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Overexpression of High Mobility Group (HMG) B1 and B2 Proteins Directly

Correlates with the Progression of Squamous Cell Carcinoma in Skin

Article  in  Cancer Investigation · October 2008

DOI: 10.1080/07357900801954210 · Source: PubMed

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 Cancer Investigation, 26:843–851, 2008
ISSN: 0735-7907 print / 1532-4192 online
Copyright 
c Informa Healthcare USA, Inc.
DOI: 10.1080/07357900801954210

ORIGINAL ARTICLE
Imaging, Diagnosis, Prognosis

Overexpression of High Mobility Group (HMG) B1 and

B2 Proteins Directly Correlates with the Progression
of Squamous Cell Carcinoma in Skin
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Ashok Sharma,1 Ruma Ray,2 and Moganty R. Rajeswari1

Departments of Biochemistry,1 and Pathology,2 All India Institute of Medical Science, New Delhi, India

ABSTRACT
High mobility group B (HMGB) chromosomal proteins, which plays important role in cancer
and inflammation, were followed at various stages of the squamous cell carcinoma of skin.
Present results were analyzed by histopathology, BrdU assay, immunohistochemistry, western
blot and RT-PCR, which indicate that at early stages of tumorigenesis, expression of HMGB (B1,
B2), raised only by about 20%. However, the advanced (≥12 weeks) tumors showed significant
(≥80%) increase in HMG levels. Using skin cancer model, we demonstrated that high levels of
HMGB directly correlate with the extent of neoplastic changes, and it appears that HMGB is an
effective stimulus for cell differentiation, tumor progression, and metastatic invasion.
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INTRODUCTION binding they bring conformational changes in DNA like un-

High mobility group B (HMGB) proteins. which consist of
HMGB1 and B2, are non-histone ubiquitous, abundant, nu-
clear proteins and have diverse functions in the cell. HMGB1,
HMGB2 are highly conserved (with more than 80% amino-acid
identify) and have indistinguishable biological properties like
binding to DNA with no-sequence specificity (1–3). However,
they recognize unusual structural element of DNA like four-
way junction, cruciform and cisplatin damaged DNA. Upon
Keywords: High Mobility Group proteins (HMGB1 and HMGB2),
Squamous Cell Carcinomas, Skin tumor, Carcinogenesis,
Benzo(a)pyrene.
We thank the Department of Science and Technology, Govt. of
India, India (Grant DST-SR/SO/AS-55/2002) and Indian Council of
Medical Research, India (Grant ICMR-45/19/2005/BMS/PHA) for
financial support. Ashok Sharma thanks Indian Council of Medical
Research (ICMR-Grant ICMR-45/19/2005/BMS/PHA) for providing
Senior Research Fellowship.
Correspondence to:
Moganty R. Rajeswari
Departmnet of Biochemistry
All India Institute of Medical Science
New Delhi 11029
India
tel: +91-11-26589760; fax: +91-11-26588663
email: mrraji@hotmail.com
winding, bending and supercoiling (4, 5). The main intranu-
clear function of HMGB proteins is to ‘distort’ the double
helix sharply so that proper physical interactions are allowed
between different transcription factors and chromatin (6, 7).
Some of the physiological functions of HMGB proteins are
that they modulate the transcriptional activity of steroid hor-
mone receptors (8), NF-kB, p53-p73transcriptional complexes
(9), homeobox containing proteins or TBP (10), and facilitates
V(D)J recombination (11). They also facilitate the integration of
‘TRANSPOSONS’, such as the Sleeping Beauty transposons
(12).
However, HMGB proteins are not always associated with
chromatin but are also found in cytoplasm and are reported to
have ‘cytokine’ like action (13). Interestingly, HMG B proteins
are shown to play important role in inflammation, and their ex-
pression is strongly correlated with metastatic potential of can-
cer (6, 8). HMGB, therefore, are suggested to be ‘late mediators
of inflammation’, ‘cytokines’ and ‘leaking proteins by necrotic
cells’ (12, 13). The extracellular HMGB proteins interact with
the receptor for advanced glycation end products (RAGE), which
is linked to cell proliferation, migration and metastasis (14). The
expression of HMGB and RAGE are greater in tumors than in
the normal surrounding epithelia as seen for a large variety of
human neoplasms, including colorectal cancer (15), hepatoma
(16), breast cancer (17), pancreatic cancer (18) and melanoma
etc (19).

843
 Skin cancer is the most common form of cancer (male hav-
ing 1:7 and female having 1:5 lifetime chance of developing
skin cancer), with the vast majority being the non-melanoma
skin cancer (97%) (20). The non-melanomas, which include
basal (BCCs) and squamous cell carcinoma (SCCs), need to
be detected early to appropriate treatment them at the right time.
SCCs caused by exposure to UV-light, carcinogens are highly
invasive in nature and unlike BCCs can also metastasize. In the
present study, Benzo(a)pyrene, a well-known ubiquitous car-
cinogen, was chosen because human contact to this agent is very
well known to cause SCCs. Therefore, we investigated the role
of HMGB1 and HMGB2 proteins in benzo(a)pyrene induced
Cancer Invest Downloaded from informahealthcare.com by Wissenschaftliche Bibliothek des Klinikums Grosshadern on 06/15/10
skin carcinogenesis in mice.
MATERIALS AND METHODS
Animals and chemicals
Random bred of 4–6 weeks old Swiss-albino mice (20–
25 g) were procured from the experimental animal facility,
All India Institute of Medical Sciences, were quarantined for
one week and then randomly housed four to five per cage in
filter-capped polycarbonate cages, and provided food and wa-
ter ad-libitum. Animals used in this study were maintained
at experimental animal facility and handled according to an-
imal ethical committee guidelines for the care of laboratory
animals. Benzo(a)pyrene and 5-bromo-2’-deoxyuridine (BrdU)
For personal use only.
were purchased from Sigma Chemical (St. Louis, MO, USA).
Anti-BrdU mouse monoclonal antibodies (Biomeda Co., Fos-
ter City, CA, USA), goat polyclonal anti-HMGB1 and anti-
HMGB2 antibodies were from BD Biosciences. The other bio-
chemicals and the company provided them are given below:
Auto Probe II Streptavidine-Biotin Horseradish Peroxidase kit
(Biomeda Co.), chemiluminescence’s western detection system,
Femto LUCENT (Geno Tech, Inc., St. Louis, MO, USA), Tri-
zol reagent (Invitrogen, Carlsbad, CA, USA), Moloney murine
leukemia virus (MMLV) reverse transcriptase RNAase H mi-
nus, NovaScript III (Life Technology, USA). The study was
performed with the approval of the Institutional Animal Care
Committee (Project clearance no.: 158/IAEC/02).
Experimental procedure
Starting at 5 weeks of age, all mice were assigned into two
groups as control (C) and test group (TA –TE ). Due to high mor-
tality rate and low survival rate, animals were distributed dif-
ferently for different groups as n = 6 (control); n = 8 (TA ),
n = 8 (TB ), n = 10 (TC ), n =14 (TD ), n = 14 (TE ) as shown
in Fig. 1. Skin tumor was induced in test group by applying
benzo(a)pyrene [B(a)P] (0.5 mg/mL acetone), topically to the
shaved areas of their backs, weekly for 20 weeks, while con-
trol group received local application of acetone only. This dose
was determined based on the previous studies (21–23). All mice
were monitored daily for their general, weighed weekly and
assessed every 1–2 weeks for tumor development, and tumors
were counted, measured, and scored as clinically apparent pa-
pillomas or clinically apparent carcinomas. Tumors were eval-
844 A. Sharma et al.
uated by visual inspection by an observer blinded to the exper-
imental group. To assess the validity of the clinically scoring
system, three types of tumor for each type (papillomas and car-
cinoma) and size category (>1 mm2 , ≥4 mm2 , ≥9 mm2 , and
≥16 mm2 ) were submitted for histologic review by a dermath-
opathologist blinded to the clinical score. For histopathological
examination, skin biopsy was taken under aseptic conditions
once every 4 weeks after†º the commencement of the carcino-
gen application. Tissue samples were fixed by immersion in 10%
formalin and embedded in paraffin by standard procedures. Five
micrometer section were cut from the paraffin blocks, stained
with hematoxylin-eosin and examined for histologic findings.
For proliferation assays, BrdU (125 mg/kg in 0.9% NaCl) was
injected (i. p) in to mice. Tumor and adjacent skin from test and
control mice were dissected 60 min. after the injection, fixed,
and processed as previously described.
BrdU labeling indices
Proliferation rate of skin tumor and control skin samples were
determined by standard BrdU assay (24). Sections were incu-
bated with anti-BrdU mouse monoclonal antibodies and visual-
ized by using the Auto Probe II Streptavidine-Biotin Horseradish
Peroxidase kit. Consecutive fields of BrdU-labeled cells were
counted throughout the section. The percentage of labeled cells
(labeling index) was determined by calculating the labeled cell
number: total cell number ×100.
Immunohistochemical analysis of murine
HMGB1 and HMGB2 proteins
Formalin fixed and paraffin-embedded tissue section (5 μm)
were deparaffinized and rehydrated followed by incubation in
0.3% H2 O2 in methanol for 30 minutes then washed with Tris-
buffer saline (pH-7.4). Sections were subjected to heat induced
antigen retrieval in 10 mM citrate buffer (pH-6.0) in a pressure
cooker for 5 min, followed by 45 min cooling at room temper-
ature. After incubation in 3% BSA in TBS, slides were then
incubated overnight at 4◦ C in a humidified chamber with the ap-
propriate antibody. The goat polyclonal anti-HMGB1 (1:100)
and anti-HMGB2 (1:100) antibodies were detected by using
the Auto Probe II Streptavidine-Biotin Horseradish Peroxidase
kit, according to the manufacturer’s instruction. Sections were
counterstained in hematoxylin, dehydrated mounted and cover-
slipped. Morphometric values were obtained by examination of
seven 0.11 mm2 sections per sample with the image analysis
system of Nikon microphoto-FxA.
Protein extraction, acid-urea gel
electrophoresis and western blotting
Total HMG protein was extracted in 5% perchloric acid
(PCA) as according to our earlier procedure (21) from the sam-
ple of control and test groups and resolved on 15% acid-urea
gel electrophoresis. Expression levels of HMGB1 and HMGB2
were determined by western blot analysis (21, 25). Eighty micro-
grams of total HMG proteins was electrophoresed on acid-urea
Cancer Invest Downloaded from informahealthcare.com by Wissenschaftliche Bibliothek des Klinikums Grosshadern on 06/15/10
Figure 1. Flow-chart showing the experimental protocol of the skin tumor induction by benzo(a)pyrene in female Swiss-albino mice with respect
to time. Each group contains at least eight animals.
gel and blotted on nitrocellulose membranes. The membranes
were blocked with 5% (w/v) non-fat dried milk powder in Tris-
For personal use only.
buffered saline containing 0.1% Tween-20 (TBS-T) and mem-
branes were incubated with the appropriate antibodies. The goat
polyclonal anti-HMGB1 (1:200) and anti-HMGB2 (1:200) were
diluted in 2%(w/v) BSA in TBS-T and incubated overnight at
4◦ C, followed by an anti-goat secondary antibody conjugated
with horseradish peroxidase. Antibody reactive bands were de-
tected using the enhanced chemiluminescence western detection
kit (Femto LUCENT). Quantitation of bands was done using
Bio-Rad gel doc system.
RNA isolation and RT-PCR
Total RNA was extracted from control and test groups using
Trizol reagent. First strand cDNA was synthesized by reverse
transcriptase. Two micrograms of total RNA in 20 μL was taken
to which 0.5 μg oligo (dT)18 and 0.25 μL (50U) of Moloney
murine leukemia virus (MMLV) reverse transcriptase RNAase
H minus were added. PCR amplification was performed in a 20
μL volume containing 1 μL of cDNA together with 10XPCR
buffer, 10 mM each deoxynucleotide triphosphates, 1 U of Taq
DNA polymerase and 10 pmol each primer. The mouse HMGB1
(164 bp) primers used were: forward primer, 5 - TGG CAA AGG
CTG ACA AGG CTC-3 and reverse primer, 5 -GGA TGC TCG
CCT TTG ATT TTG G-3 . Similarly, HMGB2 (464 bp) primers
were: forward, 5 -CCG CGA GGA GCA CAA GAA GA-3 and
reverse, 5 -TCC TGC TTC ACT TTT GCC CTT G-3 . As a
control reaction for intact RNA and cDNA, PCR amplification
of the housekeeping gene β-actin was performed on all samples
to normalize the sample amount. Forward primer, 5 -ATG ACG
Overexpression of HMGB Proteins in SCC of Skin
ATA TCG CTG CGC TGG T-3 and reverse primer 5 -AAT ACA
GCC CGG GGA GCA TCG T-3 were used for detecting mice β-
actin (98 bp). Amplification was performed for a predetermined
optimal number of cycles. The PCR products were separated
by electrophoresis on 1.8% agarose gels, which were stained
with 0.5 mg/mL ethidium bromide. To quantify the amount of
products, each gel was analyzed on a Bio-Rad densitometer.
Statistical analysis
Body weights and BrdU labeling indices were analyzed
among all groups by the unpaired Student’s t-test. Quantitative
densitometry values were also compared by unpaired Student’s
t-test (SPSS 10.0 Chicago, IL, USA).P< 0.05 were considered
statistically significant. Values were expressed as means ± SD
as indicated.
RESULTS
Tumor characterization during progression
During tumorigenesis, we noticed simultaneous appearance
of a number of tumors on the backs of mice. Mice started devel-
oping papillomas in 8 weeks time and then incidence increased
slowly but steadily until 20 weeks, when the experiment was
curtailed for ethical reasons. After 20 weeks, 97% of animals
recorded 87 tumors (mean 3.0 ± 0.85/mouse). However, the
survival rate of mice declined after 16 weeks of B(a)P treat-
ment, simultaneously with rapid progression of tumor. Table-
1 summarizes data for development of skin tumor induced by
benzo(a)pyrene. Tumor size was classified as TA > 1 mm2 , TB ≥
4 mm2 , TC ≥ 9 mm2 , and TD ≥ 16 mm2 . The papillomas in mice
845
 Table 1. Skin tumor size and morphological parameters (Pap, C/Pap ratio) at weeks 4, 8, 12, 16, and 20 after Benzo(a)pyrene application

Time (In weeks) Papa TA CA (TB ) CB (TC ) CC (TD ) CD (TE ) Total Carcinoma (C) C/Papa
4 0 2 0 (0) 0 (0) 0 (0) 0 (0) 0 0
8 6 4 2 (2) 0 (0) 0 (0) 0 (0) 2 0.33
12 15 7 12 (5) 1 (2) 0 (1) 0 (1) 13 0.87
16 40 18 4 (8) 15 (4) 18 (5) 6 (5) 43 1.08
20 41 21 1 (9) 18 (6) 26 (5) 7 (5) 50 1.12

≥TA; where 1mm2 <TB < 4 mm2 ≤TC < 9 mm2 ≤TD < 16 mm2 ≤ TE ; CA < 4 mm2 ≤CB < 9 mm2 ≤CC < 16 mm2 ≤ CD ; Papa , total papillomas;
C/Pap, carcinoma/papilloma ratio
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increased in size while retaining their overall phenotype and ir-
regularly shaped carcinomas penetrated the basement membrane
invading the dermis. On histopathological evaluation, the biopsy
of control groups show keratin layer ‘K,’ stratum corneum ‘SC,’
stratum granulosum ‘SG,’ dermis ‘D,’ hypodermis ‘H,’ along
with muscles and displaying reticulo-papillodermal appendages
into fatty subcutaneous tissues of normal skin (Fig. 2(I), A).
The pre-malignant lesion at 4 weeks was also biopsied but
did not reveal any papillomas (Fig. 2(I), B). The tumor sam-
ple biopsies at 8 week and 12 weeks showed dysplasia, hy-
poplastic hair follicles, hyperplastic sebaceous glands, advanced
hyperkeratosis, and acanthosis (Fig. 2(I), C, D). The biopsies
taken after 16 and 20 weeks tumor showed marked prolifera-
tion of squamous epithelium with typical feature of squamous
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cell carcinoma and demonstrated the proliferation of clumped
cells deep down (shown with arrow in Fig. 2(I), E), with pleo-
morphism and the atypsia and also in rapidly dividing nuclei
forming proliferating metastatic island (Fig. 2(I), F). To deter-
mine whether there is an immunological contribution to tumor
progression, the relative numbers of papillomas versus carcino-
mas were scored in test group. Tumor scored as ‘papillomas’
were well-demarcated, symmetrical, pedunculated, or dome-
shaped papules, without erosion or ulceration. Tumor scored
as ‘carcinomas’ were poorly demarcated, asymmetric, nonpe-
dunculated, or dome-shaped papules, with erosion or ulceration
(Fig. 2(I)). The tumor with size < 4 mm2 (Fig. 2(I), B) did not
show characteristics of carcinoma and so carcinomas as per our
classification as: CA ≥4 mm2 , CB ≥9 mm2 , and CC ≥16 mm2

Figure 2. Panel (I) Histopathological analysis of benzo(a)pyrene induced skin tumor. As described in “Materials and Methods,” paraffin-embedded
section from control skin, 4, 8, 12, 16, 20 weeks old tumor were analyzed by haematoxylin-eosin staining. Biopsy of (A) control; tumor samples
of (B) 4 weeks; (C) 8 weeks; (D) 12 weeks; (E) 16 weeks; and (F) 20 weeks. Labels within photographs represents: K = keratin layer; E =
epidermis; D = dermis; H = hypodermis and M = muscles. Magnification x 400; n = 6 normal skin sample; n = 8 skin tumor sample. Panel (II)
Tumor proliferation as seen by BrdU incorporation is shown in control and test groups. Details of groups ‘A’ to ‘F’ are as explained above.

846 A. Sharma et al.

 Table 2. Tumor proliferation index after benzo(a)pyrene application growth pattern at zero time (Fig. 2(II), A, ∼ 3.8 ± 1.1, P < 0.05).
with respect to time Compared with 4 weeks test group (Fig. 2(II), B, 5.7 ± 1.1, P <
0.05) tumor proliferation rate was significantly increased in ad-
BrdU labeling Index (%) vanced tumor of 16 weeks (Fig. 2(II), E, 23.7 ± 1.9, P < 0.05),
Time (in weeks) Control group Test group 20 weeks (Fig. 2(II), F, 25.8 ± 2.8, P < 0.05). The change in
0 3.7 ± 1.2 3.6 ± 1.2 body weights of animals treated with B(a)P along with control
4 3.7 ± 1.4 5.7 ± 1.1 group were comparable throughout the study, weight increase
8 3.6 ± 1.3 9.7 ±2.4 being consistently observed with test group (Figure not shown).
12 3.8 ± 1.2 13.9 ±2.2
16 3.9 ± 1.1 23.7 ± 1.9
20 4.0 ± 1.2 25.8 ± 2.8
HMGB1 and HMGB2 protein expression
The expression of HMGB proteins in squamous cell carci-
Values represent mean ± S.D (P < 0.005)
noma of skin tumor was tested by immunohistochemistry, west-
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ern blot and RT-PCR analysis. The staining of HMGB1 and

(Table 1). The C/Pap ratio of 8 week-test group mice displayed
only 6 papillomas, whereas at 20 weeks, the same mice dis-
played 41 papillomas and 46 carcinomas, giving a maximum
“carcinoma/papillomas ratio” of 1.12 (Table 1). This suggests
that the genetic damage keeps on accumulating until a threshold
is reached which seem to be about 8 weeks time, and survival
rate declined with rapid progression of the tumor.
Skin tumor proliferation
The BrdU staining images (Fig. 2(II), A–F) and labeling in-
dex (Table 2) were examined in both control and test group.
The rate of BrdU incorporation in control group shows normal
For personal use only.
HMGB2 in immunohistochemistry was nuclear in control group
and its intensity increased in 4 weeks tumor to 12 weeks tumor
in test group (Fig. 3 (I, II), A-D). Interestingly, advanced tumor
group of 16 to 20 weeks (Fig. 3 (I, II), E, F) show intense im-
munoreactivity in both cytoplasm as well as in nucleus and also
expression of HMGB1 and B2 was drastically increased.
The identity of HMGB1 and HMGB2 protein was further
confirmed by acid-urea gel electrophoresis (data not shown) and
western blot analysis (Fig. 4). Western blot experiments show
that the HMGB1, B2 expression in control group can be seen
(Fig. 4(a, b). HMGB1 levels are elevated in tumor with 4 to
16 weeks and found maximum (≥100% increased) in 20 weeks
tumor (Fig. 4a, c). Similarly, the levels of HMGB2 was also

Figure 3. Immunohistochemical detection of HMGB1 (Panel I) and HMGB2 (Panel II) protein expression in benzo(a)pyrene induced skin tumor.
HMGB1 and B2 proteins were detected using antibodies against specific peptide. Immunoreactivity appeared in biopsy of (A) Control; (B) nuclear
staining in hyperplasia appeared in the 4 weeks; (C) moderate nuclear staining in epithelial hyperplasia with cellular a typia as seen after 8 weeks;
(D) intense nuclear staining in epithelial hyperplasia with cellular atypsia appeared after 12 weeks; (E) immunoreactivity was increased and also
appeared in cytoplasm in 16 weeks; (F) very high nuclear and cytoplasmic immunoreactivity appeared in 20 weeks tumor sample.

Overexpression of HMGB Proteins in SCC of Skin 847

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Figure 4. Western blots analysis of HMGB1 and HMGB2 protein expression in skin. Eighty micrograms of PCA extracted total HMG proteins
were separated by acid-urea gel electrophoresis and transferred to nitrocellulose membrane. Immuno-blots were probed with the specific goat
polyclonal IgG anti-HMG proteins respectively. Antibody reactivity was detected by using Femto LUCENT reagent after a 1:10000 dilution of the
secondary HRP-conjugated antibody. Lanes 1-6, corresponds to control skin, 4, 8, 12, 16, 20 weeks old skin tumor, respectively, for (a) HMGB1,
(b) HMGB2. Corresponding densitometry analysis is given in (c). The bar represents mean of ± S.D, control group (n = 6) and test groups (n =
8).
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higher in advanced tumors (>100%) (Fig. 4b, c). We found that
the mRNA expression of hmgb1 and hmgb2 gene using RT-PCR
showed upregulation in benzo(a)pyrene induced carcinogenesis,
a feature similar to that of protein. Gene expression was found
to be maximum in advanced 16 to 20 weeks old squamous cell
carcinoma. However, only moderate level of gene expression of
hmgb1, b2 was found in the control group (Fig. 5a, b).
DISCUSSION
Benzo(a)pyrene is a potent environmental carcinogen found
in cigarette smoke, charred foods, and in the out put of internal
combustion engines and power plants. (26). B(a)P is enzymati-
cally converted to active metabolite, 7,8-dihydroxy-9,10-epoxy-
7,8,9,10-tetrahydro benzo(a)pyrene, known as BPDE (27). Al-
though several stereo-isomers of BPDE can be formed, the ma-
jor stable form is the (+) –trans-anti-BPDE. BPDE interacts
with nucleophilic sites on cellular proteins, RNA and DNA.
BPDE forms adducts with DNA via BPDE- N2 - dG (28). It is
reported that generally, cells at early treatment of B(a)P showed
adducts of ‘external’ nature while cells at later stages of treat-
ment contained DNA–BPDE adducts involving ‘base-stacking.’
The base-stacked DNA-BPDE adducts are less readily reparable
and so induce more mutations in gene (29–31). In the present
study, the initial genetic damage (at 4 and 8 weeks) by BPDE
in mice is reflected in the physical characteristic like loss of
fur, poor hair growth, and increase in body weight. It is also
clear that the initial tumor type (before 8 weeks) in B(a)P in-
duced murine skin are predominantly benign papillomas which
are due to keratinization (Fig. 2, I).
The accumulation of genetic damage after 8 weeks of B(a)P
treatment is reflected as a lesions and inflammation which are
noticed in 12 to 20 weeks old tumors. The survival rate of mice
decreased as tumor progressed and at the end of 20 weeks of
B(a)P treatment, only 40% of mice survived (data not shown).
However, the body weight increased as there is tumor prolif-
eration rate (Fig. 2(II), E, F) and tumor acites are formed in
advanced tumor (Fig. 2(I), E, F). The manifestation of advanced
metastatic squamous cell carcinoma is apparently, dependent on
dose and time-span of B(a)P treatment. Tumors of 16, 20 weeks
old (Fig. 2(I), E, F) reveal epidermal hyperplasia, high mitotic
indices and increased density of dark and pyknotic cells and
high number of giant keratinocytes. Obviously, BPDE causes
DNA damage and mutagenecity leads to activation of a num-
ber of genes like p53 (31), proto-oncogenes such as c-Ha-Ras
(32). HMG proteins being nuclear proteins are expected to play
an important function on the B(a)P-damaged DNA. We reported
earlier that B(a)P induced skin tumors show high level of HMGA
family of proteins (21, 25). HMGA family is also known to act
as transcriptional regulators, and their high expression is seen
during embryogenesis and neoplastic transformation (33, 34)
but not in normal adult. However, unlike HMGA, HMGB are

848 A. Sharma et al.

Cancer Invest Downloaded from informahealthcare.com by Wissenschaftliche Bibliothek des Klinikums Grosshadern on 06/15/10
Figure 5. RT-PCR analysis of the hmgb1 and hmgb2 gene expression in B(a)P induced skin tumor. (a) The products of HMGB1 and HMGB2
were analyzed on 1.8% agarose gel. β-actin products (90 bp) are shown as a positive control for cDNA integrity. Lanes 1–6, represents control
and 4, 8, 12, 16, 20 weeks old tumor, respectively. (b) Densitometry analysis of the bands seen in (a).The other experimental conditions are
same as in Figure 4.
constitutively expressed and are abundant in chromatin. How-
ever, because of their dual existence in nucleus and in cytosol
For personal use only.
(also in extracellular region) HMG got a lot of attention. Bianchi
et. al. have done extensive work on HMG proteins (2, 6, 7, 8, 35,
36, 41, 45). HMGB proteins, in the recent past, have been given
several names, ‘potent inflammatory mediators,’ ‘cytokines,’
‘leaking proteins.’ Therefore, the cellular function of HMGB
is getting curieser and curieser day by day (35). The overex-
pression of HMGB corresponds to infection, inflammation and
in cancers including colorectal adenocarcinoma, hepatocellular,
gastric, leukemia, breast cancer (35–39). In melanoma, upreg-
ulation of HMGB1 is shown to closely relate to melanoma in-
hibitory activity (MIA) expression (40). However, HMGB role
in squamous cell carcinomas, which correspond to 80% of non-
melanoma skin cancers, is not being studied.
Squamous cell carcinoma at benign stage (up to 8 weeks of
B(a)P) showed only a small increase (by about 25%) in HMGB1,
B2 as compared to expression in control group as seen by west-
ern blot for protein (Fig. 4) and RT-PCR for mRNA (Fig. 5). IHC
indicates that HMGB1 and B2 are confined to the nucleus (Fig. 3,
A–C). However, the advanced tumors showed a significant rise
which is more than 100% in the levels of HMGB1 and B2 (Figs.
4 and 5). Interestingly, the HMGB expression in metastatic skin
tumor is also seen in cytoplasm (Fig. 3, D–F). It may be recalled
that the HMGB protein binding to chromatin is highly dependent
on its extent of acetylation; hyperacetylation leads to leakage
from nucleus, and HMGB, thus, spilled from chromatin induces
several proinflammatory responses (41). This extracellularly re-
leased HMGB protein interacts with the receptor for advanced
glycation end products (RAGE), a multi-ligand member of the
immunoglobulin superfamily of cell surface molecules (42-44).
Overexpression of HMGB Proteins in SCC of Skin
RAGE triggers the activation of key cell signaling pathways,
such as p21ras, MAP kinases, NF-kB and cdc42/rac, which leads
to cellular migration and tumor progression (8, 45–47). Further,
B(a)PDE can also directly induce AP1 activation, which perhaps
leads to transcriptional activation of HMGB1, B2 gene through
PI-3K/Akt/JNK pathways (26, 27). Thus, strategies that target
HMGB proteins with specific antibodies or antagonists have po-
tential for treating cancer and other lethal systemic inflamma-
tory diseases. In conclusion, our data reveal that HMGB1 and
B2 proteins act as key regulators in promotion of squamous cell
carcinomas of skin. Finally, better understanding of the molec-
ular mechanisms underlying HMGB function could yield novel
therapeutic approaches to anti-cancer strategies.
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