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Failure of wild-type or a mutant form of protein kinase C-alpha to transform fibroblasts
C Borner1, I Filipuzzi, I B Weinstein
1Comprehensive Cancer Center, Columbia University, New York, New York 10032.
Abstract:
A mutant form of the alpha-isoform of protein kinase C (PKC) was recently isolated from an ultraviolet radiation-induced murine fibrosarcoma cell line and reported to transform mouse BALB/c 3T3 fibroblasts on transfection. Four point mutations in the regulatory domain were assumed to be responsible for its oncogenicity and unusual preference for membrane localization. Here, we report that overexpression of the reported mutant PKC alpha complementary DNA in three fibroblast cell lines, including BALB/c 3T3, does not enable these cells to grow in soft agar or nude mice. In addition, this mutant PKC alpha form seems to be indistinguishable from the wild-type PKC alpha with respect to its dependence on cofactors, phorbol ester binding, subcellular distribution and its effects on growth and morphology. These results fail to confirm the previous study and indicate that overexpression of either the wild-type or the reported mutant form of PKC alpha does not transform rodent fibroblasts.
Insights
A previously reported oncogenic mutant protein kinase C alpha (PKCα) did not transform fibroblasts in new experiments. Overexpression of wild-type or mutant PKCα did not induce cancerous growth, challenging prior findings.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- A mutant form of protein kinase C alpha (PKCα) was previously isolated from a murine fibrosarcoma.
- This mutant was reported to cause oncogenic transformation of fibroblasts due to four point mutations.
- Its unusual membrane localization and oncogenicity were attributed to these mutations.
Purpose of the Study:
- To investigate the oncogenic potential of the reported mutant PKCα.
- To determine if overexpression of mutant PKCα transforms rodent fibroblasts.
- To compare the characteristics of mutant PKCα with wild-type PKCα.
Main Methods:
- Overexpression of mutant PKCα complementary DNA in BALB/c 3T3 and other fibroblast cell lines.
- Assessing cellular transformation through soft agar growth assays and nude mouse xenografts.
- Evaluating cofactor dependence, phorbol ester binding, subcellular distribution, and effects on cell growth and morphology.
Main Results:
- Overexpression of the mutant PKCα did not induce soft agar colony formation or tumor growth in nude mice.
- The mutant PKCα exhibited indistinguishable cofactor dependence, phorbol ester binding, and subcellular distribution compared to wild-type PKCα.
- No significant differences in growth or morphology were observed between cells overexpressing wild-type or mutant PKCα.
Conclusions:
- The results fail to confirm the oncogenic transformation potential of the previously reported mutant PKCα.
- Overexpression of either wild-type or the reported mutant PKCα does not transform rodent fibroblasts.
- This study challenges the oncogenic nature attributed to the specific mutant PKCα form.