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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 26, 2002 as doi:10.1096/fj.01-0602fje. |
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Department of Experimental Pathology and Oncology, University of Florence, 50134, Firenze, Italy; and
* Department of Hematology, Careggi Hospital, 50139, Firenze, Italy
2Correspondence: Department of Experimental Pathology and Oncology, Viale G. B. Morgagni 50, 50134, Firenze, Italy. E-mail: fcplt{at}unifi.it
SPECIFIC AIMS
Our aim was to identify compounds capable of removing the developmental block occurring in leukemia cells and reprogramming them to terminal division toward the mature phenotype. Attention focused especially on modulators of platelet-activating factor receptor (PAF-r) that has been reported to be expressed in many tumor cells and inferred to play an important role in tumor growth and dissemination. Identification and characterization of new powerful inducers of differentiation are of special significance to basic tumor biology and may contribute prospectively to the development of anticancer drugs for pharmacological approach to neoplasia by differentiation therapy.
PRINCIPAL FINDINGS
The present study demonstrated that the powerful PAF antagonist WEB-2086, originally developed as an anti-inflammatory agent, is an effective inducer of differentiation in vitro, able to commit leukemia cells toward terminal maturation. This novel cytodifferentiation property of WEB-2086 (and in part of its analog WEB-2170) (Fig. 1
) initially identified and characterized in murine erythroleukemia cells (MELC), has been confirmed in human models such as erythroleukemia K562 and HEL cells and in promyelocytic leukemia HL60 cells, which could be massively prompted to maturation.
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1. Detection and modulation of PAF-r mRNA and protein levels during induced differentiation of MELC
MELC have been examined for PAF-r expression by reverse-transcriptase polymerase chain reaction (PCR), which revealed PCR products with the expected size of 235 bp. Uninduced MELC were found to express relatively low constitutive levels of PAF-r mRNA that underwent significant increase after induction to terminal division. However, the increase in PAF-r gene expression was not paralleled by comparable changes in the amount of total PAF-r protein; in fact, cytofluorimetric profiles of the receptor varied only slightly during induced differentiation, suggesting that other events, possibly post-transcriptional modifications, are involved in regulating PAF-r protein levels.
2. Dose-response effects of the PAF agonist (carbamyl-PAF) and antagonist (WEB-2086) on MELC growth and erythrodifferentiation
The occurrence of PAF-r in both uninduced and induced MELC prompted us to investigate whether cell growth and differentiation could be modulated by 1) the PAF agonist carbamyl-PAF (c-PAF), a nonmetabolizable analog of the physiological mediator, and 2) the synthetic, highly specific PAF antagonist WEB-2086, used in vitro and in vivo to abrogate competitively PAF-mediated effects. MELC incubation with increasing amounts of c-PAF (up to 500 nM) led to a stimulation of cell proliferation. A statistically significant dose-dependent increase in cell mass in the interval 0–50 nM agonist was observed in the logarithmic phase of growth (day 2–3), but there were no appreciable variations among treated and untreated cultures by day 5. Stimulation of MELC growth by 50 nM c-PAF was virtually abolished by the concomitant addition in culture of 50 µM WEB-2086 to antagonize PAF-mediated effects. On the other hand, MELC treated with the PAF antagonist WEB-2086 (0–1 mM) showed a proportionate decline in cell mass to yield at 1 mM antagonist a decrease of 
50% vs. control cultures at day 5. WEB-2086-induced arrest of MELC proliferation was accompanied by a dose-dependent increase in percentage of benzidine-positive (B+) cells at day 5 (Fig. 2
) to denote hemoglobin (Hb) accumulation (110 µg Hb/mg cell protein with 1 mM WEB-2086). Activation of erythrodifferentiation in WEB-2086-treated MELC began to be detected (B+
12%) at a concentration of 0.25 mM, appeared to be definitively triggered (B+55%) at 0.5 mM, and peaked at 1 mM WEB-2086 (B+85%) with no sign of cell degeneration.
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3. MELC commitment to terminal division by WEB-2086
To ascertain whether WEB-2086 could irreversibly activate MELC differentiation, cells were incubated with a fixed amount of drug (1 mM) for variable lengths of time for up to 6 days, then transferred to a fresh medium without the drug to monitor their fate in terms of growth rates and percentages of B+ cells. A 3.5-day treatment with WEB-2086 was sufficient to commit the bulk of MELC population to terminal division; cells remained growth-arrested even after transfer into the fresh medium and amounts of B+ cells attained at day 3.5 (50%) increased progressively during subsequent days without the drug to approach levels (B+ cells>80%) as recorded in cultures, which were maintained steadily with the inducer. MELC selected for resistance to HMBA were insensitive to induction by WEB-2086.
4. WEB-2086-induced differentiation of MELC is antagonized by the effects of either phorbol 12-myristate 13-acetate or c-PAF
The process of WEB-2086-mediated differentiation of MELC was found to be sensitive to the action of phorbol 12-myristate 13-acetate (PMA), an inhibitor of HMBA-induced MELC maturation known to antagonize the decrease in PKC activity. Addition of PMA (100 ng/ml) to WEB-2086-induced cultures prevented most cells from commitment; moreover, WEB-2086-induced differentiation of MELC was strikingly inhibited also by c-PAF, although at fairly high agonist concentrations (15 µM).
5. WEB-2086-induced MELC differentiation is accompanied by changes in gene expression that apparently do not involve histone acetylation
Some molecular changes occurring in MELC induced to differentiation by WEB-2086 and concerning gene expression of PAF-r, 
-, and ß-globin and c-Myb have been evaluated. MELC incubated for 5 days with 1 mM WEB-2086 showed augmented levels of PAF-r mRNA and large increases in - and ß-globin mRNA compared with uninduced cultures, whereas c-Myb transcription was drastically reduced according to a pattern closely recalling that obtained with 5 mM HMBA. Electrophoretic analysis of histones isolated from WEB-2086-treated MELC showed that early effects of induction did not involve (as reported for HMBA-treated cells) histone H4 acetylation. Instead, several faint acetylated forms of histone H4 appeared concomitant with a decrease of the H4 band in the profile of MELC treated with tricostatin A, which is known to act as MELC inducer through the inhibition of histone deacetylases.
6. Induction of differentiation in human leukemia cell lines by WEB derivatives
Cytodifferentiation activity of WEB-2086 is not confined to the MELC system. The two human erythroleukemia cell lines K562 and HEL proved to be very sensitive to the action of 1 mM WEB-2086 (Fig. 3
, left) and reached within 5 days of incubation B+ cell levels usually exceeding 75% of the population. The morphology of K562 and HEL cells changed significantly after 5 days incubation with 1 mM WEB-2086; induced cells showed a decrease in cell size, nuclear condensation, and reduced basophilia suggestive of maturation and hemoglobin accumulation (Fig. 3
, right). Comparable levels of K562 and HEL differentiation were obtained with 0.5 mM WEB-2170 to indicate a relatively higher efficacy of this analog vs. WEB-2086. Both WEB derivatives were approximately twofold more efficient as inducers than 1.5 mM n-butyric acid. WEB derivatives also induced myeloid differentiation in HL60 cells, which upon treatment were able to reduce NBT to formazan and showed several indented nuclei as reported for differentiation mediated by retinoids. Cytofluorimetric analyses revealed that a large portion (>70%) of HL60 cells treated with 1 mM WEB-2086 for 3 days was CD11b+ and CD14- to denote a clear inclination of induced cells toward granulocyte differentiation.
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CONCLUSIONS
The present study demonstrated that the specific PAF antagonist WEB-2086, commonly known as an anti-inflammatory agent, is effective as an inducer of cell differentiation in vitro. This novel property of WEB-2086, shared in part by its analog WEB-2170, was initially characterized in the MELC system, then confirmed in other models such as human erythroleukemia K562 and HEL cells and promyelocytic HL60 cells, which were massively prompted to differentiation.
Optimal WEB-2086 concentration for MELC cytodifferentiation is 1 mM, fivefold lower than reported for HMBA. Otherwise WEB-2086 seemed to be fairly comparable to HMBA as to the time and extent of MELC commitment, lack of effect on histone deacetylases, and molecular changes that involved increased transcription of - and ß-globin genes and marked down-regulation of c-Myb. In contrast, other inducers such as trichostatin A and members of the suberoylanilide hydroxamic acid family are known to powerfully induce MELC differentiation by acting as inhibitors of histone deacetylases and without suppressing c-Myb expression. The functional similarities of WEB-2086 and HMBA have inferred that, despite their structural diversity, the two agents might share common action mechanisms involving the depression of PKC activity as one of the obligatory steps of MELC terminal division.
The slight stimulatory effect of nanomolar c-PAF concentrations on MELC growth is consistent with activation of PAF-mediated signal transduction pathways and in keeping with data on K562 and other transformed cells lines, where PAF-r activation through autocrine and/or paracrine loops contributed in sustaining cell proliferation. The competitive inactivation of PAF-r by WEB-2086 might abrogate the c-PAF-mediated proliferative signal, then potentially favor MELC differentiation. This process, however, was triggered at millimolar WEB-2086 concentrations, which are higher by far than those compatible with a receptor-mediated effect. Therefore, whereas specific binding of micromolar WEB-2086 to PAF-r could exert a cytostatic effect, MELC differentiation by millimolar WEB-2086 concentrations may develop, conceivably through low-affinity PAF-r or even PAF-r-independent pathways. The association of anti-PAF and differentiation activities in a single molecule represents a distinctive feature of WEB-2086 vs. other known inducers of differentiation in vitro and might be important especially in vivo, where WEB-mediated inactivation of PAF-r could help in reducing cancer cell motility and inhibiting angiogenesis in surrounding tissues. Incidentally, WEB derivatives have been widely used, primarily as anti-inflammatory drugs, in experimental animal models and in humans showing high tolerability.
As for other inducers, mechanisms of WEB-2086-mediated differentiation are still unclear and need to be explored further. However, the identification of this novel property of WEB-2086 and its broad activity on murine and human leukemia cells, of erythroid and myeloid origin may lead to the development of a new class of potentially valuable inducers to be used, prospectively, for clinical purposes in the treatment of cancer by differentiation therapy.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0602fje; to cite this article, use FASEB J. (March 26, 2002) 10.1096/fj.01-0602fje 
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), WEB-2086 (•), WEB-2170 (
), and n-butyric acid (
). Results are the means (±SD) of 3 separate experiments. Cell morphology (right panel) of typical uninduced and WEB-2086-induced populations was assessed on cytospin or cell smears stained with May-Grünwald-Giemsa (x650) for K562 or HEL, respectively.