Yoshihiro Hashimoto

CRITICAL ROLE FOR THE 310 HELIX REGION OF P57KIP2 IN CYCLIN-DEPENDENT KINASE 2 INHIBITION AND GROWTH SUPPRESSION

Although crystal structural analysis of cyclin A/cyclin-dependent kinase 2 (Cdk2)/p27 (Russo, A. A., Jeffrey, P. D., Pattern, A. K., Massague, J., and Pavletich, N. P. (1996) Nature 382, 325–331) has suggested that the 310 helix region in Cdk inhibitors of the Cip/Kip family may be involved in the inhibition of cyclin/Cdk activities, there is no biochemical evidence supporting this hypothesis. In the present study, we demonstrated that cyclin and Cdk binding domains of p57 were necessary but were not sufficient in themselves for the inhibition of cyclin A/Cdk2 and cyclin E/Cdk2, and that the 310 helix region of this protein is indispensable for the inhibition of these complexes. In contrast, the 310 helix regions of p21 and p27 were not required, and cyclin- and Cdk-binding domains alone were sufficient for the inhibition of all cyclin/Cdk complexes examined. Site-directed mutagenesis identified phenylalanine 79 and tyrosine 80 within the 310 helix region of p57 as crucial residues for kinase inhibition, supporting the structural evidence that the 310 helix binds deep inside the catalytic cleft of Cdk2, mimicking ATP. Mutations within the 310helix region of the p57 molecule completely abolished the ability to arrest the cell cycle at G1 in vivo. These results indicate that this region is specifically utilized by p57 in selectively inhibiting cyclin A or E/Cdk2 activities. Thus the 310 helix motif may confer a specific regulatory mechanism by which p57 differentially regulates Cdk2 and Cdk4 activities.

Efficacy of Endoscopic Combined Intrarenal Surgery in the Prone Split-Leg Position for Staghorn Calculi

UNLABELLED Abstract Purpose: To evaluate the efficacy of endoscopic combined intrarenal surgery (ECIRS) using retrograde flexible ureteroscopy and miniature percutaneous nephrolithotomy (PNL) for the treatment of patients with staghorn calculi in the prone split-leg position. PATIENTS AND METHODS We retrospectively reviewed the records of 42 patients with staghorn calculi (45.8±3.2 mm) who underwent ECIRS using retrograde flexible ureteroscopy and miniature PNL in the prone split-leg position for the treatment of staghorn calculi in our center between December 2010 and August 2013. A flexible ureteroscope with a laser fiber was inserted through a ureteral access sheath, and lithoclast lithotripsy was performed through a mini-percutaneous tract. Both procedures were performed simultaneously by two urologists. Surgical parameters, including surgical time, stone-free (SF) rates, modified Clavien complication grades, and risk factors for residual stones, were analyzed. RESULTS Fifteen patients (35.7%) had complete staghorn calculi. Among the 42 staghorn calculi treated, 23 had 0 to 5 stone branches, 14 had 6 to 10 stone branches, and 5 had ≥11 stone branches. All procedures were performed successfully using a single lithotripsy tract with the patient in the prone split-leg position. The mean surgical time was 143.2±9.2 minutes. The initial SF rate was 71.4%, and the final SF rate was 83.3% after further treatment. One patient required a blood transfusion (2.4%), but no patient experienced a ≥3 Clavien grade complication. Risk factors for residual stones were stone size, stone surface area, complete staghorn calculi, and the number of stone branches. CONCLUSIONS ECIRS for staghorn calculi in the prone split-leg position is a safe, efficient, and versatile method for the effective management of staghorn calculi without the creation of multiple percutaneous tracts.

Induction of KAI-1 expression in metastatic cancer cells by phorbol esters

KAI-1 is a tumor suppressor gene whose down-regulation has been shown to be associated with the development of metastases of cancer cells. Here, we demonstrated that KAI-1 expression was induced by activating protein kinase C even in metastatic prostate cancer cell lines in which its expression was significantly down-regulated. KAI-1 expression was enhanced in a dose-dependent manner by PMA, and its induction is at least in part due to transcriptional activation. Pretreatment with calphostin C abrogated its induction by PMA. Our findings may provide useful information for developing a novel drug capable of inducing KAI-1 expression and thereby inhibiting metastasis.

Inverse Relationship Between Obesity and Serum Prostate-Specific Antigen Level in Healthy Japanese Men: A Hospital-Based Cross-Sectional Survey, 2004-2006

OBJECTIVES To confirm an inverse relationship between obesity and serum prostate-specific antigen (PSA) levels in Japanese men with a smaller body mass index (BMI) than white and African-American men. METHODS We analyzed 5246 apparently healthy Japanese men aged >20 years who visited our medical center for a health checkup from January 2004 to December 2006. The men were divided into 6 groups by age decade, and the BMI was categorized into 5 groups. The body fat percentage (BFP) was also used and was grouped into quartiles. The Mantel-Haenszel chi(2) test was used to check for trends in proportions of subjects with abnormal PSA values for each cutoff point (2.5 and 4.0 ng/mL) in these groups. The relationships between the PSA levels and BMI or BFP were examined using multivariate analysis. RESULTS The median age, BMI, BFP, and PSA level was 46 years, 23.2 kg/m(2), 21.5%, and 0.78 ng/mL, respectively. The proportion of subjects with an abnormal PSA value increased significantly with age (P for trend < .0001); however, no trends were found across the BMI or BFP categories. The geometric mean PSA level increased significantly with age (P for linear trend < .0001) and decreased with BMI and BFP categories (P for linear trend = .001 and P for linear trend = .002, respectively). CONCLUSIONS Our findings have demonstrated an inverse relationship between obesity and PSA levels even in Japanese men with a low prevalence of obesity, such as was previously reported for American men. Therefore, in prostate cancer screening, obesity, which can affect the accuracy of PSA testing, independent of race and ethnicity, should be taken into account.

Identification and characterization of Nek6 protein kinase, a potential human homolog of NIMA histone H3 kinase

In Aspergillus nidulans, the kinase activity of NIMA (never in mitosis, gene A) is critical for the initiation of mitosis. NIMA regulates mitotic chromatin condensation through phosphorylation of histone H3 at serine 10. In the present study, we identified human Nek6 (hNek6), a member of the mammalian NIMA-related kinases. The predicted hNek6 protein is comprised of 338 amino acids. Northern blot analysis revealed that hNek6 transcripts are ubiquitously expressed with the highest expression found in the heart and skeletal muscle. Lower cell cycle-dependent expression of hNek6 transcripts was observed in the early G1 phase. GFP-fused hNek6 protein showed both nuclear and cytoplasmic localizations in HeLa cells. Fluorescence in situ hybridization using full-length hNek6 cDNA as a probe showed that the hNek6 gene is localized to human chromosome 9q33-34, a region at which the loss of heterozygosity is associated with transitional cell carcinomas. Importantly, recombinant hNek6 protein produced in insect cells effectively phosphorylated histones H1 and H3, but not casein. Thus, these results suggest that, unlike other mammalian NIMA-related kinases, Nek6 is a mitotic histone kinase which regulates chromatin condensation in mammalian cells.

Essential role of Chk1 in S phase progression through regulation of RNR2 expression.

Chk1 is an essential kinase for maintaining genome integrity and cell cycle checkpoints through phosphorylating several downstream targets. Recently, we demonstrated that Chk1 is also required for cell proliferation in somatic cells under unperturbed condition through regulating transcription of several genes. Here, we show that Chk1 is required for S phase progression and RNR2 is a critical downstream target of genes transcriptionally regulated by Chk1. Hence, although RNR2 expression reached maximum at S phase in the presence of Chk1, Chk1 depletion arrested the cell cycle at S phase and reduced RNR2 expression at both mRNA and protein levels. Ectopic expression of RNR2 failed to rescue the S phase arrest observed in Chk1 depleted cells, suggesting the presence of an additional Chk1-target(s) for completion of S phase other than RNR2. Therefore, our results suggest that Chk1 is required for DNA replication at least through regulating RNR2 gene transcription.

Pitfalls with MRI Evaluation of Prostate Cancer Detection

Introduction: To assess differences between MRI findings and histopathologically defined prostate cancer localization, we compared clinical results with mapping of radical prostatectomy specimens, and conducted a retrospective MRI cancer localization re-assessment by a urologist-technician after surgery. Methods: We performed MRI for a total of 37 suspected prostate cancer patients. Subsequently, all underwent retropubic radical prostatectomy after prostate biopsy for confirmation of the diagnosis. All the specimens were studied histopathologically with serial sectioning using a whole organ approach. Results: Of the 37 patients, 26 had positive MRI findings. All the surgical specimens contained cancerous lesions, and 23 had multiple foci. Twenty-four of the MRI-positive cases (92.3%) demonstrated coincidence of both MRI and histopathologically defined lesions. In the single focus cases, 78.6% (11/14) demonstrated exact coincidence, but in the multifocal cases there were no cases with exact coincidence of MRI and histopathological findings (0/23). Conclusion: MRI evaluation cannot be considered an effective diagnostic tool in itself for detection of prostate cancers because sensitivity is far from satisfactory, especially in multi-focal cases.

SPECIFIC DETECTION OF KAPPA LIGHT CHAIN IN URIC ACID STONES

Abstract Proteins were extracted from uric acid stones with 6M guanidine chloride (pH 8.5), which were successively developed by 12% polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Amino acid sequence analysis of each band on SDS-PAGE revealed that major components in uric acid stones were immunoglobulin α heavy and κ light chains. Although immunoglobulin heavy chain (γ and μ, as well as α) and a κ light chain were clearly detected in uric acid stones by Western blotting using their specific antibodies, no λ chains whatsoever could be detected. The results suggest that immunoglobulins selectively containing κ light chain might have specific functions in uric acid stone formation as stone matrices.

Evaluation of operative complications related to laparoscopic radical prostatectomy.

Objectives:  In this decade, there have emerged many alternatives for the therapy of localized prostate cancer, such as brachytherapy, intensity modulated radiation therapy, high intensity focused ultrasound, and retropubic radical prostatectomy. In this retrospective study, we reviewed cases of complications related to laparoscopic radical prostatectomy (LRP) from our institution only, and we evaluated whether this procedure was minimally invasive or not.

Carbohydrate antigen 19‐9‐positive prostatic ductal adenocarcinoma effectively treated with cisplatin and gemcitabine

Abstract:  We performed a radical retropubic prostatectomy on a 59‐year‐old male patient who was diagnosed with prostatic carcinoma. Pathological findings revealed a ductal adenocarcinoma in the left lobe of the prostate, and a conventional well‐differentiated adenocarcinoma in the right lobe. Immunohistochemistry revealed that CA19‐9 was positive in the ductal adenocarcinoma, and prostate‐specific antigen was positive in the conventional well‐differentiated adenocarcinoma. Since there was an increase in the level of serum CA19‐9, which had decreased postoperatively, along with the appearance of local recurrence and bone metastasis, we treated the patient with cisplatin and gemcitabine. As a result, the level of serum CA19‐9 was normalized and a reduction of the metastatic focus was observed.