Search

Taking advantage of mice deficient in γ-glutamyl transpeptidase that are unable to metabolize glutathione (GSH), we have identified two previously unrecognized urinary metabolites of arsenite:  arsenic triglutathione and methylarsenic diglutathione. Following administration of sodium arsenite to these mice, ~60−70% of urinary arsenic is present as one of these GSH conjugates. We did not detect the dimethyl derivative, dimethyl arsenic GSH; however, dimethyl arsenic (DMA^V) represented approximately 30% of urinary arsenic. Administration of buthionine sulfoximine, an inhibitor of GSH synthesis, to wild-type mice reduced urinary arsenic excretion by more than 50%, indicating the GSH dependence of arsenic metabolism, transport, or both. Rodents deficient in three known ABC family transporters (MRP1, MRP2, and MDR1a/1b) exhibited urinary arsenic levels similar or greater than those in wild-type rodents; however, administration of MK571, an MRP inhibitor, reduced urinary arsenic excretion by almost 50%. MK571-treated mice showed ~50% reduction of As^III, MMA^V, and As^V as compared to untreated wild-type controls, while DMA^V levels were unchanged. These findings suggest that arsenic excretion is in part dependent on GSH and on an MRP transporter other than MRP1 or 2.

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.

We studied the effects of a zinc-inducible metallothionein-ras fusion gene (MTrasT24) in cultured rat liver epithelial (RLE) cells on expression of two genes induced during liver carcinogenesis in vivo: gamma-glutamyltransferase [(5-glutamyl)-peptide:amino acid 5-glutamyltransferase, EC 2.3.2.2] and glutathione S-transferase-P (RX:glutathione R-transferase, EC 2.5.1.18). Expression of MTrasT24 increased steady-state RNA levels of gamma-glutamyltransferase and glutathione transferase-P 6- to 100-fold and 1.6- to 6-fold, respectively; in contrast, levels of alpha-tubulin RNA fell slightly or were unchanged. RNA gel blots verified that gamma-glutamyltransferase and glutathione transferase-P RNAs were of the appropriate size, and results from immunocytochemistry on transfected cells demonstrated that RLE cells carrying MTrasT24 synthesized immunoreactive, appropriately localized gamma-glutamyltransferase and glutathione transferase-P. Zinc induction studies indicated that gamma-glutamyltransferase and glutathione transferase-P RNA levels were directly dependent on MTrasT24 RNA levels. These data suggest that expression of gamma-glutamyltransferase and glutathione transferase-P expression are part of a reorientation of cellular gene expression during carcinogenesis and that activated ras expression, like chemical carcinogens, can bring about this change.

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.

The rate and extent of disappearance of two DNA lesions (pyrimidine dimers and covalently bound acetylaminofluorene), both thought to be removed by the so-called wide-patch (approximately 100 nucleotides) repair process, were studied in a variety of cultured mammalian cells. With the exception of mouse cells, dimers were removed more rapidly and extensively than covalently bound acetylaminofluorene. In human cells, for example, about 50% of the dimers were excised from DNA in 1 hr while only 25-50% of the chemically induced lesions were excised from DNA after 48 hr. Surprisingly mouse cells, which remove few dimers, were about as competent as control human fibroblasts at removing acetylaminofluorene lesions; however, xeroderma pigmentosum cells (group D) removed fewer N-acetoxy-2-acetylaminofluorene-induced lesions than control human cells. Our data raise the possibility of separate repair processes for these two types of lesions and suggest that their expression may be under similar genetic control in human cells.

We have recently identified a mouse enzyme termed gamma-glutamyl leukotrienase (GGL) that converts leukotriene C4 (LTC4) to leukotriene D4 (LTD4). It also cleaves some other glutathione (GSH) conjugates, but not GSH itself (Carter, B. Z., Wiseman, A. L., Orkiszewski, R., Ballard, K. D., Ou, C.-N., and Lieberman, M. W. (1997) J. Biol. Chem. 272, 12305-12310). We have now cloned a full-length mouse cDNA coding for GGL activity and the corresponding gene. GGL and gamma-glutamyl transpeptidase constitute a small gene family. The two cDNAs share a 57% nucleotide identity and 41% predicted amino acid sequence identity. Their corresponding genes have a similar intron-exon organization and are located 3 kilobases apart. A search of Genbank and reverse transcription-polymerase chain reaction analysis failed to identify additional family members. Mapping of the GGL transcription start site revealed that the GGL promoter is TATA-less but contains an initiator, a control element for transcription initiation. Northern blots for GGL expression were negative. As judged by ribonuclease protection, in situ hybridization, and measurement of enzyme activity, spleen had the highest level of GGL expression. GGL is also expressed in thymic lymphocytes, bronchiolar epithelial cells, pulmonary interstitial cells, renal proximal convoluted tubular cells, and crypt cells of the small intestine as well as in cerebral, cerebellar, and brain stem neurons but not in glial cells. GGL is widely distributed in mice, suggesting an important role for this enzyme.

We have investigated the metabolism of leukotriene C4 (LTC4) in gamma-glutamyl transpeptidase (GGT)-deficient mice (Lieberman, M. W., Wiseman, A. L., Shi, Z-Z., Carter, B. Z., Barrios, R., Ou, C-N., Chevez-Barrios, P., Wang, Y., Habib, G. M., Goodman, J. C., Huang, S. L., Lebovitz, R. M., and Matzuk, M. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7923-7926) and have found substantial conversion of LTC4 to leukotriene D4 by high performance liquid chromatography and continuous flow fast atom bombardment-tandem mass spectrometric analyses. LTC4-converting activity has a tissue distribution different from GGT with highest activity in spleen followed by small intestine, kidney, and pancreas and lower activity in liver and lung. The activity is membrane-bound and is inhibited by acivicin, a known inhibitor of GGT. The enzyme was partially purified from the small intestine of GGT-deficient mice by papain treatment and gel filtration chromatography. The partially purified fragment released by papain has an apparent molecular mass of 65-70 kDa and the same substrate specificity as the tissue homogenate. In addition to LTC4, S-decyl-GSH is also cleaved. GSH itself, oxidized GSH, and the synthetic substrates used to analyze GGT activity (gamma-glutamyl-p-nitroanilide and gamma-glutamyl-4-methoxy-2-naphthylamide) are not substrates for this newly discovered enzyme. These data demonstrate that in addition to GGT at least one other enzyme cleaves LTC4 in mice. To reflect this enzyme's preferred substrate, we suggest that it be named gamma-glutamyl leukotrienase.

We have used inhibitors to identify the DNA polymerases which are involved in DNA excision repair induced in confluent diploid human fibroblasts by several DNA damaging agents: UV radiation, N-acetoxy-2-acetylaminofluorene, N-methyl-N-nitrosourea, and bleomycin. We find that DNA repair synthesis involves both DNA polymerase alpha and a non-alpha DNA polymerase, probably polymerase beta. The fraction of repair synthesis mediated by each of the two polymerases is dependent on which DNA-damaging agent is administered and on the dose of damaging agent. Low doses of DNA damage induce DNA repair synthesis which is mediated to a great extent by a non-alpha DNA polymerase, and with an increasing dose of damage there is increasing participation of DNA polymerase alpha in repair synthesis. At high doses of damage, the fraction of repair synthesis mediated by DNA polymerase alpha reaches a maximal level which is dependent on the damaging agent; the maximal level of polymerase alpha involvement is about 80% for UV radiation and N-acetoxy-2-acetylaminofluorene, about 70% for N-methyl-N-nitrosourea, and about 40% for bleomycin.

Using the technique for separating DNA fragments containing excision-repair sites from total genomic DNA as described in the previous paper (Cohn, S. M., and Lieberman, M. W. (1984) J. Biol. Chem. 259, 12456-12462), we have developed a method for directly determining the distribution of excision-repair sites in the genome. DNA was prepared from confluent, diploid human fibroblasts which had been irradiated with ultraviolet light and incubated in the presence of 5-bromo-2'-deoxyuridine (BrdUrd), repaired fragments were isolated, and the dependence of the fraction of total DNA fragments containing excision-repair sites on DNA fragment length was determined by electrophoretic analysis. The observed dependence was compared to the relationship expected for a random distribution of repair sites. At 36 h following 3 J/m2 UV, the distribution of repair sites was indistinguishable from a random distribution; however, at doses of UV above 6 J/m2, the observed dependence indicated that the distribution of repair sites was nonrandom. A time course of the distribution of repair sites following 12 J/m2 UV was clearly nonrandom from 4 h after irradiation until at least 36 h following irradiation. By 72 h, however, the distribution had become random. In cells treated with hydroxyurea, a reduced number of excision-repair sites were present, but the distribution of repair sites was also nonrandom. Autoradiographic analysis of the amount of unscheduled DNA synthesis in individual nuclei suggested that the nonrandom distribution of repair sites did not result from variable extents of repair synthesis in different cell populations or from cell death.

We have developed an immunological method for isolation and identification of DNA sequences containing 5-bromo-2'-deoxyuridine (BrdUrd) incorporated during UV-induced excision-repair synthesis. DNA fragments containing BrdUrd incorporated during repair synthesis were incubated with goat anti-BrdUrd and rabbit anti-goat IgG, and the antibody-DNA complexes were separated from bulk DNA by nitrocellulose filter binding. With this method, 80% of DNA sequences containing BrdUrd-labeled excision-repair sites were recovered, contaminated with less than 1% of DNA fragments devoid of excision-repair sites. Recovery of DNA fragments containing repair sites was independent of size from 2 to 20 kilobases. We have used this method in conjunction with blot hybridization to demonstrate that repair synthesis occurs in human ribosomal gene sequences in cells treated with UV.

The distribution of UV-induced DNA repair synthesis within chromatin was measured in confluent human fibroblasts that were pulse-labeled with [3H]dThd (10 or 90 min) immediately after irradiation and chased in nonradioactive medium for different time periods. Initially (i.e., at the end of the pulse period), most of the repair synthesis occurs in staphylococcal nuclease-sensitive regions. With increasing chase times the nucleotides inserted during repair synthesis become progressively more nuclease resistant. Gel electrophoresis data indicate that nuclease resistance is conferred on these nucleotides by their appearance in core DNA. The kinetics of this rearrangement process are biphasic: greater than 85% of the repair synthesis sites undergo rapid rearrangement (4--5 hr); the remaining sites ( less than 15%) rearrange much more slowly, if at all. The time courses of nucleosome rearrangement and repair synthesis are similar, suggesting that nucleosome rearrangement may be induced by the repair process or that the rate of repair synthesis may be regulated by nucleosome rearrangement.

We have examined the accumulation of newly synthesized nuclear proteins into nuclei during DNA repair synthesis in confluent WI-38 human diploid fibroblasts damaged with ultraviolet radiation or N-acetoxy-2-acetylaminofluroene. In contrast to a marked stimulation of DNA repair synthesis, stimulation of amino acid incorporation into histone polypeptides or into the various molecular weight classes of nonhistone nuclear proteins was not observed. These results suggest that detectable stimulation of newly synthesized nuclear protein incorporation into nuclei does not accompany DNA repair synthesis induced by ultraviolet radiation or a direct acting chemical carcinogen. At least for the special case of repair, DNA synthesis may be uncoupled from histone synthesis.

Membrane-bound dipeptidase (MBD) participates in the degradation of glutathione by cleaving the cysteinyl-glycine bond of cystinyl bisglycine (oxidized cysteinyl-glycine) following removal of a gamma-glutamyl group by gamma-glutamyl transpeptidase (GGT). In the mouse, MBD RNA is most abundant in small intestine, kidney, and lung and is represented by four distinct RNA species. These are generated by transcription from two promoters located 6 kilobases apart in the 5' flanking region of the gene and by the use of two different poly(A) addition sites. Promoter I is used primarily in small intestine and kidney, whereas promoter II is most active in lung and kidney. We found a discordance in the expected co-expression of MBD and GGT; as expected, MBD and GGT are both expressed at high levels in the kidney and small intestine. However, in the lung, MBD is expressed at high levels, whereas GGT is almost undetectable. The reverse is true in the seminal vesicles and fetal liver. Thus, although both enzymes may function in concert to metabolize glutathione in kidney and small intestine, in other tissues they appear to act independently, suggesting that they have independent roles in other biological processes.

Using a filter binding assay, we have detected and partially purified a protein from human placenta that has a high affinity for N-acetoxy-2-acetylaminofluorene-modified double-stranded DNA (AAF-[3H]DNA) of bacteriophage T7. This protein has been partially purified from a 1 M NaCl extract of a crude nuclear fraction by a combination of ion-exchange and nucleic acid affinity chromatography. With AAF-[3H]DNA as the substrate, the binding reaction reached equlibrium within 1 hr at 4 degrees C, and the extent of binding ws proportional to the amount of protein added. Complex formation was dependent on both pH and salt concentration and was unaffected by the presence of sulfhydryl-blocking agents. The purest protein fraction also recognizes DNA modified with methylmethane-sulfonate or methylnitrosourea. It shows little or no recognition of single-stranded DNA, double-stranded DNA, supercoiled bacteriophage phiX174 DNA, partially depurinated DNA, glucosylated bacteriophage T4DNA, or UV-irradiated DNA. No endo- or exonuclease activity, DNA polymerase activity, or glucosylase activity for AAF-DNA was detectable in the preparation.

A marked induction of DNA replication was observed in confluent human diploid fibroblast cultures treated with low relatively nontoxic doses of UV radiation, N-methyl-N-nitrosourea (MNU), and N-acetoxy-2-acetylaminofluorene (AAAF). Isopycnic CsCl density gradient analysis of newly synthesized DNA labeled with BrdUrd indicated that most of the synthesis was semiconservative. The rate of semiconservative DNA synthesis was maximal 24 hr after damage. This induction of DNA replication was greatest at approximately equal to 3 J/m2 UV, 0.5 mM MNU, or 1.0 microM AAAF; was inhibited by hydroxyurea and aphidicolin; and also occurred in repair-deficient xeroderma pigmentosum fibroblasts. Autoradiographic examination of both confluent cultures and serum-arrested cultures showed a large increase in the fraction of densely labeled (S phase) cells after UV treatment. These densely labeled cells retain the capacity for cell division and subsequent proliferation. We conclude that low doses of at least three different DNA damaging agents are capable of recruiting quiescent cells into a state of DNA replication similar to that observed in the normal cell cycle.

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.

Studies from several laboratories have shown that ATP is required for DNA excision repair in UV-irradiated mammalian cells. Using permeable human fibroblasts, we have investigated this ATP requirement in detail. We find that ATP is required for specific incision of UV-damaged DNA in permeable cells. No ATP-dependent incision is seen in UV-irradiated permeable xeroderma pigmentosum (complementation group G) fibroblasts, indicating that the ATP-dependent incision observed in normal cells is part of the normal excision repair process. We conclude that, in mammalian cells, ATP is required for specific incision of UV-damaged DNA or for some obligatory step preceding incision in the excision repair pathway. ATP also protects the permeable cells from loss of the capacity to perform excision repair, probably in a nonspecific fashion. The actual synthesis of repair patches can proceed in the absence of ATP; however, our data do not exclude the possibility that ATP can also stimulate repair synthesis directly.