Investigation of the physiological response of radiation-induced cystitis patients using hyperbaric oxygen.

Introduction: In this pilot study we have taken a novel functional approach to assess whether differences exist in the activity of key genes involved in the response to radiation and oxidative stress between patients with radiation cystitis. Materials and methods: Arm 1 consisted of patients who had previously been treated for prostate cancer and who had received definitive radiation treatment and had subsequently developed cystitis and/or proctitis and were being treated by hyperbaric oxygen (HBO). Arm 2 consisted of patients who had never been treated by radiation but who were scheduled for HBO treatment for another pathology. The genes chosen for the study were HMOX1, NOS2, SOD2, TNFα, IL-6 and TGFß. Blood and urine was collected pre and post HBO treatment. Results: Gene expression showed a significant difference in NOS2 (p = 0.0178) and TNFα (p = 0.037) between the control and cystitis patients. The plasma levels of VEGF-A were significantly elevated in cystitis patients and there was a strong trend for significant overexpression in urine. Comparing pre and post-dive samples showed little difference in both groups of patients except for VEGF-A which was reduced after the dive in plasma from cystitis patients. Conclusions: This study uncovered some physiological differences in patients with radiation-induced cystitis using HBO treatment as a stimulus to induce mild oxidative stress. Further research is ongoing to assess whether the acute exposure to HBO might be a physiological screening tool to identify patients susceptible to chronic radiation toxicity.

Association of human papillomavirus integration with better patient outcomes in oropharyngeal squamous cell carcinoma.

BACKGROUND: The molecular drivers of human papillomavirus-related head and neck squamous cell carcinoma (HPV + HNSCC) are not entirely understood. This study evaluated the relationship between HPV integration, expression of E6/E7, and patient outcomes in p16+ HNSCCs. METHODS: HPV type was determined by HPV PCR-MassArray, and integration was called using detection of integrated papillomavirus sequences polymerase chain reaction (PCR). We investigated whether fusion transcripts were produced by reverse transcriptase polymerase chain reaction (RT-PCR). E6/E7 expression was assessed by quantitative RT-PCR. We assessed if there was a relationship between integration and E6/E7 expression, clinical variables, or patient outcomes. RESULTS: Most samples demonstrated HPV integration, which sometimes resulted in a fusion transcript. HPV integration was positively correlated with age at diagnosis and E6/E7 expression. There was a significant difference in survival between patients with vs without integration. CONCLUSIONS: Contrary to previous reports, HPV integration was associated with improved patient survival. Therefore, HPV integration may act as a molecular marker of good prognosis.

Global Signaling Profiling in a Human Model of Tumorigenic Progression Indicates a Role for Alternative RNA Splicing in Cellular Reprogramming.

Intracellular signaling is controlled to a large extent by the phosphorylation status of proteins. To determine how human breast cells can be reprogrammed during tumorigenic progression, we profiled cell lines in the MCF10A lineage by phosphoproteomic analyses. A large cluster of proteins involved in RNA splicing were hypophosphorylated as cells progressed to a hyperplastic state, and then hyperphosphorylated after progression to a fully metastatic phenotype. A comprehensive transcriptomic approach was used to determine whether alterations in splicing factor phosphorylation status would be reflected in changes in mRNA splicing. Results indicated that the degree of mRNA splicing trended with the degree of tumorigenicity of the 4 cell lines tested. That is, highly metastatic cell cultures had the greatest number of genes with splice variants, and these genes had greater fluctuations in expression intensities. Genes with high splicing indices were mapped against gene ontology terms to determine whether they have known roles in cancer. This group showed highly significant associations for angiogenesis, cytokine-mediated signaling, cell migration, programmed cell death and epithelial cell differentiation. In summary, data from global profiling of a human model of breast cancer development suggest that therapeutics should be developed which target signaling pathways that regulate RNA splicing.

Characterization of clear cell renal cell carcinoma by gene expression profiling.

OBJECTIVES: Use global gene expression to characterize differences between high-grade and low-grade clear cell renal cell carcinoma (ccRCC) compared with normal and benign renal tissue. METHODS: Tissue samples were collected from patients undergoing surgical resection for ccRCC. Affymetrix gene expression arrays were used to examine global gene expression patterns in high- (n = 16) and low-grade ccRCC (n = 13) as well as in samples from normal kidney (n =14) and benign kidney disease (n = 6). Differential gene expression was determined by analysis of variance with a false discovery rate of 1% and a 2-fold cutoff. RESULTS: Comparing high-grade ccRCC with each of normal and benign kidney resulted in 1,833 and 2,208 differentially expressed genes, respectively. Of these, 930 were differentially expressed in both comparisons. In order to identify genes most related to progression of ccRCC, these differentially expressed genes were filtered to identify genes that showed a pattern of expression with a magnitude of change greater in high-grade ccRCC in the comparison to low-grade ccRCC. This resulted in the identification of genes such as TMEM45A, ceruloplasmin, and E-cadherin that were involved in cell processes of cell differentiation and response to hypoxia. Additionally changes in HIF1α and TNF signaling are highly represented by changes between high- and low-grade ccRCC. CONCLUSIONS: Gene expression differences between high-grade and low-grade ccRCC may prove to be valuable biomarkers for advanced ccRCC. In addition, altered signaling between grades of ccRCC may provide important insight into the biology driving the progression of ccRCC and potential targets for therapy.

Gene Expression Characterization of HPV Positive Head and Neck Cancer to Predict Response to Chemoradiation.

Human papillomavirus (HPV) has been shown to have a causal role in the development of head and neck squamous cell carcinoma. While HPV-positive head and neck cancer is associated with a better response to treatment in the majority of patients, there is a subset who does not respond favorably to current therapy. Identification of these patients could prevent unnecessary morbidity and indicate the need for alternative therapeutic options. Tissue samples were obtained from 19 patients with HPV-positive head and neck squamous carcinoma treated with chemoradiation therapy. HPV status was confirmed by polymerase chain reaction analysis through detection of HPV16 E7 in both DNA and RNA. RNA was isolated from tissue samples and subjected to microarray gene expression analysis. In addition to identification of potential genetic biomarkers (including LCE3D, KRTDAP, HMOX1, KRT19, MDK, TSPAN1), differentially expressed genes associated with genomic stability, cell cycle, and DNA damage were detected between responders and non-responders. These results were further validated with publicly available gene expression studies. This pilot study suggests prospective biomarkers that predict response to therapy. The importance of genes involved with genomic stability is highlighted in both development and progression of head and neck squamous cell carcinoma but also recurrence. Potential development of an assay may prove beneficial to clinicians, assisting them to provide alternative care sooner thus lowering morbidity.

Gene expression differences predict treatment outcome of merkel cell carcinoma patients.

Due to the rarity of Merkel cell carcinoma (MCC), prospective clinical trials have not been practical. This study aimed to identify biomarkers with prognostic significance. While sixty-two patients were identified who were treated for MCC at our institution, only seventeen patients had adequate formalin-fixed paraffin-embedded archival tissue and followup to be included in the study. Patients were stratified into good, moderate, or poor prognosis. Laser capture microdissection was used to isolate tumor cells for subsequent RNA isolation and gene expression analysis with Affymetrix GeneChip Human Exon 1.0 ST arrays. Among the 191 genes demonstrating significant differential expression between prognostic groups, keratin 20 and neurofilament protein have previously been identified in studies of MCC and were significantly upregulated in tumors from patients with a poor prognosis. Immunohistochemistry further established that keratin 20 was overexpressed in the poor prognosis tumors. In addition, novel genes of interest such as phospholipase A2 group X, kinesin family member 3A, tumor protein D52, mucin 1, and KIT were upregulated in specimens from patients with poor prognosis. Our pilot study identified several gene expression differences which could be used in the future as prognostic biomarkers in MCC patients.

Urine kidney injury molecule-1: a potential non-invasive biomarker for patients with renal cell carcinoma.

BACKGROUND: KIM-1 staining is upregulated in proximal tubule-derived renal cell carcinoma (RCC) including clear renal cell carcinoma and papillary renal cell carcinoma, but not in chromophobe RCC (distal tubular tumor). This study was designed to prospectively examine urine KIM-1 level before and 1 month after removal of renal tumors. PATIENTS AND DESIGN: A total of 19 patients were eventually enrolled in the study based on pre-operative imaging studies. Pre-operative and follow-up (1 month) urine KIM-1 levels were measured. The urine KIM-1 levels (uKIM-1) were then normalized to urine creatinine levels (uCr). Renal tumors were also stained for KIM-1 using immunohistochemical techniques. RESULTS: The KIM-1-negative staining group included 7 cases, and the KIM-1-positive group consisted of 12 cases. The percentage of KIM-1-positive staining RCC cells ranged from 10 to 100 %, and the staining intensity ranged from 1+ to 3+. In both groups, serum creatinine levels were both significantly elevated after nephrectomy. In the KIM-1-negative group, uKIM-1/uCr remained at a similar level before (0.37 ± 0.1 ng/mg Cr) and after nephrectomy (0.32 ± 0.01 ng/mg Cr). However, in the KIM-1-positive group, elevated uKIM-1/uCr at 1.20 ± 0.31 ng/mg Cr was significantly reduced to 0.36 ± 0.1 ng/mg Cr, which was similar to the pre-operative uKIM-1/uCr (0.37 ± 0.1 ng/mg Cr) in the KIM-1-negative group. CONCLUSION: Our small but prospective study showed significant reduction in uKIM-1/uCr after nephrectomy in the KIM-1 positive group, suggesting that urine KIM-1 may serve as a surrogate biomarker for kidney cancer and a non-invasive pre-operative measure to evaluate the malignant potential of renal masses.

SPIN: Development of sample-specific protein integrity numbers as an index of biospecimen quality.

It is widely accepted that variable biorepository specimen handling conditions can significantly alter outcomes of clinical research studies, suggesting the need for a metric for sample analyte protein integrity. In line with the National Cancer Institute (NCI) Best Practices, it is vital that the integrity of specimens used for biomarker studies are of the highest standard to ensure validity of the data they generate and confidence in the application of new findings to clinical management. We describe the creation of a program to discover proteins in biorepository samples that can be utilized to assess the integrity of stored specimens for protein-based biomarker studies, similar to the universally accepted quality metric for RNA, the RNA Integrity Number, or RIN. The study mimics potential variation in pre-analytical conditions which may result in proteolysis and other proteome-associated changes and employs surface-enhanced laser desorption time-of-flight mass spectrometry (SELDI-TOF MS) to assess changes in multiple proteins and peptides in a high-throughput manner. Candidate peaks from SELDI spectra of representative sample types (e.g., serum, urine, tissue extracts) which demonstrate differing but reproducible sensitivity to suboptimal processing and storage were selected and quantified in a series of specimens stored in the BioBank within the Beaumont Health System. We then assigned a relative index known here as Sample-specific Protein Integrity Number, or SPIN, which is derived from a ratio of nonstable vs. stable proteins for each sample type in the investigation. This methodology can be applied to every sample type and, once refined and established, the SPIN could be used by any biobank or laboratory using biobanked samples without specialized equipment and irrespective of the sample pre-analytical collection conditions.

Isolation and genomic characterization of stem cells in head and neck cancer.

BACKGROUND: This study investigated the use of 3 different established cell-sorting strategies to isolate and characterize stem cells from head and neck cancer cell lines. METHODS: Five low-passage cell lines were subjected to cell sorting based on Hoechst side population, Aldefluor, and CD44 expression. Isolated cell populations were studied for gene expression, radiosensitivity, and chemosensitivity to cisplatin and paclitaxel. RESULTS: Each sorting method identified a different set of genes associated with different gene ontology categories, with mitosis being the only common category. CD44-associated gene changes were almost exclusively associated with cell cycle and in particular mitosis. There were no significant differences in radiosensitivity or cisplatin sensitivity of stem or non-stem cells, but CD44-isolated stem cells were more resistant to paclitaxel. CONCLUSIONS: This study suggested that CD44 may be the most promising cell-sorting strategy to isolate and investigate the impact of stem cells in head and neck squamous cell cancer (HNSCC).

Gene expression changes associated with the progression of intraductal papillary mucinous neoplasms.

OBJECTIVES: The diagnosis of high-grade intraductal papillary mucinous neoplasm (IPMN) is difficult to distinguish from low-grade IPMN. The aim of this study was to identify potential markers for the discrimination of high-grade and invasive (HgInv) IPMN from low- and moderate-grade dysplasia IPMN. METHODS: Laser capture microdissection was used to isolate distinct foci of low-grade, moderate-grade, high-grade, and invasive IPMN from paraffin-embedded archival tissue from 14 patients who underwent resection for IPMN. Most samples included multiple grades in the same specimen. Affymetrix Human Exon microarrays were used to compare low- and moderate-grade dysplasia IPMN with HgInv IPMN. RESULTS: Sixty-two genes were identified as showing significant changes in expression (P ≤ 0.05 and a 2-fold cutoff), including up-regulation of 41 in HgInv IPMN. Changes in gene expression are associated with biological processes related to malignant behavior including cell motion, cell proliferation, response to hypoxia, and epithelial-to-mesenchymal transition. In addition, altered signaling in several transforming growth factor ß-related pathways was exhibited in the progression of IPMN to malignancy. CONCLUSIONS: This study identifies a set of genes associated with the progression of IPMN to malignancy. These genes are potential markers that could be used to identify IPMN requiring surgical resection.

Tryptophan hydroxylase 2 aggregates through disulfide cross-linking upon oxidation: possible link to serotonin deficits and non-motor symptoms in Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopamine neurons of the nigrostriatal system, resulting in severe motor disturbances. Although much less appreciated, non-motor symptoms are also very common in PD and many can be traced to serotonin neuronal deficits. Tryptophan hydroxylase (TPH) 2, the rate-limiting enzyme in the serotonin biosynthesis, is a phenotypic marker for serotonin neurons and is known to be extremely labile to oxidation. Therefore, the oxidative processes that prevail in PD could cause TPH2 misfolding and modify serotonin neuronal function much as is seen in dopamine neurons. Oxidation of TPH2 inhibits enzyme activity and leads to the formation of high molecular weight aggregates in a dithiothreitol-reversible manner. Cysteine-scanning mutagenesis shows that as long as a single cysteine residue (out of a total of 13 per monomer) remains in TPH2, it cross-links upon oxidation and only cysteine-less mutants are resistant to this effect. The effects of oxidants on TPH2 catalytic function and cross-linking are also observed in intact TPH2-expressing HEK293 cells. Oxidation shifts TPH2 from the soluble compartment into membrane fractions and large inclusion bodies. Sequential non-reducing/reducing 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting confirmed that TPH2 was one of a small number of cytosolic proteins that form disulfide-bonded aggregates. The propensity of TPH2 to misfold upon oxidation of its cysteine residues is responsible for its catalytic lability and may be related to loss of serotonin neuronal function in PD and the emergence of non-motor (psychiatric) symptoms.

Differential tissue distribution of tryptophan hydroxylase isoforms 1 and 2 as revealed with monospecific antibodies.

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of the neurotransmitter serotonin. Once thought to be a single-gene product, TPH is now known to exist in two isoforms-TPH1 is found in the pineal and gut, and TPH2 is selectively expressed in brain. Heretofore, probes used for localization of TPH protein or mRNA could not distinguish between the TPH isoforms because of extensive homology shared by them at the nucleotide and amino acid level. We have produced monospecific polyclonal antibodies against TPH1 and TPH2 using peptide antigens from nonoverlapping sequences in the respective proteins. These antibodies allow the differentiation of TPH1 and TPH2 upon immunoblotting, immunoprecipitation, and immunocytochemical staining of tissue sections from brain and gut. TPH1 and TPH2 antibodies do not cross-react with either tyrosine hydroxylase or phenylalanine hydroxylase. Analysis of mouse tissues confirms that TPH1 is the predominant form expressed in pineal gland and in P815 mastocytoma cells with a molecular weight of 51 kDa. TPH2 is the predominant enzyme form expressed in brain extracts from mesencephalic tegmentum, striatum, and hippocampus with a molecular weight of 56 kDa. Antibody specificity against TPH1 and TPH2 is retained across mouse, rat, rabbit, primate, and human tissues. Antibodies that distinguish between the isoforms of TPH will allow studies of the differential regulation of their expression in brain and periphery.

Mouse tryptophan hydroxylase isoform 2 and the role of proline 447 in enzyme function.

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of the neurotransmitter serotonin (5-HT). Once thought to be a single gene product, TPH is now known to exist in two isoforms. Isoform 1 (TPH1) is found in the pineal gland and gut, and isoform 2 (TPH2) is selectively expressed in brain. A single-nucleotide polymorphism in TPH2 results in a proline-to-arginine mutation at residue 447 and substantially lowers catalytic activity. In view of the importance of TPH in determining brain 5-HT function, we cloned TPH2 and produced the P447R mutant to assess the importance of this proline in enzyme function. Catalytically active TPH2 and the P447R mutant were expressed at the predicted subunit molecular mass of 56 kDa. The P447R mutant expressed less than 50% of the activity of TPH2. Mutation of this conserved proline in TPH1 (P403R) also resulted in an enzyme with significantly lower activity than the wild-type enzyme. The P447R mutant had a V(max) 50% lower than that of TPH2. The P447R mutation did not alter the oligomeric assembly of the protein, nor change its responsiveness to cysteine modification. The P447R mutation did not alter enzyme substrate specificity or stability, but conferred slightly enhanced sensitivity to inhibition by dopamine and diminished sensitivity to iron in catalysis. The conserved proline in TPH (residue 447 in TPH2 and 403 in TPH1) plays an important role in enzyme function by regulating V(max) of the catalytic reaction.

S-thiolation of tyrosine hydroxylase by reactive nitrogen species in the presence of cysteine or glutathione.

Tyrosine hydroxylase (TH) is the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine. Peroxynitrite (ONOO-) and nitrogen dioxide (NO2) inhibit TH catalytic function and cause nitration of protein tyrosine residues. Exposure of TH to either ONOO- or NO2 in the presence of cysteine (or glutathione) prevents tyrosine nitration and results in S-thiolation instead. TH catalytic activity is suppressed by S-thiolation. Dithiothreitol prevents and reverses the modification of TH by S-thiolation, and returns enzyme activity to control levels. S-Nitrosothiols, which are known to S-thiolate proteins, can be formed in the reaction of cysteine or glutathione with reactive nitrogen species. Therefore, S-nitrosoglutathione (GSNO) was tested for its ability to modify TH. Fresh solutions of GSNO did not modify TH, whereas decomposed GSNO resulted in extensive S-thiolation of the protein. Dimedone, a sulfenic acid trap, prevents S-thiolation of TH when included with GSNO during its decomposition. Taken together, these results show that TH is S-thiolated by ONOO- or NO2 in the presence of cysteine. S-Thiolation occurs at the expense of tyrosine nitration. Glutathione disulfide S-oxide, which forms spontaneously in the decomposition of GSNO and which is found in tissue undergoing oxidative stress, may be the species that S-thiolates TH.

Tetrahydrobiopterin prevents nitration of tyrosine hydroxylase by peroxynitrite and nitrogen dioxide.

Tyrosine hydroxylase (TH) is the initial and rate-limiting enzyme in the synthesis of the neurotransmitter dopamine. TH is inhibited and nitrated at tyrosine residues in vitro by the reactive nitrogen species peroxynitrite and nitrogen dioxide (NO2) and in vivo by drugs that damage dopamine neurons. Tetrahydrobiopterin, which is the essential cofactor for TH and is concentrated in dopamine neurons, completely blocks nitration of tyrosine residues in TH caused by peroxynitrite or NO2. Various tetrahydro- and dihydro-analogs of tetrahydrobiopterin, including 6,7-dimethyl-tetrahydropterin, 6-methyl-tetrahydropterin, 6-hydroxymethyl-tetrahydropterin, tetrahydropterin, 7,8-dihydrobiopterin, 7,8-dihydroxanthopterin, and sepiapterin, also prevent nitration of tyrosines caused by the reactive nitrogen species. Biopterin and pterin, the fully oxidized forms of the pterin molecule, fail to block peroxynitrite- or NO2-induced nitration of TH. Reduced pterins prevent neither the inhibition of TH activity nor cysteine modification caused by peroxynitrite or NO2, despite blocking tyrosine nitration. However, dithiothreitol prevents and reverses these effects on TH of tetrahydrobiopterin and reactive nitrogen species. Using an enhanced green fluorescent protein-TH fusion construct as a real-time reporter of intracellular tyrosine nitration, tetrahydrobiopterin was found to prevent NO2-induced tyrosine nitration in intact cells but to leave TH activity inhibited. These results indicate that tetrahydrobiopterin prevents the tyrosine-nitrating properties of peroxynitrite and NO2. Tetrahydrobiopterin-derived radical species formed by reaction with reactive nitrogen species may account for inhibition of TH via mechanisms that do not involve tyrosine nitration.

Dopamine prevents nitration of tyrosine hydroxylase by peroxynitrite and nitrogen dioxide: is nitrotyrosine formation an early step in dopamine neuronal damage?

Peroxynitrite and nitrogen dioxide (NO2) are reactive nitrogen species that have been implicated as causal factors in neurodegenerative conditions. Peroxynitrite-induced nitration of tyrosine residues in tyrosine hydroxylase (TH) may even be one of the earliest biochemical events associated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced damage to dopamine neurons. Exposure of TH to peroxynitrite or NO2 results in nitration of tyrosine residues and modification of cysteines in the enzyme as well as inactivation of catalytic activity. Dopamine (DA), its precursor 3,4-dihydroxyphenylalanine, and metabolite 3,4-dihydroxyphenylacetic acid completely block the nitrating effects of peroxynitrite and NO2 on TH but do not relieve the enzyme from inhibition. o-Quinones formed in the reaction of catechols with either peroxynitrite or NO2 react with cysteine residues in TH and inhibit catalytic function. Using direct, real-time evaluation of tyrosine nitration with a green fluorescent protein-TH fusion protein stably expressed in intact cells (also stably expressing the human DA transporter), DA was also found to prevent NO2-induced nitration while leaving TH activity inhibited. These results show that peroxynitrite and NO2 react with DA to form quinones at the expense of tyrosine nitration. Endogenous DA may therefore play an important role in determining how DA neurons are affected by reactive nitrogen species by shifting the balance of their effects away from tyrosine nitration and toward o-quinone formation.

Reduced nicotinamide nucleotides prevent nitration of tyrosine hydroxylase by peroxynitrite.

Tyrosine hydroxylase (TH) is the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine (DA). TH activity is inhibited by peroxynitrite (ONOO(-)) by a mechanism that involves nitration of tyrosine residues and oxidation of cysteine residues in the enzyme. Reduced forms of the nicotinamide adenine dinucleotide cofactors, NADH and NADPH, protect TH from inhibition by ONOO(-) and prevent nitration of tyrosine residues. NAD, the oxidized form of the cofactors, neither protects TH from ONOO(-)-induced inhibition nor prevents the nitration of tyrosine residues in the enzyme. These results suggest that the redox status of the nicotinamide nucleotide cofactors could influence the ability of ONOO(-) to modify proteins that are important to the function of DA neurons.