Toshihiko Izutsu

Telomerase activity in human chorionic villi and placenta determined by TRAP and in situ TRAP assay

Telomerase activity (TA) was analysed in human chorionic villi and placenta in normal and abnormal pregnancy using the telomeric repeat amplification protocol (TRAP) and in situ TRAP assay. Twenty chorionic villi specimens and 25 placenta specimens from normal pregnancies were examined as well as placenta specimens from 10 cases of intrauterine growth retardation (IUGR; nine asymmetric and one symmetric). TA was detected in 18 of the 20 (90 per cent) chorionic villi specimens and in 18 of the 25 (72 per cent) placenta specimens from normal pregnancy. However, no or only weak TA was exhibited in the placenta specimens of the nine asymmetric IUGR cases. In situ TRAP assay detected TA in trophoblastic cells from normal pregnancy, but not in trophoblastic cells from cases of asymmetric IUGR.

Telomerase and proliferative activity in placenta from women with and without fetal growth restriction

OBJECTIVE To analyze telomerase and proliferative activity in placenta from women with and without fetal growth restriction (FGR). METHODS Telomerase activity was analyzed in 30 first-trimester chorionic villi specimens (group A) and in 28 second- and third-trimester placenta specimens (group B) from women without FGR. Telomerase activity also was analyzed in 11 placenta specimens from women with asymmetric FGR (group C). The proliferative activity of these 69 specimens was assessed by immunohistochemical staining, using the MIB-1 monoclonal antibody. RESULTS Telomerase activity was detected in 28 (93.3%) of 30 chorionic villi specimens and in 18 (64.3%) of 28 placenta specimens without FGR. In contrast, no telomerase activity was exhibited in the placenta specimens from any of the 11 women with asymmetric FGR by telomeric repeat amplification protocol assay. Telomerase activity also was detected by in situ telomeric repeat amplification protocol assay in trophoblastic cells from women without FGR but not in trophoblastic cells from women with asymmetric FGR. Thus, telomerase activity was detected significantly more often in groups A and B than in group C (P < .01). The rate of proliferative activity, evident as positive MIB-1 staining in trophoblastic cells, in groups A and B (28.1+/-1.7% and 7.0 +/-2.9%, respectively) was significantly higher than that in group C (1.9+/-0.6%; P < .01). CONCLUSION Telomerase and proliferative activity were minimal in placenta from women with asymmetrical FGR, suggesting placental senescence with asymmetrical FGR.

Triple Marker Screening for Trisomy 21, Trisomy 18 and Open Neural Tube Defects in Singleton Pregnancies of Native Japanese Pregnant Women

Objective: To report the results of prenatal triple marker screening on a population of Japanese pregnant women.

Numerical and structural chromosome abnormalities in an ovarian fibrothecoma

Cytogenetic analysis of a fibrothecoma of ovary revealed numerical and structural chromosome abnormalities, i.e., 44,XX, dup(1)(p13p31),del(3)(p14) add (10p), -16, -22. This is the first report of numerical and structural abnormalities in a fibrothecoma of the ovary.

Comparative Cytogenetic Studies of Benign, Borderline, and Malignant Epithelial Ovarian Tumors

Comparative cytogenetic studies were performed in 40 cases of untreated epithelial ovarian tumors. Of these 40 tumors, 13 were classified as benign, 3 as borderline, and 24 as malignant, according to the WHO classification for ovarian tumors. Of 13 benign ovarian tumors, 4 (30.8%) showed chromosomal abnormalities. Of 4 ovarian tumors, 3 (75%) had single chromosomal abnormalities, and the remaining tumor (25%) retained multiple chromosomal abnormalities.

Expression of human telomerase reverse transcriptase and correlation with telomerase activity in placentas with and without intrauterine growth retardation

Background. Human telomerase reverse transcriptase has been found in telomerase‐positive tumor tissues, but not in telomerase‐negative nonmalignant somatic cells. Methods. Thirty‐two first‐trimester chorionic villi specimens (Group A), 33 second‐ and third‐trimester placenta specimens without asymmetric intrauterine growth retardation (Group B) and 13 specimens of placenta tissue from cases with intrauterine growth retardation (Group C) were examined for telomerase activity and expression of human telomerase reverse transcriptase by reverse transcription polymerase chain reaction and quantitative reverse transcription polymerase chain reaction. Results. Telomerase activity was detected in 29 of the 32 specimens (90.6%) in Group A, in 20 of the 33 specimens (60.6%) in Group B and none of the 13 specimens (0.0%) in Group C. Human telomerase reverse transcriptase was identified in all 32 specimens of Group A (100%), all 33 specimens of Group B (100%) and 2 of the 13 specimens in Group C (15.4%) by nested reverse transcription polymerase chain reaction. Copy numbers of human telomerase reverse transcriptase were 202.3±73.0 (n=32), 8.8±2.9 (n=33) and 0 (n=13) in Groups A, B, and C, respectively. Significant differences were observed between Groups A and B, Groups A and C, and Groups B and C (p<0.01, p<0.01, and p<0.01, respectively). Conclusions. Our findings indicate that human telomerase reverse transcriptase expression is the rate‐limiting determinant of telomerase activity in chorionic villi during the first trimester. Telomerase activity was not detected in placentas with intrauterine growth retardation, whereas human telomerase reverse transcriptase was expressed in some placentas with intrauterine growth retardation.

Rapid interphase cytogenetics with fluorochrome-directly-labeled probes.

細胞遺伝学的知識の集積に伴って染色体分析の必要性と需要が増し, 簡便で迅速な検査法の確立が望まれている.しかし, 分裂した細胞のみを対象とする従来の染色体分析法では熟練と時間が要求され, また培養に伴う細胞群の選択がおこる.蛍光インサイツーハイブリダイゼーション (FISH法) は間期細胞核においても染色体分析を可能にした (間期細胞遺伝学).ハプテン化染色体特異的プローブを用いたFISH法は, 染色体数の計算と数的異常の検出に汎用されてきたが, 約48時間を要し, ハプテン検出に伴う煩雑な免疫細胞化学的処理過程が必要である.この煩雑さを解消するため, 蛍光色素直接標識セントロメアプローブを用いて, 18トリソミー, 21トリソミーおよびXXYの染色体異常患者の末梢リンパ球にFISH法を適用したところ, おのおのの症例に対応するコピー数が得られた.しかも所要時間を2時間まで短縮できたうえ, 操作段階を半減することが可能であった.このように, 蛍光色素直接標識プローブによるFISH法は, 染色体の数的異常の, 迅速かつ手技的間違いのない簡便な検査法として, 染色体数異常疾患の診断に有用であることを明らかにした.

Detection of S-phase cells and tumor marker in ovarian carcinoma during chemothrapy. Immunohistochemical double staining with anti-BrdU monoclonal antibody.

アミラーゼ産生卵巣癌 (漿液性嚢胞腺癌) IV期症例にBromodeoxyuridine (BrdU) のinvivo標識を行い, 化学療法前後の摘出組織および捺印細胞標本について, 抗BrdUモノクローナル抗体を使用した酵素抗体法によりS期細胞の同定を行った. その結果, 組織における標識率は治療前11.6%, 治療後1%以下となり, 化学療法に伴ってS期細胞の著明な減少を認めた. これに伴って, 血中アミラーゼ値は化学療法中に正常値となったが, 治療後も残存組織中にアミラーゼ陽性細胞が検出され, 再燃時でもアミラーゼがマーカーとなることが示唆された. BrdU陽性細胞とアミラーゼ陽性細胞の分布を連続切片により検討したところ, 両者が同時に陽性を示した細胞は確認できず, BrdUとアミラーゼはそれぞれ独立したパラメーターとして使用できると思われた. 組織標本および捺印細胞標本に酵素抗体二重染色法を行ったがBrdUは核内に, アミラーゼは胞体内に, よいコントラストを示して染めわけられ組織と細胞の染色所見はよく一致した. 核の活動性の目安としてのBrdU染色と胞体の形質発現としての腫瘍マーカーを同一標本で分染できる二重染色は, 多数の均質な標本を得難い細胞診の場合にも有用な検査法であることが明らかになった.

Flow cytometric study on the cells of ovarian cysts and cystic carcinoma.

卵巣に発生する悪性腫瘍のおよそ75%は, 続発性腺癌で占められ, この生存率は, なお35%前後にとどまっている.「続発性」の意義は, 正常卵巣にまず発生した卵巣嚢胞の壁を構成する上皮細胞の増殖と2次的悪性化を示すものであり, この癌化過程を追求することは早期診断のため, 多くの知見をもたらすものと思われる.そこでわれわれは, 上皮性卵巣腫瘍を良性群, 中間群, 悪性群にわけ, その細胞をFlow cytometryによってDNAヒストグラムから解析, 比較検討し, 次のような結果を得た.1.良性群では漿液性嚢胞腺腫よりも, ムチン性嚢胞腺腫の方が高い増殖能を示した.2.中間群では, 低悪性ムチン性嚢胞腺腫, 腹膜偽粘液腫はムチン性嚢胞腺腫とほぼ同様の増殖能であった.3.悪性群は, 良性群, 中間群に比べ, より高い増殖能を示した.また悪性群のうち, 漿液性嚢胞腺癌はムチン性嚢胞腺癌より高い増殖能を示した.4.進行卵巣癌DNAでは, しばしば2峰性のDNAヒストグラムが得られ, cyclingをもつ細胞のなかにも, aneuploidyの存在が示唆された.5.化学療法実施前後のDNAヒストグラムの比較により, 治療効果判定に活用できる可能性が得られた.