Behavioural Effects of Using Sulfasalazine to Inhibit Glutamate Released by Cancer Cells: A Novel target for Cancer-Induced Depression.

Despite the lack of robust evidence of effectiveness, current treatment options for cancer-induced depression (CID) are limited to those developed for non-cancer related depression. Here, anhedonia-like and coping behaviours were assessed in female BALB/c mice inoculated with 4T1 mammary carcinoma cells. The behavioural effects of orally administered sulfasalazine (SSZ), a system xc- inhibitor, were compared with fluoxetine (FLX). FLX and SSZ prevented the development of anhedonia-like behaviour on the sucrose preference test (SPT) and passive coping behaviour on the forced swim test (FST). The SSZ metabolites 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP) exerted an effect on the SPT but not on the FST. Although 5-ASA is a known anti-inflammatory agent, neither treatment with SSZ nor 5-ASA/SP prevented tumour-induced increases in serum levels of interleukin-1ß (IL-1ß) and IL-6, which are indicated in depressive disorders. Thus, the observed antidepressant-like effect of SSZ may primarily be attributable to the intact form of the drug, which inhibits system xc-. This study represents the first attempt at targeting cancer cells as a therapeutic strategy for CID, rather than targeting downstream effects of tumour burden on the central nervous system. In doing so, we have also begun to characterize the molecular pathways of CID.

Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine.

Previously, we have reported the use of a recombinant adenovirus (Ad)-based host cell reactivation (HCR) assay to examine nucleotide excision repair (NER) of UVC-induced DNA lesions in several mammalian cell types. The recombinant non-replicating Ad expresses the Escherichia coli ß-galactosidase (ß-gal) reporter gene under control of the cytomegalovirus immediate-early enhancer region. We have also used methylene blue plus visible light (MB + VL) to induce the major oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG) in the recombinant Ad-encoded reporter gene in order to study base excision repair (BER). The reported variability regarding 8-oxoG's potential to block transcription by RNA polymerase II and data demonstrating that a number of factors play a role in transcriptional bypass of the lesion led us to examine the repair of 8-oxoG in the Ad reporter and its relationship to HCR for expression of the reporter gene. We have used Southern blotting to examine removal of UVC- and MB + VL-induced DNA damage by loss of endonuclease-sensitive sites from the Ad-encoded ß-gal reporter gene in human and rodent cells. We show that repair of MB + VL-induced 8-oxoG via BER and UVC-induced cyclobutane pyrimidine dimers (CPDs) via NER is substantially greater in human SV40-transformed GM637F skin fibroblasts compared to hamster CHO-AA8 cells. We also show that HCR for expression of the MB + VL-damaged and the UVC-damaged reporter gene is substantially greater in human SV40-transformed GM637F skin fibroblasts compared to hamster CHO-AA8 cells. The difference between the human and rodent cells in the removal of both 8-oxoG and CPDs from the damaged reporter gene was comparable to the difference in HCR for expression of the damaged reporter gene. These results suggest that the major factor for HCR of the MB + VL-treated reporter gene in mammalian cells is DNA repair in the Ad rather than lesion bypass.

Reduced host cell reactivation of oxidatively damaged DNA in ageing human fibroblasts.

Many reports have linked oxidative damage to DNA and the associated avoidance and/or repair processes to carcinogenesis, ageing and neurodegeneration. Cancer incidence increases with age and there is evidence that oxidative stress plays a role in human ageing and neurodegeneration. Several reports have suggested that the accumulation of unrepaired DNA lesions plays a causal role in mammalian ageing. Since base excision repair (BER) is the main pathway for the repair of oxidative DNA lesions, the relationship of BER to human ageing and carcinogenesis is of considerable interest. The aim of the present study was to examine the relationship between donor age and increasing time of cells in tissue culture and the repair of oxidative DNA damage in primary human skin fibroblasts. Methylene blue (MB) acts as a photosensitizer and after excitation by visible light (VL) produces reactive oxygen species that result in oxidative damage to DNA. MB+VL produce predominantly 8-hydroxyguanine as well as other single base modifications in DNA that are repaired by BER. We used host cell reactivation (HCR) of a non-replicating recombinant human adenovirus, Ad5CMVlacZ, which expresses the ß-galactosidase (ß-gal) reporter gene, to measure BER of MB+VL-damaged DNA. HCR of ß-gal activity for the MB+VL-treated reporter gene was examined in 10 fibroblast strains from normal donors of ages 2 to 82. The effect of cell passage number on HCR was also examined in human skin fibroblasts from 2 normal donors. We found a significant reduction in HCR with increasing cell passage number, indicating that BER decreases with increasing time of cells grown in tissue culture. We also found a significant correlation of donor age with HCR of the MB+VL-treated reporter gene for high passage number, but not for low passage number fibroblasts. The present study provides evidence that a decrease in BER of oxidatively damaged DNA may play a role in carcinogenesis, ageing and neurodegeneration.

Expression of an adenovirus encoded reporter gene and its reactivation following UVC and oxidative damage in cultured fish cells.

PURPOSE: Recombinant human adenovirus, AdCA35lacZ, was used to examine expression of a reporter gene and its reactivation following UVC (200-280 nm) and oxidative damage in fish cells. MATERIALS AND METHODS: AdCA35lacZ is a recombinant nonreplicating human adenovirus, which expresses the beta-galactosidase (beta-gal) reporter gene. UVC light produces DNA damage repaired by nucleotide excision repair (NER). In contrast, methylene blue plus visible light (MB+VL) produces oxidative DNA damage, mainly 8-oxoguanine, that is repaired by base excision repair (BER). We examined expression of the reporter gene and host cell reactivation (HCR) of the UVC-treated and MB+VL-treated reporter gene in fish cells. RESULTS: AdCA35lacZ infection of Chinook salmon cells (CHSE-214), eel cells (PBLE) and four rainbow trout cell lines (RTG-2, RT-Gill, RTS-34st and RTS-pBk), but not zebrafish (ZEB) or carp (EPC) cells resulted in expression of beta-gal. HCR of UVC-treated AdCA35lacZ in fish cells varied from that obtained in NER-deficient xeroderma pigmentosum group A fibroblasts to greater than that for NER-proficient normal human fibroblasts. HCR of UVC-treated AdCA35lacZ correlated with beta-gal expression levels for untreated AdCA35lacZ. Exposure of cells to fluorescent light (400-700 nm) increased expression of the undamaged reporter gene in normal human fibroblasts and in all fish cells except PBLE and increased HCR of the UVC-damaged reporter gene in fish cells but not in human fibroblasts. HCR of the MB + VL-treated reporter gene was similar to that in human cells for PBLE, CHSE-214, RTG-2 and RTS-pBk, but was reduced in RT-Gill and RTS-34st cells. CONCLUSIONS: These results indicate the detection of functional photoreactivation (PR) of UVC-induced DNA damage in fish cells but not in normal human fibroblasts and a link between NER and transcription of the reporter gene in the fish cells in the absence of PR. We show also efficient BER of the reporter gene in several fish cell lines.

XPF with mutations in its conserved nuclease domain is defective in DNA repair but functions in TRF2-mediated telomere shortening.

TRF2, a telomere-binding protein, is a crucial player in telomere length maintenance. Overexpression of TRF2 results in telomere shortening in both normal primary fibroblasts and telomerase-positive cancer cells. TRF2 is found to be associated with XPF-ERCC1, a structure-specific endonuclease involved in nucleotide excision repair, crosslink repair and DNA recombination. XPF-ERCC1 is implicated in TRF2-dependent telomere loss in mouse keratinocytes, however, whether XPF-ERCC1 and its nuclease activity are required for TRF2-mediated telomere shortening in human cells is unknown. Here we report that TRF2-induced telomere shortening is abrogated in human cells deficient in XPF, demonstrating that XPF-ERCC1 is required for TRF2-promoted telomere shortening. To further understand the role of XPF in TRF2-dependent telomere shortening, we generated constructs containing either wild type XPF or mutant XPF proteins carrying amino acid substitutions in its conserved nuclease domain. We show that wild type XPF can complement XPF-deficient cells for repair of UV-induced DNA damage whereas the nuclease-inactive XPF proteins fail to do so, indicating that the nuclease activity of XPF is essential for nucleotide excision repair. In contrast, both wild type XPF and nuclease-inactive XPF proteins, when expressed in XPF-deficient cells, are able to rescue TRF2-mediated telomere shortening. Thus, our results suggest that the function of XPF in TRF2-mediated telomere shortening is conserved between mouse and human. Furthermore, our findings reveal an unanticipated nuclease-independent function of XPF in TRF2-mediated telomere shortening.

Involvement of the cynABDS operon and the CO2-concentrating mechanism in the light-dependent transport and metabolism of cyanate by cyanobacteria.

The cyanobacteria Synechococcus elongatus strain PCC7942 and Synechococcus sp. strain UTEX625 decomposed exogenously supplied cyanate (NCO-) to CO2 and NH3 through the action of a cytosolic cyanase which required HCO3- as a second substrate. The ability to metabolize NCO- relied on three essential elements: proteins encoded by the cynABDS operon, the biophysical activity of the CO2-concentrating mechanism (CCM), and light. Inactivation of cynS, encoding cyanase, and cynA yielded mutants unable to decompose cyanate. Furthermore, loss of CynA, the periplasmic binding protein of a multicomponent ABC-type transporter, resulted in loss of active cyanate transport. Competition experiments revealed that native transport systems for CO2, HCO3-, NO3-, NO2-, Cl-, PO4(2-), and SO4(2-) did not contribute to the cellular flux of NCO- and that CynABD did not contribute to the flux of these nutrients, implicating CynABD as a novel primary active NCO- transporter. In the S. elongatus strain PCC7942 DeltachpX DeltachpY mutant that is defective in the full expression of the CCM, mass spectrometry revealed that the cellular rate of cyanate decomposition depended upon the size of the internal inorganic carbon (Ci) (HCO3- + CO2) pool. Unlike wild-type cells, the rate of NCO- decomposition by the DeltachpX DeltachpY mutant was severely depressed at low external Ci concentrations, indicating that the CCM was essential in providing HCO3- for cyanase under typical growth conditions. Light was required to activate and/or energize the active transport of both NCO- and Ci. Putative cynABDS operons were identified in the genomes of diverse Proteobacteria, suggesting that CynABDS-mediated cyanate metabolism is not restricted to cyanobacteria.

Enhanced expression from the human cytomegalovirus immediate-early promoter in a non-replicating adenovirus encoded reporter gene following cellular exposure to chemical DNA damaging agents.

We have examined expression from the human cytomegalovirus (CMV) promoter of a reporter gene encoded in a replication-deficient adenovirus following cellular exposure to heat shock and chemical DNA damaging agents. Expression of the reporter gene was enhanced following prior treatment of cells with cisplatin and N-acetoxy-acetylaminofluorine, but not heat shock. This enhancement was more pronounced and induced by lower chemical concentrations in xeroderma pigmentosum (XP) and Cockayne syndrome fibroblasts that are deficient in the transcription-coupled repair (TCR) pathway of nucleotide excision repair (NER) compared to that in TCR-proficient XP-C and normal strains. This is consistent with an induction of expression from the CMV promoter mediated by persistent (unrepaired) DNA damage in active genes. We show also that expression of the CMV-driven reporter is enhanced following treatment of several human tumour cell lines. This later finding has implications for combined chemotherapy and gene therapy using CMV-driven expression vectors.

Human cells deficient in transcription-coupled repair show prolonged activation of the Jun N-terminal kinase and increased sensitivity following cisplatin treatment.

PURPOSE: The Jun N-terminal kinases (JNKs) are activated by many biological, physical, and chemical stimuli, including the chemotherapeutic agent cisplatin. The primary pathway that repairs cisplatin-DNA adducts is nucleotide excision repair (NER). Xeroderma pigmentosum (XP) cells from complementation group C (XP-C) are competent in the transcription-coupled repair (TCR) pathway of NER but deficient in global genomic repair (GGR), Cockayne's syndrome (CS) cells are deficient in TCR but have normal GGR, and XP-A cells are deficient in both TCR and GGR. We used NER-deficient human fibroblasts to study the role of DNA damage in the activation of JNK and cell death following cisplatin treatment. MATERIALS AND METHODS: JNK-1 activity and clonogenic survival were examined in normal and NER-deficient human fibroblasts following cisplatin treatment. RESULTS: Cisplatin induced a transient increase in JNK-1 activity of about tenfold in normal and XP-C fibroblasts, which declined to about two- to threefold 24 h after treatment. In contrast, the activation of JNK-1 was persistent in XP-A and CS fibroblasts at 24 h after treatment and CS cells and XP-A cells, but not XP-C cells, were more sensitive to cisplatin than normal cells. CONCLUSIONS: These results suggest that a deficiency in the TCR pathway of NER results in amplified and prolonged JNK activation due to persistent damage within the transcribed strand of active genes. Further, it is this amplified and prolonged JNK activation that correlates with cisplatin-induced cell death.