Transgenic Mouse Model for Synergistic Effects of Nuclear Oncogenes and Growth Factors in Tumorigenesis: Interaction of c-myc and Transforming Growth Factor α in Hepatic Oncogenesis Hiroko Murakami, Nancy D. Sanderson, Peter Nagy, et al. Cancer Res 1993;53:1719-1723. Published online April 1, 1993.
Updated Version
Citing Articles
E-mail alerts Reprints and Subscriptions Permissions
Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/53/8/1719
This article has been cited by 39 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/53/8/1719#related-urls
Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at
[email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at
[email protected].
Downloaded from cancerres.aacrjournals.org on July 23, 2011 Copyright © 1993 American Association for Cancer Research
(CANCER RESEARCH 53, 1719-1723, April 15, 1993]
Advances in Brief
Transgenic Mouse Model for Synergistic Effects of Nuclear Oncogenes and Growth Factors in Tumor igenesis: Interaction of c-myc and Transforming Growth Factor a in Hepatic Oncogenesis Hiroko Murakami, Nancy D. Sanderson, Peter Nagy, Pamela A. Marino, Glenn Merlino, and Snorri S. Thorgeirsson1 Laboratories 20892
of Experimental
Carcinogenesis
[H, M.. N, D. S.. P. N,. P. A. M., S. S. TJ and Molecular Biologv ¡G.M.j. National Cancer Institute, NIH, Bethe.fda. Maryland
Deregulation of c-myc expression is frequently observed in experi mentally induced hepatocellular carcinoma in rodents, as well as in primary human liver tumors (10-13). Similarly, expression of TGF-a,2 a potent hepatotrophic mitogen synthesized in hepatocytes
Abstract Double transgenic mice bearing fusion genes consisting of mouse albu min enhancer/promoter-mouse c-myc complementary DNA and mouse metallothionein 1 promoter-human transforming growth factor a comple mentary DNA were generated to investigate the interaction of these genes in hepatic oncogenesis and to provide a general paradigm for character izing the interaction of nuclear oncogenes and growth factors in tumorigenesis. Coexpression of c-myc and transforming growth factor a as trans-
during regeneration, is frequently observed in both human tumors and in transformed cultured cells (14), including those of the liver (15, 16). TGF-a is structurally and functionally related to epidermal growth
genes in the mouse liver resulted in a tremendous acceleration of neoplastic development in this organ as compared to expression of either of these transgenes alone. The two distinct cellular reactions that occurred in the liver of the double transgenic mice prior to the appearance of liver tumors were dysplastic and apoptotic changes in the existing hepatocytes followed by emergence of multiple focal lesions composed of both hyperplastic and dysplastic cell populations. These observations suggest that the interaction of c-myc and transforming growth factor a, and possibly other combinations of nuclear oncogenes and growth factors, during develop ment of hepatic neoplasia contributes to the selection and expansion of the preneoplastic cell populations which consequently increases the probabil ity of malignant conversion.
Introduction Neoplastic development is a multistep phenomenon involving al teration in the genetic material of the cells (1, 2). The genetic alter ations that predispose to the development of neoplasia may involve activation of protooncogenes, inactivation of tumor suppressor genes, and/or inappropriate expression of growth factors (3). Accumulation of these genetic changes eventually leads to the emergence of malig nant cell populations capable of devastating the host organism. Whether there exists a preferred sequence of genetic alterations in tumor development (4) or a number of nonordered combinations of genetic changes that may be capable of generating the same or a similar malignant phenotype remains to be determined. The transgenic mouse system provides an unique model for assess ing the effects of oncogenes acting either alone or in combination with other oncogenes within the organism (5, 6). It therefore provides a research tool for exploring interactions of some of the genetic alter ations observed in the fully developed tumor phenotypes. This may be particularly important in the interaction of growth factors and nuclear oncogenes (3). The myc gene family (c-myc, N-myc, and L-mvc) is an important member of the nuclear oncogenes. The myc oncoproteins act as sequence-specific transcription factors that regulate a variety of genes important in normal cellular growth and differentiation pro cesses (7, 8). Aberrant expression of all three members of the myc gene family has been implicated in the development of a wide variety of both experimentally induced and naturally occurring tumors (9).
factor and exerts it effects via the epidermal growth factor receptor (17-19). Liver tumors occur in c-myc transgenic mice as a result of the selective expression of c-myc in the liver directed by the albumin enhancer/promoter or the a-1-antitrypsin promoter (20, 21). However, the tumor incidence in these c-myc transgenic mice is relatively low with a long latency period (20, 21). Similarly, transgenic mice in which TGF-a is under the transcriptional control of the metallothio nein promoter and is expressed at high levels in the liver and other tissues develop liver tumors after a latency period of 10 to ¡5months (22-24). To define the interaction, as well as the possible synergistic effects of c-myc and TGF-a during hepatocarcinogenesis, transgenic mice bearing a chimeric gene consisting of the mouse albumin enhancer/promoter and a mouse c-myc cDNA (Alb/c-mvc) that directs the expression of c-myc to the liver (25) were generated and crossed to a second transgenic mouse bearing a fusion gene consisting of the mouse metallothionein 1 promoter and a human TGF-a cDNA (MT/ TGF-a; Ref. 22; Fig. ÌA). Materials and Methods Recombinant DNA Constructs. Mouse c-myc cDNA clone, pMc-myc54 (26), was digested with Xhol. The obtained 1.4-kilobase Xhol fragment con tains full length coding sequence, and 62 base pairs of the 5' and 11 base pairs of the 3' noncoding sequences of c-myc cDNA. Both ends of the fragment were then filled up with Klenow enzyme and ligated with BamHl linker. After digestion with BamHl, the c-myc fragment was inserted into a BamHl site which is located downstream of the mouse serum albumin promoter of 2335A-1 (25). Plasmids which had the c-myc fragments in the same orientation with the albumin promoter were isolated. Lastly, the rabbit ß-globin3' noncoding sequence from pBBB (27) was inserted between the BamHl site at the 3' end of c-myc cDNA and the Kpn\ site of the 2335A-1 to obtain the final construct, pLEC 1. Transgenic Mice. A 4.5-kilobase vector free fragment was obtained by digesting pLECl with Nael and Kpnl, purified, and used to microinject into one-cell mouse embryos obtained from mating of hybrid (C57B1/6JXCBA/J) Fl mice as described (28). Transgenic offspring were identified by DNA dot blot and Southern analysis of tail DNA using nick-translated c-myc probe. The resulting founder transgenic mice were maintained as hétérozygotes in the B6XCBA Fl background. The generation of the metallothionein TGF-a trans genic mouse line MT42 was as previously described (22). These mice were generated in a CD-I genetic background. Heterozygous male Alb-c-mvt trans genic mice (line 166.8) were crossed with female mice homozygous MT/TGF-a transgene (line MT42; Ref. 22).
Received 12/30/92; accepted 3/8/93. 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. 1To whom requests for reprints should be addressed.
for the
2 The abbreviations used are: TGF-a, transforming growth factor a; cDNA, comple mentary DNA; PBS, phosphate buffered saline: Alb. albumin: MT, metallothionein. 1719
Downloaded from cancerres.aacrjournals.org on July 23, 2011 Copyright © 1993 American Association for Cancer Research
INTERACTION
SpXi ' B.mH
L-t
OF c-myc AND TGF-a
(B/Bg)
IN HEPATIC ONCOGENESIS
saponin in PBS for 2 h,followed by digestion with 0. 1% trypsin in PBS for 10-15 min. After rinsing in PBS. sections were stained for TGF-a by the avidin-biotin-peroxidase method (29) using the ABC kit from Vector Labora tories. The antibody (a gift from Dr. L. Gentry) was a rabbit anti-rat pro-TGF-a antiserum (1:1000 dilution) raised against residues 137-159 of the COOHterminal peptide of the cytoplasmic domain of pro-TGF-a (30). The demonstration of TGF-a transcripts by in silu hybridization was ac complished by using antisense and sense human TGF-a riboprobes derived from a 310-base pair fragment of the 5' end of the cDNA inserted into the
Kpnl
Alb-c-myc (pLECI)
pGEM3 transcription vector (Promega), as earlier described (22). This probe hybridized with human TGF-a but only weakly with the murine gene product. Similarly, c-myc antisense and sense riboprobes were constructed by inserting a 870-base pair fragment of murine c-myc cDNA into the pGErn4z transcrip tion vector (Promega). The in silu analysis of TGF-a and c-mvc transcripts were performed with the appropriate antisense and sense riboprobes as de scribed (31-33).
B
Results Generation of Alb/c-myc and Alb/c-/wyc-MT/TGF-a Double Transgenic Mice. Eight founder animals bearing the Alb/c-myc construct were identified from which four lines were developed (Fig. Iß).Three of the four lines (166.8, 178.3, and 181.2) contained 5-10 head-to-tail copies of the transgene, whereas line 179.2 had 1-2 copies of the transgene (data not shown). High levels of the expected transcript size ( —¿ 1.9 kilobases) of the Alb/c-mvc transgene were
- 2.3 Kb —¿ 1.9 Kb
found in livers of the 3 high copy number lines, whereas expression of the transgene was considerably lower in line 179.2 bearing the 1-2 copies of the Alb/c-myc construct (Fig. Iß).Expression of the Alb/c-myc transgene was not detected in tissues other than liver (data not shown). The double transgenic mice were generated by crossing the Alb/c-myc mice (lines 166.8, 178.3, 181.2, and 179.2) with the MT/TGF-a mice (line MT42; Ref. 22). Development of Hepatic Neoplasia and Expression of Transgenes. The male mice were selected from the double Alb/c-mvc-MT/TGF-a transgenic
I i I
mice and divided into two groups. One group was maintained on 50 min of ZnCli in the drinking water starting at 3 weeks of age, while the other was kept without the zinc supplement. The animals were then sacrificed at 3, 6, 10, and 16 weeks after starting the zinc supplement. A minimum of 12 animals were analyzed for each time point. The data reported here are those from a cross between line 166.8 and MT42, but all the lines bearing multiple Alb/c-myc
c-myc
copies gave essentially the same results when crossed with the MT42 line. After 3 weeks of zinc treatment the first histológica! indication of neoplastic development became evident and was characterized by the appearance of huge dysplastic hepatocytes around blood vessels displaying compact nuclei and condensed chromatin, indicative of apoptosis (Fig. 2C). By the sixth week 80% of the mice displayed these lesions. Foci of distinct dysplastic cells displaying numerous mitotic figures were commonly found throughout the liver lobule by week 10 (Fig. ID). At this time intravascular spread of the tumor cells was first observed in close association with these dysplastic foci (Fig. 2E), but no distant metastasis was detected. After 16 weeks of zinc treatment, over 70% of the double transgenic male mice carried single or multifocal nodules of which 25% histologically consisted of well differentiated hepatocellular carcinomas displaying either pseudoglandular or trabecular pattern (Fig. 2F). No neoplas tic lesions were observed in either the Alb/c-myc or the MT/TGF-a single
—¿1.9 Kb
TGF-o
—¿ 1.4kB
Fig. I. A, Structure of transgenes. Plusmid constructs are described in (22) and in "Materials and Methods." B. transgene expression in four of the Alb-c-mvc' mouse lines. Northern blot analysis was performed on 20 ug of total RNA isolated from liver and electrophoresed through 0. 97c agarose gel containing 12% formaldehyde, probed with nick-translated c-mvr mouse cDNA. and washed as previously described (31). C. expres sion of c-mvi' and TGF-a transgenes in a hepatocellular carcinoma and adjacent grossly normal liver. Northern blot analysis was performed as described in B; nick-translated cloned human TGF-a cDNA was used to probe for the TGF-a transgene expression (32). Equal gel loading was confirmed by ethidium bromide staining of the gels (data not shown). Kb. kilobases.
transgenic mice during the corresponding time period. In contrast, the appear ance of preneoplastic and neoplastic lesions in the double transgenic mice maintained without the zinc treatment was delayed by 6 to 8 weeks (data not shown). Different patterns of expression of the TGF-a and myc transgenes were observed. Whereas expression of the Alb/c-myc transgene coincided with that of albumin (Fig. 3£)(33, 34). MT/TGF-a expression was most intense in the hepatocytes surrounding the central vein (Fig. 3, A and ß).Similarly, TGF-a was frequently expressed strongly in the dysplastic cells and tumors (Figs. 1C and 3. C and D ), while expression of the Alb/c-myc transgene was either absent or greatly reduced in the tumors (Fig. 1C). However, high expression of the Alb/c-myc gene was occasionally seen in the early focal lesions (Fig. ÌF).
Immunohistochemistry and in Situ Hybridization. Immunohistochemical localization of the TGF-a protein in the liver was performed on deparaffinized sections. After inactivation of the endogenous peroxidase (30-min
Discussion
treatment with 0.3% H2O2in methanol), the sections were treated with 0.05%
TGF-a transgenes in mouse liver leads to a tremendous acceleration
In this report
we demonstrate
that coexpression
1720
Downloaded from cancerres.aacrjournals.org on July 23, 2011 Copyright © 1993 American Association for Cancer Research
of the c-myc and
INTERACTION
OF c-myc AND TGF-a
IN HEPATIC ONCOGENESIS
•¿ „¿:/
i -•> ?
