Huatao Huang

APC mutations in sporadic medulloblastomas

The cerebellar medulloblastoma (WHO Grade IV) is a highly malignant, invasive embryonal tumor with preferential manifestation in children. Several molecular alterations appear to be involved, including isochromosome 17q and the p53, PTCH, and beta-catenin gene mutations. In this study, 46 sporadic medulloblastomas were screened for the presence of mutations in genes of the Wnt signaling pathway (APC and beta-catenin). Single-strand conformational polymorphism (SSCP) analysis followed by direct DNA sequencing revealed 3 miscoding APC mutations in 2 (4.3%) medulloblastomas. One case contained a GCA-->GTA mutation at codon 1296 (Ala-->Val), and another case had double point mutations at codons 1472 (GTA-->ATA, Val-->Ile) and 1495 (AGT-->GGT, Ser-->Gly). Miscoding beta-catenin mutations were detected in 4 tumors (8.7%). Three of these were located at codon 33 (TCT -->TTT, Ser-->Phe) and another at codon 37 (TCT-->GCT, Ser-->Ala). Adenomatous polyposis coli (APC) gene and beta-catenin mutations were mutually exclusive and occurred in a total of 6 of 46 cases (13%). Although germline APC mutations are a well established cause of familial colon and brain tumors (Turcot syndrome), this study provides the first evidence that APC mutations are also operative in a subset of sporadic medulloblastomas.

β-Catenin Mutations Are Frequent in Human Hepatocellular Carcinomas Associated with Hepatitis C Virus Infection

Hepatocellular carcinoma (HCC) is one of the most common fatal cancers worldwide. Hepatitis B virus and hepatitis C virus infections, exposure to aflatoxin, and excessive intake of alcohol have been identified as major risk factors. However, the molecular mechanisms underlying their development are still poorly understood. Recently, β-catenin, one of the key components of the Wnt signaling pathway, has been found to be mutated in about 20% of HCCs, suggesting a role of the Wnt pathway in their development. In this study, we examined β-catenin and APC mutations in 22 HCCs associated with HCV infection, using single-strand conformation polymorphism (SSCP) followed by direct DNA sequencing. β-Catenin mutations were found in nine (41%) cases, but no APC mutations were found. β-Catenin immunohistochemistry revealed nuclear accumulation of β-catenin protein in all nine tumors with a β-catenin mutation and two additional tumors without a mutation. These results suggest that activation of the Wnt signaling pathway by β-catenin mutation contributes significantly to the hepatocellular carcinogenesis associated with HCV infection.

Null Mutation of DNA Strand Break-Binding Molecule Poly(ADP-ribose) Polymerase Causes Medulloblastomas in p53−/− Mice

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.

IDENTIFICATION IN HUMAN BRAIN TUMORS OF DNA SEQUENCES SPECIFIC FOR SV40 LARGE T ANTIGEN

Simian virus 40 (SV40) sequences have recently been identified in a variety of human neoplasms, including mesothelioma, osteosarcoma, and brain tumors, but significant discrepancies exist regarding the frequency at which this occurs. The SV40 genome is 70% homologous to JC and BK, two related polyomaviruses that are highly prevalent in humans and which may cause in immune‐compromised patients progressive multifocal leukoencephalopathy (PML) and cystitis, respectively. We have established a specific and sensitive method to identify SV40 sequence in DNA extracted from histological sections, using PCR followed by Southern hybridization to probes specific to the large T region. We found SV40 largeT antigen sequences in all brain tumor types investigated. High frequencies were found in low‐grade astrocytomas, anaplastic astro‐cytomas and secondary glioblastomas derived thereof (13/22, 59%) while somewhat lower frequencies were found in gemistocytic astrocytomas (9/28, 32%) and oligodendrogliomas (3/12, 25%). Primary glioblastomas, giant cell glioblastomas, and gliosarcomas, which clinically develop de novo, contained SV40 sequences in 11–25% of cases. Presence of viral DNA was also observed in pediatric brain tumors, including ependymomas (9/16, 56%), choroid plexus papillomas (6/16, 38%), and medulloblastomas (5/17, 29%). In 8 tumor biopsies with SV40 sequences, the adjacent normal brain tissue was also analyzed but was devoid of viral DNA in all but one case. BK and JC virus sequences were rarely detected, the overall frequencies being 3% and 2%, respectively. It remains to be shown whether the presence of SV40 contributes significantly to malignant transformation or whether certain human neoplasms provide a microenvironment that favors viral replication in humans with latent SV40 infection.

Astrocytomas and choroid plexus tumors in two families with identical p53 germline mutations.

Germline p53 mutations carry an increased risk of development of breast cancer, soft tissue and osteosarcomas, brain tumors, leukemia and adrenocortical carcinomas. Cerebral neoplasms are usually of astrocytic lineage and occur in 40% of affected families. This report presents clinical, neuropathological and molecular genetic data from 2 families in France with an identical p53 germline mutation in codon 248 (CGG->TGG; Arg->Trp) and a clustering of CNS tumors. The youngest patient in each family developed a malignant choroid plexus tumor while several young adults of both kindred succumbed to low-grade astrocytoma, anaplastic astrocytoma or glioblastoma. The only non-neural neoplasm was an adrenocortical carcinoma in a boy aged 4 years who developed an anaplastic choroid plexus papilloma 2 years later. Of 2 previously reported inherited choroid plexus tumors, 1 occurred in a family which also carried a germline mutation in codon 248. It remains to be shown whether this unusual pattern of CNS tumors is due to an organ-specific effect of this particular p53 mutation or whether it reflects the genetic background of the affected families.

Methylation of the p73 gene in gliomas.

Abstract. The p73 gene encodes a protein structurally and functionally homologous to TP53, and maps to chromosomal band 1p36.33, where loss of heterozygosity has been observed in up to 90% of oligodendrogliomas and in 10–25% of diffuse astrocytomas. We assessed the methylation status of the CpG islands in the promoter region of the p73 gene by methylation-specific PCR in 117 glioma biopsies. Methylation was detected in 5 out of 28 (18%) glioblastomas and in 4/26 (15%) anaplastic oligodendrogliomas (WHO grade III) but not in grade II oligodendrogliomas, low-grade diffuse astrocytomas (grade II), and anaplastic astrocytomas (grade III). To assess whether p73 methylation leads to loss of expression, we carried out reverse transcription-PCR and methylation-specific PCR in 10 glioblastoma cell lines. Two lines (U87MG and T98G) showed p73 methylation. U87MG had no unmethylated p73 (complete methylation), and showed loss of expression. T98G had methylated and unmethylated p73 (partial methylation), and retained p73 expression. A third cell line (LN-308) showed loss of p73 expression without p73 methylation. These results suggest that complete p73 methylation is associated with loss of expression, but that additional mechanisms may cause loss of p73 expression. Analysis of a polymorphic site in exon 2 further showed that p73 was mono-allelically expressed in 6 out of 7 primary gliomas with heterozygous GC/TA polymorphism.

Effect of Intragastric Application of N-Methylnitrosourea in p53 Knockout Mice

Nullizygous p53 knockout (p53−/−) mice are highly susceptible to spontaneous tumorigenesis, in particular malignant lymphomas at an early age. Heterozygous p53 knockout (p53+/−) mice develop spontaneous tumors less frequently but may show increased susceptibility to chemical carcinogens. In this study, p53−/−, p53+/−, and p53 wild‐type (p53+/+) mice were treated with N‐methylnitrosourea (MNU) by gastric intubation (5 μg/g body weight) three times per week for 5 wk, starting at 5–6 wk of age. The surviving mice were killed when they were 56–57 wk old. All eight p53−/− mice treated with MNU developed malignant lymphomas with a shorter latent period (mean age  =  16.4±0.5 wk) than their spontaneous tumors (61%, at age 23.3±1.4 wk). In p53+/− mice treated with MNU, malignant lymphomas developed at a higher frequency (eight of 27, 30%) than did spontaneous lymphomas (5%). Development of sarcomas in p53−/− and p53+/− mice was also significantly enhanced by treatment with MNU. All eight thymic lymphomas and three sarcomas in the p53+/− mice showed a loss of the remaining wild‐type p53 allele. These results indicate that intragastric MNU treatment significantly enhanced spontaneous development of malignant lymphomas and sarcomas in both p53−/− and p53+/− mice. In the stomachs of 12 p53+/− mice, that were killed at the end of the experiment, two adenomas, one carcinoma in situ, and four adenocarcinomas were observed. In the stomachs of 31 p53+/+ mice, eight adenomas and one carcinoma in situ were detected. The overall incidence of tumorous changes in the stomachs of p53+/− (seven of 12, 58%) and p53+/+ (nine of 31, 29%) mice were not significantly different (P = 0.090). However, adenocarcinomas invading the submucosa were observed in p53+/− mice (four of 12, 33%) but not in p53+/+ mice (zero of 31; P = 0.004), suggesting a slightly higher susceptibility to gastric carcinogenesis induced by MNU in p53+/− mice. Mol. Carcinog. 28:97–101, 2000.