Download human lymphoblastoid cells. and unbalanced translocation in p53

Illegitimate recombination leading to allelic loss
and unbalanced translocation in p53-mutated
human lymphoblastoid cells.
M Honma, L S Zhang, M Hayashi, K Takeshita, Y Nakagawa, N
Tanaka and T Sofuni
Mol. Cell. Biol. 1997, 17(8):4774.
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MOLECULAR AND CELLULAR BIOLOGY, Aug. 1997, p. 4774–4781
0270-7306/97/$04.0010
Copyright © 1997, American Society for Microbiology
Vol. 17, No. 8
Illegitimate Recombination Leading to Allelic Loss and
Unbalanced Translocation in p53-Mutated Human
Lymphoblastoid Cells
MASAMITSU HONMA,1* LI-SHI ZHANG,1† MAKOTO HAYASHI,1 KENJI TAKESHITA,1
YUZUKI NAKAGAWA,2 NORIHO TANAKA,2 AND TOSHIO SOFUNI1
Division of Genetics and Mutagenesis, National Institute of Health Sciences, Setagaya, Tokyo 158,1 and Department of
Cell Biology, Hadano Research Institute, Food and Drug Safety Center, Hadano, Kanagawa 257,2
Allelic loss and translocation are critical mutational events in human tumorigenesis. Allelic loss, which is
usually identified as loss of heterozygosity (LOH), is frequently observed at tumor suppressor loci in various
kinds of human tumors. It is generally thought to result from deletion or mitotic recombination between
homologous chromosomes. In this report, we demonstrate that illegitimate (nonhomologous) recombination
strongly contributes to the generation of allelic loss in p53-mutated cells. Spontaneous and X-ray-induced LOH
mutations at the heterozygous thymidine kinase (tk) gene, which is located on the long arm of chromosome 17,
from normal (TK6) and p53-mutated (WTK-1) human lymphoblastoid cells were cytogenetically analyzed by
chromosome 17 painting. We observed unbalanced translocations in 53% of LOH mutants spontaneously
arising from WTK-1 cells but none spontaneously arising from TK6 cells. We postulate that illegitimate
recombination was occurring between nonhomologous chromosomes after DNA replication, leading to allelic
loss and unbalanced translocations in p53-mutated WTK-1 cells. X-ray irradiation, which induces DNA
double-strand breaks (DSBs), enhanced the generation of unbalanced translocation more efficiently in WTK-1
than in TK6 cells. This observation implicates the wild-type p53 protein in the regulation of homologous
recombination and recombinational DNA repair of DSBs and suggests a possible mechanism by which loss of
p53 function may cause genomic instability.
a genotoxic insult would escape apoptosis and continue to
divide, thereby accumulating mutations (19, 25).
Inactivation of p53 enhances the gene amplification rate
(28), produces structural and numerical chromosome changes
(1, 6, 8), and deregulates centrosomal replication, leading to
abnormal chromosomal segregation (12), while it does not
affect the accumulation of point mutations (37). It is likely that
a mutator phenotype associated with p53 inactivation would
contribute to gross structural changes such as chromosomal
alterations but not small DNA changes such as point mutations
(30). Recently, we (17) and Xia et al. (48) characterized a
mutator phenotype caused by mutant p53 protein. We compared the mutation frequencies and mutational spectra at the
heterozygous thymidine kinase (tk) locus between normal
(TK6) and p53-mutated (WTK-1) human lymphoblastoid cells.
Both cell lines are derived from the same progenitor cell line,
WI-L2, but WTK-1 cells have a homozygous point mutation at
codon 237 of the p53 gene, resulting in the overproduction of
mutant p53 protein (27, 49). The spontaneous mutation frequency at the tk locus in WTK-1 cells (101.1 3 1026) was 30
times higher than it was in TK6 cells (3.5 3 1026) (17). WTK-1
cells were more resistant to the cytotoxic damage and more
sensitive to the mutagenic effect of X rays than were TK6 cells
(2, 17). X-ray doses yielding 37% survival in TK6 and WTK-1
cells were 0.8 and 2.0 Gy, respectively. A dose of 2 Gy induced
TK-deficient mutants at frequencies of 28 3 1026 in TK6 cells
and 2,400 3 1026 in WTK-1 cells. Thus, the WTK-1 cells
presented the typical mutator phenotype described above. Interestingly, molecular analysis revealed that WTK-1 cells preferably produced allelic loss (loss of heterozygosity [LOH])type mutations at the tk locus (17, 48). LOH is frequently
observed at critical loci, where tumor suppressor genes should
be located, in a variety of human cancers, and it is an important
Human tumorigenesis is a multistep process in which multiple genetic alterations accumulate, ultimately producing the
neoplastic phenotype (11, 29). These genetic alterations vary
and include point mutations, gene amplifications, rearrangements, translocations, and deletions of specific genes. It has
been hypothesized that genomic instability provides a driving
force for the acquisition of these multiple genetic alterations
(34, 47). If genomic instability increases the rate at which these
alterations occur, then the accumulation of changes and subsequent selection for growth advantage may lead to tumorigenesis. Although the molecular mechanisms leading to increased genomic instability are not fully understood, they
could be initiated by alterations in genes involving DNA replication, DNA repair, telomere stability, or chromosome segregation (32).
The tumor suppressor protein p53 is involved in controlling
genomic stability (23). It appears to mediate certain cellular
responses to DNA damage and to be required for G1 arrest
and apoptosis (19, 25, 45). It has been hypothesized that G1
arrest allows cells time to repair DNA damage prior to DNA
replication, while apoptosis serves to eliminate potentially oncogenic cells (22, 38). This hypothesis has led to the “genome
guardian” model of p53 protein (23). One prediction based on
this model is that p53-deficient cells would provide a mutator
phenotype because cells having unrepaired DNA damage after
* Corresponding author. Mailing address: Division of Genetics and
Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga,
Setagaya-ku, Tokyo 158, Japan. Phone: 81-3-3700-1141, ext. 434. Fax:
81-3-3700-2348. E-mail: [email protected].
† Present address: School of Public Health, West China University
of Medical Sciences, Chengdu 610041, People’s Republic of China.
4774
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Received 13 February 1997/Returned for modification 11 April 1997/Accepted 29 April 1997
VOL. 17, 1997
ILLEGITIMATE RECOMBINATION IN p53-MUTATED CELLS
4775
MATERIALS AND METHODS
Cells and culture conditions. The TK6 and WTK-1 human lymphoblastoid cell
lines were kindly provided by J. B. Little, Harvard School of Public Health,
Boston, Mass. The cells were grown in RPMI 1640 medium (Gibco-BRL, Life
Technology Inc., Grand Island, N.Y.) supplemented with 10% heat-inactivated
horse serum (JRH Biosciences, Lenexa, Kans.).
Isolation of TK-deficient mutants. Cultures were cleansed of preexisting mutants by treatment with CHAT medium (10 mM deoxycytidine, 200 mM hypoxanthine, 0.1 mM aminopterin, 17.5 mM thymidine) for 2 days. To ensure the
isolation of independent mutants, cultures were divided into aliquots immediately after the cleansing procedure and irradiated with X rays. X-ray irradiation
was performed with a Toshiba EX-260 GH-1A generator operating at 5 mA with
1-mm Be and 0.8-mm Al filters. The dose rate was 100 rads/min, as determined
by a model 500 Vectoreen ionizing chamber. Irradiated and nonirradiated cultures were incubated at 37°C for a week to allow expression of TK-deficient
mutants and then were plated into 96-well microtiter plates at 2 3 104 (TK6) or
103 (WTK-1) cells/well in the presence of trifluorothymidine (TFT; 2.0 mg/ml).
The TFT-resistant colonies were observed after 13 days, and one colony from
each culture was randomly isolated. Cloned tk mutant cells were screened to
confirm their phenotype and then Southern analyzed.
Southern hybridization analysis. Southern analysis of the tk locus was done as
described previously (18). Briefly, DNA was extracted from mutant cells, quantified, and digested with SacI. The digested DNAs were electrophoresed in 0.8%
agarose gels, transferred to nylon membranes, and hybridized with 32P-labeled
probes. The probe for the tk locus, pTK11, was provided by P. Deininger (Louisiana State University Medical Center, New Orleans, La.). A b-actin probe
(Oncor Inc., Gaithersburg, Md.) was used as an internal control for densitometric analysis.
Karyotype analysis and SCE. Chromosomes were G banded with Wright’s
stain. Karyotype analysis was done as described in reference 31. For sister
chromatid exchange (SCE) analysis, the culture was incubated with 20 mM
5-bromodeoxyuridine for 28 h in the dark. Colcemid (0.2 mg/ml) was added 2 h
prior to harvesting mitotic cells. Metaphase preparation and SCE analysis were
performed as described previously (16). Fifty randomly selected metaphase cells
were scored for SCE frequency in each cell line.
Chromosome painting analysis. Chromosome painting analysis using fluorescence in situ hybridization was performed as described previously (51). A whole
chromosome 17-specific biotinylated probe (Clontech Laboratories Inc., Palo
Alto, Calif.) was used. Briefly, chromosomal DNA on slides was denatured with
70% formamide with 23 SSC (13 SSC is 0.15 M NaCl plus 0.015 M sodium
citrate) at 70°C for 2 min followed by dehydration through an ethanol series (70,
85, and 100%). The hybridization mixture containing denatured DNA probe (10
ml) was placed on one spot of the chromosome preparation. After overnight
hybridization at 37°C, the slides were washed at 45°C with 23 SSC–50% formamide solution three times and then 23 SSC three times. The slides were
incubated with fluorescein avidin-DCS (Vector Laboratories Inc., Burlingame,
Calif.) solution at 37°C for 30 min and then washed with 43 SSC containing 0.1%
Tween 20. For signal amplification, the slides were incubated with biotinylated
goat anti-avidin D (Vector Laboratories) at 37°C for 30 min followed by a second
fluorescein avidin-DCS treatment. The slides were washed, counterstained with
propidium iodide solution, and mounted. For fluorescence microscopy, an Olympus model BX50 equipped with a DM-FI/PI dual-band pass filter was used. At
least 50 metaphases for each slide were examined, and the abnormalities recorded.
FIG. 1. A model for mechanisms of mutation at the heterozygous tk locus in
TK6 and WTK-1 cells and typical results of Southern analysis performed with a
human tk cDNA probe (pTK11). SacI polymorphic site exists in the 59-flanking
region of the human tk gene. Southern hybridization with the pTK11 probe
provided 14.8-kb (B) and 8.4-kb (A) allelic pair bands corresponding to the
functional and nonfunctional tk alleles, respectively, in both cell types. X indicates a point mutation leading to disfunctioning TK activity. Point mutations did
not change the band pattern, and other intragenic mutations such as deletions
and insertions gave migration of the functional 14.8-kb band. LOH was brought
about by deletion of the entire functional tk allele or by interallelic recombination. Quantitation of the nonfunctional band (8.4 kb) can allow distinction
between the mechanisms.
RESULTS
LOH mutation at the tk locus. Spontaneously arising and
X-ray-induced TK-deficient mutant clones were isolated from
parental TK6 and WTK-1 cells by TFT selection. Two distinct
phenotypic classes of TK mutants were observed. Normally
growing (NG) mutants grew at the same rate as the wild type
(doubling time, 13 to 17 h), and slowly growing (SG) mutants
grew with a doubling time of .21 h. The difference is thought
to be due to a putative growth-regulating gene near the tk locus
(3). It is believed that NG mutants result mainly from mutations within the tk locus, while SG mutants result from gross
structural changes involving the growth-regulating gene outside the tk locus. We previously reported that the SG mutants
contributed to 65% of spontaneous TK mutants from TK6 cells
and 80% from WTK-1 cells and that X rays elevated the
fraction of SG mutants in both cells (17).
The TK-deficient mutants were analyzed by Southern hybridization using the cDNA probe of the human tk gene,
pTK11 (Fig. 1). A SacI polymorphic site exists in the 59-flanking region of the human tk gene, and hybridization with the
pTK11 probe provides 14.8- and 8.4-kb allelic pair bands,
which correspond to the functional and nonfunctional alleles,
respectively, in both cells. Changes in this band pattern reflect
three classes of mutational mechanisms. The first consists of
mutants that show no apparent changes or migration of the
functional 14.8-kb band, which presumably result from point
mutations, deletions, or insertions within the functional tk
gene. The other two classes are LOH mutations that become
either hemizygous or homozygous for the nonfunctional allele;
each results from a different mechanism. Hemizygous LOH
probably results from deletion of the entire allele, while homozygous LOH is produced through mitotic recombination
between homologous alleles. They can be distinguished by
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mutational event in multistep human tumorigenesis (24). Preferable induction of LOH mutations in p53-mutated cells may
help to explain the sequential accumulation of genetic changes
involving LOH during multistep tumorigenesis after p53 mutate.
In this study, to investigate the mechanism of preferable
induction of LOH in p53-mutated cells, we analyzed LOH
mutations at tk loci in TK6 and WTK-1 cells at the cytogenetic
level. Although LOH is generally thought to result from deletions or mitotic recombination between homologous chromosomes, we demonstrated that illegitimate (nonhomologous)
recombination is a major mechanism causing LOH in p53mutated cells; it also leads to an unbalanced translocation.
Because illegitimate recombination was induced by X-ray irradiation, an inducer of double-strand breaks (DSBs), wild-type
p53 may participate in the regulation of recombinational DNA
repair of such breaks. We propose model mechanism for eliciting LOH and the rule of p53 in maintaining genomic stability.
4776
HONMA ET AL.
MOL. CELL. BIOL.
TABLE 1. Chromosome 17 painting analysis of LOH mutants at the tk locus of TK6 cells
Type of cell
Normal
Polyploid
117
NG
Hetero
100
100
0
TKNG1
TKNG13
TKNG29
TKNG14
TKNG26
TKSG42
TKSG43
TKSG56
TKSG3
TKSG4
TKSG5
TKSG6
TKSG61
0/13 (0)
NG
NG
NG
NG
NG
SG
SG
SG
SG
SG
SG
SG
SG
Hemi
Hemi
Hemi
Homo
Homo
Hemi
Hemi
Hemi
Homo
Homo
Homo
Homo
Homo
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
TXNG4
TXNG5
TXNG6
TXSG14
TXSG19
TXSG20
TXSG33
TXSG15
TXSG21
TXSG22
TXSG28
2/11 (18)
NG
NG
NG
SG
SG
SG
SG
SG
SG
SG
SG
Hemi
Hemi
Hemi
Hemi
Hemi
Hemi
Hemi
Homo
Homo
Homo
Homo
50
50
50
50
50
50
50
50
50
50
50
50
50
50
0
50
50
50
50
50
50
0
Clone
Frequency of rearranged
clones (%)
X-ray induced
Frequency of rearranged
clones (%)
a
b
Translocationb
I
II
III
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
46
0
0
0
0
0
0
11
0
0
0
4
0
0
0
0
0
0
36
0
0
0
0
0
0
0
0
0
0
0
Determined by densitometric analysis of nonfunctional 8.4-kb band. Hemi, hemizygous; Homo, homozygous.
I, 217, der(17)t(17;?); II, der(17)t(17;?); III, others (complicated types).
densitometric analysis. The intensity of the nonfunctional
8.4-kb band was measured, and the relative ratio of its intensity
was normalized to an internal control DNA amount by stripping and rehybridizing the same filter with b-actin cDNA (data
not shown). If the normalized intensity was more than 1.5
times that of the wild-type allele, which corresponds to about
two copies of the tk gene, the LOH mutant was classified as
homozygous (Table 1). We previously reported the mutational
spectra of TK mutants from TK6 and WTK-1 cells (17). Almost all TK mutants spontaneously arising and induced by
X-rays in WTK-1 cells contained homozygous LOH mutations,
while a considerable portion of TK mutants in TK6 cells contained hemizygous LOH mutations and point mutations. These
findings indicate that WTK-1 cells were prone to LOH mutations, presumably via a recombinational process. As a next
step, to investigate the mechanism preferably generating LOH
mutations in WTK-1 cells, some LOH mutants were examined
at the cytogenetic level by using chromosome painting analysis.
Karyotype of TK6 and WTK-1 cells. As an initial investigation, we analyzed the karyotype of the parental TK6 and
WTK-1 cells from which the TK-deficient mutants derived
(data not shown). A representative karyotype of TK6 cells was
47, XY, 131, t(14;?), t(21;?). These karyotypic features are
essentially identical with those found in previous analyses (15,
21). On the other hand, WTK-1 commonly showed 47, XY,
131, t(21;?), 142, del(1)(pter-q42), t(12;15), 51, but some
metaphases contained other abnormality including t(X;10),
del(3), del(8), t(3;10), t(1;8), or t(2;15). In addition, approximately 8% of metaphases in WTK-1 cells showed polyploid.
The karyotype of WTK-1 cells was more heterogeneous and
unstable than that of the TK6 cells. Chromosome 17 painting
analysis revealed that there were no structural abnormalities
involving chromosome 17 in either cell line (Fig. 2a and b),
while a chromosome 17 numerical change (trisomy of chromosome 17) was observed only in WTK-1 cells at low frequency
(2%).
Analysis of spontaneous LOH mutants. Thirteen spontaneous LOH TK6 cells (five NG and eight SG mutants) were
chosen for chromosome 17 painting analysis. Densitometric
analysis revealed that two NG and five SG mutants were homozygous at the tk locus. No apparent abnormalities, including
chromosomal deletions and translocations, with respect to the
parental TK6 cells were observed in any 13 LOH mutants
(Table 1). Similarly, we analyzed 17 LOH mutant WTK-1 cells
(6 NG and 11 SG mutants), among which 4 NG and 10 SG
mutants were homozygous mutants. Interestingly, we observed
a translocation involving chromosome 17 in every metaphase
for 9 of 11 SG mutants (Table 2). The translocations were
interpreted as two types of unbalanced translocations. The first
type was der(17) t(17q;?), which lost genetic material on the
distal end of the long arm, including the tk locus, and was
replaced by another chromosome (type I [Fig. 2c]). The second
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No. of
metaphases
analyzed
Parent
Mutants
Spontaneous
No. of changes of chromosome 17
tk locus by
Southern
analysisa
Type of
growth
VOL. 17, 1997
ILLEGITIMATE RECOMBINATION IN p53-MUTATED CELLS
4777
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FIG. 2. Examples of chromosome 17 painting in TK6 and WTK-1 cells following hybridization of a whole chromosome 17 DNA probe with metaphase
chromosomes. The labeled chromosome 17s appear yellow, and other chromosomes are red from the counterstain propidium iodide. Translocated chromosomes are
indicated by arrows. (a) Metaphase of parental TK6 cells; (b) metaphase of parental WTK-1 cells; (c) metaphase of clone WTSG29, type I [217, der(17)t(17;?)]; (d)
metaphase of clone WTSG28, type II [der(17)t(17;?)]; (e) metaphase of clone WXSG30, type III (complicated); (f) metaphase of clone WXSG29, type III
(complicated).
4778
HONMA ET AL.
MOL. CELL. BIOL.
TABLE 2. Chromosome 17 painting analysis of LOH mutants at the tk locus of WTK-1 cellsa
Type of cell
Parent
Mutant
Spontaneous
X-ray induced
Frequency of rearranged
clones (%)
a
No. of
metaphases
analyzed
Normal
Polyploid
117
NG
Hetero
100
90
8
WTNG38
WTNG41
WTNG1
WTNG2
WTNG3
WTNG4
WTSG12
WTSG14
WTSG17
WTSG26
WTSG28
WTSG29
WTSG56
WTSG62
WTSG64
WTSG70
WTSG75
9/17 (53)
NG
NG
NG
NG
NG
NG
SG
SG
SG
SG
SG
SG
SG
SG
SG
SG
SG
Hemi
Hemi
Homo
Homo
Homo
Homo
Hemi
Homo
Homo
Homo
Homo
Homo
Homo
Homo
Homo
Homo
Homo
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
29
41
45
45
41
47
0
47
44
0
0
0
0
0
0
0
0
WXNG1
WXNG5
WXNG9
WXNG10
WXNG2
WXSG21
WXSG29
WXSG30
WXSG31
WXSG41
WXSG48
9/11 (82)
NG
NG
NG
NG
NG
SG
SG
SG
SG
SG
SG
Homo
Homo
Homo
Homo
Homo
Homo
Homo
Homo
Homo
Homo
Homo
50
50
50
50
50
50
50
50
50
50
50
45
0
0
46
0
0
0
0
0
0
0
Clone
Translocationb
I
II
III
2
0
0
0
19
9
5
5
7
3
4
3
6
8
4
7
8
12
5
4
14
2
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
21
0
0
22
1
22
22
14
3
8
16
0
0
0
0
0
0
25
0
0
20
45
21
20
24
42
38
20
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
5
5
4
4
11
4
9
3
2
7
10
0
0
0
0
0
0
0
0
0
0
0
0
30
14
0
19
0
0
9
26
15
10
0
15
32
0
20
0
0
28
22
28
30
0
0
0
0
0
46
41
10
0
0
0
For details, see the footnotes to Table 1.
possessed another chromosome 17 in addition to the translocation, resulting in partial trisomy of chromosome 17 (type II
[Fig. 2d]). Every translocated chromosome 17 possessed its
centromere region. These two types of translocations were
heterogeneous, observed in the metaphases of the mutants as
mosaics. Polyploid and trisomy of chromosome 17 were observed in some LOH mutants as well as in parental WTK-1
cells (data not shown). These numerical changes, however,
seemed independent of the translocational events.
Analysis of X-ray-induced LOH mutants. We also investigated 11 X-ray-induced TK6 LOH (2 Gy) and 11 WTK-1 (4
Gy) mutants. The doses provided almost equivalent cytotoxic
damage to both cell types (2 to 5% relative survival). Two TK6
SG mutants showed an unbalanced translocation that was similar to one observed in spontaneous WTK-1 LOH mutants. We
also detected an unbalanced translocation in two of four NG
mutants and all of the seven SG mutants of WTK-1. Some
revealed unusual chromosomal changes that were difficult to
categorize, although most changes were type I or II translocations. Clone WXSG21 and WXSG30 had some metaphases
that showed two translocated chromosomes 17 with a normal
chromosome 17 (Fig. 2e). Clone WXSG29 had additional
chromosome 17 numerical changes with type I and type II
translocations in some metaphases (Fig. 2f). These changes
were categorized as type III (others). The results of chromosome painting analysis and the frequencies of LOH mutants
showing the translocations are shown in Tables 1 and 2.
SCE. The frequencies (means 6 standard deviations) of
spontaneous SCE were 2.36 6 1.32/cell (0.052/chromosome) in
TK6 cells and 2.16 6 1.49/cell (0.047/chromosome) in WTK-1
cells. There was no statistically significant difference in SCE
frequency between cell lines.
DISCUSSION
Allelic loss, which is observed as LOH at tumor suppressor
gene loci in a variety of human cancers, is an important mutational event in tumorigenesis. Although LOH could be due
to chromosome loss, chromosome loss followed by duplication
(or aberrant segregation), deletion of an entire allele, or mitotic recombination between homologous alleles, the last two
appear to be the major mechanisms associated with loss of
tumor suppressor functions (24, 39).
We previously reported that p53-mutated WTK-1 cells exhibit a mutator phenotype and preferably produce LOH-type
gene mutations, presumably because of their abnormally high
recombinational activity (17). Xia et al. (48) also demonstrated
hyperrecombinational activity in WTK-1 cells that increased
Downloaded from http://mcb.asm.org/ on April 26, 2014 by PENN STATE UNIV
Frequency of rearranged
clones (%)
No. of changes of chromosome 17
tk locus by
Southern
analysisa
Type of
growth
VOL. 17, 1997
ILLEGITIMATE RECOMBINATION IN p53-MUTATED CELLS
FIG. 3. Model for mechanisms eliciting loss of heterozygosity via homologous recombination, nonhomologous recombination, and nonhomologous recombination followed by mitotic nondisjunction. Nonhomologous recombination
can lead to unbalanced translocation.
11 (which harbors the mouse tk gene). Recently the L5178Y
cell line was shown to have a point mutation at codon 170 in
the p53 gene and to overproduce mutant p53 protein (42). This
finding supports the idea that overproducing mutant p53 protein contributes to a high frequency of unbalanced translocations. It is possible that overproduction of p53 protein enhances recombinational activity and that the mutant form of
the protein causes low recombination fidelity, leading to recombination between nonhomologous DNAs. This quantitative and qualitative abnormality of p53 protein may disorder
the recombinational machinery in mammalian cells, resulting
in a high frequency of LOH mutations and unbalanced translocations. Thus, p53 may directly regulate processes of homologous recombination in mammalian cells. p53 may be also
responsible for monitoring for homologous recombination. It
is possible that if nonhomologous recombination as well as
homologous recombination usually occur in mammalian cells,
p53 can detect and eliminate abnormal cells which have undergone nonhomologous recombination.
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the frequency of plasmid-based intermolecular homologous
recombinations. These findings suggest that the mutant p53
protein might enhance or deregulate the recombinational machinery, leading to genomic instability. It is possible, however,
that nonhomologous recombinations or other complicated
DNA rearrangements were associated with LOH in WTK-1
cells, because the mutant p53 protein might bring about inaccurate homologous recombination or other types of genomic
instability. The chromosomal abnormality observed in p53deficient and -mutated cells may be due to deregulated recombinational machinery (28, 30, 50). We therefore decided to
analyze LOH mutants at the cytogenetic level by using a chromosome painting technique.
According to the mechanisms of LOH depicted in Fig. 1,
hemizygous LOH mutations by deletion should show a shortened chromosome 17, and homozygous LOH mutations
caused by homologous recombination should show no structural alteration. Indeed, we observed no structural or numerical change in chromosome 17 in any homozygous LOH mutants arising spontaneously from TK6 cells, consistent with the
hypothesis that the homozygous LOH mutants resulted from
mitotic recombinations between homologous chromosomes.
We also found no abnormality in any hemizygous LOH mutants arising spontaneously from TK6 cells, suggesting that
deletional events occurring in SG mutants, as well as in NG
mutants, are limited to a relatively small region that cannot be
detected by usual cytogenetic analysis (presumably less than 10
Mb). Hence, spontaneous LOH mutations in TK6 cells were
probably generated through models described in Fig. 1. On the
other hand, we unexpectedly found unbalanced translocations
in 53% (9 of 17) of spontaneous LOH mutants from WTK-1
cells (Fig. 2; Table 2). The translocated chromosome 17 lost
the distal end of the long arm that includes the tk locus and is
fused to an unidentified chromosome. Because all spontaneous
mutants showing translocations were SG mutants, the putative
gene associated with cell growth control located distal to the tk
locus may be lost by the unbalanced translocation. We postulated that the unbalanced translocation resulted from illegitimate (nonhomologous) recombination between chromosome
17 and another chromosome. Figure 3 shows a model for
mechanisms eliciting LOH mutation via homologous and nonhomologous recombinational processes. According to this
model, a clone losing a functional tk allele by nonhomologous
recombination becomes resistant to TFT with an unbalanced
translocation. In addition to the unbalanced translocation, we
found partial trisomy of chromosome 17 in some metaphases
of the mutants (type II). Because these numerical chromosome
changes are not clonal in the mutants, they must not be directly
associated with tk gene mutation. Actually, this phenomenon
was observed some parental WTK-1 cells (Table 2). It is likely
that chromosome 17 trisomy found in type II translocations
resulted from a gain of chromosome 17 after the translocational event, because loss of p53 function increases the probability of generating aneuploidy through mitotic nondisjunction (aberrant segregation) (1, 12). This provides an
explanation for the densitometric data. LOH WTK-1 mutants
showing translocations were identified as homozygous by densitometric analysis because of an increased copy number of the
nonfunctional tk allele. Chromosome painting analysis, however, demonstrated that the increased copy number was a
result of mitotic nondisjunction following nonhomologous recombination, not of homologous recombination.
We observed a similar phenomenon in a study of the tk gene
mutation in L5178Y mouse lymphoma cells (51). Almost all
TK-deficient L5178Y cells showed unbalanced translocations
and other complicated rearrangements involving chromosome
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HONMA ET AL.
genomic instability, which brings about the sequential accumulation of alterations in some oncogenes and tumor suppressor
genes that correlate with more malignant phenotypes.
In conclusion, our observations are consistent with the hypothesis that (i) p53 participates in the regulation of homologous recombination and recombinational DNA repair processes and (ii) loss of wild-type p53 function deregulates the
process and causes gross structural changes in the genome.
This is one of the roles of p53 as a molecular guardian of the
genome, maintaining genomic stability in mammalian cells.
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