This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ping Siu, L. L. Articles by Kwong, Y.-L. Search for Related Content PubMed PubMed Citation Articles by Ping Siu, L. L. Articles by Kwong, Y.-L.

(American Journal of Pathology. 2002;160:59-66.)
© 2002 American Society for Investigative Pathology

Short Communications

Specific Patterns of Gene Methylation in Natural Killer Cell Lymphomas

p73 Is Consistently Involved

Lisa Lai Ping Siu^*, John Kwok Cheung Chan, Kit-Fai Wong and Yok-Lam Kwong^*

From the Department of Medicine,^*
Queen Mary Hospital, Hong Kong, and Department of Pathology,
Queen Elizabeth Hospital, Hong Kong, People’s Republic Of China

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Aberrant methylation of promoter CpG regions is a putative mechanism whereby tumor suppressor genes are inactivated. We used a candidate gene approach to investigate the patterns and significance of this epigenetic change in natural killer (NK) cell malignancies. Thirty-three patients were studied for promoter methylation in five putative tumor suppressor genes by methylation-specific polymerase chain reaction (MSP), which has a sensitivity of 10^-3. The p73 gene was methylated in 94% of cases, a frequency that is the highest known for any human malignancy. In the NK cell lymphoma line NK92, p73 was also completely methylated, and the p73 transcript was correspondingly not detectable by quantitative polymerase chain reaction. Treatment of the cell line with 5-azacytidine, a demethylation reagent, led to demethylation of the p73 promoter and reinduction of p73 gene expression. These results suggested that promoter CpG methylation might be an important mechanism in suppressing p73 gene expression in NK cells. Other methylated genes included hMLH1 (63%), p16 (63%), p15 (48%), and RARß (47%). Methylation of two or more genes occurred in 88% of cases. With promoter methylation as a molecular marker, MSP identified two cases of occult marrow metastasis. Interestingly, the primary tumor and metastasis showed different methylation patterns, implying that separate clonal evolutions might have occurred at these sites. Furthermore, MSP also identified tumor infiltration in random oropharyngeal biopsies in a case where histological examination could not show evidence of tumor involvement. We conclude that NK cell malignancies show a specific pattern of promoter methylation, with p73 being consistently involved. These results suggest that p73 may be an important target in the neoplastic transformation of NK cells, and the demonstration of its methylation may serve as a potential molecular tool for NK cell lymphoma detection.

Few genetic alterations are known in NK cell lymphomas. Recurrent chromosomal translocations have not been identified so far.⁶ Previous studies using comparative genomic hybridization⁷ and loss of heterozygosity analysis⁸ showed that deletions of chromosomes 6q, 11q, 13q, and 17p might be important in the initiation and progression of this tumor. Although these genetic alterations may give useful information on lymphomagenesis, the techniques for their recognition cannot be used for the sensitive detection of the tumor cells. Therefore, the definition of minimal tumor involvement/infiltration of organs and the monitoring of treatment by molecular techniques has not been possible. Recently, epigenetic silencing of gene expression by CpG island hypermethylation has been shown to be important in cancer formation.⁹ Experimental data from the study of numerous tumors have shown that when these CpG islands are methylated, the expression of the corresponding genes is suppressed. Therefore, DNA methylation may be an alternative mechanism to mutations or deletions in disrupting tumor suppressor gene function in carcinogenesis.

In this study, we investigated the promoter methylation of five putative tumor suppressor genes in a series of NK cell lymphomas, with a view to defining the patterns of methylation, as well as exploring the use of aberrantly methylated genes as molecular markers for tumor detection.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Patients

A total of 33 (22 nasal, 6 non-nasal, 5 aggressive) cases of NK cell lymphomas were investigated. All tumors were CD2⁺, CD3^-, CD3⁺ and CD56⁺, with EBV demonstrable by in situ hybridization in all but one case (case 27). Two NK cell lymphoma lines, HANK1 and NK92, were also studied.

Bisulfite Conversion of DNA Samples

DNA was extracted from frozen tumor tissues with standard phenol-chloroform protocols. For archival paraffin sections, the QIAamp DNA mini kit (Qiagen, Hilden, Germany) was used for DNA extraction. DNA was modified by the bisulfite reaction using the CpGenome DNA Modification Kit (Intergen, Purchase, NY). After completion of the reaction, all unmethylated cytosines are deaminated and converted to uracil, while methylated cytosines remain unchanged. Thus, methylated and unmethylated genomic regions following bisulfite conversion could be distinguished by sequence-specific polymerase chain reaction (PCR) primers.¹⁰

Methylation-Specific Polymerase Chain Reaction

The methylation status of the promoter region of the genes studied was determined by methylation specific polymerase chain reaction (MSP) using bisulfite modified DNA.¹⁰ Primer sequences for the methylated and unmethylated alleles were as published for p15, p16,¹⁰ p73,¹¹ hMLH1, and RARß.¹² The forward and reverse primer sequences for the methylated (M_F and M_R) and the unmethylated alleles (U_F and U_R) were shown in Table 1 . MSP was performed in a final volume of 50 µl containing 5 µl bisulfite modified DNA, 250 µmol/L dNTP (Life Technologies, Gaithersburg, MD), 1 µmol/L of each primer (Genosys, Cambridgeshire, UK), 1.5 to 3 mmol/L MgCl_2, 1x PCR gold buffer, and 2U AmpliTaq Gold (PE Biosystems, Foster City, CA), in an MJ PTC-200 thermocycler (MJ Research, Waltham, MA) with the following cycling parameters: 95°C for 12 minutes, 32 to 40 cycles of 94°C for 1 minute, specific annealing temperature for 1 minute, 72°C for 1 minute, and a final extension step at 72°C for 10 minutes. PCR products were separated on 7.5% nondenaturing polyacrylamide gel and visualized by ethidium bromide staining. Normal and methylated DNA (Intergen) were used to optimize the MSP conditions, and included as normal and positive controls in every experiment.

MSP was performed in duplicate, except in cases with insufficient DNA (Table 2) . As controls, amplifications for the unmethylated/methylated alleles on bisulfite-modified normal/methylated DNA, and no template control were performed. To test the sensitivity of the MSP, the NK92 DNA showing complete methylation at the p73 locus was serially diluted in normal DNA, bisulfite treated, and amplified with primers for the methylated allele.

PCR products were electrophoresed in 2% agarose gel. The specific band was excised and purified by the Qiaex II gel extraction kit (Qiagen). PCR products were sequenced directly in both directions with the same primers used for MSP. Each sequencing reaction contained 5 µl of purified DNA, 3.2 pmol of primer, 4 µl of sequencing mix (dRhodamine Terminator Cycle Sequencing Ready Reaction Kit, PE Biosystems) in a final volume of 10 µl, and was performed according to the manufacturer’s instructions. Sequencing products were purified by the DyeEx Spin Kit (Qiagen) and analyzed by an automated DNA sequencer (ABI Prism 377, PE Biosystems).

5-Azacytidine Treatment of NK92 Cell Line

The NK92 lymphoma cell line was cultured in RPMI 1640 medium with 10% fetal bovine serum (Life Technologies) supplemented with interleukin-2 (IL-2) (300 U/ml). The demethylation agent 5-azacytidine (5-AC) (Sigma, St. Louis, MO) was used to reinduce the expression of methylated genes. NK92 cell lines were cultured in the absence and presence of 5-AC at 1 µmol/L and 3 µmol/L for 3 to 9 days.

Quantification of p73 Gene Expression

Total cellular mRNA was extracted from the cultured NK92 cell pellets with the QIAamp RNeasy kit (Qiagen). The expression of the p73 gene was quantified by a real-time quantitative PCR (Q-PCR) technique with an automated DNA sequence detector (ABI Prism 7700 Sequence Detector, PE Biosystems). The primer sequences for the p73 gene (p73 Q_F and p73 Q_R), and the TaqMan probe (p73 probe) dual-labeled at the 5' end with 6-carboxyfluorescein (FAM) and the 3' end with 6-carboxytetramethylrhodamine (TAMRA), were designed by the Primer Express software (PE Biosystems), to span at least one intron to prevent amplification of contaminating DNA (Table 3) . The gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was similarly amplified as an internal control for RNA amount and integrity (Table 3) . First strand cDNA synthesis and the subsequent Q-PCR were performed by the Taqman EZ RT-PCR kit (PE Biosystems). Real-time PCR amplification data were collected continuously and analyzed with the Sequence Detection System (ABI Prism 7700 Sequence Detector, PE Biosystems). The threshold cycle (C_T) at which a significant increase in fluorescence signal was first detected was set at a minimum of 10 standard deviations above the mean baseline fluorescence. The copy number of target sequences in the tested samples was inversely proportional to and hence could be deduced from the C_T.

	Results

Top Abstract Materials and Methods Results Discussion References

The specificity of MSP was shown by the inclusion of appropriate positive and negative controls (Figure 1) . DNA methylation was only detected in methylated DNA but not in normal DNA samples. Sequencing of the PCR products also confirmed the expected patterns of bisulfite-induced changes (Figure 1) . When performed in duplicate, all of the patient samples gave concordant results. Finally, the MSP had a sensitivity of 10^-3 (Figure 1) .

View larger version (48K): [in this window] [in a new window]   Figure 1. MSP results of NK cell malignancies. A: Sequencing of PCR product obtained with primers for the methylated allele of p73. The 3' end of the product is shown, with the sequence of the wild-type p73 aligned for comparison. In the MSP product, methylated cytosine residues remaining unchanged are underlined in blue, and the unmethylated cytosine residues that have become uracil/thymidine are underlined in red. B: Sensitivity of MSP, showing detection of the methylated p73 at 10-3. Lane MW: molecular weight marker; lanes 1 to 6: serial dilution of bisulfite-modified DNA of NK92 cell line from 100 to 10-5; N: bisulfite-modified normal DNA; W: water control. C: MSP of p73. PCR product in lane U (unmethylated) indicates the presence of the unmethylated allele, and in lane M (methylated) the methylated allele. MW: molecular weight marker; W: water control; N: normal control DNA; P: methylated control DNA. Case number is indicated at the top. p73 methylation was found in cases 33 and 2, but not in case 13. U, 69 base pairs (bp); M, 60 bp. D: MSP of hMLH1, showing gene methylation in cases 12 and 20, but not in case 2. U, 107 bp; M, 106 bp. E: MSP of p16, showing gene methylation in cases 25 and 3, but not in case 33. U, 151 bp; M, 150 bp. F: MSP of p15, showing gene methylation in cases 32 and 4, but not in case 33. U, 154 bp; M, 148 bp. G: MSP of RARß, showing gene methylation in cases 2 and 31, but not in case 12. U, 95 bp; M, 93 bp.

Of the five genes examined, p73 was the most frequently methylated, occurring in 31 of 33 (94%) of cases (Table 2) . Both NK cell lymphoma lines, HANK1 and NK92, showed methylation of the p73 gene. The two other more commonly methylated genes were the DNA mismatch repair gene hMLH1 (20 of 32, 63%) and the cyclin-dependent kinase gene inhibitor p16 (20 of 32, 63%). p15 and RARß were less frequently methylated (12 of 25, 48% and 15 of 32, 47% respectively).

Reinduction of p73 Expression with Promoter Demethylation

The effect of promoter methylation on gene expression was tested for p73, since it was the most frequently methylated gene and therefore an important candidate in NK cell lymphomagenesis. The NK92 cell line was fully methylated at the p73 gene, as MSP did not show any PCR product with the unmethylated primers (Figure 2A) . Accordingly, no p73 transcript could be detected with Q-PCR (Figure 2, B and C) . Treatment of the cell line with 5-AC for 1 to 3 days resulted in demethylation of the p73 gene, as shown by appearance of positive amplification with the unmethylated primers (Figure 2A) . There was also reinduction of p73 gene expression as shown by Q-PCR (Figure 2, B and C) , confirming the functional importance of methylation in epigenetic silencing of the p73 gene expression in NK lymphoma cells. Furthermore, the growth rate of the NK92 cell line was retarded after reinduction of p73 expression with 5-AC (data not shown).

View larger version (30K): [in this window] [in a new window]   Figure 2. Reinduction of p73 gene expression in NK92 cell line by 5-AC at 1 µmol/L and 3 µmol/L. A: MSP of p73 on the NK92 cell line before and after 5-AC treatment. With the untreated cell line (day 0), PCR product was obtained only from the methylated primers (lane M) but not from the unmethylated primers (lane U). This indicates that the NK92 cell line is fully methylated at the p73 gene. Treatment of the cell line with 5-AC for 1 to 3 days resulted in de-methylation of the p73 gene. Consequently, PCR product was obtained from the unmethylated primers (lane U) as well. Amplification with the methylated primers (lane M) was weak, indicating that the p73 gene was de-methylated in the majority of the cells. MW: molecular weight marker. B: Amplification plot of real-time Q-PCR on p73 gene expression using the NK92 cell line before and after 5-AC treatment at 1 µmol/L. The y axis represents the change in Rn, which is a ratio of the fluorescence signal of the reporter dye (FAM) to that of the passive reference dye (ROX). The x axis is the threshold cycle (CT). The copy number of the target sequences in the tested sample is inversely proportional to the CT value. The p73 gene expression in the untreated cell line was undetectable (below the baseline). On addition of 1 µmol/L 5-AC, the CT values were 28.5, 27.5, and 26.5 for day 3, day 6, and day 9, respectively. This shows that 5-AC reinduces the expression of p73. The amplification profile of GAPDH, however, was similar before and after 5-AC treatment at around CT 17. C: Amplification plot of real-time Q-PCR on p73 gene expression using the NK92 cell line before and after 5-AC treatment at 3 µmol/L. The p73 gene expression in the untreated cell line was undetectable. On addition of 3 µmol/L 5-AC, the CT values were 27, 25.5, and 26.5 for days 3, 6, and 9, respectively.

There was no discernable difference in gene methylation in the different subtypes of NK cell malignancies. However, tumors at different sites were available for the analysis of methylation patterns in five cases. A differential pattern of gene methylation was found in the primary and metastatic lesions in two cases (cases 1 and 2). In both cases, the marrow was morphologically normal, but occasional EBV positive cells were found in case 2. MSP showed aberrant gene methylation in both cases, suggesting occult infiltration by lymphoma cells; which in fact became clinically manifest later in the course of disease (Table 2) . In both cases, p73 was methylated in the primary tumor and marrow. On the other hand, p16 and RARß were methylated only in the primary tumor but not in the marrow in case 1, and RARß methylated only in the primary tumor and p16 in the marrow in case 2. In another two cases (cases 3 and 33), the marrow and peripheral blood were negative by MSP. Case 3 never developed any disease outside the nose, and case 33 did not show involvement of the peripheral blood at any stage of the illness. In the last case (case 28), the patient was referred for treatment of a relapse at the oropharyngeal region. After chemotherapy and radiotherapy, morphological examination in a series of random biopsies could not definitely show the presence of tumor, due to the extensive necrosis in the small biopsies. However, p73 methylation could be detected unambiguously in the tonsil, oropharynx, and the tongue base, suggesting persistent disease. The lymphoma ultimately relapsed in the Waldeyer’s ring, at sites where p73 methylation was detected.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Genomic abnormalities in NK cell malignancies have remained largely unknown, owing to the rarity of the disease. To define the genetic aberrations in NK cell lymphomas, we have used a candidate gene approach¹³ to investigate a set of putative tumor suppressive genes that may undergo epigenetic inactivation via promoter CpG methylation. Such an approach has demonstrated that characteristic profiles of gene hypermethylation exist for different malignancies, in which some genes are shared while others might be cancer type-specific.¹³

In this study, we have shown that p73 is methylated in over 90% of NK cell lymphomas. This frequency is the highest known for any human malignancy.¹³ The p73 protein shares substantial homology and functional similarity with p53,¹⁴ both being involved in gene transactivation and induction of apoptosis or cell cycle arrest.¹⁵ It has been shown that while E2F1 activates p53 indirectly via CDKN2A, p73 can be regulated directly by E2F1 through the recognition and transactivation of the p73 promoter, leading to activation of p53-responsive target genes and apoptosis. It has therefore been postulated that p73 is a candidate tumor suppressor gene.¹⁴ Interestingly, mutation or structural alterations of the p73 gene locus are rare in hematological malignancies.¹⁶ Limited data have shown, however, that promoter methylation may be a mechanism of inactivation of p73 gene function. In one study, p73 methylation was reported in approximately 30% of acute lymphoblastic leukemia and Burkitt’s lymphoma.¹¹ In two other studies where lymphoid leukemia or lymphoma cell lines instead of primary tumor materials were examined, p73 methylation was found in 16% to 32% of cell lines investigated.^16,17 Hence, although extensive studies have not been performed, existing data indicate that p73 methylation is found only in about one third of lymphoid malignancies, in contrast to our findings of p73 methylation in almost all of NK cell malignancies. Furthermore, we have shown that in the NK lymphoma cell line NK92, complete p73 methylation led to abolition of gene expression, and demethylation of p73 resulted in reinduction of gene expression. Therefore, promoter methylation may represent a critical mechanism for the inactivation of p73 in NK cells. The very high frequency of p73 methylation found in NK cell lymphomas strongly suggests that it may be a major target gene in NK cell transformation.

The cyclin-dependent kinase gene inhibitors p16 and p15 were methylated in 63% and 48% of cases, which was in close concordance with those observed in lymphomas¹³ and other hematological malignancies.¹⁸ The DNA mismatch repair gene hMLH1 was methylated in 63% of cases. This frequency is much higher than the 6% reported in leukemia,¹³ although data for lymphoma are lacking. In solid tumors, methylation of hMLH1 is associated with loss of gene expression and microsatellite instability (MSI).¹⁹ However, MSI is rare in hematological malignancies, and, accordingly, methylations or mutations of hMLH1 are rarely found.²⁰ Furthermore, nine of the 32 cases included in this study have been investigated in a previous study of loss of heterozygosity with microsatellite markers,⁸ and MSI was not observed (Table 2) . Further investigations will therefore be required to elucidate the functional significance of hMLH1 methylation in NK cell lymphomas. The RARß gene is important in the growth regulation of epithelial cells, and its aberrant methylation has been observed in cancers of the breast and lung.^21,22 In this study, RARß gene methylation was found in nearly half of the cases, implying that dysregulation of RARß function may also be important in NK cell transformation.

Whether promoter hypermethylation initiates or is merely a reflection of gene inactivation²³ remains debatable. This controversy notwithstanding, CpG methylation may still be a sensitive molecular marker independent of its functional implications.²⁴ To test this potential application, we examined the marrow (cases 1 to 3) and peripheral blood (case 33) by MSP in some patients. Aberrant gene methylation correctly detected/ruled out occult involvement in these cases. Furthermore, an interesting observation was that gene methylation appeared to be different between the primary lesion and the marrow in cases 1 and 2. p73 was methylated in the primary tumor and marrow in both cases. However, p16 and RARß were methylated only in the primary tumor but not in the marrow in case 1, and RARß methylated only in the primary tumor and p16 in the marrow in case 2. This difference could be a reflection of progressive methylation of different genes in a multi-step carcinogenic process,²⁵ with separate clonal evolutions at different sites from a common tumor progenitor cell. That p73 methylation was commonly found in the primary/metastatic site suggested that this might be an early step in NK cell transformation. In case 28, morphological examination of biopsies at different sites failed to definitively localize tumor cells. This is a common problem in the diagnosis of NK cell lymphoma, where zonal necrosis in the tumor sometimes makes morphological examination difficult even for the experienced pathologist. Owing to the small size of the biopsies, we had only performed MSP for the p73 gene. p73 methylation could be detected in the tongue base, oropharynx, and tonsil, which were subsequently the sites of disease recurrence. The results showed that gene promoter methylation could be a sensitive tool for the detection of residual tumor in NK cell malignancies, and may therefore be a useful ancillary tumor marker for the definition of minimal tumor involvement before and after treatment.

In conclusion, aberrant gene methylation occurs commonly and with a specific pattern in NK cell malignancies. Methylation of p73 is nearly always observed, implying that functional perturbation of p73 may be important in malignant NK cell transformation, and that it may serve as a useful molecular marker for disease detection.

	Acknowledgements

Top Abstract Materials and Methods Results Discussion References

 
We thank Dr. Y. Kagami (Aichi Cancer Center Hospital, Nagoya, Japan) for providing the HANK1 cell line and valuable advice, and Eunice Chan and Alfa Bai for technical assistance.

	Footnotes

Top Abstract Materials and Methods Results Discussion References

 
Address reprint requests to Dr. Y.L. Kwong, University Department of Medicine, Professorial Block, Queen Mary Hospital, Pokfulam Road, Hong Kong, PRC. E-mail: ylkwong{at}hkucc.hku.hk

Supported by the Kadoorie Charitable Foundation.

Accepted for publication October 12, 2001.

	References

Top Abstract Materials and Methods Results Discussion References

 

1. Chan JK, Sin VC, Wong KF, Ng CS, Tsang WY, Chan CH, Cheung MM, Lau WH: Non-nasal lymphoma expressing the natural killer cell marker CD56: a clinicopathologic study of 49 cases of an uncommon aggressive neoplasm. Blood 1997, 89:4501-4513[Abstract/Free Full Text]
2. Kwong YL, Chan ACL, Liang R, Chiang AKS, Chim CS, Chan TK: CD56+ NK lymphomas: clinicopathologic features and prognosis. Br J Haematol 1997, 97:821-829[Medline]
3. Cheung MMC, Chan JKC, Lau WH, Foo W, Chan PT, Ng CS, Ngan RKC: Primary non-Hodgkin’s lymphoma of the nose and nasopharynx: clinical features, tumor immunophenotype, and treatment outcome in 113 patients. J Clin Oncol 1998, 16:70-77[Abstract/Free Full Text]
4. Jaffe ES, Harris NL, Stein H, Vardiman JW (eds): World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. IARC Press, Lyon, France, 2001, pp 189–230
5. Chan JKC: Natural killer cell neoplasms. ASCP Rev Pathol (Anat Pathol) 1998, 3:77-145
6. Wong KF, Chan JKC, Kwong YL: Identification of del(6)(q21q25) as a recurring chromosomal abnormality of putative NK cell lymphoma/leukaemia. Br J Haematol 1997, 98:922-926[Medline]
7. Siu LLP, Wong KF, Chan JKC, Kwong YL: Comparative genomic hybridization analysis of natural killer cell lymphoma/leukemia: recognition of consistent patterns of genetic alteration. Am J Pathol 1999, 155:1419-1425[Abstract/Free Full Text]
8. Siu LLP, Chan V, Chan JKC, Wong KF, Liang R, Kwong YL: Consistent patterns of allelic loss in natural killer cell lymphoma. Am J Pathol 2000, 157:1803-1809[Abstract/Free Full Text]
9. Baylin SB, Herman JG: DNA hypermethylation in tumorigenesis. Trends Genet 2000, 16:168-174[Medline]
10. Herman JG, Graff JR, Myöhänen S, Nelkin BD, Baylin SB: Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 1996, 93:9821-9826[Abstract/Free Full Text]
11. Corn PG, Kuerbitz SJ, van Noesel MM, Esteller M, Compitello N, Baylin SB, Herman JG: Transcriptional silencing of the p73 gene in acute lymphoblastic leukemia and Burkitt’s lymphoma is associated with 5'CpG island methylation. Cancer Res 1999, 59:3352-3356[Abstract/Free Full Text]
12. Ueki T, Toyota M, Sohn T, Yeo CJ, Issa JPJ, Hruban RH, Goggins M: Hypermethylation of multiple genes in pancreatic adenocarcinoma. Cancer Res 2000, 60:1835-1839[Abstract/Free Full Text]
13. Esteller M, Corn PG, Baylin SB, Herman JG: A gene hypermethylation profile of human cancer. Cancer Res 2001, 61:3225-3229[Abstract/Free Full Text]
14. Kaghad M, Bonnet H, Yang A, Creancier L, Biscan JC, Valent A, Minty A, Chalon P, Lelias JM, Dumont X, Ferrara P, Mckeon F, Caput D: Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers. Cell 1997, 90:809-819[Medline]
15. Soengas MS, Lowe SW: p53 and p73: seeing double? Nat Genet 2000, 26:391-392[Medline]
16. Kawano S, Miller CW, Gombart AF, Bartram CR, Matsuo Y, Asou H, Sakashita A, Said J, Tatsumi E, Koeffler HP: Loss of p73 gene expression in leukemias/lymphomas due to hypermethylation. Blood 1999, 94:1113-1120[Abstract/Free Full Text]
17. Liu M, Taketani T, Li R, Takita J, Taki T, Yang HW, Kawaguchi H, Ida K, Matsuo Y, Hayashi Y: Loss of p73 gene expression in lymphoid leukemia cell lines is associated with hypermethylation. Leuk Res 2001, 25:441-447[Medline]
18. Baur AS, Shaw P, Burri N, Delacrétaz F, Bosman FT, Chaubert P: Frequent methylation silencing of p15^INK4b (MTS2) and p16^INK4a (MTS1) in B-cell and T-cell lymphomas. Blood 1999, 94:1773-1781[Abstract/Free Full Text]
19. Herman JG, Umar A, Polyak K, Graff JR, Ahuja N, Issa JPJ, Markowitz S, Willson JKV, Hamilton SR, Kinzler KW, Kane MF, Kolodner RD, Vogelstein B, Kunkel TA, Baylin SB: Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc Natl Acad Sci USA 1998, 95:6870-6875[Abstract/Free Full Text]
20. Auner HW, Olipitz W, Hoefler G, Bodner C, Konrad D, Crevenna R, Linkesch W, Sill H: Mutational analysis of the DNA mismatch repair gene hMLH1 in myeloid leukaemias. Br J Haematol 1999, 106:706-708[Medline]
21. Widschwendter M, Berger J, Hermann M, Muller HM, Amberger A, Zeschnigk M, Widschwendter A, Abendstein B, Zeimet AG, Daxenbichler G, Marth C: Methylation and silencing of the retinoic acid receptor-ß2 gene in breast cancer. J Natl Cancer Inst 2000, 92:826-832[Abstract/Free Full Text]
22. Virmani AK, Rathi A, Zochbauer-Muller S, Sacchi N, Fukuyama Y, Bryant D, Maitra A, Heda S, Fong KM, Thunnissen F, Minna JD, Gazdar AF: Promoter methylation and silencing of the retinoic acid receptor-ß gene in lung carcinomas. J Natl Cancer Inst 2000, 92:1303-1307[Abstract/Free Full Text]
23. Fearon ER: BRCA1 and E-cadherin promoter hypermethylation and gene inactivation in cancer: association or mechanism? J Natl Cancer Inst 2000, 92:515-517[Free Full Text]
24. Baylin SB, Belinsky SA, Herman JG: Aberrant methylation of gene promoters in cancer: concepts, misconcepts, and promise. J Natl Cancer Inst 2000, 92:1460-1461[Free Full Text]
25. Kang GH, Shim YH, Jung HY, Kim WH, Ro JY, Rhyu MG: CpG island methylation in premalignant stages of gastric carcinoma. Cancer Res 2001, 61:2847-2851[Abstract/Free Full Text]

This article has been cited by other articles:

				S.-i. Maruya, J.-P. J. Issa, R. S. Weber, D. I. Rosenthal, J. C. Haviland, R. Lotan, and A. K. El-Naggar Differential Methylation Status of Tumor-Associated Genes in Head and Neck Squamous Carcinoma: Incidence and Potential Implications Clin. Cancer Res., June 1, 2004; 10(11): 3825 - 3830. [Abstract] [Full Text] [PDF]

				S. S. Liu, R. C.-Y. Leung, K. Y.-K. Chan, P.-M. Chiu, A. N.-Y. Cheung, K.-F. Tam, T.-Y. Ng, L.-C. Wong, and H. Y.-S. Ngan p73 Expression Is Associated with the Cellular Radiosensitivity in Cervical Cancer after Radiotherapy Clin. Cancer Res., May 15, 2004; 10(10): 3309 - 3316. [Abstract] [Full Text] [PDF]

				C.-S. Chim, S.-Y. Ma, W.-Y. Au, C. Choy, A. K. W. Lie, R. Liang, C.-C. Yau, and Y.-L. Kwong Primary nasal natural killer cell lymphoma: long-term treatment outcome and relationship with the International Prognostic Index Blood, January 1, 2004; 103(1): 216 - 221. [Abstract] [Full Text] [PDF]

				Y Kaneko, S Sakurai, M Hironaka, S Sato, S Oguni, Y Sakuma, K Sato, K Sugano, and K Saito Distinct methylated profiles in Helicobacter pylori dependent and independent gastric MALT lymphomas Gut, May 1, 2003; 52(5): 641 - 646. [Abstract] [Full Text]

				E. C. Chan, S. Y. Lam, K. W. Tsang, B. Lam, J. C. M. Ho, K. H. Fu, W. K. Lam, and Y. L. Kwong Aberrant Promoter Methylation in Chinese Patients with Non-Small Cell Lung Cancer: Patterns in Primary Tumors and Potential Diagnostic Application in Bronchoalevolar Lavage Clin. Cancer Res., December 1, 2002; 8(12): 3741 - 3746. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ping Siu, L. L. Articles by Kwong, Y.-L. Search for Related Content PubMed PubMed Citation Articles by Ping Siu, L. L. Articles by Kwong, Y.-L.