TABLE 2.

p16^INK4a genomic analysis of keratinocyte cell lines expressing hTERT

Cell line	Deletion analysisa				SSCP mutation analysisb				Sequencing resultc				Promoter methylationd
	D9S126	D9S741	D9S1748	Multiplex PCR	Exon 1	Exon 2(A)	Exon 2(B)	Exon 3	Exon	Codon	Mutation (or polymorphism)	Amino acid change
LP9 (23 PD, mid-life span)	12	12	12	N	N	N	N	N					U
LP9/TERT-1 (90 PD, RDI)	12	12	12	N	N	N	N	N					U
N (42 PD, late life span)	12	NI	12	N	N	N	N	N					U
N/TERT-1 (100 PD, RDI)	12	NI	12	N	N	N	N	N					U
LiF-Ep (25 PD, mid-life span)	NI	12	12	N	N	N	S+N	Se	2	148	(GCG→ACG)	Ala→Thr	U
									3		nt 499 C→G	3′ UTR
LiF-Ep/TERT-1 (49 PD, RDI)	NI	LOH	LOH	N	N	N		Se	2	148	(GCG→ACG)	Ala→Thr	U
									3		nt 499 C→G	3′ UTR
OKF6 (42 PD, late life span)	12	12	12	N	N	N	N	N					U
OKF6/TERT-1 (42 PD, early SGP)	12	12	12	ND	ND	ND	ND	ND					ND
OKF6/TERT-1 (63 PD, RDI)	LOH	LOH	LOH	N	N	N	N	N					U
OKF6/TERT-1R (65 PD, RDI)	12	12	12	ND	ND	ND	ND	ND					ND
OKF6/TERT-2 (52 PD, RDI)	12	12	(HD)	HD	(HD)	(HD)	(HD)	(HD)					(HD)

^aChanges 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. 

^bNote 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 p16^INK4a (reference 42; note that the old codon numbering system in this paper designated codon 148 as 140). 

^cnt, nucleotide; UTR, untranslated region. 

^dDetermined by methylation-sensitive PCR to detect methylation of the CpG island in the p16^INK4a promoter most commonly found to be hypermethylated in human cancer cells, as described in Materials and Methods. U, unmethylated; M, only methylated sequences detected. 

^eC→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.