The comet assay is traditionally used to detect DNA damage (Ostling and Johanson,
1984; Fairbairn et al, 1995), and has been modified to detect more complex DNA lesions
(Collins et al, 1993; Duthie and McMillan, 1997; McKelvey-Martin et al, 1998). We
have described the combination of the alkaline comet method with pulse labelling of
replicating DNA with the thymidine analogue bromodeoxyuridine (BrdUrd), and immunolocalisation
of BrdUrd within the heads and tails of comets, so as to assess replicative integrity:
that is, the efficiency of completion of DNA replication on a single cell basis (McGlynn
et al, 1999). A label incorporated into DNA during a brief pulse of BrdUrd is initially
close to strand discontinuities, and is seen as a fluorescently labelled comet tail
upon electrophoresis. During pulse-chase experiments, as the replicating forks move
away from the labelled regions, the label is incorporated into high molecular weight,
continuous DNA and is observed as a comet head. The % BrdUrd label in the comet tail
is therefore indicative of the proportion of recently replicated, imperfectly matured
DNA in the nucleus. Using this BrdUrd comet assay, we were able to confirm that the
known hypersensitivity of SVM84 cells to UV irradiation is because of a deficiency
in UV postreplication repair (Pillidge et al, 1986; McGlynn et al, 1999). Although
postreplication repair in this case, and in other mammalian cells, has been studied
by biochemical methods (Brown, 1980; Clark and Hanawalt, 1984), these techniques are
time-consuming, and require both radioactive labelling of DNA and the use of quite
large cell populations (ca 105 per data point) for which only an average figure can
be obtained. The BrdUrd comet assay, on the other hand, can be simply and reproducibly
performed using very small numbers of cells, and gives measurements on individual
cells (McGlynn et al, 1999). We now describe the application of the BrdUrd comet assay
to small numbers of epithelial cells derived from very small human biopsies.
Replication error positive (RER+) colorectal tumorigenesis occurs in individuals with
hereditary nonpolyposis colorectal cancer (HNPCC), accounting for up to 5% of all
colorectal cancer cases (Lynch et al, 1993) and in some sporadic tumours. How far
replicative integrity and DNA maturation is defective in precancerous colon tissue,
or varies between individuals, is unknown. The development of a method for the disaggregation
of small human endoscopic biopsies, and the assessment of DNA metabolism at the single
cell level, is of increasing importance. Here we describe such a method, together
with pilot BrdUrd comet data from normal and diseased colonic mucosa. This development
deems the BrdUrd comet assay suitable as a molecular end point that can be measured
easily, hence facilitating trial investigations of diet- or environment-related factors
affecting colorectal cancer.
MATERIALS AND METHODS
Tissue collection
Colonic biopsy specimens were obtained at endoscopy under a protocol approved by the
ethics committees of the Federated Voluntary Dublin Hospitals. The study was approved
by the ethics committee of the University of Ulster. Prior to endoscopy, patient details
including age, sex and medical history were recorded (Table 1
Table 1
Details of patients specifically mentioned in the text including those diagnosed as
histologically abnormal. Histological details of the study specimens are given together
with the histological patient diagnosis following tissue analysis for clinical purposes
Pt ID
Sex/age
FM Hx/diagnosis/current scope
Histology
Comet biopsy
4
M/70
Rectal tumour
Adenoma moderate dysplasia, Dukes' B or
D9B1 tumour
T3NOMX
D9B2 normal adjacent tissue
18
M/73
Diarrhoea
Routine bx – otherwise normal
Normal dsc
Laxative atonic gut–some tissue changes
23
M/70
Polyp r/v '98 Polyps in trans+divert dx
Tissue-trans-adenom-dysplastic-invasive
Normal dsc
25
No details
No details
No details
Site 1: normal asc
Site 2: normal dsc
26
F/45
No details
No details
Site 1: normal asc
Site 2: normal asc
28
M/67
Adenomatous mucosa polyps
Polyp-dsc-mod dys-adenomatous Dsc tissue–mild dysplasia
Normal dsc
29 Follow-up from 28
M/68
Polyp r/v This scope-polyp found at 50 cm (dsc/sig)
Routine bx –polyp base-normal histology
Normal sigmoid
35 Rpt of 23
M/71
Tubular adenoma in ileocaecal valve
Adenoma with low grade dysplasia
Site 1: adenoma
Site 2: adjacent normal tissue
Site 3: 10 cm asc colon – normal tissue
37
M/57
Normal
None done
Normal asc
Normal trans
Normal dsc
41
F/73
Fmhx crc Divert, HT
Polyp sigmoid-abnormal-tubular adenoma
Site 1: normal tissue distal sigmoid 10 cm
+ mild dysplasia
Site 2: adenomatous rectosigmoid junction
Site 3: normal tissue rectum 10 cm distal
Samples classified as normal are biopsies collected from subjects with no evidence
of malignancy. Samples classified as abnormal are biopsies taken from polyp or adenoma
tissue.
Asc=ascending, dsc=descending, caec=caecum, trans=transverse, r/v=review, divert=diverticulitis,
dx=diagnosis, fm hx=family history, pr bleeding=peri-rectal bleeding, oeso=oesophagus,
bx=biopsy, dys=dysplasia, crc=colorectal cancer.
).
Isolation of epithelial cells from small colonic biopsies
Epithelial cell isolation was carried out using a method derived from that described
by Meenan et al (1997). Briefly, two to four endoscopic biopsies from each area of
interest were taken into calcium- and magnesium-free Hank's balanced salt solution
(Life Technologies, Paisley, Scotland) supplemented with 0.3% bovine serum albumin
(0.3%) and antibiotics (HBSSsup) and then transferred into HBSSsup containing 100 μ
M BrdUrd at 37°C for 15 min. Subsequently, the specimens were processed for epithelial
cell disintegration immediately or, in chase experiments, transferred into medium
containing each of 200 μ
M thymidine, 2′-deoxycytidine, 2′-deoxyguanosine and 2′-deoxyadenosine (Sigma, Dorset,
UK). Biopsies were then transferred to fresh HBSSsup containing 0.75 mM dithiothreitol
(Sigma, Dorset, UK) and were allowed to stand at room temperature for 3 h before being
transferred to fresh HBSSsup containing 2 mM EDTA and placed on a rotating table,
inclined at 45°, for 1 h at 37°C. The resulting suspension was washed and pelleted
in HBSS, and after assessment of viability using ethidium bromide/acridine orange,
cells were suspended stored in phosphate-buffered saline, at a concentration of 2
× 105 cells ml−1, prior to comet slide preparation.
The purity of the cell preparation was confirmed using flow cytometry and a monoclonal
antibody for the detection of epithelial cell antigen (Ber-Ep-4; Dako, Glostrup, Denmark)
as described (Meenan et al, 1997) (data not shown).
Cell culture
The established colonic cell line SW620 (Papadoupolous et al, 1995) was purchased
from the ATCC (Rockville, MD, USA) and maintained in L-15 medium supplemented with
10% foetal bovine serum and antibiotics in a humidified incubator at 37°C, 5% CO2.
Prior to BrdUrd pulsing (20 μ
M for 20 mins), cells were plated at a density of 5 × 104 cells per 60 mm dish for
48 h. For chase experiments, cells labelled with BrdUrd were incubated for 1 h in
complete medium containing each of 200 μ
M thymidine, 2′-deoxycytidine, 2′-deoxyguanosine and 2′-deoxyadenosine (Sigma, Dorset,
UK). BrdUrd incorporated into DNA during a brief pulse is initially close to strand
discontinuities in the comet tail, but during pulse-chase experiments in cells capable
of normal DNA maturation, the BrdUrd is incorporated into high molecular weight, continuous
DNA represented by the comet head.
Comet slide preparation and BrdUrd comet assay
The BrdUrd comet assay was performed as previously described (McGlynn et al, 1999).
Briefly, cells were washed in cold PBS, and the cell pellet was resuspended in 100 μl
of 0.75% low melting point (LMP) agarose at 37°C. The cell suspension was spread onto
a standard comet assay slide (Singh et al, 1988) and lysed in a neutral lysis buffer
containing 0.1% LiDS, pH 8.0, and 0.03 mg ml−1 proteinase K, at 37°C overnight followed
by alkaline lysis, pH 10 for 1 h at 4°C.
DNA unwinding (40 min) and electrophoresis (20 min at 25 V and 300 mA) were carried
out under alkaline conditions (0.3 M NaOH, 1 mM EDTA, pH 13). Following electrophoresis,
the gels were neutralised (0.4 M Tris) and washed with PBS prior to immunostaining.
The gels were incubated with 25 μl per gel of mouse monoclonal anti-BrdUrd (10 μg ml−1;
BO Biosciences, Oxford, UK) in the dark at room temperature for 1 h. The primary antibody
was gently washed off with three changes of PBS and one wash with PBS/0.1% BSA, before
the addition of 25 μl per gel of secondary antibody (5 μg ml−1 sheep anti-mouse IgG,
fluorescein conjugated: Bioscience, Oxford, UK), which was incubated and washed off
as before. The gels were then counterstained with 25 μl of propidium iodide (0.75 μg ml−1;
Sigma, Dorset, UK) and covered with a coverslip (22 × 50 mm2) for image analysis.
Comet analysis
For biopsy specimens, comet slides were analysed using a single-blind approach in
that patient details were not made available until comet analysis was completed. Comet
analysis was carried out as previously described (McGlynn et al, 1999) using a final
magnification of × 400 (Nikon × 40 Fluor lens) and Komet 4.0 software (Kinetic Imaging
Ltd, Liverpool, UK). The % DNA in the comet tail for each cell was measured. Results
were expressed as mean % comet tail DNA in 25 cells on each of duplicate comet slides.
Data are collected as the mean and standard deviation of the 50 comets scored per
individual patient (as shown in Figure 1
Figure 1
Reproducible BrdUrd comet measurements from cells derived from human colonic biopsies.
Results are presented as mean % BrdUrd comet tail DNA with patient ID numbers given
in the legend. Representative examples are shown demonstrating the reproducibility
of adjacent sites in the ascending colon, patient ID 26, and of different sites in
the colon; ascending vs descending, patient ID 25; ascending vs transverse vs descending,
patient ID 37. Follow-up data for one patient is also presented comparing the initial
sample (patient ID 28) with a sample taken 4 months later (patient ID 29).
). The variance or dispersion from the mean of % tail DNA of 50 comet measurements
from each duplicate pair of slides was also noted.
RESULTS
Mucosal epithelial cell isolation
Using the ion-chelation method described, an epithelial cell population of greater
than 95% purity was obtained, as assessed by flow cytometry using epithelial cell
antigen positive expression. Contaminating lymphocytes could be excluded on the basis
of size during comet analysis.
Cell yield ranged from 1.0 to 2.0 × 105 cells per single biopsy. Cell viability, as
assessed using acridine orange and ethidium bromide, ranged from 60 to 80%, and was
found to be much greater than that found using enzymatic digestion, either alone or
in combination with ion chelation. Specimens from the ascending colon and caecum produced
higher cell yields and greater viability.
Highly reproducible comet data demonstrates little variation of replicative integrity
among normal subjects
Inter- and intrapatient reproducibility of the BrdUrd comet assay was assessed. Comet
measurements on duplicate endoscopic biopsies, which were collected from up to three
different colorectal sites in each patient (n=36 sites), showed no significant difference
between sites, demonstrating the reproducibility of the BrdUrd comet assay applied
to colonic biopsy cells (Figure 1). Furthermore, a sequential follow-up specimen was
analysed from one patient undergoing adenomatous polyp review. Upon first analysis,
the patient displayed dysplastic polyps and had a mean % comet tail of 50±11 in comets
derived from normal descending colon, upon reanalysis 4 months later, the patient
was again diagnosed with adenomatous polyps and had a mean % comet tail of 53±9 in
comets derived from histologically normal sigmoid tissue (Figure 1). This indicates
that follow-up clinical studies using the BrdUrd-comet assay may be possible. Further
statistical analysis is necessary to confirm these observations. No significant difference
in mean % comet tail was observed between duplicate comet slides (results not shown).
A total of 52 biopsies were collected from 35 patients who displayed no evidence of
malignancy (normals) upon endoscopic investigation. Little variation in mean % BrdUrd
comet tail (mean comet tail of 52 biopsies 46, s.d. 7.1) was observed among this sample
group (Figure 2
Figure 2
BrdUrd comet analysis of colonic mucosal cells disaggregated from human colonic biopsies.
Results presented as a scatterplot of mean % BrdUrd comet tail DNA. Patient ID numbers
(n=46) are given in the legend and correspond to patient details in Table 1. Multiple
samples from the same patient are labelled a, b and c.
).
Aberrant BrdUrd comets of colon cells from subjects with colonic polyps or adenoma
Pilot data revealed that biopsies of polyp or tumour tissue have a significantly higher
mean % comet tail DNA (mean of 63±2.7, collective comet data for n=4, P<0.001) compared
to those from tissue collected from normal subjects (collective comet mean 46±7.1,
n=52 normal samples) (Figure 2). This indicates a detectable deficiency in DNA replication
in these samples.
The abnormal samples included tumour tissue from a Dukes stage B invasive rectal adenoma
(patient ID 4), polyp tissue from a patient diagnosed with tubulovillus adenoma with
mild dysplasia (patient ID 3), adenomatous tissue from a tubular adenoma in the ileocaecal
valve (patient ID 35) and adenomatous tissue from a tubular adenoma in the rectosigmoid
junction (patient ID 41). Histologically normal tissue from patients with polyps/adenoma
(n=15) also showed a trend towards an elevated mean % comet tail DNA compared to normals
in most cases (collective comet mean of 50±7.1, n=15), although these results did
not reach significance.
BrdUrd comet analysis of multiple samples from patients with malignancy
Multiple biopsies were collected from various sites within the colon of patients diagnosed
with malignancy in order to assess the degree to which the deficiency in DNA replication
found in histologically abnormal samples extends into the histologically normal tissue
surrounding a polyp/adenoma. In the first patient (patient ID 35), biopsies were collected
from a tubular adenoma of the caecum and from histologically normal tissue both adjacent
to and 10 cm away from the adenoma. Cells from both the adenomatous tissue and the
adjacent histologically normal tissue showed elevated comet measurements as distinct
from the tissue 10 cm away, which showed normal comet measurements (Figure 3
Figure 3
Evidence of aberrant BrdUrd comet analysis in tumour and adjacent normal colon but
not in distal normal colon. Biopsies were collected from multiple sites – from the
adenoma, histologically normal-appearing mucosa adjacent to the adenoma and histologically
normal-appearing mucosa 10 cm away from the adenoma – and disaggregated before pulsing
with BrdUrd for 15 min and processing for the BrdUrd comet assay. Results are expressed
as frequency distribution plots of BrdUrd percentage of tail DNA and mean + standard
deviation, s.d.
, patient ID 35).
Similar results were found in a second subject (Figure 3, patient ID 4), where the
normal adjacent tissue showed similar elevated comets to that of the rectal adenomatous
tumour. Interestingly, the rectal tumour tissue displayed both an elevated mean %
comet tail DNA and a high variance in % tail DNA (variance=360 compared to overall
average variance for normal samples of 219±88, n=52 samples). On the other hand, while
the histologically normal tissue adjacent to the tumour showed a mean % comet tail
DNA similar to that of the tumour, it did not display observable comet variation (variance=232).
This variability in comet formation was seen in head/tail DNA content, tail length
and degree of BrdUrd incorporation, which is possibly indicative of the high degree
of tumour anaplasia (Figure 4
Figure 4
Variable appearance of BrdUrd comets derived from rectal tumour biopsy compared with
adjacent colon tissue. BrdUrd comet images from a single slide prepared from a rectal
tumour (A) compared with those from a single slide prepared from an adjacent normal
tissue (B). The tumour-derived comets show increased variation in comet appearance
in relation to head/tail DNA content, tail length and degree of BrdUrd incorporation
as compared with the more homogeneous normal comets.
, patient ID 4).
In a third patient diagnosed with tubular adenoma and mild dysplasia at the rectosigmoid
junction (Figure 3, patient ID 41), samples were collected from the adenomatous tissue
and from two sites 10 cm distal to the adenoma in the sigmoid colon and rectum. As
with the data from patient ID 35, the adenomatous tissue displayed an elevated % comet
tail DNA, but the two sites distal from the adenoma showed reduced comet tails (Figure
3, patient ID 41).
Defective DNA replicative integrity is detectable using BrdUrd pulse-chase in colonic
cells
For pulse-chase experiments, entire mucosal biopsies were transferred to chase medium
following BrdUrd labelling. To calibrate the assessment of DNA replicative integrity
using BrdUrd comet pulse-chase experiments, a transformed colonic cell line was used
(SW620; Figure 5
Figure 5
BrdUrd pulse-chase in colonic cells reveals inherent defects in DNA replicative integrity.
BrdUrd comet analysis of colonic mucosal cells disaggregated from human colonic biopsies
and control cultured cell lines. Cells were given a pulse of BrdUrd and either processed
immediately for the BrdUrd comet assay (A) or chased with 200 μM of all four deoxynucleotides
for 1 h prior to comet processing. (B) SW620 colon cell line, (C) normal colonic mucosa
(patient ID 18) and mucosal cells from a patient presenting with adenomatous polyps
and moderate dysplasia (patient ID 29). Results are expressed as frequency distribution
plots of BrdUrd percentage of tail DNA labelled as BrdUrd pulse alone or BrdUrd pulse
with chase.
) which showed some DNA maturation after the chase period. Mucosal cells derived from
a normal biopsy (patient ID 18) displayed more proficient DNA maturation after a chase
period of only 1 h (P<0.01). However, mucosal cells from a single patient presenting
with adenomatous polyps and moderate dysplasia (patient ID 29) demonstrated a significant
delay in DNA maturation compared to patient ID 18 (P>0.05, t-test), evidenced by a
failure of the comet tails to move into the head (Figure 5).
DISCUSSION
There is substantial evidence that the formation of a tumour is preceded by a series
of somatically inherited changes, that is mutations or epigenetic events such as DNA
methylation (Baylin, 1997; Bernstein et al, 2000). These changes lead to abnormalities
of cell growth such as increased cell proliferation and genomic instability. The identification
of these early molecular defects or markers in cells with normal morphology is important
for identifying subjects at high risk of developing cancer. These molecular markers
may also be used as biomarkers in studies to identify risk factors for carcinogenesis.
We have recently developed a novel assay for the detection of DNA synthesis and maturation
in single cells, which we have calibrated with cell lines grown in culture (McGlynn
et al, 1999).
Molecular studies of colon carcinogenesis have been performed using measures of proliferative
compartments of colonic crypts (Terpstra et al, 1987; de Leon et al, 1988), ploidy
assessment of colonic carcinomas (Jarvis et al, 1987; Cianchi et al, 1999) and cell
cycle kinetics of colonic cells in vivo using flow cytometry (Michel et al, 2000).
Each of these methods indicates that faulty cell division in the colon occurs in regions
adjacent to polyps or carcinomas, and is therefore taken to be responsible for the
progression from early neoplastic lesions to the development of an adenomatous polyp
to an invasive carcinoma. (Strictly speaking, these data are equally compatible with
the hypothesis that cells in the later stages of carcinogenesis exert a malign influence
on the surrounding, normal tissues, an issue discussed below.)
The novel BrdUrd comet is entirely separate from DNA methodologies measuring BrdUrd-labelled
DNA with flow cytometry, in that it measures precisely the efficiency of DNA maturation
in colonic cells, whether from histologically normal or abnormal tissue. An increase
in comet tail length is indicative of a delay in DNA maturation, because of stalling
of the replication fork at DNA lesions or defects in postreplicative repair.
In the present study, the BrdUrd comet assay was successfully applied to single colonic
epithelial cells derived from disaggregated human biopsies; we show that defective
DNA replication may be suitable as a molecular marker for early stages of colon carcinogenesis.
The comet assay was originally designed to detect DNA damage; therefore the choice
of method for tissue disaggregation to maintain cell viability and intact DNA is imperative.
In this study, the ion-chelation method of biopsy disaggregation proved superior to
enzymatic digestion on the basis of combined cell yield and viability (two-fold in
each case). Minimal DNA damage was induced in cells isolated in this way compared
with enzymatic digestion as determined by propidium-iodide-labelled comet tails. This
ion-chelation method was adapted from that described in detail by Meenan et al (1997)
and designed to isolate epithelial colonocytes from the heterogeneous population of
cells of the intestinal mucosa. We have demonstrated that the BrdUrd comet assay is
suitable for the measurement of DNA maturation and integrity in very small numbers
of primary epithelial cells derived from biopsy specimens of human colonic mucosa.
By exposing the cells to a short pulse of BrdUrd alone and measurement of BrdUrd comet
tails, much information regarding the status of DNA maturation can be gained. The
assay was shown to be reproducible in that duplicate specimens of colonic mucosa gave
very similar results regardless of the site of biopsy. This was shown to be the case
in both normal mucosa and in biopsied adenomatous polyp tissue (Figure 1). Furthermore,
results from a cohort of histologically normal colonic tissue samples showed a low
level of variability within this population (Figure 2). On the other hand, this study
shows that it is possible to detect elevated BrdUrd comets from histologically abnormal
tissue (rectal tumour, polyp tissue from a patient with tubulovillus adenoma and tubular
adenomas) when compared with that from normal mucosa, by an increase in % tail DNA
(P<0.001), indicative of stalled maturation (Figure 2). This may be because of a high
level of intrinsic DNA damage and/or defective postreplication repair machinery in
the tumour cells (Johnson et al, 1994).
Interestingly, increased BrdUrd comet tails were also found in samples of histologically
normal tissue from patients with an abnormal diagnosis, although these data did not
reach statistical significance. This would be compatible with a decrease in replicative
integrity in histologically normal tissue with an underlying molecular defect and
progression towards malignancy, or, as mentioned above, with a malign effect of tumour
cells on their neighbours. This is supported by data from patients IDs 23 and 35,
diagnosed with tubular adenoma (Figure 3), where there is an increase in the mean
% BrdUrd comet tail of histologically normal tissue upon follow-up investigation.
In order to investigate further the role played by replicative status in tumour formation
and progression in the colon, multiple biopsies were examined from neoplastic tissue,
histologically normal tissue adjacent to the tumour and normal tissue distal to the
tumour from patient IDs 4, 35 and 41. Interestingly, elevated BrdUrd comets were found,
not only in the tumour but also in the adjacent tissue, whereas normal levels were
found in the tissue 10 cm distal to the tumour (Figure 3). Although the Dukes' stage
B rectal tumour and adjacent tissue of patient ID 4 both displayed elevated BrdUrd
comets, a high degree of variability in comet appearance was associated with the tumour
only, possibly reflecting the high degree of anaplasia of the tumour, not yet seen
in the adjacent tissue (Figure 4).
Taken together, these results support the hypothesis that the underlying defective
replication exists in histologically normal colonic tissue, which may predispose to
neoplasia, or, conversely, that neoplastic tissues malignly affect normal neighbouring
cells. Further, these results are in agreement with previous findings showing changes
in cell proliferation in normal mucosa of colorectal cancer patients (Terpstra et
al, 1987) and may indicate that aberrant replicative integrity is an early event in
neoplasia in premalignant cells. It is possible that the adenomas in patient IDs 35
and 41 developed in an area of the colon with replicative abnormalities but that defective
replication was not a colon-wide phenomenon.
A further development of the BrdUrd comet assay is the introduction of a pulse-chase
step, to allow the maturation of DNA over time (McGlynn et al, 1999). A reduction
in tail DNA was evident in normal mucosal epithelial cells in a 1-h chase period,
which was more efficient than in either epithelial cells taken from a dysplastic mucosa
(patient ID 29) or in a cultivated colon tumour line (Figure 5). These results further
support the ability of the BrdUrd comet assay to assess DNA maturation and determine
replicative integrity in human colonic biopsies.
The present data on the application of the BrdUrd comet assay demonstrate that DNA
maturation and postreplicative repair can now be determined in small numbers of cells
derived from human colonic biopsies taken during routine endoscopy. Studies to investigate
the effects of diet and other factors on DNA replication in the colon, using this
assay, are under way.