PubMed Health⌕ Search

Biomedical subjects

M R Boyd

Publications and source records attributed to M R Boyd.

At least 181 records · Page 10Linked to original sources
PubMed1991

Practical spontaneous metastasis model for in vivo therapeutic studies using a human melanoma.

In vivo studies aimed at therapy of spontaneous human tumor metastases have been hampered by the lack of practical experimental models. The LOX amelanotic melanoma model described here represents a transplantation model which rapidly and reproducibly results in spontaneous pulmonary metastasis following s.c. inoculation into athymic mice. Pulmonary lesions can be detected using a simple bioassay procedure which is useful for estimation of metastatic cell killing. Using this model we demonstrate that systemic therapy with cyclophosphamide or dacarbazine can produce metastatic cell killing consistent with complete eradication of established pulmonary metastases. This model may also prove useful for future experimental therapeutic studies aimed at prevention of metastases by manipulating tumor staging interval and treatment schedule.

Animals↗
PubMed1991

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.

Carcinoma, Renal Cell↗
PubMed1991

Effect of modulators of glutathione synthesis on the hepatotoxicity of 2-methylfuran.

Treatment of male Sprague-Dawley rats with buthionine sulfoximine (BSO), prior to administration of carbon-14(14C)-labelled 2-methylfuran (2MF) caused a marked decrease in the covalent binding of 14C-labelled 2MF metabolites to both DNA and protein, although there was no apparent change in the distribution of the labelled parent 2MF. BSO pretreatment also protected against hepatotoxicity of 2MF, as indicated by lower serum glutamic pyruvic transaminase (GPT) levels. Pretreatment with BSO offered protection only if administered 1.5 hr before 2MF dosage. Administration of 2MF, 4 and 6 hr after BSO resulted in manifestation of the hepatotoxicity of 2MF. Prior treatment with diethylmaleate (DEM), increased covalent binding of [14C]2MF to liver proteins and also elevated serum GPT levels. Thus, depletion of tissue glutathione (GSH) by two different chemicals acting by different mechanisms produced opposite effects on the covalent binding and toxicity of 2MF. Pretreatment with L-2-oxothiazolidine-4-carboxylate (OTZ), a promoter of GSH biosynthesis, increased the hepatic covalent binding of [14C]2MF and potentiated hepatotoxicity. However, administration of OTZ and BSO prior to an i.p. dose of 100 mg/kg of 2MF, decreased the hepatic covalent binding of [14C]2MF and decreased the hepatoxicity. The marked instability of the GSH conjugate of the reactive metabolite of 2MF may account for the potentiation of hepatotoxicity of 2MF by OTZ. A single s.c. dose of BSO, caused a transient increase in plasma cystine levels concurrent with the depletion of liver GSH. Administration of 2MF, 1.5 hr after BSO, significantly decreased plasma cystine levels as compared to control animals that received vehicle alone. Pretreatment with BSO also resulted in increased excretion of urinary metabolites in 2MF treated animals as compared to animals receiving 2MF alone. Thus, BSO probably protects against hepatoxicity of 2MF by indirectly causing more detoxification of the reactive metabolite of 2MF, as it does not alter the distribution of unmetabolized 2MF and does not have any apparent effect on the microsomal mixed-function oxidase which mediates the activation of 2MF. The enhanced detoxification of 2MF in BSO treated animals appears independent of the depleted GSH levels; it may result from increased availability of a better alternative nucleophile (i.e. cysteine), capable of conjugating with acetyl acrolein. Acetyl acrolein (AA) appears to be the principal reactive metabolite of 2MF which binds covalently to tissues. Previous in vitro studies have shown that cysteine is a better trapping agent of AA than GSH or N-acetyl-cysteine.

Alanine Transaminase↗
PubMed1991

Morphometric and colorimetric analyses of human tumor cell line growth and drug sensitivity in soft agar culture.

Previous studies have demonstrated the suitability of image analysis of tetrazolium-stained colonies to assess growth and drug sensitivity of human tumor cells cultivated in soft agar culture. In the present study, the potential utility of colorimetric analysis to expedite experimental drug evaluations using human tumor cell lines was investigated. The same culture dishes were assessed by image analysis and by formazan colorimetry for purposes of comparing multiple methods of measuring growth as well as growth inhibition. Replicate cultures treated with 2-(p-iodonitrophenyl)-3-p-nitrophenyl-5-phenyltetrazolium chloride or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide exhibited nearly identical colony count and volume indices as well as excellent correlation in colorimetric end points. Colony-forming unit volume analysis versus colorimetric assessment of the same cultures following dimethyl sulfoxide extraction of protamine sulfate-rinsed, dried soft agar cultures exhibited excellent linear correlation for both growth (Pearson r ranging from 0.95 to 1.00) and drug sensitivity (Pearson r ranging from 0.90 to 0.99, and Spearman r ranging from 0.82 to 0.97) and similar drug sensitivity profiles. Results of the current investigation indicate that end points of soft agar culture remain stable for a period of at least 2 weeks following assay termination. In addition, a colorimetric detection range of 1.3-2.2 log units permits determinations of survival levels ranging from 100 to 5% of respective control levels. Colorimetric analysis is anticipated to expedite soft agar colony formation assay evaluations (a) by reducing the need to use the more rigorous and time-consuming image analysis procedures to measure activity in preliminary drug sensitivity assays and (b) by permitting the determination of effective concentration ranges of new experimental agents for subsequent, more detailed investigations.

Bleomycin↗
PubMed1991

A semiautomated multiparameter approach for anti-HIV drug screening.

We are implementing a series of complementary assays for initial follow-up confirmation and prioritization of new active anti-HIV compounds identified by the U.S. National Cancer Institute's large-scale in vitro primary anti-HIV screen. Two different kinds of cellular viability assays, in addition to specific assays for total cellular DNA content, supernatant reverse transcriptase activity, p24 core antigen production and the synthesis of infectious HIV virions are all performed from a single well of a 96-well microtiter plate containing human host cells infected with HIV. Antiviral activities of several known prototype HIV inhibitors including 3'-azido,3'-deoxythymidine, 2',3'-dideoxycytidine, dextran sulfate and phorbol myristate acetate were compared in these multiparameter assays as a means of validation. Procedures to automate the method optimally, as well as to maximize the safety of the technicians working with HIV and HIV-infected cells have been emphasized. The resulting semiautomated, highly reproducible battery of assays yields a maximum amount of antiviral and cytotoxicity information from a minimum amount of sample. This is especially crucial when analyzing new synthetic compounds and natural product extracts or fractions where the available amounts of sample may be very limited.

Antiviral Agents↗
PubMed1991

Synthesis and antiviral activity of 9-alkoxypurines. 2. 9-(2,3-Dihydroxypropoxy)-, 9-(3,4-dihydroxybutoxy)-, and 9-(1,4-dihydroxybut-2-oxy)purines.

Reaction of alkenoxyamines (3,5) or (R,S)-, (R)-, and (S)-hydroxy-protected derivatives of hydroxyalkoxyamines (20a,b, 37a-c) with 4,6-dichloro-2,5-diformamidopyrimidine (4) and cyclization of the resultant 6-[(alkenoxy)amino]-and 6-(alkoxyamino)pyrimidines (6,7,21a,b, 38a,b,c) by heating with diethoxymethyl acetate afforded 9-alkenoxy- and 9-alkoxy-6-chloropurines (9,10,22a,b, 39a-c, 40a). These were subsequently converted to 9-(2,3-dihydroxypropoxy), 9-(3,4-dihydroxybutoxy), and 9-(1,4-dihydroxybut-2-oxy) derivatives of guanine and 2-aminopurine (13-16, 25-28, 41a-c, 42a). A 2-amino-6-methoxypurine derivative (17) was also prepared. The racemic guanine derivative 13 showed potent and selective activity against herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), but was less active against varicella zoster virus (VZV). Its antiviral activity is attributable to the S isomer (28), which was found to be more active than acyclovir against HSV-1 and HSV-2 and about 4 times less active than acyclovir against VZV. The S enantiomer of 9-(1,4-dihydroxybut-2-oxy)guanine (41c) also showed noteworthy antiviral activity in cell culture. Although this acyclonucleoside (41c) is only weakly active against HSV-1 and inactive against HSV-2, it is about twice as active as acyclovir against VZV.

Antiviral Agents↗
PubMed1990

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.

Biotransformation↗
PubMed1990

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.

Adenocarcinoma↗
PubMed1990

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.

Antineoplastic Agents↗
PubMed1990

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.

Animals↗
PubMed1990

Synthesis and antiviral activity of 9-alkoxypurines. 1. 9-(3-Hydroxypropoxy)- and 9-[3-hydroxymethyl)propoxy]purines.

Reaction of hydroxyl-protected derivatives of hydroxyalkoxyamines (3a,b,c) with either 4,6-dichloro-2,5-diformamidopyrimidine (5) or 4,6-dichloro-5-formamidopyrimidine (31) and subsequent cyclization of the resultant 6-(alkoxyamino)pyrimidines (6, 17, 32, 35) by heating with diethoxymethyl acetate afforded 9-alkoxy-6-chloropurines (7, 18, 33, 36), which were converted subsequently to 9-(3-hydroxypropoxy)- and 9-[3-hydroxy-2-(hydroxymethyl)propoxy] derivatives of guanine, 2-amino-6-chloropurine, 2-amino-6-alkoxypurines, 2-aminopurine, 2,6-diaminopurine, adenine, hypoxanthine, and 6-methoxypurine (8, 12, 13, 19-21, 23-26, 34, 37-39). Carboxylic acid esters (9-11, 14-16, 27-29) and a cyclic phosphate derivative (22) of the 9-(hydroxyalkoxy)guanines (8, 21) and 2-amino-9-(hydroxyalkoxy)purines (13, 26) were also prepared. The guanine derivatives (8, 21) showed potent and selective activity against herpes simplex virus types 1 and 2 and varicella zoster virus in cell cultures and 8 is more active than acyclovir. Although without significant antiviral activity in cell cultures, the 2-aminopurines (13, 14-16, 26-29) and 2-amino-6-alkoxypurines (12, 23-25) are well absorbed after oral administration to mice and are converted efficiently to the antiviral guanine derivatives (8, 21) in vivo.

Acyclovir↗
PubMed1990

Interactive laser cytometric analysis of retroviral protein expression in HIV-infected lymphocytic cell lines.

We have used interactive laser cytometry to investigate the expression of human immunodeficiency virus (HIV) envelope glycoproteins gp160, gp41, gp120, and the core protein p24 in the HIV-infected human lymphocyte cell lines H-9, CEM-SS, and C8166. This method allowed for the ultrasensitive detection of fluorescence signals at the single cell level and, when combined with specific anti-HIV antibodies, permitted unique quantitative detection of HIV antigens. Indirect immunofluorescence assays with monoclonal antibodies directed against gp120 revealed that a large proportion of lymphocytic cells expressed increased gp120-associated fluorescence consistent with HTLV-IIIRF infection. Certain monoclonal and polyclonal antibodies were also effective in quantifying gp160, gp41, and p24 expression. Expression of these antigens was found to vary significantly within 48 h. Significant loss (greater than or equal to 50%) of gp120 expression was observed when cells were treated with 1.0 microM AZT. The expression of the HIV-associated protein markers gp160, gp41, and p24 was detectable 24 h after infection of C8166, a cord blood lymphocytic cell line. C8166 cells expressed an additional 6- to 10-fold increase in gp120 in 48 h as well as a 3- to 4-fold increase in gp160, gp41, and p24. AZT (0.01 and 0.1 microM) decreased the expression of gp120, gp160, and p24 in a dose-dependent fashion. This new application of interactive laser cytometry permits early, sensitive, and statistically based distinctions in the expression of HIV-associated antigens in infected target cells at the single-cell level, and allows detection of important changes in HIV-associated antigen expression and the kinectics thereof.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line↗
PubMed1989

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.

Adenocarcinoma↗
PubMed1989

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.

Antiviral Agents↗
PubMed1989

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.

Animals↗
PubMed1989

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.

Antineoplastic Agents↗