EXPERIMENTAL CELL RESEARCH 237, 7–13 (1997)ARTICLE NO. EX973824

MINIREVIEW

The Tumor Suppressor Protein p16INK4a

Manuel Serrano1

Department of Immunology and Oncology, Centro Nacional de BiotecnologıB a, CSIC, Campus de Cantoblanco, Madrid E-28049, Spain

frequencies broadly ranging from 25 to 70%; this is theThe tumor suppressor protein p16INK4a (inhibitor of case with cancers of the head and neck, esophagus,

CDK4) is one of the most direct links between cell- biliary tract, lung, bladder, colon, and breast; leuke-cycle control and cancer. The p16INK4a gene is fre- mias; lymphomas; and glioblastomas (reviewed in Refs.quently inactivated in human tumors, and inheritance [7–9]; see also [10–12]. The most impressive case re-of mutant alleles results in susceptibility to several ported so far is that of pancreatic carcinomas in whichtypes of cancer. p16INK4a is part of a cell-cycle regula- 98% of the tumors have inactivated the p16INK4 genetory pathway that converges in the tumor suppressor (48% by homozygous deletion, 34% by hemizygous dele-protein Rb. The mechanisms that regulate p16INK4a are tion and intragenic mutation, and 16% by hemizygousstarting to be characterized. q 1997 Academic Press deletion and methylation-mediated silencing) [13].

Germline transmission of mutant p16INK4a alleles re-sults in hereditary predisposition to the development

ISOLATION AND CHARACTERIZATION OF p16INK4aof melanomas and cancers of the pancreas and liver(reviewed in [9]; see also Ref. [14]). Finally, p16-nullThe p16INK4a protein was first identified as a protein mice develop sporadic tumors at an early age and areassociated with the cyclin D-dependent kinase CDK4 very susceptible to carcinogenic treatments [15]. Alto-by coimmunoprecipation experiments [1]. p16INK4a was gether, these observations and others discussed in thiscloned by exploiting its interaction with CDK4, and it review unambiguously define p16INK4a as a tumor sup-was demonstrated to be a specific inhibitor of the pressor gene.CDK4/cyclin D kinases [2]. At that time, it was known Here, I review the information presently availablethat the activity of CDK4/cyclin D was critical for entry on the molecular biology of p16INK4a.into the cell cycle and that cyclin D1 had oncogenic

properties (reviewed in Ref. [3]). Accordingly, it wasTHE INK4 FAMILYproposed that p16INK4a was a negative regulator of cell

proliferation [2].p16INK4a is the founder member of a family of proteinsThe p16INK4a gene was independently isolated as a

with the ability to inhibit CDK4 and the CDK4-relatedcandidate tumor suppressor located at chromosomalkinase CDK6. The INK4 family is composed of fourposition 9p21 [4, 5]. Region 9p21 was suspected to con-members in mammalian organisms: p16INK4a, p15INK4b,tain a tumor suppressor gene because it is deleted inp18INK4c, and p19INK4d [2, 16–19]. An INK4 gene hasmany human tumors and it is linked to hereditary sus-been reported in the fish Xiphophorus maculatus, andceptibility to melanoma. The p16INK4a gene was foundit has been linked to hereditary susceptibility to UV-inactivated in a large percentage of tumor cell lines,induced melanomas [20]. The four mammalian INK4suggesting that it was indeed a tumor suppressor geneproteins have similar biochemical properties: all of[4–6]. Since then, considerable effort has been put intothem bind to CDK4 and CDK6 and inhibit the kinasedetermining the extent of p16INK4a inactivation in pri-activity of the CDK4-6/cycD complexes (reviewed inmary tumors. There are three main mechanisms of ge-Ref. [21]). The relevant feature that distinguishes eachnetic inactivation of p16INK4a: deletion of both alleles,INK4 protein seems to be their different transcrip-deletion of one allele and intragenic mutation of thetional regulation. In particular, p16INK4a is upregulatedremaining allele, and deletion of one allele and methyl-during cellular senescence and in response to certaination-mediated silencing of the remaining allele. Inac-oncogenic stimuli (discussed below), the p15INK4b pro-tivation of p16INK4a occurs in most tumor types withmoter responds to the growth-inhibitory factor TGFb[16], and p18INK4c and p19INK4d expression is regulated

1 Fax: /34-1-372 0493. E-mail: mserrano@cnb.uam.es. periodically during the cell cycle with a maximum at

7 0014-4827/97 $25.00Copyright q 1997 by Academic Press

All rights of reproduction in any form reserved.

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8 MANUEL SERRANO

S-phase [18, 19]. Thus, unless otherwise proven, the inactivating mutations severely affect the stability ofthe tertiary structure of p16INK4a [32, 37, 38]. A shortfour mammalian INK4 proteins are not functionally

redundant. peptide derived from the third ankyrin repeat ofp16INK4a has been selected for its ability to bind andAnother family of CDK inhibitors is formed by pro-

teins p21WAF1/CIP1, p27KIP1, and p57KIP2 (reviewed in Ref. inhibit CDK4, suggesting that this region could be di-rectly involved in the interaction with CDK4 [39].[21]). These inhibitors contain a conserved CDK/cyclin-

inhibitory domain plus additional domains specific for Regarding CDK4, several amino acids that are im-portant for binding to p16INK4a have been identifiedeach member. In contrast to the INK4 inhibitors, the

p21WAF1/CIP1-type inhibitors bind and inhibit multiple [40–42]. It has been proposed that p16INK4a binds to adiscrete surface on CDK4 that overlaps with the p27KIP1CDK/cyclin complexes, including those containing

CDK2 [21]. binding site [42]. This is in agreement with the obser-vation that binding of p27KIP1 and p15INK4b to CDK4 ismutually exclusive [43].TWO GENES IN THE SAME LOCUS: p16INK4a AND P19ARF

The relation between p16INK4a and cyclin D with re-The INK4a locus exhibits an unusual feature that spect to their binding to CDK4 seems complex. The

consists in the presence of two overlapping genes, each p16INK4a binding site in CDK4 is distant from the pre-regulated by its own promoter (reviewed in Ref. [22]). dicted cyclin D binding site [42, 44], and it is possibleOne promoter produces a transcript that is formed by to obtain ternary complexes in vitro of the form INK4/exons 1a–2–3 and encodes p16INK4a, while the other CDK4/cycD [18, 43]. However, in addition to this, INK4promoter produces a transcript that is formed by exons proteins destabilize CDK4/cycD1 complexes in vitro1b–2–3 and encodes p19ARF (alternative reading [45, 46], and mutations in CDK4 that impair p16INK4a

frame). The two transcripts share identical exons 2 and binding also reduce the affinity for cyclin D1 [41]. Alto-3, but the reading frames are different and, therefore, gether, these observations suggest that the bindingthe amino acid sequences of p16INK4a and p19ARF are sites for cyclin D and p16INK4a in CDK4 do not overlapcompletely unrelated. The transcription start sites of but are conformationally communicated. p16INK4a couldthe p16INK4a promoter have been identified, as well as inhibit CDK4 by stabilizing a catalytically inactive con-a region of 71 bp important for promoter activity and formation with a lower affinity for cyclin D.located approximately 500 bp upstream of the tran-scription start site [23]. THE p16INK4a/CDK4/cycD1/Rb PATHWAY

Ectopic expression of p19ARF induces cell-cycle arrest[24], but its biochemical activity and its connection The retinoblastoma susceptibility protein, Rb, andwith the cell cycle are still unknown. Some intragenic the related proteins, p107 and p130, bind and inhibitmutations in the INK4a locus exclusively affect the several transcription factors required for proliferationamino acid sequence of p16INK4a; however, others affect (reviewed in Ref. [47]). Progression through G1 re-the sequence of both p16INK4a and p19ARF. Interest- quires the phosphorylation of the Rb-type proteins andingly, these mutations selectively impair the cell-cycle the ensuing liberation of the transcription factors asso-inhibitory activity of p16INK4a, but not of p19ARF, sug- ciated with them. In this manner, the Rb-type proteinsgesting that p16INK4a is the main target of these intra- constitute cell-cycle blocks that are relieved by phos-genic mutations [25]. In the case of tumors with a ho- phorylation. The specific functions of each member ofmozygous deletion of the INK4a locus, it cannot be the Rb family remain to be elucidated, although thereexcluded that loss of p19ARF may contribute to the ma- is evidence for significant differences between Rb andlignant phenotype. the other two members, p107 and p130 (see, for exam-

ple, Ref. [48]). Remarkably, Rb is the only member ofthe family that is inactivated in human cancers.THE p16INK4a PROTEIN

The CDK4-6/cycD kinases are critical in the phos-phorylation of the Rb-type proteins and, therefore,The most remarkable feature of the INK4 proteins

is the presence of four or five contiguous ankyrin motifs their activity is essential for the proliferation of normalcells. Moreover, the only essential function of the[2]. Ankyrin repeats are present in many proteins and

are generally involved in protein–protein interactions. CDK4-6/cycD kinases appears to be the phosphoryla-tion of Rb because Rb-negative cells do not requireRecently, the three-dimensional structure of murine

p19INK4d was solved by NMR [26]. The structure of CDK4-6/cycD activity to proliferate (reviewed in Ref.[47]). Accordingly, overexpression of p16INK4a in cellsp19INK4d is similar to that of 53BP2, a protein that in-

teracts with p53 through an ankyrin-repeat domain with functional Rb results in G1 arrest, but it is incon-sequential in Rb-negative cells [17, 28, 29, 49, 50].[27]. Most tumor-specific mutations in the INK4a locus

impair the functionality of protein p16INK4a [28–36], These functional relations have come to be known asthe p16INK4a/CDK4/cycD1/Rb pathway (Fig. 1).and, in those cases in which it has been analyzed, these

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9THE TUMOR SUPPRESSOR PROTEIN p16INK4a

[58–60]. The p16INK4a mRNA is extremely stable (half-life ú24 h) [23], and the half-life of the protein rangesfrom 8 to 18 h, much longer than that of cyclin D1 (12min) [61]. The remarkable stability of the mRNA andthe protein suggests that the biological role of p16INK4a

is not in controlling short-term responses like cell-cycletransitions.

Accumulation of p16INK4a mRNA and protein hasbeen reported in response to a short list of stimuli orconditions: cellular senescence, oncogenic Ras, and in-activation of Rb (Fig. 2).

Upregulation of p16INK4a during Cellular Senescence

Cell senescence in culture consists in loss of prolifera-FIG. 1. The p16INK4a/CDK4/cycD1/Rb pathway and its involve-tive potential after the accumulation of a number of cellment in the regulation of G1.doublings characteristic for each species. The view ofsenescence as a simple ‘‘wearing out’’ of the cell is con-tradicted by the fact that there are specific genetic alter-

Parallel pathways of the type (INK4a,b,c, d)/ ations that bypass senescence and facilitate immortali-(CDK4,6)/(cycD1,2,3)/(Rb,p107,p130) must exist, with zation (reviewed in Ref. [62]). Upon senescence, cellsspecific or overlapping biological roles that still remain arrest at G1 with a specific pattern of cell-cycle proteinsto be determined. Of all these possible pathways, the (reviewed in Ref. [63]). Senescent cells exhibit increasedone formed by p16INK4a/CDK4/cycD1/Rb and, to a lesser p53 DNA binding activity and upregulate the cell-cycleextent, the one formed by p15INK4b/CDK4/cycD1/Rb inhibitors p16INK4a and p21WAF1/CIP1 [23, 60, 61, 64–69].seem of special importance in tumorigenesis. p16INK4a

Interestingly, cells expressing SV40 T-antigen accumu-and Rb are tumor suppressors inactivated by point mu- late p16INK4a and p21WAF1/CIP1 at the same rate as normaltations, deletion, and promoter methylation, whereas cells despite their failure to enter senescence [61, 67].CDK4 and cyclin D1 are oncogenes activated by diverse This suggests that the upregulation of p16INK4a andmechanisms, including translocation, amplification, p21WAF1/CIP1 is not a consequence of the entry into senes-and point mutation. There is evidence suggesting that cence, but rather it is directly linked to the accumula-the p16INK4a/CDK4/cycD1/Rb pathway behaves as a sin- tion of cell doublings. In addition, loss of p16INK4a facili-gle mutagenic target during tumorigenesis. For exam- tates immortalization in many cellular systems [15, 60,ple, inactivation of p16INK4a or Rb are mutually exclu- 68–70], and the viral oncoprotein Tax from HTLV-1sive alterations in glioblastomas and in lung cancers inactivates p16INK4a by direct physical interaction and[51, 52]; also, alterations in each constituent of the facilitates immortalization [71, 72]. Altogether, thesep16INK4a/CDK4/cycD1/Rb pathway usually occur in a results strongly support the notion that p16INK4a is ac-mutually exclusive manner in gliomas and sarcomas tively involved in establishing cellular senescence.[53–55]. The analysis of familial melanoma kindreds In contrast to cellular senescence, aging is a processelegantly illustrates these functional relations. Ap- that occurs at the level of the organism and that proba-proximately 50% of these families carry inactive mu- bly involves more factors than just the accumulationtant alleles of p16INK4a [56], whereas Ç5% carry a par- of cell doublings. Importantly, it has been reported thatticular mutation in CDK4, Arg24 to Cys, which selec- a significant increase in the levels of both p16INK4a

tively disrupts the interaction of CDK4 with INK4 mRNA and protein occurs in aged mice [60].inhibitors [40, 57] (stillÇ45% of the families carry mu-tations in unknown genes). Altogether, the p16INK4a/ Upregulation of p16INK4a by Oncogenic RasCDK4/cycD1/Rb pathway has emerged as a critical con-

The activity of the Ras proto-oncogene is essentialtrol of the cell cycle that is deregulated in the largeboth for entry into the cell cycle (G0/G1 transition) andmajority of human tumors.for progression through G1 (reviewed in Refs. [73, 74]).At G1, Ras triggers the phosphorylation of Rb by a mech-REGULATION OF p16INK4a

anism that involves upregulation of cyclin D1 and down-regulation of p27KIP1 [75, 76]. In agreement with this,The main regulation of the intracellular levels of pro-

tein p16INK4a occurs at the transcriptional level. The Rb-negative cells do not require Ras activity during G1and, similarly, p16INK4a-negative cells are partially inde-abundance of p16INK4a mRNA is very low in most tis-

sues, but it is possible to detect p16INK4a mRNA ubiqui- pendent of Ras for G1 progression (in both cases Ras isstill necessary for the G0/G1 transition) [75, 77].tously by RT-PCR in both human and mouse tissues

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10 MANUEL SERRANO

FIG. 2. Regulation of p16INK4a. The scheme indicates three biological situations that induce the accumulation of p16INK4a. The possibilitythat a common mechanism could mediate the three responses is proposed as a hypothesis. The accumulation of cell doublings and thepresence of oncogenic Ras result in cell-cycle arrest mediated in part by p16INK4a. Proliferation in the absence of functional Rb results inp16INK4a-induction, but this induction has no effect on proliferation because Rb is constitutively inactive.

In contrast to the physiological effects of the Ras cells [23, 82] (although not in rodent cells [61]). How-ever, the link between p16INK4a and Rb is more compli-proto-oncogene during the normal cell cycle, expression

of oncogenic Ras in primary fibroblasts provokes a de- cated than was initially thought because p16INK4a ex-pression does not simply reflect the status of Rb. Forlayed G1 arrest accompanied by a significant increase

in the levels of p16INK4a and p53 [78]. The cell-cycle example, the expression of p16INK4a remains constantduring the transition from quiescence to proliferationarrest elicited by oncogenic Ras occurs several days

after introduction of Ras (6–8 days) and exhibits many (see Refs. [23, 58]), and during progression through thecell cycle [85]. Moreover, overexpression of the tran-features typical of cellular senescence. Accordingly,

this phenomenon has been termed ‘‘premature senes- scription factor E2F in human mammary epithelialcells does not induce p16INK4a [84]. It is possible thatcence’’ ([78]; reviewed in Ref. [79]). The causal role of

p16INK4a and p53 in this anti-proliferative response is the induction of p16INK4a expression occurs only afterprolonged proliferation in the absence of functional Rb.demonstrated by the fact that cells deficient in either

p16INK4a or p53 are permissive to oncogenic Ras and, Candidate mechanisms that could mediate this typeof response are chromatin accessibility changes in theindeed, become neoplastically transformed [78].

In other cellular systems, the simultaneous induc- INK4a locus or genomic instability.Similar to the inactivation of Rb, the inactivation oftion of p16INK4a and p53 can result in apoptotic death

instead of cellular senescence. In particular, ectopic ex- the tumor suppressor p53 can also result in upregula-tion of p16INK4a expression. In particular, cells con-pression of both p53 and p16INK4a in some tumor cell

lines induces apoptosis, whereas p16INK4a alone results taining the HPV oncoprotein E6 exhibit high levels ofp16INK4a expression in human fibroblasts [82, 83], butin cell-cycle arrest [80].not in human mammary epithelial cells [84]. Interest-ingly, E6-containing fibroblasts continue proliferatingUpregulation of p16INK4a by Inactivation of Rbdespite the induction of p16INK4a, suggesting either that

The concept that p16INK4a expression is regulated by these cells have become independent of the Rb check-Rb originated from the observation that the levels of point or that the levels of p16INK4a are not high enoughp16INK4a RNA and protein are significantly elevated in to completely eliminate the CDK4/cycD activity.human cells either carrying inactivating mutations ofRb or expressing viral oncoproteins, such as HPV-16 CONCLUDING REMARKE7, SV40 T-antigen, or adenovirus E1a [1, 2, 23, 45,81–84]. In addition, restauration of Rb in Rb-negative Since the isolation of p16INK4a in 1993 there has been

significant progress in firmly establishing p16INK4a ascells results in downregulation of p16INK4a in human

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11THE TUMOR SUPPRESSOR PROTEIN p16INK4a

sor gene in normal and transformed human tissues correlatesa tumor suppressor and in characterizing the p16INK4a/with gene silencing. Cancer Res. 55, 4531–4535.CDK4/cycD1/Rb pathway as a critical regulator of cell

13. Scutte, M., Hruban, R. H., Geradts, J., Maynard, R., Hilgers,proliferation. It is tempting to speculate that all theW., Rabindran, S. K., Moskaluk, C. A., Hahn, S. A., Scwarte-known inducers of p16INK4a expression (senescence, on- Waldhoff, I., Schmiegel, W., Baylin, S. B., Kern, S. E., and Her-

cogenic Ras, and inactivation of the Rb or p53 check- man, J. G. (1997). Abrogation of the Rb/p16 tumor-suppressivepoints) could be just different manners of inducing a pathway in virtually all pancreatic carcinomas. Cancer Res. 57,

3126–3130.common mitogenic stress (Fig. 2). The next challenge14. Chaubert, P., Gayer, R., Zimmermann, A., Fontolliet, C.,is to define the molecular events that mediate the in-

Stamm, B., Bosman, F., and Shaw, P. (1997). Germ-line muta-duction of p16INK4a.tions of the p16INK4(MTS1) gene occur in a subset of patientswith hepatocellular carcinoma. Hepatology 25, 1376–1381.

The author is very grateful to Dr. Ignacio Palmero for critically 15. Serrano, M., Lee, H.-W., Chin, L., Cordon-Cardo, C., Beach, D.,reading the manuscript, and to Dr. David Beach for his help and and DePinho, R. A. (1996). Role of the INK4a locus in tumorencouragement. The author is supported by a grant from the Minis- suppression and cell mortality. Cell 85, 27–37.terio de Educacion y Cultura, Spain. The Department of Immunology

16. Hannon, G. J., and Beach, D. (1994). p15INK4b is a potentialand Oncology was founded and is supported by the Spanish Researcheffector of cell cycle arrest mediated by TGF-b. Nature 371,Council and by Pharmacia and Upjohn.257–261.

17. Guan, K., Jenkins, C. W., Li, Y., Nichols, M. A., Wu, X., O’Keefe,C. L., Matera, A. G., and Xiong, Y. (1994). Growth suppressionREFERENCESby p18, a p16INKN4/MTS1- and p14INK4/MTS2-related CDK6 inhibitor,correlates with wild-type pRb function. Genes Dev. 8, 2939–1. Xiong, Y., Zhang, H., and Beach, D. (1993). Subunit rearrange-2952.ment of the cyclin-dependent kinases is associated with cellular

18. Hirai, H., Roussel, M. F., Kato, J., Ashmun, R. A., and Sherr,transformation. Genes Dev. 7, 1572–1583.C. J. (1995). Novel INK4 proteins, p19 and p18, are specific2. Serrano, M., Hannon, G. J., and Beach, D. (1993). A new regula-inhibitors of cyclin D-dependent kinases CDK4 and CDK6. Mol.tory motif in cell-cycle control causing specific inhibition ofCell. Biol. 15, 2672–2681.cyclin D/CDK4. Nature 366, 704–707.

19. Chan, F. K. M., Zhang, L., Chen, D. N., and Winoto, A. (1995).3. Sherr, C. J. (1993). Mammalian G1 cyclins. Cell 73, 1059–1065.Identification of human/mouse p19, a novel cdk4/cdk6 inhibitor

4. Kamb, A., Gruis, N. A., Weaver-Feldhaus, J., Liu, Q., Harsh- with homology to p16ink4. Mol. Cell. Biol. 15, 2682–2688.man, K., Tavtigian, S. V., Stockert, E., Day, R. S., III, Johnson,

20. Nairn, R. S., Kazianis, S., McEntire, B. B., Della Coletta, L.,B. E., and Skolnick, M. H. (1994). A cell cycle regulator poten-Walter, R. B., and Morizot, D. C. (1996). A CDKN2-like poly-tially involved in genesis of many tumor types. Science 264,morphism in Xiphophorus LG V is associated with UV-B-in-436–440.duced melanoma formation in platyfish–swordtail hybrids.

5. Nobori, T., Miura, K., Wu, D. J., Lois, A., Takabayashi, K., and Proc. Natl. Acad. Sci. USA 93, 13042–13047.Carson, D. A. (1994). Deletions of the cyclin-dependent kinase-

21. Sherr, C. J., and Roberts, J. M. (1995). Inhibitors of mammalian4 inhibitor gene in multiple human cancers. Nature 368, 753–G1 cyclin-dependent kinases. Genes Dev. 9, 1149–1163.756.

22. Sidransky, D. (1996). Two tracks but one race? Curr. Biol. 6,6. Okamoto, A., Demetrick, D. J., Spillare, E. A., Hagiwara, K.,523–525.Hussain, S. P., Bennett, W. P., Forrester, K., Gerwin, B., Ser-

23. Hara, E., Smith, R., Parry, D., Tahara, H., Stone, S., and Peters,rano, M., Beach, D. H., and Harris, C. C. (1994). Mutations andG. (1996). Regulation of p16CDKN2 expression and its implica-altered expression of p16INK4 in human cancer. Proc. Natl. Acad.tions for cell immortalization and senescence. Mol. Cell. Biol.Sci. USA 91, 11045–11049.16, 859–867.7. Hirama, T., and Koeffler, H. P. (1995). Role of the cyclin-depen-

24. Quelle, D. E., Zindy, F., Ashmun, R. A., and Sherr, C. J. (1995).dent kinase inhibitors in the development of cancer. Blood 86,Alternative reading frames of the INK4a tumor suppressor gene841–854.encode two unrelated proteins capable of inducing cell cycle8. Palmero, I., and Peters, G. (1997). Perturbations of cell cyclearrest. Cell 83, 993–1000.regulators in human cancer. Cancer Surv. 27, 351–367.

25. Quelle, D. E., Cheng, M., Ashmun, R. A., and Sherr, C. J. (1997).9. Foulkes, W. D., Flanders, T. Y., Pollock, P. M., and Hayward,Cancer-associated mutations at the INK4a locus cancel cell cy-N. K. (1997). The CDKN2A (p16) gene and human cancer. Mol.cle arrest by p16INK4a but not by the alternative reading frameMed. 3, 5–20.protein p19ARF. Proc. Natl. Acad. Sci. USA 94, 669–673.

10. Merlo, A., Herman, J. G., Mao, L., Lee, D. J., Gabrielson, E.,26. Luh, F. Y., Archer, S. J., Domaille, P. J., Smith, B. O., Owen, D.,Burger, P. C., Baylin, S. B., and Sidransky, D. (1995). 5* CpG

Brotherton, D. H., Raine, A. R. C., Xu, X., Brizuela, L., Brenner,island methylation is associated with transcriptional silencingS. L., and Laue, E. D. (1997). Structure of the cyclin dependentof the tumour suppressor p16/CDKN2/MTS1 in human can-kinase inhibitor p19INK4d. Nature 389, 999–1002.cers. Nature Med. 1, 686–690.

27. Gorina, S., and Pavletich, N. P. (1996). Structure of the p5311. Herman, J. G., Merlo, A., Mao, L., Lapidus, R. G., Issa, J. P.,tumor suppressor bound to the ankyrin and SH3 domains ofDavidson, N. E., Sidransky, D., and Baylin, S. B. (1995). Inacti-53BP2. Science 274, 1001–1005.vation of the CDKN2/p16/MTS1 gene is frequently associated

with aberrant DNA methylation in all common human cancers. 28. Lukas, J., Parry, D., Aagaard, L., Mann, D. J., Bartkova, J.,Strauss, M., Peters, G., and Bartek, J. (1995). Retinoblastoma-Cancer Res. 55, 4525–4530.protein-dependent cell-cycle inhibition by the tumour suppres-12. Gonzalez-Zulueta, M., Bender, C. M., Yang, A. S., Nguyen, T.,sor p16. Nature 375, 503–506.Beart, R. W., Van Tornout, J. M., and Jones, P. A. (1995). Meth-

ylation of the 5* CpG island of the p16/CDKN2 tumor suppres- 29. Koh, J., Enders, G. H., Dynlacht, B. D., and Harlow, E. (1995).

AID ECR 3824 / 6i29$$$683 11-14-97 10:56:01 eca

12 MANUEL SERRANO

Tumour-derived p16 alleles encoding proteins defective in cell- protein, increases p15INK4B-cdk4 complexes, and inhibits cyclinD1-cdk4 association in human mammary epithelial cells. Mol.cycle inhibition. Nature 375, 506–510.Cell. Biol. 17, 2458–2467.30. Ranade, K., Hussassian, C. J., Sikorski, R. S., Varmus, H. E.,

47. Weinberg, R. A. (1995). The retinoblastoma protein and cell cy-Goldstein, A. M., Tucker, M. A., Serrano, M., Hannon, G. J.,cle control. Cell 81, 323–330.Beach, D., and Dracopoli, N. C. (1995). Mutations associated

with familial melanoma impair p16INK4 function. Nature Genet. 48. Hurford, R. K., Jr., Cobrinik, D., Lee, M.-H., and Dyson, N.10, 114–116. (1997). pRb and p107/p130 are required for the regulated ex-

pression of different sets of E2F responsive genes. Genes Dev.31. Reymond, A., and Brent, R. (1995). P16 proteins from melanoma11, 1447–1463.families are deficient in binding to Cdk4. Oncogene 11, 1173–

49. Serrano, M., Gomez-Lahoz, E., DePinho, R. A., Beach, D., and1178.Bar-Sagi, D. (1995). Inhibition of ras-induced proliferation and32. Wick, S. T., Dubay, M. M., Imanil, I., and Brizuela, L. (1995).cellular transformation by p16INK4. Science 267, 249–252.Biochemical and mutagenic analysis of the melanoma tumor

50. Medema, R. H., Herrera, R. E., Lam, F., and Weinberg, R. A.suppressor gene product p16. Oncogene 11, 2013–2019.(1995). Growth suppression by p16ink4 requires functional reti-33. Yang, R. A., Gombart, A. F., Serrano, M., and Koeffler, H. P.noblastoma protein. Proc. Natl. Acad. Sci. USA 92, 6289–6293.(1995). Mutational effects on the p16INK4a tumor suppressor pro-

51. Shapiro, G. I., Edwards, C. D., Kobzik, L., Godleski, J., Rich-tein. Cancer Res. 55, 2503–2506.ards, W., Sugarbaker, D. J., and Rollins, B. J. (1995). Reciprocal34. Yang, R., Serrano, M., Slater, J., Leung, E., and Koeffler, H. P.Rb inactivation and p16INK4 expression in primary lung cancers(1996). Analysis of p16INK4a and its interaction with CDK4. Bio-and cell lines. Cancer Res. 55, 505–509.chem. Biophys. Res. Commun. 218, 254–259.

52. Ueki, K., Ono, Y., Henson, J. W., Efird, J. T., von Deimling, A.,35. Lilischkis, R., Sarcevic, B., Kennedy, C., Warlters, A., and Suth-and Louis, D. N. (1996). CDKN2/p16 or RB alterations occurerland, R. L. (1996). Cancer-associated mis-sense and deletionin the majority of glioblastomas and are inversely correlated.mutations impair p16INK4 CDK inhibitory activity. Int. J. Can-Cancer Res. 56, 150–153.cer 66, 249–254.

53. Schmidt, E. E., Ichimura, K., Reifenberger, G., and Collins,36. Arap, W., Knudsen, E. S., Wang, J. Y. J., Cavenee, W. K., andV. P. (1994). CDKN2 (p16/MTS1) gene deletion or CDK4 ampli-Huang, H.-J. S. (1997). Point mutations can inactivate in vitrofication occurs in the majority of glioblastomas. Cancer Res. 54,and in vivo activities of p16INK4a/CDKN2A in human glioma.6321–6324.Oncogene 14, 603–609.

54. He, J., Olson, J. J., and James, C. D. (1995). Lack of p16INK437. Tevelev, A., Byeon, I.-J. L., Selby, T., Ericson, K., Kim, H.-J., or retinoblastoma protein (pRb), or amplification-associated

Kraynov, V., and Tsai, M. D. (1996). Tumor suppressor p16INK4A: overexpression of cdk4, is observed in distinct subsets of malig-Structural characterization of wild-type and mutant proteins nant glial tumors and cell lines. Cancer Res. 55, 4833–4836.by NMR and circular dichroism. Biochemistry 35, 9475–9487.

55. Maelandsmo, G. M., Berner, J.-M., Florenes, V. A., Forus, A.,38. Zhang, B., and Peng, Z. (1996). Defective folding of mutant Hovig, E., Fodstad, O., and Myklebost, O. (1995). Homozygous

p16(INK4) proteins encoded by tumor-derived alleles. J. Biol. deletion frequency and expression levels of the CDKN2 geneChem. 271, 28734–28737. in human sarcomas: Relationship to amplification and mRNA

39. Fahraeus, R., Paramio, J. M., Ball, K. L., LaıB n, S., and Lane, levels of CDK4 and CCND1. Br. J. Cancer 72, 393–398.D. P. (1996). Inhibition of pRb phosphorylation and cell-cycle 56. Hussussian, C. J., Struewing, J. P., Goldstein, A. M., Higgins,progression by a 20-residue peptide derived from p16CDKN2/INK4A. P. A. T., Ally, D. S., Sheahan, M. D., Clark, W. H., Jr., Tucker,Curr. Biol. 6, 84–91. M. A., and Dracopoli, N. C. (1994). Germline p16 mutations in

40. Wolfel, T., Hauer, M., Schneider, J., Serrano, M., Wolfel, C., familial melanoma. Nature Genet. 8, 15–21.Klehmann-Hieb, E., De Plaen, E., Hankeln, T., Meyer zum 57. Zuo, L., Weger, J., Yang, Q., Goldstein, A. M., Tucker, M. A.,Buschenfelde, K.-H., and Beach, D. (1995). A p16INK4a-insensi- Walker, G. J., Hayward, N., and Dracopoli, N. C. (1996). Germ-tive CDK4 mutant targeted by cytolytic T lymphocytes in a line mutations in the p16INK4a binding domain of CDK4 in famil-human melanoma. Science 269, 1281–1284. ial melanoma. Nature Genet. 12, 97–99.

41. Coleman, K. G., Wautlet, B. S., Morrisey, D., Mulheron, J., Sed- 58. Stone, S., Jiang, P., Dayananth, P., Tavtigian, S. V., Katcher,man, S. A., Brinkley, P., Price, S., and Webster, K. R. (1997). H., Parry, D., Peters, G., and Kamb, A. (1995). Complex struc-Identification of CDK4 sequences involved in cyclin D1 and p16 ture and regulation of p16 (MTS1) locus. Cancer Res. 55, 2988–binding. J. Biol. Chem. 272, 18869–18874. 2994.

42. Peters, R., Hyberts, S., Xu, R.-M., Havel, T., Beach, D., Wagner, 59. Quelle, D. E., Ashmun, R. E., Hannon, G. J., Rehberger, P. A.,G., and Serrano, M. (1997). Identification of a p16INK4a-binding Trono, D., Richter, K. H., Walker, C., Beach, D., Sherr, C. J.,interface in CDK4. Submitted for publication. and Serrano, M. (1995). Cloning and characterization of murine

43. Reynisdottir, I., and Massague, J. (1997). The subcellular loca- p16INK4a and p15INK4b genes. Oncogene 11, 635–645.tions of p15Ink4b and p27Kip1 coordinate their inhibitory inter- 60. Zindy, F., Quelle, D. E., Roussel, M. F., and Sherr, C. J. (1997).actions with cdk4 and cdk2. Genes Dev. 11, 492–503. Expression of the p16INK4a tumor suppressor versus other INK4

family members during mouse development and aging. Onco-44. Russo, A. A., Jeffrey, P. D., Patten, A. K., Massague, J., andgene 15, 203–211.Pavletich, N. P. (1996). Crystal structure of the p27Kip1 cyclin-

dependent-kinase inhibitor bound to the cyclin A-Cdk2 complex. 61. Palmero, I., McConnell, B., Parry, D., Brookes, S., Hara, E.,Nature 382, 325–331. Bates, S., Jat, P., and Peters, G. (1997). Accumulation of

p16INK4a in mouse fibroblasts as a function of replicative senes-45. Parry, D., Bates, S., Mann, D. J., and Peters, G. (1995). Lackcence and not of retinoblastoma gene status. Oncogene 15, 495–of cyclin D-Cdk complexes in Rb-negative cells correlates with503.high levels of p16INK4/MTS1 tumour suppressor gene product.

EMBO J. 14, 503–511. 62. Votja, P. J., and Barrett, J. C. (1995). Genetic analysis of cellu-lar senescence. Biochim. Biophys. Acta 1242, 29–41.46. Sandhu, C., Garbe, J., Bhattacharya, N., Daksis, J., Pan,

C.-H., Yaswen, P., Koh, J., Slingerland, J. M., and Stampfer, 63. Stein, G. H., and Dulic, V. (1995). Origins of G1 arrest in senes-cent human fibroblasts. Bioessays 17, 537–543.M. R. (1997). Transforming growth factor b stabilizes p15INK4B

AID ECR 3824 / 6i29$$$683 11-14-97 10:56:01 eca

13THE TUMOR SUPPRESSOR PROTEIN p16INK4a

64. Atadja, P., Wong, H., Garkavtsev, I., Veillette, and Riabowol, the extracellular environment, cytoplasmic signalling cascadesand the nuclear cell-cycle machinery. FEBS Lett. 410, 11–16.K. (1995). Increased activity of p53 in senescing fibroblasts.

Proc. Natl. Acad. Sci. USA 92, 8348–8352. 75. Peeper, D. S., Upton, T. M., Ladha, M. H., Neuman, E., Zalvide,J., Bernards, R., DeCaprio, J. A., and Ewen, M. E. (1997). Ras65. Noda, A., Ning, Y., Venable, S. F., Pereira-Smith, O. M., andsignalling linked to the cell-cycle machinery by the retinoblas-Smith, J. R. (1994). Cloning of senescent cell-derived inhibitorstoma protein. Nature 386, 177–181.of DNA synthesis using an expression screen. Exp. Cell Res.

76. Aktas, H., Cai, H., and Cooper, G. M. (1997). Ras links growth211, 90–98.factor signalling to the cell cycle machinery via regulation of66. Alcorta, D. A., Xiong, Y., Phelps, D., Hannon, G., Beach, D.,cyclin D1 and the Cdk inhibitor p27KIP1. Mol. Cell. Biol. 17,and Barrett, J. C. (1996). Involvement of the cyclin-dependent3850–3857.kinase inhibitor p16 (INK4a) in replicative senescence of nor-

77. Mittnacht, S., Paterson, H., Olson, M. F., and Marshall, C. J.mal human fibroblasts. Proc. Natl. Acad. Sci. USA 93, 13742–(1997). Ras signalling is required for inactivation of the tumour13747.suppressor pRb cell-cycle control protein. Curr. Biol. 7, 219–

67. Tahara, H., Sato, E., Noda, A., and Ide, T. (1995). Increase in 221.expression level of p21sdi1/cip1/waf1 with increasing division

78. Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D., and Lowe,age in both normal and SV40-transformed human fibroblasts.S. W. (1997). Oncogenic ras provokes premature cell senescenceOncogene 10, 835–840.associated with accumulation of p53 and p16INK4a. Cell 88, 593–

68. Reznikoff, C. A., Yeager, T. R., Belair, C. D., Savelieva, E., Pu- 602.thenveettil, J. A., and Stadler, W. M. (1996). Elevated p16 at 79. Weinberg, R. A. (1997). The cat and mouse games that genes,senescence and loss of p16 at immortalization in human papillo- viruses, and cells play. Cell 88, 573–575.mavirus 16 E6, but not E7, transformed human uroepithelial 80. Sandig, V., Brand, K., Herwig, S., Lukas, J., Bartek, J., andcells. Cancer Res. 56, 2886–2890. Strauss, M. (1997). Adenovirally transferred p16INK4/CDKN2 and

69. Loughran, O., Malliri, A., Owens, D., Gallimore, P. H., Stanley, p53 genes cooperate to induce apoptotic tumor cell death. Na-M. A., Ozanne, B., Frame, M. C., and Parkinson, E. K. (1996). ture Med. 3, 313–319.Association of CDKN2/p16INK4a with human head and neck 81. Tam, S. W., Shay, J. W., and Pagano, M. (1994). Differentialkeratinocyte replicative senescence: Relationship of dysfunction expression and cell cycle regulation of the cyclin-dependent ki-to immortality and neoplasia. Oncogene 13, 561–568. nase 4 inhibitor p16Ink4. Cancer Res. 54, 5816–5820.

82. Li, Y., Nichols, M. A., Shay, J. W., and Xiong, Y. (1994). Tran-70. England, N. L., Cuthbert, A. P., Trott, D. A., Jezzard, S., Nobori,scriptional repression of the D-type cyclin-dependent kinase in-T., Carson, D. A., and Newbold, R. F. (1996). Identification ofhibitor p16 by the retinoblastoma susceptibility gene producthuman tumour suppressor genes by monochromosome transfer:pRb. Cancer Res. 54, 6078–6082.Rapid growth-arrest response mapped to 9p21 is mediated

solely by the cyclin-D-dependent kinase inhibitor gene, CDKN2 83. Xiong, Y., Kuppuswamy, D., Li, Y., Livanos, E. M., Hixon, M.,(p16INK4a). Carcinogenesis 17, 1567–1575. White, A., Beach, D., and Tlsty, T. D. (1996). Alteration of cell

cycle kinase complexes in human papillomavirus E6- and E7-71. Suzuki, T., Kitao, S., Matsushime, H., and Yoshida, M. (1996).expressing fibroblasts precedes neoplastic transformation. J.HTLV-1 Tax protein interacts with cyclin-dependent kinase in-Virol. 70, 999–1008.hibitor p16INK4a and counteracts its inhibitory activity towards

84. Khleif, S. N., DeGregori, J., Yee, C. L., Otterson, G. A., Kaye,CDK4. EMBO J. 15, 1607–1614.F. J., Nevins, J. R., and Howley, P. M. (1996). Inhibition of72. Low, F. G., Dorner, L. F., Fernando, D. B., Grossman, J., Jeang,cyclin D-CDK4/CDK6 activity is associated with an E2F-medi-K.-T., and Comb, M. J. (1997). Human T-cell leukemia virusated induction of cyclin kinase inhibitor activity. Proc. Natl.type 1 Tax releases cell cycle arrest induced by p16INK4a. J.Acad. Sci. USA 93, 4350–4354.Virol. 71, 1956–1962.

85. Soucek, T., Pusch, O., Hengstschlager-Ottnad, E., Wawra, E.,73. Downward, J. (1997). Routine role for Ras. Curr. Biol. 7, 258– Bernaschek, G., and Hengstschlager, M. (1995). Expression of

260. the cyclin-dependent kinase inhibitor p16 during the ongoingcell cycle. FEBS Lett. 373, 164–169.74. Peeper, D. S., and Bernards, R. (1997). Communication between

Received August 14, 1997Revised version received September 14, 1997

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