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Carcinogenesis vol.19 no.6 pp.973–978, 1998
Effects of carcinogenic agents upon different mechanisms for
intragenic recombination in mammalian cells
Thomas Helleday
1
, Catherine Arnaudeau and
Dag Jenssen
Department of Genetic and Cellular Toxicology, Wallenberg Laboratory,
Stockholm University, S-106 91 Stockholm, Sweden
1
To whom correspondence should be addressed
Email: helleday@genetics.su.se
A growing body of carcinogens are known to affect genetic
recombination in mammalian cells and to thereby interfere
with the process of carcinogenesis. In order to screen for
recombinogenic effects of chemical and physical agents a
variety of in vitro assay systems utilizing mammalian cells
have been developed. However, the effects of potential
carcinogens differ in these different systems. In order to
investigate this phenomenon further, we have employed
two different assay procedures, involving spontaneous
duplication mutants in mammalian cells, which respond
to homologous or non-homologous recombination. Four
carcinogens were investigated, i.e. Aroclor 1221, benzene,
methylmethanesulphonate (MMS) and thiourea, as were γ-
and UV-irradiation. With the exception of thiourea all of
these factors resulted in elevated frequencies of homologous
recombination. On the other hand, only UV-irradiation
affected the rate of non-homologous recombination. These
results indicate that substrate length and/or the recombina-
tion mechanism may influencetherecombinogenicresponse
of mammalian fibroblasts to carcinogenic factors. Thus,
procedures for recombinogenic effects of carcinogens
should consider the different pathways of recombination
occurring in mammalian cells.
Introduction
An increasing number of in vitro assays for screening the
effects of chemical or physical agents on recombination have
been developed (1). In this connection, various assay systems
have been constructed, not only in lower organisms such as
bacteria and yeast, but also in higher organisms, i.e. Drosophila
and mammalian cell lines. The effects of different agents on
recombination have been extensively screened in Drosophila
(2), and a collaborative study on induced recombination has
recently been reviewed (3).
Employing mammalian cell lines, the effects of exposure to
different agents on recombination have been studied in con-
structs involving tandem duplications of the thymidine kinase
(tk) and neo genes (4,5), among other types of constructs (6)
(see also ref. 7 for a review). More rarely have endogenous
markers been used, i.e., studies on the reversion of duplication
*Abbreviations: 6TG,6-thioguanine; hprt/HPRT, hypoxanthine guanine phos-
phoribosyltransferase; HMEM, Hank’s minimal essential medium; HBSS,
Hank’s balanced salt solution; DMSO, dimethylsulphoxide; HAsT, hypox-
anthine-
L
-azaserine-thymidine; MMS, methylmethanesulphonate; neo, neo-
mycin resistance; PBS, phosphate-buffered saline; PCB, polychlorinated
biphenyl; SV40, simian virus 40; tk, thymidine kinase.
© Oxford University Press
973
mutants in cell lines (8,9). Recently, markers in mice have
been utilized to detect effects of exposure to carcinogens on
recombination in vivo (10,11).
We have previously studied intrachromosomal recombina-
tion in mammalian cells by taking advantage of an endogenous
marker sequence present in the Sp5 cell line (12) isolated from
V79 Chinese hamster cells (13). The hypoxanthine guanine
phophoribosyltransferase (hprt*) duplication mutant Sp5 was
shown to be unstable, reverting spontaneously at high frequen-
cies (1310
–5
/cell generation) to the wild type phenotype. The
reversion frequency of Sp5 can be further elevated upon
exposure to chemical or physical agents, thus providing an
excellent tool for screening agents as possible inducers of
intrachromosomal recombination (8). An analogous in vitro
assay system based on the human GM6804 lymphoblastoid
cell line, derived from a Lesch–Nyhan patient (14), has been
used for similar studies (9).
Comparisons of induced recombination in mammalian cells
reveal discrepancies between different systems which cannot
easily be explained (8). It has been speculated that the
underlying mechanism of recombination could affect the results
obtained in a significant manner (7).
In order to investigate this question further, we have studied
the effects of several selected carcinogens having different
modes of action in two different assay systems, the Sp5/V79
recombination assay and a newly-developed procedure based
on the SPD8 cell line (15). These two cell lines utilize different
recombination mechanisms to revert to a functional hprt gene.
The occurrence of non-homologous recombination in the Sp5
cell line and homologous in the SPD8 cell line, allows
determination of how each of these mechanisms is affected
by carcinogens. Furthermore, we have addressed the question
as to whether the length of the duplicated sequence is of
importance for induced recombination. The results are analysed
and discussed in comparison to data reported by others (9).
Materials and methods
Cell lines
The Sp5 cell line investigated here was isolated from V79 Chinese hamster
cells, being obtained from a set of mutants used to study spontaneously
occurring mutation spectra (16). Southern blot data revealed that a 2.1-kb
fragment of the hprt gene, including exon 2 and flanking intron sequences,
was inserted into intron 1, 3.7 kb upstream of exon 2, i.e. the duplication was
shown to be displaced (see Figure 1b and ref. 12). The duplicated DNA
sequence resulted in a non-functional HGPRT protein containing only 54
amino acids. An in vitro procedure for investigating effects of exposure to
chemical and physical agents on recombination has been developed with this
cell line (8).
The SPD8 cell line was also isolated from V79 Chinese hamster cells.
Analysis of its hprt gene by Southern blotting and sequencing revealed a 5-kb
duplication of exon 7, intron 6 and the 39 portion of exon 6, which was
located in tandem within the hprt locus (15) (T.Helleday et al., in preparation;
see also Figure 1c). At the mRNA level only exon 7 is duplicated, which
introduces a stop codon immediately downstream from the duplicated exon,
resulting in a truncated HGPRT protein containing 180 amino acids and
without activity.
T.Helleday, C.Arnaudeau and D.Jenssen
Fig. 1. (a) Structure of the Chinese hamster hprt gene according to Rossiter et al. (50). The hprt gene consists of nine exons which span 36 kb and the HPRT
protein is encoded by 654 bp. (b) The structure of the hprt gene in the Sp5 cell line. The 2.1-kb displaced duplication of the exon 2 fragment with flanking
intron sequences (shown in gray) is inserted 3.7 kb upstreams exon 2 in intron 1 (15). (c) The hprt gene in the SPD8 cell line. The 5-kb tandem duplication
of exon 7 and the 39 portion of exon 6 is indicated as a gray area (15, Helleday et al., in preparation).
Cell culture
The Sp5 and SPD8 cell lines were cultured under the same conditions as the
parent V79 cell line, except that the medium was supplemented with 6-
thioguanine (6TG) (5 µg/ml) in order to reduce the frequency of spontaneous
reversion prior to treatments (12). The addition of 6TG at this concentration
affects neither the growth rate of these mutants nor the assay procedure.
Treatment conditions
Treatments for 24 h were performed in Hank’s minimum essential medium
(HMEM), containing Hank’s salt, with the addition of 8% fetal calf serum
and penicillin-streptomycin (90 U/ml). For treatments of shorter duration,
cells were maintained in Hank9s balanced salt solution containing HEPES
buffer (20 mM) (HBSS
11
). Agents not soluble in water were dissolved in
dimethylsulphoxide (DMSO) at a final concentration of 0.2%. UV irradiations
(with an intensity of 0.30 W/s) were performed on cells grown in a Petri
dish containing 3 ml phosphate-buffered solution (PBS). γ-irradiations were
performed in HMEM using a
137
Cs source and intensity of 0.85 Gy/min.
Reversion assay
The protocol for the reversion assay using Sp5 cells has been described
previously (12). The protocol for the reversion assay with SPD8 cells involved
inoculation of flasks (75 cm
2
) with 1.5 3 10
6
cells 4 h prior to the 24-h
treatment period. For shorter treatments 1.5 3 10
6
cells were inoculated in
HMEM 24 h before rinsing in 10 ml HBSS
11
, after which treatment was
commenced. After treatments, the cells were rinsed three times with 10 ml
HBSS
11
and 30 ml HMEM was added to allow recovery for 48 h. Selection
of revertants was performed by plating three dishes/group (3310
5
cells/
dish) in the presence of hypoxanthine-
L
-azaserine-thymidine (HAsT; 50 µM
hypoxanthine, 10 µM
L
-azaserine, 5 µM thymidine). Two dishes containing
500 cells each were plated for cloning. The cloning plates were harvested
after 7 days of growth and the colonies fixed and stained with methylene
blue/methanol (4 g/1). The cells on the selection plates were grown for 10
days before fixation.
Toxicity assay for revertant SPD8 and SPD8 cells
Spontaneous revertant clones from the SPD8 cell line have been isolated
previously (15). Equal numbers of cells from four revertant clones isolated
separately were pooled (in order to reconstruct the mean toxicity on revertant
clones), and used in the toxicity assay. This assay involved inoculation of
5310
5
cells in HMEM into flasks (25 cm
2
) 4 h before 24-h treatment. After
treatment, the flasks were rinsed three times with 5 ml HBSS
–
(Hank’s
balanced salt solution without Ca
21
or Mg
21
) and trypsinized. Two dishes
were plated with 500 cells each for each dose. The plates were fixed, stained
and counted after 7 days of growth.
Chemicals
Benzene and thiourea were obtained from Sigma (Stockholm, Sweden)
and methylmethanesulphonate (MMS) from Merck, Schuchardt (Mu
¨
nchen,
Germany). Aroclor 1221 was a gift from S.Jenssen (Stockholm, Sweden) and
its quality analysed by GC-ECD (GC-Electron Capture Detector).
Results and discussion
The effects of the treatments of Sp5 and SPD8 cells with
different agents are presented in Tables I and II. The only
974
Table I. Effects by Aroclor 1221, benzene and thiourea on non-homologous
recombination, as detected by reverse mutations in Sp5 cells. The mean and
standard error of two independent experiments are indicated
Agent Dose Treatment Survival rf/10
5
cells
(µg/ml) time (h) (% control)
Aroclor 1221 0 24 100 6 1 4.1 6 0.2
224 9660 2.5 6 0.3
524 9861 3.8 6 0.3
10 24 90 6 2 3.4 6 0.9
15 24 82 6 2 3.9 6 0.2
20 24 79 6 1 3.6 6 1.8
30 24 62 6 2 3.9 6 0.2
Benzene 0 24 100 6 10 3.6 6 0.0
150 24 92 6 4 4.0 6 0.7
200 24 79 6 0 4.1 6 1.1
300 24 98 6 2 2.4 6 0.0
400 24 81 6 11 4.6 6 1.7
500
a
24 87 6 7 3.4 6 0.3
Thiourea 0 24 100 6 12 3.6 6 0.4
524 8764 6.2 6 1.6
10 24 70 6 7 4.1 6 1.5
25 24 57 6 7 5.2 6 3.8
rf 5 Reversion frequency.
a
Precipitate was observed.
agent that caused a statistically significant increase in the
reversion frequency in both cell lines was UV irradiation. In
addition, increases in the reversion frequency for SPD8 were
caused by Aroclor 1221, benzene, γ-rays and MMS. Thiourea
did not induce any significant increase in reversion frequency
in either of the cell lines.
A comparison between the recombinogenic effects seen in
the two assay systems employed here and literature data on
the mutagenic effect at the same locus in V79 cells is shown
in Table III. It should be noted that relatively stable compounds
such as polychlorinated biphenyls (PCBs) and benzene, which
have known carcinogenic properties (17–19), can be recognized
as recombinogens in SPD8 cells, but are reported to be non-
mutagenic towards the V79 cell line (20,21).
In order to rule out the possibility that observed increases
in reversion frequency are due to phenotypic selection, we
examined the cytotoxicity of Aroclor 1221 and of benzene on
SPD8 (hprt)
–
cells, as well as on revertant cells (hprt)
1
derived
Effects upon intragenic recombination mechanisms
Table II. Effects of Aroclor 1221, benzene, γ-irradiation, MMS, thiourea
and UV-irradiation on homologous recombination, as detected by reverse
mutations in SPD8 cells. The mean and standard error of two independent
experiments are shown
Agent Dose Treatment Survival rf/10
5
cells
time (h) (% control)
Aroclor 1221
a
0 µg/ml 24 100 6 15 1.8 6 0.5
2 µg/ml 24 85 6 13 2.1 6 0.0
5 µg/ml 24 83 6 17 3.8 6 0.6
10 µg/ml 24 72 6 11 4.0 6 1.4
20 µg/ml 24 58 6 12 4.3 6 0.3***
Benzene 0 µg/ml 24 100 6 4 2.1 6 0.2
12.5 µg/ml 24 93 6 4 1.6 6 0.1
25 µg/ml 24 65 6 16 4.6 6 0.1**
50 µg/ml 24 54 6 12 3.7 6 0.7
MMS 0 mM 0.5 100 6 2 2.4 6 0.6
0.1 mM 0.5 68 6 17 6.1 6 0.8
0.2 mM 0.5 44 6 2 17.9 6 4.1
0.4 mM 0.5 24 6 14 15.3 6 3.1*
γ-rays 0 Grey – 100 6 3 2.7 6 0.4
0.5 Grey – 70 6 3 2.6 6 0.1
1 Grey – 57 6 16 3.4 6 0.6
2 Grey – 52 6 16 4.1 6 0.3
3 Grey – 37 6 15 6.2 6 0.4**
Thiourea 0 µg/ml 24 100 6 1 1.8 6 0.7
5 µg/ml 24 85 6 1 2.2 6 1.0
10 µg/ml 24 72 6 13 2.6 6 0.2
25 µg/ml 24 49 6 16 2.9 6 0.6
UV 0 J/m
2
– 100 6 1 1.5 6 0.3
2 J/m
2
–7966 3.2 6 0.8
5 J/m
2
–5362 4.1 6 0.8
10 J/m
2
–3163 7.8 6 2.9*
*Statistically significantly from control using Student’s t-test. 0.05 . P .
0.01.
**Statistically significantly from control using Student’s t-test. 0.01 . P .
0.001.
***Statistically significantly from control using Student’s t-test. P , 0.001.
a
Mean and standard errors of three independent experiments are indicated.
rf 5 Reversion frequency.
from the SPD8 cell line. These two compounds were chosen
since the magnitude of induction of recombination they caused
was low. There were no differences in cytotoxicity of Aroclor
1221 and benzene towards these two genotypically different
cell types (data not shown).
Since mitotic recombination has been shown to be a signific-
ant step in tumor development (22), it is important to consider
the different types of recombination mechanisms known to
occur in mammalian cells and how these respond to treatment
with carcinogens. In this respect, differences in the responses of
the different cell lines employed here may provide mechanistic
information.
In theory, there are many different pathways for intragenic
recombination (23,24), as illustrated in Figure 2. Data from
several reports indicate that unequal sister chromatid exchange
involving homologous recombination between Alu sequences
is a mechanism for generating both duplications (25,26) and
deletions (27) in the hprt locus of human cells. However, the
involvement of Alu sequences in producing duplications in
Chinese hamster cells has not yet been demonstrated. Sister
chromatid conversion has been observed in yeast (28) and
seems to be an important pathway for intrachromosomal
recombination in mammalian cells (29–31).
975
Table III. Comparison of the data on homologous and non-homologous
recombination and on mutations caused by six classes of carcinogenic
agents. The numbers in parentheses indicate the relevant references
Agent Recombinogenic effect in Mutagenicity
at hprt locus
Sp5 SPD8
Methylmethanesulphonate – (8) 11 11 (52)
Benzene – 1 – (21)
γ-rays – (8) 111(52)
UV 11 (8) 11 111 (52)
Thiourea – – (51)
PCB (Aroclor 1221) – 1 nt
–, No increase in reversion/mutation frequency.
1, Up to three-fold increase in reversion/mutation frequency, statistically
significant at the level of P , 0.05.
11, a three- to ten-fold increase in reversion/mutation frequency,
statistically significant at the level of P , 0.05.
111, More than 10-fold increase in reversion/mutation frequency,
statistically significant at the level of P , 0.05.
nt, Not tested.
However, these two types of recombination mechanisms
(Figure 2a–b) involve recombination between sister chromatids
(at least in yeast) in the S or G2 phase of the cell cycle and
would give rise to the same genetic constitution in the
revertants. Intrachromatid exchange could, in theory, occur at
any time in the cell cycle and is an alternative pathway for
recombination (Figure 2c). It should be noted that interchromo-
somal recombination [which is a rare event in mammalian
cells (30)] cannot be responsible for reversion to a functional
gene, since the hprt gene is localized on the X chromosome
and V79 cells originate from a male Chinese hamster.
If a displaced duplication sequence is eliminated by either
of the recombination pathways discussed above, the DNA
sequence between the duplicated fragment and the parental
sequence (shown dashed in Figure 2) is expected to be lost
(Figure 2a–c). In this case, the sequence of the wild-type hprt
gene will not be restored in the revertant cells, even though
the wild-type protein will be fully expressed. In order to obtain
information about this, revertant clones from the SPD8 and
Sp5 cell lines were isolated and characterized at the molecular
level (12,15).
In Southern blotting analysis, the SPD8 and Sp5 cell lines
demonstrated a change in the restriction pattern, reflecting the
duplicated areas, upon digestion with Bam HI, Bgl II, Eco RI,
Hin dIII or Pst I. Analysis of revertant clones from SPD8 by
Southern blotting revealed the wild-type restriction pattern,
indicating that the hprt gene had been completely restored
(15). These results were further confirmed by DNA sequencing.
Since the SPD8 duplication is localized in tandem, a crossing
over between misaligned homologous sequences in connection
with homologous recombination, would restore the wild-type
sequence.
In the case of the Sp5 cell line, revertant clones were
characterized by Southern and Northern blotting. The Southern
blot revealed that the restriction pattern in the hprt gene of all
revertant clones was the same as the wild type (12,15), i.e.
these revertants still exhibit the wild-type sequence between
the duplicated sequences, as was subsequently confirmed by
sequencing. Since Sp5 revertant clones always retain the
fragment between the duplicated sequences, recombination
pathways a–c in Figure 2 cannot be involved. Therefore,
T.Helleday, C.Arnaudeau and D.Jenssen
Fig. 2. Schematic illustration of pathways for reversion of a duplication mutant which would restore a functional hprt gene. In a displaced duplication, there
is a fragment between the duplicated sequences (dashed) which can either be lost, as in the revertants from pathways a to c, or retained, as in the revertants
from pathways d to e. The SPD8 cell line reverts via one of the pathways a to c, while the Sp5 cell line seems to revert via a non-homologous recombination
pathway (12, Helleday et al., in preparation).
reversions of the Sp5 cell line most likely occur by non-
homologous recombination, i.e. via pathway d in Figure 2.
In theory, Sp5 cells may revert by homologous recombina-
tion, since there is full homology over 2.1 kb. Nevertheless,
this was not found to be the case, which might be explained
by the fact that a loss of the DNA fragment between the
duplicated areas may impair the function of the gene product.
Recent studies have indicated that non-homologous re-
combination may be an important pathway in mammalian
cells, in contrast to what has been found in yeast, where
homologous recombination seems to be the major pathway
(30,32,33). Site-specific recombination in lymphocyte cells,
involving V(D)J recombination, has also been shown to be
involved in deletion of parts of the hprt gene (34,35). Other
pathways of recombination may involve DNA polymerase
slippage, as indicated in pathway e in Figure 2. Such a
mechanism of recombination may involve palindrome
sequences generating hairpin structures, although this mechan-
ism has only been reported to be involved in producing small
deletions at the hprt locus (36). Larger deletions have, however,
been suggested to be produced in the α-galactosidase A gene
by DNA polymerase slippage (37,38).
Upon comparing the findings for the cell lines examined
here, it is of interest to note that the relatively stable, non-
mutagenic carcinogens benzene and PCB (Aroclor 1221) are
recombinogens in the SPD8 cell line, but not in Sp5 cells.
This could imply that homologous recombination is more
vulnerable to non-reactive carcinogens than is non-homologous
recombination. This difference might reflect a mechanism in
which lipophilic compounds may interfere with the homo-
logous pairing of chromatids, a process required for homo-
logous, but not for non-homologous recombination.
Upon comparison of the results obtained here with the data
on the same agents reported by Aubrecht and co-workers (9),
who used the human GM6804 cell line, it appears that the
most sensitive system for detection of increased recombination
is the one involving GM6804 cells, followed by the SPD8 and
Sp5 assay systems. Thiourea was found to be positive in the
GM6804 assay procedure, but not in the Sp5 and SPD8
976
procedures. Benzene causes a statistically significant increase
in recombination in SPD8 cells, but not in GM6804 or Sp5
cells. Although the comparison here is based on a limited
number of compounds, the pattern observed may reflect the
fact that the recombination target in the GM6804 cell line is
larger (13.7 kb) than that in the SPD8 (5 kb) and Sp5 (2.1
kb) cell lines. The reversion pathway in GM6804 cells is
homologous recombination, as in the SPD8 cell line, since the
sequence between the duplicated fragments (dashed in Figure
2) is lost in the GM6804 revertants (39,40).
In conclusion, we suggest that the Sp5 and SPD8 cell lines
revert by non-homologous and homologous recombination,
respectively. Our results imply that certain agents act as
recombinogens in a specific manner. As indicated in Table III
Aroclor 1221, γ-rays, MMS, and benzene induce homologous,
but not non-homologous recombination. Furthermore, these
considerations might also explain apparent discrepancies found
earlier, when comparing the effects of the same agents in assay
systems involving duplication of different genes (8).
Chromatin structure has been shown to influence recombina-
tion in yeast (41,42). The structure may play an important role
in the accessibility of genes to the recombination machinery,
which has, in fact, been indicated in the case of V(D)J
recombination (43). Duplications involving exons 2 and 3 of
the hprt locus seem to be the targets for such a recombinogenic
event more often than are other exons, both in human and
hamster cells (15,26) (H.Vrieling, personal communication).
This observation might reflect reversion of the GM6804
cell line only, which might express the proteins for V(D)J
recombination, an activity occurring in pre-lymphocytes, but
not in fibroblasts.
The Sp5 and SPD8 cell lines are derived from the V79
Chinese hamster cell line (lung cells) while the GM6804 cell
line is derived from human lymphoblastoid cells (14). This
might explain why there is an effect of thiourea on GM6804
cells, but not on the SPD8 or Sp5 cell lines. The effect of
Aroclor 1221 is greater in the GM6804 assay system than in
the Sp5 and SPD8 systems, which might reflect that the fact
that the V79 cell line is known to have a different pattern of
Effects upon intragenic recombination mechanisms
expression of various genes, e.g. low cytochrome P-450-
catalysed activity. The GM6804 cell line may express more
cytochrome P-450, and thereby be able to metabolize PCB
into more recombinogenic compounds. There are reports that
metabolites of PCB may give rise to recombinogenesis in the
eye mosaic test in Drosophila (44).
Recent reports imply that p53 is involved in recombination
(45–48) and that a defective p53 is associated with an increased
frequency of spontaneous intrachromosomal recombination
(45). In addition, it has been reported that over-expression of
the simian virus 40 (SV40) large-T antigen, which binds the
p53 protein, stimulates reversion of the duplication in the
GM6804 cell line (40). Since the p53 gene has been found to
be non-functional in V79 cells (49), such reversion might also
be stimulated in the SPD8 and Sp5 cells used here. If so, this
might lead to enhanced background reversion frequencies in
the SPD8 and Sp5 assay systems, thus explaining their lower
sensitivity as compared to the GM6804 system.
Thus, species, organ and/or cell line differences are other
possible reasons for the differences in the responses to the
carcinogenic agents investigated here. If so, this could be a
problem when testing for recombinogenesis, since a battery of
assay systems will be required. At the same time, this may
actually provide more information on the mechanism of action
of each carcinogen tested.
In conclusion, our data suggest that the effects of different
carcinogenic agents on recombination may differ in different
assay systems, due to a number of the factors discussed
above. A central question is how different mechanisms for
recombination in mammalian cells may be involved in the
aetiology of cancer and how such involvement can be predicted.
It will also be of special interest to investigate to what extent
non-mutagenic carcinogens are recombinogenic in mamma-
lian cells.
Acknowledgements
We thank Anders Olsson for the GC-ECD analysis of Aroclor 1221. This
investigation was supported by the Swedish Cancer Society.
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Received on October 27, 1997; revised on January 6, 1998; accepted on
February 4, 1998
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