CD44s signals the acquisition of the mesenchymal phenotype required for anchorage-independent cell survival in hepatocellular carcinoma.

BACKGROUND: Circulating tumour cells (CTCs) have an important role in metastatic processes, but details of their basic characteristics remain elusive. We hypothesised that CD44-expressing CTCs show a mesenchymal phenotype and high potential for survival in hepatocellular carcinoma (HCC). METHODS: Circulating CD44(+)CD90(+) cells, previously shown to be tumour-initiating cells, were sorted from human blood and their genetic characteristics were compared with those of tumour cells from primary tissues. The mechanism underlying the high survival potential of CD44-expressing cells in the circulatory system was investigated in vitro. RESULTS: CD44(+)CD90(+) cells in the blood acquired epithelial-mesenchymal transition, and CD44 expression remarkably increased from the tissue to the blood. In Li7 and HLE cells, the CD44(high) population showed higher anoikis resistance and sphere-forming ability than did the CD44(low) population. This difference was found to be attributed to the upregulation of Twist1 and Akt signal in the CD44(high) population. Twist1 knockdown showed remarkable reduction in anoikis resistance, sphere formation, and Akt signal in HLE cells. In addition, mesenchymal markers and CD44s expression were downregulated in the Twist1 knockdown. CONCLUSIONS: CD44s symbolises the acquisition of a mesenchymal phenotype regulating anchorage-independent capacity. CD44s-expressing tumour cells in peripheral blood are clinically important therapeutic targets in HCC.

Preoperative serum hyaluronic acid level as a prognostic factor in patients undergoing hepatic resection for hepatocellular carcinoma.

BACKGROUND: Hyaluronic acid (HA) probably plays a critical role in tumorigenesis. The clinical significance of serum HA concentration in patients with hepatocellular carcinoma (HCC) remains to be elucidated. This study analysed the relationship between preoperative serum HA levels and prognosis after hepatic resection in patients with HCC. METHODS: Consecutive patients who underwent hepatic resection for HCC between September 1999 and March 2012 were included in this retrospective study. Serum HA levels were measured within 4 weeks before surgery by an immunoturbidimetric automated latex assay. The cut-off level for preoperative serum HA was validated using a time-dependent receiver operating characteristic (ROC) curve analysis. The prognostic impact of preoperative serum HA levels was analysed using Cox proportional hazards models. RESULTS: A total of 506 patients of median age 66 years (405 men, 80·0 per cent) were analysed. The median length of follow-up was 32 months. High serum HA levels (100 ng/ml or above) were associated with shorter recurrence-free survival (P < 0·001) (hazard ratio (HR) 1·50, 95 per cent confidence interval 1·17 to 1·93; P = 0·002) and overall survival (P = 0·001) (HR 1·46, 1·03 to 2·07; P = 0·033). In patients with HCC without severe liver fibrosis, serum HA level was correlated with multiple tumours (P = 0·039), early recurrence (P = 0·033), and poor recurrence-free (P < 0·001) and overall (P = 0·024) survival. CONCLUSION: High preoperative serum HA levels predict poor prognosis in patients with HCC after hepatic resection, and may serve as a future biomarker.

Role for caspase-mediated cleavage of Rad51 in induction of apoptosis by DNA damage.

We report here that the Rad51 recombinase is cleaved in mammalian cells during the induction of apoptosis by ionizing radiation (IR) exposure. The results demonstrate that IR induces Rad51 cleavage by a caspase-dependent mechanism. Further support for involvement of caspases is provided by the finding that IR-induced proteolysis of Rad51 is inhibited by Ac-DEVD-CHO. In vitro studies show that Rad51 is cleaved by caspase 3 at a DVLD/N site. Stable expression of a Rad51 mutant in which the aspartic acid residues were mutated to alanines (AVLA/N) confirmed that the DVLD/N site is responsible for the cleavage of Rad51 in IR-induced apoptosis. The functional significance of Rad51 proteolysis is supported by the finding that, unlike intact Rad51, the N- and C-terminal cleavage products fail to exhibit recombinase activity. In cells, overexpression of the Rad51(D-A) mutant had no effect on activation of caspase 3 but did abrogate in part the apoptotic response to IR exposure. We conclude that proteolytic inactivation of Rad51 by a caspase-mediated mechanism contributes to the cell death response induced by DNA damage.

Role for E2F in DNA damage-induced entry of cells into S phase.

Mammalian cells respond to ionizing radiation (IR) with transient cell cycle arrest and induction of apoptosis. Here we show that IR increases the expression of the E2F-1 transcription factor and the entry of cells into S phase. E2F-1 transactivation function is inhibited by cyclin A-kinase to ensure orderly progression through S phase. However, in contrast to proliferating cells, IR treatment results in down-regulation of cyclin A-kinase. Expression of a dominant negative form of the E2F heterodimeric partner DP-1 confirmed the involvement of E2F in IR-induced S-phase entry. These findings also support opposing signals involving the induction of E2F and the down-regulation of cyclin A-kinase in the IR response.

Activation of protein kinase C delta by the c-Abl tyrosine kinase in response to ionizing radiation.

The c-Abl protein tyrosine kinase is activated by ionizing radiation (IR) and certain other DNA-damaging agents. The present studies demonstrate that c-Abl associates constitutively with protein kinase C delta (PKCdelta). The results show that the SH3 domain of c-Abl interacts directly with PKCdelta. c-Abl phosphorylates and activates PKCdelta in vitro. We also show that IR treatment of cells is associated with c-Abl-dependent phosphorylation of PKCdelta and translocation of PKCdelta to the nucleus. These findings support a functional interaction between c-Abl and PKCdelta in the cellular response to genotoxic stress.

Function for p300 and not CBP in the apoptotic response to DNA damage.

The cellular response to ionizing radiation (IR) includes the induction of apoptosis. The p300/CBP proteins possess histone acetyltransferase activity and function as transcriptional coactivators of p53. We have prepared cells deficient in p300 or CBP to define the roles of these proteins in the cellular response to DNA damage. The present results demonstrate that p300, but not CBP, contributes to IR sensitivity of cells. The results also demonstrate that IR-induced apoptosis is impaired in the p300-, but not CBP-, deficient cells. These findings indicate that p300 functions in the apoptotic response to DNA damage.

Pro-apoptotic effect of the c-Abl tyrosine kinase in the cellular response to 1-beta-D-arabinofuranosylcytosine.

Treatment of cells with the antimetabolite 1-beta-D-arabinofuranosylcytosine (ara-C) and other genotoxic agents is associated with activation of the c-Abl protein tyrosine kinase. The functional role of c-Abl in the response to DNA damage, however, remains unclear. The present studies demonstrate that cells expressing a dominant negative, kinase-inactive c-Abl (K-R) are resistant to killing by ara-C. The expression of c-Abl (K-R) blocked ara-C-induced apoptosis by a mechanism that is at least in part independent of the p53 tumor suppressor. Cells null for c-Abl also exhibited resistance to induction of apoptosis. These findings provide support for a pro-apoptotic function of c-Abl in the response to certain genotoxic drugs.

Constitutive IL-8 expression in cancer cells is associated with mutation of p53.

We previously reported that most cancer cell lines constitutively express various cytokines including IL-8. But how IL-8 gene expression is regulated in cancer cells is still unclear. p53 tumor suppressor gene plays an important role in the regulation of transcription and is mutated in cancer cell lines. We investigated whether p53 status affects the constitutive expression of IL-8 in human cancer cells. SUIT-2 and RERF-LCOK cancer cells constitutively produced high levels of IL-8 in culture medium. Both cell lines were shown to carry a p53 mutation, and constitutive NF-kappaB transcriptional activity. To analyze whether p53 status mediates IL-8 expression, the effect of wild-type p53 (wt-p53) gene transfer on activation of NF-kappaB was determined in both cell lines. ELISA showed that the IL-8 concentration in medium decreased dose dependently by transient expression of wt-p53. Western-blot analysis showed no marked change in NF-kappaB protein levels in cell nuclei. EMSA showed no repression of NF-kappaB binding activity after transient expression of wt-p53. In contrast, luciferase reporter studies indicated that transcriptional activity of NF-kappaB is suppressed by transfection of wt-p53. These results show that wt-p53 gene transfer inhibits IL-8 production and NF-kappaB transcription activity in cancer cells and suggest that constitutive IL-8 production in cancer cells is associated with mutation of p53.

Five jumps per day increase bone mass and breaking force in rats.

The effects of jump training on bone morphological and mechanical properties were investigated in immature bones of female Fischer 344 rats. Five-week-old rats were divided into control or five jump-trained groups comprised of 5-, 10-, 20-, 40-, and 100-jump groups, representing the number of jumps per day. The rats were jump-trained 5 days/week for 8 weeks, and the height of jump was increased to 40 cm progressively. The femur and tibia in the 5-jump group had significantly greater fat-free dry weights per body weight and maximum loads at the fracture tests than those in the control group. The tibia in the 5-jump group also had significantly larger cortical area at the cross-sectional analysis. Although a slight tendency toward increase according to the number of jumps per day was observed, there were few differences in bone morphological and mechanical parameters among the 10-, 20-, and 40-jump groups. The present results indicate that a large number of strains per day is not necessary for bone hypertrophy to develop in rats.

Fibroblast-tumor interaction changes constitutive expression of IL-6 and IL-8 in human carcinoma-cells.

To examine whether expression of interleukin-6 (IL-6) and interleukin-8 (IL-8) in human carcinoma cells can be influenced by host fibroblasts, we investigated the expression of IL-6 and IL-8 mRNA in carcinoma cells cocultured with fibroblasts. Four human cancer cell lines, NUGC3 and GaCa gastric carcinoma cells, RERF-LCOK lung carcinoma cells, and GBK-1 gallbladder carcinoma cells, which constitutively express large amounts of both IL-6 and IL-8, were cocultured with murine 3T3 fibroblasts, in which IL-6 and IL-8 were not detected, under the same conditions. By Northern blot analysis, the expression of IL-6 mRNA was significantly decreased in NUGC3, GBK-1, and RERF-LCOK cells but was increased in GaCa cells. The expression of IL-8 mRNA was significantly increased in GaCa and GBK-1 cells but decreased in RERF-LCOK cells. Using cell-free conditioned medium, only the NUGC3-3T3 culture supernatant showed little effect on IL-6 and IL-8 mRNA expression in the NUGC3 cells, thereby suggesting that changes in IL-6 and IL-8 mRNA expression observed in the coculture experiment depended mainly on 3T3-NUGC3 contact and not on soluble factors. Similar changes in IL-6 and IL-8 mRNA expression were noted when NUGC3 cells were cultured with paraformaldehyde-fixed 3T3 cells or the 3T3 membrane fraction, thereby supporting this notion. Northern blot analysis of transplanted NUGC3 tumors in nude mice showed a decrease in IL-6 mRNA expression and augmentation of IL-8 mRNA expression. Sera from the NUGC3-bearing mice showed only small differences in IL-6 and IL-8. The downregulation of IL-6 mRNA was reversed 20 hours after NUGC3 cancer cells were separated from the in vivo grown NUGC3 tumors and cultured in vitro. These results suggest that tumors producing IL-6 or IL-8 can be differently modulated by the host cell-mediated pathways, such as contact between fibroblast and tumor cells.

IL-6 and IFN-gamma regulation of IL-10 production by human colon carcinoma cells.

IL-10 has been shown to play a crucial role in immunosuppression in cancer patients. We explored the regulation of IL-10 production by TNF-alpha, IL-1 beta, IL-6, IL-8, and IFN-gamma in human colon carcinoma COLO205 cells. Northern analysis revealed a marked expression of IL-10 mRNA after stimulation by IL-6, and a marginal but significant expression by TNF-alpha, IL-1 beta or IFN-gamma. No IL-10 mRNA expression was observed when cells were untreated or incubated with IL-8. IL-10 in the culture supernatants showed good agreement with mRNA expression. In addition, IFN-gamma dose-dependently inhibited this IL-6-induced production of IL-10. MTT assay revealed that low dose IFN-gamma (1-10 ng/ml) had no effect on growth of COLO205 cells, but that high dose IFN-gamma (>100 ng/ml) significantly inhibited their proliferation. Northern analysis of COLO205 cells pretreated with IFN-gamma demonstrated that the IL-6R alpha chain was down-regulated. These results suggest that, in certain colon carcinoma cells, tumor-derived IL-10 production is directly regulated by systemic or local production of pro-inflammatory cytokines, such as IL-6 and IFN-gamma.

Carcinoma-cells express IL-8 and the IL-8 receptor - their inhibition attenuates the growth of carcinoma-cells.

We investigated the expression of IL-8 and the IL-8 receptor (IL-8R) in human carcinoma cells and the role of IL-8 and IL-8R in the growth of carcinoma cells. IL-8 mRNA was detected in 16 of 20 (80%) carcinoma cell lines and 20 of 24 (83.3%) cancer tissues by Northern blot analysis. IL-8R mRNA was expressed in 7 of 11 (63.6%) carcinoma cell lines by reverse transcriptase polymerase chain reaction (RT-PCR). Neutrophil chemotactic activity in the culture supernatant of carcinoma cell lines correlated with immunoreactive IL-X concentration. Growth of carcinoma cells was significantly inhibited in the presence of anti-IL-8 antibody or IL-8R antisense oligonucleotide. These results revealed that IL-8 and IL-8R are expressed in the majority of carcinoma cells and suggest that they might play a role in the growth of carcinoma cells.

Humoral factors derived from glial cells protect cultured neuroblastoma cells against glutamate toxicity.

The protective effects of glial cells against glutamate cytotoxicity on neuronal cells were studied using clonal neuroblastoma cells and two types of glial cells. Neuronal cells treated with glia-conditioned medium became able to tolerate glutamate toxicity. It is suggested that the preparatory processes against glutamate toxicity might be developed in neurons by the humoral factor(s) released from glial cells.

Comparison of sympatho-adrenal activity during endurance exercise performed under high- and low-carbohydrate diet conditions.

Effects of varied carbohydrate (CHO) content in the diet on sympatho-adrenal activity to endurance exercise during which blood sugar was kept over a preexercise level were studied in five male physical education students. The CHO loading was used and consisted of a 7-day low CHO diet (30% CHO, 50% fat, 20% protein) followed by a 7-day high CHO diet (70% CHO, 20% fat, 10% protein). The results obtained from the present study were as follows: (1) plasma epinephrine (E) was almost the same between the low and the high CHO diets before and at 30 min of the exercise, while plasma norepinephrine (NE) level at 30 min of the exercise was significantly higher in the low (959 +/- 98 pg/ml) than in the high CHO diet (679 +/- 64 pg/ml) (p less than 0.05); (2) serum free fatty acid (FFA) level was significantly higher in the low than in the high CHO diet before (p less than 0.05) and at 30 min of the exercise (p less than 0.01); (3) a negative correlation was found between muscle glycogen and plasma NE (p less than 0.05). In all the subjects, increase in serum FFA accompanied by increase in plasma NE was detected in the low CHO diet. In conclusion, sympathetic activity to endurance exercise during which blood sugar was kept over a preexercise level was elevated more in the low than in the high CHO diet. It was suggested that the more elevated sympathetic nervous activity would have resulted from glycogen depletion in the working muscle due to the low CHO diet and would have increased FFA mobilization from the adipose tissue.

Characteristic feature of oxygen cost at simulated laboratory triathlon test in trained triathletes.

BACKGROUND: The present study was carried out in order to investigate the respiratory and circulatory features during a simulated laboratory triathlon test in trained triathletes. EXPERIMENTAL DESIGN: Sixteen male triathletes were divided into superior (n = 8) and slower triathletes (n = 8) according to their race time. These subjects performed both maximal exercise tests and a simulated laboratory triathlon test (ST). The latter test consisted of flume-pool swimming for 30 min, ergometer cycling for 75 min and treadmill running for 45 min as a continuous task. The exercise intensity was 60% of VO2 max during swimming, cycling and running, respectively. RESULTS: In slower triathletes, VO2, minute ventilation (VE), heart rate (HR) and temperature of external auditory canal were increased from an earlier stage compared with those in superior athletes. The percent increase (delta) of VO2, VE and HR between the 10th and last min of cycling and running stages in superior triathletes were significantly smaller than those in slower athletes. The oxygen cost (oxygen uptake/running velocity) of running stage was significantly lower in superior triathletes (0.220 +/- 0.020 ml.kg-1.m-1) compared with slower athletes (0.264 +/- 0.014 ml.kg-1.m-1). CONCLUSIONS: These results suggest that superior triathletes performed ST more economically than slower athletes and had excellent thermoregulatory adaptation.

Total hip arthroplasty: the relationship between posterolateral reconstruction, abductor muscle strength, and femoral offset.

PURPOSE: To evaluate the relationship between posterolateral reconstruction, abductor muscle strength, and femoral offset following total hip arthroplasty. METHODS: Of 28 patients (56 limbs) we assessed, 12 underwent posterolateral reconstruction (reconstruction group) and 16 did not (non-reconstruction group). Isometric abductor muscle strength was measured with a handheld dynamometer. Each patient's muscle strength was converted into a force to body weight ratio, and this ratio was used in the comparisons. RESULTS: The reconstruction group showed a higher value in abductor muscle strength than the non-reconstruction group (p<0.05). Significant correlation between abductor muscle strength and femoral offset was found in the reconstruction group (p=0.016; r=0.674). CONCLUSION: Posterolateral reconstruction and appropriate reconstruction of femoral offset following total hip arthroplasty are important to improve the abductor muscle strength.

[Decline of autopsy rates in Japan and the significance of autopsy in gastric carcinoma].

In recent years, the significance of autopsy seems to have gradually declined, because of advances in diagnostic technology. However, rediscovery of the value of autopsy has been recently emphasized by many foreign authors, and in some reports concerning diagnostic accuracy, no significant difference was observed between the three decades of the Sixties, Seventies and Eighties. Comparing the autopsy rates in 1977 and 1986 in Japan, one notes a decrease from 37.8% to 28.5%. And this declining tendency will continue for the foreseeable future. In order to confirm the value of autopsy, 100 sequential autopsy cases of gastric carcinoma in Kanto Teishin Hospital were reviewed. The autopsy rate of gastric carcinoma was 66.1% over the past decade. In 38 autopsy cases, some notable findings were provided by postmortem examination, and they may be summarized as follows. Clinically incorrect diagnosis for primary organ in 2 cases; unexpected spread of carcinoma in 2; improper cancer therapy in 8; favourable effects of anticancer drug in 4; underestimation of clinical observation in 11; latent tumors in 7; confirmation of other pathological findings than cancer in 4. The results suggest that autopsy plays an important part in the progress of medical care. In order to stop this declining trend, the department of pathology must be legally recognized as an essential unit in a hospital. Thus the autopsies should be performed not only with a view to research, but as one of the routine tasks in medical care. And cooperative efforts by both clinicians and pathologists are necessary to maintain the autopsy rates.