Toshihiko Tsujii

Evidence That Pituitary Adenylate Cyclase Activating Polypeptide Suppresses Follicle-Stimulating Hormone-β Messenger Ribonucleic Acid Levels by Stimulating Follistatin Gene Transcription1

There is accumulating evidence to suggest that pituitary adenylate cyclase-activating polypeptide (PACAP) may be an important modulator ofgonadotrope function. One of the actions of PACAP identified previously is to decrease FSHbeta messenger RNA (mRNA) levels. In the present series of experiments we demonstrate that PACAP-induced suppression of FSHbeta mRNA correlates with a rise in follistatin mRNA levels in primary pituitary cell cultures. Transient transfection of gonadotrope-derived alphaT3-1 cells with a rat follistatin promoter-luciferase reporter plasmid reveals that PACAP stimulates follistatin gene transcription. PACAP stimulation of LUC activity was maximal at concentrations as low at 1 nM. Furthermore, in alphaT3-1 cells PACAP activation of the follistatin promoter appears to be via the cAMP-dependent protein kinase A pathway. Accordingly, we propose that PACAP stimulates follistatin transcription, which neutralizes activin activity and thereby reduces FSHbeta mRNA. Since PACAP and follistatin are colocalized in multiple tissues including the brain, adrenals, and gonads, our findings may reflect a broadly distributed autocrine/paracrine mechanism for modification of activin effects that is under PACAP control.

Effects of pulsatile pituitary adenylate cyclase activating polypeptide (PACAP) on gonadotropin secretion and subunit mRNA levels in perifused rat pituitary cells

The effects of pituitary adenylate cyclase activating polypeptide (PACAP) administered intermittently on gonadotropin secretion and subunit mRNA levels in anterior pituitary cells from 7wk old intact and orchidectomized rats were studied. Cells were perifused for 9 h with hourly pulses of 2.5 nM GnRH or 10 nM PACAP, or with medium alone. Pulsatile PACAP initiated episodes of LH, FSH and alpha-subunit secretion, but was much less effective than GnRH. Responsiveness declined with repeated pulses of PACAP, although interpulse secretion increased gradually throughout the experiment. PACAP was a slightly more effective stimulator of LH release by pituitary cells from castrated than intact rats. At the completion of perifusion, pituitary RNA from castrated rats was extracted for measurement of gonadotropin subunit mRNA levels. Pulsatile PACAP stimulated alpha-subunit and LHbeta mRNA levels but did not affect FSHbeta mRNA. By contrast, continuous PACAP increased alpha-subunit mRNA levels, but suppressed FSHbeta mRNA without affecting LHbeta mRNA. We conclude that pulsatile PACAP is a relatively ineffective stimulator of gonadotropin secretion when administered alone, and regulates gonadotopin subunit gene expression quite differently than continuous PACAP. We propose that the mode of PACAP secretion in vivo may be a determinant of the differential expression of the gonadotropin subunit genes.

Glucocorticoid repression of gonadotropin-releasing hormone gene expression and secretion in morphologically distinct subpopulations of GT1-7 cells

Two morphologically distinct subpopulations of GT1-7 cells have been characterized and examined for their responsiveness to glucocorticoids. Type I cells have a neuronal phenotype, extending many lengthy processes, and express neuronal, but not glial, markers. Type II cells show weaker or negative immunostaining for neuronal markers and exhibit fewer processes. The effect of glucocorticoids on gonadotropin-releasing hormone (GnRH) secretion and gene expression was compared in type I and type II GT1-7 cells. For secretion studies, cells were attached to Cytodex beads and perifused with control medium or medium containing dexamethasone (dex). The high level of GnRH secreted by type I cells was slightly enhanced in the presence of dex, whereas dex rapidly and profoundly decreased the already low level of GnRH secreted by type II cells. Immunocytochemistry for GnRH showed dark reaction product in the cell bodies and processes of type I cells and little or no immunoreactivity in type II cells. Both the endogenous mouse GnRH mRNA and the transcriptional activity of a mouse GnRH promoter luciferase reporter gene plasmid were suppressed to a greater extent in type II cells than in type I. In electrophoretic mobility shift assays, there was no difference between type I and type II nuclear extracts in the pattern of protein-DNA complexes formed on two previously identified negative glucocorticoid response elements located at -237 to -201 and -184 to -150 bp of the mouse promoter. Both cell types contained glucocorticoid receptors (GR) by Western blot analysis. Cytosols from type I or type II cells were incubated with [3H]dex to obtain GR binding parameters. Binding data were consistent with a one-site model for dex binding in each case. Small differences in Kd (1.7 nM, type I; 3.1 nM, type II) or Bmax (approximately 3600 sites/cell, type I; approximately 1800 sites/cell, type II) were not likely to account for the differential sensitivity to dex treatment. In conclusion, nuclear alterations in type II cells leading to greater transcriptional susceptibility to dex, coupled with low GnRH storage levels, may be reflected in exquisite sensitivity of GnRH secretion to glucocorticoid repression. This represents the first example of a steroid hormone acting directly on GnRH-producing cells to alter GnRH secretion.

Regulation of α-subunit mRNA transcripts by pituitary adenylate cyclase-activating polypeptide (PACAP) in pituitary cell cultures and α T3-1 cells

Pituitary adenylate cyclase activating polypeptide (PACAP) increases glycoprotein hormone α-subunit mRNA levels suggesting a role for PACAP in maintaining the high levels of α-subunit protein characteristic of the pituitary. The present study used primary pituitary cell cultures and the α T3-1 pituitary cell line to investigate how PACAP affects α-subunit mRNA transcripts. Stimulation of cultured pituitary cells with 10 nM PACAP38, 10 nM GnRH, or the combination, for 24 h increased α-subunit mRNA levels 1.5-fold, whereas GnRH more effectively (P < 0.01) stimulated α-subunit protein release than did PACAP38 (3.2- vs. 2.0-fold). α-Subunit mRNA levels in α T3-1 cells were also increased by PACAP38 and by GnRH to maximum values at 12 h (P < 0.05), and α-subunit protein secretion rose proportionately and in parallel with α-subunit MRNA levels. PACAP38 was a 100-fold more potent stimulator of α-subunit mRNA than was VIP, and a VIP-antagonist failed to block the stimulatory effect of PACAP38, suggesting an effect via type PACAP 1 receptors. Type I receptor mRNA transcripts were identified by Northern analysis in α T3-1 cells. Depletion of PKC activity by PMA failed to block the stimulatory effect of PACAP38, but prevented GnRH from increasing α-subunit mRNA levels and α-subunit secretion. PACAP38, like 8Br-cAMP and forskolin, stimulated (P < 0.05) luciferase (LUC) activity in α T3-1 cells transfected with a plasmid containing the first 846 or 180 base pairs of the 5′-flanking region of the human α-subunit gene linked upstream to a LUC reporter gene. Finally, experiments using the transcription inhibitor DRB reveal that PACAP does not appreciably change α-subunit mRNA half-life. These findings are consistent with the proposal that PACAP contributes to the high levels of α-subunit protein characteristic of the pituitary by activating Type I receptors and stimulating α-subunit gene transcription in part by the cAMP/PKA pathway.

Young Age as Favorable Prognostic Factor for Cancer-specific Survival in Localized Renal Cell Carcinoma

OBJECTIVES To evaluate the prognostic effect of age in patients with localized renal cell carcinoma (RCC) and investigate the incidence of Xp11 translocation RCC in young patients who developed recurrence. METHODS From 1990 to 2007, 2403 Japanese patients underwent nephrectomy for presumed RCC at 9 institutions. Of those, 1143 patients had localized RCC (Stage pT1-2N0M0). Their clinical data were retrospectively reviewed. In the present study, 131 patients (11%) were considered young (≤45 years at diagnosis). In the young patients with recurrence, the nephrectomy specimens were immunostained with TFE3 to determine the incidence of Xp11 translocation RCC. RESULTS During the median follow-up of 47 months, 3 cancer deaths (2.2%) occurred among young patients and 51 (5.0%) among older patients. The 5-year cancer-specific survival (CSS) rate was significantly better for the younger patients than for the older patients (P = .049). Multivariate analysis showed that age was significantly associated with CSS, as were the pathologic T stage, tumor grade, and symptoms at diagnosis. The hazard ratio of young age was 0.31 (95% confidence interval 0.077-0.87). The recurrence-free survival curves revealed no difference between these 2 groups. Of the 74 patients with recurrence, the CSS after recurrence was significantly better in the younger patients than in the older patients (P = .0010). Of the 8 young patients with recurrence, 4 had Xp11 translocation RCC, and 3 survived for >5 years after recurrence. CONCLUSIONS Compared with the older patients, the young patients with RCC had similar recurrence-free survival rates but better CSS rates. This might have been because significant numbers of the young patients had Xp11 translocation RCC.

Equivalent survival and improved preservation of renal function after distal ureterectomy compared with nephroureterectomy in patients with urothelial carcinoma of the distal ureter: A propensity score-matched multicenter study

To investigate the oncological and functional outcome of distal ureterectomy compared with nephroureterectomy in the management of distal ureteral urothelial carcinoma.

Ureteral Involvement Is Associated with Poor Prognosis in Upper Urinary Tract Urothelial Carcinoma Patients Treated by Nephroureterectomy: A Multicenter Database Study

BACKGROUND The prognostic significance of tumor location for patients with upper urinary tract urothelial carcinoma (UUT-UC) has been disputed. Several papers have reported that ureteral cancer is associated with worse prognosis. OBJECTIVE To investigate the prognostic significance of the presence of ureteral tumors in UUT-UC patients who underwent radical nephroureterectomy (RNU). DESIGN, SETTING, AND PARTICIPANTS In this multicenter retrospective study, 1068 eligible patients (median follow-up: 40 mo [interquartile range: 17-77 mo]) were divided into three groups based on tumor location: renal pelvic, ureteral, and both-regional (having both renal pelvic and ureteral tumors). The ureteral and both-regional groups were subsequently integrated into the ureteral involvement group to evaluate its prognostic impact. INTERVENTION All patients underwent RNU. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS The prognostic impact of tumor location on survival was analyzed. RESULTS AND LIMITATIONS The renal pelvic, ureteral, and both-regional groups consisted of 507 (47.5%), 430 (40.3%), and 131 (12.3%) patients, respectively. The ureteral and both-regional groups had a higher rate of lymphovascular invasion and lymph node metastasis compared with the renal pelvic group. The renal pelvic and both-regional tumors presented more frequently with locally advanced stages (pT3/T4) compared with the ureteral tumors. The 5-yr cancer-specific survival (CSS) and progression-free survival (PFS) rates of patients in the ureteral (70.5% and 66.7%, respectively) and both-regional groups (64.8% and 57.8%, respectively) were significantly worse than those in the renal pelvic group (81.9% and 78.1%, respectively). In a multivariate analysis, the presence of ureteral involvement was a significant prognostic factor for CSS (hazard ratio [HR]: 1.50; p=0.006) and PFS (HR: 1.35; p=0.023). This study is inherently limited by the biases associated with its retrospective and multicenter design. CONCLUSIONS The presence of ureteral involvement had a significant impact on the survival of surgically treated UUT-UC patients associated with a poor prognosis. PATIENT SUMMARY We demonstrated that the ureteral involvement was associated with poor survival compared with patients with renal pelvic tumor only in upper urinary tract urothelial patients treated by nephroureterectomy.

MP72-14 IMPACT OF CHRONIC KIDNEY DISEASE AND HYPERTENSION ON DECLINE IN REMAINING KIDNEY FUNCTION AFTER RADICAL NEPHRECTOMY

1 versus>1). Recommended surveillance schedules balance estimated riskof non-cancerdeathwith recurrence riskwhereallowable recurrence is up to the risk of non-cancer death. Surveillance intervals were calculated based on each 1%, 3%, and 5% recurrence risk increase to amaximum10 years. Intervals shorter than 3 months were not allowed, with the next risk increase calculated from the date of the adjusted interval. RESULTS: AFT models for recurrence and death for stage, age, and comorbidity risk groups were generated (tables). Recommended interval schedules (e.g. 1%, 3%, or 5%) are based on the predicted risk of non-cancer death for a given age and CCI group. For example, patients <70 years with a CCI score 1 would be followed on a 1% risk of recurrence schedule for the duration of 10 years of followup regardless of stage. Conversely, patients 60-69 years old with a CCI >1, would follow a 1% interval for the first year only, then switch to 3% interval until year 6, at which point they would follow a 5% schedule. CONCLUSIONS: Using an AFT model for estimating risk of recurrence and risk of death we were able to generate risk-adapted screening intervals for surgical managed patients with RCC.

687 BIMODAL PATTERN OF THE IMPACT OF BODY MASS INDEX ON CANCER-SPECIFIC SURVIVAL OF UPPER URINARY TRACT UROTHELIAL CARCINOMA PATIENTS: MULTI-CENTER STUDY IN A 1114-CASE TOKYO METROPOLITAN DATABASE OF UROLOGIC DISEASE (TMDU) COHORT

UTUC database and the UTUC collaboration (n 4137). After excluding the nine patients who we previously reported on [5], 28 patients (0.7%) with pT0 tumor were included in this report. All patients had undergone cystoscopy, urine cytology, intravenous urography and/or abdominalpelvic CT-scans, and diagnostic ureteroscopy prior to RNU. None of the patients underwent preoperative radiotherapy and none of the patients had metastasis at time of RNU. Due to the low number of patients, only descriptive results are shown. RESULTS: The median age at diagnosis was 68 years (IQR: 57-74) and the male-to-female ratio was 2:1. Six patients had evidence of non-organ confined UTUC on pre-RNU imaging and received neoadjuvant cisplatin-based combination chemotherapy. Regional lymphadenectomy was performed in the six patients who had received neoadjuvant chemotherapy (median: 10 lymph nodes, IQR 6-14). On final pathology, all patients had pT0N0 except one patient who had two positive renal hilar lymph nodes (pT0 N1). Overall, 4 patients experienced disease recurrence (median time to recurrence : 38 months, IQR: 24-48). Three of these patients developed metastatic disease and one had a relapse in the operative field. The three patients with metastasis died from UTUC within a median time of 10 months from their disease recurrence. Within a median follow-up of 40 months (IQR: 22-64), nine patients developped intravesical recurrence (median time: 35 months, IQR: 14-54). The 5-year recurrence-free and cancer-specific survival rates were 77% (95% CI, 72-81.3) and 78% (95% CI, 69-83.8), respectively. CONCLUSIONS: pT0 UTUC may be the result from effective neoadjuvant chemotherapy, complete endoscopic treatment, misinterpretation of the pathologic specimen (i.e, false pT0), or overtreatment. Our limited data shows that post-RNU prognosis of pT0 patients is highly variable as some patients experience disease progression needing more stringent follow-up.