Methotrexate resistance in vitro is achieved by a dynamic selectionprocess of tumor cell variants emerging during treatment.

Genetic instability leads to tumor heterogeneity, which in turn provides a source of cell variants responsible for drug resistance. However, the source of resistant cells during the process of acquired resistance is poorly understood. Our aim has been to characterize the mechanism by which acquired resistance to methotrexate emerges during the course of cancer cell treatment in vitro. We recently demonstrated that, in vitro, HT-29 colon cancer cells become transiently sensitive to methotrexate by depleting the extracellular milieu of survival factors; on the other hand, the cell population under treatment can reversibly adapt to grow below a critical cell density in the presence of the drug. Here, we show that this adapted cell population gives rise to permanent resistant populations through repeated cycles of cell death and growth. This increased cell turnover, but not merely cell proliferation, is required for the appearance of increasing degrees of stable resistance that are progressively selected by drug pressure. Such a process, taking place in multiple steps, is here designated "dynamic selection." The analysis of sensitive and resistant HT-29 cell populations revealed that methotrexate induces genomic instability--characterized by centrosome amplification and aberrant chromosome recombination--leading to a low-level amplification of the 5q chromosome arm as one of the earliest genetic events selected during treatment. Therefore, this model provides a mechanism by which a tumor cell population lacking resistant subpopulations before treatment is able to acquire the genetic changes required for stable drug resistance.