Synergistic interaction between lipid-loading and doxorubicin exposure in Huh7 hepatoma cells results in enhanced cytotoxicity and cellular oxidative stress: implications for acute and chronic care of obese cancer patients.

There has been a dramatic increase in the number of clinically obese individuals in the last twenty years. This has resulted in an increasingly common scenario where obese individuals are treated for other diseases, including cancer. Here, we examine interactions between lipid-induced steatosis and doxorubicin treatment in the human hepatoma cell line Huh7. The response of cells to either doxorubicin, lipid-loading or a combination were examined at the global level by DNA microarray, and for specific endpoints of cytotoxicity, lipid-loading, reactive oxygen species, anti-oxidant response systems, and apoptosis. Both doxorubicin and lipid-loading caused a significant accumulation of lipid within Huh7 cells, with the combination resulting in an additive accumulation. In contrast, cytotoxicity was synergistic for the combination compared to the individual components, suggesting an enhanced sensitivity of lipid-loaded cells to the acute hepatotoxic effects of doxorubicin. We demonstrate that a synergistic increase in reactive oxygen species and deregulation of protective anti-oxidant systems, most notably metallothionein expression, underlies this effect. Transcriptome analysis confirms synergistic changes at the global level, and is consistent with enhanced pro-inflammatory signalling in steatotic cells challenged with doxorubicin. Such effects are consistent with a potentiation of progression along the fatty liver disease spectrum. This suggests that treatment of obese individuals with doxorubicin may increase the risk of both acute (i.e. hepatotoxicity) and chronic (i.e. progress of fatty liver disease) adverse effects. This work highlights the need for more study in the growing therapeutic area to develop risk mitigation strategies.

Intergenic transcription in the human beta-globin gene cluster.

Our previous studies on nascent transcription across the human beta-globin gene cluster revealed the presence of intergenic transcripts in addition to the expected genic transcripts. We now show that transcription into the beta-globin locus control region (LCR) begins within an ERV9 endogenous retroviral long terminal repeat upstream of DNase I hypersensitive site 5. However, in a transgenic mouse, which has the human beta-globin LCR but lacks the ERV9 LTR, transcription begins upstream of the transgenic locus. We postulate that in this transgenic mouse nearby endogenous mouse promoters are activated by the LCR. Intergenic transcription is also detected across the whole transgenic globin gene locus independently of the stage of erythroid development. Intergenic transcription in the beta-globin cluster is erythroid specific; however, it can be induced in nonerythroid cells by several means: by transinduction with a plasmid transcribing part of the cluster, by exogenous addition of transcription factors, and by treatment with the histone deacetylase inhibitor trichostatin A.

A novel G472R mutation in a Turkish family with X-linked Alport syndrome.

Alport syndrome (AS) is a hereditary disorder of progressive nephritis. Most cases are X-linked, but autosomal forms have been reported. The X-linked form is associated with mutations in the COL4A5 gene that encodes the alpha 5 chain of type IV collagen. More than 200 mutations have been reported in X-linked AS. We report a novel 1616 G > A mutation resulting in glycine substitution to arginine at position 472 in a Turkish family with a severely affected man and several variably affected women. This is the first Turkish family in whom the molecular basis of the disease has been reported.

Detection of mutations in COL4A5 in patients with Alport syndrome.

Alport syndrome (AS) can be caused by mutations in COL4A5, one of the six type IV collagen genes. For the purposes of confirming diagnoses, carrier screening and correlating genotype to phenotype, we have screened all 51 exons of this gene by SSCP analysis in 153 families with suspected AS. Mutations were identified in 77 families (of which 20 have previously been reported) and are reported with all available clinical information. All types of mutation were found (missense, nonsense, splicing, small and large deletions and insertions), with the commonest type being those affecting glycine residues in the collagen triple helix. Our 50% detection rate is similar to that of other groups and may imply the presence of mutations outside of the COL4A5 coding region or the existence of a second X-linked AS gene.

Stimulation of RNA 3' processing by flanking DNA in Xenopus oocytes.

We have previously shown that the second poly(A) signal of the Xenopus laevis alpha-tubulin gene X alpha T14, which contains the rare hexanucleotide CAUAAA, requires a surprisingly large amount of 3' flanking DNA to be used efficiently in Xenopus oocytes. To investigate the nature of the interaction between the X alpha T14 3' flank and upstream 3' processing sites, we have developed a modified oocyte assay based on the stimulation of processing at a single poly(A) signal. We mutated both the hexanucleotide and GU/U-rich components of a strong synthetic poly(A) signal (SPA) in order to weaken it severely. We found that efficient use of the mutant signal could be fully restored by the addition of 1.2 kb of X alpha T14 3' flank, but only in its natural orientation. Functional dissection of the X alpha T14 3' flank defined two separate regions that were each capable of partially restoring processing efficiency, presumably because they contain multiple, relatively weak processing enhancers. We discuss how the stimulation of 3' processing by flanking regions in oocytes could be explained by mechanisms that operate on the processing machinery directly or by indirect effects mediated by transcriptional pausing.

Genes encoding isoforms of transcription elongation factor TFIIS in Xenopus and the use of multiple unusual RNA processing signals.

We have identified cDNAs encoding three related forms of transcription elongation factor TFIIS (S-II) in Xenopus laevis ovary. Comparison of Xenopus and mammalian sequences identifies likely diagnostic amino acids that distinguish classes of vertebrate TFIIS. The diversity of TFIIS polypeptides in Xenopus is due partly to the presence of two diverged genes in this tetraploid genome. We isolated genomic clones containing one of the genes, xTFIIS.oA, and, unlike a previously described vertebrate TFIIS gene, found that it contains introns. Alternative splicing at a CAG/CAG motif containing the 3' splice site of intron 4 produces the third form of xTFIIS, which differs from one of the others simply in lacking Ser109. Intron 6 of xTFIIS.oA contains splice and branch site consensus sequences conforming to those of the minor class of AT-AC introns and this was confirmed for the homeologous xTFIIS.oB gene by genomic PCR. Other unusual but functional variants of RNA processing signals were found in xTFIIS genes at the 5' splice site of intron 8 and the polyadenylation hexanucleotides. Utilization of multiple unusual processing signals may make the generation of mature xTFIIS.o mRNAs inefficient and the possible regulatory consequences of this are discussed.