Each human cell contains hundreds to several thousand copies of the 16.5 kb human
mitochondrial genome. This closed circular genome encodes 13 polypeptides of the respiratory
chain complexes, as well as 22 transfer RNAs and two ribosomal RNAs used in mitochondrial
protein synthesis. Compared to nuclear DNA, mitochondrial DNA (mtDNA) is highly susceptible
to damage because it is not associated with protective histones, it is continually
exposed to high levels of reactive oxygen species (ROS) generated by oxidative phosphorylation,
and there is a limited capacity for mtDNA repair (Wallace, 1992). The complete mtDNA
sequence was determined in 1981 (Anderson et al, 1981) and resequenced in 1999 (Andrews
et al, 1999). A growing collection of reported mtDNA mutations and rearrangements
has been associated with muscle and neurodegenerative diseases (Brierley et al, 1998;
Chinnery et al, 1999).
Mitochondria have been implicated in the carcinogenic process because of their role
in apoptosis (Kroemer and Reed, 2000) and other aspects of tumour biology; in particular,
somatic mutations of mtDNA have been observed in a wide variety of human tumours (reviewed
in Penta et al, 2001). These findings together with the fact that ultraviolet radiation
(UVR) is important in the development of nonmelanoma skin cancer (NMSC), and has been
shown to induce mtDNA damage in human skin (Birch-Machin et al, 1998; Ray et al, 2000),
which led us to perform the first detailed study of mtDNA damage in human NMSC. This
type of skin cancer consists of basal cell and squamous cell carcinoma (i.e. BCC and
SCC, respectively). BCCs are the commonest form of skin cancer and occur mainly on
sun-exposed body sites in elderly or middle-aged subjects. They arise from the basal
keratinocytes of the epidermis, are locally invasive but rarely metastasise. SCCs
are derived from moderately differentiated keratinocytes. They mainly occur in people
over 55 years (y) of age and are found on sun-exposed sites. In contrast to BCCs,
the SCCs are derived from precursor lesions such as actinic keratoses and importantly
the SCCs may metastasise.
This study is the first of its kind in the skin cancer literature as it provides a
detailed investigation of the different types of mtDNA damage in both SCCs and BCCs
compared to matched histologically normal perilesional tissue. We have investigated
the entire spectrum of large-scale deletions, the incidence of the common deletion
and tandem duplications, and the distribution of single base changes in the mitochondrial
genome.
MATERIALS AND METHODS
Patient samples and DNA extraction
Tumour and matched perilesional skin samples were taken with informed consent from
patients undergoing excision of an NMSC, namely BCC (n=5, age range 55–89 y, mean
78 y) or an SCC (n=5, age range 70–87 y, mean 78 y) at the Out-Patients Clinic, Royal
Victoria Infirmary, Newcastle, UK. The perilesional skin was split into dermis and
epidermis, and DNA extracted as described previously (Birch-Machin et al, 1998).
Long template PCR
The deletion spectrum of large-scale deletions was investigated by amplification of
almost the entire 16.5 kb mitochondrial genome in two fragments, using long template
PCR as described previously (Ray et al, 2000). To ensure reproducibility and to prevent
any preferential amplification, the amount of DNA was standardised for each sample.
3-primer PCR
A competitive, radioactive, PCR assay was used to quantify the ratios of both deleted
and wild-type mtDNA (Sciacco et al, 1994). Wild-type and deleted mtDNAs are demonstrated
by the presence of a 755 and a 470 bp PCR product, respectively.
‘Back-to-Back’ primer assay
This methodology has been previously described to screen for the presence of tandem
duplications in the D-loop (Brockington et al, 1993). The primers were L336 (nucleotide
position (np) 336–355) and H335 (np 335–316). Under the designated PCR conditions,
the orientation of these primers prevents the generation of a product from wild-type
mtDNA but permits a product from mitochondrial genomes harbouring a 200 bp or a 260 bp
tandem duplication in the D-loop. The nucleotide positions of the tandem duplication
breakpoints have been previously described (Brockington et al, 1993; Lee et al, 1994).
DNA sequence analysis
Direct DNA sequencing of ND1, ND5, 16 s RNA and the noncoding D-loop region of the
mitochondrial genome was performed as previously described (Andrews et al, 1999).
A review of the current literature shows that these four mtDNA regions appear to be
highly susceptible to mutations (Penta et al, 2001). Automated DNA sequencing was
performed by MWG Biotech (Ebersberg, Germany) and the resulting sequences compared
to the revised Cambridge mtDNA reference sequence (MITOMAP). Confirmation of unreported
base changes was performed by sequencing in the reverse direction on independent PCR
products.
RESULTS
The spectrum of large-scale mtDNA deletions
The deletion spectrum was investigated in the mitochondrial genomes from the tumour
samples and the histologically normal perilesional dermis and epidermis samples from
each NMSC patient (total sample number is 30). Almost the entire mitochondrial genome
was amplified in two fragments, namely 11 and 5.5 kb. The spectrum of deletions is
visualised as a DNA ladder of PCR products on an agarose gel (Figure 1
Figure 1
MtDNA deletion spectrum of tumour and perilesional skin. A1 and A2: 11 kb PCR profile
for BCC and SCC, respectively. B1 and B2: 5 kb PCR profile for BCC and SCC, respectively.
Each panel of three lanes represents a single NMSC patient; lane 1=tumour, lane 2=dermis,
lane 3=epidermis. The observed deletion profiles were reproducible over three independent
PCR experiments. Molecular weight markers (Hyperladder I-range 10 kb–200 bp, Bioline
Ltd, London UK) are the single lanes at the ends of each row of panels.
). The 11 kb PCR fragment encompasses the major deletion arc where the vast majority
of mtDNA deletions are reported (MITOMAP). There were 20 different-sized deletions
observed in both the tumour and perilesional skin, with BCCs harbouring more deletions
than SCCs (Figure 1, A1 and A2, respectively). Within each triplet of patient samples,
the number of deletions in the epidermis never exceeded that observed in the corresponding
tumour and dermis. Interestingly, the observed pattern of deletions in the tumour
was often very different compared to that observed in the matched perilesional skin.
Despite this, there is not a clear deletion pattern that is characteristic of a particular
tumour type.
The 5.5 kb PCR fragment encompasses the minor deletion arc of the mitochondrial genome,
where fewer deletions are reported (MITOMAP). A total of only four different-sized
deletion species were observed but, like the 11 kb PCR results, the greatest number
of total deletions was observed in the BCC as opposed to the SCC tumour samples (Figure
1, B1 and B2, respectively). Interestingly, a PCR fragment of approximately 1.5 kb
(equivalent to a 4 kb deletion) was found to be common to all those samples that harboured
any deletion(s).
Quantification of the 4977 bp common mtDNA deletion
Low levels of the common deletion, typically much less than 0.2%, have been associated
with ageing (Nagley and Wei, 1998). To ensure that the effects of ageing do not confound
our results for the common deletion, we have presented data representing only high
levels (i.e. >2%) of deletion (a typical example is shown in Figure 2A
Figure 2
Quantification of the 4977 bp common deletion and detection of tandem duplications.
(A) An example of a phosphorimage from the 3-primer PCR assay showing the PCR products
that represent wild-type (WT) and deletion products (CD) in the tumour (T), and perilesional
dermis and epidermis (D and E, respectively) from the 89-y-old BCC patient detailed
in part B. (B) The triplet of samples representing tumour (T), perilesional dermis
(D) and epidermis (E) from each NMSC patient were screened for the common deletion
and duplications. nd=not detected (duplications); levels below 2% (common deletion).
). We have shown previously that these high deletion levels are not associated with
chronological ageing, and this is further confirmed by the data in Figure 2B that
is arranged in ascending order of age. The high levels of the common deletion varied
in the NMSC samples from 2.3 to 23.4% but were not observed in the perilesional epidermis
from the BCC patients. Four of the 10 NMSC patients harbour high deletion levels in
both the tumour and the corresponding perilesional skin. In three of these four patients,
it was the tumour that harboured the highest level of deletion by a factor of approximately
2–4-fold. Even in the outlying sample (i.e. BCC, 89 y) the levels of deletion in the
tumour and perilesional skin were similarly high at 13.4 and 14.9%, respectively.
Interestingly, in a manner similar to the spectrum of deletions, the observed patterns
of the common deletion in the tumour was often very different compared to that observed
in the matched perilesional skin.
Detection of tandem duplications within the D-loop
A total of four samples, representing three different NMSC patients, were found to
harbour a 200 or 260 bp tandem duplication. We found one BCC patient with a 260 bp
duplication and three SCC patients with a 200 bp duplication (Figure 2B). It has been
proposed previously that there is a link between the presence of duplications and
the common deletion (Nagley and Wei, 1998). In this respect, three of the four samples
with a duplication also contained the common deletion in the corresponding sample.
MtDNA base changes
Comparative DNA sequence analysis was performed on four regions of the mitochondrial
genome from the tumour samples and the histologically normal perilesional dermis and
epidermis samples from each NMSC patient. A total of 81 homoplasmic and one heteroplasmic
base substitutions were identified (Table 1
Table 1
Summary of the 82 different mtDNA base changes identified in the tumour and perilesional
skin samples. (A) Base changes that have been previously unreported in the MITOMAP
database. (B) Previously reported changes that alter an amino acid. (C) Previously
reported synchronous changes that do not alter the amino acid
Base change
Map locus
Amino-acid change
Base change
Map locus
Amino-acid change
(A)
C438Ta
OHR/LSP/TFL
Noncoding
C12348T
ND5
—
A574G
D-loop
Noncoding
C12633A
ND5
—
C494A
D-loop
Noncoding
C13251T
ND5
—
T1694C
RNR2
–
A13528G
ND5
Thr–Ala
A2294G
RNR2
–
C13565T
ND5
Ser–Phe
G3531A
ND1
–
A13614G
ND5
—
C4832T
ND2
–
A16183G
HV1/7SDNA
Noncoding
Base change
Map locus
Amino-acid change
Base change
Map locus
Amino-acid change
(B)
T4216C
ND1
Tyr–His
G13708A
ND5
Ala–Thr
A13105G
ND5
Iso–Val
C13934T
ND5
Thr–Met
A13117G
ND5
Iso–Val
Base change
Map locus
Base change
Map locus
(C)
A73G
HV2/7SDNA
A12308G
ND2
C150T
HV2/OHR/7SDNA
G12372A
ND5
T152C
HV2/OHR/7SDNA
A12612G
ND5
G185A
HV2/OHR/7SDNA
C12813T
ND5
T195C
HV2/OHR
G13368A
ND5
G228A
HV2/OHR/CSB1
A14139G
ND5
T239C
HV2/TFX/OHR
C14167T
ND5
A263G
HV2/OHR
G15928A
TT
C295T
HV2/TFY/OHR
C16069T
7SDNA
n(C)303
HV2/CSB2/TFY/OHR
T16093C
7SDNA
n(C)311
HV2/CSB2/OHR
T16126C
7SDNA
C456T
D-loop
A16162G
HV1/TAS/7SDNA
C462T
D-loop
A16163G
HV1/TAS/7SDNA
T489C
D-loop
A16166G
HV1/TAS/7SDNA
C497T
D-loop
n(C)16184
HV1/7SDNA
G499A
D-loop
C16186T
HV1/7SDNA
n(CA)514
D-loop
T16189C
HV1/7SDNA
n(C)568
D-loop
T16209C
HV1/7SDNA
G709A
RNR1
T16224C
HV1/7SDNA
T1189C
RNR1
C16256T
HV1/7SDNA
A1438G
RNR1
C16286T
HV1/7SDNA
A1811G
RNR2
C16294T
HV1/7SDNA
G1888A
RNR2
T16304C
HV1/7SDNA
A2706G
RNR2
T16311C
HV1/7SDNA
G3010A
RNR2
A16343G
HV1/7SDNA
T3290C
TL1
T16356C
HV1/7SDNA
A3480G
ND1
T16362C
HV1/7SDNA
G3915A
ND1
G16390A
7SDNA
T4646C
ND2
C16400T
7SDNA
T4703C
ND2
A16482G
7SDNA
A4727G
ND2
T16519C
7SDNA
A4769G
ND2
The abbreviations for the map loci are detailed in MITOMAP.
a
All changes are homoplasmic and occur in both tumour and perilesional skin except
this heteroplasmic somatic mutation.
). In those cases where a homoplasmic base substitution was detected in the tumour,
the same DNA change was also observed in both the perilesional dermis and epidermis.
This contrasts with the distribution of the deletions in these samples, as described
in the previous section.
Unlike the homoplasmic single base changes, the single heteroplasmic base substitution
was detected in the tumour only (i.e. SCC from a 77-y-old patient). This is particularly
interesting, as it is strongly suggestive of a somatic mutation. It has been previously
shown that approximate levels of heteroplasmy can be determined from the sequence
electropherogram (Taylor et al, 2001). Using this methodology, an approximate value
of 70% mutant mtDNA in the SCC sample was estimated. The heteroplasmic change is a
C to T transition at np 438 in the noncoding D-loop (Figure 3
Figure 3
Somatic heteroplasmic mutation in skin mtDNA from a 77-y-old SCC patient. A heteroplasmic
C→T mutation at np 438 was identified in the tumour only. The matched histologically
normal perilesional epidermis and dermis show only the wild-type sequence.
), and furthermore it has not been previously reported (MITOMAP). Similarly, 13 of
the 81 homoplasmic base changes have not been previously reported (
Table 1A). Three of these homoplasmic changes were located in the noncoding D-loop
and 10 in the coding region. Two of these 10 single base transitions occur in the
same BCC patient and alter amino acids within the ND5 subunit of the complex I. The
A to G transition at np 13528 changes a threonine to an alanine, whereas the C to
T transition at np 13565 alters a serine to phenylalanine.
Five of the 68 previously reported base changes (MITOMAP) alter an amino acid in the
ND5 (four out of five changes) and ND1 (one out of five changes) genes (
Table 1B). The T to C transition at np 4216 in the ND1 gene was in a BCC (89 y) as
well as in an SCC (76 y) patient. Two of the four base transitions within the ND5
gene at np 13105 and 13708 (
Table 1B) are both present in the same BCC patient (76 y). The two remaining base
transitions, at np 13117 and np 13934, were observed in a BCC patient (88 y) and an
SCC patient (70 y), respectively. Details of the 63 reported base changes that do
not alter an amino acid are summarised in
Table 1C. Further analysis of these and the other homoplasmic base changes reported
in
Table 1 showed a lack of correlation with age or category of NMSC (results not shown).
DISCUSSION
For the first time in the skin cancer literature, we have presented a detailed study
of the distribution of multiple forms of mtDNA damage in NMSC.
The spectrum of large-scale mtDNA deletions
This has been rarely investigated fully in previous tumour studies and certainly not
in skin (Penta et al, 2001). A total of 20 different deletion species were observed
in the major deletion arc and four species were observed in the minor deletion arc.
This contrasts markedly with a single study in colorectal carcinomas, which reported
an absence of large-scale deletions using a similar long PCR strategy (Penta et al,
2001). Although the absolute sample numbers are small, our data clearly show that
more deletions occur in the skin from BCC rather than SCC patients. This may be explained
through different selection pressures exerted on the deletions. For example, part
of the process by which precursor lesions progress to SCCs may represent a mtDNA mutation
bottleneck (reviewed in Chinnery et al, 2000) where deleterious mutations are selected
against. Within each patient, the lowest number of differently sized deletions was
observed in the epidermis, which agrees with our previous study that compared dermis
with epidermis from normal skin (Ray et al, 2000). Interestingly, in contrast to the
distribution of the homoplasmic base substitutions described in our study, the observed
pattern of deletions in the tumour was often very different compared to that observed
in the matched perilesional skin. This may be suggestive of different mutation and
segregation events thereby giving rise to different deletion spectra between the tumour
and perilesional skin. Interestingly, one of these deletions, derived from the minor
deletion arc and approximately 4 kb in size, was present in every skin sample that
harboured a deletion. The identity of this deletion may be the 3895 bp species that
has been reported in the minor deletion arc spanning nucleotides 547–5443 (MITOMAP),
and is associated with Kearns Sayre Syndrome and Chronic Progressive External Ophthalmoplegia.
The 4977 bp common mtDNA deletion and tandem duplications
The common deletion has been reported in 52% of 32 gastric carcinomas, which is in
marked contrast to the low incidence of large-scale deletions in other tumours (Penta
et al, 2001). The authors of the gastric study have explained their observations by
the use of a more sensitive PCR technique, which detects low levels of the common
deletion (Maximo et al, 2001). It is well known, however, that studies of low levels
(i.e. typically <0.2%) of mtDNA deletions can be confounded by the effects of ageing
(Birch-Machin et al, 1998; Nagley and Wei, 1998; Chinnery et al, 1999). This scenario
was avoided in our study by simply presenting only high levels (>2%) of the common
deletion. We have previously used this approach to show that this threshold value
does not reflect chronological ageing (Birch-Machin et al, 1998).
The present study is the first quantitative investigation of the incidence of the
common deletion in tumours. Higher deletion levels in tumour were observed for three
of the four patients who harboured high levels of the deletion in both tumour and
perilesional skin. The observed pattern of the common deletion contrasts once again
with the distribution of the homoplasmic base substitutions between tumour and perilesional
skin. Unlike a previous gastric study (Maximo et al, 2001), we could find no evidence
of an inverse relation between the incidence of the common deletion and single base
changes (results not shown).
There has been a suggested link between the presence of the common deletion and tandem
duplications in the D-loop (Brockington et al, 1993). Two of the seven NMSC patients,
who harboured high levels of the common deletion, also contained either a 200 or 260 bp
tandem duplication. To our knowledge, this is the first report of tandem duplications
of this type in tumours. It is known that the tandem duplication encompasses several
regulatory regions, including the light- and heavy-strand promoters, binding sites
for mitochondrial transcription factor 1 and most of the conserved sequence box II.
The significance of these findings is unclear until more work is performed within
the field of tandem duplications and skin (Krishnan and Birch-Machin, 2002).
MtDNA base changes
A total of 81 homoplasmic and one heteroplasmic single base changes were identified
in the skin from the NMSC patients. Perhaps, the most interesting of these is the
previously unreported heteroplasmic transition at np 438 that was observed in an SCC
but was absent from the histologically normal perilesional tissue. This is highly
suggestive of a somatic mutation of the kind that has been previously reported in
other tumour types (Penta et al, 2001). The somatic mutation occurs within a region
of the D-loop, which encompasses the origin of H-strand replication, the light-strand
promoter and a binding site for mitochondrial transcription factor 1. This suggests
that the base alteration at np 438 may have a profoundly deleterious effect on mitochondrial
function. The mechanism by which the heteroplasmic state of the np 438 has reached
a value of 70% is unknown. It has been shown that a selective cellular growth advantage
can be conferred by mutations in the mitochondrial genome that can result not only
in a high level of heteroplasmy but also homoplasmy for the mutation (Polyak et al,
1998). A contrasting model has recently been put forward to explain this phenomenon
(Coller et al, 2001). The authors suggest that high levels of a heteroplasmic, and
indeed a homoplasmic, mitochondrial mutation in a tumour can result from random segregation
of mutant genomes in the many cell generations that occur during tumour development.
Each of the 81 identified homoplasmic substitutions were observed both in the tumour
and perilesional skin. In all, 13 of these 81 changes are unreported in the MITOMAP
database. More than half (44 out of 81) of the single base changes was observed in
the noncoding D-loop region. There are several possible explanations for the presence
of these 81 homoplasmic changes in both tumour and perilesional skin. The first explanation
may simply be benign polymorphisms completely unrelated to the tumour formation. In
support of this, it has been suggested that sequencing the mitochondrial genomes of
random Europeans would reveal approximately 10–12 single base changes between individuals
(personal communication, Dr Neil Howell, MitoKor, San Diego, USA). An alternative
explanation may be the fact that all the skin samples used in our study are taken
from constant sun-exposed body sites and as such will have potentially received the
same mutagenic dose of UVR. This could be resolved through sequencing mtDNA isolated
from sun-protected sites, but current ethical permission for the project does not
allow the recall of the skin cancer patients for this intended purpose.
We have identified seven base changes that introduce an amino-acid change within the
genes for ND5 (n=6) and ND1 (n=1). Two of these at np 4216 (ND1) and 13708 (ND5) have
been correlated with Lebers Hereditary Optic Neuropathy (MitoAnalyzer; MITOMAP, National
Institute of Standards and Technology, Gaithersburg, MD, USA). In addition, two further
base substitutions at np 13528 and 13565 were previously unreported, and both of these
alter an amino acid with a polar side chain to one with a nonpolar side chain. The
functional significance, if any, of these alterations is at present unclear, particularly
as these amino acid changes are present both in the tumour and the perilesional skin.
However, like all the other previous mtDNA and cancer studies in the literature, the
data are derived from automated DNA sequencing that will reliably detect mutant loads
only as low as 30% (Taylor et al, 2001). Therefore, below the 30% threshold value
there might be differences in mutagenic load between tumour and perilesional skin.
This is important for two reasons. First, there is a threshold effect that describes
the critical ratio between wild-type and mutant mtDNA, which must be exceeded before
phenotypic expression of the mutant becomes apparent. For protein coding mtDNA genes,
such as those described in our study, the threshold value is around 70% and above
(Chomyn et al, 1992). Secondly, given the large number of cell divisions necessary
for tumour development, estimated at 600 by Coller et al, (2001), an alteration in
mitochondrial function that is exceedingly subtle in terms of its biochemical or physiological
manifestation may be all that is necessary to alter significantly the probability
of tumour formation (Augenlicht and Heerdt, 2001).
An increased incidence of C insertions/deletions, within a homopolymeric C-stretch
between np 303 and 315 of the D-loop (i.e. D310 tract), has been recently reported
in several cancers (Parrella et al, 2001; Sanchez-Cespedes et al, 2001). These studies
have proposed that instability in the D310 tract may be a useful diagnostic marker
for cancer development. In skin however, we have observed that instability in the
D310 tract is present not only in the tumour but also in the histologically normal
perilesional skin. Both skin sites receiving the same mutagenic dose of UVR may explain
this phenomenon. However, D310 instability is observed regularly in mitochondrial
genomes from noncancerous skeletal muscle and brain (personal communication, Dr R
Taylor, Mitochondrial Gene Therapy Group, Newcastle, UK). Given these observations,
more work is therefore needed before D310 instability can be claimed to be a reliable
and universal diagnostic marker for cancer.
In conclusion, we provide the first detailed study of the distribution of multiple
forms of mtDNA damage in NMSC and histologically normal perilesional tissue. We present
the first entire spectrum of deletions found between different types of skin tumours
and perilesional skin. In addition, we provide the first quantitative data for the
incidence of the common deletion as well as the first report of specific tandem duplications
in tumours from any tissue. This work shows that there are differences in the distribution
of deletions between the tumour and the histologically normal perilesional skin. DNA
sequencing of four mutation ‘hotspot’ regions of the mitochondrial genome identified
a previously unreported somatic heteroplasmic mutation in an SCC patient. In addition,
81 previously unreported and reported homoplasmic single base changes were identified
in the other NMSC patients. Unlike the distribution of deletions and the heteroplasmic
mutation, these homoplasmic mutations were present in both tumour and perilesional
skin. This suggests that the traditional use of histologically normal perilesional
skin in NMSC studies may have several limitations. This is important when one considers
that the majority of studies involving nuclear DNA damage and skin cancer/skin disease
often use perilesional skin as a control tissue. Currently, it is unclear whether
mtDNA damage has a direct link to skin cancer or it may simply reflect an underlying
nuclear DNA instability.