In vivo gene therapy for prostate cancer: preclinical evaluation of two different enzyme-directed prodrug therapy systems delivered by identical adenovirus vectors.

Advanced prostate cancer is invariably lethal once it becomes androgen independent (AI). With the aim of developing a new treatment we have used the human androgen-independent prostate cancer cell line, PC-3, to evaluate the effectiveness of two enzyme-directed prodrug therapy (EPT) systems as a novel means for promoting tumor cell destruction in vivo. We have confined our study to the use of a PSA promoter, in a preliminary attempt to achieve prostate specificity. The two EPT systems used were the HSVTK/GCV and PNP/6MPDR systems. These were chosen for their differential dependence on DNA replication for their mechanism of action. In the present work, either the HSVTK or PNP gene, each controlled by a PSA promoter fragment, was delivered by an E1-, replication-deficient human adenovirus (Ad5) into PC-3 tumors growing subcutaneously in BALB/c nude mice. Tumors were injected with a single dose of recombinant Ad5 and mice were treated intraperitoneally with the appropriate prodrug, twice daily, for 6 days thereafter. The growth of established PC-3 tumors was significantly suppressed and host survival increased with a single course of HSVTK/GCV or PNP/6MPDR treatment. HSVTK/GCV-treated PC-3 tumor growth was 80% less than that of control treatments on day 33, while PNP/6MPDR-treated tumor growth was approximately 75% less than that of control treatments on day 52. Survival data showed that 20% of HSVTK/GCV- or PNP/6MPDR-treated animals lived >45 and >448 days, respectively, longer than control animals. These results demonstrate that both HSVTK/GCV and PNP/6MPDR therapies interrupt the growth of an aggressive human prostate cancer cell line in vivo.

Use of polymerase chain reaction to diagnose a natural outbreak of mouse hepatitis virus infection in nude mice.

The enormous cost of eliminating mouse hepatitis virus (MHV) from a mouse colony demands that a confirmed etiologic diagnosis be made to justify the necessary remedial action. We describe an outbreak of MHV in nude mice in which histopathologic findings provided a presumptive diagnosis, but results of serologic testing of affected nude mice and immunocompetent sentinels were negative. Results of transmission electron microscopy of liver specimens from affected mice were equivocal. Confirmation of the etiopathogenesis was eventually provided by reverse transcriptase-polymerase chain reaction (RT-PCR), using primers with nested sequences directed to two separate but highly conserved regions of the MHV genome. This procedure detected MHV in the liver of an affected nude mouse and in a sentinel, although in the latter a positive result was obtained only because of the increased sensitivity of nested primers used in a second round of amplification. Virus was not detected in cell lines that had been injected into the mice, and the source of the outbreak was not found. These results indicate the applicability of RT-PCR for detecting MHV in a field situation while also illustrating that conventional, complementary techniques still have an essential role in reaching a diagnosis. It is recommended that specimens should be taken for histologic examination and serologic testing, as well as for molecular studies when MHV infection is suspected.

Serum growth hormone binding protein and hepatic GH binding sites in the Lewis dwarf rat: effects of IGF-I and GH.

A radioimmunoassay (RIA) for the rat growth hormone binding protein (GHBP) was developed using a synthetic peptide (corresponding to the hydrophilic carboxyl-terminal sequence of mouse GHBP) as standard and a monoclonal antibody (MAb 4.3) reactive with this peptide as the primary antibody. The values for GHBP concentration obtained for normal rats using this assay compare favourably with those obtained by gel filtration and ELISA methods. The concentration of GHBP in normal male rats at 11 weeks of age (680 +/- 30 ng/ml, SEM, n = 9) was significantly less than the concentration in normal females (943 +/- 47 ng/ml, SEM, n = 25). In 11-week-old dwarf male rats the concentration of GHBP was 423 +/- 35 ng/ml (n = 8); less than in dwarf females (542 +/- 32, P < 0.05, n = 9) and normal males (680 +/- 30, P < 0.001, n = 9). The GHBP concentration in dwarf rats was not age-dependent, whereas in normal females the concentration of GHBP increased with age. The availability of an RIA which is not susceptible to interference by endogenous GH, will facilitate further studies on hormonal and nutritional regulation of the rat GHBP. The assay was applied to studying the effects of IGF-I infusion (240 micrograms/day for 1 week) and GH injection (65 micrograms/100 g body weight, twice daily for 1 week and 4 weeks) on the serum concentration of GHBP in 11-week-old Lewis dwarf rats. Hepatic GH binding sites were also measured in desaturated membranes from the same animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolactin receptor expression in the gastrointestinal tract: characterization of the prolactin receptor of gastric mucosa.

There is evidence that prolactin (PRL) influences gastrointestinal function. However, the sites at which prolactin exerts these effects are not known. A monoclonal antibody was therefore generated against the rabbit mammary gland prolactin receptor (MAb 218) and used to study the distribution of the prolactin receptor in the rabbit gastrointestinal tract (GIT) by immunohistochemistry. MAb 218 is an IgG1 kappa-precipitating antibody which precipitates major affinity cross-linked mammary gland prolactin receptor subunits of molecular masses 45 and 80 kDa. It has an affinity of 0.8 x 10(9) mol/l for the prolactin receptor and does not react with GH or insulin receptors in precipitation assays. MAb 218 immunoreactivity was observed in classical prolactin target cells such as mammary gland epithelium, and this immunoreactivity was abolished by preincubation of MAb 218 with purified prolactin receptor but not by preincubation with purified GH receptor. In the GIT, the most intense immunoreactivity was associated with the oesophageal epithelium, chief (zymogenic) cells of the fundic mucosa, pancreatic islets of Langerhans and surface epithelial cells of the duodenum and jejunum. Other specific elements of the GIT were immunoreactive at lower levels or were immunonegative. No immunoreactivity was observed in these locations with a control monoclonal antibody (MAb 50.8) of identical isotype to 218. To support the immunohistochemical findings, rabbit gastric mucosal membranes were used to show the presence of lactogen-specific binding. Scatchard analysis of 125I-labelled human GH binding to the gastric mucosal membranes with rat prolactin as displacing ligand yielded an affinity constant (Ka) of 1.0 +/- 0.2 x 10(9) mol/l with a capacity of 3.5 +/- 0.4 fmol/mg protein. Affinity cross-linking and sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the gastric receptor revealed lactogenic hormone-binding subunits of molecular masses 43, 68 and 83 kDa. The 68 kDa subunit was not seen in rabbit mammary gland or ovarian tissue, and may be unique to gastric mucosa. In conclusion, we have demonstrated the presence of a high affinity lactogenic receptor in specific epithelial cell subpopulations of the GIT. This localization of the prolactin receptor in the GIT will assist in further functional assignment of prolactin to gastrointestinal physiology.

Localization and ontogeny of growth hormone receptor gene expression in the central nervous system.

There is literature evidence that both growth hormone (GH) and its mediator, insulin-like growth factor 1 (IGF-1), are able to act upon neuronal and glial cells in the brain. We report here the location of the GH receptor in the brain of the rat and rabbit. Receptor distribution was determined by immunohistochemistry with GH receptor/binding protein (BP) specific monoclonal antibodies and by in situ hybridization with a [35S]riboprobe. GH receptor/BP immunoreactivity in the rat was most prominent in the neonate and declined with postnatal age. Receptor immunoreactivity was generalised with variation in immunoreactivity in regional areas. In the rat, strongest immunoreactivity was seen in layers 2, 3, 5 and especially layer 6 of the cerebral cortex, in neurones of the thalamus and hypothalamus, in Purkinje cells of the cerebellum, in neurones of the trapezoid body of the brainstem, and in retinal ganglion cells. Glial cells, notably astrocytes were also strongly reactive, along with ependyma of the choroid plexus, ventricular lining and pia mater. In the neonatal rabbit, strongest immunoreactivity was evident in layers 2 and 3 of the cerebral cortex, in pyramidal cells of the hippocampus, and in neurones of the inferior and superior colliculi, brain stem reticular formation, dorsal thalamus and hypothalamus. A similar distribution of GH receptor mRNA was seen by in situ hybridization. The ontogeny of GH receptor/BP mRNA in whole rat brain was quantified by solution hybridization-RNAse protection assay. Contrary to its ontogeny in the liver (Endocrinology, 113 (1983) 1325-1329) receptor mRNA decreased with postnatal age.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of growth hormone receptor by immunocytochemistry in rat molar root formation and alveolar bone remodeling.

Growth hormone (GH) may regulate tooth formation and bone remodeling associated with tooth eruption. This study reports the distribution of growth hormone receptor/binding protein in developing rat molars and adjacent alveolar bone by immunocytochemistry using well-characterized anti-growth hormone receptor monoclonal antibodies. These tissues represent an excellent model for studying the ontogenic changes that occur in odontogenic and osteogenic cells, as these cells are found in linear arrays displaying the various stages of morphological and functional differentiation, and differentiated function. Immunoreactivity was first seen in precementoblasts in contact with the epithelial root sheath, and preodontoblasts. However, growth hormone receptor immunoreactivity was associated primarily with the cytoplasm of odontogenic and osteogenic cells forming their respective matrices. Thus, cementoblasts and odontoblasts at sites of new matrix formation showed intense immunoreactivity whereas cementocytes and mature odontoblasts at later stages of tooth development were nonreactive. Osteoblasts engaged in intramembranous ossification in the alveolar bone were positive, although osteocytes and endosteal cells were immunonegative. Osteoclasts at sites of alveolar bone remodeling resorption were also immunopositive. These patterns of receptor expression parallel the ontogenic sequences of odontogenic and osteogenic cells and suggest that GH promotes the functional state of these cells. Our results also imply that GH may influence differentiation or differentiated functions associated with odontogenesis, osteogenesis, and bone remodeling independent of systemic insulin-like GF-I.

Growth hormone (GH) regulation of gastric structure and function in the GH-deficient rat: up-regulation of intrinsic factor.

In a recent study we identified GH receptor/binding protein in cells of the gastric mucosa. In order to define the role of the GH receptor/binding protein in gastric function, we have investigated the effect of GH on gastric structure and function in the GH-deficient Lewis (dwarf) rat. Bovine GH, 65 micrograms/100 g body wt, was administered twice daily to adult male dwarf rats for 6 days (DW+) while control animals received vehicle only (DW-). Administration of GH produced a significant increase in body wt (P less than 0.001), stomach wt (P less than 0.01), and stomach to body wt ratio (P less than 0.05). GH administration also resulted in increased total gastric DNA, RNA, and protein content but did not produce significant differences in DNA, RNA, or protein content when normalized to stomach wt. Morphometric analysis of the gastric mucosa revealed a significantly (P less than 0.05) increased gastric epithelial height and mucosal surface area along with an increase in the proportion of nuclei with multiple nucleoli (P less than 0.01). The number of gastric mucosal cells in S-phase was determined by immunohistochemical detection of nuclear 5'-bromo-2'-deoxyuridine (BrdU) incorporated during a 2 h pulse of BrdU. GH treatment resulted in a 74% increase (P less than 0.05) in the number of BrdU-labeled nuclei/mm2 mucosa relative to vehicle-injected control animals. A modification of Zimmerman's method for the differential staining of gastric mucosa was used to delineate cell type for morphometric analysis. This showed that the density of differentiated (parietal and chief) cell types was not significantly different between DW- and to DW+ animals. Soluble extracts of gastric mucosa were prepared for estimation of pepsinogen content and [57Co]cyanocobalamin (vitamin B12) binding. GH administration produced no significant change in pepsinogen content per mg protein and did not affect the relative levels of pepsinogen isoenzymes as determined by polyacrylamide gel electrophoresis. GH administration did however result in an 86% increase (P less than 0.01) in [57Co]cyanocobalamin binding per mg protein. The increase in binding was totally displaceable by 1 microgram/ml unlabeled cyanocobalamin but not by 1 microgram/ml cobinamide dicyanide indicating it was the result of increased intrinsic factor rather than R protein. Sephadex S-300 gel filtration of mucosal extracts revealed an elution profile for [57Co]cyanocobalamin identical to that of purified porcine intrinsic factor and different from that of human salivary R protein. In conclusion, we have demonstrated that GH stimulates proliferation and enlargement of the gastric mucosa without significant alteration in cellular composition.(ABSTRACT TRUNCATED AT 400 WORDS)

Cellular localization of the growth hormone binding protein in the rat.

In the rat a GH-binding protein (GHBP) exists that is derived from the GH receptor gene by an alternative messenger RNA splicing mechanism such that the transmembrane and intracellular domains of the GH receptor are replaced by a hydrophilic carboxy terminus. Previous immunohistochemical studies detailing the localization of the GH receptor binding protein (BP) have used monoclonal antibodies that recognize extracellular region-specific epitopes common to both the GH receptor and GHBP. In this study we have used a monoclonal antibody (MAb 4.3) specific for the carboxy terminus of the rat GHBP to map its somatic distribution in the rat and have compared this distribution with that of a MAb recognizing both the BP and the GH receptor. A variety of tissues including the skeletal and muscular systems, the gastrointestinal tract and derivatives, the male and female reproductive systems, skin, central and peripheral nervous systems, and the 18 day gestation fetus were investigated. The distribution of GHBP immunoreactivity (MAb 4.3) was widespread and identical to that previously reported for the extracellular region of the GH receptor (MAbs 263 and 43). Immunoreactivity was both cytoplasmic and nuclear, indicating a possible role for the GHBP in intracellular function. GHBP immunoreactivity was predominantly associated with epithelial/endothelial cell subtypes and with mesenchymal elements such as muscle, chondrocytes, and osteoblasts, as previously described for the GH receptor extracellular region. We also report here the distribution of the GH receptor/GHBP in the kidney, cardiovascular, and respiratory systems. The most prominent immunoreactivity (MAbs 4.3 and 263) was associated with the distal convoluted tubules and collecting ducts of the kidney, with the epithelium and smooth muscle of the broncho-alveolar tree (including type I and II pneumocytes), with the Purkinje and myocardial fibers of the heart and with the endothelium and smooth muscle of blood vessels. Thus we have identified sites of direct GH action in the cardiovascular, renal, and respiratory systems. In conclusion, the extensive cellular distribution of the GHBP in the rat indicates physiological function(s) other than the binding of GH in plasma. Since GHBP mRNA has also been reported in a number of tissues, it may be that the GHBP is synthesized locally to mediate intracellular transport of GH and/or transcriptional regulation by GH in a variety of target tissues.

Prenatal expression of the growth hormone (GH) receptor/binding protein in the rat: a role for GH in embryonic and fetal development?

Although fetal growth is generally considered to be independent of pituitary growth hormone (GH), it is possible that pituitary GH plays a modulatory role in organ development or that a GH-like substance of non pituitary origin may influence fetal growth through the GH receptor. Accordingly, we have used immunohistochemistry, northern blot analysis, the reverse transcriptase-polymerase chain reaction and solution hybridization to study the ontogeny of the GH receptor/binding protein (BP) from the 12-day-old embryo (E12) to the E18 rat fetus. GH receptor/BP immunoreactivity was observed in all major organ systems of the E18 rat fetus and was not preferentially associated with any germ layer derivative. A general increase in GH receptor/BP immunoreactivity was evident from E12 to E18, with a marked increase occurring between E16 and E18. Hemangioblastic tissue was, however, strongly or intensely immunoreactive at all stages of development, as was the placenta. Most noteworthy of the other tissues expressing GH receptor/BP immunoreactivity by day 18 were skeletal and smooth muscle, chondroprogenitor cells, epithelial lining cells, neuronal ganglia, ependymal cells and the adrenal cortex. In the placenta, the most prominent immunoreactivity was associated with decidual cells. Total RNA was isolated from E12 to E18 rat fetuses and adult rat liver. Northern hybridization with a 35S-labelled rat GH receptor cRNA probe revealed that 3.9 kb and 1.2 kb transcripts complementary to the rat GH receptor riboprobe are present from at least E16. The existence of GH receptor mRNA at E12 and E14 was demonstrated by the polymerase chain reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of the growth hormone receptor/binding protein in skin.

Acromegaly is characterized by coarsening of facial features, acanthosis nigricans, hypertrichosis and oily skin. To determine the site through which GH exerts these effects, we have used immunohistochemistry to localize the GH receptor/binding protein (BP) in rat, rabbit and human skin. Three monoclonal antibodies (MAb 1, 43, 263) were immunoreactive in identical locations, whereas no immunoreactivity was evident when control monoclonal antibodies (MAb 50.8 and MAb 7 (rat] were used. Skin from neonatal and adult animals was used to determine whether GH receptor/BP expression was developmentally regulated. Immunoreactivity of the GH receptor/BP in the three species was consistently localized in the stratum basale and stratum spinosum. Intermittent staining was observed in the stratum granulosum. Scattered basal epidermal cells often displayed more intense immunoreactivity. This distribution was observed at all maturational stages examined. Intense GH receptor/BP immunoreactivity was observed in all histological layers of the lower one-third of hair follicles and in hair matrix cells of the dermal papillae. Immunoreactivity was also detected in the outer epithelial root sheath of the upper two-thirds of hair follicles, in sebaceous glands and in fibroblasts of the connective tissue sheath surrounding the follicle. GH receptor/BP immunoreactivity was also present in the secretory duct and myoepithelial cells of human eccrine sweat glands. Fibroblasts, Schwann cells of peripheral nerve fascicles, skeletal muscle cells and adipocytes of the dermis were also immunoreactive as were medial smooth muscle and endothelial cells of arteries. These results provide evidence that GH acts locally on the epidermis and epidermal appendages concordant with our recent localization of GH receptor/BP to epithelial cell types of the gastrointestinal and reproductive systems.

Uterine oxytocin receptors in an Australian marsupial, the brushtail possum, Trichosurus vulpecula.

1. Oxytocin receptors in the uterus of the brushtail possum (T. vulpecula) were characterized by radioreceptor assay and compared with those of the sheep and rat uterus. 2. A single oxytocin binding site was found with an affinity (Kd) and receptor concentration (Ro) of 3.0 +/- 0.8 nmol/l and 200 +/- 60 fmol/mg protein, respectively (SEM; n = 5). The receptor was stable at -20 degrees C; divalent ions were required for optimum binding. 3. Competitive displacement curves with related peptides showed the following order of specificity: vasotocin greater than oxytocin greater than mesotocin = arginine-vasopressin = [Thr4, Gly7]-oxytocin greater than lysine-vasopressin = isotocin much greater than [d(CH2)5, D-Phe2, Ile4, Ala9-NH2]-AVP. 4. It was concluded that oxytocin receptors in the possum have similar characteristics to those of placental mammals.