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Insights into the mechanism of cell death induced by saporin delivered into cancer cells by an antibody fusion protein targeting the transferrin receptor 1
... The mechanism of cell death induced by ch128.1Av conjugated to this toxin was shown to be a result of the effects of the toxin and not iron starvation [34,35], confirming the ability of ch128.1Av to deliver active anti-cancer agents into TfR1 overexpressing malignant cells. ch128.1Av ...
... Importantly, no toxicity to hematopoietic stem/early progenitor cells was observed upon treatment with the ch128.1Av complexed with biotinylated saporin [35], which is consistent with the lack of TfR1 expression on these cells [36][37][38]. ...
... It is also important to note that hematopoietic stem cells lack TfR1 expression [36][37][38] and are not vulnerable to the effects of ch128.1Av alone or conjugated to a toxin [33,35]. Therefore, if B-cell depletion does occur upon treatment, this cell population can be regenerated from the hematopoietic stem cells. ...
AIDS related non-Hodgkin's lymphoma (AIDS-NHL) remains a significant clinical problem in the era of effective multiple-agent highly active anti-retroviral therapy (HAART). While an overall decrease in the incidence of AIDS-NHL has been seen in the HAART era, the risk for AIDS-NHL remains elevated, and not all AIDS-NHL subtypes have decreased in incidence, with the incidence of Burkitt's lymphoma (BL) remaining unchanged. In fact, AIDS-NHL is now the most common AIDS-related cancer in developed countries, where NHL accounts for 23-30% of all AIDS-related deaths. We have previously developed an antibody-avidin fusion protein (ch128.1Av) specific for the human transferrin receptor 1 (TfR1/CD71), a receptor that is overexpressed on cancer cells due to their high demand for iron. This fusion protein exhibits an increased in vitro cytotoxicity against malignant hematopoietic cells compared to the parental antibody without avidin (ch128.1). This cytotoxicity is due to the ability of ch128.1Av to decrease cell surface TfR1, triggering its intracellular sequestration and degradation, with the subsequent induction of lethal iron deprivation. However, both ch128.1Av and ch128.1 confer protection in murine models of human disseminated multiple myeloma. ch128.1Av is also a universal delivery system and its cytotoxicity can be further enhanced by its conjugation with biotinylated drugs making it a unique molecule capable of a two-pronged attack against malignant cells through drug delivery and direct cytotoxic activity through iron starvation. The main goal of this study is to evaluate the potential use of ch128.1Av alone and as a delivery vehicle as a possible AIDS-NHL therapy. We found that, when used alone, ch128.1Av exhibits significant cytotoxic activity against two representative AIDS-NHL cell lines: 2F7 (AIDS-Burkitt's NHL) and RRBL (AIDS-diffuse large B-cell lymphoma). Importantly, the level of cytotoxicity was similar to that observed using the highly sensitive human B lymphoblastoid cell line IM-9. In addition, the cytotoxicity dramatically increases when this fusion protein is conjugated to biotinylated saporin 6 (b-SO6), a plant ribosome inactivating protein that blocks protein synthesis, resulting in an immunotoxin. We also found that non-activated (resting) B cells isolated from the peripheral blood of healthy individuals are resistant to ch128.1Av, but sensitive to ch128.1Av complexed to b-SO6, which can be explained by the different mechanisms of action of the fusion protein alone (iron starvation) and that of the immunotoxin (protein synthesis inhibition). We have previously reported ch128.1Av alone, or complexed to b-SO6, are not toxic to normal human hematopoietic stem cells, which can be explained by the lack of TfR1 expression on such cells. Our results suggest the potential use of anti-TfR1 antibody-mediated approaches as therapeutic interventions against AIDS-NHL.
Citation Format: Tracy R. Daniels-Wells, Lai Sum Leoh, Daniel Widney, Dharma R. Thapa, Jose Leon Merino, Larry Magpantay, Otoniel Martínez-Maza, Manuel L. Penichet. A flexible antibody-based strategy targeting CD71 for the treatment of AIDS-related non-Hodgkin's lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1240. doi:10.1158/1538-7445.AM2013-1240
... The mechanism of cell death induced by ch128.1Av conjugated to this toxin was shown to be due to the effects of the toxin and not iron starvation [35], suggesting the ability of ch128.1Av to deliver active anti-cancer agents into TfR1 overexpressing malignant cells. ch128.1Av ...
... Importantly, no toxicity to hematopoietic stem/early progenitor cells was observed upon treatment with the ch128.1Av complexed with biotinylated saporin [35], which is consistent with the lack of TfR1 expression on these cells [36][37][38]. ...
... It is also important to note that hematopoietic stem cells lack TfR1 expression [36][37][38] and are not vulnerable to the effects of ch128.1Av alone or conjugated to a toxin [33,35]. Therefore, if B cell depletion does occur upon treatment, this cell population can be regenerated from the hematopoietic stem cells. ...
We previously developed an antibody-avidin fusion protein (ch128.1Av) specific for the human transferrin receptor 1 (TfR1; CD71) to be used as a delivery vector for cancer therapy and showed that ch128.1Av delivers the biotinylated plant toxin saporin-6 into malignant B cells. However, due to widespread expression of TfR1, delivery of the toxin to normal cells is a concern. Therefore, we explored the potential of dual targeted lentiviral-mediated gene therapy approaches to restrict gene expression to malignant B cells. Targeting occurs through the use of ch128.1Av or its parental antibody without avidin (ch128.1) and through transcriptional regulation using an immunoglobulin promoter.
Flow cytometry was used to detect the expression of enhanced green fluorescent protein (EGFP) in a panel of cell lines. Cell viability after specific delivery of the therapeutic gene FCU1, a chimeric enzyme consisting of cytosine deaminase genetically fused to uracil phosphoribosyltransferse that converts the 5-fluorocytosine (5-FC) prodrug into toxic metabolites, was monitored by an MTS assay.
We found that EGFP was specifically expressed in a panel of human malignant B cells, but not in human T cell lines. EGFP expression was observed in all cell lines when a ubiquitous promoter was used. Furthermore, we show the decrease of cell viability in malignant plasma cells in the presence of 5-FC.
These studies demonstrate that gene expression can be restricted to malignant B cells and suggest that this dual targeted gene therapy strategy may help to circumvent the potential side effects of certain TfR1-targeted protein delivery approaches. This article is protected by copyright. All rights reserved.
... It must be underlined that these results were obtained with HeLa cells and they cannot automatically be extended to any other cell type. The nuclear localization of saporin-S6 has been postulated by another recent paper in U266 and IM-9 cells [34]. However, in this paper, the RIP was biotinylated and delivered as a complex with an antibody-avidin fusion protein, which could have altered the intracellular trafficking of the toxin as compared to that of saporin-S6 alone. ...
... To unify the intriguing results printed above, the following hypothesis may be put forward. Cell death by saporin-S6 may be induced by various cell injuries, including the inhibition of protein synthesis through RIP activity and DNA damage, either via N-glycosylase activity or as a result of oxidative stress-induction [34], or by other types of damage to the cell machinery that leads to apoptosis or different types of cell death (i.e., autophagy or necroptosis) (see Figure 3). In particular, the activation of the autophagic pathway may promote cell death as a result of cellular atrophy or leading to the execution of apoptotic or necrotic cell death programs [43]. ...
... A global gene expression analysis of two human malignant B-cell lines revealed that an IT containing saporin-S6 could upregulate eleven genes involved in the cellular response to oxidative stress/DNA damage (KLF6, TXNIP, NFKBIE, CDC14B, BHLHB2, GADD45B, HIST2H4, TSC22D3, RGS1) or involved in mRNA processing (THUMPD2, FYTTD1) [34]. The authors suggested that saporin-S6 induces in cells a transcriptional response dependent on oxidative stress/DNA damage, resulting in signal transduction blockage, cell cycle arrest and, consequently, apoptosis. ...
Thirty years ago, the type 1 ribosome-inactivating protein (RIP) saporin-S6 (also known as saporin) was isolated from Saponaria officinalis L. seeds. Since then, the properties and mechanisms of action of saporin-S6 have been well characterized, and it has been widely employed in the construction of conjugates and immunotoxins for different purposes. These immunotoxins have shown many interesting results when used in cancer therapy, particularly in hematological tumors. The high enzymatic activity, stability and resistance to conjugation procedures and blood proteases make saporin-S6 a very useful tool in cancer therapy. High efficacy has been reported in clinical trials with saporin-S6-containing immunotoxins, at dosages that induced only mild and transient side effects, which were mainly fever, myalgias, hepatotoxicity, thrombocytopenia and vascular leak syndrome. Moreover, saporin-S6 triggers multiple cell death pathways, rendering impossible the selection of RIP-resistant mutants. In this review, some aspects of saporin-S6, such as the chemico-physical characteristics, the structural properties, its endocytosis, its intracellular routing and the pathogenetic mechanisms of the cell damage, are reported. In addition, the recent progress and developments of saporin-S6-containing immunotoxins in cancer immunotherapy are summarized, including in vitro and in vivo pre-clinical studies and clinical trials.
... It binds to amino acid residues between serine 324 and serine 368 in the apical domain of TfR1 (147). The ch128.1Av antibody fusion protein was originally developed and has been used as a universal delivery system for biotinylated anti-tumor agents, including biotinylated lentiviral vectors and the plant toxin saporin (145,(148)(149)(150)(151). Surprisingly, ch128.1Av ...
... Interestingly, treatment of HSC with 10 nM of the mouse/human chimeric IgG3 ch128.1Av fusion protein alone (or even complexed with the potent plant toxin saporin) showed no toxicity to this population of cells due to the lack of expression of TfR1 (149). In contrast, 2.5 nM ch128.1Av ...
The transferrin receptor 1 (TfR1), also known as cluster of differentiation 71 (CD71), is a type II transmembrane glycoprotein that binds transferrin (Tf) and performs a critical role in cellular iron uptake through the interaction with iron-bound Tf. Iron is required for multiple cellular processes and is essential for DNA synthesis and, thus, cellular proliferation. Due to its central role in cancer cell pathology, malignant cells often overexpress TfR1 and this increased expression can be associated with poor prognosis in different types of cancer. The elevated levels of TfR1 expression on malignant cells, together with its extracellular accessibility, ability to internalize, and central role in cancer cell pathology make this receptor an attractive target for antibody-mediated therapy. The TfR1 can be targeted by antibodies for cancer therapy in two distinct ways: (1) indirectly through the use of antibodies conjugated to anti-cancer agents that are internalized by receptor-mediated endocytosis or (2) directly through the use of antibodies that disrupt the function of the receptor and/or induce Fc effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). Although TfR1 has been used extensively as a target for antibody-mediated cancer therapy over the years, interest continues to increase for both targeting the receptor for delivery purposes and for its use as direct anti-cancer agents. This review focuses on the developments in the use of antibodies targeting TfR1 as direct anti-tumor agents.
... This latter observation argues for the likely importance of the NK cell as the major cytotoxic effector responsible for ADCC augmentation of IT cytotoxicity in the SCID-HSB-2 model consequent to the established reduced functional activity of NK cells in NOD/SCID mice [6]. These observations also support our working hypothesis that the divergent apoptotic pathways initiated by NK cell delivered granzymes [7] and antibody-mediated delivery of the ribosome inactivating protein (rip) saporin [8,9] act synergistically to achieve a greater level of target cell killing. We hypothesised that stimulating ADCC activity could well increase the therapeutic efficacy of IT treatment further still and the present study was conducted to explore this concept. ...
... We propose a mechanistic model to explain this, which speculates that perforin-dependent delivery of granzymes to target HSB-2 cells by cytotoxic effector cells during ADCC synergise with the ribotoxic effect of IT-mediated saporin-mediated protein synthesis inhibition. GrzA and GzB delivered by NK-cell-mediated killing invoke apoptotic pathways that are radically divergent from each other [7], which in turn are also different from the saporin-mediated apoptotic pathway(s) [8,9]. It is easy to envisage that these different pathways converge on different and/or common death substrates to achieve a more efficient target cell killing. ...
We have previously shown that antibody-dependent cellular cytotoxicity (ADCC) cooperates with immunotoxin (IT)-mediated killing of human leukaemia cells in an severe combined immunodeficient (SCID) mouse model of human T-cell acute lymphoblastic leukaemia (SCID-HSB-2 mice), but not in an equivalent non-obese diabetic (NOD)/SCID mouse model. In these earlier studies, we reasoned that diminished ADCC due to the functional deficit in natural killer (NK) cell activity in NOD/SCID mice resulted in a failure of effective perforin/granzyme-mediated cytotoxicity necessary for the delivery of the augmentative effect. Poly-inosinic-cytidylic acid [poly (I:C)] is a synthetic dsRNA toll-like receptor 3 (TLR3) agonist that possesses a number of biological properties that includes the in vivo activation of NK cells. We show here that intravenous (i.v.) injection of SCID mice with [poly (I:C)] results in characteristic time-related changes in serum interleukin 2 (IL-2), IL-12, and interferon γ (INFγ) cytokine levels that are consistent with TLR3 driven activation of SCID mouse NK cells. Concomitantly, there are changes in the expression levels of CD2, CD16/32 (FcγRII/RIII), CD161 (NK1.1), and F4/80 in the bulk splenocyte population. These observed changes correlate with an increase in the in vitro lytic capabilities of putative NK cells from within the splenocyte population of [poly (I:C)] treated SCID mice. We demonstrate that the in vivo activation of NK cells with [poly (I:C)] in SCID mice bearing disseminated human T-cell leukaemia xenografts resulted in a significant improvement in the therapeutic activity exerted by an intact murine monoclonal antibody against human CD7. This was also seen for a saporin-based immunotoxin constructed with the same intact antibody (HB2-SAPORIN), but not with an F(ab')2 derivative of the same antibody or of an IT constructed with the same F(ab')2 HB2 antibody derivative. This study further demonstrates the previously reported reinforcing role of ADCC for the therapeutic activity of IT in an SCID mouse model of human TALL and the potential to significantly boost this further with [poly (I:C)]. Our study provides the rationale to justify the exploration of the clinical utility of IT based therapeutics in combination with TLR3 agonists, such as [poly (I:C)], for the treatment of haematological, and possibly other, malignancies.
... This result is in agreement with the previous report on the effect of saporin on tumour cells by ch128.1Av/b-SO6 conjugate [27]. PNPLA8 gene is stimulated via c-Jun N-terminal kinases pathways [28] linked with MAP kinase activity and Lauber et al. [29] had reported the participation of PNPLA8 in the clearing of apoptotic cells by caspase-3-dependent mechanism. ...
... The apoptosis assay has clearly proved that the cells have undergone early apoptosis, late apoptosis and necrosis in WERI RB1 cells and MCF-7 cells. The results were in agreement with the Tracy et al., where the saporin-antibody conjugate has yielded a high amount of late apoptosis and necrotic populations in IM9 and U266 cell lines [27]. MTT assay also revealed the minimum inhibitory concentration of 0.8-1 µg of the conjugate to target tumour cells efficiently. ...
Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein involved in cell proliferation and differentiation. Ribosomal inactivating proteins derived from plants specifically target ribosomes and irreversibly inhibit protein synthesis. EpCAM antibody and saporin were conjugated using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide chemistry. The mass of the conjugates were characterised using matrix-assisted laser desorption ionisation (MALDI). The saporin-EpCAM (SAP-EpAB) conjugates were tested in-vitro against MCF-7 (breast cancer cells), WERI-Rb1 (retinoblastoma) cells. The flow cytometry and fluorescence microscopy were performed to show the binding efficiency of SAP-EpAB conjugate. Whole transcriptome changes of sap-conjugate treated cells were studied using affymetrix microarrays. MALDI-TOF analysis and polyacrylamide gel electrophoresis confirmed the conjugation of SAP with EpCAM antibody. Flow cytometry and fluorescent microscopy analysis revealed the binding of SAP-EpAB conjugates to the MCF-7, WERI-Rb1 cells. Apoptosis assay by annexin-V has shown an increased apoptotic and necrotic population in conjugate treated cells. MTT assay confirmed the tumour cell death and had shown the IC50 value of 0.8 μg for conjugate in MCF-7 (breast cancer cells), and 1 μg for WERI-Rb1 (retinoblastoma) cells. The microarray analysis revealed downregulation of the tumourigenic genes and upregulation of pro-apoptotic genes leading to apoptosis of tumour cells.
... Transferrin receptor (CD71) is a cell surface molecule that regulates uptake of iron-bound transferrin by receptor-mediated endocytosis, is expressed in dividing cells and is a marker of B cell endocytosis (18,47,(48)(49)(50)). Highly exposed individuals had increased frequencies of CD71 + aaMBCs compared to nonexposed individuals (Figure 4A), but no other association with malaria exposure was found for CD71 expression in the remaining switched B cell subsets (Table S2 in Supplementary Material). ...
... We analyzed the expression of CD71 as a marker of B cell endocytosis (18) and observed that CD71 + aaMBCs were more frequent in exposed individuals, suggesting that aaMBCs recognize and internalize fewer antigens than other switched B cell subsets (18). The correlation between the number of cell surface CD71 molecules and the rate of cell proliferation is well known (47), as seen in numerous oncogenic cells (31,39,(47)(48)(49)(50). The increased frequencies of aaMBCs expressing CD71 in malaria-exposed pregnant women suggests that aaMBCs may proliferate at a higher rate in malaria-exposed individuals and is consistent with the overall higher frequencies of aaMBC in these individuals. ...
In persistent infections that are accompanied by chronic immune activation, such as human immunodeficiency virus, hepatitis C virus, and malaria, there is an increased frequency of a phenotypically distinct subset of memory B cells lacking the classic memory marker CD27 and showing a reduced capacity to produce antibodies. However, critical knowledge gaps remain on specific B cell changes and immune adaptation in chronic infections. We hypothesized that expansion of atypical memory B cells (aMBCs) and reduction of activated peripheral marginal zone (MZ)-like B cells in constantly exposed individuals might be accompanied by phenotypic changes that would confer a tolerogenic profile, helping to establish tolerance to infections. To better understand malaria-associated phenotypic abnormalities on B cells, we analyzed peripheral blood mononuclear cells from 55 pregnant women living in a malaria-endemic area of Papua Nueva Guinea and 9 Spanish malaria-naïve individuals using four 11-color flow cytometry panels. We assessed the expression of markers of B cell specificity (IgG and IgM), activation (CD40, CD80, CD86, b220, TACI, and CD150), inhibition (PD1, CD95, and CD71), and migration (CCR3, CXCR3, and CD62l). We found higher frequencies of active and resting aMBC and marked reduction of MZ-like B cells, although changes in absolute cell counts could not be assessed. Highly exposed women had higher PD1+-, CD95+-, CD40+-, CD71+-, and CD80+-activated aMBC frequencies than non-exposed subjects. Malaria exposure increased frequencies of b220 and proapoptotic markers PD1 and CD95, and decreased expression of the activation marker TACI on MZ-like B cells. The increased frequencies of inhibitory and apoptotic markers on activated aMBCs and MZ-like B cells in malaria-exposed adults suggest an immune-homeostatic mechanism for maintaining B cell development and function while simultaneously downregulating hyperreactive B cells. This mechanism would keep the B cell activation threshold high enough to control infection but impaired enough to tolerate it, preventing systemic inflammation.
... ch128.1Av has been shown to deliver biotinylated saporin (a plant toxin) and a biotinylated lentiviral vector containing a therapeutic gene into malignant B cells in vitro [9][10][11]. The cancer targeting potential of the lentiviral vector was further enhanced through the use of an immunoglobulin promoter that limits the expression of the therapeutic gene to B cells. ...
... This molecule was originally designed as a delivery vehicle, but also has direct cytotoxic activity against certain malignant cells in vitro through the disruption of the TfR1 cycling, degradation of the receptor, and ultimately iron starvation [7,22]. Importantly, ch128.1Av is not cytotoxic to normal hematopoietic stem cells even when conjugated to the plant toxin saporin due to their lack of TfR1 expression [10]. This observation suggests that this important cell population would be spared in therapies targeting TfR1 and also suggests that this strategy can be used for ex vivo approaches such as purging of cancer cells in autologous bone marrow and stem cell transplantation protocols. ...
The ch128.1Av chimeric fusion protein mentioned above has the advantage of having human constant regions. This molecule was originally designed as a delivery vehicle, but also has direct cytotoxic activity against certain malignant cells in vitro through the disruption of the TfR1 cycling, degradation of the receptor, and ultimately iron starvation [7,22]. Importantly, ch128.1Av is not cytotoxic to normal hematopoietic stem cells even when conjugated to the plant toxin saporin due to their lack of TfR1 expression [10]. This observation suggests that this important cell population would be spared in therapies targeting TfR1 and also suggests that this strategy can be used for ex vivo approaches such as purging of cancer cells in autologous bone marrow and stem cell transplantation protocols. The parental antibody without avidin (ch128.1) also exhibits cytotoxicity in vitro, although less than that of ch128.1Av [7]. However, in xenograft models of human multiple myeloma (MM) and non-Hodgkin lymphoma (NHL) ch128.1 has demonstrated significant anticancer efficacy, which in the case of the MM models is greater than that of the fusion protein [23,24]. Interestingly, in vivo antitumor activity is observed even against malignant cells that are not sensitive to the cytotoxic activity of the antibody in vitro. Importantly, this antibody triggers Fc effector functions, which may play a role in its antitumor activity in vivo [25]. Antibodies of complete human nature targeting TfR1 have also been developed including a human scFv targeting TfR1, which antagonizes malignant hematopoietic cell growth in vitro [26] and a fully human IgG antibody that has shown antitumor efficacy in xenograft models of human oral squamous cell carcinoma in mice [27].
... In fact, previous studies using an immunotoxin consisting of an avidin fusion protein of ch128.1/IgG3 conjugated to a biotinylated form of the ribosomal inhibitory plant toxin saporin showed no effects on this cell population [55]. Thus, the toxic effects on erythroid progenitor cells, if present, is expected to be transient. ...
Simple Summary
There is an increased risk for the development of certain cancers, including non-Hodgkin lymphoma (NHL), in individuals infected with the human immunodeficiency virus (HIV). Therefore, NHL is considered to be an acquired immunodeficiency syndrome (AIDS)-related malignancy (AIDS-NHL). There are several subtypes of NHL, but the majority of these cancers are of B-cell origin. HIV infection leads to the chronic activation of B cells, which results in high expression of a protein called the transferrin receptor 1 (TfR1), B-cell dysfunction, and ultimately the development of AIDS-NHL. TfR1 is responsible for iron uptake by cells. Iron is needed for multiple cellular processes, including DNA synthesis, which is required for cell proliferation. Thus, targeting this receptor is a meaningful strategy for treatment of AIDS-NHL. This study shows that antibodies targeting TfR1 prolong survival in two mouse models of AIDS-NHL, suggesting that these antibodies have potential as a therapy for AIDS-NHL.
Abstract
Transferrin receptor 1 (TfR1), also known as CD71, is a transmembrane protein involved in the cellular uptake of iron and the regulation of cell growth. This receptor is expressed at low levels on a variety of normal cells, but is upregulated on cells with a high rate of proliferation, including malignant cells and activated immune cells. Infection with the human immunodeficiency virus (HIV) leads to the chronic activation of B cells, resulting in high expression of TfR1, B-cell dysfunction, and ultimately the development of acquired immunodeficiency syndrome-related B-cell non-Hodgkin lymphoma (AIDS-NHL). Importantly, TfR1 expression is correlated with the stage and prognosis of NHL. Thus, it is a meaningful target for antibody-based NHL therapy. We previously developed a mouse/human chimeric IgG3 specific for TfR1 (ch128.1/IgG3) and showed that this antibody exhibits antitumor activity in an in vivo model of AIDS-NHL using NOD-SCID mice challenged intraperitoneally with 2F7 human Burkitt lymphoma (BL) cells that harbor the Epstein-Barr virus (EBV). We have also developed an IgG1 version of ch128.1 that shows significant antitumor activity in SCID-Beige mouse models of disseminated multiple myeloma, another B-cell malignancy. Here, we aim to explore the utility of ch128.1/IgG1 and its humanized version (hu128.1) in mouse models of AIDS-NHL. To accomplish this goal, we used the 2F7 cell line variant 2F7-BR44, which is more aggressive than the parental cell line and forms metastases in the brain of mice after systemic (intravenous) administration. We also used the human BL cell line JB, which in contrast to 2F7, is EBV-negative, allowing us to study both EBV-infected and non-infected NHL tumors. Treatment with ch128.1/IgG1 or hu128.1 of SCID-Beige mice challenged locally (subcutaneously) with 2F7-BR44 or JB cells results in significant antitumor activity against different stages of disease. Treatment of mice challenged systemically (intravenously) with either 2F7-BR44 or JB cells also showed significant antitumor activity, including long-term survival. Taken together, our results suggest that targeting TfR1 with antibodies, such as ch128.1/IgG1 or hu128.1, has potential as an effective therapy for AIDS-NHL.
... Moreover, pluripotent non-committed hematopoietic stem cells show little to no expression of hTfR1 25,29,30 . Even treatment with a fusion protein consisting of an IgG3 version of ch128.1 and avidin conjugated to a biotinylated version of the plant toxin saporin is not toxic to these pluripotent stem cells 49 . Thus, any erythroid progenitors that are lost can be repopulated by the stem cell compartment. ...
Five New World mammarenaviruses (NWMs) cause life-threatening hemorrhagic fever (HF). Cellular entry by these viruses is mediated by human transferrin receptor 1 (hTfR1). Here, we demonstrate that an antibody (ch128.1/IgG1) which binds the apical domain of hTfR1, potently inhibits infection of attenuated and pathogenic NWMs in vitro. Computational docking of the antibody Fab crystal structure onto the known structure of hTfR1 shows an overlapping receptor-binding region shared by the Fab and the viral envelope glycoprotein GP1 subunit that binds hTfR1, and we demonstrate competitive inhibition of NWM GP1 binding by ch128.1/IgG1 as the principal mechanism of action. Importantly, ch128.1/IgG1 protects hTfR1-expressing transgenic mice against lethal NWM challenge. Additionally, the antibody is well-tolerated and only partially reduces ferritin uptake. Our findings provide the basis for the development of a novel, host receptor-targeted antibody therapeutic broadly applicable to the treatment of HF of NWM etiology.
... It is therefore possible that the ribotoxic stress response induced by 28S rRNA damage may be part of a general cellular response sensing both RNA and standard DNA damages, as suggested also by the close similarity to RIP response with gene expression changes induced by azacitidine and TSA treatments (Zhong et al., 2007). Similarly, it was previously shown that KLF2 expression sensitizes cells to DNA damage-induced apoptosis , and several RIPs were also thought to induce signaling pathways linked to DNA-damage responses (Bolognesi et al., 2012;Daniels-Wells et al., 2013;Kumar et al., 2007;Polito et al., 2013). ...
Gene-regulatory networks reconstruction has become a very popular approach in applied biology to infer and dissect functional interactions of Transcription Factors (TFs) driving a defined phenotypic state, termed as Master Regulators (MRs). In the present work, cutting-edge bioinformatic methods were applied to re-analyze experimental data on leukemia cells (human myelogenous leukemia cell line THP-1 and acute myeloid leukemia MOLM-13 cells) treated for 6 h with two different Ribosome-Inactivating Proteins (RIPs), namely Shiga toxin type 1 (400 ng/ml) produced by Escherichia coli strains and the plant toxin stenodactylin (60 ng/ml), purified from the caudex of Adenia stenodactyla Harms. This analysis allowed us to identify the common early transcriptional response to 28S rRNA damage based on gene-regulatory network inference and Master Regulator Analysis (MRA). Both toxins induce a common response at 6 h which involves inflammatory mediators triggered by AP-1 family transcriptional factors and ATF3 in leukemia cells. We describe for the first time the involvement of MAFF, KLF2 and KLF6 in regulating RIP-induced apoptotic cell death, while receptor-mediated downstream signaling through ANXA1 and TLR4 is suggested for both toxins.
... Several studies have indicated that different cell death pathways may be involved in RIP-induced cytotoxicity. However, very few reports have investigated the early response to RIP-induced ribosomal damage (Korcheva et al., 2005;Polito et al., 2009;Bora et al., 2010;Leyva-Illades et al., 2010;Horrix et al., 2011;Daniels-Wells et al., 2013;Pervaiz et al., 2016). The present study was designed to evaluate the early response to stenodactylin in a cellular model of blood neoplasia. ...
Stenodactylin, a highly toxic type 2 ribosome-inactivating protein purified from the caudex of Adenia stenodactyla Harms, is a potential anticancer drug candidate. Previous studies demonstrated that stenodactylin induces apoptosis and necroptosis in treated cells, involving the production of reactive oxygen species. We analyzed the effect of stenodactylin on Raji and Ramos (Human Burkitt’s lymphoma cells) and MOLM-13 (acute myeloid leukemia cells). Moreover, we focused on the early events in MOLM-13 cells that characterize the cellular response to the toxin by whole-genome microarray analysis of gene expression. Treatment with stenodactylin induced the depurination of 28S rRNA within 4 h and increased the phosphorylation of p38 and JNK. A time-dependent activation of caspase 1, 2, 8, 9, 3/7 was also observed. Genome-wide gene expression microarray analysis revealed early changes in the expression of genes involved in the regulation of cell death, inflammation and stress response. After 4 h, a significant increase of transcript level was detectable for ATF3, BTG2, DUSP1, EGR1, and JUN. Increased upstream JUN signaling was also confirmed at protein level. The early response to stenodactylin treatment involves inflammatory and apoptotic signaling compatible with the activation of multiple cell death pathways. Because of the above described properties toward acute myeloid leukemia cells, stenodactylin may be a promising candidate for the design of new immunoconjugates for experimental cancer treatment.
... To evaluate targeting cancer cells with our ADC, we selected CSPG4 high-expressing melanoma cells (A375, A2058) and CSPG4 low-expressing melanoma (SBCL-2) and breast cancer (SKBR-3) cell lines. To confirm that the antibody was internalized by cancer cells, a reporter assay was employed for which the anti-CSPG4 IgG1 was linked to streptavidin and then conjugated to biotinylated Saporin (anti-CSPG4-SB-Saporin). Saporin is a 30 kDa ribosome-inhibitor unable to cross a cell membrane unaided, however Saporin is only toxic once taken up by cells, a process known to happen when it is conjugated to an internalizing antibody, as previously described [34,35]. Treatment with anti-CSPG4-SB-Saporin for 4 days decreased tumor cell viability in CSPG4-high A375 and A2058 melanoma cell lines, while it had low toxic effects on the CSPG4-low SBCL-2 melanoma and SKBR-3 breast cancer cells. ...
Despite emerging targeted and immunotherapy treatments, no monoclonal antibodies or antibody-drug conjugates (ADCs) directly targeting tumor cells are currently approved for melanoma therapy. The tumor-associated antigen chondroitin sulphate proteoglycan 4 (CSPG4), a neural crest glycoprotein over-expressed on 70% of melanomas, contributes to proliferative signaling pathways, but despite highly tumor-selective expression it has not yet been targeted using ADCs. We developed a novel ADC comprising an anti-CSPG4 antibody linked to a DNA minor groove-binding agent belonging to the novel pyrridinobenzodiazepine (PDD) class. Unlike conventional DNA-interactive pyrrolobenzodiazepine (PBD) dimer payloads that cross-link DNA, PDD-based payloads are mono-alkylating agents but have similar efficacy and substantially enhanced tolerability profiles compared to PBD-based cross-linkers. We investigated the anti-tumor activity and safety of the anti-CSPG4-(PDD) ADC in vitro and in human melanoma xenografts. Anti-CSPG4-(PDD) inhibited CSPG4-expressing melanoma cell growth and colony formation and triggered apoptosis in vitro at low nanomolar to picomolar concentrations without off-target Fab-mediated or Fc-mediated toxicity. Anti-CSPG4-(PDD) restricted xenograft growth in vivo at 2 mg/kg doses. One 5 mg/kg injection triggered tumor regression in the absence of overt toxic effects or of acquired residual tumor cell resistance. This anti-CSPG4-(PDD) can deliver a highly cytotoxic DNA mono-alkylating payload to CSPG4-expressing tumors at doses tolerated in vivo.
... The multiple death pathways induced by saporin-S6 containing ITs including apoptosis activation, autophagy, necroptosis, oxidative stress and the inhibition of protein synthesis have all been discussed in the literature [40]. A global gene expression analysis of two human malignant B-cell lines revealed that an IT containing saporin-S6 upregulated eleven genes involved in the cellular response to oxidative stress and DNA damage [41] suggesting to these authors that saporin-S6 induced a transcriptional response in cells that was dependent on oxidative stress/DNA damage leading to signal transduction blockage, cell cycle arrest and apoptosis. ...
Triterpenoid saponins from Saponinum album (SA) significantly augment the cytotoxicity of saporin-based immunotoxins but the mechanism of augmentation is not fully understood. We investigated the effects of six small molecule pharmacological agents, which interfere with endocytic and other processes, on SA-mediated augmentation of saporin and saporin-based immunotoxins (ITs) directed against CD7, CD19, CD22 and CD38 on human lymphoma and leukaemia cell lines. Inhibition of clathrin-mediated endocytosis or endosomal acidification abolished the SA augmentation of saporin and of all four immunotoxins tested but the cytotoxicity of each IT or saporin alone was largely unaffected. The data support the hypothesis that endocytic processes are involved in the augmentative action of SA for saporin ITs targeted against a range of antigens expressed by leukaemia and lymphoma cells. In addition, the reactive oxygen species (ROS) scavenger tiron reduced the cytotoxicity of BU12-SAP and OKT10-SAP but had no effect on 4KB128-SAP or saporin cytotoxicity. Tiron also had no effect on SA-mediated augmentation of the saporin-based ITs or unconjugated saporin. These results suggest that ROS are not involved in the augmentation of saporin ITs and that ROS induction is target antigen-dependent and not directly due to the cytotoxic action of the toxin moiety. Key Contribution: We have shown that endocytic processes are involved in the augmentative action of SA for saporin ITs directed against a range of lineage restricted antigens expressed by malignant lymphoid cells supplementing earlier research in a different tumour and antigen system. In addition, we have shown that reactive oxygen species (ROS) do not appear to play a mechanistic role in the augmentative effect of SA for a range of saporin-based ITs with different target molecule specificities.
... Even though committed, hematopoietic progenitor cells express high levels of the TfR1 and are vulnerable to the effects of anti-TfR1 Abs, hematopoietic pluripotent stem cells lack TfR1 expression (10,(76)(77)(78). In fact, we have shown that this cell population is not affected by an immunotoxin consisting of an Ab-avidin fusion protein that is composed of ch128.1 genetically fused to chicken avidin and conjugated to the plant toxin saporin (79). One phase I clinical trial using an anti-TfR1 Ab has been reported (80). ...
The transferrin receptor 1 (TfR1) is an attractive target for Ab-mediated cancer therapy. We previously developed a mouse/human chimeric IgG3 Ab (ch128.1) targeting human TfR1, which exhibits direct in vitro cytotoxicity against certain human malignant B cells through TfR1 degradation and iron deprivation. ch128.1 also demonstrates exceptional antitumor activity against the B cell malignancy multiple myeloma (MM) in xenograft models of SCID-Beige mice bearing either disseminated ARH-77 or KMS-11 cells in an early disease setting. Interestingly, this activity is observed even against KMS-11 cells, which show no sensitivity to the direct cytotoxic activity of ch128.1 in vitro. To understand the contributions of the Fc fragment, we generated a ch128.1 mutant with impaired binding to FcγRs and to the complement component C1q, which retains binding to the neonatal Fc receptor. We now report that this mutant Ab does not show antitumor activity in these two MM models, indicating a crucial role of the Fc fragment in the antitumor activity of ch128.1, which can be attributed to effector functions (Ab-dependent cell-mediated cytotoxicity, Ab-dependent cell-mediated phagocytosis, and/or complement-dependent cytotoxicity). Interestingly, in the KMS-11 model, complement depletion does not affect protection, whereas macrophage depletion does. Consistent with this observation, we found that ch128.1 induces Ab-dependent cell-mediated cytotoxicity and Ab-dependent cell-mediated phagocytosis against KMS-11 cells in the presence of murine bone marrow-derived macrophages. Finally, we found that ch128.1 therapy effectively increases survival in a late MM disease setting. Our results suggest that macrophages play a major role in ch128.1-mediated antitumor protection in our models and that ch128.1 can be effective against human B cell malignancies such as MM.
... These findings demonstrate that it is possible to transform an antibody specific for a cell-surface receptor that exhibits minimal inhibitory activity into a novel drug with significant intrinsic cytotoxic activity against selected cells by fusing it with avidin. More importantly, since the antibody fusion protein is a delivery system, this property can also be exploited to deliver biotinylated therapeutics into cancer cells, rendering cells that were resistant to the antibody fusion protein alone sensitive to treatment[171,172]. Further development of this technology may lead to effective therapeutics for the in vivo eradication of hematological malignancies and the ex vivo purging of cancer cells in autologous transplantation. ...
Antibodies are proteins produced by the immune system to combat pathogens and have
long been appreciated for their exquisite specificity. The development of the hybridoma
technology made it possible to immortalize single B cells, resulting in the production of
unlimited quantities of antibodies of a single, well-defined antigen-binding specificity,
known as monoclonal antibodies. However, the initial hybridoma-derived monoclonal
antibodies were murine and highly immunogenic in humans. Advances in genetic engi-
neering and expression systems have been used to overcome problems of the immuno-
genicity of rodent-produced antibodies, and to improve the ability of the antibodies
to trigger human immune effector activity. The development of chimeric, humanized,
and totally human antibodies, as well as antibodies with novel structures and functional
properties, has further expanded the potential use of monoclonal antibodies as targeted
therapeutics. As a consequence, recombinant antibody-based therapies are now used
to treat a variety of diverse conditions that include infectious diseases, inflammatory
disorders, and cancer. Today, these therapies are one of the fastest growing classes of
biopharmaceutical therapeutics. The different strategies for developing recombinant
antibodies and their derivatives are summarized and compared in this review.
... Oxidative stress was identified as being involved in cell death induced by a saporin-based conjugate directed against TfR1 [42]. Recently, it was reported that also the type 2 RIP stenodactylin is able to induce the early formation of ROS molecules in a neuroblastoma cell line and that several ROS scavengers can protect cells from RIP intoxication [40]. ...
Immunotoxins (ITs) are hybrid proteins combining the binding specificity of antibodies with the cytocidal properties of toxins. They represent a promising approach to lymphoma therapy. The cytotoxicity of two immunotoxins obtained by chemical conjugation of the plant toxin saporin-S6 with the anti-CD20 chimeric antibody rituximab and the anti-CD22 murine antibody OM124 were evaluated on the CD20-/CD22-positive cell line Raji. Both ITs showed strong cytotoxicity for Raji cells, but the anti-CD22 IT was two logs more efficient in killing, probably because of its faster internalization. The anti-CD22 IT gave slower but greater caspase activation than the anti-CD20 IT. The cytotoxic effect of both immunotoxins can be partially prevented by either the pan-caspase inhibitor Z-VAD or the necroptosis inhibitor necrostatin-1. Oxidative stress seems to be involved in the cell killing activity of anti-CD20 IT, as demonstrated by the protective role of the H2O2 scavenger catalase, but not in that of anti-CD22 IT. Moreover, the IT toxicity can be augmented by the contemporary administration of other chemotherapeutic drugs, such as PS-341, MG-132, and fludarabine. These results contribute to the understanding of the immunotoxin mechanism of action that is required for their clinical use, either alone or in combination with other drugs.
... Consistent with our findings, TFR1 has been detected in hepatoma [19] and colorectal cancer [20,21]. More interestingly, TFR1 has been extensively studied as a delivery vehicle for anti-cancer drugs, either through its ligand, transferrin, or its monoclonal antibodies [7,[22][23][24][25][26][27][28][29]. Thus, our proposal to utilize AF20 mAb for the treatment of HCC is validated by findings from others [2][3][4]. ...
We previously isolated AF20, a murine monoclonal antibody that recognizes a cell surface glycoprotein of approximately 90–110 kDa. The AF20 antigen is specifically expressed in human hepatoma and colon cancer cell lines, and thus could serve as a cancer biomarker. To uncover the molecular identity of the AF20 antigen, a combination of ion-exchange chromatography, immunoprecipitation, and SDS—polyacrylamide gel electrophoresis was employed to purify the AF20 antigen followed by trypsin digestion and mass spectrometry. Surprisingly, three host proteins were thus purified from human hepatoma and colon cancer cell lines: transferrin receptor 1 (TFR1), heat shock protein 90 (HSP90), and Na⁺/K⁺ ATPase or Mg⁺⁺ ATPase. Co-immunoprecipitation followed by Western blot analysis confirmed interaction among the three proteins. However, only the cDNA encoding TFR1 conferred strong cell surface staining by the AF20 antibody following its transient transfection into a cell line lacking endogenous AF20. In support of the molecular identity of AF20 as TFR1, diferric but not iron-free transferrin could prevent AF20 antigen-antibody interaction during immunoprecipitation. Moreover, very similar patterns of AF20 and TFR1 overexpression was documented in colon cancer tissues. In conclusion, AF20 is glycosylated TFR1. This finding could explain the molecular structure of AF20, its cell surface localization, as well as overexpression in cancer cells. Glycosylated TFR1 should serve as a usefulness target for anti-cancer therapy, or a vehicle for delivery of anti-tumor drugs with high affinity and specificity. The biological significance of the complex formation between TFR1, HSP90, and/or transporting ATPase warrants further investigation.
... These data coincide with a past study that showed improvement in proliferation of human osteoblast cells (CRL 1543) when the culture medium was supplemented with 0.0195% Tualang honey [30]. The underlying mechanism for the observed improvements is unknown, but it could be related to the active compounds present in Tualang honey or to the presence of H 2 O 2 , a ROS that is essential for modulating stem cell behaviours at the physiological level [45][46][47]. Pan et al. (2011) recently suggested that low levels of H 2 O 2 (10-50 mM) could promote rabbit corneal epithelial cell attachment, mobility, and wound repair [48]. This contradicts our findings using human cells that H 2 O 2 showed significant cytotoxicity at 50 mM and no noticeable changes in cell number at 10 and 20 mM when compared with the untreated control. ...
... As originally designed, ch128.1Av has been successfully used to deliver biotinylated toxin saporin, a ribosome inactivating protein Daniels-Wells et al., 2013), and lentivirus for gene therapy (Leoh et al., 2014;Morizono et al., 2009). Importantly, significant anti-tumor protection against xenograft models of the human B-cell malignancy multiple myeloma in SCID-Beige mice was observed using ch128.1 or ch128.1Av ...
The transferrin receptor 1 (TfR1) is involved in cellular iron uptake and regulation of cell proliferation. The increased expression of TfR1 observed in malignant cells, compared to normal cells, together with its extracellular accessibility, make this receptor an attractive target for antibody-mediated cancer therapy. We have developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1), which shows anti-tumor activity against certain malignant B cells in vitro through TfR1 degradation and iron deprivation, and in vivo through a mechanism yet to be defined. To further explore potential mechanisms of action of ch128.1, we examined its ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC). We now report that ch128.1 is capable of mediating ADCC and CDC against malignant B cells, which is consistent with its ability to bind FcγRI, FcγRIIIa, and the complement component C1q. To delineate the residues involved in these effector functions, we developed a panel of three constructs with mutations in the lower hinge region and CH2 domain: 1) L234A/L235A, 2) P331S, and 3) L234A/L235A/P331S. The triple mutant consistently displayed a significant reduction in ADCC, while the L234A/L235A mutant exhibited less reduction in ADCC, and the P331S mutant did not show reduced ADCC. However, all three mutants exhibited impaired binding to FcγRI and FcγRIIIa. These results suggest that all three residues contribute to ADCC, although to different degrees. The P331S mutant showed drastically decreased C1q binding and abolished CDC, confirming the critical role of this residue in complement activation, while the other residues play a less important role in CDC. Our study provides insights into the effector functions of human IgG3 in the context of an antibody targeting TfR1.
Copyright © 2015 Elsevier Ltd. All rights reserved.
... In addition to these GADD genes, induction of pro-apoptotic (BAX and BAK) and inhibition of antiapoptotic (BCL-2 and BCL-XL) genes were also noticed in microarray-based genetic profiling of MDA-MB-231 cells. Induction of GADD genes and modulation of pro-/ anti-apoptotic genes by RIPs, involving ER/mitochondrial arms, have also been supported in other studies (Daniels-Wells et al. 2013;Horrix et al. 2011;Li et al. 2012;Lyu et al. 2002;Narayanan et al. 2005;Xie et al. 2011). ...
Riproximin, a type II ribosome-inactivating protein (RIP), has shown significant cytotoxic effects in diverse types of cancer cells. To better understand its therapeutic potential, elaborated investigations on the mechanistic aspects of riproximin deem crucial. In this study, we focused on riproximin-mediated changes in cellular properties and corresponding molecular pathways in breast cancer cells.
Cytotoxicity of riproximin was determined by MTT assay, while the clonogenic and migratory effects were determined by colony formation, migration, and scratch assays. Cytostatic and apoptotic effects were studied by flow cytometry and nuclear staining procedures. Alterations at molecular levels were scrutinized by means of microarray and qRT-PCR methodologies.
Riproximin induced significant cytotoxic effects in the selected human breast cancer cells MDA-MB-231 and MCF-7. Profound inhibition of migration and colony formation were observed in both cell lines in response to riproximin exposure. Concomitantly, a significant arrest in S phase and nuclear fragmentation were observed as causes for its cytostatic and apoptotic effects, respectively. Genetic profiling revealed pronounced induction of the anticancer cytokine IL24/MDA-7 and ER-stress-related GADD genes. In addition, prominent inhibition of the genes relevant to migration (RHO GTPases), anti-apoptotic activities (BCL family), and cell cycle (cyclins) was also noticed.
Riproximin, with its significant antineoplastic effects, modulates multiple cytostatic and apoptotic pathways in breast cancer cells. Results from these investigations highlight the future therapeutic potential of this naturally occurring compound for breast cancer.
... Other monoclonal or recombinant antibodies have been developed to target TfR1/CD71 for delivering chemotherapeutic drugs, protein toxins, radionuclides, liposomes, modified viral particles, and nanoparticles to kill malignant cells [87-89, 107, 108] (Figure 5). The combinations of such antibodies against TfR on human tumor cells have been demonstrated to have antiproliferative effects both in vitro and in vivo [106,[109][110][111][112][113][114]. ...
Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human cancers. Actually, ATC is refractory to conventional therapies, including surgery, chemotherapy, radiotherapy, and radioiodine (
131
I) therapy. Accordingly, genetic and molecular characterizations of ATC have been frequently and periodically reviewed in order to identify potential biological markers exploitable for target therapy. This review briefly focuses on main molecular events that characterize ATC and provides an update about preclinical studies. In addition, the overexpression of transferrin receptor 1 (TfR1/CD71) by neoplastic cells of ATC is emphasized in that it could represent a potential therapeutic target. In this regard, new therapeutic approaches based on the use of monoclonal or recombinant antibodies, or transferrin-gallium-TfR1/CD71 molecular complexes, or lastly small interfering RNAs (siRNAs) are proposed.
... These data coincide with a past study that showed improvement in proliferation of human osteoblast cells (CRL 1543) when the culture medium was supplemented with 0.0195% Tualang honey [30]. The underlying mechanism for the observed improvements is unknown, but it could be related to the active compounds present in Tualang honey or to the presence of H 2 O 2 , a ROS that is essential for modulating stem cell behaviours at the physiological level [45][46][47]. Pan et al. (2011) recently suggested that low levels of H 2 O 2 (10-50 mM) could promote rabbit corneal epithelial cell attachment, mobility, and wound repair [48]. This contradicts our findings using human cells that H 2 O 2 showed significant cytotoxicity at 50 mM and no noticeable changes in cell number at 10 and 20 mM when compared with the untreated control. ...
Stem cells with enhanced resistance to oxidative stress after in vitro expansion have been shown to have improved engraftment and regenerative capacities. Such cells can be generated by preconditioning them with exposure to an antioxidant. In this study we evaluated the effects of Tualang honey (TH), an antioxidant-containing honey, on human corneal epithelial progenitor (HCEP) cells in culture. Cytotoxicity, gene expression, migration, and cellular resistance to oxidative stress were evaluated. Immunofluorescence staining revealed that HCEP cells were holoclonal and expressed epithelial stem cell marker p63 without corneal cytokeratin 3. Cell viability remained unchanged after cells were cultured with 0.004, 0.04, and 0.4% TH in the medium, but it was significantly reduced when the concentration was increased to 3.33%. Cell migration, tested using scratch migration assay, was significantly enhanced when cells were cultured with TH at 0.04% and 0.4%. We also found that TH has hydrogen peroxide (H2O2) scavenging ability, although a trace level of H2O2 was detected in the honey in its native form. Preconditioning HCEP cells with 0.4% TH for 48 h showed better survival following H2O2-induced oxidative stress at 50 µM than untreated group, with a significantly lower number of dead cells (15.3±0.4%) were observed compared to the untreated population (20.5±0.9%, p<0.01). Both TH and ascorbic acid improved HCEP viability following induction of 100 µM H2O2, but the benefit was greater with TH treatment than with ascorbic acid. However, no significant advantage was demonstrated using 5-hydroxymethyl-2-furancarboxaldehyde, a compound that was found abundant in TH using GC/MS analysis. This suggests that the cellular anti-oxidative capacity in HCEP cells was augmented by native TH and was attributed to its antioxidant properties. In conclusion, TH possesses antioxidant properties and can improve cell migration and cellular resistance to oxidative stress in HCEP cells in vitro.
... Treatment with the 192 IgG saporin leads to the loss of a large portion of the BFCNs (Ha et al., 1999; Johnson et al., 2002; Pappas and Sherren, 2003; Ricceri et al., 2004; Robertson et al., 1998; Szigeti et al., 2013). Although it is known that saporin is able to kill cells by inactivating ribosomes and thereby shutting down protein synthesis (Daniels-Wells et al., 2013), it is not yet clear how a partial, sublethal (to the cell) dose may affect the cell. The present data suggest that smaller doses of saporin, even when targeted to the specific population of BFCNs by the 192 IgG saporin, may impact a population of cells so that they appear atrophic but are still viable. ...
Transferrin (Tf), widely known for its role as an iron-binding protein, exemplifies multitasking in biological processes. The role of Tf in iron metabolism involves both the uptake of iron from Tf by various cells, as well as the endocytosis mediated by the complex of Tf and the transferrin receptor (TfR). The direct conjugation of the therapeutic compound and immunotoxin studies using Tf peptide or anti-Tf receptor antibodies as targeting moieties aims to prolong drug circulation time and augment efficient cellular drug uptake, diminish systemic toxicity, traverse the blood-brain barrier, restrict systemic exposure, overcome multidrug resistance, and enhance therapeutic efficacy with disease specificity. This review primarily discusses the various biological actions of Tf, as well as the development of Tf-targeted nano-based drug delivery systems. The goal is to establish the use of Tf as a disease-targeting component, accentuating the potential therapeutic applications of this protein.
Osteosarcoma is a kind of primary bone malignant tumors. Its cure rate has been stagnant in the past decade years. Curcin C belongs to type I ribosome inactivating proteins, extracted from the cotyledons of post-germinated Jatropha curcas seeds. It can inhibit the proliferation of several tumor lines including U2OS cells with extraordinary efficiency. The treated U2OS cells were arrested in both S and G2/M phase, showed typical apoptosis morphological characteristic, formed autophagosomes and increase the ratio of LC3II to LC3I. Meanwhile, the level of ROS in the treated cells was found increasing significantly, with the change of mitochondrial membrane potential and decreased antioxidant enzyme activities. The application of ROS scavenger NAC not only significantly inhibited the toxicity of Curcin C but also prevented the happen of apoptosis and autophagy to some extent. These results suggested that Curcin C may function through ROS pathway. In addition, the Curcin C treatment could activate JNK and inhibit ERK signal pathway. Sp600125, an inhibitor of JNK signaling pathway, can prevent subsequent apoptosis and autophagy events, suggesting that JNK pathway was at least one of the pathways of Curcin C action. Moreover, the relevant including antagonistic among autophagy, apoptosis and cell cycle arresting induced by Curcin C also was found. In summary, it can be speculated that Curcin C may induce S, G2/M phase arrest, apoptosis and autophagy of human osteosarcoma U2OS cells through activating JNK signal pathway and blocking ERK signal pathway by promoting ROS accumulation in cell, thus finally reflected in the effect of inhibiting tumor cell proliferation.
Apoptosis, also known as programed cell death, is a disease-related pathway leading to cellular dysfunction and death. Apoptosis can occur through an intrinsic or extrinsic pathway, with the intrinsic pathway mediated through the mitochondria. The apoptosis pathway is activated by physiological stress, particularly oxidative stress. N-acetylcysteine (NAC), an antioxidant and precursor to glutathione, the major intrinsic antioxidant in our body, theoretically and empirically reduces apoptosis in several disease states and in experimental models. This chapter reviews the evidence for the effect of NAC on regulating apoptosis pathways though review of disease specific animal and cellular models and through review of experiments that examine particular pathways. NAC has been used in cancer studies for showing that apoptosis resulting from chemotherapy agents involves oxidative stress and that non-cancer cells can be protected against the apoptotic effects of chemotherapy by mitigating oxidative stress. NAC has been shown to protect against apoptosis in many disease models of injury, including those specific to cardiac, gastrointestinal, renal, pulmonary, and neurological tissues. NAC has a role in protecting cells from apoptosis through reducing oxidative stress, but improvements in apoptosis also appear to be associated with pathways not typically involved in oxidative stress such as receptor-mediated apoptosis signaling, modulation of regulation of cellular growth and differentiation pathways, as well as reducing inflammatory mediators. Overall, there is significant evidence that NAC can provide protection against apoptosis in many cellular and animal models of disease.
Iron is essential for cell growth and is imported into cells in part through the action of transferrin (Tf), a protein that binds its receptor (TfR1 or CD71) on the surface of a cell, and then releases iron into endosomes. TfR1 is a single pass type-II transmembrane protein expressed at basal levels in most tissues. High expression of TfR1 is typically associated with rapidly proliferating cells, including various types of cancer. TfR1 is targeted by experimental therapeutics for several reasons: its cell surface accessibility, constitutive endocytosis into cells, essential role in cell growth and proliferation, and its overexpression by cancer cells. Among the therapeutic agents used to target TfR1, antibodies stand out due to their remarkable specificity and affinity. Clinical trials are being conducted to evaluate the safety and efficacy of agents targeting TfR1 in cancer patients with promising results. These observations suggest that therapies targeting TfR1 as direct therapeutics or delivery conduits remain an attractive alternative for the treatment of cancers that overexpress the receptor.
Iron is essential for cell growth and is imported into cells in part through the action of transferrin (Tf), a protein that binds its receptor (TfR1 or CD71) on the surface of a cell, and then releases iron into endosomes. TfR1 is a single pass type-II transmembrane protein expressed at basal levels in most tissues. High expression of TfR1 is typically associated with rapidly proliferating cells, including various types of cancer. TfR1 is targeted by experimental therapeutics for several reasons: its cell surface accessibility, constitutive endocytosis into cells, essential role in cell growth and proliferation, and its overexpression by cancer cells. Among the therapeutic agents used to target TfR1, antibodies stand out due to their remarkable specificity and affinity. Clinical trials are being conducted to evaluate the safety and efficacy of agents targeting TfR1 in cancer patients with promising results. These observations suggest that therapies targeting TfR1 as direct therapeutics or delivery conduits remain an attractive alternative for the treatment of cancers that overexpress the receptor.
Metal-based photosensitizers are of interest as their absorption and chemical binding properties can be modified via the use of different ligands. Ru2+ based photosensitizers are known to be effective photodynamic therapy (PDT) agents against bacteria, whereas use for oncological indications in vivo has not been demonstrated with the same level of evidence. We present data showing that premixing the Ru2+-complex TLD1433 with transferrin increases the molar extinction coefficient, including longer activation wavelengths, reduces photo-bleaching rates, reduces the toxicity of the complex and improving overall PDT efficacy. As the transferrin receptor is unregulated in most malignancies, premixing of the Ru2+ complex with transferrin converts the active pharmaceutical ingredient TLD1433 into a drug of potentially considerable clinical utility.
The transferrin receptor 1 (TfR1) is involved in cellular iron uptake and regulation of cell proliferation. The increased expression of TfR1 observed in malignant cells, compared to normal cells, together with its extracellular accessibility, make this receptor an attractive target for antibody-mediated cancer therapy. We have developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1), which shows anti-tumor activity against certain malignant B cells in vitro through TfR1 degradation and iron deprivation, and in vivo through a mechanism yet to be defined. To further explore potential mechanisms of action of ch128.1, we examined its ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC). We now report that ch128.1 is capable of mediating ADCC and CDC against malignant B cells, which is consistent with its ability to bind FcRI, FcRIIIa, and the complement component C1q. To delineate the residues involved in these effector functions, we developed a panel of three constructs with mutations in the lower hinge region and C H 2 domain: 1) L234A/L235A, 2) P331S, and 3) L234A/L235A/P331S. The triple mutant consistently displayed a significant reduction in ADCC, while the L234A/L235A mutant exhibited less reduction in ADCC, and the P331S mutant did not show reduced ADCC. However, all three mutants exhibited impaired binding to FcRI and FcRIIIa. These results suggest that all three residues contribute to ADCC, although to different degrees. The P331S mutant showed drastically decreased C1q binding and abolished CDC, confirming the critical role of this residue in complement activation, while the other residues play a less important role in CDC. Our study provides insights into the effector functions of human IgG3 in the context of an antibody targeting TfR1.
The thioredoxin system helps maintain a reducing environment in cells, but thioredoxin functions as more than simply an antioxidant. Thioredoxin functions depend on the protein's redox state, as determined by two conserved cysteines. Key biologic activities of thioredoxin include antioxidant, growth control, and antiapoptotic properties, resulting from interaction with target molecules including transcription factors. Mechanisms by which thioredoxin regulates cell growth include binding to signaling molecules such as apoptosis signal-regulating kinase-1 (ASK-1) and thioredoxin-interacting protein (Txnip). The molecular interplay between thioredoxin, ASK-1, and Txnip potentially influences cell growth and survival in diverse human diseases such as cancer, diabetes, and heart disease. In this review, we focus on the structure of thioredoxin and its functional regulation of cell growth through the interactions with signaling molecules.
The oxygen paradox tells us that oxygen is both necessary for aerobic life and toxic to all life forms. Reactive oxygen species (ROS) touch every biological and medical discipline, especially those involving proliferative status, supporting the idea that active oxygen may be increased in tumor cells. In fact, metabolism of oxygen and the resulting toxic byproducts can cause cancer and death. Efforts to counteract the damage caused by ROS are gaining acceptance as a basis for novel therapeutic approaches, and the field of prevention of cancer is experiencing an upsurge of interest in medically useful antioxidants. Apoptosis is an important means of regulating cell numbers in the developing cell system, but it is so important that it must be controlled. Normal cell death in homeostasis of multicellular organisms is mediated through tightly regulated apoptotic pathways that involve oxidative stress regulation. Defective signaling through these pathways can contribute to both unbalance in apoptosis and development of cancer. Finally, in this review, we discuss new knowledge about recent tools that provide powerful antioxidant strategies, and designing methods to deliver to target cells, in the prevention and treatment of cancer.
Saporin-S6 is a single-chain ribosome-inactivating protein (RIP) that has low toxicity in cells and animals. When the protein is bound to a carrier that facilitates cellular uptake, the protein becomes highly and selectively toxic to the cellular target of the carrier. Thus, saporin-S6 is one of the most widely used RIPs in the preparation of immunoconjugates for anti-cancer therapy. The endocytosis of saporin-S6 by the neoplastic HeLa cells and the subsequent intracellular trafficking were investigated by confocal microscopy that utilises indirect immunofluorescence analysis and transmission electron microscopy that utilises a direct assay with gold-conjugated saporin-S6 and an indirect immunoelectron microscopy assay. Our results indicate that saporin-S6 was taken up by cells mainly through receptor-independent endocytosis. Confocal microscopy analysis showed around 30% co-localisation of saporin-S6 with the endosomal compartment and less than 10% co-localisation with the Golgi apparatus. The pathway identified by the immunofluorescence assay and transmission electron microscopy displayed a progressive accumulation of saporin-S6 in perinuclear vesicular structures. The main findings of this work are the following: i) the nuclear localisation of saporin-S6 and ii) the presence of DNA gaps resulting from basic sites in HeLa nuclei after intoxication with saporin-S6.
Ribosome-inactivating proteins (RIPs) are EC3.2.32.22 N-glycosidases that recognize a universally conserved stem-loop structure in 23S/25S/28S rRNA, depurinating a single adenine (A4324 in rat) and irreversibly blocking protein translation, leading finally to cell death of intoxicated mammalian cells. Ricin, the plant RIP prototype that comprises a catalytic A subunit linked to a galactose-binding lectin B subunit to allow cell surface binding and toxin entry in most mammalian cells, shows a potency in the picomolar range. The most promising way to exploit plant RIPs as weapons against cancer cells is either by designing molecules in which the toxic domains are linked to selective tumor targeting domains or directly delivered as suicide genes for cancer gene therapy. Here, we will provide a comprehensive picture of plant RIPs and discuss successful designs and features of chimeric molecules having therapeutic potential.
A number of antibodies have been developed that induce lethal iron deprivation (LID) by targeting the transferrin receptor 1 (TfR1/CD71) and either neutralizing transferrin (Tf) binding, blocking internalization of the receptor and/or inducing its degradation. We have developed recombinant antibodies targeting human TfR1 (ch128.1 and ch128.1Av), which induce receptor degradation and are cytotoxic to certain malignant B-cells. We now show that internalization of TfR1 bound to these antibodies can lead to its sequestration and degradation, as well as reduced Tf uptake, and the induction of a transcriptional response consistent with iron deprivation, which is mediated in part by downstream targets of p53. Cells resistant to these antibodies do not sequester and degrade TfR1 after internalization of the antibody/receptor complex, and accordingly maintain their ability to internalize Tf. These findings are expected to facilitate the rational design and clinical use of therapeutic agents targeting iron import via TfR1 in hematopoietic malignancies.
Mitochondrial processes play an important role in tumor initiation and progression. In this review, we focus on three critical processes by which mitochondrial function may contribute to cancer: through alterations in glucose metabolism, the production of reactive oxygen species (ROS) and compromise of intrinsic apoptotic function. Alterations in cancer glucose metabolism include the Warburg effect, leading to a shift in metabolism away from aerobic respiration toward glycolysis, even when sufficient oxygen is present to support respiration. Such alterations in cellular metabolism may favor tumor cell growth by increasing the availability of biosynthetic intermediates needed for cellular growth and proliferation. Mutations in specific metabolic enzymes, namely succinate dehydrogenase, fumarate hydratase and the isocitrate dehydrogenases, have been linked to human cancer. Mitochondrial ROS may contribute to cancer via DNA damage and the activation of aberrant signaling pathways. ROS-dependent stabilization of the transcription factor hypoxia-inducible factor (HIF) may be a particularly important event for tumorigenesis. Compromised function of intrinsic apoptosis removes an important cellular safeguard against cancer and has been implicated in tumorigenesis, tumor metastasis, and chemoresistance. Each of the major mitochondrial processes is linked. In this review, we outline the connections between them and address ways these mitochondrial pathways may be targeted for cancer therapy.
Glioblastoma multiforme (GBM) is the most common primary brain tumour in adults, with a median survival of ~12-18 months post-diagnosis. GBM usually recurs within 12 months post-resection, with poor prognosis. Thus, novel therapeutic strategies to target and kill GBM cells are urgently needed. The marked difference of tumour cells with respect to normal brain cells renders glioblastoma a good candidate for selective targeted therapies. Recent experimental strategies focus on over expressed cell surface receptors. Targeted toxins represent a new class of selective molecules composed by a potent protein toxin and a carrier ligand. Targeted toxins approaches against glioblastoma were under investigation in phase I and II clinical trials with several immunotoxins (IT)/ligand toxins such as IL4-Pseudomonas aeruginosa exotoxin A (IL4-PE, NBI-3001), tumour growth factor fused to PE38, a shorter PE variant, (TGF)alpha-TP-38, IL13-PE38, and a transferrin-C diphtheriae toxin mutant (Tf-CRM107). In this work, we studied the effects of the plant ribosome-inactivating saporin and of its chimera transferrin-saporin against two different GBM cell lines. The data obtained here indicate that cell proliferation is affected by the toxin treatments but that different mechanisms are used, directly linked to the presence of an active or inactive p53. A model is proposed for these alternative intracellular pathways.
The Cdc14 dual-specificity phosphatase plays a key role in the mitotic exit of budding yeast cells. Mammals have two homologues,
Cdc14a and Cdc14b. Unlike the yeast counterpart, neither Cdc14a nor Cdc14b seems to be essential for mitotic exit. To determine
the physiological function of Cdc14b, we generated mice deficient in the phosphatase. The mutant mice were viable and did
not display overt abnormalities. However, these mice developed signs of aging at much younger ages than the wild-type mice.
At the cellular level, the Cdc14b-deficient mouse embryonic fibroblasts (MEFs) grew more slowly than the controls at later
passages as a result of increased rates of senescence. Consistent with these premature-aging phenotypes, Cdc14b-deficient
cells accumulated more endogenous DNA damage than the wild-type cells, and more Cdc14b-deficient MEFs entered senescence than
control MEFs in response to exogenous DNA damage. However, no deficiencies in DNA damage checkpoint response were detected
in Cdc14b mutant cells, suggesting that the function of Cdc14b is required for efficient DNA damage repair.
A recent study suggested that human Cdc14B phosphatase has a central function in the G2 DNA damage checkpoint. In this study, we show that chicken DT40, human HCT116, and human telomerase reverse transcription-immortalized retinal pigment epithelial cells deleted for the Cdc14A or Cdc14B gene are DNA damage checkpoint proficient and arrest efficiently in G2 in response to irradiation. Cdc14A knockout (KO) or Cdc14B-KO cells also maintain normal levels of Chk1 and Chk2 activation after irradiation. Surprisingly, however, irradiation-induced gamma-H2A.X foci and DNA double-strand breaks persist longer in Cdc14A-KO or Cdc14B-KO cells than controls, suggesting that Cdc14 phosphatases are required for efficient DNA repair.
Messenger RNA (mRNA) export adaptors play an important role in the transport of mRNA from the nucleus to the cytoplasm. They couple early mRNA processing events such as 5' capping and 3' end formation with loading of the TAP/NXF1 export receptor onto mRNA. The canonical adaptor REF/ALY/Yra1 is recruited to mRNA via UAP56 and subsequently delivers the mRNA to NXF1 [1]. Knockdown of UAP56 [2, 3] and NXF1 [4-7] in higher eukaryotes efficiently blocks mRNA export, whereas knockdown of REF only causes a modest reduction, suggesting the existence of additional adaptors [8-10]. Here we identify a new UAP56-interacting factor, UIF, which functions as an export adaptor, binding NXF1 and delivering mRNA to the nuclear pore. REF and UIF are simultaneously found on the same mRNA molecules, and both proteins are required for efficient export of mRNA. We show that the histone chaperone FACT specifically binds UIF, but not REF, via the SSRP1 subunit, and this interaction is required for recruitment of UIF to mRNA. Together the results indicate that REF and UIF represent key human adaptors for the export of cellular mRNAs via the UAP56-NXF1 pathway.
The genetic basis of myotonic dystrophy type I (DM1) is the expansion of a CTG tract located in the 3' untranslated region of DMPK. Expression of mutant RNAs encoding expanded CUG repeats plays a central role in the development of cardiac disease in DM1. Expanded CUG tracts form both nuclear and cytoplasmic aggregates, yet the relative significance of such aggregates in eliciting DM1 pathology is unclear. To test the pathophysiology of CUG repeat encoding RNAs, we developed and analyzed mice with cardiac-specific expression of a beta-galactosidase cassette in which a (CTG)(400) repeat tract was positioned 3' of the termination codon and 5' of the bovine growth hormone polyadenylation signal. In these animals CUG aggregates form exclusively in the cytoplasm of cardiac cells. A key pathological consequence of expanded CUG repeat RNA expression in DM1 is aberrant RNA splicing. Abnormal splicing results from the functional inactivation of MBNL1, which is hypothesized to occur due to MBNL1 sequestration in CUG foci or from elevated levels of CUG-BP1. We therefore tested the ability of cytoplasmic CUG foci to elicit these changes. Aggregation of CUG RNAs within the cytoplasm results both in Mbnl1 sequestration and in approximately a two fold increase in both nuclear and cytoplasmic Cug-bp1 levels. Significantly, despite these changes RNA splice defects were not observed and functional analysis revealed only subtle cardiac dysfunction, characterized by conduction defects that primarily manifest under anesthesia. Using a human myoblast culture system we show that this transgene, when expressed at similar levels to a second transgene, which encodes expanded CTG tracts and facilitates both nuclear focus formation and aberrant splicing, does not elicit aberrant splicing. Thus the lack of toxicity of cytoplasmic CUG foci does not appear to be a consequence of low expression levels. Our results therefore demonstrate that the cellular location of CUG RNA aggregates is an important variable that influences toxicity and support the hypothesis that small molecules that increase the rate of transport of the mutant DMPK RNA from the nucleus into the cytoplasm may significantly improve DM1 pathology.
Proteolysis of cellular substrates by caspases (cysteine-dependent aspartate-specific proteases) is one of the hallmarks of apoptotic cell death. Although the activation of apoptotic caspases is considered a 'late-stage' event in apoptosis signaling, past the commitment stage, one caspase family member, caspase-2, splits the cell death community into half - those searching for evidence of an apical initiator function of this molecule and those considering it as an amplifier of the apoptotic caspase cascade, at best, if relevant for apoptosis at all. This review screens past and present biochemical as well as genetic evidence for caspase-2 function in cell death signaling and beyond.
The human transferrin receptor (hTfR) is a target for cancer immunotherapy due to its overexpression on the surface of cancer cells. We previously developed an antibody-avidin fusion protein that targets hTfR (anti-hTfR IgG3-Av) and exhibits intrinsic cytotoxicity against certain malignant cells. Gambogic acid (GA), a drug that also binds hTfR, induces cytotoxicity in several malignant cell lines. We now report that anti-hTfR IgG3-Av and GA induce cytotoxicity in a new broader panel of hematopoietic malignant cell lines. Our results show that the effect of anti-hTfR IgG3-Av is iron-dependent whereas that of GA is iron-independent in all cells tested. In addition, we observed that GA exerts a TfR-independent cytotoxicity. We also found that GA increases the generation of reactive oxygen species that may play a role in the cytotoxicity induced by this drug. Additive cytotoxicity was observed by simultaneous combination treatment with these drugs and synergy by using anti-hTfR IgG3-Av as a chemosensitizing agent. In addition, we found a concentration of GA that is toxic to malignant hematopoietic cells but not to human hematopoietic progenitor cells. Our results suggest that these two compounds may be effective, alone or in combination, for the treatment of human hematopoietic malignancies.
Relapse is the overwhelming cause of treatment failure after autologous transplantation for multiple myeloma (MM). For patients with a syngeneic donor, twin transplants provide a healthy graft that is free of myeloma. The relative impact of the graft on posttransplant relapse can be estimated by comparing risk of relapse after hematopoietic cell transplantation from genetically identical twins versus autotransplants because confounding differences in minor or major histocompatibility antigens are absent in the syngeneic transplant setting. Outcomes of 43 subjects who received twin transplants for MM were compared to 170 matched autotransplant recipients reported to the Center for International Blood and Marrow Transplant Research (CIBMTR). Multivariate analysis was performed by fitting a Cox model stratified on matched pairs. The matched transplant patients studied were similar with respect to subject-, disease-, and transplant-related characteristics. Cumulative incidence of relapse/progression was significantly lower, and progression-free survival (PFS) was significantly higher following twin transplants. In multivariate analysis, the probability of relapse/progression was lower in twins (relative risk [RR] = 0.49, 95% confidence interval [CI] 0.28-0.86, P = .011). Twin transplants have a significantly lower relapse risk than autotransplants in MM, suggesting that graft composition may impact outcomes following high-dose chemotherapy.
The fate of phenotypically defined human hematopoietic stem cells (hHSCs) in culture and the link between their surface marker expression profile and function are still controversial. We studied these aspects of hHSC biology by relating the expression of the early lineage markers (ELM) CD33, CD38, and CD71 on the surface of human umbilical cord blood (UCB) CD34+ cells to their long-term nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse repopulation activity (LT-SRA). In uncultured UCB samples, LT-SRA was largely confined to the small CD34+ELM− cell fraction. CD34+ cells expressing ELM markers at their surface usually lacked LT-SRA. After culturing UCB CD34+ cells for 6 days in serum-free medium and on a feeder layer of Rat2 cells, the number of CD34+ELM− cells stayed roughly the same or showed a slight increase and the LT-SRA was preserved, suggesting a close association between LT-SRA and the CD34+ELM− phenotype. Indeed, transplantation of CD34+ELM− cells isolated from cultured UCB CD34+ cells resulted in long-term hematopoietic reconstitution of conditioned NOD/SCID mice, whereas CD34+ELM+ cells derived from the same cultures were devoid of LT-SRA. Remarkably, roughly 1% of the cells recovered from cultures initiated with isolated CD34+ELM+ cells had lost ELM surface expression. Concurrently, the cultured CD34+ELM+ cells acquired LT-SRA, suggesting that hematopoietic stem cells (HSCs) may arise by the dedifferentiation of early hematopoietic progenitor cells. The latter finding challenges the paradigm of unidirectional hematopoietic differentiation and opens new opportunities for HSC expansion prior to transplantation.
Disclosure of potential conflicts of interest is found at the end of this article.
To directly study the biological properties of purified hematopoietic colony-forming cell precursors, cells with a CD34+ CD45RAlo CD71lo phenotype were purified from human bone marrow using density separation and fluorescence-activated cell sorting, and were cultured in serum-free culture medium supplemented with various cytokines. In the presence of interleukin 3 (IL-3), IL-6, erythropoietin, and mast cell growth factor (a c-kit ligand), cell numbers increased approximately 10(6)-fold over a period of 4 wk, and the percentage of cells that expressed transferrin receptors (CD71) increased from less than 0.1% at day 0 to greater than 99% at day 14. Interestingly, the absolute number of CD34+ CD71lo cells did not change during culture. When CD34+ CD71lo cells were sorted from expanded cultures and recultured, extensive cell production was repeated, again without significant changes in the absolute number of cells with the CD34+ CD71lo phenotype that were used to initiate the (sub)cultures. These results document that primitive hematopoietic cells can generate progeny without an apparent decrease in the size of a precursor cell pool.
We have developed an antibody fusion protein (anti-rat TfR IgG3-Av) with the ability to deliver different molecules into cancer cells. It consists of avidin genetically fused to the C(H)3 region of a human IgG3 specific for the rat transferrin receptor. It forms strong, noncovalent interactions with biotinylated molecules such as glucose oxidase and beta-galactosidase, and delivers them into the rat myeloma cell line Y3-Ag1.2.3 through receptor-mediated endocytosis. Importantly, the beta-galactosidase retains activity after internalization. Furthermore, we have unexpectedly discovered that anti-rat TfR IgG3-Av, but not a recombinant anti-rat TfR IgG3 or a nonspecific IgG3-Av, possesses proapoptotic activities against Y3-Ag1.2.3 and the rat T cell lymphoma cell line C58 (NT) D.1.G.OVAR.1. These activities were not observed in two rat cell lines of nonhematopoietic lineage (bladder carcinoma BC47 and gliosarcoma 9L). Anti-human TfR IgG3-Av also demonstrated proapoptotic activity against the human erythroleukemia cell line K562. Studies showed that anti-rat TfR IgG3-Av exists as a dimer, suggesting that cross-linking of the surface transferrin receptor may be responsible for the cytotoxic activity. These findings demonstrate that it is possible to transform an antibody specific for a growth factor receptor that does not exhibit inhibitory activity into a drug with significant intrinsic cytotoxic activity against selected cells by fusing it with avidin. The antitumor activity may be enhanced by delivering biotinylated therapeutics into cancer cells. Further development of this technology may lead to effective therapeutics for in vivo eradication of hematological malignancies, and ex vivo purging of cancer cells in autologous transplantation.
Saporin-6 produced by the plant Saponaria officinalis belongs to the family of single chain ribosome-inactivating proteins. It potently inhibits protein synthesis in eukaryotic cells, by cleaving the N-glycosidic bond of a specific adenine in 28 S rRNA, which results in the cell death. Saporin-6 has also been shown to be active on DNA and induces apoptosis. In the current study, we have investigated the roles of rRNA depurination and the activity of saporin-6 on genomic DNA in its cytotoxic activity. The role of putative active site residues, Tyr(72), Tyr(120), Glu(176), Arg(179), and Trp(208), and two invariant residues, Tyr(16) and Arg(24), proposed to be important for structural stability of saporin-6, has been investigated in its catalytic and cytotoxic activity. These residues were mutated to alanine to generate seven mutants, Y16A, R24A, Y72A, Y120A, E176A, R179A, and W208A. We show that for the RNA N-glycosidase activity of saporin-6, residues Tyr(16), Tyr(72), and Arg(179) are absolutely critical; Tyr(120) and Glu(176) can be partially dispensed with, whereas Trp(208) and Arg(24) do not appear to be involved in this activity. The residues Tyr(72), Tyr(120), Glu(176), Arg(179), and Trp(208) were found to be essential for the genomic DNA fragmentation activity, whereas residues Tyr(16) and Arg(24) do not appear to be required for the DNA fragmentation. The study shows that saporin-6 possesses two catalytic activities, namely RNA N-glycosidase and genomic DNA fragmentation activity, and for its complete cytotoxic activity both activities are required.
Kruppel-like factor 6 (KLF6) is a tumor suppressor gene inactivated in prostate and colon cancers, as well as in astrocytic gliomas. Here, we establish that KLF6 mediates growth inhibition through an interaction with cyclin D1, leading to reduced phosphorylation of the retinoblastoma protein (Rb) at Ser(795). Furthermore, introduction of KLF6 disrupts cyclin D1-cyclin-dependent kinase (cdk) 4 complexes and forces the redistribution of p21(Cip/Kip) onto cdk2, which promotes G(1) cell cycle arrest. Our data suggest that KLF6 converges with the Rb pathway to inhibit cyclin D1/cdk4 activity, resulting in growth suppression.
Chronic sun exposure can lead to severe skin disorders such as carcinogenesis. The cell death process triggered by ultraviolet B (UVB) irradiation is crucial because it protects the surrounding tissue from the emergence and the accumulation of cells that bear the risk of becoming transformed. Here, we show that repression of NF-kappaB and Egr-1 expression drastically inhibits UVB-mediated cell death. Furthermore, we demonstrate that Egr-1 is induced upon UVB irradiation through NF-kappaB activation and the binding of p65/RelA within the Egr-1 promoter. We show that Egr-1 contributes to the regulation of the Gadd45a and Gadd45b genes, which are involved in the control of cell cycle, DNA repair and apoptosis, by direct binding to their promoter. Our study demonstrates for the first time a signaling cascade involving sequential activation of NF-kappaB, Egr-1 and Gadd45 to induce UVB-mediated cell death. Failure in the induction of each protagonist of this pathway alters the UVB-mediated cell death process. Therefore, impairment of the cascade could be at the onset of skin carcinogenesis mediated by genotoxic stress.
The E box sequence (5'-CANNTG-3') is found in the transcriptional regulatory region of a number of genes. Of the basic helix-loop-helix (bHLH) proteins binding to the E box sequence, class B of bHLH proteins, BHLHB2 (also referred to as the DEC1/Eip1/SHARP-2/Stra13/Clast5) and BHLHB3 (also referred to as the DEC2/SHARP-1/SHARP1), are transcription factors that contain a unique orange domain. These transcription factors repress the transcription of target genes not only via binding to the E box sequence but also via protein-protein interactions with other transcription factors. Both the BHLHB2 and BHLHB3 genes are widely expressed in both embryonic and adult tissues. Their gene expressions are regulated in a cell type-specific manner by various extracellular stimuli, such as growth factors, serum starvation, hypoxia, hormones, nutrient, cytokines, light, and infection. Therefore, these transcription factors play pivotal roles in multiple signaling pathways that impact many biological processes including development, cell differentiation, cell growth, cell death, oncogenesis, immune systems, circadian rhythm, and homeostasis. The structural features, functions, and biological roles of the novel bHLH transcription factors, BHLHB2 and BHLHB3, are discussed along with the mechanisms in which the genes encoding these factors are regulated.
A model has been presented for retrograde transport of certain toxins and viruses from the cell surface to the ER that suggests an obligatory interaction with a glycolipid receptor at the cell surface. Here we review studies on the ER trafficking cholera toxin, Shiga and Shiga-like toxins, Pseudomonas exotoxin A and ricin, and compare the retrograde routes followed by these protein toxins to those of the ER trafficking SV40 and polyoma viruses. We conclude that there is in fact no obligatory requirement for a glycolipid receptor, nor even with a protein receptor in a lipid-rich environment. Emerging data suggests instead that there is no common pathway utilised for retrograde transport by all of these pathogens, the choice of route being determined by the particular receptor utilised.
We have previously reported that an anti-human transferrin receptor IgG3-avidin fusion protein (anti-hTfR IgG3-Av) inhibits the proliferation of an erythroleukemia-cell line. We have now found that anti-hTfR IgG3-Av also inhibits the proliferation of additional human malignant B and plasma cells. Anti-hTfR IgG3-Av induces internalization and rapid degradation of the TfR. These events can be reproduced in cells treated with anti-hTfR IgG3 cross-linked with a secondary Ab, suggesting that they result from increased TfR cross-linking. Confocal microscopy of cells treated with anti-hTfR IgG3-Av shows that the TfR is directed to an intracellular compartment expressing the lysosomal marker LAMP-1. The degradation of TfR is partially blocked by cysteine protease inhibitors. Furthermore, cells treated with anti-hTfR IgG3-Av exhibit mitochondrial depolarization and activation of caspases 9, 8, and 3. The mitochondrial damage and cell death can be prevented by iron supplementation, but cannot be fully blocked by a pan-caspase inhibitor. These results suggest that anti-hTfR IgG3-Av induces lethal iron deprivation, but the resulting cell death does not solely depend on caspase activation. This report provides insights into the mechanism of cell death induced by anti-TfR Abs such as anti-hTfR IgG3-Av, a molecule that may be useful in the treatment of B-cell malignancies such as multiple myeloma.
Thioredoxin binding protein (TXNIP) has multiple functions and plays an important role in redox homeostasis. TXNIP increases the production of reactive oxygen species (ROS), and oxidative stress, resulting in cellular apoptosis. It has been identified as a tumor suppressor gene (TSG) in various solid tumors and hematological malignancies. In the present review, we will first provide an overview of TXNIP protein and function, followed by a summary of the major studies that have demonstrated the frequent repression of TXNIP in cancers. Functional characterization of TXNIP knockout mouse model is summarized. We will then discuss the use of small molecular inhibitors to reactivate TXNIP expression as a novel anticancer strategy.
Saporins are ribosome-inactivating proteins (RIPs) extracted from different tissues of the soapwort plant (Saponaria officinalis L.). While the biosynthesis of these proteins and their roles in planta have received little attention, saporins have been
extensively used for the production of targeted toxins for therapeutical and research applications. The biochemical features
of one group of closely related saporin isoforms, collectively named SO6, have been characterized in considerable detail.
In this chapter, we summarize available information on the saporin family of proteins, including their catalytic activity,
3D-structure, and biosynthetic and intoxication pathway(s), emphasizing the differences between the different family members
and the characteristics that distinguish saporin from the catalytic subunit of the prototype Type II RIP ricin. The use of
heterologous systems for the production of saporin and saporin-based chimeric toxins is also described.
Sequence profile searches were used to identify an ancient domain in ThiI-like thiouridine synthases, conserved RNA methylases, archaeal pseudouridine synthases and several uncharacterized proteins. We predict that this domain is an RNA-binding domain that adopts an α/β fold similar to that found in the C-terminal domain of translation initiation factor 3 and ribosomal protein S8.
Ozone (O(3)) uptake by plants leads to an increase in reactive oxygen species (ROS) in the intercellular space of leaves and induces signalling processes reported to involve the membrane-bound heterotrimeric G-protein complex. Therefore, potential G-protein-mediated response mechanisms to O(3) were compared between Arabidopsis thaliana L. lines with null mutations in the α- and β-subunits (gpa1-4, agb1-2 and gpa1-4/agb1-2) and Col-0 wild-type plants. Plants were treated with a range of O(3) concentrations (5, 125, 175 and 300 nL L(-1)) for 1 and 2 d in controlled environment chambers. Transcript levels of GPA1, AGB1 and RGS1 transiently increased in Col-0 exposed to 125 nL L(-1) O(3) compared with the 5 nL L(-1) control treatment. However, silencing of α and β G-protein genes resulted in little alteration of many processes associated with O(3) injury, including the induction of ROS-signalling genes, increased leaf tissue ion leakage, decreased net photosynthesis and stomatal conductance, and increased peroxidase activity, especially in the leaf apoplast. These results indicated that many responses to O(3) stress at physiological levels were not detectably influenced by α and β G-proteins.
Traditional cancer therapy can be successful in destroying tumors, but can also cause dangerous side effects. Therefore, many targeted therapies are in development. The transferrin receptor (TfR) functions in cellular iron uptake through its interaction with transferrin. This receptor is an attractive molecule for the targeted therapy of cancer since it is upregulated on the surface of many cancer types and is efficiently internalized. This receptor can be targeted in two ways: 1) for the delivery of therapeutic molecules into malignant cells or 2) to block the natural function of the receptor leading directly to cancer cell death.
In the present article we discuss the strategies used to target the TfR for the delivery of therapeutic agents into cancer cells. We provide a summary of the vast types of anti-cancer drugs that have been delivered into cancer cells employing a variety of receptor binding molecules including Tf, anti-TfR antibodies, or TfR-binding peptides alone or in combination with carrier molecules including nanoparticles and viruses.
Targeting the TfR has been shown to be effective in delivering many different therapeutic agents and causing cytotoxic effects in cancer cells in vitro and in vivo.
The extensive use of TfR for targeted therapy attests to the versatility of targeting this receptor for therapeutic purposes against malignant cells. More advances in this area are expected to further improve the therapeutic potential of targeting the TfR for cancer therapy leading to an increase in the number of clinical trials of molecules targeting this receptor. This article is part of a Special Issue entitled Transferrins: molecular mechanisms of iron transport and disorders.
We previously developed an antibody-avidin fusion protein (ch128.1Av) targeting the human transferrin receptor 1 (TfR1, also known as CD71), which demonstrates direct in vitro cytotoxicity against malignant hematopoietic cells. This cytotoxicity is attributed to its ability to decrease the level of TfR1 leading to lethal iron deprivation. We now report that ch128.1Av shows the ability to bind the Fcγ receptors and the complement component C1q, suggesting that it is capable of eliciting Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-mediated cytotoxicity. In addition, in 2 disseminated multiple myeloma xenograft mouse models, we show that a single dose of ch128.1Av results in significant antitumor activity, including long-term survival. It is interesting to note that the parental antibody without avidin (ch128.1) also shows remarkable in vivo anticancer activity despite its limited in vitro cytotoxicity. Finally, we demonstrate that ch128.1Av is not toxic to pluripotent hematopoietic progenitor cells using the long-term cell-initiating culture assay suggesting that these important progenitors would be preserved in different therapeutic approaches, including the in vitro purging of cancer cells for autologous transplantation and in vivo passive immunotherapy. Our results suggest that ch128.1Av and ch128.1 may be effective in the therapy of human multiple myeloma and potentially other hematopoietic malignancies.
Markers that could accurately predict responses to the general kinase inhibitor sorafenib are needed to better leverage its clinical applications. In this study, we examined a hypothesized role in the drug response for the growth arrest DNA damage-inducible gene 45β (GADD45β), which is commonly underexpressed in hepatocellular carcinoma (HCC) where sorafenib may offer an important new therapeutic option. The anticancer activity of sorafenib-induced GADD45β expression was tested in a panel of HCC cell lines and xenograft models. We found that GADD45β mRNA and protein expression were induced relatively more prominently in HCC cells that were biologically sensitive to sorafenib treatment. GADD45β induction was not found after treatment with either the mitogen-activated protein kinase-extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 or the Raf inhibitor ZM336372, suggesting that GADD45β induction by sorafenib was independent of Raf/MEK/ERK signaling activity. However, c-Jun NH2-terminal kinase (JNK) kinase activation occurred preferentially in sorafenib-sensitive cells. Small interfering RNA-mediated knockdown of GADD45βor JNK kinase limited the proapoptotic effects of sorafenib in sorafenib-sensitive cells. We defined the -339/-267 region in the GADD45β promoter containing activator protein-1 and SP1-binding sites as a crucial region for GADD45β induction by sorafenib. Together, our findings suggest that GADD45β induction contributes to sorafenib-induced apoptosis in HCC cells, prompting further studies to validate its potential value in predicting sorafenib efficacy.
Glucocorticoid-induced leucine zipper (GILZ) is a glucocorticoid-induced protein, the reported molecular interactions of which suggest that it functions to inhibit inflammation. However, the role of endogenous GILZ in the regulation of inflammation in vivo has not been established. This study was undertaken to examine the expression and function of GILZ in vivo in collagen-induced arthritis (CIA), a murine model of rheumatoid arthritis (RA), and in RA synoviocytes.
GILZ expression was detected in mouse and human synovium by immunohistochemistry and in cultured cells by real-time polymerase chain reaction and permeabilization flow cytometry. GILZ function was assessed in vivo by small interfering RNA (siRNA) silencing using cationic liposome-encapsulated GILZ or control nontargeting siRNA and was assessed in vitro using transient overexpression.
GILZ was readily detectable in the synovium of mice with CIA and was up-regulated by therapeutic doses of glucocorticoids. Depleting GILZ expression in vivo increased the clinical and histologic severity of CIA and increased synovial expression of tumor necrosis factor and interleukin-1 (IL-1), without affecting the levels of circulating cytokines or anticollagen antibodies. GILZ was highly expressed in the synovium of patients with active RA and in cultured RA synovial fibroblasts, and GILZ overexpression in synovial fibroblasts inhibited IL-6 and IL-8 release.
Our findings indicate that GILZ functions as an endogenous inhibitor of chronic inflammation via effects on cytokine expression and suggest that local modulation of GILZ expression could be a beneficial therapeutic strategy.
The GADD45 family of proteins consists of three small proteins, GADD45A, GADD45B, and GADD45G, implicated in modulating the cellular response to genotoxic/physiological stressors. Despite similarities in sequence, structure and function, each gadd45 gene is induced differentially by different stress stimuli. Studies on stress-mediated induction of the gadd45 genes have predominantly focused on gadd45a, with knowledge of gadd45b and gadd45g regulation lacking. To generate a more complete understanding of the regulation of gadd45 genes, a comprehensive analysis of stress-mediated induction of human gadd45b has been carried out using human RKO colorectal carcinoma cells as a model system. Novel data indicate that gadd45b induction in RKO cells is regulated by distinct mechanisms in a stress-specific manner. Methylmethane sulfonate (MMS), a DNA alkylating agent, induces gadd45b transcription through a cohort of both constitutive and inducible bound factors, including NFY, Sp1 and Egr1. In contrast, in a hyperosmotic environment generated with sorbitol, gadd45b mRNA is induced exclusively by mRNA stabilization. These findings indicate that the stress-mediated induction of gadd45b is largely distinct from gadd45a. Furthermore, data obtained provide a novel paradigm for stress-response gene induction, indicating that gadd45b induction by distinct stressors, in the same cell type and under the same experimental settings, is differentially regulated at the level of mRNA transcription or mRNA stability. Importantly, this study also provides the groundwork to further examine the regulation of gadd45b expression in in vivo settings using animal models and tissues obtained from normal individuals and cancer patients prior to and after chemotherapeutic intervention.
Cell death and differentiation is a monthly research journal focused on the exciting field of programmed cell death and apoptosis. It provides a single accessible source of information for both scientists and clinicians, keeping them up-to-date with advances in the field. It encompasses programmed cell death, cell death induced by toxic agents, differentiation and the interrelation of these with cell proliferation.
Abrin is a plant glycoprotein toxin, classified as ribosome inactivating protein (RIP) due to its property of damaging ribosomes in an irreversible manner. Many RIPs have direct DNA damaging activity. The objective of the present study was to evaluate the oxidative stress and DNA damaging potential of abrin in U937 human myeloleukemic cells. Cells were treated with abrin at IC50 of 8 ng/ml for 24h. Abrin induced a time dependent increase in reactive oxygen species and levels of antioxidant enzymes. There was significant depletion of reduced glutathione levels. DNA damage was assessed by comet assay in terms of percent head DNA, tail DNA, tail length and Olive tail moment. DNA damage was more pronounced at 4 and 8h at IC50 concentration. Abrin at 4, 8, 16 and 32 ng/ml concentration induced significant DNA damage at 4h. There was time dependent increase in levels of 8-OHdG in abrin treated cells indicating the oxidative stress mediated DNA damage. N-Acetylcysteine pretreatment at 10nM for 1h, considerably reversed the abrin induced DNA damage at 16 and 32 ng/ml. Our results clearly show oxidative DNA damage potential of abrin at low concentration.
Most recently reported methods to select early hematopoietic cells basically rely on the depletion of committed progenitors. This task is generally accomplished by laborious procedures, which are sometimes difficult to reproduce. To simplify the selection method, we took advantage of the expression of the transferrin receptor (CD71) by proliferating committed progenitors and the lack of CD71 on noncycling immature progenitors. A monoclonal antibody (MAB) reactive with CD71 has been conjugated to the Saponaria officinalis seed ribosome-inactivating protein (SO6). The immunotoxin (IT) complex was used at increasing concentrations on normal non-phagocytizing bone marrow cells. A complete and reproducible killing effect on myeloid (colony-forming unit-granulocyte/macrophage [CFU-GM]) and erythroid (burst-forming unit-erythroid [BFU-E]) progenitors was observed for IT concentrations of 1 x 10(-7) M. Unconjugated SO6 or anti-CD71 MAB had no effect on cell growth and viability. IT-resistant cells were able to generate CFU-GM after 7, 14, and 21 days of suspension culture in the presence of 5637 CM. Maximal CFU-GM values were obtained at day 21 and nearly approached the pretreatment values (mean 2587 vs. 3877 CFU-GM/mL). Growth factor enhancement of CFU-GM yield was obtained only by stem cell factor (SCF) at day 7; SCF, as well as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), had an enhancing effect at days 14 and 21. IT toxicity on highly immature progenitors was ruled out by evaluating the growth of long-term culture-initiating cells (LTC-IC) from IT-treated cultures. LTC-IC frequency was found to be 1 out of 1506 seeded cells, which is within the range of normal untreated BM cells. In conclusion, anti-CD71 IT allows a simple and complete depletion of committed progenitors while sparing immature hematopoietic cells. The high CD71 expression by leukemic cells makes the procedure potentially suitable for in vitro purging.
The single-chain ribosome-inactivating proteins (scRIPs) from plant origin are antiviral and antiproliferative agents employed in the preparation of immunotoxins. Similarly to the A-chains of ricin, sc-RIPs act as rRNA N-glycosidases. We demonstrate here that dianthin 30, saporin 6 and gelonin exert a specific nuclease activity on supercoiled DNA. Four specific sites of cleavage introduced by dianthin 30 and by saporin 6 and two specific sites of cleavage introduced by gelonin have been identified and mapped in pBR322.
We have isolated a human cDNA which encodes a novel I kappa B family member using a yeast two-hybrid screen for proteins able to interact with the p52 subunit of the transcription factor NF-kappa B. The protein is found in many cell types and its expression is up-regulated following NF-kappa B activation and during myelopoiesis. Consistent with its proposed role as an I kappa B molecule, I kappa B-epsilon is able to inhibit NF-kappa B-directed transactivation via cytoplasmic retention of rel proteins. I kappa B-epsilon translation initiates from an internal ATG codon to give rise to a protein of 45 kDa, which exists as multiple phosphorylated isoforms in resting cells. Unlike the other inhibitors, it is found almost exclusively in complexes containing RelA and/or cRel. Upon activation, I kappa B-epsilon protein is degraded with slow kinetics by a proteasome-dependent mechanism. Similarly to I kappa B-alpha and I kappa B, I kappa B-epsilon contains multiple ankyrin repeats and two conserved serines which are necessary for signal-induced degradation of the molecule. A unique lysine residue located N-terminal of the serines appears to be not strictly required for degradation. Unlike I kappa B- alpha and I kappa B-beta, I kappa B-epsilon does not contain a C-terminal PEST-like sequence. I kappa B-epsilon would, therefore, appear to regulate a late, transient activation of a subset of genes, regulated by RelA/cRel NF-kappa B complexes, distinct from those regulated by other I kappa B proteins.
Saporin, a monomeric protein extracted from the seeds of Saponaria officinalis, is an enzyme capable of specific depurination of the eukaryotic ribosomes. Because of its toxicity, saporin proved useful for the synthesis of immunotoxins, chimeric conjugates of a toxin and an antibody specifically directed against cancer cells or other targets. In this paper we report a study of the structural properties of saporin in the presence of denaturing agents and/or proteolytic enzymes. We found that saporin is extremely resistant to denaturation by urea or guanidine (up to 4 M), even at relatively high temperature (up to 55 degrees C). Moreover a structural change detected as a reduction of the fluorescence emission of the single Trp residue is reversible and is not paralleled by changes of the far UV CD spectrum, suggesting that even under harsh experimental conditions unfolding is limited. In good agreement with these results, guanidine-treated saporin is not attacked by proteolytic enzymes. The remarkable resistance of saporin to denaturation and proteolysis suggests this protein as an ideal candidate for biotechnological applications.
A novel member of the I kappaB family has been identified as a protein that associated with the p50 subunit of NF-kappaB in
a yeast two-hybrid screen. Similar to previously known I kappaB proteins, this member, I kappaB epsilon, has six consecutive
ankyrin repeats. I kappaB epsilon mRNA is widely expressed in different human tissues, with highest levels in spleen, testis,
and lung. I kappaB epsilon interacts with different NF-kappaB proteins, including p65 (RelA), c-Rel, p50, and p52, in vitro
and in vivo and inhibits the DNA-binding activity of both p50-p65 and p50-c-Rel complexes effectively. Endogenous and transfected
NF-kappaB (RelA-dependent) transcriptional activation is inhibited by I kappaB epsilon. I kappaB epsilon mRNA is expressed
at different levels in specific cell types and is synthesized constitutively in transformed B-cell lines. It also displays
differential induction in response to tumor necrosis factor alpha, interleukin-1, or phorbol ester stimulation compared to
I kappaB alpha in non-B-cell lines. Therefore, I kappaB epsilon represents a novel I kappaB family member which provides an
alternative mechanism for regulation of NF-kappaB-dependent transcription.
Our current understanding of human haematopoietic stem cell biology is based in part on the characterization of human CD34+ bone marrow cell differentiation in vitro. CD34 is highly expressed on early stem cells and haematopoietic progenitor cells with clonogenic potential and is gradually lost during differentiation and commitment. However, CD71 (transferrin receptor) is expressed at low levels on early stem cells and generally increases during haematopoietic progenitor cell proliferation. We reasoned that the combination of these surface markers would provide a useful framework for the simultaneous analysis of multiple lineage differentiation of CD34+ haematopoietic progenitor cells in liquid culture. In this report, we identify the phenotype of distinct subpopulations of myeloid, erythroid and lymphoid cells in liquid suspension culture using differential expression of CD34 vs. CD71 in combination with specific lineage markers. Freshly isolated human CD34+ bone marrow cells were introduced into suspension culture and monitored over a 6-d period using 3-colour flow cytometry. This is the first demonstration that differential expression of CD34 vs. CD71 can be used to simultaneously monitor differentiation of multiple haematopoietic cell lineages in liquid suspension culture, facilitating the study of cytokine-, drug- or chemical-induced alterations in haematopoietic progenitor cell differentiation in vitro.
Interleukin 6 (IL-6) is the major survival factor for myeloma tumor cells and induces signaling through the STAT proteins. We report that one STAT family member, Stat3, is constitutively activated in bone marrow mononuclear cells from patients with multiple myeloma and in the IL-6-dependent human myeloma cell line U266. Moreover, U266 cells are inherently resistant to Fas-mediated apoptosis and express high levels of the antiapoptotic protein Bcl-xL. Blocking IL-6 receptor signaling from Janus kinases to the Stat3 protein inhibits Bcl-xL expression and induces apoptosis, demonstrating that Stat3 signaling is essential for the survival of myeloma tumor cells. These findings provide evidence that constitutively activated Stat3 signaling contributes to the pathogenesis of multiple myeloma by preventing apoptosis.
Ribosome-inactivating proteins (RIPs) remove a specific adenine from 28S rRNA leading to inactivation of ribosomes and arrest of translation. Great interest as to a possible second physiological substrate for RIPs came from the observation that in vitro RIPs remove adenine from DNA. This paper addresses the problem of nuclear lesions induced by RIPs in human endothelial cells susceptible to the bacterial RIP Shiga toxin 1 and the plant RIP ricin. With both toxins, nuclear DNA damage as evaluated by two independent techniques (alkaline-halo assay and alkaline filter elution) appears early, concomitant with (ricin) or after (Shiga toxin 1) the inhibition of protein synthesis. At this time, the annexin V binding assay, caspase 3 activity, the formation of typical < or = 50 Kb DNA fragments, and changes in morphology associated with apoptosis were negative. Furthermore, a block of translation comparable to that induced by RIPs, but obtained with cycloheximide, did not induce nuclear damage. Such damage is consistent with the enzymatic activity (removal of adenine) of RIPs acting in vitro on RNA-free chromatin and DNA. The results unequivocally indicate that RIPs can damage nuclear DNA in whole cells by means that are not secondary to ribosome inactivation or apoptosis.
High-dose chemotherapy with hematopoietic stem-cell rescue is increasingly being used in the treatment of multiple myeloma. Bone marrow and also peripheral blood stem-cell (PBSC) grafts contain measurable quantities of plasma cells, the biological significance of which is unknown.
Patients with multiple myeloma were mobilized with chemotherapy and filgrastim. The number of CD38++/CD138+ cells/kg in the grafts for autologous transplantation was determined by flow cytometry. Patients were stratified into two groups (threshold 4.5 x 10(5) plasma cells kg(-1)) of reinfused plasma cells in the first autologous graft.
The median statistical progression-free survival was 14 months (4-34 months) in the high-contamination group (>4.5 x 10(5) plasma cells kg(-1)) compared to 26 months (4-43 months) in the low-contamination group (<4.5 x 10(5) plasma cells kg(-1), P =0.0096). Patients with 13q deletion were more frequently found to have a high contamination of the stem-cell graft with malignant plasma cells.
Patients with graft contamination of more than 4.5 x 10(5) plasma cells kg(-1) had a high risk of early disease progression after high-dose chemotherapy. In vivo tumor cell purging prior to mobilization chemotherapy might be one strategy to improve the time to progression of high-risk patients.
Recent interest in the health consequences of ricin as a weapon of terrorism has led us to investigate the effects of ricin on cells in vitro and in mice. Our previous studies showed that depurination of the 28S rRNA by ricin results in the inhibition of translation and the coordinate activation of the stress-activated protein kinases JNK and p38 MAPK. In RAW 264.7 macrophages, ricin induced the activation of ERK, JNK, and p38 MAPK, the accumulation of mRNA encoding tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, the transcription factors c-Fos, c-Jun, and EGR1, and the appearance of TNF-alpha protein in the culture medium. Using specific inhibitors of MAPKs, we demonstrated the nonredundant roles of the individual MAPKs in mediating proinflammatory gene activation in response to ricin. Similarly, the intravenous administration of ricin to mice led to the activation of ERK, JNK, and p38 MAPK in the kidneys, and increases in plasma-borne TNF-alpha, IL-1beta, and IL-6. Ricin-injected mice developed the hallmarks of hemolytic uremic syndrome, including thrombotic microangiopathy, hemolytic anemia, thrombocytopenia, and acute renal failure. Microarray analyses demonstrated a massive proinflammatory transcriptional response in the kidneys, coincidental with the symptoms of hemolytic uremic syndrome. Therapeutic management of the inflammatory response may affect the outcome of intoxication by ricin.
The mechanism of ricin-induced apoptosis in human cervical cancer cell line HeLa was studied. The present study demonstrated that ricin induces apoptosis of human cervical cancer cells (HeLa) in a time dependent manner with an IC(50) for cell viability of 1 microg/ml. Ricin treatment resulted in a time dependent increase in LDH leakage, DNA fragmentation, percent apoptotic cells, generation of reactive oxygen species and depletion of intracellular glutathione levels. DNA agarose gel electrophoresis showed typical oligonucleosomal length DNA fragmentation. Additionally, DNA diffusion assay was performed to confirm DNA damage and apoptosis. Ricin activated caspase-3 as evidenced by both proteolytic cleavage of procaspase-3 into 20 and 18 kDa subunits, and increased protease activity. Caspase activity was maximum at 4h and led to the cleavage of 116 kDa poly(ADP-ribose) polymerase (PARP), resulting in the 85 kDa cleavage product. Ricin-induced caspase-3 activation also resulted in cleavage of DNA fragmentation factor-45 (DFF45/ICAD) and DFF40 or caspase-activated DNase in HeLa cells. Activation of caspase-3, cleavage of PARP and DNA fragmentation was blocked by pre-treatment with caspase-3 specific inhibitor Ac-DEVD-CHO (100 microM) and broad-spectrum caspase inhibitor Z-VAD-FMK (40 microM). Ricin-induced DNA fragmentation was inhibited by pre-treatment with PARP inhibitors 3-aminobenzamide (100 microM) and DPQ (10 microM). Our results indicate that ricin-induced cell death was mediated by generation of reactive oxygen species and subsequent activation of caspase-3 cascade followed by down stream events leading to apoptotic mode of cell death.
The single-chain ribosome-inactivating proteins (RIPs) from plant origin, including Saporin 6 from the seeds of Saponaria officinalis, are ribotoxins known to act as N-glycosidases which depurinate the conserved alpha sarcin loop of large rRNAs. As a consequence, the eukaryotic ribosomes become inactivated, thereby arresting the protein synthesis at the elongation step. RIPs are currently under study as antiviral and antiproliferative agents. Additional in vitro activities of RIPs against either RNA or DNA have been recently described. A specific nuclease activity on plasmidic DNA was demonstrated by either purified or bacterial-recombinant molecules. We report here that human mitochondrial DNA (mtDNA) is a new specific target of Saporin 6 nuclease activity. A unique site of cleavage has been identified and mapped within the most variable part of the D-loop region of the covalently closed circular mtDNA molecule.
Caspase-2 is one of the best conserved caspases across species. This enzyme is unique among caspases in that it has features of both initiator and effector caspases. Caspase-2 appears to be necessary for the onset of apoptosis triggered by several insults, including DNA damage, administration of TNF, and different pathogens and viruses. In several experimental systems, a link has been shown between the p53 family proteins and caspase-2 activation leading to cell death. In this review, current knowledge concerning the structure of this protease and its function in cell physiology and cell death, particularly cell death triggered by DNA damage, is summarized and discussed.
Several protein toxins, such as the potent plant toxin ricin, enter mammalian cells by endocytosis and undergo retrograde transport via the Golgi complex to reach the endoplasmic reticulum (ER). In this compartment the catalytic moieties exploit the ER-associated degradation (ERAD) pathway to reach their cytosolic targets. Bacterial toxins such as cholera toxin or Pseudomonas exotoxin A carry KDEL or KDEL-like C-terminal tetrapeptides for efficient delivery to the ER. Chimeric toxins containing monomeric plant ribosome-inactivating proteins linked to various targeting moieties are highly cytotoxic, but it remains unclear how these molecules travel within the target cell to reach cytosolic ribosomes. We investigated the intracellular pathways of saporin, a monomeric plant ribosome-inactivating protein that can enter cells by receptor-mediated endocytosis. Saporin toxicity was not affected by treatment with Brefeldin A or chloroquine, indicating that this toxin follows a Golgi-independent pathway to the cytosol and does not require a low pH for membrane translocation. In intoxicated Vero or HeLa cells, ricin but not saporin could be clearly visualized in the Golgi complex using immunofluorescence. The saporin signal was not evident in the Golgi, but was found to partially overlap with that of a late endosome/lysosome marker. Consistently, the toxicities of saporin or saporin-based targeted chimeric polypeptides were not enhanced by the addition of ER retrieval sequences. Thus, the intracellular movement of saporin differs from that followed by ricin and other protein toxins that rely on Golgi-mediated retrograde transport to reach their retrotranslocation site.
The Escherichia coli verotoxin 1 (VT1) inhibits protein synthesis, cell proliferation, and damages endothelial cell in the hemolytic uremic syndrome. VT1 can specifically bind and act on endothelial cells as well as on many tumor cells because these cells express its high affinity receptor, globotriaosylceramide. This indicates that VT1 may have both antiangiogenic and antineoplastic activities. We investigated this potential of VT1 by incubating several colon cancer cell lines with VT1 for different time periods and found that HCT116 cells were especially sensitive to VT1. A combination of morphological studies, flow cytometry, DNA laddering and annexin V staining confirmed that VT1 irreversibly arrests these cells in S phase within 24 h and prolonged incubation triggers DNA fragmentation. Concomitant to the activation of the S phase checkpoint, increased levels of mRNA and proteins of growth arrest and DNA damage-inducible gene family that include GADD34, GADD45alpha, and GADD45beta was observed. Interestingly, no significant changes in expression of key cell cycle related proteins such as cdk2, cdk4, p21, p27, and p53 was found during the S phase arrest and apoptosis. We therefore suggest that GADD proteins might play an important role in VT1 induced S phase arrest and programmed cell death in HCT116 cells.
The transferrin receptor (TfR) is a cell membrane-associated glycoprotein involved in the cellular uptake of iron and in the regulation of cell growth. Iron uptake occurs via the internalization of iron-loaded transferrin (Tf) mediated by the interaction with the TfR. In addition, the TfR may also contain other growth regulatory properties in certain normal and malignant cells. The elevated levels of TfR in malignancies, its relevance in cancer, and the extracellular accessibility of this molecule make it an excellent antigen for the treatment of cancer using antibodies. The TfR can be targeted by monoclonal antibodies specific for the extracellular domain of the receptor. In this review, we summarize advancements in the basic physiology of the TfR including structure, function, and expression. We also discuss the efficacy of targeting the TfR using cytotoxic antibodies that inhibit cell growth and/or induce apoptosis in targeted malignant cells.