Effects of Antimetabolite Induced Cellular Growth Arrest on Fibroblast-Fibroblast Interactions

Exp. Eye Res. (1999) 69, 117–127 Article No. exer.1999.0684, available online at http :\\www.idealibrary.com on

Effects of Antimetabolite Induced Cellular Growth Arrest on Fibroblast-Fibroblast Interactions J U L I E T. D A N I E L Sa, *, N I C H O L A S L. O C C L E S T O Na, †, J O N A T H A N G. C R O W S T ONa    P E N G T. K H A Wa, b a

Wound Healing Research Unit, Institute of Ophthalmology, University College London, U.K. and b Moorfields Eye Hospital, London, UK (Received Lund 24 November 1998 and accepted in revised form 12 March 1999) The use of single five-minute applications of antimetabolites during glaucoma filtration surgery has significantly reduced the occurrence of post-operative scarring and bleb failure. However, surgery for some patients is still unsuccessful, despite the use of antiproliferative agents, due to formation of scar tissue at the drainage site. It is not known if cells growth arrested in the treated area with a single application of antimetabolites influence the activity of adjacent non-treated cells. We hypothesise that the activity of non-treated cells recruited to the wound site may be involved in post-operative scarring. The aim of this study was to investigate the effects of antimetabolite induced cellular growth arrest on cell–cell interactions using in vitro techniques. Tenon’s capsule fibroblast cultures were growth arrested by exposure for 5 minutes to mitomycin-C (0n001, 0n01 and 0n1 mg ml−"), 5-fluorouracil (0n25, 2n5 and 25 mg ml−") or phosphate buffered saline (PBS). Following a period of serum-starvation, conditioned media (CM) were subsequently collected from the cells at intervals up to 29 days post-treatment. Correction for cell number was made prior to supplementation of serum-free medium with CM. CM were assessed for ability to support or inhibit normal non-treated fibroblast proliferation, migration and collagen contraction. Conditioned media collected from cells growth arrested with MMC or 5FU stimulated normal fibroblast proliferation, migration and collagen contraction in excess of non-conditioned serum-free medium. Peaks of fibroblast activity in CM differed according to which drug and concentration had originally been given to the treated cells. This study has demonstrated that CM collected from fibroblasts treated for 5 minutes with a range of concentrations of antimetabolites can differentially influence normal non-treated fibroblast activity. This in vitro data suggests that despite entering growth arrest, fibroblasts may still influence the behaviour of other cells via soluble mediators. They may have implications in the clinical setting, in that it may not be sufficient to suppress proliferation alone to prevent fibroblast behaviour associated with scarring after glaucoma filtration surgery. # 1999 Academic Press Key words : antimetabolites ; cell–cell interactions ; glaucoma ; growth arrest.

1. Introduction During tissue repair the processes of cell proliferation, migration, deposition of new extracellular matrix (ECM) and tissue contraction lead to wound closure and healing. Numerous proteins including growth factors, growth factor receptors and ECM regulate these processes (Clark and Henson, 1988). The success of surgical treatment of glaucoma is dependent upon the wound healing response and in particular the potential scarring activity of fibroblasts. The use of antimetabolites has reduced the degree of postoperative scarring. Examples of these treatments include single intraoperative applications of mitomycin-C (MMC) or subconjunctival injections of 5fluorouracil (5FU ; Skuta et al., 1992 ; The fluorouracil filtering study group, 1996). The rationale of these treatments is to reduce the healing response, primarily * Address for correspondence : Julie T. Daniels, Wound Healing Research Unit, Department of Pathology, Institute of Ophthalmology, 11–43 Bath Street, London, EC1V 9EL, U.K. † Present address : Pfizer Ltd, Sandwich, Kent, U.K.

0014–4835\99\010117j11 $30.00\0

by suppressing fibroblast proliferation, for the first few weeks following surgery. It has been demonstrated both in vitro (Khaw et al., 1992a, 1992b) and in vivo (Khaw et al., 1993) that fibroblasts can remain in growth arrest for up to 36 days following a single treatment with MMC or 5FU, depending upon the agent used and its concentration. This effect is focal, leaving surrounding areas relatively unaffected. However, clinically some high-risk group patients still fail surgery even after antiproliferative treatment (Skuta et al., 1992). In many cases the avascular area treated with antimetabolites may be surrounded by a vascularised active area that may begin to invade the treated area, with scar formation in this peripheral adjacent area. The reasons for this are currently unclear. It has been shown recently that fibroblasts, growth arrested with antimetabolites, remain capable of migration and produce several regulatory proteins including growth factors\receptors and extracellular matrix molecules (Occleston et al., 1997), indicating that these cells may still be able to contribute to a # 1999 Academic Press

118

scarring response. However, the actual biological effects of these factors were not determined. The most common method for culturing human epithelial cells involves a growth arrested 3T3 fibroblast feeder layer supporting epithelial colony growth (Rheinwald and Green, 1975). One of the methods for growth arresting a 3T3 or normal fibroblast feeder layer is treatment with MMC. It has been argued that growth arrested feeder layers support epithelial cells by direct contact (Yaeger, Stiles and Rollins, 1991), however, other reports have suggested that the feeder cells can release soluble mediators of keratinocyte growth, endothelial cell angiogenesis and epithelial anti-apoptotic activity into culture medium (Blacker et al., 1987 ; Montesano, Pepper and Orci, 1993 ; Tseng et al., 1996). The mitomycin dosing time recommended for feeder cell growth arrest can vary from 2 hr and upward (Limat et al., 1989 ; Daniels, Kearney and Ingham, 1996). This current investigation was designed to determine the biological effects of conditioned medium collected from Tenon’s fibroblasts growth arrested for just 5 minutes with antimetabolites upon non-arrested (subsequently referred to as normal) fibroblasts. 2. Materials and Methods Cell Culture Human fibroblasts were cultured from Tenon’s capsule tissue from patients undergoing surgery at Moorfields Eye Hospital, London, U.K., in Dulbecco’s modification of Eagle’s medium (DMEM ; supplied by Gibco Life Technologies, Paisley, Scotland) supplemented with new-born calf serum (NBCS) as previously described (Khaw et al., 1992a). Cultures were used between passages 3 and 5 for experiments. Antimetabolite Treatment of Fibroblast Monolayers Fibroblasts were seeded at 1i10% cm−# into 175 cm# and 25 cm# tissue culture treated flasks and incubated overnight to produce actively growing subconfluent cultures. Culture medium was then removed and the monolayers were treated with mitomycin C (MMC ; 0n001, 0n01 and 0n1 mg ml−" ; Kyowa Ltd., Essex, U.K.), 5FU (0n25, 2n5 and 25 mg ml−" ; David Bull Laboratories, Warwick, U.K.) or phosphate buffered saline (PBS controls) for 5 minutes. The monolayers were then washed three times with PBS and incubated with 25 ml DMEM supplemented with 10 % (v\v) NBCS. Collection of Conditioned Medium At 2, 8, 15, 22 and 29 days post-treatment, conditioned medium was collected as previously described (Occleston et al., 1997). To do this, the culture medium was removed and replaced with serum-free DMEM containing 1 % (w\v) bovine serum albumin (BSA ; Sigma, Dorset, U.K.) and the cells were

J. T. D A N I E L S E T A L.

incubated at 37mC twice (4 hr per incubation) to remove any residual serum. The cells were then cultured for a further 24 hr in DMEM plus 1 % (w\v) BSA. Upon collection, the conditioned medium was centrifuged, aliquoted and stored at k70mC until use and the cultures were returned to full culture medium. At each time point, cells from 25 cm# sister flasks were counted using a Coulter Counter (Coulter Electronics, Bedfordshire, U.K.) to determine the cell numbers present in the larger flasks (175 cm#) used for conditioned medium collection. It was noted that the cell numbers in the control and growth arrested cultures differed at each time point. To accurately determine the comparative effects of each conditioned medium sample, these differences were taken into account. Pilot experiments were conducted 3 times to determine the optimal dilution of conditioned medium from control cultures (exposed to PBS) at each time point, to stimulate proliferation, migration and collagen contraction. The dilution of conditioned medium from growth arrested cultures, to use in subsequent experiments, were then normalised for cell number prior to use. For example, if the optimal dilution from control cultures was determined as 1 : 10 and they contained twice as many cells as a growth arrested culture, then the dilution of conditioned medium used from this growth arrested culture was 1 : 5. All experiments with the collected conditioned medium were repeated 3 times. Proliferation Assay Normal (untreated) fibroblasts were seeded into 48 well plates at a density of 5i10$ cells well−" and serum starved overnight. A volume of 500 µl of conditioned medium or serum-free unconditioned DMEM containing 1 % (v\v) BSA (to give baseline data) was added to the wells in sextuplicate. The plates were incubated for 24 hr, and the total number of cells in 5 random fields of view per well were counted using a i20 objective of an Olympus CK2 (Olympus, London, U.K.) phase contrast microscope. Migration Assay Migration assays were carried out using Transwell chambers (Costar, Buckinghamshire, U.K.). Normal fibroblasts were seeded in 100 µl serum-free DMEM (without BSA) into the upper chambers of Transwell inserts resting in 24 well tissue culture plates at a density of 1i10% per insert. A volume of 600 µl of conditioned medium sample or baseline control, in sextuplicate, was added to the lower chambers. Migration through the membrane was allowed to proceed for 16 hr only to ensure that proliferation did not skew the results. The membranes were washed with PBS, fixed in 95 % (v\v) methanol for 10 minutes, stained with Harris’s haematoxylin for 30 minutes and rinsed in tap water. Settled cells were wiped from

G R O W T H A R R E S T A N D F I B R O B L A S T I N T E R A C T I O NS

119

F. 1. Proliferation of Tenon’s capsule fibroblasts, from which conditioned medium was collected at intervals, following growth arrest with concentrations of (A) MMC : X, 0n001 mg ml−" ; , 0n01 mg ml−" ; 4, 0n1 mg ml−" and (B) 5FU X, 0n25 mg ml−" ; , 2n5 mg ml−" ; 4, 25 mg ml−" ; $, PBS. Error bars represent 95 % confidence intervals (n l 6).

the upper surface of the membrane and the total number of migrated cells on the underside of the membrane was counted. Collagen Contraction Assay To assess the influence of the conditioned medium upon fibroblast matrix re-organisation, fibroblastpopulated collagen gels were prepared in 48 well plates. Firstly, fibroblasts were re-suspended at density of 3n3i10' ml−" in serum-free, concentrated DMEM culture medium (i2n5) supplemented with final concentrations of the following : -glutamine (0n05 m ; Gibco Life Technologies, U.K.), penicillin (250 I.U ml−") and streptomycin (250 µg ml−" ; Gibco Life Technologies, U.K.), fungizone (6 µg ml−" ; Sigma, Dorset, U.K.) and buffered by sodium bicarbonate (Sigma, Dorset, U.K.). Gel mixture volumes were prepared according to experimental requirements. For example, to prepare 6 gels, 0n35 ml concentrated medium was mixed with 0n6 ml type I rat tail collagen dissolved in 0n1 % (v\v) acetic acid (5 mg ml−" ; Sigma, Dorset, U.K.). Sodium hydroxide (0n1 ) was then added drop-wise to the solution to precipitate the collagen fibrils and to return the mixture to physiological pH. Aliquots of gel mixture (150 µl) were added to the wells of a 48 well plate. Sextuplicate gels were overlaid with 500 µl conditioned medium or baseline control medium and detached from the wells. The media overlying the gels was replaced 3 days postseeding. On day 7 the gels were photographed using a QV100 digital camera (Casio Computer Co. Ltd., Tokyo, Japan). Photographs were digitised and the gel areas calculated by image analysis (Image Tool for Windows). Data Analysis One-way analysis of variance (ANOVA) was performed on all generated data using computer software (SPSS for Windows ; SPSS Inc., Chicago, U.S.A.).

Values acquired for each assay with day 2 conditioned medium were compared with values obtained with media collected at subsequent time points. The observed significance levels were adjusted with the Bonferroni test for multiple comparisons. A P value of 0n05 was considered significant. Data means were plotted graphically together with 95 % confidence intervals. Trends in fibroblast activity were plotted separately for each drug concentration for clarity. 3. Results Antimetabolite Induced Growth Arrest Figure 1 shows the profiles of fibroblast proliferation following 5-minute treatments with concentrations of MMC or 5FU. The PBS treated control cells increased in number most rapidly between days 8 and 15. Cell number, from then onwards, increased at a reduced rate. Fibroblasts treated with low dose MMC (0n001 mg ml−") were virtually recovered from growth arrest by day 29, while those cells treated with high dose MMC (0n1 mg ml−") remained non-proliferative throughout. A similar trend was seen following treatment with concentrations of 5FU. However, less recovery of proliferative potential was achieved by day 29 in the low dose 5FU (0n25 mg ml−") than low dose MMC (P 0n05). As cell counts differed between the groups with time, a correction for cell number was made during preparation of conditioned medium to be tested. This allowed the potency of each conditioned medium to be assessed on a per cell basis, indicating trends in activity at intervals post-treatment. Proliferation Figures 2(A) and 2(B) demonstrate proliferation of normal fibroblasts in the presence of conditioned medium collected from cells treated with concentrations of MMC, 5FU and PBS. All conditioned media (except 5FU, 2n5 mg ml−", day 2) supported prolifer-

120

J. T. D A N I E L S E T A L.

(B) (A)

F. 2. For legend see facing page.

G R O W T H A R R E S T A N D F I B R O B L A S T I N T E R A C T I O NS

ation above the baseline (dotted lines) maintained by the unconditioned serum-free medium (plus 1 % w\v BSA) alone. The solid line represents the mean cell number present per well at time zero before the addition of conditioned medium to the cells in Fig. 2(A) and 2(B). The ability of conditioned medium collected from cells treated with PBS to support normal fibroblast proliferation decreased with time (P 0n05). Peak proliferative activity with low dose MMC occurred at 15 days and decreased significantly after this (P 0n05). Conditioned medium collected from fibroblasts treated with high dose MMC (0n1 mg ml−") supported a significant increase in fibroblast proliferation over the time course (P 0n05). Proliferation associated with medium from intermediate dosing (0n01 mg ml−") fluctuated less with time [Fig. 2(A)]. Figure 2(B) shows similar proliferation data for the conditioned media collected from cells treated with concentrations of 5FU. By day 29 the proliferative potential of medium collected from fibroblasts treated with low dose 5FU (0n25 mg ml−") significantly decreased (P 0n05). Proliferation with conditioned medium from the intermediate dose (2n5 mg ml−") increased between days 2 and 8 and did not really change subsequently. The high dose 5FU (25 mg ml−") conditioned medium supported maximum fibroblast proliferation by day 15 with activity decreasing back to day 2 levels at the end of the time course. Migration The graphs in Fig. 3(A) and 3(B) demonstrate fibroblast chemotactic migration towards conditioned medium. The dotted lines represent normal fibroblast baseline migration in response to unconditioned serum-free medium (plus 1 % w\v BSA). The ability of conditioned medium collected from PBS treated cells to stimulate normal fibroblast migration decreased over the time course [Fig. 3(A) ; P 0n05]. Peak migratory activity was observed in the presence of conditioned medium collected from low dose MMC treated cells at 8 days, where as the intermediate and high dose medium supported maximum migration at 15 days (P 0n05). By day 29, migratory activity in the conditioned medium from each treatment group decreased significantly from the day 2 data (P 0n05). Figure 3(B) demonstrates a gradual yet significant decrease in chemotactic activity with time post-growth arrest compared with conditioned medium collected

121

on day 2 in the low dose 5FU group (P 0n05). Peak migratory activity with 2n5 and 25 mg ml−" 5FU conditioned media groups occurred with samples collected on days 8 and 15, respectively (P 0n05). Collagen Contraction Figure 4(A) and 4(B) show the effect of conditioned medium upon fibroblast-mediated collagen contraction. The solid lines show the starting gel area before addition of conditioned medium. The dotted lines indicate baseline decrease in gel area (contraction) associated with unconditioned serum-free medium (plus 1 % w\v BSA). With time, the conditioned medium produced by the cells treated with PBS decreased in ability to stimulate fibroblast-mediated collagen contraction (P 0n05). No trend in collagen contraction associated with low and intermediate dose conditioned media was as apparent with time as it had been for proliferation and migration. Conditioned medium collected from fibroblasts treated with high dose MMC (0n1 mg ml−") stimulated the greatest contraction by day 29. The results with conditioned media collected from cells treated with 5FU are shown in Fig. 4(B). Contraction associated with conditioned medium collected from cells treated with 0n25 mg ml−" 5FU gradually decreased over the time course (P 0n05). No change in contraction occurred with intermediate dose conditioned medium, while a significant increase in contractile activity occurred with high dose 5FU conditioned medium collected between 2 and 8 days (P 0n05). After this time point no further contraction occurred. Statistical Significance of Data Table I indicates significant differences by analysis of variance between fibroblast proliferation, migration or collagen contraction in the presence of conditioned medium. Values acquired with media collected on day 2 were compared with values obtained with media collected at subsequent time points within each treatment group (P 0n05). 4. Discussion The use of antimetabolites has improved the success of glaucoma filtration surgery for many patients, although not all (Skuta et al., 1992 ; The fluorouracil filtering surgery group, 1996). The scarring activity of

F. 2. (A) Proliferation of normal fibroblasts in the presence of conditioned medium collected from cells at intervals post treatment with PBS and concentrations of MMC. Error bars represent 95 % confidence intervals (n l 6). The solid line represents the number of cells present at time zero and the dotted line represents proliferation associated with serum-free medium (plus 1 % w\v BSA) alone. (B) Proliferation of normal fibroblasts in the presence of conditioned medium collected from cells at intervals post treatment with PBS and concentrations of 5FU. Error bars represent 95 % confidence intervals (n l 6). The solid line represents the number of cells present at time zero and the dotted line represents proliferation associated with serum-free medium (plus 1 % w\v BSA) alone.

122

J. T. D A N I E L S E T A L.

(A)

(B)

F. 3. For legend see facing page.

G R O W T H A R R E S T A N D F I B R O B L A S T I N T E R A C T I O NS

fibroblasts is responsible for the obstruction of aqueous flow and hence surgical failure in some cases. Cell–cell interactions play a very important role in tissue homeostasis and wound healing. This work was carried out to study the potential influence of soluble factors released by growth arrested fibroblasts on normal, non-arrested cells. Concentrations of 0n25, 2n5 and 25 mg ml−" 5FU and 0n001, 0n01 and 0n1 mg ml−" MMC were used to growth arrest fibroblasts. The more clinically relevant doses of 0n2– 0n5 mg ml−" MMC could not be used in this study as these concentrations were previously found to induce apoptosis and cell death in fibroblasts in vitro resulting in no cells remaining over the course of the experiments (Crowston et al., 1998). This may indeed be part of the mechanism of action of MMC. However, it is likely that in clinical practice less penetration occurs, so the 0n1 mg ml−" dose may be mimicking some of the cellular effects of slightly higher clinical doses such as 0n2–0n4 mg ml−". The approach of using conditioned medium was chosen as previous studies have demonstrated paracrine activity in this system. For example, conditioned medium from 3T3 fibroblast cultures has been shown to have corneal epithelial cell anti-apoptotic activity (Tseng et al., 1996). Since the number of cells from which conditioned medium was collected varied according to the concentration of antimetabolite treatment, corrected dilutions of conditioned medium were prepared prior to addition to normal cells. This permitted assessment of conditioned medium activity on a per cell basis. Although the exact mechanisms controlling the production of biologically active factors by growth arrested cells were not determined in this study, there was a marked increase in the stimulatory capacity of conditioned medium from cells exposed to higher concentrations of antimetabolites, compared to PBS. Conditioned medium collected from PBS treated cells which had become confluent and beyond (between 15 and 29 days) supported less proliferation, migration or collagen contraction of experimental fibroblasts. In general, the conditioned medium collected from antimetabolite treated fibroblasts was more stimulatory to experimental cell activity than medium collected from PBS treated cells. High cell density can induce cell cycle arrest by inactivation of the cyclin Ddependent kinase, as demonstrated in the rat cell line 3Y1 (Kato et al., 1997a, 1997b). Our data may reflect different responses to growth arrest induced by contact inhibition or antimetabolites causing fibroblasts to

123

either shut down or up-regulate potential factors of paracrine activity. In a previous study we demonstrated that fibroblasts growth arrested with single exposures to different concentrations of MMC or 5FU were still capable of producing a variety of growth factors. These included fibroblast growth factor (FGF) and transforming growth factor-beta (TGF-β), as well as extracellular matrix molecules including collagen types I\III and fibronectin. In addition, these cells expressed growth factor receptors (Occleston et al., 1997). We suggested the possibility that these activities may enable growth arrested cells to participate in a scarring response. However, we did not determine whether the factors produced were biologically functional. This data has demonstrated that medium conditioned by fibroblasts growth arrested with antimetabolites can stimulate normal fibroblast proliferation, migration and collagen contraction. The degree of activity induced by the conditioned medium seemed to reflect the concentration of antimetabolite used to induce growth arrest in the first place in culture, however the opposite is observed clinically. Our data probably reflects the large differences in cell numbers that survive after exposure to antimetabolites which produced dramatically different dilutions of conditioned media. In addition, in vivo the actual concentration of growth factors released after exposure to high concentrations of antimetabolites would likely be less than the concentration released from fibroblasts exposed to low concentrations of antimetabolites because more cells are undergoing apoptosis or necrosis after exposure to high concentrations of antimetabolites. Nonetheless, these results indicate that the conditioned medium collected from growth arrested fibroblasts probably contains biologically active growth factors. As we previously suggested, the production of these factors may be due to the degree of sublethal injury elicited upon these cells by antimetabolites (Occleston et al., 1997). The diagram in Fig. 5 shows potential interactions leading to scarring that may occur between fibroblasts focally growth arrested with antimetabolites and surrounding normal untreated fibroblasts. The fact that growth arrested fibroblasts can stimulate normal fibroblasts appears to suggest that a larger surface area of treatment may be beneficial (Cordeiro et al., 1997). A larger area growth arrests more cells (Khaw et al., 1993), leaving a smaller area of peripheral cells that can be activated by the growth arrested cells, thus reducing migration into the treated

F. 3. (A) Migration of normal fibroblasts in the presence of conditioned medium collected from cells at intervals post treatment with PBS and concentrations of MMC. Error bars represent 95 % confidence intervals (n l 6). The dotted line represents migration associated with serum-free medium (plus 1 % BSA) alone. (B) Migration of normal fibroblasts in the presence of conditioned medium collected from cells at intervals post treatment with PBS and concentrations of 5FU. Error bars represent 95 % confidence intervals (n l 6). The dotted line represents migration associated with serum-free medium (plus 1 % BSA) alone.

124

J. T. D A N I E L S E T A L.

(A)

(B)

F. 4. (A) Collagen contraction by normal fibroblasts in the presence of conditioned medium collected from cells at intervals post treatment with PBS and concentrations of MMC. Error bars represent 95 % confidence intervals (n l 6). The solid line represents the initial size of the gel. The dotted line represents the decrease in gel area associated serum-free medium (plus 1 % w\v BSA) alone. (B) Collagen contraction by normal fibroblasts in the presence of conditioned medium collected from cells at intervals post treatment with PBS and concentrations of 5FU. Error bars represent 95 % confidence intervals (n l 6). The solid line represents the initial size of the gel. The dotted line represents the decrease in gel area associated with serum-free medium (plus 1 % w\v BSA) alone.

G R O W T H A R R E S T A N D F I B R O B L A S T I N T E R A C T I O NS

125

T I The table indicates significant differences by analysis of variance between fibroblast proliferation, migration or collagen contraction in the presence of conditioned medium from one set of representative experiments. Values acquired with media collected on day 2 were compared with values obtained with media collected at subsequent time points within each treatment group (P 0n05) Day 2 Sample Proliferation PBS MMC 0n001 mg ml−" MMC 0n01 mg ml−" MMC 0n1 mg ml−" 5FU 0n25 mg ml−" 5FU 2n5 mg ml−" 5FU 25 mg ml−" Migration PBS MMC 0n001 mg ml−" MMC 0n01 mg ml−" MMC 0n1 mg ml−" 5FU 0n25 mg ml−" 5FU 2n5 mg ml−" 5FU 25 mg ml−" Collagen contraction PBS MMC 0n001 mg ml−" MMC 0n01 mg ml−" MMC 0n1 mg ml−" 5FU 0n25 mg ml−" 5FU 2n5 mg ml−" 5FU 25 mg ml−" * Indicates a significant difference (P

Day 8

Day 15

Day 22

Day 29

* * *

* * * *

*

* *

* *

* * * * * *

* *

*

* * * * *

* * * * *

* * * * * * *

* * * * *

*

* *

* * * * * *

* * *

*

*

0n05).

area and also proliferation and contraction peripherally which may result in bleb encapsulation and ultimately contraction. Previous reports have suggested that patients in high risk groups undergoing filtration surgery can fail due to post-operative scarring even following focal antimetabolite treatment (Skuta et al., 1992). In some cases, the filtration bleb area, which has been exposed to antimetabolites, develops a ring of scar tissue around the edge rather than a central area. The fact that scar tissue is deposited around the focally growth arrested area indicates that the non-treated cells at the edges of this area may still be stimulated to exhibit aspects of wound healing behaviour by the population of growth arrested cells. We have found previously that exogenous growth factors such as TGFβ may stimulate growth arrested fibroblasts to upregulate collagen production (Khaw et al., 1994). Injection of exogenous TGFβ into a rabbit model following antimetabolite treatment, can reverse the anti-scarring effects of the treatment and part of this effect may induce stimulation of growth arrested fibroblasts (Doyle et al., 1997). The findings above, in conjunction with those of this current study, strongly suggest that growth arrested fibroblasts should not be considered as cells that cannot contribute to the scarring

response. It may not be sufficient just to suppress proliferation alone, which has previously been the main target of antimetabolite use. Other aspects of cellular function may also have to be suppressed, preferably without large-scale cell death seen with high doses of antimetabolites such as MMC. Finally, these findings may have important clinical implications with respect to antimetabolite treatment regimes in that fine-tuning of the tissue concentrations of antimetabolites is required to provide optimal suppression of both proliferation and stimulatory factor production. This may ideally result in decreased scar tissue formation and increased surgical success in all patient groups. Acknowledgements This work was supported by Fight for Sight (Mr and Mrs Magnus Bequest), the International Glaucoma Association (all London U.K.) and in part by the Wellcome Trust.

References Blacker, K. L., Williams, M. L. and Coldyne, M. (1987). Mitomycin C-treated 3T3 fibroblasts used as feeder layers for human keratinocyte culture retain the capacity to generate eicosanoids. J. Invest. Dermatol. 89(6) ; 536–9.

126 J. T. D A N I E L S E T A L.

F. 5. Diagrammatic representation of possible interactions between fibroblasts growth arrested with antimetabolites and normal untreated fibroblasts that may lead to scarring.

G R O W T H A R R E S T A N D F I B R O B L A S T I N T E R A C T I O NS

Clark, R. A. F. and Henson, P. M. eds (1988). The molecular and cellular biology of wound repair. pp. 243–78. Plenum Press : New York, U.S.A. Cordeiro, M. F., Constable, P. H., Alexander, R. A., Bhattacharya, S. S. and Khaw, P. T. (1997). Effect of varying the mitomycin-C treatment area in glaucoma filtration surgery in the rabbit. Invest. Ophthalmol. Vis. Sci. 38, 1639–46. Crowston, J. G., Akbar, A. N., Constable, P. H., Occleston, N. L., Daniels, J. T. and Khaw, P. T. (1998). Antimetabolite-induced apoptosis in Tenon’s capsule fibroblasts. Invest. Ophthalmol. Vis. Sci. 39, 449–54. Daniels, J. T., Kearney, J. N. and Ingham, E. (1996). Human keratinocyte isolation and cell culture : a survey of current practices in the UK. Burns 22(1), 35–9. Doyle, J. W., Smith, M. F., Garcia, J. A., Schultz, G. and Sherwood, M. B. (1997). Treatment of bleb leaks with transforming growth factor-beta in the rabbit model. Invest. Ophthalmol. Vis. Sci. 38, 1630–4. Kato, A., Takahashi, H., Takahashi, Y. and Matsushime, H. (1997a). Inactivation of the cyclin D-dependent kinase in the rat fibroblast cell line, 3Y1, induced by contact inhibition. J. Biol. Chem. 272(12), 8065–70. Kato, A., Takahashi, H., Takahashi, Y. and Matsushime, H. (1997b). Contact inhibition-induced inactivation of the cyclin D-dependent kinase in rat fibroblast cell line, 3Y1. Leukemia 11(3), 361–2. Khaw, P. T., Ward, S., Porter, A., Grierson, I., Hitchings, R. A. and Rice, N. S. (1992a). The long-term effects of 5-fluorouracil and sodium butyrate on human Tenon’s fibroblasts. Invest. Ophthalmol. Vis. Sci. 33, 2043–52. Khaw, P. T., Sherwood, M. B., MacKay, S. L., Rossi, M. J. and Schultz, G. (1992b). Treatments with fluorouracil, floxuridine and mitomycin have long-term effects on human Tenon’s capsule fibroblasts. Arch. Ophthalmol. 110, 1150–4. Khaw, P. T., Doyle, J. W., Sherwood, M. B., Grierson, I., Schultz, G. and McGorray, S. (1993). Prolonged

127

localised effects from 5-minute exposures to fluorouracil and mitomycin C. Arch. Ophthalmol. 111, 263–7. Khaw, P. T., Occleston, N. L., Schultz, G., Grierson, I., Sherwood, M. B. and Larlin, G. (1994). Activation and suppression of fibroblast function. Eye 8, 188–95. Limat, A., Hunzinker, T., Boillat, C., Bayreuther, K. and Noser, F. (1989). Post-mitotic human dermal fibroblasts efficiently support the growth of human follicular keratinocytes. J. Invest. Dermatol. 92, 758–62. Montesano, R., Pepper, M. S. and Orci, L. (1993). Paracrine induction of angiogenesis in vitro by Swiss 3T3 fibroblasts. J. Cell Sci. 105, 1013–24. Occleston, N. L., Daniels, J. T., Tarnuzzer, R. W., Sethi, K. K., Alexander, R. A., Bhattacharya, S. S., Schultz, G. S. and Khaw, P. T. (1997). Single exposures to antiproliferative : long-term effects on fibroblast wound healing behaviour. Invest. Ophthalmol. Vis. Sci. 38, 1998–2007. Rheinwald, J. G. and Green, H. (1975). Serial cultivation of strains of human epidermal keratinocytes : the formation of keratinising colonies from single cells. Cell 6, 331–43. Skuta, G. L., Beeson, C. C., Higginbotham, E. J., Lichter, P. R., Musch, D. C., Bergstrom, J. K., Klein, T. B. and Falck, F. Y. Jr. (1992). Intraoperative mitomycin versus postoperative 5-fluorouracil in high-risk filtering surgery. Ophthalmology 99, 438–44. The Fluorouracil Filtering Surgery Study Group. (1996). Five-year follow-up of the fluorouracil filtering surgery study. Am. J. Path. 121, 349–66. Tseng, S. C. G., Kruse, F. E., Merritt, J. and Li, D-Q. (1996). Comparison between serum-free and fibroblast-cocultured single-cell clonal culture systems : evidence showing that epithelial anti-apoptotic activity is present in 3T3 fibroblast-conditioned medium. Curr. Eye Res. 15, 973–84. Yaeger, P. C., Stiles, C. D. and Rollins, B. J. (1991). Human keratinocyte growth-promoting activity on the surface of fibroblasts. J. Cell. Physiol. 149, 110–16.