The Proof

So the link is there, but where’s the proof?

To test their hypothesis that PKB activation could be linked to tobacco-related carcinogenesis, Dennis et Al (West, K. A., et al. 2003) examined:

1. If nicotine and NNK could activate PKB.
2. If PKB activated by nicotine and NNK could activate downstream targets.
3. Which nAchR subunits were involved in activation of PKB.
4. If apoptosis was inhibited in cells treated with nicotine or NNK.
5. If cell proliferation patterns were altered in cells treated with nicotine or NNK.
6. If PKB was active in lung cancers of both mice and humans.

Figure 12. Antibody staining shows phosphorylation of PKB (called Akt here), at both sites S473 and T303, by nicotine or NNK in NHBEs or SAECs. (a) shows nicotine activation in a time-dependent manner (the presence and darkness of the bands show protein presence). (b) shows dose-dependent activation of PKB by nicotine. (c) shows time-dependent activation by NNK, and (d) shows dose-dependent activation by NNK.

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Figure 13. PKB kinase activity and its effects on downstream substrates. (b) shows that with nicotine (nic) or NNK added, the activation of downstream substrates increases. Antibodies for the phosphorylated (active) forms of each protein were used to determine if substrates were activated.

Nicotine and NNK caused phosphorylation of PKB, but to ensure that this resulted in a fully active protein, Dennis et al. tested the activation of downstream targets of PKB. They found that nicotine and NNK both increased the kinase activity of PKB, demonstrating that the normal pathways of PKB (progression of cell cycle, etc) are activated by nicotine or NNK treatment (West, K. A., et al. 2003).

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To test which nicotinic acetylcholine receptor subunits were involved in the activation of PKB by nicotine, Dennis et al.(West, K. A., et al. 2003) first looked at which subunits were expressed in NHBEs and SAECs. SAECs selectively express a₂ and a₄ and NHBEs selectively express a₃ and a₅. Both cell types express a₇-a₁₀, b₂ and b₄ subunits. To see which of these are involved in PKB activation, NHBEs and SAECs were treated with drugs that selectively blocked each type of subunit, and were then treated with nicotine or NNK to see if PKB could be activated with that specific receptor subunit inactivated.

Those receptors that contained a₃ or a₄ subunits were required for nicotinic activation, while a₇ subunits were required for NNK activation of PKB. This suggests that nicotine and NNK activate PKB through separate pathways (West, K. A., et al. 2003).

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To test if nicotine or NNK activation of PKB could inhibit apoptosis or change cell proliferation, NHBEs and SAECs were treated with nicotine and/or NNK and then treated with chemicals known to cause DNA damage or UV radiation (which also damages DNA). They were then tested to see if cell growth would arrest or apoptosis would occur as in normal cells undergoing DNA damage. Nicotinic activation of PKB promoted survival of damaged cells, increasing the chance of cancer formation. In addition, nicotine-treated cells increased proliferation and decreased dependence on growth factors and attachment to the extra-cellular matrix, improving their ability to metastasize (West, K. A., et al. 2003).

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