EDITORIAL FOCUS
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
 |
ARTICLE |
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)-1
and tumor necrosis factor (TNF)-
. 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-
and IL-1
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-
and IL-1
. 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-
and IL-1
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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Am J Physiol Lung Cell Mol Physiol 280(5):L839-L840
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