Receptor-specific targeting with complementary peptide nucleic acids conjugated to peptide analogs and radionuclides

Summary Genomic sequencing makes it possible to identify all the genes of an organism, now including Homo sapiens. Yet measurement of the expression of each gene of interest still presents a daunting prospect. Northern blots, RNase protection assays, as well as microarrays and related technologies permit measurement of gene expression in total RNA extracted from cultured cells or tissue samples. It would be most valuable, however, to quantitate gene expression noninvasively in living cells and tissues. Unfortunately, no reliable method has been available to measure levels of specific mRNAs in vivo. Peptide nucleic acids (PNAs) display superior ruggedness and hybridization properties as a diagnostic tool for gene expression, and could be used for this purpose. On the down side, they are negligibly internalized by normal or malignant cells in the absence of conjugated ligands. Nevertheless, we have observed that Tc-99m-peptides can delineate tumors, and PNA-peptides designed to bind to IGF-1 receptors on malignant cells are taken up specifically and concentrated in nuclei. We have postulated that antisense Tc-99m-PNA-peptides will be taken up by human cancer cells, will hybridize to complementary mRNA targets, and will permit scintigraphic imaging of oncogene mRNAs in human cancer xenografts in a mouse model. The oncogenes cyclin D1, ERBB2, c-MYC, K-RAS, and tumor suppressor p53 are being probed initially. These experiments provide a proof-of-principle for noninvasive detection of oncogene expression in living cells and tissues. This scintigraphic imaging technique should be applicable to any particular gene of interest in a cell or tissue type with characteristic receptors.     

Receptor-specific targeting with complementary peptide nucleic acids conjugated to peptide analogs and radionuclides

Genomic sequencing makes it possible to identify all the genes of an organism, now including Homo sapiens. Yet measurement of the expression of each gene of interest still presents a dauntingprospect. Northern blots, RNase protection assays, as well as microarrays and related technologies permit measurement of gene expression in total RNA extracted from cultured cells or tissue samples. It would be most valuable, however, to quantitate gene expression noninvasively in living cells and tissues. Unfortunately,no reliable method has been available to measure levels of specificmRNAs in vivo. Peptide nucleic acids (PNAs) display superior ruggedness and hybridization properties as a diagnostic tool for gene expression, and could be used for this purpose. On the down side, they are negligibly internalized by normal or malignant cells in the absence of conjugated ligands. Nevertheless,we have observed that Tc-99m-peptides can delineate tumors, and PNA-peptides designed to bind to IGF-1 receptors on malignant cellsare taken up specifically and concentrated in nuclei. We have postulated that antisense Tc-99m-PNA-peptides will be taken up by human cancer cells, will hybridize to complementary mRNA targets, and will permit scintigraphic imaging of oncogene mRNAsin human cancer xenografts in a mouse model. The oncogenes cyclinD1, ERBB2, c-MYC, K-RAS, and tumor suppressor p53 are being probed initially. These experimentsprovide a proof-of-principle for noninvasive detection of oncogeneexpression in living cells and tissues. This scintigraphic imaging technique should be applicable to any particular gene of interest in a cell or tissue type with characteristic receptors.     

TUMOR-TARGETING PEPTIDE-PNA-PEPTIDE CHIMERAS FOR IMAGING OVEREXPRESSED ONCOGENE mRNAS

We have optimized a method involving continuous solid phase synthesis of chelator-peptide-PNA-peptide probes in order to noninvasively image oncogene mRNAs overexpressed in tumors. The PNA (peptide nucleic acid) probes carry cyclized peptide ligand analogs specific for receptors overexpressed on malignant breast or colorectal cancer cells, and chelators to bind radioactive metal ions, or a fluorophore. In vivo scintigraphic imaging of MCF7 xenografts in immunocompromised mice indicated that CCND1 and MYC [^99mTc]chelator-PNA-D(CSKC) probes concentrated in MCF7 cells up to 7 times more than the corresponding mismatch controls.     

Endothelial progenitor cells (EPCs) as gene carrier system for rat model of human glioma

Background

Due to their unique property to migrate to pathological lesions, stem cells are used as a delivery vehicle for therapeutic genes to tumors, especially for glioma. It is critically important to track the movement, localization, engraftment efficiency and functional capability or expression of transgenes of selected cell populations following transplantation. The purposes of this study were to investigate whether 1) intravenously administered, genetically transformed cord blood derived EPCs can carry human sodium iodide symporter (hNIS) to the sites of tumors in rat orthotopic model of human glioma and express transgene products, and 2) whether accumulation of these administered EPCs can be tracked by different in vivo imaging modalities.

Methods and Results

Collected EPCs were cultured and transduced to carry hNIS. Cellular viability, differential capacity and Tc-99m uptake were determined. Five to ten million EPCs were intravenously administered and Tc-99-SPECT images were acquired on day 8, to determine the accumulation of EPCs and expression of transgenes (increase activity of Tc-99m) in the tumors. Immunohistochemistry was performed to determine endothelial cell markers and hNIS positive cells in the tumors. Transduced EPCs were also magnetically labeled and accumulation of cells was confirmed by MRI and histochemistry. SPECT analysis showed increased activity of Tc-99m in the tumors that received transduced EPCs, indicative of the expression of transgene (hNIS). Activity of Tc-99m in the tumors was also dependent on the number of administered transduced EPCs. MRI showed the accumulation of magnetically labeled EPCs. Immunohistochemical analysis showed iron and hNIS positive and, human CD31 and vWF positive cells in the tumors.

Conclusion

EPC was able to carry and express hNIS in glioma following IV administration. SPECT detected migration of EPCs and expression of the hNIS gene. EPCs can be used as gene carrier/delivery system for glioma therapy as well as imaging probes.     

AC133+ progenitor cells as gene delivery vehicle and cellular probe in subcutaneous tumor models: a preliminary study

Background  

Despite enormous progress in gene therapy for breast cancer, an optimal systemic vehicle for delivering gene products to the target tissue is still lacking. The purpose of this study was to determine whether AC133+ progenitor cells (APC) can be used as both gene delivery vehicles and cellular probes for magnetic resonance imaging (MRI). In this study, we used superparamagentic iron oxide (SPIO)-labeled APCs to carry the human sodium iodide symporter (hNIS) gene to the sites of implanted breast cancer in mouse model. In vivo real time tracking of these cells was performed by MRI and expression of hNIS was determined by Tc-99m pertechnetate (Tc-99m) scan.     

Tumor-targeting peptide-PNA-peptide chimeras for imaging overexpressed oncogene mRNAs

We have optimized a method involving continuous solid phase synthesis of chelator-peptide-PNA-peptide probes in order to noninvasively image oncogene mRNAs overexpressed in tumors. The PNA (peptide nucleic acid) probes carry cyclized peptide ligand analogs specific for receptors overexpressed on malignant breast or colorectal cancer cells, and chelators to bind radioactive metal ions, or a fluorophore. In vivo scintigraphic imaging of MCF7 xenografts in immunocompromised mice indicated that CCND1 and MYC [99sTc] chelator-PNA-D (CSKC) probes concentrated in MCF7 cells up to 7 times more than the corresponding mismatch controls.     

Ribozyme as an approach for growth suppression of human pancreatic cancer

Ribozymes (catalytic RNAs, RNA enzymes) are effective modulators of gene expression because of their simple structure, site-specific cleavage activity, and catalytic potential, and have potentially important implications for cancer gene therapy. Point mutations in the K-ras oncogene are found in approx 90% of human pancreatic carcinomas, and can be used as potential targets for specific ribozyme-mediated reversal of the malignant phenotype. In this study, we focused on in vitro manipulation of ribozyme targeting of the mutated K-ras oncogene in a human pancreatic carcinoma cell line. We evaluated the efficacy of an anti-K-ras hammerhead ribozyme targeted against GUU-mutated codon 12 of the K-ras gene in cultured pancreatic carcinoma cell lines. The anti-K-ras ribozyme significantly reduced cellular K-ras mRNA level (GUU-mutated codon 12) when the ribozyme was transfected into the Capan-1 pancreatic carcinoma cells. The ribozyme inhibited proliferation of the transfected Capan-1 cells. These results suggested that this ribozyme is capable of reversing the malignant phenotype in human pancreatic carcinoma cells.     

Identification of suitable endogenous control genes for microRNA gene expression analysis in human breast cancer

The discovery of microRNAs (miRNAs) added an extra level of intricacy to the already complex system regulating gene expression. These single-stranded RNA molecules, 18–25 nucleotides in length, negatively regulate gene expression through translational inhibition or mRNA cleavage. The discovery that aberrant expression of specific miRNAs contributes to human disease has fueled much interest in profiling the expression of these molecules. Real-time quantitative PCR (RQ-PCR) is a sensitive and reproducible gene expression quantitation technique which is now being used to profile miRNA expression in cells and tissues. To correct for systematic variables such as amount of starting template, RNA quality and enzymatic efficiencies, RQ-PCR data is commonly normalised to an endogenous control (EC) gene, which ideally, is stably-expressed across the test sample set. A universal endogenous control suitable for every tissue type, treatment and disease stage has not been identified and is unlikely to exist, so, to avoid introducing further error in the quantification of expression data it is necessary that candidate ECs be validated in the samples of interest. While ECs have been validated for quantification of mRNA expression in various experimental settings, to date there is no report of the validation of miRNA ECs for expression profiling in breast tissue. In this study, the expression of five miRNA genes (let-7a, miR-10b, miR-16, miR-21 and miR-26b) and three small nucleolar RNA genes (RNU19, RNU48 and Z30) was examined across malignant, benign and normal breast tissues to determine the most appropriate normalisation strategy. This is the first study to identify reliable ECs for analysis of miRNA by RQ-PCR in human breast tissue.     

Effect of semax and its C-terminal peptide PGP on expression of neurotrophins and their receptors in rat brain during incomplete global ischemia

Neurotrophins regulate key functions of nervous tissue cells. Analysis of neurotrophin mRNA expression is an appropriate tool to assess therapeutic efficiency of antistroke drugs. We have analyzed the effect of synthetic peptide semax and its C-terminal Pro-Gly-Pro tripeptide on mRNA expression of neurotrophins Ngf, Bdnf, and Nt-3 and their receptors TrkA, TrkB, TrkC, and p75 in rat frontal cortex, hippocampus, and cerebellum after bilateral common carotid artery occlusion. The animals were decapitated at 30 min and 1, 2, 4, 8, 12, and 24 h after the operation. The mRNA expression of neurotrophins and their receptors was assessed by relative quantification using real-time RT-PCR. Our results demonstrated that ischemia caused a significant decrease in gene expression in the hippocampus. Semax and PGP treatment affected the expression of neurotrophins and their receptors predominantly in the frontal cortex and hippocampus of the ischemized animals. In the frontal cortex, Semax treatment resulted in a decrease of mRNA level of neurotrophin receptors, while PGP treatment increased the level of these mRNA. Maximal neuroprotective effect of both peptides was observed in the hippocampus 12 h after occlusion. A decrease of gene expression of neurotrophins and their receptors caused by the occlusion was overcome by Semax and PGP. These results clarify the mechanism of Semax action and reveal certain features of mRNA expression of neurotrophins and their receptors under experimental conditions.     

The value of 99mTc-MIBI scan in the detection of malignancy potential of hypermetabolic thyroid incidentalomas of 18F-FDG PET/CT

Aim of the studyIncreasingly use of PET/CT leads to discovery of incidental findings. Hypermetabolic thyroid nodules are one of the unexpected lesions in PET/CT imaging with an increased risk of thyroid cancers. Our study aims to determine the malignant potential of incidentally detected 18F-FDG avid thyroid nodules by using Tc-99m MIBI imaging.Materials and methodsPET/CT scans were performed for nonthyroidal purposes and were evaluated for the presence of hypermetabolic thyroid nodules. Tc-99m MIBI scans and ultrasonography-guided fine needle aspiration biopsies were subsequently performed for all patients.ResultsPrimary thyroid malignancies were identified in 25% of patients with increased focal FDG uptake at definitive diagnosis. Among the patients with FDG avid thyroid nodules, Tc-99m MIBI scan showed true-positive results in all thyroid carcinomas (n:7) with a 36.3% (4/11) false-positivity rate. In three patients with indeterminate cytology results, Tc-99m MIBI scan findings were also negative. The sensitivity, specificity, positive predictive value of Tc-99m MIBI scan in predicting the malignancy of FDG-positive thyroid nodules were 100%, 77%, 63.6%, respectively.ConclusionThe implementation of ^99mTc-MIBI scan performed by dual phase and SPECT/CT modality might be a helpful cost-effective approach in addition to FNAB in patients with ¹⁸F-FDG-positive thyroid nodules and indeterminate cytology to improve the patients’ prognosis and reduce unnecessary thyroid operations with associated use of FNAB.     

Tc-99m-Labeling of Modified RNA

Abstract

The synthesis of Tc-99m-labeled, modified RNA is reported. This new class of radiopharmaceuticals is of potential interest as target specific imaging agents. The preparation of N₃S-RNA was achieved by coupling protected MAG₂-units to amino modified ON's. The N₃S-RNA was Tc-99m-labeled with 90–95% radiochemical yield and specific activities of 37MBq/nmol leading to 1:1-Tc-99m-N₃S-aptamers.     

An Exploratory Study on 99mTc-RGD-BBN Peptide Scintimammography in the Assessment of Breast Malignant Lesions Compared to 99mTc-3P4-RGD2

Purpose

This study aimed to explore the diagnostic performance of single photon emission computed tomography / computerized tomography (SPECT/CT) using a new radiotracer ^99mTc-RGD-BBN for breast malignant tumor compared with ^99mTc-3P4-RGD₂.

Methods

6 female patients with breast malignant tumors diagnosed by fine needle aspiration cytology biopsy (FNAB) who were scheduled to undergo surgery were included in the study. ^99mTc-3P4-RGD₂ and ^99mTc-RGD-BBN were performed with single photon emission computed tomography (SPECT) at 1 hour after intravenous injection of 299 ± 30 MBq and 293 ± 32 MBq of radiotracers respectively at separate day. The results were evaluated by the Tumor to non-Tumor ratios (T/NT). ^99mTc-RGD-BBN and ^99mTc-3P4-RGD₂ SPECT/CT images were interpreted independently by 3 experienced nuclear medicine physicians using a 3-point scale system. All of the samples were analyzed immunohistochemically to evaluate the integrin αvβ3 and gastrin-releasing peptide receptor (GRPR) expression. The safety, biodistribution and radiation dosimetry of ^99mTc-RGD-BBN were also evaluated in the healthy volunteers.

Results

No serious adverse events were reported in any of the patients during the study. The effective radiation dose entirely conformed to the relevant standards. A total of 6 palpable malignant lesions were detected using ^99mTc-RGD-BBN SPECT/CT with clear uptake. All malignant lesions were also detected using ^99mTc-3P4-RGD₂ SPECT/CT. The results showed that five malignant lesions were with clear uptake and the other one with barely an uptake. 4 malignant cases were found with both αvβ3 and GRPR expression, 1 case with only GRPR positive expression (integrin αvβ3 negative) and 1 case with only integrin αvβ3 positive expression (GRPR negative).

Conclusion

^99mTc-RGD-BBN is a safe agent for detecting breast cancer. ^99mTc-RGD-BBN may have the potential to make up for the deficiency of ^99mTc-3P4-RGD₂ in the detection of breast cancer with only GRPR positive expression (integrin α_vβ₃ negative). The preliminary application of ^99mTc-RGD-BBN has demonstrated its powerful potential in breast cancer diagnosis and therapy.     

Circumventing senescence is associated with stem cell properties and metformin sensitivity

Most cancers arise in old individuals, which also accumulate senescent cells. Cellular senescence can be experimentally induced by expression of oncogenes or telomere shortening during serial passage in culture. In vivo, precursor lesions of several cancer types accumulate senescent cells, which are thought to represent a barrier to malignant progression and a response to the aberrant activation of growth signaling pathways by oncogenes (oncogene toxicity). Here, we sought to define gene expression changes associated with cells that bypass senescence induced by oncogenic RAS. In the context of pancreatic ductal adenocarcinoma (PDAC), oncogenic KRAS induces benign pancreatic intraepithelial neoplasias (PanINs), which exhibit features of oncogene‐induced senescence. We found that the bypass of senescence in PanINs leads to malignant PDAC cells characterized by gene signatures of epithelial‐mesenchymal transition, stem cells, and mitochondria. Stem cell properties were similarly acquired in PanIN cells treated with LPS, and in primary fibroblasts and mammary epithelial cells that bypassed Ras‐induced senescence after reduction of ERK signaling. Intriguingly, maintenance of cells that circumvented senescence and acquired stem cell properties was blocked by metformin, an inhibitor of complex I of the electron transport chain or depletion of STAT3, a protein required for mitochondrial functions and stemness. Thus, our studies link bypass of senescence in premalignant lesions to loss of differentiation, acquisition of stemness features, and increased reliance on mitochondrial functions.     

Expression of a magainin-type antimicrobial peptide gene (MSI-99) in tomato enhances resistance to bacterial speck disease

MSI-99 is a synthetic analog of magainin II (MII), a small cationic peptide highly inhibitory to a wide spectrum of microbial organisms. Tomato plants were transformed to express a gene encoding the MSI-99 peptide and tested for possible enhancement of resistance to important pathogens of this crop. Thirty-six tomato transformants carrying an MSI-99 expression vector designed to target the peptide into extracellular spaces were obtained by Agrobacterium tumefaciens-mediated transformation. Expression of MSI-99 caused no obvious cytotoxic effects in these plants. In the tests with Pseudomonas syringae pv. tomato (bacterial speck pathogen) at 10⁵ CFU/ml, several MSI-99-expressing lines developed significantly fewer disease symptoms than controls. However, MSI-99-expressing lines were not significantly different from controls in their responses to the fungal pathogen Alternaria solani (early blight) and the oomycete pathogen Phytophthora infestans (late blight). These findings are in accordance with our previous in vitro inhibition tests, which showed that the MSI-99 peptide is more inhibitory against bacteria than against fungi and oomycetes. Additional in vitro inhibition assays showed that MSI-99 loses its antimicrobial activity in the total or extracellular fluids from leaflets of non-transformed tomato plants; however, P. syringae pv. tomato could not multiply in the extracellular fluid from an MSI-99-expressing line. Our results suggest that expression strategies providing continuous high expression of MSI-99 will be necessary to achieve significant enhancement of plant disease resistance.Abbreviations AMP Antimicrobial peptide - CFU Colony forming unit - ECF Extracellular fluid - gus -glucuronidase gene - nptII Neomycin phosphotransferase II - SP Signal peptide - TF Total fluidCommunicated by S. Gleddie     

Infrared laser‐mediated local gene induction in medaka,zebrafish and Arabidopsis thaliana

Heat shock promoters are powerful tools for the precise control of exogenous gene induction in living organisms. In addition to the temporal control of gene expression, the analysis of gene function can also require spatial restriction. Recently, we reported a new method for in vivo, single‐cell gene induction using an infrared laser‐evoked gene operator (IR‐LEGO) system in living nematodes (Caenorhabditis elegans). It was demonstrated that infrared (IR) irradiation could induce gene expression in single cells without incurring cellular damage. Here, we report the application of IR‐LEGO to the small fish, medaka (Japanese killifish; Oryzias latipes) and zebrafish (Danio rerio), and a higher plant (Arabidopsis thaliana). Using easily observable reporter genes, we successfully induced gene expression in various tissues in these living organisms. IR‐LEGO has the potential to be a useful tool in extensive research fields for cell/tissue marking or targeted gene expression in local tissues of small fish and plants.     

WT1 Promotes Cell Proliferation in Non-Small Cell Lung Cancer Cell Lines through Up-Regulating Cyclin D1 and p-pRb In Vitro and In Vivo

The Wilms’ tumor suppressor gene (WT1) has been identified as an oncogene in many malignant diseases such as leukaemia, breast cancer, mesothelioma and lung cancer. However, the role of WT1 in non-small-cell lung cancer (NSCLC) carcinogenesis remains unclear. In this study, we compared WT1 mRNA levels in NSCLC tissues with paired corresponding adjacent tissues and identified significantly higher expression in NSCLC specimens. Cell proliferation of three NSCLC cell lines positively correlated with WT1 expression; moreover, these associations were identified in both cell lines and a xenograft mouse model. Furthermore, we demonstrated that up-regulation of Cyclin D1 and the phosphorylated retinoblastoma protein (p-pRb) was mechanistically related to WT1 accelerating cells to S-phase. In conclusion, our findings demonstrated that WT1 is an oncogene and promotes NSCLC cell proliferation by up-regulating Cyclin D1 and p-pRb expression.     

Strict regulation of gene expression from a high-copy plasmid utilizing a dual vector system

High-copy plasmids are useful for producing large quantities of plasmid DNA, but are generally inadequate for tightly regulating gene expression. Attempts to suppress expression of genes on high-copy plasmids often results in residual or “leaky” production of protein. For stringent regulation of gene expression, it is often necessary to excise the gene of interest and subclone it into a low-copy plasmid. Here, we report a dual plasmid technique that enables tight regulation of gene expression driven by the lac promoter in a high-copy vector. A series of plasmids with varying copies of the lacI^q gene have been constructed to permit titration of the LacI protein. When a high-copy plasmid is transformed along with the appropriate lacI^q-containing plasmid, tight gene regulation is achieved, thus eliminating the need to subclone genes into low-copy plasmids. In addition, we show that this dual plasmid technique enables high-copy gene expression of a protein lethal to Escherichia coli, the ccdB protein. In principle, this technique can be applied to any high-copy plasmid containing the popular pUC replication of origin and provides an easier means of obtaining rigid control over gene expression.     

Isomer-specific effects of conjugated linoleic acid on gene expression in RAW 264.7

Conjugated linoleic acid (CLA) is a mixture of dietary fatty acids that has various beneficial effects including decreasing cancer, atherosclerosis, diabetes and inflammation in animal models. Some controversy exists on the specific isomers of CLA that are responsible for the benefits observed. This study was conducted to examine how different CLA isomers regulate gene expression in RAW 264.7. A mouse macrophage cell line, RAW 264.7, was treated with five different CLA isomers (9E,11E-, 9Z,11E-, 9Z,11Z-, 10E,12Z- and 11Z,13E-CLA). Gene expression microarrays were performed, and several significantly regulated genes of interest were verified by a real-time polymerase chain reaction (PCR). Examination of the biological functions of various significantly regulated genes by the five CLA isomers showed distinct properties. Isomers 9E,11E-, 9Z,11Z-, 10E,12Z- and 11Z,13E-CLA decreased production of proinflammatory cytokines such as interleukin (IL)-1α, IL-1β and IL-6. Many of CLA's effects are believed to be mediated by the fatty acid receptors such as the peroxisome proliferator-activated receptors (PPAR) and retinoid-X-receptors (RXR). Using PPAR and RXR specific antagonists and coactivator recruitment assays, it was evident that multiple mechanisms were responsible for gene regulation by CLA isomers. Coactivator recruitment by CLA isomers showed their distinct properties as selective receptor modulators for PPARγ and RXRα. These studies demonstrate distinct isomer differences in gene expression by CLA and will have important ramifications for determining the potential therapeutic benefit of these dietary fatty acids in prevention of inflammation-related diseases.     

Generation and characterization of an estrogen receptor alpha‐iCre knock‐in mouse

Two estrogen receptors, ESR1 and ESR2, are responsible for the classical actions of estrogens in mammalian species. They display different spatiotemporal expression patterns and nonoverlapping functions in various tissues and physiological conditions. In this study, a novel knock‐in mouse line that expresses codon‐improved Cre recombinase (iCre) under regulation of the natural Esr1 promoter (Esr1–iCre) was developed. Functional characterization of iCre expression by crossing them with reporter lines (ROSA26‐lacZ or Ai9‐RFP) showed that iCre is faithfully expressed in Esr1‐lineage cells. This novel transgenic mouse line will be a useful animal model for lineage‐tracing Esr1‐expressing cells, selective gene ablation in the Esr1‐lineage cells and for generating global Esr1 knockout mice.