Volume 116, Issue 2, Pages 394-400 (February 1999) p16INK4 is inactivated by extensive CpG methylation in human hepatocellular carcinoma  Yasunobu Matsuda, Takafumi Ichida, Jun Matsuzawa, Kazuhito Sugimura, Hitoshi Asakura  Gastroenterology  Volume 116, Issue 2, Pages 394-400 (February 1999) DOI: 10.1016/S0016-5085(99)70137-X Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Immunohistochemical patterns for p16. (A) Strong nuclear reactivity for p16 was detected in a normal liver. Cytoplasmic staining in hepatocytes was regarded as nonspecific staining. (B) HCC exhibiting complete loss of expression of p16. Some of the admixed stromal cells in the tumor region, as an internal positive control, show nuclear reactivity (arrowhead). (C) HCC showing positive nuclear staining for p16 in most of the tumor cells and weak nonspecific labeling in the cytoplasm. (D) HCC showing weak p16-positive staining in tumor cells compared with normal cells (original magnification 50×). Gastroenterology 1999 116, 394-400DOI: (10.1016/S0016-5085(99)70137-X) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Genetic analysis of p16 in HCC. (A) Representative results of the differential PCR assay from the DNA of HCCs (lanes 1–4) and of normal liver (lane 5) (245 bp for the p16 gene amplicon and 169 bp for β-actin). Lanes 6–8, control PCR samples using a mixture of DNA of normal lymphocytes and Jurkat cells with known homozygous deletion of the p16 gene. The relative percentage of Jurkat DNA in the mixture is 100% in lane 6, 40% in lane 7, and 20% in lane 8. *ΦX174-HaeIII-digested DNA marker. (B) Mutations in p16 obtained from the DNA of HCC samples showing SSCP shifts. Tumor samples obtained from cases 3, 8, and 9 exhibited a point mutation in codons 134 (C to T), 108 (A to G), and 136 (G to A), respectively. In a tumor sample from case 12, two missense mutations were detected in codons 123 (A to G) and 124 (G to A). Mutated nucleotides are indicated by arrowheads, and their respective amino acid changes are shown. Gastroenterology 1999 116, 394-400DOI: (10.1016/S0016-5085(99)70137-X) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Methylation analysis of p16 in HCC. (A) Representative MSP results are expressed as unmethylated p16-specific bands (U) or methylated p16-specific bands (M). Bisulfite-converted DNA from normal liver tissue (N) served as a negative control as indicated by the presence of the U but not the M band. The numbers of the HCC cases are shown above the corresponding lanes. Case 2, HCC showing diffuse p16-positive staining; case 4, HCC showing weak p16 staining; cases 23 and 44, HCCs showing negative p16 staining. Almost all tumor samples exhibiting the M band also showed the U band to some degree, probably because of contaminated normal tissue. *ΦX174-HaeIII-digested DNA marker. (B) Quantitative methylation analysis by Ms-SNuPE. C, methylated fraction incorporating [32P]dCTP; T, unmethylated fraction incorporating [32P]TTP. DNA from the T24 cell line (T24) and normal liver (N) served as positive and negative control, respectively. The mean percentage of CpG methylation is indicated below each lane. All cases of HCC with negative p16 immunostaining (cases 21, 23, and 24) exhibited a high level of CpG methylation (60%–85%), whereas HCC cases 4, 11, and 38, with weak p16-positive immunostaining, exhibited <25% methylation. Gastroenterology 1999 116, 394-400DOI: (10.1016/S0016-5085(99)70137-X) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Methylation analysis of p16 in HCC. (A) Representative MSP results are expressed as unmethylated p16-specific bands (U) or methylated p16-specific bands (M). Bisulfite-converted DNA from normal liver tissue (N) served as a negative control as indicated by the presence of the U but not the M band. The numbers of the HCC cases are shown above the corresponding lanes. Case 2, HCC showing diffuse p16-positive staining; case 4, HCC showing weak p16 staining; cases 23 and 44, HCCs showing negative p16 staining. Almost all tumor samples exhibiting the M band also showed the U band to some degree, probably because of contaminated normal tissue. *ΦX174-HaeIII-digested DNA marker. (B) Quantitative methylation analysis by Ms-SNuPE. C, methylated fraction incorporating [32P]dCTP; T, unmethylated fraction incorporating [32P]TTP. DNA from the T24 cell line (T24) and normal liver (N) served as positive and negative control, respectively. The mean percentage of CpG methylation is indicated below each lane. All cases of HCC with negative p16 immunostaining (cases 21, 23, and 24) exhibited a high level of CpG methylation (60%–85%), whereas HCC cases 4, 11, and 38, with weak p16-positive immunostaining, exhibited <25% methylation. Gastroenterology 1999 116, 394-400DOI: (10.1016/S0016-5085(99)70137-X) Copyright © 1999 American Gastroenterological Association Terms and Conditions