Oxidative stress, mitochondrial dysfunction and calcium overload in human lamina cribrosa cells from glaucoma donors.

PURPOSE: Oxidative stress is implicit in the pathological changes associated with glaucoma. The purpose of this study was to compare levels of oxidative stress in glial fibrillary acid-negative protein (GFAP) lamina cribrosa (LC) cells obtained from the optic nerve head (ONH) region of 5 normal (NLC) and 4 glaucomatous (GLC) human donor eyes and to also examine mitochondrial function and calcium homeostasis in this region of the ONH. METHODS: Intracellular reactive oxygen species (ROS) production was examined by a thiobarbituric acid reactive substances (TBARS) assay which measures malondialdehyde (MDA), a naturally occurring product of lipid peroxidation and is used as an indicator of oxidative stress. Mitochondrial membrane potential (MMP) and intracellular calcium ([Ca(2+)](i)) levels were evaluated by flow cytometry using the JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetrabenzimidazolecarbocyanine iodide) and fluo-4/AM probes respectively. Anti-oxidant and Ca(2+) transport system gene and protein expression were determined by real time polymerase chain reaction (RT-PCR) using gene-specific primer/probe sets and western immunoblotting, respectively. RESULTS: Intracellular ROS production was increased in GLC compared to NLC (27.19 ± 7.05 µM MDA versus 14.59 ± 0.82 µM MDA, p < 0.05). Expression of the anti-oxidants Aldo-keto reductase family 1 member C1 (AKR1C1) and Glutamate cysteine ligase catalytic subunit (GCLC) were significantly lower in GLC (p = 0.02) compared to NLC control. MMP was lower in GLC (57.5 ± 6.8%) compared to NLC (41.8 ± 5.3%). [Ca(2+)](i) levels were found to be higher (p < 0.001) in GLC cells compared to NLC. Expression of the plasma membrane Ca(2+)/ATPase (PMCA) and the sodium-calcium (NCX) exchangers were lower, while intracellular sarco-endoplasmic reticulum Ca(2+)/ATPase 3 (SERCA) expression was significantly higher in GLC compared to NLC. Subjection of NLC cells to oxidative stress (200 µM H(2)0(2)) reduced expression of Na(+)/Ca2(+) exchanger 1 (NCX 1), plasma membrane Ca2+ ATPase 1 (PMCA 1), and PMCA 4 as determined by RT-PCR. CONCLUSIONS: Our data finds evidence of oxidative stress, mitochondrial dysfunction and impaired calcium extrusion in GLC cells compared to NLC cells and suggests their importance in the pathological changes occurring at the ONH in glaucoma. Future therapies may target reducing oxidative stress and / or [Ca(2+)](i).

The effect of an angiostatic steroid on neovascularization in a rat model of retinopathy of prematurity.

PURPOSE: The inhibition of angiogenesis by angiostatic steroids has been demonstrated in a variety of systems, including rabbit and rat cornea. There is considerable interest in the therapeutic potential of this class of compounds for angiogenic ocular conditions such as diabetic retinopathy, macular degeneration, and retinopathy of prematurity (ROP). This study was designed to test the capacity of an angiostatic steroid, anecortave acetate, to inhibit retinal neovascularization using a rat model of ROP and to investigate the mechanism of the effect. METHODS: At birth, rats were placed in an atmosphere of varying oxygen that produces retinal neovascular changes that approximate human ROP. The rats then received intravitreal injections of either anecortave acetate or vehicle at varying times, and all were subsequently placed in room air. Retinas were assessed for plasminogen activator inhibitor (PAI)-1 mRNA level by RNase protection assay at 1, 2, and 3 days after injection and for normal and abnormal blood vessel growth 3 days later. RESULTS: A significant reduction in the severity of abnormal retinal neovascularization was observed in the steroid-treated eyes compared with vehicle-injected eyes in ROP rats, yet the extent of normal total retinal vascular area was not significantly different. The drug had no effect on either retinal vascular area or neovascularization when tested in room air-raised control rats. Drug-injected eyes demonstrated a six- to ninefold increase in PAI-1 mRNA at 1 to 3 days after injection. CONCLUSIONS: This study represents the first therapeutic effect of an angiostatic steroid in an animal model of neovascular retinopathy. Additionally, the induction of PAI-1 indicates a mechanism of action for this class of compounds, and this is a novel finding in vivo. Because anecortave acetate significantly inhibited pathologic retinal angiogenesis in this model, while not significantly affecting normal intraretinal vessels, it holds therapeutic potential for a number of human ocular conditions in which angiogenesis plays a critical pathologic role.

Non-secretion of mutant proteins of the glaucoma gene myocilin in cultured trabecular meshwork cells and in aqueous humor.

Until recently, very little was known about the molecular mechanisms responsible for the development of glaucoma, a leading cause of blindness worldwide. Mutations in the glaucoma gene myocilin (MYOC, GLC1A) are associated with elevated intraocular pressure and the development of autosomal dominant juvenile glaucoma and a subset of adult-onset glaucoma. MYOC is expressed in the trabecular meshwork (TM), a tissue responsible for drainage of aqueous humor from the eye, and the tissue involved in elevated intraocular pressure associated with glaucoma. To better understand the role of MYOC in glaucoma pathogenesis, we examined the expression of normal and mutant myocilin in cultured ocular (TM) and non-ocular cells as well as in the aqueous humor of patients with and without MYOC glaucoma. Normal myocilin was secreted from cultured cells, but very little to no myocilin was secreted from cells expressing five different mutant forms of MYOC. In addition, no mutant myocilin was detected in the aqueous humor of patients harboring a nonsense MYOC mutation (Q368X). Co-transfection of cultured cells with normal and mutant myocilin led to suppression of normal myocilin secretion. These studies suggest that MYOC glaucoma is due either to insufficient levels of secreted myocilin or to compromised TM cell function caused by congestion of the TM secretory pathway.

The use of proteomics in ophthalmic research.

The goal of molecular ophthalmology is the early detection and therapeutic treatment of eye disease. Genomic technologies have profoundly enhanced the discovery of ocular disease candidate genes. Proteomics, the protein cognate of genomic technology, offers a means to monitor changes in the expression of a given ocular protein(s) and its post-translational modification, identify novel therapeutic targets and evaluate pharmacological effects on a given metabolic pathway. Using both tissue and cultured cells, numerous laboratories have begun to catalogue changes in ocular protein expression in normal, diseased and ageing subjects. Herein, we review published proteomic literature in the broad context of ophthalmic diseases involving various tissues of the eye.

Age-related permeability changes in rabbit corneas.

The present study was designed to determine whether the corneal penetration of test compounds is altered in aging. Experiments were carried out by means of passive transport under steady-state conditions using in vitro diffusion cells. Permeabilities of a variety of compounds with different physicochemical properties were measured in young (six weeks) and old (three to four years) intact and denuded (wounded) rabbit corneas. There was a marked difference in penetration of compounds between young and aged corneas. A significant decrease in permeability with age was observed. The degree of difference depended on the lipophilicity and molecular weight of the compound and the integrity of the epithelial cell layer. The difference was more pronounced for large hydrophilic than small lipophilic compounds in the intact corneas. The difference in permeabilities of test compounds between young and old denuded corneas was essentially the same (about 2-fold). These studies provide clues to the fundamental biochemical difference in young and old corneas and better enables the development of rationales for efficient drug and nutrient delivery across the cornea.

Angiostatic activity of steroids in the chick embryo CAM and rabbit cornea models of neovascularization.

Ocular neovascular diseases represent a major cause of blindness in the world. Angiostatic steroids are a unique class of compounds which inhibit the formation of new blood vessels in various models, including ocular models of angiogenesis. In search of potent new anti-angiogenic agents for the treatment of ocular neovascular disease, a large group of steroids were evaluated for angiostatic activity in the chick embryo CAM model. Angiostatic activity was found among all steroid classes included in the study. There was a good correlation between the angiostatic efficacies of 15 diverse steroids tested in the chick CAM and in the rabbit LPS-induced corneal pocket models of neovascularization (r=0.76, p=0.01). These studies show that potent angiostatic steroids inhibit neovascularization in two different animal models, suggesting a common mechanism of action. Glucocorticoid therapy is sometimes associated with ocular side effects. Two of the most potent angiostatic steroids, AL-3789 and AL-4940, were evaluated for glucocorticoid-mediated antiinflammatory activity in the in vitro U937 cell model of LPS-induced IL-1 induction and found to be devoid of glucocorticoid activity. Angiostatic steroids which lack glucocorticoid activity should be attractive drug candidates for treating ocular neovascular disease.

Inhibition of intraocular tumor growth by topical application of the angiostatic steroid anecortave acetate.

PURPOSE: This study examined the effect of an angiostatic agent on the growth of a highly vascularized intraocular tumor. METHODS: A murine uveal melanoma cell line (99E1) was transplanted intracamerally into athymic nude BALB/c mice. Mice were treated topically three times per day beginning on the day of tumor transplantation and continuing through day 28. Groups included (a) 1% anecortave acetate, (b) vehicle control, or (c) no treatment. Tumor growth was scored clinically according to the volume of anterior chamber occupied by tumor. Intraocular tumor weights were determined on days 10, 14, 21, and 28. The effect of the test agents on tumor cell proliferation was examined in vitro by [3H]thymidine incorporation. RESULTS: Tumors grew progressively in untreated mice and mice treated with the vehicle; tumors filled the entire eye by day 20 and frequently perforated the globe by day 21. By contrast, tumors treated with anecortave acetate grew significantly slower (P < 0.025) and did not perforate the eye. On days 21 and 28 the net tumor weight of the AL-3789-treated animals was 40% to 30% of controls (P < 0.05). Tumor inhibition was presumably due to the angiostatic properties of anecortave acetate because the compound did not affect tumor cell proliferation in vitro. CONCLUSIONS: The topical ocular administration of anecortave acetate restricted the growth of a highly vascularized angiogenic intraocular tumor.

Fas-activated apoptosis and apoptosis mediators in human trabecular meshwork cells.

A gradual loss of cells occurs within the human trabecular meshwork during normal aging and appears to be increased in patients with primary open-angle glaucoma. The exact mechanism by which cells are lost in either condition is not known, however phagocytosis, cell migration and cell death have been suggested. Apoptosis is one method by which cell death can occur. We have examined the modulators for apoptosis within the human trabecular meshwork using both cell lines and ex-vivo dissected trabecular meshwork tissues obtained from normal donors. Using RT-PCR it was shown that mRNA for several modulators of apoptosis (Fas, Bcl-2, Bcl-xl, Bax, and ICE) are expressed by both cell lines and ex-vivo tissues. Apoptosis was stimulated to occur by treating cell lines with a monoclonal antibody (IgM) to Fas. Apoptosis was verified via morphological changes to the cells, transferase-mediated dUTP nick-end labeling TUNEL Immunofluorescence, and DNA laddering. Control cells exposed to IgM did not undergo apoptosis. These results represent the first report of apoptosis modulators within the human trabecular meshwork and demonstrate that human trabecular meshwork cells can be stimulated to undergo apoptosis via the Fas/FasL pathway.

Expression of alternatively spliced growth factor receptor isoforms in the human trabecular meshwork.

PURPOSE: Growth factors act through high-affinity cell surface receptors expressed by target cells and are critical modulators of cell function. Because aqueous humor is known to contain growth factors, these molecules may play a key role in maintaining the normal function of the human trabecular meshwork (HTM). Alternate mRNA splicing is an important mechanism used by cells to generate diverse isoforms of growth factor receptors. Although previous investigators have suggested that HTM cells may express alternative isoforms of several growth factor receptors, there have been no studies to verify these preliminary findings. The objective of this study was to determine whether cultured and ex vivo HTM cells express alternate isoforms of hepatocyte, keratinocyte, and transforming growth factor beta (TGFbeta)-II receptors and to characterize the isoform molecular sequences. METHODS: To determine whether cells within the HTM express mRNA for alternate isoforms of growth factor receptors, total RNA was isolated from several well-characterized HTM cell lines that were established from donors of various ages and from fresh ex vivo HTM tissues from healthy donors. After cDNA synthesis, polymerase chain reaction was initiated using specific primers for alternate forms of the following receptors: hepatocyte growth factor (HGFR), keratinocyte growth factor (KGFR), and transforming growth factor beta receptor II (TGFbetaR-II). Specificity and characterization of the polymerase chain reaction amplification products were determined by nucleic acid sequencing. RESULTS: Amplification products of the expected size for the growth factor isoforms were expressed in cell lines and in ex vivo tissues. Nucleic acid sequencing showed that cultured HTM cells and fresh ex vivo trabecular meshwork tissues expressed specific mRNA for alternatively spliced isoforms of HGFR, KGFR, and TGFbetaR-II The HGFR alternate isoform contained a 96-bp insert in the C-terminal coding region of the cytoplasmic tyrosine kinase domain. The KGFR alternate isoform is a soluble, truncated form, because it has no transmembrane or cytoplasmic domain as does the normal membrane-associated form. The TGFbetaR-II alternate isoform contained a 75-bp insert in the N-terminal coding region of the extracellular domain. CONCLUSIONS: In vitro and ex vivo HTM cells express mRNA for alternatively spliced isoforms of HGFR, KGFR, and TGFbetaR-II. These alternatively spliced receptor isoforms may be functional within the HTM and may play a critical role in maintaining the normal microenvironment of this important tissue.

Cultured human trabecular meshwork cells express functional growth factor receptors.

PURPOSE: To compare the mRNA expression of growth factor receptors in cultured human trabecular meshwork (HTM) cells with ex vivo HTM tissues and to determine whether HTM cells generate a physiologic response after exposure to exogenous growth factors. METHODS: The reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect the expression of various growth factor receptor mRNAs using early passaged, cultured HTM cells from donors of several ages. RT-PCR on ex vivo HTM tissues from healthy donors and donors with glaucoma were also used to compare and contrast mRNA expression with cell culture results. After the exogenous administration of growth factors, cell proliferation and extracellular acidification rate studies were used to measure the functional responses of HTM cells to growth factors. RESULTS: Amplification products of the expected size for 15 growth factor receptors were detected in cultured HTM cells and in ex vivo HTM tissues. The administration of exogenous growth factors showed that (a) hepatocyte growth factor (HGF), epidermal growth factor (EGF), insulinlike growth factor (IGF)-1, tumor necrosis factor (TNF) alpha, platelet-derived growth factor (PDGF)-AA, PDGF-BB, PDGF-AB, and basic fibroblast growth factor (FGF-2) stimulated cell proliferation, whereas FGF-1 (acidic), transforming growth factor (TGF) alpha, interleukin (IL)-1alpha, nerve growth factor (NGF), and FGF-7 (keratinocyte growth factor [KGF]) had no significant influence on cell proliferation; (b) TGF-beta isoforms significantly inhibited EGF-stimulated trabecular meshwork cell proliferation; and (c) FGF-1 (acidic), TGF-alpha, EGF, IL-1alpha, IL-1beta, HGF, TNF-alpha, PDGF-AA, and IGF-1 significantly stimulated extracellular acidification, whereas FGF-2 (basic), FGF-7 (KGF), TGF-beta1-beta3 and NGF had no significant influence on extracellular acidification. CONCLUSIONS: These studies show that mRNA for numerous growth factor receptors can be detected in cultured HTM cells and in ex vivo HTM tissues. They also show that many of the receptors are functional, because exogenous growth factor administration elicits a physiologic response. In vivo, these receptors may be activated by growth factors present within the aqueous humor (aquecrine/paracrine) or by growth factors synthesized and released locally by trabecular meshwork cells themselves (autocrine). Specific growth factors acting through high-affinity receptors may be involved in maintaining the normal microenvironment of the HTM and also may be involved in the pathogenesis of primary open-angle glaucoma.

Steroid-induced cataract: new perspective from in vitro and lens culture studies.

The prevailing view regarding the mechanism of steroid cataract formation holds that glucocorticoids are covalently bound to lens proteins resulting in destabilization of the protein structure allowing further modification (i.e. oxidation) leading to cataract. Alternative hypotheses (e.g. that cataracts result from glucocorticoid receptor mediated effects) have been difficult to test since protein binding does in fact occur for many cataractogenic steroids. A glucocorticoid lacking the typical glucocorticoid hydroxy group at C21 (fluorometholone, FML), other steroids which can bind to proteins but lack glucocorticoid activity, and a glucocorticoid antagonist (RU486) have been utilized to discriminate between these two hypotheses. Purified bovine beta-crystallin incubated with three different 3H-steroids, dexamethasone (Dex), aldosterone or progesterone demonstrated that the C-21 hydroxyl group is not essential for steroid binding. Progesterone (with no C-21 OH) bound to the greatest extent. Pretreatment of the protein with aspirin to acetylate the free protein amino groups blocked this binding, demonstrating the probability of a Schiff base mechanism. Lens culture studies with the same three radiolabeled steroids demonstrated much the same result. Rat lenses cultured for 48 hr-11 days, demonstrated that loss of GSH is an early and significant effect of several glucocorticoids (Dex, prednisolone and FML) but is not seen with other non-glucocorticoid steroids. However, none of the steroids tested consistently produced lenticular opacification (i.e. cataracts) in this in vitro system, nor did they alter rubidium transport. We suggest that a mechanism other than covalent binding of steroids to lens proteins is responsible for glucocorticoid induced cataracts because: (1) non-glucocorticoids were demonstrated to bind lens proteins as well or better than the glucocorticoid Dex and (2) only glucocorticoids, and not other steroids, lowered lens reduced glutathione content which has been demonstrated to be associated with other forms of cataract.

Preliminary characterization of a transformed cell strain derived from human trabecular meshwork.

Cells isolated from the trabecular meshwork (TM) of a male glaucoma patient were transformed by transfection with an origin defective mutant of SV40 virus. Transformation dramatically increased the growth rate of these cells (designated HTM-3 cells), allowing biochemical and pharmacological characterization. The HTM-3 cells had cytoskeletal components that were reported to be present in TM tissue and non-transformed TM cells. Vimentin, tubulin and smooth muscle specific alpha-actin, but not desmin, were localized in these cells by immunocytochemistry. The extracellular matrix components collagen types I, III and IV, fibronectin and laminin were found in HTM-3 cells as well as their non-transformed parental cells. As predicted, the protein profile of the HTM-3 cells revealed by two-dimensional gel electrophoresis was different from that of the non-transformed cells, probably due to the enhanced growth characteristics of these cells. Furthermore, HTM-3 cells had various intracellular second messenger systems that responded to pharmacological agents. Forskolin, prostaglandin E2, beta-adrenergic and adenosine A2 agonists stimulated the adenylyl cyclase in these cells, whereas muscarinic, serotonergic, dopaminergic and other agonists were ineffective. Sodium nitroprusside increased the intracellular concentration of cGMP, demonstrating the presence of a functional guanylyl cyclase. Phospholipase C activity in these cells was also detected. Muscarinic agonists, histamine and bradykinin, but not adrenergic, serotonergic agonists or prostaglandins, increased phosphoinositide turnover. These drug responses of HTM-3 cells agree with published data on primary TM cells and TM tissues, suggesting that the transformed cells may be a valid substitute for certain pharmacological studies of TM.

Estrogen metabolism by primary cultures of rat ventral prostate epithelial and stromal cells.

Estrogen metabolism was examined in primary cultures of rat ventral prostate epithelial and stromal cells developed from young (approximately 3 weeks old) animals. Supraphysiologic concentrations (50 nM) of tritium-labelled estradiol (E2) and estrone (E1) were incubated separately with each cell type and the metabolites formed were measured at selected time points over a 24 h period. The metabolites were analyzed using high performance liquid chromatography. Epithelial cells exhibited an equal capability to interconvert E2 and E1 thus demonstrating the presence of similar oxidative and reductive activities for 17 beta-hydroxysteroid oxidoreductase (17 beta-HSOR) [0.45 and 0.40 pmol/3 h/microgram DNA respectively]. In contrast, stromal cells showed a 6-fold lower rate of oxidation of E2 to E1 (0.08 pmol/3 h/microgram DNA) but exhibited an approx 5-fold higher rate of reduction of E1 to E2 (1.81 pmol/3 h/microgram DNA). Estriol (E3) formation from either substrate was not detected in the two cell types. The results demonstrate that rat ventral prostate epithelial cells have similar capabilities to form or remove biologically active E2. In contrast, prostate stromal cells exhibited a preferential capability to form and possibly maintain high levels of biologically active E2. These findings are discussed with reference to the actions of estrogens on prostate epithelial-stromal cellular interactions.

Androgen-independent epithelial cells of the rat ventral prostate.

Androgen-independent cell lines have been clonally selected from primary cultures of androgen-dependent epithelial cells from the rat ventral prostate. These rapidly dividing epithelial-like cells (RDE) have altered morphology and adherence characteristics. Unlike normal prostate epithelial cells, the RDE cell lines do not require androgens for cell division or cell survival. In the presence of physiological concentrations of testosterone, the isoelectric focusing patterns of prostatic acid phosphatases are abnormal in these RDE cells, and the prostate steroid-binding protein genes are not expressed. The loss of androgen dependence is not due to the inability of RDE cells to metabolize testosterone to 5 alpha-dihydrotestosterone, the active androgen, since the RDE cell lines metabolize testosterone in a manner similar to normal androgen-dependent epithelial cells. When RDE cells are grown on collagen matrices, the cells assume ductlike structures, similar to prostatic acini, although PSBP gene expression is not induced. When seeded into soft agar these cell lines form distinct foci, suggesting that they are potentially tumorigenic.

The persistence of prescribing habits: a survey and follow-up of prescribing to chronic hospital in-patients.

This study describes a survey of prescribing to psychiatric patients on long-stay wards, together with a follow-up survey carried out after a 2-year period. Our findings show that there had been no significant improvement in polypharmacy and overprescribing of psychotropic drugs over that period, 1983-1985. Guidelines for improvement are suggested.

Disproportionate reduction of actin synthesis in hearts of starved rats.

We examined the synthesis of proteins in rat myocardium after starvation. Rates of total protein synthesis in myofibrillar and nonmyofibrillar fractions of myocardium of starved animals were reduced similarly (to 70-80% of the rates in hearts of fed animals, p less than 0.002), but rates of synthesis of some individual proteins were affected discoordinately. Radiolabeled proteins from atrial and ventricular explants, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that starvation for 2 days reduced the rate of cardiac actin synthesis to 26-38% of control levels, while the rate of myosin heavy chain synthesis in the same hearts was only moderately reduced (74-80% of control levels). This starvation-induced reduction in actin synthesis could be accounted for at least in part by disproportionately decreased levels of actin mRNA in starved hearts, as revealed by Northern blot hybridization and by in vitro translation analysis. The dramatic decrease in cardiac actin synthesis was rapidly reversible, and actin synthesis returned to normal after a single day of refeeding. The selective reduction of actin synthesis after starvation was specific for the heart: rates of myosin heavy chain and actin synthesis in skeletal muscles (soleus and extensor digitorum longus) were coordinately reduced in response to starvation. To our knowledge, this is the first example of such dramatic discoordinate regulation of myofibrillar protein synthesis in response to a physiological stimulus.

Androgen metabolism and actions in rat ventral prostate epithelial and stromal cell cultures.

The rat ventral prostate requires androgens for normal development, growth, and function. To investigate the relationship between androgen metabolism and its effects in the prostate and to examine differences between the epithelial and stromal cells, we have established a system of primary cell cultures of immature rat ventral prostate cells. Cultures of both cell types after reaching confluency (6-7 days) actively metabolized 3H-labelled testosterone (T), 5 alpha-dihydrotestosterone (5 alpha-DHT), 5 alpha-androstane-3 alpha,17 beta-diol, and 5 alpha-androstane-3 beta,17 beta-diol. The epithelial cells actively reduced T to 5 alpha-DHT and formed significant amounts of 5 alpha-androstane-3,17-dione from T, 5 alpha-DHT, and 5 alpha-androstane-3 alpha,17 beta-diol. All substrates were converted to significant amounts of C19O3 metabolites. The stromal cells also metabolized all substrates, but very little 5 alpha-androstane-3,17-dione was formed. The metabolism studies indicate that both cell types have delta 4-5 alpha-reductase, 3 alpha- and 3 beta-hydroxysteroid oxidoreductase and hydroxylase activities. The epithelial cells have significant 17 beta-hydroxysteroid oxidoreductase activity. The epithelial cells cultures grown in the presence of T have higher acid phosphatase (AP) contents (demonstrated histochemically and by biochemical assay). Tartrate inhibition studies indicate that the epithelial cells grown in the presence of T are making secretory AP. Stromal cell AP is not influenced by T. The results indicate that the cultured cells maintain differentiated prostatic functions: ability to metabolize androgens and, in the case of the epithelial cells, synthesize secretory AP.

Inter-laboratory quality control of estrogen and progesterone receptor assays in breast cancer tissue using lyophilised cytosols.

In 1981 a quality control (QC) program for estrogen and progesterone receptor assays was organized among six laboratories in Ontario, Canada. Twenty-three vials of lyophilised cytosol prepared from human breast tumor tissues were analysed by each laboratory over a two-year period. Samples of each batch of QC material were analysed at least twice: either in the same batch or on separate occasions. The present study demonstrates the stability of the QC material, defines the relative accuracy of the receptor assays, and provides estimates of within-batch and between-batch precision of the receptor assays.

Effects of tamoxifen on testosterone metabolism in postmenopausal women with breast cancer.

Testosterone is a known estrogen precursor especially in postmenopausal women. Tamoxifen, an anti-estrogen, is used in the treatment of women with breast cancer in whom metastatic disease has been demonstrated. The action of Tamoxifen is thought to be to occupy the intracellular estrogen receptor sites in target tissues and thus block the action of the biologically active estrogen, estradiol. Effects of Tamoxifen on the production and metabolism of hormones have been postulated. We studied the kinetics of testosterone metabolism by the constant infusion of 3H-testosterone in six postmenopausal women with breast cancer prior to and during Tamoxifen therapy. The Tamoxifen did not produce any significant change in the metabolic clearance rate, the plasma concentration or the calculated blood production rate of testosterone. The only significant alteration in the conversion ratio of testosterone to metabolites was the reduction (p less than 0.02) in conversion to 5 alpha-dihydrotestosterone. A significant reduction in the plasma concentrations (p less than 0.05) of dehydroepiandrosterone and of luteinizing hormone (p less than 0.02) was found. Other steroid and peptide hormones did not show any significant changes. We conclude that Tamoxifen therapy has very little effect on the kinetics of testosterone metabolism in postmenopausal women with metastatic breast cancer.

A protein activator of the plasma membrane Ca++-ATPase of heart sarcolemma.

A detergent extract of dog or beef heart sarcolemmal vesicles was prepared and found to have a stimulatory effect on the Ca++-ATPase of plasma membranes from human erythrocyte and cardiac sarcolemma. A procedure is described which enriches the activating fraction. The protein nature of the preparation is illustrated by its sensitivity to boiling and to the proteolytic enzyme(s) trypsin and chymotrypsin. SDS polyacrylamide gels indicate that the protein(s) involved have a molecular weight of 56 and 60 kDa. The sarcolemmal activator can stimulate the Ca++-ATPase activity of the isolated enzyme more than 100% in the presence of saturating amounts of calmodulin. The activation is calcium dependent, being greatest at approximately 10 microns Ca++, free, but does not change the Km for Ca++. A possible physiological role for the activator is discussed.