A Review of Principal Studies on the Development and Treatment of Epithelial Ovarian Cancer in the Laying Hen Gallus gallus

Often referred to as the silent killer, ovarian cancer is the most lethal gynecologic malignancy. This disease rarely shows any physical symptoms until late stages and no known biomarkers are available for early detection. Because ovarian cancer is rarely detected early, the physiology behind the initiation, progression, treatment, and prevention of this disease remains largely unclear. Over the past 2 decades, the laying hen has emerged as a model that naturally develops epithelial ovarian cancer that is both pathologically and histologically similar to that of the human form of the disease. Different molecular signatures found in human ovarian cancer have also been identified in chicken ovarian cancer including increased CA125 and elevated E-cadherin expression, among others. Chemoprevention studies conducted in this model have shown that decreased ovulation and inflammation are associated with decreased incidence of ovarian cancer development. The purpose of this article is to review the major studies performed in laying hen model of ovarian cancer and discuss how these studies shape our current understanding of the pathophysiology, prevention and treatment of epithelial ovarian cancer.

Ovarian cancer is the leading cause of death among female gynecologic malignancies, with a 47% 5 y relative survival rate.¹⁵⁴ Early detection of the disease is necessary for decreasing the high mortality rate. However, early detection is difficult due to the lack of known specific biomarkers and clinically detectable symptoms until the tumor reaches at an advanced stage. The disease has multiple subtypes. Epithelial ovarian cancer (EOC) is the most common type of ovarian cancer, accounting for about 90% of all reported cases.^127,164 EOC is commonly subdivided into 5 histotypes: high-grade serous (HGSOC), low-grade serous, mucinous, endometroid (EC), and clear cell. The histotypes differ in terms of tumor cell morphology, severity, systemic effect, and response to treatment. Among the different subtypes, HGSOC accounts for about 70% of cases of EOC observed in women. HGSOC has a higher mitotic index and is a more aggressive form of cancer with a worse prognosis. HGSOC and low-grade serous histotypes exhibit distinctly different presentations of the disease^82,166 and demand different treatment modalities. EC (10% to 20%), mucinous (5% to 20%), and clear cell (3% to 10%) histotypes are less common forms of the disease. The subtypes of EOC also differ in terms of 5 y survival rates of patients; that is, HGSOC (20% to 35%), EC (40% to 63%), mucinous (40% to 69%), and clear cell (35% to 50%).^20,76,148Developing a representative animal model for EOC has been challenging due to the histologic and pathologic differences among different subtypes of EOC. While developing a reliable animal model is challenging due to the vast complexity and limited understanding of the origin of the disease, laying hens naturally develop EOC that is histopathologically very similar to the human form of the disease (Figure 1).¹⁵ All the different human ovarian cancer histotypes have been observed in laying hen ovarian cancer (Figure 2). In addition, the presentation of the disease in chickens is remarkably similar to the human form of the disease, with early-stage ovarian cancer in laying hens having similar precursor lesions as occur in women.¹⁵ The laying hen develops ovarian cancer spontaneously, allowing analysis of early events and investigation into the natural course of the disease, as tumors can be examined as they progress from normal to late-stage ovarian carcinoma. The gross appearance of these stages is shown in Figure 3.Open in a separate windowFigure 1.Gross pathologic presentation of chicken compared with human ovarian cancer. The remarkably similar presentation in hens (A,B) and women (C,D) at the gross anatomic level with profuse abdominal ascites and peritoneal dissemination of metastasis. A) Ascites in abdominal cavity chicken with advanced ovarian cancer (photo credit: DB Hales); (B) Chicken ovarian cancer with extensive peritoneal dissemination of metastasis (photo credit: DB Hales); (C) Distended abdomen from ascites fluid accumulation in woman with ovarian cancer (http://www.pathguy.com/bryanlee/ovca.html) (D) Human ovarian cancer with extensive peritoneal dissemination of metastasis (http://www.pathguy.com/bryanlee/ovca.html).Open in a separate windowFigure 2.Gross anatomic appearance of different stages of ovarian cancer in the chicken The progression from the normal hen ovary to late-stage metastatic ovarian cancer. (A) Normal chicken ovary showing hierarchal clutch of developing follicles and postovulatory follicle; (B) Stage 1 ovarian cancer, confined to ovary with vascularized follicles; (C) Stage 2/3 ovarian cancer, metastasis locally to peritoneal cavity with ascites; (D) Stage 4 ovarian cancer, late stage with metastasis to lung and liver with extensive ascites (photo credits: DB Hales).Open in a separate windowFigure 3.Histologic subtypes in chicken compared with human ovarian cancers. H and E staining of formalin fixed paraffin embedded tissues from hens with ovarian cancer (A through D) and women (E through G). (A) Chicken clear cell carcinoma; (B) Chicken endometrioid carcinoma; (C) Chicken mucinous adenocarcinoma; (D) Chicken serous papillary adenocarcinoma (photo credits: DB Hales). (E) Human clear cell carcinoma; (F) Human endometrioid carcinoma; (G) Human mucinous cystadenocarcinoma; (H) Human serous adenocarcinoma (https://www.womenshealthsection.com).Over the past 2 decades, the laying hen has emerged as a valuable experimental model for EOC, in addition to other in vivo models such as Patient-Derived Xenografts (PDX) and Genetically Engineered Mouse Models (GEMMs). Comparison of the hen model with other animal models has been reviewed elsewhere.⁷² Modern-day laying hens, such as the white leghorn, have been selected from their ancestor red jungle fowl⁵⁷ for decreased broodiness and persistent ovulation, resulting in approximately one egg per day, if proper nutrition and light-dark cycles are maintained. Daily rupture and consequent repair of the ovarian surface epithelia (OSE) due to the persistent ovulation promotes potential error during rapid DNA replication. This increases the probability of oncogenic mutations, ultimately leading to neoplasia.¹³⁷ Inflammation resulting from continuous ovulation also promotes the natural development of EOC.⁸¹ By the age of 2.5 to 3 y, laying hens have undergone a similar number of ovulations as a perimenopausal woman. The risk of ovarian cancer in white leghorn hens in this time (4%) is similar to the lifetime risk of ovarian cancer in women (0.35% to 8.8%).¹²⁵ By the age of 4 to 6 y, the risk of ovarian cancer in hens rises to 30% to 60%.⁵⁴ The incidence of ovarian carcinoma in the hens, however, depends on the age, genetic strain,⁸⁰ and the egg-laying frequency of the specific breed.⁵⁴ The common white leghorn hen has routinely been employed in chicken ovarian cancer studies. On average, hens are exposed to 17 h of light per day, with lights turned on at 0500 h and turned off at 2200 h. The laying hen model of EOC does present some considerable challenges. Despite its great utility for research, the model is still used mainly by agricultural poultry scientists and a small number of ovarian cancer researchers.Comprehensive and proper vivarium support is required to conduct large-scale cancer prevention studies. Only a few facilities are available for biomedical chicken research, including University of Illinois Urbana-Champaign, Cornell University, Penn State University, NC State, Auburn University, and MS State University. Another difficulty is a lack of available antibodies specific for chicken antigens. Because of the structural dissimilarities between most human proteins and murine antigens to their chicken counterparts, cross-reactivity of available antibodies is also limited. The entire chicken genome was sequenced in 2004;⁷⁸ however, the chromosomal locus of many key genes, such as p53, are still unknown. Overall, humans and chickens share about 60% of genetic commonality, whereas humans and rats share about 88% of their genes. Specific pathway-mutated strains of chickens are not yet available, limiting the ability to study key pathways in carcinogenesis and prevention of cancer using this model. Although all 5 different subtypes of ovarian cancer are present in hens, their most predominant subtype is different from women. Close to 70% of women diagnosed with ovarian cancer have serous EOC, while the predominant subtype reported in hens is endometrioid.¹⁵ However, these comparisons are complicated because observations of cancer in hens consist of both early and late stages of the disease, wherein women, most of the data is from late stage and aggressive ovarian carcinoma.The spontaneous onset of ovarian cancer and the histologic and pathologic similarities to the human form of the disease make laying hens an excellent model for continued research on EOC. To date, a large number of studies have been performed on laying hens. Here we have divided the current studies into 2 groups— (A) studies that have described the molecular presentation of EOC to be similar to that in women; (

Nickel-based Enzyme Systems

Of the eight known nickel enzymes, all but glyoxylase I catalyze the use and/or production of gases central to the global carbon, nitrogen, and oxygen cycles. Nickel appears to have been selected for its plasticity in coordination and redox chemistry and is able to cycle through three redox states (1+, 2+, 3+) and to catalyze reactions spanning ∼1.5 V. This minireview focuses on the catalytic mechanisms of nickel enzymes, with an emphasis on the role(s) of the metal center. The metal centers vary from mononuclear to complex metal clusters and catalyze simple hydrolytic to multistep redox reactions.Seven of the eight known nickel enzymes (1). CODH² interconverts CO and CO₂; ACS utilizes CO; the nickel ARD produces CO; hydrogenase generates/utilizes hydrogen gas; MCR generates methane; urease produces ammonia; and SOD generates O₂.

TABLE 1

Nickel-containing enzymes

Molecular and Biochemical Characterization of the Protein Template Controlling Biosynthesis of the Lipopeptide Lichenysin

Lichenysins are surface-active lipopeptides with antibiotic properties produced nonribosomally by several strains of Bacillus licheniformis. Here, we report the cloning and sequencing of an entire 26.6-kb lichenysin biosynthesis operon from B. licheniformis ATCC 10716. Three large open reading frames coding for peptide synthetases, designated licA, licB (three modules each), and licC (one module), could be detected, followed by a gene, licTE, coding for a thioesterase-like protein. The domain structure of the seven identified modules, which resembles that of the surfactin synthetases SrfA-A to -C, showed two epimerization domains attached to the third and sixth modules. The substrate specificity of the first, fifth, and seventh recombinant adenylation domains of LicA to -C (cloned and expressed in Escherichia coli) was determined to be Gln, Asp, and Ile (with minor Val and Leu substitutions), respectively. Therefore, we suppose that the identified biosynthesis operon is responsible for the production of a lichenysin variant with the primary amino acid sequence l-Gln–l-Leu–d-Leu–l-Val–l-Asp–d-Leu–l-Ile, with minor Leu and Val substitutions at the seventh position.Many strains of Bacillus are known to produce lipopeptides with remarkable surface-active properties (11). The most prominent of these powerful lipopeptides is surfactin from Bacillus subtilis (1). Surfactin is an acylated cyclic heptapeptide that reduces the surface tension of water from 72 to 27 mN m⁻¹ even in a concentration below 0.05% and shows some antibacterial and antifungal activities (1). Some B. subtilis strains are also known to produce other, structurally related lipoheptapeptides (Table ​(Table1),1), like iturin (32, 34) and bacillomycin (3, 27, 30), or the lipodecapeptides fengycin (50) and plipastatin (29).

TABLE 1

Lipoheptapeptide antibiotics of Bacillus spp.

Peptidoglycan Fine Structure of the Radiotolerant Bacterium Deinococcus radiodurans Sark

Peptidoglycan from Deinococcus radiodurans was analyzed by high-performance liquid chromatography and mass spectrometry. The monomeric subunit was: N-acetylglucosamine–N-acetylmuramic acid–l-Ala–d-Glu-(γ)–l-Orn-[(δ)Gly-Gly]–d-Ala–d-Ala. Cross-linkage was mediated by (Gly)₂ bridges, and glycan strands were terminated in (1→6)anhydro-muramic acid residues. Structural relations with the phylogenetically close Thermus thermophilus are discussed.The gram-positive bacterium Deinococcus radiodurans is remarkable because of its extreme resistance to ionizing radiation (14). Phylogenetically the closest relatives of Deinococcus are the extreme thermophiles of the genus Thermus (4, 11). In 16S rRNA phylogenetic trees, the genera Thermus and Deinococcus group together as one of the older branches in bacterial evolution (11). Both microorganisms have complex cell envelopes with outer membranes, S-layers, and ornithine-Gly-containing mureins (7, 12, 19, 20, 22, 23). However, Deinococcus and Thermus differ in their response to the Gram reaction, having positive and negative reactions, respectively (4, 14). The murein structure for Thermus thermophilus HB8 has been recently elucidated (19). Here we report the murein structure of Deinococcus radiodurans with similar detail.D. radiodurans Sark (23) was used in the present study. Cultures were grown in Luria-Bertani medium (13) at 30°C with aeration. Murein was purified and subjected to amino acid and high-performance liquid chromatography (HPLC) analyses as previously described (6, 9, 10, 19). For further analysis muropeptides were purified, lyophilized, and desalted as reported elsewhere (6, 19). Purified muropeptides were subjected to plasma desorption linear time-of-flight mass spectrometry (PDMS) as described previously (1, 5, 16, 19). Positive and negative ion mass spectra were obtained on a short linear ²⁵²californium time-of-flight instrument (BioIon AB, Uppsala, Sweden). The acceleration voltage was between 17 and 19 kV, and spectra were accumulated for 1 to 10 million fission events. Calibration of the mass spectra was done in the positive ion mode with H⁺ and Na⁺ ions and in the negative ion mode with H⁻ and CN⁻ ions. Calculated m/z values are based on average masses.Amino acid analysis of muramidase (Cellosyl; Hoechst, Frankfurt am Main, Germany)-digested sacculi (50 μg) revealed Glu, Orn, Ala, and Gly as the only amino acids in the muramidase-solubilized material. Less than 3% of the total Orn remained in the muramidase-insoluble fraction, indicating an essentially complete solubilization of murein.Muramidase-digested murein samples (200 μg) were analyzed by HPLC as described in reference 19. The muropeptide pattern (Fig. ​(Fig.1)1) was relatively simple, with five dominating components (DR5 and DR10 to DR13 [Fig. 1]). The muropeptides resolved by HPLC were collected, desalted, and subjected to PDMS. The results are presented in Table ​Table11 compared with the m/z values calculated for best-matching muropeptides made up of N-acetylglucosamine (GlucNAc), N-acetylmuramic acid (MurNAc), and the amino acids detected in the murein. The more likely structures are shown in Fig. ​Fig.1.1. According to the m/z values, muropeptides DR1 to DR7 and DR9 were monomers; DR8, DR10, and DR11 were dimers; and DR12 and DR13 were trimers. The best-fitting structures for DR3 to DR8, DR11, and DR13 coincided with muropeptides previously characterized in T. thermophilus HB8 (19) and had identical retention times in comparative HPLC runs. The minor muropeptide DR7 (Fig. ​(Fig.1)1) was the only one detected with a d-Ala–d-Ala dipeptide and most likely represents the basic monomeric subunit. The composition of the major cross-linked species DR11 and DR13 confirmed that cross-linking is mediated by (Gly)₂ bridges, as proposed previously (20). Open in a separate windowFIG. 1HPLC muropeptide elution patterns of murein purified from D. radiodurans. Muramidase-digested murein samples were subjected to HPLC analysis, and the A₂₀₄ of the eluate was recorded. The most likely structures for each muroeptide as deduced by PDMS are shown. The position of residues in brackets is the most likely one as deduced from the structures of other muropeptides but could not be formally demonstrated. R = GlucNac–MurNac–l-Ala–d-Glu-(γ)→.

TABLE 1

Calculated and measured m/z values for the molecular ions of the major muropeptides from D. radiodurans

Genome-wide analysis of lipoxygenase gene family in Arabidopsis and rice

The enzymes called lipoxygenases (LOXs) can dioxygenate unsaturated fatty acids, which leads to lipoperoxidation of biological membranes. This process causes synthesis of signaling molecules and also leads to changes in cellular metabolism. LOXs are known to be involved in apoptotic (programmed cell death) pathway, and biotic and abiotic stress responses in plants. Here, the members of LOX gene family in Arabidopsis and rice are identified. The Arabidopsis and rice genomes encode 6 and 14 LOX proteins, respectively, and interestingly, with more LOX genes in rice. The rice LOXs are validated based on protein alignment studies. This is the first report wherein LOXs are identified in rice which may allow better understanding the initiation, progression and effects of apoptosis, and responses to bitoic and abiotic stresses and signaling cascades in plants.Key words: apoptosis, biotic and abiotic stresses, genomics, jasmonic acid, lipidsLipoxygenases (linoleate:oxygen oxidoreductase, EC 1.13.11.-; LOXs) catalyze the conversion of polyunsaturated fatty acids (lipids) into conjugated hydroperoxides. This process is called hydroperoxidation of lipids. LOXs are monomeric, non-heme and non-sulfur, but iron-containing dioxygenases widely expressed in fungi, animal and plant cells, and are known to be absent in prokaryotes. However, a recent finding suggests the existence of LOX-related genomic sequences in bacteria but not in archaea.¹ The inflammatory conditions in mammals like bronchial asthama, psoriasis and arthritis are a result of LOXs reactions.² Further, several clinical conditions like HIV-1 infection,³ disease of kidneys due to the activation of 5-lipoxygenase,^4,5 aging of the brain due to neuronal 5-lipoxygenase⁶ and atherosclerosis⁷ are mediated by LOXs. In plants, LOXs are involved in response to biotic and abiotic stresses.⁸ They are involved in germination⁹ and also in traumatin and jasmonic acid biochemical pathways.^10,11 Studies on LOX in rice are conducted to develop novel strategies against insect pests¹² in response to wounding and insect attack,¹³ and on rice bran extracts as functional foods and dietary supplements for control of inflammation and joint health.¹⁴ In Arabidopsis, LOXs are studied in response to natural and stress-induced senescence,¹⁵ transition to flowering,¹⁶ regulation of lateral root development and defense response.¹⁷The arachidonic, linoleic and linolenic acids can act as substrates for different LOX isozymes. A hydroperoxy group is added at carbons 5, 12 or 15, when arachidonic acid is the substrate, and so the LOXs are designated as 5-, 12- or 15-lipoxygenases. Sequences are available in the database for plant lipoxygenases (EC:1.13.11.12), mammalian arachidonate 5-lipoxygenase (EC:1.13.11.34), mammalian arachidonate 12-lipoxygenase (EC:1.13.11.31) and mammalian erythroid cell-specific 15-lipoxygenase (EC:1.13.11.33). The prototype member for LOX family, LOX-1 of Glycine max L. (soybean) is a 15-lipoxygenase. The LOX isoforms of soybean (LOX-1, LOX-2, LOX-3a and LOX-3b) are the most characterized of plant LOXs.¹⁸ In addition, five vegetative LOXs (VLX-A, -B, -C, -D, -E) are detected in soybean leaves.¹⁹ The 3-dimensional structure of soybean LOX-1 has been determined.^20,21 LOX-1 was shown to be made of two domains, the N-terminal domain-I which forms a β-barrel of 146 residues, and a C-terminal domain-II of bundle of helices of 693 residues²¹ (Fig. 1). The iron atom was shown to be at the centre of domain-II bound by four coordinating ligands, of which three are histidine residues.²²Open in a separate windowFigure 1Three-dimensional structure of soybean lipoxygenase L-1. The domain I (N-terminal) and domain II (C-terminal) are indicated. The catalytic iron atom is embedded in domain II (PDB ID-1YGE).²¹This article describes identification of LOX genes in Arabidopsis and rice. The Arabidopsis genome encodes for six LOX proteins²³ (www.arabidopsis.org) (LocusAnnotationNomenclatureA^*B^*C^*AT1G55020lipoxygenase 1 (LOX1)LOX185998044.45.2049AT1G17420lipoxygenase 3 (LOX3)LOX3919103725.18.0117AT1G67560lipoxygenase family proteinLOX4917104514.68.0035AT1G72520lipoxygenase, putativeLOX6926104813.17.5213AT3G22400lipoxygenase 5 (LOX5)LOX5886101058.86.6033AT3G45140lipoxygenase 2 (LOX2)LOX2896102044.75.3177Open in a separate window^*A, amino acids; B, molecular weight; C, isoelectric point.Interestingly, the rice genome (rice.plantbiology.msu.edu) encodes for 14 LOX proteins as compared to six in Arabidopsis (​and22). Of these, majority of them are composed of ∼790–950 aa with the exception for loci, LOC_Os06g04420 (126 aa), LOC_Os02g19790 (297 aa) and LOC_Os12g37320 (359 aa) (Fig. 2).Open in a separate windowFigure 2Protein alignment of rice LOXs and vegetative lipoxygenase, VLX-B,²⁸ a soybean LOX (AA B67732). The 14 rice LOCs are indicated on left and sequence position on right. Gaps are included to improve alignment accuracy. Figure was generated using ClustalX program.

Table 2

Genes encoding lipoxygenases in rice

Disease Mutations in the Human Mitochondrial DNA Polymerase Thumb Subdomain Impart Severe Defects in Mitochondrial DNA Replication

Forty-five different point mutations in POLG, the gene encoding the catalytic subunit of the human mitochondrial DNA polymerase (pol γ), cause the early onset mitochondrial DNA depletion disorder, Alpers syndrome. Sequence analysis of the C-terminal polymerase region of pol γ revealed a cluster of four Alpers mutations at highly conserved residues in the thumb subdomain (G848S, c.2542g→a; T851A, c.2551a→g; R852C, c.2554c→t; R853Q, c.2558g→a) and two Alpers mutations at less conserved positions in the adjacent palm subdomain (Q879H, c.2637g→t and T885S, c.2653a→t). Biochemical characterization of purified, recombinant forms of pol γ revealed that Alpers mutations in the thumb subdomain reduced polymerase activity more than 99% relative to the wild-type enzyme, whereas the palm subdomain mutations retained 50–70% wild-type polymerase activity. All six mutant enzymes retained physical and functional interaction with the pol γ accessory subunit (p55), and none of the six mutants exhibited defects in misinsertion fidelity in vitro. However, differential DNA binding by these mutants suggests a possible orientation of the DNA with respect to the polymerase during catalysis. To our knowledge this study represents the first structure-function analysis of the thumb subdomain in pol γ and examines the consequences of mitochondrial disease mutations in this region.As the only DNA polymerase found in animal cell mitochondria, DNA polymerase γ (pol γ)³ bears sole responsibility for DNA synthesis in all replication and repair transactions involving mitochondrial DNA (1, 2). Mammalian cell pol γ is a heterotrimeric complex composed of one catalytic subunit of 140 kDa (p140) and two 55-kDa accessory subunits (p55) that form a dimer (3). The catalytic subunit contains an N-terminal exonuclease domain connected by a linker region to a C-terminal polymerase domain. Whereas the exonuclease domain contains essential motifs I, II, and III for its activity, the polymerase domain comprising the thumb, palm, and finger subdomains contains motifs A, B, and C that are crucial for polymerase activity. The catalytic subunit is a family A DNA polymerase that includes bacterial pol I and T7 DNA polymerase and possesses DNA polymerase, 3′ → 5′ exonuclease, and 5′-deoxyribose phosphate lyase activities (for review, see Refs. 1 and 2). The 55-kDa accessory subunit (p55) confers processive DNA synthesis and tight binding of the pol γ complex to DNA (4, 5).Depletion of mtDNA as well as the accumulation of deletions and point mutations in mtDNA have been observed in several mitochondrial disorders (for review, see Ref. 6). mtDNA depletion syndromes are caused by defects in nuclear genes responsible for replication and maintenance of the mitochondrial genome (7). Mutation of POLG, the gene encoding the catalytic subunit of pol γ, is frequently involved in disorders linked to mutagenesis of mtDNA (8, 9). Presently, more than 150 point mutations in POLG are linked with a wide variety of mitochondrial diseases, including the autosomal dominant (ad) and recessive forms of progressive external ophthalmoplegia (PEO), Alpers syndrome, parkinsonism, ataxia-neuropathy syndromes, and male infertility (tools.niehs.nih.gov/polg) (9).Alpers syndrome, a hepatocerebral mtDNA depletion disorder, and myocerebrohepatopathy are rare heritable autosomal recessive diseases primarily affecting young children (10–12). These diseases generally manifest during the first few weeks to years of life, and symptoms gradually develop in a stepwise manner eventually leading to death. Alpers syndrome is characterized by refractory seizures, psychomotor regression, and hepatic failure (11, 12). Mutation of POLG was first linked to Alpers syndrome in 2004 (13), and to date 45 different point mutations in POLG (18 localized to the polymerase domain) are associated with Alpers syndrome (9, 14, 15). However, only two Alpers mutations (A467T and W748S, both in the linker region) have been biochemically characterized (16, 17).During the initial cloning and sequencing of the human, Drosophila, and chicken pol γ genes, we noted a highly conserved region N-terminal to motif A in the polymerase domain that was specific to pol γ (18). This region corresponds to part of the thumb subdomain that tracks DNA into the active site of both Escherichia coli pol I and T7 DNA polymerase (19–21). A high concentration of disease mutations, many associated with Alpers syndrome, is found in the thumb subdomain.Here we investigated six mitochondrial disease mutations clustered in the N-terminal portion of the polymerase domain of the enzyme (Fig. 1A). Four mutations (G848S, c.2542g→a; T851A, c.2551a→g; R852C, c.2554c→t; R853Q, c.2558g→a) reside in the thumb subdomain and two (Q879H, c.2637g→t and T885S, c.2653a→t) are located in the palm subdomain. These mutations are associated with Alpers, PEO, mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), ataxia-neuropathy syndrome, Leigh syndrome, and myocerebrohepatopathy (POLG mutationDiseaseGeneticsReferenceG848SAlpers syndromeIn trans with A467T, Q497H, T251I-P587L, or W748S-E1143G in Alpers syndrome15, 35, 43–50Leigh syndromeIn trans with R232H in Leigh syndrome49MELASIn trans with R627Q in MELAS38PEO with ataxia-neuropathyIn trans with G746S and E1143G in PEO with ataxia50PEOIn trans with T251I and P587L in PEO51, 52T851AAlpers syndromeIn trans with R1047W48, 53In trans with H277CR852CAlpers syndromeIn trans with A467T14, 48, 50In cis with G11D and in trans with W748S-E1143G or A467TAtaxia-neuropathyIn trans with G11D-R627Q15R853QMyocerebrohepatopathyIn trans with T251I-P587L15Q879HAlpers syndrome with valproate-induced hepatic failureIn cis with E1143G and in trans with A467T-T885S35, 54T885SAlpers syndrome with valproate-induced hepatic failureIn cis with A467T and in trans with Q879H-E1143G35, 54Open in a separate windowOpen in a separate windowFIGURE 1.POLG mutations characterized in this study. A, the location of the six mutations characterized is shown in red in the primary sequence of pol γ. Four mutations, the G848S, T851A, R852C, and R853Q, are located in the thumb domain, whereas two mutations, the Q879H and T885S, are in the palm domain of the polymerase region. B, sequence alignment of pol γ from yeast to humans. The amino acids characterized in this study are shown in red. Yellow-highlighted amino acids are highly conserved, and blue-highlighted amino acids are moderately conserved.     

Phosphoprotein Secretome of Tumor Cells as a Source of Candidates for Breast Cancer Biomarkers in Plasma

Breast cancer is a heterogeneous disease whose molecular diversity is not well reflected in clinical and pathological markers used for prognosis and treatment selection. As tumor cells secrete proteins into the extracellular environment, some of these proteins reach circulation and could become suitable biomarkers for improving diagnosis or monitoring response to treatment. As many signaling pathways and interaction networks are altered in cancerous tissues by protein phosphorylation, changes in the secretory phosphoproteome of cancer tissues could reflect both disease progression and subtype. To test this hypothesis, we compared the phosphopeptide-enriched fractions obtained from proteins secreted into conditioned media (CM) derived from five luminal and five basal type breast cancer cell lines using label-free quantitative mass spectrometry. Altogether over 5000 phosphosites derived from 1756 phosphoproteins were identified, several of which have the potential to qualify as phosphopeptide plasma biomarker candidates for the more aggressive basal and also the luminal-type breast cancers. The analysis of phosphopeptides from breast cancer patient plasma and controls allowed us to construct a discovery list of phosphosites under rigorous collection conditions, and second to qualify discovery candidates generated from the CM studies. Indeed, a set of basal-specific phosphorylation CM site candidates derived from IBP3, CD44, OPN, FSTL3, LAMB1, and STC2, and luminal-specific candidates derived from CYTC and IBP5 were selected and, based on their presence in plasma, quantified across all cell line CM samples using Skyline MS1 intensity data. Together, this approach allowed us to assemble a set of novel cancer subtype specific phosphopeptide candidates for subsequent biomarker verification and clinical validation.Breast cancer (BC)¹ is a heterogeneous disease whose molecular complexity and diversity is not well reflected in current clinical and pathological markers. Therefore, there is a critical need to increase the number of clinically suitable biomarkers that better reflect the many molecular subtypes of BC (1–3). BC can be categorized by gene expression profiling and molecular pathology into three major clinical types, each with different natural histories and therapeutic recommendations, and exhibiting significant molecular and clinical heterogeneity. First, luminal estrogen receptor (ER) positive breast cancers exist in luminal A and B subtypes; they are the most numerous and clinically diverse of all breast cancers, with luminal A tumors having the more favorable prognosis because of their responsiveness to targeted endocrine therapy compared with the more proliferative luminal B tumors. Second, human epidermal growth factor receptor-2 (HER2/ErbB2) amplified breast cancers, despite having poor prognosis in the absence of any systemic adjuvant therapy, can now be successfully treated with HER2-targeted agents. Third, basal-like breast cancers are among the most aggressive tumors, and are further subdivided. Those with BRCA1-like features are modeled by basal-A breast cancer cell lines, and those with mesenchymal and stem/progenitor-cell features are modeled by basal-B breast cancer cell lines (4). This latter subtype of basal-like tumors include triple negative breast cancers (TNBC), lacking expression of ER, progesterone receptor (PR), and HER2, and therefore not susceptible to more advanced targeted treatment options and requiring aggressive chemotherapy with otherwise very poor prognosis (5).BC is the leading cause of adult female mortality worldwide, caused by recurrent spread of metastatic disease that is thought to have seeded prior to the time of primary tumor excision (6). Thus, blood-based biomarkers that are highly specific as well as capable of detecting BC prior to primary tumor diagnosis offer the potential to decrease BC morbidity as well as identify the most appropriate treatment options (7). As cancer cells are known to secrete proteins into the extracellular microenvironment that modify cell adhesion, intercellular communication, motility, and invasiveness (8), it is expected that some will enter the blood stream and become suitable targets for early noninvasive diagnosis or monitoring of treatment progression.It is well recognized that blood contains hormones, cytokines, and other nonhormonal proteins, as well as a tissue leakage products and secretions from diseased tissues and tumors (9). Secreted proteins are often in the low abundance range of plasma protein concentrations, and likely contain proteins specific for distinct tumor and/or tissue types. Because tumorogenesis is known to involve changes in cellular signaling pathways involving protein kinases, protein phosphorylation is a particularly promising target for the detection of such activated pathways in BC (10). For example, almost half of the tyrosine kinases of the human “kinome” are implicated in human cancers (11) as well as numerous serine-threonine kinases, including Akt and mTOR (12, 13). Kinases participating in signal transduction pathways phosphorylate their substrates altering their conformation, localization, and activity, which in turn modulates downstream protein effectors and alters cellular processes. Like other posttranslational modifications, changes in the phosphorylation status of a protein do not directly correlate with changes in expression, and are therefore not accounted for in most gene expression or protein array analyses (14). Therefore, we hypothesized that phosphoproteins secreted or shed by cancer cells constitute a largely overlooked source of biomarker candidates that could be correlated with BC subtypes and/or disease status (15, 16).To test this hypothesis, we analyzed the conditioned media (CM) from human cancer cell lines, a well-established model for the discovery of disease-specific biomarkers (17, 18). Breast cancer cell lines derived from primary tumors or pleural effusions are a good model of BC, mirroring molecular characteristics of primary breast tumors (19). The use of CM is also advantageous in that it provides sufficient amounts of sample to identify candidates that can subsequently be targeted in more limited breast cancer patient plasma samples. To examine the phosphorylation status of secreted proteins, we examined a panel of five luminal and five basal type BC cell lines thought to emulate the molecular characteristics of most primary breast tumor types, including four basal-B subtypes corresponding to TNBC (19). A mass spectrometry-based proteomic approach was used that employed HILIC fractionation, TiO₂ affinity enrichment of phosphopeptides, and final mass spectrometric analysis by reverse-phase liquid chromatography and label-free quantification (Fig. 1). MS1 Filtering in Skyline (20, 21) was used to quantify relative differences in site-specific protein phosphorylation between secretomes of BC cell lines derived from breast tumor subtypes to discern luminal or basal tumor specificity. Lastly, plasma obtained from breast cancer patients and controls were analyzed in an optimized workflow suitable to both preserve and identify phosphopeptides, and to qualify a subset of biomarker candidates selected from the CM analysis (Fig. 1). Overall, we identified 107 phosphorylation sites specific for basal-type tumors derived from 84 proteins and 95 phosphorylation sites specific for luminal-type tumors derived from 64 proteins. Moreover, we qualified the presence of seven basal type specific and two luminal specific phosphosites derived from eight phosphoproteins in BC patient and control plasma.

Table I

Luminal and basal breast cancer cell lines

Genome-wide analysis of thioredoxin fold superfamily peroxiredoxins in Arabidopsis and rice

A broad range of peroxides generated in subcellular compartments, including chloroplasts, are detoxified with peroxidases called peroxiredoxins (Prx). The Prx are ubiquitously distributed in all organisms including bacteria, fungi, animals and also in cyanobacteria and plants. Recently, the Prx have emerged as new molecules in antioxidant defense in plants. Here, the members which belong to Prx gene family in Arabidopsis and rice are been identified. Overall, the Prx members constitute a small family with 10 and 11 genes in Arabidopsis and rice respectively. The prx genes from rice are assigned to their functional groups based on homology search against Arabidopsis protein database. Deciphering the Prx functions in rice will add novel information to the mechanism of antioxidant defense in plants. Further, the Prx also forms the part of redox signaling cascade. Here, the Prx gene family has been described for rice.Key words: antioxidant defense, chloroplast, gene family, oxidative stress, reactive oxygen speciesThe formation of free radicals and reactive oxygen species (ROS) occur in several enzymatic and non-enzymatic reactions during cellular metabolism. The accumulation of these reactive and deleterious intermediates is suppressed by antioxidant defense mechanism comprised of low molecular weight antioxidants and enzymes. In photosynthetic organisms, the defense against the damage from free radicals and oxidative stress is crucial. For instance, the ROS production occurs in photosystem II with generation of singlet oxygen (¹O₂) and hydrogen peroxide (H₂O₂),^1,2 photosystem I from superoxide anion radicals (O₂⁻),³ and during photorespiration with generation of H₂O₂.⁴ ROS production may exceed under environmental stress conditions like excess light, low temperature and drought.⁵The antioxidant defense mechanism is activated by antioxidant metabolities and enzymes which detoxify ROS and lipid peroxides. The detoxification of ROS can occur in various cellular compartments such as chloroplasts, mitochondria, peroxisomes and cytosol.⁶ The enzymes like ascorbate peroxidase, catalase, glutathione peroxidase and superoxide dismutase are prominent antioxidant enzymes.⁶ The peroxiredoxins (Prx) emerged as new components in the antioxidant defense network of barley.^7,8 Later, Prx were studied in other plants.^9–14Prx can be classified into four different functional groups, PrxQ, 1-Cys Prx, 2-Cys Prx and Type-2 Prx.^15,16 They are members of the thioredoxin fold superfamily.^17,18 In this study, the prx genes found in Arabidopsis and rice genomes are been identified. The Arabidopsis genome encodes 10 prx genes classified into four functional categories, 1-Cys Prx, 2-Cys Prx, PrxQ and Type-2 Prx.¹³ Of these, one each of 1-Cys Prx and PrxQ, two of 2-Cys Prx (2-Cys PrxA and 2-Cys PrxB) and six Type-2 Prx (PrxA–F) are identified¹³ (LocusAnnotationSynonymA^*B^*C^*AT1G481301-Cysteine peroxiredoxin 1 (ATPER1)1-Cys Prx21624081.36.603AT1G60740Peroxiredoxin type 2Type-2 PrxD16217471.95.2297AT1G65970Thioredoxin-dependent peroxidase 2 (TPX2)Type-2 PrxC16217413.95.2297AT1G65980Thioredoxin-dependent peroxidase 1 (TPX1)Type-2 PrxB16217427.84.9977AT1G65990Type 2 peroxiredoxin-relatedType-2 PrxA55362653.66.4368AT3G06050Peroxiredoxin IIF (PRXIIF)Type-2 PrxF20121445.29.3905AT3G116302-Cys Peroxiredoxin A (2CPA, 2-Cys PrxA)2-Cys PrxA26629091.77.5686AT3G26060ATPRX Q, periredoxin QPrxQ21623677.810.0565AT3G52960Peroxiredoxin type 2Type-2 PrxE23424684.09.572AT5G062902-Cysteine Peroxiredoxin B (2CPB, 2-Cys PrxB)2-Cys PrxB27329779.55.414Open in a separate window^*A, amino acids; B, molecular weight; C, isoelectric point.In rice (rice.plantbiology.msu.edu/), there are 11 genomic loci which encode for Prx proteins (​and33). Interestingly, a new prx gene (LOC_Os07g15670) annotated as “peroxiredoxin, putative, expressed” is identified making the tally of prx genes to eleven in rice as compared to ten in Arabidopsis (​and22). The BLAST search has identified its counterpart in Arabidopsis which has been annotated as “antioxidant/oxidoreductase” (AT1G21350) in the TAIR database (www.arabidopsis.org). The rice LOC_Os07g15670 and Arabidopsis AT1G21350 share protein homology %68/78 for 236 amino acids (ChromosomeLocus IdPutative function/AnnotationA^*B^*C^*1LOC_Os01g16152peroxiredoxin, putative, expressed19920873.68.22091LOC_Os01g24740peroxiredoxin-2E-1, chloroplast precursor, putative10711591.56.79061LOC_Os01g48420peroxiredoxin, putative, expressed16317290.85.68282LOC_Os02g09940peroxiredoxin, putative, expressed22623179.56.5352LOC_Os02g33450peroxiredoxin, putative, expressed26228096.95.77094LOC_Os04g339702-Cys peroxiredoxin BAS1, chloroplast precursor, putative, expressed12213410.24.37056LOC_Os06g09610peroxiredoxin, putative, expressed2662892610.50976LOC_Os06g42000peroxiredoxin, putative, expressed23323688.39.20597LOC_Os07g15670peroxiredoxin, putative, expressed25327684.69.85457LOC_Os07g44440peroxiredoxin, putative, expressed22124232.65.36187LOC_Os07g44430peroxiredoxin, putative25627785.36.8544Open in a separate window^*A, amino acids; B, molecular weight; C, isoelectric point.

Table 3

Identification of rice homologs of peroxiredoxins in A. thaliana

Stress-induced flowering

Many plant species can be induced to flower by responding to stress factors. The short-day plants Pharbitis nil and Perilla frutescens var. crispa flower under long days in response to the stress of poor nutrition or low-intensity light. Grafting experiments using two varieties of P. nil revealed that a transmissible flowering stimulus is involved in stress-induced flowering. The P. nil and P. frutescens plants that were induced to flower by stress reached anthesis, fruited and produced seeds. These seeds germinated, and the progeny of the stressed plants developed normally. Phenylalanine ammonialyase inhibitors inhibited this stress-induced flowering, and the inhibition was overcome by salicylic acid (SA), suggesting that there is an involvement of SA in stress-induced flowering. PnFT2, a P. nil ortholog of the flowering gene FLOWERING LOCUS T (FT) of Arabidopsis thaliana, was expressed when the P. nil plants were induced to flower under poor-nutrition stress conditions, but expression of PnFT1, another ortholog of FT, was not induced, suggesting that PnFT2 is involved in stress-induced flowering.Key words: flowering, stress, phenylalanine ammonia-lyase, salicylic acid, FLOWERING LOCUS T, Pharbitis nil, Perilla frutescensFlowering in many plant species is regulated by environmental factors, such as night-length in photoperiodic flowering and temperature in vernalization. On the other hand, a short-day (SD) plant such as Pharbitis nil (synonym Ipomoea nil) can be induced to flower under long days (LD) when grown under poor-nutrition, low-temperature or high-intensity light conditions.^1–9 The flowering induced by these conditions is accompanied by an increase in phenylalanine ammonia-lyase (PAL) activity.¹⁰ Taken together, these facts suggest that the flowering induced by these conditions might be regulated by a common mechanism. Poor nutrition, low temperature and high-intensity light can be regarded as stress factors, and PAL activity increases under these stress conditions.¹¹ Accordingly, we assumed that such LD flowering in P. nil might be induced by stress. Non-photoperiodic flowering has also been sporadically reported in several plant species other than P. nil, and a review of these studies suggested that most of the factors responsible for flowering could be regarded as stress. Some examples of these factors are summarized in 12^–14

Table 1

Some cases of stress-induced flowering