Article Figures & Data
Figures
- Figure 1.
A, Western blot analysis of DNA mismatch repair proteins in human tumor spheroids (S) and subconfluent monolayer (M) cultures. Cells were grown for 1 to 4 d and lysed, and proteins from each sample were blotted for PMS2, MLH1, PTEN, ERK, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-actin. ERK was subsequently used as a loading control in Western blots. B, examples of Western blotting analysis of DNA mismatch repair proteins in human cancer cell lines grown as spheroids or monolayers (Mono). Cells were lysed at the indicated times, and for each sample, proteins were tested for the expression of PMS2 and MLH1 as well as other DNA mismatch repair proteins, such as MSH6, MSH3, and MSH2. MDA-MB-435.TO.1 is a Taxol-resistant variant of the MDA-MB-435 breast cancer cell line. C and D, Western blot analysis of DNA mismatch repair proteins in human tumor spheroids and subconfluent monolayer cultures. Cells were grown for 1 to 4 d and lysed, and protein lysates were blotted for PMS2, MLH-1, and MSH-2. ERK was used as loading control.
- Figure 2.
Growth of EMT-6/P spheroids and Western blotting analysis of EMT-6/P as monolayers or spheroids treated with or without 5-azacytidine (5′Aza). Cells were plated in 96-well plates precoated with agarose. For some samples, after growth as spheroids for 2 d, cells were treated with 5-azacytidine (1 μmol/L final concentration) or PBS (control) and left for 2 more days. At the indicated times (in days), cells were photographed under ×40 magnification and lysed, and protein from each sample was probed for expression of PMS2, MLH1, MSH6, and MSH2. Bar, 0.5 mm. ERK was used as a loading control.
- Figure 3.
A, morphology of EMT-6 spheroids in the absence of alkylating agent treatment. Examples of EMT-6P and drug-resistant variants grown as spheroids following treatment with 5-azacytidine or hyaluronidase. i, spheroids of EMT-6/P (parental) and Thiotepa-resistant variants after 24 h in tissue culture with 5-azacytidine (5-Aza, 2 μmol/L final concentration). ii, EMT-6/Thio-resistant variants were pretreated as monolayer cultures with PBS (control) or 5-azacytidine (2 μmol/L final concentration), trypsinized, and replated as spheroids in medium supplemented with PBS (control) or 5-azacytidine (2 μmol/L final concentration). Control EMT-6/Thio cells were also plated as spheroids in medium containing hyaluronidase (HA, 2 mg/mL final concentration) as a positive control for spheroid disruption. iii, EMT-6/CTX-resistant variants were pretreated as monolayer cultures with PBS (control), 5-azacytidine (2 μmol/L final concentration), or 6-azacytidine (6-Aza, 2 μmol/L final concentration), trypsinized, and replated as spheroids in medium supplemented with PBS (control), 5-azacytidine (2 μmol/L final concentration), or 6-azacytidine (2 μmol/L final concentration). Cells were photographed after 24 h of plating spheroid cultures. Magnification, ×40. Bar, 0.5 mm. B, Western blotting analysis of DNA mismatch repair proteins in EMT-6 drug-resistant variants (EMT-6/Thio and EMT-6/CTX) grown as monolayers or spheroids (Sph) in the presence or absence of 5-azacytidine, 6-azacytidine, or hyaluronidase (2 mg/mL) as described above. Lysates of monolayer cultures were included as a control. For 5-azacytidine- and 6-azacytidine-treated groups, cells were first pretreated as monolayers for 10 h with either 5-azacytidine or 6-azacytidine (2 μmol/L final concentration) and replated as spheroids in medium containing 5-azacytidine or 6-azacytidine (2 μmol/L final concentration). After 24 h of spheroid culture, cells were lysed and protein lysates were blotted for expression of PMS2 and MLH1. ERK was used as a loading control.
- Figure 4.
EMT-6 spheroid compaction and Western blotting analysis of EMT-6/P-resistant (parental) and EMT-6/cisplatin-resistant (EMT-6/DDP) variants. Cells were plated as spheroid cultures for 24 h in 96-well plates precoated with agarose. Numbers in brackets, approximate number of cells plated per well. Cells were photographed after 1 to 4 d of plating. Magnification, ×40. EMT-6/DDP cells formed highly cohesive spheroids compared with the parental cell line. Bar, 0.5 mm. For Western blotting, EMT-6/DDP monolayer cultures were lysed and compared with lysates of EMT-6/DDP spheroids grown for 1 to 4 d. After 4 d of spheroid culture, several EMT-6/DDP spheroids were dispersed by trypsin treatment, replated for 48 h as monolayers (Mono*), lysed, and included in the Western blotting analysis of PMS2 and MLH1. ERK was used as a loading control.
- Figure 5.
Effect of 5′-azacytidine and trichostatin A on EMT-6 spheroids. A, trichostatin A (Tricho) did not cause loss of spheroid compaction in EMT-6/DDP cells compared with medium alone or DMSO controls (diluted 1:11,000 in medium). No effect was observed in DMSO alone compared with medium alone. B, in contrast, 5′-azacytidine or 2′-deoxy-5′-azacytidine (2-deoxyAza) both caused the edge of EMT-6/DDP spheroids to grow in a loose fashion. C, trichostatin A treatment of EMT-6/P cells led to increased spheroid compaction in contrast to the relatively loose spheroids observed in cells treated with 5′-azacytidine or controls.
- Figure 6.
A, analysis of human MDA-MB-231 (breast), MDA-MB-435 (breast), and PC3 (prostate) tumor cell lines and variant cell lines derived from tumors relapsed to metronomic cyclophosphamide (CTX) treatment in vivo (MDA-MB-231/CTX, MDA-MB-435/CTX, and PC3/CTX), grown in vitro as tumor spheroids. B, Western blot analysis of PMS2 and MLH1 in PC3 tumor fragments from parental PC3 tumor grown in nude mice and PC3 tumors relapsing to metronomic cyclophosphamide treatment (PC3-CTX). C, Western blot analysis for PMS2 and MLH1 in parental and in vitro derived drug-resistant variants. EMT-6/P (parental) cells were selected for resistance in vitro to cisplatin present in the growth medium to a final concentrations of 1 μmol/L (1-CDDP) and 2 μmol/L (2-CDDP). EMT-6 cells were also compared with variants selected in vitro for Adriamycin (AR 10), vincristine (VR 1.0), and methotrexate (MTX 1.0) resistance. Human H69 lung cancer cells were compared with variants selected in vitro for resistance to vincristine (VCR) or cisplatin (CPR). ERK was used as a loading control.
- Figure 7.
Analysis of human melanoma WM9 and WM239 monolayers and spheroids. Cells were plated as spheroids for 1 to 4 d and then photographed at ×40 magnification. Bar, 0.5 mm. Western blotting analysis was carried out for PMS2 and MLH1 expression in monolayer and spheroids grown for 1 to 4 d and for WM239 spheroids disrupted by trypsin treatment and replated as monolayers for 48 h (Mono*). ERK was used as a loading control.
- Figure 8.
A and B, Southern blot analysis for MLH1 promoter methylation probed for a region of the human MLH1 promoter [as described by Strathdee et al. (22)]. Human RKO, WM9, and WM239 cells were grown as monolayers or spheroids for 4 d. Genomic DNA was extracted and treated with XbaI and EcoRI to excise a fragment of the MLH1 promoter and was then further digested with no enzyme, HpaII, or MspI (both enzymes cut the same sequence, but HpaII is ineffective at digesting methylated sequences), showing no obvious methylation of this region of the human MLH1 promoter. C, analysis of DNA from RKO, WM9, and WM239 cells grown as monolayers or spheroids for 4 d using the Transignal Methylation kit. DNA samples were subjected to bisulfate treatment followed by PCR reactions specific for methylated (m) or unmethylated (u) region of the human MLH1 promoter.
- Figure 9.
Effect of 5-azacytidine on EMT-6/cisplatin-resistant variants (EMT-6/DDP). A, EMT-6/DDP cells were pretreated (as monolayers) for 10 h with 5-azacytidine (2 μmol/L final concentration) or PBS (control). Cells were then rinsed and replated as spheroids for 24 h, at which point spheroids were photographed at ×40 magnification, lysed, and tested for relative expression of PMS2 and MLH1. Bar, 0.5 mm. ERK was used as a loading control. B, in vitro survival of EMT-6/DDP cells pretreated (as monolayers) for 10 h with 5-azacytidine (2 μmol/L final concentration) or PBS (control) followed by treatment for 1 h with different concentrations of cisplatin. Cells were then washed, plated as spheroids for 24 h, trypsinized, counted, and plated for a colony formation assay. Points, averages; bars, SD. The colony formation data are significantly impaired by 5-azacytidine pretreatment. P < 0.5 (paired t test).
- Figure 10.
Proposed model for suppression of DNA mismatch repair activity as observed in low-level microsatellite instability tumors and in EMT-6 variants selected in vivo for resistance to alkylating agents as a consequence of three-dimensional tumor growth and/or hypoxic conditions.
Volume 4, Issue 10
