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Updated: Aug 4, 2026
A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide
Published on: October 18, 2014
Connexin expression and cell coupling fail to reverse the v-src transformed growth characteristics of a Cx43-/- cell
A Chandrasekhar1, M Merritt, S J Huh
1Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.
Abstract:
Gap junctions, composed of connexins, have been shown to suppress transformation in a variety of malignancies and transformed cell types. In addition, transforming factors such as the src oncogene have been shown to directly phosphorylate some connexins (e.g., Cx43) and inhibit coupling. To investigate the role of gap junctions in cell transformsation by v-src, we utilized a clonal cell line derived from Cx43 knockout mice (KoA) that was immortalized, but not transformed. Transfection by v-src induced a marked transformed phenotype characterized by growth in low serum and anchorage-independent conditions. Subsequent transfections by Cx43, Cx32 or vector alone were then tested for their effects on growth. Activity of pp60v-src was confirmed in all transfectants as well as the ability of pp60v-src to phosphorylate Cx43 in several clones. Despite the documented effect of pp60v-src on Cx43 channel closure, modest coupling was still retained in many of the Cx43 and Cx32 transfectants. However, none of the four Cx43 transfected clones showed significant inhibitory effects on proliferation in either anchorage-independent or low serum growth conditions. Of the Cx32 clones, only one in five showed effects on growth in both assays, which was the same ratio observed for the control transfectants. Thus, based on the levels of expression achieved, which were comparable to endogenous levels in established cell lines, neither Cx43 nor Cx32 serve as effective suppressors of the transformed growth phenotype of this v-src expressing cell line.
Insights
Gap junctions, formed by connexins, typically inhibit cell transformation. However, this study found that connexin 43 (Cx43) and connexin 32 (Cx32) did not suppress v-src-induced transformation in knockout mice cells.
Area of Science:
- Cell Biology
- Molecular Oncology
- Biochemistry
Background:
- Gap junctions, comprised of connexins, are known to suppress cellular transformation.
- Oncogenic factors like v-src can phosphorylate connexins, inhibiting their function.
Purpose of the Study:
- To investigate the role of gap junctions in v-src-mediated cell transformation.
- To determine if connexin 43 (Cx43) or connexin 32 (Cx32) can suppress the transformed phenotype induced by v-src.
Main Methods:
- Utilized a Cx43 knockout immortalized cell line (KoA).
- Induced transformation using v-src transfection.
- Re-introduced Cx43, Cx32, or vector control via transfection.
- Assessed proliferation under low serum and anchorage-independent conditions.
Main Results:
- v-src expression induced a transformed phenotype in KoA cells.
- pp60v-src phosphorylated Cx43, but some coupling persisted.
- Neither Cx43 nor Cx32 re-expression significantly inhibited proliferation in most clones.
- One of five Cx32 clones showed minor growth inhibition, similar to controls.
Conclusions:
- Cx43 and Cx32, at levels comparable to endogenous expression, did not effectively suppress v-src-induced transformation.
- The study suggests limitations in the tumor-suppressive role of these specific connexins in this context.
