Synthesis and evaluation of 18F-labeled choline as an oncologic tracer for positron emission tomography: initial findings in prostate cancer.

The up-regulation of rates of choline uptake and phosphorylation in certain malignancies has motivated the development of positron-labeled choline analogues for noninvasive detection of cancer using positron emission tomography (PET). The choline analogue, no-carrier-added [18F]fluoromethyl-dimethyl-2-hydroxyethyl-ammonium (FCH), was synthesized through the intermediate [18F]fluorobromomethane. FCH was evaluated in relationship to 2-[18F]fluoro-2-deoxyglucose (FDG) as an oncological probe in cultured PC-3 human prostate cancer cells, a murine PC-3 human prostate cancer xenograft model, and in PET imaging studies of patients with prostate cancer. FCH was synthesized in 20-40% radiochemical yield and >98% radiochemical purity. Accumulation of FCH and FDG were comparable in cultured prostate cancer cells, whereas only FCH was inhibited (90%) by hemicholinium-3, a specific inhibitor of choline transport and phosphorylation. FCH showed similar biodistribution to [14C]choline in the tumor-bearing mouse, with prominent renal and hepatic uptake. Tumor uptake of FCH was similar to choline and FDG in the mouse model, although tumor:blood ratios were moderately higher for FCH. Initial PET imaging studies in prostate cancer patients showed high uptake of FCH in advanced prostate carcinoma and detection of osseous and soft tissue metastases. FCH uptake by tumors was markedly reduced in patients rescanned during androgen deprivation therapy. It is concluded that FCH closely mimics choline uptake by normal tissues and prostate cancer neoplasms. FCH is potentially useful as a PET tracer for detection and localization of prostate cancer and monitoring effects of therapy.

Alpha 1-adrenergic receptor regulation: basic science and clinical implications.

Adrenergic receptors (ARs) are members of the G-protein-coupled receptor family, which includes alpha 1ARs, alpha 2ARs, beta 1ARs, beta 2ARs, beta 3ARs, adenosine, muscarinic, angiotensin, endothelin receptors, and many others that are responsible for a large variety of physiologic effects through G-protein coupling. This review focuses on alpha 1ARs and their regulation at both the mRNA and protein levels. Currently, three alpha 1AR subtypes have been characterized both pharmacologically and at the gene level: alpha 1aAR, alpha 1bAR, and alpha 1dAR. These are expressed in a species- and tissue-dependent manner. Mutagenesis approaches have been extremely valuable in the identification of key residues that govern alpha 1AR ligand binding and signaling. These studies reveal that alpha 1ARs have evolved an exquisitely sensitive regulation of their activity in which any disruption of the native structure has profound effects on subsequent function and effector coupling. Significant advances have also been made in the elucidation of signaling pathway components, resulting in the identification of novel pathways that can lead to pathologic conditions. Specific topics include mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and G-protein-coupled receptor cross-talk pathways. Within this context, recent studies identifying underlying transcriptional mechanisms involved in the regulation of the alpha 1AR subtypes are also discussed. Finally, given the potentially important role of alpha 1ARs in the vasculature, as well as in the pathology of many diseases, such as myocardial hypertrophy and benign prostatic hyperplasia, the clinical relevance of alpha 1AR distribution, pharmacology, and therapeutic intervention is reviewed.

Zinc alpha-2-glycoprotein is expressed by malignant prostatic epithelium and may serve as a potential serum marker for prostate cancer.

Zinc alpha-2-glycoprotein (ZAG) is a M(r) 41,000 glycoprotein secreted by a variety of normal epithelia. ZAG was recently shown to stimulate lipolysis in adipocytes, leading to the development of cachexia in animals with ZAG-producing tumors. To understand the possible contribution of ZAG to the development of cachexia in men with prostate cancer, ZAG production by normal and malignant prostate tissue was investigated using immunohistochemical assays. Anti-ZAG monoclonal antibodies reacted strongly with normal prostate epithelium but not with other components of prostate or seminal vesicles. The majority of prostate cancers tested (35 of 48; 73%) also reacted with anti-ZAG antibodies. High-grade tumors expressed significantly less ZAG than moderate-grade tumors (mean ZAG score 1.1 versus 1.9; P < 0.01). Men with ZAG-producing prostate carcinomas had elevated levels of serum ZAG relative to their normal age- and race-matched controls (P < 0.02). Furthermore, s.c. growth of human ZAG-producing murine tumors in syngeneic mice and orthotopic growth of ZAG-producing human prostate carcinomas in nude rats resulted in readily detectable levels of human ZAG in the serum. Taken together, these studies show that ZAG production by prostate cancer can lead to systemically elevated serum ZAG levels that may be useful diagnostically. The effects of elevated systemic ZAG on cachexia-associated complications in patients with advanced prostate cancer deserves additional investigation.

Preliminary data on DSM-III associated features of major depressive disorder in children and adolescents.

Fifty-nine subjects 5-16 years old with major depressive disorder were assessed for the presence of separation anxiety and antisocial behavior, mentioned in DSM-III as associated features of the disorder in children and adolescents. The findings of the study support the existence of these associated features.

Redox control of vascular nitric oxide bioavailability.

NO is an important component of vascular homeostasis and abnormal NO bioactivity has been implicated in number of disease states with important public health implications. One clear mechanism of impaired NO bioactivity and vascular disease is excess vascular oxidative stress. There is now a wealth of developing data that manipulation of vascular antioxidant stress is the considerable influence of the biologic activity of endothelium-derived NO. It remains to be seen if this influence can be exploited in a manner that truly alters the course of human disease.

Synthesis and evaluation of (18)F-labeled choline analogs as oncologic PET tracers.

UNLABELLED: Elevated levels of choline (trimethyl-2-hydroxyethylammonium) and choline kinase (CK) activity in neoplasms have motivated the development of positron-labeled choline analogs for noninvasive detection of cancer using PET. The aim of this study was to further evaluate [(18)F]fluorocholine (fluoromethyl-dimethyl-2-hydroxyethylammonium [FCH]) as an oncologic probe in comparison with several other closely related molecules. METHODS: FCH, [(18)F]fluoromethyl-methylethyl-2-hydroxyethylammonium (FMEC), [(18)F]fluoroethyl-dimethyl-2-hydroxyethylammonium (FEC), and [(18)F]fluoropropyl-dimethyl-2-hydroxyethylammonium (FPC) were synthesized through [(18)F]fluoroalkylation reactions. In vitro phosphorylation rates of the (18)F-labeled choline analogs and [methyl-(14)C]choline (CH) were studied using yeast CK. Several choline radiotracers were also evaluated in cultured PC-3 human prostate cancer cells. Data on chemical stability, radiation dosimetry, and toxicity of FCH were obtained. PET studies with FCH were performed on a patient with prostate cancer and a patient with a brain tumor. RESULTS: FCH and FMEC revealed in vitro phosphorylation by CK that was similar to that of choline, whereas rates of phosphorylation of FEC and FPC were 30% (P < 0.01) and 60% (P < 0.01) lower, respectively. Accumulations of FCH, CH, and FPC in cultured PC-3 cancer cells were comparable, whereas uptake of FEC was approximately one fifth that of FCH. Dosimetry estimates using FCH biodistribution data in mice indicated that the kidneys are radiation-dose-critical organs for FCH. PET images of a patient with recurrent prostate cancer showed uptake of FCH in the prostatic bed and in metastases to lymph nodes. FCH PET showed uptake in malignancies in a patient with metastatic breast cancer. PET revealed FCH uptake in biopsy-proven recurrent brain tumor with little confounding uptake by normal brain tissues. CONCLUSION: The fluoromethyl choline analog FCH may serve as a probe of choline uptake and phosphorylation in cancer cells, whereas fluoroethyl (FEC) and fluoropropyl (FPC) analogs appear to have relatively poorer biologic compatibility. Preliminary PET studies on patients with prostate cancer and with breast cancer and brain tumor support further studies to evaluate the usefulness of FCH as an oncologic probe.

Comparison of cardiovascular adaptations to long-term arm and leg exercise in wheelchair athletes versus long-distance runners.

The effect of long-term arm exercise on cardiac morphology and function is unknown. To study these effects, highly trained wheelchair athletes were compared with long-distance runners and controls. In addition, the wheelchair athletes were compared with the long-distance runners to determine if long-term leg exercise confers a training effect during the performance of dynamic arm exercise. The study included 31 male subjects (mean age of 33+/-5 years), who comprised 3 groups matched for age and weight: wheelchair athletes (n = 9), long-distance runners (n = 12), and healthy controls (n = 10). All underwent echocardiography at rest and arm ergometry exercise testing with expiratory gas analysis. The peak work rate during arm exercise was highest among the wheelchair athletes, and was significantly higher in both groups of trained athletes compared with the control group (p<0.001). Runners demonstrated a significantly lower submaximal heart rate response to arm exercise compared with wheelchair and control subjects. Wheelchair athletes had increased left ventricular (LV) volume and mass by echocardiography compared with controls, but not to the same degree as that of runners. Although chamber dimensions and wall thickness did not differ among the groups, the LV volume index tended to be largest in the runners. Doppler indexes of diastolic LV filling were similar between the trained and untrained subjects. These data demonstrate that both long-term arm and leg exercise yield increases in LV volume and mass compared with untrained control subjects, although to a lesser degree in arm-trained athletes. Runners demonstrated a transfer of training effect in the performance of dynamic arm exercise, as demonstrated by their ability to achieve a higher peak work rate than controls, and showed a lower heart rate response to submaximal exercise than the wheelchair athletes and control subjects.

Vesical endometriosis: report of two cases and review of the literature.

Endometriosis is a common gynecologic disease in which endometrial tissue is deposited outside the normal confines of the uterine cavity. In rare instances, endometriosis involves the urinary tract, with the bladder the most frequent organ affected. Classic presenting symptoms include cyclic irritative voiding symptoms and suprapubic discomfort with or without hematuria. Both medical and surgical management have been advocated, but surgical extirpation is probably more efficacious. Two cases of endometriosis involving the the bladder are presented and contrasted in terms of pathophysiology. Contemporary management of this condition is reviewed, and guidelines for diagnosis and treatment are proposed.

Effects of androgen deprivation on prostate alpha 1-adrenergic receptors.

OBJECTIVES: Benign prostatic hyperplasia (BPH), the most common benign tumor in men, consists of two components-static (enlargement regulated by androgens) and dynamic (smooth muscle contraction through alpha 1-adrenergic receptors [alpha 1-ARs]). Because medical therapy of BPH involves tissue androgen deprivation, we studied the influence of androgen deprivation and replacement on regulation of rat ventral prostate alpha 1-ARs. METHODS: Prostate weight, alpha 1-AR density, autoradiographic images, histologic features, and cell-specific protein were examined before and after castration and androgen replacement. RESULTS: Castration decreases ventral prostate wet weight, a process reversed by testosterone administration. In contrast, there is an apparent increase in alpha 1-AR density (29 +/- 4 versus 65 +/- 6 fmol/mg total protein, mean +/- SEM) after castration, returning to baseline with testosterone replacement; alpha 1-AR density remains constant in control liver membranes. Alpha 1-ARs predominate in stroma throughout androgen deprivation therapy. Epithelially derived cells decrease (83% to 67%) after castration, resulting in a relative doubling in stroma (17% to 33%); the protein content of epithelial and stromal cells remains identical. Therefore, prostate-specific increases in alpha 1-ARs appear to result from relative increases in the ratio of smooth muscle to epithelium after castration rather than from direct upregulation of alpha 1-AR protein. CONCLUSIONS: Because alpha 1-AR density does not decrease with androgen deprivation, these studies suggest that alpha 1-AR antagonists remain an important component in BPH therapy, even when 5-alpha-reductase inhibitors are utilized.

Outcome analysis in patients with primary necrotizing fasciitis of the male genitalia.

OBJECTIVES: To characterize patients with primary necrotizing fasciitis of the male genitalia (Fournier's gangrene) and to identify risk factors and prognostic variables of survival. METHODS: Fifty consecutive patients with primary necrotizing fasciitis of the male genitalia treated at our institution during a 15-year period between 1984 and 1998 were retrospectively analyzed. Of these patients, 44 (88.0%) were found to be eligible for analysis of the outcome parameters. Univariate survival analysis was performed using the Kaplan-Meier algorithm followed by multivariate analysis of statistically significant variables. Six patients (12.0%) who were severely immunocompromised were studied separately. RESULTS: Medical comorbidities were prevalent, with diabetes being the most common condition (50%). The overall mortality rate was 20% (10 of 50). Three statistically significant predictors of outcome were identified among the variables analyzed. These were the extent of the infection (P = 0.0262), the depth of the necrotizing infection (P = 0.0107), and treatment with hyperbaric oxygen (P = 0.0115). Multivariate regression analysis of these variables identified the extent of the infection (P = 0.0234) as the only statistically significant, independent predictor of outcome in the presence of other covariables. CONCLUSIONS: The involved body surface area appears to be the most important prognostic variable, with a significant impact on outcome. Given the high mortality of the disease entity and a trend toward the improved survival of patients receiving hyperbaric oxygen, this treatment form appears indicated in more severe cases. Immunocompromised patients, who frequently have an atypical and fulminant clinical course, appear to constitute a separate group with a dismal prognosis.

Distribution of beta 3-adrenoceptor mRNA in human tissues.

The beta 3-adrenoceptor is a G protein-coupled receptor which mediates metabolic functions of the endogenous catecholamines epinephrine and norepinephrine. Questions exist regarding distribution of the beta 3-adrenoceptor in human tissue. In order to examine the distribution of beta 3-adrenoceptor mRNA in human tissues, we used sensitive and specific RNase protection assays without previous PCR amplification in an extensive list of human tissues. We confirm the presence of beta 3-adrenoceptor mRNA in human white fat from several locations, gall bladder, and small intestine, as well as extend the distribution of beta 3-adrenoceptor mRNA to previously uncharacterized human tissues such as stomach and prostate. The presence of beta 3-adrenoceptor mRNA in human white adipose tissue has important implications regarding possible use of beta 3-adrenoceptor selective agonists as anti-obesity agents, and the demonstration of beta 3-adrenoceptor mRNA in a number of gastrointestinal tissues and prostate raises the question of the role of the beta 3-adrenoceptor in motility and secretory processes.

Down-regulation of transforming growth factor beta receptor type I, II, and III during liver regeneration.

BACKGROUND: After partial hepatectomy (PH), it has been shown that hepatocytes are resistant to the mitoinhibitory effects of transforming growth factor beta (TGF-beta). Three types of TGF-beta receptors have been characterized in mammals. MATERIALS AND METHODS: In this study, changes in the protein and mRNA expression of TGF-beta types I, II, and III receptors relative to DNA synthesis were studied as a function of time after PH in the rat. RESULTS: There was a significant decrease in the protein and mRNA for all three receptor subtypes immediately after PH. All three receptors reached a nadir at 24 hours after PH, which correlated to the time of peak DNA synthesis. The type II receptor recovered to preoperative levels by 120 hours after PH, whereas the types I and III receptors remained at 60% of prehepatectomy levels. Sham-operated rats did not experience a significant drop in receptor protein or mRNA levels. CONCLUSIONS: This decrease in TGF-beta type II receptor expression may account for the reduction in the sensitivity of hepatocytes to TGF-beta after PH. Furthermore, the TGF-beta receptors appear to represent a class of immediate-early genes that are negatively, rather than positively, regulated after PH.

Factor V Leiden mutation and the risks for thromboembolic disease: a clinical perspective.

BACKGROUND: A single point mutation in the gene coding for coagulation factor V results in a form of factor Va that is resistant to degradation by activated protein C and leads to a relative hypercoagulable state. This mutation, factor V Leiden, is found in 4% to 6% of the U.S. population. PURPOSE: To review clinical data on factor V Leiden mutation, with emphasis on prevalence of and risks for thromboembolism and implications for screening and management. DATA SOURCES: A MEDLINE search of the English-language literature published between 1993 and April 1997 and an extensive bibliography review. STUDY SELECTION: Case-control and prospective cohort studies were reviewed if clinical features of thromboembolic disease associated with factor V Leiden mutation or resistance to activated protein C were presented. Original research articles were reviewed if they addressed the identification of the laboratory abnormality of activated protein C or factor V Leiden mutation. Case reports and case series were reviewed when no analytic data were available. DATA EXTRACTION: Review of the identified articles. DATA SYNTHESIS: Factor V Leiden mutation is associated with three- to sixfold increases in risks for primary and recurrent venous thromboembolism, especially in patients without transient risk factors, such as surgery or trauma. Risks for venous thromboembolism in genetically affected persons are substantially higher among patients with coexistent predispositions for thrombosis, such as advanced age, use of oral contraceptives, hyperhomocystinemia, and deficiencies of protein C and protein S. Factor V Leiden mutation does not seem to increase risks for arterial thrombosis. Whether patients with the mutation would benefit from more intense or prolonged anticoagulation is unknown. CONCLUSIONS: The presence of factor V Leiden mutation predisposes patients to venous thromboembolism, but screening for this disorder is of uncertain utility. Decisions about whether to screen for the mutation will depend on the results of clinical trials designed to evaluate the benefit-to-risk ratio of long-term anticoagulation in the secondary prevention of venous thromboembolism in patients with resistance to activated protein C.

PIP: The factor V Leiden mutation, present in 4-6% of the US population, makes the activated form of factor V relatively resistant to degradation by activated protein C, in turn producing resistance to activated protein C. Clinical studies have suggested that factor V Leiden mutation increases the risk of venous thrombosis during pregnancy and in oral contraceptive (OC) users, but the benefit-to-risk ratio of screening for this mutation is unclear. This paper reviews English-language articles published in 1993-97 on resistance to activated protein C or the factor V Leiden mutation with regard to laboratory diagnosis, prevalence, risks for thromboembolic disease, screening, and management. Included were case-control studies, prospective cohort studies, and case reports. The literature suggests that factor V Leiden mutation is associated with 3- to 6-fold increases in risks for primary and recurrent venous thromboembolism. In genetically affected persons, this risk is substantially higher among those with co-existent predispositions for thrombosis, including advanced age, OC use, hyperhomocystinemia, and deficiencies of proteins C and S. Any decisions about screening for factor V Leiden must await clinical trials designed to evaluate the benefit-to-risk ratio of long-term anticoagulation in the secondary prevention of venous thromboembolism in patients with resistance to activated protein C.

Mitogenic signaling in androgen sensitive and insensitive prostate cancer cell lines.

PURPOSE: To investigate the role of a specific mitogen activated protein kinase, extracellular signal-regulated kinase (ERK), in regulating cell proliferation induced by three potentially important prostate cancer mitogens that signal via different classes of receptors. MATERIALS AND METHODS: Androgen sensitive (LNCaP) and insensitive (PC-3) prostate cancer cell lines were used in these studies. Epidermal growth factor (EGF), lysophosphatidic acid (LPA), and dihydrotestosterone (DHT) were the mitogenic stimulants and AG1478, a receptor tyrosine kinase inhibitor, and PD98059, an inhibitor of MEK, were the chemical inhibitors used in this study. Cell proliferation was measured using the WST-1 assay and ERK expression and activation was determined by immunoblotting for phospho- and total ERK. RESULTS: In androgen-sensitive LNCaP cells, epidermal growth factor (EGF) and dihydrotestosterone (DHT) both enhanced cell proliferation. EGF-stimulation dramatically increased ERK phosphorylation while DHT did not. In the androgen-insensitive cell line, PC-3, EGF- and LPA-induced ERK phosphorylation and cell proliferation. Inhibition of EGF- and LPA- induced ERK activation with the EGF receptor inhibitor, AG1478, or the MEK inhibitor, PD98059, attenuated their proliferative effects. Neither inhibitor had an effect on DHT stimulated cell proliferation. CONCLUSIONS: These data demonstrate heterogeneity of mitogenic signaling in prostate cancer cells, and support the hypothesis that androgens and growth factors utilize divergent signaling pathways in prostate cancer to induce proliferation.

Localization of mRNA for three distinct alpha 1-adrenergic receptor subtypes in human tissues: implications for human alpha-adrenergic physiology.

alpha 1-Adrenergic receptors (alpha 1ARs) are virtually ubiquitous in human tissues and mediate important physiological functions as diverse as smooth muscle contraction, glycogenolysis, and myocardial inotropy. At least three alpha 1AR subtypes (alpha 1A/D, alpha 1B, and alpha 1C) have been described using molecular and pharmacological techniques. The identification of species heterogeneity (rat versus rabbit) in alpha 1AR subtype distribution has made it imperative to determine the distribution of alpha 1AR subtypes in human tissues. Accordingly, RNA extracted from human tissues was analyzed using RNase protection assays to determine alpha 1AR subtype expression. Of the cloned alpha 1ARs, alpha 1CAR mRNA predominates in many human tissues (heart, liver, cerebellum, and cerebral cortex), in contrast to its restricted distribution in both rats and rabbits. alpha (1B)AR mRNA is present in highest concentrations in human spleen, kidney, and fetal brain. alpha 1A/DAR mRNA is present in highest concentrations in human aorta and cerebral cortex. Hence, alpha 1AR subtype mRNA distribution is tissue selective and differs from that reported for rats and rabbits. These results have potentially significant implications for understanding human adrenergic physiology and are important for the rational development of alpha 1AR subtype-selective drugs.

Immortalization of a human prostate stromal cell line using a recombinant retroviral approach.

PURPOSE: We established an immortalized human prostate stromal cell line with retained markers of cell differentiation and alpha1-adrenergic receptor expression. MATERIALS AND METHODS: Primary human prostate stromal explants were infected with an amphotrophic retrovirus encoding the E6/E7 open reading frame of the human papillomavirus type 16. Immunohistochemistry was used to verify the expression of prostate stromal markers. alpha1-Adrenergic receptor expression was investigated using ribonuclease protection assays and radioligand binding. Cell proliferation was measured by the WST-1 assay and cell counting. RESULTS: Clonal isolates of individual prostate stromal cells were isolated and passed in selection media. E6 and E7 expression was verified using reverse transcriptase polymerase chain reaction in the selected cell line. The new prostate stromal cell line PS30 was established which maintains the expression of alpha-smooth muscle actin and expresses 22 fmol./mg. of protein of alpha 1-adrenergic receptors, approximately equal to native human prostate alpha 1-adrenergic receptor expression. However, at a subtype level alpha 1a-adrenergic receptor expression is down-regulated and not detectable by ribonuclease protection assays or radioligand binding, while alpha 1b and alpha 1d-adrenergic receptor expression is enhanced. From a physiological prospective PS30 cells do not form tumors in nude mice and stimulation with phenylephrine does not increase cell proliferation. CONCLUSIONS: We successfully established and characterized an in vitro human prostate stromal cell line. This cell line should facilitate studies designed to characterize the role of the adrenergic nervous system in the regulation of prostate growth.

Inhibition of telomerase is related to the life span and tumorigenicity of human prostate cancer cells.

PURPOSE: Telomerase, the enzyme that catalyzes the elongation of telomeres, is illegitimately activated in the majority of cancers, including that of the prostate, where it may greatly extend the life span of malignant cells. The inhibition of telomerase by molecular intervention has been shown to lead eventually to cell death in several tumor or in vitro immortalized cell lines and in 1 case prevent tumor growth in vivo. Therefore, we tested whether a similar strategy may be used to limit the tumorigenic potential of late stage prostate cancer cells. MATERIALS AND METHODS: PC-3, LNCaP and DU-145 human prostate cancer cells were infected with a retrovirus encoding a dominant-negative version of the catalytic subunit of telomerase (DN-hTERT). Subclones or polyclonal populations were assayed for DN-hTERT expression, telomerase activity, telomere length, cell life span and in most cases tumorigenicity in nude mice. RESULTS: DN-hTERT expression levels directly correlated with cell life span and tumorigenic growth. PC-3 cells expressing high levels of DN-hTERT died rapidly and failed to form tumors in nude mice, whereas cells expressing the lowest levels proliferated the longest and generated tumors that later spontaneously regressed. Similarly the inhibition of telomerase activity in LNCaP cells was greater than in DU-145 cells and correspondingly LNCaP cells had a shorter life span. CONCLUSIONS: DN-hTERT expression limits the life span and tumorigenic potential of human prostate cancer cells, although the onset of these effects appears to be dictated by the expression level of DN-hTERT. Therefore, telomerase represents an attractive target for potentially managing prostate cancer. Nevertheless, effective means of inhibiting the enzyme may be required for a therapeutically useful outcome.