Defective high-affinity thiamine transporter leads to cell death in thiamine-responsive megaloblastic anemia syndrome fibroblasts

Abstract

We have investigated the cellular pathology of the syndrome called thiamine-responsive megaloblastic anemia (TRMA) with diabetes and deafness. Cultured diploid fibroblasts were grown in thiamine-free medium and dialyzed serum. Normal fibroblasts survived indefinitely without supplemental thiamine, whereas patient cells died in 5-14 days (mean 9.5 days), and heterozygous cells survived for more than 30 days. TRMA fibroblasts were rescued from death with 10-30 nM thiamine (in the range of normal plasma thiamine concentrations). Positive terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL) staining suggested that cell death was due to apoptosis. We assessed cellular uptake of [3H]thiamine at submicromolar concentrations. Normal fibroblasts exhibited saturable, high-affinity thiamine uptake (Km 400-550 nM; Vmax 11 pmol/min/10(6) cells) in addition to a low-affinity unsaturable component. Mutant cells lacked detectable high-affinity uptake. At 30 nM thiamine, the rate of uptake of thiamine by TRMA fibroblasts was 10-fold less than that of wild-type, and cells from obligate heterozygotes had an intermediate phenotype. Transfection of TRMA fibroblasts with the yeast thiamine transporter gene THI10 prevented cell death when cells were grown in the absence of supplemental thiamine. We therefore propose that the primary abnormality in TRMA is absence of a high-affinity thiamine transporter and that low intracellular thiamine concentrations in the mutant cells cause biochemical abnormalities that lead to apoptotic cell death.

Figure 1

Reversible toxicity in TRMA^–/– cells maintained in thiamine-depleted medium. Fibroblasts from a patient with TRMA were grown for 9 days without thiamine (a) or with 3 mM supplemental thiamine (b). Without thiamine, all cells in thi– medium die by day 10 (c). Addition of 3 mM thiamine at day 9 rescues the remaining viable cells within 24 h (d). ×20 (a, b, d); ×40 (c). Thi– medium, α-MEM minus thiamine; TRMA, thiamine-responsive megaloblastic anemia.

Figure 2

Uptake of [³H]thiamine by TRMA fibroblasts. (a) Time course of uptake. Cells were washed and placed in thi– medium 1 day before assay. Uptake was measured for times shown, at 37°C, in serum-free medium supplemented with 66nM thiamine as described in Methods (mutant, –/–; heterozygote, +/–; normal, +/+). Means of duplicate wells are given. Values are corrected for cell-associated radioactivity at 0°C. (b) Defective specific uptake in TRMA^–/– cells, assessed after 30 min incubation at 37°C. Specific uptake was calculated by subtracting radioactivity incorporated in the presence of 3 mM unlabeled thiamine. Means of duplicate wells from one subject of each genotype are represented. Error bars represent ranges for duplicate determinations.

Figure 3

Concentration dependence of high-affinity thiamine uptake in normal fibroblasts. Cells were labeled with 66 nM [³H]thiamine diluted with unlabeled thiamine for 30 min at 37°C. Values are corrected for nonspecific uptake by subtracting counts associated with the presence of 500-fold excess unlabeled compound. Results are shown for two separate experiments (solid line with triangles and dotted line with circles), done on different days with different wild-type cell lines. Inset: double reciprocal plot; the 800-nM point was eliminated as an outlier. All other data from the rectangular plot were included. The least-squares regression line gives an apparent K_m of 550 nM; apparent V_max for uptake is 11 pmol/10⁶ cells/30 min. Each point is the mean of duplicate wells ± range (error bars).

Figure 4

Apoptosis of fibroblasts subjected to thi– medium. TUNEL stain was performed as described in Methods. Normal (a and b) or TRMA mutant cells (c and d) were incubated for 10 days in the presence (a and c) or absence (b and d) of thiamine. (a) Wild-type cells, thiamine-replete. (b) Wild-type cells, thiamine-depleted. (c) TRMA mutant cells with thiamine. (d) TRMA fibroblasts, thiamine-depleted. ×40. TUNEL, deoxynucleotide transferase–mediated dUTP nick end-labeling.

Figure 5

Complementation of TRMA fibroblasts defects with yeast thiamine transporter gene THI10. Cells transfected with the THI10 gene and vector alone were grown in the presence or absence of thiamine as described in Methods. TRMA fibroblasts transfected with vector alone and grown in thi– medium died after 13 days. The experiment was terminated after 44 days. (a) TRMA fibroblasts transfected with THI10 gene, thiamine-replete. (b) TRMA fibroblasts transfected with vector alone, thiamine-replete. (c) TRMA fibroblasts transfected with THI10 gene, thi– medium. (d) TRMA fibroblasts transfected with vector alone, thi– medium.