Changing expression of chloride channels during preimplantation mouse development.

Plasma membrane chloride channels (ClCs) play important roles in a broad range of cellular processes including cell volume regulation, proliferation, and transepithelial transport, all of which are critical during preimplantation embryonic development. In this study, the molecular and functional expression of voltage-gated ClCs was analyzed throughout preimplantation development of the mouse conceptus. mRNA transcripts for all Clcn genes were detected. Only Clcn1 mRNA showed differential expression in the blastocyst, being detected in the trophectoderm but not in the inner cell mass. CLCN3 protein was detected at low levels in the cytoplasm and plasma membrane in 4-cell embryos and was localized to the apical plasma membrane of the trophoblasts in the blastocyst. Whole-cell patch-clamp recordings demonstrated the presence of a DIDS-sensitive, outwardly rectifying Cl(-) current throughout development, with this conductance being large at the 1-cell, morula and blastocyst stages. A second DIDS-insensitive Cl(-) current, which was inactivated by membrane depolarization, was present in cells differentiating into the trophoblast lineage and during blastocyst expansion. Inhibition of the DIDS-sensitive current and the DIDS-insensitive current, with 9-AC, prevented blastocyst expansion.

Interleukin 13 (IL-13)-regulated expression of the chondroprotective metalloproteinase ADAM15 is reduced in aging cartilage.

OBJECTIVE: The adamalysin metalloproteinase 15 (ADAM15) has been shown to protect against development of osteoarthritis in mice. Here, we have investigated factors that control ADAM15 levels in cartilage. DESIGN: Secretomes from wild-type and Adam15 -/- chondrocytes were compared by label-free quantitative mass spectrometry. mRNA was isolated from murine knee joints, either with or without surgical induction of osteoarthritis on male C57BL/6 mice, and the expression of Adam15 and other related genes quantified by RT-qPCR. ADAM15 in human normal and osteoarthritic cartilage was investigated similarly and by fluorescent immunohistochemistry. Cultured HTB94 chondrosarcoma cells were treated with various anabolic and catabolic stimuli, and ADAM15 mRNA and protein levels evaluated. RESULTS: There were no significant differences in the secretomes of chondrocytes from WT and Adam15 -/- cartilage. Expression of ADAM15 was not altered in either human or murine osteoarthritic cartilage relative to disease-free controls. However, expression of ADAM15 was markedly reduced upon aging in both species, to the extent that expression in joints of 18-month-old mice was 45-fold lower than in that 4.5-month-old animals. IL-13 increased expression of ADAM15 in HTB94 â€‹cells by 2.5-fold, while modulators of senescence and autophagy pathways had no effect. Expression of Il13 in the joint was reduced with aging, suggesting this cytokine may control ADAM15 levels in the joint. CONCLUSION: Expression of the chondroprotective metalloproteinase ADAM15 is reduced in aging human and murine joints, possibly due to a concomitant reduction in IL-13 expression. We thus propose IL-13 as a novel factor contributing to increased osteoarthritis risk upon aging.

Two doses of prostaglandin improve pregnancy rates to timed-AI in a 5-day progesterone-based synchronization protocol in beef cows.

The objective was to compare reproductive performance of Angus-cross beef cows synchronized with GnRH, a progesterone-based intravaginal insert (Controlled Internal Drug Release, CIDR) for 5-d, and one dose of either dinoprost (PGF) or cloprostenol (CLP, a PGF analogue) or two doses of PGF on the day of CIDR withdrawal. All cows (N=830) at six locations received 100microg of GnRH and a CIDR on Day 0. Within farm, cows were randomly allocated to receive 25mg of PGF at the time of CIDR insert removal on Day 5 (1xPGF; N=277), two 25mg doses of PGF, the first given on Day 5 at the time of CIDR removal and the second 7h later (2xPGF; N=282), or 500microg of CLP at the time of CIDR removal on Day 5 (1xCLP; N=271). All cows were given 100microg of GnRH on Day 8 (72h after CIDR removal) and concurrently inseminated (5-d CO-Synch+CIDR). Cows were fitted with a pressure-sensitive estrus detection device at the time of CIDR withdrawal. Timed-AI pregnancy rates were greater (P<0.0001) in the 2xPGF (69.0%) than the 1xPGF (52.0%) and 1xCLP (54.3%) treatments. However, breeding-season pregnancy rates were not different among treatments (87.0% for 1xPGF, 92.9% for 2xPGF and 87.5% for 1xCLP; P>0.1). In conclusion, cows that received two doses of PGF on the day of CIDR removal in a 5-d CO-Synch+CIDR synchronization protocol had excellent timed-AI pregnancy rates that were greater than in cows receiving a single treatment with either PGF or CLP.

Decreasing the interval between GnRH and PGF2alpha from 7 to 5 days and lengthening proestrus increases timed-AI pregnancy rates in beef cows.

Four experiments were conducted in postpartum beef cows to evaluate the influence of reducing the interval from GnRH to PGF(2alpha) from 7 to 5d in a Select-Synch + CIDR or CO-Synch + CIDR estrous synchronization program. In Expt 1, cows (n=156) were treated with either a 7 or 5d Select-Synch + CIDR program. A second PGF(2alpha) treatment was given to all cows in all experiments at 12h after the initial PGF(2alpha) (to ensure that luteolysis occurred with the 5d program). Estrous response, interval to estrus, conception rate, and first service AI pregnancy rates were similar between treatments. In Expt 2, cows (n=223) were treated with either a 7 or 5d CO-Synch + CIDR program, with timed-AI concomitant with GnRH at 60 h after PGF(2alpha). Timed-AI pregnancy rates were similar between treatments. In Expt 3 (n=223) and 4 (n=400) cows were treated with either a 7 or 5d CO-Synch + CIDR program with timed-AI concurrent with GnRH at either 60 h (7d) or 72 h (5d) after CIDR withdrawal. Timed-AI pregnancy rates were 13.3% (P<0.05; Expt 3) and 9.1% (P<0.05; Expt 4) greater for the 5 than 7d program. In conclusion, timed-AI pregnancy rates were improved with a 5d CO-Synch + CIDR program with timed-AI at 72 h after CIDR withdrawal, compared to a 7d CO-Synch + CIDR program with timed-AI at 60 h after CIDR withdrawal.

Impact of a timed-release FSH treatment from 2 to 6 months of age in bulls II: Endocrinology, puberty attainment, and mature sperm production in Holstein bulls.

The use of genomic testing in the cattle industries has renewed an interest in hastening bull puberty. In prepubertal males, FSH facilitates Sertoli cell proliferation and testis maturation. The aim of this study was to determine the effect of prepubertal administration of a timed-release FSH (delivered in a hyaluronan solution) on hormone secretion, puberty attainment, and mature sperm production in Holstein bulls in an AI center. Bulls (n = 29) were randomly assigned to one of two treatment groups based on birth date and pedigree. Beginning at 62 days of age (Day 62), bulls were injected im every 3.5 days with either 30 mg FSH (Folltropin-V; NIH-FSH-P1 units) in a 2% hyaluronan solution (FSH-HA, n = 17) or saline (control, n = 12) until Day 170.5. Blood samples to assess FSH, activin A, and testosterone were collected prior to each treatment. Scrotal circumference (SC) and BW were measured monthly. Puberty assessment (ability to ejaculate 5 × 107 sperm, 10% motile) was initiated at Day 244. Average mature daily sperm production (3× wk collection, combined 2 ejaculates) was assessed from Day 571-627. In blood collected every 3.5 days, FSH concentrations within FSH-HA bulls were increased (P < 0.05) over initial Day 62 concentration from Day 93.5-170.5. Concentrations of FSH did not differ between treatments from Day 62-93.5, but were greater (P < 0.05) in FSH-HA than control bulls from Day 97-170.5. Concentrations of activin A assessed for Day 62, 86.5, 107.5, 139, and 170.5 were greater (P < 0.05) in FSH-HA than control bulls on Day 86.5 and 107.5. Treatments did not differ (P > 0.1) in testosterone, BW, or SC. FSH-HA bulls attained puberty at a younger age than control bulls (278 ± 7.7 vs. 303 ± 9.1 days of age, P < 0.05), but mature daily sperm production was not different when measured from Day 571-627 (average 5.84 ± 0.11 billion cells/day, P = 0.5). In summary, FSH administration every 3.5 days from Day 62-170.5 resulted in an increase in FSH concentration beginning at 97 days of age and a hastened age of puberty. We propose this exogenous FSH delivered in hyaluronan initiates a positive feedback loop that includes an increase in activin A production observed on Day 86.5 and 107.5. However, differences in mature sperm production were not realized in this experiment.

Impact of a timed-release FSH treatment from 2 to 6 months of age in bulls I: Endocrine and testicular development of beef bulls.

In prepubertal males, FSH facilitates Sertoli cell proliferation and testis maturation. The study aimed to determine the effect of an exogenous FSH treatment on hormone secretion and testis development in Angus bulls. Bulls (n = 22) weaned at 53 ± 3.8 days of age were randomized into two treatment groups based on age and pedigree. Beginning at Day 59, bulls were injected im every 3.5 days with either 30 mg FSH (Folltropin-V; NIH-FSH-P1 units) in a 2% hyaluronan solution (FSH-HA, n = 11) or saline (control, n = 11) until Day 167.5. Blood samples to assess FSH, activin A, and testosterone were collected prior to each treatment. To determine how FSH profiles surrounding treatment were affected, three intensive blood sampling periods, each encompassing two treatment administrations, began at Day 66, 108, and 157, and blood was collected at 0, 6, 12, 18, 24, 36, 60, and 84 h respective to time of treatment. Scrotal circumference (SC) and BW were measured monthly. Bulls were castrated at Day 170 to measure testis size, seminiferous tubule diameter, and the number of Sertoli and germ cells per tubule cross-section. During intensive FSH sampling, FSH-HA bulls experienced an increase (P < 0.05) in FSH over control bulls for at least 18 h post-injection in all instances. In blood collected every 3.5 days, FSH concentrations in FSH-HA bulls were increased (P < 0.05) over initial Day 59 concentration from Day 97.5-167.5. FSH concentrations did not differ between treatments from Day 59-90.5, but were greater (P < 0.05) in FSH-HA from Day 94-167.5. Concentrations of activin A assessed for Day 59, 83.5, 94, 129, and 167.5 were greater (P < 0.05) in FSH-HA than control bulls on Day 83.5 and 94. The treatments did not differ (P > 0.1) in testosterone, BW, SC, testis size, tubule diameter, or number of germ cells per tubule. However, the number of Sertoli cells per tubule was greater in FSH-HA than control bulls (45.2 ± 1.4 vs. 41.6 ± 0.9 cells, P < 0.05). In summary, FSH-HA treatment every 3.5 days from Day 59-167.5 maintained elevated FSH for a minimum of 18 h post-injection, likely attributable to the addition of HA. We propose the exogenous FSH-HA treatment initiates a positive feedback loop that includes an increased density of Sertoli cells per tubule cross-section, which is related to increased activin A concentrations on Day 83.5 and 94. Furthermore, this activin A increase preceded an increase in endogenous FSH from Day 94-167.5 in FSH-HA bulls.

Role of progesterone concentrations during early follicular development in beef cattle: I. Characteristics of LH secretion and oocyte quality.

Objective was to investigate the effect of different progesterone (P4) concentrations during early follicular development on luteinizing hormone (LH) secretion and oocyte characteristics in beef cows. Primiparous cows (n = 24) were estrous pre-synchronized and follicular ablation was performed (d 0) 6 days following the time of ovulation. At the time of follicular ablation, cows were assigned to either: 1) high P4 treatment - HiP4; a new CIDR was inserted on d 0 to supplement P4 from the existing corpus luteum [CL], or 2) low P4 treatment - LoP4; a previously-used CIDR and two doses of PGF 8 to 12 h apart were given on d 0. Concentrations of P4 were greater (P < 0.01) in the cows of the HiP4 than LoP4 group on d 1.5, 2.5, and 3.5. Peripheral concentrations of E2 were greater (P < 0.05) in the cows of the LoP4 than HiP4 group on d 2.5 and 3.5. Frequency of LH pulses was greater (P <  0.05) in the LoP4 than HiP4 group on d 2.5, but mean LH concentration and pulse amplitude did not differ between treatments. Number of follicles aspirated per cow, total oocytes recovered, recovery rate, percentage of oocytes graded 1 to 3, oocyte diameter, percentage BCB+ oocytes, and relative abundance of oocyte mRNA for FST did not differ (P >  0.10) between treatments. In conclusion, lower P4 concentrations during early follicular development resulted in increased LH pulse frequency and E2 concentrations, but did not affect characteristics of oocyte developmental competence.

Role of progesterone concentrations during early follicular development in beef cattle: II. Ovulatory follicle growth and pregnancy rates.

Two experiments were conducted to investigate the role of relatively lesser and greater progesterone (P4) concentrations during early follicular development on ovulatory follicle growth and pregnancy rate in beef cattle. In Experiment 1, time of ovulation was synchronized with the 5 d CO-Synch + CIDR (Controlled Internal Drug Release) program in multiparous cows (n = 241). Six days after the 2nd GnRH injection of the pre-synchronization program (d 0), ablation of follicles ≥ 5 mm in the ovaries was performed and cows were assigned to receive either a previously used CIDR and 2x-25 mg PGF2α doses 8 h apart (LoP4), or a new CIDR (HiP4). On d 5, CIDR were removed from all cows, 2x-25 mg PGF2α were administered, and estrous detection tail paint was applied. Timed artificial insemination (TAI) was performed on d 8. On d 5, P4 concentrations were greater (P <  0.01) in the HiP4 (4.9 ± 0.13 ng/mL) than LoP4 (1.0 ± 0.06 ng/mL) treatment group. Conversely, d 5 estradiol (E2) concentrations and follicular diameter were greater (P <  0.01) in the LoP4 (5.0 ± 0.23 pg/mL and 8.9 ± 0.20 mm) than HiP4 (1.5 ± 0.12 pg/mL and 7.4 ± 0.15 mm) treatment group. Follicular diameter at TAI (12.0 ± 0.12 mm, Table 1) and TAI pregnancy rate did not differ (P >  0.10) between treatment groups. In Experiment 2, a new follicular wave was induced with estradiol benzoate on d -7, and cows (n = 275) were assigned on d 0 to receive 25 mg PGF2α and either have the CIDR replaced with a new CIDR (HiP4) or the used CIDR was left in place (LoP4).Furthermore, all cows received GnRH on d 0. The CIDRs were removed from all cows on d 5 and two doses of -25 mg PGF2α were administered. Estrous detection combined with AI 12 h later (Estrus-AI) was performed for 60 h after CIDR removal with TAI coupled with GnRH administration at 72 h if estrus was not detected. The concentrations of P4 on d 5 were greater (P <  0.01) in the HiP4 (2.8 ± 0.10 ng/ml) than LoP4 (1.7 ± 0.05 ng/mL) treatment group. For cows that were detected in estrus after PGF2α administration, estrous response (83.5%) and interval to estrus (55.0 ± 0.5 h) did not differ between treatment groups. Pregnancy rate (combined Estrus-AI and TAI) that resulted from breeding at the time of the synchronized time of estrus was similar between treatment groups (HiP4: 77.1%; LoP4: 82.3%). In conclusion, differences in P4 concentrations during early follicular development do not effect pregnancy rate in beef cows when the cows are inseminated at the time of a synchronized estrus if the cows have similar intervals of proestrus.

Human polyomavirus BKV transcriptionally activates DNA methyltransferase 1 through the pRb/E2F pathway.

Many DNA tumor virus oncogenes are capable of activating and highjacking the host cell's DNA replication machinery for its own reproduction purposes through targeting and inactivation of the retinoblastoma pocket protein family. Pocket proteins function to regulate cell cycle progression and DNA synthesis through inhibitory interactions with the E2F transcription factors. The interaction of viral oncogenes with the pocket proteins is crucial for their transforming activity. We recently demonstrated that the DNA methyltransferase 1 (DNMT1) gene is an E2F target gene that is transcriptionally activated in cells lacking the retinoblastoma gene (Rb-/-). Overexpression of DNMT1 is implicated in tumor suppressor gene hypermethylation which is associated with tumorigenesis. Given that viral oncogenes potently stimulate E2F activity, we hypothesized that viral infection might activate DNMT1 and thereby promote transformation. Herein, we demonstrate that DNMT1 is strongly activated by the human polyomavirus BKV large T antigen (TAg) and adenovirus E1a. Viral oncogene mutants incapable of binding the pocket proteins are ineffective at activating DNMT1 compared to their wild-type counterparts. Additionally, mutation of the E2F sites within the DNMT1 promoters dramatically abrogates transcriptional activation. These data suggest that viral induction of DNMT1 through modulation of the pRB/E2F pathway may be involved in viral transformation.

Developmental changes in the rat atriopeptin hormonal system.

We undertook a study of fetal synthesis, storage, and release of atriopeptin (AP). Plasma levels of both atriopeptin immunoreactivity (APir) and the NH2-terminal fragment of the prohormone immunoreactivity (NTFir) were very high in the fetus (4 and 20 times the maternal plasma, respectively). However, the atrial content of the AP was low, but surprisingly, ventricular content of AP was quite high (relative to the adult) in the fetus and fell postnatally. Atrial AP messenger RNA (mRNA) increased with postnatal age, whereas ventricular mRNA was extremely high in the fetus and fell rapidly after birth. High fetal plasma peptide levels may derive from the mother since infusion of exogenous atriopeptin 24 into the mother resulted in parallel increases in fetal and maternal peptide levels. Fetal plasma APir and NTFir levels partially reflect the markedly reduced total renal metabolic capacity compared with that of the adult. Plasma levels fell progressively after birth; whereas neonatal atrial content rose substantially. Plasma AP and NTF were simultaneously elevated in both the maternal and fetal circulation after vasopressin injection of the mother. The fetus can also respond to exogenous stimuli (vasopressin or indomethacin--presumably via ductal closure) and promptly release substantial amounts of peptide into its circulation. Thus, it appears that the AP hormonal system is functional during fetal life and responds avidly to increases in intracardiac pressure as does the mature animal.

Steroidogenic changes and steady state amount of messenger RNA encoding steroidogenic enzymes, gonadotropin receptors and cell-death signalling in the dominant ovarian follicle during estradiol-induced atresia in cattle.

Changes in steroidogenic function and associated gene expression were characterized in dominant ovarian follicles (DF) of cattle where follicles were induced to become atretic by systemic administration of estradiol benzoate (EB). In experiment 1, follicular fluid (FF) steroid concentrations in the DF were measured at 12-hourly time points for 48 h in heifers treated with 1 mg EB i.m./500 kg body weight (EB; n=20) as compared with untreated controls (C; n=19). Treatment with EB promoted a transient reduction in circulating FSH, a rapid (12 h) and sustained reduction in FF estradiol, a rapid (12 h) but transient reduction in FF progesterone and a delayed (36 h) increase in FF testosterone concentrations. In experiment 2, whole follicular wall tissue was collected from DF of mature non-lactating cows allocated to a 0 h control group (0 HC: n=7), a 24h control group (24 HC; n=7) or an EB-treated group where tissue was collected 24 h after administration of 1 mg EB i.m./500 kg body weight (EB; n=8). As for experiment 1, EB promoted a transient reduction in circulating FSH, a pronounced reduction in FF estradiol and a smaller but significant reduction in FF progesterone concentrations. Semi-quantitative RT-PCR on follicular wall tissue revealed that the loss in estrogen activity at 24 h after EB was associated with two-fold reduction in aromatase mRNA, with an apparent acceleration in loss of 17alpha-hydroxylase mRNA. Expression of genes for gonadotropin receptors (LHR and FSHR) and a cell-death signalling pathway (Fas antigen and Fas ligand) were unchanged during the initial 24h of EB-induced atresia. These results suggest that EB initiates atresia in dominant ovarian follicles through a rapid suppression of follicular estradiol synthesis, an effect associated with down-regulation of the aromatase gene. A transient suppression in circulating FSH following administration of EB appears to have initiated these events, and it is suggested that subsequent processes involved in atresia follow this loss in estrogenic function.

Impact of a timed-release follicle-stimulating hormone treatment from one to three months of age on endocrine and testicular development of prepubertal bulls.

In prepubertal bulls, FSH facilitates testis maturation and a transient proliferation of Sertoli cells. Two experiments examined the effects of exogenous FSH on hormone secretion and testis development in Angus bulls. Exogenous FSH treatment consisted of an intramuscular injection (i.m.) of 30 mg FSH (Folltropin-V) in a 2% hyaluronic acid solution (FSH-HA). In Exp. 1, bulls (50 ± 6.5 d of age) received either FSH-HA ( = 5) or saline (control; = 5) on d 50 and 53.5. Blood samples were collected via jugular venipuncture to assess FSH concentrations every 6 h for 24 h after treatment and every 12 h until 84 h. After each treatment, peripheral FSH concentrations were greater ( < 0.05) in the FSH-HA-treated bulls than in the control bulls 6 h after treatment and tended to be greater ( ≤ 0.08) 12 h after treatment. The FSH concentration from 18 to 84 h after treatment did not differ between treatments. In Exp. 2, bulls were treated with FSH-HA ( = 11) or saline (control; = 11) every 3.5 d from 35 to 91 ± 2 d of age. Blood samples were collected before each treatment to quantify FSH, testosterone, and activin A concentrations. Scrotal circumference (SC) and BW were measured weekly. Bulls were castrated at 93 ± 2 d of age. Seminiferous tubule diameter, testis composition, and the number of Sertoli cells per tubule cross section (GATA-4 positive staining) were determined from fixed and stained histological sections. Follicle-stimulating hormone concentrations within the FSH-HA-treated bulls increased ( < 0.05) on d 70 from prior sampling and remained elevated. The FSH concentration did not differ between treatments from 35 to 66.5 d of age but were greater ( < 0.05) in the FSH-HA-treated bulls than in the control bulls from 70 to 91 d of age. Serum concentration of activin A on d 35, 70, and 91 did not differ between treatments. The FSH-HA and control bulls did not differ ( > 0.1) in BW, SC, testis weight, testis volume, percent of parenchyma composed of tubules, tubule diameter, and concentration of testosterone. The number of Sertoli cells per tubule cross section was greater in the FSH-HA-treated bulls than in the control bulls (33.35 ± 0.9 vs. 28.27 ± 0.9 cells; ˂ 0.05). In summary, the FSH-HA treatment from 35 to 91 d of age resulted in increased endogenous FSH from 70 to 91 d and increased numbers of Sertoli cells at 93 d of age. Exogenous FSH altered endocrine mechanisms regulating endogenous FSH secretion and augmented Sertoli cell proliferation in young bulls, but this effect was apparently not caused by increased activin A concentration in the FSH-HA-treated bulls.

Fibulin-3 promotes muscle-invasive bladder cancer.

Urothelial carcinoma is the most common type of bladder cancer and can be categorized as either non-muscle-invasive (Ta-T1) or muscle-invasive (⩾T2). The majority of bladder cancers are non-muscle-invasive at presentation; however, the recurrence rate for these tumors is high and a subset can progress to T2. In this study, we aimed to identify genes differentially expressed between T1 vs T2 bladder cancer to identify key regulators of bladder cancer progression and/or invasion. We performed RNA-Seq on T1 and T2 bladder cancer tissues and used publicly available bladder cancer profiling studies to prioritize differentially expressed genes for validation and functional assessment. This integrative approach nominated an extracellular matrix glycoprotein, fibulin-3 (FBLN3, also known as EFEMP1), as being highly expressed in T2 vs T1 bladder cancer and aggressive vs indolent disease. We confirmed the overexpression of fibulin-3 in ⩾T2 vs non-muscle-invasive bladder cancer (NMIBC) by quantitative reverse transcriptase-PCR. Consistent with these findings, fibulin-3 expression level correlated with the invasive ability of several bladder cancer cell lines and modulation of fibulin-3 expression directly affected invasion. Fibulin-3 knockdown in bladder cancer cells decreased the incidence of MIBCs in a murine orthotopic bladder cancer model and decreased the expression of insulin-like growth factor-binding protein-5 (IGFBP5). Restoring IGFBP5 in these cells rescued their invasive and migratory potential. These results indicate that fibulin-3 serves as a pro-invasive factor in bladder cancer, which may be mediated through modulation of IGFBP5 expression. This also suggests fibulin-3 and IGFBP5 may have potential as biomarkers of aggressive bladder cancer or therapeutic targets.

Prostatic nerve growth factor inducible A gene binds a novel element in the retinoblastoma gene promoter.

We are investigating the role of the early response transcription factor, nerve growth factor inducible A gene (NGFI-A), as a modulator of retinoblastoma (RB) gene transcription in prostate cells. Examination of the RB promoter reveals a novel element GCGGGGGAG located at nucleotides 152-144 upstream of the methionine initiation codon. This sequence shares strong homology with the consensus NGFI-A binding element GCGGGGGCG varying by a single nucleotide. In DNA binding assays, an NGFI-A fusion protein and the native protein product of the NGFI-A gene purified from prostate cancer cells bound specifically to an oligonucleotide containing the RB promoter element. Gene expression studies in rat ventral prostate demonstrated a 1.9-fold increase in RB mRNA following castration that parallels a 2.7-fold induction of NGFI-A mRNA. In summary, the in vitro DNA binding data and the transient coregulation of rat NGFI-A and RB following castration suggests that the RB gene may be transcriptionally regulated by NGFI-A in prostate cells.

Overexpression of bcl-2 protects prostate cancer cells from apoptosis in vitro and confers resistance to androgen depletion in vivo.

Normal (nonneoplastic) human prostatic secretory epithelial cells do not express the bcl-2 protein. However, a recent immunohistochemical survey of neoplastic human prostate tissues showed that a fraction of primary untreated prostate adenocarcinoma cells expressed this apoptosis-suppressing oncoprotein at significant levels (Colombel et al., Am. J. Pathol., 143: 390-400, 1993). Additionally, a number of hormone-refractory prostatic adenocarcinomas obtained from hormonally-treated patients (subsequent to surgical or drug castration therapy) were examined and were found to be uniform in their elevated expression of bcl-2 oncoprotein. The results of this preliminary survey imply that bcl-2 expression distinguishes a subgroup of primary human prostate cancers and that the expression of this protein might be a factor enabling prostate cancer cells to survive in an androgen-deprived environment. The current study was undertaken to determine the degree to which overexpression of bcl-2 can protect human prostate cancer cells from apoptotic stimuli in vitro and in vivo. Human prostate cancer cells (LNCaP) were transfected with a neomycin-selectable eucaryotic expression vector containing cDNA encoding human bcl-2. Transfected clonal variants that express bcl-2 protein (LNCaP/bcl-2) were unaltered with regard to their basal growth rate in 10% serum-containing medium, or with regard to their expression of the differentiated human prostate cell gene products prostate-specific antigen or androgen receptor protein. The bcl-2-transfected clones were altered, however, with regard to their growth rate in charcoal-stripped serum lacking dihydrotestosterone. Additionally, in contrast to the parental or control-transfected cell lines, LNCaP/bcl-2 cells were highly resistant to a variety of apoptotic stimuli in vitro including serum starvation and 10 nM phorbol ester (phorbol 12-myristate 13-acetate) supplementation of the medium. Lastly, the overexpression of bcl-2 by these prostate cancer cells altered their tumorigenic potential in a nude mouse assay. s.c. injections of 10(6) LNCaP/bcl-2 cells into male nude mice resulted in earlier and larger tumor formation compared to an equivalent injection of parental or control-transfected LNCaP cells. When these variant cell lines were injected into castrated male nude mice, only the LNCaP/bcl-2-transformed cells gave rise to tumors. Moreover, LNCaP/bcl-2 tumors grown in intact male nude mice were refractory to the growth-inhibiting effects of castration demonstrated by parental LNCaP cells. Data obtained in this study demonstrate that the bcl-2 oncoprotein can protect prostate cancer cells from apoptotic stimuli in vitro and suggest that such protection correlates with the ability to form hormone-refractory prostate tumors in vivo.

Paclitaxel-associated multimininucleation is permitted by the inhibition of caspase activation: a potential early step in drug resistance.

The chemotherapeutic agent paclitaxel disrupts microtubule dynamics causing mitotic arrest, which leads to cell death. However, in paclitaxel-resistant tumor cells, treatment with paclitaxel induces abnormal progression through prophase resulting in a multimininucleated phenotype. Multimininucleation and subsequent polyploidization have been correlated with paclitaxel resistance. Paclitaxel treatment of HeLa cells resulted in cell death via typical activation of the apoptotic machinery, whereas treatment of the relative paclitaxel-resistant prostate cancer cell line PC-3 induced an attenuated caspase activation and multimininucleation. The multimininucleated phenotype could be mimicked in HeLa cells treated with paclitaxel and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), a peptide caspase inhibitor. Interestingly, we observed no discernible difference in the pattern of cdc-2 kinase activation or phosphorylation of bcl-2-like proteins in PC-3 and HeLa cells treated with paclitaxel, which demonstrated that these molecules could not be used as indicators for the degree of caspase activation. In this study, we establish a connection between relative paclitaxel resistance, caspase attenuation/inhibition, and the multimininucleated phenotype.

Posttranslational truncation and inactivation of human E-cadherin distinguishes prostate cancer from matched normal prostate.

An essential event in the progression of adenocarcinoma is the loss of organized epithelial attachment (both to the basement membrane and to adjoining epithelial cells). The E-cadherin cell adhesion molecule has an established function in maintaining normal phenotype and tissue homeostasis, and loss of E-cadherin function has been implicated in tumorigenesis. Aberrations in E-cadherin are associated with prostate cancer progression; however, these aberrations are not simply a result of prodigious allelic loss. We have previously demonstrated a novel posttranslational truncation within the cytosolic domain of native Mr 120,000 E-cadherin to a membrane-bound Mr 97,000 species. We hypothesize that truncation of E-cadherin is an inactivating event that is significantly increased in localized prostate tumors and that it represents a novel molecular event that may distinguish prostate cancer from adjacent normal tissue. E-cadherin was characterized by Western blot analysis in matched normal and cancer tissue from 18 prostate cancer patients. Imaging and densitometry software were used to quantify the truncation of E-cadherin by measuring the ratio of Mr 97,000 E-cadherin to Mr 120,000 E-cadherin, which was significantly increased in the tumor aspect of the prostate gland. Herein, we report the first experiment comparing case-matched human normal and cancerous prostate tissue in the context of E-cadherin truncation.

Activated in prostate cancer: a PDZ domain-containing protein highly expressed in human primary prostate tumors.

Critical events in prostate tumorigenesis and metastasis likely include the abnormal activation and expression of specific genes. Using RNA expression profiling techniques, we have identified a transcript originating from the activated in prostate cancer (AIPC) gene, the expression of which is preferentially up-regulated in several cultured prostate tumor cell lines and human primary prostate tumors. Sequence analysis revealed that the AIPC protein encodes six PDZ domains, which are protein-protein binding domains likely involved in protein clustering and scaffolding. Immunohistochemical analysis of a tissue microarray comprising 158 tumor, 18 high-grade prostatic intraepithelial neoplasia, and 91 normal prostate specimens with an anti-AIPC antibody demonstrated abundant AIPC protein expression in 75% of tumors, 83% of prostatic intraepithelial neoplasia lesions, and 3% of normal tissues (P < 0.0001). These data suggest that the accumulation of AIPC protein may be closely associated with the initiation or early promotion of prostate tumorigenesis.

Sex hormone-induced carcinogenesis in Rb-deficient prostate tissue.

The retinoblastoma (Rb) gene product is a prototypic tumor suppressor. Mice lacking the Rb gene are not viable and die in utero at approximately 13 days of gestation. In this study, we have rescued Rb-/- prostates by grafting pelvic organ rudiments from Rb-/- mouse embryos under the renal capsule of adult male nude mouse hosts. Grafts of embryonic pelvic organs developed into functional prostatic tissue. Some of the prostatic tissue generated was further used to construct chimeric prostatic tissue recombinants by combining wild-type rat urogenital mesenchyme (rUGM) with Rb-/- and Rb+/+ prostatic epithelium (PRE). The tissue recombinants were grown as subcapsular renal grafts and treated from the time of grafting with Silastic capsules containing 25 mg of testosterone plus 2.5 mg of estradiol. During 5-8 weeks of hormone treatment, rUGM+Rb+/+PRE tissue recombinants developed prostatic hyperplasia, whereas PRE in rUGM+Rb-/-PRE tissue recombinants developed hyperplasia, atypical hyperplasia, and carcinoma. During carcinogenesis in rUGM+Rb-/-PRE tissue recombinants, prostatic epithelial cells of the basal lineage disappeared, whereas the luminal cells underwent carcinogenesis. Epithelial E-cadherin almost totally disappeared. In all cases, epithelial PCNA labeling was elevated in tissue recombinants containing Rb-/- versus Rb+/+ epithelium. These epithelial changes were associated with almost total loss of smooth muscle cells in the stroma. In contrast, in untreated hosts rUGM+Rb+/+PRE tissue recombinants developed normally, and rUGM+Rb-/-PRE tissue recombinants developed mild epithelial hyperplasia. The results of this study demonstrate that Rb-/- prostatic tissue can be rescued from embryonic lethal mice and used to test its susceptibility to hormonal carcinogenesis. Deletion of the Rb gene predisposes prostatic epithelium to hyperplasia and increases proliferative activity Susceptibility to hormonal carcinogenesis in response to exogenous testosterone + estradiol is manifested in the progression from atypica hyperplasia to carcinoma. Thus, these findings demonstrate that the absence of the Rb tumor suppressor gene may predispose prostatic epithelial cells to carcinogenesis. Rescue of organs from Rb-/- embryos not only provides an opportunity to analyze the Rb gene pathway in the development and progression of prostate cancer but also provides an opportunity for specifically evaluating the role of the Rb pathway in development and carcinogenesis in other organs, such as the mammary gland and colon. Because rUGM greatly stimulates prostatic epithelial proliferation, the tissue recombinant model is a particularly useful tool for assessing the functional role of other genes in prostatic carcinogenesis through use of the appropriate transgenic or gene knockout mice.

Effects of dietary energy on sexual maturation and sperm production in Holstein bulls.

In prepubertal bulls and heifers of dairy and beef breeds, puberty can be induced to occur earlier than typical with targeted high-energy diets due to precocious activation of the endocrine mechanisms that regulate puberty. Precocious activation of puberty in bulls intended for use in the AI industry has the potential to hasten and perhaps increase sperm production. It was hypothesized that feeding bulls a high-energy diet beginning at 8 wk of age would advance the prepubertal rise in LH and lead to advanced testicular maturation and age at puberty. From 58 to 230 ± 0.3 d of age, Holstein bulls received either a high-energy diet (HE;n = 9; targeted ADG 1.5 kg/d) or a control diet (CONT;n = 10; targeted ADG 0.75 kg/d). Thereafter, all bulls were fed a similar diet. The HE treatment increased LH secretion at 125 d of age, testosterone concentrations from 181 to 210 d, and scrotal circumference (SC) from 146 to 360 d of age relative to the CONT treatment. Beginning at 241 ± 5 d of age, semen collection (artificial vagina) was attempted every 14 d in bulls from the HE (n = 8) and CONT (n = 7) treatment until each bull attained puberty (ejaculate containing 50 × 10 spermatozoa with 10% motility). To assess semen production as mature bulls, semen was collected thrice weekly beginning at 541 ± 5 d of age until slaughter at 569 ± 5 d of age. After slaughter, epididymal and testicular measurements were collected and testicular tissue was fixed to determine seminiferous tubule diameter. Age at puberty did not differ between treatments (310 ± 35 d). Although testis and epididymal weight and testis volume were greater (P < 0.05) in the HE than the CONT treatment, sperm production of mature bulls did not differ between treatments. Diameter of seminiferous tubules also did not differ between treatments. We conclude that the HE advanced aspects of sexual maturation and increased testes size, but this was not reflected in hastened puberty or sperm production in the present experiment.