Nesterova Maria, Bossis Ioannis, Wen Feng, Horvath Anelia, Matyakhina Ludmila, Stratakis Constantine A
Section on Endocrinology and Genetics, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Clin Endocrinol Metab. 2008 Feb;93(2):565-71. doi: 10.1210/jc.2007-1902. Epub 2007 Dec 4.
Inactivating mutations of PRKAR1A, the regulatory subunit type 1A (RIalpha) of protein kinase A (PKA), are associated with tumor formation.
Our objective was to evaluate the role of PKA isozymes on proliferation and cell cycle.
A cell line with RIalpha haploinsufficiency due to an inactivating PRKAR1A mutation (IVS2+1 G-->A) was transfected with constructs encoding PKA subunits. Genetics, PKA subunit mRNA and protein expression and proliferation, aneuploidy, and cell cycle status were assessed. To identify factors that mediate PKA-associated cell cycle changes, we studied E2F and cyclins expression in transfected cells and E2F's role by small interfering RNA; we also assessed cAMP levels and baseline and stimulated cAMP signaling in transfected cells.
Introduction of PKA subunits led to changes in proliferation and cell cycle: a decrease in aneuploidy and G(2)/M for the PRKAR1A-transfected cells and an increase in S phase and aneuploidy for cells transfected with PRKAR2B, a known PRKAR1A mutant (RIalphaP), and the PKA catalytic subunit. There were alterations in cAMP levels, PKA subunit expression, cyclins, and E2F factors; E2F1 was shown to possibly mediate PKA effects on cell cycle by small interfering RNA studies. cAMP levels and constitutive and stimulated cAMP signaling were altered in transfected cells.
This is the first immortalized cell line with a naturally occurring PRKAR1A-inactivating mutation that is associated in vivo with tumor formation. PKA isozyme balance is critical for the control of cAMP signaling and related cell cycle and proliferation changes. Finally, E2F1 may be a factor that mediates dysregulated PKA's effects on the cell cycle.
蛋白激酶A（PKA）的调节亚基1A（RIα）即PRKAR1A的失活突变与肿瘤形成有关。
我们的目的是评估PKA同工酶在细胞增殖和细胞周期中的作用。
用编码PKA亚基的构建体转染因PRKAR1A失活突变（IVS2 +1 G→A）导致RIα单倍体不足的细胞系。评估遗传学、PKA亚基mRNA和蛋白表达以及增殖、非整倍体和细胞周期状态。为了确定介导PKA相关细胞周期变化的因素，我们研究了转染细胞中E2F和细胞周期蛋白的表达以及通过小干扰RNA研究E2F的作用；我们还评估了转染细胞中的cAMP水平以及基础和刺激后的cAMP信号传导。
引入PKA亚基导致增殖和细胞周期发生变化：PRKAR1A转染细胞的非整倍体和G(2)/M期减少，而用已知的PRKAR1A突变体（RIαP）即PRKAR2B和PKA催化亚基转染的细胞的S期和非整倍体增加。cAMP水平、PKA亚基表达、细胞周期蛋白和E2F因子存在改变；小干扰RNA研究表明E2F1可能介导PKA对细胞周期的影响。转染细胞中的cAMP水平以及组成型和刺激后的cAMP信号传导发生改变。
这是首个具有自然发生的PRKAR1A失活突变的永生化细胞系，该突变在体内与肿瘤形成有关。PKA同工酶平衡对于控制cAMP信号传导以及相关的细胞周期和增殖变化至关重要。最后，E2F1可能是介导PKA对细胞周期失调作用的一个因素。
