Mutations in the glutathione synthetase gene cause 5-oxoprolinuria.

5-Oxoprolinuria (pyroglutamic aciduria) resulting from glutathione synthetase (GSS) deficiency is an inherited autosomal recessive disorder characterized, in its severe form, by massive urinary excretion of 5-oxoproline, metabolic acidosis, haemolytic anaemia and central nervous system damage. The metabolic defect results in low GSH levels presumably with feedback over-stimulation of gamma-glutamylcysteine synthesis and its subsequent conversion to 5-oxoproline. In this study, we cloned and characterized the human GSS gene and examined three families with four cases of well-documented 5-oxoprolinuria. We identified seven mutations at the GSS locus on six alleles: one splice site mutation, two deletions and four missense mutations. Bacterial expression and yeast complementation assays of the cDNAs encoded by these alleles demonstrated their functional defects. We also characterized a fifth case, an homozygous missense mutation in the gene in an individual affected by a milder-form of the GSS deficiency, which is apparently restricted to erythrocytes and only associated with haemolytic anaemia. Our data provide the first molecular genetic analysis of 5-oxoprolinuria and demonstrate that GSS deficiency with oxoprolinuria and GSS deficiency without 5-oxoprolinuria are caused by mutations in the same gene.

Formation and distribution of nuclear pore complexes in interphase.

The possibility of nuclear pore formation in the interphase nucleus was investigated in control and phytohemagglutinin (PHA) stimulated lymphocytes by the freeze-etching technique. 48 hr after the addition of PHA, the newly formed blasts which had not as yet divided had at least twice the number of pores per nucleus as controls. This clearly demonstrates that in lymphocytes nuclear pore formation can take place during interphase. It has generally been assumed that the distribution of nuclear pore complexes in somatic animal cells is random. However, we have utilized freeze etched rat kidney cells and a computer program to evaluate pore distribution. We find a minimum pore center-to-center spacing of approximately 1300 A and multiples thereof with high frequency. This is strong evidence for a nonrandom distribution of nuclear pores. The nonrandomness may be related to an underlying chromosomal organization in interphase. Using three criteria for identifying prospective pore sites (membrane specialization, nonrandomness, and alteration of heterochromatin distribution), we have found forming pores in sectioned material from cultured human melanoma cells. While nuclear pore formation may take place in conjunction with reformation of the nuclear membrane, a mechanism also exists for their formation during interphase.

Time sequence of nuclear pore formation in phytohemagglutinin-stimulated lymphocytes and in HeLa cells during the cell cycle.

The time sequence of nuclear pore frequency changes was determined for phytohemagglutinin (PHA)-stimulated human lymphocytes and for HeLa S-3 cells during the cell cycle. The number of nuclear pores/nucleus was calculated from the experimentally determined values of nuclear pores/micro(2) and the nuclear surface. In the lymphocyte system the number of pores/nucleus approximately doubles during the 48 hr after PHA stimulation. The increase in pore frequency is biphasic and the first increase seems to be related to an increase in the rate of protein synthesis. The second increase in pores/nucleus appears to be correlated with the onset of DNA synthesis. In the HeLa cell system, we could also observe a biphasic change in pore formation. Nuclear pores are formed at the highest rate during the first hour after mitosis. A second increase in the rate of pore formation corresponds in time with an increase in the rate of nuclear acidic protein synthesis shortly before S phase. The total number of nuclear pores in HeLa cells doubles from approximately 2000 in G(1) to approximately 4000 at the end of the cell cycle. The doubling of the nuclear volume and the number of nuclear pores might be correlated to the doubling of DNA content. Another correspondence with the nuclear pore number in S phase is found in the number of simultaneously replicating replication sites. This number may be fortuitous but leads to the rather speculative possibility that the nuclear pore might be the site of initiation and/or replication of DNA as well as the site of nucleocytoplasmic exchange. That is, the nuclear pore complex may have multiple functions.

Genomic hypomethylation and far-5' sequence alterations are associated with carcinogen-induced activation of the hamster thymidine kinase gene.

We have investigated the mechanism of activation of an inactive but functionally intact hamster thymidine kinase (TK) gene by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine. Following carcinogen treatment of TK- RJK92 Chinese hamster cells, aminopterin-resistant (HATr) colonies appeared at a frequency 50-fold higher than in untreated controls. More than 80% of these HATr variants expressed TK enzymatic activity and were divided into high- and low-activity classes. In all TK+ variants, TK expression was correlated with demethylation in the 5' region of the TK gene and the appearance a 1,400-nucleotide TK mRNA. Using high-performance liquid chromatography to measure the level of genomic methylation, we found that four of five high-activity lines demonstrated extensive genomic hypomethylation (approximately 25% of normal level) that was associated with demethylation of all TK gene copies. Restriction endonuclease analysis of 15 low-activity lines revealed four instances of sequence alterations in the far-5' region of the TK gene and one instance of a tandem low-copy amplification. In these lines, the structurally altered gene copy was demethylated. Thus, we propose that a chemical carcinogen can activate TK expression by several different mechanisms. Focal demethylation with or without gene rearrangement was associated with low TK activity, whereas demethylation throughout the genome was associated with high TK activity.

Disruption of gamma-glutamyl leukotrienase results in disruption of leukotriene D(4) synthesis in vivo and attenuation of the acute inflammatory response.

To study the function of gamma-glutamyl leukotrienase (GGL), a newly identified member of the gamma-glutamyl transpeptidase (GGT) family, we generated null mutations in GGL (GGL(tm1)) and in both GGL and GGT (GGL(tm1)-GGT(tm1)) by a serial targeting strategy using embryonic stem cells. Mice homozygous for GGL(tm1) show no obvious phenotypic changes. Mice deficient in both GGT and GGL have a phenotype similar to the GGT-deficient mice, but approximately 70% of these mice die before 4 weeks of age, at least 2 months earlier than mice deficient only in GGT. These double-mutant mice are unable to cleave leukotriene C(4) (LTC(4)) to LTD(4), indicating that this conversion is completely dependent on the two enzymes, and in some organs (spleen and uterus) deletion of GGL alone abolished more than 90% of this activity. In an experimental model of peritonitis, GGL alone is responsible for the generation of peritoneal LTD(4). Further, during the development of peritonitis, GGL-deficient mice show an attenuation in neutrophil recruitment but not of plasma protein influx. These findings demonstrate an important role for GGL in the inflammatory response and suggest that LTC(4) and LTD(4) have distinctly different functions in the inflammatory process.

Inherent tumorigenic and metastatic properties of rat-1 and rat-2 cells.

Rat-1 and Rat-2 cells have been used in many studies of in vitro transformation and are widely assumed to be nontumorigenic because of their low incidence of focus formation, their poor growth in soft agar, and their reported failure to form tumors in animals. We examined more carefully the relationship between the in vitro and in vivo behavior of these cells and found that in spite of their in vitro characteristics, injection of these cells into Fischer rats invariably produced invasive tumors which frequently metastasized. When cells from primary tumors or metastases were cultured in vitro, the resultant cell lines were usually morphologically indistinguishable from parental cells and neither formed foci nor grew in soft agar. Thus, in vitro growth patterns do not correlate well with in vivo behavior in these cells and their inherent tumorigenicity warrants caution in the interpretation of results of in vitro transformation studies.

Nonspecific inhibition of DNA repair synthesis by tumor promoters in human diploid fibroblasts damaged with N-acetoxy-2-acetylaminofluorene.

The effects of selected tumor-promoting agents and their nonpromoting analogs on DNA repair synthesis were examined in human diploid fibroblasts (WI-38) damaged with N-acetoxy-2-acetylaminofluorene. Over a range of doses, three promoters (croton oil, 12-O-tetradecanoylphorbol-13-acetate, and anthralin) were found to inhibit DNA repair synthesis while their nonpromoting analogs (phorbol and 1,8-dihydroxyanthraquinone) had little effect. Another tumor promoter, phenol, inhibited DNA repair synthesis only at very high concentrations while an analog, 4-nitrophenol, produced inhibition of DNA repair synthesis at molar concentrations at which phenol had no effect. To investigate the specificity of this phenomenon, the effects of these agents on DNA-replicative synthesis, RNA synthesis, protein synthesis, and cell morphology were evaluated. At equimolar concentrations, tumor promoters were found to inhibit DNA-replicative synthesis as effectively as repair synthesis. RNA and protein synthesis were similarly inhibited over the same range of concentrations. Extensive morphological changes, interpreted as evidence of toxicity, were seen at concentrations of promoters that inhibited the macromolecular syntheses studied. The nonpromoting analogs, with the exception of nitrophenol, had little effect on these processes and showed only slight morphological damage. Thus tumor-promoting agents appeared to inhibit a number of macromolecular synthetic events, including DNA repair synthesis. It is suggested that the effect of tumor promoters on DNA repair synthesis is part of a general response to cellular injury rather than a selective response involving a single metabolic pathway. Furthermore, it is unlikely that the inhibition of repair synthesis represents the major mode of action of promoting agents in the carcinogenic process.

Two genes associated with liver cancer are regulated by different mechanisms in rasT24 transformed liver epithelial cells.

We compared the regulation of gamma-glutamyl transferase (gamma GT) and glutathione-S-transferase-P (GST-P) expression in rat liver epithelial cells (228 cells) and a line derived from them (C5 cells) by stable transfection with a metallothionein-activated ras fusion gene (MTrasT24). Earlier studies demonstrated that steady state RNA levels of these genes are increased after transformation of liver cells by MTrasT24 (Proc. Natl. Acad. Sci., 85, 344-348, 1988). In the present study, we found that the rate of gamma GT transcription increased approximately 20 fold after transformation by MTrasT24 while the rate of GST-P transcription increased no more than two fold. However, the stability of GST-P RNA was increased about 3 fold in these cells. Comparisons of gamma GT RNA stability were not possible since nontransformed liver cells (228) contain little or no gamma GT RNA. Thus, the accumulation of gamma GT RNA in C5 cells is heavily dependent on increased rates of transcription while the more modest increases in GST-P RNA levels result in large part from increased RNA stability. In ras transformed cells both transcriptional and post-transcriptional events contribute to the increased steady state RNA levels of cellular genes.

Early and late responses to induction of rasT24 expression in Rat-1 cells.

We have used a series of Rat-1 cell lines carrying a Zn-inducible human c-Ha-ras oncogene construction (MTrasT24) to evaluate the effect of varied ras oncogene expression on the expression of genes and proteins related to morphologic transformation in vitro. In response to the expression of the ras oncogene, at least two different classes of events occur. These events, referred to as 'early and late' events, are dependent on distinctively different accumulated levels of the ras oncoprotein. Relatively low levels of activated c-Ha-ras p21 protein (1.5-2.5 times the proto-oncogene level) stimulate rapid entry of quiescent (G0) cells into the cell cycle and result in increased steady state c-myc and glucose transporter mRNA levels which are detectable as early as 3-6 h after zinc addition. In contrast, morphologic transformation develops more slowly and does not appear until 72-96 h after Zn++ stimulation in cells with very low basal levels of activated p21 (MR4 cells) and 24-48 h in cells with higher basal levels (MR5 cells). These morphologic changes depend on the accumulation of significant amounts of the ras oncoprotein (greater than 4 to 5 times the proto-oncogene level) and are accompanied by large increases in the steady state mRNA levels of transin and TGF-alpha and decreases in PDGF-receptor mRNA and fibronectin protein and mRNA levels. In addition, the level of a novel cytoplasmic protein species (referred to as p29), which is stained by a monoclonal antibody for ras, is dramatically reduced in response to these levels of activated ras protein. Thus changes in morphology and gene expression induced by rasT24 occur sequentially and are quantitatively dependent on activated ras expression.

Regulation of a metallothionein-rasT24 fusion gene by zinc results in graded alterations in cell morphology and growth.

We constructed fusion genes consisting of the mouse metallothionein I (MT) 5' region and the coding region of either the human H-ras gene (c-rasP3) or a mutated allele (c-rasT24); both ras genes lacked the initial (non-coding) exon and the first 30 bp of the non-coding region of the second exon. Transfection of Rat-1 cells produced foci only with pMT-rasT24, and selection in soft agar yielded clones in which MT-rasT24 expression was zinc-regulatable. In response to increasing concentrations of ZnSO4, these lines showed increasingly altered morphology (conversion to fusiform or spheroidal morphology), progressively higher maximal cell density, and an increasingly greater fraction of cells in the S + G2 + M portion of the cell cycle at high density. MT-rasT24 RNA levels in zinc-responsive lines were increased between 4- and 6-fold by the addition of ZnSO4 (final concentration = 100 microM) to the medium. Replating cells in the absence of zinc reversed the biological effects and resulted in reduction in MT-rasT24 RNA levels. Thus, graded alterations in phenotype result from increasing levels of MT-rasT24 gene expression.

Effect of mercuric and silver ions on cesium sulfate equilibrium buoyant densities of synthetic polydeoxyribonucleotides.

The effect of Hg2+ and Ag+ on the buoyant density (rho) of four synthetic DNA polymers, poly[d(A-T)]; poly(dA) - poly(dT); oikt[d(G-C)]; and poly(dG) - poly(dC), was investigated. The buoyant density of poly[d(A-T)] in Cs2SO4 increased dramatically after complexing with Hg2+, but little change in the buoyant density of other polymers resulted except at very high molar ratios of Hg2+/DNA-P (rf). Hg2+ raised the thermal transition temperature (Tm) of alternating polymers and lowered the Tm of homopolymers. Measurements in the preparative ultracentrifuge indicated that lowered Tm correlated with Hg2+-induced strand separation of one homopolymer [poly(dA) - poly(dT)], but strand separation was not observed with another homopolymer [poly(dG) - poly(dC)] complexed with Hg2+. When Ag+ was mixed with the polymers, the buoyant density of poly(dG) - poly(dC) increased most markedly. A substantial increase in the buoyant density of poly[d(A-T)] and a small increase in the buoyant density of poly[d(G-C)] were also observed. The Tm changes induced by Ag+ were not related in any obvious way to buoyant density changes. These findings indicate that nucleotide sequence as well as overall base composition is of importance in understanding the buoyant density changes induced by metal ions. Although these data do not allow construction of a detailed molecular model of polymer-metal ion interactions, they may be used to explain much of the behavior of naturally occurring DNA sequences, such as heterochromatic satellite sequences and 5 S and rRNA sequences, in Hg2+/Cs2SO4 and Ag+/Cs2SO4 gradients.

Elimination of the differential chemoresistance between the murine B-cell lymphoma LY-ar and LY-as cell lines after arsenic (As2O3) exposure via the overexpression of gsto1 (p28).

PURPOSE: Arsenic, in the form of As(2)O(3), has gained therapeutic importance because it has been shown to be very effective clinically in the treatment of acute promyelocytic leukemia (APL). Via numerous pathways arsenic induces cellular alterations such as induction of apoptosis, inhibition of cellular proliferation, stimulation of differentiation, and inhibition of angiogenesis. Responses vary depending on cell type, dose and the form of arsenic. GSTO1, a member of the glutathione S-transferase superfamily omega, has recently been shown to be identical to the rate-limiting enzyme, monomethyl arsenous (MMA(V)) reductase which catalyzes methylarsonate (MMA(V)) to methylarsenous acid (MMA(III)) during arsenic biotransformation. In this study, we investigated whether arsenic trioxide (As(2)O(3)) induces apoptosis in both chemosensitive and chemoresistant cell lines that varied in their expression of p28 (gsto1), the mouse homolog of GSTO1. METHODS: The cytotoxicity of arsenic in the gsto1- and bcl-2-expressing chemoresistant and radioresistant LY-ar mouse lymphoma cell line, was compared with that of the LY-ar's parental cell line, LY-as. LY-as cells are radiosensitive, apoptotically permissive, and do not express gsto1 or bcl-2. Cell survival, glutathione (GSH) levels, mitochondrial membrane potential, and stress-activated kinase status after arsenic treatment were examined in these cell lines. RESULTS: As(2)O(3) induced an equivalent dose- and time-dependent increase in apoptosis in these cell lines. Cellular survival, as measured after a 24-h exposure, was also the same in each cell line. Reduced GSH was modulated in a similar time- and dose-dependent manner. Apoptosis was preceded by loss of mitochondrial membrane potential that triggered caspase-mediated pathways associated with apoptosis. With a prolonged exposure of As(2)O(3), both cell lines showed decreased activation of ERK family members, ERK1, ERK2 and ERK5. As(2)O(3) enhanced the death signals in LY-ar cells through a decrease in GSH, loss of mitochondrial membrane potential, and abatement of survival signals. This effect is similar to that seen when LY-ar cells are treated with thiol-depleting agents or by the removal of methionine and cysteine (GSH precursor) from the growth medium. This response is also completely contrary to that seen for radiation, actinomycin D, VP-16 and other agents, where LY-ar cells do not succumb to apoptosis. CONCLUSIONS: The overexpression of gsto1 in normally chemoresistant and radioresistant LY-ar cells renders them vulnerable to the cytotoxic effects of As(2)O(3), despite the 30-fold overexpression of the survival factor bcl-2. Gsto1 and its human homolog, GSTO1, may serve as a marker for arsenic sensitivity, particularly in cells that are resistant to other chemotherapeutic agents.

Reproductive defects in gamma-glutamyl transpeptidase-deficient mice.

Mice deficient in gamma-glutamyl transpeptidase (GGT) are growth retarded as a result of cysteine deficiency secondary to excessive glutathione excretion in urine and display coat color defects and cataracts. Although GGT is widely expressed throughout the mouse reproductive axis, little is known about its role in reproduction. Here, we present an analysis of the reproductive phenotypes of GGT-deficient mice. Mutant male mice have reduced testis and seminal vesicle size and suppressed serum insulin-like growth factor I and FSH levels and are infertile. Although these mice are severely oligospermic, histological analysis of testes reveals grossly normal stages of spermatogenesis, including late stage spermatids, but the tubule diameter is reduced. GGT-deficient female mice are also hypogonadal and infertile. At 6 weeks of age, the ovaries of mutant mice are histologically indistinguishable from those of its wild-type counterpart. However, the absence of antral follicles and corpora lutea and follicular degeneration are apparent by 11-13 weeks. In addition, immature female mutant mice (at 21-23 days) are insensitive to exogenous gonadotropin administration and fail to superovulate, suggesting an intraovarian defect. Consistent with these mutant phenotypes, HPLC analysis of adult mutant testes and ovaries showed a reduction in intracellular cysteine levels. Administration of N-acetylcysteine in the drinking water beginning on day 21 to mutant mice for 2 weeks restored testis, seminal vesicle, and ovary sizes to values comparable to those in wild-type mice. Furthermore, N-acetylcysteine-fed (continuously) mutant male and female mice were fertile and produced normal numbers of offspring when mated to wild-type control mice. These results demonstrate that GGT itself is not necessary for reproductive function. However, GGT plays an important role in cysteine homeostasis within the mouse reproductive axis.

Mouse leukotriene A4 hydrolase is expressed at high levels in intestinal crypt cells and splenic lymphocytes.

LTA4 hydrolase (EC 3.3.2.6) is a dual-function enzyme that is essential for the conversion of leukotriene A4 (LTA4) to leukotriene B4 (LTB4) and also possesses an aminopeptidase activity. To characterize the expression of this unusual enzyme, we have cloned the mouse LTA4 hydrolase cDNA. The deduced amino acid sequence revealed 92% identity with the human sequence. Cloning and analysis of genomic sequences of mouse LTA4 hydrolase indicated that it is a single-copy gene spanning over 40kb and containing 20 exons. LTA4 hydrolase is widely expressed, with the highest levels of expression occurring in the small intestine, followed by the spleen. In situ hybridization revealed that LTA4 hydrolase is localized in the crypt cells of the small intestine, white pulp of the spleen, bronchiolar epithelium of the lung, myocardium, adrenal cortex, epithelium of the seminal vesicles, proximal tubules and the collecting ducts of the kidney, and occasional hepatocytes. Thus the widespread distribution of LTA4 hydrolase in various cell types in the tissues suggests that LTB4 may possess biological activities other than those known at present. It is also plausible that the widespread occurrence of LTA4 hydrolase in various tissues may correspond more with its function as an aminopeptidase than its function as an LTA4 hydrolase.

Cloning of cDNA and genomic structure of the mouse gamma-glutamyl transpeptidase-encoding gene.

We have isolated and characterized cDNA and genomic clones containing the coding region for the mouse gamma-glutamyl transpeptidase (GGT). The sequences of the full-length cDNAs for three of the seven known mouse Ggt RNAs (types I, II and III) were determined and found to be identical in the coding region. Comparisons of the deduced amino-acid sequence of mouse GGT with that of rat and human reveal 95 and 79% overall identities, respectively. The mouse Ggt gene has 12 coding exon and spans approx. 12 kb. We have also re-analyzed rat genomic Ggt clones previously isolated by us and found that the rat and mouse genes share the same intron/exon boundaries. Our findings are of interest because they define the structure of the mouse and rat Ggt genes and will allow comparison with human GGT genes which, recent findings suggest, have diverged substantially from rodents.

N-acetylcysteine as an antidote in methylmercury poisoning.

Methylmercury is a ubiquitous environmental pollutant and potent neurotoxin. Treatment of methylmercury poisoning relies almost exclusively on the use of chelating agents to accelerate excretion of the metal. The present study demonstrates that oral administration of N-acetylcysteine (NAC), a widely available and largely nontoxic amino acid derivative, produces a profound acceleration of urinary methylmercury excretion in mice. Mice that received NAC in the drinking water (10 mg/ml) starting at 48 hr after methylmercury administration excreted from 47 to 54% of the 203Hg in urine over the subsequent 48 hr, as compared to 4-10% excretion in control animals. When NAC-containing water was given from the time of methylmercury administration, it was even more effective at enhancing urinary methylmercury excretion and at lowering tissue mercury levels. In contrast, excretion of inorganic mercury was not affected by oral NAC administration. The ability of NAC to enhance methylmercury excretion when given orally, its relatively low toxicity, and is wide availability in the clinical setting indicate that it may be an ideal therapeutic agent for use in methylmercury poisoning.

Cyclosiloxanes produce fatal liver and lung damage in mice.

To examine the toxicity of cyclosiloxanes (CSs), the predominant low molecular weight cyclic silicones found in breast implants, we injected female CD-1 mice intraperitoneally with different doses of distillate (3.5-35 g/kg body weight) containing cyclosiloxane D3 (hexamethylcyclotrisiloxane; CS-D3), cyclosiloxane D4 (octamethylcyclotetrasiloxane; CS-D4), cyclosiloxane D5 (decamethylcyclopentasiloxane; CS-D5), and cyclosiloxane D6 (dodecamethylcyclohexasiloxane; CS-D6). The distillate was found to be lethal and all the mice injected with 35 g/kg died within 5-8 days. The median lethal dose (LD50) for distillate was estimated to be approximately 28 g/kg. These mice developed inflammatory lesions of the lung and liver as well as liver cell necrosis with elevated serum levels of alanine aminotransferase, aspartate aminotransferase, and lactic acid dehydrogenase. Administration of CS-D4 alone also produced lethality in these mice with an LD50 of 6-7 g/kg. CS-D4-treated mice also exhibited pulmonary and hepatic lesions and elevated serum enzymes. Analysis of LD50 data indicates that CS-D4 is about as toxic as carbon tetrachloride or trichloroethylene. We measured hydroxyl radical formation in CS-D4-treated mice and found increases of approximately 20-fold in liver and approximately 7-fold in lung on day 4 following injection. Our findings are significant because in vitro experiments have demonstrated that CSs can migrate out of breast implants, and in mouse experiments CSs have been shown to be widely distributed in many organs after a single subcutaneous injection and to persist for at least a year.

Analysis of the role of 5' regulatory mutations in the activation of quiescent metallothionein genes after carcinogen treatment.

S49 are mouse thymic lymphoma cells which do not express the two closely linked mouse metallothionein (MT) genes; however, previous studies demonstrated that treatment of S49 cells with chemical carcinogens or ultraviolet irradiation can activate these quiescent genes. To determine if activation of MT-I or MT-II in these variants is a result of cis-acting mutations, we amplified and sequenced the immediate 5' regions of 19 cadmium resistant S49 variants: MT-I+/MT-II-, MT-I-/MT-II+, MT-I+/MT-II+, and MT-I-/MT-II-. None of the variants contained mutations in the analyzed regions. Thus, the observed changes in MT expression must result from mutations at other sites or from non-mutational mechanisms.

Suramin inhibits wound healing following filtering procedures for glaucoma.

BACKGROUND: Trabeculectomies are the most frequently performed procedures in surgically treating eyes with glaucoma. Failures are caused by fibrosis in the external ostium of the filtering procedure. In order to inhibit the fibrotic wound healing reaction, a new pharmacological approach using suramin, which inhibits a variety of important growth factors was used. METHODS: Pigmented rabbits were used and filtering procedures performed. Suramin was applied with concentrations ranging from 10 mg/ml to 333 mg/ml once during surgery and four times following surgery. The success of the filtering procedure was assessed by intraocular pressure measurements. To evaluate possible intraocular toxic effects, treated eyes were histopathologically evaluated after 4 weeks, and the ciliary body adjacent to the site of application was examined using electron microscopy. RESULTS: With concentrations of suramin of 200 mg/ml and 333 mg/ml, the trabeculectomies were patent longer than in the controls and in eyes operated with mitomycin C, which currently is the most frequently used antiproliferative drug to enhance the outcome of surgery in humans. No severe toxic effects to the ciliary epithelium were seen in suramin treated eyes. CONCLUSIONS: This study demonstrates for the first time the efficiency of a substance that broadly inhibits the action of growth factors on target cells in the setting of ocular wound healing. In this in vivo model, suramin has been shown to be highly effective in preventing scarring and in having fewer toxic side effects than usually used antimetabolites. These results therefore may suggest a new approach to the surgical treatment of glaucoma.

The influence of ras oncogene expression on radiation response in the Rat-1 cell.

We evaluated the effect of H-ras oncogene expression on resistance to ionizing radiation in cultured rat fibroblasts. The Rat-1 cell line, and two Rat-1 derivatives, MR4 and MR7, carrying a ZN-regulatable metallothionein-rasT24 fusion gene were used to study the effects of the ras oncogene on radiation sensitivity. Cells were irradiated with a 137Cs source (450 cGY/min) in the presence or absence of ZnSO4. Multiple cell survival studies did not show an appreciable difference in sensitivity to radiation among the lines in the presence or absence of ras oncogene expression.