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p14^ARF Expression Increases Dihydrofolate Reductase Degradation and Paradoxically Results in Resistance to Folate Antagonists in Cells with Nonfunctional p53

¹ Joan and Sanford I. Weill Graduate School of Medical Sciences of Cornell University, New York, New York; ² Memorial Sloan Kettering Cancer Center, New York, New York; and ³ The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, New Brunswick, New Jersey

The p14^ARF protein, the product of an alternate reading frame of the INK4A/ARF locus on human chromosome 9p21, disrupts the ability of MDM2 to target p53 for proteosomal degradation and causes an increase in steady-state p53 levels, leading to a G₁ and G₂ arrest of cells in the cell cycle. Although much is known about the function of p14^ARF in the p53 pathway, not as much is known about its function in human tumor growth and chemosensitivity independently of up-regulation of p53 protein levels. To learn more about its effect on cellular proliferation and chemoresistance independent of p53 up-regulation, human HT-1080 fibrosarcoma cells null for p14^ARF and harboring a defective p53 pathway were stably transfected with p14^ARF cDNA under the tight control of a doxycycline-inducible promoter. Induction of p14^ARF caused a decrease in cell proliferation rate and colony formation and a marked decrease in the level of dihydrofolate reductase (DHFR) protein. The effect of p14^ARF on DHFR protein levels was specific, because thymidylate kinase and thymidylate synthase protein levels were not decreased nor were p53 or p21WAF1 protein levels increased. The decrease in DHFR protein was abolished when the cells were treated with the proteasome inhibitor MG132, demonstrating that p14^ARF augments proteasomal degradation of the protein. Surprisingly, induction of p14^ARF increased resistance to the folate antagonists methotrexate, trimetrexate, and raltitrexed. Depletion of thymidine in the medium reversed this resistance, indicating that p14^ARF induction increases the reliance of these cells on thymidine salvage.

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