Dexamethasone-enhanced sensitivity of mouse hippocampal HT22 cells for oxidative stress is associated with the suppression of nuclear factor-kappaB.

Glucocorticoids (GCs) exacerbate various insults to the hippocampus but the exact molecular mechanisms of this GC activity is not known. GCs can suppress the activity of the redox-sensitive nuclear factor NF-kappaB, which potentially serves neuroprotective functions. Employing electrophoretic mobility shift assays and transfection assays using a NF-kappaB-dependent reporter plasmid, we demonstrate that the increased oxidative stress sensitivity of clonal mouse hippocampal HT22 cells caused by GCs is associated with the suppression of NF-kappaB. GCs increased the expression of IkappaBalpha, the physiological inhibitor of NF-kappaB. Downregulation of NF-kappaB activity after overexpression of a dominant-negative mutant form of IkappaBalpha results in an increased sensitivity to oxidative stress. We conclude that the suppression of the basal NF-kappaB activity contributes to the enhanced vulnerability of neuronal cells to oxidative stress caused by GCs.

Mutagenic activity of ambient oxygen and mitomycin C in Fanconi's anaemia cells.

Cellular evidence suggests that Fanconi's anaemia (FA) might be a condition of increased oxygen sensitivity. In order to test this hypothesis, a common shuttle vector assay with the plasmid pZ189 was utilized. We transfected intact, circular plasmid into FA and control lymphoblast and fibroblast host cells maintained at 5 and 20% O2 (v/v). In parallel experiments, host cells were exposed to different concentrations of mitomycin C (MMC), a cross-linking agent towards which FA cells are known to be hypersensitive. Baseline mutation frequencies at 20% oxygen were significantly higher in plasmids passaged through FA lymphoblasts or FA fibroblasts in comparison with passage through the corresponding control cells. Lowering the oxygen concentration during the 48 h transfection period to 5% resulted in a significant decrease of mutation frequencies in plasmids passaged through FA cells. Sequence analysis of plasmids recovered from FA lymphoblasts revealed a mutation hot spot (22% of point mutations with G:C to A:T base substitutions) at base 117 of the supF tRNA gene. This hot spot was present only at 20% oxygen. 59% of the base changes at the hot spot and 39% of the changes elsewhere in the supF gene were C to T transitions (the corresponding figures are 0 and 27% at 5% oxygen), the most common type of base change induced by oxygen. The mutation spectrum observed suggests a role for 8-hydroxydeoxyguanosine in G:C to A:T base substitutions: at 20% oxygen, FA cells displayed 4 times as many G:C to T:A transversions than FA cells kept at 5% O2. In MMC treated cells the decrease in plasmid survival is dose dependent and more pronounced in FA than control cells. Mutation analysis shows similar rates of deletions for both control and FA cells. However, FA cells generate a specific type of deletion whose breakpoint involves an indirect repeat that corresponds to a heptamer signal sequence commonly seen at recombination sites. Together our data provide compelling evidence that the genetic defect in FA causes oxygen sensitivity and recombinational types of DNA lesions following exposure to MMC.

p53 activates Fanconi anemia group C gene expression.

The tumor suppressor protein p53 (wtp53) can bind to specific target sequences and activate transcription of genes adjacent to these DNA elements. Two p53 binding sites are present in the gene coding for the Fanconi anemia complementation group C (FAC), one in the promoter region (from -1295 to -1266) and one in the coding region of FAC (from +1828 to +1848). Gel shift experiments show that wtp53 binds to the p53 target sequence in the promoter region of the FAC gene. We have investigated whether binding of p53 to these target sites may affect expression of the FAC gene. Transfection experiments show that overexpression of wtp53 in human diploid fibroblasts and lymphoblasts augments transcription of the FAC gene up to three-fold. The transfection efficacy was approximately 15% for both cell types. The FAC expression activity per transformed cell was stimulated to an estimated level of 18- to 21-fold upon overexpression of p53. The tumor-derived p53 mutants, His175 and His273, that fail to bind DNA showed only a reduced stimulatory activity on FAC transcription. Luciferase assays demonstrated that interaction of p53 with its target site in the FAC promoter does not modulate the promoter activity. We suggest that the p53 binding site contributes to, but may not be an absolute prerequisite for p53-directed transcriptional activation. We conclude that the FAC gene can be added to the list of genes that interact with p53.

A family of hexosephosphate mutases in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae PGM1 and PGM2 genes encoding two phosphoglucomutase isoenzymes have been isolated and sequenced. The derived protein sequences are closely related to one another and show distinct sequence similarities to the human and rabbit phosphoglucomutases, especially in the region supposed to constitute the active site. PGM1 and PGM2 are located on chromosomes XI and XIII, respectively, just upstream of the known genes YPK1 and YKR2 coding for a pair of closely related putative protein kinases. These observations suggest that an extended region of DNA arose by the process of gene duplication. Cells deleted for both, PGM1 and PGM2, could not grow on galactose. No residual phosphoglucomutase activity could be measured in crude extracts or in permeabilized cells of pgm1/2 double mutants. Unexpectedly, growth with glucose was not impaired and the mutant cells were still able to accumulate trehalose and glycogen, although at a reduced level. Two further genes could be isolated and characterized which when over-expressed on a multi-copy plasmid could restore growth on galactose of the pgm1/2 double deletion mutant. Multi-copy complementation was due to a sharply increased level of phosphoglucomutase activity. Partial sequencing and characterization of the two genes revealed one of them to be SEC53 encoding phosphomannomutase. No extended sequence similarities could be found in the databases for the second gene. However, part of the derived amino acid sequence contained a region of high similarity to the active-site consensus sequence of hexosephosphate mutases from different organism. Further investigations suggest that a complex network of mutases exist in yeast which interact and can partially substitute for each other.