Bulletin of Mathematical Biology (1999) 61, 379398 Article No. bulm.1998.0077 Available online at http://www.idealibrary.

com on

Replication Potential of Cells via the Protein Kinase C-MAPK pathway: Application of a Mathematical Model
Hisham A. El-Masri
Novigen Sci, Inc., 1730 Rhode Island Avenue NW, Suite 1100, Washington, DC 20036, U.S.A.
E-mail: helmasri@novigensci.com

Christopher J. Portier
Laboratory of Computational Biology and Risk Analysis, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, U.S.A.
A mechanistically based mathematical model is used to investigate some of the important factors in priming hepatocytes to enter the G1 phase of the cell cycle. The model considers all of the relevant biochemical mechanisms from signal-receptor binding to the elevation of AP-1(activation protein transcription factor) levels. Focus is centered on the chain of biochemical events governing the sequential activation of protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and AP-1. Factors such as amplitude and duration of growth factors signals, the kinetics of guanosine diphosphate (GDP) to guanosine triphosphate (GTP) conversion, and the negative feedback control mechanisms governing initial steps in cellular replication were theoretically examined. The results of our theoretical assessments support the nding that specic mutations along the PKC-AP1 pathways can have a critical effect on the rate at which cells enter the division cycle. c 1999 Society for Mathematical Biology

1.

I NTRODUCTION

Several mathematical models have been developed to describe the crucial events in the cell cycle. Recently, Novak et al. (1998) developed a mathematical model of ssion yeast growth and division. Their model considered the biochemical checkpoint controls at the G1/S and G2/M with specic emphasis on the temporal uctuation effects of a single cyclin-dependent protein kinase Cdc13/Cdc2 heterodimer and its interactions with other kinases and phosphotases. A previous mathematical model discussed essential features of the mechanisms that are responsible for characteristic properties of Start control in ssion yeast (Novak and Tyson, 1997). Furthermore, Tyson et al. (1995) developed models that relate physiological properties of the cell cycle to molecular checkpoints at the G1 (Start) and G2 phases. Other models focused on the role of the maturation promotion factor (MPF) in induction of
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cellular movement to the mitotic phase (Norel and Agur, 1991). They specically considered the interaction of MPF and cyclin in early embryo development. Their model examined how MPF multiple-cycle activity can be produced and how the internal clock that determines durations and number of cycles can be adjusted by modulating the rate of change in MPF or cyclin concentrations. Similarly, Thron (1991) discussed other models relating to the mitotic clock of cells. Additional mathematical models concentrated on G1 events of the cell cyle as was illustrated by Obeyesekere et al. (1997). They develpoed a mathematical model which integrated the roles of cyclin D, cdk4, cyclin E, cdk2, E2F and RB (retinoblastoma protein). The above models focused on specic biochemical events in relation to the crucial checkpoints of the cycle for cells that are already primed to replicate. However, celluar priming to division is a complex process that is mediated by external and internal stimuli. Analysis of these stimuli is vital because loss of control over cellular replication is a detrimental step in the process of transforming a cell from a normal functioning state to carcinogenesis. Cell replication is a process that is mediated by many cascading phosphorylation and dephosphorylation events. It is also complicated by positive and negative feedback loops, the purpose of which is the tight control of replication. In many instances, cellular replication is stimulated by an external signal to the tissues. One possible scenario is the down ow of the external signal through protein kinase C (PKC) and the mitogen-activated protein kinase (MAPK) to result in the transfer of quiescent cells into the G1 phase of the cell cycle (Boulikas, 1995). In this mechanism, growth factors are implicated in signaling the initiation of cellular replication via binding to cellular receptors. This binding initiates many possible activities including the activation of PKC. Activation of PKC is initiated by a build up of activated guanosine triphosphate (GTP) binding proteins which leads to a sudden activation of phospholipid lipase C (PLC), followed by diacylglycerol (DAG) and mobilization of calcium. Negative feedback processes switch off the calcium rise and brings the PKC down to its steady-state level (Cuthbertson and Chay, 1991). One workable scenario for the negative down regulation of PKC is through the action of active PKC itself which causes an increase in the degradation of GTP. This, in all likelihood, represents the initial negative feedback checkpoint for initiation of replication via PKC pathways. Figure 1 illustrates the biochemical events leading to activation of PKC. Activation of PKC is transferred into the nucleus by multiple steps involving phosphorylation and dephosphorylation of MAPK (Yamaguchi et al., 1995). Initially, PKC activates Raf kinases which then activates MAP kinase-kinase (MAPKK) to ultimately increase nuclear MAPK activity (Boulikas, 1995; Kyriakis et al., 1992). However, this cascade of activation of kinases is controlled by the negative feedback effect of MAPK in deactivating Raf (Ueki et al., 1994). This represents the second possible checkpoint for entry into cellular replication as shown in Fig. 2.

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GGDP Growth Factor Receptor PLC*

GGTP

PLC InsP3 & DAG

(+) (+)

PKC

PIP2

Ca (ER)

Ca2+ (free)

Figure 1. Protein kinase C (PKC) activation mechanism adopted from Cuthbertson and Chay (1991). Growth factor signal binds to a receptor which activates GTP. GTP and diacylglcyerol (DAG) results in activating phospholipase C (PLC) which degrades on phsphatidyl inositol (4,5) bisphosphate (PIP2 ) into DAG and inositol (1,4,5) trisphosphate (InsP3 ). InsP3 production results in release of intercellular calcium (Ca) which along with DAG activates PKC. The cycle is shut down when active PKC degrades active GTP.

RAF (+)

PKC

RAF* () MAPKK MAPK MAPKK* MAPK*

Figure 2. Chain of phosphorylation reactions leading to the activation of MAPK. Active PKC phosphorylates RAF into an active form. Active RAF phosphorylates MAPK kinase (MAPKK) which then phosphorylates MAPK. The chain of events is controlled by the negative feedback loop of active RAF deactivation by active MAPK (Kyriakis et al., 1992; Ueki et al., 1994).

Activation of MAPK leads to elevation of nuclear levels of jun and fos (Boulikas, 1995). The process by which the expression of these proteins is elevated are quite different. In the case of fos, MAPK activates the binding of two other proteins: p62TCF (P62) and the serum response factor (SRF). The hetrodimer of phosphorylated P62 and SRF binds with the fos promoter region on DNA to activate transcription (Boulikas, 1995; Gille et al., 1992). In the case of jun, expression is initially controlled by its phosphorylation status and subsequently through an interaction of jun itself with fos. The prevalent form of jun in tissues is phosphorylated at three specic sites (Thr-239, Ser-243, and Ser-249) which are in proximity to the DNA binding domain (Boyle et al., 1991; Lin et al., 1992). Phosphorylation of jun at these site inhibits binding to DNA. This restraint is removed when jun is partially dephospho-

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MAPK*

jun

junPO4

jundPO4 + DNA

P62

P62*

SRF

SRF*

(+)

(+)

jun DNA
+

AP-1

+ fos

P62*SRF* + DNA

Figure 3. Mechanisms of jun, fos and AP-1. Active MAPK activates P62 and SRF to form a complex which binds to DNA to produce fos. Additionally, active MAPK partially dephosphorylates jun. This results in an initial increase of jun production which is phosphorylated under normal situations. Jun binds with fos to form AP-1 which, upon binding with DNA, results in additional AP-1 formation.

rylated at theses sites, a process which is mediated by active MAPK. Furthermore, jun binds with fos to form AP-1 which induces the expression of jun gene and primes cells to replicate (Curran and Franza, 1988; Angel et al., 1988). Figure 3 depicts the complex chain of events leading to the formation of AP-1 as discussed above. In many situations, the complex mechanisms described above have been explored individually, without reference to their complex interdependencies. This limitation can be overcome by detailed analysis of the collective mechanistic data to form mathematical descriptions. These detailed mathematical expressions, which we denote the replication potential model, allow us to study the broader issue of overall control of cellular replication in its totality. Our purpose is to use this replication potential model to simulate and assess key modications to this mechanism which might affect the early stages in cellular priming. Specically, the replication potential model is used to investigate the effects of factors such as amplitude and duration of growth factor signals, the kinetics of GDP to GTP activation, and the role of the negative feedback loops provided by PKC and the MAPK pathways on the hepatocyte priming process. 2. T HE M ODEL

2.1. Overall model formulation. The overall model consists of three sections that sequentially describe the chain of events initiated by growth factor binding eventually resulting in AP-1 production. The rst section, activation of PKC, focuses on the initial interpretation of the external signal to replicate. The second

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section, activation of MAPK kinases, covers the steps involved in translating the growth signal from the cytoplasm to DNA. The nal section, expression of AP-1, covers the nuclear events leading to expression of this signaling protein. One issue in the estimation of the rate parameters used in this model is worth noting. Most of the available data on the steady-state levels for proteins and their associated mRNAs are expressed between groups of cells as fold increases above steady-state levels. This represented a challenging obstacle to modeling the rates of formation for these cellular proteins. To estimate steady-state levels, this problem was overcome by setting the mass balance rate equations for the production of proteins (or their mRNAs) to zero in most cases. Whenever possible, steady-state levels were also calculated as a ratio of initial concentrations. These approaches added further constraints in the model parameters through the restriction that the model be able to simulate folds of unity magnitudes at steady-state conditions in addition to the requirement to simulate relevant data. Although many of the described biochemical events may occur as reversible reactions, in many situations the model emphasizes the forward direction of these reaction as more salient; especially under external indications. The overall assumptions used in the model are explained in the following detailed description of mathematical equations. Note that, in what follows, where applicable, we will distinguish between the active and inactive fraction of a protein by adding a superscript * to the active fraction only. For example, Raf is the concentration of activated Raf whereas Raf is the concentration of the inactive form of Raf. Also note that the italicized terms are quantities from the model (generally concentrations) and non-italicized terms are abbreviated names for the molecules. Finally, the ratio of the inactive or active fraction of a protein to the total concentration of that protein is denoted by putting an R in front of the respective molecule. Thus, continuing with the example, RRaf denotes the ratio of active Raf to total Raf. 2.2. PKC activation section. Figure 1 is a depiction of this portion of the replication potential model; it was adopted from an earlier calcium model with minor modications (Cuthbertson and Chay, 1991). Although many of the equations in this section can be found in the Cuthbertson and Chay (1991) document, we decided to list them here again for the sake of overall model completeness. The process by which PKC is activated starts with the binding of a growth factor to a receptor which then activates the activation of GDP to GTP as given by the following equation: dGTP = K gf GF GTP hg RPKC dt (1)

where dGTP/dt is the rate of GDP (nM min1 ) activation, K gf is a rst-order constant (min1 ) of growth factor binding to receptor, GF is the concentration (nM) of growth factor (nM), and hg is a rst-order rate constant (min1 ) of GTP inactivation. The rst negative feedback mechanism (see description above) is given

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by the hg constant which is multiplied by the ratio of the active form of PKC to total PKC present (RPKC). Following transition of GDP to GTP, active GTP and DAG stimulate phospholipase c (PLC) as shown by the following equation: RPLC = (DAG/ K d )n (GTP/ K g )n , (DAG/ K d )n + 1 (GTP/ K g )n + 1 (2)

where RPLC is the ratio of active to total PLC, DAG is the concentration (nM) of diaceylglycerol, K d is the Hill binding coefcient (nM) of DAG to PLC, K g is the Hill binding coefcient (nM) of GTP to PLC, and n and m are Hill coefcients for DAG and GTP binding to PLC, respectively. Activated PLC metabolizes plasma membrane phospholipids to form DAG and inositol (1,4,5) trisphosphate (InsP3 ). In the model, the rate of DAG formation is assumed to be rst-order and is governed by the following equation: dDAG = K DAG RPLC hd DAG + ld dt (3)

where dDAG/dt is the rate (nM min1 ) of DAG formation, K DAG is the rate (nM min1 ) of DAG formation by RPLC, hd is a rst-order constant (min1 ) for the breakdown of DAG, and ld is the background release rate (nM min1 ) of DAG. InsP3 concentrations were assumed to be similar to DAG (Cuthbertson and Chay, 1991). InsP3 has been shown to control the release of intracellular stores of calcium (Cuthbertson and Chay, 1991). Control of this release depends on the binding of InsP3 to the receptor subunit of the intracellular channel as described: dCa (InsP3 / K s )n = hc Ca + lc dt (InsP3 / K s )n + 1 (4)

where dCa/dt is the rate (nM min1 ) of calcium release, InsP3 is the concentration (nM) of InsP3 , K s is the Hill binding coefcient of InsP3 to intracellular calcium membrane (controlling release), hc is a rst-order constant (min1 ) of calcium uptake, and lc is the background rate of calcium leakage rate (nM min1 ) from intracellular storage compartments. The release of calcium (Ca) and DAG initiates the activation of PKC through a simple binding of each molecule to inactive PKC as given by the following equation: RPKC = DAG + TPA Ca + RPKCi , K P + DAG + TPA Ca + K C (5)

where RPKC is the fraction of PKC that is active, K P is the binding coefcient for DAG to PKC, K c is the binding coefcient for calcium to PKC, TPA is the concentration of phorbol esters if present in an in vitro experiment being modeled, and RPKCi is the background active portion of PKC.

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2.3. MAPK activation. The second section of the model describes the activation of Raf, MAPKK and MAPK as depicted in Fig. 2. Raf is phosphorylated by active PKC into an active form; assuming this reaction rate is rst-order, the rate of change in activated Raf is given as follows. dRaf = Vraf RPKC Raf K dc Raf RMAPK , dt (6)

where dRaf /dt is the rate (nM min1 ) of active Raf formation, Vraf is the rstorder phosphorylation constant (min1 ) of Raf, and K dc is the rst-order constant (min1 ) representing the second negative feedback mechanism as given by the deactivation of Raf via the active portion of MAPK (RMAPK ). Inactive Raf mass balance equation (dRaf /dt ) is negative of equation (6). The ratio of active Raf (RRaf ) is given by division of Raf by its steady-state concentration (Raf s ). This steady-state concentration is dened as a ratio (1 ) of initial Raf. Similarly, concentration of active MAPKK is calculated by rst describing the phosphorylation of MAPKK. Again, assuming the reaction is governed by rstorder kinetics, cellular concentrations of active MAPKK are calculated by dMAPKK = Vmapkk MAPKK RRaf Vdmapkk MAPKK dt (7)

where dMAPKK /dt is the rate (nM min1 ) of production of active MAPKK production, Vmapkk is the rst-order phosphorylation rate constant (min1 ) of MAPKK, and Vdmapkk is the rst-order rate constant (min1 ) of MAPKK dephosphorylation. The mass balance equation of inactive MAPKK is the negative of equation (7). The ratio of active MAPKK (RMAPKK ) is given by MAPKK /MAPKK s where MAPKK s is the steady-state concentration of MAPKK and is given as a ratio (2 ) of the initial MAPKK. Active MAPKK is then available to phosphorylate MAPK, again using rst-order kinetics as follows: dMAPK = Vmapk MAPK RMAPKK Vdmapk MAPK , dt (8)

where dMAPK /dt is the rate (nM min1 ) of active MAPK production, Vmapk is the rst-order phosphorylation rate constant (min1 ) of MAPK, and Vdmapk is the rst-order rate constant (min1 ) of MAPK* dephosphorylation. The mass balance equation of inactive MAPK is the negative of equation (8). The ratio of active to total MAPK (RMAPK ) is given by MAPK /MAPK s where MAPK s is the steady-state concentration of MAPK and is given as a ratio (3 ) of the initial MAPK. 2.4. Increase of AP-1 levels section. The third section describes the production of AP-1 through the signal cascade starting with active MAPK phosphorylating jun, P62 and SRF. This portion of the model is illustrated in Fig. 3.

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The initial steps in this signal cascade describe the phosphorylation of P62 and SRF which we describe mathematically as dP62 = Vp62 RMAPK P 62 Vdp62 P 62 Kbind RSRF RP62 (9) dt dP62syn dP62 = Vdp62 P 62 Vp62 RMAPK P 62 + (10) dt dt where Vp62 is the rst-order rate constant (min1 ) for phosphorylation of P62 by active MAPK, Vdp62 is the rst-order dephosphorylation rate constant (min1 ) of the phosphorylated form P62 ( P 62 ), Kbind is the rate at which P62 and SRF bind (nM min1 ), and RP62 and RSRF are ratios of active P62 and SRF to their respective steady-state levels. Steady-states levels of both P62 and SRF were derived as a factor (4 ) of their initial levels. To maintain steady-state levels of P62 in the cell after the initiating signal disappears, the term dP62syn /dt (nM min1 ) is included in equation (10) as the rate at which P62 is synthesized to replace P62 changed into fos mRNA. This term is set equal to the rate of degradation of fos mRNA (shown later). SRF activation and mass balance are modeled according to the following equations: dSRF = Vsrf RMAPK SRF Vdsrf SRF Kbind RSRF RP62 (11) dt dSRF syn dSRF = Vdsrf SRF Vsrf RMAPK SRF + (12) dt dt where Vsrf is the rst-order phosphorylation rate constant (min1 ) governing the activation SRF (SRF ), Vdsrf is rst-order rate constant (min1 ) for dephosphorylation of active SRF, and dSRF syn /dt(nM min1 ) is the rate of synthesis of SRF due to loss of SRF during the production of fos mRNA. Both active forms of SRF and P62 bind to form a complex ( P S ) as given below. dPS = Kbind RSRF RP62 Vdna PS dt (13)

where PS is the dimer concentration (nM) and Vdna (min1 ) is the rst-order rate constant for the binding of P62-SRF dimer (PS) to DNA. Following binding of the dimer molecule to DNA, fos mRNA (RNAfos ) is produced (rst-order process) and degraded (also rst-order) as given below: dRNAfos = Vdna PS K drna RNAfos dt where K drna is the rst-order rate constant (min1 ) for mRNA degradation. (14)

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Rfosrna is calculated as the ratio of fos mRNA concentration over its steady-state level in order to simulate published data which are usually reported as fold increase of fos mRNA. Steady-state levels of fos mRNA are given when equation (14) is set to zero as K drna PSss RNAfosss = (15) Vdna where PSss is the steady-state levels of PS when equation (13) is set to zero. Equation (15) then leads to the formula for the production of fos from its message as dfos = K fos RNAfos K dfos fos dt (16)

where K fos (scaled to match fold increase) and K dfos are the rst-order constants (min1 ) of fos synthesis and degradation, respectively, and fos is the concentration of fos in tissue. Most of the constitutively expressed jun is in the phosphorylated form. However, activation of MAPK results in partial dephosphorylations of jun at specic sites which enables jun to bind to DNA and produce additional jun mRNA (Boyle et al., 1991). Hence, the mass balance equation for native jun (non-phosphorylated form) describes its synthesis from basal, partially dephosphorylated jun, and AP-1 controlled mRNA levels in addition to its native phosphorylation rate; described mathematically djun = Vjunsyn RNAjun + Vjunsyn1 RNAjun1 Vjunphos jun dt +Vjunsyn2 RNAjun2 K djun junext ,

(17)

where djun/dt is the mass balance rate (nM min1 ) of native (non-phosphorylated) jun, Vjunsyn is the rst-order rate constant (min1 ) for jun synthesis from basal levels of jun mRNA (RNAjun ), Vjunsyn1 is the rate constant (min1 ) of jun synthesis resulting from the binding of partially dephosphorylated jun to DNA (RNAjun1 ), Vjunphos is the rst-order rate constant (min1 ) for the phosphorylation of native jun, Vjunsyn2 is the rst-order rate constant (min1 ) for the synthesis of native jun from mRNA produced by AP-1 binding to DNA (RNAjun2 ), K djun is the rate of degradation of additional jun formed above its steady-state levels (junext ). Steadystate level of jun (junss ) were assumed to be a ratio (5 ) of its initial value. Following its synthesis, native jun undergoes phosphorylation at the rate djunPO4 = Vjunphos jun Vdjunp junPO4 , dt (18)

where junPO4 is the concentration (nM) of the phosphorylated form of jun, Vdjunp is the rst-order rate constant (min1 ) for the ambient breakdown of junPO4 . To keep

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the steady-state levels of jun and junPO4 constant over time, the rate of basal (not signal induced) jun mRNA production is set as follows: dRNAjun = Vdjunp junPO4 Vjunsyn RNAjun , dt (19)

which leads to the calculation of steady-state levels of jun mRNA when equation (19) is set to zero: Vdjunp junPO4ss RNAjunss = , (20) Vjunsyn where jun PO4ss is the steady-state concentration (nM) of jun PO4 calculated using junss when equation (18) is set to zero. In response to active MAPK, partial dephosphorylation of junPO4 leads to the production of extra jun mRNA as given by djundPO4 = Vdjun1 RMAPK junPO4 Vdna1 jundPO4 , dt (21)

where jundPO4 is the concentration (nM) of the partially dephosphorylated junPO4 , Vdjun1 is the rst-order rate constant (min1 ) for partial dephosphorylation of jun, and Vdna1 is the rst-order constant (min1 ) for binding of jundPO4 to DNA. This binding results in the production of extra jun mRNA (RNAjun1 ) as follows. dRNAjun1 = Vdna1 jundPO4 Vjunsyn1 RNAjun1 . dt (22)

Additional jun mRNA is produced by the binding of AP-1 to DNA. This additional mRNA (RNAjun2 ) is produced linearly as follows. dRNAjun2 = Vdna2 RMAPK AP1 Vjunsyn2 RNAjun2 , dt (23)

where Vdna2 is the RMAPK induced rst-order rate constant for binding of AP-1 to DNA, and AP1 is the concentration (nM) of AP-1 protein derived from the binding of jun to fos. The rate equation for AP-1 production is given by dAP1 = Vap1syn jun fos Vdna2 AP1, dt (24)

where Vap1syn is the second-order rate constant (min1 nM1 ) for synthesis of AP-1. The fold increase of AP-1 at any time (RAP1) is calculated by RAP1 = AP1 , AP1ss (25)

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where and AP1ss is the steady-state amount of AP-1 calculated as AP1ss = Vap1syn fosss junss Vdna2 , (26)

where fosss is the steady-state levels of fos derived from setting equation (16) to zero, and junss is taken as a ratio (5 ) of initial jun. Finally, the overall fold increase of jun mRNA (Rjunr na ), used to simulate available literature data, is given by the following equation: Rjunrna = (RNAjun + RNAjun1 + RNAjun2 ) RNAjunss (27)

2.5. Model parameters. Table 1 lists the estimated model parameters. Many of the parameters used in the model could not be uniquely identied because the available data did not provide actual mRNA or protein levels. However, literature exists on fold increase for all of these mRNAs/proteins allowing for the collective estimation of the parameters. In addition, the parameters are constrained to yield folds of unity magnitude at steady-state conditions. When compared to available literature data and the constraints on steady-state concentration folds applied, simulations of the model allowed the estimations of all parameters except for the ones in the PKC activation section. With the exception of RPKCi , parameters in the PKC section were obtained from an earlier paper by Cuthbertson and Chay (1991). RPKCi and the remaining parameters were determined by optimizing the model simulations to literature results. These optimizations are described below. Ueki et al. (1994) applied 100 ng ml1 of the phorbol ester 12- O -tetradecanoylphorbol-13-acetate (TPA) to Chinese hamster ovary (CHO) cells and measured fold increases of Raf, MAPKK and MAPK activities. Using our model, an equivalent TPA (MW = 616.92) dose of 162 nM was applied as described in equation (5). The parameters in the MAPK activation section were estimated from these data using nonlinear least-squares optimizations. Figure 4 compares the simulated behavior of the model, using the estimated parameter values, to the relevant data. The predicted three-fold activations of Raf, MAPKK and MAPK are consistent with observations made in other cell systems such as cultured primary hepatocytes (Gines et al., 1995). There were insufcient data to formally estimate all model parameters in the AP-1 activation section. This clearly indicates a parameter identiability problem which must be addressed at a later stage. One way of moving forward in cases on nonidentibility is to use scientic judgement on values for the model parameters and to adjust the parameters to match the data visually. This does not replace objective estimation methods, but allows for the use of the model in studying how the signaling cascade works. For the AP-1 activation section, visual methods were used to obtain the model to match fold levels of unity at steady-state situations and to simulate fos

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Table 1. List of estimated model parameters.

Parameter Value Units MAPK activation sectiona RPKCi 0.006 (unitless) Vraf 1.6 min1 K dc 0.04 min1 Vmapkk 0.02 min1 Vdmapkk 2 min1 Vmapk 0.01 min1 Vdmapk 1.1 min1 1 0.1 unitless 0.1 unitless 2 0.1 unitless 3 fos sectionb Vp62 0.01 min1 Vdp62 0.05 min1 Kbind 0.5 nM min1 Vsrf 0.1 min1 Vdsrf 0.01 min1 Vdna 0.3 min1 K drna 10 min1 K fos 1 min1 K dfos 0.17 min1 4 0.05 unitless jun sectionb Vjunsyn 1 min1 Vjunsyn1 1 min1 Vjunsyn2 0.1 min1 Vjunphos 1 min1 K djun 10 min1 Vdjunp 1 min1 Vdjun1 0.1 min1 Vdna1 1 min1 Vdna2 5 min1 5 0.083 unitless AP-1 sectionc Vap1syn 0.43 min1 nM1 a Statistically optimized using data of Ueki et al. (1994). b Visually optimized using data of Westwick et al. (1995). c Visually estimated to yield unity fold activity of AP-1 at steady state.

and jun mRNA fold increase literature data (Westwick et al., 1995). In order to reproduce their experiment mathematically, the model was run presuming a partial hepatectomy. To model this, a signal of an early (rst 30 min) and delayed (30 min to 6 hr) prolactin peak of magnitude 50 ng ml1 (2 nM) was introduced as the growth

Cellular Replication Potential Mathematical Model

3.5 Fold RAF activity Fold RAF activity 3 2.5 2 1.5 1 0.5 1000 1010 1020 1030 1040 1050 Time (min) 3.5 Fold RAF activity 3 2.5 2 1.5 1 0.5 1000 1010 1020 1030 1040 1050 Time (min)
Figure 4. Model simulations in comparison to time course data of (a) RAF, (b) MAPKK, and (c) MAPK activities. Data were obtained from in vitro experiments by Ueki et al. (1994). The model had to be run for 1000 simulated minutes to achieve steady-state before the signal was introduced.

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(b)

3.5 (a) 3 2.5 2 1.5 1 0.5 1000 1010 1020 1030 1040 1050 Time (min)

(c)

factor (GF ) in equation (1) in agreement with previously published data produced by Buckley et al. (1991). Figure 5 illustrates the t of the model simulations for fos and jun mRNA levels to their respective data. Finally, the two parameters governing AP-1 production (Vap1syn , Vdna2 ) were adjusted simultaneously with the parameters for fos and jun production to yield a fold value of unity for AP-1 under steady-state conditions after long (1000 min) simulation runs.

3.

R ESULTS AND D ISCUSSION

3.1. fos and jun mRNA levels. Figure 5 shows an increase of fos mRNA to about 2.5 fold of its steady-state levels almost exactly matching the peak values observed earlier (Westwick et al., 1995). For jun mRNA levels, the model predicts a maximum increase to about three fold over its steady-state levels occurring earlier than the documented 3.5 fold increase (Westwick et al., 1995). There is an extremely fast decline in fos mRNA which may be attributed to the sharp decrease of MAPK

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(a) Fold c-jun mRNA 2 3.5 3 (b)

Fold c-fos mRNA

2.5 2 1.5 1

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0.5 1000

1050

1100

1150

1200

1250

0.5 1000

1050

1100

1150

1200

1250

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Time (min)

Figure 5. Model simulations in comparison to time course data of (a) fos mRNA, (b) jun mRNA induction. Data were obtained from Westwick et al. (1995). The model had to be run for 1000 simulated minutes to achieve steady state before the signal was introduced.

2.6 2.4 2.2 2 Fold 1.8 1.6 1.4 1.2 1 1000 1020 1040 1060 Time (min)
Figure 6. Model simulations of fos (solid line), jun (dashed line) and AP-1 (dotted line) in response to an assumed signal of amplitude 100 nM and duration of 10 min. The model had to be run for 1000 simulated minutes to achieve steady state before the signal was introduced.

c-jun c-fos AP-1

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activity caused by the negative feedback effect of active Raf. Initially, levels of jun mRNA decline at slower rates because of the positive feedback inherent in jun production. This feedback dampens the sharp decline of the activating MAPK. This positive feedback is lacking from the fos production kinetics which returns to its steady-state levels once active MAPK does. The rapid drop in jun mRNA at 50 min following its peak is due to the loss of fos production. 3.2. AP-1 levels and replication potential simulations. As shown in Fig. 6, mean levels of fos, jun and AP-1 were estimated by model simulations using a hypothetical growth factor signal of magnitude 100 nM and 10 min duration [substituted into GF in equation (1)]. AP-1 peaks after fos and jun triggering the cell to enter the

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cell cycle and proceed to replication. This is a mean value for AP-1, and, in a large collection of cells, represents the potential for the cell to enter the cell cycle. The greater the quantity of AP-1 produced in a given period of time, the greater the potential for cell to cycle is. A useful quantity for measuring this replication potential is the integrated (over time) ratio (RAP1) of active to steady-state AP-1. In this manner, the effect of amplitude and duration of the AP-1 curve are lumped together. We dened replication potential as the magnitude of this integral. 3.3. Effects of growth factor signaling on replication potential. Continued increase of AP-1 above steady-state levels is considered to be a possible cause for the high replication rate in colonies of cells mutated by some chemical initiator (Brenner et al., 1989). Conversely, inhibition of AP-1 mediated transcriptional activities was shown to be sufcient in some cases to suppress the tumorigenic phenotype of malignant cells (Bowden et al., 1994). The model can be used to test the effect of various biochemical modications (mutations) on replication potential. Consider rst the effect of mutations on growth-factor signaling. Signal amplitude and duration are the two variables that relate to mutations in the production of growth factors, changes in signal amplitude and duration will be investigated for their effects on replication potential. To facilitate this analysis, signals were forced to be of a single pulse shape with amplitude () and duration ( t ). This growth factor signal is then multiplied by the binding rate constant K gf [see equation (1)] which, without loss of generality, was set to unity (min1 ). An initial signal of = 0.1 nM and t = 0.1 was tested and did not elicit any response (replication potential equal to zero). Gradual increases in the signal duration were considered with xed at 0.1 nM; results are shown in Fig. 7(a). Conversely, signal amplitude was increased gradually for a xed duration ( t = 0.1 min) to generate Fig. 7(b). Both gures illustrate a nonlinear (threshold-like) relationship between signal amplitude and duration with replication potential. This result is due to the pulsing nature of intracellular calcium release kinetics, and the eventual activation of PKC (Cuthbertson and Chay, 1991). Further analysis of the effect of signal duration was assessed at higher signal amplitudes = 100 (nM). The duration of the growth factor pulse was varied from t = 0.560 min. Variations of signal duration showed a gradual increase in replication potential, but at this magnitude of the signal, no strong nonlinear behavior was observed. Signal amplitude was varied from a range of = 0.1100 (nM) for a xed t = 10 min. This variation of amplitude showed a slight gradual increase from no effect at = 0.1 to a saturable response. To investigate the effect of signaling on replication further, a case of no effect ( = 0.1 nM, t = 10 min) was investigated by changing it to a constant signal of same amplitude by keeping = 0.1 and setting t = . The results of this simulation showed an increase of replication potential up to a saturable effect level equal to 62 min. These simulations indicate that the accumulation of low amplitude signals is eventually able to trigger the

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Signal duration (min) 25.0 Replication potential 20.0 15.0 10.0 5.0 0 0 0.2 0.4 0.6 0.8 1.0 1.2 Signal amplitude (mM) (b)

Figure 7. Effects of signal duration and amplitude on replication potential. (a) Signal duration is varied for xed signal amplitude of 0.1 nM. (b) Signal amplitude is varied for a xed signal duration of 0.1 min. Dots are results of model simulations in both gures.

system, perhaps as an after effect of the accumulations of jun in response to its positive feedback loops. 3.4. Effect of mutations in the GDP to GTP activation rate on replication potential. To study the effect of specic mutations along the PKC pathway on replication potential, the initiating signal was xed at = 100 nM and t = 10 min. These conditions gave a replication potential of 62 min in the baseline model. Mutations leading to an increased rate of GTP binding will result in replication potential similar to those seen for the increased signal amplitude. However, when a mutation blocks or destroys the negative feedback effect on GTP activation by PKC [effectively setting to hg = 0 in equation (1)], replication potential increases in magnitude up to a higher saturable value. No further increase of replication potential ensued because of the down stream negative feedback loop of Raf. These results indicate it is possible for a single mutation that increases GTP activation to increase the saturable level of replication potential, but by itself, it is not sufcient to cause a sustainable increase in replication potential.

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Figure 8. Model simulations of proliferation potential in response to loss of MAPK negative feedback checkpoint. The model had to be run for 1000 simulated minutes to achieve steady-state before the signal was introduced.

3.5. Alterations of negative feedback checkpoints. Two checkpoints against sustained cellular replication exist in the model based upon the deactivation of GTP by PKC (hg) and the deactivation of active Raf by MAPK ( K dc ). Several scenarios exist for testing the effects of mutations in these checkpoints. Two examples are given here based on two GF signals covering the range from no effect on replication potential to saturation of replication potential. The rst example uses a constant signal of = 0.1 and t = which produces replication potential saturating at 62 min. The second example uses a single pulse signal of = 0.1 and t = 10 which did not produce any replication potential. For the constant low amplitude signal, removing the effect of PKC negative feedback (hg = 0) resulted in an increase in the replication potential from 62 to 200 min. However, this increase reached saturation and no further increase above 200 was detected. Alternatively, when the negative effect of Raf activation is removed ( K dc = 0), replication potential has increased linearly as shown in Fig. 8. Another example was used for a high amplitude signal ( = 100 nM, and t = 10) which indicated that replication potential increases to a saturable value of 262 min when hg = zero and a linear sustained increase when K dc is set to zero. In both scenarios, removal of the PKC checkpoint increased replication potential to a saturable level, and removal of the MAPK checkpoint resulted in a linear sustainable increase of replication potential. 4. C ONCLUSION Theoretical deliberations with the model suggest that cellular replication via the PKC and MAPK pathways require a signal of minimum duration and amplitude to start. Once red it will increase linearly with signal duration and saturably with

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amplitude. Alterations of the GTP activation kinetics follow that of the signal amplitude prole concurring with saturable receptorligand mechanisms. Furthermore, cellular replication is controlled by two important negative feedback mechanisms; upstream deactivation of GTP by active PKC and downstream deactivation of Raf by active MAPK. When both mechanisms were investigated under different assumptions, the negative action of MAPK is the only one that could elicit sustainable increase in replication potential. Although it may seem that loss of MAPK checkpoint is responsible for uncontrollable replication, loss of negative feedback of PKC could not be ignored. An increase of replication may be detrimental if not offset by a matched level of cellular loss to maintain cellular homeostasis. However, loss of MAPK activity results in a linear and continuing increase in replication potential which demands apoptosis to also increase at the same rate if tissue size is to be maintained constant. This places an additional burden on tissues to maintain homeostasis. The empirical mechanistic model presented in this paper was constructed for investigating the effects of selected variables on priming a cell to enter replication. The focus of this investigation was on the signaling pathway by which the PKCMAPK activation mechanism produces AP-1 as the priming agent. Although, other mechanisms are involved in AP-1 production, our selected mechanisms are based on the early events by which tissues respond to proliferative signals (Buckley et al., 1987; Fausto and Webber, 1993). In general, further extensions of the model to eventual cellular division would require the inclusion of other biochemical factors such as ras and c-myc. Analysis of factors inuencing entry of a cell into G1 phase of the cell cycle is prudent since this process is a necessary, but not sufcient, condition for replication. The mathematical model presented here sets a complete picture of the documented priming process that enables the identication of some crucial inuencing factors. The model can also be used to design experiments along critical points. Interestingly, the possibility of continually increasing cellular replication by inhibiting the MAPK negative feedback loop was demonstrated to take place under low, but continuous, growth factor signaling. This low continuos signal can be applied with inhibitors of the MAPK negative feedback to induce sustained replication potential, and probably replication, without having to resort to surgical (partial hepatectomy) or chemical (carbon tetrachloride) methods. The experimental value of such methods for detecting increased carcinogenic potential of chemicals is evident. R EFERENCES
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Received 21 June 1998 and accepted 17 January 1999