Addition Of Farnesyl Pyrophosphate Research Articles

Abstract 1218: Synthetic lethal targeting of p53 mutant cells with prenylation inhibitors

Abstract Introduction: TP53 is mutated in 30-40% of breast cancers and is associated with poor prognosis. Mutation of TP53 causes increased cellular proliferation, migration and invasion, and downstream activation of multiple pathways. HMG CoA reductase inhibitors, such as simvastatin, inhibit tumorigenic properties induced by TP53 mutation. Mechanism of this response is an important question in targeted cancer therapy. It is well known that HMG CoA reductase inhibitors block cholesterol production and prenylation. Therefore, we hypothesized that prenylation inhibitors would target p53 mutant cells primarily by inhibiting activation of the ras family induced by TP53 mutation. Method: We generated 5 MCF10A stably transduced cell lines over-expressing each of the common TP53 point mutations, R273H, G245S, R248Q, Y234C, or wild type p53. Total Ras, Ras-GTP, total RhoA and RhoA-GTP was measured by immunoprecipitation and immunoblot. Proliferation was measured using CellTiterGlo. The functional effects of a panel of prenylation inhibitors was measured using a caspase3 reporter assay, and migration was measured with a scratch assay. Illumina mRNA sequencing was performed to measure gene expression of mutant and wild type cells before and after simvastatin treatment (2.5µl, 48 h). The RNAseq was analyzed with the DAVID Enrichment tool to evaluate ras pathway activation induced by TP53 mutation. Results: Mutation in TP53 was associated with significant activation of both wild-type Ras and RhoA. Proliferation of cells expressing mutant TP53 was sensitive to a panel of prenylation inhibitors, including simvastatin. Growth inhibition by simvastatin correlated with induction of apoptosis, and could be fully rescued by addition of farnesylpyrophosphate or geranylgeranylpyrophosphate, suggesting that simvastatin functionally blocks both prenylation pathways which are activated in the presence of mutant p53 (IC50(wt/mut) = 4). RNAseq analysis confirms that Ras signaling pathways, including GAPs and GEFs are enriched in the presence of mutant TP53, and FOXO signaling is significantly targeted post statin treatment (P<0.01). Conclusion: p53 mutant breast cancer cells are highly sensitive to prenylation inhibition due to activation of both ras and rhoA. Simvastatin inhibits both farnesylation and geranylgeranylation, effectively blocking ras pathway activation and induction of proliferation in p53 mutant cell lines. A gene expression panel associated with ras pathway activation was identified and may be predictive biomarkers for sensitivity to statin therapy. Citation Format: Shay R. Ferdosi, Benjamin Katchman, Jia Loo, Harneet Grewal, Seron Eaton, Shanshan Yang, Jin Park, Joshua Labaer, Karen S. Anderson. Synthetic lethal targeting of p53 mutant cells with prenylation inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1218. doi:10.1158/1538-7445.AM2017-1218

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Objectives Preclinical studies suggest pravastatin is a candidate therapeutic for preeclampsia, leading to clinical trials. Surprisingly, the amount of functional interrogation of pravastatin (indeed all statins) on primary gestational tissues published thus far is very limited. The objective of this study was to determine the effect of statin administration to primary gestational tissues in vitro. Methods Pravastatin, simvastatin and rosuvastatin (HMG Co-A reductase inhibitors) were administered to primary endothelial cells (HUVECs), primary trophoblasts and preterm preeclamptic placental explants and the effect on sFlt1 and sEng secretion assessed. To identify the mechanism involved, farnesyl pyrophosphate, a HMG Co-A reductase pathway activator was added and sFlt1 and sENG was measured. Endothelial dysfunction was induced by addition of TNF α or trophoblast conditioned media to HUVECs, and the effect of statins on VCAM and monocyte adhesion determined. Finally HUVEC migration in the presence of sFlt1 ± statins was measured using the xCELLigence system (measures in real time). Results Statins significantly reduced sFlt1 release, reducing mRNA expression of the human placental specific variant, sFlt1-e15a and up-regulating heme-oxygenase-1. Pravastatin also significantly reduced sFlt1 release from preeclamptic placental explants. Concerning however, all three statins significantly increased secretion of sEng in a dose dependent manner. Addition of farnesyl pyrophosphate blocked statin-induced sFlt-1 reduction and sEng increase from HUVEC cells, indicating these changes are mediated via inhibition of HMG Co-A reductase. Primary trophoblast conditioned media and TNF α significantly upregulated markers of endothelial dysfunction, VCAM and Endothelin-1, which were quenched by adding statins. Statins also decreased monocyte adhesion to primary HUVECs, enhanced HUVEC tube forming and restored sFlt1 impaired HUVEC migration towards VEGF. Conclusions We characterized the effect of three statins on primary human endothelial cells and trophoblast cells. Our data confirms statins reduce sFlt1 and improve endothelial dysfunction. However, of potential concern is that statins increase sEng release. Disclosures F.C. Brownfoot: None. T.J. Kaitu’u-Lino: None. N. Hannan: None. R. Hastie: None. P. Cannon: None. K. Onda: None. S. Tong: None.

Statin-induced apoptosis via the suppression of ERK1/2 and Akt activation by inhibition of the geranylgeranyl-pyrophosphate biosynthesis in glioblastoma

BackgroundStatins are inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis. The inhibition of this key enzyme in the mevalonate pathway leads to suppression of cell proliferation and induction of apoptosis. However, the molecular mechanism of apoptosis induction by statins is not well understood in glioblastoma. In the present study, we attempted to elucidate the mechanism by which statins induce apoptosis in C6 glioma cells.MethodsThe cytotoxicity of statins toward the C6 glioma cells were evaluated using a cell viability assay. The enzyme activity of caspase-3 was determined using activity assay kits. The effects of statins on signal transduction molecules were determined by western blot analyses.ResultsWe found that statins inhibited cell proliferation and induced apoptosis in these cells. We also observed an increase in caspase-3 activity. The apoptosis induced by statins was not inhibited by the addition of farnesyl pyrophosphate, squalene, ubiquinone, and isopentenyladenine, but by geranylgeranyl-pyrophosphate (GGPP). Furthermore, statins decreased the levels of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt.ConclusionsThese results suggest that statins induce apoptosis when GGPP biosynthesis is inhibited and consequently decreases the level of phosphorylated ERK1/2 and Akt. The results of this study also indicate that statins could be used as anticancer agents in glioblastoma.

Differential effects of ibandronate, docetaxel and farnesol treatment alone and in combination on the growth of prostate cancer cell lines

Ibandronate, one of the most potent bisphosphonates, has been shown to inhibit growth of various cancer cell lines. In contrast, little is known about the effects of ibandronate on prostate cancer cells. Therefore the aim of our study was to characterize the effects of ibandronate alone and in combination with docetaxel on the growth of prostate cancer cell lines and to identify the underlying signalling pathways. Material and methods. The prostate cancer cell lines LNCaP and PC-3 were treated with increasing concentrations of ibandronate and docetaxel alone and in combination. Viable cell number was measured after five days using a hemocytometer and the MTT-method. The effects of ibandronate were tentatively antagonized by addition of farnesyl-pyrophosphate (FPP) or farnesol (FOH). Results. Ibandronate inhibits growth of both prostate cancer cell lines in a dose dependent manner. In combination with docetaxel, synergistic effects are found as evidenced by a combination index (CI) of <1. Addition of FOH and FPP completely antagonized the growth inhibitory effects of ibandronate on both cell lines. Surprisingly, in combination with ibandronate and docetaxel, FOH further increased growth inhibition instead of antagonizing the growth inhibitory effects of ibandronate. Furthermore, FOH alone appeared to be a potent inhibitor of tumor cell growth. Discussion. Ibandronate effectively inhibits growth of prostate cancer cell lines via inhibition of the farnesyl-IPP-synthase and exhibits synergistic effects with docetaxel. In addition, FOH is a potent inhibitor of prostate cancer cell lines and may display an interesting treatment option for patients with CRPC.

Protein geranylgeranylation controls collagenase expression in osteoarthritic cartilage

Statins possess anti-inflammatory properties. This study was undertaken to characterize the mechanism of action of statin drugs on collagenase expression in primary human osteoarthritic cartilage tissue. Human articular chondrocytes and cartilage explants from osteoarthritic donors were exposed to simvastatin in the presence or absence of interleukin-1 beta (IL-1beta). Collagenase expression was determined by quantifying levels of matrix metalloproteinase 13 (MMP-13) and MMP-1 mRNA and MMP-13 protein. The mechanism of statin action was tested by addition of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) or by using inhibitors of farnesyl transferase (FT) and geranylgeranyl transferase (GGT-1). Treatment of osteoarthritic chondrocytes with simvastatin decreased mRNA levels of MMP-13 and MMP-1 whether under basal conditions or during stimulation with IL-1beta. MMP-13 protein secreted into the culture media was also decreased. Genes involved in cartilage synthesis (type II collagen and aggrecan) were not down-regulated by simvastatin. Exogenous addition of GGPP completely reversed the statin-mediated decrease in MMP-13 mRNA and protein levels whereas FPP partially reversed the statin-mediated effect. An inhibitor of GGT-1 mimicked the simvastatin-mediated reduction in MMP-13 expression by chondrocytes. Finally, consistent with impacts on MMP-13 and MMP-1 expression, simvastatin as well as the GGT-1 inhibitor both blocked type II collagen degradation in primary human articular cartilage explants. These results suggest that statins modulate chondrocyte metabolism by reducing prenylation of key signaling molecules that control the expression of collagen-degrading enzymes. Our results strongly support the hypothesis that protein prenyltransferases including geranylgeranyl transferase regulate chondrocyte collagenase expression in osteoarthritis.

Simvastatin inhibits growth via apoptosis and the induction of cell cycle arrest in human melanoma cells

Competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (the statins) that inhibit the synthesis of mevalonic acid are in wide use for treatment of hypercholesterolemia. Although antitumor effects on a variety of cell types have been reported for statins, the effect of simvastatin (one of the statins) on human melanoma cell lines is not known. Here, we report antitumor effects of simvastatin on human melanoma cell lines. We treated human melanoma cell lines, A375M, G361, C8161, GAK, and MMAc with simvastatin in various concentrations for 1 to 3 days. To investigate the antitumor effect of simvastatin, we analyzed cell viability, morphologic changes, reversibility of inhibition by geranylgeranyl pyrophosphate and farnesyl pyrophosphate, apoptosis and the cell cycle. Simvastatin treatment reduced cell viability in all five melanoma cell lines. The different melanoma cell lines, however, displayed different sensitivities to simvastatin. The addition of geranylgeranyl pyrophosphate to A375M and G361 cells in the presence of simvastatin completely restored the viability of cells, but the addition of farnesyl pyrophosphate did not. DNA fragmentation assay showed that simvastatin induced apoptosis in A375M and G361 cells. Simvastatin caused a G1 arrest in G361 and MMAc cells. Consistent with the cell cycle arrest, simvastatin caused an increase in the mRNA levels of p21 and p27 on G361 and MMAc cells. We conclude that simvastatin has an antitumor effect on human melanoma cells in vitro via apoptosis and cell cycle arrest. These results suggest that simvastatin may be an effective anticancer drug for malignant melanoma.

RhoA and p38 MAPK mediate apoptosis induced by cellular cholesterol depletion

Cholesterol is essential for cell viability, and homeostasis of cellular cholesterol is crucial to various cell functions. Here we examined the effect of cholesterol depletion on apoptosis and the mechanisms underlying this effect in NIH3T3 cells. We show that chronic cholesterol depletion achieved with lipoprotein-deficient serum (LPDS) and 25-hydroxycholesterol (25-HC) treatment resulted in a significant increase in cellular apoptosis and caspase-3 activation. This effect is not due to a deficiency of nonsterol isoprenoids, intermediate metabolites of the cholesterol biosynthetic pathway, but rather to low cholesterol levels, since addition of cholesterol together with LPDS and 25-HC nearly abolished apoptosis, whereas addition of farnesyl pyrophosphate or geranylgeranyl-pyrophosphate did not reverse the cell viability loss induced by LPDS plus 25-HC treatment. These effects were accompanied by an increase in ERK, JNK and p38 MAPK activity. However, only the inhibition of p38 MAPK with the specific inhibitor SB203580 or the overexpression of a kinase defective MKK6 resulted in a significant decrease in apoptosis and caspase-3 cleavage induced by cholesterol depletion. Furthermore, LPDS plus 25-HC increased RhoA activity, and this effect was reversed by addition of exogenous cholesterol. Finally, overexpression of the dominant negative N19RhoA inhibited p38 MAPK phosphorylation and apoptosis induced by low cholesterol levels. Together, our results demonstrate that cholesterol depletion induces apoptosis through a RhoA- and p38 MAPK-dependent mechanism.

Nitrogen-Containing Biphosphonates Inhibit the Mevalonate Pathway and Prevent Post-Translational Prenylation of GTP-Binding Proteins, Including Ras

Abstract Bisphosphonates are currently the most important class of antiresorptive drugs used for the treatment of metabolic bone diseases. Although the molecular targets of bisphosphonates have not been identified, these compounds inhibit bone resorption by mechanisms that can lead to osteoclast apoptosis. Bisphosphonates also induce apoptosis in mouse J774 macrophages in vitro, probably by the same mechanisms that lead to osteoclast apoptosis. We have found that, in J774 macrophages, nitrogen-containing bisphosphonates (such as alendronate, ibandronate, and risedronate) inhibit post-translational modification (prenylation) of proteins, including the GTP-binding protein Ras, with farnesyl or geranylgeranyl isoprenoid groups. Clodronate did not inhibit protein prenylation. Mevastatin, an inhibitor of 3-hydroxy-3-methylglutatyl (HMG)-CoA reductase and hence the bio-synthetic pathway required for the production of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, also caused apoptosis in J774 macrophages and murine osteoclasts in vitro. Furthermore, alendronate-induced apoptosis, like mevastatin-induced apoptosis, could be suppressed in J774 cells by the addition of farnesyl pyrophosphate or geranylgeranyl pyrophosphate, while the effect of alendronate on osteoclast number and bone resorption in murine calvariae in vitro could be overcome by the addition of mevalonic acid. These observations suggest that nitrogen-containing bisphosphonate drugs cause apoptosis following inhibition of post-translational prenylation of proteins such as Ras. It is likely that these potent antiresorptive bisphosphonates also inhibit bone resorption by preventing protein prenylation in osteoclasts and that enzymes of the mevalonate pathway or prenyl protein transferases are the molecular targets of the nitrogen-containing bisphosphonates. Furthermore, the data support the view that clodronate acts by a different mechanism.

Mevastatin induces apoptosis in HL60 cells dependently on decrease in phosphorylated ERK

Mevastatin which is an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol synthesis, suppress cell proliferation and induce apoptosis. However, the molecular mechanism of apoptosis induction is not well understood. So, in the present study, we attempted to clarify the mechanism by which mevastatin induces apoptosis in HL60 cells. It was found that mevastatin induced apoptosis. At that time, we observed an increase in caspase-3 activity and morphological fragmentation of the nuclei. The apoptosis induced by mevastatin was not inhibited by the addition of farnesyl pyrophosphate (FPP), squalene, ubiquinone, and isopentenyladenine, but was inhibited by the addition of geranylgeranyl pyrophosphate (GGPP). When we examined the survival signals at the time of apoptotic induction, we also observed that the administration of mevastatin had caused a remarkable decrease in the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). However, other survival signals, such as nuclear factor kappa B (NF-kappaB), protein kinase B (Akt), and p38 mitogen-activated protein kinase (p38), exhibited no change. In addition, no quantitative change was observed in Bcl-2, which was an anti-apoptosis protein. It was also observed that apoptosis was induced when U0126, an MEK inhibitor, was added to the cells to inhibit ERK. These results suggested that mevastatin induced apoptosis when it inhibited GGPP biosynthesis and consequently decreased the level of phosphorylated ERK, which was a survival signal; moreover, at that time, there was no influence on NF-kappaB, Akt, p38, and Bcl-2. The results of this study also suggested that mevastatin could be used as an anticancer agent.

A new bisphosphonate, YM529 induces apoptosis in HL60 cells by decreasing phosphorylation of single survival signal ERK

It is believed that bisphosphonates (BPs) induce apoptosis in cells such as myeloma cells, as they inhibit prenylation of G-proteins. However, the details of the apoptosis-inducing mechanism remain obscure. In the present study, we attempted to clarify the mechanism by which YM529, a new bisphosphonate, induces apoptosis. YM529 induced cell deaths in HL60 cells in a concentration-dependent manner. At that time, we observed an increase in Caspase-3 activity and morphological fragmentation of the nuclei. We could confirm that these cell deaths were evidence of apoptosis. The apoptosis induced by YM529 was not inhibited by the addition of farnesyl pyrophosphate (FPP), but was by the addition of geranylgeranyl pyrophosphate (GGPP). When we examined the survival signals at the time of apoptotic induction, we also observed that the administration of YM529 caused a remarkable decrease in the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). However, other survival signals such as nuclear factor kappa B (NF-κB), protein kinase B (Akt), and p38 mitogen-activated protein kinase (p38) exhibited no change. In addition, no quantitative change was observed in Bcl-2, which is an anti-apoptosis protein. It was also observed that apoptosis was induced when U0126, an MEK inhibitor, was added to the cells to inhibit ERK. These results suggest that YM529, the new bisphosphonate, induced apoptosis when inhibit GGPP synthase and consequently decreased the levels of phosphorylated ERK, which is a survival signal; moreover, during this process, there is no influence on NF-κB, Akt, p38, and Bcl-2. The results of this study also suggest that YM529 can be used as an anticancer agent, in addition to its use as a therapeutic agent to treat osteoporosis.

Probing the Conformational Change of Escherichia coliUndecaprenyl Pyrophosphate Synthase during Catalysis Using an Inhibitor and Tryptophan Mutants

Undecaprenyl pyrophosphate synthase (UPPS) catalyzes the consecutive condensation reactions of eight isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate C(55) undecaprenyl pyrophosphate (UPP). In the present study, site-directed mutagenesis, fluorescence quenching, and stopped-flow methods were utilized to examine the substrate binding and the protein conformational change. (S)-Farnesyl thiopyrophosphate (FsPP), a FPP analogue, was synthesized to probe the enzyme inhibition and events associated with the protein fluorescence change. This compound with a much less labile thiopyrophosphate shows K(i) value of 0.2 microm in the inhibition of Escherichia coli UPPS and serves as a poor substrate, with the k(cat) value (3.1 x 10(-7) s(-1)) 10(7) times smaller than using FPP as the substrate. Reduction of protein intrinsic fluorescence was observed upon addition of FPP (or FsPP) to the UPPS solution. Moreover, fluorescence studies carried out using W91F and other mutant UPPS with Trp replaced by Phe indicate that FPP binding mainly quenches the fluorescence of Trp-91, a residue in the alpha3 helix that moves toward the active site during substrate binding. Using stopped-flow apparatus, a three-phase protein fluorescence change with time was observed by mixing the E.FPP complex with IPP in the presence of Mg(2+). However, during the binding of E.FsPP with IPP, only the fastest phase was observed. These results suggest that the first phase is due to the IPP binding to E.FPP complex, and the other two slow phases are originated from the protein conformational change. The two slow phases coincide with the time course of FPP chain elongation from C(15) to C(55) and product release.

Nitrogen-containing bisphosphonates inhibit the mevalonate pathway and prevent post-translational prenylation of GTP-binding proteins, including Ras.

Bisphosphonates are currently the most important class of antiresorptive drugs used for the treatment of metabolic bone diseases. Although the molecular targets of bisphosphonates have not been identified, these compounds inhibit bone resorption by mechanisms that can lead to osteoclast apoptosis. Bisphosphonates also induce apoptosis in mouse J774 macrophages in vitro, probably by the same mechanisms that lead to osteoclast apoptosis. We have found that, in J774 macrophages, nitrogen-containing bisphosphonates (such as alendronate, ibandronate, and risedronate) inhibit post-translational modification (prenylation) of proteins, including the GTP-binding protein Ras, with farnesyl or geranylgeranyl isoprenoid groups. Clodronate did not inhibit protein prenylation. Mevastatin, an inhibitor of 3-hydroxy-3-methylglutatyl (HMG)-CoA reductase and hence the biosynthetic pathway required for the production of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, also caused apoptosis in J774 macrophages and murine osteoclasts in vitro. Furthermore, alendronate-induced apoptosis, like mevastatin-induced apoptosis, could be suppressed in J774 cells by the addition of farnesyl pyrophosphate or geranylgeranyl pyrophosphate, while the effect of alendronate on osteoclast number and bone resorption in murine calvariae in vitro could be overcome by the addition of mevalonic acid. These observations suggest that nitrogen-containing bisphosphonate drugs cause apoptosis following inhibition of post-translational prenylation of proteins such as Ras. It is likely that these potent antiresorptive bisphosphonates also inhibit bone resorption by preventing protein prenylation in osteoclasts and that enzymes of the mevalonate pathway or prenyl protein transferases are the molecular targets of the nitrogen-containing bisphosphonates. Furthermore, the data support the view that clodronate acts by a different mechanism.

Solubilization and characterization of the long chain prenyltransferase involved in dolichyl phosphate biosynthesis.

Membranes from Ehrlich ascites tumor cells possess an activity which, in the presence of farnesyl pyrophosphate, incorporates [14C]isopentenyl pyrophosphate into a product which is soluble in chloroform/ methanol and retained by DEAE-cellulose. The product co-migrated with authentic dolichyl phosphate on thin layer chromatography but was degraded to neutral labeled compounds when subjected to mild acid hydrolysis, suggesting the presence of an unsaturated alpha-isoprene unit. Triton X-100 (2%) liberated the activity from the membranes and the resulting solubilized preparation was further characterized. The enzyme was found to be sensitive to sulfhydryl reagents and stimulated by ionic strength. A strong dependence of activity on the addition of farnesyl pyrophosphate was observed, allowing several compounds to be tested for their ability to serve as potential primers. Geranyl pyrophosphate, neryl pyrophosphate, all-trans farnesyl pyrophosphate and all-trans geranylgeranyl pyrophospate were found to be effective substrates, although to different extents. Citronellyl pyrophosphate (which has a saturated alpha-isoprene) was inactive as a substrate. The chain length of the products generated was investigated by using a double label isotope procedure and by reverse-phase high performance liquid chromatography. All active primers yielded a product containing 16-19 isoprene units; the distribution of the individual isoprene species was essentially identical regardless of the primer used as substrate. These findings indicate that the specificity lies in the absolute chain length of the product released and not the number of isoprenes added. High performance liquid chromatography of the [14C]polyprenol resulting from enzymatic dephosphorylation of the reaction product indicated the presence of an unsaturated alpha-isoprene unit in a cis-configuration. It is proposed that the enzyme is the long chain cis-prenyltransferase involved in the biosynthesis of dolichyl phosphate.

Enzymatic alkylation of menaquinone-o to menaquinones by microsomes from chick liver

1. The alkylation of menaquinone-o to menaquinone-4 occurs in chick liver. Studies have been carried out to localize the enzyme in a cell particulate and demonstrate its activity in vitro. Injection of [6,7- 3H 2]menaquinone-o into vitamin K-deficient chicks resulted in the biosynthesis of menaquinone-4 within 15 min. After 3 h, the distribution among mitochondria, microsomes, and cytosol were 24, 76 and 0%, respectively. Liver cell fractions were incubated in phosphate buffer with [6,7- 3H 2]menaquinone-o or [6,7- 3H 2]menaquinol-o with or without geranyl pyrophosphate, farnesyl pyrophosphate, or geranylgeranyl pyrophosphate for 1 h. Menaquinones formed were identified by thin-layer chromatography and gas-liquid chromatography. With geranylgeranyl pyrophosphate, the incorporation into cell particulates were 0.14, 0.68, and 0.09% for mitochondria, microsomes, and cytosol. The addition of farnesyl pyrophosphate, geranyl pyrophosphate, and geranylgeranyl pyrophosphate to microsomes increased the overall synthesis of menaquinones from 0.03 to 0.55, 0.24 and 0.10 nmoles, respectively. It appears that the homologue synthesized is controlled by isoprenyl pyrophosphate available for condensation.