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Up-regulation of Cx43 expression and GJIC function in acute leukemia bone marrow stromal cells post-chemotherapy
Leukemia Research 34 (2010) 631–640
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Up-regulation of Cx43 expression and GJIC function in acute leukemia bone marrow stromal cells post-chemotherapy Yao Liu a , Xi Zhang a , Zhong-jun Li b , Xing-hua Chen a,∗ a b
Department of Hematology, Xinqiao Hospital, The Third Military Medical University, Shapingba District, Xinqiao Street, Chongqing 400037, China Department of Blood Transfusion, Xinqiao Hospital, The Third Military Medical University, Chongqing, China
a r t i c l e
i n f o
Article history: Received 26 June 2009 Received in revised form 10 September 2009 Accepted 14 October 2009 Available online 12 November 2009 Keywords: Bone marrow stromal cells Connexin43 Gap junction intercellular communication Acute leukemia Complete remission
a b s t r a c t Gap junction intercellular communication (GJIC) among bone marrow stromal cells (BMSCs) most frequently occurs through a channel composed of connexin43 (Cx43). Dysregulation of connexin expression is believed to have a role in carcinogenesis. In earlier work, we found that in acute leukemia BMSCs, expression of Cx43 and functioning GJIC declined. However, there has been no evaluation of whether GJIC in BMSCs in complete remission (CR) post-chemotherapy is different from GJIC pre-chemotherapy. We studied Cx43 expression and tested GJIC function in human bone marrow cultures under different physiological and pathological conditions. To assay Cx43 expression we used immunocytochemistry, laser scan confocal microscopy (LSCM), flow cytometry and RT-PCR. The results showed that the expression level of Cx43 and its mRNA in acute leukemia BMSCs post-chemotherapy was significantly higher and similar to normal levels than in primary acute leukemia BMSCs (p < 0.01). Functional tests in cultures using dye transfer and fluorescence recovery after photobleaching (FRAP) assays showed that the function of GJIC in acute leukemia BMSCs was significantly improved following effective chemotherapy. Our findings suggest Cx43 and GJIC might be involved in the courses of occurrence, development and termination of acute leukemia, and effective chemotherapy could improve Cx43 expression and GJIC function that were dysfunctional prior to treatment. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction The hematopoietic microenvironment controls the growth and development of hematopoietic stem/progenitor cells, and bone marrow stromal cells (BMSCs) play a major role in this process [1]. Marrow stroma regulates and controls hematopoiesis via paracrine hematopoietic growth factors [2,3] and frequent communication between stromal cells and hematopoietic cells [4]. Both of these regulatory pathways are based on receptor–ligand interactions outside the cells. However, there is another form of cell-to-cell communication based on direct transport of molecules between cells coupled by gap junctions (GJ) [5]. GJ are specializations of the cell membrane composed of members of a conserved family of transmembrane proteins, termed connexins (Cxs), which allow the passage of signaling molecules smaller than 1 kDa between the cytoplasm of two adjacent cells [6]. To date, at least 21 different human Cxs have been identified [7]. Each Cx shows tissueor cell-specific expression, and Cx43 is the major component of hematopoietic tissue GJ [8]. Study [9] shows that gap junction intercellular communication (GJIC) mediated by Cx43 involved in
∗ Corresponding author. Tel.: +86 023 68755609; fax: +86 023 65200687. E-mail address: [email protected] (X.-h. Chen). 0145-2126/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2009.10.013
variety of interactions between bone marrow stromal cells (BMSCs) as well as between BMSCs and hematopoietic cells. The functional importance of GJIC and Cxs can be illustrated by the broad array of diseases caused by Cx mutations, including deafness, various skin disorders, and Charcot-Marie-Tooth disease [10]. Increasing evidence suggests that the dysregulation of Cx expression and dysfunction of GJIC are related to uncontrolled proliferation and malignant phenotypes and may be two of the genetic events involved in tumorigenesis [11]. The functional status of GJIC in stromal cells has a direct impact on the proliferation and differentiation of hematopoietic stem/progenitor cells. In bone marrow, Cx43 expression was found to be highly up-regulated in newborn mice and after regeneration/recovery from cytotoxic treatment [12,13]. Other studies also showed that restoring Cx gene expression and GJIC by gene therapy in Cx-deficient tumor cells can decrease tumor cell growth [14–17]. All of these findings suggest an important function for Cx and GJIC in tumor formation. Our previous studies have provided evidence that the expression of Cx43 and function of GJIC in acute leukemia were significantly lower than in normal BMSCs [18]. The function of BMSCs in acute leukemia was sensitive to chemotherapeutic drugs and could be restored along with the recovery of hematopoiesis after effective chemotherapy [19]. However, there is little information available on the changes of Cx and GJIC in acute leukemia BMSCs post-chemotherapy.
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Fig. 1. Expression of Cx43 in BMSCs through immunocytochemistry assay. Cells were stained with Cx43 antibody and visualized using the phase-contrast-equipped epifluorescence microscope. In normal (A) and post-chemotherapy (B) BMSCs, Cx43 could be seen in the cell membrane, appearing as areas of large brown adhesion plaque, and could also be found in cytoplasm, frequently only one side of cell. In the group of initial acute leukemia samples (C), Cx43 was only expressed in a few cells, and there was no expression in the negative control group (D). As viewed through density scanning for immunocytochemistry results, the average optical values of Cx43 protein in normal, pre- and post-chemotherapy BMSCs were 121.0 ± 16.28, 48.3 ± 11.69 and 93.3 ± 14.38 respectively, and the differences among groups were statistically significant (p < 0.01). Images were taken at 400× magnification. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Here we studied Cx43 expression and GJIC function in BMSCs in normal, primary acute leukemia and acute leukemia postchemotherapy samples. We found that (1) expression of Cx43 and function of GJIC in acute leukemia BMSCs post-chemotherapy are similar to normal BMSCs, (2) expression levels of Cx43 and its mRNA in acute leukemia BMSCs post-chemotherapy are significantly higher than in primary acute leukemia BMSCs, and (3) the function of GJIC in acute leukemia BMSCs post-chemotherapy is significantly improved compared to primary acute leukemia. Therefore, expression of Cx43 and the function of gap junctions in acute leukemia bone marrow stromal cells are extremely plastic and effective chemotherapy may improve the expression of Cx43 and function of GJIC in acute leukemia. 2. Materials and methods
first CR. Samples of normal BMSCs were taken from normal healthy donors. The cases involved in this study were diagnosed at the Department of Pathology of the Xinqiao Hospital on the basis of clinical data, tissue/cell morphology, and immunophenotype. The investigation was approved by the ethics committee of Xinqiao Hospital, and subjects gave written informed consent for the study. 2.2. Cell culture Human BMSCs were isolated as described previously [20], and were plated in T75 flasks for continuous passage in DMEM (Gibco BRL) medium supplemented with 12.5% FBS (Gibco BRL), 12.5% HS (Gibco BRL) and 10−5 mol/L hydrocortisone (Sigma, USA). Medium was changed twice weekly, and cells were passaged into fresh culture flasks at a ratio of 1:4 upon reaching confluence using trypsin (ZhongShan, China).
Table 1 The expression of Cx43 in normal BMSCs/CR-BMSCs/leukemia BMSCs by RT-PCR. Groups
2.1. Sample source Samples of bone marrow were taken from 29 primary acute leukemia patients from the Department of Hematology of Xinqiao Hospital between March and November in 2008. Patients included 16 males and 13 females, with a median age of 34. Among them 8 patients were with ALL-L1, 11 patients with ALL-L2, 4 patients with AML-M3, 3 patients with AML-M5, and 3 patients with AML-M6. Complete remission (CR) acute leukemia samples were taken from above patients after their
Normal BMSCs Leukemia BMSCs CR-BMSCs *
Relative amount of Cx43 mRNA (Cx43 OD value/-actin OD value) 1
2
3
x¯ ± z
0.661 0.515 0.634
0.705 0.529 0.652
0.740 0.511 0.648
0.702 ± 0.040 0.621 ± 0.084* 0.645 ± 0.095
p < 0.01 vs. normal BMSCs and CR-BMSCs.
Y. Liu et al. / Leukemia Research 34 (2010) 631–640 Cultures were incubated at 37 ◦ C in a humidified incubator (Biorad, USA) with 5% CO2 . 2.3. Immunocytochemistry and fluorescent immunostaining Human bone marrow stromal cells cultured on glass cover-slips in six-well plates were grown to 80% confluence. Cells were washed with PBS (ZhongShan, China) and fixed with 40 mg/L paraformaldehyde (ZhongShan, China) for 20 min. After washing, samples were blocked with 10% goat serum albumin (ZhongShan, China) for 20 min. For immunocytochemistry, the cells were reacted overnight at 4 ◦ C with a drop of 1:500 diluted rabbit anti-human Cx43 polyclonal antibody (Zymed, USA), then washed and incubated with anti-IgG fluorescein-isothiocyanate-labeled antibody (Sigma, USA) for 1 h. Analysis was performed under a phase-contrastequipped epifluorescence microscope (Carl Zeiss, Germany), using PBS instead of Cx43 antibody as negative control. For fluorescent immunostaining, a drop of DAPI staining solution (ZhongShan, China, 1 mg/mL) was added and allowed to
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stain for 5 min. Layers were washed with PBS and a drop of 1:500 diluted rabbit anti-human Cx43 polyclonal antibody was added and incubated for 24 h at 4 ◦ C. Samples were then warmed to room temperature for 1 h before washing with PBS and the addition of 1 drop of 1:60 diluted FITC labeled goat anti-rabbit IgG (Jingmei, China), followed by incubation for 1 h at room temperature. After washing with PBS, the film was sealed using 60% buffering glycerol (ZhongShan, China) and detected by a laser confocal microscope (Leica, Germany) with a fast scanning mode. 2.4. Flow cytometry Monolayers of 80% fusional BMSCs in culture flasks were removed using 0.25% trypsin and a single cell suspension was prepared. Cells were centrifuged at 250 × g for 5 min and the supernatant was removed. Cells were then washed twice in PBS by centrifugation at 250 × g for 10 min. Cells were re-suspended at 1 × 106 mL−1 and 4 g/mL rabbit anti-human Cx43 polyclonal antibody was added before incubation
Fig. 2. Expression of Cx43 in BMSCs. Cells were stained with Cx43 antibody and visualized using the laser scanning microscope. Green fluorescence was located throughout the cell membrane and cytoplasm in normal BMSCs (A) and CR-BMSCS (B); yet in leukemia BMSCs (C), we saw only noncontiguous small mass-like and dot-like fluorescence on the cell membrane and in the cytoplasm. The pixel densities of Cx43 in normal, pre-chemotherapy and CR-BMSCs after chemotherapy were 81.04% (±8.84), 34.10% (±17.91) and 70.33% (±4.90) respectively. Nuclei were stained with DAPI. Images were taken at 800× magnification.
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for 1 h at 4 ◦ C. PBS instead of primary antibody was added to the control samples. Then 0.1 mg/mL FITC labeled goat anti-rabbit IgG was added and protected from light for 1 h at 4 ◦ C. Samples were then analyzed on a flow cytometer (Beckman Coulter, USA). 2.5. RT-PCR Total RNA was isolated using Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions. cDNA synthesis and amplification from 2 g mRNA via PCR was performed using the RevertAidTM First Strand cDNA Synthesis Kit (Fermentas, USA) using a GeneAmp PCR System 2700 Thermal Cycler (Applied Biosystems, USA). Gene expression (from 100 ng cDNA) was measured by real-time PCR using the DyNAmo Flash SYBR Green qPCR Kit (Finnzymes, Oy, Finland) and custom primers for Cx43 (NM 000165): F: 5 -AACCTGGTTGTGAAAATGTC-3 ; R: 5 -GCAAGTGTAAACAGCACTCA-3 and -actin (NM 001101): F: 5 CCTGTGGCATCCACGAAACT-3 ; R: 5 -GAGCAATGATCCTGATCTTC-3 . The PCR reaction and detection were performed with the MJ Research Chromo 4 PTC200 (MJ Research, USA). The expression levels were calculated and normalized to -actin. 2.6. Dye transfer assay Treated cells grown on sterile glass cover-slips in a 24-well dish were assayed for gap junction-mediated intercellular coupling by dye transfer assay as previously described [21]. In brief, the culture medium from a confluent monolayer was removed and saved. The cells were rinsed three times with HBSS (with calcium and magnesium) containing 1% BSA, before a 27-gauge needle was used to create multiple scratches through the cell monolayer in the presence of PBS containing 0.5% Lucifer yellow (LY) (Gibco BRL). After exactly 1 min, the culture was rinsed three times with HBSS and then incubated for an additional 10 min in the saved culture medium to allow the loaded dye to transfer to adjoining cells. The cells were then rinsed and fixed with 4% paraformaldehyde and immediately imaged on the confocal microscope using the argon laser at 485 nm. The level of GJIC was quantified as the average distance traveled (m) by the LY dye, from six different sites in each sample, after 10 min of dye loading. The efficiency (as a percentage) was calculated by normalizing to the average distance traveled by control and mock control. 2.7. FRAP assay GJIC was quantitatively assessed in living cells by FRAP assay as described previously [22]. Human bone marrow stromal cells cultured on glass cover-slips in six-well plates were grown to 80% confluence. The cells were then loaded at 37 ◦ C with CFDA (10 mol/L, 15 min) (Sigma, USA). After the loading process,
the cell culture slides were washed three times with DMEM medium to remove the fluorochrome-ester and prevent further dye loading during subsequent measurements. The cells were subjected to FRAP analysis. Clusters of five to six cells were selected under the microscope with the 20× objective lens. A cell contacted by four to five neighboring cells within a cluster was photobleached to 10% of the original fluorescence intensity using an argon laser beam. The bleached cell was then monitored for transfer of fluorescent dye from neighboring cells and examined for recovery of fluorescence by scanning at intervals of 50 s for a total period of 400 s. The data on the maximum intensity of recovered fluorescence (It ) at the 400th s was collected as the functional index of GJIC. At least three such clusters were selected from each dish. The analyzed fluorescence recovery index is expressed as: R = (It − I0 )/(I − I0 ) × 100%, where I0 is the intensity of the photobleached fluorescence and I is the intensity of pre-bleached fluorescence. 2.8. Statistical analysis Data were represented as mean values with standard deviation. Statistical significance was analyzed by Student’s t test. P values less than 0.05 were considered significant. The assessment of GJIC was performed using the Pearson Chi Squared test.
3. Results 3.1. Expression of Cx43 Immunocytochemistry assays demonstrated that expression of Cx43 in normal, pre- and post-chemotherapy BMSCs cultured in vitro could be found but that intensity of expression varied. In normal and post-chemotherapy BMSCs, Cx43 could be seen in the cell membrane appearing as large areas of brown adhesion plaque; it could also be found in the cytoplasm, frequently on just one side of cell. But, in samples from initial acute leukemia patients, Cx43 only expressed in a few cells, and there was no expression in the negative control group (Fig. 1A–D). The distribution of Cxs can be studied by immunofluorescence assay. In this study, Cx43 is found along appositional membranes of adjacent cells; these sites exhibit intense immunoreactivity
Fig. 3. Expression of Cx43 mRNA in BMSCs. Messenger RNA was isolated from 80% fusional monolayers of BMSCs from the 3 test groups and cDNA produced as described above. M: marker; 1–3: normal BMSCs; 4–6: CR-BMSCs; 7–9: leukemia BMSCs. BMSCs from leukemia patients expressed less Cx43 mRNA when compared to the other BMSC groups. Associated densitometric analysis demonstrated that the relative amount of Cx43 mRNA in leukemia BMSCs (0.621 ± 0.084%) was significantly lower than that in NBMSCs (0.072 ± 0.040%) and ABMSCs-CR (0.645 ± 0.095%) (p < 0.01).
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with anti-connexin antibodies. In some cells, a substantial amount of connexin can also be detected by immunofluorescence in the cytoplasm; this represents a storeroom of unassembled connexins within the biosynthetic pathway. Fluorescence of Cx43 was not detected in the cell nucleus. In normal BMSCs and CR-BMSCs, we could see green fluorescence located throughout the cell membrane and cytoplasm; we saw only noncontiguous small mass-like and dot-like fluorescence on the cell membrane and cytoplasm in acute leukemia BMSCs (Fig. 2A–C). Taken together, these results indicate that the level of Cx43 expression in CR-BMSCs was significantly higher than in primary acute leukemia BMSCs (p < 0.01), although lower than in normal BMSCs. The above results were confirmed by flow cytometry. Expression in normal BMSCs was the strongest (76.51 ± 13.15%). Expression in the CR-BMSCs group after chemotherapy (71.98 ± 7.5%) was slightly lower than expression in the normal group, although not statistically significant, and was significantly higher than that in primary acute leukemia (38.75 ± 23.95%) (p < 0.01). RT-PCR results showed that the stromal cells of all the three groups express Cx43 mRNA. As expected, the expression level of the initial acute leukemia group was lower than that of the normal and CR-BMSCs groups (p < 0.01, Table 1 and Fig. 3).
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3.2. Function of GJIC In normal BMSCs, LY fluorescence was transmitted to more than 10 cell lines through gap junctions over the time allowed in this study (Fig. 4A). LY fluorescence in the BMSCs of initial acute leukemia patients dispersed to cells nearby in 1–2 lines (Fig. 4B). The LY fluorescence in CR-BMSCs had dispersed to cells in 8–9 lines (Fig. 4C) indicating that communication between acute leukemia BMSCs was defective and had increased following chemotherapy. Furthermore, the stain could go through normal and CR-BMSCs within 1–2 min while it took more than 8 min to go through the acute leukemia BMSCs. The results demonstrated that function of GJIC in acute leukemia post-chemotherapy was significantly improved not only in its propagative scope but also in its propagative velocity compared to primary acute leukemia. FRAP results showed that the fluorescence intensity of normal BMSCs could recover quickly after photobleaching: 50% could recover in 1 min while 84.29% (±2.93) could recover within 400 s (Fig. 5A:1–3). Fluorescence intensity of CR-BMSCs could also recover quickly, and 78.94% (±2.46) could recover within 400 s (Fig. 5B:1–3). However, fluorescence intensity in BMSCs from acute leukemia recovered only about 20% in 1 min, and the maximal recovery percentage was 38.30% (±2.95) (Fig. 5C:1–3), which was
Fig. 4. Transmission of LY in BMSCs. LY transmission between (A) normal BMSCs; (B) leukemia BMSCs; and (C) CR-BMSCs were assessed. LY dye was allowed to diffuse through gap junctions for 10 min before assessment by confocal microscopy. BMSCs from leukemia patients showed decreased transmission of LY dye compared to normal and CR-BMSCs. The difference in the transmission of the LY stain between normal BMSCs and acute leukemia BMSCs was significant (Pearson Chi Squared = 93.600, v = 5, p < 0.01), as was the difference between acute leukemia BMSCs compared to CR-BMSCs (Pearson Chi Squared = 87.692, v = 5, p < 0.01). Images were made with 100× magnification.
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significantly different from the other two groups (p < 0.01). The average recovery rates of normal and CR-BMSCs were 19.20% (±1.39) min−1 and 13.52% (±1.65) min−1 respectively, significantly higher than that of acute leukemia BMSCs: 5.58% (±1.00) min−1 (p < 0.01) (Fig. 5D). 4. Discussion Occurrence of malignancy and malignant phenotypes of tumors may be closely related to gene expression defects in gap junction proteins and to inhibition of GJIC function [23–25]. Although gap junction proteins have been described in healthy human BMSCs [9,26,27], the functional status of GJIC in abnormal BMSCs is
unclear. In our previous work [18], decreased expression of Cx43 and GJIC dysfunction were observed in acute leukemia BMSCs. These results are consistent with the findings of previous solid tumor studies [28–30], which support the hypothesis that abnormal or paired cell-to-cell communication via gap junctions may be involved in the pathogenesis of tumors including acute leukemia. As a kind of hematopoietic stem cells malignant disease, abnormal hematopoietic stem cell generates not only abnormal hematopoietic cells but also abnormal stromal cells [31,32]. Bone marrow stromal cells also responded to therapy and, when undergoing effective chemotherapy, recovered their ability to construct and support blood-forming activity in acute leukemia BMSCs [33]. Then, we are more concerned about alteration of Cx43 expression and
Fig. 5. Results of FRAP in BMSCs. Fluorescence intensity of normal BMSCs could recover quickly after photobleaching and approximately 85% fluorescence could recover within 400 s (A1–3, 1, pre-photobleaching; 2, 1.5 s post-photobleaching; 3, 400 s post-photobleaching). Fluorescence intensity of CR-BMSCs could also recover quickly and approximately 80% fluorescence could recover within 400 s (B1–3, 1, pre-photobleaching; 2, 1.5 s post-photobleaching; 3, 400 s post-photobleaching). Fluorescence intensity of BMSCs of acute leukemia recovered more slowly and approximately 40% fluorescence could recover within 400 s (C1–3, 1, pre-photobleaching; 2, 1.5 s postphotobleaching; 3, 400 s post-photobleaching). Comparison of fluorescence recovery among NBMSCs/ABMSCs-CR/ABMSCs was summarized in (D). Images were made with 200× magnification.
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Fig. 5. (Continued )
GJIC function in acute leukemia BMSCs that have been effectively treated. Using 29 cases of primary acute leukemia of various types, we cultured BMSCs, compared the change in Cx43 expression pre- and post-chemotherapy, and compared the results to normal BMSCs as controls. The results showed that all three groups can express Cx43 in cell membranes and cytoplasm, but not in the cell nucleus. Interestingly, after effective chemotherapy, the amount of Cx43 in CR-BMSCs increased and was similar to that of normal BMSCs, and mRNA and protein levels were significantly higher than in primary acute leukemia (p < 0.01). Results showed that Cx43 expression in acute leukemia BMSCs is in a state of flux, and that factors involved in the process of treatment, including drugs, may affect its expression. Cxs expression in many tissues can be regulated by both in vitro and in vivo factors [34,35]. Cx43 is a major Cx in blood-forming tissues, and its expression within the BM is crucial in the development
of an efficient response to hematopoiesis [36]. Expression level of Cx43 fluctuated with the body’s need for hematopoiesis, improving the expression of this protein when such a need was increased [37,38]. Ongoing studies in our laboratory have demonstrated that Cx43 expression in acute leukemia BMSCs is up-regulated by retinoic acid (Liu et al., in preparation), showing that Cx43 in acute leukemia BMSCs is sensitive to drugs. This positive association between connexin expression and retinoic acid has been detected in other cell systems [39–41]. Recent studies verified that some hormones, cytokines and drugs could affect the expression of Cxs between coupling cells [42–45]. The dynamics of connexin expression in acute leukemia BMSCs is a complex process involving perturbations of connexin gene expression and connexin protein synthesis and degradation, as well as rearrangements in the spatial distribution of the protein. Further studies are required to identify changes that occur following treatment. Because of the half-lives of Cx are generally between 2 and 3 h
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Fig. 5. (Continued ).
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[46–49], that allow for regulation of GJIC within a few hours. This regulation occurs through alterations in connexin turn-over and degradation and by remodeling of gap junctions. It seems likely, because the internalized Cxs presumably cannot be recycled, so that up-regulation depends on synthesis of Cx43 de novo [50]. This process was possibly favored by the recovery of the BMSCs after effective treatment. On the other hand, the drugs that we used to cure leukemia or humoral mediators of the hematopoietic microenvironment can influence the synthesis, assembly and expression of Cx43 in BMSCs. Cx proteins can have antitumor functions independent of GJIC. Cx43 was shown to reduce proliferation in human glioblastoma cells without increasing GJIC [51]. Cx26 and Cx43 have also been shown to reduce growth of human breast cancer cells without altering GJIC [52]. These findings have led to investigation into whether GJIC function in acute leukemia BMSCs is altered following the increased expression of Cx43. Results of dye transfer and FRAP assays suggested that the function of GJIC was reconstructed by increasing Cx43 expression. It was shown that after effective treatment, both the quantity of Cx43 and the ability to form effective cell-to-cell communications increased. Whether the recovery of GJIC function post-chemotherapy came about through the direct effect of drugs (such as participating in the assembly of Cx) or through indirect regulation by induction of other cytokines, or both, remains unknown. Rivedal and Leithe [53] demonstrated that recovery of GJIC during continuous TPA (a kind of tumor promoter) exposure in normal and transformed rat liver epithelial cells is dependent on the synthesis of Cx43 protein de novo, as well as the transport of already synthesized Cx43 from intracellular pools to the plasma membrane. Confirmed by other recent studies on changes of GJIC function and some drugs [53,54], the results suggest that communication among cells is a dynamic process, which could be affected by other mediators. As GJIC was a tumor suppressor, improvement of its functions would have played an important role in inhibiting tumor formation [55]. At the same time, due to GJIC’s involvement in blood formation within the hematopoietic microenvironment [36,56], improving its function may be required for support of hematopoiesis of stromal cells. It is likely that Cx43 expression and GJIC function are affected when one or more chemotherapy drugs are used for different clinical programs, in turn affecting recovery of hematopoietic function and the development process of tumors in treated individuals. By extension, this study implicated GJIC as a potential therapeutic target and the ability to up-regulate GJIC may be an important indicator of chemotherapy potential. In conclusion, this paper described for the first time the change of Cx43 and GJIC among various BMSCs; expression of Cx43 and function of GJIC in acute leukemia BMSCs postchemotherapy increased and improved. These changes in connexin expression and GJIC function appear to reflect the changing requirements of hematopoiesis. The results also suggested that Cx43 expression and GJIC function in BMSCs is likely to be a dynamic process, regulated by many factors in vivo and in vitro. An in-depth study on the trend of change and relevant influencing factors of GJIC in tumorigenesis and tumor development can provide a theoretical and experimental basis for understanding GJIC in hematopoietic injury reconstruction and drug treatment for hematological malignancies, including leukemia.
Conflict of interest The authors declare that they have no potential conflicts of interest.
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Acknowledgments Xi Zhang contributed equally to this study. Yao Liu and Xi Zhang took responsibility in conception and design, collection and analysis of data, manuscript writing and final approval of manuscript; Zhong-jun Li has participated in the design and execution of the study; Xing-hua Chen has primary responsibility for the study design. We are grateful to Drs. Lei Gao, Li Gao and Cheng Zhang for bone marrow samples. We also thank Dr. Shi-cang Yu (Institute of Pathology, Southwest Hospital, Third Military Medical University) for the English writing. This work was supported in part by a grant-in-aid from the National Natural Science Foundation of China (No. 30670890), Chongqing Key Discipline of Medical Science (No. 2006C028) and special foundation for the “1520 project” of Xinqiao Hospital of Third Military Medical University.
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Leukemia Research journal homepage: www.elsevier.com/locate/leukres
Up-regulation of Cx43 expression and GJIC function in acute leukemia bone marrow stromal cells post-chemotherapy Yao Liu a , Xi Zhang a , Zhong-jun Li b , Xing-hua Chen a,∗ a b
Department of Hematology, Xinqiao Hospital, The Third Military Medical University, Shapingba District, Xinqiao Street, Chongqing 400037, China Department of Blood Transfusion, Xinqiao Hospital, The Third Military Medical University, Chongqing, China
a r t i c l e
i n f o
Article history: Received 26 June 2009 Received in revised form 10 September 2009 Accepted 14 October 2009 Available online 12 November 2009 Keywords: Bone marrow stromal cells Connexin43 Gap junction intercellular communication Acute leukemia Complete remission
a b s t r a c t Gap junction intercellular communication (GJIC) among bone marrow stromal cells (BMSCs) most frequently occurs through a channel composed of connexin43 (Cx43). Dysregulation of connexin expression is believed to have a role in carcinogenesis. In earlier work, we found that in acute leukemia BMSCs, expression of Cx43 and functioning GJIC declined. However, there has been no evaluation of whether GJIC in BMSCs in complete remission (CR) post-chemotherapy is different from GJIC pre-chemotherapy. We studied Cx43 expression and tested GJIC function in human bone marrow cultures under different physiological and pathological conditions. To assay Cx43 expression we used immunocytochemistry, laser scan confocal microscopy (LSCM), flow cytometry and RT-PCR. The results showed that the expression level of Cx43 and its mRNA in acute leukemia BMSCs post-chemotherapy was significantly higher and similar to normal levels than in primary acute leukemia BMSCs (p < 0.01). Functional tests in cultures using dye transfer and fluorescence recovery after photobleaching (FRAP) assays showed that the function of GJIC in acute leukemia BMSCs was significantly improved following effective chemotherapy. Our findings suggest Cx43 and GJIC might be involved in the courses of occurrence, development and termination of acute leukemia, and effective chemotherapy could improve Cx43 expression and GJIC function that were dysfunctional prior to treatment. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction The hematopoietic microenvironment controls the growth and development of hematopoietic stem/progenitor cells, and bone marrow stromal cells (BMSCs) play a major role in this process [1]. Marrow stroma regulates and controls hematopoiesis via paracrine hematopoietic growth factors [2,3] and frequent communication between stromal cells and hematopoietic cells [4]. Both of these regulatory pathways are based on receptor–ligand interactions outside the cells. However, there is another form of cell-to-cell communication based on direct transport of molecules between cells coupled by gap junctions (GJ) [5]. GJ are specializations of the cell membrane composed of members of a conserved family of transmembrane proteins, termed connexins (Cxs), which allow the passage of signaling molecules smaller than 1 kDa between the cytoplasm of two adjacent cells [6]. To date, at least 21 different human Cxs have been identified [7]. Each Cx shows tissueor cell-specific expression, and Cx43 is the major component of hematopoietic tissue GJ [8]. Study [9] shows that gap junction intercellular communication (GJIC) mediated by Cx43 involved in
∗ Corresponding author. Tel.: +86 023 68755609; fax: +86 023 65200687. E-mail address: [email protected] (X.-h. Chen). 0145-2126/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2009.10.013
variety of interactions between bone marrow stromal cells (BMSCs) as well as between BMSCs and hematopoietic cells. The functional importance of GJIC and Cxs can be illustrated by the broad array of diseases caused by Cx mutations, including deafness, various skin disorders, and Charcot-Marie-Tooth disease [10]. Increasing evidence suggests that the dysregulation of Cx expression and dysfunction of GJIC are related to uncontrolled proliferation and malignant phenotypes and may be two of the genetic events involved in tumorigenesis [11]. The functional status of GJIC in stromal cells has a direct impact on the proliferation and differentiation of hematopoietic stem/progenitor cells. In bone marrow, Cx43 expression was found to be highly up-regulated in newborn mice and after regeneration/recovery from cytotoxic treatment [12,13]. Other studies also showed that restoring Cx gene expression and GJIC by gene therapy in Cx-deficient tumor cells can decrease tumor cell growth [14–17]. All of these findings suggest an important function for Cx and GJIC in tumor formation. Our previous studies have provided evidence that the expression of Cx43 and function of GJIC in acute leukemia were significantly lower than in normal BMSCs [18]. The function of BMSCs in acute leukemia was sensitive to chemotherapeutic drugs and could be restored along with the recovery of hematopoiesis after effective chemotherapy [19]. However, there is little information available on the changes of Cx and GJIC in acute leukemia BMSCs post-chemotherapy.
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Fig. 1. Expression of Cx43 in BMSCs through immunocytochemistry assay. Cells were stained with Cx43 antibody and visualized using the phase-contrast-equipped epifluorescence microscope. In normal (A) and post-chemotherapy (B) BMSCs, Cx43 could be seen in the cell membrane, appearing as areas of large brown adhesion plaque, and could also be found in cytoplasm, frequently only one side of cell. In the group of initial acute leukemia samples (C), Cx43 was only expressed in a few cells, and there was no expression in the negative control group (D). As viewed through density scanning for immunocytochemistry results, the average optical values of Cx43 protein in normal, pre- and post-chemotherapy BMSCs were 121.0 ± 16.28, 48.3 ± 11.69 and 93.3 ± 14.38 respectively, and the differences among groups were statistically significant (p < 0.01). Images were taken at 400× magnification. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Here we studied Cx43 expression and GJIC function in BMSCs in normal, primary acute leukemia and acute leukemia postchemotherapy samples. We found that (1) expression of Cx43 and function of GJIC in acute leukemia BMSCs post-chemotherapy are similar to normal BMSCs, (2) expression levels of Cx43 and its mRNA in acute leukemia BMSCs post-chemotherapy are significantly higher than in primary acute leukemia BMSCs, and (3) the function of GJIC in acute leukemia BMSCs post-chemotherapy is significantly improved compared to primary acute leukemia. Therefore, expression of Cx43 and the function of gap junctions in acute leukemia bone marrow stromal cells are extremely plastic and effective chemotherapy may improve the expression of Cx43 and function of GJIC in acute leukemia. 2. Materials and methods
first CR. Samples of normal BMSCs were taken from normal healthy donors. The cases involved in this study were diagnosed at the Department of Pathology of the Xinqiao Hospital on the basis of clinical data, tissue/cell morphology, and immunophenotype. The investigation was approved by the ethics committee of Xinqiao Hospital, and subjects gave written informed consent for the study. 2.2. Cell culture Human BMSCs were isolated as described previously [20], and were plated in T75 flasks for continuous passage in DMEM (Gibco BRL) medium supplemented with 12.5% FBS (Gibco BRL), 12.5% HS (Gibco BRL) and 10−5 mol/L hydrocortisone (Sigma, USA). Medium was changed twice weekly, and cells were passaged into fresh culture flasks at a ratio of 1:4 upon reaching confluence using trypsin (ZhongShan, China).
Table 1 The expression of Cx43 in normal BMSCs/CR-BMSCs/leukemia BMSCs by RT-PCR. Groups
2.1. Sample source Samples of bone marrow were taken from 29 primary acute leukemia patients from the Department of Hematology of Xinqiao Hospital between March and November in 2008. Patients included 16 males and 13 females, with a median age of 34. Among them 8 patients were with ALL-L1, 11 patients with ALL-L2, 4 patients with AML-M3, 3 patients with AML-M5, and 3 patients with AML-M6. Complete remission (CR) acute leukemia samples were taken from above patients after their
Normal BMSCs Leukemia BMSCs CR-BMSCs *
Relative amount of Cx43 mRNA (Cx43 OD value/-actin OD value) 1
2
3
x¯ ± z
0.661 0.515 0.634
0.705 0.529 0.652
0.740 0.511 0.648
0.702 ± 0.040 0.621 ± 0.084* 0.645 ± 0.095
p < 0.01 vs. normal BMSCs and CR-BMSCs.
Y. Liu et al. / Leukemia Research 34 (2010) 631–640 Cultures were incubated at 37 ◦ C in a humidified incubator (Biorad, USA) with 5% CO2 . 2.3. Immunocytochemistry and fluorescent immunostaining Human bone marrow stromal cells cultured on glass cover-slips in six-well plates were grown to 80% confluence. Cells were washed with PBS (ZhongShan, China) and fixed with 40 mg/L paraformaldehyde (ZhongShan, China) for 20 min. After washing, samples were blocked with 10% goat serum albumin (ZhongShan, China) for 20 min. For immunocytochemistry, the cells were reacted overnight at 4 ◦ C with a drop of 1:500 diluted rabbit anti-human Cx43 polyclonal antibody (Zymed, USA), then washed and incubated with anti-IgG fluorescein-isothiocyanate-labeled antibody (Sigma, USA) for 1 h. Analysis was performed under a phase-contrastequipped epifluorescence microscope (Carl Zeiss, Germany), using PBS instead of Cx43 antibody as negative control. For fluorescent immunostaining, a drop of DAPI staining solution (ZhongShan, China, 1 mg/mL) was added and allowed to
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stain for 5 min. Layers were washed with PBS and a drop of 1:500 diluted rabbit anti-human Cx43 polyclonal antibody was added and incubated for 24 h at 4 ◦ C. Samples were then warmed to room temperature for 1 h before washing with PBS and the addition of 1 drop of 1:60 diluted FITC labeled goat anti-rabbit IgG (Jingmei, China), followed by incubation for 1 h at room temperature. After washing with PBS, the film was sealed using 60% buffering glycerol (ZhongShan, China) and detected by a laser confocal microscope (Leica, Germany) with a fast scanning mode. 2.4. Flow cytometry Monolayers of 80% fusional BMSCs in culture flasks were removed using 0.25% trypsin and a single cell suspension was prepared. Cells were centrifuged at 250 × g for 5 min and the supernatant was removed. Cells were then washed twice in PBS by centrifugation at 250 × g for 10 min. Cells were re-suspended at 1 × 106 mL−1 and 4 g/mL rabbit anti-human Cx43 polyclonal antibody was added before incubation
Fig. 2. Expression of Cx43 in BMSCs. Cells were stained with Cx43 antibody and visualized using the laser scanning microscope. Green fluorescence was located throughout the cell membrane and cytoplasm in normal BMSCs (A) and CR-BMSCS (B); yet in leukemia BMSCs (C), we saw only noncontiguous small mass-like and dot-like fluorescence on the cell membrane and in the cytoplasm. The pixel densities of Cx43 in normal, pre-chemotherapy and CR-BMSCs after chemotherapy were 81.04% (±8.84), 34.10% (±17.91) and 70.33% (±4.90) respectively. Nuclei were stained with DAPI. Images were taken at 800× magnification.
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for 1 h at 4 ◦ C. PBS instead of primary antibody was added to the control samples. Then 0.1 mg/mL FITC labeled goat anti-rabbit IgG was added and protected from light for 1 h at 4 ◦ C. Samples were then analyzed on a flow cytometer (Beckman Coulter, USA). 2.5. RT-PCR Total RNA was isolated using Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions. cDNA synthesis and amplification from 2 g mRNA via PCR was performed using the RevertAidTM First Strand cDNA Synthesis Kit (Fermentas, USA) using a GeneAmp PCR System 2700 Thermal Cycler (Applied Biosystems, USA). Gene expression (from 100 ng cDNA) was measured by real-time PCR using the DyNAmo Flash SYBR Green qPCR Kit (Finnzymes, Oy, Finland) and custom primers for Cx43 (NM 000165): F: 5 -AACCTGGTTGTGAAAATGTC-3 ; R: 5 -GCAAGTGTAAACAGCACTCA-3 and -actin (NM 001101): F: 5 CCTGTGGCATCCACGAAACT-3 ; R: 5 -GAGCAATGATCCTGATCTTC-3 . The PCR reaction and detection were performed with the MJ Research Chromo 4 PTC200 (MJ Research, USA). The expression levels were calculated and normalized to -actin. 2.6. Dye transfer assay Treated cells grown on sterile glass cover-slips in a 24-well dish were assayed for gap junction-mediated intercellular coupling by dye transfer assay as previously described [21]. In brief, the culture medium from a confluent monolayer was removed and saved. The cells were rinsed three times with HBSS (with calcium and magnesium) containing 1% BSA, before a 27-gauge needle was used to create multiple scratches through the cell monolayer in the presence of PBS containing 0.5% Lucifer yellow (LY) (Gibco BRL). After exactly 1 min, the culture was rinsed three times with HBSS and then incubated for an additional 10 min in the saved culture medium to allow the loaded dye to transfer to adjoining cells. The cells were then rinsed and fixed with 4% paraformaldehyde and immediately imaged on the confocal microscope using the argon laser at 485 nm. The level of GJIC was quantified as the average distance traveled (m) by the LY dye, from six different sites in each sample, after 10 min of dye loading. The efficiency (as a percentage) was calculated by normalizing to the average distance traveled by control and mock control. 2.7. FRAP assay GJIC was quantitatively assessed in living cells by FRAP assay as described previously [22]. Human bone marrow stromal cells cultured on glass cover-slips in six-well plates were grown to 80% confluence. The cells were then loaded at 37 ◦ C with CFDA (10 mol/L, 15 min) (Sigma, USA). After the loading process,
the cell culture slides were washed three times with DMEM medium to remove the fluorochrome-ester and prevent further dye loading during subsequent measurements. The cells were subjected to FRAP analysis. Clusters of five to six cells were selected under the microscope with the 20× objective lens. A cell contacted by four to five neighboring cells within a cluster was photobleached to 10% of the original fluorescence intensity using an argon laser beam. The bleached cell was then monitored for transfer of fluorescent dye from neighboring cells and examined for recovery of fluorescence by scanning at intervals of 50 s for a total period of 400 s. The data on the maximum intensity of recovered fluorescence (It ) at the 400th s was collected as the functional index of GJIC. At least three such clusters were selected from each dish. The analyzed fluorescence recovery index is expressed as: R = (It − I0 )/(I − I0 ) × 100%, where I0 is the intensity of the photobleached fluorescence and I is the intensity of pre-bleached fluorescence. 2.8. Statistical analysis Data were represented as mean values with standard deviation. Statistical significance was analyzed by Student’s t test. P values less than 0.05 were considered significant. The assessment of GJIC was performed using the Pearson Chi Squared test.
3. Results 3.1. Expression of Cx43 Immunocytochemistry assays demonstrated that expression of Cx43 in normal, pre- and post-chemotherapy BMSCs cultured in vitro could be found but that intensity of expression varied. In normal and post-chemotherapy BMSCs, Cx43 could be seen in the cell membrane appearing as large areas of brown adhesion plaque; it could also be found in the cytoplasm, frequently on just one side of cell. But, in samples from initial acute leukemia patients, Cx43 only expressed in a few cells, and there was no expression in the negative control group (Fig. 1A–D). The distribution of Cxs can be studied by immunofluorescence assay. In this study, Cx43 is found along appositional membranes of adjacent cells; these sites exhibit intense immunoreactivity
Fig. 3. Expression of Cx43 mRNA in BMSCs. Messenger RNA was isolated from 80% fusional monolayers of BMSCs from the 3 test groups and cDNA produced as described above. M: marker; 1–3: normal BMSCs; 4–6: CR-BMSCs; 7–9: leukemia BMSCs. BMSCs from leukemia patients expressed less Cx43 mRNA when compared to the other BMSC groups. Associated densitometric analysis demonstrated that the relative amount of Cx43 mRNA in leukemia BMSCs (0.621 ± 0.084%) was significantly lower than that in NBMSCs (0.072 ± 0.040%) and ABMSCs-CR (0.645 ± 0.095%) (p < 0.01).
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with anti-connexin antibodies. In some cells, a substantial amount of connexin can also be detected by immunofluorescence in the cytoplasm; this represents a storeroom of unassembled connexins within the biosynthetic pathway. Fluorescence of Cx43 was not detected in the cell nucleus. In normal BMSCs and CR-BMSCs, we could see green fluorescence located throughout the cell membrane and cytoplasm; we saw only noncontiguous small mass-like and dot-like fluorescence on the cell membrane and cytoplasm in acute leukemia BMSCs (Fig. 2A–C). Taken together, these results indicate that the level of Cx43 expression in CR-BMSCs was significantly higher than in primary acute leukemia BMSCs (p < 0.01), although lower than in normal BMSCs. The above results were confirmed by flow cytometry. Expression in normal BMSCs was the strongest (76.51 ± 13.15%). Expression in the CR-BMSCs group after chemotherapy (71.98 ± 7.5%) was slightly lower than expression in the normal group, although not statistically significant, and was significantly higher than that in primary acute leukemia (38.75 ± 23.95%) (p < 0.01). RT-PCR results showed that the stromal cells of all the three groups express Cx43 mRNA. As expected, the expression level of the initial acute leukemia group was lower than that of the normal and CR-BMSCs groups (p < 0.01, Table 1 and Fig. 3).
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3.2. Function of GJIC In normal BMSCs, LY fluorescence was transmitted to more than 10 cell lines through gap junctions over the time allowed in this study (Fig. 4A). LY fluorescence in the BMSCs of initial acute leukemia patients dispersed to cells nearby in 1–2 lines (Fig. 4B). The LY fluorescence in CR-BMSCs had dispersed to cells in 8–9 lines (Fig. 4C) indicating that communication between acute leukemia BMSCs was defective and had increased following chemotherapy. Furthermore, the stain could go through normal and CR-BMSCs within 1–2 min while it took more than 8 min to go through the acute leukemia BMSCs. The results demonstrated that function of GJIC in acute leukemia post-chemotherapy was significantly improved not only in its propagative scope but also in its propagative velocity compared to primary acute leukemia. FRAP results showed that the fluorescence intensity of normal BMSCs could recover quickly after photobleaching: 50% could recover in 1 min while 84.29% (±2.93) could recover within 400 s (Fig. 5A:1–3). Fluorescence intensity of CR-BMSCs could also recover quickly, and 78.94% (±2.46) could recover within 400 s (Fig. 5B:1–3). However, fluorescence intensity in BMSCs from acute leukemia recovered only about 20% in 1 min, and the maximal recovery percentage was 38.30% (±2.95) (Fig. 5C:1–3), which was
Fig. 4. Transmission of LY in BMSCs. LY transmission between (A) normal BMSCs; (B) leukemia BMSCs; and (C) CR-BMSCs were assessed. LY dye was allowed to diffuse through gap junctions for 10 min before assessment by confocal microscopy. BMSCs from leukemia patients showed decreased transmission of LY dye compared to normal and CR-BMSCs. The difference in the transmission of the LY stain between normal BMSCs and acute leukemia BMSCs was significant (Pearson Chi Squared = 93.600, v = 5, p < 0.01), as was the difference between acute leukemia BMSCs compared to CR-BMSCs (Pearson Chi Squared = 87.692, v = 5, p < 0.01). Images were made with 100× magnification.
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significantly different from the other two groups (p < 0.01). The average recovery rates of normal and CR-BMSCs were 19.20% (±1.39) min−1 and 13.52% (±1.65) min−1 respectively, significantly higher than that of acute leukemia BMSCs: 5.58% (±1.00) min−1 (p < 0.01) (Fig. 5D). 4. Discussion Occurrence of malignancy and malignant phenotypes of tumors may be closely related to gene expression defects in gap junction proteins and to inhibition of GJIC function [23–25]. Although gap junction proteins have been described in healthy human BMSCs [9,26,27], the functional status of GJIC in abnormal BMSCs is
unclear. In our previous work [18], decreased expression of Cx43 and GJIC dysfunction were observed in acute leukemia BMSCs. These results are consistent with the findings of previous solid tumor studies [28–30], which support the hypothesis that abnormal or paired cell-to-cell communication via gap junctions may be involved in the pathogenesis of tumors including acute leukemia. As a kind of hematopoietic stem cells malignant disease, abnormal hematopoietic stem cell generates not only abnormal hematopoietic cells but also abnormal stromal cells [31,32]. Bone marrow stromal cells also responded to therapy and, when undergoing effective chemotherapy, recovered their ability to construct and support blood-forming activity in acute leukemia BMSCs [33]. Then, we are more concerned about alteration of Cx43 expression and
Fig. 5. Results of FRAP in BMSCs. Fluorescence intensity of normal BMSCs could recover quickly after photobleaching and approximately 85% fluorescence could recover within 400 s (A1–3, 1, pre-photobleaching; 2, 1.5 s post-photobleaching; 3, 400 s post-photobleaching). Fluorescence intensity of CR-BMSCs could also recover quickly and approximately 80% fluorescence could recover within 400 s (B1–3, 1, pre-photobleaching; 2, 1.5 s post-photobleaching; 3, 400 s post-photobleaching). Fluorescence intensity of BMSCs of acute leukemia recovered more slowly and approximately 40% fluorescence could recover within 400 s (C1–3, 1, pre-photobleaching; 2, 1.5 s postphotobleaching; 3, 400 s post-photobleaching). Comparison of fluorescence recovery among NBMSCs/ABMSCs-CR/ABMSCs was summarized in (D). Images were made with 200× magnification.
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Fig. 5. (Continued )
GJIC function in acute leukemia BMSCs that have been effectively treated. Using 29 cases of primary acute leukemia of various types, we cultured BMSCs, compared the change in Cx43 expression pre- and post-chemotherapy, and compared the results to normal BMSCs as controls. The results showed that all three groups can express Cx43 in cell membranes and cytoplasm, but not in the cell nucleus. Interestingly, after effective chemotherapy, the amount of Cx43 in CR-BMSCs increased and was similar to that of normal BMSCs, and mRNA and protein levels were significantly higher than in primary acute leukemia (p < 0.01). Results showed that Cx43 expression in acute leukemia BMSCs is in a state of flux, and that factors involved in the process of treatment, including drugs, may affect its expression. Cxs expression in many tissues can be regulated by both in vitro and in vivo factors [34,35]. Cx43 is a major Cx in blood-forming tissues, and its expression within the BM is crucial in the development
of an efficient response to hematopoiesis [36]. Expression level of Cx43 fluctuated with the body’s need for hematopoiesis, improving the expression of this protein when such a need was increased [37,38]. Ongoing studies in our laboratory have demonstrated that Cx43 expression in acute leukemia BMSCs is up-regulated by retinoic acid (Liu et al., in preparation), showing that Cx43 in acute leukemia BMSCs is sensitive to drugs. This positive association between connexin expression and retinoic acid has been detected in other cell systems [39–41]. Recent studies verified that some hormones, cytokines and drugs could affect the expression of Cxs between coupling cells [42–45]. The dynamics of connexin expression in acute leukemia BMSCs is a complex process involving perturbations of connexin gene expression and connexin protein synthesis and degradation, as well as rearrangements in the spatial distribution of the protein. Further studies are required to identify changes that occur following treatment. Because of the half-lives of Cx are generally between 2 and 3 h
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Fig. 5. (Continued ).
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[46–49], that allow for regulation of GJIC within a few hours. This regulation occurs through alterations in connexin turn-over and degradation and by remodeling of gap junctions. It seems likely, because the internalized Cxs presumably cannot be recycled, so that up-regulation depends on synthesis of Cx43 de novo [50]. This process was possibly favored by the recovery of the BMSCs after effective treatment. On the other hand, the drugs that we used to cure leukemia or humoral mediators of the hematopoietic microenvironment can influence the synthesis, assembly and expression of Cx43 in BMSCs. Cx proteins can have antitumor functions independent of GJIC. Cx43 was shown to reduce proliferation in human glioblastoma cells without increasing GJIC [51]. Cx26 and Cx43 have also been shown to reduce growth of human breast cancer cells without altering GJIC [52]. These findings have led to investigation into whether GJIC function in acute leukemia BMSCs is altered following the increased expression of Cx43. Results of dye transfer and FRAP assays suggested that the function of GJIC was reconstructed by increasing Cx43 expression. It was shown that after effective treatment, both the quantity of Cx43 and the ability to form effective cell-to-cell communications increased. Whether the recovery of GJIC function post-chemotherapy came about through the direct effect of drugs (such as participating in the assembly of Cx) or through indirect regulation by induction of other cytokines, or both, remains unknown. Rivedal and Leithe [53] demonstrated that recovery of GJIC during continuous TPA (a kind of tumor promoter) exposure in normal and transformed rat liver epithelial cells is dependent on the synthesis of Cx43 protein de novo, as well as the transport of already synthesized Cx43 from intracellular pools to the plasma membrane. Confirmed by other recent studies on changes of GJIC function and some drugs [53,54], the results suggest that communication among cells is a dynamic process, which could be affected by other mediators. As GJIC was a tumor suppressor, improvement of its functions would have played an important role in inhibiting tumor formation [55]. At the same time, due to GJIC’s involvement in blood formation within the hematopoietic microenvironment [36,56], improving its function may be required for support of hematopoiesis of stromal cells. It is likely that Cx43 expression and GJIC function are affected when one or more chemotherapy drugs are used for different clinical programs, in turn affecting recovery of hematopoietic function and the development process of tumors in treated individuals. By extension, this study implicated GJIC as a potential therapeutic target and the ability to up-regulate GJIC may be an important indicator of chemotherapy potential. In conclusion, this paper described for the first time the change of Cx43 and GJIC among various BMSCs; expression of Cx43 and function of GJIC in acute leukemia BMSCs postchemotherapy increased and improved. These changes in connexin expression and GJIC function appear to reflect the changing requirements of hematopoiesis. The results also suggested that Cx43 expression and GJIC function in BMSCs is likely to be a dynamic process, regulated by many factors in vivo and in vitro. An in-depth study on the trend of change and relevant influencing factors of GJIC in tumorigenesis and tumor development can provide a theoretical and experimental basis for understanding GJIC in hematopoietic injury reconstruction and drug treatment for hematological malignancies, including leukemia.
Conflict of interest The authors declare that they have no potential conflicts of interest.
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Acknowledgments Xi Zhang contributed equally to this study. Yao Liu and Xi Zhang took responsibility in conception and design, collection and analysis of data, manuscript writing and final approval of manuscript; Zhong-jun Li has participated in the design and execution of the study; Xing-hua Chen has primary responsibility for the study design. We are grateful to Drs. Lei Gao, Li Gao and Cheng Zhang for bone marrow samples. We also thank Dr. Shi-cang Yu (Institute of Pathology, Southwest Hospital, Third Military Medical University) for the English writing. This work was supported in part by a grant-in-aid from the National Natural Science Foundation of China (No. 30670890), Chongqing Key Discipline of Medical Science (No. 2006C028) and special foundation for the “1520 project” of Xinqiao Hospital of Third Military Medical University.
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