null mice spontaneously developed pituitary intermediate lobe tumors (>75%) (5
) or, at a lower penetrance, T-cell lymphomas (~10%) (15
) late in life, with mean latencies of about 12 and 14 months, respectively. Simultaneous loss of three or four alleles of the p18
genes accelerates tumor growth, resulting in tumor-induced animal death by the ages of 9 and 4 months, respectively (6
). We examined the susceptibility of p18
mutant mice to tumorigenesis after continuous low-dose exposure to the chemical carcinogen DMN, which targets mainly the liver and lung (2
). As shown in Fig. , p18−/−
mice and p18+/−
mice are more susceptible to tumor-induced mortality after DMN treatment than wild-type mice (P
< 0.001). No p18−/−
mice in a cohort of 17 survived beyond 45 weeks of exposure to DMN; the mean survival time was 35 weeks. All p18+/−
= 21) died within 58 weeks after exposure to DMN, with a mean survival time of 45 weeks. In contrast, 7 of 11 wild-type mice survived beyond 60 weeks after DMN treatment, and the mean survival time of the 4 mice that died from tumors was 56 weeks. In the untreated control cohorts monitored during the same 60 weeks of the experimental period, four out of nine p18−/−
and two out of nine p18+/−
mice died. We were unable to establish the exact cause of death of these mice. We detected no malignant tumors in these mice, and only one p18−/−
mouse developed a pituitary adenoma. None of 16 wild-type mice died. These results provide further support for the function of the p18
gene in tumor suppression and demonstrate that loss of p18 function sensitizes mice to carcinogen-induced tumorigenesis. Notably, loss of one allele of p18
, while not exhibiting an evident tumorigenic effect in untreated mice (5
), effectively predisposes hemizygous mice to carcinogenesis.
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 (more ...)
Necropsy of DMN-treated p18 mutant animals revealed tumor development predominantly in three tissues: adenoma or carcinoma in the intermediate lobe of the pituitary, adenoma or carcinoma from lung bronchiolar or alveolar cells, and hemangiosarcoma from the sinusoidal endothelial cells of the liver (Table and Fig. to ). In addition, DMN treatment also caused increased incidence of hyperplasia in these tissues, as well as several other tissues in adrenal, thyroid, and testis in both p18 null and heterozygous mice (Table ). Liver hemangiosarcoma developed at a higher penetrance (50%) than either lung (35%) or pituitary tumors (25%) (Table ). The rate of tumor growth was much faster in p18 null mice (averaging 38 weeks for hemangiosarcomas and 41 weeks for lung tumors, respectively) and p18 heterozygous mice (43 and 44 weeks) than in wild-type mice (60 and 58 weeks) (data not shown).
Spontanous and DMN-induced tumor and hyperplasia incidence
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 (more ...)
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 (more ...) p18
null mice spontaneously develop hyperplasia and adenomas in the intermediate lobe of the pituitary (5
). DMN treatment accelerated pituitary adenoma growth, from a mean latency of 50 weeks in untreated p18
null mice to 32 weeks in treated mice (Table ) (data not shown). The cells from the pituitary adenomas stained strongly for ACTH, confirming their intermediate lobe origin (data not shown). A carcinogenic effect of DMN has not been previously reported in the pituitary. This result indicates a broader tumorigenic effect of DMN in nontarget tissues sensitized by other events, such as tumor suppressor gene mutation. Pituitary adenomas that developed in DMN-treated p18
null mice were also more aggressive, often invading the adjacent anterior lobe and obliterating the neurohypophysis (Fig. ). Although no pituitary tumors were found in the DMN-treated p18+/−
mice when they succumbed to liver tumors before the age of 56 weeks, their intermediate lobes were often enlarged compared with those of untreated p18+/−
mice (Fig. ).
Lung bronchiolar or alveolar adenomas or adenocarcinomas were observed in 7 of 20 (35%) p18−/− mice, 6 of 20 (30%) p18+/− mice, and 2 of 13 (15%) wild-type mice exposed to DMN between 35 and 58 weeks (Table and Fig. ). In untreated control cohorts, lung tumors were found in 3 of 14 p18−/− mice (21%), but no wild-type (n = 18) or p18+/− (n = 12) mice. DMN treatment also caused an increased incidence of lung bronchiolar or alveolar hyperplasia (Fig. ). The lung adenomas were composed of atypical cells with nuclear pleomorphism and were characterized by the compression of adjacent alveolar tissue (Fig. ). Bronchiolar and alveolar adenocarcinomas invaded adjacent alveolar tissue (Fig. ). Loss of one or both alleles of p18 not only sensitized the mutant mice to DMN-induced lung tumor development, but also increased both the number and the size of lung tumors in each mouse. On average, 4.6 ± 2.7 and 8.1 ± 3.9 lung tumors developed in each DMN-treated p18+/− and p18−/− mouse, comparing with 1 tumor in each wild-type mouse (Fig. ). The average tumor sizes were 1.3 ± 0.3 and 1.9 ± 0.5 mm in DMN-treated p18+/− and p18−/− mice, compared with 0.8 ± 0.5 mm in wild-type mice (Fig. ).
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 (more ...)
Neither pituitary nor pulmonary tumors were considered to be the cause of death in mice bearing them. The increased mortality of p18 mutant mice was mainly attributable to hemorrhage from hepatic hemangiosarcomas. When clinically normal DMN-treated mice of three genotypes were sacrificed and examined at 12 weeks of age, multiple liver hemangiosarcomas were present in p18 null and p18 hemizygous mice, although they were small and displayed no evidence of internal hemorrhaging (Fig. ). At 34 weeks of age, most p18 null mice were debilitated, while p18+/− mice remained relatively healthy despite the development of multiple hemangiosarcomas and retained normal liver histology (Fig. ). Only one DMN-treated wild-type mouse developed hemangiosarcomas by 34 weeks of age (Fig. ). The livers in 34-week-old DMN-treated p18 null mice were pale, necrotic, and contained multiple hemangiosarcomas, and normal liver tissue was difficult to observe (Fig. ). These hemangiosarcomas formed poorly delineated vascular channels lined by pleomorphic endothelial cells with frequent mitotic figures. Many liver hemangiosarcomas had progressed to become large blood-filled cavernous tumors that distended the liver capsule (Fig. ). Tumors frequently ruptured, causing massive hemorrhage into the body cavity. In addition to the liver, hemangiosarcomas also developed in the adrenal glands, abdominal cavity, and pancreas (Table ).
To determine the status of the p18 gene in DMN-induced tumors, we microscopically dissected tumor cells from paraffin-embedded samples and analyzed the p18 gene by PCR. Of 15 tumors from p18 hemizygous mice (8 liver hemangiosarcomas and 7 lung adenomas) and 2 liver hemangiosarcomas from wild-type mice, the wild-type allele was retained in all 17 tumors (Fig. ). To determine whether p18 expression was silenced in tumors, we determined p18 protein expression by immunohistochemistry with an affinity-purified antibody to p18INK4c. Of the same set of 15 tumors from p18 hemizygous mice and 2 liver hemangiosarcomas from wild-type mice, p18 staining was evident in all of the tumors (a representative tumor of each organ is shown in Fig. ). In addition, we have also examined p18INK4c protein expression in two abdominal sarcomas (Fig. ) and one adrenal hemangiosarcoma (data not shown) arising from p18+/− mice and observed positive p18 expression by immunohistochemical staining. To determine whether expressed p18 alleles suffered mutations during tumor development, we amplified p18 from genomic DNA prepared from microscopically dissected tumor cells from all 17 liver and lung tumors induced in either wild-type or p18 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 from p18 hemizygous mice and 1 liver hemangiosarcoma from a wild-type mouse (data not shown).
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 (more ...)