AbstractIn this study we investigated the relevance of the oncogenic protein NPM-ALK in regulating cellular pH (pHi) through the modulation of Na+/H+ exchanger 1 (NHE1)-activity and the consequences of pHi pharmacological manipulation in cells expressing NPM-ALK.
We have recently reviewed the biological relevance of ALK and related fusion proteins in oncogenesis.1 A recent study has pointed out that the Na/H exchanger (NHE)1-dependent alkaline pHi is an important event in the malignant-transformation associated phenotype.2 NHE1 is a commonly expressed plasma membrane protein that regulates pHi through an exchange of extracellular Na for intracellular H We have recently shown that troglitazone (TRO) induces prolonged cellular acidosis by inhibiting NHE1 activity and glutamine (GLU) oxidation via the transamination pathway forming alanine (ALA), shifting glutamate (GLU) into ammoniogenesis via oxidative deamination by alanine aminotransferase (ALT), and glutamate dehydrogenase (GDH).4,5 Hence tumor cells express negligible or low levels of GDH. Their dependency upon GLU for rapid growth and proliferation is entirely dependent upon the transamination-pathway.5 To test whether TRO would be effective in arresting proliferation of ALK/ALCL (anaplastic large cell lymphoma)-derived L82 cells by inducing cellular acidosis and inhibiting GLU oxidation, cells were incubated for 18 h in the presence of 40 μM TRO or vehicle (DMSO), and GLU metabolism and DNA synthesis were determined as previously described.4,5 TRO-treated cells utilized far less GLU (883±789 vs 3190±270 nmoL/mg protein, p<0.05) as a result of marked inhibition (p<0.01) of the pH-dependent transamination reaction (ALA production decreasing from 1293±278 to 218±54 nmol/mg protein). On the other hand, ammonium production equalled GLU utilization consistent with high glutaminase activity of tumor cells, but it failed to increase with TRO (2038±537 vs 3340±637 nmoL/mg for control) reflecting the low activity of GDH in tumor cells. We have previously shown that TRO-induced cellular acidosis (peroxisome proliferator-activated receptor γ(PPARγ)-independent) is associated with reduced DNA synthesis in breast cancer-derived cells.4 As shown in Figure 1A, left side, H-thymidine incorporation was decreased in TRO-L82 cells [3252±482 vs L82-control cells 5745±353 (p<0.005)]. We also measured the caspase 3/7 activity since we have recently shown that TRO-induced cellular acidosis is associated with increased mitochondrial permeability.6 L82 cells incubated with 40 μM TRO showed a significant increase of caspase activity measured with CellProbeHT Caspase 3/7 Whole Cell Assay, Beckman Coulter, as compared to control at 18 hours (Figure 1A, right side, TRO-L82 cells 6306±134 vs L82-control cells 2063±70, p<0.001). Given the relevance of the consequences of pharmacological manipulation of pHi in ALK cells L82, we assessed whether NPM-ALK played any role in regulating the pHi. For this purpose, we stably transfected NIH3T3 fibroblasts with a pSRoMSVtkneo vector expressing NPM-ALK (a gift from Dr. S. Morris, St. Jude Children’s Research Hospital, Memphis, TN, USA) as previously described.7 A striking change in cell morphology was observed in ALK-NIH3T3 cells that transformed from a single monolayer (controls) to elongated cells with long pseudopodia (Figure 1B, inset). ALK-NIH3T3 cells proliferated at a faster rate than parental cells, with a 1.5 fold increase in cell number over a period of 72 h (Figure 1B, p<0.02). Ratio of ammonia (NH4) to alanine (ALA) was used as a surrogate marker of pHi as previously described.5 Analysis of the conditioned media from control and ALK-NIH3T3 cells showed a 4-fold decrease in the ratio of ammonia to ALA (NH4/ALA 16.3 control vs 4.0 for transfectants) consistent with an alkaline pHi and increased proliferation as previously shown.2,5 Subsequently, NHE1 activity was determined as the rate of pHi recovery after an ammonia-chloride (NH4Cl) load and assayed using (2,7)-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) as previously described.4,5 As shown in Figure 1C, the untransformed NIH3T3 cells (left side) have modest NHE1 activity (ΔpH/Δt) compared to the ALK-NIH3T3 cells (right side) in response to the NH4Cl load as represented by the difference in the recovery phase. Baseline pHi in ALK-NIH3T3 cells of 7.45 confirmed that as expected these cells were more alkaline than the control at 7.34 (Figure 1C). Our data support a pH-mediated oncogenic activity of NPM-ALK protein through the regulation of the NHE1 activity that favors alkaline conditions by increasing the extrusion of cellular H. The increased NPM-ALK mediated alkaline conditions favor growth and proliferation that are reversed by TRO-induced cellular acidosis in ALK-ALCL-derived L82 cells. Our results support the study of drugs that induce cellular acidosis either by interfering with acid extrusion or acid production or, ultimately, by a combination of both in tumors induced by oncogenic alkaline fusion proteins.8
- Funding: Feist-Weiller Cancer Center and Louisiana Gene Therapy Consortium Inc. Funds (F. Turturro).
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