Induction of medulloblastomas in p53-null mutant mice by somatic inactivation of Rb in the external granular layer cells of the cerebellum.
Journal: 2000/May - Genes and Development
ISSN: 0890-9369
PUBMED: 10783170
Abstract:
Medulloblastomas are among the most common malignancies in childhood, and they are associated with substantial mortality and morbidity. The molecular pathogenesis as well as the ontogeny of these neoplasms is still poorly understood. We have generated a mouse model for medulloblastoma by Cre-LoxP-mediated inactivation of Rb and p53 tumor suppressor genes in the cerebellar external granular layer (EGL) cells. GFAP-Cre-mediated recombination was found both in astrocytes and in immature precursor cells of the EGL in the developing cerebellum. GFAP-Cre;Rb(LoxP/LoxP);p53(-/- or LoxP/LoxP) mice developed highly aggressive embryonal tumors of the cerebellum with typical features of medulloblastoma. These tumors were identified as early as 7 weeks of age on the outer surface of the molecular layer, corresponding to the location of the EGL cells during development. Our results demonstrate that loss of function of RB is essential for medulloblastoma development in the mouse and strongly support the hypothesis that medulloblastomas arise from multipotent precursor cells located in the EGL.
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Genes Dev 14(8): 994-1004

Induction of medulloblastomas in <em>p53</em>-null mutant mice by somatic inactivation of <em>Rb</em> in the external granular layer cells of the cerebellum

Division of Molecular Genetics and Centre of Biomedical Genetics, The Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands
Corresponding author.
Received 2000 Jan 21; Accepted 2000 Mar 10.

Abstract

Medulloblastomas are among the most common malignancies in childhood, and they are associated with substantial mortality and morbidity. The molecular pathogenesis as well as the ontogeny of these neoplasms is still poorly understood. We have generated a mouse model for medulloblastoma by Cre–LoxP-mediated inactivation of Rb and p53 tumor suppressor genes in the cerebellar external granular layer (EGL) cells. GFAP–Cre-mediated recombination was found both in astrocytes and in immature precursor cells of the EGL in the developing cerebellum. GFAP–Cre;Rb;p53 mice developed highly aggressive embryonal tumors of the cerebellum with typical features of medulloblastoma. These tumors were identified as early as 7 weeks of age on the outer surface of the molecular layer, corresponding to the location of the EGL cells during development. Our results demonstrate that loss of function of RB is essential for medulloblastoma development in the mouse and strongly support the hypothesis that medulloblastomas arise from multipotent precursor cells located in the EGL.

Keywords: Cre–LoxP system, medulloblastoma, Rb, GFAP, p53, astrocyte
Abstract

Astrocytes are the most abundant non-neuronal cells in the central nervous system (CNS) and participate in a wide variety of physiologic and pathologic processes. In humans this cell type most frequently undergoes neoplastic transformation in the adult brain. Although some genetic events in the neoplastic transformation of astrocytes have been identified, relatively little is known about the pathways involved in the development and progression of glial tumors.

The glial fibrillary acidic protein (GFAP) gene encodes an intermediate filament cytoskeletal protein that is restricted primarily to astroglia in the adult CNS (Bignami et al. 1972). Mouse GFAP promoter sequences have been extensively characterized and used to express heterologous genes in transgenic mice (Toggas et al. 1994; Johnson et al. 1995). We reasoned that expressing the Cre recombinase of the Cre–LoxP system (for review, see Rajewsky et al. 1996) under control of this promoter would enable us to study the role of the tumor suppressor genes involved in the pathogenesis of gliomas.

There is evidence that loss of function of the tumor suppressor gene P53 is one of the initiator events in the neoplastic transformation of astrocytes. Families with germ line mutations of P53 have an incidence of CNS tumors of 13%, most of which (73%) are astrocytomas (Kleihues et al. 1997). The pattern of mutations found in sporadic and inherited brain tumors is similar (Fulci et al. 1998). p53 knockout mice have been generated and extensively characterized (Donehower et al. 1992; Jacks et al. 1994), but no increased incidence of glial tumors have been reported in the short life span of these highly tumor prone mice. Astrocytes derived from these p53 knockout mice, however, undergo spontaneous immortalization (Bogler et al. 1995; Yahanda et al. 1995).

The retinoblastoma gene was the first tumor suppressor gene to be cloned (Dryja et al. 1986; Friend et al. 1986; Lee et al. 1987a,b) and has been found mutated in a variety of human sporadic and hereditary tumors (for review, see Weinberg 1995). The retinoblastoma gene product, RB, is not only required for regulating entry into the cell cycle but also for terminal differentiation of different cell types, including CNS precursors (Lee et al. 1994). There is evidence from in vitro studies for the involvement of RB in differentiation and maturation of neurons and glia. Retinoic acid treated p19 embryonal carcinoma cells failed to differentiate into neurons and glia upon functional inactivation of RB by E1A (Slack et al. 1995). Moreover, loss of RB has been implicated in glial malignancies. A proportion of children with the inherited form of retinoblastoma will develop brain tumors later in life, and alterations of chromosome 13 have been reported in glioma cell lines as well as in about one-third of malignant gliomas (Hamel et al. 1993).

Although inactivation of the Rb gene in the mouse germ line leads to embryonic lethality at day 12.5 of gestation (Clarke et al. 1992; Jacks et al. 1992; Lee et al. 1992) as a result of major defects in the hematopoietic and nervous system, mice heterozygous for Rb or mice chimeric for wild-type and Rb-deficient cells develop pituitary tumors early in life (Maandag et al. 1994; Williams et al. 1994). Therefore, the consequences of lack of RB on glial differentiation and neoplastic transformation cannot be studied in these mice.

We have chosen to investigate the role of RB in the neoplastic transformation of astrocytes using the Cre–LoxP system. To assess a possible cooperation in tumorigenesis between RB and p53, we examined the effects of GFAP–Cre-mediated Rb inactivation in a p53-null background. Moreover, to overcome the limitations linked to the short life span of the p53 knockout mice, we used p53 conditional mutant mice, and we studied the selective inactivation of this tumor suppressor gene in astrocytes either alone or in combination with Rb.

We report here that RB is not required for the normal maturation of astrocytes and that the independent inactivation of p53 or RB is not sufficient to cause the neoplastic transformation of these cells in vivo. However, we found that expression of the GFAP–Cre transgene is not restricted to mature astrocytes. Precursor cells located in the external granular layer (EGL) of the cerebellum also express Cre recombinase driven from the GFAP promoter. Lack of Rb in these cells in combination with either a somatic or a germ-line p53 inactivation leads to medulloblastomas, highly aggressive embryonal tumors of the cerebellum. No glial tumors have been found in these mice.

The total number of mice analyzed for each genotype and the occurrence of medulloblastomas at different time points is presented.

(N.D) Not determined.

Acknowledgments

We thank Karin van Veen and Frank Matthesius for assisting in the generation and genotyping of the mice, Nell Bosnie and Fina van der Ahe for the daily care of the mice, and Dennis Hoogervorst for the preparation of histological slides. L. Mucke and L. Donehower kindly provided the GFAP promoter construct and the p53 knockout mice, respectively. S.M. is a recipient of a Marie Curie Research Fellowship of the European Community (ERBFMBICT972620).

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC section 1734 solely to indicate this fact.

Acknowledgments

Footnotes

E-MAIL ln.ikn@snrebt; FAX 31 20 5122011.

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