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.
The major polycyclic aromatic hydrocarbon inducible-cytochrome P4501A1 gene (CYP1A1) is presumed to be important in pulmonary carcinogenesis and toxicology because its product, the cytochrome P4501A1-dependent (CYP1A1-dependent) monooxygenase, transforms selected xenobiotics (including polycyclic aromatic hydrocarbon procarcinogens in cigarette smoke) to potent carcinogenic metabolites. CYP1A1 messenger RNA (mRNA) expression has not, however, been previously demonstrated in human pulmonary tissue. This report defines CYP1A1 gene expression in normal lung tissue and primary pulmonary carcinoma tissue obtained at thoracotomy from 56 patients with lung cancer. When Northern blot hybridization analyses were performed, 17 of 19 (89%) and zero of five (0%) samples of normal lung tissue from active cigarette smokers and nonsmokers, respectively, expressed the normal 2.8-kilobase CYP1A1 mRNA. In addition, a time-dependent decrease in expression of the CYP1A1 gene was noted in normal lung tissue from individuals who were former smokers, with a decrease in expression occurring as early as 2 weeks following cessation of cigarette smoking. Expression became undetectable in all patients who had stopped smoking more than 6 weeks prior to study. When CYP1A1 gene expression was evaluated in lung cancers, mRNA levels were detectable in one of four (25%) tumors from nonsmokers; two of 24 (8%) tumors from former smokers; and seven of 15 (47%) tumors from cigarette smokers. In addition, an approximately 10-kilobase CYP1A1 RNA species, which was not detectable in normal lung tissue, was observed in five of ten (50%) of the lung cancers that expressed the CYP1A1 gene.(ABSTRACT TRUNCATED AT 250 WORDS)
Obesity is an independent risk factor for early pregnancy loss. This risk is, in part, related to the lower number of collected oocytes in obese women.
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