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Heterozygous Mice for the Transforming Growth Factor-ß Type II Receptor Gene Have Increased Susceptibility to Hepatocellular Carcinogenesis

Laboratory of Cell Regulation and Carcinogenesis [Y-H. I., H. T. K., I. Y. K., K-B. H., M. A., J-J. J., K. F., S-J. K.] and Laboratory of Experimental Carcinogenesis [V. M. F., S. S. T.], National Cancer Institute, Bethesda, Maryland 20892; Pathology/Histotechnology Laboratory, SAIC-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 [D. C. H.]; and Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Victoria 3050, Australia [A. S.]

	ABSTRACT

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The transforming growth factor-ß (TGF-ß) receptor complex and its downstream signaling intermediates constitute a tumor suppressor pathway. In many cancers, expression of TGF-ß type II receptor (TßR-II) is markedly decreased. In the present study, we show that the hepatocytes isolated from 15-day-old, but not 9-month-old, mice heterozygous for the deletion of the TßR-II gene are slightly less sensitive to the growth-inhibitory effect of TGF-ß when compared with wild-type littermates of same age. In addition, the proliferation index of hepatocytes as indicated by bromodeoxyuridine incorporation is mildly increased in the heterozygous mice. These subtle changes in cellular phenotype did not result in either gross or microscopic abnormality of the liver. The treatment of these mice with the chemical carcinogen, diethylnitrosamine, results in a significantly enhanced tumorigenesis in the liver when compared with the wild-type littermates. Our results demonstrate the gene-dosage effect of TßR-II and indicate that the reduced expression of TßR-II in mice increases susceptibility to tumorigenesis in the liver.

	Introduction

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TGF-ß⁵ is a multifunctional growth factor expressed by many cell lines and tissue types. Although this growth factor has been shown to regulate immune response, angiogenesis, chondrogenesis, myogenesis, and production of extracellular matrix proteins, it usually acts as a potent inhibitor of growth in most cells, especially those of the epithelial lineage (1 , 2) . TGF-ß exerts its effects through an interaction with TßR-I and TßR-II (3 , 4) . Initially, the ligand binds to TßR-II (5) . Then, TßR-I is recruited into the complex and initiates the signal transduction cascade after cross-phosphorylation by TßR-II at the highly conserved GS domain (5) . During carcinogenesis, the disruption of TGF-ß signaling has been shown to be critical. The underlying mechanism of resistance to the growth-inhibitory effect of TGF-ß in malignant cells involves altered expression of either the receptors or the signaling molecules. Evidence to date, though, suggests that the most prevalent mechanism involves the receptors. The loss of expression of TßR-II and TßR-I in association with resistance to the growth-inhibitory effect of TGF-ß has been reported in various types of cancers, including gastric, colon, breast, thyroid, and prostate (6, 7, 8, 9, 10) . Because the loss of TßR-II expression is frequently observed in many different types of cancer, TßR-II has been proposed to be a tumor suppressor. In individuals with genetic predisposition to malignancies, tumor suppressors usually have a germ-line mutation in one allele followed by a local somatic inactivation of the remaining WT allele. Thus, the HT state for a tumor suppressor gene represents an increased risk of carcinogenesis. To determine whether or not TßR-II follows this standard paradigm of tumor suppressors, the present study investigated the rate of malignant transformation in the liver in TßR-II^+/- (HT) mice after treatment with DEN and phenobarbital. We report that TßR-II is a suppressor of hepatocarcinogenesis.

	Materials and Methods

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TßR-II^+/- Mice and Chemical Carcinogenesis.
TßR-II^+/- mice (HT) have been generated by gene targeting. The TßR-II gene was targeted using a 15.5-kb genomic fragment derived from a mouse 129 SV40 genomic library. Exon 2 of the TßR-II was disrupted using a phosphoglycerate kinase-neo cassette, and the resulting construct was electroporated into mouse ES cells (D6 subclone). After successful selection with neomycin, the resulting ES cells were then analyzed using PCR and Southern analysis, and ES clones containing a successfully targeted type II receptor gene were injected into C57BL/6J mouse blastocysts and implanted into pseudopregnant carrier mice. The resulting chimeric mice were then analyzed, and HT mice were bred.

For the carcinogenesis study, WT and HT littermates were generated by mating the HT. Mice were housed in a pathogen-free barrier facility and cared for according to NIH and AAALAC guidelines. At 15 days of age, male mice of both genotypes were given either a single i.p. injection of 5 mg/kg DEN in 0.1 ml of saline or saline alone. Animals were euthanized after 9 months. Tissues were analyzed microscopically and histologically for evidence of tumors by a board-certified veterinary pathologist using established diagnostic criteria.

RNA Isolation and Northern Blot Analysis.
The harvested mouse liver tissues were immediately frozen in liquid nitrogen and subsequently processed for RNA isolation using the TRIzol reagent (Life Technologies, Inc., Grand Island, NY) according to the manufacturer’s recommended protocol. Once total RNA was isolated, 15 µg were separated by electrophoresis and transferred onto Zeta-Probe blotting membrane (Bio-Rad Laboratories, Hercules, CA). Prehybridization, hybridization, and washing of the filters was described (8) . The TßR-II and ß-actin probes were labeled using the PRIME-IT random primer labeling kit (Stratagene, La Jolla, CA).

TGF-ß Receptor Cross-linking Assay.
Cell monolayers at 50–60% confluency were incubated with 200 pM ¹²⁵I-labeled TGF-ß1 (93 µCi/µg; DuPont/NEN, Boston, MA) at 4°C for 4 h with gentle shaking. Freshly prepared disuccinimidyl suberate (Pierce Chemical Co., Rockford, IL) at a final concentration of 0.6 mM was used to cross-link the bound ¹²⁵I-labled TGF-ß1 to the receptors at 4°C for 1 h. After solubilization in lysis buffer [1% Triton X-100, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, and 1 µg/ml leupeptin], protein samples were separated by SDS-PAGE on a 10% gel under reducing conditions, and cross-linked complexes of ¹²⁵I-labeled TGF-ß1 bound to the TGF-ß receptors were visualized by autoradiography.

In Vitro Growth Inhibition Assay.
Primary hepatocyte cultures were established from the mice as described previously (11) . Primary hepatocytes were plated in 24-well dishes at a density of 0.3 x 10⁶ cells/well in 0.5 ml of DMEM/F12 supplemented with 0.02% BSA; 18 mM HEPES (pH 7.4); 5 mM sodium pyruvate; 6 mM sodium bicarbonate; 1 mg/ml galactose; 30 µg/ml L-proline; 2 mM glutamine; 1% insulin, transferrin, and selenium+ (Collaborative Biomedical Products, Bedford, MA); 0.1% gentamicin; and 10% fetal bovine serum. After incubating for 73 h for cell attachment, media were changed to serum-free media containing supplements as above and 10 ng/ml epidermal growth factor (Upstate Biotechnology, Lake Placid, NY), and then TGF-ß1 (5 ng/ml) was added into the media 1 h after media change. Cells were then incubated in the presence of TGF-ß for 20 h, and 5 µCi of [³H]thymidine was added into the media. The cells were cultured for additional 4 h in the presence of TGF-ß. [³H]thymidine incorporation was measured in a liquid scintillation counter and normalized for DNA content.

In Vivo Proliferation Assay.
Mice were injected with 150 mg BrdUrd/kg 1 h before sacrifice. Ethanol-fixed sections were immunostained using an anti-BrdUrd monoclonal antibody (Dako, Carpinteria, CA) and vector M. O. M. kit (Vector Laboratories, Burlingame, CA). After counterstaining with hematoxylin, the number of labeled hepatocytes in 50 high power fields was determined for each liver sample.

Immunoblot Analysis.
Frozen liver tissues from the mice were homogenized with PBS at 4°C, and the protein concentration was determined. Samples were placed in sample buffer (0.0625 M Trizma base, 2% SDS, and 5% 2-mercaptoethanol) and boiled for 5 min. Electrophoresis was carried out under reducing conditions using 100 µg of total protein in each lane. After electrophoresis, protein was transferred to a 0.2-µm nitrocellulose membrane and incubated for 1 h in blocking buffer (5% nonfat dry milk, Tris-buffered saline, and 1% Tween). Subsequently, the membrane was incubated with the appropriate antibody overnight at room temperature. After washing with 0.1% Tween-Tris-buffered saline, the membrane was incubated in the presence of appropriate horseradish peroxidase-labeled secondary antibody (Bio-Rad Laboratories) at a dilution of 1:5000 for 1 h at room temperature. After washing three times, immunoreactive bands were visualized by enhanced chemiluminescence (Pierce Chemical Co.). Primary antibodies used were as follows: anti-p27 (C19), anti-cdc 25A (#144), anticyclin D1 (C-20), anti-cdk 4 (M2), and anti-cdk 2 (M2) from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-c-myc (Ab-2) from Oncogene Research Products (Cambridge, MA).

Immunohistochemistry.
Tissues were fixed in 10% neutral buffered formalin overnight. Subsequently, they were processed and embedded in paraffin blocks. Immunochemical staining was performed using an indirect immunoperoxidase protocol (Vectastain Elite kit; Vector Laboratories). Once in paraffin blocks, the tissues were sectioned at a thickness of 5 µm, deparaffinized in Hemo-de (Fisher Scientific Co., Pittsburgh, PA), and rehydrated in PBS (pH 7.2). The endogenous peroxidase activity was inactivated by incubation in 0.3% H₂O₂ for 10 min, and normal goat serum was used to block nonspecific sites. Next, the sections were incubated with primary antibodies for 18 h at 4°C in humidified chambers. TßR-II was detected by polyclonal antihuman TßR-II (C-16; Santa Cruz Biotechnology). TGF-ß1 is detected with the rabbit polyclonal antibody LC(1–30; Ref. 12 ). Antigenic binding sites were visualized by incubation with biotinylated secondary antibody, followed by avidin-biotin-horseradish peroxidase complex and diaminobenzidine tetrachloride before counterstaining with Gills’ hematoxylin. Negative control sections were processed in an identical manner after substituting the primary antibodies with rabbit IgG fraction.

	Results

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Levels of TßR-II mRNA and Protein in the HT Mice.
In the WT and HT mice, the relative levels of expression of TßR-II mRNA in the liver were investigated by Northern blot analysis. In both the 15-day and the 9-month-old HT mice, the levels of expression of TßR-II mRNA were 50% of those of the WT mice (Fig. 1a) . Interestingly, among the WT mice, the level of expression of TßR-II mRNA was reduced in the liver tissues of the 9-month-old mice when compared with that of the 15-day-old mice.

View larger version (43K): [in this window] [in a new window]   Fig. 1. Expression of TßR-II in liver tissues of TßR-II+/- mice. a, Northern blot analysis; b, TGF-ß receptor cross-linking assay. Note that the levels of expression of TßR-II mRNA and protein were decreased in HT mice when compared with those of WT mice.

Proliferation in the HT Mice.
TGF-ß inhibits cell growth in hepatocytes (13) . Therefore, we measured the proliferation rate in the livers of immature mice using BrdUrd (Fig. 2a) . The rate of DNA synthesis was increased by 1.7-fold in the livers of 15-day-old HT mice when compared with their WT littermates. Next, the growth-inhibitory activity of TGF-ß was investigated in the primary cultures of hepatocytes isolated from the livers of the 15-day and the 9-month-old HT mice (Fig. 2b) . Primary hepatocytes obtained from the liver of WT littermates were used as the control. Upon the treatment with 5 ng/ml TGF-ß, hepatocytes isolated from the HT 15-day-old mice showed a decrease in the magnitude of growth inhibition when compared with the WT mice of similar age (85.8 versus 92.9%). In the 9-month-old mice, there was a tendency toward a decreased level of growth inhibition in the primary hepatocytes obtained from HT mice, but there was no statistically significant difference. These results suggest that the sensitivity to TGF-ß is reduced in the liver of the HT mice.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Rate of proliferation in hepatocytes of HT mice. a, in vivo proliferation assay using BrdUrd. The number of labeled nuclei per high power field significantly increased in the liver of HT mice. b, in vitro growth-inhibition assay. When treated with 5 ng/ml TGF-ß1, the magnitude of growth inhibition was significantly decreased in primary hepatocyte cultures obtained from the HT mice when compared with the WT mice at 15 days of age. At 9 months of age, there was a tendency toward a decreased level of growth inhibition in the hepatocytes obtained from the HT mice; this difference, though, was not statistically significant.

View larger version (72K): [in this window] [in a new window]   Fig. 3. Immunoblot analysis for cell cycle-associated proteins. Of the seven cell cycle proteins investigated in the present study, c-myc was significantly decreased, whereas cyclin A was unaffected in the HT mice at 15 days but increased at 9 months.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Tumor incidence in the liver of HT and WT mice after treatment with DEN. Male mice (15 days old) were treated with DEN and were killed and analyzed grossly and histologically for tumors 9 months after treatment. Mice with any tumor (macroscopic or microscopic and adenoma or carcinoma) and mice with malignant tumors (carcinoma) were shown. Statistical significance was determined using Fisher’s exact probability test: two tailed.

View larger version (49K): [in this window] [in a new window]   Fig. 5. Immunohistochemistry of TßR-II and TGF-ß1 in HT tumors. a, TßR-II; b, TGF-ß1; c, control rabbit IgG fraction. Arrows, the junction between normal liver tissue (left of arrow) and tumor (right of arrow). As shown, the level of expression of TßR-II was significantly decreased in the tumor when compared with the surrounding normal tissue. However, no observable difference in the level of expression of TGF-ß1 was seen between the normal and malignant tissues.

	Discussion

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The magnitude of growth inhibition in the hepatocytes obtained from the HT mice was significantly smaller than that of the cells obtained from the WT littermates. However, this decreased sensitivity to TGF-ß did not result in hyperplasia. Because organ development and homeostasis involve a tightly controlled network of proliferation, differentiation, and apoptosis, perturbation in one pathway is likely not sufficient for gross phenotypic changes. Such hypothesis is consistent with previously published reports in which TGF-ß HT mice exhibited no microscopic or gross phenotypic changes (15) .

Spontaneous tumorigenesis was not detected in both the HT and the WT mice 1 year of age. However, upon treatment with the chemical carcinogen DEN, the rate of tumorigenesis in the liver was significantly higher in the HT mice when compared with WT littermates. In addition, the developed tumors were also significantly larger in the HT mice when compared with that of the WT mice. These results indicate that TßR-II genotype is a significant factor during progression of the hepatocellular carcinoma. But above all, these results demonstrate that the endogenous TßR-II has a tumor suppressor activity in the liver. Such a conclusion is consistent with the report published by Markowitz et al. (9) . According to this work, a subset of colon cancer cell lines with microsatellite instability has an initiating event that results in an increased rate of error during replication. Then, this error leads to mutation of the TßR-II gene and to tumor progression. The mechanism responsible for the increased risk of liver tumorigenesis in the HT mice remains to be elucidated. It is likely that the decreased sensitivity to TGF-ß from the loss of one allele of TßR-II is the most significant factor. However, the possibility exists that the loss of the remaining WT allele may have occurred as a result of the exposure to the carcinogens. In the present study, though, the status of the remaining TßR-II WT allele in the tumors was not investigated because of the difficulty in completely separating the malignant from the benign cells.

The precise mechanism for the inactivation of TßR-II in tumor tissues de novo is not clear. As with established tumor suppressor genes such as p53 and Rb, mutations may be one possibility. Consistent with this hypothesis, it has been reported that TßR-II is frequently mutated in a subset of colon cancer cells (9) . Alternatively, transcriptional repression is another potential mechanism. In fact, evidence to date suggests that the transcriptional repression of the TßR-II gene may be the major mechanism of inactivating TßR-II in malignant cells, e.g., the loss of TßR-II expression as demonstrated by immunohistochemistry and Northern blot analysis was clearly demonstrated without mutation or loss of heterozygosity in cancers of the breast, prostate, bladder, and stomach (9 , 18 , 19) .

In conclusion, results of the present study demonstrated that the HT mice for the TßR-II gene have subtle changes in proliferation in the liver. However, these changes were not sufficient for gross or microscopic phenotypic changes. When challenged with liver carcinogens, the HT mice exhibited increased incidence of liver tumorigenesis when compared with the WT littermates. Taken together, these observations demonstrate the gene-dosage effect of TßR-II and suggest that TßR-II is a tumor suppressor in the liver. Future work will focus on determining the precise mechanism for the repression of TßR-II transcription in liver tumors.

	ACKNOWLEDGMENTS

 
We thank M. Shrader and D. Logsdon for their technical expertise in animal breeding and carcinogenesis and L. Wakefield for helpful discussions.

	FOOTNOTES

 
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.

¹ Y-H. I. and H. T. K. equally contributed to this work.

² Present address: Division of Hematology/Oncology, Department of Internal Medicine, Samsung Medical Center, Sungkyunkwan University College of Medicine, Kangnam-ku, Seoul 135-710, Korea.

³ Present address: Department of Gastroenterology, Ajou University School of Medicine, Suwon 442-721, Korea.

⁴ To whom requests for reprints should be addressed, at Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Building 41, Room B1106, NIH, Bethesda, MD 20892-5055. Phone: (301) 496-8350; Fax: (301) 496-8395; E-mail: kims{at}dce41.nci.nih.gov

⁵ The abbreviations used are: TGF-ß, transforming growth factor-ß; TßR-II, TGF-ß type II receptor; HT, heterozygous; DEN, diethylnitrosoamine; WT, wild-type; BrdUrd, bromodeoxyuridine.

Received 2/26/01. Accepted 7/30/01.

	REFERENCES

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 

1. Roberts A. B. Sporn M. B. eds. . Peptide Growth Factors and Their Receptors. Handbook of Experimental Pharmacology, : 419-472, Springer-Verlag Heidelberg, Germany 1990.
2. Massagué J. TGF-ß signal transduction. Annu. Rev. Biochem., 67: 753-791, 1998.[Medline]
3. Franzen P., ten Dijke P., Ichijo H., Yamashita H., Schulz P., Heldin C., Miyazono K. Cloning of a TGF-ß type I receptor that forms a heteromeric complex with the TGF-ß type II receptor. Cell, 75: 681-692, 1994.
4. Lin H. Y., Wang X., Ng-Eaton E., Weinberg R. A., Lodish H. F. Expression cloning of the TGF-ß type II receptor. A functional transmembrane serine/threonine kinase. Cell, 68: 775-785, 1992.[Medline]
5. Wrana J. L., Attisano L., Wieser R., Ventura F., Massague J. Mechanism of activation of the TGF-ß receptor. Nature (Lond.), 370: 341-347, 1994.[Medline]
6. Kim S-J., Im Y-H., Markowitz S. D., Bang Y-J. Molecular mechanisms of inactivation of TGF-ß receptors during carcinogenesis. Cytokine Growth Factor Rev., 11: 159-168, 2000.[Medline]
7. Kim I. Y., Ahn H-J., Zelner D. J., Shaw J. W., Sensibar J. A., Kim J-H., Kato M., Lee C. Genetic change in transforming growth factor ß (TGF ß) receptor type I gene correlates with insensitivity to TGF-ß1 in human prostate cancer cells. Cancer Res., 56: 44-48, 1996.[Abstract/Free Full Text]
8. Park K., Kim S. J., Bang Y. J., Park J. G., Kim N. K., Roberts A. B., Sporn M. B. Genetic changes in the transforming growth factor ß (TGF-ß) type II receptor gene in human gastric cancer cells: correlation with sensitivity to growth inhibition by TGF-ß. Proc. Natl. Acad. Sci. USA, 91: 8772-8776, 1994.[Abstract/Free Full Text]
9. Markowitz S., Wang J., Meyeroff L., Parsons R., Sun L., Lutterbaugh J., Fan R. S., Zborowska E., Kinzler K. W., Vogelstein B., Brattain M., Willson J. K. V. Inactivation of the TGF-ß type II receptor in colon cancer cells with microsatellite instability. Science (Wash. DC), 268: 1336-1338, 1995.[Abstract/Free Full Text]
10. Hahm K-B., Cho K., Lee C., Im Y-H., Choi S-G., Sorensen P. H. B., Thiele C. J., Kim S-J. The EWS-FLI1 oncogene of Ewing sarcoma represses TGF-ß type II receptor gene expression. Nat. Genet., 23: 222-227, 1999.[Medline]
11. Kao C-Y., Factor V. M., Thorgeirsson S. S. Reduced growth capacity of hepatocytes from c-myc and c-myc/TGF-ß transgenic mice in primary culture. Biochem. Biophys. Res. Commun., 222: 64-70, 1996.[Medline]
12. Flanders K. C., Thompson N. L., Cissel D. S., Van obberghen-Schilling E., Baker C. C., Kass M. E., Ellingsworth L. R., Roberts A. B., Sporn M. B. Transforming growth factor-ß1: histochemical localization with antibodies to different epitopes. J. Cell Biol., 108: 653-660, 1989.[Abstract/Free Full Text]
13. Dixon M., Agius L., Yeaman S. J., Day C. P. Inhibition of rat hepatocyte proliferation by transforming growth factor ß and glucagon is associated with inhibition of ERK2 and p70 S6 kinase. Hepatology, 29: 1418-1424, 1999.[Medline]
14. Ravitz M. J., Wenner C. E. Cyclin-dependent kinase regulation during G1 phase and cell cycle regulation by TGF-ß. Adv. Cancer Res., 71: 165-207, 1997.[Medline]
15. Iavarone A., Massague J. Repression of the CDK activator Cdc 25A and cell cycle arrest by cytokine TGF-ß in cell lacking the CDK inhibitor p15. Nature (Lond.), 387: 417-422, 1997.[Medline]
16. Tang B., Bottinger E. P., Jakowlew S. B., Bagnall K. M., Mariano J., Anver M. R., Letterio J. J., Wakefield L. M. Transforming growth factor-ß1 is a new form of tumor suppressor with true haploid insufficiency. Nat. Med., 4: 802-807, 1998.[Medline]
17. Shimizu I., Yasuda M., Mizobuchi Y., Ma Y-R., Liu F., Shiba M., Horie T., Ito S. Suppressive effect of oestradiol on chemical hepatocarcinogenesis in rats. Gut, 42: 112-119, 1998.[Abstract/Free Full Text]
18. Takenoshita S., Mogi A., Tani M., Osawa H., Sunaga H., Kakegawa H., Yanagita Y., Koida T., Kimura M., Fujita K. I., Kato H., Kato R., Nagamachi Y. Absence of mutations in the analysis of coding sequences of the entire transforming growth factor-ß type II receptor gene in sporadic human breast cancers. Oncol. Rep., 5: 367-371, 1998.[Medline]
19. Kim I. Y., Ahn H. J., Zelner D. J., Shaw J. W., Lang S., Kato M., Oefelein M. G., Miyazono K., Nemeth J. A., Kozlowski J. M., Lee C. Loss of expression of transforming growth factor ß type I and type II receptors correlates with tumor grade in human prostate cancer tissues. Clin. Cancer Res., 2: 1255-1261, 1996.[Abstract]

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