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(Journal of Leukocyte Biology. 2001;69:794-802.)
© 2001 by Society for Leukocyte Biology

Modulation of death receptor-mediated apoptosis in differentiating human myeloid leukemia HL-60 cells

Jan Vondrácek*, Michael A. Sheard2, Pavel Krejcí3, Katerina Minksová*, Jirina Hofmanová* and Alois Kozubík*

* Institute of Biophysics,
{dagger} Masaryk Memorial Cancer Institute, and
{ddagger} Laboratory of Molecular Embryology, Mendel University, Brno, Czech Republic

Correspondence: Jan Vondrácek, Institute of Biophysics, Královopolská 135, 612 65 Brno, Czech Republic. E-mail: hivrisek{at}ibp.cz


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Differentiating myeloid cells may become resistant to various apoptotic stimuli. In the present study, dimethyl sulfoxide (DMSO) and all-trans retinoic acid (ATRA) were found to modulate the sensitivity of HL-60 cells to death receptor-mediated apoptosis in a time-dependent manner. During the early stages of differentiation, DMSO treatment increased the response of HL-60 cells to tumor necrosis factor {alpha}; (TNF-{alpha}), but enhanced responsiveness was lost during later differentiation stages. In contrast, ATRA treatment induced resistance to TNF-{alpha}-induced apoptosis. HL-60 cells were resistant to Fas-mediated apoptosis but were sensitized by culturing in serum-free conditions. Similar to its effect on TNF-{alpha} sensitivity, DMSO pretreatment augmented the response to Fas-mediated signaling, which coincided with increased expression of Fas on DMSO-pretreated cells. However, during the later stages of DMSO-induced differentiation, sensitivity to anti-Fas antibody-induced apoptosis declined significantly, although Fas expression was still elevated. The reduced sensitivity to anti-Fas treatment partially correlated with increased Fas-associated phosphatase-1 mRNA expression. Thus, regardless of either Fas up-regulation or potentiation of TNF-{alpha}-mediated apoptosis during early DMSO-induced differentiation, a slow increase in resistance to apoptosis mediated through these death receptors occurs during DMSO-induced differentiation, which contrasts with the rapid induction of resistance following treatment with ATRA.

Key Words: all-trans retinoic acid • dimethyl sulfoxide • Fas/CD95/APO-1 • TNF-{alpha} • Bcl-2 • FAP-1


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis, or programmed cell death, represents an active process that is critical for both development and tissue homeostasis [1 ]. It is characterized by membrane blebbing; cytoplasmic, nuclear, and chromatin condensation; activation of caspases; and cleavage of DNA into multiple intranucleosomal fragments [2 , 3 ]. The initiation of apoptosis is controlled by integration of both proapoptotic and antiapoptotic signal transduction pathways that mediate stimuli such as the deprivation of survival factors, cell-damaging stress, the action of growth factors and cytokines, or signals through death receptors [4 ].

Death receptors such as tumor necrosis factor (TNF) receptor type 1 (TNFR1), Fas, TRAMP, TRAIL-R1, TRAIL-R2, and others belong to the TNF receptor superfamily. Each member contains a death domain sequence—an intracellular region of about 80 amino acids that is essential for triggering cell death in target cells [3 , 5 6 7 ]. The signaling pathways by which these receptors induce apoptosis exhibit many similarities, including recruitment of death domain-containing adapter proteins and activation of downstream effector caspases via caspase 8 (FLICE) or 10 (FLICE-2) [3 , 5 , 8 ]. However, other signaling pathways seem to be functionally linked to death receptor-mediated apoptosis as well, including sphingomyelinases, c-Jun N-terminal kinase, NF-{kappa}B-inducing kinase, and reactive oxygen intermediates [for recent reviews, see references 3 , 6 , and 9 ].

Elucidation of the mechanisms involved in the regulation of apoptosis in both normal and malignant hematopoietic cells may contribute to the development of improved therapeutic strategies in the treatment of leukemia [10 ]. Cellular differentiation can affect apoptosis sensitivity, and it provides a successful strategy for the treatment of acute promyelocytic leukemia [11 ]. However, the exact relationship between differentiation and apoptosis remains unclear [12 , 13 ]. While terminal differentiation of myeloid cells may result in apoptosis [14 ], it has been shown that differentiating myeloid cells can become resistant to various apoptotic stimuli. This phenomenon has been observed during differentiation induced by phorbol esters [15 16 17 ], all-trans retinoic acid (ATRA) [12 , 13 , 18 ], vitamin D3 [13 , 18 , 19 ], or dimethyl sulfoxide (DMSO) [20 ]. Although the relative importance of differentiation-mediated resistance to apoptosis in vivo is still unclear, apoptosis of acute promyelocytic leukemia cells after ATRA therapy or in combination with chemotherapy in vivo has been demonstrated [21 ]. Thus, the relationship between apoptosis and differentiation deserves more attention, especially given the possible use of differentiation therapy in combination with chemotherapy [22 ].

The present study elucidated whether and, if so, how two types of inducers of granulocytic differentiation (DMSO and ATRA) might modulate the sensitivity of HL-60 cells to TNF-{alpha} or anti-Fas monoclonal antibody (mAb) as they progress through different stages of differentiation. Induction of apoptosis by death receptors appears to be regulated by a number of antiapoptotic mechanisms, such as by proteins of the Bcl-2 family [3 , 4 , 9 ] or Fas-associated phosphatase-1 (FAP-1), which has been suggested to act as a negative regulator that binds to the cytoplasmic region of TNF receptor superfamily members [23 , 24 ]. Therefore, the expression of Bcl-2 and FAP-1 was investigated during our study.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and treatments
Human leukemia HL-60 cells were obtained from the European Collection of Animal Cell Cultures (Porton Down, Salisbury, UK). Cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and gentamicin (50 µg/mL) (referred to as full medium) and maintained in a humidified incubator at 37°C in 5% CO2. Apoptosis was induced using either CH-11 mAb (100 or 500 ng/mL final concentration), which was reported to induce apoptosis by cross-linking Fas [25 ], or human recombinant TNF-{alpha} (1 or 10 ng/mL final concentration). In all experiments, cells were incubated with apoptosis inducers for 16 h and harvested for detection of apoptosis. Three different experimental settings were used throughout the study: (1) cells were incubated in full medium with apoptosis inducers for 16 h in the presence of either 1.25% DMSO or 1 µM ATRA (0 + 16 h group); (2) for DMSO and ATRA pretreatment, exponentially growing cells were seeded at 2 x 105 cells per mL of full medium supplemented with either 1.25% DMSO or 1 µM ATRA for 48 or 96 h, and after various time periods, apoptosis inducers were added for another 16 h (48 + 16 h and 96 + 16 h groups); and (3) when investigating Fas-mediated apoptosis, cells were pretreated with differentiation inducers for 48 or 96 h (48 + 16 h and 96 + 16 h groups), washed three times with fresh medium without serum, and seeded at 2 x 105 cells per mL of serum-free medium supplemented with insulin-transferrin-sodium selenite medium supplement (referred to as serum-free medium) (Sigma Chemical Co., St. Louis, MO).

Reagents
Human recombinant TNF-{alpha}, RPMI 1640 medium, ribonuclease A, propidium iodide (PI), DMSO, ATRA, and secondary anti-murine immunoglobulin G (IgG) antibody conjugated with horseradish peroxidase were purchased from Sigma. Anti-human Fas CH-11 and UB2 (fluorescein isothiocyanate [FITC] conjugated), anti-human CD11b-FITC, and IgG1-FITC isotype control antibodies were from Immunotech (Marseilles, France). Mouse anti-human antibodies against TNFR1 and TNFR2 [phycoerythrin (PE) conjugated] and the IgG1-PE isotype control were from Caltag Laboratories (Burlingame, CA). Anti-Bcl-2 mAb was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). FBS was from PAN Systems (Nürnberg, Germany). 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Fluka (Buchs, Switzerland). MOWIOL® 40-88 was from Aldrich Chemicals (Milwaukee, WI).

Analyses of expression of Fas, TNFR1, and TNFR2
Following 24, 48, 72, or 96 h of differentiation in full medium, cells were counted with a Coulter Counter (Coulter Corp., Hialeah, FL), and their viability was checked by the eosin exclusion method. Cells were washed twice with modified Hanks’ balanced salt solution without calcium or magnesium but containing 0.2% bovine serum albumin and 0.1% NaN3 and stained with FITC-labeled anti-Fas or PE-labeled anti-TNFR1 or PE-labeled anti-TNFR2 mAbs. After two washes, cells were resuspended and analyzed using an EPICS XL flow cytometer (Coulter Corp.). Dead cells were excluded from analysis by the PI exclusion method. At minimum, 10,000 events were collected. Values for mean fluorescence intensity (MFI) index were calculated using the following formula: MFI index = MFI of specific mAb-stained cells/MFI of isotype control mAb-stained cells.

Detection of apoptosis
Following 16 h of incubation with apoptosis inducers, cells were harvested and prepared for DNA labeling with PI or DAPI. For PI staining, 106 cells were washed once with phosphate-buffered saline (PBS) and fixed in cold 70% ethanol. Fixed cells were washed twice with PBS, and low-molecular-weight DNA was extracted with citric acid buffer [26 ]. Cells were then resuspended in PBS containing 20 µg/mL of PI and 5 Kunitz U/mL of ribonuclease A and incubated for 30 min at room temperature. Cells were analyzed with a FACS®Calibur (Becton Dickinson, San Jose, CA). At minimum, 15,000 events were collected per sample. For DAPI staining, 5 x 105 cells were resuspended in 30 µL of methanol containing 1 µg/mL of DAPI (final concentration) and incubated for 30 min at room temperature. After incubation, cells were mixed with 30 µL of MOWIOL solution and mounted for counting with a fluorescence microscope. At least 200 cells were counted per sample.

Detection of differentiation
Following a 96-h incubation with 1 µM ATRA or 1.25% DMSO, cells were counted with a Coulter Counter and assayed for cell oxidative burst as described previously [27 28 ]. Briefly, cells (106 from each experimental group) were harvested and resuspended in cultivation medium without FBS. Cells were induced with zymosan opsonized by human serum. Luminol (5 x 10-4 M final concentration)-dependent chemiluminescence was measured for 60 min (20 cycles) at 37°C with an LKB Wallac 1251 luminometer (Pharmacia, Turku, Finland).

For CD11b detection, 106 cells were incubated with FITC-conjugated CD11b mAb and washed twice with PBS–1% FBS–0.1% NaN3. Cells were resuspended in the same solution and were analyzed with a FACS®Calibur. At least 10,000 events were collected.

Expression of Bcl-2 protein
Whole-cell lysates (105 cells) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% gel) and transferred to polyvinylidene difluoride membranes (Immobilon®; Millipore, Bedford, MA). After incubation with primary and secondary antibodies, detection was performed with the ECL Plus Western blotting detection system (Amersham Pharmacia Biotech, Little Chalfont, UK).

Reverse transcriptase-polymerase chain reaction
Total RNA was isolated from cells by using an RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Randomly primed complementary DNA was synthesized from 500 ng of total RNA by using Moloney murine leukemia virus reverse transcriptase (Top-Bio, Prague, Czech Republic). The primer pair used for specific PCR amplification of the FAP-1 sequence was 5'-GAATACGAGTGTCAGACATGG –3' (forward) and 5'-AGGTCTGCAGAGAAGCAAGAATAC-3' (reverse), and the product size was 607 bp [23 ]. The primer pair used for specific PCR amplification of the ß-actin sequence was 5'-GACGAGGCCCAGAGCAAGAG-3' (forward) and 5'-GGGCCGGACTCATCGTACTC –3' (reverse); the product size was 935 bp.

All PCRs were performed with Taq DNA polymerase (Top-Bio) as follows: 30 cycles of denaturation at 94°C for 15 s, annealing at 63°C for 30 s, and synthesis at 72°C for 45 s. In the last 15 cycles, synthesis time was elongated by 5 s, and the last synthesis time period was 5 min. The products were subjected to 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining. Authenticity of the FAP-1 PCR product was verified by digesting with EcoRI and XhoI endonucleases (MBI Fermentas, Vilnius, Lithuania).

Statistics
Student’s t-test and one-way analysis of variance were used to compare results.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DMSO potentiates, while ATRA blocks, TNF-{alpha}-induced apoptosis
Both DMSO and ATRA induced differentiation in HL-60 cells, as assessed by the luminol-enhanced chemiluminescence assay and expression of the CD11b marker 96 h after addition of either DMSO or ATRA (Table 1 ). To examine the effect of DMSO on sensitivity to TNF-{alpha}, a morphological analysis of DAPI-stained cells was performed, revealing a significantly higher percentage of nuclei with typical apoptotic features in cells treated with both DMSO and TNF-{alpha} (either 1 or 10 ng/mL) than in cells treated with TNF-{alpha} alone (Fig. 1 ;Table 2 ). Although DMSO-pretreated cells were sensitive to TNF-{alpha} treatment after 48 h, a decrease in sensitivity to TNF-{alpha}-induced apoptosis was observed 96 h after initiation of differentiation with DMSO (in contrast to control cells, which were more sensitive to TNF-{alpha} than freshly seeded cells after 48 h of incubation). The results of this morphological analysis were confirmed by an increase in the subdiploid population in PI-stained cells after extraction of low-molecular-weight DNA with citric acid buffer (Fig. 2 ). These results indicate that DMSO potentiated TNF-{alpha}-induced apoptosis in HL-60 cells in a time-dependent manner. Contrary to this finding, incubation of cells with ATRA inhibited TNF-{alpha}-induced apoptosis both at the beginning and after 48 h of differentiation (Fig. 1 and 2 ; Table 2 ). The 96-h interval is not shown because a significant number of ATRA-treated cells had already undergone apoptosis at that time point (n = 3; data not shown).


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  		Table 1. Differentiation of HL-60 Cells After 96 h of Treatment with DMSO (1.25%) or ATRA (1 µM);a>

 


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  		Figure 1. DMSO potentiates, while ATRA blocks, TNF-{alpha}-induced apoptosis in differentiating HL-60 cells. The 96 h + 16 h control group was omitted from the study due to the effects of high cellular density on cell behavior (>3 x 106 cells/mL). The ATRA 96 h + 16 h group is not shown because a significant number of ATRA-treated cells had already undergone apoptosis at that time point. Cells were stained with DAPI as described in Materials and Methods and observed by fluorescence microscopy. Results are representative of three independent experiments.

 

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  		Table 2. TNF-{alpha}- and CH-11 mAb-Induced Apoptosis in HL-60 Cells in Full Medium and Under Serum-Free Conditions

 


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  		Figure 2. Representative DNA content analyses (n = 3) for HL-60 cells treated with DMSO or ATRA, and TNF-{alpha}. Cells were stained and analyzed as described in Materials and Methods. y-axis, counts; x–axis, PI staining. Gates denote subdiploid fraction.

 
In marked contrast to cellular sensitivity to TNF-{alpha}, undifferentiated, DMSO-treated, and ATRA-treated cells were all insensitive to treatment with an agonistic anti-Fas mAb (Table 2) .

HL-60 cells become sensitive to anti-Fas mAb treatment in serum-free conditions, and DMSO pretreatment potentiates this effect
To reduce the potential effects of serum-derived pleiotropic survival signals on cellular sensitivity to Fas-mediated apoptosis, we incubated control and ATRA- or DMSO-pretreated cells with anti-Fas mAb in serum-free medium. As shown in Figure 3 and Table 2 , 48 h of DMSO-induced differentiation rendered cells significantly more sensitive to Fas-mediated apoptosis than either control or ATRA-pretreated cells maintained in serum-free conditions. As with TNF-{alpha} sensitivity, sensitivity to anti-Fas mAb had decreased back to untreated levels after a 96-h differentiation period (Fig. 3 ; Table 2 ). These results were confirmed by DNA content analyses (Fig. 4 ).



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  		Figure 3. Apoptosis in HL-60 cells preincubated with ATRA for 48 h or with DMSO for 48 and 96 h in serum-containing medium and then exposed to 500 ng/mL of anti-Fas mAb CH-11 for 16 h in serum-free medium. Cells were stained with DAPI as described in Materials and Methods and observed by fluorescence microscopy. Results are representative of three independent experiments.

 


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  		Figure 4. Representative DNA content analyses (n = 4) for HL-60 cells pretreated with DMSO or ATRA for the indicated time period and then incubated with anti-Fas mAb CH-11 for 16 h in serum-free medium. Cells were stained and analyzed as described in Materials and Methods. y-axis, counts; x-axis, PI staining. Gates denote subdiploid fraction.

 
The kinetics of changes in BCL-2 expression are different in ATRA- and DMSO-treated cells
To investigate the mechanism regulating DMSO-induced potentiation of both TNFR1- and Fas-mediated apoptosis, Bcl-2 expression levels were examined. Bcl-2 expression was down-regulated 48–96 h after treatment with ATRA, whereas a slight down-regulation after DMSO treatment was first detectable only after 72 h and beyond (Fig. 5 ). These results indicate that regulation of Bcl-2 expression cannot explain DMSO-induced potentiation of death receptor-mediated apoptosis.



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  		Figure 5. Effect of DMSO- and ATRA-induced differentiation on Fas, TNFR1, and -2 expression in HL-60 cells. Cells were prepared and stained with respective antibodies as described in Materials and Methods. Data represent means ± standard deviations of values from a minimum of three independent experiments. #, significantly different from the respective control group (P < 0.01).

 
DMSO, but not ATRA, induces up-regulation of Fas, while TNFR2 is up-regulated during ATRA-induced differentiation of HL-60 cells.
To examine whether the observed effects of DMSO and ATRA on apoptosis could be explained by differential expression of either Fas or TNF receptors on the cell surface, Fas and TNFR expression was analyzed by flow cytometry. Significant up-regulation of Fas was observed on the cell surface 48–96 h after treatment with DMSO, while significant modulation of Fas expression was not observed after ATRA treatment (Fig. 6 ). None of the treatments modulated the expression of TNFR1. However, ATRA was found to up-regulate TNFR2 expression significantly by 72–96 h after the onset of differentiation (Fig. 6) .



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  		Figure 6. Bcl-2 expression in ATRA- and DMSO-differentiated cells. Total protein was extracted at the indicated time points to assess Bcl-2 protein expression. Results are representative of three independent experiments.

 
Both DMSO and ATRA induce up-regulation of FAP-1 mRNA during the later stages of differentiation of HL-60 cells
In the present study, the expression of FAP-1 was investigated at different time intervals after treatment of HL-60 cells with DMSO or ATRA. As shown in Figure 7 , FAP-1 mRNA expression had increased by 72 and 96 h after ATRA treatment and by 96 h after DMSO treatment, with ATRA being the more potent inducer. Thus, the increased resistance to death receptor-mediated apoptosis coincided with an increase in FAP-1 expression.



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  		Figure 7. Reverse transcriptase (RT)-PCR analysis of FAP-1 mRNA expression in control and in DMSO- or ATRA-treated HL-60 cells at the indicated time periods. The efficacy of the reverse transcription and the amount of RNA used in the RT-PCR were verified by detection of human ß-actin mRNA. Last lane: molecular size markers (100-bp DNA ladder). Human breast carcinoma MCF-7 cells were used as a positive control for FAP-1 expression (right). Results are representative of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although it is believed that terminal differentiation of myeloid cells ultimately leads to apoptosis in vitro [12 , 29 ], the exact relationship between cell maturation and apoptosis is unclear. Terminally differentiated HL-60 cells display typical morphological features of apoptosis and characteristic DNA laddering [12 , 14 , 30 ]. On the other hand, a study of NB4 myeloid cells has shown that retinoids may induce cell differentiation that is insufficient to allow terminal apoptosis; retinoids may also induce apoptosis in the absence of differentiation [31 ]. Moreover, it has been reported that myeloid cells may become resistant to various apoptotic stimuli during the process of differentiation. This phenomenon has been observed during differentiation induced by phorbol esters [15 16 17 ], ATRA [12 , 13 , 18 ], vitamin D3 [13 , 18 , 19 ], or DMSO [20 ].

DMSO was found to potentiate TNF-{alpha}-mediated apoptosis at the onset of differentiation, in agreement with previous reports, which demonstrated that DMSO could potentiate TNF-{alpha}-mediated cytotoxicity in human myeloid cell lines [32 , 33 ]. This phenomenon has not been observed with other inducers of apoptosis, such as irradiation, cytostatics, or hyperthermia. Instead, preincubation with DMSO leads to a reduction of the extent of apoptosis when these effectors are used or has no effect [17 , 20 ]. However, we have found that the effect of DMSO on TNF-{alpha}-induced apoptosis is dependent on the degree of differentiation of HL-60 cells; at the beginning of DMSO treatment, TNF-{alpha}-induced apoptosis is potentiated, but this effect is lost after a 96-h incubation period.

The modulatory effect of ATRA treatment on HL-60 cells follows a different time course than with DMSO treatment. ATRA inhibited TNF-{alpha}-induced apoptosis both at the onset of differentiation and after 48 h of treatment (Figs. 1 and 2) . We found that ATRA rapidly induces resistance to TNF-{alpha} in HL-60 cells, without a requirement for longer preincubation as reported previously [18 ]. Our observation of inhibition of TNF-{alpha}-induced apoptosis by ATRA treatment is similar to the previous finding that ATRA induces resistance to irradiation and cytostatics in HL-60 cells [12 , 13 ]. Thus, unlike the effect of DMSO, the effect exerted by ATRA on death receptor-mediated apoptosis is similar to that on programmed cell death induced by cytostatics. This finding is consistent with a report that ATRA induces resistance to treatment with either TNF-{alpha} or anti-Fas in U937 cells [18 ].

The observed effects did not correlate with either TNFR1 or TNFR2 expression. We did not observe any changes in TNFR1 expression during either ATRA- or DMSO-induced differentiation. TNFR2 expression was not modulated in DMSO-treated cells, although it was substantially increased 72 h after ATRA treatment, as previously reported [34 ]. Thus, no significant changes in expression of either TNFR1 or TNFR2 were detectable during the first 48 h of treatment with differentiation inducers, when their effects on apoptosis were most prominent.

Although HL-60 cells maintained in serum-containing medium were found to express Fas on their surface, they were resistant to anti-Fas mAb treatment in the present study. Treatment with DMSO resulted in up-regulation of Fas expression but had no effect on sensitivity to Fas-mediated apoptosis in the absence of sensitizing (serum-free) conditions. Maintenance in serum-free conditions rendered HL-60 cells partially sensitive to anti-Fas treatment, and a 48-h pretreatment in DMSO further potentiated Fas-mediated apoptosis (Fig. 3) . However, after 96 h, the potentiated response to Fas-mediated signaling had declined back to untreated levels. The sensitivity of HL-60 cells to Fas-mediated apoptosis and even the expression of Fas in these cells are matters of controversy; while some authors have reported sensitivity of HL-60 cells to Fas-cross-linking antibodies [35 36 37 38 ], others have observed resistance to either agonist antibody or FasL [39 40 41 42 43 ]. Similarly, while HL-60 cells have been reported to express Fas [36 37 38 , 42 , 44 , 45 ], others have reported Fas negativity [39 , 43 ]. It has been reported that widespread use of MCF-7 cells in research has led to establishment of variants of this breast cancer cell line that differ in (1) their susceptibility to TNF-{alpha}-induced apoptosis, (2) TNFR expression, (3) ceramide generation, (4) expression of Bcl-2 family members, and (5) protease activation [46 ]. Like MCF-7, HL-60 cells are among the cell lines most frequently used in cell biology research. Discrepancies in the literature concerning the Fas sensitivity of HL-60 cells might be explained by the existence of cell variants exhibiting different sensitivities to anti-Fas mAb and different levels of Fas expression.

Although both DMSO and ATRA are known to induce granulocytic differentiation of HL-60 cells [47 , 48 ], the differentiation programs elicited by DMSO and ATRA are different and promote different degrees of maturation, with ATRA being a more complete and effective inducer [49 ]. Thus, the slower onset of resistance to apoptosis after DMSO treatment that was observed in our study could be related to the dissimilar kinetics of differentiation-associated processes. One of the proteins that has been shown to be regulated in a differentiation-linked manner is Bcl-2. Induction of differentiation by ATRA in HL-60 cells leads to a decrease in Bcl-2 protein levels [50 ]. Down-regulation of Bcl-2 occurred with rapid kinetics in ATRA-treated cells, but it was slow and barely detectable in DMSO-differentiated cells (Fig. 5) . This rapid down-regulation of Bcl-2 by ATRA is not necessarily the cause of the massive apoptosis observed after 96 h of incubation with ATRA alone, since it has been shown that down-regulation of Bcl-2 by ATRA in NB4 cells is not immediately followed by apoptosis [31 ]. However, down-regulation of Bcl-2 by ATRA could render HL-60 cells sensitive to the cytotoxic activity of retinoids [29 ]. Other studies have similarly shown that the down-regulation of Bcl-2 expression induced by DMSO in HL-60 cells is weaker and occurs later than that induced by other differentiation inducers, such as phorbol esters or ATRA [50 51 52 ]. Bcl-2 expression levels did not correlate with sensitivity to TNF-{alpha}- or Fas-mediated apoptosis after ATRA or DMSO treatment in our study, indicating that regulation of Bcl-2 expression does not explain the observed changes in sensitivity of DMSO-treated HL-60 cells to death receptor-mediated apoptosis. Nevertheless, changes in Bcl-2 expression appear to correspond to the rate of differentiation in differentiating ATRA- and DMSO-treated HL-60 cells.

It has been suggested that FAP-1 can act as a negative regulator that binds to the cytoplasmic region of TNF receptor superfamily members, such as Fas and the common neurotrophin receptor p75NTR [23 , 24 ]. Thus, changes in FAP-1 levels could play a role in regulating cell sensitivity to Fas-mediated apoptosis [23 , 24 , 53 , 54 ], although it has been reported that FAP-1 may not play a key role in the Fas-mediated programmed cell death pathway [38 , 55 56 57 ]. Recently, it has been shown that TNF-{alpha} treatment can down-regulate FAP-1 expression in HL-60 cells [58 ]. Our results show that both DMSO and ATRA can up-regulate FAP-1 mRNA expression in the later stages of HL-60 cell differentiation, with ATRA being the more potent inducer. These data suggest that increased FAP-1 expression after 96 h of DMSO treatment could play a role in the observed decline in the DMSO-potentiated response to Fas-mediated signaling.

In conclusion, although both DMSO and ATRA induce granulocytic differentiation, we have shown that these agents differentially modulate the sensitivity of HL-60 cells to death receptor-mediated apoptosis. At least three different phenomena seem to interplay to produce the observed results: (1) up-regulation of Fas during DMSO-induced differentiation, (2) a rapid but transient potentiation of TNF-{alpha}-mediated apoptosis by DMSO, and (3) a slowly increasing resistance to apoptotic stimuli during DMSO-induced differentiation (in contrast to the rapid induction of resistance by ATRA). The increased resistance to death receptor-mediated apoptosis during the later stages of HL-60 cell differentiation coincides with an increase in FAP-1 expression.


   ACKNOWLEDGEMENTS
 
This work was supported by grants 312/98/P011 and 524/99/0694 from the Grant Agency of the Czech Republic. The authors thank T. G. Cotter (University College, Cork, Ireland) for helpful comments on the manuscript and Petr Dvorák (Laboratory of Molecular Embryology, Mendel University, Brno, Czech Republic) for support.

Received February 11, 2000; revised December 10, 2000; accepted December 12, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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