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Transforming activity of retroviral genomes encoding Gag-Axl fusion proteins
Q K Zhang1, S Boast, K de los Santos
1Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
Abstract:
Retroviral genomes encoding a portion of the Moloney murine leukemia virus Gag protein fused to portions of the murine axl cDNA were constructed so as to mimic naturally occurring transforming viruses. Virus MA1 retained 5 amino acids of the extracellular domain and the complete transmembrane and intracellular domains of Axl; virus MA2 retained only the intracellular Axl sequences beginning 33 amino acids downstream of the transmembrane region. Although both viruses could transform NIH 3T3 cells, they induced different morphological changes. MA1 transformants became elongated and assumed a cross-hatched pattern, while MA2 transformants were round and very refractile and grew to high density. Gag-Axl and Glyco-Gag-Axl proteins were detected in both types of transformed cells and were predominantly localized to the cytoplasmic compartment. When cell-free v-axl virus supernatants were introduced into wild-type BALB/c neonates, Rag-2-deficient mice, or c-myc transgenic mice, they did not cause tumors in a 3-month period. However, MA2-transformed NIH 3T3 cells, but not MA1 or control cells, could establish sarcomas by subcutaneous or intraperitoneal injection into BALB/c neonates. These results show that the transforming potential of the axl gene can be activated by truncation of the extracellular domain of the receptor and fusion of the remaining sequence to the gag gene.
Insights
Truncating the Axl receptor
Area of Science:
- Molecular Biology
- Oncology
- Virology
Background:
- The Axl receptor tyrosine kinase plays a role in cell proliferation and survival.
- Activating mutations or aberrant expression of Axl can contribute to cancer development.
- Understanding how Axl's structure influences its transforming potential is crucial for cancer research.
Purpose of the Study:
- To investigate the transforming potential of the Axl gene when fused to the Moloney murine leukemia virus Gag protein.
- To determine how different truncations of the Axl extracellular domain affect its oncogenic activity.
- To assess the in vivo tumor-forming capacity of retroviruses encoding truncated Axl.
Main Methods:
- Construction of retroviral vectors encoding Gag-Axl fusion proteins with varying Axl truncations.
- Cell transformation assays using NIH 3T3 fibroblasts.
- Analysis of Gag-Axl protein expression and localization.
- In vivo tumorigenesis studies in neonatal mice.
Main Results:
- Both MA1 (partial extracellular domain) and MA2 (intracellular domain only) Gag-Axl viruses transformed NIH 3T3 cells, inducing distinct morphologies.
- Gag-Axl proteins were detected and localized to the cytoplasm in transformed cells.
- MA2-transformed cells, but not MA1 or control cells, formed sarcomas upon injection into neonatal BALB/c mice.
- Cell-free virus supernatants did not induce tumors in various mouse models.
Conclusions:
- Truncation of the Axl extracellular domain and fusion to Gag can activate its transforming potential.
- The intracellular domain of Axl is sufficient for oncogenic transformation and tumor formation in vivo.
- Specific structural alterations in Axl influence its oncogenic properties and cellular transformation characteristics.