Polyploidization and exit from cell cycle as mechanisms of cultured melanoma cell resistance to methotrexate

Abstract

Numerous malignant neoplasias are found to contain varying proportions of high-ploidy cells. Although the role they play in the tumor is poorly understood, several lines of evidence suggest that these cells could be especially resistant to various aggressions, a possibility of great interest in cancer treatment. In the present study, we tested this hypothesis through the analysis of the presence of high-ploidy cells following the administration of the chemotherapeutic agent methotrexate. We also determined the expression of two proliferation markers, PCNA and CDK1, after methotrexate-treatment. Cultured cells from the murine melanoma B16F10 were treated with high doses of methotrexate for seven days prior to determination of DNA content and proliferation markers. Our results showed an obvious increase in the mean ploidy of this population. Specifically, there was a dramatic reduction in the proportion of tetraploid cells (predominant in the original population), and an increase in the proportion of cells with higher ploidies, particularly those whose DNA content was greater than 8c, including some cells with ploidies greater than 16c. Furthermore, there was a reduction in the number of PCNA-expressing cells and the reduction was much more marked in the case of CDK1 that was almost absent in the modal-ploidy treated cells. These alterations concerning ploidy and expression of proliferation markers had completely reverted two weeks after withdrawal of the drug. Our results indicate that methotrexate at a high dosage selects a cell population heterogeneous concerning its ploidy level, composed of one subpopulation of high-ploidy cells and another of modal-ploidy cells that, considering its lack of CDK1 expression, would remain in a latent state to evade the effects of the drug.