNA replication rate alteration in senescent human cells are discussed.     

Expression of the neuron-specific protein, 14-3-2, and steroid sulfatase in neuroblastoma cell hybrids

Three series of neuroblastoma X fibroblast hybrid clones were isolated from crosses between mouse or human fibroblasts and mouse or human neuroblastoma cell lines by virus-mediated cell fusion. The expression of 14-3-2 protein (an acidic protein specific to neurons) and steroid sulfatase activity was studied in parental and hybrid cell lines. Steroid sulfatase was extinguished in hybrids when only one parent expressed the enzyme, but was expressed in one hybrid combination in which both parents expressed the enzyme. The neuron-specific 14-3-2 protein, on the other hand, continued to be expressed in all three series of neuroblastoma x fibroblast hybrids. In most cases where these pheno-types were expressed, they also exhibited temporal modulation; that is, specific activity is low during logarithmic growth and increases markedly during stationary phase. The glial-specific protein S-100 is absent from all parents and hybrids. The results are discussed in terms of mechanisms of regulation of differentiated phenotypes in mammalian cells.     

Changes in the number of sister chromatid exchanges in cultured Chinese hamster cells with limited proliferation. Additional studies

During "stationary phase ageing" of cultured Chinese hamster cells (B11dii-FAF28 line, 2372a clone), i. e. while decreasing the proliferation rate and in the stationary growth phase the frequency of spontaneous sister chromatid exchanges (SCE) progressively increases (from 2- to 23-day "age"); the frequency of thiophosphamide-induced (1h) SCE increases from 2- to 23-day "age" by the same value as the frequency of spontaneous SCE; the cells deepen into the R-phase of the cell cycle.     

Aging in vitro and D-glucose uptake kinetics of diploid human fibroblasts

By use of a rapid technique, initial rates of D-glucose transport were obtained during the lifespan in vitro of a commercially available strain of human embryo lung fibroblasts (Flow 2000). The apparent Km of the D-glucose carrier did not change during senescence in vitro: x̄ = 1.8 mM (range 1.3–2.3) in phase II, x̄ = 1.8 mM (range 1.5–2.2) in phase III. Transport rates remained constant in stationary phase II cultures, which had completed between 30% and 80% of their replicative lifespan. A wide variation, however, was observed in terminally differentiated cells (phase III), which showed a two- to threefold increase in average cell size and protein content. In some senescent cultures, glucose transport calculated on a per cell basis was also two-to threefold increased, while it was strongly decreased (-75%) in others. When calculated per unit of cell water, protein, and surface area, respectively, transport rates in phase III cultures ranged from values established for stationary phase II cultures down to very low values. Detaching cells flushed off from senescent cultures did not show measurable rates of glucose transport into the inulin impermeable cell space. Present evidence argues against the idea that an impairment of D-glucose transport might precede loss of replicative potential in aging human fibroblasts. Instead our data indicate that the transport capacity of cell membrane finally decreases during postreplicative senescence in terminally differentiated cells.     

Changes in the activity of enzymes involved in purine "salvage" and nucleic acid degradation during the growth of Catharanthus roseus cells in suspension culture

Changes during growth in the activity of several enzymes involved in purine "salvage", adenine phosphoribosyltransferase (EC 2.4.2.7), guanine phosphoribosyl-transferase (EC 2.4.2.8), hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) and adenosine kinase (EC 2.7.1.20), the enzymes which catalyze the conversion of nucleoside monophosphate to triphosphate, nucleoside monophosphate kinase (EC 2.7.4.4) and nucleoside diphosphate kinase (EC 2.7.4.6), and several degradation enzymes, deoxyribonucleae(s), ribonuclease(s). phosphatase(s), nucleosidase (EC 3.2.2.1), 3'-nucleotidase (EC 3.1.3.6) and 5'-nucleotidase (EC 3.1.3.5) were examined in cells of Catharanthus roseus (L.) G. Don cultured in suspension. In addition, the incorporation of [8-¹⁴C] adenine, [8-¹⁴C] adenine, [8-¹⁴C]hypoxanthine. [8-¹⁴C] adenosine and [8-¹⁴C]inosine into nucleotides and nucleic acids was also determined using intact cells.
The activities of all purine "salvage" enzymes examined and those of nucleoside monophosphate and diphosphate kinases increased rapidly during the lag phase and decreased during the following cell division and cell expansion phases. The rate of incorporation of adenine, guanine, hypoxanthine, and adenosine into nucleotides and nucleic acids was higher in the lag phase cells than during the following three phases. The highest rate of [8-¹⁴C]inosine incorporation was observed in the stationary phase cells. The activity of all degradation enzymes examined decreased when the stationary phase cells were transferred to a new medium.
These results indicated that the increased activity of purine "salvage" enzymes observed in the lag phase cells may contribute to an active purine "salvage" which is required to initiate a subsequent cell division.     

Scleral fibroblasts of the chick embryo proliferate by an autocrine mechanism in protein-free primary cultures: Differential secretion of growth factors depending on the growth state

Scleral fibroblasts of the chick embryo in primary culture proliferated in a protein-free medium. Conditioned medium (CdM) from the culture contained plural growth-promoting factors, which were active to the same cell type. The activity of one of the growth-promoting factors (SAF-I) was heat-resistant and the rest (SAF-II) were heat-sensitive. SAF-I accumulated in the CdM only during the growing phase; on the other hand, SAF-II accumulated in the CdM during the stationary phase. SAF-I showed the same time course of DNA synthesis-promoting activity as human PDGF. However, the activity of the SAF-I was not neutralized by anti-human PDGF. On the other hand, a part of the SAF-II (SAF-II a) showed a strong affinity for heparin.     

Regulation of synthesis of N-linked glycoproteins and their lipid intermediates in human T lymphoblastoid cells

The synthesis of N-linked glycoproteins and their lipid intermediates was investigated in cell-free preparations of human T lymphoblastoid cells during two phases of cell growth. The incorporation of 14C-labeled Man into glycoproteins and dolichol-linked oligosaccharides was greater during the logarithmic growth phase than the stationary phase. The incorporation of 14C-labeled GlcNAc into dolichol derivatives was increased in the logarithmic phase. However, the synthesis of Dol-P-Man was not significantly different. These data suggest that the differences are due, at least partially, to the increased synthesis of Dol-P-P-GlcNAc.     

Analysis of the kinetic growth patterns of cultured cells. III. The effect of inoculation density, of a geroprotector-antioxidant and of stationary-phase aging

With the help of a previously devised model of cultured cell growth kinetics it was shown that the "plateau" level on the growth curve of human embryo diploid fibroblasts increased, if the cells were grown with Epigid geroprotector-antioxidant (equal results with two drug concentrations: 10(-5) and 10(-7) M, resp.). It was also found that the "plateau" level on the growth curve of cultured "3 days aged" Chinese hamster cells (subcultivated to a fresh medium 3 days after a 1:6 subcultivation) lays higher than the "plateau" level of "14 days aged" cells but lower than that of "7 day aged" cells. Furthermore it was shown that an increase in inoculation density of Chinese hamster cells increased proportionately the rate of cell population growth but had a little effect on the "plateau" level of the growth curve. The data obtained are discussed in terms of some "proliferative" theories of cellular ageing.     

Collagen synthesis in growing human skin fibroblasts

Collagen and noncollagen protein synthesis in cultured human skin fibroblasts was studied in relation to different growth phases. In order to quantify collagen synthesis, we determined the release of incorporated radioactivity using purified bacterial collagenase. Collagen as well as noncollagen protein synthesis markedly decreased during fibroblast growth. On the other hand, we found a 3-fold increase in relative collagen synthesis (i.e. collagen synthesis compared to total protein synthesis) comparing cells in the log growth phase with cells in the stationary growth phase.     

Protein SH-group concentration and RNA synthesis during the process of induction of proliferation in the stationary phase of cell culture growth]

The dynamics of intracellular protein SH-group (PSH) content was studied cytochemically in the course of stimulation of cell proliferation in stationary cultures of an established Chinese hamster cell line and of human diploid embryo fibroblasts. The results were compared with the pattern of RNA synthesis during the prereplicative period. In Chinese hamster cells immediately after medium changing in stationary cultures there is an augmentation of PSH content in parallel withe the increase in RNA synthesis rate. Later on, the rate of RNA synthesis and PSH content are seen decreasing followed by a new increase in the rate of RNA synthesis correlated with the second rise in PSH content. In stationary cultures of human diploid fibroblasts, there is also an increase in the rate of RNA synthesis and in the content of SH after medium changing, but the second wave of RNA synthesis and the second rise in PSH content are not pronounced. The variation in PSH content reflects the shift in the cell metabolism during the prereplicative period and is not attributed to changes in cell protein content.     

Does capacity of DNA replication change during in vitro ageing?

We described elsewhere how a lack of change in the rate of DNA chain elongation occurred during in vitro ageing of human diploid fibroblasts. Here we further examined the rate of actual incorporation of tritiated thymidine, the center-to-center distance of replicons and the length of each phase of the cell cycle in order to extend our previous results to the other aspects of DNA replication. The results obtained showed that the rate of net DNA synthesis, the replicon size and the duration of S phase did not change during in vitro ageing. Our findings indicated that the reason why the greater part of the cell population at high population doubling levels becomes incapable of proliferating might not be the gradual decline in the ability of DNA replication. The regulation system(s) of DNA replication may alter during the period of culturing without any change in the capacities of the DNA replication machinery and, consequently, the non-cycling cells increase.     

Atmospheric oxygen accelerates the induction of a post-mitotic phenotype in human dermal fibroblasts: the key protective role of glutathione

It has been proposed that ageing of human dermal fibroblasts occurs as a multi-stage process during which cells progress from a mitotic to a post-mitotic state. We describe the development of a simple and novel cell-cloning model for identifying and quantifying the different fibroblast morphotypes associated with the induction of post mitotic behaviour. We have found that under atmospheric (20%) oxygen tension a significant proportion of human dermal fibroblasts are rapidly induced to switch from a mitotic to a post-mitotic phenotype. In contrast, under more physiological (4%) oxygen conditions, the induction of a post-mitotic phenotype is largely prevented. Increasing oxidative stress by addition of hydrogen peroxide or depletion of glutathione also induced a switch from a mitotic to a post-mitotic phenotype in these cells, whereas addition of the anti-oxidant N-acetylcysteine under atmospheric (20%) oxygen tension potently inhibited this process. In addition, a statistically significant correlation was observed between the magnitude of intracellular glutathione depletion and the reduction in the population of mitotic cells in this model. We propose that the switch from a mitotic to a post-mitotic phenotype represents a process of cellular ageing and that standard atmospheric oxygen tension imposes a substantial oxidative stress on dermal fibroblasts which accelerates this process in culture. The data also suggest that intracellular glutathione levels strongly influence the induction of a post-mitotic phenotype and that, by implication, depletion of glutathione may play a significant role in the progression of cellular ageing in human skin.     

Biosynthesis of isoprenoids in intact cells of Escherichia coli

Upon rehydration of lyophilized Escherichia coli cells with phosphate buffer containing [14C]isopentenyl pyrophosphate (IPP), 14C was incorporated into the cells. Radioactivity was found in ubiquinone-8, an unidentified precursor of ubiquinone-8, demethylmenaquinone-8 and phosphate esters of all-trans-octaprenol and cis, trans-polyprenols. On rehydration of the cells with the buffer containing geranyl pyrophosphate or farnesyl pyrophosphate in combination with [14C]IPP, higher radioactivity was incorporated into the above products and some radioactivity was found in free prenols. Fractionation of the 14C-labeled cells by sucrose-density gradient centrifugation before and after recultivation indicated that the size of 14C-labeled cells had changed during the recultivation. This shows that radioactivity of [14C]IPP was incorporated into live cells but not into dead cells. The metabolism of the radioactive products in the recultivated cells was examined. It was found that the unidentified precursor was converted to ubiquinone-8, but demethylmenaquinone-8 was not converted to menaquinone-8. "Lipid intermediates" in peptidoglycan synthesis increased in the logarithmic growth phase and decreased in the stationary phase. In the stationary phase, however, an increase in cis,trans-polyprenyl monophosphates was observed. These observations suggest the operation of the lipid cycle of peptidoglycan synthesis.     

The use of Ultroser G as a serum substitute in the culture of human skin fibroblasts

Growth of human skin fibroblasts was dramatically enhanced when serum in the culture medium was replaced by Ultroser G. Compared to the same cells cultured in the presence of serum, alterations in glucose and lipid metabolism and an increase in the activity of prolidase (EC 3.4.13.9) and prolinase (EC 3.4.13.8) were also observed. Consequently, we advise extreme caution in the use of Ultroser G in metabolic studies, especially for periods of culture exceeding 10 days. However, Ultroser G can help to produce a large number of cells and so facilitate purification of the proteins produced during the stationary growth phase.     

Reciprocal changes in the levels of functionally related folate enzymes during the culture cycle in human fibroblasts

The activities of 6 folate enzymes were measured in extracts of human diploid skin fibroblasts during the lag, log and stationary phases of the culture cycle. The levels of 4 folate enzymes involved in nucleic acid biosynthesis, $\text{viz.}$, folate reductase, serine hydroxymethyltransferase, thymidylate synthetase and 10-formyl-THF synthetase, increased from 2–20 fold during the log phase of growth. In contrast, the levels of 2 enzymes, $\text{viz.}$, methylene-THF reductase and 5-methyl-THF: homocysteine methyltransferase, involved in regulating the levels of 5-methyl-THF, the major tissue and serum folate compound, decreased 3–4 fold during log growth, returning to high levels again only after the cells had been in the stationary phase for 5 and 20 days respectively. This reciprocal pattern of change is consistent with the known or postulated functions of these folate enzymes.     

Methylation of the Cellular Lipid of Methionine-requiring Agrobacterium tumefaciens

Mutants of Agrobacterium tumefaciens requiring methionine for growth on a solid basal medium were induced by the use of N-methyl-N'-nitro-N-nitrosoguanidine. In addition to the difference of mutant strains, the extent of methionine dependency differed in a liquid basal medium and in the presence of aspartate or fumarate. When ((14)C-methyl)-methionine was added to strain WM-11 growing in a prescribed basal medium, incorporation of (14)C into the cellular "residue" fraction and polar "N-methylated" lipid fraction depended strictly on cellular growth and on external methionine concentration. However, a net synthesis of the "cyclopropane" fatty acid fraction occurred even during the maximal stationary phase if excess methionine was present.     

Stimulation of sugar uptake in cultured fibroblasts by epidermal growth factor (EGF) and EGF-binding arginine esterase.

Mouse epidermal growth factor causes a rapid increase in 2-deoxyglucose uptake in stationary phase mouse (3T3) cells or human fibroblasts. Maximum effect is approximately two fold over control levels for 3T3 cells and about 50% over controls for human fibroblasts. Maximum effect on 3T3 cells is seen about two hours after addition of 10 ng/ml EGF to the culture medium. Stimulation is easily measureable within the first fifteen minutes after addition of the hormone and may be detected at hormone concentrations as low as 0.1 ng/ml. The EGF-binding arginine esterase found associated with EGF in the mouse submaxillary gland causes an enhancement of the EGF effect. In serum-free medium, the EGF effect is still readily observed, but no enhancement by the esterase is seen. SV40 virus-transformed 3T3 cells show no effect on deoxyglucose uptake after addition of 10 ng/ml EGF to the culture medium, but a response may be demonstrated after these cells are incubated for 12 hours or more in serumless medium. EFG stimulates transport of 3-O-methylglucose in stationary phase 3T3 and human fibroblasts but no EGF stimulation of alpha-amino-isobutyrate uptake in 3T3 cells is seen under conditions is reproted to inhibit intracellular degradation of human EGF by human fibroblasts, does not diminish the EGF effect on deoxyglucose uptake in human fibroblasts.