Henry R. V. Arnstein

The structural mimicry of membrane sterols by tamoxifen: evidence from cholesterol coefficients and molecular-modelling for its action as a membrane anti-oxidant and an anti-cancer agent

The anti-cancer drug tamoxifen is a potent inhibitor of lipid peroxidation induced by Fe(III)-ascorbate in ox-brain phospholipid liposomes. Similar anti-oxidant effects, but with varying potencies, are also shown by 4-hydroxy-tamoxifen, cholesterol, ergosterol and 17-beta-oestradiol. We now describe a computer-graphic fitting technique that demonstrates a structural similarity between the five compounds. In addition, we have quantified the differences (relative to cholesterol) between the anti-oxidant activities of the compounds in terms of a novel expression referred to here as the cholesterol coefficient (Cc) Finally, we discuss how the inhibitory effect of tamoxifen on lipid peroxidation may result from a membrane stabilization that is associated with a decrease in membrane fluidity. This action may be related to the anti-proliferative effect exerted by tamoxifen on cancer and fungal cells.

Quantitation of globin mRNA levels during erythroid development in the rabbit and discovery of a new β-related species in immature erythroblasts

Abstract Globin mRNA is present in proerythroblasts isolated from anemic rabbit bone marrow by unit gravity sedimentation. Its level has been quantitated in these cells and in the other cells of the erythroid lineage by RNA excess hybridization to highly labeled complementary globin DNA. Globin mRNA levels per cell remain fairly constant at about 10,000 copies per cell until cell division has stopped permanently, when this value rises to approximately 23,000. The rate of accumulation of globin mRNA in nondividing erythroblasts was found to be compatible with the accepted rates of in vivo gene transcription. We have also found evidence for the presence of a β-related globin mRNA in proerythroblasts and basophilic erythroblasts which comprises about 50% of their total globin mRNA. It was detected by the diphasic melting curves of the hybrids between globin cDNA and RNA from these immature erythroblasts. The mismatched component melted 3°C lower than the true hybrid. It was identified as being β- rather than α-related by determining the melting temperatures of hybrids between RNA from basophilic erythroblasts and cDNA transcribed from either α- or β-globin mRNA species (separated by electrophoresis in formamide). Melting curves of hybrids between erythroblast RNA and tritiated DNA copied from rabbit β-globin plasmid DNA, donated by Dr. B. Mach, confirmed that the new globin mRNA is β-related and that it is absent in late orthochromatic erythroblasts.

Enhancement by tamoxifen of the membrane antioxidant action of the yeast membrane sterol ergosterol: relevance to the antiyeast and anticancer action of tamoxifen.

The anticancer drug tamoxifen inhibits lipid peroxidation in ox-brain phospholipid liposomes, and is a good antiyeast agent, with clinical potential. We now report that the ergosterol-containing lipid fraction derived from yeast microsomal membranes (and the ergosterol separated from it) inhibited lipid peroxidation when introduced into ox-brain phospholipid liposomes. Inhibition of lipid peroxidation by the lipid fraction was greatly enhanced when yeast cell growth was inhibited with tamoxifen prior to lipid extraction. The ability of tamoxifen to enhance the membrane antioxidant ability of ergosterol is expressed in terms of a tamoxifen enhancement coefficient. Enhancement by tamoxifen of the membrane antioxidant action of ergosterol is discussed in relation to the antifungal and anticancer actions of tamoxifen.

Tamoxifen inhibits lipid peroxidation in cardiac microsomes. Comparison with liver microsomes and potential relevance to the cardiovascular benefits associated with cancer prevention and treatment by tamoxifen.

Tamoxifen and 4-hydroxytamoxifen were both good inhibitors of iron-dependent lipid peroxidation in rat cardiac microsomes. Tamoxifen was also a good inhibitor of lipid peroxidation in liposomes prepared from the phospholipid obtained from rat liver microsomes. In a modified rat liver microsomal system containing a sufficiently low amount of peroxidizable phospholipid to make it comparable with the rat cardiac microsomal system, tamoxifen and 4-hydroxytamoxifen were of similar effectiveness as in the cardiac system. Tamoxifen is known to lower serum cholesterol levels, and the findings reported here indicate that the drug might also protect heart cell membranes against peroxidative damage. Potential cardioprotective and antiatherosclerotic benefits of tamoxifen are discussed in relation to the drugs use in cancer prevention and treatment.

Droloxifene (3-hydroxytamoxifen) has membrane antioxidant ability: potential relevance to its mechanism of therapeutic action in breast cancer

Droloxifene (3-hydroxytamoxifen), is a triphenylethylene derivative recently developed for the treatment of breast cancer. Droloxifene was found to exhibit a membrane antioxidant ability in that it inhibited Fe(III)-ascorbate dependent lipid peroxidation in rat liver microsomes and ox-brain phospholipid liposomes. It also inhibited microsomal lipid peroxidation induced by Fe(III)-ADP/NADPH. Droloxifene was a better inhibitor of lipid peroxidation than tamoxifen, but was less effective than 17 beta-oestradiol in the two microsomal systems and in the preformed liposomal system. When introduced into ox-brain phospholipid liposomes, droloxifene inhibited Fe(III)-ascorbate induced lipid peroxidation to approximately the same extent as similarly introduced cholesterol and tamoxifen, although to a lesser extent than 17 beta-oestradiol. This inhibition of lipid peroxidation by droloxifene may result from a membrane stabilization that could be associated in cancer cells with decreased plasma membrane fluidity. This mechanism may be related to the clinically important antiproliferative action of droloxifene on cancer cells.

Regulation of erythroid cell differentiation by haemin.

The differentiation of immature erythroblasts, isolated from anaemic rabbit bone marrow by density centrifugation to bovine serum albumin gradients, is accelerated by the addition of 10(-5)-10(-4) M haemin to the culture medium. Both the proportion of benzidine-positive cells and the synthesis of haemoglobin relative to the total protein were increased, whereas cell growth and DNA synthesis were decreased. Some of these changes were detected within 4 h and were maximal after 18-40 h. It is suggested that haem may have a physiological role in regulating in vivo erythropoiesis during haemolysis by accelerating terminal erythroid cell differentiation.

Stimulation by reticulocyte initiation factors of protein synthesis in a cell-free system from Krebs II ascites cells

Abstract 1. We have studied the effect of a crude initiation factor preparation, the 0.5 M KCl extract of reticulocyte ribosomes, on the translation of messenger RNAs in a cell-free system from Krebs II ascites cells. 2. Addition of these factors promotes the translation of lens and globin mRNAs and also stimulates the translation of a number of endogenous messengers. 3. Translation of encephalomyocarditis virus (EMC-) RNA is stimulated when protein synthesis is performed at high K + concentrations. At lower K + levels there is no effect or a slight inhibition. Poly(U)-directed incorporation is unaffected. 4. The reticulocyte factors enhance the incorporation of formylmethionine (fMet) from fMet-tRNA Met f into protein, and also the formation of fMet-puromycin, showing that their action is at the level of initiation. Results obtained with pactamycin and double-stranded RNA, both of which are inhibitors of initiation, confirm this conclusion. 5. The reticulocyte factor preparation also contains small amounts of globin mRNA which directs the synthesis of globin containing an excess of α chains. This mRNA can be inactivated by pancreatic ribonuclease without loss of the stimulatory activities of the initiation factors themselves.

The role of cAMP and calcium in the stimulation of proliferation of immature erythroblasts by erythropoietin.

The hypothesis that cAMP or calcium are the second messengers of erythropoietin (Epo) was tested on fractionated, Epo-responsive immature erythroblasts from anemic rabbit bone marrow by examining whether the proliferative effects of the hormone could be mimicked by agents that increase the intracellular concentration of cAMP or Ca2+. None of the compounds tested (including 10(-6)-10(-4) M db-cAMP, forskolin, isoprenaline or 10(-7)-10(-6) M of the calcium ionophore A23187) alone or in combination could either initiate or potentiate the mitogenic action of the hormone. Furthermore, addition of 0.2 U/ml erythropoietin produced no permanent or transient increase in the uptake of 45Ca2+ by erythroblasts at 37 degrees C. However, cells cultured with imidazole or cordycepin (which reduce the level of intracellular cAMP), or with the calcium chelator EGTA, or the drugs verapamil or TMB-8 (which interfere with the utilization of extracellular or intracellular calcium) showed a decreased stimulation of DNA synthesis by Epo. Finally, the tumour promoter phorbol ester TPA could partially mimic the action of Epo when added to cultures containing more immature progenitor cells. We conclude then that an artificial increase in the cytoplasmic concentration of either cAMP or Ca2+ is not sufficient to elicit the proliferation of Epo-responsive cells.

Intracellular distribution of ribonuclease activity during erythroid cell development

Five ribonuclease activities, separable by polyacrylamide gel electrophoresis, have been detected in erythroid bone marrow cells from anaemic rabbits. Their intracellular distribution has been investigated and compared with that of the ribonucleases in reticulocytes. Both the acid and alkaline ribonuclease activities of reticulocytes are much lower (30--50 fold) than those of bone marrow erythroid cells. The most marked decrease in enzyme activity occurs in the fractions containing ribosomes and mitochondria plus lysosomes. In these subcellular organelles there was also a qualitative change in the ribonuclease electrophoretic pattern, whereas the cytosol enzymes of marrow erythroid cells and reticulocytes remained largely unchanged. Several ribonucleases released from reticulocyte membranes with urea were similar to those present in the lysosomal plus mitochondrial fraction, as shown by detection of enzyme activity after polyacrylamide gel electrophoresis. The decline in ribonuclease activity was found to begin in the orthochromatic cells, which have a highly condensed nucleus and are no longer active in DNA and RNA synthesis, and to coincide with a decrease in acid phosphatase activity and loss of lysosomes.

The nonhistone proteins of developing mammalian erythroid cells

Anaemic rabbit bone marrow cells, labelled with [35S]methionine, were separated at unit gravity. Chromatin was isolated from these cells and proteins were separated from DNA, using urea/salt extraction. Two-dimensional gel electrophoresis of the nonhistone proteins showed that these proteins appeared to change quantitatively but not qualitatively, with one important exception, as cell development proceeded. The one protein that did change had a molecular weight of approximately 20,000 and was very basic. This protein was synthesised at low levels in the early cells, but its synthesis was seen to increase at the polychromatic stage of cell development, just prior to nuclear condensation. Treatment of bone marrow cells with sodium butyrate was shown to increase the synthesis of this protein in the early cells.