Novel expression of kallikreins, kallikrein-related peptidases and kinin receptors in human pleural mesothelioma

Malignant mesothelioma is an aggressive cancer of the pleura that is causally related to exposure to asbestos fibres. The kallikrein serine proteases [tissue (hK1) and plasma (hKB1) kallikreins, and kallikrein-related peptidases (KRP/hK2–15)] and the mitogenic kinin peptides may have a role in tumourigenesis. However, it is not known whether hK1, hKB1, KRP/hK proteins or kinin receptors are expressed in pleural mesotheliomas. The expression of hK1, hKB1, KRP/hK2, 5, 6, 7, 8 and 9, and kinin B1 and B2 receptors was assessed in archived selected normal tissue and mesothelioma tumour sections by immunoperoxidase and immunofluorescence labelling. hK1, hKB1 and kinin B1 and B2 receptors were expressed in malignant cells of the epithelioid and sarcomatoid components of biphasic mesothelioma tumour cells. The percentage of cells with cytoplasmic and nuclear labelling and the intensity of labelling were similar for hK1, hKB1 and the kinin receptors. KRP/hK2, 6, 8 and 9 were also expressed in the cytoplasm and nuclei of mesothelioma cells, whereas KRP/hK5 and hK7 showed predominantly cytoplasmic localisation. This is a first report, but further studies are required to determine whether these proteins have a functional role in the pathogenesis of mesothelioma and/or may be potential biomarkers for pleural mesothelioma.

Mapping photoreceptor and postreceptoral labelling patterns using a channel permeable probe (agmatine) during development in the normal and RCS rat retina

The aim of this study was to determine whether agmatine, a channel permeable probe, can identify photoreceptor dysfunction in the Royal College of Surgeons (RCS) retina at an earlier stage to that shown by apoptosis or anatomical markers, and also characterize the neurochemical development of the inner retina in the normal and degenerating rat. We used isolated retinas at different ages incubated in physiological media containing agmatine. Subsequently, postembedding immunocytochemistry was used to determine the number of labelled photoreceptors and the labelling pattern within postreceptoral neurons. Agmatine labelling patterns revealed a sequential development of retinal neurons beginning at postnatal day (PND)11/12 with most horizontal cells, a few ganglion and amacrine cells, showing a strong signal. The neurochemical development progressed rapidly, and reflects to a large part the known distribution of glutamate receptors, with inner nuclear labelling being evident by PND14, continuing with the same pattern of labelling in adulthood for the control retina. The RCS retina showed markedly reduced agmatine labelling in the inner retina at PND20. A rapid increase in photoreceptor AGB labelling was evident during the degeneration phase. Multiple samples at PND14 and PND16 confirmed a significant increase of labelled photoreceptors in the RCS retina.

Detection of genome impairment in bovine early embryos by autoradiography: a methodological note

The applicability of Pavlok's method characterising the nuclear status of early preimplantation bovine embryos by nuclear labelling pattern after a short pulse of [5-3H]uridine (revealing in situ detection of RNA transcription at the onset of the major embryonic transcription) was tested on experimentally irradiated 8- to 16-cell bovine embryos. After [5-3H]uridine labelling the semi-thin sections of these embryos were analysed by autoradiography for intranuclear distribution of newly synthesised RNA expected to be influenced by increasing doses of irradiation by gamma rays from a 60Co source. In control embryos, the labelling was homogeneously distributed in nucleoplasm and in nucleoli. The expected effects were clearly detected already in embryos irradiated with a dose of 2 Gy, in which low-level RNA synthesis was localised mostly at the periphery of the nucleus, the nuclear centre being without labelling. A detailed analysis of consecutive sections of embryos from all groups of irradiated and control embryos, using an arbitrary scale considering these effects, confirmed the detectability of the threshold level of genome impairment.

Antisense rescue defines specialized and generalized functional domains for c-Fos protein

Serum induces the expression of a number of proteins with similar transcriptional properties, including those encoded by the proto-oncogenes c-fos and c-jun. This study employs a novel antisense rescue method to determine whether antisense-resistant genes (constructed by deletion of antisense RNA target sequences) can replace c-fos expression during serum-induced DNA synthesis. Immunoprecipitation studies and nuclease protection assays demonstrated that anti-fos RNA inhibited endogenous c-fos expression but did not inhibit expression of transfected antisense-resistant mutant c-fos genes. The results of nuclear-labelling and cellular-proliferation studies indicated that C terminally truncated Fos mutants, including FBR v-fos, could not rescue endogenous Fos, although full-length and minimally truncated c-fos expression vectors could restore serum-induced DNA synthesis in cells expressing anti-fos RNA. Overexpression of c-Jun protein (Jun) could not restore serum-induced DNA synthesis to cells expressing inducible anti-fos RNA despite equivalent transactivation of an AP-1 target gene. Thus, the antisense rescue method defines a specialized function for c-Fos protein which is distinct from the function(s) of Jun and/or transforming FBR v-Fos proteins.

Antisense rescue defines specialized and generalized functional domains for c-Fos protein.

Serum induces the expression of a number of proteins with similar transcriptional properties, including those encoded by the proto-oncogenes c-fos and c-jun. This study employs a novel antisense rescue method to determine whether antisense-resistant genes (constructed by deletion of antisense RNA target sequences) can replace c-fos expression during serum-induced DNA synthesis. Immunoprecipitation studies and nuclease protection assays demonstrated that anti-fos RNA inhibited endogenous c-fos expression but did not inhibit expression of transfected antisense-resistant mutant c-fos genes. The results of nuclear-labelling and cellular-proliferation studies indicated that C terminally truncated Fos mutants, including FBR v-fos, could not rescue endogenous Fos, although full-length and minimally truncated c-fos expression vectors could restore serum-induced DNA synthesis in cells expressing anti-fos RNA. Overexpression of c-Jun protein (Jun) could not restore serum-induced DNA synthesis to cells expressing inducible anti-fos RNA despite equivalent transactivation of an AP-1 target gene. Thus, the antisense rescue method defines a specialized function for c-Fos protein which is distinct from the function(s) of Jun and/or transforming FBR v-Fos proteins.

Analysis of prostatic bud induction by brief androgen treatment in the fetal rat urogenital sinus

ABSTRACT The androgen dependency of prostatic bud formation in fetal rat urogenital sinuses was studied using brief treatments with androgen, and the incorporation of androgens by the sinus mesenchyme was followed by steroid autoradiography. Urogenital sinuses from 16·5-day fetuses of both sexes were grown in organ culture and treated with androgens for periods ranging from 4 to 72 h and then transferred to control medium. A minimum treatment of 24 h was required to induce prostatic buds in male sinuses and of 36 h in all female sinuses. This difference in response disappeared after more prolonged treatment. In both sexes the number of prostatic buds increased with the time of exposure to androgens. Prostatic bud formation continued for 24–36 h after transfer to control medium. Steroid autoradiographic analysis showed that the labelled androgen was concentrated in the mesenchymal nuclei. The rate of incorporation rose steeply during the first 12 h and then more slowly. After transfer to control medium the amount of labelled androgen decreased rapidly to half within 12 h and then decreased more slowly. In the competition experiments a 200-fold excess of unlabelled testosterone or dihydrotestosterone in the labelling medium greatly reduced the nuclear labelling with [3H]testosterone. J. Endocr. (1986) 110, 467–470

Autoradiographic studies of androgen-binding sites in the rat urogenital sinus and postnatal prostate

ABSTRACT Binding sites of [3H]testosterone and [3H]dihydrotestosterone in the rat fetal urogenital sinus and postnatal prostate and vagina grown in vitro were examined by steroid autoradiography. Distinct nuclear incorporation of both androgens appeared between 14·5 and 16·5 days of gestation in rat fetuses. Nuclear labelling in the sinus was restricted to the mesenchyme surrounding the epithelium which showed no nuclear labelling. A similar distribution of labelled cells was observed in male and female sinuses up to 18·5 days of gestation. By 20·5 days of gestation, the labelling in the ventral mesenchyme of female urogenital sinuses became less intense but persisted in the mesenchyme of the dorsal sinus wall from which the vagina is formed. In the postnatal prostate, the epithelium showed nuclear [3H]testosterone labelling at 10 days coinciding with the onset of its functional differentiation. Epithelial labelling became more intensive at 4 weeks post partum while that of the mesenchyme declined. The results suggest two phases of androgen action: formation of the prostatic buds mediated by the androgen-activated mesenchyme of the fetal urogenital sinus and the differentiation of the postnatal prostatic epithelium directly stimulated by androgens. J. Endocr. (1985) 104, 87–92

Sexual differentiation of the urogenital tract in the chicken embryo: autoradiographic localization of sex-steroid target cells during development

The determinant role ascribed to steroid hormones in sexual differentiation of the reproductive tract of the embryo implies the presence of target cells for sex steroids. An autoradiographic technique adapted for diffusible compounds was employed to characterize and localize cells which concentrate either [3H]oestradiol (E2) or [3H]dihydrotestosterone (DHT) in their nuclei. This paper describes the topographical distribution of cells containing receptor sites for oestrogen or androgen in various tissues of the reproductive tract of chicken embryos from day 6 to 15 of incubation. Receptor sites for oestradiol are present in the mesenchyme of the cloaca and in urodeum and vascular body. In the lower part of the Wolffian duct, only epithelial cells display nuclear labelling. In the Müllerian duct, nuclear receptor sites for [3H]oestradiol are observed not before day 15. Receptor sites for DHT are localized in the mesenchyme of the cloacal region from day 7 to 15. The Wolffian, but not the Müllerian duct contains receptor sites for DHT in the nuclei of epithelial and mesenchymal cells. Cross-competition experiments between [3H]E2 or [3H]DHT and unlabelled DHT or E2 respectively, show that 2 different types of receptor sites exist. The observations indicate: (a) complementary roles for oestrogenic and androgenic hormones in embryonic sexual differentiation; (b) precocity of receptors for sex hormones during embryonic development; (c) importance of mesenchyme in differentiation processes which are sex-steroid dependent.

Somatomedin and analogues of cyclic AMP increase the number of cells synthesizing DNA in cartilage from hypophysectomized rats

The effects of somatomedin and certain nucleotides on nuclear labelling of cartilage cells with [3H] thymidine were determined by autoradiography. Segments of costal cartilage from hypophysectomized rats were incubated for 24 h in a basal medium with or without additions and then pulsed for 2 h with [3H]thymidine in the basal medium. Both somatomedin (0.1 U/ml) and Bt2cAMP (10−4m) increased the number of labelled nuclei, and the combined effects were more than additive. A parallelism between the effects of these agents on nuclear labelling and their effects on total thymidine incorporation into DNA was demonstrated. The 8-bromated derivative of cAMP (10−4m) also enhanced chondrocyte nuclear labelling, but neither 8-Br5'-AMP (10 −4m) nor 8-Br-cGMP (10−4m) exhibited actions of the cAMP analogues. It is concluded that in cartilage obtained from hypophysectomized rats and incubated under the specified conditions (1) both somatomedin and cAMP analogues increase the number of cells synthesizing DNA as well as total thymidine incorporation into DNA, (2) the effects of the hormone and cyclic nucleotide in combination are synergistic, and (3) the increased incorporation of labelled thymidine into DNA reflects increased DNA synthesis and not merely an alteration of the specific activity of the intracellular thymidine nucleotide pool.