Xuening Wang

Activation of extracellular signal‐regulated kinases (ERKs) defines the first phase of 1,25‐dihydroxyvitamin D3‐induced differentiation of HL60 cells

Activation of ERK1 and ERK2 protein kinases has been implicated in diverse cellular processes, including the control of cell proliferation and cell differentiation (Marshall [ 1995 ] Cell 80:179). In human myeloblastoid leukemia HL60 cells rapid (ca. 15 min) but transient activation of ERK1/2 has been reported following induction of macrophage/monocyte differentiation by phorbol esters, or by very high (10−6 M) concentrations of 1,25‐dihydroxyvitamin D3 (1,25D3), while retinoic acid‐induced granulocytic differentiation was accompanied by sustained activation of ERK1/2. We report here that monocytic differentiation of HL60 cells induced by moderate (10−9 to 10−7 M) concentrations of 1,25D3 could be divided into at least two stages. In the first phase, which lasts 24–48 h, the cells continued in the normal cell cycle while expressing markers of monocytic phenotype, such as CD14. In the next phase the onset of G1 cell cycle block became apparent and expression of CD11b was prominent, indicating a more mature myeloid phenotype. The first phase was characterized by high levels of ERKs activated by phosphorylation, and these decreased as the cells entered the second phase, while the levels of p27/Kip1 increased at that time. Serum‐starved or PD98059‐treated HL60 cells had reduced growth rate and slower differentiation, but the G1 block also coincided with decreased levels of activated ERK1/2. The data suggest that the MEK/ERK pathway maintains cell proliferation during 1,25D3‐induced monocytic differentiation of HL60 cells, but that ERK1/2 activity becomes suppressed during the later stages of differentiation, and the consequent G1 block leads to “terminal” differentiation. J. Cell. Biochem. 80:471–482, 2001.

MicroRNAs181 regulate the expression of p27Kip1 in human myeloid leukemia cells induced to differentiate by 1,25-dihydroxyvitamin D3.

Human myeloid leukemia cells exposed to 1,25-dihydroxyvitamin D3 (1,25D), a major cancer chemopreventive agent, acquire features of normal monocytes and arrest in the G1 phase of the cell cycle, due to the upregulation of p27Kip1 and p21Cip1, but the mechanism is not clear. Here evidence is provided that an exposure of HL60 and U937 cells to low (1-10 nM) concentrations of 1,25D decreases the expression of miR181a and miR181b in a concentration and time-dependent manner. Since the predicted miR181 targets include the 3’-UTR of p27Kip1, we expressed pre-miR181a in these cells, and found that the elevation of cellular miR181a levels abrogates the 1,25D-induced increase in p27Kip1 at both mRNA and protein levels. In contrast, transfection of pre-miR181a resulted in a slight elevation of p21Cip1 expression. Importantly, transfection of pre-miR181a blunted the effect of 1,25D on the expression of monocytic differentiation markers, and reduced the G1 block in 1,25D-treated cells, while transfection of anti-miR181a increased 1,25D-induced differentiation. Together, these data show that miR181a participates in 1,25D-induced differentiation of HL60 and U937 cells, and suggest that a high constitutive expression of members of miR181 family may contribute to the malignant phenotype in the myeloid lineage.

Inhibition of p38MAP kinase potentiates the JNK/SAPK pathway and AP‐1 activity in monocytic but not in macrophage or granulocytic differentiation of HL60 cells

Monocytic differentiation of HL60 cells induced by 1,25‐dihydroxyvitamin D3 (1,25 D3) has been recently shown (Exp Cell Res 258, 425, 2000 ) to be enhanced by an exposure to SB203580 or to SB202190, specific inhibitors of p38MAP kinase, with concomitant up‐regulation of the c‐jun N terminal kinase (JNK) pathway. In the present study we inquired if this enhancement and the JNK up‐regulation are limited to 1,25 D3‐induced differentiation, or if they occur more generally in HL60 cell differentiation. We found that dimethylsulfoxide (DMSO)‐induced differentiation, and to a lesser extent tetradecanoylphorbol acetate (TPA)‐induced macrophage differentiation were also potentiated by the p38MAPK inhibitors, but that granulocytic differentiation in response to all‐trans retinoic acid (RA) was not. The enhancement of differentiation by p38MAPK inhibitors was accompanied by an activation of the JNK MAPK pathway, as shown by the phosphorylation levels of these kinases and by AP‐1 binding, but only in 1,25 D3‐treated cells. This shows that an up‐regulation of the JNK stress pathway during 1,25 D3‐induced monocytic differentiation occurs selectively in this lineage of differentiation and is not necessary for the expression of the differentiated phenotype. J. Cell. Biochem. 82: 68–77, 2001.

Jun N-terminal kinase pathway enhances signaling of monocytic differentiation of human leukemia cells induced by 1,25-dihydroxyvitamin D3

Recent studies revealed that the MEK/ERK module of the mitogen‐activated protein kinase (MAPK) signaling cascades is up‐regulated in the early stages of 1α,25‐dihydroxyvitamin D3 (1,25D3)‐induced monocytic differentiation of human leukemia cells HL60. In the present study, we investigated whether another MAPK module, the JNK pathway, also participates in this form of differentiation. We found that the dependence on the concentration of the inducer, the vitamin‐hormone 1,25D3, in two types of human leukemia cells, HL60 and U937, and the kinetics of monocytic differentiation in HL60 cells, parallel the degree of the activation of the JNK pathway. A blockade of JNK signaling by a stable expression of dominant negative (dn) JNK1 mutant in U937 cells resulted in reduced c‐jun phosphorylation, and the differentiation of these cells was markedly decreased. Similarly, inhibition of JNK1 and JNK2 activities by the selective inhibitor SP600125 led to both dose‐dependent reduction of c‐jun and ATF‐2 phosphorylation, and of the differentiation of HL60 cells. In addition, we found that JNK activity is essential for the AP‐1 DNA binding induced by 1,25D3 in HL60 and U937 cells. The results indicate that in cultured human leukemia cells, the JNK pathway participates in the induction of monocytic differentiation by 1,25D3, probably by activating the AP‐1 transcription factor.

The rationale for deltanoids in therapy for myeloid leukemia: Role of KSR–MAPK–C/EBP pathway

The evidence for the promising potential for derivatives of Vitamin D (deltanoids) in the treatment of myeloid leukemias is increasing, but currently is not matched by the understanding of the precise mechanisms by which these anti-neoplastic effects are achieved. Unlike solid tumors in which growth retardation by deltanoids appears to result from inhibition of cell proliferation and the promotion of cell death by apoptosis, control of myeloid leukemia proliferation by deltanoids results from the induction of differentiation of the immature myelo-monocytic cells towards functional monocytic cells. We present here the accumulating evidence that a pathway that is initiated by deltanoid activation of Vitamin D receptor (VDR) and leads to monocytic differentiation of human myeloblastic HL60 cells, includes the MEK-ERK and JNK mitogen-activated protein kinases (MAPKs), their positive and negative regulators and a downstream effector C/EBPbeta. As in other cells, the abundance of VDR protein increases shortly after an exposure of HL60 cells to 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2) D(3)). Other early events include a parallel upregulation of kinase suppressor of Ras (KSR-1) and the activation of the ERK MAPK pathway and data suggest that KSR-1 acts to amplify the signal provided by low concentrations of 1alpha,25(OH)(2) D(3). Maintenance of monocytic differentiation may be enhanced by JNK, but diminished by p38, MAPK signaling. Downstream, one of the targets of these pathways is C/EBPbeta, which can directly interact with the promoter for CD14, a gene characteristically expressed in monocytes. Importantly, in freshly obtained acute myeloid leukemia (AML)-M2 cells exposed to PRI-2191, a novel deltanoid with a modified side chain, upregulation of C/EBPbeta paralleled the induction of monocytic differentiation. These data provide a basis for the hypothesis that deltanoid-induced upregulation of C/EBPbeta bypasses the block to granulocytic differentiation in myeloid leukemia cells by redirecting the cells to monocytic differentiation.

Downregulation of telomerase activity in HL60 cells by differentiating agents is accompanied by increased expression of telomerase-associated protein

Telomerase activity provides a mechanism for the unlimited division potential of neoplastic cells. Induced differentiation of these cells was found to be associated with repression of telomerase activity irrespective of the inducing agent. We have employed a series of sublines of human promyelocytic leukemia line HL60 with differing degrees of resistance to differentiation to determine how tightly the expression of the differentiated phenotype is coupled to the downregulation of telomerase activity and to the expression of the recently identified telomerase‐associated protein 1 (TP1). As expected, in the 1,25D3‐dihydroxyvitamin D3 (1,25D3)–resistant subclones (20A–100A cells), telomerase activity was not significantly downregulated by 1,25D3 and, in most cases, by all‐trans retinoic acid (atRA), to which these cells were cross‐resistant, but telomerase activity was repressed by dimethylsulfoxide (DMSO) and phorbol‐12‐myristate‐13‐acetate (TPA), to which the sublines were in general sensitive. However, there were exceptions; in some instances telomerase activity was repressed in the absence of the expression of markers of differentiation. Also, there was an inverse relationship between telomerase activity and the cellular levels of TP1 transcripts. We conclude that in HL60 cells downregulation of telomerase is loosely associated with upregulation of differentiation markers and with other cellular changes which include an upregulation of TP1. J. Cell. Biochem. 67:13–23, 1997.

Inhibition of Cot1/Tlp2 oncogene in AML cells reduces ERK5 activation and upregulates p27Kip1 concomitant with enhancement of differentiation and cell cycle arrest induced by silibinin and 1,25-dihydroxyvitamin D3

Acute myelogenous leukemia (AML) is a disease characterized by dysregulated cell proliferation associated with impaired cell differentiation, and current treatment regimens rarely save the patient. Thus, new mechanism-based approaches are needed to improve prognosis of this disease. We have investigated in preclinical studies the potential anti-leukemia use of the plant-derived polyphenol Silibinin (SIL) in combination with 1,25-dihydroxyvitamin D3 (1,25D). Although most of the leukemic blasts ex vivo responded by differentiation to treatment with this combination, the reasons for the absence of SIL-1,25D synergy in some cases were unclear. Here we report that failure of SIL to enhance the action of 1,25D is likely due to the SIL-induced increase in the activity of differentiation-antagonizing cell components, such as ERK5. This kinase is under the control of Cot1/Tlp2, and inhibition of Cot1 activity by a specific pharmacological inhibitor 4-(3-chloro-4-fluorophenylamino)-6-(pyridin-3-yl-methylamino-3-cyano-[1–7]-naphthyridine, or by Cot1 siRNA, increases the differentiation by SIL/1,25D combinations. Conversely, over-expression of a Cot1 construct increases the cellular levels of P-ERK5, and SIL/1,25D-induced differentiation and cell cycle arrest are diminished. It appears that reduction in ERK5 activity by inhibition of Cot1 allows SIL to augment the expression of 1,25D-induced differentiation promoting factors and cell cycle regulators such as p27Kip1, which leads to cell cycle arrest. This study shows that in some cell contexts SIL/1,25D can promote expression of both differentiation-promoting and differentiation-inhibiting genes, and that the latter can be neutralized by a highly specific pharmacological inhibitor, suggesting a potential for supplementing treatment of AML with this combination of agents.

Kinase suppressor of RAS (KSR) amplifies the differentiation signal provided by low concentrations 1,25-dihydroxyvitamin D3.

The activity of kinase suppressor of ras (KSR), a kinase or a molecular scaffold upstream from Raf‐1, is involved in the MEK/ERK MAP kinase cascade which can signal cell growth, survival, or differentiation, depending on the cellular context. We provide evidence here that KSR is upregulated in HL60 cells undergoing differentiation induced by low (0.3–3 nM) concentrations of 1,25‐dihydroxyvitamin D3 (1,25D3), and an antisense oligo (AS), but not a sense oligo, to KSR inhibits this differentiation. The inhibition of differentiation by AS–KSR oligo was less apparent when the concentration of 1,25D3 was increased, suggesting that at the higher concentrations of 1,25D3 KSR is not essential for the signaling of the differentiated phenotype. The reduced differentiation of HL60 cells exposed to AS–KSR was paralleled by reduced phosphorylation of Raf‐1 Ser 259, and of p90RSK, used here as read‐out for MAPK cascade activity. Conversely, ectopic expression of Flag‐tagged wild type KSR potentiated the differentiation‐inducing effects of low concentrations of 1,25D3. Additional data suggest that the kinase activity of KSR is required for these effects, as transfection of a kinase inactive KSR construct did not significantly increase the 1,25D3‐induced differentiation. Enzyme assays performed with KSR immunoprecipitated from 1,25D3‐treated cells showed kinase activity when recombinant Raf‐1 was used as the substrate, but not when the 1,25D3‐treated cells were pretreated with AS–KSR oligos. Taken together, these data suggest that KSR participates in signaling of monocytic differentiation by augmenting the strength of the signal transmitted through Raf‐1 to downstream targets. J. Cell. Physiol. 198: 333–342, 2004© 2003 Wiley‐Liss, Inc.

Raf-1 signaling is required for the later stages of 1,25-dihydroxyvitamin D3-induced differentiation of HL60 cells but is not mediated by the MEK/ERK module

We are interested in determining the signaling pathways for 1,25‐dihydroxyvitamin D3 (1,25D)‐induced differentiation of HL60 leukemic cells. One possible candidate is Raf‐1, which is known to signal cell proliferation and neoplastic transformation through MEK, ERK, and downstream targets. It can also participate in the regulation of cell survival and various forms of cell differentiation, though the precise pathways are less well delineated. Here we report that Raf‐1 has a role in monocytic differentiation of human myeloid leukemia HL60, which is not mediated by MEK and ERK, but likely by direct interaction with p90RSK. Specifically, we show that Raf‐1 and p90RSK are increasingly activated in the later stages of differentiation of HL60 cells, at the same time as activation of MEK and ERK is decreasing. Transfection of a wild‐type Raf‐1 construct enhances 1,25D‐induced differentiation, while antisense Raf‐1 or short interfering (si) Raf‐1 reduces 1,25D‐induced differentiation. In contrast, antisense oligodeoxynucleotides (ODN) and siRNAs to MEK or ERK have no detectable effect on differentiation. In late stage differentiating cells Raf‐1 and p90RSK are found as a complex, and inhibition of Raf‐1, but not MEK or ERK expression reduces the levels of phosphorylated p90 RSK. These findings support the thesis that Raf‐1 signals cell proliferation and cell differentiation through different intermediary proteins. J. Cell. Physiol. 209: 253–260, 2006.

Induction of differentiation of human leukemia cells by combinations of COX inhibitors and 1,25-dihydroxyvitamin D3 involves Raf1 but not Erk 1/2 signaling

Differentiation therapy of cancer is being explored as a potential modality for treatment of myeloid leukemia, and derivatives of vitamin D are gaining prominence as agents for this form of therapy. Cyclooxygenase (COX) inhibitors have been reported to enhance 1,25-dihydroxyvitamin D3 (1,25D)-induced monocytic differentiation of promyeloblastic HL60 cells, but the mechanisms of this effect are not fully elucidated, and whether this potentiation can occur in other types of myeloid leukemia is not known. We found that combination treatment with 1,25D and non-specific COX inhibitors acetyl salicylic acid (ASA) or indomethacin can robustly potentiate differentiation of other types of human leukemia cells, i.e. U937, THP-1, and that ASA +/- 1,25D is effective in primary AML cultures. Increased cell differentiation is paralleled by arrest of the cells in the G1 phase of the cell cycle, and by increased phosphorylation of Raf1 and p90RSK1 proteins. However, there is no evidence that this increase in phosphorylation of Raf1 is transmitted through the ERK module of the MAPK signaling cascade. Transfection of small interfering (si) RNA to Raf1 decreased differentiation of U937 cells induced by a combination of ASA or indomethacin with 1,25D. However, phosphorylation levels of ERK1/2, though not of p90RSK, were increased when P-Raf1 levels were decreased by the siRNA, suggesting that in this system the ERK module does not function in the conventional manner. Identification of the strong antiproliferative activity of ASA/1,25D combinations associated with monocytic differentiation has implications for cancer chemoprevention in individuals who have a predisposition to myeloid leukemia.