Null mutation of DNA strand break-binding molecule poly(ADP-ribose) polymerase causes medulloblastomas in p53(-/-) mice

Abstract

Medulloblastoma is an invasive embryonal tumor of the cerebellum with predominant neuronal differentiation. Although several genes have been implicated in medulloblasoma formation, such as Patched (Ptc1) and the adenomatous polyposis coli gene (Apc), the majority of these tumors cannot be explained by mutations in these genes. The cellular origin as well as the genetic and molecular changes involved in the genesis and progression of human medulloblastomas remain largely unknown. Here we show that disruption of poly(ADP-ribose) polymerase (PARP-1) causes a high incidence (49%) of aggressive brain tumors in p53 null mice, with typical features of human cerebellar medulloblastomas. At as early as 8 weeks of age, lesions started on the outer surface of the cerebellum from remnant granule cell precursors of the developmental external germinal layer. Progression of these tumors is associated with the re-activation of the neuronal specific transcription factor Math1, dysregulation of Shh/Ptc1 signaling pathway, and chromosomal aberrations, including triradial and quadriradial chromosomes. The present study indicates that the loss of function of DNA double-strand break-sensing and repair molecules is an etiological factor in the evolution of the cerebellar medulloblastomas. These PARP-1/p53 double null mice represent a novel model for the pathogenesis of human medulloblastomas.

Figure 1.

Incidence of brain tumors in PARP-1^−/−p53^−/− mice. A high frequency of brain tumors was only observed in PARP-1/p53 double mutant mice. All brain tumors were identified by macro- and microscopic examinations. The groups of various genotypes were monitored for 24 weeks and the curves show only brain tumors in various genotypes. The incidence of lymphomas and soft tissue sarcomas were not shown.

Figure 2.

Gross and histological examinations of representative cerebellar tumors in PARP-1/p53 double null mice. A: Gross appearance of normal adult mouse brain at 20 weeks of age and a sagittal section (H&E) of a normal cerebellum is shown in insert. B: Gross appearance of a cerebellar tumor (arrow) in a PARP-1^−/−p53^−/− mouse at 15 weeks of age. A sagittal section of this tumor stained by H&E and is shown as an inset. Bars in (A) and (B) represent 0.5 cm. C: H&E staining of an early neoplastic lesion, spreading from the cerebellar surface through the molecular layer. D: Advanced medulloblastoma (H&E), consisting of densely packed cells with hyperchromatic nuclei and brisk mitotic activity (arrow). A neuroblastic rosette is shown as insert. (E–G). Early neoplastic lesions are immunoreactive for NeuN (E) and in advanced tumors, most cells express NeuN (F). G: MAP-2 cytoplasmic immunostaining shows a filamentous pattern through out the tumor (Tu). (H) Infiltration by a cerebellar medulloblastoma (Tu) into the fourth ventricle. mo, molecular layer. gr, granule cell layer. CP, choroid plexus. Original magnification of C, D, E, F, and G is ×20 and H is ×10.

Figure 3.

A: Re-activation of neuron-specific transcription factor Math1 in medulloblastomas. Northern blot analysis of math1 expression in a normal cerebellum (N) at postnatal day 6 (P6), P10, P21, and at 6 weeks (W6) in adult mice, and medulloblastomas at the age of 15 to 22 weeks (T1–T4). The math1 transcript (2.5 kb) was detected in the cerebellum at P6 and P10, but was absent at postnatal day 21 and in adult cerebella (W6 and N). However, high expression levels of math1 mRNA were detected in medulloblastomas (T1–T4). Ethidium staining of 18S and 28S rRNA was used to control loading. Immunohistochemistry of Math1 in the normal cerebellum (B) and medulloblastomas (C–E). B: A normal cerebellum from a PARP-1^−/−p53^−/− mouse (16 weeks old) is devoid of Math1 staining in the molecular (mo) and granule (gr) layers, similar to wild-type animals. C: Tumor cells from an early neoplastic lesion were located in the outer surface of the cerebellar molecular layer (arrows). D: An advanced cerebellar tumor showing a diffused staining for Math1 (see also insert with Math1-positive cells in the granule cell layer). E: Math1-positive cells were also detected in tumor cells infiltrated into the molecular layer at advanced stages. Original magnification of B and E is × 10 and C and D is × 20.

Figure 4.

Dysregulation of the Shh/ptc1 signaling pathway in medulloblastomas derived from PARP-1/p53 double null mice. A: Representative Northern blot analysis for ptc1 and gli1 expression in normal adult cerebellum and medulloblastomas, and a quantitation of expression levels of ptc1 (B) and gli1 (C) is shown. Reduced levels of the ptc1 transcript (8 kb) were detected in all tumor samples when compared to non-tumor cerebellar tissue of adult (N4) and 6-week-old (W6) mice. In contrast, weak expression of gli1 (4.0 kb) was seen in the normal cerebellum (W6 and N4), whereas higher expression levels of gli1 were present in medulloblastomas (T1–T4). The β-actin probe is used to control loading.

Figure 5.

Cytogenetic analysis of medulloblastoma cells derived from PARP-1/p53 double null mice. Metaphase chromosomes were prepared from primary medulloblastomas and stained with the telomeric FISH probe (D, F, H, J, and L) then counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) (B, C, E, G, I, and K). A: Summary of chromosomal aberrations in primary medulloblastoma cells. RL, Robertsonian-like fusions; Dic, dicentric chromosomes. Radial chromosomes include triradial and quadriradial chromosomes. Representative metaphase spread from medulloblastoma cells is shown in (B) and chromosomal abnormalities include Robertsonian-like fusions (C and D), chromosomal break (E and F), fragmentations (G–J), triradial (I and J), and quadriradial (K and L) chromosomes.