close

Вход

Забыли?

вход по аккаунту

?

89

код для вставкиСкачать
Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
Int. J. Cancer: 70, 551–555 (1997)
r 1997 Wiley-Liss, Inc.
DNA REPAIR CAPACITY AND CISPLATIN SENSITIVITY
OF HUMAN TESTIS TUMOUR CELLS
Beate KÖBERLE1, Keith A. GRIMALDI2, Andrew SUNTERS2, John A. HARTLEY2, Lloyd R. KELLAND3 and John R.W. MASTERS1*
1Institute of Urology and Nephrology, London, UK
2CRC Drug-DNA Interactions Research Group, Department of Oncology, University College London, London, UK
3CRC Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey, UK
Metastatic testicular germ cell tumours can be cured using
cisplatin-based combination chemotherapy. To investigate
the role of DNA repair in cisplatin sensitivity, we measured
the formation and removal of cisplatin adducts in the whole
genome and in specific genomic regions in 3 testis and 3
bladder cancer cell lines. Following a 1 hr exposure to 50 µM
cisplatin, the mean level of DNA platination was lower in the
testis tumour cell lines. During a 72 hr post-treatment
incubation period, the 3 bladder cell lines removed platinum
from the overall genome, whereas 2 of the testis tumour cell
lines showed relatively little reduction of DNA platination.
The third testis tumour cell line, SuSa, showed an intermediate capacity to remove cisplatin. Cisplatin-induced damage
and repair in selected regions of the actively transcribed N-ras
gene and the inactive CD3d gene were measured using
quantitative PCR. The data were in agreement with those
obtained with atomic absorption spectroscopy for the whole
genome, showing that the bladder lines were repair-proficient: 2 of the testis tumour cell lines showed no repair, and
the third testis line, SuSa, showed an intermediate level of
repair in these 2 genes. Our findings confirm that reduced
capacity to repair cisplatin-damaged DNA may contribute to
the hypersensitivity of testis tumour cells to DNA-damaging
agents. Int. J. Cancer 70:551–555, 1997.
r 1997 Wiley-Liss, Inc.
Metastatic testicular germ cell tumours are cured in over 80% of
patients using cisplatin-based combination chemotherapy (Einhorn, 1990). In contrast, most other types of metastatic cancer
rarely are cured using chemotherapy. Therefore, the sensitivity of
testicular germ cell tumours to chemotherapy may have more
general relevance as it may provide clues to improving therapy for
other types of cancer.
Cell lines derived from testis tumours are also more sensitive to
chemotherapeutic drugs than those derived from other types of
cancer, indicating that these cells retain their relative sensitivity to
DNA-damaging agents in vitro, reflecting the clinical response. We
have compared the sensitivity of cell lines derived from testicular
germ cell tumours with those derived from bladder cancers.
Cisplatin is also the most effective single agent for bladder cancer,
but only 40–50% of metastatic bladder cancers respond to combination chemotherapy and long-term survival is rare (Seidman and
Scher, 1991). Testis tumour cell lines are more sensitive than
bladder cancer cells to the cytotoxic effects of irradiation (Parris et
al., 1988) and many chemotherapeutic drugs (Walker et al., 1987;
Masters et al., 1993; Pera et al., 1995).
Cells derived from certain DNA repair disorders are also more
sensitive to chemotherapeutic drugs and ionising radiation. Testis
tumour cells are similar to cells derived from patients with
xeroderma pigmentosum (XP) and ataxia telangiectasia in their
sensitivities to UV and gamma radiation (Pera et al., 1987) and to
excision repair-deficient rodent cell mutants in their sensitivity to
cisplatin (Lee et al., 1993). In keeping with this analogy, studies on
repair in the whole genome indicate that some testis tumour cell
lines are repair-deficient (Bedford et al., 1988; Hill et al., 1994;
Sark et al., 1995; Kelland et al., 1992). This deficiency may
contribute to their sensitivity to DNA-damaging agents.
The rate of DNA repair is not uniform throughout the genome.
There are 2 repair pathways: one deals with the rapid and efficient
removal of lesions from transcribed genes, while the other accom-
plishes the slower and less efficient repair of bulk DNA (Bohr,
1991; Link et al., 1991). It has been suggested that the repair of
certain essential genes may be more critical for survival than repair
of the whole genome (Bohr et al., 1986). In repair-deficiency
disorders the defect can affect either one or both of the repair
pathways. In XP group C the repair defect is limited to the overall
genome repair (Venema et al., 1990a), whereas in Cockayne
syndrome cells the repair defect is limited to preferential genespecific repair (Venema et al., 1990b).
The published descriptions of DNA repair capacity in testis
tumour cells (Bedford et al., 1988; Hill et al., 1994; Sark et al.,
1995; Kelland et al., 1992) are restricted to repair in the whole
genome. The aim of our study was to extend these studies to
investigate the potential contribution of repair in active and
inactive genes. To confirm the results on DNA repair in the whole
genome, we compared DNA repair after treatment with cisplatin in
3 testis tumour cell lines with that in 3 bladder tumour cell lines in
the whole genome using atomic absorption spectroscopy. To
investigate repair in selected genes, we measured damage induction
and repair in defined regions of the actively transcribed N-ras gene
and the inactive CD3d gene using quantitative (Q-PCR) (Grimaldi
et al., 1994).
MATERIAL AND METHODS
Cell culture and drug treatment
Details of the origins of the cell lines have been described
previously (Masters et al., 1993). All cell lines were grown
routinely as monolayers in 25 cm2 tissue culture flasks in RPMI
1640 medium supplemented with 10% heat-inactivated FCS and 2
mM 1-glutamine at 36.5°C in a humidified atmosphere of 5% CO2
in air. Each cell line was used over a maximum of 15 passages to
minimize changes that might occur during long-term culture.
Cisplatin (Sigma, Poole, UK) was prepared immediately before use
by dissolving in water to a concentration of 1 mg/ml and
sterilization through a 0.22 µM filter.
Cisplatin sensitivity measurements
Cytotoxicity was determined by plating 2,500 cells (testis
tumour cell lines) or 750 cells (bladder tumour cell lines) in 60 3
15 mm Petri dishes containing 5 ml medium. Four dishes were set
up for untreated controls and each concentration of cisplatin.
Following incubation overnight at 36.5°C, cells were treated with a
range of concentrations of cisplatin for 1 hr, then washed twice with
PBS and incubated in fresh medium for 10–14 days. Colonies were
fixed with methanol, stained with 10% Giemsa and those consisting
of 50 or more cells counted. Inhibition of colony formation in
Contract grant sponsor: the Cancer Research Campaign; contract grant
number: SP 1997.
*Correspondence to: Institute of Urology and Nephrology, University
College London, 67 Riding House Street, 3rd Floor, London W1P 7PN,
UK. Fax: 004401716377076. E-mail: Regnjrm@Ucl.ac.uk
Received 19 August 1996.
552
KÖBERLE ET AL.
treated dishes was expressed as a percentage of colony formation in
untreated controls.
Measurement of platinum binding to DNA
Between 5 3 107 and 108 cells were grown to near confluence in
175 cm2 culture flasks, exposed to cisplatin (50 µM) for 1 hr and
harvested either immediately after drug exposure or after recovery
periods of 24, 48 and 72 hr. DNA was isolated by treatment with
proteinase K, 0.5% SDS and lysis buffer (150 mM NaCl, 10 mM
Tris [pH 8.0], 10 mM EDTA [pH 8.0]) overnight at 37°C. After
phenol/chloroform extraction (23) and chloroform extraction
(13), the DNA solution was treated with RNase (10 mg/ml) at
37°C for 1 hr followed by phenol/chloroform (13) and chloroform
extraction (13). DNA was precipitated with a 1/10 vol of sodium
acetate (3 M) and 2 vol of cold ethanol (absolute), washed twice
with ethanol (70%), dried and dissolved in 0.1 M nitric acid. The
amount of platinum bound to DNA was measured by atomic
absorption spectroscopy and the amount of DNA determined by
measuring the absorbance at 260 nm. To correct for dilution due to
DNA synthesis during the recovery period, cells were labelled with
50 nCi/ml [ 14C]thymidine for 24 hr before drug treatment. Dilution
factors (specific activity of DNA at time point/specific activity of
DNA at 0 hr) were then determined for the 24, 48 and 72 hr repair
periods. The reported values are the mean of up to 6 independent
experiments corrected for dilution due to DNA synthesis.
Damage and repair of cisplatin-induced lesions in specific genes
Cell culture and drug treatment were performed in 6-well plates
(Nunc, Roskilde, Denmark): 106 cells were treated with a range of
concentrations of cisplatin for 5 hr, after which cells were washed
with PBS and either trypsinised for DNA isolation or incubated for
another 24 hr in fresh medium for repair studies. DNA isolation and
Q-PCR were performed according to Grimaldi et al. (1994).
Briefly, cells were lysed with a solution composed of 340 µl lysis
buffer (400 mM Tris-HCl [pH 8.0], 60 mM EDTA, 150 mM NaCl,
1% w/vol SDS) and 100 µl 5 M sodium perchlorate. After
vortexing, the cell lysate was incubated on a shaking platform at
37°C for 20 min and then at 65°C for 20 min; 580 µl of chloroform
were added to the solution and the mixture was rotated for 20 min
at room temperature and centrifuged at 11,500g in a microfuge; 330
µl of the upper layer were transferred to a separate Eppendorf tube
and the DNA precipitated with 660 µl ethanol (absolute), washed
twice with ethanol (70%), dried and resuspended in 400 µl H2O.
Then, PCR was performed with 25 µl DNA suspension (equivalent
to 5 3 104 cells) in a 100 µl reaction mixture containing 50 pmoles
of each primer (Pharmacia, Uppsala, Sweden); 2 units Red Hot
DNA polymerase (Advanced Biotechnologies, Columbia, MD);
120 µM each dATP, dCTP, dGTP and dTTP (Pharmacia); 0.75 mM
MgCl2, buffer IV (Advanced Biotechnologies); and 1 µCi (a32P)dCTP (Amersham, Aylesbury, UK). The CD3d gene fragment
was amplified using the forward primer 58-TGA GGA CAG AGT
GTT TGT GAA-38 and the reverse primer 58-AGA GTA ACT CCC
AGC TGA GAC-38. The amplification product was 934 bp in
length and contained part of exon 2, intron 2 and exon 3.
Amplification of the N-ras gene, which gave a 1,053 bp fragment
covering the first intron, was performed with the forward primer
58-GCC TGG TTA CTG TGT CCT GT-38 and the reverse primer
58-GCC AGC CAC ATC TAC AGT AC-38. DMSO (5%) was added
to the reaction mixture for amplification of the fragment of the
N-ras gene. The mixture was overlaid with 100 µl mineral oil. PCR
was carried out in a Perkin-Elmer 480 Thermal Cycler (Norwalk,
CT) under the following conditions: the CD3d gene fragment was
amplified after an initial denaturation step of 5 min at 96°C by 25
cycles of 96°C for 1 min, 60°C for 1 min and 70°C for 1 min,
followed by a final incubation of 4 min at 70°C at the end of the
cycling; the N-ras fragment was amplified after an initial denaturation step of 2 min at 94°C, followed by 25 cycles of 94°C for 1
min, 59°C for 1 min and 72°C for 1 min, with a final incubation of 4
min at 72°C at the end of the cycling. Preliminary experiments
ensured that under these conditions the PCR was still in exponential phase when the PCR reaction stopped. The PCR product was
measured by precipitating 40 µl of PCR mixture with 1 ml TCA
(5% w/v TCA, 20 mM tetrasodium pyrophosphate). The precipitate
was captured on Whatman (Maidstone, UK) filter discs held in a 12
position vacuum manifold (Millipore, Bedford, MA). Filters were
washed free of unincorporated a-32P-dCTP with 10 ml TCA (5%)
and 10 ml absolute ethanol, placed in vials containing 5 ml
scintillation fluid (Ecoscint; National Diagnostics, Manville, NJ)
and counted on a Beckman (Palo Alto, CA) LS1800 scintillation
counter. The lesions per region were determined according to the
formula: lesions per region 5 2ln (Ad/A), where A is the PCR
product from the undamaged template and Ad is the product from
the damaged template. To demonstrate that the PCR reaction was
specific, resulting in only 1 product, 10 µl of PCR product were
subjected to electrophoresis on a 1.5% agarose gel overnight at 42V
and the gel was dried and exposed to autoradiographic film (Kodak,
Rochester, NY), to visualize the band.
RESULTS
Survival curves of the 3 testis (SuSa, 833K, GCT27) and 3
bladder (MGH-U1, RT112, HT1376) tumour cell lines following a
1 hr exposure to a range of concentrations of cisplatin are shown in
Figure 1. Comparing the mean IC50 values, the testis tumour cell
lines were on average 4.1-fold more sensitive to cisplatin than the
bladder tumour cell lines (Table I).
The amount of platinum bound to DNA directly after exposure to
cisplatin (50 µM) for 1 hr is shown in Table I. There was no
significant difference in the initial platination in the 3 testis tumour
cell lines and one of the bladder cancer cell lines (MGH-U1).
However, the other 2 bladder cancer cell lines contained approximately twice the levels of DNA platination of the testis tumour cell
lines.
Removal of platinum from the DNA of the 3 testis and the 3
bladder tumour cell lines is shown in Figure 2. The amount of DNA
platination of the 3 bladder tumour cell lines was reduced by
50–60% within 24 hr. In contrast, 2 of the 3 testis tumour cell lines
FIGURE 1 – Clonogenic cell survival curves of testis tumour cell
lines (open symbols) and bladder cancer cell lines (closed symbols)
after exposure to cisplatin for 1 hr.
DNA REPAIR IN TESTIS TUMOUR CELLS
553
TABLE I – CISPLATIN SENSITIVITY AND INITIAL PLATINATION OF TESTIS AND
BLADDER CANCER CELL LINES FOLLOWING A 1 HR EXPOSURE TO CISPLATIN
Cell line
Testis
SuSa
833K
GCT27
Bladder
MGH-U1
RT112
HT1376
IC50 (µM)
DNA platination
(nmol Pt/gDNA)
5.6 6 0.3
4.3 6 0.4
6.3 6 0.9
41.8 6 14.9
55.1 6 17.7
67.0 6 19.7
14.2 6 1.7
16.1 6 2.9
20.1 6 3.9
48.3 6 10.4
120.4 6 17.5
108.1 6 33.2
(833K and GCT27) showed less removal of platinum (approximately 30%) during the post-incubation time (Fig. 2). The third
testis line, SuSa, removed platinum at an intermediate rate at 24 hr
and to the same extent as the bladder cancer cells at 48 and 72 hr.
To investigate damage induction and repair in single-gene
regions, cells were treated with cisplatin at concentrations ranging
from 25 to 200 µM for the bladder tumour cells and from 25 to 200
µM for the testis tumour cells for 5 hr. Under these conditions, testis
tumour cell lines showed minimal new DNA synthesis (,1%) at
cisplatin concentrations of 25 µM (833K, SuSa) or 50 µM (GCT27)
as measured by incorporation of 14C-thymidine. With the bladder
tumour cell lines there was less than 5% new DNA synthesis at 50
µM (HT1376, RT112) or 100 µM (MGH-U1) cisplatin. Cells were
harvested either directly after treatment or after a 24 hr recovery
period. Using Q-PCR, the cisplatin-induced lesions were measured
in a 934 bp fragment of the CD3d gene and a 1,053 bp fragment of
the N-ras gene. Some of these data were also used in a study of the
repair of cisplatin-induced DNA damage in cisplatin-sensitive
parental lines compared with their cisplatin-resistant sublines
(Köberle et al., 1996).
Figure 3 shows an example of an autoradiograph of PCR
products of the CD3d gene region of HT1376 cells directly after
treatment or after a 24 hr recovery period. There is only one band,
reflecting the specificity of the PCR reaction. With increasing
cisplatin concentration the intensity of the band decreases, reflecting an increase in damage. This is reversed after 24 hr, showing
repair of the lesion.
The data for the bladder and testis tumour cell lines are
summarized in Tables II and III, respectively. Comparing the 2
gene fragments, there was no significant difference in the initial
frequency of lesions in the CD3d gene fragment when compared
with the N-ras gene fragment in all 6 tumour cell lines examined.
The repair studies showed that all 3 bladder tumour cell lines
removed cisplatin-induced damage from both gene fragments.
Comparing the repair rates after treatment with cisplatin at a
concentration of 100 µM for 5 hr, almost complete repair of the
CD3d and N-ras fragments of MGH-U1 cells after 24 hr was
observed (Table II). RT112 and HT1376 showed proportionately
less repair compared with MGH-U1, particularly at the higher
concentrations of 150 and 200 µM cisplatin. Moreover, in both cell
lines, cisplatin-induced lesions were repaired less efficiently from
the transcriptionally active N-ras gene compared with the inactive
CD3d gene (Table II). However, RT112 and HT1376 contained
more lesions per fragment following exposure to 100 µM than
MGH-U1. Therefore, when the number of lesions repaired was
calculated, it became apparent that the amount of repair was almost
identical for both genes in all 3 cell lines.
In contrast to the bladder cancer cell lines, neither 833K nor
GCT27 showed repair in the N-ras or the CD3d gene but showed
an increase in damage after 24 hr (Table III). However, the other
testis tumour cell line, SuSa, removed cisplatin-induced damage
from the CD3d gene and, to a lesser extent, from the N-ras gene,
but its repair capacity was lower than that of the 3 bladder tumour
cell lines (Table III).
FIGURE 2 – Removal of platinum from total genomic DNA of testis
tumour cell lines (open symbols) and bladder cancer cell lines (closed
symbols) measured by atomic absorption spectroscopy, expressed as
fraction of that present immediately following a 1 hr exposure to
cisplatin (50 µM).
FIGURE 3 – Autoradiograph of PCR products of HT1376 cells
treated with a range of cisplatin concentrations. C, untreated control;
Oh, directly after treatment; 24 h, after a 24 hr recovery period.
DISCUSSION
Our results have shown that testis tumour cells have relatively
little capacity to repair cisplatin-damaged DNA and that this
deficiency can be detected at the level of specific genes.
Following cisplatin exposure, the levels of DNA platination
were the reverse of what might be expected. DNA platination was,
on average, lower in the testis tumour cells, despite their greater
sensitivity to cisplatin. This lack of correlation between cisplatin
sensitivity and the level of DNA platination has previously been
observed in ovarian cancer cell lines (Johnson et al., 1994; Shellard
et al., 1991), lung carcinoma cell lines (Shellard et al., 1993), colon
carcinoma cell lines (Sark et al., 1995) and testis tumour cell lines
(Pera et al., 1987; Hill et al., 1994; Bedford et al., 1988). DNA
damage does not appear to be associated with inherent sensitivity to
cisplatin. However, cisplatin-resistant sublines of ovarian cancer
cells (Zhen et al., 1992) and testis tumour cells (Timmer-Bosscha et
al., 1993) showed less initial DNA platination than their parental
lines.
One of the testis tumour cell lines (SuSa) showed overall
genome repair but at a decreased efficiency compared to the
bladder tumour cell lines. This suggests that, although DNA repair
deficiency might contribute to the hypersensitivity of testis tumour
cell lines, this might not be the sole mechanism. Our findings,
however, are in direct contrast to previous results, where SuSa was
KÖBERLE ET AL.
554
TABLE II – NUMBER OF LESIONS IN SELECTED REGIONS OF THE CD3 GENE AND N-ras GENE IN BLADDER TUMOUR
CELL LINES AFTER TREATMENT WITH CISPLATIN
CD3
N-ras
MGH-U1 lesions/kg
RT112 lesions/kb
HT1376 lesions/kb
Cisplatin
(µM)
0 hr
24 hr
0 hr
24 hr
0 hr
24 hr
25
50
100
150
200
25
50
100
150
200
0.58 6 0.37
0.80 6 0.52
1.28 6 0.38
1.81 6 0.14
2.35 6 0.14
0.35 6 0.42
0.65 6 0.52
0.90 6 0.28
1.36 6 0.32
1.69 6 0.26
0.0 6 0.0
0.0 6 0.0
0.0 6 0.0
0.24 6 0.24
0.64 6 0.29
0.0 6 0.0
0.0 6 0.0
0.02 6 0.02
0.23 6 0.32
0.56 6 0.13
0.69 6 0.22
1.28 6 0.59
1.87 6 0.07
1.81 6 0.29
2.03 6 0.14
0.42 6 0.06
0.98 6 0.37
1.56 6 0.04
1.73 6 0.19
1.92 6 0.32
0.06 6 0.01
0.32 6 0.14
0.59 6 0.16
1.81 6 0.14
2.20 6 0.14
0.09 6 0
0.42 6 0.06
0.84 6 0.18
1.64 6 0.06
2.06 6 0.39
1.17 6 0.53
1.87 6 0.97
2.84 6 0.11
3.31 6 0.0
3.10 6 0.67
0.51 6 0.46
1.15 6 0.44
1.90 6 0.44
2.21 6 0.46
2.58 6 0.59
0.33 6 0.32
0.80 6 0.22
1.60 6 0.48
2.46 6 0.52
2.99 6 0.44
0.0 6 0.0
0.40 6 0.28
1.14 6 0.37
1.69 6 0.56
2.49 6 0.59
TABLE III – NUMBER OF LESIONS IN SELECTED REGIONS OF THE CD3 GENE AND N-ras GENE IN TESTIS TUMOUR
CELL LINES AFTER TREATMENT WITH CISPLATIN
CD3
N-ras
GCT27 lesions/kg
833K lesions/kb
SuSa lesions/kb
Cisplatin
(µM)
0 hr
24 hr
0 hr
24 hr
0 hr
24 hr
25
50
100
150
200
25
50
100
150
200
0.16 6 0.21
0.37 6 0.07
0.38 6 0.21
1.07 6 0.14
1.49 6 0.0
0.23 6 0.32
0.51 6 0.46
1.09 6 0.28
1.31 6 0.13
1.83 6 0.19
0.67 6 0.84
1.60 6 1.28
1.07 6 1.07
2.08 6 1.58
1.71 6 0.44
0.0 6 0.0
1.17 6 0.32
1.88 6 0.18
2.06 6 0.39
2.02 6 0.06
0.56 6 0.05
0.80 6 0.33
0.98 6 0.25
1.51 6 0.19
2.14 6 0.44
0.65 6 0.52
1.26 6 0.19
1.64 6 0.37
2.03 6 0.37
2.30 6 0.19
1.42 6 0.26
1.41 6 0.19
1.73 6 0.37
2.16 6 0.32
1.97 6 0.07
1.92 6 0.46
2.39 6 0.19
2.39 6 0.23
2.53 6 0.32
2.49 6 0.06
0.18 6 0.02
0.69 6 0.22
1.19 6 0.27
1.45 6 0.34
1.81 6 0.29
0.08 6 0.01
0.47 6 0.13
1.03 6 0.09
1.31 6 0.39
1.5 6 0.39
0.32 6 0.14
0.37 6 0.07
0.74 6 0.53
1.20 6 0.53
0.58 6 0.37
0.46 6 0.38
0.56 6 0.13
0.75 6 0.09
1.12 6 0.39
0.89 6 0.19
unable to repair platinated DNA while 833K proved to be
repair-proficient (Bedford et al., 1988). The identity of the lines
was checked using locus-specific probes, and the SuSa cell line was
shown to be different from 833K but identical to a cisplatinresistant subline of SuSa we had developed previously. We
therefore have no explanation for these earlier data, which were
obtained in a different laboratory.
An increase in cisplatin-induced damage during a postincubation period, as observed for 833K and GCT27 at the level of
the gene, has also been reported at the level of the whole genome in
testis tumour cell lines (Hill et al., 1994; Sark et al., 1995).
Cisplatin binds to DNA, forming mainly intrastrand cross-links
and, in a slower reaction, interstrand cross-links (Eastman, 1983).
The increase in damage, therefore, may be due to the continuous
reaction leading to interstrand cross-links. However, as interstrand
cross-links account for only 1% of cisplatin-induced damage, this
does not account for all of the observed increase. The increase in
damage observed at the level of the gene in 833K and GCT27
might in addition be due to degradation of DNA. This would result
in less template for the PCR reaction, which would be reflected as
damage. In contrast to the other testis tumour cell lines, SuSa
repaired cisplatin-induced damage in both gene regions, in agreement with the data for the whole genome. The CD3d gene was
repaired more efficiently compared with the N-ras gene, as was
seen with the bladder tumour cell lines.
Interestingly, the removal of damage was more efficient from the
inactive CD3d gene than from the transcriptionally active N-ras
gene. In a number of studies it has been found that actively
transcribed genes are repaired faster and more efficiently than
inactive genes and bulk DNA after treatment with a number of
DNA-damaging agents, including cisplatin (Islas et al., 1991;
Jones et al., 1991; Zhen et al., 1993). The removal of damage from
actively transcribed genes has been associated with transcription
coupled repair, which seems to be completed after a few hours,
whereas repair in the whole genome takes up to 24 hr (Venema et
al., 1992). Therefore, it is likely that a preference in repair in the
actively transcribed gene would have been shown after a shorter
time period. In addition, the discrepancy with published results
may be explained by the fact that we investigated repair in only a
fragment of the respective genes, while in the other studies repair in
the whole gene (10–20 kb) was investigated using Southern
blotting techniques. Alternatively, a more open structure of the
CD3 gene could allow better accessibility to repair enzymes and,
therefore, better repair. The fact that N-ras seems to be expressed at
lower levels (Futscher and Erickson, 1990) than genes often used to
study gene-specific damage and repair, such as DHFR and c-myc,
may also be a contributory factor.
These results indicate that the hypersensitivity of testis tumour
cells to cisplatin can be related to a deficiency in DNA repair.
Differential sensitivity of testis tumour cells and other types of cell
to DNA-damaging agents has been associated with an increased
propensity to undergo apoptosis (Lowe et al., 1993; Chresta et al.,
1996). Further studies investigating which steps in the repair
process are defective in testis tumour cells will help to clarify
whether testis tumours die as a result of a defect in DNA repair or
because of their propensity to undergo apoptosis.
REFERENCES
BEDFORD, P., FICHTINGER-SCHEPMAN, A.M.J., SHELLARD, S.A., WALKER,
M.C., MASTERS, J.R.W. and HILL, B.T., Differential repair of platinum-DNA
adducts in human bladder and testicular tumour continuous cell lines.
Cancer Res., 48, 3019–3024 (1988).
mammalian cells correlates with efficient DNA repair in essential genes.
Proc. nat. Acad. Sci. (Wash.), 83, 3830–3833 (1986).
BOHR, V., Gene specific DNA repair. Carcinogenesis, 12, 1983–1992
(1991).
CHRESTA, C.M., MASTERS, J.R.W. and HICKMAN, J.A., Hypersensitivity of
human testicular tumors to etoposide-induced apoptosis is associated with
functional p53 and high Bax:Bcl-2 ratio. Cancer Res., 56, 1834–1841
(1996).
BOHR, V., OKUMOTO, D.S. and HANAWALT, P.C., Survival of UV-irradiated
EASTMAN, A., Characterization of the adducts produced in DNA by
DNA REPAIR IN TESTIS TUMOUR CELLS
cis-diamminedichloroplatinum(II) and cis-dichloro(ethylenediamine)platinum(II). Biochemistry, 22, 3927–3933 (1983).
EINHORN, L.H., Treatment of testicular cancer: a new and improved model.
J. clin. Oncol., 8, 1777–1781 (1990).
FUTSCHER, B.W. and ERICKSON, L.C., Changes in c-myc and c-fos expression in a human tumour cell line following exposure to bifunctional
alkylating agents. Cancer Res., 50, 62–66 (1990).
GRIMALDI, K.A., BINGHAM, J.P., SOUHAMI, R.L. and HARTLEY, J.A., DNA
damage by anticancer agents: mapping in cells at the subgene level with
quantitative PCR. Analyt. Biochem., 222, 236–242 (1994).
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).
ISLAS, A.L., VOS, J.M.H. and HANAWALT, P.C., Differential induction and
repair of psoralen photoadducts to DNA in specific human genes. Cancer
Res., 51, 2867–2873 (1991).
JOHNSON, S.W., PEREZ, R.P., GODWIN, A.K., YEUNG, A.T., HANDEL, L.M.,
OZOLS, R.F. and HAMILTON, T.C., Role of platinum-DNA adduct formation
and removal in cisplatin resistance in human ovarian cancer cell lines.
Biochem. Pharmacol., 47, 689–697 (1994).
JONES, J.C., ZHEN, W., REED, E., PARKER, R.J., SANCAR, A. and BOHR, V.A.,
Gene specific formation and repair of cisplatin intrastrand adducts and
interstrand crosslinks in Chinese hamster ovary cells. J. biol. Chem., 266,
7101–7107 (1991).
KELLAND, L.R., MISTRY, P., ABEL, G., FREIDLOS, F., LOH, S.Y., ROBERTS, J.J.
and HARRAP, K.R., Establishment and characterization of an in vitro model
of acquired resistance to cisplatin in a human testicular nonseminomatous
cell line. Cancer Res., 52, 1710–1716 (1992).
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).
LEE, K.B., PARKER, R.J., BOHR, V., CORNELISON, T. and REED, E., Cisplatin
sensitivity/resistance in UV repair deficient Chinese hamster ovary cells of
complementation groups 1 and 3. Carcinogenesis, 14, 2177–2180 (1993).
LINK, C.J., BURT, R.K. and BOHR, V.A., Gene specific repair of DNA
damage induced by UV irradiation and cancer chemotherapeutics. Cancer
Cells, 3, 427–436 (1991).
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).
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).
PARRIS, C.N., ARLETT, C.F., LEHMANN, A.R., GREEN, M.H.L. and MASTERS,
555
J.R.W., Differential sensitivities to gamma radiation of human bladder and
testicular tumour cell lines. Int. J. Rad. Biol., 53, 599–608 (1988).
PERA, M.F., FRIEDLOS, F., MILLS, J. and ROBERTS, J.J., Inherent sensitivity of
cultured human embryonal carcinoma cells to adducts of cis-diamminedichloroplatinum(II) on DNA. Cancer Res., 47, 6810–6813 (1987).
PERA, M.F., KÖBERLE, B. and MASTERS, J.R.W., Exceptional sensitivity of
testicular germ cell tumour cell lines to the new anti-cancer agent,
temozolamide. Brit. J. Cancer, 71, 904–906 (1995).
SARK, M.W.J., TIMMER-BOSSCHA, H., MEIJER, C., UGES, D.R.A., SLUITER,
W.J., PETERS, W.H.M., MULDER, N.H. and DEVRIES, E.G.E., Cellular basis
for differential sensitivity to cisplatin in human germ cell tumour and colon
carcinoma lines. Brit. J. Cancer, 71, 684–690 (1995).
SEIDMAN, A.D. and SCHER, H.I., The evolving role of chemotherapy for
muscle infiltrating bladder cancer. Semin. Oncol., 18, 585–595 (1991).
SHELLARD, S.A., FICHTINGER-SCHEPPMAN, A.M.J., LAZO, J.S. and HILL, B.T.,
Evidence of differential cisplatin-DNA adduct formation, removal and
tolerance of DNA damage in three human lung carcinoma cell lines.
Anticancer Drugs, 4, 491–500 (1993).
SHELLARD, S.A., HOSKING, L.K. and HILL, B.T., An anomalous relationship
between cisplatin sensitivity and the formation and removal of platinumDNA adducts in two human ovarian carcinoma cell lines in vitro. Cancer
Res., 51,4557–4564 (1991).
TIMMER-BOSSCHA, H., TIMMER, A., MEIJER, C., DE VRIES, E.G.E., DE JONG,
B., OOSTERHUIS, J.W. and MULDER, N.H., cis-Diamminedichloroplatinum(II) resistance in vitro and in vivo in human embryonal carcinoma cell lines.
Cancer Res., 53, 5705–5713 (1993).
VENEMA, J., BARTOSOVA, Z., NATARAJAN, A.T., VAN ZEELAND, A.A. and
MULLENDERS, L.H.F., Transcription affects the rate but not the extent of
repair of cyclobutane pyrimidine dimers in the human adenosine deaminase
gene. J. biol. Chem., 267, 8852–8856 (1992).
VENEMA, J., MULLENDERS, L.H.F., NATARAJAN, A.T., VAN ZEELAND, A.A.
and MAYNE, L.V., The genetic defect in Cockayne syndrome is associated
with a defect in repair of UV-induced damage in transcriptionally active
DNA. Proc. nat. Acad. Sci. (Wash.), 87, 4707–4711 (1990b).
VENEMA, J., VAN HOFFEN, A., NATARAJAN, A.T., VAN ZEELAND, A.A. and
MULLENDERS, L.H.F., The residual repair capacity of xeroderma pigmentosum complementation group C fibroblasts is highly specific for transcriptionally active DNA. Nucleic Acids Res., 18, 443–448 (1990a).
WALKER, M.C., PARRIS, C.N. and MASTERS, J.R.W., Differential sensitivities
to chemotherapeutic drugs between testicular and bladder cancer cells. J.
nat. Cancer Inst., 79, 213–216 (1987).
ZHEN, W., EVANS, M.E., HAGGERTY, C.M. and BOHR, V.A., Deficient gene
specific repair of cisplatin-induced lesions in xeroderma pigmentosum and
Fanconi’s anemia cells. Carcinogenesis, 14, 919–924 (1993).
ZHEN, W., LINK, C.J., O’CONNOR, P.M., REED, E., PARKER, R., HOWELL, S.
and BOHR, V.A., Increased gene specific repair of cisplatin interstrand
crosslinks in cisplatin resistant human ovarian cancer cell lines. Mol. cell.
Biol., 12, 3689–3698 (1992).
Документ
Категория
Без категории
Просмотров
3
Размер файла
96 Кб
Теги
1/--страниц
Пожаловаться на содержимое документа