Takeshi Takayama

Inhibition of peritoneal dissemination of ovarian cancer by tyrosine kinase receptor inhibitor SU6668 (TSU‐68)

SU6668 (TSU‐68) is a small‐molecule synthetic inhibitor of the angiogenic related receptor tyrosine kinases Flk‐1/KDR, PDGFRβ, and FGFR1. Using a mouse model of peritoneally disseminated ovarian cancer, we investigated whether SU6668 inhibits peritoneal dissemination and prolongs survival time. BALB/c nude mice were intraperitoneally (i.p.) inoculated with SHIN‐3 (VEGF‐hypersecretory) or KOC‐2S (PDGF‐hypersecretory) ovarian serous adenocarcinoma cells with marked peritoneal dissemination ability. From the day after i.p. inoculation of tumor cells, SU6668 was orally administered 6 times weekly at a daily dose of 100 mg/kg or 400 mg/kg. The SU6668‐administered group and the vehicle‐administered control group were compared for the number of tumor vascular endothelial cells, weight of peritoneally disseminated tumors, amount of ascitic fluid and survival time. As a result, these 3 parameters were significantly smaller in the SHIN‐3‐inoculated, SU6668‐administered mice than in the control group (p = 0.03, p = 0.002, and p = 0.02, respectively). The mean survival time was significantly longer, at 58.1 ± 11.2 days, in the SU6668‐administered mice than that (34.5 ± 8.8 days) in the control group (p = 0.002). Similarly, in the KOC‐2S‐inoculated mice, the oral administration of SU6668 significantly reduced these 3 parameters (p = 0.04, p = 0.04, and p = 0.03, respectively), and significantly prolonged survival (16.6 ± 1.7 days vs. 11.0 ± 0.7 days, p = 0.008). Thus, the oral administration of SU6668 inhibited angiogenesis and peritoneal dissemination and prolonged survival in mice with peritoneally disseminated ovarian cancer. These effects were observed with both the VEGF‐ and PDGF‐hypersecretory cell lines. Our results suggest that molecular targeting with oral SU6668 will become a new therapeutic strategy targeting peritoneally disseminated ovarian cancer.

Identification, Cloning, and Initial Characterization of a Novel Mouse Testicular Germ Cell-Specific Antigen

Abstract A monoclonal antibody, designated TES101, was raised by immunizing BALB/c mice with an allogenic mouse testicular homogenate followed by immunohistochemical selection as the initial screening method. By searching the expressed sequence tag (EST) database with the N-terminal amino acid sequence of TES101 reactive protein, we found that the predicted amino acid sequence encoded by a mouse testicular EST clone matched the TES101 protein sequence. Sequence analysis of the clone revealed no homologous molecule in the DNA/protein database. Based on data obtained from N-terminal amino acid analysis of the TES101 protein, the derived amino acid sequence contained a signal peptide region of 25 amino acids and a mature protein region of 225 amino acids, which translated into a protein with a molecular weight of 24 093. Northern blot analysis showed that mRNA of the TES101 protein was found in testis but not in any other mouse tissues examined. Western blot analysis revealed that TES101 reacted with a 38-kDa band on SDS-PAGE under nonreducing conditions, and this reactivity was abrogated under reducing conditions. Immunoelectron microscopic studies demonstrated that the molecule was predominantly located on the plasma membrane of spermatocytes and spermatids but not in Sertoli cells or interstitial cells, including Leydig cells. Thus, the TES101 protein is a novel molecule present primarily on the surface of developing male germ cells. TES101 protein may play a role in the processes underlying male germ cell formation.

Sexually Dimorphic Expression of the Novel Germ Cell Antigen TEX101 During Mouse Gonad Development

Abstract Prospermatogonia, or gonocytes, are the cells that differentiate from primordial germ cells to the first mature type of spermatogonia in the developing testis. Although prospermatogonia play a central role in this stage (i.e., prespermatogenesis), the details regarding their characterization have not been fully elucidated. Recently, we identified a novel mouse testicular germ cell-specific antigen, TES101 reactive protein (TES101RP), in the adult mouse testis. The protein TES101RP is also designated as protein TEX101. In the present study, we investigated the expression of TEX101 on germ cells in developing mouse gonads using histochemical techniques (i.e., immunohistochemistry, BrdU labeling, and TUNEL staining) and reverse transcription-polymerase chain reaction. TEX101 appeared on germ cells in both male and female gonads after the pregonadal period. In the testis, TEX101 was expressed constitutively on surviving prospermatogonia during prespermatogenesis. After the initiation of spermatogenesis, the prospermatogonia differentiated into spermatogonia. TEX101 expression disappeared from the spermatogonia, but reappeared on spermatocytes and spermatids. In the ovary, TEX101 was expressed on germ cells until the start of folliculogenesis; TEX101 was not detected on oocytes that were surrounded by follicular cells. These findings indicate that TEX101 is a specific marker for both male and female germ cells during gonadal development. Because the on and off switching of TEX101 expression in germ cells almost parallels the kinetics of gametogenesis, TEX101 may play an important physiological role in germ cell development.

TEX101 is shed from the surface of sperm located in the caput epididymidis of the mouse

It is generally believed that cell-to-cell cross-talk and signal transduction are mediated by cell surface molecules that play diverse and important regulatory roles in spermatogenesis and fertilization. Recently, we identified a novel plasma membrane-associated protein, TES101-reactive protein (TES101RP, or TEX101), on mouse testicular germ cells. In this study, we investigate Tex101 mRNA expression in the adult mouse testis using in situ hybridization, and we examine the fate of TEX101 during sperm transport by immunohistochemical and Western blot analyses. Tex101 mRNA was expressed in a stage-specific manner in spermatocytes and in step 1-9 spermatids of the testis, but not in spermatogonia. Although the TEX101 protein remained on the cell surfaces of step 10-16 spermatids and testicular sperm, it was shed from epididymal sperm located in the caput epididymidis. The results of this study provide additional information on germ cell-specific TEX101 expression during spermatogenesis and post-testicular sperm maturation.

Suppression of ovarian cancer by muscle-mediated expression of soluble VEGFR-1/Flt-1 using adeno-associated virus serotype 1-derived vector

Vascular endothelial growth factor (VEGF) is known to play a major role in angiogenesis in a variety of tumors. A soluble form of Flt‐1 (sFlt‐1), a VEGF receptor, is potentially useful as an antagonist of VEGF, and accumulating evidences suggest the applicability of sFlt‐1 in tumor suppression by means of anti‐angiogenesis. We previously demonstrated the efficacy of sflt‐1 gene expression in situ to suppress tumor growth and ascites in ovarian cancer. Here, we demonstrate the therapeutic applicability of muscle‐mediated expression of sFlt‐1 in tumor‐bearing mice. Initially, tumor suppressive action was confirmed by inoculating sFlt‐1‐expressing ovarian cancer (SHIN‐3) cells into mice, both subcutaneously and intraperitoneally. To validate the therapeutic efficacy in a more clinically relevant model, adeno‐associated virus vectors encoding sflt‐1 were introduced into mouse skeletal muscles and were subsequently inoculated with tumor cells. As a result, high serum sFlt‐1 levels were constantly observed, and the growth of both subcutaneously‐ and intraperitoneally‐inoculated tumors was significantly suppressed. No delay in wound healing or adverse events of neuromuscular damage were noted, body weight did not change, and laboratory data, such as those representing liver and renal functions, were not affected. These results indicate that sFlt‐1 suppresses growth and peritoneal dissemination of ovarian cancer by the inhibition of angiogenesis, and thus suggest the usefulness of gene therapy for ovarian cancer.

Overexpression of PTEN in ovarian cancer cells suppresses i.p. dissemination and extends survival in mice

The main mode of progression of ovarian cancer is peritoneal dissemination, and its inhibition may lead to improved outcome. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) reportedly inhibits the proliferation, migration, and invasion of cancer cells. The purpose of this study is to explore the possibility of PTEN gene therapy for ovarian cancer. We transfected the ovarian cancer cell line SHIN-3 [vascular endothelial growth factor (VEGF)–hypersecretory cell line] with PTEN or luciferase (LUC)–expressing plasmid. After selection, PTEN-overexpressing cells (SHIN-3/PTEN) and control cells (SHIN-3/LUC) were obtained. SHIN-3/PTEN implanted s.c. into nude mice was examined for the change in tumor diameter and the number of new blood vessels. Mice with peritoneally disseminated tumors created by i.p. inoculation of the same cells were examined for changes in body weight and abdominal circumference and for survival time. The growth of s.c. SHIN-3/PTEN was significantly lower than that of control (P < 0.001). Compared with controls, mice with i.p. inoculated SHIN-3/PTEN showed significantly smaller increases in the body weight and abdominal circumference (P < 0.01) and a significantly longer survival time (P < 0.05). VEGF concentration in the supernatant of SHIN-3/PTEN was about half that of controls (P < 0.05). The number of new blood vessels in SHIN-3/PTEN was significantly smaller than that in controls (P < 0.001). Overexpression of PTEN suppressed tumor growth and peritoneal dissemination of VEGF-hypersecretory ovarian cancer cells and prolonged the survival time of the mice with peritoneal disseminated tumor. PTEN gene therapy could have therapeutic potential for ovarian cancer and exerts some of this effect by inhibiting angiogenesis. [Mol Cancer Ther 2008;7(3):704–11]

Profiling of proteins phosphorylated or dephosphorylated during hyperactivation via activation on hamster spermatozoa

Background and AimsIt has been widely accepted that sperm hyperactivation is regulated by protein phosphorylations. But, the sperm hyperactivation phosphorylation pathway is not well understood yet because several different proteins have been detected in other studies. In order to understand the phosphorylation pathway that regulates hyperactivation, we established how to extract sperm protein completely and detected proteins that were phosphorylated during hyperactivation.MethodsProtein phosphorylation of hamster spermatozoa was detected by western blotting using antiphospho-amino acid monoclonal antibodies or the SELDI ProteinChip system with IMAC-Ga(III).ResultsWe detected 75 protein/peptide phosphoryations using the method established in the present study. Tyrosine phosphorylations occurred during hyperactivation. Serine or threonine phosphorylations occurred for 30 min. Furthermore, four of the serine or threonine phosphorations were phosphorylated by A-kinase. As for peptides, 15 peptides were dephosphorylated for 30 min. Other peptides were phosphorylated during hyperactivation.ConclusionsBecause most of the proteins detected in the present study have been described previously, we could detect comprehensive protein phosphorylations. Moreover, we also detected many novel phosphopeptides. Although we did not understand the role of peptide, it was likely that motility was basically regulated by serine/threonine phosphorylations and hyperactivation was mainly regulated by tyrosine phosphorylations. (Reprod Med Biol 2006; 5: 123–135)

Elevation of the Serum Uric Acid Level Preceding the Clinical Manifestation of Preeclampsia in Twin Pregnancies

To assess changes in the serum uric acid level in the third trimester of twin pregnancies, a total of 152 consecutive women with twin pregnancies were examined. Serum uric acid levels were analyzed in the women at varying gestational weeks in the presence or absence of preeclampsia. A receiver operating characteristic curve was used to determine the optimal cutoff value of serum uric acid between 30 and 31 weeks of gestation predicting subsequent development of preeclampsia. Forty-four women (29%) developed preeclampsia (preeclampsia group) at 33.2 +/- 1.9 weeks (mean +/- SD) and gave birth at 35.4 +/- 1.5 weeks of gestation. The remaining 108 women (71%) gave birth at 35.6 +/- 1.7 weeks of gestation (control group without preeclampsia). Serum uric acid levels rose gradually with advancing gestation in both groups. In the preeclampsia group, they were already increased at 30-31 weeks of gestation and corresponded to those seen in the control group at 37 weeks. The cutoff value at 30-31 weeks was 5.5 mg/dl, with a sensitivity of 73% and a specificity of 74%. These results suggest that an elevation in serum urate preceded the onset of preeclampsia. Determination of the serum level of uric acid between 30 and 31 weeks of gestation was useful for detecting a higher risk of late-onset preeclampsia in twin pregnancies.

Risks Associated with Cesarean Sections in Women with Placenta Previa

Objective: To investigate the risks associated with cesarean section performed because of placenta previa.

Profiling of proteins phosphorylated or dephosphorylated during hyperactivation via activation on hamster spermatozoa: Phosphoprotein profiling on hyperactivation

Background and Aims:  It has been widely accepted that sperm hyperactivation is regulated by protein phosphorylations. But, the sperm hyperactivation phosphorylation pathway is not well understood yet because several different proteins have been detected in other studies. In order to understand the phosphorylation pathway that regulates hyperactivation, we established how to extract sperm protein completely and detected proteins that were phosphorylated during hyperactivation.