Hidenori Kawashima

A novel steroid receptor co-activator protein (SRAP) as an alternative form of steroid receptor RNA-activator gene: expression in prostate cancer cells and enhancement of androgen receptor activity

We have cloned a cDNA coding for a novel steroid receptor co-activator protein termed SRAP from a rat prostate library. Although the nucleotide sequence of the SRAP has 78.2% identity to that of the human steroid receptor RNA activator (SRA), a novel RNA molecule which was reported to act as an RNA transcript without being translated into protein [Lanz, McKenna, Onate, Albrecht, Wong, Tsai, Tsai and OMalley (1999) Cell 97, 17-27], the cDNA of SRAP is capable of generating a functional protein. Glutathione S-transferase pull-down assays showed that SRAP associates with the partial androgen receptor (AR) protein composed of a DNA-binding domain and an activation function 2. Luciferase assays demonstrated that SRAP enhances the transactivation activity of the AR, the glucocorticoid receptor and the peroxisome proliferator-activated receptor gamma(1) in a ligand-dependent manner. Using a green fluorescent protein (GFP) fusion-protein construct, we demonstrated in vivo translation of the GFP-SRAP fusion protein in HeLa cells co-transfected with pSG5AR and reporter gene in the presence of 5 alpha-dihydrotestosterone (DHT). Co-transfection of the GFP-SRAP fusion protein expression plasmid enhanced the transactivation activity of AR whereas incorporation of mutations in SRAP of the fusion protein resulted in loss of enhancement of the transactivation activity. Northern blot analysis and reverse transcriptase PCR assays showed that SRAP and SRA are expressed in rat and human prostate cancer cell lines respectively. In HeLa cells and the human prostate cancer cells line DU-145, co-transfected with SRAP, the DHT-dependent transactivation activities of AR were not completely inhibited by the anti-androgen flutamide, but the transactivation activities still remained high even in the presence of 5 microM flutamide, suggesting that SRAP may play an important role in enhancing AR activity in prostate cancer.

“Decoy” of androgen‐responsive element induces apoptosis in LNCaP cells

In an androgen‐dependent manner, the androgen receptor (AR) binds to the androgen‐responsive element (ARE) in the regulatory region of target genes. We hypothesize that an “ARE decoy,” a double‐stranded oligonucleotide containing the same DNA sequence as ARE, can inhibit prostatic proliferation by competitive inhibition of AR transcriptional activity.

Purification and NH2-terminal amino acid sequences of human and rat kidney fatty acid ω-hydroxylases

A cytochrome P-450 (P-450), designated P-450HK omega, has been isolated and purified from human kidney microsomes to a specific content of 13 nmoles of P-450/mg of protein. P-450HK omega showed an apparent molecular weight of 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Absolute spectra of the oxidized form indicated that this P-450 was largely in the low-spin state and partly in the high-spin state. It catalyzed the omega- and (omega-1)-hydroxylation of fatty acids such as laurate, myristate, and palmitate, with no activity toward prostaglandin A1, benzphetamine, 7-ethoxycoumarin, or 7-ethoxyresorufin. The first 35 NH2-terminal amino acid sequence of P-450HK omega had about 70% homology with those of rabbit kidney fatty acid omega-hydroxylases of the P-450 IVA gene subfamily, P-450ka-1, P-450ka-2, and P-450kd, except for four undetermined residues. Moreover, Western blot and immuno-inhibition studies showed that P-450HK omega reacted with an antibody against the rabbit kidney fatty acid omega-hydroxylase. The results suggest that P-450HK omega is a member of the same P-450 gene family (IVA subfamily) as the rabbit enzymes. In addition, the terminal sequence of P-450HK omega also showed 54% homology with that of P-450k-2, a fatty acid omega-hydroxylase from rat kidney microsomes. To our knowledge, this is the first time that a P-450 specific for fatty acid omega-hydroxylase activity has been isolated to homogeneity from human tissues.

A novel approach to successful ABO-incompatible high-titer renal transplantation.

BACKGROUNDnCurrently the long-term outcome among recipients of ABO-incompatible renal transplantations is excellent in Japan. However, previous reports have documented poor outcomes in patients with high (> 1:256) anti-A/B antibody titers pretreatment. The immunosuppressive protocol for ABO-incompatible high-titer renal transplantation has remained a medical challenge.nnnMETHODSnWe treated 3 patients with high (> 1:512) anti-A/B antibody titers prior to ABO-incompatible renal transplantation. Our immunosuppressive protocol was initiated 1 month prior to surgery and included mycophenolate mofetil (1 g/d) and low-dose steroid (methylprednisolone [8 mg/d]). Two doses of the anti-CD20 antibody rituximab, (150 mg/m2) were administered 2 weeks before and on the day of transplantation. We performed antibody removal with 6 to 8 sessions of plasmapheresis (plasma exchange or double-filtration plasmapheresis) before transplantation. Splenectomy was also performed on the day of transplantation. Postoperative immunosuppression followed the same regimen as ABO-compatible cases, in which calcineurin inhibitors were initiated 3 days before transplantation combined with 2 doses of basiliximab.nnnRESULTnWith this protocol, the anti-A/B antibody was reduced to below 1:16 in all cases. All 3 patients underwent successful transplantation with a mean current serum creatinine of 1.32 mg/dL (range, 1.22-1.50 mg/dL). There were no episodes of antibody-mediated rejection. No serious complications or side effects were encountered.nnnCONCLUSIONSnA preconditioning protocol consisting of rituximab infusions, splenectomy, plasmapheresis, and pharmacologic immunosuppression enabled ABO-incompatible renal transplantation in patients with high (> 1:512) anti-A/B antibody titer.

Involvement of estrogen receptors in prostatic diseases.

Accumulating evidence shows that estrogens participate in the pathogenesis and development of benign prostatic hyperplasia and prostate cancer by activating estrogen receptoru2003α. In contrast, estrogen receptoru2003β is involved in the differentiation and maturation of prostatic epithelial cells, and thus possesses antitumor effects in prostate cancer. However, the natural ligands of estrogen receptoru2003β are not fully understood, and its mode of action according to its ligands and the binding sites located in the promoter regions of downstream genes remains to be elucidated. Here, we review recent experimental investigations of estrogen receptors and their urological relevance. Estrogen receptor‐mediated signaling in the prostate is essential together with the androgen receptor‐mediated pathway, providing a new therapeutic target for prostatic diseases.

Hyperbaric oxygen therapy for painful bladder syndrome/interstitial cystitis resistant to conventional treatments: long-term results of a case series in Japan

BackgroundThere is no confirmed strategy for treating painful bladder syndrome/interstitial cystitis (PBS/IC) with unclear etiology. Therefore, a pilot study was carried out to evaluate the efficacy and safety of hyperbaric oxygen (HBO) therapy in treatment-resistant PBS/IC patients.MethodsHBO treatment (2.0 ATA for 60 minutes/day × 5 days/week for 2 or 4 weeks) was performed on 11 patients with severe symptoms that had not been improved by previous therapy regimens between December 2004 and July 2009.ResultsSeven of the 11 patients demonstrated persistent improvement in symptoms during the 12 months after HBO treatment. These responders demonstrated a decrease in the pelvic pain scale and urgency scale from 7.7 ± 1.0 and, 6.6 ± 0.9 to 3.4 ± 2.5 and 4.3 ± 2.4 after 12 months, respectively (p < 0.05). The total score of the interstitial cystitis symptom index and 24-hour urinary frequency demonstrated a significant sustained decrease from the baseline. Two responders, who received an additional course of HBO 12 and 13 months after initial treatment, respectively, did not suffer impairment for more than two years. There was one case of transient eustachian tube dysfunction and three cases of reversible exudative otitis media as a consequence of HBO treatment.ConclusionsHBO is a potent treatment for PBS/IC patients resistant to conventional therapy. It was well tolerated and provided maintained amelioration of pain, urgency and urinary frequency for at least 12 months.

Effect of anti-estrogens on the androgen receptor activity and cell proliferation in prostate cancer cells.

Although some anti-estrogens have been reported to inhibit the proliferation of prostate cancer cells, few studies on the mechanism by which they suppress the growth of prostate cancer have been reported. We investigated, for the first time, whether anti-estrogens modulate the transactivation activity of the androgen receptor (AR) in prostate cancer cells. In DU-145 cells transfected with AR, the transactivation activity of AR was inhibited by tamoxifen and toremifene, even in the presence of 10xa0nM of DHT. On the other hand, in LNCaP cells having an endogenous AR mutation at codon 877, the activity of AR was suppressed by faslodex in the presence of 10xa0nM DHT, whereas it was not inhibited by tamoxifen nor toremifene. In PC-3 cells, both the cell growth and the AR activity were remarkably inhibited by tamoxifen at 50xa0μM. Faslodex and toremifene inhibited AR activity to some extent, but they seemed to function as agonists at higher concentrations. In PC-3 cells, the inhibition of cell growth by flutamide, faslodex and toremifene was much less than their suppression of AR activity. We also demonstrated that a synthetic estrogen diethylstilbestrol and progesterone-related drugs such as chlormadinone acetate and allylestrenol dose-dependently inhibited the activity of AR in DU-145 and PC-3 cells. These results highlight the anti-androgenic aspect of anti-estrogens and estrogens in regard to the AR-mediated transcription of the relevant genes in prostate cancer.

NEDD9 crucially regulates TGF-β-triggered epithelial-mesenchymal transition and cell invasion in prostate cancer cells: involvement in cancer progressiveness.

NEDD9 is one of the Crk‐associated substrate (Cas) family proteins that mediate downstream signaling processes including cytoskeletal organization, cell‐cycle and tumorigenesis. While NEDD9 plays a crucial role in epithelial–mesenchymal transition (EMT), the functional mechanism underlying NEDD9‐mediated EMT in prostate cancer (PCa) remains uncertain.

Renal allograft arteriovenous fistula and large pseudoaneurysm

Abstract:u2002 The patient was a 51‐year‐old female. Post‐biopsy arteriovenous fistula (AVF) and pseudoaneurysm in a renal allograft were diagnosed 5u2003yr and 4u2003months after she received a renal transplantation. Four years after the diagnosis, interventional treatment for the AVF and pseudoaneurysm was performed because of a high risk of pseudoaneurysm rupture. Although the longitudinal diameter of the pseudoaneurysm was more than 5u2003cm, this AVF and pseudoaneurysm were treated successfully by a percutaneous transluminal embolization, and renal function has remained stable after embolization. A selective interventional procedure proved effective for the large pseudoaneurysm in the renal allograft. Therefore, when a transcutaneous needle biopsy of the renal allograft is performed, although there are no apparent symptoms or signs of vascular complications during the clinical course, periodical examinations such as echo‐Doppler imaging should be made on the allograft.

The importance of studying pressure-flow for predicting postoperative voiding difficulties in women with stress urinary incontinence: a preliminary study that correlates low Pdet×Qave with postoperative residual urine

We evaluated the parameters of preoperative pressure-flow for predicting the postoperative voiding difficulties in women with stress urinary incontinence. The preoperative urodynamic study records of 14 women treated using the tension-free vaginal tape (TVT) procedure were retrospectively analyzed. Of the patients treated with the TVT procedure, urinary retention occurred in one patient, and three had a residual urine volume of more than 30xa0ml. All patients became completely free of stress urinary incontinence postoperatively. The lowest Pdet max (5xa0cmH2O) in the preoperative pressure-flow study was found in a patient with a remarkable postoperative residual urine volume of more than 50xa0ml. The second lowest Pdet max value (8xa0cmH2O) was seen in a patient with postoperative urinary retention, whose residual urine volume, however, decreased to almost zero 1xa0year after the operation. The preoperative Pdet max×Qave values were remarkably low for these three patients, including the one with the lowest Pdet max, with a post-void residual urine volume of more than 30xa0ml. The plots of Pdet max×Qave versus the age of patients show that the Pdet max×Qave values tend to decrease with aging. The preoperative Pdet max×Qave value can be an important parameter for predicting increased residual urine after TVT sling surgery.