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D Grunberger

Publications and source records attributed to D Grunberger.

At least 37 records · Page 2Linked to original sources
PubMed1993

A mutational hot spot induced by N-hydroxy-aminofluorene in dihydrofolate reductase mutants of Chinese hamster ovary cells.

Eighteen mutants deficient in dihydrofolate reductase (DHFR) activity were induced with 0.5 microM N-hydroxy-aminofluorene in four separate experiments. This carcinogen dose killed approximately 80% of the treated cells and resulted in a mutational frequency approximately 3 x 10(-6). The nature of the induced changes in each of the mutants was determined by direct sequencing following polymerase chain reaction amplification, or in one instance, by Southern blot analysis. Nearly all (15/17) of the mutations were single base changes. Consistent with the binding specificity of this chemical, all mutations were targeted to guanine bases. The predominant change was G:C-->T:A transversion which was evident in 11/15 mutants. A single dG-AF mutational hotspot was noted at a site in the DHFR coding sequence of exon 4; one-third of the induced point mutations arose at this position. These results are compared with our previous analyses of mutants induced with the related aromatic amine, N-2-acetoxy-2-acetyl-aminofluorene.

Acetoxyacetylaminofluorene↗
PubMed1993

Splicing mutants and their second-site suppressors at the dihydrofolate reductase locus in Chinese hamster ovary cells.

Point mutants induced with a variety of mutagens at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary (CHO) cells were screened for aberrantly spliced dhfr mRNA by RNase protection and/or reverse transcriptase coupled with cDNA amplification by the polymerase chain reaction (PCR). Of 115 mutants screened, 28 were found to be affected in splicing. All exhibited less than 1% correct splicing, probably because the selection procedure was stringent. All 26 unique mutations were located within the consensus splice sequences; changes were found at 9 of 10 possible sites in this 25-kb six-exon gene. Mutations at the sites flanking the first and last exons resulted in the efficient recruitment of a cryptic site within each exon. In contrast, mutations bordering internal exons caused predominantly exon skipping. In many cases, multiple exons were skipped, suggesting the clustering of adjacent exons prior to actual splicing. Six mutations fell outside the well-conserved GU and AG dinucleotides. All but one were donor site single-base substitutions that decreased the agreement with the consensus and resulted in little or no correct splicing. Starting with five of these donor site mutants, we isolated 31 DHFR+ revertants. Most revertants carried a single-base substitution at a site other than that of the original mutation, and most had only partially regained the ability to splice correctly. The second-site suppression occurred through a variety of mechanisms: (i) a second change within the consensus sequence that produced a better agreement with the consensus; (ii) a change close to but beyond the consensus boundaries, as far as 8 bases upstream in the exon or 28 bases downstream in the intron; (iii) mutations in an apparent pseudo 5' site in the intron, 84 and 88 bases downstream of a donor site; and (iv) mutations that improved the upstream acceptor site of the affected exon. Taken together, these second-site suppressor mutations extend the definition of a splice site beyond the consensus sequence.

Alternative Splicing↗
PubMed1992

DNA strand-specific repair of (+-)-3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene adducts in the hamster dihydrofolate reductase gene.

We evaluated the formation and removal of (+-)-3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene (BcPHDE)-DNA adducts in two Chinese hamster ovary (CHO) cell lines. One line of repair-proficient cells (MK42) carries a stable 150-fold amplification of the dihydrofolate reductase (DHFR) locus. The other line of repair-deficient cells (UV-5) is diploid for this gene and is defective in excision of bulky DNA lesions. Two methods were used to quantitate adduct levels in treated cells: Escherichia coli UvrABC excision nuclease cleavage and 32P-postlabeling. DNA repair was examined in the actively transcribed DHFR gene, in an inactive region located 25 kilobases downstream, and in the overall genome. Between 8 and 24 hr after BcPHDE exposure, preferential repair of the DHFR gene compared to the noncoding region was apparent in MK42 cells. This gene-specific repair was associated with adduct removal from the DHFR transcribed strand. However, UV-5 cells showed no lesion reduction from this strand of the gene. By both quantitation methods, regions accessible to repair in MK42 cells showed a 2-fold reduction in DNA adduct levels by 24 hr. That the decline in adducts reflects genomic repair was demonstrated by the constant damage level remaining in UV-5 cells. Since BcPHDE-induced mutations in DHFR apparently arise from adducted purines on the nontranscribed strand, results from the present study support the idea that a consequence of strand-specific repair is strand-biased mutations.

Animals↗
PubMed1992

Growth inhibition and modulation of antigenic phenotype in human melanoma and glioblastoma multiforme cells by caffeic acid phenethyl ester (CAPE)

The active component of the honeybee hive product propolis, caffeic acid phenethyl ester (CAPE), has been shown to display increased toxicity toward various oncogene-transformed cell lines in comparison with their untransformed counterparts (Su et al., 4: 231-242, 1991). This observation provides support for the concept that it is the transformed phenotype which is specifically sensitive to CAPE. In the present study, we have determined the effect of CAPE on the growth and antigenic phenotype of a human melanoma cell line, HO-1, and a human glioblastoma multiforme cell line, GBM-18. For comparison, we have also tested the effects of mezerein (MEZ), mycophenolic acid (MPA) and retinoic acid (RA), which can differentially modulate growth, differentiation and the antigenic phenotype in these human tumor cell lines. Growth of both cell lines was suppressed by CAPE in a dose-dependent fashion, with HO-1 cells being more sensitive than GBM-18 cells. The antiproliferative effect of CAPE was enhanced in both cell types if CAPE and MEZ were used in combination. Growth suppression was associated with morphological changes in H0-1 cells, suggesting induction of a more differentiated phenotype. CAPE also differentially modulated the expression of several antigens on the surface of the two tumor cell lines. These results suggest a potential role for CAPE as an antitumor agent, an antigenic modulating agent and possibly a differentiation inducing agent.

Antigens, Neoplasm↗
PubMed1991

DNA strand-specific mutations induced by (+/-)-3 alpha,4 beta-dihydroxy- 1 alpha,2 alpha-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene in the dihydrofolate reductase gene.

We previously showed that the preferred mutation induced by (+/-)-3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy- 1,2,3,4-tetrahydrobenzo[c]phenanthrene (BcPHDE) in the dihydrofolate reductase gene in Chinese hamster ovary cells was a purine to thymine transversion on the nontranscribed strand at the sequence 5'-RRR-3' (R is a purine and the mutated base is underlined). To determine whether the observed mutational strand specificity was due to bias in the phenotypic selection, we designed a nonsense-codon reversion assay in which a triple purine target was present on both strands and all R----T transversion mutations yielded amino acid substitutions that were compatible with dihydrofolate reductase enzyme activity. From the size of the targets, a 2:1 ratio of mutations at the purines on the nontranscribed strand was expected if the DNA strands were mutationally equivalent. We isolated a total of 66 BcPHDE-induced revertants of two mutants that carry point mutations at either the 5' or the 3' end of the gene. All reversions at the 5' end arose by substitution on the nontranscribed strand; those at the 3' end showed a strand bias that favored this strand by 7:1. For both mutants, R----T transversions accounted for 88% of all the induced base changes. Thus, in this system, mutational strand bias is independent of the selection for phenotype. The results are consistent with the model of preferential repair of the transcribed strand as proposed by others. The involvement of RNA polymerase in the selective repair recruitment is discussed.

Animals↗
PubMed1991

Suppression of adenovirus type 5 E1A-mediated transformation and expression of the transformed phenotype by caffeic acid phenethyl ester (CAPE).

Viral transformation and DNA-transfection assays were employed to investigate the differential toxic effect of caffeic acid phenethyl ester (CAPE), an extract of the honeybee hive product propolis, on adenovirus type 5 (Ad5)-transformed cloned rat embryo fibroblast (CREF) cells. CAPE inhibited, in a dose-dependent manner, both de novo and carcinogen-enhanced transformation of CREF cells by H5hr1, the cold-sensitive (cs) host-range mutant of Ad5. CAPE had a selective inhibitory effect on Ad5-induced transformation when a wild-type (wt) Ad5 E1A gene or a cs Ad5 E1A gene (at 37 degrees C, but not at 32 degrees C) was cotransfected into CREF cells with a dominant-acting bacterial hygromycin-resistance gene. A requirement for the expression of Ad5 E1A-encoded mRNAs and transforming proteins and sensitivity to CAPE was demonstrated using CREF cells stably transformed by a cs Ad5 E1A gene and an Ad5 E1A gene under the transcriptional control of a mouse mammary tumor virus promoter. To distinguish between the effects of the two Ad5 E1A-encoded proteins of 289 amino acids (aa) and 243 aa, CREF cells were stably transformed with cDNAs encoding either the 13S or the 12S E1A mRNA. CREF cells expressing the 13S E1A-encoded 289-aa protein were more sensitive to the growth-suppressing effect of CAPE than cells producing only the 12S E1A-encoded 243-aa protein. However, the growth-suppressing and toxic effects of CAPE were greatest in cells expressing both E1A-encoded transforming proteins. Analysis of the effect of CAPE on E1A and beta-actin gene expression in wt and cs E1A and H5hr1-transformed CREF cells indicated that low levels of CAPE, which were growth suppressive, did not selectively suppress E1A expression. These results demonstrated that cellular changes induced in CREF cells by the 13S E1A-encoded 289-aa protein of Ad5, when expressed alone or in combination with the 12S E1A-encoded 243-aa protein, rendered transformed cells sensitive to the growth-suppressing and toxic effects of CAPE.

Actins↗
PubMed1990

Restriction fragment length polymorphism of chromosome 3 (3p) and the epidermal growth factor receptor gene in human non-small cell lung carcinomas.

We have examined the possible loss of 3p alleles in lung tumor samples from 28 patients with non-small cell lung cancers (non-SCLC), using tumor adjacent lung tissue from the same patients as controls. Of the 14 patients with squamous cell carcinoma only 2 (14%) displayed constitutional heterozygosity at the 3p locus and the tumors of both of these cases did not show reduction to homozygosity. Of the 14 patients with adenocarcinomas, 50% had constitutional heterozygosity, and two of the tumors displayed a loss of heterozygosity. We have also examined restriction fragment length polymorphisms (RFLPs) of the epidermal growth factor (EGF) receptor gene in 29 non-SCLC tumor samples and in the tumor adjacent lung tissue samples obtained from the same cases. Digestion of the DNA samples with the BstEII enzyme and hybridization to a HER-A64-3 probe revealed four different types of polymorphic patterns. We did not, however, detect significant differences in the specific polymorphic bands between tumor and paired non-tumor lung tissues or between the different types of carcinomas.

Blotting, Southern↗
PubMed1990

Expression of cloned human reticulocyte 15-lipoxygenase and immunological evidence that 15-lipoxygenases of different cell types are related.

Cloned 15-lipoxygenase has been expressed for the first time in eukaryotic and prokaryotic cells. Transfection of osteosarcoma cells with a mammalian expression plasmid containing the cDNA for human reticulocyte 15-lipoxygenase resulted in cell lines that were capable of oxidizing body arachidonic acid and linoleic acid. The lipoxygenase metabolites were identified by reverse-phase and straight-phase high pressure liquid chromatography, ultraviolet spectroscopy, and direct mass spectrometry, verifying that the cDNA for 15-lipoxygenase encodes an enzyme with authentic 15-lipoxygenase activity. Incubation of the transformed cells with arachidonic acid generated 15-hydroxyeicosatetraenoic acid (HETE) and 12-HETE in a ratio of 8.6 to 1, demonstrating that 15-lipoxygenase can also perform 12-lipoxygenation. Lesser amounts of 15-keto-ETE, four isomers of 8,15-diHETE, and one isomer of 14,15-diHETE were observed. Incubation with linoleic acid generated predominantly 13-hydroxy linoleic acid. The reaction was inhibited by eicosatetraynoic acid but not by indomethacin. Antibodies to a peptide corresponding to a unique region of the predicted amino acid sequence were generated and shown to react with one major band of 70 kDa on immunoblots of human leukocyte 15-lipoxygenase. To obtain antibodies to the full length enzyme, the cDNA was subcloned into a bacterial expression vector and was expressed as a fusion with the CheY protein. The overexpressed protein was readily purified from bacteria and was shown to be immunoreactive to the peptide-derived antibody. Antibodies raised to this recombinant enzyme did not cross-react with human leukocyte 5-lipoxygenase but did identify 15-lipoxygenase in rabbit reticulocytes, human leukocytes, and tracheal epithelial cells, suggesting that the 15-lipoxygenases from these different cell types are structurally related.

Amino Acid Sequence↗
PubMed1990

Splicing mutations in the CHO DHFR gene preferentially induced by (+/-)-3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene.

Cultured Chinese hamster ovary (CHO) cells were treated with the polycyclic aromatic hydrocarbon racemic 3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene. Mutants deficient in dihydrofolate reductase activity were isolated. A carcinogen treatment at 0.1 microM yielded at 46% survival of the treated population and an induced frequency of mutation of 1.7 x 10(-4), 10(3)-fold greater than the spontaneous rate. By polymerase chain reaction amplification and direct DNA sequencing, we determined the base changes in 38 mutants. Base substitutions accounted for 78% (30/38) of the mutations. We obtained, in addition, four frameshift and four complex mutations. The preferred type of mutation was transversion (A.T----T.A and G.C----T.A) occurring in 69% of the analyzed mutants. A purine was on the 3' side of the putative adduct site in every mutant. Mutations were favored at sequences AGG, CAG, and AAG (the underlined base is the target). Surprisingly, 42% of the mutations created mRNA splicing defects (16/38), especially at splice acceptor sites for each of the five introns. Thus, this chemical carcinogen may recognize some aspect of DNA structure in regions corresponding to pre-mRNA splice sites.

Animals↗
PubMed1990

DNA base changes in benzo[a]pyrene diol epoxide-induced dihydrofolate reductase mutants of Chinese hamster ovary cells.

We previously treated Chinese hamster ovary (CHO) cells with benzo[a]pyrene diol epoxide (BPDE) and mutants at the dihydrofolate reductase (dhfr) locus were isolated. On the basis of Southern blotting and RNA heteroduplex mapping experiments, 14 of the 15 mutants were presumed to carry point mutations. Two restriction fragment length polymorphism mutants were cloned and sequenced; one carried a point mutation, the other a -1 frameshift mutation. Using polymerase chain reaction techniques and direct sequencing of amplified mutant DNA, we have now determined the induced changes in the remaining 12 cell lines. All changes occurred at guanine bases; all target guanines except one were on the non-transcribed coding strand. Most mutants (79%) contained base substitutions; the rest (3/14) carried frameshift mutations. Of the point mutations, all but one (91%) were GC----TA transversions either in the dhfr coding sequence or at splice sites. The single exception was a GC----AT transition. Of the frameshift mutations, two were deletions of a GC pair and the other was an insertion of an AT pair. Four different mutations (29%) were clustered in a 3 bp region in exon 4. Tandem guanine bases adjacent to adenine were favored sites for mutation occurring in 9/14 cases (64%). These results are consistent with data previously obtained by others in the supF shuttle vector system and the CHO aprt gene.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1989

NMR and computational characterization of the N-(deoxyguanosin-8-yl)aminofluorene adduct [(AF)G] opposite adenosine in DNA: (AF)G[syn].A[anti] pair formation and its pH dependence.

This paper reports on a combined two-dimensional NMR and energy minimization computational characterization of the conformation of the N-(deoxyguanosyl-8-yl)aminofluorene adduct [(AF)G] positioned across adenosine in a DNA oligomer duplex as a function of pH in aqueous solution. This study was undertaken on the d[C1-C2-A3-T4-C5-(AF)G6-C7-T8-A9-C10-C11].[G12-G13-T14 -A15-G16-A17-G18- A19-T20-G21-G22] complementary undecamer [(AF)G 11-mer duplex]. The modification of the single G6 on the pyrimidine-rich strand was accomplished by reaction of the oligonucleotide with N-acetoxy-2-(acetylamino)fluorene and subsequent deacetylation under alkaline conditions. The HPLC-purified modified strand was annealed with the unmodified purine-rich strand to generate the (AF)G 11-mer duplex. The exchangeable and nonexchangeable protons are well resolved and narrow in the NMR spectra of the (AF)G 11-mer duplex so that the base and the majority of sugar nucleic acid protons, as well as several aminofluorene ring protons, have been assigned following analysis of two-dimensional NOESY and COSY data sets at pH 6.9, 30 degrees C in H2O and D2O solution. The NOE distance constraints establish that the glycosidic torsion angle is syn at (AF)G6 and anti at A17, which results in the aminofluorene ring being positioned in the minor groove. A very large downfield shift is detected at the H2' sugar proton of (AF)G6 associated with the (AF)G6[syn].A17[anti] alignment in the (AF)G 11-mer duplex. The NMR parameters demonstrate formation of Watson-Crick C5.G18 and C7.G16 base pairs on either side of the (AF)G6[syn].A17[anti] modification site with the imino proton of G18 more stable to exchange than the imino proton of G16. Several nonexchangeable aminofluorene protons undergo large downfield shifts as do the imino and H8 protons of G16 on lowering of the pH from neutrality to acidic values for the (AF)G 11-mer duplex. Both the neutral and acidic pH conformations have been defined by assigning the NOE constraints in the [C5-(AF)G6-C7].[G16-A17-G18] segment centered about the modification site and incorporating them in distance constrained minimized potential energy calculations in torsion angle space with the DUPLEX program. A series of NOEs between the aminofluorene protons and the DNA sugar protons in the neutral pH conformation establish that the aminofluorene ring spans the minor groove and is directed toward the G16-A17-G18 sugar-phosphate backbone on the partner strand.(ABSTRACT TRUNCATED AT 400 WORDS)

2-Acetylaminofluorene↗
PubMed1989

DNA base changes and RNA levels in N-acetoxy-2-acetylaminofluorene-induced dihydrofolate reductase mutants of Chinese hamster ovary cells.

Formerly, we isolated a series of dihydrofolate reductase-deficient Chinese hamster ovary cell mutants that were induced by N-acetoxy-2-acetylaminofluorene. Deletions and complex gene rearrangements were detected in 28% of these mutants; 72% contained putative point mutations. In the present study, we have localized the putative point mutations in the 25,000 base dhfr gene by RNase heteroduplex mapping. Assignment of a position for each mutation was successful in 16 of 19 mutants studied. We cloned DNA fragments containing the mapped mutations from nine mutants into a bacteriophage lambda vector. In the case of 11 other mutants, DNA was amplified by the polymerase chain reaction procedure. Sequence analysis of cloned and amplified DNA confirmed the presence of point mutations. Most mutants (90%) carried base substitutions; the rest contained frameshift mutations. Of the point mutations, 75% were G.C to T.A transversions in either the dhfr coding sequence or at splice sites; transition G.C to A.T mutations were found in two mutants (10%). In one of these transition mutants, the base substitution occurred at the fifth base of the third intron. Of the frameshift mutations, one was a deletion of G.C pair and the other was an insertion of an A.T pair. Of the mapped mutants, 38% exhibited greatly reduced (approximately 10-fold) steady-state levels of dhfr mRNA. All eight sequenced mutants displaying this phenotype contained premature chain termination codons. Normal levels of dhfr mRNA were observed in five missense mutants and in five mutants carrying nonsense codons in the translated portion of exon VI. Taken together with the results of other mutagens at this locus, we conclude that the low dhfr mRNA phenotype is correlated with the presence of nonsense codons in exons II to V but not in the last exon of the dhfr gene.

2-Acetylaminofluorene↗
PubMed1989

Point mutation analysis in a mammalian gene: rapid preparation of total RNA, PCR amplification of cDNA, and Taq sequencing by a novel method.

We have developed a very rapid procedure for DNA sequence analysis of induced mutations in a typically large mammalian gene. We are able to determine the nature of chemical carcinogen-induced point mutations in the 25 kb Chinese hamster ovary (CHO) cell dihydrofolate reductase gene within two days starting with 5 to 10 x 10(6) cells. The approach is based on the use of rapidly prepared total RNA from which a 730 bp dhfr cDNA is synthesized by reverse transcriptase and amplified by the polymerase chain reaction (PCR) procedure. Genomic DNA can simultaneously be prepared from the same cells. The amplified double-stranded cDNA is then sequenced directly by the dideoxy method using Taq polymerase in the Thermal Cycler (Perkin Elmer/Cetus). We have previously shown that nonsense codons in the dhfr coding sequence often result in greatly reduced steady-state levels of dhfr mRNA (2). The methods described are suitable for mutants of this type which contain only 1 to 2 copies of mRNA per cell. This approach is readily adaptable to other selectable genes and other cell types provided the necessary primers can be prepared.

Animals↗
PubMed1988

Molecular cloning and primary structure of human 15-lipoxygenase.

A full-length cDNA encoding 15-lipoxygenase has been isolated from a human reticulocyte cDNA library. The predicted primary structure of the enzyme exhibits a sequence similarity of 61% and 45% with human 5-lipoxygenase and the soybean lipoxygenase isoenzyme I, respectively. When all three lipoxygenases are aligned, there are two distinct regions of significant sequence identity including a cluster of five histidine residues conserved in all three lipoxygenases. Because histidines can serve as ligands for the enzymatically active iron, this region may be critical to enzymatic function. These results provide a basis for exploring functional domains of lipoxygenases.

Amino Acid Sequence↗
PubMed1988

Probing the surface of Z-DNA with anti-nucleoside antibodies.

Antibodies specific for cytidine (C) and guanosine (G) were used to probe the surface of two Z-DNA conformers. When tested by ELISA, anti-G reacted with poly(dG-dC).poly(dG-dC) treated with bromine water [Br-poly(dG-dC).poly(dG-dC)] but anti-C did not. A weak reaction with anti-C was detected by dot immunobinding. In contrast, anti-C reacted strongly with poly(dG-dC).poly(dG-dC) treated with N-acetoxy-2-(acetylamino)fluorene [AAF-poly(dG-dC).poly(dG-dC)]; anti-G reacted weakly, despite the fact that most G residues had not been substituted with AAF. Neither antinucleoside bound to the B conformation of poly(dG-dC).poly(dG-dC). In competition experiments, GMP was the most efficient competitor of the reaction of anti-G with Br-poly(dG-dC).poly(dG-dC); AMP and TMP were 100-fold less efficient, and CMP did not compete to a significant extent. In contrast, the reaction of anti-Z with Br-poly(dG-dC).poly(dG-dC) was not inhibited by nucleotides. Of five possible sites recognized on guanosine by anti-G antibodies (N1, C6, O6, N7, and C8), AMP and TMP share three or their equivalent and CMP only one. The binding of anti-C to AAF-poly(dG-dC).poly(dG-dC) was inhibited best by CMP; AMP was 8 times less efficient; GMP and TMP were about 35-fold less efficient than CMP. Thus, although the amino group on the C4 position of CMP appears to be immunodominant, the capacity of GMP and TMP to inhibit the reaction indicates that other sites are also recognized in AAF-poly(dG-dC).poly(dG-dC), e.g., the exposed C5 position.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗
PubMed1988

Inhibition of c-H-ras oncogene induced transformation of rat embryo fibroblasts by cotransfected polynucleotides containing alternating purine-pyrimidine sequences.

When Rat 6 cultures were cotransfected with an activated c-H-ras oncogene (pT24) and poly(dG-m5dC), a synthetic polymer that has the potential to form Z DNA, there was marked inhibition of cell transformation. Cotransfection of pT24 DNA with poly(dG-dC) caused somewhat less inhibition, poly(dA-dC). (dG-dT) caused moderate inhibition, and poly(dG). (dC) exerted negligible inhibition. Evidence was obtained that the inhibition seen with poly(dG-m5dC) was not simply due to an inhibition of cellular uptake of the pT24 DNA. Our results suggest that certain polymers that have the potential to form Z DNA can inhibit the integration and expression of a transfected oncogene.

Animals↗