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Oncogenes in human tumor cell lines: molecular cloning of a transforming gene from human bladder carcinoma cells
Abstract:
The presence of dominant transforming genes in human tumor cell lines has been investigated. High molecular weight DNAs isolated from cell lines established from carcinomas and sarcomas of various organs as well as from a glioblastoma and two melanomas were utilized to transfect NIH/3T3 mouse fibroblasts. The DNAs of T24 and A2182, two cell lines derived from a bladder and a lung carcinoma, respectively, and of HT-1080, a cell line established from a fibrosarcoma, were able to transform recipient NIH/3T3 cells. First-cycle transformants exhibited anchorage-independent growth and were tumorigenic in athymic and immunocompetent mice. Moreover, they contained human DNA sequences and were able to transmit their malignant phenotype in additional cycles of transfection. Southern blot analysis of T24-derived transformants showed that a single fragment of human DNA specifically cosegregated with the malignant phenotype, suggesting that it contained the T24 oncogene. Therefore, these human sequences were molecularly cloned with lambda Charon 9A as the cloning vector. The resulting recombinant DNA molecule, designated lambda T24-15A, was shown to contain a 15-kilobase-pair EcoRI insert of human cellular DNA. lambda T24-15A DNA (either intact or EcoRI digested) transformed NIH/3T3 fibroblasts with a specific activity of 20,000 focus-forming units per pmol of cloned DNA. Our results indicate that we have molecularly cloned a biologically active oncogene present in T24 human bladder carcinoma cells.
Insights
Researchers identified a dominant transforming gene, the T24 oncogene, from human bladder carcinoma cells. This oncogene was molecularly cloned and shown to be biologically active, causing malignant transformation in NIH/3T3 mouse fibroblasts.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Human tumor cell lines contain dominant transforming genes.
- These genes can induce malignant transformation in recipient cells.
Purpose of the Study:
- To investigate the presence of dominant transforming genes in human tumor cell lines.
- To molecularly clone and characterize a transforming gene from human bladder carcinoma cells.
Main Methods:
- Transfection of NIH/3T3 mouse fibroblasts with high molecular weight DNA from human tumor cell lines.
- Analysis of transformants for anchorage-independent growth and tumorigenicity.
- Southern blot analysis to identify human DNA sequences cosegregating with the malignant phenotype.
- Molecular cloning of human DNA sequences using lambda Charon 9A vector.
Main Results:
- DNAs from T24 (bladder), A2182 (lung), and HT-1080 (fibrosarcoma) cell lines transformed NIH/3T3 cells.
- First-cycle transformants exhibited anchorage-independent growth and tumorigenicity.
- A single human DNA fragment from T24 cells cosegregated with the malignant phenotype.
- Molecular cloning yielded lambda T24-15A, containing a 15-kilobase-pair human DNA insert, which efficiently transformed NIH/3T3 cells.
Conclusions:
- A biologically active oncogene, the T24 oncogene, has been molecularly cloned from human bladder carcinoma cells.
- The cloned oncogene is capable of inducing malignant transformation in NIH/3T3 fibroblasts.
- This study provides a molecular basis for understanding oncogenesis in human bladder cancer.