A bile acid-induced apoptosis assay for colon cancer risk and associated quality control studies.
(73/8454)
Bile acids are important in the etiology of colorectal cancer. Bile acids induce apoptosis in colonic goblet cells at concentrations comparable to those found in fecal water after high-fat meals. Preliminary evidence indicated that cells of the normal-appearing (nontumorous) portion of the colon epithelium of colon cancer patients are more resistant to bile salt-induced apoptosis than are cells from normal individuals. In the present study, 68 patients were examined, and biopsies were taken at 20 cm from the anal verge, cecum, and descending colon. The patients included 17 individuals with a history of colorectal cancer, 37 individuals with adenomas, and 14 individuals who were neoplasia free. The mean bile salt-induced apoptotic index among normal individuals was 57.6 +/- 3.47 (SE), which differed significantly (P < 0.05) from the mean value of 36.41 +/- 3.12 in individuals with a history of colon cancer. The correlation between independent observers was 0.89 (P < 0.001), indicating good interobserver reliability. Components of variance comparing interindividual versus intraindividual sources of variation suggested that site-to-site variability, both between regions of the colon and for adjacent biopsies, was larger than the interpatient variability for individuals with a history of neoplasia. Therefore, there was "patchiness" of the susceptibility of regions of the colon to bile acid-induced apoptosis in individuals with a history of neoplasia (a patchy field effect). There was no obvious correlation of low-apoptotic index regions with regions in which previous neoplasias had been found and removed. On the other hand, for normal, i.e., neoplasia-free, individuals, there was relatively less intraindividual variation compared to interindividual variation. Our assay shows an association between resistance to bile acid-induced apoptosis, measured at 20 cm from the anal verge, and colon cancer risk. Thus, this assay may prove useful as a biomarker of colon cancer risk. (+info)
Expression and up-regulation of retinoic acid receptor-beta is associated with retinoid sensitivity and colony formation in esophageal cancer cell lines.
(74/8454)
Retinoids exhibit chemotherapeutic and chemopreventive activities, possibly due to their ability to modulate cell growth, differentiation, and apoptosis. These effects are thought to be mediated by nuclear retinoic acid (RA) receptors (RARs) and retinoid X receptors, each of which includes three subtypes (alpha, beta, and gamma) that act as transcription factors. To determine whether RARs play a role in mediating the effects of RA on human esophageal cancer (HEC) cells, we analyzed the effects of RA on: (a) the growth, differentiation, and apoptosis in seven HEC cell lines; (b) receptor expression; (c) receptor modulation by RA; and (d) expression of receptors in 20 surgical HEC specimens. RA inhibited the growth of five of seven cell lines and also the constitutive expression of the squamous differentiation markers cytokeratin 1 and transglutaminase I in all cell lines. The growth inhibition by RA was due to the induction of apoptosis in the five cell lines. All seven cell lines expressed RAR-alpha and RAR-gamma, and four cell lines showed some changes by RA, but not associated with apoptosis. In contrast, RAR-beta was expressed in five of seven cell lines and up-regulated by RA in these five cell lines, which were associated with apoptosis. Two cell lines that failed to express RAR-beta showed no growth inhibition or apoptosis and no RAR-beta inducibility. Interestingly, only these two cell lines were able to form colonies in soft agar. RAR-alpha, RAR-beta, and RAR-gamma mRNAs were expressed in all 20 adjacent normal esophageal tissues. The expression of RAR-alpha and RAR-gamma remains positive in HEC specimens, but RAR-beta expression was detected in only 6 of 20 HEC specimens. These data suggest that the expression of RAR-beta is associated with response of HEC cells to RA and that the loss of RAR-beta expression may be associated with HEC development. (+info)
In vivo responses to antimalarials by Plasmodium falciparum and Plasmodium vivax from isolated Gag Island off northwest Irian Jaya, Indonesia.
(75/8454)
There is renewed interest in the rich nickel and cobalt deposits of Pulau Gag, an isolated but malarious island off the northwest coast of Irian Jaya. In preparation for an expanded workforce, an environmental assessment of malaria risk was made, focusing upon malaria prevalence in the small indigenous population, and the in vivo sensitivity of Plasmodium falciparum and P. vivax to chloroquine (CQ) and sulfadoxine/pyrimethamine (S/P), the respective first- and second-line drugs for uncomplicated malaria in Indonesia. During April-June 1997, mildly symptomatic or asymptomatic malaria infections were found in 24% of 456 native residents. Infections by P. falciparum accounted for 60% of the cases. Respective day 28 cure rates for CQ (10 mg base/kg on days 0 and 1; 5 mg/kg on day 2) in children and adults were 14% and 55% (P < 0.005). Type RII and RIII resistance characterized only 5% of the CQ failures. Re-treatment of 36 P. falciparum CQ treatment failures with S/P (25 mg/kg and 1.25 mg/kg, respectively) demonstrated rapid clearance and complete sensitivity during the 28-day follow-up period. More than 97% of the P. vivax malaria cases treated with CQ cleared parasitemia within 48 hr. Three cases of P. vivax malaria recurred between days 21 and 28, but against low drug levels in the blood. The low frequency of RII and RIII P. falciparum resistance to CQ, the complete sensitivity of this species to S/P, and the absence of CQ resistance by P. vivax are in contrast to in vivo and in vitro test results from sites on mainland Irian Jaya. (+info)
Synechococcus mutants resistant to an enamine mechanism inhibitor of glutamate-1-semialdehyde aminotransferase.
(76/8454)
An enamine mechanism-based inactivator of mammalian delta-aminobutyric acid aminotransferase, 4-amino 5-fluoropentanoic acid is a potent inhibitor of cell growth and pigment formation in the cyanobacterium Synechococcus PCC 6301. It was demonstrated that 4-amino 5-fluoropentanoic acid inhibits the aminolaevulinate synthesis at glutamate 1-semialdehyde aminotransferase and that in the mutant obtained by exposing cells to 40 microM 4-amino 5-fluoropentanoic acid, this enzyme was insensitive to the inhibitor. The specific activity of glutamate 1-semialdehyde aminotransferase in cell extracts was lower in the mutant, although the cell growth rate was unaffected. The decrease in sensitivity to 4-amino 5-fluoropentanoic acid in the mutant is due to a structural gene mutation, a single base change in the hemL gene resulting in a S162T substitution in the gene product. (+info)
Cellular balance of glutathione levels through the expression of gamma-glutamylcysteine synthetase and glutathione thiol transferase genes in human hepatic cells resistant to a glutathione poison.
(77/8454)
Buthionine sulfoximine (BSO) is a synthetic amino acid that irreversibly inhibits glutathione biosynthesis and deranges reduced glutathione (GSH) metabolism in liver cells. We isolated two BSO-resistant lines, HLE/BSO2-1 and HLE/BSO2-2, from human hepatic HLE/WT cells. Cellular levels of the Pi class glutathione thiol transferase (GSTP1) were 3-fold lower in BSO-resistant lines than in HLE/WT cells. By contrast, gamma-glutamylcysteine synthetase (GCS) heavy subunit (GCSh) mRNA levels were markedly decreased in HLE/BSO2-1 and HLE/BSO2-2 as compared with HLE/WT. The expression of a dominant-negative mutant of c-Jun inhibited the GCSh promoter activity in HLE/WT, but not in HLE/BSO2-1. Cellular levels of AP-1, however, were not decreased in either BSO-resistant cell line. Transfection of GCSh promoter of various lengths driven reporter constructs showed no sequence-specific increase in the promoter activities in HLE/BSO2-1. However, transfection of GSTP1 cDNA into HLE/BSO2-1 and HLE/BSO2-2 restored the levels of GCSh mRNA and the GCSh promoter activity to those of HLE/WT. Sequences between -315 and -241 bp of the 5' region contained an AP-1 site responsible for the enhanced GCSh promoter activity in GSTP1 transfectants of HLE/BSO2-1. In vivo footprint analysis showed a specific protection of the AP-1 site on GCSh promoter in GSTP1 transfected HLE/BSO2-1. GSH homeostasis thus appears to be maintained by an interaction between GSTP1 and GCS in human hepatic cells resistant to the GSH poison. (+info)
Induced p21waf expression in H1299 cell line promotes cell senescence and protects against cytotoxic effect of radiation and doxorubicin.
(78/8454)
The CDK inhibitor p21waf is a principal mediator of p53 function but can also be transactivated by many p53-independent stimuli leading to cell growth arrest or differentiation. In order to study the function of p21waf in a p53-deficient environment, we established an inducible expression of p21waf in the p53-null lung cancer cell line H1299, based on the muristerone-regulated system. Overexpression of p21waf led cells to growth arrest which after several days became irreversible and the arrested cells acquired a senescent phenotype as judged by cell shape, the senescence-associated beta-gal marker and inhibition of colony formation. The effect of p21waf overexpression, in the absence of p53, on the cytotoxicity caused by irradiation, doxorubicin and taxol was studied. Expression of p21waf provided protection against the cytotoxic effect of radiation and doxorubicin but not of taxol. These results are relevant to treatment of cancer when p53 is inactive. (+info)
Batrachotoxin-resistant Na+ channels derived from point mutations in transmembrane segment D4-S6.
(79/8454)
Local anesthetics (LAs) block voltage-gated Na+ channels in excitable cells, whereas batrachotoxin (BTX) keeps these channels open persistently. Previous work delimited the LA receptor within the D4-S6 segment of the Na+ channel alpha-subunit, whereas the putative BTX receptor was found within the D1-S6. We mutated residues at D4-S6 critical for LA binding to determine whether such mutations modulate the BTX phenotype in rat skeletal muscle Na+ channels (mu1/rSkm1). We show that mu1-F1579K and mu1-N1584K channels become completely resistant to 5 microM BTX. In contrast, mu1-Y1586K channels remain BTX-sensitive; their fast and slow inactivation is eliminated by BTX after repetitive depolarization. Furthermore, we demonstrate that cocaine elicits a profound time-dependent block after channel activation, consistent with preferential LA binding to BTX-modified open channels. We propose that channel opening promotes better exposure of receptor sites for binding with BTX and LAs, possibly by widening the bordering area around D1-S6, D4-S6, and the pore region. The BTX receptor is probably located at the interface of D1-S6 and D4-S6 segments adjacent to the LA receptor. These two S6 segments may appose too closely to bind BTX and LAs simultaneously when the channel is in its resting closed state. (+info)
Isolate resistance of Blastocystis hominis to metronidazole.
(80/8454)
Isolates of Blastocystis hominis from infected immigrant workers from Indonesia, Bangladesh and infected individuals from Singapore and Malaysia were assessed for growth pattern and degree of resistance to different concentrations of metronidazole. Viability of the cells was assessed using eosin-brillian cresyl blue which stained viable cells green and nonviable cells red. The Bangladeshi and Singaporean isolates were nonviable even at the lowest concentration of 0.01 mg/ml, whereas 40% of the initial inoculum of parasites from the Indonesian isolate at day one were still viable in cultures with 1.0 mg/ml metronidazole. The study shows that isolates of B. hominis of different geographical origin have different levels of resistance to metronidazole. The search for more effective drugs to eliminate th parasite appears inevitable, especially since surviving parasites from metronidazole cultures show greater ability to multiply in subcultures than controls. (+info)