Download Up-regulation of CIR1/CROC1 expression upon cell

Oncogene (1998) 17, 1321 ± 1326
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SHORT REPORT
Up-regulation of CIR1/CROC1 expression upon cell immortalization and
in tumor-derived human cell lines
Libin Ma1,4, Stacey Broom®eld2, Carol Lavery1, Stanley L Lin3, Wei Xiao2 and Silvia Bacchetti1
1
Cancer Research Group, Department of Pathology, HSC-4H30, McMaster University, 1200 Main Street West, Hamilton, Ontario
L8N 3Z5, Canada; 2Department of Microbiology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK S7N 5E5,
Canada; 3Department of Psychiatry, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey
08854, USA
Acquisition of the immortal phenotype by tumor cells
represents an essential and potentially rate-limiting step
in tumorigenesis. To identify changes in gene expression
that are associated with the early stages of cell
immortalization, we compared genetically matched pairs
of pre-immortal and immortal human cell clones by
mRNA di€erential display. Two transcripts, denoted
CIR1 and CIR2, were identi®ed which were up-regulated
in immortal cells. Sequence analysis revealed CIR1 to be
identical to the recently cloned CROC1/UEV-1 gene,
whereas CIR2 corresponds to an as yet uncharacterized
1.2 kb mRNA. A 5 ± 6-fold elevation in CIR1/CROC1
expression and a 2 ± 3-fold elevation in CIR2 expression
were observed in SV40-transformed human enbryonic
kidney cells immediately following proliferative crisis,
suggesting a potential role for these genes in immortalization. Expression of CIR1/CROC1 was found to be
elevated also in a variety of immortal human tumorderived cell lines, as compared to their normal tissue
counterparts. These results are compatible with induction
of CIR1/CROC1 being an early event in the acquisition
of immortality and with a role for this gene in the
immortal phenotype of tumor cells.
Keywords: CIR1; CROC1; UEV-1; immortalization;
tumorigenesis di€erential display
Normal somatic human cells have a ®nite replicative
life span and are ultimately subject to an irreversible
arrest of cell division, called cellular senescence
(Hay¯ick and Moorhead, 1961; Goldstein, 1990),
whereas many human tumor cells display an unlimited
cell division capacity when grown in vitro, i.e. they
exhibit an immortal phenotype (Stamps et al., 1992).
Acquisition of immortality by pre-neoplastic or
neoplastic cells is critical to continued tumor growth
and metastatic spread in neoplasia. Transformation of
human cells in vitro, by DNA tumor viruses or other
agents, can eciently abrogate senescence and confer
an extended life span (Girardi et al., 1965; Shay et al.,
1991; Ozer et al., 1996). However, transformed cells
eventually enter a proliferative crisis, from which rare
clonal populations of immortal cells arise (Girardi et
Correspondence: S Bacchetti
Received 21 February 1998; revised 21 April 1998; accepted 21 April
1998
al., 1965). Cell culture models indicate that in order to
become immortal human cells must overcome the
barriers of both senescence and crisis (or Mortality
stages 1 and 2) (Shay et al., 1991).
Genetic analysis has suggested that immortalization
is the result of loss of gene function(s) that are essential
for senescence. Cell hybrids between immortal and
normal cells revealed the recessive nature of immortalization, and pair-wise cell fusion analysis between
tumor-derived and immortalized human cells suggested
the existence of di€erent complementation groups
(Smith and Pereira-Smith, 1996). Re-introduction of
individual human chromosomes into immortal cells can
result in growth inhibition and the re-appearance of a
senescent-like morphology (Smith and Pereira-Smith,
1996). In some cases, more than one chromosome has
been shown capable of abrogating the immortal
phenotype in a given cell line (Sasaki et al., 1994),
suggesting multiple pathways of cell senescence and
immortalization. On the other hand, transfection of
oncogenes into cells which undergo senescence has
been shown to confer immortality (Edwards, 1993;
Harrington et al., 1994), indicating that de novo or
elevated expression of speci®c genes could also be
involved in immortalization. Moreover, elevated
expression of immortalization-related genes is needed
for either the prevention of the decline in, or the
maintenance of proliferative capacity. For example,
immortality is usually associated with the acquisition
of telomerase activity which compensates with the
proliferation-related decline in telomere length (Counter et al., 1992; Kim et al., 1994; Bacchetti, 1996;
Harley and Sherwood, 1997). Identi®cation of such
genes and their regulation can be expected to provide
insight into mechanisms of cell immortalization and
aging.
Several laboratories, using di€erent approaches such
as subtractive hybridization, have identi®ed a number
of genetic alterations in immortal cells (Imai and
Takano 1992; Satoh et al., 1994; Pardinas et al., 1997),
most of them of unknown function in immortalization.
These studies focused primarily on the identi®cation of
genes that are lost or down-regulated after immortalization. We have used mRNA di€erential display
(Liang and Pardee, 1992; Liang et al., 1993) to
compare gene expression in genetically matched preimmortal and immortal human cells. We report the
identi®cation of two transcripts, designated CIR1 and
CIR2 (for Cell Immortalization-Related genes), whose
expression is up-regulated in immortal cells. While
CIR2 represents a novel cDNA, CIR1 is identical to
CIR1/CROC1 over-expression in immortal cells
L Ma et al
1322
the recently cloned gene, CROC1 (also called UEV-1),
which was originally identi®ed on the basis of its
capacity to activate c-fos gene transcription (Rothofsky
and Lin, 1997), and as a transcript that was downregulated upon di€erentiation of the human HT-29-M6
colon carcinoma cell line (Sancho et al., 1998). The
location of this gene at 20q13.2 (Sancho et al., 1998), a
region that is frequently highly ampli®ed in many types
of human cancers (Tanner et al., 1994), suggests that
its overexpression may be instrumental to tumorigenesis. Consistent with a role in immortalization, we have
observed elevated expression of CIR1/CROC1/UEV1
in several tumor-derived cell lines.
Results and discussion
Strategy for di€erential display of cell immortalizationrelated (CIR) genes
To identify cellular genes that are regulated during the
process of cell immortalization and potentially contribute to the immortal phenotype, we have utilized
di€erential display of mRNAs (Liang and Pardee,
1992; Liang et al., 1993) to compare pre-immortal
clonal populations of SV40-transformed human
embryonic kidney (HEK) cells with their corresponding immortal clones isolated following proliferative
crisis (Stewart and Bacchetti, 1991; Counter et al.,
1992; S Bacchetti, unpublished results). Such matched
pairs of pre-immortal and immortal cells are particularly suitable for the comparison of gene expression,
since they were derived from the same transformed cell
and thus should be essentially isogenic. Previously, we
had shown that in the pre-immortal HA1 clone,
telomere length declines with cell division and
telomerase remains inactive until the cells reach
proliferative crisis, while in immortal HA1 cells
telomere length stabilizes and telomerase is activated
(Counter et al., 1992; Avilion et al., 1996). Recently,
we also found that up-regulation of the mRNA
encoding the catalytic subunit of human telomerase,
hTERT (Meyerson et al., 1997; Weinrich et al., 1997;
Nakamura and Cech, 1998), coincides with the
emergence of immortal HA1 cells (Meyerson et al.,
1997).
Stringent criteria were utilized to de®ne and select
immortalization-related genetic alterations. First, we
chose to compare pre-immortal and immortal cell
populations just prior to and immediately after
proliferative crisis, to eliminate from the analysis
genetic alterations that occurred upon transformation
but are not necessarily related to cell immortalization.
To further minimize random changes in gene
expression that may arise during growth of transformed cells, we included in the analysis three HA1
subclones (A33, A34 and C12) that were obtained by
limiting dilution at very early times after transformation. Like the parental cells, these subclones underwent proliferative crisis and eventually gave rise to
immortal cells (S Bacchetti, unpublished results).
Lastly, only changes in gene expression that were
consistent in all four clones, when analysed by
di€erential display and/or Northern hybridization,
were considered as indicative of potential Cell
Immortalization-Related (CIR) genes.
Up-regulation of CIR1 and CIR2 expression in immortal
cells
Using 16 arbitrary primers in combination with three
one-base anchored primers (see Figure 1), an average of
*100 ± 120 cDNA bands were displayed in each lane.
Thus, a total of approximately 5000 cDNAs from each
cell population, representing roughly 10 ± 15% of the
transcribed genes (Liang and Pardee, 1992), were
directly compared on display gels. We identi®ed 16
candidate bands that di€ered in intensity between preimmortal and immortal HA1 and A33 populations (e.g.
see Figure 1). Of these, only two cDNA fragments,
designated CIR1 and CIR2 (Figure 1a,b), were
consistently up-regulated in immortal cells on display
gels and were di€erentially expressed in all four cell lines
as judged by Northern analysis (Figure 2, compare lanes
1, 3, 5 and 7 to lanes 2, 4, 6 and 8). Di€erential
expression of the remaining cDNAs could not be
con®rmed on Northern blots: these cDNAs either did
not produce detectable signal, or were not di€erentially
expressed, or exhibited di€erential expression only in a
subset of cell lines (data not shown). Others have also
reported a comparable high percentage of false positive
signals on display gels (Liang et al., 1993).
On display gels, the CIR1 band (*650 bp in size) was
visible in pre-immortal HA1 and A33 cell populations
but its intensity increased in immortal cells (Figure 1a,
lanes 1 and 2 versus 3 and 4). The CIR2 band
(*500 bp) was barely visible in pre-immortal cells but
became readily detectable after immortalization (Figure
1b, lanes 1, 2 versus 3, 4). Northern blot analysis
con®rmed these observations (Figure 2). The CIR1
fragment detected a doublet of mRNA species of
approximately 2.0 ± 2.1 kb, which corresponds to two
isoforms of the CROC1 gene (see below) and the CIR2
probe hybridized to a single mRNA species of &1.2 kb
(Figure 2). Based on quantitation by PhosphorImager
analysis and normalization for RNA loading by
GAPDH control hybridization, we estimated the
increase in expression level upon immortalization to
be 5 ± 6-fold for CIR1 and 2 ± 3-fold for CIR2. The
extent of up-regulation of CIR1 and CIR2 in immortal
cells was reproducibly observed on independent Northern blots (data not shown).
CIR1 is identical to CROC1
The CIR1 and CIR2 fragments were cloned (see Figure
3); sequencing of four independent clones of each
revealed that they contain identical CIR1 or CIR2
inserts. When used as probes on Northern blots, the
cloned fragments detected the same bands and revealed
the same pattern of di€erential expression in preimmortal versus immortal cell populations (data not
shown), thus ensuring the authenticity of the cloned
fragments. The sequences of the CIR1 and CIR2 clones
are shown in Figure 3. Both fragments were ¯anked by
sequences corresponding to the one-base anchored
oligo(dT) primer and the arbitrary primer used for
di€erential display, and putative polyadenylation signal
sequences were identi®ed in both cases. Homology
searches through the NIH GenBank and the EMBL
databases, using the BLAST and FASTA programs,
revealed no signi®cant homology with any published
genes in the case of the CIR2 sequence, although a
CIR1/CROC1 over-expression in immortal cells
L Ma et al
number of human expressed sequence tags (ESTs) of
unknown function that are identical or almost identical
to CIR2 were identi®ed (e.g., Genbank accession nos.
AA156251, AA075294 and W39392). Further analysis
of CIR2 was not pursued. On the other hand, the CIR1
sequence was found to perfectly match the 3'-end
sequences of two GeneBank entries previously identi®ed as the CROC1 gene (CROC-1A, accession no.
U39360, and CROC-1B, accession
(Rothofsky and Lin, 1997).
no.
U39361)
Expression of CIR1/CROC1 in human tissues and
tumor-derived cell lines
The CROC1 genes were originally isolated on the basis
of their ability to mediate transcriptional activation of
Figure 1 Representative banding patterns on mRNA di€erential display gels showing up-regulated PCR fragments in immortal
cells. CIR1 (a) and CIR2 (b) are indicated by arrowheads. Lanes 1 and 2, RNA from pre-immortal A33 and HA1 cells; lanes 3 and
4, RNA from immortal HA1 and A33 cells. Genetically matched pairs of pre-immortal and immortal HA1 cells were established
from SV40-transformed human embryonic kidney (HEK) cells and cultured as described previously (Stewart and Bacchetti, 1991;
Counter et al., 1992). Three HA1 subclones, A33, A34, and C12 were obtained by limiting dilution of HA1 cells at a very early stage
after SV40 transformation (S Bacchetti, unpublished data). Total cellular RNA was isolated using the TRIzol reagent according to
the manufacturer's guidelines (Gibco/BRL Life Technologies) and was treated with RNase-free DNase I (Pharmacia) to remove
contaminating DNA (Liang et al., 1993). RNA was quantitated by UV absorbance. Di€erential display was performed essentially as
described (Liang and Pardee, 1992; Liang et al., 1993). Brie¯y, an aliquot of DNA-free total RNA (0.2 mg in 20 ml) was reverse
transcribed with 100 units of Mo-MLV reverse transcriptase for 1 h at 378C, in the presence of 20 mM deoxynucleotide triphosphates
and 0.2 mM H-T11M (5'-AAGCTTTTTTTTTTTM-3', where M is G, C, or A). Two ml of the resultant cDNA was ampli®ed by PCR
in a 20 ml reaction volume, with 2.0 mM deoxynucleotide triphosphates, 0.2 mM downstream H-T11M primer, 0.2 mM upstream
arbitrary primers (H-AP1-8 and H-AP41-48; RNAimage kits No. 1 and 6; GeneHunter, Nashville, TN), in the presence of
[a-33P]dATP (2000 Ci/mmol, DuPont/NEN) and 1 unit of Taq DNA polymerase (Boehringer Mannheim). The 5' arbitrary primer
that gave CIR1 and CIR2 products was H-AP42 (5'-AAGCTTTGCACCG-3') and 3' anchored primers were H-T11A and H-T11C,
respectively. PCR conditions were 948C for 5 min, followed by 40 cycles at 948C for 30 s, 408C for 2 min and 728C for 30 s. A ®nal
extension was carried out at 728C for 5 min. An aliquot of each PCR reaction (5 ml) was analysed on a 6% polyacrylamide DNA
sequencing gel. Bands on the display gels that showed di€erential intensity in pre-immortal and immortal cells were visualized by
exposing the dried gels to X-ray ®lms, excised from the gel, and soaked in water to elute the DNA, which was then reampli®ed by
PCR using the same primer set (Liang et al., 1993)
1323
CIR1/CROC1 over-expression in immortal cells
L Ma et al
1324
Figure 2 Up-regulation of CIR1 and CIR2 in immortal cells.
The blot was sequentially hybridized to reampli®ed PCR
fragments corresponding to CIR1 (upper) and CIR2 (middle)
and to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
cDNA. Lanes 1, 3, 5 and 7, RNA from pre-immortal HA1,
A33, A34 and C12 cells; lanes 2, 4, 6 and 8, RNA from immortal
HA1, A33, A34 and C12 cells. Fifteen mg of total RNA was size
fractionated on a 1.2% agarose-formaldehyde gel and transferred
onto ZetaProbe GT membranes (BioRad) by standard capillary
blotting techniques in 106SSC. Speci®c cDNA probes were
labeled with [a-32P]dCTP using a random priming DNA labeling
kit (Boehringer Mannheim). The ®lters were hybridized overnight
at 428C in 50% formamide, 56SSC, 0.5 M sodium phosphate
bu€er and 1% SDS, washed twice in 26SSC/0.1% SDS for
30 min followed by 0.16SSC/0.1% SDS for 15 ± 30 min at 558C.
Quantitation of mRNA levels was done by PhosphorImager
(Molecular Dynamics). Total counts of the CIR1 or CIR2 band
(minus background) were divided by the total count of the
GAPDH band (minus background) to normalize for di€erences in
lane loading
Figure 3 Nucleotide sequences of CIR1 and CIR2 cDNAs.
Primers originally used for di€erential display are depicted in
lowercase for CIR1 (a) and CIR2 (b) (Genbank accession no.
AFO 49671 and AFO 49672, respectively). Putative polyadenylation signal sequences are in bold. The reampli®ed CIR1 and CIR2
fragments were cloned into pBluescript at the EcoRV site after the
addition of T overhangs. Four independent clones of each were
sequenced and all four contained identical CIR1 or CIR2 inserts
the human c-fos promoter from its direct repeat
enhancer sequences (Rothofsky and Lin, 1997).
CROC-1A and CROC-1B are predicted to encode
two proteins of 170 and 221 amino acids, respectively,
that are identical in their carboxy-terminal 140 residues
(Rothofsky and Lin, 1997). An isoform of CROC1,
called UEV-1, was recently identi®ed as a transcript
that was down-regulated upon di€erentiation of a
human colon carcinoma cell line (Sancho et al., 1998).
CROC1/UEV-1 represents a class of newly de®ned
ubiquitin-conjugating E2 enzyme variant proteins,
which share signi®cant sequence similarity and predicted sedondary and tertiary structure with the
ubiquitin-conjugating enzymes, but which lack ubiquitin-conjugating activity (Sancho et al., 1998). We have
also identi®ed a Saccharomyces cerevisiae gene, MMS2
(orf YGL087c), that is closely related to CIR1/CROC1
(48% identity in a 136 amino-acid overlap) (Sancho et
al., 1998; Broom®eld et al., 1998; LM and S Bacchetti,
unpublished data) and found that haploid yeast strains
carrying deletions of MMS2 were viable, suggesting
that this gene is not essential for yeast cell survival.
We have examined the levels of CIR1/CROC1
mRNA in human tissues and tumor-derived cell
lines. Northern analysis of human multiple tissue
blots revealed two major mRNA species of about
2.0 ± 2.1 kb (Figure 4), which represent the two forms
of CROC1 (Rothofsky and Lin, 1997). CIR1/CROC1
mRNA was present in all normal tissues but levels
of expression were highly variable (Figure 4a,b,d).
We examined CIR1/CROC1 expression in eight
di€erent tumor cell lines (Figure 4c). All were
positive for CIR1/CROC1 expression and levels of
mRNAs were consistently higher than in most
normal human tissues, and in particular substantially
higher than in the corresponding normal tissues (e.g.,
compare A549 versus lung, SW480 versus colon, and
three leukemia cell lines HL-60, K562 and MOLT-4
versus peripheral blood leukocytes and bone marrow). The CROC1/UEV1-1 gene is located on
chromosome 20q13.2 (Sancho et al., 1998), a region
frequently ampli®ed in many types of human cancer
(Tanner et al., 1994). Others have reported that
SW480 cells contain aberrant chromosomes harboring
large portions of 20q (Brinkmann et al., 1996), a
®nding that may account, at least in part, for the
high level of CIR1/CROC1 mRNA present in this
cell line.
In conclusion, by means of di€erential display of
mRNAs we have identi®ed two species, CIR1 and
CIR1/CROC1 over-expression in immortal cells
L Ma et al
1325
Figure 4 Expression of CIR1/CROC1 in human tissues and tumor cell lines. Northern blots containing 2 mg of poly(A)-selected
mRNA from multiple human tissues and cancer cell lines were purchased from Clontech (Palo Alto, CA) and are as follows: (a)
Human MTN blot, (b) Human III MTN blot, (c) Human Cancer Line MTN blot, and (d) Human II MTN blot. Tissues and cell
lines from which RNA was isolated are indicated. Top and bottom panels contain the results after hybridization with the 0.86 kb
SalI ± BsaBI fragment of the CROC-1B cDNA (Rothofsky and Lin, 1997) and with a b-actin probe (Clontech), respectively. All four
membranes were hybridized with the CROC1 probe and exposed to X-ray ®lm under identical conditions. Hybridization of Human
III MTN blot (b) and Human Cancer Line MTN blot (c) with b-actin was performed together, whereas Human MTN blot (a) and
Human II MTN (d) were hybridized on separate occasions
CIR2, that are up-regulated early during cell
immortalization. Our ®ndings extend the available
information on transcriptional alterations that are
associated with the immortal phenotype, although it
remains to be established whether enhanced expression
of CIR1 and CIR2 is causal to or merely a
consequence of cell immortalization. Sequence analysis revealed that CIR1 is identical to the recently
cloned CROC1/UEV-1 gene (Rothofsky and Lin,
1997; Sancho et al., 1998) which de®nes a novel
subfamily of highly conserved, inactive variants of the
ubiquitin-conjugating E2 enzymes (Sancho et al.,
1998). Several observations suggest a potential
involvement of CIR1/CROC1/UEV-1 in cell proliferation and di€erentiation: (i) CROC1 is a nuclear
protein that is capable of activating transcription of
the c-fos promoter direct enhancer sequences, implicating the protein in the RAF-mediated signal
transduction pathway (Rothofsky and Lin, 1997); (ii)
constitutive expression of UEV-1 inhibits the capacity
of HT-29-M6 cells to di€erentiate upon reaching
con¯uence and has profound inhibitory e€ects on
the activity of the mitotic kinase cdk1, resulting in
DNA endoreduplication and accumulation of cells in
G2-M (Sancho et al., 1998). Inhibition of differentiation would be a prerequisite for the maintenance of
proliferative capacity of immortalized cells; (iii)
CROC1/UEV-1 maps to chromosome 20q13.2 (Sancho et al., 1998), a region which is moderately
ampli®ed in many types of cancer (e.g. Kallioniemi
et al., 1994; Muleris et al., 1994; Tanner et al., 1994,
1995; Brinkman et al., 1996) and highly ampli®ed in
others, e.g. breast cancer, where it correlates with
aggressiveness and poor prognosis (Tanner et al.,
1995). There is also a strong association between
20q13.2 ampli®cation and genomic instability in HPVtransformed uroepithelial cells and between high
frequency of 20q gain and cell immortalization
(Savelieva et al., 1997; Yeager et al., 1998). Indeed,
20q gain is thought to re¯ect one genetic alteration
required by tumor cells to overcome senescence
(Savelieva et al., 1997; Yeager et al., 1998). Lastly,
we have documented that expression of CIR1/CROC1
is up-regulated early in cell immortalization and is
common in tumor-derived cell lines. Taken together,
these ®ndings suggest that CIR1/CROC1 might be
implicated in the immortalization process and consequently also in human carcinogenesis.
Acknowledgements
We thank Richard Austin for help in setting up di€erential
display, Brenda Andrews for help with the yeast experiments, BL Chow for technical assistance, Afra van der
Beek and Ping Wang for helpful discussions and Afra van
der Beek and Frank Graham for critical reading of the
manuscript. This work was supported by grants from the
Medical Research Council (MRC) and the National Cancer
Institute of Canada (NCIC) to S Bacchetti and WX. LM is
a postdoctoral fellow of the Dutch Cancer Society and was
also supported by a MRC postdoctoral fellowship for six
months during this work. WX is a Research Scientist of the
NCIC and S Bacchetti is a Terry Fox Cancer Research
Scientist of the NCIC.
CIR1/CROC1 over-expression in immortal cells
L Ma et al
1326
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