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TPR-MET oncogenic rearrangement: detection by polymerase chain reaction amplification of the transcript and
Abstract:
Activation of the MET protooncogene by a rearrangement involving the fusion of TPR and MET specific gene sequences has been observed in a human osteosarcoma cell line (HOS) treated in vitro with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). No information has been available about the possible occurrence of this rearrangement in human tumors. To facilitate rapid screening of human cell lines and tumor samples for this specific gene rearrangement, we developed a sensitive detection method based on polymerase chain reaction (PCR) amplification of TPR-MET mRNA. cDNA was generated from cellular transcripts by using one of the PCR primers, which was then used as a template for PCR amplification of a 205-base-pair region carrying the breakpoint. An end-labeled internal probe was hybridized in solution to an aliquot of the PCR product for detecting amplification. Cells could be directly screened by the assay without prior isolation of RNA. A 205-base-pair DNA fragment characteristic of the TPR-MET rearrangement was detected in cell lines previously known to contain this altered sequence. The rearrangement was also detected at very low levels in the parental (nontransformed) cell line, HOS TE-85. A preliminary survey of cell lines derived from a variety of human tumors indicates that TPR-MET rearrangement occurred and was expressed at very low frequencies by cells from 7 of 14 tumors of nonhematopoietic origin.
Insights
A new method detects the TPR-MET gene rearrangement, crucial in osteosarcoma, using polymerase chain reaction (PCR). This technique enables rapid screening of human tumors for this specific genetic alteration.
Area of Science:
- Molecular Biology
- Oncology
- Genetics
Background:
- The MET protooncogene can be activated by rearrangements, such as the TPR-MET fusion gene.
- This rearrangement has been identified in human osteosarcoma cell lines but its occurrence in human tumors remains largely uncharacterized.
Purpose of the Study:
- To develop a sensitive and rapid method for detecting the TPR-MET gene rearrangement in human cell lines and tumor samples.
- To screen various human tumor-derived cell lines for the presence of the TPR-MET rearrangement.
Main Methods:
- Development of a polymerase chain reaction (PCR) assay to amplify TPR-MET mRNA.
- Utilized cDNA generated from cellular transcripts for PCR amplification of the breakpoint region.
- Employed solution hybridization with an end-labeled internal probe for sensitive detection of the PCR product.
Main Results:
- The developed PCR assay successfully detected the characteristic 205-base-pair DNA fragment of the TPR-MET rearrangement in known positive cell lines.
- The rearrangement was detected at low levels in the parental HOS TE-85 cell line.
- A preliminary survey revealed TPR-MET rearrangement in 7 out of 14 nonhematopoietic human tumor-derived cell lines.
Conclusions:
- The developed PCR-based method is sensitive and efficient for screening TPR-MET gene rearrangement without prior RNA isolation.
- The TPR-MET rearrangement occurs at low frequencies in a subset of human nonhematopoietic tumors, suggesting its potential role in tumorigenesis.
- This assay facilitates rapid screening of clinical samples for this specific genetic alteration.