In vivo imaging of proteolytic enzyme activity using a novel molecular reporter.

The single biggest challenge facing in vivo imaging techniques is to develop biocompatible molecular beacons that are capable of specifically and accurately measuring in vivo targets at the protein, RNA, or DNA level. Our efforts have focused on developing activatable imaging probes to measure specific enzyme activities in vivo. Using cathepsin D as a model target protease, we synthesized a long-circulating, synthetic graft copolymer bearing near-infrared (NIR) fluorochromes positioned on cleavable substrate sequences. In its native state, the reporter probe was essentially nonfluorescent at 700 nm due to energy resonance transfer among the bound fluorochromes (quenching) but became brightly fluorescent when the latter were released by cathepsin D. NIR fluorescence signal activation was linear over at least 4 orders of magnitude and specific when compared with scrambled nonsense substrates. Using matched rodent tumor models implanted into nude mice expressing or lacking the targeted protease, it could be shown that the former generated sufficient NIR signal to be directly detectable and that the signal was significantly different compared with negative control tumors. The developed probes should find widespread applications for real-time in vivo imaging of a variety of clinically relevant proteases, for example, to detect endogenous protease activity in disease, to monitor the efficacy of protease inhibitors, or to image transgene expression.

Sequence and factor requirements for faithful in vitro transcription of human 7SL DNA.

We have analysed the transcription of a functional human 7SL gene by RNA polymerase III (RNAPIII) in S100 extracts in vitro. Accurate and efficient synthesis of 7S L RNA depends on the presence of (i) an upstream sequence and (ii) an internal promoter element located within the first 22 bp of the gene. These findings were substantiated by DNase I footprinting. Mutations of the internal promoter identified the doublet CG [nucleotide (nt) +15/+16] outside the A-box homologue (nt +5 to +14) as being essential for both proper promoter function in the in vitro transcription assay and competition in the template-exclusion assay. Fractionation of S100 extracts identified two fractions required in addition to RNAPIII for faithful transcription of the gene. Each of these two fractions gave rise to one of two footprints observed in DNase I protection experiments, indicating that at least two DNA-binding factors are involved.

Imaging of tumour neovasculature by targeting the TGF-beta binding receptor endoglin.

In vivo imaging of endothelial markers in intact tumour neovasculature would have applications in assessing the efficacy of anti-angiogenic agents in clinical trials. Although a variety of different endothelial markers have been described, few have been evaluated as imaging markers. The transforming growth factor-beta (TGF-beta) binding receptor endoglin is a proliferation-associated endothelial marker. We hypothesised that endoglin would be an ideal target for imaging since it is strongly upregulated in proliferating endothelial cells of the tumour neovasculature. We used a radiolabelled monoclonal anti-endoglin antibody and compared its neovascular binding, accumulation and in vivo behaviour to an isotype-matched control IgG(2a). Our data show that the probe binds specifically and rapidly within minutes in vivo and that correlative autoradiography and immunohistology support the in vivo imaging findings. Imaging of abundantly expressed endothelial targets circumvents delivery barriers normally associated with other tumour targeting strategies, and can potentially be used to quantitate molecular angiogenic markers.

Diurnal variations of interleukin-1 beta mRNA and beta-actin mRNA in rat brain.

Interleukin-1 beta (IL-1 beta) is posited to play an important physiological role in brain functions in addition to its better defined role in pathology. The experiments described herein were performed to determine if IL-1 beta mRNA and beta-actin display diurnal rhythms in various areas of brain. Rats were sacrificed at 4 h intervals across a 12:12 h light/dark cycle. Hypothalamic, hippocampal and cortical IL-1 beta mRNA peaked just after lights were turned on, declined slightly during the remaining light period and stayed low in the dark. There were no significant changes in IL-1 beta mRNA in brain stem or cerebellum samples. beta-actin mRNA levels were relatively constant across the day in the hypothalamus, brain stem and cerebellum. However, beta-actin mRNA levels were lower during the day than during the night in the hippocampus and cortex.

Prolactin and rapid eye movement sleep regulation.

During the past few years data have accumulated suggesting the involvement of prolactin (PRL) in rapid eye movement sleep (REMS) regulation. Pituitary PRL secretion seems to be, at least in part, sleep-dependent. PRL is also found in the central nervous system. PRL-containing neurons in the hypothalamus project to various structures in the brain. Systemic injection of PRL promotes REMS in rats, cats and rabbits. Intracerebroventricular injection of PRL enhances REMS in rats. Stimulation of endogenous PRL secretion by vasoactive intestinal peptide (VIP) also promotes REMS. Immunoneutralization of blood-borne PRL slightly reduces REMS. Various observations (hypoprolactinemic and hyperprolactinemic rats) indicate that PRL may act on REMS via modulating the diurnal rhythms of REMS. It is likely that hypothalamic PRL is more important for sleep regulation than circulating PRL. Hypothalamic PRL is likely involved in the mediation of the REMS-promoting activity of VIP. We conclude that PRL has a role in REMS regulation.

Hypothalamic growth hormone-releasing hormone mRNA varies across the day in rats.

Experiments were performed to determine whether growth hormone-releasing hormone (GHRH) mRNA displays diurnal variations in the hypothalamus and cortex of the rat. Levels of GHRH and beta-actin mRNA were measured from hypothalamic and cortical extracts using the reverse transcriptase polymerase chain reaction method in rats sacrificed at 4 h intervals across a 12:12 h light:dark cycle. Hypothalamic GHRH mRNA peaked around the light onset, declined during the light period, and stayed low in the dark. Variations in hypothalamic beta-actin and cortical GHRH mRNA levels were not observed. beta-Actin mRNA expression in the cortex was higher in the dark than in the light period. The results demonstrate that hypothalamic GHRH mRNA displays diurnal variations.

Increase of prolactin mRNA in the rat hypothalamus after intracerebroventricular injection of VIP or PACAP.

Vasoactive intestinal peptide (VIP), the structurally homologous pituitary adenylate cyclase-activating peptide (PACAP) and the pituitary hormone, prolactin (PRL) enhance rapid eye movement sleep (REMS). VIP and PACAP are both inducers of PRL gene expression and release in the pituitary gland. Little is known about PRL regulation in the brain although it is hypothesized that the REMS-promoting activity of i.c.v. administered VIP may be mediated via the activation of cerebral PRL. To test whether VIP or PACAP in fact increase intracerebral mRNA, the peptides (VIP: 30 or 300 pmol; PACAP: 220 pmol) were injected i.c.v. into rats at dark onset. 1 h later, cDNA was synthesized from purified hypothalamic mRNA. Standardized amounts were analysed for PRL using the polymerase chain reaction followed by Southern blotting and hybridization. Compared with beta-actin mRNA levels, both VIP and PACAP increased PRL mRNA levels in a dose-dependent fashion though VIP was more effective on a molar basis. The previously reported alternatively spliced PRL mRNA (lacking exon 4) was not detected. The data support the hypothesis that the REMS-promoting activity of central VIP and PACAP might be mediated by cerebral PRL.

Sleep in rats rendered chronically hyperprolactinemic with anterior pituitary grafts.

A hyperprolactinemic rat model [rats bearing anterior pituitary grafts under the capsule of the kidney (AP-grafted rats)] was used to study sleep-wake activity and cortical brain temperature (T(crt)). Fisher 344 male rats (n = 24) were implanted with anterior pituitaries from rat pups; the control rats (n = 12) were sham-operated. Sleep-wake activity and T(crt) were recorded for 2 days between weeks 3 and 7 after surgery. The hyperprolactinemic state of the rats was confirmed by plasma prolactin (PRL) assays on week 7 and by determination of PRL mRNA levels in the anterior pituitary of the AP-grafted rats. Neither growth hormone plasma concentration nor pituitary mRNA levels were affected by the pituitary grafts. Duration of non-rapid eye movement sleep (NREMS) was slightly enhanced in the AP-grafted rats. A large increase in rapid eye movement sleep (REMS) during the 12-h light period was the major effect of the implantation of the extra pituitaries. Both the duration and the frequency of the REMS episodes increased and persisted for weeks 4-7 post-implantation. The nocturnal states of vigilance, T(crt), and intensity of NREMS (EEG slow wave activity) were not altered. The results clearly indicate that the enhancements in REMS persist during hyperprolactinemia, and support the hypothesis that PRL possesses REMS-promoting activity.

The seemingly identical 7SK and U6 core promoters depend on different transcription factor complexes.

Fractions obtained from HeLa cell extracts were used to study RNA polymerase III-catalyzed transcription from the human 7SK and mouse U6 RNA promoters in vitro. Although both genes depend on two almost identical core promoter elements (TATA box and PSE), different fractions were required. The 7SK promoter revealed full activity with the phosphocellulose B fraction alone. In contrast, efficient transcription from the U6 promoter depended on the additional presence of the C or D fraction. The analysis of the b1 and b2 subfractions (obtained by DEAE-Sephadex chromatography) revealed that for both promoters the b1 and the phosphocellulose D fraction were mutually interchangeable. However, while both fractions were fully equivalent for the 7SK promoter, the U6 promoter revealed an additional requirement for the C fraction in the presence of the b1 fraction. Since the b1 and the D fractions enclose two different complexes of the TATA-binding protein (TBP), B-TFIID and D-TFIID, our results indicate that functionally these two complexes are responsible for the observed differences in transcription of the 7SK and U6 genes.

Preparation of a cathepsin D sensitive near-infrared fluorescence probe for imaging.

A variety of proteases are overexpressed or activated during pathogenesis and represent important targets for therapeutic drugs. We have previously shown that optical imaging probes sensitive in the near-infrared fluorescence (NIRF) spectrum can be used for in vivo imaging of enzyme activity. In the current study, we show that these probes can be designed with specificity for specific enzymes, for example, cathepsin D which is known to be overexpressed in many tumors. A NIR cyanine fluorochrome served as the optical reporter and was attached to the amino terminal of an 11 amino acid peptide sequence with specificity for cathepsin D. The peptides were subsequently attached to a synthetic graft copolymer for efficient tumoral delivery. The close spatial proximity of the multiple fluorochromes resulted in quenching of fluorescence in the bound state. A 350-fold signal amplification was observed post cleavage during in vitro testing. Cell culture experiments using a rodent tumor cell line stably transfected with human cathepsin D confirmed enzyme specific activation within cells. This sequence but not a scrambled control sequence showed enzyme specificity in vitro. We conclude that activatable NIRF optical probes can be synthesized to potentially probe for specific enzymes in living organisms.

A model system to quantitate tumor burden in locoregional lymph nodes during cancer spread.

In order to quantitate the metastatic burden of secondary tumor deposits in locoregional lymph nodes, we produced a green fluorescent protein (GFP)-expressing murine cell line (B16F1) metastatic to lymph nodes in immunocompetent mice. When implanted into the hindleg of mice, all animals developed paraaortic lymph node metastases. Fluorescence microscopy, immunohistochemistry, and RT-PCR confirmed the presence of metastatic cells in lymph nodes. Tumoral deposits occurred preferentially in marginal sinuses and spread superficially to the subcortical area. Western blot analysis showed that the local tumor burden could be quantitated in individual lymph nodes. This model system should be useful for quantitating metastatic invasion particularly of micrometastases and aid in the development of lymphotropic drugs to detect and/or treat lymph node metastases in advanced cancers.

DNA binding chelates for nonviral gene delivery imaging.

Noninvasive in vivo monitoring of gene delivery would provide a critically important information regarding the spatial distribution, local concentration, kinetics of removal and/or biodegradation of the expression vector. We developed a novel approach to noninvasive gene delivery imaging using heterobifunctional peptide-based chelates (PBC) bearing double-stranded DNA-binding groups and a technetium-binding amino acid motif. One of such chelates: Gly-Cys(Acm)-Gly-Cys(Acm)-Gly-Lys(4)-Lys-(N-epsilon-[4-(psoralen-8-yloxy)]butyrate)-NH(2) has been characterized and labeled with reduced (99m)Tc pertechnetate (oxotechnetate). The psoralen moiety (a DNA binding group of PBC) allowed linking to double-stranded DNA upon short-term irradiation with the near UV range light (>320 nm). Approximately 30-40% of added (99m)Tc-labeled PBC was nonextractable and co-eluted with a model pCMV-GFP vector during the gel-permeation chromatography. Nuclear imaging of "naked" DNA and DNA complexes with lipid-based transfection reagents ("lipoplexes") has been performed after systemic or local administration of (99m)Tc-PBC-labeled DNA in mice. Imaging results were corroborated with the biodistribution using (99m)Tc-PBC and (32)P-labeled DNA and lipoplexes. A markedly different biodistribution of (99m)Tc PBC-labeled DNA and lipoplexes was observed with the latter being rapidly trapped in the liver, spleen and lung. (99m)Tc PBC-DNA was used as an imaging tracer during in vivo transfection of B16 melanoma by local injection of "naked" (99m)Tc PBC-DNA and corresponding lipoplexes. As demonstrated by nuclear imaging, (99m)Tc PBC-DNA lipoplexes showed a slower elimination from the site of injection than (99m)Tc PBC-DNA alone. This result correlated with a higher expression of marker mRNA and green fluorescent protein as determined using RT-PCR and immunohistochemistry, respectively.

Expression of a human 7S K RNA gene in vivo requires a novel pol III upstream element.

The 5'-flanking sequences required for expression of a human 7S K RNA gene have been defined by mutant analysis. A -111 upstream deletion mutant showed full activity when analysed by in vitro transcription with HeLa cell extracts. In contrast, upon transfection into intact cells, this mutant only revealed a basal level activity of approximately 6% as compared to the wild-type promoter up to position -252. The deleted upstream sequence element acts as a transcriptional activator in vivo, in a strictly position and orientation-dependent manner. Two octamer-like binding motifs observed within this upstream sequence were both dispensable for proper function of this RNA polymerase III promoter in vivo. Instead, a detailed analysis of this region identified a CACCC-box element, together with its surrounding base pairs, as the essential upstream element required for expression of this 7S K RNA gene in vivo. Furthermore, this CACCC-box is centered within a footprint obtained with HeLa cell nuclear proteins.

Synthetic influenza viral double-stranded RNA induces an acute-phase response in rabbits.

Numerous studies have characterized the physiological effects of synthetic, high-molecular-weight, homopolymeric, double-stranded RNA (dsRNA), particularly polyriboinosinic.polyribocytidylic acid [Carter and De Clercq (1974): Science 186:1172-1178], but limited information exists regarding the physiological effects of dsRNA of viral composition and size. In this report, we determined sleep and fever responses of rabbits to intracerebroventricular injection of different doses of synthetic viral dsRNA (either 108 base pairs or 661 base pairs) derived from the N-terminal sequence of gene segment 3 of the A/PR/8/34-H1N1 (PR8) influenza virus. Both the108-mer and the 661-mer dsRNAs increased nonrapid eye movement sleep, suppressed rapid eye movement sleep, and induced fever. The 661-mer dsRNA had more potent somnogenic and pyrogenic effects than the 108-mer dsRNA on the basis of weight. Neither single-stranded RNA from the corresponding sequences had significant effects on sleep or brain temperature. These results demonstrate for the first time that low-molecular-weight, viral dsRNA has the stability in vivo that is required to induce the fever and sleep changes found in natural viral infections, and the hypothesis is supported that virus-associated dsRNA may be responsible for initiating the acute-phase response during viral infections.

Spontaneous release of stable viral double-stranded RNA into the extracellular medium by influenza virus-infected MDCK epithelial cells: implications for the viral acute phase response.

The viral factor responsible for triggering the acute phase response, or 'flu' syndrome, associated with many acute viral infections is not defined. One candidate viral factor is double-stranded RNA (dsRNA) generated during viral replication. In this report we demonstrate by reverse-transcriptase polymerase-chain reaction that nuclease-stable viral RNA was released from influenza-infected MDCK epithelial cells at the time of cell lysis. Removal of virion-associated RNA by ultracentrifugation left equal amounts of positive- and negative-strand viral RNA in the medium that resisted degradation by endogenous RNase in the medium and by exogenous RNase added prior to phenol extraction. These data are the first demonstration that viral RNA with characteristics of dsRNA is spontaneously released from dying influenza virus-infected cells, and thus is available to amplify cytokine induction and contribute to systemic disease.

Diurnal variations of tumor necrosis factor alpha mRNA and alpha-tubulin mRNA in rat brain.

The experiments described herein were designed to determine whether tumor necrosis factor alpha (TNF-alpha) displays a diurnal variation in various areas of the normal rat brain. TNF-alpha mRNA transcripts were detected by reverse-transcriptase polymerase chain reaction. To monitor diurnal changes in TNF-alpha and alpha-tubulin expression, rats were sacrificed every 4 h for 24 h starting 1 h after light onset; relative mRNA levels were determined for the cerebellum, cortex, hippocampus, hypothalamus and brainstem. TNF-alpha mRNA was higher during the light than in the dark phase in the hypothalamus and hippocampus. alpha-Tubulin mRNA exhibited a similar diurnal variation in the hypothalamus, hippocampus and cortex. In contrast, beta-actin mRNA was lower during the light phase than the dark phase in the hippocampus and cortex. The observed diurnal variations in TNF-alpha mRNA are consistent with the hypothesis that TNF has a physiological role in the brain.

Optical imaging of matrix metalloproteinase-2 activity in tumors: feasibility study in a mouse model.

PURPOSE: To develop an optical imaging method to determine the expression level of tumoral matrix metalloproteinase-2 (MMP-2) in vivo. MATERIALS AND METHODS: An optical contrast agent was developed that was highly activatable by means of MMP-2-induced conversion. Signal characteristics of the probe were quantified ex vivo with a recombinant enzyme. Animal tumor models were established with MMP-2-positive (human fibrosarcoma cell line, n = 4) and MMP-2-negative (well-differentiated mammary adenocarcinoma, n = 4) tumor cell lines. Both tumors were implanted into nude mice and were optically imaged after intravenous administration of the MMP-2-sensitive probe. RESULTS: The MMP-2-sensitive probe was activated by MMP-2 in vitro, producing up to an 850% increase in near-infrared fluorescent signal intensity. This activation could be blocked by MMP-2 inhibitors. MMP-2-positive tumors were easily identified as high-signal-intensity regions as early as 1 hour after intravenous injection of the MMP-2 probe, while contralateral MMP-2-negative tumors showed little to no signal intensity. A nonspecific control probe showed little to no activation in MMP-2-positive tumors. CONCLUSION: It is feasible to image MMP-2 enzyme activity in vivo by using near-infrared optical imaging technology and "smart" matrix metalloproteinase-sensitive probes.

MR imaging and scintigraphy of gene expression through melanin induction.

PURPOSE: To determine whether an expression vector that encodes for human tyrosinase, the key enzyme in the melanin production pathway, can be used to image gene expression with magnetic resonance (MR) imaging and scintigraphy. MATERIALS AND METHODS: Mouse fibroblasts and human embryonal kidney cells were transfected with an expression vector that contained a complete complementary DNA sequence that encodes the human tyrosinase gene (pcDNA3tyr). Transfected cells were assayed for messenger RNA presence, melanin staining, and indium-111 binding; scintigraphy and MR imaging were performed. RESULTS: Transfected cells contained tyrosinase messenger RNA and stained positively for melanin. Transfected cells had a higher In-111 binding capacity than nontransfected cells, a difference readily detectable with scintigraphy. MR imaging showed transfected cells to have markedly higher signal intensity after gene transfer than nontransfected cells. CONCLUSION: Gene transfer and expression in cell culture can be detected with MR imaging and scintigraphy. The proposed strategy of using an imaging marker gene may have a substantial effect on the noninvasive imaging of gene therapy.

In vivo assessment of vascular endothelial growth factor-induced angiogenesis.

To determine whether vascular endothelial growth factor (VEGF)-induced tumor microvascularity is detectable by in vivo NMR imaging, an experimental study was conducted in nude mice. Human breast cancer cells (MCF-7) and MCF-7 cells stably transfected with the cDNA for the VEGF165 isoform (MV165) were grown in nude mice and models were characterized by RT-PCR, Western blotting, ELISA, immunohistochemistry and NMR imaging using a novel synthetic protected graft copolymer (PGC) as a vascular probe. MV165 tumors showed a 1.6-fold higher microvascular density by histology. Both tumors showed identical MR signal intensities on non-contrast and Gd-DTPA enhanced images. PGC enhanced MR imaging of tumoral vascular volume fraction (VVF), however, revealed significant differences between the 2 tumor types (MV165: 8.9 +/- 2.1; MCF-7: 1.7 +/- 0.5; p < 0.003), as expected from histology. VVF changes were more heterogeneous in the MV165 model both among tumors as well as within tumors as determined 3-dimensionally at submillimeter resolutions. Our results have potential applications for non-invasive assessment of angiogenesis by in vivo imaging and for clinical monitoring during angiogenic therapies.

Activating-transcription-factor (ATF) regulates human 7S L RNA transcription by RNA polymerase III in vivo and in vitro.

The gene-external part of the human 7S L promoter was analyzed by transcription in vitro and in vivo. Compared to the wild type promoter (-178), a -66 5'deletion mutant revealed full activity in vitro but was inefficiently transcribed in vivo. Further deletion to -37 reduced template activity to 50% in vitro and to basal level expression in vivo (below 5%). A DNase I footprint observed around position -50 protected an ATF-like binding site ('TGACGT'). With respect to 7S L transcription regulation, the functionality of this ATF-like binding site was confirmed in competition experiments and by mutation analysis. Furthermore, S100 extracts of cells pretreated with forskolin in vivo to induce the cAMP system, revealed significantly increased transcription of 7S L RNA in vitro, with no effect on a 7S K RNA gene, lacking such an ATF binding site. Thus, the 7S L RNA gene too is controlled by a regulatory element originally defined in class II promoters and represents another rare example where a specific type of transcription regulation in vivo can be mimicked with cell-free extracts in vitro.