Activation of p21-Dependent G1/G2 Arrest in the Absence of DNA Damage as an Antiapoptotic Response to Metabolic Stress

Abstract

The folate enzyme, FDH (10-formyltetrahydrofolate dehydrogenase, ALDH1L1), a metabolic regulator of proliferation, activates p53-dependent G1 arrest and apoptosis in A549 cells. In the present study, we have demonstrated that FDH-induced apoptosis is abrogated upon siRNA knockdown of the p53 downstream target PUMA. Conversely, siRNA knockdown of p21 eliminated FDH-dependent G1 arrest and resulted in an early apoptosis onset. The acceleration of FDH-dependent apoptosis was even more profound in another cell line, HCT116, in which the p21 gene was silenced through homologous recombination (p21 -/-cells). In contrast to A549 cells, FDH caused G2 instead of G1 arrest in HCT116 p21 +/+ cells; such an arrest was not seen in p21-deficient (HCT116 p21 -/-) cells. In agreement with the cell cycle regulatory function of p21, its strong accumulation in nuclei was seen upon FDH expression. Interestingly, our study did not reveal DNA damage upon FDH elevation in either cell line, as judged by comet assay and the evaluation of histone H2AX phosphorylation. In both A549 and HCT116 cell lines, FDH induced a strong decrease in the intracellular ATP pool (2-fold and 30-fold, respectively), an indication of a decrease in de novo purine biosynthesis as we previously reported. The underlying mechanism for the drop in ATP was the strong decrease in intracellular 10-formyltetrahydrofolate, a substrate in two reactions of the de novo purine pathway. Overall, we have demonstrated that p21 can activate G1 or G2 arrest in the absence of DNA damage as a response to metabolite deprivation. In the case of FDH-related metabolic alterations, this response delays apoptosis but is not sufficient to prevent cell death.

Rho GTPases RhoA and Rac1 Mediate Effects of Dietary Folate on Metastatic Potential of A549 Cancer Cells through the Control of Cofilin Phosphorylation

“…Four reduced folate forms represent the majority of the folate pool in mammalian cells (tetrahydrofolate (THF), 5-methyltetrahy- drofolate (5-CH 3 -THF), 5,10-methylenetetrahydrofolate (5,10-CH 2 -THF), and 10-formyltetrahydrofolate (10-CHO-THF)), whereas in plasma, 5-CH 3 -THF comprises more than 90% of the reduced folate (41). We measured reduced folate pools in blood, lung, and liver using a technique allowing the measurement of major folate forms independent of their polyglutamylation status (30,31). We observed that the total folate in blood, represented mostly by 5-CH 3 -THF, was decreased 2.2-fold in mice kept on folate-free chow compared with mice on a typical folate diet (Fig.…”

“…Lysates were chilled on ice and centrifuged for 5 min at 17,000 ϫ g at 4°C. Folate pools were measured in tissue lysates by the ternary complex assay method as we described previously (30,31). Folate levels were calculated per milligram of protein measured by Bradford assay or per milliliter of whole blood.…”

Folate Stress Induces Apoptosis via p53-dependent de Novo Ceramide Synthesis and Up-regulation of Ceramide Synthase 6

“…This downregulation is achieved through extensive methylation of the ALDH1L1 promoter and apparently serves the purpose of relieving transformed cells from one of the proliferation controlling mechanisms (34). In agreement with this mechanism, reconstitution of Aldh1l1 in Aldh1l1-deficient cancer cells produces strong antiproliferative effects including cell cycle arrest (35,36), inhibition of motility (37), and apoptosis (35). The antiproliferative effect of Aldh1l1, resulting from the decrease in the intracellular purine levels (38) and impaired folate-dependent homocysteine remethylation (39), is mediated by numerous downstream effectors including p53, p21, PUMA, JNK1/2, c-Jun, caspases 3, 8, and 9, protein phosphatase 1/protein phosphatase 2A, and cofilin (36 -38, 40, 41).…”

“…Transfections were performed following the manufacturer's protocol. Silencing of PUMA by siRNA was carried out as we described previously (36). Tet-On A549/Aldh1l1 cells (1.2 ϫ 10 5 ) were transfected with 25 nmol of Stealth RNAi (Invitrogen) using 5-10 l of Lipofectamine 2000.…”

“…Induces Ceramide Accumulation-Our previous studies demonstrated that Aldh1l1 expression in deficient cancer cells induces strong metabolic alterations and apoptosis (35,36,38). Because involvement of proapoptotic ceramide signaling in these effects has not been studied previously, we investigated whether Aldh1l1-induced folate stress leads to ceramide generation.…”