Role of multifunctional transcription factor TFII-I and putative tumour suppressor DBC1 in cell cycle and DNA double strand damage repair.

BACKGROUND: In multicellular organisms, precise control of cell cycle and the maintenance of genomic stability are crucial to prevent chromosomal alterations. The accurate function of the DNA damage pathway is maintained by DNA repair mechanisms including homologous recombination (HR). Herein, we show that both TFII-I and DBC1 mediate cellular mechanisms of cell-cycle regulation and DNA double strand damage repair. METHODS: Regulation of cell cycle by TFII-I and DBC1 was investigated using Trypan blue dye exclusion test, luciferase assay, and flow cytometry analysis. We also analysed the role of TFII-I and DBC1 in DNA double strand damage repair after irradiation by immunofluorescence study, clonogenicity assay, and HR assay. RESULTS: Flow cytometry analysis revealed a novel function that siRNA-mediated knockdown of endogenous DBC1 resulted in G2/M phase arrest. We also have shown that both endogenous TFII-I and DBC1 activate DNA repair mechanisms after irradiation because irradiation-induced foci formation of TFII-I-γH2AX was observed, and the depletion of endogenous TFII-I or DBC1 resulted in the inhibition of normal HR efficiency. CONCLUSION: These results reveal novel mechanisms by which TFII-I and DBC1 can modulate cellular fate by affecting cell-cycle control as well as HR pathway.

Multifunctional transcription factor TFII-I is an activator of BRCA1 function.

BACKGROUND: The TFII-I is a multifunctional transcriptional factor known to bind specifically to several DNA sequence elements and to mediate growth factor signalling. A microdeletion at the chromosomal location 7q11.23 encoding TFII-I and the related family of transcription factors may result in the onset of Williams-Beuren syndrome, an autosomal dominant genetic disorder characterised by a unique cognitive profile, diabetes, hypertension, anxiety, and craniofacial defects. Hereditary breast and ovarian cancer susceptibility gene product BRCA1 has been shown to serve as a positive regulator of SIRT1 expression by binding to the promoter region of SIRT1, but cross talk between BRCA1 and TFII-I has not been investigated to date. METHODS: A physical interaction between TFII-I and BRCA1 was explored. To determine pathophysiological function of TFII-I, its role as a transcriptional cofactor for BRCA1 was investigated. RESULTS: We found a physical interaction between the carboxyl terminus of TFII-I and the carboxyl terminus of BRCA1, also known as the BRCT domain. Endogenous TFII-I and BRCA1 form a complex in nuclei of intact cells and formation of irradiation-induced nuclear foci was observed. We also showed that the expression of TFII-I stimulates the transcriptional activation function of BRCT by a transient expression assay. The expression of TFII-I also enhanced the transcriptional activation of the SIRT1 promoter mediated by full-length BRCA1. CONCLUSION: These results revealed the intrinsic mechanism that TFII-I may modulate the cellular functions of BRCA1, and provide important implications to understand the development of breast cancer.

Identification of DBC1 as a transcriptional repressor for BRCA1.

BACKGROUND: DBC1/KIAA1967 (deleted in breast cancer 1) is a putative tumour-suppressor gene cloned from a heterozygously deleted region in breast cancer specimens. Caspase-dependent processing of DBC1 promotes apoptosis, and depletion of endogenous DBC1 negatively regulates p53-dependent apoptosis through its specific inhibition of SIRT1. Hereditary breast and ovarian cancer susceptibility gene product BRCA1, by binding to the promoter region of SIRT1, is a positive regulator of SIRT1 expression. METHODS: A physical interaction between DBC1 and BRCA1 was investigated both in vivo and in vitro. To determine the pathophysiological significance of DBC1, its role as a transcriptional factor was studied. RESULTS: We found a physical interaction between the amino terminus of DBC1 and the carboxyl terminus of BRCA1, also known as the BRCT domain. Endogenous DBC1 and BRCA1 form a complex in the nucleus of intact cells, which is exported to the cytoplasm during ultraviolet-induced apoptosis. We also showed that the expression of DBC1 represses the transcriptional activation function of BRCT by a transient expression assay. The expression of DBC1 also inhibits the transactivation of the SIRT1 promoter mediated by full-length BRCA1. CONCLUSION: These results revealed that DBC1 may modulate the cellular functions of BRCA1 and have important implications in the understanding of carcinogenesis in breast tissue.

The oncogenic mutation in the pleckstrin homology domain of AKT1 in endometrial carcinomas.

BACKGROUND: The phosphatidylinositol 3'-kinase (PI3K)-AKT pathway is activated in many human cancers and plays a key role in cell proliferation and survival. A mutation (E17K) in the pleckstrin homology domain of the AKT1 results in constitutive AKT1 activation by means of localisation to the plasma membrane. The AKT1 (E17K) mutation has been reported in some tumour types (breast, colorectal, ovarian and lung cancers), and it is of interest which tumour types other than those possess the E17K mutation. METHODS: We analysed the presence of the AKT1 (E17K) mutation in 89 endometrial cancer tissue specimens and in 12 endometrial cancer cell lines by PCR and direct sequencing. RESULTS: We detected two AKT1 (E17K) mutations in the tissue samples (2 out of 89) and no mutations in the cell lines. These two AKT1 mutant tumours do not possess any mutations in PIK3CA, PTEN and K-Ras. INTERPRETATION: Our results and earlier reports suggest that AKT1 mutations might be mutually exclusive with other PI3K-AKT-activating alterations, although PIK3CA mutations frequently coexist with other alterations (such as HER2, K-Ras and PTEN) in several types of tumours.

Changes of phylloquinone and menaquinone-4 concentrations in rat liver after oral, intravenous and intraperitoneal administration.

To study the metabolism of K Vitamins (VK) in the liver, two types of natural VK, phylloquinone (K1) and menaquinone-4 (MK-4), were administered to male Wistar rats orally (P.O.), intravenously (I.V.) and intraperitoneally (I.P.). Blood and a small portion of the liver (and ascites by I.P.) were collected 8 times up to 72 h (P.O.) or 24 h (I.V. and I.P.). A modified assay procedure followed by high performance liquid chromatography (HPLC) was developed to detect VK from small amounts of liver tissue. After oral administration of both K1 and MK-4 (10 mumol/kg-P.O.), their concentrations in the liver increased from 1 h then reached a maximum at 6 h (10 nmol/g v.s. 0.35 nmol/g). After intravenous or intraperitoneal administration of K1 and MK-4 (0.5 mumol/kg-I.V. and I.P.), MK-4 concentrations in the liver reached a maximum faster than those of K1 (1.2 nmol/g -3 h vs. 1.3 nmol/g -0.5 h I.V. and 0.97 nmol/g -6 h vs. 0.47 nmol/g -1 h I.P.). MK-4 also increased in the liver from 6 h to 12 h (0.11 nmol/g -12 h) after oral administration of K1 (P.O.). These results indicate that K1 stays in plasma and liver longer than MK-4 and orally administered K1 might be transformed partially into MK-4 in the liver.

Changes of K vitamins in portal and femoral venous plasma of rats after oral administration of phylloquinone and menaquinone-4.

To clarify the mechanism of absorption and metabolism of K vitamins (VK), we administered phylloquinone (K1) and menaquinone-4 (MK-4) orally to male Wistar rats whose portal and femoral veins were cannulated. Blood was collected 9 times up to 120 min later, and 50 microliters plasma was used for VK analysis by high performance liquid chromatography (HPLC) using a modification of our previous method. There were no significant differences between portal and femoral venous VK concentrations throughout all the experiments. When K1 and MK-4 were administered simultaneously, K1 appeared in plasma at 15 min, and MK-4 at 10 min. The concentration of MK-4 was significantly higher than that of K1 up to 60 min. In contrast to the single administration, the MK-4 concentration was reduced after 60 min. After K1 single administration, the MK-4 was not detected in either portal or femoral venous plasma up to 120 min. These results suggested that (1) the main absorption route of both VK may be the extra-portal pathway, (2) MK-4 is absorbed faster than K1 (3) some interactions may exist between K1 and MK-4 and (4) the intestinal cells might not be the major site to transform K1 into MK-4.

Distribution of K vitamins (phylloquinone and menaquinones) in human placenta and maternal and umbilical cord plasma.

It is a well-known fact that neonatal and infantile vitamin K deficiencies cause melena neonatorum and intracranial hemorrhagic disorders. These disorders occur more frequently in breast-fed babies than in bottle-fed babies, for reasons that are still obscure. Endogenous concentrations of K vitamins (phylloquinone and menaquinones) in human placentas and in paired specimens of maternal and umbilical cord plasma were detected by high-performance liquid chromatography with fluorometric detection and postcolumn reduction. Phylloquinone (K1) and menaquinones (MK-4, MK-6, and MK-7) were found in placentas and maternal plasma. K1 and MK-4 were found in umbilical cord plasma. The experimental subjects were 13 pregnant women with normal diets (group A) and two pregnant women with diets supplemented with fermented soybeans (group B), in which MK-7 is abundant. Samples from their placentas and maternal and umbilical cord plasma were collected just after delivery. The placentas and maternal plasma of group B contained higher concentrations of MK-7 (placentas = 10.82 ng/gm and maternal plasma = 3.55 ng/ml) than did group A (placentas = 1.08 ng/gm, maternal plasma = 0.70 ng/ml). However, MK-7 was not found in umbilical cord plasma of any of the subjects. The mean K1 values of all 15 patients in placentas and maternal and umbilical cord plasma were 1.28 ng/gm, 1.54 ng/ml, and 0.11 ng/ml, respectively; mean MK-4 values were 1.18 ng/gm, 0.05 ng/ml, and 0.04 ng/ml, respectively. A high concentration of MK-4 was detected in placentas.

[Fundamental and clinical studies of T-1982 (cefbuperazone) in the field of obstetrics and gynecology].

Human pharmacokinetics and clinical studies of T-1982 (cefbuperazone) were carried out and the following results were obtained. Transference into pelvic cavity fluid T-1982 level in pelvic cavity fluid showed the peak level of 48.0 micrograms/ml at 30 minutes after 2 hours drip infusion of 1 g in 1 case. The concentration tended to be more sustained than that of venous blood. Clinical study T-1982 was administered to 2 cases with pelveoperitonitis, 2 cases with puerperal fever and 1 case with adnexitis at a dose of 1 g twice a day for a period of 4 or 5 days. The clinical effect was excellent in 3 and good in 2 cases. Neither side effects nor abnormalities of laboratory findings were observed.

[Fundamental and clinical studies of piperacillin in the field of obstetrics and gynecology].

Human pharmacokinetics and clinical studies of piperacillin (PIPC) were carried out and the following results were obtained. 1. Transference into various uterine tissues PIPC transference into various uterine tissues was 27.3 approximately 67.9% (the ratio with respect to uterine artery blood level) at 1 hour after the completion of PIPC 1 or 2g dripping infusion for 1 hour in 5 cases. 2. Transference into retroperitoneal space exudate PIPC level in retroperitoneal space exudate showed the peak level 38.0 micrograms/ml at 30 minutes after the completion of PIPC 2g dripping infusion for 1 hour in 1 case. The pattern of the concentration trends was more continuous than that of venous blood level. 3. Clinical study We administered PIPC to 5 patients with pelveoperitonitis in 3 cases, puerperal fever in 1 case and wound infection in 1 case at a dose of 4 g per day (twice a day) for a period of 5 approximately 9 days. The clinical effect was excellent in 1, good in 3 and poor in 1 case. Neither side effects nor abnormality of laboratory findings were observed.