A. Pacini

Critical Appraisal of the MTT Assay in the Presence of Rottlerin and Uncouplers

Rottlerin is a natural product isolated from Mallotus philippinensis. This polyphenolic compound, originally described as a selective inhibitor of PKCδ, can inhibit many other PKC-unrelated kinases and has a number of biological actions, including mitochondrial uncoupling effects. We recently found that Rottlerin inhibits the transcription factor nuclear factor κB in different cell types, causing downregulation of cyclin D1 and growth arrest. The present study was carried out to clarify the surprising lack of effect of Rottlerin on MCF-7 cell viability, assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. We found that Rottlerin causes overestimation of the MTT test, leading to inconsistent results between cell number and cell viability. Rottlerin, however, strongly differs from other antioxidant polyphenols, which directly reduce tetrazolium salts, since it does not exhibit any reactivity toward the tetrazolium salts in vitro nor does it modulate lactate dehydrogenase activity. The interference in the MTT assay occurred only in cultured cells, concomitantly with a decrease in the energy charge. Because the same MTT overestimation was observed in the presence of uncoupling agents, we conclude that the Rottlerin artifact is linked to its uncoupling action that, by accelerating oxidative chain, accidentally results in enhanced MTT reduction. These results suggest caution in the use of the MTT assay in the presence of Rottlerin and uncouplers in general.

The role of liver in the catabolism of human alpha- and beta-interferon.

The susceptibility of human leukocyte (alpha), fibroblast (beta) and recombinant alpha-2-interferons to clearance by the isolated and perfused rabbit liver has been evaluated. Human leukocyte and recombinant alpha-2-interferons were stable and their initial levels were maintained in the perfusate even if they had been treated with neuraminidase, thus suggesting that alpha-interferons have no exposed sugars recognizable by hepatic binding proteins. On the other hand, native, and particularly desialylated human beta-interferon, underwent marked hepatic uptake confirming the importance of the liver as a catabolic site for glycosylated interferons.

Pulmonary catabolism of interferons: alveolar absorption of 125I-labeled human interferon alpha is accompanied by partial loss of biological activity

The catabolism of interferon was examined in isolated rabbit lungs which were ventilated and perfused with homologous blood. Natural human interferon-alpha (HuIFN-alpha) from lymphoblastoid Namalwa cells or recombinant DNA-derived HuIFN-alpha 2 were labeled with 125I, mixed with an excess of the respective cold interferons and added to the perfusion blood. Protein-bound and acid-soluble radioactivity, as well as antiviral activity, were measured at regular time intervals. During the first 3 h of perfusion, only very small fractions of the interferons disappeared from the perfusate, irrespective of whether lungs were inserted in the perfusion system. This indicated that catabolism of interferons in the pulmonary circulation was negligible. On the other hand, when the interferons were instilled into the bronchial-alveolar tree, absorption of antiviral activity differed from that of acid-precipitable protein-associated radioactivity. While most of the radioactivity was transferred into the perfusate, only 2% of antiviral activity of natural HuIFN-alpha and 30% of that of HuIFN-alpha 2 were recovered in the perfusate. In both cases acid-soluble radioactivity in the system reached about 10%. Since radioiodide, instilled in the bronchial-alveolar tree, was transported rapidly into the perfusate, this type of analysis did not help in locating the site(s) of degradation. Alveolar macrophages did not catabolize or inactivate interferons in vitro.

Catabolic sites of human interferon-gamma.

Recombinant human interferon-gamma (HuIFN-gamma) injected into rabbits disappeared from the circulation more rapidly than natural IFN-gamma. The latter displayed an initial decay curve more rapid than that for natural HuIFN-alpha although 4 h after injection plasma levels were similar. This result suggests that IFN-gamma has pharmacokinetic properties different to those of IFN-alpha which may be explained by considerable and simultaneous hepatic and renal catabolism. Surprisingly, the hepatic uptake of recombinant (unglycosylated) IFN-gamma was more marked than uptake of natural IFN-gamma. Moreover, both IFN-gamma preparations were cleared by the isolated and perfused kidney and once again the recombinant IFN disappeared more rapidly. This result does not conform with the suggestion that IFN-gamma exists as a tetramer which would not be filtered by the glomerulus, but is consistent with the pharmacokinetic behaviour shown in vivo.

Rottlerin inhibits the nuclear factor κB/Cyclin-D1 cascade in MCF-7 breast cancer cells

In the course of a project aimed to clarify the molecular mechanisms by which phorbol 12-myristate 13-acetate (PMA)-activated forms of protein kinase C (PKC) promote growth arrest in an MCF-7 cell line, we found that the PKCdelta inhibitor Rottlerin was able by itself to block cell proliferation. In the current study, we investigated further the antiproliferative response to Rottlerin. Western blotting analysis of cytoplasmic/nuclear extracts showed that the drug did not prevent either extracellular signal-regulated kinase (ERK) activation by PMA or Akt phosphorylation, but did interfere with the NFkappaB activation process (both basal and PMA-stimulated), by lowering the levels of phospho-IkappaBalpha and preventing p65 nuclear migration. The growth arrest evoked by Rottlerin was not mediated by cell-cycle inhibitors p21 and p27 but was accompanied by a dramatic fall in the cyclin-D1 protein, the levels of which were not altered by the pan-PKC inhibitor GF 109203X, thus excluding a PKC-mediated mechanism in the Rottlerin effect. The parallel drop in cyclin-D1 mRNA suggested a down-regulation of the gene caused by the inhibition of nuclear factor-kappa B (NFkappaB), which occurs via a PKC-, Akt-, ERK- and mitochondrial uncoupling-independent mechanism. We provide preliminary evidence that the interference on the NFkappaB activation process likely occurs at the level of calcium/calmodulin-dependent protein kinase II (CaMKII), a known Rottlerin target. Indeed the drug prevented calcium-induced CaMKII autophosphorylation which, in turn, led to decreased NFkappaB activation.

Metabolism of interferon: hepatic clearance of native and desialylated interferon.

The effect of removal of sialic acid on the survival of rabbit serum and urinary interferon (IF) has been investigated in isolate, perfused rabbit liver preparations. In contrast with native IF, which may be already partially desialylated. IF freed of 80 to 90% of its sialic acid was rapidly cleared from the perfusate of normal livers, or livers pre-treated with actinomycin D. The results suggest that the mechanism of IF catabolism by the liver is similar to that reported for several other circulating glycoproteins.

Rottlerin inhibits the nuclear factor kappaB/cyclin-D1 cascade in MCF-7 breast cancer cells.

In the course of a project aimed to clarify the molecular mechanisms by which phorbol 12-myristate 13-acetate (PMA)-activated forms of protein kinase C (PKC) promote growth arrest in an MCF-7 cell line, we found that the PKCdelta inhibitor Rottlerin was able by itself to block cell proliferation. In the current study, we investigated further the antiproliferative response to Rottlerin. Western blotting analysis of cytoplasmic/nuclear extracts showed that the drug did not prevent either extracellular signal-regulated kinase (ERK) activation by PMA or Akt phosphorylation, but did interfere with the NFkappaB activation process (both basal and PMA-stimulated), by lowering the levels of phospho-IkappaBalpha and preventing p65 nuclear migration. The growth arrest evoked by Rottlerin was not mediated by cell-cycle inhibitors p21 and p27 but was accompanied by a dramatic fall in the cyclin-D1 protein, the levels of which were not altered by the pan-PKC inhibitor GF 109203X, thus excluding a PKC-mediated mechanism in the Rottlerin effect. The parallel drop in cyclin-D1 mRNA suggested a down-regulation of the gene caused by the inhibition of nuclear factor-kappa B (NFkappaB), which occurs via a PKC-, Akt-, ERK- and mitochondrial uncoupling-independent mechanism. We provide preliminary evidence that the interference on the NFkappaB activation process likely occurs at the level of calcium/calmodulin-dependent protein kinase II (CaMKII), a known Rottlerin target. Indeed the drug prevented calcium-induced CaMKII autophosphorylation which, in turn, led to decreased NFkappaB activation.

Renal Metabolism of Rabbit Serum Interferon

Summary Three different approaches have been used to evaluate the catabolism of rabbit serum interferon (IFN) by the kidneys. Firstly, in normal rabbits the disappearance of exogenous IFN from plasma was very rapid, whereas it was significantly slower after bilateral nephrectomy. Secondly, the IFN level in arterial blood was always higher than in renal venous blood: the mean renal extraction rate of IFN in the rabbit, with a renal plasma flow of 9 ml/min, was about 1 ml/min. Thirdly, a selective and reversible tubular damage induced by maleate before intravenous administration of IFN significantly inhibited luminal uptake of IFN and markedly increases the interferonuria. All of these results support the view that the kidneys have a preponderant role in IFN filtration, catabolism and excretion.

Studies of factors regulating the ageing of human erythrocytes--I. The role of pH and of divalent cations.

Abstract 1. 1. Human erythrocytes incubated in vitro in a protein-free medium containing calcium and magnesium but without glucose, undergo a rapid process of ageing demonstrated by ATP depletion, discocyte-echinocyte transformation and membrane shedding. 2. 2. Lowering the pH from 7.2 to 6.8 increases the release of sialic acid-components from the membrane. 3. 3. In absence of glucose, calcium increases strongly this release that can be blocked by addition of a chelator. 4. 4. Erythrocytes incubated in heparinized plasma similarly loose sialic acid from the membrane but in small amounts because of the protective action of plasma factors. 5. 5. Loss of free sialic acid from the membrane is surprisingly little while the bulk is lost as bound to glycopeptides and to vesicles.

Catabolism of native and denatured serum 131I-proteins by polymorphonuclear leucocytes☆

The physiological role of the peritoneal polymorphonuclear leucocytes (PMNL) in serum protein breakdown has been investigated by incubating PMNL in the presence of serum protein fractions labelled with 131I and used as unscreened, or after screening in vivo or, in the case of γ-globulin, also as aggregates (gga). PMNL incubated either in stationary or in suspended cultures have been found capable of breaking down very small proportions of molecules altered during the fractionation and iodination procedures. On the other hand, no liberation of non-protein 131I was found when using screened samples. The conclusion has been reached that, in the experimental conditions here used, PMNL do not catabolise 131I-proteins that behave like native ones. Rapid breakdown occurred when homologous 131I-γ-globulin was presented to the PMNL as aggregates. The catabolism proceeds almost linearly with time and can be stopped by excluding the cells.