Overexpresson of RII_β, Regulatory Subunit of Protein Kinase A in Human Colon Carcinoma Cell Induces Growth Arrest and Phenotypic Changes that are Abolished by Site‐Directed Mutation of RII_β

Abstract

-EJB 95 l670/1 I S 1 74T human colon carcinoma cells that contain approximately equal amounts of CAMP-dependent protein kinase (PKA) isozymes, PKA-I and PKA-11, were infecled with retroviral vectors coding for regulatory (R) and catalytic (C) subunits of human PKA. In cells overexpressing RIIcr, Rllij, and RTT, P (a RII,, mutant at the autophosphorylation site), PKA-JT levels increased whilc PKA-I levels decreased. PKA-I was almost completely eliminated in cells overexpressing RII,{ or RIT,,-P. In contrast, overexpression of either RI,, or C, had little or no effect on PKA isozyme levels. Although all infectants expressed high levels of PKA subunit inRNAs in accordance with gene introduction, the R subunit protein expression was reflected in PKA isozyme levels rather than in subunit mRNA levels. Only RII, infectants demonstrated marked growth inhibition in monolayer C U~~L I I .~, reduced thymidine incorporiition into DNA, and inability to grow in semisolid medium or in serum-free medium. Conversely, it11 other infectants displayed growth propcrties similar to uninfected parental cells. The growth-retardation properties of RIT, infectants were reflected i n their altered phenotypic appearances. Our findings that the mutant RIl,rP could not mimic the growth-inhibitory effect of RII, suggest thc functional importance of the autophosphurylation site in RII,,. Our results suggest a role for KIT,, in the suppression of neoplastic cell growth, and thus abnormal expression of R subunit isoforms of PKA may bc involved in neoplastic transformation.

Dual Anticancer Activity of 8-Cl-cAMP: Inhibition of Cell Proliferation and Induction of Apoptotic Cell Death

“…4B), as reported in other cancer cell lines (7)(8)(9). However, RII␤-overexpressing cells did not show any spontaneous apoptosis, and underwent apoptosis with the same degree to the parental cells upon treatment with 8-Cl-cAMP (Fig.…”

“…A group of site-selective cAMP analogues inhibited cell growth in a variety of cancer cells and induced differentiation in leukemic cells (5,6). In addition, introduction of RII␤ subunit of PKA, which resulted in the increase of type II PKA and the downregulation of type I PKA, suppressed growth and transformed phenotype of transformed or cancer cells (7)(8)(9).…”

Protein kinase A-Iα subunit-directed antisense inhibition of ovarian cancer cell growth: crosstalk with tyrosine kinase signaling pathway

“…The total RNAs isolated from untreated control (C) and antisense (AS) or control mismatched oligonucleotide (MO)-treated (for 5 days at 150 nM) cells were subjected to Northern blotting analysis using 32 P-labeled cDNA probes as indicated. Total cellular RNA preparation, Northern blot analysis and hybridization of RNA with 32 P-labeled DNA probes were as described earlier (Nesterova et al, 1996). DNA was labeled with a-32 P-dCTP according to a standard protocol for nick translation reactions using an Amersham nick translation kit.…”

“…I, PKA-I; II, PKA II. The PKA-I and PKA-II activities were calculated by the area estimation of the peak I and II (i.e., activity in (+) cAMP minus activity in (7) cAMP Protein kinase A antisense inhibition of ovarian cancer cell growth O È Alper et al shown previously in other cells (Figure 3) (Nesterova et al, 1996). The antisense treatment (150 nM, for 5 days) selectively eliminated PKA-I, the RIa-containing holoenzyme without changing PKA-II activity ( Figure 3).…”

“…The control cells contained PKA-II with its peak eluted at 230 mM NaCl, whereas the antisense-treated cells contained the PKA-II with its peak eluted at 280 mM NaCl prominently (Figure 3). The PKA elution pro®le observed at a wide salt concentration range (200 ± 300 mM NaCl) has been associated with the presence of multiple kinase species including either RII 2 C trimer or RII 2 C 2 tetramer with nonsaturating amounts of cAMP bound and cAMP unbound form (Cobb et al, 1987), the phosphorylated versus unphosphorylated (Nesterova et al, 1996;Budillon et al, 1995) and the RIIa versus RIIb-containing PKA-II (Otten et al, 1991;Nesterova et al, 1996). The RIIa-containing PKA-II elutes at a higher salt concentration than the RIIb-containing PKA-II, and PKA-II containing the mutant RIIb lacking the autophosphorylation site elutes at a lower salt concentration than the wild-type RIIb-containing PKA-II (Budillon et al, 1995).…”