Prostate cancer has the highest prevalence of any nonskin cancer in the human body, with similar likelihood of neoplastic foci found within the prostates of men around the world regardless of diet, occupation, lifestyle, or other factors. Essentially all men with circulating androgens will develop microscopic prostate cancer if they live long enough. This review is a contemporary and comprehensive, literature-based analysis of the putative risk factors for human prostate cancer, and the results were presented at a multidisciplinary consensus conference held in Crystal City, Virginia, in the fall of 2002. The objectives were to evaluate known environmental factors and mechanisms of prostatic carcinogenesis and to identify existing data gaps and future research needs. The review is divided into four sections, including 1) epidemiology (endogenous factors [family history, hormones, race, aging and oxidative stress] and exogenous factors [diet, environmental agents, occupation and other factors, including lifestyle factors]); 2) animal and cell culture models for prediction of human risk (rodent models, transgenic models, mouse reconstitution models, severe combined immunodeficiency syndrome mouse models, canine models, xenograft models, and cell culture models); 3) biomarkers in prostate cancer, most of which have been tested only as predictive factors for patient outcome after treatment rather than as risk factors; and 4) genotoxic and nongenotoxic mechanisms of carcinogenesis. The authors conclude that most of the data regarding risk relies, of necessity, on epidemiologic studies, but animal and cell culture models offer promise in confirming some important findings. The current understanding of biomarkers of disease and risk factors is limited. An understanding of the risk factors for prostate cancer has practical importance for public health research and policy, genetic and nutritional education and chemoprevention, and prevention strategies.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/cncr.20408

Human prostate cancer risk factors.

The literature describing 31P, 1H, 13C, 23Na and 19F MRS in vivo in human cancers is reviewed. Cancers have typical metabolic characteristics in 31P and 1H MRS including high levels of phospholipid metabolites and a cellular pH more alkaline than normal. These alone are not specific for cancer but are diagnostic in appropriate clinical settings. Some metabolic characteristics appear to be prognostic indices and correlation with treatment response is emerging as an important potentially cost-effective use of MRS in oncology. 19F MRS examines pharmacokinetics of 5-fluorouracil and by demonstrating its retention predicts response of a cancer to treatment. Current needs include improvement of diagnostic specificity by use of techniques like multivoxel MRS, proton decoupling of 31P, short echo time and fat-suppressed 1H MRS, 13C MRS direct or via 1H-observe, and statistical analysis of multiple spectral features. Trials in large populations in well defined clinical settings are needed to determine if MRS can provide independent prognostic indices useful in cancer management.

Studies of human tumors by MRS: a review.

Three-dimensional tissue culture models in cancer biology.

Paracrine and autocrine effects of nitric oxide on myocardial function.

NMR methods are being applied to study phospholipid metabolism of cancer cells by monitoring the resonances which appear in the 31P spectrum. This review, aside from considering the applicability of NMR to this specific pathway, raises the question of whether the phospholipid metabolite peaks observed by MR are indicators of cancer cell function or tumor response to treatment. After assessing the results from many investigations, it is concluded that there is no clear correlation and that a combination of techniques, including in vitro and extract studies, will be necessary for a more comprehensive evaluation of the in vivo data.

Phospholipid metabolites as indicators of cancer cell function.

OBJECTIVE: Inducible nitric oxide synthase (iNOS) activity, as measured by conversion of L-14C-arginine to L-14C-citrulline is significantly increased in infarcted rabbit myocardium as compared to healthy myocardium 48 h after coronary occlusion. The aim of this study was to localise the nitric oxide synthase (NOS) isoforms in normal myocardium and compare these findings with NOS activity in cells of the infarcted region. METHODS: Activities of NOS isoforms were enzymatically assayed in normal and infarcted myocardium. Localisation of NOS was performed on identical sections using specific monoclonal IgG antibodies against endothelial constitutive (cNOS) and macrophage inducible (iNOS) nitric oxide synthase. In addition, macrophages were identified using fluorescein conjugated ChromPure rabbit IgG, Fc fragment. RESULTS: iNOS activity increased significantly in infarcted as compared to normal myocardium [0.42(SEM 0.03) v 0.85(0.08) fmol.microgram-1.min-1, P = 0.02, respectively]. However, cNOS did not increase significantly in infarcted regions [0.18(0.04) v 0.24(0.06) fmol.microgram-1.min-1; P = 0.16, respectively]. cNOS was immunohistochemically localised in endothelial and endocardial cells in normal and infarcted tissues. The presence of iNOS activity in macrophages in infarcted myocardium was identified immunohistochemically. Cardiomyocytes and neutrophils did not label with the antibodies to cNOS and iNOS. CONCLUSIONS: (1) Infiltrating macrophages are the main site of increased iNOS activity in infarcted rabbit myocardium. (2) cNOS activity is not significantly increased in infarcted tissues as compared to normal myocardium. (3) Neutrophils and cardiomyocytes do not express NOS immunoreactivity in infarcted and normal rabbit myocardium.

Immunohistochemistry in the identification of nitric oxide synthase isoenzymes in myocardial infarction

A serum-free medium (HMRI-2) has been developed for the outgrowth and subculture of epithelial cells from normal adult human ureter and bladder. Medium HMRI-2 consists of Ham’s MCDB 152 with double the amounts of the essential amino acids in Stock 1, low Ca2+ (0.06 mM) and is supplemented with epithelial growth factor, 5 ng/ml; transferrin, 5 μg/ml; insulin, 5 μg/ml; ethanolamine and phosphoethanolamine, 0.1 mM each; hydrocortisone, 2.8×10−6
 M; and bovine pituitary extract, 126 μg protein/ml. The cultured cells showed ultrastructural markers of epithelial cells (prekeratin fibers, tonofilaments, surface microvilli with glycocalyx), exhibited ABO antigens, and had a normal human diploid karyotype. Primary cultures could be subcultured and also cryopreserved in HMRI-2 in liquid nitrogen. Cells in mass cultures showed a population doubling time of 40.5±4.5 h and had a maximum in vitro life span of 20 to 25 population doublings. It was observed that primary outgrowths, secondary cultures, and even cryopreserved cells all retained the capacity to respond to high Ca2+ and serum by differentiation and desquamation. This study has resulted in the availability of easily obtainable serum-free epithelial cultures from normal adult human ureter and bladder. The useful in vitro life span of these cultures may be important in future studies of carcinogenesis.

Selective growth of normal adult human urothelial cells in serum-free medium

A simple technique permitting long term 31P magnetic resonance spectroscopy of cultured mammalian cells is described. Cells are encapsulated in calcium alginate gels and perfused with a new phosphate-fre medium designed for maintenance of high cell viability. Intracellular pH and beta-NTP/Pi ratios, studied in three cell types, remained stable over a 16-20 h NMR monitoring period. The techniques described may be useful for monitoring cell metabolism with a variety of cell types.

31P NMR of mammalian cells encapsulated in alginate gels utilizing a new phosphate‐free perfusion medium

This study is concerned with the use of freshly harvested bovine endothelial cells attached to microcarrier beads in the production of the endothelium-derived relaxing factor (EDRF). The results are compared to production of EDRF by endothelial cells grown in tissue cultures. We found that freshly harvested cells attach themselves to microcarrier beads within minutes. This results in large surface/area volume ratio and permits superfusion of cells suspension on a filter (pore size of 25-30 microns), resulting in cell free filtrate. When superfusing an endothelium-deprived pulmonary artery strip, the effluent causes relaxation; the response depends on the number of superfused endothelial cells. The number of viable freshly harvested cells attached to microcarrier beads in 5 ml Krebs-Henseleit solution is small (30%), as compared to almost 100% for cultured cells. Despite this difference, percent relaxation induced for the same number of viable cells is identical for both groups. Scanning electromicrographs confirm anchorage of endothelial cells to microcarrier beads. While cultured cells cover the entire surface and are individually attached, freshly harvested cells are anchored as cell aggregates leaving some of the surface free. Attachment of freshly harvested endothelial cells to microcarrier beads offers an alternative for the study of the role of endothelial cells in the production of vasoactive substances.

The use of microcarrier beads in the production of endothelium-derived relaxing factor by freshly harvested endothelial cells.

Serum acid phosphatase has been considered a marker enzyme for prostatic cancer for a number of years.‘,* Measurement of this enzyme has been advocated for diagnostic screening, ~ t ag ing .~ and treatment managemer~t.~ “Prostatic” acid phosphatase is also considered to be a marker for cultured cells derived from the prostate or prostatic cancer.s Within the last five years, two cell lines (PC-3. DU 145) have been established from metastatic prostatic Both have been reported to have low hut detectable prostatic acid phosphatase activity as well as lysosomal acid phosphatase.’ RecentIy, we have reported the long-term growth of both PC-3 and DU 145 in a defined medium (PFMR-4) free of serum, hormones, or other growth factor^.^ The availability of a suitable defined medium made it possible to investigate the effect of serum on the expression of acid phosphatase activity in prostatic carcinoma cell cultures.

K. Shankar Narayan

Papers

Immunohistochemistry in the identification of nitric oxide synthase isoenzymes in myocardial infarction

Selective growth of normal adult human urothelial cells in serum-free medium

31P NMR of mammalian cells encapsulated in alginate gels utilizing a new phosphate‐free perfusion medium

The use of microcarrier beads in the production of endothelium-derived relaxing factor by freshly harvested endothelial cells.

Control of acid phosphatase activity in human prostatic carcinoma cell cultures by serum