Table 1.

Low-dose methotrexate (MTX) changes endogenous and exogenous formate utilization for nucleotide biosynthesis.

(A) Purine synthesis from L-[3-13C]serine derived formate and exogenous [13C]formate 1,2.
	Endogenous Formate from L-[3-13C]serine			Exogenous [13C]formate Incorporation
	dA+1 3	dA+2 3	dA (MIA) 4,6	dA+1 3	dA+2 3	dA (MIA) 4,6
CTL 4	0.310 ± 0.003	0.179 ± 0.003	1.155 ± 0.021	0.319 ± 0.002	0.121 ± 0.003	0.756 ± 0.025
MTX 4	0.025 ± 0.014	0.008 ± 0.002	0.648 ± 0.154	0.324 ± 0.005	0.142 ±0.003	0.876 ± 0.007
p-value2	0.001	0.000	0.044	0.281	0.022	0.023
%change5	−92.06%	−95.54%	−50.73%	1.65%	+17.72%	+11.94%
	dG+1 3	dG+2 3	dG (MIA) 4,6	dG+1 3	dG+2 3	dG (MIA) 4,6
CTL 4	0.321 ± 0.015	0.171 ± 0.007	1.066 ± 0.042	0.323 ± 0.008	0.119 ± 0.001	0.736 ± 0.013
MTX 4	0.025 ± 0.010	0.001 ± 0.002	0.120 ± 0.169	0.336 ± 0.006	0.135 ± 0.002	0.802 ± 0.001
p-value2	0.002	0.001	0.017	0.199	0.011	0.019
%change5	−92.34%	−99.14%	−94.59%	4.27%	+13.70%	+6.64%
(B) Relative incorporation of L-[3-13C]serine derived formate and exogenous [13C]formate in purines 1,2.
	Endogenous Formate from L-[3-13C]serine			Exogenous [13C]formate Incorporation
	Ser+1 3	dA+1 3	dA+1/Ser+1 7	Ser+1 3	dA+1 3
CTL 4	0.515 ± 0.016	0.310 ± 0.003	0.602 ± 0.006	0.189 ± 0.007	0.319 ± 0.002
MTX 4	0.475 ± 0.008	0.025 ± 0.014	0.052 ± 0.029	0.166 ± 0.000	0.324 ± 0.005
p-value2	0.087	0.001	0.001	0.046	0.281
%change5	−7.60%	−92.06%	−55.05%	−12.50%	1.65%
	Ser+1 3	dG+1 3	dG+1/Ser+1 7	Ser+1 3	dG+1 3
CTL 4	0.515 ± 0.016	0.321 ± 0.015	0.624 ± 0.029	0.189 ± 0.007	0.323 ± 0.008
MTX 4	0.475 ± 0.008	0.025 ± 0.010	0.052 ± 0.021	0.166 ± 0.000	0.336 ± 0.006
p-value2	0.087	0.002	0.002	0.046	0.199
%change5	−7.60%	−92.34%	−57.21%	−12.50%	4.27%
(C) Thymidine synthesis from L-[3-13C]serine derived formate and exogenous [13C]formate 1,2.
	Endogenous Formate from L-[3-13C]serine			Exogenous [13C]formate Incorporation
	Ser+1 3	dT+1 3	dT+1/Ser+1 7	Ser+1 3	dT+1 3
CTL 4	0.515 ± 0.016	0.452 ± 0.003	0.879 ± 0.006	0.189 ± 0.007	0.384 ± 0.002
MTX 4	0.475 ± 0.008	0.198 ± 0.022	0.417 ± 0.047	0.166 ± 0.000	0.397 ± 0.001
t-Test 2	0.087	0.004	0.005	0.046	0.017
%change 5	−7.60%	−56.14%	−46.14%	−12.50%	+3.42%

¹ To investigate formate utilization, L02 cells cultured in minimum essential medium (MEM) media were incubated with addition glycine, vitamin B12, and L-[3-¹³C]serine to reach a final concentration of 0.667 mM glycine, 0.001 mM B12, and 0.25 mM serine. In a parallel experiment, 0.25 mM [¹³C]formate was used instead of serine. ² Enrichment of isotopic tracers with or without MTX treatment is expressed as mean ± SD (n = 2–3). Data are compared by student’s t-test. Bold values indicate statistically significant differences (p < 0.05). Italic values indicated a trend of difference (p < 0.05, p < 0.1). ³ dA+1: deoxyadenosine enrichments; dG+1: deoxyguanosine; dT+1: deoxythymidine; Ser+1: serine enrichments from L-[3-¹³C]serine or from [¹³C]formate. ⁴ CTL: control; MTX: methotrexate; MIA: mass isotopomer analysis. ⁵ The % change of the isotopic enrichments in MTX treated cells compared to control cells. ⁶ Determined from the ratio of the M+1 and M+2 isomers of dA and dG. A value of 1.0 would indicate that 100% of the C-2 and C-8 carbons of the purine ring were derived from [¹³C]formate. ⁷ The relative enrichment in deoxythymidine (dT+1) synthesis or purine (dA+1 or dG+1) from L-[3-¹³C]serine tracer was calculated and abbreviated as dT+1/Ser+1, dA+1/Ser+1, dG+1/Ser+1, respectively.