A mutation in the c-myc-IRES leads to enhanced internal ribosome entry in multiple myeloma: a novel mechanism of oncogene de-regulation

The 5' untranslated region of the proto-oncogene c-myc contains an internal ribosome entry segment (IRES) (Nanbru et al., 1997; Stoneley et al., 1998) and thus c-myc protein synthesis can be initiated by a cap-independent as well as a cap-dependent mechanism (Stoneley et al., 2000). In cell lines derived from patients with multiple myeloma (MM) there is aberrant translational regulation of c-myc and this correlates with a C-T mutation in the c-myc-IRES (Paulin et al., 1996). RNA derived from the mutant IRES displays enhanced binding of protein factors (Paulin et al., 1998). Here we show that the same mutation is present in 42% of bone marrow samples obtained from patients with MM, but was not present in any of 21 controls demonstrating a strong correlation between this mutation and the disease. In a tissue culture based assay, the mutant version of the c-myc-IRES was more active in all cell types tested, but showed the greatest activity in a cell line derived from a patient with MM. Our data demonstrate that a single mutation in the c-myc-IRES is sufficient to cause enhanced initiation of translation via internal ribosome entry and represents a novel mechanism of oncogenesis.     

Patterns of methylation of the c-myc gene in human colorectal cancer progression.

Over-expression and abnormal intracellular location of the product of the oncogene c-myc in colonic dysplasia and neoplasia may be related to alterations in epigenetic mechanisms controlling the functioning of this gene. We have investigated the methylation patterns of the c-myc oncogene in human colorectal tissue representing various stages of dysplasia and neoplasia, including metastasis to liver, omentum and lymph node. Comparison of normal and neoplastic tissues from the same patient showed a decrease in methylation in a specific CCGG site in the third exon of c-myc through the progression from normal via dysplastic to neoplastic and metastatic tissue. Quantitative analysis revealed that in colonic adenocarcinomas an average of 66.1% and in metastatic deposits 83.1% of the myc gene DNA was hypomethylated at this site, as compared to a value of 9.2% in normal colonic mucosa. Adenomatous polyps showed an average value of 50.5% and hyperplastic polyps, 24.8%. The results suggest that partial hypomethylation of the c-myc gene third exon is associated with cell proliferation, and that deregulation of proliferation may be linked to the high levels of hypomethylation, presumably involving both copies of the gene in some cells, which occur at a relatively early stage in neoplastic progression.     

Interaction of the v-and c-Myb proteins with regulatory sequences of the human c-myc gene.

Eight c-Myb-binding sites have been identified in the regulatory region of the human c-myc gene using gel retardation and DNAase I footprint assays with purified bacterially expressed full-length and carboxy-terminally truncated c-Myb proteins. These binding sites exhibit different affinities whereby strong binding correlates better with conservation of the palindromic sequences, AACXGTT or AACGTT, than the previously described consensus sequence. Flanking AT-rich sequences further increase the binding affinity. The c-Myb-binding sites are arranged in pairs consisting of one high- and one low-affinity binding site. Binding of the Myb proteins to these sites is non-cooperative. The v-Myb protein protects two nucleotides fewer than the c-Myb protein. Co-transfection of reporter CAT genes, containing upstream human c-myc sequences including exon 1, with c-Myb-expressing constructs resulted in positive transactivation, which was eightfold with full-length Myb and 14-fold with the truncated Myb. This result suggests that the Myb protein could participate in regulation of human c-myc gene expression.     

芳香化酶mRNA在肺癌组织中表达的初步研究

目的 探讨芳香化酶Ｐ４５０ ｍＲＮＡ在肺癌组织中的表达及其定位分布情况。方法 以芳香化酶ｃＲＮＡ作探针,采用原位杂交检测２３例肺癌及其相对应的癌旁组织和７例肺良性疾病及其相应正常肺组织中芳香化酶ｍＲＮＡ的表达。结果 １１例肺鳞癌、６例腺癌和２例腺鳞癌芳香化酶ｍＲＮＡ表达均为阳性,而３例小细胞肺癌和１例肺泡细胞癌均为弱阳性。阳性信号主要位于癌细胞的胞浆和胸核内,肿瘤的间质细胞和血管内皮细胞也有少量表     

Investigation of human brain tumors for the presence of polyomavirus genome sequences by two independent laboratories

JC virus (JCV), BK virus (BKV) and simian virus 40 (SV40) may be associated with human brain tumors. These polyomaviruses have been shown to induce brain tumors in experimentally infected animals. Several studies have found polyomavirus genomic sequences in human brain tumor tissues by using polymerase chain reaction (PCR), while others have not. Inconsistencies in previous findings may be due in part to small sample sizes and differences in underlying patient populations, laboratory techniques and quality control measures. To assess the role of polyomaviruses in human brain tumors and address inconsistencies of previous reports, we investigated the prevalence of viral sequences in a series of 225 brain tumor tissue specimens in 2 independent laboratories. PCR followed by Southern hybridization was performed at the National Institute of Neurological Disorders and Stroke (NINDS). Real-time quantitative PCR was performed on the same tissues at Johns Hopkins University (JHU). Only those tumors with amplifiable DNA were tested further for polyomavirus sequences. Positive and negative control tissues were included, and all specimens were masked. Amplifiable DNA was detected in 225/225 (100%) tumors at NINDS, 9 (4%) of which contained polyomavirus sequences (3 JCV-positive, 3 BKV-positive and 3 SV40-positive). The JHU laboratory amplified DNA from 165/225 (73%) tumors, of which 1 tumor tested positive (for SV40). No tumors tested positive in both laboratories. Results for masked quality control tissues were concordant between laboratories. Nucleotide sequences for JCV, BKV and SV40 are rarely present in a large series of adult and pediatric brain tumors.     

Increased expression of human ribosomal phosphoprotein P0 messenger RNA in hepatocellular carcinoma and colon carcinoma.

To search for differentially expressed gene products in selected cancers of endodermal origin, cDNA libraries derived from mRNA in human hepatocellular carcinoma and adjacent grossly normal tissue were generated. From these parent libraries, subtracted cDNA libraries of tumor minus normal and normal minus tumor tissues were constructed. After screening these subtracted libraries by +/- hybridization, a cDNA clone that is overexpressed in hepatocellular carcinoma and encodes the human acidic ribosomal phosphoprotein P0 (P0) was identified. We then evaluated the expression of this phosphoprotein P0 in human colon carcinoma samples. Surgical specimens of primary tumors and liver metastases were examined by Northern hybridization of total RNA with one of 2 32P-labeled P0 probes. The mRNA level of the P0 was greater in primary colon carcinoma than in paired adjacent normal colonic epithelium in 36 of 38 cases; the mean tumor/normal ratio was 2.7 (range, up to 13). The tumor/normal ratio, when plotted against the Dukes' stage of disease, gave evidence for increasing P0 expression with increasing stage of colon carcinoma (P = 0.02). In all 8 cases of paired colon carcinoma metastatic to liver and 2 cases of paired primary hepatocellular carcinoma, the P0 mRNA level was greater in tumor than in adjacent normal liver tissue. The mean tumor/normal ratio was 4.0 (range, up to 11) for the colon cancers metastatic to liver and 4.2 for the primary hepatocellular carcinoma samples. These findings support a common increased expression of selected gene products in different tumors of endodermal origin and suggest that increased P0 expression, in line with certain other ribosomal proteins, may be associated with human colorectal cancer progression and biological aggressiveness.     

N-ras oncogene expression changes the growth characteristics of human melanoma in two independent SCID-hu mouse models

Fifteen percent of all human melanomas carry mutations in ras genes, the majority of which are located in codon 61 of the N-ras gene. However, the biological significance of these mutations is as yet unknown. In this study, we investigated the influence of N-ras oncogene products mutated in codon 61 on the growth characteristics of human melanoma in vivo by establishing 2 SCID-hu mouse xenotransplantation models. Tumors grown in SCID mice injected with human melanoma carrying activated N-ras genes were significantly larger (p < 0.004) than tumors grown in animals injected with the appropriate control transfectants. Additionally, tumors with N-ras point mutations clearly showed a more pleomorphic phenotype than the control groups. Our results, obtained in 2 independent SCID-hu xenotransplantation models, suggest that mutated N-ras oncogene expression may be an important factor influencing growth characteristics of human melanoma without altering metastatic potential. These novel in vivo model systems provide a tool for further study of the biology of mutated ras in melanoma and should also prove useful for testing new and improved treatment strategies for human melanoma carrying mutated ras genes. © 1996 Wiley-Liss, Inc.