Associations between tissue-specific DNA alkylation, DNA repair and cell proliferation in the colon and colon tumour yield in mice treated with 1,2-dimethylhydrazine
Peta E. Jackson1,2,
Peter J. OConnor1,
Donald P. Cooper1,3,
Geoffrey P. Margison1 and
Andrew C. Povey1,4
1 Cancer Research UK Carcinogenesis Group, Paterson Institute for Cancer Research, Manchester, M20 9BX, UK
2 Current address: Department of Environmental Health Sciences, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, MD 21205, USA
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Abstract
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Putative risk factors (DNA damage) and risk modifying factors (DNA repair and cell proliferation) were examined in an experimental mouse model in which treatment with dimethylhydrazine (6.8 mg/kg DMH i.p. once weekly) for up to 20 weeks induces colon tumours in a site specific manner with 0, 43 and 87% of animals having proximal, mid and distal colon tumours respectively at the highest cumulative dose. Levels of the pro-carcinogenic DNA adduct, O6-methylguanine (O6-MeG), in colonic DNA were found to vary with time after final treatment and with location within the colon but not with total DMH dose. O6-MeG levels were generally lowest in proximal colon DNA and highest in distal colon DNA. Steady state O6-MeG levels were obtained at the highest cumulative DMH dose with O6-MeG levels in mid and distal colon DNA being 5 and 10 times higher those in proximal colon DNA. O6-alkylguanine-DNA alkyltransferase (MGMT) activity, and cell proliferation indices in the colon were also found to vary with time after final treatment but not with either location within the colon or total DMH dose. O6-MeG levels, MGMT activity and cell proliferation indices at specific time points as well as basal MGMT activity were not associated with differences in tumour yield within the colon. However tumour yield was associated with the cumulative amount of O6-MeG present in DNA over the treatment period and with the treatment induced cumulative increase in cell proliferation, particularly within regions of the colon crypt where stem cells reside but not with cumulative changes in MGMT activity. Results are consistent with an increased cancer risk arising from an increased mutation load in the target stem cell population due to increased adduct formation/persistence and cell proliferation but also suggest that other cell specific factors may help to determine tumourigenic response.
Abbreviations: DMH, dimethylhydrazine; O6-MeG, O6-methylguanine.
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Introduction
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The induction of colon tumours by dimethylhydrazine (DMH) is a widely studied experimental model because of a number of factors including the pronounced organotropic properties of the carcinogen (1), the wide variation in tumour response in different rodent strains (25), the dietary induced modulation in tumour incidence (6), the similar distribution of tumours within the human colon and the colon of treated animals (7,8) and the similarity between animal and human pathology (9). Implicit in such work is the assumption that DNA alkylation may also be important in human colon cancer formation (1012) so that by understanding the mechanisms through which DMH induces colon tumours, new approaches to identifying high risk populations or preventing human colon cancer may be developed.
DMH produces predominantly distal colon tumours but at widely varying rates depending upon the strain of the animal (24,1315). This differential susceptibility has been ascribed to variations in the ability to metabolize DMH (16,17) or in proliferative responses to the carcinogen (18). Furthermore a number of susceptibility loci have been reported including Ccs1 (2), Scc1 and Scc2 (3), Scc3-Scc5 (4) and Scc 69 (5) but their biological role remains to be further elucidated. Following DMH administration, susceptible mouse strains can have higher amounts of DNA strand breaks and DNA adducts than non-susceptible strains (19,20) but such observations are not consistently reported (21,22). DNA alkylation is also detected in tissues other than the colon (e.g. liver, kidney) and the specific induction of colon tumours has been ascribed to tissue specific differences in the persistence of certain DNA adducts (21,2325). Other factors such as whether damaged stem cells, at positions 1 and 2 within the crypt, are selectively removed by apoptosis or suffer deleterious mutations may also be important (26). Levels of alkyl adducts in DNA in colon tissue are higher in the areas where colon tumours subsequently appear (27) though intra-colonic differences in baseline proliferative parameters (crypt length, labelling index and proliferative zone) have also been reported and are associated with the tumour formation (27,28). Such potential risk factors have been examined in model systems in which tumour induction is modified by various agents. Reduced tumour incidence has generally been associated with either reduced adduct levels (e.g. 25), decreases in cellular proliferation (e.g. 29,30) or increases in apoptosis (e.g. 29). However, it has also been reported that increased proliferation can be associated with decreased tumour induction (e.g. 31), that tumour yield can be reduced without a reduction in cell proliferation (32) and that cell proliferation can be reduced without a decrease in tumour incidence (33). DMH-induced tumour incidence can also be reduced by supplemental dietary calcium without altering DNA adduct levels, DNA repair activity or cellular proliferation parameters in the rat colon (27).
Such results are generally, but not totally, consistent with a simple model for the induction of tumours by chemical carcinogens that involves the following factors: (a) the formation of DNA adducts within the target tissue and target cells; (b) the lack of repair of DNA adducts by active repair systems; (c) a decrease in cell loss due to apoptosis; and (d) a subsequent round of DNA synthesis which results in the fixation of mutations at the sites of DNA adducts that have persisted. To further clarify the respective roles of DNA alkylation, DNA repair and cell proliferation, in inducing colon tumours, levels of the pro-carcinogenic adduct, O6-methylguanine (O6-MeG) in DNA, the activity of O6-alkylguanine-DNA alkyltransferase (MGMT) which repairs O6-MeG adducts in DNA, and cell proliferation have been measured within the colon following administration of DMH once weekly for up to 20 weeks. This regimen has been shown to induce tumours within the distal colon but not in the proximal colon (34).
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Materials and methods
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Treatment of animals
1,2-DMH.2HCl was dissolved in 0.9% saline containing 1 mM EDTA and 10 mM sodium citrate and the pH adjusted to 6.5 using 0.25 M NaOH. Female SWR mice (89 weeks old, purchased from Harlan Olac, Blackthorn, Bicester, UK) were given once weekly i.p. injections of 15 mg 1,2-DMH.2HCl/kg body weight for either 1, 5, 10 or 20 weeks resulting in total doses of 1,2-DMH of 6.8, 34, 68 and 136 mg/kg respectively. Animal experiments were performed in accordance with the Animals (Scientific Procedures) Act of the UK Parliament, 1986. Groups of five mice were killed 8, 56 and 152 h after the final injection of DMH and the large bowel was removed and divided into thirds (proximal, mid and distal colon). Tissues taken for the measurement of O6-MeG levels and MGMT activity were placed in Eppendorf tubes, frozen on dry ice and stored at 20°C until DNA or tissue extracts were prepared. Saline control animals were given weekly i.p. injections of the vehicle, 0.9% (w/v) saline containing 1 mM EDTA and 10 mM sodium citrate (pH 6.5). Control tissues for measurement of O6-MeG levels were taken at 8, 56 and 152 h after saline treatment: control tissues for MGMT measurement were taken at 56 h after saline treatment.
To determine the extent of DNA synthesis in separate groups of mice, each mouse received 20 µCi of [3H]thymidine (1 mCi/ml) in PBS in a volume of 0.1 ml by i.p. injection 40 min prior to sacrifice. All animals receiving [3H]-thymidine for cell proliferation studies were killed between 46 p.m. to reduce the effect of diurnal variation on this parameter. Tissue samples, from the proximal and distal colon, taken for analysis of DNA synthesis were fixed in 70% (v/v) ethanol overnight before embedding and sectioning.
Quantitation of O6-MeG in DNA
The tissue of interest from mice in each group was pooled and the DNA extracted using a phenol-based method. It was then dissolved in buffer I (50 mM TrisHCl pH 8.3, 1 mM EDTA, 3 mM dithiothreitol). O6-MeG levels were then measured in a recombinant human O6-alkyl-guanine-DNA alkyltransferase inhibition assay using calf thymus DNA methylated in vitro with N-nitroso-N-[3H]-methylurea ([3H]-methylated CT-DNA) as the substrate (35).
Quantitation of MGMT activity
Tissue from the mice within each group was pooled, minced finely on ice, resuspended in 1 ml of ice-cold buffer I containing 5 µg leupeptin/ml and 10 µl phenylmethylsulphonylfluoride (50 mM PMSF in ethanol). The sample was sonicated for 3 x 10 s and a further 10 µl PMSF was added to the sample immediately after the final sonication. The extract was then separated from the cell debris by centrifugation at 15 000 g, 4°C for 10 min and the supernatant transferred to a fresh tube on ice; the MGMT activity was then measured immediately using [3H]-methylated CT-DNA as the substrate (36).
Autoradiography of tissue sections
Paraffin embedded sections (3 µm) were dewaxed in xylene overnight, rehydrated and the slide covered with K5-contact emulsion. After drying, the slides were placed in double light-tight boxes containing silica gel, and exposed at 4°C for 10 days before developing. Sections were counterstained with haematoxylin and eosin. Intact longitudinal sectioned crypts with a lumen were counted: 200 half crypts (40 from each of five mice per group) were scored as labelled or unlabelled beginning at the base of the crypt and ending at the lumen of the colon. Cells with more than five grains over the nuclei were considered to be positively [3H]thymidine-labelled.
Data analysis
The time and dose-dependent variation in O6-MeG levels and MGMT activity were analysed using ANOVA followed by Dunnetts test. Data generated from tissue autoradiography was analysed by the CRYPTS program written by Dr S.Roberts (Biomathematics and Computing Unit, PICR) which provides the percentage labelling index (i.e. the number of labelled cells divided by the total number of cells; %LI) and LI distribution curves (presented smoothed over three cell positions). Time and dose-dependent variations in labelling index were analysed using ANOVA followed by Dunnetts test after calculating the percentage increase in labelling index, i.e. the difference in mean labelling indices following treatment with DMH or saline divided by the control saline labelling index.
To examine associations between subsequent tumour induction and cumulative changes in potential risk factors, the cumulative sums of (i) O6-MeG levels (CSUMO6-MeG); (ii) the difference between MGMT levels in treated and untreated animals (CSUMMGMT); and (iii) the difference between labelling indices in treated and untreated animals (CSUMCPI) and more specifically within crypt cell positions 1 and 2 were calculated in two stages. At each experimental time point (i.e. 1, 5, 10 or 20 weeks which were equivalent to total DMH doses of 6.8, 34, 68 and 136 mg/kg), the area under the curve (AUC) was determined from the change in the variable with the time since the last injection. For MGMT (AUCMGMT) and cell proliferation indices (AUCCPI) the time period examined was 0152 h whereas for O6-MeG (AUCO6-MeG ) the time covered was 8152 h. The cumulative sum at each of the sampling times (1, 5, 10 and 20 weeks) was then calculated as the sum of each individual weekly time course using the individual weekly values obtained at 1, 5, 10 and 20 weeks and assuming that values at weeks 24, 69 and 1119 were equivalent to those at weeks 1, 5 and 10, respectively. For example: cumulative O6-MeG levels for up to 20 weeks =
week 1 (AUC 8152 h) +
week 2 (AUC 8152 h) + ....
week 20 (AUC 8152 h) where
week 2 (AUC 8152 h) =
week 3(AUC 8152 h) =
week 4(AUC 8152 h) =
week 1(AUC 8152 h) etc.
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Results
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Associations between biomarkers and DMH exposure
O6-MeG levels in DNA.
O6-MeG levels were generally highest in the distal colon, lower in the mid colon and lowest in the proximal colon following DMH injections for up to 20 weeks (Figure 1
). O6-MeG levels in the proximal colon at 8, 56 and 152 h after the last DMH dose ranged between 0.71.8, 0.20.9 and 0.30.5 fmol O6-MeG/µg DNA (Figure 1A
). In the mid-colon, O6-MeG levels ranged between 4.38.6, 0.44.8 and <0.14.0 fmol O6-MeG/µg DNA at 8, 56 and 152 h after the last DMH dose (Figure 1B
). In the distal colon, O6-MeG levels ranged between 816.1, 0.97.7 and <0.16.4 fmol O6-MeG/µg DNA at 8, 56 and 152 h after the last DMH dose (Figure 1C
). O6-MeG levels in the proximal and mid colon were not associated with total DMH dose but in the distal colon there was a significant association between O6-MeG levels 152 h after the last DMH dose and the total DMH dose (r2 = 0.97, P = 0.02: Figure 1F
).
O6-MeG levels decreased with time following the last injection in all treatment groups except for those animals that received the highest dose (136 mg/kg DMH) where the adduct appeared persistent (Figure 1
). At this high dose, adduct levels in the colon appeared to achieve a steady state, with relative O6-MeG levels in the proximal, mid- and distal colon being 1.0, 5.4 and 10.6 respectively. After 6.8 mg/kg DMH, the t1/2 value for O6-MeG levels (i.e. 50% of the 8 h level) was reached after
2532 h in all regions of the colon. After 34 mg/kg DMH, this was
93113 h, whereas after 68 mg/kg t1/2 was
25 h in the proximal colon, 57 h in the mid colon and 96 h in distal colon. In those animals treated with 136 mg/kg DMH, it was not possible to estimate t1/2 as O6-MeG levels had not fallen to <50% (of the 8 h level) by 152 h after the final dose.
O6-alkylguanine DNA alkyltransferase levels.
In saline treated SWR mice (controls), colonic MGMT levels were measured over the time period of the study and were found to vary between 0.452.09, 0.932.53, 0.831.65 fmol/µg DNA in the proximal, mid- and distal colon respectively (Figure 2
). Following DMH administration, MGMT activity was lowest at 8 h, but had recovered to pre-treatment levels in most cases by 152 h. In the proximal colon, MGMT activity fell to between 15100% of the saline control level 8 h after treatment (Figure 2A
) and recovered to 50% of the control level after a further 1529 h. In the mid colon, MGMT activity fell to 853% of that in the saline control 8 h after treatment (Figure 2C
) and recovered to 50% of the control after a further 3354 h (depending on the dose). In the distal colon, MGMT activity fell to 1127% of the saline control level 8 h after treatment (Figure 2E
) and recovered to 50% of the control after a further 2147 h (depending on the dose). There were no associations between total DMH dose and MGMT activity in the proximal (Figure 2B
), mid (Figure 2D
) or distal colon (Figure 2F
).
Cell proliferation
Cell proliferation in the proximal and distal colon was altered by treatment with DMH. The percentage increase in labelling index following treatment with DMH is shown in Figure 3
and this increase varied with time after final treatment but not with total DMH dose or tissue. Specifically, labelling was increased 8 and 56 h after DMH treatment but not 152 h.
The position of labelled cells within the crypt also varied in a time dependent manner (data for 136 mg/kg DMH is shown in Figure 4
). In the proximal colon, there was initially a general increase in the numbers of labelled cells but there was no expansion of the labelling zone after treatment with 136 mg/kg DMH (at 8 h, Figure 4A
). After 56 h there was no difference in either the numbers of cells or the size of the labelling zone (Figure 04B
) and by 152 h after DMH treatment, the numbers of labelled cells had decreased to below the control level (Figure 4C
). In contrast, in the distal colon, treatment with 136 mg/kg DMH resulted in both an increase in the number of labelled cells and an upward shift in the labelling zone that was detectable at both 8 h and markedly at 56 h (Figure 4D and E
). After 152 h these parameters had returned to levels found in control animals (Figure 4F
).

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Fig. 4. Time dependent variation in labelling index with crypt cell position in the proximal colon (A, B and C) and the distal colon (D, E and F) following treatment with a total dose of 136 mg/kg DMH (D, E and F). Labelling index measured at 8, 56 and 152 h following the final DMH dose or saline treatment. DMH treatment (______), saline (vehicle) treatment (.............).
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Associations between biomarkers and tumour yield
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Previously we have shown that in this model distal colon tumours were induced in 26%, 67% and 87% of SWR mice treated with a total cumulative dose of 34, 68 and 136 mg/kg DMH respectively (34). In the mid colon tumours were induced with cumulative doses of 68 and 136 mg/kg DMH in 24 and 43% of animals respectively and no tumours were detected in the proximal colon (34). The risk factor data described in the previous section together with additional data derived from these risk factor results (AUC; CSUM), were then used to examine whether subsequent tumour yield was associated with:- O6-MeG levels at specific time points following DMH treatment as these levels provide a snapshot of the mutagenic potential at any specific time (and would be equivalent to a single adduct measurement in human DNA samples).
- The cumulative O6-MeG sum (CSUMO6-MeG) as this would be a better marker of total adduct formation (and hence total mutagenic potential) than a single adduct measurement.
- Basal MGMT levels as these levels would help to determine the extent of repair of O6-MeG adducts following the initial DMH dose.
- Induced MGMT levels as these levels would help to determine the extent of repair of O6-MeG adducts at subsequent DMH doses.
- Cumulative changes in MGMT activity (CSUM MGMT) as this would provide a better indicator of DNA repair activity (and hence removal of O6-MeG) over the time course of the experiment.
- Cumulative changes in labelling index (CSUMCPI) as this would provide an indicator of the proliferative changes and hence the potential for mutation fixation over the course of the experiment not only within the whole crypt but also within specific regions of the crypt where stem cells are believed to reside.
Adduct formation and persistence
There was no direct association between tumour formation and the 8 h O6-MeG level (Figure 5A
): similar O6-MeG levels (
8 µmol/mol dG) were associated with a tumour yield ranging from 070%. Similar results were obtained using the 56 h O6-MeG levels (data not shown). At 152 h, O6-MeG levels above 1 µmol/mol dG were associated with tumour formation but not in a doseresponse manner (Figure 5B
). In addition, O6-MeG levels below this level were not associated with tumour formation except in the mid colon where tumours developed although O6-MeG was not detectable 152 h after the final treatment (Figure 5B
).
In the proximal colon, the AUCO6-MeG varied little with increasing DMH dose and was significantly lower than those observed in either the mid or distal colon (Figure 5C
). In the mid colon, the AUCO6-MeG was constant between a total DMH dose of 34136 mg/kg (Figure 5C
). In the distal colon, the AUCO6-MeG were generally higher than those in the mid and proximal colon: the relative AUC values were 1:5:10 for the proximal, mid and distal colon, respectively.
The cumulative sum of the weekly amounts of total O6-MeG formed (CSUM O6-MeG ) was associated with tumour yield in both the distal and mid colon (Figure 5D
). Low CSUM O6-MeG levels (
23000 units) were associated with tumours in the distal but not the mid or proximal colon. At higher CSUM O6-MeG levels (>5000 units), the tumour yield was
50% higher in the distal colon than in the mid colon.
DNA repair capacity
There was no association between tumour yield and basal MGMT levels as MGMT levels in the mid and distal colon of untreated animals varied between 1.22.3 and 0.63.4 times the level in proximal colon, respectively (data not shown). Induced changes in MGMT activity were then examined by comparing the ratio of MGMT activity in treated mice to that in saline control mice 56 h after the last treatment. There was no clear association between the MGMT treated/MGMT non-treated ratio (Figure 6A
). There were no associations between this MGMT ratio and subsequent tumour yield (Figure 6B
): the MGMT ratio in the distal colon was higher after treatments which induced tumours but this inverse association was non-significant (r2 = 0.78; P = 0.12: Figure 6B
).
The cumulative difference in MGMT levels (CSUMMGMT) between treated and control animals is shown in Figure 6C
. In the distal colon, there was a non-linear association between CSUMMGMT and tumour yield and the greater the CSUMMGMT level the higher the tumour yield but there was no such association in the mid colon (Figure 6C
). Furthermore, comparable CSUMMGMT levels, which were associated with the formation of tumours in the mid and distal colon, were also found in the proximal colon where no tumours were produced (Figure 6C
).
Cell proliferation
The AUCCPI for the distal and proximal colon derived from all cell positions or specifically from cell positions 1 and 2 (derived from Figure 4
) are shown in Figure 7
. AUCsCPI were not associated with total DMH dose but were associated with time after final treatment and with location (i.e. distal or proximal colon).
There was a clear association between CSUMCPI in the distal colon derived from the total labelling index (based on data used in Figure 3
) and for cell positions 1 and 2 (from Figure 4
) and subsequent tumour yield (Figure 8
). Comparable CSUMCPI levels derived from the total labelling index were associated with the formation of tumours in the distal colon but not the proximal colon. However, there was greater discrimination in CSUMCPI levels in cell positions 1 and 2 in that CSUM CPI levels in the proximal colon were at least 50% lower than those associated with tumour formation in the distal colon (Figure 8B
).
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Discussion
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Understanding the role of various risk factors in chemical carcinogenesis is clearly of critical importance in identifying populations at increased cancer risk (37). Increasingly, DNA from different human tissues has been shown to contain DNA adducts, arising both from exogenous and endogenous exposures to chemical carcinogens (38). These adducts are present in widely different amounts and have widely differing toxic, mutagenic and carcinogenic potential. The biological significance of these adduct levels remains to be clarified but potentially, individuals with high DNA adduct levels may be at increased cancer risk. So far, there is little evidence to support this hypothesis directly from human studies, although individuals with high levels of urinary aflatoxin-guanine adducts were at increased liver cancer risk compared with individuals with low levels (39).
Associations between DNA adduct levels and tumour induction have been observed in experimental models in which carcinogens are administered chronically to rodents, though the precise nature of this relationship can vary (40). To further evaluate the relative biological importance of DNA adduct formation, we treated mice with DMH, a known colon carcinogen, over a prolonged period. This model, in principle, mimics the chronic exposures that humans may encounter (41) and we have examined the colonic levels of a putative risk factor (O6-MeG) and two risk modifying factors (MGMT activity and cell proliferation indices). Consistent with previous studies (1833), DMH treatment resulted in (i) the formation of O6-MeG in colonic DNA in a time and location specific manner; (ii) a concomitant loss in colonic MGMT activity with subsequent recovery; and (iii) marked transient alterations (both increases and decreases) in total cell labelling and positional cell labelling in both the proximal and distal colon.
We then investigated whether such changes were associated with subsequent tumour induction within the same region of the colon. Individual measurements of DNA damage, induced or basal DNA repair activity or cell proliferation were not in general associated with tumour induction. Persistent (steady state) levels of DNA damage (i.e. O6-MeG levels 152 h post treatment) were generally higher in regimes that induced tumours but there was no doseresponse relationship. Hence a single measurement of target tissue DNA damage may bear little association to subsequent risk. This may also be the situation for human studies where adduct levels may not be in steady state and can be measured in non-target tissues.
In contrast to these individual measurements, cumulative estimates of total O6-MeG levels and cell proliferation indices, particularly within crypt cell positions 1 and 2, where stem cells are believed to reside, were associated with tumour induction in the distal colon. These results are then consistent with an increased cancer risk arising from a higher mutational load due to increased adduct formation/persistence and increased cell proliferation amongst the stem cell population. However other factors, not measured in this study, are also of importance. Increased cell death (e.g. through apoptosis) could explain the lack of linearity in the (cumulative) doseresponse curves, observed in the distal colon (42,43). Such mechanisms could also potentially explain the difference in tumour yield despite similar cumulative O6-MeG levels in the distal and mid colon.
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Notes
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3 Current address: Micromass UK Limited, Floats Road, Wythenshawe, Manchester, M23 9LZ, UK 
4 To whom correspondence should be addressed at: School of Epidemiology and Health Sciences, Medical School, The University of Manchester, Oxford Road, Manchester, M13 9PT, UK Email: apovey{at}man.ac.uk 
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Acknowledgments
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The authors wish to thank Cancer Research UK for their support.
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Received December 14, 2001;
revised September 9, 2002;
accepted October 25, 2002.