Carlo M. Veneziale

Androgen receptor binding activity in human prostate cancer.

Androgen binding (cytosol and nucleus) was measured in tissue obtained from 223 untreated patients with proven prostate cancer (199 primary tumor, 24 malignant lymph nodes), 19 patients with hormone refractory cancer, and 46 patients with benign prostatic hyperplasia (BPH). The mean binding in both the cytosol and nucleus was significantly higher for patients with cancer than for those with BPH. Binding appeared to correlate with tumor stage. Androgen binding in malignant nodes can differ from that in the primary tissue and can vary from node to node in the same patient. Results obtained from an assay using a single saturating concentration of R1881 correlated well with those calculated from a full six‐point Scatchard analysis when an adequate amount (500 mg) of tissue was available. However, binding results obtained from a single‐point analysis performed on needle biopsy specimens (about 50 mg) obtained before complete surgical removal of the prostate correlated poorly with those derived from a full six‐point analysis performed on tissue (500–1000 mg) removed from the center of the malignancy. Androgen binding in nuclear extracts of histologically benign tissue adjacent to the malignancy was significantly higher than in nuclear extracts of BPH tissue. Cytosolic androgen binding in tissue removed from patients who were refractory to hormonal therapy was higher than in tissue from untreated cancer patients. The binding of estradiol by extracts of benign and malignant prostate tissue was low or absent and, thus, did not appear to be a significant phenomenon.

Relationship between androgen receptor binding activity in human prostate cancer and clinical response to endocrine therapy

The authors investigated the ability of androgen receptor binding in prostate cancer tissue to predict the response of prostate cancer patients to endocrine therapy. The clinical response of 37 previously untreated patients with various grades and stages of prostate cancer was correlated with androgen receptor binding and detailed histologic data obtained before treatment. All patients underwent cold‐punch transurethral resection of the prostate and received endocrine therapy. The association between time to progression and cytosolic androgen binding was not significant. However, the associations of time to progression to nuclear binding and to total androgen binding were significant (P = 0.029 and 0.038, respectively). The authors found no association between clinical stage and time to progression, but did find an association between time to progression and pathologic grade (P = 0.003); grade 4 lesions were the least responsive to hormone therapy. When grade 4 lesions were excluded (N = 3), binding levels were still predictive of progression independently of grade and stage. The authors conclude that nuclear receptor binding activity in localized and metastatic prostate cancer tissue is predictive of response to hormonal manipulation.

Concentration of skeletal and cardiac muscle pyruvate kinase determined by specific radioimmunoassay

Abstract A specific radioimmunoassay has been developed for rabbit skeletal muscle pyruvate kinase (ATP:pyruvate 2- O -phosphotransferase, EC 2.7.1.40) and applied to extracts of skeletal and cardiac muscle from rabbits subjected to differing dietary states and alloxan diabetes. In animals fed Purina Checkers ® rabbit chow the concentration was 11.1 ± 3.3 μ M is skeletal and 3.1 ± 0.6 μ M (μmol/1000 g wet weight) in heart muscle. Compared to the chow-fed animals the enzyme concentration increased approximately 2-fold in both tissues after a 4 day fast or after fat feeding, without any change in enzyme activity. Using slab gel electrophoresis, we demonstrated that fat feeding and fasting evidently cause the muscle cell to accumulate inactive or less active, tetrameric pyruvate kinase. Alloxan diabetes had no influence on the concentration of the enzyme in chow-fed animals. Pyruvate kinase activity was measurement in the same extracts by a conventional assay. From the data specific activities were calculated: for example, 40 units/nmol skeletal muscle enzyme and 19 units/nmol heart muscle enzyme in chow-fed animals. It is only by the combined application of activity and chemical concentration assays to tissue extracts could such data be obtained. Thus, in addition to measuring enzyme concentration an approach is provided to detect differences in the catalytic state of the enzyme. Our data show that assayable activity of this enzyme does not always reflect enzyme concentration and that regulation of enzyme concentration and enzyme activity can occur independently.

The Growth of Individual Seminal Vesicle Epithelial Cells and Their Proliferation

Abstract Histologically seminal vesicle epithelium (WE) of the intact adult guinea pig is a discrete and segregated monolayer of highly specialized tall columnar cells. The epithelial layer is so sharply demarcated from its attached stroma (primarily smooth muscle), that blunt dissection alone is sufficient to separate epithelium from muscle. After castration the epithelial cells decrease in both size and number so that by the fifth day, the surviving cells are greatly involuted structurally and comprise only about 12% of the original numerical population normally present in one seminal vesicle. Injected testosterone leads to restructuring of individual cells followed by cell replenishment. The major goal of this effort was to elaborate upon the processes of individual cell growth and cell replenishment during restoration of the tissue to normal cell size and number. The two separate processes were studied using light and electron microscopy, [3H]thymidine incorporation, and Northern blots with labeled histone gene probes. By approximately 48 hr of hormone repletion, parenchymal cell size had returned to normal as the result of a dramatic anabolic process of individual cell growth while cell number remained unchanged. During the subsequent 48-hr period of hormone repletion, the cell population was restored to normal as cell replenishment became the predominant process. Microscopic analysis at intervals throughout the 96-hr period failed to disclose any mitotic events to account for cell replenishment even when Colcemid had been administered. Nor could the increase in cell numbers be correlated with a great increase in [3H]thymidine incorporation or in histone mRNA synthesis. Thus, we could provide no evidence that mitotic division of the parenchymal cells themselves is responsible for cell replenishment. During the 24- to 48-hr interval of hormone repletion, electron microscopic examination disclosed the presence of small epithelial cells lying in a basal position.

Dexamethasone effects on liver pyruvate kinase

Dexamethasone in the medium perfusing isolated rabbit livers caused a fast-acting and reversible effect on liver pyruvate kinase. The effect was to lower the assayable V activity (units/g tissue) without changing the concentration (nmol/g enzyme protein). In effect, glucocorticoid lowered the specific activity (units/nmol of enzyme) by direct action on liver. The effect on liver pyruvate kinase is mediated by a relatively stable alteration; 30 min after perfusate (with steroid) was replaced by perfusate (without steroid), the effect remained strongly evident.

The fate of the label during gluconeogenesis from [1-14C]pyruvate in isolated rat liver

Abstract When livers from fasted rats were perfused with [1- 14 C]pyruvate, the mean ratios of specific radioactivities were: phosphoenolpyruvate to malate, 0.42 (S.D., 0.06; N = 6), 3-phosphoglycerate to malate, 1.15 (S.D., 0.17; N = 6); and 3-phosphoglycerate to phosphoenolpyruvate, 2.86 (S.D., 0.70; N = 6). Aspartate was labeled to such a limited extent relative to malate as to make it appear an insignificant intermediate in the conversion of pyruvate to glucose. These observations fail to support current concepts of gluconeogenesis from pyruvate and are consistent with the view that undiscovered enzyme reactions or compartmentation phenomena, or both, are also operative.

Glucose formation by the 27,500g supernatant fraction of rat liver homogenate☆

The gluconeogenic properties of the 27,500g supernatant fraction of a 20% rat liver homogenate were investigated. This cell-free system, prepared from a salt solution simulating the ionic composition of intracellular fluid, was found to be capable of forming glucose from added glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-diphosphate, and dihydroxyacetone phosphate. No metabolism of l-α-glycerophosphate occurred. Fructose 1,6-diphosphate and dihydroxyacetone phosphate were metabolized also in the direction of glycolysis; 3-phosphoglycerate was metabolized exclusively in this direction. Inhibition of the enolase reaction with NaF prevented metabolism of 3-phosphoglycerate unless both ATP and NADH were added to the system, in which case l-α-glycerophosphate was formed. The addition of ATP and NADH to the system in the absence of NaF resulted in the formation of both lactate and l-α-glycerophosphate from 3-phosphoglycerate. The conversion of fructose 1,6-diphosphate and fructose 6-phosphate to glucose was effectively prevented by the addition of ATP to the system, which caused it to metabolize these substrates by glycolysis.

Insulin and dexamethasone effects on liver fructose bisphosphatase.

Insuling perfusion of isolated diabetic rabbit liver and glucocorticoid perfusion of isolated normal rabbit liver resulted in an increased accumulation of immunoreactive fructose bisphosphatase (nmol/g). The insulin effect was maximal at 30 min with concentration (nmol/g) and specific activity (units/nmol) returning to normal within 2 h after insulin removal. Similarly, dexamethasone perfusion increased enzyme concentration and decreased its specific activity; however, the effects were maximal at 60-120 min. The results parallel those previously demonstrated in whole animals and establish that both hormones influence the enzyme by direct effects on the liver.

Can Androgens Alone Fully Restore Seminal Vesicle Epithelium

Abstract The purpose of this work was to determine if injected testosterone alone could restore the involuted seminal vesicle epithelium of hypophysectomized guinea pigs. Adult male guinea pigs were castrated, hypophysectomized, or hypophysectomized and castrated. Seminal vesicle epithelium was then isolated, weighed, analyzed for cytoplasmic protein content, and studied for its ability to synthesize four soluble, cell-specific, secretory proteins in vitro using immunoprecipitation techniques. Hypophysectomy, like castration, caused profound involution of this androgen-dependent tissue including loss of its protein-synthesizing capability. Testosterone replacement therapy restored wet weight, general cytoplasmic protein content, and in vitro protein synthesis to values found in castrated guinea pigs given testosterone. Thus, with regard to the parameters studied, the tissue responded to testosterone alone without apparent need for a pituitary hormone. Under the experimental conditions the administration of ovine prolactin simultaneously with testosterone did not enhance the action of testosterone in hypophysectomized-castrated guinea pigs.

Intracellular Concentration of Hepatic Glycerol-3-phosphate Dehydrogenase in Normal, Diabetic, and Hormonally Manipulated Rabbits

Abstract A radioimmunoassay (RIA) for cytosolic glycerol-3-phosphate dehydrogenase (GPDH) was developed as a means of determining enzyme concentration independent of assayable activity. The GPDH concentration (nmole/g wet wt), activity (units/g wet wt), specific activity (units/nmole GPDH), total liver concentration (nmole/liver/kg body wt), and total liver activity (units/liver/kg body wt) were determined under conditions favorable and unfavorable to gluconeogenesis. Fasted rabbits showed a decrease in total liver nanomoles of GPDH per killigram of body weight. Cortisone, triamcinolone, and thyroxine administration caused increases in total liver units and total liver nanomoles of GPDH per killigram of body weight in the fed animal. The increases or decrease observed correlated directly with changes in liver size. Diabetic, insulin-controlled diabetic, and glucagon-treated fed animals showed neither GPDH activity nor concentration changes. There was no change in the specific activity of GPDH under any conditions. The constant specific activity observed indicates a single catalytic state for the enzyme. Thus, changes in total liver enzyme rather than in specific activity become critical in the regulation of the GPDH reaction.