Frequent inactivation of the p73 gene by abnormal methylation or LOH in non-Hodgkin's lymphomas

Authors


Abstract

p73 is a candidate tumor suppressor and imprinted gene that shares significant homology with the p53 gene. It is located on 1p36, a region frequently deleted in neuroblastoma and other tumors. To investigate the pattern of inactivation of this gene in human lymphomas, we studied 59 tumors to identify abnormal methylation in exon 1 and loss of heterozygosity (LOH) at this locus. p73 was methylated in 13/50 (26%) B cell lymphomas. There was no evidence of p73 methylation in the 9 T cell lymphomas analyzed. Burkitt's lymphomas showed the highest proportion of methylated cases (36%), although this alteration also affected other aggressive lymphomas such as diffuse large cell and some marginal zone lymphomas. LOH at the p73 locus was detected in 4/34 (11%) B and 1/9 (11%) T cell lymphomas. The p73 expression analysis showed absence or low level of p73 product in methylated lymphomas, whereas p73 was always detected in unmethylated tumors. We found monoallelic expression in normal peripheral blood samples, consistent with imprinting. None of the tumors showed LOH and methylation of the remaining allele simultaneously, suggesting that alteration of the expressed allele could lead to the total inactivation of the gene. Our results show that deletion or methylation of the p73 gene could be important mechanisms in suppressing p73 expression in B cell non-Hodgkin's lymphomas. © 2002 Wiley-Liss, Inc.

p73 is a candidate tumor suppressor gene that shares significant aminoacid homology with the p53 gene throughout its DNA-binding, transactivation and homo-oligomerization domains.1, 2 Moreover, forced overexpression of p73 can activate p53-responsive promoters and induce apoptosis in p53-deficient cells.3

The p73 gene was mapped within a chromosomal region frequently deleted in many types of human tumors (1p32–pter) where the existence of tumor suppressor genes had been suggested.4, 5, 6, 7, 8, 9 Location of p73 at 1p36.3 suggested that it could be an important tumor suppressor gene. In contrast to the p53 gene, however, mutations of p73 are rarely found in most human cancers.10, 11, 12, 13, 14, 15

Abnormal hypermethylation constitute another inactivating mechanism that commonly affects tumor suppressor genes. Transcriptional silencing of the p73 gene through methylation has been demonstrated in some leukemias and lymphomas,16, 17, 18, 19 although it seems not to be associated with other solid tumors.16p73 gene encodes different transcripts as a result of alternative splicing.20, 21 In addition, expression studies have found that the p73 gene is differently expressed in various human tissues, showing that monoallelic or biallelic pattern of expression could be tissue dependent. Evidences of monoallelic expression of this gene have been described in neuroblastoma, normal lymphocytes and other normal tissues such as kidney and lung.10, 22, 23 Biallelic expression was observed, however, in normal bladder, normal breast epithelium and in some tumors such as those of bladder or lung.23, 24, 25 These findings suggest that p73 could be an imprinted suppressor gene that in some tumors could be inactivated by a single event affecting the expressed allele16, 25 whereas in others it could be altered through loss of imprinting (LOI), leading to the activation of the silent allele.22, 26

To elucidate the involvement of p73 alterations in human lymphomas, in the present study we have analyzed the incidence of loss of heterozygosity (LOH), abnormal methylation of the p73 promoter region and expression of this gene in a group of different subtypes of T and B cell lymphomas, and the coexistence of these alterations with p53 gene mutations.

MATERIAL AND METHODS

Tumor samples

DNA and RNA were extracted from 59 lymph node tissue samples or bone marrow from patients with B cell or T cell non-Hodgkin's lymphoma (NHL). Tumors were obtained from the Pathology Department of the Fundación Jiménez Díaz Hospital and the Haematology Department of the San Pablo y Santa Cruz Hospital after histological diagnosis. Six tonsil samples were used as normal lymphoid tissue. Lymphomas were classified according to the WHO classification and included 50 B cell (3 mantle cell lymphoma, MCL, 4 follicular lymphomas, FL, 17 diffuse large cell lymphomas DLCL, 4 marginal zone lymphomas, MZL, and 22 Burkitt's lymphomas, BL) and 9 T cell nodal lymphomas. Details of the histological subtypes are described in Tables I and II. Forty-three tumor samples had been previously characterized by us to know the status of different tumor suppressor genes.27, 28 Moreover, we analyzed previously these tumors and their corresponding normal samples for the presence of LOH in the 1p36 region and in p73 gene.29

Table I. Distribution of The p73 LOH and Abnormal Methylation Together With p53 Mutations in the Lymphoma Samples1
CaseDiagnosisp73 LOHp73 Methylationp53 Mutation
  • 1

    PTL, peripheral T-cell lymphomas; AIL, angioimmunoblastic lymphoma; MCL, mantle cell lymphomas; FL, follicular lymphomas; DLCL, diffuse large cell lymphomas; CB, centroblastic; ALC, anaplastic; MZL, marginal zone lymphomas; MALT, mucosa associated lymphoid tissue; BL, Burkitt lymphomas; Nd, not done.

  • 2

    Coexistence with carcinoma.

1PTL
2PTL
3PTL lymphoepiteioid
4PTL lymphoepiteioidLOH
5PTL pleomorphic
6PTL
7PTL AIL
8PTL lymphoepiteioid
9PTL pleomorphic
10MCL
11MCL
12MCL
13FL
14FL
15FL
16FL
17DLCL
18DLCL CBMUT
19DLCL CB
20DLCL CB
21DLCL CB
22DLCL ALC
23DLCL ALC
24DLCL ALCMET
25DLCL ALC
26DLCL ALC
27DLCL ALC
28DLCL ALC
29DLCL ALCMET
30DLCL post MCLMET
31DLCL post MCLMET 
32DLCLMUT
33DLCL
34MZL gastric MALT
35MZL gastric MALT2LOHMUT
36MZL gastric MALTMET
37MZL gastric MALT
38BURKITTLOH
39BURKITTMETMUT
40BURKITT
41BURKITTMUT
42BURKITTLOH
43BURKITTLOHMUT
44BURKITTNdMETMUT
45BURKITTNdMUT
46BURKITTNdMUT
47BURKITTNd
48BURKITTNd
49BURKITTNdMET
50BURKITTNdMET
51BURKITTNd
52BURKITTNdMET
53BURKITTNdMET
54BURKITTNd
55BURKITTNdMET
56BURKITTNd
57BURKITTNd
58BURKITTNdMUT
59BURKITTNdMET
Table II. Clinical Features of Cases With p73 LOH or Methylation1
CaseDiagnosisStage of diseaseAgeGenderLocalizationRelapse samplesEvolution (months)Actual statusP73 gene status
  • 1

    BM, bone marrow; CR, complete remission; PR, partial remission; NA, not available.

4Peripheral T lymphomaIII B48MSpleenYesNANALOH
24DLBCL AnaplasticII B24MMediastinoYesNAAliveMET
29DLBCL AnaplasticII A21MLymph nodeNoCR 48AliveMET
30DLBCL post MCLIV B72FSpleenYes216AliveMET
31DLBCL post MCLIV B64FSpleen/lymph nodeYes9DeadMET
35MZL gastric MALT + carcinomaI EA60FStomachNoPR 4DeadLOH
36MZL gastric MALTI EA35MStomachNo48AliveMET
38BurkittIV B68MLymph nodesYesCR 14DeadLOH
39BurkittIV B14MLymph nodes/testisNoPR 12DeadMET
42BurkittIV BNAMLymph nodesNANANALOH
43BurkittIV A27FOvaryNANANALOH
44BurkittIII8FNANoCR 96AliveMET
49Burkitt/LAL-L3IV21MBMYes2DeadMET
50BurkittIV34M Yes6DeadMET
52BurkittIV80FIntestinalYes3DeadMET
53Burkitt/LAL-L3IV70MBMYes1DeadMET
55BurkittIV16FBMYes16DeadMET
59Burkitt/LAL-L3IV68MBMYes2DeadMET

Methylation analysis of p73 gene

The methylation status of the p73 exon 1 was determined by methylation-specific PCR (MSP) after the treatment of the DNA with sodium bisulfite as described previously.30, 31 Specific primer sequences for unmethylated and methylated fragments had been described by Corn et al..16 Unmethylated and methylated fragments were amplified under different PCR reactions, as follows: 40 cycles of 20 sec at 94°C, 20 sec at 65°C and 20 sec at 72°C for the unmethylated products, whereas methylated products were amplified in 40 cycles of 20 sec at 94°C and 20 sec at 72°C. In both cases an initial denaturation of 7 min at 94°C and a final extension of 5 min at 72°C was carried out. PCR products were resolved in 12% polyacrylamide gels and visualized after ethidium bromide staining. As positive controls of the methylated reaction we included a methylated sample or a bisulfite modified DNA from the HL-60 cell line, which had been described as being methylated.16 A control without DNA was included in all reactions.

Expression analysis

Expression of p73 gene was detected by RT-PCR analysis using previously described primers located in exons 1 and 3.13p73 expression was studied in 6 additional tumors and in 1 tonsil sample in which RNA material was available and in which methylation analysis was also carried out. Moreover, to establish the presence of monoallelic or biallelic p73 expression in normal lymphocytes, we analyzed the pattern of expression of 8 peripheral blood samples heterozygous for the already described polymorphism in exon 2.20 One microgram of RNA was reverse-transcribed to generate cDNA using AMV reverse transcriptase and random hexamers in a reaction volume of 40 μl. Then expression of p73 was detected using 5 μl of the cDNA to amplify the corresponding fragment of the gene. Those samples showing expression products were sequenced to detect the polymorphism at exon 2 and thereby the existence of uniallelic or biallelic expression of the gene. Expression of the abl gene was carried out beforehand to test the quality of the cDNA.

Screening for mutations of p53 gene

PCR-SSCP analysis was carried out to detect mutations in the entire coding sequence of p53 gene by methods described previously.32 The PCR products were electrophoresed in 6–8% polyacrilamide/10% glycerol gels. All cases showing abnormal SSCP pattern were then sequenced to characterize the mutations.

RESULTS

Abnormal methylation of the p73 exon 1 was detected only in B cell lymphomas, in which 13/50 lymphomas (26%) were methylated. In contrast, none of the 9 T cell lymphomas analyzed were methylated. Loss of heterozygosity was found in 5/43 lymphomas (11%), including 4/34 (11%) B cell and 1/9 (11%) T cell lymphomas (Table I). Interestingly, p73 abnormalities were found in general in aggressive tumors with bad response to conventional polychemotherapy and included 11/22 Burkitt's lymphomas (50%), 4/17 diffuse large cell lymphomas (DLCL) (23%), 2/4 marginal zone lymphomas (MZL) of gastric MALT and 1/9 T cell lymphoma (Table I). Five of 18 cases with p73 LOH or methylation corresponded to relapse samples and 6/18 relapsed during the evolution of the disease (Table II), indicating a possible relation with the lack of response to therapy and tumor recurrence. LOH of p73 gene was found in Case 4 that corresponded to a sample at relapse but this alteration was not present in the initial tumor, indicating that the loss of p73 is acquired during the evolution of the disease. There was, however, no evidence of methylation in the 6 normal tonsil samples or in the peripheral blood from all the lymphoma patients included in the study.

All methylated lymphomas showed evidence for both methylated and unmethylated p73 products (Fig. 1). This fact could be due to the presence of contamination with normal lymphocytes in these primary tumors, but it could also be that methylation affects only one of the p73 alleles and the other remains unmethylated. Using methylation-specific PCR, the HL-60 cell line showed the methylated p73 fragment and also a very weak signal of the unmethylated fragment, as described previously.16

Figure 1.

Methylation analysis of p73 gene in lymphoma samples by MSP. Top panel: using methylation specific primers (M), 2 cases (1 and 6) appeared methylated, together with the positive control (C+). Bottom panel: amplification products obtained using the unmethylated specific primers (U). All cases showed the unmethylated fragment excepting the positive control. Line 8 corresponds to a control without DNA.

To better understand the mechanism of inactivation of the p73 gene in lymphomas we correlated the methylation results with LOH data that had been obtained previously by using 3 markers located in or near the p73 gene.29 None of the tumors showing LOH displayed abnormal methylation in the other allele, suggesting that deletion or methylation inactivate only one of the alleles. Taken together, abnormal methylation or LOH affected 18/59 (30%) lymphomas, but these alterations never occurred in the same tumor sample.

Because p73 shares some functional characteristics with p53, we also examined whether p53 mutations occurred together with methylation or LOH of p73. Among 10 p53-mutated lymphomas, 2 cases were also methylated in p73 and 2 others additionally presented LOH at p73 locus (Table I). These findings indicate that although both genes have several functional similarities and may be found simultaneously altered in a tumor sample.

The relatively high incidence of abnormal methylation or LOH in this group of lymphomas (30%) suggests that inactivation of p73 may be an important event in the etiology of these tumors. Because methylation is an inactivating mechanism of several tumor suppressor genes, we analyzed p73 expression in 6 tumor samples in which RNA material was available (Table III). Two methylated lymphomas correlated with the absence or very low level of expression of p73, whereas in 4 unmethylated lymphomas and in the normal tonsil, p73 expression was always detected (Fig. 2). These results suggest that DNA methylation is involved in p73 inactivation in NHLs. Because p73 could be an imprinted gene, we also analyzed the pattern of p73 expression, monoallelic or biallelic, in normal peripheral blood samples. We selected 8 samples heterozygous for the polymorphism in exon 2, and monoallelic expression was detected in all of them (Fig. 2).

Table III. Correlation Between Methylation Status and Expression of the p73 Gene in 6 Lymphomas and in a Normal Lymphoid Tissue Sample
SampleMethylationExpressionAllele1
  • 1

    Expressed allele of the polymorphism at exon 2.

T1YesLowG/C
T2Yes 
T3No+G/C
T4No+G/C
T5No+G/C
T6No+G/C
TonsilNo+A/T
Figure 2.

Expression analysis of p73 gene. (a) RT-PCR to detect p73 expression. Line 1: 1 Kb ladder molecular weigh marker (Gibco, Grand Island, NY). Samples in Lines 2, 4, 5, 7 and 8 correspond to a tonsil and the 4 unmethylated samples showing p73 expression. Lines 3 and 6 show 2 methylated tumors. p73 expression was completely absent in Line 3, whereas tumor in Line 6 showed low expression of this gene. Line 9: control without DNA. (b) Monoallelic expression in a normal peripheral blood sample. At the top, the sequence of the genomic DNA of a heterozygous sample at the polymorphism in exon 2 is shown. At the expression level (bottom sequence), this sample only showed 1 of the alleles.

DISCUSSION

p73 has been postulated to be a candidate tumor suppressor and imprinted gene.20 The mechanisms of inactivation of this gene in different tumor types have not been yet established. Mutations in the coding sequence have rarely been found10, 11, 14, 15 and the fact that the p73 gene has monoallelic or biallelic expression in different tissue types makes it difficult to understand the role of this gene in cancer. In lymphoid malignancies, the p73 gene seems to be inactivated in a significant fraction of tumors because 33% of ALL/B-NHL cell lines had negative expression of this gene17 and have been described in a significant fraction of Burkitt's and natural killer cell lymphomas.16, 18 We describe the methylation status, LOH and expression of this gene in a series of B cell and T cell NHLs.

The methylation analysis showed a relatively high incidence of p73 methylation among B cell lymphomas, mainly in Burkitt's lymphomas (36%). Corn et al.16 also found an important percentage of p73 methylation in Burkitt's lymphomas (30%), although they explained that this alteration was absent in other primary non-Hodgkin's lymphomas. Our results, however, showed p73 methylation also in other subtypes of B NHL, such as DLCL, or some MZL, indicating that p73 methylation, although more frequent, is not only restricted to Burkitt's but also affects other aggressive B cell NHL. Abnormal methylation was not detected in T cell lymphomas. Although these tumors showed a high number of tumoral cells (>70%) that allows the detection of methylation, we do not have evidence of p73 methylation in these samples. The number of T cell tumors was too small, however, to allow conclusions about a different implication of the alterations in p73 between B cell and T cell lymphomas. Abnormal methylation was never found in the normal peripheral blood samples of lymphoma patients, indicating that this alteration specifically occurred in the tumors and could be an important inactivating mechanism, especially in Burkitt's lymphomas. Most of the samples showing any of the p73 alterations corresponded to aggressive tumors, mainly Burkitt's and diffuse lymphomas, and in 11 cases p73 alterations were found in relapsed samples or in cases that relapsed in their evolution, suggesting a relation between p73 inactivation and the aggressiveness of these tumors. The T cell lymphoma showing LOH corresponded to a sample at relapse and the LOH was not detected in the primary tumor. Two of the DLCL with p73 methylation arose after a mantle cell lymphoma, suggesting an involvement of p73 in the transformation to a more aggressive phenotype of the tumors. Burkitt's or DLCL lymphomas constitute aggressive entities among the lymphoid tumors. Moreover, p73 methylation has been described to be consistently involved in other aggressive lymphomas as natural killer cell malignancies.18 A more detailed study of other clinical parameters remains to be done to establish the clinicopathological relevance of p73 alterations.

To analyze the pattern of inactivation of the p73 gene in lymphomas further, we studied the presence of LOH in this locus in the same tumors. Interestingly, we found no correlation between LOH and methylation of the remaining allele. None of the samples analyzed showed both mechanisms simultaneously (Table I), suggesting that alteration in one of the p73 alleles could lead to the total inactivation of the gene. This would be possible if p73 were an imprinted gene resulting in expression of only one allele in normal cells. This monoallelic expression of p73 was proposed in several normal tissues, including normal lymphocytes and some tumors,20, 22 although biallelic expression has also been observed in other tissue types. For this reason, p73 could be a tissue-specific imprinted gene. Some controversial results about mono or biallelic expression have been reported, for example, in normal and tumoral lung samples.10, 23 Kaghad et al.20 found monoallelic expression in normal lymphocytes, whereas another study reported biallelic expression in normal peripheral blood although they described unbalanced expression between the 2 p73 alleles.17 In our experience, using the same polymorphic site as Kaghad et al.,20 monoallelic p73 expression was found in peripheral blood samples, as would be expected if this gene were imprinted in normal lymphocytes.

In all methylated lymphomas the unmethylated product was always detected. The expression analysis in 2 of these cases showed the absence or a very low level of the p73 transcript (Fig. 2), indicating that the unmethylated allele was not expressed, probably because it would be imprinted. The weak signal detected in 1 of these tumors could be due to contamination by normal cells in the sample (Table III). The fact that only 1 of the alleles was expressed in normal peripheral blood samples heterozygous for the polymorphism analyzed, suggests that the p73 gene is monoallelically expressed in normal lymphocytes and that the alteration of the expressed allele could lead to the total inactivation of this gene.

p73 and p53 genes have a comparable ability to induce cell growth arrest and apoptosis.3 For this reason it is possible that p73 is altered more frequently in cases without p53 mutations. In our series, 14/18 (77%) lymphomas had p73 alteration in the absence of p53 mutations (Table I). Several DNA damage agents that induce p53 response do not induce p73, however, indicating differential regulation of these genes.33 The differences between p73 and p53 regulation imply different mechanisms of pathogenesis. Alteration of both genes, as occurred in 4 of our lymphomas (1 MZL and 3 BL), indicate that dysfunction of both p53 and p73 are not exclusive, and suggests that those tumors may have additional defects that confer an increased selective growth advantage.

In summary, our results suggest that the p73 gene is altered mainly by abnormal methylation or deletion in a relatively high proportion of NHLs (30%), indicating that it could be an important tumor suppressor gene involved in the development of some types of lymphoma. As p73 seems to be an imprinted gene with monoallelic expression in normal lymphocytes, alteration of the expressed allele probably leads to its total inactivation. The similarities that the p73 and p53 gene shares suggest that p73 could be an important target in cases without p53 mutations, or could increase the malignant phenotype when both these genes are altered.

Acknowledgements

We would like to thank Dr. M. Esteller for kindly provided the positive control for p73 methylation analysis, and for his critical reading of the manuscript. B.M. is a fellowship holder of the Comunidad Autonoma de Madrid, and M.C. has a grant from the Pharmaceutical College.

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