Transient Nutlin-3 treatment of diploid HCT116 clones induces the appearance of cells with>4N DNA content.

<p><b>A</b>) Diploid HCT116 clones D3 and D8 were untreated (NT) or exposed to Nutlin (NUT 10 µM) for 24 hrs, followed by Nutlin removal. The cells were harvested at the indicated times after Nutlin removal. Fixed cells were stained with propidium iodide (25 µg/ml) and subjected to flow cytometry analysis. <b>B</b>) Cells were untreated (NT) or exposed to Nutlin (NUT 10 µM) for 24 hrs, followed by Nutlin removal. Cell lysates were collected at the indicated time points and analyzed by immunoblotting with the indicated antibodies. Actin was as a loading control. <i>P-Cdc2</i>, phosphor-Cdc2 (Tyr-15).</p

Stable tetraploid clones were isolated from diploid D3 and D8 clones after endoreduplication.

<p><b>A</b>) Shown is the procedure to isolate diploid and tetraploid clones from cells transiently exposed to Nutlin. D3 and D8 were untreated (NT) or treated with 10 µM Nutlin (NUT) for 24 hrs, followed by Nutlin removal for an additional 16 hrs. The cells were then live-stained with Hoechst 33342 (5 µg/ml). Cell sorting was performed on a MoFlo cytometer equipped with a UV excitation wavelength laser. Sorted 2N and 8N cells were plated at low density in normal medium (minus Nutlin) and individual clones isolated. <b>B</b>) <i>Top</i>, the comparison of the DNA profiles between D3 and a representative tetraploid clone isolated from D3. <i>Bottom</i>, comparison of the DNA profiles between a representative diploid clone (D81B) isolated from D8 cells, and a representative tetraploid clone (TD6) isolated from D8 cells. <b>C</b>) Representative metaphase spread from diploid D3 cells and tetraploid T3 cells. The number in the bottom right indicates the number of chromosomes counted. <b>D</b>) FISH analysis with chromosome 17 (Chr 17) and p53-specific probes shows tetraploid (T3) cells contain 4 copies of p53 and Chr 17, while diploid (D3) cells contain 2 copies of p53 and Chr 17.</p

Tetraploid clones express more p53-MDM2 complexes and more p53 after Nutlin treatment than diploid counterparts.

<p><b>A</b>) P53 and MDM2 mRNA levels were compared in untreated tetraploid clones (T3, TD6) and their diploid counterparts (D3, D81B). <b>B</b>) Diploid (D3, D81B) and tetraploid (T3, TD6) clones were untreated or treated with proteasome inhibitor MG132 (10 µM) for 8 hrs. <i>Upper</i> Levels of MDM2, p53, and actin (loading control) in untreated and MG132 treated cells are shown. <i>Lower</i> To determine levels of p53-MDM2 complexes in diploid and tetraploid cells, protein lysates were immunoprecipitated with anti-p53 antibody, followed by immunoblotting for MDM2. <b>C</b>) Diploid (D3, D81B) and tetraploid (T3, TD6) clones were untreated or treated with Nutlin (10 µM) for 24 hrs. p53 and MDM2 protein levels were detected by immunoblotting and quantified using Image-J software. The relative amount of p53 and MDM2 protein in the untreated diploid clones was given a value of “1.0”. Numbers indicate the relative level of each protein. Actin was used as a loading control.</p

Tetraploid clones show resistance to cisplatin (CP) and ionizing radiation (IR)-induced apoptosis.

<p><b>A</b>) Five diploid clones isolated from Nutlin treated D3 cells (D3 Diploid) and D8 cells (D8 Diploid), and five tetraploid clones isolated from Nutlin treated D3 cells (D3 Tetraploid) and D8 cells (D8 Tetraploid) were untreated (NT) or exposed to CP (20 µM) or IR (10 Gy) for 48 hrs. The percentage of cells with sub-G1 DNA was determined. Shown are the mean results from three separate experiments, <i>bars</i>, Standard error (SE). * significance value (P<0.05). <b>B</b>) Tetraploid clones (T3, TD6) and their diploid counterparts (D3, D81B) were untreated (NT) or exposed to CP (20 µM) or IR (10 Gy) for 72 hrs. The percentage of cells with sub-G1 DNA (propidium iodide staining) was determined. Shown are the mean results from three separate experiments, <i>bars</i>, Standard error (SE). * significance value (P<0.05). <b>C</b>) The indicated diploid and tetraploid clones were untreated (NT) or exposed to CP (20 µM) or IR (10 Gy) for 24 hrs. p53, p21, and MDM2 protein levels were determined by immunoblotting and quantified. Numbers indicate the relative level of each protein. Actin was used as a loading control. <b>D</b>) The indicated diploid and tetraploid clones untreated (NT) or exposed to CP (20 µM) or IR (10 Gy) for 48 hrs. Cleaved PARP and Caspase-3 protein levels were determined by immunoblotting and quantified relative to the untreated. <b>E</b>) qRT-PCR was used to determine mRNA levels for the indicated genes in diploid (D3, D81B) and tetraploid (T3, TD6) clones that were either untreated (NT) or exposed to CP (20 µM) or IR (10 Gy) for 24 hrs. The level of each mRNA transcript in untreated diploid clones (D3 NT, D81B NT) was considered “1.0”, and all other values are plotted relative to it.</p

Nutlin-3 causes a p53-dependent tetraploid G1-arrest in diploid HCT116 clones.

<p><b>A</b>) HCT116 diploid clones D3 and D8, and HCT116 that express wild-type p53 (p53+/+) or are p53-null (p53-/-) were untreated or treated with Nutlin (NUT 10 µM) for 24 hrs, and cell cycle profile determined by flow cytometry. <b>B</b>) Expression of the indicated proteins was monitored by immunoblot in cells untreated or treated with Nutlin (NUT 10 µM) for 24 hrs. <b>C</b>) mRNA levels for the indicated factors in untreated cells or cells treated with Nutlin (NUT 10 µM) for 24 hrs was determined by qRT-PCR.</p

Tetraploid clones are more susceptible to Nutlin-induced cell cycle arrest and apoptosis.

<p><b>A</b>) Five diploid clones isolated from Nutlin treated D3 cells (D3 Diploid) and D8 cells (D8 Diploid), and five tetraploid clones isolated from Nutlin treated D3 cells (D3 Tetraploid) and D8 cells (D8 Tetraploid) were untreated or treated with increasing Nutlin (1.0–5.0 µM) for 24 hrs. The percent G1 and S-phase cells was determined by flow cytometry, and the fold change of G1/S ratio is plotted. Untreated diploid clones G1/S ratio was given a value of 1.0. Shown is the average of three separate experiments, +/- SE. <b>B</b>) Tetraploid clones (T3, TD6) and diploid counterparts (D3, D81B) were untreated or treated with Nutlin (1.0–5.0 µM) for 24 hrs. The fold change of G1/S ratio is plotted. Shown is the average of three separate experiments, +/- SE. <b>C</b>) Diploid (D3, D81B) and tetraploid (T3, TD6) clones were untreated or treated with increasing Nutlin (1.0–5.0 µM) 24 hrs. P53 and p21 protein levels were quantified using Image-J software. Numbers indicate the relative levels of each protein. P53 and p21 levels in untreated diploid clones was given a value of “1.0”. Actin was used as loading control. <b>D</b>) Five diploid clones isolated from Nutlin treated D3 cells (D3 Diploid) and D8 cells (D8 Diploid), and five tetraploid clones isolated from Nutlin treated D3 cells (D3 Tetraploid) and D8 cells (D8 Tetraploid) were untreated (NT) or treated with Nutlin (20 µM) 72 hrs and apoptosis determined. Shown are the mean results from three separate experiments, <i>bars</i>, Standard error (SE). *(P<0.05). <b>E</b>) Representative diploid (D3, D81B) and tetraploid (T3, TD6) clones were untreated (NT) or treated with Nutlin (20 µM) for 72 hrs. The percentage of cells with sub-G1 DNA content (propidium iodide staining) was determined. Shown are the mean results from three separate experiments, <i>bars</i>, Standard error (SE). * significance value (P<0.05). <b>F</b>) The indicated diploid and tetraploid clones were untreated (NT) or exposed to Nutlin (20 µM) for 48 hrs. Cleaved PARP and Caspase-3 protein levels were determined by immunoblotting and quantified relative to the untreated.</p

Novel Roles for P53 in the Genesis and Targeting of Tetraploid Cancer Cells

<div><p>Tetraploid (4N) cells are considered important in cancer because they can display increased tumorigenicity, resistance to conventional therapies, and are believed to be precursors to whole chromosome aneuploidy. It is therefore important to determine how tetraploid cancer cells arise, and how to target them. P53 is a tumor suppressor protein and key regulator of tetraploidy. As part of the “tetraploidy checkpoint”, p53 inhibits tetraploid cell proliferation by promoting a G1-arrest in incipient tetraploid cells (referred to as a tetraploid G1 arrest). Nutlin-3a is a preclinical drug that stabilizes p53 by blocking the interaction between p53 and MDM2. In the current study, Nutlin-3a promoted a p53-dependent tetraploid G1 arrest in two diploid clones of the HCT116 colon cancer cell line. Both clones underwent endoreduplication after Nutlin removal, giving rise to stable tetraploid clones that showed increased resistance to ionizing radiation (IR) and cisplatin (CP)-induced apoptosis compared to their diploid precursors. These findings demonstrate that transient p53 activation by Nutlin can promote tetraploid cell formation from diploid precursors, and the resulting tetraploid cells are therapy (IR/CP) resistant. Importantly, the tetraploid clones selected after Nutlin treatment expressed approximately twice as much <i>P53</i> and <i>MDM2</i> mRNA as diploid precursors, expressed approximately twice as many p53-MDM2 protein complexes (by co-immunoprecipitation), and were more susceptible to p53-dependent apoptosis and growth arrest induced by Nutlin. Based on these findings, we propose that p53 plays novel roles in both the formation and targeting of tetraploid cells. Specifically, we propose that 1) transient p53 activation can promote a tetraploid-G1 arrest and, as a result, may inadvertently promote formation of therapy-resistant tetraploid cells, and 2) therapy-resistant tetraploid cells, by virtue of having higher <i>P53</i> gene copy number and expressing twice as many p53-MDM2 complexes, are more sensitive to apoptosis and/or growth arrest by anti-cancer MDM2 antagonists (e.g. Nutlin).</p></div

Tetraploid clones are more resistant than diploid clones to Cisplatin (CP) and ionizing radiation (IR) and more sensitive to Nutlin in a long term survival assay.

<p><b>A</b>) Diploid (D3, D81B) and tetraploid (T3, TD6) clones were untreated or treated with CP (10 µM) for 24 hrs. Cells were rinsed and re-fed with drug free media and stained after 2–3 weeks. The colonies were counted and normalized with plating efficiency of untreated controls. Shown are the mean results from three separate experiments. <b>B</b>) Indicated clones were untreated or exposed to IR (3 Gy). Colonies were counted 2–3 weeks later and normalized with plating efficiency of untreated controls. Shown are the mean results from three separate experiments. <b>C</b>) Diploid and tetraploid clones were grown in normal medium (minus Nutlin) or grown in continuous Nutlin (1 µM) and stained after 2–3 weeks. Colony number was normalized with the plating efficiency of untreated controls. <b>D</b>) Indicated clones were untreated or treated with Nutlin (20 µM) for 72 hrs after which the cells were rinsed and re-fed with normal medium (minus Nutlin) for 2–3 weeks. The colonies were then counted and normalized with the plating efficiency of untreated controls. Shown are the mean results from three separate experiments. <i>bars</i> indicate standard error (SE). * significance value (P<0.05).</p

HCT116 clones vary in sensitivity to cisplatin.

<p><b>A)</b> HCT116 cells were plated at single cell density and ten individual clones (designated D1–D10) were isolated and expanded. The clones were exposed to 15 µM Cisplatin for 72 hrs, and apoptosis determined by the percentage of cells with decreased mitochondrial membrane potential (% low ΛΨm), sub-G1 DNA content, or Annexin-V positive staining. (H) refers to the parental HCT116 cell population. Data represent the average of 3 experiments +/− s.e.m. <b>B)</b> Clones D3 and D6 were treated with 5 or 15 µM cisplatin for 24 hrs, followed by cisplatin removal. Percent colony formation was determined 2 weeks after cisplatin removal. Plotted is the average of 3 separate experiments +/− s.e.m.</p

Cisplatin induces a tetraploid G1 arrest that is p53 and p21 dependent.

<p><b>A)</b> HCT116 clones D6 and D7 were treated with 15 µM cisplatin (CP) for 24 or 48 hrs, followed by immunoblotting for the indicated proteins. Actin levels were used as a loading control. <b>B)</b> HCT116 clones D6 and D7 mock transfected (MT) or transfected with control siRNA or siRNA targeting p53 or p21. Twenty four hrs after transfection, the cells were treated with cisplatin (15 µM) for an additional 48 hrs. Protein lysates examined by immunoblotting.</p