Chapter 6 Cell Cycle Kinetic Profiles for Rapamycin and Wortmannin Effect on TGF-β1-Mediated DNA Synthesis, Protein Synthesis, and System A Neutral Amino Acid Transport in C3H-10T½ Murine Embryonic Fibroblasts

Introduction

Understanding metabolic integration is important in investigating the biochemical basis of cancer. This macroscopic perspective sets the stage for examining how extracellular signals lead to specific cellular consequences. Such biochemical approaches provide valuable insights into regulatory paradigms that impact global cellular state.

Radiolabeled substrates are useful for monitoring cellular responses without extensive disruption of physiologic processes. While handling and disposing radioactive materials pose limitations in experimental design, radioactive bioassays uncover cellular responses to mitogens and growth inhibitors based on measurements of DNA synthesis, protein synthesis, and substrate transport. Cellular solubilization for liquid-scintillation counting allows quantification of radiolabeling results at the molecular level if the specific activities of radiolabeled substrates are known. While autoradiography provides visual information this is not an easily quantifiable method.

The following experiments present information on the effects of transforming growth factor- (TGF-) β1 on cell cycle progression in C3H-10T½ murine embryonic fibroblasts at the level of macromolecular synthesis and transport. DNA synthesis was measured by tritiated-thymidine incorporation, protein synthesis by tritiated-leucine incorporation, and amino acid transport by a ¹⁴C-labeled substrate.

Materials and Methods

Cell culture and growth factor treatment

Cells were cultured and treated with growth factor or growth inhibitors according to Materials and Methods, Chapter 2.

Radiolabeling Assay and Preparation of Radiolabeling Solution

Confluent cells treated with TGF-β1 or inhibitors were radiolabeled for analysis of macromolecular synthesis (incorporation) or substrate transport as described in Materials and Methods, Chapter 2.

Results

TGF-β1 dose response in C3H-10T½ murine embryonic fibroblasts: ³H-thymidine uptake and incorporation

To establish the optimal growth factor concentration for stimulating cell cycle progression in murine fibroblasts, deoxyribonucleic acid (DNA) synthesis was monitored as a function of TGF-β1 concentration by measuring tritiated-thymidine (³H-Thd) incorporation. Optimal concentration of transforming growth factor-beta 1 (TGF-β1) for stimulating DNA synthesis was determined to be 10 nanograms per milliliter (10 ng/ml) of media vehicle supplemented with 0.5% FBS, Figure 6-1B. Increased thymidine incorporation at higher TGF-β1 concentrations may be due to increased cellular thymidine pools. Thymidine uptake was shown to reach an elevated steady state under TGF-β1 stimulation as compared with control cells not receiving TGF-β1 and this correlated with increased DNA synthesis, Figure 6-1A. Tritiated-thymidine incorporation into cellular DNA may be primed by increased substrate uptake during G0 or G1. Correlation between uptake and incorporation therefore depended on the temporal distance between G0-G1 phases and S-phase. This observation underscores the importance of consistent cell staging to achieve synchrony by growth arrest and serum deprivation and therefore in developing cell-based assays.

Kinetics of proliferation assay response to TGF-β1: ³H-thymidine incorporation

To establish the time of cell-cycle entry induced by TGF-β1 in 10T½ fibroblasts, confluent cells were staged (serum-deprived) and treated with 10 ng/ml of TGF-β1. Stimulated fibroblasts were pulse-labeled with tritiated-thymidine for 1-hour intervals spanning 60 hours. TGF-β1 initiated cell-cycle entry kinetics at 16 hours, with an optimal stimulation window at 24-36 hours. Thymidine incorporation decreased at 40 hours. At 48 hours, a second wave of stimulation appeared to cease at 56 hours, Figure 6-2A. While stimulation at the later time points did not reach peak levels observed at 36 hours, this second wave of stimulation may contribute to the biological activity of TGF-β1 over an extended-treatment period in therapeutic applications of this growth factor. Mechanisms governing this second window of stimulation are unknown. Based on these observations, biochemical assays measuring S-phase entry in C3H 10T½ fibroblasts were designed to reflect delayed cell cycle kinetics characteristic of TGF-β1 by extending treatment period beyond 24 hours of growth-factor incubation.

Cell cycle entry kinetics stimulated by TGF-β1 was compared with those by TGF-α, a growth factor that signals through the EGF receptor. Like EGF, TGF-α displayed early cell-cycle entry with peak DNA synthesis occurring at 14 hours, Figure 6-2B. By 24 hours, DNA synthesis had ceased to basal levels. A second and slight increase in stimulation by TGF-α occurred at 28 hours. These results demonstrate that TGF-α and TGF-β1 display different cell cycle entry kinetics.

Early kinetics of TGF-β stimulated leucine uptake and protein synthesis

An early effect of growth factor addition is pronounced amino acid transport. Cell cycle progression intimately reflects cellular physiological state and may therefore be expressed as a function of energy profiles including glycolysis and oxidative phosphorylation. Induction of components involved in energetic pathways thus plays an important role in driving G1/S phase transition.

To examine how TGF-β1 affected early amino acid transport events, tritiated-leucine was administered at the time of growth-factor addition. Cellular accumulation of radioactive leucine was measured. Presentation of tritiated-leucine to TGF-β1-treated cells induced a sharp peak in uptake followed by a depression during the first 20 minutes of exposure. Control cells receiving no growth factor exhibited a slow but steady increase in uptake, Figure 6-3. The "wave-like" phenomenon in uptake observed in TGF-β1-stimulated cells may be due to a sharp up-regulation of growth factor-dependent transport activity (initial overshoot) that was rapidly compensated by acute adaptation. Chronic adaptation may subsequently increase substrate uptake. This observation of leucine uptake in TGF-β1-treated cells and later in control cells suggested that leucine uptake may not occur in a steady manner but rather in a punctuated manner. In addition, the uptake "wavelength" of control cells was longer than that for stimulated cells, suggesting that the mechanisms of adaptation may be enhanced during stimulation.

In addition to measuring DNA synthesis, determining amino acid availability (transport functions) and incorporation (protein synthesis) also reflects cell cycle progression. The transformed Chinese hamster fibroblast line V79-8 is an illustration of the importance of protein synthesis in initiating DNA synthesis: under normal growth conditions, these fibroblasts lacked a detectable G1 phase, while partial inhibition of protein synthesis with cycloheximide induced a G1 phase (Liskay et al, 1980). This supported a strong requirement for continued protein synthesis towards S-phase progression.

Early kinetics of TGF-β1-mediated protein synthesis showed that while protein synthesis occurred when tritiated leucine was presented to both control cells and TGF-β1-treated cells, significant induction of protein synthesis was observed in TGF-β1-stimulated fibroblasts by four hours, Figure 6-4. Compared with control cells, TGF-β1-stimulated cells consistently accumulated more leucine. A threshold of amino acid availability may be required for protein synthesis to activate. Whereas TGF-β1-stimulated S-phase entry monitored by DNA synthesis was observed to begin as late as 16 hours, the induction of protein synthesis by TGF-β1 occurred earlier at 4 hours. This observation supports the increased functional demand hypothesis (Novi, 1976), and in this particular model system the addition of leucine to quiescent fibroblasts activated pre-existing translational components in both control cells and cells receiving TGF-β1. The presence of TGF-β1 induced de novo synthesis of additional components required in achieving critical cell mass for G1/S phase progression.

Modified HBSS-depletion test against trans-inhibition by TGF-β1 stimulated intracellular neutral amino acid pools

TGF-β1 profoundly affects cellular metabolic activities by influencing transporter functions and utilization pathways of metabolic intermediates. An early effect of TGF-β1 response is increased activity of the system A neutral amino acid transporter - a Na⁺/K⁺ ATPase-driven transporter that fuels protein synthesis under growth conditions. TGF-β1-mediated effects on transporter activity were blocked by cycloheximide, a general protein synthesis inhibitor, supporting the requirement for de novo protein synthesis (Boerner et al, 1985; Dixon and Wilson, 1992).

Cellular amino acid pools were depleted by a 10-minute incubation in modified-Hanks balanced salt solution. This relatively short depletion time was sufficient for eliminating trans-inhibition caused by internal amino acid pools without compromising cell integrity since 10 ng/ml TGF-β1 showed increased system A transport over control cells, Figure 6-5. When cells were not depleted of amino acids with HBSS prior to ¹⁴C-radiolabeling, TGF-β1-treated samples showed no uptake due to trans-inhibition by amino acids in the cells.

To examine the effects of TGF-β1 on system A transport, radiolabeling of substrate uptake was measured at a 5-minute pulse using ¹⁴C-methy-aminoisobutyrate (MeAIB), a nonmetabolizable- and specific substrate of the system A transporter. Increase in System A activity was proportional to increase in TGF-β1 concentration, Figure 6-6. Since cells have been pre-treated with TGF-β1 for 24 hours before measuring for system A activity, the five-minute pulse measurement may reflect an increased number of transporters, increased uptake efficiency, or both.

Effect of rapamycin on TGF-β1-stimulated cell cycle responses

The cytoplasmic signaling events mediating TGF-β1 stimulation of cell cycle entry is unclear. Rapamycin was selected to probe signaling pathways to determine whether the mTOR (mammalian Target of Rapamycin)/FRAP (FKBP-12 and Rapamycin-Associated Protein)/p70 S6-kinase pathway mediates the TGF-β1 response. To investigate whether rapamycin affected TGF-β1-stimulated cell cycle entry in C3H-10T½ fibroblasts, staged cells were pre-incubated with 25 nM rapamycin before treatment with TGF-β1. Tritiated-thymidine incorporation data showed that rapamycin inhibited TGF-β1-stimulated DNA synthesis, Figure 6-7. Incubation with rapamycin alone appeared to affect basal-level DNA synthesis as thymidine incorporation was lower than that observed for control cells. Cells treated with a higher concentration of TGF-β1 (25 ng/ml) were still inhibited by rapamycin. These results suggest that TGF-β1-mediated DNA synthesis was rapamycin-sensitive.

The effects of rapamycin on TGF-β1-mediated thymidine uptake and incorporation were examined from the TCA-soluble fraction and NaOH-soluble fraction respectively, Figures 6-8 and 6-9. Inhibition of thymidine uptake by rapamycin corresponded to inhibition of DNA synthesis. A high concentration of rapamycin (1000 nM) suppressed thymidine uptake to control levels but affected DNA synthesis more significantly to below basal levels, indicating a cytotoxic effect at high concentrations. Based on these observations, rapamycin appeared to be a specific inhibitor of a TGF-β1-mediated pathway controlling DNA synthesis.

Similarly, the dose-response effects of rapamycin on TGF-β1-mediated amino acid uptake and protein synthesis were examined. To determine whether rapamycin inhibited TGF-β1-stimulation of protein synthesis and amino acid uptake, fibroblasts were staged in low serum media overnight before pre-incubation with 25 nM of rapamycin. Upon administration of 10 ng/ml TGF-β1, tritiated leucine was added to monitor uptake and incorporation over time.

Figures 6-10 and 6-11 showed that while TGF-β1 stimulated at least a 10-fold increase in leucine incorporation at 48 hours, rapamycin was able to inhibit stimulated protein synthesis in a dose-response manner. Inhibition of protein synthesis by rapamycin correlated with inhibition of leucine uptake, suggesting that limiting substrate contributed in part to the rapamycin effect.

Since TGF-β1-mediated leucine incorporation was apparently maintained by elevated steady-state levels of available leucine, the ability of rapamycin to change this steady state was examined. Early leucine uptake kinetics under rapamycin treatment suggested a contribution to substrate availability in rapamycin inhibition. Rapamycin repressed leucine-uptake by TGF-β1 stimulated cells in a dose-dependent manner, Figure 6-12. The initial overshoot caused by TGF-β1 was also blunted in a dose-dependent manner. Rapamycin repression of subsequent uptake but not the initial overshoot suggested that partial repression of the TGF-β1 effect may be contributed by a "leakiness" at initial influx point stimulated by the growth factor. This leakiness may consist of compensating transport pathways parallel to the rapamycin-sensitive pathway against TGF-β1.

Increasing rapamycin concentration significantly repressed protein synthesis after 3 hours, Figure 6-13. TGF-β1 was previously observed to induce protein synthesis at 4 hours and rapamycin inhibited this induction. Reducing cellular steady state of leucine by rapamycin may be similar to the mechanism by which control cells were precluded from continued protein synthesis beyond 4 hours - in other words, rapamycin prevented substrate accumulation to a threshold requirement for activating protein synthesis.

The system A neutral amino acid transporter is a sodium-dependent transporter that is regulated by metabolic changes and cell cycle progression. System A activation has been observed to correlate with peptide growth-factor stimulation and is an important parameter in determining bioenergetics toward cell cycle progression. System A inhibition frequently corresponds to G1 arrest. To determine whether rapamycin inhibited system A transport, cells were stimulated with growth factor for 24 hours before treating with rapamycin. Based on cell cycle entry kinetics, TGF-β1 would have initiated G1-S transition by this time. Drug treatment thus coincides with the optimal TGF-β1 stimulation-window (32-48 hours). Rapamycin inhibited system A , Figure 6-14.

Cell cycle synchrony ("staging") ensures fidelity of growth factor responses as well as allowing dissection of phase-sensitivity to chemical transformation. Traditional cell cycle synchronizing methods were based upon physical properties (mitotic shake) and selectively activated biochemical processes during cell cycle progression (chemical arrest). Early investigations with C3H-10T½ fibroblasts demonstrated cell cycle-dependence in oncogenesis by some chemical carcinogens; for example, sensitivity towards oncogenic transformation induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) rested between the G1/S transition whereas sensitivity towards cytosine arabinoside (1-beta-D-arabinofuranosylcytosine, ara-C) was observed in S-phase (Bertram et al, 1975; Jones et al, 1977).

Cell cycle inhibitors including lovastatin and staurosporine had been used successfully as staging agents (Lin et al, 1992). An important parameter in evaluating staging agents is the ability of stimulatory signals to release cells from drug-inhibition. Since rapamycin and wortmannin had been shown to arrest various cell systems at G1, these inhibitors were evaluated for feasibility as a staging agent for TGF-β1 stimulation. Rapamycin was administered to high density fibroblasts for 24 hours before adding increasing concentrations of TGF-β1, Figures 6-15 and 6-16. Since residual inhibitor in the media was aspirated before stimulating with TGF-β1, any inhibition must therefore occur independently of residual inhibitory signaling and represents irreversible events against growth factor signaling. To ensure biologic activity of TGF-β1, control samples were pre-incubated in staging media without inhibitors, and by 48 hours, TGF-β1 effectively stimulated thymidine incorporation as expected.

Whereas 10 ng/ml TGF-β1 stimulated DNA synthesis Figure 6-16, pre-incubation with 25 nM of rapamycin eliminated TGF-β1 stimulation. This suggests rapamycin irreversibly inhibited pathways or components required for TGF-β1 stimulation. Rapamycin treatment did not affect the steady state thymidine pool, Figure 6-15; therefore rapamycin inhibition was not accountable by reducing substrate availability. Limiting transport efficiency may be one mechanism by which rapamycin prevented TGF-β1 stimulatory responses if inhibition depended upon the continual presence of rapamycin. Based on irreversibility of the rapamycin-block, rapamycin was not an ideal staging agent for assessing TGF-β1 stimulation, but rather a potent inhibitory probe of pathways mediating TGF-β1 signaling.

Effects of wortmannin on TGF-β1-stimulated cell cycle responses

Wortmannin partially inhibited TGF-β1-stimulated DNA synthesis and did not significantly affect the steady state accumulation of thymidine pools, Figures 6-17 and 6-18. When staged cells were stimulated with TGF-β1 and monitored for leucine uptake and protein synthesis, wortmannin did not significantly affect TGF-β1 stimulation at doses below 1000 nM. These results suggest that wortmannin was not an effective inhibitor of TGF-β1-mediated responses in these fibroblasts and wortmannin-sensitive pathways probably diverged from rapamycin-sensitive pathways.

In 3T3-L1 cells, PI 3-kinase activation may play a role in insulin-regulation of system A: inhibition of the Ras/MAPK- and p70S6k pathways did not affect insulin stimulation of system A but inhibition of PI 3-kinase pathways by wortmannin precluded insulin stimulation of system A transport (Su et al, 1998). To examine whether PI 3-kinase activity may be involved in TGF-β1-stimulation of system A transport, TGF-β1-stimulated fibroblasts were depleted of intracellular amino acids before treating with wortmannin and monitored for ¹⁴C-MeAIB uptake at 5 minute pulse-labeling, Figure 6-19. Low doses of wortmannin affected basal level system A transport and inhibited inducible system A transport. Although the system A transporter is cell cycle sensitive, inhibition by wortmannin did not correlate with inhibition of DNA- and protein synthesis.

Wortmannin was investigated as a potential staging agent for TGF-β1 stimulation. High-density fibroblasts were staged in wortmannin overnight before replenishing with wortmannin-free low serum-media and treating with TGF-β1. In wortmannin-staged cells, TGF-β1-stimulation of thymidine accumulation and DNA synthesis were enhanced, suggesting a potentiation effect of TGF-β1 stimulation by wortmannin, Figures 6-20 and 6-21. These results suggest a divergence between wortmannin-sensitive pathways from rapamycin-sensitive pathways against TGF-β1-mediated cell cycle progression, resulting in differential inhibition against TGF-β1 for DNA synthesis, protein synthesis, and System A amino acid transport.

A possible mechanism for the observed differences between wortmannin and rapamycin effects on TGF-β1 may include rapamycin-sensitive intermediates regulating a major stimulatory pathway through which TGF-β1 signals are transduced. Wortmannin-sensitive intermediates may control a feedback regulatory pathway that was activated by TGF-β1 as a negative feedback signal. Rapamycin inhibition prevented TGF-β1 signaling, and the presence of an active PI 3-K pathway may further contribute to an inhibitory response. Wortmannin staging potentiated the TGF-β1 response by removing negative feedback signals to enhance substrate uptake and DNA synthesis. If PI 3-kinase pathways were indeed within a negative feedback framework, wortmannin would relieve the PI 3-kinase block to potentiate the TGF-β1 effect.

When both rapamycin and wortmannin were used to stage the cells, thymidine uptake was not significantly reduced whereas DNA synthesis was inhibited, Figures 6-24 and 6-25. Whereas staging with wortmannin potentiated TGF-β1-mediated DNA synthesis, the presence of rapamycin in the staging media precluded this potentiation by wortmannin. These results suggest that at some point, rapamycin signaling converged with wortmannin signaling. Rapamycin-sensitive pathways may also preside over wortmannin-sensitive pathways.

Effect of rapamycin and wortmannin on TGF-β1-stimulated cell volume differential

In rat primary skeletal muscle cells, volume-induced changes in amino acid transport were PI 3-kinase dependent but p70 S6 kinase-independent. System A transport behavior was sensitive to wortmannin-inhibition of cell volume differential, suggesting a role of PI 3-kinase in allowing muscle cells to rapidly adapt amino acid transport to volume changes (Low et al, 1997). Cell cycle progression stimulated by growth factors often correspond with increases in cell volume. Insulin increased hepatocytic cell volume, and, in the liver, changes in cell volume correlated with changes in protein metabolism and may physiologically fine-tune cell metabolism (Zhande and Brownsey, 1996). TGF-β1 increased cell volume in a rapamycin-sensitive and wortmannin-resistant manner, Figures 6-22 and 6-23.

Discussion

Compared with peptide growth factors such as EGF and PDGF that stimulated early-entry cell cycle kinetics, the delayed-cell cycle entry characteristic of TGF-β is important in the context of modulating other peptide growth factors. Historic transformation studies in NRK fibroblasts demonstrated that a "sarcoma growth factor" was actually the combined effects of transforming growth factors-alpha (TGF-α) and -beta (TGF-β; Anzano et al, 1983). While TGF-β1 signaled through a serine/threonine-kinase-dependent pathway, TGF-α signaled through an EGF-receptor-dependent pathway. Differences in cell cycle kinetics between TGF-α (early ) and TGF-β1 (delayed) suggested that divergent signaling pathways probably mediate the mitogenic potential of these structurally-unrelated transforming growth factors.

TGF-β1 was previously shown to alter System A transport activity where, depending on cellular context, TGF-β1 may either down-regulate or up-regulate system A transport. In hepatocytes, TGF-β1 suppressed IL-6 induced system A- and system L transport. However, in normal resting rat kidney (NRK) cells, TGF-β1 stimulated both system A and system L transport. A potential mechanism of chemical transformation may be at the turnover rate of System A: MCA-transformed 10T½ cells were observed to exhibit lower turnover rates than that for the normal C3H-phenotype (Dr. Kirk Leister, unpublished data). Increased transporter stability may contribute to elevated uptake activities.

Taken together, the differential effects of rapamycin and wortmannin on TGF-β1-mediated DNA synthesis, protein synthesis, and inducible amino acid transport suggest a role for rapamycin-sensitive pathways in mediating positive growth effects of TGF-β1 in C3H fibroblasts, and implicated a negative regulation by wortmannin-sensitive pathways on the TGF-β response.

Implication of rapamycin-sensitive pathways in TGF-β1 signaling also came from observations of the association of FKBP-12 (FK506 binding protein-12) with the type I TGF-β receptor (Wang et al, 1994). Binding of FKBP-12 to TbRI prevented transactivation by the type II receptor (Chen et al, 1997). Further investigation of this interaction via protein-receptor fusion showed that the fusion construct was activated by FKBP-12 antagonists and may represent an inhibitory role of FKBP-12 for TβRI (Stockwell and Schreiber, 1998). Since FKBP-12 appeared to be dispensable for TGF-β1-mediated signaling and the interaction between this protein and TGF-β1 receptors was not conserved across species, the role of rapamycin-sensitive pathway in TGF-β1-signaling remains controversial (Shou et al, 1998).

This thesis presented evidence for the dissection of growth factor- and inhibitor signaling to include examination of the kinetics of macromolecular synthesis rather than of isolated protein factors or pathways. The effectiveness of an inhibitory agent may depend upon the inhibition of collective events including DNA synthesis, protein synthesis, and amino acid transport. Rapamycin was demonstrated to be an excellent inhibitor of TGF-β1-mediated DNA synthesis, protein synthesis, and amino acid transport. Wortmannin was not an effective inhibitor of TGF-β1-mediated cell cycle progression but showed promise as a potentiator of TGF-β1 signaling. Although initial investigations suggested the roles of p70 S6 kinase phosphorylation and eIF-2α induction in differentiating the effects of these inhibitors against TGF-β1 signaling, future experiments are warranted to confirm these observations.

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