close

Вход

Забыли?

вход по аккаунту

?

980

код для вставкиСкачать
Int. J. Cancer: 73, 592–599 (1997)
r 1997 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
LACK OF CORRELATION BETWEEN CISPLATIN-INDUCED APOPTOSIS, p53
STATUS AND EXPRESSION OF Bcl-2 FAMILY PROTEINS IN TESTICULAR GERM
CELL TUMOUR CELL LINES
Herman BURGER, Kees NOOTER*, Antonius W.M. BOERSMA, Christine J. KORTLAND and Gerrit STOTER
Department of Medical Oncology, University Hospital Rotterdam and Rotterdam Cancer Institute (Daniel den Hoed Kliniek),
Rotterdam, The Netherlands
We investigated the role of p53 and of the Bcl-2 family
proteins in the apoptotic response of a panel of testicular
tumour cell lines (NT2, NCCIT, S2 and 2102 EP). The p53
gene status and the capacity of the p53 protein to transactivate the p21/WAF/CIP gene were determined, and we examined the correlation between p53 status and the susceptibility
to cisplatin-induced apoptosis. In contrast to wild-type p53containing NT2 and 2102 EP cells, NCCIT (mutant p53) and
S2 (no p53 protein) cells were shown to be p53-transactivation defective. However, NCCIT and S2 cells with nonfunctional p53 were readily triggered into apoptosis by cisplatin, whereas p53-transactivation competent 2102 EP cells
failed to undergo cisplatin-induced apoptosis. The defective
apoptotic pathway in 2102 EP cells was reflected by a 4-fold
decreased sensitivity to cisplatin in the MTT assay. We
further demonstrated that the p53-independent differential
cisplatin sensitivity among the testicular germ cell tumour
(TGCT) cell lines was not due to differences in cellular
cisplatin accumulation or DNA platination. The pattern of
endogenous expression levels of Bax, Bcl-2, Bcl-x and Bak,
which was not modulated by cisplatin treatment, demonstrated that these Bcl-2 family proteins are not involved in
drug-induced apoptosis in the TGCT cell lines. Our results
suggest a lack of correlation between cisplatin-induced apoptosis, p53 status and expression of Bcl-2 family proteins in our
panel of TGCT cell lines. We conclude that the cisplatininduced apoptotic pathway in TGCT cell lines might be
p53-independent and is probably not associated with differences in the Bcl-2/Bax rheostat. Int. J. Cancer 73:592–599,
1997.
r 1997 Wiley-Liss, Inc.
Testicular germ cell tumours (TGCTs) comprise a versatile
group of neoplasms often showing complex histological features.
They are classified into seminomas (SE), which are composed of
the neoplastic counterparts of primordial germ cells, and nonseminomatous (NS) TGCTs, which are neoplastic caricatures of
early embryonic development. TGCTs represent one of the few
tumour types that are curable by chemotherapy and radiotherapy,
with an overall cure rate of approximately 80% (Einhorn, 1990). As
yet, the nature of the exceptional sensitivity of testicular tumours to
cytoreductive therapy has not been defined. Because it has now
been established that ionizing radiation and a large variety of
anti-cancer drugs exert their cytotoxic action through the induction
of apoptosis, and that inhibition of the apoptotic pathway may lead
to cytotoxic drug resistance (reviewed by Fisher, 1994), one might
speculate that TGCTs are hypersensitive to treatment-induced
apoptosis.
Several lines of evidence show that the product of the p53
tumour suppressor gene plays a pivotal role in the sensitivity of
tumour cells to apoptosis induced by chemotherapy or radiation
therapy (Lowe et al., 1993; Clark et al., 1993; Fisher, 1994; Harris,
1996). In some circumstances, functional inactivation of p53 by
mutations or interactions with cellular or viral proteins can lead to
resistance to genotoxic agents commonly used in anti-cancer
therapies. Indeed, cells from transgenic mice homozygous for p53
null alleles were shown to be more resistant to induction of
apoptosis by chemotherapeutic agents than cells from their normal
wild-type p53 (wtp53) littermates (Lowe et al., 1993). Another line
of evidence for a role of wtp53 in apoptosis comes from in vitro
studies which show that mutations in the p53 gene may render cells
resistant to induction of apoptosis by ionizing radiation and
chemotherapeutics (reviewed by Fisher, 1994).
Members of the Bcl-2 family of proteins are involved in the
control of apoptosis in a range of different cell types (reviewed by
Reed et al., 1996). The bcl-2 gene was first identified at the t(14;18)
translocation commonly found in human follicular lymphoma
(Korsmeyer, 1992). Several cellular and viral homologs of Bcl-2
have been identified that belong to this rapidly expanding Bcl-2
protein family (Reed et al., 1996). Bcl-2 family proteins can
function either as inhibitors (e.g., Bcl-2, Bcl-xL, Mcl-1, A1 and
Bag) or promoters (e.g., Bax, Bcl-xS, Bad and Bak) of cell death
and can physically interact with each other and subsequently
modulate apoptosis (Reed et al., 1996). For instance, Bcl-2 and Bax
form homodimers and heterodimers, and the balance between the
respective dimers (i.e., Bcl-2/Bcl-2; Bcl-2/Bax; Bax/Bax) determines the extent to which apoptosis is induced or suppressed. It has
been proposed that Bax homodimers promote apoptosis and that
the Bax-mediated cell death is counteracted by Bcl-2/Bax heterodimerization. Thus, the ratio of Bcl-2/Bax represents one
cell-autonomous rheostat that determines the cell’s fate; bcl-2 and
bax are immediate early response genes of the p53 tumour
suppressor gene (Miyashita et al., 1994). The promoter of the bax
gene contains several p53 consensus binding sites, and wtp53, but
not mutant p53 (mtp53), can transactivate the expression of the bax
gene. In contrast, the bcl-2 gene contains a p53-dependent negative
response element through which the p53 protein can function as a
repressor of bcl-2 expression. Thus, Bcl-2 and Bax are independently regulated by p53, and its effects on bcl-2 and bax gene
expression may determine the vulnerability of cells to apoptotic
stimuli. Accordingly, in a panel of human cells with defined p53
status, it has been shown that DNA damage-induced upregulation
of Bax and downregulation of Bcl-2 was dependent on the presence
of wtp53 (Zhan et al., 1994). Although it is generally accepted that
the presence of wtp53 increases the susceptibility of cells to
induction of apoptosis by a wide variety of genotoxic agents,
probably by modulation of the Bcl-2/Bax rheostat, an increasing
amount of evidence indicates that genotoxic insults can trigger
apoptosis also via p53-independent mechanisms (Clarke et al.,
1993). Some of these p53-independent apoptotic routes, but not all,
are also regulated by members of the Bcl-2 protein family. The
existence of p53-independent apoptotic pathways likely implies
that the regulatory role of p53 in apoptosis is influenced by the
particular cellular context in which the product of the p53 tumour
suppressor gene is expressed. Hence, p53 is apparently not the only
regulator of DNA damage-associated checkpoint(s) of apoptosis.
Contract grant sponsor: Dutch Cancer Society; Contract grant number:
DDHK 94-846.
*Correspondence to: Department of Medical Oncology, University
Hospital Rotterdam, Room D337, Dr. Molewaterplein 40, 3015 GD,
Rotterdam, The Netherlands. Fax: (31) 10 463 4627. E-mail:
[email protected]
Received 21 May 1997; Revised 8 July 1997
CISPLATIN-INDUCED APOPTOSIS IN TGCT CELL LINES
Clinical data revealed that the vast majority of TGCTs has no
mutations in their p53 alleles (Peng et al., 1993); this lack of p53
mutations has been implicated in the high response rate of this
neoplasm to combination chemotherapy. Several human NS TGCT
cell lines have been established that retain their relative sensitivity
to cytotoxic agents, demonstrating that these cell lines are representative models of chemo-sensitive tumours. In the present study, we
investigated whether the presence of functional wtp53 is a prerequisite for the onset of apoptosis in TGCTs. We studied the role of p53
and the Bcl-2 family proteins in drug-induced apoptosis in a panel
of well-defined TGCT cell lines. Our data suggest that the
cisplatin-induced apoptotic pathway in TGCT cell lines is p53independent and most likely not correlated with inherent or
drug-induced differences in the Bcl-2/Bax rheostat.
MATERIAL AND METHODS
TGCT cell lines and culture conditions
Four established TGCT cell lines were analyzed. The TGCT cell
line NT2 (ATCC CRL-1973) and the 2102 EP cell line (Wang et al.,
1981) have been derived from NS, whereas S2 (a gift from A. von
Keitz, Marburg, Germany) and NCCIT are 2 cell lines that exhibit
some SE-like characteristics (Damjanov et al., 1993). The cell lines
were grown as monolayer and maintained at 37°C in a humidified
cell culture incubator with 8.5% CO2 in HEPES-buffered RPMI
1640 supplemented with 10% FCS (GIBCO BRL, Paisley, UK),
100 IU/ml penicillin (Sigma-Aldrich, Zwijndrecht, the Netherlands), 100 µg/ml streptomycin (Sigma) and 2 mM L-glutamine
(GIBCO BRL).
Drug sensitivity assay
The MTT colorimetric assay, which measures the number of
viable cells capable of reducing the tetrazolium compound (SigmaAldrich, Zwijndvecht, The Netherlands) to a blue formazan
product, was used to quantitate the chemosensitivity of the cell
lines to cisplatin (cis-diamminedichloroplatinum II). Briefly, cells
were harvested during the exponential growth phase and seeded
into 96-well (3000 cells/well) tissue culture plates (Microtest III,
Falcon 3072, Beckton Dickinson, Lincoln Park, NJ). After overnight pre-incubation at 37°C, serial dilutions of cisplatin (Platosin,
Pharmachemie, Haarlem, The Netherlands) were added to quadruplicate wells, and the cells were exposed to the drug for an
additional 4 days. Processing of the 96-well microtiterplates and
absorbance measurements were performed according to standard
procedures. The IC50 and IC90 values, defined as the cisplatin
concentration that reduced the absorbance with 50% or 90%,
respectively, were estimated graphically from the concentration
response curves.
Intracellular platinum (Pt) accumulation
Triplicate 75-cm2 tissue culture flasks with exponentially growing cells were exposed to 33 µM (10 µg/ml) and 100 µM (30 µg/ml)
of cisplatin for 2 hr. Following exposure to drugs, the cells were
immediately washed to remove free cisplatin, harvested by trypsinization, washed with ice-cold PBS (3 3 10 ml) and lysed on ice in
500 µl of 0.2% (w/v) Triton-X-100 (Sigma)/H2O. Protein concentration in the resulting lysates was determined using a Bio-Rad
(Veenendaal, The Netherlands) protein assay kit. Total Pt content
was determined in duplicate by atomic absorption spectrometry
(AAS) using a flameless Perkin-Elmer (Foster City, CA) 4110 ZL
spectrometer. Intracellular Pt levels were expressed as µg of Pt per
mg of protein (µg Pt mg21 protein).
Determination of Pt bound to DNA
Following cisplatin incubation, triplicate monolayer cultures
were washed with PBS (3 3 20 ml), and the cells were lysed in the
tissue culture flasks at 37°C for 16 hr with 10 ml of DNA lysis
buffer containing 0.5% SDS, 10 mM Tris-HCl (pH 8.2), 400 mM
NaCl, 2 mM EDTA and 0.5 mg/ml proteinase K (Boehringer
Mannheim, Germany). The salting out procedure for extracting
DNA (Miller et al., 1988) was used to prepare genomic DNA.
Subsequently, the DNA samples were sonicated at 4°C for 1 hr, and
593
DNA content (absorbance at 260 nm) and Pt-DNA adducts (AAS)
were determined. The DNA platination levels were expressed as pg
of Pt per µg of DNA (pg Pt µg21 DNA).
Induction of apoptosis in TGCT cell lines by cisplatin
Cells from exponential phase cultures were used for the induction of apoptosis by cisplatin. The cells were seeded at a density of
106 cells per 75 cm2 in tissue culture flasks and 24 hr later incubated
with various concentrations (3.1 µM, 6.3 µM, 12.5 µM and 25 µM)
of cisplatin. After incubation with drug (2 hr at 37°C in culture
medium), the cells were washed with culture medium and further
cultured in drug-free medium for 24, 48 or 72 hr.
Microscopical detection of apoptotic cells
Apoptotic cells were recognized by the appearance of condensed
nuclear chromatin and fragmented nuclei. For the visualization of
these features in apoptotic cells, 2 DNA stains Hoechst 33342 and
PI were used (both obtained from Calbiochem, La Jolla, CA).
Hoechst 33342 is a fluorescent dye that is used to stain DNA
structures in viable cells. Because PI can only enter cells with a
disrupted cell membrane, this dye can be used both as a DNA stain
and concomitantly to determine the integrity of the cell membrane.
Cells were simultaneously incubated with Hoechst 33342 (0.5
µg/ml) and PI (2.5 µg/ml) for 20 min and viewed under a
fluorescence microscope (Carl Zeiss, Weesp, The Netherlands);
micrographs were taken according to standard procedures.
Quantification of apoptotic cells by annexin V labeling
Single cell suspensions for annexin V labeling were obtained by
trypsinization, washed with culture medium containing 10% FCS
and thereafter washed twice with culture medium without phenol
red. Annexin V labeling of cells was performed as described
previously (Boersma et al., 1996). Briefly, floating and adherent
cells were incubated for 1 hr at 37°C in HEPES (10 mM
HEPES/NaOH, pH 7.4) buffered RPMI (GIBCO BRL) culture
medium (without phenol red) supplemented with 0.5 µg/ml FITCconjugated annexin V (BioWhittaker, Verviers, Belgium), washed
twice in culture medium and kept on ice until further processing.
Flow cytometry was performed on a FACScan flow cytometer
(Beckton Dickinson, San Jose, CA) tuned at 488 nm. The FITC
fluorescence (515–545 nm) was measured in logarithmic mode,
whereas forward light scatter (FLS) and perpendicular light scatter
(PLS) were measured in linear mode. Cell debris was excluded
from analysis by appropriate FLS threshold setting.
Irradiation
To investigate the effect of irradiation on p53 and p21/CIP/WAF
expression, TGCT cell lines were g-irradiated (10 Gy) in 75 cm2
tissue culture flasks positioned between 2 opposing 137Cs sources
(Gamma Cell 40, Atomic Energy of Canada, Ottawa, Canada) at a
dose rate of 1.06–1.08 Gy/min. Cells were seeded at a density of
2 3 106 cells/tissue culture flask and 24 hr later used for the
irradiation experiment. Irradiated cells were incubated for an
additional 6–24 hr and thereafter further processed for Western and
Northern blot analysis.
Immunoblotting
Expression of p53, Bcl-2, Bcl-x, Bak and Bax proteins was
assessed in the TGCT cell lines by Western blot analysis. After
exposure to cisplatin, both detached and adherent cells were
combined and washed with ice-cold PBS (3 3 10 ml). Cell pellets
were resuspended in 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5
mM EDTA, 1% Triton-X-100 and the protease inhibitors phenylmethylsulfonyl fluoride (1 mM PMSF; Boehringer Mannheim),
aprotinin (0.23 U/ml; Sigma) and leupeptin (10 µM; Boehringer
Mannheim). Samples containing 20 µg of protein in loading buffer
(50 mM Tris-HCl; pH 6.8, 0.1% bromophenol blue, 2% SDS, 10%
glycerol, 100 mM dithioerythritol) were boiled for 3 min, subjected
to SDS-PAGE (12%) and transferred to Immobilon-P transfer
membrane (Millipore, Bedford, MA) using a semi-dry blotting
system (Hoefer, San Francisco, CA) with a continuous buffer
BURGER ET AL.
594
system (39 mM glycine, 48 mM Tris, 0.0375% SDS and 20%
methanol) at 0.8 mA/cm2 for 90 min. Membranes were preincubated in 5% (w/v) non-fat dry milk (Protifar, Nutricia, Zoetermeer, the Netherlands) in TBS (50 mM Tris, pH 7.5; 150 mM
NaCl) for 1 hr, washed and subsequently incubated overnight at
4°C in TBST (2% Tween 20 in TBS) supplemented with specific
monoclonal antibody (MAb) or polyclonal antibody (PAb). Bcl-2
specific mouse IgG1 MAb (100), p53 specific mouse IgG2a MAb
(DO-1), Bax specific rabbit IgG PAb (N-20), Bcl-xS/L specific
rabbit IgG PAb (S-18) and Bak specific rabbit IgG PAb (G-23) were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and
used at a dilution of 1:5,000 (Bax) or 1:1,000 (p53, Bcl-2, Bcl-xS/L,
and Bak). Poly(ADP-ribose) polymerase (PARP) specific mouse
MAb (C-2-10) and Bax specific mouse IgG2b MAb were purchased
respectively from BIOMOL (Plymouth Meeting, PA) and Immunotech (Coulter, Mijdrecht, The Netherlands). MAb were both used at
a dilution of 1:5,000. Immunological complexes were visualized by
enhanced chemiluminescence (Pierce, Cambridge, UK) using
horseradish peroxidase-conjugated goat anti-mouse or goat antirabbit IgG (1:4,000; Santa Cruz Biotechnology).
Isolation and sequencing of p53 cDNAs
To establish the p53 DNA sequences of the 4 studied TGCT cell
lines, total cellular RNA was isolated according to standard
procedures and reverse-transcribed using antisense p53 primers.
The single stranded p53 cDNA was subsequently amplified by the
polymerase chain reaction using 4 different sets of p53-specific
primers. The 4 p53 cDNA fragments (fragment 1: nt 137–486
corresponding to aa 1–117; fragment 2: nt 411–762 corresponding
to aa 93–202; fragment 3: nt 694–1029 corresponding to aa
188–291; fragment 4: nt 993–1317 corresponding to aa 287–387)
were completely sequenced according to the chain-termination
method, and p53 nt and aa positions are as described by ZakutHouri et al. (1985).
Northern blot analysis
Total cytoplasmic RNA was isolated and subsequently equal
amounts of RNA (20 µg/lane) were loaded on 1% agarose gels,
size-fractionated by gel electrophoresis and blotted onto Hybond
N1 nylon membranes (Amersham, Aylesbury, UK) according to
standard procedures. Blots were hybridized with [a-32P] dATP
(Amersham) radiolabeled cDNA probes according to the recommendation of the Random Primed DNA Labeling Kit (Boehringer
Mannheim). The used cDNA probes were the NotI fragment
purified from plasmid pCEP-WAF-1 and the b-actin probe.
RESULTS
Sensitivity to cisplatin and growth characteristics
The sensitivity of the 4 TGCT cell lines for cisplatin-induced
growth inhibition was estimated by the MTT assay. The sensitivity
of these cell lines for cisplatin expressed in IC50 values ranged
between 1.17 6 0.59 µM (mean 6 standard deviation [SD]) of
cisplatin for the NT2 cell line and 4.01 6 0.75 µM for the 2102 EP
cell line (Table I). Based on these IC50 values, 2102 EP cells were
approximately 4-fold less sensitive to cisplatin compared with NT2
cells. The IC90 values for these TGCT cell lines show an identical
pattern of cisplatin sensitivity. We further investigated whether
differences in growth characteristics could account for the differential chemosensitivity of these cell lines. The in vitro doubling times
of the TGCT cell lines were in the same order of magnitude (Table
I), and cell cycle analysis of exponentially growing cells showed no
differences in the distribution over the different cell cycle phases
(G1, S, G2/M) between the TGCT cell lines (data not shown).
Apparently, the differential cisplatin sensitivity of the TGCT cell
lines is not due to differences in their growth characteristics.
Cisplatin accumulation and DNA platination
To investigate whether differences in cisplatin accumulation
could account for the differential chemosensitivity among the
TGCT cell lines, we determined the intracellular Pt accumulation
for the different TGCT cell lines (Table I). Cells were incubated for
2 hr with 10 µg/ml (33.3 µM) or 30 µg/ml (100 µM) of cisplatin,
and the intracellular Pt concentration was quantitated by AAS. All
cell lines display an approximately linear correlation between
incubation dose and accumulation. NT2 and 2102 EP cells
accumulated about equal amounts of cisplatin, while these cell
lines showed a 4-fold difference in cisplatin sensitivity as determined by the MTT assay. The intracellular Pt concentration of
cisplatin-treated S2 and NCCIT cells were, respectively, approximately 1.5- and 2-fold higher than in NT2 or 2102 EP cells. Thus,
the intracellular Pt levels were not correlated with the cisplatin
sensitivity of these TGCT cell lines.
We also determined the total DNA platination in the TGCT cell
lines (Table I). Similar to the accumulation experiments, cells were
incubated with 10 or 30 µg/ml of cisplatin and analyzed for
Pt-DNA adduct formation. The DNA platination data show that the
small differences in accumulation are completely paralleled by
similar differences in Pt-DNA adduct formation. As for the
intracellular Pt accumulation, no clear correlation between total
DNA lesions in the overall genome and cisplatin sensitivity of these
TGCT cell lines was noted. Moreover, equimolar doses of cisplatin
in NT2 and 2102 EP cells were found to be equi-damaging with
respect to Pt-DNA adduct formation but showed a 4-fold difference
in drug sensitivity as measured by the MTT assay. These results
indicate that the differential cisplatin sensitivity of the TGCT cell
lines is not attributed to differences in DNA platination.
Induction of apoptosis
To evaluate whether observed differences in cisplatin sensitivity
of these TGCT cell lines are related to their proneness to
drug-induced apoptosis, we determined the threshold of susceptibility for induction of apoptosis by cisplatin. Apoptosis was examined
morphologically by Hoechst/PI staining, assessed by immunoblot
analysis of proteolytic cleavage of PARP and quantitated flow
cytometrically by annexin V labeling.
TABLE I – CHARACTERISTICS OF TGCT CELL LINES
Cell line
DT (h)
NT2
NCCIT
S2
2102 EP
33.5 6 9.8
32.4 6 9.5
30.7 6 6.0
24.1 6 1.4
IC50
(µM) 1
1.17 6 0.59
1.99 6 0.64
2.75 6 0.62
4.01 6 0.75
IC90 (µM) 1
Cisplatin accumulation (ng Pt mg21 protein) 2
DNA platination (pg Pt µg21 DNA) 3
p534
8.49 6 4.43
7.04 6 0.28
15.18 6 2.94
27.24 6 3.63
26.0 6 3.1 (85.5 6 8.5)
39.6 6 5.1 (117.3 6 1.8)
52.0 6 6.3 (193.6 6 9.8)
23.6 6 2.8 (84.8 6 3.7)
33.1 6 3.1 (76.3 6 8.2)
30.8 6 6.7 (98.4 6 9.7)
37.2 6 4.9 (132.9 6 8.9)
24.6 6 8.3 (79.2 6 6.7)
wt/wt
mt/2
wt/wt
wt/wt
1IC and IC are those drug concentrations effective in inhibiting 50% and 90%, respectively, of the cell growth measured by MTT assay after
50
90
4 days of continuous exposure to the drug. The MTT data were derived from 3 independent experiments.–2Total intracellular Pt concentration
(mean 6 SD) after 2-hr exposure of the cells to 10 µg/ml of cisplatin. Values in parentheses are the accumulation data of 30 µg/ml of cisplatin
exposure. The Pt accumulation data were derived from 3 independent experiments.–3Total amount of Pt bound to DNA measured by AAS after
2-hr exposure of the cells to 10 µg/ml of cisplatin. Values in parentheses are the accumulation data of 30 µg/ml of cisplatin exposure. The Pt/DNA
adducts data were derived from 3 independent experiments.–4The p53 gene status of the TGCT cell lines was determined by sequencing their
complete p53 cDNA. Sequence data revealed that NCCIT cells are hemizygous for p53 containing 1 mutated allele carrying 1 bp deletion.–DT, In
vitro doubling time.
CISPLATIN-INDUCED APOPTOSIS IN TGCT CELL LINES
The induction of apoptosis was recognized by morphological
changes such as chromatin condensation and nuclear fragmentation. These typical nuclear changes, visualized by staining the cells
with Hoechst 33342 (blue) and PI (pink), were monitored with a
fluorescence microscope and were apparent 48–72 hr after cisplatin
treatment. Spontaneous apoptosis in TGCT cell cultures was low
(,3%). Micrographs of representative samples illustrate that the
vast majority of cisplatin-treated NT2, NCCIT and S2 cells have an
apoptotic appearance (Fig. 1a). In contrast, cisplatin-treated 2102
EP cells did not differ from untreated cells; even at a dose of 25 µM,
only approximately 8% of cells exhibited apoptotic nuclei.
A family of cysteine proteases showing structural homology to
interleukin-1b-converting enzyme (ICE) have been implicated in
the initiation of the active phase of apoptosis through specific
proteolytic cleavage of essential substrates including PARP, a
nuclear enzyme involved in DNA repair (Martin and Green, 1995).
Therefore, we studied the proteolytic cleavage of PARP using a
PARP specific MAb (C-2-10) that recognizes both 116 kDa PARP
and the 85 kDa apoptosis-related cleavage fragment. The presence
of 85 kDa proteolytic PARP fragment was demonstrated by
Western blot analysis in cell lysates of cisplatin-treated NT2,
NCCIT and S2 cultures (Fig. 1b). Although the intact 116 kDa
PARP protein was readily detected, the apoptosis-specific 85 kDa
PARP fragment was not present in drug-treated 2102 EP cells. The
apparent absence of proteolytic cleavage of PARP confirmed the
morphological data and indicates that the 2102 EP TGCT cell line
is relatively resistant to chemotherapy-induced apoptosis.
An established approach for the quantification of apoptotic cells
is based on the fact that the surface of apoptotic cells differs from
normal cells in that phosphatidylserine (PS) is aberrantly exposed
on the external face of the cell membrane. Annexin V binds
preferentially to PS, which can be used to quantitate the early
phases of the apoptotic process (Boersma et al., 1996). Representative FITC frequency histograms of the 4 TGCT cell lines (Fig. 1c)
show that untreated control cells had a mean fluorescence intensity
of approximately 20–30 arbitrary units (a.u.) and illustrate that the
apoptotic cells are represented by an additional distinct subpopulation with a fluorescence intensity $100 a.u. (Boersma et al., 1996).
Nearly all NT2, NCCIT and S2 cells that were still present in the
drug-treated culture were annexin-V–positive, suggesting massive
induction of apoptosis in these TGCT cell lines. In contrast, hardly
any of the treated 2102 EP cells showed increased ($100 a.u.)
annexin V staining, suggesting that the vast majority of 2102 EP
cells had failed to undergo drug-induced apoptosis. At 72 hr after
cisplatin treatment (compared with 48 hr), the percentage of
annexin-V–positive 2102 EP did not increase, suggesting that the
apoptotic response in 2102 EP was not simply delayed. In
summary, morphological studies, analysis of PARP cleavage and
annexin V labeling results support the concept that the 2102 EP cell
line, compared with the other TGCT cell lines, is significantly less
sensitive to cisplatin-induced apoptosis.
p53 status (wt vs. mt) of the TGCT cell lines
The p53 gene status of the TGCT cell lines was determined by
sequence analysis of the complete p53 cDNA. The sequence data
revealed that NT2, S2 and 2102 EP cells are homozygous for wtp53
(Table I). In contrast, NCCIT cells were shown to be hemizygous
for p53 containing 1 mutated allele carrying a 1 bp deletion. This
deletion (G at nt position 949; codon 272) results in a frameshift
mutation leading to altered downstream codons and eventually to a
stopcodon at position 1114. This mutated p53 allele encodes a
truncated p53 protein of 347 amino acids. Concomitantly, NCCIT
p53 protein migrates faster than wtp53 protein as detected by
Western blot analysis using the DO-1 MAb (Fig. 2a). NT2 and
2102 EP cells express similar basal levels of wtp53 protein.
Surprisingly, no endogenous expression of p53 protein was detected in S2 cells despite the presence of wtp53 alleles.
595
Effects of cisplatin on p53 protein accumulation and
p21/WAF/CIP expression
The p53 response to genotoxic insults was investigated by
monitoring p53 protein levels in cisplatin-treated TGCT cells using
Western blot analysis (Fig. 2b). In contrast to NT2 and 2102 EP
cells that show a normal p53 response, NCCIT and S2 cells did not
show a significant increase in the level of p53 protein after cisplatin
treatment.
The presence of transactivation-competent p53 protein was
determined by its ability to activate the p21/WAF/CIP gene, whose
expression is regulated at the transcriptional level by the wtp53 but
not the mtp53 protein (Harris, 1996). Indeed, p21/WAF/CIP mRNA
was found to be significantly upregulated 6–12 hr following
g-irradiation (10 Gy) in wtp53-expressing NT2 and 2102 EP cells
(Fig. 3). No increased p21/WAF/CIP mRNA levels were detected in
p53 non-expressing S2 cells, and a complete absence of p21/WAF/
CIP mRNA expression was found in mtp53-containing NCCIT
cells. A study on the time course of p21/WAF/CIP expression
demonstrated that the absence of detectable p21/WAF/CIP mRNA
levels at 6 hr after irradiation was not simply the result of an
inappropriate sampling time. Apparently, NCCIT and S2 cells are
p53-transactivation deficient.
Expression of different members of the Bcl-2 protein family
Several studies have implicated members of the rapidly expanding Bcl-2 protein family as important components of the apoptosis
pathway in a wide range of different cell types (Reed et al., 1996).
As an initial approach to determine the possible involvement of the
Bcl-2 family proteins in the apoptotic pathway of TGCT cells, we
examined the constitutive protein expression of Bax, Bcl-2, Bcl-x
and Bak by Western blot analysis (Fig. 4). Rather high but
comparable endogenous levels of Bax protein were found in all
TGCT cell lines. Identical results were found with a MAb directed
against the Bax protein that became available only recently. No
Bcl-2 protein expression was found in NCCIT cells, whereas
comparable Bcl-2 protein levels were found in the other TGCT cell
lines. The endogenous protein levels of Bcl-xL, the long form of
Bcl-x that can heterodimerize with Bax and consequently influences the Bcl-2/Bax rheostat, were also shown to be comparable. In
addition, the short form of Bcl-x (Bcl-xS ) was never detected. Bak
expression was found to be somewhat more heterogeneous among
the studied TGCT cell lines but showed no apparent association
with induction of apoptosis. These studies on the intrinsic expression of Bcl-2 family proteins revealed no correlation between the
Bcl-2/Bax ratio and the induction of apoptosis.
The constitutive expression levels of death-suppressing and
death-promoting genes are inherent to the cell. Because genotoxic
stress-induced DNA damage may modulate these expression
levels, we examined the effect of cisplatin treatment on the
expression of the Bcl-2 family proteins. The TGCT cells were
incubated for 2 hr with different doses of cisplatin (3.1–12.5 µM);
at 6, 12, 24 and 48 hr after treatment, the expression level of these
death-related proteins was determined by Western blot analysis
(Fig. 4). Notably, the endogenous expression of Bax, Bcl-2, Bcl-x
and Bak was not affected by cisplatin at all time points studied.
DISCUSSION
TGCTs represent one of the few types of cancer that are curable
by chemotherapy and radiotherapy (Einhorn, 1990). Consistently,
most TGCT cell lines display an unusually high sensitivity to
cytotoxic agents. Analysis of potentially relevant parameters,
including cellular detoxification mechanisms (e.g., the glutathione
and the metallothionein system), Pt accumulation, DNA platination
and repair as well as topoisomerases activity, did not elucidate the
nature of the exceptional sensitivity of TGCTs to cyto-reductive
therapy (Masters et al., 1993; Sark et al., 1995). Most TGCT cell
lines are prone to drug-induced apoptosis that may be an important
determinant in the chemotherapeutic response of this type of cancer
(Cresta et al., 1996). However, the cellular components that
596
BURGER ET AL.
FIGURE 1
CISPLATIN-INDUCED APOPTOSIS IN TGCT CELL LINES
determine the low threshold for apoptosis induction have not been
defined, although it has been suggested that p53 status plays a
pivotal role (Peng et al., 1993; Cresta et al., 1996).
In the present study, we investigated the role of p53 and
members of the Bcl-2 protein family in drug-induced apoptosis in a
panel of TGCT cell lines. The induction of apoptosis, recognized
morphologically by chromatin condensation and nuclear fragmentation, was apparent 48–72 hr after cisplatin treatment in cultures of
NT2, NCCIT and S2 cells, but not in 2102 EP cells (Fig. 1a).
Accordingly, the apoptosis-associated proteolytic cleavage of PARP
was readily detected in drug-treated NT2, NCCIT and S2 cells,
although it was absent in 2102 EP cells (Fig. 1b). Quantification of
the apoptotic process by annexin V labeling demonstrated that in
contrast to the other TGCT cell lines, the vast majority of 2102 EP
cells are resistant to the induction of apoptosis (Fig. 1c). The
insensitivity of 2102 EP cells to drug-induced apoptosis correlates
with the MTT data (Table I). Cisplatin resistance has been
associated with reduced intracellular accumulation. However,
chemosensitive NT2 and chemoresistant 2102 EP cells accumulated approximately equal amount of cisplatin and showed no
difference in DNA platination. The observed intracellular Pt levels
in these TGCT cell lines excluded the possibility that increased
efflux as a result of functional overexpression of an ATP-dependent
glutathione S-conjugate pump (Ishikawa et al., 1994) accounted for
the differential chemosensitivity among these TGCT cell lines. The
hypersensitivity of TGCTs may be related to a defective capacity to
remove (repair) Pt-DNA adducts (Hill et al., 1994). However, the
identical DNA platination in chemosensitive NT2 and chemoresistant 2102 EP cells was accompanied by an identical capacity to
remove the Pt lesions from the genome (data not shown), indicating
that differential DNA repair is presumably not involved. In line
with our observations, the absence of a correlation between DNA
repair and chemosensitivity in TGCT cell lines has been reported
by others as well (Köberle et al., 1996). From these data we
concluded that the observed differential cisplatin sensitivity of the
TGCT cell lines was not associated with differences in growth
characteristics, Pt accumulation, Pt-DNA adduct formation or DNA
repair, but probably has to be attributed merely to different
thresholds to drug-induced apoptosis.
The product of the p53 tumour suppressor gene is involved in
multiple cellular processes, including gene transcription, DNA
repair, genomic stability, cell cycle control and apoptosis (reviewed
by Harris, 1996). The status of p53 is thought to be an important
mediator in the cellular response to chemotherapy. With respect to
testicular tumours, it has been suggested that the absence of
mutations in the p53 gene accounts for the hypersensitivity of
FIGURE 1 – Induction of apoptosis in TGCT cell lines by cisplatin.
(a) Typical morphological changes associated with apoptosis were
examined by Hoechst/PI staining and visualized by fluorescence
microscopy analysis. Representative micrographs of untreated (top)
and cisplatin-treated (12.5 µM cisplatin, 2 hr at 37°C) TGCT cell
cultures (bottom) at 72 hr after treatment are shown. The different
TGCT cell lines are indicated. Scale bar 5 30 µm. (b) Western blot
analysis of cisplatin-induced cleavage of PARP in TGCT cell lines.
Cells were incubated in the absence (2) and presence (1) of drug (12.5
µM cisplatin, 2 hr at 37°C). At 48 hr after treatment, the integrity of
PARP was monitored by immunoblotting. The different TGCT cell
lines are indicated. A Western blotting control (C) to detect specific
PARP cleavage was included and represents cell extracts of untreated
(2) and etoposide-treated (1) human HL60 leukemia cells. The results
of 1 experiment are typical of 3 independent replicates. (c) Annexin V
binding histograms of cisplatin-treated TGCT cells. At 48 hr after drug
treatment (12.5 µM cisplatin, 2 hr at 37°C), the cells were stained with
FITC-conjugated annexin V. The binding of annexin V was quantitated
by flow cytometry, and the fluorescence intensity was expressed in
arbitrary units (a.u.). Solid lines represent untreated (control) cells, and
dotted lines represent drug-treated cells. Apoptotic cells with a high
fluorescence intensity ($100 a.u.) are indicated by the shaded areas.
The different TGCT cell lines are indicated. The data shown are
representative univariate histograms.
597
FIGURE 2 – Western blot analysis of p53 protein levels in TGCT cell
lines. Basal levels of p53 protein (a) and the effects of cisplatin
treatment (12.5 µM cisplatin, 2 hr at 37°C) on endogenous p53 protein
levels (b) are shown. Cells were incubated in the absence (2) and
presence (1) of cisplatin; at 48 hr after drug treatment, the p53 protein
levels were monitored by immunoblotting. The different TGCT cell
lines are indicated.
FIGURE 3 – Northern blot analysis of endogenous (2) and radiationinduced p21/WAF/CIP mRNA levels (1) in TGCT cell lines. Total
RNA was isolated from the cells at 6 hr after g-irradiation (10 Gy).
Equal amounts of RNA (10 µg/lane) were size fractionated on 1%
agarose gels, transferred onto a nylon membrane and probed with the
NotI fragment of the human p21/WAF/CIP cDNA. The different TGCT
cell lines are indicated. Equal intensity of ethidium bromide staining of
ribosomal RNA bands and the hybridizing results of b-actin confirmed
the presence of equal amounts of RNA within each lane. Autoradiographs were exposed for 16 hr with intensifying screens.
TGCT cells to genotoxic agents (Cresta et al., 1996). Therefore, we
evaluated the role of p53 in drug-induced apoptosis in our panel of
TGCT cell lines. In contrast to wtp53-containing NT2, S2 and 2102
EP cells, NCCIT cells were found to be hemizygous for p53
containing 1 mutated allele carrying a 1 bp deletion at position 949
of codon 272. Consequently, NCCIT cells express a truncated p53
protein of 347 amino acids that lacks the tetramerization, nuclear
localization and DNA damage recognition site (Harris, 1996). The
carboxy-terminus encoded by exons 9–11 of the p53 protein, which
is absent in the truncated p53 protein of NCCIT cells, has been
implicated in the induction of apoptosis (Harris, 1996). However,
598
BURGER ET AL.
FIGURE 4 – Western blot analysis of endogenous and cisplatin-induced Bax, Bcl-2, Bcl-x and Bak protein levels in TGCT cell lines. Cells were
incubated in the absence (c) and presence of cisplatin (3.1, 6.3, 12.5 µM); at 48 hr after drug treatment, the Bcl-2 family protein levels were
monitored by immunoblotting. The Bak PAb reacted, apart from the Bak specific band (Mr: approx. 24,000), with an Mr: approx. 21,000 band that
possibly represents a proteolytic degradation product or an alternatively spliced variant of Bak. Coomassie staining of duplicate blots showed that
equivalent amounts of protein were present in all samples analyzed. The different TGCT cell lines are indicated. Molecular weight markers are
indicated in kDa at the left, and the arrows indicate the respective position of the Bcl-2 family protein.
we clearly demonstrated that mtp53 NCCIT and wtp53 NT2 cells
are equally sensitive to drug-induced apoptosis. Although no basal
level of p53 protein was detected in S2 cells, despite the presence
of p53 mRNA, drug-induced apoptosis was evident. Whether S2
cells have an impaired translational machinery or whether the p53
protein is specifically inactivated by cellular or viral proteins
remains to be elucidated. In contrast to wtp53 NT2 and 2102 EP
cells, the endogenous p53 protein levels of NCCIT and S2 cells
were not affected by cisplatin treatment or g-irradiation, indicating
that NCCIT and S2 cells have a general defect in their p53 response
to genotoxic insults. Consistently, NT2 and 2102 EP cells normally
transactivated the expression of p21/WAF/CIP after DNAdamaging treatment, although no increase was observed in NCCIT
and S2 cells (Fig. 3). These results indicate that NT2 and 2102 EP
cells express functional wtp53, whereas NCCIT and S2 cells are
deficient in p53-mediated transactivation. Obviously, no correlation between p53 status and induction of apoptosis was detected,
suggesting the presence of a p53-independent apoptotic pathway in
TGCTs. Previously, p53-independent apoptosis has been observed
in a variety of tumour cell types. Although it is clear that
chemotherapy- and radiation-induced apoptosis can proceed despite the absence of functional p53, little is known about the
activation and signaling pathway involved in p53-independent
apoptosis. Our data might imply that the onset of apoptosis in
TGCTs is p53-independent. In contrast to this hypothesis, Cresta et
al. (1996) concluded that the hypersensitivity of TGCT cell lines to
etoposide-induced apoptosis was associated with functional p53.
However, this conclusion was primarily based on differential
apoptosis induction between chemo-sensitive wtp53 TGCT cell
lines and chemo-resistant bladder cell lines expressing mtp53,
rather than differential sensitivity among TGCT cell lines with
different p53 status. Alternatively, although less likely, the onset of
cisplatin- and etoposide-induced apoptosis may proceed through
different apoptotic pathways. Notably, preliminary results showed
that the p53-independent induction of apoptosis in TGCT cell lines
is not cisplatin-specific. Both the NCCIT cell line expressing
transactivation-deficient mtp53 and the NT2 cell line endowed with
wtp53 alleles were shown to be highly susceptible to radiationinduced apoptosis, whereas the p53-transactivation competent
2102 EP cell line appeared to be resistant to induction of apoptosis
by g-radiation.
Members of the Bcl-2 protein family have been implicated as
important components of the apoptosis pathway in a wide range of
different cell types (reviewed by Reed et al., 1996). Based on the
capacity of these proteins to form dimers, a simple competitive
binding model has been established in which the Bcl-2/Bax ratio
determines the extent to which apoptosis is induced or suppressed
(Reed et al., 1996). Our data, as presented in Figure 4, reveal that
the endogenous protein levels of the studied members of the Bcl-2
protein family (Bcl-2, Bax, Bcl-x and Bak) were not associated
with the differential susceptibility to drug-induced apoptosis.
Moreover, the expression of the Bcl-2 family proteins was not
affected by genotoxic stress, indicating that the Bcl-2/Bax rheostat
is not involved in the p53-independent induction of apoptosis in
TGCT cell lines. Although no Bax upregulation was apparent by
immunoblotting, we previously demonstrated heterogeneous Bax
expression and upregulation within the apoptotic NT2 population
CISPLATIN-INDUCED APOPTOSIS IN TGCT CELL LINES
by flow cytometry (Boersma et al., 1997). However, no Bax
upregulation could be detected by flow cytometry in the other
TGCT cell lines (data not shown). In line with our results, Bax
protein was not increased after etoposide treatment in any of the 6
TGCT cell lines studied by Cresta et al. (1996). In addition, high
levels of ectopically expressed Bcl-2 protein in S2 cells transfected
with the complete bcl-2 cDNA were not sufficient to inhibit
cisplatin-induced apoptosis in this TGCT cell line (data not shown).
Our findings in TGCT cell lines are in accordance with a previous
report demonstrating that differential sensitivity of Burkitt’s lymphoma cell lines to g-irradiation is independent of the status of p53
and the expression of Bax, Bcl-2 and Bcl-x (Khanna et al., 1996).
In contrast, it has been suggested that inherent protein levels of Bax
and Bcl-2 in TGCT cell lines determine the threshold of susceptibility to apoptosis induction by etoposide (Cresta et al., 1996).
However, this premise was primarily based on differences in Bcl-2
expression between chemosensitive TGCT cell lines and chemoresistant bladder cell lines.
From our present results on the cisplatin-induced apoptotic
pathway in TGCT cell lines, we concluded that the susceptibility to
apoptosis induction does not appear to be related to p53 status, to
endogenous protein levels of the members of the Bcl-2 protein
family and to drug-induced modulation of the expression of Bcl-2
family proteins. This report demonstrates that drug-induced apoptosis in TGCT cell lines is not regulated by Bcl-2 family proteins and
can proceed in the absence of functional wtp53. The results of our
study and the finding that alterations of the p53 gene in carcinoma
599
in situ of the testis are more common than previously thought
(Kuczyk et al., 1996) may indicate that the presence of functional
wtp53 is not required for the successful treatment of TGCTs.
Furthermore, p53 inactivation in both experimental and human
tumorigenesis is believed to be a late event and superimposed on a
series of progressive genetic abnormalities that may all influence
the outcome of anti-cancer therapies (Fisher, 1994). The existence
of many Bcl-2 family proteins and the fact that new members are
still being identified may suggest that a unified model of apoptosis
is more complex than the rather simple Bcl-2/Bax competitive
binding model or that multiple models of apoptosis exist. Apparently, distinct cellular thresholds for induction of apoptosis exist
among different tumour types. Thus, further unraveling of the
apoptotic pathway and identification of key determinants of
chemo-sensitivity in TGCTs may lead to more appropriate and
successful anti-cancer treatment modalities for tumour types that
are now considered to be drug resistant.
ACKNOWLEDGMENTS
We thank Dr. B. Vogelstein (The Johns Hopkins University
School of Medicine, Baltimore, MD) for providing the pCEPWAF-1 plasmid. We also thank Mr. E.C.E. Brouwer for assistance
with Pt determinations by AAS and Mr. R.H.A.M. Vossen (Laboratory of Anthropogenetics, Sylvius Laboratories, Leiden University,
Leiden, The Netherlands) for his help with p53 sequence determinations.
REFERENCES
BOERSMA, A.W.M., NOOTER, K., BURGER, H., KORTLAND, C.J. and STOTER,
G., Bax upregulation is an early event in cisplatin-induced apoptosis in
human testicular germ cell tumour cell line NT2, as quantitated by flow
cytometry. Cytometry, 27, 275–282 (1997).
BOERSMA, A.W.M., NOOTER, K., OOSTRUM, R.G. and STOTER, G., Quantification of apoptotic cells with FITC-labeled Annexin V in CHO cells treated
with cisplatin. Cytometry, 24, 123–130 (1996).
CLARKE, A.R., PURDIE, C.A., HARRISON, D.J., MORRIS, R.G., BIRD, C.C.,
HOOPER, M.L. and WYLLIE, A.H., Thymocyte apoptosis induced by
p53-dependent and independent pathways. Nature (Lond.), 362, 849–852
(1993).
CRESTA, C.M., MASTERS, J.R.W. and HICKMAN, J.A., Hypersensitivity of human
testicular tumours to etoposide-induced apoptosis is associated with functional
p53 and a high Bax:Bcl-2 ratio. Cancer Res., 56, 1834–1841 (1996).
DAMJANOV, I., HORVAT, B. and GIBAS, Z., Retinoic acid-induced differentiation of the developmentally pluripotent human germ cell tumour-derived
cell line, NCCIT. Lab. Invest., 68, 220–232 (1993).
EINHORN, L.H., Treatment of testicular cancer: a new and improved model.
J. clin. Oncol., 8, 1777–1781 (1990).
FISHER, D.E., Apoptosis in cancer therapy: crossing the threshold. Cell, 78,
539–542 (1994).
HARRIS, C.C., Structure and function of the p53 tumour suppressor gene:
clues for rational cancer therapeutic strategies. J. nat. Cancer Inst., 88,
1442–1452 (1996).
HILL, B.T., SCANLON, K.J., HANSSON, J., HARSTRICK, A., PERA, M.,
FICHTINGER-SCHEPMAN, A.M.J. and SHELLARD, S.A., Deficient repair of
cisplatin-DNA adducts identified in human testicular teratoma cell lines
established from tumours from untreated patients. Europ. J. Cancer, 30A,
832–837 (1994).
ISHIKAWA, T., WRIGHT, C.D. and ISHIZUKA, H., GS-X pump is functionally
overexpressed in cis-diamminnedichloroplatinum(II)-resistant human leukemia HL-60 cells and down-regulated by cell differentiation. J. biol.
Chem., 269, 29085–29093 (1994).
KHANNA, K.K., WIE, T., SONG, Q., BURROWS, S.R., MOSS, D.J., KRAJEWSKI,
S., REED, J.C. and LAVIN, M.F., Expression of p53, bcl-2, bax, bcl-x2 and
c-myc in radiation-induced apoptosis in Burkitt’s lymphoma cells. Cell
Death Differentiation, 3, 315–322 (1996).
KÖBERLE, B., PAYNE, J., GRIMALDI, K.A., HARTLEY, J.A. and MASTERS,
J.R.W., DNA repair in cisplatin-sensitive and resistant human cell lines
measured in specific genes by quantitative polymerase chain reaction.
Biochem. Pharmacol., 52, 1729–1734 (1996).
KORSMEYER, S.J., Bcl-2 initiates a new category of oncogenes: regulators of
cell death. Blood, 80, 879–886 (1992).
KUCZYK, M.A., SERTH, J., BOKEMEYER, C., JONASSEN, J., MACHTENS, S.,
WERNER, M. and JONAS, U., Alterations of the p53 tumour suppressor gene
in carcinoma in situ of the testis. Cancer, 78, 1958–1966 (1996).
LOWE, S.W., RULEY, H.E., JACKS, T. and HOUSMAN, D.E., p53-Dependent
apoptosis modulates the cytotoxicity of anticancer agents. Cell, 74,
957–967 (1993).
MARTIN, S.J. and GREEN, D.R., Protease activation during apoptosis: death
by a thousand cuts? Cell, 82, 349–352 (1995).
MASTERS, J.R.W., OSBORNE, E.J., WALKER, M.C. and PARRIS, C.N., Hypersensitivity of human testis tumour cell lines to chemotherapeutic drugs. Int.
J. Cancer, 53, 340–346 (1993).
MILLER, S.A., DYKES, D.D. and POLESKY, H.F., A simple salting out
procedure for extracting DNA from human nucleated cells. Nucl. Acids
Res., 16, 1215 (1988).
MIYASHITA, T., KRAJEWSKI, S., KRAJEWSKI, M., WANG, H.G., LIN, H.K.,
LIEBERMANN, D.A., HOFMANN, B. and REED, J.C., Tumour suppressor p53 is
a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene,
9, 1799–1805 (1994).
PENG, H.Q., HOGG, D., MALKIN, D., BAILEY, D., GALLIE, B.L., BULBUL, M.,
JEWETT, M., BUCHANAN, J. and GROSS, P.E., Mutations of the p53 gene do
not occur in testis cancer. Cancer Res., 53, 3574–3578 (1993).
REED, J.C., MIYASHITA, T., TAKAYAMA, S., WANG, H.-G., SATO, T.,
KRAJEWSKI, S., AIMÉ-SEMPÉ, C., BODRUG, S., KITADA, S. and HANADA, M.,
Bcl-2 family proteins: regulators of cell death involved in the pathogenesis
of cancer and resistance therapy. J. cell. Biochem., 60, 23–32 (1996).
SARK, M.W.J., TIMMER-BOSSCHA, H., MEIJER, C., UGES, D.R.A., SLUITER,
W.J., PETERS, W.H.M., MULDER, N.H. and DE VRIES, E.G.E., Cellular basis
for differential sensitivity to cisplatin in human germ cell tumour and colon
carcinoma cell lines. Brit. J. Cancer, 71, 684–690 (1995).
WANG, N., PERKINS, K.L., BRONSON, D.L. and FRALEY, E.E., Cytogenetic
evidence for premeiotic transformation of human testicular cancers. Cancer
Res., 41, 2135–2140 (1981).
ZAKUT-HOURI, R., BIENZ-TADMOR, B., GIVOL, D. and OHREN, M., Human
p53 cellular tumour antigen: cDNA sequence and expression in COS cells.
EMBO J., 4, 1251–1255 (1985).
ZHAN, Q., FAN, S., BAE, I., GUILLOUF, C., LIEBERMANN, D.A., O’CONNOR,
P.M. and FORNACE, A.J., Induction of bax by genotoxic stress in human cells
correlates with normal p53 status and apoptosis. Oncogene, 9, 3743–3751
(1994).
Документ
Категория
Без категории
Просмотров
6
Размер файла
659 Кб
Теги
980
1/--страниц
Пожаловаться на содержимое документа