Levels of urinary inter-alpha-trypsin inhibitor trimer as a function of age and sex-hormone status in males and females not forming stones.

OBJECTIVES: To determine if levels of inter-alpha-trypsin inhibitor (I alpha TI)-trimer differ in normal individuals based on age, gender or hormonal status, as the regulation of calcium oxalate (CaOx) crystallization inhibitors, e.g. by sex steroids, could be a mechanism contributing to the differences in CaOx urolithiasis between the sexes. SUBJECTS AND METHODS: Voided urine samples were collected from normal males and females. In Experiment 1 samples were grouped by gender and age, i.e. paediatric (PED) < or = 10 years, male (M) 21, female (F) 14; young adult (YGAD) 20-30 years, M 23, F 18; adults (AD), 35-50 year, M 25, F 13; adults aged > or = 60 years (> 60), M 24, F 16 (totals, M 93, F 61). In Experiment 2 samples were grouped by gender, age and hormonal status, i.e. PED, M 24, F 17; AD, M 24, F 22; > 60 and not on hormonal therapy, M 23, F 30; M > 60 and on androgen deprivation therapy (ANDEP) 18; and F > 60 on oestrogen supplementation, F+EST, 18 (total M 89, F 85). Levels of urinary I alpha TI-trimer were determined by immunoblotting and enhanced chemiluminescence, and relative densities of the bands determined. RESULTS: In both experiments the relative levels of I alpha TI-trimer were 2-7 times higher in M-PED than in all other groups of males (P < or = 0.007). Among adult males, I alpha TI-trimer levels were similar in all groups, including ANDEP (P > or = 0.9). There were no differences in the relative levels of I alpha TI-trimer among any of the groups of females, regardless of age or hormonal status (P > or = 0.7). CONCLUSIONS: In males a decrease in I alpha TI-trimer was associated with the onset of adulthood and entry into the 'stone-forming years'. Females did not show this decrease, and neither sex showed an increase in I alpha TI-trimer in the > 60 group, when the incidence of CaOx urolithiasis is supposedly declining. While changes in urinary I alpha TI-trimer levels in males may reflect maturational changes in the kidney, overall these data do not support the hypothesis that the age-related changes in the incidence of urolithiasis are paralleled by changes in the expression I alpha TI-trimer. Additionally, the sex steroids do not appear to acutely regulate the expression of I alpha TI-trimer in adults, making differences in I alpha TI-trimer levels unlikely to be the reason for the disparity in the incidence of CaOx urolithiasis between the sexes.

Expression of proteins that inhibit calcium oxalate crystallization in vitro in the urine of normal and stone-forming individuals.

The factors precipitating clinically active calcium oxalate (CaOx) urolithiasis are not known. This study examined the relationships between urinary proteins that inhibit CaOx crystallization in vitro and the incidence of CaOx urolithiasis. The first hypothesis is that levels of urinary CaOx crystallization inhibitors differ between clinically active stone formers (SFs) and normal individuals. The second hypothesis is that lower levels of urinary CaOx crystallization inhibitors contribute to the two- to threefold greater incidence of CaOx urolithiasis in males compared with females. These hypotheses were derived from previous observations on the expression of urinary inter-alpha-trypsin inhibitor trimer (IalphaTI-trimer) in normal and stone-forming individuals. The proteins of void urine samples from normal volunteers (24 males, 19 females) and CaOx-SFs (26 males, 16 females) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoreactive IalphaTI-trimer, osteopontin, and prothrombin were detected by immunoblot plus enhanced chemiluminescence; the relative densities of the bands were then determined. With the exception of IalphaTI-trimer (P:

Chemoprevention of prostate carcinogenesis by alpha-difluoromethylornithine in TRAMP mice.

Development of effective chemopreventive agents for human consumption requires conclusive evidence of their efficacy in animal models that have relevance to human diseases. Transgenic adenocarcinoma mouse prostate (TRAMP) is an excellent model of prostate cancer that mimics progressive forms of human disease inasmuch as 100% of males develop histological PIN by 8-12 weeks of age that progress to adenocarcinoma with distant site metastases by 24-28 weeks of age. In these animals, ornithine decarboxylase (ODC) activity (>3-fold) as well as protein expression (>4-fold) was found to be markedly higher in the dorsolateral prostate as compared with the nontransgenic littermates, suggesting their suitability to determine the chemopreventive effect of alpha-difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ODC, against prostate cancer. Using male TRAMP mice, we studied the effect of oral consumption of DFMO on development of prostate carcinogenesis and surrogate end point biomarkers related to prostate cancer progression. In two independent experiments, each consisting of 8 animals on test, the cumulative incidence of prostatic cancer development at 28 weeks of age in 16 untreated TRAMP mice was 100% (16 of 16), whereas 94% (15 of 16) and 69% (11 of 16) of the animals exhibited distant site metastases to lymph nodes and lungs, respectively. Oral consumption of 1% DFMO (w/v) in the drinking water to TRAMP mice from 8 to 28 weeks of age resulted in a significant decrease in (a) weight (59%) and volume (66%) of prostate, (b) genitourinary weight (63%), and (c) ODC enzyme activity (52%) in the dorsolateral prostate. Importantly, in none of the DFMO-fed TRAMP mice were any distant metastases to lymph node and lungs observed. Furthermore, DFMO treatment resulted in the marked reduction in the protein expression of proliferation cell nuclear antigen, ODC, and probasin in the dorsolateral prostate. The protein expression of antimetastases markers, i.e., E-cadherin and alpha- and beta-catenin, was found to be restored in DFMO-fed animals as compared with the non-DFMO-fed mice. These chemopreventive effects of DFMO were further confirmed by immunohistochemical analysis of the dorsolateral prostate. Histological analysis of the dorsolateral prostate of DFMO-fed animals displayed marginal epithelial stratification, a small number of cribriform structures, elongated hyperchromatic epithelial nuclei, and a significant increase in apoptotic index. Non-DFMO-fed animals, on the other hand, displayed extensive epithelial stratification with profound cribriform structures accompanied with marked thickening, remodeling, and hypercellularity of the fibromuscular stroma. In nontransgenic littermates fed with DFMO, no significant alterations in the above parameters were evident. These data demonstrate that ODC represents a promising and rational target for chemoprevention of human prostate cancer and that TRAMP mice are excellent models for screening of novel drugs and chemopreventive regimens for potential human use.

A new human prostate carcinoma cell line, 22Rv1.

A cell line has been derived from a human prostatic carcinoma xenograft, CWR22R. This represents one of very few available cell lines representative of this disease. The cell line is derived from a xenograft that was serially propagated in mice after castration-induced regression and relapse of the parental, androgen-dependent CWR22 xenograft. Flow cytometric and cytogenetic analysis showed that this cell line represents one hyper DNA-diploid stem line with two clonal, evolved cytogenetic sublines. The basic karyotype is close to that of the grandparent xenograft, CWR22, and is relatively simple with 50 chromosomes. In nude mice, the line forms tumors with morphology similar to that of the xenografts, and like the parental CWR22 and CWR22R xenografts, this cell line expresses prostate specific antigen. Growth is weakly stimulated by dihydroxytestosterone and lysates are immunoreactive with androgen receptor antibody by Western blot analysis. Growth is stimulated by epidermal growth factor but is not inhibited by transforming growth factor-beta1.

Overexpression of ornithine decarboxylase in prostate cancer and prostatic fluid in humans.

Prostate cancer (PCA), the most commonly diagnosed cancer in males in the United States, is the second leading cause of cancer-related deaths of males in this country. Because of the poor success rate in the treatment of PCA, an intervention at an early stage may reduce the progression of small carcinoma to large metastatic lesion, thereby reducing PCA-related deaths. Concerted efforts are needed to establish mechanism-based approaches to develop: (a) the markers for early detection of the disease as well as toward monitoring the efficacy of treatment(s); and (b) novel chemopreventive strategies against PCA. Using unique samples of pair-matched benign and cancer tissue obtained from the same PCA patient, we showed that ornithine decarboxylase (ODC) activity is significantly (P < 0.001) elevated in PCA (1142 +/- 100; mean +/- SE) than in paired benign tissue (427 +/- 51; mean +/- SE). The immunoblot analysis also showed a significant elevation in the protein expression of ODC in the PCA tissues as compared with the paired benign tissue. Furthermore, our data showed that the ODC activity in the prostatic fluid obtained by a digital rectal massage from the patients with PCA (3847 +/- 162; mean +/- SE) was significantly higher than in the patients with benign prostatic hyperplasia (2742 +/- 167; mean +/- SE) or normal individuals (1244 +/- 67; mean +/- SE). This observation might be of significance because the prostatic fluid could be obtained noninvasively by digital rectal massage. We suggest that ODC could serve as a target for early detection of human PCA as well as for monitoring the efficacy of treatment(s). The development of ODC as a target for novel chemopreventive strategies against PCA is an intriguing possibility.

Overexpression of p185erbB2/neu in the NbE prostatic epithelial cell line increases cellular spreading and the expression of integrin alpha6beta1.

Overexpression of p185erbB2/neu has been demonstrated in approximately 30% of prostatic carcinomas and has been shown to induce tumorigenesis and metastasis in a prostatic epithelial cell line, NbE. To metastasize successfully, cells must be able to proliferate, degrade and be motile on a variety of substrates; thus attachment and spreading on a variety of substrates are key features of the metastatic phenotype. Using in vitro assays, we demonstrate that NbE-neuT-9/10 clones attached significantly better to specific substrates and spread significantly better on both specific and non-specific substrates as compared to the control clones. Additionally, the expression of integrin alpha6beta1, a key receptor enabling attachment and spreading on laminin, is upregulated on the metastatic clones NbE-neuT-9 and 10.

Differential expression of urinary inter-alpha-trypsin inhibitor trimers and dimers in normal compared to active calcium oxalate stone forming men.

PURPOSE: We determine if the immunoreactive profile of urinary inter-alpha-trypsin inhibitor can be used to distinguish between normal individuals and individuals with calcium oxalate stone disease. MATERIALS AND METHODS: Urinary proteins were dialyzed against water (15 kDa. molecular weight cutoff), lyophilized and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (6% acrylamide, reducing conditions) followed by Western blot. Inter-alpha-trypsin immunoreactive proteins were detected by enhanced chemiluminescence. Stone formation was confirmed to be active radiologically or passed as stone or gravel within 12 months of the sample. Stone composition was confirmed crystallographically. Normal individuals had no personal or familial history of urolithiasis and matched stone forming patients regarding race (white) and age (23 to 71 years old). Urine from a total of 101 individuals was analyzed. RESULTS: The intact inter-alpha-trypsin trimer (approximately 220 to 240 kDa.) and heavy chain (HC) 2-bikunin/HC1-bikunin dimers (approximately 115 to 130 kDa.) were detected more often in stone forming men (23 of 26 [89%] and 26 of 26 [100%], respectively) than in normal individuals (6 of 26 [23%] and 5 of 26 [19%], respectively, p < 0.0001). In those normal individuals who expressed inter-alpha-trypsin trimer and HC-bikunins the relative intensities were 5.3+/-1.4% and 16.3+/-17.1% of the stone forming controls, respectively. The identity of high molecular weight-inter-alpha-trypsin immunoreactive bands was confirmed using antibodies against the individual subunits (HC1, HC2, HC3, bikunin). In contrast to men high molecular weight-inter-alpha-trypsin's were readily detected in normal and stone forming women with equal frequency (inter-alpha-trypsin-trimer p=0.1337, HC-bikunins p=0.2836): inter-alpha-trypsin-trimer 17 of 18 [94%] and 9 of 13 [77%]; HC-bikunins 17 of 18 [94%] and 10 of 13 [85%]). Inter-alpha-trypsin-trimer and HC-bikunins, respectively, were detected in 2 and 5 of 10 patients with chronic renal disease. Expression was not related to hematuria or proteinuria. CONCLUSIONS: Immunoreactive profiles of urinary proteins may be able to be developed into a useful diagnostic tool to identify active stone formation, although a separate panel may be required for men and women. It is possible that these differences may provide clues as to why the incidence of stone disease is higher in men than women.

Metastasis induced by overexpression of p185neu-T after orthotopic injection into a prostatic epithelial cell line (NbE).

Overexpression of p185erbB2/neu has been detected in many adenocarcinomas, including prostatic cancer. In this study, a nontumorigenic cell line isolated from the rat prostatic epithelium (NbE) transfected with the activated oncogene p185neu-T was used to investigate the role of this oncogene in tumor progression. When clones overexpressing p185neu-T were injected orthotopically (1.5 to 2 x 10(6) cells) into the dorsal-lateral prostates of nude mice, prostatic tumors were detected in all mice injected and metastasis to the skeletal muscle in the rib area in 60-80% of the mice injected. Tumor and metastasis origin was confirmed by reselection with G418 and reverse transcriptase-polymerase chain reaction. Control cell lines produced no prostatic tumors or metastases. Incubation at low density (12500 cells/2 cm2) in serum-free medium revealed that clones overexpressing p185neu-T had a higher rate of [3H]thymidine incorporation than did control clones on 3, 5, and 7 d after plating (P < or = 0.0001) and constitutively overexpressed the 2.6-kb ornithine decarboxylase transcript. Additionally, clones overexpressing p185neu-T demonstrated an increased expression of epidermal growth factor receptor and p180erbB4, as judged by RNA blot analysis. Together these data support the hypothesis that overexpression of p185neu-T fosters tumor progression by several pathways, including induction of the metastatic cascade, increased proliferative capabilities, and increased expression of other members of the erbB2 gene family.

An orthotopic model for the study of growth factors in the ventral prostate of the rat: effects of epidermal growth factor and basic fibroblast growth factor.

In order to determine if epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) are capable of stimulating prostatic growth in situ, we complexed EGF and bFGF to a Matrigel (MG) vehicle that was subsequently injected orthotopically into the ventral prostate of adult rats. Three weeks following a single injection of 0.1 ng of EGF or bFGF, the wet weight of the growth factor-injected lobes of the ventral prostate was increased (P < or = 0.025) 144 +/- 14% and 138 +/- 8%, respectively compared to contralateral lobes injected with MG only. Total DNA and protein per lobe (P < or = 0.025) and the incorporation of bromodeoxyuridini (BrdUrd; P < or 0.01) were all significantly increased in lobes injected with EGF or bFGF compared to MG-injected lobes. Thus, EGF and bFGF were stimulating true growth of the rat ventral prostate in situ. The induced prostatic growth was ligand specific, because co-injection with neutralizing antibodies abolished EGF and bFGF stimulation of prostatic wet weight. The effect of a single injection of 0.1 ng of growth factor was long-lasting and elevated prostatic wet weight for 4 (P < or = 0.05; bFGF) to 6 (P < or = 0.05; EGF) weeks. Histologic evaluation did not reveal any gross changes in the ratio of stroma to epithelium in either EGF- or bFGF-injected lobes at the 0.1 ng/lobe dose. A slight hyperplasia of the prostatic epithelium was detected in lobes injected with 10 ng of EGF. Of note, the incorporation of BrdUrd was primarily localized in the luminal and basal epithelial cells, whereas incorporation into the prostatic stroma was scanty in lobes injected with either EGF or bFGF. In summary, we have developed an orthotopic model enabling the study of the roles of growth factors in prostatic physiology, function, and disease in situ. Additionally, we have demonstrated that when injected orthotopically into the rat ventral prostate, EGF and bFGF stimulate the growth of the prostatic epithelium to similar degrees.

Morphological features of principal cells in the ovine epididymis: a quantitative and qualitative study.

Functions of the epididymis differ by region, and this may be reflected in epithelial structure. Therefore, tissues from the initial segment (IS), proximal and central caput (PCap, CCap), and proximal and central corpus (PCor, CCor) epididymidis were examined by light and transmission electron microscopy. The proportion of principal cells in the epithelium was highest (p less than 0.05) in the CCap (74%) and lowest in the CCor (68%), whereas proportions of basal cells (25%), apical cells (1.4%), and white blood cells (2%) were similar in all regions. Volume density (VD) of the nucleus was lower (p less than 0.05) in principal cells in the IS (7%) than in other regions (10%). There was no regional difference in VD of the Golgi complex (14%) or endoplasmic reticulum (19%) in principal cells. The VD of mitochondria averaged 4% in the IS through CCap, but only 2.5% in PCor or CCor (p less than 0.05). The VD of clear vesicles + multivesicular bodies (8%) and dense vesicles (6%) were higher (p less than 0.05) in the CCap than in other regions (1% each), while there were more lipid droplets (12%) in the PCor than in other regions (less than or equal to 2%). Most quantitative differences in VD of organelles within principal cells were small even though significant. However, there were profound differences in the morphological features of the Golgi complex, endoplasmic reticulum, and mitochondria among regions.

Steroidogenesis and testosterone metabolism in cultured principal cells from the ram epididymis.

To determine if ram principal cells can synthesize or metabolize testosterone, or metabolize other steroids present in rete testis fluid, principal cells from the initial segment, central caput, and proximal corpus epididymidis were isolated and cultured in a floating collagen matrix with medium containing 20% dialyzed rete testis fluid. In the first experiment, each matrix was washed twice in testosterone-free medium on day 2.8, transferred into culture medium containing 100 nM of a tritiated steroid and incubated for 4 hours at 34 C. The tritiated steroids were pregnenolone, 5-androstene-3 beta,17 beta-diol, progesterone, 4-androstene-3,17-dione, testosterone, and dihydrotestosterone. Since testosterone was not formed from 5-androstene-3 beta,17 beta-diol or 4-androstene-3,17-dione, testosterone synthesis by ram principal cells is unlikely Pregnenolone and 5-androstene-3 beta,17 beta-diol were not metabolized and only slight metabolism of dihydrotestosterone occurred. Progesterone, 4-androstene-3,17-dione, and testosterone were metabolized to 5 alpha-reduced products tentatively identified as 5 alpha-pregnane-3,20-dione and 5 alpha-pregnan-3 beta-ol-20-one and/or 5 alpha-pregnan-20 alpha-ol-3-one; 5 alpha-androstane-3,17-dione and 5 alpha-androstan-3 alpha-ol-17-one, and dihydrotestosterone, respectively. The second experiment evaluated testosterone metabolism by both cultured principal cells and minced epididymal tissue. On day 1 of culture, during 12 hours the accumulation of dihydrotestosterone in medium from cells of the central caput was 48 X and 1.1 X that in medium from cells of the initial segment and proximal corpus epididymidis.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin F2 alpha as the luteolysin in swine: VI. Hormonal regulation of the movement of exogenous PGF2 alpha from the uterine lumen into the vasculature.

To test the endocrine-exocrine theory of maternal recognition of pregnancy in the pig 16 gilts were assigned randomly to a 2 X 2 factorial involving pretreatment with sesame oil (SO) or estradiol valerate (5 mg; EV) injected on Days 11 through 14 of the estrous cycle and an intrauterine injection of saline (5 ml; SA) or prostaglandin F2 alpha (50 micrograms; PGF) on Day 14. Peripheral blood samples were collected for 120 min postinjection and analyzed for 15-keto-13,14-dihydro-PGF2 alpha (PGFM). PGFM concentrations were lower in EV than SO gilts (438 vs. 844 pg/ml; p less than 0.05). There was heterogeneity of regression between EV and SO gilts (p less than 0.01), with EV gilts having a slower release of PGF from the uterine lumen into the vasculature. Prostaglandin F2 alpha did not increase mean PGFM concentrations (p greater than 0.10), but resulted in an altered temporal pattern of PGFM (p less than 0.05) compared to SA gilts. There was an interaction between the two treatments over time, with EV-PGF gilts demonstrating a slower, more gradual release of PGFM than SO-PGF gilts. To test whether prostaglandins of the E series were involved in this mechanism, gilts were assigned to two 4 X 4 latin squares balanced for residual effects and treated with saline or flunixen meglumine (Banamine). Each gilt was treated with four PGE:PGF infusion sequences (SEQ) in each uterine horn: phosphate-buffered saline (PBS; PBS-SEQ), PGE1 (50 micrograms), PGE2 (50 micrograms), and PGE1 (25 micrograms) + PGE2 (25 micrograms) (PGE-SEQ), with each infusion followed 15 min later by PGF (25 micrograms).(ABSTRACT TRUNCATED AT 250 WORDS)