Activation of protein kinase A (PKA) by 8-Cl-cAMP as a novel approach for antileukaemic therapy

Abstract

Activation of PKA by cAMP agonists, such as 8-Cl-cAMP activation, selectively causes rapid apoptosis in v-abl transformed fibroblasts by inhibiting the Raf-1 kinase. Here we investigated whether 8-Cl-cAMP is useful for the treatment of chronic myelogenous leukaemia (CML), which is hallmarked by the expression of the p210(bcr/abl) oncogene. Autologous bone marrow transplantation is a feasible alternative for patients with no suitable donor, but hampered by the risk of relapse due to the persistence of leukaemia cells in the transplant. To study the effects of 8-Cl-cAMP on primary leukaemic cells, bone marrow cells (BMCs) from eight CML patients (one at diagnosis, three in chronic and four in accelerated phase) were treated. Ex vivo treatment of BMCs obtained in chronic phase of CML with 100 microM 8-Cl-cAMP for 24-48 h led to the selective purging of Philadelphia Chromosome (Ph1 chromosome) without toxic side effects on BMCs from healthy donors as measured by colony-forming unit (CFU) assays. BMCs from patients in accelerated phase showed selective, but incomplete elimination of Ph1 chromosome positive colony forming cells. The mechanism of 8-Cl-cAMP was investigated in FDCP-mix cells transformed by p210(bcr/abl), a cell culture model for CML. The results showed that 8-Cl-cAMP reduced DNA synthesis and viability independent of Raf inhibition as Raf inhibitors had no effect. MEK inhibitors interfered with DNA synthesis, but not with viability. In summary, our results indicate that 8-Cl-cAMP could be useful to purge malignant cells from the bone marrow of patients with CML and certain other forms of leukaemias.

Figure 1

Procedure for PKA activation in bone marrow mononuclear cells (MNC). Marrow MNC were harvested, washed and resuspended in LTC medium. Separate flasks were generated for long-term culture and 8-Cl-cAMP was added as indicated. After 24 h cells were washed, and expanded in long-term cultures as described. CFU assays were setup weekly with the nonadherent cells (NADC). At the end of the long-term culture, adherent cells (ADC) and nonadherent cells (NADC) were harvested and analysed in separate CFU-assays.

Figure 2

Effect of 8-Cl-cAMP, Raf kinase inhibitors and MEK inhibitors on the proliferation of FDCP-mix (A) and p210^bcr-abl transformed FDCP-mix cells (B). Cells were cultured at the permissive temperature in Fisher's medium with 20% horse serum and increasing concentrations of IL-3 (0, 0.01, 0.1, 1, 10 ng ml⁻¹) as shown on the X-axis. 8-Cl-cAMP (100 μM), MEK inhibitors U0126 (10 μM) or PD98059 (50 μM), and Raf kinase inhibitors, Raf kinase inhibitor I (Raf KI, 10 μM) and ZM336372 (100 μM) were added and DNA synthesis was assessed by measuring [3H] thymidine incorporation after 16 h. Experiments were carried out in triplicates.

Figure 3

Effect of MEK inhibitors (A) and Raf kinase inhibitors (B) on the viability of FDCP-mix and p210^bcr-abl transformed FDCP-mix cells. Cells were cultured as in Figure 2. IL-3 was removed and inhibitors were added at the concentrations described in Figure 2. Cell viability was assessed by trypan blue exclusion 72 h after IL-3 removal. Experiments were carried out in triplicates.

Figure 4

Analysis of the effect of 8-Cl-cAMP on the viability of FDCP-mix and p210^bcr-abl transformed FDCP-mix cells. Cells were cultured as in Figure 2. IL-3 was removed and 8-Cl-cAMP (100 μM) was added. Cell viability was assessed 24, 48 and 72 h after IL-3 removal using (A) trypan blue exclusion, (B–D) Annexin and propidium iodide staining as described in the Materials and Methods section. Experiments were carried out in triplicates. The significance of changes was analysed by Student's paired T-test and significant changes are indicated in the figure along with the P-values.