Using a combination of DNA-cytophotometry and tritiated thymidine-autoradiography, we have shown that the majority of nondividing cells in serially propagated human diploid cell populations have the 2 C DNA content consistent with their being arrested in the G1 phase of the diploid cell cycle. Unlabeled 4C cells appear increasingly with time in culture. These may be arrested G2 diploids or they may be G1 tetraploids, since there is an associated increase in polyploidy in older cultures as evidenced by the appearance of labeled 8C cells.Human diploid fibroblast-like cell lines have a finite lifetime in terms of their proliferative capacity in vitro. Soon after being established in culture, these populations proliferate vigorously, but with successive serial subcultivations, the growth rate progressively declines, and ultimately the culture cannot be propagated (Hayflick and Moorhead, '61; Hayflick, '65). This phenomenon appears to be intrinsic to the culture and has been suggested as a model for the cellular expression of aging. Cristofalo ('72) has discussed this aspect of the diploid cell culture model system in a recent review.The decrease in growth rate in aging cultures appears to be due to both lengthening of the average cell cycle times '66; Absher et al.) and to a progressive increase in the nondividing fraction of the population. That the nondividing fraction increases exponentially as a function of the number of passages of the culture, is shown by the decreasing ability of single cell isolates to divide and form clones (Merz and Ross, '69), and by an exponential increase with age in the percent of cells unable to incorporate tritiated thymidine (3HdT) after a 24-to 30-hour exposure (Cristofalo and Sharf, '73).To investigate further the conversion of dividing to nondividing cells, we examined the stage of the cell cycle in which the nondividing cells were arrested. A combination of cytophotometry and autoradiography was used to determine for individual cells at various serial subcultivation levels both their proliferative status and their stage in the cell cycle. MATERIALS AND METHODSDetails of our method for culture of the human diploid lung cells, WI-38, have been described (Cristofalo and Sharf, '73). In brief, cultures were maintained in modified Eagle's MEM with 10% fetal calf serum and sub-cultivated routinely when the monolayers were confluent. Aging cultures which did not reach confluency by one week were refed at weekly intervals. Cultures were monitored periodically for mycoplasma contamination by Dr. Leonard Hayflick of Stanford University.Cellular measurements were done on cultures seeded at a density of 1.3 X lo4 cellslcm2 in Lab-Tek microscope slide chambers (Miles Laboratories, Westmont, Ill.) and incubated at 37" C in an atmosphere of 5% C02:95% air. At 24 hours Received Jan. 22, '74. Accepted May 3, '74. 1 Preliminary versions of this work were presented a t the Gerontological Society, San Juan, Puerto Rico, December, 1972, (TheGerontologist 12: 35,1972 and at the Tissue Cultu...

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With parameters of transformation other than immortality (3). Another logical possibility is that the ability to induce DNA synthesis in senescent HDC might be associated with viral transformation. Cells transformed by viruses may have gained a factor that overrides the inhibitory effect of senescent HDC, whereas carcinogen-transformed cells may have lost a normal factor necessary for the expression ofsenescence while retaining their sensitivity to the putative inhibitor when it is supplied to them in heterodikaryons. We have tested these hypotheses by fusing senescent HDC to a series of transformed human cells that have various transformed properties and were transformed in different ways. MATERIALS AND METHODSOur procedures for cell culture, cell fusion, autoradiography, and identification of heterodikaryons have been described (2,3,5).In these experiments, the senescent HDC were either IMR-90 or WI-38 fetal lung fibroblasts at 48-53 population doublings. Forty-eight hours before fusion, senescent HDC were labeled with both [3H]thymidine (0.5 ,Ci/ml; 1 Ci = 3.7 X 10'°b ecquerels) and 2-,um-diameter latex beads (Dow The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Serum-deprived quiescent human diploid cells (HDC) were fused to replicative HDC, and DNA synthesis was monitored in the resulting heterodikaryons. Quiescent Previous studies with HeLa cells suggest that entry into S phase is controlled by a positive effector in cycling cells. HeLa cells in S phase induced DNA synthesis in HeLa cells in G1 phase after cell fusion (1). In contrast, DNA synthesis was not induced in HeLa cells in G2 phase fused to cells in S phase. These data suggest that S-phase cells contain an inducer of DNA synthesis that stimulates entry into S phase in GI-phase cells. This possibility is supported by other experiments showing that when HeLa cells in early, middle, and late G1 phase were fused together in various combinations, both nuclei in the resulting binucleates entered S phase prematurely, except in early G1 x early GC fusions (2). Cells in late GC phase contributed the greatest acceleration ofentry into S phase. Therefore, these data suggest that cells in GC phase accumulate some factor that is necessary for entry into S phase, and this could be the putative inducer found in S-phase cells themselves.In contrast to these observations on cycling cells, it appears that in noncycling senescent human diploid cells (HDC), regulation of entry into S phase may be negatively controlled. When senescent HDC were fused to replicative young HDC, DNA synthesis was inhibited in the young HDC nuclei in heterodikaryons (3). Further analysis indicated that ongoing DNA synthesis in young HDC in S phase at the time of fusion was not inhibited but that entry into S phase was inhibited (4). Similar results were obtained when senescent HDC were fused to SUSM-1 chemically transformed human cells, CT-1 radiationtransformed WI-38 cells, and several human tumor cell lines (5, 6). In each case, ongoing DNA synthesis was not inhibited in the replicative cell nuclei, but entry into S phase was blocked. A simple explanation for these results is that nonreplicative senescent HDC contain an inhibitor of entry into S phase. This hypothesis is consistent with the observation that senescent HDC themselves are blocked from entering S phase (7,8).Normal human cells, such as WI-38 fetal lung fibroblasts, become quiescent when they are deprived of serum; the cells almost completely cease to proliferate and yet they maintain high viability (9). These quiescent HDC have Gj-phase DNA contents, indicating that they cannot enter S phase (10). This paper analyzes heterodikaryons formed between quiescent HDC and replicative cells to determine whether entry into S phase is positively or negatively controlled in quiescent HDC.

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ABST RAEC Cells (Chinese hamster ovary) in GI phase were fused with cells in late S phase to determine if a cell in late S phase can induce DNA synthesis in the nucleus of a GI cell and, if so, to determine if the DNA synthesis so induced in a GI phase nucleus has an autoradiographic pattern characteristic of early or of late S phase synthesis. The results indicate (i) that 89% of the GI nuclei in late-S/G1 binucleates synthesized DNA, while only 2% of the control unfused GI cells synthesized DNA, and (ii) that in all late-S/G1 binucleates the GI nucleus was induced to synthesize early S phase DNA. These results are compatible with the idea that a cytoplasmically transmissible factor initiates DNA synthesis but that an intranuclear mechanism defines the temporal order of replication.Two important problems in the study of cell reproduction are the mechanism governing the initiation of DNA replication and the mechanism responsible for the temporal ordering of DNA synthesis within the S phase. Evidence indicates that a cytoplasmically transmissible inducer of DNA synthesis is present in early S phase HeLa cells; when a Gi phase cell was fused with an early S phase cell 1 hr after its release from a double thymidine block, DNA synthesis was induced prematurely in the GI phase nucleus (1). In addition, fusion of erythrocytes (2) or macrophages (3) with S phase cells that had been selected at various times after mitosis has suggested that a cytoplasmically transmissible inducer is present throughout the S phase. However, the difficulty in positively identifying late S phase cells in these types of experiments leaves open the question of whether such an inducer of DNA synthesis is present in late S phase. If such an inducer is present in late S phase, does it induce the synthesis of early or late S phase DNA in a Gi nucleus; i.e., is DNA synthesis induced in the normal temporal order or are there specific late S phase initiators that can alter the normal replication sequence?To study these problems we fused Chinese hamster ovary (CHO) Cell Synchrony. CHO cells were grown in the presence of 2 mM deoxythymidine (dThd) for 9 hr to synchronize them coarsely in S phase (5-7). The medium was replaced, and 5-7 hr later mitotic cells were collected by the shake-synchrony procedure (8, 9). With this procedure generally 95-99% of the detached cells were in mitosis. The mitotic cells were grown in conditioned medium for 1 hr to obtain Gi phase cells. Alternatively, the mitotic cells were grown in conditioned medium containing 1 mM hydroxyurea for 9 hr to obtain early S phase cells (10, 11). The hydroxyurea was removed and the cells were allowed to proceed to late S phase.Labeling with Latex Beads. To identify fusion products between cells from S and GI phase populations we used latex beads (Dow Chemical Co., Indianapolis, IN) of two different sizes to prelabel the cytoplasms of the cells (approximately 1 Am diameter beads for the S phase cells and approximately 2 Am diameter beads for the GI phase cells; ref. 12). Cells were al...