Effect of wortmannin on the suppression of thermotolerance in tsAF8 cells.

Phosphatidylinositol 3 kinases (PI-3 kinases) play a crucial role in the inositol phospholipid signaling pathway. The effect of wortmannin, an inhibitor of PI-3 kinases, on the suppression of thermotolerance development was investigated in the temperature-sensitive cell line tsAF8. When wortmannin was added immediately after the preheating, the thermotolerance was suppressed, with this suppression being dose-dependent from 0.01 to 0.1 microM, but not dose-dependent from 0.1 to 20 microM. Our results suggest that PI-3 kinases might play an important role in thermotolerance development in tsAF8 cells.     

Aberrant methylation and histone deacetylation of cyclooxygenase 2 in gastric cancer.

Cyclooxygenase 2 plays a critical role in the development of gastrointestinal cancers in both human and animal models. About 80% of the gastric cancer showed a high level of expression of cyclooxygenase 2, but a subset of cases do not express without unknown reason. Aberrant methylation of CpG island of COX-2 was examined by using a series of gastric cancer cell lines and primary gastric cancers. Two out of 8 cell lines (25%) and 11 out of 93 (12%) primary cancers showed aberrant methylation of the 5' region of COX-2. Methylation of COX-2 was closely associated with loss of expression and treatment of methylation inhibitor, 5-deoxy-2'-azacytidine restored the expression of COX-2. A combined treatment of 5-deoxy-2'-azacytidine and a histone deacetylese inhibitor, trichostatin A, restored re-expression of the gene synergistically and chromatin immunoprecipitation analysis revealed that histone of methylated COX-2 promoter is deacetylated, indicating the role of cytosine methylation and histone deacetylation in the silencing of the gene. These results indicate that a subset of gastric cancer with COX-2 methylation evolves through the pathway that is independent of COX-2 expression and that COX-2 inhibitor may not be useful to induce apoptosis in these cases.     

Therapeutic potential of a reduced-intensity preparative regimen for allogeneic transplantation with cladribine, busulfan, and antithymocyte globulin against advanced/refractory acute leukemia/lymphoma.

PURPOSE: Cladribine (2-CdA) is a purine analogue that exhibits activity against a variety of hematological malignancies and has a potent immunosuppressive effect. We therefore performed a pilot study to evaluate the feasibility of a novel 2-CdA-based reduced-intensity stem cell transplantation (RIST) regimen. EXPERIMENTAL DESIGN: A total of 16 scheduled patients with hematological malignancies were enrolled for comparison of their data with conventional stem cell transplantation (n = 19). The regimen for RIST consisted of 2-CdA (0.11 mg/kg/day for 6 days), busulfan (4 mg/kg/day for 2 days), and rabbit antithymocyte globulin (2.5 mg/kg/day for 4, 2, or 0 days). The underlying diseases included acute myelogenous leukemia (n = 6), chronic myelogenous leukemia (n = 2), myelodysplastic syndrome (n = 6), and non-Hodgkin's lymphoma (n = 2). RESULTS: After RIST, four patients died before day 100 as a result of acute graft-versus-host disease (n = 1), bacteremia (n = 1), disseminated candidasis (n = 1) and congestive heart failure (n = 1). Another patient died of cerebral infarction on day 140. Thus, acute-phase regimen-related toxicities >grade III were observed in only one patient. Engraftment and complete donor chimerism were achieved by day 28 in 14 evaluable patients, and 6 of them (43%) experienced grade II-IV acute graft-versus-host disease. With a median follow-up of 328 days (range, 231-633 days), the actuarial 1-year overall and disease-free survival rates were 69% and 50%, respectively. Notably, among seven high-risk patients (five patients had been in complete remission two or more times and two not in complete remission with refractory disease at transplant), only two patients developed leukemia relapse after RIST. Although the recovery of CD4+ cells was significantly slower (P = 0.02) in RIST than in conventional stem cell transplantation, the incidence of clinically documented infections was not significantly different between the two groups. CONCLUSION: The results suggest that this novel regimen containing 2-CdA is well tolerated and induces early complete donor chimerism. The unexpected durable remission achieved in patients with advanced disease at transplant suggests the presence of an acceptable antileukemia/lymphoma effect, which would warrant a further clinical trial.     

Elevated level of plasma basic fibroblast growth factor in multiple myeloma correlates with increased disease activity.

Recent reports that bone marrow angiogenesis is increased in multiple myeloma prompted us to examine plasma concentrations of angiogenic growth factors and to elucidate their clinical and biological significance. In 45 cases including 36 cases of multiple myeloma and 9 cases of monoclonal gammopathies of undetermined significance (MGUS), plasma concentrations of basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) were evaluated. FGF-2 was significantly elevated in 25 out of 45 (56%) of the patients with multiple myeloma compared with control subjects (median 9.01 pg ml vs. 1.58 pg/ml, P < 0.0001). The 25 cases were all active multiple myeloma, and none of the non-active myeloma and MGUS patients showed a high FGF-2 level. VEGF level was also elevated in 26 out of 45 patients (58%) compared with control subjects (median 42.0 pg/ml vs. 15.8 pg/ml, P < 0.0001 for VEGF). VEGF concentration was high in 20 active myelomas, but also in one non-active myeloma and five MGUS. Elevation of FGF-2 level was associated with beta2-microglobulin level, anemia and bone marrow plasma cell percentage, which represent disease activity. Interestingly, none of five Bence-Jones type myelomas, including four clinically active cases, revealed a high plasma FGF-2 level, while all of them showed a high VEGF level. In all five responders, the plasma FGF-2 levels were significantly decreased after chemotherapy. FGF-2 was immunohistochemically detected in the bone marrow myeloma cells of the patients with high plasma FGF-2 level. We conclude that plasma concentration of FGF-2 can be a useful indicator of disease activity.     

Development of Hepatocellular Adenomas and Carcinomas Associated with Fibrosis in C57BL/6J Male Mice Given a Choline-deficient, L-Amino Acid-defined Diet

Development of hepatocellular carcinomas in rats caused by a choline-deficient, L-amino acid defined (CDAA) diet, usually associated with fatty liver, fibrosis, cirrhosis and oxidative DNA damage, has been recognized as a useful model of hepatocarcinogenesis caused by endogenous factors. In the present study, in order to further explore involved factors and genes, we established an equivalent model in spontaneous liver tumor-resistant C57BL/6J mice. Six-week-old males and females were continuously fed the CDAA diet and histological liver lesions and oxidative DNA damage due to 8-hydroxydeoxyguanosine (8-OHdG) were examined after 22, 65 and 84 weeks. In male mice, fatty change and fibrosis were evident at 22 weeks, and preneoplastic foci of altered hepatocytes were seen at an incidence of 8/8 (100%) and a multiplicity of 6.6±4.0 per mouse at 65 weeks. Hepatocellular adenomas and carcinomas developed at incidences of 16/24 (66.7%) and 5/ 24 (20.8%), and multiplicities of 1.421±1.32 and 0.29±0.62, respectively, at 84 weeks. The female mice exhibited resistance to development of these lesions. The CDAA diet also increased 8-OHdG levels in male but not female mice. These results indicate that a CDAA diet causes hepatocellular preneoplastic foci, adenomas and carcinomas associated with fibrosis and oxidative DNA damage in mice, as in rats, providing a hepatocarcinogenesis model caused by endogenous factors in mice.     

Phase III study of concurrent versus sequential thoracic radiotherapy in combination with cisplatin and etoposide for limited-stage small-cell lung cancer: results of the Japan Clinical Oncology Group Study 9104.

PURPOSE: To evaluate the optimal timing for thoracic radiotherapy (TRT) in limited-stage small-cell lung cancer (LS-SCLC), the Lung Cancer Study Group of the Japan Clinical Oncology Group conducted a phase III study in which patients were randomized to sequential TRT or concurrent TRT. PATIENTS AND METHODS: We treated 231 patients with LS-SCLC. TRT consisted of 45 Gy over 3 weeks (1.5 Gy twice daily), and the patients were randomly assigned to receive either sequential or concurrent TRT. All patients received four cycles of cisplatin plus etoposide every 3 weeks (sequential arm) or 4 weeks (concurrent arm). TRT was begun on day 2 of the first cycle of chemotherapy in the concurrent arm and after the fourth cycle in the sequential arm. RESULTS: Concurrent radiotherapy yielded better survival than sequential radiotherapy (P =.097 by log-rank test). The median survival time was 19.7 months in the sequential arm versus 27.2 months in the concurrent arm. The 2-, 3-, and 5-year survival rates for patients who received sequential radiotherapy were 35.1%, 20.2%, and 18.3%, respectively, as opposed to 54.4%, 29.8% and 23.7%, respectively, for the patients who received concurrent radiotherapy. Hematologic toxicity was more severe in the concurrent arm. However, severe esophagitis was infrequent in both arms, occurring in 9% of the patients in the concurrent arm and 4% in the sequential arm. CONCLUSION: This study strongly suggests that cisplatin plus etoposide and concurrent radiotherapy is more effective for the treatment of LS-SCLC than cisplatin plus etoposide and sequential radiotherapy.     

Expression of Vascular Endothelial Growth Factor and E-Cadherin in Human Ovarian Cancer: Association with Ascites Fluid Accumulation and Peritoneal Dissemination in Mouse Ascites Model

Ascites formation and peritoneal dissemination are critical problems in patients with advanced ovarian cancer. Vascular endothelial growth factor (VEGF), also known as angiogenic growth factor, is a potent mediator of peritoneal fluid accumulation and angiogenesis of tumors. E-Cadherin is an adhesion molecule that is important for cell-to-cell interaction. To elucidate the molecular mechanism of ascites formation and peritoneal dissemination of ovarian cancer, we examined the expression of VEGF and E-cadherin in different ovarian cancer cell lines and utilized nude mice to compare the biological characteristics of ovarian cancer cells. Three human ovarian cancer cell lines (AMOC-2, HNOA and HTBOA) were used in this study. Expression of genes was analyzed by northern blotting and RT-PCR methods. AMOC-2 expressed E-cadherin, but not VEGF. HNOA expressed VEGF without E-cadherin expression. HTBOA expressed both VEGF and E-cadherin. Each human ovarian cancer model revealed a specific feature. The AMOC-2 mouse had a single large peritoneal tumor without ascites or remarkable peritoneal dissemination. HTBOA and HNOA mice had bloody ascites and marked peritoneal dissemination. Introduction of VEGF antisense into HTBOA cells could inhibit the ascites formation. It is suggested that VEGF is important for the ascites formation via the increased vascular permeability effect. The deregulation of E-cadherin expression might be involved in the peritoneal dissemination. These molecules are important for the formation of specific features of advanced ovarian cancer. Ovarian cancer cell lines that had different gene expression patterns produced nude mouse human ovarian cancer models with different characteristics.     

U-shaped Effect of Drinking and Linear Effect of Smoking on Risk for Stomach Cancer in Japan

A case-control study was conducted to evaluate the relationship between smoking or drinking doses and risk for stomach cancer, and to clarify whether the relationship is dose-dependent or U-shaped. Smoking dose was categorized as 0,1–399, 400–799, or 800+ cigarette-years, and drinking dose as 0, occasional/0.1–134.9, 135–1349.9, or 1350+ alcohol-years (ml of pure alcohol intake per day multiplied by years of drinking). Helicobacter pylori status was determined by serology for adjustment. Using logistic regression, the adjusted effects of smoking and drinking doses on risk for stomach cancer were calculated for both genders. Among male subjects, the odds ratios (95% confidence intervals (CIs)) were 1.29 (0.76, 2.18) for 1–399, 1.71 (1.05, 2.80) for 400–799 and 2.46 (1.49, 4.07) for 800+ cigarette-years compared with never-smokers, and 1.89 (0.97, 3.69) for never-drinkers, 2.82 (1.63, 4.86) for 135–1349.9 and 2.84 (1.97, 4.83) for 1350.0+, compared with occasional/0.1–134.9 alcohol-years. Among female subjects, they were 0.44 (0.20, 1.00) for 1–399 and 2.471 (0.91, 6.68) for 400+ cigarette-years compared with never-smokers, and 1.54 (0.90, 2.63) for never-drinkers and 1.39 (0.66, 2.93) for 135.0+ alcohol-years. Smoking seems to exert a linear effect and drinking, a J- or U-shaped effect on risk for stomach cancer, although there might be a dip of risk in light smokers among female subjects.     

Immunohistochemical study of the kidneys after severe muscular injury

Severe muscular injury sometimes causes renal failure, and myoglobin in skeletal muscle is known to induce toxic free oxygen radicals in the kidneys. The relationship between the immunohistochemical expression of myoglobin and the scavenger copper/zinc superoxide dismutase (Cu/Zn-SOD) was investigated in kidneys taken from two autopsy groups, a group with tourniquet shock (n = 4), and a group with severely injured skeletal muscle (n = 18). Paraffin-embedded kidney sections were used for immunohistochemical staining by the avidin-biotin-complex (ABC) method using antibodies against myoglobin and Cu/Zn-SOD. Detection of the two antigens was analyzed qualitatively. In most cases of tourniquet shock in which the survival time was considered to be relatively long, myoglobin staining was positive and Cu/Zn-SOD was negative. Among the seven cases of severely injured skeletal muscle in which the survival period was considered to be relatively short, positive staining was detected immunohistochemically for both myoglobin and Cu/Zn-SOD. Moreover, in most of the cases in this group that showed acute tubular necrosis, immunohistochemical staining was negative for both markers, whereas positive staining was found for most of the cases in which the kidneys were revealed to be normal by HE staining. These findings suggest that when myoglobin enters the kidneys via the circulation, Cu/Zn-SOD reacts to eliminate free radicals, but is depleted by consumption in the long run, and that there might be a relationship between these histological findings and immunohistochemical expression. Received: 2 December 1999 / Accepted: 22 May 2000