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[Cancer Research 65, 92-98, January 1, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology and Genetics

Tumor Susceptibility of Rassf1a Knockout Mice

Stella Tommasi1, Reinhard Dammann3, Zhongqiu Zhang4, Yian Wang4, Limin Liu1, Walter M. Tsark1, Sharon P. Wilczynski2, Jie Li4, Ming You4 and Gerd P. Pfeifer1

1 Division of Biology and 2 Department of Anatomic Pathology, City of Hope National Medical Center, Duarte, California; 3 Medizinische Fakultät, Martin-Luther-Universität Halle-Wittenberg, Halle/Saale, Germany; and 4 Department of Surgery, Washington University School of Medicine, St. Louis, Missouri

Requests for reprints: Gerd P. Pfeifer, Beckman Research Institute, City of Hope National Medical Center, 1450 East Duarte Road, Duarte, CA 91010. Phone: 626-301-8853; Fax: 626-930-5366. E-mail: gpfeifer{at}coh.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The human Ras association domain family 1 (RASSF1) gene is located at 3p21.3 in an area that is believed to harbor at least one important tumor suppressor gene. The two major isoforms of RASSF1, RASSF1A and RASSF1C, are distinguished by alternative NH2-terminal exons and the two transcripts initiate in two separate CpG islands. RASSF1A is one of the most frequently inactivated genes described thus far in human solid tumors. Inactivation of RASSF1A most commonly involves methylation of the promoter and CpG island associated with the RASSF1A isoform. In contrast, RASSF1C is almost never inactivated in tumors. Here, we have derived Rassf1a knockout mice in which exon 1-{alpha} of the Rassf1 gene was deleted, leading to specific loss of Rassf1a but not Rassf1c transcripts. Rassf1a-targeted mice were viable and fertile. Rassf1a–/– mice were prone to spontaneous tumorigenesis in advanced age (18–20 months). Whereas only two tumors developed in 48 wild-type mice, six tumors were found in 35 Rassf1a+/– mice (P < 0.05) and thirteen tumors were found in 41 Rassf1a–/– mice (P < 0.001). The tumors in Rassf1a-targeted mice included lung adenomas, lymphomas, and one breast adenocarcinoma. Rassf1a–/– and wild-type mice were treated with two chemical carcinogens, benzo(a)pyrene and urethane, to induce skin tumors and lung tumors, respectively. Rassf1a–/– and Rassf1a+/– mice showed increased tumor multiplicity and tumor size relative to control animals. The data are consistent with the tumor-suppressive role of Rassf1a, which may explain its frequent epigenetic inactivation in human tumors.

Key Words: RASSFIA • tumor suppressor • DNA methylation • gene targeting • lung cancer


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Ras association domain family 1 (RASSF1) gene (1) is located at 3p21.3 within an area of common heterozygous and homozygous deletions, which occur frequently in a variety of human solid tumors (2–4). The RASSF1 locus encodes two major transcripts and several tissue-specific splice variants. The two major transcripts initiate in two CpG islands separated by about 3.5 kb. They have different NH2-terminal exons and are referred to as RASSF1A and RASSF1C (1, 6). Both RASSF1A and RASSF1C proteins contain a Ras association domain (6). RASSF1A also contains a diacylglycerol-phorbol ester binding domain near its NH2 terminus (1). Only one of the isoforms of the RASSF1 gene, RASSF1A, is inactivated by DNA methylation of its promoter CpG island at a high frequency in many human solid tumors, as observed originally in lung cancers (1, 5, 7). RASSF1A is probably the most frequently methylated gene described thus far in human cancer (8, 9). RASSF1A methylation occurs in a broad spectrum of carcinomas and other solid tumors (1. 5, 7, 9–29). In several types of tumors, RASSF1A is inactivated at a high frequency. For example, methylation of RASSF1A is found in 80% of small cell lung cancers (5, 16), over 60% of breast tumors (5, 10, 30), over 70% of prostate cancers (12, 13, 31), and in 90% of hepatocellular carcinomas (15, 29, 32). In contrast, inactivation of RASSF1C in human tumors has rarely been reported.

The biological function of RASSF1A is unknown. Transfection of RASSF1A reduces the growth of human cancer cells in vitro and in vivo supporting a role for RASSF1 as a tumor suppressor gene (1, 5, 11, 12). RASSF1A's closest homologue is the RAS effector protein NORE1A (33). RASSF1A and NORE1A form heterodimers suggesting that RASSF1A may function in signal transduction pathways involving RAS-like small GTPase proteins. Recent data indicate that RASSF1A itself binds to RAS only weakly and that binding to activated RAS may require heterodimerization of RASSF1A and NORE1 (34). There is evidence for an association of both NORE1 and RASSF1A with the proapoptotic kinase MST1. This interaction is involved in cellular processes leading to apoptosis (35). Other investigators have described the role of RASSF1A in the suppression of cyclin D accumulation and cell cycle progression (36). Recently, we presented evidence that RASSF1A is a microtubule-binding protein that can stabilize microtubules, and that overexpression of RASSF1A causes metaphase arrest (37). RASSF1A localizes to the mitotic apparatus and controls mitotic progression by modulating microtubule dynamics and/or by interacting with components of the anaphase-promoting complex (37–39).

Animal models for studying the function of RASSF1A have not yet been described. Here, we have created a mouse knockout for Rassf1a. In a model which closely mimics the situation in human tumors, the Rassf1a isoform was specifically inactivated in the mouse. We studied the tumor susceptibility of Rassf1a knockout mice, both for spontaneous tumor formation and for chemical carcinogen-induced tumor formation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of Gene-Targeted Mice. The Rassf1a knockout vector was made by high-fidelity PCR with the proofreading-capable Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA) and 129/SvImJ mouse ES cell DNA as the template. The PgK-neo cassette was flanked by 3.9 kb of genomic sequence upstream of Rassf1a exon1-{alpha} and by 2.5 kb of sequence downstream of exon 1-{alpha}. The 5' insert was generated with primers UMBLU1 (5'-GGGGCCTGCGCAGCTTCCAGCT-3') and LM10A1 (5'-TGGGCGGGGCACGGCACAA-3'), cloned into the pCR2.1 vector and verified by sequencing. The insert was released by EcoRI digestion and cloned into theEcoRI sites of the pKSneolox-cloning vector. The primers UM11AA (5'-GCGGCAGTGGAGGGCTGGTCA-3') and LM121 (5'-TGTGAGAGGTGCGCGTGCTCC-3') were utilized to clone the 3' insert into pCR2.1. The orientation and integrity of the fragment was confirmed by sequencing and it was cloned into the XhoI and KpnI sites of pKSneolox. The resultant construct replaces exon 1-{alpha} of Rassf1a with the PgK-neo cassette. Gene targeting was done in the ES cell line 129S3/SvImJ. The gene-targeting vector was linearized with KpnI and introduced into ES cells by electroporation. G418-resistant ES cell colonies were transferred to 96-well tissue culture plates for expansion and analysis. To identify homologous recombinants, Southern blot analyses using diagnostic restriction enzyme digests with probes that are external to the regions of homology included in the targeting vector were done. A 402-bp probe downstream of the 3' insert of the Rassf1a knockout was generated using primers U33 (5'-GGCGAGGCTGAAACACCTTCCT-3') and LM8109 (5'-CCCTGGCTCAACCGGTCTGTGA-3'). Genomic DNA of the ES cell colonies was digested with HindIII and separated on 0.8% TAE agarose gels, blotted, hybridized with the single-stranded labeled PCR probe, and then visualized. In all colonies with an apparent knockout allele, correct targeting was verified by Southern blots using NdeI digestion and external and internal hybridization probes.

Once the correctly targeted ES cell clones had been identified and confirmed by Southern blot analysis, they were used for injection into C57BL/6 blastocysts and generation of mouse chimeras. Male chimeras were mated to C57BL/6 females. After germ line transmission and breeding to homozygosity, the absence or presence of Rassf1a and Rassf1c transcripts, respectively, was confirmed by reverse transcription-PCR.

Reverse Transcription-PCR Analysis. Total RNA was isolated from early passage primary mouse embryo fibroblasts using the Trizol reagent (Invitrogen, Carlsbad, CA). Mouse embryo fibroblasts were derived from 13.5 days postcoitum embryos and genotyped as described below. For reverse transcription-PCR analysis, 200 ng of RNA was pre-associated with a lower primer from exon 4 of the mouse Rassf1 gene (5'-GATGAAGCCTGTGTAGGAGCCATCCT). After the reverse transcription reaction, either an upper primer from exon 1-{alpha} (5'-GTCGTGGCCACCGTTTCCAG) or an upper primer specific for exon 2-{gamma} (5'-CTGAAACACCTTCCTTCGAAATGACCT) was used in the PCR reaction to measure Rassf1a and Rassf1c mRNA levels, respectively. PCR conditions were 95°C for 30 seconds, 61°C for 30 seconds, and 68°C for 40 seconds (35 cycles), followed by a 10 minutes extension at 68°C. Glyceraldehyde-3-phosphate dehydrogenase primers were used in the same reverse transcription reactions as a control for mRNA integrity (25).

Animals and Genotyping. Animals were housed in plastic cages with hardwood bedding and dust covers, in a high-efficiency particulate air–filtered, environmentally controlled room (24 ± 1°C, 12/12 hours light/dark cycle). Animals were given Rodent LabChow #5001 (Purina, St. Louis, MO) and water ad libitum. The mice used in the study were genotyped by PCR analysis for identification of the Rassf1a genotype. The primer sequences were as follows: UMIOAI, 5'-TTGTGCCGTGCCCCGCCCA; LMIIAA, 5'-TGACCAGCCCTCCACTGCCGC; and Neo48U, 5'-GGGCCAGCTCATTCCTCCCAC. The multiplex PCR produces a 520-bp band for the wild-type allele and a 380-bp band for the knockout allele and both for heterozygous mice (see example in Fig. 1C).



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  		Figure 1. Generation of Rassf1a knockout mice. A, strategy for generating a mouse knockout of Rassf1a. The wild-type (wt) allele represents the organization of the exons (open boxes) of the human and mouse genes, which is conserved. For the mouse knockout of Rassf1a, the entire exon 1-{alpha} was deleted. The necessary homologies for the replacement vector are provided by the 5' and 3' arms, respectively. B, Southern blot analysis of ES cell clones targeted with the vector for the knockout of Rassf1a. After correct targeting, the expected size of the HindIII fragment is 3 kb. This band is present in the 13 targeted clones shown. "C" is a non-targeted clone. C, example of PCR genotyping of Rassf1a gene-targeted mice. D, reverse transcription-PCR analysis of expression of Rassf1a and Rassf1c in fibroblasts derived from the Rassf1a-targeted mice. Expression of glyceraldehyde-3-phosphate dehydrogenase was analyzed as a control.

 
Spontaneous Tumorigenesis Studies. At the age of 6 weeks, animals were assigned to three different groups (wild-type, Rassf1a heterozygous, and Rassf1a homozygous knockout, according to the genotype) and were on a 129SvJ x C57BL/6 background (roughly 50% each). At 18 to 20 months of age, the mice were euthanized by carbon dioxide asphyxiation and subjected to complete necroscopy of all major organs. Any abnormal tissues found were fixed in 10% neutral-buffered formalin followed by 70% ethanol and paraffin embedding. Tissue sections (5 µm) were stained with H&E for histopathologic evaluation. Immunohistochemistry was done using rat antibodies specific for the B cell marker CD45R/B220 (BD Biosciences, San Diego, CA) and the T cell marker CD3 (DakoCytomation, Carpinteria, CA) followed by secondary antibody, an avidin-biotin amplification system and peroxidase staining (Vector Laboratories, Burlingame, CA).

Skin Tumorigenesis Studies. A standard skin carcinogenesis study was done. Eight-week-old mice on a mixed 129 x C57BL/6 background were assigned to three groups according to the Rassf1a genotypes. All mice had their backs shaved weekly. Forty-eight hours prior to initial treatment, the dorsal skin of mice was shaved. All mice were treated topically twice weekly for 10 weeks with benzo(a)pyrene (100 nmol/mouse; Sigma Chemical, Co., St. Louis, MO) dissolved in 200 µl acetone. Skin tumors were monitored and recorded thrice a week for the duration of the studies. This bioassay was terminated at 7 months after the final benzo(a)pyrene exposure. For each mouse, the skin tumors were enumerated and three diameters were measured; only tumors with a diameter of > 2 mm were included. Part of the dorsal normal skin tissue and tumors were isolated and placed in individual tubes and were immediately frozen in liquid nitrogen. The rest of the tumors were fixed in 10% neutral-buffered formalin overnight, followed by 70% ethanol and paraffin embedding. Tissue sections (5 µm) were stained with H&E for histopathologic analysis. A gross necropsy was also done. The total tumor volumes of skin tumors were calculated by V = (4/3){pi}r3 or V = 2r{pi}h (r, radius) dependent on the tumor shape.

Lung Tumorigenesis Studies. Eight-week-old mice on a mixed 129 x C57BL background were assigned to three groups according to the Rassf1a genotypes. Animals were given two i.p. injections of urethane (1 mg/g body weight; Sigma) in 0.2 mL PBS 1 week apart. Seven months after treatment with carcinogens, animals were euthanized by carbon dioxide asphyxiation. For each mouse, portions of the tumors plus some normal lung tissue were frozen in liquid nitrogen. The remaining tissue and tumors were fixed in Tellyesniczky's solution overnight, followed by 70% ethanol treatment. Each lung was examined with the aid of a dissecting microscope to obtain the tumor count and size. Tumor volumes were determined by measuring the three-dimensional size of each tumor and by using the average of the three measurements as the diameter. Only tumors with a diameter of > 1 mm were included. The radius (diameter/2) was determined, and the total tumor volume was calculated by: volume = (4/3){pi}r3.

Rassf1a Methylation Analysis. The genomic DNA from mouse skin and lung tumors of Rassf1a+/– mice was isolated using the Trizol solution (Invitrogen) according to the manufacturer's protocols. The methylation status of the Rassf1a promoter region was determined by chemical modification of genomic DNA with sodium bisulfite and methylation-specific PCR. Bisulfite treatment converts cytosine bases to uracil bases but has no effect on 5-methylcytosine bases. The bisulfite-treated DNA was used as a template for the methylation-specific PCR reaction. Primers for the unmethylated DNA-specific reaction were: F, 5'-GGTGTTGAAGTTGTGGTTTG-3'; R, 5'-TATTATACCCAAAACAATACAC-3'. Primers for the methylated DNA-specific reaction were: F, 5'-TTTTGCGGTTTCGTTCGTTC-3'; R, 5'-CCCGAAACGTACTACTATAAC-3'. The reactions were incubated at 95°C for 1 minute, 55°C for 1minute, and 72°C for 1 minute, for 35 cycles. The PCR product obtained from methylated DNA was 213 bp, and the fragment obtained from unmethylated DNA was 204 bp. The amplified fragments were confirmed by DNA sequencing. DNA from normal lung was used as a control for unmethylated Rassf1a, and normal lung DNA treated with SssI DNA methyltransferase was used as a control for methylated Rassf1a. Water was used as a negative control, 25 µl of each PCR reaction was loaded onto a 6% nondenaturing polyacrylamide gel, which was stained with ethidium bromide, and photographed under UV light.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Two major transcripts, termed Rassf1a and Rassf1c originate at the human and mouse genomic Rassf1 loci and the organization of the exons is highly conserved between mouse and human. They code for two different proteins, RASSF1A and RASSF1C that differ in their NH2-terminal sequence but have common COOH-terminal sequences (1, 5). Although RASSF1C is almost never inactivated in human tumors, loss of RASSF1A expression is very commonly observed in a variety of human solid tumors and is accompanied by methylation of the CpG island associated with the transcription start site of RASSF1A.

In order to study the function of Rassf1a in the mouse, we have carried out gene targeting experiments in which exon 1-{alpha} of the Rassf1 gene was deleted (Fig. 1A). This targeting strategy should selectively eliminate the Rassf1a transcript but should leave expression of the Rassf1c isoform unaltered, a situation that is commonly found in human tumors. Several recombinant ES cell clones, in which exon 1-{alpha} was targeted, were identified (Fig. 1B). Two ES cell clones (#102 and #145) were used for blastocyst injection and generation of gene-targeted mice (Fig. 1B and C). Reverse transcription-PCR analysis confirmed that the Rassf1a transcript is missing and that Rassf1c is still expressed (Fig. 1D. Rassf1a homozygous knockout mice were viable and fertile, and no overt phenotype was immediately apparent. All experiments described here were subsequently carried out with the clone #145 mice. Mice were genotyped and assigned to three different groups according to genotype (wild-type, heterozygous, and homozygous Rassf1a knockout). They were kept and observed over extended periods of time. One Rassf1a–/– mouse died at 40 days of age and a splenic lymphoma was found. When the mice reached 18 to 20 months of age, they were euthanized and subjected to complete necroscopy. Only two lymphomas were found in a total of 48 wild-type mice (Tables 1 and 2). Six of thirty-five Rassf1a heterozygous mice developed tumors (two lymphomas and four lung adenomas). However, a total of 13 tumors were found in 41 Rassf1a–/– mice. The differences between wild-type and Rassf1a knockout mice were statistically significant (Table 1), in particular for the homozygous mice (P < 0.001, {chi}2 test).


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  		Table 1. Tumor susceptibility of Rassf1a knockout mice

 

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  		Table 2. Tumor types in Rassf1a knockout mice

 
The types of tumors found in the Rassf1a–/– mice were hematologic tumors, lung tumors, one adenocarcinoma of the breast, and one rectal squamous papilloma. All tumors were subjected to histopathologic analysis. Examples are shown for the breast adenocarcinoma, two lung tumors, and one lymphoma (Fig. 2). Two homozygous Rassf1a knockout animals developed both lymphomas and adenomas of the lung. Most tumors in the Rassf1a knockout mice were morphologically similar to spontaneously observed tumors (40). The breast tumor measured 1.2 cm in diameter and was composed of multiple small glands and microcysts composed of relatively uniform cuboidal cells with vesicular nuclei and mild to moderate pleomorphism. The epithelial component was embedded in an abundant myxomatous stroma with spindled cells (Fig. 2A and B). Histologically similar tumors have been classified in the mouse as breast adenocarcinoma type C. The lung tumors were well circumscribed but nonencapsulated and composed of closely packed acini of cuboidal and low columnar epithelial cells (Fig. 2C and D). Some tumors showed papillary formations and others had more nuclear pleomorphism with rare intranuclear inclusions (Fig. 2F). A few animals had both the epithelial tumor as well as lymphoma (Fig. 2E). In general, the hematopoietic tumors extensively involved both solid organs such as the lung and spleen as well as soft tissues. Morphologically, they were composed of small atypical lymphoid cells with plasmacytic differentiation. Immunologic characterization indicated that five of the six lymphomas analyzed, including the tumors in Rassf1a–/– mice, were high-grade B cell lymphomas.



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  		Figure 2. Histologic analysis of tumors found in Rassf1a–/– mice. A, breast adenocarcinoma; the tumor is well circumscribed and benign breast tissue is seen at the periphery (4x magnification). B, breast adenocarcinoma; the breast tumor is composed of rather uniform cells, forming small glands and cysts in a loose myxomatous matrix (40x magnification). C, lung adenoma; the tumor is well circumscribed and nonencapsulated (4x magnification). D, lung adenoma; the tumor is composed of closely packed acini of cuboidal to low columnar cells (40x magnification). E, a lung adenoma and multiple nodules of lymphoma in the same lung (4x magnification). F, some lung tumors were more cellular with intranuclear invaginations and moderate pleomorphism (60x magnification). Tissues were paraffin-embedded and stained with H&E stain.

 
We then conducted standard skin and lung carcinogenesis experiments to characterize the susceptibility of Rassf1a gene-targeted mice to tumor formation. In the skin carcinogenesis studies, benzo(a)pyrene was applied to the shaved dorsal skin of mice beginning at 8 weeks of age. Tumor formation was evaluated 7 months after the treatments (Fig. 3). Tumor size was significantly increased in Rassf1a knockout mice (Fig. 3A and B). However, tumor multiplicity was increased only in heterozygous Rassf1a knockout mice. Importantly, all tumors found in Rassf1a+/+ mice were skin papillomas. However, approximately 50% of the skin tumors found in either Rassf1a+/– or Rassf1a–/– mice were skin squamous cell carcinomas.



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  		Figure 3. Rassf1a gene-targeted mice are susceptible to chemical carcinogen-induced tumorigenesis. A, skin carcinogenesis; cohorts of 8-week-old mice with different genotypes (Rassf1a+/+, Rassf1a+/–, Rassf1a–/–) were exposed twice weekly for 10 weeks to the chemical carcinogen benzo(a)pyrene on the dorsal skin. Photographs were taken at 7 months after the last exposure. B and C, quantitation of the skin and lung carcinogenesis data. Skin tumors (B), were induced with benzo(a)pyrene and lung tumors (C), were induced by injection of urethane. Tumor load and tumor multiplicity were determined 7 months after the exposures. White columns, Rassf1a+/+ mice; gray columns, Rassf1a+/– mice; and black columns, Rassf1a–/– mice. The number of animals for the skin carcinogenesis study was (Rassf1a+/+, n = 14; Rassf1a+/–, n = 23; Rassf1a–/–, n = 19). The number of animals for the lung tumorigenesis study was (Rassf1a+/+, n = 8; Rassf1a+/–, n = 26; Rassf1a–/–, n = 21). Bars, SE; *, P < 0.05, t test.

 
In the lung tumorigenesis study, mice at 8 weeks of age were injected with urethane, a well-established lung carcinogen. Lungs were dissected at 7 months after the treatments. Both tumor load and tumor multiplicity were increased in Rassf1a gene-targeted mice relative to wild-type mice but the difference between heterozygous and homozygous mice was not significant (Fig. 3). All lung tumors found in either Rassf1a+/+, Rassf1a+/–, or Rassf1a–/– mice were lung adenomas.

Because the multiplicity of skin tumors was highest in the heterozygous animals and the most common mechanism for inactivation of Rassf1a is by methylation silencing, we examined if the remaining allele of Rassf1a had become methylated in the tumors. We investigated a total of 17 benzo(a)pyrene-induced skin tumors from wild-type and Rassf1a heterozygous mice as well as nine lung tumors found in Rassf1a+/– mice. No methylation was found in the tumors from wild-type mice but 3 out of 10 skin tumors arising in heterozygous animals showed the presence of methylated Rassf1a alleles (Fig. 4, samples 13, 17, and 18); Table 3). Because these mice have only one copy of Rassf1a, the presence of a band derived from the unmethylated allele is expected to arise from normal cell contamination or from partial methylation present within the tumor. No methylation was found in the lung tumors from heterozygous animals.



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  		Figure 4. Analysis of Rassf1a methylation in skin squamous cell carcinomas induced by benzo(a)pyrene. Tumors from wild-type (Rassf1a+/+) and Rassf1a heterozygous (Rassf1a+/–) mice were analyzed using methylation-specific PCR. Normal lung DNA treated with SssI DNA methyltransferase was used as a control for methylated Rassf1a (lanes 1 and 2) and untreated DNA from normal lung was used as a control for unmethylated Rassf1a (lanes 3 and 4). U, PCR product derived from unmethylated Rassf1a alleles (204 bp); M, PCR product derived from methylated Rassf1a alleles (213 bp).

 

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  		Table 3. Methylation-specific PCR analysis of Rassf1a in carcinogen-induced tumors in Rassf1a wild-type and heterozygous mice

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Methylation of the RASSF1A gene is one of the most common epigenetic aberrations described thus far in human tumors (1, 5, 7–32, 41–44) . The common epigenetic silencing and its location within an area of overlapping homozygous deletions at 3p21.3 (4) suggest that RASSF1A may be a tumor suppressor gene. Here we present evidence that mice, which lack the Rassf1a isoform, are susceptible to late stage spontaneous tumorigenesis and to formation of lung and skin tumors induced by chemical carcinogens. Although inactivation of RASSF1A in human nonmelanoma skin tumors has not yet been investigated, there are several reports documenting RASSF1A methylation in human lung tumors (1, 5, 7, 42–44). A breast adenocarcinoma was found in one Rassf1a–/– mouse and epigenetic inactivation of RASSF1A in human breast cancers is a common event and occurs in over 60% of the tumors (5, 10, 30).

Heterozygous Rassf1a knockout mice were significantly tumor-prone, both for spontaneous tumor formation and for the chemically induced tumors. This may suggest that the Rassf1a gene has the characteristics of conferring haploinsufficiency when only one allele is lost. There are now many examples demonstrating that loss of a single gene copy is sufficient to promote tumor formation (45, 46). Another possibility is that the single remaining Rassf1a allele in heterozygous mice is particularly prone to inactivation. We have tested this possibility using carcinogen-induced skin and lung tumors. For the skin tumors, tumor multiplicity was particularly high in heterozygous mice (Fig. 3). The data for skin tumors indicate that methylation was more common in tumors from heterozygous animals than in wild-type animals, although not all tumors from heterozygous mice showed methylation (Fig. 4). However, no methylation was found in lung tumors from heterozygous mice. The data from lung tumors and most skin tumors support the hypothesis of haploinsufficiency. However, the observation that Rassf1a promoter methylation is seen in a subset of skin tumors from heterozygous animals indicates that some skin tumors may not fit this hypothesis. We are pursuing immunohistochemistry studies on tumor tissue from heterozygous mice to show Rassf1a expression. However, we have not found or developed an antibody that works, and this makes this approach unfeasible at present. Further, we believe that studies on Rassf1a mutations in tumors from Rassf1a+/– mice are not likely to reveal any changes because mutational inactivation of RASSF1A in human tumors is exceedingly rare. On the other hand, loss of heterozygosity at 3p21.3 is a common and early event in the progression of several human cancers and is observed, for example, in breast and lung cancers (2, 4, 47, 48). Methylation of RASSF1A has been observed in early lesions of the lung, breast, and thyroid gland (14, 49, 50).

In summary, we provide the first genetic model to support the role of RASSF1A as a tumor suppressor gene. It will be interesting to determine if loss of RASSF1A in conjunction with the inactivation or overexpression of other genes will produce a more severe phenotype. For example, the closest homologue of RASSF1A, NORE1A, which is about 55% identical to RASSF1A, has been reported to be inactivated in human tumors as well (51, 52). Because RASSF1A has been proposed to be a negative regulator of activated Ras (53, 54), experiments in which loss of RASSF1A function is combined with expression of activated k-Ras may be informative. Finally, because RASSF1A stabilizes microtubules and plays a role in mitotic control (37, 38), the simultaneous inactivation of RASSF1A and mitotic checkpoint control genes, such as MAD2, may dramatically increase tumor susceptibility.


   Acknowledgments
 
Grant support: NIH grants (CA88873 to G.P. Pfeifer, and CA093643 to M. You) and by grant BMBF-FKZ01ZZ0104 to R. Dammann.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Donna Isbell for technical help with the mouse colony.


   Footnotes
 
Note: S. Tommasi, R. Dammann, Z. Zhang, and Y. Wang contributed equally to this work.

Received 8/ 5/04. Revised 10/ 9/04. Accepted 11/ 3/04.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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