Identification and Characterization of a New Platinum-Induced TP53 Mutation in MDAH Ovarian Cancer Cells

Abstract

Platinum-based chemotherapy is the therapy of choice for epithelial ovarian cancer (EOC). Acquired resistance to platinum (PT) is a frequent event that leads to disease progression and predicts poor prognosis. To understand possible mechanisms underlying acquired PT-resistance, we have recently generated and characterized three PT-resistant isogenic EOC cell lines. Here, we more deeply characterize several PT-resistant clones derived from MDAH-2774 cells. We show that, in these cells, the increased PT resistance was accompanied by the presence of a subpopulation of multinucleated giant cells. This phenotype was likely due to an altered progression through the M phase of the cell cycle and accompanied by the deregulated expression of genes involved in M phase progression known to be target of mutant TP53. Interestingly, we found that PT-resistant MDAH cells acquired in the TP53 gene a novel secondary mutation (i.e., S185G) that accompanied the R273H typical of MDAH cells. The double p53 S185G/R273H mutant increases the resistance to PT in a TP53 null EOC cellular model. Overall, we show how the selective pressure of PT is able to induce additional mutation in an already mutant TP53 gene in EOC and how this event could contribute to the acquisition of novel cellular phenotypes.Cells 2020, 9, 36 2 of 18 PT resistance has been linked to alterations in several processes such as drug transport, drug inactivation, DNA damage response, DNA repair, apoptosis, and autophagy [8][9][10][11]. Many studies have been done to identify genes and mechanisms directly associated to resistance to PT therapy. We have recently contributed to this issue reporting the selection and preliminary characterization of three new isogenic models of PT-resistant EOC cell lines-MDAH-2774, TOV-112D and OVSAHO. This work has pointed out a common ability of the three isogenic PT-resistant cells to resolve the PT-induced DNA damage compared to parental cells. Moreover, all PT-resistant cells displayed a change in their morphology and a higher ability to grow on mesothelium [12].In the present work, we better characterized MDAH-2774 PT-resistant (PT-res) cells reporting the appearance of a novel mutation in TP53 induced by platinum pressure that is linked to the increased resistance to PT and an altered mitotic division observed in PT-res cells. Materials and Methods Cell CultureMDAH-2774 (CRL-10303) parental and SKOV-3 (HTB-77) cells are from ATCC. Cisplatin-resistant (PT-res) isogenic cells were generated as described [12]. All cell lines were maintained in RPMI-1640 medium (Sigma-Aldrich Co., St. Louis, MO, USA) containing 10% heat-inactivated FBS, 100 U/mL penicillin and streptomycin (Sigma-Aldrich Co., St. Louis, MO, USA) at 37 • C in a 5% CO 2 atmosphere. After the selection process, PT-res cells were kept in PT-free medium. MDAH-2774 PT-res clones were obtained from PT-res pools by plating them at 0.7 cell/well dilution in a 96-multiwell plate. All cell lines were authenticated in our lab using the Cell ID TM System (Promega, Madison, WI, USA) ...

Splicing factor proline- and glutamine-rich (SFPQ) protein regulates platinum response in ovarian cancer-modulating SRSF2 activity

“…2e). Next, using these cells in time course analysis of PT response [19][20][21], we evaluated the expression of SFPQ protein during the phases of PT-induced DNA damage and repair. The results showed that higher SFPQ expression in PT-Res cells correlated with decreased and delayed apoptosis and DNA damage response, as evidenced by PARP-1 and caspase-3 cleavage and γ-H2AX expression, respectively ( Fig.…”

Purified Tea (Camellia sinensis (L.) Kuntze) Flower Saponins Induce the p53-Dependent Intrinsic Apoptosis of Cisplatin-Resistant Ovarian Cancer Cells

“…Additionally, many reports have focused on the p53-dependent anti-cancer effect of natural products [43,[58][59][60][61][62]. Recently, it has been reported that platinum chemotherapy could induce additional mutations in TP53 and further increase platinum resistance in ovarian cancer [63]. In this study, we suggested that p53 was up-regulated and phosphorylated at Ser 15 by PTFSs and played a vital function in intrinsic apoptosis stimulated through PTFSs.…”

HSP90 identified by a proteomic approach as druggable target to reverse platinum resistance in ovarian cancer

“…CDDP-resistant cancer cells (referred as Pt-res cells) were generated as previously described and include pooled resistant populations (pool) and individual clones, as indicated in the text [6][7][8].…”

“…On this regard, we previously generated and characterized three cisplatin (CDDP)-resistant isogenic high grade EOC cell lines (TOV-112D Pt-res, MDAH-2774 Pt-res and OVSAHO Pt-res) from their parental counterparts [6][7][8]. Since gene expression profiling failed to identify signaling pathways commonly altered in Pt-resistant (Pt-res) cells [6], we took advantage of a proteomic approach to investigate the possible mechanisms by which cells acquire CDDP-resistance.…”