Cell Cycle

ISSN: 1538-4101 (Print) 1551-4005 (Online) Journal homepage: http://www.tandfonline.com/loi/kccy20

-H2AX in Cancer Cells: A Potential Biomarker for

Cancer Diagnostics, Prediction and Recurrence
Olga A. Martin & William M. Bonner
To cite this article: Olga A. Martin & William M. Bonner (2006) -H2AX in Cancer Cells: A
Potential Biomarker for Cancer Diagnostics, Prediction and Recurrence, Cell Cycle, 5:24,
2909-2913, DOI: 10.4161/cc.5.24.3569
To link to this article: http://dx.doi.org/10.4161/cc.5.24.3569

Published online: 28 Nov 2006.

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Date: 04 October 2016, At: 07:03

[Cell Cycle 5:24, 2909-2913, 15 December 2006]; 2006 Landes Bioscience

Brief Report

H2AX in Cancer Cells

A Potential Biomarker for Cancer Diagnostics, Prediction and Recurrence
Abstract

*Correspondence to: Olga A. Sedelnikova; Laboratory of Molecular Pharmacology,

Center for Cancer Research, National Cancer Institute, NIH; Bldg. 37 Room 5050,
9000 Rockville Pike; Bethesda, Maryland 20892 USA; Tel.:301.402.3649; Fax:
301.402.0752; Email: sedelnio@mail.nih.gov

Previously published online as a Cell Cycle E-publication:

http://www.landesbioscience.com/journals/cc/abstract.php?id=3569

Key words

OT
D

IST

Original manuscript submitted: 10/30/06

Manuscript accepted: 10/31/06

UT
E

Laboratory of Molecular Pharmacology, Center for Cancer Research, National

Cancer Institute, NIH; Bethesda, Maryland USA

Current advances in cancer biology have identified major pathways involved in

tumorigenesis. The association of DNA damage with premalignant stages of tumor
progression, genome instability and further oncogenic transformation opens the possi
bility of using common DNA damage markers for early cancer detection, prediction,
prognosis, therapeutics and possibly for cancer prevention. Perhaps the most sensitive
DNA damage marker is gH2AX formation in the chromatin flanking the free DNA
doublestranded ends in doublestrand breaks (DSBs) and eroded telomeres, both present
during oncogenic transformation. Our group and others found elevated endogenous
levels of gH2AX in various human cancer cell lines, premalignant lesions and solid
tumors. These data suggest that increased DNA damage is a general characteristic of
cancer development. gH2AXbased assay can be applied to human biopsies, aspirates
and, possibly, to mononuclear cells of the peripheral blood. We propose that detection
of gH2AX could benefit for the early cancer screening and to ascertain the efficiency of
clinical treatment involving chemo and radiotherapeutic protocols.

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

phosphorylated histone H2AX

double-strand break
central neural system

.D

Introduction

Abbreviations
H2AX
DSB
CNS

ON

DNA damage, H2AX, genome instability,

tumor progression, cancer biomarker

Acknowledgements

20

06

LA

ND

ES

BIO

SC

We thank Dr. Yves Pommier and W.M.B.

group members for critical reading of the
manuscript, and Drs. M. Sokolov and A. Rao
for sharing their data on endogenous levels
of -H2AX in cancer cell lines. This work
was supported by the Intramural Research
Program of the National Cancer Institute,
NIH.

www.landesbioscience.com

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H2AX as a Cancer Biomarker

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.

COOH terminal sequence, reveals that gH2AX molecules appear in

discrete nuclear foci within 1 min after cellular exposure to ionizing
radiation; maximal amounts are reached by 1030 min (for a review
see refs. 68). It has been suggested that each gH2AX focus represents
one DNA DSB.911 These individual foci may be counted, making
this technique much more sensitive than other known DSBdetection
methods.1013
In this study we assessed spontaneous DNA DSBs, revealed by
the gH2AX focus formation assay, in malignant tumors of different
origins, as well as in adjacent normal tissues from cancer patients
of different ages. We propose that elevated levels of endogenous
gH2AX are a general characteristic for cultured cancer cells, tumors
and premalignant lesions and that gH2AX has a great potential to
become a common cancer biomarker with a 100% sensitivity to early
cancers.

Materials and Methods

Human Tissues. Thirty human biopsies were obtained from
the Cooperative Human Tissue Network (CHTN), MidAtlantic
Division (Charlottesville, VA) and Pediatric Division (Columbus,
OH) with IRB approval. Colon, breast, ovary, liver and kidney
tumors and normal adjacent tissues from cancer patients (age
range was from one month to 88 years) were sliced into pieces and
touchprinted and/or cryosectioned for immunofluorescent analysis.
The tissues were not removed specifically for research but were the
excess tissues not essential for routine diagnosis. Patients confidentiality was maintained.
Laser Scanning Confocal Microscopy. The frozen sections and
touchprints were dried, fixed in 2% paraformaldehyde in PBS for
20 min, washed in PBS, and touchprints were permeabilized with
cold 70% ethanol. Tissue sections were permeabilized with 1%
Triton X100 for 5 min. Then the preparations were processed for
immunofluorescent staining with a custommade rabbit gH2AX
primary antibody and Alexa488labeled goat antirabbit secondary
antibody (Invitrogen, Eugene, OR) as previously described.9 Cells
were mounted in a Vectashield mounting medium with propidium iodide (Vector, Burlingame, CA). Confocal microscopy was
performed with a Nikon PCM 2000 (Nikon Inc, Augusta, GA). The
2910

foci were counted by eye in randomly chosen 50200 cells of touch

print preparations in a blinded fashion.

Results and Discussion

Our work and others1416 indicates that gH2AX foci accumulate
in senescent human cell cultures and in germ, as well as somatic, cells
of aging mice. The numbers of gH2AX foci per cell in early passages
of interphase primary cultures of different origin (fibroblasts, epithelium, melanocytes and keratinocytes) vary in the range of 0.20.8,
usually 310 times lower than in late passages.14,17 We named these
foci of unknown origin cryptogenic foci to differentiate them from
those of known causes such as ionizing radiation. We proposed that
these persistent DNA lesions contain unrepairable DSBs and may
have a causal role in aging.14
The endogenous levels of gH2AX have been examined in tumor
cell cultures of 6 cervical cell lines, 16 cell lines randomly chosen from
the NCI60 panel of tumor cell lines and 5 melanoma cell lines.1719
These results as well as our laboratorys observations (gH2AX foci in
untreated cervix and colon carcinomas, fibrosarcoma, osteosarcoma,
glyoma and neuroblastoma cell lines) are summarized in (Table
1) and indicate that untreated cancer cells contain significantly
higher numbers of cryptogenic foci than primary young cultures.
Often these high levels reduce the sensitivity for detecting drug or
IRinduced DSBs. Interestingly, immortalized primary cell cultures
exhibit cryptogenic focal incidences close to those in senescent or
tumor cells.14
Recently, increased spontaneous levels of gH2AX have been
reported in human malignant tumors, such as lung, urinary bladder,
breast, colon and ovarian carcinomas, in melanoma, and in several
corresponding premalignant lesions.20,21 Here we present our data
on gH2AX increases in malignant tumors of various types.
We stained biopsies taken from cancer patients at the time of
surgery, with antigH2AX antibody. Figure 1 shows a comparison
of immunofluorescent staining of cryostat sections of colon tubular
adenocarcinoma, breast invasive ductal adenocarcinoma, ovary
adenocarcinoma, hepatoblastoma and Wilms tumor (nephroblastoma) (bottom panel) with that in normal tissues adjacent to the
tumors (top panel). The nuclei of tubular epithelial cells in normal

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H2AX as a Cancer Biomarker

Table 1

Variations in numbers of gH2AX foci per cell

in untreated cancer cell lines
gH2AX fpc Reference

Cancer Cell Line

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

Banath et al., 2004

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

Wartes et al., 2005

Rao, Pommier

colon, normal breast ductal epithelium, ovary epithelium, hepatocytes

and kidney epithelium contain some low gH2AX staining (top panel),
but markedly, malignant epithelium in all cases contains extensive
increases in nuclear gH2AX staining (bottom panel). Nonspecific
staining may also be outside the nuclei, regions not stained with
propidium iodide, in several tissues. This is due to crossreaction with
a mucinous compound.
Representative images of touch prints corresponding to the
frozen sections in Figure 1 are shown in Figure 2A. We performed a
www.landesbioscience.com

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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H2AX as a Cancer Biomarker

Figure 2. Incidences of gH2AX foci in touch print preparations. A)

Touch prints of human tumors corresponding to the frozen sections
in (Fig. 1) (colon, breast and ovary) and normal tissues adjacent to
tumors were immunostained with antigH2AX antibody. Each image
represents a projection of all optical sections through the nuclei so all
detected gH2AX foci would be presented. Blue, propidium iodide;
green, gH2AX. B) Focal incidences counted in the cell populations
of colon, breast and ovary tumor (gray bars) and normal (black
bars) touch print preparations. Error bars are standard errors.

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.
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