Cdkn2a, the cyclin-dependent kinase inhibitor encoding p16INK4a and p19ARF, is a candidate for the plasmacytoma susceptibility locus, Pctr1

Abstract

Plasma cell tumor induction in mice by pristane is under multigenic control. BALB/c mice are susceptible to tumor development; whereas DBA/2 mice are resistant. Restriction fragment length polymorphisms between BALB/c and DBA/2 for Cdkn2a(p16) and Cdkn2b(p15), and between BALB/c and Mus spretus for Cdkn2c(p18(INK4c)) were used to position these loci with respect to the Pctr1 locus. These cyclin-dependent kinase (CDK) inhibitors mapped to a 6 cM interval of chromosome 4 between Ifna and Tal1. C.D2-Chr 4 congenic strains harboring DBA/2 alleles associated with the Pctr1 locus contained DBA/2 "resistant" alleles of the CDK4/CDK6 inhibitors p16 and p15. On sequencing p16 and p18 cDNAs, two different allelic variants within ankyrin repeat regions of p16 were found between BALB/c and DBA/2 mice. By using an assay involving PCR amplification and restriction enzyme digestion, allelic variants were typed among several inbred strains of mice. One of the variants, G232A, was specific to two inbred strains, BALB/cAn and ABP/Le, of mice and occurred in a highly conserved amino acid in both human and rat p16. When tested with wild-type (DBA/2) p16, both A134C and G232A BALB/c-specific variants of p16 were inefficient in their ability to inhibit the activity of cyclin D2/CDK4 in kinase assays with retinoblastoma protein, suggesting this defective, inherited allele plays an important role in the genetic susceptibility of BALB/c mice for plasmacytoma induction and that p16(INK4a) is a strong candidate for the Pctr1 locus.

Figure 1

Cdkn2a, Cdkn2b, and Cdkn2c map to the mid-portion of mouse Chr 4 within the interval harboring the plasmacytoma susceptibility/resistance gene, Pctr1. (A) Haplotype analysis of 166 backcross progeny from the cross (BALB/cAnPt × DBA/2N)F₁ × BALB/cAnPt. The loci genotyped in the cross are indicated on the left. Each column represents the chromosome inherited in the backcross progeny; the number of progeny exhibiting each type of chromosome is listed at the bottom. Empty squares refer to the BALB/cAnPt allele and filled squares to the DBA/2NPt allele. (B) A map of mouse Chr 4 indicating the locations of genes mapped in the cross and derived from the haplotype data in A.

Figure 2

Amino acid sequence of the ankyrin repeat regions of DBA/2 (wild-type) p16^INK4a. Amino acid substitutions are indicated above or below the site of the polymorphic variants (ankyrin repeats 1 and 2) and tumor-specific mutations (ankyrin repeat 3). The amino acid sequences between BALB/c and DBA/2 were identical in the third and fourth ankyrin repeats.

Figure 3

Allele-specific variants of p16^INK4a exon 1 (A134C) and exon 2 (G232A) in a variety of inbred and wild-derived strains of mice. The majority of strains carried the DBA/2N allele and thus are designated as wild type.

Figure 4

Allelic variants of p16^INK4a are inefficient inhibitors of Rb phosphorylation. Extracts from baculovirus-infected Sf9 cells expressing CDK4 and cyclin D2 were mixed with increasing amounts of wild-type or variant A134C (exon 1) and G232A (exon 2) GST-p16^INK4a fusion proteins. The mixtures were incubated for either 20 or 30 min before assaying their ability to phosphorylate pRb as described in Materials and Methods.

Figure 5

Northern analysis of the Cdkn2a locus reveals that the majority of plasmacytoma cell lines do not express exon 1α transcripts of p16INK4a, but do express a 1-kb transcript corresponding to exon 1β (p19^ARF). (Left to Right) The lanes contain 5 μg of poly(A)⁺ mRNA from BALB/cAn spleen, DBA/2 spleen, a primary plasma cell tumor, and five established plasmacytoma cell lines (in order): MOPC460, TEPC2027, XRPC24, TEPC1165, and MOPC265.