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

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Exp Cell Res144(1983)

Human Diploid Fibroblasts (HDF) can Induce DNA Synthesis in Cycling HDF but not in Quiescent HDF or SenescentHDF GRETCHEN

Copyright @ 1983 by Academic has, Inc. All rights of reproduction in any form reserved

0014-4827183/040468-04$02.0010

H. STEIN

Department of Molecular, Campus Box 347, Boulder,

Cellular and Developmental CO 80309, USA

Biology,

University

of Colorado,

Summary. 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 tibroblasts (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.

The regulation of DNA synthesis in normal and transformed human cells has been analysed by fusing together cells in different replicative states. When replicative young human diploid tibroblasts (HDF) are fused to non-replicative senescent HDF, DNA synthesis is not induced in the senescent HDF nuclei [l]. Rather, the young HDF nuclei are inhibited from entering S phase in these heterodikaryons [2]. Likewise, replicative young HDF do not induce DNA synthesis in quiescent serum-deprived HDF, and the replicative nuclei are inhibited from entering S phase [3, 41. In contrast, HeLa cells induce DNA synthesis in both senescent HDF and quiescent HDF in heterodikaryons [3,5]. Thus, normal HDF and HeLa cells differ in their ability to induce DNA synthesis in nonreplicative cells. Studies on the regulation of DNA synthesis in cycling cells have been done primarily with HeLa cells or other types of transformed cells. These studies show that HeLa cells in S phase can induce DNA synthesis in cycling cells in Gl phase, but not in cycling cells in G2 phase [6]. Heterokaryons formed between HeLa cells in various stages of Gl enter S phase at an accelerated rate compared to the unfused cells, with the greatest acceleration being contributed by cells in late Gl phase [7]. These studies suggest that initiation of DNA synthesis in cycling cells is positively controlled by an inducing factor that accumulates during Gl phase and remains present during S phase. An important question is whether this hypothesis is applicable to normal HDF as well as to transformed cells such as HeLa. Because HeLa cells in S phase can induce DNA synthesis in nonreplicative cells as well as in cycling cells, whereas normal HDF cannot induce DNA synthesis in non-replicative cells, we cannot automatically assume that normal HDF in S phase will be able to induce DNA synthesis in cycling cells. This paper demonstrates that young HDF in S phase can induce DNA synthesis in cycling HDF. Furthermore, the rate of this induction is comparable to the rate at which HeLa cells in S phase induce DNA synthesis in cycling cells. Materials

and Methods

The young HDF used in these experiments were IMR-90 fetal lung fibroblasts [8] that had undergone 19-24 doublings in culture. Two days before fusion, subconfluent cultures of IMR-90 were harvested

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and seeded at 6.25x 103 cells/cm2 on coverslips in Eagle’s Basal Medium plus 10% fetal bovine serum (BME+IO% FBS). Some 15-20 mitt before fusion, a high concentration of L3Hlthymidine (6-g @i/ml) was added to the cultures in order to label cells in S phase heavily. (The t3Hlthymidine stock solution had a sp. act. of 50 Ci/mmole.) The cells were rinsed quickly with BME and immediately fused with polyethylene glycol 1000 (Koch-Light) according to the monolayer fusion procedure of Hales [9]. The cells were rinsed three times with BME and then incubated in BME+lO% FBS containing a low concentration of [3H]thymidine. The amount of [‘Hlthymidine used varied inversely with the length of the labelling period, i.e., cells were labelled with 0.1 uCi/ml for O-l h after fusion, with 0.05 uCi/ml for O-2 h, with 0.033 $X/ml for 63 h, and so forth. Consequently, the number of silver grains over the cells labelled with [‘Hlthymidine after fusion was consistently one-twentieth the number of grams over the S phase cells that incorporated r3H]thymidine before fusion. At the end of each labelling period, the cells were incubated in BME+lO% FBS+O.l mM unlabelled thymidine until g-9 h after fusion, when all the cultures were fixed with methanol: acetic acid (3 : 1). Autoradiograms were prepared as previously described [lo]. The experiments were scored in several different ways to insure that there was no investigatorinduced bias in the results. Two of the experiments were scored independently by two different investigators, two experiments were scored blind, and one experiment was scored using two sizes of latex beads to identify binucleate cells. The methods for using latex beads have been described previously [2, 31 including control experiments that show that there is negligible bead exchange by mechanisms other than cell fusion [ 111.

Results and Discussion

The goal of this study was to determine whether normal HDF in S phase can induce DNA synthesis in cycling HDF. Our basic experimental plan was to compare the rate at which non-S-phase cells entered S phase when they were unfused single cells (monokaryons), when they were fused to S-phase cells (heterodikaryons) and when they were fused to other non-S-phase cells (homodikaryons). All steps were carried out on unsynchronized populations of replicating young IMR-90 cells in order to avoid potential artifacts due to synchronization procedures, e.g., induction of a state of unbalanced growth in the cells or detachment of the cells from the substrate. Instead, we identified the S phase cells in a population of attached, replicating cells by labelling the cultures for 15-20 min with a high concentration of [3H]thymidine immediately before fusion. Hereafter, we will refer to the cells that were not in S phase at the time of fusion as non-S-phase cells, even though some of them entered S phase later in the experiment. We monitored entry into S phase in the non-S-phase nuclei by incubating the cells in a low concentration of [3H]thymidine for various times after fusion, The incorporation of [3H]thymidine was measured by autoradiography. Nuclei that were in S phase at the time of fusion were heavily labelled with silver grains and non-S-phase nuclei were either unlabelled or lightly labelled with silver grains. Fig. 1 shows the results of two separate experiments, in order to demonstrate the variability between individual experiments. The data shown are the percentages of non-S-phase nuclei that had entered S phase at various times after the fusion procedure. The non-S-phase monokaryons gradually entered S phase, as expected for an unsynchronized population of cells. The non-S-phase nuclei in heterodikaryons formed with S-phase cells entered S phase at a much faster rate than the monokaryons. This result suggests that young HDF in S phase can induce DNA synthesis in cycling non-S-phase nuclei. The non-S-phase nuclei in homodikaryons entered S phase at a rate intermediate between that of the monokaryons and heterodikaryons. A plausible interpretation of these data is

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Fig. 1. Replicative IMR-90 were fused to each other and the percentage of non-S-phase nuclei that synthesized DNA after fusion was determined for A-. A, monokaryons; C-4, homodikaryons; and O-O, heterodikaryons. The IMR-90 were at population doubling level 19 (A) and 24 (B), with 42% (A) and 50% (B) of cells in S phase at the time of fusion. Fig. 2. Replicative IMR-90 were fused and analysed as in fig. 1. The percentage induction of DNA synthesis was calculated as (percentage labelled non-S-phase nuclei in heterodikaryons minus percentage labelled non-S-phase nuclei in monokaryons) divided by percentage unlabelled non-S-phase nuclei in monokaryons. The results of three independent experiments are presented.

that when one non-S-phase nucleus in a homodikaryon enters S phase, it has the ability to induce DNA synthesis in the other non-S-phase nucleus in that cell. Hence, these results are consistent with the hypothesis that young HDF in S phase can induce DNA synthesis in cycling HDF. We compared the rate at which young HDF in S phase induce DNA synthesis in cycling HDF to the rate at which HeLa cells in S phase have been shown to induce DNA synthesis in cycling HeLa cells [6]. Fig. 2 shows the percentage induction of DNA synthesis in non-S-phase HDF nuclei as a function of time after fusion for three independent experiments. In each case, by 4 h after fusion, the amount of induction is greater than 90 % of the maximum level achieved. The half-maximal levels are reached between l-2 h after fusion. These kinetics are the same as those previously reported for fusion of S phase HeLa to Gl phase HeLa [6]. However, an important difference is that the maximum percentage induction in our experiments with HDF was approx. 35 %, whereas the maximum percentage induction in the HeLa experiments was 75-80%. We suggest that the maximum percentage induction in our HDF experiments is lower because (1) the nonS-phase HDF in interphase were distributed in both Gl phase and G2 phase; (2) only the Gl phase cells can be induced to enter S phase. This interpretation is consistent with the previous studies on HeLa cells, which indicate that G2 phase cells are refractory to the induction of DNA synthesis by fusion to S phase cells [6]. Numerous measurements of the length of Gl phase and G2 phase in young HDF have shown that on the average Gl phase is only slightly (i.e., 8%) longer than G2 phase [12]. Thus, the presence of G2 phase cells in the non-S-phase HDF population can account for a large proportion of the uninduced HDF nuclei in our experiments. In contrast, in the HeLa experiments, all of the non-S-phase cells were in Gl phase. The level of induction in our HDF experiments is also

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decreased because some of the cells in Gl phase at the time of fusion entered S phase on their own in the first few hours after fusion. Thus, the pool of inducible nuclei was decreasing as induction was taking place. In contrast, in the HeLa experiments, there was no decrease in the pool of inducible nuclei because the Gl phase cells did not begin to enter S phase until 8 h after fusion. Finally, it is possible that in the HDF experiments, some of the cells were not inducible because they were already senescent. Populations of young HDF are heterogeneous and typically contain a small (510%) subpopulation of non-cycling cells 18, 131.

In conclusion, we have shown that young HDF can induce DNA synthesis in cycling HDF with kinetics comparable to those demonstrated previously for HeLa cells. Thus, the hypothesis that initiation of DNA synthesis is positively controlled by the presence of an inducer(s) in cycling cells seemsvalid for normal cells as well as for transformed cells. On the other hand, cycling HDF cannot induce DNA synthesis in non-cycling quiescent HDF or senescent HDF; rather, the cycling HDF are inhibited from entering S phase. These data suggest that senescent HDF and quiescent HDF contain an inhibitor of entry into S phase. Taken together, these two sets of data suggest that maintenance of normal HDF in a non-replicative resting state involves a negative control mechanism, whereas the timing of initiation of DNA synthesis in cycling cells involves a positive control mechanism. I wish to thank Tina Buckel, Mary Beeson and Lena Gordon for their excellent technical assistance. This work was supported by NIH grant AGO0947 and by NIH contract NOl-CO-75380 with Litton Bionetics, Inc.

References 1. Norwood, T H, Pendergrass, W R, Sprague, C A & Martin, G M, Proc natl acad sci US 71 (1974) 2231. 2. Yanishevsky, R M &z Stein, G H, Exp cell res 126 (1980) 469. 3. Stein, G H & Yanishevsky, R M, Proc natl acad sci US 78 (1981) 3025. 4. Rabinovitch, P S & Norwood, T H, Exp cell res 130 (1980) 101. 5. Not-wood, T H, Pendergrass, W R & Martin, G M, J cell bio164 (1975) 551. 6. Rao, P N & Johnson, R T, Nature 225 (1970) 159. 7. Rao, P N, Sunkara, P S & Wilson, B A, Proc natl acad sci US 74 (1977) 2869. 8. Nichols, W W, Murphy, D G, Cristofalo, V J, Toji, L H, Greene, A E &Dwight, S A, Science 196 (1977) 60. 9. Hales, A, Somatic cell genet 3 (1977) 227. 10. Stein, G H & Yanishevsky, R, Methods enzymol 58 (1979) 279. 11. Levine, M R & Cox, R P, Somatic cell genet 4 (1978) 507. 12. Grove, G L & Cristofalo, V J, J cell physiol 90 (1977) 415. 13. Cristofalo, V J & Sharf, B B, Exp cell res 76 (1973) 419. Received September 22, 1982 Revised version received December 29, 1982