A potential H-DNA element in the MUC1 promoter does not influence transcription.

A purine/pyrimidine mirror repeat element (M-PMR3) in the MUC1 promoter has been shown to form H-DNA under in vitro conditions. We investigated this element for biological function in the regulation of transcription of this gene. Chloramphenicol acetyltransferase reporter-promoter constructs were prepared in which the mirror repeat element (PMR3) was intact, deleted, or modified, and their activities were evaluated by transient transfection assays into the cell lines Capan-2, PANC1, and HT-29. Deletion or modification of M-PMR3 increased expression of chloramphenicol acetyltransferase activity in MUC1-expressing cells; however, a role for an H-DNA structure in this activity was not supported by the results.

Potential for H-DNA in the human MUC1 mucin gene promoter.

Similar imperfect purine/pyrimidine mirror repeat (PMR) elements have previously been identified upstream of the human MUC1 mucin and CFTR genes. These elements confer S1 nuclease sensitivity on isolated plasmid DNA at low pH. We now present a detailed characterization of the non-B DNA structure responsible for S1 nuclease sensitivity upstream of the MUC1 gene. A approximately 90-base pair (bp) DNA fragment containing a 32-bp PMR element termed M-PMR3 was subcloned into a recombinant vector. This fragment conferred S1 nuclease sensitivity on the resulting supercoiled plasmid. High resolution mapping of sites reactive to S1 and P1 nucleases demonstrates that cleavage occurs within the M-PMR3 element. High resolution mapping with chemical agents selective for non-B DNA provides evidence that M-PMR3 adopts an H-DNA structure (intramolecular triple helix) in the less common H-y5 isomer at low pH. This result is observed in the presence or absence of Mg2+. Mutation of the native M-PMR3 element to create perfect homopurine/homopyrimidine mirror symmetry alters the preferred folding to the more common H-y3 triplex DNA isomer. These results demonstrate that imperfections in mirror symmetry can alter the relative stabilities of different H-DNA isomers.

A nuclear factor that binds purine-rich, single-stranded oligonucleotides derived from S1-sensitive elements upstream of the CFTR gene and the MUC1 gene.

We have identified two regions of non-random purine/pyrimidine strand asymmetry that were nearly identical in sequence in the 5' flanking (promoter) regions of the human cystic fibrosis transmembrane conductance regulator (CFTR) gene and the human MUC1 gene. These regions contain perfect mirror repeat elements, a sequence motif previously found to be associated with the formation of H-DNA conformations. In this report we demonstrate that a single-stranded non-B DNA conformation exists at low pH in supercoiled plasmids containing the similar mirror repeat elements, and that S1 nuclease digestion maps the single-stranded region to the position of the mirror repeats. In addition, we identify a nuclear protein of approximately 27 kD that binds to single-stranded oligonucleotides corresponding to the purine-rich strand of this region, but not to the pyrimidine-rich strands or to double-stranded oligonucleotides with corresponding purine/pyrimidine strand asymmetry.

[In vivo and in vitro effects of GnRH analogs on ovarian Leydig cell tumor]. / In-vivo- und In-vitro-Effekte von GnRH-Analoga auf einen ovariellen Leydigzelltumor.

A 65-year old patient, suspected to be suffering from an androgen producing ovarian tumour, was treated preoperatively with the GnRH agonist triptorelin (500 micrograms/day s.c.) for 7 days. After an initial rise, gonadotrophin levels were suppressed under this treatment. The elevated serum testosterone concentrations were reduced by approx. 50% by the triptorelin injections. After the extirpation of the tumour (histologically a Leydig cell tumour of the ovary without signs of malignancy), primary cell cultures which secreted testosterone and androstenedione were prepared. Coincubation of the tumour cells with the GnRH agonist triptorelin had no effect on their androgen secretion. Treatment of the tumour cells with high concentrations (10(-5) M) of a GnRH antagonist, however, resulted in a 100% increase of their testosterone and androstenedione secretion. GnRH-binding sites of low affinity (Ka = 0.54 x 10(5) M-1) and high capacity (B max = 1364 x 10(-12) M/mg membrane protein) were identified in the tumour. These findings suggest that GnRH analogues might modify androgen secretion of sex-cord stromal tumours of the ovary via the suppression of endogenous gonadotrophin secretion and possibly also via direct effects on the tumour cells.

Intracellular actions of gonadotropic and peptide hormones and the therapeutic value of GnRH-agonists in ovarian cancer.

During the last two decades, considerable experimental evidence has been collected indicating that epithelial ovarian cancer might be gonadotropin dependent. LH and FSH receptors have been described in some of these tumors. The proliferation of ovarian cancer cells could be stimulated in vitro by gonadotropins. Suppression of endogenous LH and FSH secretion by GnRH-agonist treatment inhibited the growth of experimental or heterotransplanted ovarian cancers in various animal models. A number of recent phase II clinical trials have shown that the application of GnRH-agonists can lead to remission or stable disease in patients with relapsed advanced ovarian cancer. At present, prospective controlled clinical studies are being performed to assess the efficacy of GnRH-agonist treatment in addition to conventional surgical and cytostatic therapy in ovarian cancer in FIGO stages III and IV. Also, direct effects of GnRH analogues on ovarian cancer seem possible: a GnRH-like protein has been found in the human ovary. Our group discovered and partially characterized a specific GnRH-binding site (mol. wt 63.2 kDa) in ovarian cancer which is very similar to other human extrapituitary GnRH-binding sites of the low affinity, high capacity type, e.g. in breast cancer or the placenta. Recently, other groups have described also high affinity GnRH-agonist binding sites in ovarian cancer as well as in other extrapituitary tissues. First results from our laboratory indicate that the proliferation of certain ovarian cancer cell lines in vitro is reduced by both agonistic and antagonistic analogues of GnRH. Other authors were able to inhibit gonadotropin-induced in vitro proliferation of ovarian cancer cell lines by co-incubation with a GnRH-agonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of estrogenic/antiestrogenic potential of antifertility substances using rat uterine peroxidase assay.

The effect of three compounds (clomiphene citrate, centchroman, embelin) and plant-derived methanolic extracts (Abutilon indicum and Butea monosperma) was studied on uterotropic and uterine peroxidase activities in ovariectomized rats. It was observed that these two parameters were highly correlated in response to treatment with these test materials and also to estradiol. It was suggested that the uterine peroxidase assay could be utilized as a biochemical parameter in the screening of new antifertility agents for their estrogenic/antiestrogenic properties.

Specific low affinity binding sites for gonadotropin-releasing hormone in human endometrial carcinomata.

Specific low-affinity high-capacity binding sites for gonadotropin-releasing hormone (GnRH) have recently been discovered in human breast and ovarian carcinomata. We checked whether similar binding sites are present in human endometrial cancer. Plasma membrane preparations were incubated with [125I,D-Ala6-desGly10]-GnRH-ethylamide in the presence or absence of unlabelled GnRH agonists or other peptides. GnRH-binding could be demonstrated in all 12 tumor samples tested. The mathematical analysis of the binding data was consistent with a single class of low affinity (Ka = (0.8-1.4) x 10(5) M-1) and high-capacity (Bmax = (134-142) x 10(-12) M/mg membrane protein) binding sites. Native GnRH had a similar affinity to the binding sites as the GnRH agonist used. Other peptides such as oxytocin, somatostatin and thyrotropin-releasing hormone did not crossreact with the binding sites. A photolabelled derivative of [D-Lys6]-GnRH was prepared with the bifunctional photolabile reagent (4-azidobenzyl)-N-hydroxysuccinimide. Photoaffinity labelling of endometrial carcinoma membranes and subsequent sodium dodecyl sulfate electrophoresis in 10% polyacrylamide gel revealed the presence of a single molecular mass component of 62 +/- 1.9 kDa. The appearance of this photolabelled binding site could be largely suppressed by the addition of unlabelled GnRH-agonist (10(-4) M) and thus represents the specific binding site for GnRH in endometrial cancer.

LHRH-receptors and LHRH-agonist treatment in ovarian cancer: an overview.

Considerable evidence exists that ovarian cancer might be gonadotrophin-dependent. Receptors for LH and FSH have been discovered in these tumors. Proliferation of ovarian cancer cells in vitro could be stimulated by gonadotrophins. Withdrawal of LH and FSH in animal models of ovarian cancer inhibited growth of these tumors. Phase-II clinical studies have shown that suppression of endogenous gonadotrophins by LHRH-agonists can be beneficial in women with advanced ovarian cancer. Respective controlled clinical trials are performed at present. Also direct effects of LHRH analogues on ovarian tumors have been reported. An LHRH like protein was found in human ovarian tissue. We discovered a specific LHRH binding site (mol. wt 63.2 kDa) in ovarian cancer tissue which is very similar to other human extrapituitary LHRH binding sites, of the low-affinity, high-capacity type, e.g. in breast cancer and the placenta. In the latter tissues, LHRH or a related substance has been proposed as an autocrine regulator of cellular function. If this was also the case in ovarian cancer, direct effects of LHRH analogs on the tumor cells could be used as additional therapeutical points of attack.

Toxicity studies with potassium embelate, a new analgesic compound.

Potassium embelate, 2,5-dihydroxy, 3-undecyl-1, 4-benzoquinone, from Embelia ribes Burm. was subjected to toxicity evaluation which included subacute, chronic, reproductive toxicity testing and teratological investigations in laboratory animals (mice, rats and monkeys). The results did not indicate adverse effects suggesting that potassium embelate is a safe compound.

Photoaffinity labelling of gonadotropin releasing hormone binding sites in human epithelial ovarian carcinomata.

A photoaffinity labelled derivative of [D-Lys6]-GnRH was prepared with a bifunctional photolabile reagent (4-azidobenzoyl)-N-hydroxysuccinimide. In rat pituitary membranes, this analog retained high binding affinity (Ka = 0.12 x 10(9) M-1) consistent with a single class of receptors. The analog was iodinated and used for the identification of GnRH binding sites in human epithelial ovarian carcinomata. By sodium dodecyl sulfate electrophoresis in 10% polyacrylamide gel the presence of two labelled components could be demonstrated: a high molecular weight component of 63,200 and a smaller component of 46,000. Competition experiments with unlabelled ligand suggest that it is the high molecular weight component which specifically binds GnRH.

Gonadotropin releasing hormone binding sites in human epithelial ovarian carcinomata.

As a first step to investigate whether gonadotropin releasing hormone (GnRH) analogs might be able to modulate directly the proliferation of human epithelial ovarian carcinomata, we checked if binding sites for GnRH are present in these malignancies. Specific binding of [125I][D-Ala6-des Gly10]-GnRH-ethylamide (GnRH agonist = GnRH-A) could be demonstrated in plasma membranes from 32 out of 40 ovarian carcinomata tested. This binding was dependent on temperature, time and plasma membrane concentration. Mathematical analysis of the binding data showed that the interaction of GnRH-A with the binding sites was consistent with a single class of low affinity, high capacity binding sites (Ka = 1.42 +/- 0.14 X 10(5) M-1; range: 0.3-3.8 X 10(5) M-1; R = 209 +/- 69 X 10(-12) M/mg membrane protein; range 16-400 X 10(-12) M/mg MP; means +/- S.E., n = 32). Native GnRH and the GnRH antagonist [D-p-Glu1, D-Phe2, D-Trp3,6]-GnRH had Ka values comparable to those of the GnRH-A used. [125I]GnRH-A binding could not be displaced by oxytocin, thyrotropin releasing hormone and corticotropin releasing factor in concentrations up to 10(-4) M. Somatostatin cross-reacted with binding sites from some carcinomata, while it did not displace GnRH-A binding in membranes from others. Though the functional role of this specific binding site for GnRH in human epithelial ovarian carcinomata is still obscure, it might be part of an autocrine regulatory system and provide a possible point of attack for therapeutic approaches using GnRH analogs in this malignancy.

Prolactin in blood plasma and milk of buffalo during estrous cycle and early pregnancy.

Concentration of prolactin was measured by radioimmunoassay for 11 primiparous and 16 multiparous buffalo. Prolactin concentration of blood plasma on day of insemination was about 200 ng/ml for primiparous and about 315 ng/ml for multiparous animals and fluctuated between 130 and 200 ng/ml for primiparous and between 250 and 345 ng/ml for multiparous pregnant animals. For the nonpregnant group prolactin fluctuated between 145 and 240 ng/ml for primiparous and between 210 and 310 ng/ml for multiparous animals with minor elevations 1 to 2 days before estrus. Concentration of prolactin of milk was not significantly different from that of plasma and was positively correlated .68 for nonpregnant and .93 for pregnant animals.

Gonadal steroid hormone level in blood plasma and milk of primiparous and multiparous nonpregnant and pregnant buffaloes.

Changes in the concentration of progesterone and estradiol-17beta were measured by radioimmunoassay in 11 primiparous and 17 multiparous buffaloes at estrus and daily post insemination and in 6 nonbred buffaloes at 6 hour intervals from 4 days before expected estrus to one day after estrus. Plasma progesterone concentration at estrus was 0.1 ng/ml which rose to a peak level of 3.47 ng/ml on day 17. It fluctuated around this level in those animals which conceived, but followed a declining trend in those which failed to do so and attained lowest values on the day of next estrus. Temporal changes of the hormone revealed that the occurrence of major decline varied between 16 and 62 h before estrus. The average concentration in milk was about three to four times higher than in plasma. The concentration of estradiol-17beta about 23.50 pg/ml at estrus and fluctuated around 10 pg/ml in animals that returned to estrus with a peak around estrus. Temporal changes of hormone revealed that peak level occurred 8-17 h before estrus. The concentration of estradiol in pregnant animals fluctuated around 10 pg/ml. The concentration in milk was about 2-3 times higher than in plasma. There was no significant (P>0.05) difference in the concentrations of progesterone and estradiol-17beta between primiparous and multiparous animals.

Hormonal milieu around parturition in buffaloes (Bubalus bubalis).

Radioimmunological measurements of prostaglandin F2 alpha, 13,14-dihydro-15-keto-PGF2 alpha (PGFM), estradiol-17 beta, progesterone, prolactin and luteinizing hormone (LH) were carried out in peripheral plasma around parturition in Murrah buffaloes. The PGF2 alpha concentration fluctuated before parturition and a peak was observed 1 day prior to parturition. PGFM, estradiol-17 beta and prolactin concentration increased gradually over the last 7 days with a significant peak (P less than 0.001) 1 day before parturition. The progesterone level declined gradually and an abrupt fall occurred 1-2 days before parturition. The estradiol-17 beta and prolactin concentrations declined to basal concentrations 1-2 days post partum, whereas PGF2 alpha and PGFM remained higher than basal concentrations. The LH concentration remained low without any significant variation. The progesterone concentration was negatively correlated with PGF2 alpha (r = 0.7039; P less than 0.05), PGFM (r = -0.8322; P less than 0.01) and estradiol-17 beta (r = -0.8896; P less than 0.001) before parturition.