Phenylbutyrate-induced apoptosis and differential expression of Bcl-2, Bax, p53 and Fas in human prostate cancer cell lines.

OBJECTIVE: To assess the mechanisms of action of phenylbutyrate (PB), an investigational chemotherapeutic agent for prostate cancer (PCa), in apoptosis induction in PCa cell lines in vitro. STUDY DESIGN: We analyzed the differential expression of different apoptosis modulators, Bcl-2, Bax, p53 and Fas, for their potential role in PB-induced apoptosis using relative quantitative flow cytometry (FCM). Both androgen-dependent (LNCaP) and androgen-independent (C-4-2, PC-3-PF and DU145) human PCa cell lines were examined. RESULTS: PB induced apoptosis in PCa cell lines in a dose-dependent manner. Fifty percent apoptosis could be induced by 5-10 mM PB. Bcl-2 was down-regulated 30-75% and the Bax:Bcl-2 ratio elevated in apoptotic PCa cell lines regardless of their androgen dependency or p53 status. FCM revealed a heterogeneous stimulatory effect on the expression of Bax and Bcl-2 in PC3-PF cells at 0.5-2.5 mM PB. In a p53-positive cell line (DU145), p53 was repressed by 70% and Fas elevated sixfold with 10 mM PB. CONCLUSION: Our data show that PB-induced PCa apoptosis is associated with the relative repression of Bcl-2 and with up-regulation of Bax and Fas proteins and that this PB-induced apoptosis is independent of p53 and androgen-dependency status of PCa cell lines.

Dose-response effect of in vivo administration of endotoxin on polymorphonuclear leukocytes oxidative burst.

In vitro, endotoxin primes polymorphonuclear leukocytes (PMNs) to respond with a greater oxidative burst. The purpose of the present study was to investigate the in vivo effect of a wide range of single endotoxin bolus doses using a rat model. PMNs were subsequently challenged in vitro with phorbol ester to produce reactive oxygen intermediates (ROI). Flow cytometric determination of ROI production by large doses induced a decrease in ROI production by the few PMNs that remained in the circulation. By 6 h after injection, ROI production had returned to basal levels after a high dose, and was still increasing after a low dose. Neutropenia occurred immediately after endotoxin injection. After 6 h, PMN counts returned to almost normal levels with a high dose, but rebound neutrophilia occurred with a small dose. In contrast to in vitro studies, in vivo injection showed a response pattern that varied widely with dose and time of observation.

Long-term primary culture of epithelial cells from rainbow trout Oncorhynchus mykiss) liver.

Long-term primary cultures of epithelial cells from rainbow trout (Oncorhynchus mykiss) liver have been established. Nearly homogenous (> 97%) populations of hepatocytes were placed into primary culture and remained viable and proliferative for at least 70 d. In addition to hepatocytes, proliferative biliary cells persisted in the cultures for at least 30 d. Finally, a third type of epithelial cell, which we have termed a "spindle cell," consistently appeared and proliferated to confluence in these cultures. The confluent cultures of spindle cells were successfully subcultured and passaged. The initial behavior, growth, and optimization of serum and media requirements for these cells is described. All three cell types proliferated as measured by thymidine incorporation, autoradiography, proliferating cellular nuclear antigen analysis, and propidium iodine staining. Further efforts to characterize the cells included western blotting and immunohistochemical staining with antibodies to cytokeratins previously reported in fish liver. From these data, it appears that all three cell populations are epithelial in nature. Furthermore, significant changes in actin organization, often indicative of transformation or pluripotent cells, were observed with increased time in primary culture.

Substance abuse during pregnancy in a rural population.

Drug abuse during pregnancy in rural populations has received less attention than that in urban populations. Urban studies have reported alarming rates, but it is unknown whether the situation is the same in rural areas. To investigate this, urine samples were collected anonymously from 181 pregnant women who presented to the University of Missouri clinics for care and who resided in communities of less than 25,000. Each urine specimen was tested for cocaine, marijuana, amphetamines, barbiturates, opiates, phencyclidine, benzodiazepines, ethanol, and nicotine. Of the 181 specimens, 83 (46%) contained nicotine, 17 (9.4%) contained marijuana, and one each (0.6%) tested positive for cocaine, barbiturates, ethanol, and benzodiazepines. No other tested substances were detected. Excluding nicotine and ethanol, 20 (11%) of the urine samples tested positive for the screened substances. Review of the prenatal records revealed that 46% of the women reported using tobacco, 15% reported using alcohol, and 8.3% reported illicit drug use during pregnancy. This study indicates that there is a substantial drug abuse problem in rural populations, and that the profile of abuse differs from that of urban populations. Tobacco, ethanol, and marijuana were the most prevalent substances abused during pregnancy, but cocaine was a minor problem. This information may help in directing resources to reduce drug abuse during pregnancy.

Immunoassay method validation for a modified EMIT phencyclidine assay.

Immunoassay drug testing methods, that have been modified from the manufacturers' recommended procedure to be used for the analysis of federally regulated specimens or other forensic samples require evaluation to ensure their scientific validity. These validation studies must demonstrate the accuracy, precision, and linearity of the modified immunoassay around the cutoff concentration, substantiate adequate rate separation, and verify the ability of the assay to differentiate positive and negative samples. Modification of the EMIT d.a.u. phencyclidine assay in order to achieve the federally mandated cutoff concentration of 25 ng/mL is common. This study describes the validation of a modified EMIT phencyclidine assay and a protocol that allows for the evaluation of similarly modified immunoassays.

Cellular F-actin levels as a marker for cellular transformation: relationship to cell division and differentiation.

Transformation is associated with profound structural and quantitative changes in the cytoskeleton. Herein we report studies using F-actin, a major cytoskeletal protein, as a quantitative marker for transformation cells, focusing on separating the effects of the cell cycle, cell differentiation, and transformation. The model system for these studies consisted of three lymphoblastic cell lines, one untransformed line (RPMI) and two transformed lines, one (HL-60) of which can be induced to differentiate and the other (Daudi) which cannot. The relation of F-actin levels to cell cycle was studied by flow cytometry with the use of fluorescein-phalloidin to label F-actin and propidium iodide to label DNA. F-actin levels in transformed Daudi and HL-60 lines were only two-thirds that of the untransformed RPMI cells. Histograms of the distribution of F-actin showed that the transformed lines consisted of two cell populations, one having an F-actin content near that of untransformed cells and the other having much less. Cell cycle analysis showed that F-actin in untransformed cells increased 10-15% as cells entered the S compartment, remaining approximately constant through G2 + M phases of the cell cycle, but in transformed cells the major increase in F-actin occurred during G2 + M phase. Double-label studies with rhodamine-phalloidin for F-actin and KI-67 monoclonal antibody for dividing cells (cells at late G1, S, G2, and M) measured with quantitative fluorescence image analysis showed that the mean F-actin content of dividing cells was twice that of nondividing cells. These results suggested that most of the cell division-related F-actin increase occurred during late G1 phase in untransformed cells. Differentiation of HL-60 cells with dimethyl sulfoxide or retinoic acid normalized the F-actin content of the nondividing cell population, but dimethyl sulfoxide and retinoic acid produced no detectable change in F-actin in the undifferentiable Daudi cells. A tumor promoter (12-O-tetradecanoylphorphol-13-acetate) inhibits differentiation of hematopoietic cells, resulted in a 32% decrease in the mean F-actin content of RPMI cells due to the appearance of a new subpopulation of low F-actin content. The 12-O-tetradecanoylphorbol-13-acetate-induced changes reversed slowly after removal of 12-O-tetradecanolyphorbol-13-acetate but more rapidly in the presence of retinoic acid. These results indicate that F-actin quantification can serve as a marker for cellular transformation and provides a tool for studying the mechanisms of cellular differentiation that may lead to a better understanding of the oncogenic process.

Flow cytometric analysis of megakaryocytes from patients with abnormal platelet counts.

Megakaryocytes (MKs) from 40 patients with quantitative platelet disorders and 19 normal volunteers were analyzed by flow cytometry for size, fine cell internal structure and granularity, membrane expression of the glycoprotein (GP) IIb/IIIa complex, and for ploidy distribution. Analysis was performed on unfractionated minimally manipulated marrows obtained from routine bone marrow aspirates. MKs were labeled with a fluorescent lineage-specific monoclonal antibody to the GPIIb/IIIa complex followed by DNA staining with propidium iodide. Eight hundred to 3,000 MKs were analyzed in each sample. The modal ploidy distribution in normals was 16N, comprising about half of the megakaryocytic population, with 22.6% of the cells less than or equal to 8N and 22.0% greater than or equal to 32N. Twelve thrombocytopenic patients with decreased marrow MKs on biopsy (mean platelet count [MPC] 44,600/microliters) showed an increase in low ploidy cells with 53.2% less than or equal to 8N (P less than .01); cell size was reduced in three patients when compared to normal cells of identical ploidy (P less than .05). Eight thrombocytopenic patients with enhanced platelet destruction (with normal or increased MKs on biopsy and shortened platelet survival; MPC 41,400/microliters) showed an increased proportion of high ploidy cells greater than or equal to 32N to 39.2% (P less than .01). Increased cell size and granularity were found in four of these patients (P less than .05). Six patients with thrombocytopenia secondary to multiple mechanisms affecting both platelet production and destruction (MPC 66,700/microliters) showed no shift in ploidy. Four patients with primary thrombocytosis (two with thrombocythemia and two with polycythemia vera; MPC 822,500/microliters) showed a marked shift toward high ploidy cells with 42.3% greater than or equal to 32N and 7.6% greater than or equal to 64N cells (P less than .01). The shift was accompanied by a marked increase in cell size and granularity in the patients with thrombocythemia. Ten patients with thrombocytosis secondary to chronic blood loss, malignant or inflammatory disorders (MPC 714,000/microliters), showed variable distributions with four patients exhibiting a shift in ploidy to the right similar to that found in the patients with increased platelet destruction. Based upon the present data, flow cytometric ploidy distribution may be diagnostically useful in thrombocytopenic patients by discriminating between disorders of platelet production and destruction. (ABSTRACT TRUNCATED AT 400 WORDS)