Exp. Eye Res. (1998) 66, 521–529

Apoptosis and Morphologic Changes in Drug-Treated Trabecular

Meshwork Cells In Vitro

SANTIAGO ANTONIO B. SIBAYANa*, MARK A. LATINAa, MARGARET E. SHERWOODa,

THOMAS J. FLOTTEa KRISTIN WHITEb

a Wellman Laboratories of Photomedicine and b Cutaneous Biology Research Center, Department

of Dermatology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, U.S.A.

(Received Columbia 10 September 1997 and accepted in revised form 11 November 1997)

Using an in vitro culture system, we investigated whether bovine trabecular meshwork cells undergoapoptosis (programmed cell death) following exposure to anti-glaucoma medications (timolol, pilocarpineand epinephrine) and known inducers of apoptosis (5-fluorouracil, mitomycin-C and dexamethasone).Third to fifth passage bovine trabecular meshwork cells were grown to confluence and incubated for 1–12days in growth media with timolol (1–1000 µ), pilocarpine (15–15000 µ), epinephrine (5–5000 µ),5-fluorouracil (10–100 µg ml−"), mitomycin-C (0±01–100 µg ml−") and dexamethasone (0±01–100 µ).The cultures were evaluated for apoptosis by phase-contrast microscopy, transmission electronmicroscopy and in situ apoptosis labeling. 5-Fluorouracil (10–100 µg ml−"), mitomycin-C (0±1–100 µgml−") and epinephrine (500–5000 µ) induced apoptosis in a dose and time-dependent manner. Timolol,pilocarpine, and dexamethasone-treated specimens did not show evidence of apoptosis at any of theconcentrations tested. Trabecular meshwork cells incubated in timolol (100–1000 µ) developedcytoplasmic granules, and specimens treated with pilocarpine (15000 µ) developed cytoplasmicvacuoles. These granules and vacuoles have the appearance of secondary lysosomes. Dexamethasone-treated cells developed an increased number of mitochondria. This study suggests that the trabecularmeshwork may undergo apoptosis following exposure to 5-fluorouracil, mitomycin-C and epinephrine.Timolol, pilocarpine and dexamethasone did not induce apoptosis. However, these drugs can incitecharacteristic morphologic changes in cultured trabecular meshwork cells.

# 1998 Academic Press LimitedKey words : apoptosis ; dexamethasone; epinephrine; 5-fluorouracil ; mitomycin-C; pilocarpine; timolol ;

trabecular meshwork.

1. Introduction

Apoptosis (programmed cell death) is a gene-regulated

process that plays an important role in the normal

physiologic turnover of cells and in various patho-

logical processes. It may occur spontaneously or in

response to physiologic stimuli. Apoptosis may also be

induced by exogenous toxic agents (Kerr, Wyllie and

Currie, 1972; Duvall and Wyllie, 1986; Kerr et al.,

1987; Schwartzman and Cidlowski, 1993).

Characteristic morphologic and biochemical

features distinguish apoptosis from necrosis. Mor-

phologic changes in necrosis include cell swelling,

degradation of organelles, rupture of cell membranes,

and spillage of cellular contents into the extracellular

space, resulting in an inflammatory response (Duvall

and Wyllie, 1986; Kerr et al., 1987; Schwartzman

and Cidlowski, 1993). In contrast, apoptosis is

characterized by condensation and margination of

chromatin, structural disorganization of the nucleus,

and formation of cell fragments or ‘apoptotic bodies ’.

Apoptotic bodies maintain intact organelles and cell

membranes, and are engulfed by neighboring phago-

cytic cells, thus no inflammatory response is elicited

* For correspondence at : Medico Building, Room 401, SanMiguel Avenue, 1600 Pasig City, Philippines.

(Kerr et al., 1972, 1987; Duvall and Wyllie, 1986;

Schwartzman and Cidlowski, 1993). Some apoptotic

bodies, however, may escape phagocytosis and under-

go swelling and degradation of intracellular content, a

process referred to as ‘secondary degeneration’ (Searle

et al., 1975; Don et al., 1977; Duvall and Wyllie,

1986; Kerr et al., 1987; Schwartzman and Cidlowski,

1993). Biochemical changes in apoptosis are charac-

terized by enzymatic cleavage of DNA by endogenous

endonuclease, often into multiples of 180–200 base

pairs (Wyllie, 1980; Duvall and Wyllie, 1986;

Schwartzman and Cidlowski, 1993). In necrosis, DNA

is cleaved into segments of random length (Afanas’ev

et al., 1986).

Trabecular meshwork (TM) cells perform a variety

of activities in maintaining the normal function of the

aqueous outflow pathway. It has been reported that

loss of TM cells occurs with age. Such loss is greater

in eyes with open-angle glaucoma, as compared

to non-glaucomatous age-matched eyes (Alvarado,

Murphy and Juster, 1984). It has also been proposed

that chronic administration of anti-glaucoma medi-

cations may promote loss and further damage of

already diseased TM cells (Watson and Grierson,

1981; Grierson et al., 1982). Whether apoptosis plays

a role in the loss of TM cells has thus far not been

investigated.

0014–4835}98}050521­09 $25.00}0}ey970458 # 1998 Academic Press Limited

522 S. A. B. SIBAYAN ET AL.

In this study, we investigated whether TM cells can

undergo apoptosis in vitro following exposure to anti-

glaucoma medications (timolol, pilocarpine and epi-

nephrine) and known inducers of apoptosis [5-

fluorouracil (5-FU) (Barry, Behnke and Eastman,

1990; Lowe et al., 1993), mitomycin-C (MMC) (Searle

et al., 1975; Ohmori et al., 1993; Rey, Scott and

Mu$ ller, 1994) and dexamethasone (Cohen and Duke,

1984; Compton and Cidlowski, 1986; Kawabori et al.,

1991; Hassan et al., 1996; Zysk et al., 1996)]. In

addition to being known inducers of apoptosis, 5-FU

and MMC are used adjunctively during glaucoma

filtration surgery (Heuer et al., 1986; Chen et al.,

1990; Palmer, 1991), and dexamethasone is used as

an anti-inflammatory agent.

2. Materials and Methods

Bovine Trabecular Meshwork Cell Cultures

Primary TM cell cultures were prepared from fresh

bovine eyes obtained from a local slaughterhouse.

Trabecular tissue was excised from the eyes within

6 hr of the animal’s death. Dissection was performed

under sterile conditions with the aid of a stereo-

microscope (Typ 355110, Wild Inc., Heerbrugg,

Switzerland). The trabecular tissue was cultured on

35 mm plastic petri dishes (Falcon}Becton-Dickinson,

Franklin Lakes, NJ, U.S.A.) and left undisturbed for 1

week to allow the cells to plate onto the dish. The

growth media consisted of Dulbecco’s Modified Eagle

Medium (D-MEM) with 10% fetal bovine serum, 1%

penicillin-streptomycin, and 1% amphotericin B

(Gibco, Buffalo, NY, U.S.A.). The cultures were

maintained in an atmosphere of 5% carbon dioxide,

95% relative humidity, at a temperature of 37°C. The

cells were subsequently trypsinized and cultured in

tissue culture flasks (Falcon}Becton-Dickinson, Frank-

lin Lakes, NJ, U.S.A.). Cells used for our experiments

were in the third to fifth passages, and were grown to

confluence in 35 mm plastic petri dishes.

Drug Solutions

Bovine TM cell cultures were incubated in the

following drugs (Sigma Chemical Co., St. Louis, MO,

U.S.A.) dissolved in the previously described culture

media at various specified concentrations : 5-FU

(10–100 µg ml−"), MMC (0±01–100 µg ml−"), dexa-

methasone (0±01–100 µ), timolol (1–1000 µ), pilo-

carpine (15–15000 µ) and epinephrine (5–

5000 µ). Cultures treated with 5-FU, MMC, pilo-

carpine and epinephrine were incubated for a maxi-

mum of 7 days, while those treated with timolol and

dexamethasone were incubated for 12 days. Cell

cultures incubated in drug-free media served as

controls. All experiments were performed in quad-

ruplicate.

The drug concentration ranges used in this study

are similar to those used by Kawa et al. (1993) in their

study on the effect of anti-glaucoma medications on

bovine TM cell morphology. The peak drug levels

achieved in the aqueous humor following topical

administration in vivo served as the basis for the

concentration ranges tested. It has been estimated that

30 minutes after applying 0±5% timolol in rabbits, the

aqueous levels reach 1–5 µ (Polansky and Alvarado,

1985; Ellis, Wu and Riegel, 1991). The concentration

of pilocarpine in aqueous humor has been determined

to reach 15–20 µ 30 minutes after administration of

2% or 4% pilocarpine (Ellis, Matsumura and Rendi,

1985; Ellis et al., 1991). One hr after topical appli-

cation of 1% epinephrine eye drops in cynomolgus

monkeys, aqueous levels can reach 40 µ (Kaufman

and Rentzhog, 1981). Ninety to 120 minutes after

administration of 0±1% dexamethasone alcohol, aq-

ueous levels approach 0±1 µ (Watson et al., 1988).

One hour after subconjuctival administration of

12±5 mg 5-FU, levels ranging from 65±7–69±5 µg

ml−" of aqueous humor have been reported (Rootman

et al., 1979; Fantes et al., 1987). MMC can reach

levels of up to 0±2 µg ml−" in the aqueous humor 30

minutes after intraoperative application of a 0±2 mg

0±5 ml−" solution (Kawase et al., 1992; Seah et al.,

1993).

Morphology Studies

TM cell cultures were evaluated daily for mor-

phologic changes by phase contrast microscopy with

the use of an inverted microscope (Model IM 35, Carl

Zeiss, Go$ ttingen, Germany). Changes in morphology

were documented photographically.

For transmission electron microscopy studies, TM

cells were plated on 1 cm glass cover slips in 24-well

cell culture plates and incubated in media as previously

described. Following incubation in the different drug

solutions, specimens were fixed in 4% glutaraldehyde

for 1 hr at room temperature, and transferred to

cacodylate buffer solution. Cells were post-fixed in 2%

osmium tetroxide in 0±1 sodium cacodylate buffer,

pH 7±4, for 1 hr at room temperature. The cell

monolayers on coverslips were processed through

ethanol dehydration, infiltrated with increasing per-

centages of Epon 812:ethanol (Electron Microscopy

Sciences, Fort Washington, PA, U.S.A.) and embedded

with inverted Beem capsules placed directly over the

coverslips. The blocks were sectioned and sub-

sequently examined with a transmission electron

microscope (Model CM-10, Philips Corp., Eindhoven,

The Netherlands).

In Situ Apoptosis Staining

TM cells were processed for in situ labeling with the

TACS 1 Klenow Apoptosis Screening Kit (Trevigen

Inc., Gaithersberg, MD, U.S.A.). This assay is a

histochemical stain that is similar in principle to the

DRUG-INDUCED APOPTOSIS IN TRABECULAR CELLS 523

TdT-mediated dUTP nick end labeling (TUNEL) assay,

except that it labels 5«-overhangs, instead of 3«-OH

ends of DNA fragments that are generated during the

process of apoptosis. These overhangs are end-labeled

with biotinylated nucleotides and are tagged with

streptavidin-horseradish peroxidase, which in turn, is

made to react with diaminobenzidine (DAB), resulting

in a brown precipitate. Non-apoptotic cells have

relatively insignificant amounts of 5«-overhangs, and

thus do not stain.

The following is a summary of the in situ staining

procedure. Following incubation with the various

drugs, the TM cell cultures were fixed in the petri

dishes for 30 minutes in 4% formaldehyde. The cells

were then washed three times in phosphate buffered

saline (PBS) for 2 minutes each wash. Cell membranes

were permeabilized by treating the samples with

0±7 mg% proteinase K solution at room temperature

for 5 minutes. Endogenous peroxidase activity was

quenched by immersing the specimens in 2% hydro-

gen peroxide at room temperature for 5 minutes. The

samples were rinsed for at least 1 minute by replacing

the hydrogen peroxide solution with Klenow labeling

buffer. The labeling buffer was aspirated, 100 µl

of Klenow labeling reaction mix were applied, and

plastic coverslips were placed over the specimens to

spread the reagent evenly. The specimens were then

placed in an incubator for 1 hr at a relative humidity

of 95% and a temperature of 37°C. The coverslips

were removed, and the labeling reaction was stopped

by immersion in stop buffer at room temperature for 5

minutes. The specimens were subsequently washed in

PBS for 2 minutes. One-hundred microliters of strepta-

vidin-horseradish peroxidase were applied on the

specimens and plastic coverslips were again applied to

spread the reagent evenly over the samples. The

specimens were incubated at room temperature for 20

minutes. The coverslips were then removed and the

samples were washed twice in PBS for 2 minutes each

wash. The specimens were immersed in DAB at room

temperature for 5–10 minutes until a satisfactory

color reaction was achieved. The specimens were

briefly rinsed in deionized water, and counterstained

by immersion in methyl green for 10 seconds. Methyl

green was then aspirated and replaced by PBS.

This assay was validated with the use of TACS Cell

Culture Control Slides (Trevigen Inc., Gaithersberg,

MD, U.S.A.). Specimens on these slides have been

ascertained to contain apoptotic (positive control) and

non-apoptotic (negative control) ML-1 cells.

Quantitation of Apoptotic Cells.

Quantitation of apoptotic cells was performed by

counting the number of apoptotic cells per 200¬field.

This figure was divided by the total number of cells

within the same field, yielding the percentage of

apoptotic cells within each specified field. Statistical

analyses were performed using the Student’s t test,

with a P value of less than 0±05 considered statistically

significant.

3. Results

5-FU (10–100 µg ml−"), MMC (0±1–100 µg ml−"),

and epinephrine (500–5000 µ) induced apoptosis in

TM cell cultures in a dose and time-dependent manner.

Specimens treated with these drugs underwent con-

densation of cytoplasm, and compaction and margi-

nation of chromatin, morphologic changes charac-

teristic of apoptosis [Fig. 1(B), (C), (D)]. They also

stained positively with the in situ apoptosis label [Fig.

2(B), (C), (D)].

Timolol, pilocarpine and dexamethasone-treated TM

cells did not show any morphologic changes consistent

with apoptosis, nor did they stain with the in situ

apoptosis label at any of the concentrations tested.

However, these drugs developed characteristic mor-

phologic changes as discussed below.

5-FU

Between 4 and 5 days of incubation in 100 µg ml−"

5-FU, 50% of cells were apoptotic (41±4% and 62±9%

at days 4 and 5 respectively). By day 7, 100% were

apoptotic. Cells incubated at lower concentrations

resulted in a lower percentage of apoptotic cells at

each time period. After 7 days of incubation in 5-FU,

all specimens had a significantly higher number of

apoptotic cells, as compared to the control (P!0±05)

[Fig. 3(A)].

Mitomycin C

After 2 days of incubation in 100 µg ml−" MMC,

almost half of the cells (46±3%) were apoptotic, and by

the third day, nearly all (96±5%) were apoptotic. It

took 6 days for the 10 µg ml−" MMC specimen to reach

100% apoptosis. Compared to the control, specimens

incubated in 0±1 µg ml−" and 1 µg ml−" had a sig-

nificantly greater number of apoptotic cells (P!0±05)

after 7 days of incubation in MMC. Control cells and

cells treated for 7 days in 0±01 µg ml−" MMC achieved

apoptosis levels of 7±37% and 8±92% respectively. The

difference between these values was statistically insig-

nificant (P¯0±09) [Fig. 3(B)].

Epinephrine

Apoptotic changes were observed in TM cell cultures

as early as 4 hr after incubation in 5000 µ epi-

nephrine. After 24 hr, 100% of cells were apoptotic.

After 2 days of incubation in 500 µ epinephrine,

23±1% of cells were apoptotic. On the third to seventh

days, a plateau was reached with 26±1–27±4% of the

total cell population being apoptotic. Apoptosis was

not observed after 7 days of incubation in 5 µ and

50 µ epinephrine [Fig. 3(C)].

524 S. A. B. SIBAYAN ET AL.

F. 1. Transmission electron micrographs of drug-treated TM cells. (A) Control cells. Cells treated with (B) 5-FU (50 µgml−"¬7 days), (C) MMC (1 µg ml−"¬7 days) and (D) epinephrine (5000 µ¬4 hr) demonstrate condensation of cytoplasm,and coalescence and margination of chromatin, morphologic features characteristic of apoptosis. These apoptotic bodies are

DRUG-INDUCED APOPTOSIS IN TRABECULAR CELLS 525

Timolol

Although TM cells incubated in timolol did not

demonstrate apoptosis, they underwent character-

istic morphologic changes. Specimens incubated in

1000 µ and 100 µ timolol became rounded, and

granules were noted to surround the nucleus [Fig.

1(E)]. Such changes began to manifest on the second

day of incubation in the cultures treated with 1000 µ

timolol and on the fifth day of incubation in those

treated with 100 µ timolol. Cells incubated in timolol

at lower concentrations did not show such changes.

None of the specimens stained with the in situ

apoptosis label [Fig. 2(E)].

Pilocarpine

No changes consistent with apoptosis were observed

in TM cells treated with pilocarpine. However, cells

incubated in 15000 µ pilocarpine were noted to

develop vacuoles by the first day of incubation [Fig.

1(F)]. By the third day, these cells began to detach

from the substrate. Some of these cells had disrupted

cell membranes and disorganized intracellular con-

tent, findings consistent with necrosis. Neither vacuo-

lation nor necrosis was observed in cells treated with

lower concentration levels (15–1500 µ) even after 7

days of incubation. In situ apoptosis staining was

negative in all specimens [Fig. 2(F)].

Dexamethasone

No morphologic changes consistent with apoptosis

were observed in any of the specimens treated with

dexamethasone. However, these cells demonstrated a

large number of mitochondria as compared to control

cells [Fig. 1(G)]. In situ apoptosis staining was negative

in all specimens [Fig. 2(G)].

4. Discussion

This study demonstrates that cultured bovine TM

cells can undergo apoptosis following exposure to 5-

FU, MMC and epinephrine. Timolol, pilocarpine and

dexamethasone did not induce apoptosis at the various

concentrations tested in vitro. However, these agents

can induce characteristic morphologic changes in

cultured bovine TM cells.

Many of the drug concentrations tested are much

higher than those observed following routine admini-

stration in vivo. Furthermore, cell cultures are

continuously bathed in drugs and are not subject to

the physiologic turnover of aqueous humor that takes

undergoing ‘secondary degeneration’, as indicated by degradation of organelles and vacuolation. (E) Timolol (1000 µ¬12days) induced development of cytoplasmic granules. (F) Pilocarpine (15000 µ¬3 days) treatment resulted in formation ofcytoplasmic vacuoles. These granules and vacuoles [as illustrated in figures (E) and (F), respectively] have the appearanceof secondary lysosomes. (G) Dexamethasone (100 µ¬12 days) treated cells exhibit an increased number of mitochondria.Magnification: (A)¯¬1450, (B)¯¬4700, (C)¯¬4900, (D)¯¬8700, (E)¯¬2000, (F)¯¬1650, (G)¯¬1700.

place in vivo. Since only a few cells undergo apoptosis

at any specific time point, and the kinetics of apoptosis

are rapid, detection of apoptosis can be difficult (Coles,

Burne and Raff, 1993). Administration of such high

drug doses may allow us to observe apoptotic changes

more readily.

A large number of cultured TM cells were needed for

these experiments, therefore bovine, rather than

human TM cells were used. Bovine cells grow faster

than human cells and are readily available from the

slaughterhouse (Kawa et al., 1993). Bovine TM cells

also appear to be a valid model for the determination

of drug effects in human cells, since their responses are

consistent with those reported for human cells (Kawa

et al., 1993).

Being known inducers of apoptosis, 5-FU and MMC

were primarily used to develop positive controls in TM

cells. However, it is known that these agents can be

detected in the aqueous humor when used adjunc-

tively glaucoma filtration surgery (Rootman et al.,

1979; Fantes et al., 1987). The observation that these

agents can induce apoptosis in TM cells may be of

limited clinical significance since glaucoma filtration

surgery is usually performed in cases of advanced

glaucoma where the TM is presumably already

profoundly compromised. Furthermore, these drugs

are not administered in a chronic fashion, and thus

may not have a sustained toxic effect.

It has been shown that agents that elevate cAMP

levels can induce apoptosis (McConkey, Orrenius and

Jondal, 1990). Tripathi, Tripathi and Millard (1989)

have shown that stimulation of beta-1 and beta-2

adrenergic receptors in TM cells in vitro causes a net

increase in cAMP levels. To our knowledge, our

current study is the first to show that epinephrine

can induce apoptosis in TM cells in vitro, presumably

by a beta-adrenergic}cAMP-mediated process. The

exact mechanism by which epinephrine induces

apoptosis in these cells will be a subject of further

investigation.

In contrast, Rinner et al. (1994) have shown that

epinephrine can trigger apoptosis in rat thymocytes in

vivo only if administered concomitantly with a beta-

blocker (propranolol). Epinephrine administered alone

or together with an alpha-blocker (phentolamine) did

not trigger an increase in apoptotic activity. These

findings suggest that apoptosis may be mediated by an

alpha-adrenergic receptor, and that beta-adrenergic

stimulation may counteract the apoptosis-inducing

activity of alpha-adrenergic receptors. Further studies

must be conducted in order to determine whether this

variation in biological response is due to a difference

between the two cell lines (rat thymocytes and bovine

526 S. A. B. SIBAYAN ET AL.

F. 2. In situ apoptosis staining of drug-treated TM cells in vitro. (A) Control cells do not exhibit staining. Positive stainingis demonstrated in cells treated with (B) 5-FU (50 µg ml−"¬7 days) (arrows), (C) MMC (1 µg ml−"¬7 days) (arrows), (D)epinephrine (5000 µ¬24 hr) (all cells). No staining is observed in cells treated with (E) timolol (1000 µ¬12 days), (F)pilocarpine (15000 µ¬3 days), and (G) dexamethasone (100 µ¬12 days). All photomicrographs, magnification¯¬200.

DRUG-INDUCED APOPTOSIS IN TRABECULAR CELLS 527

F. 3. Dose response curves illustrate the dose- and time-dependent inducement of apoptosis following treatment with(A) 5-FU, (B) MMC and (C) epinephrine.

TM cells), or a difference between cell responses in vivo

and in vitro.

If apoptosis in TM cells in vivo is mediated by beta-

adrenergic stimulation, administration of beta-

blockers may provide a protective effect in epinephrine-

treated eyes. On the other hand, if apoptosis is

mediated by an alpha-adrenergic receptor and beta-

blockade potentiates apoptosis, eyes treated concomi-

tantly with beta blockers and epinephrine may

undergo greater apoptotic changes in the TM as

compared to those that are treated with epinephrine

alone.

In addition to its use as an anti-glaucoma drug,

epinephrine is commonly used in ophthalmic surgery

as a mydriatic agent. Intraoperatively, solutions with

concentrations of up to 550 µ (1:10000 solution)

are injected directly into the anterior chamber. Using

such concentrations, the levels achieved within the

anterior chamber following intracameral administra-

tion could reach levels higher than those observed

following topical application. Intracameral adminis-

tration of epinephrine could therefore have greater

potential for acute cytotoxicity. Topical administra-

tion, on the other hand, results in sustained low levels

of epinephrine, which could result in chronic effects.

Timolol and pilocarpine-treated TM cells did not

exhibit any evidence of apoptosis. However, they

underwent characteristic morphologic changes which

have previously been described (Kawa et al., 1993).

Timolol-treated cells developed cytoplasmic granules,

and pilocarpine-treated specimens developed cyto-

plasmic vacuoles. These granules and vacuoles have

the appearance of secondary lysosomes. In addition,

we have demonstrated that high doses of pilocarpine

(15000 µ) can induce necrosis.

Steroids can induce glaucoma in certain susceptible

individuals (Bernstein and Schwartz, 1962; Armaly,

1963; Becker and Mills, 1963). Dexamethasone has

been shown to cause a decrease in the cellularity of the

TM of eyes that are prone to steroid-induced glaucoma

(Clark et al., 1995). Such a decrease could contribute

to the pathogenesis of this disease process. Steroids

have also been shown to induce apoptosis in certain

cells, such as thymocytes (Wyllie, 1980; Cohen

and Duke, 1984; Compton and Cidlowski, 1986),

eosinophils (Kawabori et al., 1991), leukemia cells

(McConkey et al., 1991) and neurons (Hassan et al.,

1996; Zysk et al., 1996). However, not all cells

are susceptible to dexamethasone-induced apoptosis

[e.g., HeLa S$

cells (Cidlowski and Cidlowski,

1981; Schwartzman and Cidlowski, 1991) and

glucocorticoid-resistant lymphocytes (Cohen, 1989)].

Although our studies did not demonstrate dexam-

ethasone-induced apoptosis in cultured TM cells, it is

possible that the cells we used were not responsive to

steroids. Studies using TM cells obtained from eyes

that have been proven susceptible to steroid-induced

glaucoma may elucidate whether apoptosis can be

induced in such cells.

The large number of mitochondria observed in

dexamethasone-treated cells is indicative of elevated

metabolic activity. These findings are consistent with

previous observations in steroid-treated TM cells

(Rohen, Linner and Witmer, 1973; Clark et al., 1995).

Whether exposure to anti-glaucoma medications

plays a role in decreased TM cellularity in vivo cannot

528 S. A. B. SIBAYAN ET AL.

be determined from this study. However, our findings

demonstrate that cultured TM cells can undergo

apoptosis when exposed to 5-FU, MMC and epi-

nephrine. While timolol, pilocarpine and dexametha-

sone did not induce apoptosis, they can incite

characteristic morphologic changes in TM cells in

vitro.

Acknowledgements

The authors thank Dr Salvador Gonza! lez for his commentson the manuscript. This study was supported by a grantfrom the Office of Naval Research (MFEL Grant No. N00014-94-I-0927).

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