of University of Medi-
cine and Dentistry of New Jersey, New Jersey, USA.
K562 is a cell line derived from chronic myeloid leu-
kaemia (CML) patient and belongs to the granulocytic
series of cell [18]. K562 cells exhibits delayed apoptotic
features such as cleavage of caspase substrate, cyto-
chrome c release, DNA fragmentation and apoptotic
morphological changes [19]. Etoposide (VP-16) was
purchsed from SIGMA. DNA Polymerase I, Large
(Klenow) fragment, T4 DNA Ligase and all the restric-
tion enzymes were purchased from New England Biolabs
(NEB, England). Phytohaemagglutinin (PHA) was pur-
chased from Gibco, USA. PCR primers were obtained
from First Base Laboratories. Phusion DNA Polymerase
was purchased from Finnzymes, Finland. Caspase-3 in-
hibitor II (Z-DEVD-FMK) was purchased from Calbio-
chem, USA. Blood and cell culture DNA Mini-Prep kit,
and QIAquick Gel Extraction Kit were obtained from
QIAGEN, Germany. DYEnamic ET Terminator Cycle
Sequencing Kit was purchased from Amersham, UK.
2.2. Cell Culture and Apoptosis Induction Assay
Cells were cultured in RPMI 1640 medium containing
10% heat-inactivated fetal bovine serum, 100 units/ml
penicillin, 100 g/ml streptomycin, and 2 mM L-gluta-
mine. Cells were constantly grown at 37˚C with 5% CO2.
CEM and K562 cells at a density of 1 × 106 cells/ml
were treated with 50 µM of VP-16 to induce apoptosis.
Treatment with 0.1% DMSO as solvent control was in-
cluded. All treatments were performed at 37˚C for 5
hours otherwise mentioned. Cells were collected for ge-
nomic DNA extraction using QIAGEN Blood and Cell
Culture DNA Mini-Prep according to manufacturer’s
protocol. The purified DNA was then digested with 100
U of BamH I in a 150 µl reaction at 37˚C for 16 hours.
2.3. Modification of Digested DNA as Template
for Inverse Polymerase Chain Reaction
(IPCR)
Figure 1 shows a summary of DNA modification as well
as the IPCR. The BamH I-digested genomic DNA con-
sists of a mixture of intact BamH I AF9 fragments and
cleaved AF9 fragments. BamH I digestion generates
sticky ends while the ends of the cleaved AF9 fragments
are believed to be either a 1-base 5’ overhang or a blunt
end [20]. Therefore, Klenow fill-in was carried out to
generate fragments with blunt ends. Two µg of DNA was
used as template for Klenow fill-in following standard
protocol [21]. Subsequently ligation/cyclisation with
2000 U of T4 DNA Ligase was performed in a 500 µl
reaction at 16˚C for 16 hours. Ethanol precipitation was
carried out with 2 volumes of cold absolute ethanol and
0.1 volume of 3 M sodium acetate, pH 5.2. DNA pellet
was washed with 70% ethanol and briefly air-dried, then
dissolved in TE buffer, pH 8.0. The dissolved DNA was
divided into two, one digested with 20 U of Kpn I and
the other digested with 20 U of Nde I. Kpn I digestion
was performed to linearise the cyclised DNA whereas
Nde I digestion was performed to eliminate amplification
of the intact BamH I AF9 fragment, thus eliminating its
competition with the cleaved AF9 fragment during IPCR.
Digested DNA was purified using QIAGEN QIAquick
Gel Extraction Kit according to manufacturer’s protocol.
2.4. Nested IPCR and DNA Sequencing
Nested IPCR was performed with 200 ng of template
DNA (Kpn I- or Nde I-digested), 0.5 mM of each primer,
200 µM of each dNTP, 1× Phusion buffer HF (containing
1.5 mM MgCl2) and 0.4 U of Phusion Polymerase. IPCR
cycle condition for the first round was: 30 seconds of
98˚C for 1 cycle, followed by 30 cycles of 98˚C for 10
seconds, 69˚C for 30 seconds, 72˚C for 15 seconds, fol-
lowed by a 1 cycle of 72˚C for 10 minutes. Second round
IPCR was performed with 2 µl of 5-times diluted first
round IPCR product with similar cycle condition, except
that the annealing temperature was 57˚C. The primers
used during the first round of IPCR were 5’-ATTCTA-
GACCCCAAAAAATTCTCAG-3’ and 5’-CTCTTAATG-
CCACTGCCATGA-3’, while the primers used in the
Copyright © 2012 SciRes. OPEN ACCESS
C. P. Nicholas, S.-P. Sim / Advances in Bioscience and Biotechnology 3 (2012) 686-694
Copyright © 2012 SciRes.
688
Figure 1. Flow chart showing DNA modification and IPCR. The arrow heads indicate the
forward and reverse primers that were designed in opposite direction. BamH I digestion
yielded a mixture of intact chromosome and cleaved chromosome. Klenow fill-in produced
blunt ended chromosome fragments which were then cyclilsed by T4 DNA ligase. The intact
chromosome will become a large circle while the cleaved chromosome will become a smaller
circle and lost one of the Nde I sites. Upon cyclisation, the primers are now in correct orienta-
tion for amplification. Kpn I digestion cleaved both circles outside the amplification region,
thus merely linearise the molecule. Nde I digestion linearise the small circle but also cleave
within the amplification region of the large circle, thus preventing amplification of the intact
chromosome. This enhances the amplification of the cleaved chromosome.
second round were 5’-ATTGGTGTCAATCAAATGC-3’
and 5’-CATATCCTTTTCATACCTGG-3’. IPCR products
were then analysed on 1.5% agarose gel in 0.5× TBE
buffer. Amplification of the intact BamH I AF9 fragment
was expected to produce a 950 base pairs IPCR product.
IPCR products smaller than 950 base pairs, which were
expected to be the amplification of cleaved AF9 frag-
ments were excised and purified using QIAGEN QIA-
quick Gel Extraction kit according to manufacturer’s
protocol. These IPCR products were cloned and se-
quenced using DYEnamic ET Terminator Cycle Se-
quencing Kit according to manufacturer’s protocol. Se-
quencing data was analysed using SeqMan DNASTAR
software (Lasergene, USA) and compared with the pub-
lished AF9 gene sequence (GenBank accession No.
AC000007).
OPEN ACCESS
2.5. Caspase Inhibition Assay in Leukaemic
Cells
CEM cells at 1 × 106 cells/ml were pre-treated with 50
M of caspase-3 inhibitor II (Z-DEVD-FMK) at 37˚C,
5% CO2 for 1 hour. DMSO was included as solvent con-
trol. Subsequently the cells were co-treated with either
50 μM of VP-16 or 0.1% DMSO for 5 hours. Genomic
DNA was extracted and purified for use as DNA tem-
plate for IPCR as described before.
2.6. VP-16-Induced Chromosome Breaks in
Cultured Normal Blood Cell and Its
Caspase Inhibition Asay
Venous blood from a normal volunteer was collected in a
lithium heparin tube. One ml of the peripheral whole
blood was cultured in 5 ml of RPMI 1640 medium con-
taining 20% heat-inactivated foetal bovine serum, 200
units/ml penicillin, 200 µg/ml streptomycin, 2 mM L-
glutamine, and 2% phytohaemagglutinin (PHA) (M-
form) [22]. Whole blood cultures were incubated at 37˚C
for 48, 54, 60, 66 and 72 hours in the presence of PHA.
Cells were then treated with 50 µM of VP-16 for 5 hours.
Subsequently genomic DNA was extracted and modified
C. P. Nicholas, S.-P. Sim / Advances in Bioscience and Biotechnology 3 (2012) 686-694 689
for IPCR analysis. For caspase inhibition assay, after the
cells have been cultured in the presence of PHA for each
time point (60, 66 and 72 hours), cells were pre-treated
for 1 hour with caspase inhibitor. Subsequently, they
were either co-treated with 50 μM of VP-16 or 0.1%
DMSO for 5 hours. Cells were then collected and proc-
essed for IPCR.
3. RESULTS
3.1. VP-16 Induces Cleavage of the AF9 BCR
CEM cells were treated with VP-16 and the extracted
genomic DNA was modified and processed for IPCR
analysis. IPCR primers were designed to detect chromo-
some breaks within the BCR1 (intron 4 telomeric end) of
AF9. As shown in Figure 2, with Kpn I digestion, only
the intact AF9 gene was amplified, represented by the
950 base pairs bands (lanes 2-4). IPCR band less than
950 base pairs representing cleaved AF9 was not de-
tected. This could be due to the competition between the
intact chromosome and the cleaved chromosome (which
is in a very small quantity) for the amplification process.
Thus, Nde I was used to eliminate the amplification of
the intact chromosome while at the same time to linearise
the cyclised cleaved fragment as shown in Figure 1.
Amplification of the intact AF9 was not eliminated but
reduced (Figure 2, lanes 5-7), and at the same time
smaller IPCR products of sizes 180 base pairs and 730
base pairs were detected upon VP-16 treatment (Figure 2,
lane 7). DNA sequencing shows that the 180 base pairs
was an amplification product of an AF9 cleavage frag-
ment that carries a breakpoint at coordinate 26805
(GenBank accession No. AC000007), while the 730 base
pairs corresponds to amplification of a cleaved AF9
fragment that carries a breakpoint at coordinate 26235
(GenBank accession No. AC000007).
3.2. Caspase Inhibitor Eliminates
VP-16-Induced Cleavage of the AF9 BCR
VP-16 is known to induce apoptosis [14], thus in order to
investigate if this VP-16-induced AF9 cleavage was me-
diated by the apoptotic nuclease CAD, caspase inhibition
assay was performed. CAD usually exists as a complex
with its inhibitor, ICAD. It is only upon apoptosis active-
tion, when caspase cleaves ICAD, then CAD will be re-
leased and be activated to cleave the genomic DNA [23].
Thus by inhibiting the caspase, CAD will not be acti-
vated and would prevent cleavage of the AF9 BCR. As
shown in Figure 3(a), in the absence of caspase inhibitor,
treatment with VP-16 resulted in cleavage of the AF9
BCR producing a 420 base pairs band (lane 5). Indeed,
pre-treatment with caspase inhibitor (Z-DEVD- FMK),
and later co-treatment with VP-16 prevented VP-16-
induced cleavage (Figure 3(a), lane 8). To ensure that
the non-amplification observed in other samples were not
due to template DNA degradation, IPCR with Kpn
I-digested template DNA was performed, and amplifica-
tion of the intact AF9 was observed as a 950 base pairs
band (Figure 3(b), lanes 2-8).
3.3. K562 Cells are Resistant to VP-16-Induced
Cleavage
K562 cells have been shown to be more resistant to
apoptosis induction compared with HL-60 cells [19].
Therefore K562 cells were used in this study to investi-
gate if VP-16-induced AF9 cleavage would be delayed.
As shown in Figure 4, with Kpn I digestion, amplifica-
tion of the intact AF9 fragment was detected (lanes 2-8).
However, with Nde I digestion, amplification of the in-
tact AF9 fragment was eliminated, but there was no de-
tection of the cleavage fragment (lanes 10-15). Note that
the treatment times for K562 (5, 7 and 24 hours) were
much longer as compared with that of CEM (5 hours) as
presented before. Even with the extended treatment time,
VP-16 failed to induce AF9 cleavage in K562 cells, fur-
ther supporting AF9 cleavage was mediated through
apoptosis.
Figure 2. VP-16 induces AF9 cleavage. CEM cells were treated
with 50 M of VP-16 (lanes 4 and 7), 0.1% DMSO (lanes 3
and 6), or left untreated as cell control (lanes 2 and 5). The cells
were then lysed, genomic DNA was extracted and modified for
IPCR as described in Materials and Methods. The modified
DNA was either digested with Kpn I (lanes 2-4) or Nde I (lanes
5-7) prior to IPCR. Lane 1 is a negative control, which does not
contain DNA template. The side arrows show the 950 bp, 730
bp, and 180 bp DNA fragments. M1: 1 kb DNA ladder. M2: 100
p DNA ladder. b
Copyright © 2012 SciRes. OPEN ACCESS
C. P. Nicholas, S.-P. Sim / Advances in Bioscience and Biotechnology 3 (2012) 686-694
Copyright © 2012 SciRes.
690
(a) (b)
Figure 3. Caspase inhibitor abolishes VP-16-induced AF9 cleavage. CEM cells were either pre-treated with
caspase inhibitor Z-DEVD-FMK for 1 hr (lanes 6-8) or left untreated (lanes 3-5). The cells were then either
co-treated with VP-16 (lanes 5 and 8), DMSO (lanes 4 and 7), or left untreated (lanes 3 and 6) to serve as cell
control. DMSO was included as a solvent control for Z-DEVD-FMK pre-treatment (lane 2). Genomic DNA
was extracted, processed and digested with Nde I (panel (a)) or Kpn I (panel (b)) prior to nested IPCR. A
negative control containing no DNA template was included as well (lane 1). The side arrows indicate the 950
bp and 420 bp DNA fragments. M1: 1 kb DNA ladder. M2: 100 bp DNA ladder.
Figure 4. VP-16 does not induce AF9 cleavage in K562 cells.
K562 cells were treated with DMSO (lanes 3-5 and 10-12) or
VP-16 (lanes 6-8 and 13-15) for 5 hr (lanes 3, 6, 10, and 13), 7
hr (lanes 4, 7, 11, and 14), and 24 hr (lanes 5, 8, 12, and 15).
Genomic DNA was extracted, processed, and digested with
either Kpn I (lanes 2-8) or Nde I (lanes 9-15). Nested IPCR was
performed. Untreated K562 cells were included as control
(lanes 2 and 9). Lane 1 is a negative control that does not con-
tain DNA template. The side arrow shows the 950 bp DNA
fragment. M1: 1 kb DNA ladder. M2: 100 bp DNA ladder.
3.4. VP-16 Induces AF9 Cleavage in Cultured
Normal Blood Cells
Our results using CEM cells suggest that VP-16 indeed
induced chromosome breaks within the AF9 BCR. As
CEM is a leukaemic cell line, it brings to the question
that if the same thing is happening in normal blood cell.
To answer this question, peripheral whole blood from a
normal volunteer was used to study VP-16-induced AF9
cleavage. For this purpose, the whole blood microculture
was performed in the presence of phytohaemagglutinin
(PHA) (M-form), which is a strong mitogen. Exposure of
fully differentiated lymphocytes in the whole blood to
PHA will transform these cells into mitotically active
cells. It is crucial that the cells are mitotically active as
VP-16 targets DNA topoisomerase II that is highly ex-
pressed in proliferating cells [24]. The whole blood cul-
ture was performed in the presence of PHA for specific
periods of time and then treated with VP-16 as described
in materials and methods. As shown in Figure 5(a), with
Kpn I digestion, only amplification of the intact AF9 was
detected (lanes 2-6). However, with Nde I digestion,
three smaller IPCR bands of 740 base pairs, 180 base
pairs and 240 base pairs were detected (Figure 5(b)).
They were detected in cells cultured in the presence of
PHA for 48 hours, 66 hours and 72 hours respectively
(Figure 5(b), lanes 2, 5 and 6). Sequencing results reveal
that the 740 base pairs band was derived from cleavage
of AF9 at coordinate 26335 while the 180 base pairs and
240 base pairs bands were amplification products of AF9
cleavage at coordinates 26817 and 26756 respectively
(GenBank accession No. AC000007).
3.5. Caspase Inhibitor Does Not Inhibit
VP-16-Induced AF9 Cleavage in Cultured
Normal Blood Cells
As discussed earlier, VP-16-induced AF9 cleavage in
CEM leukaemic cells was caspase-dependent. Thus, we
would like to know if VP-16-induced AF9 cleavage in
OPEN ACCESS
C. P. Nicholas, S.-P. Sim / Advances in Bioscience and Biotechnology 3 (2012) 686-694 691
(a) (b)
Figure 5. VP-16 treatment of cultured normal blood cells results in AF9 cleavage. Blood cells from a normal volun-
teer were cultured in the presence of PHA for 48 hr (lane 2), 54 hr (lane 3), 60 hr (lane 4), 66 hr (lane 5), and 72 hr
(lane 6). The cells were then treated with VP-16 for 5 hr. Genomic DNA was extracted and processed as described in
Materials and Methods. DNA was digested with either Kpn I (panel (a)) or Nde I (panel (b)) followed by IPCR. Lane
1 is negative control, which does not contain DNA template. The side arrows show the 950 bp, 740 bp, 240 bp, and
180 bp DNA fragments. M1: 1 kb DNA ladder. M2: 100 bp DNA ladder.
cultured normal blood cell is also caspase-dependent. As
shown in Figure 6, in the absence of caspase inhibitor,
Vp-16 treatment resulted in cleavage of the AF9 BCR, as
indicated by IPCR bands of sizes smaller than 950 base
pairs (lanes 6 and 8). However, in contrast to that ob-
served in the CEM leukaemia cells, these breaks forma-
tion was not inhibited by caspase inhibitor (lanes 14).
Interestingly, treatment with 0.1% DMSO also resulted
in AF9 cleavages that were not inhibited by caspase in-
hibitor (lanes 3, 10 and 11).
4. DISCUSSION
Treatment with etoposide (VP-16) has been correlated
with translocation of the MLL gene observed in t-AML
patients [4,5]. Multiple mechanisms of translocation had
been proposed for the MLL gene [11-13,25]. Among
them, the involvement of apoptosis, particularly the
apoptotic nuclease, CAD was proposed [14]. Apoptosis
is a natural cell death pathway, however, it has been
suggested that apoptotic cells may survive apoptosis and
eventually survive with chromosome rearrangements
[16]. Since the AF9 gene has similar structure as the
MLL gene [10], we intended to investigate if VP-16-
induced apoptosis does lead to cleavage of the AF9 gene.
Indeed, our results show that VP-16 induced chromo-
some breaks within the AF9 breakpoint cluster region
(BCR). The breakpoints identified in this study clustered
with those mapped in patients as well as those obtained
from cell lines carrying t(9, 11) [9,26-28]. Interestingly,
treatment of TK6 cells with anti-CD95 antibody, a
known apoptosis inducer, also resulted in cleavage of the
AF9 BCR [29]. The breakpoints obtained in this study
Figure 6. Caspase inhibitor does not inhibit VP-16-induced
AF9 cleavage in cultured normal blood cells. Blood cells from
a normal volunteer were cultured in the presence of PHA for 60
hr (lanes 3, 6, 9, and 12), 66 hr (lanes 4, 7, 10, and 13), and 72
hr (lanes 5, 8, 11, and 14). The cells were then either pre-
treated with caspase inhibitor for 1 hr (lanes 9-14) or left un-
treated (lanes 3-8). Subsequently, the cells were co-treated with
DMSO (lanes 3-5 and 9-11) or VP-16 (lanes 6-8 and 12-14) for
5 hr. DMSO was included as a solvent control for caspase in-
hibitor pre-treatment (lane 2). Genomic DNA was extracted,
processed and digested with Nde I prior to nested IPCR. Lane 1
is a negative control, which does not contain DNA template.
The side arrows show the 950 bp DNA fragment. M1: 1 kb
DNA ladder. M2: 100 bp DNA ladder.
are in very close proximity to those identified in the TK6
cells. From this comparison, it seems that cleavage of the
AF9 BCR is independent of cell line/patients and the
type of stimuli. However, it is worth noticing that, both
Copyright © 2012 SciRes. OPEN ACCESS
C. P. Nicholas, S.-P. Sim / Advances in Bioscience and Biotechnology 3 (2012) 686-694
692
VP-16 and anti-CD95 are apoptosis stimuli. Similar re-
sults were observed for the ML L BCR cleavage, whereby
various chemotherapeutic drugs (VP-16, 5-fluorouracil,
methotrexate, vinblastine and camptothecin) induced apo-
ptosis as well as cleavage of the MLL BCR [14].
VP-16 targets DNA topoisomerase II and results in
DNA double stranded breaks [30]. This DNA damage
may induce the apoptotic signalling pathway [31],
whereby the caspase cascade is activated. CAD is nor-
mally inhibited by ICAD. During apoptosis, caspase 3
cleaves ICAD, thus releasing the activated CAD, which
eventually cleaves the chromosomal DNA [23,32]. In our
study, Z-DEVD-FMK was used to investigate if the
VP-16-induced AF9 cleavage was mediated by CAD.
Z-DEVD-FMK is a potent, cell-permeable and irreversi-
ble inhibitor of caspase 3, 6, 7, 8 and 10. Our results in
leukaemic cell line indicated that CAD was most likely
responsible for the AF9 cleavage. This is in line with
other reports in leukaemic cell line [14] and in naso-
pharyngeal carcinoma cell line as well [33]. In apoptotic
cells, CAD associates with the nuclear matrix [34], thus
allowing it to cleave the base of the chromatin loops at-
taching to the nuclear matrix or scaffold, generating high
molecular weight (HMW) DNA during early stage
apoptosis [15,35]. This is again supported by our results
in K562 cells, where AF9 cleavage was absent despite
long period of VP-16 treatment. K562 is known to ex-
hibit delayed apoptosis induction. This delay resulted
from a delay in the signaling cascade upstream of cyto-
chrome c release and caspase activation [19]. Taken to-
gether, it seems that VP-16-induced cleavages of the AF9
were most likely to be mediated by CAD.
Our results showed that normal blood cell also exhib-
ited AF9 cleavage upon VP-16 treatment. However, these
cleavages were not inhibited by caspase inhibitor. This
contradictory observation could be due to a few possi-
bilities. Firstly, normal blood cells and leukaemic cells
may have different capacity of uptake for the caspase
inhibitor. Although the uptake of caspase inhibitor has
not been reported, it is known that the uptake capacity of
methotrexate is different between leukaemic cells and
normal leucocytes [36]. In addition, uptake of nitrogen
mustard and choline were shown to be higher in leukae-
mic lymphoid cells compared with normal lymphoid
cells [37]. Secondly, the normal blood cells might have
died via the caspase-independent pathway as demon-
strated by others [38,39]. This is seconded by the obser-
vation that mouse embryonic fibroblast (MEF) from
caspase 3-deficient mice and CAD-deficient chicken
DT40 cells were able to generate HMW DNA during
apoptosis induction [40,41]. Other than CAD, the other
possible candidate is Endonuclease G (Endo G). Endo G
is an evolutionary conserved endonuclease that usually
localised in the intermembrane space of mitochondria,
but released during apoptosis and results in caspase-in-
dependent DNA degradation [42,43]. However, based on
its cleavage preferences, Endo G most likely does not
work alone. It may work with DNase-I-like enzymes
together with exonucleases in vivo for apoptotic DNA
processing [44]. In addition, apoptosis-inducing factor
(AIF) is another enzyme that is released from mitochon-
dria during apoptosis and cause chromatin condensation
and large scale DNA fragmentation [41]. DNA topoi-
somerase II is another possible player. It was proposed to
be directly involved in the MLL BCR cleavage [45].
Poisoning of topoisomerase II by VP-16 resulted in
chromatin loop excision [46]. This is very logical as to-
poisomerase II is one of the major proteins found in the
nuclear scaffold [47]. Interestingly, CAD interacts with
topoisomerase II and enhances topoisomerase II’s de-
catenation activity in vitro [48].
Our sequencing analysis revealed that all the break-
points are mapped inside the BCR1 within close prox-
imity to one of the MAR/SAR. MAR/SAR sequences are
abundantly found at the base of chromatin loop struc-
tures [49], and excision of the chromatin loops forming
HMW DNA occurs during early apoptosis [35]. Thus our
results indicate a possibility that the apoptotic nuclease(s)
could be cleaving the base of the chromatin loop struc-
ture during early apoptosis. This is a crucial observation,
as we hypothesise that apoptotic cells escape the cell
death pathway and rescued as cells carrying chromosome
rearrangements, thus it is logical that only those at early
apoptosis could be rescued.
In conclusion, our results suggest that apoptotic nu-
clease(s) could be involved in VP-16-induced AF9
cleavage, and thus contributing to chromosome translo-
cation in t-AML.
5. ACKNOWLEDGEMENTS
We would like to acknowledge Prof. Dr. Leroy F. Liu for the K562 cell
line. We are also grateful to the Sarawak General Hospital pathology
laboratory staff for their technical advice. This work was supported by
the UNIMAS Fundamental Research Grant No. 1(4)/266/02(4) and
partly the IRPA Grant No. 09-02-09-1017 from the Ministry of Science,
Technology and Innovation.
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