Functional identification of LZTS1 as a candidate prostate tumor suppressor gene on human chromosome 8p22.

Deletions in the 8p21-22 region of the human genome are among the most common genetic alterations in prostate carcinomas. Several studies in different tumor tissues, including prostate, indicate that there are probably multiple tumor suppressor genes (TSGs) present in this region. To identify candidate TSGs on 8p22 a YAC contig spanning this region was assembled and YAC clones retrofitted with a selectable marker (neo) were transferred into rat prostate AT6.2 cells. Two overlapping YAC clones showed greatly reduced colony-forming efficiency, indicating they may carry a TSG. Two BAC clones encompassing the overlapping region also appeared to exert suppressive effects on the growth of AT6.2 cells. Database searches for genes mapped to the critical region identified a gene known as FEZ1 (LZTS1) as a potential candidate suppressor gene. Subsequent experiments showed that over-expression of LZTS1 cDNA inhibited stable colony-forming efficiencies of AT6.2, HEK-293 and LNCaP cells. In contrast, LZTS1-transfected Rat-1 and RM1 cells were growth-stimulated. Database searches also identified additional isoforms of the LZTS1 mRNA, as well as LZTS1 protein domains reminiscent of those found in transcription factors. Together these data suggest that the LZTS1 gene is involved in the regulation of cell growth and its loss of function may contribute to the development of prostatic carcinomas, as well as other cancers.

Ovarian function in superoxide dismutase 1 and 2 knockout mice.

Copper/zinc superoxide dismutase (SOD1) and manganese superoxide dismutase (SOD2) are the two major intracellular enzymes which inactivate superoxide radicals. SOD1 is present in both cytoplasmic and nuclear compartments whereas SOD2 is localized to mitochondria. Both enzymes are expressed in multiple tissues as well as ovaries of several species including humans and rodents. Dominant mutations in SOD1 are associated with amyotrophic lateral sclerosis. We have previously demonstrated that SOD2-deficient mice die within three weeks of birth due to oxidative mitochondrial injury in central nervous system neurons and cardiac myocytes. In this report, we demonstrate that female homozygous mutant mice lacking SOD1 can survive to the adult stage but are subfertile. Whereas breeding of 5 SOD1 heterozygote females produced an average of 1.0 litter/month with 8.6 offspring/litter (n = 31 litters), only 11 of 16 SOD1 homozygote mice over a 2-6 month period became pregnant averaging 0.23 litters/month with an average litter size of 2.7 (n = 21 litters). Histological analysis of the ovaries from SOD1-deficient mice often reveals many primary and small antral follicles but few corpora lutea. In addition, ovaries from postnatal SOD2-deficient mice, transplanted to the bursa of wild-type hosts, show all stages of folliculogenesis including corpora lutea and can give rise to viable offspring. These studies support an important role of SOD1 in female reproductive function and suggest that SOD2 is not essential for ovarian function.

Manganese superoxide dismutase protects nNOS neurons from NMDA and nitric oxide-mediated neurotoxicity.

Neuronal nitric oxide synthase (nNOS) neurons kill adjacent neurons through the action of NMDA-glutamate receptor activation, although they remain relatively resistant to the toxic effects of NMDA and NO. The molecular basis of the resistance of nNOS neurons to toxic insults is unknown. To begin to understand the molecular mechanisms of the resistance of nNOS neurons, we developed a pheochromacytoma-derived cell line (PC12) that is resistant to the toxic effects of NO. We found through serial analysis of gene expression (SAGE) that manganese superoxide dismutase (MnSOD) is enriched in the NO-resistant PC12 cell-derived line (PC12-R). Antisense MnSOD renders PC12-R cells sensitive to NO toxicity and increases the sensitivity to NO in the parental, NO-sensitive PC12 line (PC12-S). Adenoviral transfer of MnSOD protects PC12-S cells against NO toxicity. We extended these studies to cortical cultures and showed that MnSOD is enriched in nNOS neurons and that antisense MnSOD renders nNOS neurons susceptible to NMDA neurotoxicity, although it has little effect on the overall susceptibility of cortical neurons to NMDA toxicity. Overexpression of MnSOD provides dramatic protection against NMDA and NO toxicity in cortical cultures, but not against kainate or AMPA neurotoxicity. Furthermore, nNOS neurons from MnSOD -/- mice are markedly sensitive to NMDA toxicity. Adenoviral transfer of MnSOD to MnSOD-/- cultures restores resistance of nNOS neurons to NMDA toxicity. Thus, MnSOD is a major protective protein that appears to be essential for the resistance of nNOS neurons in cortical cultures to NMDA mediated neurotoxicity.

Release of low molecular weight silicones and platinum from silicone breast implants.

We have conducted a series of studies addressing the chemical composition of silicone gels from breast implants as well as the diffusion of low molecular weight silicones (LM-silicones) and heavy metals from intact implants into various surrounding media, namely, lipid-rich medium (soy oil), aqueous tissue culture medium (modified Dulbecco's medium, DMEM), or an emulsion consisting of DMEM plus 10% soy oil. LM-silicones in both implants and surrounding media were detected and quantitated using gas chromatography (GC) coupled with atomic emission (GC-AED) as well as mass spectrometric (GC/MS) detectors, which can detect silicones in the nanogram range. Platinum, a catalyst used in the preparation of silicone gels, was detected and quantitated using inductive argon-coupled plasma/mass spectrometry (ICP-MS), which can detect platinum in the parts per trillion range. Our results indicate that GC-detectable low molecular weight silicones contribute approximately 1-2% to the total gel mass and consist predominantly of cyclic and linear poly-(dimethylsiloxanes) ranging from 3 to 20 siloxane [(CH3)2-Si-O] units (molecular weight 200-1500). Platinum can be detected in implant gels at levels of approximately 700 micrograms/kg by ICP-MS. The major component of implant gels appears to be high molecular weight silicone polymers (HM-silicones) too large to be detected by GC. However, these HM-silicones can be converted almost quantitatively (80% by mass) to LM-silicones by heating implant gels at 150-180 degrees C for several hours. We also studied the rates at which LM-silicones and platinum leak through the intact implant outer shell into the surrounding media under a variety of conditions. Leakage of silicones was greatest when the surrounding medium was lipid-rich, and up to 10 mg/day LM-silicones was observed to diffuse into a lipid-rich medium per 250 g of implant at 37 degrees C. This rate of leakage was maintained over a 7-day experimental period. Similarly, platinum was also observed to leak through intact implants into lipid-containing media at rates of approximately 20-25 micrograms/day/250 g of implant at 37 degrees C. The rates at which both LM-silicones and platinum have been observed to leak from intact implants could lead to significant accumulation within lipid-rich tissues and should be investigated more fully in vivo.

A 346-base pair region of the mouse gamma-glutamyl transpeptidase type II promoter contains sufficient cis-acting elements for kidney-restricted expression in transgenic mice.

The mouse gamma-glutamyl transpeptidase (GGT) gene encodes seven distinct mRNAs that are transcribed from seven separate promoters. Type II mRNA is the most abundant in kidney. We have developed a cell line with features of renal proximal tubular cells which expresses GGT mRNA types with a pattern similar to that of mouse kidney. Because a 346-bp sequence from the type II promoter directed the highest level of CAT activity in these cells, this region was used to drive the expression of a beta-galactosidase reporter gene in transgenic mice. Two transgenic mouse lines expressed beta-galactosidase limited to the renal proximal tubules. Site-directed deletions within this 346-bp promoter region demonstrated that cis-elements containing the consensus binding sites for AP2, a glucocorticoid response element (GRE)-like element, and the initiator region were required for transcriptional activity and were not additive. Purified AP2 bound and footprinted the AP2 consensus region, making it likely that transcription from the GGT type II promoter is regulated in part by AP2. These data suggest that transcription of the type II promoter requires multiple protein DNA interactions involving at least an AP2 element, and probably a GRE-like element and the initiator region.

Detection and characterization of poly(dimethylsiloxane)s in biological tissues by GC/AED and GC/MS.

We have developed a sensitive method for the detection, characterization, and quantitation of low molecular weight silicones using gas chromatography coupled with atomic emission detection (GC/AED) and gas chromatography/ mass spectrometry (GC/MS). Using this approach, we have detected 12 distinct silicon-containing peaks in PDMS-V poly(dimethylsiloxane) oil by GC/AED, and we have used GC/MS analysis to identify some of the abundant peaks by MS spectral matching. Polydimethylpolysiloxanes contain 37.8% silicon; therefore, the amount of poly(dimethylsiloxane) in each peak can be calculated from its silicon content. The first three GC peaks from PDMS-V were identified as dodecamethylpentasiloxane, tetradecamethylhexasiloxane, and hexadecamethylheptasiloxane using Wiley Mass Spectral Library match (> 90%). Peaks 4-12 could not be matched unequivocally with the spectral library but showed ionic fragments characteristic of PDMS (73, 147, 221, 281, 295, and 369 amu). The detection limit for silicones using GC/AED and GC/MS systems was found to be 80 and 10 pg/microL, respectively. Studies were conducted using mouse liver homogenates spiked with varying amounts of PDMS-V, and the recovery was found to be greater than 90% over a wide range of PDMS-V concentrations. This method appears to work equally well for both linear and cyclic poly(dimethylsiloxane)s. Thus, the methodology described here has the potential to allow the measurement of less than 1 microgram of silicone/g of biological tissue. The overall goal of this research is to establish and validate a methodology by which the unequivocal identification and quantitation of poly(dimethylsiloxane)s can be accomplished.

Neurodegeneration, myocardial injury, and perinatal death in mitochondrial superoxide dismutase-deficient mice.

Manganese superoxide dismutase (SOD2) converts superoxide to oxygen plus hydrogen peroxide and serves as the primary defense against mitochondrial superoxide. Impaired SOD2 activity in humans has been associated with several chronic diseases, including ovarian cancer and type I diabetes, and SOD2 overexpression appears to suppress malignancy in cultured cells. We have produced a line of SOD2 knockout mice (SOD2m1BCM/SOD2m1BCM) that survive up to 3 weeks of age and exhibit several novel pathologic phenotypes including severe anemia, degeneration of neurons in the basal ganglia and brainstem, and progressive motor disturbances characterized by weakness, rapid fatigue, and circling behavior. In addition, SOD2m1BCM/SOD2m1BCM mice older than 7 days exhibit extensive mitochondrial injury within degenerating neurons and cardiac myocytes. Approximately 10% of SOD2m1BCM/SOD2m1BCM mice exhibit markedly enlarged and dilated hearts. These observations indicate that SOD2 deficiency causes increased susceptibility to oxidative mitochondrial injury in central nervous system neurons, cardiac myocytes, and other metabolically active tissues after postnatal exposure to ambient oxygen concentrations. Our SOD2-deficient mice differ from a recently described model in which homozygotes die within the first 5 days of life with severe cardiomyopathy and do not exhibit motor disturbances, central nervous system injury, or ultrastructural evidence of mitochondrial injury.

Growth retardation and cysteine deficiency in gamma-glutamyl transpeptidase-deficient mice.

gamma-Glutamyl transpeptidase (GGT) is an ectoenzyme that catalyzes the first step in the cleavage of glutathione (GSH) and plays an essential role in the metabolism of GSH and GSH conjugates of carcinogens, toxins, and eicosanoids. To learn more about the role of GGT in metabolism in vivo, we used embryonic stem cell technology to generate GGT-deficient (GGTm1/GGTm1) mice. GGT-deficient mice appear normal at birth but grow slowly and by 6 weeks are about half the weight of wild-type mice. They are sexually immature, develop cataracts, and have coats with a gray cast. Most die between 10 and 18 weeks. Plasma and urine GSH levels in the GGTm1/GGTm1 mice are elevated 6-fold and 2500-fold, respectively, compared with wild-type mice. Tissue GSH levels are markedly reduced in eye, liver, and pancreas. Plasma cyst(e)ine levels in GGTm1/GGTm1 mice are reduced to approximately 20% of wild-type mice. Oral administration of N-acetylcysteine to GGTm1/GGTm1 mice results in normal growth rates and partially restores the normal agouti coat color. These findings demonstrate the importance of GGT and the gamma-glutamyl cycle in cysteine and GSH homeostasis.

A single mouse glutathione synthetase gene encodes six mRNAs with different 5' ends.

To understand more about the role of glutathione (GSH) in metabolism, we have cloned both cDNA and genomic sequences for mouse glutathione synthetase (GSH syn), the enzyme that catalyzes the last step in the synthesis of glutathione. The mouse cDNA contains an open reading frame (ORF) of 474 aa and shares 64 and 95% deduced amino acid sequence identity with Xenopus cDNA and rat cDNA, respectively. The cDNA complements Schizosaccaromyces pombe strains deficient in GSH syn. The gene is a single-copy gene spanning approximately 30 kb and is composed of at least 15 exons. Steady-state RNA levels and enzyme activity levels are highest in kidney, about 3-fold lower in liver, and 8- to 10-fold lower in lung and brain. We have identified six different GSH syn RNAs: three, termed types A1, A2, and A3, have different 5' ends that localize to different sites in the gene, but appear to encode the same protein (474 aa). Types B, C1, and C2 all have unique 5' ends and type-specific ORFs, which are shorter than that for types A1, A2, and A3. In liver only type A1 GSH syn RNA is detectable, while in kidney 90% of GSH syn RNA is type A1 and types B and C account for about 10%.

Retention of p53val135 wild-type function in transgenic mice.

We targeted a mutant p53 gene (val135), previously shown to cause tumors in transgenic mice, to the kidney and eye using a gamma-glutamyltranspeptidase promoter. Although transgene RNA was expressed in both tissues, and mutant protein could be detected at high levels in the kidney and was appropriately localized to the nuclei of proximal tubules, no gross or microscopic lesions developed, even when mice were held as long as 75 weeks. When these mice were crossed with transgenic mice carrying HrasT24 (containing a codon 12 mutation) driven by the same promoter, the p53val135 transgene partially suppressed the mutant ras phenotype (proximal tubular hyperplasia and adenomas and carcinomas of the ciliary body and retinal pigment epithelium). The kidneys of double transgenic mice younger than 25 weeks showed less tubular hyperplasia and cystic change than littermates carrying gamma-glutamyltranspeptidase(I)rasT24 alone. By 33 weeks, there was no difference in the severity of the kidney lesions. The eye lesions were less aggressive, and no malignant lesions were identified. Our findings are consistent with the work of others, indicating that p53val135 is not tumorigenic under all conditions; in fact, in some circumstances, it retains some of the suppressing activity of wild-type p53.

Quantitative examination of dynamic interneuronal coupling via single-spike extracellular potassium ion transients.

A computer simulation developed to examine activity-dependent, transient variations of local extracellular potassium ion concentration in the central nervous system is extended to a consideration of depolarizing interactions between an active (primary) neuronal membrane and adjacent quiescent neuronal membranes. A class of potassium-mediated depolarizing interactions is described and quantified in relation to the local histologic fine structure. With a relatively open and rapidly equilibrating extracellular environment, the interactions are in the millivolt range and thus are largely subthreshold. However, with a sufficiently restricted extracellular microenvironment and without additional dynamic mechanisms for enhanced potassium clearance, a single action potential is shown to be capable of producing secondary suprathreshold depolarization of the primary membrane. Such potassium-mediated self-excitation evolves to "autogenic" paroxysmal discharge, that is, a burst of activity terminating in sustained depolarization block. Similar suprathreshold ionic coupling between the primary membrane and its adjacent neuronal membranes can likewise yield "cooperative" paroxysmal discharge, wherein the burst of action potentials from the referential primary membrane interdigitates with homologous bursts in adjacent neuronal elements. The partitioning of the local extracellular space by fine cellular processes, fundamental to determining the magnitude of single-spike local extracellular potassium transients, is a basic determining factor for such putative potassium-mediated suprathreshold events. Of more general relevance is the existence of a class of robust but highly localized, subthreshold potassium-mediated ionic interactions that can support non-synaptic dynamic modulation of neuronal activity. Thus, in addition to providing a quantitative theory for the initiation and propagation of non-synaptic epileptiform events, the model suggests a mechanism for potassium-mediated and activity-dependent proximity modulation of neuronal network throughout that can be significant at normal levels of neuronal activity and normal histologic fine structure.

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.

gamma-Glutamyl transpeptidase. What does the organization and expression of a multipromoter gene tell us about its functions?

gamma-Glutamyl transpeptidase is a key enzyme in glutathione (GSH) salvage, metabolism of endogenous mediators such as leukotrienes and prostaglandins, detoxification of xenobiotics including environmentally important compounds and carcinogens, and cellular processes dependent on the oxidation/reduction of glutathione. The enzyme is widely distributed, and these functions often occur in separate tissues and in response to different stimuli. Evidence indicates that gamma-glutamyl transpeptidase plays a direct role in some hepatic and renal responses to injury. In the mouse gamma-glutamyl transpeptidase is a single copy gene expressed from at least seven promoters, and many of the transcribed gamma-glutamyl transpeptidase RNAs are restricted in their expression. Studies that combine analyses of cellular processes with a knowledge of gene structure and expression hold promise for unravelling how these two different levels of function are integrated.

Expression of gamma-glutamyl transpeptidase in midgestation mouse yolk sac and mouse visceral yolk sac carcinoma cells.

gamma-Glutamyl transpeptidase (gamma GT) is a crucial enzyme for the metabolism of xenobiotics and endogenous mediators of biological functions (leukotrienes, prostaglandins, and hepoxillins). Yet little is known about its potential role during development. It is a single copy gene expressed from at least seven promoters. Using histochemistry and immunohistochemistry we demonstrate that gamma GT first appears in the midgestational yolk sacs of mouse embryos. Established cell lines with phenotypic features of yolk sac endoderm (JC-44) or embryonic stem cells were also assayed for the expression of gamma GT. Significant levels were detected in JC-44 cells and higher levels were found in JC-44-derived embryoid bodies. Because this cell line appears to be a good in vitro counterpart of yolk sac differentiation, we characterized the gamma GT mRNA types expressed in JC-44 cells. By ribonuclease protection analysis, gamma GT RNA types IV and VI represent about 80% of the total gamma GT RNA in JC-44 embryoid bodies. Reverse transcription-mediated polymerase chain reaction detected low amounts of gamma GT RNA types I, III, and V. Expression of gamma GT in yolk sac follows a pattern seen in many tissues in which one or two gamma GT RNA types dominate the expression profile; however, the reason for this tissue specificity is unknown.

Identification of a sixth promoter that directs the transcription of gamma-glutamyl transpeptidase type III RNA in mouse.

We have previously identified five promoters in the 5'-flanking region of the mouse gamma-glutamyl transpeptidase (gamma GT) gene. We now report the localization of a sixth promoter that supports the transcription of type III RNA, the major gamma GT RNA in fetal liver. We made a fetal liver cDNA library enriched for gamma GT RNA and obtained 12 gamma GT type III-specific clones. The longest clone is consistent with a transcription start site for type III RNA at a position 5' to the type IV promoter and about 5 kilobase(s) (kb) 5' to the first coding exon. We estimated by ribonuclease protection assay that about 80% of the gamma GT mRNA in fetal liver was type III. Primer extension and nuclease protection analyses mapped the 5' end of type III mRNA in fetal liver and kidney to a single cluster of potential major and minor transcription start sites. Deletion analysis using transient expression of chloramphenicol acetyltransferase constructs of the type III promoter region revealed the greatest activity with a 1-kb 5'-flanking fragment in mouse kidney proximal tubular cells and no detectable activity in NIH-3T3 fibroblasts. These studies demonstrate that the type III 5' region of the mouse gamma GT gene is organized into two distinct exons (IIIa and IIIb) and that type III RNA is expressed under the control of its own promoter.

Rat small intestine expresses primarily type VI gamma-glutamyl transpeptidase RNA.

To clarify differences between gamma-glutamyl transpeptidase (gamma GT) expression in the rat and the mouse small intestine, we cloned and sequenced 13 rat small intestine gamma GT cDNAs and compared them with those obtained from the mouse (Carter, et al. (1994) J. Biol. Chem. 269, 24581-24585). We found that all 13 were type VI, which in both species accounts for 80-90% of intestinal gamma GT. However, rat type VI gamma GT is substantially longer than mouse type VI RNA and includes at least 67 additional nucleotides at the 5' end that are non-homologous with the 5' end of the mouse type VI RNA; further, these show no homology to another mouse exon (VII) that splices to type VI RNA. In addition, a 168 nucleotide stretch is present in the middle of rat RNA that is absent in mouse type VI RNA.

Transgenic mice carrying a PSArasT24 hybrid gene develop salivary gland and gastrointestinal tract neoplasms.

BACKGROUND: Although prostate cancer is one of the most prevalent tumors in men, knowledge of its biology has been hindered by lack of animal models. We have attempted to develop a prostate cancer model utilizing transgenic mouse technology. EXPERIMENTAL DESIGN: Two lines of transgenic mice were derived from one cell stage embryos injected with a fusion gene consisting of a mutated (codon 12) ras gene driven by the human prostate specific antigen (PSA) promoter in an attempt to target the oncogene specifically to the mouse prostate gland. Nontransgenic FVB/N mice were used as controls. The animals were sacrificed for study between 4 and 55 weeks of age. RESULTS: All organs were normal except the salivary glands and gastrointestinal tracts, both of which developed carcinomas in animals older than 44 weeks. The salivary gland tumors were of ductal origin, exhibited a variable degree of differentiation, and were shown to contain abundant PSAras mRNA by in situ hybridization. The gastrointestinal tract tumors were undifferentiated but appeared to be of stromal origin. Both salivary gland and gastrointestinal tumors occasionally metastasized. No transgene expression could be demonstrated in the prostate gland by either reverse transcription-polymerase chain reaction or in situ hybridization. CONCLUSIONS: Lack of transgene expression by the prostate can be explained on the basis of the apparent species specificity previously observed for PSA. Expression in salivary gland is best attributed to identity between the nucleotide sequences of the PSA promoter and of a mouse glandular kallikrein normally secreted by the salivary gland.

p53 accumulation in benign breast biopsy specimens.

Several studies of benign breast lesions using methacran-fixed, paraffin-embedded tissues and cytological preparations have suggested that p53 accumulation in these lesions as detected by immunohistochemical (IHC) staining is rare to absent. As a result, several different investigators have suggested that p53 immunoreactivity in breast specimens infers a diagnosis of malignancy or may identify premalignant lesions. We immunostained 271 breast biopsy specimens from 271 patients with the monoclonal anti-p53 antibody BP-53-12 and found positive nuclear staining in seven of 23 malignant lesions (30%) and 39 of 248 benign biopsy specimens (16%). Of the benign lesions, 30% of fibroadenomas, nonpremalignant breast lesions, were positive. Long-term follow-up information was available on 48 patients with benign biopsy specimens and showed that 12% of those positive and 7% of those negative for p53 developed breast carcinoma. This difference was not significant (P > .2). We conclude that (1) p53 immunoreactivity in breast lesions should not be used as exclusive evidence of malignancy and (2) p53 immunoreactivity in benign breast lesions may not identify a subset of patients at increased risk for breast carcinoma.