Prognostic significance of p62/SQSTM1 subcellular localization and LC3B in oral squamous cell carcinoma.

BACKGROUND: Autophagy is a programmed cell survival mechanism that has a key role in both physiologic and pathologic conditions. The relationship between autophagy and cancer is complex because autophagy can act as either a tumour suppressor or as a tumour promoter. The role of autophagy in oral squamous cell carcinoma (OSCC) is controversial. Several studies have claimed that either a high or low expression of autophagy-related proteins was associated with poor prognosis of OSCCs. The aims of the study were to compare autophagy in OSCCs, verrucous hyperplasias, and normal oral mucosas, and to inspect the prognostic role of autophagy in OSCCs. METHODS: We used the autophagosome marker, LC3B, and autophagy flux marker, p62/SQSTM1 (p62), by using immunohistochemistry, and examined p62 mRNA by RNA in situ hybridization, to evaluate autophagy in 195 OSCCs, 47 verrucous hyperplasias, and 37 normal oral mucosas. The prognostic roles of LC3B and p62 protein expressions in OSCCs were investigated. RESULTS: We discovered that the normal oral mucosa exhibited limited LC3B punctae and weak cytoplasmic p62 staining, whereas the OSCCs exhibited a marked increase in LC3B punctae and cytoplasmic p62 expression. The expression pattern of LC3B and cytoplasmic p62 of the verrucous hyperplasias were between normal oral mucosas and OSCCs. The normal oral mucosas, verrucous hyperplasias, and OSCCs presented no differences in nuclear p62 expression and the p62 mRNA level. p62 mRNA expression was elevated in a minority of cases. High p62 mRNA expression was associated with high p62 protein expression in the cytoplasm. Increased LC3B punctae, high cytoplasmic p62, and low nuclear p62 expressions in OSCCs were associated with aggressive clinicopathologic features and unfavourable prognosis. In addition, low nuclear p62 expression was an independent prognostic factor for overall and disease-specific survival rates. Furthermore, we disclosed that high cytoplasmic p62 expression accompanied with either a low or high LC3B expression, which indicated autophagy impairment under basal or activated autophagic activity, was associated with aggressive behaviour in advanced OSCCs. CONCLUSIONS: We suggested that autophagy was altered during cancer initiation and progression. Autophagy impairment contributed to cancer progression in advanced OSCCs.

Nuclear c-Myc plays an important role in the cytotoxicity of tumor necrosis factor alpha in tumor cells.

The phosphoprotein c-Myc has the potential to kill cells by apoptosis. To investigate whether c-Myc is involved in tumor necrosis factor alpha (TNF-alpha)-mediated cell killing, we have examined two HeLa cell lines (D98 and H21) which show dramatic differences in their susceptibilities to TNF-alpha cytotoxicity. Northern (RNA) blot analyses showed that there were no significant differences between these cell lines in basal or TNF-alpha-induced mRNA expression for a variety of proteins, including manganous superoxide dismutase, A20 zinc finger protein, plasminogen activator inhibitor type 2, and hsp70, all of which are known to influence the susceptibility of certain cells to TNF-alpha killing. On the other hand, there was a dramatic increase in c-Myc mRNA expression in TNF-alpha-sensitive D98 cells, but not in TNF-alpha-resistant H21 cells, which was only observed when the cells were treated with cycloheximide. Western blot (immunoblot) analyses revealed that even in the absence of TNF-alpha or cycloheximide, c-Myc was detectable only in nuclear extracts of TNF-alpha-sensitive D98 cells, implying a role for preexisting c-Myc in TNF-alpha killing. In support of this interpretation, a c-myc antisense oligonucleotide specifically inhibited the TNF-alpha killing of D98 cells, provided that the oligonucleotide was added 6 h prior to TNF-alpha treatment. Either dexamethasone treatment or transient expression of c-myc antisense cDNA fragments decreased nuclear c-Myc in D98 cells and rendered the cells more resistant to TNF-alpha cytotoxicity. Nuclear c-Myc was also detectable in a TNF-alpha-sensitive human HT-1080 fibrosarcoma cell line, but it was undetectable in a derivative of HT-1080 (SS-HT-1080) known to be resistant to TNF-alpha killing because of overexpression of plasminogen activator inhibitor type 2. HT-1080 cells transfected with antisense c-myc cDNA had significantly less nuclear c-Myc and were resistant to TNF-alpha cytotoxicity. Together, these data indicate that a nuclear transcription factor, c-Myc, plays an important role in sensitizing two different tumor cell types to TNF-alpha cytotoxicity.

Cyclin D3 sensitizes tumor cells to tumor necrosis factor-induced, c-Myc-dependent apoptosis.

c-Myc is an important mediator of apoptosis in cytokine- or serum-deprived cells and sensitizes various cell types to tumor necrosis factor alpha (TNF) cytotoxicity. However, downstream mediators of c-Myc-dependent apoptosis are largely unknown. In this study, we investigated whether one or more cyclins which, like c-Myc, are important regulators of the cell cycle are involved in TNF-induced apoptosis downstream of c-Myc. Cyclin D3 and c-Myc levels in HeLa and fibrosarcoma cells correlated with sensitivity of these cells to TNF-induced apoptosis, as both proteins were highly expressed in TNF-sensitive HeLa D98 cells and HT-1080 fibrosarcoma cells but not in their TNF-resistant counterparts, HeLa H21 and SS-HT-1080 cells, respectively. All other cyclins tested were equally expressed in all tumor cell lines. Reduction in the expression of c-Myc by dexamethasone or inhibition of the transcriptional activity of c-Myc by introduction of a dominant negative form of c-Myc into TNF-sensitive HeLa D98 cells strongly suppressed the expression of cyclin D3 (but none of the other cyclins) and rendered the cells resistant to TNF-induced apoptosis. Conversely, introduction of the c-myc gene into TNF-resistant, c-Myc- and cyclin D3-deficient HeLa H21 cells resulted in enhanced cyclin D3 expression and TNF killing. When cyclin D3 expression in HeLa cells was altered by sense or antisense cyclin D3 cDNA, there was a concomitant alteration in their susceptibility to TNF-induced apoptosis without any change in c-Myc levels. Overall, our results show that cyclin D3 sensitizes tumor cells to TNF-induced apoptosis and indicate that the expression of c-Myc and expression of cyclin D3 in HeLa and in HT-1080 fibrosarcoma cells are closely linked.

Analysis of the involvement of cells from donor and recipient mice in adoptive transfer of antitumor immunity.

In this study, we attempted to analyze the effector cells for adoptive transfer of protective immunity directed against a P815Ys tumor. The spleen, lymph node, and peritoneal exudate cells obtained from immune mice at Day 7 to Day 10 after last challenge were tested for their in vitro cell-mediated cytotoxicity against P815Ys cells, using a 4-hr 51Cr release assay. The immune spleen lymphocytes (ISL) showed no cytotoxicity, whereas the peritoneal exudate cells exhibited marked cytotoxicity. Unexpectedly, when ISL or peritoneal exudate cells were adoptively transferred i.v. into mice bearing the P815Ys tumor, it was the ISL but not the peritoneal exudate cells that provided the hosts with significant protection. Using alloantibodies for negative depletion of cells in ISL, it was found that, after treatments with anti-Thy 1.2 or anti-Lyt 1 antiserum plus complement but not with anti-Lyt 2 or complement alone, the protective capacity of ISL can be abolished, indicating that the effector cells for conferring protective immunity to the host are Lyt 1-bearing T-cells. Moreover, culture supernatants of ISL with or without mitomycin C-treated P815Ys contain helper factor, interferon, and interleukin 2, which enhanced the in vitro generation of cell-mediated cytotoxicity against P815Ys. Taken together, these results strongly suggest that the donor helper T-cells are the effector cells responsible for adoptive transfer of protective immunity. We next examined the contribution of host cells. Syngeneic mice were made to become either T-cell (with thymectomy and irradiation)- or macrophage (with the administration of silica) depleted and were then subjected to adoptive transfer experiments. Both the thymectomized and the silica-treated mice, after receiving the ISL, showed significantly better survival times than did normal mice. Thus, the data suggest that the elimination of T-cells or inactivation of macrophages, presumably with immunosuppressive activity in the recipients, will allow further improvement of their battle for survival against tumor.

Bleomycin disposition in children with cancer.

Bleomycin kinetics were determined in 14 children after intravenous bolus and prolonged infusion doses. Plasma and urine bleomycin concentrations were determined by radioimmunoassay. After intravenous bolus, bleomycin concentrations were adequately described by a two-compartment open model with a mean t1/2 alpha and t1/2 beta of 0.3 +/- 0.1 and 3.2 +/- 0.7 hr (mean +/- SEM). Volume of the central compartment and volume of distribution at steady-state (Vss) were 4.3 +/- 0.5 and 9.9 +/- 1.1 l/m2. Total plasma (CLT) and renal (CLR) clearance were 51.8 +/- 6.1 and 33.5 +/- 2.4 ml/min/m2. Three intravenous bolus courses were given to two patients who received more than four courses of cisplatin (greater than 300 mg/m2); CLT and CLR for these courses were 18.0 +/- 3.3 and 8.2 ml/min/m2. Conversely, children under 3 yr old eliminated bleomycin more rapidly than older children. Decline in bleomycin concentrations after seven 24- or 48-hr intravenous infusions was described by a one-compartment model. Mean values for plasma t1/2, Vss, CLT, and CLR were 2.1 +/- 0.1 hr, 11.0 +/- 2.6 l/m2, 57.1 +/- 13.5 ml/min/m2, and 33.2 +/- 6.4 ml/min/m2. One patient received his bleomycin infusion when ureteral obstruction was present; CLT and CLR for this course were 4.8 and 4.1 ml/min/m2. These data indicate that young children eliminate bleomycin more rapidly than older children and that children with impaired renal function may have prolonged elevations in plasma concentration due to reduced bleomycin clearance. Bleomycin disposition in older children is as in adults.

Does elevated cardiac troponin I in patients with unstable angina predict ischemia on stress testing?

To help guide physicians in their evaluation of patients with acute coronary syndromes, we investigated whether elevated cardiac troponin I in patients presenting with unstable angina predicts ischemia on stress testing. Elevated cardiac troponin I in patients who present with chest pain and normal creatine kinase levels is associated with ischemia on stress testing, as well as with future cardiac events.

Perfluorooctyl bromide concentration in plasma and tissues of beagle dogs.

Plasma levels were determined frequently after single doses of perfluorooctyl bromide were administered to beagle dogs at doses of either 30.8 g/kg po or 3.9 g/kg intratracheally. The apparent first-order half-life during the terminal elimination phase was about 1 day after oral medication and about 7 days after intratracheal administration. Analysis of tissues revealed the highest concentrations of the compound in abdominal fat of dogs autopsied 4 weeks later.

Determination of dicumarol metabolites in bile of rats.

After intravenous administration of dicumarol-14C to rats, the bile excreted over the next 24 hr contained from 32 to 46% of the administered radioactivity. At least three primary metabolites and a small amount of unchanged dicumarol were present in the bile. Over 91% of the primary metabolites was converted to dicumarol and 7-hydroxydicumarol by hydrolysis with beta-glucuronidase. Some primary metabolites were hydrolyzed simply by acidification to pH 3 or by treatment under the acidic conditions utilized in the enzymatic hydrolysis. The three primary metabolites contain carboxylic acid groups, as indicated by their electrophoretic mobility-pH profiles, and some are simple glucuronides of dicumarol and 7-hydroxydicumarol. The possibility that others are derivatives of these compounds in which a coumarin lactone ring is opened cannot be ruled out. When the metabolites released by either acidification or enzymatic hydrolysis were chromatographed in n-butanol-3 M ammonia, artifacts were produced, presumably as a result of decomposition of 7-hydroxydicumarol. The question is raised whether a previously reported metabolite (B055) is an artifact.

Soy fiber delays disease onset and prolongs survival in experimental Clostridium difficile ileocecitis.

Clostridium difficile colitis is a disabling complication in critically ill patients who commonly receive broad-spectrum antibiotics and liquid diets. To date, there is no experimental model specifically designed to investigate the effects of liquid diets on this type of colitis. The addition of fiber to liquid diets normalizes gut structure and improves absorptive function in selected conditions of intestinal dysfunction. The purposes of this study were the following: (1) to develop a reproducible model to examine the interaction of acute C difficile-induced colitis and liquid diets, (2) to determine whether the addition of soy fiber to a liquid diet improves disease, and (3) to investigate possible mechanisms of fiber-mediated disease improvement. Syrian hamsters were pair-fed with either a polymeric liquid diet or the same diet with 1.4% soy fiber for 10 days. Animals were given either clindamycin and C difficile (to produce ileocecitis), or equivalent volumes of saline. Mean survival time and systematic stool examinations for C difficile toxin positivity, liquidity, and percent water were performed to determine the effect of soy fiber on disease. Survival time was prolonged by 34% (p < .05), and C difficile toxin positivity and stool liquidity were significantly reduced (p < .05) with fiber. Additional animals were studied to determine possible mechanisms for improved survival in fiber-supplemented animals. Cecal histology, colonic water absorption, cecal microflora, and gastric to anus transit time were measured in these animals. Colonic water absorption and gastric to anus transit time were significantly increased (p < .05) and decreased (p < .05) with fiber, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Management of gastroschisis in a peripheral hospital setting.

Ten patients (5 males and 5 females) with gastroschisis were treated in Alor Setar Hospital from January 1989 to December 1993. Two patients had associated congenital anomalies. Primary closure was possible in 9 patient while the other patient had stage closure. All patients received prophylactic antibiotics, 9 patients were ventilated electively in the post-operative period and 7 patients received parenteral nutrition. There were 9 survivors. Complications especially wound infection and breakdown were seen in 7 patients. The average hospital stay was 36 days.

Repeat curettage after evacuation of hydatidiform mole. An appraisal.

In the management of patients with molar pregnancy, a repeat uterine curettage is generally advocated after evacuation of the hydatidiform mole. To assess the usefulness of a repeat curettage, we reviewed our experience with this procedure over an 8-year period. We found that it was unnecessary in 90% of the cases and did not predict or influence the outcome in all but one case of invasive mole. We feel that the procedure is not cost-effective and should be reserved for patients with specific indications such as incomplete evacuation and abnormal uterine bleeding.

Channelling of patients taking NSAIDs or cyclooxygenase-2-specific inhibitors and its effect on interpretation of outcomes.

When new drugs with improved safety or efficacy are introduced, they may be preferentially prescribed to specific populations of patients. Safety and efficacy may be underestimated if such channelling effects are not recognized. Meloxicam and cyclooxygenase (COX)-2-specific inhibitors were developed as safer alternatives to non-steroidal anti-inflammatory drugs (NSAIDs) for the treatment of osteoarthritis and rheumatoid arthritis. Studies of the use of meloxicam and COX-2-specific inhibitors demonstrate that both of these drugs are being prescribed to patients at increased risk of gastrointestinal adverse drug events. In the case of COX-2-specific inhibitors, this channelling appears to represent a prescribing pattern consistent with current recommendations. Subsequent analysis of the data, after adjusting for channelling bias, showed that the risk of gastrointestinal toxicity for meloxicam was similar to that for other NSAIDs, while COX-2-specific inhibitors reduced the risk of developing gastrointestinal adverse drug events by approximately 60%. These studies serve as examples of observed channelling bias and highlight the need for adjusting for channelling in order to provide a valid assessment of relevant outcomes for drugs likely to be preferentially prescribed to specific populations.

Comparative tissue distribution of ceforanide, cefazolin, and cefamandole in rats.

The comparative tissue distribution of ceforanide, cefazolin, and cefamandole was determined in rats after subcutaneous doses of 100 mg/kg. Ceforanide had the longest plasma half-life, 0.9 h, versus 0.5 h for cefazolin and 0.4 h for cefamandole, and the highest area under the plasma concentration time curve, 324 micrograms x h per ml, versus 184 micrograms x h per ml for cefazolin and 42 micrograms x h per ml for cefamandole. The peak plasma concentrations of ceforanide and cefazolin were 173 and 140 micrograms/ml, respectively, and were threefold higher than that of cefamandole (49 micrograms/ml). Measureable concentrations of the three compounds were found in the liver, kidneys, lungs, submaxillary glands, cervical lymph nodes, bones, heart, abdominal muscles, eyes, and testes, with cefamandole levels being generally lower and more variable. The peak tissue levels of ceforanide and cefazolin were comparable, within the limit of data variation, and were considerably higher than that of cefamandole. The tissue half-lives of these cephalosporins were similar to the respective plasma half-lives.

The pharmacokinetics of intravenous, intramuscular, and subcutaneous nalbuphine in healthy subjects.

The pharmacokinetics of intravenously, intramuscularly, and subcutaneously administered nalbuphine were studied in three parallel groups of 12 healthy volunteers each. The subjects received single doses of 10 mg and 20 mg of nalbuphine separated by a one week washout period. Blood specimens were obtained up to 15 h after dosing for determination of nalbuphine. Mean plasma nalbuphine concentrations 5 min after intravenous administration of 10 or 20 mg were 39 and 73 ng/ml, respectively. The mean maximum plasma concentrations (Cmax) after intramuscular or subcutaneous administration of nalbuphine 10 mg were 29 and 31 ng/ml, respectively. Mean Cmax values after 20 mg doses were 60 and 56 ng/ml. Mean Cmax occurred 30 to 40 min after nalbuphine administration. The mean elimination half-lives of parenterally administered nalbuphine ranged between 2.2 and 2.6 h, regardless of dose given or route administered. The mean absolute bioavailability was 81% and 83% for the 10 and 20 mg intramuscular doses, respectively, and 79% and 76% following 10 and 20 mg of subcutaneous nalbuphine. The mean volumes of distribution (Vss) of the intravenously administered drug were 290 and 274 l and the mean systemic clearances were 1.6 and 1.5 l/min following administration of 10 and 20 mg doses, respectively. Intramuscular and subcutaneous nalbuphine appear to be interchangeable based on the similarities in Cmax, mean times until maximum concentration, mean AUC data, and absolute bioavailabilities.

Clinical pharmacology of intraperitoneal cisplatin.

The clinical pharmacology of cisplatin was determined in six patients with malignant ascites secondary to ovarian cancer, and in one patient with peritoneal mesothelioma, following intraperitoneal administration of cisplatin (25-60 mg/m2). The drug was administered in 1 liter of normal saline as a 15- to 30-min infusion. Total, and in some patients free (ultrafilterable), platinum concentrations were determined in plasma, urine, and ascitic fluid by flameless atomic absorption spectrometry. The peak total platinum concentrations in ascitic fluid at the end of infusion were related to dose, a 50 mg/m2 dose producing a 20 to 80 micrograms cisplatin/ml concentration. Filterable platinum represented between 3 and 59% of total platinum in the peritoneum at 4 to 6.5 hr following its administration. Plasma platinum concentrations ranged between 0.2 to 1.6 micrograms/ml 4 hr following administration, and reached a plateau for the next 24 to 48 hr largely in the form of protein-bound platinum. The urinary excretion of cisplatin was consistent with variation in absorption from the peritoneum. Minimal gastrointestinal, bone marrow, and renal toxicities during therapy suggest that sustained free platinum concentrations in ascites may be obtained without significant toxicity and support the intraperitoneal route of administration as an effective strategy for cisplatin therapy of intra-abdominal malignancies.

High-performance liquid chromatographic determination of plasma and urinary 1-ethyl-1,4-dihydro-4-oxo-1,8-naphthyridine-3,7-dicarboxylic acid.

A high-performance liquid chromatographic method for the analysis of 1-ethyl-1,4-dihydro-4-oxo-1,8-naphthyridine-3,7-dicarboxylic acid (I) in plasma and urine is described. A statistical evaluation of the assay technique has shown acceptable accuracy and precision at concentrations as high as 2.0 microgram/ml of plasma or 29.0 microgram/ml of urine for samples augmented with 1. As little as 0.08 microgram/ml of I in plasma or 0.42 microgram/ml of I in urine were quantitatively determined. The mean relative error for the assay of unknown concentrations of I in plasma and urine was +/- 8% and +/- 3%, respectively. This method was used for the analysis of I in the plasma and urine of rhesus monkeys following oral administration of 200 mg/kg of nalidixic acid.

Preliminary studies of the pharmacokinetics of talisomycin in the rhesus monkey.

The single dose intravenous pharmacokinetics of talisomycin (3 mg/M2) and bleomycin (18 U/M2) were determined in the rhesus monkey at non-nephrotoxic doses. Serum concentrations were analyzed by radioimmunoassay procedures. The tissue distribution of talisomycin was significantly higher and the elimination slower than bleomycin. The volume of distribution (Vdss) was 2.3 and 22.6 L/M2 for bleomycin and talisomycin, respectively. The volume of the peripheral tissue compartment (V2) of talisomycin was 17 times greater than bleomycin. The slower elimination of talisomycin was reflected by a half-life (t1/2) of 10.6 hr versus 1.6 hr for bleomycin. The slower elimination from the peripheral tissue compartments was also evidenced by a five-fold difference in the tissue transfer ratio (k12/k21) for these compounds. Similar differences, of lesser magnitude, have also been reported in the dog. The potential for higher tissue distribution and slower elimination of talisomycin could be related to the differences in in vivo antitumor activity and toxicity, and should be considered in design of the dose schedule in clinical studies.

Phase II evaluation of mitomycin C (MMC) in children with refractory solid tumors using the single high-intermittent-dose schedule.

Nine evaluable patients with refractory solid tumors received mitomycin C as a single intravenous injection at a dose of 20 mg/m2 at 6- to 8-week intervals. Toxicity was tolerable. One patient with Wilms' tumor had a transient decrease in liver size. Pharmacokinetic data on three patients have suggested that the disposition of mitomycin C were comparable to those obtained in adults.