Reinitiation of host DNA synthesis in senescent human diploid cells by infection with simian virus 40

Human diploid fibroblasts, TIG-1, cease to proliferate at about 60-62 population doubling level. In their senescent state used in this study, the percentage of nuclei labeled by [3H]thymidine for 48 h was around 1-2% in fresh medium containing 5-40% fetal bovine serum. The percentage of labelled nuclei increased up to 10-fold after infection with SV40. This increase reflects stimulation of cell DNA synthesis because: 1. The increase also occurred when ts A900 was used for infection at the non-permissive temperature, under these conditions viral DNA synthesis is inhibited; 2, the increase paralleled the stimulation of [3H]thymidine incorporation into DNA in a Hirt-precipitate fraction from SV40-infected cells. UV-irradiated SV40 had reduced ability to induce DNA synthesis. A viable deletion mutant of SV40, d1940, had almost the same activity to induce cell DNA synthesis as did wild-type SV40. Equilibrium density gradient centrifugation analysis of DNA labelled with 5-bromodeoxyuridine (BrdU) supported semiconservative replication rather than repair synthesis. We conclude that a considerable fraction of human diploid cells in a senescent population initiate host DNA replication by infection with SV40, although these cells cannot be stimulated with fetal bovine serum.     

Inhibition of DNA synthesis in proliferating human diploid fibroblasts by fusion with senescent cytoplasts

Cytoplasts were prepared from senescent human diploid fibroblasts. The cytoplasts were fused to young human diploid fibroblasts and DNA synthesis was analyzed in the fusion products. DNA synthesis was inhibited (greater than or equal to 40%) in the senescent cytoplast fusion products when compared to unfused young cells or young cytoplasts fused with young cells. These results are consistent with previous experiments that have shown the blockage of DNA synthesis in both nuclei of heterokaryons from fusions of senescent and young human diploid fibroblast cells. Furthermore, these results support the postulate that senescent cells synthesize a specific substance(s), which is present in the cytoplasm of the senescent cell that inhibits DNA synthesis.     

Correlation between the presence of T-antigen and the reinitiation of host DNA synthesis in senescent human diploid fibroblasts after SV40 infection

Senescent human diploid fibroblasts, TIG-1, had labelling indices of about 0.5-3% when labelled with [3H]thymidine for 3 days in fresh medium containing 10% fetal bovine serum. When these cells were infected with SV40, the percentage of nuclei incorporating [3H]thymidine increased by about 10-fold. The frequency of T-antigen-positive cells and that of [3H]thymidine-incorporating cells were almost the same. About 80% of T-antigen-positive cells were also positive to incorporation of [3H]thymidine, and the same result was obtained in infected young cells. These results indicated that senescent human diploid cells which are brought to synthesize T-antigen always initiate DNA synthesis as young cells do. The characteristics of senescent cells as compared with younger cells was low incidence of T-antigen-positive cells after infection. The basis of low susceptibility of senescent cells to initiate DNA synthesis by SV40 infection thus seems to be concerned with an event after the adsorption of virus, but before the synthesis of a detectable amount of T-antigen.     

Reinitiation of DNA synthesis in senescent human fibroblasts upon fusion with cells of unlimited growth potential

Postreplicative, "senescent" human fibroblasts were fused to HeLa or to SV-40 transformed human fibroblasts with Sendai virus. DNA synthesis was reinitiated in senescent nuclei in a high proportion of the heterodikaryons. The [3H]thymidine labeling index of senescent fibroblast nuclei in heteropolykaryons was a function of the ratio of HeLa to senescent nuclei.     

Evidence for endogenous polypeptide-mediated inhibition of cell-cycle transit in human diploid cells

Previous studies have shown that the senescent phenotype is dominant with respect to DNA synthesis in fusions between late passage and actively replicating human diploid fibroblasts. Brief postfusion treatments with the protein synthesis inhibitor cycloheximide (CHX) or puromycin have been found to significantly delay (by 24-48 h) the inhibition of entry into DNA synthesis of young nuclei in heterokaryons after fusion with senescent cells. A significant fraction of the senescent nuclei incorporated tritiated thymidine in CHX-treated heterokaryons. The optimal duration of exposure to CHX was 1-3 h immediately after fusion, although treatments beginning as late as 9 h after fusion elevated the heterokaryon labeling index. Prefusion treatments with CHX were without a significant effect. These results are consistent with the interpretation that regulatory cell cycle inhibitor(s) which are dependent upon protein synthesis may be present in heterokaryons between senescent and actively replicating cells.     

Stimulation of DNA synthesis in senescent human cells following incubation with plasma membranes

DNA synthesis and mitosis were increased in mitogen-stimulated senescent WI-38 cells following incubation with plasma membranes prepared from young or senescent WI-38 cells, A431 cells, 3T3 cells, or NR6 cells. The percentage of [3H]thymidine-labeled nuclei in senescent cultures was two- to fivefold greater than that seen in controls in which cells were incubated in the absence of membranes or in the presence of boiled membranes. The effect was trypsin sensitive, suggesting that a protein moiety is necessary for stimulation of DNA synthesis. As the culture age increased, basal levels of DNA synthesis, as well as maximal stimulation of DNA synthesis following incubation with plasma membranes, decreased. These observations are consistent with the hypothesis that different subpopulations exist in senescing cultures and suggest a complex pattern of inhibitory and stimulatory regulation of cell proliferation.     

Human diploid fibroblasts (HDF) can induce DNA synthesis in cycling HDF but not in quiescent HDF or senescent HDF

HeLa cells in S phase induce DNA synthesis in cycling cells, serum-deprived quiescent cells, and non-replicative senescent cells following cell fusion. In contrast normal human diploid fibroblasts (HDF) do not induce DNA synthesis in either quiescent cells or senescent cells. Instead, the replicative HDF nuclei are inhibited from entering S phase in heterokaryons formed with these two types of non-replicative cells. These differences in the inducing capabilities of normal HDF and HeLa cells raise the question whether normal HDF in S phase can induce DNA synthesis in cycling cells. This paper demonstrates that young HDF in S phase can induce DNA synthesis in cycling HDF. Thus, the hypothesis that initiation of DNA synthesis in cycling cells is positively controlled by inducer molecules appears to be valid for normal HDF as well as for transformed cells such as HeLa.     

Ongoing DNA synthesis continues in young human diploid cells (HDC) fused to senescent HDC, but entry into S phase is inhibited

Previous experiments have shown that when young human diploid cells (HDC) were fused to senescent HDC, neither nucleus synthesized DNA. This paper demonstrates that when young HDC are in S phase at the time of fusion to senescent HDC, they do make DNA in heterodikaryons; therefore, ongoing DNA synthesis is not inhibited by senescent cells. On the other hand, entry into S phase is inhibited: young HDC nuclei in G1 phase do not make DNA in heterodikaryons with senescent HDC.     

Analysis of the capacity of extracts from normal human young and senescent fibroblasts to support DNA synthesis in vitro

Cytoplasmic extracts from early-passage (young), late-passage (senescent) normal human fibroblast (HF) cultures and immortalized human cell lines (HeLa, HT-1080, and MANCA) were analyzed for their ability to support semiconservative DNA synthesis in an in vitro SV40-ori DNA replication system. Unsupplemented extracts from the three permanent cell lines were demonstrated to be active in this system; whereas young HF extracts were observed to be minimally active, and no activity could be detected in the senescent HF extracts. The activity of these extracts was compared after supplementation with three recombinant human replication factors: (1) the catalytic subunit of DNA polymerase alpha (DNA pol-alpha-cat), (2) the three subunits of replication protein A (RPA), and (3) DNA topoisomerase I (Topo I). The addition of all three recombinant proteins is required for optimum activity in the young and senescent HF extracts; the order of the level of activity is: transformed > young HF > senescent HF. Young HF extracts supplemented with RPA alone are able to support significant replicative activity but not senescent extracts which require both RPA and DNA pol-alpha-cat for any detectable activity. The necessary requirement for these factors is confirmed by the failure of unsupplemented young and senescent extracts to activate MANCA extracts that have been immunodepleted of DNA pol-alpha-cat or RPA. Immunocytochemical studies revealed that RPA, DNA pol-alpha, PCNA, and topo I levels are higher in the immortal cell types used in these studies. In the HF cells, levels of DNA pol-alpha-cat and PCNA are higher (per mg protein) in the low-passage than in the senescent cells. By contrast, RPA levels, as determined by immunocytochemical or Western blot studies, were observed to be similar in both young and senescent cell nuclei. Taken together, these results indicate that the low to undetectable activity of young HF extracts in this system is due mainly to reduced intracellular levels of RPA, while the senescent HF extracts are relatively deficient in DNA polymerase alpha and probably some other essential replication factors, as well as RPA. Moreover, the retention of RPA in the senescent HF nuclei contributes to the low level of this factor in the cytoplasmic extracts from these cells.     

Complementation in heterokaryons derived from a thymidine kinase deficient cell line and senescent human diploid cells.

Incorporation of [3H]thymidine was observed in both parental nuclei in heterokaryons resulting from the fusion of post-mitotic, "senescent" human diploid cells and a thymidine kinase-deficient murine cell line (3T3der-4E). The senescent nuclei displayed a sudden increase of activity approximately 4--6 hours after fusion. A moderate increase of thymidine incorporation was observed in 3T3der-4E cells cocultivated with but not fused to senescent cells, consistent with metabolic cooperation. Chromosome preparations of cultures fixed approximately 24 hr after fusion revealed the presence of hybrid metaphase cells containing almost the entire human complement. All of the identifiable human chromosomes were bi-armed, suggesting that the senescent nuclei were stimulated to reinitiate replicative DNA synthesis rather than repair synthesis in these heterokaryons.     

Entry into S phase is inhibited in two immortal cell lines fused to senescent human diploid cells.

Senescent human diploid cells (HDC) were fused to T98G human glioblastoma cells and to RK13 rabbit kidney cells, and DNA synthesis was analyzed in the heterodikaryons. T98G and RK13 cells are “partially transformed” cell lines that have some characteristics of normal cells, yet are transformed to immortality, i.e., they do not senesce. Previous experiments have shown that “fully transformed” HeLa and SV80 cells induce DNA synthesis in senescent HDC nuclei, whereas normal young HDC do not. Our experiments show that T98G and RK13 cells do not induce DNA synthesis in senescent HDC nuclei. These results demonstrate that the ability to induce DNA synthesis in senescent HDC is not correlated with immortality per se. Our results show further that a T98G cell in S phase at the time of fusion to a senescent HDC will continue to make DNA. However, a T98G cell in G1 phase at the time of fusion is prevented from initiating DNA synthesis. RK13 cells behave similarly to T98G. These results are consistent with the hypothesis that the molecular basis for the senescent phenotype involves a block that prevents cells in G1 phase from entering S phase. Thus, we conclude that the senescent phenotype can be dominant in heterokaryons composed of senescent HDC fused with certain immortal cell lines. To explain the different results obtained with various immortal cell lines, we present a model that suggests that T98G and RK13 cells are immortal because they have lost a normal regulatory factor, whereas HeLa and SV80 are immortal because they have gained a dominant transformation factor.     

Murine temperature-sensitive DNA polymerase α mutant displays a diminished capacity to stimulate DNA synthesis in senescent human fibroblast nuclei in heterokaryons at the nonpermissive condition

We have investigated the capacity of a murine cell line with a temperature-sensitive (ts) mutation in the DNA polymerase α (Pola) locus and a series of ts non-Pola mutant cell lines from separate complementation groups to stimulate DNA synthesis, in senescent fibroblast nuclei in heterokaryons. In the Pola mutant × senescent heterodikaryons, both human and murine nuclei display significantly diminished levels of DNA synthesis at the restrictive temperature (39.5°C) as determined by [³H]thymidine labeling in autoradiographs. In contrast, all of the non-Pola mutants, as well as the parental (wild-type) murine cells, induced similar levels of DNA synthesis in both parental nuclei at the nonpermissive and permissive temperatures. Similarly, young human fibroblasts are also able to initiate DNA synthesis in heterokaryons with the ts Pola mutant at the two temperatures. In order to determine if complementation of the non-Pola mutants requires induction of serum responsive factors in the senescent cells, fusion studies of similar design were conducted with young and old human fibroblasts incubated in low serum (0.2%) for 48 hr prior to and after cell fusion. Again, a diminished level of DNA synthesis was observed at 39.5°C in the Pola mutant x senescent cell heterokaryons. In these low-serum studies, both parental nuclei in the Pola x young cell heterokaryons and the human nuclei in heterokaryons with one of the non-Pola mutants (FT107) also displayed diminished levels of DNA synthetic activity. All of the other mutants are able to support similar levels of synthetic activity at both temperatures in the presence of reduced serum. The nature of the mutation in three of the non-Pola lines has not been determined but, like the Pola mutant cells, are inhibited in the G₁ phase of the cell cycle when incubated at the nonpermissive temperature (39.5°C). The fourth non-Pola mutant line is known to have at least one ts mutation in the cdc2 gene and is inhibited in the G₂ phase when exposed to 39.5°C. These results suggest that there may be a functional deficiency of pol α in senescent human fibroblasts, and this replication factor may be one of the rate-limiting factors involved in loss of the capacity to initiate DNA synthesis in senescent cells. © 1994 Wiley-Liss, Inc.     

DNA polymerase alpha and the regulation of entry into S phase in heterokaryons

We have previously reported that the DNA polymerase alpha activity/unit cellular protein is decreased in late-passage (senescent) human diploid fibroblast-like (HDFL) cultures due to the cellular enlargement associated with in vitro aging. In the studies described here, we have used cell fusion technology to investigate the formal kinetic relationship between the concentration of DNA polymerase alpha and the rate of reinitiation of DNA synthesis in nuclei from senescent cells. Heterokaryons were derived from the fusion of senescent cells to a series of actively dividing cell types with inherently different DNA polymerase alpha activities per cell. A kinetic analysis revealed a first-order relationship between the entry into S phase of senescent nuclei and the concentration of DNA polymerase alpha activity calculated to be in heterokaryons. This result suggests that increases in cell volume may be related to the decline in proliferative activity of late-passage HDFL cells, via "dilution" of factors essential for cellular replication.     

Reactivation of chick erythrocyte nuclei in young and senescent WI38 cells

The chromatin of the dormant chick nucleus is dispersed in the heterokaryons made by Sendai virus fusion of phase II WI38 cells with chick erythrocyte nuclei. The erythrocyte nucleus resumes RNA synthesis and enters into DNA synthesis with the host nucleus. In the heterokaryons of phase III WI38 cells and chick erythrocytes, the nuclear chromatin is not dispersed and RNA synthesis occurs at a reduced rate. The differences in the physiological state of the young and senescent cells measured by [³H]uridine incorporation into nuclear RNA is reflected in the extent of reactivation of the chick erythrocyte nuclei in the cytoplasm of these cells. The reactivation of the chick nucleus in enucleated fibroblasts parallels the nucleated cells. The results of these studies are interpreted as evidence that there is a specific loss of nuclear function in the senescent cells.     

Possible role of SV40 T antigen in cell transformation]

The effect of purified SV40 T antigen on DNA synthesis in isolated nuclei from the confluent culture of CV-1 cells was studied. In the presence of T antigen the incorporation of [3H]TTP into DNA was found to be 2 to 3 times as high as in the control nuclei. The resulting labelled DNA was subjected to alkaline sucrose gradient centrifugation, which revealed the presence of 4S DNA species, corresponding to Okazaki fragments of animal cells. The latter finding suggests a replicative mode of DNA synthesis induced by T antigen. T antigen isolated from the cells infected with SV40 tsA-mutant and kept at a nonpermissive (41 degrees) temperature fails to stimulate DNA synthesis in isolated nuclei from resting cells. On storage at 4 degrees SV40 T antigen gradually loses its ability to stimulate DNA synthesis and by the 8th day even suppresses it when tested on isolated nuclei from a growing cell culture. No effect of T antigen on the endonuclease-induced reparative synthesis of DNA could be observed. The data described suggest that T antigen is directly involved in the control of DNA synthesis in the cells infected or transformed with SV40.     

DNA polymerase α and the regulation of entry into S phase in heterokaryons

We have previously reported that the DNA polymerase α activity/unit cellular protein is decreased in latepassage (senescent) human diploid fibroblast-like (HDFL) cultures due to the cellular enlargement associated with in vitro aging. In the studies described here, we have used cell fusion technology to investigate the formal kinetic relationship between the concentration of DNA polymerase α and the rate of reinitiation of DNA synthesis in nuclei from senescent cells. Heterokaryons were derived from the fusion of senescent cells to a series of actively dividing cell types with inherently different DNA polymerase α activities per cell. A kinetic analysis revealed a first-order relationship between the entry into S phase of senescent nuclei and the concentration of DNA polymerase a activity calculated to be in heterokaryons. This result suggests that increases in cell volume may be related to the decline in proliferative activity of late-passage HDFL cells, via “dilution” of factors essential for cellular replication.     

Increase in DNA synthesis in Werner''s syndrome cells by hybridization with normal human diploid and HeLa cells

The dominance or recessiveness of the senescent phenotype in cells from patients with Werner's syndrome (WS cells) was investigated using cell fusion. The [³H]thymidine labeling index of normal human diploid fibroblast cell X WS cell heterodikaryons was considerably lower than that of normal homodikaryons, but was significantly higher than that of WS homodikaryons. The labeling index of WS cell X HeLa cell heterodikaryons was the same as that of HeLa homodikaryons. The labeling indices of heterodikaryons obtained by fusion between various strains of premature aging cells were as low as those of parental homodikaryons. These results indicate: (1) the senescent phenotype of WS cells appears to be partially recessive to the phenotype of normal cells and completely recessive to that of HeLa cells; (2) the marked inhibition of DNA synthesis in normal nuclei in heterodikaryons with WS cells could be due to ‘senescent factor(s)’ in WS cells; and (3) no complementation phenomenon was observed among genetically different premature aging cells, probably due to ‘senescent factor(s)’.     

Comparative heterokaryon study of cellular senescence and the serum-deprived state

Autoradiographic patterns of DNA replication in serum-deprived human diploid fibroblast-like cells (HDFC) and “senescent” HDFC have been compared in two types of heterokaryons. Each was fused to low passage, proliferating HDFC and, in separate experiments, to HeLa cells. Sequential 1 h pulses with [³H]thymidine were initiated at short intervals following fusion. In all hybridizations serum-deprived and senescent cells behaved identically. Upon fusion to HeLa cells, DNA synthesis in the quiescent nuclei occurred in a wave between 3 and 30 h after fusion. When either serum-deprived or senescent HDFC were fused to young proliferating HDFC, the nuclei of the latter were blocked from entering the S phase if fusion occurred at least 3 h before the G/S boundary. These findings are consistent with the interpretation that one or more crucial steps in G0 occurs 3 h before the G1/S interface. That young serum-deprived (G0) HDFC behave identically to senescent cells in these hybridization studies suggests that the mechanism of arrest in each state might share a final common pathway, and a model based on these observations is proposed.