Related Experiment Videos
A heterotrimeric Gi3-protein controls autophagic sequestration in the human colon cancer cell line HT-29
E Ogier-Denis1, A Couvineau, J J Maoret
1INSERM U410, Faculté de Médecine Xavier Bichat, Paris, France.
Abstract:
Human colon cancer HT-29 cells exhibit a differentiation-dependent autophagic-lysosomal pathway that is responsible for the degradation of a pool of newly synthesized N-linked glycoproteins in undifferentiated cells. In the present study, we have investigated the molecular control of this degradative pathway in undifferentiated HT-29 cells. For this purpose, we have modulated the function and expression of the heterotrimeric G-proteins (Gs and Gi) in these cells. After pertussis toxin treatment which ADP-ribosylates heterotrimeric Gi-proteins, we observed an inhibition of autophagic sequestration and the complete restoration of the passage of N-linked glycoproteins through the Golgi complex. In contrast, autophagic sequestration was not reduced by cholera toxin, which acts on heterotrimeric Gs-proteins. Further insights on the nature of the pertussis toxin-sensitive alpha subunit controlling autophagic sequestration were obtained by cDNA transfections of alpha i subunits. Overexpression of the alpha i3 subunit increased autophagic sequestration and degradation in undifferentiated cells, whereas overexpression of the alpha i2 subunit, the only other pertussis toxin-sensitive alpha subunit expressed in HT-29 cells, did not alter the rate of autophagy.
Insights
This study reveals that specific G-proteins control the autophagic-lysosomal pathway in colon cancer cells. Inhibiting Gi-proteins with pertussis toxin halts glycoprotein degradation, impacting cancer cell processes.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Human colon cancer HT-29 cells possess a differentiation-dependent autophagic-lysosomal pathway.
- This pathway degrades newly synthesized N-linked glycoproteins in undifferentiated cells.
Purpose of the Study:
- Investigate the molecular control of the autophagic-lysosomal degradative pathway in undifferentiated HT-29 cells.
- Modulate heterotrimeric G-proteins (Gs and Gi) to understand their role in this pathway.
Main Methods:
- Utilized pertussis toxin to ADP-ribosylate heterotrimeric Gi-proteins.
- Employed cholera toxin to target heterotrimeric Gs-proteins.
- Performed cDNA transfections of alpha i subunits (alpha i2 and alpha i3) to study their specific roles.
Main Results:
- Pertussis toxin treatment inhibited autophagic sequestration and restored N-linked glycoprotein passage through the Golgi complex.
- Cholera toxin did not reduce autophagic sequestration.
- Overexpression of the alpha i3 subunit enhanced autophagic sequestration and degradation, while alpha i2 overexpression had no effect.
Conclusions:
- Heterotrimeric Gi-proteins, particularly the alpha i3 subunit, play a crucial role in controlling autophagic sequestration in undifferentiated HT-29 colon cancer cells.
- This pathway's regulation by Gi-proteins is a potential target for understanding and treating colon cancer progression.