Regulation of redox and DNA repair genes by arsenic : low dose protection against oxidative stress?

We have evaluated the molecular responses of human epithelial cells to low dose arsenic to ascertain how target cells may respond to physiologically relevant concentrations of arsenic. Data gathered in numerous experiments in different cell types all point to the same conclusion: low dose arsenic induces what appears to be a protective response against subsequent exposure to oxidative stress or DNA damage, whereas higher doses often provoke synergistic toxicity. In particular, exposure to low, sub-toxic doses of arsenite, As(III), causes coordinate up-regulation of multiple redox and redox-related genes including thioredoxin (Trx) and glutathione reductase (GR). Glutathione peroxidase (GPx) is down-regulated in fibroblasts, but up-regulated in keratinocytes, as is glutathione S-transferase (GST). The maximum effect on these redox genes occurs after 24 h exposure to 5–10 mM As(III). This is 10-fold higher than the maximum As(III) concentrations required for induction of DNA repair genes, but within the dose region where DNA repair genes are co-ordinately down-regulated. These changes in gene regulation are brought about in part by changes in DNA binding activity of the transcription factors activating protein-1 (AP-1), nuclear factor kappa-B, and cAMP response element binding protein (CREB). Although sub-acute exposure to micromolar As(III) up-regulates transcription factor binding, chronic exposure to submicromolar As(III) causes persistent down-regulation of this response. Similar long-term exposure to micromolar concentrations of arsenate in drinking water results in a decrease in skin tumour formation in dimethylbenzanthracene (DMBA)/phorbol 12-tetradecanoate 13-acetate (TPA) treated mice. Altered response patterns after long exposure to As(III) may play a significant role in As(III) toxicology in ways that may not be predicted by experimental protocols using short-term exposures.

Constitutive expression of hZnT4 zinc transporter in human breast epithelial cells

Zinc is an essential trace element required by all living organisms. An adequate supply of zinc is particularly important in the neonatal period. Zinc is a significant component of breast milk, which is transported across the maternal epithelia during lactation. The mechanisms by which zinc becomes a constituent of breast milk have not been elucidated. The function of the zinc transporter ZnT4 in the transport of zinc into milk during lactation was previously demonstrated by studies of a mouse mutant, the ‘lethal milk’ mouse, where a mutation in the ZnT4 gene decreased the transport of zinc into milk. In the present study, we have investigated the expression of the human orthologue of ZnT4 (hZnT4) in the human breast. We detected hZnT4 mRNA expression in the tissue from the resting and lactating human breast, using reverse-transcriptase PCR. Western-blot analysis using antibodies to peptide sequences of hZnT4 detected a major band of the predicted size of 47 kDa and a minor band of 77 kDa, in extracts from the resting and lactating breast tissues. There was no difference in the hZnT4 expression levels between lactating and resting breasts. The hZnT4 protein was present in the luminal cells of the ducts and alveoli where it had a granular distribution. A cultured human breast epithelial cell line PMC42 was used to investigate the subcellular distribution of hZnT4 and this showed a granular label throughout the cytoplasm, consistent with a vesicular localization. The presence of zinc-containing intracellular vesicles was demonstrated by using the zinc-specific fluorphore Zinquin (ethyl-[2-methyl-8-p-toluenesulphonamido-6-quinolyloxy]acetate). Double labelling indicated that there was no obvious overlap between Zinquin and the hZnT4 protein, suggesting that hZnT4 was not directly involved in the transport of zinc into vesicles. We detected expression of two other members of the hZnT family, hZnT1 and hZnT3, in human breast epithelial cells. We conclude that hZnT4 is constitutively expressed in the human breast and may be one of the several members of the ZnT family involved in the transport of zinc into milk.

Epithelial - mesenchymal and mesenchymal - epithelial transitions in carcinoma progression

Like a set of bookends, cellular, molecular, and genetic changes of the beginnings of life mirror those of one of the most common cause of death--metastatic cancer. Epithelial to mesenchymal transition (EMT) is an important change in cell phenotype which allows the escape of epithelial cells from the structural constraints imposed by tissue architecture, and was first recognized by Elizabeth Hay in the early to mid 1980's to be a central process in early embryonic morphogenesis. Reversals of these changes, termed mesenchymal to epithelial transitions (METs), also occur and are important in tissue construction in normal development. Over the last decade, evidence has mounted for EMT as the means through which solid tissue epithelial cancers invade and metastasize. However, demonstrating this potentially rapid and transient process in vivo has proven difficult and data connecting the relevance of this process to tumor progression is still somewhat limited and controversial. Evidence for an important role of MET in the development of clinically overt metastases is starting to accumulate, and model systems have been developed. This review details recent advances in the knowledge of EMT as it occurs in breast development and carcinoma and prostate cancer progression, and highlights the role that MET plays in cancer metastasis. Finally, perspectives from a clinical and translational viewpoint are discussed.

Induction of epithelial to mesenchymal transition in PMC42-LA human breast carcinoma cells by carcinoma-associated fibroblast secreted factors

Background
Breast carcinoma is accompanied by changes in the acellular and cellular components of the microenvironment, the latter typified by a switch from fibroblasts to myofibroblasts.


Methods
We utilised conditioned media cultures, Western blot analysis and immunocytochemistry to investigate the differential effects of normal mammary fibroblasts (NMFs) and mammary cancer-associated fibroblasts (CAFs) on the phenotype and behaviour of PMC42-LA breast cancer cells. NMFs were obtained from a mammary gland at reduction mammoplasty, and CAFs from a mammary carcinoma after resection.


Results
We found greater expression of myofibroblastic markers in CAFs than in NMFs. Medium from both CAFs and NMFs induced novel expression of α-smooth muscle actin and cytokeratin-14 in PMC42-LA organoids. However, although conditioned media from NMFs resulted in distribution of vimentin-positive cells to the periphery of PMC42-LA organoids, this was not seen with CAF-conditioned medium. Upregulation of vimentin was accompanied by a mis-localization of E-cadherin, suggesting a loss of adhesive function. This was confirmed by visualizing the change in active β-catenin, localized to the cell junctions in control cells/cells in NMF-conditioned medium, to inactive β-catenin, localized to nuclei and cytoplasm in cells in CAF-conditioned medium.


Conclusion
We found no significant difference between the influences of NMFs and CAFs on PMC42-LA cell proliferation, viability, or apoptosis; significantly, we demonstrated a role for CAFs, but not for NMFs, in increasing the migratory ability of PMC42-LA cells. By concentrating NMF-conditioned media, we demonstrated the presence of factor(s) that induce epithelial-mesenchymal transition in NMF-conditioned media that are present at higher levels in CAF-conditioned media. Our in vitro results are consistent with observations in vivo showing that alterations in stroma influence the phenotype and behaviour of surrounding cells and provide evidence for a role for CAFs in stimulating cancer progression via an epithelial-mesenchymal transition. These findings have implications for our understanding of the roles of signalling between epithelial and stromal cells in the development and progression of mammary carcinoma.

St. Johns wort attenuates irinotecan-induced diarhea via down-regulation of intestinal pro-inflammatory cytokines and inhibition of intestinal epithelial apoptosis

Diarrhea is a common dose-limiting toxicity associated with cancer chemotherapy, in particular for drugs such as irinotecan (CPT-11), 5-fluouracil, oxaliplatin, capecitabine and raltitrexed. St. John's wort (Hypericum perforatum, SJW) has anti-inflammatory activity, and our preliminary study in the rat and a pilot study in cancer patients found that treatment of SJW alleviated irinotecan-induced diarrhea. In the present study, we investigated whether SJW modulated various pro-inflammatory cytokines including interleukins (IL-1β, IL-2, IL-6), interferon (IFN-γ) and tumor necrosis factor-α (TNF-α) and intestinal epithelium apoptosis in rats. The rats were treated with irinotecan at 60 mg/kg for 4 days in combination with oral SJW or SJW-free control vehicle at 400 mg/kg for 8 days. Diarrhea, tissue damage, body weight loss, various cytokines including IL-1β, IL-2, IL-6, IFN-γ and TNF-α and intestinal epithelial apoptosis were monitored over 11 days. Our studies demonstrated that combined SJW markedly reduced CPT-11-induced diarrhea and intestinal lesions. The production of pro-inflammatory cytokines such as IL-1β, IFN-γ and TNF-α was significantly up-regulated in intestine. In the mean time, combined SJW significantly suppressed the intestinal epithelial apoptosis induced by CPT-11 over days 5–11. In particular, combination of SJW significantly inhibited the expression of TNF-α mRNA in the intestine over days 5–11. In conclusion, inhibition of pro-inflammatory cytokines and intestinal epithelium apoptosis partly explained the protective effect of SJW against the intestinal toxicities induced by irinotecan. Further studies are warranted to explore the potential for STW as an agent in combination with chemotherapeutic drugs to lower their dose-limiting toxicities.

DNA repair deficiencies increase the resistance of Arabidopsis Thaliana to Hyaloperonospora Parasitica

We have found that UV-C treatment of Arabidopsis thaliana induces resistance to the biotrophic pathogen Hyaloperonospora parasitica, and our data suggest UV induced DNA photoproducts are involved (see accompanying abstract by K.G. McKenzie et al.). To address the potential role of DNA damage, we have examined the effect of mutations in nucleotide excision repair (uvr1-1), photoreactivation of cyclobutane pyrimidine dimers (uvr2-1) or flavonoid production (tt5) on the resistance of Arabidopsis to the pathogen with or without pre-inoculation treatment with UV-C. In the mutant backgrounds, UV-C induced pathogen resistance (as measured by decreased conidiophore formation) to the same degree as in the wildtype plants, but much lower UV doses were required (e.g., 100 Jm-2 in the mutant vs. 400 Jm-2 in the wildtype). This is the result expected if damage to DNA rather than a non DNA target is involved. Interestingly, in the absence of UV-C, the tt5 mutation alone resulted in a slight increase in resistance. However, when coupled with uvr1-1, resistance was enhanced to an even greater extent. Remarkably, the tt5 uvr1-1 uvr2-1 triple mutant was completely resistant to the pathogen. Since tt5 mutants are sensitive to reactive oxygen species, which can cause DNA damage susceptible to nucleotide excision repair, our results suggest that in addition to UV photoproducts, an accumulation of endogenous oxidative DNA damage may also trigger resistance to the pathogen. We are currently examining pathogen resistance in other DNA repair deficient mutants, and quantifying UV-C-induced DNA damage in Arabidopsis in order to assess the relationship between damage levels and the extent of resistance.

ATP7B expression in human breast epithelial cells is mediated by lactational hormones

A role for the copper transporter, ATP7B, in secretion of copper from the human breast into milk has previously not been reported, although it is known that the murine ortholog of ATP7B facilitates copper secretion in the mouse mammary gland. We show here that ATP7B is expressed in luminal epithelial cells in both the resting and lactating human breast, where it has a perinuclear localization in resting epithelial cells and a diffuse location in lactating tissue. ATP7B protein was present in a different subset of vesicles from those containing milk proteins and did not overlap with Menkes ATPase, ATP-7A, except in the perinuclear region of cells. In the cultured human mammary line, PMC42-LA, treatment with lactational hormones induced a redistribution of ATP7B from a perinuclear region to a region adjacent, but not coincident with, the apical plasma membrane. Trafficking of ATP7B was copper dependent, suggesting that the hormone-induced redistribution of ATP7A was mediated through an increase in intracellular copper. Radioactive copper (⁶⁴Cu) studies using polarized PMC42-LA cells that overexpressed mAtp7B protein showed that this transporter facilitates copper efflux from the apical surface of the cells. In summary, our results are consistent with an important function of ATP7B in the secretion of copper from the human mammary gland.

Repair and tolerance of DNA damage in higher plants

DNA repair mechanisms constitute an essential cellular response to DNA damage arising either from metabolic processes or from environmental sources such as ultraviolet radiation. Repair of these lesions may be via direct reversal, or by processes such as nucleotide excision repair (NER), a coordinated pathway in which lesions and the surrounding nucleotides are excised and replaced via DNA resynthesis. The importance of repair is illustrated by human disease states such as xeroderma pigmentosum and Cockayne's syndrome which result from defects in the NER system arising from mutations in XP- genes or XP- and CS- genes respectively Little detail is known of DNA damage repair processes in plants, despite the economic and ecological importance of these organisms. This study aimed to expand our knowledge of the process of NER in plants, largely via a polymerase chain reaction (PCR)-based approach involving amplification, cloning and characterisation of plant genomic DNA and cDNA. Homologues of the NER components XPF/RAD1 and XPD/RAD3 were isolated as both genomic and complete cDNA sequences from the model dicotyledonous plant Arabidopsis thaliana. The sequence of the 3'-untranslated region of atXPD was also determined. Comparison of genomic and cDNA sequences allowed a detailed analysis of gene structures, including details of intron/exon processing. Variable transcript processing to produce three distinct transcripts was found in the case of atXPF. In an attempt to validate the proposed homologous function of these cDNAs, assays to test complementation of resistance to ultraviolet radiation in the relevant yeast mutants were performed. Despite extensive amino acid sequence conservation, neither plant cDNA was able to restore UV-resistance. As the yeast RAD3 gene product is also involved in vivo in transcription, and so is required for viability, the atXPD cDNA was tested in a complementation assay for this function in an appropriate yeast mutant. The plant cDNA was found to substantially increase the viability of the yeast mutant. The structural and functional significance of these results is discussed comparatively with reference to yeast, human and other known homologues. Other putative NER homologues were identified in A. thaliana database sequences, including those of ERCC1/RAD10 and XPG/ERCC5/RAD2, and are now the subjects of ongoing investigations. This study also describes preliminary investigations of putative REVS and RAD30 translesion synthesis genes from A. thaliana.

Epithelial-stromal interactions in mammary lineage development and carcinogenesis

This study determined that the microenvironment influences mammary cellular development and that this microenvironment differs between normal and malignant tissue. Cancer-associated fibroblasts, loss of an extracellular protein and presence of a growth factor were demonstrated to influence cancer cell migration, presenting a basis for the understanding of breast cancer metastasis.

Arsenic induced regulation of base excision repair in human cells

Short term exposure to low levels of arsenic in human cells increased the cells' capacity to repair its DNA. In turn, cells became resistant to the toxic effects of UV radiation. However prolonged increases in principal repair proteins may actually lead to cancerous effects by destabilizing DNA repair.