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- MATERIALS AND METHODS
- LITERATURE CITED
- Supporting Information
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
Cancer stem cells (CSCs) may contribute to chemoresistant sub-fractions in a variety of malignancies (1–4) and underpin the post-therapeutic recovery potential of tumors. In mouse embryonic stem cell identification, cell surface markers, such as, CD15, CD184, and c-kit are frequently exploited in flow cytometric studies (5), whereas for CSCs in solid tumors various markers have been investigated including ABCG5, ALDH1, CD24 (HSA), CD44 (PGP1), CD90 (THY1), CD133 (promin1), EPCAM, ABCG2, and Hoechst side population (SP) phenotype (3). The ABCG2 transporter (MXR/BCRP) (5, 6) is linked with the CSC phenotype (3, 6–9) contributing to the definition of the pluripotential SP fraction using flow cytometry (10–12) and slide-cytometry (13). SP cells show efficient exclusion of the DNA minor-groove-binding ligand (MGL) Hoechst 33342, detected by changes in the fluorescence emission spectrum of nuclear bound dye (14). Hoechst 33342 has cytotoxic, mutagenic, and DNA damaging properties (15) arising from both minor groove binding and interactions with DNA topoisomerase I (16). An early study recognized the differences between MGLs in their ability to generate topoisomerase I-cleavable complexes (e.g., in order of efficiency: Hoechst 33342 and 33,258 >> distamycin A > berenil > netropsin), and that cleavable complex formation did not correlate with DNA binding efficiency alone (16). In the case of the drug tallimustine (FCE24517), which contains a benzoic acid nitrogen mustard appended to MGL distamycin A, there is evidence of a cooperative DNA binding mode in which noncovalent residence in the DNA minor groove perturbs the helix DNA structure and allows another drug molecule to alkylate the N7 position of guanines located on the periphery of distamycin A consensus sequences (17). The impact of SP-like ABCG2 expression on FCE24517 toxicity is not known.
In murine bone marrow ABCG2 expression appears to be exclusively able to allow the visualization by cytometry of the SP, although the bone marrow of Mdr1a/1b−/− mice display an elevated SP, reversible by the ABCG2 inhibition, suggesting that ABCG2 expression can over-compensate for Mdr1a/1b loss (18).
Some human cancer cell lines have SP fractions containing stem-like cancer cells (19, 20), although the study of SP resistance patterns is complicated by the need for FACS-separation of SP and nonSP fractions and the impact of dye toxicity, dye photosensitization, and clonal instability through asymmetric division (12). ABCG2 expression may reflect fast-cycling tumor progenitors in SP fractions (21), presenting a challenge for therapies that target active S-phase and comprise agents susceptible to active cellular efflux. This is highlighted in the case of the camptothecin class of S-phase targeting antineoplastic agents (e.g., topotecan; TPT) for which the ABCG2 transporter is functional (22–24). TPT acts by stabilizing a covalent topoisomerase I-DNA complex, thereby generating a hindrance to DNA replication fork progression with a subsequent formation of potentially lethal DNA lesions (25, 26). CSC/SP evasion of such cell cycle targeting may involve both modified cell cycle status and “micropharmacokinetic” protection of nuclear targets by changes in the active drug concentrations available within cellular sub-compartments (27). Further, it is not clear how additional features expressed by SP-like cells may augment such resistance to a defined agent.
Although a murine cell line cannot substitute for the likely complexity associated with SP dynamics in tumor cell populations, we hypothesized that we could assess the degree of nuclear protection minimally afforded by a transporter-driven SP phenotype, and its cellular consequences without sorting, by using cells in which the majority of the population expresses Hoechst 33342 resistance. Here we have exploited an established clonal variant murine cell line, previously derived by one-step selection resulting in resistance to the MGLs Hoechst dye 33258 (28) and the more lipophilic 33,342 (29). This approach allows for a direct assessment of the extent to which the SP phenotype recruits co-resistance to MGLs, specifically TPT, given that these agents share noncovalent DNA minor groove-binding and topoisomerase I inhibitory properties (16, 30, 31). This unique murine system obviates the need for SP isolation facilitating microarray analysis for gene expression patterns that contribute to a basal SP-like phenotype. Here we have compared the murine SP profile with the SP pattern operating in the A549 human lung cancer cell line which demonstrates a functional over-expression of the ABCG2-encoded efflux pump (19, 20) and maintains a resident CSC/SP fraction (20).
The results provide evidence of a high specific protection against noncovalent MGLs attributable to ABCG2 function in the murine system, establishing a stable clonal system for the functional definition of the SP-like phenotype and basal drug crossresistance patterns. Hoechst 33342 resistance and evasion of TPT-induced cell cycle arrest was attributable to efflux alone with no evidence of modulation of target availability, endogenous competitors for nuclear DNA binding or augmentation by any ancillary gene expression changes. A novel, nonfluorometric, photo-crosslinking method was used to demonstrate directly that residence of Hoechst 33342 molecules on DNA was greatly limited by efflux, whereas covalent linkage of another minor groove ligand circumvented the impact of efflux. The murine models reveal a strong linkage between ABCG2 function and enhanced TPT resistance via drug exclusion with implications for the predicted efficient evasion of TPT therapy by SP fractions of human tumors.
- Top of page
- MATERIALS AND METHODS
- LITERATURE CITED
- Supporting Information
The HoeR415 variant showed SP-like characteristics apparent in the majority of cells and a significantly reduced residence of MGLs on DNA. Protection is driven primarily by a selective and functional over-expression of the drug transporter ABCG2, identified in both microarray and inhibitor studies. The Hoechst 33342 clearing observed in HoeR415 was at a similar level to that achieved by the SP fraction in human A549 cell populations. Enhanced efflux recruited a moderate twofold clonogenic resistance to TPT, some fivefold less than the relative resistance achieved for continuous Hoechst 33342 exposure. Resistance permitted complete evasion of early cell cycle arrest associated with the TPT targeting of S-phase at low-drug concentrations—both Hoechst 33342 and TPT resistance being FTC-reversible. HoeR415 showed that a dramatic reduction in initial TPT loading is also reversible by FTC and again similar in extent to that achieved in A549 cell populations. The apparent stability of the HoeR415/SP phenotype allows for whole population analyses, overcoming the problem of generation of a nonSP sub-fractions shown to occur upon isolation of tumor-derived SP cells (12).
HoeR415 and parental cells had similar responses to Hoechst 33342 presensitization to UVA radiation (generating DNA–protein crosslinks; Fig. 3) and FCE24517 (a base-damaging agent; (17) indicating that the intrinsic responses to a range of genotoxic damage remains unchanged in the variant. UVA sensitization was lost more rapidly by HoeR415 at both the clonogenic survival and this paralleled the loss of Hoechst 33342 sensitization to UVA-induced DNA–protein crosslinking. The findings strongly suggest that dye residence on DNA limits MGL sensitization mechanisms. Hoechst 33342 sensitization to UVA-induced DNA–protein crosslinking paralleled the fluorometrically tracked rapid loss of dye—providing a nonfluorescence based identification of SP-like cells and a means of selection of SP cells through dye-targeted UVA-toxicity for nonSP cells. The unique murine cell system removes the need to fractionate populations (12) with implications for rapid drug screening and could provide a functional benchmark for the quantification of SP fractions by flow cytometry.
Microarray analyses revealed 56 recognized gene transcipts were over-expressed and 17 under-expressed in HoeR415 relative to parental cells. Searches were undertaken for candidate genes, whose change expression in HoeR415 might impact directly on drug resistance. Other than ABCG2, we identified ALDH, MGST1, Plk2, and Nnt. Increased MGST1 could contribute to the drug resistance phenotype via direct detoxification effects with indirect evidence previously reported of a positive correlation for camptothecin resistance (48). A minor reduction in Plk2 expression observed in HoeR415 cells is unlikely to impose resistance to the S-phase targeting agent TPT because silencing Plk2 sensitizes cells to replicative stress (49). Likewise, the reduction in Nnt expression observed in the variant is unlikely to contribute to the SP-like phenotype because this encoded mitochondrial inner membrane protein normally affords indirect protection against mitochondrial oxidative stress (50).
The increase in gene expression in HoeR415 cells of ALDH1A1 is intriguing because it is a potential candidate for a CSC-linked marker. CSCs may express collateral drug resistance through the aldehyde dehydrogenase (ALDH) (3). The enzyme family oxidizes a wide range of reactive and toxic aldehydes to their corresponding carboxylic acids (51) and facilitates resistance to cyclophosphamide, reversible by the ALDH inhibitor disulfiram (47). There is currently no evidence to functionally link the operation of the ABCG2 transporter with that of other CSC-associated markers in augmenting innate resistance to a defined antineoplastic agent. Importantly co-expression of ALDH and ABCG2, which can occur in stem-like cell fractions (3) does not appear to augment TPT export in the murine system—a view supported by our preliminary studies that show TPT accumulation in HoeR415 is not blocked by the ALDH inhibitor disulfiram and despite the additional potential for disulfiram metabolites to inactivate P-glycoprotein (52). To be able to establish that this murine cell line can be a surrogate for human SPs, comparative gene expression analyses and pharmacologic studies would be required profiling different human tumors and cell lines. However, the basal SP-like phenotype described here may impose selective MGL resistance due to the degree of expression of the transporter or potentially a mutated form. A recent report has noted that acquisition of moderate levels of enhanced transporter expression in mouse models, below those levels found in some normal tissues, can be sufficient to cause doxorubicin resistance (53).
We suggest that the basal ABCG2-driven efflux process, in underpinning the micropharmacokinetic SP phenotype, gives rise to selective resistance to noncovalent binding MGLs because they are preferred substrates and can dissociate from intracellular targets. Our results show that complete first cycle evasion of TPT action can be achieved via enhanced efflux despite the exquisite sensitivity of S-phase, with the implication that low-dose continuous exposure would favor CSC survival if primarily determined by a basal SP-like process. Higher levels of functional ABCG2 activity may be required to give rise to additional resistance for substrates, such as, mitoxantrone, that show reduced dissociation capacity from binding at intracellular targets (54) biasing their location at target. Effectively, a hierarchy of micropharmacokinetic resistance is imposed on the SP fraction together with a pharmacodynamic impact that varies according to the drug potency during residency at target. In a CSC model, the associated loss of SP characteristics during asymmetric division will generate cohorts of cells with changing drug resistance profiles with respect to different agents, even when the impact of a single transporter is considered. Intracellular stability of the α-hydroxylactone E-ring of camptothecins in SP cells may be critical if ring-opened products are favored substrates for ABCG2-mediated export. Enhanced SP-targeting using the camptothecins could be achieved by the deployment of stabilized E-ring camptothecin keto analogs (e.g., S38809 and S39625) given that such analogues can retain topoisomerase I inhibition without being substrates for either the ABCB1 (multidrug resistance-1/P-glycoprotein) or ABCG2 drug efflux transporters (55). Furthermore, the use of agents actively transported by human ABCB1A/ ABCB1B, but not by ABCG2 (BCRP1) (56), could be used to target CSC fractions carrying intrinsically drug resistant fractions, as recently demonstrated for the polyether organic anion salinomycin (57).