The transcriptional response after oxidative stress is defective in Cockayne syndrome group B cells.

Cockayne syndrome (CS) is a human hereditary disease belonging to the group of segmental progerias, and the clinical phenotype is characterized by postnatal growth failure, neurological dysfunction, cachetic dwarfism, photosensitivity, sensorineural hearing loss, and retinal degradation. CS-B cells are defective in transcription-coupled DNA repair, base excision repair, transcription, and chromatin structural organization. Using array analysis, we have examined the expression profile in CS complementation group B (CS-B) fibroblasts after exposure to oxidative stress (H2O2) before and after complete complementation with the CSB gene. The following isogenic cell lines were compared: CS-B cells (CS-B null), CS-B cells complemented with wild-type CSB (CS-B wt), and a stably transformed cell line with a point mutation in the ATPase domain of CSB (CS-B ATPase mutant). In the wt rescued cells, we detected significant induction (two-fold) of 112 genes out of the 6912 analysed. The patterns suggested an induction or upregulation of genes involved in several DNA metabolic processes including DNA repair, transcription, and signal transduction. In both CS-B mutant cell lines, we found a general deficiency in transcription after oxidative stress, suggesting that the CSB protein influenced the regulation of transcription of certain genes. Of the 6912 genes, 122 were differentially regulated by more than two-fold. Evidently, the ATPase function of CSB is biologically important as the deficiencies seen in the ATPase mutant cells are very similar to those observed in the CS-B-null cells. Some major defects are in the transcription of genes involved in DNA repair, signal transduction, and ribosomal functions.

Activation of the MEK/MAPK pathway is involved in bryostatin1-induced monocytic differenciation and up-regulation of X-linked inhibitor of apoptosis protein.

Induction of monocytic differentiation by bryostatin1 (bryo1) conferred on THP-1 leukemia cells the ability to resist Z-LLL-CHO-induced apoptosis. The mechanism of resistance developed during this process was investigated. Apoptosis resistance was associated with an enhanced expression of X-linked inhibitor of apoptosis protein (XIAP), an endogenous caspase inhibitor, in differentiated THP-1 cells. Bryo1 also increased the level of c-IAP-1, yet decreased the level of c-IAP-2 in THP-1 cells, indicating that distinct regulatory mechanisms are operative. In addition, treatment of THP-1 cells with bryo1 induced a rapid and sustained activation of MEK, prior to the upregulation of XIAP and monocytic differentiation. Pretreatment of THP-1 cells with MEK inhibitors (U0126 and PD98059) prior to bryo1 induction blocked the expression of both XIAP and the c-fms product (M-CSF receptor), a hallmark of monocytic differentiation, but not Bcl-2. In addition, the expression of XIAP in bryo1-treated cells was inhibited by CAPE, a NF-kappaB-specific inhibitor, indicating that its expression is under the transcriptional regulation of NF-kappaB downstream of the MEK/MAPK pathway. The importance of XIAP in mediating apoptosis resistance was illustrated in cells transiently transfected with XIAP, which conferred on THP-1 cells the ability to resist Z-LLL-CHO-induced apoptosis. These findings suggest that the expression of XIAP is linked to monocytic differentiation in bryo1-treated THP-1 cells and represents one of the potential antiapoptotic mechanisms acquired during this process.

Functional crosstalk between hOgg1 and the helicase domain of Cockayne syndrome group B protein.

We have previously reported that the Cockayne syndrome group B gene product (CSB) contributes to base excision repair (BER) of 8-hydroxyguanine (8-OH-Gua) and the importance of motifs V and VI of the putative helicase domains of CSB in BER of 8-OH-Gua. To further elucidate the function of CSB in BER, we investigated its role in the pathway involving human 8-OH-Gua glycosylase/apurinic lyase (hOgg1). Depletion of CSB protein with anti-CSB antibody reduced the 8-OH-Gua incision rate of wild type cell extracts but not of CSB null and motif VI mutant cell extracts, suggesting a direct contribution of CSB to the catalytic process of 8-OH-Gua incision and the importance of its motif VI in this pathway. Introduction of recombinant purified CSB partially complemented the depletion of CSB as shown by the recovery of the incision activity. This complementation could not fully recover the deficiency of the incision activity in WCE from CS-B null and mutant cell lines, suggesting that some additional factor(s) are necessary for the full activity. Electrophoretic mobility shift assays (EMSAs) showed a defect in binding of CSB null and motif VI mutant cell extracts to 8-OH-Gua-containing oligonucleotides. We detected less hOgg1 transcript and protein in the cell extracts from CS-B null and mutant cells, suggesting hOgg1 may be the missing component. Pull-down of hOgg1 by histidine-tagged CSB and co-localization of those two proteins after gamma-radiation indicated their co-existence in vivo, particularly under cellular stress. However, we did not detect any functional and physical interaction between purified CSB and hOgg1 by incision, gel shift and yeast two-hybrid assays, suggesting that even though hOgg1 and CSB might be in a common protein complex, they may not interact directly. We conclude that CSB functions in the catalysis of 8-OH-Gua BER and in the maintenance of efficient hOgg1 expression, and that motif VI of the putative helicase domain of CSB is crucial in these functions.