aphidicolin and Carcinoma--Small-Cell

Accumulating evidence indicates that small cell lung cancer (SCLC) is defective in many of the regulatory mechanisms that control cell cycle progression. The purpose of this study was to determine the effects of flavopiridol, a pan-cyclin-dependent kinase inhibitor, on growth and apoptosis of SCLC cell lines.. Cell growth was monitored using 3-(4,5dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT) and clonogenic assays. Induction of apoptosis was assessed using multiple assays, including flow cytometric determination of DNA content and mitochondrial membrane potential, terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL), and Western blot analysis of procaspase 3 and poly(ADP-ribose) polymerase cleavage.. Flavopiridol induced growth inhibition and cytotoxicity in multiple SCLC cell lines, with an IC(50) of 50-100 nM and an LD(50) of 150-200 nM in 72-h MTT assays. The cytotoxicity seen in the MTT assay proved to be apoptosis by several criteria. Interestingly, inhibition of caspase activation with the caspase inhibitor Boc-Asp(OMe)-CH(2)F reduced TUNEL labeling by 40% but did not have any effect on the loss of mitochondrial membrane potential (detected as early as 4 h after drug exposure) or cytotoxicity in MTT assays. These results suggest that the primary event in flavopiridol-induced apoptosis involves induction of mitochondrial dysfunction. Cells synchronized with aphidicolin at the G(1)-S border and treated with flavopiridol during S phase showed a marked increase in apoptosis compared with an asynchronous population or a population treated during G(2)-M. Despite the increased apoptosis, a significant proportion of synchronized cells proceeded through S, G(2)-M, and into G(1) phase in the presence of flavopiridol, demonstrating that a high-grade cell cycle arrest is not required for apoptosis. Cells synchronized at the G(1)-S border treated with a short exposure to flavopiridol also showed more than a 10-fold decrease in clonogenicity compared with asynchronous cells treated identically.. Taken together, these data demonstrate that flavopiridol potently and selectively induces SCLC apoptosis preferentially during S phase, in a manner that involves early mitochondrial dysfunction without a requirement for a high-grade block to cell cycle progression. Furthermore, clonogenicity data suggests that prior S phase synchronization could be a highly effective way of enhancing the efficacy of bolus or short infusions of flavopiridol in the clinical setting.

Topics: Aphidicolin; Apoptosis; Blotting, Western; Carcinoma, Small Cell; Caspase 3; Caspases; Cell Division; Enzyme Inhibitors; Flavonoids; Flow Cytometry; Humans; In Situ Nick-End Labeling; Lung Neoplasms; Membrane Potentials; Mitochondria; Piperidines; Poly(ADP-ribose) Polymerases; S Phase; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured; Tumor Stem Cell Assay

Among common fragile sites, fra(3)(p14.2) is the most expressed either spontaneously or after treatment with aphidicolin (APC) in lymphocyte cultures. Because recurrent chromosomal abnormalities involving the short arm of chromosome 3 in tumor tissue are present in various malignancies, including lung cancer, the induction of fra(3)(p14.2) elicited by APC was investigated with the aim of detecting possible interindividual polymorphism in its expression that might be relevant to predisposition toward cancer-related events. Thirty-four patients affected with various lung cancers (14 squamous cell carcinomas, 13 adenocarcinomas, and seven small cell carcinomas) and 14 controls (patients undergoing routine routine follow-up after coronary by-pass) were included in this study. The frequency of fra(3)(p14.2) expression was not significantly different among the patients grouped either by disease or by sex and age. It was estimated that fra(3)(p14.2) accounts for about 20% of total breakage in APC-treated lymphocyte cultures from the general population.

Topics: Adenocarcinoma; Aphidicolin; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Cells, Cultured; Chromosome Fragile Sites; Chromosome Fragility; Chromosomes, Human, Pair 3; Diterpenes; Humans; Karyotyping; Lung Neoplasms; Lymphocytes