Drug resistant tumor ''side-populations,'' enriched in cancer stem cells and identified by reduced accumulation of Hoechst 33342 under ABCG2-mediated efflux, may compromise therapeutic outcome. Side-population cells have predicted resistance to minor groove ligands, including the DNA topoisomerase I poison topotecan. We have used a stable Hoechst 33342-resistant murine L cell system (HoeR415) to study resistance patterns, removing the need for SP isolation before microarray analysis of gene expression and the tracking of cell cycle dynamics and cytotoxicity. The majority of HoeR415 cells displayed a side-population phenotype comparable with that of the side-population resident in the ABCG2 over-expressing A549 lung cancer cell line. Photo-crosslinking showed direct protection against minor groove ligand residence on DNA, driven by ABCG2-mediated efflux and not arising from any binding competition with endogenous polyamines. The covalent minor-groove binding properties of the drug FCE24517 (tallimustine) prevented resistance suggesting a mechanism for overcoming SP-related drug resistance. Hoechst 33342-resistant murine cells showed lower but significant crossresistance to topotecan, again attributable to enhanced ABCG2 expression, enabling cells to evade S-phase arrest. Hoechst 33342/TPT-resistant cells showed limited ancillary gene expression changes that could modify cellular capacity to cope with chronic stress including over-expression of Aldh1a1 and Mgst1, but under-expression of Plk2 and Nnt. There was no evidence to link the putative stem cell marker ALDH1A1 with any augmentation of the TPT resistance phenotype. The study has implications for the patterns of drug resistance arising during tumor repopulation and the basal resistance to minor groove-binding drugs of tumor side-populations. ' 2009 International Society for Advancement of Cytometry
Metallothioneins (MTs) have an important role in zinc homeostasis and may counteract the impact of oversupply. Both intracellular zinc and MT expression have been implicated in proliferation control and resistance to cellular stress, although the interdependency is unclear. The study addresses the consequences of a steady-state overexpression of MT-1 for intracellular zinc levels, cell cycle progression, and protection from zinc toxicity using a panel of cell lines with differential expression of MT-1. The panel comprised parental Chinese hamster ovary-K1 cells with low endogenous expression of MT and transfectants with enhanced expression of mouse MT-1 on an autonomously replicating expression vector with a noninducible promoter. Cell cycle progression, determined by flow cytometry and time-lapse microscopy, revealed that enhanced cytoplasmic expression of MT-1 does not impact on normal cell cycle operation, suggesting that basal levels of MT-1 expression are not limiting for background levels of oxidative stress. MT-1 overexpression correlated with a steady-state increase in cytoplasmic free Zn(2+), assessed using the fluorescent zinc-sensor Zinquin, particularly at high levels of overexpression, further suggesting that zinc availability is normally not limiting for cell cycle progression. Enhanced MT-1 expression, over a 10-fold range, had a clear impact on resistance to Cd(2+) and Zn(2+) toxicity. In the case of Zn(2+), the degree of protection afforded was less, indicating that MT-1 has a limited range and saturable capacity for effecting resistance. The results have implications for the use of cellular stress responses to exogenously supplied zinc and zinc-based systemic therapies.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.