TSC2 epigenetic defect in primary LAM cells. Evidence of an anchorage-independent survival.
Bottom Line: Moreover, LAM/TSC cells bear characteristics of stemness and secrete high amount of interleukin (IL)-6 and IL-8.Anti-EGF receptor antibodies and rapamycin affect proliferation and viability of non-adherent cells.In conclusion, the understanding of LAM/TSC cell features is important in the assessment of cell invasiveness in LAM and TSC and should provide a useful model to test therapeutic approaches aimed at controlling their migratory ability.
Affiliation: Laboratory of Pharmacology, Dept. of Health Sciences, Università degli Studi di Milano, Milano, Italy.Show MeSH
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Mentions: For reasons of the different extent of S6 phosphorylation in adherent and non-adherent cells, and the S6 role in the process of growth, we evaluated the proliferative status of LAM/TSC cells by flow cytometric analysis. We found that most adherent cells were in high replication condition with a higher DNA replication phase S compared with non-adherent LAM/TSC cells and HASMCs used as control (Fig. 5A). The floating cells were less in G0/G1 phase than adherent cells. The number of apoptotic cells in any group was low. To better analyse the replication of adherent and non-adherent LAM/TSC cells, we evaluated specific cyclin-dependent kinases (Cdks), such as Cdk 4, Cdk2 and Cdk6, which control the transition from G1 to the S phase of the cell cycle, by combining with their appropriate cyclin D . Deregulation of Cdk4 and cyclin D1 is widespread in human cancer. The expression of Cdk4 and cyclin D1 is decreased in non-adherent LAM/TSC cells compared with the adherent cells (Fig. 5B). Moreover, we analysed PCNA, essential for DNA replication and repair of DNA errors, highly expressed during G1 and S-phases that decrease in G2 and M-phases . Proliferating cellular nuclear antigen protein level and mRNA expression were significantly reduced in non-adherent LAM/TSC cells compared with adherent cells (Fig. 5B and C).
Affiliation: Laboratory of Pharmacology, Dept. of Health Sciences, Università degli Studi di Milano, Milano, Italy.