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TSC2 epigenetic defect in primary LAM cells. Evidence of an anchorage-independent survival.

Lesma E, Ancona S, Sirchia SM, Orpianesi E, Grande V, Colapietro P, Chiaramonte E, Di Giulio AM, Gorio A - J. Cell. Mol. Med. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Pharmacology, Dept. of Health Sciences, Università degli Studi di Milano, Milano, Italy.

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Proliferation of lymphangioleiomyomatosis/tuberous sclerosis complex (LAM/TSC) cells in non-adherent status and adherent condition. (A) Exemplificative linear fluorescence histograms of PI of different phases of cell cycle were analysed by flow cytometric analysis in human aortic smooth muscle cells (HASMCs,) non-adherent (NA) and adherent (A) LAM/TSC cells. Abscissa indicates relative intensity of PI fluorescence and ordinate relative number of cells. Data are expressed as mean ± SD of three independent experiments. **P < 0.01 and ***P < 0.001 versus HASMCs; ##P < 0.01 versus A LAM/TSC cells (anova followed by Bonferroni*s test). (B) Representative western blots show proliferating cellular nuclear antigen (PCNA), Cdk4 and cyclin D-1 expression in A and NA LAM/TSC using HASMCs as control. β-actin is the loading control. Relative intensity by densitometric analysis was evaluated relatively to β-actin levels. Error bars represent the SD for four experiments. *P < 0.05 versus HASMCs (anova with Bonferroni*s test). (C) PCNA mRna relative expression was analysed by RT-PCR in A and NA LAM/TSC cells considering HASMCs value as 1 unit. Error bars represent the SD for three experiments. Unpaired Student*s t-test p-values: *P < 0.05 versus HASMCs.
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fig05: Proliferation of lymphangioleiomyomatosis/tuberous sclerosis complex (LAM/TSC) cells in non-adherent status and adherent condition. (A) Exemplificative linear fluorescence histograms of PI of different phases of cell cycle were analysed by flow cytometric analysis in human aortic smooth muscle cells (HASMCs,) non-adherent (NA) and adherent (A) LAM/TSC cells. Abscissa indicates relative intensity of PI fluorescence and ordinate relative number of cells. Data are expressed as mean ± SD of three independent experiments. **P < 0.01 and ***P < 0.001 versus HASMCs; ##P < 0.01 versus A LAM/TSC cells (anova followed by Bonferroni*s test). (B) Representative western blots show proliferating cellular nuclear antigen (PCNA), Cdk4 and cyclin D-1 expression in A and NA LAM/TSC using HASMCs as control. β-actin is the loading control. Relative intensity by densitometric analysis was evaluated relatively to β-actin levels. Error bars represent the SD for four experiments. *P < 0.05 versus HASMCs (anova with Bonferroni*s test). (C) PCNA mRna relative expression was analysed by RT-PCR in A and NA LAM/TSC cells considering HASMCs value as 1 unit. Error bars represent the SD for three experiments. Unpaired Student*s t-test p-values: *P < 0.05 versus HASMCs.

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 [33]. 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 [34]. 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).


TSC2 epigenetic defect in primary LAM cells. Evidence of an anchorage-independent survival.

Lesma E, Ancona S, Sirchia SM, Orpianesi E, Grande V, Colapietro P, Chiaramonte E, Di Giulio AM, Gorio A - J. Cell. Mol. Med. (2014)

Proliferation of lymphangioleiomyomatosis/tuberous sclerosis complex (LAM/TSC) cells in non-adherent status and adherent condition. (A) Exemplificative linear fluorescence histograms of PI of different phases of cell cycle were analysed by flow cytometric analysis in human aortic smooth muscle cells (HASMCs,) non-adherent (NA) and adherent (A) LAM/TSC cells. Abscissa indicates relative intensity of PI fluorescence and ordinate relative number of cells. Data are expressed as mean ± SD of three independent experiments. **P < 0.01 and ***P < 0.001 versus HASMCs; ##P < 0.01 versus A LAM/TSC cells (anova followed by Bonferroni*s test). (B) Representative western blots show proliferating cellular nuclear antigen (PCNA), Cdk4 and cyclin D-1 expression in A and NA LAM/TSC using HASMCs as control. β-actin is the loading control. Relative intensity by densitometric analysis was evaluated relatively to β-actin levels. Error bars represent the SD for four experiments. *P < 0.05 versus HASMCs (anova with Bonferroni*s test). (C) PCNA mRna relative expression was analysed by RT-PCR in A and NA LAM/TSC cells considering HASMCs value as 1 unit. Error bars represent the SD for three experiments. Unpaired Student*s t-test p-values: *P < 0.05 versus HASMCs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig05: Proliferation of lymphangioleiomyomatosis/tuberous sclerosis complex (LAM/TSC) cells in non-adherent status and adherent condition. (A) Exemplificative linear fluorescence histograms of PI of different phases of cell cycle were analysed by flow cytometric analysis in human aortic smooth muscle cells (HASMCs,) non-adherent (NA) and adherent (A) LAM/TSC cells. Abscissa indicates relative intensity of PI fluorescence and ordinate relative number of cells. Data are expressed as mean ± SD of three independent experiments. **P < 0.01 and ***P < 0.001 versus HASMCs; ##P < 0.01 versus A LAM/TSC cells (anova followed by Bonferroni*s test). (B) Representative western blots show proliferating cellular nuclear antigen (PCNA), Cdk4 and cyclin D-1 expression in A and NA LAM/TSC using HASMCs as control. β-actin is the loading control. Relative intensity by densitometric analysis was evaluated relatively to β-actin levels. Error bars represent the SD for four experiments. *P < 0.05 versus HASMCs (anova with Bonferroni*s test). (C) PCNA mRna relative expression was analysed by RT-PCR in A and NA LAM/TSC cells considering HASMCs value as 1 unit. Error bars represent the SD for three experiments. Unpaired Student*s t-test p-values: *P < 0.05 versus HASMCs.
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 [33]. 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 [34]. 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).

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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Pharmacology, Dept. of Health Sciences, Università degli Studi di Milano, Milano, Italy.

Show MeSH
Related in: MedlinePlus