Masaomi Takayama

Human arylhydrocarbon receptor repressor (AHRR) gene: genomic structure and analysis of polymorphism in endometriosis

AbstractThe diversity of biological effects resulting from exposure to dioxin may reflect the ability of this environmental pollutant to alter gene expression by binding to the arylhydrocarbon receptor (AHR) gene and related genes. AHR function may be regulated by structural variations in AHR itself, in the AHR repressor (AHRR), in the AHR nuclear translocator (ARNT), or in AHR target molecules such as cytochrome P-4501A1 (CYP1A1) and glutathione S-transferase. Analysis of the genomic organization of AHRR revealed an open reading frame consisting of a 2094-bp mRNA encoded by ten exons. We found one novel polymorphism, a substitution of Ala by Pro at codon 185 (GCC to CCC), in exon 5 of the AHRR gene; among 108 healthy unrelated Japanese women, genotypes Ala/Ala, Ala/Pro, and Pro/Pro were represented, respectively, by 20 (18.5%), 49 (45.4%), and 39 (36.1%) individuals. We did not detect previously published polymorphisms of ARNT (D511N) or the CYP1A1 promoter (G-469A and C-459T) in our subjects, suggesting that these polymorphisms are rare in the Japanese population. No association was found between uterine endometriosis and any polymorphisms in the AHRR, AHR, ARNT, or CYP1A1 genes analyzed in the present study.

Increased heterogeneity of chromosome 17 aneuploidy in endometriosis

OBJECTIVE Endometriosis is a complex gynecologic disorder that may display features similar to malignancy, including aggressive growth and localized invasion of the myometrium or spread to various organs outside the uterus. Molecular studies of cancer have demonstrated that genomic instability involving chromosome 17 plays a role in the development and progression of various tumor types. These involve gain and/or loss, deletions, and mutations of candidate tumor suppressor genes (eg, BRCA1 and p53 ) on chromosome 17. STUDY DESIGN We used a 2-color fluorescence in situ hybridization method for analysis of endometriotic and normal archival tissue. Centromere-specific and locus-specific p53 probes localized to chromosome 17 were selected to study 8 patients with late-stage (severe) endometriosis. Single cells localized to endometriotic lesions or normal endometrial glands were analyzed and identified as normal or abnormal on the basis of the distribution of fluorescence in situ hybridization signals. RESULTS Overall, chromosome 17 aneuploidy was significantly greater (P <.05) in the endometriosis specimens (mean of 65%) than in normal endometrial cells (mean of 25%). No significant difference (P =.1071) in the distribution of fluorescence in situ hybridization signals was observed among the 5 normal endometrial specimens. However, significant differences (P <. 0001) were observed between the 8 endometriosis tissue specimens. CONCLUSION We found increased heterogeneity of chromosome 17 aneuploidy in endometriosis. These findings support a multistep pathway involving somatic genetic alterations in the development and progression of this common disease.

Differentially regulated genes as putative targets of amplifications at 20q in ovarian cancers.

Frequent amplification of DNA at 20q or part of 20q has been demonstrated by comparative genomic hybridization in ovarian cancer (OC), but the genetic target(s) of these amplification events remain unknown. We examined copy‐number changes with respect to six candidate genes, E2F1 (20q11.2), TGIF2 (20q11.2), AIB1 (20q12), PTPN1 (20q13.1), ZNF217 (20q13.2), and BTAK (20q13), and then measured transcription levels of each candidate in 18 OC cell lines. Three distinct cores of amplification were identified: 20q11.2, harboring E2F1 and TGIF2 (region I; 1 of 18 cell lines, 5.6%); 20q13.1, harboring PTPN1 (region II; 5 lines, 27.8%); and 20q13.2, harboring ZNF217 and BTAK (region III; 6 lines, 33.3%). Among the six genes examined, expression levels of PTPN1 and ZNF217 were significantly correlated with absolute copy‐number, and those of PTPN1 and TGIF2 were significantly correlated with copy‐number relative to the centromere of chromosome 20 (20cen). Among 19 primary OCs examined, moreover, we observed amplification of TGIF2, PTPN1 and ZNF217 in five (26.3%), ten (52.6%), and twelve (63.2%) tumors, respectively. Expression levels of PTPN1 and ZNF217 were significantly correlated with their copy‐numbers in those primary OCs. Our results suggest that 20q amplifications in OCs can be extensive and complex, probably due to synergistic or non‐synergistic amplification of separate regions of 20q, involving multiple, independently amplified targets.

Matrix metalloproteinase‐26 is expressed in human endometrium but not in endometrial carcinoma

The human matrix metalloproteinase (MMP)‐26, also called matrilysin‐2 or endometase, has been isolated as a matrilysin (MMP‐7) homolog. Matrix metalloproteinase‐26 was expressed in tissue samples from the placenta and endometrial tumors and its expression may be related to the development of endometrial carcinomas.

Abnormally Large Placenta Associated with Beckwith-Wiedemann Syndrome

A 37-year-old G1-P1 was diagnosed by ultrasonography at 26 weeks of gestation as having an abnormally large placenta with hemangiomas and a fetus associated with exomphalos. Placental protein 5 levels were relatively high in placental protein levels in maternal serum. The infant, delivered by cesarean section at 34 weeks, had the typical clinical features associated with Beckwith-Wiedemann syndrome. The abnormally large placenta weighed 1,492 g, measured 25 X 25 X 5.1 cm, and featured multiple hemangiomas. Microscopic placental features included edematous villi, increased fibrin deposition, intervillous thrombi, and multiple angiomatous and cellular chorangiomas.

The correlation of TERT expression with c-myc expression in cervical cancer

Recently putative catalytic telomerase subunit was identified as telomerase reverse transcriptase (TERT). Several reports showed that TERT expression correlated with telomerase activity. It has also been found that c-myc can induce telomerase activation through TERT expression. We examined expression of TERT and c-myc and their correlation in cervical cancers by reverse transcription-polymerase chain reaction (RT-PCR). It was found that TERT and c-myc expression was observed mostly in malignancies and expression of c-myc was concordant for positivity and negativity with TERT. These results support recent studies that c-myc expression is closely associated with TERT and telomerase activity. c-myc up-regulation may play an important role in activation of TERT and telomerase.

Parent cells for trophoblast hybridization II: AC1 and related trophoblast cell lines, a family of HGPRT-negative mutants of the choriocarcinoma cell line JEG-3

Summary To gain analytical and productive access to the oncofetally modified molecules in the extracellular matrix of human non-proliferative invasive trophoblast cells, we intend to immortalize these cells together with their productive capacity by fusion with their malignant trophoblast counterpart. To generate a suitable malignant fusion partner we have selected hypoxanthine-guanosin-phosphoribosyl-transferase-negative mutants of the choriocarcinoma cell line JEG-3. We obtained the cell line AC1 and its subclones AC1-1, AC1-5 and AC1-9. AC1-1 is our preferred candidate for fusion with matrixproducing invasive trophoblast cells since this subclone homogenously did not react with antibodies recognizing trophoblast matrix molecules.

Mother-to-infant transmission of TT virus in Japan

Objective: The TT virus (TTV) was detected for the first time by Nishizawa and Okamoto et al. in 1997 in the serum of a patient with post‐transfusion hepatitis of unknown origin (non‐A‐non‐G type). TTV was subsequently, also found in the serum of blood donors with no history of blood transfusion, although at a lower rate than among donors with a history of blood transfusion. In the present study, we determined the percentage of TTV carriers among pregnant women with no history of blood transfusion, and evaluated the possibility of mother‐child transmission. Methods: Blood was sampled from 300 normal pregnant women with no history of blood transfusion, 10 infants born by vaginal delivery from TTV‐positive women, 10 infants born by abdominal cesarean section from TTV‐positive women at both 5 days and 3 months after birth, and 10 infants born from TTV‐positive women at 6 months after birth. Amniotic fluid and breast milk were sampled from 10 and 30 TTV‐positive women, respectively. Informed consent was obtained from all women before sampling. TTV DNA was detected by the nested polymerase chain reaction (PCR) method. Results: (1) Of the 300 normal pregnant women with no history of blood transfusion, 60 (20%) were TTV‐positive. (2) All infants from TTV‐positive mothers were TTV‐negative at both 5 days and 3 months after birth, regardless of whether they were born by vaginal delivery or abdominal cesarean section. (3) Of the 10 infants who were born from TTV‐positive mothers and examined 6 months after birth, 4 (40%) were TTV‐positive. (4) Amniotic fluid from all 10 TTV‐positive women was TTV‐negative. (5) Breast milk from 7 (23.3%) of the 30 TTV‐positive women was TTV‐positive. Conclusion: TTV was detected in 20% of pregnant women with no history of blood transfusion, suggesting that TTV infection can occur through non‐ blood‐mediated routes. The possibility of transfer of TTV into amniotic fluid was ruled out due to its absence in amniotic fluid samples. All infants from TTV‐positive women were TTV‐negative at both 5 days and 3 months after birth, regardless of whether they were born by vaginal delivery or abdominal cesarean section, suggesting that infection in the parturient canal or the pelvis is unlikely. Because TTV was detected in breast milk from TTV‐positive women and some of their infants were TTV‐positive, breast milk was thought to be a mother–child infection route. These findings suggest that horizontal infection is more likely than vertical infection in mother–child transmission of TTV.

Increased expression of IgE-dependent histamine-releasing factor in endometriotic implants

A complex network of cytokines mediates the immunoregulatory responses leading to endometriosis. Recent intensive studies suggest that monocyte and T cell chemoattractants contribute to the inflammatory environment of endometriotic implants. The relationship between the inflammation present during endometriosis and the development of endometriotic implants in the peritoneal cavity remains unclear. On the other hand, the association between endometriosis and 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD; dioxin) exposure has been discussed in recent years, and our previous results revealed that IgE‐dependent histamine‐releasing factor (HRF) is inducible by TCDD. The present study aimed to clarify the expression, localization, and function of HRF in endometriosis. Northern blot analysis demonstrated that HRF is overexpressed in endometriotic implants. RT‐PCR with Southern blot analysis, however, showed that HRF overexpression was not always accompanied by CYP1A1 induction in endometriotic implants, suggesting that HRF is inducible in endometriosis without exposure to TCDD. HRF is also inducible by macrophage colony‐stimulating factor (M‐CSF). Immunohistochemistry showed CD68‐positive macrophages in the stroma of endometriotic implants, adjacent to regions with prominent HRF accumulation. HRF‐overexpressing cells exhibited high implantation efficiency in comparison to control cells when the cells were injected into the peritoneal cavities of nude mice. These results suggest that the accumulation of macrophages in endometriotic implants induces HRF; the overexpression of HRF accelerates the growth of endometriotic implants. Copyright

Establishment of a new human cell line (EN) with TP53 mutation derived from endometrial carcinoma

We present a new cell line, EN, established from an invasive endometrioid adenocarcinoma of the uterine corpus in 50-year-old patient. The cells show rapid growth in culture with a doubling time of 24.4 hours and high migration activity. Monolayer-cultured cells were polygonal in shape and showed a tendency to pile up without contact inhibition. Subcutaneous transplantation of the EN cells into nude mice formed solid tumors that were histologically diagnosed as adenocarcinoma, whereas no metastasis was observed. Cultured EN cells produced tissue polypeptide antigen. Genetic and molecular analyses revealed high telomerase activity and estrogen receptor beta but not alpha expression. Using the polymerase chain reaction-single strand conformation polymorphism technique, we have screened EN cells for TP53 mutation in exons 5-8. A mobility shift was observed in this cell line in exon 8. A nucleotide insertion (CGT-->CAGT) was detected at codon 273, which resulted in a creation of a stop codon at codon 308. This cell line thus appears to represent the development of a more malignant clone with divergent receptor function and growth behavior, and provides us with an interesting new tool for the study of tumorigenesis in the human endometrium.