Bis induces growth inhibition and differentiation of HL-60 cells via up-regulation of p27

Bis (Bag-3, CAIR), a Bcl-2-interacting protein, promotes the anti-apoptotic activity of Bcl-2 and increased levels of Bis have been observed in several disease models. The involvement of Bcl-2 and some Bcl-2-binding proteins in differentiation has recently been reported. However, the relevance of Bis to cellular differentiation remains unknown. The findings herein show that Bis expression is up-regulated during the differentiation of HL-60 cells. To investigate the effect of Bis expression on differentiation, we established Bis-overexpressing HL-60 cells (HL-60-bis). HL-60-bis cells have a low nuclear: cytoplasmic ratio and indented nucleus in Wright- Giemsa staining, and an increased expression of CD11b in immunofluorescence study, indicating the promotion of differentiation. The overexpression of Bis also resulted in a retarded cell growth rate, accompanied by the accumulation of HL-60 cells at the G0/G1 phase of the cell cycle, which was sustained during the differentiation process. Western blot analysis revealed that the expression of p27, a representative inducer of cell cycle arrest at the G1 phase, was increased 2.5-fold in HL-60-bis cells compared to HL-60-neo cells. These results suggest that the Bis induced growth inhibition of HL-60 cells promotes G0/G1 phase arrest via up-regulation of p27, which seems to be a prerequisite for differentiation. Further studies will be required to define the exact roles of Bis on cellular differentiation more precisely.


Introduction
Bis has been identified as a novel Bcl-2 binding protein that enhances the anti-apoptotic activity of Bcl-2 (Lee et al., 1999). Bis is also known as Bag-3 or CAIR-1, which has been reported to bind to Hsp70 or phospholipase Cγ, respectively (Takayama et al., 1999;Doong et al., 2000). The Bis protein shows no significant homology with the Bcl-2 family proteins and the transfection of bis DNA indicates that Bis itself serves as only a weak anti-apoptotic protein, but synergistically acts with Bcl-2 to protect from Bax and Fas-mediated cell death (Lee et al., 1999). The pro-survival activity of Bis is further supported by findings showing that the expression of Bis is increased in pancreatic cancer where Bcl-2 is frequently overexpressed and the down regulation of Bis protein levels using specific antisense nucleotides results in B-CLL cell apoptosis (Liao et al., 2001;Romano et al., 2003). In the brain, we previously observed that the expression levels of Bis protein were increased in reactive astrocytes, accompanied by increased Bcl-2 levels in the hippocampus after forebrain ischemia and kainic acidinduced seizure (Lee et al., 2002a;. Collectively, these results suggest that Bis modulates Bcl-2 activity under the certain patho-physiological conditions. It has recently been suggested that Bcl-2, in addition to its ability to protect against apoptosis, might also be involved in the regulation of cell differentiation such as neural differentiation and myeloid cell differentiation (Seite et al., 2000;Liang et al., 2003;Zhang et al., 2003). Immunohistochemistry experiments also revealed that Bcl-2 is highly expressed in several organs during morphogenesis and differentiation (Delia et al., 1992;Sun et al., 2002). In addition, the overexpression of Bag-1, a

Bis induces growth inhibition and differentiation of HL-60 cells via up-regulation of p27
Bcl-2 binding protein that shares the Bag domain with Bis (Takayama et al., 1995;Takayama et al., 1999), slowed the proliferation rates of CSM14.1 neuronal cells (rat nigro-striatal cell line) and accelerated neuronal differentiation (Kermer et al., 2002). Based on the findings that the Bis modulates anti-apoptotic activity of Bcl-2 in vitro (Lee et al., 1999) and Bis is co-expressed with Bcl-2 in vivo after an ischemic injury or excitotoxic brain injury (Lee et al., 2002a;Schwarz et al., 2002), it is possible that Bis also influences the differentiation process in which Bcl-2 is involved.
In the present study, therefore, we examined the Bis protein expression during the differentiation of HL-60 cells and the effect of Bis expression on the growth and differentiation of HL-60 cells to determine if a causal relationship exists between expression of Bis and differentiation.

Assessment of differentiation
Differentiation was determined by morphological observation after staining using Wright-Giemsa stain solution (Sigma-aldrich) of cytospins of HL-60-neo and HL-60-bis. Cell surface immunofluoroscence staining was also performed for both cells. Briefly, the cells were washed with cold PBS containing 1% bovine serum albumin, and labeled with PE-conjugated anti-human CD11b (BD Pharmingen, San Diego, CA) for 30 min at 4 o C in the dark. The cells were then washed twice with PBS and fixed in 1% paraformaldehyde/PBS solution. The cells were analyzed on FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA) using Cell Quest software (Becton Dickinson).

Cell growth analysis
Twenty thousand cells were plated on a 48-well plate in 200 µl of medium and incubated with various agents for the indicated times. Cell growth was monitored by the classical 2-(4,5-dimethyltriazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, 50 µl of MTT reagent (500 µg/ml) (Sigmaaldrich) was added to each well. After 3 h at 37 o C, the cell supernatants were discarded, MTT crystals were dissolved in acid isopropanol and the absorbance was measured at 570 nm using UV MR 700 (Dynatech Laboratories Inc., Chantilly, VA). All assays were performed in triplicate. The relative growth rate was defined as the percentage of the absorbance of treated cells versus untreated cells at 0 day. For the comparisons of growth rate, one-way ANOVAs were used. All results are presented as mean values ± SEM. Statistical significance was accepted at P 0.05.

Analysis of cell cycle distribution
After treatment with 1.3% aqueous DMSO, 1 × 10 6 HL-60-neo or HL-60-bis cells were harvested at the indicated times and washed twice with cold PBS and fixed in 70% ethanol at -20 o C overnight. The cells were washed with PBS and incubated with 100 µg/ml RNase A at 37 o C for 30 min. Propidium iodide (PI) solution (Sigma-aldrich) was added at a final concentration of 50 µg/ml. Analysis was performed immediately after PI staining by a FACS Calibur flow cytometer.

Expression of Bis during differentiation of HL-60 cells
To determine whether Bis expression is regulated during the HL-60 cell differentiation process, Bis protein levels were measured by Western blotting following the induction of differentiation using appropriate stimuli in HL-60 cells. Treatment of HL-60 cells with RA or DMSO is known to induce granulocytic differentiation while PMA leads to mono-cytic differentiation (Collons et al., 1978;Rovera et al., 1979;Breitman et al., 1980). As shown in Figure  1, all these three stimuli significantly increased the level of Bis expression in HL-60 cells and Bis induction was clearly detected as early as day 1 and decreased at day 5 after treatment. These results indicate that Bis is induced in the early phase of the differentiation process, raising the possibility that Bis is one of determinants of differentiation.

Effects of Bis expression on the morphology and growth of HL-60 cells
To investigate the significance of the Bis expression observed in the differentiation process, we established Bis-overexpressing HL-60 cells and examined the effect of Bis expression on differentiation. After transfection of the Bis-expressing vector or control vector, followed by three weeks of selection, the pooled populations from each preparation were analyzed for Bis expression by Western blotting.

HL-60-bis cells showed a considerable increase in
Bis expression, about a 13-fold higher level than in HL-60-neo cells (Figure 2A). Wright Giemsa staining was performed to observe whether Bis expression induces morphological changes in HL-60 cells. HL-60-neo cells were predominantly promyelocytes with large nucleoli with dispersed nuclear chromatin and the nuclear: cytoplasmic ratio, an indicator of differentiation, was relatively high ( Figure 2B). HL-60-bis cells showed a heterogeneous pattern of population. The main populations showed a low nuclear: cytoplasmic ratio and nucleoli were markedly reduced or had completely disappeared, and the nuclear membrane was indented or convoluted ( Figure 2B), suggesting that Bis expression promotes differentiation in HL-60 cells. Furthermore, the immunofluorescence study revealed that the CD11b antigen was expressed in a larger population of untreated HL-60-bis cells than in HL-60-neo cells, 14% and 1.8%, respectively as shown in Figure 2C, suggesting that phenotypic differentiation was also induced by Bis expression in HL-60 cells.
Since a differentiated phenotype is frequently associated with the transition out of the mitotic pool (Yaroslavskiy et al., 1999), we measured the growth kinetics of HL-60-bis cells. As shown in Figure 3A, HL-60-neo cells cultured for 3 days showed a 13-fold increase in viable cell populations while the HL-60-bis cells showed only a 5-fold increase for the same period, indicating that Bis expression caused an appreciable retardation in the rate of proliferation in HL-60 cells. When these two cells were induced to differentiate by RA or DMSO, HL-60-bis cells maintained their decreased growth rate, showing HL-60-neo and HL-60-bis cells were also analyzed for cell cycle progression by flow cytometry. Figure  3B demonstrates that the proportion of HL-60-bis cells in the G0/G1 phase was higher than that of HL-60-neo cells, 55.5% and 44.8%, respectively, in the absence of differentiation stimuli. When these two cells were treated with DMSO, to induce differentiation, the percentage of G0/G1 phase of cell cycle was increased in HL-60-neo cells, as observed previously (Braverman et al., 1986;Spector et al., 1993), from 44.8% at day 0 to 52.1 % at day 1 to 80.3% at day 2. In HL-60-bis cells, the G1 accumulation was persistent during differentiation, from 55.5% at day 0 to 61.2% at day 1 to 88.3% at day 2 in the presence of DMSO. These results indicate that the growth retardation in HL-60-bis cells could be attributed to the increased accumulation of cells at the G1 phase, in the absence or presence of differentiation agents.

p27 is up-regulated in HL-60-bis cells
To analyze the molecular mechanism of cell growth inhibition of Bis, we investigated the expression level of p21 and p27 proteins in HL-60-neo and HL-60-bis cells. These two proteins are representative inducers of cell cycle arrest at the G1 phase and also known to be increased by the induction of differentiation in hematopoietic cells (Taniguchi et al., 1999). A Western blot analysis showed that p27 expression was significantly increased in HL-60-bis cells, about 2.5-fold, compared to HL-60-neo cells ( Figure 3C). An increase in the expression of p21 was not detected in our experiments (data not shown). Therefore, the increased p27 may cause the cells to accumulate at G1 phase, which contributes in part to the growth inhibition of HL-60-bis cells.

Discussion
The control of cellular differentiation is a complex process that depends on a variety of endogenous and exogenous factors. In the present report, we observed that Bis, a novel Bcl-2 binding protein, is induced during the differentiation of HL-60 cells with appropriate differentiation-inducing stimuli. The maximal increase in Bis protein was observed at 3 day after the induction of differentiation when the differentiation is not fully completed, indicating that Bis induction is related to the early stage of differentiation rather than a result or an accompanying event of differentiation. Considering that an increase in Bis protein level is not confined to the differentiation of specific cell lineages, Bis might be involved in the basic mechanism of differentiation of different cell lineage. Although the functional role of Bis in the differentiation of HL-60 cells is not known, our results using Bis-expressing HL-60 cells suggest that Bis expression triggers the progress of differentiation, based on the morphological findings as well as CD11b expression. In addition, HL-60-bis cells also showed a retarded growth rate in the absence or presence of differentiation inducing stimuli, which is probably due to the accumulation of cells at the G1 phase, as evidenced by cell cycle analysis. Therefore, the increase in Bis expression during the differentiation seems to be involved in the inhibition of cell growth progress, which is known to be tightly coupled to steps forward differentiation (Defacque et al., 1999;Cabodi et al., 2000;Curtis et al., 2000;Nakajima et al., 2003).
While Bcl-2 has been recently reported to be involved in the differentiation of several cell types including neuronal cells (Mariano et al., 1992;Hanada et al., 1993;Zhang et al., 1996;Suzuki et al., 1998;Liang et al., 2003;Schuller et al., 2004), Bcl-2 levels decrease during the differentiation of myeloid cells (Naumovski et al., 1994). In addition, bcl-2-transduced HL-60 cells showed same differentiative response to RA or PMA with a prolonged survival (McCarthy et al., 1994;Park et al., 1994;Robertson et al., 1998). Thus, apoptosis and differentiation are regulated independently in myeloid cells. The induction of Bis observed in the present study is not correlated with the decreasing expression pattern of Bcl-2 in HL-60 cells (Delia et al., 1992;Naumovski et al., 1994). Therefore, the growth inhibitory activity of Bis may be a novel function of Bis which is not dependent on the presence of Bcl-2, even though Bis has been cloned as Bcl-2 binding protein (Lee et al., 1999). It is also possible that Bcl-2 negatively regulates Bis protein stability which is increased by the down-regulation of Bcl-2 during differentiation, as simply postulated by the inverse expression pattern of these two proteins. Even though the molecular mechanisms by which Bis induces growth retardation in HL-60 cells is not clear, an elevated expression of p27 in HL-60-bis cells provide a possibility that p27 functions as a mediator that delays the cell cycle progression. Our presumption is supported by a recent paper showing that HL-60 cells expressing antisense RNA for p27 revealed a significant decrease in their ability to differentiate into granulocytes (Horie et al., 2004), indicating that an increased expression of p27 is essential for cellular differentiation of HL-60 cells. However, differentiation was shown to be uncoupled from growth arrest in several cells such as primary keratinocytes or luteal cells. Therefore, it can not be excluded that Bis induces differentiation of HL-60 cells through uncharacterized mechanism which is not related with p27 (Tong et al., 1998;Dransfield et al., 2001). Consequently, an increased expression of p27 as well as the growth retardation observed in HL-60-bis cells might be an accompanying phenotype of differentiation, rather than a prerequisite for differentiation. Thus, the issue of whether p27 levels and growth rate are directly affected by Bis expression should be investigated in other types of cells to clarify the role of p27 in the differentiation of these cells in relation to Bis expression. And further studies using the systems in which Bis or p27 expression is genetically down-regulated will be necessary to define their roles in differentiation more precisely.