CRIF1 Interacting with CDK2 Regulates Bone Marrow Microenvironment-Induced G0/G1 Arrest of Leukemia Cells

Background To assess the level of CR6-interacting factor 1 (CRIF1), a cell cycle negative regulator, in patients with leukemia and investigate the role of CRIF1 in regulating leukemia cell cycle. Methods We compared the CRIF1 level in bone marrow (BM) samples from healthy and acute myeloid leukemia (AML), iron deficiency anemia (IDA) and AML-complete remission (AML-CR) subjects. We also manipulated CRIF1 level in the Jurkat cells using lentivirus-mediated overexpression or siRNA-mediated depletion. Co-culture with the BM stromal cells (BMSCs) was used to induce leukemia cell cycle arrest and mimic the BM microenvironment. Results We found significant decreases of CRIF1 mRNA and protein in the AML group. CRIF1 overexpression increased the proportion of Jurkat cells arrested in G0/G1, while depletion of endogenous CRIF1 decreased cell cycle arrest. Depletion of CRIF1 reversed BMSCs induced cell cycle arrest in leukemia cells. Co-immunoprecipitation showed a specific binding of CDK2 to CRIF1 in Jurkat cells during cell cycle arrest. Co-localization of two proteins in both nucleus and cytoplasm was also observed with immunofluorescent staining. Conclusion CRIF1 may play a regulatory role in the BM microenvironment-induced leukemia cell cycle arrest possibly through interacting with CDK2 and acting as a cyclin-dependent kinase inhibitor.


Introduction
The leukemia resistance to the chemotherapeutic drugs becomes the minimal residual disease (MRD) and is the major obstacle in the treatment of leukemia [1,2]. The bone marrow (BM) microenvironment is the sanctuary for minimal residual leukemia cells [3][4][5]. In the BM microenvironment, a proportion of the leukemia cells are insensitive to chemotherapy, which is the main cause of the drug resistance and relapse. The BM microenvironment consists of a variety of composition whose roles involved in the leukemia drug resistance remain unclear. It might protect leukemic cells from the killing of the chemotherapeutic drugs by secretion of soluble cytokines, cell adhesion, upregulating resistance gene and changing cell cycle [6][7][8][9][10].
Yoshiyuki et al [11] found that the cell cycle of leukemia cells was arrested at the G0-G1 phase when leukemia cells were cocultured with BM stromal cells of which the main feature is quiescent [12]. Recent studies have revealed cytokines such as interferon-alpha (IFNa), G-CSF, and arsenic trioxide (As2O3) to be efficient agents for promoting cycling of dormant leukemic stem cells. These activated stem cells become exquisitely sensitive to different chemotherapeutic agents [13,14]. For instance, histone deacetylase inhibitors in combination with imatinib mesylate can effectively target quiescent chronic myelogenous leukemia stem cells [15]. A growing number of studies have shown that break dormancy of leukemia cells can effectively enhance their sensitivity to chemotherapeutic drugs [16]. Therefore, exploring the mechanism that induces their dormancy would be very important for MRD treatment.
CR6-interacting factor 1 (CRIF1) is a newly discovered cell cycle negative regulatory factor. Previous study in our lab found that the cell cycle of leukemia cells was arrested at G0/G1 phase in vitro bone marrow microenvironment and the expression level of CRIF1 was also increased. Additionally, we found CRIF1 level in leukemia cells co-cultured with bone marrow stromal cells (BMSCs) also increased significantly, suggesting that co-culture induced cell cycle arrest is associated with increased expression of CRIF1 in leukemic cells [17]. We also found that CRIF1 involved in the G1 arrest and thereby the reducing drug-induced apoptosis of leukemic cells which were induced by abnormal BM microenvironment [17] in vitro. Its overexpression increased the percentage of cells in G1 and suppressed growth in NIH3T3 cells [18]. Ito K et al. [19] reported that targeting the quiescent leukemia cells would be an effective way for the future treatment of MRD.
Current treatment of MRD mainly includes chemotherapy, molecular targeted therapy, gene therapy, BM transplantation, and immunotherapy. However, none of them could completely eradicate residual leukemic cells [20,21]. Therefore, understanding the mechanism of CRIF1 in regulating of cell cycle and activating the quiescent leukemia cells might provide a new treatment strategy to enhance the drug sensitivity of leukemia cells.

Reagents
RPMI1640 medium and fetal bovine serum was purchased from HyClone. The mouse polyclonal to GADD45GIPI (CRIF1) was obtained from Abcam. The goat primary antibody to CRIF1 (sc-103445), rabbit primary antibodies against CDK2, CDK4, CDK6,Cyclin E and D, and Protein G PLUS-Agrose Immunoprecipitation Reagent were purchased from SANTA. RIPA and Trizol were purchased from BeyoECL Plus. The Taq and T4 Ligase were purchased from Takara. ONE-HOUR Complete IP-Western Kits L00232 and L00231 were purchased from Gen-Script. Plasmids pET-32a/CRIF1 and pLenti6/V5-D-TOPO vector were constructed in our laboratory previously. The CRIF1siRNA was purchased from SANTA (sc-97804).

Isolation and expansion of BMSCs
The isolation of BM stromal cells (BMSCs) and their co-culture with Jurkat cells were performed as described by Fortney et al [22]. Briefly, 2 ml of BM was mixed 4 ml 1.077 g/L Percoll and centrifuged at 1800 r/min for 15 min. Cells in the middle white layer were harvested and washed with 16 phosphate-buffered saline (PBS, pH 7.4) twice, followed by centrifugation at 1000 r/ min for 5 min. Cell pellets were resuspended and cultured in RPMI-1640 medium containing 12.5% FBS, 12.5% horse serum, 1610 26 mol/L hydrocortisone, and 10 ng/mL bFGF in an incubator with 5% CO 2 at 37uC. Culture medium was half changed at day 3 and full changed at day 5. Cells were passaged at 80% confluence. When the second generation of cells became 80% confluent they were seeded in24-well plates at density of 2610 4 cells/cm 2 . After 24-hour adherent culture, 2610 5 /ml Jurkat cells in exponential phase were added to BMSCs. After 24 hours, co-cultured cells were used for the following experiments.

Cell culture
Jurkat cells were cultured in RPMI1640 medium supplemented with 10% FBS in an incubator contained humidified 5% CO 2 and 95% oxygen at 37uC. The Jurkat cells were co-cultured with the second-generation of isolated BMSCs in vitro.

Production of CRIF1 overexpression virus
Plasmid pLenti6/V5-D-TOPO/CRIF1 was constructed by inserting PCR-amplified human CRIF1 cDNA from pET-32a/ CRIF1 into the pLenti6/V5-D-TOPO vector. The constructs were verified by restriction enzyme analysis and DNA sequencing. The production, concentration and titration of lentivirus were carried out as described previously [23]. Briefly, 5 ug pMD2.G, 15 ug psPAX2 and 20 ug vectors were co-transfected into 293 cells in a 10-cm plate using calcium phosphate transfection method. The supernatant was replaced with fresh DMEM medium 10 hours later. After additional 48 hours, the viral stocks were collected and filtered through 0.45 um filter. To concentrate the lentivirus, the stocks were centrifuged at 50000 g for 2 hours. The pellet was suspended in an indicated volume of HBSS. To titrate the virus, serial dilutions of virus were added into 293 cells and the titers were calculated by the proportion of viral infected cells, which was measured using FACS.

Transfection of CRIF1-siRNA into the Jurkat cell line
The 21-nucleotide siRNA targeting the human CRIF1 (GenBank TM accession number AF479749) was purchased from SANTA. The sequences of CRIF1 siRNA are Sense: UGCCA-CAGAUGAUUGUGAAtt and Antisense: UUCACAAUCAU-CUGU-GCAtt. The transfection procedure was performed as described in the manufactural instruction of sc-97804.

Flow cytometry
The flow cytometry (Beckman Coulter, MoFlo XDP) was used in the study to detect the cell cycle of Jurkat cells. Cells were washed with 16PBS and then fixed with ice-cold 70% ethanol. Samples were washed with 16PBS and stained with propidium iodide (60 mg/ml, Sigma) containing RNase 100 mg/ml (Sigma) for 30 min at 37uC, followed by flow cytometry analysis.

Western blot
Western Blot was used to analyze the protein expression of CRIF1 in Jurkat cells and 40 BM samples. Total protein lysates were extracted with RIPA and denatured by boiling. Protein samples were resolved on 12% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes. Membranes were blocked in PBS containing 5% (w/v) nonfat dry milk and 0.1% Tween and incubated for 2 h with appropriate antibodies.
Blots were developed using horseradish peroxidase-linked antimouse secondary antibody and developed with a chemiluminescent detection system (Phototope-HRP Western blot detection kit; New England Biolabs).

Co-immunoprecipitation
Jurkat cells were co-cultured with BMSCs for 24 h, and protein extraction of Jurkat cells was performed for co-immunoprecipitation to analyze the interaction between CRIF1 and CDK2, CDK4, CDK6, and Cyclin D and E. Samples containing 500 mg total cellular protein were transferred into a 1.5 ml microcentrifuge tube, and incubated with 1 mg primary antibody at 4uC for 1 h. 20 ml resuspended Protein G PLUS-Agrose was added and incubated at 4uC on a rocker overnight. Immunoprecipitates were collected by centrifugation at 2500 rpm at 4uC for 5 min. The pellet was washed 4 times with PBS, and resuspended in 40 ml of 16 electrophoresis sample buffer, then boiled for 3 min. The analysis of samples were used ONE-HOUR Complete IP-Western Kit L00232, L00231 (GenScript).

Immunofluorescence
Jurkat cells were washed three times in PBS after co-cultured with BMSCs for 24 h, and fixed with 4% formaldehyde for 30 min at room temperature. Cells were washed three times in PBS and incubated in blocking solution containing 5% bovine serum albumin in PBS. For permeabilization, cells were incubated in 0.2% Triton X-100. CRIF1 and CDK2 antibodies were incubated with the cells overnight at 4uC. Cells were washed five times with PBS and incubated with secondary antibodies conjugated with FTIC or Cy3 for 30 min at 37uC. The cells were washed five times in PBS. For nuclear staining, cells were incubated with DAPI for 2 min, then washed five times in PBS. Fluorescence was visualized using a confocal microscopy.

Statistics analysis
The expression levels of CRIF1 gene in the tested samples were expressed in the form of cycle threshold (CT) level; then normalized copy number (relative quantitation) was calculated using the DDCT equation as follows: DDCT = DCT of BM sample -DCT of b-actin, then the normalized copy number (relative quantitation) = 2 2DDCT . All data were presented in mean 6 s.d. and data from multi-group were analyzed using one-way ANOVA followed by post-hoc test. Results were considered significant when P,0.05.

Comparison of CRIF1 levels in human BM samples
In this study, we compared mRNA and protein levels of CRIF1 in 40 BM samples, including 13 healthy human samples, 7 AML samples, 12 IDA samples, and 8 AML-CR samples. The mRNA level of CRIF1 was detected using real-time quantitative PCR ( Figure 1A, Table 1). Results showed that the AML group had a significantly decreased (0.64 fold, p,0.05) CRIF1 mRNA level compared to the healthy group. IDA patients showed a 2.25-fold higher level of CRIF1 mRNA compared to the healthy group (P,0.05). The mRNA level of CRIF1 in the AML-CR group was not significantly different from that of the control group (0.88 fold, P.0.05). The protein levels of CRIF1 were examined using WB ( Figure 1B). The changes of CRIF1 protein level among groups showed a similar trend as that of the mRNA expression. Compared to the healthy group, AML group showed a significant decrease (0.70 fold, p,0.05), and the IDA group exhibited a 1.59fold (p,0.05) increase in CRIF1 protein level. The observation that CRIF1 expression decreased in the AML patients but increased in the IDA patients suggested that expression level of CRIF1 was associated with the different types of blood diseases.

Expression level of CRIF1 affected the cell cycle of Jurkat cells
To study whether the expression level of CRIF1 affects the cell cycle of leukemia cells, we used viral vector pLenti6/V5-D-TOPO/CRIF1 to overexpress human CRIF1 in Jurkat cells. The real-time quantitative PCR result showed a 12-fold increase in the expression level of CRIF1 mRNA at 48 hours after viral infection ( Figure 2A). Correspondingly, CRIF1 protein level showed a 4fold increase as detected by western blot ( Figure 2B). To decrease the CRIF1 level, we used siRNA against CRIF1 and the specific down-regulatory effect on CRIF1 mRNA was confirmed with non-specific mRNA ( Figure 3A and B).
The cell cycles of the control cells or cells with CRIF1 overexpression were analyzed by flow cytometry. The distributions of cells in G0/G1, G2/M and S phases were plotted for quantification ( Figure 3C). Our result showed that overexpression of Identify the protein interacting with CRIF1 in the G1/S checkpoint in Jurkat cells Cyclins and CDKs are two critical classes of molecules in determining the cell cycle progression. In order to investigate the key molecule(s) interacting with CRIF1 in the G1/S checkpoint, we used co-immunoprecipitation to detect the protein that binding to CRIF1 in Jurkat cells co-cultured with BMSCs. At 24 h after  co-cultured, the proteins from Jurkat cells were extracted for coimmunoprecipitation using antibodies against CDK2, CDK4, CDK6, Cyclin D and Cyclin E, respectively. Immunoprecipitation result showed that CRIF1 only bound to CDK2 but not other examined proteins ( Figure 4A and B).

Expression level of CRIF1 in Jurkat cells affected G1 negative regulators
CDKs play a key role in the process of G1 checkpoints. CRIF1 binding to CDK2 may arrest cell cycle through preventing the consequential signals of cell cycle. To further explore the possible involvement of the G1 negative regulators in the cell cycle arrest, we analyzed the levels of p15, p16, p21, p27 and p53 in Jurkat cells co-cultured with BMSCs. Quantitative real-time PCR result showed that BMSCs co-cultured significantly up-regulated mRNA levels of all negative regulators examined in this experiment ( Figure 5A). WB result also showed similar effect of BMSCs coculture on increasing protein levels of these regulators ( Figure 5B). Interestingly, depletion of CRIF1 using siRNA inhibited BMSCs co-culture induced up-regulation of the negative regulators ( Figure 5A and B). This result suggested that during BMSCsinduced cell cycle arrest increased CRIF1 might bind to CDK2 which resulted in the elevation of negative regulators and consequent arrest of cell cycle at the G1 phase. Depletion of CRIF1 might prevent the cell cycle arrest through inhibiting the expression of the negative regulators.

Intracellular co-localization of CRIF1 and CDK2
To explore the intracellular localization of CRIF1 and CDK2 in cells during cell cycle arrest, we performed double-immunofluorescence staining for CRIF1 and CDK2. Fluorescence signal for CRIF1 (green) and CDK2 (red) were detected using confocal microscopy in Jurkat cells co-cultured with BMSCs. The endogenous CRIF1 protein was detected in both nucleus and cytoplasm in Jurkat cells and it mainly located in the cytoplasm ( Figure 6A). The distribution of CRIF1 in Jurkat cells observed in our experiment is consistent with previous report [24], Doubleimmunofluorescence staining revealed that the majority of CDK2 expression co-localized with CRIF1 in Jurkat cells ( Figure 6A). Interestingly, similar intracellular co-localization and distribution of these two proteins were also observed in the BMSCs ( Figure 6B).

Discussion
CRIF1 was first discovered as a nuclear protein that acted as a negative regulatory factor in the cell cycle progression and cell growth [18]. Other biological functions of CRIF1 have also been reported, including its role in regulation of transcriptional activity through its DNA binding domain [25][26][27][28][29] and in promoting cell proliferation through interaction with CKII and the phosphorylation of CKBBP2/CRIF1 [30]. In this study, we reported for the first time that AML patients had a significantly decreased level of CRIF1 in their BM. Using an in vitro leukemia cell model, we found that overexpression of CRIF1 resulted in cell cycle arrest at G1 checkpoint in Jurkat cells. On the contrary, depletion of CRIF1 promoted cell cycle progress at the G0/G1 check-point and reversed BMSCs-induced cell cycle arrest in leukemia cells. This result suggested a regulatory role of CRIF1 in the progress of cell cycle in the leukemia cells. This role of CRIF1 might possibly exert through its interaction with CDK2 that consequently increased the levels of negative regulators, such as p15, p16, p21, p27 and p53, and ultimately lead to cell cycle arrest in the leukemia cells.
In this study, we included 40 BM samples from the healthy control and patients with AML or IDA. Decreased level of CRIF1 in the AML group and restored CRIF1 level in the AML-CR group suggested that the expression level of CRIF1 in the BM of patients might be associated with the disease status of AML. Compared to the healthy group, AML patients showed higher myeloproliferative activity that might be related to decreased level of CRIFI, a negative regulator of cell cycle.
Previously, we found that BMSCs co-culture can induce cell cycle arrest of Jurkat cells. This BMSCs-induced cell cycle arrest might be related to BMSCs-mediated negative regulatory mechanism. In this study, overexpression of CRIF1 resulted in the cell cycle arrest of Jurkat cells, while depletion of CRIF1 decreased cells arrested in G0/G1, indicating that inhibition of endogenous CRIF1 could promote cell cycle progression. Moreover, depletion of CRIF1 also reversed effect of BMSCs on the cell cycle arrest in leukemia cells, suggesting that CRIF1 might involve in the machinery of BMSCs co-culture induced negative regulation of G1/S check point of leukemia cells.
In the early G1 phase, cyclin D is expressed in response to a growth-promoting mitogen and subsequently binds to CDK4/6. The cyclin D/CDK4/6 complex is then activated by Cdkactivating kinase (CAK) which leads to the phosphorylation of the Rb protein. Phosphorylation of the Rb protein disrupts its association with the E2F family of transcription factors. The released E2F triggers the expression of several essential proteins for cell cycle progression, such as cyclin E and cyclin A. Cyclin E forms a complex with CDK2 and the activation of cyclin E/ CDK2 complex completes the process by phosphorylating the Rb protein on additional sites. Cyclin/CDK complexes regulate progression through the G1 phase and the initiation of DNA synthesis or entry into the S phase [31,32]. CDK is the center of cell cycle regulation and regulated by Cyclin and CKI. When combined with Cyclin, CDK is activated and promote cell proliferation, while binding with CKI prevents the cell cycle progression. Therefore, CDK is the key of G1/S regulation [33][34][35]. In this study, co-immunoprecipitation results showed that CRIF1 only interacted with CDK2, and suggested CRIF1 may act as CKI when combined with CDK2, and prevent cell cycle progression of leukemia cells. CRIF1 binding to CDK2 prevented  the consequential signals of cell cycle, which caused G1 arrested and accumulations of G1 negative regulators, such as P15, P16, P21, P27, and P53 [36,37]. Furthermore, BMSCs could upregulate expression of certain genes by secretion of soluble cytokines. For instance, BMSCs induced expression of P27 in leukemia cells [11], and this effect was weakened by inhibition of endogenous CRIF1, and at the same time G1 negative regulators, such as P15, P16, P21, and P53 were all decreased. When the endogenous CRIF1 was inhibited, BMSCs could not induce cell cycle arrest and the cell cycle was allowed to progress through G1 phase, suggesting that CRIF1 might be a downstream factor of in response to the component derived from BMSCs.
Interestingly, previous studies reported that CRIF1 was exclusively localized in the nucleus in cells transfected with pMyc-CRIF1 or pEGFP-CRIF1 [18,25,26]. However, in our study we found that CRIF1 and CDK2 co-localized in both the nucleus and cytoplasm in Jurkat cells. Similar results were also observed in BM mesenchymal stem cells. We speculated that CRIF1 may partly translocate from the nucleus to the cytoplasm where it interacts with CDK2 and induces cell cycle arrest. CRIF1 might interact with CDK2 both in the nucleus and cytoplasm.
In summary, our results showed that CRIF1 overexpression arrested the cell cycle of leukemia cells, while depletion of endogenous CRIF1 reversed the BMSCs-induced cell cycle arrest. Specific interaction of CRIF1 with CDK2 was observed in both nucleus and cytoplasm in arrested Jurkat cells. These results suggest that decreased expression of CRIF1 in leukemia cells might contribute to the sensitivity of the leukemia cells to the chemotherapy drugs. However, it remains unclear how CRIF1 regulates the activity of CDK2 and where the binding cite is. Whether CRIF1 could be a key target to break dormancy of leukemia cells still need further investigation.