Regulating macrophage phenotype and function by retinal pigment epithelial cells

Purpose: We have shown previously that macrophages/microglia accumulate in the subretinal space and express CD68 and Arginase-1 in the aging eye. Subretinal macrophages are in close contact with retinal pigment epithelial (RPE) cells. We hypothesize that RPE cells may play an important role in regulating macrophage/microglial phenotype and function. The aim of this study was to investigate the effect of RPE cells on the phenotype and function of bone marrow–derived macrophages (BM-DMs).
Methods: BM-DM from C57BL/6J mice were cultured in DMEM supplemented with 20%L929 cell supernatant for 5 days. The phenotype of BM-DMs was confirmed by flow cytometry as CD11b+F4/80+. Primary RPE cells were cultured from C57BL/6J mice and confirmed by RPE65 and cytokeratin staining. BMDMs were co-cultured with different types of RPE cells (healthy, oxidized, and apoptotic RPE) and then isolated from the co-culture system for phenotypic and functional assays.
Results: Co-culture of BM-DMs with RPE cells results in a time-dependent down-regulation of MHC-II expression and the generation of CD11b+F4/80+Ly6G+ myeloid-derived suppressor cells (MDSC). qRT-PCR analysis showed that RPE-induced MDSCs expressed high levels of IL-6, IL-1β, and Arginase-1, but lower levels of IL-12p40 and TNF-a compared to naïve BM-DMs. The expression levels of iNOS, TGF-β and Ym1 did not differ 207 between naive BMDMs and RPE-induced MDSCs. Furthermore, functional studies showed that these cells had reduced phagocytic activity and lower ability to stimulate T cell activation and proliferation. When RPE cells were pre-treated with oxidized photoreceptor outer segments before co-culturing with BMDMs, the expression of IL-1β and IL-6 in BMDMs was increased whereas the expression of Arginase-1 was decreased. 
Conclusion: Our results suggest that healthy RPE cells can convert BMDMs into myeloid-derived suppressor cells under in vitro culture conditions, RPE-induced myeloid-derived suppressor cells are CD11b+F4/80+Ly6G+MHCIIlowIL6+IL1b+Arg-1+. The ability of RPE cells is reduced when suffering from oxidative insults.

Downregulation of the Wnt inhibitor CXXC5 predicts a better prognosis in acute myeloid leukemia

The gene CXXC5 on 5q31 is frequently deleted in acute myeloid leukemia (AML) with del(5q), suggesting that inactivation of CXXC5 might play a role in leukemogenesis. Here, we investigated the functional and prognostic implications of CXXC5 expression in AML. CXXC5 mRNA was downregulated in AML with MLL rearrangements, t(8;21) and GATA2 mutations. As a mechanism of CXXC5 inactivation, we found evidence for epigenetic silencing by promoter methylation. Patients with CXXC5 expression below the median level had a lower relapse rate (45% vs 59%; P = .007) and a better overall survival (OS, 46% vs 28%; P < .001) and event-free survival (EFS, 36% vs 21%; P < .001) at 5 years, independent of cytogenetic risk groups and known molecular risk factors. In gene-expression profiling, lower CXXC5 expression was associated with upregulation of cell-cycling genes and codownregulation of genes implicated in leukemogenesis (WT1, GATA2, MLL, DNMT3B, RUNX1). Functional analyses demonstrated CXXC5 to inhibit leukemic cell proliferation and Wnt signaling and to affect the p53-dependent DNA damage response. In conclusion, our data suggest a tumor suppressor function of CXXC5 in AML. Inactivation of CXXC5 is associated with different leukemic pathways and defines an AML subgroup with better outcome.

BRCA1, a 'complex' protein involved in the maintenance of genomic stability

BRCA1 is a major breast and ovarian cancer susceptibility gene, with mutations in this gene predisposing women to a very high risk of developing breast and ovarian tumours. BRCA1 primarily functions to maintain genomic stability via critical roles in DNA repair, cell cycle checkpoint control, transcriptional regulation, apoptosis and mRNA splicing. As a result, BRCA1 mutations often result in defective DNA repair, genomic instability and sensitivity to DNA damaging agents. BRCA1 carries out these different functions through its ability to interact, and form complexes with, a vast array of proteins involved in multiple cellular processes, all of which are considered to contribute to its function as a tumour suppressor. This review discusses and highlights recent research into the functions of BRCA1-related protein complexes and their roles in maintaining genomic stability and tumour suppression.

Role played by BRCA1 in transcriptional regulation in response to therapy

BRCA1 (breast-cancer susceptibility gene 1) is a tumour suppressor, implicated in the hereditary predisposition to breast and ovarian cancer. BRCA1 has been implicated in a number of cellular processes including DNA repair and recombination, cell cycle checkpoint control, chromatin remodelling and ubiquitination. In addition, substantial data now exist to suggest a role for BRCA1 in transcriptional regulation; BRCA1 has been shown to interact with the Pol II holoenzyme complex and to interact with multiple transcription factors, such as p53 and c-Myc. We have previously identified a range of BRCA1 transcriptional targets and have linked these to specific cellular pathways, including cell cycle checkpoint activation and apoptosis. Current research is focused on the transcriptional mechanisms that underpin the association of BRCA1 deficiency with increased sensitivity to DNA damage-based chemotherapy and resistance to spindle poisons.

Uncovering BRCA1-regulated signalling pathways by microarray-based expression profiling

The introduction of microarray technology to the scientific and medical communities has dramatically changed the way in which we now address basic biomedical questions. Expression profiling using microarrays facilitates an experimental approach where alterations in the transcript level of entire transcriptomes can be simultaneously assayed in response to defined stimuli. We have used microarray analysis to identify downstream transcriptional targets of the BRCA1 (Breast Cancer 1) tumour-suppressor gene as a means of defining its function. BRCA1 has been implicated in the predisposition to early onset breast and ovarian cancer and while its exact function remains to be defined, roles in DNA repair, cell-cycle control and transcriptional regulation have been implied. In the current study we have generated cell lines with tetracycline-regulated, inducible expression of BRCA1 as a tool to identify genes, which might represent important effectors of BRCA1 function. Oligonucleotide array-based expression profiling identified a number of genes that were upregulated at various times following inducible expression of BRCA1 including the DNA damage-responsive gene GADD45 (Growth Arrest after DNA Damage). Identified targets were confirmed by Northern blot analysis and their functional significance as BRCA1 targets examined.

Uncovering functionally relevant signaling pathways using microarray-based expression profiling

The introduction of microarray technology to the scientific and medical communities has fundamentally altered the way in which we now address basic biomedical questions. Microarrays technology facilitates a more complete and inclusive experimental approach where alterations in the transcript level of entire genomes can be simultaneously assayed in response to a variety of stimuli. Conceptually different approaches to the development of microarray technology have resulted in the generation of two different array formats: oligonucleotide arrays and cDNA arrays. The application of microarray and related technologies to identify specific targets of defined genes that have clearly been implicated in cancer progression requires a specific experimental approach. The objective of tiffs approach is to define changes in transcriptional profile that occur in response to modulating the expression level of the gene to be studied. The resulting altered expression profile can then be viewed as a blueprint by which that gene effects its cellular function. We have used oligonucleotide array-based expression profiling in collaboration with Affymetrix to identify downstream transcriptional targets of the BRCA1 tumor-suppressor gene as a means of defining its function. BRCA1 has been implicated in at least three functional pathways, namely, mediating the cellular response to DNA damage, as a cell cycle checkpoint protein and in the regulation of transcription. The physiological significance of these properties and their implications for the function of BRCA1 as a tumor-suppressor gene remain to be defined.

Measuring a cell's response to stress:the p53 pathway.

Heterozygous ATM mutations do not contribute to early onset of breast cancer

Ataxia telangiectasia (AT) is a recessive syndrome, including cerebellar degeneration, immunologic defects and cancer predisposition, attributed to mutations in the recently isolated ATM (ataxia telangiectasia, mutated) gene. AT is diagnosed in 1/40,000 to 1/100,000 live births, with carriers calculated to comprise approximately 1% of the population. Studies of AT families have suggested that female relatives presumed to be carriers have a 5 to 8-fold increased risk for developing breast cancer, raising the possibility that germline ATM mutations may account for approximately 5% of all breast cancer cases. The increased risk for breast cancer reported for AT family members has been most evident among younger women, leading to an age-specific relative risk model predicting that 8% of breast cancer in women under age 40 arises in AT carriers, compared with 2% of cases between 40-59 years. To test this hypothesis, we undertook a germ-line mutational analysis of the ATM gene in a population of women with early onset of breast cancer, using a protein truncation (PTT) assay to detect chain-terminating mutations, which account for 90% of mutations identified in children with AT. We detected a heterozygous ATM mutation in 2/202 (1%) controls, consistent with the frequency of AT carriers predicted from epidemiologic studies. ATM mutations were present in only 2/401 (0.5%) women with early onset of breast cancer (P = 0.6). We conclude that heterozygous ATM mutations do not confer genetic predisposition to early onset of breast cancer.

Prevalence of germ-line mutations in p16, p19ARF, and CDK4 in familial melanoma:analysis of a clinic-based population

Five to ten percent of individuals with melanoma have another affected family member, suggesting familial predisposition. Germ-line mutations in the cyclin-dependent kinase (CDK) inhibitor p16 have been reported in a subset of melanoma pedigrees, but their prevalence is unknown in more common cases of familial melanoma that do not involve large families with multiple affected members. We screened for germ-line mutations in p16 and in two other candidate melanoma genes, p19ARF and CDK4, in 33 consecutive patients treated for melanoma; these patients had at least one affected first or second degree relative (28 independent families). Five independent, definitive p16 mutations were detected (18%, 95% confidence interval: 6%, 37%), including one nonsense, one disease-associated missense, and three small deletions. No mutations were detected in CDK4. Disease-associated mutations in p19ARF, whose transcript is derived in part from an alternative codon reading frame of p16, were only detected in patients who also had mutations inactivating p16. We conclude that germ-line p16 mutations are present in a significant fraction of individuals who have melanoma and a positive family history.

The epigenome as a therapeutic target in prostate cancer

During cancer development and progression, tumor cells undergo abnormal epigenetic modifications, including DNA methylation, histone deacetylation and nucleosome remodeling. Collectively, these aberrations promote genomic instability and lead to silencing of tumor-suppressor genes and reactivation of oncogenic retroviruses. Epigenetic modifications, therefore, provide exciting new avenues for prostate cancer research. Promoter hypermethylation is widespread during neoplastic transformation of prostate cells, which suggests that restoration of a 'normal' epigenome through treatment with inhibitors of the enzymes involved could be clinically beneficial. Global patterns of histone modifications are also being defined and have been associated with clinical and pathologic predictors of prostate cancer outcome. Although treatment for localized prostate cancer can be curative, the development of successful therapies for the management of castration-resistant metastatic disease is urgently needed. Reactivation of tumor-suppressor genes by demethylating agents and histone deacetylase inhibitors could be a potential treatment option for patients with advanced disease.

Transcriptional response of T cells to IFN-alpha:changes induced in IFN-alpha-sensitive and resistant cutaneous T cell lymphoma

Interferon-alpha (IFN-alpha) therapy is commonly used in the treatment of neoplastic and autoimmune diseases, including cutaneous T cell lymphoma (CTCL). However, the IFN-alpha response is unpredictable, and the IFN-alpha cell targets and pathways are only partially understood. To delineate the molecular mechanisms of IFN-alpha activity, gene expression profiling was performed in a time-course experiment of both IFN-alpha sensitive and IFN-alpha-resistant variants of a CTCL cell line. These experiments revealed that IFN-alpha is responsible for the regulation of hundreds of genes in both variants and predominantly involves genes implicated in signal transduction, cell cycle control, apoptosis, and transcription regulation. Specifically, the IFN-alpha response of tumoral T cells is due to a combination of induction of apoptosis in which TNFSF10 and HSXIAPAF1 may play an important role and cell cycle arrest achieved by downregulation of CDK4 and CCNG2 and upregulation of CDKN2C and tumor suppressor genes (TSGs). Resistance to IFN-alpha appears to be associated with failure to induce IRF1 and IRF7 and deregulation of the apoptotic signals of HSXIAPAF1, TRADD, BAD, and BNIP3. Additionally, cell cycle progression is heralded by upregulation of CDC25A and CDC42. A critical role of NF-kappaB in promoting cell survival in IFN-alpha-resistant cells is indicated by the upregulation of RELB and LTB.

Promoter methylation correlates with reduced Smad4 expression in advanced prostate cancer

BACKGROUND: Transforming growth factor-beta (TGF-beta) is a potent growth inhibitor in a wide range of cell types. A transducer of TGF-beta signaling known as Mothers against decapentaplegic homologue 4 (Smad4) is a known tumor suppressor found on chromosome 18q21.1 and is typically inactivated by deletion or mutation in pancreatic and colorectal cancers. The purpose of the article is to investigate Smad4 expression, gene copy number and methylation status in advanced cases of prostate cancer.

METHODS: We have employed Methylation Specific PCR (MSP) to identify methylation sites within the Smad4 promoter and combined this with quantitative real-time PCR to look for correlates between methylation status and Smad4 expression and to examine androgen receptor (AR) expression. Bacterial artificial chromosome-comparative genomic hybridization (BAC-CGH) has been used to look for genomic amplifications and deletions which may also contribute to expression changes.

RESULTS: We fail to find evidence of genomic deletions or amplifications affecting the Smad4 locus on chromosome 18 but show a correlation between promoter methylation and the loss of Smad4 expression in the same material. We confirm that the AR locus on the X chromosome is amplified in 30% of the advanced clinical samples and that this correlates with increased transcript levels as previously reported by other groups.

CONCLUSION: This indicates that epigenetic changes affect the expression of the Smad4 protein in prostate cancer and points to methylation of the promoter as a novel marker of and contributor to the disease warranting further study.

Regulation of human dUTPase gene expression and p53-mediated transcriptional repression in response to oxaliplatin-induced DNA damage

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and PPi. Although dUTP is a normal intermediate in DNA synthesis, its accumulation and misincorporation into DNA is lethal. Importantly, uracil misincorporation is a mechanism of cytotoxicity induced by fluoropyrimidine chemotherapeutic agents including 5-fluorouracil (5-FU) and elevated expression of dUTPase is negatively correlated with clinical response to 5-FU-therapy. In this study we performed the first functional characterization of the dUTPase promoter and demonstrate a role for E2F-1 and Sp1 in driving dUTPase expression. We establish a direct role for both mutant and wild-type forms of p53 in modulating dUTPase promoter activity. Treatment of HCT116 p53(+/+) cells with the DNA-damaging agent oxaliplatin induced a p53-dependent transcriptional downregulation of dUTPase not observed in the isogenic null cell line. Oxaliplatin treatment induced enrichment of p53 at the dUTPase promoter with a concomitant reduction in Sp1. The suppression of dUTPase by oxaliplatin promoted increased levels of dUTP that was enhanced by subsequent addition of fluoropyrimidines. The novel observation that oxaliplatin downregulates dUTPase expression may provide a mechanistic basis contributing to the synergy observed between 5-FU and oxaliplatin in the clinic. Furthermore, these studies provide the first evidence of a direct transcriptional link between the essential enzyme dUTPase and the tumor suppressor p53.

STAT3 regulated ARF expression suppresses prostate cancer metastasis

Prostate cancer (PCa) is the most prevalent cancer in men. Hyperactive STAT3 is thought to be oncogenic in PCa. However, targeting of the IL-6/STAT3 axis in PCa patients has failed to provide therapeutic benefit. Here we show that genetic inactivation of Stat3 or IL-6 signalling in a Pten-deficient PCa mouse model accelerates cancer progression leading to metastasis. Mechanistically, we identify p19(ARF) as a direct Stat3 target. Loss of Stat3 signalling disrupts the ARF-Mdm2-p53 tumour suppressor axis bypassing senescence. Strikingly, we also identify STAT3 and CDKN2A mutations in primary human PCa. STAT3 and CDKN2A deletions co-occurred with high frequency in PCa metastases. In accordance, loss of STAT3 and p14(ARF) expression in patient tumours correlates with increased risk of disease recurrence and metastatic PCa. Thus, STAT3 and ARF may be prognostic markers to stratify high from low risk PCa patients. Our findings challenge the current discussion on therapeutic benefit or risk of IL-6/STAT3 inhibition.

Immunohistochemistry should undergo robust validation equivalent to that of molecular diagnostics

Immunohistochemistry (IHC) is a widely available and highly utilised tool in diagnostic histopathology and is used to guide treatment options as well as provide prognostic information. IHC is subjected to qualitative and subjective assessment, which has been criticised for a lack of stringency, while PCR-based molecular diagnostic validations by comparison are regarded as very rigorous. It is essential that IHC tests are validated through evidence-based procedures. With the move to ISO15189 (2012), not just of the accuracy, specificity and reproducibility of each test need to be determined and managed, but also the degree of uncertainty and the delivery of such tests. The recent update to ISO 15189 (2012) states that it is appropriate to consider the potential uncertainty of measurement of the value obtained in the laboratory and how that may impact on prognostic or predictive thresholds. In order to highlight the problems surrounding IHC validity, we reviewed the measurement of Ki67and p53 in the literature. Both of these biomarkers have been incorporated into clinical care by pathology laboratories worldwide. The variation seen appears excessive even when measuring centrally stained slides from the same cases. We therefore propose in this paper to establish the basis on which IHC laboratories can bring the same level of robust validation seen in the molecular pathology laboratories and the principles applied to all routine IHC tests.