Supplementary Figure Legends (Bartek et al. 2004-07-21856A, re-revised)
Supplementary figure S1. Multifocal pattern of H2AX staining of human Ta lesions of
the urinary bladder. a, Confocal laser microscope image of immunofluoresence staining
on a tissue section (red), counterstained for DNA (blue). b, Example of
immunoperoxidase staining photographed at high magnification reveals multiple foci of
H2AX in tumour cell nuclei.
Supplementary figure S2. Graph summaries of Ser1981-phosphorylated ATM, Thr68phosphorylated Chk2, and classification of bladder tumours according to their genomic
instability. a, b, Frequency of constitutive activatory phosphorylation of ATM on serine
1981 (pS-ATM) detected immunohistochemically in normal tissue versus different stages
(Ta, T1, T2-4) of human urinary bladder cancerogenesis (a), and in bladder lesions of
differential genomic instability (b). c, Subclassification of 35 human bladder tumours into
three categories with increasing genomic instability, based on the frequency of allelic
imbalances between control blood DNA and the corresponding tumour DNA, as assessed
by an array analysis of single nucleotide polymorphisms (SNPs), of which around 2600
were commonly heterozygous in the blood samples. The cases marked by the circles that
touch the upper horizontal line that separates the most stable and the intermediate
categories, were assigned to the intermediate stability group, to keep the numbers within
the three categories approximately equal (see Methods for details). d, e, Reproducibility
of the immunohistochemical patterns of pT-Chk2 and pS-ATM obtained by independent
staining of the larger first series of invasive bladder T1, T2-4 tumours and the second
series that was evaluated by the SNP arrays. (d), Graphical summary of the pT-Chk2
results to compare the first series (‘Group A’: a total of 73 cases of T1, T2-T4 tumours)
and the SNP series (a total of 24 cases of T1, T2-T4 tumours). There are no statistical
differences between these two groups. (e), Summary of the pS-ATM results to compare
the subset from the first series that was examined for pS-ATM (‘Group B’: a total of 40
cases of T1, T2-T4 tumours from the group A, since not all the 73 cases from group A
were analysed for ATM) and the entire SNP series (a total of 24 cases of T1, T2-T4
tumours). There are no statistical differences between these groups.
Supplementary figure S3. Mononuclear cell infiltrate is insufficient to activate the DNA
damage response in human colon tissues. a, Examples of immunohistochemistry images
on serial sections of inflammatory Crohn’s disease (representative patterns from 26
patients) with nuclear positivity for total Chk2 protein, in contrast to the lack of staining
for the DNA damage markers pT-Chk2, pS-ATM and H2AX; (inf.) marks the areas of
dense mononuclear infiltrate, also negative for the markers except for total Chk2.
b, Serial sections of a colon tumour with adjacent pronounced mononuclear infiltrate
(inf.). The markers used are indicated next to the panels. It is apparent that both the
infiltrate and the tumour cells express Chk2 and proliferate (positive Ki67 staining), yet
lack any detectable staining for pT-Chk2 or pS-ATM.
Supplementary figure S4. Cytological detection of activated DNA damage checkpoint
markers in U-2-OS cells exposed to various hyperproliferative, proto-oncogenic stimuli.
a,b. Immunoperoxidase staining (a) and confocal microscopy (b) immunofluorescence
images of cells with cyclin E or Cdc25A induced for 4 days by removing tetracycline
from the medium (-TET). Both immunofluorescence and immunoperoxidase detection
methods provide consistent results, here exemplified by detection of pS-ATM and pTChk2 upon induction of cyclin E and Cdc25A, respectively. c, Detection of Ser15phosphorylated p53 is suppressible by an antioxidant (NAC) when the DNA damage is
induced by hydrogen peroxide (H2O2), but not when induced by overexpressed cyclin E.
Supplementary figure S5. Detection of cyclin E-activated DNA damage checkpoint
markers in normal human fibroblasts. a, Examples of immunofluorescence (confocal
microscopy) and immunoperoxidase staining (light microscopy) images of early-passage
human diploid fibroblasts upon retrovirus-mediated ectopic expression of cyclin E
(CycE), compared with cells infected with control virus (Mock). b,c, Detection of
phosphorylated pS-ATM or H2AX in cyclin E-overexpressing normal human fibroblasts
is not altered in cells treated with an antioxidant (NAC), similarly to data with U-2-OS
cells overexpressing cyclin E, Cdc25A or E2F1. Note the lack of DNA damage response
in mock-infected control fibroblasts (Mock).
Supplementary figure S6. Altered dynamics of DNA replication and chromatin
association of RPA upon induced overexpression of cyclin E in human U-2-OS cells.
a, DNA fiber analysis at 2 days after cyclin E induction. DNA fiber labeling was
performed as described35. Briefly, cells were labeled with 50 mM IdU for 20 minutes
before preparing the DNA fibers, and immunofluorescence detection of IdU. The mean
length of at least 30 replication tracks is plotted. The difference in fiber length between
cells with and without ectopic cyclin E expression is statistically significant
(P=3.19234E-08; by paired Student’s t-test).
b, Cyclin E induction leads to an increase of non-extractable RPA protein, and
hyperphosphorylation of RPA in U-2-OS cells (-TET) compared with control cells grown
in parallel (+TET). Chromatin bound: cells were incubated with extraction buffer, and
non-extractable, chromatin bound fraction loaded in increasing amounts (1x,2x,4x) and
processed for Western blotting. Total protein: total cell lysates analysed for RPA and
Mcm7 protein (loading marker). Note the pronounced mobility shift of RPA in cyclin E
expressing cells, consistent with RPA hyperphosphorylation and activated checkpoint
response36,37. The presence of extended ssDNA stretches upon cyclin E induction was
also verified by detectable BrDU incorporation without denaturation of the fixed cells
(data not shown, method reported by Raderschall et al. Proc. Natl. Acad. Sci. USA 96,
1921-1926; 1999), consistent with the enhanced chromatin association of the ssDNAbinding RPA36 (shown in the top panel).
Supplementary figure S7. Comparative immunohistochemical analysis of cyclin E and
H2AX in normal human colon and colon adenomas. a, Examples of
immunohistochemical staining patterns for -H2AX and total cyclin E in normal human
colon mucosa (Normal), and two colon adenomas. b, A summary of
immunohistochemical data documenting a correlation between -H2AX and
overexpression of cyclin E among a series of human colorectal adenomas.
Supplementary figure S8. Comparative immunohistochemical analysis of Tyr15phosphorylated Cdk1 and H2AX in normal human colon and colon adenomas.
a,b, A summary of immunohistochemical data documenting a correlation between
phosphorylated -H2AX and Tyr15-phosphorylated Cdk1 (pY-Cdk1) among a series of
human colon adenomas (a) and carcinomas (b). Note the lower frequency of cases with
the highly pronounced checkpoint response among carcinomas. c. Examples of
immunohistochemical staining patterns for -H2AX and pY-Cdk1 (top panels, case with
the pY-Cdk1 pattern similar to normal colon, and low -H2AX). For examples of pYCdk1 and -H2AX patterns in normal colon and a highly positive adenoma, see Fig. 6d.
Bottom panels: example of a colon adenoma with high expression of pY-Cdk1 and
virtual lack of proliferation, as documented by Ki67 staining (note that some stromal cells
and a group of adenoma cells proliferate, in contrast to the remaining large area of the
epithelial adenoma structures)