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Restriction Point Control of Cell Growth by a Labile Protein

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Restriction Point Control of Cell Growth by a Labile Protein Proc. NatL Acad. Sci. USA Vol. 79, pp. 436-440, January 1982 Cell Biology Restriction point control of cell growth by a labile protein: Evidence for increased stability in transformed cells (animal cells/transformation/protein degradation/cell cycle/growth control) JUDITH CAMPISI, ESTELA E. MEDRANO*, GAIL MORREO, AND ARTHUR B. PARDEEt Department of Pharmacology, Harvard Medical School, and Division ofCell Growth and Regulation, Sidney Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115 Contributed by Arthur B. Pardee, September 14, 1981 ABSTRACT It has been proposed that animal cells must ac- mouse 3T3 cells at 2-3 hr prior to the onset of DNA synthesis cumulate a labile protein(s) before they can pass the restriction (7, 8). (R) point in the G1 phase ofthe cell cycle [Rossow, P. W., Riddle, What is the biochemical nature of the R point? Two types of V. G. H. & Pardee, A. B. (1979) Proc NatL Acad Sci USA 76, experiments have led to the hypothesis that afunctionally short- 4446-4450]. Here, we present evidence that this R protein ac- lived (labile) regulatory protein(s) (R protein), whose synthesis quires increased stability in transformed 3T3 cells, thereby allow- is sensitive to environmental conditions, need accumulate to a ing these cells to continue growth under conditions that arrest critical amount before a cell can pass the R point and proceed untransformed cells. Low doses of cycloheximide or histidinol towards DNA synthesis. First, pulses ofhigh concentrations of drastically reduced the rate at which normal 3T3 (A31) fibroblasts cycloheximide (CHM), administered to synchronized cultures, in early G, could enter DNA synthesis. These drugs had less effect on entry of two tumorigenic A31 derivatives, BPA31 and SVA31, produced delays in the onset ofDNA synthesis that were longer into S, although measurement of [3H]leucine incorporation than the length of the pulses (9, 10). From the excess delays, showed that the inhibitors were equally effective in the three cell it was estimated that the half-life ofthe putative R protein was lines. The hypothesis is that the transformed lines are less sensitive 2-3 hr. Second, moderate inhibition of protein synthesis by because moderate inhibition of their R protein synthesis is com- CHM (50-70%) specifically delayed transit through that part pensated by lower rates of protein degradation. To test this idea, of G1 prior to the R point, with kinetics in quantitative agree- we completely inhibited cytoplasmic protein synthesis for several ment with the labile protein model (5). hours shortly before A31 and BPA31 cells had reached the R point. Transformed cells, which lack the stringent growth control of After removal of inhibitor, A31 cells showed delays in the onset normal cells, were relaxed with respect to the CHM-induced ofS thatwere in excess ofthe inhibitor pulse, consistentwith decay inhibition of GC transit (11). The transit rate from mitosis (M) of labile protein during the pulse. BPA31 cells showed no excess to the R point was measured as a function ofthe CHM-inhibited delays, suggesting a much more stable R protein. The half-life of rate of protein synthesis in 3T3 cells and in chemically, DNA the R protein was estimated as 2.5 hr in A31 cells, indicating that, virus-, and RNA virus-transformed 3T3 cells. The transformed in these cells, R protein synthesis starts at the beginning ofGa. In cells all continued to cycle until protein synthesis was 90% in- the BPA31 cells, the R protein showed no signs ofdecay for at least hibited, whereas the normal cells became arrested at 65% in- 8 hr. hibition (12). Our interpretation of these results is that the R protein is more stable in the transformed cells. The major regulatory events leading to proliferation in animal In this paper, we examine this possibility more closely. Using cells occur in the G1 phase of the cell cycle (1). In vivo, most moderate and severe inhibition of protein synthesis by CHM quiescent cells have a G1 DNA content. In culture, normal cells or histidinol, we characterize the lability of protein elements become arrested in G1 under a variety of conditions that ulti- necessary for the commencement of DNA synthesis in normal mately prevent or diminish proliferation: nutrient or serum and transformed 3T3 cells. Our data suggest that the R protein deprivation (2, 3), high cell density (4), and partial inhibition acquires greater stability in transformed cells. We discuss the of protein synthesis (5). possibility that the 53-kilodalton protein recently described in From the kinetics with which quiescent cells resume prolif- a variety oftransformed cells (13-15) may function as an R pro- eration, it has been proposed that a single major regulatory tein in normal cell growth. event occurs in G1 that permits a cell to complete another round of the cell cycle (1). This has been named the restriction or R MATERIALS AND METHODS point. According to this model, newly divided cells (in early G1) prepare for DNA synthesis and cell division while continuously Cell Culture. BALB/c 3T mouse fibroblast clone A31-CL7 If conditions are the (A31), benzo[a]pyrene-transformed A31 cells [BP 3T3-CL7-5 monitoring their environment. favorable, (BPA31)], and simian virus 40 (SV40)-transformed A31 cells [SV- cells will pass through the R point and become committed to A31-CL29 (SVA31)] were obtained and grown as described (11). complete the remainder of the cell cycle. If conditions are not New cultures were started from frozen stocks every 4-6 weeks. favorable, they cease proliferation, remaining with a G1 DNA Each stock was determined to be free of mycoglasma contam- content. ination by using the ratios of incorporated [ H]uridine and Earlier work with serum-stimulated chicken cells had sug- [3H]uracil (16). gested the existence ofa serum-dependent event in mid-GC (6). More recently, the R point has been temporally mapped in Abbreviations: CHM, cycloheximide; SV40, simian virus 40. * Present address: Instituto de Investigaciones Bioquimicas, Fundacion The publication costs ofthis article were defrayed in part by page charge Campomar, Obligado 2490, 1428 Buenos Aires, Argentina. payment. This article must therefore be hereby marked "advertise- t To whom requests should be addressed at the Sidney Farber Cancer ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Institute. 436 Downloaded by guest on September 26, 2021 Cell Biology: Campisi et al. Proc. Natl. Acad. Sci. USA 79 (1982) 437 Nuclear Labeling and Autoradiography. Cells were plated at 3 x 103 per cm2 in Falcon 16-mm multiwell plates. The me- 100 *dium was aspirated 48 hr later and replaced with fresh medium containing 2 AuCi (1 Ci = 3.7 X 10 ° becquerels) of [methyl- 3H]thymidine per ml and CHM or histidinol where indicated. At various times thereafter incorporation was stopped by the addition of4 drops of 1 M ascorbic acid to the appropriate well. The entire experiment was carried out in a 370C room to min- imize temperature fluctuations. At the end ofthe experiment, the cells were washed with phosphate-buffered saline, fixed with methanol, and dried. The wells were coated with Kodak NTB2 emulsion, exposed for 3-5 days, and developed. The nuclei were lightly stained with Giemsa stain and the percent labeled nuclei was determined by microscopic counting. A min- imum of 300 cells was counted for each determination. Measurements ofProtein Synthesis Rates. Cells were plated at 3 x 103 per cm2 in 35-mm culture dishes (Falcon). The me- dium was aspirated 48 hr later and replaced with fresh medium -- ~ containing inhibitors. [3H]leucinewas added as described in the legends. The amount of radioactivity incorporated into trichlo- roacetic acid-insoluble material was determined as follows: the cells were washed twice with ice-cold phosphate-buffered saline C and cold trichloroacetic acid was added. After at least 20 min at 40C, the cells were washed once with cold trichloroacetic acid dissolved in 0.5 ml of 0.2 M NaOH, and incubated for 20 min at 37VC. A portion of the NaOH was then mixed with Triton X- 20 100/toluene scintillation cocktail formeasurement ofradioactivity. Cell Synchrony and Measurement of DNA Synthesis. Cells were plated at 3 X 103 per cm2 in 16-mm multiwell plates. The 4 8 12 16 20 24 28 32 medium- was removed 24 hr later, and the cells were washed twice with serum-free medium and incubated in medium con- Time, hr taining either 0.5% (A31) or 0.2% (BPA31) serum as described FIG. 1. (A) Entry rate into S of exponentially growing A31 cells in the text. DNA synthesis was measured by the amount of exposed to OHM. *, Control; *, 0.05 ,ug/ml; 00.1 plg/mi; a, 0.2 pg/ [3H]thymidine incorporated into trichloroacetic acid-insoluble ml. (B) Entry rate into S of exponentially growing A31 cells exposed material. Incorporation was stopped by the addition of 1 M as- to histidinol. *, Control; *, 0.5 mM; 0, 1 mM; A, 2 mM. corbic acid. The cells were then washed twice with phosphate- buffered saline and precipitated with trichloroacetic acid as de- ,ug/ml). These results suggest that CHM initially blocked scribed for the incorporation of [3H]leucine. transit past R; in addition, it slowed the overall rate of M-to-R Materials. CHM and histidinol were from Sigma, [3H]leucine transit and also "set back" those cells that had not passed the and [3H]thymidine were from New England Nuclear, and R point. These cells only later regained ability to enter S. serum and powdered media were from Flow Laboratories A second inhibitor, histidinol, demonstrated the specific role (McLean, VA). of protein synthesis in the ability of A31 cells to enter S.
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