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Cdkn2a, the Cyclin-Dependent Kinase Inhibitor Encoding P16ink4a and P19arf, Is a Candidate for the Plasmacytoma Susceptibility Locus, Pctr1

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Cdkn2a, the Cyclin-Dependent Kinase Inhibitor Encoding P16ink4a and P19arf, Is a Candidate for the Plasmacytoma Susceptibility Locus, Pctr1 Proc. Natl. Acad. Sci. USA Vol. 95, pp. 2429–2434, March 1998 Genetics Cdkn2a, the cyclin-dependent kinase inhibitor encoding p16INK4a and p19ARF, is a candidate for the plasmacytoma susceptibility locus, Pctr1 SHULING ZHANG,EDWARD S. RAMSAY, AND BEVERLY A. MOCK* Laboratory of Genetics, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255 Communicated by Michael Potter, National Institutes of Health, Bethesda, MD, December 24, 1997 (received for review November 17, 1997) ABSTRACT Plasma cell tumor induction in mice by cytoma susceptibilityyresistance locus resides. The protein pristane is under multigenic control. BALByc mice are sus- products of these genes bind to the CDKs CDK4 and CDK6 (6, ceptible to tumor development; whereas DBAy2 mice are 7), which, in turn, form complexes with the D-type G1 cyclins resistant. Restriction fragment length polymorphisms be- that phosphorylate pRb to control both cell cycle and tumor tween BALByc and DBAy2 for Cdkn2a(p16) and Cdkn2b(p15), suppression (8, 9). We compared the sequences of p16 and p18 and between BALByc and Mus spretus for Cdkn2c(p18INK4c) between susceptible (BALByc) and resistant (DBAy2N) were used to position these loci with respect to the Pctr1 locus. strains of mice, and found sequence variants in p16 located in These cyclin-dependent kinase (CDK) inhibitors mapped to a two different ankyrin repeat regions of the gene. DBAy2 (wild 6 cM interval of chromosome 4 between Ifna and Tal1. type) and BALByc A134C and G232A variants of the p16 C.D2-Chr 4 congenic strains harboring DBAy2 alleles asso- genes were fused to the glutathione S-transferase (GST) gene, ciated with the Pctr1 locus contained DBAy2 ‘‘resistant’’ and purified GST fusion proteins were tested for their ability alleles of the CDK4yCDK6 inhibitors p16 and p15. On se- to inhibit cyclin D2yCDK4 activity in kinase assays with quencing p16 and p18 cDNAs, two different allelic variants full-length retinoblastoma (Rb) protein. The BALByc variants within ankyrin repeat regions of p16 were found between were defective in their ability to inhibit Rb phosphorylation, BALByc and DBAy2 mice. By using an assay involving PCR implying that these allelic variants predispose BALByc mice amplification and restriction enzyme digestion, allelic vari- for the development of plasma cell tumors. ants were typed among several inbred strains of mice. One of the variants, G232A, was specific to two inbred strains, MATERIALS AND METHODS BALBycAn and ABPyLe, of mice and occurred in a highly conserved amino acid in both human and rat p16. When tested Mice and Markers. Backcross mice were bred and main- with wild-type (DBAy2) p16, both A134C and G232A BALBy tained in conventional closed barrier conditions at PerImmune c-specific variants of p16 were inefficient in their ability to (Rockville, MD) under National Cancer Institute Contract inhibit the activity of cyclin D2yCDK4 in kinase assays with NO1-BC-21075. DNA isolation, restriction enzyme digestion, retinoblastoma protein, suggesting this defective, inherited agarose gel electrophoresis, Southern blotting, and restriction allele plays an important role in the genetic susceptibility of fragment length polymorphism analyses were performed as BALByc mice for plasmacytoma induction and that p16INK4a described (3, 10, 11). DNA probes for Ifna, Mtv13, D4Lgm1, is a strong candidate for the Pctr1 locus. D4Rck41, Ccnb1-rs10, Tal2, Gt10, and Nppa were described previously (3, 12). The probes for Cdkn2a(p16) and Cdkn2b(p15) were 1.1-kb and 1.3-kb EcoRI fragments isolated Mouse plasma cell tumors provide an animal model system from pBluescript KS (13). The probe for Cdkn2c(p18) was a relevant to several human B cell malignancies, including 510-bp BamHI–EcoRI fragment containing the entire coding human plasma cell tumors, multiple myeloma, Burkitt’s lym- sequence of p18 excised from pBluescript SK (14). Mouse map phoma, and non-Hodgkin’s lymphomas. The mouse plasma pairs (D4Mit) for microsatellite sequences (Research Genet- cell tumors are characterized by a hallmark translocation ics, Huntsville, AL) also were typed in the backcross by PCR. involving the myc oncogene on mouse chromosome (Chr) 15 PCRs were performed on 500 ng of DNA with either a and one of the Ig loci, either Igh, Igk,orIgl located on Chr 12, PTC-100 (MJ Research, Cambridge, MA) or Omnigene (Hy- 6, and 16, respectively (1). A relatively high incidence (40– y baid, Middlesex, U.K.) thermocycler by using the following 60%) of plasma cell tumors can be induced in BALB cAn mice cycling conditions: 94°C for 1 min, 55°C for 1 min, 72°C for 30 by the i.p. injection of pristane or silicone gels (2); in contrast, y sec for 28 cycles, and a final extension at 72°C for 10 min. DBA 2N mice do not develop tumors after injection with these Tumor Inductions. Primary tumors were induced with either agents that induce a state of chronic inflammation in the three 0.5-ml injections of pristane or by a single 0.2-ml peritoneal cavity. Backcross (3) and congenic strain analyses injection of pristane followed by inoculation with a retroviral (4, 5) have indicated that at least four genes determine vector containing ras and myc sequences coupled to Ig heavy susceptibility to mouse plasmacytomagenesis. One of these chain enhancer and promoter sequences (15, 16). genes, Pctr1, resides in the mid-portion of mouse Chr 4 near the Northern Analyses. Total RNA and polyadenylated mRNA interferon alpha locus (3, 4). was isolated from pulverized mouse tissues by using Trizol In the current study, we mapped the genes encoding three cyclin-dependent kinase (CDK) inhibitors, Cdkn2a(p16INK4a), INK4b INK4c Abbreviations: CDK, cyclin-dependent kinase; GST, glutathione S- Cdkn2b(p15 ), and Cdkn2c(p18 ) to the interval of transferase; Rb, retinoblastoma; Chr, chromosome; LOH, loss of mouse Chr 4 between Ifa and Tal1 where the Pctr1 plasma- heterozygosity; IL, interleukin. Data deposition: The sequences reported in this paper have been The publication costs of this article were defrayed in part by page charge deposited in the GenBank database (accession nos. AF044335 and AF044336). payment. This article must therefore be hereby marked ‘‘advertisement’’ in *To whom reprint requests should be addressed at: National Cancer accordance with 18 U.S.C. §1734 solely to indicate this fact. Institute, National Institutes of Health, Building 37, Room 2B08, 37 0027-8424y98y952429-6$0.00y0 Convent Drive, MSC 4255, Bethesda, MD 20892-4255. e-mail: bev@ PNAS is available online at http:yywww.pnas.org. helix.nih.gov. 2429 Downloaded by guest on September 23, 2021 2430 Genetics: Zhang et al. Proc. Natl. Acad. Sci. USA 95 (1998) (GIBCOyBRL) and Fast-Track (Invitrogen), respectively. Fif- fused in-frame to the GST gene in the vector pGEX-5X-3 teen micrograms of total and 5 mg of poly(A)1 mRNA were (Pharmacia), and the sequences of alleles were confirmed by separated on 1% agarose gels containing 13 MOPS and 2% dideoxy sequencing (fmol DNA Sequencing System, Pro- formaldehyde and transferred to positively charged nylon mega). Proteins were isolated with modifications to previously membranes (Hybond N1)in103 saline sodium citrate. RNA published protocols (18, 19). All proteins were checked by was covalently linked to the membrane with 300 mJoules of SDSyPAGE gels and quantified by using a Bio-Rad Protein UV radiation in a Bio-Rad crosslinker. Probes for full-length Assay Kit. p16, exon 1a (bases 32–205), and exon 1b (bases 47–206) were In vitro Kinase Assays. Sf9 insect cell lysates containing hybridized for 14 hr. Blots were washed with a final stringency CDK4 and cyclinD2 were prepared as described (20, 21). of 0.23 saline sodium citrate at 65°C for 20 min. Supernatants were precleared with protein A-Sepharose beads Reverse Transcription–PCR. Five micrograms of total or 2 (20 mlymg of lysate) for 1 hr at 4°C; beads were removed by mg of poly(A)1 mRNA was coincubated with 500 ng of centrifugation and supernatants incubated with a polyclonal oligo(dT) cellulose for 10 min at 70°C in a 6-ml volume. After anticyclin D antibody (20 mgymg lysate) (Upstate Biotechnol- incubation, the following reagents (GIBCOyBRL) were ogy) to immunoprecipitate the CDK4-cyclinD2 complex from added: 13 reverse transcriptase buffer, 5 units of RNase 200 mg of the lysate. Immunoprecipitated CDK4-cyclinD2 was inhibitor, 5 mM DTT, 1 mM dNTP, and 200 units of Moloney mixed with increasing amounts of either GST-p16INK4a wild- murine leukemia virus–reverse transcriptase. The 20.5-ml re- type (DBAy2), GST-p16INK4a A134C, G232A, or the com- action was incubated for 45 min at 37°C. PCR with an oil bination of A134C and G232A fusion proteins (250–1,500 ng) overlay was done in a 50-ml reaction volume containing 5 mlof in a total volume of 40 ml of kinase buffer for either 20 or 30 the reverse transcriptase reaction product, 10 mM Tris (pH min at 30°C. After this preincubation, the kinase reactions RB 8.3), 50 mM KCl, 1.5 mM MgCl2, 50 pmols of each primer, 100 were begun by adding 200 ng of Rb protein (p110 , full mM of each deoxyribonucleotide, and 2.5 units of Taq DNA length) (QED Bioscience, San Diego, CA) as substrate, 10 mCi polymerase (Perkin–Elmer). Each cycle of PCR amplification of g-32P ATP (4,500 CiymMol, Amersham), and 50 mM cold included 50 sec at 94°C, 45 sec at 62°C, and 1 min at 72°C. ATPto10ml of the CDK4-cyclinD2-p16 mixtures and incu- Product bands were excised after separation on 2% agarose bating for 20 min at 30°C. Sample preparation, electrophoresis gels and removed from the gel with Geneclean II (Bio101) NaI on SDSyPAGE gels, autoradiography and phosphor screen reagents.
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