Possible involvement of LKB1-AMPK signaling in non-homologous end joining.

LKB1/STK11 is a tumor suppressor gene responsible for Peutz-Jeghers syndrome, an inherited cancer disorder associated with genome instability. The LKB1 protein functions in the regulation of cell proliferation, polarization and differentiation. Here, we suggest a role of LKB1 in non-homologous end joining (NHEJ), a major DNA double-strand break (DSB) repair pathway. LKB1 localized to DNA ends upon the generation of micro-irradiation and I-SceI endonuclease-induced DSBs. LKB1 inactivation either by RNA interference or by kinase-dead mutation compromised NHEJ-mediated DNA repair by suppressing the accumulation of BRM, a catalytic subunit of the SWI/SNF complex, at DSB sites, which promotes the recruitment of an essential NHEJ factor, KU70. AMPK2, a major substrate of LKB1 and a histone H2B kinase, was recruited to DSBs in an LKB1-dependent manner. AMPK2 depletion and a mutation of H2B that disrupted the AMPK2 phoshorylation site impaired KU70 and BRM recruitment to DSB sites. LKB1 depletion induced the formation of chromosome breaks and radials. These results suggest that LKB1-AMPK signaling controls NHEJ and contributes to genome stability.

Histone acetylation by CBP and p300 at double-strand break sites facilitates SWI/SNF chromatin remodeling and the recruitment of non-homologous end joining factors.

Non-homologous end joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs) generated by ionizing radiation (IR) and anti-cancer drugs. Therefore, inhibiting the activity of proteins involved in this pathway is a promising way of sensitizing cancer cells to both radiotherapy and chemotherapy. In this study, we developed an assay for evaluating NHEJ activity against DSBs in chromosomal DNA in human cells to identify the chromatin modification/remodeling proteins involved in NHEJ. We showed that ablating the activity of the homologous histone acetyltransferases, CBP and p300, using inhibitors or small interfering RNAs-suppressed NHEJ. Ablation of CBP or p300 impaired IR-induced DSB repair and sensitized lung cancer cells to IR and the anti-cancer drug, etoposide, which induces DSBs that are repaired by NHEJ. The CBP/p300 proteins were recruited to sites of DSBs and their ablation suppressed acetylation of lysine 18 within histone H3, and lysines 5, 8, 12, and 16 within histone H4, at the DSB sites. This then suppressed the recruitment of KU70 and KU80, both key proteins for NHEJ, to the DSB sites. Ablation of CBP/p300 also impaired the recruitment of BRM, a catalytic subunit of the SWI/SNF complex involved in chromatin remodeling at DSB sites. These results indicate that CBP and p300 function as histone H3 and H4 acetyltransferases at DSB sites in NHEJ and facilitate chromatin relaxation. Therefore, inhibition CBP and p300 activity may sensitize cancer cells to radiotherapy and chemotherapy.

Unbalanced translocation, a major chromosome alteration causing loss of heterozygosity in human lung cancer.

Loss of heterozygosity (LOH) is a major genetic event causing inactivation of tumor suppressor genes in human carcinogenesis. To elucidate chromosomal mechanisms causing LOH, 201 LOHs in 10 cases of human lung cancer, which were detected by a genome-wide single nucleotide polymorphism array analysis, were investigated for responsible chromosome alterations by integrating information on breakpoints for DNA copy number changes obtained by array-comparative genome hybridization and on numerical and structural chromosomal alterations obtained by spectral karyotyping. The majority (80%) of LOHs were partial chromosome LOHs caused by structural chromosomal alterations, while the remaining (20%) were whole chromosome LOHs caused by whole chromosome deletions. Unbalanced translocation was defined as the most frequent alteration, and it accounted for 30% of all LOHs. Three other structural alterations-interstitial deletion (19%), mitotic recombination (9%) and gene conversion (6%)-also contributed to the occurrence of LOH, while terminal deletion contributed to only a small subset (1%). Since unbalanced translocation is a common chromosomal alteration in lung cancer cells, the results in the present study strongly indicate that a considerable fraction of LOHs detected in lung cancer cells are caused by unbalanced translocation.

A comparison of the University of Wisconsin solution and the modified Kawakami solution for initial flush and coronary perfusion in long-term canine heart preservation.

BACKGROUND: We compared two different solutions, the University of Wisconsin (UW) solution (intracellular-like) and the modified Kawakami (mK) solution (extracellular-like), for initial flush of coronary vascular beds before simple storage and following coronary perfusion. METHODS: After a right thoracotomy in the 4th intercostal space, the donor heart was isolated by ligating the azygos vein and venae cavae, and cross-clamping the aorta. Cardiac arrest was then obtained with a cold GIK solution. Following initial flush of coronary vascular beds, the donor heart was resected, stored utilizing a combination of simple immersion and coronary perfusion, and then transplanted. A total of 48 mongrel dogs was divided into three groups each using different solutions for the initial flush of coronary vascular beds and for coronary perfusion. In group I (n=10) the UW solution was used for both initial flush and coronary perfusion. In group II (n=7) the mK solution was used for both initial flush and coronary perfusion, and in group III (n=7) the UW solution was used for initial flush and the mK solution for coronary perfusion. Intracellular high-energy phosphate was surveyed by 31P-nuclear magnetic resonance spectroscopy. RESULTS: After 12-hour simple immersion and 1-hour coronary perfusion, phosphocreatine and adenosine triphosphate were significantly (p<0.05) higher in group III than in groups I and II. The high-energy phosphate levels of the graft tissue were better in groups I and III than in group II. Orthotopic transplantation was then performed using 10 preserved grafts in group I and seven preserved grafts of group III. After transplantation, left ventricular (LV) pressure of group I animals recovered to 82.3% and group III recovered to 95.8% of the control values. LV dp/dt of group I and III animals recovered to 76.5% and 96.7% of the control values, respectively. CONCLUSIONS: The UW solution, which is acceptable for both initial flush and simple storage, is not suitable for continuous coronary perfusion even for a short period due to its high viscosity. A combination of the UW solution both for initial flush and the following cold simple immersion and the mK solution for continuous coronary perfusion is appropriate for long-term preservation of the canine heart.

The effects of the hypothermic management of brain dead dogs on preserving graft viability in heart transplantation.

The effect of hypothermic management for brain dead dogs on preserving graft viability was evaluated through preservation and transplantation. After the occurrence of brain death, 43 dogs were divided into two groups; the normothermic group (37.2+/-0.3 degrees C) and the hypothermic group (31.8+/-0.3 degrees C) according to the esophageal temperature. After the 6-hour management of brain dead donors, the heart beat was arrested using a cardioplegic solution followed by coronary vascular bed washout. The donor heart was then harvested and preserved for 12 hours with simple immersion into the University of Wisconsin solution. Following preservation, orthotopic transplantation was performed in six grafts randomly selected from each group. During the 6-hour management of brain dead dogs; 1) heart rates, rate-pressure products, and the total amount of catecholamine were significantly (p<0.05) lower in the hypothermic group than in the normothermic group, and 2) lactate contents collected from the coronary sinus blood and O2-extraction rates of the heart tended to be lower in the hypothermic group than in the normothermic group. During 12 hours of preservation, intracellular pH and creatine phosphate contents were higher in the hypothermic group than in the normothermic group. Following orthotopic transplantation, the animals in the hypothermic group showed a significantly (p<0.05) higher recovery rate of left ventricular (LV) pressure and the maximum rate of the rise of LV pressure compared with normothermic group animals. We conclude that the hypothermic management of brain dead dogs may be effective in preserving graft viability and may provide a clinical application for heart transplantation with acceptable outcomes.

Laparoscopic minilaparotomy pancreaticoduodenectomy with lymphadenectomy using an abdominal wall-lift method.

Because of technical difficulties attributable to the lack of appropriate techniques, the high cost of laparoscopic instruments, and the need for numerous disposable stapling devices, laparoscopic pancreaticoduodenectomy has been performed in only two patients. To solve the above problems, we devised a method of laparoscopic minilaparotomy using an abdominal wall-lift method and have successfully used it to perform pancreaticoduodenectomy with lymphadenectomy in a patient with distal choledochal cancer.

Long-term heart preservation by the combined method of simple immersion and coronary perfusion.

BACKGROUND: The combined method of cold storage and coronary perfusion for prolonged preservation of donor hearts was evaluated through preservation and transplantation with the use of 10 pair of adult mongrel dogs. METHODS: In situ initial flush for cooling and coronary vascular washout was performed with a University of Wisconsin solution. The heart was then removed and immersed into a cold (4degrees C) University of Wisconsin solution. After 12-hour cold storage, 1-hour preservation was added by coronary perfusion using a 4degree C oxygenated University of Wisconsin solution. High-energy phosphate (phosphocreatine, beta-adenosine triphosphate) and phosphate were measured by 31P-nuclear magnetic resonance spectroscopy at 0-hour cold storage, 12-hour cold storage, and immediately after coronary perfusion. RESULTS: Phosphocreatine and beta-adenosine triphosphate decreased to 12.1 +/- 24.2% (mean +/ standard deviation), 37.4% +/- 25.0%, respectively, after 12-hour cold storage, and significantly increased to 94.0% +/- 48.7% (p < 0.001), 48.8% (p < 0.05), respectively after 1-hour coronary perfusion. Disorder of nuclear arrangement and edema of muscular cells, which were observed after cold storage, were histologically restored after 1-hour coronary perfusion. After transplantation of preserved grafts, left ventricular pressure and left ventricular rate of pressure rise were evaluated in the graft with 12-hour cold storage only (group A) and in the graft with 12-hour cold storage and 1-hour coronary perfusion (group B). Left ventricular pressure 2 hours after transplantation in group B recovered to 76.1% significantly (p < 0.01), compared with 51.9% in group A. Significantly higher values of left ventricular rate of pressure rise 2 hours after transplantation was observed in group B (83.0% +/ 12.7%) compared with group A (68.2% +/- 12.5%). CONCLUSIONS: These results indicate that the combined method of cold storage and coronary perfusion may be effective for myocardial protection during long-term preservation.

Laparoscopic retrograde cholecystectomy (from fundus downward) facilitated by lifting the liver bed up to the diaphragm for inflammatory gallbladder.

In order to safely and reliably perform laparoscopic cholecystectomy in severe inflammatory cases (e.g., acute or chronic cholecystitis), we have designed a method of suturing the liver bed to the diaphragm, lifting it cephalad so as to maintain a good operative field. Initially, we dissect the gallbladder fundus, fully dissecting the neck of the gallbladder from the liver and finally dissecting the cystic duct (laparoscopic retrograde cholecystectomy facilitated by lifting the liver bed up to the diaphragm; Lap-RC). This method is different from laparoscopic standard cholecystectomy (Lap-SC), in which dissection of the cystic duct is done first. One hundred and twenty-nine consecutive laparoscopic cholecystectomies for various gallbladder diseases were carried out at Nerima General Hospital between August 1991 and June 1994. Fifteen cases of Lap-RC and six cases of Lap-SC in a severe inflammatory group were comparatively evaluated. Thirteen cases of Lap-RC and 92 cases of Lap-SC in a noninflammatory group were also comparatively evaluated. The rates of conversion to laparotomy were 0% in Lap-RC cases (0/15) and 33% in Lap-SC cases (3/9) in the severe inflammatory group. The incidences of major postoperative complications were 0% in Lap-RC cases (0/15) and 17% in Lap-SC cases (1/6) in the severe inflammatory group. In conclusion, Lap-RC showed satisfactory results in terms of both safety and reliability in patients with severe inflammatory disease.

Laparoscopic and minilaparotomy proximal gastrectomy and esophagogastrostomy: technique and case report.

A laparoscopic and minilaparotomy proximal gastrectomy with esophagogastrostomy (end-to-side anastomosis) was performed on a 56-year-old woman with a leiomyoma located just below the esophagogastric junction. Gastroscopic examination revealed a leiomyoma (diameter of 2.5 cm) just below the esophagogastric junction. We considered a laparoscopic proximal gastrectomy safer than a laparoscopic partial gastrectomy because of the risk of postoperative anastomotic stenosis in this case. Therefore, the patient underwent laparoscopic minilaparotomy proximal gastrectomy with esophagogastrostomy. On postoperative day 1, she was able to walk. On postoperative day 4, she started on a clear liquid diet and was discharged on postoperative day 14. During her postoperative recovery, the patient experienced little pain and did not request narcotic analgesia.

Inflammatory responses in open versus laparoscopic herniorrhaphy.

The aim of this study is to verify whether differences could be found or not as concerns of the postoperative surgical stress between the conventional open herniorrhaphy and laparoscopic herniorrhaphy. We therefore compared the immunological and inflammatory responses to open and laparoscopic procedures in this study. The sex, age, weight, and height characteristics of group 1 (open surgery group) matched those of group 2 (laparoscopic surgery group), and no statistically significant differences were found between them. Nor were there any statistically significant differences in operating time or the postoperative hospitalization. Blood samples were collected preoperatively [postoperative day (POD)0], POD 1 and 5, and the following parameters were assessed: total leukocyte (WBC) count, including neutrophil and lymphocyte subpopulations, HLA-DR+CD3+, IL-6, C-reactive protein (CRP), serum albumin, and body temperature. No differences were detected between two groups in leukocyte, neutrophil, or lymphocyte count, HLA-DR+CD3+, IL-6, albumin, or body temperature or any of the postoperative days. The only statistically significant difference was a higher CRP value in the laparoscopy group than the open group only on POD 1 (p < 0.05). It can be concluded that the laparoscopic approach appears to offer no advantages over the open approach to herniorrhaphy from the standpoint of immunological and inflammatory responses.

Laparoscopic Billroth I gastrectomy for gastric ulcer: technique and case report.

A laparoscopic distal gastrectomy with gastroduodenostomy (Billroth I) was performed on a patient with intractable gastric ulcer. The patient was a 56-year-old man, complaining of severe epigastralgia, who had a 10-year history of peptic ulcer. Gastroscopy had revealed a UL-IV gastric ulcer on the lesser curvature at the angle of the stomach and a deformity of the bulbus. A diagnosis of intractable gastric ulcer was made, and the patient underwent laparoscopic distal gastrectomy and gastroduodenostomy. On postoperative day 1, he was able to walk. On postoperative day 4, he started on a clear liquid diet and was discharged on postoperative day 14.

Laparoscopic minilaparotomy Billroth I gastrectomy with extraperigastric lymphadenectomy for early gastric cancer using an abdominal wall-lifting method.

Laparoscopic gastrectomy with extraperigastric lymphadenectomy for early gastric cancer has never been performed because of technical difficulties attributable to the lack of appropriate techniques, the high cost of laparoscopic instruments, and the need for numerous disposable stapling devices. In order to solve these problems, we have designed a method of laparoscopic minilaparotomy using an abdominal wall-lifting method, and a patient with early gastric cancer (depth of submucosa) underwent by this laparoscopic minilaparotomy distal gastrectomy with extraperigastric lymphadenectomy. During his postoperative recovery, the patient requested no narcotic analgesic, and was discharged on postoperative day 14.

Laparoscopic and minilaparotomy Billroth I gastrectomy for gastric ulcer using an abdominal wall-lifting method.

Laparoscopic distal gastrectomy is still technically difficult because of the lack of appropriate techniques, the expensive laparoscopic instruments, and the use of numerous disposable stapling devices. In an attempt to solve these problems, we have designed a method of laparoscopic and minilaparotomy Billroth I gastrectomy using an abdominal wall-lifting method.

Diminished visceral adipose tissue in cancer cachexia.

To estimate the relationship between the visceral adipose tissue (AT) area and cancer cachexia, 13 cachectic patients (7 males, 6 females; age 65.2 +/- 11.0 years; body mass index 20.8 +/- 4.1 kg/m2) were examined by computed tomography (CT) scanning. Cachectic cancer patients who had a 10% decrease of body weight and died within 6 months because of gastrointestinal carcinoma had a significantly smaller visceral AT area than control subjects (mean +/- sd: 43.9 +/- 42.2 cm2 vs. 93.4 +/- 56.0 cm2, P < 0.05, P = 0.014). Otherwise, there were no significant differences between the visceral AT areas of cachectic cancer patients and those of cancer patients with resectable tumors treated by curative operation (mean +/- sd: 68.8 +/- 57.7 cm2) (NS, P = 0.206). There was, however, a tendency for cachectic cancer patients to have a smaller visceral AT area than those with resectable tumors. This result suggests that the visceral AT area is not preserved in the cachectic state associated with cancer.

Variables related to risk of recurrence in rectal cancer without lymph node metastasis.

BACKGROUND: There has been recent interest in the use of local excision for rectal cancer under consideration of patient's quality of life. However, local excision of the primary tumor does not remove the areas of lymphatic spread. Therefore, the decision to use this procedure must be considered carefully. METHODS: The authors retrospectively analyzed 142 patients who underwent radical resection of rectal cancer without lymph node metastasis in order to define the risk factors for recurrence. The macroscopic and microscopic pathological characteristics, immunohistochemical staining for p53, and DNA ploidy pattern of the primary tumor were examined as potential predictors of recurrence. RESULTS: The rates for 5-year disease-free survival, local control, freedom from distant metastasis, and overall survival in these 142 patients were 87%, 93%, 93%, and 91%, respectively. Factors related to recurrence and prognosis included the depth of tumor invasion, vascular/lymphatic involvement, tumor differentiation, and tumor size. However, p53 staining and DNA ploidy pattern were not useful indicators. CONCLUSIONS: Our findings suggest that adjunctive radiotherapy and chemotherapy should be considered for patients who have rectal cancer without lymph node metastasis in the following situations: tumor invasion of the serosa, vascular/lymphatic involvement, moderately differentiated adenocarcinoma, and lesions > 2 cm in diameter. Local excision should not be used in these situations, even if there are no lymph node metastases.

Extended lymphadenectomy and the quality of life in rectal cancer patients.

For a better quality of life in rectal cancer patients, high dose radiotherapy following abdominoperineal resection of the rectum with a pelvic partition is another surgical option replacing extended abdominoperineal resection. In addition to pelvic partition with polyglycolic acid mesh, the tissue expander was inserted into the pelvic cavity to support the intestine upward and the bladder forward. The mean total radiation dosage was 5040 cGy. Between 1989 and 1991, 10 patients were treated according to this method. Out of 10 patients 9 were free of recurrence, and only one had hepatic metastasis. In addition, postoperatively, the average residual urine by this method was 39.1 ml and was statistically different compared to a figure of 200 ml in conventional abdominoperineal resections (p < 0.001, "t"-test). In order to individualize the operative procedures among a variety of surgical options, the molecular biological technique was utilized. In p53 stain analysis of 114 colorectal cancer patients, patients with p53 positive staining reached a higher stage than those with p53 negative staining (p < 0.05, chi 2 analysis). Therefore, we surmised that the positivity of the p53 stain could be one of the factors gauged as an indication of postoperative high-dose radiation. In conclusion, high-dose postoperative radiotherapy was thought to be one of the treatment modalities to improve the survival and quality of life of advanced rectal cancer in selected cases.