1 Bisphosphonates are currently the most important class of antiresorptive drugs used for the treatment of diseases with excess bone resorption. On the basis of their molecular mechanism of action, bisphosphonates can be divided into two pharmacological classes; nitrogen-containing (N-BPs) and non-nitrogen-containing bisphosphonates (non-N-BP). Both classes induce apoptosis but they evoke it differently; N-BPs by inhibiting the intracellular mevalonate pathway and protein isoprenylation, and non-N-BPs via cytotoxic ATP analog-type metabolites. N-BPs are not metabolized to ATP analogs, but we report here that these bisphosphonates can induce formation of a novel ATP analog (ApppI) as a consequence of the inhibition of the mevalonate pathway in cells. We also investigated whether ApppI is involved in the apoptosis induced by N-BPs. 2 Mass spectrometry and NMR were used to identify ApppI in N-BP treated osteoclasts, macrophages and glioma cells. The potency of different bisphosphonates to promote ApppI production was tested in J774 macrophages. The effects of ApppI on ADP/ATP translocase in isolated mitochondria and its capability to induce apoptosis in osteoclasts were also studied. 3 ApppI production correlated well with the capacity of N-BPs to inhibit mevalonate pathway. ApppI inhibited the mitochondrial ADP/ATP translocase and caused apoptosis in osteoclasts. 4 In conclusion, these findings provide the basis for a new mechanism of action for N-BPs. Some of these very potent bisphosphonates, such as zoledronic acid, represent a third class of bisphosphonates that can act both via the inhibition of the mevalonate pathway and by the blockade of mitochondrial ADP/ATP translocase, which is known to be involved in the induction of apoptosis.

We have earlier shown that ␣-methylated spermidine and spermine analogues rescue cells from polyamine depletion-induced growth inhibition and maintain pancreatic integrity under severe polyamine deprivation. However, because ␣-methylspermidine can serve as a precursor of hypusine, an integral part of functional eukaryotic translation initiation factor 5A required for cell proliferation, and because ␣,-bismethylspermine can be converted to methylspermidine, it is not entirely clear whether the restoration of cell growth is actually attributable to hypusine formed from these polyamine analogues. Here, we have used optically active isomers of methylated spermidine and spermine and show that polyamine depletion-induced acute cytostasis in cultured cells could be reversed by all the isomers of the methylpolyamines irrespective of whether they served or not as precursors of hypusine. In transgenic rats with activated polyamine catabolism, all the isomers similarly restored liver regeneration and reduced plasma ␣-amylase activity associated with induced pancreatitis. Under the above experimental conditions, the (S,S)-but not the (R,R)-isomer of bismethylspermine was converted to methylspermidine apparently through the action of spermine oxidase strongly preferring the (S,S)-isomer. Of the analogues, however, only (S)-methylspermidine sustained cell growth during prolonged (more than 1 week) inhibition of polyamine biosynthesis. It was also the only isomer efficiently converted to hypusine, indicating that deoxyhypusine synthase likewise possesses hidden stereospecificity. Taken together, the results show that growth inhibition in response to polyamine depletion involves two phases, an acute and a late hypusine-dependent phase.A large number of studies have indicated that a continuous supply of the polyamines (spermidine and spermine) is required for animal cell proliferation to occur as polyamine depletion resulted either from a specific inhibition of their biosynthesis (1) or from an activation of their catabolism (2) invariably leads to growth inhibition. The molecular mechanisms involved in the requirement of polyamines for animal cell growth are largely unknown besides the fact that spermidine, but not spermine, serves as the sole biosynthetic precursor for hypusine, an unusual amino acid that is an integral component of eukaryotic translation initiation factor 5A (eIF5A) 4 (3). Because functional (hypusinated) eIF5A is required for animal cell growth (4, 5), it is often difficult to judge whether spermidine depletion-induced growth inhibition is secondary to hypusine deprivation. The finding that cytostasis resulting from an inhibition of S-adenosylmethionine decarboxylase in cultured cells is reversed by ␣-methylspermidine (MeSpd) but not by ␣,-bismethylspermine (Me 2 Spm) indicates that growth inhibition was attributable to hypusine depletion as MeSpd, but not Me 2 Spm, can serve as the biosynthetic precursor for hypusine formation (6). On the other hand, MeSpd and both singly and doubly methylated spermine deri...

Programmed cell death was induced by HSV-tk gene therapy in rat BT4C glioma cells, and metabolite changes associated with cell damage were monitored in vivo by 1 H NMR spectroscopy and ex vivo by high resolution magic angle spinning (HRMAS) 1 H NMR, and in vitro in perchloric acid extracts of tumors. Metabolite concentrations, as quantified in vivo using water as an internal reference and in vitro in extracts, were correlated with cell density. The results showed that both in vivo and in vitro glycine and creatine concentrations followed volume-averaged cell density, whereas that of total choline-containing compounds was unaffected by a cell loss approaching 60%. Meanwhile, both saturated and unsaturated 1 H NMR visible lipids increased. HR-MAS 1 H NMR spectroscopy of the tumor samples at 14.1 tesla demonstrated the presence of nucleotide peaks from adenosine and uridine nucleotides in glioma samples ex vivo. The assignment of a doublet at 7.95 ppm to UDP was confirmed by spiking experiments of tumor extracts in conjunction with 1 H and 31 P NMR spectroscopy. HRMAS also resolved the choline-containing peak at 3.2 ppm in vivo into resonances from choline (3.20 ppm), phosphocholine (3.22 ppm), glycerophosphocholine (3.24 ppm), and taurine (3.26 ppm). These resonances were uncorrelated with temporal progression through programmed cell death. Our results show that 1 H NMR-detected lipids and some of the small molecular weight metabolites respond to gene therapy. However, the choline-containing compounds are unaffected by severe decline in cell density. The latter observation supports the idea that triacylglycerols, rather than membrane phospholipids, are the key components of 1 H NMR visible lipids, and it also casts doubt on the validity of resonance of choline-containing compounds as a diagnostic marker of programmed cell death in vivo. Programmed cell death (PCD)1 involves a cascade of biochemical processes in an ATP-dependent manner, and the process is associated with substantial morphological alterations in the cell interior before phagocytosis (1-3). Recent evidence from cell culture studies points to a number of characteristic metabolic perturbations appearing in the early phase of PCD. These include affections of intermediary metabolism, such as accumulation of glycolytic intermediates fructose 1,6-bisphosphate, dihydroxy acetone phosphate, and glycerol-3-phosphate because of inhibition of glyceraldehyde-3-phosphate dehydrogenase (4), retention of CDP-choline (5) as a result of inhibition of CDP-choline:1,2-diacylglycerol choline phosphotransferase (6), and collapse of NAD(H) levels (5). The severe decline in NAD(H), leading to inhibition of glycolysis, may result from activation of poly(ADP-ribose) polymerase in apoptotic cells (5). Inhibition of CDP-choline:1,2-diacylglycerol choline phospotransferase leads to cessation of phosphatidylcholine biosynthesis (6), and it has been proposed that this might be one of the mechanisms explaining accumulation of 1 H NMR lipids into cells undergoing apoptosis (7,8). I...

The electronic nose is capable of rapidly and noninvasively discriminating prostate cancer and benign prostatic hyperplasia using urine headspace in patients undergoing surgery.

Metabolically stable polyamine derivatives may serve as useful surrogates for the natural polyamines in studies aimed to elucidate the functions of individual polyamines. Here we studied the metabolic stability of ␣-methylspermidine, ␣-methylspermine, and bis-␣-methylspermine, which all have been reported to fulfill many of the putative physiological functions of the natural polyamines. In vivo studies were performed with the transgenic rats overexpressing spermidine/spermine N 1 -acetyltransferase. ␣-Methylspermidine effectively accumulated in the liver and did not appear to undergo any further metabolism. On the other hand, ␣-methylspermine was readily converted to ␣-methylspermidine and spermidine; similarly, bis-␣-methylspermine was converted to ␣-methylspermidine to some extent, both conversions being inhibited by the polyamine oxidase inhibitor N 1 ,N 2 -bis(2,3-butadienyl)-1,4-butanediamine. Furthermore, we used recombinant polyamine oxidase, spermidine/spermine N 1 -acetyltransferase, and the recently discovered spermine oxidase in the kinetic studies. In vitro studies confirmed that methylation did not protect spermine analogs from degradation, whereas the spermidine analog was stable. Both ␣-methylspermidine and bis-␣-methylspermine overcame the proliferative block of early liver regeneration in transgenic rats and reversed the cytostasis induced by an inhibition of ornithine decarboxylase in cultured fetal fibroblasts.Although the requirement of the natural polyamines spermidine, spermine, and their precursor putrescine for the growth of mammalian cells is extremely well documented, their specific functions in proliferative processes are largely unknown (1). Some of the published data appear to assign a central role to spermidine, whereas putrescine is supposed to serve as its precursor and spermine as a storage pool convertible back to spermidine. For the elucidation of the physiological roles of individual polyamines, metabolically stable derivatives of polyamines fulfilling their specific cellular functions would be extremely valuable. Methyl derivatives of spermidine and spermine have been used as substitutes for the natural polyamines both in vitro and in vivo. ␣-Methylspermidine (MeSpd), 1 ␣-methylspermine (MeSpm), and bis-␣-methylspermine (1,12-dimethylspermine, Me 2 Spm) are equally effective as the natural polyamines in inducing the conversion of right-handed B-DNA to left-handed Z-DNA (2). In addition to spermidine and spermine, cytostasis that resulted from the inhibition of the S-adenosylmethionine decarboxylase can be reversed by MeSpd but not by Me 2 Spm (3). Spermidine, spermine (because of its conversion to spermidine), and MeSpd serve as substrates for the synthesis of deoxyhypusine (an integral part of eukaryotic initiation factor 5A), whereas Me 2 Spm does not (3). Interestingly, all of the mentioned methylated derivatives of spermidine and spermine have been reported to reverse the cytostasis induced by difluoromethylornithine, a specific inhibitor of mammalian ornithine decarboxyl...