Jay S. Hanas

Biomarker identification in human pancreatic cancer sera

Objective: The aim of this study is to identify biomarkers in sera of pancreatic cancer patients using mass spectrometry (MS) approaches. Methods: Sera from patients diagnosed with pancreatic adenocarcinoma and sera from normal volunteers were subjected to gel electrophoresis to resolve and quantify differences in protein levels. Protein bands that differed quantitatively were digested with trypsin, and peptides were identified by electrospray ionization (ESI) ıon-trap tandem MS. Mass spectra were also collected directly from pancreatic cancer sera as well as healthy control sera using ESI-MS. Results: Three large-mass proteins were found to be elevated in pancreatic cancer sera versus normal sera, &agr;-2 macroglobulin, ceruloplasmin, and complement 3C. Complement 3C is a major regulator of inflammatory responses. The ESI-MS of human pancreatic cancer sera versus normal sera revealed greater heterogeneity in cancer sera than control sera, especially in the low-mass region. Bootstrapping statistical analysis identified 20 low-mass serum peaks that correlated with control sera and 20 different peaks that correlated with pancreatic cancer sera. Conclusions: The fact that inflammation-sensitive proteins were identified as increased in pancreatic cancer sera supports the hypothesis that inflammatory-driven processes are involved in pancreatic carcinogenesis. Liquid ESI-MS analyses of sera hold promise for future pancreatic cancer blood tests as well as for understanding mechanisms of pancreatic carcinogenesis. The variability observed between the low-mass regions of normal versus pancreatic cancer spectra may aid in diagnosis and therapy.

Increased expression of alpha-1-antitrypsin, glutathione S-transferase π and vascular endothelial growth factor in human pancreatic adenocarcinoma

BACKGROUND This study was designed to investigate abnormalities in gene expression in ductal adenocarcinoma of the pancreas using cDNA arrays. METHODS Gene expression in pancreatic ductal adenocarcinoma was compared with normal pancreatic tissue controls. Specimens from 5 patients with pancreatic adenocarcinoma were taken fresh at operation and analyzed using commercially prepared cDNA arrays evaluating approximately 2,000 genes. Immunohistochemical staining was used to confirm protein expression of selected genes. RESULTS Alpha-1-antitrypsin (A1AT) and glutathione S-transferase pi (GSTP) were significantly up-regulated in all 5 tumors. Vascular endothelial growth factor (VEGF) was up-regulated in 4 of the 5 patients. Immunohistochemical staining verified the overexpression of each of these genes. CONCLUSIONS A1AT, GSTP, and VEGF are overexpressed in human pancreatic adenocarcinoma specimens taken fresh at operation. To our knowledge, this is the first study of human pancreatic ductal adenocarcinoma demonstrating the up-regulation of these genes using gene expression arrays.

Induction of cardiovascular pathology in a novel model of low-grade chronic inflammation

OBJECTIVE Epidemiological and clinical evidence indicate that inflammatory processes play a pivotal role in a number of conditions associated with aging, including osteoporosis and cardiovascular diseases. The purpose of this study was to evaluate cardiovascular pathology and select inflammatory mediators of interest in a model of low-grade inflammation-induced osteopenia. METHODS Slow-release pellets were subcutaneously implanted in male rats to deliver 0, 3.3, or 33.3 microg of lipopolysaccharide (LPS)/day for 90 days. Tail blood was collected at 1, 2, and 3 months for differential white cell counts, and at the end of the study, hearts were harvested for histological and immunohistochemical evaluation. RESULTS The low-grade inflammatory response was characterized by elevated peripheral blood neutrophils and monocytes. Histological examination of heart cross sections revealed increased fibrous tissue, infiltration of lymphocytes, accumulation of mast cells, and roughened intimal borders within the arteries and arterioles, consistent with vascular disease. Inflammatory mediators (cyclooxygenase-2, tumor necrosis factor-alpha, and interleukin-1 beta) were up-regulated, and increased expression of platelet endothelial cell adhesion molecule-1 and receptor activator for NF-kappaB ligand was localized to the microvasculature endothelium. CONCLUSIONS These findings suggest that inflammation induced by chronic exposure to LPS produces cardiovascular pathology in the smaller intramural arteries and arterioles and support the utility of this model for further mechanistic and therapeutic studies focused on the role of chronic inflammation in cardiovascular disease.

Genes for murine Y1 and Y3 Ro RNAs have Class 3 RNA polymerase III promoter structures and are unlinked on mouse chromosome 6

Murine YRNAs, which are components of the conserved Ro ribonucleoprotein (RNP) complex, have been identified by enzymatic RNA sequencing. Mouse Y1 (mY1) and Y3 (mY3; originally named mY2) RNAs share 97 and 95% identity to the human Y1 and Y3 RNAs, respectively. TATA-like sequences, Proximal Sequence Elements, and octamer sequences, which are upstream promoter element motifs indicative of Class 3 RNA Polymerase III (RNAPIII) transcribed genes, are found upstream of both the putative mY1 and mY3 coding regions. Further, these elements are strikingly conserved both in sequence and position relative to known Class 3 genes and to human YRNA genes. Inhibition of transcription in vitro by 200 micrograms/ml but not 1 microgram/ml of alpha-amanitin indicates transcription of the mouse YRNA genes by RNAPIII. Southern blot of C57BL/6J and Mus spretus murine genomic DNA with mY1 and mY3 gene-specific probes suggests that these genes are single copy in the mouse genome. Finally, gene mapping with a (C57BL/6J x SPRET/Ei)F1 x SPRET/Ei mouse interspecific backcross DNA panel localizes the mY1 gene to the distal end of mouse chromosome 6, close to the motheaten (me) autoimmunity locus. The mY3 gene maps to the proximal end of mouse chromosome 6 very close to the T cell receptor beta locus, in a region homologous to human chromosome 7 where the human YRNA genes have been mapped.

DNA Ploidy and Markovian Analysis of Neoplastic Progression in Experimental Pancreatic Cancer

Computer-assisted analysis of DNA ploidy and nuclear morphology were used to elucidate changes in the cell nucleus that occur during the development of experimental pancreatic cancer. Ductal pancreatic adenocarcinoma was induced in 49 Syrian hamsters by SC injection of N-nitrosobis (2-oxopropyl) amine; twenty hamsters served as controls. Groups of animals were sacrificed every 4 weeks for 20 weeks and adjacent sections of pancreatic tissue were H&E and Feulgen-stained for light microscopy and computer assisted cytometry. Pancreatic ductal cells were classified as normal, atypical, or malignant; tissue inflammation (pancreatitis) was also noted when present. DNA ploidy and nuclear morphology evaluation (Markovian analysis) identified an atypical cell stage clearly distinguishable from either normal or malignant cells; pancreatitis preceded this atypia. The DNA ploidy histogram of these atypical cells revealed a major diploid peak and a minor aneuploid peak. The receiver operator characteristic curve areas for a logistic regression model of normal vs atypical cells was 0.94 and for atypical vs malignant was 0.98, numbers indicative of near-perfect discrimination among these three cell types. The ability to identify an atypical cell population should be useful in establishing the role of these cells in the progression of human pancreatic adenocarcinoma.

Structure, function, evolution of transcription factor IIIA

Publisher Summary This chapter discusses the structure, function, and evolution of transcription factor (TF) IIIA. The large amount of cysteine in TFIIIA prompted an examination of the possible roles of metals in TFIIIA structure and function because cysteine is a metal chelator and not usually found in such large amounts in intracellular proteins. The zinc present in TFIIIA is loosely bound, as evidenced by complete chelation under very mild conditions. The primary structure of TFIIIA can be modeled into nine zinc binding domains, termed as “zinc finger,” in which a zinc ion is coordinated between two cysteines and two histidines. Finger region is the DNA-binding entity of the protein. TFIIIA has been initially shown to be required for the transcription of 5-S genes in cell extracts. In addition, TFIIIA promotes RNA polymerase-III-dependent transcription of 5-S genes by binding to the internal control region. In trying to understand the role of TFIIIA in the transcription of 5-S RNA genes, it is probably helpful to understand the way factors IIIC and IIIB and RNA polymerase III interact and promote the transcription of both tRNA and 5-S RKA genes. Moreover, the cloning and sequencing of TFIIIA cDNAs from other amphibian species have revealed that TFIIIA is not as highly conserved at the amino-acid level as are other DNA-binding proteins.

Mechanism of action of RNA polymerase II elongation factor elongin. Maximal stimulation of elongation requires conversion of the early elongation complex to an elongin-activable form

We previously identified and purified Elongin by its ability to stimulate the rate of elongation by RNA polymerase IIin vitro (Bradsher, J. N., Jackson, K. W., Conaway, R. C., and Conaway, J. W. (1993) J. Biol. Chem. 268, 25587–25593). In this report, we present evidence that stimulation of elongation by Elongin requires that the early RNA polymerase II elongation complex undergoes conversion to an Elongin-activable form. We observe (i) that Elongin does not detectably stimulate the rate of promoter-specific transcription initiation by the fully assembled preinitiation complex and (ii) that early RNA polymerase II elongation intermediates first become susceptible to stimulation by Elongin after synthesizing 8–9-nucleotide-long transcripts. Furthermore, we show that the relative inability of Elongin to stimulate elongation by early elongation intermediates correlates not with the lengths of their associated transcripts but, instead, with the presence of transcription factor IIF (TFIIF) in transcription reactions. By exploiting adenovirus 2 major late promoter derivatives that contain premelted transcriptional start sites and do not require TFIIF, TFIIE, or TFIIH for transcription initiation, we observe (i) that Elongin is capable of strongly stimulating the rate of synthesis of trinucleotide transcripts by a subcomplex of RNA polymerase II, TBP, and TFIIB and (ii) that the ability of Elongin to stimulate synthesis of these short transcripts is substantially reduced by addition of TFIIF to transcription reactions. Here we present these findings, which are consistent with the model that maximal stimulation of elongation by Elongin requires that early elongation intermediates undergo a structural transition that includes loss of TFIIF.

Distinguishing early-stage pancreatic cancer patients from disease-free individuals using serum profiling

This study evaluated the usefulness of electrospray mass spectrometry to distinguish sera of early-stage pancreatic cancer patients from disease-free individuals. Sera peak data were generated from 33 pancreatic cancer patients and 30 disease-free individuals. A “leave one out” cross-validation procedure discriminated stage I/II pancreatic cancer versus disease-free sera with a p value <.001 and a receiver–operator characteristic curve area value of 0.85. Predictive values for cancer stage I/II test efficiency, specificity, and sensitivity were 78%, 77%, and 79%, respectively. These studies indicate that electrospray mass spectrometry is useful for distinguishing sera of early-stage pancreatic cancer patients from disease-free individuals.

Mass profiling of serum to distinguish mice with pancreatic cancer induced by a transgenic Kras mutation

Mass spectrometry (MS) has the unique ability to profile, in an easily accessible body tissue (peripheral blood/serum,) the sizes and relative amounts of a wide variety of biomolecules in a single platform setting. Using electrospray ionization (ESI)‐MS, we distinguished individual serum from wild‐type control mice from serum of mice containing an oncogenic Kras mutation, which leads to development of pancreatic ductal adenocarcinoma (PDAC) similar to that observed in humans. Identification of differences in significant ESI‐MS sera mass peaks between Kras‐activated mice and control mice was performed using t tests and a “nested leave one out” cross‐validation procedure. Peak distributions in serum of control mice from mice with Kras‐mutant‐dependent PDAC were distinguished from those of pancreatic intraepithelial neoplasia (PanIN) lesions (p = 0.00024). In addition, Kras mutant mice with PDAC were distinguished from Kras mutant mice with PanIN alone (p = 0.0057). Test specificity, a measure of the false positives, was greater for the control vs. Kras mutated mice, and the test sensitivity, a measure of false negatives, was greater for the PDAC vs. PanIN containing mice. Receiver‐operating characteristic (ROC) curve discriminatory values were 0.85 for both comparisons. These studies indicate ESI‐MS serum mass profiling can detect physiological changes associated with pancreatic cancer initiation and development in a GEM (genetic engineered mouse) model that mimics pancreatic cancer development in humans. Such technology has the potential to aid in early detection of pancreatic cancer and in developing therapeutic drug interventions.

Serum profiling to distinguish early- and late-stage ovarian cancer patients from disease-free individuals

Sera mass spectrometry (MS) peak differences were analyzed from 35 ovarian cancer patients and 16 disease-free individuals. “Leave one out” cross validation was used to assign “% cancer peaks” in control and ovarian cancer sera samples. Sera MS discriminated stage I/II and stage III/V ovarian cancer patients versus controls with ROC curve area values of 0.82 and 0.92. Test sensitivities for ovarian cancer stage I/II and III/V were 80% and 93% respectively. These results indicate that MS is useful for distinguishing sera from early-stage ovarian cancer patients, and has potential as a test for early detection of this disease.