The biology of breast tumor progression : acquisition of hormone independence and resistance to cytotoxic drugs

Many breast tumors appear to follow a predictable clinical pattern, being initially responsive to endocrine therapy and to cytotoxic chemotherapy but ultimately exhibiting a phenotype resistant to both modalities. Using the MCF-7 human breast cancer cell line as an example of an 'early' phenotype (estrogen and progesterone receptor positive, steroid responsive, low metastatic potential), we have isolated and characterized a series of hormone-independent but hormone-responsive variants (MIII and MCF7/LCC1). However, these variants remain responsive to both antiestrogens and cytotoxic drugs (methotrexate and colchicine). MIII and MCF7/LCCl cells appear to mimic some of the critical aspects of the early progression to a more aggressive phenotype. An examination of the phenotype of these cells suggests that some hormone-independent breast cancer cells are derived from hormone-dependent parental cells. The development of a hormone-independent phenotype can arise independently of acquisition of a cytotoxic drug resistant phenotype.

Progression of human breast cancer cells from hormone-dependent to hormone-independent growth both in vitro and in vivo

We have isolated a series of sublines of the hormone-dependent MCF-7 human breast cancer cell line after selection both in vivo and in vitro for growth in the presence of subphysiological concentrations of estrogens. These sublines represent a model system for study of the processes leading to hormonal autonomy. The cells form growing tumors in ovariectomized athymic nude mice in the absence of estrogen supplementation but retain some responsivity to estrogen as determined by stimulation of the rate of tumor growth in vivo and by induction of progesterone receptor. An ovarian-independent but hormone-responsive phenotype may occur early in the natural progression to hormone-independent and unresponsive growth in breast cancer. We observed no change in the affinity or decrease in the level of expression of estrogen receptors and progesterone receptors among the sublines and the parental cells. Epidermal growth factor receptors are not overexpressed in ovarian-independent cells. Thus, altered hormone receptor expression may be a late event in the acquisition of a hormone-independent and unresponsive phenotype. Sublines isolated by in vivo but not in vitro selection are more invasive than the parental cells both in vivo and across an artificial basement membrane in vitro. Thus, as yet unknown tumor-host interactions may be important in the development of an invasive phenotype. Furthermore, acquisition of the ovarian-independent and invasive phenotypes can occur independently.

Laying equitable claims to rest : key concerns in estate litigation post death

Equitable claims now increasingly arise in Australian estate litigation, particularly in conjunction with family provision applications.

Hormone and Metabolite Profiles in Nesting Green and Flatback Turtles: Turtle Species with Different Life Histories

Herbivorous turtle, Chelonia mydas, inhabiting the south China Sea and breeding in Peninsular Malaysia, and Natator depressus, a carnivorous turtle inhabiting the Great Barrier Reef and breeding at Curtis Island in Queensland, Australia, differ both in diet and life history. Analysis of plasma metabolites levels and six sex steroid hormones during the peak of their nesting season in both species showed hormonal and metabolite variations. When compared with results from other studies progesterone levels were the highest whereas dihydrotestosterone was the plasma steroid hormone present at the lowest concentration in both C. mydas and N. depressus plasma. Interestingly, oestrone was observed at relatively high concentrations in comparison to oestradiol levels recorded in previous studies suggesting that it plays a significant role in nesting turtles. Also, hormonal correlations between the studied species indicate unique physiological interactions during nesting. Pearson correlation analysis showed that in N. depressus the time of oviposition was associated with elevations in both plasma corticosterone and oestrone levels. Therefore, we conclude that corticosterone and oestrone may influence nesting behaviour and physiology in N. depressus. To summarise, these two nesting turtle species can be distinguished based on the hormonal profile of oestrone, progesterone, and testosterone using discriminant analysis.

A comparison of lungworm faecal larval counts and trichostrongyloid faecal egg counts between Red Deer (Cervus elaphus) and Red DeerXWapiti F1 hybrids.

To determine if breed differences in susceptibility to trichostrongyloid and lungworm infection exist, two groups of weaner deer containing seven red deer and red deer X wapiti F1 hybrids were compared using faecal egg counts and faecal larval counts. All animals were run on the same pasture at the same time and treated with the same anthelmintics at the same time. Results indicated that there were significant differences between red deer and red deer X wapiti hybrids, with red deer having higher faecal lungworm counts and red deer X wapiti hybrids having significantly higher faecal egg counts. It is likely that these differences were due to breed. Differences in the efficacy of anthelmintic treatments were also noted between the two groups, with oral oxfendazole being less effective at reducing faecal lungworm counts in red deer X wapiti hybrids than red deer.

Comparative analysis reveals loss of the appetite-regulating peptide hormone ghrelin in falcons

Ghrelin and leptin are key peripherally secreted appetite-regulating hormones in vertebrates. Here we consider the ghrelin gene (GHRL) of birds (class Aves), where it has been reported that ghrelin inhibits rather than augments feeding. Thirty-one bird species were compared, revealing that most species harbour a functional copy of GHRL and the coding region for its derived peptides ghrelin and obestatin. We provide evidence for loss of GHRL in saker and peregrine falcons, and this is likely to result from the insertion of an ERVK retrotransposon in intron 0. We hypothesise that the loss of anorexigenic ghrelin is a predatory adaptation that results in increased food-seeking behaviour and feeding in falcons.

An N-ethyl-n-nitrosourea induced corticotropin-releasing hormone promoter mutation provides a mouse model for endogenous glucocorticoid excess

Cushing's syndrome, which is characterized by excessive circulating glucocorticoid concentrations, maybe due to ACTH-dependent or -independent causes that include anterior pituitary and adrenal cortical tumors, respectively. ACTH secretion is stimulated by CRH, and we report a mouse model for Cushing's syndrome due to an N-ethyl-N-nitrosourea (ENU) induced Crh mutation at -120 bp of the promoter region, which significantly increased luciferase reporter activity and was thus a gain-of-function mutation. Crh -120/+ mice, when compared with wild-type littermates, had obesity, muscle wasting, thin skin, hair loss, and elevated plasma and urinary concentrations of corticosterone. In addition, Crh-120/+ mice had hyperglycemia, hyperfructosaminemia, hyperinsulinemia, hypercholesterolemia, hypertriglyceridemia, and hyperleptinemia but normal adiponectin. Crh -120/+ mice also had low bone mineral density, hypercalcemia, hypercalciuria, and decreased concentrations of plasma PTH and osteocalcin. Bone histomorphometry revealed Crh-120/+ mice to have significant reductions in mineralizing surface area, mineral apposition, bone formation rates, osteoblast number, and the percentage of corticoendosteal bone covered by osteoblasts, which was accompanied by an increase in adipocytes in the bone marrow. Thus, a mouse model for Cushing's syndrome has been established, and this will help in further elucidating the pathophysiological effects of glucocorticoid excess and in evaluating treatments for corticosteroid-induced osteoporosis.

Mapping the regulatory sequences controlling 93 breast cancer-associated miRNA genes leads to the identification of two functional promoters of the Hsa-mir-200b cluster, methylation of which is associated with metastasis or hormone receptor status in advanced breast cancer

MicroRNAs (miRNAs) are small non-coding RNAs of 20 nt in length that are capable of modulating gene expression post-transcriptionally. Although miRNAs have been implicated in cancer, including breast cancer, the regulation of miRNA transcription and the role of defects in this process in cancer is not well understood. In this study we have mapped the promoters of 93 breast cancer-associated miRNAs, and then looked for associations between DNA methylation of 15 of these promoters and miRNA expression in breast cancer cells. The miRNA promoters with clearest association between DNA methylation and expression included a previously described and a novel promoter of the Hsa-mir-200b cluster. The novel promoter of the Hsa-mir-200b cluster, denoted P2, is located 2 kb upstream of the 5′ stemloop and maps within a CpG island. P2 has comparable promoter activity to the previously reported promoter (P1), and is able to drive the expression of miR-200b in its endogenous genomic context. DNA methylation of both P1 and P2 was inversely associated with miR-200b expression in eight out of nine breast cancer cell lines, and in vitro methylation of both promoters repressed their activity in reporter assays. In clinical samples, P1 and P2 were differentially methylated with methylation inversely associated with miR-200b expression. P1 was hypermethylated in metastatic lymph nodes compared with matched primary breast tumours whereas P2 hypermethylation was associated with loss of either oestrogen receptor or progesterone receptor. Hypomethylation of P2 was associated with gain of HER2 and androgen receptor expression. These data suggest an association between miR-200b regulation and breast cancer subtype and a potential use of DNA methylation of miRNA promoters as a component of a suite of breast cancer biomarkers.

Growth hormone resistance and somatomedins in children with end-stage liver disease awaiting transplantation

Background: The success of orthotopic liver transplantation as treatment for end-stage liver disease has prompted investigation of strategies to maintain or improve nutrition and growth in children awaiting transplantation, because malnutrition is an adverse prognostic factor. The purpose of this study was to evaluate the effect of recombinant human growth hormone therapy on body composition and indices of liver function in patients awaiting transplant. Methods: The study was designed as a placebo- controlled, double-blind, crossover trial. Patients received 0.2 U/kg growth hormone, subcutaneously, or placebo daily for 28 days during two treatment periods, separated by a 2-week washout period. Ten patients (mean age, 3.06 ± 1.15 years; range, 0.51-11.65 years, five men), with extrahepatic biliary atresia (n = 8) or two with Alagille's syndrome (n = 2), with end-stage liver disease, completed the trial while awaiting orthotopic liver transplantation. Height, weight, total body potassium, total body fat, resting energy expenditure, respiratory quotient, hematologic and multiple biochemical profile, number of albumin infusions, insulin-like growth factor-1 and 1, growth hormone binding protein (GHBP), and insulin-like growth factor binding protein-1 (IGFBP-1) and insulin-like growth factor binding protein (IGFBP-3) were measured at the beginning and end of each treatment period. Results: Growth hormone treatment was associated with a significant decline in serum bilirubin (-34.6 ± 16.5 μmol/l vs. 18.2 ± 11.59 μmol/l; p < 0.02) but there was no significant effect on any anthropometric or body composition measurements, or on any biochemical or hematologic parameters. Conclusions: These children with end-stage liver disease displayed growth hormone resistance, particularly in relation to the somatomedin axis. Exogenous growth hormone administration may be of limited value in these patients

Effect Of Administration Of Luteinizing-Hormone (Lh) And Deprivation Of Lh On The Proteins Of Smooth Endoplasmic-Reticulum Of Immature And Adult-Rat Leydig-Cells

Analysis of proteins of smooth endoplasmic reticulum (SER) of Leydig cells from immature and admit rats by two-dimensional polyacrylamide gel electrophoresis (SDS-PAGE) revealed the presence of several new proteins in the adult rats. Administration of human chorionic gonadotropin to immature rats for ten days also resulted in a significant increase as well as the appearance of several new proteins. The general pattern of SDS-PAGE analysis of the SER proteins of Leydig cells resembled that of the adult rat. SDS-PAGE analysis of the SER proteins of Leydig cells from adult rats following deprivation of endogenous luteinizing hormone by administration of antiserum to ovine luteinizing hormone resulted in a pattern which to certain extent resembled that of an immature I at. Western Blot analysis of luteinizing hormone antiserum treated rat Leydig cell proteins revealed a decrease in the 17-alpha-hydroxylase compared to the control. These results provide biochemical evidence for the suggestion that one of the main functions of luteinizing hormone is the control of biogenesis and/or turnover SER of Leydig cells in the rat.

Immunization of male rabbits with sheep luteal receptor to LH results in production of antibodies exhibiting hormone-agonistic and -antagonistic activities

Antibodies to LH/chorionic gonadotrophin receptor (LH/CG-R; molecular weight 67 000), isolated in a homogenous state (established by SDS-PAGE and ligand blotting) from sheep luteal membrane using human CG (hCG)-Sepharose affinity chromatography, were raised in three adult male rabbits (R-I, R-II and R-III). Each of the rabbits received 20-30 mu g oi the purified receptor in Freund's complete adjuvant at a time. Primary immunization was followed by booster injection at intervals. Production of receptor antibodies was monitored by (1) determining the dilution of the serum (IgG fraction) that could specifically bind 50% of I-125-LH/CG-R added and (2) analysing sera for any chance in testosterone levels. Following primary immunization and the first booster, all three rabbits exhibited a 2.5- to 6.0-fold increase in serum testosterone over basal levels and this effect was spread over a period of time (similar to 40 days) coinciding with the rise and fall of receptor antibodies. The maximal antibody titre (ED(50)) produced at this time ranged from 1:350 to 1:100 to below detectable limits for R-I, R-II and R-III respectively. Subsequent immunizations followed by the second booster resulted in a substantial increase in antibody titre (ED(50) of 1:5000) in R-I, but this was not accompanied by any change in serum testosterone over preimmune levels, suggesting that with the progress of immunization the character of the antibody produced had also changed. Two pools of antisera from R-I collected 10 days following the booster (at day 70 (bleed I) and day 290 (bleed II)) were used in further experiments. IgG isolated from bleed I but not from bleed II antiserum showed a dose-dependent stimulation of testosterone production by mouse Leydig cells in vitro, thus confirming the in vivo hormone-mimicking activity antibodies generated during the early immunization phase. The IgG fractions from both bleeds were, however, capable of inhibiting (1) I-125-hCG binding to crude sheep luteal membrane (EC(50) of 1:70 and 1:350 for bleed I and II antisera respectively) and (2) ovine LH-stimulated testosterone production by mouse Leydig cells in vitro, indicating the presence oi antagonistic antibodies irrespective of the period of time during which the rabbits were immunized. The: fact that bleed I-stimulated testosterone production could be inhibited in a dose-dependent manner by the addition of IgG from bleed II to the mouse Leydig cell in vitro assay system showed that the agonistic activity is intrinsic to the bleed I antibody. The receptor antibody (bleed II) was also capable of blocking LH action in vivo, as rabbits passively (for 24 h with LH/CG-R antiserum) as well as actively (for 130 days) immunized against LH/CG-R failed to respond to a bolus injection of LH (50 mu g). At no time, however, was the serum testosterone reduced below the basal level. This study clearly shows that, unlike with LH antibody, attempts to achieve an LH deficiency effect in vivo by resorting to immunization with hole LH receptor is difficult, as receptor antibodies exhibit both hormone-mimicking (agonistic) as well as hormone-blocking (antagonistic) activities.

Effect of diethylstilbesterol and prolactin on the induction of follicle stimulating hormone receptors in immature and cycling rats

Induction of follicle stimulating hormone receptor in the granulosa cells of intact immature rat ovary by diethylstilbesterol, an estrogen, has been studied. A single injection of 4 mg of diethylstilbesterol produced 72 h later a 3-fold increase in follicle stimulating hormone receptor concentration as monitored by [125I]-oFSH binding to isolated cells. The newly induced receptors were kinetically indistinguishable from the preexisting ones, as determined by Lineweaver-Burk plot of the binding data. The induced receptors were functional as evidenced by increased ability of the granulosa cells to incorporate [3H]-leucine into cellular proteins. Neutralization of endogenous follicle stimulating hormone and luteinizing hormone by administering specific antisera had no effect on the ability of diethylstilbesterol to induce follicle stimulating hormone receptors, whereas blockade of endogenous prolactin secretion by ergobromocryptin administration significantly inhibited (∼ 30 %) the response to diethylstilbesterol; this inhibition could be completely relieved by ovine prolactin treatment. However, ovine prolactin at the dose tried did not by itself enhance follicle stimulating hormone receptor level. Administration of ergobromocryptin to adult cycling rats at noon of proestrus brought about as measured on diestrusII, (a) a reduction of both follicle stimulating hormone (∼ 30 %) and luteinizing hormone (∼ 45 %) receptor concentration in granulosa cells, (b) a drastic reduction in the ovarian tissue estradiol with no change in tissue progesterone and (c) reduction in the ability of isolated granulosa cells to convert testosterone to estradiol in response to follicle stimulating hormone. Ergobromocryptin treatment affected only prolactin and not follicle stimulating hormone or luteinizing hormone surges on the proestrus evening. Treatment of rats with ergobromocryptin at proestrus noon followed by an injection of ovine prolactin (1 mg) at 1700 h of the same day completely reversed the ergobromocryptin induced reduction in ovarian tissue estradiol as well as the aromatase activity of the granulosa cells on diestrus II, thus suggesting a role for proestrus prolactin surge in the follicular maturation process.

Regulation of Gonadotropin-Releasing Hormone (GnRH)-Receptor Gene Expression in Tilapia: Effect of GnRH and Dopamine

The present work was designed to study certain aspects of the endocrine regulation of gonadotropin-releasing hormone receptor (GnRH-R) in the pituitary of the teleost fish tilapia. A GnRH-R was cloned from the pituitary of hybrid tilapia (taGnRH-R) and was identified as a typical seven-transmembrane receptor. Northern blot analysis revealed a single GnRH-R transcript in the pituitary of approximately 2.3 kilobases. The taGnRH-R mRNA levels were significantly higher in females than in males. Injection of the salmon GnRH analog (sGnRHa; 5–50 μg/kg) increased the steady-state levels of taGnRH-R mRNA, with the highest response recorded at 25 μg/kg and at 36 h. At the higher dose of sGnRHa (50 μg/kg), taGnRH-R transcript appeared to be down-regulated. Exposure of tilapia pituitary cells in culture to graded doses (0.1–100 nM) of seabream (sbGnRH = GnRH I), chicken II (cGnRH II), or salmon GnRH (sGnRH = GnRH III) resulted in a significant increase in taGnRH-R mRNA levels. The highest levels of both LH release and taGnRH-R mRNA levels were recorded after exposure to cGnRH II and the lowest after exposure to sbGnRH. The dopamine-agonist quinpirole suppressed LH release and mRNA levels of taGnRH-R, indicating an inhibitory effect on GnRH-R synthesis. Collectively, these data provide evidence that GnRH in tilapia can up- regulate, whereas dopamine down-regulates, taGnRH-R mRNA levels.

Egg parasitoids of the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), from south-east Queensland

Field surveys of egg parasitoids of the diamondback moth, Plutella xylostella, were conducted at Redlands and Gatton, south-east Queensland. Eggs of P. xylostella were present all year round in both localities, and parasitized eggs were consistently found between late spring and early winter. Percent parasitism in the range 30–75% was recorded on many occasions, although rates less than 10% were more common. The major parasitoids included Trichogrammatoidea bactrae Nagaraja and Trichogramma pretiosum Riley. Laboratory evaluation showed that the T. pretiosum from Gatton has a high capacity to parasitize P. xylostella eggs under suitable conditions. This study represents the first record of egg parasitoids of P. xylostella from Australia.

Isolation and characterization of binding proteins for retinol from the cytosol, nucleosol and chromatin of the oviduct magnum of laying hens

Protein fractions that bind retinol were isolated from the cytosol, nucleosol and chromatin of the oviduct magnum of laying hens. The proteins isolated from the three sources showed similar elution profiles on chromatography through Sephadex G-75 and G-50 columns, and comparable mobility during electrophoresis on sodium dodecyl sulphate/polyacrylamide gels. Their molecular weights were calculated to be around 14500. When oviducts from vitamin A-depleted and vitamin A-repleted immature chicks given oestrogen injections for 6 consecutive days were incubated with [3H]retinyl acetate, uptake of the radioactivity in the nuclei of the vitamin A-depleted tissue was severalfold higher than that in the nuclei from the vitamin A-repleted tissue.