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HONG SUG KIM,* JONG WOO LEE,* YOUNG HWA SOUNG,* WON SANG PARK,*
SU YOUNG KIM,* JONG HEUN LEE,* JIK YOUNG PARK,* YOUG GU CHO,* CHANG JAE KIM,*
SEONG WHAN JEONG,‡ SUK WOO NAM,* SANG HO KIM,* JUNG YOUNG LEE,* NAM JIN YOO,*
and SUG HYUNG LEE*
Departments of *Pathology and ‡Biochemistry, College of Medicine, The Catholic University of Korea, Seoul, Korea
Background & Aims: There has been evidence that dys- apoptosis.3,4 The caspase gene family thus far contains at
regulation of apoptosis is involved in the pathogenesis least 14 mammalian members, of which 12 human en-
of cancer development. Caspase-8 is an initiation zymes are known.3,4 From a functional perspective, the
caspase that activates the caspase cascade during ap- caspase gene family has been classified into either initi-
optosis. The aim of this study was to explore the possi- ator caspases or effector caspases. The initiator caspases
bility that mutation of the caspase-8 gene might be
would seem to be caspase-2, -8, -9, and -10, and the
involved in the development of colorectal cancer.
effector caspases would seem to be caspase-3, -6, and
Methods: We analyzed the entire coding region of the
caspase-8 gene for the detection of somatic mutations -7.3,4 The death signals originating from the death re-
in 180 colorectal tumors (98 invasive carcinomas and ceptors, such as tumor necrosis factor receptors, Fas, and
82 adenomas) by polymerase chain reaction, single- tumor necrosis factor–related apoptosis-inducing ligand
strand conformation polymorphism, and DNA sequenc- (TRAIL) receptors, are transduced through the recruit-
ing. Results: Overall, we detected a total of 5 somatic ment of pro– caspase-8 to the death-induced signaling
mutations in 98 invasive carcinomas (5.1%), but no complex (DISC) by the adaptor molecule Fas-associated
mutations were detected in 82 adenomas (0%). The death domain protein (FADD).2 The local aggregation of
frequency of caspase-8 mutation in the carcinomas was pro– caspase-8 is sufficient to allow autoprocessing or
significantly higher than that in adenomas (P < 0.05). transprocessing to produce active caspase-8, which can
The 5 mutations consisted of 1 frameshift, 1 nonsense subsequently activate downstream executioners, such as
mutation, and 3 missense mutations. We expressed the caspase-3 and -7.3,4
5 tumor-derived caspase-8 mutants and found that 3 of
The death receptors are widely expressed in normal
the 5 mutations markedly decreased apoptosis activity
and neoplastic cells,5,6 but the expression of these pro-
of caspase-8. Furthermore, expression of the inactivat-
ing caspase-8 mutants interfered with apoptosis by teins does not necessarily predict susceptibility to kill-
death receptor overexpression, indicating that these mu- ing.7,8 This can reflect the presence of inhibiting mech-
tants have dominant-negative inhibition of the death anisms of the death receptor–mediated apoptosis. The
receptor–induced apoptosis. Conclusions: The presence death receptor–mediated apoptosis can be blocked by
of caspase-8 mutation in colon carcinomas suggests several mechanisms, including mutations of the genes
that caspase-8 gene mutation might lead to the loss of involved in the death receptor–mediated apoptosis.9 –12
its apoptotic function and contribute to the pathogene- It is now believed that clonal expansion and tumor
sis of colorectal carcinomas, especially at the late stage growth are the results of the deregulation of intrinsic
of colorectal carcinogenesis. proliferation (cell division) and cell death (apoptosis).13
Failure of apoptosis could allow the survival of trans-
poptosis is a fundamental biochemical cell-death
A pathway that is essential for normal tissue ho-
meostasis and cellular differentiation and development.1
Abbreviations used in this paper: DISC, death-induced signaling
complex; FADD, Fas-associated death domain protein; 5-FU, 5-fluoro-
uracil; PARP, poly-(ADP-ribose) polymerase; PCR, polymerase chain
Proteases cause the morphological changes that are rec- reaction; SSCP, single strand conformation polymorphism; TRAIL,
ognized as apoptosis and the biochemical changes asso- tumor necrosis factor–related apoptosis-inducing ligand; TRAIL-R2,
ciated with apoptosis.1– 4 Among the proteases, a family TRAIL-receptor 2.
© 2003 by the American Gastroenterological Association
of cysteine proteases that cleave the substrates at aspar- 0016-5085/03/$30.00
tate residues, known as caspases, plays a main role during doi:10.1016/S0016-5085(03)01059-X
September 2003 CASPASE– 8 MUTATION IN COLON CANCER 709
formed cells that are prone to undergo further genetic quences obtained from Genbank (accession No. NT_005403).
damage and play an important role in the pathogenesis of Numbering of complementary DNA of caspase-8 was per-
tumors. Either inactivation of a proapoptotic pathway or formed with respect to the ATG start codon (NM_033355).
activation of an antiapoptotic pathway results in failure Radioisotope ([32P]deoxycytidine triphosphate) was incorpo-
of apoptosis, thereby promoting tumor cell survival. rated into the polymerase chain reaction (PCR) products for
detection by autoradiogram. The PCR reaction mixture was
There has been evidence that alterations of caspase par-
denatured for 1 minute at 94°C and incubated for 30 cycles
ticipate in tumor development.12,14 –20
(denaturing for 30 seconds at 94°C, annealing for 30 seconds
Somatic mutations of the genes involved in apoptosis, at 53°C– 66°C, and extending for 30 seconds at 72°C). Other
including the death receptors and caspases, have been procedures of PCR and single-strand conformation polymorphism
reported in human cancers,9 –12,14,16 –19 implying that the (SSCP) analysis were performed as described previously.9 –12
somatic mutations might not be a rare event in the After SSCP, DNAs showing mobility shifts were cut out from
apoptosis pathway. Among the caspases, mutations of the dried gel and reamplified for 30 cycles by using the same
caspase-3, -5, -8, and -10 have been reported in human primer sets. Sequencing of the PCR products was performed
cancer tissues and cell lines.12–14,16 –19 Caspase-8 muta- with the cyclic sequencing kit (Perkin-Elmer, Foster City, CA)
tions have been reported in some cancer cell lines,16 –18 according to the manufacturer’s recommendations.
suggesting the presence of caspase-8 mutation in human To analyze the allelic status of caspase-8 in the tumors, we used
cancer tissues. However, to date, data on caspase-8 mutation 6 intragenic polymorphisms. Because biallelic polymorphisms at
the coding sequence (960 A/G, dbSNP: 1045487) and the intron
in cancer tissues are lacking. In this study, to explore the
sequence (IVS9-19A/G, dbSNP: 376918) have been found in the
possibility that alterations of the caspase-8 gene might
Genbank database and because an additional biallelic polymor-
play a role in colorectal cancer development, we analyzed phism at the nucleotide position 789 A/G was detected in this
180 colon tumors for detection of caspase-8 mutation. study, SSCP analysis at these polymorphic sites was used for the
detection of loss of heterozygosity, as well as for the detection of
Materials and Methods mutations. Complete or nearly complete absence of 1 allele in
tumor DNA of informative cases, as defined by direct visualiza-
Tissue Samples, Microdissection, and
tion, was considered as loss of heterozygosity.
Plasmids
Paraffin-embedded histological sections of 82 colorec- Site-Directed Mutagenesis
tal adenomas (40 low-grade dysplasias and 42 high-grade Site-directed mutagenesis was performed by using a
dysplasias) and 98 invasive colorectal carcinomas were ob- Quick Change Site-Directed Mutagenesis kit (Stratagene, La
tained from the Catholic University of Korea–affiliated hospi- Jolla, CA) according to the manufacturer’s instructions. To
tals (Seoul, Korea). The term invasive carcinoma means, strictly, change a base, a plasmid that contained the caspase-8 gene in
a cancer that has invaded beyond the muscularis mucosa. The pcDNA3.1-Flag was used as a template. The nucleotide se-
carcinomas originated from cecum (n ⫽ 2), ascending colon quences of the mutagenized plasmids were confirmed by DNA
(n ⫽ 18), transverse colon (n ⫽ 6), descending colon (n ⫽ 3), sequencing.
sigmoid colon (n ⫽ 27), and rectum (n ⫽ 42). Approval for
this study was obtained from the Catholic University of Korea, Coimmunoprecipitations and
College of Medicine’s Institutional Review Board. Informed Immunoblotting Assay
consent was provided according to the Declaration of Helsinki. Human embryonic kidney 293T cells in log phase
Malignant and normal cells were selectively procured from were transfected in 6-well plates with expression plasmids by
H&E-stained slides by using a 30-gauge 1/2 hypodermic using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA).
needle (Becton Dickinson, Franklin Lakes, NJ) affixed to a Cells were harvested 1 day later and lysed in ice-cold Nonidet
micromanipulator, as described previously.21 DNA extraction P-40 lysis buffer supplemented with 1⫻ protease inhibitor
was performed by a modified single-step DNA extraction mix (Roche Molecular Biochemicals, Mannheim, Germany).
method, as described previously.21 The plasmid constructs Cell lysates were subjected to immunoprecipitation with aga-
encoding Fas, FADD, caspase-8, and TRAIL-receptor 2 rose-conjugated anti–Flag M2 antibodies (Sigma, St. Louis,
(TRAIL-R2) were described previously.12 MO). Immunocomplexes were fractionated by sodium dodecyl
sulfate polyacrylamide gel electrophoresis and transferred to
Single-Strand Conformation Polymorphism
nitrocellulose membranes. The resulting blots were incubated
Analysis with anti-hemagglutinin (1:1000 vol/vol; Roche Molecular
Genomic DNAs from tumor cells and corresponding Biochemicals) followed by horseradish peroxidase– conjugated
normal cells were amplified with primer pairs covering the secondary antibodies and detection by an enhanced chemilu-
entire coding region (8 exons) of the human caspase-8 gene. minescence method (Amersham Pharmacia Biotech, Bucking-
Oligonucleotide primers were designed with the program hamshire, UK). Alternatively, lysates were analyzed directly by
Oligo (National Biosciences, Plymouth, MN) by using se- immunoblotting after normalization for total protein content.
710 KIM ET AL. GASTROENTEROLOGY Vol. 125, No. 3
NI, not informative (homozygosity); HET, retention heterozygosity; TNM, tumor-node-metastasis; DED, death effector domain; p20, p20 large
subunit; p10, p10 small subunit.
aPolymorphisms at nucleotides 789 and 960 of caspase-8 complementary DNA.
bThe polymorphism at intron 7 of caspase-8.
September 2003 CASPASE– 8 MUTATION IN COLON CANCER 711
R162I, Flag-caspase-8-G366H, Flag-caspase-8-R413G, 293T cells, and compared the poly-(ADP-ribose) poly-
Flag-caspase-8-R413 stop, and Flag-caspase-8-frame- merase (PARP) cleavage in the lysates by immunoblot-
shift) by using site-directed mutagenesis. On their trans- ting. Overexpression of the mutants R413G, R413stop
fection into 293T cells, we found that the mutants and frameshift showed absence of PARP cleavage,
R413G, R413 stop, and frameshift showed significant whereas overexpression of wild-type caspase-8, caspase-
defects in apoptosis function compared with the wild- 8-R162I and Flag-caspase-8-G366H showed the discern-
type caspase-8 (Figure 2A; Dunnett multiple comparison able cleavage of PARP (Figure 3).
test; P ⬍ 0.001). Finally, we determined whether the inactivating muta-
Furthermore, to explore how the caspase-8 mutants tions of caspase-8 render colorectal cancer cells resistant to
impair apoptosis, we coexpressed each inactivating the induction of apoptosis by drugs commonly used to treat
caspase-8 mutant (R413G, R413 stop, and frameshift) colorectal cancer. To this purpose, transient transfection of
either with Fas or TRAIL-R2 in 293T cells. We found DLD-1 cells with the caspase-8 mutants or wild-type
that these mutants interfered with the death induction caspase-8 was performed. After 3 hours of the transfection,
by Fas or TRAIL-R2 overexpression, indicating that 100 – 600 mol/L 5-FU was added. Figure 4 shows de-
these mutants have dominant-negative inhibition of the creased cell death by the caspase-8-R413 stop and caspase-
death receptor–induced apoptosis (Figure 2B; Dunnett 8-frameshift compared to the wild-type caspase-8 and other
multiple comparison test; P ⬍ 0.001). Also, to deter- caspase-8 mutants after 5-FU (600 mol/L) treatment. At
mine if the caspase-8 mutants are functional, we trans- other ranges of 5-FU concentration (100 –500 mol/L),
fected the caspase-8 mutants or wild-type caspase-8 in similar differences were observed (data not shown).
712 KIM ET AL. GASTROENTEROLOGY Vol. 125, No. 3
Among the 5 mutations of caspase-8 found in this There is a death effector domain/caspase gene cluster
study, 3 showed significant defects in inducing apopto- on human chromosome 2q33–34 that contains the genes
sis. The frameshift mutation would change the last 20 for FLICE-inhibitory protein, caspase-8, and caspase-10,
amino acids in the p10 subunit. Because the active suggesting that all of these genes have arisen by tandem
caspase-8 consists of a (p20/p10)2 tetramer,22 this frame- duplication.25,26 Such duplications have been associated
shift mutation may also lead to abnormal construction of with other chromosome regions that are altered during
the tetramer. Also, 1 nonsense mutation (R413 stop) was tumorigenesis, and this may reflect the relative instabil-
identified in the coding regions of the small p10 subunit. ity of the chromosomal region.27 The occurrences of
This mutation is predicted to cause a premature termi- caspase-10 gene mutations in non-Hodgkin lymphoma
nation of caspase-8 protein synthesis in the middle of the and the caspase-8 gene mutations in colon cancer found
p10 and, hence, resembles a typical loss-of-function mu- in this study suggest that caspase-8 and caspase-10
tation. Another inactivating mutation of caspase-8 might be candidate tumor-suppressor genes at chromo-
would also change amino acid residue 413 (R413G). The some 2q33–34. Whether caspase-8 is such a tumor sup-
arginine at amino acid 413 is conserved in other caspases, pressor is unknown at this stage, but its function as an
such as caspase-1, -2, -3, -4, -5, and -6 (Genbank data- apoptosis initiator would be consistent with tumor sup-
base). The previous structural analysis of caspases showed pressor function.
that the residues constituting the binding pocket for P1 In the colon carcinomas analyzed, all of the 5 muta-
aspartic acid are conserved in caspases and are important tions of the caspase-8 gene showed evidence of retention
for the apoptosis induction of the caspases.23 The argi- of the wild-type allele (Table 1). Also, 3 of the mutations
nine 413 of caspase-8 is one of the constituting residues (cases 5, 17, and 21) showed a marked decrease of apo-
for the P1 binding pocket,24 and it seems that alteration ptosis induction (Figure 2A). Therefore, in these cases, it
of the arginine 413 decreases the protease function of is possible that the heterozygously mutated caspase-8,
caspase-7. Because 2 different mutations in unrelated which inefficiently autoprocesses, and normal caspase-8
patients would involve the same amino acid (arginine are co-recruited into the DISC and could therefore block
413) and both of these mutations resulted in significant the caspase-8 activation in a dominant-negative fashion
functional losses of caspase-8, this position may represent (Figure 2B).
a mutational hotspot in the caspase-8 coding sequence. Transcription of caspase-8 can be inhibited by DNA
In contrast to the inactivating mutations, 2 mutations methylation,20 which, in turn, blocks the production of
did not show any significant loss of apoptosis function of caspase-8 protein. To discover whether caspase-8 in colon
caspase-8 (Figure 2). However, the functional implica- cancer could be inactivated by DNA methylation, we
tions of these mutations are unknown at this stage. analyzed caspase-8 protein expression by immunohisto-
714 KIM ET AL. GASTROENTEROLOGY Vol. 125, No. 3
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