Retinoic Acid Therapy Resistance Progresses from Unilineage to Bilineage in HL-60 Leukemic Blasts

Emergent resistance can be progressive and driven by global signaling aberrations. All-trans retinoic acid (RA) is the standard therapeutic agent for acute promyelocytic leukemia, but 10–20% of patients are not responsive, and initially responsive patients relapse and develop retinoic acid resistance. The patient-derived, lineage-bipotent acute myeloblastic leukemia (FAB M2) HL-60 cell line is a potent tool for characterizing differentiation-induction therapy responsiveness and resistance in t(15;17)-negative cells. Wild-type (WT) HL-60 cells undergo RA-induced granulocytic differentiation, or monocytic differentiation in response to 1,25-dihydroxyvitamin D3 (D3). Two sequentially emergent RA-resistant HL-60 cell lines, R38+ and R38-, distinguishable by RA-inducible CD38 expression, do not arrest in G1/G0 and fail to upregulate CD11b and the myeloid-associated signaling factors Vav1, c-Cbl, Lyn, Fgr, and c-Raf after RA treatment. Here, we show that the R38+ and R38- HL-60 cell lines display a progressive reduced response to D3-induced differentiation therapy. Exploiting the biphasic dynamic of induced HL-60 differentiation, we examined if resistance-related defects occurred during the first 24 h (the early or “precommitment” phase) or subsequently (the late or “lineage-commitment” phase). HL-60 were treated with RA or D3 for 24 h, washed and retreated with either the same, different, or no differentiation agent. Using flow cytometry, D3 was able to induce CD38, CD11b and CD14 expression, and G1/G0 arrest when present during the lineage-commitment stage in R38+ cells, and to a lesser degree in R38- cells. Clustering analysis of cytometry and quantified Western blot data indicated that WT, R38+ and R38- HL-60 cells exhibited decreasing correlation between phenotypic markers and signaling factor expression. Thus differentiation induction therapy resistance can develop in stages, with initial partial RA resistance and moderate vitamin D3 responsiveness (unilineage maturation block), followed by bilineage maturation block and progressive signaling defects, notably the reduced expression of Vav1, Fgr, and c-Raf.


Introduction
For three decades, retinoic acid (RA) differentiation therapy has been tantamount to transforming acute promyelocytic leukemia (APL) from a fatal diagnosis into a manageable disease. RA induces remission in 80-90% of APL PML-RARa-positive patients [1]. However, remission is not durable and relapsed cases exhibit emergent RA resistance [2,3]. Meanwhile similar success stories have yet to be achieved for other cancer cell types. Parallel to the clinical use of RA in APL treatment, intense research has focused on understanding the source of cancer treatment relapse, and exploring the effectiveness of RA in other cancers.
Historically RA resistance in APL has been associated with mutation(s) in the PML-RARa fusion protein, rendering it unresponsive to RA. However, in some APL patients, PML-RARa mutations emerge months after termination of RA therapy, suggesting the existence of other defects [4]. In the patient-derived APL cell line NB4, RA resistance may or may not be correlated with mutant PML-RARa [4]. RA-resistant NB4 cells often remain partially RA-responsive in that they can upregulate RA-inducible differentiation markers, such as CD38 or CD18 [5]. HL-60, another patient-derived leukemia cell line, does not harbor the t(15;17) translocation pathognomonic for APL and thus lacks PML-RARa, but is nevertheless RA-responsive. Like NB4 cells, in vitro maturation of HL-60 cells is consistent with that of primary APL cells in culture and with clinical RA differentiation therapy progression [4]. Ectopic expression of RARa in RA-resistant HL-60 cells in which mutant RARa was found also does not necessarily restore RA responsiveness, again suggesting the presence of other defects [6,7].
There is great interest in employing differentiation-promoting agents in combination with RA treatment to overcome resistance, and improve therapy and prognosis in APL and other cancer types. The active form of vitamin D 3 , 1,25-dihydroxyvitamin D 3 (D 3 ), which acts through vitamin D receptor (VDR), is capable of inducing differentiation in myelo-monocytic precursor cells, but has been less widespread as a clinical treatment since D 3 also induces hypercalcemia and hyperphosphatemia. However, co-administration of RA with D 3 is a potential therapeutic strategy to mitigate the side effects and limitations of each individual inducer. Bipotent human acute myeloblastic leukemia (FAB M2) HL-60 cells can be induced to terminally differentiate in vitro along the granulocytic lineage toward neutrophil-like cells using RA, while differentiation along the monocytic lineage can be achieved with D 3 .
RA-treated HL-60 undergoing granulocytic differentiation display early increased surface expression of CD38, followed by CD11b expression. D 3 -treated HL-60 cells undergoing monocytic differentiation express CD38, higher levels of CD11b, and the monocytic surface marker CD14. Induced terminal differentiation is accompanied by G1/G0 cell cycle arrest, and the development of inducible oxidative metabolism (respiratory burst), a function of mature granulocytes and monocytes. For the RA-treated case, differentiation requires sustained activation of mitogen-activated protein kinase (MAPK) signaling along the Raf/MEK/ERK axis [8], and a cascade of signaling regulatory events involving a putative signalosome containing c-Cbl, Vav1, and the Src-family kinases Lyn and Fgr [9,10]. This is due in part to retinoic acid response elements (RAREs) in the promoter regions of CD38 and BLR1 [11,12]. Both of these proteins are rapidly upregulated by RA; CD38 is the nexus for the putative signalosome while BLR1 drives a prolonged MAPK signal though its relationship with c-Raf [12]. However, D 3 -induced differentiation also requires sustained MAPK signaling [13] and results in upregulation of CD38 and CD38-associated factors.
Onset of G1/G0 arrest and terminal differentiation is slow requiring approximately 48 h of treatment, during which HL-60 cells undergo two sequential, functionally discernible stages [14][15][16][17]. With a doubling time of approximately 20-24 h, induced HL-60 cells first become primed for differentiation (precommitment phase) and undergo early differentiation events. During the subsequent 24 h, HL-60 complete a second cell division that results in terminally differentiating cells which are committed to a specific lineage determined by the inducer present, e.g. RA or vitamin D 3 [14,16]. Although lineage-specific events, such as CD14 expression, can in fact occur during the first 24-48 h of D 3 treatment in HL-60 [13], the final inducer present is nonetheless the determining factor for lineage selection and subsequent terminal differentiation into that lineage [18]. It has also been shown that HL-60 cells treated with RA for 24 h followed by washing and no retreatment results in a still-proliferating population that retains a ''memory'' for differentiation that lasts 4-6 cell divisions [15]. During this time, cells proliferate until retreatment in which short RA doses can induce complete granulocytic differentiation.
We previously isolated two emergent RA-resistant HL-60 cell lines [19] after chronic RA exposure. These RA-resistant lines do not express CD11b, exhibit G1/G0 arrest, nor develop oxidative metabolism after RA treatment. One resistant line (R38+) retains RA-inducible CD38 expression while the other (R38-) has lost this ability. The R38-line, which sequentially emerged from R38+, thus appears to have an earlier defect which blocks the RAinduced differentiation sequence before the expression of CD38. Signaling events that define the wild-type response are compromised in both R38+ and R38-, which include RA-induced c-Raf expression and phosphorylation, c-Cbl and Vav1 expression, expression of Src-family kinases (SFKs) Lyn and Fgr and Y416 SFK phosphorylation.
In this study we examined whether the RA resistance defect segregates with lineage specificity, or with early or late stages of induced differentiation. An early defect might compromise both lineages, whereas a late defect might only affect the granulocytic lineage. Here we report that an RA-resistant cell line that retains partial RA-responsiveness (R38+) is more amenable to D 3 -induced differentiation, while the more resistant cell line (R38-) is only partially responsive to D 3 . We conclude that the defect in RA response is not necessarily compensated for by D 3 treatment to enable myeloid differentiation, and the RA defect is apparently early and late, possibly reflecting dysfunctions in proper prolonged signaling during early and late stages. The signaling dysfunction notably involves reduced Fgr, c-Raf, and Vav1 expression. Overall, the results are of potential significance to the use of differentiation-inducing agents for overcoming RA resistance.

Results
We examined if 1,25-dihydroxyvitamin D 3 (D 3 ) is able to upregulate differentiation markers in either R38+ or R38-retinoic acid (RA)-resistant HL-60 cells, and by exploiting the biphasic characteristics of HL-60 differentiation, whether D 3 could compensate for RA response dysfunction during the precommitment (first 24 h) or lineage-commitment stage. HL-60 cells were treated with one inducer (RA or D 3 ) for 24 h, then washed and retreated with either the same, different, or no inducer. The expectation was that the cells receiving the same differentiation agent will behave as if in continuous exposure, while the cells whose differentiation agent switched will reveal dependence of various differentiation markers on the precommitment vs. lineagecommitment stages. Cells receiving no retreatment will reveal what aspects of the differentiation program are precommitmentdependent and will provide a control for the retreated cases. For each treatment regimen, 1 mM RA or 0.5 mM D 3 was used as these doses were previously shown to yield comparable levels of differentiation in HL-60.
R38-exhibit diminished CD38 expression in response to D 3 compared to R38+ and WT HL-60 We assessed CD38 expression at 24 h to probe resistance in early or precommitment events prior to retreatment with a second inducer, and hence prior to the lineage-commitment phase. At 24 h, both RA and D 3 induced CD38 expression in WT HL-60 ( Fig. 1A). Three RA and D 3 cases are shown since each served as the initial 24 h treatment for the subsequent timepoints. An average of 94% and 70% of the WT cells were CD38 positive for RA and D 3 respectively. At 24 h, R38+ and R38-respond to RAtreatment as similarly reported for 48 h [19], with R38+ expressing, and R38-failing to express, CD38 after RA treatment. CD38 expression in RA-treated R38+ was similar to that of RAtreated WT HL-60, while the CD38 expression level in RAtreated R38-was similar to untreated WT HL-60, as expected. D 3 induced CD38 expression in both resistant cell lines. In R38-the expression was about 50% of the expression in the WT cells, and in R38+ the expression was significantly (p,0.05) higher than in WT. CD38 expression in D 3 -treated R38+ cells is similar to CD38 expression in RA-treated WT HL-60, despite expression in RAtreated WT HL-60 significantly exceeding D 3 -treated WT HL-60 (p,0.0001). Thus R38-cells have precommitment resistance to D 3 but R38+ cells do not, and R38+ actually have higher CD38 expression than D 3 -treated WT HL-60.
48 h and 72 h timepoints probed resistance in later events. 48 h post treatment (24 h in precommitment and 24 h in lineagecommitment stages), R38-cells treated with RA/D 3 or D 3 /D 3 have comparable CD38 expression levels (38% vs. 40%, Fig. 1B). R38-cells treated with D 3 /RA are 31% positive for CD38, indicating that in R38-, D 3 presented early or late could elicit CD38 expression at comparable levels, although it was short of that of WT cells. By 72 h, CD38 expression induced by D 3 in the resistant cells does not increase significantly (Fig. 1C). R38+ cells present two interesting behaviors. First, in the cells receiving RA in the precommitment phase and not receiving any differentiation agent in the lineage-commitment phase (RA/-), a decrease in CD38 expression is more abrupt in R38+ (75% at 48 h vs. 54% at 72 h) than in WT HL-60 (92% vs. 85%), showing an impaired ability of R38+ to maintain CD38 expression. Second, D 3 /D 3 and D 3 /-treatments in R38+ have higher levels of CD38 than WT HL-60 cells (p ,0.005 and p,0.05 respectively).
Overall, WT HL-60 cells behave as expected, with CD38 expression increasing over time during all treatment patterns, except for RA/-and D 3 /-, in which CD38 expression decreases as the cells revert to a proliferating state. R38+ have more CD38 expression than WT when treated with D 3 first (but not with RA first), and show a more rapid decrease in CD38 expression than WT during RA/-and D 3 /-. R38-have half the induced CD38 expression compared to WT during RA/D 3 and D 3 /D 3 , slightly less for D 3 /RA, no CD38 expression during RA/RA or RA/-, and decreasing CD38 expression during D 3 /-. Hence compared to R38+, R38-cells have a diminished response to D 3 in terms of CD38 expression, and they have a more pronounced early defect not apparent in R38+.
WT, R38+ and R38-HL-60 cells comparatively display decreasing D 3 -induced CD11b expression CD38 is the only marker significantly expressed during the precommitment stage (first 24 h). We next focused on 48 h and 72 h to probe resistance in late events. CD11b is an integrin component expressed in granulocytes and monocytes [20]. RA does not induce CD11b expression in either RA-resistant HL-60 cell line [19]. D 3 rescues CD11b expression in both R38+ and R38-cells when administered early or late, with R38+ being slightly more responsive (Fig. 2). The effect of D 3 on CD11b expression appeared to be more potent if administrated during the lineage-commitment stage. Comparing RA/D 3 and D 3 /RA at 48 h, p,0.004 for WT HL-60, p = 0.03 for R38+ and p = 0.01 for R38- (Fig. 2A). By 72 h, the D 3 /RA-treated R38+ and R38-cells have similar levels of CD11b to cells treated with D 3 /-, indicating that retreatment with RA was comparable to no retreatment for both the resistant cell lines (Fig. 2B). Overall, WT HL-60 behaved as expected. WT HL-60 exhibited increasing (over time) CD11b expression for all treatment regimens save for RA/-and D 3 /-, in which CD11b expression levels dropped by 72 h. Since D 3 /RA treatment, compared to D 3 /D 3 , did not fully restore a WT-like response in the RA-resistant cells, the data suggest a late RA defect(s), which is putatively more pronounced in R38-than R38+.  CD14 is a glycosylphosphatidylinositol-anchored membrane protein expressed by monocytes, but not by granulocytes [21]. CD14 is a monocytic-specific marker for detecting a differentiation response to D 3 treatment, and here its expression reveals whether defects are lineage unrestricted or restricted (i.e. early or late). We expect WT HL-60 cells to exhibit CD14 expression only during D 3 /D 3 and RA/D 3 treatments. By 72 h, all three cell lines treated with D 3 /D 3 expressed CD14 at significantly higher levels than all other treatments, with R38+ expressing slightly higher levels (43% positive cells) than the WT HL-60 cells (33% positive cells, Fig. 3B). At 72 h there is no significant difference between RA/D 3 and D 3 / D 3 treated cells for either R38+ or R382 individually, although R38+ cells had higher CD14 expression than R382 cells (Fig. 3B). This indicates that monocytic differentiation can potentially occur in the RA-resistant cells, and that monocytic differentiation can occur if the differentiation agent present during the lineagecommitment phase is D 3 . The R382 response was weaker than the R38+ response, consistent with progressive attenuation of D 3 response as cells become more resistant.

D 3 cannot rescue respiratory burst activity in either R38+ or R382
Respiratory burst (oxidative metabolism) is the ability of mature neutrophils and macrophages to respond to bacterial infections, and is considered a final functional marker of maturity. None of the treatment regimens were able to significantly rescue this late differentiation marker in the RA-resistant HL-60 ( Fig. 4, also see Discussion). Although D 3 /D 3 treatment tended to increase the respiratory burst activity, this did not reach statistical significance. The WT HL-60 behaved as expected, exhibiting a strong respiratory burst in all treatment cases except for RA/-or D 3 /-.

R38-cells comparatively display the lowest level of D 3induced G1/G0 cell cycle arrest
We examined G1/G0 cell cycle arrest, which is also a relatively late attribute of induced differentiation. In WT HL-60, all treatments except RA/-and D 3 /-induced a significant (p, 0.005) G1/G0 enrichment at 48 h (Fig. 5A). RA-treated RAresistant cells do not exhibit G1/G0 arrest. For both R38+ and R38-, the only treatments that significantly (p,0.05) increased the proportion of cells in G1/G0 were RA/D 3 and D 3 /D 3. Differences between the individual responses of R38+ and R38were not yet significant at 48 h. By 72 h, the WT cells treated with RA/RA, RA/D 3 , D 3 /D 3 or D 3 /RA were significantly (p,0.005) arrested in G1/G0 (Fig. 5B) . Also at 72 h, R38+ cells were arrested by RA/D 3 and D 3 /D 3 treatments, while the R38-resistant cells were arrested just by D 3 /D 3 treatment (p = 0.03). Thus for the R38+ cells, D 3 had to be administrated at least in the lineagecommitment stage, whereas for the more severely resistant R38cells, D 3 had to be administrated in both precommitment and lineage-commitment stages to obtain significant growth arrest by 72 h. This is consistent with a late differentiation dysfunction for R38+ and early and late dysfunction for R38-.  [8,10,[22][23][24][25][26][27][28][29]. Knowing the behavior of this ensemble during RAinduced differentiation, we sought deviations from this in the resistant cells to gain insight into the potential molecular basis of the cellular phenotypic behavior above. We examined the CD38associated proteins c-Cbl, Vav1, and Slp76; the Src-family kinases (SFKs) Lyn and Fgr, and the Y416 SFK phosphorylation site; c-  Raf and its RA-induced phosphorylation sites S259, S621 and S289/296/301; p47 phox , one of many proteins related to oxidative metabolism; and aryl hydrocarbon receptor (AhR) which we reported drives differentiation.
We first investigated proteins known to exhibit increased expression in WT HL-60 by 24 h. Fgr is upregulated by RA after 24 h in WT HL-60, but not in R38+ or R38-resistant cells. Fgr could still be upregulated by D 3 in R38+ cells at 24 h. (Fig 6A). But R382 cells were slower and required 48 h (Fig 6B, Fig 7) before Fgr upregulation was discernible. This correlates with the putative more profound resistance of R382 cells. Vav1 is upregulated by RA and D 3 in WT HL-60, and in contrast to Fgr, D 3 results in higher Vav1 expression during the first 24 h compared to RA treatment. But in resistant cells, RA did not cause any appreciable Vav1 upregulation, whereas D 3 is able to increase Vav1 expression in both RA-resistant cell lines. p47 phox is comparably induced by RA and D 3 in WT HL-60 during the first 24 h. But in R38+ cells RA induces only a small increase in p47 phox and none detectable in R382. In contrast D 3 treatment prominently increases expression of p47 phox in both RA-resistant lines. Slp76 is upregulated by both RA and D 3 in WT HL-60, but expression in resistant cells hardly changed with either RA or D 3 treatment at 24 h. Meanwhile c-Cbl does not exhibit much change by the end of the early 24 h timepoint. There are thus early defects in RAinduced upregulation of select signaling molecules such as Fgr, Vav1, and p47 phox , whose expression in the resistant cells is, to some degree, rescued by D 3 .
WT, R38+ and R382 HL-60 cells comparatively display decreasing D 3 -induced expression and phosphorylation of differentiation-associated signaling factors at 48 h We examined signaling factor expression at 48 h to probe for resistance-related aberrations during the late, lineage-commitment phase. Representative blots for 48 h signaling data are shown in Fig. 6B. To address slight variations across repeats, all repeats where quantified and average fold change 6 S.E.M. is presented in Fig. 7. Also, Figure S1 regraphs the same data separated by cell line to further clarify the expression differences. At 48 h, WT HL-60 treated with RA/RA behaved as expected, with RA exposure resulting in increased Fgr, Lyn, Vav1, c-Cbl, Slp76, c-Raf, AhR and p47 phox expression (Fig. 6B, Fig. 7) compared to untreated control. However, RA-resistant cells (R38+ and R382) treated with RA/RA displayed little to no upregulation of these signaling proteins (excluding Slp76, as found previously [19]) compared to WT HL-60. For the RA/D 3 treatment case, WT HL-60 again show upregulated expression of these proteins. Interestingly, R38+ and R382 treated with RA/D 3 exhibited increased Vav1 and p47 phox expression compared to the RA/RA case, although expression in R382 was diminished relative to R38+. R38+ cells also exhibited c-Cbl expression with RA/D 3 treatment, but R382 tended not to. Lyn and c-Raf were minimally increased in the RAresistant cells during RA/D 3 treatment. In the resistant cells, Fgr expression is only slightly increased during the RA/D 3 case, with the R38+ line exhibiting less Fgr expression compared to WT HL-60, and R382 even less compared to R38+. Overall this indicates that D 3 treatment, despite the previous RA treatment, was able to predominantly rescue expression of Vav1 and p47 phox signaling proteins in R38+ and less so in R382, consistent with the more apparent resistance to D 3 of R382 compared to R38+. As most evidenced by Fgr, WT cells treated with RA/-(i.e. RA followed by no retreatment) displayed similar but diminished expression of signaling proteins compared to the RA/RA case, consistent with a need for continuous early and late exposure to drive expression.
For D 3 /D 3 treated cells, WT and R38+ lines displayed upregulated Fgr, Lyn, Vav1, c-Cbl, c-Raf and p47 phox expression compared to untreated controls, while R382 had notably diminished expression of these proteins, again consistent with greater D 3 response dysfunction in R382 compared to R38+ (Fig 6B, Fig 7). For the D 3 /RA treatment pattern, WT HL-60 still show upregulation of these proteins. But for the RA-resistant R38+ and R382 cells, D 3 followed by RA treatment resulted in less Fgr, Vav1, c-Cbl, c-Raf and p47 phox expression compared to the D 3 / D 3 case, consistent with a putative late defect in both resistant cell lines.
During RA/RA treatment in WT HL-60, phosphorylation at c-Raf sites S259, S621, and S289/296/301 is increased (Fig. 6B,  Fig. 7). For the resistant cells, this response was largely lost. For RA/D 3 treatment, the pS259c-Raf and pS289/296/301c-Raf responses were recovered in R38+ cells, but not in R382 cells, consistent with their putative greater resistance. In both resistant cells, the pS621c-Raf response was lost in RA/D 3 treatments, consistent with the importance of this phosphorylation event in driving differentiation as suggested earlier [30]. D 3 /D 3 treatment induced phosphorylation at all c-Raf sites checked in WT and R38+ cells. Both R38+ and R382 retained S621c-Raf during D 3 / D 3 treatment, but unlike R38+, phosphorylation at S259 and S289/296/301 sites on c-Raf was lost in R382 cells. Increased loss of induced c-Raf phosphorylation thus correlated with increased resistance. c-Raf phosphorylation was generally reduced in D 3 /RA-treated RA-resistant cells compared to the D 3 /D 3 case. Overall these c-Raf phosphorylation changes did not necessarily correlate with the changes in c-Raf expression level. In the different treatment regimens, RA and D 3 , administered singly or in sequential combination, caused increased c-Raf expression in WT HL-60. The response in each instance was diminished in R38+ and even more diminished in R382. The Y416 SFK (Srcfamily kinase) site also showed increased phosphorylation in RAtreated WT HL-60 cells, and this response was largely abrogated in both resistant cells. D 3 administered early or late tended to cause, albeit much smaller, an increase in Y416 SFK phosphorylation in the R38+ but not R382 cells.
Taken together the above data motivate the notion that this ensemble of signaling molecules and events support differentiation and that progressive resistance is concomitant with their decreased expression and phosphorylation. Cluster analysis (see Discussion) reveals that in WT HL-60 cells there is a tight coupling between the responses of the ensemble of signaling molecules for different treatment regimens, but the coupling is degraded as the cells become progressively more resistant (Figs. 8B-D).

Phenotypic Differences between WT, R38+ and R382 HL-60 cells
To our knowledge, this is the first study that analyzes how RA resistance depends on early vs. late lineage-commitment events in lineage-bipotent myeloid cells and relates to lineage crossresistance. Taking the cellular response data together, the responses of R38+ and R382 RA-resistant HL-60 cells to the combinatorial sequences of RA and D 3 treatment distills to two basic results. First, in both R38+ and R382 resistant cells, D 3 /RA treatment does not restore RA response, while RA/D 3 could not fully restore D 3 response. Thus D 3 cannot necessarily abrogate the temporally segregated early or late RA defect(s). Second, as the resistance to RA became more pronounced with progression from R38+ to R382, there was progressive emergence of worse D 3 response. That is, the response to D 3 administered early or late in combination with RA, or administered both early and late, was less effective in R382 than R38+ cells. Although both R38+ and R382 cells equally failed to develop a significant oxidative metabolism, D 3 treatment can nevertheless rescue expression of the respiratory burst-associated protein p47 phox in the resistant cells, with the greatest expression occurring during D 3 /D 3 treatment and slightly lower during RA/D 3 treatment, and greater expression always occurring in R38+ compared to R382. When treated with D 3 during the lineage-commitment phase, R38+ cells always exhibited higher CD14 expression than R382 cells. R382 cells consistently displayed lower CD38 and CD11b expression, lower differentiation-associated signaling factor expression and phosphorylation, and notably had lower G1/G0 cell cycle arrest compared to both WT and R38+ HL-60. Therefore we found that RA differentiation therapy resistance can develop in stages, with initial partial RA resistance and moderate D 3 responsiveness (unilineage maturation block), followed by subsequent pronounced RA resistance and partial D 3 resistance (bilineage maturation block).
RA can inhibit monocyte/macrophage activity [31], and other differentiation programs can also be suppressed by a RARdependent process [32]. In the case of WT HL-60 cells, although the precommitment stage can be induced by RA or D 3 , the later stages of monopoiesis are inhibited by RA [15,16]. If enhancing the differentiation process toward one lineage may inhibit another, then it may be plausible that cells resistant to one induced lineage can respond more strongly to another induced lineage (i.e., the ''repressive'' pathway is removed). This could be one explanation for why early D 3 treatment induced a slightly stronger response in the R38+ RA-resistant cells than the WT cells in terms of CD38 and CD14 expression.
We performed hierarchical clustering analysis between the cell lines across all treatments and results, and interestingly found that WT and R38+ clustered more closely than R382 (Fig. 8E). Agglomerative hierarchical clustering analysis across all cell lines and treatments vs. signaling components is diagramed in Fig. 8A. The treatment cluster family for WT HL-60 separates into two clusters: those treated with RA first and those treated with D 3 first. The untreated control samples exist in a cluster with R382 RA/ RA and R382 RA/-. This is consistent with the notion that R382 is the most resistant cell line and consequentially the least dissimilar from untreated WT cells. Allowing that R382 RA/represents the least responsive case, then the cluster analysis reveals a progression of cases that become more distal to and deviate from the most unresponsive case, namely R382 RA/2, R382 RA/RA, R38+ RA/2, R38+ RA/RA, and finally the RA/ D 3 cases for both resistant lines. This clustering conforms to the anticipation that R382 are less responsive than R38+, and that RA is generally less effective than D 3 in eliciting response in the resistant cells. The cases for early D 3 -treated resistant cells group together further away in the clustering analysis, consistent with weaker resistance to D 3 compared to RA posited earlier.
When comparing both the signaling results and the cellular phenotypic results, hierarchical clustering across all treatments for WT (Fig. 8B), R38+ (Fig. 8C), and R382 (Fig. 8D) reveals the increasing distances (lower correlations) as cells become more resistant compared to the WT HL-60 cells. A progressive uncoupling of the signaling molecules thus occurs as WT HL-60 change to R38+ and then to R382. Thus the repertoire of signaling proteins surveyed may have a seminal role in effecting differentiation. Progressive degradation of the clustering of an ensemble of putative signalosome molecules as resistance increases supports the importance of an intimate co-regulated clustering of those molecules to drive differentiation. We investigated RARa and VDR protein levels at 24 and 48 h ( Figure S2) and were unable to attribute decreasing resistance to loss of either receptor.

Vav1, Fgr and c-Raf emerge as prominent differentiationassociated factors
A potential suite of molecular dysfunctions is seminal to the progression of observed resistance phenotypes. Vav1 is required for RA-induced granulocytic differentiation [33] as well as TPAinduced monocytic differentiation of HL-60 [34]. Vav1, along with c-Cbl and Slp76, exhibit increased expression and exist in a CD38-associated complex during RA-induced differentiation of WT HL-60 [9]. These signaling factors are also upregulated along with CD38 during D 3 treatment in WT HL-60, as well as in RAresistant HL-60. A cohort of molecules known to interact with CD38 is evidently expressed along with CD38 during either monocytic or granulocytic differentiation. D 3 induced Vav1 expression in R38+ and R382 during the first 24 h. If the two RA-resistant lines were retreated with D 3 , then Vav1 expression persisted. However if the second treatment was RA, Vav1 expression tended to diminish by 48 h. A similar outcome occurs for c-Cbl, and p47 phox . Thus, although ectopic overexpression of Vav1 or c-Cbl can enhance RA-induced differentiation in WT HL-60 [9,33], early-induced expression of these signaling factors in resistant cells is not enough to propel RAinduced differentiation during the lineage-commitment stage, which may reflect the co-existence of other potential defects. The data suggest that a late Vav1-dependent function may be disrupted in resistance, and lesser Vav1 expression in R382 compared to R38+ cells may contribute to the increased D 3 resistance in R382 cells.
We have previously reported that the Src-family kinases (SFKs) Lyn and Fgr are upregulated with RA treatment in WT HL-60 cells [10]. The D 3 -induced upregulation of Lyn and Fgr has been noted by us and others [35]. Lyn and Fgr are the predominant SFKs expressed in myeloid cells [35,36]. However of the two, only Lyn appears to be the predominantly active (phosphorylated) kinase in RA-induced HL-60 [10,37], as well as in RA-treated NB4 cells [38]. Lyn and Fgr have been found to exert their functional roles in distinct subcellular compartments [38]. When the aryl hydrocarbon receptor (AhR) ligand 6-formylindolo(3,2b)carbazole (FICZ) enhances Lyn and Fgr expression, as well as Vav1, c-Cbl, and p47 phox expression, it also enhances RA-induced differentiation in WT HL-60 cells [39].
In R38+ and R382 RA-resistant HL-60 cells, Fgr expression was not induced by RA at 24 h, as expected, yet was only minimally rescued either early or late by D 3 compared to WT HL-60 cells. Thus Fgr may be a signaling component important to the non-resistant phenotype, and dysfunctional early RA regulation of Fgr emerges as a prominent feature of resistance that correlates with loss of cellular phenotypic response. Phosphorylation at the  Figure 6 were quantified using ImageJ and average fold change from control was graphed in GraphPad. Error bars represent standard error. Note that the fold change axis scale may differ for each bar graph. doi:10.1371/journal.pone.0098929.g007 Y416 SFK site seems to be primarily an RA-driven event in the WT HL-60 cells, as the highest pY416 SFK phosphorylation occurs during RA/RA, RA/D 3 and D 3 /RA treatments. In contrast, results for Lyn and AhR were not as striking. Overall, there appeared to be higher Lyn expression in WT HL-60 cells across all treatment patterns. But upregulation by RA/RA, RA/ D 3 , D 3 /D 3 or D 3 /RA for all cells lines remained similar; hence it is not apparent whether Lyn expression is specific to any inducing agent or phase of differentiation. Similar results were obtained for AhR, with the exception of RA/RA treated WT HL-60, which had the highest AhR expression among all treatment cases and cell lines.
c-Raf phosphorylation appears disrupted in resistance. Phosphorylation at the putative inhibitory site S259, the stability site S621, and the functionally ambiguous S289/296/301 site has been found to be induced by RA [10,19,30]. Here we show that S259 c-Raf phosphorylation may be an early (but not late) RA driven event. Enhanced pS259c-Raf is observed in WT cells during RA/RA, RA/D 3 , and RA/2 treatment (Fig. 7A), but not during D 3 /RA treatment (despite higher phosphorylation for the D 3 /D 3 case). Also p259c-Raf is increased in the R38+ HL-60 during RA/D 3 and D 3 /D 3 , but not D 3 /RA, treatment. Thus for both the WT and R38+ cells, pS259 was higher during RA/D 3 than D 3 /RA treatment, consistent with being an early RA-driven event. Interestingly, the phosphorylation site S289/296/301 was significantly increased (as high as in WT cells) in R38+ during RA/D 3 treatment, but when D 3 was used first, R38+ had less pS289/296/301c-Raf than WT (Fig. 7A and 7B).
Overall for c-Raf expression and phosphorylation, the R382 cells tended not to show as great a response to D 3 compared to WT or R38+, whether D 3 was treated first or last (Fig. 7C). This is again indicative of the greater degree of disrupted c-Rafdependent signaling in these cells. Consistent with this, c-Raf expression was similarly progressively less during all treatments across WT, R38+ and R382 HL-60 cells. Like Vav1 and Fgr, c-Raf emerges as putatively a key component of the non-resistant phenotype. p47 phox and c-Cbl expression may be correlated with CD38 and/or CD14, since these two signaling factors were also more highly expressed in early D 3 -treated R38+ cells compared to WT HL-60. But Fgr, Vav1 and c-Raf showed decreasing (across WT, R38+, R382) induced expression for all treatments, similar to CD11b expression and G1/G0 arrest, notably implicating their dysfunction in progressive resistance.

D 3 treatments and RA resistance in other studies
Retinoic acid (RA) and the active form of vitamin D 3 , 1,25dihydroxyvitamin D 3 (D 3 ), are dietary factors that demonstrate chemotherapeutic efficacy in inducing maturation in leukemia cells. RA is the current treatment for acute promyelocytic leukemia (APL) [40], and retinoids serve preventative and therapeutic roles in other cancers and diseases [3,41,42]. D 3 is able to exert anti-proliferative effects in other myeloid cells [43] and other cancer cell types [44]. It has been shown that analogs of D 3 can induce differentiation of myeloid cells with minimal calcium toxicity [45][46][47]. Like D 3 , D 3 analogs have shown efficacy in inducing differentiation not only in myeloid lines, but in prostate and breast cancer cells [48,49]. Co-administration of RA with D 3 or analogs thereof is a potential therapeutic strategy to mitigate the side effects of each individual inducer (RA syndrome, hypercalcemia, RA or D 3 resistance).
One group found that RA and analogs of D 3 can act synergistically in WT HL-60 to promote differentiation and inhibit cell growth [50], and that a RA-resistant HL-60 cell line is more sensitive to D 3 treatment than the parental WT cells [51]. We previously developed an HL-60 cell line resistant to sodium butyrate (a monocytic inducer) that was also cross-resistant to RA; however, this line remained responsive to monocytic differentiation by D 3 [52]. Interestingly, D 3 was shown to induce granulocytic (not monocytic) differentiation in a RA-resistant APL cell line [53]. In another case, D 3 treatment did not induce differentiation in RA-resistant HL-60 [6]. RA resistance in HL-60 has been historically attributed to mutation of RARa [54,55]. However, in some RA-resistant myeloid lines where a mutation in RARa was found, expression of wild-type RARa did not fully restore RA responsiveness [6,7]. It is clear that other defects arise, which most likely vary across resistant sublines developed in different laboratories. This may account for the varying reports seen in the literature regarding a response (or lack of response) of RA-resistant cells to D 3 treatment. In fact, in a single study one group developed two RA-resistant HL-60 cell lines, one of which was D 3 -responsive and harbored a RARa mutation, while the other was D 3 -resistant and had intact RARa [56].

Conclusions
The present study shows that induced signaling and phenotypic conversion progressively degrade in discernible stages of resistance. We showed that D 3 cannot necessarily abrogate temporally segregated early or late RA-resistance defect(s). Nonetheless, D 3 can induce extensive, but not complete, functional monocytic differentiation in the RA-resistant cells compared to WT HL-60. Therefore although the segregation of unilineage vs. bilineage resistance is not exact, we show that an RA-resistant cell line that retains partial RA-responsiveness (R38+) is more amenable to D 3induced differentiation, while a sequentially emergent cell line more resistant to RA (R382) is less responsive to D 3 . We found that the emergent R382 RA-resistant HL-60 cell line was more dissimilar from WT and R38+ than WT and R38+ where from each other. An ensemble of signaling molecules that are coregulated in WT HL-60 become progressively more uncoupled as resistance becomes more pronounced, a trend involving increasing loss of response to RA and then D 3 . There was a putative early Fgr expression dysfunction and a late Vav1-dependent dysfunction correlated with progressive resistance, as well as dysfunctional c-Raf expression. HL-60 are negative for the t(15;17) mutation, making RA-induced mechanisms in these cells potentially applicable to other cancers. Overall RA resistance may thus result from dysfunction of multiple pathways, rather than single genetic defects.

Materials and Methods
Cell culture and treatments HL-60 human myeloblastic leukemia cells, derived from the original patient isolates, were a generous gift of Dr. Robert Gallagher, and were maintained in this laboratory and published previously ( [8][9][10][11][12][13][14][15][16][17] and others). HL-60 wild-type (WT), and the two RA-resistant HL-60 (R38+ and R382) cells subsequently isolated in our laboratory [19] were grown in RPMI 1640 supplemented with 5% fetal bovine serum (both: Invitrogen, Carlsbad, CA) and 1x antibiotic/antimycotic (Sigma, St. Louis, MO) in a 5% CO 2 humidified atmosphere at 37uC. Cells were cultured in constant exponential growth as previously described [57]. Viability was monitored by 0.2% trypan blue (Invitrogen, Calsbad, CA) exclusion and routinely exceeded 95%. Experimental cultures were initiated at a density of 0.2610 6 cells/ml. There were seven treatment regimens studied: (1) untreated, (2) RA/RA, (3) RA/D 3 , (4) RA/2, (5) D 3 /D 3 , (6) D 3 /RA, and (7) D 3 ,/2. The first agent, RA or D 3 , was added for the first 24 h (precommitment phase) followed by wash and retreatment with either the same, different, or no inducing agent (2)  CD38, CD11b and CD14 quantification 1610 6 cells were collected from cultures and centrifuged at 700 rpm for 5 min. Cell pellets were resuspended in 200 ml 37uC PBS containing 2.5 ml of either APC-conjugated CD11b antibody, PE-conjugated CD38 antibody, or PE-conjugated CD14 antibody (all from BD Biosciences, San Jose, CA). Following 1 h incubation at 37uC, cell surface expression levels were analyzed with a BD LSRII flow cytometer (BD Biosciences, San Jose, CA). APC fluorescence (excitation at 633 nm) was collected with a 660/20 band pass filter and PE fluorescence (excitation at 488 nm) was collected with a 576/26 band pass filter. Undifferentiated control cells were used to determine the fluorescence intensity of cells negative for the respective surface antigen. The gate to determine percent increase of expression was set to exclude 95% of the control population.
Respiratory burst quantification 1610 6 cells were collected and centrifuged at 700 rpm for 5 min. Cell pellets were resuspended in 500 ml 37uC PBS containing 5 mM 5-(and-6)-chloromethyl-29,79-dichlorodihydrofluorescein diacetate acetyl ester (H 2 -DCF, Molecular Probes, Eugene, OR) and 0.2 mg/ml 12-o-tetradecanoylphorbol-13-acetate (TPA, Sigma, St. Louis, MO). Both, H 2 -DCF and TPA stock solutions were made in DMSO at concentrations of 0.2 mg/ml and 5 mM, respectively. A control group incubated in H 2 -DCF and DMSO without TPA was included. Cells were incubated for 20 min at 37uC prior to analysis by flow cytometry. Oxidized DCF was excited by a 488 nm laser and emission collected with a 530/30 nm band pass filter. The shift in fluorescence intensity in response to TPA was used to determine the percent cells with the capability to generate inducible oxidative metabolites [58]. Gates to determine percent positive cells were set to exclude 95% of control cells not stimulated with TPA.
Cell cycle quantification 1610 6 cells were collected by centrifugation at 700 rpm for 5 min and resuspended in 200 ml of cold propidium iodide (PI) hypotonic staining solution containing 50 mg/ml propidium iodine, 1 ml/ml Triton X-100, and 1 mg/ml sodium citrate. Cells were incubated at room temperature for 1 h and analyzed by flow cytometry using 488-nm excitation and collected with a 575/26 band-pass filter. Doublets were identified by a PI signal width versus area plot and excluded from the analysis [58,59].

Statistical analysis
Treatment group means were compared using the Paired-Samples T-Test. The data represent the means of three repeats 6 S.E.M. A p-value of ,0.05 was considered significant (using GraphPad software and Excel). For agglomerative hierarchical clustering of signaling data, average quantified Western blot data was clustered using Cluster 3.0 and visualized with TreeView. To assess the correlation of the expression patterns of both the phenotypic markers and signaling molecules, a hierarchical cluster analysis [60] was conducted by single linkage method (nearest neighbor) and marker similarity metrics based on the Pearson correlation using SYSTAT 8.0 software. Three repeats of Western blot data (using whole cell lysates) were quantified using ImageJ and average fold change from control was graphed in GraphPad. Error bars represent standard error. GAPDH loading controls were also performed on each individual blot to ensure even loading (not shown). Note that the fold change axis scale may differ for each bar graph. (A) At 24 h there is minimal to no change in the expression of RARa or VDR with RA or D 3 treatment in all three cell lines. (B) Interestingly RA tended to increase VDR expression in WT HL-60 cells at 48 h. R38+ cells tended to have slightly higher receptor expression when treated with D 3 first. However, overall we found that increasing resistance of any cell line could not be attributable to significant loss of either receptor. (TIF) Author Contributions