Anti-platelet-activating factor effects of highly active antiretroviral therapy (HAART): a new insight in the drug therapy of HIV infection?

Platelet-activating factor (PAF) is a potent inflammatory mediator, which seems to play a role in the pathogenesis of several AIDS manifestations such as AIDS dementia complex, Kaposi's sarcoma, and HIV-related nephropathy. PAF antagonists have been studied in these conditions with promising results. In order to examine the possible interactions between PAF and antiretroviral therapy, we studied the effect of nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors against PAF biological activities and its basic biosynthetic enzymes dithiothreitol-insensitive PAF-cholinephosphotransferase (PAF-CPT) and lyso-PAF-acetyltransferase (Lyso-PAF-AT), as well as its main degradative enzyme PAF-acetylhydrolase, of human mesangial cell line (HMC). We also studied the effect of several backbones and highly active antiretroviral therapy (HAART) regimens against PAF activity. Among the drugs tested, several inhibited PAF-induced platelet aggregation in a concentration-depended manner, with tenofovir, efavirenz, and ritonavir exhibiting the higher inhibitory effect. In addition, when these drugs were combined in backbones and HAART regimens based on American antiretroviral therapy proposals, they also synergistically exhibited an inhibitory effect against PAF-induced platelet aggregation. Several of these drugs have also inhibited in vitro microsomal PAF-CPT activity, and concentrations of lopinavir-r or tenofovir-DF (similar to their IC(50) against PAF-induced platelet aggregation) exhibited the same effect against PAF-CPT and Lyso-PAF-AT when added in the cell medium of cultured HMC. In addition, in naïve patients treated with one of the most potent anti-PAF HAART regimens (efavirenz/emtricitabine/tenofovir-DF) for a period of 1 month, a significant reduction of the specific activity of PAF-CPT of washed human leukocytes of these patients was also observed, compared with its levels before the HAART treatment. These promising results need to be further studied and confirmed by additional in vivo tests in order to optimize HAART efficacy.

Modulation of cholinephosphotransferase activity in breast cancer cell lines by Ro5-4864, a peripheral benzodiazepine receptor agonist.

Changes in phospholipid and fatty acid profile are hallmarks of cancer progression. Increase in peripheral benzodiazepine receptor expression has been implicated in breast cancer. The benzodiazepine, Ro5-4864, increases cell proliferation in some breast cancer cell lines. Biosynthesis of phosphatidylcholine (PC) has been identified as a marker for cells proliferating at high rates. Cholinephosphotransferase (CPT) is the terminal enzyme for the de novo biosynthesis of PC. We have addressed here whether Ro5-4864 facilitates some cancer causing mechanisms in breast cancer. We report that cell proliferation increases exponentially in aggressive breast cancer cell lines 11-9-1-4 and BT-549 when treated with nanomolar concentrations of Ro5-4864. This increase is seen within 24 h of treatment, consistent with the cell doubling time in these cells. Ro5-4864 also upregulates c-fos expression in breast cancer cell lines 11-9-1-4 and BT-549, while expression in non-tumorigenic cell line MCF-12A was either basal or slightly downregulated. We further examined the expression of the CPT gene in breast cancer (11-9-1-4, BT-549) and non-tumorigenic cell lines (MCF-12A, MCF-12F). We found that the CPT gene is overexpressed in breast cancer cell lines compared to the non-tumorigenic cell lines. Furthermore, the activity of CPT in forming PC is increased in the breast cancer cell lines cultured for 24 h. Additionally, we examined the CPT activity in the presence of nanomolar concentrations of Ro5-4864. Biosynthesis of PC was increased in breast cancer cell lines upon treatment. We therefore propose that Ro5-4864 facilitates PC formation, a process important in membrane biogenesis for proliferating cells.

A lysophosphatidic acid analogue is revealed as a potent inhibitor of phosphatidylcholine synthesis, inducing apoptosis.

A previous study demonstrated that cross-desensitization experiments performed with the lysophosphatidic acid (LPA) analogues (R)- and (S)-N-palmitoyl-norleucinol 1-phosphate (PNPAs) inhibited LPA-induced platelet aggregation without any stereospecificity. Here we report opposite biological effects of the two enantiomers on mitogenesis of IMR-90 fibroblasts in relation to their respective metabolism. (R)PNPA was proliferative, while (S)PNPA induced apoptosis by specifically inhibiting phosphatidylcholine biosynthesis at the last step of the CDP-choline pathway controlled by cholinephosphotransferase. This effect was not direct but required dephosphorylation of PNPAs by ecto-lipid phosphate phosphatase before cellular uptake of the generated N-palmitoyl-norleucinols (PNOHs). Inhibition of cholinephosphotransferase by the derivative (S)PNOH was confirmed by an in vitro assay. (S)PNPA proapoptotic effects led us to clarify the mechanism linking cholinephosphotransferase inhibition to apoptosis. Three proapoptotic responses were observed: the activation of caspase-3, the production of ceramides from newly synthesized pools (as demonstrated by the inhibitor Fumonisin B1) and finally the activation of stress-activated protein kinase, p38 and c-Jun N-terminal kinases 1/2, as a result of ceramide increase. Thus our data demonstrate that synthetic analogues of LPA might display stereospecific effects leading to apoptosis independently of classical LPA-activated pathways.

The AAPT1 gene of soybean complements a cholinephosphotransferase-deficient mutant of yeast.

Aminoalcoholphosphotransferases (AAPTases) utilize diacylglycerols and cytidine diphosphate (CDP)-aminoalcohols as substrates in the synthesis of the abundant membrane lipids phosphatidylcholine and phosphatidylethanolamine. A soybean cDNA encoding an AAPTase that demonstrates high levels of CDP-choline:sn-1,2-diacylglycerol cholinephosphotransferase activity was isolated by complementation of a yeast strain deficient in this function and was designated AAPT1. The deduced amino acid sequence of the soybean cDNA showed nearly equal similarity to each of the two characterized AAPTase sequences from yeast, cholinephosphotransferase and ethanolaminephosphotransferase (CDP-ethanolamine:sn-1,2-diacylglycerol ethanolaminephosphotransferase). Moreover, assays of soybean AAPT1-encoded enzyme activity in yeast microsomal membranes revealed that the addition of CDP-ethanolamine to the reaction inhibited incorporation of 14C-CDP-choline into phosphatidylcholine in a manner very similar to that observed using unlabeled CDP-choline. Although DNA gel blot analysis suggested that AAPT1-like sequences are represented in soybean as a small multigene family, the same AAPT1 isoform isolated from a young leaf cDNA library was also recovered from a developing seed cDNA library. Expression assays in yeast using soybean AAPT1 cDNAs that differed only in length suggested that sequences in the 5'leader of the transcript were responsible for the negative regulation of gene activity in this heterologous system. The inhibition of translation mediated by a short open reading frame located 124 bp upstream of the AAPT1 reading frame is one model proposed for the observed down-regulation of gene activity.

Effects of fasting on phosphatidylcholine biosynthesis in hamster liver: regulation of cholinephosphotransferase activity by endogenous argininosuccinate.

The control of phosphatidylcholine biosynthesis in the hamster liver was examined. Livers of hamsters fasted for 24 and 48 h were perfused with labelled choline. Under both fasting conditions, the incorporation of labelled choline into phosphatidylcholine was reduced. After 48 h of fasting, the 52% reduction in phosphatidylcholine biosynthesis was caused by changes in several factors including a diminishing rate of choline uptake and severe reductions in the pool sizes of ATP and CTP (to 33-37% control values) which resulted in a decrease in the pools of choline-containing metabolites. The activation of cytidylyltransferase after 48 h of fasting might be regarded as a compensatory mechanism for the maintenance of phosphatidylcholine biosynthesis. After 24 h of fasting, a 25% reduction in phosphatidylcholine biosynthesis was observed. The ATP and CTP levels were decreased but the reduction was not severe enough to affect the choline uptake or the labelling of the phosphocholine fraction. The activities of the cytidylyltransferase remained unchanged but an accumulation of labelled CDP-choline was detected. Although choline-phosphotransferase activity was not changed in the microsomes, the enzyme activity was attenuated in the postmitochondrial fraction. Further analysis revealed that cholinephosphotransferase in the liver was inhibited by an endogenous inhibitor in the cytosol which was later identified as argininosuccinate. The level of argininosuccinate was elevated during fasting and the change quantitatively accounted for the attenuation of cholinephosphotransferase activity. The inhibition of choline-phosphotransferase by argininosuccinate, coupled with a substantial decrease in the diacylglycerol level, would provide the hamster liver with an immediate mechanism for the transient modulation of phosphatidylcholine biosynthesis during short-term fasting.