MSR1 repeats modulate gene expression and affect risk of breast and prostate cancer.

Background: MSR1 repeats are a 36-38 bp minisatellite element that have recently been implicated in the regulation of gene expression, through copy number variation (CNV). Patients and methods: Bioinformatic and experimental methods were used to assess the distribution of MSR1 across the genome, evaluate the regulatory potential of such elements and explore the role of MSR1 elements in cancer, particularly non-familial breast cancer and prostate cancer. Results: MSR1s are predominately located at chromosome 19 and are functionally enriched in regulatory regions of the genome, particularly regions implicated in short-range regulatory activities (H3K27ac, H3K4me1 and H3K4me3). MSR1-regulated genes were found to have specific molecular roles, such as serine-protease activity (P = 4.80 × 10-7) and ion channel activity (P = 2.7 × 10-4). The kallikrein locus was found to contain a large number of MSR1 clusters, and at least six of these showed CNV. An MSR1 cluster was identified within KLK14, with 9 and 11 copies being normal variants. A significant association with the 9-copy allele and non-familial breast cancer was found in two independent populations (P = 0.004; P = 0.03). In the white British population, the minor allele conferred an increased risk of 1.21-3.51 times for all non-familial disease, or 1.7-5.3 times in early-onset disease. The 9-copy allele was also found to be associated with increased risk of prostate cancer in an independent population (odds ratio = 1.27-1.56; P =0.009). Conclusions: MSR1 repeats act as molecular switches that modulate gene expression. It is likely that CNV of MSR1 will affect risk of development of various forms of cancer, including that of breast and prostate. The MSR1 cluster at KLK14 represents the strongest risk factor identified to date in non-familial breast cancer and a significant risk factor for prostate cancer. Analysis of MSR1 genotype will allow development of precise stratification of disease risk and provide a novel target for therapeutic agents.

HMG20A is required for SNAI1-mediated epithelial to mesenchymal transition.

HMG20A is a high mobility group (HMG) domain containing protein homologous to HMG20B, a core subunit of the Lys-specific demethylase 1/REST co-repressor 1 (LSD1-CoREST) histone demethylase complex. Here, we show that HMG20A can replace HMG20B and, therefore, they are mutually exclusive subunits of the complex. Both proteins interact through a coiled-coil domain with BHC80, another subunit of the LSD1-CoREST complex. To investigate the functional differences between the two proteins, we performed transcriptomic analysis of HMG20A- and HMG20B-depleted cells. Analysis of the misregulated genes in HMG20A-knockdown cells evidenced a high proportion of genes related to the epithelial-to-mesenchymal transition (EMT) process. EMT occurs during embryonic development or during the course of malignant cancer progression and consists in the dynamic and reversible transitions between epithelial and mesenchymal phenotypes. We show that HMG20A together with LSD1 are required for SNAI1-dependent repression of epithelial genes and for (transforming growth factor ß) TGF-ß-triggered EMT. Importantly, HMG20A-depleted cells displayed reduced binding of LSD1 to epithelial gene promoters and increased methylation of lysine 4 of histone H3, suggesting a role of HMG20A in recruiting or in stabilizing the complex at the chromatin. SNAI1 and the TGF-ß-related transcription factor SMAD4 were found to be associated with the LSD1-CoREST complex containing HMG20A. Furthermore, we show that HMG20A-depleted cells displayed reduced motility and invasion activity. Finally, we show that expression of HMG20A correlates positively with mesenchymal markers and negatively with epithelial markers in human tumor samples. Taken together, our data demonstrate that HMG20A is essential for the mesenchymal phenotype.

Effect of gas flow rates on the anatase-rutile transformation temperature of nanocrystalline TiO2 synthesised by chemical vapour synthesis.

Of the three crystallographic allotropes of nanocrystalline titania (rutile, anatase and brookite), anatase exhibits the greatest potential for a variety of applications, especially in the area of catalysis and sensors. However, with rutile being thermodynamically the most stable phase, anatase tends to transform into rutile on heating to temperatures in the range of 500 degrees C to 700 degrees C. Efforts made to stabilize the anatase phase at higher temperatures by doping with metal oxides suffer from the problems of having a large amorphous content on synthesis as well as the formation of secondary impurity phases on doping. Recent studies have suggested that the as-synthesised phase composition, crystallite size, initial surface area and processing conditions greatly influence the anatase to rutile transformation temperature. In this study nanocrystalline titania was synthesised in the anatase form bya chemical vapour synthesis (CVS) method using titanium tetra iso-propoxide (TTIP) as a precursor under varying flow rates of oxygen and helium. The anatase to rutile transformation was studied using high temperature X-ray diffraction (HTXRD) and simultaneous thermogravimetric analysis (STA), followed by transmission electron microscopy (TEM). It was demonstrated that the anatase-rutile transformation temperatures were dependent on the oxygen to helium flow rate ratio during CVS and the results are presented and discussed.

Clinical characterisation of a family with retinal dystrophy caused by mutation in the Mertk gene.

BACKGROUND/AIM: MERTK, a tyrosine kinase receptor protein expressed by the retinal pigment epithelium (RPE), is mutated in both rodent models and humans affected by retinal disease. This study reports a survey of families for Mertk mutations and describes the phenotype exhibited by one family. METHODS: 96 probands with retinal dystrophy, consistent with autosomal recessive segregation, were screened by direct sequencing. A family homozygous for a likely null allele was investigated clinically. RESULTS: A novel frame shifting deletion was identified in one of 96 probands. Other polymorphisms were detected. The deletion allele occurred on both chromosomes of four affected family members. Electrophysiology demonstrated early loss of scotopic and macular function with later loss of photopic function. Visual acuities and visual fields were preserved into the second decade. Perception of light vision was present in a patient in the fourth decade. A "bull's eye" appearance and a hyperautofluorescent lesion at the central macula were consistent clinical findings. CONCLUSIONS: Mutations in Mertk are a rare cause of ARRP in humans. The study extends the phenotypic characteristics of this retinal dystrophy and shows distinctive clinical signs that may improve its clinical identification. The moderate severity and presence of autofluorescence implies that outer segment phagocytosis is not entirely absent.

A novel GJA8 mutation is associated with autosomal dominant lamellar pulverulent cataract: further evidence for gap junction dysfunction in human cataract.

PURPOSE: To identify the gene responsible for autosomal dominant lamellar pulverulent cataract in a four-generation British family and characterise the functional and cellular consequences of the mutation. METHODS: Linkage analysis was used to identify the disease locus. The GJA8 gene was sequenced directly. Functional behaviour and cellular trafficking of connexins were examined by expression in Xenopus oocytes and HeLa cells. RESULTS: A 262C>A transition that resulted in the replacement of proline by glutamine (P88Q) in the coding region of connexin50 (Cx50) was identified. hCx50P88Q did not induce intercellular conductance and significantly inhibited gap junctional activity of co-expressed wild type hCx50 RNA in paired Xenopus oocytes. In transfected cells, immunoreactive hCx50P88Q was confined to the cytoplasm but showed a temperature sensitive localisation at gap junctional plaques. CONCLUSIONS: The pulverulent cataract described in this family is associated with a novel GJA8 mutation and has a different clinical phenotype from previously described GJA8 mutants. The cataract likely results from lack of gap junction function. The lack of function was associated with improper targeting to the plasma membrane, most probably due to protein misfolding.

The cytopathic 18f strain of Hepatitis A virus induces RNA degradation in FrhK4 cells.

The mechanism responsible for the induction of apoptosis by the rapidly replicating HM175/18f strain of Hepatitis A virus (HAV) was investigated. Full length HAV RNA and viral capsid protein VP1 were detected in 18f infected cells at earlier times post-infection than in HM175/clone 1 infected cells. Analysis of total cellular RNA from HM175/18f infected FrhK4 cells by denaturing agarose gel electrophoresis and Northern blot hybridization revealed extensive degradation of both the 28S and 18S ribosomal RNA (rRNA) molecules. Similar degradation was observed when these cells were infected with Human coxsackievirus B1, a fast replicating enterovirus. In contrast, the parental strain of 18f, HM175/clone 1 did not induce RNA degradation. Inhibition of RNA degradation correlated with inhibition of virus replication. The pattern of rRNA degradation resembled degradation of rRNAs by RNase L, an enzyme activated in interferon-treated cells following infection with certain viruses. Ribosomal RNA degradation was accompanied by the reduction in the levels of several cellular RNAs including those for beta-actin and glyceraldehyde-3-phosphate dehydrogenase, while the levels of c-myc and c-jun were higher. Interferon mRNAs could not be detected in either infected or mock-infected control cells, and STAT1, a key regulator of interferon action was not phosphorylated following virus infection. These results reveal a heretofore-undescribed pathway that involves the regulation of RNA degradation and apoptosis following HAV/18f replication in FrhK4 cells.

An immune response after intraocular administration of an adenoviral vector containing a beta galactosidase reporter gene slows retinal degeneration in the rd mouse.

BACKGROUND/AIMS: Retinal degenerations are a leading cause of blindness for which there are currently no effective treatments. This has stimulated interest in the investigation of gene therapy strategies for these diseases in a variety of animal models. A number of attempts have been made to prevent photoreceptor loss in the rd mouse model of retinal degeneration using adenoviral vectors containing either a copy of the missing functional gene or a gene encoding either a neurotrophic factor or an antiapoptotic factor. The authors have previously demonstrated that intraocular administration of an adenoviral vector containing a beta galactosidase gene (AV.LacZ) results in an immune response to viral gene products and beta galactosidase. Here the effect of the immune response on retinal degeneration is examined. METHODS: Juvenile rd mice were injected intravitreally with AV.LacZ and a proportion were depleted of either CD4+ or CD8+ T cells or both. Control animals were injected with PBS. The mice were sacrificed 10 and 20 days post-injection and their eyes embedded in paraffin wax and sectioned. RESULTS: 10 days after intravitreal injection of AV.LacZ, the outer nuclear layer contains an average of 2.5 rows compared with 1.5 in PBS injected animals (p<0.005). The protective effect of AV.LacZ is negated by immune suppression and does not extend beyond 20 days. CONCLUSION: An immune response to vector and transgene products is able to slow degeneration in the rd mouse. This phenomenon should be taken into account when analysing the degeneration in the rd mouse following gene transfer.

OPA1, encoding a dynamin-related GTPase, is mutated in autosomal dominant optic atrophy linked to chromosome 3q28.

Autosomal dominant optic atrophy (ADOA) is the most prevalent hereditary optic neuropathy resulting in progressive loss of visual acuity, centrocoecal scotoma and bilateral temporal atrophy of the optic nerve with an onset within the first two decades of life. The predominant locus for this disorder (OPA1; MIM 165500) has been mapped to a 1.4-cM interval on chromosome 3q28-q29 flanked by markers D3S3669 and D3S3562 (ref. 3). We established a PAC contig covering the entire OPA1 candidate region of approximately 1 Mb and a sequence skimming approach allowed us to identify a gene encoding a polypeptide of 960 amino acids with homology to dynamin-related GTPases. The gene comprises 28 coding exons and spans more than 40 kb of genomic sequence. Upon sequence analysis, we identified mutations in seven independent families with ADOA. The mutations include missense and nonsense alterations, deletions and insertions, which all segregate with the disease in these families. Because most mutations probably represent null alleles, dominant inheritance of the disease may result from haploinsufficiency of OPA1. OPA1 is widely expressed and is most abundant in the retina. The presence of consensus signal peptide sequences suggests that the product of the gene OPA1 is targeted to mitochondria and may exert its function in mitochondrial biogenesis and stabilization of mitochondrial membrane integrity.

Restoration of photoreceptor ultrastructure and function in retinal degeneration slow mice by gene therapy.

The gene Prph2 encodes a photoreceptor-specific membrane glycoprotein, peripherin-2 (also known as peripherin/rds), which is inserted into the rims of photoreceptor outer segment discs in a complex with rom-1 (ref. 2). The complex is necessary for the stabilization of the discs, which are renewed constantly throughout life, and which contain the visual pigments necessary for photon capture. Mutations in Prph2 have been shown to result in a variety of photoreceptor dystrophies, including autosomal dominant retinitis pigmentosa and macular dystrophy. A common feature of these diseases is the loss of photoreceptor function, also seen in the retinal degeneration slow (rds or Prph2 Rd2/Rd2) mouse, which is homozygous for a null mutation in Prph2. It is characterized by a complete failure to develop photoreceptor discs and outer segments, downregulation of rhodopsin and apoptotic loss of photoreceptor cells. The electroretinograms (ERGs) of Prph2Rd2/Rd2 mice have greatly diminished a-wave and b-wave amplitudes, which decline to virtually undetectable concentrations by two months. Subretinal injection of recombinant adeno-associated virus (AAV) encoding a Prph2 transgene results in stable generation of outer segment structures and formation of new stacks of discs containing both perpherin-2 and rhodopsin, which in many cases are morphologically similar to normal outer segments. Moreover, the re-establishment of the structural integrity of the photoreceptor layer also results in electrophysiological correction. These studies demonstrate for the first time that a complex ultrastructural cell defect can be corrected both morphologically and functionally by in vivo gene transfer.

TGF-beta1, -beta2, and -beta3 in vitro: biphasic effects on Tenon's fibroblast contraction, proliferation, and migration.

PURPOSE: To compare the effects of the three human transforming growth factor-beta (TGF-beta) isoforms and different concentrations of TGF-beta on human Tenon's capsule fibroblasts (HTF), with a view to delineating the role of this growth factor in the subconjunctival scarring response after glaucoma filtration surgery. METHODS: Application of recombinant human TGF-beta1, -beta2, and -beta3 (range 0-10(-8) M) was assessed using several assays of HTF function: fibroblast-mediated collagen contraction, proliferation, and migration. RESULTS: All three isoforms of TGF-beta behaved in a similar manner in vitro. They each stimulated HTF-mediated collagen contraction, proliferation, and migration with a characteristic concentration-dependent response, with peak activities at 10(-9), 10(-12), and 10(-9) M, respectively, that were significantly different from control (P<0.05). At concentrations above and below peak activities, HTF activity was reduced, demonstrating biphasic effects of TGF-beta. CONCLUSIONS: TGF-beta1, -beta2, and -beta3 have similar actions in vitro; this is demonstrated by their effects on several HTF-mediated functions. TGF-beta induces a response in HTF that is concentration-dependent, with different functions being maximally stimulated at different concentrations. This biphasic response highlights the significance of the concentration profile of TGF-beta at the wound site. These findings are important in filtration surgery, where constant changes in the local environment occur due to the passage of aqueous and the wound healing process. The varying levels of TGF-beta in the aqueous and subconjunctival tissues may thus significantly modify the conjunctival scarring response.

Refined genetic and physical positioning of the gene for Doyne honeycomb retinal dystrophy (DHRD).

Doyne honeycomb retinal dystrophy (DHRD) is a late-onset autosomal dominant disorder that causes degeneration of the retina and can lead to blindness. We have previously assigned DHRD to a 5-cM region of chromosome 2p16 between marker loci D2S2739 and D2S378. Using sequence-tagged sites (STSs), expressed sequence tags (ESTs) and polymorphic markers within the DHRD region, we have identified 18 yeast artificial chromosomes (YACs) encompassing the DHRD locus, spanning approximately 3 Mb. The YAC contig was constructed by STS content mapping of these YACs and incorporates 13 STSs, including four genes and six polymorphic marker loci. We also report the genetic mapping of two families with a dominant drusen phenotype to the DHRD locus, and genetic refinement of the disease locus to a critical interval flanked by microsatellite marker loci D2S2352 and D2S2251, a distance of approximately 700 kb. These studies exclude a number of candidate genes and provide a resource for construction of a transcriptional map of the region, as a prerequisite to identification of the DHRD disease-causing gene and genes for other diseases mapping in the region, such as Malattia leventinese and Carney complex.

Absence of p53 delays apoptotic photoreceptor cell death in the rds mouse.

PURPOSE: This study was aimed at determining whether or not apoptotic photoreceptor cell death in a mouse model of inherited retinal degeneration is p53 dependent. METHODS: A colony of p53-deficient rds mice were obtained by crossing homozygous rds mice with animals homozygous for a targeted disruption of the p53 gene and genotyping the offspring of the F1 cross. Both parental strains were on a BALB/c background. Age matched p53-deficient rds mice and controls (p53-deficient, rds and BALB/c mice), were sacrificed from day 1 to day 58 after birth. Eyes were paraffin-embedded and a modified terminal dUTP nick-end labeling (TUNEL) technique was used to detect the number of cells displaying DNA fragmentation within the sectioned retina. Eyes were also resin-embedded for semi-thin and ultra-thin sectioning. RESULTS: The peak in photoreceptor apoptosis, which occurs at 16 days in the rds mouse, was delayed by 3 days in p53-deficient rds mice. In addition, there was also a delay in the loss of photoreceptor cells between 16 and 26 days. However, absence of p53 did not prevent retinal degeneration in the rds mouse. The number of photoreceptor cells in p53-deficient rds mice at 35 days was very similar to that in the controls. CONCLUSIONS: We have demonstrated that absence of p53 delays but does not prevent photoreceptor cell loss in the rds mouse. Our results provide evidence for plasticity in the mechanism by which apoptosis proceeds in retinal degeneration.

A mutation in guanylate cyclase activator 1A (GUCA1A) in an autosomal dominant cone dystrophy pedigree mapping to a new locus on chromosome 6p21.1.

We report a mutation (Y99C) in guanylate cyclase activator 1A (GUCA1A), the gene for guanylate cyclase activating protein (GCAP1), in a family with autosomal dominant cone dystrophy. Linkage analysis excluded all the known cone and cone-rod dystrophy loci, except the chromosome 6p21.1 region. This is known to contain the RDS gene, which is associated with dominant cone-rod dystrophy. Screening of the RDS gene by heteroduplex analysis and direct sequencing failed to demonstrate sequence changes in the coding region of this gene. The gene for GCAP1, a calcium binding protein which is highly expressed in photoreceptor outer segments, is also located in 6p21.1. It was screened for mutations, and all affected individuals showed a single base pair missense mutation (A-->G) at codon 99 in exon 2 of this gene generating a tyrosine-to-cysteine change in the GCAP1 protein. This change was absent from 206 unrelated normal controls. We propose that this change would at least disrupt the EF3handof GCAP1 thereby preventing calcium binding and consequently interfere with activation. The resulting effect on cGMP production would predictably modify the number of open cGMP gated cation channels, and could explain the ultimate demise of cone photoreceptor cells.

Adeno-associated virus gene transfer to mouse retina.

Ocular gene transfer may provide a means for arresting the retinal degeneration characteristic of many inherited causes of blindness, including retinitis pigmentosa (RP). Previously, we have shown in immunodeficient animals that recombinant adeno-associated virus (rAAV) mediates transduction of photoreceptors as well as the retinal pigment epithelium (RPE) following subretinal injection. In this study we extend these observations and show that highly purified recombinant AAV vectors encoding the reporter gene LacZ transduce photoreceptors in an immunocompetent mouse strain following subretinal injection and efficiently transduce ganglion cells after intravitreal injection. Levels of transduction increase over time. Sublethal gamma-irradiation is shown to facilitate this process.

Co-injection of adenovirus expressing CTLA4-Ig prolongs adenovirally mediated lacZ reporter gene expression in the mouse retina.

There is growing interest in gene delivery to the eye in order to develop gene therapy for the many ocular disorders which may be amenable to this approach. To date, recombinant adenoviruses (AV) have been the main vector used for gene delivery to anterior and posterior segments in animal models. As with delivery to other organs, immune responses to vector and transgene limit the duration of expression in the eye. Using an E1-deleted adenoviral vector carrying a lacZ reporter gene, we have previously demonstrated that a T cell-mediated immune response reduces the level of intra-ocular transgene expression over time and limits it to around 3 weeks in mice. This report describes a strategy for prolonging gene expression by blocking the B7-CD28 interactions between antigen presenting cells (APC) and T cells in order to prevent the costimulatory signals required for T cell survival and proliferation. This was achieved by the co-injection of AV encoding a secreted immunomodulatory molecule (CTLA4-Ig) which consists of the extra-cellular domain of mouse CTLA4 fused to the Fc region of human IgG. Subretinal co-injection of AV encoding beta galactosidase with AV encoding CTLA4-Ig results in prolonged expression in retinal cells compared with subretinal injection of only adenovirus encoding beta galactosidase.

High frequency of persistent hyperplastic primary vitreous and cataracts in p53-deficient mice.

In order to investigate whether the p53 gene product plays a role in normal eye development, age matched p53-deficient mice and wild-type controls were sacrificed from day 2 to day 21 after birth. Eyes were paraffin-embedded and sectioned. Serial sections were taken at the level of the tunica vasculosa lentis and the hyaloid artery. The terminal dUTP nick-end labelling technique (TUNEL) was used to detect the number of cells displaying DNA fragmentation within these structures. Eyes were also prepared for scanning electron microscopy and resin embedded for semi-thin sections. Adult wild-type mice and p53-deficient mice were examined ophthalmoscopically in vivo. Ophthalmoscopical examination of mice completely deficient in p53 revealed them to be normal except for the persistence of the hyaloid vasculature, a structure that normally regresses during eye development. In adult animals there was also a high frequency of cataracts. Using morphological assessment and TUNEL we could show that in normal mice, regression of the primary vitreous, which includes the hyaloid artery, the vasa hyaloidea propria as well as the tunica vasculosa lentis, occurs via apoptotic cell death within 5 - 6 weeks after birth. The number of TUNEL-positive cells within these structures was significantly reduced in the p53-deficient mice in which parts of the hyaloid vasculature persisted and developed into a fibro-vascular retrolental plaque analogous to persistent hyperplastic primary vitreous (PHPV) described in humans. As in humans, PHPV in mice resulted in the development of cataracts. We have identified a role for p53-dependent apoptosis in the regression of the hyaloid vasculature and tunica vasculosa lentis. Our results provide further evidence for the importance of p53 in normal development and provide the first detailed evidence of its role in postnatal development in remodelling the developing eye.

Immune responses limit adenovirally mediated gene expression in the adult mouse eye.

In order to investigate the immunological consequences of gene transfer to the eye using viral vectors, adenovirus carrying a lacZ reporter gene (AV.LacZ) was injected either subretinally, subconjunctivally or into the anterior chamber of three groups of adult mice: immunocompetent or transiently immunosuppressed BALB/c mice and congenic immunodeficient nude mice. Adenovirally mediated lacZ expression persisted for approximately 3 weeks following injection of the vector into the anterior chamber, retina or extra ocular tissues of the conjuctiva of BALB/c mice. It appears that T cell-mediated immune responses limit the duration of AV-mediated ocular gene expression in adult mice since lacZ gene expression was detected for at least 15 weeks in T cell-deficient BALB/c nude mice, although the level of transgene expression decreased with time. Since intra-ocular AV-mediated gene expression was not significantly longer than extra-ocular expression, it appears that the eye is not normally immune-privileged with respect to viral vectors. Inflammatory cells were detected in the vitreous after anterior chamber injection and in the retina after subretinal injection of adenovirus. The presence of both CD4+ and CD8+ T cells was established by immunophenotyping. Reinjection of BALB/c mice resulted in rapid decline in reporter gene expression, but successful readministration was possible in the case of immunodeficient nude mice. However, after transient depletion of T cells, achieved by intraperitoneal injection of both CD8- and CD4-specific antibodies, the duration of expression in BALB/c mice was longer in the eye (at least 12 weeks, again with decrease in level over time), than in extra-ocular tissues (8 weeks) provided the animal was not reinjected with virus raising the possibility of partial ocular immune-privilege after transient immunosuppression.

New model of conjunctival scarring in the mouse eye.

AIMS: To establish a simple model of conjunctival wound healing in the mouse eye. METHODS: 4 week old BABL/c mouse eyes were studied over a 14 day period. Surgical procedure under general anaesthesia involved a blunt dissection of the conjunctiva performed by injection of 25 microliters of PBS via a 27 gauge needle into one eye, while the contralateral eye was used as control. Mice were assessed clinically and sacrificed at 1, 2, 3, 7, and 14 days after surgery. Enucleated eyes were prepared for histological analysis. Development of scar tissue was studied with haematoxylin and eosin, oxidation aldehyde fuchsin, and van Gieson stains, with assessment of cellularity, extracellular matrix formation, and wound characterisation. RESULTS: Histological analysis revealed a marked and characteristic healing response initiated by a predominantly granulocytic inflammatory reaction at day 1 with peak fibroblast activity 3 days after surgery. Oxytalan fibres and newly laid collagen fibres were detected early in the subconjunctival wound area and up to 7 days after surgery. Remodelling and complete organisation of scar tissue was evident by day 14. CONCLUSION: A single subconjunctival injection in the mouse eye results in a marked and consistent healing response. This represents a simple, inexpensive, and reliable animal model of conjunctival scarring. The mouse is a biologically well characterised animal model and allows the use of a wide variety of molecular tools. There are potentially significant clinical applications, in particular in investigating the effects of modulating agents such as antimetabolites, growth factors, and their antagonists on conjunctival scarring.

Single exposures to antiproliferatives: long-term effects on ocular fibroblast wound-healing behavior.

PURPOSE: To determine the long-term effects of single, 5-minute exposures to 5-fluorouracil (5FU) and mitomycin-C (MMC) on Tenon's capsule fibroblast migration, growth factor production, growth factor receptor expression, and extracellular matrix (ECM) production. METHODS: Monolayer cultures and the overlying growth medium of Tenon's capsule fibroblasts exposed to 5FU (0.25 to 25 mg/ml) or MMC (0.001 to 0.1 mg/ml) were harvested up to 48 days after treatment. The expression of growth factors and growth factor receptors, including transforming growth factor beta (TGFbeta), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF), and ECM molecules (collagen type I, collagen type III, and fibronectin) were quantitated at the mRNA and protein levels. The ability of fibroblasts exposed to 5FU and MMC to migrate to fetal calf serum was also investigated up to 48 days after treatment. RESULTS: Control cultures were found to produce the growth factors TGFbeta and bFGF but not EGF. Exposure to 5FU or MMC resulted in an initial significant increase (P < 0.05) in the production of TGFbeta and bFGF, with levels then decreasing toward those of controls. Cells exposed to 5FU or MMC exhibited an initial significant decrease (P < 0.05) in the number of TGFbeta, bFGF, and EGF growth factor receptors, with subsequent recovery toward control levels by day 48 after treatment. Both 5FU and MMC caused a significant reduction (P < 0.05) in collagen type I and fibronectin production compared to controls throughout the 48-day culture period. The production of collagen type III was initially elevated (P < 0.05) compared to controls after exposure to 5FU or MMC, production then decreasing toward control levels over the remainder of the 48-day culture period. The migration of cells exposed to 5FU or MMC was significantly reduced (P < 0.05) compared to controls up to 48 days after treatment; these cells exhibited a partial recovery of migratory ability throughout this period. CONCLUSIONS: Fibroblasts whose growth was arrested using single, short exposures to 5FU or MMC appear to be capable of performing several crucial aspects of wound healing, including the expression of growth factors and receptors and ECM molecules and the ability to migrate. These findings may help explain why in some patients treated with antiproliferatives, glaucoma filtration surgery fails because of scarring.