Should Singapore have a second medical school?

The case for establishing a second medical school in Singapore is strong. Given the recent explosive advances in medicine and biology, Singapore has identified the life sciences as an area of great economic potential, and aspires to become a regional "medical hub" capable of achieving excellence in healthcare, medical education and biomedical research. The existing medical faculty at the National University of Singapore is currently taking in 230 students per year, creating a very heavy teaching load which, coupled with even heavier clinical duties, makes it extraordinarily difficult for staff members to derive professional satisfaction and to pursue research interests. Creating a second medical school will alleviate some of these problems, which have contributed in no small way to the exodus of experienced clinicians and teachers to the private sector. Perhaps more importantly, having a second medical school will permit direct comparisons of the relative merits of different approaches to medical education, healthcare, and administrative practices. This in turn should lead to improvements in all these areas, thereby creating working environments more likely to satisfy staff aspirations, improve medical education and enhance research. Concerns about possible "unhealthy competition" and "costly duplication" with the establishment of a second medical school are largely unfounded if resources are managed appropriately.

Programmed cell death and cancer.

Programmed cell death or apoptosis, in which cells actively participate in their own self-destruction, is increasingly being recognised as a biological process of critical importance, not only in normal physiology but in the pathogenesis of diseases such as cancer. A number of genes regulating apoptosis have been identified. Mutations of these genes or their abnormal expression have been linked to the development of malignancies in animals and humans and have, in some instances, been associated with resistance of cancers to treatment. Such observations not only increase our understanding of cancer biology, but will probably lead to novel approaches to cancer therapy as well.

Lymphoma cells selected for resistance against the cytotoxic effect of oxygenated sterols are also resistant to nonsteroidal antiestrogens.

Oxygenated derivatives of cholesterol and related compounds (oxysterols) have long been known to be cytotoxic to many different cell types. The mechanism of this cytotoxic effect is not fully understood. Our laboratory has earlier reported that oxysterol cytotoxicity resembles that of nonsteroidal antiestrogens in some aspects: (i) the cytotoxic action of both types of compounds is blocked by inhibitors of protein or RNA synthesis, and (ii) both classes of compounds bind with high affinity to the microsomal antiestrogen binding site, a protein which may mediate the cytotoxicity of its ligands. We have now extended these studies by developing cell lines which are resistant to the cytotoxic action of oxysterols. Oxysterol-resistant cells were isolated by exposing two murine lymphoma cell lines, K36 and EL4, to incremental concentrations of 7-ketocholestanol. Intriguingly, the resistant cells thus obtained also exhibited considerable resistance to the cytotoxic effects of nonsteroidal antiestrogens such as tamoxifen and clomiphene, having LD50 values which were 10-100-times higher than that of the parental cells. The resistance appeared to be selective for oxysterols and antiestrogens and did not extend to non-specific toxic agents such as azide, ethanol, Triton-X100, or heat. The biochemical basis of the resistance is not clear but is not due to diminished cellular uptake or increased metabolism of the cytotoxic agents or to changes in the antiestrogen binding protein. The availability of the resistant cell lines should facilitate further studies on the mechanism of oxysterol- and antiestrogen-induced cell death.

Inhibitors of protein and RNA synthesis block the cytotoxic effects of non-steroidal antiestrogens.

Non-steroidal antiestrogens such as tamoxifen are known to exert cytotoxic effects against various cell lines in culture. When the antiestrogens are present at sufficiently high concentrations, their cytotoxicity cannot be reversed by estrogens and is demonstrable even with cell lines which lack the estrogen receptor. The mechanism of this cytotoxicity, which is clearly independent of estrogen antagonism, remains unknown. Using two murine cancer cell lines (the K36 leukemia and the EL4 lymphoma cell line), the human breast cancer cell line MCF7, and two non-steroidal antiestrogens (tamoxifen and clomiphene), our laboratory attempted to determine whether the cytotoxic action of non-steroidal antiestrogens was mediated by a mechanism requiring protein or RNA synthesis. In the case of K36 and EL4 cells, inclusion of tamoxifen or clomiphene in the culture medium regularly caused the viable call count to fall below 20-30% of control in 36-48 h. Under these conditions, the addition of inhibitors of protein or RNA synthesis consistently increased viable cell count in a dose-dependent manner. With cultures of K36 cells grown in the presence of 10 microM tamoxifen, for example, the addition of appropriate concentrations of emetine, cycloheximide, puromycin, or actinomycin D increased the percentage of viable cells to 5.0, 2.4, 4.0, and 4.0 times that of control, respectively. Additional experiments revealed that the macromolecular synthesis inhibitors, while effective in inhibiting protein or RNA synthesis to varying degrees, did not affect the cellular uptake of [3H]tamoxifen, suggesting that their ability to protect cells against antiestrogen-induced cell death was not due to an inhibition of cellular uptake of antiestrogens. In the case of MCF7 cells, however, inhibition of protein synthesis did not protect the cells against the cytotoxic effect of tamoxifen. These observations suggest that non-steroidal antiestrogens may exert their cytotoxic effect by at least two different mechanisms; only one of these require de novo protein synthesis. The effect of antiestrogens on K36 and EL4 cells may provide a useful system for the identification of proteins involved in cell death.

Psychiatric complications of family reunion after four decades of separation.

OBJECTIVE: After four decades of separation due to civil war, many people who had migrated to Taiwan from mainland China had the unexpected opportunity to briefly visit their families in mainland China. A study was conducted to examine psychiatric complications associated with these family reunions following long involuntary separation. METHOD: Eighty subjects who made the journey were given semistructured interviews investigating their psychological experiences relating to the family reunion. The variables investigated were sociodemographic characteristics, the subject's previsit emotional condition, factors related to the family reunion experience, and stress encountered during the visit. RESULTS: The survey indicated that more than one-fifth of the subjects (22.5%) developed psychiatric complications, mainly depression, immediately after the visit. Determinants found to contribute significantly to the occurrence of emotional disorders were previous ways of expressing homesickness, family members seen during the reunion, family misfortunes that the subjects discovered, family conflict encountered at the reunion, and tactics for coping with such stressors. CONCLUSIONS: The study results suggest that family reunions after long-term separation can be trauma-repairing experiences for some but for others can lead to trauma reexperiencing and psychiatric complications requiring mental health intervention.

Inhibitors of protein and RNA synthesis block the cytotoxic effects of oxygenated sterols.

Oxygenated derivatives of cholesterol are known to exhibit potent cytotoxic effects against many different cell types. The cellular basis of this cytotoxicity is not understood. Using two murine cancer cell lines (the EL4 lymphoma and the K36 leukemia cell line) and two oxygenated sterols (7-ketocholestanol and 25-hydroxycholesterol), our laboratory attempted to determine whether the cytotoxic action of oxysterols was mediated by a mechanism requiring protein or RNA synthesis. The addition of 5 microM 7-ketocholestanol or 25-hydroxycholesterol to the culture medium regularly caused the viable cell count to fall below 10-20% of control within 48-72 h. In the presence of inhibitors of protein or RNA synthesis, however, cell viability was consistently and significantly increased in a dose-dependent manner. For cultures of EL4 cells grown in the presence of 5 microM 7-ketocholestanol, for example, the addition of appropriate concentrations of cycloheximide, puromycin, emetine, and actinomycin increased the percentage of viable cells from a control value of less than 6% to 66%, 28%, 76% and 42%, respectively. Qualitatively similar results were obtained with the K36 cell line. Additional studies revealed that macromolecular synthesis inhibitors, while effective in inhibiting protein or RNA synthesis to varying degrees, did not affect the cellular uptake of 7-keto[3H]cholestanol, suggesting that their ability to protect cells against oxysterol-induced cytotoxicity was not due to an inhibition of the cellular oxysterol uptake. These observations suggest that the cytotoxicity of oxygenated sterols may be mediated by mechanisms requiring de novo protein or RNA synthesis and that oxysterol-induced cytotoxicity may provide a useful system for the identification of proteins involved in cell death.

Biological activities of oxygenated sterols: physiological and pathological implications.

Oxygenated derivatives of cholesterol (oxysterols) are widely distributed in nature, being found in the blood and tissues of animals and man as well as in foodstuff. They exhibit many biological activities which are of potential physiological, pathological or pharmacological importance. Many oxysterols have been found to be potent inhibitors of cholesterol biosynthesis and one or more oxysterols may play a role as the physiologic feedback regulator of cholesterol synthesis. Oxysterols also inhibit cell replication and have cytotoxic properties, effects which suggest that these sterols may participate in the regulation of cell proliferation and may be potentially useful as therapeutic agents for cancer. Furthermore, there is considerable evidence that oxysterols may be involved in the pathogenesis of atherosclerosis. Although the mechanism of action of oxysterols in all these instances is not well understood, the existence of cytosolic and microsomal proteins which bind oxysterols with high affinity and specificity suggests that this group of compounds may represent a family of intracellular regulatory molecules.

Factors affecting anti-estrogen binding site concentration in rat liver.

It is known that synthetic anti-estrogens such as tamoxifen bind to specific high affinity anti-estrogen binding sites (AEBS), which are distinct from estrogen receptors. These binding sites are widely distributed in animal and human tissues, the highest concentrations being found in the liver. The physiological role of these intracellular binding sites, which are located predominantly in the microsomal fraction, is currently unknown, as is the nature and identity of their endogenous ligands. In an attempt to gain information which may provide clues to the possible physiological role of these binding sites, studies were carried out to determine whether the concentration of these binding sites in rat liver was affected by a number of physiological variables. The results of these studies indicated that in the rat (i) liver AEBS increased progressively with age; (ii) liver AEBS concentration tended to be higher among females than males after 100 days of age; (iii) there was no significant variation in liver AEBS level with different phases of the estrous cycle; (iv) liver AEBS level was not significantly affected by castration in both males and females or by estradiol replacement in castrated females; (v) liver AEBS concentration increased significantly with increases in ambient temperature; (vi) there was no clearly detectable alteration in liver AEBS levels with changes in the light:dark cycle; (vii) starvation for 24, 48, and 72 hr increased liver AEBS by approximately 1.5-, 3-, and 2-fold, respectively, while refeeding decreased its level; and (viii) liver AEBS was not affected by increasing dietary fat content from 0.5% to 20% (w/w), but was increased modestly by the addition of cholesterol (2% w/w) to the diet. These observations identify several physiological variables which are associated with changes in liver AEBS concentration and suggest possible avenues for future studies to define the physiological role of these binding sites.

Antiproliferative effects of oxygenated sterols: positive correlation with binding affinities for the antiestrogen-binding sites.

Oxygenated derivatives of cholesterol have long been known to exhibit antiproliferative properties but the mechanism of this effect remains incompletely understood. Following up on a recent observation in our laboratory that certain oxysterols bind with high affinity to the microsomal antiestrogen-binding site, we attempted to determine if a relationship existed between the antiproliferative effect of oxysterols and their binding affinities for the antiestrogen-binding site. Using the human breast cancer cell line, MCF7, and 14 different compounds (4 nonsteroidal antiestrogens, 6 oxysterols with appreciable binding affinities for the antiestrogen-binding site, and 4 other sterols with little or no affinity for this site), we showed that for compounds which bound to the antiestrogen-binding site, there was a clear positive correlation between their relative cytotoxic potencies and their affinities for this binding site. Two sterols, namely 25-hydroxycholesterol and 20 alpha-hydroxycholesterol, were exceptions to this general pattern, both clearly inhibited cell proliferation without having significant binding affinity for the antiestrogen-binding site. The same general order of antiproliferative potency of the oxysterols seen with MCF7 cells was also observed with the murine lymphoma cell line EL4. These findings suggest the possibility that the antiestrogen-binding site may be involved in mediating the antiproliferative effects of nonsteroidal antiestrogens and oxygenated sterols.

High-affinity binding sites for oxygenated sterols in rat liver microsomes: possible identity with antiestrogen binding sites.

Oxygenated derivatives of cholesterol are known to exhibit a number of biological activities including the inhibition of cholesterol biosynthesis and of cell proliferation, but their mechanism of action remains unclear. Previous studies have identified a cytosolic protein which binds 25-hydroxycholesterol, as well as several other oxysterols, with high affinity, possibly mediating some of their effects. We now report the existence of a high-affinity oxysterol binding site in rat liver microsomes which is distinct from the cytosolic binding protein. Among the oxygenated sterols examined, 5 alpha-cholestan-3 beta-ol-7-one (7-ketocholestanol) had the highest affinity for this microsomal binding site (Kd = 2.7 nM). Using 7-keto[3H]cholestanol as the radioactive ligand, we found that binding of this oxysterol to the microsomal binding site was saturable and reversible and was displaceable by the following oxysterols in descending order of potency: 7-ketocholestanol greater than 6-ketocholestanol greater than 7 beta-hydroxycholesterol = 7-ketocholesterol greater than cholesten-3 beta,5 alpha, 6 beta-triol = 7 alpha-hydroxycholesterol greater than 4-cholesten-3-one. All other sterols studied, including, notably, 25-hydroxycholesterol, had little or no inhibitory effect on 7-keto[3H]cholestanol binding. Additional studies revealed that the microsomal oxysterol binding site was probably identical to the antiestrogen binding site described by other workers. First, saturation analysis and kinetic studies demonstrated that the antiestrogen tamoxifen competed directly with 7-keto[3H]cholestanol for the same binding site. Second, the ability of different oxysterols and antiestrogens to inhibit 7-keto[3H]cholestanol binding to the microsomal binding site paralleled their ability to inhibit [3H]tamoxifen binding to the antiestrogen binding site. Third, the tissue distribution of binding sites for 7-keto[3H]cholestanol was similar to that of the antiestrogen binding site. We conclude that: (1) in rat liver microsomes there are high-affinity oxysterol binding sites whose ligand specificity is different from that of the cytosolic oxysterol binding protein; and (2) the microsomal oxysterol binding site is probably identical to the antiestrogen binding site. The biological significance of these observations remains to be explored.

Antiestrogen-binding sites in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors: relation to growth.

We have detected specific high-affinity binding sites for nonsteroidal antiestrogens in 98% of 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors. Since recent studies have suggested that these binding sites may be involved in the regulation of cell growth and proliferation, we attempted to define a possible relationship between the growth of these hormone-dependent tumors and their antiestrogen-binding site content. Rats bearing such tumors were either treated with haloperidol (to increase prolactin secretion and stimulate tumor growth) or oophorectomized (to reduce circulating estrogen concentration and suppress tumor growth). Compared with controls, haloperidol treatment clearly enhanced tumor growth while oophorectomy induced tumor regression, but neither procedure had any effect on the antiestrogen-binding site concentration. Furthermore, tumors which responded to endocrine manipulation had similar antiestrogen-binding site concentrations as tumors which did not respond. We conclude that (1) the alterations in tumor growth induced by these endocrine manipulations are probably not mediated through a change in antiestrogen-binding site concentration, and (2) the tumor concentration of these binding sites is not under estrogen or prolactin control.

Interactions of sterols with antiestrogen-binding sites: structural requirements for high-affinity binding.

Animal and human tissues contain a microsomal protein that binds nonsteroidal antiestrogens with high affinity and specificity. The functions of these binding sites and the identity of their natural ligands are unknown. Following a report that certain sterols inhibit [3H]tamoxifen binding to this site, we attempted to define the structural requirements for maximal inhibition of [3H]tamoxifen binding to rat liver antiestrogen-binding sites. Our studies identified 5 alpha-cholestan-3 beta-ol-7-one (7-ketocholestanol) as the most potent sterol, having an inhibitory activity that was 12% that of unlabeled tamoxifen and an equilibrium dissociation constant of 6.3 nM. Structural features that appeared important for the inhibitory activity of this sterol include the presence of i) a hydrocarbon side chain at C17; ii) an oxygen function at C7; iii) a hydroxyl group at C3; and iv) the absence of a double-bond between C5 and C6. Saturation analysis and kinetic studies of [3H]tamoxifen binding in the presence of varying concentrations of 7-ketocholestanol clearly indicated that this sterol competed directly with tamoxifen for the antiestrogen-binding site. Unlike tamoxifen, this sterol did not bind to the estrogen receptor. These features make 7-ketocholestanol a potentially valuable tool for studying the properties and functions of this site.

Murine antiestrogen-binding protein: characterization, solubilization and modulation by lipids.

The properties of the antiestrogen-binding protein have been examined in mouse tissues, a species in which nonsteroidal antiestrogens are virtually pure agonists. As in other species studied, this protein was distributed in all tissues - highest levels being in the liver. Subcellular fractionation of mouse liver showed that 82% of the antiestrogen-binding protein was associated with the rough endoplasmic reticulum where it was confined to the membranous component. The antiestrogen-binding protein was also present in smooth endoplasmic reticulum, nuclei and cytosol. Its concentration in intact nuclei was at least 10-times higher than levels previously reported in intact rat liver nuclei. Binding of [3H]tamoxifen to the murine antiestrogen-binding protein was of high affinity (Kd = 1 nM) and was inhibited by unsaturated fatty acids and 7-ketocholesterol. In general, cis-isomers of unsaturated fatty acids were more effective binding inhibitors than trans-isomers. The antiestrogen-binding protein solubilized from rough endoplasmic reticulum membranes by the zwitterionic detergent CHAPS, had a molecular mass of approx. 700 kDa and a sedimentation coefficient of about 19 S. [3H]Tamoxifen binding capacity of the solubilized protein was abolished by trypsin and nonspecific proteinases but not by clostripain or Staphylococcus aureus V8 proteinase, suggesting that lysine residue(s) may be involved in [3H]tamoxifen binding.

Interaction of unsaturated fatty acids with anti-oestrogen-binding sites.

Specific high-affinity binding sites for non-steroidal anti-oestrogens such as tamoxifen have been identified in many animal and human tissues. The function of these binding sites and the nature of their endogenous ligands are currently unknown. Our laboratory has previously reported that unsaturated fatty acids at micromolar concentrations inhibited [3H]tamoxifen binding to the anti-oestrogen-binding sites in rat liver, raising the possibility that fatty acids might represent endogenous ligands for these sites. These studies have now been extended to examine the mechanism by which fatty acids inhibit [3H]tamoxifen binding to the anti-oestrogen-binding site. Saturation analysis revealed that increasing concentrations of oleic acid progressively decreased the apparent binding affinity of these sites for [3H]tamoxifen without decreasing the total number of binding sites; however, the apparent dissociation constant did not vary linearly with the prevailing oleic acid concentration, suggesting that the inhibition of [3H]tamoxifen binding by fatty acid was not competitive in nature. Kinetic studies of [3H]tamoxifen binding showed that oleic acid did not affect the rate of association, but increased the rate of dissociation of [3H]tamoxifen from the anti-oestrogen-binding site; the latter finding would not be expected if oleic acid acted as a competitive inhibitor. Furthermore, incubation of a rat microsomal fraction with [3H]oleic acid in the absence and presence of excess non-radioactively labelled tamoxifen also failed to demonstrate direct competition between oleic acid and tamoxifen for the same binding site. It is concluded that oleic acid, and presumably other unsaturated fatty acids, do not compete for the anti-oestrogen-binding site and probably reduce its tamoxifen-binding affinity by some other mechanism, such as perturbation of the lipid environment of the binding site. The biological significance of this interaction of unsaturated fatty acids with the anti-oestrogen-binding site remains to be elucidated.

Unsaturated fatty acids as endogenous inhibitors of tamoxifen binding to anti-oestrogen-binding sites.

It is known that triphenylethylene anti-oestrogens such as tamoxifen bind to specific high-affinity anti-oestrogen-binding sites, which are distinct from oestrogen receptors. These binding sites are widely distributed in human and animal tissues, but their function and endogenous ligands are unknown. By using [3H]tamoxifen and a rat liver microsomal fraction, a radio-ligand-binding assay was developed in an attempt to identify endogenous ligands for the anti-oestrogen-binding sites in the rat. An ether extract of rat serum inhibited [3H]tamoxifen binding to rat liver binding sites in a dose-dependent manner. Identification of the active serum constituents that inhibited [3H]tamoxifen binding was achieved by g.l.c.-mass spectrometry after preliminary purification of a rat serum extract by silica-gel t.l.c. Three unsaturated fatty acids (oleic, linoleic and arachidonic) accounted for about 50% of the total inhibiting activity of the serum extract. The concentrations of these fatty acids required to inhibit [3H]tamoxifen binding were in the range of 10-100 microM, comparable with those found in the rat circulation under physiological conditions. Saturated fatty acids present in rat serum (palmitic and stearic) did not inhibit [3H]tamoxifen binding. A survey of other fatty acids revealed that, in general, unsaturated fatty acids were far more potent than saturated fatty acids in inhibiting [3H]tamoxifen binding. These studies demonstrate that unsaturated fatty acids are quantitatively the most important circulating inhibitors of [3H]tamoxifen binding to the anti-oestrogen-binding sites. The biological significance of their interaction with these sites, however, remains to be clarified.

Effect of oral contraceptives on serum prolactin: a longitudinal study in 126 normal premenopausal women.

A prospective longitudinal study was carried out in 126 normal premenopausal women aged 18-34 years to determine the effect of oral contraceptives containing less than 50 micrograms of oestrogen on serum prolactin concentration. The mean prolactin concentration in patients receiving oral contraceptives (n = 80) rose from a control level of 8.9 ng/ml to 10.2 ng/ml at 3 months (P less than 0.05) and 10.9 ng/ml at 12 months (P less than 0.001). In a parallel group of patients (n = 46) who used intrauterine devices for contraception, no significant change in serum prolactin concentration was observed over the same period. It is concluded that currently available oral contraceptives have a small but significant stimulatory effect on prolactin secretion in normal women.

Tungstate stimulates adenylate cyclase.

Tungstate stimulates adenylate cyclase (E.C. 4.6.1.1) in ovarian homogenates of the rat; maximal stimulation (approximately 3-fold) is achieved at a concentration of 1 mM. This stimulation of cAMP production cannot be explained by the inhibition of phosphodiesterase activity or of ATP hydrolysis. Activation of adenylate cyclase by tungstate is rapid and reversible. The effects of tungstate and hCG on cyclase activity are additive, but tungstate does not augment fluoride-stimulated activity. At higher concentrations (5 and 10 mM), tungstate inhibits basal as well as hCG- and fluoride-stimulated cyclase activity in an irreversible manner. After solubilization by Lubrol-PX, the cyclase enzyme is inhibited by tungstate at concentrations ranging from 0.1-10 mM. Tungstate also activates adenylate cyclase in the brain, heart, lungs, kidneys, and liver of the rat. This suggests that tungstate activation of adenylate cyclase is a general phenomenon and may be mediated by a similar mechanism in different tissues. Tungstate provides an additional tool by which the molecular basis of adenylate cyclase activation can be probed. (Endocrinology 108: 435, 1981)