Effect of the anti-neoplastic drug doxorubicin on XPD-mutated DNA repair-deficient human cells

Doxorubicin (DOX), a member of the anthracycline group, is a widely used drug in cancer therapy. The mechanisms of DOX action include topoisomerase II-poisoning, free radical release, DNA adducts and interstrand cross-link (ICL) formation. Nucleotide excision repair(NER) is involved in the removal of helix-distorting lesions and chemical adducts, however, little is known about the response of NER-deficient cell lines to anti-tumoral drugs like DOX. Wild type and XPD-mutated cells, harbouring mutations in different regions of this gene and leading to XP-D, XP/CS or TTD diseases, were treated with this drug and analyzed for cell cycle arrest and DNA damage by comet assay. The formation of DSBs was also investigated by determination of gamma H2AX foci. Our results indicate that all three NER-deficient cell lines tested are more sensitive to DOX treatment, when compared to wild type cells or XP cells complemented by the wild type XPD cDNA, suggesting that NER is involved in the removal of DOX-induced lesions. The cell cycle analysis showed the characteristic G2 arrest in repair-proficient MRC5 cell line after DOX treatment, whereas the repair-deficient cell lines presented significant increase in sub-G1 fraction. The NER-deficient cell lines do not show different patterns of DNA damage formation as assayed by comet assay and phosphorylated H2AX foci formation. Knock-down of topoisomerase II alpha with siRNA leads to increased survival in both MRC5 and XP cells, however, XP cell line still remained significantly more sensitive to the treatment by DOX. Our study suggests that the enhanced sensitivity is due to DOX-induced DNA damage that is subject to NER, as we observed decreased unscheduled DNA synthesis in XP-deficient cells upon DOX treatment. Furthermore, the complementation of the XPD-function abolished the observed sensitivity at lower DOX concentrations, suggesting that the XPD helicase activity is involved in the repair of DOX-induced lesions. (C) 2009 Elsevier B.V. All rights reserved.

Germline CYBB mutations that selectively affect macrophages in kindreds with X-linked predisposition to tuberculous mycobacterial disease

Germline mutations in CYBB, the human gene encoding the gp91(phox) subunit of the phagocyte NADPH oxidase, impair the respiratory burst of all types of phagocytes and result in X-linked chronic granulomatous disease (CGD). We report here two kindreds in which otherwise healthy male adults developed X-linked recessive Mendelian susceptibility to mycobacterial disease (MSMD) syndromes. These patients had previously unknown mutations in CYBB that resulted in an impaired respiratory burst in monocyte-derived macrophages but not in monocytes or granulocytes. The macrophage-specific functional consequences of the germline mutation resulted from cell-specific impairment in the assembly of the NADPH oxidase. This `experiment of nature` indicates that CYBB is associated with MSMD and demonstrates that the respiratory burst in human macrophages is a crucial mechanism for protective immunity to tuberculous mycobacteria.

Screening of ARHSP-TCC Patients Expands the Spectrum of SPG11 Mutations and Includes a Large Scale Gene Deletion

Autosomal recessive spastic paraplegia with thinning of corpus callosum (ARHSP-TCC) is a complex form of HSP initially described in Japan but subsequently reported to have a worldwide distribution with a particular high frequency in multiple families from the Mediterranean basin. We recently showed that ARHSP-TCC is commonly associated with mutations in SPG11/KIAA1840 on chromosome 15q. We have now screened a collection of new patients mainly originating from Italy and Brazil, in order to further ascertain the spectrum of mutations in SPG11, enlarge the ethnic origin of SPG11 patients, determine the relative frequency at the level of single Countries (i.e., Italy), and establish whether there is one or more common mutation. In 25 index cases we identified 32 mutations; 22 are novel, including 9 nonsense, 3 small deletions, 4 insertions, 1 in/del, 1 small duplication, 1 missense, 2 splice-site, and for the first time a large genomic rearrangement. This brings the total number of SPG11 mutated patients in the SPATAX collection to 111 cases in 44 families and in 17 isolated cases, from 16 Countries, all assessed using homogeneous clinical criteria. While expanding the spectrum of mutations in SPG11, this larger series also corroborated the notion that even within apparently homogeneous population a molecular diagnosis cannot be achieved without full gene sequencing. (C) 2008 Wiley-Liss, Inc.