A novel transformation suppressor, Pdcd4, inhibits AP-1 transactivation but not NF-kappaB or ODC transactivation.

Pdcd4 is a novel transformation suppressor that is highly expressed in promotion-resistant (P-) mouse epidermal JB6 cells but not in susceptible (P+) cells. Overexpression of pdcd4 cDNA in stably transfected P+ cells rendered cells resistant to tumor promoter-induced transformation, indicating that elevated expression of Pdcd4 protein is sufficient to suppress neoplastic transformation. To determine whether Pdcd4 suppresses neoplastic transformation through inhibiting known transformation required events, we examined the possibility that pdcd4 inhibited the activation of AP-1 or NF-kappaB dependent transcription or of ornithine decarboxylase (ODC) activity. Activation of AP-1-dependent transcriptional activity was inhibited by pdcd4 expression in a concentration dependent manner. In contrast, Pdcd4 slightly increased NF-kappaB-dependent transcription and did not alter ODC enzymatic activity. Previous studies suggested that activation of AP-1 was required for P+ cell transformation as well as for tumor promotion in vivo. These results indicate that Pdcd4 functions as a transformation suppressor, possibly through inhibiting AP-1 activation in combination with other factors such as enhancing NF-kappaB activation. Pdcd4 may thus constitute a useful molecular target for cancer prevention.

cDNA cloning and mapping of mouse pleckstrin (Plek), a gene upregulated in transformation-resistant cells.

Changes that occur during tumor promotion, the rate-limiting phase of multistep carcinogenesis, may offer the best targets for prevention of cancer or reversal of early disease. The murine epidermal JB6 promotion-sensitive (P+) and -resistant (P-) cell lines provide a cell culture model for tumor promoter-induced neoplastic transformation ideally suited to the identification of molecular events that mediate or inhibit transformation. A differential display comparison of P+ and P- cell mRNAs yielded seven differentially expressed sequences. One of the sequences preferentially expressed in P- cells identified an approximately 3. 6-kb message that was induced to higher levels in P- cells following exposure to the tumor promoter 12-O-tetradecanoylphorbol acetate than in P+ cells. The message was detected in mRNA from heart, lung, and spleen. cDNA cloning of the P- preferential sequence revealed a high degree of identity to human pleckstrin (PLEK), the major PKC substrate in platelets (Tyers et al., 1988, Nature 333: 470). We report the complete mouse cDNA sequence of pleckstrin and the localization of the gene to chromosome 11, its expression in a nonhematopoetic cell line, and its potential role in blocking neoplastic transformation.

Differentially expressed protein Pdcd4 inhibits tumor promoter-induced neoplastic transformation.

An mRNA differential display comparison of mouse JB6 promotion-sensitive (P+) and -resistant (P-) cells identified a novel gene product that inhibits neoplastic transformation. The JB6 P+ and P- cells are genetic variants that differ in their transformation response to tumor promoters; P+ cells form anchorage-independent colonies that are tumorigenic, and P- cells do not. A differentially displayed fragment, A7-1, was preferentially expressed in P- cells at levels >/=10-fold those in P+ cells, making its mRNA a candidate inhibitor of neoplastic transformation. An A7-1 cDNA was isolated that was identical to murine Pdcd4 gene cDNAs, also known as MA-3 or TIS, and analogous to human H731 and 197/15a. Until now, the function of the Pdcd4 protein has been unknown. Paralleling the mRNA levels, Pdcd4 protein levels were greater in P- than in P+ cells. Pdcd4 mRNA was also expressed at greater levels in the less progressed keratinocytes of another mouse skin neoplastic progression series. To test the hypothesis that Pdcd4 inhibits tumor promoter-induced transformation, stable cell lines expressing antisense Pdcd4 were generated from parental P- cells. The reduction of Pdcd4 proteins in antisense lines was accompanied by acquisition of a transformation-sensitive (P+) phenotype. The antisense-transfected cells were reverted to their initial P- phenotype by overexpression of a Pdcd4 sense fragment. These observations demonstrate that the Pdcd4 protein inhibits neoplastic transformation.

Tumor promoter induces high mobility group HMG-Y protein expression in transformation-sensitive but not -resistant cells.

Elevated levels of high mobility group (HMG) nonhistone chromosomal proteins I and Y, alternatively spliced members of the HMG-I(Y) family of architectural transcription factors, have been linked with human cancer and with neo-plastic and metastatic phenotypes in model systems. To investigate whether HMG-I(Y) proteins may influence susceptibility to neoplastic transformation, HMG-I(Y) mRNA and protein levels were compared in the JB6 murine model of neoplastic progression. HMG-I(Y) mRNAs were expressed at very low levels in preneoplastic, transformation-resistant (P-) cell lines and were constitutively expressed at much higher levels in both transformation-sensitive (P +) and transformed (Tx) tumorigenic cell lines. HMG-I(Y) mRNAs were induced to higher levels by the tumor promoter 12-O-tetradecanoylphorbol acetate (TPA) and were sustained longer in P+ than in P- cells. Nevertheless, in both P- and P+ cells, primer extension analysis revealed that the same four major HMG-I(Y) gene transcription start sites were utilized with or without TPA treatment. RT-PCR revealed that there was always slightly more Y than I form mRNA present in all of the variant JB6 cell lines. Immunoblotting indicated that both HMG-I and -Y proteins increased in P + cells in response to TPA treatment. Remarkably, in P- cells treated with TPA, only HMG-I (and not HMG-Y) protein levels increased. This unique differential TPA-induction of the HMG-Y protein in JB6 variants suggests a role for HMG-Y in mediating tumor promoter-induced neoplastic transformation. Furthermore, these results demonstrate that HMG-I and Y protein translation and/or stability is differently regulated in JB6 P- cells and provide the first indication that I and Y proteins may have different functions.

Preferential primary-response gene expression in promotion-resistant versus promotion-sensitive JB6 cells.

The 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible sequence (TIS) genes are a set of primary response genes induced in Swiss 3T3 cells by TPA. They include three transcription factors, a prostaglandin synthase, and three proteins of unknown function. To ascertain which, if any TIS genes might be involved in tumor promotion, we examined the expression of these genes in response to tumor promoters in transformation promotion-sensitive (P+) and -resistant (P-) JB6 murine epidermal cells, a model used to identify events relevant to promotion. A subset of TIS genes (TIS1, TIS10, and TIS21) was preferentially induced by TPA in P-cells. In addition, TIS1 and TIS21 mRNAs were preferentially induced in P-cells by epidermal growth factor, another transformation promoter that distinguishes P+ from P-cells. TIS1 and TIS21 protein levels were also greater in TPA-treated P-cells than P+ cells. Forskolin, a cAMP-elevating anti-promoter, increased TPA-induced levels of TIS1, TIS10, and TIS21 mRNAs in P+ cells, ruling in potential roles for these genes in modulating tumor promotion. The anti-promoters fluocinolone acetonide, retinoic acid, and superoxide dismutase did not enhance TPA-induced levels of TIS1 and TIS21 mRNAs in P+ cells, suggesting that these inhibitors may act on other promotion-relevant genes. TIS1 encodes a member of the steroid receptor superfamily. TIS1 encodes a protein of unknown function with strong sequence similarity to BTG1, a proposed "anti-proliferative gene" (Rouault JP, Rimokh R, Tessa C, et al., EMBO J 11:1663-1670, 1992). Preferential induction by multiple promoters of these TIS genes in P-cells and enhancement of their induction in P+ cells by the anti-promoter forskolin make TIS1 and TIS21 candidates for promotion suppressor genes.

Differential transformation efficiency but not AP-1 induction under anchorage-dependent and -independent conditions.

The JB6 mouse epidermal cell system has been extensively used as an in vitro model for the study of tumor promotion. The present study aimed to assess the relevance of monolayer measurements to the process of transformation, which is induced more efficiently under anchorage-independent (AI) conditions. Although it would be ideal to use identical conditions for studying tumor promoter-induced transformation and biochemical and molecular events that may cause the process, it is not feasible in the case of soft agar conditions because cells cannot be readily recovered. In the present report, we used liquid medium over agar as an AI condition that permitted efficient recovery of cells. Responses to tumor promoter have been compared with those in monolayer and semisolid agar. Results indicate that 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced similar magnitude concentration-dependent transformation of JB6 cells under both of the AI conditions, namely soft agar and over-agar. Under anchorage-dependent (AD) conditions of exposure to TPA, the transformation efficiency was much lower than that seen under AI conditions. Mechanical detachment of monolayer cells after 5-10 days TPA exposure enriched the transformed phenotype. Activator protein 1 transcriptional activity measured at 12 h was induced equally under AD and AI conditions, and thus is not an early limiting event that could explain the lower transformation efficiency seen under AD conditions. To summarize, the over-agar and monolayer assays described in this study can be considered valid for the study of early biochemical and molecular events relevant to the promotion of transformation measured in soft agar.

Mutation spectrum and sequence alkylation selectivity resulting from modification of bacteriophage M13mp18 DNA with S-(2-chloroethyl)glutathione. Evidence for a role of S-(2-N7-guanyl)ethyl)glutathione as a mutagenic lesion formed from ethylene dibromide.

The major DNA adduct (greater than 95% total) resulting from the bioactivation of ethylene dibromide by conjugation with GSH is S-(2-(N7-guanyl)ethyl)GSH. The mutagenic potential of this adduct has been uncertain, however, because the observed mutagenicity might be caused by other adducts present at much lower levels, e.g. S-(2-N1-adenyl)ethyl)GSH. To assess the formation of other potential adducts, S-(2-(N3-deoxycytidyl)ethyl)GSH, S-(2-(O6-deoxyguanosyl)ethyl)GSH, and S-(2-(N2-deoxyguanosyl)ethyl)GSH were prepared and used as standards in the analysis of calf thymus DNA modified by treatment with [1,2-14C]ethylene dibromide and GSH in the presence of rat liver cytosol; only minor amounts (less than 0.2%) were found. A forward mutation assay in (repair-deficient) Salmonella typhimurium TA100 and sequence analysis were utilized to determine the type, site, and frequency of mutations in a portion of the lacZ gene resulting from in vitro modification of bacteriophage M13mp18 DNA with S-(2-chloroethyl)GSH, an analog of the ethylene dibromide-GSH conjugate. An adduct level of approximately 8 nmol (mg DNA)-1 resulted in a 10-fold increase in mutation frequency relative to the spontaneous level. The spectrum of spontaneous mutations was quite varied, but the spectrum of S-(2-chloroethyl)GSH-induced mutations consisted primarily of base substitutions of which G:C to A:T transitions accounted for 75% (70% of the total mutations). All available evidence implicates S-(2-(N7-guanyl)ethyl)GSH as the cause of these mutations inasmuch as the levels of the minor adducts are not consistent with the mutation frequency observed in this system. The sequence selectivity of alkylation was determined by treatment of end-labeled lac DNA fragments with S-(2-chloroethyl)GSH, cleavage of the DNA at adduct sites, and electrophoretic analysis. Comparison of the sequence selectivity with the mutation spectrum revealed no obligate relationship between the extent of adduct formation and the number of mutations which resulted at different sites. We suggest that the mechanism of mutagenesis involves DNA sequence-dependent alterations in the interaction of the polymerase with the (modified) template and incoming nucleotide.

Neighboring nucleotide interactions during DNA sequencing gel electrophoresis.

Electrophoretic separation of oligonucleotides in denaturing polyacrylamide gels is primarily a function of length-dependent mobility. The 3' terminal nucleotide sequence of the oligonucleotide is a significant, secondary determinant of mobility and separation. Oligomers with 3'-ddT migrate more slowly than expected on the basis of length alone, and thus are better separated from the preceding, shorter oligomers in the sequencing ladder. Oligomers with 3'-ddC are relatively faster than expected, and are therefore less separated. At the 3' penultimate position, -dC- increases and -dT- reduces separation. Purines at the 3' terminal or penultimate positions of oligonucleotides affect separation less than the pyrimidines. These results suggest specific interactions among neighboring nucleotides with important effects on the conformation of oligonucleotides during electrophoresis. These interactions are compared to compression artifacts, which represent more extreme anomalies of length-dependent separation of oligonucleotides. Knowledge of base-specific effects on electrophoretic behavior of DNA oligomers supplements the usual information available for determination of sequences; additionally it provides an avenue to thermodynamic and hydrodynamic investigations of DNA structure.

Comparison of the DNA-alkylating properties and mutagenic responses of a series of S-(2-haloethyl)-substituted cysteine and glutathione derivatives.

The mutagenicity of 1,2-dibromoethane is highly dependent upon its conjugation to glutathione by the enzyme glutathione S-transferase. The conjugates thus formed can react with DNA and yield almost exclusively N7-guanyl adducts. We have synthesized the S-haloethyl conjugates of cysteine and glutathione, as well as selected methyl ester and N-acetyl derivatives, and compared them for ability to produce N7-guanyl adducts with calf thymus DNA. The cysteine compounds were found to be more reactive toward calf thymus DNA and yielded higher adduct levels than did the glutathione compounds. Adduct levels tended to be suppressed when there was a net charge on the compound and were not affected by substitution of bromine for chlorine, as expected for a mechanism known to involve an intermediate episulfonium ion. Sequence-selective alkylation of fragments of pBR322 DNA was investigated. The compounds produced qualitatively similar patterns of alkylation, with higher levels of alkylation at runs of guanines. The compounds were also tested for their ability to act as direct mutagens in Salmonella typhimurium TA98 and TA100. None of the compounds caused mutations in the TA98 frameshift mutagenesis assay. In the strain TA100, where mutation of a specific guanine by base-pair substitution produces reversion, all compounds were found to produce mutations, but the levels of mutagenicity did not correlate at all with the levels of DNA alkylation. The ratio of mutations to adducts varied at least 14-fold among the various N7-guanyl adducts examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Selectivity of rat and human glutathione S-transferases in activation of ethylene dibromide by glutathione conjugation and DNA binding and induction of unscheduled DNA synthesis in human hepatocytes.

The major DNA adduct formed by the carcinogen ethylene dibromide (EDB) is S-[2-(N7-guanyl)ethyl]glutathione. This adduct results from the glutathione S-transferase (GST)-catalyzed conjugation of EDB with glutathione (GSH), which generates an episulfonium ion capable of reacting with cellular nucleophiles. Purified rat and human GST enzymes were compared for their ability to conjugate EDB with GSH and displayed high selectivity. Of the six forms of rat GST tested, conjugation was catalyzed by the alpha class enzyme 2-2 and, to a lesser extent, by the mu class enzyme 3-3. Of the three classes of cytosolic human GST, EDB conjugation was catalyzed by the alpha class enzymes. Three dimers of the human alpha class (alpha x-alpha x, alpha x-alpha y, and alpha y-alpha y) were separated by chromatofocusing. The alpha x-alpha x preparation demonstrated the highest specific activity. Rat microsomal GST had negligible activity for the conjugation of EDB with GSH. The levels of EDB-DNA adducts formed in rat and human hepatocytes were compared. DNA was isolated from both rat and human hepatocytes incubated with 0.5 mM EDB, and the level of DNA adduct formation in the human samples was about 40% of that in the rat hepatocytes. EDB concentration-dependent unscheduled DNA synthesis was demonstrated in isolated human hepatocytes. Concurrent treatment of the hepatocytes with diethylmaleate to deplete intracellular GSH inhibited EDB-induced unscheduled DNA synthesis. These results indicate that EDB alkylates DNA in human hepatocytes and that enzymatic repair of adducts may occur. The results of experiments done in rat and human systems using both purified GST enzymes and intact hepatocytes imply that the genotoxic pathway of EDB metabolism in rats and humans is similar.

DNA-glutathione adducts derived from vic-dihaloalkanes: mechanisms of mutagenesis.

The conjugation of the prototype dihaloalkane ethylene dibromide (EDB) with glutathione (GSH) yields S-(2-bromoethyl)GSH, which gives rise to S-[2-(N7-guanyl)ethyl]GSH as the major DNA adduct (greater than or equal to 95%). All reaction steps have SN2 character. Another minor DNA and RNA adduct is S-[2-(N1-adenyl)ethyl]GSH, formed in vitro and in vivo. These adducts have similar half-lives in vivo. Enhancement of GSH conjugation or inhibition of cytochrome P-450 IIE1 oxidation enhances DNA adduct levels in vivo and GSH depletion lowers levels. The mercapturic acid N-acetyl-S-[2-(N7-guanyl)ethyl]cysteine is excreted in urine and may find use as a biomarker. A series of compounds of the general structure RSCH2CH2Cl has been used to alkylate Salmonella typhimurium TA100. The ratio of (guanyl) base-pair mutations to N7-guanyl adducts varies dramatically, with S-(2-chloroethyl)GSH apparently producing the most potent guanyl adduct. This mutagenicity is not due to SOS response or alkylation specificity. Physical studies with modified oligonucleotides indicate that the N7-guanyl substitution weakens G-C pairing but does not in itself alter the selectivity of pairing to C in an isolated oligomer.

Activation of dihaloalkanes by glutathione conjugation and formation of DNA adducts.

Ethylene dibromide (1,2-dibromoethane, EDB) can be activated to electrophilic species by either oxidative metabolism or conjugation with glutathione. Although conjugation is generally a route of detoxication, in this case it leads to genetic damage. The major DNA adduct has been identified as S-[2-(N7-guanyl)ethyl]glutathione, which is believed to arise via half-mustard and episulfonium ion intermediates. The adduct has a half-life of about 70 to 100 hr and does not appear to migrate to other DNA sites. Glutathione-dependent DNA damage by EDB was also demonstrated in human hepatocyte preparations. The possible relevance of this DNA adduct to genetic damage is discussed.

Metabolism of pyrilamine maleate in Fischer 344 rats, Part I: Activity excretion profiles.

Male and female Fisher 344 rats (12 per group) were dosed by gavage with either 2 or 10 mg (based on the free amine) pyrilamine maleate containing about 12 and 6 muCi 14C-pyrilamine maleate, respectively, to determine excretion of the activity as a function of dose and sex with time. Urine and feces were collected at timed intervals through 144 h. Most of the dose (about 70%) was eliminated within 48 h through the urine and feces, but only about 80% of the total dose was recovered during the experiment. Less than 1% of the total dose remained in the rats at the end of the test period. In an additional experiment to determine the location of the remainder of the dose (about 20%), male rats were dosed with 2 mg pyrilamine maleate containing 14C-pyrilamine maleate. After 144 h, exhaustive washing of the cages resulted in recovery of approximately 20% of the dose, thus identifying its location. There were no significant sex or dose related differences observed in the total amount of 14C that was eliminated through the urine or feces and recovered. Urine and feces are the major routes of elimination of pyrilamine maleate in the Fischer 344 rat. The urinary route of elimination was more predominant than the fecal route in both sexes at either dose.

Covalent binding of 1,2-dihaloalkanes to DNA and stability of the major DNA adduct, S-[2-(N7-guanyl)ethyl]glutathione.

The major DNA adduct formed from the carcinogen ethylene dibromide (1,2-dibromoethane, EDB) is S-[2-(N7-guanyl)ethyl]glutathione, resulting from the reaction of guanyl residues with the half-mustard S-(2-bromoethyl)glutathione, which is generated by glutathione S-transferase-catalyzed conjugation of EDB with glutathione. The half-life of the alkylating species [putative S-(2-bromoethyl)glutathione or the derived episulfonium ion] was estimated to be less than 10 s. However, the stability was enough for approximately half of the alkylating metabolites to leave isolated rat hepatocytes before reacting with nucleic acids. Treatment of isolated rat hepatocytes with diethylmaleate decreased covalent binding of EDB to DNA, but treatment with 1-phenylimidazole did not, consistent with the view that conjugative metabolism is of greater importance than oxidation with regard to DNA binding. When EDB was administered to rats in vivo, only one major adduct, S-[2-(N7-guanyl)ethyl]glutathione, was formed in liver or kidney. S-[2-(N7-Guanyl)ethyl]glutathione was found in liver and kidney DNA of rats treated with 1,2-dichloroethane, but other adducts were also present. The gamma-glutamyl transpeptidase inhibitor AT-125 [L-(alpha-(5S)-alpha-amino-S-chloro-4,5-dihydro-5-isoxazoleacetic acid] did not affect the level of EDB bound to DNA by glutathione-fortified rat kidney homogenates or bound to liver or kidney DNA in vivo. The in vitro half-life of S-[2-(N7-guanyl)ethyl]glutathione in calf thymus DNA was 150 h; the half-life of the adduct in rat liver, kidney, stomach, and lung was between 70 and 100 h. Isolated S-[2-(N7-guanyl)ethyl]glutathione did not react with DNA to form new adducts. These results provide a further basis for understanding the carcinogenic action of 1,2-dihaloethanes.

Metabolism of doxylamine succinate in Fischer 344 rats. Part I: Distribution and excretion.

Experiments were conducted with male and female rats (12 per group) dosed by gavage with 2 or 20 mg (based on the free amine) doxylamine succinate containing about 10 microCi 14C-doxylamine succinate to determine distribution and excretion of the activity as a function of dose and sex with time. Urine and feces were collected at intervals up to 72 hr. Most of the dose (approximately equal to 70%) was eliminated in the first 24 hr after dosing and 95 to 100% of the dose was recovered during the 72-hr course of the experiments with both sexes and dose levels. Less than 1% of the total dose remained in the rats at the end of the test period. The urinary route of elimination was more predominant than the fecal route in both sexes given the 20-mg dose. The fecal route predominates in low-dose males whereas there is no significant difference between urinary and fecal routes of elimination in low-dose females. Preliminary characterization of urinary metabolite form using extraction techniques shows 99% of the metabolites to be in the polar conjugated form.