Mol Biol Rep DOI 10.1007/s11033-014-3159-9

Association of mutation and hypermethylation of p21 gene with susceptibility to breast cancer: a study from north India Naseem Akhter • Md. Salman Akhtar • Md. Margoob Ahmad Shafiul Haque • Sarah Siddiqui • Syed Ikramul Hasan • Nootan K. Shukla • Syed Akhtar Husain

•

Received: 16 June 2013 / Accepted: 13 January 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract p21 gene located at chromosome 6p21.2 is a possible tumour suppressor gene involved in the pathogenesis of breast cancer. Both genetic and epigenetic alterations in p21 have been implicated in breast carcinoma. In the present study, our main aim was to study the impact of these two kinds of alterations of p21 gene in Indian female breast cancer patients. A total of 150 female breast cancer patients of north India were screened by PCR-SSCP followed by direct sequencing and methylation specific PCR. Mutational screening of p21 gene revealed significant amount of mutations [32.66 % (49/150)] in exon 2, whereas p21 promoter was found hypermethylated in 42 of 150 (28 %) breast cancer patients in our population. The intriguing feature of the study was the G[T transition (GAG[TAG) at codon 107 and the A[C transition (AGC[CGC) at codon 146 possibly rendering p21 completely ineffective in its anti- proliferative activity. Our results suggest a significant association between the mutational and hypermethylation profile of p21 gene. N. Akhter  Md. S. Akhtar  Md. M. Ahmad  S. Siddiqui  S. I. Hasan  S. A. Husain (&) Department of Biotechnology, Jamia Millia Islamia, New Delhi, India e-mail: [email protected] Present Address: N. Akhter Department of Laboratory Medicine, Faculty of Applied Medical Sciences, Albaha University, Albaha, Saudi Arabia S. Haque Faculty of Pharmacy, Centre for Drug Research (CDR), Viikki Biocentre 2, University of Helsinki, Helsinki, Finland N. K. Shukla Department of Surgical Oncology, Dr. BRA-IRCH, All India Institute of Medical Sciences, New Delhi, India

Therefore, we show for the first time that the significant association of p21 mutation and hypermethylation leads to the complete inactivation of p21 gene in Indian female breast cancer patients. Complete silencing of the p21 gene seems to be the result not only of genetic alterations but also of epigenetic modification. Keywords p21  Breast cancer  North India  Mutation  Hypermethylation

Introduction Globally, breast cancer is the most frequent cancer among women with an estimated 1.38 million new cancer cases diagnosed in 2008 (23 % of all cancers) [1]. An increasing incidence of breast cancer is observed in urban areas of India [2]. Breast cancer is a disease of abnormal gene/pathway function caused by specific genetic and epigenetic alterations in the genome [3]. Genetic abnormalities associated with cancer genes may occur as germline and/or somatic events. Somatic mutations include small sequence changes that activate/inactivate gene function, gross chromosomal rearrangements (resulting in translocations, deletions, duplications and amplifications) and altered gene expression, while epigenetic changes in tumors predominantly result in inappropriate gene silencing [4]. One of the key engines that drive cellular transformation is the loss of proper control of the mammalian cell cycle. The cyclin-dependent kinase inhibitor p21 (also known as p21waf1/Cip1) promotes cell cycle arrest in response to many stimuli [5]. It is well positioned to function as both a sensor and an effector of multiple anti-proliferative signals [6]. Since the cell cycle governs the proliferation and growth of cells, alterations in the cell cycle genes and their products could predispose to tumors.

123

Mol Biol Rep

p21 acts as a mediator of p53 tumor suppressor activity and as an inhibitor of cell cycle progression owing to its ability to inhibit the activity of CDK-cyclin complexes and proliferating cell nuclear antigen (PCNA) [7]. The tumor suppressor activity of p21 stems from its role in inducing growth arrest, differentiation or senescence [6] and its mutation and polymorphism has been studied as a risk factor in various cancers [8–18] although conflicting research exists [19–22]. Aberrant DNA hypermethylation has been recognized as one of the most common molecular alterations in cancer incidence [23]. Hypermethylation of CpG islands in gene promoter regions of many tumor suppressor and DNA repair genes is associated with chromatin condensation, delayed replication, inhibition of initiation of transcription and silencing of genes [24]. In the case of breast tumors, there is evidence of hypermethylation of functionally important genes including those involved in DNA repair (BRCA1) [24], cell cycle regulation (p16) [25], cell adhesion (E-cadherin) [26], hormone and receptor-mediated cell signaling receptors including ER (estrogen receptor) and RAR-b2 (retinoic acid binding receptor-b2), regulation of cell transcription (HOXA5 (homeo box A5). p21 is less frequently mutated or deleted in tumors when compared to other cyclin-dependent kinase inhibitor (CDKI) genes while an alternative mechanism of p21 inactivation with promotor hypermethylation is demonstrated in some hematologic and solid tumors [27–29]. Keeping aforesaid facts in view, the present study aims to evaluate the frequency of p21 gene abnormalities in Indian breast cancer patients along with the association of the abnormalities with clinicopathological characteristics of cancer.

Conventional PCR-SSCP assay DNA was extracted from fresh tumor and normal adjacent tissue samples as described earlier [31]. A typical PCR amplification was performed in a 25 ll reaction volume (10 mM Tris–HCl pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 200 lM of each dNTPs, 100–500 ng of tumor DNA, 0.5 U Taq polymerase and 5 pmol of each oligonucleotide primer) along with negative controls. Exon 2 of p21 gene (codons 1–148) that contained approximately 90 % of the coding region was analyzed using primers from Kawasaki, 1996 [10]. Amplification conditions were, initial denaturation 95 °C for 4 min, 30 cycles of denaturation at 95 °C for 30 s, annealing 55 °C for 30 s and extension 72 °C for 30 s, final extension for 7 min at same temperature. The PCR products were separated by electrophoresis using EtBr-stained 3 % agarose gel in 1X TAE using the Gel doc system (Bio-Rad). High quality DNA was screened for mutations in the p21 gene by Single strand conformation polymorphism (SSCP) analysis performed according to the method described earlier [32]. Briefly, 8 ll of PCR product was added to 2 ll of SSCP gel loading buffer [95 % formamide, 20 mM EDTA (pH 8.0), 0.05 % each of xylene cyanol and bromophenol blue] and denatured for 5 min at 95 °C and immediately chilled on ice for 5 min. 4 ll of this product was subjected to non-denaturing electrophoresis in a 6 % polyacrylamide sequencing gel with 0.5X TBE (Tris– borate EDTA) buffer at 200 V for 12 h at 17 ± 1 °C in SequiGen GT sequencing gel apparatus (Bio-Rad). The gels were stained using a non-radioactive silver staining method and bands were visualized and photographed. Samples that showed band-shifts different from wild-type bands were identified as mutants, and subjected to SSCP analysis.

Materials and methods

Automatic sequencing

Biological specimens

The samples which showed variant band-shifts in SSCP were re-amplified for sequencing. The PCR products were extracted using a Gel Extraction Kit (Qiagen) and then sequenced. DNA sequencing was carried out by The Centre for Genomic Applications, New Delhi, India. To minimize sequencing artifacts, products from at least two different PCRs were sequenced using forward and reverse primers. The sequence analysis was done using chromas 231 (en.bio-soft.net/dna/chromas.html). The BLAST tool was employed for pairwise nucleotide sequence alignment.

One hundred and fifty (n = 150) tissue specimens of different grades of adeno- or infiltratory duct carcinoma of breast cancer and adjacent normal tissues (5–10 mm size), not infiltrated by tumors as confirmed by a pathologist, were collected from B.R. Ambedkar-Institute Rotary Cancer Hospital (BRA–IRCH), AIIMS, New Delhi, India. The biopsies were collected directly into sterile collection vials containing chilled phosphate buffer saline (PBS; pH 7.2), and stored in liquid nitrogen/-70 °C. Breast cancer cases were classified and graded according to WHO criteria and staged according to the criteria of International Federation of Gynecology and Obstetrics [30]. Prior informed consent was obtained from all participating patients. The study was approved by the Ethics Committee and Institutional Review Board of Jamia Millia Islamia, New Delhi, India and All India Institute of Medical Sciences, New Delhi, India.

123

Bisulfite modification of DNA and methylation specific PCR (MSP) The methylation status of the promoter region of p21 gene was determined using the MSP method [33]. Briefly, genomic DNA was modified by treatment with Sodium bisulfite using Methylation direct kit (ZymoResearch). This

Mol Biol Rep

converts unmethylated cytosine to uracil, while methylated cytosine remained unchanged. Modified DNA was stored at -70 °C. For MSP, two sets of primers were used. One pair bound to a sequence in which CpG sites were unmethylated (bisulfite modified to UpG), and a second that recognized a sequence in which CpG sites were methylated (unmodified by bisulfite). PCR was performed using 150–200 ng of modified DNA as a template, 25 ll of reaction mixture containing 2 ll of modified DNA, 1X PCR buffer, 0.25 lM of each primer, 250 lM of dNTPs mix, and 2 units of Ampgold Taq DNA polymerase. Cycling conditions were, initial denaturation 95 °C for 5 min, 40 cycles of denaturation 95 °C for 30 s, annealing 60 °C (for methylated primers) and 65 °C (for unmethylated primers) for 30 s, extension 72 °C for 30 s, and final extension 72 °C for 7 min. Amplicons were checked on 6 % EtBr stained non-denaturing polyacrylamide gels. Protein extraction The biological specimen (about 1 mm3) was thawed at 4 °C in 2 ml PBS containing 1 mmol/litre phenyl methyl sulphonyl fluoride (PMSF) and washed three times using the same buffer. The tissue was then minced-up very finely (\1 mm) in the PBS/PMSF buffer. The tissue sludge was suspended in 0.5 ml Tris-HCI pH 8.0 (10 mmol/l) buffer containing 0.5 % (v/v) Triton X-100 and 1 mmol/l PMSF by vortexing for 1 min. Tissue was disrupted first by vortexing followed by sonication for 30 min. The extract was finally subjected to centrifugation (13,0009g for 30 min at 4 °C) prior to the estimation of protein content in the supernatant using BCA protein assay kit (Pierce, Thermo Scientific). Western blot Equivalent amounts of tissue extracts (15 lg) were loaded onto 10 % SDS-PAGE, and proteins were separated. The separated proteins were electroblotted on 0.45 lm nitrocellulose membranes (Bio-Rad). The membranes were blocked with 5 % non-fat dry milk powder and probed with anti-p21 (Mouse mAb #2946S, Cell signaling) or anti-b-actin (Mouse mAb #A5316, Sigma-Aldrich) primary antibodies for 12 h at 4 °C (1:1000 dilution). After washing, membranes were incubated for 1 h at 20 °C with anti-mouse IgG conjugated to horseradish peroxidase (HRP) secondary antibody (1:3000 dilution). The membranes were treated with enhanced chemiluminescent reagent, and the bands visualized using QuantityOne Software on a Gel doc system (Bio-Rad). Statistical analysis Pearson’s two proportions test was used to compare the mutational and methylation status of p21 gene with various

clinical parameters. Differences with p \ 0.05 were accepted as statistically significant. Statistical analysis was done using SPSS (version 21.0) program.

Results Mutational analysis of p21 gene Mutational analysis of all (n = 150) breast cancer patients’ samples showed that 49 (32.66 %) cases had mutations in exon 2 of p21 gene in north Indian population. Direct sequencing of p21 detected 49 mutations, out of which 27 were mis-sense, 10 were non-sense and 12 were silent (Fig. 1; Table 1). An important alteration at codon 107 and at codon 146 in the carboxy-terminal domain of p21 gene was detected. Codon 146 is involved in binding to PCNA. The G[T transition (GAG[TAG) at codon 107 results in the substitution of glutamic acid by stop codon. Methylation analysis of p21 gene The hypermethylation status of the p21 gene promoter was analyzed in all breast cancer samples. Results showed that the promoter was methylated in 28 % (42/150) of the samples. However, few of the normal control samples also demonstrated methylation of p21 gene (Fig. 2). Majority of the samples showing promoter hypermethylation were from the tumor clinical stage III and IV, i.e., 67 % (28/42) and tumor size pT13 (\15), i.e., 55 % (23/42) (Table 2), indicating a possible relationship between promoter hypermethylation and the progression and poor prognosis of breast cancer. Expression profile of p21 gene in breast carcinoma Protein expression analysis revealed a down-regulation of p21 gene expression in breast cancer samples having missense and non-sense mutations. Samples carrying both hit- mutation as well as hypermethylation revealed a significant loss in p21 protein expression (Fig. 3). Clinicopathological association Breast tumors carrying the mutations were statistically significant for pre-menopausal status. There was however no significant association with age. Breast tumors that were found hypermethylated for the p21 gene promoter were significantly associated with parameters such as pre-menopausal status and poorly differentiated histological grade (Table 2).

123

Mol Biol Rep Fig. 1 Representative image of SSCP and partial electropherogram. a SSCP analysis of p21 gene showing mobility shifts in tumor samples, b , c electropherogram showing the wild type (indicated by arrows) (above) along with its mutant sequence (below)

p21 gene mutation and hypermethylation status: two hit 21.3 % (32/150) of the cancer samples harbored both hitmutation (i.e., inactivation of p21 tumor suppressor gene) and epigenetic silencing (promoter hypermethylation leading to complete inactivation of p21 gene) (Tables 3,4).

Discussion p21 is a putative tumor suppressor gene and its mutations have been studied as a risk factor in various cancers [9]. In the present study, mutational analysis has been carried out to understand the p21 spectrum of mutation and expression patterns. Early reports showed that p21 codon 149 polymorphism is associated with increased cancer risk [14, 15] whereas another report failed to deduce any correlation between p21 mutations and the incidence of oral cancer [16]. We discovered alteration at codon 107 and at codon 146 in the carboxy-terminal domain of the p21 gene, which

123

is involved in binding to PCNA. The binding of p21 to PCNA has been shown to result in G1 and G2 cell cycle arrest in p53 deficient cells [34]. The PCNA binding motif is located in the carboxy-terminal part of the p21 protein between residues 144–151 [35, 36] and alterations in this region results in structural changes in the protein product and leads to differences in binding capacity to PCNA, which are ultimately required for DNA replication and repair. Crystal structure studies of human PCNA complexed with a 22 residue PCNA binding peptide containing this motif have revealed that the p21 carboxy-terminal domain interacts with the inter-domain connector loop of PCNA and is likely to prevent the interaction of PCNA with other components of the polymerase assembly [37] and thus contribute to the antiproliferative activity of p21. The G [ T transition (GAG[TAG) at codon 107 resulted in the substitution of glutamic acid by a stop codon as observed in this present study. This will likely lead to the expression of a truncated protein without the entire C-terminal located PCNA binding region of p21 and thus

Mol Biol Rep Table 1 Details of p21 mutations in exon 2 of Indian female breast cancer patients Codon position

Codon change

Amino acid change

Effect

30 31

CTG[CTT

Leu[Leu

Silent

AGC[AGA

Ser[Arg

Mis-sense/ Polymorphism

70

GGC[AGC

Gly[Ser

Mis-sense

73

CTG[GTG

Leu[Val

Mis-sense

75

AAG[GAC

Lys[Aspartic acid

Mis-sense

78

CTT[GTT

Leu[Val

Mis-sense

79

CCA[TTA

Pro[Leu

Mis-sense

82

CCC[CCA

Pro[Pro

Silent

84

CGA[CAA

Arg[Glutamine

Mis-sense

88

GAG[GAC

Mis-sense

93

AGG[CGT

Glutamic acid[Aspartic acid Arg[Arg

94

CGG[TGG

Arg[Try

Mis-sense

95

CCT[GCT

Pro[Ala

Mis-sense

106

GCA[GCC

Ala[Ala

Silent Non-sense

Silent

107

GAG[TAG

Glutamic Acid[Stop

132

GGT[TGT

Glycine[Cysteine

Mis-sense

146

AGC[CGC

Ser[Arg

Mis-sense

Fig. 2 Methylation status of p21 gene in normal and tumor tissues from the same patient (74 bp). lane N normal breast tissue, T tumor tissue, lane M methylated, lane U unmethylated

likely rendering it ineffective in its antiproliferative activity. Similarly, the A [ C transition (AGC[CGC) at codon 146 resulted a substitution of arginine by serine. This will change the net charge of the domain causing conformational changes which may inhibit p21/PCNA interaction. Therefore, it can be speculated that the p21 mutations at codon 107 and 146 may decrease the antiproliferative activity of p21 and account for increased susceptibility for the development of breast cancer in the Indian population. The G[T transition (GAG[TAG) at codon 107 and concomitant loss of p21 activity presumably has an association with the onset and progression of breast cancer. The presumption is in accordance with earlier reports stating that the somatic deletion of p21 is associated with growth proliferative response to tamoxifen in immortalized human breast epithelial cell lines [38]. Wang et al., further highlighted the role of p21 as a critical mediator of G1 arrest in

DU145 prostate cancer cells by showing that knock down of p21 by siRNA abolishes G1 arrest [39]. While studying the effects of new antiprogestin lonaprisan on the breast cancer cell line, it has been noticed that elevated p21 expression inhibits the cell proliferation and arrests the G0/ G1 phase [40]. However, earlier reports suggest that p21 mutation is rare and infrequent but still significant in a variety of human malignancies [9, 10, 17]. Non-sense as well as mis-sense mutations are observed in the present study. In stage III and IV tumors exhibiting poorly differentiated histological grade. Lymph node positive and ER& PR- negative tumor cell presence are seemingly associated with malignancy and poor prognosis. We found significant association between p21 mutations and premenopause stage that suggests that pre-menopausal women are at a greater risk of developing breast cancer. Current observations are in agreement with the age incidence rates of breast cancer in India suggesting that the disease peaks at a younger age than in western countries [1]. Silent mutations found in the current study do not have any apparent association with clinicopathological variables; however, their function in alternative splicing cannot be ruled out. Silencing of tumor suppressor genes through promoter hypermethylation is a frequent and early event in carcinogenesis and occurs as frequently as genetic mutations in somatic cells thus contributing to overall genetic instability of the tumor [41]. The silencing of tumor suppressor genes, DNA repair genes, and genes related to cancer metastasis and invasion leads to a shift of cells from a normal cellular cycle to a state of high proliferation that favors tumor development and progression [42], but the diagnostic potential of DNA methylation profiling remains largely unexplored. Earlier studies reported the detection of DNA methylation in different cancers and highlighted the potential of DNA hypermethylation for early diagnosis and disease management [43–45]. The CDKI p21 promoter was found hypermethylated in 28 % of breast cancer patient samples which is similar to the mutation frequency observed in this study suggesting that hypermethylation may significantly contribute to genetic instability by altering gene expression patterns [27]. Our findings are thus congruent with previous studies [27–29, 46–49], which suggests that hypermethylation is an alternative mechanism for p21 inactivation. In all promoter hypermethylation breast cancer samples, 67 % (28/42) of tumors showed lymph node positivity and ER- & PR- negativity in clinical stage III and IV tumors. The finding suggests a possible relationship between promoter hypermethylation and the progression of breast cancer in Indian females. It can be speculated that methylation and somatic mutations may be the major loss of function pathways for p21 gene, and methylation might be an early stage event in breast

123

123

69

9

89

pT13(\15)

59

91

PR status Positive

Negative

60

90

I ? II

III & IV

Clinical stage TNM

54

96

Positive

Negative

ER status

61

pT1 (\2) ? pT2 (\5)

Tumor size

141

IDC

ILC

60.0

40.0

60.7

39.3

64.0

36.0

59.3

40.7

6.0

94.0

46.0

54.0

34.7

52

81

65.3

PD

Histological status

62.7 37.3

98

MD?WD

Histological grading

Negative

Nodal status Positive

94

56

68.7

103

Pre-menopausal

31.3

Percentage

47

Total (n = 150)

Post-menopausal

Menstrual status

\50

[50

Age

Variable

33

16

34

15

33

16

28

21

3

46

28

21

20

29

31

18

36

13

Mutants (n = 49)

67.3

32.7

69.4

30.6

67.3

32.7

57.1

42.9

6.1

93.9

57.1

42.9

40.8

59.2

63.3

36.7

73.5

26.5

Percentage

1.375; 0.400 (0.700-2.696)

1.470; 0.310 (0.742-2.907)

1.160; 0.732 (0.589-2.282)

0.914; 0.868 (0.478-1.746)

1.022; 1.000 (0.288-3.664)

1.565; 0.191 (0.821-2.984)

1.30; 0.494 (0.675-2.506)

2.891; 0.002 (1.490-5.607)

1.264; 0.593 (0.619-2.576)

OR; p value (CI 95 %)

Table 2 Effect of p21 mutation and hypermethylation pattern in Indian female breast cancer patients

28

14

27

15

24

18

23

19

2

40

29

13

20

22

24

18

27

15

Methylated (n = 42)

66.7

33.3

64.3

35.7

57.1

42.9

54.8

45.2

4.8

95.2

69.0

31.0

47.6

52.4

57.1

42.9

64.3

35.7

Percentage

1.333; 0.477 (0.654-2.714)

1.167; 0.722 (0.577-2.356)

0.750; 0.472 (0.376-1.494)

0.830; 0.600 (0.419-1.642)

0.783; 1.000 (0.184-3.376)

2.619; 0.009 (1.274-5.374)

1.713; 0.150 (0.863-3.404)

2.238; 0.033 (1.124-4.454)

0.821; 0.582 (0.403-1.672)

OR; p-value (CI 95 %)

Mol Biol Rep

Mol Biol Rep

Fig. 3 Detection of p21 in normal and tumor tissue. Lysates of tumors carrying mutation as well as hypermethylation (two hit) showed least p21 expression. b-actin served as loading control

reports showing that epigenetic silencing is a common mechanism for loss of function for several tumor suppressor genes including p16 [51], BRCA1 [25], APC, BIN1, BMP6, BRCA1, CST6, ESR-b, GSTP1, p16, p21, TIMP3 [52], and p21 [27]. According to Knudson’s proposed two hit hypothesis, disruption of both copies of a given gene is required for the complete loss of function of a tumor suppressor gene [53]. We conclude that our study showed a significant association of p21 mutation with promoter hypermethylation in 32 samples (Table 3). The findings correlate with Knudson’s model, and matched previous studies of VHL syndrome [54], BRCA1 [25] and serine/threonine kinase 11 (STK11) genes [55]. Abnormal methylation of promoters of tumor suppressor genes have been established as the second hit, with intragenic mutations and loss of chromosomal material acting being the first hit [50]. Statistically significant tumor grade was found in relation to mutation and methylation status, (Table 4) suggesting that both of these modifications contribute substantially to the higher grade of breast carcinoma in the Indian population.

Table 3 Statistical relation between promoter hypermethylation and mutation of p21 gene

Conclusion

p21 promoter hypermethylation versus p21 mutation p21 methylation p21 mutation

Unmethylated (n = 108)

Methylated (n = 42)

OR; p-value (CI 95 %)

Wild type (n = 101)

91

10

17.129; 0.000 (7.175-40.851)

Mutant (n = 49)

17

32

Table 4 Correlation of tumor grade versus mutation versus hypermethylation status p21 gene status

Tumor Low grade I ? II

Grade

Fisher’s exact test p-value High grade III ? IV

Double hit (n = 32)

5

27

Single hit (mutation only) n = 17

8

9

Single hit (methylation only) n = 10

4

6

0.0499

The p21 gene, a downstream effector of the p53 tumor suppressor gene is involved in breast carcinogenesis and in other malignancies. The mutational and hypermethylation profile of the p21 gene in the north Indian population revealed that they are associated with breast cancer development and progression. Genetic and epigenetic modifications of p21 gene led to its complete inactivation and the eventual development of breast carcinoma. The epigenetic modification of the p21 gene is an important clinical discovery because of possible reverse epigenetic changes and restoration of the cells gene function. p21 can thus be targeted for therapeutic interventions in addition to its utility as a diagnostic and prognostic marker. Future studies should however involve larger cohorts and longer follow-up periods. Acknowledgments The manuscript was thankfully reviewed by Dr. Shaun Cochrane, Ph.D. graduate from NIMR, MRC, London.The financial support for this research work was provided by the University Grants Commission, New Delhi, India under Grant No. F. No. 34- 219/2008 (SR)/14.01.2009 Conflict of interest

cancer pathogenesis. Interestingly, some normal samples were found hypermethylated for the p21 gene promoter. These samples although not cancerous but may not actually have been normal and might represent appearance of premalignant lesions. Our findings support the revised Knudson two-hit theory [50], and is in agreement with several

The authors declare no conflict of interest.

References 1. Ferlay J, Shin HR, Bray F, Forman D, Mathers C et al (2010) Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 127:2893–2917

123

Mol Biol Rep 2. Steward BW, Kleihues P (2003) World cancer report. International agency for research on cancer (IARC) 3. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674 4. Jones PA, Baylin SB (2002) The fundamental role of epigenetic events in cancer. Nat Rev Genet 3:415–428 5. Cazzalini O, Scovassi AI, Savio M, Stivala LA, Prosperi E (2010) Multiple roles of the cell cycle inhibitor p21(CDKN1A) in the DNA damage response. Mutat Res 704:12–20 6. Abbas T, Dutta A (2009) p21 in cancer: intricate networks and multiple activities. Nat Rev Cancer 9:400–414 7. Waga S, Hannon GJ, Beach D, Stillman B (1994) The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature 369:574–578 8. Mousses S, Ozcelik H, Lee PD, Malkin D, Bull SB et al (1995) Two variants of the CIP1/WAF1 gene occur together and are associated with human cancer. Hum Mol Genet 4:1089–1092 9. Watanabe H, Fukuchi K, Takagi Y, Tomoyasu S, Tsuruoka N et al (1995) Molecular analysis of the Cip1/Waf1 (p21) gene in diverse types of human tumors. Biochim Biophys Acta 1263:275–280 10. Kawasaki T, Tomita Y, Bilim V, Takeda M, Takahashi K et al (1996) Abrogation of apoptosis induced by DNA-damaging agents in human bladder-cancer cell lines with p21/WAF1/CIP1 and/or p53 gene alterations. Int J Cancer 68:501–505 11. Heinzel PA, Balaram P, Bernard HU (1996) Mutations and polymorphisms in the p53, p21 and p16 genes in oral carcinomas of Indian betel quid chewers. Int J Cancer 68:420–423 12. Sjalander A, Birgander R, Rannug A, Alexandrie AK, Tornling G et al (1996) Association between the p21 codon 31 A1 (arg) allele and lung cancer. Hum Hered 46:221–225 13. Facher EA, Becich MJ, Deka A, Law JC (1997) Association between human cancer and two polymorphisms occurring together in the p21Waf1/Cip1 cyclin-dependent kinase inhibitor gene. Cancer 79:2424–2429 14. Ralhan R, Agarwal S, Mathur M, Wasylyk B, Srivastava A (2000) Association between polymorphism in p21(Waf1/Cip1) cyclin-dependent kinase inhibitor gene and human oral cancer. Clin Cancer Res 6:2440–2447 15. Bahl R, Arora S, Nath N, Mathur M, Shukla NK et al (2000) Novel polymorphism in p21(waf1/cip1) cyclin dependent kinase inhibitor gene: association with human esophageal cancer. Oncogene 19:323–328 16. Mitra S, Sikdar N, Misra C, Gupta S, Paul RR et al (2005) Risk assessment of p53 genotypes and haplotypes in tobacco-associated leukoplakia and oral cancer patients from eastern Idia. Int J Cancer 117:786–793 17. Knappskog S, Chrisanthar R, Staalesen V, Borresen-Dale AL, Gram IT et al (2007) Mutations and polymorphisms of the p21B transcript in breast cancer. Int J Cancer 121:908–910 18. Roh JW, Kim BK, Lee CH, Kim J, Chung HH et al (2010) P53 codon 72 and p21 codon 31 polymorphisms and susceptibility to cervical adenocarcinoma in Korean women. Oncol Res 18:453–459 19. Milner BJ, Brown I, Gabra H, Kitchener HC, Parkin DE et al (1999) A protective role for common p21WAF1/Cip1 polymorphisms in human ovarian cancer. Int J Oncol 15:117–119 20. Ressiniotis T, Griffiths PG, Keers SM, Chinnery PF, Birch M (2005) A polymorphism at codon 31 of gene p21 is not associated with primary open angle glaucoma in caucasians. BMC Ophthalmol 5:5 21. Taghavi N, Biramijamal F, Abbaszadegan MR, Khademi H, Sotoudeh M et al (2010) P21(waf1/cip1) gene polymorphisms and possible interaction with cigarette smoking in esophageal squamous cell carcinoma in northeastern Iran: a preliminary study. Arch Iran Med 13:235–242 22. Tornesello ML, Buonaguro L, Cristillo M, Biryahwaho B, Downing R et al (2011) MDM2 and CDKN1A gene polymorphisms and risk

123

23. 24.

25.

26.

27.

28.

29.

30. 31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41. 42.

of kaposi’s sarcoma in african and caucasian patients. Biomarkers 16:42–50 Esteller M (2008) Epigenetics in cancer. N Engl J Med 358:1148–1159 Esteller M (2000) Epigenetic lesions causing genetic lesions in human cancer: promoter hypermethylation of DNA repair genes. Eur J Cancer 36:2294–2300 Esteller M, Silva JM, Dominguez G, Bonilla F, Matias-Guiu X et al (2000) Promoter hypermethylation and BRCA1 inactivation in sporadic breast and ovarian tumors. J Natl Cancer Inst 92: 564–569 Graff JR, Herman JG, Myohanen S, Baylin SB, Vertino PM (1997) Mapping patterns of CpG island methylation in normal and neoplastic cells implicates both upstream and downstream regions in de novo methylation. J Biol Chem 272:22322–22329 Roman-Gomez J, Castillejo JA, Jimenez A, Gonzalez MG, Moreno F et al (2002) 50 CpG island hypermethylation is associated with transcriptional silencing of the p21(CIP1/WAF1/SDI1) gene and confers poor prognosis in acute lymphoblastic leukemia. Blood 99:2291–2296 Zhu WG, Srinivasan K, Dai Z, Duan W, Druhan LJ et al (2003) Methylation of adjacent CpG sites affects Sp1/Sp3 binding and activity in the p21(Cip1) promoter. Mol Cell Biol 23:4056–4065 Bott SR, Arya M, Kirby RS, Williamson M (2005) p21WAF1/ CIP1 gene is inactivated in metastatic prostatic cancer cell lines by promoter methylation. Prostate Cancer Prostatic Dis 8:321–326 Devitt JE (1967) The clinical stages of breast cancer—what do they mean? Can Med Assoc J 97:1257–1262 Sambrook J, Russell DW (2001) Molecular cloning : a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T (1989) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci USA 86:2766–2770 Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 93: 9821–9826 Cayrol C, Knibiehler M, Ducommun B (1998) p21 binding to PCNA causes G1 and G2 cell cycle arrest in p53-deficient cells. Oncogene 16:311–320 Chen J, Jackson PK, Kirschner MW, Dutta A (1995) Separate domains of p21 involved in the inhibition of Cdk kinase and PCNA. Nature 374:386–388 Podust VN, Podust LM, Goubin F, Ducommun B, Hubscher U (1995) Mechanism of inhibition of proliferating cell nuclear antigen-dependent DNA synthesis by the cyclin-dependent kinase inhibitor p21. Biochemistry 34:8869–8875 Gulbis JM, Kelman Z, Hurwitz J, O’Donnell M, Kuriyan J (1996) Structure of the C-terminal region of p21(WAF1/CIP1) complexed with human PCNA. Cell 87:297–306 Abukhdeir AM, Vitolo MI, Argani P, De Marzo AM, Karakas B et al (2008) Tamoxifen-stimulated growth of breast cancer due to p21 loss. Proc Natl Acad Sci USA 105:288–293 Wang Z, Lee HJ, Chai Y, Hu H, Wang L et al (2010) Persistent p21Cip1 induction mediates G(1) cell cycle arrest by methylseleninic acid in DU145 prostate cancer cells. Curr Cancer Drug Targets 10:307–318 Busia L, Faus H, Hoffmann J, Haendler B (2011) The antiprogestin Lonaprisan inhibits breast cancer cell proliferation by inducing p21 expression. Mol Cell Endocrinol 333:37–46 Jones PA, Baylin SB (2007) The epigenomics of cancer. Cell 128:683–692 Esteller M (2005) Aberrant DNA methylation as a cancerinducing mechanism. Annu Rev Pharmacol Toxicol 45:629–656

Mol Biol Rep 43. Syeed N, Husain SA, Sameer AS, Chowdhri NA, Siddiqi MA (2011) Mutational and promoter hypermethylation status of FHIT gene in breast cancer patients of kashmir. Mutat Res 707:1–8 44. Raish M, Dhillon VS, Ahmad A, Ansari MA, Mudassar S et al (2009) Promoter hypermethylation in tumor suppressing genes p16 and FHIT and their relationship with estrogen receptor and progesterone receptor status in breast cancer patients from northern india. Transl Oncol 2:264–270 45. Naqvi RA, Hussain A, Raish M, Noor A, Shahid M et al (2008) Specific 500 CpG island methylation signatures of FHIT and p16 genes and their potential diagnostic relevance in Indian breast cancer patients. DNA Cell Biol 27:517–525 46. Chen B, He L, Savell VH, Jenkins JJ, Parham DM (2000) Inhibition of the interferon-gamma/signal transducers and activators of transcription (STAT) pathway by hypermethylation at a STAT-binding site in the p21WAF1 promoter region. Cancer Res 60:3290–3298 47. Shin JY, Kim HS, Park J, Park JB, Lee JY (2000) Mechanism for inactivation of the KIP family cyclin-dependent kinase inhibitor genes in gastric cancer cells. Cancer Res 60:262–265 48. Go JH (2003) Methylation analysis of cyclin-dependent kinase inhibitor genes in primary gastrointestinal lymphomas. Mod Pathol 16:752–755

49. Moreira PR, Guimaraes MM, Guimaraes AL, Diniz MG, Gomes CC et al (2009) Methylation of P16, P21, P27, RB1 and P53 genes in odontogenic keratocysts. J Oral Pathol Med 38:99–103 50. Jones PA, Laird PW (1999) Cancer epigenetics comes of age. Nat Genet 21:163–167 51. Shima K, Nosho K, Baba Y, Cantor M, Meyerhardt JA et al (2011) Prognostic significance of CDKN2A (p16) promoter methylation and loss of expression in 902 colorectal cancers: cohort study and literature review. Int J Cancer 128:1080–1094 52. Radpour R, Barekati Z, Kohler C, Lv Q, Burki N et al (2011) Hypermethylation of tumor suppressor genes involved in critical regulatory pathways for developing a blood-based test in breast cancer. PLoS ONE 6:e16080 53. Knudson AG Jr, Hethcote HW, Brown BW (1975) Mutation and childhood cancer: a probabilistic model for the incidence of retinoblastoma. Proc Natl Acad Sci USA 72:5116–5120 54. Herman JG, Latif F, Weng Y, Lerman MI, Zbar B et al (1994) Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. Proc Natl Acad Sci USA 91:9700–9704 55. Esteller M, Avizienyte E, Corn PG, Lothe RA, Baylin SB et al (2000) Epigenetic inactivation of LKB1 in primary tumors associated with the Peutz-Jeghers syndrome. Oncogene 19: 164–168

123