Abstract
Abstract 3623
Many genetic alterations have been detected in various tumors, and their expression levels have also been investigated concerning cancer development or progression. Gene expressions have so far been examined by Northern blotting, reverse-transcriptase-polymerase chain reaction (RT-PCR), microarray and real-time PCR. There are several papers reporting that the expression level of genes, such as EVI1 and BAALC, are associated with clinical outcome in some types of hematologic malignancies, however, we do not know whether the genes detected are structurally normal or abnormal, because the point mutation, fusion gene and alternative splicing could not be detected by conventional method without sequencing. We previously found recurrent genomic copy number variations at 18q21–22 in malignant lymphoma (ML) and multiple myeloma (MM) cell lines by oligonucleotide array analysis. When we analyzed the several cancer-related genes a t 18q21–22, strange expressions of DCC gene were found in some MM cell lines by chance. The expression of DCC gene were analyzed in 21 MM and 11 ML cell lines by RT-PCR using 15 primer pairs covering the full length of DCC, and we found 4 MM cell lines which did not express the transcripts containing exon 1 but those containing other exons. This result made us predict the abnormal form of DCC transcripts fused to unknown sequences instead of exon 1, therefore, we tried to clone them using cDNA bubble PCR method. Some products were obtained and sequence analysis revealed that the 2 kinds of bubble PCR products contained unknown sequences fused to exon 2 of DCC. BLAST search revealed that both unknown sequences were different parts of intron 1 of DCC, which are not connected to exon 2 normally. Sequential analysis of DCC expression by RT-PCR revealed that both normal (containing exon 1) and abnormal (lacking exon 1 and containing intron 1) transcripts were detected in 16/21 (76.2%) MM cell lines, 8/33 (24.2%) MM cases, and 0/11 (0%) ML cases, and that only abnormal transcripts were in 4 (19.0%) MM cell lines, 10 (30.3%) MM cases, and 5 (45.5%) ML cases. Furthermore, another abnormal DCC transcripts which lacked both exon 1 and intron 1 were detected in 12 (36.4%) MM cases, 1 (9.1%) ML cell line, and 4 (36.4%) ML cases. In total, abnormal DCC transcripts were detected in 20 (95%) MM cell lines, 30 (90.9%) MM cases, 1 (9.1%) ML cell line, and 9 (81.8%) ML cases. One (4.8%) MM cell line, 3 (9.1%) MM cases, 10 (90.9%) ML cell lines, and 2 (18.2%) ML cases did not express normal or abnormal transcripts. We next performed Western blotting to detect DCC protein. After immunoprecipitation with the anti-C-terminus of DCC antibody, the DCC protein was detected in 2 MM cell lines with abnormal transcript of DCC, which also expressed normal of DCC clearly. On the other hand, no DCC protein was detected in MM cell lines with abnormal DCC transcripts, which expressed normal DCC transcript weakly. These findings suggest that DCC protein detected by Western blotting is derived from the normal transcript, and that abnormal DCC transcripts result in the defect of their function. DCC, which encodes a receptor for netrin-1, would control apoptosis and be concerned in oncogenesis. Loss of one allele or decreased expression of DCC has been reported in many malignancies including solid tumors, leukemia and ML. It has been considered that DCC inactivation by these mechanisms constitutes a critical event in the development of these tumors. In this study, although partial expression of DCC could be detected by RT-PCR in many MM and ML cases, most of them were abnormal DCC, which would result in lacking DCC function. In addition, our data indicated that the creation of aberrant transcript by possibly abnormal alternative splicing may be another mechanism of tumor suppressor gene inactivation, therefore, analysis of gene expression should be performed in view of the genomic structure of genes.
Disclosures:
No relevant conflicts of interest to declare.
Genetic variation in the first intron and exon of the myostatin gene in several Indonesian cattle populations
