Shohei Miyata

HORMONAL REVERSAL AND THE GENETIC CONTROL OF SEX DIFFERENTIATION IN XENOPUS

Abstract Administration of exogenous estradiol between stages 50 and 52 completely feminized the developing gonads of Xenopus laevis. However, when tadpoles were injected or cultured during the critical period with an inhibitor (CGS 16949A) of aromatase that prevents synthesis of estradiol from androgen, there were no detectable effects on the sexual differentiation of the gonads. Aromatase transcription in Xenopus gonads was then studied by the reverse-transcription polymerase chain reaction (PCR) method. In embryos at the beginning of the estradiol-sensitive period (stages 49 and 50), expression of the aromatase gene was not detected in the gonad. These results show that the period between stages 50 and 52 is the time when Xenopus is sensitive to sex reversal by estradiol and critical for sex determination, although estradiol synthesis may not be naturally involved in the gonad at this step.

Sequence analysis and expression of the P450 aromatase and estrogen receptor genes in the Xenopus ovary

Recent studies point to a key role for the estrogen synthesizing enzyme P450 aromatase (P450 arom) in ovary determination in fish, birds and reptiles. It is unclear whether estrogen synthesis is important in sex determination of Xenopus gonad. To determine whether the aromatase gene is transcribed in the gonads of Xenopus tadpoles during the sex determination, we cloned a P450 arom cDNA and examined the level of P450 arom and estrogen receptor (ER) gene expression in association with estrogen activity. cDNA clones for P450 arom were isolated from a Xenopus ovarian cDNA library. There was an open reading frame (ORF) of 1500 bp from the ATG start to TAA stop codons encoding 500 predicted amino acids. cDNAs for P450 arom have previously been cloned from various vertebrates. The homology between the Xenopus P450 aromatase and the human P450 arom was higher. The expression of the P450 arom gene was mainly limited to reproductive organs. To determine the beginning of estrogen activity in gonads of embryos, expression of the aromatase and ER gene was also examined by RQ-RT-PCR. Both Xenopus aromatase and ER mRNA was detected at stage 51 in gonads. These observations are consistent with estrogens having a key role in ovarian development in various other vertebrates.

SRY-related genes in Xenopus oocytes☆

SRY-related genes are known as Sox (Sry-box) genes. Two Sox cDNAs from Xenopus oocytes were analyzed. The deduced product of the Xenopus Sox gene (xSox-11) consisted of the standard domains of an HMG box, glycine/alanine-rich region and glutamic acid/aspartic acid-rich region and may be involved in the control of transcription. The other Sox gene (xSox-11-D) had a deletion of 262 nucleotides at one end of the HMG box in the xSox-11 cDNA. The deletion resulted in a frame-shift and in variations in base pair composition and the length of the trinucleotide repeat in the C-terminal coding region. The amino acid sequence of the C-terminal domain encoded by xSox-II-D was highly basic and might be involved, together with the HMG box, in the binding to DNA.

Selective inhibition of the growth of cancer cells by diterpenes selected with embryonic cells of Xenopus

We used Xenopus embryo cells with a cell cycle of 20–30 min to detect inhibitory effects on cell proliferation. Inhibition of proliferation was observed when isolated embryonic cells were incubated for 16 h in a simple salt solution containing the well‐known anticancer drugs 5‐fluorouracil and adriamycin. In addition, three diterpene compounds isolated from the anticancer herbal medicine kansui: kansuinin B, 20‐OD‐ingenol Z, and 20‐OD‐ingenol E specifically inhibited the proliferation of isolated embryonic cells. The inhibitory compounds selected using the embryonic cells also inhibited proliferation of certain mammalian cell types.

Analysis of inhibition of topoisomerase IIα and cancer cell proliferation by ingenolEZ

We previously reported that many ingenol compounds derived from Euphorbia kansui exhibit topoisomerase inhibitory activity and/or inhibitory activity of cell proliferation. The inhibitory effects of 20‐O‐(2′E,4′Z‐decadienoyl) ingenol and 3‐O‐(2′E,4′Z‐decadienoyl)‐ingenol among these compounds on topoisomerase II activity and on the cell proliferative activity and arrest phase of the cell cycle were studied using a mouse breast cancer (MMT) cell line. Although 20‐O‐ingenolEZ exerted inhibitory effects on both topoisomerase II activity and cell proliferative activity, 3‐O‐ingenolEZ exerted inhibitory activity on neither. The 20‐O‐ingenolEZ‐induced cell arrest of MMT‐cell proliferation led to a cell cycle arrest in the G2/M phase. Topoisomerase II inhibition can be divided into the poison and catalytic inhibitor types. A checkpoint mechanism is activated when cells are treated with these topoisomerase II inhibitors. Poison‐type inhibition occurs via induction of the DNA damage checkpoint and the catalytic‐type inhibition occurs via induction of the DNA‐decatenation checkpoint, suggestive of distinct checkpoint reactions. 20‐O‐ingenolEZ inhibited topoisomerase IIα activity through inhibition of ATPase, and induced DNA‐decatenation checkpoint without signaling for phosphorylation of H2AX. (Cancer Sci 2010; 101: 374–378)

Effects on properties of a thiol protease from Xenopus embryos of changes in substrate and assay conditions.

A protease was purified from Xenopus embryos. Proteolytic activity of the protease against BSA had an optimum pH of 3.8 in acetate buffer and was not detectable at neutral pH. However, when embryonic proteins were used as substrates and digested in phosphate buffer, proteolysis of embryonic proteins was enhanced and was detectable from pH 5.0 to pH 7.0. Digestion of three proteins were mainly detected in digestion of total embryonic proteins. The proteins digested had the same mobilities (on SDS polyacrylamide gel) as yolk proteins. The protease was present in the cytoplasm and around yolk granules. We propose that this protease mainly cleaves a certain yolk proteins in the cytoplasm of Xenopus embryos.

Mechanism of the inhibition of leukemia cell growth and induction of apoptosis through the activation of ATR and PTEN by the topoisomerase inhibitor 3EZ, 20Ac-ingenol.

The PI3K/Akt signaling pathway is constitutively activated in various leukemias. In the present study, the topoisomerase inhibitor, 3EZ, 20Ac-ingenol, was more effective in inhibiting the growth of BALL-1 cells than that of normal lymphocyte cells. ATM/ATR protein levels were increased, PTEN protein was upregulated, and p-Akt protein was downregulated at early time points after treatment with 3EZ, 20Ac-ingenol. In further experiments, p53 protein expression was increased, and H2AX phosphorylation and p21 protein expression were induced after treatment with 3EZ, 20Ac-ingenol. Moreover, the activation of caspase 3 followed decrease in the Bcl-2/Bax ratio after treatment with 3EZ, 20Ac-ingenol, and accumulation of sub-G1 phase cells was observed in flow cytometry analyses. These data suggest that 3EZ, 20Ac-ingenol-induced DNA damage downregulates p-Akt and upregulates ATR leading to cell cycle arrest and increased apoptosis in BALL-1 cells.

Isolation of novel isoforms of estrogen receptor genes from Xenopus gonad and brain.

Abstract We isolated four variants of ER α mRNA, two each from the brain and gonad of the Xenopus embryo (brain, bER α 1 and bER α 2; gonad, gER α 1 and gER α 2). The N-terminal-domain of ER α differed between the brain and gonad. While the C-terminal region downstream of the DNA-binding domain of ER α differed between ER α 1 and ER α 2 in both the brain and gonad, each of ER α 1 and ER α 2 contained the same sequence sets (bER α 1 and gER α 1; bER α 2 and gER α 2) between the brain and gonad. Both bER α 1 and gER α 1 contained the same full-length, C-terminal estrogen receptor, whereas the C terminus of bER α 2 and gER α 2 lacked half of the DNA-binding, ligand-binding, and transcriptional activation domain. Although gER α was mainly expressed in the gonad, both bER α and gER α were expressed in the brain of the Xenopus embryo at stage 50. These isoforms might be transcribed by alterative splicing of a single gene. Two gonad ER β s differing in the length of the N-terminal sequence were isolated from the Xenopus gonad. While ER β was expressed in the gonad, it was not expressed in the brain of the Xenopus embryo at stage 50.

3EZ,20Ac-ingenol, a catalytic inhibitor of topoisomerases, downregulates p-Akt and induces DSBs and apoptosis of DT40 cells

We have previously reported that many ingenol compounds derived from Euphorbia kansui exhibit topoisomerase (topo) II inhibitory activity. Of these compounds, 3EZ,20Ac-ingenol inhibited topo I activity. Camptothecin, which inhibits the religation activity of topo I without interfering with the binding of topo I to DNA and induces topo I-mediated DNA cleavage, was used as a positive control. In this study, we found that 3EZ,20Ac-ingenol did not hamper the binding of topo I to DNA in the same manner as camptothecin but affected the inhibition of cleavage of one DNA strand. 3EZ,20Ac-ingenol inhibited cell proliferation by blocking cell cycle progression in the G2/M phase. To define the mechanism of inhibition of DT40 cell proliferation, the change in Akt activity was observed because Akt activity is regulated in response to DNA damage. Western blot analysis revealed that 3EZ,20Ac-ingenol downregulated the expression of p-Akt, and apoptosis was detected by the presence of DNA double-strand breaks and caspase 3 activation.

20-O-IngenolEZ, a catalytic topoisomerase II inhibitor, specifically inhibits cell proliferation and induces double-strand DNA breaks in BLM-/-cells

Bis-dioxopiperazines have been termed catalytic inhibitors to distinguish them from topoisomerase poisons that induce DNA double-strand breaks (DSBs). However, it has been reported that ICRF-193 acts as a poison in cells containing mutated genes related to checkpoint mechanisms. We also showed previously that 20-O-ingenolEZ acts as a catalytic inhibitor to inhibit the ATPase activity of topoisomerase IIα, inducing the G2 arrest of mouse mammary tumor (MMT) cells. In this study, we observed the effects of 20-O-ingenolEZ on cells containing a mutation in the RecQ helicase gene. 20-O-IngenolEZ completely inhibited the proliferation of BLM-/-cells in BLM-/- and WRN-/-DT40 cells and wild-type DT40 cells. This inhibition induced the phosphorylation of H2AX in response to agents that introduce topoisomerase II-mediated DSBs. Following DNA damage, the induction of apoptosis in the BLM-/-cells by 20-O-ingenolEZ showed the characteristics of a topoisomerase II poison.