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Published on: March 5, 2013
Functional identification and molecular cloning of a rat gene mediating growth inhibition and programmed cell death
I Schwarte-Waldhoff1, W Martin, K Willecke
1Department of Pathology, University of Zürich, Switzerland.
Abstract:
We wished to identify DNA sequences conferring suppression of proliferation and transformed phenotypes. Thus, we have transfected DNA from normal rat cells, covalently linked to neo DNA coding for neomycin resistance into a tumorigenic, HRAS transformed rat cell line. Phenotypic revertants were selected after the first cycle of transfection by enrichment procedures that served to eliminate transformed cells. The revertant clones continued to express the HRAS oncogene, but exhibited a lower tumorigenicity, loss of anchorage-independent proliferation, flat morphology, and retardation of growth in monolayer culture. The reverted phenotype could be transferred in a second cycle of transfection into the HRAS transformed rat cells. Neo DNA ligated to genomic donor DNA was used as a tagging sequence to molecularly clone the transferred DNA sequence in a recombinant phage. Fragments of the cloned DNA detect a 2.5 kb transcript in parental cells and revertants. Thus, the recombinant phage harbors a putative growth inhibitory gene, designated trg, that is expressed at a higher level in rat embryo fibroblasts and in the REF52 cell line. Introduction of recombinant phage DNA into established 208F and Rat-2 cells and into HRAS-, v-fgr-, v-fms- and v-raf-transformed rat cell lines resulted in inhibition of growth and induction of programmed cell death.
Insights
Researchers identified a novel gene, designated trg, that suppresses tumor cell growth and proliferation. This gene, when transferred into cancer cells, reversed their transformed phenotype, inhibiting growth and inducing cell death, offering potential therapeutic strategies.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- Tumorigenic cell lines often exhibit uncontrolled proliferation and altered morphology.
- Identifying genes that regulate cell growth is crucial for understanding cancer development.
Purpose of the Study:
- To identify DNA sequences that suppress proliferation and transformed phenotypes in cancer cells.
- To isolate and characterize a gene responsible for reversing oncogenic transformation.
Main Methods:
- Transfection of normal rat cell DNA into HRAS-transformed rat cells.
- Selection of phenotypic revertants using enrichment procedures.
- Molecular cloning of transferred DNA using a neomycin resistance gene as a tag.
- Analysis of gene expression via transcript detection.
Main Results:
- Revertant clones showed reduced tumorigenicity, loss of anchorage-independent growth, and normalized morphology.
- The reverted phenotype was transferable to other transformed cells.
- A putative growth inhibitory gene, trg, was cloned and found to be expressed at higher levels in normal cells.
- Introduction of trg into various transformed rat cell lines inhibited growth and induced apoptosis.
Conclusions:
- A novel gene, trg, confers a non-transformed phenotype and suppresses proliferation in cancer cells.
- Trg acts as a tumor suppressor by inhibiting growth and inducing programmed cell death.
- The findings suggest trg as a potential therapeutic target for cancer treatment.
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