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Brief Report
Key words
OT
D
IST
UT
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RIB
Olga A. Sedelnikova*
William M. Bonner
CE
To detect tumors and assess the response to antitumor therapy, clinicians make use
of substances, called cancer biomarkers, which are produced by the body or tumors in
response to tumor growth. Cancer biomarkers are helpful for early tumor diagnostics,
prediction of tumor development and individual tumors response to therapy, cancer
management, as well as for prognosis of tumor outcome and recurrence (reviewed in
ref. 1 and 2). Many widely known biomarkers, such as carcinoembrionic antigen (CEA),
prostatespecific antigen (PSA), CA199 for colorectal and pancreatic cancer, CA15.3
for breast cancer, CA125 for ovarian cancer and others became reliable indicators of
tumors and brought a dramatic increase in their early detection.2 Discovery of new cancer
biomarkers has become a major focus of cancer research.
However, there are some limitations in using cancer biomarkers in clinical practice.
Single biomarkers are not very efficient, most biomarkers detect late stages of tumor development. There is often a crossreactivity with other types of tumors, as well as with normal
tissues; some tumors do not express common biomarkers. As it has been pessimistically
noted in one review, it seems unlikely that any single marker will ever possess 100% sensitivity and specificity.2 Combinations of selected markers may provide the most effective
means for diagnostics and predicting disease outcome. Recently, a multibiomarker blood
test has been reported to be able to detect nonsmall lung cancer about 35 years before
the tumor reaches the conventional size limit needed for diagnosis by current radiographic
screening methods (0.5 mm).3
The integrity of chromosomal DNA is essential to its informational transfer functions
such as replication and transcription as well as to the mechanical segregation functions
of chromosomes during mitosis and meiosis. Thus, a single unrejoined DSB in the DNA
may be an oncogenic and even lethal lesion; evidence is accumulating that defects in
maintenance of DNA integrity play major roles in tumorogenesis. However, DNA DSBs
do arise, accidentally from metabolic errors, environmental insults and intentionally
during several important cellular functions such as V(D)J and meiotic recombination.4,5
One of the first responses when DSBs are introduced into the DNA of eucaryotic
cells is the immediate and massive phosphorylation of serine 139 at the COOH terminus
of histone H2AX, yielding a specific modified form named gH2AX in mammals. An
antibody against a synthetic phosphorylated peptide containing the mammalian gH2AX
IEN
.D
Introduction
Abbreviations
H2AX
DSB
CNS
ON
Acknowledgements
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Figure 1. Endogenous gH2AX levels in tissues. Frozen sections of human tumors (colon tubular adenocarcinoma, breast ductal adenocarcinoma, ovary
adenocarcinoma, hepatoblastoma and Wilms tumor), bottom and adjacent normal surgical tissue samples, top, immunostained with antigH2AX anti
body. White arrows indicate nonspecific crossreaction with mucine; gH2AX staining in nuclei is pointed with red arrows. Blue, propidium iodide; green,
gH2AX.
Cell Cycle
Table 1
Origin
HeLa
Cervix
20
Caski
Cervix
25
MS751
Cervix
13
C33A
Cervix
SW756
Cervix
46
SiHa
Cervix
A549
Non-small cell
lung
1.9
MCF7
CCRFCEM
HCT116
ACHN
Breast
2.1
Leukemia
2.4
Colon
3.7
Renal
3.9
OVCAR8
Ovarian
4.0
HL60
Leukemia
4.4
SF539
CNS
4.5
PC3
Prostate
4.7
DU145
Prostate
4.9
SKMEL5
Melanoma
5.1
SKOV3
Ovarian
5.7
Colon
6.9
Non-small cell
lung
7.3
HT29
NCIH322M
SF295
CNS
8.5
MDAMB231
Breast
10.6
Yu et al., 2006
HACaT
Melanoma
14
YUSIT
Melanoma
6.5
YUGEN
Melanoma
10
LOX
Melanoma
11
YUSAC
Melanoma
17
Cervix
2.3
HT1080
Fibrosarcoma
1.5
Sedelnikova,
U2 OS
Osteosarcoma
2.1
Sokolov
MO59J
CNS
1.2
MO59K
CNS
1.1
KBV1
SF268
CNS
3.1
HCT15
Colon
2.1
HCC2998
Colon
5.5
COLO205
Colon
2.9
SW620
Colon
7.1
KM12
Colon
4.8
Rao, Pommier
quantitative analysis of cryptogenic gH2AX foci in touch print preparations of several cases of colon, breast and ovary carcinomas and
adjacent normal tissues (Fig. 2B). Although gH2AX levels in normal
cells were quite variable among patients and organs, we detected a
significant increase in focal incidences (in most cases 27 fold) in
all corresponding tumor samples. Thus, the increase of endogenous
gH2AX marking DNA damage may be a general process in tumor
cell cultures and tissues.
DNA damage initiates a signaling cascade designed to arrest the
cell cycle through checkpoint activation.22 Large amounts of publications suggest that H2AX phosphorylation acts as a checkpoint
maintenance factor while its dephosphorylation is a signal for
resumption of the cell cycle and, therefore, H2AX is essential for
genome stability.5,2327
gH2AX increases in premalignant lesions have been associated with
DNA damage checkpoint activation via stimulated cell proliferation
and replication stress. DNA damage with H2AX phosphorylation
and other damage sensors along with dysfunctional telomeres, can
act as a common mechanism to prevent malignant progression.20,21
The existence of this preventive pathway has recently been confirmed
by in vivo evidence of oncogeneinduced senescence, which is a
tumorsupressor mechanism, that occurs in diverse precancerous
tissues of both human and mouse.28 On the other hand, it has
been shown that some cells can spontaneously escape senescence
and acquire genomic changes that predict oncogenic evolution,29
or DNA damage can lead to allelic imbalances that induce cells to
progress to full malignancy.21
Until now, studies in the cancer biomarker research field mostly
have been concentrated around tumorspecific antigens. Now, the
concept replication stress g DNA damage g senescence/precancer
g cancer is applied to the general process of oncogenic transformation. Recent reviews suggest using markers of cell proliferation,
oncogeneinduced senescence, telomerase, DNA damage repair,
and corresponding epigenetic markers as general cancer biomarkers,
which are powerful and promising for cancer prediction, prognosis,
therapeutics, and possibly cancer prevention.3035 Since DNA
damage is an upstream event in this concept, H2AX phosphorylation
in response to DNA damage is a crucial step in the process of tumorigenesis. This makes detection of gH2AX an attractive biomarker that
could serve as an early cancer indicator. The gH2AXbased assay has
advantages over standard methods; it is the most sensitive modern
method for DNA DSB detection. gH2AX is already coming to
clinics as a therapeutic target for monitoring computed tomography
and radiation therapy,3638 as well as for drug treatment evaluation.
For example, it can be used for assessment of DNA or chromatin
targeted agents such as topoisomerase inhibitors.39 gH2AX can
be detected in human biopsies (tissue sections, touch prints and
smears), aspirates and mononuclear cells of the peripheral blood
(Dr. C. Redon, unpublished results). Fluorescent and peroxidase
immunohistochemical techniques, both qualitative and quantitative,
show reliable results. The method offers a broad range of possibilities
for improving efficiency in early diagnostics, prediction, prognosis,
therapeutics and recurrence of malignant neoplasias. Since the blood
levels of tumor markers usually correlate with the tumor size and the
disease stage, gH2AX has a potential to become a common tumor
biomarker that could be easily detected in patients blood at the early
stage of tumor development. In addition, a phenomenon known as
the bystander effect is now well established in which irradiated cells
transmit signals into their surroundings or through their membranes
that induce DNA damage in their unirradiated neighbors.13 We
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have shown that unirradiated tumor cells also induce DNA damage
in normal cells that come in contact with media from tumor cells or
communicate via gapjunctions (unpublished data).
Further investigations are required to elucidate the benefits of
gH2AX use in initial cancer screening, drug and radiation treatment
protocols and monitoring patients in remission to determine whether
its levels remain stable. It is important to include gH2AX in the panel
of new cancer biomarkers for validation and proper evaluation40 of
its impact on cancer prevention.
References
1. Conley B, Taube SE. Prognostic and predictive markers in cancer. Disease Markers 2004;
20:3543.
2. Chatterjee SK, Zetter BR. Cancer biomarkers: Knowing the present and predicting the
future. Future Oncology 2005; 1:3750.
3. Zhong L, Coe SP, Stromberg AJ, Khattar NH, Jett JR, Hirschowitz EA. Profiling tumorassociated antibodies for early detection of nonsmall cell lung cancer. J Thor Oncol 2006;
1:5138.
4. FernandezCapetillo O, Mahadevaiah SK, Celeste A, Romanienko PJ, CameriniOtero RD,
Bonner WM, Manova K, Burgoyne P, Nussenzweig A. H2AX is required for chromatin
remodeling and inactivation of sex chromosomes in male mouse meiosis. Dev Cell 2003;
4:497508.
5. Bassing CH, Suh H, Ferguson DO, Chua KF, Manis J, Eckersdorff M, Gleason M, Bronson
R, Lee C, Alt FW. Histone H2AX: A dosagedependent suppressor of oncogenic translocations and tumors. Cell 2003; 114:35970.
6. Redon C, Pilch D, Rogakou E, Sedelnikova O, Newrock K, Bonner W. Histone H2A variants H2AX and H2AZ. Cur Opin Gen Devel 2002; 12:1629.
7. Pilch DR, Sedelnikova OA, Redon C, Celeste A, Nussenzweig A, Bonner WM.
Characteristics of gH2AX foci formation at DNA doublestrand break sites. Biochem Cell
Biol 2003; 81:1239.
8. Sedelnikova OA, Pilch DR, Redon C, Bonner WM. Involvement of H2AX in the DNA
damage and repair response. Cancer Biol Ther 2003; 2:2335.
9. Rogakou EP, Boon C, Redon C, Bonner WM. Megabase chromatin domains involved in
DNA doublestrand breaks in vivo. J Cell Biol 1999; 146:90515.
10. Sedelnikova OA, Rogakou EP, Panyutin IG, Bonner WM. Detection of 125IUdRinduced
DNA doublestrand breaks with gammaH2AX antibody. Rad Res 2002; 158:493504.
11. Rothkamm K, Lobrich M. Evidence for a lack of DNA doublestrand break repair in human
cells exposed to very low xray doses. Proc Natl Acad Sci USA 2003; 100:505762.
12. Sedelnikova OA, Panyutin IV, Bonner WM, Panyutin IG. Assessment of intracellular DNA
damage produced by 125Itriplexforming oligonucleotides in cells. Int J Rad Biol 2004;
80:92731.
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13. Sokolov MV, Smilienov LB, Hall EJ, Panyutin IG, Bonner WM, Sedelnikova OA.
Radiation induces DNA doublestrand breaks in unirradited primary human fibroblasts.
Oncogene 2005; 24:725765.
14. Sedelnikova OA, Horikawa I, Zimonjic DB, Popescu NC, Bonner WM, Barrett JC.
Senescing human cells and ageing mice accumulate DNA lesions with unrepairable
doublestrand breaks. Nat Cell Biol 2004; 6:16870.
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Saretzki G, Carter NP, Jackson SP. A DNA damage checkpoint response in telomere-initiated senescence. Nature 2003; 426:1948.
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dysfunctioninduced stress response in fully senescent cells. Cancer Res 2004; 64:374852.
17. Warters RL, Adamson PJ, Pond CD, Leachman SA. Melanoma cells express elevated levels
of phosphorylated histone H2AX foci. J of Invest Dermatol 2005; 124:80717.
18. Banath JP, MacPhail SH, Olive PL. Radiation sensitivity, H2AX phosphorylation, and
kinetics of repair of DNA strand breaks in irradiated cervical cancer cell lines. Cancer Res
2004; 64:71449.
19. Yu Y, MacPhail SH, Banath JP, Klokov D, Olive PL. Endogenous expression of phosphorylated histone H2AX in tumors in relations to DNA doublestrand breaks and genomic
instability. DNA Repair 2006; 5:93546.
20. Bartkova J, Horejsi Z, Koed K, Kramer A, Tort A, Zieger K, Guldberg P, Sehested M,
Nesland JM, Lukas C, Orntoft T, Lukas J, Bartek J. DNA damage response as a candidate
anticancer barrier in early human tumorigenesis. Nature 2005; 434:86470.
21. Gorgoulis VG, Vassillou LVF, Karakaidos P, Zacharatos P, Kotsinas A, Liloglou T, Venere
M, Ditullio Jr RA, Kastrinakis NG, Levy B, Kletsas D, Yoneta A, Herlyn M, Kittas C,
Halazonetis TD. Activation of DNA damage checkpoints and genomic instability in human
precancerous lesions. Nature 2005; 434:90713.
22. Downey M, Durocher D. gH2AX as a checkpoint maintenance signal. Cell Cycle 2006;
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23. Celeste A, Petersen S, Romanienko PJ, FernandezCapetillo O, Chen HT, Sedelnikova
OA, ReinaSanMartin B, Coppola V, Meffre E, Difilippantonio MJ, Redon C, Pilch DR,
Olaru A, Eckhaus M, CameriniOtero RD, Tessarollo L, Livak F, Manova K, Bonner WM,
Nussenzweig MC, Nussenzweig A. Genomic instability in mice lacking histone H2AX.
Science 2002; 296:9227.
24. Celeste A, FernandezCapetillo O, Kruhlak MJ, Pilch DR, Staudt DW, Lee A, Bonner RF,
Bonner WM, Nussenzweig A. Histone H2AX phosphorylation is dispensable for the initial
recognition of DNA breaks. Nat Cell Biol 2003; 5:6759.
25. Stucki M, Clapperton JA, Mohammad D, Yaffe MB, Smerdon SJ, Jackson SP. MDC1
directly binds phosphorylated histone H2AX to regulate cellular responses to DNA
doublestrand breaks. Cell 2005; 123:121326.
26. Chowdhury D, Keogh MC, Ishii H, Peterson CL, Buratowski S, Lieberman J. GammaH2AX
dephosphorylation by protein phosphatase 2A facilitates DNA doublestrand break repair.
Mol Cell 2005; 20:8019.
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