AbstractThe sections in this article areIntroductionEndoplasmic ReticulumGolgi ApparatusVacuolesSecreted Proteins fromArabidopsisMembrane ProteinsThe Mechanism of Membrane Trafficking in CellsFuture Directions

The chemotherapeutic agents doxorubicin (dox) or 5-fluorouracil (5FU) are used to treat cancer cells as they cause irreparable DNA damage, inducing these aberrant cells to undergo cell death. The mediator of this process is presumed to be in part the tumor suppressor p53 which regulates genes involved in DNA repair and cell death. When MCF-7 breast cancer cells are treated with these drugs, we observed that the level of p53 and the p53 negative regulator, Mdm2, increased, as seen by others. But contrary to some reports, we observed minimal phosphorylation of p53 at serine 15 in MCF-7 cells after drug treatment. Interestingly, we determined that there was differential regulation of the kinases ATM and Chk2 with the drug treatments, likely the cause for the lack of phosphorylation of p53. We found a dramatic drop in p53 DNA binding affinity for p21 and other gene response elements (RE) after drug treatment. To determine if the p53 that accumulated in the drug treated cells was functionally active, we monitored changes in the protein products of two p53-regulated genes following drug treatment with and without the addition of a p53-specific siRNA. In response to 5FU, both p21 and Mdm2 proteins increased and that increase was alleviated if a p53-specific siRNA was added. This effect was not seen with the addition of dox. Thus, the phosphorylation at serine 15 is not necessary for the functional activation of this transcription factor. We propose a new model for the regulation of p53, Mdm2, and MdmX after drug treatment.

The ability to monitor biomolecular recognition such as DNA hybridization and enzymatic reactivity in solutions with high sensitivity is important for developing effective bioassay strategies. Surface enhanced Raman scattering (SERS) based on use of solid substrates to produce the SERS effect for the detection often requires substrate preparation which is ineffective for rapid monitoring. This report describes a new strategy exploiting a gold nanoparticle (AuNP) based interparticle "hot-spot" for SERS monitoring of DNA mediated assembly and enzyme induced cleavage of the assembly in solution phase. The DNAs consist of two different complementary DNA strands with a thiol modification for attachment to AuNPs of selected sizes. In a solution containing AuNPs conjugated with one of the single-stranded (ss) DNA and other AuNPs labeled with a Raman reporter molecule, 4-mercaptobenzoic acid (MBA), the introduction of the complementary DNA strand leads to a linkage of the two types of AuNPs, producing double-stranded (ds) DNA-AuNP assembly (ds-DNA-AuNPs) with an interparticle "hot-spot" for SERS detection of the diagnostic bands of the reporter. Upon introducing a restriction enzyme (e.g. MspI) into the ds-DNA-AuNP assembly solution, the removal of the interparticle "hot-spot" due to restriction enzyme cleavage of the ds-DNA leads to a decrease of the SERS signals. While the detailed cleavage process may depend on the reaction time and the amount of enzyme, the viability of using gold nanoparticle "hot-spot" based SERS monitoring of DNA assembly and enzyme cleavage is clearly demonstrated, which has important implications for developing new strategies for bioassays.

Screening and analysis of collections of DNA molecules is a standard aspect of many DNA computing approaches. We describe the use of three different polymerase chain reaction (PCR) detection methods to screen specific members of a 5-site, 2-variable DNA computing library previously created using parallel overlap assembly of unique sequences generated from the SynDCode program. The three PCR methods (conventional gel-based PCR, SYBR Green real-time PCR and TaqMan real-time PCR) could all successfully identify individual library members separately or in a mixture. The TaqMan approach was also able to identify members in the original library we had not yet sequenced, providing more evidence supporting our hypothesis that the DNA library we generated may be complete. We expect these three approaches will be useful in future screening of other DNA computing libraries and structures.

Abstract Mutations in the p53 tumor suppressor affect protein accumulation, localization and DNA binding. We undertook to determine these properties of p53 in breast cancer cells and to see how those attributes change following oxidative stress. We analyzed the p53 in 8 different breast cancer cell lines, with either wild-type or mutant p53 protein. By ELISA and western blots, we found that the levels of the p53 protein were generally low for cells carrying wild-type protein and high in cells having a mutation in the TP53 gene. This was contrasted to the case in the HCC2157 cell line which has wild-type p53 but accumulates the protein to high levels. Using biotinylated DNA and streptavidin magnetic beads, we found that wild-type p53 protein from MCF-7 and ZR-75-1 cells binds with different affinity to 12 gene sequences covering several pathways regulated by p53. Treatment of MCF-7 cells with H2O2 caused an increase in this binding affinity. The p53 from the HCC2157 cells and all of the mutant p53 proteins had minimal to weak binding to these sequences even after treatment with H2O2. We also analyzed the phosphorylation of serine 15 on p53 in 4 of the cell lines. The p53 protein from the HCC2157 cells and two other cell lines showed phosphorylated protein at this site, but we found no correlation between that modification and the levels, localization, or binding affinity of the protein. From this and other work, it appears that the mutation status of the TP53 gene alone can not predict the activity of this tumor suppressor since cell lines with the same genetic information do not show the same properties of this protein. We argue that the analysis of DNA binding or transcription activity must be done in order to correlate the functionality of p53 with clinical outcome in tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1186. doi:1538-7445.AM2012-1186

BACKGROUND/AIM:Multidrug resistance poses a serious challenge in cancer therapy. To address this problem, we designed and synthesized Adva-27a, a novel non-ester GEM-difluorinated C-glycoside derivative of podophyllotoxin. MATERIALS AND METHODS:Adva-27a activity was evaluated in a variety of assays including inhibition of topoisomerase IIα, cytotoxic activity in drug-sensitive and drug-resistant cancer cell lines, metabolic stability in human liver microsomes and pharmacokinetic properties in rats. RESULTS:Adva-27a exhibited dose-dependent human topoisomerase IIα inhibitory activity and dose-dependent growth inhibitory activity in several drug-sensitive and two multidrug-resistant cancer cell lines. In the multidrug-resistant cell lines, MCF-7/MDR (breast cancer) and H69AR (small-cell lung cancer), Adva-27a was significantly more potent than etoposide. The metabolic stability of Adva-27a in human liver microsomes and its pharmacokinetic properties in rats were better than those of etoposide. CONCLUSION:Our studies have identified Adva-27a as a novel topoisomerase II inhibitor with superior cytotoxic activity against multidrug-resistant human cancer cells and more desirable pharmacokinetic properties than etoposide.

A novel non-ester GEM-difluorinated C-glycoside derivative of podophyllotoxin, Adva-27a, had been designed and synthesized. The growth inhibitory properties of this drug compared to the currently used Topoisomerase II inhibitor etoposide was evaluated in MCF-7 breast cancer cells and in a multi-drug resistant (MDR) variant of this cell line. These cells were cultured in the presence of the drugs at different concentrations (0.1-100μM) for various times (1-9 days) and analyzed for viability using a mitochondrial metabolic activity dye, Alamar blue versus solvent (DMSO) treated cells. The MCF-7 cells were sensitive to Adva-27a and etoposide, resulting in an IC50 of less than 10μM for etoposide and 20μM for Adva-27a, while the IC50 for the MDR MCF-7 cells was 25μM with Adva-27a, but greater than 100μM for etoposide. The timing for killing by Adva-27a of the MDR MCF-7 cells appeared significantly different than for the MCF-7 parent cells. In this case, 100μM drug resulted in less than 50% of viability at day 3 for the MDR MCF-7 cells, while the same concentration of drug required 5-6 days to cause the same level of killing of the parent cells. To determine the mechanism of action of the drug, we analyzed the Adva-27a treated cells by flow cytometry using propidium iodide staining. We found that after 24 hours with 30μM of the drug, both MCF-7 and the MDR MCF-7 cells were accumulating in the G2/M phase of the cell cycle. Both cells are similarly killed after 7 days by this concentration of the drug. Etoposide is known to cause accumulation of cells at a similar phase, implying the Adva-27a has a similar drug target. Thus, the mechanism for the difference in sensitivity of the MDR MCF-7 cells for Adva-27a relative to etoposide is not yet understood, but will be explored in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1758. doi:1538-7445.AM2012-1758

Abnormalities in several important cellular pathways and processes are often a major contributing factor to the progression of cancer. The cell cycle is regulated by checkpoint proteins, including the cyclins and cyclin dependent protein kinases (cdk), which play an important role in the prevention of aberrant cell division. Normal cells have the ability to stop cell division and initiate DNA repair or apoptosis (programmed cell death) when genomic abnormalities cannot be repaired. Apoptotic signaling is necessary for the elimination of unwanted cells arising from exposure to stress or toxins or as a function of normal tissue development and senescence. Proteins regulating these pathways are critical to maintain genomic integrity by controlling normal cell division and cell death; one of the most important of these is the tumor suppressor p53. The p53 protein plays a critical role in maintaining genomic integrity through control of cell division, apoptosis, DNA repair and angiogenesis, and thus is known as the “guardian of the genome” (Lane, 1992). Cancer cells have evolved the ability to bypass the cell cycle checkpoints, to favor anti-apoptotic pathways and thus, proliferate uncontrollably. Many anti-tumor drugs and treatments such as radiation are designed to target the induction of pro-apoptotic pathways in cancer cells in an effort to stop cell division and ultimately kill these aberrant cells. In many cases, these drugs and treatments activate p53 which can then regulate the expression of genes controlling the cell cycle and apoptotic pathways. Understanding the role of the tumor suppressor p53 in the regulation of these processes is the focus of many laboratories around the world.

Stress treatment generally causes the post-translational modification and accumulation of the p53 protein, although the role of these aspects has not been always understood in relation to this protein's tumor suppressor activity. We analyzed these attributes of p53 in eight different breast cancer cell lines, with either wild-type or mutant p53 protein, in response to oxidative stress. We found that the wild-type p53 protein from MCF-7 and ZR-75-1 cells binds with different affinity to 12 gene sequences covering several pathways regulated by p53. Treatment of MCF-7 cells with H2O2 caused an increase in this binding affinity while this same treatment of ZR-75-1 cells caused the p53 protein to lose binding affinity to several genes. The mutant p53 proteins from all cell lines had minimal to weak binding to these sequences even after treatment with H2O2. The p53 protein from the ZR-75-1 cells and three cell lines with mutant p53 showed serine 15 phosphorylated protein, but we found no correlation between that modification and the levels or localization of this protein although DNA binding affinity of wild-type protein might be affected by this modification. From this and other work, it appears that the mutation status of the TP53 gene alone cannot predict the activity of this tumor suppressor since cell lines with the same genetic information do not show the same properties of this protein.

Abstract The DNA repair enzyme Topoisomerase II (TopoII) has been recognized in the oncology field as a clinically important therapeutic target for a variety of cancers. The class of agents known as etoposides were specifically designed with this target in mind. Etoposides lacked the major problem of cardiac toxicity associated with the anthracyclines class of drugs (e.g. doxorubicin). Sunshine Biopharma has recognized that the reduced efficacy and high toxicity of etoposide are due to instability of the molecule leading to a conversion of the drug which then functions as a tubulin inhibitor rather than a TopoII inhibitor (unpublished observations). Accordingly, the company set out to develop a true TopoII inhibitor and these efforts have yielded two compounds called Adva-27a and Adva-32a. These drugs and their derivatives have been described in US Patent Application Number: 20090318675. Using cell lines derived from a variety of human cancers, Adva-27a is almost always a more potent inhibitor of cell growth compared to etoposide, and cells expressing the MDR-drug resistance protein are very sensitive to this drug. Furthermore, consistent with the literature, amplification of the TOP2α gene is associated with increased sensitivity to growth inhibition. The preliminary data show that in all but the brain tumor cell line, increased TOP2α copy number is associated with even greater percent inhibition by Adva-27a versus etoposide regardless of the tissue of origin of the tumor cell lines. This data and plans for further analysis of these drugs will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-201. doi:10.1158/1538-7445.AM2011-LB-201

Measurement of the level of a specific protein can be an important parameter to discern as that can change and reflect disease status. A number of methods have been developed to quantitate the level of a protein, some amenable to high throughput screening. A method is described to measure the total level of the tumor suppressor p53 using scintillation proximity assay (SPA) beads and radiolabeled streptavidin. Three different cell extracts were used, with one used to develop the standard curve for the amount of p53. This method allows the specific detection of p53 in the range of 50 to 300 pg in 10 mu l of an extract. While this detection is less than what can be detected by commercially available enzyme linked immunosorbent assay (ELISA) kits, the SPA compares favorably on time required and cost. This new assay also has the potential to be coupled with measurements for p53 DNA binding, a unique aspect of this approach. (C) 2010 Elsevier B.V. All rights reserved.

This work-in-progress paper describes the math and engineering module of the Go Green Institute at Binghamton University. This institute is a 10-day summer program for middle school students and was developed in an effort to increase the level of understanding of the scientific aspects of environmental sustainability and to promote interest in science, technology, engineering, and math careers. The Go Green Institute is comprised of three course modules; (1) Biology/Life Science, (2) Chemistry/Physical Science, and (3) Math/Engineering, all with a focus on climate change and sustainability issues. Along with these course modules the institute also included field trips, guest speakers, and team projects that were all related to the institute's goals. The focus of this paper is on the math and engineering module of the institute. The curriculum of this module will be presented and its effectiveness will be discussed. In addition, a plan for an assessment of the effect of the curriculum on the student's interest in the field of engineering will be presented.

A fluorescent probe has been attached to the carboxy terminus of the α-subunit of α,β-tubulin by an enzymatic reaction followed by a chemical reaction. The unnatural amino acid 3-formyltyrosine is attached to the carboxy terminus of α-tubulin through the use of the enzyme tubulin tyrosine ligase. The aromatic aldehyde of the unnatural amino acid serves as an orthogonal electrophile that specifically reacts with a fluorophore containing an aromatic hydrazine functional group, which in this case is 7-hydrazino-4-methyl coumarin. Conditions for covalent bond formation between the unnatural amino acid and the fluorophore are mild, allowing fluorescently labeled tubulin to retain its ability to assemble into microtubules. A key feature of the labeling reaction is that it produces a red shift in the fluorophore’s absorption and emission maxima, accompanied by an increase in its quantum yield; thus, fluorescently labeled protein can be observed in the presence of unreacted fluorophore. Both the enzymatic and coupling reaction can occur in living cells. The approach presented here should be applicable to a wide variety of in vitro systems.

Abstract Cell cycle regulation is a function of several regulatory genes such as cyclin G, p21, etc. controlled by p53. Several other genes such as pcna, puma, noxa involved in DNA repair and apoptotic pathways are also coordinated by p53. It is thus understandable that mutations in p53 could result in de-regulation of cell cycle progression, DNA repair, apoptosis, etc. Any imbalance in the above mentioned processes normally culminates in cancer. Our present focus of study is breast cancer where the cells harbor either wild-type or mutant p53. Progression of cancer in cell lines with wild-type p53 indicates a different regulatory switch/pathway responsible for the establishment of this disease. In an effort to understand the functional effect of both wild-type and mutant p53, we have done p53 DNA binding with nuclear extracts from several breast cancer cell lines using two assays. The DNA sequences being considered as target for p53 binding are from genes involved in cell cycle regulatory pathways, DNA repair, apoptosis and p53 regulation. We have observed differences in binding of p53 to some of these gene sequences. Cell lines with wild-type p53 and functional estrogen receptor (ER) bind more to the promoter for pcna and cyclin G than cell lines with mutant p53. The binding to the promoter of the puma gene is more similar between cell lines with mutant and wild-type p53 than the other genes analyzed. We anticipate a certain trend in DNA binding based on the functional status of p53 and its interaction with other proteins such as ER. This trend of p53 binding to different gene sequences may be utilized to better understand the causes of breast cancer, its subsequent progression as well as for designing treatment protocols. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-9.

Cancer-associated mutations in the p53 gene often change amino acids in the protein's DNA binding domain. We used three different binding assays specifically gel shift, DNA binding scintillation proximity assay and a streptavidin magnetic bead assay to analyze the DNA binding of the tumor suppressor p53 from 4 human cell lines with different DNA sequences from the mdm2, p21 and cyclin G genes and a mutant form of the cyclin G sequence. Treatment of MCF-7 cells having wild-type p53 with hydrogen peroxide increased the binding of p53 to DNA as detected using all three assays, but to different extents. The p53 proteins from the thyroid cancer cell lines with different p53 mutations (ARO, WRO and NPA) have comparable binding reactions in the three assays, but show different specificities for the sequences. Here we show that multiple different binding assays allow us to generate a more complete picture of the function of DNA transcription factors in diseases such as cancer.

Using a universal and parallel battery of PCR reactions, we give a nonadaptive group testing method for identifying the individual strands in a pooled sample of several different DNA sequences taken from a DNA library. The method discussed here has potential applications to DNA taggants, DNA memory and DNA computing.

The ability to manipulate and intervene in the processes of assembly and disassembly of DNAs and nanoparticles is important for the exploitation of nanoparticles in medical diagnostics and drug delivery. This report describes the results of an investigation of a strategy to intervene in the assembly and disassembly processes of DNAs and gold nanoparticles based on two approaches. The first approach explores the viability of molecular intervention to the assembly-disassembly-reassembly process. The temperature-induced assembly and disassembly processes of DNAs and gold nanoparticles were studied as a model system to illustrate this approach. The introduction of a molecular recognition probe leads to intervention in the assembly-disassembly process depending on its specific biorecognition. This process was detected by monitoring the change in the optical properties of gold nanoparticles and their DNA assemblies. The second approach involves the disassembly of the DNA-linked assembly of nanoparticles using restriction enzymes (e.g., MspI). The presence of the double stranded DNAs in the nanoparticle assembly was also substantiated by a Southern blot. Implications of the results to exploration of the molecular intervention for fine-tuning interfacial reactivities in DNA-based bioassays are also discussed.