Relationship between RNA content and progression of lymphocytes through S phase of cell cycle.

Abstract

ABSTRACr A flow cytometric technique for simultaneous measurements of RNA and DNA in individual cells has been applied to correlate the content of cellular RNA with the rate of progression of cells through the S phase. Human peripheral lymphocytes stimulated with phytohemagglutinin were blocked at the G1/S phase boundary by hydroxyurea or 5-fluorodeoxyuridine treatment. Cells in the G1 phase as well as cells blocked at the G1/S phase boundary showed high heterogeneity with respect to stainable RNA content. After release from the block, the cells traversed the S phase at rates proportional to the quantity of stainable RNA per cell. Cells with the highest RNA content completed DNA replication 5 hr after release from the block; the cells with minimal RNA traversed the S phase at one-fifth of this rate. The large intercellular variation in stainable RNA and length of the S phase may be due to functional heterogeneity in the lymphocyte population. Our results suggest a correlation between the number of ribosomes and the rate of DNA replication in lymphocytes. Peripheral blood lymphocytes are quiescent (Go) cells that may be stimulated in culture by mitogens to enter the cell cycle (for reviews, see refs. 1 and 2). After stimulation, lymphocytes asynchronously leave the Go state, traverse the G1 phase, and then initiate DNA replication (1, 2). In contrast to most eukaryotic cell types, which have an S period of relatively constant duration (6-9 hr), stimulated lymphocytes are characterized by a large intercellular variation in the length of the S phase (3-5). Duration of the S phase in these cells has been shown to vary from 6 to as much as 30 hr (3-6), with mean values ranging between 9 and 12 hr (7,8 Lymphocytes. Cell collection, separation, and culture methods were described in detail (10,12,13). Briefly, the blood was collected by venipuncture of healthy volunteers, and the leukocytes were separated by gradient centrifugation on Ficoli-Isopaque (Lymphoprep; Nyegaardo, Oslo, Norway). The mononuclear cells were suspended in Eagle's basal medium containing 15% fetal calf serum and subcultured on plastic dishes to remove most of the monocytes (13). The nonadhering cells were then suspended in Eagle's basal medium containing penicillin, streptomycin, and 15% fetal calf serum and incubated at 370C in the presence of phytohemagglutinin (12, 13). To synchronize the cells at the GI/S phase border, the cultures incubated with phytohemagglutinin for 24 hr were treated with 1 mM hydroxyurea (Sigma) or 0.1 tM 5-fluorodeoxyuridine (Sigma) for the next 18-24 hr.Cell Staining. Portions (0.2 ml) of suspension cultures, containing approximately 1-4 X 105 cells, were mixed with 0.4 ml of 0.08 M HCl/0.15 M NaCl/0.1% Triton X-100 (Sigma) at 4VC. The cells were stained 30 sec later by the addition of 1.2 ml of a solution containing 0.2 M Na2HP04/0.1 M citric acid buffer (pH 6.0), 1 mM Na EDTA, 0.15 M NaCl, and AO (chromatographically purified; Polysciences, Inc., Warrington, PA) at 6 ,tg/ml. Considering cell number per sample, the aver...

New cell cycle compartments identified by multiparameter flow cytometry

“…It should be emphasized, however, that cells arrested in the G1 phase by hydroxyurea, or 5-fluorodeoxyuridine display an increased RNA content (33,34). Thus, in contrast to the effect of serum deprivation or cell crowding, which block cells in GIA (subsequently moving them into the G 1~ compartment), hydroxyurea or 5-fluorodeoxyuridine arrest cells specifically in GIB phase, or more precisely at the border between G I~ and s (33,34).…”

“…Thus, in contrast to the effect of serum deprivation or cell crowding, which block cells in GIA (subsequently moving them into the G 1~ compartment), hydroxyurea or 5-fluorodeoxyuridine arrest cells specifically in GIB phase, or more precisely at the border between G I~ and s (33,34).…”

“…G1Q celk are distinctly different from their cycling counterparts since there is no overlap in RNA and at values between cells in these two states. Nonstimulated peripheral blood lymphocytes are the most typical examples of GIQ cells (25,28,(30)(31)(32)(33). 3T3 cells or normal cells in primary cultures maintained at confluency for an extended period of time also approach the G~Q state.…”

“…However, in quiescent diploid cell cultures there are always some cells present (about 5%) with RNA and at values that overlap those of the cycling populations. When GIQ cells are stimulated to proliferate (e.g., lymphocytes by mitogens) the inductive phase preceeding DNA replication is long, varying between 24-48 hr (25,32,33).…”

“…However, their further progression through S does involve a gradual alteration in chromatin (increase in at) which is evidenced by the nonvertical distribution of total fluorescence values for S-phase cells when plotted against at (Fig. 2B) (25,33,34). The point we want to stress here is t h e distinction between noncycling quiescent lymphocytes (GIQ cells) and the cycling population, with special reference t o the heterogeneity of the GI population.…”