br rate dehydrogenase PHGDH and through
rate dehydrogenase (PHGDH) and through successive
steps into serine. Serine is then converted by serine hy-
droxymethyltransferase 2 (SHMT2) into glycine. The
carbon unit is incorporated into the folate cycle which
transfers the carbon unit to homocysteine to generate
methionine. The asymmetric hydrolysis of glucose
causes two isotopomers of serine, Ser+4 and Ser+5.
Decarboxylation of serine by SHMT results in a single
isotopomer of glycine, Gly+2. The carbon unit from serine has two deuterium and a 13C; however, it is oxi- dized to formate causing a loss of a deuterium before
incorporation into the folate cycle and ultimately gen-
Fig. 4. Representative Expanded SIM ion chromatograms and mass spectra of amino acids in A375 + PHGDH cells.
A. SIM ion chromatogram of glycine;
B. SIM ion chromatogram of methionine;
C. SIM ion chromatogram of serine;
D. SIM mass spectrum of glycine;
E. SIM mass spectrum of methionine;
F. SIM mass spectrum of serine.
The SIM of an amino ML-210 (Gly, Met or Ser) includes the ions corresponding to the isotope standard, glucose-derived labeled amino acid, unlabeled media amino acids.
Fig. 5. Measurement of glucose-derived amino acids in cancer cells that differentially express
A. Western-blot analysis of PHGDH and SHMT2 in U251, A375 and A375 overexpressing PHGDH (A375 + PHGDH) cells; loading controls were GAPDH; B. Concentrations of Ser+4 and Ser+5 in the cells measured by GC-MS;
C. Concentrations of Gly+2 in the cells measured by GC-MS;
D. Concentrations of Met+2 in the cells measured by GC-MS.
enriched internal standards. This method allows for monitoring how this pathway may be altered by growth conditions and as well as to test the selectivity and efficacy of potential chemotherapy drugs targeted at PHGDH, SHMT2, or other enzymes in this pathway.
4.1. De novo serine pathway used to make intermediates for nucleic acid synthesis rather than produce methionine
The rate-limiting step in the synthesis of serine, glycine, and me-thionine is the conversion of 3-phosphoglycerate to 3-phosphohydrox-ypyruvate by PHGDH. We show that production of these amino acids in U251 cell line is minimal due to low endogenous levels of PHGDH,
while over-expression of PHGDH in the PHGDH overexpressing A375 cell line provides the greatest conversion to Ser+4/Ser+5. All three cell lines had measurable SHMT2 protein, and all generated si-milar amounts of Met+2. We note that levels of Met+2 are substantially lower than levels of either Gly+2 or Ser+4/Ser+5, ~50-fold. This result can likely be attributed to alternative fates of methylated folate analogs in that once a labeled methyl group is transferred to tetrahydrofolate, it can transfer to thymidylate or two positions of the purine ring, in competition to synthesis of methionine. Because Met+2 is only 1–2% of total intracellular methionine and levels are much lower than the other labeled amino acids, we believe the predominance of these carbon units are being incorporated into nucleic acid synthesis. Our data is further
Fig. 6. Measuring serine and methionine in-tracellular pools in three different cell lines and the fraction produced from de novo synthesis from glycolytic precursors.
Fig. 7. Measurement of glucose-derived
amino acids in cancer cells treated with
PHGDH or SHMT2 inhibitors.
A375+PHGDH cells treated with PHGDH in-
supported by Snell et al. who first demonstrated that serine was directly incorporated into DNA and increased PHGDH activity preceded a rapid increase in cell proliferation . Together these data support that extracellular serine and serine derived from glucose can be used for cell proliferation in cancer.
However, typical culture conditions with DMEM contain 200 μM methionine which is at least 20-fold excess of what we measured in-tracellularly. While this may be the standard in the field, our mea-surements suggest that this is in vast excess. Further studies need to be done on optimal cell culturing conditions to better reflect in vivo con-ditions and how these metabolic pathways might respond. Perhaps under high methionine conditions cells can rapidly proliferate by shunting carbon units to nucleic acid synthesis while under other conditions those carbon units may instead be directed towards me-thionine. This may also explain why tumors may have an addiction to methionine as it may free up carbon units that would otherwise be diverted.
4.2. Behavior of amino acid pools under different inhibitors