Chris D. Green

Enrichment of lymphocytes with cholesterol and its effect on lymphocyte activation

Since the development of the concept of the cell membrane as a mosaic of lipid bilayer and globular protein [ 1,2] , there has been much interest in the relationship between cell properties associated with the plasma membrane and the composition and physical state of the membrane lipids. One such property is the activation of lymphocytes to blast cells by antigens and lectins such as concanavalin A (Con. A) or phytohaemagglutinin. This has been associated with the movement of receptor molecules which are probably partially embedded in the plasma membrane. These receptors are normally randomly distributed over the surface, but rapidly aggregate into patches and caps after antigen or lectin binding [3-61. This does not occur at low temperatures where the decreased fluidity of the membrane lipids presumably restricts the motion of proteins embedded in them. As Con. A does not have to cross the cell membrane to stimulate the lymphocyte [7], information must be transmitted across it and it is for this reason that the movement of surface-bound activating molecules is of interest. We have studied, amongst other processes, the effects of increasing the cholesterol content of the lymphocyte upon its activation by lectins. The sterol content of the cells was raised by incubation with liposomes of high cholesterol content. The extra cholesterol was present, at least in part, in the plasma membrane. Such treatment does not alter the ability of the cells to bind Con. A but it markedly suppresses their subsequent activation to blast cells.

Interaction between estradiol and growth factors in the regulation of specific gene expression in MCF-7 human breast cancer cells.

The response of two endogenous, estrogen-induced genes, LIV-1 and pS2, to growth factor stimulation of MCF-7 cells was examined. Epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha) and insulin-like growth factor-1 (IGF-1) were each able to induce an increase in the two mRNAs in the absence of estradiol, and their effects were additive to that of an optimally inducing concentration (10(-8) M) of the hormone. Induction by EGF and TGF alpha, but not by IGF-1, were also additive to induction by a saturating concentration (2 microg/ml) of insulin. TGFbeta, an antimitogenic growth factor for MCF-7 cells, did not induce LIV-1 or pS2 mRNA but inhibited induction by estradiol. Increases in mRNA were shown to reflect increases in specific gene transcription. Induction by growth factors, but not by estradiol, was dependent upon protein synthesis. Induction by both growth factors and estradiol was inhibited by the pure antiestrogen, ICI 164384 (ICI), and by the mixed agonist/antagonist, tamoxifen. Despite differences in patterns of expression in vivo and in vitro, both LIV-1 and pS2 appeared to be responsive to growth factors via a mechanism distinct from that of estradiol but requiring the estrogen receptor.

Effects of oestrogen on the expression of a 4.4 kb mRNA in the ZR-75-1 human breast cancer cell line

A cDNA library has been constructed from the poly(A)+ mRNA of oestrogen-stimulated ZR-75-1 human breast cancer cells. Screening by differential hybridization has identified eight clones which are stimulated between 4- and 16-fold by oestrogen. Two clones (pLIV-1) that are stimulated 4-fold, hybridize to three different mRNA species. A further five recombinants encode for a mRNA 600 bp long which is induced greater than 16-fold and have been shown to cross-hybridize to the oestrogen-responsive clone, pS2, isolated from the MCF-7 breast cancer cell line. Oestradiol was shown to be without detectable effect upon the expression of mRNA for dihydrofolate reductase, which is reported to be oestrogen regulated in MCF-7 cells. Actin gene expression is also unresponsive to oestradiol in ZR-75-1 cells. These results suggest that pLIV-1 represents a previously unidentified mRNA that may be involved in the oestrogen-regulated growth of ZR-75-1 human breast cancer cells.

The rate of cholesterol ‘flip-flop’ in lipid bilayers and its relation to membrane sterol pools

The movement of lipid molecules from one side of a lipid bllayer to the other (the so-called ‘flip-flop’) was first studied in films of stearate [I]. At 25°C stearate molecules transferred with a halftime of 25 min in the absence of Ca2 + and 50 min in its presence. Of more relevance to cellular membranes is the ‘flipflop’ of the major structural lipids, phospholipids and unesterified sterols. Phospholipid transfer has been investigated by a spin-labelling technique in liposomes of egg lecithin [2] . The half-time at 30°C was 6.5 hr and the process is at least eight orders of magnitude slower than translational migration within the plane of the bilayer [3]. We have now utilized the ability of iodide ions added to one side of a lipid bilayer to quench the fluorescence of the sterol sterophenol (l-methyl19nor-cholesta-1,3,5( 1 O)trien-3-01; fig. 1) on that side of the bilayer, to measure sterol ‘flipflop’ in liposo mes. The process is much faster than that of phospho lipids, with a half-time at 3O’C of 72 min. This finding is of relevance to studies of cholesterol exchange between plasma lipoproteins and cell membranes and to the finding in such studies [4,5] of more than one pool of membrane sterol.

PARTICLES WITH PROPERTIES OF RETROVIRUSES IN MONOCYTES FROM PATIENTS WITH BREAST CANCER

An agent with the properties of a retrovirus has been detected regularly in monocytes from patients with breast cancer. In 97% of breast cancer patients the cell-free culture medium (CFCM) in which the monocytes had been cultured possessed reverse transcriptase (RT) activity. In contrast, RT activity was detected in the CFCM from only 11% of age and sex matched controls (p less than 0.0001; Wilcoxon rank sum test). The RT activity was associated with particles having a buoyant density of between 1.165 and 1.18 g/ml, similar to that of retroviruses. Treatment of the samples with non-ionic detergent abolished the peaking of the activity in this fraction. Enveloped particles (100-120 nm in diameter) with a fringed surface resembling murine mammary tumour virus were found on negative-stain electron microscopy in CFCM obtained from patients with breast cancer. Retrovirus-like particles were also observed in the cytoplasm of giant cells formed by monocytes from these patients, and also in macrophages in breast cancer tissue; however, no such particles were detected in the tumour cells. These findings strongly suggest the presence of a retrovirus in the monocytes from patients with breast cancer. The importance of these observations in the pathophysiology of carcinoma of the breast remains to be established.

Insulin/IGF-1 modulation of the expression of two estrogen-induced genes in MCF-7 cells

Estrogen responses of human breast cancer cell lines have frequently been shown to be promoted by insulin. We have examined the action of insulin, and its interaction with estradiol, in regulating the expression of the estrogen-induced genes, LIV-1 and pS2. Both hormones cause increases in mRNA levels of the two genes but do so by distinct mechanisms. The concentration of insulin required to produce this effect suggests that it is acting via its ability to bind to the IGF-1 receptor. Both insulin and estradiol exert their effects at the level of transcription. Induction by insulin is dependent upon continued protein synthesis whereas induction by estradiol is not. Induction by both insulin and estradiol is prevented by the pure antiestrogen. ICI 164384, indicating the requirement for an activatable estrogen receptor. Insulin does not stimulate LIV-1 expression via the androgen receptor. These results demonstrate that both estradiol and insulin can stimulate the transcription of these estrogen-inducible genes, by separate mechanisms both of which involve the estrogen receptor.

Placental alkaline phosphatase activity is inversely related to cell growth rate in HeLaS3 cervical cancer cells

Placental alkaline phosphatase is an inducible enzyme, expressed in HeLaS3 cells, which has been shown to possess protein phosphotyrosine phosphatase activity. Since phosphotyrosine levels are known to increase in actively dividing cells we sought an inverse correlation between PLAP activity and growth rate in HeLaS3 cells. We found that PLAP inducers, Na‐butyrate, dexamethasone, bromodeoxyuridine and dibutyryl cAMP caused a dose‐dependent reduction in growth rate. Mimosine, an agent that blocks the cell cycle in Gl, caused an increase in PLAP activity whilst the mitogen EGF caused a corresponding decrease in PLAP activity. PLAP activity may therefore be related to cell proliferation rate.

INTERACTION BETWEEN ESTRADIOL AND CAMP IN THE REGULATION OF SPECIFIC GENE EXPRESSION

The mRNA levels of LIV-1 and pS2, two estrogen-responsive genes, are increased by the agents, cholera toxin (CT) plus 3-isobutyl-l-methylxanthine (IBMX), which cause an increase in cAMP in MCF-7 human breast cancer cells. The simultaneous addition of estradiol and CT/IBMX results in a synergistic induction of the two mRNAs. The changes in mRNA reflect changes in transcription of the two genes. Interestingly, the addition of CT/IBMX to estradiol not only causes a greater increase in transcription rate but the increase is longer-lasting that seen with the hormone alone. Stimulation of mRNA levels by CT/IBMX, but not by estradiol, was prevented by cycloheximide. Stimulation by both estradiol and by CT/IBMX was prevented by the antiestrogen, ICI 164387. Transcription of LIV-1 and pS2 genes is by both estradiol and cAMP, via separate mechanisms both requiring the estrogen receptor.

Incorporation of plant sterols into membranes and its relation to sterol absorption

Studies of the specificity of structure needed for the incorporation of steroid molecules into cellular membranes and model membranes (liposomes, composed of bilayers of lecithin) have established that the natural sterols can be replaced by closely related compounds [1 ,2] . A further investigation of the role of the side chain of the sterol molecule has now been made using the major plant sterols present in the diet, campesterol and sitosterol, which differ from cholesterol only in the possession, respectively, of an extra methyl and ethyl group in the side chain. These, and other plant and fungal sterols, are poorly absorbed in the intestine compared with animal sterols and this specificity of absorption was attributed to the ability of sterols to enter the membranes and soluble lipoproteins of the intestinal cell [3, 4]. The present paper shows that in vitro the ability of sterols to enter liposomes and erythrocyte membranes is in the order cholesterol > campesterol > sitosterol and that their behaviour in this system is similar to that seen during intestinal absorption. It also shows that the incorporation of these sterol molecules into erythrocyte membranes can be related to the relative proportions of lecithin and sphingomyelin present.

Polyamines and growth regulation of cultured human breast cancer cells by 17β-oestradiol

The growth of ZR-75-1 cells, a line of human breast cancer cells in culture, is stimulated by oestradiol and inhibited by anti-oestrogens. Changes in growth rate caused by these agents are accompanied by changes in activity of ornithine decarboxylase, a rate-limiting enzyme for polyamine synthesis. Furthermore, the growth inhibition caused by tamoxifen, an anti-oestrogen, can be reversed by the addition of spermine, spermidine or putrescine to the cells. Insulin can also stimulate ZR-75-1 cell growth and this is again accompanied by an increase in ODC activity. The reduced cell growth rate observed when the cells become confluent is associated with a marked decrease in ornithine decarboxylase activity. Experiments performed with DFMO, a specific and irreversible inhibitor of ODC, show that this compound can prevent the stimulation of growth by oestradiol and that this may be overcome by the addition of putrescine to the cells. It would appear that increased ODC activity and polyamine synthesis are necessary components of the stimulation of breast cancer cell growth by oestradiol but that other growth regulatory stimuli also may act via this enzyme.