Retinoic acid-induced blr1 expression promotes ERK2 activation and cell differentiation in HL-60 cells

Retinoids are known to induce the differentiation and cell cycle arrest of human myeloid leukemia cells in vitro. Differential display was used to identify putative early regulatory genes that are differentially expressed in HL-60 human promyelocytic leukemia cells treated with retinoic acid. One of the cDNAs cloned encodes sequences identifying Burkitt's lymphoma receptor 1 (BLR1), a recently described chemokine receptor. Northern blot analysis demonstrates that blr1 mRNA expression increases within 9 h of retinoic acid treatment, well before functional differentiation or G(1)/G(0) growth arrest at 48 h or onset of morphological changes, suggesting a possible regulatory function. The expression of blr1 mRNA is transient, peaking at 72 h when cells are differentiated. blr1 mRNA also is induced by other differentiation-inducing agents, 1alpha,25-dihydroxyvitamin D(3) and DMSO. Induction of blr1 mRNA by retinoic acid is not blocked by the protein synthesis inhibitor cycloheximide. In HL-60 cells stably transfected with blr1 cDNA, ectopic expression of blr1 causes an increase in ERK2 MAPK activation and promotes retinoic acid-induced G(1)/G(0) growth arrest and cell differentiation. The early expression of blr1 mRNA during differentiation, its ability to increase ERK2 activation, and its enhancement of retinoic acid-induced differentiation suggest that blr1 expression may be involved in retinoic acid-induced HL-60 differentiation.     

Retinoic acid increases amount of phosphorylated raf; Ectopic expression of cFMS reveals that retinoic acid-induced differentiation is more strongly dependent on ERK2 signaling than induced go arrest is

Summary Retinoic acid is known to cause the myeloid differentiation and G1/0 cell cycle arrest of HL-60 cells in a process that requires mitogen-activated protein/extracellular signal regulated kinase (MEK)-dependent extracellular signal regulated kinase (ERK)2 activation. It has also been shown that ectopic expression of cFMS, a platelet-derived growth factor (PDGF)-family transmembrane tyrosine kinase receptor, enhances retinoic acid-induced differentiation and G1/0 arrest. The mechanism of how the retinoic acid and cFMS signaling pathways intersect is not known. The present data show that the ectopic expression of cFMS results in the differential loss of sensitivity of retinoic acid-induced differentiation or G1/0 arrest to inhibition of ERK2 activation. PD98059 was used to inhibit MEK and consequently ERK2. In wild-type HL-60 cells, PD98059 blocked retinoic acid-induced differentiation; but in cFMS stable transfectants, PD98059 only attenuated the induced differentiation, with the resulting response resembling that of retinoic acid-treated wild-type HL-60. In wild-type HL-60, PD98059 greatly attenuated the retinoic acid-induced G1/0 arrest allied with retinoblastoma (RB) hypophosphorylation; but in cFMS stable transfectants, PD98059 had no inhibitory effect on RB hypophosphorylation and G1/0 arrest. This differential sensitivity to PD98059 and uncoupling of retinoic acid-induced differentiation and G1/0 arrest in cFMS transfectants is associated with changes in mitogen-activated protein kinase signaling molecules. The cFMS transfectants had more activated ERK2 than did the wild-type cells, which surprisingly was not attributable to enhanced mitogen-activated protein-kinase-kinase-kinase (RAF) phosphorylation. Retinoic acid increased the amount of activated ERK2 and phosphorylated RAF in both cell lines. But PD98059 eliminated detectable ERK2 activation, as well as inhibited RAF phosphorylation, in untreated and retinoic acid-treated wild-type HL-60 and cFMS transfectants, consistent with MEK or ERK feedback-regulation of RAF, in all four cases. Since PD98059 blocks the cFMS-conferred enhancement of the retinoic acid-induced differentiation, but not growth arrest, the data indicate that cFMS-enhanced differentiation acts primarily through MEK and ERK2, but cFMS-enhanced G1/0 arrest allied with RB hypophosphorylation depends on another cFMS signal route, which by itself can effect G1/0 arrest without activated ERK2. Ectopic expression of cFMS and differential sensitivity to ERK2 inhibition thus reveal that retinoic acid-induced HL-60 cell differentiation and G1/0 arrest are differentially dependent on ERK2 and can be uncoupled. A significant unanticipated finding was that retinoic acid caused a MEK-dependent increase in the amount of phosphorylated RAF. This increase may help sustain prolonged ERK2 activation.     

Transformation-Defective Polyoma Middle T Antigen Mutants Defective in PLCγ, PI-3, or src Kinase Activation Enhance ERK2 Activation and Promote Retinoic Acid-Induced, Cell Differentiation Like Wild-Type Middle T

In HL-60 human myeloblastic leukemia cells, retinoic acid is known to cause cFMS, RAF, MEK, and ERK2 dependent myeloid cell differentiation and G0 arrest associated with RB tumor suppressor protein hypophosphorylation, implicating receptor tyrosine kinase signal transduction in propelling these retinoic acid-induced cellular effects. Furthermore, ectopic expression of polyoma middle T antigen, which activates similar early signal transduction molecules as PDGF class receptors such as cFMS, accelerates these retinoic acid-induced effects. To determine if this depends on middle T's ability to activate PLCγ, PI-3 kinase, and src-like kinases, stable transfectants of HL-60 cells expressing either the polyoma middle T dl23 mutant, which is defective for PLCγ and PI-3 kinase activation, or the Δ205 mutant, which in addition has greatly attenuated src-like kinase activation ability, were created and compared to wild-type middle T-transfected HL-60. The transgenes were under control of the retinoic acid (or 1,25-dihydroxy vitamin D3) inducible Moloney murine leukemia virus LTRs. Expression of the dl23 or Δ205 mutant accelerated retinoic acid-induced cell differentiation. The effects of the mutants were comparable to those of the wild-type middle T. Likewise, retinoic acid-induced G0 arrest of mutant transfected cells and wild-type middle T transfected cells was similar. The same was true for 1,25-dihydroxy vitamin D3-induced monocytic differentiation as for retinoic acid-induced myeloid differentiation. The mutants did not cause the same slight shortening of the cell cycle as wild-type middle T. Both the mutants and the wild-type middle T caused a similar increase in the cellular basal level of activated ERK2 MAPK. Since retinoic acid increases ERK2 activation, which is necessary for differentiation, the data suggest that mutant and wild-type middle T enhanced the retinoic acid effects by increasing basal levels of ERK2 activation. Consistent with this, the polyoma-induced foreshortening of the time for differentiation coincided with the time for retinoic acid to significantly increase ERK2 activation. As in wild-type HL-60, retinoic acid induced the early down-regulation of RXRα in mutant transfectants similar to wild-type middle T transfectants, consistent with no loss or gain of relevant functions due to the mutations. In contrast, vitamin D3 did not down-regulate RXRα in HL-60 or transfectants. Polyoma middle T and these transformation-defective mutants thus enhanced ERK2 activation to have an early effect in promoting retinoic acid-induced differentiation without a strong dependence on activating PLCγ, PI-3 kinase, or src-like kinase.     

Transformation-defective polyoma middle T antigen mutants defective in PLCgamma, PI-3, or src kinase activation enhance ERK2 activation and promote retinoic acid-induced, cell differentiation like wild-type middle T

In HL-60 human myeloblastic leukemia cells, retinoic acid is known to cause cFMS, RAF, MEK, and ERK2 dependent myeloid cell differentiation and G0 arrest associated with RB tumor suppressor protein hypophosphorylation, implicating receptor tyrosine kinase signal transduction in propelling these retinoic acid-induced cellular effects. Furthermore, ectopic expression of polyoma middle T antigen, which activates similar early signal transduction molecules as PDGF class receptors such as cFMS, accelerates these retinoic acid-induced effects. To determine if this depends on middle T's ability to activate PLCgamma, PI-3 kinase, and src-like kinases, stable transfectants of HL-60 cells expressing either the polyoma middle T dl23 mutant, which is defective for PLCgamma and PI-3 kinase activation, or the Delta205 mutant, which in addition has greatly attenuated src-like kinase activation ability, were created and compared to wild-type middle T-transfected HL-60. The transgenes were under control of the retinoic acid (or 1, 25-dihydroxy vitamin D3) inducible Moloney murine leukemia virus LTRs. Expression of the dl23 or Delta205 mutant accelerated retinoic acid-induced cell differentiation. The effects of the mutants were comparable to those of the wild-type middle T. Likewise, retinoic acid-induced G0 arrest of mutant transfected cells and wild-type middle T transfected cells was similar. The same was true for 1, 25-dihydroxy vitamin D3-induced monocytic differentiation as for retinoic acid-induced myeloid differentiation. The mutants did not cause the same slight shortening of the cell cycle as wild-type middle T. Both the mutants and the wild-type middle T caused a similar increase in the cellular basal level of activated ERK2 MAPK. Since retinoic acid increases ERK2 activation, which is necessary for differentiation, the data suggest that mutant and wild-type middle T enhanced the retinoic acid effects by increasing basal levels of ERK2 activation. Consistent with this, the polyoma-induced foreshortening of the time for differentiation coincided with the time for retinoic acid to significantly increase ERK2 activation. As in wild-type HL-60, retinoic acid induced the early down-regulation of RXRalpha in mutant transfectants similar to wild-type middle T transfectants, consistent with no loss or gain of relevant functions due to the mutations. In contrast, vitamin D3 did not down-regulate RXRalpha in HL-60 or transfectants. Polyoma middle T and these transformation-defective mutants thus enhanced ERK2 activation to have an early effect in promoting retinoic acid-induced differentiation without a strong dependence on activating PLCgamma, PI-3 kinase, or src-like kinase.     

Retinoic acid selectively activates the ERK2 but not JNK/SAPK or P38 map kinases when inducing myeloid differentiation

Summary Among the three major mitogen-activated protein kinase (MAPK) cascades—the extracellular signal regulated kinase (ERK) pathway, the c-JUN N-terminal/stress-activated protein kinase (JNK/SAPK) pathway, and the reactivating kinase (p38) pathway—retinoic acid selectively utilizes ERK but not JNK/SAPK or p38 when inducing myeloid differentiation of HL-60 human myeloblastic leukemia cells. Retinoic acid is known to active ERK2. The present data show that the activation is selective for this MAPK pathway. JNK/SAPK or p38 are not activated by retinoic acid. Presumably because it activates relevant signaling pathways including MAPK, the polyoma middle T antigen, as well as certain transformation defective mutants thereof, is known to promote retinoic acid-induced differentiation, although the mechanism of action is not well understood. The present results show that consistent with the selective involvement of ERK2, ectopic expression of either the polyoma middle T antigen or its dl23 mutant, which is defective for PLCγ and PI-3 kinase activation, or the Δ205 mutant, which in addition is also weakened for activation of src-like kinases, caused no enhanced JNK/SAPK or p38 kinase activity that promoted the effects of retinoic acid. However, all three of these polyoma antigens are known to enhance ERK2 activation and promote differentiation induced by retinoic acid. Polyoma-activated MAPK signaling relevant to retinoic acid-induced differentiation is thus restricted to ERK2 and does not involve JNK/SAPK or p38. Taken together, the data indicate that among the three parallel MAPK pathways, retinoic acid-induced HL-60 myeloid differentiation selectively depends on activating ERK but not the other two MAPK pathways, JNK/SAPK or p38, with no apparent cross talk between pathways. Furthermore, the striking ability of polyoma middle T antigens to promote retinoic acid-induced differentiation appears to utilize ERK, but not JNK/SPK or p38 signaling.     

Ectopic expression of CXCR5/BLR1 accelerates retinoic acid- and vitamin D(3)-induced monocytic differentiation of U937 cells

The product of the blr1 gene is a CXC chemokine receptor (CXCR5) that regulates B lymphocyte migration and has been implicated in myelomonocytic differentiation. The U937 human leukemia cell line was used to study the role of blr1 in retinoic acid-regulated monocytic leukemia cell growth and differentiation. blr1 mRNA expression was induced within 12 hr by retinoic acid in U937 cells. To determine whether the early induction of blr1 might regulate inducible monocytic cell differentiation, U937 cells were stably transfected with blr1 (U937/blr1 cells). Ectopic expression of blr1 caused no significant cell cycle or differentiation changes, but caused the U937/blr1 cells to differentiate faster when treated with either retinoic acid or 1alpha,25-dihydroxyvitamin D(3). Treated with retinoic acid, U937/blr1 cells showed a greater increase in the percentage of CD11b expressing cells than vector control cells. Retinoic acid also induced a higher percentage of functionally differentiated blr1 transfectants as assessed by nitroblue tetrazolium reduction. U937/blr1 cells underwent moderate growth inhibition on treatment with retinoic acid. Similar results occurred with 1alpha,25-dihydroxyvitamin D(3). Because blr1 was induced early during cell differentiation and because its overexpression accelerated monocytic differentiation, it may be important for signals controlling cell differentiation.     

Relationships between the cell cycle and the expression of c-myc and transferrin receptor genes during induced myeloid differentiation

We examined the relationship of cellular oncogene c-myc and transferrin receptor (TfR) gene expression to cell proliferation and cell cycle progression during myeloid differentiation in the HL-60 myeloid leukemia cell line. In order to determine levels of mRNA for these genes in HL-60 cells induced to differentiate along the myeloid pathway, RNA was isolated from HL-60 cells incubated with retinoic acid for 24 h and Northern blots were probed with labeled cDNAs for c-myc and TfR. c-myc mRNA decreased within 3 h of retinoic acid addition, and TfR mRNA decreased after 9 h; both mRNAs continued to decrease over 24 h. RNA was also isolated from HL-60 cells separated by centrifugal elutriation into cell cycle phases. TfR and c-myc cDNA probes hybridized equally to RNA from uninduced cells in all phases of the cell cycle. However, after 24 h incubation with the differentiation inducer retinoic acid, TfR mRNA was expressed substantially less in the G1 stage, whereas c-myc mRNA was still expressed equally in all cell cycle phases. These data indicate that, although TfR and c-myc expression are both associated with cell proliferation in the HL-60 line, TfR is down-regulated specifically in G1 upon induction of terminal differentiation whereas c-myc expression is disassociated from cell cycle control in these cells.     

Precommitment states induced during HL-60 myeloid differentiation: possible similarities of retinoic acid- and DMSO-induced early events

The possible relationship of the pathways by which two inducers, retinoic acid and DMSO, cause myeloid differentiation of HL-60 promyelocytic leukemia cells was studied. HL-60 cells were first exposed to retinoic acid and then washed free of it. As reported previously, this brief exposure results in no subsequent G0 growth arrest or phenotypic differentiation. When these cells were subsequently exposed to DMSO, onset of G1/0 growth arrest but not phenotypic differentiation occurred within 24 h. Since in these cells retinoic acid or DMSO normally requires 48 h of continuous exposure for onset of significant G0 growth arrest and phenotypic differentiation, it appears that retinoic acid and DMSO induce similar early cellular events needed for subsequent G0 growth arrest but not for phenotypic differentiation. While onset of growth arrest and differentiation occur together when the cells are exposed for 48 h to retinoic acid, the present results indicate that their occurrence can be uncoupled by this split dosage to inducers. The results are discussed in terms of a previously hypothesized model of cellular response to the inducers.     

Induction by retinoic acid of NAD+-glycohydrolase activity of myelomonocytic cell lines HL-60, THP-1 and U-937, and fresh human acute promyelocytic leukemia cells in primary culture

HL-60, a human promyelocytic leukemia cell line, is induced to differentiate by retinoic acid to mature granulocytes. We have now found that after the addition of 1 μM retinoic acid to HL-60 cultures an increase in NAD⁺-glycohydrolase (NADase) activity is detected by 6 hr and after a 33-fold increase in activity reaches a plateau by 24 hr. Cycloheximide inhibits completely the retinoic acid-induced increase in NADase activity indicating that enzyme induction requires protein synthesis $\text{de}\text{,}\text{novo}$. An increase of NADase activity was found not only in HL-60 cells but also in two human monoblast cell lines (U-937 and THP-1) and fresh cells in primary culture from two patients with acute promyelocytic leukemia. An increase in synthesis $\text{de}\text{,}\text{novo}$ of NADase does not appear to be obligatory for differentiation of HL-60 because there was no increase of NADase activity in HL-60 cells induced to differentiate with either dimethylsulfoxide, hypoxanthine, butyrate, or 1, 25-dihydroxycholecalciferol and there were marked increases in NADase activity at concentrations of retinoic acid having little or no effect on differentiation.     

Retinoic acid-induced CD38 expression in HL-60 myeloblastic leukemia cells regulates cell differentiation or viability depending on expression levels

Retinoic acid-induced expression of the CD38 ectoenzyme receptor in HL-60 human myeloblastic leukemia cells is regulated by RARalpha and RXR, and enhanced or prevented cell differentiation depending on the level of expression per cell. RARalpha activation caused CD38 expression, as did RXR activation but not as effectively. Inhibition of MAPK signaling through MEK inhibition diminished the induced expression by both RARs and RXRs. Expression of CD38 enhanced retinoic acid-induced myeloid differentiation and G0 cell cycle arrest, but at higher expression levels, induced differentiation was blocked and retinoic acid induced a loss of cell viability instead. In the case of 1,25-dihydroxyvitamin D3, induced monocytic differentiation was also enhanced by CD38 and not enhanced by higher expression levels, but without induced loss of viability. Expression levels of CD38 thus regulated the cellular response to retinoic acid, either propelling cell differentiation or loss of viability. The cellular effects of CD38 thus depend on its expression level.     

Sodium Valproate Facilitates the Propagation of Granulocytic Ehrlichiae (Anaplasma phagocytophilum) in HL-60 Cells

As already shown, some inducers of the differentiation of promyelocytic cells along the granulocytic pathway, such as dimethylsulphoxide (DMSO) or all-trans retinoic acid, can enhance propagation of granulocytic ehrlichiae in HL-60 cell cultures. This study was conducted to prove whether sodium valproate, a salt of di-n-propylacetic acid (VPA) known to trigger cellular differentiation in several solid and hematopoietic malignancies is similarly efficient in ehrlichial cultures. Two cell lines derived from HL-60, that is, low-passage undifferentiated HL-60 (HL-60F) and high-passage HL-60 spontaneously differentiated towards monocytic phenotype (HL-60J) were grown in RPMI 1640 medium supplemented with 10% FBS. The respective HL-60F and HL-60J IC₅₀-values for NaVPA were estimated to be 0.8 and 2.2 mM under these culture conditions; to stimulate the differentiation, the respective doses of 0.3 and 1.2 mM were then applied. When the NaVPA-treated cells of both lines were challenged with an ehrlichial laboratory strain (HGE), maintained in splenectomized NMRI mice, the respective 1–2 and ≤0.1% primary infection rates in HL-60F and HL-60J cultures were observed 3 days post-inoculation. In comparison, only rare (≤0.1%) infected HL-60F and no infected HL-60J cells were recorded under the same experimental conditions in untreated control cultures. HGE continuously propagated in NaVPA-supplemented HL-60F cultures remained infectious to mice at least up to the 95th passage (12 months). NaVPA can thus facilitated continuous propagation of granulocytic ehrlichiae in cell cultures without a substantial loss of infectiveness. This revised version was published online in July 2006 with corrections to the Cover Date.     

All-trans-retinoic acid-mediated modulation of p53 during neural differentiation in murine embryonic stem cells

All-trans-retinoic acid (RA) plays an important physiological role in embryonic development and is teratogenic in large doses in almost all species. p53, a tumor suppressor gene encodes phosphoproteins, which regulate cellular proliferation, differentiation, and apoptosis. Temporal modulation of p53 by retinoic acid was investigated in murine embryonic stem cells during differentiation and apoptosis. Undifferentiated embryonic stem cells express a high level of p53 mRNA and protein followed by a decrease in p53 levels as differentiation proceeds. The addition of retinoic acid during 8–10 days of differentiation increased the levels of p53 mRNA and protein, accompanied by accelerated neural differentiation and apoptosis. Marked increase in apoptosis was observed at 10–20 h after retinoic acid treatment when compared with untreated controls. Retinoic acid-induced morphological differentiation resulted in predominantly neural-type cells. Maximum increase in p53 mRNA in retinoic acid-treated cells occurred on day 17, whereas maximum protein synthesis occurred on days 14–17, which coincided with increased neural differentiation and proliferation. Increased p53 levels did not induce p21 transactivation, interestingly a decrease in p21 was observed on day 17 on exposure to retinoic acid. The level of p53 declined by day 21 of differentiation. The results demonstrated that retinoic acid-mediated apoptosis preceded the changes in p53 expression, suggesting that p53 induction does not initiate retinoic acid-induced apoptosis during development. However, retinoic acid accelerated neural differentiation and increased the expression of p53 in proliferating neural cells, corroborated by decreased p21 levels, indicating the importance of cell type and stage specificity of p53 function. This revised version was published online in July 2006 with corrections to the Cover Date.     

Coordinate expression of OPN and associated receptors during monocyte/macrophage differentiation of HL-60 cells

Promyelocytic leukemia HL-60 cells promoted by PMA to differentiate along the monocyte pathway adhere to tissue culture plates. To explore the regulation of adhesion molecules in cells promoted to differentiate, the expression and secretion of osteopontin (OPN) and expression of associated cell surface receptors, CD44 and integrin subunits α_v, β₃, β₁, were examined. Results were as follows: (1) PMA induced OPN mRNA and OPN secretion into media; (2) untreated cells expressed β₁ and CD44 mRNA, and PMA induced α_v and β₃ mRNA and increased β₁ and CD44 mRNA expression; (3) PMA increased levels of α_v, β₃, β₁ and CD44 protein on the cell surface; and (4) retinoic acid, which promotes granulocytic differentiation of HL-60 cells, did not affect OPN, α_v, β₃, β₁, or CD44 mRNA or protein expression. These data suggest that induction of OPN and associated receptors may play a role during monocytic differentiation of HL-60 cells. J. Cell. Physiol. 175:229–237, 1998. © 1998 Wiley-Liss, Inc.     

EGFR inhibitors exacerbate differentiation and cell cycle arrest induced by retinoic acid and vitamin D3 in acute myeloid leukemia cells

By means of an unbiased, automated fluorescence microscopy-based screen, we identified the epidermal growth factor receptor (EGFR) inhibitors erlotinib and gefitinib as potent enhancers of the differentiation of HL-60 acute myeloid leukemia (AML) cells exposed to suboptimal concentrations of vitamin A (all-trans retinoic acid, ATRA) or vitamin D (1α,25-hydroxycholecalciferol, VD). Erlotinib and gefitinib alone did not promote differentiation, yet stimulated the acquisition of morphological and biochemical maturation markers (including the expression of CD11b and CD14 as well as increased NADPH oxidase activity) when combined with either ATRA or VD. Moreover, the combination of erlotinib and ATRA or VD synergistically induced all the processes that are normally linked to terminal hematopoietic differentiation, namely, a delayed proliferation arrest in the G₀/G₁ phase of the cell cycle, cellular senescence, and apoptosis. Erlotinib potently inhibited the (auto)phosphorylation of mitogen-activated protein kinase 14 (MAPK14, best known as p38^MAPK) and SRC family kinases (SFKs). If combined with the administration of ATRA or VD, the inhibition of p38^MAPK or SFKs with specific pharmacological agents mimicked the pro-differentiation activity of erlotinib. These data were obtained with 2 distinct AML cell lines (HL-60 and MOLM-13 cells) and could be confirmed on primary leukemic blasts isolated from the circulation of AML patients. Altogether, these findings point to a new regimen for the treatment of AML, in which naturally occurring pro-differentiation agents (ATRA or VD) may be combined with EGFR inhibitors.     

Lipids of human promyelocytic leukemia cell HL-60: increasing levels of ether-linked phospholipids during retinoic acid-induced differentiation

Cells of the human promyelocytic leukemia cell line HL-60 as well as HL-60 granulocytes induced in vitro by retinoic acid were examined for lipid composition. One of our original aims was to clarify how human granulocyte (differentiated HL-60 cells) synthesized enough precursors of lipid mediators, such as prostaglandins and/or platelet activating factor. Comparison studies yielded the following results. 1) After granulocyte differentiation, total phospholipid of HL-60 cells decreased to about 70% of that of untreated cells, while the content of triglyceride increased to about 200% of the original level. 2) The subclass composition of ethanolamine-containing glycerophospholipid was greatly altered during differentiation; 1-alkenyl-2-acyl glycerophosphoethanolamine (GPE) increased to 166% of that in the untreated cells, while 1,2-diacyl GPE decreased to 46% of the original value. The resultant profile became very similar to that of human peripheral polymorphonuclear leukocytes. 3) During differentiation, the amount of arachidonic acid stored in both phospholipid and triglyceride of retinoic acid-treated HL-60 cells significantly increased. Its distribution was also modified; arachidonic acid in 1,2-diacyl GPE decreased to 63%, while those of 1-alkenyl-2-acyl GPE, choline-containing glycerophospholipids, and phosphatidylinositol increased to 169, 154, and 153%, respectively. These results suggested that the regulatory mechanism of lipid turnover in HL-60 cells was modified during retinoic acid-induced granulocyte differentiation. The alterations were not enough to explain fully the capability of differentiated HL-60 cells to produce lipid mediators upon stimulation, but they were probably one of the factors that regulate these reactions.     

Retinoic acid-induced expression of tissue transglutaminase in human promyelocytic leukemia (HL-60) cells

Addition of retinoic acid to human promyelocytic leukemia cells results in a dramatic increase in cellular transglutaminase activity. This increase is due to the induction of a specific intracellular transglutaminase, tissue transglutaminase. Retinoic acid-induced expression of tissue transglutaminase is potentiated by analogues of cyclic AMP. The induction of the enzyme can be detected within 6 h of the addition of the retinoid to the cell and results in increases of the enzyme of at least 50-fold. The induction of HL-60 transglutaminase is a specific response of the cells to retinoic acid and is not seen with other agents that induce HL-60 differentiation. We believe that the induction of tissue transglutaminase is a useful index of the early events in retinoid-regulated gene expression in both normal and transformed cells.     

Kindlin-2 is required for myocyte elongation and is essential for myogenesis

Background

Retrodifferentiation and regained proliferative capacity of growth-arrested human leukemic cells after monocyte-like differentiation requires proteolytic activities together with distinct regulatory factors. The AAA ATPase valosin-containing protein (VCP/p97) contributes to protein degradation and cell cycle regulation, respectively, and it was of interest to study a possible role of VCP/p97 during this myelomonocytic differentiation and retrodifferentiation.

Results

Separation of autonomously proliferating human U937 myeloid leukemia cells by centrifugal elutriation demonstrated unaltered VCP/p97 expression levels throughout distinct phases of the cell cycle. However, phorbol ester-induced G₀/G₁ cell cycle arrest in differentiating human U937 leukemia cells was associated with a significantly increased protein and mRNA amount of this AAA ATPase. These elevated VCP/p97 levels progressively decreased again when growth-arrested U937 cells entered a retrodifferentiation program and returned to the tumorigenic phenotype. Whereas VCP/p97 was observed predominantly in the cytosol of U937 tumor and retrodifferentiated cells, a significant nuclear accumulation appeared during differentiation and G₀/G₁ growth arrest. Analysis of subcellular compartments by immunoprecipitations and 2D Western blots substantiated these findings and revealed furthermore a tyrosine-specific phosphorylation of VCP/p97 in the cytosolic but not in the nuclear fractions. These altered tyrosine phosphorylation levels, according to distinct subcellular distributions, indicated a possible functional involvement of VCP/p97 in the leukemic differentiation process. Indeed, a down-modulation of VCP/p97 protein by siRNA revealed a reduced expression of differentiation-associated genes in subsequent DNA microarray analysis. Moreover, DNA-binding and proliferation-associated genes, which are down-regulated during differentiation of the leukemic cells, demonstrated elevated levels in the VCP/p97 siRNA transfectants.

Conclusion

The findings demonstrated that monocytic differentiation and G₀/G₁ growth arrest in human U937 leukemia cells was accompanied by an increase in VCP/p97 expression and a distinct subcellular distribution to be reverted during retrodifferentiation. Together with a down-modulation of VCP/p97 by siRNA, these results suggested an association of this AAA ATPase in the differentiation/retrodifferentiation program.