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Nuclear Medicine in the Era of Genomics and Proteomics: Lessons from Annexin V
Abstract
In the past decade, genomics and proteomics have begun to develop many new targets for potential diagnostic and therapeutic agents. Among the life sciences, nuclear medicine is also deeply involved in the field of clinical investigation. Experience with radiolabeled annexin V highlights the many steps required to translate a good basic-science concept into the clinical setting. This model also emphasizes the value of synergy between basic and medical specialties in developing and optimizing a clinically useful product initially derived from basic investigation.
... However, non-apoptotic PS exposure in the extracellular leaflet has been described during macrophage-mediated phagocytosis or immune cell activation [19], which could result in false positive signals. Thus, the in vivo performance of Annexin V requires improvement in terms of the signal-to-background ratio and specificity for apoptotic over non-apoptotic cells [20]. ...
... Although Annexin V is the standard reference for detecting apoptotic cells, it still needs improvement because of limited efficiency of delivery to target tissues, low signal-tobackground ratio, and low specificity for apoptotic over nonapoptotic cells [19,20,28]. To assess whether C ApoPep-1 can be a suitable substitute for Annexin V in vitro, apoptosis was induced in Jurkat T cells by treatment with STS and the proportion of apoptotic cells as a function of STS concentration was measured using Annexin V and C ApoPep-1. ...
... Although quantitative measurement of apoptosis is important for diagnosing the pathophysiological stages of RA, there are no useful probes for both in vivo imaging and ex vivo diagnosis [12,20]. To determine whether ApoPep-1 can be used as an imaging probe for real-time in vivo monitoring of apoptosis, we first tested whether it can detect apoptotic cells in tissue. ...
Apoptosis plays an essential role in the pathophysiologic processes of rheumatoid arthritis. A molecular probe that allows spatiotemporal observation of apoptosis in vitro, in vivo, and ex vivo concomitantly would be useful to monitoring or predicting pathophysiologic stages. In this study we investigated whether cyclic apoptosis-targeting peptide-1 (CApoPep-1) can be used as an apoptosis imaging probe in inflammatory arthritis. We tested the utility of CApoPep-1 for detecting apoptotic immune cells in vitro and ex vivo using flow cytometry and immunofluorescence. The feasibility of visualizing and quantifying apoptosis using this probe was evaluated in a murine collagen-induced arthritis (CIA) model, especially after treatment. CApoPep-1 peptide may successfully replace Annexin V for in vitro and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for ex vivo in the measurement of apoptotic cells, thus function as a sensitive probe enough to be used clinically. In vivo imaging in CIA mice revealed that CApoPep-1 had 42.9 times higher fluorescence intensity than Annexin V for apoptosis quantification. Furthermore, it may be used as an imaging probe for early detection of apoptotic response in situ after treatment. The CApoPep-1 signal was mostly co-localized with the TUNEL signal (69.6% of TUNEL⁺ cells) in defined cell populations in joint tissues of CIA mice. These results demonstrate that CApoPep-1 is sufficiently sensitive to be used as an apoptosis imaging probe for multipurpose applications which could detect the same target across in vitro, in vivo, to ex vivo in inflammatory arthritis.
... For this reason, radiolabeled annexin V has been evaluated as an imaging agent for the noninvasive detection of therapyinduced apoptosis. [10][11][12] However, the labeled protein is rapidly cleared from the body with a predominant renal excretion and a very short blood half-life (, 7 minutes). 11,12 On the other hand, complete annexin V binding requires times up to 1 hour and approximately 2.5 mM of extracellular Ca 2+ . ...
... [10][11][12] However, the labeled protein is rapidly cleared from the body with a predominant renal excretion and a very short blood half-life (, 7 minutes). 11,12 On the other hand, complete annexin V binding requires times up to 1 hour and approximately 2.5 mM of extracellular Ca 2+ . 13,14 Given its large molecular weight (35 kDa) and slower diffusion rate compared with low-molecular-weight compounds, there is concern whether optimal imaging of apoptotic cells in solid tumors is achievable with annexin V directly labeled with radioisotopes. ...
... However, the contrast of images in terms of signal to background ratio was clearly not optimal. 11 An alternative solution to optimize the imaging properties of radiolabeled annexin V is to use a longcirculating PEGylated annexin V. 15,16 Although improved visualization of apoptotic response in solid tumors was observed, a significant amount of the injected dose of annexin V was distributed to the liver and the kidneys. Other promising approaches toward optimizing biodistribution of annexin V for imaging applications include the use of mutant forms of annexin V with endogenous sites for the ( 99m Tc) labeling, 17 site-specific labeling, 18 and labeling with 18 F-fluorodeoxyglucose. 19 Small-molecular-weight peptides are a potential alternative to proteins for diagnostic applications. ...
Phosphatidylserine (PS) is a well-characterized biomarker for apoptosis. Ligands that bind to PS can be used for noninvasive imaging of therapy-induced cell death, particularly apoptosis. In this study, we screened a random 12-mer peptide phage library on liposomes prepared from PS. One clone displaying the peptide SVSVGMKPSPRP (designated as PS3-10) bound to PS approximately 4-fold better than its binding to phosphatidylcholine and 18-fold better than to bovine serum albumin in a solid-phase binding assay. In addition, the binding of the corresponding PS3-10 peptide to PS was significantly higher than that of a scrambled peptide. PS3-10 phages, but not a control 4-2-2 phage, bound to aged red blood cells that had PS exposed on their surface. Binding of PS3-10 phages and PS3-10 peptide to TRAIL-induced apoptotic DLD1 cells was 3.2 and 5.4 times higher than their binding to untreated viable cells, respectively. Significantly, immunohistochemical staining confirmed selective binding of PS3-10 phages to apoptotic cells. Our data suggest that panning of phage display libraries may allow the selection of suitable peptide ligands for apoptotic cells and that PS3-10 peptide may serve as a template for further development of molecular probes for in vitro and in vivo imaging of apoptosis.
... The most common method of detecting PS on a cell surface is to use the Ca 2+dependent, PS-binding protein Annexin V [172 -174]. For in vitro assays, the 35 kDa protein is typically labeled with a fluorescent dye, whereas radioactive and MRI contrast agents are employed for in vivo imaging [175,176]. Although widely utilized, the labeled protein is expensive and has a limited shelf life. ...
... Previously, we discovered that zinc dipicolylamine (Zn 2+ -DPA) complexes have a strong affinity for bilayer membranes that are enriched with anionic phospholipids [176]. ...
... & Sang Hyun Park parksh@kaeri.re.kr based imaging of apoptosis [16]. 99m Tc-labeled annexin-V has been used for the SPECT based detection of diverse medical disorders, such as acute cardiac rejection, myocardial infarction [17], organ transplant rejection studies [18], monitoring therapeutic effects of many anticancer drugs [19][20][21][22], chemotherapy effects, and the detection of cardiomyocyte death [23]. ...
Apoptosis is one of the fundamental phenomena behind successful radiation and chemotherapy treatments. Non-invasive imaging of apoptosis can offer an early diagnosis of disease and the true efficiency of an ongoing treatment procedure. The present study describes an attempt to develop 99mTc-labeled 2-methyl-2-pentylmalonic acid ([99mTc] 8) as a new SPECT based apoptosis imaging agent. An optimized chemical and radiosynthesis procedure provided desired product [99mTc] 8 with high radiochemical yield (84%, n = 3) and radiochemical purity (>99%) as determined by radio HPLC. Biodistribution data indicated that the radiotracer has a rapid clearance from blood and other background tissues. High testes accumulation confirmed the ability of the radiotracer to detect testicular apoptosis in mice.
... Proteomics, the study of proteomes on a large scale, promises to transform biology and medicine [42,43]. The study of proteomes in the large scale is defined as proteomics. ...
This paper presents a review of the importance and role of precision medicine and molecular imaging technologies in cancer diagnosis with therapeutics and diagnostics purposes. Precision medicine is progressively becoming a hot topic in all disciplines related to biomedical investigation and has the capacity to become the paradigm for clinical practice. The future of medicine lies in early diagnosis and individually appropriate treatments, a concept that has been named precision medicine, i.e. delivering the right treatment to the right patient at the right time. Molecular imaging is quickly being recognized as a tool with the potential to ameliorate every aspect of cancer treatment. On the other hand, emerging high-throughput technologies such as omics techniques and systems approaches have generated a paradigm shift for biological systems in advanced life science research. In this review, we describe the precision medicine, difference between precision medicine and personalized medicine, precision medicine initiative, systems biology/medicine approaches (such as genomics, radiogenomics, transcriptomics, proteomics, and metabolomics), P4 medicine, relationship between systems biology/medicine approaches and precision medicine, and molecular imaging modalities and their utility in cancer treatment and diagnosis. Accordingly, the precision medicine and molecular imaging will enable us to accelerate and improve cancer management in future medicine.
... The Zn 2+ coordination complexes allow users to identify apoptotic cells under Ca 2+ -free conditionsand with fast binding kinetics, which broadens the scope of the PS-based method for apoptosis detection. Indeed, the low molecular weight, non-protein probes presented may be adaptable to other imaging techniques, such as radiography and magnetic resonance spectroscopy.108 The first step toward development of such imaging reagents was achieved here by direct conjugation of a Zn 2+ -DPA PS affinity group to a phospholipidencapsulated quantum dot. ...
... Annexin V is a protein with a molecular weight (MW) of 36 kDa and has a high-affinity to phosphatidyserine (PS) that was exposed on the surface of apoptotic cells. It has been reported, however, that the in vivo performance of annexin V still needs improvement in terms of signal to background ratio and specificity to apoptotic cells over non-apoptotic cells [3,4]. ...
In vivo imaging of apoptosis could allow monitoring of tumor response to cancer treatments such as chemotherapy. Using phage display, we identified the CQRPPR peptide, named ApoPep-1(Apoptosis-targeting Peptide-1), that was able to home to apoptotic and necrotic cells in tumor tissue. ApoPep-1 also bound to apoptotic and necrotic cells in culture, while only little binding to live cells was observed. Its binding to apoptotic cells was not dependent on calcium ion and not competed by annexin V. The receptor for ApoPep-1 was identified to be histone H1 that was exposed on the surface of apoptotic cells. In necrotic cells, ApoPep-1 entered the cells and bound to histone H1 in the nucleus. The imaging signals produced during monitoring of tumor apoptosis in response to chemotherapy was enhanced by the homing of a fluorescent dye- or radioisotope-labeled ApoPep-1 to tumor treated with anti-cancer drugs, whereas its uptake of the liver and lung was minimal. These results suggest that ApoPep-1 holds great promise as a probe for in vivo imaging of apoptosis, while histone H1 is a unique molecular signature for this purpose.
The therapeutic efficacy of radioiodine (131I) therapy has been reported to be variable among cancer patients and even between metastatic regions in the same patients. Because the expression level of sodium iodide symporter (NIS) cannot reflect the efficacy of therapy, other strategies are required to predict the precise therapeutic effect of 131I therapy. In this research, we investigated the correlation between iodine (I) uptake, apoptosis imaging, and therapeutic efficacy. Two HT29 cell lines, cytomegalovirus (CMV)-NIS (or NIS+++) and TERT-NIS (or NIS+), were established by retroviral transfection. I uptake was estimated by I-uptake assay and gamma camera imaging. Apoptosis was evaluated by confocal microscopy and a Maestro fluorescence imaging system (CRi Inc., Woburn, MA) using ApoFlamma (BioACTs, Seoul, Korea), a fluorescent dye-conjugated apoptosis-targeting peptide 1 (ApoPep-1). Therapeutic efficacy was determined by tumor size. The CMV-NIS showed higher I uptake and ApoFlamma signals than TERT-NIS. In xenograft models, CMV-NIS also showed high 99m technetium signals and ApoFlamma signals. Tumor reduction had a stronger correlation with apoptosis imaging signals than with gamma camera imaging signals, which reflect I uptake. Higher NIS-expressing tumors showed increased apoptosis and I uptake, resulting in a significant tumor reduction. Moreover, tumor reduction showed a strong correlation with ApoFlamma imaging compared to I-uptake imaging.
For the preparation of novel organogel–carbon nanotube nanocomposites, pristine single-walled carbon nanotubes (SWNT) were incorporated into physical gels formed by an L-alanine based low molecular mass organogelator (LMOG). The gelation process and the properties of the resulting nanocomposites were found to depend on the kind of SWNTs incorporated in the gels. With pristine SWNTs, only a limited amount could be dispersed in the organogels. Attempted incorporation of higher amounts of pristine SWNTs led to precipitation from the gel. To improve their solubility in the gel matrix, a variety of SWNTs functionalized with different aliphatic and aromatic chains were synthesized. Scanning electron microscope images of the nanocomposites showed that the texture and organization of the gel aggregates were altered upon the incorporation of SWNTs. The microstructures of the nanocomposites were found to depend on the kind of SWNTs used. Incorporation of functionalized SWNTs into the organogels depressed the sol to gel transition temperature, with the n-hexadecyl chain functionalized SWNTs being more effective than the n-dodecyl chain functionalized counterpart. Rheological investigations of pristine SWNT containing gels indicated that the flow of nanocomposites became resistant to applied stress at a very low wt% of SWNT incorporation. Again more effective control of flow behavior was achieved with functionalized SWNTs possessing longer hydrocarbon chains. This happens presumably via effective interdigitation of the pendant chains with the fatty acid amides of L-alanine in the gel assembly. Remarkably, using near IR laser irradiation at 1064 nm for a short duration (1 min) at room temperature, it was possible to selectively induce a gel-to-sol phase transition of the nanocomposites, while prolonged irradiation (30 min) of the organogel under identical conditions did not cause gel melting.
Programmed cell death also called apoptosis is a universal phenomenon that rules key-biological processes in living organisms. Externalization of phosphatidylserine is one of the early cell membrane events in the apoptotic cascade, which occurs before bleb formation and DNA degradation. The annexin A5, an endogenous 36 KDa protein, binds with high affinity to cells with exposed phosphatidylserine in the presence of calcium ions. Accordingly, the radiolabeling of annexin A5 was proposed as molecular probe to trace apoptotic cells non invasively. The present review synthesizes the current knowledge on the experimental and clinical use of the radiolabeled annexin A5. Clinical perspectives are also suggested regarding future applications.
There is an increasing need to develop non-invasive molecular imaging strategies for visualizing and quantifying apoptosis status of diseases (especially for cancer) for diagnosis and monitoring treatment response. Since externalization of phosphatidylserine (PS) is one of the early molecular events during apoptosis, Annexin B1 (AnxB1), a member of Annexins family with high affinity toward the head group of PS, could be a potential positron emission tomography (PET) imaging probe for imaging cell death process after labeled by positron-emitting nuclides, such as 18F. In the present study, we investigated a novel PET probe, 18F-labeled Annexin B1 ( 18F-AnxB1), for apoptosis imaging. 18F-AnxB1 was prepared reliably by conjugating AnxB1 with a 18F-tag, N-succinimidyl 4-[ 18F]fluorobenzoate ([18F]SFB), in a radiolabeling yield of about 20 % within 40 min. The in vitro binding of 18F-AnxB1 with apoptotic cells induced by anti-Fas antibody showed twofold increase compared to those without treatment, confirmed by flow cytometric analysis with AnxV-FITC/PI staining. Stability tests demonstrated 18F-AnxB1 was rather stable in vitro and in vivo without degradation. The serial 18F-AnxB1 PET/CT scans in healthy rats outlined its biodistribution and pharmacokinetics, indicating a rapid renal clearance and predominant accumulation into kidney and bladder at 2 h p.i. 18F-AnxB1 PET/CT imaging was successfully applied to visualize in vivo apoptosis sites in tumor induced by chemotherapy and in kidney simulated by ischemia-reperfusion injury. The high-contrast images were obtained at 2 h p.i. to delineate apoptotic tumor. Apoptotic region could be still identified by 18F-AnxB1 PET 4 h p.i., despite the high probe retention in kidneys. In summary, we have developed 18F-AnxB1 as a PS-specific PET probe for the apoptosis detection and quantification which could have broad applications from disease diagnosis to treatment monitoring, especially in the cases of cancer.
The balance between proliferation and programmed cell death - apoptosis - is essential for multicellular organisms which use apoptosis to regulate and maintain the number and type of their cells during embryogenesis, growth and homeostasis. Increased cell proliferation or enhanced cell loss can be caused by dysregulated apoptosis and are observed in various diseases: in clinical scenarios such as neurodegenerative disorders, myocardial infarction and stroke the rate of apoptosis is upregulated compared to the physiological situation, while in clinical scenarios such as cancer or autoimmune diseases which are connected with pathological proliferation, apoptosis is often downregulated. Therefore, non-invasive imaging of apoptosis is of great clinical interest as patients would clearly benefit from the diagnosis of cell loss post infarction or from monitoring apoptosis triggered by chemotherapy or radiation therapy of tumours. Several biochemical transformations occur in apoptotic cells offering different biological targets for the development of specific molecular biomarkers of apoptosis. Key steps that occur during apoptosis have already been evaluated; among these are the externalisation of phospholipid phosphatidylserine to the outer leaflet of the cell membrane, which can be visualized by labeled annexin A5 and the activation of caspases, especially effector caspase-3, which can be addressed by labeled enzyme substrates or synthetic caspase inhibitors. Here, recent advances in tracer development for the molecular imaging techniques PET, SPECT and optical imaging are presented, the discussion of breakthroughs is involved, drawbacks and methodological issues of apoptosis imaging are highlighted.
Cancer therapy and cancer imaging are scientific and clinical companions. Most cancers are initially detected by an imaging technique, either through the routine screening of at-risk populations or by the evaluation of patients presenting with symptoms. Imaging the human body began as part of routine clinical care with the development of X-ray imaging by Roentgen. While X-rays still play an important role in cancer evaluation, the imaging technologies have expanded greatly over the past 30 years. Computerized tomographic (CT) imaging has added immeasurably to the clinician’s ability to find, measure, and monitor cancers. The algorithms originally developed by Hounsfield to produce tomographic images with X-rays have also been extended to nuclear medicine for use with positron emission tomography (PET) and single photon emission computed tomography (SPECT). The development of magnetic resonance imaging (MRI) has provided high levels of contrast with superb resolution in many areas of the body. These techniques have been complemented by ultrasound (US) imaging and more recently by the introduction of new optical approaches. Each technique has its strengths and limitations, leading clinicians to compare the results and data from the various approaches to determine the optimal imaging modalities to use for a given indication. Clinical presentation often leads a physician to obtain a series of images as an evaluation progresses or changes. For example, one might initially detect a suspicious lesion with a simple chest X-ray, more completely evaluate its characteristics and extent with a CT scan, and use PET to help stage the tumor within the thorax and abdomen or MRI to detect lesions within the brain. In fact, new devices are beginning to combine modalities, such as PET and CT, to take advantage of the strengths of each approach (Fig. 1)
.
an important role in cancer drug discovery for more than 60 years. The same models have proven critical as tools for the elucidation of the molecular basis of neoplastic transformation, the processes involved in tumor progression and metastasis, and the determinants of therapeutic success or failure. More recently, transgenic models in particular have been used to “validate” and prioritize new strategies for therapeutic intervention. In vivo cancer models can be considered to fall within two broad classes, transplantable models, and in situ models, each with a number of subtypes (Fig. 1). For pragmatic reasons, transplantable models as a group are the most commonly used for drug evaluation, while in situ models such as cancer-prone transgenic mice provide a rich source of information on cancer etiology. It should be noted that each transplantable model represents the tumor of a single patient, not a tumor type. This discussion is centered on the application of both model types, and the potential impact of imaging technologies for cancer drug discovery. However, with recent advances in preclinical imaging technologies, these models are also proving useful in the development and testing of new imaging techniques and contrast agents. Increasingly, with the expanding role of drugs tied to specific molecular targets, these models are also being used to optimize and validate clinical imaging strategies. Finally, molecular imaging techniques are finding a critical role preclinically in the simultaneous confirmation of mechanism of action and assessment of efficacy. This is particularly true in orthotopic or transgenic model systems.
Fig. 1.
Schematic representation of the broad categories of preclinical cancer models in use today. In situ tumor models can be subcategorized by the method for induction of the tumor. Transplantable tumor models are commonly subcategorized according to whether the tumor is implanted in the organ in which the cell line originated (orthotopic versus ectopic) and in the species in which it originated (syngeneic versus xenogeneic).
Computed tomography (CT) remains one of the mainstay techniques for anatomical evaluation of tumor growth and response to
therapy, both in clinical practice and drug trials (see Chapter 3). Computed tomography measurements of perfusion can be added
to a conventional CT examination to provide a functional response assessment within the same examination. This combined approach
can overcome some of the limitations of a purely structural evaluation, such as slow response to therapy and residual nonmalignant
masses, while avoiding the inconvenience to the patient and additional cost of further examinations using different imaging
modalities. Furthermore, as positron emission tomography (PET) systems are now commonly integrated with CT, functional CT
techniques can be combined with PET evaluations of tumor physiology using a single imaging device.
Over the past decade positron emission tomography (PET) has become the fastest growing medical imaging technology. This is
primarily based on its performance as a diagnostic tool in oncology. In particular, its sensitivity for detecting metastases
using whole body scans and 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) as a tracer is unrivaled, resulting in reimbursement for a steadily increasing number of indications. The development
of PET/computed tomography (CT) scanners has further stimulated the use of PET, as investment in such a scanner is now also
feasible for smaller hospitals.
Many diseases are associated with an abnormal increase or decrease in apoptosis (programmed cell death). A variety of newer
drugs have been designed to enhance apoptosis (such as cancer therapy) or decrease programmed cell death (such as agents interrupting
the immune inflammatory pathway in arthritis). Until recently apoptosis could be assessed only in vitro or by histological assay of biopsied material. To determine the effectiveness of therapy, it would be very helpful to have
an in vivo imaging technique. Characteristic changes of apoptosis can be identified by ultrasound, magnetic resonance, and radionuclide
approaches. The status of these approaches is outlined in this chapter with possible clinical uses listed in Table 1.
Positron emission tomography (PET) is a highly sensitive noninvasive functional imaging modality that can provide quantitative
in vivo tissue data with high temporal resolution. Such information can be exploited to assess the behavior of a drug within the
body (pharmacokinetics) and its effects on the biosystem (pharmacodynamics). Knowledge of in vivo drug pharmacology is especially invaluable in the assessment and development of anticancer agents and is likely to play a
significant role in the development of novel targeted therapies. Specifically, PET pharmacokinetic studies can provide information
on drug access, kinetics, and drug concentration in tumors and normal tissues, all of which have a bearing on drug activity,
tissue toxicity, and drug scheduling. In addition, such studies can provide proof of principle of the in vivo mechanism of drug action and in vivo targeting of molecular therapeutic targets. In this review, basic principles of PET drug kinetic measurement techniques are
discussed, followed by examples of PET kinetic studies performed at various stages of the drug developmental process and their
utility and contribution to anticancer drug development are highlighted.
Imaging is an essential step in the diagnostic pathway for patients with clinical signs pointing to the presence of a tumor.
Anatomical imaging is routinely applied to uncover the presence of a suspicious lesion. Then a biopsy specimen, taken of this
lesion, is analyzed by histopathology, which is the gold standard for the confirmation of the presence of cancer and its further
identification. Imaging methods have become an important complementary element in the differentiation between malignant and
benign lesions and in the determination of the local or metastatic extent of the tumor. Moreover, if cancer tissue is present,
newer (bio)imaging methods may help to establish the type, grade, and stage of the tumor. All this information is crucial
in the further management of disease, such as treatment planning, prediction of progression, and response to treatment. Magnetic
resonance (MR) is often the major modality in the imaging of cancer patients. In routine clinical practice MR is commonly
used to obtain anatomical information, but this technique is very versatile and offers many possibilities to acquire more
functional information, in particular, information of a physiological and metabolic nature. To increase the specificity and
sensitivity of common magnetic resonance imaging (MRI), more functional approaches are increasingly being included in clinical
examinations of patients with tumors. For instance, these may include perfusion weighted imaging (PWI) to assess vascular
functionality, diffusion weighted imaging (DWI) to assess tissue characteristics associated with water movement, and MR spectroscopy
(MRS) to assess tissue metabolism and physiology.
One of the most difficult obstacles to early treatment of cancer is lack of sound methodologies for detection of the disease.
By the time cancer is detected it has frequently progressed to the metastatic state. Because of this, the normal course of
treatment, in addition to surgery, often consists of aggressive chemotherapy, radiation therapy, or both with potential for
undesirable side effects (1)–(6).
While the ultimate goal of cancer treatment is to eliminate all evidence of the tumor’s presence, the more immediate goal
of treatment with chemotherapy, radiation, and biological agents is to decrease the tumor’s ability to replicate and increase
the death rate of cancer cells. This basis has inspired researchers to design and develop imaging approaches to assess tumor
proliferation and, more importantly, a tumor’s response to new treatments. Such approaches, while still in development, may
complement the routine imaging of tumor size now done as part of standard clinical care. In general, successful therapy should
lead to declines in the size of tumors as reflected by techniques such as computed tomography (CT) and magnetic resonance
imaging (MRI) (1,2). While anatomic imaging is relatively straightforward and readily available, it has a number of limitation. First, it measures
the size of the mass, but does not determine the cellularity or growth rate of the tumor. Second, after treatment, the tumor
may be left with a fibrotic mass that can persist even after successful treatment. While eventually a mass may decline in
size after therapy, this can take many weeks to months for the cells to lyse and finally to be absorbed. Finally, when therapy
is unsuccessful, it may take months for the failure of treatment to become apparent, since tumors may grow very slowly. In
fact, the doubling time of most tumors is generally from 1 to 3 months (3). It is for these reasons that imaging cell proliferation is attractive to assess treatment response and for potential use
to steer or change a course of therapy. This approach to positron emission tomography (PET) imaging may also complement imaging
other aspects of tumor metabolism, such as energetics, protein and membrane synthesis, and apoptosis.
Several approaches to magnetic resonance (MR) measurements of tumor perfusion (i.e., the nutritive blood flux through tissues
at the capillary level, alternatively termed tumor blood flow) and other properties of the tumor vasculature have been used
in both preclinical and clinical studies. This chapter will first describe the principal approaches that have been used for
measuring tumor perfusion and vascularity, including the data analysis methods used to extract quantitative information relating
to the underlying tumor physiology. The remainder of the chapter will focus on dynamic contrast-enhanced MRI (DCE-MRI), which
is currently the most widely used of these approaches. The methodological considerations for data acquisition, consensus approaches
for data analysis, potential advances in DCE-MRI, and applications representative of how it is being used in drug development
will be considered.
The concept of using gene therapy for cancer treatment was initially met with enthusiasm. The possibility of replacing or
altering damaged genes, the introduction of suicide genes into cancer cells, and the alteration of cell function as a consequence
of exogenous gene expression were advocated. Therapeutic genes can be transferred to patients through a variety of vehicles.
These include retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, lentiviruses, baculoviruses, liposomes,
bacterial hosts, naked DNA, DNA precipitates, and protein-DNA conjugates (Table 1) (1,2). However, the practical application of gene therapy to treat cancer has been somewhat disappointing so far. Major obstacles
remain, including the inability to target appropriate tissues and deliver therapeutic genes to a sufficient number of target
cells, the inability to monitor the level of expression of the therapeutic gene, the loss of therapeutic gene expression over
time, and the inability to correlate the level and duration of gene expression with therapeutic outcome.
One of the most important applications of positron emission tomography (PET) in clinical oncology is monitoring and assessing
tumor response to treatment (as discussed in Chapter 1). In contrast to conventional anatomic imaging, which is insensitive
for early assessment of therapeutic response, molecular imaging by PET can provide unique information about the likelihood
of tumor responsiveness to therapy before initiation of treatment or early during a course of treatment. Additionally, PET
is more sensitive and specific than conventional imaging for defining a complete or near-complete response after a course
of treatment has ended. One of the most challenging aspects of managing patients with breast cancer is determining the most
appropriate therapeutic regimen for an individual patient, based on the tumor’s biological features. Additionally, it is difficult
to identify patients who are not responding to the selected therapy shortly after starting treatment, so that an alternative
therapy can be initiated. The traditional criteria for assessing therapeutic response in breast cancer are based on changes
in size of measurable disease or on decline of tumor markers. Unfortunately, these criteria are not particularly useful in
distinguishing responders from nonresponders early after therapy is begun. Additionally, patients with predominantly or only
osseous metastatic disease represent a particularly difficult problem for treating physicians, as their disease is not usually
measurable or easily evaluable, and several months of therapy are needed before radiographs or bone scintigraphy can be used
to determine the effectiveness of therapy. The disease status of these patients can be monitored by changes in clinical symptoms,
in particular pain, and in tumor markers. However, not all such patients are symptomatic nor do all have abnormal tumor marker
levels to monitor.
The need to monitor cancer therapy-induced cellular and tissue changes using noninvasive imaging techniques continues to stimulate both basic and clinical research. Monitoring changes in cellular proliferative capacity that occur after treatment with radiation and/or chemotherapy has the potential to provide longitudinal information on the cellular dynamics of tumors before, during, and after therapeutic intervention. Cells can lose their reproductive potential through one of several mechanisms, including apoptosis and autophagy (which are forms of programmed cell death), premature senescence, or necrosis. When a tumor responds to therapy, current imaging methods do not provide information about the exact mechanism of cell death executed. We are now beginning to develop the molecular imaging tools that will enable us to noninvasively image cell death mechanisms both in experimental models and in the clinical cancer environment. Studies with these imaging tools will contribute to a better understanding of therapeutic responses and assist in the design and evaluation of more effective treatments. This review examines the state-of-the-art in the use of (radio)tracers for the purpose of imaging mechanisms of tumor cell inactivation (cell death) in animal models and in clinical trials.
The balance between proliferation and programmed cell death--apoptosis--is essential for multicellular organisms which use apoptosis to regulate and maintain the number and type of their cells during embryogenesis, growth and homeostasis. Increased cell proliferation or enhanced cell loss can be caused by dysregulated apoptosis and are observed in various diseases: in clinical scenarios such as neurodegenerative disorders, myocardial infarction and stroke the rate of apoptosis is upregulated compared to the physiological situation, while in clinical scenarios such as cancer or autoimmune diseases which are connected with pathological proliferation, apoptosis is often downregulated. Therefore, non-invasive imaging of apoptosis is of great clinical interest as patients would clearly benefit from the diagnosis of cell loss post infarction or from monitoring apoptosis triggered by chemotherapy or radiation therapy of tumours. Several biochemical transformations occur in apoptotic cells offering different biological targets for the development of specific molecular biomarkers of apoptosis. Key steps that occur during apoptosis have already been evaluated; among these are the externalisation of phospholipid phosphatidylserine to the outer leaflet of the cell membrane, which can be visualized by labeled annexin A5 and the activation of caspases, especially effector caspase-3, which can be addressed by labeled enzyme substrates or synthetic caspase inhibitors. Here, recent advances in tracer development for the molecular imaging techniques PET, SPECT and optical imaging are presented, the discussion of breakthroughs is involved, drawbacks and methodological issues of apoptosis imaging are highlighted.
Programmed cell death also called apoptosis plays a pivotal role in many physiological and pathological conditions. In the multi-step process of drug development, a number of medications are being designed to target strategic checkpoints of the apoptotic cascade either to induce or to inhibit programmed cell death. Conceptually, the assessment of programmed cell death in response to various therapeutic interventions appears to be critical for evaluating the efficacy of many drugs that act through apoptotic pathways. In the last decade, nuclear medicine techniques provided proofs of principle for the imaging of apoptosis both in vitro and in vivo. The purpose of this article was to review current knowledge on the imaging of apoptosis with radiolabelled annexin A5 in various pre-clinical and clinical models, and beyond that, to assess the potential integration of such a dedicated technology into translational research.
The phospholipid bilayer surrounding animal cells is made up of four principle phospholipid components, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM). These four phospholipids are distributed between the two monolayers of the membrane in an asymmetrical fashion, with PC and SM largely populating the extracellular leaflet and PE and PS restricted primarily to the inner leaflet. Breakdown in this transmembrane phospholipid asymmetry is a hallmark of the early to middle stages of apoptosis. The consequent appearance of PS on the extracellular membrane leaflet is commonly monitored using dye-labeled Annexin V, a 36 kDa, Ca2+-dependent PS binding protein. Substitutes for Annexin V are described, including small molecules, nanoparticles, cationic liposomes, and other proteins that can recognize PS in a membrane surface. Particular attention is given to the use of these reagents for detecting apoptosis.
Novel bifunctional chelating agents bearing an aromatic rigid backbone have been synthesized and characterized on the basis of spectroscopic techniques. These macrocyclic multidentate chelating agents were conjugated with monoclonal antibody which forms stable complexes with 99mTc with high radiochemical purity.
The appearance of phosphatidylserine on the membrane surface of apoptotic cells (Jurkat, CHO, HeLa) is monitored by using a family of bis(Zn2+-2,2'-dipicolylamine) coordination compounds with appended fluorescein or biotin groups as reporter elements. The phosphatidylserine affinity group is also conjugated directly to a CdSe/CdS quantum dot to produce a probe suitable for prolonged observation without photobleaching. Apoptosis can be detected under a wide variety of conditions, including variations in temperature, incubation time, and binding media. Binding of each probe appears to be restricted to the cell membrane exterior, because no staining of organelles or internal membranes is observed.
Atherosclerosis, a major cause of disease and death from cardiovascular disease (CVD), is an inflammatory disease characterized by T cell and monocyte/macrophage infiltration in the intima of large arteries. During recent years and with improved treatment of acute disease manifestations, it has become clear that the risk of CVD is very high in systemic lupus erythematosus (SLE), often considered a prototypic autoimmune disease. A combination of traditional and non-traditional risk factors, including dyslipidemia, inflammation, antiphospholipid antibodies (aPL) and lipid oxidation are related to CVD in SLE. aPL are highly thrombogenic, and possible mechanisms include direct effects of aPL on endothelial and other cells, and interference with coagulation reactions. More than a thousand proteins of the annexin-superfamily are expressed in eukaryotes. Annexins are ubiquitous, highly conserved, predominantly intracellular proteins, widely distributed in tissues. Annexin A5 (ANXA5) is an important member of the annexin family due to its antithrombotic properties. These are believed to be caused by it forming a two-dimensional protective shield, covering exposed potentially thrombogenic cell surfaces. Recently, ANXA5 has been implicated in SLE since aPL interfere with ANXA5 binding to placental trophoblasts, causing microthrombosis and miscarriage, a rather common complication in SLE. We recently demonstrated that ANXA5 may play a role in CVD and is abundant in late-stage atherosclerotic lesions. Sera from SLE-patients with a history of CVD inhibited ANXA5 binding to endothelium, caused by IgG antibodies, to a significant degree aPL. This review will focus on potential involvement of ANXA5 in pathogenesis of CVD, particularly caused by underlying atherosclerosis and atherothrombosis.
The appearance of anionic phosphatidylserine (PS) in the outer monolayer of the plasma membrane is a universal indicator of the early/intermediate stages of cell apoptosis. The most common method of detecting PS on a cell surface is to use the protein annexin V; however, in certain applications there is a need for alternative reagents. Recent research indicates that rationally designed zinc 2,2'-dipicolylamine (Zn2+-DPA) coordination complexes can mimic the apoptosis sensing function of annexin V. Here, a series of fluorescently-labelled, tri- and pentapeptides with side chains containing Zn2+-DPA are prepared and shown to selectively bind to anionic vesicle membranes. Fluorescein-labelled versions of the peptides are used to detect apoptotic cells by fluorescence microscopy and flow cytometry.
Apoptosis (programmed cell death) plays a key role in the pathogenesis of many disorders including cerebral and myocardial ischemia, autoimmune and neurodegenerative diseases, infections, organ and bone marrow transplant rejection, and tumor response to chemotherapy and/or radiotherapy. Apoptosis in itself represents a complex mechanism where numerous (pro-apoptotic and anti-apoptotic) molecules interact in an elaborate manner. Since the original description by Kerr et al. in 1972, clinical assessment of apoptosis has always required biopsies or aspirated material for in vitro investigations. Several well-established methods are available for in vitro tests using tissue specimens. However, a non-invasive detection of apoptosis would be of great benefit for many patients in various situations. Today, non-invasive techniques for direct in vivo detection of apoptotic cells are rare and urgently need improvement. The early in vivo detection of apoptotic cells can provide the physician with important information to develop further therapeutic strategies in chemotherapy or radiotherapy of tumors, in transplantation of organs, or in healing of infarct areas. In some preliminary publications, several authors reported on the in vivo use of caspase-inhibitors and annexin V, labeled with indium-111, technetium-99m, iodine-123, iodine-124 or fluoride-18. In the present paper, we review the current applicability of both techniques for in vivo apoptosis imaging, and discuss the methodical problems.
Annexin V binds phosphatidylserine moieties on apoptotic cells. This study reports the initial experience at Stanford University Medical Center with 99mTc-labeled annexin V imaging as a noninvasive measure of apoptosis in acute cardiac rejection. Ten cardiac transplant patients had 99mTc Annexin V imaging and endomyocardial biopsy (EMB) performed within 24 h. No complications related to 99mTc annexin V administration occurred. Eight patients had ISHLT grade of acute rejection of 1A or less. Five patients had two or more areas of uptake noted in the right ventricle on imaging studies. Two of these patients had positive biopsies: one patient had grade 2 rejection with two focal uptake areas and another had grade 3A rejection with three foci. An additional five patients had either one or zero hot spot areas and corresponding negative EMBs. 99mTc-annexin V appears to be well tolerated and may identify patients with acute cardiac rejection.
Proteins of the annexin/lipocortin family act as in vitro anticoagulants by binding to anionic phospholipid vesicles. In this study, we investigated whether annexin V (placental anticoagulant protein I) would bind to human platelets. Annexin V bound to unstimulated platelets in a reversible, calcium-dependent reaction with an apparent Kd of 7 nM and 5000-8000 sites/platelet. Additional binding sites could be induced by several platelet agonists in the following order of effectiveness: A23187 greater than collagen + thrombin greater than collagen greater than thrombin. However, neither ADP nor epinephrine induced additional binding sites. Three other proteins of the annexin family (annexins II, III, and IV) competed for annexin V platelets binding sites with the same relative potencies previously observed for binding to phospholipid vesicles. Phospholipid vesicles containing phosphatidylserine completely inhibited binding of annexin V to platelets. Annexin V completely blocked binding of 125I-factor Xa to thrombin-stimulated platelets. These results support the hypothesis that phosphatidylserine exposure occurs during platelet activation and may be necessary for assembly of the prothrombinase complex on platelet membranes.
One of the earliest events in programmed cell death is the externalization of phosphatidylserine, a membrane phospholipid
normally restricted to the inner leaflet of the lipid bilayer. Annexin V, an endogenous human protein with a high affinity
for membrane bound phosphatidylserine, can be used in vitro to detect apoptosis before other well described morphologic or nuclear changes associated with programmed cell death. We
tested the ability of exogenously administered radiolabeled annexin V to concentrate at sites of apoptotic cell death in vivo. After derivatization with hydrazinonicotinamide, annexin V was radiolabeled with technetium 99m. In vivo localization of technetium 99m hydrazinonicotinamide-annexin V was tested in three models: fuminant hepatic apoptosis induced
by anti-Fas antibody injection in BALB/c mice; acute rejection in ACI rats with transplanted heterotopic PVG cardiac allografts;
and cyclophosphamide treatment of transplanted 38C13 murine B cell lymphomas. External radionuclide imaging showed a two-
to sixfold increase in the uptake of radiolabeled annexin V at sites of apoptosis in all three models. Immunohistochemical
staining of cardiac allografts for exogenously administered annexin V revealed intense staining of numerous myocytes at the
periphery of mononuclear infiltrates of which only a few demonstrated positive apoptotic nuclei by the terminal deoxynucleotidyltransferase-mediated
UTP end labeling method. These results suggest that radiolabeled annexin V can be used in vivo as a noninvasive means to detect and serially image tissues and organs undergoing programmed cell death.
The purpose of this study was to develop an imaging technique to measure and monitor tumor cells undergoing programmed death caused by radiation and chemotherapy using 99mTc-EC-annexin V. Annexin V has been used to measure programmed cell death both in vitro and in vivo. Assessment of apoptosis would be useful to evaluate the efficacy and mechanisms of therapy and disease progression or regression.
Ethylenedicysteine (EC) was conjugated to annexin V using sulfo-N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-HCl as coupling agents. The yield of EC-annexin V was 100%. In vitro cellular uptake, pre- and post-radiation (10-30 Gy) and paclitaxel treatment, was quantified using 99mTc-EC-annexin V. Tissue distribution and planar imaging of 99mTc-EC-annexin V were determined in breast tumor-bearing rats at 0.5, 2, and 4 hrs. To demonstrate in vivo cell apoptosis that occurred during chemotherapy, a group of rats was treated with paclitaxel and planar imaging studies were conducted at 0.5-4 hrs. Computer outlined region of interest (ROI) was used to quantify tumor uptake on day 3 and day 5 post-treatment.
In vitro cellular uptake showed that there was significantly increased uptake of 99mTc-EC-annexin V after irradiation (10-30 Gy) and paclitaxel treatment. In vivo biodistribution of 99mTc-EC-annexin in breast tumor-bearing rats showed increased tumor-to-blood, tumor-to-lung and tumor-to-muscle count density ratios as a function of time. Conversely, tumor-to-blood count density ratios showed a time-dependent decrease with 99mTc-EC in the same time period. Planar images confirmed that the tumors could be visualized clearly with 99mTc-EC-annexin. There was a significant difference of ROI ratios between pre- and post-paclitaxel treatment groups at 2 and 4 hrs post injection.
The results indicate that apoptosis can be quantified using 99mTc-EC-annexin and that it is feasible to use 99mTc-EC-annexin to image tumor apoptosis.
The progress in diagnostic nuclear medicine over the years since the discovery of 99mTc is indeed phenomenal. Over 80% of the radiopharmaceuticals currently being used make use of this short-lived, metastable radionuclide, which has reigned as the workhorse of diagnostic nuclear medicine. The preeminence of 99mTc is attributable to its optimal nuclear properties of a short half-life and a gamma photon emission of 140 keV, which is suitable for high-efficiency detection and which results in low radiation exposure to the patient. 99mTcO4-, which is readily available as a column eluate from a 99Mo/99mTc generator, is reduced in the presence of chelating agents. The versatile chemistry of technetium emerging from the 8 possible oxidation states, along with a proper understanding of the structure-biologic activity relationship, has been exploited to yield a plethora of products meant for morphologic and functional imaging of different organs. This article reviews the evolution of 99mTc dating back to its discovery, the development of 99Mo/99mTc generators, and the efforts to exploit the diverse chemistry of the element to explore a spectrum of compounds for diagnostic imaging, planar, and single photon emission computed tomography. A brief outline of the 99mTc radiopharmaceuticals currently being used has been categorically presented according to the organs being imaged. Newer methods of labeling involving bifunctional chelating agents (which encompass the "3 + 1" ligand system, Tc(CO)3(+1)-containing chelates, hydrazinonicotinamide, water-soluble phosphines, and other Tc-carrying moieties) have added a new dimension for the preparation of novel technetium compounds. These developments in technetium chemistry have opened new avenues in the field of diagnostic imaging. These include fundamental aspects in the design and development of target-specific agents, including antibodies, peptides, steroids, and other small molecules that have specific receptor affinity.
As we emerge into the post-genome era, proteomics finds itself as the driving force field as we translate the nucleic acid information archive into understanding how the cell actually works and how disease processes operate. Even so, the traditionally held view of proteomics as simply cataloging and developing lists of the cellular protein repertoire of a cell are now changing, especially in the sub-discipline of clinical proteomics. The most relevant information archive to clinical applications and drug development involves the elucidation of the information flow of the cell; the "software" of protein pathway networks and circuitry. The deranged circuitry of the cell as the drug target itself as well as the effect of the drug on not just the target, but also the entire network, is what we now are striving towards. Clinical proteomics, as a new and most exciting sub-discipline of proteomics, involves the bench-to-bedside clinical application of proteomic tools. Unlike the genome, there are potentially thousands of proteomes: each cell type has its own unique proteome. Moreover, each cell type can alter its proteome depending on the unique tissue microenvironment in which it resides, giving rise to multiple permutations of a single proteome. Since there is no polymerase chain reaction equivalent to proteomics- identifying and discovering the "wiring diagram" of a human diseased cell in a biopsy specimen remains a daunting challenge. New micro-proteomic technologies are being and still need to be developed to drill down into the proteomes of clinically relevant material. Cancer, as a model disease, provides a fertile environment to study the application of proteomics at the bedside. The promise of clinical proteomics and the new technologies that are developed is that we will detect cancer earlier through discovery of biomarkers, we will discover the next generation of targets and imaging biomarkers, and we can then apply this knowledge to patient-tailored therapy.
Proteomics is a promising approach in the identification of proteins and biochemical pathways involved in carcinogenesis. Proteomic technologies are now being incorporated in oncology in the post-genomic era. Cancer involves alterations in protein expression and provides a good model not only for detection of biomarkers but also their use in drug discovery. Proteomics has an impact on diagnostics as well as drug discovery. Genomics still remains an important approach but the value of proteomics lies in the fact that most of the diagnostics and drugs target proteins. The importance of application of proteomics in oncology is recognized by the publication of this special issue of TRCT.
Radiolabeled annexin V may allow for repetitive and selective in vivo identification of apoptotic cell death without the need for invasive biopsy. This study reports on the relationship between quantitative technetium-99m- (99mTc-) 6-hydrazinonicotinic (HYNIC) radiolabeled annexin V tumor uptake, and the number of tumor apoptotic cells derived from histologic analysis.
Twenty patients (18 men, two women) suspected of primary (n = 19) or recurrent (n = 1) head and neck carcinoma were included. All patients underwent a spiral computed tomography (CT) scan, 99mTc-HYNIC annexin V tomography, and subsequent surgical resection of the suspected primary or recurrent tumor. Quantitative 99mTc-HYNIC annexin V uptake in tumor lesions divided by the tumor volume, derived from CT, was related to the number of apoptotic cells per tumor high-power field derived from terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assays performed on sectioned tumor slices.
Diagnosis was primary head and neck tumor in 18 patients, lymph node involvement of a cancer of unknown primary origin in one patient, and the absence of recurrence in one patient. Mean percentage absolute tumor uptake of the injected dose per cubic centimeter tumor volume derived from tomographic images was 0.0003% (standard deviation [SD], 0.0004%) at 1 hour postinjection (PI) and 0.0001% (SD, 0.0000%) at 5 to 6 hours PI (P =.012). Quantitative 99mTc-HYNIC annexin V tumor uptake correlated well with the number of apoptotic cells if only tumor samples with no or minimal amounts of necrosis were considered.
In the absence of necrosis, absolute 99mTc-HYNIC annexin V tumor uptake values correlate well with the number of apoptotic cells derived from TUNEL assays.
Apoptosis is common in advanced human atheroma and contributes to plaque instability. Because annexin V has a high affinity for exposed phosphatidylserine on apoptotic cells, radiolabeled annexin V may be used for noninvasive detection of apoptosis in atherosclerotic lesions.
Atherosclerotic plaques were produced in 5 rabbits by deendothelialization of the infradiaphragmatic aorta followed by 12 weeks of cholesterol diet; 5 controls were studied without manipulation. Animals were injected with human recombinant annexin V labeled with technetium-99m before imaging. Aortas were explanted for ex vivo imaging, macroautoradiography, and histological characterization of plaque. Radiolabeled annexin V cleared rapidly from the circulation (T1/2, alpha 9 and beta 46 minutes). There was intense uptake of radiolabel within lesions by 2 hours; no uptake was seen in controls. The results were confirmed in the ex vivo imaging of the explanted aorta. Quantitative annexin uptake was 9.3-fold higher in lesion versus nonlesion areas; the lesion-to-blood ratio was 3.0+/-0.37. Annexin uptake paralleled lesion severity and macrophage burden; no correlation was observed with smooth muscle cells. DNA fragmentation staining of apoptotic nuclei was increased in advanced lesions with evolving necrotic cores, predominantly in macrophages; the uptake of radiolabel correlated with the apoptotic index.
Because annexin V clears rapidly from blood and targets apoptotic macrophage population, it should constitute an attractive imaging agent for the noninvasive detection of unstable atherosclerotic plaques.
Hydrazino nicotinate (Hynic) is one of the most attractive bifunctional agents designed for the labeling of proteins with (99m)Tc. Recently, a (99m)Tc-labeled Hynic-Annexin V derivative has been described and successfully evaluated in animal models of apoptosis. Prior to a phase I human study, the preparation of (99m)Tc-Hynic-Annexin V has been optimized. The influence of the Hynic-load of Annexin V, amount of protein, nature and amount of reducing agent, activity and co-ligand on the labeling yield were evaluated using ITLC and size-exclusion FPLC. Optimal labeling yields were obtained when 60-90 microgram Hynic-Annexin V was labeled with up to 1.11 GBq (30 mCi) (99m)TcO(4)-using 10-20 microgram SnCl(2).2H(2)O as reducing agent and 1.5 mg tricine as the co-ligand. Biodistribution in normal mice was comparable to literature data.
During normal tissue remodeling, macrophages remove unwanted cells, including those that have undergone programmed cell death, or apoptosis. This widespread process extends to the deletion of thymocytes (negative selection), in which cells expressing inappropriate Ag receptors undergo apoptosis, and are phagocytosed by thymic macrophages. Although phagocytosis of effete leukocytes by macrophages has been known since the time of Metchnikoff, only recently has it been recognized that apoptosis leads to surface changes that allow recognition and removal of these cells before they are lysed. Our data suggest that macrophages specifically recognize phosphatidylserine that is exposed on the surface of lymphocytes during the development of apoptosis. Macrophage phagocytosis of apoptotic lymphocytes was inhibited, in a dose-dependent manner, by liposomes containing phosphatidyl-L-serine, but not by liposomes containing other anionic phospholipids, including phosphatidyl-D-serine. Phagocytosis of apoptotic lymphocytes was also inhibited by the L isoforms of compounds structurally related to phosphatidylserine, including glycerophosphorylserine and phosphoserine. The membranes of apoptotic lymphocytes bound increased amounts of merocyanine 540 dye relative to those of normal cells, indicating that their membrane lipids were more loosely packed, consistent with a loss of membrane phospholipid asymmetry. Apoptotic lymphocytes were shown to express phosphatidylserine (PS) externally, because PS on their surfaces was accessible to derivatization by fluorescamine, and because apoptotic cells expressed procoagulant activity. These observations suggest that apoptotic lymphocytes lose membrane phospholipid asymmetry and expose phosphatidylserine on the outer leaflet of the plasma membrane. Macrophages then phagocytose apoptotic lymphocytes after specific recognition of the exposed PS.
A critical event during programmed cell death (PCD) appears to be the acquisition of plasma membrane (PM) changes that allows phagocytes to recognize and engulf these cells before they rupture. The majority of PCD seen in higher organisms exhibits strikingly similar morphological features, and this form of PCD has been termed apoptosis. The nature of the PM changes that occur on apoptotic cells remains poorly defined. In this study, we have used a phosphatidylserine (PS)-binding protein (annexin V) as a specific probe to detect redistribution of this phospholipid, which is normally confined to the inner PM leaflet, during apoptosis. Here we show that PS externalization is an early and widespread event during apoptosis of a variety of murine and human cell types, regardless of the initiating stimulus, and precedes several other events normally associated with this mode of cell death. We also report that, under conditions in which the morphological features of apoptosis were prevented (macromolecular synthesis inhibition, overexpression of Bcl-2 or Abl), the appearance of PS on the external leaflet of the PM was similarly prevented. These data are compatible with the notion that activation of an inside-outside PS translocase is an early and widespread event during apoptosis.
[99mTc]Annexin V can be used to image organs undergoing cell death during cancer chemotherapy and organ transplant rejection. To simplify the preparation and labeling of annexin V for nuclear-medicine studies, we have investigated the addition of peptide sequences that will directly form endogenous chelation sites for 99mTc. Three mutant molecules of annexin V, called annexin V-116, -117, and -118, were constructed with N-terminal extensions of seven amino acids containing either one or two cysteine residues. These molecules were expressed cytoplasmically in Escherichia coli and purified to homogeneity with a final yield of 10 mg of protein/L of culture. Analysis in a competitive binding assay showed that all three proteins retained full binding affinity for erythrocyte membranes with exposed phosphatidylserine. Using SnCl2 as reducing agent and glucoheptonate as exchange agent, all three proteins could be labeled with 99mTc to specific activities of at least 50−100 μCi/μg. The proteins retained membrane binding activity after the radiolabeling procedure, and quantitative analysis indicated a dissociation constant (Kd) of 7 nmol/L for the annexin V-117 mutant. The labeling reaction was rapid, reaching a maximum after 40 min at room temperature. The radiolabeled proteins were stable when incubated with phosphate-buffered saline or serum in vitro. Proteins labeled to a specific activity of 25−100 μCi/μg were injected intravenously in mice at a dose of 100 μg/kg, and biodistribution of radioactivity was determined at 60 min after injection. Uptake of radioactivity was highest in kidney and liver, consistent with previous results obtained with wild-type annexin V. Cyclophosphamide-induced apoptosis in vivo could be imaged with [99mTc]annexin V-117. In conclusion, annexin V can be modified near its N-terminus to incorporate sequences that form specific chelation sites for 99mTc without altering its high affinity for cell membranes. These annexin V derivatives may be useful for in vivo imaging of cell death.
Either inadequate or excessive apoptosis (programmed cell death) is associated with many diseases. A method to image apoptosis
in vivo, rather than requiring histologic evaluation of tissue, could assist with therapeutic decision making in these disorders.
Programmed cell death is associated with a well-choreographed series of events resulting in the cessation of normal cell function,
and the ultimate disappearance of the cell. One component of apoptosis is signaling adjacent cells that this cell is committing
suicide by externalizing phosphatidylserine to the outer leaflet of the cell membrane. Annexin V, a 32-kDa endogenous human
protein, has a high affinity for membrane-bound phosphatidylserine. We have coupled annexin V with the bifunctional hydrazinonicotinamide
reagent (HYNIC) to prepare technetium-99m HYNIC-annexin V and demonstrated localization of radioactivity in tissues undergoing
apoptosis in vivo. In this report we describe the results of a series of experiments in mice and rats to characterize the
biologic behavior of 99mTc-HYNIC- annexin V. Biodistribution studies were performed in groups of rats at 10–180 min after intravenous injection of
99mTc-HYNIC-annexin V. In order to estimate the degree of apoptosis required for localization of 99mTc-annexin V in vivo, mice were treated with dexamethasone at doses ranging from 1 to 20 mg/kg, 5 h prior to 99mTc-HYNIC-annexin V administration, to induce thymic apoptosis. Thymus was excised 1 h after radiolabeled HYNIC-annexin V injection;
thymocytes were isolated, incubated with Hoechst 33342 followed by propidium iodide, and analyzed on a fluorescence-activated
cell sorter. Each sorted cell population was counted in a scintillation counter. To test 99mTc-HYNIC-annexin V as a tracer for external radionuclide imaging of apoptotic cell death, radionuclide imaging of Fas-defective
mice (lpr/lpr mice) and wild-type mice treated with the antibody to Fas (anti-Fas) was carried out 1 h post injection. Rat biodistribution
studies demonstrated a blood clearance half-time of less than 10 min for 99mTc-HYNIC-annexin V. The kidneys had the highest concentration of radioactivity at all time points. Studies in the mouse thymus
demonstrated a 40-fold increase in 99mTc-HYNIC-annexin V concentration in apoptotic thymocytes compared with the viable cell population. A correlation of r=0.78 was found between radioactivity and flow cytometric and histologic evidence of apoptosis. Imaging studies in the lpr/lpr and wild-type mice showed a substantial increase of activity in the liver of wild-type mice treated with anti-Fas, while
there was no significant change, irrespective of anti-Fas administration, in lpr/lpr mice. Excellent images of hepatic apoptosis were obtained in wild-type mice 30 min after injection of 99mTc-HYNIC-annexin V. The imaging results were consistent with histologic analysis in these animals. In conlusion, these studies
confirm the value of 99mTc-HYNIC-annexin V uptake as a marker for the detection and quantification of apoptotic cells in vivo.
Apoptosis, also known as programmed cell death, is an indispensable component of normal human growth and development, immunoregulation
and homeostasis. Apoptosis is nature’s primary opponent of cell proliferation and growth. Strict coordination of these two
phenomena is essential not only in normal physiology and regulation but in the prevention of disease. Programmed cell death
causes susceptible cells to undergo a series of stereotypical enzymatic and morphologic changes governed by ubiquitous endogenous
biologic machinery encoded by the human genome. Many of these changes can be readily exploited to create macroscopic images
using existing technologies such as lipid proton magnetic resonance (MR) spectroscopy, diffusion-weighted MR imaging and radionuclide
receptor imaging with radiolabeled annexin V. In this review the cellular phenomenon of apoptotic cell death and the imaging
methods which can detect the process in vitro and in vivo are first discussed. Thereafter an outline is provided of the role
of apoptosis in the pathophysiology of clinical disorders including stroke, neurodegenerative diseases, pulmonary inflammatory
diseases, myocardial ischemia and inflammation, myelodysplastic disorders, organ transplantation, and oncology, in which imaging
may play a critical role in diagnosis and patient management. Objective imaging markers of apoptosis may soon become measures
of therapeutic success or failure in both current and future treatment paradigms. Since apoptosis is a major factor in many
diseases, quantification and monitoring the process could become important in clinical decision making.
Two crystal forms (P6(3) and R3) of human annexin V have been crystallographically refined at 2.3 A and 2.0 A resolution to R-values of 0.184 and 0.174, respectively, applying very tight stereochemical restraints with deviations from ideal geometry of 0.01 A and 2 degrees. The three independent molecules (2 in P6(3), 1 in R3) are similar, with deviations in C alpha positions of 0.6 A. The polypeptide chain of 320 amino acid residues is folded into a planar cyclic arrangement of four repeats. The repeats have similar structures of five alpha-helical segments wound into a right-handed compact superhelix. Three calcium ion sites in repeats I, II and IV and two lanthanum ion sites in repeat I have been found in the R3 crystals. They are located at the convex face of the molecule opposite the N terminus. Repeat III has a different conformation at this site and no calcium bound. The calcium sites are similar to the phospholipase A2 calcium-binding site, suggesting analogy also in phospholipid interaction. The center of the molecule is formed by a channel of polar charged residues, which also harbors a chain of ordered water molecules conserved in the different crystal forms. Comparison with amino acid sequences of other annexins shows a high degree of similarity between them. Long insertions are found only at the N termini. Most conserved are the residues forming the metal-binding sites and the polar channel. Annexins V and VII form voltage-gated calcium ion channels when bound to membranes in vitro. We suggest that annexins bind with their convex face to membranes, causing local disorder and permeability of the phospholipid bilayers. Annexins are Janus-faced proteins that face phospholipid and water and mediate calcium transport.
Annexin V (placental anticoagulant protein I) binds tightly to anionic phospholipid vesicles in the presence of calcium. Four mutant proteins were expressed in Escherichia coli in which Ala replaced one of the following residues in the third repeat of annexin V: Arg-200, His-204, Arg-206, or Lys-207. In a competitive fluorescence quenching assay, the wild-type recombinant protein had the same affinity for phosphatidylserine-containing vesicles as the placentally derived protein. The affinity of the four mutant proteins for phosphatidylserine-containing vesicles was unchanged relative to wild-type protein. We conclude that His-204 and adjacent basic residues, including the highly conserved Arg-200 residue, are not required for high-affinity phospholipid binding.
We characterized the region of human chromosome 4q26-q28 that contains the gene encoding annexin V (placental anticoagulant protein I), a member of a family of calcium-dependent phospholipid binding proteins. A total of 14.5 kb, containing 9 introns, could be directly amplified from genomic DNA; the remainder was characterized from genomic clones in phage lambda and a yeast artificial chromosome. The gene was mapped with restriction enzymes BamHI, EcoRI, HindIII, SacI, StuI, and XbaI; the transcribed region spans 28 kb and contains 13 exons (44 tp 530 bp in size) and 12 introns (0.23 to 8.8 kb in size). Several putative transcription factor binding sites are present in the 5'-region, but the promoter has no recognizable TATA box. This study will facilitate further analysis of the functions of annexin V and its role in disease.
The growing awareness of the frequency and importance of physiological cell death (PCD) has resulted from two related developments, an increasing understanding of the biochemistry of PCD and a utilization of that understanding to develop sensitive methods for detecting PCD. The realization that this form of cell death is generally accompanied by internucleosomal DNA cleavage led to a variety of techniques for detecting this process, including DNA electrophoresis, flow cytometry, filtration assays, and in situ nick end labeling. Recent studies have demonstrated that PCD is accompanied by loss of phospholipid asymmetry across the plasma membrane as well as activation of various proteases, particularly members of the interleukin-1/3 converting enzyme (ICE) family. This chapter briefly describes the biochemical basis for each of these techniques and then attempts to summarize the strengths and weaknesses of each approach. In this regard Inhibitors of proteases (and other enzymes) are potentially useful tools when employed properly. Because the selectivity of these inhibitors is not fully known, however, experiments must be carefully designed, suitably controlled, and cautiously interpreted. Moreover, multiple alternative techniques (including antisense approaches and genetic knockouts) must be employed to confirm or refute conclusions suggested by inhibitor studies.
The goal of proteomics is a comprehensive, quantitative description of protein expression and its changes under the influence of biological perturbations such as disease or drug treatment. Quantitative analysis of protein expression data obtained by high-throughput methods has led us to define the concept of "regulatory homology" and use it to begin to elucidate the basic structure of gene expression control in vivo. Such investigations lay the groundwork for construction of comprehensive databases of mechanisms (cataloguing possible biological outcomes), the next logical step after the soon to be completed cataloguing of genes and gene products. Mechanism databases provide a roadmap towards effective therapeutic intervention that is more direct than that offered by conventional genomics approaches.
Apoptosis, or programmed cell death, has been suggested as a mechanism of immunologic injury during cardiac allograft rejection. We tested the hypothesis that technetium Tc 99m annexin V, a novel radiopharmaceutical used to detect apoptosis, can be used to detect cardiac allograft rejection by nuclear imaging.
Untreated ACI rats served as recipients of allogeneic PVG rat (n = 66) or syngeneic ACI rat (n = 30) cardiac grafts. Untreated recipient animals underwent 99mTc-annexin V imaging daily for 7 days. Region of interest analysis was used to quantify the uptake of 99mTc-annexin V. Immediately after imaging grafts were procured for histopathologic analysis and terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate-biotin nick-end labeling of apoptotic nuclei. One group was treated with 10 mg/kg/d cyclosporine (INN: ciclosporin) commencing on day 4 after transplantation (n = 6).
Untreated allografts showed histologic signs of rejection 4 days after transplantation. Apoptotic nuclei could be demonstrated in myocytes, endothelial cells, and graft-infiltrating cells of all rejecting allografts. Nuclear imaging revealed a significantly greater uptake of 99mTc-annexin V in rejecting allogeneic grafts than in syngeneic grafts on day 4 (P = .05), day 5 (P < .001), day 6 (P < .001), and day 7 (P = .013) after transplantation. A correlation between the histologic grade of acute rejection and uptake of 99mTc-annexin V was observed (r2 = 0.87). After treatment of rejection with cyclosporine, no apoptotic nuclei could be identified in allografts and uptake of 99mTc-annexin V decreased to baseline.
Apoptosis occurs during acute cardiac allograft rejection and disappears after treatment of rejection. 99mTc-annexin V can be used to detect and monitor cardiac allograft rejection.
Early detection and treatment of lung transplant rejection is critical for preservation of pulmonary graft function. Damage to pulmonary allografts is mediated by apoptotic cell death induced by the alloreactive T lymphocytes that infiltrate lung grafts. Previous studies demonstrate that acute cardiac allograft rejection can be visualized using radiolabeled annexin V. This study was done to determine whether this technique could visualize acute rejection in a rodent model of unilateral orthotopic lung transplantation.
Eighteen Sprague-Dawley ACI rats underwent removal of their left lung followed by orthotopic transplant of either an allogeneic (PVG, immunologically mismatched; N = 10) or a syngeneic (ACI, immunologically matched) pulmonary graft (N = 8). Animals were imaged 1 h after IV injection of 1 mCi (37.0 MBq) of (99m)Tc-annexin V 1 to 7 days after transplantation.
Lungs receiving the allograft demonstrated moderate to marked mononuclear infiltration of the perivascular, interstitial, and peribronchial tissues. No mononuclear infiltrates were noted in the native right lungs nor in the syngeneic transplants. Region of interest image analysis revealed significant (p < 0.0005) increases of transplant to normal lung activity ratios 3 to 7 days after allograft surgery. The increased annexin V uptake in these lungs was confirmed at biodistribution assay (allograft 151% greater than isograft activity, p < 0.005).
Acute experimental lung transplant rejection can be noninvasively identified using (99m)Tc-annexin V. Radiolabeled annexin V may be a clinically useful noninvasive screening tool for acute rejection.
To assess the value of imaging rejection-induced apoptosis with technetium 99m and annexin V, a human protein-based radiopharmaceutical used in the diagnosis of acute rejection of a liver transplant, in a well-characterized rodent model of orthotopic liver transplantation.
99mTc-radiolabeled annexin V was intravenously administered to six allografted (immunologically mismatched) and five isografted (immunologically matched) recipient rats on days 2, 4, and 7 after orthotopic liver transplantation. Animals were imaged 1 hour after injection of 0.2-2.0 mCi (8.0-74.0 MBq) of radiolabeled annexin V by use of clinical nuclear scintigraphic equipment.
All animals in the allografted group demonstrated marked increases of 55% and 97% above the activity in the isografted group in hepatic uptake of annexin V on days 4 and 7, respectively. Severe acute rejection was histologically detected in all allografted livers on day 7. There was no histologic evidence of acute rejection in isografted animals. Dynamic hepatobiliary imaging with 99mTc and mebrofenin, an iminodiacetic acid derivative, demonstrated no correlation with the presence or absence of acute rejection or with annexin V uptake.
Noninvasive imaging with radiolabeled annexin V is more sensitive and specific than imaging with 99mTc-mebrofenin in the diagnosis of acute rejection of a liver transplant.
In-vivo visualisation and quantification of the extent and time-frame of cell death after acute myocardial infarction would be of great interest. We studied in-vivo cell death in the hearts of patients with an acute myocardial infarction using imaging with technetium-99m-labelled annexin-V-a protein that binds to cells undergoing apoptosis.
Seven patients with an acute myocardial infarction and one control were studied. All patients were treated by percutaneous transluminal coronary angioplasty (six primary and one rescue), resulting in thrombolysis in myocardial infarction (TIMI) III flow of the infarct-related artery. 2 h after reperfusion, 1 mg annexin-V labelled with 584 MBq Tc-99m was injected intravenously. Early (mean 3.4 h) and late (mean 20.5 h) single-photon-emission computed tomographic (SPECT) images of the heart were obtained. Routine myocardial resting-perfusion imaging was also done to verify infarct localisation.
In six of the seven patients, increased uptake of Tc-99m-labelled annexin-V was seen in the infarct area of the heart on early and late SPECT images. No increased uptake was seen in the heart outside the infarct area. All patients with increased Tc-99m-labelled annexin-V uptake in the infarct area showed a matching perfusion defect. In a control individual, no increased uptake in the heart was seen.
Increased uptake of Tc-99m-labelled annexin-V is present in the infarct area of patients with an acute myocardial infarction, suggesting that programmed cell death occurs in that area. The annexin-V imaging protocol might allow us to study the dynamics of reperfusion-induced cell death in the area at risk and may help to assess interventions that inhibit cell death in patients with an acute myocardial infarction.
Annexin V was radiolabelled with iodine-123 in order to develop a SPECT-ligand for imaging atherosclerosis and apoptosis. Iodination by means of electrophylic substitution resulted in radiochemical yields up to 70% and specific activities of 7.4-92.5 MBq/microg protein. Binding experiments with blood platelets indicated that 123I-labelled annexin V remained its biological activity.
In developed countries, ischemic stroke is one of the leading causes of death and neurological impairment. The two most important therapeutic approaches in patients with acute cerebral ischemia consist of improving cerebral blood flow and blocking the biochemical and metabolic changes at the ischemic cascade level. The significant advances made in the past decade in the knowledge of the physiopathological mechanisms of cerebral ischemia, and the development of new drugs have given rise to true expectations regarding treatment and the rejection of nihilist attitudes. In the past 15 years, based on the excellent results obtained in experimental models of ischemia, many clinical trials have been conducted with different neuroprotective drugs. The results obtained in most studies have been negative, or the studies were terminated early owing to side effects. However, some drugs (citicoline, clomethiazole, piracetam and ebselen) have shown a certain degree of clinical efficacy, limited to subgroups of patients, and with a narrow therapeutic window, longer-lasting in the case of citicoline. The design of new clinical trials with neuroprotective drugs requires adequate preclinical assessment and the use of the new magnetic resonance techniques for the selection of patients and the assessment of the efficacy of treatment. The new trends in neuroprotection in focal cerebral ischemia and the results of the clinical trials published to date are reviewed.
The sequencing of the human genome was only made possible by the massively parallel use of automated high-throughput technologies. These technologies have required the development and interfacing of new hardware and software in a way which would have been hardly conceivable only ten years ago. As a consequence, an unbroken trend to more 'industrialized science' is apparent. The wealth of information generated by intensive sequencing efforts is now being further exploited by using complex tools to comprehensively analyze complex systems at the DNA, RNA and protein level. A landmark innovation was the introduction of the biochip principle, best exemplified with the development of the DNA chip, mainly used for RNA expression profiling. The chip principle, together with miniaturization, has now become the dominating theme for a number of new genomics and proteomics technologies, culminating in the lab-on-a-chip concept which, in the next five to ten years, could advance at a comparable rate to that of computers over the last 50 years. Some of the new technologies are already used for comprehensive analysis of clinical samples in an attempt to describe disease and disease risk at the molecular level. However, all of these technologies are far from routine in clinical use and it is also too early to decide whether molecular fingerprints or signature profiles will have the diagnostic and prognostic power currently predicted.
Efforts in genomics over the last decade have created a stream of opportunities for drug discovery. High-throughput DNA sequencing has forced a re-definition of the paradigm for identification and validation of targets for drug development. One purpose of this review is to delineate the different approaches to sequence data generation and to establish their various uses for the definition of gene function. There still remain crucial dilemmas for the pharmaceutical industry. The multitude of potential targets can each absorb enormous validation costs and the vast majority are likely to prove academically interesting but useless for drug development. An additional dimension arises from the importance of sequence variation between different individuals. These differences can determine response to therapy and must inform both the drug development process and healthcare delivery. This presents great challenges and opportunities for drug companies, their customers and society as a whole. I will review the technological aspects in some detail and give my view of the legal and social aspects. The field of bioinformatics is at the core of functional and pharmacogenomics and advances will depend on the continuing evolution of tools to interpret data. For the most part this evolution is reviewed in the context of specific application areas rather than as a discrete field, in recognition of its all-pervasive effects.
The purpose of this study was to determine the biodistribution and the associated radiation dose of technetium-99m 4,5-bis(thioacetamido)pentanoyl-annexin-V (99mTc-Apomate), a tracer proposed for the study of apoptosis. Eight patients (including two females) with normal kidney and liver functions were included in the study. An activity of 580 +/- 90 MBq of 99mTc-Apomate was injected intravenously, immediately followed by a dynamic study of 30 frames of 1 min each. At about 1 h, 4 h and 20 h p.i., whole-body scans were acquired. All activity distributions were measured using a dual-head gamma camera. Before injection of activity, a transmission scan with a cobalt-57 flood source had been performed to determine patient attenuation. Blood samples were taken every 10 min during the first hour after injection, and at about 4 and 20 h. Urine and faeces were collected during the first 20 h. Organ uptake was estimated after correction for body background activity, attenuation and scatter. Residence times were calculated from the dynamic and whole-body studies and used as input in the Mirdose 3.1 program to obtain organ doses and effective dose. It was found that radioactivity strongly accumulated in the kidneys and the liver [at 70 min p.i., 28% +/- 8% and 20% +/- 4% of the injected dose (ID), respectively]. Uptake in the target tissues (lymphomas or heart) was negligible from a dosimetric point of view. Extrapolating data from the first 20 h, one finds that approximately 73% of the ID will be excreted in the urine, and 27% in the faeces. The biological half-life of the activity in the total body was 16 +/- 7 h. Some organ doses +/- standard deviation (SD) in microGy/MBq were: kidneys 63 +/- 22, urinary bladder 20 +/- 6, spleen 15 +/- 3, liver 13 +/- 3, upper large intestine 12 +/- 6, lower large intestine 8 +/- 4, testes 6 +/- 2 and red bone marrow 4 +/- 0.7. The effective dose was 7.6 +/- 0.5 microSv/MBq, corresponding to a total effective dose of 4.6 +/- 0.3 mSv for a nominal injected activity of 600 MBq. In conclusion, 99mTc-Apomate has a high uptake in the kidneys and liver--in fact a factor of 1.3-1.6 higher than that found for the previously studied 99mTc-(n-1-imino-4-mercaptobutyl)-annexin-V. The biological half-life is shorter, however, but still long compared with the physical half-life of 99mTc. The faster appearance of activity in the intestines may preclude imaging of apoptosis in the abdomen. The effective dose is within the lower range of values reported for typical 99mTc compounds.
New technologies designed to facilitate the comprehensive analyses of genomes, transcriptomes and proteomes in health and disease are poised to exert a dramatic change on the pace of cancer research and to impact significantly on the care of cancer patients. These approaches have already demonstrated the power of molecular medicine in discriminating among disease subtypes that are not recognizable using traditional pathological criteria and in identifying specific genetic events involved in cancer progression. This review outlines the current status of these technologies and highlights recent studies in which they have been applied in the context of carcinogenesis.
Heart transplant rejection is characterized pathologically by myocyte necrosis and apoptosis associated with interstitial mononuclear cell infiltration. Any one of these components can be targeted for noninvasive detection of transplant rejection. During apoptotic cell death, phosphatidylserine, a phospholipid that is normally confined to the inner leaflet of cell membrane bilayer, gets exteriorized. Technetium-99m-labeled annexin-V, an endogenous protein that has high affinity for binding to phosphatidylserine, has been administered intravenously for noninvasive identification of apoptotic cell death. In the present study of 18 cardiac allograft recipients, 13 patients had negative and five had positive myocardial uptake of annexin. These latter five demonstrated at least moderate transplant rejection and caspase-3 staining, suggesting apoptosis in their biopsy specimens. This study reveals the clinical feasibility and safety of annexin-V imaging for noninvasive detection of transplant rejection by targeting cell membrane phospholipid alterations that are commonly associated with the process of apoptosis.
Now that the sequencing of the human genome has been completed, the basic challenges are finding the genes, locating their coding regions and predicting their functions. This will result in a new understanding of human biology as well as in the design of new molecular structures as potential novel diagnostic or drug discovery targets. The assessment of gene function may be performed using the tools of the genome program. These tools represent high-throughput methods used to evaluate changes in the expression of many or all genes of an organism at the same time in order to investigate genetic pathways for normal development and disease. This will lead to a shift in the scientific paradigm: In the pre-proteomics era, functional assignments were derived from hypothesis-driven experiments designed to understand specific cellular processes. The new tools describe proteins on a proteome-wide scale, thereby creating a new way of doing cell research which results in the determination of three-dimensional protein structures and the description of protein networks. These descriptions may then be used for the design of new hypotheses and experiments in the traditional physiological, biochemical and pharmacological sense. The evaluation of genetically manipulated animals or newly designed biomolecules will require a thorough understanding of physiology, biochemistry and pharmacology and the experimental approaches will involve many new technologies, including in vivo imaging with single-photon emission tomography and positron emission tomography. Nuclear medicine procedures may be applied for the determination of gene function and regulation using established and new tracers or using in vivo reporter genes such as enzymes, receptors, antigens or transporters. Pharmacogenomics will identify new surrogate markers for therapy monitoring which may represent potential new tracers for imaging. Also, drug distribution studies for new therapeutic biomolecules are needed, at least during preclinical stages of drug development. Finally, new biomolecules will be developed by bioengineering methods which may be used for isotope-based diagnosis and treatment of disease.
Annexins are Ca2+ and phospholipid binding proteins forming an evolutionary conserved multigene family with members of the family being expressed throughout animal and plant kingdoms. Structurally, annexins are characterized by a highly alpha-helical and tightly packed protein core domain considered to represent a Ca2+-regulated membrane binding module. Many of the annexin cores have been crystallized, and their molecular structures reveal interesting features that include the architecture of the annexin-type Ca2+ binding sites and a central hydrophilic pore proposed to function as a Ca2+ channel. In addition to the conserved core, all annexins contain a second principal domain. This domain, which NH2-terminally precedes the core, is unique for a given member of the family and most likely specifies individual annexin properties in vivo. Cellular and animal knock-out models as well as dominant-negative mutants have recently been established for a number of annexins, and the effects of such manipulations are strikingly different for different members of the family. At least for some annexins, it appears that they participate in the regulation of membrane organization and membrane traffic and the regulation of ion (Ca2+) currents across membranes or Ca2+ concentrations within cells. Although annexins lack signal sequences for secretion, some members of the family have also been identified extracellularly where they can act as receptors for serum proteases on the endothelium as well as inhibitors of neutrophil migration and blood coagulation. Finally, deregulations in annexin expression and activity have been correlated with human diseases, e.g., in acute promyelocytic leukemia and the antiphospholipid antibody syndrome, and the term annexinopathies has been coined.
Many therapeutically active anticancer treatments exert their effect by the induction of apoptosis and necrosis. Serial biopsies in breast cancer patients have suggested that response to therapy correlates with early posttreatment increases in tumor apoptotic index. Radiolabeled technetium Tc 99m-recombinant human (rh) annexin V provides a noninvasive technique for imaging treatment-induced cell death. Annexin V is a naturally occurring human protein that binds avidly to membrane-associated phosphatidylserine (PS). PS is normally found only on the inner leaflet of the cell membrane double layer, but it is actively transported to the outer layer as an early event in apoptosis and becomes available for annexin binding. Annexin also gains access to PS as a result of the membrane fragmentation associated with necrosis. In vitro studies of apoptosis using fluorescein annexin have shown good correlation with assessments of apoptosis documented by nuclear DNA degradation and caspase activation. In vivo localization of intravenously administered Tc 99m-annexin V has been demonstrated in numerous preclinical models of apoptosis, including anti-Fas-mediated hepatic apoptosis, rejection of allogeneic heterotopic cardiac allografts, cyclophosphamide treatment of murine lymphoma, cyclophosphamide-induced apoptosis in bone marrow, and leukocyte apoptosis associated with abscess formation. Scintigraphic studies in humans using Tc 99m-rh annexin V have demonstrated the feasibility of imaging cell death in acute myocardial infarction, in tumors with a high apoptotic index, and in response to anti-tumor chemotherapy of non-small cell lung cancer, small-cell lung cancer, breast cancer, lymphoma, and sarcoma. Increased localization of Tc 99m-rh annexin V within 1 to 3 days of chemotherapy has been noted in some, but not all, subjects with these tumors. To date, most subjects showing increased Tc 99m-rh annexin V uptake after the first course of chemotherapy have shown objective clinical responses. A single site study in 15 subjects with 1-year follow-up has suggested that increased posttreatment Tc 99m-rh annexin uptake is associated with improved time to progression of disease and survival time. In vivo imaging of cell death may have the potential to improve the treatment of cancer patients by allowing rapid, objective, patient-by-patient assessment of the efficacy of tumor cell killing.
With the human genome sequence now determined, the field of molecular medicine is moving beyond genomics to proteomics. In the field of cancer research, the key question is: how can oncologists best use techniques of proteomics in basic research and clinical application? In the postgenomic era, proteomics promises the discovery of biomarkers and tumor markers for early detection and diagnosis, novel protein-based drug targets for anticancer therapy, and new endpoints for the assessment of therapeutic efficacy and toxicity. This review paper will explore key themes in proteomics and their application in clinical cancer research.
Annexin-V is a calcium-dependent protein that binds with high affinity to phosphaditylserine exposed during apoptosis. The aim of this study was to radiolabel annexin-V with iodine-124 for use as a potential probe of apoptosis by positron emission tomography. Annexin-V was radioiodinated directly using the cyclotron-produced positron emitter iodine-124 by the chloramine-T (CAT) method and indirectly by the pre-labelled reagent N-succinimidyl 3-[124I]iodobenzoate ([124I]m-SIB). Some reaction parameters of the CAT method such as reaction time and pH were optimised to give radiochemical yields of 22.3 +/- 2.6%(n = 3, gel-filtration). After incubation with [124I]m-SIB, radiolabelled annexin-V was obtained in 14% and 25% yield by FPLC and gel-filtration, respectively. The radiochemical purities from direct and indirect labelling were 97.7 +/- 1.0%(n = 3) and 96.7 +/- 2.1%(n = 3), respectively. The new radiotracers could be stored for up to four days without significant de-iodination. The biological activity of radiolabelled annexin-V was tested in control and camptothecin-treated (i.e. apoptotic) human leukaemic HL60 cells. A significantly higher (21%) binding in treated cells was observed with [125I]m-SIB-annexin-V. The binding of [125I]m-SIB labelled annexin-V to camptothecin treated cells was blocked (68%) by a 100-fold excess of unlabelled annexin-V.
Inflammation and cell death are two important components of myocarditis. We evaluated the distribution of inflammation and apoptotic cell death in rats with autoimmune myocarditis using two radiotracers - technetium-99m Hynic-annexin V ((99m)Tc-annexin) as a marker of apoptotic cell death and carbon-14 deoxyglucose ((14)C-DG) as a marker of inflammation - in comparison with histologic findings. Three, 7 and 14 weeks after immunization with porcine cardiac myosin (acute, subacute, and chronic phases, respectively) (99m)Tc-annexin and (14)C-DG were injected. The uptake in the total heart was determined as the percentage of injected dose per gram (% ID/g) by tissue counting. Dual-tracer autoradiography with (99m)Tc-annexin and (14)C-DG was performed. The distribution of each of these agents was compared with the results of hematoxylin and eosin staining to identify areas of inflammation, and TUNEL staining to identify areas of apoptosis. Total cardiac uptake of (99m)Tc-annexin in the acute phase of myocarditis was significantly higher than that in normal rats (1.28%+/-0.30% vs 0.46%+/-0.01%; P<0.0001); it then decreased in the subacute phase and reached normal levels (0.56%+/-0.08% vs 0.60%+/-0.08%; P=NS). Total cardiac uptake of (14)C-DG in the acute phase of myocarditis was significantly higher than that in normal rats (2.78%+/-0.95% vs 1.02%+/-0.25%; P<0.0001); it then decreased in the subacute phase, but still remained higher than in controls (2.06%+/-0.52% vs 1.37%+/-0.46%; P<0.05). Using autoradiography and staining of tissue specimens, it was found that most histologic inflammatory foci corresponded to areas of high (14)C-DG uptake; some also corresponded to areas of high (99m)Tc-annexin uptake in the acute phase of myocarditis. (99m)Tc-annexin localization was strongly correlated with the number of TUNEL-positive cells (P<0.0001, r=0.83), but the uptake of (14)C-DG showed no relationship with it. There is a marked difference in the distribution of inflammation and apoptotic cell death in the myocardium of animals with immune myocarditis. These changes are mirrored by the localization of (14)C-DG and (99m)Tc-annexin. Sites of inflammation and zones of apoptotic cell death change over the course of immune myocarditis.
In this report, we describe the synthesis of 4-[18F]-fluorobenzoyl-annexin V (4-[18F]FBA). In a four-step procedure, 4-[18F]FBA was synthesised with a microcomputer controlled, automated module within 90min. The radiochemical yield was in the range of 15-20% (corrected for decay) with a specific activity of more than 35GBq/micromol. The specific binding was confirmed by studies of 4-[18F]FBA with phosphatidylserine-containing liposomes. The biological activity of 4-[18F]FBA was verified by measuring its binding to Jurkat T-cell lymphoblasts after induction of apoptosis as compared to control cells without apoptosis. 4-[18F]FBA seems to be a suited tracer to measure apoptotic activity in vivo.
Proteomics is the study of the function of all expressed proteins. Tremendous progress has been made in the past few years in generating large-scale data sets for protein-protein interactions, organelle composition, protein activity patterns and protein profiles in cancer patients. But further technological improvements, organization of international proteomics projects and open access to results are needed for proteomics to fulfil its potential.
We have developed a one-step procedure to introduce both polyethylene glycol (PEG) and the metal chelator diethylenetriaminepentaacetic acid (DTPA) to proteins through a heterofunctional PEG precursor. The PEG precursor contains DTPA at one end and an amine-reactive isothiocyanate (SCN-) functional group at the other end. It was obtained as lyophilized powder and could be stored at 4 degrees C for several months. Protein conjugation was achieved by simply mixing the proteins and the PEG precursor SCN-PEG-DTPA in an aqueous solution. As exemplified by the PEGylation and radiolabeling of annexin V, the resulting conjugate 111In-DTPA-PEG-annexin V showed selective binding to apoptotic cells in vitro and increased blood half-life in vivo. The PEGylated, radiolabeled annexin V may be useful in the noninvasive imaging of apoptosis.
Proteomics, the systematic evaluation of changes in the protein constituency of a cell, is more than just the generation of lists of proteins that increase or decrease in expression as a cause or consequence of disease. The ultimate goal is to characterize the information flow through protein pathways that interconnect the extracellular microenvironment with the control of gene transcription. The nature of this information can be a cause or a consequence of disease processes. Clinical applications of proteomics involve the use of proteomic technologies at the bedside. The analysis of human cancer as a model for how proteomics can have an impact at the bedside is now employing several new proteomic technologies that are being developed for early detection, therapeutic targeting and finally, patient-tailored therapy.
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PR049968A
Lessons from Annexin V
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