Promoter hypermethylation of the cyclin-dependent kinase inhibitor (CDKI) gene p21^{WAF1/CIP1/SDI1} is rare in various lymphomas and carcinomas

The cyclin-dependent kinase inhibitor (CDKI) p21 can act as a tumor suppressor to inhibit tumor cell growth.¹  In contrast to other CDKI genes, p21 is rarely mutated or deleted in tumors.¹  Alternative mechanisms of p21 inactivation have been suspected, with p21 hypermethylation demonstrated in some hematologic and solid tumors.^1-9  Therefore, we read with great interest the 2 contrasting reports on p21 methylation in Blood,^2,3  and we would like to share our results of p21 methylation.

CpG islands (CGIs) are frequently silenced by methylation in tumors.¹⁰  The p21 promoter and exon 1 are within a typical CGI (Figure 1A). We examined its expression and methylation in 46 tumor cell lines (6 Hodgkin disease [HD], 1 leukemia, 33 carcinomas, 6 Burkitt lymphoma [BL]) and 12 normal peripheral blood mononuclear cell (PBMC) samples. p21 was readily expressed in all the samples, except silenced in Rael (Figure 1B). Methylation analysis of p21 in 58 cell lines, 12 normal PBMCs, and 10 normal tissues, using methylation-specific polymerase chain reaction (MSP),¹⁰  showed that this promoter was consistently unmethylated in all the samples (Table 1), except it was weakly methylated in Raji and strongly methylated in Rael.

We further examined p21 methylation in 187 primary tumors (lymphomas and carcinomas). Only 3 lymphomas showed methylation (Figure 1C). We also examined p21 methylation in more detail by bisulfite genomic sequencing (BGS; Figure 1E-F).¹⁰  Consistent with our MSP analysis, the results showed that, in Rael, 93% CpG sites were methylated, whereas only 37% CpG sites (most of them outside the core promoter) were methylated in Raji. Only a few scattered CpG sites were methylated in other samples. The abundant expression of p21 in CA46 suggested that the patchy methylation of p21 in intron 1, but not the core promoter and exon 1, does not affect its expression.

We also treated Rael with 5-aza-2′-deoxycytidine. p21 expression was restored after 24 hours of treatment, and more profoundly at 48 hours and 96 hours. Concomitantly, unmethylated p21 alleles were detected after the treatment (Figure 1D). Therefore, this promoter could be demethylated and activated by 5-aza-2′-deoxycytidine alone, indicating that methylation directly mediates its suppression.

Furthermore, we have reviewed all literature about p21 methylation (Table 1). p21 methylation is rare in tumors in general but does occur in certain tumors. The reports using restriction enzyme digestion–based assays, which only detect the methylation of very few CpG sites at specific restriction sites, tend to detect relatively high frequencies of p21 methylation. The CpG within a signal transducers and activators of transcription (STAT) site at –692 in the distal promoter was reported frequently methylated in Rhabdomyosarcoma and normal tissues¹ ; however, we did not detect any methylation in any normal tissue or PBMCs. Studies using bisulfite-modification–based methods (MSP and combined bisulfite restriction analysis [COBRA]) tend to detect little if any methylation. The different results of the 2 recent reports may also be due to their different techniques, or a geographic/ethnic variation as suggested by the authors.^2,3  With this precaution, we have used both MSP and BGS to verify our results, and recruited samples from all over the world.

In summary, we found that p21, unlike p16 and p15,⁵  is rarely inactivated by methylation in lymphomas and carcinomas. However, our study still does not rule out the possibility of epigenetic repression of this gene, to some extent, through chromatin/histone structure changes, since histone deacetylase inhibitors trichostalin A (TSA), phenylbutyrate, and subercylanilide hydroxamic acid (SAHA) can also activate p21 expression.¹¹  5-aza-2′-deoxycytidine can also activate p21 expression through methylation-independent mechanisms.

We thank Drs Sen-Tien Tsai, Thomas Putti, Guiyuan Li, Ya Cao, Bert Vogelstein, Dolly Huang, Luke Tan, Boon Cher Goh, and Soo Chin Lee for cell lines and samples, and Chaiyen Lim and Li Fu for technical support. This project was supported by an A*STAR research grant to Johns Hopkins Singapore (Q.T. and R.A.).