Lens Epithelial Cell Proliferation in Response to Ionizing Radiation.

Lens epithelial cell proliferation and differentiation are naturally well regulated and controlled, a characteristic essential for lens structure, symmetry and function. The effect of ionizing radiation on lens epithelial cell proliferation has been demonstrated in previous studies at high acute doses, but the effect of dose and dose rate on proliferation has not yet been considered. In this work, mice received single acute doses of 0.5, 1 and 2 Gy of radiation, at dose rates of 0.063 and 0.3 Gy/min. Eye lenses were isolated postirradiation at 30 min up until 14 days and flat-mounted. Then, cell proliferation rates were determined using biomarker Ki67. As expected, radiation increased cell proliferation 2 and 24 h postirradiation transiently (undetectable 14 days postirradiation) and was dose dependent (changes were very significant at 2 Gy; P = 0.008). A dose-rate effect did not reach significance in this study (P = 0.054). However, dose rate and lens epithelial cell region showed significant interactions (P < 0.001). These observations further our mechanistic understanding of how the lens responds to radiation.

Dotting the eyes: mouse strain dependency of the lens epithelium to low dose radiation-induced DNA damage.

PURPOSE: Epidemiological evidence regarding the radiosensitivity of the lens of the eye and radiation cataract development has led to changes in the EU Basic Safety Standards for protection of the lens against ionizing radiation. However, mechanistic details of lens radiation response pathways and their significance for cataractogenesis remain unclear. Radiation-induced DNA damage and the potential impairment of repair pathways within the lens epithelium, a cell monolayer that covers the anterior hemisphere of the lens, are likely to be involved. MATERIALS AND METHODS: In this work, the lens epithelium has been analyzed for its DNA double-strand break (DSB) repair response to ionizing radiation. The responses of epithelial cells located at the anterior pole (central region) have been compared to at the very periphery of the monolayer (germinative and transitional zones). Described here are the different responses in the two regions and across four strains (C57BL/6, 129S2, BALB/c and CBA/Ca) over a low dose (0-25 mGy) in-vivo whole body X-irradiation range up to 24 hours post exposure. RESULTS: DNA damage and repair as visualized through 53BP1 staining was present across the lens epithelium, although repair kinetics appeared non-uniform. Epithelial cells in the central region have significantly more 53BP1 foci. The sensitivities of different mouse strains have also been compared. CONCLUSIONS: 129S2 and BALB/c showed higher levels of DNA damage, with BALB/c showing significantly less inter-individual variability and appearing to be a more robust model for future DNA damage and repair studies. As a result of this study, BALB/c was identified as a suitable radiosensitive lens strain to detect and quantify early low dose ionizing radiation DNA damage effects in the mouse eye lens specifically, as an indicator of cataract formation.

Heat shock proteins are differentially expressed in brain and spinal cord: implications for multiple sclerosis.

Multiple sclerosis (MS) is a chronic neurodegenerative disease characterized by demyelination, inflammation and neurodegeneration throughout the central nervous system. Although spinal cord pathology is an important factor contributing to disease progression, few studies have examined MS lesions in the spinal cord and how they differ from brain lesions. In this study we have compared brain and spinal cord white (WM) and grey (GM) matter from MS and control tissues, focusing on small heat shock proteins (HSPB) and HSP16.2. Western blotting was used to examine protein levels of HSPB1, HSPB5, HSPB6, HSPB8 and HSP16.2 in brain and spinal cord from MS and age-matched non-neurological controls. Immunohistochemistry was used to examine expression of the HSPs in MS spinal cord lesions and controls. Expression levels were quantified using ImageJ. Western blotting revealed significantly higher levels of HSPB1, HSPB6 and HSPB8 in MS and control spinal cord compared to brain tissues. No differences in HSPB5 and HSP16.2 protein levels were observed, although HSPB5 protein levels were higher in brain WM versus GM. In MS spinal cord lesions, increased HSPB1 and HSPB5 expression was observed in astrocytes, and increased neuronal expression of HSP16.2 was observed in normal-appearing GM and type 1 GM lesions. The high constitutive expression of several HSPBs in spinal cord and increased expression of HSPBs and HSP16.2 in MS illustrate differences between brain and spinal cord in health and upon demyelination. Regional differences in HSP expression may reflect differences in astrocyte cytoskeleton composition and influence inflammation, possibly affecting the effectiveness of pharmacological agents.

THE IMPACT OF CIRCADIAN RHYTHMS ON MEDICAL IMAGING AND RADIOTHERAPY REGIMES FOR THE PAEDIATRIC PATIENT.

Daily rhythmic changes are found in cellular events in cell cycle, DNA repair, apoptosis and angiogenesis in both normal and tumour tissue, as well as in enzymatic activity and drug metabolism. In this paper, we hypothesize that circadian rhythms need to be considered in radiation protection and optimization in personalized medicine, especially for paediatric care. The sensitivity of the eye lens to ionizing radiation makes the case for limiting damage to the lens epithelium by planning medical radio-imaging procedures for the afternoon, rather than the morning. Equally, the tumour and normal tissue response to radiotherapy is also subject to diurnal variation enabling optimization of time of treatment.

The lens of the eye: exposures in the UK medical sector and mechanistic studies of radiation effects.

The recommendation from the International Commission on Radiological Protection that the occupational equivalent dose limit for the lens of the eye should be reduced to 20 mSv year(-1), averaged over 5 years with no year exceeding 50 mSv, has stimulated a discussion on the practicalities of implementation of this revised dose limit, and the most appropriate risk and protection framework to adopt. This brief paper provides an overview of some of the drivers behind the move to a lower recommended dose limit. The issue of implementation in the medical sector in the UK has been addressed through a small-scale survey of doses to the lens of the eye amongst interventional cardiologists and radiologists. In addition, a mechanistic study of early and late post-irradiation changes in the lens of the eye in in-vivo-exposed mice is outlined. Surveys and studies such as those described can contribute to a deeper understanding of fundamental and practical issues, and therefore contribute to a robust evidence base for ensuring adequate protection of the eye while avoiding undesirable restrictions to working practices.

Regulation of contractility by Hsp27 and Hic-5 in rat mesenteric small arteries.

The regulation of small artery contractility by vasoconstrictors is important for vascular function, and actin cytoskeleton remodeling is required for contraction. p38 MAPK and tyrosine kinases are implicated in actin polymerization and contraction through heat shock protein 27 (Hsp27) and the cytoskeletal protein paxillin, respectively. We evaluated the roles of downstream targets of p38 MAPK and tyrosine kinases in cytoskeletal reorganization and contraction and whether the two signaling pathways regulate contraction independent of each other. We identified the expression of the paxillin homologue hydrogen peroxide-inducible clone-5 (Hic-5) and showed its activation by norepinephrine (NE) in a Src-dependent manner. Furthermore, we demonstrated a NE-induced interaction of proline-rich tyrosine kinase-2 (PYK2) but not Src or p125 focal adhesion kinase with Hic-5. This interaction was Src dependent, suggesting that Hic-5 was a substrate for PYK2 downstream from Src. The activation of Hic-5 induced its relocalization to the cytosol. The parallel activation of Hsp27 by NE was p38 MAPK dependent and led to its dissociation from actin filaments and translocation from membrane to cytosol and increased actin polymerization. Both Hsp27 and Hic-5 activation resulted in their association within the same time frame as NE-induced contraction, and the inhibition of either p38 MAPK or Src inhibited the interaction between Hsp27 and Hic-5 and the contractile response. Furthermore, combined p38 MAPK and Src inhibition had no greater effect on contraction than individual inhibition, suggesting that the two pathways act through a common mechanism. These data show that NE-induced activation and the association of Hsp27 and Hic-5 are required for the reorganization of the actin cytoskeleton and force development in small arteries.

Human keratin 8 mutations that disturb filament assembly observed in inflammatory bowel disease patients.

We have identified miss-sense mutations in keratin 8 in a subset of patients with inflammatory bowel disease (Crohn disease and ulcerative colitis). Inflammatory bowel diseases are a group of disorders that are polygenic in origin and involve intestinal epithelial breakdown. We investigated the possibility that these keratin mutations might contribute to the course of the disease by adversely affecting the keratin filament network that provides mechanical support to cells in epithelia. The mutations (Gly62 to Cys, Ile63 to Val and Lys464 to Asn) all lie outside the major mutation hotspots associated with severe disease in epidermal keratins, but using a combination of in vitro and cell culture assays we show that they all have detrimental effects on K8/K18 filament assembly in vitro and in cultured cells. The G62C mutation also gives rise to homodimer formation on oxidative stress to cultured intestinal epithelial cells, and homodimers are known to be polymerization incompetent. Impaired keratin assembly resulting from the K8 mutations found in some inflammatory bowel disease patients would be predicted to affect the maintenance and re-establishment of mechanical resilience in vivo, as required during keratin cytoskeleton remodeling in cell division and differentiation, which may lead to epithelial fragility in the gut. Simple epithelial keratins may thus be considered as candidates for genes contributing to a risk of inflammatory bowel disease.

Tropomodulin binds to filensin intermediate filaments.

Tropomodulin (Tmod) is an actin filament pointed end capping protein found in the membrane skeleton of lens fiber cells. We demonstrate that Tmod4 is able to bind the lens-specific intermediate filament protein, filensin, in either co-sedimentation or solid phase binding assays in a saturable fashion, but with low affinity and stoichiometry. Furthermore, Tmod4 does not bind the 53 kDa rod domain of filensin, nor to CP49, the obligate assembly partner of filensin. Finally, the binding of filensin to Tmod4 does not inhibit the actin capping activity of Tmod4 in vitro, suggesting that the two functions are not mutually exclusive.

Aniridia-associated translocations, DNase hypersensitivity, sequence comparison and transgenic analysis redefine the functional domain of PAX6.

The transcription factor PAX6 plays a critical, evolutionarily conserved role in eye, brain and olfactory development. Homozygous loss of PAX6 function affects all expressing tissues and is neonatally lethal; heterozygous null mutations cause aniridia in humans and the Small eye (Sey) phenotype in mice. Several upstream and intragenic PAX6 control elements have been defined, generally through transgenesis. However, aniridia cases with chromosomal rearrangements far downstream of an intact PAX6 gene suggested a requirement for additional cis-acting control for correct gene expression. The likely location of such elements is pinpointed through YAC transgenic studies. A 420 kb yeast artificial chromosome (YAC) clone, extending well beyond the most distant patient breakpoint, was previously shown to rescue homozygous Small eye lethality and correct the heterozygous eye phenotype. We now show that a 310 kb YAC clone, terminating just 5' of the breakpoint, fails to influence the Sey phenotypes. Using evolutionary sequence comparison, DNaseI hypersensitivity analysis and transgenic reporter studies, we have identified a region, >150 kb distal to the major PAX6 promoter P1, containing regulatory elements. Components of this downstream regulatory region drive reporter expression in distinct partial PAX6 patterns, indicating that the functional PAX6 gene domain extends far beyond the transcription unit.

Antiischemic effects of SB203580 are mediated through the inhibition of p38alpha mitogen-activated protein kinase: Evidence from ectopic expression of an inhibition-resistant kinase.

The aim of the present study was to determine whether the attenuation of myocardial ischemic injury by SB203580 is due to the inhibition of p38 mitogen-activated protein kinase (MAPK) or to other documented nonspecific effects of the drug. We made adenoviral vectors encoding the alpha isoform of p38 MAPK with or without site-directed mutations to prevent SB203580 binding and inhibition. In embryonal rat heart-derived cells and adult rat cardiocytes expressing wild-type p38alpha MAPK, injury was reduced significantly by SB203580 present during simulated ischemia. In contrast, SB203580 did not protect cells expressing the SB203580-resistant form of p38alpha MAPK. These observations suggest that SB203580-mediated protection depends on the inhibition of p38alpha MAPK.

Alpha-B crystallin gene (CRYAB) mutation causes dominant congenital posterior polar cataract in humans.

Congenital cataracts are an important cause of bilateral visual impairment in infants. In a four-generation family of English descent, we mapped dominant congenital posterior polar cataract to chromosome 11q22-q22.3. The maximum LOD score, 3.92 at recombination fraction 0, was obtained for marker D11S898, near the gene that encodes crystallin alpha-B protein (CRYAB). By sequencing the coding regions of CRYAB, we found in exon 3 a deletion mutation, 450delA, that is associated with cataract in this family. The mutation resulted in a frameshift in codon 150 and produced an aberrant protein consisting of 184 residues. This is the first report of a mutation, in this gene, resulting in isolated congenital cataract.

Expression of individual lamins in basal cell carcinomas of the skin.

In this study we used a unique collection of type specific anti-lamin antibodies to study lamin expression patterns in normal human skin and in skin derived from patients with basal cell carcinomas (BCCs). Lamin expression in serial sections from frozen tissue samples was investigated by single and double indirect immunofluorescence. In normal skin, lamin A was expressed in dermal fibroblasts and in suprabasal epithelial cells but was absent from all basal epithelial cells. Lamin C was expressed in dermal fibroblasts, suprabasal epithelial cells and a majority of basal epithelial cells. However, lamin C was not expressed in quiescent basal epithelial cells. Lamin B1 was expressed in all epithelial cells but was not expressed in dermal fibroblasts. Finally, lamin B2 was expressed in all epithelial cells but was not expressed in dermal fibroblasts. Finally, lamin B2 was expressed in all cell types in normal skin. Lamin expression was also investigated in a collection of 16 BCCs taken from a variety of body sites. Based upon patterns of lamin expression the BCCs were classified into four groups: A-negative (10/16 tumours), C-negative (5/16 tumours), A/C-negative (1/16 tumours) and A/B2-negative (1/16 tumours). Lamin expression was also compared to cell proliferation index by staining serial sections with the proliferation marker Ki67. 9/10 of the lamin A negative tumours were highly proliferative, whereas 4/5 of the lamin C negative tumours were slow growing. Thus as a general rule absence of lamin A was correlated with rapid growth within the tumour, while absence of lamin C was correlated with slow growth within the tumour. Our data supports the hypothesis that lamin A has a negative influence on cell proliferation and its down regulation may be a requisite of tumour progression.

Antagonistic action of Six3 and Prox1 at the gamma-crystallin promoter.

Gamma-crystallin genes are specifically expressed in the eye lens. Their promoters constitute excellent models to analyse tissue-specific gene expression. We investigated murine CRYGE/f promoters of different length in lens epithelial cell lines. The most active fragment extends from position -219 to +37. Computer analysis predicts homeodomain and paired-domain binding sites for all rodent CRYGD/e/f core promoters. As examples, we analysed the effects of Prox1 and Six3, which are considered important transcription factors involved in lens development. Because of endogenous Prox1 expression in N/N1003A cells, a weak stimulation of CRYGE/f promoter activity was found for PROX1. In contrast, PROX1 stimulated the CRYGF promoter 10-fold in CD5A cells without endogenous PROX1. In both cell lines Six3 repressed the CRYGF promoter to 10% of its basal activity. Our cell transfection experiments indicated that CRYG expression increases as Six3 expression decreases. Prox1 and Six3 act antagonistically on regulation of the CRYGD/e/f promoters. Functional assays using randomly mutated gammaF-crystallin promoter fragments define a Six3-responsive element between -101 and -123 and a Prox1-responsive element between -151 and -174. Since Prox1 and Six3 are present at the beginning of lens development, expression of CRYGD/e/f is predicted to remain low at this time. It increases as Six3 expression decreases during ongoing lens development.

Up-regulation of novel intermediate filament proteins in primary fiber cells: an indicator of all vertebrate lens fiber differentiation?

The early embryonic development and expression patterns of the eye lens specific cytoskeletal proteins, CP49 and CP95, were determined for the chick and were found to be similar in both human and mouse. These proteins, as well as their homologs in other species, are obligate polymerization partners which form unique filamentous structures termed "beaded filaments." CP49 and CP95 appeared as protein products after 3 days of embryonic development in the chick during the elongation of primary fiber cells. Although limited data were obtained for human embryos at these early developmental timepoints, they were consistent with the interpretation that the up-regulation of these lens specific proteins began only after the initiation of lens vesicle closure. In situ hybridization with the mouse lens confirmed that message levels for beaded filament proteins were greatly elevated in differentiating primary fiber cells. Nuclease protection assays established that mRNA levels for CP49 remained relatively constant while CP95 mRNA levels increased once the process of secondary fiber formation was under way. Although present in relatively low abundance, the mRNA for a unique splice variant of CP49, CP49(INS), was also detected early in embryonic development and into adulthood. Peptide-specific antibodies directed against unique predicted sequences were able to confirm the protein expression of CP49(INS) in both embryonic and adult chick lens cells. These data present the first detailed study of the expression of CP49 and CP95 during early lens development. They suggest that the up-regulated expression of CP49 and CP95 could serve as pan-specific markers for all vertebrate lens fiber development.

Mapping of the human CP49 gene and identification of an intragenic polymorphic marker to allow genetic linkage analysis in autosomal dominant congenital cataract.

The CP49 protein is an intermediate filament protein expressed specifically in the lens fibre cells of the lens, where it is an important cytoplasmic structural component. Dominant-negative mutations in other intermediate filament proteins, such as keratins, cause disorders characterised by dense cytoplasmic aggregates in specific cell types. The CP49 gene is therefore a good candidate for dominantly inherited forms of cataract. To allow genetic linkage analysis of families with autosomal dominant cataract with respect to CP49, a highly polymorphic intragenic microsatellite marker for this gene has been developed. In addition, both low and high resolution radiation hybrid mapping of the CP49 gene has been completed, placing it very close to microsatellite marker D3S1290 on human chromosome 3q. Furthermore, using the intragenic CP49 microsatellite, linkage was excluded in four families with genetically uncharacterized forms of autosomal dominant congenital cataract.

The cardiomyopathy and lens cataract mutation in alphaB-crystallin alters its protein structure, chaperone activity, and interaction with intermediate filaments in vitro.

Desmin-related myopathy and cataract are both caused by the R120G mutation in alphaB-crystallin. Desmin-related myopathy is one of several diseases characterized by the coaggregation of intermediate filaments with alphaB-crystallin, and it identifies intermediate filaments as important physiological substrates for alphaB-crystallin. Using recombinant human alphaB-crystallin, the effects of the disease-causing mutation R120G upon the structure and the chaperone activities of alphaB-crystallin are reported. The secondary, tertiary, and quaternary structural features of alphaB-crystallin are all altered by the mutation as deduced by near- and far-UV circular dichroism spectroscopy, size exclusion chromatography, and chymotryptic digestion assays. The R120G alphaB-crystallin is also less stable than wild type alphaB-crystallin to heat-induced denaturation. These structural changes coincide with a significant reduction in the in vitro chaperone activity of the mutant alphaB-crystallin protein, as assessed by temperature-induced protein aggregation assays. The mutation also significantly altered the interaction of alphaB-crystallin with intermediate filaments. It abolished the ability of alphaB-crystallin to prevent those filament-filament interactions required to induce gel formation while increasing alphaB-crystallin binding to assembled intermediate filaments. These activities are closely correlated to the observed disease pathologies characterized by filament aggregation accompanied by alphaB-crystallin binding. These studies provide important insight into the mechanism of alphaB-crystallin-induced aggregation of intermediate filaments that causes disease.

Gap junctions containing alpha8-connexin (MP70) in the adult mammalian lens epithelium suggests a re-evaluation of its role in the lens.

A missense mutation in one of the three lens connexins, alpha8-connexin, has been recently shown to be the genetic basis of the zonular pulverant lens cataract. This connexin had been considered to be expressed only in lens fibre cells. The present studies show that alpha8-connexin is also expressed in the lens epithelial cell layer. For this study, the distribution of gap junctions in the adult bovine lens has been investigated by confocal immunofluorescence microscopy using antibodies against alpha8-connexin (MP70) and alpha1-connexin (Cx43). In addition to the anticipated localisation of alpha8-connexin to the broad faces of lens fibre cells as reported in other species, alpha8-connexin was also found colocalized with alpha1-connexin at plaques in the lateral epithelial-epithelial plasma membranes of the bovine lens. These data suggest that mixed alpha8-connexin/alpha1-connexin plaques are between epithelial cells at their apico-lateral plasma membranes, rather than between epithelial and fibre cells. Indeed, freeze fracture analyses of the epithelial-fibre cell interface failed to reveal gap junctions connecting the epithelium and the underlying fibre cells. Importantly, microdissection and subsequent immunoblotting of lens epithelium samples confirmed the immunolocalisation results. The data suggest mature mammalian lens epithelial cells could form either heteromeric, heterotypic and/or mixed homomeric-homotypic gap junctional complexes with unique physiological properties, an important point when considering the role of epithelial cell connexins in cataractogenesis.

Use of a drug-resistant mutant of stress-activated protein kinase 2a/p38 to validate the in vivo specificity of SB 203580.

Stress-activated protein kinase 2a, also called p38, is inhibited by SB 203580 and this drug has been used widely to implicate this enzyme in the regulation of many physiological processes. Here, we introduce a novel method of general application, which can be used to establish whether the effects of SB 203580 are mediated via inhibition of stress-activated protein kinase 2a/p38 or whether they result from 'non-specific' effects. Four events thought to occur upon activation of stress-activated protein kinase 2a/p38 have been established unequivocally. These are the activation of mitogen-activated protein kinase-activated protein kinase-2 and mitogen- and stress-activated protein kinase-1 and the phosphorylation of their presumed substrates, heat shock protein 27 and the transcription factor cyclic AMP response element binding protein, respectively. In contrast, the SB 203580-induced activation of c-Raf is independent of stress-activated protein kinase 2a/p38 inhibition.

Intermediate filament interactions can be altered by HSP27 and alphaB-crystallin.

HSP27 and alphaB-crystallin are both members of the small heat shock protein family. alphaB-crystalllin has been proposed to modulate intermediate filaments and recently a mutation in alphaB-crystallin has been identified as the genetic basis of desmin related myopathy. This disease is characterised in its pathology by aggregates of intermediate filaments associated with alphaB-crystallin. Here we report that HSP27 like alphaB-crystallin is associated with glial fibrillary acidic protein and vimentin intermediate filament networks in unstressed U373MG astrocytoma cells. HSP27 is also associated with keratin filaments in MCF7 cells, indicating that this association is not restricted to a particular intermediate filament type. The association of sHSPs with both the soluble and filamentous intermediate filament fractions of U373 cells was demonstrated biochemically. Heat shock or drug treatments induced a co-collapse of intermediate filaments and associated small heat shock proteins. These data show that the presence of HSP27 or alphaB-crystallin could not prevent filament collapse and suggest that the purpose of this association is more than just filament binding. Indeed, in U373MG cells the intermediate filament association with small heat shock proteins is similar to that observed for another protein chaperone, HSC70. In order to discern the effect of different chaperone classes on intermediate filament network formation and maintenance, several in vitro assays were assessed. Of these, falling ball viscometry revealed a specific activity of small heat shock proteins compared to HSC70 that was apparently inactive in this assay. Intermediate filaments form a gel in the absence of small heat shock proteins. In contrast, inclusion of alphaB-crystallin or HSP27 prevented gel formation but not filament assembly. The transient transfection of GFAP into MCF7 cells was used to show that the induction of a completely separate network of intermediate filaments resulted in the specific association of the endogenous HSP27 with these new GFAP filaments. These data lead us to propose that one of the major functions of the association of small heat shock proteins with intermediate filaments is to help manage the interactions that occur between filaments in their cellular networks. This is achieved by protecting filaments against those non-covalent interactions that result when they come into very close proximity as seen from the viscosity experiments and which have the potential to induce intermediate filament aggregation as seen in some disease pathologies.

Molecular chaperones: small heat shock proteins in the limelight.

Small heat shock proteins have been the Cinderellas of the molecular chaperone world, but now the crystal structure of a small heat shock protein has been solved and mutation of two human homologues implicated in genetic disease. Intermediate filaments appear to be one of the key targets of their chaperone activity.