[CANCER RESEARCH 54, 6353-6358, December 15, 19941 Advances in Brief Deletion ofpl6 and pl5 Genes in Brain Tumors1 Jin Jen, J. Wade Harper, Sandra H. Bigner, Darell D. Bigner, Nickolas Papadopoulos, Sanford Markowitz, James K. V. Wilson, Kenneth W. Kinzler, and Bert Vogelstein2 The Johns Hopkins Oncology Center, Baltimore, Maryland 21231 Ii. J., N. P., K. W. K., B. V.]; the Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030 Ii. W. H.]; the Department of Pathology. Duke University School of Medicine, Durham, North Carolina 27710 (5. H. B., D. D. B.]; and the Department of Medicine and Ireland Cancer Center, University Hospital of Cleveland and Case Western Reserve University, Cleveland, Ohio 44106 (S. M.. J. K. V. W.] Abstract We have used molecular genetic methods to examine the status of cell cycle-inhibitory genes in human brain tumors. We found that p16 and a neighboring gene, pi5, were often homozygously deleted in glioblastoma multiformes but not in medulloblastomas or ependymomas. The deletions occurred in both primary tumors and their derived xenografts, but no Intragenic mutations In either of the two genes were found. TheplS gene was expressed in a more widespread pattern in normal tissues than pl6, but the products of both genes had similar capacities to bind to cydlin D-dependent kinases 4 and 6. These data suggest that the target of deletion in glioblastoma multiforme includes both p15 and p16 genes. The reason that homozygous deletions, rather than intragenic mutations, are so corn mon in these tumors rnay be that deletion is a more efficient mechanism for simultaneous inactivation of both genes. Introduction The Cdks3 are essential enzymes with activities that are tightly regulated by protein phosphorylation and association with other pro teins, particularly the cyclins (i, 2). Sequentiai activation of the cy ciin-Cdk complexes is thought to be responsible for orderly transitions through the cell cycle. The abnormal activation of Cdk activity, through a variety of mechanisms, is expected to underlie part of the uncontrolled growth that characterizes cancer. Two classes of genes whose products can inhibit Cdks have re cently been identified. The first class consists of p21 [also called dpi, WAFJ, SDIi,CAP2O (3—8)] andp27[alsocalledKIPi,(9—il)] which have a high degree of sequence similarity and can inhibit a variety of Cdk subtypes. The expression of p2i is regulated by p53, providing a link between tumor suppressor genes and cell cycle regulation (5). The second class is represented by pitS (also called MTSJ), which encodes a protein with ankyrin-like repeats that can inhibit Cdk4 and Cdk6 but not other Cdks (12, i3). Cdk4 and Cdk6 are closely related in primary sequence and are both activated by D type cyclins during G1 (14, 15). Previous studies have demonstrated that many tumors, particularly melanomas, leukemias, and gliomas, contain hemizygous or homozy gous deletions of chromosome 9p2i (16—18). Additionally, a gene responsible for melanoma predisposition has been mapped to this same chromosomai region by linkage analysis (19). The pitS gene Received 10/11/94; accepted 11/3/94. The costs of publication of this article were defrayed in part by the payment of page charges. This articie must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Preuss Foundation; the Clayton Fund; the National Foundation for Cancer Research; Baylor SPORE in Prostate Cancer; NIH Grants CA 58204 (J. W. H.), AG-i1085 (S. J. E., J. W. H.), CA-43733 and CA-51504 (S. H. B.), CA-i1898 and NS-20023 (D. D. B.), CA-57208 (S. M.), CA-51183 (J. W.), CA-4i183 (B.V.),andCA-09243(J.J.).B.V.is anAmericanCancerSocietyResearchProfessor. 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: Cdk, cyclin-dependent kinase; MTS, multiple tumor suppressor gene; CIP1, Cdk-interacting protein; WAF1, wild-type p53-activated fragment; SDI1, senescent cell-derived inhibitor; CAP1, Cdk-associated protein; KIP1, Cdk-inhibitory protein; RT, reverse transcriptase; GM, glioblastoma multiforme; ORF, open reading frame. resides within the deleted 9p2i region within these tumors (20, 2i). Subtle mutations of the pi6 gene have been reported in a variety of tumor cell lines and in some primary tumors (20—23). Moreover, germline alterations of the pitS gene have been found in a subset of patients with familial melanoma (21, 24, 25). There has been consid erabie controversy over the role of pitS in neopiasia, in part because of conflicting results that have been reported (20—29). In this study, we have searched for genetic aiterations of p16 in human brain tumors. During the course of this work, we were led to characterize a neighboring gene, p15, that proved to have biochemical properties similar to those of pi6. Our results suggest that both of these genes may play a role in gliobiastoma tumorigenesis. Materials and Methods Tumor and Nucleic Acid Sources. Brainand colorectaltumorxenografts were established in nude mice as described previously (30). DNA from primary and xenografted tumors were prepared by standard methods (31). RNA from normal human adult tissues and GM xenografts were purified by a guanidine isothiocyanate based method (Promega) following the manufactur er's instructions. Placenta and HeLa cell polyadenylated mRNA were pur chased from Stratagene (San Diego, CA). PCR Amplification and Sequencing of Tumor DNA. Primersused to amplify pi6 exons are listed in Table 1. All PCR reactions were carried out in 50-pA reactions using 100 ng genomic DNA. The PCR solutions have been described (32), and the reaction used a 95°C,2-mm initial denaturation step followed by 30 to 35 cycles at 95°C for 30 s, 58°C for 1 mm, and 70°C for 1 min. The PCR products were separated by electrophoresis on 1.5% agarose or 10% nondenaturing polyacrylamide gels. Mouse spleen DNA was used to ensure that the PCR primers were specific for human pitS. Sequencing of the PCR products was performed with Sequimerm Polymerase (Epicentre) as described by the manufacturer, using the primers listed in Table 1. Amplifi cation of pi5 was carried out similarly, using the primers described in Table 1. A 500-base pair genomic Cdk4 fragment(33) was amplified in parallel PCR experiments to control for the integrity of DNA samples. p's cDNACloning. The5'endofthepi5cDNAwasobtained through the rapid amplification of cDNA ends procedure, using placenta cDNA as template (34, 35). The rapid amplification of cDNA ends anchor primer (Clontech) was combined with either primer P15-2R or primer P15-54 (Table 1) for the first and second rounds of PCR amplification, respectively. A PCR product of about 800 base pairs generated after the second PCR amplification was cloned directly into the pCRII vector (Invitrogen). The sequences of the clones were determined using 17 and SP6 primers as well as additional primers from the p15 gene as its sequence was generated. The sequence was confirmed by direct sequencing of a P1 clone (P2420) containing the entire pi5 and p16 genes usingpi5-specific primers. The P2420 clone was isolated by PCR screening of a human P1 genomic library (Genome Systems, Inc.) Functional Analyses of p15. RT-PCR products containing the complete coding sequence of the pi6 and pi5 genes were generated from HeLa and placenta mRNA, respectively, using primers containing signals for in vitro transcription by 11 polymerase and in vitro translation by reticulocyte lysates (Table 1, primers TNT-Pi6 and P16-S13 for pi6 and primers TNT-P15 and P15-S2R for pi5). The p16 and p15 proteins for in vitro binding assays were synthesized using these RT-PCR products and an in vitro transcription-trans lation kit (Promega) as described previously for other genes (36, 37). 6353 Research. on November 15, 2015. © 1994 American Association for Cancer cancerres.aacrjournals.org Downloaded from