Expression of apoptosis-related genes in human head and neck squamous cell carcinomas undergoing p53-mediated programmed cell death.

Human head and neck squamous cell carcinoma (HNSCC) lines infected with a replication-defective Ad5CMV-p53 vector bearing a wild-type human p53 gene were used to examine alterations in the production of proteins implicated in regulating apoptosis. Because HNSCC lines express abundant levels of c-myc, and simultaneous expression of c-myc and p53 is known to trigger apoptosis in other cells, cooperation between these two genes was examined. Surprisingly, levels of c-myc mRNA and protein were rapidly and profoundly suppressed after infection with wild-type p53. Suppression of c-myc using antisense oligodeoxynucleotides (in the absence of p53) was sufficient to trigger apoptosis in Tu-138 cells, raising the possibility that the reduction of c-myc may be involved in at least one of the cell death pathways mediated by p53. Expression of a panel of Bcl-2 homology proteins was also examined in HNSCC lines undergoing p53-mediated apoptosis. No changes in Bcl-2, Bak, or Bcl-xS were found after p53 expression. Increased levels of the apoptosis-accelerating protein Bax were found in HNSCC lines after infection with Ad5CMV-p53. Induction of the apoptosis-inhibiting protein Bcl-xL was observed in Tu-167 cells and may account for the delayed onset of apoptosis in these cells. These studies suggest that multiple pathways may regulate apoptosis after transient overexpression of p53.

UV-induced photoproducts of 5-methylcytosine in a DNA sequence context.

In order to detect possible m5C photoproducts, highly purified rat liver DNA-cytosine methyltransferase was used to specifically generate m5C with a radioactive methyl group. When these DNAs were subjected to a large dose (10 kJ/m2) of 254 nm or 302 nm ultraviolet light (UVB) to enhance the yield, two labeled photoproducts were detected and isolated by reverse phase HPLC after formic acid hydrolysis. Further studies using acetone as a triplet state sensitizer and UVB irradiation suggested that photoproduct II was activated via a triplet state while the more polar photoproduct I was not. Photoreversion of the purified photoproducts with 10 kJ/m2 254 nm light demonstrated the following reactions: Photoproduct I regenerated m5C, while photoproduct II is split and regenerated m5C and photoproduct I. These results suggest that photoproduct I is monomeric while photoproduct II dimeric, and from the latter's elution position possibly a cyclobutyl type dimer arising from a reaction with an adjacent cytosine. Using d[TTG] and d[Cm5CG] as models of typical sequences, irradiation with 10 kJ/m2 254 nm or 302 nm, respectively, gave rise to a small component having altered mobility in sequencing gels. The altered mobility trinucleotides were resistant to degradation by PI and micrococcal nucleases as expected from photodimerization of the pyrimidine bases. Furthermore, oligonucleotide substrates containing m5C were synthesized and shown to be susceptible to T4 endonuclease v action at locations consistent with d[Cm5C] photodimer formation when irradiated in the UVB range.

Particle size-dependent leakage and losses of aerosols in respirators.

Measuring particle size-dependent leakage into and losses inside a respirator reveals the deposition mechanisms occurring at the leak site and the flow dynamics inside the respirator. This study investigated particle size-dependent leakage and deposition within the mask by examining the leakage into the mask for different hole locations, probe locations, hole shapes, hole lengths and hole sizes. The shape of the leak has an effect on particle size-dependent leakage. Probe and leak location tests indicated that not only does the total measured leakage change but also the size-dependence of the leakage changes depending on the leak and probe locations. When the leak site is in the chin area, the clean air entering through the filters at the chin helps to carry the inward leakage into the breathing zone. Particle size-dependent leakage does occur and is due to both inertial entry losses at the leak site and within the mask, and diffusional losses within the mask and leak site. Particle size-dependent curves change shape as the hole size changes with relatively more larger particles entering through the small hole size.

The effect of aerosol size distribution and measurement method on respirator fit.

The particle size-dependent leakage into a respirator was examined by measuring the leakage of particle sizes between 0.07 to 4.4 microns through three hole sizes in a negative-pressure half-mask respirator worn by a human subject. This investigation showed that the size distribution of an aerosol test agent and the measurement method have an effect on the leakage measured in a quantitative fit test. For instance, the ratio of percent leakage measured by light scattering between test aerosols with count median diameters of 2.2 and 0.28 microns can be as large at 5:1. Likewise, the ratio of the percent leakage measured by a particle count method vs. a mass method of detection of the same polydisperse aerosol with a count median diameter equal to 2.2 microns can be as high as 4:1. The mass leakage into a mask with a leak is also greater for an exposure aerosol with a count median diameter between 0.15 to 0.30 micron compared to exposure aerosols with larger count median diameters for aerosols with the same mass concentration.

Perturbation of maintenance and de novo DNA methylation in vitro by UVB (280-340 nm)-induced pyrimidine photodimers.

The effect of pyrimidine photodimers on transmethylation reactions catalyzed by a highly purified rat liver DNA (cytosine-5-)-methyltransferase (EC 2.1.1.37) that exhibits maintenance and de novo methylation activities was studied in vitro, using the viral substrates M13 mp9 replicative form (RF) DNA and the hemimethylated analog formed from primed synthesis of phage DNA in the presence of 2'-deoxy-5-methylcytidine 5'-triphosphate. These DNAs were irradiated with UVB (280-340 nm) at 900-3600 J/m2 in the presence of the triplet-state sensitizers acetone or 3-dimethylaminopropiophenone. Under these conditions of irradiation, which approximate solar UV, pyrimidine cyclobutane photodimers were introduced without producing any evidence of single-strand breaks or alkali-sensitive sites [i.e., no (6-4)pyrimidine-pyrimidone photoproducts]. This was confirmed by gel analysis, a T4 UV endonuclease nicking assay specific for cyclobutane-type dimers, and HPLC analysis of the photoproducts. The methylation of irradiated templates by DNA methyltransferase was inhibited in an approximately linear fashion as a function of increasing UVB dose. This inhibition was correlated with the number of lethal photoproducts detected by the simultaneous measurement of the surviving fraction of infectious phage DNA. For approximately the same number of pyrimidine cyclobutane photoproducts introduced, de novo methylation activity was approximately 2-fold more sensitive than the maintenance mode of methylation. The ability of these putatively carcinogenic, pyrimidine photoproducts to inhibit DNA methylation suggests a common mechanism of action with several chemical carcinogens that are known to modify bases.

Acid secretion by guinea-pig isolated stomach.

An isolated stomach preparation from the guinea-pig is described. 2. Both histamine acid phosphate (1-4 mug/ml.) and theophylline hydrate (0-2-3-2 mg/ml.) separately stimulated hydrochloric acid, HCl, secretion from the guinea-pig stomach preparation. A linear dose-response relationship was obtained for theophylline. 3. Addition of theophylline (0-2 and 1-6 mg/ml.) during maximal response to histamine increased the secretion further, whereas addition of histamine during maximal response to theophylline did not cause further secretion. 4. The secretory activities of Nalpha-MeH (0-3-5-0 muM), Nalpha-Me2H (1-2-9-5 muM) and 5-MeH (1-5-12 muM) were compared with histamine (0-9-13 muM) on a threshold background secretion induced by theophylline (0-2 mg/ml.). Linear log.-dose response relationships were obtained for each test drug. The results confirm that Nalpha-MeH is a more potent secretagogue than histamine. 5. Pentagastrin (0-3-1-0 mug/ml.) stimulated HCl secretion in approximately half the experiments. The response was often transitory. In the other experiments, pentagastrin had no effect on HCl secretion although subsequent administration of histamine did stimulate HCl secretion.

Can the actions of adrenoceptor agonists and antagonists on pentagastrin-induced gastric secretion be due to their effects of histamine formation?

1 The effects of some adrenoceptor agonists and antagonists which have been reported to affect histamine formation in leucocytes (Assem & Feigenbaum, 1972) have been investigated on gastric secretion in conscious dogs with Heidenhain pouches.2 Submaximal secretion in response to pentagastrin was enhanced by propranolol (0.1-1.0 mg/kg i.v.) and phenylephrine (1.0 mug kg(-1) min(-1) i.v. for 20 min), which increase histamine formation, and was decreased by phentolamine (2 mg/kg i.v.) and isoprenaline (0.05-0.2 mug kg(-1) min(-1) i.v. for 30 min), which decrease histamine formation. Practolol (2 mg/kg i.v.), which has no effect on histamine formation, had no effect on secretion.3 Acid secretion in response to histamine was either unaffected or affected in the opposite direction by these drugs.4 The effects of the drugs on pentagastrin-induced secretion were not secondary to changes in mucosal blood flow (radioactive aniline clearance).5 The results are consistent with the hypothesis that acid secretion in response to pentagastrin involves the formation of endogenous histamine.

The measurement of dog gastric mucosal blood flow by radioactive aniline clearance compared with amidopyrine clearance.

1. Methods for the estimation of radioactive aniline in body fluids are described. The recovery of aniline added to blood, plasma and gastric juice was over 90% of the recovery from saline.2. In the doses used aniline caused methaemoglobinaemia of 5-11% of total haemoglobin. No other effect was detected. Gastric secretion was also unaffected.3. Aniline clearance increased in parallel with acid secretion from Heidenhain pouches in conscious dogs and in anaesthetized dogs. In conscious dogs the ratio of aniline clearance to acid secretion was significantly higher for histamine stimulation than for pentagastrin stimulation.4. Aniline and amidopyrine clearances were compared simultaneously in the same dogs. Aniline clearance was about 80% of amidopyrine clearance.5. The proportion of aniline bound to plasma proteins was measured by two methods and found to be 25%. When aniline clearance was corrected for plasma binding, aniline and amidopyrine clearances were equal.

The effect of betahistine on gastric acid secretion and mucosal blood flow in conscious dogs.

In 3 conscious dogs, betahistine (2-(2'-methyl aminoethyl pyridine)) (80 or 160mug kg(-1) min(-1)) increased acid secretion from Heidenhain pouches to 8.8% and 17.6% respectively of the maximal response to histamine. Betahistine also increased mucosal blood flow (radioactive aniline clearance). The ratio of mucosal blood flow to secretion was greater for betahistine than for histamine but the difference between betahistine and histamine was significant in only one of the dogs.

The effects of isoprenaline and noradrenaline on pentagastrin-stimulated gastric acid secretion and mucosal blood flow in the dog.

1. Isoprenaline (0.02 to 2.0 mug kg(-1) min(-1)) inhibited gastric secretion in response to pentagastrin in both conscious and anaesthetized dogs and in response to feeding in conscious dogs.2. The inhibition was unaffected during cardiac beta-adrenoceptor blockade by propranolol.3. The inhibition was not due to decreased mucosal blood flow.4. This effect of isoprenaline is different from its effect on histamine-induced gastric secretion.5. Noradrenaline (0.05-2.0 mug kg(-1) min(-1)) also decreased gastric secretion but it was less effective than isoprenaline.6. The mechanism of action of noradrenaline is probably a decrease in mucosal blood flow.