p14ARF Expression Increases Dihydrofolate Reductase Degradation and Paradoxically Results in Resistance to Folate Antagonists in Cells with Nonfunctional p53

Abstract

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 G 1 and G 2 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 doxycyclineinducible 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 p14ARF 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.

Low-Level Expression of MicroRNAs let-7d and miR-205 Are Prognostic Markers of Head and Neck Squamous Cell Carcinoma

“…The activity of p14 ARF results in increased degradation of DHFR and resistance to folate antagonists in cells with nonfunctional p53. 30 Our results suggest that these pathways may intersect and that miR-205 may be involved in the regulation of these events that determine these cancer-related phenotypes. Further dissection of these pathways and direct experimental determination of miR-205 target genes in HNSCC cell lines and confirmation of these targets in specific head and neck clinical samples should help to understand mechanisms involved in these responses.…”

Imaging Colon Cancer Response Following Treatment with AZD1152: A Preclinical Analysis of [18F]Fluoro-2-deoxyglucose and 3′-deoxy-3′-[18F]Fluorothymidine Imaging

“…AZD1152 is effective in inhibiting the growth of two human colorectal/colonic cancer cell lines (HCT116 and SW620) in culture and xenografts in nude mice (9, 13, 23) (Figs 3E and 4E). Both cell lines were sensitive to AZD1152-HQPA in the low nano-molar range.…”

“…The cell number vs. time profiles were plotted and fitted to a single exponential equation to estimate doubling time. Immunoblots using antibodies against human thymidine kinase (TK) and thymidylate synthetase (TS) o btained from Santa Cruz Biotech, Santa Cruz,CA were performed as previously described (23). …”

The former annotated human pseudogene dihydrofolate reductase-like 1 ( DHFRL1 ) is expressed and functional

“…These posttranslational modifications are thought to be important for its localization at specific phases of the cell cycle. DHFR has also been reported to be regulated posttranslationally by p14 ARF by an unknown indirect mechanism that affects protein stability (18).…”