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Is the A(G0) state time-independent?
J. theor. Biol. (1979) 79, 259-262
Is the A(Go) State Time-independent? Cells which cease cycling can be considered to enter the Go (Lajtha, 1963) or A (Smith & Martin, 1973) state. According to the transition probability model (Smith & Martin, 1973; Burns & Tannock, 1970), such cells have a certain probability-the transition probability-of re-entering the mitotic cycle (B phase) and continuing towards cell division [Fig. 1(a)]. This model has evoked serious criticism from several authors. Pledger and co-workers
B phase
Gel I -death
FIG. 1. (a) A standard view of the cell cycle. P is the transition probability. (b) A modified view of the cell cycle. X, Y and 2 represent successivepoints in time in the A state. P,, P, and P, are the transition probabilities at times X, Y and Z. T,, Tr and T, are the lengths of time taken for the transltion P, < P, < P, and T, > T, > T,. At point Z, P, = 0 and T, = co. The cell is irreversibly trapped in G,(A) and proceeds towards cell death. 259 OO22-5193/79/140259+04 $02.00/o :Q 1979 Academic Press Inc. (London) Ltd.
260
C.
H.
EVANS
(Pledger, Stiles, Antoniades & Scher, 1978) among others, have demonstrated that an ordered sequence of different steps, rather than one “master-switch” triggered at random, precedes commitment to DNA synthesis and subsequent cell division. Nevertheless, the passage of cells into the mitotic cycle does follow first order kinetics and the model does describe the entry of cells into the B phase; whether or not it controls it is controversial and not the subject of this communication. The matter has been well reviewed by several authors (e.g. Gelfant, 1977). Experiments conducted to test the transition probability hypothesis have employed conditions where cells spend a relatively short time in the A state (e.g. Shields, 1977 ; Shields & Smith, 1977). It is assumed by the model in particular, and by many cell biologists in general, that the quescent cell is time-independent. Thus the .4 state has been described as a “sort of limbo” (Shields & Smith, 1977) such that a cell “may remain in this state for any length of time throughout which its probability of entering the B phase is constant” (Smith & Martin, 1973). Here I suggest that the A state is neither time-independent nor indefinitely reversible. Mammalian cells maintained in long term quiescence in vitro undergo time-dependent alterations. The pre-replicative phase following mitogenic stimulation of resting, human, diploid fibroplasts increases with the time of quiescence (Augenlicht & Baserga, 1974). Mouse fibroblasts also show this (C.H.E., unpub.), as does a cell-cycle mutant of Chinese hamster ovary cells which reversibly arrests in Go (Crane & Thomas, 1976). With mammalian fibroblasts (Augenlicht & Baserga, 1974; C.H.E., unpub.), an increasing fraction of non-dividing cells fails to respond at all to mitogenic stimuli, suggesting that the processes occurring during G, which result in diminished mitotic response proceed to a situation where the cell is irreversibly trapped in a non-dividing state. At this point, the transition probably is zero. Thus the transition probability itself may decrease as the time spent in the A state increases. Data pertaining to resting cells in vivo support this contention. Following partial hepatectomy, the liver cells of adult rats, which normally divide only rarely, are stimulated to proliferate. The pre-replicative period before this response increases with the time these cells have remained in the nondividing state, i.e. the adult age of the rat. Cells newly formed after such partial hepatectomy respond to subsequent stimulation much quicker. Lymphocytes also have extended inter-mitotic periods in rive. Their response to the mitogen polyhaemagglutanin declines with age, cells becoming blocked in G, (Preumont, Van Gansen & Brachet, 1978). A more realistic view of the cell cycle might therefore be that shown in Fig. l(b). Several of the changes that accompany long-term quiescence resemble
LETTERS
TO
THE
EDITOR
261
those occurring during the ageing of cells both in viro and in vitro: if the A state is indeed time-dependent, cells therein are, by definition, ageing. Alterations in the morphology and ultrastructure (Brunk, Ericsson & Ponten, 1973), chromatin (Evans. Van Gansen & Rasson, 1978; Rossini, Lin Sr Baserga, 1975) and pattern of transcription (Evans, Van Gansen & Rasson, 1978) of cells “senescing” in the classical in vitro manner parallel those found in cells maintained for extended periods in a non-mitotic state. As primary cultures of mammalian fibroblasts age, an increasing proportion of cells either cease to divide, or have extended inter-mitotic periods. The DNA content of such cells is compatible with their arrest in Go. A similar case can be made for cells ageing in riro. This is discussedfully elsewhere (Evans, 1979). Grove & Cristofalo (1976) have attempted to analyze ageing cultures of human, diploid fibroblasts by application of the transition probability. without success.However, on their premises, we would predict this result. In their article the authors consider the loss of replicative ability of diploid fibroblasts in the light of differentiational (Cristofalo, 1972) or “error” (Orgel, 1963) events. The transition probability hypothesis does not address itself to differentiation; neither can it be held to describe the results of the missynthesisof proteins, mutations, etc., that might curtail mitosis in ageing fibroblasts. Hence in neither case would agreement with the model be anticipated. Furthermore, their analysis assumes,as do all such treatments. that the A state and transition probability are independent of time. The data of Grove & Cristofalo (1976) provide evidence for multiple transition probabilities in late passagecultures. This is consistent with my model of a heterogenous population of cells which are independently undergoing ageing in the non-mitotic state and proceeding towards cell death, as shown in Fig. l(b) (Evans, 1979). CHRISTOPHER Department of Orthopaedic Surgery, University qf Pittsburgh Medical School, Pittsburgh. PA 15261, U.S.A. (Recewed 14 August 1978, and in revised form 28 November 1978)
REFERENCES AUCEN!XTHT, BRUNK. J., BURNS, F. CRANE. M. CRISTOFALO, EVANS. C. EVANS, C.
L. H. & BASERGA, R. (1974). E.\rpt. Cell Res. 80, 255. ERICS~N. L. E. & PONTEN, J. (1973). E.Ypt. Cell Res. 70. 1. J. & TANNOCK. I. F. (1970). Cell Tk. Kinet. 3, 321, ST. J. & THOMAS, D. B. (1976). Nature 261, 205. V. J. (1972). Adv. Gerontol. Rex. 4, 45.
H. (1979). H.,
Med.
VAN GANSEN.
H.vporh.
5, 53.
P. & RASSON,
I. ( 1978).
Biol.
Cell. 33. 117
H.
.
EVANS
262 GELFANT,
C
S. (1971).
Cancer
H
EVANS
Rex 37, 3845.
GROVE.
G. L. & CRISTOFALO. V. J. (1976). Cell Ti,rs. Kiner. 9. 395. LAJTHA. L. G. (1963). J. Cell Comp. Physiol. 60, suppl. 1, 143. ORGEL, L. E. ( 1963). Proc,. natn. Ac,ad. Sci. U.S.A. 70. 1263. PLEDC;ER, W. J., STILES, C. D., ANTONIADES. H. N. & SCHER, C. D. ( 1978)
(1.S.A. PREUMONT, ROSSINI. SHIELDS. SHIELDS, SMITH. J.
Proc. nafn. Acad. Sd
75, 2839. A. M.. VAN GANSEN. P. & BRACHET, J. (1978). Mech. Age. Develop. M.. LIN. J. C. & BASERGA. R. (1975). J. c,ell. Physiol. 88, 1. R. (1977). Nuture 267. 704. R. & SMITH. J. A. ( 1977). J. c,ell. Physiol. 91, 345. A. & MARTIN. L. (1973). Prw. noin. Ac,ad. Sci. U.S.A. 70, 1263.
7, 25.
Is the A(Go) State Time-independent? Cells which cease cycling can be considered to enter the Go (Lajtha, 1963) or A (Smith & Martin, 1973) state. According to the transition probability model (Smith & Martin, 1973; Burns & Tannock, 1970), such cells have a certain probability-the transition probability-of re-entering the mitotic cycle (B phase) and continuing towards cell division [Fig. 1(a)]. This model has evoked serious criticism from several authors. Pledger and co-workers
B phase
Gel I -death
FIG. 1. (a) A standard view of the cell cycle. P is the transition probability. (b) A modified view of the cell cycle. X, Y and 2 represent successivepoints in time in the A state. P,, P, and P, are the transition probabilities at times X, Y and Z. T,, Tr and T, are the lengths of time taken for the transltion P, < P, < P, and T, > T, > T,. At point Z, P, = 0 and T, = co. The cell is irreversibly trapped in G,(A) and proceeds towards cell death. 259 OO22-5193/79/140259+04 $02.00/o :Q 1979 Academic Press Inc. (London) Ltd.
260
C.
H.
EVANS
(Pledger, Stiles, Antoniades & Scher, 1978) among others, have demonstrated that an ordered sequence of different steps, rather than one “master-switch” triggered at random, precedes commitment to DNA synthesis and subsequent cell division. Nevertheless, the passage of cells into the mitotic cycle does follow first order kinetics and the model does describe the entry of cells into the B phase; whether or not it controls it is controversial and not the subject of this communication. The matter has been well reviewed by several authors (e.g. Gelfant, 1977). Experiments conducted to test the transition probability hypothesis have employed conditions where cells spend a relatively short time in the A state (e.g. Shields, 1977 ; Shields & Smith, 1977). It is assumed by the model in particular, and by many cell biologists in general, that the quescent cell is time-independent. Thus the .4 state has been described as a “sort of limbo” (Shields & Smith, 1977) such that a cell “may remain in this state for any length of time throughout which its probability of entering the B phase is constant” (Smith & Martin, 1973). Here I suggest that the A state is neither time-independent nor indefinitely reversible. Mammalian cells maintained in long term quiescence in vitro undergo time-dependent alterations. The pre-replicative phase following mitogenic stimulation of resting, human, diploid fibroplasts increases with the time of quiescence (Augenlicht & Baserga, 1974). Mouse fibroblasts also show this (C.H.E., unpub.), as does a cell-cycle mutant of Chinese hamster ovary cells which reversibly arrests in Go (Crane & Thomas, 1976). With mammalian fibroblasts (Augenlicht & Baserga, 1974; C.H.E., unpub.), an increasing fraction of non-dividing cells fails to respond at all to mitogenic stimuli, suggesting that the processes occurring during G, which result in diminished mitotic response proceed to a situation where the cell is irreversibly trapped in a non-dividing state. At this point, the transition probably is zero. Thus the transition probability itself may decrease as the time spent in the A state increases. Data pertaining to resting cells in vivo support this contention. Following partial hepatectomy, the liver cells of adult rats, which normally divide only rarely, are stimulated to proliferate. The pre-replicative period before this response increases with the time these cells have remained in the nondividing state, i.e. the adult age of the rat. Cells newly formed after such partial hepatectomy respond to subsequent stimulation much quicker. Lymphocytes also have extended inter-mitotic periods in rive. Their response to the mitogen polyhaemagglutanin declines with age, cells becoming blocked in G, (Preumont, Van Gansen & Brachet, 1978). A more realistic view of the cell cycle might therefore be that shown in Fig. l(b). Several of the changes that accompany long-term quiescence resemble
LETTERS
TO
THE
EDITOR
261
those occurring during the ageing of cells both in viro and in vitro: if the A state is indeed time-dependent, cells therein are, by definition, ageing. Alterations in the morphology and ultrastructure (Brunk, Ericsson & Ponten, 1973), chromatin (Evans. Van Gansen & Rasson, 1978; Rossini, Lin Sr Baserga, 1975) and pattern of transcription (Evans, Van Gansen & Rasson, 1978) of cells “senescing” in the classical in vitro manner parallel those found in cells maintained for extended periods in a non-mitotic state. As primary cultures of mammalian fibroblasts age, an increasing proportion of cells either cease to divide, or have extended inter-mitotic periods. The DNA content of such cells is compatible with their arrest in Go. A similar case can be made for cells ageing in riro. This is discussedfully elsewhere (Evans, 1979). Grove & Cristofalo (1976) have attempted to analyze ageing cultures of human, diploid fibroblasts by application of the transition probability. without success.However, on their premises, we would predict this result. In their article the authors consider the loss of replicative ability of diploid fibroblasts in the light of differentiational (Cristofalo, 1972) or “error” (Orgel, 1963) events. The transition probability hypothesis does not address itself to differentiation; neither can it be held to describe the results of the missynthesisof proteins, mutations, etc., that might curtail mitosis in ageing fibroblasts. Hence in neither case would agreement with the model be anticipated. Furthermore, their analysis assumes,as do all such treatments. that the A state and transition probability are independent of time. The data of Grove & Cristofalo (1976) provide evidence for multiple transition probabilities in late passagecultures. This is consistent with my model of a heterogenous population of cells which are independently undergoing ageing in the non-mitotic state and proceeding towards cell death, as shown in Fig. l(b) (Evans, 1979). CHRISTOPHER Department of Orthopaedic Surgery, University qf Pittsburgh Medical School, Pittsburgh. PA 15261, U.S.A. (Recewed 14 August 1978, and in revised form 28 November 1978)
REFERENCES AUCEN!XTHT, BRUNK. J., BURNS, F. CRANE. M. CRISTOFALO, EVANS. C. EVANS, C.
L. H. & BASERGA, R. (1974). E.\rpt. Cell Res. 80, 255. ERICS~N. L. E. & PONTEN, J. (1973). E.Ypt. Cell Res. 70. 1. J. & TANNOCK. I. F. (1970). Cell Tk. Kinet. 3, 321, ST. J. & THOMAS, D. B. (1976). Nature 261, 205. V. J. (1972). Adv. Gerontol. Rex. 4, 45.
H. (1979). H.,
Med.
VAN GANSEN.
H.vporh.
5, 53.
P. & RASSON,
I. ( 1978).
Biol.
Cell. 33. 117
H.
.
EVANS
262 GELFANT,
C
S. (1971).
Cancer
H
EVANS
Rex 37, 3845.
GROVE.
G. L. & CRISTOFALO. V. J. (1976). Cell Ti,rs. Kiner. 9. 395. LAJTHA. L. G. (1963). J. Cell Comp. Physiol. 60, suppl. 1, 143. ORGEL, L. E. ( 1963). Proc,. natn. Ac,ad. Sci. U.S.A. 70. 1263. PLEDC;ER, W. J., STILES, C. D., ANTONIADES. H. N. & SCHER, C. D. ( 1978)
(1.S.A. PREUMONT, ROSSINI. SHIELDS. SHIELDS, SMITH. J.
Proc. nafn. Acad. Sd
75, 2839. A. M.. VAN GANSEN. P. & BRACHET, J. (1978). Mech. Age. Develop. M.. LIN. J. C. & BASERGA. R. (1975). J. c,ell. Physiol. 88, 1. R. (1977). Nuture 267. 704. R. & SMITH. J. A. ( 1977). J. c,ell. Physiol. 91, 345. A. & MARTIN. L. (1973). Prw. noin. Ac,ad. Sci. U.S.A. 70, 1263.
7, 25.