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J M Gudas

Publications and source records attributed to J M Gudas.

29 records · Page 2Linked to original sources
PubMed1991

Growth-responsive expression from the murine thymidine kinase promoter: genetic analysis of DNA sequences.

As a first step toward elucidating the biochemical basis of gene regulation at the G1-S boundary of the cell cycle, we have identified regions of the murine thymidine kinase (TK) promoter sufficient to confer appropriately growth-responsive expression to a heterologous gene. Using a series of TK promoter-chloramphenicol acetyltransferase (CAT) gene fusion constructs, we have identified sequences located between -174 base pairs upstream and +159 base pairs downstream of the TK translation initiation site that are sufficient to drive efficient S phase-specific expression of the CAT reporter gene in transfected murine fibroblasts. Both deletion analysis and site-specific mutagenesis experiments indicated that an Sp1 consensus binding site is critical to the activity of this promoter. Synchronized populations of BALB/c 3T3 cells stably transfected with either TK promoter-CAT fusion constructs or TK promoter-beta-globin fusion constructs expressed their respective reporter genes in an S phase-specific manner following serum stimulation. In each case, reporter gene expression was reduced during quiescence and G1 and rose upon entry of cells into S phase. The TK sequences included in these constructs therefore contained information sufficient to confer S phase-specific regulation to these two reporter genes. These results set the stage for a more detailed analysis of the sequences and trans-acting factors responsible for regulating murine TK gene expression and may lead to insights into the control of proliferation in normal and transformed cells.

Animals↗
PubMed1990

Ordered splicing of thymidine kinase pre-mRNA during the S phase of the cell cycle.

Concomitant with the onset of S phase, a series of thymidine kinase (TK) splicing intermediates as well as mature TK mRNA accumulates in the nucleus of BALB/c 3T3 cells. Most of the TK splicing intermediates are retained by oligo(dT)-cellulose chromatography, and, therefore, 3' end formation and polyadenylation probably precede the splicing of TK pre-mRNAs. We have further characterized the TK pre-mRNAs that are present in the nuclei of S-phase cells by using specific probes derived from each of the six TK intervening sequences. Based on the sizes of the pre-mRNAs and their patterns of hybridization with these intron probes, we propose a pathway for intron removal from nascent TK transcripts. Intron excision occurred by a preferred, but not necessarily obligatory, order which appears to have been conserved in mouse and Chinese hamster cells.

Animals↗
PubMed1988

A third genetic locus affecting the Ah (dioxin) receptor.

We have isolated a new benzo(a)pyrene-resistant clone, c35, of the mouse hepatoma line, Hepa-1. Cytochrome P1-450 mRNA and P1-450-dependent aryl hydrocarbon hydroxylase (AHH) activity are no longer inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin in c35. The phenotype of c35 is unstable in long-term culture. A subclone, c35-1, having partially restored AHH inducibility, was studied in detail. The concentration of dioxin required to give half-maximal induction of AHH activity was 16-fold greater in c35-1 than in Hepa-1. Scatchard analysis showed that c35-1 contains reduced levels of the Ah (dioxin) receptor, which mediates induction of P1-450, but that the affinity of the receptor for dioxin is unaltered. In vivo assays confirmed that c35-1 possesses reduced levels of receptor but showed that it is even more severely affected in nuclear translocation of the receptor. Somatic cell hybridization experiments demonstrated that c35 is recessive and belongs to a new, third complementation group of mutants defective in Ah receptor activity. We propose that c35 is mutated either in the ligand-binding Ah receptor polypeptide or in another polypeptide required for receptor function and that in c35-1 partial reversion has occurred to generate a polypeptide which is still impaired in its role in promoting nuclear translocation and/or DNA binding.

Animals↗
PubMed1988

Nuclear posttranscriptional processing of thymidine kinase mRNA at the onset of DNA synthesis.

The posttranscriptional regulatory mechanism(s) underlying thymidine kinase (TK) mRNA accumulation was investigated in BALB/c 3T3 cells during their progression from G0 into S phase of the cell cycle. Very little TK mRNA could be detected in either the nuclear or the cytoplasmic compartment from cells harvested in G0 or G1. At the onset of S phase, however, the level of nuclear TK mRNA precursors and mature TK mRNAs increased dramatically. The high molecular weight TK heterogeneous nuclear RNA species detected in the nuclei of S-phase cells were polyadenylylated and hybridized to intron sequences derived from the TK gene. A series of high molecular weight precursors could be chased to lower molecular weight species in the presence of actinomycin D, suggesting an ordered removal of intron sequences with the kinetics of a precursor-product relationship. These results demonstrate a striking change in the nuclear posttranscriptional processing of TK heterogeneous nuclear RNA at the G1-S boundary and, furthermore, define a model system for the examination of RNA-processing events in vivo.

Animals↗
PubMed1987

Regulation of cytochrome P-450c in differentiated and dedifferentiated rat hepatoma cells: role of the Ah receptor.

The induction of cytochrome P 450c mRNA and associated aryl hydrocarbon hydroxylase (AHH) activity is mediated by the Ah receptor in rodent liver and hepatic cells in vitro. In the present study we have investigated the underlying mechanisms responsible for the regulation of AHH activity in differentiated and dedifferentiated variants of the rat hepatoma cell line H4IIEC3. All of the dedifferentiated variants expressed inducible cytochrome P-450c mRNA and AHH activity following treatment with polycyclic aromatic hydrocarbons or the compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Most of the differentiated derivatives, however, were not inducible for either of these functions. Somatic cell hybridization studies revealed that the differentiated cells were AHH negative due to a defect that corresponded to the Ah receptor D gene product. 5-Azacytidine and sodium butyrate, but not mutagens, reactivated a functional Ah receptor in the differentiated line Fao, indicating that a requisite gene had been silenced by an epigenetic mechanism in this strain. Since many of the 5-azacytidine-induced revertant clones resembled dedifferentiated derivatives with respect to morphology and/or diminished expression of hepatic traits, our results support a correlation between coexpression of the dedifferentiated phenotype and AHH inducibility in these hepatoma cells.

Animals↗
PubMed1987

Cell-cycle-specific interaction of nuclear DNA-binding proteins with a CCAAT sequence from the human thymidine kinase gene.

Induction of thymidine kinase parallels the onset of DNA synthesis. To investigate the transcriptional regulation of the thymidine kinase gene, we have examined whether specific nuclear factors interact in a cell-cycle-dependent manner with sequences upstream of this gene. Two inverted CCAAT boxes near the transcriptional initiation sites were observed to form complexes with nuclear DNA-binding proteins. The nature of the complexes changes dramatically as the cells approach DNA synthesis and correlates well with the previously reported transcriptional increase of the thymidine kinase gene.

Binding Sites↗
PubMed1986

Intracellular location of the Ah receptor.

The subcellular distribution of the Ah receptor from the mouse hepatoma line, Hepa-1, was investigated following cytochalasin B treatment and cell enucleation. Probing the resultant cytoplast and nucleoplast fractions with radiolabelled tetrachlorodibenzo-p-dioxin (TCDD) revealed the presence of a specifically bound peak of receptor only in the cytoplast fraction. However, the quantity of receptor recovered in these experiments was only 10-12% of the expected value. We therefore undertook an investigation to determine the fate of the Ah receptor in the presence of cytochalasin B. Incubation of Hepa-1 cells with this compound resulted in a rapid loss or inactivation of cytosolic binding activity with a concomitant decrease in the amount of receptor partitioned into the nucleus at all time periods examined. Control experiments indicated that cytochalasin B did not compete with TCDD for binding to the Ah receptor and furthermore, that its mechanism of action could not be attributed to a non-specific effect on all cytosolic proteins. The results obtained are discussed in relation to the proposed models for induction by the estrogen and glucocorticoid binding receptors.

Animals↗
PubMed1986

Reversible inactivation of the Ah receptor associated with changes in intracellular ATP levels.

We have previously demonstrated that incubation of Hepa-1 cells in the presence of cytochalasin B (CB) results in a time- and temperature-dependent loss of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binding activity (Gudas et al., 1986). We show here that this loss of binding activity is probably attributable to a CB induced inhibition of glucose transport, as incubation of cells in the presence of glycolytic inhibitors or in glucose free medium caused a similar effect. All conditions leading to loss of binding also caused a marked reduction in cellular ATP concentration, suggesting that ATP (or perhaps another energy-dependent molecule) is required for maintaining the receptor in the active state. Inactivation of the Ah receptor occurred in the cytosol but not when it had translocated to the nucleus. Reactivation of receptor binding activity occurred readily in vivo and did not require de novo protein synthesis. However, attempts to restore receptor binding activity in vitro were not successful. To our knowledge this is the first reported evidence indicating that the TCDD binding Ah receptor can exist in both an inactive and an active form, with the amount present in the active ligand binding form being coupled to the energy state of the cells.

Adenosine Triphosphate↗
PubMed1986

DNA-mediated restoration of aryl hydrocarbon hydroxylase induction in a mouse hepatoma mutant defective in nuclear translocation of the Ah receptor.

In order to study the induction mechanism of aryl hydrocarbon hydroxylase (AHH), non-inducible mutants have previously been isolated from the mouse hepatoma cell line, Hepa-1. With the ultimate goal of isolating the corresponding genes, restoration of AHH inducibility to representative mutants by means of DNA-mediated gene transfer has been set out. The successful transfection of a C- mutant, which is defective in nuclear translocation of the Ah receptor-inducer complex, is described here, using rat genomic DNA as donor material. Primary and secondary rat transfectants were obtained, and they were assayed for hydroxylase activity, receptor translocation, and homology with a rat repetitive DNA sequence.

Animals↗
PubMedDate not supplied

Disruption of cell cycle kinetics by benzo[a]pyrene: inverse expression patterns of BRCA-1 and p53 in MCF-7 cells arrested in S and G2.

The effects of a ligand of the aromatic hydrocarbon receptor (AhR), benzo[a] pyrene (B[ a]P), and its metabolite, BPDE (7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene), on BRCA-1 levels and cell cycle kinetics were determined in MCF-7 breast cancer cells. Exposure of asynchronous MCF-7 cells for 72 hours to a non-cytotoxic dose of 0.5 microM B[a]P triggered a three-fold reduction in BRCA-1 protein. In MCF-7 cells resistant (20% to 30%) to genotoxic concentrations of B[a]P (1 to 5 microM), the loss of BRCA-1 protein was coupled with pausing in S-phase and G2/M, and accumulation of p53, mdm2 and p21. Treatment of MCF-7 cells synchronized in S-phase (72%) with B[a]P prolonged the arrest in S-phase, although this checkpoint was transient since cells resumed to G2/M after 12 hours with reduced levels of BRCA-1. In these cells, levels of p53 were increased, whereas the cellular content of p21 remained unaltered. In contrast, the co-treatment with the AhR antagonist, alpha-naphthoflavone (ANF), abrogated the deleterious effects of B[a]P on BRCA-1 expression, while preventing the accumulation of p53 and disruption of cell cycle profile. These findings suggest that the AhR mediated the inverse expression patterns of BRCA-1 and p53 upon exposure to B[a]P. The treatment with BPDE induced S-phase arrest and reduced BRCA-1 mRNA levels. The negative effects of BPDE on BRCA-1 expression were not transient since removal of BPDE did not allow complete reversal of the repression. These cumulative data suggest that the B[a]P metabolite, BPDE, may play a key role in disruption of BRCA-1 expression and cell cycle kinetics in breast epithelial cells.

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