Haploinsufficiency of p18INK4c Sensitizes Mice to Carcinogen-Induced Tumorigenesis

The INK4 family of cyclin-dependent kinase (CDK) inhibitorsnegatively regulates cyclin D-dependent CDK4 and CDK6 and therebyretains the growth-suppressive function of Rb family proteins.Mutations in the CDK4 gene conferring INK4 resistance are associatedwith familial and sporadic melanoma in humans and result ina wide spectrum of tumors in mice. Whereas loss of functionof other INK4 genes in mice leads to little or no tumor development,targeted deletion of p18^INK4c causes spontaneous pituitary tumorsand lymphoma late in life. Here we show that treatment of p18null and heterozygous mice with a chemical carcinogen resultedin tumor development at an accelerated rate. The remaining wild-typeallele of p18 was neither mutated nor silenced in tumors derivedfrom heterozygotes. Hence, p18 is a haploinsufficient tumorsuppressor in mice.

INTRODUCTION

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Introduction

The Rb family proteins, pRB, p107, and p130, play critical rolesin controlling mammalian G₁ cell cycle progression (33). Emergingfrom mitosis or present in quiescent cells as hypophosphorylatedforms, these proteins negatively regulate the activity of E2Ftranscription factors to prevent S-phase entry. Extracellularmitogens induce the expression of D-type cyclins and activatecyclin D-dependent kinases CDK4 and CDK6, leading to phosphorylationand functional inactivation of Rb proteins. Conversely, inhibitionof CDK4 and CDK6, resulting from either lack of cyclin D synthesisor binding with an INK4 protein, retains Rb proteins in theirgrowth-suppressive states and prevents the G₁/S transition.Disruption of this pathway, consisting of INK4-cyclin D/CDK4/6-Rb-E2F,deregulates G₁ cell cycle progression and is believed to bea common event for the development of most types of cancer (29).

Mutational analysis of human tumors has provided compellingevidence supporting the critical importance of this pathwayin tumor suppression. The Rb gene is frequently mutated in familialretinoblastomas and osteosarcomas, as well as in sporadic lung,prostate, bladder, and breast cancers (reviewed in references20 and 33). The p16^INK4a locus is frequently deleted or mutatedin wide range of human tumors (13, 22; compiled in reference27). Chromosomal translocation and genomic amplification ofcyclin D1 and CDK4 genes leading to elevated level of eitherprotein and its associated kinase activities have been observedin cancers of the breast, head and neck, esophagus, lymphoidsystem, parathyroid, and brain (reviewed in references 9, 10,and 29). Finally, missense mutations in the CDK4 locus renderingresistance to INK4 binding have been observed in familial andsporadic melanomas (35, 41).

Genetic study with targeted mice provides further support fora critical function of this pathway in tumor suppression. Miceheterozygous for Rb mutation spontaneously develop tumors inseveral neuroendocrine organs, including the characteristicintermediate lobe of the pituitary (12, 19, 21, 26, 34). Engineeringof transgenic mice to overexpress cyclin D1 in mammary glandscauses hyperplasia and carcinoma (32), and conversely ablationof cyclin D1 renders mice resistant to breast cancer development(36). Mice carrying an INK4-insensitive mutation (R24C) in theCDK4 locus develop a wide spectrum of tumors, including tumorsin several neuroendocrine organs (Leydig cells of testes, granulosacells of ovaries, pancreatic islet cells, the intermediate lobeof the pituitary, and thyroid) as well as lung adenomas or adenocarcinomasand hepatocellular tumors (30).

However, genetic analysis of mutant mice has yet to clarifythe role of individual INK4 genes in tumor suppression. Micelacking p16^INK4a perplexingly display a tumor phenotype thatis considerably weaker and different from that predicted fromhuman tumor analysis. Only about one-quarter of mice carryinga deletion of exon 1 ${alpha}$ of p16^INK4a developed spontaneous tumors—mostlysarcomas and lymphomas late in life (16, 28). Such phenotypicdiscrepancy between CDK4^R24C and INK4a^exon1 mice suggests thatan additional INK4 gene or genes, although not frequently mutatedin human tumors, must be involved in regulating CDK4 (and possiblyCDK6) activity and the Rb pathway in tumor suppression in mice.No obvious tumor phenotypes were detected in mice lacking eitherp15^INK4b (18) or p19^INK4d (40). Several observations suggestthat p18^INK4c may play a major role in mediating the tumor suppressionfunction of the Rb pathway. Mice lacking p18^INK4c spontaneouslydevelop intermediate lobe pituitary tumors late in life (5,18), those that lack both p18^INK4c and p27^KIP1 develop tumorsin several neuroendocrine organs by 3 months of age, and p18^INK4cand p21^CIP1/WAF1 double knockout mice develop gastric neuroendocrinehyperplasia and lung bronchioalveolar tumors late in life (6).To further evaluate the function of p18^INK4c in tumor suppression,we examined the rate and spectrum of tumor development in p18^INK4cmutant mice exposed to a chemical carcinogen. Our studies indicatethat loss or decrease of p18^INK4c function predisposes miceto tumorigenesis and that p18^INK4c is haploinsufficient in tumorsuppression.

MATERIALS AND METHODS

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Materials and Methods

Mice. p18^INK4c mutant mice were initially generated in a mixed C57BL/6/129SV/D2genetic background (5). p18^INK4c mutant mice with an enrichedC57BL/6 genetic background were generated after six successivegenerations of backcrosses with C57BL/6 wild-type mice (15)and were used in this study. Littermates of different genotypeswere produced from interbreeding of heterozygotes. All micewere genotyped by PCR as previously described (5). All animalswere maintained in accordance with institutional guidelinesand were observed daily for up to 15 months.

Carcinogenesis. A cohort of 50 p18^+/+, 45 p18^+/-, and 45 p18^-/- animals wereproduced from interbreeding of p18 heterozygotes and used forthis study. Twenty-seven wild-type, 30 heterozygous, and 20p18 homozygous mice were treated continuously with 0.0005% dimethylnitrosamine(DMN) in drinking water beginning at the age of 8 weeks as describedpreviously. Mice were monitored daily over the course of theexperiment, and all genotypes consumed roughly the same amountof water on a daily basis. Twenty-three p18^+/+, 25 p18^-/-, and15 p18^+/- mice were monitored in parallel with the treated mice.Cohorts of treated mice were killed and necropsied at 3 and6 months of age (at least three mice for each genotype and eachtime point). The remaining mice were necropsied at death orwere sacrificed after reaching a moribund state. Tissues ofmost organs, as well as apparent tumor tissues, were removed,fixed in 10% neutral buffered formalin, and examined histologicallyby two pathologists after hematoxylin-eosin staining. Lesionswere photographed, and additional sections were taken for immunohistochemicalanalyses.

Immunohistochemistry. Specimens clearly representative of their respective pathologicaltypes were examined immunohistologically with an affinity-purifiedantibody specific to p18 (2 µg/ml) (7, 8) or to adrenocorticotropichormone (ACTH; 12 µg/ml) (no. A0579; DAKO). Tissue sectionswere blocked with normal goat serum in phosphate-buffered saline(PBS), and incubated with primary antibodies for 1 h and biotin-conjugatedsecondary antibody for 30 min. Immunocomplexes were detectedwith the Vectastain ABC alkaline phosphatase kit according tothe manufacturer's instructions (Vector Laboratories).

Tumor microdissection and genomic DNA amplification. Tumor microdissection and DNA isolation procedures were describedbefore (1). Ten-micrometer sections adhered on slides were deparaffinizedand lightly stained with hematoxylin. Under microscopic observation,the lesions that were clearly separated from normal tissueswere scraped from the slides. Particular care was taken to avoidcontamination by surrounding tissue. Scraped tissue sampleswere transferred into a sterile microcentrifuge tube. Fortymicroliters of digestion buffer (200 µM proteinase K perml, 0.5% Tween 20, 1 mM EDTA, 20 mM Tris-HCl [pH 7.4]) was addedto each tube, and the mixture was then incubated at 42°Cfor 2 days and then incubated in boiling water for 6 to 7 min.Thirty-five microliters of the supernatant was stored at -20°Cuntil used for PCR assays (1). The murine INK4c gene containsthree exons: a noncoding exon, 1, corresponding to the 5'-untranslatedregion and two coding exons comprising the entire coding sequences(24). Coding exons 2 and 3 were amplified and sequenced withtwo pairs of primers: p18-E2FW (5'-AGCCTGATTAGGAGCAAAGG-3')and p18-E2RV (5'-GCTGTCATTTTAGAAACCCAGGC-3') for exon 2 andp18-E3FW (5'TTGTTGTGGCTCAAGAAGCTG-3') and p18-E3RV (5'TAGTGAAACGGACAGCCAAC-3')for exon 3. Reaction products were resolved on a 1.2% agarosegel or subjected to direct DNA sequencing after purificationthrough a QIAquick mini column (QIAGEN). For genotyping, wild-typeand targeted exon 3 were amplified with a mixture of three primers:p18E3FW3 (5'-ACTACACAGGTCTTTGTGAAAG-3'), p18E3REV3 (5'-TCTCCGGATTTCCAAGTTTC-3'),and 5'-CCAGCCTCTGAGCCCAGAAAGCGAAGG-3',a primer correspondingto a neo sequence. The PCR conditions were 95°C for 45 s,55°C for 45 s, and 72°C for 45 s for each of 30 cycles.

Statistical analyses. The survival rate was calculated by the CA-Cricket Graph IIImethod, and statistical significance was assessed with Wilcoxon'slog-rank test. The range of tumor numbers and sizes (mean ±standard error) were compared with those from an unpaired ttest. P < 0.05 was considered to be statistically significant.

RESULTS

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Results

p18^INK4c null mice spontaneously developed pituitary intermediatelobe tumors (>75%) (5, 6, 18) or, at a lower penetrance,T-cell lymphomas ( $~$ 10%) (15) late in life, with mean latenciesof about 12 and 14 months, respectively. Simultaneous loss ofthree or four alleles of the p18 and p27 genes accelerates tumorgrowth, resulting in tumor-induced animal death by the agesof 9 and 4 months, respectively (6). We examined the susceptibilityof p18 mutant mice to tumorigenesis after continuous low-doseexposure to the chemical carcinogen DMN, which targets mainlythe liver and lung (2). As shown in Fig. 1, p18^-/- mice andp18^+/- mice are more susceptible to tumor-induced mortalityafter DMN treatment than wild-type mice (P < 0.001). No p18^-/-mice in a cohort of 17 survived beyond 45 weeks of exposureto DMN; the mean survival time was 35 weeks. All p18^+/- mice(n = 21) died within 58 weeks after exposure to DMN, with amean survival time of 45 weeks. In contrast, 7 of 11 wild-typemice survived beyond 60 weeks after DMN treatment, and the meansurvival time of the 4 mice that died from tumors was 56 weeks.In the untreated control cohorts monitored during the same 60weeks of the experimental period, four out of nine p18^-/- andtwo out of nine p18^+/- mice died. We were unable to establishthe exact cause of death of these mice. We detected no malignanttumors in these mice, and only one p18^-/- mouse developed apituitary adenoma. None of 16 wild-type mice died. These resultsprovide further support for the function of the p18 gene intumor suppression and demonstrate that loss of p18 functionsensitizes mice to carcinogen-induced tumorigenesis. Notably,loss of one allele of p18, while not exhibiting an evident tumorigeniceffect in untreated mice (5, 6, 18), effectively predisposeshemizygous mice to carcinogenesis.

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FIG. 1. Tumor-free survival in untreated wild-type and p18 mutant mice and in mice exposed to DMN. p18^+/+, p18^+/-, and p18^-/- mice were either untreated or fed with DMN (0.0005%) in their drinking water continuously from the age of 8 weeks. Statistically significant differences in survival were detected between cohorts of DMN-treated p18^-/- and DMN-treated p18^+/- mice (P < 0.001) and between DMN-treated p18^+/- and DMN-treated p18^+/+ mice (P < 0.05).

Necropsy of DMN-treated p18 mutant animals revealed tumor developmentpredominantly in three tissues: adenoma or carcinoma in theintermediate lobe of the pituitary, adenoma or carcinoma fromlung bronchiolar or alveolar cells, and hemangiosarcoma fromthe sinusoidal endothelial cells of the liver (Table 1 and Fig.2 to 4). In addition, DMN treatment also caused increased incidenceof hyperplasia in these tissues, as well as several other tissuesin adrenal, thyroid, and testis in both p18 null and heterozygousmice (Table 1). Liver hemangiosarcoma developed at a higherpenetrance (50%) than either lung (35%) or pituitary tumors(25%) (Table 1). The rate of tumor growth was much faster inp18 null mice (averaging 38 weeks for hemangiosarcomas and 41weeks for lung tumors, respectively) and p18 heterozygous mice(43 and 44 weeks) than in wild-type mice (60 and 58 weeks) (datanot shown).

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TABLE 1. Spontanous and DMN-induced tumor and hyperplasia incidence

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FIG. 2. DMN treatment accelerated pituitary tumorigenesis in p18-deficient mice. Pituitary glands (pointed by arrows) from p18^+/+, p18^+/-, and p18^-/- mice, either untreated or exposed to DMN, were microscopically examined at 6 months (A to L) and 9 months (M to P) of age either directly (top two rows; bars, 1 mm) or after hematoxylin and eosin (H/E) staining (lower two rows; bars, 200 µm). WT, wild type. Anterior lobe (A), intermediate lobe (I), neurohypophysis (N), and tumor (T) are indicated.

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FIG. 4. Mutation in the p18 gene sensitized mice to DMN-induced liver tumors. (A to F) Livers from p18^-/-, p18^+/-, and p18^+/+ mice exposed to DMN were examined at the ages of 12 weeks (A to C) and 34 weeks (D to F). Normal gross liver architecture was retained in DMN-treated mutant mice at a young age despite the development of multiple hemangiosarcomas (red spots pointed by the arrows), but was not observed in p18 null mice after 34 weeks of age. WT, wild type. (G) Microscopic examination of hemangiosarcoma that developed in the liver of a 40-week-old p18^-/- mouse exposed to DMN. Note that irregular vascular spaces have destroyed the normal hepatic parenchyma. (H) Higher magnification of the boxed area in (G) showing proliferating malignant endothelial cells replacing normal hepatic parenchyma.

p18 null mice spontaneously develop hyperplasia and adenomasin the intermediate lobe of the pituitary (5, 6, 18). DMN treatmentaccelerated pituitary adenoma growth, from a mean latency of50 weeks in untreated p18 null mice to 32 weeks in treated mice(Table 1) (data not shown). The cells from the pituitary adenomasstained strongly for ACTH, confirming their intermediate lobeorigin (data not shown). A carcinogenic effect of DMN has notbeen previously reported in the pituitary. This result indicatesa broader tumorigenic effect of DMN in nontarget tissues sensitizedby other events, such as tumor suppressor gene mutation. Pituitaryadenomas that developed in DMN-treated p18 null mice were alsomore aggressive, often invading the adjacent anterior lobe andobliterating the neurohypophysis (Fig. 2K and O). Although nopituitary tumors were found in the DMN-treated p18^+/- mice whenthey succumbed to liver tumors before the age of 56 weeks, theirintermediate lobes were often enlarged compared with those ofuntreated p18^+/- mice (Fig. 2G).

Lung bronchiolar or alveolar adenomas or adenocarcinomas wereobserved in 7 of 20 (35%) p18^-/- mice, 6 of 20 (30%) p18^+/-mice, and 2 of 13 (15%) wild-type mice exposed to DMN between35 and 58 weeks (Table 1 and Fig. 3). In untreated control cohorts,lung tumors were found in 3 of 14 p18^-/- mice (21%), but nowild-type (n = 18) or p18^+/- (n = 12) mice. DMN treatment alsocaused an increased incidence of lung bronchiolar or alveolarhyperplasia (Fig. 3D). The lung adenomas were composed of atypicalcells with nuclear pleomorphism and were characterized by thecompression of adjacent alveolar tissue (Fig. 3E). Bronchiolarand alveolar adenocarcinomas invaded adjacent alveolar tissue(Fig. 3F). Loss of one or both alleles of p18 not only sensitizedthe mutant mice to DMN-induced lung tumor development, but alsoincreased both the number and the size of lung tumors in eachmouse. On average, 4.6 ± 2.7 and 8.1 ± 3.9 lungtumors developed in each DMN-treated p18^+/- and p18^-/- mouse,comparing with 1 tumor in each wild-type mouse (Fig. 3G). Theaverage tumor sizes were 1.3 ± 0.3 and 1.9 ± 0.5mm in DMN-treated p18^+/- and p18^-/- mice, compared with 0.8± 0.5 mm in wild-type mice (Fig. 3H).

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FIG. 3. Mutation in p18 gene sensitized mice to DMN-induced lung tumors. (A to C) The rate of lung tumor development varied between age-matched (11 months) p18^+/+, p18^+/-, and p18^-/- mice after DMN exposure. Gross alveolar and bronchiolar adenomas (white spots) are identified by arrows. Note that p18 mutant lungs developed more tumors. (D to F) Alveolar or bronchiolar origin of lung tumor development. Lungs of DMN-treated mice at different ages were microscopically examined after hematoxylin-eaosin staining and exhibited alveolar or bronchiolar hyperplasia (D), papillary alveolar or bronchiolar adenoma with mucinous cell differentiation (E), and alveolar or bronchiolar carcinoma (F). The inset in panel F is a higher magnification showing tumor infiltration of adjacent pulmonary parenchyma. (G) The number of lung tumors that developed in each mouse exposed to DMN increases with p18 gene mutation. A statistically significant difference in tumor multiplicity was seen in mice of a different p18 genotype following exposure to DMN. WT, wild type. (H) The average size of DMN-induced lung tumors was larger in p18 mutant mice than in wild-type mice.

Neither pituitary nor pulmonary tumors were considered to bethe cause of death in mice bearing them. The increased mortalityof p18 mutant mice was mainly attributable to hemorrhage fromhepatic hemangiosarcomas. When clinically normal DMN-treatedmice of three genotypes were sacrificed and examined at 12 weeksof age, multiple liver hemangiosarcomas were present in p18null and p18 hemizygous mice, although they were small and displayedno evidence of internal hemorrhaging (Fig. 4B and C). At 34weeks of age, most p18 null mice were debilitated, while p18^+/-mice remained relatively healthy despite the development ofmultiple hemangiosarcomas and retained normal liver histology(Fig. 4E). Only one DMN-treated wild-type mouse developed hemangiosarcomasby 34 weeks of age (Fig. 4D). The livers in 34-week-old DMN-treatedp18 null mice were pale, necrotic, and contained multiple hemangiosarcomas,and normal liver tissue was difficult to observe (Fig. 4F).These hemangiosarcomas formed poorly delineated vascular channelslined by pleomorphic endothelial cells with frequent mitoticfigures. Many liver hemangiosarcomas had progressed to becomelarge blood-filled cavernous tumors that distended the livercapsule (Fig. 4). Tumors frequently ruptured, causing massivehemorrhage into the body cavity. In addition to the liver, hemangiosarcomasalso developed in the adrenal glands, abdominal cavity, andpancreas (Table 1).

To determine the status of the p18 gene in DMN-induced tumors,we microscopically dissected tumor cells from paraffin-embeddedsamples and analyzed the p18 gene by PCR. Of 15 tumors fromp18 hemizygous mice (8 liver hemangiosarcomas and 7 lung adenomas)and 2 liver hemangiosarcomas from wild-type mice, the wild-typeallele was retained in all 17 tumors (Fig. 5A and B). To determinewhether p18 expression was silenced in tumors, we determinedp18 protein expression by immunohistochemistry with an affinity-purifiedantibody to p18^INK4c. Of the same set of 15 tumors from p18hemizygous mice and 2 liver hemangiosarcomas from wild-typemice, p18 staining was evident in all of the tumors (a representativetumor of each organ is shown in Fig. 5). In addition, we havealso examined p18^INK4c protein expression in two abdominal sarcomas(Fig. 5I) and one adrenal hemangiosarcoma (data not shown) arisingfrom p18^+/- mice and observed positive p18 expression by immunohistochemicalstaining. To determine whether expressed p18 alleles sufferedmutations during tumor development, we amplified p18 from genomicDNA prepared from microscopically dissected tumor cells fromall 17 liver and lung tumors induced in either wild-type orp18 heterozygous mice and sequenced the entire coding region.No mutation was detected in the 10 tumor samples we examined,including 6 liver hemangiosarcomas and 3 lung adenomas fromp18 hemizygous mice and 1 liver hemangiosarcoma from a wild-typemouse (data not shown).

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FIG. 5. p18 is a haploinsufficient tumor suppressor. (A and B) Retention of the remaining wild-type p18 allele in liver (A) and lung (B) tumors from wild-type and p18 heterozygous mice. Genomic DNA was extracted from microdissected tumor cells or tails of p18^+/- or p18^-/- mice and subjected to PCR amplification with two pairs of primers flanking either the wild-type (WT) or mutated (mt) allele of the p18 gene. PCR products were resolved by agarose gel (1.2%) electrophoresis. The entire coding regions of p18 from liver tumor samples 2 to 11 and from lung tumor samples 2 to 8 were sequenced, and no mutation was found. (C to J) The expression of p18^INK4c protein in three different types of tumors (liver hemangiosarcoma, lung adenoma, and abdominal sarcoma) from mice of three different p18 genotypes was determined by immunohistological staining with an affinity-purified polyclonal antibody specific to p18^INK4c protein (8). Bars in all panels are 50 µm.

DISCUSSION

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Discussion

In this study, we provide further evidence supporting the roleof p18^INK4c gene in tumor suppression in mice; mutation in oneor both alleles of p18 gene predisposed mice to carcinogen-inducedtumorigenesis. Our results also establish that the p18 geneis haploinsufficient in tumor suppression. In both liver andlung tumors where we could isolate relatively pure tumor samples(17 samples), we found no loss of the wild-type p18 allele ormutations in the p18 coding region. Clear p18 protein expressionwas seen in tumor cells.

p18 joins a small number of tumor suppressor genes that havebeen definitively demonstrated to be haploinsufficient throughthe analysis of genetically targeted mice. These include p53(31), p27 (4), Dmp1 (11), Nf1 (39), and Pten (17). How widespreadhaploinsufficiency is among tumor suppressor genes is a topicof current interest (3, 25). The determination of this issuenot only may influence our view on the notion that inactivationmutations in tumor suppressor genes are recessive, but alsohas a clinical implication. While both alleles of a haplosufficienttumor suppressor gene are inactivated in tumors, a haploinsufficienttumor suppressor gene likely retains at least one allele intumors and thus represents a potential target of therapeuticintervention. A single wild-type allele, according to the classicaltwo-hit model of tumor suppressor genes, is sufficient to suppresstumor development and must be inactivated for tumorigenesis(14). This model is supported by the observations that germline inactivation mutations have occurred in cancer-prone families,and tumors that developed in these family members are frequentlyassociated with inactivation mutations of the remaining wild-typeallele. Although experimentally suitable for identifying tumorsuppressor genes with hereditary mutations in human tumors,the criterion of biallelic or complete functional loss excludesgenes that are haploinsufficient, such as p18. For these haploinsufficienttumor suppressor genes, genomic mutations in human tumors maybe rare, and hemizygous loss and decrease of expression levelare difficult to determine. Our results add to the call forfurther investigations into genes that are not frequently mutatedin human tumors, but exhibit clear tumor suppression functionin mice.

Various degrees of haploinsufficiency have been seen with demonstratedtumor suppressor genes, with p27 and Dmp1 exhibiting eitherno or rare loss of the remaining wild-type allele in tumorsfrom heterozygous mice, and thus a strong haploinsufficiency,and p53 and Pten displaying a noticeable frequency of loss ofthe wild-type allele and a weak effect. Moreover, the mutationrate in human tumors correlates with the various degrees ofhaploinsufficiency observed in mice. While both p53 and Ptenare frequently mutated in human tumors, mutations in p18, p27,and Dmp1 appear to be rare. Is p18 a completely haploinsufficienttumor suppressor gene? It is important to note that loss oftwo alleles of p18 is clearly more tumorigenic than loss ofone. The mean survival times of DMN-treated p18^-/- and p18^+/-mice are 35 and 45 weeks, respectively. The average numbersand size of tumors developed in DMN-treated p18^-/- mice are8.1 tumors per lung and 1.9 mm per tumor, compared with 4.6tumors per lung and 1.3 mm per tumor in DMN-treated p18^+/- mice.These results indicate that the haploinsufficiency of p18 intumor suppression is incomplete and there must be a gradientof p18 tumor suppression activity, with expression of one alleleexhibiting an intermediate effect between full function andcomplete functional loss. The biochemical basis for such gradienthaploinsufficiency of p18 is not clear, but likely relates toa change in the level of CDK4 and CDK6 kinase activity, becausethese two enzymes are the only identified targets of p18 function.

Biochemically, all four INK4 proteins inhibit CDK4 and CDK6almost indistinguishably, implying that members of the INK4gene family, through activating or retaining the growth suppressionactivity of pRb proteins, could have a function in tumor suppressionand are candidate tumor suppressors. Yet, mutations in humantumors have only been found in the p16^INK4a gene and are rarein the other three INK4 genes (23, 37, 38). The finding thatp18 is a haploinsufficient tumor suppressor may explain thelack of widespread mutations in the p18 gene in human tumors.It suggests a need for quantitative examination of p18 proteinlevels in order to accurately assess the role of p18 in thesuppression of human tumors. Loss of function of p18 alone exhibiteda relative weak tumor phenotype; about half of p18 null micedeveloped pituitary tumors, and 10% developed T-cell lymphomasafter 1 year of age, but no obvious tumor phenotype developedin p18 heterozygous mice (5, 15). A clear tumor suppressionfunction for p18 was revealed by the analysis of p18 p27 doublemutant mice (6) and after carcinogen exposure (this study).These findings suggest the possibility that the role of otherINK4 genes in tumor suppression could have been underestimated,and further investigations are needed to more completely explorethis issue.

ACKNOWLEDGMENTS

We thank Chad McCall for reading the manuscript and Joe He forhelping with figure preparation.

Y.X. is supported in part by a U.S. Department of Defense CareerDevelopment Award (DAMD17-99-1-9574). This study was supportedby NIH grants CA65572 and CA68377 to Y.X.

FOOTNOTES

* Corresponding author. Mailing address: Lineberger Comprehensive Cancer Center, Campus Box 7295, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295. Phone: (919) 962-2142. Fax: (919) 966-8799. E-mail: yxiong{at}email.unc.edu.

REFERENCES

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