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Fishing for inflammatory cytokine-inducible genes with an old trick

Reen Wu and Yin Chen

Center for Comparative Respiratory Biology and Medicine, University of California, Davis, California 95616


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DIVERSIFIED RESPONSES in respiratory inflammation are normally a reflection of differential gene expression by various cell types in response to inflammatory cytokines such as interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha . Airway epithelium is one of the primary targets of these inflammatory cytokines. The identification of these genes that are altered in inflammatory airway epithelium can lead to a better understanding of how these cells react and behave. With this in mind, Cooper et al. (3) have an article in this issue of the American Journal of Physiology-Lung Cellular and Molecular Physiology describing their use of an old trick approach to identify genes in which expression in cultured human bronchial epithelial cells is induced by a TNF-alpha and IL-1beta mixture. The approach is relatively high throughput, with a finding of 93 genes potentially induced in vitro in one round of screening. Among these genes, 23 of 31 (74%) tested were confirmed by "virtual" Northern blot hybridization and 13 of 19 (68%) tested were confirmed by traditional Northern blot hybridization. Thus it is possible that 70% of these 93 genes identified in a single round of screening are induced in airway epithelial cells by TNF-alpha and IL-1beta . This is probably the first big catch for this type of gene-fishing expedition. The method is a cDNA representational difference analysis, which was introduced in 1994 (4). This method is a PCR-based amplification and subtraction approach, with different adapters for tester and driver. The trick is these adapters that have been specifically designed and must be purified by HPLC (11). Normally, it takes two rounds of enrichment to generate a difference product for further screening on gene filters (e.g., GeneFilter series and other array products) or for a direct sequencing of each individual clone. Accordingly, it is claimed that the method is able to identify an induced message at 1 copy/cell. Thus this method is a powerful tool for various gene-fishing studies.

Among those sequences selected and positively identified as inducible genes in this report (3), several have been reported before. These genes are related to protease/antiprotease, membrane receptor, apoptosis, and cytokines, which include the induction of TNF-alpha and IL-1beta themselves. These results would confirm the validity of this approach. The most interesting finding is the identification of three novel genes induced by inflammatory cytokines. One sequence is in the expressed sequence tag (EST) database, whereas the other two sequences (EXER102 and EXR107) have not been reported before. Apparently, these novel genes are all expressed in the lungs. It will be very informative if the expression of these genes is located in the inflammatory airway tissues. Obviously, more study is needed to elucidate the functions of these novel genes in airway inflammation. However, there are also some drawbacks in this study. Some inducible genes, such as IL-8, knowingly induced by inflammatory cytokines, were not found. The most serious drawback is that 20% of the selected genes are false positive. This is because these genes contain adapter sequences such as R-Bgl-24 (3, 11), which are PCR by-products. This is obviously of great concern.

The development of an efficient method to isolate and identify these differentially expressed genes will help in understanding the complexity and diverse response associated with respiratory inflammation. The same is true for various biological processes. Conventional approaches such as differential (2, 9) and subtractive (5) hybridization techniques have been successfully used to isolate genes of differential expression. However, the screening procedures are very labor intensive and time consuming. The mRNA differential display (mDD) technique (6, 7) has been used successfully to identify differentially expressed gene transcripts by directly comparing reverse-transcribed RNA species. The major drawback for mDD is the relatively high percentage of false-positive results that may be alleviated by the difficulty in designing primers and the PCR conditions for various transcripts. The other drawback for mDD is the short DNA fragment obtained, which is largely at the 3'-end. Further cloning and DNA sequencing are required to obtain the basic information about the nature of the selected gene. For last several years, high-density cDNA arrays on glass slides (8, 10, 12) or nylon membranes (1) have been developed for high-throughput differential hybridization. The concept is based on hybridization kinetics if the hybridization is carried out in such a small area with excessive targeted DNA molecules. Under such conditions, the first-order kinetics of hybridization can be achieved and the expression level for all the messages can be quantified according to their abundance. Such a concept may be feasible in the future. With completion of the human genomic sequence and gene identification, it is possible to develop such a "gene chip" containing all the human genes in a small designated surface. Thus one can analyze the expression of all the human genes simultaneously in a single hybridization step. The amount of information generated will be enormous. Such a development will further advance our knowledge of understanding how cells respond and behave in such a complex interaction associated with airway inflammation.


    FOOTNOTES

Address for reprint requests and other correspondence: R. Wu, Center for Comparative Respiratory Biology and Medicine, Univ. of California, Davis, CA 95616 (E-mail: rwu{at}ucdavis.edu).


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1.   Chen, JJW, Wu R, Yang PC, Huang JY, Sher YP, Han MH, Kao WC, Lee PJ, P, Chiu TF, Chang F, Chu YW, Wu CW, and Peck K. Profiling expression patterns and isolating differentially expressed genes by cDNA microarray system with colorimetry detection. Genomics 51: 313-324, 1998[ISI][Medline].

2.   Cochran, BH, Reffel AC, and Stiles CD. Molecular cloning of gene sequences regulated by platelet-derived growth factor. Cell 33: 939-947, 1983[ISI][Medline].

3.   Cooper, P, Potter S, Mueck B, Yousefi S, and Jarai G. Identification of genes induced by inflammatory cytokines in airway epithelium. Am J Physiol Lung Cell Mol Physiol 280: L841-L852, 2001[Abstract/Free Full Text].

4.   Hubank, M, and Schatz DG. Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Res 22: 5640-5648, 1994[Abstract].

5.   Lee, SW, Tomasetto R, and Sager R. Positive selection of candidate tumor-suppressor genes by subtractive hybridization. Proc Natl Acad Sci USA 88: 2825-2829, 1991[Abstract].

6.   Liang, P, Averboukh L, and Pardee A. Distribution and cloning of eukaryotic mRNAs by means of differential display: refinements and optimization. Nucleic Acids Res 21: 3269-3275, 1993[Abstract].

7.   Liang, P, and Pardee AB. Differential display of eukaryotic messenger RNA by means of polymerase chain reaction. Science 257: 967-971, 1992[ISI][Medline].

8.   Lockhart, DJ, Dong H, Byrne MC, Follettie MT, Gallo MV, Chee MS, Mittmann M, Wang C, Kobayashi M, Horton H, and Brown EL. Expression monitoring by hybridization to high-density oligonucleotide arrays. Nat Biotechnol 14: 1675-1680, 1996[ISI][Medline].

9.   Luo, G, An G, and Wu R. A PCR differential screening method for rapid isolation of clones from a cDNA library. Biotechniques 16: 672-675, 1994.

10.   Nguyen, C, Rocha D, Granjeaud S., Baldit M, Bernard K, Naquet P, and Jordon BR. Differential gene expression in the murine thymus assayed by quantitative hybridization of arrayed cDNA clones. Genomics 29: 207-215, 1995[ISI][Medline].

11.   O'Neill, MJ, and Sinclair AH. Isolation of rare transcripts by representational difference analysis. Nucleic Acids Res 25: 2681-2686, 1997[Abstract/Free Full Text].

12.   Schena, M, Shalon D, Davis RW, and Brown PO. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270: 467-470, 1995[Abstract].


Am J Physiol Lung Cell Mol Physiol 280(5):L839-L840
1040-0605/01 $5.00 Copyright © 2001 the American Physiological Society




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