Aya Naiki-Ito

Necessary and Sufficient Role for a Mitosis Skip in Senescence Induction

Senescence is a state of permanent growth arrest and is a pivotal part of the antitumorigenic barrier in vivo. Although the tumor suppressor activities of p53 and pRb family proteins are essential for the induction of senescence, molecular mechanisms by which these proteins induce senescence are still not clear. Using time-lapse live-cell imaging, we demonstrate here that normal human diploid fibroblasts (HDFs) exposed to various senescence-inducing stimuli undergo a mitosis skip before entry into permanent cell-cycle arrest. This mitosis skip is mediated by both p53-dependent premature activation of APC/C(Cdh1) and pRb family protein-dependent transcriptional suppression of mitotic regulators. Importantly, mitotic skipping is necessary and sufficient for senescence induction. p16 is only required for maintenance of senescence. Analysis of human nevi also suggested the role of mitosis skip in in vivo senescence. Our findings provide decisive evidence for the molecular basis underlying the induction and maintenance of cellular senescence.

Gpx2 Is an Overexpressed Gene in Rat Breast Cancers Induced by Three Different Chemical Carcinogens

Gene expression alterations are essential for the process of carcinogenesis. A carcinogen may have specific mechanisms for inducing tumors, which may involve inducing characteristic gene expression alterations. In this study, we attempted to identify genes crucial for mammary carcinogenesis. For this purpose, we used human c-Ha-ras proto-oncogene transgenic rats (Hras128), which are highly sensitive to mammary carcinogens including N-methyl-N-nitrosourea, 7,12-dimethyl benz[a]anthracene, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. DNA microarray analysis revealed that glutathione peroxidase 2 (Gpx2) was commonly up-regulated in the mammary carcinomas induced by the three different carcinogens, and its up-regulation was confirmed by quantitative reverse transcriptase-PCR and Western blotting analysis. In addition, expression of GPX2 was recognized in all 41 immunohistochemically examined cases of human breast cancer. Forced suppression of GPX2 expression by siRNA resulted in significant growth inhibition in both rat and human mammary carcinoma cell lines with wild-type p53 cells. Thus, these data suggested that GPX2 may be involved in mammary carcinogenesis and cell proliferation in both rats and humans, indicating that GPX2 may be a novel target for the prevention and therapy of breast cancer.

Gap junction dysfunction reduces acetaminophen hepatotoxicity with impact on apoptotic signaling and connexin 43 protein induction in rat.

Acetaminophen (APAP) is a widely used antipyretic and analgesic agent. However, overdosing and sometimes even a recommended dose can lead to serious and conceivably fatal liver toxicity. Therefore, it is important to clarify understand mechanisms of hepatotoxicity induced by APAP. Gap junctions, formed by connexin, have important roles in maintenance of tissue homeostasis and control of cell growth and differentiation. In the liver, Cx32 is a major gap junction protein whose expression is known to gradually decrease with chronic liver disease progression. In the present study, acute hepatotoxic effects of APAP were found to be reduced in Cx32 dominant negative transgenic rats lacking normal gap junctional intercellular communication in the liver. In littermate wild-type rats, the injured centrilobular hepatocytes were positive for TUNEL staining and featured elevated expre ssion of cleaved caspase-3 and Cx43, which is not expressed in normal hepatocytes. These results suggest that APAP hepatotoxicity involves apoptosis, and induction of Cx43 expression may play an important role in the apoptotic signaling. Moreover, gap junctional functions of Cx32 can play important roles in removing damaged hepatocytes by apoptosis for liver tissue homeostasis.

Napsin A is a specific marker for ovarian clear cell adenocarcinoma

Ovarian clear cell adenocarcinoma has a relatively poor prognosis among the ovarian cancer subtypes because of its high chemoresistance. Differential diagnosis of clear cell adenocarcinoma from other ovarian surface epithelial tumors is important for its treatment. Napsin A is a known diagnostic marker for lung adenocarcinoma, and expression of napsin A is reported in a certain portion of thyroid and renal carcinomas. However, napsin A expression in ovarian surface epithelial tumors has not previously been examined. In this study, immunohistochemical analysis revealed that in 71 of 86 ovarian clear cell adenocarcinoma patients (83%) and all of the 13 patients with ovarian clear cell adenofibroma, positive napsin A staining was evident. No expression was observed in 30 serous adenocarcinomas, 11 serous adenomas or borderline tumors, 19 endometrioid adenocarcinomas, 22 mucinous adenomas or borderline tumors, 10 mucinous adenocarcinomas, or 3 yolk sac tumors of the ovary. Furthermore, expression of napsin A was not observed in the normal surface epithelium of the ovary, epithelia of the fallopian tubes, squamous epithelium, endocervical epithelium, or the endometrium of the uterus. Therefore, we propose that napsin A is another sensitive and specific marker for distinguishing ovarian clear cell tumors (especially adenocarcinomas) from other ovarian tumors.

Glutathione S-transferase Pi mediates proliferation of androgen-independent prostate cancer cells

Prostate cancers generally acquire an androgen-independent growth capacity with progression, resulting in resistance to antiandrogen therapy. Therefore, identification of the genes regulated through this process may be important for understanding the mechanisms of prostate carcinogenesis. We here utilized androgen-dependent/independent transplantable tumors, newly established with the ‘transgenic rat adenocarcinoma in prostate’ (TRAP) model, to analyze their gene expression using microarrays. Among the overexpressed genes in androgen-independent prostate cancers compared with the androgen-dependent tumors, glutathione S-transferase pi (GST-pi) was included. In line with this, human prostate cancer cell lines PC3 and DU145 (androgen independent) had higher expression of GST-pi compared with LNCaP (androgen dependent) as determined by semiquantitative reverse transcription–polymerase chain reaction analysis. To investigate the roles of GST-pi expression in androgen-independent human prostate cancers, GST-pi was knocked down by a small interfering RNA (siRNA), resulting in significant decrease of the proliferation rate in the androgen-independent PC3 cell line. In vivo, administration of GST-pi siRNA–atelocollagen complex decreased GST-pi protein expression, resulting in enhanced numbers of TdT mediated dUTP-biotin nick-end labering (TUNEL)-positive apoptotic cells. These findings suggest that GST-pi might play important roles in proliferation of androgen-independent human prostate cancer cells.

Ellagic acid, a component of pomegranate fruit juice, suppresses androgen-dependent prostate carcinogenesis via induction of apoptosis

Ellagic acid (EA), a component of pomegranate fruit juice (PFJ), is a plant‐derived polyphenol and has antioxidant properties. PFJ and EA have been reported to suppress various cancers, including prostate cancer. However, their chemopreventive effects on development and progression of prostate cancer using in vivo models have not been established yet.

GPX2 overexpression is involved in cell proliferation and prognosis of castration-resistant prostate cancer

There is a need for exploration of new therapeutic strategies that target distinct molecular mechanisms of castration-resistant prostate cancer (CRPC) because its emergence following androgen deprivation therapy is a major clinical problem. In this report, we investigated the role of glutathione peroxidase 2 (GPX2) in CRPC. GPX2 expression was analyzed in rat and human CRPC cells. Next, we determined the proliferation rate and level of reactive oxygen species (ROS) in GPX2-small interfering RNA (siRNA)-transfected CRPC cells. For in vivo analysis, siRNA-transfected cells were subcutaneously implanted into normal and castrated nude mice. Further, immunohistochemical and prognostic analyses of GPX2 were performed using human specimens. Silencing of GPX2 caused significant growth inhibition and increased intracellular ROS in both rat (PCai1) and human (PC3) CRPC cells. Flow cytometry and western blot analyses revealed that the decrease in proliferation rate of the GPX2-silenced cells was due to cyclin B1-dependent G2/M arrest. Furthermore, knockdown of Gpx2 inhibited tumor growth of PCai1 cells in castrated mice. Immunohistochemical analyses indicated that expression of GPX2 was significantly higher in residual cancer foci after neoadjuvant hormonal therapy than in hormone naive cancer foci. Moreover, patients with high GPX2 expression in biopsy specimen had significantly lower prostate-specific antigen recurrence-free survival and overall survival than those with no GPX2 expression. These findings suggest that GPX2 is a prognostic marker in CRPC and affects proliferation of prostate cancer under androgen depletion partially through protection against ROS signaling.

SCF Fbxo22 -KDM4A targets methylated p53 for degradation and regulates senescence

Recent evidence has revealed that senescence induction requires fine-tuned activation of p53, however, mechanisms underlying the regulation of p53 activity during senescence have not as yet been clearly established. We demonstrate here that SCFFbxo22-KDM4A is a senescence-associated E3 ligase targeting methylated p53 for degradation. We find that Fbxo22 is highly expressed in senescent cells in a p53-dependent manner, and that SCFFbxo22 ubiquitylated p53 and formed a complex with a lysine demethylase, KDM4A. Ectopic expression of a catalytic mutant of KDM4A stabilizes p53 and enhances p53 interaction with PHF20 in the presence of Fbxo22. SCFFbxo22-KDM4A is required for the induction of p16 and senescence-associated secretory phenotypes during the late phase of senescence. Fbxo22−/− mice are almost half the size of Fbxo22+/− mice owing to the accumulation of p53. These results indicate that SCFFbxo22-KDM4A is an E3 ubiquitin ligase that targets methylated p53 and regulates key senescent processes.

Induction of apoptosis in the LNCaP human prostate carcinoma cell line and prostate adenocarcinomas of SV40T antigen transgenic rats by the Bowman-Birk inhibitor.

The soybean‐derived serine protease inhibitor, Bowman–Birk inhibitor (BBI), has been reported as a potent chemoprevention agent against several types of tumors. The present study was undertaken to evaluate the effects of BBI on androgen‐sensitive/dependent prostate cancers using a human prostate cancer cell (LNCaP) and the transgenic rats developing adenocarcinoma of the prostate (TRAP) model. Treatment of LNCaP prostate cancer cells with 500 µg/mL BBI resulted in inhibition of viability measured on WST‐1 assays, with induction of connexin 43 (C×43) and cleaved caspase‐3 protein expression. Feeding of 3% roughly prepared BBI (BBIC) to TRAP from the age 3 weeks to 13 weeks resulted in significant reduction of the relative epithelial areas within the acinus and multiplicity of the adenocarcinomas in the lateral prostate lobes. C×43‐ and terminal deoxynucleotidyl transferase mediated dUTP‐biotin end labeling of fragmented DNA (TUNEL)‐positive apoptotic cancer cells were more frequently observed in the lateral prostates treated with BBIC than in the controls. These in vivo and in vitro results suggest that BBI possesses chemopreventive activity associated with induction of C×43 expression and apoptosis.

Apocynin, an NADPH oxidase inhibitor, suppresses rat prostate carcinogenesis

Recent evidence suggests that oxidative stress contributes to the pathogenesis of prostate cancer. The present study focused on the effect of apocynin, an inhibitor of NADPH oxidase, on prostate carcinogenesis using the transgenic rat for adenocarcinoma of prostate (TRAP) model. There were no toxic effects with apocynin treatment. The percentages and numbers of carcinomas in both the ventral and lateral prostate were significantly reduced by apocynin treatment, with dose dependence. Reduction of reactive oxygen species by apocynin was confirmed by immunohistochemistry of 8‐OHdG and dihydroethidium staining. Positivity of Ki67 was significantly reduced by apocynin treatment, and downregulation of clusterin expression, as well as inactivation of the MEK‐ERK1/2 pathway, was a feature of the apocynin treated groups. In human prostate cancer cell line LNCaP, apocynin also inhibited reactive oxygen species production and blocked cell growth by inducing G0/G1 arrest with downregulation of clusterin and cyclin D1. These data suggest that apocynin possesses chemopreventive potential against prostate cancer.