Trade-off between pulse rate and radiation dose during modified barium swallow examination: what is the reality?

AIM: To evaluate the effect of modification of dose mode and frame rate on patient radiation dose during modified barium swallow (MBS) examinations. MATERIALS AND METHODS: A retrospective review was undertaken of consecutive MBS examinations performed over 6 months in the inpatient setting. Patients were divided into two cohorts: pre-implementation of the MBS Impairment Profile (MBSImP; low rate, normal dose) and post-implementation (high rate, low dose). Prior to implementation, pulse rate and dose testing were performed on multiple phantoms. RESULTS: Four hundred and forty-nine patients were included in the pre-implementation cohort and 378 in the post-implementation cohort. Phantom dose testing demonstrated no significant difference in dose on either phantom between low rate/normal dose and high rate/low dose modes. Prior to MBS standardisation, the mean radiation dose was 5.86 (±4.35) mGy. Following standardisation, the mean radiation dose was 4.72 (±3.77) mGy (p<0.0001). The mean fluoroscopy time for MBS prior to standardisation was 83.8 (±44.4) seconds and the mean fluoroscopy time for MBS after standardisation was 82.3 (±39.8) seconds (p=0.62). The dose rate for MBS prior to standardisation was 4.35 (±2.42) and the dose rate for MBS after standardisation was 3.55 (±2.41) mGy/s (p<0.0001). CONCLUSION: Adjustments made to lower the dose mode and the increase in fluoroscopy frame rate decreased the patient radiation dose and did not increase fluoroscopy time.

Will peripherally restricted kappa-opioid receptor agonists (pKORAs) relieve pain with less opioid adverse effects and abuse potential?

WHAT IS KNOWN AND OBJECTIVE: Optimal utilization of opioid analgesics is significantly limited by the central nervous system adverse effects and misuse/abuse potential of currently available drugs. It has been postulated that opioid-associated adverse effects and abuse potential would be greatly reduced if opioids could be excluded from reaching the brain. We review the basic science and clinical evidence of one such approach - peripherally restricted kappa-opioid receptor (KOR) agonists (pKORAs). METHODS: Published and unpublished literature, websites and other sources were searched for basic science and clinical information related to the potential benefits and development of peripherally restricted kappa-opioid receptor agonists. Each source was summarized, reviewed and assessed. RESULTS: The historical development of pKORAs can be traced from the design of increasingly KOR-selective agonists, elucidation of the pharmacologic attributes of such compounds and strategies to restrict passage across the blood-brain barrier. Novel compounds are under development and have progressed to clinical trials. WHAT IS NEW AND CONCLUSIONS: The results from recent clinical trials suggest that peripherally restricted opioids can be successfully designed and that they can retain analgesic efficacy with a more favourable adverse effect profile.

Reactive metabolites from the bioactivation of toxic methylfurans.

An important mechanism of toxicity of furans involves the cytochrome P-450 monooxygenase-catalyzed bioactivation of the compound in situ directly within the target tissues to highly reactive electrophilic products. The unsaturated aldehydes acetylacrolein and methylbutenedial have been identified as the principal reactive intermediates of 2- and 3-methylfuran, respectively, that are produced and bound covalently to tissue macromolecules in hepatic and pulmonary microsomal systems in vitro.

New carbon dioxide-independent basal growth medium for culture of diverse tumor and nontumor cells of human and nonhuman origin.

A eukaryotic growth medium (Program Development Research Group Basal Growth Medium) was developed for CO2-independent maintenance and propagation of human and nonhuman tumor cell lines representing diverse histologies (e.g., cancers of the brain, colon, lung, ovary, and kidney, as well as leukemia and melanoma). It was also shown to be suitable for the maintenance and propagation of nontumor cells of human and nonhuman derivation. The medium derives its buffering capacity primarily from beta-glycerophosphate, exhibits a stable physiologic pH of 7.3-7.4, and is optimized to facilitate growth in atmospheric CO2. It is also useful in cellular growth and cytotoxicity assays based on either the metabolic reduction of tetrazolium reagents or protein staining. The 50% inhibitory concentration values obtained with carmustine, doxorubicin, and tamoxifen in cell lines maintained in the new medium under atmospheric CO2 were closely comparable to those obtained with these drugs against cells maintained in RPMI-1640 under a 5% CO2 environment.

New soluble-formazan assay for HIV-1 cytopathic effects: application to high-flux screening of synthetic and natural products for AIDS-antiviral activity.

We have developed an effective and optimally safe microculture method for rapid and convenient assay of the in vitro cytopathic effects of human immunodeficiency virus (HIV-1) on human lymphoblastoid or other suitable host cells. The assay procedure is applicable to the evaluation of drug effects on in vitro infections induced directly in cultured host cells by cell-free HIV-1 or by coculture with H9 cells chronically infected with HIV-1. The assay uses a newly developed tetrazolium reagent that is metabolically reduced by viable cells to yield a soluble, colored formazan product measurable by conventional colorimetric techniques. This simple microassay minimizes the number of plate manipulations typically required with other assay methods and, coupled with computerized data collection and analysis, facilitates large-scale screening of agents for potential antiviral activity. To support and enhance the discovery of new anti-HIV-1 agents, the National Cancer Institute is offering investigators worldwide the opportunity to submit new candidate agents for anti-HIV-1 screening with this method.

AIDS-antiviral sulfolipids from cyanobacteria (blue-green algae).

A recently developed tetrazolium-based microculture assay was used to screen extracts of cultured cyanobacteria (blue-green algae) for inhibition of the cytopathic effects of the human immunodeficiency virus (HIV-1), which is implicated as a causative agent of AIDS. A number of extracts were found to be remarkably active against the AIDS virus. A new class of HIV-1-inhibitory compounds, the sulfonic acid-containing glycolipids, was discovered through the use of the microculture assay to guide the fractionation and purification process. The pure compounds were active against HIV-1 in cultured human lymphoblastoid CEM, MT-2, LDV-7, and C3-44 cell lines in the tetrazolium assay as well as in p24 viral protein and syncytium formation assays.

Display and analysis of patterns of differential activity of drugs against human tumor cell lines: development of mean graph and COMPARE algorithm.

The objective of this study was to develop and investigate an approach to optimally detect, rank, display, and analyze patterns of differential growth inhibition among cultured cell lines. Such patterns of cellular responsiveness are produced by substances tested in vitro against disease-oriented panels of human tumor cell lines in a new anticancer screening model under development by the National Cancer Institute. In the first phase of the study, we developed a key methodological tool, the mean graph, which allowed the transformation of the numerical cell line response data into graphic patterns. These patterns were particularly expressive of differential cell growth inhibition and were conveniently amenable to further analyses by an algorithm we devised and implemented in the COMPARE computer program.

4-Ipomeanol: a novel investigational new drug for lung cancer.

4-Ipomeanol (IPO) is the first agent to undergo preclinical development at the National Cancer Institute (NCI) based principally on a specific biochemical-biological rationale for clinical investigation as an antineoplastic agent targeted against lung cancer. This disease-specific development of IPO was initially stimulated by observations that the compound was activated by metabolism, preferentially within the mammalian lung, specifically within bronchiolar Clara cells, and that its predominant toxicity was to the lung in most species. IPO is inactive or only minimally active against most conventional antitumor test systems. However, some human lung cancer cell lines, as well as a variety of fresh human lung tumor biopsy specimens, have been shown to be capable of mediating the in situ biotransformation of IPO to a potentially cytotoxic intermediate. In this report, the biochemistry, metabolism, preclinical pharmacology, and toxicology of IPO are reviewed and the clinical development plans for this unique and challenging new agent are presented.

Altered regulation of the cytochrome P4501A1 gene: novel inducer-independent gene expression in pulmonary carcinoma cell lines.

The cytochrome P450 (CYP) systems catalyze the metabolic transformation of a wide variety of xenobiotics including procarcinogens present in cigarette smoke condensate as well as atmospheric pollutants. The CYP1A1 isoenzyme is of particular interest because it has been implicated as a risk factor in the etiology of lung cancer in heavy cigarette smokers. The identification and expression of the structural CYP1A1 gene in either normal human lung or lung cancer cells has not been reported. Because of its potential significance in human lung cancer, we investigated the expression of the CYP1A1 structural gene in 24 established human lung cancer cell lines including 15 non-small cell (eight adenocarcinomas, three large cell undifferentiated carcinomas, two bronchioloalveolar cell carcinomas, and two squamous cell carcinomas) and nine small cell lung carcinomas. CYP1A1 mRNA was detected in 14 of 15 (93%) of the non-small cell lung carcinoma cell lines examined following 24-hour treatment with benz[a]anthracene (BA) and in nine of 15 (60%) of the non-small cell lines cultured without an inducer in the medium. When the small cell lung cancer lines were evaluated for CYP1A1 gene expression, two of nine (22%) expressed detectable CYP1A1 mRNA in both BA-induced cell cultures and constitutive (control) cultures. A positive correlation was noted between BA-induced CYP1A1 mRNA levels and the corresponding aryl hydrocarbon hydroxylase activity expressed as absolute BA-induced enzyme activity (r = 0.74; P less than .01; n = 24), which further demonstrated that CYP1A1 mRNA expression reflects CYP1A1 enzyme activity in the individual cell lines. These observations represent the first known demonstration of constitutive (non-induced) CYP1A1 gene expression in human cells and suggest altered regulation of the CYP1A1 gene in selected lung cancer cell lines. These human pulmonary carcinoma cell lines, which have documented regulatory defects, could be useful for further identification of the mechanisms associated with CYP1A1 gene regulation.

New colorimetric cytotoxicity assay for anticancer-drug screening.

We have developed a rapid, sensitive, and inexpensive method for measuring the cellular protein content of adherent and suspension cultures in 96-well microtiter plates. The method is suitable for ordinary laboratory purposes and for very large-scale applications, such as the National Cancer Institute's disease-oriented in vitro anticancer-drug discovery screen, which requires the use of several million culture wells per year. Cultures fixed with trichloroacetic acid were stained for 30 minutes with 0.4% (wt/vol) sulforhodamine B (SRB) dissolved in 1% acetic acid. Unbound dye was removed by four washes with 1% acetic acid, and protein-bound dye was extracted with 10 mM unbuffered Tris base [tris (hydroxymethyl)aminomethane] for determination of optical density in a computer-interfaced, 96-well microtiter plate reader. The SRB assay results were linear with the number of cells and with values for cellular protein measured by both the Lowry and Bradford assays at densities ranging from sparse subconfluence to multilayered supraconfluence. The signal-to-noise ratio at 564 nm was approximately 1.5 with 1,000 cells per well. The sensitivity of the SRB assay compared favorably with sensitivities of several fluorescence assays and was superior to those of both the Lowry and Bradford assays and to those of 20 other visible dyes. The SRB assay provides a colorimetric end point that is nondestructive, indefinitely stable, and visible to the naked eye. It provides a sensitive measure of drug-induced cytotoxicity, is useful in quantitating clonogenicity, and is well suited to high-volume, automated drug screening. SRB fluoresces strongly with laser excitation at 488 nm and can be measured quantitatively at the single-cell level by static fluorescence cytometry.

Comparison of in vitro anticancer-drug-screening data generated with a tetrazolium assay versus a protein assay against a diverse panel of human tumor cell lines.

The National Cancer Institute (NCI) is implementing a large-scale in vitro drug-screening program that requires a very efficient automated assay of drug effects on tumor cell viability or growth. Many laboratories worldwide have adopted a microculture assay based on metabolic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). However, because of certain technical advantages to use of the protein-binding dye sulforhodamine B (SRB) in a large-scale screening application, a detailed comparison of data generated by each type of assay was undertaken. The MTT and SRB assays were each used to test 197 compounds, on simultaneous days, against up to 38 human tumor cell lines representing seven major tumor categories. On subsequent days, 38 compounds were retested with the SRB assay and 25 compounds were retested with the MTT assay. For each of these three comparisons, we tabulated the differences between the two assays in the ratios of test group values to control values (T/C) for cell survival; calculated correlation coefficients for various T/C ratios; and estimated the bivariate distribution of the values for IC50 (concentration of drug resulting in T/C values of 50%, or 50% growth inhibition) for the two assays. The results indicate that under the experimental conditions used and within the limits of the data analyses, the assays perform similarly. Because the SRB assay has practical advantages for large-scale screening, however, it has been adopted for routine use in the NCI in vitro antitumor screen.

Metabolic activation of 4-ipomeanol in human lung, primary pulmonary carcinomas, and established human pulmonary carcinoma cell lines.

4-Ipomeanol (IPO) is a pulmonary-specific toxin that is metabolically activated by a cytochrome P450 pathway in lung tissue. In this study, IPO metabolism, as determined by measurement of [14C]IPO covalent binding, was evaluated in a diverse sampling of 18 established, human lung cancer cell lines as well as in normal lung tissue and primary lung carcinoma tissue obtained at the time of thoracotomy from 56 patients with lung cancer. [14C]IPO covalent binding in lung cancer cell lines ranged from 248 to 1,047 pmol of bound [14C]IPO per milligram of protein per 30 minutes (mean +/- SE = 547 +/- 62.2). IPO metabolism in normal lung tissue ranged from 12 to 2,007 pmol of covalently bound [14C]IPO per milligram of protein per 30 minutes (mean +/- SE = 549 +/- 60). In lung cancer tissue, values ranged from 0 to 2.566 pmol of covalently bound [14C]IPO per milligram of protein per 30 minutes (mean +/- SE = 547 +/- 60, P greater than .3). When patients were divided into smokers and current non-smokers (no tobacco products smoked for greater than 6 mo), no effects of cigarette smoking were observed for either normal lung tissue or lung tumor tissue (P greater than .1 in all instances). A wide range of IPO metabolic activity was observed among different histological classifications of lung cancer cell lines and of fresh lung cancer tissues. IPO metabolism was simultaneously compared in normal lung tissue and lung cancer tissue from individual patients, but no positive correlation was observed (r = .10; P greater than .30). The results clearly demonstrate a wide range of IPO metabolism in both normal and lung cancer cells and indicate that a wide diversity of human lung cancers possess the metabolic enzyme system(s) necessary for the bioactivation of IPO to a potentially cytotoxic intermediate. Therefore, the continued exploration for any possible therapeutic potential of IPO in patients with lung cancer appears warranted.

Activity of mitozolomide (NSC 353451), a new imidazotetrazine, against xenografts from human melanomas, sarcomas, and lung and colon carcinomas.

The chemosensitivity of human tumor xenografts to mitozolomide, 8-carbamoyl-3-(2-chloroethyl)imidazo[5-1-d]-1,2,3,5-tetrazin-4(3H) -one, was studied in 3 different assay systems. In concentrations of 1 to 500 micrograms/ml, mitozolomide completely inhibited the colony-forming ability in soft agar of cell suspensions from sarcomas, melanomas, lung and colon cancers, and a mammary carcinoma. When a panel of tumors of the different histological types was tested for its sensitivity to mitozolomide in vitro, in the 6-day subrenal capsule assay in conventional mice, and, in some cases, as s.c. growing tumors in nude mice, good agreement between the different assay systems was seen. In most cases, a very pronounced antitumor effect was observed. The efficacy of mitozolomide was as good or better than that of the drugs clinically used against the tumor types tested. Tumor size measurements and histological examinations indicated that nude mice carrying a melanoma, a small cell lung cancer, and an osteosarcoma were cured of their tumors. The approach here used for evaluating the effect of a new drug on human cancers may be useful for selecting the tumor types which primarily should be studied in clinical trials. The results indicate that clinical responses to mitozolomide may be anticipated in sarcoma, melanoma, small cell lung cancer, and possibly in colon cancer.

Evidence for thromboxane biosynthesis in established cell lines derived from human lung adenocarcinomas.

Thromboxane B2 (TxB2) is the stable nonenzymatic hydrolysis product of thromboxane A2, a substance implicated in the initiation of the facilitative role of thrombocytes in the metastatic process. TxB2 was isolated from protein-free culture medium of cell lines Calu-3, Calu-6, A549, and A549/Asc-1, derived from human lung adenocarcinomas. TxB2 and other 20-carbon fatty acid cyclooxygenase products synthesized from exogenous and endogenous arachidonic acid were identified by their characteristic retention indices and fragmentation of electron-capture derivatives of unlabeled and deuterium-labeled products during combined capillary gas chromatography-mass spectrometry. TxB2 comprised 2 to 6% of 20-carbon fatty acid cyclooxygenase products biosynthesized from endogenous arachidonic acid in calcium ionophore A23187-stimulated Calu-6 and A549/Asc-1 cells and 16 to 25% of these products in Calu-3 and A549 cells. The addition of 10(-5) M exogenous arachidonic acid to the cultured cells resulted in a 2- to 3-fold increase in TxB2 and bisenoic prostanoid production with no significant alterations in the proportion of TxB2 production. Prostaglandin E2 and prostaglandin F2 alpha, two prostanoids that can be formed either enzymatically or nonenzymatically from prostaglandin H2, accounted for greater than 75% of isolatable 20-carbon fatty acid cyclooxygenase products synthesized from endogenous and exogenous arachidonic acid.

Profiles of prostaglandin biosynthesis in sixteen established cell lines derived from human lung, colon, prostate, and ovarian tumors.

The profiles of prostanoid biosynthesis from endogenous arachidonic acid in 16 established cell lines derived from 4 histological classes of human carcinomas were determined by capillary gas chromatography-mass spectrometry. Detectable quantities of prostanoids were isolated from the culture medium of cell lines representative of the different histological classes of human tumors: colorectal adenocarcinomas (one of three cell lines); ovarian adenocarcinomas (one of three cell lines); prostate adenocarcinomas (zero of two cell lines); non-small cell carcinomas of the lung (four of five cell lines); and small cell carcinomas of the lung (zero of three cell lines). Prostaglandins E2 and F2 alpha were the only prostanoids synthesized in detectable quantities. Prostaglandin E2 biosynthesis (mean +/- SD), pmol/10(6) cells, n = 4) in cell lines exhibiting positive prostaglandin H synthase activity was: LoVo (colorectal adenocarcinoma, 0.4 +/- 0.1); A2780 (ovarian adenocarcinoma, 1.3 +/- 0.3); NCI-H322 (bronchioloalveolar cell carcinoma, 8.4 +/- 3.1); NCI-H358 (bronchioloalveolar cell carcinoma, 7.8 +/- 2.4); EKVX (adenocarcinoma of the lung, 21.3 +/- 5.5); and A427 (large cell undifferentiated carcinoma of the lung, 12.6 +/- 2.8). Prostaglandin F2 alpha production (pmol/10(6) cells +/- SD) was: LoVo (0.3 +/- 0.1); NCI-H322 (0.6 +/- 0.2); NCI-H358 (0.4 +/- 0.1); EKVX (1.8 +/- 0.4); and A427 (11.1 +/- 3.1). These findings suggest that within certain limitations cultured tumor cells provide simplified experimental systems for determination of prostaglandin biosynthetic characteristics of human tumors and that prostanoid biosynthesis may be particularly characteristic of certain non-small cell carcinomas of the lung.

Metabolic activation and cytotoxicity of 4-ipomeanol in human non-small cell lung cancer lines.

In the normal lungs of many animal species, 4-ipomeanol is transformed to a highly reactive metabolite preferentially in pulmonary bronchiolar Clara cells and to a lesser extent in alveolar type II cells, potentially leading to damage or destruction of these cell types. Since Clara cells and type II cells are suspected sites of origin of certain "non-small cell" lung cancers, the metabolic activation of 4-ipomeanol (measured by the metabolism-dependent covalent binding of 4-ipomeanol to cellular macromolecules) was compared in two human non-small cell carcinoma derived cell lines (NCI-H322 and NCI-H358) and two human small cell carcinoma derived cell lines (NCI-H128 and NCI-H69). Metabolic activation of 4-ipomeanol was evident in the non-small cell lines; the production of covalently bound metabolite was somewhat greater in NCI-H322 (morphology related to Clara cells) compared to NCI-H358 (morphology related to alveolar type II cells), but was entirely undetectable in the small cell lines. The activation pathway was concentration (4-ipomeanol) and time dependent and followed Michaelis-Menten kinetics. Metabolism to the reactive intermediate required oxygen and was strongly inhibited by carbon monoxide. Covalent binding was enhanced in the non-small cell lines by prior incubation with beta-naphthoflavone and by supplementation of the incubate with exogenous reduced nicotinamide adenine dinucleotide phosphate. 4-Ipomeanol was more cytotoxic to the non-small cell lines than to the small cell lines under the in vitro growth conditions used. These studies indicate that certain human non-small cell lung cancers have metabolic characteristics of normal bronchiolar Clara cells and alveolar type II cells; these results would therefore be consistent with an origin of these tumors from Clara cells or type II cells, respectively. The present studies indicate that the further preclinical testing and development of 4-ipomeanol is warranted, with a view toward possible clinical evaluation against human lung cancers.

Lack of correlation between natural killer activity and tumor growth control in nude mice with different immune defects.

To elucidate the in vivo role of natural killer (NK) cells, the growth of several murine and human tumors was studied in four variants of athymic, nude mice with different levels of NK activity. Beige-nude mice, homozygous for both the beige and the nude genes, had very low levels of NK activity, and their response to the B-cell mitogen, bacterial lipopolysaccharide, was lower than that of high-NK, adult NIH nude mice. Young and adult NIH nudes had different NK levels and showed different response in assays for K-cell, T-cell, and B-cell activity. The B-cell-defective NIH-II mice had slightly lower NK levels than adult NIH animals, but much lower response in the antibody-dependent cell-mediated cytotoxicity assay. No correlation was found between host NK activity and the s.c. growth of various human (LOX, CEM, K562) and murine (YAC-1) tumor cells. Low NK activity was not associated with increased lung colony formation in a metastasis model using i.v.-injected human (LOX) and murine (B16F10) melanoma cells. No relationship was found between host NK activity and the rate of elimination of i.v.-injected 5-iodo-2'-deoxyuridine-labeled LOX, B16F10, and YAC-1 cells from lungs, liver, or spleen. The results fail to support the view that NK cells exert significant direct effects on tumor cells in vivo.

Extrapulmonary, tissue-specific metastasis formation in nude mice injected with FEMX-I human melanoma cells.

FEMX-I human malignant melanoma cells, originating from a lymph node metastasis in a patient, uniquely and selectively produced extrapulmonary metastases after i.v. injection of cells prepared from xenografts into adult, nude mice. After a lag time of approximately 50 days, metastases were observed in s.c. sites at the back and front of the neck, and in axilla and inguinal regions. Tumor colony formation in lungs were never detected. The interscapular tumors showed a close relationship to brown fat, partly infiltrating this tissue, whereas the other s.c. tumors seemed to be localized to lymph nodes. Mesenterial and mediastinal lymph node metastases were frequently found, together with retroperitoneal tumors along the spine. The normal cells of the adrenal medulla were often replaced by melanoma cells, whereas the cortical tissue was not affected. The conclusion that FEMX-I cells possess an inherent ability for tissue-specific metastasis formation is supported by the metastatic pattern seen after i.p. and intrasplenic injection, as well as after inoculation of the cells in the footpads of the mice. The relatively slowly growing FEMX-I tumors showed the same differentiated morphology as the patient's tumor, independent of the site of growth and the number of passages in the animals. The FEMX-I tumor was easily established as a cell line in vitro. Such cells showed a strongly reduced metastatic capacity, indicating that the in vitro growth conditions had induced alterations in the FEMX-I cells influencing their ability to form site-specific metastases, changes that were shown to be reversible. It is suggested that structures on the surface of the tumor cells, as well as growth factors in the host tissues, may be of importance for the observed tissue specificity. The FEMX-I melanoma, which, as a human tumor in nude mice, has a unique metastatic pattern, offers possibilities for investigating mechanisms involved in site-specific metastasis formation, as well as for testing effects of antimetastatic, chemotherapeutic, and immunotherapeutic agents against human extrapulmonary micro- and macrometastases.

Metabolism of arachidonic acid in human lung cancer cell lines.

The metabolism of arachidonic acid (AA) was studied in two pulmonary bronchioloalveolar-carcinoma cell lines (NCI-H322 and NCI-H358) and two small cell lung carcinoma cell lines (NCI-H69 and NCI-H128). Exogenous AA was metabolized only in the NCI-H322 and NCI-H358 cells. There was no detectable metabolism of AA in NCI-H69 or NCI-H128 cells, either in the presence or the absence of the calcium ionophore A23187. The major metabolite of AA isolated from both NCI-H322 and NCI-H358 cells was prostaglandin E2 (PGE2). Prostaglandin endoperoxide synthase activities, expressed as immunoreactive PGE2 (pmol/min/mg protein), were 10.3 +/- 0.28 (SD) and 4.8 +/- 0.48 in NCI-H358 and NCI-H322 cells, respectively. The rate of production of PGE2 by both NCI-H358 and NCI-H322 cells was linear up to 10 min. Production of PGE2 in both cell lines was dependent upon substrate concentration and was maximal above 17 microM AA. Moreover, PGE2 did not undergo further metabolism by either the NCI-H358 or the NCI-H322 cells. Aspirin (0.1 mM), a cyclooxygenase inhibitor, decreased PGE2 production by 77 and 60% in NCI-H358 and NCI-H322 cells, respectively. In the presence of exogenous AA the calcium ionophore, A23187 (20 microM), stimulated PGE2 production in NCI-H322 cells by almost 2-fold, although it did not affect PGE2 production in the NCI-H358 cells. In contrast, A23187 stimulated the endogenous production of PGE2 in both NCI-H322 and NCI-H358 cells by 4- and 9-fold respectively. In addition, both the NCI-H358 and NCI-H322 cell lines were susceptible to the cytotoxic effects of the anticancer agent mitoxantrone in both a time and concentration dependent manner. In contrast, the two cell lines lacking detectable prostaglandin synthesis activity, NCI-H69 and NCI-H128 were unaffected by treatment with mitoxantrone. These results illustrate that there are major differences in the abilities of human lung cancer cell lines to biosynthesize and release PGE2. It is conceivable that such differences might have exploitable diagnostic and/or therapeutic implications.

Tetrazolium-based assays for cellular viability: a critical examination of selected parameters affecting formazan production.

The hydrogen acceptor 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) is commonly utilized to estimate cellular viability in drug screening protocols. The present investigation was prompted, in part, by observations that reduction of MTT to its colored reaction product, MTT formazan, varied between cell lines and with culture age. A correlation was established between the D-glucose concentration of the culture medium at the time of assay and the production of MTT formazan for cell lines representing seven tumor histologies. A decrease in the concentration of D-glucose from culture medium was accompanied by a decrease in MTT specific activity (MTT formazan/microgram cell protein) for a number of cell lines. Cells which extensively metabolized D-glucose exhibited the greatest reduction in MTT specific activity. Further evidence that the D-glucose concentration of the culture medium played an important role in MTT reduction was provided by experiments which demonstrated that transfer of cells to a glucose-free medium (L-15) was accompanied by an immediate decrease in MTT reduction which was pH independent. These studies suggested that cellular transport and constant metabolism of glucose were required for maximum MTT reduction. Decreases in the cellular concentration of the reduced pyridine nucleotides NADH and NADPH were accompanied by concomitant decreases in MTT formazan production. MTT formazan varied significantly among cell lines in both the kinetics of its formation and the degree of saturability exhibited. Apparent IC50 values for Adriamycin varied, in a cell line-specific manner, with MTT exposure time. These results indicate that MTT specific activity is significantly influenced by a number of parameters and suggest that assay conditions should be established which minimize their effects.