Figure 1.
Figure 1. Expression and methylation status of p21 in various cell lines and primary tumors. (A) The CpG island in the p21 promoter includes the core promoter, exon 1 (with 2 splicing variants) and part of intron 1. A DNA region with an observed/expected CpG ratio of more than 0.6 and a GC content of more than 50% is considered a CpG island.10 The transcription start site is indicated by bent arrows (based on NCBI database). The 2 discrete MSP regions and one BGS region analyzed in the p21 CGI are indicated. Region 1 corresponds to the area studied by Roman-Gomez et al2 and Shen et al,3 while region 2 has also been studied by Shen et al. MSP primers used are as follows, for region 1 (methylated), p21m1: 5′-TTAGGTTTAGTTGGTTCGGC, p21m2: 5′-ACTAACGCAACTCAACGCG; for region 2 (methylated), p21bm1: 5′-GTGAACGTAGTATATATTCGC, p21bm2: 5′-ATAAAACCGAAACTAAACGCG; and for region 2 (unmethylated), p21bu1: 5′-TTGTGAATGTAGTATATATTTGT, p21bu2: 5′-TTATAAAACCAAAACTAAACACA. Primers for BGS are as follows: p21BGS1, 5′-AGGGAAGTGTTTTTTTGTAGT and p21BGS2, 5′-TAACCAAAAATTCCTATACTTA. MSP primers have been tested for not amplifying any unbisulfited DNA. MSP and BGS were performed as previously described.10 (B) Representative semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR) and MSP results of p21 in cell lines. GAPDH was used as a control for RT-PCR.10 M indicates methylated; U, unmethylated; EsCa, esophageal carcinoma; NPC, nasopharyngeal carcinoma; BrCa, breast carcinoma. (C) Representative MSP results in several primary BLs. (D) Demethylation and activation of p21 in Rael after treatment with 5 μM 5-aza-2′-deoxycytidine (Aza). Hours of treatment are indicated by 12 h, 24 h, 48 h, and 96 h. (E) Partial sequence of the p21 promoter CGI. CpG sites are bolded. The transcription start site is marked as a bent arrow. The TATA box is capitalized. Six Sp1 binding sites and some other regulatory elements (E2F, STAT) within this sequence are underlined. MSP primers for region 1 are framed. (F) High-resolution methylation analysis of the p21 promoter by BGS, which reveals the methylation status of every CpG site in the studied region. A 576-bp region spanning the p21 promoter with 64 CpG sites was analyzed, with the 2 MSP regions labeled. The p21 exon 1 is labeled by a dot-framed box. Each CpG site is shown at the top row as a number. BGS results of 4 lymphoma cell lines (Rael, Raji, CA46, and L540), 2 normal PBMCs, and 1 primary BL are shown. Each row in the grid, next to the sample name, represents an individual allele of the p21 promoter analyzed by BGS in that sample.10 Filled circles are methylated CpG sites and open circles are unmethylated CpG sites. % M is the percent of methylated CpG site of all CpG sites analyzed. M indicates methylated; U, unmethylated; (M), weakly methylated; (U), weakly unmethylated; NA, not available.

Expression and methylation status ofp21in various cell lines and primary tumors. (A) The CpG island in the p21 promoter includes the core promoter, exon 1 (with 2 splicing variants) and part of intron 1. A DNA region with an observed/expected CpG ratio of more than 0.6 and a GC content of more than 50% is considered a CpG island.10  The transcription start site is indicated by bent arrows (based on NCBI database). The 2 discrete MSP regions and one BGS region analyzed in the p21 CGI are indicated. Region 1 corresponds to the area studied by Roman-Gomez et al2  and Shen et al,3  while region 2 has also been studied by Shen et al. MSP primers used are as follows, for region 1 (methylated), p21m1: 5′-TTAGGTTTAGTTGGTTCGGC, p21m2: 5′-ACTAACGCAACTCAACGCG; for region 2 (methylated), p21bm1: 5′-GTGAACGTAGTATATATTCGC, p21bm2: 5′-ATAAAACCGAAACTAAACGCG; and for region 2 (unmethylated), p21bu1: 5′-TTGTGAATGTAGTATATATTTGT, p21bu2: 5′-TTATAAAACCAAAACTAAACACA. Primers for BGS are as follows: p21BGS1, 5′-AGGGAAGTGTTTTTTTGTAGT and p21BGS2, 5′-TAACCAAAAATTCCTATACTTA. MSP primers have been tested for not amplifying any unbisulfited DNA. MSP and BGS were performed as previously described.10  (B) Representative semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR) and MSP results of p21 in cell lines. GAPDH was used as a control for RT-PCR.10  M indicates methylated; U, unmethylated; EsCa, esophageal carcinoma; NPC, nasopharyngeal carcinoma; BrCa, breast carcinoma. (C) Representative MSP results in several primary BLs. (D) Demethylation and activation of p21 in Rael after treatment with 5 μM 5-aza-2′-deoxycytidine (Aza). Hours of treatment are indicated by 12 h, 24 h, 48 h, and 96 h. (E) Partial sequence of the p21 promoter CGI. CpG sites are bolded. The transcription start site is marked as a bent arrow. The TATA box is capitalized. Six Sp1 binding sites and some other regulatory elements (E2F, STAT) within this sequence are underlined. MSP primers for region 1 are framed. (F) High-resolution methylation analysis of the p21 promoter by BGS, which reveals the methylation status of every CpG site in the studied region. A 576-bp region spanning the p21 promoter with 64 CpG sites was analyzed, with the 2 MSP regions labeled. The p21 exon 1 is labeled by a dot-framed box. Each CpG site is shown at the top row as a number. BGS results of 4 lymphoma cell lines (Rael, Raji, CA46, and L540), 2 normal PBMCs, and 1 primary BL are shown. Each row in the grid, next to the sample name, represents an individual allele of the p21 promoter analyzed by BGS in that sample.10  Filled circles are methylated CpG sites and open circles are unmethylated CpG sites. % M is the percent of methylated CpG site of all CpG sites analyzed. M indicates methylated; U, unmethylated; (M), weakly methylated; (U), weakly unmethylated; NA, not available.

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