The EMBO Journal Vol.17 No.11 pp.3052–3065, 1998
A homologue of Drosophila aurora kinase is
oncogenic and amplified in human colorectal
James R.Bischoff, Lee Anderson1,
Yingfang Zhu, Kevin Mossie, Lelia Ng,
Brian Souza, Brian Schryver,
Peter Flanagan, Felix Clairvoyant,
Charles Ginther1, Clarence S.M.Chan2,
Mike Novotny, Dennis J.Slamon1and
SUGEN, Inc., 351 Galveston Drive, Redwood City, California 94063,
1Division of Hematology-Oncology and Jonsson Comprehensive
Cancer Center, UCLA School of Medicine, 10833 Le Conte Avenue,
Los Angeles, CA 90095 and2Department of Microbiology,
University of Texas, Austin, TX 78712, USA
Genetic and biochemical studies in lower eukaryotes
have identified several proteins that ensure accurate
segregation of chromosomes. These include the Droso-
phila aurora and yeast Ipl1 kinases that are required
for centrosome maturation and chromosome segre-
gation. We have identified two human homologues of
these genes, termed aurora1 and aurora2, that encode
cell-cycle-regulated serine/threonine kinases. Here we
demonstrate that the aurora2 gene maps to chromo-
some 20q13, a region amplified in a variety of human
cancers, including a significant number of colorectal
malignancies. We propose that aurora2 may be a target
of this amplicon since its DNA is amplified and its
RNA overexpressed, in more than 50% of primary
colorectal cancers. Furthermore, overexpression of
aurora2 transforms rodent fibroblasts. These observ-
ations implicate aurora2 as a potential oncogene in
many colon, breast and other solid tumors, and identify
centrosome-associated proteins as novel targets for
Keywords: 20q13 amplicon/centrosome/colon cancer/
Chromosomal abnormalities are a hallmark of human
cancer, reflecting the deleterious consequences of the gain
or loss of genetic information (Boveri, 1929; Hartwell
and Kastan, 1994; Mitelman et al., 1997). Some of these
defects may have a causal role in cellular transformation
due to loss of a negative growth regulator or a gene
responsible for maintenance of genome integrity, or
through the amplification, overexpression or mutational
activation of an oncogene (Hunter, 1997; Kinzler and
Vogelstein, 1997). Alternatively, these abnormalities may
be a consequence of tumor progression, where disruption
of mitotic checkpoints can result in abnormal nuclei,
missegregated chromosomes and aneuploidy (Elledge,
1996; Sherr, 1996).
© Oxford University Press
A direct connection between the cell cycle and cancer
was first established with the observation that the cyclin
D1 gene was amplified in a subset of human cancers
(Motokura et al., 1991; Hunter and Pines, 1994). The
subsequent discovery that the tumor suppressor p53
regulates p21, an inhibitor of cyclin-dependent kinases
(el-Deiry et al., 1993; Xiong et al., 1993), as well as
kinase inhibitor—as a major tumor suppressor gene
(Elledge, 1996; Sherr, 1996), has firmly entrenched the
view that misregulation of the cell cycle machinery can
have enormous impact on cellular proliferation. Based
on the prevalence of genetic abnormalities in human
cancer, it is plausible that proteins involved in main-
taining the integrity of chromosome segregation may
also play a role, directly or indirectly, in cellular
transformation. The fidelity of chromosome segregation
is monitored by mitotic checkpoints that delay entry
into mitosis until a functional centrosome is present, or
delay progression beyond anaphase until the chromo-
somes are aligned on the metaphase plate by the mitotic
spindle. In normal cells, centrosomes play an important
role in coordinating the changes required for the onset
of mitosis, serving as an anchor for reorganization of
the cytoplasmic microtubules into a mitotic spindle
apparatus and for recruitment of numerous structural,
motor and catalytic proteins to the centrosome complex.
Proper execution of this process ensures that each
daughter cell receives the correct number of chromo-
somes. Recent studies suggest that a G2/M checkpoint
may exist to ensure the integrity of this process of
centrosome maturation (Nigg et al., 1996).
Genetic and biochemical studies in yeast and Droso-
phila have identified several proteins involved in
chromosome segregation and spindle assembly. Disrup-
tion of these proteins results in chromosome missegre-
gation, monopolar or disrupted spindles and/or abnormal
nuclei. Several of these proteins represent distinct
families of protein serine/threonine kinases, including:
Cdc2, a cyclin-dependent kinase conserved from yeast
to mammals that is required for centrosome separation
and formation of a bipolar spindle (Sherr, 1994, 1996;
Morgan, 1995; Elledge, 1996); Mps1, a Saccharomyces
cerevisiae dual specificity kinase required for spindle
pole body duplication (Weiss and Winey, 1996); Bub1,
a S.cerevisiae and mammalian mitotic checkpoint kinase
(Hoyt et al., 1991; Taylor and McKeon, 1997); PLK1,
a mammalian homologue of polo, Cdc5p and plo1
kinases from Drosophila, budding and fission yeast,
respectively, that communicates the presence of a
functional centrosome to the Cdk/cyclin complex prior
to entry into mitosis (Lane and Nigg, 1996, 1997); and
the Ipl1 and aurora kinases from S.cerevisiae and
Drosophila, respectively, that are required for centrosome
Aurora2 oncogene amplified in colorectal cancers
Fig. 1. Homology between human, Xenopus, Drosophila and yeast auroras. (A) The sequences for human aurora1 and aurora2 were deduced from
full-length cDNA clones isolated from normal duodenum, pancreatic carcinoma and primary colorectal carcinoma libraries. Xenopus p46B
(PIR:S53343), Drosophila aurora (PIR:A56220) and S.cerevisiae Ipl1 (SWISS-PROT:P38991) are included in the alignment. The alignment was
generated by also including the two murine (DDBJ/EMBL/GenBank accession Nos D21099 and GB:U80932), an additional Xenopus (PIR:S53342)
and two C.elegans (DDBJ/EMBL/GenBank accession Nos U53336 and U97196) sequences as input into msa, a parallel-coded multiple sequence
alignment program that was run on MasPar MP2216 supercomputer. Boxed residues are common to three or more of the sequences; shaded residues
represent regions of amino acid similarity between two or more sequences; overlines correspond to the conserved Aurora Box1 and Aurora Box2
sequences; the arrow denotes the start of the C-terminal serine/threonine kinase domain; the circled residue indicates the location of a single
nucleotide polymorphism described in the text; solid circles correspond to the location of various yeast and Drosophila mutants (Franscisco et al.,
1994; Glover et al., 1995); and stars denote the site of the kinase-inactivating and -activating point mutants described in the text. (B) Schematic
domain structure of human aurora1 and aurora2.
separation and chromosome segregation (Francisco et al.,
1994; Glover et al., 1995). Among these kinases, only
PLK1 has been shown to be transforming (Smith et al.,
1997), although many are implicated to play a role in
the genotypic changes associated with immortalized
cells, possibly due to the presence of a compromised
checkpoint (Hoyt et al., 1991; Lane and Nigg, 1996,
1997; Taylor and McKeon, 1997).
J.R.Bischoff et al.
Here, we describe the identification and characteriza-
tion of two human homologues of Drosophila aurora
and yeast Ipl1, that we have named aurora1 and aurora2.
Structural comparison of aurora homologues
We initiated a PCR-based screen in order to identify novel
colon cancer-associated kinases. One of these clones
encoded a protein with homology to the aurora protein
kinase from Drosophila melanogaster and the Ipl1 kinase
from S.cerevisiae (Francisco et al., 1994; Glover et al.,
1995). While using this fragment to screen for a full-length
cDNA clone, we also identified a weakly hybridizing clone
that was found to encode a related kinase. We refer to
these genes as aurora1 and aurora2, to reflect their
homology to each other and to the Drosophila aurora
Fig. 2. The catalytic domain of aurora2, but not aurora1, partially complements the yeast ipl1-1 mutation. (A) Map of various yeast transformants of
strain CCY464-1D streaked onto SC-URA plates. Clockwise from top: vector, empty expression vector; Ipl1/A1KM, N-terminal domain of Ipl1
fused with the C-terminal portion of a kinase-dead aurora1 construct; Ipl1/A1, N-terminal domain of Ipl1 fused with the C-terminal portion of wild-
type aurora1; Ipl1/A2, N-terminal domain of Ipl1 fused with the C-terminal portion of wild-type aurora2; Ipl1/A2KM, N-terminal domain of Ipl1
fused with the C-terminal portion of a kinase-dead aurora2 construct; and Ipl1, wild-type Ipl1. (B) Plate grown at the permissive temperature of
26°C. (C) Plate grown at the restrictive temperature of 34°C. (D) Plate grown at the restrictive temperature of 37°C.
Fig. 3. Aurora1 and aurora2 proteins are cell cycle-regulated and localized to mitotic structures. Exponentially growing HeLa cells were
synchronized at the G1/S transition by a double thymidine/aphidicolin block. Separate plates (10 cm) were harvested for FACS analysis, RNA
isolation, protein quantitation and kinase assays at the indicated times. (A) FACS analysis was performed on exponentially growing HeLa cells, as
well as cells harvested at 0, 4, 9, 10 and 12 h after release. (B) Northern blots of synchronized HeLa cells probed with a32P-labeled aurora1 cDNA
(top panel), a32P-labeled aurora2 cDNA (middle panel), and a32P-labeled actin cDNA (bottom panel). Equal amounts of total RNA (10 µg) were
loaded in each lane. (C) Immunoblots probed with protein A-purified anti-aurora1 antibodies (top panel), anti-aurora2 antibodies (middle panel) or
anti-p34cdc2antibodies (bottom panel). Equal amounts of total cellular protein (50 µg) were loaded in each lane. (D) In vitro kinase assays with anti-
aurora1 immune complexes (top panel) using GST2TK (PKA phosphorylation site) as a substrate, with anti-aurora2 immune complexes (middle
panel) using myelin basic protein (MBP) as an artificial substrate, or anti-p34cdc2immune complexes (bottom panel) using histone H1 as a substrate.
Equal amounts of total HeLa cell protein (500 µg) were used for each immunoprecipitation. (E) Aurora1 is localized to the midzone and post-mitotic
bridge. HeLa cells at various stages of mitosis were stained for DNA, α-tubulin and aurora1. Top panels, DAPI staining of DNA; middle panels,
α-tubulin immunostaining; bottom panel, aurora1 immunostaining. (F) Aurora2 is localized to the mitotic spindle of metaphase and anaphase cells.
HeLa cells at various stages of mitosis were stained for DNA, α-tubulin and aurora2. Top panels, DAPI staining of DNA; middle panels, α-tubulin
immunostaining; bottom panel, aurora2 immunostaining.
kinase. The aurora1 cDNA contained a 1032 bp open
reading frame that encodes a 344 amino acid polypeptide
with a predicted molecular mass of 39.3 kDa. The aurora2
cDNA contained a 1209 bp open reading frame that
encodes a 403 amino acid polypeptide with a predicted
molecular mass of 45.8 kDa. Two additional human aurora
pseudogenes were also identified as expressed transcripts
that are each contained on single exons and maintain
striking DNA homology to either aurora1 or 2, yet exhibit
preparation of this manuscript, a partial sequence of BTAK
(Sen et al., 1997), a breast tumor-associated kinase, was
reported that appears to be a fragment of human aurora2.
A second paper reported the sequence of human aik
(Kimura et al., 1997), a cell cycle-regulated protein
localized to spindle pole bodies. The published sequence
shares 92% amino acid identity with our sequence of
Aurora2 oncogene amplified in colorectal cancers
J.R.Bischoff et al.
human aurora2. We believe that aurora2 and aik are
identical and six frameshifts resulting from sequencing
errors explain the small differences in the published
sequence. Three additional papers provide the sequence
of AYK1 (Yanai et al., 1997), a meiotic-regulated gene
and IAK1 (Gopalan et al., 1997), both of which appear to
be the murine orthologues of aurora2, and AIM-1 (Terada
et al., 1998) which is a rat orthologue of aurora1. The
current report describes the first complete sequence for
both human aurora1 and aurora2.
The deduced amino acid sequences of human aurora1
and aurora2 are presented in Figure 1A, aligned with the
yeast and Drosophila homologues Ipl1 and aurora and an
additional homologue p46B from Xenopus laevis. Human
aurora2 protein shares 57%, 63%, 43% and 41% identity
over its entire length with human aurora1, Xenopus p46B,
Drosophila aurora and Ipl1, respectively. The four
sequences contain a C-terminal domain with all the
subdomains characteristic of a serine/threonine kinase.
Thekinase domainof humanaurora2 shares74%,62% and
49% amino acid identity with human aurora1, Drosophila
aurora and Ipl1, respectively, and 83.5% identity with
two amphibian homologues present in Xenopus [p46A
(p46Eg22, PIR:S53342) and p46B (p46Eg265, PIR:
S53343)]. Drosophila aurora is most related to human
aurora1 whereas yeast Ipl1 is most related to aurora2.
Whereas a single aurora-like kinase is present in yeast, at
least two members are present in Caenorhabditis elegans
(DDBJ/EMBL/GenBank accession No. U53336, gene
K07C11.2 and U97196, gene B0207.4). The deduced
catalytic domains of these C.elegans proteins share 55%
and 64% amino acid sequence identity to the human
aurora2 kinase domain. We predict that an additional
aurora homologue will ultimately be identified in Droso-
phila as characterization of its genome nears completion.
The 129 and 73 amino acid N-terminal domains of
human aurora2 and aurora1 share limited homology with
each other and with the analogous 160 and 100 amino acid
domains of Drosophila aurora and yeast Ipl1. However, the
N-terminal regions of human and mouse aurora2 share
54% identity to each other and 28–30% identity to the two
Xenopus proteins and together help define two distantly
conserved motifs present in the non-catalytic region of all
auroras (Figure 1A and B). These motifs are composed
predominantly of conserved hydrophobic and polar
residues. The first motif spans 18–37 amino acids (Aurora
Box1), with aurora1 and yeast Ipl1 lacking the central
portion and the second motif spans 21 amino acids (Aurora
Box 2; see overlines in Figure 1A). Several potential
serine and threonine phosphorylation sites are also con-
served among these proteins, including a protein kinase A
phosphorylation motif RRXT in the activation loop of the
kinase. A temperature-sensitive mutant of the yeast Ipl1
gene has a threonine to alanine substitution at this site
(Francisco et al., 1994), suggesting that phosphorylation
on this threonine residue within the activation loop may
be biologically relevant. Additional mutations in the yeast
(Francisco et al., 1994) and Drosophila (Glover et al.,
to the kinase domain, except for a single Drosophila
mutant (Glover et al., 1995) that changes an aspartate to
an alanine at residue 47 within the N-terminal Aurora
Box1. Since these mutations result in abnormal nuclei,
chromosome missegregation and monopolar spindles,
these findings suggest that the catalytic activity of the
auroras may play an important role in centrosome biology.
Aurora2 partially complements Ipl1
To determine whether human aurora1 and/or aurora2 are
functionally equivalent to their S.cerevisiae homologue
Ipl1, we attempted to complement the ipl1-1 temperature-
sensitive mutant strain, CCY464-1D (Francisco et al.,
1994) with expression plasmids encoding the aurora pro-
teins. The CCY464-1D strain is inviable above 34°C due
to a mutation in Ipl1 (Francisco et al., 1994). Neither
aurora1 nor aurora2 was able to complement the ipl1-1
mutation at 37°C, probably due to an inhibition of cell
growth on overexpression of the unique N-terminal
domains of these proteins (unpublished observation). To
plasmids, Ipl1/A1 and Ipl1/A2, containing the unique N-
terminal domain of Ipl1 (amino acids 1–101), fused to the
C-terminal catalytic domain of aurora1 (amino acids 75–
344) or aurora2 (amino acids 131–403), respectively.
Additional Ipl1/aurora fusions were made in which the
essential lysine at residue 106 (K106) of aurora1 or residue
162 (K162) of aurora2 was mutated to a methionine
resulting in catalytically inactive forms of both proteins.
These kinase-dead constructs were designated Ipl1/A1KM
and Ipl1/A2KM, respectively. These coding regions were
subcloned into a CEN vector (Sikorski and Hieter, 1989)
under control of the native Ipl1 promoter. The wild-type
Ipl1 construct complemented the ipl1-1 mutation at 37°C,
whereas no growth was observed with either the wild-
type or kinase-dead fusions of Ipl1/aurora1 or Ipl1/aurora2
(Figure 2D). However, at the less restrictive temperature
of 34°C, the Ipl1/A2 fusion partially complemented the
ipl1-1 mutation, whereas the kinase-dead Ipl1/A2KM and
all aurora1 constructs failed to rescue the mutation (Figure
2C). Thus, in support of the conclusions derived from
analysis of the primary amino acid sequence of these
proteins, it appears that the aurora2 kinase is structurally
and functionally equivalent to Ipl1, whereas aurora1
exhibits a biologically distinct activity.
Aurora1 and aurora2 are cell cycle regulated
Based on the predicted involvement of Drosophila aurora
and yeast Ipl1 in centrosome separation and/or chromo-
some segregation, we investigated whether human aurora1
and aurora2 are cell cycle regulated. HeLa cells were
synchronized at the G1/S transition by a double thymidine/
aphidicolin block (Golsteyn et al., 1994) and followed
through the completion of mitosis. After release from the
G1/S transition, the cells were analyzed for aurora1 and
aurora2 RNA expression, protein expression and kinase
activity. The DNA content at each time point was analyzed
by flow cytometry (Figure 3A). Aurora1 and aurora2 RNA
levels were low at the G1/S transition (time ? 0) and
gradually increased as the cells progressed through S phase
(time ? 2–6 h) (Figure 3B, upper and middle panels) and
through G2and mitosis (time ? 8–10 h). Aurora1 RNA
levels were highest at 8–10 h after release, corresponding
to the G2and M phases of the cell cycle (Figure 3B,
upper panel). The amount of aurora2 RNA peaked at 8
and 9 h post-release as the cells progressed from G2into
mitosis and returned to low levels as the cells re-entered
Aurora2 oncogene amplified in colorectal cancers
G1at 12 h after release from the block (Figure 3B, middle
panel). Actin RNA served as a loading control (Figure
3B, bottom panel). As expected, aurora1 and aurora2
proteins also varied during the cell cycle. Aurora1 and
aurora2 proteins peaked at 8–11 h and 8–10 h after release,
respectively (Figure 3C, upper and middle panels). p34cdc2
protein levels served as a loading control (Figure 3C,
bottom panel). We also examined aurora1 and aurora2
kinase activity during cell cycle progression. The aurora1
kinase activity was maximal during mitosis at 10–11 h
after release (Figure 3D, top panel). Aurora2 kinase
activity peaked at 9 h after release (Figure 3D, middle
panel). p34cdc2kinase activity, which served as a marker
for mitosis, peaked at 10 h after release (Figure 3D,
bottom panel). Thus, both aurora1 and aurora2 RNA,
protein and kinase activity were cell cycle-regulated, all
being maximal during G2and mitosis. Aurora2 kinase
activity was highest just prior to maximal activation of
aurora1 and p34cdc2. These data suggest that aurora2
function precedes that of aurora1 in mitosis.
Aurora1 and aurora2 are localized to mitotic
The subcellular location of endogenous aurora1 and
aurora2 was determined by indirect immunofluoresence.
Exponentially growing HeLa cells were fixed with meth-
anol and probed with a monoclonal antibody to α-tubulin
and with protein A affinity-purified antibodies to either
aurora1 (Figure 3E, bottom panel) or aurora2 (Figure 3F,
bottom panel). The aurora1 and aurora2 antibodies only
stained structures in mitotic cells and did not stain any
recognizable structures or compartments in interphase
cells. This is understandable given that the proteins are
most abundant at this stage of the cell cycle (Figure 3C).
In anaphase and early telophase, the aurora1 antibodies
stained the midzone and telophase disc (Andreassen et al.,
1991), whereas in late telophase and early G1, they stained
the post-mitotic bridge (Figure 3E, bottom panel). In
metaphase and anaphase, the aurora2 antibodies stained
the centrosome, spindle poles and the spindle (Figure 3F,
bottom panel), whereas in telophase cells the aurora2
antibodies primarily stained the spindle pole (Figure 3F,
bottom panel). The aurora2 immunostaining is consistent
with that reported elsewhere (Gopalan et al., 1997; Kimura
et al., 1997). The subcellular localization of aurora1 and
aurora2 suggests that aurora1 may function later in mitosis
than aurora2. This supports the observation that, in syn-
chronized cells, aurora2 kinase activity precedes that of
aurora1 (Figure 3D). In addition, the localization of
aurora1 and aurora2 to mitotic structures confirms that
they are indeed likely to be involved in chromosome
Expression of aurora1 and aurora2 RNA
Northern blot analysis of mRNA isolated from normal
adult human tissues demonstrates that aurora2 expression
is primarily restricted to testis, thymus and fetal liver
(Figure 4A), with very weak expression in bone marrow,
lymph node and spleen, and no detectable expression in
all other adult tissues examined. Human aurora1 was also
expressed at highest levels in normal thymus and fetal
liver, with a moderate level of expression in lung and
small intestine (Figure 4A).
Fig. 4. Expression of human aurora1 and aurora2. (A) Northern blot
containing poly(A)?mRNA (2 µg per lane) from normal human tissue
hybridized with an aurora1 or aurora2 DNA probe. (B) Aurora2
Northern blots containing total RNA (20 µg) from human tumor cell
lines. The single 2.4 kb aurora2 transcript is marked. RNA from the
lung cancer cell line NCI-H23 was included as a standard for the
tumor blots. Cell lines included are: 1, HT-29; 2, HCC-2998; 3, COLO
205; 4, HCT-15; 5, KM012; 6, UO-31; 7, SN12C; 8, CAKI-1; 9,
RXF393; 10, ACHN; 11, 786-0; 12, TK-10; 13, LOX IMVI; 14, SK-
MEL-2; 15, SK-MEL-5; 16, SK-MEL-28; 17, UACC-62; 18, UACC-
257; 19, M14; 20, MCF-7/ADR-RES; 21, HS 578T; 22, MDA-MB-
435; 23, MDA-N; 24, T-47D. (C) Aurora2 Northern blot containing
total RNA (10 µg) from cultured primary human endothelial and
epithelial cells. The single 2.4 kb aurora2 transcript is marked. RNA
from the lung cancer cell line NCI-H23 was included as a standard for
the blots. Primary cell RNAs are: 1, coronary artery endothelial cells;
2, pulmonary artery endothelial cells; 3, lung microvascular
endothelial cells; 4, dermal microvascular endothelial cells; 5,
mammary epithelial cells; 6, renal proximal tubule epithelial cells; and
7, renal cortex epithelial cells.
Since aurora2 was highly represented in the initial
PCR screen of primary colon tumors, we examined the
expression of aurora2 RNA in a panel of 25 human tumor
cell lines of lung, colon, renal, melanoma and breast
origin. The 2.4 kb aurora2 transcript was expressed at
high levels in 96% (24 of 25) of these transformed cell
lines (Figure 4B), with the only exception being the UO-
31 renal carcinoma cell line. Our preliminary analysis
revealed that the 1.4 kb aurora1 transcript was also
expressed in the same 24 tumor cell lines (unpublished
data), although we have yet to examine this in more detail.
We also saw modest, but detectable, expression of aurora2
in a panel of RNAs isolated from cultured primary
epithelial and endothelial cells (Figure 4C). We conclude
that aurora2 is preferentially expressed in all rapidly
J.R.Bischoff et al.
dividing cells, but its levels are significantly up-regulated
in a broad range of tumor cell lines.
Amplification and overexpression of aurora2 in
primary human colorectal cancers
The aurora2 gene was mapped using the Stanford Human
is located on chromosome 20q13.2 (LOD score of 17.26
to linked marker SHGC-3245). Mapping was also con-
firmed by hybridization to a human–rodent somatic cell
hybrid panel (Coriell Cell Repository, Camden, NJ).
Aurora2 maps adjacent to the vitamin D hydroxylase
(CYP24) gene and the cosmid probe RMC20C001 that lie
at 0.825–0.83 Flpter (fractional length from pter) on
chromosome 20 (Tanner et al., 1994, 1996). Both of these
markers have been characterized for their presence in the
20q13 amplicon common to many human malignancies,
particularly those from breast, bladder and colon cancers
(Muleris et al., 1987; Bigner et al., 1988; Yaseen et al.,
1990; Kallioniemi et al., 1994; Tanner et al., 1994, 1996;
Iwabuchi et al., 1995; Schlegel et al., 1995; Bockmuhl
et al., 1996; Courjal et al., 1996; Reznikoff et al., 1996;
Solinas-Toldo et al., 1996; James et al., 1997; Larramendy
et al., 1997).
Since the aurora2 gene maps to a prevalent tumor
amplicon, we questioned whether the aurora2 gene was
also amplified in a cohort of primary human colorectal
tumors and matched normal colorectal tissue from the
same patients. Southern blot hybridization was performed
using an aurora2 cDNA probe along with a control probe
for the CYP24 gene that serves as a marker of the amplicon
(Tanner et al., 1994, 1996). The aurora2 probe hybridized
to PstI fragments of 5.8, 3.7, 3.3, 2.8, 2.5 and 1.3 kb. The
5.8, 3.3, 2.8 and 2.5 kb bands are specific to aurora2, while
the 3.7 and 1.3 kb bands represent cross-hybridization to
the aurora3 and aurora4 pseudogenes which map to
chromosomes 1 and 10, respectively. Only the aurora2-
specific bands showed amplification in the tumor samples.
Aurora2 DNA was amplified in (52%) 41 of 79 of the
primary colorectal tumors for which suitable DNA was
available for genotyping (Figure 5B). The CYP24 gene
was found to be co-amplified with aurora2 in (90%) 37
of 41 matched pairs and was found only once to be
amplified in the absence of aurora2 amplification.
Aurora2 RNA levels were characterized by Northern
blot analysis of samples from the same panel of matched
tumor/normal tissues. Approximately 54% (22 of 41) of
the tumors showed increased expression of the 2.4 kb
aurora2 transcript as compared with the normal colon
control. Aurora2 RNA showed 4- to 28-fold overexpres-
sion in tumor versus normal tissue. Representative North-
ern andSouthern data from12 matchedtumor/normal pairs
are shown in Figure 5, where nine samples demonstrated
amplification of aurora2 DNA in the range of 2- to 8-fold
in the tumors compared with normal tissue (2164, 2172,
2193, 3204, 2255, 3189, 3191, 3193 and 2176) and three
samples (1985, 2175 and 2257) showed no amplification.
This level of aurora2 amplification is consistent with
other reports of 1.5- to 10-fold increases in copy number of
20q13 in primary tumors and tumor cell lines (Kallioniemi
et al., 1994; Tanner et al., 1996; Sen et al., 1997). Sample
3193 still shows a relative level of DNA amplification
after adjusting for unequal sample loading. One of the
samples (1985) clearly demonstrates RNA overexpression
in the absence of DNA amplification, whereas the other
11 show a direct correlation between DNA amplification
and RNA overexpression. We obtained complete data for
analysis from 37 matched sets of RNA and DNA from
both normal and tumor samples. Data in Table I show a
high correlation (ρ ? 0.695, Pearson correlation;
P ?0.00003, Fisher’s exact test) between aurora2 DNA
amplification and RNA overexpression with only one
discordant result. In the single case of aurora2 DNA
amplification in the absence of RNA overexpression,
aurora2 RNA was actually elevated in both the normal
and tumor specimens, compared with other tumor/normal
pairs. It is conceivable that high expression of aurora2
RNA in this normal colon sample may represent an
early predisposing lesion. Conversely, five paired samples
showed increased RNA expression in the absence of DNA
amplification, possibly due to transcriptional activation. If
these five pairs are excluded from the analysis, the
correlation between aurora2 DNA amplification and RNA
overexpression increases to ρ ? 0.939. These data suggest
that DNA amplification is a mechanism for aurora2
activation and also implicates aurora2 as an oncogene at
20q13 whose high level amplification correlates with poor
clinical outcome in breast cancer (Isola et al., 1995).
To determine if the aurora2 sequence from the 20q13
amplicon was the same as that from normal sources,
we performed direct sequencing of RT–PCR products
encompassing the complete aurora2 coding region from
10 primary colorectal tumor samples. Eight samples,
including tissues with both normal and amplified levels
of the 20q13 amplicon, confirmed the aurora2 sequence.
A single base change was identified in two samples (1985
and 2193)resulting in aphenylalanine toisoleucine change
at residue 31 in the N-terminal Aurora Box1 (circled in
Figure 1A). Experiments are planned to determine if this
is simply a polymorphism or whether this change affects
aurora2 activity. Nonetheless, these analyses demonstrate
that the 20q13 amplicon typically contains increased
copies of the intact, unmutated aurora2 coding region.
Detection of aurora2 protein in primary human
colon cancer samples
To determine whether the amplification of aurora2 gene
and message resulted in overexpression of the protein,
anti-aurora2 antibodies were used to probe blots of protein
lysates made from cryostat sections of primary human
colon carcinomas or from adjacent normal tissue isolated
from the same patient. As shown in Figure 6A, the aurora2
antibodies detected a protein of ~46 kDa in the two
primary human colon carcinomas, but not in samples
derived from the adjacent normal tissue. Due to the limited
amount of tissue available, we were unable to determine
if aurora2 was amplified in these samples. These anti-
bodies also detected overexpression of aurora2 protein in
various cultured tumor cell lines derived primarily from
colorectal carcinomas (Figure 6B). While most tumor cell
lines examined expressed detectable levels of aurora2
protein, others including A549 cells do not (Figure 6B,
Aurora2 transforms Rat1 fibroblasts
If aurora2 is a relevant target on the 20q13 amplicon, one
might expect that overexpression of aurora2 would be
Aurora2 oncogene amplified in colorectal cancers
Fig. 5. Aurora2 RNA overexpression and DNA amplification in primary human colorectal cancers. (A) Northern blots containing total RNA (6 µg
per lane) from primary human colorectal cancers and from matched normal colon controls were hybridized with an aurora2 DNA probe. Blots were
stripped and reprobed with a human β-actin probe to confirm equivalency and quality of RNA loading. (B) Southern blots containing 5 µg of PstI-
digested DNA per lane from primary human colorectal cancers and from matched normal colon controls were hybridized with an aurora2 DNA
probe (pSG19). The location of the 2.8 and 2.5 kb aurora2 fragments are marked. Blots were stripped and reprobed with a human β-globin probe to
confirm equal loading. Patient numbers are shown for each of the matched sets of normal (N) and tumor (T) tissue.
Table I. Amplification of aurora2 DNA correlates with RNA
Sample Amount DNA amplified or RNA overexpressed
DNA and RNA
DNA w/o RNA
RNA w/o DNA
Matched tumor/normal pairs of colorectal tissue were analyzed by
Southern blot for amplification of aurora2 DNA and by Northern blot
for overexpression of aurora2 RNA. Data are presented for the 79
DNA samples and 41 RNA samples analyzed. Both RNA and DNA
samples were available for 37 matched tumor/normal pairs. DNA w/o
RNA, refers to DNA amplification without RNA overexpression.
transforming. To examine this question, we established
stable NIH 3T3 and Rat1 cell lines that express human
aurora2. Rat1 cells were infected with retroviruses that
express a hemagglutinin (HA)-tagged (Pati, 1992) wild-
type aurora2 or a kinase-inactive mutant where the essen-
tial lysine at residue 162 (Figure 1A) was changed to a
methionine (K162M). In addition, an activating mutation
was made in which the threonine at residue 288 (Figure
1A) in the activation loop was changed to an aspartic
acid (T288D). This mutation was designed to mimic
constitutive phosphorylation at this site and the recom-
binant T233D aurora2 protein demonstrated increased
specific activity when expressed in bacteria or baculovirus-
infected insect cells (unpublished data). Several clones
expressing each construct were selected that expressed
similar amounts of aurora2 protein (Figure 7A, lanes 2–4).
To characterize the transforming potential of aurora2,
we performed soft agar assays with the Rat1 clones. The
vector control, K162M, wild-type and T288D aurora2-
expressing Rat1 cells were plated in soft agar and scored
for growth after 4 weeks. As shown in Figure 7B, cells
expressing the wild-type and the T288D aurora2 formed
colonies in soft agar, in contrast to the lack of growth by
cells expressing the kinase-inactive aurora2. Ten of 13
Fig. 6. Aurora2 protein levels are elevated in tissue and cell lines
derived from colon carcinomas. (A) Total protein lysates were
prepared from matched samples of tumor and adjacent normal tissue.
Equal amounts of protein from each pair was loaded on a 12% gel and
immunoblotted with affinity-purified antibodies to aurora2. Lane 1,
72 µg of total protein isolated from normal colon epithelium adjacent
to tumor HT374; lane 2, 72 µg of total protein isolated from tumor
HT374; lane 3, 90 µg of total protein isolated from normal tissue
adjacent to tumor HT376; lane 4, 90 µg of total protein isolated from
tumor HT376. N, normal tissue; T, tumor tissue. (B) Total protein
lysates were prepared from six colon adenocarcinoma cell lines
(LS180, HCT-15, HT-29, COLO 205, SW480 and SW948), one rectal
adenocarcinoma cell line (SW837), one primary cecum carcinoma cell
line (SNU-C2B) and one lung carcinoma cell line (A549). 50 µg of
total protein were loaded in each lane on a 12% gel and
immunoblotted with affinity-purified antibodies to aurora2.
wild-type clones and six of 12 T288D clones grew in soft
agar, compared with one of 11 vector and K162M clones.
We quantitated the number of colonies formed in soft agar
J.R.Bischoff et al.
Fig. 7. Aurora2 transforms Rat1 fibroblasts. (A) Rat1 cells were transfected with either pLXSN (vector), HA-tagged kinase-inactive (K162M)
aurora2, HA-tagged wild-type aurora2, or HA-tagged activated (T288D) aurora2. Cell lysates from stable Rat1 clones were immunoprecipitated
with a monoclonal antibody specific to the HA tag. Immune complexes were resolved directly on a 12% SDS–polyacrylamide gel, transferred to a
nylon membrane and probed with protein A-purified antibodies to aurora2. Lane 1, vector control; lane 2, kinase-inactive aurora2 (K162M); lane 3,
wild-type aurora2; lane 4, activated aurora2 (T288D). (B) Stable Rat1 cell lines expressing either kinase-inactive (K162M), wild-type or activated
(T288D) aurora2 were plated on soft agar and incubated at 37°C for 4 weeks. ‘Vector’ represents Rat1 cells stably infected with pLXSN.
(C) NIH 3T3 cells expressing the kinase-inactive (K162M) or activated aurora2 (T288D) constructs were injected subcutaneously between the
scapula of nu/nu mice. Tumor size was measured at 4, 6 and 8 weeks post-implantation.
from two independent clones of each of the transfections.
The average number of colonies per 200 000 cells plated
were: K162M, 32 colonies; wild-type, 470 colonies; and
T288D, 250 colonies. Although the T288D Rat1 stables
formed fewer colonies than the wild-type aurora2 Rat1
stables, the T288D colonies in general grew to larger size
(Figure 7B). None of the Rat1 stables showed altered
growth characteristics as compared with the vector control;
however, the NIH 3T3 T288D clones did grow to a higher
density than the vector control, wild-type and K166M
clones. The T288D aurora2 mutant was also able to
transform NIH 3T3 cells, as measured by growth in soft
agar (unpublished data) and growth as tumors in nude
mice (Figure 7C). Apparently, aurora2 kinase activity was
required for cellular transformation, as the kinase-inactive
aurora2 (K162M) failed to stimulate the growth of Rat1
or NIH 3T3 cells in soft agar or as tumors in nude mice.
In this report we characterize a new family of two human
serine/threonine kinases designated aurora1 and aurora2.
Using drug release protocols to synchronize HeLa cells,
we found that aurora1 and aurora2 RNA levels, protein
levels and kinase activities were low during interphase,
but increased as cells proceed into mitosis. These activity
profiles are similar to that of cell cycle-regulated p34cdc2;
however, aurora2 activity peaks 1 h before p34cdc2, and
the aurora1 kinase activity persists for 1 h after p34cdc2.
The activity of p34cdc2has been shown to maximal in
metaphase, suggesting that aurora2 kinase activity peaks
in prometaphase while aurora1 kinase activity is high
from metaphase through telophase. Increased aurora2
protein levels cannot completely account for its increased
kinase activity. Immunoblot analysis demonstrates equiva-
lent amounts of aurora2 protein in samples prepared 9
and 10 h after release from a double thymidine/aphidicolin
block (Figure 3C, middle panel), but the amount of aurora2
kinase activity is three times greater at the earlier time
point (Figure 3D, middle panel). This raises the question
of whether aurora2 kinase activity is also regulated by a
post-translational mechanism, possiblyby phosphorylation
of Thr288 in the activation loop.
The aurora1 protein was localized to the midzone of
cells in anaphase and to the post-mitotic bridge and
midbody during telophase, while aurora2 protein was
localized to the mitotic spindle of cells in metaphase and
anaphase. These findings support the biochemical studies
which suggest that both are cell-cycle-regulated proteins
and that aurora2 functions earlier in mitosis than aurora1.
A recent manuscript on AIM-1, the rat orthologue of
aurora1, also describes its localization to the midbody
region during telophase and presents evidence to suggest
that aurora1 is required for cytokinesis, since its disruption
results in polyploidy and decreased viability (Terada
et al., 1998).
We have shown that aurora2 RNA is expressed in a
variety of human tumor cell lines while having limited
expression in normal human tissue. The aurora2 gene
was mapped to chromosome 20q13, a region frequently
Aurora2 oncogene amplified in colorectal cancers
amplified in human tumors. Aurora2 DNA was found to
be amplified, and its RNA overexpressed, in 52% of a
we demonstrate that overexpression of the aurora2 kinase
transforms rodent fibroblasts.
Gene amplification in tumor cells is often characterized
by the presence of cytogenetic aberrations including
heterogeneous staining regions (HSRs) and double minute
chromosomes. Recently, the technique of comparative
genomic hybridization (CGH) has provided a sensitive
means by which to identify tumor-associated amplicons
(Kallioniemi et al., 1992; Tanner et al., 1994, 1996).
While several oncogenes have been shown to be amplified
in human tumors (Slamon et al., 1987; van de Vijver,
1993), including HER2 (17q12), myc (8q24) and cyclin D
(11q13), most of these genes are not contained within
some of the more prevalent amplicons (Tanner et al.,
The most common regions of high-copy amplification
in human breast cancer have been localized to 17q22 and
20q13.2 (Kallioniemi et al., 1994; Tanner et al., 1994,
1996). Low-level amplification of 20q has been described
in 6–18% of primary breast cancers and 40% of breast
cancer cell lines and the incidence increases to 60% in
BRCA2-positive breast cancers (Kallioniemi et al., 1994;
Tanner et al., 1994, 1996; Tirkkonen et al., 1997). High
levels of 20q amplification also correlate with poor
prognosis for patients with node-negative breast cancer
(Isola et al., 1995). Low-level amplification of 20q has
also been noted in colon cancer, ovarian cancer, bladder
cancer, gliomas, medulloblastomas, chondrosarcomas,
pancreatic tumors and head and neck cancers (Muleris
et al., 1987; Bigner et al., 1988; Yaseen et al., 1990;
Iwabuchi et al., 1995; Schlegel et al., 1995; Bockmuhl
et al., 1996; Courjal et al., 1996; Reznikoff et al., 1996;
Solinas-Toldo et al., 1996; James et al., 1997; Larramendy
et al., 1997). Relevant to this manuscript, several studies
have found chromosomal gains of 20q in ~60% of primary
colorectal carcinomas (Muleris et al., 1987; Yaseen et al.,
1990; Schlegel et al., 1995). Cell culture models have
suggested that low-level amplification of 20q is associated
with immortalization and subsequent high-level amplifica-
tion correlates with chromosomal instability (Savelieva
et al., 1997).
Recently, the CAS (cellular apoptosis susceptibility)
gene has been localized to the 20q13.2 amplicon (Brink-
mann et al., 1996). CAS is a human homologue of the
yeast CSE1 gene and functions as a transport factor to
bind importin-α and mediate its transport from the nucleus
back into the cytoplasm (Kutay et al., 1997). While CAS
is amplified or translocated in a variety of tumor cell
lines, it is amplified in only a restricted number of primary
tumors and does not appear to be the primary target of
the amplicon (Tanner et al., 1994, 1996).
Aurora2 maps adjacent to the CYP24 gene and the
cosmid probe RMC20C001 that have been proposed to
reside within the center of the 20q13.2 amplicon (Tanner
et al., 1994, 1996). Southern analysis confirms the tight
linkage of aurora2 to the amplicon. However, unlike
CYP24 and CAS, the amplification of aurora2 DNA also
correlates with overexpression of its transcript. A recent
report describes a search for expressed transcripts encoded
by the 20q amplicon in a panel of three breast cancer cell
lines, leading to the identification of a fragment of the
aurora2 gene (Sen et al., 1997). This report further
confirms our localization of aurora2 to this critical region.
Furthermore, we demonstrate the transforming effects of
the aurora2 kinase, making it a strong candidate for an
oncogene on the 20q13 amplicon which is predicted to
play a role in a wide variety of epithelial tumors. We
show here that the wild-type aurora2 transforms Rat1
fibroblasts. However, this same construct has no effect on
NIH 3T3 transformation, whereas the activated aurora2
mutant was able to transform both cell lines. A similar
selectivity in cellular transformation has been reported for
the serine/threonine kinase Pak1, which plays a role in
ras-dependent transformation of Rat1 fibroblasts, but not
of NIH 3T3 cells (Tang et al., 1997). It is conceivable
that the presence of normal cell cycle checkpoints in NIH
3T3 cells prevents the activation of the aurora2 protein.
Alternatively, it is possible that overexpression of aurora2
is transforming only in the presence of co-amplification
or mutation in additional genes.
Identification of aurora2 as a transforming protein
expands a rather limited list of serine/threonine kinase
oncogenes have been found to encode tyrosine kinases,
only a few are found to be STKs including: raf, mos,
pim1, cot, mek, akt and PLK1 kinases (Van Beveren et al.,
1981; Selten et al., 1986; Beck et al., 1987; Miyoshi
et al., 1991; Bellacosa et al., 1993; Cowley et al., 1994;
Smith et al., 1997). Many of these proteins are involved
in signaling through MAP kinase pathways (raf, mos, cot
and mek), whereas pim1 cooperates with c-Myc and akt
may be involved in the oncogenic signal transduced by
PI3 kinase (Hunter, 1997; Kinzler and Vogelstein, 1997).
Both PLK1 and aurora2 are cell-cycle-regulated STKs
and provide the first example to suggest a link between
centrosome integrity and cellular transformation.
cell cycle progression and in the process of centrosome
separation and chromosome segregation (Hoyt et al., 1991;
Elledge, 1996; Sherr, 1996; Weiss and Winey, 1996;
Taylor and McKeon, 1997). Until recently, no compelling
connection has been made between the proteins involved
in this process and cancer. The observation that mRNA
levels of human PLK1 are elevated in a majority of non-
small cell lung carcinomas and that high levels correlate
with poor prognosis (Wolf et al., 1997), implies either a
causal or symptomatic role in cancer for proteins involved
in the centrosome regulation. Microinjection of anti-PLK1
antibodies into HeLa cells and normal diploid Hs68
fibroblasts results in an inhibition of centrosome matura-
tion and a block in mitosis (Lane and Nigg, 1996, 1997).
In addition, the disruption of PLK1 by these antibodies
leads to the formation of abnormal nuclei in HeLa cells,
but not in Hs68 cells, suggesting that a checkpoint which
monitors centrosome maturation in normal cells may be
absent in tumor cells (Lane and Nigg, 1996, 1997).
Furthermore, constitutive overexpression of PLK1 trans-
forms NIH 3T3 cells (Smith et al., 1997). The genetics
of Drosophila aurora and the yeast Ipl1 protein kinases
suggest they may be involved in PLK1/polo/Cdc5p path-
way, or in a related parallel cascade. Indeed, the cell cycle
regulation and the intracellular localization of the human
aurora1 and aurora2 kinases are similar to those of
J.R.Bischoff et al.
PLK1. It is conceivable that these three proteins form a
centrosome-associated kinase cascade whose disruption
leads to genomic instability and chromosome segregation
defects. It will be important to determine whether aurora2
amplification results in a compensatory increase in PLK1
or aurora1 expression and if any of these proteins serve
as substrates for the others.
Materials and methods
Degenerate oligonucleotide primers were designed for PCR cloning
based on kinase domains I and IX of CCK4 (DDBJ/EMBL/GenBank
accession No. U33635) (Mossie et al., 1995), a receptor tyrosine kinase
expressed in a wide range of normal and transformed epithelial cells. The
sense primer was 5?-GARTTYGGNGARGTNTTYYTNGC-3?, encoding
the amino acids EFGEVFLA and the antisense primer was 5?-AGNACN-
CCRAANGCCCACACRTC-3?, encoding the complementary strand of
amino acids DVWAFGVL. These primers were applied to sscDNA
generated from RNA isolated from several colon cancer cell lines as
well as other tumor sources. PCR products of 500–600 bp were subcloned
and sequenced, revealing a fragment related to Drosophila aurora. This
fragment was used to probe a lambda library constructed from a pool
of several human pancreatic cancer cell line RNAs, leading to isolation
of full-length clones for human aurora1. Two weakly hybridizing clones
were also isolated and sequence analysis revealed that they represented
a related but distinct cDNA termed aurora2. Full-length clones were
also isolated for both genes from normal human duodenum cDNA. All
clones were sequenced on both strands with internal oligonucleotide
primers using both T7 polymerase manual sequencing and using dye-
terminator cycle sequencing with AmpliTaq DNA polymerase on an
ABI Prism 377. The complete aurora2 coding sequence was also
confirmed from 10 primary colorectal tumor samples. Primers
5?-CGCCTTTGCATCCGCTCCTG-3? and 5?-GATTTGCCTCCTGTG-
AAGAC-3? were used in an RT–PCR with sscDNA generated from the
tumor RNAs. The PCR products were purified by GeneClean and
sequenced directly by dye-terminator cycle sequencing with several
oligonucleotide primers. While no sequence differences were observed
between clones isolated from normal or tumor sources, we did identify
a single nucleotide polymorphism in two of the tumor samples which
would encode an F to I change at residue 31. Abbreviations for the
amino acid residues are: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G,
Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R,
Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. Abbreviations for
degenerate nucleotide residues are: R ? A or G; Y ? C or T; N ? A,
C, G or T.
Generation of Ipl1/aurora fusion constructs
DNA encoding the N-terminal 101 amino acids of S.cerevisiae Ipl1was
fused to a fragment encoding the C-terminal 270 amino acids (75–344)
of human aurora1, or to the C-terminal 273 amino acids (131–403) of
human aurora2, to generate plasmids Ipl1/A1 (pSG125) and Ipl1/A2
(pSG121), respectively. Kinase-dead forms of both plasmids were
constructed by oligonucleotide-directed mutagenesis to convert a Lys to
Met at the ATP binding site in the catalytic domains, generating
Ipl1/A1KM (pSG129) and Ipl1/A2KM (pSG1260). A full-length Ipl1
construct (pSG128) and the IPL/aurora fusions were subcloned into a
low-copy CEN URA3 plasmid (Sikorski and Hieter, 1989) under the
control of the Ipl1 native promoter. A hemagglutinin epitope (Pati, 1992)
was fused to the C-terminus of all the constructs. Cell lysates were
prepared from the wild-type yeast strain, CY184 (Zhu et al., 1995)
transformed with the individual expression plasmids and recombinant
proteins were detected by Western analysis with an anti-HA antibody.
Complementation in yeast
The Ipl1 and the AUR expression plasmids were transferred into the
ipl1-1 ts mutant strain, CCY464-1D (Francisco et al., 1994) and selected
on synthetic medium minus uracil (SC-URA) (Sherman et al., 1974).
Single transformants were isolated and restreaked in triplicate onto the
same medium and incubated for three days at 26°C, 34°C or 37°C.
amino acids of aurora1 conjugated to KLH or a purified GST fusion
protein containing the entire coding sequence of aurora2. Bleeds were
tested for their ability to immunoprecipitate in vitro-translated
35S-labeled aurora1 and aurora2, respectively. Aurora1- or aurora2-
specific immune sera were partially purified by protein A affinity
chromatography and frozen in small aliquots. The aurora1 antisera
immunoprecipitated a ~40 kDa protein from HeLa cells, close to the
predicted molecular weight of 39.4 kDa, and was competed by the
aurora1 peptide (unpublished data). The aurora1 antisera also recognized
a 40 kDa protein by immunoblotting of total HeLa cell lysates (unpub-
lished data). The aurora2 antisera immunoprecipitated a ~48 kDa protein
from HeLa cells, close to the predicted molecular weight of 45.8 kDa,
that was not detected by the preimmune serum. The aurora2 antisera
also recognized a 48 kDa protein by immunoblotting of total HeLa cell
lysates. In vitro kinase assays demonstrate that the aurora1 and aurora2
immune complexes both contained a kinase capable of phosphorylating
MBP, α-casein and protein kinase A (GST2TK, a GST vector containing
a PKA phosphorylation site), but not histone H1 (unpublished data).
Cell cycle analysis
Ten cm tissue culture dishes were seeded at a density of 3?106with
exponentially growing HeLa cells. The following day, thymidine (Sigma)
was added to the media to a final concentration of 2 mM and the plates
were incubated for 14 h at 37°C. The plates were then washed three
times with phosphate-buffered saline (PBS) and normal growth media
was added. Following 11 h at 37°C, aphidicolin (Sigma) was added to
a final concentration of 1 µg/ml and the plates were incubated at 37°C
for an additional 14 h. Plates were then washed three times with PBS
followed by the addition of normal growth media. This time point was
designated time zero. Flow cytometry was as previously described
(Bischoff et al., 1990). Total RNA was resolved on a 1.2% agarose gel
and transferred to a nylon membrane (Amersham). Full-length aurora1
and aurora2 cDNAs were32P-labeled by random priming (Stratagene)
and used to probe Northern blots. Total protein for immunoblots and
kinase assays were isolated as follows: HeLa cells were solubilized in
kinase lysis buffer (50 mM HEPES pH 7.4, 100 mM KCl, 25 mM NaF,
0.5% NP-40, 1 mM Na3VO4, 1 mM DTT and protease inhibitors) for
15 min on ice, spun in a microfuge at 10 000 g for 10 min at 4°C. The
resulting supernatant was transferred to a clean tube and the total protein
concentration was determined by Bradford analysis. Equal amounts of
protein, 50 µg and 500 µg, were loaded on gels for immunoblots or
immunoprecipitated for kinase assay, respectively. The immune com-
plexes were washed three times with kinase lysis buffer followed by
three washes with kinase buffer (without [γ-32P]ATP and artificial
substrate) and resuspended in 30 µl of 1? kinase buffer [20 mM HEPES
pH 7.4, 150 mM KCl, 5 mM MnCl2, 5 mM NaF, 1 mM DTT, 50 µM
ATP, 20 µCi [γ32P]ATP and 0.5 mg/ml myelin basic protein, GST2TK
protein (Pharmacia) or histone H1 (Boehringer Mannheim)]. In vitro
kinase reactions were carried out for 20 min at 37°C and stopped by the
addition of 30 µl of 2? Laemmli SDS sample buffer. Samples were
incubated for 5 min at 95°C and resolved on 14% SDS–polyacryl-
HeLa cells were plated on coverslips at ~25% confluency. The following
day the cells were washed once with ice-cold PBS and fixed with
methanol at –20°C overnight. The cells were washed three times with
ice-cold PBS followed by a 5 min incubation at room temperature in
PBS containing 0.05% Triton X-100. The permeabilized cells were
washed three times with ice-cold PBS and then covered with a solution
of 10% non-fat milk in PBS and incubated for 30 min at 37°C in a
humidified chamber. The antibodies, α-aurora1 and α-aurora2 and
α-tubulin, were diluted in PBS containing 10% non-fat milk, placed as
a drop on the coverslips, and incubated for 30 min at 37°C in a
humidified chamber. The coverslips were then washed six times with
PBS and covered with a solution containing goat anti-rabbit-FITC (Santa
Cruz Biotech), goat anti-mouse Rhodamine (Sigma) and 1 µg/ml DAPI
(Boehringer Mannheim) for 30 min in the dark at 37°C in a humidified
chamber. The coverslips were then washed six times with PBS and
attached to slides with clear nail polish.
Cell pellets from cultured tumor cell lines were provided by Nick
Scuidero (Developmental Therapeutics Program, NCI) and are part of the
NCI tumor panel (see website listing at http://epnws1.ncifcrf.gov:2345/
lial cells were obtained from Clontech. Normal human tissue samples
were obtained from the Cooperative Human Tissue Network (Cleveland,
Aurora2 oncogene amplified in colorectal cancers
were obtained from Los Angeles area hospitals including UCLA-Harbor,
Wadsworth and Cedars Sinai from 1988 to 1997. Tumor histology was
confirmed prior to preparing RNA, DNA and protein lysates from each
sample. Total cell or tissue RNA was isolated using the guanidine salts/
phenol extraction protocol of Chomczynski and Sacchi (1987). Northern
blotting was performed using standard techniques (Peles et al., 1997)
with a random-labeled 586 bp BamHI–SspI fragment of the human
aurora2 cDNA. A multiple tissue Northern blot and a human immune
system blot (Clontech) containing 2 µg poly(A)?mRNA per lane were
also probed for aurora2 expression. A human β-actin cDNA probe
(Clontech) was used to confirm equivalent loading of intact RNA. RNA
(10 µg) from the NCI-H23 lung cancer cell line served as an internal
standard for detection of aurora2 expression on each blot. Blots were
quantitated usinga phosphorimagerand ImageQuantsoftware (Molecular
Dynamics, Mountain View, CA).
The Stanford G3 radiation hybrid panel was obtained from Research
Genetics (Huntsville, Alabama). Aurora2 primers used for radiation
hybrid mapping were: 5?-CAGGGCTGCCATATAACCTGA-3? and
5?-CTAGCACAGGCTGACGGGGC-3?. The aurora2 primers amplify a
255 bp fragment from the 3? UTR following a 25-cycle PCR with a
54°C annealing temperature. The raw score for aurora2 against the
SHGCR G3 panel is: 100000000010001010000001000100100000000
Genomic DNA was isolated from the human colorectal tissue samples
by standard methods (Proteinase K digestion, phenol:chloroform extrac-
tion and ethanol precipitation). Southern blots were prepared by digesting
5 µg of DNA with PstI, separating the fragments on 1% agarose gels,
blotting onto nylon membranes (Nytran-Plus, Schleicher & Schuell) and
probing sequentially with a random primer-labeled 1044 bp aurora2
cDNA fragment (pSG19) and a 1700 bp cloned fragment of the CYP24
gene (pKS-h24; from J.Omdahl, University of New Mexico). A probe
for human β-globin was used to confirm equivalent sample loading.
Final washes were at 0.1? SSC, 0.1% SDS, 60°C. Autoradiographs were
quantitated relative to β-globin using ImageQuant software (Molecular
Dynamics, Mountain View, CA).
Statistical significance of the correlation between DNA amplification
and RNA overexpression was calculated using Pearson correlation and
the one-tailed Fisher’s exact test using SAS Release 6.12.
Matched human tissue samples from primary colorectal carcinomas and
adjacent normal tissue were obtained from the Cooperative Human
Tissue Network (Cleveland, OH) and Pathology Associates International
(Frederick, MD). Thirty µm cryostat sections of OCT-embedded tissue
was lysed directly in 25 µl of ice-cold RIPA buffer (50 mM Tris–Cl pH
8.0, 150 mM NaCl, 1.0% NP-40, 0.5% deoxycholate, 0.1% SDS, 1 mM
DTT and protease inhibitors) by gentle mixing on ice for 20 min. The
lysate was then spun for 10 min at 10 000 g in a microfuge at 4°C. The
resulting supernatant was transferred to a clean tube and the total protein
concentration determined by Bradford analysis. Equal amounts of total
gel, transferred to a nylon membrane (BioRad) and probed with a 1:2000
dilution of protein A-purified antibodies to aurora2. The immunoblot
was developed with ECL reagent (Amersham). Lysates from tumor cell
lines were prepared and analyzed as described above.
HA-tagged (Pati, 1992) versions of wild-type, kinase-dead (K162M) and
activated (T288D) aurora2 were subcloned into the expression vector
pLXSN. These constructs were transfected into the amphotropic pack-
aging cell line PA317 and the supernatants were harvested and used to
infect the producer cell line GP?E-86 (Markowitz et al., 1988).
Neomycin-resistant clones were selected and assayed for aurora2 protein
expression (unpublished data). Supernatants from the positive producer
cell lines were used to infect Rat1 and NIH 3T3 cells. Stable clones
were selected for in the presence of neomycin and assayed for aurora2
protein expression by immunoblotting.
Soft agar assays
A 3% solution of agar (at 56°C) was diluted to a final concentration of
0.6% with growth medium (at 56°C), pipetted into tissue culture dishes
and allowed to solidify at room temperature for 20–30 min. At this time,
2?105cells in a volume of 50 µl were mixed with 0.3% agar (diluted
with growth medium at 40°C), pipetted gently onto the bottom agar
layer and allowed to solidify for 20–25 min at room temperature. Once
solidified, the plates were incubated at 37°C in a 5% CO2atmosphere.
Fresh top agar was added once a week. After 4 weeks the plates were
stained with neutral red.
In vivo tumor growth
Animal experiments in 4- to 6-week-old male athymic Balb/c nu/nu
mice were carried out in accordance with both institutional and federal
animal care regulations. NIH 3T3 cells expressing the kinase-inactive
(K162M) or activated kinase (T288D) construct were grown in DMEM
supplemented with 10% FBS. Cells were harvested by trypsinization,
centrifuged at 300 g for 5 min, washed twice and resuspended in sterile
PBS. 1?106cells in 0.2 ml were injected subcutaneously between the
scapula of each mouse. Tumor volumes were estimated by caliper
measurements at 4, 6 and 8 weeks.
We thank Sara Courtneidge for her critical review and continued
encouragement with this project and T.Kerlavage for providing access
to the multiple sequence alignment program, msa. D.J.S was supported
in part by a grant from the Revlon/UCLA Women’s Cancer Research
Program, and C.S.M.C. by grant GM45185 from the NIH, and grant
#4496 from The Council for Tobacco Research.
Andreassen,P.R., Palmer,D.K., Wener,M.H. and Margolis,R.L. (1991)
Telophase disc: a new mammalian mitotic organelle that bisects cell
with a possible function in cytokinesis. J. Cell Sci., 99, 523–534.
Beck,T.W., Huleihel,M., Gunnell,M., Bonner,T.I. and Rapp,U.R. (1987)
The complete coding sequence of the human A-raf-1 oncogene and
transforming activity of a human A-raf carrying retrovirus. Nucleic
Acids Res., 15, 595–609.
Bellacosa,A., Franke,T.F., Gonzalez-Portal,M.E., Datta,K., Taguchi,T.,
Gardner,J., Cheng,J.Q., Testa,J.R. and Tsichlis,P.N. (1993) Structure,
expression and chromosomal mapping of c-akt: relationship to v-akt
and its implications. Oncogene, 8, 745–754.
Bigner,S.H., Mark,J., Friedman,H.S., Biegel,J.A. and Bigner,D.D. (1988)
Structural chromosomal abnormalities in human medulloblastoma.
Cancer Genet. Cytogenet., 30, 91–101.
Bischoff,J.R., Friedman,P.N., Marshak,D.R., Prives,C. and Beach,D.
(1990) Human p53 is phosphorylated by cyclin B-cdc2 and p60-cdc2.
Proc. Natl Acad. Sci. USA, 87, 4766–4770.
Bockmuhl,U., Petersen,I., Schwendel,A. and Dietel,M. (1996) Genetic
screening of head-neck carcinomas using comparative genomic
hybridization (CGH). Laryngorhinootologie, 75, 408–414.
Boveri,T. (1929) The Origin of Malignant Tumors. Williams & Wilkins,
Brinkmann,U., Gallo,M., Polymeropoulos,M.H. and Pastan,I. (1996) The
human CAS (cellular apoptosis susceptibility) gene mapping on
chromosome 20q13 is amplified in BT474 breast cancer cells and part
of aberrant chromosomes in breast and colon cancer cell lines. Genome
Res., 6, 187–194.
Chomczynski,P. and Sacchi,N. (1987) Single-step method of RNA
extraction. Anal. Biochem., 162, 156–159.
Courjal,F., Cuny,M., Rodriguez,C., Louason,G., Speiser,P., Katsaros,D.,
Tanner,M.M., Zeillinger,R. and Theillet,C. (1996) DNA amplifications
at 20q13 and MDM2 define distinct subsets of evolved breast and
ovarian tumours. Br. J. Cancer, 74, 1984–1989.
Cowley,S., Paterson,H., Kemp,P. and Marshall,C.J. (1994) Activation of
MAP kinase kinase is necessary and sufficient for PC12 differentiation
and for transformation of NIH 3T3 cells. Cell, 77, 841–852.
el-Deiry,W.S. et al. (1993) WAF1, a potential mediator of p53 tumor
suppression. Cell, 75, 817–825.
Elledge,S.J. (1996) Cell cycle checkpoints: preventing an identity crisis.
Science, 274, 1664–1672.
Francisco,L., Wang,W. and Chan,C.S. (1994) Type 1 protein phosphatase
segregation. Mol. Cell. Biol., 14, 4731–4740.
J.R.Bischoff et al.
Glover,D.M., Leibowitz,M.H., McLean,D.A. and Parry,H. (1995)
Mutations in aurora prevent centrosome separation leading to the
formation of monopolar spindles. Cell, 81, 95–105.
Golsteyn,R.M., Schultz,S.J., Bartek,J., Ziemiecki,A., Ried,T. and
Nigg,E.A. (1994) Cell cycle analysis and chromosomal localization
of human Plk1, a putative homologue of the mitotic kinases Drosophila
polo and Saccharomyces cerevisiae Cdc5. J. Cell Sci., 107, 1509–1517.
Gopalan,G., Chan,C.S.M. and Donovan,P.J. (1997) A novel mammalian,
segregation regulators. J. Cell Biol., 138, 643–656.
Hartwell,L.H. and Kastan,M.B. (1994) Cell cycle control and cancer.
Science, 266, 1821–1828.
Hoyt,M.A., Totis,L. and Roberts,B.T. (1991) S. cerevisiae genes required
for cell cycle arrest in response to loss of microtubule function. Cell,
Hunter,T. (1997) Oncoprotein networks. Cell, 88, 333–346.
Hunter,T. and Pines,J. (1994) Cyclins and cancer. II: Cyclin D and CDK
inhibitors come of age. Cell, 79, 573–582.
Isola,J.J., Kallioniemi,O.P., Chu,L.W., Fuqua,S.A., Hilsenbeck,S.G.,
Osborne,C.K. and Waldman,F.M. (1995) Genetic aberrations detected
by comparative genomic hybridization predict outcome in node-
negative breast cancer. Am. J. Pathol., 147, 905–911.
Iwabuchi,H., Sakamoto,M., Sakunaga,H., Ma,Y.Y., Carcangiu,M.L.,
Pinkel,D., Yang-Feng,T.L. and Gray,J.W. (1995) Genetic analysis of
benign, low-grade and high-grade ovarian tumors. Cancer Res., 55,
Comparative genomic hybridisation of ductal carcinoma in situ of the
breast: identification of regions of DNA amplification and deletion in
common with invasive breast carcinoma. Oncogene, 14, 1059–1065.
Kallioniemi,A., Kallioniemi,O.P., Sudar,D., Rutovitz,D., Gray,J.W.,
Waldman,F. and Pinkel,D. (1992) Comparative genomic hybridization
for molecular cytogenetic analysis of solid tumors. Science, 258,
Kallioniemi,A. et al. (1994) Detection and mapping of amplified DNA
sequences in breast cancer by comparative genomic hybridization.
Proc. Natl Acad. Sci. USA, 91, 2156–2160.
Kimura,M., Kotani,S., Hattori,T., Sumi,N., Yoshioka,T., Todokoro,K.
and Okano,Y. (1997) Cell cycle-dependent expression and spindle
pole localization of a novel human protein kinase, Aik, related to
Aurora of Drosophila and yeast Ipl1. J. Biol. Chem., 272, 13766–
Kinzler,K.W. and Vogelstein,B. (1997) Cancer-susceptibility genes.
Gatekeepers and caretakers. Nature, 386, 761–763.
Kutay,U., Bischoff,F., Kostka,S., Kraft,R. and Go ¨rlich,D. (1997) Export
of importin alpha from the nucleus is mediated by a specific nuclear
transport factor. Cell, 90, 1061–1071.
Lane,H.A. and Nigg,E.A. (1996) Antibody microinjection reveals an
essential role for human polo-like kinase 1 (Plk1) in the functional
maturation of mitotic centrosomes. J. Cell Biol., 135, 1701–1713.
Lane,H.A. and Nigg,E.A. (1997) POLO-like kinases join the outer circle.
Trends Cell Biol., 7, 63–68.
Larramendy,M.L., Tarkkanen,M., Valle,J., Kivioja,A.H., Ervasti,H.,
Karaharju,E., Salmivalli,T., Elomaa,I. and Knuutila,S. (1997) Gains,
losses and amplifications of DNA sequences evaluated by comparative
genomic hybridization in chondrosarcomas. Am. J. Pathol., 150,
Markowitz,D., Goff,S. and Bank,A. (1988) Construction and use of a
safe and efficient amphotropic packaging cell line. Virology, 167,
Mitelman,F., Mertens,F. and Johansson,B. (1997) A breakpoint map of
recurrent chromosomal rearrangements in human neoplasia. Nature
Genet., 15, 417–474.
Miyoshi,J., Higashi,T., Mukai,H., Ohuchi,T. and Kakunaga,T. (1991)
Structure and transforming potential of the human cot oncogene
encoding a putative protein kinase. Mol. Cell. Biol., 11, 4088–4096.
Mossie,K., Jallal,B., Alves,F., Sures,I., Plowman,G.D. and Ullrich,A.
(1995) Colon carcinoma kinase-4 defines a new subclass of the
receptor tyrosine kinase family. Oncogene, 11, 2179–2184.
Motokura,T., Bloom,T., Kim,H.G.,
Kronenberg,H.M. and Arnold,A. (1991) A novel cyclin encoded by a
bcl1-linked candidate oncogene. Nature, 350, 512–515.
and Dutrillaux,B. (1987) Characteristic chromosomal imbalances in
18 near-diploid colorectal tumors. Cancer Genet. Cytogenet., 29,
Nigg,E.A., Blangy,A. and Lane,H.A. (1996) Dynamic changes in nuclear
architecture during mitosis: on the role of protein phosphorylation in
spindle assembly and chromosome segregation. Exp. Cell Res., 229,
Pati,U.K. (1992) Novel vectors for expression of cDNA encoding
epitope-tagged proteins in mammalian cells. Gene, 114, 285–288.
Peles,E., Nativ,M., Lustig,M., Grumet,M., Schilling,J., Martinez,R.,
Plowman,G.D. and Schlessinger,J. (1997) Identification of a novel
contactin-associated transmembrane receptor with multiple domains
implicated in protein–protein interactions. EMBO J., 16, 978–988.
Reznikoff,C.A., Belair,C.D., Yeager,T.R., Savelieva,E., Blelloch,R.H.,
Puthenveettil,J.A. and Cuthill,S. (1996) A molecular genetic model
of human bladder cancer pathogenesis. Semin. Oncol., 23, 571–584.
Waldman,F. and Reznikoff,C.A. (1997) 20q gain associates with
immortalization: 20q13.2 amplification correlates with genome
instability in human papillomavirus 16 E7 transformed human
uroepithelial cells. Oncogene, 14, 551–560.
Schlegel,J., Stumm,G., Scherthan,H., Bocker,T., Zirngibl,H., Ruschoff,J.
and Hofstadter,F. (1995) Comparative genomic in situ hybridization of
colon carcinomas with replication error. Cancer Res., 55, 6002–6005.
Selten,G., Cuypers,H.T., Boelens,W., Robanus-Maandag,E., Verbeek,J.,
Domen,J., van Beveren,C. and Berns,A. (1986) The primary structure
of the putative oncogene pim-1 shows extensive homology with
protein kinases. Cell, 46, 603–611.
Sen,S., Zhou,H. and White,R.A. (1997) A putative serine/threonine
kinase encoding gene BTAK on chromosome 20q13 is amplified and
overexpressed in human breast cancer cell lines. Oncogene, 14,
Sherman,F., Fink,G. and Laurence,C. (1974). Methods in Yeast Genetics.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Sherr,C.J. (1996) Cancer cell cycles. Science, 274, 1672–1677.
Sikorski,R.S. and Hieter,P. (1989) A system of shuttle vectors and
yeast host strain designed or efficient manipulation of DNA in
Saccharomyces cerevisiae. Genetics, 122, 19–27.
McGuire,W.L. (1987) Human breast cancer: correlation of relapse and
survival with amplification of the HER-2/neu oncogene. Science, 235,
and Ferris,D.K. (1997) Malignant transformation of mammalian cells
initiated by constitutive expression of the polo-like kinase. Biochem.
Biophys. Res. Commun., 234, 397–405.
Solinas-Toldo,S., Wallrapp,C., Muller-Pillasch,F., Bentz,M., Gress,T. and
Lichter,P. (1996) Mapping of chromosomal imbalances in pancreatic
carcinoma by comparative genomic hybridization. Cancer Res., 56,
Tang,Y., Chen,Z., Ambrose,D., Liu,J., Gibbs,J.B., Chernoff,J. and Field,J.
(1997) Kinase-deficient Pak 1 mutants inhibit ras transformation of
Rat-1 fibroblasts. Mol. Cell. Biol., 17, 4454–4464.
Tanner,M.M. et al. (1994) Increased copy number at 20q13 in breast
cancer: defining the critical region and exclusion of candidate genes.
Cancer Res., 54, 4257–4260.
Tanner,M.M. et al. (1996) Independent amplification and frequent
co-amplification of three nonsyntenic regions on the long arm of
chromosome 20 in human breast cancer. Cancer Res., 56, 3441–3445.
Taylor,S.S. and McKeon,F. (1997) Kinetochore localization of murine
Bub1 is required for normal mitotic timing and checkpoint response
to spindle damage. Cell, 89, 727–735.
AIM-1: a mammalian midbody-associated protein required for
cytokinesis. EMBO J., 17, 677–676.
Tirkkonen,M. et al. (1997) Distinct somatic genetic changes associated
with tumor progression in carriers of BRCA1 and BRCA2 germ-line
mutations. Cancer Res., 57, 1222–1227.
Van Beveren,C., Galleshaw,J.A., Jonas,V., Berns,A.J., Doolittle,R.F.,
Donoghue,D.J. and Verma,I.M. (1981) Nucleotide sequence and
formation of the transforming gene of a mouse sarcoma virus. Nature,
van de Vijver,M.J. (1993) Molecular genetic changes in human breast
cancer. Adv. Cancer Res., 61, 25–56.
Weiss,E. and Winey,M. (1996) The Saccharomyces cerevisiae spindle
pole body duplication gene Mps1 is part of a mitotic checkpoint.
J. Cell Biol., 132, 111–123.
Aurora2 oncogene amplified in colorectal cancers Download full-text
Wolf,G., Elez,R., Doermer,A., Holtrich,U., Ackermann,H., Stutte,H.J.,
Altmannsberger,H.M., Rubsamen-Waigmann,H. and Strebhardt,K.
(1997) Prognostic significance of polo-like kinase (PLK) expression
in non-small cell lung cancer. Oncogene, 14, 543–549.
Xiong,Y., Hannon,G.J., Zhang,H., Casso,D., Kobayashi,R. and Beach,D.
(1993) p21 is a universal inhibitor of cyclin kinases. Nature, 366,
Yanai,A., Arama,E., Kilfin,G. and Motro,B. (1997) Ayk1, a novel
mammalian gene related to Drosophila aurora centrosome separation
kinase, is specifically expressed during meiosis. Oncogene, 14,
Yaseen,N.Y., Watmore,A.E., Potter,A.M., Potter,C.W., Jacob,G. and
Rees,R.C. (1990) Chromosome studies in eleven colorectal tumors.
Cancer Genet. Cytogenet., 44, 83–97.
Zhu,Y., Peterson,C.L. and Christman,M.F. (1995) HPR1 encodes a global
positive regulator of transcription in Saccharomyces cerevisiae. Mol.
Cell. Biol., 15, 1698–1708.
Received February 16, 1998; revised and accepted March 27, 1998