[Cancer Research 60, 4697-4700, September 1, 2000]
© 2000 American Association for Cancer Research
Aberrant Transcripts of the Cyclin-dependent Kinase-associated Protein Phosphatase in Hepatocellular Carcinoma1
Chau-Ting Yeh2,
Su-Chuan Lu,
Tse-Ching Chen,
Cheng-Yuan Peng and
Yun-Fan Liaw
Liver Research Unit [C-T. Y., S-C. L., C-Y. P., Y-F. L.] and Department of Pathology [T-C. C.], Chang Gung Memorial Hospital and Chang Gung University School of Medicine, Taipei, Taiwan
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ABSTRACT
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The cyclin-dependent kinase (Cdk)-associated protein phosphatase (KAP)
is a human dual specificity protein phosphatase that
dephosphorylates Cdk2 on threonine 160 in a
cyclin-dependent manner. To investigate whether mutations of this
enzyme occur in hepatocellular carcinoma (HCC), KAP mRNA was analyzed
by reverse transcription-PCR (RT-PCR), followed by cloning and
sequencing. Eight of 14 biopsy tissues obtained from advanced HCC, 6 of
13 surgically removed HCC tissues, and 2 of the adjacent noncancerous
tissues contained aberrant KAP transcripts. Using the yeast two-hybrid
system, five of seven representative KAP mutants were shown to be
defective in interacting with Cdk2. These data suggest a possible role
of KAP mutations in multiple-step hepatocarcinogenesis.
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Introduction
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Activation of
Cdk23
requires binding of the Cdk to a corresponding cyclin and
phosphorylation of Cdk2 at a conserved threonine residue (T160 in Cdk2;
Refs. 1
and 2
). The activity of an activated,
T160 phosphorylated Cdk2 can be inhibited by phosphorylation of the
other two conserved residues in the catalytic cleft (tyrosine 15 and
threonine 14; Refs. 3
and 4
). Such regulatory
phosphorylation pathways are conserved among species, from yeast to
humans (5)
. Several kinases have been shown to
phosphorylate T160, such as Cdk7-cyclin H, which is present in the
transcription factor TFIIH in humans and Cdk-activating kinase 1 in
budding yeast (6
, 7)
. On the other hand, dephosphorylation
of T160 can be achieved by enzymes such as protein phosphatase 2A and a
dual-specific protein phosphatase, i.e., KAP (8
, 9)
. Because of their capabilities of phosphorylating or
dephosphorylating T160, these enzymes are believed to play a role in
regulating the Cdk activity. KAP, also named cyclin dependent kinase
interactor 1, is expressed at the G1-to-S
transition of the cell cycle and forms stable complexes with Cdk2.
Overexpression of wild-type KAP delays progression through the cell
cycle in yeast and HeLa cells, and the delay is dependent on its
phosphatase activity (10)
. This enzyme contains the
HCXX-XXGR motif characteristic of protein tyrosine phosphatases, but it
can dephosphorylate substrates containing other phosphorylated amino
acid residues (11)
. In native monomeric Cdk2, KAP was able
to dephosphorylate only T160 but not Y15. The binding of cyclin A to
Cdk2 inhibited the dephosphorylation but did not preclude the binding
of KAP to the cyclin A-Cdk2 complex. The dephosphorylation of T160 by
KAP inhibits Cdk2 activity upon subsequent association with cyclin A
(9)
. It is therefore believed that KAP binds to Cdk2 and
dephosphorylates T160 to inactivate Cdk2 when the associated cyclin
subunit is degraded. Although the function of KAP was well
characterized in vitro, its contribution in cell cycle
regulation is still uncertain in vivo (8)
.
Mutations of several tumor suppresser genes have been identified in
HCC. According to the in vitro experiments mentioned above,
KAP might function similarly to a tumor suppresser. In this study, we
investigated whether mutations occurred in KAP genes
in HCC by analyzing the nucleotide sequence of its mRNA. Strikingly,
several aberrant transcripts of this gene were identified. The possible
role of these mutants in multiple-step hepatocarcinogenesis was
discussed.
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Patients and Methods
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Patients.
Three groups of patients were included:
Group 1, 12 randomly selected patients who received liver biopsy
because of chronic hepatitis B or C, and the pathological examination
showed minimal hepatitis using the Knodell scoring system
(12)
.
Group 2, 14 randomly selected patients (accrued December 1997 to
December 1998) who received liver biopsy under the clinical
impression of advanced HCC, and the diagnosis was subsequently
confirmed by pathological examination. None of these patients received
surgical treatment because of intrahepatic spread of the tumors (in 11
cases), huge size of tumor (in 1 case), or both (in 2 cases).
Group 3, 13 patients (accrued June 1997 to December 1998) who received
partial hepatectomy for HCC. These tumors were 2.8–5.2 cm in size,
except for tumor T3, which was 14 cm in size. Cancerous and
noncancerous parts of HCCs were separated by the surgeons and examined
by a pathologist. All collected tissues were frozen in liquid nitrogen
until analysis. The sex, age, and serological markers for hepatitis B
or C are listed in Table 1
.
Serology.
Hepatitis B surface antigen was assayed using a commercially available
RIA (Austria II; Abbott Laboratories, Chicago, IL). Antibody to
hepatitis C virus (anti-hepatitis C virus) was assayed using an enzyme
immunoassay (Abbott HCV EIA III; Abbott Laboratories).
Extraction of Total Cellular RNA.
Total RNA was isolated from both cancerous and noncancerous liver
tissues using TRI Reagent (Molecular Research Center, Inc., Cincinnati,
OH) according to the manufacturer’s instructions. To eliminate
contaminated DNA, the extracted RNA was dissolved in diethyl
pyrocarbonate-treated water containing 10 mmol/l of
MgCl2 and incubated with 100 µg/ml of
RNase-free DNase I for 30 min at 37°C. EDTA was added to a final
concentration of 30 mmol/l, and the mixture was heated at 95°C for 5
min to stop the reaction.
RT-PCR, Cloning, and Sequencing.
Total RNA derived from tumors or noncancerous liver tissues was
subjected to RT-PCR. The primers were designed according to a published
KAP sequence: 5'-ACTGGTCTCGACGTGGGGCG-3' (nucleotides 25–44, sense)
and 5'-GTTGATAACACTGGTGGTTTC-3' (nucleotides 780–760, antisense; Ref.
11
). The resulting PCR fragment contained the entire
coding sequence of KAP. To confirm the short stretches of nucleotide
deletions identified in some cases, two additional primers were
synthesized: 5'-CCAGCGATGAAGCCGCCCAG-3' (nucleotides 46–65, sense) and
5'-AGCTGGCTATGTCAGGAGTC-3' (nucleotides 406–387, antisense). The
antisense primer was used for reverse transcription. The details of
RT-PCR were described elsewhere (13
, 14)
. After RT-PCR,
the product was gel purified and cloned into a vector, pCR2.1-TOPO
(Invitrogen, Carlsbad, CA). If multiple bands were found in the agarose
gel, each band was gel purified and cloned into the vector separately.
At least five clones were selected and sequenced for each PCR product.
Nucleotide sequencing was performed using an automatic DNA sequencer
(CEQ 2000; Beckman Instruments, Inc., Fullerton, CA).
Interaction Assay by the Yeast Two-Hybrid System.
Protein-protein interaction between Cdk2 and KAP mutants was evaluated
using the yeast two-hybrid system (Matchmaker LexA two-hybrid system;
Clontech Laboratories, Inc., Palo Alto, CA). Primers containing an
engineered EcoRI or XhoI site were first
synthesized: P1,
5'-GGAATTCATGGAGAACTTCCAAAAGG-3' (sense);
P2, 5'-ACTCGAGGATGGGGTACTGGCTTGG-3' (antisense); P3,
5'-GGAATTCATGAAGCCGCCCAGTTCAATAC-3' (sense); and P4,
5'-CCTCGAGTCTTGATACAGATCTTGATTG-3' (antisense). The
engineered sites were underlined. P1 and P2 were designed according to
the 5' and 3' ends of Cdk2 coding region, whereas P3 and P4 were
designed according to the 5' and 3' ends of the KAP coding region. The
Cdk2 coding region was then amplified by RT-PCR using normal liver mRNA
(purchased from Invitrogen), verified by nucleotide sequencing,
digested with restriction enzymes, and inserted (in-frame) into
EcoRI-XhoI sites of pLexA, a plasmid encoding the
202-residue LexA protein and HIS3 marker. The wild-type or
mutant KAP coding region was amplified from the RT-PCR-derived clone
used for mutation analysis (see the above section). The amplified
coding region was verified by nucleotide sequencing and inserted
(in-frame) into the EcoRI-XhoI sites of pBD42AD,
a plasmid encoding the 88-residue B42 acidic activator and
TRP1 marker. To demonstrate the presence of interaction
between the two proteins, the two plasmids were cotransfected into
EGY48[p8op-lacZ] yeast cells, a yeast strain (EGY48) transformed with
the autonomously replicating plasmid p8op-lacZ (containing
URA3 marker). This system allows double selection with both
leucine prototrophy and lacZ reporter. Cotransformants were selected in
SD/-His/-Trp/-Ura medium. Protein-protein interaction was demonstrated
by growth of the cotransformants as blue colonies on an
SD/Gal/Raf/-His/-Trp/-Ura/-Leu plate containing
5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside.
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Results
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The basic data of the three groups of patients are summarized in
Table 1
. They were not significantly different in the number of
patients, sex, and the distribution of hepatitis B and C markers. Group
2 patients were, however, significantly older than the other two
groups. The tissues in group 2 were named BX-01 to BX-14. The samples
in group 3 were named NT1 to NT13 (noncancerous tissues) and T1 to T13
(cancerous tissues). The short stretches of nucleotide deletions in
nucleotides 6 to 52 were confirmed by RT-PCR using a second pair of
primers (see "Materials and Methods"). The nucleotide sequence data
were deposited with GenBank (accession nos. AF213033 to AF213035). The
interpreted amino acid sequences of mutants are listed in Fig. 1
.
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Fig. 1. The predicted amino acid sequences of KAP mutants. The
nucleotide sequence data of these mutants were deposited with GenBank
(accession nos. AF213033 to AF213035). A prototype sequence was listed
at the top row as a reference (11)
. Single-letter symbols
were used. -, the same amino acid residue as in the reference
sequence; /, amino acid deletion; #, stop codon.
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All 12 patients in group 1 had wild-type KAP sequence, whereas 8 of 14
and 6 of 13 cancerous tissues of group 2 and 3, respectively, had
mutant KAP sequences. Of group 2 tissues, 4 had nucleotide
substitutions leading to either premature stop codons (Mpst; BX-03 and
07) or amino acid substitutions (Maas; BX-01, 05, 07 and 10), 1 had a
single nucleotide insertion leading to a truncated reading frame
(Mtrf-i; BX-11), and 2 had short stretches of deletions leading to
either truncated reading frames (Mtrf-d; BX-02) or in-frame amino acid
deletions (Mifd; BX-09). In group 2 tissues harboring KAP mutants, only
one form of mutant was detected in each sample. A mixture of wild and
mutant type sequences were detected in two samples: BX-11 and BX-03.
Of the cancerous parts of the 13 paired samples in group 3, none had
Mpst and Mtrf-i mutants but 2 (T4 and T9) showed Maas, 2 (T6 and T7)
showed Mtrf-d, and 4 (T2, T3, T4, and T6) showed Mifd mutants,
respectively. Two different kinds of Mifd mutants were present in T4
(T4-1 and T4-2). Otherwise, only one kind of mutant was detected in
other cancerous tissues of group 3. Wild-type KAP sequences coexisted
with the mutants as a mixture in all of these cancerous tissues except
for T3.
In the noncancerous parts of the paired tissues, mutations were
detected in four samples. In one particular patient, a single amino
acid substitution was found in the noncancerous tissue (NT1), but only
wild-type sequence was present in the cancerous tissue (T1). In NT4
tissue, two amino acid substitutions were identified, which were also
present in T4-1 and T4-2. In the remaining two cases, the noncancerous
parts contained not only the mutants identical to the cancerous parts
(NT2-1/T2 and NT3-1/T3) but also an additional distinct mutant for each
sample (NT2-2 and NT3-2). In all these noncancerous tissues, wild-type
KAP sequences coexisted with the mutants.
Using the yeast two-hybrid system, the interactions between Cdk2 and
KAP proteins were studied. The wild-type KAP and seven KAP mutants
(BX-02, BX-07, T2, T3, T4-2, T7 and NT2-2) were tested. Only wild-type
KAP, T4-2, and T7 mutants were capable of interaction with Cdk2 protein
(Table 2)
.
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Discussion
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KAP is capable of dephosphorylating T160 of Cdk2 and thus
inhibiting its function in vitro (9)
. Although
it is seemingly an important regulator in the cell cycle, solid
evidence demonstrating its crucial role in vivo is lacking
(8)
. In this report, we discovered that multiple forms of
KAP mutants were present in HCC but not in hepatitis tissues.
The patterns of KAP mutants were somewhat different between the
advanced HCC group (group 2) and the resectable HCC group (group 3):
(a) there were two cases with Mpst mutation in group 2 but
none in group 3, whereas there was only one case with Mifd mutation in
group 2 but four in group 3; and (b) except for 1 cancerous
sample, all tissues harboring mutants in group 3 also possessed
wild-type KAP sequences, whereas only two samples in group 2 contained
a mixture of wild- and mutant-type KAP. Taken together, there seemed to
be a trend that in the precancerous/early stage of HCC, multiple forms
of KAP mutants, with a majority in the forms of Mifd, developed. These
mutants coexisted with the wild-type KAP sequence. When the tumor grew
to an advanced stage, the cell clones with truncated reading frames
either caused by Mpst, Mtrf-d, or Mtrf-i mutation were gradually
selected, whereas the clones with only wild-type sequence or Mifd were
lost. The process of selection was reflected in NT2/T2 and NT3/T3, in
which two forms of mutants were present in the noncancerous parts,
whereas only one form was found in the tumor part. The presence of
single but not mixed forms of mutants at the advanced stage possibly
attributed to deletion of one of the KAP alleles (loss of
heterozygosity). Establishment of a valid assay is needed to verify
this point. Although this was the likely scheme observed in our
patients with KAP mutants, about half of our samples harbored only
wild-type KAP sequence. It is still possible that one of the alleles
was totally deleted, and thus only the remaining wild-type gene was
detected, or the regions matched to our detecting primers were deleted
so that the transcripts cannot be amplified. Alternatively, in such
cases, other mechanisms unrelated to the disruption of the
KAP gene were involved in hepatocarcinogenesis.
Because most of the mutants coexisted with the wild-type KAP, it is
possible that such mutants interfere with the dephosphorylation ability
of wild-type KAP through a competition mechanism. For instance, mutants
such as T4-2 and T7 might still bind to Cdk2 but fail to
dephosphorylate T160. On the other hand, all but one in-frame amino
acid deletion occurred in amino acids 6–52, suggesting that a common
mechanism was used. These mutants could be functionally defective,
thereby facilitating the growth of tumor cells. Although it is possible
to assay the phosphorylation status of T160 in HCC tissues, the
results could be very difficult to decipher. Factors other than KAP
were shown to regulate phosphorylation of T160 (see
"Introduction"). It is therefore difficult to correlate the
presence of KAP mutations and the status of T160 phosphorylation in HCC
tissues. In contrast, using the yeast two-hybrid system, the
interaction between Cdk2 and KAP mutants can be clearly demonstrated.
Our results indicated that some KAP mutants failed to bind to Cdk2 and
were thus functionally defective.
In summary, we have discovered that in HCC, multiple forms of aberrant
KAP transcripts were present. In two cases, the aberrant transcripts in
the cancerous tissues were also present in the nearby noncancerous
tissues, suggesting that the mutations occurred in a precancerous stage
and likely contributed to the process of multiple-step
hepatocarcinogenesis.
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ACKNOWLEDGMENTS
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We thank Dr. Chen Min-Fu (Department of Surgery, Chang Gung
Memorial Hospital) for providing the surgical specimens.
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Grant CMRP-800 from
the Chang Gung Medical Research Council. 
2 To whom requests for reprints should be
addressed, at Liver Research Unit, Chang Gung Memorial Hospital, 199,
Tung Hwa North Road, Taipei, Taiwan. Phone: 886-3-3281200, extension
8120; Fax: 886-3-3282824; E-mail: catyeh{at}ms14.hinet.net
3 The abbreviations used are: Cdk2,
cyclin-dependent kinase 2; Cdk, cyclin-dependent kinase; T160,
threonine 160; KAP, Cdk-associated protein phosphatase; HCC,
hepatocellular carcinoma; RT-PCR, reverse transcription-PCR.
Received 1/ 6/00.
Accepted 7/18/00.
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ABSTRACT
Introduction
