James R. Hocker

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.

Mercuric ion inhibition of eukaryotic transcription factor binding to DNA.

Mercury has harmful effects in both rodents and humans. In rodent tissue culture cells exposed to HgCl(2), the metal ions were observed to concentrate in cell nuclei and to associate with chromatin. Thus, transcription factors and other proteins associated with chromatin are possible targets of mercuric ion toxicity. In this study, mercuric ions were found to inhibit the DNA binding activity of the Cys(2)His(2) zinc finger proteins transcription factor IIIA (TFIIIA) and Sp1. These factors are prototypes of the largest eukaryotic protein superfamily. Neither the presence of excess zinc ions nor beta-mercaptoethanol prevented inhibition by mercuric ions. Mercuric ions also inhibited DNA binding by the non-zinc finger protein AP2. Zinc finger-DNA binding was inhibited when both TFIIIA/5S RNA complex and TFIIIA alone were preincubated with concentrations as low as 15 microM mercuric ion. Inhibition occurred in less than 1 min and was not readily reversible. Mercuric ions also inhibited the digestion of DNA by the restriction enzymes BamHI or EcoRI. Inhibition of transcription factors as well as potentially other DNA binding proteins by micromolar concentrations of mercuric ion suggests additional biochemical mechanisms for mercury toxicity in promoting disease via alterations in gene transcription patterns.

Mechanisms of inhibition of zinc-finger transcription factors by selenium compounds ebselen and selenite.

The anti-inflammatory selenium compounds, ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one) and selenite, were found to alter the DNA binding mechanisms and structures of cysteine-rich zinc-finger transcription factors. As assayed by DNase I protection, DNA binding by TFIIIA (transcription factor IIIA, prototypical Cys(2)His(2) zinc finger protein), was inhibited by micromolar amounts of ebselen. In a gel shift assay, ebselen inhibited the Cys(2)His(2) zinc finger-containing DNA binding domain (DBD) of the NF-kappaB mediated transcription factor Sp1. Ebselen also inhibited DNA binding by the p50 subunit of the pro-inflammatory Cys-containing NF-kappaB transcription factor. Electrospray ionization mass spectrometry (ESI-MS) was utilized to elucidate mechanisms of chemical interaction between ebselen and a zinc-bound Cys(2)His(2) zinc finger polypeptide modeled after the third finger of Sp1 (Sp1-3). Exposing Sp1-3 to micromolar amounts of ebselen resulted in Zn(2+) release from this peptide and the formation of a disulfide bond by oxidation of zinc finger SH groups, the likely mechanism for DNA binding inhibition. Selenite was shown by ESI-MS to also eject zinc from Sp1-3 as well as induce disulfide bond formation through SH oxidation. The selenite-dependent inhibition/oxidation mechanism differed from that of ebselen by inducing the formation of a stable selenotrisulfide bond. Selenite-induced selenotrisulfide formation was dependent upon the structure of the Cys(2)His(2) zinc finger as alteration in the finger structure enhanced this reaction as well as selenite-dependent zinc release. Ebselen and selenite-dependent inhibition/oxidation of Cys-rich zinc finger proteins, with concomitant release of zinc and finger structural changes, points to mechanisms at the atomic and protein level for selenium-induced alterations in Cys-rich proteins, and possible amelioration of certain inflammatory, neurodegenerative, and oncogenic responses.

Doublecortin-like kinase 1 is elevated serologically in pancreatic ductal adenocarcinoma and widely expressed on circulating tumor cells.

Doublecortin-like kinase 1 (DCLK1) is a putative pancreatic stem cell marker and is upregulated in pancreatic cancer, colorectal cancer, and many other solid tumors. It marks tumor stem cells in mouse models of intestinal neoplasia. Here we sought to determine whether DCLK1 protein can be detected in the bloodstream and if its levels in archived serum samples could be quantitatively assessed in pancreatic cancer patients. DCLK1 specific ELISA, western blotting, and immunohistochemical analyses were used to determine expression levels in the serum and staining intensity in archived tumor tissues of pancreatic ductal adenocarcinoma (PDAC) patients and in pancreatic cancer mouse models. DCLK1 levels in the serum were elevated in early stages of PDAC (stages I and II) compared to healthy volunteers (normal controls). No differences were observed between stages III/IV and normal controls. In resected surgical tissues, DCLK1 expression intensity in the stromal cells was significantly higher than that observed in tumor epithelial cells. Circulating tumor cells were isolated from KPCY mice and approximately 52% of these cells were positive for Dclk1 staining. Dclk1 levels in the serum of KPC mice were also elevated. We have previously demonstrated that DCLK1 plays a potential role in regulating epithelial mesenchymal transition (EMT). Given the increasingly recognized role of EMT derived stem cells in cancer progression and metastasis, we hypothesize that DCLK1 may contribute to the metastatic process. Taken together, our results suggest that DCLK1 serum levels and DCLK1 positive circulating tumor cells should be further assessed for their potential diagnostic and prognostic significance.

Inhibition of zinc finger protein-DNA interactions by sodium selenite.

Sodium selenite and sodium selenate were analyzed for their ability to alter the DNA binding mechanisms of the Cys(2)His(2) zinc finger proteins, transcription factor IIIA (TFIIIA) and Sp1. TFIIIA is a positive regulator of 5S ribosomal RNA synthesis, and Sp1 is involved in cell proliferation and invasiveness. As assayed by DNase I protection, the interaction of the DNA binding domain of TFIIIA with the 5S ribosomal gene was inhibited by 25 microM selenite ions but not by 250 microM selenate ions. Selenite inhibition kinetics of TFIIIA progressed to completion in about 5 min. Preincubation of free TFIIIA with selenite resulted in DNA binding inhibition, whereas preincubation of a TFIIIA/5S RNA complex with selenite did not. Since 5S RNA binds to the TFIIIA DNA binding domain, this result is consistent with an inhibition mechanism via selenite binding to that region of this protein. Inhibition was not readily reversible and occurred in the presence of an excess of beta-mercaptoethanol; elevated amounts of dithiothreitol mitigated the inhibitory effect. Significantly less selenite (2.5-5 microM) inhibited the specific DNA binding of transcription factor Sp1 to the simian virus 40 (SV40) early promoter/enhancer. The selenite inhibition kinetics of Sp1 were fast, going to completion in about 1 min. SV40 DNA binding by the non-zinc finger transcription factor AP-2 was not inhibited by selenite. Inhibition of Cys(2)His(2) zinc finger proteins by micromolar amounts of selenite points to additional mechanisms for selenite-induced diminution of cell growth and anticancer activity.

cDNA cloning, DNA binding, and evolution of mammalian transcription factor IIIA.

cDNA for rat transcription factor IIIA (TFIIIA) was cloned by degenerate PCR and rapid amplification of cDNA ends. This cDNA coded for a protein with nine Cys(2)His(2) zinc fingers and a non-finger C-terminal tail; 63% amino acid (aa) sequence identity was observed with the Xenopus TFIIIA zinc finger region. Recombinant rat protein containing only the nine fingers afforded DNase I protection of the identical nucleotides protected by Xenopus laevis native TFIIIA on the Xenopus 5S RNA gene internal control region. A putative mouse TFIIIA clone was identified in an expressed sequence tag database by sequence similarity to rat TFIIIA. Recombinant nine-finger protein from this clone afforded DNase I protection of the Xenopus 5S rRNA gene like the native frog protein as did a recombinant nine-finger form of a putative human TFIIIA clone. These DNA binding results demonstrate that these clones code for the respective mammalian TFIIIAs. Rodent and human TFIIIAs share about 87% aa sequence identity in their zinc finger regions and have evolved to about the same extent as X. laevis and Xenopus borealis TFIIIAs. A monoclonal antibody against human p53 tumor suppressor bound to rat and mouse TFIIIA but not to human TFIIIA in Western blots. The N-terminal regions of rodent and human TFIIIA do not contain the oocyte-specific initiating Met and accompanying conserved residues found in fish and amphibian TFIIIAs. In their non-finger C-terminal tails, mammalian and amphibian TFIIIAs share a conserved transcription activation domain as well as conserved nuclear localization and nuclear export signals.

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.

Zinc Finger Interactions with Metals and Other Small Molecules

Zinc fingers encompass a wide variety of compact protein domains that are stabilized by a structural zinc ion which minimally interacts with a cysteine-rich coordination sphere. The selectivity for zinc ion binding is governed by coordinating amino acid side chains and by thermodynamic parameters. Since metal coordination spheres in zinc finger proteins are susceptible to chemical attack (principally at thiolates) and because zinc finger proteins have prominent roles in many cellular processes including the regulation of gene expression and signal transduction, an underlying mechanism for a number of cellular dysfunctions is likely to be the disruption of zinc coordination spheres by a variety of metals and other small molecules. For instance, a number of toxicity mechanisms are likely to be the consequence of zinc replacement by xenobiotic metals resulting in changes in polypeptide conformation and the concomitant loss of protein function. Zinc finger disruption could also occur by oxidation and modification of critical cysteine and histidine amino acids in the zinc coordination sphere resulting in zinc release and alteration of conformation. The chemical reactivity of metal coordination spheres of zinc finger proteins are utilized in normal physiological processes by providing regulatory sites for signal transduction via small molecules like nitric oxide and oxygen and their reactive intermediates. In addition, zinc finger proteins and their metal binding sites are promising targets for specific drug design to help ameliorate major diseases.

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.