生水 真紀夫

Objective: The aim of this study was to assess clinically the adequacy of vessel-contouring-based pelvic radiotherapy with regard to nodal coverage for uterine cervical cancer. Methods: Fifty patients with Stages I-III cervical cancer, treated with vessel-contouring-based three-dimensional radiotherapy since August 2002, were entered into the study (median age: 54, 47 received concurrent daily cisplatin). All patients were treated with external beam radiotherapy using a four-field box technique with or without brachytherapy. Pelvic blood vessels were identified and contoured on computed tomography simulation images. A generous margin was set outside these vessels outlined on digitally reconstructed radiograph accounting for normal size lymph nodes, patient's motion and set-up uncertainty. Multi-leaf collimator (MLC) was inserted and adjusted manually. Patterns of recurrence were clinically evaluated. Results: Distance between major vessels and MLC edges varied inter- and intra-individually. Median distance in the mid-iliosacral joint level was 25 mm (left) and 24 mm (right). The maximum and the minimum distances ranged from 25 to 45 mm (median, 32) and 9 to 27 mm (median, 15) for left side and 24 to 41 mm (median, 30) and 7 to 28 mm (median, 15) for right side, respectively. With a median follow-up of 43 months, 10 patients developed recurrence. However, no marginal recurrence was occurred just lateral to the contoured vessels. All three patients who developed regional recurrence had recurred at the internal iliac node or the obturator node medial to contoured vessels. Conclusions: Contoured vessels can be used as surrogate markers for location of the pelvic lymph nodes. © The Author (2009). Published by Oxford University Press. All rights reserved.

アロマターゼは、エストロゲン合成活性をもつ唯一の酵素である。顆粒膜細胞・黄体細胞・合胞体栄養膜細胞に強く発現するほか、脳・前脂肪細胞・平滑筋細胞・骨芽細胞・皮膚線維芽細胞などにも発現している。多重エクソンI構造をもつCYP19A1遺伝子によりコードされており、臓器ごとに特異的なプロモーターによって発現が制御されている。通常、転写は抑制的に制御されており、特定の誘導刺激によって転写が開始される。(著者抄録)

Microarray studies have identified many genes that are down-regulated in uterine leiomyoma compared with myometrium, including early growth response gene-1 (EGR1). However, the mechanisms underlying coordinated down-regulation of this gene cohort remain unknown. To address the transcriptional role of EGR1 in leiomyoma, EGR1 binding to promoter sequences on target genes was assessed by chromatin immunoprecipitation (ChIP) assay in leiomyoma tissues and myometrium-derived KW cells. Computer analysis demonstrated that 50 out of 135 genes listed as down-regulated in array reports possessed potential binding sites for EGR1 within 1 kb promoter sequence. ChIP assay was performed for a random selection of 13 genes possessing potential binding sites for EGR1 (Group A), 3 genes known as EGR1 targets in other tissues (Group B), and 4 control genes. Decreased EGR1 bindings were significant for 11 out of 16 genes (Group A+B) in leiomyoma tissues compared with myometrium, and mRNA levels in tissue samples were actually decreased for 7 out of the 11 genes. ChIP analyses performed on KW cells showed induction of EGR1 binding to the promoter region of all genes except one Group A+B gene, but for none of the control genes. These results indicate that EGR1 is a key player in coordinated down-regulation of genes in leiomyoma. Application of ChIP-quantitative PCR assay with the aid of computer-assisted analysis of genome databases appears useful for the comprehensive interpretation of array data.

OBJECTIVE: The purpose of this study was to investigate the diagnostic impact of preoperative serum p53 antibody (S-p53 Ab) in patients with endometrial cancer. STUDY DESIGN: A hospital-based series of 67 patients, comprising 58 endometrioid adenocarcinomas (EA) and 9 serous adenocarcinomas (SA) between 1998-2002 were included. First, preoperative pathology was compared with final pathology in terms of histologic classification and tumor grade. Second, S-p53 Ab and CA125 were measured using preoperative serum samples, and immunohistochemical staining for p53 protein was assessed using hysterectomy specimens. RESULTS: There were discrepancies between preoperative and final pathology in terms of histologic classification (7%) and tumor grade in EAs (30%); other objective tests, therefore, were needed to minimize the diagnostic problems. S-p53 Ab titers varied from 0.27-786 (mean, 124) in SAs and from 0.1-7.47 (mean, 0.46) U/mL in EAs, respectively, and were positive in 6 (67%) SAs and in 3 (6%) EAs using 1.3 U/mL as cut-off. S-p53 Ab-positive rate was significantly correlated with SA histology and grade 3 EA tumor (odds ratio, 40; P = .005; 95% confidence interval, 3.04-525.43) with higher sensitivity and higher specificity than p53 staining and CA125, respectively. CONCLUSION: S-p53 Ab could conveniently and specifically identify high-risk endometrial cancer.

Production of appropriate quantities of estrogen in various tissues is essential for human physiology. A single gene (CYP19), regulated via tissue-specific promoters, encodes the enzyme aromatase, which catalyzes the key step in estrogen biosynthesis. Aromatase excess syndrome is inherited as autosomal dominant and characterized by high systemic estrogen levels, short stature, prepubertal gynecomastia and testicular failure in males, and premature breast development and uterine pathology in females. The underlying genetic mechanism is poorly understood. Here, we characterize five distinct heterozygous rearrangements responsible for aromatase excess syndrome in three unrelated families and two individuals (nine patients). The constitutively active promoter of one of five ubiquitously expressed genes located within the 11.2 Mb region telomeric to the CYP19 gene in chromosome 15q21 cryptically upregulated aromatase expression in several tissues. Four distinct inversions reversed the transcriptional direction of the promoter of a gene (CGNL1, TMOD3, MAPK6 or TLN2), placing it upstream of the CYP19 coding region in the opposite strand, whereas a deletion moved the promoter of a fifth gene (DMXL2), normally transcribed from the same strand, closer to CYP19. The proximal breakpoints of inversions were located 17-185 kb upstream of the CYP19 coding region. Sequences at the breakpoints suggested that the inversions were caused by intrachromosomal nonhomologous recombination. Splicing the untranslated exon downstream of each promoter onto the identical junction upstream of the translation initiation site created CYP19 mRNA encoding functional aromatase protein. Taken together, small rearrangements may create cryptic promoters that direct inappropriate transcription of CYP19 or other critical genes.

BACKGROUND: There are therapeutic dilemmas regarding conservative management of endometrial cancer in young women. METHODS: We planned a prospective study to conservatively treat women aged under 40 years with clinical stage 1A, grade 1 endometrioid adenocarcinoma from 1999 to 2005. There were nine women (aged 28-40) who fulfilled the criteria, and medroxyprogesterone acetate (400 mg/day) was continued for 6 months. Curettage materials were pathologically evaluated according to our criteria including partial response (PR) (a small amount of cancer tissue with remarkable hormonal effects or atypical hyperplasia). To predict complete response (CR) to progestin, immunohistochemical staining for insulin-like growth factor type 1 receptor, phosphatase and tensin homolog deleted on chromosome ten, progesterone receptor (PgR), estrogen receptor and Ki67 were assessed. RESULTS: Seven (78%) and two cases presented complete and PRs, respectively. Two patients developed recurrent disease 10 and 22 months after the last dilatation and curettage, and both had synchronous ovarian cancer. However, all nine patients were alive and disease-free for a mean of 39 months. Of eight married patients, four (50%) conceived and three delivered full-term singletons. CR was related to positive expression of PgR (P=0.008). CONCLUSIONS: Patients with an initial PR can obtain CR after further treatment, and the PgR may be useful in predicting CR to fertility-preserving treatment in young women with endometrial cancer.

BACKGROUND: Malignant transformation of adenomyosis is a very rare event. Only about 30 cases of this occurrence have been documented till now. CASE PRESENTATION: The patient was a 57-year-old woman with a slightly enlarged uterus, who underwent total hysterectomy and unilateral adnexectomy. On gross inspection, the uterine wall displayed a single nodule measuring 5 cm and several small gelatinous lesions. Microscopic examination revealed a common leiomyoma and multiple adenomyotic foci. A few of these glands were transformed into a moderately differentiated adenocarcinoma. The endometrium was completely examined and tumor free. The carcinoma was, therefore, considered to be an endometrioid adenocarcinoma arising from adenomyosis. Four months later, an ultrasound scan revealed enlarged pelvic lymph nodes: a cytological diagnosis of metastatic adenocarcinoma was made. Immunohistochemical studies showed an enhanced positivity of the tumor site together with the neighbouring adenomyotic foci for estrogen receptors, aromatase, p53 and COX-2 expression when compared to the distant adenomyotic glands and the endometrium. We therefore postulate that the neoplastic transformation of adenomyosis implies an early carcinogenic event involving p53 and COX-2; further tumor growth is sustained by an autocrine-paracrine loop, based on a modulation of hormone receptors as well as aromatase and COX-2 local expression. CONCLUSION: Adenocarcinoma in adenomyosis may be affected by local hormonal influence and, despite its small size, may metastasize.

10年間にLEEP円錐切除を施行した子宮頸部初期病変症例について、後方視的に術前診断としての有用性と子宮温存治療としての有用性を検討し、円錐切除の適応と限界について考察を行った。その結果、1)パンチ生検診と円錐切除診の病理学的診断が一致したのは346例中226例(65.3%)であった。2)CIN IIの症例での切除断端陽性率は2.5%と低く、その後の観察で腫瘍の残存が確認された症例は認められなかった。3)子宮頸部初期扁平上皮病変に対するLEEP円錐切除は、診断的価値が高いことが確認された。またIa1期で脈管侵襲を認めた症例やIa2およびIb1期の症例では腫瘍残存率は高く、子宮温存には慎重に対応する必要性が考えられた。

Synovial sarcoma, a malignant mesenchymal neoplasm, occurs mostly near the joints of the extremities and occasionally outside the joint such as lung. We report a case of soft tissue sarcoma arising in the fallopian tube origin that showed characteristic pathological appearance of biphasic synovial sarcoma. Molecular analysis detected a fusion gene transcript of synovial sarcoma translocation (SYT) gene from chromosome 18 and synovial sarcoma X chromosome breakpoint 1 (SSX1) gene, which is believed to pathognomonic for synovial sarcoma of joint origin. Recurrent abdominal tumor, observed at 12 month after the initial surgery and following chemotherapy using doxorubicin, cisplatin and ifosfamide, partially responded to chemotherapy using paclitaxel and carboplatin and, then, optimal surgery was performed. This is the first report of a synovial sarcoma arising in the fallopian tube.

最近の研究で、父性インプリンティングを受けるp57KIP2の免疫染色により雄核発生の証明ができることが報告され、同染色を行えば全奇胎と部分・顕微鏡的奇胎とを鑑別しうると考えられている。今回、先行妊娠が部分奇胎または顕微鏡的奇胎と診断されていた存続絨毛症11例を対象に、先行妊娠の除去手術時に採取された組織標本を用いてp57KIP2免疫染色を行い、先行妊娠が本当に部分・顕微鏡的奇胎であったのか否かを検討した。結果、全例で栄養膜細胞と絨毛間質細胞がp57KIP2陰性を示したことから、全例とも本当は部分・顕微鏡的奇胎ではなく全奇胎であった可能性が高いと思われた。

1996〜2005年に経験した卵巣外原発性腹膜癌7例について臨床的検討を行った。年齢は47〜77歳(中央値66歳)、臨床進行期はII期3例、III期4例であった。治療法は3例に初回治療として手術が施行され、他の4例は術前化学療法後に手術が行われた。手術方法は全例、腹式単純子宮全摘+両側付属器切除+大網部分切除が施行され、2例には後腹膜切除、1例には直腸切除が併施された。初回治療として手術を選択された3例はいずれも術前検査で腹水がなく画像上卵巣の明らかな腫大を認めなかったためにダグラス窩腫瘍または子宮体癌が疑われたケースであり、3例とも原発部位・組織型の確定とoptimal debulking surgeryを目的に手術が行われた。術後の再発は初回手術群で2例(67%)、術前化学療法群で1例(25%)に認めた。Kaplan-Meier法による無病生存期間は両群間に有意差を認めなかった。

OBJECTIVE: To evaluate the inhibitory effect of danazol on estrogen (E) production in endometriosis. DESIGN: Prospective randomized study. SETTING: Academic research unit of the department of obstetrics and gynecology in a university hospital. PATIENT(S): Thirteen patients with endometriosis. INTERVENTION(S): Danazol was added to the culture of endometriosis-derived stromal cells or suspensions of microsomes prepared from chocolate cysts. MAIN OUTCOME MEASURE(S): The aromatase activities as well as mRNA and protein levels of aromatase in endometriosis-derived stromal cells or microsomes of endometriosis were examined. RESULT(S): Danazol treatment with a concentration greater than 10(-6) M significantly suppressed aromatase activity of endometriosis-derived stromal cells under basal and prostaglandin E(2) (PGE(2))-stimulated conditions. Danazol (10(-5) M) did not affect mRNA and protein levels of aromatase. Danazol competitively inhibited aromatase activity (by 1.7 x 10(-6) M of calculated Ki and 2.9 x 10(-5) M of Ki') of endometriosis microsomes. CONCLUSION(S): Danazol competitively inhibited aromatase activity in endometriosis-derived stromal cells without affecting either the mRNA or protein levels of aromatase. These results indicate the efficacy of local application of danazol to endometriotic lesions.

OBJECTIVE: To evaluate the efficacy of a drug delivery system composed of danazol-loaded hyaluronic acid for local application to endometriosis. DESIGN: Prospective, randomized study. SETTING: Academic research unit of the department of obstetrics and gynecology in a university hospital. PATIENT(S): Adult female Sprague-Dawley rats. INTERVENTION(S): Danazol-loaded hyaluronic acid hydrogel (DZ-HA gel) was injected into the rat endometriosis model. MAIN OUTCOME MEASURE(S): Size and histological changes in experimental endometriosis, the concentration of danazol in the cyst wall and plasma, and estrous cycles were examined. RESULT(S): Histologically, DZ-HA gel-treated cysts displayed marked atrophy of the endometrial epithelium. Increased numbers of apoptotic cells and decreased numbers of proliferative cells were noted with 10 mg/mL DZ-HA gel. Size of treated cysts decreased to approximately 60% at 9 weeks after injection. The estrous cycles were not disturbed during DZ-HA gel treatment. CONCLUSION(S): Local injection of DZ-HA gel achieved endometrial atrophy of an experimental model of endometriosis without disturbing the sexual cycle. These results suggest that local application of DZ using this drug delivery system may prove useful for treating endometriosis.

The intestinal-carriage rates of Clostridium difficile in neonates hospitalized in the University Hospital's Center for Perinatal and Reproductive Health and in infants and children enrolled in two day-nurseries and a kindergarten were examined. Swab samples from the floors of these facilities were also analyzed to determine the extent of environmental contamination by this organism. C. difficile was found in the stool of only one of 40 neonates during the normal 1-week stay in the hospital after delivery. The isolate from the neonate was identical to that of her mother, as determined by PCR ribotyping, pulsed-field gel electrophoresis analysis, and toxin gene type, suggesting that the C. difficile-positive neonate acquired the organism from her mother rather than from the environment. By contrast, 47 (48.0%) of the 98 infants and children, comprising 50 enrolled in two day-nurseries who were >= 3 years old and 48 enrolled in a kindergarten who were 2-5 years old, carried C. difficile. The carriage rate in infants under 2 years of age was much higher (84.4%) than in children 2 years old and older (30.3%). When analyzed according to age group, the carriage rates were 100, 75.0, 45.5, 24.0, 38.5, and 23.5% in infants and children 0, 1, 2, 3, 4, and 5 years old, respectively. The observation that several children were colonized with the same type of C. difficile strain in each day-care facility, and that the floors of day-nursery A and kindergarten C were contaminated with C. difficile strains identical to those colonizing the intestines of children enrolled in those facilities suggests that cross-infection of C. difficile among children occurs through C. difficile-carrying children or their contaminated environments.

Purpose and Experimental Design: To assess the prognostic significance of intratumoral aromatase in endometrioid endometrial cancer, sections from 55 patients with endometrial cancer were evaluated for expression of aromatase using immunohistochemistry, and the correlation between aromatase expression and clinicopathologic parameters were analyzed. Results: Immunohistochemical staining for aromatase was positive for 32 (58%), 20 (36%), and 19 (34%) patients in cancer epithelial cells, stromal cells, and myometrial cells around the flank invasion, respectively. In situ hybridization also detected aromatase mRNA in all three types of cells. RT-PCR analysis revealed that aromatase mRNA was 2.5 +/- 1.0 amol/mu g total RNA (mean +/- SE; n = 7) in tumor tissue. Western blot analysis detected the expected aromatase protein size of 58 kDa in cancer tissues more abundantly than in cancer-free endometrium (n = 3). The immunoreactivity in stromal cells correlated positively with advanced surgical stage and poor survival. Survival analysis revealed that the immunoreactivity of stromal cells was a significant prognostic factor, independent of histologic grade, muscular invasion, and lymph node metastasis, but dependent on surgical stage. By contrast, the immunoreactivity of aromatase both in cancer epithelial cells and myometrial cells did not correlate with prognosis. Conclusions: To the best of our knowledge, this is the first evidence associating intratumoral aromatase expression in stromal cells and poor survival in endometrioid endometrial cancer. This positive linkage indicates that local expression of aromatase plays a role in tumor progression through the formation of in situ estrogens. In situ expression of aromatase may offer a potential target for management of endometrial cancers.

Expression of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) was compared between leiomyoma and myometrium. Cytosolic fractions from leiomyoma homogenate displayed 5-fold higher activity ( estrone to estradiol), compared with surrounding myometrium (n = 6, P < 0.05), whereas microsomal fractions showed no difference. Oxidative activity ( estradiol to estrone) did not differ between leiomyoma and myometrium. Levels of mRNA for 17 beta-HSDs were then measured using real-time PCR techniques. Among the eight different types of 17 beta-HSDs (types 1-5, 7, 8, and 10), type 1 was the only enzyme displaying differential expression between leiomyoma and myometrium. Mean concentration of type 1 17 beta-HSD mRNA was 4-fold higher in leiomyoma than in surrounding myometrium (n = 20, P < 0.05). Type 1 transcript levels correlated significantly with reductive activity in individual samples (n = 6, P < 0.05). Northern blot analysis of leiomyoma and myometrium tissues detected 2.3- and 1.0-kb transcripts of type 1 enzyme, whereas the major 1.3-kb transcript for 17 beta-HSD in placenta-derived JEG-3 cells was not detected. None of the factors increasing mRNA levels for type 1 enzyme in placenta increased mRNA levels in leiomyoma. These results indicate that leiomyoma tissues overexpress type 1 17 beta-HSD, resulting in high conversion of estrone to estradiol. In situ expression of type 1 17 beta-HSD may play a role in self-supported growth of leiomyoma cells.

Expression of early growth response (Egr)-1, a transcriptional factor implicated in growth regulation, is suppressed in several malignant tumors. The present study investigated the expression of Egr-1 and related genes in uterine leiomyoma and normal myometrium to determine possible contributions of Egr-1 to neoplastic growth in leiomyoma cells. Levels of Egr-1 transcripts were decreased in all leiomyomas (n = 20) to approximately 10% of levels in corresponding myometrium, where basal expression was high. Preoperative leuprorelin acetate therapy increased levels of Egr-1 mRNA in normal myometrium only. Northern blot analysis using additional sample sets (n 5) revealed the full-length Egr-1 transcript. Western blot analysis (n 5) confirmed decreased expression of Egr-1 protein. Southern blot analysis of the Egr-1 gene and microsatellite analysis of the chromosomal location at 5q31 (D5S414, D5S500, and D5S476) revealed neither DNA recombination nor loss of heterozygosity in leiomyomas. Moreover, Egr-1 retained identical responsiveness to phorbol 12-myristate 13-acetate in primary cultures derived from both leiomyoma and normal tissues. Electrophoretic mobility shift analysis revealed that phorbol 12-myristate 13-acetate-induced Egr-1 in leiomyoma cells retained DNA binding ability. Egr-1 thus appears functionally intact in leiomyoma cells. Finally, consistent with the role of Egr-1 in growth inhibition, transfection of Egr-1 expression vector into a myometrial cell line (KW) that expresses low levels of Egr-1 and displays rapid growth inhibited thymidine uptake in these cells. Egr-1 may display tumor-suppressing activity and offers a potential target for leiomyoma management.

Aim: A number of studies for the measurement of cell-free fetal DNA in maternal blood have been reported; however, their clinical significance has remained unclear. We proposed to clarify the relationship between fetal DNA levels and obstetrical disorders. Methods: One hundred and eighty-five cases of normal pregnancy, ranging from 8 to 40 weeks' gestation, and 70 cases of abnormal pregnancy were included. SRY levels in maternal plasma were quantified with a real-time quantitative polymerase chain reaction. Results; Sex-determining region Y (SRY) levels and the number of patients with positive levels peaked at 3336 weeks in normal pregnancy The SRY levels in threatened abortion (11.6 +/- 4.8 copies/mL to 0 +/- 0, P < 0.05) and threatened preterm labor (44.6 +/- 16.1 copies/mL to 15.9 +/- 6.2, P < 0.01) were significantly higher than those of the normal group. In pre-eclamptic patients, SRY levels were markedly higher than those of the normal group (173.2 +/- 94.8 copies/mL to 22.4 +/- 8.9, P < 0.05). Patients with premature separation of the placenta (266.8 +/- 137.1 copies/mL to 4.9 +/- 3.7, P < 0.05) and placenta previa (167.7 +/- 32.4 copies/mL to 37.0 +/- 17.3, p< 0.01) also showed elevated SRY levels. Conclusion: Sex-determining region Y levels in maternal plasma were elevated in patients with an abnormal pregnancy, particularly those with placental injury of damage. These results suggested that increased SRY levels are consistently caused by the leak of fetal components, and thus the measurement of SRY levels in maternal plasma is useful for the evaluation of placental injuries.

In leiomyoma of the uterus, both aromatase and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type I are overexpressed compared with myometrium. This suggests that leiomyoma cells convert circulating androstenedione into estrone (via aromatase), then into the active form of estrogen, estradiol (via 17beta-HSD type 1). In vitro experiments and several clinical findings support the notion that in situ estrogen plays a role in leiomyoma growth under hypoestrogenemic conditions, such as natural menopause and therapy with gonadotropin-releasing hormone (GnRH) agonists. GnRH agonists abolish estrogen production both in situ in leiomyoma and in the ovary, leading to quick and profound regression of the leiomyoma. Aromatase inhibitors also inhibit estrogen synthesis in both leiomyoma and the ovary and may be used therapeutically. Certain doses of competitive aromatase inhibitors would completely inhibit estrogen production in leiomyoma, whereas ovarian production of estrogen would continue at reduced levels. This may lead to advantageous therapeutic conditions in which leiomyoma regresses without adverse symptoms related to estrogen depletion because levels of ovarian estrogen would be insufficient to support leiomyoma growth but sufficient to prevent symptoms associated with deficiency. This article discusses the potential uses of aromatase inhibitors.

Several methods for detection of fetal components in maternal blood have been reported. However, few have proven clinically useful for determining the treatment in cases of placental injuries. Here, we report a case of extensive intervillous hematoma diagnosed at 25 weeks of gestation with severe intrauterine growth restriction and oligohydramnios. Marked elevation of fetal DNA levels was observed in maternal blood. Fetal DNA levels decreased after 27 weeks of gestation, concurrent with recovery of fetal growth. We conservatively managed this case until 30 weeks of gestation, when a male infant was delivered. He weighed 508 g and displayed Apgar scores of 7 at 1 minute and 9 at 5 minutes. Histological examination of the placenta revealed intervillous thrombosis without infarction or inflammatory changes. In this case, decreasing fetal DNA levels in maternal plasma correlated with recovery of fetal growth and provided useful information for fetal management as well as insight into the pathogenesis of placental injuries.

The human CYP19 (P450arom) gene is located in the chromosome 15q21.2 region and is comprised of a 30 kb coding region and a 93 kb regulatory region. The Internet-based Human Genome Project data enabled us to elucidate its complex organization. The unusually large regulatory region contains 10 tissue-specific promoters that are alternatively used in various cell types. Each promoter is regulated by a distinct set of regulatory sequences in DNA and transcription factors that bind to these specific sequences. In most mammals, P450arom expression is under the control of gonad- and brain-specific promoters. In the human, however, there are at least eight additional promoters that seemed to have been recruited throughout the evolution possibly via alterations in DNA. One of the key mechanisms that permit the recruitment of such a large number of promoters seems to be the extremely promiscuous nature of the common splice acceptor site, since activation of each promoter gives rise splicing of an untranslated first exon onto this common junction immediately upstream of the translation start site in the coding region. These partially tissue-specific promoters are used in the gonads, bone, brain, vascular tissue, adipose tissue, skin, fetal liver and placenta for physiologic estrogen biosynthesis. The most recently characterized promoter (I.7) was cloned by analyzing P450arom mRNA in breast cancer tissue. This TATA-less promoter accounts for the transcription of 29-54% of P450arom mRNAs in breast cancer tissues and contains endothelial-type cis-acting elements that interact with endothelial-type transcription factors, e.g. GATA-2. We hypothesize that this promoter may upregulate aromatase expression in vascular endothelial cells. The in vivo cellular distribution and physiologic roles of promoter I.7 in healthy tissues, however, are not known. The gonads use the proximally located promoter II. The normal breast adipose tissue, on the other hand, maintains low levels of aromatase expression primarily via promoter I.4 that lies 73 kb upstream of the common coding region. Promoters I.3 and II are used only minimally in normal breast adipose tissue. Promoters II and I.3 activities in the breast cancer, however, are strikingly increased. Additionally, the endothelial-type promoter I.7 is also upregulated in breast cancer. Thus, it appears that the prototype estrogen-dependent malignancy breast cancer takes advantage of four promoters (II, I.3, I.7 and I.4) for aromatase expression. The sum of P450arom mRNA species arising from these four promoters markedly increase total P450arom mRNA levels in breast cancer compared with the normal breast.

BACKGROUND: Gynecomastia of prepubertal onset may result from increased estrogen owing to excessive aromatase activity in extraglandular tissues. A gene in chromosome 15q21.2 encodes aromatase, the key enzyme for estrogen biosynthesis. Several physiologic tissue-specific promoters regulate the expression of aromatase, giving rise to messenger RNA (mRNA) species with an identical coding region but tissue-specific 5'-untranslated regions in placenta, gonads, brain, fat, and skin. METHODS: We studied skin, fat, and blood samples from a 36-year-old man, his 7-year-old son, and an unrelated 17-year-old boy with severe gynecomastia of prepubertal onset and hypogonadotropic hypogonadism caused by elevated estrogen levels. RESULTS: Aromatase activity and mRNA levels in fat and skin and whole-body aromatization of androstenedione were severely elevated. Treatment with an aromatase inhibitor decreased serum estrogen levels and normalized gonadotropin and testosterone levels. The 5'-untranslated regions of aromatase mRNA contained the same sequence (FLJ) in the father and son and another sequence (TMOD3) in the unrelated boy; neither sequence was found in control subjects. These 5'-untranslated regions normally make up the first exons of two ubiquitously expressed genes clustered in chromosome 15q21.2-3 in the following order (from telomere to centromere): FLJ, TMOD3, and aromatase. The aromatase gene is normally transcribed in the direction opposite to that of TMOD3 and FLJ. Two distinct heterozygous inversions reversed the direction of the TMOD3 or FLJ promoter in the patients. CONCLUSIONS: Heterozygous inversions in chromosome 15q21.2-3, which caused the coding region of the aromatase gene to lie adjacent to constitutively active cryptic promoters that normally transcribe other genes, resulted in severe estrogen excess owing to the overexpression of aromatase in many tissues.

Objective: To assess the management of symptomatic leiomyomas using a nonsteroidal aromatase inhibitor in perimenopausal women. Design: Case report. Setting: Academic clinical practice. Patient(s): A 53-year-old woman suffering from recurrent urinary retention secondary to a uterine leiomyoma. Intervention(s): Fadrozole, orally, 2 mg daily for 8 weeks and then 1 mg daily for 4 weeks. Main Outcome Measure(s): Measurements of leiomyoma volume, and levels of serum E-2, LH, and FSH. Result(s): Urinary retention resolved after 2 weeks of treatment and did not recur. Leiomyoma volume estimated by ultrasonography revealed a 71% reduction after 8 weeks of treatment. Conclusion(s): Fadrozole was useful for the management of a symptomatic leiomyoma without transient deterioration of symptoms. Clinical trials are warranted. (Fertil Steril((R)) 2003;79:628-31. (C) 2003 by American Society for Reproductive Medicine.).

Intratumoral expression of aromatase P450 (P450arom) promotes the growth of breast tumors via increased local estrogen concentration. We cloned a novel 101-bp untranslated first exon (1.7) that comprises the 5'-end of 29-54% of P450arom transcripts isolated from breast cancer tissues (n = 7). The levels of P450arom transcripts with exon 1.7 were significantly increased in breast tumor tissues and adipose tissue adjacent to tumors. We identified a promoter immediately upstream of exon 1.7 and mapped this to about 36 kb upstream of ATG translation start site of the CYP19 (aromatase cytochrome P450) gene. Sequence analysis of 1.7 revealed a TATA-less promoter containing an initiator, two consensus GATA sites, and cis-regulatory elements found in megakaryocytes and endothelial type promoters. Luciferase activity directed by the promoter 1.7 sequence (-299/+81 bp) was 4-fold greater than a minimum length promoter sequence (-35/+81 bp) in human microvascular endothelial cells (HMEC-1), but only 2-fold greater in MCF-7 breast malignant epithelial cells. There was no promoter activity in primary breast adipose fibroblasts. Site-directed mutations demonstrated that maximal basal promoter activity required two GATA motifs at -146/-141 bp and -196/-191 bp. Gel shift and deoxyribonuclease I footprinting assays demonstrated the binding of GATA-2 transcription factor but not GATA-1 to the -196/-191-bp region. Overexpression of GATA-2 in HMEC-1 cells increases promoter 1.7 activity by 5-fold. In conclusion, promoter 1.7 is a GATA-2-regulated endothelial promoter of the human CYP19 gene and may increase estrogen biosynthesis in vascular endothelial cells of breast cancer. The activity of this promoter may also be important for intracrine and paracrine effects of estrogen on blood vessels.

Endometriosis is an estrogen-dependent disease of women of reproductive age. Recent studies demonstrate that endometriosis per se express high levels of estrogen synthetase (aromatase P450). The resulting estrogen synthesized in situ may play a role in the development and exacerbation of the disease. For ovarian endometrioma, previous studies have been conducted ex vivo using cells obtained from endometrioma and have demonstrated that steroidogenic factor-1 is involved in the expression of aromatase. The aim of the present study was to provide in vivo evidence that steroidogenic factor-1 plays an important role in the regulation and overexpression of aromatase P450 in situ. First, promoter use of aromatase P450 in endometrioma tissue was determined using quantitative methods. Ovarian endometrioma tissue was chopped into small pieces, and two exon 1-specific transcripts of aromatase P450 (PII-specific and I.4-specific transcripts) were quantified using competitive RT-PCR. PII-specific transcript was more abundant than the I.4-specific transcript in 13 of the 15 endometriomas and less abundant in the remaining two. Spatial distribution of aromatase P450 transcripts in these endometrioma tissues revealed heterogeneous expression in the cyst wall, demonstrating wide variability even in the same endometrioma. Two possible regulators of aromatase expression (steroidogenic factor-1 and IL-1beta) were then measured in all endometrioma samples and the correlation between aromatase P450 transcripts and these possible regulators in the endometrioma samples were tested using Spearman's rank order correlation test. Levels of steroidogenic factor-1 transcript were found to correlate closely with levels of PII-specific transcript in eight of nine endometriomas examined. On the other hand, the level of IL-1beta weakly correlated with I.4-specific transcripts in three of the nine endometriomas. We next histologically examined samples of four endometriomas in which complete sets of tissue samples corresponded to the RNA samples. We could not identify any specific pathology to explain the heterogenous expression of PII-specific transcripts of aromatase P450, although the number of CD-68 positive macrophages in the tissue sections weakly correlated with the level of I.4-specific transcript in two of four endometriomas. These results provide strong evidence that promoter II is the predominant promoter of aromatase P450 in endometrioma. tissues in vivo and that steroidogenic factor-1 in situ is a major determinant of aromatase P450 overexpression in endometrioma tissues in vivo.

The CYP19 gene encoding aromatase P450 (estrogen synthetase) is expressed in several extragonadal sites and regulated in a tissue-specific fashion, which is achieved by alternative use of the seven different promoters (and corresponding exons 1) of the CYP19 gene. Previously, we demonstrated that aromatase P450 is overexpressed in leiomyoma tissue and that in situ estrogen synthesized in leiomyoma tissues possibly plays a role in leiomyoma growth. To elucidate the mechanism of overexpression of aromatase P450, we determined the promoter use of aromatase P450 in leiomyomas. 5'-Rapid amplification of cDNA ends analysis revealed that of six leiomyoma nodules tested, four nodules contained I. 4-specific transcript of aromatase P450 alone, one nodule contained PII-specific transcript alone, and the remaining nodule contained both 1.4- and PII-specific transcripts simultaneously. The levels of aromatase transcripts were then quantified by competitive RT-PCR assay. Among 21 leiomyomas, I.4-specific transcript and PII-specific transcript were predominant in 18 and 2 leiomyomas, respectively, whereas the remaining leiomyoma was negative for aromatase P450 expression. We next compared the aromatase activity of leiomyoma cells stimulated by promoter-specific regulatory factors. A combination of IL-1beta and dexamethasone, known as a potent inducer of promoter I.4-driven transcription, effectively increased aromatase activity. A combination of (Bt)(2)cAMP, 3-isobutyl-1-myethylxanthine, and PGE(2), known as inducers of promoter II-driven transcription, also increased aromatase activity, but the increases found were smaller than that induced by dexamethasone and IL-1beta. The transcriptional ability of the promoter 1.4 sequence was confirmed by transient transfection assay using primary cells released from leiomyomas and established cells from normal myometrium (KW cells). Luciferase vectors containing promoter I.4 sequence (-340/+14 or longer) showed a significant increase in luciferase activity in response to dexamethasone. Deletion or mutation of a putative glucocorticoid-responsive element in the promoter 1.4 sequence eliminated promoter activity. These results indicate that promoter 1.4 is the major promoter responsible for overexpression of aromatase P450 in leiomyomas and that a glucocorticoid-responsive element within it plays a substantial role in the expression of aromatase P450.