Minichromosome maintenance 2 bound with retroviral Gp70 is localized to cytoplasm and enhances DNA-damage-induced apoptosis

Abstract

The interaction of viral proteins with host-cellular proteins elicits the activation of cellular signal transduction pathways and possibly leads to viral pathogenesis as well as cellular biological events. Apoptotic signals induced by DNA-damage are remarkably up-regulated by Friend leukemia virus (FLV) exclusively in C3H hosts; however, the mechanisms underlying the apoptosis enhancement and host-specificity are unknown. Here, we show that C3H mouse-derived hematopoietic cells originally express higher levels of the minichromosome maintenance (MCM) 2 protein than BALB/c- or C57BL/6-deriverd cells, and undergo more frequent apoptosis following doxorubicin-induced DNA-damage in the presence of the FLV envelope protein gp70. Dual transfection with gp70/Mcm2 reproduced doxorubicin-induced apoptosis even in BALB/c-derived 3T3 cells. Immunoprecipitation assays using various deletion mutants of MCM2 revealed that gp70 bound to the nuclear localization signal (NLS) 1 (amino acids 18-24) of MCM2, interfered with the function of NLS2 (amino acids 132-152), and suppressed the normal nuclear-import of MCM2. Cytoplasmic MCM2 reduced the activity of protein phosphatase 2A (PP2A) leading to the subsequent hyperphosphorylation of DNA-dependent protein kinase (DNA-PK). Phosphorylated DNA-PK exhibited elevated kinase activity to phosphorylate P53, thereby up-regulating p53-dependent apoptosis. An apoptosis-enhancing domain was identified in the C-terminal portion (amino acids 703-904) of MCM2. Furthermore, simultaneous treatment with FLV and doxorubicin extended the survival of SCID mice bearing 8047 leukemia cells expressing high levels of MCM2. Thus, depending on its subcellular localization, MCM2 plays different roles. It participates in DNA replication in the nucleus as shown previously, and enhances apoptosis in the cytoplasm.

Figure 1. In vivo assessment of doxorubicin-induced apoptosis and the associated changes in mRNA expression in FLV-infected mice.

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 (−)” (*p<0.01 and ^# p<0.01). Data represent the mean and 95% confidence intervals (CI) of 3 independent experiments. (D) Quantitative RT-PCR analysis of Mcm2 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 Mcm2 in all groups compared to the corresponding groups of BALB/c and C57BL/6 mice (*p<0.01, for each group). (E) Quantitative RT-PCR analysis of Mcm2 mRNA expression in the spleen of uninfected and FLV-infected BALB/c, C57BL/6, and C3H mice. Spleen Mcm2 expression is higher in the “FLV (+), Doxorubicin (−)” and “FLV (+), Doxorubicin (+)” C3H mice than in the corresponding groups of BALB/c and C57BL/6 mice (*p<0.01 and ^# p<0.01, respectively). In C3H mice, FLV-infection induces higher levels of Mcm2 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 Mcm-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 Mcm2 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 Mcm 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). Mcm family members, Dna-pk, caspase-3 (Casp3), Ppp2ac, and Ppp6 exhibit gene expression patterns similar to that of Mcm2.

Figure 2. Dual transfection of gp70 and Mcm2 enhances DNA-damage-induced apoptosis in 3T3 cells.

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

Figure 3. Direct interaction of MCM2 with gp70.

(A) 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 HA-tagged Mcm2 mutants along with FLAG-tagged gp70, and either left untreated (B) or treated with 1 µM doxorubicin for 24 h (C). The expression of the MCM2 mutants (B, C, left upper) and FLAG-gp70 (B, C, 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, C, right panel).

Figure 4. The C-terminal portion of MCM2 is important for apoptosis enhancement.

3T3 cells were co-taransfected with gp70 and Mcm2-FL or the mutants (A, B) or transfected with Mcm2-FL or the mutants (C, D) and treated with 1 µM doxorubicin for 24 h. Cell survival (A, C) and apoptotic cell ratios (B, D) were determined using the MTT assay and annexin V-staining, respectively. Asterisks (*) indicate p<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 gp70/Mcm2-FL- and gp70/mutant-transfected 3T3 cells (E) and Mcm2-FL- and mutant-transfected 3T3 cells (F) 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. (G) 3T3 cells co-transfected with gp70/Mcm2-FL or gp70/mutants and (H) 3T3 cells transfected with Mcm2-FL 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 (G and H, bottom) compared to the levels in the absence of NU7026 (E and F, respectively). Whole cell lysates from gp70- and Mcm2-FL-transfected 3T3 cells after doxorubicin treatment are shown as a positive control (PC, G and H, bottom). Apoptotic cell ratios were determined with annexin V-staining (G and H, upper graph). In both panels, data represent the mean and 95% CI of three independent experiments.

Figure 5. MCM2 (FL and mutants) interacts with PP2A.

(A) The Mcm2-FL- or mutant-transfected 3T3 cells (left) and gp70/Mcm2-FL- or gp70/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) 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 p<0.01 for control vs. mutant-transfected cells. Data represent the mean and 95% CI of 3 independent experiments. (C) 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.

Figure 6. Subcellular localization of MCM2 and the role of the NLS domains in enhancing doxorubicin-induced apoptosis.

HA-Mcm2-FL and HA-mutant-transfected 3T3 cells (A), and FLAG-gp70/HA-Mcm2-FL and FLAG-gp70/HA-mutant-transfected 3T3 cells (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. (C) Schematic diagram of the NLS deletion mutants MCM2-ΔNLS1, MCM2-ΔNLS2, and MCM2-ΔNLS1-2. (D) Mcm2-NLS 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 Mcm2-ΔNLS2- or Mcm2-ΔNLS 1-2-transfected cells (*p<0.01). Data represent the mean and 95% CI of 3 independent experiments.

Figure 7. In vivo anti-tumor effects of gp70 expression and DNA-damage on the C3H-derived cells in SCID mice.

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. (A) Quantitative RT-PCR analysis of gp70 mRNA expression in the liver of SCID mice with multiple foci of leukemic infiltration. The samples from FLV-infected mice exhibit higher levels of gp70 than those from uninfected mice (*p<0.01). Data represent the mean and 95% CI of from 10 mice in each group and are representative of 2 independent experiments. (B) Microscopic features of TUNEL-positive cells in hepatic nodules and (C) 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 (*p<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. (D) 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. (E) The cell counts for cytoplasmic localization of MCM2. Cell counts are shown as the number of cells per 10 high-power fields (HPF). [# p<0.01 compared with tumor cells of “FLV (−), doxorubicin (−)” mice; *p<0.001 compared with “FLV (−) doxorubicin (−)” mice and p<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. (F) 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 [p<0.01 compared with “FLV (−), doxorubicin (−)” and “FLV (−), doxorubicin (+)” mice] and in mice with FLV-infection and doxorubicin-treatment [p<0.001 compared with “FLV (−), doxorubicin (−)” and “FLV (−), doxorubicin (+)” mice, p<0.01 compared with “FLV (+), doxorubicin (−)” mice]. The survival curves represent data from 10 mice in each group.

Figure 8. Schematic illustration of the structure of MCM2 and its functions in the cytoplasm and nucleus.

(A) The various functional domains of the MCM2 protein are shown, and the domains and regions required for the activities are indicated. (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).