Particle radiation alters expression of matrix metalloproteases resulting in ECM remodeling in human lens cells.

Relatively low doses of space radiation have been correlated with an increased incidence and earlier appearance of cataracts in space travelers. The lens is a radiosensitive organ of the body with a very obvious late end point of radiation damage--cataract. However, many molecular changes occur in the lens soon after radiation exposure and long before the appearance of an opacification. The goal of our research is to elucidate early mechanisms associated with particle radiation-induced cataractogenesis, with the ultimate goal of developing countermeasures. Normal, cultured non-immortalized human lens cells were grown on matrix-coated plastic tissue culture vessels and irradiated with particle beams at Lawrence Berkeley National Lab (LBNL) or at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Lab. Samples were harvested at different times after radiation exposure. Using a focused genetic approach, total RNA and protein extracts from control and irradiated samples were processed and probed for the expression of genes associated with extracellular matrix (ECM) proteases. Matrix metalloproteinases (MMPs) have previously been studied in adult postmortem human lenses, in post-cataract intraocular lens (IOL) surgery capsular bags and with immortalized human lens cell cultures. Significant differences exist in the expression pattern with these various model systems. We have evidence for the cell stage-specific expression of MMP family of genes during lens fiber differentiation, and for radiation-induced alterations in the misregulation of MMP expression. Our data indicate that radiation exposure may lead to differences in the expression of radiation stress responses, which may impact selective ECM remodeling and cell differentiation.

Effects of iron ions, protons and X rays on human lens cell differentiation.

We have investigated molecular changes in cultured differentiating human lens epithelial cells exposed to high-energy accelerated iron-ion beams as well as to protons and X rays. In this paper, we present results on the effects of radiation on gene families that include or are related to DNA damage, cell cycle regulators, cell adhesion molecules, and cell cytoskeletal function. A limited microarray survey with a panel of cell cycle-regulated genes illustrates that irradiation with protons altered the gene expression pattern of human lens epithelial cells. A focus of our work is CDKN1A (p21(CIP1/WAF1)), a protein that we demonstrate here has a role in several pathways functionally related to LET-responsive radiation damage. We quantitatively assessed RNA and protein expression in a time course before and after single 4-Gy radiation doses and demonstrated that transcription and translation of CDKN1A are both temporally regulated after exposure. Furthermore, we show qualitative differences in the distribution of CDKN1A immunofluorescence signals after exposure to X rays, protons or iron ions, suggesting that LET effects likely play a role in the misregulation of gene function in these cells. A model of molecular and cellular events is proposed to account for precataractous changes in the human lens after exposure to low- or high-LET radiations.

Modulation of lens cell adhesion molecules by particle beams.

Cell adhesion molecules (CAMs) are proteins which anchor cells to each other and to the extracellular matrix (ECM), but whose functions also include signal transduction, differentiation, and apoptosis. We are testing a hypothesis that particle radiations modulate CAM expression and this contributes to radiation-induced lens opacification. We observed dose-dependent changes in the expression of beta 1-integrin and ICAM-1 in exponentially-growing and confluent cells of a differentiating human lens epithelial cell model after exposure to particle beams. Human lens epithelial (HLE) cells, less than 10 passages after their initial culture from fetal tissue, were grown on bovine corneal endothelial cell-derived ECM in medium containing 15% fetal bovine serum and supplemented with 5 ng/ml basic fibroblast growth factor (FGF-2). Multiple cell populations at three different stages of differentiation were prepared for experiment: cells in exponential growth, and cells at 5 and 10 days post-confluence. The differentiation status of cells was characterized morphologically by digital image analysis, and biochemically by Western blotting using lens epithelial and fiber cell-specific markers. Cultures were irradiated with single doses (4, 8 or 12 Gy) of 55 MeV protons and, along with unirradiated control samples, were fixed using -20 degrees C methanol at 6 hours after exposure. Replicate experiments and similar experiments with helium ions are in progress. The intracellular localization of beta 1-integrin and ICAM-1 was detected by immunofluorescence using monoclonal antibodies specific for each CAM. Cells known to express each CAM were also processed as positive controls. Both exponentially-growing and confluent, differentiating cells demonstrated a dramatic proton-dose-dependent modulation (upregulation for exponential cells, downregulation for confluent cells) and a change in the intracellular distribution of the beta 1-integrin, compared to unirradiated controls. In contrast, there was a dose-dependent increase in ICAM-1 immunofluorescence in confluent, but not exponentially-growing cells. These results suggest that proton irradiation downregulates beta 1-integrin and upregulates ICAM-1, potentially contributing to cell death or to aberrant differentiation via modulation of anchorage and/or signal transduction functions. Quantification of the expression levels of the CAMs by Western analysis is in progress.

Growth and differentiation of human lens epithelial cells in vitro on matrix.

PURPOSE: To characterize the growth and maturation of nonimmortalized human lens epithelial (HLE) cells grown in vitro. METHODS: HLE cells, established from 18-week prenatal lenses, were maintained on bovine corneal endothelial (BCE) extracellular matrix (ECM) in medium supplemented with basic fibroblast growth factor (FGF-2). The identity, growth, and differentiation of the cultures were characterized by karyotyping, cell morphology, and growth kinetics studies, reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence, and Western blot analysis. RESULTS: HLE cells had a male, human diploid (2N = 46) karyotype. The population-doubling time of exponentially growing cells was 24 hours. After 15 days in culture, cell morphology changed, and lentoid formation was evident. Reverse transcription-polymerase chain reaction (RT-PCR) indicated expression of alphaA- and betaB2-crystallin, fibroblast growth factor receptor 1 (FGFR1), and major intrinsic protein (MIP26) in exponential growth. Western analyses of protein extracts show positive expression of three immunologically distinct classes of crystallin proteins (alphaA-, alphaB-, and betaB2-crystallin) with time in culture. By Western blot analysis, expression of p57(KIP2), a known marker of terminally differentiated fiber cells, was detectable in exponential cultures, and levels increased after confluence. MIP26 and gamma-crystallin protein expression was detected in confluent cultures, by using immunofluorescence, but not in exponentially growing cells. CONCLUSIONS: HLE cells can be maintained for up to 4 months on ECM derived from BCE cells in medium containing FGF-2. With time in culture, the cells demonstrate morphologic characteristics of, and express protein markers for, lens fiber cell differentiation. This in vitro model will be useful for investigations of radiation-induced cataractogenesis and other studies of lens toxicity.

Particle irradiation induces FGF2 expression in normal human lens cells.

Particle Irradiation Induces FGF2 Expression in Normal Human Lens Cells. Particle radiations, including both proton and helium-ion beams, have been used to successfully treat choroidal melanoma, but with the complication of radiation-induced cataract. We have investigated a role for radiation-induced changes in the expression of basic fibroblast growth factor (FGF2) gene expression as part of the mechanism(s) underlying lens cell injury associated with cataract. Normal human lens epithelial (HLE) cells were cultured in vitro on extracellular matrix (ECM) originated from bovine corneal endothelial cells. This study reports evidence for rapid but transient induction of FGF2 transcripts, an increase of between 5- and 8-fold, within 0.5 h after exposure to particle radiation, followed by another wave of increased transcription at 2-3 h postirradiation. Immunofluorescence results confirm the enhanced levels of FGF2 protein rapidly after exposure to protons or helium ions, followed by another wave of increased activity unique to helium at 6 h postirradiation. This second wave of increased immunoreactivity was not observed in the proton-irradiated samples. Total FGF2 protein analysis after helium-ion exposures shows induced expression of three FGF2 isoforms, with an increase of up to 2-fold in the 18-kDa low-molecular-weight species. Studies of the effects of protons on individual FGF2 protein isoforms are in progress. Several mechanisms involving a role for FGF2 in radiation-induced cataract are discussed.

Human glioblastoma cell lines: levels of low-density lipoprotein receptor and low-density lipoprotein receptor-related protein.

The status of the low-density lipoprotein (LDL) receptor and LDL receptor-related protein (LRP) in seven human glioma cell lines was evaluated to extend our knowledge of human glioblastoma multiforme tumor metabolism for future drug design. Cell lines SF-767, SF-763, A-172, U-87 MG, U-251 MG, U-343 MG, and SF-539 were used. Binding of 125I-labeled LDL to these cells at 4 degrees C was carried out to determine the number of LDL receptors on cells and the affinity of LDL for these receptors. The content of LRP was measured by immunoblotting. The presence of specific saturable LDL receptors was proven in six of the cell lines investigated. SF-767 cells revealed high-affinity LDL binding (equilibrium dissociation constant, Kd = 7 nM) and maximum binding capacity approximating 300,000 receptors/cell. Most of the remaining cell lines had relatively lower affinity (Kd = 38-62 nM) but also had very high numbers of receptors (128,000-950,000/cell). All cell lines exhibited LRP, but the expression was variable. The cell lines SF-539, U-87 MG, and U-343 MG were particularly rich in this protein. The data suggest that glioblastoma cells have high numbers of LDL receptors; however, there is considerable variation in binding affinity. Overall, this finding suggests that LDL receptors on glioblastoma cells could potentially be useful for targeting antitumor agents. LRP, a multifunctional receptor expressed on glioblastoma cells, also has the possibility for serving as a therapeutic target.

Boronated protoporphyrin (BOPP): localization in lysosomes of the human glioma cell line SF-767 with uptake modulated by lipoprotein levels.

PURPOSE: Boronated protoporphyrin (BOPP) is a candidate for use in both boron neutron capture therapy (BNCT) and photodynamic therapy (PDT) of glioblastoma multiforme (GBM). Our objectives are to identify factors that influence the uptake and retention of BOPP in vitro and to determine BOPP distribution in a human glioma cell line in vitro. This information will aid the development of compounds and treatment strategies that increase the effectiveness of BNCT therapy for GBM. METHODS AND MATERIALS: The amount, distribution pattern, and site of internalization of BOPP were assessed using fluorescence microscopy. Living human glioma (SF-767) cells were imaged after a 24-h exposure to BOPP (20-135.6 microg/ml, normal serum). Dose-dependent uptake of BOPP was determined using both fluorescence microscopy of individual living cells and inductively-coupled plasma-atomic emission spectroscopy (ICP-AES) analysis of cell pellets. Lysosome- or mitochondria-specific fluorescent probes were used to identify the cellular compartment containing BOPP. Two human fibroblast cell lines, AG-1522 (LDL receptor-positive) and GM019-15C (LDL receptor-deficient), were used to investigate LDL receptor-dependent BOPP uptake. The dependence of BOPP uptake on lipoproteins in the media was determined by exposing each of the three cell types to BOPP in medium containing either normal (NS) or lipoprotein deficient serum (LPDS). RESULTS: BOPP accumulated in the lysosomes of human glioma cells in vitro, and not in the mitochondria, as reported for C6 rat glioma cells in vitro. BOPP uptake was concentration-dependent and was also dependent on the amount of lipoproteins in the medium. Over the range of incubation concentrations studied and at the single exposure duration time point investigated (24 h), all cells retained a similar amount of BOPP. At the lowest incubation concentration (20 microg/ml, NS), the amount of boron retained was near 10(9) atoms per cell (15 microg B/g cells). Lysosomes containing high concentrations of BOPP were randomly distributed throughout the cytoplasm; however, larger lysosomes containing BOPP were concentrated around the cell nucleus. Little or no BOPP accumulated in the cell nucleus. At incubation concentrations of 20 and 40 microg/ml (24-h time point), BOPP uptake in SF-767 cells was reduced in LPDS compared with NS (66% reduction). A similar result was observed for normal human fibroblasts (AG-1522 cells, 40 microg/ml, 24 h). At 40 microg/ml, in both NS and LPDS at 24 h, BOPP accumulation in LDL receptor-deficient human fibroblasts (GM019-15C cells) was reduced relative to AG-1522 cells. BOPP accumulation in GM019-15C cells (40 microg/ml, 24 h) was not affected by serum lipoprotein levels. CONCLUSION: In cell culture, BOPP is taken up by human glioma cells via the LDL pathway and is compartmentalized into cellular lysosomes. Knowledge of this mechanism of BOPP uptake and retention will be important in attempts to modify toxicity and efficacy of this drug.

Involvement of protein synthesis in the development of thermotolerance using a CHO temperature-sensitive mutant.

A Chinese hamster ovary temperature-sensitive mutant (CHO-tsH1) with defective leucyl-tRNA synthetase at temperatures greater than 39 degrees C was used to examine the importance of protein synthesis in the development of thermotolerance. Its wild-type parent CHO-SC1 was used as the control. At temperatures of 41.5 degrees C, 42 degrees C and 42.5 degrees C, SC1 showed the classical biphasic thermotolerant response while tsH1 showed no thermotolerance. When both cell lines were heated for 15 min at 45 degrees C, then allowed to incubate at the permissive temperature of 35 degrees C and finally challenged with another 25 min treatment at 45 degrees C, tolerance was expressed in both cell lines. When the development incubation temperature was raised from 35 degrees C to the non-permissive temperature of 40 degrees C, tolerance was also observed. Although both cell lines expressed tolerance under these conditions, the magnitude and duration of response of the mutant cell line were reduced. Heat-shock protein analysis using one-dimensional gel electrophoresis showed that, under permissive conditions, the mutant cell was able to express the full spectrum of heat-shock proteins as seen in the wild-type cells. Under non-permissive conditions, little or no detectable proteins were synthesized in the mutant cell. We therefore postulate that the synthesis of new cytosol proteins is not required for the initial onset of thermotolerance but is necessary for the sustenance of tolerance.

The REC46 gene of Saccharomyces cerevisiae controls mitotic chromosomal stability, recombination and sporulation: cell-type and life cycle stage-specific expression of the rec46-1 mutation.

The recessive hyperrecombination mutation rec46-1, isolated by ultraviolet light mutagenesis of the MAT alpha n+1 chromosome VII disomic strain LBL1 (Esposito et al. 1982), enhances the mitotic rates of spontaneous gene conversion, intergenic recombination and restitution of haploidy (due to chromosomal loss or mitotic nondisjunction) in MAT alpha n+1 chromosome VII disomic strains. The rec46-1 mutation does not prevent HO directed homothallic interconversion of mating types. MATa/MaT alpha rec46-1/rec46-1 diploids exhibit the same degree of hyperrecombinational activity as MAT alpha rec46-1 n+1 chromosome VII disomics with respect to gene conversion and intragenic recombination resulting in prototrophy. When compared to MAT alpha rec46-1 n+1 disomics however, MATa/MAT alpha rec46-1/rec46-1 diploids exhibit a ten fold reduced level of hyperrecombinational activity with respect to intergenic recombination and present no evidence of chromosomal loss or nondisjunction resulting in 2n-1 monosomic segregants. MATa/MAT alpha rec46-1/rec46-1 diploids are sporulation-deficient. The results obtained demonstrate that the REC46 gene product modulates mitotic chromosomal stability and recombination and is essential for sporulation (meiosis and ascospore formation).

Simultaneous detection of changes in chromosome number, gene conversion and intergenic recombination during mitosis of Saccharomyces cerevisiae: spontaneous and ultraviolet light induced events.

We have employed a hyperhaploid strain of Saccharomyces cerevisiae disomic for chromosome VII to monitor spontaneous and ultraviolet light induced restitution of haploidy (chromosomal loss and/or nondisjunction), mitotic gene conversion and mitotic intergenic recombination. The disomic chromosomal pair incorporates six heterozygous markers, including cyh2 (r), distributed on both sides of the centromere. Cycloheximide resistant segregants of spontaneous origin were analyzed to calculate the spontaneous mitotic rates of restitution of haploidy, intergenic recombination and gene conversion that result in expression of the cyh2 (r) mutation. Restitution of haploidy was found to be the most common source of spontaneously arising cycloheximide resistant segregants. In contrast, those induced by ultraviolet light resulted most frequently from gene conversion of CYH2 (s) to cyh2 (r). The chromosome VII hyperhaploid system provides a sensitive method to detect the aneugenic and recombinagenic effects of suspect chemical and physical agents.