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(Chest. 2004;125:110S-111S.)
© 2004 American College of Chest Physicians

Improvement and Application of Fluorescence Inter-Simple Sequence Repeat Polymerase Chain Reaction for the Study of Subclonal Growths in Lung Epithelial Cell Populations*

Tao Lu, PhD and Walter N. Hittelman, PhD

* From the Department of Experimental Therapeutics, University of Texas MD Anderson Cancer Center, Houston, TX.

Correspondence to: Tao Lu, PhD, Department of Experimental Therapeutics, Box 19, University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030; e-mail: tlu{at}mdanderson.org

Chromosome in situ hybridization and loss of heterozygosity analyses on bronchial biopsy specimens of current and former smokers have demonstrated the presence of clonal and subclonal outgrowths throughout the exposed lung epithelium. Since high frequencies of clonal outgrowths have been detected in the normal/premalignant epithelium adjacent to lung tumors, it is postulated that the frequency of subclonal outgrowths may provide a risk marker for lung cancer development. We therefore examined a quantitative technique with sufficient dynamic range (ie, inter-simple sequence repeat polymerase chain reaction [PCR]) for its ability to detect subclonal outgrowths in lung epithelial cell populations.

To improve the reproducibility and quantitative aspects of this method, we used fluorescent dye-labeled primers for the inter-simple sequence repeat (FISSR)-PCR reaction, separated and quantified the PCR products on a sequencer (model ABI 377l), and analyzed the results using computer software (GeneScan; Foster City, CA). To test the reproducibility of the sequencer, aliquots of single FISSR-PCR reactions were run in separate lanes and were shown to give reproducible band patterns. To test the reproducibility of the FISSR-PCR reaction, we carried out triplicate reactions with the same DNA source and showed reproducible band patterns. To determine its sensitivity for detecting clonal evolution during lung tumorigenesis, we quantified the number of genetic alterations detected by FISSR-PCR in a bronchial epithelial cell progression model system consisting of four evolved lung cell lines, including large T-antigen immortalized normal human bronchial epithelial cells (BEAS-2B cells), grown in vivo (1799 cells), treated with cigarette smoke condensate (1198 cells), and evolved to cancer (1170I cells) [originally derived by Klein-Szanto et al1]. Using two groups of primers, we identified nine alterations between the BEAS2B and 1799 cells, 3 additional changes to the 1198 cells, and 4 additional changes to tumorigenic 1170I cells. To determine the ability to detect subclonal populations, we isolated single-cell clones from the BEAS2B and 1170I populations, and demonstrated the presence of distinct subclones. To determine the sensitivity of this technique for detecting subclones, we carried out mixing experiments of subclonal fractions, and demonstrated a quantitative relationship between relative peak height and subclonal fraction.

These results suggest that FISSR-PCR has promise for quantifying the extent of subclonal outgrowth in cell populations, and that it may prove useful in the assessment of lung cancer risk when applied to the analysis of the bronchial epithelium of current and former smokers.


   Footnotes
 
Abbreviations: FISSR = fluorescent dye-labeled primers for the inter-simple sequence repeat; PCR = polymerase chain reaction

This research was supported in part by grants DAMD17–02-1–0706, CA-68437, and CA70907.


   References
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 References
 

  1. Klein-Szanto, AJP, Iizasa, T, Momiki, S, Garcia-Plazzo, I, Caamano, J, Metcall, R, Welsh, J, Harris, CC (1992) A tobacco-specific N-nitrosamine or cigarette smoke condensate causes neoplastic transformation of xenotransplanted human bronchial epithelial cells. Proc Natl Acad Sci U S A 89,6693-6697[Abstract/Free Full Text]




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