Table 2.

p16INK4a genomic analysis of keratinocyte cell lines expressing hTERT

Cell lineDeletion analysisaSSCP mutation analysisbSequencing resultcPromoter methylationd
D9S126D9S741D9S1748Multiplex PCRExon 1Exon 2(A)Exon 2(B)Exon 3ExonCodonMutation (or polymorphism)Amino acid change
LP9 (23 PD, mid-life span)121212NNNNNU
LP9/TERT-1 (90 PD, RDI)121212NNNNNU
N (42 PD, late life span)12NI12NNNNNU
N/TERT-1 (100 PD, RDI)12NI12NNNNNU
LiF-Ep (25 PD, mid-life span)NI1212NNNS+NSe 2148(GCG→ACG)Ala→ThrU
3nt 499 C→G3′ UTR
LiF-Ep/TERT-1 (49 PD, RDI)NI LOH LOH NNNSe 2148(GCG→ACG)Ala→ThrU
3nt 499 C→G3′ UTR
OKF6 (42 PD, late life span)121212NNNNNU
OKF6/TERT-1 (42 PD, early SGP)121212NDNDNDNDNDND
OKF6/TERT-1 (63 PD, RDI) LOH LOH LOH NNNNNU
OKF6/TERT-1R (65 PD, RDI)121212NDNDNDNDNDND
OKF6/TERT-2 (52 PD, RDI)1212(HD) HD (HD)(HD)(HD)(HD)(HD)
  • a Changes from the parent cell line are noted in boldface. PCR primer sets were used to amplify the polymorphic microsatellite sequences D9S126, D9S741, and D9S1748 to detect loss of heterozygosity (LOH) in the 9p21 region. 12, two alleles detected; NI, not informative; LOH, allelic loss compared with parent cell line or normal cells from same donor. Multiplex PCR was performed with exon 2 primers and primers that amplified sequences from a control locus as described in Materials and Methods. N, exon 2 sequences detected; HD, homozygous deletion; ND, not determined; (HD), no PCR products observed because of HD.

  • b Note that two overlapping regions of exon 2 (designated A and B) were amplified separately. N, PCR product migrating as expected for wild-type allele; S, shift in migration compared with that of the wild-type allele. The codon 148 missense G-to-A alteration in LiF-Ep, identified as shifted in the SSCP analysis, is a polymorphism that does not result in altered biological activity of p16INK4a (reference 42; note that the old codon numbering system in this paper designated codon 148 as 140).

  • c nt, nucleotide; UTR, untranslated region.

  • d Determined by methylation-sensitive PCR to detect methylation of the CpG island in the p16INK4apromoter most commonly found to be hypermethylated in human cancer cells, as described in Materials and Methods. U, unmethylated; M, only methylated sequences detected.

  • e C→G substitution in nucleotide 494 of the 3′ untranslated region was identified in the allele of LiF-Ep that was retained by LiF-Ep/TERT-1.