The key role of stem cell factor/KIT signaling in the proliferation of blast cells from Down syndrome-related leukemia.

Transient leukemia (TL) has been observed in approximately 10% of newborn infants with Down syndrome (DS). Although treatment with cytarabine is effective in high-risk TL cases, approximately 20% of severe patients still suffer early death. In this study, we demonstrate abundant KIT expression in all 13 patients with GATA1 mutations, although no significant difference in expression levels was observed between TL and acute myeloid leukemia. Stem cell factor (SCF) stimulated the proliferation of the TL cells from five patients and treatment with the tyrosine kinase inhibitor imatinib suppressed the proliferation effectively in vitro. To investigate the signal cascade, we established the first SCF-dependent, DS-related acute megakaryoblastic leukemia cell line, KPAM1. Withdrawal of SCF or treatment with imatinib induced apoptosis of KPAM1 cells. SCF activated the RAS/MAPK and PI3K/AKT pathways, followed by downregulation of the pro-apoptotic factor BIM and upregulation of the anti-apoptotic factor MCL1. Although we found novel missense mutations of KIT in 2 of 14 TL patients, neither mutation led to KIT activation and neither reduced the cytotoxic effects of imatinib. These results suggest the essential role of SCF/KIT signaling in the proliferation of DS-related leukemia and the possibility of therapeutic benefits of imatinib for TL patients.

Physical association of the patient-specific GATA1 mutants with RUNX1 in acute megakaryoblastic leukemia accompanying Down syndrome.

Mutations of the GATA1 gene on chromosome X have been found in almost all cases of transient myeloproliferative disorder and acute megakaryoblastic leukemia (AMKL) accompanying Down syndrome (DS). Although most GATA1 mutations lead to the expression of GATA1s lacking the N-terminal activation domain, we recently found two novel GATA1 proteins with defects in another N-terminal region. It has been suggested that loss of the N-terminal portion of GATA1 might interfere with physiological interactions with the critical megakaryocytic transcription factor RUNX1, and this would imply that GATA1s is not able to interact properly with RUNX1. However, the interaction domain of GATA1 remains controversial. In this study, we show that GATA1 binds to RUNX1 through its zinc-finger domains, and that the C-finger is indispensable for synergy with RUNX1. All of the patient-specific GATA1 mutants interacted efficiently with RUNX1 and retained their ability to act synergistically with RUNX1 on the megakaryocytic GP1balpha promoter, whereas the levels of transcriptional activities were diverse among the mutants. Thus, our data indicate that physical interaction and synergy between GATA1 and RUNX1 are retained in DS-AMKL, although it is still possible that increased RUNX1 activity plays a role in the development of leukemia in DS.

Cerebral venous thrombosis in adult nephrotic syndrome due to systemic amyloidosis.

Although venous thrombosis is one of the common complications in nephrotic patients, cerebral venous thrombosis (CVT) is rarely reported. CVT is so difficult to be detected by conventional diagnostic methods that it is sometimes overlooked despite its potential severity. We report a 79-year-old female with nephrotic syndrome due to systemic amyloidosis who suddenly altered mental status during her hospitalization. The underlying etiology had been not identified by physical examinations, various laboratory data, and repeated computed tomography, and finally she died. The post-mortem examination showed a massive thrombus impacted in intracranial left-sided transverse and sigmoid sinus. This case suggests that CVT can occur in a nephrotic patient who presents unexplained neurological signs and symptoms, which might not be detected only through conventional diagnostic tests.

Demonstration of focal p53 expression without genetic alterations in endometriotic lesions.

Their monoclonal origin (as indicated by recent investigations) indicates the neoplastic nature of most endometriotic lesions. p53, a representative tumor suppressor, regulates cell proliferation, and genetic alterations in p53 are involved in carcinogenesis in a wide variety of human cancers. The aim of this study was to examine endometriotic lesions for p53 expression and genetic alterations in p53. An immunohistochemical study revealed that 20% (13/64) of endometriotic lesions showed focal p53 expression in the epithelial cells. Using serial paraffin sections, we employed a microdissection method to extract DNA from the endometriotic tissues that showed p53 expression. No mutations were found in exons 5-8 in p53 by cleavase fragment length polymorphism scanning and polymerase chain reaction-DNA sequencing. Moreover, neither loss of heterozygosity nor microsatellite instability was detected at the microsatellite marker sites of p53. These results suggest that the focal p53 expression recognized in the endometriotic epithelia may be due to overproduction of wild-type p53 protein.

Genetic alterations in microsatellite marker sites among tumor suppressor genes in endometriosis.

Four endometriotic lesions were examined for the presence of genetic alterations in microsatellite marker sites among eight tumor suppressor genes. For this, a microdissection method was used on paraffin sections. Only one instance of loss of heterozygosity was detected at the PTCH locus. Heterozygosity was retained (indicating the absence of both loss of heterozygosity and microsatellite instability) at the other seven tumor suppressor gene loci in all the cases. Among the tumor suppressor genes examined, genetic defects in these microsatellite regions are certainly not ubiquitous in endometriosis and may be uncommon.

Desferrioxamine, an iron chelator, upregulates cyclooxygenase-2 expression and prostaglandin production in a human macrophage cell line.

Prostaglandins (PGs) play regulatory roles in a variety of physiological and pathological processes, including the immune response, cytoprotection and inflammation. Desferrioxamine (DFX), an iron chelator, is known to reduce free radical-mediated cell injury and to upregulate certain inflammatory mediators. We investigated the effects of DFX on the production of PGs and the expression of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in the synthesis of PGs, using a human macrophage cell line, U937. Our results showed that COX-2 expression and PGE(2) production are upregulated by DFX treatment and that this upregulation is dependent on both COX-2 promoter activity and alteration of mRNA stability. COX-2 promoter activity may be, at least in part, mediated by activation of the extracellular signal-regulated kinase pathway. These findings suggest that iron metabolism may regulate inflammatory processes by modulating PGs as well as other inflammatory mediators.

Transcription factor BACH1 is recruited to the nucleus by its novel alternative spliced isoform.

The transcription factor Bach1 is a member of a novel family of broad complex, tramtrack, bric-a-brac/poxvirus and zinc finger (BTB/POZ) basic region leucine zipper factors. Bach1 forms a heterodimer with MafK, a member of the small Maf protein family (MafF, MafG, and MafK), which recognizes the NF-E2/Maf recognition element, a cis-regulatory motif containing a 12-O-tetradecanoylphorbol-13-acetate-responsive element. Here we describe the gene structure of human BACH1, including a newly identified promoter and an alternatively RNA-spliced truncated form of BACH1, designated BACH1t, abundantly transcribed in human testis. The alternate splicing originated from the usage of a novel exon located 5.6 kilobase pairs downstream of the exon encoding the leucine zipper domain, and produced a protein that contained the conserved BTB/POZ, Cap'n collar, and basic region domains, but lacked the leucine zipper domain essential for NF-E2/Maf recognition element binding. Subcellular localization studies using green fluorescent protein as a reporter showed that full-length BACH1 localized to the cytoplasm, whereas BACH1t accumulated in the nucleus. Interestingly, coexpression of BACH1 and BACH1t demonstrated interaction between the molecules and the induction of nuclear import of BACH1. These results suggested that BACH1t recruits BACH1 to the nucleus through BTB domain-mediated interaction.

Epithelioid trophoblastic tumor metastatic to the vagina: an immunohistochemical and ultrastructural study.

We describe an epithelioid trophoblastic tumor (ETT) metastatic to the vagina in a 30-year-old Japanese woman. A polypoid tumor in the vaginal orifice was composed of nests of intermediate trophoblastic cells that showed a striking epithelioid appearance. In the hysterectomy specimen, a tumor infiltrated through the myometrium and showed histologic findings similar to those of the vaginal tumor. The tumor cells were positive for cytokeratin, inhibin-alpha, and melanoma cell adhesion molecule (Mel-CAM, CD146) but were only focally positive for human placental lactogen. Electron microscopic examination revealed bundles of well-developed, intermediate-type filaments surrounding the nuclei.

Proliferative activity and genetic alterations in TP53 in endometriosis.

In the secretory phase of the menstrual cycle, proliferative activity and expression levels of cell cycle-regulatory molecules are higher in endometriotic lesions than in the corresponding eutopic endometrium. In some endometriotic lesions, proliferative activity remains high in the postmenopausal period. By immunostaining, one can recognize TP53 overexpression in epithelial cells in a considerable number of endometriotic lesions. In those endometriotic lesions that show TP53 overexpression, neither TP53 point mutations (exons 5 to 8) nor microsatellite alterations (microsatellite instability or loss of heterozygosity) has been detected. Thus, the TP53 overexpression demonstrated in endometriotic epithelia seems to result from an overproduction of wild-type TP53 protein. The biological role of TP53 in endometriotic lesions is still unclear.

Enhancement of antitumor effect of bleomycin by low-voltage in vivo electroporation: a study of human uterine leiomyosarcomas in nude mice.

Uterine leiomyosarcoma is an extremely malignant neoplasm with high rates of distant metastasis, and systemic chemotherapy is not particularly effective. Thus, the introduction of more active anticancer agents, or of a new drug delivery system, is urgently needed. Recently, electrochemotherapy has been introduced as a way of enhancing the cytotoxic effects of chemotherapeutic agents. This involves administering the drug in combination with electric pulses (which permeabilize tumor cell membranes and allow the drug to enter the cells). In particular, bleomycin (BLM) cannot cross the plasma membrane efficiently, but its cytotoxicity can be enhanced by electropermeabilization. The aim of the present study was to investigate the effect of low-voltage electroporation (EP) in combination with local BLM injection on the growth of uterine leiomyosarcoma in nude mice. Human uterine leiomyosarcoma cells (SK-LMS-1) were implanted subcutaneously into nude mice. Tumor growth in mice treated with EP (100 V/cm) plus BLM was compared with that in mice receiving BLM alone, EP alone, or no treatment (controls). Tissue BLM concentrations and histological analysis (including mitotic counts) were evaluated in tumor tissues. There was a significant reduction in tumor growth in mice that received EP with BLM. One hour after the treatment, the local BLM concentration was 10 times higher in the tumors that received EP with BLM than in those receiving only BLM. Moreover, the mitotic count was lower in the tumors that received EP plus BLM than in the controls. These results demonstrate the possible therapeutic value of low-voltage EP with BLM in human uterine leiomyosarcoma.

Functional characterization of the two alternative promoters of human p45 NF-E2 gene.

OBJECTIVE: The transcription factor NF-E2, a heterodimeric protein complex composed of p45 and small Maf family proteins, is considered crucial for the proper differentiation of erythrocytes and megakaryocytes in vivo. We report the results of studies aimed at understanding the regulatory mechanisms controlling p45 gene expression in erythroid cells. MATERIALS AND METHODS: Human p45 mRNAs have two alternative isoforms, aNF-E2 and fNF-E2, and these isoforms are transcribed from the alternative promoters. We investigated lineage-specific expression of both isomers in human erythroid and megakaryocytic cells by reverse transcriptase polymerase chain reaction or Northern blot analysis. For functional characterization of both promoters, plasmids in which reporter genes were placed under the control of a series of truncated or mutated promoter fragments were transfected to human hematopoietic cell lines. RESULTS: When CD34(+) cells isolated from human cord blood were induced to unilineage erythroid or megakaryocytic differentiation in liquid suspension culture, both transcripts, although barely detected at day 0, were induced in both erythroid and megakaryocytic cultures. fNF-E2 mRNA was found to be more abundant in erythroid cells than megakaryocytic cells at day 7 of culture. Although both isomers were expressed in human erythroid-megakaryocytic cell lines, megakaryocytic maturation with loss of erythroid phenotype induced by phorbol 12-myristate 13-acetate (PMA) resulted in exclusive downregulation of fNF-E2, suggesting that fNF-E2 promoter is more erythroid specific. Functional analysis of fNF-E2 promoter showed that the promoter is active only in erythroid-megakaryocytic cells and that the double GATA site in the proximal region is necessary for its efficient activity. CONCLUSION: These results suggest that GATA proteins, which govern the differentiation of erythroid lineage cells, are required for full promoter activity of the p45 gene.

Immunohistochemical analysis of CA125, CA19-9, and Ki-67 in stage III or IV endometriosis: positive correlation between serum CA125 level and endometriotic epithelial cell proliferation.

BACKGROUND: Serum levels of CA125 and CA 19-9 are often elevated in patients with endometriosis, but the clinical or biological significance of this is not well established. The aim of the present study was to compare serum and tissue levels of CA125 and CA19-9, and to examine the correlation between these levels and cell proliferation using immunohistochemical analysis in stage III or IV endometriosis. METHODS: Forty-five cases diagnosed histologically as endometriosis were analyzed (26 cases were stage III and 19 were stage IV using the revised American Fertility Society classification). The preoperative serum levels of CA125 and CA19-9 were measured by immunoradiometric assay. Immunohistochemical analysis was performed using antibodies against CA125, CA199, and Ki-67 (a representative marker of cell proliferation). RESULTS: The serum levels of CA125 and CA19-9 were elevated (over the cutoff values) in 25 cases and 21 cases, respectively. There was no significant correlation between serum CA125 and serum CA19-9 levels (correlation coefficient [q]=0.19). The serum CA19-9 level correlated well with the degree of immunostaining for CA19-9 (q=0.57), but not with the Ki-67 labeling index. The serum CA125 level did not show a strong correlation with CA125 staining (q=0.41), but it correlated well with the Ki-67 labeling index (q=0.53). CONCLUSIONS: The present study indicates that the serum CA125 level may correlate with the proliferative activity of epithelial cells in endometriotic lesions.

Endometrial carcinoma remaining after term pregnancy following conservative treatment with medroxyprogesterone acetate.

BACKGROUND: Successful pregnancies after conservative progestin treatment to young women with endometrial carcinoma have recently been reported. However, it is not known for certain whether the lesion is completely eradicated in such patients. We present a case of residual endometrial carcinoma after term pregnancy which had been treated conservatively before the pregnancy began. CASE: A 28-year-old woman with endometrial carcinoma received conservative treatment with high-dose medroxyprogesterone acetate (MPA) and then conceived. After delivery at term, atypical cells were found in the endometrial curettage specimen. A hysterectomy was performed 6 months after delivery and revealed the presence of a small focus of intramucosal, grade 1, endometrioid-type adenocarcinoma. Immunohistochemically, the tumor cells were positive for estrogen and progesterone receptors. CONCLUSION: We concluded that while MPA treatment had been effective, it had not completely eradicated the carcinomatous lesion, which remained during and after the term pregnancy.

A comparative analysis of cross sectional imaging techniques in minimal deviation adenocarcinoma of the uterine cervix.

A pre-operative diagnosis of minimal deviation adenocarcinoma of the cervix is often difficult. We assessed transvaginal sonography, computed tomography and magnetic resonance imaging in 16 women with histologically confirmed minimal deviation adenocarcinoma. Increased fluid accumulation was frequently observed within the endometrial cavity and/or vagina by all three techniques. A multicystic cervical lesion was occasionally detected by transvaginal sonography or by computed tomography. On T2-weighted magnetic resonance images, a noncystic fine villous or multicystic lesion was noted in most cases. Among the three imaging techniques used, T2-weighted magnetic resonance images showed the most detailed features and the best correlation with the histology.

Cloning and expression of human B cell-specific transcription factor BACH2 mapped to chromosome 6q15.

The transcription factor Bach2, a member of the BTB-basic region leucine zipper (bZip) factor family, binds to a 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive element and the related Maf-recognition element (MARE) by forming homodimers or heterodimers with Maf-related transcription factors. Bach2 regulates transcription by binding to these elements. To understand the function in hematopoiesis, we isolated a cDNA clone for human Bach2 (BACH2) encoding a protein of 841 amino acid residues with a deduced amino acid sequence having 89.5% identity to mouse homolog. Among human hematopoietic cell lines, BACH2 is expressed abundantly only in some B-lymphocytic cell lines. RT-PCR analysis of hematopoietic cells revealed that BACH2 mRNA is expressed in primary B-cells. Enforced expression of BACH2 in a human Burkitt cell line, RAJI that does not express endogenous BACH2, resulted in marked reduction of clonogenic activity, indicating that BACH2 possesses an inhibitory effect on cell proliferation. By fluorescent in situ hybridization, the BACH2 gene was localized to chromosome 6q15. Because deletion of the long arm of chromosome 6 (6q) is one of the commonest chromosomal alterations in human B-cell lymphoma, we examined for the loss of heterozygosity (LOH) of the BACH2 gene in human B-cell non-Hodgkin's lymphomas (NHL). Among 25 informative cases, five (20%) showed LOH. These results indicate that BACH2 plays important roles in regulation of B cell development.

HCG promotes proliferation of uterine leiomyomal cells more strongly than that of myometrial smooth muscle cells in vitro.

Uterine myomas often enlarge rapidly during pregnancy. This rapid increase in size may imply that human chorionic gonadotrophin (HCG) influences cell proliferation in uterine leiomyomata. To assess the direct effect of HCG on normal uterine smooth muscle and uterine leiomyomata, we investigated cell proliferation and the expression of cell cycle-related proteins in these cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that HCG/LH receptor was present in both cultured myometrial and leiomyomal cells. Treatment with HCG significantly increased cell proliferation in both myometrial and leiomyomal cells (P < 0.03), especially at an early phase in the 9 day culture. The increase in the viable cell number induced by HCG treatment was significantly greater in leiomyoma cells than in myometrial cells on day 3 in culture (P < 0.03). In leiomyomal cells, the expression of proliferating cell nuclear antigen (PCNA), cyclin E and cdc2 was significantly increased by HCG treatment (P < 0.05) even at the lowest concentration used (3 nmol/l). In myometrial cells, the expression of cyclin E and cdc2 was significantly increased by HCG treatment (P < 0.05) only at the highest concentration used (30 nmol/l). These results suggest that HCG directly promotes the proliferation of myometrial and leiomyomal cells, with the latter showing the greater response of the two.

Immunohistochemical detection of the Wilms' tumor gene (WT1) in epithelial ovarian tumors.

The Wilms' tumor gene WT1 plays multiple roles in the development of the genitourinary organs and Wilms' tumors. The aims of this study were to immunohistochemically evaluate WT1 expression in normal female genital tissues and in epithelial ovarian tumors and to look for correlations between WT1 expression and histologic subtypes and cell proliferation in epithelial ovarian tumors. In normal female genital organs, WT1 expression was recognized in ovarian surface epithelium, the lining of inclusion cysts, and tubal epithelium, but not in the cervical or endometrial epithelium. In epithelial ovarian tumors, serous tumors generally revealed a high WT1 expression. Among adenocarcinomas, serous carcinoma revealed a significantly higher WT1 expression than the other histologic subtypes. There were no significant correlations between the WT1 labeling index and the Ki-67 labeling index, and no significant difference in survival between those showing high and low WT1 expression among the malignant cases. These results suggest that WT1 expression may be related to cell differentiation, and that the histologic subtypes of epithelial ovarian carcinomas may differ considerably in their biological characteristics.

Standard radial bone mineral density and physical factors in ordinary Japanese women.

To prevent osteoporosis, which is expected to increase in incidence in this rapidly aging society, in recent years bone mineral density (BMD) has frequently been measured as a predisposition index. However, these measurements are made on different sites with different apparatus, and the results are independently studied by different institutions. In our present investigation, to establish the standard radius BMD as determined by dual-energy X-ray absorptiometry (DXA), we carried out a general population survey in 29 municipalities and prefectures on 11,252 locally residing females aged 15 to 83 years (mean, 35.61 +/- 12.85 years). Their YAM (young adult mean) BMD was estimated at 0.664 +/- 0.054 g/cm2, which was almost the same as the figure given in the 1996 version of the diagnostic criteria for primary osteoporosis. We further studied the relationships of BMD to age and physical factors known to be influential to BMD. It was found that BMD was correlated negatively to age and positively to body mass index (BMI). The average values we obtained for age and physique groups appeared to have provided reliable indices for the primary prevention of osteoporosis.