Biomedical subjects

Daniel J Brackett

Publications and source records attributed to Daniel J Brackett.

16 recordsLinked to original sources

PubMed2007

Expression profiling identifies microRNA signature in pancreatic cancer.

microRNAs are functional, 22 nt, noncoding RNAs that negatively regulate gene expression. Disturbance of microRNA expression may play a role in the initiation and progression of certain diseases. A microRNA expression signature has been identified that is associated with pancreatic cancer. This has been accomplished with the application of real-time PCR profiling of over 200 microRNA precursors on specimens of human pancreatic adenocarcinoma, paired benign tissue, normal pancreas, chronic pancreatitis and nine pancreatic cancer cell lines. Hierarchical clustering was able to distinguish tumor from normal pancreas, pancreatitis and cell lines. The PAM algorithm correctly classified 28 of 28 tumors, 6 of 6 normal pancreas and 11 of 15 adjacent benign tissues. One hundred microRNA precursors were aberrantly expressed in pancreatic cancer or desmoplasia (p < 0.01), including microRNAs previously reported as differentially expressed in other human cancers (miR-155, miR-21, miR-221 and miR-222) as well as those not previously reported in cancer (miR-376a and miR-301). Most of the top aberrantly expressed miRNAs displayed increased expression in the tumor. Expression of the active, mature microRNA was validated using a real-time PCR assay to quantify the mature microRNA and Northern blotting. Reverse transcription in situ PCR showed that three of the top differentially expressed miRNAs (miR-221, -376a and -301) were localized to tumor cells and not to stroma or normal acini or ducts. Aberrant microRNA expression may offer new clues to pancreatic tumorigenesis and may provide diagnostic biomarkers for pancreatic adenocarcinoma.

Adenocarcinoma↗

PubMed2005

Cocaine up-regulates Fra-2 and sigma-1 receptor gene and protein expression in brain regions involved in addiction and reward.

Sigma receptors have recently been implicated in the actions of cocaine, and antagonists of these receptors prevent many acute and subchronic cocaine effects. A previous study revealed that the immediate early gene fra-2 is up-regulated after cocaine administration, and this effect is prevented by the sigma-1 receptor antagonist BD1063 [1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine]. In the present study, the effects of cocaine and BD1063 on the expression of six fos and jun genes were evaluated in mouse brains using cDNA microarrays. Several of these genes were altered by cocaine, but only the alteration in fra-2 was prevented by BD1063. The time courses of fra-2 and sigma-1 receptor gene and protein expression in different brain regions were also determined. Cocaine up-regulated fra-2, which was followed by a later up-regulation of sigma-1 receptors. The cocaine-induced up-regulation of fra-2 and sigma-1 receptor genes and proteins were detected in whole brain, striatum, and cortex, but not in cerebellum. All of these cocaine-induced effects were prevented by BD1063. The interaction between cocaine, fra-2, and sigma-1 receptors involves brain regions that are established components of the neural circuit for reward, suggesting that they may contribute to the enduring changes that underlie the cellular basis of drug abuse.

Actins↗

PubMed2005

[1,2-13C2]-D-glucose profiles of the serum, liver, pancreas, and DMBA-induced pancreatic tumors of rats.

OBJECTIVES: In vitro stable isotope glucose tracer studies indicate that undifferentiated cells of the pancreas use glucose primarily through the nonoxidative reactions of the pentose cycle for nucleic acid ribose synthesis, whereas normal or less transformed cells primarily use the oxidative branch of the cycle. METHODS: The pancreatic heads of 4 groups (5/group) of male rats were implanted with time-release pellets designed to deliver placebo or 7,12-dimethylbenzanthracene (DMBA) at 11, 33, or 56 mg/d. Four weeks after pancreatic exposure to DMBA, [1,2-C2]-D-glucose tracer (1 g/kg) was injected intraperitoneally followed by sera collection at 1 and 2 hours and harvest of tumors, adjacent pancreatic tissue, and sera at 3 hours. RESULTS: Tumors (2-9 mm) were found across DMBA groups, with the largest in the high-dose group (> or =5 mm). Selective monitoring by gas chromatography-mass spectrometry of the doubly-labeled [1,2-C2]-D-ribose of RNA, which requires nonoxidative synthesis in the pentose cycle, showed a 2.8-, 2.9-, and 5.7-fold increase in pancreatic tumors. Liver and adjacent pancreas preferentially produced [1-C1]-D-ribose through the oxidative reactions of the cycle. Tumor-bearing animals also cleared and recycled tracer glucose at a faster rate. CONCLUSIONS: Simultaneous selective positional ion monitoring of C-labeled metabolites and their mass isotopomers in tissues and blood opens new avenues for the early detection and response to therapy testing of pancreatic cancer using GC-MS and/or magnetic resonance imaging-based methods. This study emphasizes the benefits of stable isotope-based dynamic metabolic profiling, when applied in vivo, and the several advantages it offers to positron emission tomography.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2005

Medicinal chemistry, metabolic profiling and drug target discovery: a role for metabolic profiling in reverse pharmacology and chemical genetics.

Comprehensive analysis of the metabolome can contribute to mechanism of action studies for small molecules discovered in phenotypic screens. Examples are presented in this overview of the rapidly developing field of "metabolic profiling." These examples include the use of NMR in gene function analysis, GC-based studies on the identification of metabolic pathways affected by PPAR-gamma agonists, applications of Fourier-transform MS and the use of stable isotope-based metabolic profiling (SIDMAP) to investigate metabolic adaptive changes induced by effective anticancer agents.

Animals↗

PubMed2005

Diverse gene expression pattern during 5-fluorouridine-induced apoptosis.

The purpose of this study was to establish experimental conditions to produce apoptosis by the fluorinated pyrimidine 5-fluorouridine and to examine the changes in gene expression that occurred during cell death. HCT-116 colorectal carcinoma cells were exposed to 10 microM 5-fluorouridine alone or in the presence of 1 mM uridine, 30 microM thymidine or both uridine and thymidine. A time-dependent increase in the percentage of apoptotic cells and a decrease in the percentage of viable cells were observed when the cells were treated with 5-fluorouridine in the absence of uridine (p < 0.001) but not in the presence of uridine. cDNA microarray analysis was used to study the expression of 1,200 different genes during apoptosis by 5-flurouridine. The expression of 33 genes was upregulated by 5-fold or greater at 16 and 24 h of 5-fluorouridine exposure. The largest cluster of upregulated genes included a group of genes classified as growth factors, cytokines and chemokines (e.g. interleukin-3, interleukin-4, B-cell growth factor 1 and stem cell growth factor). The expression of MIC-1 increased up to 100-fold during 5-flurouridine exposure. One hundred and twenty-four genes were downregulated by 5-fold or greater following exposure to 5-fluorouridine. The downregulated genes were distributed throughout the six different classifications on the array. Our data demonstrate a diverse pattern of gene expression during the fluorouridine-induced apoptosis and suggest that mechanisms besides a global inhibition of RNA synthesis/ processing contribute to the RNA-directed cytotoxicity of fluoropyrimidines.

Analysis of Variance↗

PubMed2004

Expression of prohibitin 3' untranslated region suppressor RNA alters morphology and inhibits motility of breast cancer cells.

The prohibitin 3' untranslated region (3'UTR) belongs to a novel class of non-coding regulatory RNAs. It arrests cell cycle progression by blocking G1-S transition in breast and other cancers. Our previous studies comparing MCF7 derived clones constitutively expressing a common allelic form of prohibitin RNA (UTR/C) to various controls demonstrated that it functions as a tumor suppressor. Here, we further characterized the morphology and motility of these transgenic breast cancer cells when grown in cell culture and on nude mice. In contrast to empty vector (EV) cells, UTR/C cells were observed to grow in an organized manner with more cell-cell contact and differentiate into structures with a duct-like appearance. Computer assisted cytometry to evaluate differences in nuclear morphology was performed on UTR/C and EV tissues from nude mice. Receiver operator curve areas generated using a logistic regression model were 0.8, indicating the ability to quantitatively distinguish UTR/C from EV tissues. Keratinocyte growth factor-induced motility experiments showed that migration of UTR/C cells was significantly reduced (80-90%) compared to EV cells. Together, these data indicate that this novel 3'UTR influences not only the tumorigenic phenotype but also may play a role in differentiation and migration of breast cancer cells.

3' Untranslated Regions↗

PubMed2003

Tumor suppression by the prohibitin gene 3'untranslated region RNA in human breast cancer.

Prohibitin is a candidate tumor suppressor gene located on human chromosome 17q21, a region of frequent loss of heterozygosity in breast cancers. We showed previously that microinjection of RNA encoded by the prohibitin gene 3'untranslated region (3'UTR) blocks the G(1)-S transition causing cell cycle arrest in several human cancer cell lines, including MCF7. Two allelic forms (C versus T) of the prohibitin 3'UTR exist, and carriers of the less common variant (Tallele) with a family history of breast cancer exhibited an increased risk of breast cancer. In the present study, we examined the tumor suppressor activity of the prohibitin 3'UTR in human breast cancer cells. Stable clones of MCF7 cells expressing either the C allele or the T allele RNA under the control of the cytomegalovirus promoter were isolated and compared with empty vector clones. Clones expressing the C allele RNA (UTR/C) exhibited significant suppression of growth in cell proliferation assays, inhibition of colony formation in soft agar assays, and suppression of xenograft tumor growth when implanted on nude mice, compared with either T allele expressing or empty vector clones. Immunohistochemical analyses with Ki67 staining confirmed a significant reduction in proliferation of UTR/C tumors. Thus, the C allele of prohibitin 3'UTR produces a functional RNA, whereas a single nucleotide polymorphism creates a null allele (T allele) of which the RNA product has lost activity. Our data demonstrate for the first time that an RNA molecule functions as a tumor suppressor in human breast cancer.

3' Untranslated Regions↗

PubMed2003

Sigma receptors: potential medications development target for anti-cocaine agents.

The ability of cocaine to interact with sigma receptors suggests a viable target for medications development. Recently, numerous novel compounds and antisense oligodeoxynucleotides targeting sigma receptors have been synthesized and shown to prevent the behavioral toxicity and psychomotor stimulant effects of cocaine in animals. Protective doses of sigma receptor antagonists have also been shown to prevent changes in gene expression that are induced by cocaine. Together, the studies provide insight and promising future directions for the development of potential medications for the treatment of cocaine addiction and overdose.

Animals↗

PubMed2003

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.

Adenocarcinoma↗

PubMed2003

Metabolic biomarker and kinase drug target discovery in cancer using stable isotope-based dynamic metabolic profiling (SIDMAP).

Tumor cells respond to growth signals by the activation of protein kinases, altered gene expression and significant modifications in substrate flow and re-distribution among biosynthetic pathways. This results in a proliferating phenotype with altered cellular function. These transformed cells exhibit unique anabolic characteristics, which includes increased and preferential utilization of glucose through the non-oxidative steps of the pentose cycle for nucleic acid synthesis but limited de novo fatty acid synthesis and TCA cycle glucose oxidation. This primarily non-oxidative anabolic profile reflects an undifferentiated highly proliferative aneuploid cell phenotype and serves as a reliable metabolic biomarker to determine cell proliferation rate and the level of cell transformation/differentiation in response to drug treatment. Novel drugs effective in particular cancers exert their anti-proliferative effects by inducing significant reversions of a few specific non-oxidative anabolic pathways. Here we present evidence that cell transformation of various mechanisms is sustained by a unique disproportional substrate distribution between the two branches of the pentose cycle for nucleic acid synthesis, glycolysis and the TCA cycle for fatty acid synthesis and glucose oxidation. This can be demonstrated by the broad labeling and unique specificity of [1,2-(13)C(2)]glucose to trace a large number of metabolites in the metabolome. Stable isotope-based dynamic metabolic profiles (SIDMAP) serve the drug discovery process by providing a powerful new tool that integrates the metabolome into a functional genomics approach to developing new drugs. It can be used in screening kinases and their metabolic targets, which can therefore be more efficiently characterized, speeding up and improving drug testing, approval and labeling processes by saving trial and error type study costs in drug testing.

Biomarkers, Tumor↗

PubMed2003

Gene activity associated with cancers treated by surgical oncologists.

A significant component of the general surgeon's practice is treatment of cancer that most commonly involves breast, colorectal, pancreas, and hepatic surgeries. The prognosis for these patients is largely dependent upon the stage of the disease at the time of diagnosis. There is convincing evidence that the induction and progression of cancer occurs when the function of genes that regulate cell proliferation, cell death and DNA repair becomes altered. These molecular disturbances result from ongoing, tumor-promoting activity long before symptoms of the disease become apparent. The role of many genes and the extent of their involvement in this deleterious, molecular process is unique to the organ. However, the tumor suppressor genes p53 and p16, the oncogene c-myc, and the inflammation-associated enzyme COX-2 are implicated in all four of the tumors. The roles of the oncogenes ras and HER-2/erbB-2/neu are significant in all of the tumors except hepatocellular carcinoma. The systematic establishment of a comprehensive data base relating gene expression activity to the initiation and development of cancer will provide molecular markers for detection and targets for treatment. This overview presents the current status of information associating gene expression activity with cancers typically encountered by general surgeons.

Breast Neoplasms↗

PubMed2002

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.

Amino Acid Sequence↗

PubMed2002

Increased expression of alpha-1-antitrypsin, glutathione S-transferase pi 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.

Adenocarcinoma↗

PubMed2002

Antitumor activity of SS(dsFv)PE38 and SS1(dsFv)PE38, recombinant antimesothelin immunotoxins against human gynecologic cancers grown in organotypic culture in vitro.

PURPOSE: Mesothelin, a cell surface glycoprotein overexpressed in ovarian cancer, mesotheliomas, and some squamous cell carcinomas, is an attractive candidate for targeted therapy because it is not shed in significant amounts into the bloodstream and is not present in significant amounts on normal human tissues except for mesothelial cells. The objective of this study was to determine the antitumor activity of SS1(dsFv)PE38, a recombinant antimesothelin immunotoxin, against human gynecologic tumors grown in short-term culture in vitro. EXPERIMENTAL DESIGN: Tumor cells obtained from primary cultures of five ovarian and one cervical tumor were mixed with an equal proportion of NIH-3T3 fibroblasts and plated inside collagen gels in tissue culture plates. After 4-7 days of growth, these organotypic cultures were treated with media alone, SS1(dsFv)PE38, and a control immunotoxin RFB4(dsFv)PE38, which targets the CD22 antigen not present on gynecologic tumors, every other day x 3. The organotypic culture gels were then formalin fixed, paraffin embedded, and evaluated for immunotoxin sensitivity using light microscopic examination of H&E-stained slides and also evaluated for apoptosis using the terminal deoxynucleotidyl transferase-mediated nick end labeling assay. RESULTS: Tumors expressing mesothelin showed a significant dose-dependent sensitivity to SS1(dsFv)PE38 even at concentrations as low as 1 ng/ml, whereas no antitumor activity was seen at 100 ng/ml in tumors that did not express mesothelin. This activity was specifically attributable to mesothelin targeting because RFB4 (dsFv)-PE38 had no activity against mesothelin-expressing tumors. CONCLUSIONS: These results demonstrate that ovarian and cervical tumor cells obtained from patients can be grown in short-term culture using an organotypic culture model. Our results also show low concentrations of an immunotoxin targeting mesothelin is cytotoxic to mesothelin-expressing human tumors by inducing apoptosis.

3T3 Cells↗

PubMedDate not supplied

Antisense KGFR oligonucleotide inhibition of KGF-induced motility in breast cancer cells.

BACKGROUND: The metastasis of breast cancer is known to be directly associated with the motility of breast cancer cells. We have previously shown that keratinocyte growth factor (KGF) enhances the motility of estrogen receptor (ER)-positive breast cancer cells and that this motility response is associated with cellular levels of the KGF receptor (KGFR). Further, we observed that KGF treatment enhanced KGFR gene expression in MCF-7 cells. The objective of the present study was to examine the influence of antisense KGFR oligonucleotide treatment on the KGF-induced motility response in breast cancer cells. MATERIALS AND METHODS: Both time-lapse video microscopy (TLVM) and culture wounding experiments were used to quantify cell motility. KGFR antisense effects on the expression of KGFR were determined by Western blotting, real time PCR and immunocytochemistry. RESULTS: Antisense KGFR treatment significantly reduced both KGFR mRNA and protein expression. In addition, the antisense KGFR abolished the KGF-mediated cell motility response as early as 2 h following KGF treatment as observed by TLVM and lasting for up 48 h as observed by culture wounding. CONCLUSIOS: The results of this study indicate that KGFR activation and intracellular signaling mediates the KGF motility effect and suggests that KGFR may be an important new therapeutic target for the treatment or prevention of metastatic progression in breast cancer.

Base Sequence↗

PubMedDate not supplied

Targeting a methioninase-containing fusion protein to breast cancer urokinase receptors inhibits growth and migration.

BACKGROUND: We previously reported that a novel fusion protein (consisting of an amino-terminal fragment of urokinase which binds to the urokinase receptor, and L-methioninase which depletes methionine and arrests the growth of methionine-dependent tumors) inhibited MCF-7 breast cancer cells in vitro. MATERIALS AND METHODS: We produced this fusion protein, L-methioninase, and a mutated fusion protein without L-methioninase activity by recombinant methods. MCF-7 cell proliferation and mobility were measured in vitro in a culture wounding assay. Protein binding to MCF-7 cells was measured by immunocytochemical localization. MCF-7 tumor xenograft growth was measured in nude mice. RESULTS: The fusion protein was significantly more effective than L-methioninase in either the in vitro or in vivo assays. The binding assay showed that the unmutated and mutated fusion protein bound to the cells, but L-methioninase did not. CONCLUSION: Our results suggest that this fusion protein has potential as a therapeutic agent for cancer treatment.

Animals↗