Seven-to-eleven-week-old Balb/c and nude (Severe combined immunodeficient, SCID) mice (Huafukang Technology Corp., Beijing, China), CD19^cre mice and Foxo1^F/F (Nanjing Biomedical Research Institute of Nanjing University, Nanjing, China) were bred in our animal facilities under specific pathogen-free conditions. Mice with loxP sites flanking exon 2 of Foxo1 (Foxo1^F/F) were crossed to mice expressing Cre recombinase under control of the CD19 promoter (CD19^Cre) to delete Foxo1 in B cells. Care, use and treatment of mice in the present study were in strict agreement with international guidelines for the care and use of laboratory animals. This study was approved by the Animal Ethics Committee of the Beijing Institute of Basic Medical Sciences.

APC-conjugated anti-mouse CD138 (281-2) was from BD Biosciences (San Jose, CA, USA). FITC or PE conjugated anti-mouse Grm3 (bs-12012R) was from Bioss Corp. (Woburn, MA, USA). Anti-Grm3 (ab13193) and anti-β-actin (ab8227) were from Abcam (Cambridge (MA, USA). Anti-mouse Foxo1 (L27 and C29H4) was from Cell Signaling Technology (Danvers, MA, USA). Apoptosis detection kits (70-AP101-100) contained FITC or APC-conjugated Annexin V and PI were from MultiSciences. Grm3 shRNA (MSH042669-HIVU6) and LV81/Grm3 (EX-Mm13188-Lv81) were from GeneCopoeia (Rockville, MD, USA). Foxo1 shRNA (sc-35383-V) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). FK506 (F4679), nocodazole (M1404), monastrol (M8515), rapamycin (37094) and LPS (L2880) were from Sigma-Aldrich (St. Louis, MO, USA). Bortezomib (M1686) was from Abmole Bioscience Inc. (Houston, TX, USA).

Human B-cell leukemia cell line Nalm-6 cells and mouse myeloma cell line SP 2/0 cells and human embryonic kidney HEK 293T cells were from the American Type Culture Collection (ATCC; Rockville, MD, USA). Primary B cells were sorted from from CD19^cre/+Foxo1^F/+ and CD19^cre/+Foxo1^F/F mice by using B220 microbeads (autoMACS; Miltenyi Biotec, San Diego, CA, USA). All cells were cultured in RPMI-1640 supplemented with 10% fetal calf serum (FCS) and 50 μM 2-mercaptoethanol at 37°C under a 5% CO₂. In some experiments, different concentrations of FK506, nocodazole, monastrol, rapamycin and LPS were added into the culture.

Annexin V/PI staining was performed as previously described (25). Briefly, cells were centrifuged at 335 × g for 10 min and resuspended in 2 ml 1X phosphate-buffered saline (PBS) −/− (no calcium, no magnesium). Cells were centrifuged at 335 × g for 10 min and resuspended in 1 ml 1X Annexin V binding buffer. A total of 5 μl Alexa Fluor 488-conjugated Annexin V was added and the tubes incubated in the dark for 15 min at room temperature. A total of 100 μl of 1X Annexin V binding buffer was added to each reaction tube (final volume: ~200 μl). PI (4 μl) was diluted 1:10 in 1X Annexin V binding buffer and a final PI concentration of 2 μg/ml was added in each sample. Tubes were incubated in the dark for 15 min at room temperature. 1X Annexin V binding buffer (500 μ) was added to wash the cells. Then the samples were ready to be analyzed by flow cytometry (FACS).

Cytometric analysis has been described in our previous studies (24,26). Briefly, cells (1×10⁶ cells/sample) were washed with fluorescence-activated cell sorting staining buffer (phosphate-buffered saline, 2% fetal bovine serum or 1% bovine serum albumin, 0.1% sodium azide). All samples were incubated with anti-Fc receptor Ab (BD Biosciences), prior to incubation with other Abs diluted in fluorescence-activated cell sorting buffer supplemented with 2% anti-Fc receptor Ab. The samples were filtered immediately before analysis or cell sorting to remove any clumps. Data collection and analyses were performed on a FACSCalibur flow cytometer using CellQuest software.

Quantitative PCR analysis was described in our previous studies (24). Briefly, total RNA was extracted from B cells with TRIzol (Invitrogen-Life Technologies, Carlsbad, CA, USA). The final RNA pellets were dissolved in 0.1 mM EDTA (2 μl/mg original wet weight). Reverse transcription reactions were carried out on 22 μl of sample using sSuperScript II RNAse H-Reverse Transcriptase (Invitrogen-Life Technologies) in a reaction volume of 40 μl. All samples were diluted in 160 μl nuclease-free water. qPCR was employed to quantify mouse Grm3 gene expression from the cDNA samples. Sequences of primer pairs are available upon request. Mouse Grm3 mRNA expression was normalized to the levels of the β-actin gene.

Western blot analysis was described in our previous studies (24). Briefly, whole-cell lysates were prepared for western blotting and blots were probed with anti-mouse β-actin and anti-mouse Grm3 antibody. Preimmune serum was used in parallel as controls and HRP-conjugated secondary F(ab′)2 (Zymed Laboratories, San Francisco, CA, USA) were used in concert with the ECL detection system (Amersham Life Science, Arlington Heights, IL, USA).

SP 2/0 cells were infected with control, Grm3 or Foxo1-specific shRNA using standard methods as described in our previous studies (24,26). Briefly, in a 6-well tissue culture plate, 2×10⁵ cells/well were seeded in 2 ml antibiotic-free normal growth medium supplemented with FBS. Cells were incubated for 24 h at 37°C in a CO₂ incubator until 60–80% confluent. 1× 10⁶ infectious units of virus (IFU) of control, Grm3 or Foxo1-specific shRNA-expressing lentivirus and 10 μg/ml polybrene were added into the culture. On day 1 after the infection, the transfection mixture was removed and 1X normal growth medium was added into the culture.

On days 3 after the infection, cells were sorted using multicolor flow cytometry. All flow cytometry data were acquired with FACSCanto, FACSCanto II, or FACSAria (BD Biosciences), gated on live lymphocyte-sized cells on the basis of forward and side scatter, and analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA).

To evaluate tumor growth in mouse models, 200 μl of cell suspension from 5×10⁶ SP 2/0 expressing GFP/Grm3 shRNA and SP 2/0 cells expressing GFP/control shRNA were subcutaneously injected into the left and right sides of the back of each Balb/c or nude mouse. Mice were sacrificed on day 10 after the injection. Tumor volumes were determined by measuring the major (L) and minor (W) diameters with an electronic caliper. The tumor volume was calculated according to the following formula: Tumor volume = π/6 × L × W².

cDNA encoding Foxo1 (1-149), Foxo1 (1-400) and Foxo1 (1-655) corresponding to amino acids 1-149, 1-400, 1-655 of Foxo1 was amplified from cDNA encoding Foxo1 (Addgene, Cambridge, MA, USA) by PCR and subcloned into plasmid pEGFP-N1. SP 2/0 cells were transfected with the appropriate amount of plasmid (5–20 μg) using Lipofectamine 2000 according to the manufacturer’s instructions.

Statistics were generated using t-test in GraphPad Prism version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) and values are represented as mean ± standard error of the mean (SEM). Results were considered statistically significant at P<0.05.

To explore whether Grm3 play an important role in the apoptosis in B-cell-related tumor, we first determined whether B-cell-related tumor cell lines can express Grm3. Human B-cell leukemia Nalm-6 cells and mouse myeloma SP 2/0 cells were selected because Pre-B Nalm-6 cells and plasma SP 2/0 cells represent two different stages of B-cell development and two different species (human and mouse). This is proved by our data suggesting that SP 2/0 cells but not Nalm-6 cells express CD138, a marker on the surface of plasma cells (Fig. 1). As expected, we found that both Nalm-6 cells and SP 2/0 cells could express Grm3 by flow cytometry (FACS) (Fig. 1). However, Grm3 was not expressed in CD138⁺ SP 2/0 cells (Fig. 1), making us to propose that Grm3 would be expressed in apoptotic cells.

To determine whether Grm3 is expressed in apoptotic cells, we used Annexin V and phospholine iodide (PI) staining to mark the apoptotic cells. As expected, we found that Grm3 was expressed in Annexin V⁺ and PI⁺ cells of SP 2/0 (Fig. 2A) and Nalm-6 (Fig. 2B) cells, whereas live cells did not express Grm3. These results suggest that Grm3 is expressed in apoptotic cells.

To examine which apoptotic stage Grm3 expression emerged, we used Annexin V and PI co-staining assay to distinguish different apoptotic stages. We analyzed mean fluorescence intensity (MFI) of Grm3 expression in Annexin V⁻PI⁻, Annexin V⁺PI⁻, Annexin V⁺PI^lo, Annexin V⁺PI^hi, Annexin V-PI^hi cell subpopulations (Fig. 3, left panel). The data demonstrated that Grm3 MFIis the highest in Annexin V⁺PI^lo cell subpopulation of SP 2/0 (Fig. 3A) and Nalm-6 (Fig. 3B) cells. These results suggest that Grm3 is expressed mainly in the middle stage of apoptosis.

To determine whether apoptosis-induced drugs can effectively induce Grm3 expression, we first used FK506 (tacrolimus), a generally applied immunosuppressant in organ transplantation and recently reported to induce apoptosis in prostate cancer (27). We found that 2.5 μg/ml FK506 could effectively induce SP 2/0 cell apoptosis (Fig. 4A). Furthermore, qPCR (Fig. 4B) and western blot analysis (Fig. 4C and D) assays demonstrated that apoptosis significantly upregulated Grm3 mRNA and protein expression in SP 2/0 cells. In addition, FACS analysis showed that FK506 dose-dependently induced Grm3 expression in SP 2/0 cells (Fig. 4E). Compared with SP 2/0 cells, human embryonic kidney 293T cells could not effectively be induced to express Grm3 (Fig. 4F). These results are in line with our previous study suggesting that Grm3 expression was induced mainly in B cells (24).

Low dose of bortezomib, first-line drug in patients with newly diagnosed multiple myeloma, could effectively induce apoptosis and Grm3 expression (Fig. 5). Antineoplastic agents such as nocodazole and monastrol could also effectively induce apoptosis and Grm3 expression (Fig. 5). However, rapamycin, a drug which prevents activation of T cells and B cells by inhibiting the production of interleukin-2 (IL-2) in organ transplantation, could not induce SP 2/0 cell apoptosis (Fig. 5). Therefore, it could not effectively induce Grm3 expression (Fig. 5). Together, our data suggest that apoptosis-induced drugs can effectively induce Grm3 expression in SP 2/0 cells.

To explore the role of Grm3 in B-cell-related tumor cell apoptosis, we first used Grm3-specific shRNA to knock down the Grm3 expression in SP2/0 cells. We found that the level of EGFP expression is negatively associated with cell apoptosis in Grm3 shRNA-infected SP 2/0 cells, whereas the level of EGFP expression is positively associated with cell apoptosis in control shRNA-infected SP 2/0 cells (Fig. 6A). In addition, FACS analysis demonstrated that Grm3 shRNA effectively depleted Grm3 expression in EGFP⁺ cells (Fig. 6B). Together, our data suggest that Grm3 deficiency suppresses SP 2/0 cell apoptosis. Furthermore, we found that Grm3 deficiency could effectively reduce FK506-, rapamycin-, monastrol- or nocodazole-induced SP 2/0 cell apoptosis (Fig. 6C–E).

To further determine the effect of Grm3 deficiency on in vivo tumor progression, SP 2/0 xenograft mouse models were developed in Balb/c and nude mice. SP 2/0 cells were infected with control- or Grm3-specific shRNA (with EGFP)-expressing lentivirus and sorted by FACS based on EGFP expression. EGFP⁺ cells/mouse (5×10⁶) were subcutaneously injected into Balb/C (5 mice/group) or nude (2 mice/group) mice. Our data demonstrated that Grm3 deficiency promoted in vivo tumor progression in Balb/c and nude mice (Fig. 7A). Tumor volumes and average tumor weights from each group were also measured. The results showed that Grm3 deficiency upregulated tumor volumes and weights in Balb/c, although the difference was not significant, whereas Grm3 deficiency significantly upregulated in vivo tumor volumes and weights in nude mice (Fig. 7B and C). Together, our results suggest that Grm3 deficiency promotes SP 2/0 xenograft tumor progression.

To further explore the role of Grm3 in SP 2/0 cell apoptosis, we overexpressed Grm3 in SP 2/0 cells. We used control- or Grm3-expressing Lv81 (with EGFP) lentivirus to infect SP 2/0 cells. We analyzed cell apoptosis in EGFP⁺ cells by FACS. The results demonstrated that Grm3 overexpression upregulates cell apoptosis (Fig. 8).

Grm3 are linked to the inhibition of the cyclic AMP cascade (24) and cAMP-PKA has been shown to phosphorylate FoxO1 (28). These results suggest that Grm3 may promote Foxo1 expression by suppressing Foxo1 phosphorylation. Thus, we proposed that Foxo1 mediated mechanisms underlie Grm3-induced SP 2/0 cell apopotosis. We first examined the effect of Grm3 deficiency on Foxo1 expression in SP 2/0 cells. The data demonstrated that compared with control, or Grm3-specific shRNA-infected SP 2/0 cells reduced Foxo1 expression in SP 2/0 cells (Fig. 9A). The results suggest that Grm3 deficiency reduce Foxo1 expression in SP 2/0 cells. To explore direct effect of Foxo1 on SP2/0 cells, SP 2/0 cells were transfected with plasmids pEGFP-N1 expressing Foxo1 (1-149), Foxo1 (1-400) or Foxo1 (1-655). The data showed that full-length Foxo1 could upregulate SP 2/0 cell apoptosis (Fig. 9B). In addition, we used Foxo1-specific shRNA to reduce Foxo1 expression in SP 2/0 cells. The results suggest that Foxo1-deficiency reduced FK506-induced SP 2/0 cell apoptosis (Fig. 9C). The effect of Foxo1-deficiency on cell apoptosis was approved by using B cells from CD19^cre/+Foxo1^F/+ and CD19^cre/+Foxo1^F/F (Fig. 9D). Together, our data suggest that Grm3 mediates cell apoptosis by regulating Foxo1 expression.