Induction of apoptosis in mouse thymocytes by microcolin A and its synthetic analog

Life Sciences, Vol. 64, No. 12, pp. 1013-1028, 1999 Copyright 0 1999 Ekevicr Science Inc. Printed in the USA. All rights memd OO24-3205/99/S--see front matter

PI1 SOO24-3205(99)00028~

ELSEVIER

INDUCTION OF APOPTOSIS IN MOUSE THYMOCYTES BY MICROCOLIN A AND ITS SYNTHETIC

ANALOG

Ling-Hua Zhang and Ross E. Longley *

Division of Biomedical Marine Research, Harbor Branch Oceanographic 5600 US 1 N., Fort Pierce, FL 34946, USA

(Received

in final form December

Institution,

Inc.

7, 198)

Summary

Microcohn A (Mic-1), a marine-derived compound, has been shown to be a novel antiproliferative and immunosuppressive agent. We investigated the ability of Mm-1 and its chemosynthetic analog, microcolin A3 (Mic3), to induce apoptosis in murine thymocytes. Following incubation of the cells with Mic-1 (lo-100 nM) or Mic-3 (1 O-100 nM), intemucleosomal DNA tkgmentation in apoptotic cells was detected by agarose gel electrophoresis and the diphenylamine (DPA) assay; the presence of hypodiploid nuclei assessed by propidium iodide (PI) staining; and the percentages of apoptotic and necrotic cells quantified by morphological observation and fluorescein labeled annex&V binding. Our results show that both Mic-1 and Mic-3 am potent inducers of apoptosis in thymocytes depending on drug concentration and time of exposure, with Mic-3 being more potent than Mic-1 in the induction of apoptosis. Furthermore, flow cytometric analysis using monoclonal antibodies specific to thymocyte subpopulations showed that the proportion of the early immature CD4+CDs+ T-cell subpopulation in thymocytes was selectively decreased by both agents with a corresponding increase of other subpopulations, indicating that CD;CDs+ T cells are the most likely targets of Mic-1 and Mic-3. These in vitro results suggest that the antiproliferative and immunosuppressive properties of both compounds are possibly associated with apoptosis-inducing events and imply that they may have additional potential value as antineoplastic agents. Key Words:

apoptosis,

microcolin

l To whom correspondence e-mail: [email protected]

A, microcolin

A3, mouse,

should be addressed.

Phone:

thymocytes

(561) 465.2400,

extension

486; Fax: (561) 465.1523;

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Considerable experimental evidence has demonstrated that apoptosis is particularly important for the physiological development and function of the immune system (1). Apoptosis appears to play a pivotal role during the course of thymic development, when selection events occur in the thymus to eliminate autoreactive T cells and to retain T-cells which are reactive to foreign antigens (2). Murphy, ef al. (3) and MacDonald, et al. (4) reported that intrathymic clonal deletion can be achieved via apoptosis, affecting either immature / intermediate (CDd’CDs+) or mature (CD.++CDs) thymocytes. Only thymocytes that fail to recognize self antigens are allowed to proceed in their development; the rest undergo apoptosis and are destroyed. Experimentally, thymic apoptosis not only can be induced by various treatments such as glucocorticoids (5), irradiation (6), lipopolysaccharide (7), heat shock (8), HIV (9), nitric oxide (lo), or antimetabolites like arabinosylcytosine and analogs (1 l), but also can be induced by immunomodulators such as cyclosporine A (12, 13), FKS06 (14, 15), linomide (16) and the monoclonal antibody OKT3 (17). There is accumulating evidence that the efficacy of immunomodulators is possibly related to the ability of the immune cells to respond to these agents by apoptosis. In this paper, we investigate the in vitro effects of microcolin A on thymocyte apoptosis. Microcolin A (Mic-1), a marine-derived lipopeptide originally isolated by our group from the marine blue gteen alga, Lyngbyu majusculu, has been shown to have in vitro antiproliferative and immunosuppressive activities more potent than the widely-used immunosuppressant cyclosporine A. It has a median concentration (100) ranging from 2 to 50 r&l for inhibition of murine T and B cell proliferation, and acts in a reversible and dose-dependent manner (18). The compound also activates macrophages by synergizing with IFN-y to induce the production of nitric oxide, the expression of inducible nitric oxide synthase (iNOS, EC1.14.13.39) and the killing of tumor target cells in mouse peritoneal macrophages and macrophage-like RAW264.7 cells (Zhang, ef al, submitted). To further understand the mechanism of action of Mic-1 and to evaluate a potent microcolin analog, we investigated the comparative effects of Mic-1 and its semisynthetic analog microcolin A3 (Mic-3) (Fig.1) on the induction of apoptosis in murine primary thymocytes, using the apoptosis-inducing glucocorticoid, dexamethasone (Dex), as a reference standard. In addition, to clarify the potential specificity of apoptosis induction in thymocytes, the effects of Mic-1 and Mic-3 incubation on the percentages of T cell subpopulations of murine thymocytes were also examined.

Materials and Methods

Reagents RPMI-1640 tissue culture medium (TCM) supplemented with I-glutamine, fetal calf serum (FCS), and non-essential amino acids, 1 kb DNA ladder were obtained from GIBCO Co.(Grand Island, NY). Diphenylamine (DPA), ribonuclease A (RNase, type III-A), acridine orange, ethidium bromide and propidium iodide (PI) were purchased from Sigma Chemical Co. (St. Louis, MO). Amrexin V Apoptosis Detection Kits were kindly supplied by R&D Systems Inc. (Minneapolis, MN). R-phycoerythrin (PE) conjugated rat anti-mouse CD4 monoclonal antibody (MAb) and fluorescein isothiocyanate (FITC) conjugated rat anti-mouse CDs MAb were products of PharMingen Co. (San Diego, CA).

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Drugs Microcolin A (M&l), mol. wt. 747, was isolated from frozen samples of Lyngbyu majuscula according to the previously published method (19). Its synthetic analog microcolin A3 (Mic-3) was kindly provided by Dr. Carl P. Decicco, DuPont Merck, Inc. (Wilmington, DE). Stock solutions (lmg/ml)

of both compounds

were prepared

in absolute

ethanol and stored at -70°C.

Working

solution were prepared by diluting stock solutions with fully supplemented tissue culture medium (TCM) to give the required test concentration. The final concentration of ethanol in the vehicle control was l/20,000 (v/v), the highest concentration in test groups. Dexamethasone (Dex) was purchased from Sigma Chemical Co.

microcolin

microcolin

A (Mic-1).

C39H6509NJ, mol. wt. 747

A3 (Mic-3).

Cj9HsJ09NS, mol. wt. 747

Fig. 1 Chemical structures of microcolin A (Mic-I)

and its synthetic

analog microcolin

A3 (Mic_-3)

Animais Pathogen-free male &BL/6 inbred mice, 4 to 8 weeks old, were purchased from Harlan Sprague Dawley Inc. (Indianapolis, IN). They were housed in sterile cages with sterile bedding in wellventilated rooms at room temperature and fed a standard diet and sterilized water ad libitum.

Preparation of murine thymocytes and cell culture suspensions were prepared as described elsewhere (20). Briefly, several mice were sacrificed by cervical dislocation, thymuses excised, and single cell suspensions prepared by grinding the thymuses with the plastic end of a sterile plunger of a 10 ml syringe against a nylon mesh in 10 ml of cold 10% FCS RPMI- 1640 TCM. Debris was removed with a 70 pm Falcon cell Thymocyte

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Microcolin A Induces Apoptosis

strainer (Becton-Dickinson,

NY) and the cells were recovered by centrifugation

Vol. 64, No. 12, 1999

at 200 x g. RBC

were lysed with 0.89% buffered NH&l. Cell viability was confirmed to be at least 95% by trypan blue dye exclusion test and the suspensions were adjusted to give the required number of viable thymocytes / ml. Thymocytes were inoculated in 24-well flat-bottom cell culture plates and incubated for indicated periods at 37 “C in a humidiied

incubator in an atmosphere

of 5% CO* in

the absence or presence of test agents. Analysis of internucleosomal

DNA pagmentation

For DNA gel electrophoresis, treated cells were harvested by centrimgation at 200 x g for 10 min, then lysed in ice-cold TTE buffer (10 mM Tris, pH 8.0, 1 mM EDTA and 0.2% Triton-100). The lysate was centrifuged for 10 min at 13,000 x g at 4°C to separate the fragmented DNA (soluble) from intact chromatin (pellet). The supematant was treated with 0.1 ml cold NaCl(5M) and 0.7 ml cold isopropanol, was centrifuged

vortexed and then kept at -20°C overnight to precipitate

DNA. The precipitate

and the pellet was washed with 70% cold ethanol, air dried and dissolved

in 36 ~1

TE buffer (10 mM Tris, 1 mM EDTA, pH8.0). Approximately 10 pg DNA was loaded into the wells of a 1% agarose gel. Electrophoresis was carried out in TBE buffer (2 mM EDTA, 89 mM boric acid, 89 mM Tris, pH8.4) and the gel stained by ethidium bromide. DNA-intercalated ethidium fluorescence was visualized on a UV (302 nm) transilluminator and photographed on Polaroid film 667 (PM) (Sigma Co.) using an orange wratten B filter. Subsequently, the resulting negative was developed for the DNA profile. For the DPA assay, the fractions of the fragmented DNA and the intact chromatin were separated as described above, precipitated overnight in 25% TCA, pelleted at 13,000 x g for 10 min at 4OC, and hydrolyzed

in 80 ~15% TCA at 90°C for 15 min. After 4 hr incubation

with 160 ~1 of DPA

reagent (0.088 M DPA, 98% VN glacial acetic acid, 1.5 VN sulfuric acid, and 0.5% VN of 1.6% paraldehyde) at 37°C the amount of DNA in each sample was quantified from its absorbance at 570 nm in a plate reader. Results are reported as amount of fragmented DNA divided by total DNA, and expressed as a percentage. Flow cytometric analysis of hypodiploid nuclei Cytofluorometric analysis of apoptosis was performed by PI staining of nuclei as reported by Nicoletti et al. (21). Briefly, following incubation of thymocytes with test agents for various intervals, the 200 x g cell pellet was fixed in 2 ml cold absolute ethanol at 4°C for 1 h, and then washed twice with cold PBS. 200 l.tl RNase (1 mg /ml in PBS), 40 ul PI (2.5 mg/rnl in PBS) and 1.76 ml PBS was added to the pelleted cells and the cell suspensions incubated in the dark for 15 min and kept at 4°C until analyses. The PI fluorescence of individual nuclei was determined using an EPICS Elite Flow Cytometer (Coulter Co., Hialeah, FL) with 488 nm excitation and measurement of emission at 675 nm. The percentage of apoptotic nuclei was determined on the basis of the proportion of cells comprising the sub-diploid DNA peak in the DNA fluorescence histogram. The Multicycle program (Phoenix Flow System, San Diego, CA) was used for the analysis of cell cycle distribution.

Microcolin

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FITC-Annexin/PI

1017

double staining

Double staining for FITC-Annexin the protocol

A Induces Apoptosis

of the manufacturer.

V binding and for DNA using PI was performed Briefly,

after washing

twice with PBS,

according to

1~10~ cells were

resuspended in binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCI, 2.5 mM CaQ). FITC-Annexin V was added to a final concentration of 1 mg/ml. PI (10 mg/ml in binding buffer) was added to produce a fmal concentration of 1 mg PI/ml cell suspension. The mixture was incubated for 15 min in the dark at room temperature and then measured by an EPICS Elite Flow Cytometer, using 488 run excitation and measurement of emission at 525 nm (for FITC) and 675 nm (for PI). Morphological determination of apoptotic nuclei Briefly, 25 ~1 aliquots of 4~10~ cells/ml were mixed with 1 ~1 of PBS containing acridine orange for determination

of nuclear morphology

100 l.@nl of

plus 100 pg/ml of ethidium bromide to

assess viability, and then observed under an epifluorescence microscope using fluorescein filters (22). Apoptosis was expressed as a percentage calculated from the number of cells with apoptotic nuclear morphology divided by the total number of cells examined. For each sample, at least 100 cells were scored. Characterization

of thymocyte subpopulations

The treated thymocytes were harvested and then washed with cold PBS containing 1% FCS, resuspended in 0.1 ml of diluted FITC-conjugated MAb to mouse CD4 and PE-conjugated MAb to mouse CDs. After 30 min incubation at 4°C in the dark, cells were washed three times and resuspended with 0.5 ml ice-cold 1% FCS-PBS. Cell fluorescence was analyzed by flow cytometry with excitation set at 488 run. Unstained thymocytes, treated similarly, were used as autofluorescence

controls. Data were collected on 1~10~ cells.

Statistical analysis The results are expressed as mean f S.D. Comparisons are made with the appropriate controls employing a two-tail Student’s t-test. Differences were considered significant at P c 0.05. Results Effect of MC-1 and MC-3 on DNA j?agmentation The major biochemical halhnark of apoptotic cell death is the cleavage of chromosomal DNA at intemucleosomal sites into fragments or multiples of 180-200 base pairs (23). To test whether Mic- 1 and Mic-3 induced DNA cleavage in mouse thymocytes, equal numbers of thymocytes were incubated at 37°C for 4 h in the presence of medium alone, Dex, Mic-I or Mic-3. The low mol. wt DNA fragments were isolated from thymocytes and assayed by gel electrophoresis. As shown in Fig. 2, ethidium bromide staining of the nucleosomal DNA ladder which is characteristic of apoptosis, is much more intense in the lane containing low mol. wt DNA from thymocytes continually exposed for 4 h to Dex (100 nM), Mic-1 (100 nMj or Mic-3 (50, 100 r&l) than in the control lane containing low mol. w-t DNA isolated from an equal number of untreated thymocytes

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(lanes 2, 5, 7, 8 vs. lane 1). These results strongly suggested that these test agents induced an increased DNA kagrnentation in thymocytes. The fact that thymocytes are known to undergo a certain amount of spontaneous apoptosis in the thymus and in vitro cultures (3) accounts for the presence of the diiy stained nucleosomal-sized DNA fkagments in the control (lane 1). By contrast, DNA isolated from thymocytes which were treated with Mic-1 (10 or 50 nM) or Mic-3 (10 nM) for 4 h displayed faintly detectable ladder patterns which appeared only slightly more intense than that of untreated control cells (lanes 3, 4, 6 vs. lane 1). From these experiments, we conclude that Mic-1 and Mic3 induced significant thymocyte apoptosis only at higher concentrations and that Mic-3 appeared more potent than Mic-1 .

Ml2345678 Fig. 2. Agarose gel electrophoresis

of nuclear

DNA fragments of the hypodiploid thymocytes, showing the ladder pattern characteristic of apoptosis. Mouse thymocytes were incubated in presence or absence of test agents for 4 h. DNA in the eight lanes was purified from cells treated as follows: Lane 1, medium control (untreated cells); Lane 2, Dex (100 nM); Lane 3-5, Mic- 1 (10, 50, 100 nM); Lane 6-8: Mic-3 (IO, 50, 100 nM). Lane M contains the Ikb marker DNA ladder.

To confirm the induction of thymocyte apoptosis by Mic-1 and Mic-3, intact chromosome-length DNA and fkgmented DNA were quantified using a DPA calorimetric assay. As depicted in Fig. 3a, DNA fragmentation in thymocytes incubated for 4 h was induced by Mic-1 and Mic-3 in the concentration range of 10 to 100 nM in a dose-dependent manner. An 18% increase in DNA fragmentation (relative to the medium control) was induced by Mic-1 at 100 nh$ whereas a 32% increase in DNA fragmentation was induced by Mic3 at 100 nM. The results from the time kinetic study shown in Fig. 3b demonstrate that DNA fragmentation in untreated control thymocytes gradually increased in a time-dependent manner when cells were incubated in vifro. Exposure to a concentration (50 nM) of Dex, Mic-1 or Mic-3 for a period from 2 to 8 h, significantly increased the content of fragmented DNA in apoptotic thymocytes. In agreement with the results of the gel electrophoresis study, the results of the DPA assay confirmed that Mic-3 is more potent than Mic- 1 in induction of thymocyte apoptosis.

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1019

60

a

10 -

0-I

0

I

1

a

I

I

20

40

60

60

100

Concentration (nM) 100 -

80 -

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Mic-1 50 nM

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a9 20 b 0’

I 0

I 2

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, 6

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incubation time (h) Fig. 3. Dose-dependent and time-kinetic effects of Mic-1 and Mic-3 on percentage of DNA fragmentation. Thymocyks were incubated in the presence OTabsence of test agents at concentrations ranging from 10 to 100 nM for 4 h (a) or 50 nM of test agents for 0 - 8 h (b) at 37°C in complete RPMI-1640 medium DNA fragmentation was determined using the DPA assay. Each of these results is representative of three imkpencknt experiments. The data shown are the mean k S.D. of triplicate measurements.

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Effect of Mic-1 and Mic-3 on the percentage of hypodiploid nuclei in thymocytes To further quantify the apoptotic activity of Mic-1 and Mic-3, the number of apoptotic thymocytes was determined by measuring the percentages of hypodiploid nuclei using PI staining and flow cytometric methods. As shown in Fig. 4a and 4b, the DNA histogram of untreated thymocytes following a 4 h incubation reveals one DNA peak, of a high, narrow, diploid form (relative nuclei number: GdGr 69.0%, S 7.6% and Glh4 9X%), and a low hypodiploid apoptotic peak (Ap: 10.6%) corresponding to naturally apoptotic cell nuclei. Following a 4 h incubation of thymocytes with Mic-1, or Mic-3, however, a measurable increase in the percentage of hypodiploid nuclei was observed which was dose-dependent and did not result in a significant change in the cell cycle distribution of these cells (Mic- 1 100 nM-treated cells: hypodiploid nuclei 23.1%, GdG, 59.7%, S 6.2% and Gz/M 7.4%; Mic-3 100 nM -treated cells: hypodiploid nuclei 27.6%, GdG, 53.6%, S 10.3% and G$M 9.2%). Dex (100 nM) induced a dramatic increase in the percentage of hypodiploid nuclei (Ap: 45.4%) which was associated with a significant change in cell cycle distribution characterized by losses of GdGl phase cells (40.3 %) and G2IM phase cells (5.1%). The effects of Mic- 1, Mic-3 and Dex were even more pronounced after 20 h incubation, with all three agents inducing a marked increase in the percentages of hypodiploid nuclei (Data not shown). These observations suggest that untreated thymocytes are able to undergo a spontaneous apoptosis in vitro, which is accelerated by Mic-1 and Mic-3. Moreover, Mic-3 appeared to be slightly more potent than Mic-1, and similar effects were observed for both compounds and were concentration related. However, neither Mic-1 nor Mic-3 was as potent as the control positive DeX.

Eflect of Mic-1 and Mic-3 on the percentage of apoptotic and necrotic cells in thymocytes Fluorescein labeled annexin V binding in conjunction with the PI dye exclusion test was also used to discriminate intact cells (FITC/PI-), apoptotic cells (FITC‘IPI-) and necrotic cells (FITC’LPI’) in thymocytes (24). Fig. 5a shows the results of bivariate FITC-Annexin V/PI flow cytometric analysis of thymocytes. Exposure of freshly isolated thymocytes to Mic- 1 (lo- 100 r&l), Mic-3 (IO-100 nM) and Dex (100 nM) for 4 h generally resulted in a significant increase in the percentage of apoptotic cells in a concentration-dependent manner compared to the medium control. This change of the percentage of apoptotic cells was accompanied by a significant reduction (P < 0.05) in the percentage of intact cells in a concentration-dependent manner. However, no significant change in the percentage of necrotic cells (~10%; P > 0.05, compared to untreated control cells ) was observed, indicating that the cell viability of thymocytes treated for 4 hours with these doses of test agents was not significantly reduced (P > 0.05). In addition, thymocyte control cultures also underwent spontaneous apoptosis: as shown in Fig. 5a, 17.1% of control thymocytes in this assay were apoptotic when incubated for 4 h. These apoptotic-inducing effects of Mic-1 and Mic-3 were also confirmed by morphological observations. Those changes, characteristic of apoptosis, were visualized by staining cell nuclei with a&dine orange and ethidium bromide. As shown in Fig. 5b, unlike normal nuclei, which are large and show mixed light and dark areas due to variable condensation of chromatin, apoptotic nuclei consist of one or more small featureless beads: dead or necrotic cells are distinguished by their orange-red fluorescence. Fig. 5c demonstrated that bc& IMic- 1 and Mic-3 induced significant

Microcolin A Induces Apoptosis

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o@w

T

~1(40*)

Control

1021

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400

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Fig. 4. Effects of Mic-1. Mic-3 and Dex on percentage of apqtotic nuclei and cell cycle distribution of mouse thymocytm. Cells were incubated in the presence or absence of test agents at indicated concentrations for 4 h at 37% Cell cycle was analyzed by PI staining and flow cytometry. The cell cycle distribution shown (a) is representative of three independent experiments. ‘Re data of apoptotic nuclei shown (b) are the mean f S.D. of triplicate measurements.

1022

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90 80 70 E!!

80-

d

50 -

= (j

4030 20 10 -

A Induces

Apoptosis

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60

g

1023

7

40

v) = 8

.g

30

0 8 $

20

10

+ 0

r

0

~

1

20

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-1

40

80

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100

Concentration (rhl)

Fig. 5. Effects of Mic-1, Mic-3 and Dex on apoptosis induction of mouse thymocytes. Thymocytes were incubated in the presence or absence of test agents at indicated concentrations for 4 h at 37°C in complete RFMI-1640 medium. The apoptotic cell percentages were measured by bivariate FITC-Annexin V/PI flow cytometric analysis (a) and also determined using acridine orange and ethidium bromide staining (b, c). Each of these results is representative of three independent experiments. The data shown are the mean + S.D. of triplicate measurements.

percentages of cells with apoptotic morphology in a dose-dependent manner (P < 0.05, compared to untreated control cells). Increases of approximately 16%, 23%, and 33% of apoptotic cells (relative to untreated control cells, which were -10% apoptotic) were observed when cells were treated with a same concentration (100 nM) of Mic-1 ; Mic3 and Dex, respectively. Effect of MC-1 and Mic-3 on T cell subpopulations

in thymocytes

To determine which thymocyte populations are the targets of the in vitro apoptosis-inducing effects of Mic-1 and Mic-3, we studied the number of CDdCDs-, CDd+CDs+, CD4’CDsS and CD4CDs+ cells following the thymocyte treatment with Mic-1 (10, 50 and 100 t&I), Mic-3 (10, 50 and 100 nM) or Dex (100 nM) for 20 h. Fig. 6 show the results of the flow cytometric analysis of double-labeled (FITC-anti-CD4 and PE-anti-CDs) thymocytes after exposure to test agents for 20 h. Mic-I and Mic-3 treatment led to a dose-dependent decrease in the percentage of CD4’CDs’ cells associated with corresponding increases of other T-cell subpopulations in thymocytes. A substantial increase in the ratio of CDd+/CDs’ cells was also observed in the treated cells (data not

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Microcolin A Induces Apoptosis

shown).

Dex-treatment

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in a more substantial reduction of CD4’CDsc T-cell compared to treatment with Mic-1 or Mic-3. Mic-3 was more

resulted

subpopulations in thymocytes effective than Mic-1 in modifying

the relative number of T-cell subpopulations.

1.c

0.8 .-

0 5

0.6

!

2

=

0.4

6 0.2

0

Fig. 6. Cell number distribution

of T-cell

marker flow cytometry phenotype absence of test agents at indicated medium. Approximately 1 x lo4 representative of three independent

in mouse thymocytes determined by double analysis. Thymocytes were incubated in the presence or concentrations for 20 h at 37°C in complete RPMI- 1640 cells were collected for phenotype analysis. The result is experiments.

subpopulations

Discussion Apoptosis, a preprogrammed cellular ‘suicide’ achieved by activating endogenous nucleases that cause DNA fragmentation, has been recognized as an important mechanism to achieve a balanced expansion and elimination of immune effector cells (23). Many more thymocytes are produced than are needed as mature T cells. The bulk of the evidence obtained to date suggests that cells which fail the selection process are eliminated intrathymically (25). Both undifferentiated and mature T-lymphocytes undergo apoptosis under many physiologic and experimental conditions. Importantly, failure of apoptosis may result in autoimmunity as illustrated by the Zpr gene mutation which leads to deficient expression of the cell surface APO- 1/Fas antigen in several mouse strains (26,27). In the animal model, experimental autoimmune encephalomyelitis, apoptosis of Tcells terminates inflammation within the central nervous system (28). There is also evidence

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102.5

suggesting tight associations between disturbed cell death programs and systemic human autoimmune disorders. Some evidence in animals and humans indicates that extended survival of autoreactive cells is implicated in at least two chronic autoimmune syndromes: systemic lupus erythematosus and rheumatoid arthritis (29). The efficacy of many immunotherapeutic agents has been proposed to be related to the ability of the immune cells to respond to these agents by apoptosis. In this paper, we sought to understand the possible mechanism of a novel antiproliferative and immunosuppressive agent microcolin A (Mic-1) and its chemosynthetic analog microcolin A3 (Mic-3). Several observations have been made concerning their effects on murine thymocytes by the induction of apoptosis. Since Dex in vitro has long been used as a model for endogenous glucocorticoid-mediated thymocyte apoptosis (30), it was used as a reference standard in our experiments. The most commonly identified biochemical characteristic of apoptosis is the induction of doublestranded DNA fragmentation at linker regions between nucleosomes. Agarose gel electrophoresis displays the intemucleosomal DNA fragments Tom apoptotic cells in a characteristically ordered, ladder-like pattern, whereas DNA cleavage in necrotic cells is random and is seen as a DNA “smear” (3 I). Our elecizophoresis results of the nuclear DNA present in the cytoplasm of mouse thymus cells exposed to Mic- 1 or Mic-3 for 4 h revealed detectable oligo-nucleosomal fragments of DNA. These initial results qualitatively indicated that the two compounds appear to be potent apoptosis inducing agents and Mic-3 is found to be more potent than Mic-1 in this activity. In addition, the DPA assay was utilized to investigate apoptotic effects of Mic-1 and Mic-3. These quantitative results demonstrated that both Mic-1 and Mic-3 induced thymocyte apoptosis in a dose- and time-dependent manner. Flow cytometric analysis of nuclear DNA from drug-treated thymocytes, showed that the percentage of hypodiploid nuclei, or fragments of apoptotic nuclei, increased with the incubating dose of Mic-1 or Mic-3 ranging from 10 to 100 nM. As observed with gel electrophoresis and the DPA assay, Mic-3 appeared more potent than Mic-I, but not as potent as Dex in inducing hypodiploid nuclei at the same concentration. Using fluorescein labeled annexin V binding in conjunction with the PI exclusion test, we were able to discriminate intact cells, apoptotic cells and necrotic cells in thymocytes. Unfixed, viable cells exclude PI while those undergoing apoptosis transport phosphatidylserine from the inside membrane surface to exposure to the outside which can then bind to FITC labeled annex&V. Thus, cells which are undergoing apoptosis (FITC+) but whose membrane integrity is still intact (PI‘) can readily be distinguished from those cells undergoing necrosis (FITC’/PI+) using two color immunofluorescence (24, 32). Our results showed that thymocytes which were continually exposed to Mic- 1 or Mic-3, underwent an appreciable amount of apoptosis associated with a dosedependent reduction of intact cells, while the percentage of necrotic cells (or cell viability) remained relatively unchanged. Thymocytes which were treated for 4 h showed a significant increase of apoptotic cells when compared to untreated cells. More apoptotic cells were induced when treated with Mic-3 than with Mic-1 . When thymocytes were exposed to these agents for 24 h, Mic- 1 or Mic-3 treatment resulted in a high percentage of apoptotic cells similar to that obtained with Dextreatment, accompanying a significant reduction of cell viability (Data not shown). In addition. our

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morphological observation of thymocytes stained with acridiie orange and ethidium further confirmed the dose-dependent apoptotic effects of both compounds.

bromide

Moreover, consistent with prior results (12, 13, 30), our present study showed that untreated primary thymocytes incubated for 4 h underwent spontaneous apoptosis (lo-17% apoptotic cells) and displayed significant apoptotic characteristics: an appreciable percentage of hypodiploid nuclei (-10%) and a detectable ladder-like nucleosomal pattern of fragmented DNA, suggesting that the untreated thymocytes incubated in vilro have a strong propensity to proceed to apoptotic death. Previous studies have shown that clonal deletion in the thymuses can be achieved via the apoptotic death of cells with either CDb’CDs+ or CDd’CDs- surface phenotype (3, 4). Loss of cell volume, increase in cell density, membrane blebbing, nuclear collapse concomitant with chromatin damage, and down-regulation of CD4 and CDs molecules are characteristic features of thymocytes undergoing apoptosis (33). Our results show that the percentage of CDd+CDs+ cells was substantially decreased in Mic-1 or Mic-3 treated thymocytes, while other thymocyte subpopulations were relatively increased. Our data suggest that the immunosuppressive agents Mic- 1 and Mic-3 may delete the immature CDd’CDs+ thymocytes via apoptosis. This capacity of these compounds may be associated, at least in part, with their other immunologic effects. In addition, the efficacy of many antineoplastic chemotherapeutic agents is related to. the induction of apoptosis of the target tumor cells (34, 35). Anti-apoptosis has been proposed as a fundamental mechanism of action of many tumor promoters (36). Overexpression of anti-apoptotic genes and their encoded proteins (such as bcl-2, etc.) occurs frequently in human cancers and contributes to tumor chemoresistance by blocking apoptosis (37). Thus, pro-apoptotic effects of Mic-1, Mic-3 have been extended to some cancer cells, such as mouse T- hybridoma DO 11.10 cells and human GI-101 A breast carcinoma cells (data not shown). These properties imply that both compounds may have potential value as antineoplastic agents. Additional studies of these compounds will be required to investigate the relationship between their pro-apoptotic, immunosuppressive and potential antitumor activity. In conclusion, our present findings have demonstrated a dose- and time-dependent inducing effect of Mic-1 and its analog, Mic-3, on apoptosis of mouse thymocytes. Mic-3 appeared to have greater apoptosis-inducing effects compared to its natural product counterpart Mic-I . These results suggest that antiproliferative and immunosuppressive properties of both compounds are mediated by novel mechanisms worthy of further investigation. The molecular mechanism of the observed in vitro apoptosis-inducing effects of Mic-1 and the greater activity of the synthetic analog, Mic3, as well as whether these compounds elicit similar responses in thymocytes when treated in vivo, remain to be elucidated.

Acknowledgments We thank Ms. Pat Linley for her critical review of the manuscript. L.-H. Z. is a postdoctoral fellow supported by the Cancer Research Institute of New York. This is Harbor Branch Oceanographic Institution Contribution No. 1268.

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