Vitamin B6 Deficiency Promotes Loss of Heterozygosity (LOH) at the Drosophila warts (wts) Locus

Abstract

The active form of vitamin B6, pyridoxal 5'-phosphate (PLP), is a cofactor for more than 200 enzymes involved in many metabolic pathways. Moreover, PLP has antioxidant properties and quenches the reactive oxygen species (ROS). Accordingly, PLP deficiency causes chromosome aberrations in Drosophila, yeast, and human cells. In this work, we investigated whether PLP depletion can also cause loss of heterozygosity (LOH) of the tumor suppressor warts (wts) in Drosophila. LOH is usually initiated by DNA breakage in heterozygous cells for a tumor suppressor mutation and can contribute to oncogenesis inducing the loss of the wild-type allele. LOH at the wts locus results in epithelial wts homozygous tumors easily detectable on adult fly cuticle. Here, we found that PLP depletion, induced by two PLP inhibitors, promotes LOH of wts locus producing significant frequencies of wts tumors (~7% vs. 2.3%). In addition, we identified the mitotic recombination as a possible mechanism through which PLP deficiency induces LOH. Moreover, LOH of wts locus, induced by PLP inhibitors, was rescued by PLP supplementation. These data further confirm the role of PLP in genome integrity maintenance and indicate that vitamin B6 deficiency may impact on cancer also by promoting LOH.

Keywords: Drosophila; loss of heterozygosity (LOH); mitotic recombination; pyridoxal 5′ phosphate (PLP); vitamin B6; warts.

Figure 1

PLP inhibitors induce wts tumors. (A) Examples of wts tumors in wts/+ flies treated with different concentrations of PLP inhibitors (arrowed). (B) Quantification of the results. Each column represents the mean value ± SEM. For each condition, 300–700 flies from three different experiments were examined. (C) Percentage of flies reaching the adult stage. Each column represents the mean value ± SEM. For each condition, about 500 flies from three different experiments were examined. s.m. = standard medium; 4DP = 4-deoxypyiridoxine; GT = ginkgotoxin; ns = non-significant Chi-square test. **, *** significant Chi-square test with p < 0.01 and <0.001, respectively.

Figure 2

PLP supplementation rescues wts tumors. Each column represents the mean value ± SEM. For each condition, at least 500 flies were examined in three different experiments. *, *** significant Chi-square test with p < 0.05 and <0.001, respectively. ns = non-significant Chi-square test; s.m. = standard medium; PLP = pyridoxal 5′-phosphate; 4DP = 4-deoxypyridoxine; GT = ginkgotoxin.

Figure 3

Mitotic recombination as a possible mechanism of LOH. The TM3 balancer chromosome prevents LOH. Each column represents the mean value ± SEM. For each condition, more than 500 flies were examined in three different experiments. *** significant Chi-square test with p < 0.001; ns = non-significant Chi-square test. s.m. = standard medium; 4DP = 4 deoxypyridoxine.

Figure 4

Effect of the combined treatment HSD + 4DP on LOH at the wts locus. (A) Quantification of tumors in wts/+ flies reared in the indicated media. For each condition, more than 500 flies were examined in three different experiments. Each column represents the mean value ± SEM. (B) Percentage of flies that reach the adult stage. Each column represents the mean value ± SEM obtained by examining about 500 flies per condition in three different experiments. s.m. = standard medium HSD = high sugar diet; 4DP = 4 deoxypyridoxine; ns = non-significant Chi-square test. *** Significantly different in Chi-square test, with p < 0.001.