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ABSTRACTOur studies on the role of cholesterol homeostasis in the pathogenesis of scrapie revealed abnormal accumulation of cholesterol esters in ex vivo peripheral blood mononuclear cells (PBMCs) and skin fibroblasts from healthy and scrapie-affected sheep carrying a scrapie-susceptible genotype compared to sheep with a resistant genotype. Similar alterations were observed in mouse neuroblastoma N2a cell lines persistently infected with mouse-adapted 22L and RML strains of scrapie that showed up to threefold-higher cholesterol ester levels than parental N2a cells. We now report that proteinase K-resistant prion protein (PrPres)-producing cell populations of subclones from scrapie-infected cell lines were characterized by higher cholesterol ester levels than clone populations not producing PrPres. Treatments with a number of drugs known to interfere with different steps of cholesterol metabolism strongly reduced the accumulation of cholesterol esters in ex vivo PBMCs and skin fibroblasts from scrapie-affected sheep but had significantly less or no effect in their respective scrapie-resistant or uninfected counterparts. In scrapie-infected N2a cells, inhibition of cholesterol esters was associated with selective antiprion activity. Effective antiprion concentrations of cholesterol modulators (50% effective concentration [EC50] range, 1.4 to 40 μM) were comparable to those of antiprion reference compounds (EC50range, 0.6 to 10 μM). These data confirm our hypothesis that abnormal accumulation of cholesterol esters may represent a biological marker of susceptibility to prion infection/replication and a novel molecular target of potential clinical importance.

Previous studies from our laboratories have shown a stimulation of HMP-shunt, cholesterol metabolism and DNA synthesis during cell proliferation. In order to understand the co-ordinated regulation of these pathways during cell growth, the above metabolic pathways were studied in: 1) liver regeneration after partial hepatectomy, 2) lead-induced liver hyperplasia, 3) liver cell proliferation induced by insulin in streptozotocin-diabetic rats, 4) liver cell proliferation in fasted rats after refeeding and, 5) hepatocyte nodules induced by a selection procedure. The results indeed indicate that changes in HMP-shunt and cholesterol metabolism occur at a very early stage during the process of normal as well as preneoplastic cell growth. The coordinated regulation between cell growth and changes in these metabolic pathways needs further study.

CEM and MOLT4 are human T-cell lines isolated from patients with acute cell leukaemia. In culture they show important differences in cholesterol metabolism, CEM being less efficient at synthesizing cholesterol and having a lower activity of 3-hydroxy-3-methylglutaryl-CoA (HMGCoA) reductase. To investigate further the relationship between regulation of intracellular cholesterol metabolism at various steps and rate of cell growth, cholesterol synthesis, esterification and efflux were evaluated in CEM and MOLT4 cells at different times during exponential and stationary growth in vitro. It was shown that, although CEM cells have a lower rate of cholesterol synthesis, they grow at a faster rate than MOLT4 cells. However, CEM cells exhibit an increased capacity to esterify cholesterol associated with a decreased efflux of newly synthesized cholesterol into the medium. These results provide evidence for an association between the capability to synthesize and retain cell cholesterol esters and the growth rate potential.

1. Serum lipids and lipoprotein profiles were determined in children affected by different types of malignancies (leukaemias or lymphomas and solid tumours) both before any treatment and after remission of the disease following chemical or surgical therapy. 2. At the time of diagnosis, children bearing tumours showed hypertriglyceridaemia and reduced concentrations of plasma high-density lipoprotein cholesterol levels, the decrease being particularly prominent in patients with haematological tumours. Children bearing solid tumours displayed an increase of total cholesterol, while those with haematological cancer showed decreased phospholipid levels; low-density lipoprotein cholesterol in neoplastic patients was not significantly different from control values. High triacylglycerol and low high-density lipoprotein cholesterol levels were also evident in cancer patients divided according to age into three groups (0–5, 6–10 and 11–15 years) when compared with age-matched control subjects. Similarly, high triacylglycerol and low high-density lipoprotein cholesterol levels were also observed in both male and female children when patients were divided according to sex and compared with corresponding controls. 3. Clinical remission after therapy was accompanied by an increase of high-density lipoprotein cholesterol levels compared with values observed at diagnosis. In contrast, post-treatment levels of triacylglycerol were higher than those observed before therapy. These results support the hypothesis that alterations of high-density lipoprotein cholesterol levels may be related, at least in part, to the rate of tumour growth, while modifications of triacylglycerol levels may be mediated by different mechanisms.

Cholesterol esterification by acyl‐CoA:cholesterol acyltransferase (ACAT) and proliferation of vascular smooth muscle cells (VSMC) are key events in vascular proliferative diseases. Here we performed experiments to ascertain the role of cholesterol ester pathway in the control of human aortic VSMC cycle progression. Results showed that serum‐induced VSMC proliferation was preceded by an increased ability of the cells to esterify cholesterol as well as by an increased expression of ACAT and multidrug resistance (MDR1) mRNAs and extracellular related kinases 1/2 (ERK1/2), whereas caveolin‐1 levels were markedly decreased. Cell cycle analyses performed in the presence of two inhibitors of cholesterol esterification, directly inhibiting ACAT (Sandoz 58–035) or the transport of cholesterol substrate from plasma membrane to endoplasmic reticulum (progesterone), indicate that each inhibitor suppressed the serum‐induced DNA synthesis by accumulation of VSMCs in the G1phase. The effect was associated with a rapid inhibition of ERK1/2 mitogenic signaling pathway; a down‐regulation of cyclin D1, ACAT, and MDR1 mRNA; and an up‐regulation of caveolin‐1. These data provide a plausible link between cholesterol esterification and control of cell cycle G1/S transition, supporting the hypothesis that cholesterol esterification may accelerate the progression of human vascular proliferative diseases by modulating the rate of the VSMC proliferation.

Checking the resident 1,868 males found to be G6PD deficient out of 15,964 examined, we observed the percentage of G6PD deficiency in male population largely varying among the various Sardinian towns ranging from 1 to over 50%. As preliminary analysis, in 24 towns out of 39 in which the sample was sufficiently suggestive of their whole resident male population, we calculated Standardized Mortality Rates (SMR) for all cancers and linear regression between SMRs and frequency of G6PD deficiency in the same towns. Previous observations suggested the hypothesis of a negative correlation between frequency of G6PD deficiency and incidence of cancer, as we found in the preliminary analysis of this study. The results are not sufficient either to accept or to refuse this hypothesis.