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CORRESPONDENCE Two human stromal cell lines, HS-5 and HS-27a, represent
functionally distinct components of the bone marrow
microenvironment.1,2 HS-27a supports cobblestone area
formation by early hematopoietic progenitors, whereas HS-5 secretes
multiple cytokines that support the proliferation of committed
progenitors. These cell lines, which are available from the American
Type Culture Collection (ATCC, Manassas, VA), have been distributed to
research groups worldwide for use as a tool to understand interactions
between hematopoietic cells and their microenvironment. We have
recently used DNA microarray technology to characterize and compare the expression of over 17 000 genes in these cell lines. Briefly, microarray construction and hybridization protocols were
modified from Marton et al.3 The microarrays were
constructed using a set of more than 17 000 sequence-verified clones
from Research Genetics (Huntsville, AL). Of the 17 761 features
(spots) on the microarray, 186 are control nonexpressed or nonhuman
sequences or housekeeping genes. UniGene cluster IDs could be assigned
by GenBank to 16 592 of the features as of June 1, 2001 (UniGene Build
133), indicating that they are representatives of nonredundant unique
genes. Many have been functionally characterized, and chromosomal location and tissue expression patterns are known for others. Total RNA
was isolated from semiconfluent cultures and reverse-transcribed into
cDNA in a nucleotide mix containing amino-allyl deoxyuracil triphosphate (dUTP). The cDNA from stromal cells and Universal RNA
(Stratagene, La Jolla, CA) was covalently coupled separately with Cy5
and Cy3 monoreactive fluors, respectively. The Universal RNA consists
of a mixture of RNA from 10 different human cell lines with a broad
expression coverage of over 80% of the sequences on the array,
allowing comparison of expression patterns of multiple different
samples of different origin. The Cy5- and Cy3-labeled cDNAs were
combined for hybridization to the microarray. Fluorescent array images
were collected for both Cy3 and Cy5, and image-intensity data
were extracted and analyzed to obtain expression ratios to Universal
RNA for each stromal cell line. From these the expression in the 2 lines could be compared. Here, we present tables identifying the genes with greater than
10-fold and significant differences (Student t test,
P < .05) in expression between the 2 cell lines (Tables
1 and
2). The interleukin-7 receptor, among
the genes with much higher expression in HS-27a, is the subject of a
separate manuscript appearing in this issue (Iwata et al, page
1318).4 A summary table presenting the complete
expression profile for each cell line, as well as a comparison between
them, for all 17 000 sequences, is available at
http://parma.fhcrc.org/MIwata. This site also presents technical details of array spotting, hybridization, cDNA synthesis, and fluor-coupling procedures. The entire raw data set, consisting of 4 microarrays per cell line, is publicly available at
http://www.ncbi.nlm.nih.gov/geo/.
Gene expression differences in cytokines/chemokines, G-protein signaling molecules, and multiple extracellular matrix proteins add to the known protein and functional characterization of the lines, leading to new insight into the differences in their support function for hematopoietic progenitors.
Lynn Graf, Mineo Iwata, and Beverly Torok-Storb Supported in part by grants HL62923, CA15704, and DK56465 from the National Institutes of Health, Bethesda, MD. References
1.
Roecklein BA, Torok-Storb B.
Functionally distinct human marrow stromal cell lines immortalized by transduction with the human papilloma virus E6/E7 genes.
Blood.
1995;85:997-1005 2. Torok-Storb B, Iwata M, Graf L, Gianotti J, Horton H, Byrne MC. Dissecting the marrow microenvironment. Ann N Y Acad Sci. 1999;872:164-170[CrossRef][Medline] [Order article via Infotrieve]. 3. Marton MJ, Derisi JL, Bennett HA, et al. Drug target validation and identification of secondary drug target effects using DNA microarrays. Nat Med. 1998;4:1293-1301[CrossRef][Medline] [Order article via Infotrieve].
4.
Iwata M, Graf L, Awaya N, Torok-Storb B.
Functional interleukin-7 receptors (IL-7Rs) are expressed by marrow stromal cells: binding of IL-7 increases levels of IL-6 mRNA and secreted protein.
Blood.
2002;100:1318-1325   CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
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