extfieldsofstudy: Chemistry Medicine

text:
Tropomyosin-dependent filament formation by a polymerization-defective mutant yeast actin (V266G,L267G).

A major function of tropomyosin (TPM) in nonmuscle cells may be stabilization of F-actin by binding longitudinally along the actin filament axis. However, no clear evidence exists in vitro that TPM can significantly affect the critical concentration of actin. We previously made a polymerization-defective mutant actin, GG (V266G, L267G). This actin will not polymerize alone at 25 degrees C but will in the presence of phalloidin or beryllium fluoride. With beryllium fluoride, but not phalloidin, this polymerization rescue is cold-sensitive. We show here that GG-actin polymerizability was restored by cardiac tropomyosin and yeast TPM1 and TPM2 at 25 degrees C with rescue efficiency inversely proportional to TPM length (TPM2 > TPM1 > cardiac tropomyosin), indicating the importance of the ends in polymerization rescue. In the presence of TPM, the apparent critical concentration of actin is 5.5 microm, 10-15-fold higher than that of wild type actin but well below that of the GG-actin alone (>20 microm). Non N-acetylated TPMs did not rescue GG-actin polymerization. The TPMs did not prevent cold-induced depolymerization of GG F-actin. TPM-dependent GG-actin polymerization did not occur at temperatures below 20 degrees C. Polymerization rescue may depend initially on the capture of unstable GG-F-actin oligomers by the TPM, resulting in the strengthening of actin monomer-monomer contacts along the filament axis.

Tropomyosins are a family of highly conserved eukaryotic actin-binding proteins with molecular masses ranging from 19 kDa for yeast TPM2 1 (1) to 40 kDa for rat fibroblast TPM1 (2). Tropomyosins possess a dimeric ␣-helical coiled-coil structure along virtually the entire length of the protein, and heptad repeat motifs with hydrophobic residues at positions 1 and 4 are found along the entire molecule. These quasirepeat regions appear to match up with actin monomers when tropomyosin binds to F-actin along the filament axis and may serve as weak actin binding regions. Tropomyosins have a basic amino terminus, which is usually N-acetylated, and for most tropomyosins, the acetyl group is believed to be important for preservation of the helical structure at the end of the protein (3). Adjacent tropomyosins form head-to-tail overlapping interactions along the length of the actin filament (4), and these overlaps are believed to be the major site of interaction between F-actin and tropomyosin (5,6). Recombinant skeletal muscle tropomyosin with either an unacetylated N terminus (7) or with a C-terminal deletion (5) loses the ability to bind to actin. The interaction between actin and the internal quasirepeat regions of tropomyosin is believed to be weaker but still critical for cooperative binding to F-actin actin (8), and there is a hierarchy of importance among these quasirepeat regions (9).
The role of tropomyosin, along with troponin, as a mediator of calcium regulation of thin filament function in sarcomeric muscle is well established. However, its role in nonmuscle cells, where there is no troponin, is poorly understood. Based on its ability to interact simultaneously with a number of actin monomers along the actin helix, it has been hypothesized that tropomyosin functions as a stabilizer of actin filaments in nonmuscle cells. Even a modest effect of TPM on filament stability would be biologically significant because actin monomer binding proteins in the cell act to amplify any change in stability such as the critical concentration (10). Studies in vitro addressing this question, however, do not yield consistent results. Experiments in vitro using muscle actin and muscle tropomyosins showed that tropomyosin can alter the monomer association and dissociation rates at both ends of the filament, resulting in a small change in critical concentration (11). Broschat et al. (12,13) demonstrated that nonmuscle and muscle tropomyosins slowed depolymerization at the pointed end of the filament, while another study reported that tropomyosin prevented filament fragmentation but did not alter elongation rate (14). These differing results may derive from differences in muscle versus nonmuscle tropomyosin or from differences in experimental methodology. It is also possible that the low critical concentration and the stability of muscle actin filaments in vitro render the filament-stabilizing properties of tropomyosin difficult to measure reproducibly. This situation would also make it very difficult for one to determine the relative contribution of different parts of the tropomyosin molecule to actin filament stabilization.
A valuable system for the assessment of tropomyosin in nonmuscle cells has been the budding yeast Saccharomyces cerevisiae. This yeast contains two tropomyosins, each encoded by a separate gene. TPM1, which contains five internal quasirepeat regions, constitutes about 85% of the cell's tropomyosin and is present in sufficient quantities to saturate the Factin present in the cell (15). TPM2, which contains only four quasirepeat regions, comprises 15% of the cell's tropomyosin (1). Both bind to actin in a saturable fashion, with the binding of TPM2 less sensitive to higher salt than TPM1. The simultaneous deletion of both TPM genes results in cell death, whereas deletion of the TPM1 gene, alone, results in a mild ts phenotype, and disappearance of actin cables. These phenotypes can be partially rescued by overexpression of TPM2. Deletion of TPM2 alone produces no recognizable phenotype.
We previously generated a mutant yeast actin, GG, with a polymerization defect in vitro. Cells expressing this as their only actin are viable but sick (16). In this actin, two hydrophobic residues, Val 266 and Leu 267 , were simultaneously altered to glycines. These residues are at the amino-terminal end of a hydrophobic loop between actin subdomains 3 and 4, which, according to a model advanced by Holmes and colleagues (17), interacts with a hydrophobic surface composed of two neighboring monomers on the opposing strand of the actin filament. Such an interaction would impart cross-strand stabilization to the filament structure. The model predicts that interference with this hydrophobic interaction should destabilize the filament, and we showed that this mutant actin would not polymerize at concentrations as high as 19 M. Phalloidin, which forms a bridge between the two strands of the filament (18,19), restores polymerization of GG-actin (20). Beryllium fluoride (BeFx) also restores polymerizability to the actin. BeFx occupies the position on the actin monomer vacated by the inorganic phosphate that is slowly released following hydrolysis of ATP by actin during polymerization of the protein (21). The binding of BeFx converts the filament to a more stable ADP-P i -like state by strengthening intermonomer contacts along the filament axis such that the increased stability can compensate for the loss in cross-strand stabilization caused by the GG mutation (20). Hydrophobic interactions are predicted to be cold-sensitive (22). However, while phalloidin rescue is not cold-sensitive, BeFx rescue is; the GG-actin depolymerizes as the temperature decreases to 4°C. Sac6p, the yeast homologue of the actin filament bundling protein fimbrin, binds to the actin filament between neighboring actin monomers along the filament axis, and in agreement with our BeFx results, this protein also rescues GG polymerization in a temperature-sensitive fashion (23). The viability of GG-actin-expressing cells, despite the polymerization defect exhibited by the actin in vitro, implies that another protein within the yeast can rescue GG-actin function.
A candidate for such a protein is tropomyosin due to its mode of binding to the actin filament and to the amount of TPM1 relative to the amount of actin in the cell. Although the binding of tropomyosin to actin depends on the integrity of the ends of TPM, the contribution of the internal repeats to formation of a stable tropomyosin-actin complex is still under debate. The side-binding nature of TPM suggests that it should rescue GG-actin polymerizability in much the same way that Sac6p does, and the availability of tropomyosins of different lengths allows us to also address the relative contribution of the ends of TPM versus the internal quasirepeat regions in actin filament stabilization. In this paper, we assess the ability of N-acetylated and non-N-acetylated yeast TPM1 and TPM2 and bovine cardiac tropomyosin (CTPM) to rescue GG-actin polymerization at different temperatures.

EXPERIMENTAL PROCEDURES
Materials-Affi-Gel 10 activated resin was purchased from Bio-Rad, and DNase I (grade D) was purchased from Worthington. DNase I affinity columns were made as described previously (24). ATP was purchased from Sigma. Yeast cakes were obtained from a local food store. All other chemicals used were reagent grade quality. Recombinant yeast TPM1 and TPM2, isolated from Escherichia coli and therefore possessing an unblocked NH 2 terminus, were the generous gift of Dr. Larry Tobacman (University of Iowa). Recombinant chicken striated muscle ␣-tropomyosin produced from E. coli was a generous gift of Sarah Hitchcock-DeGregori (University of Medicine and Dentistry of New Jersey).
Actin Purification-Yeast wild type and mutant GG-actins were purified in the Ca 2ϩ form by a DNase I affinity chromatography protocol as described (20,24) and stored in Ca 2ϩ -G-buffer (10 mM Tris-HCl, pH 7.5, containing 0.2 mM ATP, 0.2 mM CaCl 2 , and 0.1 mM dithiothreitol). Actins were converted to their Mg 2ϩ form at 4°C according to Strzelecka-Golaszewska et al. (25) and used within 12 h.
Tropomyosin Purification-Native bovine cardiac tropomyosin was prepared from an acetone powder of heart tissue according to the procedure of Butters et al. (5) based on heat fractionation, salt fractionation, and DEAE-cellulose chromatography. To produce yeast TPM1, we first constructed a ⌬tpm2 haploid yeast strain (yPAR-5) and transformed it with a TPM1 overexpression plasmid in which expression of TPM1 was under control of the GAL1 promoter. For TPM2 production, a haploid ⌬tpm1 strain (yPAR-6) was constructed and transformed with a TPM2 overexpression plasmid, also under galactose regulation. The TPM1 and TPM2 plasmids were kindly provided by A. Bretscher (Cornell University). TPM1 and TPM2 were purified according to a modification of the protocol of Drees et al. (1). Following induction of the plasmids with galactose for 12 h, cell lysates were made from 4-liter cultures and subjected to heat and salt fractionation followed by ion exchange chromatography on a Pharmacia MonoQ column. These two tropomyosins were subjected to an additional gel filtration step on Sephacryl 200 in 10 mM Tris-HCl, pH 7.5, containing 150 mM KCl and 1 mM dithiothreitol. The resulting protein was then dialyzed against the same buffer without the KCl. In each case, single bands were obtained following SDS gel electrophoresis. Tropomyosin concentrations (dimers) were determined by UV absorbance at 280 nm using the following extinction coefficients: ⌬⑀ CTPM ϭ 0.01536 cm -1 ⅐M Ϫ1 , ⌬⑀ TPM1 ϭ 0.02788 cm Ϫ1 ⅐M Ϫ1 , and ⌬⑀ TPM2 ϭ 0.03068 cm Ϫ1 ⅐M Ϫ1 . Yields of the tropomyosins were 8.1 mg of TPM1 and 4 mg of TPM2 from 10 g of each cell type.
Actin Polymerization-Actin polymerization was assessed by following the increase in light scattering as a function of time. Experiments were performed in a 120-l cuvette in a thermostatted cell chamber of a Spex Fluorolog 3 fluorescence spectrometer with excitation and emission wavelengths set at 360 nm. For experiments at 25°C, tropomyosin of the desired type and concentration was mixed with Mg 2ϩ -GG-actin in Mg 2ϩ -G buffer (10 mM Tris-HCl, pH 7.5, 0.2 mM MgCl, and 0.1 mM dithiothreitol). Polymerization was initiated by the addition of MgCl 2 and KCl to final concentrations of 2 and 50 mM, respectively. The steep deflection observed at the beginning of the kinetic trace is due to the opening and closing of the sample chamber during the addition of the salt. Actin polymerization data are normalized against the net increase in light scattering caused by the polymerization of 19 M WT actin in the presence of saturating amounts of CTPM. Temperature was regulated with a thermostatted water bath attached to the cuvette chamber. In those cases where the temperature was lowered from 25°C, the rate of decrease was about 2°C per 3 min. In cases where we increased the temperature from 4°C, the temperature was raised 5°C over a 2-min period, after which the sample was allowed to remain at the new temperature for 10 min.
Electron Microscopy-Samples containing 19 M actin were deposited on carbon-coated Formvar grids, negatively stained with 1.5% uranyl acetate, and observed on a Hitachi 7000 transmission electron microscope in the University of Iowa Central Electron Microscopy Facility.

RESULTS
Tropomyosin Restores Polymerization of GG-actin-We first assessed the ability of different tropomyosins to restore the polymerizability of the Mg 2ϩ form of GG-actin at 25°C (Fig. 1). As reported previously, the addition of salt in the absence of tropomyosin causes an initial small increase in light scattering followed by a slow decrease toward base-line values. This change occurs without the formation of stable filaments as judged by electron microscopy (Fig. 3) and may involve an abortive attempt at filament formation following salt-induced nucleation. At saturating levels of tropomyosin, the three tropomyosins tested, CTPM, TPM1, and TPM2 (Fig. 1, A-C) all induced the same increase in light scattering in the presence of 19 M GG-actin, although the rate of increase for the two yeast tropomyosins was faster than that observed for the cardiac protein. In each of these cases, electron microscopy showed that this stable increase in light scattering was accompanied by F-actin formation and did not merely result from salt-induced aggregation. Filaments were also observed (data not shown) at subsaturating tropomyosin concentrations in which the final extent of increased light scattering was reduced by about 60%.
We wished to determine whether the ability of tropomyosin to restore GG-actin polymerizability depended on the nature of the divalent cation bound in the high affinity site at the base of the cleft separating actin's two domains. Although the physiologically relevant form of actin has Mg 2ϩ bound at this site, the protein is often prepared and studied in the Ca 2ϩ form because of its enhanced stability. Previous work has shown that there are distinct differences in the dynamic properties of these two forms of actin. We thus determined the ability of TPM1 to induce polymerization of the Ca 2ϩ form of GG-actin (Fig. 2). With Ca 2ϩ actin, the initial rise in light scattering seen in the absence of tropomyosin does not occur, in contrast to the situation with the Mg 2ϩ form of actin, in agreement with previous results (20). The addition of saturating TPM1 induced the same degree of light scattering increase observed with the Mg 2ϩ form, and EM demonstrated again that this increased light scattering resulted from formation of F-actin (Fig. 3). However, the rate of increase for the Ca 2ϩ form of actin is slower than that observed with the Mg 2ϩ form and may simply reflect the faster rate of polymerization of the Mg 2ϩ form of WT actin alone in comparison with its Ca 2ϩ form.
The Importance of Tropomyosin N-terminal Acetylation in the Rescue of Mg 2ϩ -GG-actin Polymerizability-It had been shown previously that removal of the N-terminal acetyl group from sarcomeric tropomyosins prevented their binding to Factin. We therefore assessed the effect of this post-translational modification on the ability of muscle and both yeast tropomyosins to rescue the polymerization of GG-actin. The unacetylated proteins were obtained from E. coli that had been transformed with plasmids carrying the tropomyosin coding sequences. We utilized chick skeletal muscle ␣-tropomyosin instead of the CTPM because of its availability as a recombinant protein. None of these nonacetylated tropomyosins were able to restore GG-actin polymerization at concentrations where the rescue by the acetylated proteins was maximal (Fig.  4), and EM confirmed the absence of actin filaments in these preparations (Fig. 3, F-H). It had previously been demonstrated that extension of the N terminus of a nonacetylated sarcomeric tropomyosin by addition of an Ala-Ser restored the ability of the tropomyosin to bind to F-actin and to restore thin filament regulation (26). This tropomyosin also restored GGactin polymerization although to a lesser extent than WT acetylated tropomyosin (data not shown). These experiments underscore the importance of the tropomyosin N terminus in the rescue of GG-actin polymerization and suggest that tropomyosin end-end overlap plays an important role in the establishment of GG-actin filament stability.
GG-actin Polymerization as a Function of Tropomyosin Concentration-To further establish the relative importance of the  Fig. 5 and are presented on a weight basis with respect to the amount of tropomyosin used. The use of weight rather than molarity allows one to account for differences in the molecular weights of the three different tropomyosins used. Because of this molecular weight difference, approximately the same weight of each of the tropomyosins, not the same number of molecules, should be bound per length of actin filament at saturating concentrations. The results show that the efficiency of rescue of GG-actin polymerization varies inversely with the length of the tropomyosin used. Saturation was approximately achieved with 360 g/ml of CTPM, 225 g/ml of TPM1, and 171 g/ml of TPM2. A plot of these concentrations versus the number of quasirepeats in the tropomyosin yields a straight line (Fig. 6), suggesting that the number of ends per length of actin is a central factor in rescue efficiency.
Apparent Critical Concentration of GG-actin in the Presence of Tropomyosin-The incomplete polymerization of GG-actin produced in the presence of saturating tropomyosin concentrations ( Figs. 1 and 8) indicates that the critical concentration of GG-actin in the presence of tropomyosin is much higher than the value for wild type actin, 0.4 M (27). To determine an approximate value for the critical concentration of GG-actin, we incubated the G form of Mg 2ϩ -GG-actin at concentrations between 1 and 19 M with 7.5 M CTPM, TPM1, or TPM2 in F-buffer at 25°C and recorded the final increase in light scattering. This concentration of tropomyosin is well above that needed for saturation for each of the tropomyosins based on the results of Fig. 7. The final net increase in light scattering was then plotted as a function of actin concentration. Fig. 7 shows that for each of the three tropomyosins, the critical concentration for Mg 2ϩ -GG-actin was approximately 5.5 M. Thus, although each of the tropomyosins tested can lower the critical concentration of actin from Ͼ Ͼ20 to about 5 M, the final value is still 10 -15 times that observed for the critical concentration of WT actin in the absence of tropomyosin.
Temperature Dependence of the Rescue of GG-actin Polymerization by Tropomyosin-The side-binding nature of the tropomyosin-F-actin interaction predicts that tropomyosin's ability to rescue GG-actin polymerization should display a cold sensitivity similar to that seen with BeFx or Sac6p-induced rescue. To test this hypothesis, 19 M GG-actin was polymerized in the presence of 500 g/ml CTPM, TPM1, or TPM2 at 25°C (Fig.  8A). This concentration was well above saturating levels for each of the tropomyosins in question. The results show a TPMdependent acceleration of polymerization with TPM2 Ͼ TPM1 Ͼ CTPM. The temperature was then lowered to 4°C over about a 30-min period with continuous monitoring of the light scattering of the sample. With each of the three tropomyosins, we observed a decrease in light scattering, which reached a plateau at between 4 and 8% above background (Fig.  8B). Electron microscopic examination of these samples revealed mostly small aggregates with occasional filament-like structures observed (Fig. 9). The relative rates of depolymerization were much more similar for these three samples than were the relative polymerization rates. Additionally, the fila- ment stability order was reproducibly different as well from what we observed for polymerization efficiency: CTPM Ͼ TPM2 Ͼ TPM1. We then determined whether this cold-induced depolymerization was reversible and, if so, what the temperature dependence of repolymerization was. No light scattering increase was observed when any of the samples were raised to 10 or 15°C (Fig. 8C). However, elevation of the sample to 20°C resulted in an increased light scattering (Fig. 8D), which EM showed was due to repolymerization of the actin (Fig. 9). These last results are remarkable for two reasons. First, the plateau level reached is significantly lower that that achieved initially at 25°C, demonstrating the extreme temperature sensitivity of GG F-actin stability in the presence of tropomyosin. Second, the order of repolymerization efficiency with respect to tropomyosin is identical to the polymerization efficiency order seen originally at 25°C. However, the relative differences between the ability of these three tropomyosins to induce polymerization are much greater at lower temperatures, suggesting that as the temperature is lowered, the tropomyosin end interactions become more crucial in the rescue process. If the sample is jumped to 25°C (data not shown), the light scattering reaches the same plateau that we observed in Fig. 8A, demonstrating the total reversibility of this process. DISCUSSION The focus of this work was to determine whether or not tropomyosin, because of its ability to bind F-actin along the filament axis, could restore polymerizability to GG-actin, a polymerization-defective mutant yeast actin. If so, then tropomyosin would become a prime candidate for a protein that is required for GG-actin to function in vivo in a manner compatible with cell viability. Our results clearly demonstrate the ability of not only both yeast tropomyosins but also a sarcomeric tropomyosin to restore GG-actin polymerizability. All three tropomyosins do this in a saturable manner by a mechanism that apparently requires the ability of the ends of tropomyosin to interact with one another, based on our results with the nonacetylated proteins. Furthermore, all three tropomyosins confer the same apparent critical concentration on the actin, about 5.5 M. In the absence of TPM, the critical concentration is so high that stable polymers will not form at 25°C at concentrations as high as 19 M GG-actin. Therefore, the interaction of the TPM with the actin monomers at or near the end of the filament must change their conformations via strengthened intermonomer contacts such that the addition of a new monomer to the filament end becomes favorable. We believe this is, if not the first, then certainly one of the clearest demonstrations of tropomyosin's ability to act as an F-actin stabilizer by significantly lowering the critical concentration of the actin as opposed to protecting the filament against severing.
As impressive as this rescue is, it is much less efficient than the ability of yeast fimbrin to restore GG-actin polymerizability. At 25°C, saturating concentrations of the yeast fimbrin, Sac6p, at any of the actin concentrations we examined, drove all of the GG-actin into filament bundles, implying that the critical concentration for actin in this case was equal to or less than that exhibited by WT actin. We believe this difference in rescue efficiency reflects the very different modes by which Sac6p and tropomyosin bind to the actin filament. For tropomyosin, it has been hypothesized that the strongest interactions with actin monomers involve the interacting ends of adjacent TPMs with weaker interactions involving the internal quasirepeat regions. Thus, with the tropomyosins we employed, there would be a strong contact between every four and seven actin monomers depending on the tropomyosin. On the other hand, the actin binding domain of Sac6p binds between adjacent monomers, forming a very tight bridge between them, and we have shown that one Sac6p contact per two actin monomers was needed for stable filament formation. Furthermore, at 25°C with Sac6p in vitro, one does not observe single filaments but ordered bundles due to the cross-linking nature of the protein. This added stabilization may well play a significant role in the enhanced ability of the Sac6p to restore GGactin polymerization. At very low temperatures, isolated single actin filaments were observed in the presence of Sac6p, presumably due to the effect of the lowered temperature on the ability of the protein to cross-link filaments.
Tropomyosin's longitudinal stabilization of monomers within the F-actin helix predicts that tropomyosin-dependent rescue should exhibit the same cold sensitivity observed with BeFxand Sac6p-dependent rescue of GG-actin polymerization. In terms of the Holmes model of the actin filament, the hydrophobic plug, which was mutated to form GG-actin, is involved in a cross-strand stabilization of F-actin by virtue of its ability to contact a hydrophobic surface made up by the interface of two monomers on the opposing strand (17,19). Because hydrophobic interactions are cold-sensitive, elimination of two-thirds of the hydrophobicity in this plug reduces this interaction to such an extent that filament stability can no longer be maintained. Enough residual stabilization remains at 25°C such that M GG-actin was combined with 500 g/ml of CTPM (curve 1), TPM1 (curve 2), or TPM2 (curve 3), and polymerization was allowed to go to completion at 25°C (A). The thermostat was then lowered to 4°C, and the decrease in light scattering for each sample was followed over time (B). When the decrease in light scattering reached a plateau, the temperature was first raised to 10°C and then to 15°C, and each temperature was maintained for 10 min (C). The temperature of the sample was then raised to 20°C, and the increase in light scattering was monitored over time (D). Polymerization was normalized to the amount of light scattering achieved with an equivalent amount of WT actin in the presence of saturating CTPM at 25°C. strengthening longitudinal monomer-monomer contacts can at least partially overcome this plug deficit. However, lowering the temperature reduces this residual cross-strand stabilization to the point where it can no longer be compensated for by increased longitudinal contacts, and the filament disassembles.
To a first approximation, our data support this cold sensitivity prediction. However, there are significant differences between the sensitivity of the GG-actin to depolymerization in the presence of Sac6p versus tropomyosin. Possibly because of the enhanced longitudinal stabilization or the additional stabilization due to filament bundling, lowering the temperature to 4°C in the presence of Sac6p did not result in complete depolymerization based on light scattering and what looked like the appearance of small aggregates under EM. Subsequent raising of the temperature produced no additional increase in light scattering until 15°C, with further increase observed until 25°C when recovery was complete. EM showed that this increased light scattering represented F-actin formation. Sac6p is a Ca 2ϩ -sensitive bundling protein, and when the residual assemblies at 4°C were eliminated by treatment of the samples with Ca 2ϩ followed by removal of the Ca 2ϩ with EGTA, then polymerization was not observed until Ն20°C. We interpreted this difference in temperature response to reflect the elongation of the filament (Ն15°C) versus nucleation (Ն20°C). With tropomyosin, depolymerization appeared complete at 4°C, and repolymerization, based on light scattering, did not occur until Ն20°C. This temperature dependence again reflects the relative lack of efficiency of tropomyosin in allowing GG-actin repolymerization, and it also implies that tropomyosin-induced repolymerization requires de novo polymer nucleation.
Two of our observations provide further insight into what might be the mechanism by which tropomyosin facilitates the polymerization of GG-actin: the requirement for N-terminal acetylation of tropomyosin and the inverse relationship between the number of tropomyosin internal quasirepeats and the kinetic efficiency with which the tropomyosin affects rescue of polymerization. Rescue clearly depends heavily on the length of the tropomyosin and the ability of neighboring tropomyosins to interact with one another through their ends. The binding of a single tropomyosin molecule to a stable F-actin filament, let alone three or four actin monomers, is very weak, and if the actin filament itself is inherently unstable, this is not likely to occur to any significant extent. We suggest that the addition of salt induces nucleation and subsequent elongation, leading to the generation of unstable GG F-actin oligomers of different lengths. Since stable filaments do not form with GG-actin alone, the distribution of these oligomers will probably be skewed toward those with shorter rather than longer lengths. The extended structure of tropomyosin together with its quasirepeat structure makes it somewhat like a template, suggesting that a stable interaction between the two proteins requires a long enough oligomer to span the length of the tropomyosin. The relatively greater efficiency of rescue exhibited by the shorter tropomyosin could then be attributed to a greater number of oligomers available to interact with the shorter tropomy-osin molecules during the polymerization process than the longer ones. Subsequent filament formation could then result from elongation and stabilization by tropomyosin or by annealing of these tropomyosin-stabilized oligomers. This oligomer capture model is supported by other experiments in our laboratory. 2 This work shows that exposure of GG-actin to F-buffer, in the absence of a stabilizing factor, induces an F-actin-like ATPase activity that reflects a need for oligomerization despite the inability of the protein to form stable actin filaments.
Actin function in vivo in yeast requires the involvement of individual actin filaments, not bundles like those formed by Sac6p. Our work clearly demonstrates that tropomyosin can allow GG-actin to form individual filaments by significantly affecting the critical concentration of the actin. The question remains as to whether or not tropomyosin is required to function in this manner in GG-actin-expressing cells and to what extent tropomyosin functions this way in general in nonmuscle cells as has so often been hypothesized. We are currently addressing this question in yeasts expressing GG-actin as their sole actin.


extfieldsofstudy: Biology Medicine

text:
Stable Expression of a Dominant Negative Mutant of CCAAT Binding Factor/NF-Y in Mouse Fibroblast Cells Resulting in Retardation of Cell Growth and Inhibition of Transcription of Various Cellular Genes*

The heterotrimeric CCAAT-binding factor CBF specifically interacts with the CCAAT motif present in the proximal promoters of numerous mammalian genes. To understand the in vivo function of CBF, a dominant negative mutant of CBF-B subunit that inhibits DNA binding of wild type CBF was stably expressed in mouse fibroblast cells under control of tetracycline-responsive promoter. Expression of the mutant CBF-B but not the wild-type CBF-B resulted in retardation of fibroblast cell growth. The analysis of cell growth using bromodeoxyuridine labeling showed that expression of the mutant CBF-B decreased the number of cells entering into S phase, and also delayed induction of S phase in the quiescent cells after serum stimulation, thus indicating that the inhibition of CBF binding prolonged the progression of S phase in fibroblasts. These results provide direct evidence for the first time that CBF is an important regulator of fibroblast growth. The inhibition of CBF binding reduced expression of various cellular genes including the α2(1) collagen, E2F1, and topoisomerase IIα genes which promoters contain the CBF-binding site. This result implied that expression of many other genes which promoters contain CBF-binding site was also decreased by the inhibition of CBF binding, and that the decreased expression of multiple cellular genes possibly caused the retardation of fibroblast cell growth.

The heterotrimeric CCAAT-binding factor CBF specifically interacts with the CCAAT motif present in the proximal promoters of numerous mammalian genes. To understand the in vivo function of CBF, a dominant negative mutant of CBF-B subunit that inhibits DNA binding of wild type CBF was stably expressed in mouse fibroblast cells under control of tetracycline-responsive promoter. Expression of the mutant CBF-B but not the wild-type CBF-B resulted in retardation of fibroblast cell growth. The analysis of cell growth using bromodeoxyuridine labeling showed that expression of the mutant CBF-B decreased the number of cells entering into S phase, and also delayed induction of S phase in the quiescent cells after serum stimulation, thus indicating that the inhibition of CBF binding prolonged the progression of S phase in fibroblasts. These results provide direct evidence for the first time that CBF is an important regulator of fibroblast growth. The inhibition of CBF binding reduced expression of various cellular genes including the ␣2(1) collagen, E2F1, and topoisomerase II␣ genes which promoters contain the CBFbinding site. This result implied that expression of many other genes which promoters contain CBF-binding site was also decreased by the inhibition of CBF binding, and that the decreased expression of multiple cellular genes possibly caused the retardation of fibroblast cell growth.
Eukaryotic RNA polymerase II promoters consist of multiple DNA elements that together control the transcriptional activity of the promoters. Among these various elements, several are present in many different polyerase II promoters. In higher eukaryotic promoters, the CCAAT box is most often found between 80 and 100 base pairs upstream of the transcription start site, and the sequences around the CCAAT box of a specific gene are usually highly conserved in different species (1)(2)(3)(4).
The mammalian CCAAT binding factor CBF 1 (also called NF-Y and CP1) which is a ubiquitous transcription factor, consists of three different subunits (CBF-A, CBF-B, and CBF-C), all of which are needed to form a CBF-DNA complex (5)(6)(7). Human, mouse, and rat CBF subunits show more than 95% amino acid sequence identity among the different mammalian species (8). Portions of the amino acid sequences of CBF-A, CBF-B, and CBF-C are also evolutionarily conserved, with a high degree of sequence identity with segments of yeast HAP3, HAP2, and HAP5, respectively. The products of these HAP genes, which are components of the yeast multimeric CCAATbinding protein, are necessary for expression of genes encoding components of the electron transport chain and thus regulate the respiratory function of yeast (9,10). Previous studies demonstrated that CBF-A and CBF-C interact with each other to form a stable heterodimer and that CBF-B only interacts with the CBF-A/CBF-C heterodimer, not with CBF-A or CBF-C alone, to form a heterotrimeric CBF molecule which then binds to DNA (7). The conserved segments of CBF-A and CBF-C that are necessary for formation of a CBF-DNA complex show amino acid sequence homology with each other. Interestingly, the amino acid sequences of these two CBF segments are similar to the histone-fold motifs of histones H2B and H2A, respectively, and to the archaebacterial histone-like protein Hmf-2 (11)(12)(13)(14). In contrast, the conserved segment of CBF-B that is needed for formation of the CBF-DNA complex shows no homology with the histone-fold motif or with any of the known dimerization motifs found in other heteromeric DNA-binding proteins (15). This analysis classified CBF as a unique DNA-binding protein.
In an in vitro reconstituted transcription system, CBF stimulates transcription of several different mammalian promoters (16). This showed that the heterotrimeric CBF contains two transcription activation domain, one present in CBF-B and the other in CBF-C. The two CBF activation domains are rich in glutamine and hydrophobic residues and show similarities to the glutamine-rich activation domain of SP1 transcription factor. The specific DNA sequences that are required for CBF binding have been defined by using a PCR-mediated random binding selection method. This showed that specific sequences flanking to the CCAAT are required for high-affinity CBF binding and that the CCAAT motifs of various mammalian promoters are very similar to the high-affinity CBF-binding site (17).
Role of CBF in the in vivo transcription of various mammalian genes is still unclear. The CBF-binding sites in promoters have mostly been studied by in vitro transcription assay or by DNA transfection experiments in different cell lines. Generally, mutation in the CBF-binding site results in decreased basal promoter activity. Previous studies of CBF binding in the promoters of serum albumin and major histocompatibility complex class II genes showed that CBF facilitates formation of stable transcription complexes together with tissue-specific transcription factors. This indicated that CBF might regulate the transcription of various promoters by cooperative interaction with promoter-specific transcription factors (18,19). More recently, studies of the Xenopus hsp70 promoter showed that CBF binding to this promoter disrupts local chromatin structures and facilitates transcription within the chromatin (20). This demonstrated that the Xenopus CBF interacts with p300 acetyltransferase, which modifies chromatin by acetylation, thus suggesting that CBF could regulate the in vivo transcription of various promoters by chromatin modification.
To understand in vivo function of CBF in mammalian cells, in this study a dominant negative CBF-B mutant was expressed stably in mouse fibroblast cells under the control of a tetracycline-responsive promoter. The consequent expression of the mutant CBF-B inhibited the DNA binding of cellular CBF and interestingly resulted in the retardation of fibroblast cell growth. Our results also showed that the expression of the mutant CBF-B delayed progression of S phase and reduced the expression of several genes which promoters contain CBFbinding site. This result implied that the retardation of cell growth is possibly caused by decreased expression of various cellular genes.

MATERIALS AND METHODS
Plasmids-The pTet-Off vector (CLONTECH) which was originally developed by Gossen and Bujard (21), expresses a tetracycline-responsive transcription activator (tTA) under the control of immediate early promoter of cytomegalovirus (CMV) and also contains a neomycin resistance gene. The pTRE vector (CLONTECH) contains multiple cloning sites immediately downstream of the tetracycline responsive (TRE) promoter consisting of seven copies of a tetracycline operator sequence followed by the minimal immediate early CMV promoter. To introduce more unique restriction enzyme sites for subsequent use in cloning genes downstream of the TRE promoter, a double-stranded oligonucleotide was cloned between the SacII and BamHI sites located in the multiple cloning site of the pTRE vector. This modified multiple cloning site contained unique SacII, EcoRV, BglII, NcoI, NdeI, EcoRI, XbaI, and BamHI restriction enzyme sites. A double-stranded oligonucleotide (upper strand 5Ј-CATGGACTACAAAGACGATGACGATAAACA-3Ј; lower strand 5Ј-TATGTTTATCGTCATCGTCTTTGTAGTC-3Ј) that encoding a FLAG epitope peptide (Met-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) was cloned between the NcoI and NdeI sites of the multiple cloning site. This constructed plasmid was designated pTRE-FLAG. The DNA corresponding to the full-length wild-type CBF-B which was isolated by digesting pCITE-CBF-B (14) with NdeI and XbaI was cloned in-frame with the FLAG epitope between the NdeI and XbaI sites of the pTRE-FLAG. The mutant CBF-B, here designated Bmut and containing amino acid substitution mutations at positions 304 and 311 (15), was also cloned in-frame with the FLAG epitope between the NdeI and XbaI sites of pTRE-FLAG. The constructed plasmids containing the wildtype and the mutant CBF-B were designated pTRE-flagB and pTRE-flagBmut, respectively. The pTRE-Luc plasmid containing a luciferase reporter gene under the control of the TRE promoter and the pTK-Hyg plasmid, which was used for the selection of hygromycin resistance stable cell clones, were purchased from CLONTECH.
The DNA probes of mouse topoisomerase and E2F1 genes for Northern blot analysis were generated by PCR using double-stranded cDNA of mouse chondrocyte cell mRNA as a template. The PCR primers mtopo 4273 (5Ј-GACCGTGGGAGTGAAGAAGACAGCAAC-3Ј) and mtopo 4594 (5Ј-AATTGGCTTCCTCGCCCGGTCAGATTTGGCT-3Ј) were based on the sequence of mouse topoisomerase II␣ cDNA (22) and were used to amplify nucleotides 4273-4594 of the topoisomerase II␣ cDNA. The PCR primers mE2F 877 (5Ј-CTCCTGAGACCCAACTA-CAAGCTGTGGA-3Ј) and mE2F 1320 (5Ј-CCTCCTCGAGACCAAAGT-GATAGTCAAG-3Ј) were based on the mouse E2F1 sequence (23) and were used to amplify nucleotides 877-1320 of the E2F1 cDNA. The amplified DNA fragments were cloned into the TA cloning vector pCR2.1-TOPO (InVitrogen) and checked by DNA sequencing. The DNA probe of mouse ␣2(1) collagen gene used in the Northern blot hybridization was described previously (24).
Cell Culture, Transfection, and Isolation of Stable Fibroblast Cell Clones Expressing Wild-type and Mutant CBF-B-Mouse fibroblast NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin. To select stable cell clones expressing tTA, the NIH3T3 cells were transfected with the pTet-Off plasmid using the calcium phosphate precipitation method (25). Several individual cell clones grown in the presence of 800 g/ml G418 were isolated. To examine the expression of tTA in the selected clones, each clone was transfected with the pTRE-Luc plasmid using Lipo-fectAMINE reagent (Life Technologies, Inc.), and expression of the luciferase gene was measured using luciferin as substrate with a luminometer (26). In one cell clone luciferase expression was high when the clone was cultured in the absence of tetracycline but reduced 100-fold when cultured in the presence of 2 g/ml tetracycline. This cell clone was then subsequently used to establish stable cell clones expressing either the wild-type or the mutant CBF-B protein. In brief, this cell clone was co-transfected with either the pTRE-flagB or the pTRE-flagBmut plasmid together with the pTK-Hyg plasmid in a 9:1 ratio. After transfection, individual cell clones were selected and isolated in the presence of 400 g/ml G418, 200 g/ml hygromycin, and 2 g/ml tetracycline in the culture medium. Each isolated cell clone was subsequently maintained in culture medium containing 400 g/ml G418, 100 g/ml hygromycin, and 2 g/ml tetracycline. To examine the expression of CBF-B, each cell clone was cultured at high cell density in the presence and absence of tetracycline for 3 days and then subjected to Western blot analysis.
Western Blot Analysis-For initial screening to identify cell clones expressing CBF-B, the individual cell clones were grown in 6-well plates in the presence and absence of tetracycline. The cells in each well were washed twice with phosphate-buffered saline; resuspended in 20 ml of buffer containing 50 mM Tris, pH 6.8, 2% SDS, and 100 mM dithiothreitol; and then extracted by incubating in a boiling water bath for 5 min. The extracts were then centrifuged for 5 min at high speed in a microcentrifuge. Next, 10 ml of the supernatant of each cell extract was fractionated on a 12% SDS-polyacrylamide gel and then electroblotted onto a nitrocellulose membrane (Schleicher & Schuell). Both wild-type and mutant CBF-B protein in the blot was detected by the ECL Western blotting detection system (Amersham Pharmacia Biotech) using anti-FLAG M2 monoclonal antibody (Eastman Kodak) diluted 1:1500.
For quantitative Western blot analysis which is presented in Fig. 2, monolayer fibroblast cultures grown in 100-mm dishes were harvested by trypsin treatment and collected by centrifugation. The cells were then washed twice with phosphate-buffered saline, resuspended in a buffer (40 l of buffer per 1 ϫ 10 6 cells) containing 1% sodium deoxycholate, 1% Triton X-100, 50 mM Tris, pH 7.5, 150 mM sodium chloride, 10 g/ml pepstatin, 10 g/ml leupeptin, 20 g/ml aprotinin, 10 mM sodium pyrophosphate, 50 mM sodium fluoride, and 0.5 mM sodium orthovanadate, and lysed by sonication. The protein in the cell extracts was quantified using a Dc protein assay kit (Bio-Rad). Thirty micrograms of each cell extract was fractionated on a 12% SDS-polyacrylamide gel and then electroblotted onto a nitrocellulose membrane. The CBF-B protein in each blot was detected by the ECL Western blot method as described earlier.
Growth Rate Determination, Cell Cycle Analysis, and Serum Stimulation-To determine cell growth, 2 ϫ 10 5 cells of each stable clone were seeded onto a 100-mm dish in the absence or presence of 2 g/ml tetracycline. Fresh culture medium was added to each dish at intervals of 2 days. Cells were collected by trypsinization at different time intervals, stained with trypan blue, and counted with a hemacytometer. This experiment was performed independently three times.
For cell cycle analysis, cells were plated and maintained in the same way as for the growth rate experiment and then analyzed by flow cytometry using propidium iodide staining as described by Tate et al. (27), or analyzed by 5-bromo-2Ј-deoxyuridine (BrdUrd) labeling to determine the number of proliferating cells (or the cells going through the S phase of the cell cycle). For flow cytometry analysis, cells were harvested after 72 h in culture by trypsinization, resuspended in 10 ml of Dulbecco's modified Eagle's medium containing 20% calf serum, and collected by centrifugation. The cell pellet was then resuspended gently in a solution containing 50 mg/ml propidium iodide, 4 mM sodium citrate, pH 7.8, 30 units/ml boiled RNase, and 0.1% Triton X-100 and incubated at 37°C for 10 min. After incubation, NaCl was added to the cell suspension to a final concentration of 0.25 M. Finally, cells were analyzed in a fluorescence-activated cell sorter (FACScan, Becton Dickinson) and the percentages of cells in different phases of the cell cycle were determined by using the Lysys II version 1.2 program. To determine the number of proliferating cells, the cells were labeled with 10 M BrdUrd for 30 min in culture and the BrdUrd-labeled cells were detected by the immunochemical method using the in situ cell proliferation kit, FLUOS (Roche Molecular Biochemicals) and followed by flow cytometric analysis. The details of the incubation procedure are described in the instruction manual. Briefly, after BrdUrd labeling the cells were harvested by trypsinization, resuspended in phosphate-buffered saline, and then fixed with 70% ethanol. The fixed cells were incubated with 4 N HCl to denature cellular DNA, and then mixed with 5 volumes of phosphate-buffered saline containing 0.5% Triton X-100 (PBT). The cells were collected by centrifugation, incubated in a solution containing fluorescein-conjugated anti-BrdUrd monoclonal antibody, and then analyzed in a fluorescence-activated cell sorter as described earlier. The percentage of BrdUrd-labeled cells were determined using the Lysys II program.
For serum stimulation experiments, 10 6 cells of each cell clone were seeded in 150-mm dish and grown in culture media with regular serum (10% fetal calf serum ) for 24 h. Next, the cells were starved by incubating them in media containing a low concentration of serum (0.5% fetal calf serum) for 48 h and then stimulated for growth in a media containing 10% serum. The cells were harvested at different times after serum stimulation for use in preparing nuclear extracts and total RNA. To determine the number of proliferating cells, the cells were labeled with BrdUrd after serum stimulation and then analyzed by flow cytometry as described earlier.
For cell death analysis, 2 ϫ 10 5 cells were seeded onto a slide chamber in the absence or presence of tetracycline and grown for 72 h. The apoptotic cell death in the culture chamber was determined by TUNEL reaction (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) using in situ cell death detection kit (Roche Molecular Biochemicals). The details of the procedure are described in the instruction manual. Briefly, after 72 h in culture the cells were fixed with 4% paraformaldehyde, incubated in a permeabilization solution containing 0.1% Triton X-100, and then labeled by TUNEL reaction with a fluorescein-dUTP. The labeled cells were detected under the fluorescence microscope. For positive control, after incubation with the permeabilization solution the cells were first treated with 50 g/ml micrococcal nuclease and then labeled by the TUNEL reaction. Most of the cells in the positive control were labeled with the fluorescein-dUTP.
Nuclear Extracts and DNA Binding Assay-Nuclear extracts were prepared from 1 ϫ 10 6 or more cells using a method described by Schreiber et al. (28). The CBF binding activity in the nuclear extracts was determined using a radiolabeled oligonucleotide containing the CBF-binding site of the mouse ␣2(1) collagen promoter (6). The DNA binding of CTF/NF-1 and OCT1 were assayed as described by Coustry et al. (29) and Lefebvre et al. (30), respectively.
Differential Display-The differential display reverse transcriptionpolymerase chain reaction (DDRT-PCR) was performed using the RNAimage kit (GenHunter) essentially according to the instruction manual. Briefly, the cells from two stable cell clones expressing wildtype or mutant CBF-B were grown in selection medium with or without tetracycline (2 g/ml). After 72 h, total RNAs were isolated from the cells and then used to synthesize first-strand cDNAs primed by using one of the three different 1-base-anchored H-T11M primers (where M may be G, A, or C). The cDNAs were amplified in PCRs with the H-T11M primer which was used to synthesize the cDNA, in combination with one of the eight different arbitrary primers (H-AP1, H-AP2, H-AP3, H-AP4, H-AP5, H-AP6, and H-AP8). The PCR amplified DNAs were radiolabeled with [ 35 S]dATP, separated by electrophoresis in a 6% urea-polyacrylamide gel, and finally detected by autoradiography. Then the differentially displayed cDNA bands were cut out, eluted, and amplified again using the same primers. The reamplified bands were then cloned into the TA cloning vector pCR2.1-TOPO (InVitrogen) and identified by DNA sequencing. In this experiment, three different partial cDNAs were isolated: Mts1, containing nucleotides 39 to 423 of the Mts1 cDNA (31); Egr1, containing nucleotides 202 to 814 of the Egr1 cDNA (32); and S6, containing nucleotides 202 to 814 of the S6 cDNA (33). These cDNAs were used as probes in Northern blot analyses which showed that both Mts1 and Egr1 but not S6 were differentially expressed in the Bmut cell clone.
RNA Isolation and Northern Blot Analysis-Total RNA was isolated from about 5 ϫ 10 6 cells by disrupting the cells with guanidinium thiocyanate followed by centrifugation of the cell extracts in a cesium chloride cushion (34). The resulting RNA pellet was resuspended in 0.3 M sodium acetate, pH 6, and then precipitated by ethanol. Ten micrograms of RNA from each cell clone was then separated on a 1% agaroseformaldehyde gel for Northern blot analysis using standard procedures. Briefly, RNAs were transferred to a ZetaProbe blotting membrane (Bio-Rad) and fixed by UV cross-linking. Membranes were hybridized 12-16 h at 65°C with 1-2 ng of radiolabeled probe/ml in 0.3 M sodium phosphate buffer, pH 7.2, and 7% SDS. The radiolabeled DNA probes were prepared by random priming. After hybridization, the membranes were washed at 65°C once in 2 ϫ SSC (1 ϫ SSC contained 150 mM NaCl, 15 mM sodium citrate, pH 7.0) and 0.1% SDS for 30 min and twice in 0.2 ϫ SSC and 0.1% SDS for 15 min. The radioactive bands in the membranes were detected by autoradiography and quantitated using a PhosphorImager (Molecular Dynamics).

Stable Expression of Wild-type and Mutant CBF-B in Mouse
Fibroblast Cells-Previous studies had shown that a DNAbinding domain mutant of CBF-B which interacted with CBF A/CBF-C but did not bind DNA, inhibited DNA binding of wild-type CBF subunits in vitro. Those studies also indicated that the mutant CBF-B acted as a competitive inhibitor of interaction between wild-type CBF-B and CBF-A/CBF-C and formed an inactive complex with wild-type CBF-A/CBF-C (35,36). Thus, to inhibit the DNA binding of cellular CBF in vivo, the mutant CBF-B and, as a control, the wild-type CBF-B were expressed stably under the control of a tetracycline-responsive promoter in NIH3T3 cells. Each of the wild-type and mutant CBF-B cDNA was inserted into a pTRE vector as in-frame fusion with DNA encoding the FLAG epitope ( Fig. 1). We then used a two-step selection procedure to isolate the stable fibroblast cell clones. In the first step, cell clones that stably expressed the tetracycline-regulated transactivator tTA protein were isolated by transfecting NIH3T3 cells with the pTet-Off vector. To monitor expression of the tTA protein, the cell clones were transfected with the pTRE-Luc vector, which contains a luciferase reporter gene under the control of the tetracyclineresponsive promoter. In one cell clone luciferase expression was repressed in the presence of tetracycline but induced 100-fold in the absence of tetracycline (data not shown). In the second step, this cell line was transfected with the CBF-B cDNA constructs. Expression of CBF-B protein in the cell clones was then detected by Western blot analysis using anti-FLAG antibodies.
Three cell clones expressing wild-type CBF-B (B-1, B-2, and B-3), and three cell clones expressing mutant CBF-B (Bmut-a, Bmut-b, and Bmut-c) were isolated (Fig. 2). Each of the cell clones expressed CBF-B protein in the absence of tetracycline (lanes 2, 4, 6, 8, 10, and 12), but not in the presence of tetracycline (lanes 1, 3, 5, 7, 9, and 11), indicating that expression of CBF-B in these cell clones is tightly regulated by tetracycline. Nuclear extracts were prepared from each of the cell clones and were examined for CBF binding activity by electrophoretic mobility shift assay (Fig. 3A). As a control, DNA binding activ-FIG. 1. Schematic of wild-type and mutant CBF-B constructs in a tetracycline-regulated expression vector. The mutant CBF-B, here designated as Bmut and containing an alanine substitution mutation at amino acids (aa) 304 and 311, was generated previously (15). Both the wild-type and mutant CBF-B DNAs were inserted into the pTRE vector under the control of a promoter containing a TRE upstream of the minimal CMV promoter (PminCMV). Each DNA was cloned in-frame with the DNA of the FLAG epitope to code for a FLAG-CBF-B polypeptide.
ity of the Oct1 transcription factor was also assayed by electrophoretic mobility shift assay (Fig. 3B). No significant change in CBF binding activity was observed in nuclear extracts of B-1, B-2, or B-3 cells in the presence or absence of tetracycline (Fig.  3A, lanes 1-6). In contrast, CBF binding activity was decreased significantly in nuclear extracts of Bmut-a, Bmut-b, and Bmut-c cells in the absence of tetracycline (Fig. 3A, lanes 8, 10,  and 12) compared with the activity in the presence of tetracycline (Fig. 3A, lanes 7, 9, and 11). The DNA binding activity of the Oct1 transcription factor was very similar in the nuclear extracts of the Bmut cells in the presence and absence of tetracycline (Fig. 3B, lanes 7-12). Together, these results indicated that the mutant CBF-B protein which was expressed in the Bmut cell lines in the absence of tetracycline inhibited the DNA binding of cellular CBF protein. The DNA binding of cellular CBF was less inhibited in the Bmut-c cell line, which expressed less of the mutant CBF-B protein than did the Bmut-a and Bmut-b cell clones (see Fig. 2). Thus, as expected in light of the in vitro DNA binding results, the mutant CBF-B protein acted as a dominant negative mutant in vivo in the mouse fibroblasts.
Expression of Mutant but Not Wild-type CBF-B Results in Cell Growth Arrest-Each stable cell clone was analyzed for its growth in culture with or without tetracycline. The B-1, B-2, and B-3 cells grew at almost equal rates in the presence and absence of tetracycline, indicating that the expression of excess wild-type CBF-B had no effect on the cell growth rate (Fig. 4A). In contrast, almost no growth of the Bmut-a and Bmut-b cells and little growth of the Bmut-c cells was observed in the absence of tetracycline, while normal growth was seen in the presence of tetracycline (Fig. 4, A and B). This result indicated that the expression of mutant CBF-B, which inhibited the DNA binding of cellular CBF, resulted in inhibition of cell growth. The cell growth of the Bmut-c cells was less inhibited than that of the Bmut-a and Bmut-b cells, which is consistent with the observation that less of the mutant CBF-B protein was expressed in the Bmut-c cells than in the Bmut-a and Bmut-b cells.
To determine the cell cycle distribution of the growth-inhibited cells, we used flow cytometry to analyze the Bmut-b cells grown in the presence and absence of tetracycline. This analysis showed that the cell cycle distributions of Bmut-b growing cells in the presence of tetracycline and of Bmut-b growtharrested cells in the absence of tetracycline were very similar (data not shown), thus suggesting that the expression of mutant CBF-B in the Bmut cells did not result in growth arrest in a specific cell cycle stage, but likely resulted in growth retardation in all stages of the cell cycle. To detect specifically the number of cycling cells or the S phase cells, we first labeled the Bmut-b cells at different times in culture by short incubation with BrdUrd and then the percentage of BrdUrd-labeled cells was determined using the immunochemical method followed by flow cytometry. The BrdUrd which is a thymidine analogue, incorporated into the genomic DNA of the DNA synthesizing cells (ϭ the S phase cells) but not the resting cells. The percentage of BrdUrd-labeled Bmut-b cells in the absence of tetracycline is significantly lower than that of the BrdUrd-labeled Bmut-b cells in the presence of tetracycline (Fig. 4C), thus indicating that the expression of mutant CBF-B reduced the number of cells entering into S phase.
It is possible that the growth retardation of the Bmut cells in the absence of tetracycline may be caused partly by cell death or apoptosis. To test this possibility we analyzed the Bmut-b cells by in situ cell death detection method using TUNEL reaction (TdT-mediated dUTP nicked labeling) which determines genomic DNA cleavage that generally occurs during apoptosis. In this experiment very few apoptotic cells were observed in the culture chamber containing the Bmut-b cells grown in the presence of tetracycline, and no increase in the Bmut-a, Bmut-b, and Bmut-c were isolated after transfection of the pTRE-flagBmut vector. Whole cell extracts were prepared from each clone which was grown in the presence (ϩ) and absence (Ϫ) of tetracycline (Tc) for 60 -72 h. The extracts were analyzed by the Western blot method to monitor expression of CBF-B protein using anti-FLAG antibodies. In each cell clone, the 41-kDa CBF-B polypeptide was expressed in the absence (Ϫ), but not in the presence (ϩ) of tetracycline. A 40-kDa polypeptide band visible below the CBF-B band in each lane, was a nonspecific band whose appearance was possibly due to nonspecific interaction with the anti-FLAG antibodies. This band also appeared in extracts of NIH3T3 cells without the transgenes.

FIG. 3. Inhibition of DNA binding of cellular CBF by expression of mutant CBF-B in Bmut cell clones.
Nuclear extracts were prepared from each cell clone which was grown in the presence (ϩ) or absence (Ϫ) of tetracycline (Tc) for 60 -72 h. Three micrograms of nuclear extract from each clone was added in a DNA binding reaction along with either a radiolabeled oligonucleotide containing the CBFbinding site of the mouse ␣2(1) collagen promoter (A) or a radiolabeled oligonucleotide containing the consensus binding site for the Oct1 transcription factor (B). apoptotic cell numbers were observed in the Bmut-b cells grown in the absence of tetracycline (data not shown). Thus this result indicates that the growth retardation of the Bmut-b cells in the absence of tetracycline is not caused by apoptosis.
To determine cell cycle progression, the B-2 and the Bmut-b cells were first synchronized by serum starvation and then stimulated to grow by adding serum. Analysis of DNA content of the serum-starved cells by flow cytometry demonstrated that about 80% of the cells entered into quiescence state (G 0 /G 1 phase) after the starvation period (data not shown). Nuclear extracts were prepared from cells at different time points after serum addition and assayed for CBF binding activity using electrophoretic mobility shift assay. No change in CBF binding activity was observed in the nuclear extracts of B-2 cells at different time points after serum addition, and as expected, the CBF binding activity was similar in the nuclear extracts of the cells grown in the presence and absence of tetracycline (data not shown). The CBF binding activity in the nuclear extracts of Bmut-b cells in the presence of tetracycline was very similar at different time points after serum addition (Fig. 5, lanes 1, 3, 5,  7, and 9). However, the CBF binding activity in the nuclear extracts of the cells in the absence of tetracycline was strongly inhibited before serum addition and also at each different time point after serum addition (lanes 2, 4, 6, 8, and 10). As a control, the DNA binding activity of CTF/NF-1 which is known in the literature as another CCAAT-binding transcription factor, was measured and found to be unchanged in the nuclear extracts of Bmut-b cells in the presence and absence of tetracycline (lanes 11-20). These results indicate that the mutant CBF-B was expressed in the serum-starved Bmut-b cells and specifically inhibited CBF binding activity before and at different time points after serum addition. The cell numbers were counted at different time points during serum stimulation. This showed that the doubling time of the B-2 clone was very similar (about 28 h) in the presence and absence of tetracycline (data not shown). In contrast, the doubling time of the Bmut-b clone was 26 and 51 h in the presence and absence of tetracycline, respectively (Fig. 6A). To determine S phase of the Bmut-b cells during serum stimulation, we used the BrdUrd labeling method as described earlier. This showed that the and absence (Ϫ) of tetracycline (Tc). After 48 h, the cells were stimulated to grow with complete medium containing 10% serum. The cells were then harvested at 0, 3, 6, 12, and 24 h after serum addition and were used to isolate nuclear extracts. Three micrograms of each nuclear extract was used in a DNA binding reaction with a radiolabeled oligonucleotide containing either the CBF-binding site (lanes 1-10) or a consensus CTF/NF-1-binding site (lanes [11][12][13][14][15][16][17][18][19][20]. percentage of BrdUrd-labeled Bmut-b cells in the presence of tetracycline started to increase to a high level at 12 h and reached a maximum level at 16 h after serum stimulation (Fig.  6B). In contrast, the percentage of BrdUrd-labeled Bmut-b cells in the absence of tetracycline increased very little at 12 h and reached a maximum level at 20 h after serum stimulation. Furthermore, the maximum level of the BrdUrd-labeled cells in the absence of tetracycline is significantly lower than that of the BrdUrd-labeled cells in the presence of tetracycline. These results indicate that expression of the mutant CBF-B in the Bmut-b cells delayed entry into S phase from G 1 phase, and also reduced the percentage of cells entering into S phase.
Identification of Genes whose Expression Is Altered as a Consequence of Inhibition of CBF Binding-Previously using an in vitro reconstituted transcription system we demonstrated that recombinant CBF activates transcription of the ␣2(1) collagen promoter which contained a CBF-binding site, and that mutation in the CBF-binding site of the promoter abolished the CBF-dependent transcription activation (16), indicating that the CBF binding to the promoter is required for the in vitro transcription activation of this promoter. The ␣2(1) collagen which is a subunit of the type I collagen, is expressed at high level in fibroblast cells. To analyze expression of this gene in the B and Bmut cells, total RNAs were isolated from these cell clones in the presence and the absence of tetracycline and then used in the Northern blot analysis. As a control expression of the ribosomal protein S6-encoding gene (S6) was measured. The level of expression of the collagen gene is very similar in B-2 or B-3 cells in the presence and the absence of tetracycline (Fig. 7A, lanes 1 and 2; the data for B-2 cells are not shown). In contrast, the collagen gene was expressed about 6-fold lower in the Bmut-b or Bmut-a cells in the absence of tetracycline than in the presence of tetracycline (Fig. 7A, lanes 3 and 4; data for Bmut-b cells are not shown), indicating that the inhibition of CBF binding in the Bmut cells resulted in reduction of expression of the ␣2(1) collagen gene. Thus as consistent with our previous in vitro studies, the present analysis indicated that the CBF binding is required for the in vivo transcription of the ␣2(1) collagen gene. FIG. 6. Analysis of serum stimulated growth of quiescent Bmut-b cells using the BrdUrd labeling method. The Bmut-b cells were first synchronized in the presence (ϩ) and absence (Ϫ) of tetracycline (Tc), and then stimulated to grow by serum addition as described in Fig. 5. A, the cells were harvested at 0, 12, 24, 36, and 48 h after serum stimulation and then counted as described in the legend to Fig.  4A. B, at different time points after serum stimulation the cells were labeled with BrdUrd for 30 min, and the percentage of the BrdUrdlabeled cells was measured using the method described in the legend to  7. Inhibition of CBF binding reduced expression of E2F1, topoisomerase II␣, and ␣2(1) collagen genes. A, total RNAs were isolated from equal numbers of cells of B-3 and Bmut-a clones grown 72 h in the presence and absence of tetracycline. Ten micrograms of each RNA sample was separated on a 1% agarose-formaldehyde gel and analyzed for expression of different genes by the Northern blot method. B and C, B-2 and Bmut-b cells were first synchronized and then stimulated to grow as described in the legend to Fig. 5. The cells were harvested at different time intervals and were used to isolate total RNAs. Ten micrograms of each RNA sample was used in the Northern blot analysis. The total RNAs isolated after 12 and 15 h of serum stimulation were used to detect expression of the E2F1 gene (B), and the total RNAs isolated after 18 and 21 h of serum stimulation were used to detect expression of the topoisomerase II␣ and ␣2(1) collagen genes (C). In each experiment, expression of the S6 gene, which encodes ribosomal S6 protein, was measured as a control. The 18 S and 28 S RNA bands of each loaded RNA sample are shown at the bottom of the figures. The radiolabeled bands were quantitated with a PhosphorImager and adjustments were made for the amount of total RNA loaded in each lane, which was measured in relation to the amount of S6 band and the intensity of 28 S and 18 S RNAs.
Recent studies have indicated that promoters of several genes including E2F-1, topoisomerase II␣ (topo II␣), thymidine kinase, and protein phosphatase cdc25C whose transcriptions are regulated during the cell cycle contain multiple CBF-binding sites, and that expression of many of these genes are crucial for cell cycle progression (4,(37)(38)(39)(40). For example, the mRNA level of the E2F1 gene, a member of the E2F of family transcription factors, is increased severalfold at the G 1 /S phase of cell cycle, and the expression of E2F1 in quiescent cells can activate transcription of several G 1 /S-phase-regulated promoters (38,41), suggesting that E2F1 plays a critical role in cell cycle progression through G 1 to S phase. In the case of topo II␣ which is one of the two isoforms of the DNA topoisomerase II enzyme that plays an essential role in several cellular processes such as replication, recombination, and chromosome condensation and segregation (39), the level of its mRNA varies through the cell cycle: low in G 1 and increasing through S phase to maximal in G 2 /M phase. If CBF acts as an upstream positive regulator of transcription of both the E2F1 and topo II␣ genes, then inhibition of CBF binding in vivo should result in decreased expression of these critical genes and lead to cell growth retardation. To test this possibility, we analyzed the expression of the E2F1 and topo II␣ genes in B-2 and Bmut-b cells. Total RNAs were isolated at different time points after serum addition in the synchronized B-2 and Bmut-b cells grown with and without tetracycline, and then subjected to Northern blot analyses to measure expression of the E2F1 and topo II␣ genes. As expected from previous studies, in these cell clones E2F1 and topo II␣ were expressed at maximal level at 15 and 21 h after serum addition, respectively (Fig. 7, B and C). The level of expression of these genes were very similar in the B-2 cells in the presence and absence of tetracycline. In contrast, both the E2F1 and the topo II␣ genes were expressed about 2.5-fold lower in the Bmut-b cells in the absence of tetracycline than in the presence of tetracycline. These results indicated that inhibition of CBF binding in the Bmut-b cells resulted in reduction of expression of both E2F1 and topo II␣ genes that occurred during cell cycle progression. In this experiment, the expression of the ␣2(1) collagen gene which transcription is not regulated during cell growth was reduced 6-fold by the inhibition of CBF binding (Fig. 7C), similar to the earlier experiment in the unsynchronized cells in Fig. 7A. Altogether these results showed that the inhibition of CBF binding decreased expression of the E2F1, topo II␣, and ␣2(1) collagen genes which promoters contained the CBF-binding site, thus implicating that CBF directly regulates expression of these genes in vivo in mouse fibroblasts.
Since the CBF-binding site is present in the promoters of many mammalian class II genes, the inhibition of CBF binding may have altered the transcription of many other cellular genes. To test this possibility, we monitored the mRNA profiles of B-2 and Bmut-b cells using the DDRT-PCR method (42). The DDRT-PCR method is widely used to identify differential gene expression. In brief, total RNAs were isolated from each of the B-2 and Bmut-b cells in the presence and absence of tetracycline, and then used in DDRT-PCRs as described under "Materials and Methods." The amplified cDNAs generated from the RNAs of the B-2 and Bmut-b cells were separated on a denaturing polyacrylamide gel. The patterns of cDNA bands were then compared to identify differentially expressed genes in the Bmut-b cells grown in the absence of tetracycline (Fig. 8). Surprisingly, the majority of the cDNA bands for the Bmut-b cells grown with and without tetracycline were very similar in intensity (Fig. 8, A-C, lanes 3 and 4). Moreover, these cDNA bands were also comparable in intensity to the cDNA bands of B-2 cells grown with and without tetracycline (Fig. 8, A-C,  lanes 1 and 2). These results suggested that the levels of many different mRNAs were not changed significantly by the expression of mutant CBF-B in the growth-inhibited Bmut-b cells. Nonetheless, the DDRT-PCR analysis identified two cDNA bands whose levels were specifically decreased in the Bmut-b cells but not in the B-2 cells grown in the absence of tetracycline (indicated by arrows in Figs. 8, A and B). These cDNA bands were isolated from the gel, reamplified by PCR, subcloned, and then identified by DNA sequencing. The DNA sequences of the two clones thus isolated from the cDNA bands of Figs. 8, A and B, matched completely with the sequences of the Egr1 gene and Mts1 gene, respectively. Subsequent Northern blot analysis showed that the levels of both the Egr1 and Mts1 mRNAs were decreased by about 30 and 65%, respectively, in the Bmut-b cells but not in the B-2 cells in absence versus in the presence of tetracycline (Fig. 8D). This indicated that expression of the Egr1 and Mts1 genes was decreased as a result of the inhibition of CBF binding in the Bmut-b cells. The Egr1 gene (also known as Krox-24) encodes a zinc finger-containing DNA-binding protein and is one of the early growth response genes (32). The Mts1 gene, which encodes a calciumbinding protein, was initially isolated from metastatic tumors in which it was expressed at much higher level than in normal tissue. The Mts1 gene is also expressed in various established cell lines and appears to be regulated during cell growth (31). Altogether our results showed that the inhibition of CBF binding decreased the expression of multiple genes including several apparently cell cycle regulated genes. DISCUSSION Our results indicated that stable expression of mutant CBF-B inhibits the DNA binding of cellular CBF and thus acts as a dominant negative mutant in vivo. Interestingly, expression of the mutant but not the wild-type CBF-B resulted in arrest of fibroblast cell growth, which in turn correlated well with the inhibition of CBF binding, thus indicating that the DNA binding activity of CBF is required for normal fibroblast cell growth. The nature of the cell growth arrest was revealed by our flow cytometry, BrdUrd labeling, and TUNEL assay analyses. The flow cytometry and TUNEL assay indicated that the growth arrest is not due to either the growth arrest in specific cell cycle stage or the apoptotic cell death. In contrast, the BrdUrd labeling analysis which measured specifically the DNA synthesizing or the S phase cells, showed that expression of the mutated CBF-B resulted in reduction of BrdUrd-labeled cells indicating that the inhibition of CBF binding decreased the number of cells going through the S phase of cell cycle. In the case of serum starvation/stimulation experiment, the mutant CBF-B was expressed in the Bmut-b cells in the absence of tetracycline during the starvation period and inhibited almost 90% of the cellular CBF binding activity in the quiescent cells. The inhibition of CBF binding in the quiescent Bmut cells did not completely inhibit the serum-stimulated cell growth. However, the doubling time of growth of the Bmut cells expressing mutant CBF-B is two times longer than that of the Bmut cells not expressing mutant CBF-B, thus indicating that the inhibition of CBF binding reduced significantly the rate of progression of cell growth. The BrdUrd labeling analysis of the quiescent cells at different times after serum stimulation clearly showed that the expression of the mutant CBF-B delayed the progression of S phase and also reduced the percentage of cells progressing through the S phase. Thus the inhibition of CBF binding results in inhibition of both the rate of progression of S phase and the amount of cells entering into the S phase which is most likely cause of the retardation of cell growth.
The inhibition of CBF binding decreased significantly ex- pression of the ␣2(1) collagen gene which promoter contained a CBF-binding site. This result is consistent with previous studies which showed that mutation of the CBF-binding site in the collagen promoter decreased in vivo transcription activity of the promoter in a transient transfection experiment in fibroblast cells (26). This result indicated that expression of the ␣2(1) collagen gene is directly regulated by CBF in fibroblasts. Our results also indicated that the inhibition of CBF binding decreased expression of both the E2F1 and topo II␣ genes that are activated at different stages of the cell cycle. The promoters of the mouse E2F1 and topo II␣ genes consist of multiple CBF-binding sites which are also conserved in the corresponding human promoters (37)(38)(39), suggesting that these genes are also directly regulated by CBF. Recent studies of the E2F1 promoter showed that the transcriptional activity of this promoter, like the expression of the endogenous E2F1, is stimulated strongly at the G 1 /S phase of the cell cycle (38). The E2F1 promoter consists of multiple DNA elements, including binding sites for E2F, SP1, and CBF transcription factors, that regulate repression of the promoter in G 0 and activation in S phase. Those studies revealed that mutation of the E2F site increased the promoter activity in G 0 but had little effect in S phase; in contrast, mutation of the CBF-binding site reduced promoter activity only in S phase. These results are consistent with our present observation that the inhibition of CBF binding decreased the activated expression of E2F1 at 15 h after serum stimulation. This indicated that CBF participated in the transcription activation of the E2F1 gene during the period when this gene was stimulated by the growth signal. Since the CBF binding is not regulated during cell growth, it is possible that the CBF-mediated activation of the E2F1 gene is repressed during G 0 phase and that, in S phase, CBF activates this gene when the repression is relieved. Recent studies of the mouse topo II␣ promoter demonstrated that transcription of this promoter is low in the G 1 phase and is stimulated through S phase to a maximal level at G 2 /M phase of the cell cycle, similar to the case for endogenous topo II␣ gene (37). However, the role of the multiple CBF-binding sites in the promoter activity is not known. The human topo II␣ promoter also contains multiple CBF-binding sites that, when deleted, resulted in significant loss of the promoter activity, indicating that the CBF-binding sites are part of an upstream activator sequence (39). We speculate that activation of the mouse topo II␣ is regulated during the cell cycle by derepression of the CBF-mediated activation. Recent studies showed that transcription activation of several cell cycle-regulated promoters, including the promoters of the cyclin A, Cdc2, and CDC25C genes, appeared to be mediated by a derepression mechanism (40). These promoters consisted of a cell cycle-regulated repressor element and an upstream activator sequence element which mediate repression and activation of the promoter, respectively. The CBFbinding site is found in the upstream activator sequence of these cell cycle-regulated genes, suggesting that inhibition of CBF binding possibly decreased expression of these genes similar to the E2F1 and topo II␣ genes.
The inhibition of CBF binding results in a modest decrease of expression of both the E2F1 and topo II␣ genes compare with that of the ␣2(1) collagen gene. The modest decrease of E2F1 and topo II␣ expression is somewhat consistent with the observation that the inhibition of CBF binding only delayed but did not completely block the progression of S phase. It is possible that the decreased expression of the ␣2(1) collagen gene may partly contribute to the retardation of cell growth. The ␣2(1) collagen is a subunit of the type I collagen which is part of the extracellular matrix protein. Recent studies showed that the extracellular matrix protein and the mitogens together regulate induction of cyclin D and cyclin A in mouse and human fibroblasts, and play an important role in the anchorage-dependent cell cycle progression (43). However, a specific role of the type I collagen in the cell growth process is not known. Although the collagen promoter contains a single CBF-binding site, the inhibition of CBF binding reduced the expression of the collagen gene more than that of the E2F1 and topo II␣ genes which promoters contain multiple CBF-binding sites. This indicates that the in vivo transcription of these promoters containing CBF-binding sites are not equally dependent on CBF activity. It seems likely that in case of E2F1 and topo II␣, the contribution of CBF in the in vivo transcription is minor and possibly regulated by other transcription factors which bind to these promoters.
Our differential display experiments indicated that the inhibition of CBF binding did not result in a significant change in expression of many cellular genes. This experiment, however, identified two genes, Egr1 and Mts1 whose expression is specifically decreased by the inhibition of CBF binding. Analysis of the mouse Egr1 and Mts1 promoters revealed that none of the promoters contain consensus CBF-binding site in the proximal promoter region (31,44), thus indicating that these genes are not directly regulated by CBF. However, it is possible that the CBF-binding site in these promoters may be located in the distal promoter regions which are not yet identified. Altogether our results showed that the inhibition of CBF binding decreased expression of multiple cellular genes, which is consistent with the previous studies that showed that CBF binding to the various promoters is required for optimal transcription activity of the promoters (4). Since expression of none of the genes except the collagen gene are decreased at high levels, it is possible that the inhibition of CBF binding could decrease a smaller amount of expression of many other genes which are required for cell proliferation, and such a smaller decrease of multiple genes could result in significant reduction of the rate of cell growth progression. However, we cannot rule out a possibility that the inhibition of CBF binding could decrease significantly expression of specific gene(s) which are required for the progression of fibroblast growth. Further studies are needed to test all these possibilities.
The function of CBF homologues in yeast was studied extensively which showed these genes were specifically required for expression of several nuclear genes involved in mitochondrial function (9,10). Our present results provide direct evidence for the first time that CBF is an important regulator for the normal growth of mouse fibroblast cells, and that it is required for expression of various mouse genes. However, it is possible that CBF may also be required for expression of nuclear genes involved in mitochondrial function in mouse cells, and that the inhibition of CBF binding in fibroblast could disrupt the mitochondrial function, and thus cannot produce adequate energy, which could be an important cause for the retardation of fibroblast growth.
The function of CBF homologues in other eukaryotes was only revealed recently. For example, disruption of the CBF-C homologue aab1 in Neurospora crassa results in very slow growth and altered morphology, thus indicating pleiotropic effects on growth and development (45); and in the higher plant, Arabidopsis thaliana, the CBF-A homologue LEC1 functions at an early developmental stage and activates transcription of genes required for both embryo morphogenesis and cellular differentiation (46). Altogether these results indicated that the function of CBF has diverged during evolution and that this transcription factor likely plays an important role in growth and development of higher eukaryotes.


s2fieldsofstudy: Biology Chemistry Medicine

text:
Functional Brain Receptor Imaging with Positron Emission Tomography

: A new cocaine derivative for imaging the dopamine transporter has been developed. Measurements of radioligand binding of 11C-(+)-McN-5652 in vivo with PET suggests that ecstasy interacts directly with the serotonin reuptake sites and that a single oral dose of ecstasy (1.5 mg/kg) does not cause any changes in the serotonin transporter density in the human brain. Finally, a number of epibatidine derivatives have been developed as ligands to study the central nAChRs in vivo, however, toxicity studies prevented further clinical use.

Introduction
In this paper, we present the activities of two groups that are involved in the development of radioligands for imaging brain functions within the Center for Radiopharmaceutical Science of ETH, Paul Scherrer Institute and University Hospital of ZUrich. The groups are the Radiotracer Synthesis Group and the Radiopharmacy Division Group in ZUrich. The aim of both research groups is the development of selective and high affinity radioligands for diagnosis using non-evasive positron emission tomography (PET). Research is on the dopaminergic, serotonergic, glutamatergic and the nicotinergic acetylcholine neurotransmission systems, however, the last two neurotransmission systems are currently the main focus of research. The research activities cover all aspects of preclinical research and BAG-approved clinical investigations. Some of our research efforts on the dopaminergic, serotonergic, and the nicotinergic acetylcholine neurotrans-mIsSIOn systems are discussed here. It should be mentioned that extensive research efforts are also being made to develop PET radioligands for the glutamatergic neurotransmission system for which currently no PET radioligand exists.
PET is an imaging modality that allows the study of physiological, biochemical and pharmacological functions at a molecular level. Its strength is the ability to obtain direct quantitative information on the pharmacokinetics and pharmacodynamics of a biomolecule in living animals and humans. PET offers a specific advantage when studying biological molecules that can be labelled with positron emitting radioisotopes such as IIC, 13N, and 150 with half-lives of 2.1,10 and 20.3 min, respectively. Since the main constituents of biological molecules are carbon, nitrogen and oxygen, the incorporation of 150, 13N or IIC into pharmaceuticals leads to radiopharmaceuticals which are chemically indistinguishable from their non-labelled counterparts. Apart from negligible isotopic effects, the radiolabeled pharmaceutical possesses the same pharmacological properties as the non-labelled compound. 18F with a half-life of 110 min is another frequently used positron-emitting radionuclide. However, only a few bioorganic compounds contain either a fluorine atom or a halogen. 18F may be substituted for hydrogen in many compounds with no substantial change in their biological activities. Because most PET radioligands are produced at high specific radioactivity, the PET technique employs trace amounts (few nanomoles) of the pharmaceuticals so that the risk of untoward effect or chemical toxicity remains also negligible.
Establishing the usefulness of a PET radioligand for human applications requires the expertise of several scientists including radiochemists, pharmacologists and clinicians who work in a close collaboration in an interdisciplinary environment. There are a number of criteria that must be fulfilled in order to obtain a brain receptor ligand that can be useful in vivo. The conditions are briefly discussed below.
High Affinity for the Receptor (K d :5;10-9 M) The equilibrium dissociation constant (K d ) of a drug receptor complex is the concentration of drug that occupies or binds to 50 % of available receptor population. By definition, affinity is the reciprocal of equilibrium dissociation constant. Considering that the concentration of binding sites (B max ) for most brain receptors is rather low (nano-to femtomoles per milligram tissue), PET radio-ligands should have binding affinities in the subnanomolar range. This can be established in vitro either by a direct or competitive binding assay.

Specific Radioactivity
Specific radioactivity refers to the amount of radioactivity per unit mass of a radiopharmaceutical. Unlike radioligands used only for in vitro binding assays, radioligands used for in vivo brain receptor imaging must be prepared in high specific radioactivities so that only a small percentage of the total number of available binding sites are occupied by the radioligand. Specific radioactivities H I3-CPPIT should be greater than 3700GBq/mmol. Thus, saturation of binding sites and consequent pharmacological effects are avoided.

Metabolism and Route of Application
Whereas many drugs are given orally, PET radioligands are administered in most cases by an intravenous injection. The oral administration of a PET radiopharmaceutical would result in too rapid a metabolism and a slow uptake of radioligand into plasma. These pharmacodynamic properties would not be compatible with the relatively short half-lives of I H CHIMIA 2000, 54. No. 11 commonly used PET radionuclides. Because PET cannot discriminate between signals from parent radioligand and radiolabeled metabolites, care must be taken to ensure that radioactive metabolites that are formed in the course of the studies do not contaminate the PET signals. It has to be verified separately for each PET radioligand whether the PET signals originate from a single radiolabeled chemical entity or not.

Blood-Brain Barrier Permeability
For a central nervous system (eNS) radiopharmaceutical to be useful as an

Neuronal Nicotinic Acetylcholine Receptors (nAChRs)
Nicotinic receptors are involved in a broad variety of physiological and patho-strated in chronic MDMA users. Using PET and S-methyl labelled IIC_(+)-McN-5652, we examined whether a single acute oral dose of ecstasy had any effects on the serotonin transporter density in healthy volunteers. Distribution volume was used as a parameter for radioligand binding.
The results (Fig. 4) showed that a single oral dose of ecstasy (1.5 mglkg) given 90 min prior to PET measurement, significantly reduced "C-( +)-McN-5652 binding in subcortical regions compared to baseline scan. The reduction in IIC_(+)-McN-5652 binding suggests that ecstasy interacts directly with the serotonin reuptake sites in the human brain. A second question of the study was whether four weeks after a single oral dose of ecstasy, probable alterations in the density of the serotonin transporter would persist and still be detectable. Four weeks after the second scan, a third PET scan was performed in the same volunteers. No significant effects of the single oral dose of ecstasy on the binding of lIC-(+)-McN-5652 were observed. These results corroborate and extend numerous findings in animals that one or a few single doses of ecstasy (1.5 mglkg) are unlikely to cause alterations in serotonin transporter density indicative for serotonergic neurotoxicity.   The compound, (+)-McN-5652 (Fig.  3), is an exceedingly potent and selective blocker of the serotonin transporter and "C labelled trans-( +)-McN-5652 is generally accepted as the most successful ligand for visualising the serotonin transporters in vivo using PET. We have recently improved on the synthesis of trans-( +)-McN-5652 and have also established a method to prepare the enantiomerically pure (+)-thioester precursor in high reproducible yields.
The human use of methylenedioxymethamphetamine (MDMA or ecstasy) as a recreational drug is of a major concern due to the fact that ecstasy is a selective neurotoxin that causes damage to serotonin neurons. A decrease in serotonin transporter density has been demon-Changes in the brain neurotransmission have been implicated in the pathophysiology of several brain disorders and therefore receptors and reuptake sites of the various neurotransmission systems have become targets for radiopharmaceutical design in the last years. With regard to the dopaminergic neurotransmitter system, we have radiolabeled cocaine analogues such as B-CIT (2B-carbomethoxy-3~-4-iodophenyl)tropane and FP-~-ClT(N -fluoropropy 1-2~-carbomethoxy-3P-4-iodophenyl)nortropane with IIC. Notable disadvantages associated with these compounds are non-selective binding to the serotonin transporter and the late binding equilibrium. Until recently, we have been using [IIC]FP-CIT on routine basis to examine changes in dopamine transporter density in Parkinson patients. A rather new compound, 3~-( 41-chlorophenyl)-2~-(3'-phenyloxazolon-5'-)tropane (CPPIT) (Fig. 1), a cocaine analogue that lacks the 2~-ester group has been radiolabeled with IIC by N-methylation and evaluated in mice and in five healthy volunteers. Blockade studies in mice confirmed the in vitro selectivity reported for CPPIT. The uptake of radioactivity in the striatum, a region with the highest densities of the dopamine transporter, was significantly reduced by preinjecting GBR 12909 (5 mglkg), a dopamine transporter antagonist. PET studies of [IIC]CPPIT in healthy volunteers indicated high accumulation of the radioligand in the striatum (Fig. 2). Results obtained so far indicate that [IIC]CPPIT has the potential to be used as a radioligand for imaging the dopamine transporter in humans.

Dopaminergic System
imaging agent, it must be lipid soluble and should pass the blood-brain barrier (BBB). The octanollwater partition coefficient, P, is often used as a predictor for BBB penetration. The P values can be computed or experimentally measured and log P values between 2 and 3 are generally considered optimal.

Proteins
Also of importance are rapid clearance rates from blood and non-specific binding sites. A low binding of radioligand to plasma proteins is also essential because only the unbound fraction of radioligand in plasma is available for diffusion out of the vascular tissue. CHIMIA 2000.54. No. 11 Received: September 6,2000 concepts to develop a useful nAChR radio-ligand.
The N-[IIC]methyl compounds show impressive images of the thalamus: the site of highest nicotine receptor density; cytisine-blocked (-) and the (+)-isomer images show no specific brain uptake (Fig. 6). The eyes also accumulate radioligand, but this is only visible after cytisine block and with the (+)-isomer, where no specific brain uptake occurs.
To determine the concentration of the unchanged IIC radioligand during the PET -study, a solid phase extraction was done and tissue tracer kinetics were analysed according to a two or three compartment model. As expected, the (-)enantiomers revealed in contrast to the (+)-enantiomers high levels of specific binding. In addition, the in vivo kinetics revealed high affinity, high specificity and reversible transport. Thus, these ligands proved to be suitable ligands to study the central nAChRs in vivo. Despite these encouraging results, detailed toxicity studies prevented further clinical use: even at levels where no carrier was added to the IIC compounds, the toxicity of the compounds was too high for routine human use.
In conclusion epibatidine and its analogues are excellent ligands for the nAChRs and the IIC-methylated derivatives have ideal pharmacokinetic properties as PET ligands. However, their extreme toxicity prevents human application.
Collaborations exist outside the Center for Radiopharmaceutical Science among others with Prof. Gustav K: von Schulthess, University Hospital ZUrich, Prof. Daniel Bertrand, University of Geneva, Associate Prof. Franz W. Vollenweider, Psychiatric Hospital ZUrich, and pharmaceutical industries.

Recent references from our PET-
Tracer research ly reported. With the exception of PABH and 4PABH, all compounds were separated into their enantiomers via chiral high-performance liquid chromatography as described recently. Optical purities higher than 98% ee were obtained. For in vivo studies and further investigations with PET EPB, DCI-EPB, 2PABH and HEPB were radiolabeled with IIC, which was introduced by selective methylation of the 7-aza or 8-aza position.
Rats were investigated in a PET scanner in such a way that a dynamic data set was acquired. Experiments with rats gave excellent results. Using this method, the (11C]methylated enantiomers of HEPB, DCIEPB and 2PABH were compared with N-[IIC]methyl-(+)-EPB and (-) -EPB. Promising results were obtained for N-[IIC]methyl-(-)-DCIEPB and N-[11C]methyl-(-)-HEPB. Both radioligands showed high brain uptake with high levels of specific binding and thus revealed a similar in vivo behaviour as N-[IIC]methyl-(-)-EPB. In contrast, the labelled 2PABH derivatives did not display good brain uptake curves. As a result the 'DCIEPB-' and the 'HEPBpathway' represented two encouraging The development of new drugs acting as cholinergic channel modulators might be beneficial for the treatment of Alzheimer's disease, Parkinson's disease, attention deficit hyperactivity disorder, Tourette's syndrome, schizophrenia, depression, anxiety, smoking cessation, and for the treatment of pain, and neurogenic inflammation. Furthermore, nicotinic receptor subtypes are involved in learning, memory, and cognitive performance. Subtype selective ligands of nicotinic nAChR may be useful in the treatment of these CNS disorders. Beside a possible benefit as therapeutically useful substances, the new ligands also might be useful as radiotracers in biological research and for the diagnosis of above mentioned CNS disorders. The syntheses of all ligands followed a convergent path: the azabicyclo[2.2.1] heptene system was constructed and the pyridine moiety was attached (Heck reaction). Variations about the pyridine ring of EPB and modification of the bicyclomoiety resulted in novel ligands (Fig. 5).
The syntheses of EPB, HEPB, DCI-EPB, 2PABH, and 4PABH, were recent- Fig. 6. Pig brain images of N-[11C]methyl-(-)-HEPB (without and with blockade by cytisine) and of N_[11 C]methyl-(-)-H EPB; summation of all frames from 3D-90 min. All pictures are coronal brain slices of the same position. Beside the brain the eyes are visible as two lateral activity spots. The eye uptake is the same in all cases, but the images of the eyes are clearer in the blocking experiment and with N-[11C]methyl-(+)-HEPB because the intensity scale is normalised on the most active spots in the image.