An immortalized human cell line bearing a PRKAR1A-inactivating mutation: effects of overexpression of the wild-type Allele and other protein kinase A subunits

Abstract

Context: Inactivating mutations of PRKAR1A, the regulatory subunit type 1A (RIalpha) of protein kinase A (PKA), are associated with tumor formation.

Objective: Our objective was to evaluate the role of PKA isozymes on proliferation and cell cycle.

Methods: 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.

Results: 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.

Conclusion: 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.

Figure 1

Characterization of PKA subunit gene expression in parental and transfected cells. A, Expression of PKA subunit proteins. Cell extracts were prepared as described earlier and 10 μg protein subjected to Western blot analysis (8). B, Cell proliferation in monolayer culture. Cells were plated at 2 × 10³ cells in 60-mm culture dishes, harvested at the indicated times by trypsinization, and counted using a Z1 Coulter Counter (Beckman Coulter). Results are expressed as the mean cell number per milliliter ± sd.

Figure 2

Differential expression of PKA subunits affected expression of E2F genes. A, Western blot analysis of parental and RIα- and RIIβ-transfected cells. Cell lysates were prepared, subjected to SDS-PAGE, and then transferred to nitrocellulose membranes and immunoblotted. B, Differential expression of PKA subunits affected the levels of E2F1 and E2F4. Cell extract was prepared from parental and transfected cells and analyzed by Western blotting. C, Down-regulation of expression of E2Fs affected the growth of parental cells. Cells were transfected with E2F siRNA, and after 24 h, they were harvested and counted, and their extract was subjected to Western blot analysis with E2F-specific antibodies. Results are expressed as the mean cell number per milliliter ± sd. *, Statistical significance.

Figure 3

Characterization of biochemical changes in parental and transfected cells. A, Levels of cAMP in parental and transfected cells; B, expression of somatostatin promoter-CAT gene. Cells were transfected with the Δ-77-CAT-plasmid. After 48 h, cells were treated with 10 μm forskolin for 4 h and harvested. Cell extract was prepared and assayed for CAT gene expression. Results are expressed as the mean ± sd. *, Statistical significance; NS, nonsignificant.