Drosophila RecQ5 is involved in proper progression of early spermatogenesis.

RecQ5, a member of the conserved RecQ DNA helicase family, is required for the maintenance of genome stability. The human RECQL5 gene is expressed ubiquitously in almost all tissues, with strong expression in the testes (Shimamoto et al., 2000). However, it remains to be elucidated in which cells RecQ5 is expressed and how RecQ5 functions in the testes. In this present study we analyzed the expression of RecQ5 in Drosophila testes. The RecQ5 protein was specifically expressed in germline cells in larval, pupal, and adult testes. Drosophila RecQ5 was localized in nuclei of male germline stem cells, spermatogoniablasts, spermatogonia, and early spermatocytes. As growth of the early spermatocyte proceeded, the amount of RecQ5 increased in the nuclei. However, before maturation of the spermatocyte, the level of RecQ5 declined. Thus, RecQ5 expression was regulated. Furthermore, we compared recq5 mutant testes with the wild-type ones. The most conspicuous alterations were swelling of the apical region of and an increase in the number of spermatocytes in the recq5 testis, suggesting a relative accumulation of spermatocytes in the recq5 mutant testes. Therefore, Drosophila RecQ5 may contribute to the proper progression from germline stem cells to spermatocytes for maintenance of genome stability.

RecQ5 protein translocation into the nucleus by a nuclear localization signal.

RecQ5, a member of the RecQ helicase family, maintains genome stability via participation in many DNA metabolic processes including DNA repair, DNA resolution, and RNA transcription, processes occurring in the nucleus. Previously, we reported that RecQ5 and Rad51, also involved in DNA repair, become co-localized in nuclei when co-expressed in cultured cells. Nuclear localization of RecQ5 appears to be important for cellular function along with Rad51. However, little is known about the nuclear localization of RecQ5. Here, we generated enhanced green fluorescent protein (EGFP)-tagged RecQ5 transgenic flies and analyzed localization of this protein in early embryos by live imaging. In syncytial embryos, RecQ5 was localized synchronously in interphase nuclei, and spread repeatedly over the embryos in mitosis. Thus, RecQ5 was transported into nuclei at the early interphase. Furthermore, we examined the subcellular localization of a series of truncated forms of Drosophila RecQ5 in cultured cells to determine the nuclear localization signal (NLS). Entire coding or deleted RecQ5 sequences of various sizes were ligated into EGFP vectors, which were then used to transfect cultured Drosophila cells. The region responsible for nuclear localization of Drosophila RecQ5 contained a short stretch of positively charged basic amino acids, 2 of which were particularly important for the nuclear localization. This stretch was sufficient for nuclear localization when fused with EGFP. Although the NLS of Drosophila RecQ5 was distinct from that of human RECQL5 in terms of position and amino acid sequence, this fly RecQ5 protein was translocated into the nucleus by an NLS.

LC/MS/MS of steroids having vicinal diol as electrospray-active boronates.

A derivatization procedure with (3-dimethylaminophenyl)dihydroxyborane (DAPB) was introduced to enhance the detectability of steroids having a vicinal diol in LC/electrospray ionization (ESI)-MS/MS. DAPB reacted with the vicinal diol on the steroids [4ß-hydroxycholesterol (4-HCh), pregnanetriol (PT) and 20R,22R-dihydroxycholesterol] in pyridine at 50°C within 1 h. The resulting DAPB-derivatives were highly responsive in ESI-MS operating in the positive-ion mode and gave characteristic product ions during MS/MS, which enabled sensitive detection using a selected reaction monitoring mode; the detection responses of the DAPB-derivatives were increased by 20-160-fold over those of the intact steroids and the limits of detection were in the low femtomole or attomole range. The derivatization procedure was successfully applied to biological sample analysis; the derivatization followed by LC/ESI-MS/MS enabled the specific detection of trace amounts of 4-HCh in human plasma and PT in human urine with a small sample volume, simple pretreatment and short chromatographic run time.

Significant prognostic factors in patients with Stage IV gastric cancer with special reference to the curability of surgery.

PURPOSES: The purpose of this study was to determine an effective treatment strategy for patients with Stage IV gastric cancer. METHODS: We analyzed the significant prognostic factors in 74 patients who underwent surgery between 1989 and 2005, and were finally determined to have Stage IV gastric cancer. These patients were classified as curability A (n = 0), B (n = 29) and C (n = 45) according to the criteria outlined by Japanese Gastric cancer society. Anti-tumor drugs were used after surgery in some cases. There were 32 patients who received either no treatment or an oral anti-tumor drug, and 42 patients who received new chemotherapeutic regimens. RESULTS: According to a univariate analysis, the postoperative mean survival times were significantly different; tumor size ≤ 12 cm, a tumor without lymphatic involvement, more than D2 lymphadenectomy, and classification as curability B were favorable prognostic factors. The multivariate analysis revealed that tumor size, lymphadenectomy and curability were independent prognostic factors. In curability B patients, venous involvement was an independent prognostic factor. In curability C patients, both the tumor size and postoperative chemotherapy affected their prognosis. CONCLUSIONS: In patients with curable Stage IV gastric cancer, at least a D2 gastrectomy to reduce the absolute volume of tumor cells, followed by adjuvant chemotherapy, may be essential to improve their prognosis. In incurable cases, aggressive new chemotherapeutic regimens should be the treatment of choice for the prolongation of survival.

RecQ5 interacts with Rad51 and is involved in resistance of Drosophila to cisplatin treatment.

RecQ5 is a member of the RecQ family of DNA helicases. There are 5 RecQ members in humans. Defects in 3 of them, i.e., BLM, WRN, and RTS, cause Bloom, Werner, and Rothmund-Thomson syndromes, respectively. RECQL1 and RECQL5 have not been associated with any human disease, and their precise roles are unknown. Our previous study suggests that the lack of RecQ5, which is the Drosophila homolog of RECQL5, leads to the accumulation of DNA double-stranded breaks (DSBs). It is possible that RecQ5 is involved in DSB repair. However, little is known about this possible function of RecQ5 in DSB repair. Here, we report that Rad51 protein, which plays a critical role in DSB repair, interacted with RecQ5 in vitro and in vivo in Drosophila. The Rad51 protein interacted with the C-terminal region of RecQ5, as shown by the yeast two-hybrid method. Moreover, the C-terminal region of the RecQ5 protein and the central region of Rad51 interacted directly and specifically when examined by the glutathione-S-transferase pull-down method. Consistent with these results, when RecQ5 and Rad51 were co-expressed in Drosophila cells in culture, they became co-localized in nuclei and could be co-immunoprecipitated. Furthermore, RecQ5-deficient flies (recq5) were more sensitive to the chemotherapeutic agent cisplatin compared with wild-type ones. Also, Rad51 mutants (rad51) were more sensitive to cisplatin, with sensitivity similar to that of recq5 rad51 double mutants. These data suggest that RecQ5 and Rad51 in Drosophila functioned for survival after the flies had been treated with cisplatin.

The effect of overweight status on the short-term and 20-y outcomes after hepatic resection in patients with hepatocellular carcinoma.

BACKGROUND: We aimed to evaluate the impact of body mass index (BMI) on the short- and long-term outcomes of hepatic resection in patients with hepatocellular carcinoma (HCC). METHODS: We performed 371 hepatic resections in HCC patients whom we categorized into two groups based on BMI: BMI ≥ 25 (n = 77) and BMI <25 (n = 294). We compared surgical outcomes between groups. RESULTS: The incidence of postoperative complications in the BMI ≥ 25 group was comparable to those in the BMI <25 group. However, patients in the BMI <25 group showed a significantly worse long-term prognosis than those in the BMI ≥ 25 group (P < 0.01). The results of multivariate analyses showed that BMI <25 was an independent and prognostic indicator of long-term outcome after hepatic resection in HCC patients. CONCLUSIONS: A BMI ≥ 25 is not a risk factor for mortality or postoperative complications, and is considered to provide a better long-term prognosis (>20 y) than a BMI <25 in patients with HCC after hepatic resection. Further studies are needed to determine whether these results apply to other patient populations outside Japan where BMI ≥ 30 is more prevalent.

Efficacy of surgical microwave therapy in patients with unresectable hepatocellular carcinoma.

BACKGROUND: We aimed to evaluate the efficacy and long-term outcome in surgical microwave therapy (MW) for patients with unresectable hepatocellular carcinoma (HCC). METHODS: An institutional review board approved and single-institutional study of surgical MW of unresectable HCC was conducted from May 2003 to December 2010. The median follow-up period was 19 months (range 1-77 months). RESULTS: A total of 60 patients underwent 143 surgical MW for unresectable HCC. Of these, 15 patients had initial HCC and 45 had recurrent HCC. The median tumor size of HCC was 1.95 cm (range 0.8-3.3 cm). The median numbers of nodules that underwent surgical MW were 2 (range 1-9). Multinodular type was found in 33 patients (55%). Morbidity was 18.3%, and there was zero mortality. Also, 3 patients (5%) had incomplete MW. Of the 60 patients, 39 (65%) had recurrence, and 7 (11.6%) had local recurrence. The 1- and 3-year recurrence-free survival rates of the patients who underwent surgical MW for initial HCC were 55.1 and 36.7%, respectively, and those for recurrent HCC were 41.6% and 8.8%, respectively. A tumor size ≥ 2.0 cm and multiple nodules were selected as independent and significant indicators for recurrence of the disease. The 1-, 3-, and 5-year overall survival rates after the surgical MW procedure were 93.9, 53.8, and 43.1%, respectively. A level of des-gamma carboxyprothrombin (DCP) was an independent and significant indicator for overall survival. CONCLUSIONS: Surgical MW is an effective method for treating initial or recurrent unresectable HCC, and it can be undergone safely.

Anaphase DNA bridges induced by lack of RecQ5 in Drosophila syncytial embryos.

Drosophila melanogaster RecQ5, a member of the RecQ family, is expressed in early embryos. The loss of maternally-derived RecQ5 leads to spontaneous mitotic defects in syncytial embryos. We demonstrate that the mitotic defects are derived from anaphase DNA bridges. Pairs of daughter nuclei that had been linked by the bridges concurrently exited from the cycle and were eliminated by Chk2-dependent centrosome inactivation. These results suggest that the lack of RecQ5 leads to spontaneous double-stranded DNA breaks (DSBs). RecQ5 may function in the resolution of anaphase DNA bridges during mitosis or in DSB repair during interphase in syncytial Drosophila embryos.

Loss of RecQ5 leads to spontaneous mitotic defects and chromosomal aberrations in Drosophila melanogaster.

RecQ5 belongs to the RecQ DNA helicase family that includes genes causative of Bloom, Werner, and Rothmund-Thomson syndromes. Although no human disease has been genetically linked to a mutation in RecQ5, Drosophila melanogaster RecQ5 is highly expressed in early embryos, suggesting an important role for it in the DNA metabolism of the early embryo. In this present study, we generated RecQ5 mutants in D. melanogaster. Embryos lacking maternally derived RecQ5 contained irregular nuclei in early embryogenesis. These irregular nuclei emerged in nuclear cycle 11-13, lost cell-cycle markers, and were located below the surface monolayer of nuclei. By time-lapse microscopy, these irregular nuclei were observed not to divide, whereas all neighboring nuclei proceeded through normal mitotic division with synchrony. These data suggest that the irregular nuclei exited from the nuclear division cycle. This phenotype is reminiscent of the effect of X-ray irradiation on wild-type embryos and was rescued by expression of RecQ5. Thus, the maternal supply of RecQ5 is important for the nuclear cycles in syncytical embryos. Furthermore, the frequencies of spontaneous and induced chromosomal aberrations were increased in RecQ5 mutant neuroblasts. These data imply that DNA damage accumulates spontaneously in RecQ5 mutants. Therefore, endogenous genomic damage may be produced in Drosophila development, and RecQ5 would be involved in the maintenance of genomic stability by suppressing the accumulation of DNA damage.

Factors influencing the development of small intestinal obstruction following gastrectomy for early gastric cancer.

BACKGROUND/AIMS: The factors influencing the development of small intestinal obstruction following gastrectomy for early gastric cancer are controversial. METHODOLOGY: Univariate and multivariate analyses were carried out using data from 136 patients with early gastric cancer who underwent gastrectomy. The mean follow-up interval was 5 years and 11 months. RESULTS: Of these 136 patients, 15(11.0%) presented mechanical obstruction in the small intestine postoperatively. Re-operation for repair of the related ileus was required in 8 patients, 4 of whom had been treated with total gastrectomy, 7 with resection of the greater omentum, and 3 with concomitant resection of other organs. Development of an obstruction was not related to wide resectional procedures such as extended lymph node dissection or combined resection of other organs, but was significantly correlated with total gastrectomy and resection of the greater omentum (p < 0.05). In multivariate logistic regression analysis, total gastrectomy proved to be a significant risk factor related to the development of small intestinal obstruction. CONCLUSIONS: In patients with early gastric cancer, total gastrectomy should not be attempted to prevent postoperative ileus and to ensure a fairly sustained quality of life.

Appraisal of hepatic resection in the treatment of hepatocellular carcinoma with severe thrombocytopenia.

BACKGROUND: Hepatic resection for hepatocellular carcinoma (HCC) patients with liver cirrhosis and severe hypersplenic thrombocytopenia is risky and controversial. METHODS: From 1989 to 2005, 341 patients underwent hepatic resection for HCC in our hospital. Of these, 15 patients were concomitant with severe thrombocytopenia (platelet count,

Extended hemi-hepatectomy with portal vein reconstruction in a patient with situs ambiguous.

We report a case of far-advanced hepatocellular carcinoma (HCC) with situs ambiguous, complex visceral and vascular anomalies, who was successfully managed by extended hemi-hepatectomy. A 67-year-old man was referred to our hospital with a large liver mass. Abdominal ultrasonography, computed tomography and angiography revealed HCC with a diameter of 10 cm, with tumor thrombus in the main and first branch of the portal vein. Multiple complex anomalies in the abdomen were determined preoperatively. He had right-sided spleens-stomach-duodenum, liver at midline, inferior vena cava interruption with azygous continuation, and hepatic arterial anomaly. Extended left lobectomy of the liver with reconstruction of the portal vein was performed. Postoperatively, the patient recovered without major complications, and he was discharged on postoperative day 21. We report the first successful extended hepatectomy with portal vein reconstruction for HCC in a patient with rare situs anomalies.

Ten-year survival of patients with hepatocellular carcinoma after hepatectomy.

BACKGROUND/AIMS: The characteristics in patients with hepatocellular carcinoma who survive more than 10 years after hepatectomy remain unclear. METHODOLOGY: Eighty-five cases of hepatocellular carcinoma were retrospectively identified as short-term survivors (S-group: <5 years, n=41), medium-term survivors (M-group: > or =5 and <10 years, n=25), and long-term survivors (L-group: > or =10 years, n=19) to examine the clinicopathologic factors for the 10-year survival after curative hepatectomy. RESULTS: In the L-group, platelet count and albumin level were higher and total bilirubin level was lower than those in the S-group. In the S-group, the total bilirubin level was higher and vascular invasion was more frequent than those in the M-group. Multiple regression analysis revealed that only host-related factors such as age, albumin level, and total bilirubin level were selected as significant factors to determine the 10-year survival. However, no tumor-related factor was selected. CONCLUSIONS: The present study suggests that the important factor linked to the 10-year survival of hepatocellular carcinoma patients is the host-related factor, but not the tumor-related factor. Especially, younger age, higher albumin level, and lower total bilirubin level are quite important to determine the 10-year survival.

Growth stimulation of non-small cell lung cancer cell lines by antibody against epidermal growth factor receptor promoting formation of ErbB2/ErbB3 heterodimers.

Antibodies are the most rapidly expanding class of human therapeutics, including their use in cancer therapy. Monoclonal antibodies (mAb) against epidermal growth factor (EGF) receptor (EGFR) generated for cancer therapy block the binding of ligand to various EGFR-expressing human cancer cell lines and abolish ligand-dependent cell proliferation. In this study, we show that our mAb against EGFRs, designated as B4G7, exhibited a growth-stimulatory effect on various human cancer cell lines including PC-14, a non-small cell lung cancer cell line; although EGF exerted no growth-stimulatory activity toward these cell lines. Tyrosine phosphorylation of EGFRs occurred after treatment of PC-14 cells with B4G7 mAb, and it was completely inhibited by AG1478, a specific inhibitor of EGFR tyrosine kinase. However, this inhibitor did not affect the B4G7-stimulated cell growth, indicating that the growth stimulation by B4G7 mAb seems to be independent of the activation of EGFR tyrosine kinase. Immunoprecipitation with anti-ErbB3 antibody revealed that B4G7, but not EGF, stimulated heterodimerization between ErbB2 and ErbB3. ErbB3 was tyrosine phosphorylated in the presence of B4G7 but not in the presence of EGF. Further, the phosphorylation and B4G7-induced increase in cell growth were inhibited by AG825, a specific inhibitor of ErbB2. These results show that the ErbB2/ErbB3 dimer functions to promote cell growth in B4G7-treated cells. Changes in receptor-receptor interactions between ErbB family members after inhibition of one of its members are of potential importance in optimizing current EGFR family-directed therapies for cancer.

Relationships of Drosophila melanogaster RECQ5/QE to cell-cycle progression and DNA damage.

Members of the RecQ family of DNA helicases are involved in the cellular response to DNA damage and are regulated in the cell-cycle. However, little is known about RecQ5, one of these members. The level of RECQ5/QE, Drosophila melanogaster RecQ5, was increased after the exposure of cultured cells to methyl-methanesulfonate. Transgenic flies that overexpressed RECQ5/QE in their developing eye primordia showed mild roughening of the ommatidial lattice. DNA-damaging agents and the mei-41 mutation enhanced the phenotype caused by RECQ5/QE overexpression. Overexpression of RECQ5/QE perturbed the progression of the cell-cycle in response to DNA damage in the eye imaginal discs. These results suggest that RECQ5/QE interacts with components of the cell-cycle during its progression in response to DNA damage.

The impact of preoperative serum C-reactive protein on the prognosis of patients with hepatocellular carcinoma.

BACKGROUND: The authors evaluated the significance of the preoperative serum C-reactive protein (CRP) level as a prognostic indicator in patients with hepatocellular carcinoma (HCC). METHODS: One hundred forty-one patients who underwent curative resection for HCC were reviewed retrospectively. Clinicopathologic variables were compared between patients with serum CRP levels >/= 1.0 mg/dL (n = 22 patients; the CRP-positive group) and patients with serum CRP levels < 1.0 mg/dL (n = 119 patients; the CRP-negative group). Univariate and multivariate analyses were conducted to identify factors that affected survival and disease recurrence. RESULTS: There was a significant correlation between the preoperative serum CRP level and tumor size. Invasion to the portal vein in the CRP-positive group was significantly more frequent than that in the CRP-negative group. Even after they underwent curative resection, 75.3% of patients in the CRP-positive group experienced recurrence within 1 year. The overall survival and recurrence-free survival rates in the CRP-positive group were significantly lower compared with the rates in the CRP-negative group. On multivariate analysis, the preoperative serum CRP level was selected as one of the unfavorable indicators regarding survival and recurrence. When CRP levels, albumin levels, and platelet counts that were available before surgery were scored as a combined index, the total score demonstrated a good stratification value for survival after hepatic resection. CONCLUSIONS: The current results showed that the preoperative serum CRP level is an independent and significant indicator predictive of poor prognosis and early recurrence in patients with HCC. The new CRP-based scoring system offers reliable information for predicting survival.

Roles of the HSP70-subunit in a eukaryotic multi-site-specific endonuclease, Endo.SceI: autophosphorylation and heat stability.

The 70 kDa heat shock proteins (HSP70) are a family of molecular chaperones that bind transiently to unfolded proteins in an ATP/ADP dependent manner. Endo.SceI comprises a unique example for mitochondrial HSP70, which exists in a stable complex with a nucleolytic subunit as a multi-site specific DNase. The HSP70-subunit in Endo.SceI was autophosphorylated by ATP in vitro. The autophosphorylation was higher in the Endo.SceI complex form than in the free form. Although the autophosphorylation had no significant effect on the endonucleolytic activity of Endo.SceI, the factors favoring autophosphorylation protected the endonucleolytic activity of Endo.SceI against heat inactivation. ATP, adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), and ADP not only protected the endonucleolytic activity against heat inactivation in the presence of Ca(2+) ions, but also reduced the labeling of the HSP70-subunit by [gamma-(32)P]ATP in Endo.SceI. These findings suggest that the HSP70-subunit shields Endo.SceI from heat inactivation through ATP/ADP binding.

The possible roles of the DNA helicase and C-terminal domains in RECQ5/QE: complementation study in yeast.

DmRECQ5/QE is a member of the RECQ5 subfamily, which shares homology with the Escherichia coli RecQ DNA helicase. Although the DNA helicase activity of RECQ5/QE has been characterized in vitro, the in vivo function of RECQ5/QE was essentially unknown. To investigate the cellular role of RECQ5, the potential of RECQ5/QE was evaluated by substitution of the only RecQ-like helicase, Sgs1, in budding yeast. RECQ5/QE can complement several phenotypes of sgs1, including the synthetic growth defect with srs2, the hypersensitivity to hydroxyurea and methyl methanesulfonate, and the elevated frequency of homologous recombination and sister chromatid exchange (SCE), but poorly complemented the suppression of slow growth in top3. These data suggested that RECQ5/QE exhibits an evolutionarily conserved RecQ function in vivo. The RECQ5/QE domain necessary for the yeast complementation was determined. The helicase domain and helicase activity were required to complement both the sgs1srs2 and sgs1top3 phenotypes. In contrast, the C-terminal domain was dispensable for complementing the sgs1srs2 phenotype, but was required for the sgs1top3 phenotype. These results suggested that the RECQ5/QE helicase activity is important for cellular function and that the C-terminal domain has a specific function in the absence of Top3.

Drosophila melanogaster RECQ5/QE DNA helicase: stimulation by GTP binding.

The Drosophila melanogaster RECQ5/QE gene encodes a member of the DNA helicase family comprising the Escherichia coli RecQ protein and products of the human Bloom's, Werner's, and Rothmund-Thomson syndrome genes. The full-length product of RECQ5/QE was expressed in the baculovirus system and was purified. Gel filtration experiments indicated that RECQ5/QE was present in an oligomeric state. The RECQ5/QE protein hydrolyzed ATP and even more actively GTP in the presence of single-stranded DNA. ATP drove the DNA helicase activity of RECQ5/QE, whereas GTP had little effect. GTP exhibited a stimulatory effect on DNA unwinding when it was used together with ATP. This effect was more apparent with non-hydrolyzable GTP analogs, such as GTPgammaS and GMPPNP. These results indicate that GTP binding to RECQ5/QE triggers its DNA helicase activity. GTP binding increased the rate of strand separation without affecting the S(0.5) (K(m)) values for the substrates during the DNA helicase reaction. The data collectively suggest that the RECQ5/QE protein is activated upon GTP binding through the ATP-binding site.