589055

research-article2015
CMSXXX10.1177/1203475415589055Journal of Cutaneous Medicine & SurgeryEl Tawdy et al

Original Article

Journal of Cutaneous Medicine and Surgery

Assessment of Tissue Level of Histone 2016, Vol. 20(1) 40­–43

© The Author(s) 2015
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Deactylase-2 (HDAC-2) in Patients With sagepub.com/journalsPermissions.nav
DOI: 10.1177/1203475415589055

Mycosis Fungoides jcms.sagepub.com

Amira El Tawdy1, Iman Amin1, Rania Abdel Hay1,

Laila Rashed1, and Zeiad Gad2

Abstract
Background: Histone deactylases (HDAC) have a role in the pathogenesis of mycosis fungoides (MF) through their actions
on different apoptosis pathways.
Objective: To assess the possible role played by HDAC-2 in MF by estimating the tissue expression of HDAC2 mRNA in
different stages of MF.
Methods: This study included 28 MF patients and 30 controls. The HDAC-2 levels were detected by real-time polymerase
chain reaction (PCR). Correlations of HDAC-2 levels with clinical presentation and different stages of MF were analyzed.
Results: Mean HDAC-2 level was significantly higher in patients (P < .001) than in controls. HDAC-2 highest mean value was
significantly detected in patients with stage IIb, and the lowest mean value was detected in patients with stage Ia (P < .001).
Conclusion: Up-regulation of tissue HDAC-2 in MF patients might develop a new approach in the understanding of the
pathogenesis of MF. Histone deactylases are important targets for molecular cancer therapeutics.

Keywords
apoptosis, cancer therapeutics, cell cycle, HDAC-2, mycosis fungoides

Introduction antiapoptotic proteins or transcriptional activation of pro-

Mycosis fungoides (MF) is a clonal proliferative disorder of
skin-trafficking T-cells. Epigenetics might play an important
role in the pathogenesis of MF.1 Histones bind to negative-
charged DNA through the positive charge of lysine in the tail
of histones. Modifications of lysine by deacetylation and
acetylation are thought to be crucial in transcription repres-
sion and activation. The removal of the acetyl group of lysine
is done by the histone deactylases (HDACs).2
Eighteen HDACs are identified in humans and are subdi-
vided into 4 classes: HDACs class I (HDACS 1, 2, 3, and 8),
class II (HDACs 4, 5, 6, 7, 9, and 10), and class III and class
IV (HDAC 11).3 Global hypoacetylation of histone is a com-
mon hallmark of cancer, and altered expression of individual
HDACs in tumor samples has been reported.4
HDACs are involved in uncontrolled cell cycle and pro-
hibited differentiation through the cell cycle inhibitor p21/
WAF1. Oncogenic B-cell lymphoma 6 (BCL6) is a transcrip-
tion factor overexpressed in diffuse large B-cell lymphomas.
BCL6 recruits HDAC2 to repress CDKN1A, which encodes
p21/WAF1.5
HDACs can either promote or repress the expression of
some proteins involved in apoptosis. Correspondingly,
HDAC inhibitors (HDACis) can activate the mitochondrial
apoptosis pathway, either by transcriptional repression of
apoptosis proteins.6
Absence of apoptosis and high expression of bcl-2
together with a low expression of apoptosis-inducing gran-
zyme B in the reactive lymphocytes has been reported in MF.
This could explain the chronic nature of the disease and the
poor response to therapy.7 Hence, we hypothesized a possi-
ble role of HDACs in the pathogenesis of MF through its
actions on different apoptosis pathways.
No previous studies investigated the tissue expression of
any HDACs in patients of MF in spite of the reported thera-
peutic success of HDACis in resistant MF patients.8,9
Accordingly, the aim of the present study was to assess
the possible role played by HDAC-2 in MF, which was ful-
filled by estimating the tissue expression of HDAC-2 mRNA
by real-time polymerase chain reaction (PCR) technique, in
different stages of MF.
1
Faculty of Medicine, Cairo University, Cairo, Egypt
2
National Cancer Institute, Cairo University, Cairo, Egypt
Corresponding Author:
Rania Abdel Hay, Faculty of Medicine, Cairo University, 13th Abrag
Othman, Kournish el Maadi, Cairo, 11431, Egypt.
Email: raniamounir@kasralainy.edu.eg
El Tawdy et al 41
Methods
Twenty-eight MF patients and 30 age- and sex-matched
apparently healthy volunteers were enrolled in the present
study. All subjects were recruited from the Dermatology
Outpatient Clinic, Kasr Al-Ainy Hospital, Cairo University,
and each individual signed a written informed consent.
Diagnosis was done based on clinical basis and confirmed
by skin biopsy. Patients suffering from any stage of MF and
not receiving any systemic therapy for at least 6 weeks were
included in the study. Patients receiving any topical or sys-
temic treatment were excluded.
Patients were subjected to thorough history taking and
physical and dermatological examination to determine the
distribution, clinical variant, and extent of lesions. Routine
laboratory investigations, lymph node assessment, chest
x-ray, and abdominal ultrasound were done for proper stag-
ing of the disease.
Staging was based on revised criteria proposed by the
International Society for Cutaneous Lymphomas (ISCL) and
the cutaneous lymphoma task force of the European
Organization of Research and Treatment of Cancer (EORTC).10
Methods
A 4 mm punch skin biopsy was obtained from lesional skin
of patients and from normal skin of controls. The biopsies
were stored at -70°C for measurement of HDAC-2 mRNA.
Detection of HDAC-2 Gene Expression Level
Using Real-Time PCR
RNA extraction and cDNA synthesis.  Total RNA was extracted
from skin tissue using Trizol reagent (Invitrogen, Karlsruhe,
Germany). The quantity and purity of extracted RNA was
assessed by measuring absorbance at 260 nm and the ratio
A260/A280 in a UV-spectrophotometer (NanoDrop Inc,
Wilmington, DE, USA). Only samples with an A260/A280
ratio up to 1.8 were considered valid for real-time PCR.
Reverse transcription of 1 μg of RNA into cDNA was per-
formed using Takara first strand cDNA synthesis kit
(Takara, Bio Inc, Shiga, Japan), according to manufacturer’s
instructions.
Real-time PCR.  The expressions of target gene (HDAC-2)
were quantified using the comparative threshold cycle (Ct)
method where the amount of target mRNA was normalized
to an internal control (glyceraldehyde-3-phosphate dehydro-
genase [GAPDH]). The real-time PCR was performed using
the LightCycler FastStart DNA SYBR-Green I kit (Roche
Applied Sciences, RAS, Mannheim, Germany) according
to the protocol provided in the kit, briefly 10 μL amplifica-
tion mixtures containing equivalent to 8 ng of reverse-tran-
scribed RNA and 300 nM primers; the sequences of PCR
Table 1.  Primer Sequences Used for Real-Time Polymerase
Chain Reaction.
Primer Sequence
HDAC Forward: 5-ATAAAGCCACTGCCGAAGAA-3
Reverse: 5-TCCTCCAGCCCAATTAACAG-3
GAPDH Forward: 5-GGATTTGGTCGTATTGGG-3
Reverse: 5-GGAAGATGGTGATGGGATT-3
Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
HDAC, histone deactylase.
primer pairs used for each gene are shown in Table 1. Reac-
tions were run on an ABI PRISM 7900 HT detection system
(Applied Biosystems, Carlsbad, CA, USA); PCR reactions
consisted of 95°C for 10 minutes (1 cycle), 94°C for 15 sec-
onds, and 60°C for 1 minute (40 cycles). Data were analyzed
with the ABI Prism sequence detection system software and
quantified using the v1·7 Sequence Detection Software from
PE Biosystems (Foster City, CA, USA). Relative expres-
sion of studied genes was calculated using the comparative
threshold cycle method. All values were normalized to the
GAPDH genes.11
Statistical Analysis
Statistical Package for Social Science (SPSS) program
version 17 was used for analysis of data. Data were sum-
marized as mean, standard deviation (SD), number (N),
and percentage (%). The t test was used for analysis of
quantitative data, and 1-way analysis of variance
(ANOVA) test was used for analysis of more than 2 quan-
titative variables. Pearson’s correlation was done to test
linear relation between quantitative variables. P value is
considered significant if <.05, and chi-square test was
used for analysis of qualitative data.
Results
This case control study included 28 MF patients and 30
healthy age- and sex-matched individuals serving as con-
trols. The MF patients were 11 males (39%) and 17 females
(61%), their age ranged from 25 to 62 years (mean, 34.32 ±
17.31 years). The duration of their disease ranged from 0.08
to 15 years (4.42 ± 3.86 years). Twenty patients (71.4%) pre-
sented clinically with patch stage, while 8 patients (28.6%)
presented clinically with plaque stage.
According to the staging of MF, 6 patients (21.4%) had
stage Ia, 6 patients (21.4%) had stage Ib, 8 patients (28.6%)
had stage IIa, and 8 patients (28.6%) had stage IIb.
The controls group included 30 age- and sex-matched
healthy individuals (P = .178 and .118, respectively). They
were 12 (40%) males and 18 females (60%), with age range
from 36 to 55 years (41.80 ± 12.20 years).
42 Journal of Cutaneous Medicine and Surgery 20(1)
The tissue levels of HDAC-2 in MF patients (range, 0.63-
2.70; 1.57 ± 0.59) were statistically significantly higher than
those of controls (range, 0.08-0.22; 0.14 ± 0.05) (P < .001).
In the patients group, there was a significant positive cor-
relation between age of patients and disease duration (r =
0.021; P = .011); however, there were nonsignificant positive
correlations between the tissue levels of HDAC-2 with both
age of patients (r = 0.109; P = .580) and disease duration (r =
0.023; P = .906).
Comparing the tissue levels of HDAC-2 in relation to the
sex difference in MF patients revealed a nonsignificant dif-
ference (P = .961) between their mean value in male patients
(1.58 ± 0.65) when compared to female patients (1.57 ±
0.59).
Comparing the tissue levels of HDAC-2 in relation to the
different clinical presentations in MF patients revealed that
their mean value was higher in patients with plaque stage (1.79
± 0.69) than in patients with patch stage (1.49 ± 0.56). However,
this finding was statistically nonsignificant (P = .232).
On comparing the tissue levels of HDAC-2 in relation to
the different staging of MF in the patient group, we found a
statistically significant difference (P < .001) with the high-
est mean value detected in patients with stage IIb and the
lowest mean value detected in patients with stage Ia (Table 2,
Figure 1).
Discussion
The current study pointed to a possible pathogenic role of
HDAC-2 in MF. The mean tissue level of HDAC-2 mRNA
was significantly higher in MF patients than the controls in
the current work. It has been reported that modulation of his-
tone and protein acetylation alters pathways that promote
proliferation, angiogenesis, and survival in cancer cells. A
common finding in malignant cells is the high level of
expression of HDAC isoenzymes and a corresponding hypo-
acetylation of histones.12 Although we didn’t do immunohis-
tochemical or mRNA in situ hybridization analysis for
accurate HDAC-2 localization, we suggest its expression in
the neoplastic MF lymphocytes.
Altered expression of individual HDACs in several tumor
samples has been previously reported. HDACs play impor-
tant roles in cell cycle progression, cell proliferation, and dif-
ferentiation.13 Thus, HDACs are important targets for
molecular cancer therapeutics.14
To the best of our knowledge, this is the first study done
to estimate the tissue level of HDAC-2 mRNA in different
stages of MF.
In the current study, on comparing the tissue levels of
HDAC-2 mRNA in relation to the different clinical presenta-
tions in MF patients, we reported higher levels in patients
with plaque stage than in patients with patch stage. This
could be explained by the presence of more malignant cells
in MF plaques compared to MF patches. However, this
Table 2.  Tissue Histone Deacetylase Levels (mean ± SD) in
Different Mycosis Fungoides (MF) Staging.
MF Staging Ia (n = 6) Ib (n = 6) IIa (n = 8) IIb (n = 8)
Histone 0.84 ± 0.11 1.17 ± 0.19 1.7 ± 0.2 2.3 ± 0.27
deacetylase
P valuea <.001*
a
P < .05 is statistically significant.
Figure 1.  Mean tissue histone deacetylase levels in different
mycosis fungoides (MF) staging.
finding was statistically nonsignificant. This could be attrib-
uted to the small sample size in our study.
While comparing the tissue levels of HDAC-2 mRNA in
relation to the different staging of MF, we found a statisti-
cally significant difference, with the highest mean value
detected in patients with stage IIb and the lowest mean value
detected in patients with stage Ia. This emphasizes the active
role of HDAC-2 in various stages, especially the advanced
stages, of MF.
Similarly, a previous study done on head and neck cancer
cell lines reported that HDAC-2 expressions in oral squa-
mous cell carcinoma (OSCC) had been implicated mainly in
advanced stages. They suggested a possible link between the
migration/invasion potential of oral cancer cells and the
prevalent expression of HDAC-2.15
Another study done in gastric carcinoma also noted that
HDAC-2 expression appeared to be associated with tumor
aggressiveness as HDAC-2 expression was observed to be
statistically significant in advanced gastric cancer and in
positive lymph node metastasis. Taken together, they sug-
gested that HDAC-2 may play an important role in the
aggressiveness of gastric cancer.4
The use of HDACis in the treatment of various tumors has
been growing over the past years. Histone deactylase inhibi-
tors exhibit potent anti-proliferative effects on tumor cells by
increasing the expression of pro-apoptotic genes and down-
regulating the expression of anti-apoptotic genes to induce
cell cycle arrest and apoptosis.16,17
El Tawdy et al 43
Romidepsin and Vorinostat have been FDA approved for
treatment of refractory MF cases. Both inhibit HDAC I and
II isoforms.9,18 Histone deactylase-2 belongs to class I
HDAC, thus its inhibition might contribute to the reported
therapeutic efficacy of those drugs.
This study lacks the immunohostochemical or the mRNA
in situ hybridization analysis for proper HDAC-2 localiza-
tion. Further studies on HDACs are recommended to delin-
eate more their future promising impact on treating several
types of malignancies. Also, comparing the expression of
HDAC in MF (as neoplastic lymphocytes) and psoriasis (as
inflammatory lymphocytes) is our ongoing research in order
to estimate the role of HDAC in neoplastic versus non-neo-
plastic, inflammatory cells and assess their levels semiquan-
titatively. Comparing involved skin to normal skin in MF
patients is also recommended for better understanding of the
possible role played by HDAC in MF.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, author-
ship, and/or publication of this article.
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