Promoter hypermethylation of p73 and p53 genes in cervical cancer patients among north Indian population

Promoter hypermethylation of p73 and p53 genes in cervicalcancer patients among north Indian population

Abhimanyu Kumar Jha • Mohsen Nikbakht •

Veena Jain • Alka Sehgal • Neena Capalash •

Jagdeep Kaur

Received: 17 November 2011 / Accepted: 9 June 2012 / Published online: 24 June 2012

� Springer Science+Business Media B.V. 2012

Abstract Hypermethylation of CpG islands leads to

transcriptional silencing and it is the predominant mecha-

nism of tumor suppressor gene inactivation in many

tumors. Methylation-specific polymerase chain reaction

was performed to analyse the methylation status of the

promoter region of the tumor suppressor genes. Hyper-

methylation of the 50 CpG island of the p21CIP1, p27KIP1,

p57KIP2, p53, p73 and RB 1 gene promoter were found in

8.8, 8.8, 11.2, 12, 25.6 and 4.8 % of 125 cervical cancer

samples from north Indian population, respectively.

Methylation of p73 was significantly (P \ 0.001) associ-

ated with the cervical cancer cases in comparison to con-

trols. Significant correlation was also observed between the

methylation of p73 gene and increase in the risk of cervical

cancer among passive smokers. Promoter hypermethyla-

tion of p53 gene was also observed to be significant among

oral contraceptive users and cervical cancer patients having

age at first sexual intercourse \20 years whereas hyper-

methylation of other genes was not found to be significant

in the present study. This is the first report showing sig-

nificant hypermethylation of p73 and p53 genes among

cervical cancer patients in north Indian population. This is

also the first report on significant p53 hypermethylation in

cervical cancer in any population. Our findings did not

show any correlation between promoter methylation of p73

and the other genes under study with clinicopathological

parameters, including human papillomavirus infection and

stage of the disease. The frequency of aberrant methylation

of p73 and p53 gene promoter was unchanged according to

the age of patients.

Keywords Hypermethylation � MSP � CpG island �AFSI � HPV

Introduction

Cervical cancer is one of the most common cancers that

affect a woman’s reproductive organs [1]. In frequency, it

is the seventh cancer overall and third among women. In

developing countries, cervical cancer is often the most

common cancer in women and constitutes up to 25 % of all

cancers in female [2]. The risk factors of cervical cancer

include HPV infection and other cofactors like age,

smoking, oral contraceptives, low age at first sexual

intercourse (AFSI), multiple pregnancy, sexual history of

the woman’s male partner, deficient diet and the family

history of cervical cancer.

In quiescent cells, CIP/KIP cyclin dependent kinase

inhibitors (CDKIs) bind to and inhibit the activity of

CDK2, resulting in cell cycle arrest. In cycling cells,

CDKIs of the CIP/KIP family detach from CDK2/cyclin E

complex and bind to CDK4/6 instead, resulting in dere-

pression (and thus activation) of CDK2 and further acti-

vation of CDK4/6, resulting in commitment of the cell to

transit the G1–S cell cycle checkpoint [3]. The CIP family

includes p21CIP1, p27KIP1 and p57KIP2 genes. Increased

Neena Capalash and Jagdeep Kaur contributed equally for this study.

A. K. Jha � M. Nikbakht � N. Capalash � J. Kaur (&)

Department of Biotechnology, Panjab University,

Chandigarh 160014, India

e-mail: [email protected]

V. Jain

Department of Gynaecology, Mohan Dai Oswal Cancer

Treatment and Research Foundation, Ludhiana, India

A. Sehgal

Department of Gynaecology, Government Medical

College and Hospital, Chandigarh, India

123

Mol Biol Rep (2012) 39:9145–9157

DOI 10.1007/s11033-012-1787-5

expression of CDKIs has been recognized as a general

mechanism for cell cycle arrest, and CDKIs have also been

considered to be implicated in tumorigenesis as possible

tumor-suppressor genes [4, 5].

p21CIP1 gene is a downstream effector of p53. Expres-

sion of p21CIP1 is induced by wild-type p53 in the presence

of DNA damage, leading to apoptosis or cell cycle arrest at

the G1 checkpoint [6, 7]. The possible role of p21CIP1 in

oncogenesis has been considered on the basis of its tran-

scriptional control by p53 [7, 8]. It has been reported that

p21CIP1 plays an inhibitory role in the proliferation of acute

lymphoblastic leukemia (ALL) cells [9]. Down-regulation

of p21 transcription has been correlated with tumor pro-

gression [10, 11]. According to several reports, mutations

or deletions of p21CIP1 has not been detected [12–14].

p27KIP1, considered as a tumor suppressor gene, is

located at chromosome 12p13. It is a regulator of the

mammalian cell cycle [15]. Distinct altered patterns of

p27KIP1 protein expression are found in a variety of human

carcinomas, and p27KIP1 expression levels usually correlate

directly with disease-free survival. The mechanism by

which p27Kip1 expression is reduced or lost during

tumorigenesis remains unclear [15]. p57 gene locus is

subject to imprinting, with preferential expression of the

maternal allele that is associated with various cancers. This

establishes its important role in carcinogenesis [16].

Among the various tumor suppressor genes, p53 is more

commonly undergoing changes in most human neoplasia

than any other single gene reflecting its control of critical

cellular activities [17, 18]. It is expressed in almost all

tissues as a constitutive repressed protein. p53 is activated

by DNA damaging agents (genotoxic stress), constitutive

activation of growth signaling cascade (oncogenic stress)

and depletion of ribonucleotides or hypoxia. p53 mutations

are present in the coding region with predominance in

exons 4–9 (DNA binding domain––central core region) and

less often in exon 2 (transactivation domain) and exon 11

(C-terminal domain). The most common mutations occur

in the DNA binding domain at six ‘‘hotspots’’ (codons 175,

245, 248, 249, 273, 282) [19].

The p73 gene is located on chromosome 1p36, a region

frequently deleted in neuroblastoma, melanoma and breast

cancer [20]. It has been considered as a candidate for tumor

suppressor and encodes protein which is structurally sim-

ilar to p53 protein within DNA binding domain and olig-

omerization and transactivating domain. p73 could

promote the programmed cell death (apoptosis) in associ-

ation with cell cycle gene p21 [21]. The p73 gene is rarely

mutated in tumors [22].

Retinoblastoma (Rb) is a neoplasm that can be sporadic or

hereditary and arises from retinal cells. RB gene was the first

tumor suppressor to be identified and was initially discov-

ered in a malignant tumor of the retina known as

retinoblastoma [23, 24].The hyperphosphorylation of Rb1

can inactivate either through aberrant activation of cyclin D1

and cyclin dependent kinases or loss of function of cdk4/6

inhibitors such as p16INK4a causes Rb1 to be hyperphos-

phorylated and this leads to its inactivation [25].

Epigenetic changes are equally responsible as genetic

changes in the development and progression of cancer.

Tumor suppressor genes like p53 can be inactivated not only

through structural changes, like deletion, mutation etc. or by

HPV- E6 but also by lack of expression through promoter

hypermethylation [26]. Aberrant promoter hypermethyla-

tion of tumor suppressor genes has been shown to be

involved in human neoplasia [27]. Of the hypermethylation

events studied in association with carcinogenesis, promoter

CpG island hypermethylation has been frequently investi-

gated in many human cancers, including cervical cancer [25,

28]. CpG islands of a large number of genes, which are un-

methylated in normal tissue, are methylated to varying

extents in many human cancers, and these methylations are a

potential means of tumor suppressor gene inactivation [29].

Materials and method

Sample collection

Biopsy and blood samples were collected with informed

consent of patients diagnosed with cervical cancer after

obtaining the necessary ethical clearance from Mohan Dai

Oswal Cancer Treatment and Research Foundation, Lu-

dhiana, Punjab, India. Biopsy samples (125) were collected

from patients diagnosed with cervical cancer in sterile

water. The blood samples (100) from the healthy females

(control) were collected in tubes with anticoagulant (hep-

arin). The paired biopsy samples (10) as controls were

obtained from the females in which hysterectomy had been

carried out but the cervix was normal. The control samples

were age matched with the patients. The samples were

further used for the extraction of DNA.

Extraction of DNA

Cells obtained from tissue biopsies and blood samples were

lysed in digestion buffer (Tris–HCl 10 mM, pH 8.0, EDTA

10 mM, NaCl 150 mM, and SDS 2 %) containing pro-

teinase K (0.2 mg/ml). DNA was then purified using the

standard phenol–chloroform extraction and ethanol pre-

cipitation [30].

HPV infection and HPV 16 typing

HPV consensus primers, MY09 and MY11 (Table 1), were

used in the PCR assay to amplify an approximately 450-bp

9146 Mol Biol Rep (2012) 39:9145–9157

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fragment from the L1 regions of HPV-16 and 18 [31].

Reaction mixture with no DNA served as a negative con-

trol and 100 ng of HPV-16 viral genomes cloned into

pBR322 was used as the positive control. HPV-16: pBR322

plasmid DNA was a gift from E. M. DeVilliers of Deuts-

ches Krebsforschungszentrum, Heidelberg, Germany.

For typing of HPV-16, the isolated DNA was mixed

with PCR buffer (50 mM KCl, 4 mM MgCl2, 10 mM

Tris–Cl, pH 8.3), HPV-16 E6 primers (Table 1),

200 lM dNTP, 100 mg/ml gelatin, and 2.5 units Taq

polymerase. The reaction mix was subjected to 40 cycles of

amplification, consisting of 1 min at 94 �C, 1 min at 45 �C,

and 1 min at 72 �C using a thermocycler (Bio-Rad). Primer

sets for HPV-16 typing were taken from the published

literature [32]. The size of the amplified product was

206 bp (Fig. 1).

Methylation-specific PCR (MSP)

DNA isolated from biopsy (125) and blood (100) samples

was modified with sodium bisulphite and MSP was carried

out [33] using specific primers for methylation and un-

methylation for the tumor suppressor genes (Table 2). The

amplified products were run on 2 % agarose gel.

Bisulfite sequencing

For sequencing, MS-PCR product was purified by gel

purification kit (Sigma–Aldrich, USA) according to man-

ufacturer manual. The sequencing was carried out by 3100

ABI sequencer and finally the sequence of DNA was col-

lected using the chromatogram.

Statistical analysis

The obtained data was stratified and classified for better

interpretation. The association between hypermethylation

of all gene promoters and with the risk of cervical cancer

was estimated by computing ORs (Odds Ratio) and 95 %

CI using Chi-square, Fisher’s exact test and multivariate

logistic regression analysis, which included several

potential confounding variables (e.g., age as continuous

variables to find out any difference between cases and

controls). The reported odds ratio could be interpreted as

age-adjusted estimates of the relative risk of developing

cervical cancer with methylation of studied genes. Analysis

of continuous data like age was done by one way ANOVA.

Statistical analysis was performed using SPSS, version

11.5 and Epical, version 3.2.c-Coefficient was calculated to

correlate the hypermethylation of tumor suppressor genes

with increase in stage of cervical cancer. P value \ 0.05

was considered as significant.

Results

HPV infection and HPV-16 typing

HPV infection has been shown to be the most important

risk factor for cervical cancer. MY09 and MY11 consensus

primers were used to detect HPV infection. For HPV 16

typing primers from the E6 region were used. The repre-

sentative gels for HPV infection and HPV 16 typing has

been shown in Fig. 1.

It was observed that 116 out of 125 (92.8 %) cervical

cancer patients were HPV positive. In the control group,

95 % samples were HPV negative. Typing was carried out

for HPV-16, which showed that out of the HPV infected

patients, 69.8 % were found to be containing HPV16

(Table 3).

Study of the methylation status of CIP/KIP family

Bisulfite treated DNA from the 125 cervical cancer patients

were analyzed for promoter hypermethylation. The stan-

dard MSP technique was employed to analyze the status of

promoter methylation (Fig. 2). Unmethylation and meth-

ylation specific primers used for MSP analysis were located

within the promoter associated CpG island near the major

transcription site.

Hypermethylation of p21CIP1 and p27KIP1 was observed in

8.8 % of cervical cancer patients whereas hypermethylation

of p57KIP2 was observed in 8 % of cervical cancer patients

(Fig. 3). Statistically significant correlation and risk of cer-

vical cancer was not observed in association with methylation

of these three genes (P value [ 0.05) (Table 4; Fig. 3).

Statistically significant correlation between hyperme-

thylation of the three genes of CIP/KIP family and increase in

the risk of cervical cancer with respect to HPV infection was

not observed (P value [ 0.05) (data not shown). Methyla-

tion of p21CIP1, p27KIP1 and p57KIP2 was not found to be

statistically significant in patients of both the age groups i.e.

Age B 45 years and Age [ 45 years (P value [0.05). The methylation of p21CIP1, p27KIP1 and p57KIP2

showed marginal but insignificant risk of developing

cervical cancer in passive smokers among patients

Table 1 Primer sequences for HPV infection and HPV 16 typing

Primers Sequence

MY09 50-CGTCCMARRGGAWACTGATC-30

MY11 50-GCMCAGGGWCATAAYAATGG-30

HPV16 typing E6 Forward: 50-AAGGCGTAACCGAAATCGG-30

Reverse: 50-CATATACCTCACGTCGCA-30

Where M = A ? C, R = A ? G, W = A ? T and Y = C ? T

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Table 2 Sequences and conditions of MS-PCR primers

S. no. Gene Primer pair and PCR programme Size (bp) References

1 p21-MSP Methylated Forward: 50-TTG GGC GCG GAT TCG TC-30

Reverse: 50-CTA AAC CGC CGA CCC GA-30100 [34]

Initial denaturation 95 �C 5 min

35 cycles

Denaturation 95 �C 45 s

Annealing 62 �C 45 s

Extension 72 �C 1 min

Final extension 72 �C 7 min

2 p21-MSP Unmethylated Forward: 50-CAT GTA CGT TGC TAT CCA GGC-30

Reverse: 50-CTC CTT AAT GTC ACG CAC GAT-30110 [34]


35 cycles





3 p27-MSP Methylated Forward: 50-AAG AGG CGA GTT AGC GT-30

Reverse: 50-AAA ACG CCG CCG AAC GA-30195 [35]


35 cycles



Extension 72 �C 30 s


4 p27-MSP Unmethylated Forward: 50-ATG GAA GAG GTG AGT TAG T-30

Reverse: 50-AAA ACC CCA ATT AAA AAC A-30212 [35]


35 cycles





5 p57-MSP Methylated Forward: 50-CGC GGT CGT TAA TTA GTC GC-30

Reverse: 50-ACA CAA CGC ACT TAA CCT ATA A-3095 [36]


35 cycles





6 p57-MSP Unmethylated Forward: 50-TTT GTT TTG TGG TTG TTA ATT AGT TGT-30

Reverse: 50-ACA CAA CGC ACT TAA CCT ATA A-3095 [36]


35 cycles





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Table 2 continued

S. no. Gene Primer pair and PCR programme Size (bp) References

7 p53-MSP Methylated Forward: 50-ATTTACGGTATTAGGTCGGC-30

Reverse: 50-ACACGCTCCCAACCCGAACG-30285 [37]


35 cycles


Annealing 62 �C 1 min



8 p53-MSP Unmethylated Forward: 50-TTTAAAATGTTAGTATTTATGGTATTAGGTTGGT-30

Reverse: 50-CATCATAAAAAACACACTCCCAACCCAAACA-30310 [37]


35 cycles





9 p73-MSP Methylated Forward: 50-GGA CGT AGC GAA ATC GGG GTT C-30

Reverse: 50-ACC CCG AAC ATC GAC GTC CG-3060 [38]


35 cycles

Denaturation 94 �C 1 min




10 p73-MSP Unmethylated Forward: 50-AGG GGA TGT AGT GAA ATT GGG GTT T-30

Reverse: 50-ATC ACA ACC CCA AAC ATC AAC ATC CA-3069 [38]


35 cycles

Denaturation 94 �C 1 min




11 RB1-MSP Methylated Forward: 50-GGGAGTTTCGCGGACGTGAC-30

Reverse: 50-ACGTCGAAACACGCCCCG-30172 [39]


35 cycles





12 RB1-MSP Unmethylated Forward: 50-GGGAGTTTTGTGGATGTGAT-30

Reverse: 50-ACATCAAAACACACCCCA-30172 [39]


35 cycles





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(P value [ 0.05). Marginal and insignificant risk of cervical

cancer in association with hypermethylation of p21CIP1,

p27KIP1 and p57KIP2 with respect to OC users was observed

(P value [ 0.05). No significant association between meth-

ylation of p21CIP1, p27KIP1 and p57KIP2 and risk of cervical

cancer in association with the AFSI was observed (data not

shown).

Hypermethylation of p53, p73 and RB1

Methylation of p53 was found in 12 % of patients (Figs. 4

and 5). Significant risk of cervical cancer development was

observed in patients with hypermethylated p53 promoter

(P value \ 0.05; OR = 1.78, 95 % CI = 1.49–2.13).

Methylation of p73 was found in 25.6 % of patients and

4 % of controls (Figs. 4 and 5). Statistically significant

difference in methylation of p73 (P value \ 0.001)

between patients and controls was observed. Risk of cer-

vical cancer increased by 1.81 fold with hypermethylated

p73 promoter (OR = 1.81, 95 % CI = 1.50–2.17)

(Table 5). Statistically, significant difference in methyla-

tion of RB1 (P value [ 0.05) between patients and controls

was not observed. The methylation of RB1 did not increase

the risk of cervical cancer in patients (Table 5; Fig. 5).

Statistically significant correlation between hyperme-

thylation of p53, p73 and RB1 and increase in the risk of

cervical cancer with respect to HPV infection was not

Fig. 1 HPV infection and HPV 16 typing a HPV infection was

confirmed by the PCR product of 450 bp. Lanes 1–4 Amplified

product (450 bp) showing HPV infection in the samples. Lane 5positive control. Lane 6 negative control. Lane 7 100 bp ladder.

b HPV-16 typing was confirmed by the PCR product of 206 bp. Lane1 100 bp ladder. Lanes 2–4 Amplified product (206 bp) showing

HPV16 in the samples. Lane 5 HPV 16 negative sample. Lane 6 HPV

16 positive control. Lane 7 negative control

Table 3 HPV infection and HPV 16 typing in cervical cancer

patients and samples

Patients Control

HPV positive

(?) ve 116 (92.8) 5 (5)

(-) ve 9 (7.2) 95 (95)

HPV 16 Typing

HPV 16 (?) ve 81 (69.8) 3 (60)

HPV 16 (-) ve 35 (30.1) 2 (40)

Fig. 2 A representative agarose gel showing MS-PCR product of

a p21CIP1, b p27KIP1, c p57KIP2 (N = sample from normal patient;

MA Marker 100 bp; M Methylated primer; U Unmethylated primer;

NC Negative control)

Table 4 Frequency of methylation of CIP/KIP family genes with

relative risk of cervical cancer between patients and healthy controls

Patient Control OR (95 % CI) P value

n = 125 (%) n = 100 (%)

Methylation of

p21CIP1 11 (8.8) 2 (2) 1.57 (1.21–2.05) 0.059

p27KIP1 11 (8.8) 2 (2) 1.57 (1.21–2.05) 0.059

p57KIP2 10 (8) 3 (3) 1.42 (1.03–1.96) 0.19

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observed (P value [ 0.05) (data not shown). Hypermethy-

lation of p53 and p73 was found to be significant in patients

with age group B 45 years (P value \ 0.05) as well as age

group [ 45 years (P value \ 0.05). The risk of cervical

cancer was increased around 1.87 fold with respect to p73

hypermethylation in association with age group [ 45 years

(OR = 1.87, 95 % CI = 1.48–2.36). Significant risk of

cervical cancer development was also detected in asso-

ciation with age group B 45 years (OR = 1.74, 95 %

CI = 1.30–2.32) (Table 6). Statistically significant relation

was not detected among these two groups (P value [ 0.05)

with respect to hypermethylation of RB1.

The marginal but insignificant risk of cervical cancer

was observed with respect to p53 and RB1methylation in

case of passive smokers (P value [ 0.05) (Table 7). p 73

hypermethylation was found to be statistically significant

in case of passive smokers (P value \ 0.05). Statistically

significant risk of cervical cancer was observed in case of

passive smokers (OR = 1.75, 95 % CI = 1.27–2.41) as

compared to non-smokers (Tables 7 and 8).

Significant risk of cervical cancer was observed with

respect to the methylation of p53 gene in OC users

(P value \ 0.05; OR = 1.80, 95 % CI = 1.50–2.15) as

compared to those not using OC (Tables 9 and 10). But,

statistically significant correlation with risk of cervical

Fig. 3 The percentage of methylation of CIP/KIP family of genes in

patients and controls

Fig. 4 A representative agarose gel showing MS-PCR product of

a p53, b p73, c RB1 (N = sample from normal patient; MA Marker

100 bp; M Methylated primer; U Unmethylated primer; NC Negative

control)

Table 5 Frequency of methylation of p53, p73 and RB1 genes with

relative risk of cervical cancer between patients and healthy controls

Patient Control OR (95 % CI) P value

n = 125 (%) n = 100 (%)

Methylation of

p53 15 (12) 1 (1) 1.78 (1.49–2.13) 0.003

p73 32 (25.6) 4 (4) 1.81 (1.50–2.17) 0.000

RB1 6 (4.8) 3 (3) 1.21 (0.75–1.95) 0.73

Fig. 5 The percentage of methylation of p53, p73 and RB1 genes in

patients and controls

Table 6 Frequency of methylation of p53, p73 and RB1 genes and estimated relative risk with age

Age B 45 years OR (95 % CI) P Age [ 45 years OR (95 % CI) P

Patient

n = 63 (%)

Control

n = 55 (%)

Patient

n = 62 (%)

Control

n = 45 (%)

Methylation of

p53 8 (12.6) 1 (1.8) 1.76 (1.31–2.37) 0.03 7 (11.2) 0 (0) 1.82 (1.52–2.17) 0.02

p73 15 (23.8) 3 (5.4) 1.74 (1.30–2.32) 0.012 17 (27.4) 1 (2.2) 1.87 (1.48–2.36) 0.001

RB1 3 (4.7) 2 (3.6) 1.04 (0.50–2.16) 1.0 3 (4.8) 1 (1.8) 1.41 (0.78–2.55) 0.62

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cancer in association with p73 hypermethylation was not

observed in case of OC users (P value [ 0.05) (Tables 9 and

10). Statistically significant correlation between methylation

of RB1 and risk of cervical cancer was not found in case of

OC users (P value [ 0.05) (Tables 9 and 10).

Statistically significant relation with respect to p53

methylation was detected among patients in the group

AFSI B 20 years (P value \ 0.05). Significant risk of

cervical cancer was detected with respect to the methyla-

tion of p53 gene in patients with AFSI B 20 years

(P value \ 0.05; OR = 1.71, 95 % CI = 1.41–2.08).

Marginal but insignificant risk of cervical cancer devel-

opment was detected in patients with AFSI [ 20 years

(P value [ 0.05) (Table 11). Statistically significant rela-

tion was detected with respect to p73 hypermethylation

among patients with AFSI [ 20 years as well as

AFSI B 20 years (p value \ 0.05). Significant risk of

cervical cancer was observed with the methylation of p73

gene in patients with AFSI B 20 years (OR = 1.76, 95 %

CI = 1.36–2.26) as well as AFSI [ 20 years (OR = 1.78,

95 % CI = 1.35–2.35) (Table 11). Statistically significant

relation was not detected among these two groups in

association with hypermethylation of RB1 gene

(P value [ 0.05).

Bisulfite sequencing

The bisulfite sequencing of 2 samples which were hyper-

methylated in their promoter region of the tumor suppres-

sor genes as compared with wild type (unmethylated

promoter region) revealed conversion of unmethylated

cytosine but not methylated cytosine which could down

regulate the expression of the genes (Fig. 6).

Discussion

Cervical cancer is the third most common cancer in

women, and the seventh overall, with an estimated

5,29,000 new cases in 2008 [1]. It may constitute up to

25 % of all female cancers in developing countries [2].

HPV infection is a cause of almost all the cases of cervical

cancer and this was reflected in the present study also.

The initiation and progression of cancer, is now realized

to involve epigenetic abnormalities along with genetic

alterations. Epigenetics refers to the change in gene

expression without the change in the sequence of the gene

[40]. The epigenetic changes include DNA hypermethyla-

tion, global hypomethylation and histone modifications

[41]. Much of the contemporary research is focused on the

study of epigenetic changes, like promoter hypermethyla-

tion resulting in many types of neoplasia [42]. The present

study was carried out to investigate whether epigenetic

changes like promoter hypermethylation of tumor sup-

pressor genes like p21, p27, p57, p53, p73 and RB 1 with

the different risk factors of cervical cancer, like HPV

infection, age, passive smoking, oral contraceptive use and

AFSI.


respect to passive smoking in cervical cancer patients and healthy

controls

Passive smoking OR (95 % CI) P

Patient Control

n = 26 (%) n = 12 (%)

Methylation of

p53 4 (15.3) 0 (0) 1.55 (1.21–1.98) 0.28

p73 10 (38.4) 0 (0) 1.75 (1.27–2.41) 0.01

RB1 2 (7.6) 0 (0) 1.50 (1.19–1.89) 1.0

Table 8 Frequency of methylation of p73 gene(as this was the only

gene found to be significantly hypermethylated with respect to pas-

sive smoking) among non smokers in cervical cancer patients and

healthy controls

Non smokers OR (95 % CI) P

Patient Control

n = 99 (%) n = 88 (%)

Methylation of

p73 22 (22.2) 4 (4.5) 1.60 (1.29–1.98) 0.04


respect to the use of oral contraceptives in cervical cancer patients

and healthy controls

OC users OR (95 % CI) P

Patient Control

n = 62 (%) n = 43 (%)

Methylation of

p53 8 (12.9) 0 (0) 1.80 (1.50–2.15) 0.01

p73 9 (14.5) 3 (6.9) 1.45 (0.98–2.13) 0.23

RB1 4 (6.4) 2 (4.6) 1.14 (0.63–2.05) 1.0

Table 10 Frequency of methylation of the p53 gene (as this was the

only gene found to be significantly hypermethylated with respect to

the use of oral contraceptives) among OC non users in cervical cancer

patients and healthy controls

OC nonusers OR (95 % CI) P

Patient Control

n = 63 (%) n = 57 (%)

Methylation of

p53 7 (11.1) 1 (1.7) 1.67 (1.22–2.28) 0.07

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Investigations of hematological malignancies and solid

tumors for p21CIP1 promoter hypermethylation, an important

mechanism of epigenetic silencing, have shown variable

results [14, 43, 44]. Loss of expression of p21 has been shown

to be associated with disease progression in patients with

solid tumors, like mantle-cell lymphoma [10, 11]. Hyper-

methylation of p21CIP1 was not found to be significant in

cervical cancer in the population under the study. Significant

correlation pattern of aberrant methylation of p21CIP1 pro-

moter in either of the age groups was not observed

(age B 45 years and age [ 45 years). In addition to this,

hypermethylation of p21 was not found to be significant in

association with passive smoking, oral contraceptives and

AFSI. The results in the present study indicated that p21CIP1

was not observed to be significantly methylated in the

present population and hence, it could not be used as a

diagnostic biomarker at any stage in this disease.

Down-regulation of transcription of p27KIP1 mRNA as

well as protein expression of p27KIP1 protein in hepato-

cellular carcinoma (HCC) has been reported [45–47].

Several reports have shown its inactivation or inhibition to

be involved in tumor development [48, 49]. Low frequency

of hypermethylation of p27KIP1 has been reported in oral

squamous cell carcinoma (4.1 %) and malignant astrocy-

tomas (6 %) [50, 51]. p27 hypermethylation was observed

in 9 % of metastatic malignant melanoma, suggesting that

p27 methylation might be a cause of monoallelic p27

silencing in a small fraction of malignant melanomas [15].

DNA methylation was correlated inversely with the

expression of p27 gene products in pituitary tumor cell

lines, so increased DNA methylation is an important

mechanism for the silencing of p27 gene in the pituitary

tumors [50]. p27 hypermethylation was detected in 11 % of

primary central nervous system lymphomas, and 22 %

were negative for p27 expression [51]. High frequency of

p27 methylation (90 %) was observed in dental follicles,

however, some odontogenic keratocystic (OKC) lesions

(10 %) and normal mucosa samples (33 %) were also

methylated [52]. Due to the role of p27KIP1 in maintaining

cellular quiescence, loss of its expression can be an indi-

cator of the aggressiveness of cancer. p27KIP1 promoter

hypermethylation has been reported in some subtypes of

hematological malignancies, with lymphoma cases repor-

ted to be positive for p27KIP1 methylation in association

with absence of p27KIP1 protein [53, 54]. To the best of our

knowledge no report is available that could directly relate

hypermethylation of p27KIP1 to cervical cancer.

No significant risk of cervical cancer was observed in

association with hypermethylation of p27KIP1 in the present

study. No significant correlation of aberrant methylation of

p27KIP1 promoter was observed with age, HPV infection,

passive smoking, use of oral contraceptives and AFSI. This

result demonstrates that p27KIP1 is an insignificant tumor

suppressor gene in pathogenesis of cervical cancer in north

Indian population. Therefore, the methylation status of the

p27KIP1 gene could not be a diagnostic biomarker at any

stage in this disease.

p57 is an additional biological marker for predicting

increased overall survival in patients with diffuse large B

cell lymphoma (DLBCL) [55]. Hypermethylation mediated

down-regulation of p57KIP2 was reported in different types

of neoplasia like CLL, AML and ALL. In low-grade

MALT lymphomas, p57KIP2 was methylated in 29.2 % of

the cases. In diffuse large B cell lymphomas, methylation

of p57KIP2 was found in 36.4 % of the cases [55]. But, no

report implicates p57 hypermethylation in cervical cancer.

In the present population of study also, p57 hypermethy-

lation was not found to be significant. Hypermethylation

pattern of p57KIP2 in cervical cancer with respect to HPV

infection was not significant (P value [ 0.05). No signifi-

cant correlation of aberrant methylation of p57KIP2 pro-

moter with the increase in the risk of cervical cancer was

observed with age and AFSI. In addition to that, hyper-

methylation of p57KIP2 was not observed to be significantly

increasing the risk of cervical cancer among passive

smokers and OC users. The down-regulation of mRNA

expression of p57KIP2 by its aberrant promoter methylation

is not a common inactivating event in cervical cancer. This

result suggests that p57KIP2 is not a commonly hyperme-

thylated tumor suppressor gene in cervical cancer in north

Indian population. p57KIP2 gene could not be used as a

diagnostic biomarker at any stage in this disease.

Table 11 Frequency of methylation of p53, p73 and RB1 genes with respect to AFSI in cervical cancer patients in comparison to healthy

controls

AFSI B 20 years OR (95 % CI) P AFSI [ 20 years OR (95 % CI) P

Patient Control Patient Control

n = 51 (%) n = 30 (%) n = 74 (%) n = 70 (%)

Methylation of

p53 9 (17.6) 0 (0) 1.71 (1.41–2.08) 0.02 6 (8.1) 1 (1.4) 1.73 (1.22–2.44) 0.11

p73 17 (33.3) 1 (3.3) 1.76 (1.36–2.26) 0.004 15 (20.2) 3 (4.2) 1.78 (1.35–2.35) 0.008

RB1 4 (7.8) 2 (6.6) 1.06 (0.59–1.92) 1.00 2 (2.7) 1 (1.4) 1.31 (0.58–2.95) 1.00

Mol Biol Rep (2012) 39:9145–9157 9153

123

Fig. 6 Bisulfite sequencing of

a p21CIP1, b p27KIP1, c p57KIP2,

d p73, e RB1

9154 Mol Biol Rep (2012) 39:9145–9157

123

The hypermethylation status of the promoter region of

p53 gene was detected in 3 of 26 cases in a study on breast

carcinoma. Methylation in the p53 promoter region is

found in the breast cancer irrespective of the status of

invasion, and that the hypermethylation in the p53 pro-

moter region is an alternative pathway to tumorigenesis

where there is no p53 gene mutation [56]. Mutations in p53

gene are generally associated with majority of cancers and

p53 promoter methylation has been observed in oral

squamous cell carcinoma (OSCC) [37]. Since, cervical

cancer is mainly squamous cell carcinoma, it is important

to study the methylation status of this gene with respect to

cervical cancer. It was observed that the effect of p53

hypermethylation was found to be significant in cervical

cancer in north Indian population (P value \ 0.05). How-

ever, statistically significant correlation between hyper-

methylation of p53 and increase in the risk of cervical

cancer with respect to HPV infection was not observed.

Age was not found to be a factor for increase in risk of

cervical cancer with respect to hypermethylation of p53

gene in the present population of study. In addition to that

hypermethylation of p53 promoter was not found to be

significantly increasing the risk of cervical cancer among

passive smokers but significant increase in risk of cervical

cancer was observed in OC users (P value \ 0.05).

AFSI B 20 years was found to have significant impact on

methylation pattern of p53 in the present population of

study (P value \ 0.05). The present result is the first report

with respect to methylation of p53 in cervical cancer

patients. To the best of our knowledge, this is the first

report which demonstrates the correlation between AFSI

and OC use with respect to p53 promoter methylation in

cervical cancer patients in any population.

Hypermethylation of p53 was found to be at low fre-

quency in the present population of study but since it was

also observed to have significant correlation with some of

the risk factors this study needs to be extended to a larger

population size to ascertain whether p53 can be used as a

significant or reliable marker in cervical cancer in the north

Indian population.

A relatively high incidence of p73 methylation has been

observed among B cell lymphomas, mainly in leukemias

and lymphomas [57, 58]. In hematological malignancies

like ALL and Burkitt’s lymphomas, p73 was observed to

be hypermethylated. The p73 gene encodes a protein

structurally and functionally homologous to p53, and maps

to chromosomal band 1p36.33, where loss of heterozy-

gosity has been observed in up to 90 % of oligodendro-

gliomas and in 10–25 % of diffuse astrocytomas [59]. A

recent study found high incidence of p73 hypermethylation

(38.8 %) in cervical cancer and proposed that epigenetic

modification of p73 via CpG-island hypermethylation

represents a critical alternative mechanism for inactivation

of this gene in cervical cancer [60]. In the present study, an

attempt was made to study the involvement of p73 in

development and progression of cervical cancer in north

Indian population. Significant trend towards increasing risk

of cervical cancer was observed with respect to methyla-

tion of p73 gene in patient samples in comparison to

healthy controls (P value \ 0.001).

Significant trend towards increasing risk of cervical can-

cer was not observed with methylation of p73 gene in asso-

ciation with HPV infection in patient samples in comparison

to healthy control. Significant correlation pattern of aberrant

methylation of p73 promoter was observed in both the age

groups (age B 45 years and age [ 45 years).In addition to

that, hypermethylation of p73 was found to be significantly

increasing the risk of cervical cancer among passive smok-

ers. However no risk was observed in case of OC users. AFSI

was not found to have significant impact on methylation

pattern of p73 in the present population of study as the risk of

cervical cancer was increased significantly in association

with both the groups. In the present population of study,

69.1 % down-regulation of p73 transcription was observed.

In the quantitative study of transcription with real time PCR,

4.5 fold down-regulation of p73 was observed (data not

shown). The analysis of result of semi quantitative and

quantitative RT-PCR showed the significant effect of hy-

permethylated promoter in down-regulation of p73.It was

observed that the down-regulation of expression of p73 by its

aberrant promoter methylation is an important event in cer-

vical cancer patients. The result of the present study support

the existing reports that p73 methylation is significant in

pathogenesis of cervical cancer [60]. Therefore, because of

significant frequency in the present population of study and

also significant correlation with risk factors in this study, it

may be used as a significant and reliable biomarker for

diagnosis of cervical cancer.

The hypermethylation of RB1 is an independent mech-

anism which selectively target RB1 for inactivation, it

further demonstrated the critical role of Rb1 in governing

various aspects of cell cycle. Methylation of RB1 can be

considered as one of prognosis indicator for progression

and development of bladder cancer [61]. According to a

report, out of a panel of 16 genes selected for a study in

cervical cancer, RB1 did not show promoter methylation

[62]. An attempt was made to study the involvement of

RB1 in development and progression of cervical cancer in

north Indian population.No significant effect of RB1 hy-

permethylation was observed in north Indian population.

Our results support the existing report that RB1 methyla-

tion is insignificant in pathogenesis of cervical cancer [61].

Therefore, because of very low frequency in population of

study and also insignificant correlation with risk factors in

this research, it could not be used as a significant and

reliable biomarker for diagnosis of cervical cancer.

Mol Biol Rep (2012) 39:9145–9157 9155

123

No regular pattern of increase or decrease in methylation

was observed in case of p21CIP1, p27KIP1 and p57KIP2 with

the stage of cervical cancer. Similar pattern was found in

case of p53 and RB1. In case of p73 gene, however,

increase in methylation was observed with increase in the

stage. An increasing trend for methylation of p73 was

observed with increasing pathological change in the pres-

ent study which further supports their candidature to be

used as biomarkers. c-coefficient was also found to be

significant for p73 (P value \ 0.05) (data not shown).

The outcome of the present study indicates that hyper-

methylation pattern of p73 gene is a common phenomenon

in cervical cancer. A significant association was observed

between hypermethylation of p73 gene and increase in the

risk of cervical cancer among passive smokers. Although

p53 methylation has not been reported so far in cervical

cancer, it was observed in the present population under

study, although at a low frequency. Promoter hyperme-

thylation of p53 was also observed to be significantly

associated with oral contraceptive users and cervical cancer

patients with AFSI \ 20 years. This is the first report

showing significant hypermethylation of p73 and p53 genes

among cervical cancer patients in north Indian population.

The present findings need to be extended to a larger series.

Acknowledgments The authors acknowledge the financial assis-

tance provided by CSIR (Council of Scientific and Industrial

Research), India to AKJ.

Conflict of interest The authors report no conflict of interest.

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Promoter hypermethylation of p73 and p53 genes in cervical cancer patients among north Indian population