Proc. Natl. Acad. Sci. USA Vol. 95, pp. 2429–2434, March 1998 Genetics

Cdkn2a, the -dependent kinase inhibitor encoding p16INK4a and p19ARF, is a candidate for the plasmacytoma susceptibility , 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 susceptibility͞resistance locus resides. The pristane is under multigenic control. BALB͞c mice are sus- products of these bind to the CDKs CDK4 and CDK6 (6, ceptible to tumor development; whereas DBA͞2 mice are 7), which, in turn, form complexes with the D-type G1 resistant. Restriction fragment length polymorphisms be- that phosphorylate pRb to control both and tumor tween BALB͞c and DBA͞2 for Cdkn2a() and Cdkn2b(p15), suppression (8, 9). We compared the sequences of p16 and p18 and between BALB͞c and Mus spretus for Cdkn2c(p18INK4c) between susceptible (BALB͞c) and resistant (DBA͞2N) 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 repeat regions of the . DBA͞2 (wild 6 cM interval of 4 between Ifna and Tal1. type) and BALB͞c A134C and G232A variants of the p16 C.D2-Chr 4 congenic strains harboring DBA͞2 alleles asso- genes were fused to the glutathione S-transferase (GST) gene, ciated with the Pctr1 locus contained DBA͞2 ‘‘resistant’’ and purified GST fusion were tested for their ability alleles of the CDK4͞CDK6 inhibitors p16 and p15. On se- to inhibit cyclin D2͞CDK4 activity in kinase assays with quencing p16 and p18 cDNAs, two different allelic variants full-length retinoblastoma (Rb) protein. The BALB͞c variants within regions of p16 were found between were defective in their ability to inhibit Rb phosphorylation, BALB͞c and DBA͞2 mice. By using an assay involving PCR implying that these allelic variants predispose BALB͞c mice amplification and restriction 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 BALB͞cAn and ABP͞Le, 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 (DBA͞2) p16, both A134C and G232A BALB͞ 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 D2͞CDK4 in kinase assays with NO1-BC-21075. DNA isolation, restriction enzyme digestion, , 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 BALB͞c 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– ͞ 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, ͞ 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 0027-8424͞98͞952429-6$0.00͞0 Convent Drive, MSC 4255, Bethesda, MD 20892-4255. e-mail: bev@ PNAS is available online at http:͞͞www.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)

(GIBCO͞BRL) 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 ␮g of poly(A)ϩ mRNA were (Pharmacia), and the sequences of alleles were confirmed by separated on 1% agarose gels containing 1ϫ 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 Nϩ)in10ϫ saline sodium citrate. RNA published protocols (18, 19). All proteins were checked by was covalently linked to the membrane with 300 mJoules of SDS͞PAGE gels and quantified by using a Bio-Rad Protein UV radiation in a Bio-Rad crosslinker. Probes for full-length Assay Kit. p16, 1␣ (bases 32–205), and exon 1␤ (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 0.2ϫ 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 ␮l͞mg of lysate) for 1 hr at 4°C; beads were removed by ␮g of poly(A)ϩ 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-␮l volume. After anticyclin D antibody (20 ␮g͞mg lysate) (Upstate Biotechnol- incubation, the following reagents (GIBCO͞BRL) were ogy) to immunoprecipitate the CDK4-cyclinD2 complex from added: 1ϫ reverse transcriptase buffer, 5 units of RNase 200 ␮g 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-␮l re- type (DBA͞2), 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-␮l reaction volume containing 5 ␮lof in a total volume of 40 ␮l 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 ␮M of each deoxyribonucleotide, and 2.5 units of Taq DNA length) (QED Bioscience, San Diego, CA) as substrate, 10 ␮Ci polymerase (Perkin–Elmer). Each cycle of PCR amplification of ␥-32P ATP (4,500 Ci͞mMol, Amersham), and 50 mM cold included 50 sec at 94°C, 45 sec at 62°C, and 1 min at 72°C. ATPto10␮l 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 SDS͞PAGE gels, autoradiography and phosphor screen reagents. Primers (F, R) for p16 were: ACTGAATCTCCG- imaging were done as described (20, 21). The kinase assays CGAGGAAAGCGAACT,AGACACGCTAGCATCGCTA- were repeated four times with the exception that the assay GAAGTGAAGC. Primers (F, R) for mP18 were: CACCA- involving 1.5 ␮g of the various p16 allelic variants was per- CTGTGAACAAGGGACCCTAAAGA,CAGTGTGAGGT- formed once. Densitometric analyses were averaged to calcu- CAGTGAGAGAGACCTCTACA. Primers (F, R) for mP19 late the percent of wild-type activity for each of the BALB͞ were: GTCGCAGGTTCTTGGTCACTGTGA,AGACACG- c-specific variants. CTAGCATCGCTAGAAGTGAAGC. Sequencing. Reverse transcription–PCR products were RESULTS cloned into TA vector pCR 2.1 (Invitrogen) according to manufacturer’s recommendations. Dideoxy sequencing (fmol Cdkn2a, Cdkn2b, and Cdkn2c Map in the Pctr1 Interval. DNA Sequencing System, Promega) was performed on 5 ␮lof Restriction fragment length polymorphisms were used to map plasmid DNA isolated with Wizard reagents (Promega). The each of the three CDK inhibitors. For Cdkn2a (p16), PvuII thermocycler repeated extension program was 35 cycles of digestion identified 3.4-, 5.1-, and 5.9-kb fragments in BALB͞ 95°C for 15 sec, 60°C for 45 sec, and 72°C for 20 sec. Sequence cAnPt and 3.7-, 5.1-, and 6.4-kb fragments in DBA͞2N. For primers included vector M13F and M13R, the reverse tran- Cdkn2b (p15), TaqI digestion identified 400-bp and 1-kb scription–PCR primers, and internally chosen primers. Prod- fragments in BALB͞cAnPt and 380- and 600-bp fragments in ucts were run on denaturing 6% polyacrylamide-urea gels for DBA͞2N. Cdkn2a and Cdkn2b were hybridized to a panel of ͞ ϫ ͞ ϫ ͞ 2–3 hr, followed by drying and autoradiography. 166 (BALB c DBA 2)F1 BALB c backcross progeny that Allele-Specific PCRs. Several inbred and wild strains of mice had previously been typed for both plasmacytoma suscepti- were genotyped for their allelic variants of exon 1 (A134C) and bility and a number of other DNA markers distributed exon 2 (G232A). Exon 1 primers used to amplify the region throughout the genome (3). The allele distribution patterns containing the A134C variant were: (F) ACTGAATCTCCG- were linked to those of several markers on mouse Chr 4. Based CGAGGAAAGCGAACT and (R) GAATCGGGGTACGA- on these analyses Cdkn2a and Cdkn2b cosegregated with each CCGAAAGAGT. Exon 2 primers used to amplify the region other as predicted from their physical locations (13, 22, 23). containing the G232A variants were: (F) GTGATGATGAT- These two CDK inhibitors mapped distal to the interferon GGGCAACGTTCA and (R) GGGCGTGCTTGAGCTGA- alpha locus on mouse Chr 4 (Fig. 1A). The gene order, AGCTA. PCR products containing 1 and 2 were di- recombination fraction (recombinants͞total) and distance (in gested with NlaIII and BsaAI, respectively, to produce allele- cM) between markers were as follows: specific bands for detection of the polymorphic variants. Ifna –(3͞166) 1.8 Ϯ 1.03–Cdkn2a, b–(4͞166) 2.4 Ϯ 1.2– Cycling conditions for the amplification of exon 1 were 94°C D4Rck12–(6͞166) 3.6 Ϯ 1.4–Mtv13–(1͞166) 0.6 Ϯ 0.6–Tal2. for 1 min, followed by 35 cycles of 94°C for 1 min, 62°C for 45 Cdkn2c(p18) was not polymorphic between BALB͞c and sec, 72°C for 2 min, and for exon 2, they were 94°C for 1 min, DBA͞2N; however, a PvuII digestion revealed a 3.8-kb frag- followed by 35 cycles of 94°C for 30 sec and 71°C for 45 sec and ment in BALB͞c and a 2.5-kb fragment in Mus spretus. When a final extension of 72°C for 10 min for both exon 1 and exon Cdkn2c was hybridized to PvuII-digested DNA from 66 ͞ ϫ ϫ ͞ 2 PCRs. (BALB c M. spretus)F1 BALB c backcross progeny, its Construction of GST-Fusion Proteins. DBA͞2 (wild type) allele distribution patterns cosegregated completely (0͞66) p16INK4a was cloned into pBluescript KS. BALB͞c allelic with those for Tal2, indicating that the two genes were tightly variants A134C and G232A p16INK4a clones were constructed linked. A genetic map showing the locations of the three CDK by PCR-directed mutagenesis with Pfu polymerase (Strat- inhibitors relative to the Chr 4 markers and the Pctr1 locus is agene) (17). Primers used to generate allelic variants were: shown in Fig. 1B. In previous studies, C.D2-Lgm1A and (A134C F: TGTGCCTGACGTGCGGGCACT and R: AGT- C.D2-Lgm1H congenic strains of mice had been shown to GCCCGCACGTCAGGCACA) and (G232A F: GGCAAC- carry DBA͞2 alleles of genes across this same interval of GTTCACATAGCAGCTCTTC and R: GAAGAGCTGCTA- mouse Chr 4 and were relatively resistant to plasma cell tumor TGTGAACGTTGCC). The wild-type and variant genes were induction (3, 4), thus establishing the fact that the CDK Downloaded by guest on September 23, 2021 Genetics: Zhang et al. Proc. Natl. Acad. Sci. USA 95 (1998) 2431

FIG. 1. Cdkn2a, Cdkn2b, and Cdkn2c map to the mid-portion of mouse Chr 4 within the interval harboring the plasmacytoma susceptibility͞ resistance gene, Pctr1.(A) Haplotype analysis of 166 backcross progeny from the cross (BALB͞cAnPt ϫ DBA͞2N)F1 ϫ BALB͞cAnPt. 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 BALB͞cAnPt allele and filled squares to the DBA͞2NPt 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.

inhibitors, Cdkn2a,b,c, reside in the same interval as the inbred strains; these PCR products were digested with en- plasma cell tumor susceptibility͞resistance gene, Pctr1. Both of zymes whose restriction sites were destroyed by the base these resistant congenic strains of mice carried DBA͞2 alleles change. The distribution of allele specific variants is shown in of p15 and p16; p18 was not polymorphic between BALB͞c Fig. 3. BALB͞c and ABP͞Le mice were the only inbred strains and DBA͞2, but mapped at the border of the Pctr1 interval. with the G232A variant of p16INK4a. This variant also was Sequence Variants of Cdkn2a in Susceptible and Resistant present in two wild-derived strains of mice. Mice. p16 and p18 cDNAs from BALB͞c and DBA͞2 were BALB͞c Allelic Variants of p16INK4a Are Inefficient Inhib- cloned and their sequences compared. The Cdkn2c(p18) cD- itors of Rb Phosphorylation. To test whether BALB͞c and NAs showed no differences between BALB͞c and DBA͞2. DBA͞2 allelic variant p16 proteins differed in their ability to The Cdkn2a locus encodes two distinct proteins that result inactivate the -CDK4 complex, we performed in vitro from of either exon 1␣ to make p16INK4a or kinase assays using baculovirus-expressed cyclin D2-CDK4 exon 1␤ to make p19ARF to exon 2. For Cdkn2a, two sense complexes with full-length Rb as substrate. As shown in Fig. 4 (T75C in exon 1␤ of p19ARF and C142A in exon 1␣ of P16INK4a (representative of one of the four experiments), 1 ␮gof and two missense (A134C in exon 1␣ of p16 and G232A in exon DBA͞2 (wild type) p16 was capable of fully inhibiting Rb 2ofp16(ϭ G257A in p19ARF because p16 and p19 share the phosphorylation. In contrast, 1 ␮gofBALB͞c A134C, G232A, same exon 2) allelic variants were found between BALB͞c and and A134C with G232A variant proteins were significantly DBA͞2 (Fig. 2). Three additional cDNAs from BALB͞c compromised in their ability to inhibit Rb phosphorylation plasmacytoma cell lines TEPC2027, TEPC1165, and X24 also (Fig. 4). Densitometry revealed the following percent of were sequenced and found to contain the BALB͞c-specific wild-type activity for 1 ␮g of each variant tested: A134C (0%), variants. The two variants of p16 common in BALB͞c mice, G232A (25%), and the combination of A134C and G232A A134C in exon 1␣ and G232A in exon 2, produce amino acid (0%). In addition, 1.5 ␮g of each of these variant proteins were changes from histidine to proline and valine to isoleucine, also inefficient in their inhibition of the cyclin D2-CDK4 respectively. G257A in p19ARF results in an amino acid change complex. from arginine to histidine. The A134C change in p16INK4a Northern Analysis of Cdkn2a. When the full-length probe occurs within the first ankyrin repeat (Fig. 2). The G232A for p16 was hybridized to total RNA from BALB͞c and variant within the second ankyrin repeat (Fig. 2) of p16INK4a DBA͞2 kidneys, spleens, livers, and intestines, a normal is of interest in that the valine in the DBA allelic form at this transcript of approximately 990 bp corresponding to an mRNA position is also present in humans and rats. Two of the tumor that uses exon 1␣ was evident (Fig. 5). Mature B cells purified cell lines also carried additional somatic mutations within the from C.B6-interleukin (IL) 6 (N12) transgenic mice also third ankyrin repeat of p16 (Fig. 2). These constituted single expressed the 990-bp transcript, indicating the use of exon 1␣. base substitutions: G301A, C304T, and A350G, which pro- However, among 10 established plasmacytoma cell lines, only duced amino acid changes from valine to methionine, histidine two tumor cell lines expressed the exon 1␣ transcript (XRPC24 to tyrosine, and histidine to arginine, respectively in p16INK4a. and MOPC265). Eighty percent of the cell lines (TEPC1165, The sequence variants for p16 observed in BALB͞c and TEPC2027, BPC4, MOPC460, MOPC31C, MOPC315, B9, and tumors are illustrated in Fig. 2. Allele-specific primers were SP2͞0) expressed a 1.1- to 1.2-kb transcript, typical of the used to amplify the two allelic variants in a number of different alternatively spliced variant of p16INK4a that uses the longer

FIG. 2. Amino acid sequence of the ankyrin repeat regions of DBA͞2 (wild-type) p16INK4a. 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 BALB͞c and DBA͞2 were identical in the third and fourth ankyrin repeats. Downloaded by guest on September 23, 2021 2432 Genetics: Zhang et al. Proc. Natl. Acad. Sci. USA 95 (1998)

FIG. 3. Allele-specific variants of p16INK4a exon 1 (A134C) and exon 2 (G232A) in a variety of inbred and wild-derived strains of mice. The majority of strains carried the DBA͞2N allele and thus are designated as wild type.

exon1 ␤, yielding the protein referred to as p19ARF (Fig. 5). In G232A was a rare variant occurring in only two of the 14 inbred addition, one primary tumor and one established cell line, strains examined. Both variants, coding for different amino TEPC 1198, did not express a transcript for exon 1␣ or ␤. acids at the two positions, are old polymorphisms as indicated Assessment of Loss of Heterozygosity (LOH) at Cdkn2a. by the presence of these variants in wild-derived strains of Second-generation transplanted tumors derived from a series mice. ͞ ϫ ͞ of 16 (BALB c C57BL 6)F1 primary tumors induced with The in vitro kinase assays show that BALB͞cAn p16 is an pristane were examined for evidence of LOH at the Cdkn2a inefficient inhibitor of Rb phosphorylation by Cdk4͞cyclin D2. locus; only one tumor showed LOH. This tumor has shown When compared with wild-type (DBA͞2) p16, the BALB͞cAn LOH at all loci examined from Chr 4 (data not shown). In specific variant proteins (A134C, G232A, and the combination addition, LOH at Cdkn2a was not seen in a series of 14 primary of A134C and G232A) were defective in their ability to inhibit tumors induced with pristane plus retroviral vectors contain- Rb phosphorylation. Because BALB͞c mice make a less ͞ ϫ ͞ ing Ha-ras and Myc sequences in (BALB c DBA 2)F1 efficient or defective p16 protein, it is likely that the negative hybrids. feedback loop between p16 and Rb (24) is ineffective, and transformed B cells are allowed to continue through the cycle DISCUSSION instead of becoming growth arrested in G1. Several p16 point mutations were found linked to familial These studies have strongly implicated Cdkn2a as a candidate (25). At least five of the point mutations observed ͞ gene for one of the plasmacytoma tumor susceptibility in melanoma kindreds also were shown to be less efficient in resistance loci, Pctr1. Pctr1 maps distal to the Ifa locus (3, 4) their inhibition of Rb phosphorylation relative to wild-type p16 in a region of mouse Chr 4 very near the boundary of linkage (21). Homozygous deletion of p16 is the most common form with either human 9p21 or 1p31. In the current study of alteration seen in human tumors and most mouse tumors we mapped the Cdkn2a and Cdkn2b loci (human 9p21) 1.8 cM examined (26, 27). In general, BALB͞c plasma cell tumors do distal to the Ifa locus, placing these two CDK inhibitors near not show evidence of p16 deletions. Only one of the 30 primary the Jun oncogene (human 1p31) and well within the Pctr1 tumors induced in F1 hybrids showed evidence of LOH and interval. only one established plasmacytoma cell line lacked both ␣ and Sequence variants in the Cdkn2a gene were found between ␤ ͞ ͞ transcripts. tumor-resistant DBA 2 mice and tumor-susceptible BALB Northern analyses of primary and established tumor cell cAn mice. The majority of inbred strains were found to carry ͞ ͞ lines shows a relatively complex pattern of p16 p19 expression. the DBA 2 allele at both sequence variants. Furthermore, In normal mouse tissues (spleen, kidney, and purified popu- lations of B cells), p16 mRNA predominated, indicating tran- scription initiation at exon 1␣. In contrast, almost all estab- lished plasmacytoma cell lines exhibited expression of p19ARF, indicating the initiation of transcription at the upstream exon 1␤. The majority of plasma cell tumors did not express exon 1␣-containing mRNA for p16INK4a. Preliminary experiments (data not shown) have suggested that p16 is silenced by

FIG. 4. Allelic variants of p16INK4a are inefficient inhibitors of Rb FIG. 5. Northern analysis of the Cdkn2a locus reveals that the phosphorylation. Extracts from baculovirus-infected Sf9 cells express- majority of plasmacytoma cell lines do not express exon 1␣ transcripts ing CDK4 and cyclin D2 were mixed with increasing amounts of of p16INK4a, but do express a 1-kb transcript corresponding to exon wild-type or variant A134C (exon 1) and G232A (exon 2) GST- 1␤ (p19ARF). (Left to Right) The lanes contain 5 ␮g of poly(A)ϩ mRNA p16INK4a fusion proteins. The mixtures were incubated for either 20 or from BALB͞cAn spleen, DBA͞2 spleen, a primary plasma cell tumor, 30 min before assaying their ability to phosphorylate pRb as described and five established plasmacytoma cell lines (in order): MOPC460, in Materials and Methods. TEPC2027, XRPC24, TEPC1165, and MOPC265. Downloaded by guest on September 23, 2021 Genetics: Zhang et al. Proc. Natl. Acad. Sci. USA 95 (1998) 2433

methylation in a large number of plasmacytomas, similar to synthesis of p18INK4c and stabilize its interaction with CDK6 in that observed in a growing number of human tumors (28–30), B cells (55). It is intriguing to propose a similar role for IL-6 INK4a͞ ARF including multiple myeloma (31). in the up-regulation of p16 p19 , leading to G1 growth In addition to regulation by both deficient (loss of function) arrest. allelic variation and methylation, p16INK4a is likely to be One scenario is that an inefficient or nonfunctional p16 impaired in plasma cell tumors by additional somatic mutation. allele in BALB͞c mice permits hyperphosphorylation of Rb by The Cdkn2a locus was sequenced in three plasmacytoma cell Cdk4͞Cdk6. This could lead to unchecked plasma cell prolif- lines; in addition to carrying the BALB͞c allelic variants of eration, further somatic mutation in the Cdkn2a locus, and p16, two of the tumor lines carried additional point mutations. possibly even to a reduction in and IL-6 production Interestingly, two of the tumor-specific mutations, H75Y and by macrophages. The fact that at least two additional genes on H90R, were observed at two conserved histidines, H83 and Chr 4 also are involved in the development of pristane-induced H98 in humans. These same histidines also have been mutated plasma cell tumors in BALB͞c mice underscores the complex- in pancreatic adenocarcinomas (H83Y) (32), primary astro- ity of this genetic disease and its usefulness as a model for cytic tumors (H83Y) (33), primary (H98P) (34), human B cell malignancies. and gliomas (H98Y) (35). H98P was found to be functionally defective in its ability to inhibit both Rb phosphorylation (34, We thank C. J. Sherr who provided baculoviruses containing CDK4 36) and promotion of G1 growth arrest (34); furthermore, and cyclin D2, as well as probes for p15, p16, and p18. We appreciate protein folding studies revealed that the H98P protein had a the assistance of V. Bliskovsky with in vitro mutagenesis design. We disrupted secondary structure and backbone folding (36). On appreciate discussions with R. DePinho concerning knockout mice. the other hand, H98Y was as effective as wild-type p16 in The following were especially helpful in discussions concerning the in inhibiting Rb phosphorylation (35). It remains to be tested vitro kinase assays: X. Zou, K. Calame, M. Serrano, F. Kaye, G. whether a histidine to arginine change, as seen in plasma cell Otterson, S. Huang, W. Kaelin, and P. Hamel. We also thank N. Huang tumors, inactivates the inhibitor or not. Human wild-type p16 and M. Potter for providing us with purified B cells from C.B6-IL6 has been found to be a relatively unstable protein with a free transgenic mice, G. Jones for providing RNA from silicone-induced energy of unfolding of 1.9 kcal͞mol (37). Perhaps this explains tumors, and R. Nordan and E. Mushinski for providing several why so many mutations (including a sense mutation) disrupt plasmacytoma cell lines. In addition, we thank W. DuBois and J. Wax for the breeding and tumor inductions in F hybrid mice. We thank M. the structure and folding of the molecule. Therefore, it seems 1 ͞ Potter, F. Mushinski, and B. Gerwin for their helpful comments plausible to hypothesize that BALB cAn mice are carrying concerning the manuscript. defective alleles of Cdkn2a (Pctr1) and that tumors arising in these mice are susceptible to somatic mutation that may 1. Potter, M. & Wiener, F. (1992) Carcinogenesis 13, 1681–1697. INK4a ARF further alter or inactivate Cdkn2a (p16 and p19 ) 2. Potter, M. & Morrison, S. (1996) Curr. Top. Microbiol. Immunol. function. 210, 397–407. Our observations further support a role for Cdkn2a in B cell 3. Mock, B. A., Krall, M. A. & Dosik, J. K. (1993) Proc. Natl. Acad. malignancy. Studies of p16 and p19 knockout mice (with Sci. USA 90, 9499–9503. abnormal extramedullary hematopoiesis and development of 4. Potter, M., Mushinski, E. B., Wax, J. S., Hartley, J. & Mock, B. B cell lymphomas) (38, 57), non-Hodgkin’s lymphomas of B (1994) Cancer Res. 54, 969–975. cell origin (14% of 42 patients had p16 mutations) (39), 5. Mock, B. A., Hartley, J., Le Tissier, P., Wax, J. S. & Potter, M. multiple myeloma (hypermethylation observed in 75% of 12 (1997) Blood 90, 4092–4098. patients) (31), and precursor B acute lymphoblastic leukemias 6. Xiong, Y., Zhang, H. & Beach, D. (1993) Genes Dev. 7, 1572– 1583. [15% of 81 primary tumors revealed homozygous deletions 7. Serrano, M., Hannon, G. J. & Beach, D. 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When Rb is hypophosphorylated, it can bind to and 13. Quelle, D. E., Ashmun, R. A., Hannon, G. J., Rehberger, P. A., sequester members of the transcription factor family Trono, D., Richter, K. H., Walker, C., Beach, D., Sherr, C. J. & Serrano, M. (1995) Oncogene 11, 635–645. (reviewed in refs. 45 and 46). In this manner Rb acts to inhibit 14. Hirai, H., Roussel, M. F., Kato, J.-Y., Ashmun, R. A. & Sherr, E2f activity, thereby blocking the transactivation of genes (e.g., C. J. (1995) Mol. Cell. Biol. 15, 2672–2681. Myc, Myb, Dhfr, and DNA pol a) essential for pro- 15. Clynes, R. J., Wax, J., Staton, L. W., Smith-Gill, S., Potter, M. & gression and cellular proliferation (45–47). Marcu, K. B. (1988) Proc. Natl. Acad. Sci. USA 85, 6067–6071. In its hypophosphorylated state, Rb also can bind the 16. Mock, B., Wax, J., Clynes, R., Marcu, K. B. & Potter, M. (1988) nuclear transcription factor, NF-IL-6, albeit, with a 10-fold Curr. Top. Microbiol. Immunol. 141, 125–127. lower affinity than E2F1 (48, 49). In this situation, Rb acts 17. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R. positively to increase both the DNA binding and transcrip- (1989) Gene 77, 51–59. tional activities of NF-IL-6 (48). NF-IL-6 (CCAAT͞enhancer- 18. Smith, D. B. & Johnson, K. S. (1988) Gene 67, 31–40. binding protein beta, Cebpb) has several roles germane to 19. Kaelin, W. G., Pallas, D. C., DeCaprio, J. A., Kaye, F. J. & plasma cell proliferation, growth, and differentiation. It has Livingston, D. M. (1991) Cell 643, 521–532. been shown to promote cellular differentiaton in B cells (48), 20. Kato, J.-Y., Matsushime, H., Hiebert, S. W., Ewen, M. E. & Genes Dev. 7, induce apoptosis in the SP2͞0 hybridoma when overexpressed Sherr, C. J. (1993) 331–342. 21. Ranade, K., Hussussian, C. J., Sikorski, R. S., Varmus, H. E., by retroviral transfection (50), and regulate the transcription Goldstein, A. M., Tucker, M. 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