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Analysis of the fibroblast transformation potential of GTPase-deficient gip2 oncogenes
S K Gupta1, C Gallego, J M Lowndes
1Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206.
Abstract:
Expression of GTPase-deficient Gi2 alpha subunit (alpha i2) mutant polypeptides and overexpression of the wild-type alpha i2 polypeptide in Rat 1a, Swiss 3T3, and NIH 3T3 fibroblasts altered normal growth regulation and induced a loss of contact inhibition. In Rat 1a cells (but not in NIH 3T3 or Swiss 3T3 cells), expression of the GTPase-deficient alpha i2 mutant polypeptides allowed colony formation in soft agar, which correlated with a loss in anchorage dependence and a decreased serum requirement. The altered growth regulatory properties of Rat 1a cells induced by expression of alpha i2 mutant polypeptides was not significantly inhibited by cotransfection with a dominant negative Ha-ras mutant polypeptide (Asn-17rasH), indicating that the activated Gi2 membrane signal transduction protein is uniquely capable of altering the regulation of Rat 1a cell growth by a predominantly c-ras-independent mechanism. The results show that GTPase-deficient alpha i2 mutant polypeptides have the properties of an oncogene that can induce the phenotypic characteristics of transformation in Rat 1a cells but that only a subset of these changes is observed with NIH 3T3 and Swiss 3T3 cells.
Insights
Overexpression of a mutated Gi2 alpha subunit (alpha i2) protein disrupts normal cell growth regulation and causes loss of contact inhibition in fibroblasts. This oncogenic protein promotes anchorage-independent growth in Rat 1a cells via a ras-independent pathway.
Area of Science:
- Cell Biology
- Molecular Biology
- Oncogenesis
Background:
- Cellular communication and growth are tightly regulated processes.
- Aberrant signaling pathways can lead to uncontrolled cell proliferation and cancer.
- G-protein coupled receptors and their downstream effectors play crucial roles in cell signaling.
Purpose of the Study:
- To investigate the role of the Gi2 alpha subunit (alpha i2) in cell growth regulation.
- To determine if GTPase-deficient alpha i2 mutants possess oncogenic properties.
- To elucidate the signaling pathways involved in alpha i2-mediated cell transformation.
Main Methods:
- Expression of wild-type and GTPase-deficient Gi2 alpha subunit (alpha i2) mutant polypeptides in Rat 1a, Swiss 3T3, and NIH 3T3 fibroblasts.
- Assessing cell growth regulation, contact inhibition, colony formation in soft agar, anchorage dependence, and serum requirement.
- Cotransfection with a dominant-negative Ha-ras mutant polypeptide (Asn-17rasH) to investigate pathway dependency.
Main Results:
- Expression of alpha i2 mutants and wild-type alpha i2 altered normal growth regulation and induced loss of contact inhibition in fibroblasts.
- Rat 1a cells expressing alpha i2 mutants showed colony formation in soft agar, reduced anchorage dependence, and decreased serum requirement.
- These effects in Rat 1a cells were largely independent of Ha-ras signaling.
- NIH 3T3 and Swiss 3T3 cells exhibited a subset of these transformation characteristics.
Conclusions:
- GTPase-deficient Gi2 alpha subunit (alpha i2) mutant polypeptides exhibit oncogenic properties.
- Activated Gi2 signaling can induce phenotypic transformation in Rat 1a cells through a predominantly c-ras-independent mechanism.
- Cellular context influences the extent of transformation induced by alpha i2 mutations.