Introduction

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Excessive bone resorption is the major pathological feature of a number of common bone diseases, including Paget's disease, tumor-associated osteolysis, and postmenopausal osteoporosis (Mundy, 1995). Postmenopausal osteoporosis affects approximately 30% of women over the age of 50 and therefore is of particular social and economic importance. Bisphosphonates are among the most important drugs used in the clinical management of these diseases, because they are powerful inhibitors of bone resorption (Fleisch, 1991).

Bisphosphonates are a class of pyrophosphate analogs containing a phosphate-carbon-phosphate (P-C-P) backbone. This structure confers the ability to chelate calcium ions and, consequently, the ability to target to bone mineral in vivo. The geminal carbon of the P-C-P group has two side chains, R₁ and R₂. The R₁ side chain is usually a hydroxyl group, because this enhances the affinity of the compounds for bone mineral but has little influence on the antiresorptive potency (van Beek et al., 1994; Rogers et al., 1995). The major determinant of antiresorptive potency is the structure and conformation of the R₂ side chain (van Beek et al., 1994; Rogers et al., 1995). First generation of bisphosphonate drugs have a short R₂ side chain, such as ---CH₃ (as in etidronate) or ---Cl (as in clodronate). These bisphosphonates, together with tiludronate (which has a chlorophenylthiomethylene R₂ side chain), are 10- to 1000-fold less potent than the second-generation bisphosphonates, which have an R₂ side chain containing a primary amino group (as in pamidronate and alendronate). The third-generation bisphosphonate ibandronate is an analog of pamidronate that contains a tertiary amino group and is 10,000-fold more potent than the first-generation compounds (Geddes et al., 1994) (Table 1).

Bisphosphonates seem to inhibit bone resorption by directly affecting bone-resorbing osteoclasts (Flanagan and Chambers, 1991), preventing processes required for resorption (Sato et al., 1991; Murakami et al., 1995), or by promoting osteoclast apoptosis (Hughes et al., 1995). Bisphosphonates may also inhibit bone resorption by preventing the formation of osteoclasts from hematopoietic precursors (Boonekamp et al., 1986) or through effects on osteoblasts (Sahni et al., 1993). Until recently, however, the molecular basis for these effects was unclear. We have been using the J774 macrophage-like cell line to study the molecular properties of bisphosphonates, because these cells also undergo apoptosis after treatment with some bisphosphonates (Rogers et al., 1996b). Apoptosis in J774 cells induced by alendronate involves the activation of caspase-3-like enzymes (Coxon et al., 1998). Caspases are a family of cysteine proteases that have recently been shown to play an important role in the initiation and execution of apoptosis (Cohen, 1997). Caspases can be divided into two major groups depending on their homology to interleukin-1-converting enzyme (caspase-1) or CPP32 (caspase-3), which differ in their ability to cleave the fluorogenic peptide substrates N-acetyl-Tyr-Val-Ala-Asp-aminomethylcoumarin (Ac-YVAD-AMC) and N-acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin (Ac-DEVD-AMC), respectively (Cohen, 1997). This permits quantification of active caspase-1-like or caspase-3-like proteases in cell lysates.

Several studies have suggested that aminobisphosphonates have a molecular mechanism of action different from or in addition to that of the nonaminobisphosphonates (Reitsma et al., 1982; Boonekamp et al., 1986). We found previously (Rogers et al., 1992, 1994a; Frith et al., 1997) that clodronate could be metabolized intracellularly by eukaryotic cells in vitro to a nonhydrolyzable analog of ATP (AppCCl₂p), which could be detected in cell extracts by using fast-performance liquid chromatography (FPLC). More recently, we developed a highly sensitive technique to identify bisphosphonate metabolites in cell extracts, combining HPLC and tandem mass spectrometry (MS) (Auriola et al., 1997). Using the latter procedure, we confirmed that clodronate could be metabolized to AppCCl₂p (Auriola et al., 1997). This clearly raises the possibility that some bisphosphonates could act because of the accumulation of AppCp-type metabolites. By contrast with clodronate, we recently proposed that the amino-bisphosphonate alendronate inhibits enzymes of the mevalonate pathway, resulting in the loss of isoprenoid intermediates [farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP)] required for post-translational modification (isoprenylation) of small GTP-binding proteins (Coxon et al., 1998; Luckman et al., 1998). It is likely that apoptosis induced by alendronate is the consequence of loss of isoprenylated proteins (Coxon et al., 1998). Consistent with this notion, we found that apoptosis induced by alendronate could be partially suppressed by the addition of farnesyl pyrophosphate and geranylgeranyl pyrophosphate (Luckman et al., 1998).

Taken together, these observations suggest that bisphosphonates may be tentatively classified into two groups with distinct molecular mechanisms of action; those that can be metabolically incorporated into analogs of ATP (the nonaminobisphosphonates) and those that inhibit protein isoprenylation (the aminobisphosphonates). To examine this hypothesis, we directly compared the effects of three nonaminobisphosphonates [dichloromethylene-1,1-bisphosphonate (CLO), 1-hydroxyethylidene-1,1-bisphosphonate (ETI), and chloro-4-phenylthiomethylene-1,1-bisphosphonate (TIL)] and three aminobisphosphonates [4-amino-1-hydroxybutylidene-1,1-bisphosphonate (ALN), 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (PAM), and 1-hydroxy-3(methylpentylamino)propylidene-1,1-bisphosphonate (IBA)] on J774 macrophages. Specifically, we compared 1) the ability of the bisphosphonates to activate caspase-3- or caspase-1-like proteases and to induce apoptosis; 2) the ability of cell-permeable analogs of FPP and GGPP, farnesol (FOH), and geranylgeraniol (GGOH), respectively (Crick et al., 1997), to suppress caspase activation and apoptosis; 3) the ability of bisphosphonates to inhibit protein isoprenylation; and 4) the ability of J774 cells to metabolize these compounds to AppCp-type analogs of ATP.

	Materials and Methods

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Reagents. ALN, IBA, PAM, CLO, ETI, and TIL were kindly provided by Procter and Gamble Pharmaceuticals (Cincinnati, OH). Stock solutions (10 mM or 50 mM) were prepared in PBS, pH 7.4, then filter-sterilized using a 0.2-µm filter. Caspase substrates (Ac-DEVD-AMC and Ac-YVAD-AMC) were purchased from Alexis (Zurich, Switzerland) and caspase-3 inhibitor (Z-DEVD-fmk) was from Calbiochem (Nottingham, UK). Stock solutions (10 mM) of the caspase substrates and caspase inhibitor were prepared in dry dimethyl sulfoxide. [¹⁴C]Mevalonolactone was purchased from Amersham (Aylesbury, UK). Mevastatin was purchased from Sigma Chemical Co. (Poole, UK) and prepared as described previously (Luckman et al., 1998). All other reagents were purchased from Sigma Chemical Co. (Poole, UK).

Cell Culture. J774 cells were obtained from the European Collection of Animal Cell Cultures (Salisbury, UK). Cells were cultured at 37°C in Dulbecco's modified Eagle's medium (GIBCO, Paisley, UK) supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1 mM L-glutamine in a 5% CO₂ atmosphere.

Determination of Caspase Activity. Caspase-3- and caspase-1-like enzyme activity was measured by proteolytic cleavage of the fluorogenic substrates Ac-DEVD-AMC and Ac-YVAD-AMC, respectively. J774 cells were seeded in 6-well plates at a density of 7 × 10⁵ cell/well and left to adhere for 24 h. Cells were treated with 100 µM ALN, IBA, or PAM or 750 µM CLO, ETI, or TIL for 16 h. Adherent and nonadherent cells were then harvested and pooled, washed in PBS, and lysed in 70 µl of lysis buffer as described previously (Coxon et al., 1998). For the caspase assay, 50 µl of cell lysates were made up to 1.5 ml with lysis buffer containing 5 mM cysteine and the respective substrate (40 µM) and incubated at 37°C for 1 h. The release of amino-4-methylcoumarin was determined on a Perkin-Elmer fluorometer (Perkin-Elmer, Norwalk, CT) using an excitation wavelength of 380 nm and an emission wavelength of 460 nm. The results were corrected for protein content of the samples (bicinchoninic acid assay; Pierce, Rockford, IL) and expressed as percentage of control.

Quantification of Apoptosis. J774 cells were seeded into 24-well tissue culture plates (1 × 10⁵ cells/well) and left overnight to adhere. The cells were then treated for up to 48 h with 100 µM ALN, 100 µM IBA, or 100 µM PAM alone or in combination with 50 µM GGOH or 50 µM FOH (quadruplicate wells for each treatment). After 24 and 48 h, adherent and nonadherent cells in each well were pooled and centrifuged (3200g, 5 min) and the percentage of apoptotic cells was assessed based on nuclear morphology after staining nuclei with 4,6-diamidino-2-phenylindole, as described previously (Rogers et al., 1996b).

Incorporation of [¹⁴C]Mevalonate into Isoprenylated Proteins. The ability of amino- and nonaminobisphosphonates to affect protein isoprenylation in J774 cells was investigated by measuring metabolic incorporation of [¹⁴C]mevalonolactone into proteins that had been post-translationally modified with farnesyl and geranylgeranyl groups, using the method of Luckman et al. (1998). In short, cells were depleted of mevalonate by incubation with 5 µM mevastatin for 4 h. Cells were then treated for 24 h with 7.5 µCi/ml [¹⁴C]mevalonolactone (specific activity, 57 mCi/mmol); 5 µM mevastatin; and either 100 µM ALN, IBA, or PAM or 750 µM CLO, ETI, or TIL. Cells were lysed in radioimmunoprecipitation assay buffer and 50 µg of protein (as determined by Pierce bicinchoninic acid assay) of each sample were electrophoresed on 12% polyacrylamide-SDS gels. These were then stained with Coomassie blue to confirm equal loading of wells and dried. Radiolabeled bands were visualized using a Bio-Rad Personal FX Imager (Bio-Rad, Hercules, CA).

Identification of Bisphosphonate Metabolites by HPLC-Tandem MS. Approximately 2.5 × 10⁶ J774 cells were seeded into 162-cm² tissue culture flasks (one flask per treatment). The cells were cultured until approximately 70% confluent and then treated for 24 h with either 250 µM CLO, 750 µM ETI, 100 µM TIL, 100 µM ALN, 100 µM IBA, 100 µM PAM, or an equivalent volume of PBS. These concentrations were selected because they either cause a significant reduction in J774 cell viability as assessed by the MTT assay (for CLO, ETI, TIL, data not shown) or induce a significant increase in apoptosis in cultures of J774 cells (for ALN, IBA, PAM; Fig. 4). After treatment, the cells were scraped from the flask, centrifuged (220g, 5 min) and washed in cold PBS. The cell pellets were kept on ice and were resuspended in 300 µl of acetonitrile (ACN). Within 2 min, 200 µl of ice-cold distilled water was added, the extracts were centrifuged (14,000g, 2 min, 4°C), and the supernatants were transferred to fresh Eppendorf tubes. The ACN was evaporated under a stream of nitrogen (approximately 5 min) and the aqueous samples that remained were dried down in a SpeedVac concentrator (Savant, Holbrook, NY) and then stored at 20°C.

Statistical Analysis. Changes in the percentage of apoptotic cells in treated cultures were analyzed by the Mann-Whitney U test or by ANOVA.

	Results

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Aminobisphosphonates Cause Activation of Caspase-3-Like Proteases in J774 Cells. After treatment of cultures of J774 macrophages with 100 µM ALN, IBA, or PAM (the aminobisphosphonates) for 16 h, a 12- to 15-fold increase in caspase-3-like activity (determined by the cleavage of the fluorogenic substrate Ac-DEVD-AMC) could be detected in cell lysates (Fig. 1A). By contrast, treatment of J774 macrophages with 750 µM CLO, ETI, or TIL (the nonaminobisphosphonates) for 16 h did not cause activation of caspase-3-like proteases (Fig. 1A).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   A, the effect of three aminobisphosphonates (100 µM ALN, IBA, or PAM) and three nonaminobisphosphonates (750 µM CLO, ETI, or TIL) on caspase-3-like activity (cleavage of Ac-DEVD-AMC) in J774 cells. B, the effect of the bisphosphonates on caspase-1-like activity (cleavage of Ac-YVAD-AMC) in J774 cells. Values are the mean ± S.E.M. (n = 3) expressed as a percentage of caspase activity in control cells.

Neither Aminobisphosphonates nor Nonamino-Bisphosphonates Cause Activation of Caspase-1-Like Proteases in J774 Cells. After treatment of cultures of J774 macrophages for 16 h with 100 µM ALN, IBA, or PAM (the aminobisphosphonates) or 750 µM CLO, ETI, or TIL (the nonaminobisphosphonates), there was no detectable increase in caspase-1-like activity in cell lysates (determined by the cleavage of the fluorogenic substrate Ac-YVAD-AMC) (Fig. 1B).

Inhibition of Caspase-3-Like Proteases Suppresses Alendronate-Induced Apoptosis. Treatment for 30 h with 100 µM ALN caused a significant increase in the proportion of cells with morphologically apoptotic nuclei (14.6%) compared with cells from control cultures (1.0%), in accord with our previous observations (Rogers et al., 1996b). When cultures were coincubated with 100 µM ALN and 200 µM caspase-3 inhibitor (Z-DEVD-fmk), the proportion of apoptotic cells was significantly reduced to 9.2% (Fig. 2). After coincubation of J774 cells with 100 µM ALN and 200 µM Z-DEVD-fmk for 24 h, however, there was no increase in caspase-3-like activity in subsequent cell lysates (data not shown). These observations suggest that Z-DEVD-fmk is both cell permeable and an effective inhibitor of caspase-3-like proteases but did not completely prevent apoptosis.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Percentage of apoptotic J774 cells after culture for 30 h without bisphosphonates (CTL), with 100 µM alendronate, or with 100 µM alendronate and 200 µM Z-DEVD-fmk. The proportion of apoptotic cells was assessed by changes in nuclear morphology. Values are expressed as the mean ± S.E.M. (n = 4). * denotes values significantly different from treatment with alendronate alone (p < .05, ANOVA).

FOH and GGOH Prevent Activation of Caspase-3-Like Proteases and Induction of Apoptosis by Amino-bisphosphonates. As described above, treatment of J774 macrophages for 24 h with the aminobisphosphonates ALN, IBA, or PAM caused a substantial increase in caspase-3-like activity compared with control. Coincubation of J774 cells with 100 µM ALN, IBA, or PAM and 50 µM FOH for 24 h, significantly reduced the increase in caspase-3-like activity (Fig. 3). Similarly, addition of 50 µM GGOH to amino-bisphosphonate-treated cultures effectively reduced caspase-3-like activity, to the levels observed in untreated cultures in the case of ALN + GGOH, or IBA + GGOH (Fig. 3). After 48 h of treatment, GGOH was still effective at preventing activation of caspase-3-like proteases. However, FOH was completely ineffective (data not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   The effect of 50 µM FOH or 50 µM GGOH on caspase-3-like activity induced in J774 cells after 24-h treatment with 100 µM ALN, IBA, and PAM. Values are the mean ± S.E.M. (n = 3) expressed as a percentage of caspase activity in control cells.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   The effect of (A) 50 µM FOH or (B) 50 µM GGOH on J774 apoptosis induced by 100 µM ALN, IBA, and PAM. Values are the mean ± S.E.M. (n = 4). * denotes values significantly different from treatment with bisphosphonate alone (p < .05, Mann-Whitney U test).

Aminobisphosphonates Inhibit Protein Isoprenylation, whereas Nonaminobisphosphonates Do Not. After metabolically labeling J774 cells with [¹⁴C]mevalonolactone for 24 h, radiolabeled isoprenylated proteins in cell lysates could be separated by electrophoresis into major bands of 21 to 26 kDa (mostly geranylgeranylated small GTP-binding proteins, but also farnesylated Ras proteins) and 60 to 70 kDa (farnesylated lamin B and prelamin A), in accord with previous studies (Luckman et al., 1998). When J774 cells were treated with ALN, IBA, or PAM (all at 100 µM) for the 24-h labeling period, the incorporation of [¹⁴C]mevalonolactone into the protein bands (especially the small GTP-binding proteins) was markedly inhibited (a representative gel is shown in Fig. 5). ALN and IBA seemed to be consistently more effective than PAM. In addition, ALN, IBA, and PAM inhibited the incorporation of [¹⁴C]mevalonolactone into the major band at the dye front, which we previously suggested consists of low-molecular-mass isoprenoid intermediates of the mevalonate pathway, such as GGPP (Luckman et al., 1998). By contrast, when J774 cells were incubated with 750 µM CLO or ETI for the 24-h labeling period, there was no marked effect on the incorporation of radiolabel into the isoprenylated protein bands or into the low-molecular-mass compounds at the dye front. TIL (750 µM) slightly inhibited protein isoprenylation (although this concentration, unlike CLO or ETI, substantially reduced cell viability) but did not reduce the incorporation of label into isoprenoid compounds at the dye front.

View larger version (98K): [in this window] [in a new window]   Fig. 5.   The effect of aminobisphosphonates and nonaminobisphosphonates on incorporation of [14C]mevalonolactone into isoprenylated proteins. Cell lysates were from control cells (CTL) or cells treated for 24 h with 100 µM ALN, IBA, and PAM or with 750 µM CLO, ETI, and TIL. Wells were equally loaded with 50 µg of protein. The positions of molecular mass markers are also indicated.

Nonaminobisphosphonates Are Metabolized to Nonhydrolyzable Analogs of ATP, but Aminobisphosphonates Are Not. A concentration of aminobisphosphonates was chosen (100 µM) that induces significant increases in the level of apoptosis in cultures of J774 cells (Fig. 4). When extracts of J774 cells previously treated with the aminobisphosphonates (ALN, IBA, or PAM) were analyzed by HPLC-ESI-MS, there were no peaks containing ions with the predicted molecular weights for AppCp-type metabolites (m/z 577, 647, and 563, respectively) that were not present in control cell extracts (Fig. 6). This demonstrated that these bisphosphonates were not metabolized to AppCp-type metabolites.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Extracted HPLC ion chromatograms of ACN extracts from J774 cells previously treated with 100 µM ALN (scan region of m/z 577) (A), 100 µM IBA (scan region of m/z 647) (B), and 100 µM PAM (scan region of m/z 563) (C) compared with control extracts (bottom lines).

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Analysis of ACN extracts of J774 cells previously treated with 250 µM CLO (A) showing i) extracted HPLC ion chromatogram of m/z 571-576, ii) full-scan MS of the peak at 6.08 min, and iii) MS/MS of m/z 571-576; 750 µM etidronate (B) showing i) extracted HPLC ion chromatogram of m/z 534, ii) full-scan MS of the peak at 5.83 min, and iii) MS/MS of m/z 534; and 100 µM tiludronate (C) showing i) extracted HPLC ion chromatogram of m/z 646, ii) full-scan MS of the peak at 9.90 min, and iii) MS/MS of m/z 643-649.

	Discussion

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Ever since the first use of bisphosphonates to inhibit bone resorption (Fleisch et al., 1969; Smith et al., 1971), efforts have been made to identify the molecular mechanisms involved. It is now clear that bisphosphonates inhibit bone resorption by affecting osteoclasts, the specialized multinucleated bone resorbing cells. It is also possible that effects on cells of the osteoblast lineage or on osteoclast precursors may contribute to the overall inhibition of bone resorption in vivo (Ebetino et al., 1998; Fleisch, 1998).

More than 10 years ago, Boonekamp et al. (1986) proposed that bisphosphonates containing a nitrogen group in the R₂ side chain (such as PAM) had a mode of action in addition to that of the bisphosphonates that do not contain a nitrogen group (such as CLO). More specifically, they proposed that CLO affected only mature osteoclasts, whereas PAM at low concentrations could affect osteoclast precursors. Higher concentrations of PAM could affect both mature osteoclasts and osteoclast precursors. Similar results were later obtained with another nitrogen-containing bisphosphonate (Boonekamp et al., 1987; Lowik et al., 1988). Reitsma et al. (1982), also presented evidence that CLO and PAM differed in their cytotoxic effects on macrophages. However, the molecular basis for these differences remained unknown.

We recently demonstrated that CLO can be metabolically incorporated into a nonhydrolyzable analog of ATP (AppCCl₂p) in the cytoplasm of J774 macrophages in vitro (Auriola et al., 1997; Frith et al., 1997). The intracellular accumulation of the metabolite is the likely cause of the growth inhibitory effect and cytotoxicity of these bisphosphonates toward macrophages (Frith et al., 1997). Metabolites of aminobisphosphonates could not be detected by UV absorbance using the technique of FPLC to separate nucleotides in cell extracts (Frith et al., 1997). However, we recently found that amino-bisphosphonates could inhibit enzymes of the mevalonate pathway in J774 macrophages, thus leading to loss of isoprenoid intermediates (FPP and GGPP) required for the post-translational modification of GTP-binding proteins with isoprenoid groups (Luckman et al., 1998). Loss of isoprenylated proteins is the likely cause of apoptosis induced by these bisphosphonates, involving the activation of caspase-3-like proteases (Coxon et al., 1998). In this study, we directly compared the ability of three nonaminobisphosphonates and three aminobisphosphonates to cause activation of caspases in J774 macrophages. Clearly, all of the aminobisphosphonates caused an increase in the activity of caspase-3-like proteases, whereas the nonaminobisphosphonates did not (even at concentrations known to reduce cell viability or inhibit cell proliferation). This is consistent with our previous studies, which found that aminobisphosphonates were much more effective at causing apoptosis of J774 cells than nonaminobisphosphonates (Rogers et al., 1996b). Neither aminobisphosphonates nor nonaminobisphosphonates caused an increase in the activity of caspase-1-like proteases.

Activation of caspase-3-like enzymes is thought to be the irreversible step in the pathway leading to apoptotic cell death and to be required for some of the morphological and biochemical changes associated with the process (Cohen, 1997). Accordingly, we found that an inhibitor of caspase-3 significantly reduced the proportion of apoptotic cells coincubated with ALN. The caspase-3 inhibitor did not completely prevent apoptosis. However, treatment with the inhibitor did completely prevent the increase in activity of caspase-3-like proteases. Thus, caspase-3 or caspase-3-like proteases seem to be involved in, but not essential for, apoptosis induced by aminobisphosphonates. Studies with caspase inhibitors and other apoptosis-inducing agents have led to similar conclusions (Inayat-Hussain et al., 1997).

FOH and GGOH, cell permeable analogs of the mevalonate-derived compounds FPP and GGPP, respectively, effectively prevented the activation of caspase-3-like proteases by all three of the aminobisphosphonates. Similarly, FOH and GGOH effectively prevented apoptosis induced by these bisphosphonates and were more effective than FPP or GGPP (Luckman et al., 1998). Interestingly, in a recent preliminary report (Reszka et al., 1998), GGOH was also shown to prevent the ALN-induced and PAM-induced activation of Mst 1, a kinase that is cleaved and activated in osteoclasts by caspase-3-like proteases. These observations are consistent with our recent hypothesis that aminobisphosphonates cause apoptosis as a consequence of the loss of farnesylated and/or geranylgeranylated proteins (Luckman et al., 1998). It is likely that the addition of exogenous FOH or GGOH rescues the cells from apoptosis by replenishing the cytosolic pool of isoprenoid substrates required for protein isoprenylation. FOH and GGOH can be converted to FPP and GGPP, respectively, and consequently used for protein farnesylation and geranylgeranylation (Crick et al., 1997). Although both FOH and GGOH were effective at preventing caspase activation and apoptosis after 24 h of treatment with aminobisphosphonates, only GGOH was effective after 48 h of treatment. This suggests that geranylgeranylated proteins (such as Rho and Rac) rather than farnesylated proteins (such as Ras) may be particularly important for preventing apoptosis. It is possible that the protective effect of FOH after 24 h may result from the conversion of some FOH to GGPP via FPP, although it is currently thought that FOH cannot be converted to GGPP (Crick et al., 1997). Alternatively, it is possible that proteins normally geranylgeranylated were farnesylated (using FOH), resulting in temporary rescue of cells from apoptosis.

Because the nonaminobisphosphonates CLO, ETI, and TIL did not cause activation of caspase-3-like proteases in J774 cells, we could not examine any suppressive effect of FOH or GGOH. However, we previously demonstrated that CLO can cause necrotic cell death and a reduction in total cell viability, which can be detected using the MTT assay (Frith et al., 1997). In the present study, we also found that treatment for 48 h with 100 µM TIL, CLO, and ETI reduced total cell viability to 3, 44, and 81% of control, respectively. Furthermore, cotreatment with 50 µM FOH or GGOH had no significant effect on the reduction in cell viability (data not shown), supporting the view that these bisphosphonates do not act by preventing protein isoprenylation.

To confirm that aminobisphosphonates, but not nonaminobisphosphonates, inhibit protein isoprenylation, J774 macrophages were metabolically labeled with [¹⁴C]mevalonate. After a 24-h labeling period in the presence of 100 µM each of the aminobisphosphonates, the incorporation of [¹⁴C]mevalonolactone into both farnesylated and geranylgeranylated small GTP-binding proteins was substantially inhibited. ALN and IBA seemed to be slightly more effective than PAM, consistent with the difference in antiresorptive potency of these compounds. By contrast, the nonaminobisphosphonates CLO and ETI did not inhibit the incorporation of [¹⁴C]mevalonate into small GTP-binding proteins, even at the much higher concentration of 750 µM. This concentration of TIL slightly inhibited protein isoprenylation (probably the result of reduced cell viability) but did not inhibit the formation of low-molecular-mass isoprenoid compounds. Together with the evidence that FOH and GGOH prevent aminobisphosphonate-induced apoptosis and caspase activation, these data clearly demonstrate that aminobisphosphonates, but not nonaminobisphosphonates, inhibit enzymes of the mevalonate pathway required for protein isoprenylation.

We previously suggested that nonaminobisphosphonates and aminobisphosphonates also differ in their ability to be metabolized to AppCp-type nucleotides (Rogers et al., 1996a). In previous studies, we were unable to detect metabolites of the aminobisphosphonates ALN and PAM on the basis of UV absorbance after FPLC separation (Rogers et al., 1994a, 1996a; Frith et al., 1997). However, this did not rule out the possibility that metabolites of these bisphosphonates could be formed but at concentrations below the limit of detection by FPLC. More recently, we developed a more sensitive technique for detecting bisphosphonate metabolites using HPLC-MS (Auriola et al., 1997). In the present study, we optimized the procedure for preparation of cell extracts for HPLC-MS analysis and directly compared the ability of nonaminobisphosphonates and aminobisphosphonates to be metabolized. Using this technique, we found that all three of the nonaminobisphosphonates examined could be metabolically incorporated into AppCp-type nucleotides. However, no AppCp-type metabolites of any of the three aminobisphosphonates could be detected using the sensitive HPLC-MS technique. It is unlikely that the lack of detection of metabolites of aminobisphosphonates was attributable to the lower concentration of aminobisphosphonates used to treat the J774 cultures (100 µM aminobisphosphonates versus 100 µM TIL, 250 µM CLO, and 750 µM ETI). We previously showed that metabolites of aminobisphosphonates cannot be detected even when cell-free lysates are incubated with 500 µM aminobisphosphonates (conditions in which 500 µM nonaminobisphosphonates are metabolized; Rogers et al., 1996a). Thus, we conclude that aminobisphosphonates are not metabolized by J774 macrophages.

These observations provide conclusive evidence for two pharmacological classes of bisphosphonates. Those that lack an amino group in the R₂ side chain can be metabolically incorporated into nonhydrolyzable nucleotide analogs, which may compete with ATP in enzymatic reactions. It is likely that the growth-inhibitory and cytotoxic effects of these bisphosphonates on macrophages and other cells, including osteoclasts (Flanagan and Chambers, 1991; Mönkkönen et al., 1994; Rogers et al., 1994b; Frith et al., 1997), are the result of the cytoplasmic accumulation of the metabolites. By contrast, the amino-containing bisphosphonates are not metabolized but can inhibit enzymes of the mevalonate pathway, indirectly preventing the isoprenylation (and hence the function) of small GTP-binding proteins. These proteins are involved in signaling pathways regulating apoptosis and processes vital for osteoclastic bone resorption, such as organization of the actin cytoskeleton (Zhang et al., 1995), membrane ruffling (Ridley et al., 1992), and vesicular trafficking (Olkkonen and Stenmark, 1997). Therefore, it is likely that this is the mechanism by which aminobisphosphonates cause morphological changes to macrophages and osteoclasts, inhibit osteoclast function (Sato et al., 1991; Murakami et al., 1995), and cause apoptosis (Hughes et al., 1995; Rogers et al., 1996b). This is supported by our finding that caspase activation and macrophage apoptosis induced by all three aminobisphosphonates tested could be prevented by addition of FOH and, in particular, GGOH, which can be used as substrates for protein isoprenylation. Although the exact enzymes of the mevalonate pathway that are inhibited by these bisphosphonates remain to be identified, it is likely that the presence of a nitrogen function in the R₂ side chain is crucial for the ability of these bisphosphonates to interact with enzymes of the mevalonate pathway.

Nonaminobisphosphonates and aminobisphosphonates are known to differ in their ability to cause an acute phase response in patients receiving the drugs for the first time (Adami et al., 1987; Sauty et al., 1996). In addition, studies in vitro and in animal models have suggested that nonamino-bisphosphonates seem to have anti-inflammatory properties at concentrations that are not cytotoxic (Pennanen et al., 1995; Makkonen et al., 1996; Nakamura et al., 1996), whereas aminobisphosphonates may be proinflammatory (Pennanen et al., 1995; Nakamura et al., 1996). Identification of the molecular mechanism of action of these two classes of bisphosphonates may help in understanding the basis for these differences in clinical practice.