Affiliation of authors: Department of Pathology and Laboratory Medicine, UMDNew Jersey Medical School, Newark.
Correspondence to: George P. Studzinski, M.D., Ph.D., Department of Pathology and Laboratory Medicine, UMDNew Jersey Medical School, 185 S. Orange Ave., C543, Newark, NJ 07103 (e-mail: studzins{at}umdnj.edu).
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several compounds other than ATRA can induce differentiation of human leukemia cells in vitro, but the translation of these findings to the clinic has so far not been successful. For instance, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces monocytemacrophage differentiation of HL60 human leukemia cells (5) as well as megakaryocytic differentiation of K562 leukemia cells (6,7); however, since this phorbol ester promotes carcinogenesis [e.g., (8,9)], clinical use of TPA cannot be contemplated. Similarly, dimethyl sulfoxide (DMSO) has diverse differentiation-inducing effects on leukemia cells, including morphologic granulocytic differentiation of HL60 cells (10), but it is a well-known chemical solvent unsuitable for internal human use. More promising is the potential use of the natural body metabolite, the vitaminhormone 1,25-dihydroxyvitamin D3 (1
,25[OH]2D3), and its chemically modified analogues, which induce monocyte-like differentiation of several human myeloid and myelomonocytic leukemia cell lines but which elicit no general cytotoxicity (11,12). However, even in this case, their clinical use is limited by systemic hypercalcemia resulting from the increased intestinal absorption of calcium and the calcium-mobilizing properties of 1
,25(OH)2D3.
One approach to overcoming the problem of vitamin D-induced hypercalcemia, as well as the possible immunosuppressive effects of its analogues, is to combine relatively low doses of 1,25(OH)2D3 with the administration of another agent that augments the differentiation-inducing action of 1
,25(OH)2D3 but that does not enhance the levels of circulating calcium and is not immunosuppressive. An example of such an approach is the combination of ketoconazole with 1-desoxy analogues of vitamin D3, which strongly potentiated the differentiation of HL60 cells in vitro but produced only minor changes in intracellular calcium homeostasis (13). While proof of principle was demonstrated, ketoconazole has substantial toxicity on its own and, so far, has not been introduced as a component of therapy for leukemia.
More recently, some plant-derived dietary antioxidants have been found not only to reduce the risk of cardiovascular disease and cancer in general (14) but also to enhance the differentiation of HL60 cells. Lycopene, a carotenoid present in fruits and vegetables but primarily present in tomatoes, can induce differentiation and can reduce the rate of growth of HL60 cells; however, more remarkably, lycopene cooperates with 1,25(OH)2D3 to produce these effects (15). Also, carnosic acid, an antioxidant polyphenol derived from the plant rosemary (Rosmarinus officinalis), augments the inhibition of growth and the induction of differentiation of HL60 and U937 leukemia cells by 1
,25(OH)2D3 and ATRA (16). Since carnosic acid is a major component of rosemary extracts that are used widely as additives for the preservation of certain foods [e.g., (17)] and, therefore, appears to be safe for human administration, we investigated the nature and mechanisms of induction of differentiation of HL60 cells by using a combination of carnosic acid and low concentrations of several inducers of monocytic or granulocytic differentiation.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Carnosic acid was purchased from Alexis Biochemicals (Läufenfingen, Switzerland). 1,25(OH)2D3 was a gift from Dr. Milan Uskokovic (Hoffmann-La Roche Inc., Nutley, NJ). ATRA, TPA, and DMSO were purchased from Sigma Chemical Co. (St. Louis, MO). Stock solutions of carnosic acid (10 mM), 1
,25[OH]2D3 (0.25 mM), ATRA (1 mM), and TPA (2 mM) were prepared in absolute ethanol. The antibodies against vitamin D receptor (VDR) (i.e., C-20), retinoid X receptor (RXR)-
(i.e., D-20), cyclin D1 (i.e., R-124), early growth response gene (EGR)-1 (i.e., 558), Cdk5 (i.e., DC17), and p35Nck5a (i.e., C-19) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-cyclin E (Ab-1) and anti-p27Kip1 antibodies were obtained from Calbiochem-Novabiochem Corp. (La Jolla, CA) and BD BiosciencesPharMingen (San Diego, CA), respectively. Phycoerythrin-conjugated anti-CD14 (MY4-RD-1) and fluorescein isothiocyanate-conjugated anti-CD11b (MO1-FITC) antibodies were obtained from Coulter Corp. (Miami, FL). Anti-calreticulin antibody (PA3-900) was purchased from Affinity BioReagents Inc. (Golden, CO). Horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) and anti-mouse IgG were obtained from Santa Cruz Biotechnology Inc.
Cell culture and proliferation assay.
HL60-G cells (18), a subclone of human promyeloblastic leukemia HL60 cells (19), were cultured routinely at 37 °C in RPMI-1640 medium (Mediatech, Herndon, VA) supplemented with 1% glutamine and 10% heat-inactivated, iron-enriched bovine calf serum (HyClone Laboratories Inc., Logan, UT). The cultures were passaged two or three times weekly to maintain a log-phase growth. If not indicated otherwise, the cells were seeded at 1.0 x 105 cells/mL in 25-cm2 tissue culture flasks and incubated for 96 hours with carnosic acid, 1,25(OH)2D3, ATRA, TPA, DMSO, or combinations of carnosic acid with the above differentiation inducers. To demonstrate the enhancement of differentiation induced by 1
,25(OH)2D3 or ATRA, we used low (1 nM) concentrations of these inducers, whereas we used 100 nM 1
,25(OH)2D3 or 1 µM ATRA to illustrate the maximal effects of these inducers. Cell growth was estimated by counting the cells with a Coulter counter after dilution in Isoton-II (Coulter Electronics, Hialeah, FL). Cell viability was determined with the use of trypan blue dye (0.25%) exclusion.
Determination of markers of differentiation.
Aliquots of 1 x 106 cells were harvested, washed twice with phosphate-buffered saline (PBS), and suspended in 10 µL 1x PBS. The cell suspensions were incubated for 45 minutes at room temperature with 0.5 µL MY4-RD-1 and 0.5 µL MO1-FITC (1 : 20 dilution of the stock antibodies) to analyze the expression of the cell surface markers CD14 and CD11b, respectively. The cells were then washed three times with ice-cold 1x PBS and resuspended in 1 mL of PBS. Two-parameter analysis was performed with the use of an FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Isotypic mouse IgG1 was used to set threshold parameters. Monocytic differentiation was also monitored by cytochemical determination of the activity of monocytic serine esterase (MSE), also known as nonspecific esterase (NSE), as described previously (13).
Cell cycle distribution.
The cells (1 x 106) were washed twice with ice-cold 1x PBS and were fixed in 75% ethanol at 20 °C overnight. They were then washed twice with 1x PBS and incubated in 1 mL of a solution containing 1 µg (100 U/mL) ribonuclease (RNase) (BMB, Indianapolis, IN) and 10 µg/mL propidium iodide (Sigma Chemical Co.) for 1 hour at 4 °C in the dark. The cell DNA content was determined with the use of an FACSCalibur flow cytometer, and the cell cycle distribution was analyzed by a ModFit LT computer program (Verity Software House; AMPL Software, Turramurra, Australia), as described previously (20).
Cell extracts and western blotting.
The cells (approximately 1 x 107) were washed twice with ice-cold 1x PBS, snap-frozen in liquid N2, and stored at 80 °C. Whole-cell extracts were made by mixing the thawed cell pellets with an extraction buffer, i.e., 20 mM TrisHCl, 0.25 M sucrose, 10 mM ethylene glycol-O,O`-bis-[2-amino-ethyl]-N,N,N`,N`-tetraacetic acid, 2 mM ethylenediaminetetraacetic acid (EDTA) (pH 7.5), 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.02% leupeptin, and 10 µg/mL aprotinin, followed by vigorous vortexing for 10 seconds. The extracts were mixed with an equal amount of 3x sodium dodecyl sulfate (SDS) sample buffer (i.e., 150 mM Tris, 30% glycerol, 3% SDS, 1.5 mg/mL bromophenol blue dye, and 100 mM dithiothreitol [DTT]). Equal amounts of extracts (40 µg of protein) were separated with the use of SDSpolyacrylamide gel electrophoresis (PAGE) (12% polyacrylamide gel containing SDS) and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech Inc., Piscataway, NJ). The membranes were blocked with 5% milk in Tris-buffered saline containing 0.1% Tween 20 for 1 hour and blotted for 1 hour with the specified primary antibody and then for 1 hour with a horseradish peroxidase-conjugated secondary antibody. The protein bands were detected with the use of a chemiluminescence assay system (Amersham Pharmacia Biotech Inc.) and visualized on Kodak X-Omat LS film. The optical density (OD) of each band was quantified with the use of an image quantitator (Molecular Dynamics, Sunnyvale, CA). The blots were stripped according to the manufacturer's protocol (Amersham Pharmacia Biotech Inc.) and successively reprobed with different antibodies and finally for calreticulin that is present constitutively.
Preparation of nuclear extracts.
The nuclear extracts used for gel mobility shift assays were prepared by the procedure described previously (21). All steps were performed at 4 °C. Briefly, 2 x 107 cells were harvested, washed twice with PBS, and resuspended in 0.2 mL of cell extraction buffer (i.e., 10 mM HEPESKOH [pH 7.9], 1.5 mM MgCl2, 10 mM KC1, 0.5 mM DTT, 0.2 mM PMSF, and 10 µg/mL aprotinin). The cells were kept on ice for 10 minutes, vortexed for 10 seconds, and centrifuged at 4 °C at 16 000g for 30 seconds. The pellet was resuspended in 2040 µL of nuclear extraction buffer (i.e., 20 mM HEPESKOH [pH 7.9], 25% glycerol, 420 mM NaCl, 1.5 mM MgC12, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, and 10 µg/mL aprotinin), placed on ice for 20 minutes, and centrifuged at 4 °C at 16 000g for 2 minutes. The supernatant was saved as the nuclear extract and stored at 80 °C.
Gel mobility shift assay.
The vitamin D response element (VDRE) sequenceoligonucleotide 5'-CTAGTGCTCGGGTAGAGGTCAAGGAGGTCACTCGAC-3'and its complement were synthesized and annealed, and the double-stranded DR3the response elements are typically composed of two hexameric half-sites organized as directed repeats (DR), and the binding specificity is provided by the spacing between each half-site; following this rule, the consensus VDRE, which has a half-site spacing of three nucleotides (DR3), is called VDRE-DR3was 5'-end phosphorylated by T4 polynucleotide kinase (Life Technologies Inc. [GIBCO BRL], Rockville, MD) in the presence of [-32P]adenosine triphosphate (Amersham Pharmacia Biotech Inc.). The mutant VDRE-DR3 oligo, 5'-AGCTTCAGaaCAAGGAGaaCAGAGAGC-3' (residues changed in the DR3 sequence in the mutant oligonucleotides are indicated in lower case type; the bold letters are the consensus sequences), was used as a nonspecific competitor in the competition assay. The gel mobility shift assay was carried out with the use of the Gel Shift Assay Kit (Stratagene, La Jolla, CA). Nuclear extract proteins (10 µg) and the labeled VDRE probe (0.8 ng) were incubated for 30 minutes at room temperature; then the samples were separated on a 4% polyacrylamide gel with a constant current of 22 mA for 3 hours. The gel was dried and exposed to Kodak X-Omat LS film. Supershift assays were performed by adding 1 µg of anti-VDR (C-20, rabbit polyclonal antibody at dilution [1 : 25] of stock antibody [1 µg/µL] was used) and 1 µg of anti-RXR-
(D-20, rabbit polyclonal antibody at dilution [1 : 25] of stock antibody [1 µg/µL] was used) antibodies to the nuclear protein sample and incubating the mixture for 20 minutes at room temperature. The reaction was then continued for 30 minutes. This assay detects supershifted bands when VDR and RXR-
form a heterodimeric complex.
Reverse transcriptionpolymerase chain reaction (RTPCR).
Total RNA was extracted from 5 x 106 cells with the use of the RNeasy Kit (Quiagen, Valencia, CA) according to the manufacturer's recommended procedure. The RNA concentration was determined spectrophotometrically. For RTPCR, a 20-µL master mix of RT was prepared as follows: 5 mM MgCl2, PCR Buffer II, 1 mM deoxyguanosine triphosphate, 1 mM deoxyadenosine triphosphate, 1 mM deoxycytidine triphosphate, 1 mM deoxythymidine triphosphate, 1 U/µL RNase inhibitor, 2.5 µM random hexamers, a 1-µg sample of RNA, and diethyl pyrocarbonate-treated distilled water. The master mix was incubated in the Perkin-Elmer GeneAmp PCR system 9600 (Roche, Branchburg, NJ) at 42 °C for 15 minutes and then at 99 °C for 5 minutes and at 5 °C for 5 minutes. After RT, 78 µL of PCR master mix containing 2 mM MgCl2, PCR Buffer II, 2.5 U/µL AmpliTaq DNA polymerase, and the following primers was added at a 0.15 µM concentration: EGR-1, upstream primer (5'-AGATGATGCTGCTGAGCAAC-3'), downstream primer (5'-AGTAAATGGGACTGCTGTCG-3'); RXR-, upstream primer (5'-TCACCTATGAACCCCGTCAG-3'), downstream primer (5'-TCGACTCCACCTCATTCTCG-3'); VDR, upstream primer (5'-AGGCCTTGAAGGACAGTCTG-3'), downstream primer (5'-GGTCTCTGAATCCTGGTATC-3'); 24-hydroxylase, upstream primer (5'-CCTTGACAAACCAACGGTT-3'), downstream primer (5'-TCCACAGGTTCATTGTCTGT-3'); human osteocalcin (hOC), upstream primer (5'-GAGCCCTCACACTCCTCGCCCTATT-3'), downstream primer (5'-GTAGAAGCGCCGATAGGCCTCCTGA-3'); and
-actin, upstream primer (5'-TGACGGGGTCACCCACACTGTGCCCAGCTA-3'), downstream primer (5'-CTAGAAGCATTTGCCGGTGGACGATGGAGGG 3'). The complementary DNAs (cDNAs) in samples were amplified in the GeneAmpPCR System 9600 as follows: 105 seconds at 95 °C as an initial step, followed by 35 cycles of 15 seconds each at 95 °C and 30 seconds each at 60 °C and finally 7 minutes at 72 °C. The RTPCR products were separated on 2% agarose gel. The intensity of the bands corresponding to VDR, RXR-
, and EGR-1 was measured with the use of the Image QuaNT Program (Molecular Dynamics, Sunnyvale, CA).
Statistical methods.
All experiments were repeated at least three times (n = 38). Measurement of cell number and viability as well as counting of cells stained for MSE (Table 1) in each experiment was carried out in triplicate. The statistical analysis was performed with the GraphPad Prism 3.0 Program (GraphPad Software, San Diego, CA). Data are reported as means ± 95% confidence intervals. Statistically significant differences among the multiple groups (see Figs. 2, 3, 6, and 7
and Table 1
) were tested with the use of the nonparametric KruskalWallis one-way analysis-of-variance test, followed by Dunn's adjustment for individual groups versus control. Two compounds (A and B) were considered to show enhancement in the particular experiment if the effect of their combination (AB) was larger than the sum of their individual effects (AB>A + B), the data being compared after subtraction of the respective control values from A, B, and AB. Statistically significant differences between AB and A + B were estimated with the use of the nonparametric Wilcoxon matched pairs test. A P value less than .05 was considered to be statistically significant.
|
|
|
|
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It has already been reported that the plant antioxidant carnosic acid enhances the effects of 1,25(OH)2D3 to induce in wild-type HL60 cells the expression of the CD14 marker of monocytic differentiation as well as the oxidative burst activity and expression of chemotactic peptide receptors (16). In this study, we used a subline of HL60 cells, HL60-G cells established in our laboratory (18). Figs. 1 and 2
illustrate that dual labeling of HL60-G cells with antibodies against CD14 and a general myeloid differentiation marker CD11b can demonstrate increased expression of the CD11b following a 96-hour treatment of these cells with either 1
,25(OH)2D3 or ATRA at low concentrations (both at 1 nM), as compared with the control cells (P<.001). The expression of CD14 was increased appreciably in the 1
,25(OH)2D3-treated cells (P<.001), but it was minimal in cells treated with ATRA, a finding consistent with the granulocytic lineage of differentiation induced by ATRA (10). TPA at 1 nM caused only a marginal but statistically not significant increase in the expression of CD11b (P = .074) and did not affect the CD14 expression (Fig. 2
).
|
Preferential Increase of Monocyte-Specific Markers by Carnosic Acid in Differentiating HL60-G Cells
The monocytic phenotype is characterized by the high expression of the CD14 marker (22), as exemplified by cells exposed to 1,25(OH)2D3 (Figs. 1 and 2
) and by the expression of the cytoplasmic enzyme MSE (23). In view of the well-established consensus that ATRA induces granulocytic lineage of differentiation in HL60 cells (10), the unexpected synergistic increase in CD14 expression by the carnosic acidATRA combination (Figs. 1 and 2
) prompted us to further analyze this system. The results showed that there was a marked increase in cells doubly positive for CD14 and CD11b (Fig. 2
, B) when carnosic acid was added along with either 1
,25(OH)2D3 (P<.001) or ATRA (P = .012) and in cells positive for MSE (P<.001 for both 1
,25(OH)2D3 and ATRA, Table 1
column A). A smaller increase in the CD14/CD11b- positive (P = .039; Fig. 2
, B) and the MSE-positive (P = .043; Table 1
column A) cells by the carnosic acidTPA combination was noted, whereas no augmentation by the polyphenol was seen in the DMSO-treated cells (Fig. 2
, B; Table 1
column A). These data indicate that carnosic acid promotes the monocytic phenotype when it is combined with some, although not all, inducers of monocytic differentiation.
Effects of Carnosic Acid on Survival, Proliferation, and Cell Cycle Traverse of Differentiating Cells
Carnosic acid at a concentration of 10 µM, either alone or in combination with other differentiating agents, had no detectable cytotoxicity, as determined by microscopic examination of the nuclear morphology of the stained cells (data not shown) or exclusion of the trypan blue dye (Table 1 column B). When added to HL60-G cells for 96 hours, carnosic acid alone only slightly affected cell proliferation (Table 1
column C) compared with untreated control cells, but it statistically significantly reduced the rate of cell growth when it was combined with 1
,25(OH)2D3 (P<.001), ATRA (P<.001), or TPA (P = .008). However, the enhancing effect of carnosic acid was statistically significant only when it was added together with 1 nM 1
,25(OH)2D3 (P = .015) or 1 nM ATRA (P = .025) (Table 1
column C). In view of the reduced rate of proliferation resulting from the exposure to carnosic acid together with differentiation agents, we examined the parameters of the cell cycle under these conditions. Small but statistically significant blocks of the G1- to S-phase transition manifested by the increase in the G0/G1 and a decrease in the S-phase cell populations were produced by both 1
,25(OH)2D3 (P = .021 and P = .017 for G0/G1 and S, respectively) and ATRA (P = .019 and P = .026 for G0/G1 and S, respectively), whereas TPA and DMSO were without statistically significant effects at the concentrations used here (Fig. 3
, A). It is interesting that, while it had no effect on the cell cycle progression when added alone, carnosic acid enhanced this increase in the G0/G1 phase (P = .004) and a decrease in the S phase (P = .027) induced by 1
,25(OH)2D3 (Fig. 3
, A). In contrast, carnosic acid did not statistically significantly alter the effects of ATRA (P = .074 and P = .136 for G0/G1 and S phases, respectively) or TPA (P = .215 and P = .236 for G0/G1 and S phases, respectively) and had no enhancing effect when it was combined with DMSO (P = .455 and P = .332 for G0/G1 and S phases, respectively) in these experiments (Fig. 3
, A).
Further examination revealed that growth inhibition induced by the combination of carnosic acid and 1,25(OH)2D3 increased considerably with time, leading to a complete growth arrest after 168 hours of treatment (P<.001), without a statistically significant decrease in cell viability (mean [95% confidence intervals] for treated = 96.0% [93.4% to 98.6%] versus control = 98.1% [96.8% to 99.4%] at 168 hours; n = 3) (Fig. 3
, B). This effect was accompanied by an increasingly higher G1/S ratio (Fig. 3
, C), signifying a marked G1- to S-phase block (P<.001 at 168 hours). (For Fig. 3
, B, = .032 and P = .018 at 96 and 120 hours, respectively; for Fig. 3
, C, P = .012 and P<.001 at 96 and 120 hours, respectively.).
Consistent with the G1 block, the cyclin-dependent kinase inhibitor p27/Kip1, which controls the G1- to S-phase transition in 1,25(OH)2D3-treated HL60 cells (24,25), showed markedly elevated levels in cells treated with the carnosic acid1
,25(OH)2D3 combination. However, in cells treated with the carnosic acidATRA, the carnosic acidTPA, or the carnosic acidDMSO combinations, the changes in the p27/Kip1 levels were less marked or were not detectable, as shown by representative immunoblots (Fig. 4
, A).
|
Carnosic Acid-Induced Increases in Levels of Functional Nuclear Receptors VDR and RXR- and of Monocyte-Specific Proteins
The nuclear receptors VDR and RXR- heterodimerize to form a transcription factor that binds to the promoters of 1
,25(OH)2D3-regulated genes, while RARs heterodimerize with RXRs to control ATRA-responsive genes (12). The heterodimers have low or absent transcription-activating activities that are increased by their ligands 1
,25(OH)2D3 or ATRA, respectively. We therefore determined the protein abundance of VDR, RXR-
, and RAR-
in HL60-G cells. We found that carnosic acid increased the levels of VDR and RXR-
and increased the levels of VDR, RXR-
, and RAR-
further in the presence of 1
,25(OH)2D3 and ATRA (Fig. 4
, B). When added with carnosic acid, TPA, but not DMSO, also markedly increased the levels of VDR and RAR-
. RXR-
levels were increased by TPA and DMSO; however, in those cases, no enhancement was detectable in the presence of carnosic acid when added alone (Fig. 4
, B).
Evaluation of the presence of active receptor complexes in nuclear extracts showed that carnosic acid treatment of HL60-G cells slightly increased the binding of active complexes to VDRE and potentiated the effects of 1,25(OH)2D3 and, to a lesser extent, the effects of ATRA when it was used (Fig. 5
). This observation suggested that the increased levels of the VDRE-binding proteins VDR and RXR-
(Fig. 4
, B) had functional significance and were examined at the level of expression of VDRE-activated genes, the 25-dihydroxyvitamin D3 24-hydroxylase (27), and the hOC gene (28). As shown in Fig. 6
, the modest stimulation of 24-hydroxylase expression by carnosic acid was not statistically significant (P = .155), but carnosic acid did elevate the expression of hOC (P<.001). These disparate results on the activation of two VDRE-activated genes may be due to the lower sensitivity of the RTPCR procedure used here to detect changes in 24-hydroxylase messenger RNA (mRNA) levels. Alternatively, it is possible that carnosic acid alters the composition or configuration of transcription factors or cofactors on the VDRE in a manner that depends on the context of surrounding sequences, resulting in gene-specific activation.
|
Transcriptional Regulation of VDR, RXR-, and EGR-1 Abundance by Carnosic Acid
Determination of specific mRNA levels in cell extracts showed that treatment with carnosic acid alone resulted in a statistically significant increase in the expression of VDR (P = .007), RXR- (P = .011), and EGR-1 (P = .009) genes (Fig. 7
), indicating that the increased expression of these genes by carnosic acid occurs at least in part at the transcriptional level. DMSO and 1
,25(OH)2D3 also statistically significantly increased the transcription of VDR (P = .021) and RXR-
(P = .032), respectively, whereas all of the differentiation-inducing agents induced increased expression of the EGR-1 gene (P = .011 to P = .018) (Fig. 7
), as was reported previously for the increased expression of EGR-1 by TPA (29). Statistical analysis revealed an enhancement by carnosic acid of the 1
,25(OH)2D3-induced increase in the expression of VDR (P = .034). In addition, carnosic acid produced additive effects with DMSO on VDR expression (P = .097) and with 1
,25(OH)2D3 on EGR-1 expression (P = .076), which were not statistically significant (Fig. 7
). However, in general, the cooperation of carnosic acid with differentiation inducers at the mRNA level was less marked than at the protein level, suggesting both transcriptional and post-transcriptional controls.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It was reported some time ago that HL60/MR1 cells, a subline of HL60 cells, derived from a transplantable HL60 tumor established in athymic nude mice, differentiate to monocytic rather than to granulocytic cells when they are cultured with ATRA in vitro (34). It is interesting that the cells from this xenotransplant-derived HL60 subline also showed other properties observed by us in carnosic acid-treated HL60 cells; the cells had markedly enhanced sensitivity to ATRA, and the differentiation pathways induced by TPA and DMSO were the same as those in the parental HL60 cells. More recently, M2 leukemic blast cells were found to undergo monocytic differentiation when they were treated with ATRA, which induces functionally active VDR in these cells (35). Thus, it appears that ATRA-induced granulocytic differentiation involves signaling circuits similar to those that are active in monocytic differentiation; however, in ATRA-treated HL60 cells, there is a lack of an additional input required for the monocytic form of differentiation. This input may be EGR-1, Cdk5 activated by p35Nck5a, or both of these, mediated by VDRRXR--induced transcription. Taken together, our data support the notion that granulocytic phenotype results from a default pathway in myeloid lineage differentiation.
The effects of carnosic acid on differentiation induced by low concentrations of TPA or DMSO were less striking. An increase in differentiation resulting from the combination of TPA with carnosic acid was noted by the determination of MSE and CD11b markers, and this increase was statistically significant but was not apparent when CD14 was used as the marker. Conversely, a combination of carnosic acid and DMSO at several concentrations was less than additive (Table 1, A; Fig. 2
; and data not shown). Consistent with this finding, there was no detectable enhancement by carnosic acid of VDR or RXR-
protein levels induced by DMSO. The enhancement by carnosic acid of a TPA-induced increase in RAR-
expression was marked, but at present its implications are unclear. In view of the low potential of TPA or DMSO for human use, this situation was not analyzed further; however, it seems that, while differentiation induced by the physiologically occurring compounds 1
,25(OH)2D3 and ATRA is markedly promoted by carnosic acid, differentiation induced by exogenous xenobiotics is at best marginal.
The data also suggest that carnosic acid enhances 1,25(OH)2D3-induced and probably also ATRA-induced mid- to late-G1-phase cell cycle arrest. The support for this hypothesis is derived from the observation that cyclin D1 levels and cyclin E levels parallel the levels of p27Kip1 (Fig. 4
, A), which have been shown previously to be regulating the G1-phase arrest in this system (24,25), and from the knowledge that the maximal expression of cyclin D1 is in mid-G1, while the maximal expression of cyclin E is at the G1/S-phase boundary (26). The increased levels of cyclin D1 in cells that are arresting in G1 phase (Fig. 3
) can be explained by a progressive accumulation of cells at the point of G1, probably just prior to or at the R point (36), where they have maximal expression of cyclin D1. It appears that carnosic acid induces such an arrest in this window of the G1 phase and does not interfere with the normal expression of cyclin D1 or cyclin E. It is also possible that some or all of the other increases in expression in proteins studied here become elevated as a result of the same mechanism, i.e., arrest in mid- to late-G1 phase, already shown to exhibit maximal sensitivity to the differentiation-inducing activity of 1
,25(OH)2D3 (37).
The fulfillment of the promise for the use of 1,25(OH)2D3 as an agent for differentiation therapy for leukemia is blocked currently by the hypercalcemia attendant on its use in vivo (11). The approaches employed to overcome this limitation have included the use of analogues of 1
,25(OH)2D3 (3840), also referred to as "deltanoids" [e.g., (41)], or combinations of these deltanoid analogues with chemicals or cytokines to enhance differentiation [e.g., (13)]. For instance, the inhibitors of p38MAP kinase SB203580 and SB202190 were found to markedly potentiate differentiation of HL60 induced by low concentrations of 1
,25(OH)2D3 (42). However, the potential of carnosic acid as a component of differentiation therapy regimen appears to be greater than that of other chemicals because of its anticipated safety and its great potency in enhancing the pro-differentiation of effects of low concentrations of the naturally occurring vitamin/hormone 1
,25(OH)2D3 and the regulator of embryonic development ATRA.
In this study, we have focused on the mechanistic aspects of the action of carnosic acid that can identify the effectors of the expression of the monocytic phenotype induced by carnosic acid in combination with natural hormones/morphogens. Whether antioxidant properties of carnosic acid have any relevance to the increased expression of 1,25(OH)2D3-responsive genes and the enhancement of the monocytic phenotype is an open question, and the upstream regulators of the carnosic acid-induced increased abundance of the nuclear receptors VDR and RXR-
, the transcription factor EGR-1, or the Cdk5/p35 complex remain to be identified in future studies. However, the identification of these downstream regulators of monocytic differentiation, with its attendant G1 block, should provide new approaches to combined chemoprevention or differentiation therapy for myeloid leukemia.
![]() |
NOTES |
---|
Supported by Public Health Service grant CA44722 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services.
We thank Dr. Yan Ji (New Jersey Medical School, Newark) for valuable help with reverse transcriptionpolymerase chain reaction experiments. We also thank Dr. J. Levy and Dr. Y. Shavoni (Ben-Gurion University) for their support.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1 Huang ME, Ye YC, Chen SR, Chai JR, Lu JX, Zhoa L, et al. Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia. Blood 1988;72:56772.[Abstract]
2 Castaigne S, Chomienne C, Daniel MT, Ballerini P, Berger R, Fenaux P, et al. All-trans retinoic acid as a differentiation therapy for acute promyelocytic leukemia. I. Clinical results. Blood 1990;76:17049.[Abstract]
3 Warrell RP Jr, Frankel SR, Miller WH Jr, Scheinberg DA, Itri LM, Hittelman WN, et al. Differentiation therapy of acute promyelocytic leukemia with tretinoin (all-trans-retinoic acid). N Engl J Med 1991;324:138593.[Abstract]
4 Zhang JW, Wang JY, Chen SJ, Chen Z. Mechanisms of all-trans retinoic acid-induced differentiation of acute promyelocytic leukemia cells. J Biosci 2000;25:27584.[Medline]
5 Rovera G, O'Brien TG, Diamond L. Induction of differentiation in human promyelocytic leukemia cells by tumor promoters. Science 1979;204:86870.[Medline]
6 Tabilio A, Pelicci PG, Vinci G, Mannoni P, Civin CI, Vainchenker W, et al. Myeloid and megakaryocytic properties of K-562 cell lines. Cancer Res 1983;43:456974.[Abstract]
7 Tetteroo PA, Massaro F, Mulder A, Schreuder-van Gelder R, von dem Borne AE. Megakaryoblastic differentiation of proerythroblastic K562 cell-line cells. Leuk Res 1984;8:197206.[Medline]
8 Armuth V, Berenblum I. Phorbol as a possible systemic promoting agent for skin carcinogenesis. Z Krebsforsch Klin Onkol Cancer Res Clin Oncol 1976;85:7982.[Medline]
9 Oesch F, Schafer A, Wieser RJ. 12-O-tetradecanoylphorbol-13-acetate releases human diploid fibroblasts from contact-dependent inhibition of growth. Carcinogenesis 1988;9:131922.[Abstract]
10 Collins SJ, Ruscetti FW, Gallagher RE, Gallo RC. Normal functional characteristics of cultured human promyelocytic leukemia cells (HL-60) after induction of differentiation by dimethylsulfoxide. J Exp Med 1979;149:96974.[Abstract]
11 Studzinski GP, McLane JA, Uskokovic MR. Signaling pathways for vitamin D-induced differentiation; implications for therapy of proliferative and neoplastic diseases. Crit Rev Eukaryot Gene Expr 1993;3:279312.[Medline]
12 Uskokovic MR, Studzinski GP, Reddy SG. In: Feldman D, Glorieux FH, Pike JW, editors. Vitamin D. San Diego (CA): Academic Press; 1997. p. 104570.
13
Wang X, Gardner JP, Kheir A, Uskokovic MR, Studzinski GP. Synergistic induction of HL60 cell differentiation by ketoconazole and 1-desoxy analogues of vitamin D3. J Natl Cancer Inst 1997;89:1199206.
14
Rao AV, Agarwal S. Role of antioxidant lycopene in cancer and heart disease. J Am Coll Nutr 2000;19:5639.
15 Amir H, Karas M, Giat J, Danilenko M, Levy R, Yermiahu T, et al. Lycopene and 1,25-dihydroxyvitamin D3 cooperate in the inhibition of cell cycle progression and induction of differentiation in HL-60 leukemic cells. Nutr Cancer 1999;33:10512.[Medline]
16 Danilenko M, Shteiner M, Amir H, Priel I, Giat J, Levy J, et al. Dietary antioxidants enhance growth inhibition and differentiation induced by retinoic acid and 1,25-dihydroxyvitamin D3 in leukemic cells [abstract]. Proc Am Assoc Cancer Res 2000;41:55.
17 Smith C, Halliwell B, Aruoma OI. Protection by albumin against the pro-oxidant actions of phenolic dietary components. Food Chem Toxicol 1992;30:4839.[Medline]
18
Studzinski GP, Reddy KB, Hill HZ, Bhandal AK. Potentiation of 1--D-arabinofuranosylcytosine cytotoxicity to HL-60 cells by 1,25-dihydroxyvitamin D3 correlates with reduced rate of maturation of DNA replication intermediates. Cancer Res 1991;51:34515.[Abstract]
19 Gallagher R, Collins S, Trujillo J, McCredie K, Ahearn M, Tsai S, et al. Characterization of the continuous, differentiating myeloid cell line (HL-60) from a patient with acute promyelocytic leukemia. Blood 1979;54:71333.[Abstract]
20 Studzinski GP, Rathod B, Rao J, Kheir A, Wajchman HL, Zhang F, et al. Transition to tetraploidy in 1,25-dihydroxyvitamin D3-resistant HL60 cells is preceded by reduced growth factor dependence and constitutive up-regulation of Sp1 and AP-1 transcription factors. Cancer Res 1996;56:551321.[Abstract]
21 Andrews NC, Faller DV. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res 1991;19:2499.[Medline]
22 Griffin JD, Ritz J, Nadler LM, Schlossman SF. Expression of myeloid differentiation antigens on normal and malignant myeloid cells. J Clin Invest 1981;68:93241.[Medline]
23 Yam LT, Li CY, Crosby WH. Cytochemical identification of monocytes and granulocytes. Am J Clin Pathol 1971;55:28390.[Medline]
24 Wang QM, Jones JB, Studzinski GP. Cyclin-dependent kinase inhibitor p27 as a mediator of the G1-S phase block induced by 1,25-dihydroxyvitamin D3 in HL60 cells. Cancer Res 1996;56:2647.[Abstract]
25 Wang QM, Chen F, Luo X, Moore DC, Flanagan M, Studzinski GP. Lowering of p27Kip1 levels by its antisense or by development of resistance to 1,25-dihydroxyvitamin D3 reverses the G1 block but not differentiation of HL60 cells. Leukemia 1998;12:125665.[Medline]
26
Sherr CJ. The Pezcoller lecture: cancer cell cycles revisited. Cancer Res 2000;60:368995.
27
Ozono K, Liao J, Kerner SA, Scott RA, Pike JW. The vitamin D-responsive element in the human osteocalcin gene. Association with a nuclear proto-oncogene enhancer. J Biol Chem 1990;265:218818.
28
Ohyama Y, Ozono K, Uchida M, Shinki T, Kato S, Suda T, et al. Identification of a vitamin D-responsive element in the 5'-flanking region of the rat 25-dihydroxyvitamin D3 24-hydroxylase gene. J Biol Chem 1994;269:1054550.
29 Nguyen HQ, Hoffman-Liebermann B, Liebermann DA. The zinc finger transcription factor Egr-1 is essential and restricts differentiation along the macrophage lineage. Cell 1993;72:197209.[Medline]
30
Krishnaraju K, Hoffman B, Liebermann DA. The zinc finger transcription factor Egr-1 activates macrophage differentiation in M1 myeloblastic leukemia cells. Blood 1998;92:195766.
31
Chen F, Studzinski GP. Expression of the neuronal cyclin-dependent kinase 5 activator p35Nck5a in human monocytic cells is associated with differentiation. Blood 2001;97:37637.
32 Chen F, Rao J, Studzinski GP. Specific association of increased cyclin-dependent kinase 5 expression with monocytic lineage of differentiation of human leukemia HL60 cells. J Leukoc Biol 2000;67:55966.[Abstract]
33 Zhuang SH, Schwartz GG, Cameron D, Burnstein KL. Vitamin D receptor content and transcriptional activity do not fully predict antiproliferative effects of vitamin D in human prostate cancer cell lines. Mol Cell Endocrinol 1997;126:8390.[Medline]
34 Imaizumi M, Uozumi J, Breitman TR. Retinoic acid-induced monocytic differentiation of HL60/MRI, a cell line derived from a transplantable HL60 tumor. Cancer Res 1987;47:143440.[Abstract]
35
Manfredini R, Trevisan F, Grande A, Tagliafico E, Montanari M, Lemoli R, et al. Induction of a functional vitamin D receptor in all-trans-retinoic acid-induced monocytic differentiation of M2-type leukemic blast cells. Cancer Res 1999;59:380311.
36 Duo QP, Levin AH, Zhao S, Pardee AB. Cyclin E and cyclin A as candidates for the restriction point protein. Cancer Res 1993;53:12937.[Abstract]
37 Studzinski GP, Bhandal AK, Brelvi ZS. Cell cycle sensitivity of HL-60 cells to the differentiation-inducing effects of 1-alpha,25-dihydroxyvitamin D3. Cancer Res 1985;45:3898905.[Abstract]
38 Zhou JY, Norman AW, Akashi M, Chen DL, Uskokovic MR, Aurrecoechea JM, et al. Development of a novel 1,25(OH)2-vitamin D3 analog with potent ability to induce HL-60 cell differentiation without modulating calcium metabolism. Blood 1991;78:7582.[Abstract]
39 Abe J, Nakano T, Nishii, Y, Matsumoto T, Ogata E, Ikeda K. A novel vitamin D3 analog, 22-oxa-1,25-dihydroxyvitamin D3, inhibits the growth of human breast cancer in vitro and in vivo without causing hypercalcemia. Endocrinology 1991;129:8327.[Abstract]
40 Abe-Hashimoto J, Kikuchi T, Matsumoto T, Nishii Y, Ogata E, Ikeda K. Antitumor effect of 22-oxa-calcitriol, a noncalcemic analogue of calcitriol in athymic mice implanted with human breast carcinoma and its synergism with tamoxifen. Cancer Res 1993;53:25347.[Abstract]
41 Sporn MB. New agents for chemoprevention of prostate cancer. Eur Urol 1999;35:4203.[Medline]
42 Wang X, Rao J, Studzinski GP. Inhibition of p38 MAP kinase activity up-regulates multiple MAP kinase pathways and potentiates 1,25-dihydroxyvitamin D3-induced differentiation of human leukemia HL60 cells. Exp Cell Res 2000;258:42537.[Medline]
Manuscript received December 27, 2000; revised June 12, 2001; accepted June 22, 2001.
This article has been cited by other articles in HighWire Press-hosted journals:
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |