Serial analysis of gene expression identifies putative metastasis-associated transcripts in colon tumour cell lines.

Abstract

We have used serial analysis of gene expression (SAGE) to identify gene expression differences between a primary colon tumour cell line (SW480) and an isogenic lymph-node metastasis cell line (SW620). Differential expression was confirmed for the following genes: keratin K5, cystatin S, serum amyloid A, the human homologue of yeast ribosomal S28 and the p32 subunit of human pre-mRNA splicing factor SF2. Expression of confirmed differences were also analysed in other metastatic cell lines.

Full text

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We have used the method of serial analysis of gene expression
(SAGE) (Velculescu et al, 1995) to identify changes in gene
expression during colorectal cancer progression. Although SAGE
has already been applied to an analysis of colorectal cancer (Zhang
et al, 1997), that study focused on gene expression differences
between normal and tumour tissues/cell lines. Our study focused
on identifying expression differences among highly expressed
genes between a primary colorectal tumour cell line (SW480) and
an isogenic lymph-node metastasis cell line (SW620) isolated
from the same patient (Leibovitz et al, 1976), thus providing infor-
mation on the gene expression changes which tumour cells may
undergo in order to become metastatic.
MATERIALS AND METHODS
Cell culture
The cell lines SW480, SW620, SW1116, LoVo, Colo201, T84
(ATCC, USA) and 498LI (Lieberman et al, 1991) were cultured
under conditions recommended by the supplier.
Serial analysis of gene expression (SAGE)
SAGE was carried out on the isogenic cell lines SW480 and
SW620 essentially as previously described (Velculescu et al, 1997;
Zhang et al, 1997). Clones containing at least 10–20 tags were
sequenced and analysed on an ALFexpress™
sequencer (Pharmacia, UK). Sequence files were analysed using
the SAGE software version 1.0 (Velculescu et al, 1995).
automated
Confirmation of differential expression
Semi-quantitative RT-PCR amplifications were based on
the method of He et al (1995) with modifications including a
Cy5-labelled primer in all PCR amplifications. Gene-specific
primer sequences are available from the authors upon request.
Cy5-labelled PCR products were analysed on an ALFexpress™
automated sequencer (Pharmacia). Northern blotting was carried
out by resolving total RNA on a 1% denaturing formaldehyde gel
and transferring onto Hybond N+(Amersham, UK) nylon
membrane by capillary blotting. Transcript sizes were estimated
by migration relative to 28S and 18S ribosomal RNA. Sequential
hybridizations were carried out overnight at 65°C with purified
cDNA fragments radiolabelled with [α-32P] dCTP by the random
prime method using the Rediprime® labelling kit II (Amersham,
UK).
RESULTS
Generation and analysis of SAGE libraries
SAGE was applied to the cell lines SW480 and SW620 as candi-
date pre-metastatic and metastatic cell populations. The total
number of ‘tags’ sequenced included 5079 tags from SW480 and
5044 from SW620. Comparison of tag abundances between the
two libraries showed that the majority of genes were expressed at
comparable levels between the two. As the number of tags
sequenced only numbered approximately 5000 from each cell line,
the significance of tag abundance differences had to be confirmed
by an independent method. We examined the expression of ten
putative differentially expressed genes identified from the SAGE
data (Table 1); only half were confirmed as true differences
between SW480 and SW620 by semi-quantitative RT-PCR or
Northern blot.
Analysis of expression differences by semi-quantitative
RT-PCR and Northern blotting
Analysis of the expression of myosin light-chain, high-mobility
group box protein (SSRP1), β-tubulin, hnRNP L and hnRNP C by
semi-quantitative RT-PCR revealed no significant differences in
expression between SW480 and SW620 (data not shown) and
thus were not analysed further. The human homologue of yeast
Serial analysis of gene expression identifies putative
metastasis-associated transcripts in colon tumour cell
lines
A Parle-McDermott, P McWilliam, O Tighe, D Dunican and DT Croke
Department of Biochemistry, Royal College of Surgeons in Ireland, 123 St Stephen’s Green, Dublin 2, Ireland
Summary We have used serial analysis of gene expression (SAGE) to identify gene expression differences between a primary colon tumour
cell line (SW480) and an isogenic lymph-node metastasis cell line (SW620). Differential expression was confirmed for the following genes:
keratin K5, cystatin S, serum amyloid A, the human homologue of yeast ribosomal S28 and the p32 subunit of human pre-mRNA splicing
factor SF2. Expression of confirmed differences were also analysed in other metastatic cell lines. © 2000 Cancer Research Campaign
Keywords: SAGE; colon; differential; metastasis; expression
725
Received 20 October 1999
Revised 11 April 2000
Accepted 11 May 2000
Correspondence to: DT Croke
British Journal of Cancer (2000) 83(6), 725–728
© 2000 Cancer Research Campaign
doi: 10.1054/ bjoc.2000.1330, available online at http://www.idealibrary.com on

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ribosomal S28, the p32 subunit of human pre-mRNA splicing
factor SF2, keratin type II K5, cystatin S and serum amyloid A
(SAA) all showed differential expression in the metastatic cell line
(SW620) relative to the primary tumour cell line (SW480) (Figure
1A, lanes 1 and 2) (Table 2). The cDNA products generated from
the RT-PCR analysis were purified and used for Northern blot
analysis (Figure 2). All five cDNA probes hybridized to transcripts
of the expected size and differential expression was also further
confirmed.
Analysis of expression in other cell lines
We examined the level of expression of the five confirmed differ-
entially expressed genes in other colon carcinoma cell lines by
semi-quantitative RT-PCR. These included a pair of cell lines:
SW1116, a human colon adenocarcinoma line and 498LI
(Lieberman et al, 1991), a metastatic variant of SW1116 selected
in vitro (Figure 1B, lanes 1 and 2) (Table 2). Three metastatic cell
lines, LoVo, Colo201 and T84, all originating from primary colon
tumours, were also examined (Figure 1A, lanes 3–5). The expres-
sion data is summarized in Table 2.
DISCUSSION
Our SAGE analysis has allowed a comparison between highly
expressed genes and, thus far, we have further examined the
expression of ten genes showing differential expression. We have
found that a SAGE analysis based on approximately 5000 tags per
cell type results in a number of false-positives, despite the ‘house-
keeping’ genes GAPDH and β-actin (Cleveland et al, 1980; Fort
et al, 1985) showing a similar tag number (Table 1). All of the
false-positives that we identified were represented by <10 tags in
726
A Parle-McDermott et al
British Journal of Cancer (2000) 83(6), 725–728
© 2000 Cancer Research Campaign
A
B
M - 1 2 3 4 5 M 1 2 3 4 5 M 1 2 3 4 5
20 2326
M - 1 2 3 4 5 M 1 2 3 4 5 M 1 2 3 4 5
20
M - 1 2 1 2 1 2
2225 28
202326
M - 1 2 1 2 1 2
2326
- 1 2 3 4 5 M 1 2 3 4 5 1 2 3 4 5
222528
SAA
SFp32
140bp
Keratin K5
146bp
Cystatin S
141bp
SAA
140bp
136bp
M - 1 2 3 4 5 M 1 2 3 4 5 M 1 2 3 4 5
1618 20
S28
homologue
M - 1 2 3 4 5 M 1 2 3 4 5 M 1 2 3 4 5
18
M - 1 2 1 2 1 2
18 2022
18 20 22
M - 1 2 1 2 1 2
20 22
- 1 2 3 4 5 M 1 2 3 4 5 M 1 2 3 4 5
18
20
22
S28
homologue
151bp
β-actin
151bp
SFp32
136bp
β-actin
109bp
109bp
M T
Keratin K5
Cystatin S
Serum Amyloid A
Homologue of
yeast ribosomal
S28
SFp32
β-actin
Figure 1
numbers written in bold refer to the cycle numbers sampled for each PCR amplification. β-actin was amplified in parallel as a control. (A) 1: SW480, 2: SW620,
3: LoVo, 4: Colo201, 5: T84. (B) 1: SW1116, 2: 498LI. Note: keratin K5 and cystatin S were not detected in SW1116 or 498LI and thus are not shown here.
Semi-quantitative RT-PCR products resolved by electrophoresis on 12% polyacrylamide gels. M: molecular weight marker; – : negative control. The
Figure 2
genes identified by SAGE. T refers to the primary tumour cell line SW480
and M refers to the metastatic cell line SW620. The blot was probed with β-
actin to control for equal loading and transfer.
Northern blot analysis of the confirmed differentially expressed

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abundance. Therefore, increasing the number of tags sequenced is
likely to reduce or eliminate the number of false-positives.
We have confirmed differential expression of five genes
between SW480 and SW620 (Table 2). Cystatin S and keratin type
II K5 were not expressed at a significant level in the four other
metastatic cell lines that we examined (LoVo, Colo201, T84 and
498LI) or in the primary tumour cell line SW1116. Cystatin S is a
member of the cysteine proteinase inhibitor superfamily (Bobek
et al, 1991) and other members, such as cystatin M, have been
previously associated with metastatic spread (Sotiropoulou et al,
1997). Keratin K5 is a type II cytoskeletal intermediate filament
protein (Fuchs and Weber, 1994) and the cytoskeletal structures
are known to play a major role in cell motility and invasion, prolif-
eration, differentiation and in the transduction of extracellular
signals (Sherbet and Lakshmi, 1997).
Serum amyloid A (SAA) showed varying degrees of loss of
expression in all of the metastatic cell lines that we examined.
SAA is an acute-phase reactant and is found in the circulation as
an apolipoprotein bound to HDL (high density lipoprotein)
(apoSAA) (Strachan et al, 1989). The potential role that SAA may
have in metastasis is currently unclear but the observed increase in
expression of SAA in response to p53-induced apoptosis (Polyak
et al, 1997) may be significant.
We also observed increased expression of the p32 subunit
precursor of pre-mRNA splicing factor SF2, and the human homo-
logue of yeast ribosomal S28 in the metastatic cell line(s) relative
to their primary tumour cell line counterpart(s) (Table 2). The
interaction of p32 with the pre-mRNA splicing factor SF2 (Krainer
et al, 1991; Honore et al, 1993) and its ability to bind hyaluronic
acid (HA) (Deb and Datta, 1996) suggests a potential role in
metastasis. Increased expression of the human homologue of yeast
ribosomal S28 has also been found in colon tumours as compared
to normal tissue (Zhang et al, 1997). This indicates that increased
expression of S28 may be correlated with colon cancer progres-
sion.
In conclusion, we have shown here that analysis of a moderate
number of SAGE tags (approximately 10 000 in total) has allowed
us to identify a number of differentially expressed genes that may
be involved in colon tumour metastasis. However, our analysis has
also shown that, with a moderate level of SAGE tags, comparison
of the gene expression profiles must be interpreted with caution as
tag abundances <10 do not consistently represent true differences
in expression. This agrees with another recent SAGE analysis of
endothelial cells which consisted of over 12 000 tags (de Waard et
al, 1999). The five differentially expressed genes that we have
confirmed have not been implicated in colon tumour progression
Differential gene expression in colon tumour cell lines
727
British Journal of Cancer (2000) 83(6), 725–728
© 2000 Cancer Research Campaign
Table 1
Subset of differentially expressed tags further analysed
SAGE Tag T/MGeneAccession Number
CTGTTGGTG
GCCCCTGCT
GTACACACA
GTGCTGAAT
GTGCGGAGG
TACTCTTGG
TGAGGCCAG
ATAGACATA
AACGACCTC
TCAAATGCA
*TACCATCAA
*GCTTTATTT
1/17
18/1
15/1
8/1
7/0
0/7
6/0
1/6
1/6
0/5
32/26
9/8
Human homologue of yeast ribosomal S28
Keratin type II K5
Cystatin S
Myosin light-chain mRNA
Serum amyloid A
hnRNPL
High-mobility group box (SSRP1) mRNA
p32 subunit of Splicing Factor 2
β-tubulin
hnRNP C
Glyceraldehyde 3-phosphate dehydrogenase
β-actin
D14530
M21389
X54667
U02629
X51439
X16135
M86737
M69039
V00599
M16342
J02642
X00351
T/M is the ratio of tags from the primary tumour cell line SW480 (T) compared to the metastatic cell line SW620 (M).
Genes written in bold were confirmed by independent methods (see text). *The ‘housekeeping’ genes GAPDH and β-
actin showed a similar tag abundance as expected
Table 2
Fold differences in expression between primary tumour and metastatic cell lines
Gene transcript SW480
SW620LoVo Colo201T84
SW1116
498LI
Fold decreased expression in metastatic cell linesa
Keratin type II K5
Cystatin S
Serum amyloid A
Fold increased expression in metastatic cell linesb
p32 subunit of splicing factor 2
Human homologue of yeast ribosomal S28
1.0
1.0
1.0
nd
nd
nd
nd
nd
16
nd
nd
8.6
nd
nd
nd
nd
nd
1.0
nd
nd
1.4
1.0
1.0
2.0
2.2
1.6
6.2
0.8
0.9
0.7
0.6
1.0
1.0
2.2
0.7
Cell lines written in bold are metastatic and those written in roman are the primary colon tumour cell lines. Fold differences were calculated based on β-
actin ratios and relative to expression in primary tumour cell lines. The results are based on repeated experiments with an overall average standard error
of the mean of 4.1%. Cell lines SW620, LoVo, Colo201 and T84 are described relative to SW480, and 498LI is described relative to SW1116; nd = not
detected; aFold differences are described as decreased expression in the metastatic cell lines relative to the primary tumour cell lines; bFold differences
are described as increased expression in the metastatic cell lines relative to the primary tumour cell lines

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previously and may contribute to our understanding of metastasis.
However, it is clear that our study requires further analysis in order
to show a direct association between the identified differentially
expressed genes and the metastatic phenotype.
ACKNOWLEDGEMENTS
We wish to thank Drs Jill Powell (ICRF, UK) and Victor Veculescu
(Johns Hopkins, MD, USA) for helpful advice in implementing the
SAGE protocol. We also thank Dr Noel Williams (PA, USA) for his
continued interest and support of the project and Dr Meenhard
Herlyn (Wistar Institute, PA, USA) for the kind gift of 498LI. This
work was funded by the Health Research Board of Ireland.
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© 2000 Cancer Research Campaign