Subcellular localization of MCM2 and the role of the NLS domains in enhancing doxorubicin-induced apoptosis.

<p><i>HA-Mcm2-FL</i> and <i>HA-</i>mutant-transfected 3T3 cells (<b>A</b>), and <i>FLAG-gp70</i>/<i>HA-Mcm2-FL</i> and <i>FLAG-gp70</i>/<i>HA</i>-mutant-transfected 3T3 cells (<b>B</b>) were treated with 1 µM doxorubicin for 24 h. HA-positive cells containing the MCM2-derived proteins are shown in red (TRITC), and DAPI-stained nuclei are shown in blue. Images were acquired using a BZ-9000 microscope (KEYENCE) with a 400× objective. (<b>C</b>) Schematic diagram of the NLS deletion mutants MCM2-ΔNLS1, MCM2-ΔNLS2, and MCM2-ΔNLS1-2. (<b>D</b>) <i>Mcm2-NLS</i> deletion mutant-transfected 3T3 cells were treated with 1 µM doxorubicin for 24 h, and apoptotic cell ratios were determined with annexin V-staining. Data represent the mean and SD of 3 experiments. The asterisks (*) indicate significant differences between the control and <i>Mcm2-ΔNLS2</i>- or <i>Mcm2-ΔNLS 1-2</i>-transfected cells (*<i>p</i><0.01). Data represent the mean and 95% CI of 3 independent experiments.</p

Dual transfection of <i>gp70</i> and <i>Mcm2</i> enhances DNA-damage-induced apoptosis in 3T3 cells.

<p>(<b>A</b>) Quantitative RT-PCR analysis of <i>Mcm2</i> mRNA expression in untreated and doxorubicin-treated BALB/c-derived BaF3 and 3T3 cells, and primary cultured fibroblasts, and C3H-derived 8047 and 32D cells, and primary cultured fibroblasts. Data represent the mean and 95% CI of 3 independent experiments. (<b>B</b>) Cell survival (% of control) measured with the MTT assay in <i>gp70</i> and/or <i>Mcm2</i>-transfected 3T3 cells after treatment with doxorubicin for 24 h. Cell survival is significantly different between control cells “<i>gp70</i> (−), <i>Mcm2</i> (−)” and <i>gp70/Mcm2</i>-transfected cells “<i>gp70</i> (+), <i>Mcm2</i> (+)” (#<i>p</i><0.01). Data represent the mean and 95% CI of 3 independent experiments. (<b>C</b>) Apoptotic cell ratios in <i>gp70</i> and/or <i>Mcm2</i>-transfected 3T3 cells were determined with annexin V-staining after treatment with 1 µM doxorubicin for 24 h. The ratios in the control cells “<i>gp70</i> (−), <i>Mcm2</i> (−)” and <i>gp70/Mcm2</i>-transfected cells “<i>gp70</i> (+), <i>Mcm2</i> (+)” are significantly different (#<i>p</i><0.01). Data represent the mean and 95% CI of 3 independent experiments. (<b>D</b>) Western blot analysis of <i>gp70</i> and/or <i>Mcm2-FL</i>-transfected 3T3 cells after treatment with 1 µM of doxorubicin for 24 h. Gp70 and MCM2 protein levels are similar in all groups. (<b>E</b>) Expression of endogenous <i>gp70</i> mRNA in BaF3, 3T3, 8047, and 32D cells. G<i>p70</i> mRNA expression (ng) was normalized to that of <i>GAPDH</i>. Note the significantly higher expression of <i>gp70</i> mRNA in 32D cells compared to that in the other cells (*<i>p</i><0.01). Data show the mean and 95% CI of three independent experiments. (<b>F</b>) <i>Mcm2</i> knockdown in BaF3 and 32D cells using siRNA. Quantitative RT-PCR (upper) was performed to confirm <i>si-Mcm2</i>-induced reduction of <i>Mcm2</i> mRNA expression. Apoptotic cell ratios were determined with annexin V-staining after treatment with doxorubicin for 24 h (bottom). Note the significant decrease in the apoptotic cell ratio of 32D cells treated with <i>si-Mcm2,</i> compared to that of cells treated with <i>si-Control</i> (*<i>p</i><0.01). Data show the mean and 95% CI of 3 independent experiments.</p

Schematic illustration of the structure of MCM2 and its functions in the cytoplasm and nucleus.

<p>(<b>A</b>) The various functional domains of the MCM2 protein are shown, and the domains and regions required for the activities are indicated. (<b>B</b>) Schematic of the novel role of MCM2 in apoptosis enhancement. Normally, MCM2 is recruited into the nucleus for participation in DNA replication. As a result, cellular proliferation is upregulated (proliferation signal). However, when gp70 is present in the cytoplasm, it binds to MCM2 and inhibits its nuclear entry. Furthermore, cytoplasmic gp70-MCM2-complex interacts with PP2A and inhibits its interaction with DNA-PK. Consequently, hyperphosphorylated DNA-PK enhances DNA-damage-induced apoptosis via a P53-related pathway (apoptosis signal).</p

<i>In vivo</i> anti-tumor effects of <i>gp70</i> expression and DNA-damage on the C3H-derived cells in SCID mice.

<p>Two weeks after transplantation, mice were inoculated (i.p.) with FLV. Seven days later, the mice were treated with 1.5 mg/kg of doxorubicin or PBS. (<b>A</b>) Quantitative RT-PCR analysis of <i>gp70</i> mRNA expression in the liver of SCID mice with multiple foci of leukemic infiltration. The samples from FLV-infected mice exhibit higher levels of <i>gp70</i> than those from uninfected mice (*<i>p</i><0.01). Data represent the mean and 95% CI of from 10 mice in each group and are representative of 2 independent experiments. (<b>B</b>) Microscopic features of TUNEL-positive cells in hepatic nodules and (<b>C</b>) TUNEL-positive cell ratio in each group of mice. Note the significant increase in apoptotic 8047 cells in mice with FLV infection and doxorubicin treatment (*<i>p</i><0.01 compared with the tumor cells of “FLV (−), doxorubicin (−) mice”). Data represent the mean and 95% CI of from 10 mice in each group and are representative of 2 independent experiments. (<b>D</b>) Subcellular localization of MCM2 in 8047 cells of the liver demonstrated by immunohistochemistry. Images were captured with a microscope at 1,000× magnification power. Note the nuclear and/or cytoplasmic localization of MCM2 in the 8047 cells from each group of mice. (<b>E</b>) The cell counts for cytoplasmic localization of MCM2. Cell counts are shown as the number of cells per 10 high-power fields (HPF). [# <i>p</i><0.01 compared with tumor cells of “FLV (−), doxorubicin (−)” mice; *<i>p</i><0.001 compared with “FLV (−) doxorubicin (−)” mice and <i>p</i><0.05 compared with “FLV (+), doxorubicin (−)” mice]. Data represent the mean and 95% CI of from 10 mice in each group and are representative of 2 independent experiments. (<b>F</b>) Kaplan-Meier survival curves for 8047-transplanted SCID mice with/without FLV-infection and doxorubicin-treatment. Note the significant elongation of survival time in mice with FLV-infection [<i>p</i><0.01 compared with “FLV (−), doxorubicin (−)” and “FLV (−), doxorubicin (+)” mice] and in mice with FLV-infection and doxorubicin-treatment [<i>p</i><0.001 compared with “FLV (−), doxorubicin (−)” and “FLV (−), doxorubicin (+)” mice, <i>p</i><0.01 compared with “FLV (+), doxorubicin (−)” mice]. The survival curves represent data from 10 mice in each group.</p

Direct interaction of MCM2 with gp70.

<p>(<b>A</b>) Schematic diagram of full-length MCM2 (MCM2-FL) and MCM2 deletion mutants, MCM2-ΔC (aa 1–703), MCM2-ΔN (aa 156–703), MCM2-N (aa 1–155) and MCM2-C (aa 704–904). The NLS domains are shown in black, and the Zn-finger domains are gray. 3T3 cells were transfected with <i>HA</i>-tagged <i>Mcm2</i> mutants along with <i>FLAG</i>-tagged <i>gp70</i>, and either left untreated (<b>B</b>) or treated with 1 µM doxorubicin for 24 h (<b>C</b>). The expression of the MCM2 mutants (<b>B</b>, <b>C</b>, left upper) and FLAG-gp70 (<b>B</b>, <b>C</b>, left middle) was confirmed in 3T3 cells. Cell lysates were subjected to a pull-down assay to detect the binding of MCM2-FL or MCM2 mutants to FLAG-gp70 (<b>B</b>, <b>C</b>, right panel).</p

MCM2 (FL and mutants) interacts with PP2A.

<p>(<b>A</b>) The <i>Mcm2-FL</i>- or mutant-transfected 3T3 cells (left) and <i>gp70</i>/<i>Mcm2-FL-</i> or <i>gp70</i>/mutants-transfected 3T3 cells (right) were treated with 1 µM doxorubicin for 24 h. Cell lysates were subjected to a pull-down assay to detect the binding of MCM2-FL or the mutants to PP2A. (<b>B</b>) 3T3 cells were pre-incubated with 10 nM okadaic acid (OA) and 10 µM NU7026 for 2 h, and treated with 1 µM doxorubicin for 24 h. The apoptotic cell ratio was determined with annexin V-staining. Asterisk (*) indicates <i>p</i><0.01 for control vs. mutant-transfected cells. Data represent the mean and 95% CI of 3 independent experiments. (<b>C</b>) Western blot analysis of 3T3 cells to detect phospho-DNA-PK. Note the significantly increased levels of DNA-PK-p2053 in OA-treated 3T3 cells, and the complete abrogation by NU7026.</p

<i>In vivo</i> assessment of doxorubicin-induced apoptosis and the associated changes in mRNA expression in FLV-infected mice.

<p>Uninfected or FLV-infected BALB/c (A), C57BL/6 (B), and C3H (C) mice were intraperitoneally (i.p.) administrated with 1.5 mg/kg of doxorubicin or PBS, and the apoptotic cell ratios in the bone marrow (gray bars) and spleen cells (black bars) were determined 24 h later with annexin V-staining. Note the significant increase in the proportion of annexin V-positive cells in the bone marrow and spleen of FLV-infected C3H mice after the doxorubicin treatment compared to that in the bone marrow and spleen cells of uninfected mice “FLV (−), Doxorubicin (−)” (*<i>p</i><0.01 and ^#<i>p</i><0.01). Data represent the mean and 95% confidence intervals (CI) of 3 independent experiments. (D) Quantitative RT-PCR analysis of <i>Mcm2</i> mRNA expression in the bone marrow of uninfected and FLV-infected BALB/c, C57BL/6, and C3H mice. The bone marrow cells of the C3H strain exhibit higher levels of <i>Mcm2</i> in all groups compared to the corresponding groups of BALB/c and C57BL/6 mice (*<i>p</i><0.01, for each group). (E) Quantitative RT-PCR analysis of <i>Mcm2</i> mRNA expression in the spleen of uninfected and FLV-infected BALB/c, C57BL/6, and C3H mice. Spleen <i>Mcm2</i> expression is higher in the “FLV (+), Doxorubicin (−)” and “FLV (+), Doxorubicin (+)” C3H mice than in the corresponding groups of BALB/c and C57BL/6 mice (*<i>p</i><0.01 and ^#<i>p</i><0.01, respectively). In C3H mice, FLV-infection induces higher levels of <i>Mcm2</i> expression compared to the expression in uninfected mice. Data represent the mean and 95% CI from 5 mice in each group and are representative of 2 independent experiments. The GeneChip data for <i>Mcm</i>-associated and apoptosis-associated genes were analyzed using the Percellome method. Forty-eight male C57BL/6 and C3H mice were divided into 16 groups of 3 mice each. Uninfected or FLV-infected C57BL/6 and C3H mice were administered (i.p.) with 15 mg/kg (high dose) or 1.5 mg/kg (low dose) of doxorubicin, and the spleen was sampled 0, 1, 6, and 24 h after administration. The spleen transcriptome was measured using the Affymetrix Mouse 430-2 GeneChip. (F) The Percellome data were plotted on 3-dimensional graphs for average, +1 SD, and −1 SD surfaces as demonstrated in the left schema. The scale of expression (vertical axis) is the copy number per cell. The x-axis of the 3-dimensional graph shows the experimental groups, including the C3H and C57BL/6 mice with doxorubicin treatment (high and low doses) with or without FLV-infection. The y-axis shows the time course (0, 1, 6, and 24 h) after treatment with doxorubicin and the z-axis (vertical) indicates the intensity of mRNA expression of each gene. The data of each point are connected to form a surface illustration. The expression patterns of genes are compared using the surface images. (G) The <i>Mcm2</i> expression pattern is shown in the upper right box. Of the lower columns, the first column (H) shows the data for the genes of representative <i>Mcm</i> family members, the second column (I), PI3K members, the third column (J), p53-associated genes, the fourth column (K), caspase members and fifth column (L), protein phosphatase members (PPs). <i>Mcm</i> family members, <i>Dna-pk</i>, <i>caspase-3</i> (<i>Casp3</i>), <i>Ppp2ac,</i> and <i>Ppp6</i> exhibit gene expression patterns similar to that of <i>Mcm2</i>.</p

The C-terminal portion of MCM2 is important for apoptosis enhancement.

<p>3T3 cells were co-taransfected with <i>gp70</i> and <i>Mcm2-FL</i> or the mutants (<b>A</b>, <b>B</b>) or transfected with <i>Mcm2-FL</i> or the mutants (<b>C</b>, <b>D</b>) and treated with 1 µM doxorubicin for 24 h. Cell survival (<b>A</b>, <b>C</b>) and apoptotic cell ratios (<b>B</b>, <b>D</b>) were determined using the MTT assay and annexin V-staining, respectively. Asterisks (*) indicate <i>p</i><0.01 for control vs. mutant-transfected cells. In all panels, data represent the mean and 95% CI of 3 independent experiments. Western blot analysis of <i>gp70</i>/<i>Mcm2-FL-</i> and <i>gp70</i>/mutant-transfected 3T3 cells (<b>E</b>) and <i>Mcm2-FL-</i> and mutant-transfected 3T3 cells (<b>F</b>) after treatment with 1 µM doxorubicin for 24 h. The levels of DNA-PK, phospho-DNA-PK (pS2053), P53, phospho-P53, and cleaved caspase-3 are elevated in the groups with elevated apoptotic ratios. (<b>G</b>) 3T3 cells co-transfected with <i>gp70</i>/<i>Mcm2-FL</i> or <i>gp70</i>/mutants and (<b>H</b>) 3T3 cells transfected with <i>Mcm2-FL</i> or the mutants were pre-incubated with 10 µM NU7026, a DNA-PK-inhibitor, for 2 h and treated with 1 µM doxorubicin for 24 h. DNA-PK-pS2053 levels are substantially reduced in cells treated with the DNA-PK-inhibitor (<b>G</b> and <b>H</b>, bottom) compared to the levels in the absence of NU7026 (<b>E</b> and <b>F</b>, respectively). Whole cell lysates from <i>gp70-</i> and <i>Mcm2-FL</i>-transfected 3T3 cells after doxorubicin treatment are shown as a positive control (PC, <b>G</b> and <b>H</b>, bottom). Apoptotic cell ratios were determined with annexin V-staining (<b>G</b> and <b>H,</b> upper graph). In both panels, data represent the mean and 95% CI of three independent experiments.</p