Changes of serum-induced ornithine decarboxylase activity and putrescine content during aging of IMR-90 human diploid fibroblasts

The roles of ornithine decarboxylase (ODC, EC 4.1.1.17) and polyamines in cellular aging were investigated by examining serum-induced changes of these parameters in quiescent IMR-90 human diploid fibroblasts as a function of their population doubling level (PDL) and in human progeria fibroblasts. Serum stimulation caused increases of ODC and DNA synthesis in IMR-90 human diploid fibroblasts, with maximal values occurring, respectively, 10 hr and 22 hr after serum stimulation. Both serum-induced ODC activity and DNA synthesis in IMR-90 cells were found to be inversely related to their PDL. Maximal ODC activity and DNA synthesis in young cells (PDL = approximately 18-22) were, respectively, five-fold and six-fold greater than that in old cells (PDL = approximately 50-55), which in turn were comparable or slightly higher than that in progeria fibroblasts. Polyamine contents (putrescine, spermidine, and spermine) in quiescent IMR-90 cells did not show significant PDL-dependency. The putrescine and spermine contents in quiescent progeria cells were comparable to those in quiescent IMR-90 cells. The spermidine content in quiescent progeria cells, however, was extremely low, less than half of that in quiescent IMR-90 cells. Serum stimulation caused a marked increase in putrescine content in young cells but not in old cells or in progeria cells. The spermidine and the spermine content in IMR-90 cells, either young or old, and in progeria cells did not change significantly after serum stimulation. Our study indicated that aging of IMR-90 human diploid fibroblasts was accompanied by specific changes of polyamine metabolism, namely, the serum-induced ODC activity and putrescine accumulation. These changes were also observed in progeria fibroblasts derived from patients with Hutchinson-Gilford syndrome.     

Changes in histone H3 composition and synthesis pattern during lymphocyte activation

Freshly isolated human lymphocytes were found to synthesize histones at a significant rate even though no DNA was being synthesized. The synthesis pattern of histone variants in resting lymphocytes is similar to that found in other quiescent cells and different from that found in S-phase cells. For this reason, the histone synthesis in resting lymphocytes cannot be attributed to contamination by S-phase cells. Stimulation by the mitogen phytohemagglutinin resulted in a dramatic switch in the histone H3 variant synthesis pattern as well as a readily apparent change in the histone H3 mass pattern. Thus, the chromatin of activated lymphocytes has a different histone H3 variant composition than resting or quiescent lymphocytes. It is suggested that the proportion of H3.3 in the mass pattern of the chromatin of a cell may be related solely to how long that cell has been quiescent. Inducing resting lymphocytes to synthesize DNA by UV irradiation did not qualitatively change the histone variant synthesis pattern. No S-phase H3 variants were induced by the repair process. Furthermore, the quantity of histone synthesized neither increased nor decreased after treatment with UV light.     

Specific alterations in the pattern of histone-3 synthesis during conversion of human leukemic cells to terminally differentiated cells in culture

The presence of nano- to micromolar concentrations of 12-0-tetradecanoyl-phorbol-13-acetate (TPA) in suspension cultures of human promyelocytic leukemia cells, HL-60, or human monocytic leukemia cells, THP-1, resulted in the appearance of macrophage-like cells attached to the substratum. The terminally TPA-differentiated cells continued to synthesize histones at a low rate even though DNA replication had ceased. The pattern of synthesis of histone variants in differentiated cells differed from that in undifferentiated cells and resembled that of quiescent or density-arrested cells. In undifferentiated cells, all three histone-H3 variants are synthesized, while in quiescent cells, only the H3.3 variant is synthesized. When TPA-differentiated macrophages were placed in normal medium, the pattern of histone synthesis was not altered, thus substantiating previous findings that the differentiation is irreversible. Further, TPA-differentiated macrophages and macrophages isolated from a normal human donor exhibited identical pattern of histone synthesis. Altogether, the results indicate that changes in the synthetic rates of histones during the TPA-induced maturation of human leukemic cells is not directly due to TPA or terminal cell differentiation per se but is due to the cessation of cell proliferation and DNA replication.     

Coordinate regulation of histone mRNAs during growth and differentiation of rat myoblasts

To determine whether histone genes are coordinately regulated, histone mRNA concentrations were measured in exponentially growing L6 myoblasts, S-phase synchronized myoblasts and in differentiating myoblasts. The levels of various histone mRNA subspecies declined rapidly and coordinately once myoblasts were given the signal to differentiate. mRNA levels were reduced on average to 1-5% of the amount observed in exponentially growing cells by 48 h after the signal to differentiate. The reductions occurred in concert with the cessation of DNA synthesis as the cells differentiated. Inhibition of DNA synthesis by treating myoblasts with Ara-C or hydroxyurea resulted in a histone mRNA half-life of 10-13 min for each of the histones examined. One example of non-coordinate regulation was observed however among the H4 mRNA subspecies in S-phase synchronized cells. The levels of two major subspecies of H4 mRNA increased coordinately in S-phase compared to levels observed in cells growing exponentially. A third subspecies of H4 mRNA on the other hand was found to decline by 50%. These studies suggest that the majority of histone mRNA subspecies are under coordinate control, although one exception has been noted among the subspecies of histone H4.     

Quiescence in 9L cells and correlation with radiosensitivity and PLD repair

The onset of quiescence, changes in X-ray sensitivity, and changes in capacity for potentially lethal damage (PLD) repair of unfed plateau-phase 9L44 cell cultures have been systematically investigated. The quiescent plateau phase in 9L cells was the result of nutrient deprivation and was not a cell contact effect. Eighty-five to 90% of the plateau-phase cells had a G1 DNA content and a growth fraction less than or equal to 0.15. The cell kinetic shifts in the population were temporally correlated with a developing radioresistance, which was characterized by a larger shoulder in the survival curve of the quiescent cells (Dq = 5.71 Gy) versus exponentially growing cells (Dq = 4.48 Gy). When the quiescent plateau-phase cells were refed, an increase in radiosensitivity resulted which approached that of exponentially growing 9L cells. Delayed plating experiments after irradiation of exponentially growing cells, quiescent plateau-phase cells, and synchronized early to mid-G1-phase cells indicated that while significant PLD repair was evident in all three populations, the quiescent 9L cells had a higher PLD repair capacity. Although data for immediate plating indicated that 9L cells may enter quiescence in the relatively radioresistant mid-G1 phase, the enhanced PLD repair capacity of quiescent cells cannot be explained by redistribution into G1 phase. When the unfed quiescent plateau-phase 9L cells were stimulated to reenter the cell cycle by replating into fresh medium, the first G1 was extended by 6 h compared with the G1 of exponentially growing or refed plateau-phase 9L cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deoxyribonucleic Acid Synthesis During the Division Cycle of Escherichia coli: a Comparison of Strains B/r, K-12, 15, and 15T− Under Conditions of Slow Growth

The rates of deoxyribonucleic acid (DNA) synthesis during the division cycles of the Escherichia coli strains B/r, K-12 3000, 15T(-), and 15 have been measured in synchronous cultures, under several conditions of slow growth. These synchronous cultures were obtained by sucrose gradient centrifugation of exponentially growing cultures, after which the smallest cells were removed from the gradient and allowed to grow. Sucrose gradient centrifugation did not adversely affect the cell cycle, since an experiment in which an exponentially growing culture was pulsed with [(3)H]thymidine prior to the periodic separation and assay of the smallest cells resulted in the same conclusions, as given below. In the strains of E. coli that were studied, a decreased rate of [(3)H]thymidine incorporation was seen late in the cell cycle, prior to cell division. No decrease in the rate of [(3)H]thymidine incorporation was seen at or near the beginning of the cell cycle. Thus, all these strains appear to regulate DNA synthesis in a similar fashion during slow growth. In addition, a correlation between the appearance of cells with visible cross-walls and the start of a new round of DNA synthesis was seen, indicating that these two events might be related.     

Centriole ciliation and cell cycle variability during G1 phase of BALB/c 3T3 cells

Although variability in the duration of the cell cycle is thought to reflect growth-regulatory processes that control cell cycle progression, the precise timing of the variable period within the G1 phase of the cell cycle has not been defined. In particular, the timing of cell cycle variability in relation to the cell's commitment (R point) to the initiation of DNA synthesis remains controversial. In order to investigate cell cycle variability, indirect immunofluorescence was used to measure the formation of the primary cilium as a possible marker of G1 events in both stimulated quiescent and exponentially growing cells. The primary cilium, an internal "9 + 0" nonmotile structure formed by one of the interphase centrioles, was first detected in postmitotic BALB/c 3T3 cells 5 hr before the initiation of DNA synthesis, an interval similar to that for the reassembly of the primary cilium in serum-stimulated quiescent fibroblasts. This similarity in the timing of ciliation suggests that serum-stimulated quiescent cells reenter the cell cycle in early G1 and recapitulate much of G1. Moreover, the rate of cilia formation in both postmitotic and serum-stimulated quiescent cells was identical to the rate of DNA synthesis initiation. Thus, cell cycle variability occurs before ciliation in both stimulated quiescent and exponentially growing cells. Furthermore, since ciliation also precedes the R point, variability in the centriole cycle occurs before the R point and thus may reflect processes controlling the cell's commitment to the initiation of DNA synthesis.     

Histone gene expression and chromatin structure in mammalian cell hybrids

DNA isolated from mammalian cell nuclear reveals discrete size patterns when partially digested with micrococcal nuclease. The DNA repeat lengths from different tissues within a species or from different species may vary. These differences have been attributed to the presence of different species of histone H1. To examine the nature of regulation of DNA repeat lengths and their possible relationship to histone H1, we have selected several mouse and human cell lines that differ in their DNA repeat lengths and examined them and their cell hybrids. 24 mouse X human and five mouse X mouse hybrid cell lines were analyzed. All the interspecific hybrids exhibited the repeat pattern characteristic of the murine parent. The mouse intraspecific hybrids had a repeat pattern of only one of the parents. We conclude that the partial human chromosome complements retained in the hybrids assume the repeat lengths exhibited by the mouse cells. Because H1 histones have been implicated in the determination of DNA repeat lengths, we also investigated the regulation of H1 histone expression in these cell hybrids. Purified H1 histones were radioactively labeled in vitro, and individual subfractions were subjected to proteolysis followed by gel electrophoresis. The resulting partial peptide maps off H1 histone subfractions A and B were distinguishable from one another and from different cell lines. In the mouse X human hybrids analyzed, only the mouse H1 histones were detected. These observations were extended to H2b by analysis of the hybrid cell histone by Triton-acid-urea gels. Neither the DNA repeat length nor histone expression is affected by the presence of any specific human chromosome. The fact that human genes are expressed in these hybrids suggests that the H1 histones of one species is able to interact with the chromatin of another species in a biologically funtional conformation. Analysis of the intraspecific PG19 X B82 (mouse X mouse) hybrids reveals the presence of H1 histone subfractions of the B82 mouse cells. Because these hybrids exhibit the nucleosome repeat length only of the PG19 cells, it appears that if histone H1 plays a role in determining the repeat length it does so in consort with other nonhistone chromosomal proteins.     

Dexyribonucleic acid polymerases of BHK-21/C13cells. Relationship to the physiological state of the cells, and to synchronous indution of synthesis of deoxyribonuleic acid.

BHK-21/C13 cells were grown in culture under conditions that provided exponentially growing cells and quiescent cells, by modifying the concentration of serum in the growth medium. The high-molecular-weight DNA polymerase (DNA polymerase I) from exponentially growing cells accounted for 90% of the total polymerase activity; the low-molecular-weight DNA polymerase (DNA polymerase II) accounted for the remaining 10%. In quiescent cells, DNA polymerase I contributed only 39% of the total polymerase activity and DNA polymerase II 61%. The total amount of DNA polymerase I in exponentially growing cells was 11.3-fold greater than that in quiescent cells, whereas the amount of DNA polymerase II appeared to be relatively independent of the physiological state of the cells. In an extension of these experiments, cells in a quiescent state (Go cells) were stimulated by the 'serum-step-up' method of Burk (1970) to grow and to enter a synchronous wave of DNA synthesis (S-phase cells), 87% of the cells synthesizing DNA at 20 h after the 'serum-step-up'. During the synchrony experiment, the total cytoplasmic and total nuclear DNA polymerase activities each increased about 4-fold in parallel with the increase in the rate of DNA synthesis. Cytoplasmic polymerase activity was always greater than nuclear polymerase activity. The increases observed were maximal at 20 h after 'serum step-up'. By 26 h, there was a decrease in enzyme activity (8% for cytoplasmic polymerase and 16% for nuclear polymerase, both relative to the maximum at 20 h), but the rate of DNA synthesis had declined by 37% relative to the maximum at 20 h. In Go cells, DNA polymerase II (mol.wt. 46000 +/- 4000) was the predominant species, there being twice as much of it as of the total DNA polymerase I. In these cells there was little DNA polymerase IC and ID; the amounts of IA (mol.wt. 900 times 10(3)-1100 times 10(3)) and IB (mol.wt. 460 times 10(3)-560 times 10(3)) were about equal but small.     

Cytomatrix synthesis in MDCK epithelial cells

Detailed information regarding the synthesis rates of individual protein components is important in understanding the assembly and dynamics of the cytoskeletal matrix of eukaryotic cells. As an approach to this topic, the dual isotope technique of Clark and Zak (J. Biol. Chem., 256:4863-4870, 1981), was employed to measure fractional synthesis rates (FSRs) in growing and quiescent cultures of MDCK epithelial cells. Cell protein was labeled to equilibrium with [14C]leucine over several days and then pulse-labeled for 4 hours with [3H]leucine. FSRs (as percent per hour) were calculated from the 3H/14C ratio of cell extracts or individual proteins separated by two-dimensional polyacrylamide gel electrophoresis and the 3H/14C ratio of free leucine in the medium. Synthesis of total cell protein rose from approximately 1.4%/hour in quiescent cells to 3.5%/hour in the growing cultures. The latter rate was sufficient to account for the rate of protein accumulation and a low level of turnover in the growing cultures. The FSR of the buffered-Triton soluble extract was higher and the cytoskeletal FSR significantly lower than that for total protein in quiescent monolayers. This difference, however, was not observed in growing cultures. A distinct pattern of differences was seen in the FSRs of individual cytoskeletal proteins in the quiescent cultures. Vimentin synthesis was significantly lower than that of the keratins and the keratin FSRs were not obviously matched in pairwise fashion. Unexpectedly, the FSRs of alpha- and beta-tubulin diverged in quiescent cells with alpha-tubulin turnover exceeding beta-tubulin. Likewise, components of the microfilament lattice showed unequal fractional synthesis rates, myosin and alpha-actinin being faster than actin. In addition, the FSR for globular actin exceeded that of the cytoskeletal associated form. The results suggest that metabolic coupling between individual cellular filament systems is not strict. The data are, however, consistent with models that predict that assembly of a subcellular structure influences the turnover of its component proteins.     

Thymidine triphosphate synthesis in senescent WI38 cells : Relationship to loss of replicative capacity

The effect of in vitro age on thymidine triphosphate (TTP) synthesis was assessed in WI38 cultures according to the following measurements: (1) thymidine kinase activity of broken cell preparations; (2) in situ incorporation of [3H]thymidine into acid-soluble material; and (3) total intracellular TTP content as determined by an enzymatic assay. All three parameters were maximal in exponentially proliferating populations and minimal in quiescent monolayers; no significant differences between young and old cultures were observed despite the reduced replicative capacity of the latter. The addition of serum to density-arrested cultures induced both TTP synthesis and DNA replication after a lag of approx. 12 h; although a greater percentage of young cells initiated replication as compared with old, pool sizes expanded to a similar extent in both populations. Pool expansion did not require entry into S phase; the pool sizes of control and cytosyl arabinoside-treated cultures were comparable. These findings suggest that senescent cells retain the ability to synthesize TTP, even though they are incapable of replicating DNA. Because TTP synthesis is a cell cycle-dependent event that normally begins in late G1, senescent cells might be blocked in the latter portion of the prereplicative phase and not in G0 as are quiescent cells.     

K12-biotinylated histone H4 is enriched in telomeric repeats from human lung IMR-90 fibroblasts

Covalent modifications of histones play a role in regulating telomere attrition and cellular senescence. Biotinylation of lysine (K) residues in histones, mediated by holocarboxylase synthetase (HCS), is a novel diet-dependent mechanism to regulate chromatin structure and gene expression. We have previously shown that biotinylation of K12 in histone H4 (H4K12bio) is a marker for heterochromatin and is enriched in pericentromeric alpha satellite repeats. Here, we hypothesized that H4K12bio is also enriched in telomeres. We used human IMR-90 lung fibroblasts and immortalized IMR-90 cells overexpressing human telomerase (hTERT) in order to examine histone biotinylation in young and senescent cells. Our studies suggest that one out of three histone H4 molecules in telomeres is biotinylated at K12 in hTERT cells. The abundance of H4K12bio in telomeres decreased by 42% during telomere attrition in senescent IMR-90 cells; overexpression of telomerase prevented the loss of H4K12bio. Possible confounders such as decreased expression of HCS and biotin transporters were formally excluded in this study. Collectively, these data suggest that H4K12bio is enriched in telomeric repeats and represents a novel epigenetic mark for cell senescence.     

H2A.X. a histone isoprotein with a conserved C-terminal sequence, is encoded by a novel mRNA with both DNA replication type and polyA 3'' processing signals.

A full length cDNA clone that directs the in vitro synthesis of human histone H2A isoprotein H2A.X has been isolated and sequenced. H2A.X contains 142 amino acid residues, 13 more than human H2A.1. The sequence of the first 120 residues of H2A.X is almost identical to that of human H2A.1. The sequence of the carboxy-terminal 22 residues of H2A.X is unrelated to any known sequence in vertebrate histone H2A; however, it contains a sequence homologous with those of several lower eukaryotes. This homology centers on the carboxy-terminal tetrapeptide which in H2A.X is SerGlnGluTyr. Homologous sequences are found in H2As of three types of yeasts, in Tetrahymena and Drosophila. Seven of the nine carboxy-terminal amino acids of H2A.X are identical with those of S. cerevisiae H2A.1. It is suggested that this H2A carboxy-terminal motif may be present in all eukaryotes. The H2A.X cDNA is 1585 bases long followed by a polyA tail. There are 73 nucleotides in the 5' UTR, 432 in the coding region, and 1080 in the 3' UTR. Even though H2A.X is considered a basal histone, being synthesized in G1 as well as in S-phase, and its mRNA contains polyA addition motifs and a polyA tail, its mRNA also contains the conserved stem-loop and U7 binding sequences involved in the processing and stability of replication type histone mRNAs. Two forms of H2A.X mRNA, consistent with the two sets of processing signals were found in proliferating cell cultures. One, about 1600 bases long, contains polyA; the other, about 575 bases long, lacks polyA. The short form behaves as a replication type histone mRNA, decreasing in amount when cell cultures are incubated with inhibitors of DNA synthesis, while the longer behaves as a basal type histone mRNA.     

Low molecular weight mitogenic factor produced by BRL-3A cultured rat liver cells

A new mitogenic factor has been isolated from medium conditioned by BRL-3A rat liver cells. The factor has been partially purified by a two step procedure involving ion exchange chromatography on Dowex 50 followed by gel filtration chromatography on Sephadex G-75 in 1 M acetic acid. The factor is eluted from the Sephadex G-75 column in the low molecular weight region, behin three peaks of multiplication stimulating activity. The factor is inactivated by treatment with trypsin and dithiothreitol, suggesting that it is a peptide that contains a disulfide linkage. Unlike multiplication stimulating activity, the new factor only weakly stimulates DNA synthesis in quiescent chick fibroblasts, whereas it strongly stimulates DNA synthesis in quiescent NIL8 hamster cells, $\text{BALB}\text{c}$ 3T3 cells, and IMR-90 human fibroblasts.     

Different quiescence states of three culture cell lines detected by acridine orange staining of cellular RNA

Three cell lines (CHO, L-929, and R1H) were investigated for their growth kinetics and the difference of exponential and quiescent state of monolayers in medium with and without serum (L-929). The noncycling populations of L-929 and R1H in medium with serum contained increased G1-phase percentages but also considerable proportions of SQ and G2Q cells. Although about 90% of the cells excluded trypan blue, the viability tested by colony assay was clearly lower than for exponentially growing cultures. CHO cells showed similar fractions of cells in G1-, S-, and G2-Q compartments but in addition considerable cell loss. The RNA content of these cells was reduced in plateau phase by 7-48% depending on cell type and residence time in the noncycling state. The data suggest that the cells suffered from nutrition depletion and were arrested in all phases of the cycle. In contrast, L-929 cells in medium without serum reduced their RNA content down to one-third that of proliferating cells and still retained the full viability as shown by the same plating efficiency in a colony assay. Since about 90% of the cells had G1 DNA content, these cells resemble true G1Q or G0 cells controlled by growth factors rather than nutritional depletion.     

Arginine deprivation in KB cells: I. Effect on cell cycle progress

When exponentially growing KB cells were deprived of arginine, cell multiplication ceased after 12 h but viability was maintained throughout the experimental period (42-48 h). Although tritiated thymidine ([(3)H]TdR) incorporation into acid-insoluble material declined to 5 percent of the initial rate, the fraction of cells engaged in DNA synthesis, determined by autoradiography, remained constant throughout the starvation period and approximately equal to the synthesizing fraction in exponentially growing controls (40 percent). Continous [(3)H]TdR-labeling indicated that 80 percent of the arginine-starved cells incorporated (3)H at some time during a 48-h deprivation period. Thus, some cells ceased DNA synthesis, whereas some initially nonsynthesizing cells initiated DNA synthesis during starvation. Flow microfluorometric profiles of distribution of cellular DNA contents at the end of the starvation period indicated that essentially no cells had a 4c or G2 complement. If arginine was restored after 30 h of starvation, cultures resumed active, largely asynchronous division after a 16-h lag. Autoradiographs of metaphase figures from cultures continuously labeled with [(3)H]TdR after restoration indicated that all cells in the culture underwent DNA synthesis before dividing. It was concluded that the majority of cells in arginine-starved cultures are arrested in neither a normal G1 nor G2. It is proposed that for an exponential culture, i.e. from most positions in the cell cycle, inhibition of cell growth after arginine with withdrawal centers on the ability of cells to complete replication of their DNA.     

Galectin-3 Stimulates Cell Proliferation

Galectin-3, a β-galactoside-binding protein, has been shown to be involved in multiple biological processes through interaction with its complementary glycoconjugates. Here we provide the first evidence of galectin-3 as a mitogen. Incubation of quiescent cultures of normal human lung fibroblast IMR-90 cells with recombinant galectin-3 (rgalectin-3) stimulated DNA synthesis as well as cell proliferation in a dose-dependent manner. This mitogenic activity was dependent on the lectin property of galectin-3, as it could be significantly inhibited by lactose, a disaccharide competitive for carbohydrate-binding by galectin-3. Chemical cross-linking and affinity-purification experiments identified binding of rgalectin-3 to cell surface glycoproteins, which were not recognized by antibodies directed against lysosome-associated membrane proteins (LAMPs), putative cellular ligands for galectin-3. Moreover, pulse–chase analysis revealed no secretion of galectin-3 by IMR-90 cells. These results indicate that galectin-3 is a mitogen capable of stimulating fibroblast cell proliferation in a paracrine fashion through interaction with cell surface glycoconjugates different from LAMPs and suggest a possible involvement of galectin-3 in tissue remodeling.     

Regulation of nucleosomal core histone variant levels in differentiating murine erythroleukemia cells

During hexamethylenebis(acetamide)-induced terminal differentiation of murine erythroleukemia (MEL) cells in vitro, the histone variant proportions undergo changes similar to those observed in vivo in terminally differentiating cells of the young mouse. Thus, there is a rapid increase in the relative amounts of the variants H2A.1 and H2B.2 in parallel with the increase in the number of hemoglobin-producing cells and the sharp decrease in the growth rate. We show that the changes in variant proportions are not associated with slower growth per se but are most likely due to differential changes in the rates of variant synthesis as a result of commitment to terminal differentiation. In addition, we observed an inducer-specific increase in the rate of synthesis and the relative amount of the minor H2A variant 4, well before hemoglobin accumulation. We also present evidence that H2A and H2B histones are synthesized and incorporated into chromatin at a significant rate even when DNA synthesis is inhibited, suggesting turnover of these histones. H2A and H2B turnover can be detected directly even in exponentially growing cells. H2A.1 and H2B.2 have higher turnover rates than H2A.2 and H2B.1, respectively, in exponentially growing cells, a difference which is even more pronounced in induced cells. The magnitude of the differential turnover is not sufficient to account for the changes in the histone variant proportions in the short life of induced MEL cells but could explain the slow accumulation of H2A.2, H2B.1, and H3.3 in nondividing adult tissues of the mouse.