colo320dm cell line  (BioResource International Inc)

Journal: The Journal of Biological Chemistry

Article Title: Mitotic chromatin compaction tethers extrachromosomal DNA to chromosomes and prevents their mis-segregation into micronuclei

doi: 10.1016/j.jbc.2025.111081

Figure Lengend Snippet: The prometaphase spread technique untethers ecDNA and chromosomes by decompacting mitotic chromatin. A , schematic of an ecDNA-containing cell undergoing mitosis (from left to right : prophase, metaphase, anaphase, telophase). Acentric ecDNA ( red ) have been observed to tether to or “hitchhike” on chromosomes ( blue ) during mitosis to ensure their proper segregation and inheritance by daughter cells. B , prometaphase spreads performed on colcemid-arrested COLO320DM cells with the conventional hypotonic solution (75 mM KCl) and higher osmolarity solutions (100 mM and 125 mM KCl), producing varying amounts of chromosome (DAPI, blue ) individualization and ecDNA ( MYC FISH, red ) untethering. C , quantification of panel B . Left : boxplots quantifying chromosome individualization in prometaphase spreads performed with varying solution osmolarity; from left to right , n = 3, 3, 3 biological replicates and 95, 92, 105 cells; one-way ANOVA, F = 100.7, p < 0.001; ∗∗ p < 0.01 by Tukey’s honestly significant difference (HSD), ns = not significant. Right : quantification of ecDNA untethering; one-way ANOVA, F = 48.3, p < 0.001. Chromosome individualization is represented by the number of connected components identified as chromosomes by ecSeg. ecDNA untethering is represented by the number of ecDNA unattached to chromosomes divided by the total number of ecDNA not completely surrounded by chromosomes. D , prometaphase spreads performed with incubation in 1× PBS using COLO320DM cells pretreated for 8 h with vehicle (0.1% DMSO) or varying concentrations of Trichostatin A (TSA). E , quantification of panel D . Left : quantification of chromosome individualization in prometaphase spreads performed with 1× PBS on cells pretreated with TSA; n = 3, 3, 3, 3, 3, 3 biological replicates and 88, 113, 96, 80, 76, 84 cells; one-way ANOVA, F = 38.5, p < 0.001; ∗∗ p < 0.01 by Tukey’s HSD, ns = not significant. Right : quantification of ecDNA untethering; one-way ANOVA, F = 166.5, p < 0.001. F , linear regression analysis of median chromosome individualization v er s us ecDNA untethering of prometaphase spread conditions in panels B – E (n = 9). G , schematic: electrostatic/hydrophobic mitotic compaction forces at the level of nucleosomes tether ecDNA ( red ) to mitotic chromosomes ( blue ). In panels B–E , scale bar = 10 μm and each data point in graphs represents one cell.

Article Snippet: Human colorectal cancer cell lines COLO320DM and COLO320HSR were purchased from ATCC.

Techniques: Incubation

Journal: The Journal of Biological Chemistry

Article Title: Mitotic chromatin compaction tethers extrachromosomal DNA to chromosomes and prevents their mis-segregation into micronuclei

doi: 10.1016/j.jbc.2025.111081

Figure Lengend Snippet: Hypotonic conditions and HDAC inhibition untether ecDNA. A , fixed metaphase COLO320DM cells cultured on glass coverslips treated for 15 min with 1× media, 0.75× media (not shown), 0.5× media, 1:1 mix of 1× PBS with 1× media (1× PBS-media, not shown), and 1:1 mix of 1.5× PBS with 1× media (1.25× PBS-media); dashed outlines indicate metaphase plate chromosomes (DAPI, blue ), arrowheads indicate untethered ecDNA (identified by MYC FISH signal, red ). B , boxplots quantifying untethered ecDNA per cell from panel A , represented by % of MYC FISH signal in a cell unattached to chromosomes aligned at the metaphase plate (non-overlapping pixels); from left to right, n = 4, 6, 6, 7, 4 biological replicates and 427, 672, 691, 532, 686 cells; one-way ANOVA, F = 882.3, p < 0.001; ∗ p < 0.05, ∗∗ p < 0.01 by Tukey’s HSD. C , live imaging of COLO320DM cells expressing H2B-emiRFP670 (chromatin, blue ) and tetR-mNeonGreen which binds to a tetO-96mer repeat sequence inserted near MYC loci ( MYC , red ). Cells were arrested at metaphase with 10 μM MG132 and placed in hypotonic 0.5× media at t = 0 min to decompact chromatin; arrows indicate untethered ecDNA; after image acquisition at t = 15 min, cells were placed in relatively normotonic 1× PBS-media to recompact chromatin; arrows indicate ecDNA-ecDNA tethering. Representative images of n = 5 biological replicates and >50 cells. D , fixed metaphase COLO320DM cells cultured on glass coverslips treated for 8 h with vehicle (0.1% DMSO) or indicated concentrations of TSA; dashed outlines indicate metaphase plate chromosomes (DAPI, blue ), arrowheads indicate untethered ecDNA (identified by MYC FISH signal, red ). E , boxplots quantifying untethered ecDNA per cell from panel D ; from left to right , n = 3, 3, 3, 3 biological replicates and 98, 144, 161, 246 cells; one-way ANOVA, F = 34.9, p < 0.001; ∗∗ p < 0.01 by Tukey’s HSD. F , modified volcano plot summarizing the results of the targeted screen for drugs with the ability to untether ecDNA and chromosomes, quantified by the area of the convex hull of the prometaphase spread produced without hypotonic solution incubation. TSA (0.5 μM) was included as positive control. padj = adjusted two-tailed Student’s t test p value using Bonferroni multiple test correction (44 total comparisons were made). Each data point represents the average of all cells for each condition. G , metaphase COLO320DM cells cultured on glass coverslips treated for 24 h with vehicle (0.1% DMSO) or indicated concentrations of LMK235; dashed outlines indicate metaphase plate chromosomes, arrowheads indicate untethered ecDNA. H , quantification of untethered ecDNA per cell from panel G; n = 11, 3, 7, 4, 3, 3 biological replicates and 1278, 501, 875, 472, 571, 388 cells; one-way ANOVA, F = 735.1, p < 0.001; ∗∗ p < 0.01 by Tukey’s HSD, ns = not significant. In all panels, scale bar = 10 μm. Except in panel F , each data point in graphs represents one cell.

Article Snippet: Human colorectal cancer cell lines COLO320DM and COLO320HSR were purchased from ATCC.

Techniques: Inhibition, Cell Culture, Imaging, Expressing, Sequencing, Modification, Produced, Incubation, Positive Control, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Mitotic chromatin compaction tethers extrachromosomal DNA to chromosomes and prevents their mis-segregation into micronuclei

doi: 10.1016/j.jbc.2025.111081

Figure Lengend Snippet: Hypotonic conditions and HDAC inhibition lead to mis-segregation of ecDNA into micronuclei. A , live cell imaging of COLO320DM cells undergoing mitosis (chromatin labeled by H2B-emiRFP670, blue ) and ( MYC loci labeled by tetR-mNeonGreen, red ). Mitotic cells were placed in 0.5× media at t = 0 min; dashed outlines indicate chromosomes/primary nuclei, arrowheads indicate untethered ecDNA, arrows indicate micronuclei. Representative image of n = 5 biological replicates and >50 cells. B , fixed newly-divided daughter COLO320DM cells, as identified by the presence of Aurora B staining, treated for 6 h with 1× media, 0.75× media (not shown), 0.5× media, 1× PBS-media (not shown), and 1.25× PBS-media; dashed outlines indicate primary nuclei, arrowheads indicate micronuclei. C , quantification of panel B . Left : quantification of the percentage of daughter cell pairs with micronuclei; chi-squared, ∗ p < 0.05, ∗∗ p < 0.01. Right : quantification of the percentage of all MYC FISH signal per daughter cell pair inside micronuclei as opposed to the primary nuclei (symlog scale, linear ≤2, log >2); one-way ANOVA, F = 151.4, p < 0.001, ∗ p < 0.05, ∗∗ p < 0.01; n = 3, 3, 3, 6, 4 biological replicates and 601, 233, 148, 881, 836 daughter cell pairs. D and E , fixed newly-divided daughter COLO320HSR cells, treated similarly as in panels B and C . E , left : chi-squared, ∗∗ p < 0.01, ns = not significant. Right : one-way ANOVA, F = 0.9, p = 0.49; n = 3, 3, 3, 5, 3 biological replicates and 680, 256, 179, 578, 487 daughter cell pairs. F , quantification of pan-Histone H3 acetylation (pan-H3ac) IF in cytospin preparations of COLO320DM prometaphase spreads with chromosome and ecDNA segmentation by ecSeg (see C ); cells were treated in vehicle or LMK235 for 24 h; n = 2, 2, 2, 2 biological replicates and 52, 44, 75, 84 cells; one-way ANOVA, chromosomes: F = 67.6, p < 0.001, ecDNA: F = 81.3, p < 0.001, ∗ p < 0.05, ∗∗ p < 0.01 by Tukey’s HSD. G , newly-divided daughter COLO320DM cells, as identified by the presence of Aurora B staining (not shown), treated for 24 h with vehicle (0.1% DMSO) or indicated concentrations of LMK235; dashed outlines indicate primary nuclei, arrowheads indicate micronuclei. H , quantification of panel G . Left : quantification of the percentage of daughter cell pairs with micronuclei; chi-squared, ∗∗ p < 0.01. Right : quantification of the percentage of all MYC FISH signal per daughter cell pair inside micronuclei (symlog scale, linear ≤2, log >2); one-way ANOVA, F = 151.4, p < 0.001, ∗ p < 0.05, ∗∗ p < 0.01; n = 5, 5, 4 biological replicates and 713, 683, 420 daughter cell pairs. I , same as panel F , for COLO320HSR cells (chromosomes only; see E ); n = 2, 2, 2, 2 biological replicates and 85, 85, 63, 99 cells; one-way ANOVA, F = 204.1, p < 0.001, ∗∗ p < 0.01 by Tukey’s HSD. J and K , newly-divided daughter COLO320HSR cells, treated similarly as in panels G and H. K , left : chi-squared. Right : one-way ANOVA, F = 0.1, p = 0.88; n = 4, 3, 3 biological replicates and 486, 383, 335 daughter cell pairs. In all panels, scale bar = 10 μm and each data point in graphs represents one cell or daughter cell pair.

Article Snippet: Human colorectal cancer cell lines COLO320DM and COLO320HSR were purchased from ATCC.

Techniques: Inhibition, Live Cell Imaging, Labeling, Staining

Journal: The Journal of Biological Chemistry

Article Title: Mitotic chromatin compaction tethers extrachromosomal DNA to chromosomes and prevents their mis-segregation into micronuclei

doi: 10.1016/j.jbc.2025.111081

Figure Lengend Snippet: Ki67 gain-of-function untethers ecDNA from chromosomes. A , fixed COLO320DM cells cultured on coverslip and incubated in 0.5× media for 15 min to visualize and colocalize individual ecDNA ( MYC FISH) with Ki67 IF ( arrowheads ). B , line profile of MYC FISH and Ki67 IF intensity along the line indicated in panel A; polynomial curves were fitted to the line profiles. C , metaphase COLO320DM cells cultured on glass coverslips 2 to 4 days post mCherry-only or Ki67-mCherry expression plasmid transfection; cells were categorized based on mCherry fluorescence: - indicates lack of mCherry expression, ++ indicates top 20% tile mCherry expression by fluorescence intensity, + indicates the remaining cells that express mCherry; dashed outlines indicate chromosomes aligned at the metaphase plate, arrowheads indicate untethered ecDNA. D , boxplots quantifying ecDNA untethering in plasmid transfected cells from panel C ; mCh = mCherry expression category, Plas = plasmid transfected; from left to right ; n = 4, 4, 4, 4, 4, 4 biological replicates and 20, 63, 22, 116, 221, 86 total cells; two-way ANOVA, mCherry expression category (- v er s us + v er s us ++): F = 123.9, p < 0.001; plasmid transfected (mCherry-only v er s us Ki67-mCherry): F = 40.5, p < 0.001; interaction: F = 33.8, p < 0.001; ∗∗ p < 0.01 by Tukey’s HSD, ns = not significant. E , newly-divided daughter COLO320DM cells, as identified by the presence of Aurora B staining (not shown), 2 to 4 days post mCherry-only or Ki67-mCherry expression plasmid transfection (mCherry ++ cells are shown); dashed outlines indicate primary nuclei, arrowheads indicate micronuclei. F , quantification of panel E . Left : quantification of the percentage of daughter cell pairs with micronuclei; chi-squared, ∗∗ p < 0.01. Right: quantification of the percentage of all MYC FISH signal per daughter cell pair inside micronuclei (symlog scale, linear ≤2, log >2); two-way ANOVA, mCherry expression category (- v er s us + v er s us ++): F = 7.6, p < 0.001; plasmid transfected (mCherry-only v er s us Ki67-mCherry): F = 7.6, p = 0.0058; interaction: F = 4.9, p = 0.0073; ∗∗ p < 0.01; n = 7, 7, 7, 7, 7, 7 biological replicates and 93, 312, 104, 179, 400, 147 daughter cell pairs. G and H , newly-divided daughter COLO320HSR cells, transfected similarly as in panels E and F (mCherry ++ cells are shown). H , left : chi-squared, ∗ p < 0.05. Right : two-way ANOVA, mCherry expression category (- v er s us + v er s us ++): F = 0.7, p = 0.49; plasmid transfected (mCherry-only v er s us Ki67-mCherry): F = 0.1, p = 0.77; interaction: F = 0.9, p = 0.39; n = 4, 4, 4, 4, 4, 4 biological replicates and 168, 245, 107, 202, 227, 111 daughter cell pairs. In all panels, scale bar = 10 μm. Except in panel B , each data point in graphs represents one cell or daughter cell pair.

Article Snippet: Human colorectal cancer cell lines COLO320DM and COLO320HSR were purchased from ATCC.

Techniques: Cell Culture, Incubation, Expressing, Plasmid Preparation, Transfection, Fluorescence, Staining

Journal: The Journal of Biological Chemistry

Article Title: Mitotic chromatin compaction tethers extrachromosomal DNA to chromosomes and prevents their mis-segregation into micronuclei

doi: 10.1016/j.jbc.2025.111081

Figure Lengend Snippet: Ki67 loss-of-function decreases ecDNA untethering from chromosomes. A , metaphase COLO320DM wildtype (NT) and MKI67 knockout cells (gRNA #1 and #2), incubated for 15 min in 1× media (not shown), 0.75× media, and 0.5× media (not shown); dashed outlines indicate chromosomes aligned at the metaphase plate, arrowheads indicate untethered ecDNA. B , quantification of ecDNA untethering of COLO320DM wildtype and MKI67 knockout clones from panel A (each data point represents the weighted average of at least 95 cells from each clone) after 15 min incubation in 1× media ( left ; n = 4, 6, 5 clones; one-way ANOVA, F = 1.7, p = 0.23), 0.75× media ( middle ; n = 4, 6, 5 clones; one-way ANOVA, F = 12.4, p = 0.001), and 0.5× media ( right ; n = 4, 6, 5 clones; one-way ANOVA, F = 0.1, p = 0.99); ∗∗ p < 0.01 by Tukey’s HSD, ns = not significant; error bars = mean ± standard deviation. C , metaphase COLO320DM wildtype (NT) and MKI67 knockout cells (gRNA #1 and #2), treated for 24 h with vehicle (not shown), 0.2 μM LMK235, and 0.5 μM LMK235 (not shown); dashed outlines indicate chromosomes aligned at the metaphase plate, arrowheads indicate untethered ecDNA. D , quantification of ecDNA untethering of COLO320DM wildtype and MKI67 knockout clones from panel C (each data point represents the weighted average of at least 60 cells from each clone) after 24 h treatment with vehicle (0.1% DMSO, left ; n = 4, 3, 3 clones; one-way ANOVA, F = 3.9, p = 0.073), 0.2 μM LMK235 ( middle ; n = 4, 3, 3 clones; one-way ANOVA, F = 100.8, p < 0.001, ∗∗ p < 0.01 by Tukey’s HSD), and 0.5 μM LMK235 ( right ; n = 4, 3, 3 clones; one-way ANOVA, F = 0.8, p = 0.51); error bars = mean ± standard deviation; MKI67 knockout clones are gRNA #1 clones 1, 4, and 5, and gRNA #2 clones 1, 4, and 5 (see , A – C ). E , schematic: the biological surfactant Ki67 ( green ) coats the surface of mitotic ecDNA ( red ) and chromosomes ( blue ), helping to prevent tethering by electrostatic repulsion and steric hindrance. In all panels, scale bar = 10 μm.

Article Snippet: Human colorectal cancer cell lines COLO320DM and COLO320HSR were purchased from ATCC.

Techniques: Knock-Out, Incubation, Clone Assay, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Mitotic chromatin compaction tethers extrachromosomal DNA to chromosomes and prevents their mis-segregation into micronuclei

doi: 10.1016/j.jbc.2025.111081

Figure Lengend Snippet: HDAC inhibition leads to a loss of oncogene copy number in COLO320DM cells, but not in COLO320HSR cells. A and B , quantification of MYC DNA expression in COLO320DM ( A ) and COLO320HSR ( B ) cells treated with LMK235; qPCR, 2 -ΔΔCT analysis (normalized to LINE1 copy number and vehicle control); x-axis = symlog scale, linear ≤2, log >2. Error bars = mean ± 95% confidence interval. Drug-containing media was replaced every 2 to 3 days. A , treatment with vehicle (0.1% DMSO), 0.2 μM, 0.3 μM, or 1 μM LMK235 for 1 day (one-way ANOVA, F = 4.1, p = 0.036, n = 4, 4, 4, 3), 2 days (F = 31.3, p < 0.001, n = 4, 4, 4, 3), 10 days (F = 5.4, p = 0.017, n = 6, 6, 6, no 1 μM), 30 days (F = 19.8, p = 0.0023, n = 3, 3, 3, no 1 μM); 0 days: pretreatment (one-way ANOVA, F = 0.4, p = 0.77, n = 3, 3, 3, 3); # p < 0.05, ## p < 0.01 (0.2 μM LMK235 v er s us vehicle); ∗ p < 0.05, ∗∗ p < 0.01 (0.3 μM LMK235 v er s us vehicle); ˆˆ p < 0.01 (1 μM LMK235 v er s us vehicle) by Tukey’s HSD; B , treatment with vehicle, 0.3 μM LMK235, or 1 μM LMK235 for 2 days (one-way ANOVA, F = 2.2, p = 0.17, n = 4, 4, 4), 5 days (F = 0.5, p = 0.62, n = 4, 4, 4), 10 days (F = 10.1, p = 0.019, n = 4, 4), and 30 days (F = 2.7, p = 0.15, n = 4, 4); 0 days: pretreatment (one-way ANOVA, F = 0.8, p = 0.48); ∗ p < 0.05 (0.3 μM LMK235 v er s us vehicle). C , schematic, top : under normal mitotic conditions, ecDNA tether to chromosomes throughout mitosis to ensure their segregation into the primary nuclei of divided daughter cells; bottom : under conditions that decompact chromatin (hypotonic conditions and HDAC inhibition) or prevent ecDNA-chromosome interaction at their surfaces (Ki67 overexpression), ecDNA untether from chromosomes, leading some to be mis-segregated into micronuclei. D and E , schematics of a colloidal and surface chemistry-based framework for approaching ecDNA and chromosome compaction and tethering during mitosis. Arrow 1 represents biophysical changes to the colloidal particles (nucleosomes and chromatin molecules) or the solution/suspension medium (cytosol), such as alterations to the intracellular ionic strength or to the acetylation state of chromatin. Arrow 2 represents changes in the surfactant (Ki67) concentration of the system. Both sets of changes affect particle-particle and particle-solution interactions.

Article Snippet: Human colorectal cancer cell lines COLO320DM and COLO320HSR were purchased from ATCC.

Techniques: Inhibition, Expressing, Control, Over Expression, Suspension, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Radiosensitizer Effect of β-Apopicropodophyllin against Colorectal Cancer via Induction of Reactive Oxygen Species and Apoptosis

doi: 10.3390/ijms222413514

Figure Lengend Snippet: Anti-cancer effects of APP in human colorectal cancer cells. ( A ) Chemical structure of APP. ( B – E ) IC50 values determination for APP. The CRC cell lines HCT116, DLD-1, SW480, and COLO320DM cells were treated with 6.25, 12.5, 25, 50, or 100 nM APP for 48 or 72 h, and cell viability was measured by the MTT assay. IC50 values of APP for CRC cell lines were calculated as described in the Materials and Methods. ( F ) Immunoblot assays for the detection of γH2AX activation in cells treated with APP. HCT116, DLD-1, SW480, and COLO320DM cells were incubated with or without 7.5 nM APP for 24 h prior to harvesting. The relative band densities were determined via densitometry using ImageJ software (NIH, Bethesda, MD, USA) and then normalized to that of each control. All experiments were repeated in triplicate, and statistical analyses were performed with Student’s t -test; * p < 0.05, ** p < 0.01. Bands in the figures show representative data.

Article Snippet: HCT116, DLD-1, SW480, and COLO320DM cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA).

Techniques: MTT Assay, Western Blot, Activation Assay, Incubation, Software, Control

Journal: International Journal of Molecular Sciences

Article Title: Radiosensitizer Effect of β-Apopicropodophyllin against Colorectal Cancer via Induction of Reactive Oxygen Species and Apoptosis

doi: 10.3390/ijms222413514

Figure Lengend Snippet: Radiosensitizer effect of APP. ( A ) Clonogenic assays. Clonogenic assays for DLD-1 and HCT116 cells were performed as described in the Materials and Methods. ‘DMSO’, DMSO-treated mock control; ‘APP’, cells treated with 7.5 nM APP. ( B – E ) Cell counting assay. Cell counting assays were performed as described in the Materials and Methods. ‘Con’, DMSO-treated mock control cells; ‘APP 7.5 nM’, cells treated with 7.5 nM APP; ‘IR 3Gy’, cells treated with 3Gy IR; ‘IR + APP’, cells treated with 7.5 nM APP and 3Gy IR. The lower panel shows microscopic images prior to cell detachment. Experiments were repeated in triplicate, and the results indicate the mean of triplicate assays [ , ]. Each bar in the pictures indicates 500 μm. ( F ) Immunoblot assay for γH2AX. HCT116, DLD-1, SW480, and COLO320DM cells were incubated with or without 7.5 nM APP, exposed to 3 Gy IR, and incubated for 72 h prior to harvest. Each graph in the right panel indicates the statistical analysis of immunoblot bands. The relative band densities were determined via densitometry using ImageJ software (NIH, Bethesda, USA) and then normalized to the density of each control. All experiments were repeated in triplicate, and statistical analyses were performed with Student’s t -test; * p < 0.05, ** p < 0.01, *** p < 0.001. Bands in the figures show representative data.

Article Snippet: HCT116, DLD-1, SW480, and COLO320DM cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Control, Cell Counting, Western Blot, Incubation, Software