Thymosin Fraction-5 PossessesAntiproliferative Properties in HL-60Human Promyelocytic Leukemia Cells

Characterization of an Active Peptide

BRYAN L. SPANGELO,a JOSEPH D. ROACH,a FREIDUN HADI,a

ALI A. DAMAVANDY,b JORDAN PLIESKATT,c

AND MAHNAZ BADAMCHIANb

aDepartment of Chemistry, University of Nevada Las Vegas, Las Vegas,Nevada, USAbDepartments of Biochemistry and Molecular Biology, and Microbiology,The George Washington University School of Medicine and Health Sciences,Washington, DC, USAcDepartment of Microbiology, Immunology, and Tropical Medicine, The GeorgeWashington University School of Medicine and Health Sciences, Washington,DC, USA

ABSTRACT: Thymosin fraction-5 (TF5) is a protein preparation of thebovine thymus. TF5 stimulates many assays of T cell—mediated immu-nity. We found that TF5 substantially suppressed proliferation of therat C6 glioma and MMQ pituitary adenoma cell lines. Our current re-search using the promyelocytic cell line HL-60 suggests that TF5 alsoprevents proliferation of human myeloid leukemia cells. Our objectiveis the purification and chemical characterization of TF5 peptide com-ponents responsible for inhibition of HL-60 proliferative capacity. Usingthe inhibition of HL-60 cell proliferation, we have chemically character-ized TF5 using fast protein liquid chromatography (FPLC), reversed-phase high-performance liquid chromatography (RP-HPLC), and high-performance capillary electrophoresis (HPCE). Vital dye-exclusion, ox-idative metabolism of chromogenic dyes, and clonogenic growth pro-files were used to determine rates of HL-60 proliferation. Our resultsidentified an ∼6000 Da component of TF5 capable of inducing HL-60growth arrest. Synchronized HL-60 cells exposed to TF5 and its variousconstituents were subjected to cytometric analysis by flow cytometry.TF5-treated HL-60 cells had an increased subdiploid faction (i.e., sub-G1) compared to control cells. TF5 also increased Annexin V staining in

Address for correspondence: Bryan L. Spangelo, Department of Chemistry, University of NevadaLas Vegas, 4505 Maryland Parkway, Las Vegas, NV 89154-4003. Voice: 702-895-3797; fax: 702-895-4072.

bryan.spangelo@unlv.edu

Ann. N.Y. Acad. Sci. 1112: 305–316 (2007). C© 2007 New York Academy of Sciences.doi: 10.1196/annals.1415.022

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randomly cycling HL-60 cells. Thus, a TF5 subfraction possesses growth-suppressive activity for human myeloid neoplasms. Our results indicatethat this effect is characterized by at least one hallmark of apoptosis.Future clinical management strategies for certain leukemias may involvethe use of thymic peptides.

KEYWORDS: HL-60 cells; human promyelocytic leukemia cells; thymicpeptides; thymosin fraction 5; thymosin �1; thymosin �4; apoptosis

INTRODUCTION

Thymus-mediated immunity is essential for normal immune function. Thy-mus gland involution is accompanied by parallel decreases in levels of thymus-derived hormones in thymic epithelium and in the periphery.1,2 This postulatedthymic hormone decline is coincidental with a decrease in immune reactivity,an increase in the occurrence of age-related disease, and increases in the inci-dence of cancers.3 Thymic hormones act within both the thymus and peripheryto enhance T-lymphocyte differentiation and immune function. Efforts to es-tablish an immunological role of thymic peptides have been performed using aclassic protein preparation of the bovine thymus designated thymosin fraction-5 (TF5).

TF5 is composed of 99% protein, with insignificant amounts of carbohydrateand purine nucleotides.4 Amino acid analysis indicates a composition highin acidic residues (∼50%), with very few aromatic residues (∼3%) and notryptophan. Several component polypeptides of TF5 have been described, butnot all of the observed biological effects of TF5 can be ascribed to any singleone of these individual constituents. TF5 contains approximately 40 heat-stablepolypeptides with molecular weights in the range of 1.2–14.0 kDa. SeveralTF5-derived peptides have been purified and sequenced, including thymosin�1 (T�1), thymosin �4 (T�4), and MB-35.4,5 TF5 itself is active in severalbiological assays that assess immune reactivity.6 Exogenous TF5 substitutesfor endogenous thymic function in thymectomized or athymic animals. Peptideconstituents of TF5, such as T�1, circulate in human serum, as determined bydirect radioimmunoassay.2,7

Our previous results have demonstrated that TF5 effectively inhibits prolif-eration of rat neuroendocrine tumor cells in vitro.8,9 This preparation alsoinhibits growth of several neoplastic cell lines in vitro, including murineDunning monocytoid leukemia cells,10 M-109 murine lung carcinoma cells,11

L-1210 murine lymphoblastic leukemia cells,12 and human acute lymphoblas-tic T-leukemia cells.13,14 Given that TF5 generally restores immunological de-ficiency and substantially restricts the proliferative activity of some neoplasticcell types, we determined whether TF5 inhibits the growth of the human HL-60promyelocytic leukemia cell in vitro. We found that a component activity inTF5 exerts an antiproliferative effect in human myeloid leukemia cells. Our

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studies identified an HL-60 growth-suppressive TF5 subfraction that is uniquefrom previously characterized thymosin peptides.15

HL-60 CELL LINE

Leukemias are a large proportion of hematological diseases, occurring asacute or chronic malignancies. Early hematopoietic progenitors do not re-spond to appropriate stimuli within the bone marrow microenvironment, thusallowing proliferation to high numbers and export to vascular compartments.Leukemia cells do not undergo apoptosis and fail to differentiate. The degree ofdifferentiation is inversely related to malignant potential, suggesting that differ-entiation is uncoupled from cellular growth control mechanisms in leukemiccells. Thus, attempts to induce differentiation should reduce or prevent themalignant potential of leukemic cells.

Several leukemic cell lines exist, including the human promyelocyticleukemic HL-60, human lymphocytic leukemic U937, and the human leukemicK562. These cell lines grow in suspension culture as homogeneous cell pop-ulations unless forced to differentiate into various phenotypes. The HL-60cell line was developed from cells isolated from peripheral blood of a patientwith acute promyelocytic leukemia in 1978.16 Differentiation of these cellsinto neutrophils is possible with several agents including DMSO, retinoic acid,5-azacytidine, or tunicamycin.17 Tumor promoters, such as phorbol myristateacetate, cause differentiation of HL-60 cells to a macrophage–monocyte celltype. Interestingly, tumor necrosis factor-� (TNF-�) induces differentiation ofHL-60 cells into the macrophage–monocyte lineage coincidental with cytosta-sis.17 A variety of other treatments cause induction of an eosinophil phenotypeor one with distinct granulocyte characteristics. Granulocytic maturation isaccompanied with an irreversible differentiation and loss of proliferation. Acharacteristic of terminally differentiated HL-60 cells is the failure to formcolonies in soft agar.

Induction of Apoptosis in HL-60 Cells

Elucidation of programmed cell death or apoptosis pathways in HL-60 cellshas revealed a role for sphingophospholipid-derived messengers. Undifferen-tiated HL-60 cells are particularly sensitive to the addition of low micromolarconcentrations of ceramide, resulting in an increase in double-stranded DNAbreaks.18 In addition, increased intracellular diglyceride concentrations in-hibit ceramide stimulation of apoptotic DNA damage in HL-60 cells. Becausediglyceride activates protein kinase C (PKC), a cytoprotective role for this ki-nase family has been postulated. The sphingoid base sphingosine inhibits PKCactivity and reduces proliferative capacity and viability in HL-60 cells. Sph-ingosine also augments the apoptotic capacity of ceramide.19 Thus, reciprocalinfluences of sphingoid bases and diglyceride on PKC coordinately modulateceramide-mediated apoptosis in HL-60 cells.

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TABLE 1. Chemical and biological properties of thymosin peptides tested

Thymosin �1 (T�1)Ac-S-D-A-A-V-D-T-S-S-E-I-T-T-K-D-L-K-E-K-K-E-V-V-E-E-A-E-N28 amino acids; 3108 Da; pI 4.2Increases MHC class 1 expression of lymphoid cellsIncreases expression of IL-2 receptorsIncreases production of IL-2 in lymphocytesAntagonism of apoptosis in immature thymocytesEnhances response rates in hepatitis B patientsIncreases antibody response to influenza vaccine

Thymosin �4 (T�4)Ac-S-D-K-P-D-M-A-E-I-E-K-F-D-K-S-K-L-K-K-T-E-T-Q-E-K-N-P-L-P-S-K-E-T-I-E-

Q-E-K-Q-A-G-E-S43 amino acids; 4963 Da; pI 5.1Inhibits actin polymerizationDecreases F-actin contentIncreases wound-healing ratesStimulates aortic ring sprouting and angiogenesisReduces septic shock severityDownregulates inflammatory cytokines and chemokines

THYMIC PEPTIDES: CHEMISTRYAND BIOLOGICAL PROPERTIES

The thymus produces a family of hormonal-like peptides that can modulateboth immune and nonimmune physiologic processes. Some of the humoralmessengers first isolated from the thymus, such as T�1, thymulin (FTS-Zn),thymic humoral factor (THF-�), and thymopoietin (ITP-5), are primarily im-munostimulating agents whereas the action of others, such as T�4, thymosin�10 (T�10), and MB35, possess multiple biological activities. The physiologicprocesses that these thymic peptides influence include neuroendocrine modu-lation, stimulation or suppression of immune responses, wound healing, andangiogenesis. Genes for thymic derived peptides, such as T�4, are upregulatedas cells differentiate and following tissue injury. T�4 plays an important rolein the remodeling of tissues and wound healing by virtue of its ability to regu-late the polymerization and depolymerization of actin and by downregulatinga number of inflammatory chemokines and cytokines.20–22

Over 25 biologically active peptides from the thymus gland have been chem-ically characterized. Most of these molecules belong to the thymosin familyand are small peptides with molecular weights of less than 10,000 Da. Thechemical and select biological properties of T�1 and T�4 are presented inTABLE 1. Certain TF5-derived peptides are part of larger precursor molecules.This is true of T�1, a 28-amino acid peptide that is also contained within theNH2-terminus of a larger 109-amino acid peptide termed prothymosin � (ProT�). Although T�1 and Pro T� are present in significant amounts in thymus,they are also present in other tissues. Other peptides, such as T�4 (first isolated

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from TF5), are present in much higher concentrations in blood platelets andin tissues other than the thymus. T�4 is the major actin-sequestering pep-tide in mammalian cells. It is a 43-amino acid peptide and a member of the�-thymosin family of small, water-soluble peptides. It plays an important rolein wound repair and remodeling of tissues following injury. Another isolatedpeptide from TF5 is MB-35, which is a basic peptide composed of 35 aminoacid residues with a molecular weight of 3756. Peptide MB-35 is a potentstimulator of prolactin (PRL) and growth hormone (GH) release from anteriorpituitary cells in vitro. Peptide MB-35 is identical to a fragment of nuclearprotein histone H2A isolated from human, rat, chicken, and bovine thymus,which suggests that a fragment of this nucleoprotein is involved in the modula-tion of PRL and GH responses.5,21,23 Thus, thymosins are important biologicalresponse modifiers (BRMs) that demonstrate therapeutic potential in a rangeof clinical situations.23

T�1 and T�4, the best characterized thymic peptides, act at different stagesof T cell development. T�4 affects early T cell progenitors in the bone mar-row and thymus. In contrast, T�1 targets thymocytes at intermediate stagesof development and mature T cells in peripheral circulation and tissues. Theimmunomodulatory activity of these peptides may provide a means of safelyaugmenting (or suppressing) T-lymphocyte function in patients with compro-mised or overactive thymic-dependent immunity, including cancer patients, theelderly, patients with autoimmune diseases, and patients with infections andother acquired immunodeficiencies. Additionally, T�1 facilitates viral clear-ance in animal models of hepatitis, suggesting an antiviral function.23 Recentwork with T�4 clearly demonstrates an ability to regulate the polymerization ofactin, suggesting a role in the treatment of wounds. T�4 also has great potentialas a novel anti-inflammatory agent. The use of T�1 and T�4 in combinationwith other BRMs or drugs may result in useful therapeutic applications ofthese molecules. Ongoing experimental and clinical studies have confirmedthis possibility.23,24

THYMOSIN PEPTIDES AS ADJUVANTSFOR MYELOPROLIFERATIVE DISEASE

HL-60 Cells and TF5

We previously reported that TF5 substantially restricts the proliferative ca-pacities of neuroendocrine tumor cells.8 The MMQ cell line is a PRL-secretinganterior pituitary adenoma sensitive to dopamine.25 TF5 completely preventedMMQ proliferation in cell culture suspensions in a concentration- and cellnumber-dependent fashion. Similarly, TF5 suppressed proliferation of the ratC6 astrocytoma cell line in vitro.8 Neither of the well-characterized thymosinpeptides, T�1 and T�4, had any effect on MMQ or C6 cell proliferation.

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Interestingly, the effects of TF5 on neuroendocrine tumor cell proliferationwere not accompanied by large decreases in cellular viability. These prelimi-nary data were extended to human promyelocytic leukemia cells.

We hypothesized that a circulating thymic peptide may exert an antiprolif-erative effect on certain lymphoid neoplasms. This potential immune surveil-lance mechanism was examined using HL-60 and U937 cells. We obtained theHL-60 and U937 cell lines from the American Type Tissue Culture Collection(ATCC, Rockville, MD). These myeloid neoplasms are frequently used forinvestigations of apoptosis and cytostasis. We examined the effect of TF5 onHL-60 and U937 cells and found that this preparation exerts antiproliferativeeffects in both myeloid neoplasms.15

As for MMQ and C6 cells, TF5 prevented HL-60 cell proliferation in aconcentration- and cell number-dependent manner.15 The EC50 for TF5 was∼400 �g/mL as determined in cell suspension density assays and the MTTmetabolism assay. Conditioning of TF5 solutions in HL-60 cell cultures re-moved or possibly metabolized the activity within 24 h. This “cell-inactivated”TF5 had no effect in treatment-naı̈ve HL-60 cultures. Extending these resultsto HL-60 colony formation in soft agar, we reported that TF5 demonstratedprofound suppressions in clonogenic potential following 4 h of pretreatment(i.e., 30–60%); these reductions in colony formation were noted after 14 days,revealing the long-term effects of TF5.15

In contrast to our reported lack of effect of T�1 and T�4 on MMQ or C6 cellproliferation, Huang et al.26 demonstrated that T�4 suppressed proliferationand [3H]-thymidine incorporation in HL-60 cells. Therefore, we examined theeffects of T�1 and T�4 on HL-60 proliferation to determine if either were re-sponsible for the effects of TF5. As shown in FIGURE 1, T�4 (1 nM to 1 �M) didnot affect HL-60 cell proliferation as determined by the MTT assay. In similartrials, T�1 did not alter HL-60 proliferation rates (data not shown). In contrast,TF5 (200 �g/mL to 800 �g/mL) consistently produces a striking inhibitionof HL-60 cell growth (FIG. 1). Therefore, we further purified TF5 using a fastpeptide liquid chromatography (FPLC) system. We injected ∼100 mg TF5onto a Superose gel filtration column and collected four fractions. Thymosinsubfraction 2 substantially restricted HL-60 proliferation in cell suspension by71%. Thus, approximately 40 �g/mL of subfraction 2 was more effective than800 �g/mL TF5 (i.e., 71% versus 49% suppression).15

In contrast to apoptosis, mechanisms underlying induction of cytostasis arenot well described in HL-60 cells. Shen et al.27 reported that neither TNF-�or IL-1� had any effect on [3H]-thymidine incorporation in HL-60 cells. Incontrast, both cytokines potently inhibited IGF-I induction of DNA synthesis.Thus, these authors ascribe cytostatic properties to TNF-� and IL-1� onlyin the presence of IGF-I. We have reported that TF5 reduced the clonogenicsurvival of U937 and HL-60 cells.15 These reductions in proliferation rateswere unaccompanied by any change in apoptotic DNA degradation or theappearance of cytological features characteristic of apoptosis in U937 cells.

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FIGURE 1. T�4 does not suppress HL-60 cell proliferation in vitro. Cultured HL-60cells were exposed to either vehicle (HL-60 culture medium), T�4 (0.1–1000 nM) or TF5(200–800 �g/mL), for 72 h. Cell proliferation was determined by the MTT viability assay.(A) T�4 did not significantly affect HL-60 proliferation. (B) All concentrations of TF5significantly suppressed HL-60 cell proliferation compared to control (P< 0.01). Nonpara-metric statistical analysis consisted of analysis of variance (ANOVA), with significanceconfirmed using the Bonferroni test for multiple comparisons (GraphPad InStat, version3.0; GraphPad Software, Inc. San Diego, CA). The data are expressed as the mean ± SEMof groups consisting of four observations.

Because cytofluorometric analysis of TF5-treated HL-60 cells also revealed anincrease in the subdiploid fraction (i.e., sub-G1), we suggested that TF5 maydeliver an effective G1/S phase cell cycle block resulting in the accumulationof subdiploid DNA.15

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FIGURE 2. TF5 induces apoptosis in HL-60 cells in vitro. Cultured HL-60 cells(1 × 106/5 mL RPMI-1640) were exposed to either vehicle or TF5 (200–800 �g/mL)for 6 h. HL-60 cells (0.25 × 106) were stained with Annexin V-FITC (BD Biosciences, SanJose, CA) and analyzed using a FACSCaliber Flow Cytometry (Benton Dickenson, FranklinLakes, NJ). Acquired data were evaluated using CellQuest program (BD Biosciences). TF5induced a concentration-responsive increase in the appearance of apoptotic cells.

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FIGURE 3. RP-HPLC fraction 6 CE analysis. A P/ACETM MDQ Series CapillaryElectrophoresis System (Beckman Coulter) was used to compare fraction 6 behavior onCE to T�1 and T�4. The retention differences and peak shape identify fraction 6 as ahomogeneous fraction that is distinct from both T�1 and T�4.

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We next examined the effects of TF5 on randomly cycling HL-60 cells in anAnnexin V and propidium iodine (PI) assay using flow cytometry. In contrast tothe lack of cytological features of apoptosis in U937 cells, TF5-treated HL-60cells had a dramatic increase in apoptosis as determined by positive AnnexinV staining (FIG. 2). Thus, TF5 may exhibit either measurable cytostatic orapoptotic effects in vitro depending on the target human leukemia cell line.

Isolation and Chemical Characterization of the Active Thymosin Principle

As noted above, TF5 and FPLC subfraction 2 prevented HL-60 prolifera-tion in the MTT assay.15 We have continued the isolation of a novel cytostaticpeptide from subfraction 2. We first examined FPLC subfraction 2 on SDS-PAGE (data not shown). The apparent molecular weight of the major bandrevealed by silver staining was ∼6000 Da. Subfraction 2 was then subjectedto reversed-phase high-performance liquid chromatography (RP-HPLC), re-sulting in a homogeneous absorption peak with antiproliferative activity forHL-60 cells (i.e., fraction 6). We injected fraction 6 onto a Beckman Coul-ter (Fullerton, CA) high-performance capillary electrophoresis (HPCE) andcompared it to T�1 and T�4. As shown in FIGURE 3, the fraction 6 elutiontime is different from those of T�1 or T�4. In addition, RP-HPLC fraction6 exhibits a single peak in HPCE, suggesting apparent homogeneity. Thus,we are currently in the final stages of characterization of this novel thymosinantiproliferative peptide.

NEOPLASIA AND IMMUNE SURVEILLANCE

Our studies have identified an approximately 6000 Da thymic peptide in TF5,which prevents HL-60 cell proliferation through the induction of apoptosis. Wesuggest that this new thymosin peptide may participate in an immune surveil-lance mechanism of neoplastic suppression. This novel biological property ofthe thymosin peptide family may be useful in a combinatorial immunothera-peutic treatment strategy, resulting in the suppression of proliferation of humanmyelogenous neoplasms.

ACKNOWLEDGMENTS

These studies were supported by an NIH grant 1R15NS051198-01A toB.L.S., the INBRE Flow Cytometry Core Lab at UNLV, and a gift fund fromTransdermal Biotechnology to M.B. The authors thank Shirley Shen for herexcellent technical support of the Flow Cytometer.

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