Genistein and coumestrol reduce MCF-7 breast cancer cell viability and inhibit markers of preferential metastasis, bone matrix attachment and tumor-induced osteoclastogenesis.

The propensity of breast cancer to preferentially metastasize to the skeleton is well known. Once established in bone metastatic breast cancers have a poor prognosis due to their ability to promote extensive bone loss which augments tumor burden. Unfortunately, current anti-resorptive therapies for skeletal metastasis are typically prescribed after secondary tumors have formed and are palliative in nature. One group of compounds with the potential to reduce both tumor burden and osteolysis are phytoestrogens (PE), but the mechanisms mediating a beneficial effect are unclear. Therefore, the current study examined the effect of genistein and coumestrol alone or in combination on breast cancer cell number, expression of mediators of preferential skeletal metastasis, bone matrix attachment and tumor-induced osteoclast formation. Results showed that genistein and coumestrol significantly reduced viable cell number in an estrogen receptor dependent manner (p < 0.05), whereas combinations of PE had no effect. In addition, genistein and coumestrol significantly reduced expression of genes driving epithelial to mesenchymal transition (snail), bone attachment (CXCR4 and integrin αV) and osteolysis (PTHrP and TNF-α). In keeping with this genistein and coumestrol significantly suppressed attachment of breast cancer cells to bone matrix and inhibited tumor and RANKL-induced osteoclast formation. Our data suggests that phytoestrogens not only decrease breast cancer cell viability but also antagonize essential tumor bone interactions that establish and drive the progression of skeletal metastasis.

Dynamic real-time optical microscopy of oral mucosal lesions using confocal laser endomicroscopy.

OBJECTIVES: Confocal laser endomicroscopy (CLE) is a novel non-invasive point-of-care optical biopsy technology that enables real-time in vivo microscopic visualisation of cellular and tissue architecture. In this study, we assessed the diagnostic accuracy of a hand-held fluorescence single-fibre distal-scanning CLE (fsdCLE) platform for diagnosing oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: Forty-seven patients presenting with 63 distinct oral mucosal lesions were subjected to optical biopsy using a miniaturised fsdCLE system (ViewnVivo®, Optiscan Imaging Ltd) and topical exogenous acriflavine hydrochloride contrast agent before undergoing tissue biopsy and histopathological consensus review by four pathologists. CLE images were captured in vivo in real-time during clinical examination and assessed on-the-fly for the presence of cellular and architectural features of OED/OSCC offering an instantaneous diagnosis. Predicted optical diagnoses were compared to definitive consensus tissue histopathology. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy were calculated for the presence/absence of dysplasia/malignancy on optical biopsy. Percentage agreement, Fleiss' kappa, and intraclass correlation coefficient (ICC) were calculated for each assessment stage during the consensus histopathology process. RESULTS: Diagnostic accuracy was extremely high at 88.9%. Other metrics were sensitivity 86.8%, specificity 92%, PPV 94.3% and NPV 82.1%. One hundred percent of carcinoma cases were detected accurately using CLE in the clinic. CONCLUSION: fsdCLE is a highly accurate, easy-to-use, rapid and slide-free point-of-care in vivo optical technology for diagnosing OED/OSCC and discriminating between dysplastic and non-dysplastic pathology. It demonstrates near-perfect agreement with traditional consensus histopathology without the need for physical tissue biopsy.

A Novel Preclinical In Vitro 3D Model of Oral Carcinogenesis for Biomarker Discovery and Drug Testing.

This study aimed to develop an in vitro three-dimensional (3D) cell culture model of oral carcinogenesis for the rapid, scalable testing of chemotherapeutic agents. Spheroids of normal (HOK) and dysplastic (DOK) human oral keratinocytes were cultured and treated with 4-nitroquinoline-1-oxide (4NQO). A 3D invasion assay using Matrigel was performed to validate the model. RNA was extracted and subjected to transcriptomic analysis to validate the model and assess carcinogen-induced changes. The VEGF inhibitors pazopanib and lenvatinib were tested in the model and were validated by a 3D invasion assay, which demonstrated that changes induced by the carcinogen in spheroids were consistent with a malignant phenotype. Further validation was obtained by bioinformatic analyses, which showed the enrichment of pathways associated with hallmarks of cancer and VEGF signalling. Overexpression of common genes associated with tobacco-induced oral squamous cell carcinoma (OSCC), such as MMP1, MMP3, MMP9, YAP1, CYP1A1, and CYP1B1, was also observed. Pazopanib and lenvatinib inhibited the invasion of transformed spheroids. In summary, we successfully established a 3D spheroid model of oral carcinogenesis for biomarker discovery and drug testing. This model is a validated preclinical model for OSCC development and would be suitable for testing a range of chemotherapeutic agents.

Dysplastic oral leukoplakia is molecularly distinct from leukoplakia without dysplasia.

OBJECTIVE: The molecular mechanisms underlying the development of dysplasia in leukoplakia are unknown. We used RNA sequencing to examine the molecular and biological pathway differences in oral leukoplakia with and without oral epithelial dysplasia. MATERIALS AND METHODS: Excisional biopsy specimens (25) were taken from 24 patients with oral leukoplakia diagnosed histopathologically as either oral epithelial dysplasia (13) or epithelial hyperplasia and keratosis without dysplasia (12). Transcriptome analysis used RNA sequencing, differential expression and hierarchical clustering. Biological signalling was examined by gene ontology, pathway and protein-protein interaction analysis. RESULTS: Differential expression analysis showed distinction between the two groups identifying 47 genes as altered in leukoplakia with dysplasia, including SAA1, SAA2, KRT31, KRT37, KRT76, ROBO2, DNAJB5 and DNAJA4. Using hierarchical clustering, dysplastic leukoplakia readily segregated from leukoplakia without dysplasia. Pathway and ontology enrichment analysis provided evidence that downregulation of extracellular matrix (ECM) pathways was a feature of dysplastic lesions. CONCLUSION: Our results suggest that there are detectable changes in the molecular profile of oral leukoplakia exhibiting dysplasia including downregulated ECM as a distinguishing feature of dysplastic lesions. This suggests that reactive changes in stroma may be an early manifestation of dysplastic development. Our study also demonstrates the feasibility of detecting such molecular changes in oral leukoplakia, providing avenues for further investigation of molecular mechanisms of oral dysplasia.

Delivery of expression constructs of secreted frizzled-related protein 4 and its domains by chitosan-dextran sulfate nanoparticles enhances their expression and anti-cancer effects.

In malignant mesothelioma (MM) cells, secreted frizzled-related protein 4 (SFRP4) expression is downregulated by promoter methylation. In this study, we evaluated the effect of encapsulated chitosan-dextran (CS-DS) nanoparticle formulations of SFRP4 and its cysteine-rich domain (CRD) and netrin-like domain (NLD) as means of SFRP4-GFP protein delivery and their effects in JU77 and ONE58 MM cell lines. CS-DS formulations of SFRP4, CRD, and NLD nanoparticles were prepared by a complex coacervation technique, and particle size ranged from 300 nm for empty particles to 337 nm for particles containing the proteins. Measurement of the zeta potential showed that all preparations were around 25 mV or above, suggesting stable formulation and good affinity for the DNA molecules. The CS-DS nanoparticle formulation maintained high integrity and entrapment efficiency. Gene delivery of SFRP4 and its domains showed enhanced biological effects in both JU77 and ONE58 cell lines when compared to the non-liposomal FUGENE® HD transfection reagent. In comparison to the CRD nanoparticles, both the SFRP4 and NLD nanoparticles significantly reduced the viability of MM cells, with the NLD showing the greatest effect. The CS-DS nanoparticle effects were observed at an earlier time point and with lower DNA concentrations. Morphological changes in MM cells were characterized by the formation of membrane-associated vesicles and green fluorescent protein expression specific to SFRP4 and the NLD. The findings from our proof-of-concept study provide a stepping stone for further investigations using in vivo models.

Transcriptome changes induced in vitro by alcohol-containing mouthwashes in normal and dysplastic oral keratinocytes.

BACKGROUND: The role of alcohol-containing mouthwash as a risk factor for the development of oral cancer is a subject of conflicting epidemiological evidence in the literature despite alcohol being a recognised carcinogen. The aim of this study was to use in vitro models to investigate mechanistic and global gene expression effects of exposure to alcohol-containing mouthwash. METHODS: Two brands of alcohol-containing mouthwash and their alcohol-free counterparts were used to treat two oral cell lines derived from normal (OKF6-TERT) and dysplastic (DOK) tissues. Genotoxicity was determined by Comet assay. RNA-seq was performed using the Ion Torrent platform. Bioinformatics analysis used R/Bioconductor packages with differential expression using DEseq2. Pathway enrichment analysis used EnrichR with the WikiPathways and Kegg databases. RESULTS: Both cell lines displayed dose-dependent DNA damage in response to acute exposure to ethanol and alcohol-containing mouthwashes as well as alcohol-free mouthwashes reconstituted with ethanol as shown by Comet assay. The transcriptomic effects of alcohol-containing mouthwash exposure were more complex with significant differential gene expression ranging from >2000 genes in dysplastic (DOK) cells to <100 genes in normal (OKF6-TERT) cells. Pathway enrichment analysis in DOK cells revealed alcohol-containing mouthwashes showed common features between the two brands used including DNA damage response as well as cancer-associated pathways. In OKF6-TERT cells, the most significantly enriched pathways involved inflammatory signalling. CONCLUSIONS: Alcohol-containing mouthwashes are genotoxic in vitro to normal and dysplastic oral keratinocytes and induce widespread changes in gene expression. Dysplastic cells are more susceptible to the transcriptomic effects of mouthwash.

Structure of a Potential Therapeutic Antibody Bound to Interleukin-16 (IL-16): MECHANISTIC INSIGHTS AND NEW THERAPEUTIC OPPORTUNITIES.

Interleukin-16 (IL-16) is reported to be a chemoattractant cytokine and modulator of T-cell activation, and has been proposed as a ligand for the co-receptor CD4. The secreted active form of IL-16 has been detected at sites of TH1-mediated inflammation, such as those seen in autoimmune diseases, ischemic reperfusion injury (IRI), and tissue transplant rejection. Neutralization of IL-16 recruitment to its receptor, using an anti-IL16 antibody, has been shown to significantly attenuate inflammation and disease pathology in IRI, as well as in some autoimmune diseases. The 14.1 antibody is a monoclonal anti-IL-16 antibody, which when incubated with CD4(+) cells is reported to cause a reduction in the TH1-type inflammatory response. Secreted IL-16 contains a characteristic PDZ domain. PDZ domains are typically characterized by a defined globular structure, along with a peptide-binding site located in a groove between the αB and ßB structural elements and a highly conserved carboxylate-binding loop. In contrast to other reported PDZ domains, the solution structure previously reported for IL-16 reveals a tryptophan residue obscuring the recognition groove. We have solved the structure of the 14.1Fab fragment in complex with IL-16, revealing that binding of the antibody requires a conformational change in the IL-16 PDZ domain. This involves the rotation of the αB-helix, accompanied movement of the peptide groove obscuring tryptophan residue, and consequent opening up of the binding site for interaction. Our study reveals a surprising mechanism of action for the antibody and identifies new opportunities for the development of IL-16-targeted therapeutics, including small molecules that mimic the interaction of the antibody.

Therapeutic approach to target mesothelioma cancer cells using the Wnt antagonist, secreted frizzled-related protein 4: Metabolic state of cancer cells.

Malignant mesothelioma (MM) is an aggressive cancer, characterized by rapid progression, along with late metastasis and poor patient prognosis. It is resistant to many forms of standard anti-cancer treatment. In this study, we determined the effect of secreted frizzled-related protein 4 (sFRP4), a Wnt pathway inhibitor, on cancer cell proliferation and metabolism using the JU77 mesothelioma cell line. Treatment with sFRP4 (250 pg/ml) resulted in a significant reduction of cell proliferation. The addition of the Wnt activator Wnt3a (250 pg/ml) or sFRP4 had no significant effect on ATP production and glucose utilisation in JU77 cells at both the 24 and 48 h time points examined. We also examined their effect on Akt and Glycogen synthase kinase-3 beta (GSK3ß) phosphorylation, which are both important components of Wnt signalling and glucose metabolism. We found that protein phosphorylation of Akt and GSK3ß varied over the 24h and 48 h time points, with constitutive phosphorylation of Akt at serine 473 (pAkt) decreasing to its most significant level when treated with Wnt3a+sFRP4 at the 24h time point. A significant reduction in the level of Cytochrome c oxidase was observed at the 48 h time point, when sFRP4 and Wnt3a were added in combination. We conclude that sFRP4 may function, in part, to reduce/alter cancer cell metabolism, which may lead to sensitisation of cancer cells to chemotherapeutics, or even cell death.

Secreted frizzled related proteins: Implications in cancers.

The Wnt (wingless-type) signaling pathway plays an important role in embryonic development, tissue homeostasis, and tumor progression becaluse of its effect on cell proliferation, migration, and differentiation. Secreted frizzled-related proteins (SFRPs) are extracellular inhibitors of Wnt signaling that act by binding directly to Wnt ligands or to Frizzled receptors. In recent years, aberrant expression of SFRPs has been reported to be associated with numerous cancers. As gene expression of SFRP members is often lost through promoter hypermethylation, inhibition of methylation through the use of epigenetic modifying agents could renew the expression of SFRP members and further antagonize deleterious Wnt signaling. Several reports have described epigenetic silencing of these Wnt signaling antagonists in various human cancers, suggesting their possible role as tumor suppressors. SFRP family members thus come across as potential tools in combating Wnt-driven tumorigenesis. However, little is known about SFRP family members and their role in different cancers. This review comprehensively covers all the available information on the role of SFRP molecules in various human cancers.

Catechols and 3-hydroxypyridones as inhibitors of the DNA repair complex ERCC1-XPF.

Catechol-based inhibitors of ERCC1-XPF endonuclease activity were identified from a high-throughput screen. Exploration of the structure-activity relationships within this series yielded compound 13, which displayed an ERCC1-XPF IC50 of 0.6 µM, high selectivity against FEN-1 and DNase I and activity in nucleotide excision repair, cisplatin enhancement and γH2AX assays in A375 melanoma cells. Screening of fragments as potential alternatives to the catechol group revealed that 3-hydroxypyridones are able to inhibit ERCC1-XPF with high ligand efficiency, and elaboration of the hit gave compounds 36 and 37 which showed promising ERCC1-XPF IC50 values of <10 µM.

N-Hydroxyimides and hydroxypyrimidinones as inhibitors of the DNA repair complex ERCC1-XPF.

A high throughput screen allowed the identification of N-hydroxyimide inhibitors of ERCC1-XPF endonuclease activity with micromolar potency, but they showed undesirable selectivity profiles against FEN-1. A scaffold hop to a hydroxypyrimidinone template gave compounds with similar potency but allowed selectivity to be switched in favour of ERCC1-XPF over FEN-1. Further exploration of the structure-activity relationships around this chemotype gave sub-micromolar inhibitors with >10-fold selectivity for ERCC1-XPF over FEN-1.

Osteoclastic resorptive capacity is suppressed in patients receiving hyperbaric oxygen therapy.

BACKGROUND AND PURPOSE: Hypoxia, necrosis, and bone loss are hallmarks of many skeletal diseases. Hyperbaric oxygen therapy (HBO) is often used as an adjunctive therapy in these cases. However the in vivo effect of HBO on osteoclast formation has not been fully established. We therefore carried out a longitudinal study to examine the effect of HBO on osteoclast formation and bone resorptive capacity in patients who were referred to the Plymouth Hyperbaric Medical Centre. METHODS: Osteoclast precursors were isolated from peripheral blood prior to and following 10 and 25 daily hyperbaric treatments (100% O2 at 2.4 atmospheres absolute ATA for 90 min) to determine osteoclast formation and resorptive capacity. The expression of key regulators of osteoclast differentiation RANK, Dc-STAMP, and NFATc1 was also assessed by quantitative real-time PCR. RESULTS: HBO reduced the ability of precursors to form osteoclasts and reduced bone resorption in a treatment-dependent manner. The initial suppressive effect of HBO was more pronounced on mononuclear osteoclast formation than on multinuclear osteoclast formation, and this was accompanied by reduction in the expression of key regulators of osteoclast formation, RANK and Dc-STAMP. INTERPRETATION: This study shows for the first time that in vivo, HBO suppresses the ability of monocytic precursors to form resorptive osteoclasts.

Genome-wide characterization of the mutational landscape of proliferative verrucous leukoplakia.

OBJECTIVES: Proliferative verrucous leukoplakia (PVL) is a rare but highly aggressive variant of oral leukoplakia that almost inevitably progresses to oral squamous cell carcinoma (OSCC). The aims of this study were to perform whole exome sequencing of a cohort of patients diagnosed with PVL and identify potential mutational profiles and pathways in this disorder. STUDY DESIGN: A total of 12 oral cavity mucosal biopsies from 6 patients with oral lesions clinically compatible with PVL were used. Of these, 9 were diagnosed as dysplasia, 1 OSCC, and 2 hyperkeratosis/hyperplasia. Exome sequencing used the Ion AmpliSeq Exome platform. Ion Reporter software was used for variant calling, annotation, and filtering. Analysis and visualization of somatic mutations was carried out using the MAFtools R package. RESULTS: Following exome sequencing and mutational profiling, we analyzed the profiles for cancer associated genes and signatures. Genes previously associated with OSCC, including HYDIN, MUC16, MAML3, CDKN2A, FAT1, and CASP8, were mutated in multiple samples. Several DNA damage repair genes including PARP1 were mutated in PVL samples. NOTCH and Hippo pathways were the most frequently impacted by mutation. CONCLUSIONS: This genome wide characterization of premalignant PVL identifies both known and potentially novel oncogenic mechanisms in this disorder.

Global gene expression profile of proliferative verrucous leukoplakia and its underlying biological disease mechanisms.

BACKGROUND: Proliferative verrucous leukoplakia (PVL) is a rare and enigmatic oral potentially malignant disorder which almost invariably results in oral squamous cell carcinoma (OSCC). The aims of this project were to use transcriptome profiling to characterise PVL gene expression patterns for biomarker identification and gain insight into the molecular aetiopathogenesis of PVL. METHODS: Forty-three oral cavity mucosal biopsies from 32 patients with oral lesions clinically compatible with either PVL or non-PVL conventional oral leukoplakia (OLK) underwent transcriptome profiling by RNA sequencing. Data was analysed by hierarchical clustering, differential gene expression, functional enrichment and network analysis, sparse partial least squares discriminant analysis sPLS-DA, and immune cell phenotypic estimation. RESULTS: We found 464 genes significantly differentially expressed at least 2-fold between PVL and non-PVL OLK (193 up and 271 down). HOX genes, including HOXA1 and HOXB7, keratin-associated proteins (KRTAPs) and olfactory receptor G proteins (OR) were significantly upregulated in PVL. Other upregulated genes in PVL included FOS, WNT16 and IFNA1. Pathway analysis showed that there was a significant downregulation of connective tissue signalling in PVL. Classifying multivariate models based upon 22 genes discriminated PVL from non-PVL OLK. Bioinformatic profiling showed that immune cell profiles in PVL and OLK were similar except that fibroblast markers were reduced in PVL. CONCLUSION: These results demonstrate that PVL and conventional OLK are molecularly distinct with upregulation of many cancer-associated genes. They provide insight into the pathogenesis of PVL and show that biomarker based molecular diagnostics is feasible to discriminate and inform diagnosis and management.

Spatial transcriptomics reveals discrete tumour microenvironments and autocrine loops within ovarian cancer subclones.

High-grade serous ovarian carcinoma (HGSOC) is genetically unstable and characterised by the presence of subclones with distinct genotypes. Intratumoural heterogeneity is linked to recurrence, chemotherapy resistance, and poor prognosis. Here, we use spatial transcriptomics to identify HGSOC subclones and study their association with infiltrating cell populations. Visium spatial transcriptomics reveals multiple tumour subclones with different copy number alterations present within individual tumour sections. These subclones differentially express various ligands and receptors and are predicted to differentially associate with different stromal and immune cell populations. In one sample, CosMx single molecule imaging reveals subclones differentially associating with immune cell populations, fibroblasts, and endothelial cells. Cell-to-cell communication analysis identifies subclone-specific signalling to stromal and immune cells and multiple subclone-specific autocrine loops. Our study highlights the high degree of subclonal heterogeneity in HGSOC and suggests that subclone-specific ligand and receptor expression patterns likely modulate how HGSOC cells interact with their local microenvironment.

Suppression of T cell-induced osteoclast formation.

Inhibition of T cell derived cytokine production could help suppress osteoclast differentiation in inflammatory skeletal disorders. Bisphosphonates are typically prescribed to prevent inflammatory bone loss but are not tolerated by all patients and are associated with an increased risk of osteonecrosis of the jaw. In light of this other anti-resorptives such as phytoestrogens are being considered. However the effect of phytoestrogens on T cell-induced osteoclast formation is unclear. The effect of genistein and coumestrol on activated T cell-induced osteoclastogenesis and cytokine production was therefore examined. Concentrations of genistein and coumestrol (10(-7)M) previously shown to directly inhibit osteoclast formation also suppressed the formation of TRAP positive osteoclast induced by con A activated T cells, which was dependent on inhibition of T cell derived TNF-α. While both reduced osteoclast formation their mechanism of action differed. The anti-osteoclastic effect of coumestrol was associated with a dual effect on con A induced T cell proliferation and activation; 10(-7)M coumestrol significantly reducing T cell number (0.36) and TNF-α (0.47), IL-1ß (0.23) and IL-6 (0.35) expression, whereas genistein (10(-7)M) had no effect on T cell number but a more pronounced effect on T cell differentiation reducing expression of TNF-α (0.49), IL-1ß (0.52), IL-6 (0.71) and RANKL (0.71). Phytoestrogens therefore prevent the pro-osteoclastic action of T cells suggesting they may have a role in the control of inflammatory bone loss.

Expression profile and function of Wnt signaling mechanisms in malignant mesothelioma cells.

Malignant mesothelioma (MM) is an uncommon and particularly aggressive cancer associated with asbestos exposure, which currently presents an intractable clinical challenge. Wnt signaling has been reported to play a role in the neoplastic properties of mesothelioma cells but has not been investigated in detail in this cancer. We surveyed expression of Wnts, their receptors, and other key molecules in this pathway in well established in vitro mesothelioma models in comparison with primary mesothelial cultures. We also tested the biological response of MM cell lines to exogenous Wnt and secreted regulators, as well as targeting ß-catenin. We detected frequent expression of Wnt3 and Wnt5a, as well as Fzd 2, 4 and 6. The mRNA of Wnt4, Fzd3, sFRP4, APC and axin2 were downregulated in MM relative to mesothelial cells while LEF1 was overexpressed in MM. Functionally, we observed that Wnt3a stimulated MM proliferation while sFRP4 was inhibitory. Furthermore, directly targeting ß-catenin expression could sensitise MM cells to cytotoxic drugs. These results provide evidence for altered expression of a number of Wnt/Fzd signaling molecules in MM. Modulation of Wnt signaling in MM may prove a means of targeting proliferation and drug resistance in this cancer.

Chemotherapy broadens the range of tumor antigens seen by cytotoxic CD8(+) T cells in vivo.

Cytotoxic chemotherapies may expose the immune system to high levels of tumor antigens and expand the CD8(+) T-cell response to include weak or subdominant antigens. Here, we evaluated the in vivo CTL response to tumor antigens using a murine mesothelioma tumor cell line transfected with a neotumor antigen, ovalbumin, that contains a known hierarchy of epitopes for MHC class I molecules. We show that as tumors progress, effector CTLs are generated in vivo that focus on the dominant epitope SIINFEKL, although a weak response was seen to one (KVVRFDKL) subdominant epitope. These CTLs did not prevent tumor growth. Cisplatin treatment slowed tumor growth, slightly improved in vivo SIINFEKL presentation to T cells and reduced SIINFEKL-CTL activity. However, the CTL response to KVVRFDKL was amplified, and a response to another subdominant epitope, NAIVFKGL, was revealed. Similarly, gemcitabine cured most mice, slightly enhanced SIINFEKL presentation, reduced SIINFEKL-CTL activity yet drove a significant CTL response to NAIVFKGL, but not KVVRFDKL. These NAIVFKGL-specific CTLs secreted IFNγ and proliferated in response to in vitro NAIVFKGL stimulation. IL-2 treatment during chemotherapy refocused the response to SIINFEKL and simultaneously degraded the cisplatin-driven subdominant CTL response. These data show that chemotherapy reveals weaker tumor antigens to the immune system, a response that could be rationally targeted. Furthermore, while integrating IL-2 into the chemotherapy regimen interfered with the hierarchy of the response, IL-2 or other strategies that support CTL activity could be considered upon completion of chemotherapy.

Subcellular analysis of starch metabolism in developing barley seeds using a non-aqueous fractionation method.

Compartmentation of metabolism in developing seeds is poorly understood due to the lack of data on metabolite distributions at the subcellular level. In this report, a non-aqueous fractionation method is described that allows subcellular concentrations of metabolites in developing barley endosperm to be calculated. (i) Analysis of subcellular volumes in developing endosperm using micrographs shows that plastids and cytosol occupy 50.5% and 49.9% of the total cell volume, respectively, while vacuoles and mitochondria can be neglected. (ii) By using non-aqueous fractionation, subcellular distribution between the cytosol and plastid of the levels of metabolites involved in sucrose degradation, starch synthesis, and respiration were determined. With the exception of ADP and AMP which were mainly located in the plastid, most other metabolites of carbon and energy metabolism were mainly located outside the plastid in the cytosolic compartment. (iii) In developing barley endosperm, the ultimate precursor of starch, ADPglucose (ADPGlc), was mainly located in the cytosol (80-90%), which was opposite to the situation in growing potato tubers where ADPGlc was almost exclusively located in the plastid (98%). This reflects the different subcellular distribution of ADPGlc pyrophosphorylase (AGPase) in these tissues. (iv) Cytosolic concentrations of ADPGlc were found to be close to the published K(m) values of AGPase and the ADPGlc/ADP transporter at the plastid envelope. Also the concentrations of the reaction partners glucose-1-phosphate, ATP, and inorganic pyrophosphate were close to the respective K(m) values of AGPase. (v) Knock-out of cytosolic AGPase in Riso16 mutants led to a strong decrease in ADPGlc level, in both the cytosol and plastid, whereas knock-down of the ADPGlc/ADP transporter led to a large shift in the intracellular distribution of ADPGlc. (v) The thermodynamic structure of the pathway of sucrose to starch was determined by calculating the mass-action ratios of all the steps in the pathway. The data show that AGPase is close to equilibrium, in both the cytosol and plastid, whereas the ADPGlc/ADP transporter is strongly displaced from equilibrium in vivo. This is in contrast to most other tissues, including leaves and potato tubers. (vi) Results indicate transport rather than synthesis of ADPGlc to be the major regulatory site of starch synthesis in barley endosperm. The reversibility of AGPase in the plastid has important implications for the regulation of carbon partitioning between different biosynthetic pathways.