ABSTRACT
Asparagine and glutamine depletion operated by the drug Asparaginase (ASNase) has revolutionized therapy in pediatric patients affected by Acute Lymphoblastic Leukemia (ALL), bringing remissions to a remarkable 90 % of cases. However, the knowledge of the proproliferative role of asparagine in adult and solid tumors is still limited. We have here analyzed the effect of ASNase on three adenocarcinoma cell lines (A549, lung adenocarcinoma, MCF-7, breast cancer, and 786-O, kidney cancer). In contrast to MCF-7 cells, 786-O and A549 cells proved to be a relevant target for cell cycle perturbation by asparagine and glutamine shortage. Indeed, when the cell-cycle was analyzed by flow cytometry, A549 showed a canonical response to asparaginase, 786-O cells, instead, showed a reduction of the percentage of cells in the G1 phase and an increase of those in the S-phase. Despite an increased number of PCNA and RPA70 positive nuclear foci, BrdU and EdU incorporation was absent or strongly delayed in treated 786-O cells, thus indicating a readiness of replication forks unmatched by DNA synthesis. In 786-O asparagine synthetase was reduced following treatment and glutamine synthetase was totally absent. Interestingly, DNA synthesis could be recovered by adding Gln to the medium. MCF-7 cells showed no significant changes in the cell cycle phases, in DNA-bound PCNA and in total PCNA, but a significant increase in ASNS and GS mRNA and protein expression. The collected data suggest that the effect observed on 786-O cells following ASNase treatment could rely on mechanisms which differ from those well-known and described for leukemic blasts, consisting of a complete block in the G1/S transition in proliferating cells and on an increase on non-proliferative (G0) blasts.
ABSTRACT
BACKGROUND: DNA-Damaged Binding protein 2 (DDB2) is a protein involved in the early step of Nucleotide Excision Repair. Recently, it has been reported that DDB2 is involved in epithelial-to-mesenchymal transition (EMT), key process in tumour invasiveness and metastasis formation. However, its role is not completely known. METHODS: Boyden chamber and cell adhesion assays, and ICELLigence analysis were performed to detect HEK293 adhesion and invasion. Western blotting and gelatine zymography techniques were employed to assess the EMT protein levels and MMP enzymatic activity. Immunofluorescence analysis and pull-down assays facilitated the detection of NF-kB sub-cellular localization and interaction. RESULTS: We have previously demonstrated that the loss of DDB2-PCNA binding favours genome instability, and increases cell proliferation and motility. Here, we have investigated the phenotypic and molecular EMT-like changes after UV DNA damage, in HEK293 clones stably expressing DDB2Wt protein or a mutant form unable to interact with PCNA (DDB2PCNA-), as well as in HeLa cells transiently expressing the same DDB2 constructs. Cells expressing DDB2PCNA- showed morphological modifications along with a reduced expression of E-cadherin, an increased activity of MMP-9 and an improved ability to migrate, in concomitance with a significant upregulation of EMT-associated Transcription Factors (TFs), whose expression has been reported to favour tumour invasion. We observed a higher expression of c-Myc oncogene, NF-kB, both regulating cell proliferation and metastatic process, as well as ZEB1, a TF significantly associated with tumorigenic potential and cell migratory ability. Interestingly, a novel interaction of DDB2 with NF-kB was detected and found to be increased in cells expressing the DDB2PCNA-, suggesting a direct modulation of NF-kB by DDB2. CONCLUSION: These results highlight the role of DDB2-PCNA interaction in counteracting EMT since DDB2PCNA- protein induces in HEK293 transformed cells a gain of function contributing to the acquisition of a more aggressive phenotype.