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Carcinogenesis |
Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75235
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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XP-G) and a variant form
(XP-V) of the disease have been identified (1, 2, 3, 4)
. In
recent years, several mouse models of human XP gene defects have been
generated by targeted gene disruption (5)
. These mice have
provided informative model systems for studying various aspects of NER,
as well as the molecular pathology of cancer predisposition associated
with defects in this DNA repair process. In previous studies, we generated Xpc mutant mice and demonstrated that cells from such mice are deficient for NER (6) . These mice are highly predisposed to UVB radiation-induced skin cancer (7) and to tumors of the liver and lungs after administration of the chemical carcinogen acetylaminofluorene (8) . In both humans and mice, XPC protein is specifically required for NER of base damage in transcriptionally silent regions of the genome, as well as the nontranscribed strand of transcriptionally active genes (9 , 10) . The protein is not required for NER of the transcribed strand of transcriptionally active genes (9 , 10) . Recent studies (11) suggest that XPC protein complexed to hHR23B protein may play a specific role in the recognition of base damage during NER, but that when this process occurs in transcriptionally active regions of the genome, this role is subserved by some other moiety, perhaps the arrested transcription machinery.
The p53 (mouse Trp53) gene is one of the most commonly mutated genes in human cancers (12) and is a frequent target for mutation in UVB radiation-induced skin cancer (13) . The p53 protein is integral to several cellular responses to DNA damage (12 , 14 , 15) . After insult by chemical or physical agents that damage DNA, p53-dependent mechanisms are invoked that inhibit cell cycle progression and activate apoptosis (12 , 14 , 15) . Additionally, several studies have suggested that p53 protein may directly modulate the activity of DNA repair pathways (16, 17, 18) . To examine the consequences of a combined deficiency in NER and Trp53 gene function, we made genetic crosses between Xpc and Trp53 mutant strains and reported previously that the time of appearance of skin cancer in Xpc-/- mice is significantly reduced if they are additionally heterozygous or homozygous mutant for Trp53 (7 , 19) .
We also extended our investigations to include the role of defects in the BER pathway in UV radiation-induced carcinogenesis. Inactivation of the mouse AP-endonuclease (Apex; human HAP1 = Ref-1) gene in embryonic stem cells results in embryonic lethality (20 , 21) .6 However, Apex+/- mice are viable. When crossed to Xpc-/- mice, the loss of a single Apex allele increased the predisposition of these animals to UV radiation-induced skin cancer (19) . This effect apparently operates genetically through Trp53 function, because the Apex+/- state did not further increase the predisposition to skin cancer in either Xpc-/- Trp53+/- or Xpc-/-Trp53-/- mutant mice, i.e., the Apex+/- and Trp53+/- states are epistatic (19) . Independent studies have demonstrated that human HAP-1 (Ref-1) protein is required for activation of p53 protein in vitro (22) . Hence, in addition to its indispensable role in BER, HAP-1 (Ref-1) protein also influences p53-dependent processes. HAP-1 (Ref-1) protein is additionally essential for the redox-dependent activation of various transcription factors (23) , possibly including p53 (22 , 24 , 25) . In view of these multiple functions of the HAP1 (Ref-1) protein, it is not surprising that ablation of the gene in mice results in early embryonic lethality (20 , 21) .6
In the present studies, we document a comprehensive analysis of UVB radiation-induced skin cancer in Xpc-/- and Xpc+/- animals and in various Xpc, Trp53, and Apex mutant combinations using larger cohorts of animals and longer periods of observation. We demonstrate the predisposition of Xpc+/- animals to UVB radiation-induced skin cancer and present evidence suggesting that this predisposition is the result of haploinsufficiency of the Xpc gene. We also present evidence suggestive of a contribution of haploinsufficiency of the Apex gene to skin cancer predisposition in the presence of defective NER. Finally, we show that the pathology of UVB radiation-induced skin cancer is influenced by the specific genotype of afflicted mice. In the accompanying report (26) , we document the results of a detailed analysis of the spectrum of Trp53 mutations in skin tumors from mice with various Xpc, Trp53, and Apex genotypes.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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70% 129/Sv and 30%
C57Bl/6. Results obtained with animals originating from the two
different crosses (Xpc x Trp53 or
Xpc, Trp53 x Apex)
were analyzed independently. All skin tumor incidence curves represent
study and control animals from identical genetic backgrounds. Results
were only pooled when background differences were found to have no
effect on cancer incidence curves.
UV-induced Skin Cancer.
The dorsal skin of mice, 8–12 weeks of age, was shaved and irradiated
for 5 days/week at a dose rate of 120 J/m2/min
for 14 min, using two FS20 erythemal (UVB) lamps (Phillips) filtered by
Kodacel sheeting (Kodak, Rochester, NY). Mice were irradiated until
either skin tumors were visible to the naked eye or for a maximum of 18
weeks. The mice were monitored for the presence of skin tumors visible
to the naked eye at least once a week for up to 100 weeks. Skin tumors
were biopsied, and a portion of each tumor was fixed in 10%
neutral-buffered formalin and prepared for routine and special
histology. Skin tumors were graded and classified by standard
histopathological criteria.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In our earlier studies, we observed no significant differences in
skin cancer predisposition in Xpc wild-type and heterozygous
mutants monitored for 30 weeks after the onset of daily doses of UVB
radiation (7
, 19)
. We now show that when animals were
monitored for longer periods, the latency time for the appearance of
skin cancer was reduced in Xpc+/- mice
compared with Xpc+/+ litter mates.
Xpc+/+ mice manifested a 50% incidence of
skin cancer by 92 weeks after the onset of 18 weeks of daily UV
irradiation (Fig. 1)
. In contrast, Xpc+/-
mice suffered a 50% incidence at 50 weeks after irradiation (Fig. 1)
.
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, Xpc+/+ Trp53+/-
mice are more cancer prone than wild-type or
Xpc+/- Trp53+/+
litter mates. However, double heterozygous mutants
(Xpc+/-Trp53+/-) are even more cancer prone and
manifest a 50% skin cancer incidence about 3 weeks earlier than
Xpc+/+ Trp53+/-
mice (Fig. 2)
, despite the fact that (as indicated above) increased
cancer predisposition in Xpc+/-Trp53+/+ mice does not manifest until 50
weeks after the onset of irradiation. One obvious explanation for the
predisposition to skin cancer in Xpc+/-
mice compared with appropriate controls is that the single remaining
Xpc allele is frequently mutated as a result of exposure to
UVB radiation. However, direct examination of Xpc cDNA
identified mutations in the coding region of the Xpc gene in
only 2 of 16 skin tumors examined. One of these, from an
Xpc+/- Trp53+/+
animal, was a CT transition at a dipyrimidine site in codon 724,
which did not alter the encoded amino acid (leucine). The second
mutation, from an Xpc+/-Trp53+/- animal, was also a CT transition at
a dipyrimidine site. This mutation is expected to substitute leucine
with proline at codon 319. Because proline is the amino acid encoded by
the corresponding codon in the human XPC gene, we predict
that this mutation is functionally silent in the mouse gene. Although
we cannot formally exclude the (unlikely) possibility of mutations in
the promoter regions of the single Xpc gene in all these
tumors or that epigenetic effects may have altered the expression of
the gene, our results suggest that the increased predisposition to UVB
radiation-induced skin cancer in Xpc+/-
mice results from haploinsufficiency of the Xpc gene.
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. Sequence analysis of Apex cDNA from 12 skin tumors derived
from Xpc-/-Apex+/- mice failed to reveal mutations in a
single case. In contrast, we detected mutations in Trp53 in
all cases (26)
. These observations lead us to the
suggestion that, as is the case for the Xpc heterozygous
state, the effect of heterozygous deletion of the Apex gene
reflects haploinsufficiency with respect to predisposition to UVB
radiation-induced skin cancer.
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. The same is true for the comparison between
Xpc+/- Trp53+/-Apex+/+ and
Xpc+/- Trp53+/-Apex+/- animals, and between
Xpc+/- Trp53+/+Apex+/+ and
Xpc+/- Trp53+/+Apex+/- animals (Fig. 4)
. Indeed, in the latter
case, the Apex+/- state actually
protected older Xpc+/-Trp53+/+ animals from skin cancer. Hence, the
skin cancer predisposition associated with the
Apex+/- state only manifested when NER
was defective (Xpc-/- state; Fig. 3
).
One possible explanation for this observation is that in the absence of
NER, there is a critical requirement for BER of damaged pyrimidine
mononucleotides of the type generated by exposure to UV radiation or to
reactive oxygen species (e.g., thymine glycols; Ref.
1
), and that haploinsufficiency of the Apex
gene impairs BER of these photoproducts. This explanation is consistent
with independent studies showing enhanced sensitivity to killing of
cells from Apex+/- mice after exposure to
agents that are known to promote oxidative damage to
DNA.7
However, in light of the fact that the Apex protein also modulates the
activity of a number of transcription factors and activates Trp53
protein by both redox-dependent and redox-independent mechanisms
(22, 23, 24, 25)
, other explanations are possible for the
observation that the skin cancer predisposition associated with the
Apex+/- state only manifests when NER is
defective. The complex regulatory functions of Apex protein may also
account for the curious paradox noted above that older
Xpc+/- Trp53+/+Apex+/- animals are less skin cancer prone than
Xpc+/- Trp53+/+Apex+/+ controls (Fig. 4)
.
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. In contrast, a significant number of the skin tumors from
Xpc+/+ Trp53+/+
and Xpc+/-Trp53+/+ mice were poorly differentiated (grade
3–4) squamous cell carcinomas (Fig. 5)
. The observation that
Xpc-/- mice are more predisposed to skin
cancer but suffer a less aggressive form of the disease may relate to
the observation that when these mice are exposed to an acute dose of
UVB radiation, the epithelium of the skin rapidly undergoes marked
thickening because of hyperplasia and hyperkeratosis (7)
.
UVB radiation only penetrates 70 µm of the skin (27)
.
Hence, the increased epidermal thickening and limited penetration of
the radiation may increase the fraction of well-differentiated target
cells that are progenitors for skin cancer. In contrast, the absence of
epidermal hyperplasia in Xpc+/+ and
Xpc+/- mice (7)
may allow
deeper penetration of the UVB radiation, resulting in skin cancers that
derive from less well-differentiated progenitor cells of the basal
layer of epidermis and/or from deeper skin appendage structures.
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. Once again, in general carcinomas in
Xpc-/- mice were better differentiated
than in Xpc+/+ and
Xpc+/- animals. However, in
Xpc-/- mice that were also
Trp53-/-, a significant fraction of the
tumors was poorly differentiated squamous cell carcinomas,
undifferentiated spindle cell tumors, sarcomas, and lymphomas (Fig. 6)
.
These observations are consistent with the increased epidermal
dysplasia observed in Trp53 mutant mice after exposure to
acute doses of UVB radiation (7)
. We observed several
unusual skin tumors in Xpc+/-Trp53+/- mice, including acantholytic granular
cell variants and tumors of dermal appendage structures. Skin cancer
could not be examined in Trp53-/- mice
that were additionally Xpc+/- or
Xpc+/+ because the animals died of other
Trp53-/--related tumors before
they had time to manifest skin cancer.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present studies provide the first documented evidence in mammals of phenotypes associated with the Xpc heterozygous state. Xpc+/- mice are at a greater risk for UVB radiation-induced skin cancer than wild-type litter mate controls. Additionally, such mice show an increased risk of skin cancer when one allele of the Trp53 gene is inactivated compared with Trp53 heterozygous mutants alone. In both of these genotypic states, we found no evidence of mutations in the coding region of the remaining Xpc allele in skin tumors. Although we cannot exclude the possibility of mutations or epigenetic effects that altered expression of this allele, we are led to the notion that loss of one Xpc allele results in haploinsufficiency. On the basis of this conclusion, we suggest that it may be prudent to alert obligate XP heterozygous human individuals (of whom there may be as many as 2 to 3/1000 in the general population) of a possible increased risk for cancer associated with exposure to sunlight and to other known carcinogens. We are presently screening skin cancers from humans, especially older individuals, for heterozygosity in one or more XP genes.
Our previously published and present studies demonstrate additive effects with respect to skin cancer predisposition in mice defective in both NER and Trp53 functions. Conceivably, both parameters operate independently in promoting genomic instability in the presence of DNA damage. Alternatively, or additionally, inactivation of the Trp53 gene may compromise residual repair of the transcribed strand of transcriptionally active genes in Xpc mice. Direct evidence for the involvement of p53 in such repair has been provided by other studies (16 , 17) . The observation that Xpc-/-Trp53-/- mice are more cancer prone than Xpc-/- Trp53+/- animals suggests that in the latter group the remaining Trp53 allele is frequently inactivated in the tumors. Evidence that this is indeed the case is presented in an accompanying report, which also details the mutational spectrum in the Trp53 gene (26) .
Mice that are defective in one Apex allele also manifest haploinsufficiency with respect to skin cancer predisposition. However, this is only observed in the presence of defective NER. On the basis of the results of studies in human and mouse cells, normal levels of Apex protein are apparently required for optimal activation of Trp53 protein (19 , 22) . Hence, the increased cancer predisposition observed in Xpc-/-Apex+/- mice may derive from reduced activity of Trp53 protein. Alternatively, defective NER associated with the Xpc-/- state may require optimal levels of Apex protein for BER of specific photoproducts. Both brain cells and fibroblasts from Apex+/- mice are abnormally sensitive to agents that cause oxidative damage to DNA.7
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 These studies were supported by Research Grant
CA44247 (to E. C. F.) and by postdoctoral fellowships from the
American Cancer Society (to D. L. C.) and The Friends of the Center
for Human Nutrition, University of Texas Southwestern Medical Center. 
2 The contributions of these authors are
considered equal.
3 Present address: Life Technologies, Inc.,
Rockville, MD 20894-6482.
4 To whom requests for reprints should be
addressed at, Southwestern Medical School, 5323 Harry Hines Boulevard,
Dallas, TX 75235-9072. E-mail: friedberg.errol{at}pathology.swmed.edu
5 The abbreviations used are: XP, xeroderma
pigmentosum; Xpc, xeroderma pigmentosum group C gene;
NER, nucleotide excision repair; BER, base excision repair.
6 L. B. Meira and E. C. Friedberg, unpublished
observations.
7 L. B. Meira, G. Kisby, and E. C. Friedberg,
unpublished observations.
Received 8/31/99. Revised 12/ 6/99. Accepted 1/19/00.
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REFERENCES |
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,
Xpc+/+ (n = 23); 
, Xpc+/-
(n = 20); 
,
Xpc-/- (n = 32).
,
Xpc+/- Trp53+/-
(n = 36).
, Trp53+/+Apex+/- (n = 26);
, Trp53+/-Apex+/- (n = 20);
, Trp53+/-Apex+/+ (n = 25).
,
Xpc+/+ Trp53+/+
or Xpc+/-Trp53+/+ (n = 11);
,
Xpc+/+ Trp53+/-
(n = 17); 
, Xpc-/-Trp53-/- (n = 14).