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Dual localization or dual targeting refers to the phenomenon by which identical, or almost identical, proteins are localized to two (or more) separate compartments of the cell. From previous work in the field, we had estimated that a third of the mitochondrial proteome is dual-targeted to extra-mitochondrial locations and suggested that this abundant dual targeting presents an evolutionary advantage. Here, we set out to study how many additional proteins whose main activity is outside mitochondria are also localized, albeit at low levels, to mitochondria (eclipsed). To do this, we employed two complementary approaches utilizing the α-complementation assay in yeast to uncover the extent of such an eclipsed distribution: one systematic and unbiased and the other based on mitochondrial targeting signal (MTS) predictions. Using these approaches, we suggest 280 new eclipsed distributed protein candidates. Interestingly, these proteins are enriched for distinctive properties compared to their exclusively mitochondrial-targeted counterparts. We focus on one unexpected eclipsed protein family of the Triose-phosphate DeHydrogenases (TDH) and prove that, indeed, their eclipsed distribution in mitochondria is important for mitochondrial activity. Our work provides a paradigm of deliberate eclipsed mitochondrial localization, targeting and function, and should expand our understanding of mitochondrial function in health and disease.

Several tumor suppressor genes do not follow the canonical function of cell cycle repressors. For example, fumarate hydratase (FH) is an evolutionary conserved TCA cycle enzyme that reversibly catalyzes the hydration of fumarate to L-malate and has a moonlight function in the DNA damage response (DDR). Interestingly, despite this enzyme’s essential role in central carbon metabolism, FH is inactive or absent in several tumors, such as hereditary leiomyomatosis and renal cell cancer (HLRCC). Accordingly, FH has a contradictory cellular function, as it is pro-survival through its role in the TCA cycle, yet its loss can drive tumorigenesis. These observations have supported the role of FH as a tumor suppressor. Here, we solved this contradiction by determining the molecular mechanisms that allow the cells to survive and even proliferate upon FH loss. We found that the cells’ response to FH loss is separated into two distinct time frames based on cell proliferation and DNA damage repair. During the early stages of FH loss, the cells’ proliferation and DNA damage repair are inhibited. However, over time the cells overcome the FH loss and form knockout clones, indistinguishable from WT cells in their proliferation rate. Due to the FH loss effect on DNA damage repair, we assumed that the recovered cells bear adaptive mutations. Therefore, we applied whole-exome sequencing to identify such mutated genes systematically. Indeed, we identified recurring mutations in genes belonging to the central oncogenic signaling pathways, such as JAK/STAT, which we validated to be impaired in FH-KO clones. Intriguingly, we demonstrated that these adaptive mutations are responsible for FH-KO cell proliferation under TCA cycle malfunction.

To overcome the lack of specificity of cancer therapeutics and thus create a more potent and effective treatment, we developed a novel chimeric protein, IL2-Smurf2. Here, we describe the production of this chimeric IL2-Smurf2 protein and its variants, with inactive or over-active killing components. Using Western blots, we demonstrated the chimeric protein’s ability to specifically enter target cells alone. After entering the cells, the protein showed biological activity, causing cell death that was not seen with an inactive variant, and that was shown to be apoptotic. The chimeric protein also proved to be active as an E3 ligase, as demonstrated by testing total ubiquitination levels along with targeted ubiquitination for degradation. Finally, we tested IL2-Smurf2 and its variants in an in vivo mouse model of leukemia and demonstrated its potential as a drug for the targeted treatment of cancer cells. In the course of this work, we established for the first time the feasibility of the use of Smurf2 as a killing component in chimeric targeting proteins. Utilizing the IL2 cytokine to target cells overexpressing IL-2R and Smurf2 to cause protein degradation, we were able to produce a chimeric protein with dual functionality which causes targeted cell death.

The intricate neuronal wiring during development requires cytoskeletal reorganization orchestrated by signaling cues. Because cytoskeletal remodeling is a hallmark of cell migration, we investigated whether metastatic cancer cells exploit axon guidance proteins to migrate. Indeed, in breast cancer patients, we found a significant correlation between mesenchymal markers and the expression of dihydropyrimidinase-like 2 (DPYSL2), a regulator of cytoskeletal dynamics in growing axons. Strikingly, DPYSL2 knockout in mesenchymal-like breast cancer cells profoundly inhibited cell migration, invasion, stemness features, tumor growth rate, and metastasis. Next, we decoded the molecular mechanism underlying this phenomenon and revealed an interaction between DPYSL2 and Janus kinase 1 (JAK1). This binding is crucial for activating signal transducer and activator of transcription 3 (STAT3) and the subsequent expression of vimentin, the promigratory intermediate filament. These findings identify DPYSL2 as a molecular link between oncogenic signaling pathways and cytoskeletal reorganization in migrating breast cancer cells.

Pathogenesis of neurodegenerative diseases involves dysfunction of mitochondria, one of the most important cell organelles in the brain, with its most prominent roles in producing energy and regulating cellular metabolism. Here we investigated the effect of transferring active intact mitochondria as a potential therapy for Alzheimer's disease (AD), in order to correct as many mitochondrial functions as possible, rather than a mono-drug related therapy. For this purpose, AD-mice (amyloid-β intracerebroventricularly injected) were treated intravenously (IV) with fresh human isolated mitochondria. One to two weeks later, a significantly better cognitive performance was noticed in the mitochondria treated AD-mice relative to vehicle treated AD-mice, approaching the performance of non-AD mice. We also detected a significant decrease in neuronal loss and reduced gliosis in the hippocampus of treated mice relative to untreated AD-mice. An amelioration of the mitochondrial dysfunction in brain was noticed by the increase of citrate-synthase and cytochrome c oxidase activities relative to untreated AD-mice, reaching activity levels of non-AD-mice. Increased mitochondrial activity was also detected in the liver of mitochondria treated mice. No treatment-related toxicity was noted. Thus, IV mitochondrial transfer may possibly offer a novel therapeutic approach for AD.

The intricate neuronal wiring during development requires cytoskeletal reorganization orchestrated by signaling cues. Considering that cytoskeletal remodeling is a hallmark of cell migration, we inquired whether metastatic cancer cells exploit the axon guidance proteins to migrate. Indeed, in breast cancer patients, we found a significant correlation between the mesenchymal markers and the expression of dihydropyrimidinase-like 2 (DPYSL2), a regulator of cytoskeletal dynamics in growing axons. Strikingly, DPYSL2 knockout in mesenchymal-like cells profoundly inhibited cell migration, invasion, stemness features, tumor growth rate, and metastasis. Next, we aimed to decode the molecular mechanism underlying this phenomenon and revealed an interaction between DPYSL2 and Janus kinase 1 (JAK1). This binding is crucial for triggering signal transducer and activator of transcription 3 (STAT3) and subsequently expressing vimentin, the pro-migratory intermediate filament. Collectively, we identified DPYSL2 as a molecular link between oncogenic signaling pathways and cytoskeletal reorganization in migrating breast cancer cells.Statement of significanceThis study shows that the axon guidance adaptor protein DPYSL2 is essential for promoting breast cancer migration. Specifically, this protein interacts with JAK1 to govern STAT3 signaling and subsequently vimentin expression.

Much effort has been dedicated in the recent decades to find novel protein/enzyme-based therapies for human diseases, the major challenge of such therapies being the intracellular delivery and reaching sub-cellular organelles. One promising approach is the use of cell-penetrating peptides (CPPs) for delivering enzymes/proteins into cells. In this review, we describe the potential therapeutic usages of CPPs (mainly trans-activator of transcription protein, TAT) in enabling the uptake of biologically active proteins/enzymes needed in cases of protein/enzyme deficiency, concentrating on mitochondrial diseases and on the import of enzymes or peptides in order to destroy pathogenic cells, focusing on cancer cells.

Mitochondrial Targeting Sequences (MTSs) are responsible for trafficking nuclear-encoded proteins into mitochondria. Once entering the mitochondria, the MTS is recognized and cleaved off. Some MTSs are long and undergo two-step processing, as in the case of the human frataxin (FXN) protein (80aa), implicated in Friedreich's ataxia (FA). Therefore, we chose the FXN protein to examine whether nuclear-encoded mitochondrial proteins can efficiently be targeted via a heterologous MTS (hMTS) and deliver a functional protein into mitochondria. We examined three hMTSs; that of citrate synthase (cs), lipoamide deydrogenase (LAD) and C6ORF66 (ORF), as classically MTS sequences, known to be removed by one-step processing, to deliver FXN into mitochondria, in the form of fusion proteins. We demonstrate that using hMTSs for delivering FXN results in the production of 4-5-fold larger amounts of the fusion proteins, and at 4-5-fold higher concentrations. Moreover, hMTSs delivered a functional FXN protein into the mitochondria even more efficiently than the native MTSfxn, as evidenced by the rescue of FA patients' cells from oxidative stress; demonstrating a 18%-54% increase in cell survival; and a 13%-33% increase in ATP levels, as compared to the fusion protein carrying the native MTS. One fusion protein with MTScs increased aconitase activity within patients' cells, by 400-fold. The implications form our studies are of vast importance for both basic and translational research of mitochondrial proteins as any mitochondrial protein can be delivered efficiently by an hMTS. Moreover, effective targeting of functional proteins is important for restoration of mitochondrial function and treatment of related disorders.

Metabolic reprogramming as a downstream result of oncogenic signaling pathways has been described as a hallmark of cancer. Here, we describe a reverse scenario in which a metabolic enzyme regulates cancer cell behavior by triggering a signaling pathway. We find that glutathione peroxidase 8 (GPX8), a poorly characterized redox enzyme that resides in the endoplasmic reticulum, is upregulated during the epithelial-to-mesenchymal (EMT) program in HMLE and A549 cells. In cancer patients, high tumor levels of GPX8 correlate with mesenchymal markers and poor patient outcome. Strikingly, GPX8 knockout in mesenchymal-like cells results in an epithelial-like morphology, downregulation of EMT characteristics, loss of cancer stemness features, and impeded tumor initiation in mice. We determine the mechanism governing this reduction in cancer aggressiveness is through the repression of crucial autocrine factors, in particular, interleukin-6 (IL-6). Specifically, GPX8 knockout impairs IL-6-driven activation of the JAK-STAT3 signaling pathway, a critical regulator of a cancer-aggressive state. Altogether, we uncover the GPX8-IL-6 axis as a novel metabolic-inflammatory pathway that acts as a robust EMT activator and program to induce aggressive cancer cell characteristics.

Although much has been done to understand how rearrangement of the Igκ locus is regulated during B-cell development, little is known about the way the variable (V) segments themselves are selected. Here we show, using B6/Cast hybrid pre-B-cell clones, that a limited number of V segments on each allele is stochastically activated as characterized by the appearance of non-coding RNA and histone modifications. The activation states are clonally distinct, stable across cell division and developmentally important in directing the Ig repertoire upon differentiation. Using a new approach of allelic ATAC-seq, we demonstrate that the Igκ V alleles have differential chromatin accessibility, which may serve as the underlying basis of clonal maintenance at this locus, as well as other instances of monoallelic expression throughout the genome. These findings highlight a new level of immune system regulation that optimizes gene diversity., B cell development involves sequential rearrangement of the immunoglobulin chains, but fine control over the selection process remains a mystery. Here the authors show that individual alleles in pre-B cells are clonally unique and result from stochastic activation of V gene segments to induce optimal generation of a diverse repertoire.

Cancer-dependent metabolic rewiring is often manifested by selective expression of enzymes essential for the transformed cells&rsquo, viability. However, the metabolic variations between normal and transformed cells are not fully characterized, and therefore, a systematic analysis will result in the identification of unknown cellular mechanisms crucial for tumorigenesis. Here, we applied differential gene expression transcriptome analysis to examine the changes in metabolic gene profiles between a wide range of normal tissues and cancer samples. We found that, in contrast to normal tissues which exhibit a tissue-specific expression profile, cancer samples are more homogenous despite their diverse origins. This similarity is due to a &ldquo, proliferation metabolic signature&rdquo, (PMS), composed of 158 genes (87 upregulated and 71 downregulated gene sets), where 143 are common to all proliferative cells but 15 are cancer specific. Intriguingly, the PMS gene set is enriched for genes encoding rate-limiting enzymes, and its upregulated set with genes associated with poor patient outcome and essential genes. Among these essential genes is ribulose-5-phosphate-3-epimerase (RPE), which encodes a pentose phosphate pathway enzyme and whose role in cancer is still unclear. Collectively, we identified a set of metabolic genes that can serve as novel cancer biomarkers and potential targets for drug development.

Significance Even though DNA methylation is known to be correlated with gene repression, it has never been demonstrated that this modification must indeed be removed from a gene in order for it to become activated during cell differentiation in vivo. In this paper, we inactivated the enzymes responsible for the demethylation reaction in the B-cell lineage and in this manner have shown that this epigenetic mark plays a critical role in development, independently of the many specific transcription factors that direct the selection of genes involved in cell differentiation. Our study is the first to our knowledge to causally connect all of the molecular components necessary to prove the link between the Tet enzymes, CpG demethylation, expression, and phenotype.

Designing a chimeric protein and developing a procedure for its stable production as a biologically active protein, are key steps in its potential application to clinical trails. IL2-Caspase3 chimeric protein designed to target activated T lymphocytes was found to be a promising molecule for targeted treatment, however was found to be difficult to produce as a biological active molecule. Thus, we designed a new version of the molecule, IL2-Caspase3s, in which six amino acids (aa 29-34) from the N-terminus of the large subunit of caspase 3 were excluded. Repeated expressions, productions, and partial purifications of the IL2-Caspase3s yielded reproducible batches with consistent results. We found that IL2-Caspase3s causes cell death in a specific, dose-, and time-dependent manner. Cell death due to IL2-Caspase3s is caused by apoptosis. This improved and biologically stable IL2-Caspase3s chimeric protein may be developed in the future for clinical trails as a promising therapy for several pathologies involving activated T-cells. Moreover, this truncated caspase 3 sequence, lacking the N-terminal six amino acids of its large subunit, may be used in other caspase 3-based chimeric proteins targeted against various human diseases, using the appropriate targeting moiety.

One of the main problems of conventional anticancer therapy is multidrug resistance (MDR), whereby cells acquire resistance to structurally and functionally unrelated drugs following chemotherapeutic treatment. One of the main causes of MDR is overexpression of the P-glycoprotein transporter. In addition to extruding the chemotherapeutic drugs, it also inhibits apoptosis through the inhibition of caspases. To overcome MDR, we constructed a novel chimeric protein, interleukin (IL)-2 granzyme A (IGA), using IL-2 as a targeting moiety and granzyme A as a killing moiety, fused at the cDNA level. IL-2 binds to the high-affinity IL-2 receptor that is expressed in an array of abnormal cells, including malignant cells. Granzyme A is known to cause caspase 3-independent cell death. We show here that the IGA chimeric protein enters the target sensitive and MDR cancer cells overexpressing IL-2 receptor and induces caspase 3-independent cell death. Specifically, after its entry, IGA causes a decrease in the mitochondrial potential, triggers translocation of nm23-H1, a granzyme A-dependent DNase, from the cytoplasm to the nucleus, where it causes single-strand DNA nicks, thus causing cell death. Moreover, IGA is able to overcome MDR and kill cells resistant to chemotherapeutic drugs. We believe that overcoming MDR with targeted molecules such as IGA chimeric protein that causes caspase-independent apoptotic cell death could be applied to many other resistant types of tumors using the appropriate targeting moiety. Thus, this novel class of targeted molecules could open up new vistas in the fight against human cancer.

IL2-caspase3 chimeric protein was designed to target and kill cells expressing the high affinity IL-2 receptor. Its effects on lymphocyte reactivity and on experimental autoimmune encephalomyelitis (EAE), a T-cell mediated disease, were tested in this study. Our data show that IL2-caspase3 promoted cell specific apoptosis both in vitro and in vivo. Cell lines preferentially expressing the IL-2R alpha chain and encephalitogenic lymphocytes derived from EAE-induced mice were highly sensitive to the chimeras' activity. This was demonstrated by increased DNA fragmentation and annexin labeling together with reduced specific T-cell proliferation in response to IL2-casepase3 treatment. Furthermore, IL2-caspase3 treatment of EAE-induced mice caused a significant delay in disease onset together with a reduction in disease burden. The efficacy of IL2-caspase3 treatment was dependent on the time at which treatment begun, with the chimera ameliorating EAE only when administered at maximal activation of peripheral lymphocytes. According to our findings we suggest that the chimeric protein IL2-caspase3 may provide a novel approach for the treatment of a variety of autoimmune disorders, such as multiple sclerosis, as well as for other pathological conditions that involve uncontrolled expansion of activated T cells.

Background: Inflammatory bowel disease (IBD) comprises primarily the two disorders ulcerative colitis (UC) and Crohn9s disease (CD) that involve deregulated T cell responses. The ever-increasing incidence rate of CD and UC during recent decades, combined with the limited efficacy and potential adverse effects of current treatments, explain the real need for seeking more specific and selective methods for treating these diseases. Aim: To investigate the ability of IL2-Caspase3 chimeric protein, designed to target activated T lymphocytes that express the high affinity IL2 receptor, to ameliorate the clinical symptoms of acute murine experimental colitis, using the DSS-induced mouse model. Methods: Mice with DSS-induced colitis were treated with IL2-Caspase3 for 7 days and disease severity was assessed in parallel to control, non-treated mice, receiving only daily injections of PBS. IL2-Caspase3 was tested both for its ability to prevent the development of colitis, and for its therapeutic potential to cure on-going, active acute disease. In addition, colon tissue samples were used for MPO assays and RNA isolation followed by PCR to determine mRNA expression levels of specific genes. Results: Treatment with IL2-Caspase3 dose-dependently ameliorated the Disease Activity Index (DAI) of mice colitis. We achieved up to 78% improvement in DAI with i.v. injections of 15 μg/mouse/day. Furthermore, IL2-Caspase3 decreased neutrophil and macrophage infiltration to the inflamed tissue by up to 57%. IL2-Caspase3 was proven as a therapeutic reagent in another model, where treatment begins only after disease onset. Here we demonstrated a 70% decrease in DAI when compared to non-treated sick mice. A reduction in mRNA expression levels of both IL-1β and TNF-α was found in lysates of total colon tissue of treated mice, as compared to sick, untreated mice. We also found that expression levels of BCL2 were significantly decreased after treatment, while BAX was up-regulated in comparison to non-treated mice. Moreover, the BCL2/BAX ratio, which is elevated in both experimental colitis and in human Crohn9s Disease, was decreased dramatically after treatment. Conclusions: IL2-Caspase3 chimeric protein may provide a novel approach to the therapy of human IBD, and a possible suggested treatment for other pathological conditions that involve uncontrolled expansion of activated T cells.

Plants have been used for medical purposes since the beginning of human history and are the basis of modern medicine. Most chemotherapeutic drugs for cancer treatment are molecules identified and isolated from plants or their synthetic derivatives. Our hypothesis was that whole plant extracts selected according to ethnobotanical sources of historical use might contain multiple molecules with antitumor activities that could be very effective in killing human cancer cells. This study examined the effects of three whole plant extracts (ethanol extraction) on human tumor cells. The extracts were fromUrtica membranacea(Urticaceae),Artemesia monosperma(Asteraceae), andOriganum dayi post(Labiatae). All three plant extracts exhibited dose- and time-dependent killing capabilities in various human derived tumor cell lines and primary cultures established from patients’ biopsies. The killing activity was specific toward tumor cells, as the plant extracts had no effect on primary cultures of healthy human cells. Cell death caused by the whole plant extracts is via apoptosis. Plant extract 5 (Urtica membranacea) showed particularly strong anticancer capabilities since it inhibited actual tumor progression in a breast adenocarcinoma mouse model. Our results suggest that whole plant extracts are promising anticancer reagents.

mRNA export is mediated by RNA-binding proteins which shuttle between the nucleus and cytoplasm. Using an in vitro unidirectional export assay, we observe that the shuttling mRNA-binding protein, hnRNP A1, is exported only extremely slowly unless incubations are supplemented with snRNA-specific oligonucleotides which inhibit splicing. In vivo microinjection experiments support this conclusion. Like many examples of nucleocytoplasmic transport, export of hnRNP A1 requires energy and is sensitive to the presence of wheat germ agglutinin. It does not, however, require supplementation with cytoplasmic proteins. Although the exportin, Crm1, is needed for export of several varieties of RNA, both the in vitro assay and in vivo assays show that it is not required for export of hnRNP A1. In vitro and in vivo studies also show that inhibition of transcription allows continued shuttling of hnRNP A1 and in fact accelerates its export. Judging from the stimulatory effects of targeted destruction of snRNAs, this is likely to reflect completion of the covalent maturation of the RNAs with which hnRNP A1 associates. These observations therefore provide a simple explanation of why multiple RNA-binding proteins relocate to the cytoplasm upon inhibition of transcription in vivo.

Gaucher disease, the most common glycolipid storage disease, is caused by glucocerebrosidase deficiency, resulting in accumulation of glucocerebrosides within the macrophages of the reticuloendothelial system. The disease is characterized by great phenotypic heterogeneity, which can be explained only in part by the various mutations in the glucocerebrosidase gene, and by the amount of storage material in affected organs and tissues. Therefore, it has been postulated that some of the biochemical and clinical features may be related to the fact that "Gaucher" cells, as activated macrophages, express and release cytokines such as IL-1beta, IL-8, IL-6 and TNF-alpha which play a role in different physiological processes. In the present study, cytokine mRNA expression was measured in monocytes isolated from Gaucher patients and from healthy controls, using RT-PCR methodology with semiquantitative analysis. We found significantly increased expression of IL-1beta mRNA, as well as a trend to elevated TNF-alpha mRNA in Gaucher patients relative to healthy individuals. There were no statistically significant differences between Gaucher disease patients and controls with respect to two other tested cytokines (IL-6 and IL-8).

Disorders of the oxidative phosphorylation (OXPHOS) system frequently result in a severe multisystem disease with the consequence of early childhood death. Among these disorders, isolated complex I deficiency is the most frequently diagnosed, accounting for one-third of all cases of respiratory chain deficiency. We chose to focus on complex I deficiency, caused by mutation in the assembly factor chromosome 6, open reading frame 66 (C6ORF66; NADH dehydrogenase [ubiquinone] complex I assembly factor 4 [NDUFAF4]) protein. We used the approach of cell- and organelle-directed protein/enzyme replacement therapy, with the transactivator of transcription (TAT) peptide as the moiety delivery system. This step will enable us to deliver the wild-type assembly factor C6ORF66 into patient cells and their mitochondria, leading to the proper assembly and function of complex I and, as a result, to a functional OXPHOS system. We designed and constructed the TAT-ORF fusion protein by gene fusion techniques, expressed the protein in an Escherichia coli expression system and highly purified it. Our results indicate that TAT-ORF enters patients’ cells and their mitochondria rapidly and efficiently. TAT-ORF is biologically active and led to an increase in complex I activity. TAT-ORF also increased the number of patient cells and improved the activity of their mitochondria. Moreover, we observed an increase in ATP production, a decrease in the content of mitochondria and a decrease in the level of reactive oxygen species. Our results suggest that this approach of protein replacement therapy for the treatment of mitochondrial disorders is a promising one.

We studied the molecular mechanism of demethylation and its role in kappa chain gene regulation. Following transfection into B cell cultures, this gene undergoes regional demethylation in a process that is developmentally regulated in a lineage- and stage-specific manner. Although a germline V kappa promoter is not required for the demodification activity, a fragment containing the intronic kappa chain transcriptional enhancer and the nearby matrix attachment region is essential. In its natural location downstream to the J kappa 5 sequence, this element induces bidirectional demodification of plasmid constructs in a distance- and orientation-independent manner. When this enhancer is placed in an upstream position, however, the kappa gene remains modified and transcriptionally inactive, demonstrating that demethylation is required for kappa chain activation. These studies suggest that the kappa enhancer plays a dual role in regulating B cell differentiation by inducing demethylation and by promoting tissue-specific transcription.

Tissue-specific animal cell genes are usually fully methylated in the germ line and become demethylated in those cell types in which they are expressed. To investigate this process, we inserted a methylated IgG K gene into fibroblasts and lymphocytes at various stages of development. The results show that this gene undergoes demethylation only in the mature lymphocytes and therefore suggest that the ability to demethylate a gene is developmentally regulated. These studies were supported by similar experiments using the rat Insulin I gene, and in this case it appears that the cis -acting elements that control demethylation may be different from those responsible for gene activation. The ability to demethylate the housekeeping gene APRT is also under developmental control, because this occurs only in embryonic cells, both in tissue culture and in transgenic mice.

Since early diagnosis of many types of cancer greatly improves the chances for successful treatment, high-quality methods for cancer detection are necessary. Our laboratory develops chimeric proteins for targeted therapy, such as gonadotropin releasing hormone (GnRH)-based chimeric proteins for the targeted therapy of adenocarcinomas in humans. For chimeric proteins to cause specific cell death, they must first recognize specific receptors/binding sites expressed on the surface of target cells. Thus, we examined whether we could exploit these binding sites not only as targets for the killing of specific cells but also as a diagnostic marker for identifying adenocarcinomas, using the same chimeric proteins. In this report, we show that one such GnRH-based chimeric protein, GnRH-Caspase3, can indeed serve as a diagnostic tool. GnRH-Caspase3 was able to specifically bind adenocarcinoma cells, as measured by FACS analysis and demonstrated with the aid of confocal microscopy and specific antibodies. Moreover, we found a correlation between cell sensitivity to treatment and the binding level of the chimeric protein to the cells. Hence, we suggest that in addition to their therapeutic potential, GnRH-based chimeric proteins can be used as a diagnostic tool for the detection of adenocarcinomas.

The rising rate of cancer-related diseases and mortality underscores the need for new approaches to directly target and light cancer. This review summarizes one of these new promising treatments, the use of targeted chimeric proteins. Chimeric proteins are two proteins fused at the DNA level in such a way that, once expressed, they result in a single polypeptide chain consisting of two moieties: a targeting moiety (usually a cytokine or growth lactor) and a killing moiety (usually a hacterial or plant texio). Many chimeric proteins have been constructed and developed over the years for the treatment of a variety of malignancies and these molecutes are the main scope of this review. Moreover, this review presents new approaches for battling cancer, such as recruiting the apoptotic machinery via chimeric proteins, the use of receptor-mediated delivery of toxin-DNA, or T-cells as vehicles for delivering immunotoxins, all of which are trying to develop specific, efficient, nontoxic and non-immunogenic reagents for targeted cancer treatment.