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Cells deficient in base-excision repair reveal cancer hallmarks originating from adjustments to genetic instability.

Markkanen E, Fischer R, Ledentcova M, Kessler BM, Dianov GL - Nucleic Acids Res. (2015)

Bottom Line: Nevertheless, experimental evidence for this model remains inconsistent and elusive.Here, we performed a proteomic analysis of BER deficient human cells using stable isotope labelling with amino acids in cell culture (SILAC), and demonstrate that BER deficiency, which induces genetic instability, results in dramatic changes in gene expression, resembling changes found in many cancers.We observed profound alterations in tissue homeostasis, serine biosynthesis, and one-carbon- and amino acid metabolism, all of which have been identified as cancer cell 'hallmarks'.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Roosevelt Drive, Oxford OX3 7DQ, United Kingdom.

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Increased expression of serine biosynthesis pathway enzymes after XRCC1 knockdown. (A) mRNA levels of MTHFD2 after XRCC1 KD, quantified by qPCR and normalised to GAPDH. Values are mean ± SD from four independent experiments. The P-value was calculated using Student's t-test. (B) Measurement of GSH levels in cells using fluorescence of Dibromobimane normalised to fluorescence of Hoechst 33342. Control or XRCC1 KD cells were seeded 24 h prior to measurement at the indicated densities into 24-well plates. Values are mean ± SD from three independent experiments with four replicates each. The P-values were calculated using Student's t-test.
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Figure 5: Increased expression of serine biosynthesis pathway enzymes after XRCC1 knockdown. (A) mRNA levels of MTHFD2 after XRCC1 KD, quantified by qPCR and normalised to GAPDH. Values are mean ± SD from four independent experiments. The P-value was calculated using Student's t-test. (B) Measurement of GSH levels in cells using fluorescence of Dibromobimane normalised to fluorescence of Hoechst 33342. Control or XRCC1 KD cells were seeded 24 h prior to measurement at the indicated densities into 24-well plates. Values are mean ± SD from three independent experiments with four replicates each. The P-values were calculated using Student's t-test.

Mentions: The serine biosynthesis pathway (Supplementary Figure S2) provides direct inputs into the one-carbon metabolism, which consists of the folate and the methionine cycles, as well as the transsulfuration pathway. By donating a carbon unit from its side chain to tetrahydrofolate, serine feeds directly into the folate cycle. The folate cycle can provide building blocks for nucleotide synthesis, and additionally fuels the methionine cycle and the transsulfuration pathway. As a ‘side product’ of the carbon donation step, serine itself is converted to glycine, which in turn can again directly feed the folate cycle. Interestingly, the tetrahydrofolate salvage pathway involving the enzyme MTHFD2 was significantly upregulated upon XRCC1 KD both on protein (Table 1, rank 12) and mRNA levels (Figure 5A), suggesting that this metabolic branch is indeed in a state of enhanced activity. This was verified with a second siRNA sequence against XRCC1 (Supplementary Figure S1). Of note, ATF4−/- cells have been found to display decreased levels of MTHFD2 (24), suggesting that MTHFD2 up-regulation in XRCC1 KD cells may be a consequence of increased ATF4 expression observed in our experiments (Figure 3D). In addition to the increase in intracellular synthesis of amino acids by the serine biosynthesis pathway, we also observed an increase in expression of a variety of amino acid transporters of the solute carrier family (Table 3, light red). Interestingly, SLC1A4, SLC3A2, SLC7A1 and SLC7A5 are all regulated by ATF4 as well (24,26). These changes further support the notion that XRCC1 KD promotes changes in cellular metabolism toward an anabolic state via the ATF4-mediated ISR pathway.


Cells deficient in base-excision repair reveal cancer hallmarks originating from adjustments to genetic instability.

Markkanen E, Fischer R, Ledentcova M, Kessler BM, Dianov GL - Nucleic Acids Res. (2015)

Increased expression of serine biosynthesis pathway enzymes after XRCC1 knockdown. (A) mRNA levels of MTHFD2 after XRCC1 KD, quantified by qPCR and normalised to GAPDH. Values are mean ± SD from four independent experiments. The P-value was calculated using Student's t-test. (B) Measurement of GSH levels in cells using fluorescence of Dibromobimane normalised to fluorescence of Hoechst 33342. Control or XRCC1 KD cells were seeded 24 h prior to measurement at the indicated densities into 24-well plates. Values are mean ± SD from three independent experiments with four replicates each. The P-values were calculated using Student's t-test.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4402536&req=5

Figure 5: Increased expression of serine biosynthesis pathway enzymes after XRCC1 knockdown. (A) mRNA levels of MTHFD2 after XRCC1 KD, quantified by qPCR and normalised to GAPDH. Values are mean ± SD from four independent experiments. The P-value was calculated using Student's t-test. (B) Measurement of GSH levels in cells using fluorescence of Dibromobimane normalised to fluorescence of Hoechst 33342. Control or XRCC1 KD cells were seeded 24 h prior to measurement at the indicated densities into 24-well plates. Values are mean ± SD from three independent experiments with four replicates each. The P-values were calculated using Student's t-test.
Mentions: The serine biosynthesis pathway (Supplementary Figure S2) provides direct inputs into the one-carbon metabolism, which consists of the folate and the methionine cycles, as well as the transsulfuration pathway. By donating a carbon unit from its side chain to tetrahydrofolate, serine feeds directly into the folate cycle. The folate cycle can provide building blocks for nucleotide synthesis, and additionally fuels the methionine cycle and the transsulfuration pathway. As a ‘side product’ of the carbon donation step, serine itself is converted to glycine, which in turn can again directly feed the folate cycle. Interestingly, the tetrahydrofolate salvage pathway involving the enzyme MTHFD2 was significantly upregulated upon XRCC1 KD both on protein (Table 1, rank 12) and mRNA levels (Figure 5A), suggesting that this metabolic branch is indeed in a state of enhanced activity. This was verified with a second siRNA sequence against XRCC1 (Supplementary Figure S1). Of note, ATF4−/- cells have been found to display decreased levels of MTHFD2 (24), suggesting that MTHFD2 up-regulation in XRCC1 KD cells may be a consequence of increased ATF4 expression observed in our experiments (Figure 3D). In addition to the increase in intracellular synthesis of amino acids by the serine biosynthesis pathway, we also observed an increase in expression of a variety of amino acid transporters of the solute carrier family (Table 3, light red). Interestingly, SLC1A4, SLC3A2, SLC7A1 and SLC7A5 are all regulated by ATF4 as well (24,26). These changes further support the notion that XRCC1 KD promotes changes in cellular metabolism toward an anabolic state via the ATF4-mediated ISR pathway.

Bottom Line: Nevertheless, experimental evidence for this model remains inconsistent and elusive.Here, we performed a proteomic analysis of BER deficient human cells using stable isotope labelling with amino acids in cell culture (SILAC), and demonstrate that BER deficiency, which induces genetic instability, results in dramatic changes in gene expression, resembling changes found in many cancers.We observed profound alterations in tissue homeostasis, serine biosynthesis, and one-carbon- and amino acid metabolism, all of which have been identified as cancer cell 'hallmarks'.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Roosevelt Drive, Oxford OX3 7DQ, United Kingdom.

Show MeSH
Related in: MedlinePlus