A novel method to investigate drug resistance in the chronic lymphocytic leukemia (CLL) microenvironment: Analysis of CLL Cellular Environment and Response (ACCER).

Microenvironments such as lymph nodes allow chronic lymphocytic leukemia (CLL) cells to survive and become drug resistant. There are limited methods to study the to study the contribution of the stromal microenvironment. We have adapted a solid tumor microenvironment cell culture system that provides elements of the CLL microenvironment called Analysis of CLL Cellular Environment and Response (ACCER). We optimized the cell number for patient's primary CLL cells and HS-5 human bone marrow stromal cell line that will give sufficient cell number and viability with the ACCER. We then determined the amount of collagen type 1 to give the best extracellular matrix to seed CLL cells to the membrane. Finally, we determined that ACCER provide CLL cell protection against cell death following treatment with fludarabine and ibrutinib compared to co-culture conditions. This describes novel microenvironment model to investigate factors that promote drug resistance in CLL.

Truncated forms of BNIP3 act as dominant negatives inhibiting hypoxia-induced cell death.

BNIP3 (Bcl-2/adenovirus E1B Nineteen Kilodalton Interacting Protein) is a pro-cell death member of the Bcl-2 family of proteins. Its expression is induced by the transcription factor Hypoxia Inducible Factor-1 (HIF-1) under conditions of low oxygen (hypoxia) and is found over expressed in hypoxic regions of many tumors. When over expressed, BNIP3 induces cell death through induction of mitochondrial dysfunction that is dependent on the presence of BNIP3's TM domain. Herein, we have determined that the SkOv3 ovarian cancer cell line expresses a truncated BNIP3 protein, which results in the elimination of the transmembrane domain. Truncation that eliminates all four domains of BNIP3 protein also inhibits hypoxia-induced cell death in SkOv3, HEK293, U251 and MCF-7 cells. Three different mutations in a BNIP3 expression vector that lead to a truncated BNIP3 protein, lacking TM domain only, or lacking CD, BH3, and TM domains resulted in inhibition of hypoxia-induced cell death when transfected into HEK293 cells. We found that truncated BNIP3 failed to associate with the mitochondria and the truncated BNIP3 lacking all four domains can bind to wild type BNIP3. Taken together, truncation of BNIP3 could be a novel mechanism for cancer cells to avoid hypoxia-induced cell death mediated by BNIP3 over expression.

Prostate Cancer Cells Are Sensitive to Lysosomotropic Agent Siramesine through Generation Reactive Oxygen Species and in Combination with Tyrosine Kinase Inhibitors.

BACKGROUND: Prostate cancer is the most common cancer affecting men often resulting in aggressive tumors with poor prognosis. Even with new treatment strategies, drug resistance often occurs in advanced prostate cancers. The use of lysosomotropic agents offers a new treatment possibility since they disrupt lysosomal membranes and can trigger a series of events leading to cell death. In addition, combining lysosomotropic agents with targeted inhibitors can induce increased cell death in different cancer types, but prostate cancer cells have not been investigated. METHODS: We treated prostate cancer cells with lysosomotropic agents and determine their cytotoxicity, lysosome membrane permeabilization (LMP), reactive oxygen species (ROS) levels, and mitochondrial dysfunction. In addition, we treated cells with lysosomotropic agent in combination with tyrosine kinase inhibitor, lapatinib, and determined cell death, and the role of ROS in this cell death. RESULTS: Herein, we found that siramesine was the most effective lysosomotropic agent at inducing LMP, increasing ROS, and inducing cell death in three different prostate cancer cell lines. Siramesine was also effective at increasing cell death in combination with the tyrosine kinase inhibitor, lapatinib. This increase in cell death was mediated by lysosome membrane permeabilization, an increased in ROS levels, loss of mitochondrial membrane potential and increase in mitochondrial ROS levels. The combination of siramesine and lapatinib induced apoptosis, cleavage of PARP and decreased expression of Bcl-2 family member Mcl-1. Furthermore, lipid peroxidation occurred with siramesine treatment alone or in combination with lapatinib. Treating cells with the lipid peroxidation inhibitor alpha-tocopherol resulted in reduced siramesine induced cell death alone or in combination with lapatinib. The combination of siramesine and lapatinib failed to increase cell death responses in normal prostate epithelial cells. CONCLUSIONS: This suggests that lysomotropic agents such as siramesine in combination with tyrosine kinase inhibitors induces cell death mediated by ROS and could be an effective treatment strategy in advanced prostate cancer.

Autophagy inhibition by TSSC4 (tumor suppressing subtransferable candidate 4) contributes to sustainable cancer cell growth.

Cancer cell growth is dependent upon the sustainability of proliferative signaling and resisting cell death. Macroautophagy/autophagy promotes cancer cell growth by providing nutrients to cells and preventing cell death. This is in contrast to autophagy promoting cell death under some conditions. The mechanism regulating autophagy-mediated cancer cell growth remains unclear. Herein, we demonstrate that TSSC4 (tumor suppressing subtransferable candidate 4) is a novel tumor suppressor that suppresses cancer cell growth and tumor growth and prevents cell death induction during excessive growth by inhibiting autophagy. The oncogenic proteins ERBB2 (erb-b2 receptor tyrosine kinase 2) and the activation EGFR mutant (EGFRvIII, epidermal growth factor receptor variant III) promote cell growth and TSSC4 expression in breast cancer and glioblastoma multiforme (GBM) cells, respectively. In EGFRvIII-expressing GBM cells, TSSC4 knockout shifted the function of autophagy from a pro-cell survival role to a pro-cell death role during prolonged cell growth. Furthermore, the interaction of TSSC4 with MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) via its conserved LC3-interacting region (LIR) contributes to its inhibition of autophagy. Finally, TSSC4 suppresses tumorsphere formation and tumor growth by inhibiting autophagy and maintaining cell survival in tumorspheres. Taken together, sustainable cancer cell growth can be achieved by autophagy inhibition via TSSC4 expression.Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; CQ: chloroquine; EGFRvIII: epidermal growth factor receptor variant III; ERBB2: erb-b2 receptor tyrosine kinase 2; GBM: glioblastoma multiforme; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule Associated protein 1 light chain 3; TSSC4: tumor suppressing subtransferable candidate 4.

Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells.

The epidermal growth factor receptor (EGFR) family member erb-b2 receptor tyrosine kinase 2 (ERBB2) is overexpressed in many types of cancers leading to (radio- and chemotherapy) treatment resistance, whereas the underlying mechanisms are still unclear. Autophagy is known to contribute to cancer treatment resistance. In this study, we demonstrate that ERBB2 increases the expression of different autophagy genes including ATG12 (autophagy-related 12) and promotes ATG12-dependent autophagy. We clarify that lapatinib, a dual inhibitor for EGFR and ERBB2, promoted autophagy in cells expressing only EGFR but inhibited autophagy in cells expressing only ERBB2. Furthermore, breast cancer database analysis of 35 genes in the canonical autophagy pathway shows that the upregulation of ATG12 and MAP1LC3B is associated with a low relapse-free survival probability of patients with ERBB2-positive breast tumors following treatments. Downregulation of ERBB2 or ATG12 increased cell death induced by chemotherapy drugs in ERBB2-positive breast cancer cells, whereas upregulation of ERBB2 or ATG12 decreased the cell death in ERBB2-negative breast cancer cells. Finally, ERBB2 antibody treatment led to reduced expression of ATG12 and autophagy inhibition increasing drug or starvation-induced cell death in ERBB2-positive breast cancer cells. Taken together, this study provides a novel approach for the treatment of ERBB2-positive breast cancer by targeting ATG12-dependent autophagy.

Misoprostol treatment prevents hypoxia-induced cardiac dysfunction through a 14-3-3 and PKA regulatory motif on Bnip3.

Systemic hypoxia is a common element in most perinatal emergencies and is a known driver of Bnip3 expression in the neonatal heart. Bnip3 plays a prominent role in the evolution of necrotic cell death, disrupting ER calcium homeostasis and initiating mitochondrial permeability transition (MPT). Emerging evidence suggests a cardioprotective role for the prostaglandin E1 analog misoprostol during periods of hypoxia, but the mechanisms for this protection are not completely understood. Using a combination of mouse and cell models, we tested if misoprostol is cardioprotective during neonatal hypoxic injury by altering Bnip3 function. Here we report that hypoxia elicits mitochondrial-fragmentation, MPT, reduced ejection fraction, and evidence of necroinflammation, which were abrogated with misoprostol treatment or Bnip3 knockout. Through molecular studies we show that misoprostol leads to PKA-dependent Bnip3 phosphorylation at threonine-181, and subsequent redistribution of Bnip3 from mitochondrial Opa1 and the ER through an interaction with 14-3-3 proteins. Taken together, our results demonstrate a role for Bnip3 phosphorylation in the regulation of cardiomyocyte contractile/metabolic dysfunction, and necroinflammation. Furthermore, we identify a potential pharmacological mechanism to prevent neonatal hypoxic injury.

Antihistamines are synergistic with Bruton's tyrosine kinase inhibiter ibrutinib mediated by lysosome disruption in chronic lymphocytic leukemia (CLL) cells.

Lysosomes in chronic lymphocytic leukemia (CLL) cells have previously been identified as a promising target for therapeutic intervention in combination with targeted therapies. Recent studies have shown that antihistamines can induce lysosomal membrane permeabilization (LMP) in a variety of cell lines. Furthermore, our previous data indicates that lysosomotropic agents can cause synergistic cell death in vitro when combined with some tyrosine kinase inhibitors (TKI). In the current study, we have shown that three over-the-counter antihistamines, clemastine, desloratadine, and loratadine, preferentially induce cell death via LMP in CLL cells, as compared to normal lymphocytes. We treated primary CLL cells with antihistamines and found clemastine was the most effective at inducing LMP and cell death. More importantly, the antihistamines induced synergistic cytotoxicity when combined with the tyrosine kinase inhibitor, ibrutinib, but not with chemotherapy. Moreover, the synergy between clemastine and ibrutinib was associated with the induction of reactive oxygen species (ROS), loss of mitochondrial membrane potential and decreased Mcl-1 expression leading to apoptosis. This study proposes a potential novel treatment strategy for CLL, repurposing FDA-approved allergy medications in combination with the targeted therapy ibrutinib to enhance drug efficacy.

The BH3 only Bcl-2 family member BNIP3 regulates cellular proliferation.

The BH3-only family member BNIP3 has been described as either promoting cell survival or cell death. This depends upon the level of BNIP3 expression and its cellular localization. Increased BNIP3 expression under hypoxia contributes to cell death through increased mitochondrial dysfunction. Furthermore, mice lacking BNIP3 show inhibition of ischemic cardiomyocyte apoptosis. In contrast, nuclear localization of BNIP3 contributes to blockage of apoptosis in glioma cells through repression of pro-apoptotic genes. We have discovered that mouse embryonic fibroblasts (MEFs) lacking BNIP3 expression show increased proliferation and cell number compared to wild-type cells. Furthermore, the cells lacking BNIP3 showed increased MAPK activation. Increased proliferation was not due to decreased cell death as oxidative stress induced cell death in BNIP3 null MEFs. In addition, we isolated astrocytes from wild-type or embryonic mice lacking expression of BNIP3. There was increased density and cell number in the astrocytes lacking BNIP3 expression. To confirm these results in human cells, we inducibly expressed BNIP3 in human embryonic kidney (HEK293) cells and found that induced BNIP3 reduced cell proliferation and failed to change background cell death levels. Transient over-expression of BNIP3 in the nucleus of HEK293 cells also reduced DNA synthesis. Finally, to determine whether this increased proliferation occurs in mice lacking BNIP3, we isolated brains from wild-type mice or those lacking BNIP3 expression. The mice lacking BNIP3 had increased cellularity in the brain of embryonic and adult mice. Taken together, our study describes a new function for BNIP3 in the regulation of cellular proliferation.

Surviving cell death through epidermal growth factor (EGF) signal transduction pathways: implications for cancer therapy.

There is a balance between cell death and survival in living organisms. The ability of cells to sense their environment and decide to survive or die is dependent largely upon growth factors. Epidermal growth factor (EGF) is a key growth factor regulating cell survival. Through its binding to cell surface receptors, EGF activates an extensive network of signal transduction pathways that include activation of the PI3K/AKT, RAS/ERK and JAK/STAT pathways. These pathways predominantly lead to activation or inhibition of transcription factors that regulate expression of both pro- and anti-apoptotic proteins effectively blocking the apoptotic pathway. In cancer, EGF signaling pathways are often dysfunctional and targeted therapies that block EGF signaling have been successful in treating cancers. In this review, we will discuss the EGF survival signaling network, how it cross-talks with the apoptotic signaling pathways and the therapeutic drugs targeting the EGF survival pathway used to treat cancers.

Herceptin sensitizes ErbB2-overexpressing cells to apoptosis by reducing antiapoptotic Mcl-1 expression.

PURPOSE: Monoclonal antibodies, such as herceptin and trastuzumab, against the epidermal growth factor receptor ErbB2 (also known as HER2/neu) are an effective therapy for breast cancer patients with overexpression of ErbB2. Herceptin, in combination with standard chemotherapy, such as taxol or etoposide, gives a synergistically apoptotic response in breast tumors. EXPERIMENTAL DESIGN: The mechanism underlying this synergy between chemotherapy and herceptin treatment is not well understood. Herein, we have determined that addition of herceptin, sensitized breast cancer cell lines MDA-MB-231 and MCF-7 to etoposide- or taxol-induced apoptosis. RESULTS: This treatment resulted in reduced expression of ErbB2 and the antiapoptotic Bcl-2 family member Mcl-1 in MDA-MB-231 cells. Using antisense oligonucleotides against Mcl-1, MDA-MB-231 cells were rendered sensitive to etoposide-induced apoptosis similar to herceptin, but combined treatment of antisense against Mcl-1 and herceptin failed to give a significant increase in apoptosis. In 29 human breast tumors immunostained for ErbB2 and Mcl-1, we found that when ErbB2 was overexpressed, there was a corresponding increase in Mcl-1 expression. DISCUSSION: Using murine fibroblasts that express human ErbB2, but no other ErbB family member (NE2), these cells showed resistance to both taxol- and etoposide-induced apoptosis compared with parental cells. In addition, NE2 cells preferentially express the antiapoptotic Bcl-2 family member Mcl-1 compared with parental cells, and treatment with herceptin reduces Mcl-1 expression. Taken together, these results suggest that herceptin sensitizes ErbB2-overexpressing cells to apoptosis by reducing antiapoptotic Mcl-1 protein levels.

EGFR Family Members' Regulation of Autophagy Is at a Crossroads of Cell Survival and Death in Cancer.

The epidermal growth factor receptor (EGFR) signaling pathways are altered in many cancers contributing to increased cell survival. These alterations are caused mainly through increased expression or mutation of EGFR family members EGFR, ErbB2, ErbB3, and ErbB4. These receptors have been successfully targeted for cancer therapy. Specifically, a monoclonal antibody against ErbB2, trastuzumab, and a tyrosine kinase inhibitor against EGFR, gefitinib, have improved the survival of breast and lung cancer patients. Unfortunately, cancer patients frequently become resistant to these inhibitors. This has led to investigating how EGFR can contribute to cell survival and how cancer cells can overcome inhibition of its signaling. Indeed, it is coming into focus that EGFR signaling goes beyond a single signal triggering cell proliferation and survival and is a sensor that regulates the cell's response to microenvironmental stresses such as hypoxia. It acts as a switch that modulates the ability of cancer cells to survive. Autophagy is a process of self-digestion that is inhibited by EGFR allowing cancer cells to survive under stresses that would normally cause death and become resistant to chemotherapy. Inhibiting EGFR signaling allows autophagy to contribute to cell death. This gives new opportunities to develop novel therapeutic strategies to treat cancers that rely on EGFR signaling networks and autophagy. In this review, we summarize the current understanding of EGFR family member regulation of autophagy in cancer cells and how new therapeutic strategies could be developed to overcome drug resistance.

Lysosomes as Oxidative Targets for Cancer Therapy.

Lysosomes are membrane-bound vesicles that contain hydrolases for the degradation and recycling of essential nutrients to maintain homeostasis within cells. Cancer cells have increased lysosomal function to proliferate, metabolize, and adapt to stressful environments. This has made cancer cells susceptible to lysosomal membrane permeabilization (LMP). There are many factors that mediate LMP such as Bcl-2 family member, p53; sphingosine; and oxidative stress which are often altered in cancer. Upon lysosomal disruption, reactive oxygen species (ROS) levels increase leading to lipid peroxidation, mitochondrial dysfunction, autophagy, and reactive iron. Cathepsins are also released causing degradation of macromolecules and cellular structures. This ultimately kills the cancer cell through different types of cell death (apoptosis, autosis, or ferroptosis). In this review, we will explore the contributions lysosomes play in inducing cell death, how this is regulated by ROS in cancer, and how lysosomotropic agents might be utilized to treat cancers.

Ferroptosis and autophagy induced cell death occur independently after siramesine and lapatinib treatment in breast cancer cells.

Ferroptosis is a cell death pathway characterized by iron-dependent accumulation of reactive oxygen species (ROS) within the cell. The combination of siramesine, a lysosome disruptor, and lapatinib, a dual tyrosine kinase inhibitor, has been shown to synergistically induce cell death in breast cancer cells mediated by ferroptosis. These treatments also induce autophagy but its role in this synergistic cell death is unclear. In this study, we determined that siramesine and lapatinib initially induced ferroptosis but changes to an autophagy induced cell death after 24 hours. Furthermore, we found that intracellular iron level increased in a time dependent manner following treatment accompanied by an increase in ROS. Using the iron chelator deferoxamine (DFO) or the ROS scavenger alpha-tocopherol decreased both autophagy flux and cell death. We further discovered that decreased expression of the iron storage protein, ferritin was partially dependent upon autophagy degradation. In contrast, the expression of transferrin, which is responsible for the transport of iron into cells, is increased following treatment with lapatinib alone or in combination with siramesine. This indicates that ferroptosis and autophagy induced cell death occur independently but both are mediated by iron dependent ROS generation in breast cancer cells.

Epidermal growth factor protects epithelial-derived cells from tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by inhibiting cytochrome c release.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in combination with chemotherapeutic drugs induces a synergistic apoptotic response in cancer cells. TRAIL death receptors have also been implicated in chemotherapeutic drug-induced apoptosis. This has lead to TRAIL being proposed as a potential cancer treatment. In nude mice injected with human tumors, TRAIL reduces the size of these tumors without toxic side effects. We demonstrate that the epidermal growth factor (EGF) stimulation of human embryonic kidney cells HEK 293 and breast cancer cell line MDA MB 231 effectively protects these cells from TRAIL-induced apoptosis in a dose-dependent manner. This stimulation blocks apoptosis by inhibiting TRAIL-mediated cytochrome c release from the mitochondria and caspase 3-like activation. EGF survival response involves the activation of AKT. Expression of activated AKT was sufficient to block TRAIL-induced apoptosis, and kinase-inactive AKT expression blocked EGF-protective response. In contrast, inhibition of EGF stimulation of extracellular-regulated kinase (ERK) activity did not affect EGF protection. These findings indicate that EGF receptor activation provides a survival response against TRAIL-induced apoptosis by inhibiting mitochondrial cytochrome c release that is mediated by AKT activation in epithelial-derived cells.

Tyrosine kinase receptor EGFR regulates the switch in cancer cells between cell survival and cell death induced by autophagy in hypoxia.

Autophagy is an intracellular lysosomal degradation pathway where its primary function is to allow cells to survive under stressful conditions. Autophagy is, however, a double-edge sword that can either promote cell survival or cell death. In cancer, hypoxic regions contribute to poor prognosis due to the ability of cancer cells to adapt to hypoxia in part through autophagy. In contrast, autophagy could contribute to hypoxia induced cell death in cancer cells. In this study, we showed that autophagy increased during hypoxia. At 4 h of hypoxia, autophagy promoted cell survival whereas, after 48 h of hypoxia, autophagy increased cell death. Furthermore, we found that the tyrosine phosphorylation of EGFR (epidermal growth factor receptor) decreased after 16 h in hypoxia. Furthermore, EGFR binding to BECN1 in hypoxia was significantly higher at 4 h compared to 72 h. Knocking down or inhibiting EGFR resulted in an increase in autophagy contributing to increased cell death under hypoxia. In contrast, when EGFR was reactivated by the addition of EGF, the level of autophagy was reduced which led to decreased cell death. Hypoxia led to autophagic degradation of the lipid raft protein CAV1 (caveolin 1) that is known to bind and activate EGFR in a ligand-independent manner during hypoxia. By knocking down CAV1, the amount of EGFR phosphorylation was decreased in hypoxia and amount of autophagy and cell death increased. This indicates that the activation of EGFR plays a critical role in the switch between cell survival and cell death induced by autophagy in hypoxia.

Bcl-2 family member Mcl-1 expression is reduced under hypoxia by the E3 ligase FBW7 contributing to BNIP3 induced cell death in glioma cells.

Mcl-1 is an anti-apoptotic Bcl-2 family member that is often over-expressed in the malignant brain tumor glioblastoma (GBM). It has been previously shown that epidermal growth factor receptors up-regulate Mcl-1 contributing to a cell survival response. Hypoxia is a poor prognostic marker in glioblastoma despite the fact that hypoxic regions have areas of necrosis. Hypoxic regions of GBM also highly express the pro-cell death Bcl-2 family member BNIP3, yet when BNIP3 is overexpressed in glioma cells, it induces cell death. The reasons for this discrepancy are unclear. Herein we have found that Mcl-1 expression is reduced under hypoxia due to degradation by the E3 ligase FBW7 leading to increased hypoxia induced cell death. This cell death is reduced by EGFR activation leading to increased Mcl-1 expression under hypoxia. Conversely, BNIP3 is over-expressed in hypoxia at times when Mcl-1 expression is decreased. Knocking down BNIP3 expression reduces hypoxia cell death and Mcl-1 expression effectively blocks BNIP3 induced cell death. Of significance, BNIP3 and Mcl-1 are co-localized under hypoxia in glioma cells. These results suggest that Mcl-1 can block the ability of BNIP3 to induce cell death under hypoxia in GBM tumors.

Cyclin D expression in chronic lymphocytic leukemia.

Although the majority of circulating leukemic cells in chronic lymphocytic leukemia (CLL) are in G0/early G1, recent studies have shown that these cells have undergone multiple cell divisions. In this study, we have determined whether there are abnormalities in cell cycle control in CLL by examining the three cyclin D isoforms in 43 patients and correlating the findings with clinical features. Cyclin D mRNA was measured by a sensitive RNase protection assay and the order of expression in CLL cells was D3 > D2 > D1. The mean cyclin D1 and D3 mRNA levels were 4 to 6-fold higher in CLL cells than in normal peripheral blood B cells. In contrast, the levels of cyclin D2 mRNA were similar in CLL and normal B cells. Expression of the cyclin D isoforms was two- to four-fold greater in normal T cells than B cells, and the order of expression for both cell types was D2 > D3 > D1. The relative overexpressions of cyclins D1 and D3 in CLL were unrelated to gene amplification, as assessed by Southern blotting, but structural changes in the genes were seen in four patients. Both cyclin D1 and D3 mRNA levels correlated positively with lymphocyte doubling time (LDT) and inversely with Rai stage and duration of disease. In addition, a significant correlation was observed between cyclin D mRNA levels and survival, with patients having high levels of cyclin D1, and to a lesser extent cyclin D3, mRNA having the best survival. Thus, cyclin D1 and D3 are relatively overexpressed in CLL cells and patients with higher levels have low stage disease, long LDT and prolonged survival. Further studies should evaluate the predictive value of cyclin D measurements in comparison to other prognostic markers in CLL.

Estrogen regulation of anti-apoptotic Bcl-2 family member Mcl-1 expression in breast cancer cells.

Estrogen is implicated as an important factor in stimulating breast cancer cell proliferation, and presence of estrogen receptor (ER) is an indication of a good prognosis in breast cancer patients. Mcl-1 is an anti-apoptotic Bcl-2 family member that is often over expressed in breast tumors, correlating with poor survival. In breast cancer, it was been previously shown that epidermal growth factor receptors up-regulate Mcl-1 but the role of estrogen in increasing Mcl-1 expression was unknown. In ERα positive cell lines MCF-7 and ZR-75, estrogen treatment increased Mcl-1 expression at both the protein and mRNA level. In two ERα negative cell lines, SK-BR-3 and MDA-MB-231, estrogen failed to increase in Mcl-1 protein expression. We found that ERα antagonists decreased estrogen mediated Mcl-1 expression at both the protein and mRNA level. Upon knockdown of ERα, Mcl-1 mRNA expression after estrogen treatment was also decreased. We also found that ERα binds to the Mcl-1 promoter at a region upstream of the translation start site containing a half ERE site. Streptavidin-pull down assay showed that both ERα and transcription factor Sp1 bind to this region. These results suggest that estrogen is involved in regulating Mcl-1 expression specifically through a mechanism involving ERα.

Altered immune responses in rhesus macaques co-infected with SIV and Plasmodium cynomolgi: an animal model for coincident AIDS and relapsing malaria.

BACKGROUND: Dual epidemics of the malaria parasite Plasmodium and HIV-1 in sub-Saharan Africa and Asia present a significant risk for co-infection in these overlapping endemic regions. Recent studies of HIV/Plasmodium falciparum co-infection have reported significant interactions of these pathogens, including more rapid CD4+ T cell loss, increased viral load, increased immunosuppression, and increased episodes of clinical malaria. Here, we describe a novel rhesus macaque model for co-infection that supports and expands upon findings in human co-infection studies and can be used to identify interactions between these two pathogens. METHODOLOGY/PRINCIPAL FINDINGS: Five rhesus macaques were infected with P. cynomolgi and, following three parasite relapses, with SIV. Compared to macaques infected with SIV alone, co-infected animals had, as a group, decreased survival time and more rapid declines in markers for SIV progression, including peripheral CD4+ T cells and CD4+/CD8+ T cell ratios. The naïve CD4+ T cell pool of the co-infected animals was depleted more rapidly than animals infected with SIV alone. The co-infected animals also failed to generate proliferative responses to parasitemia by CD4+ and CD8+ T cells as well as B cells while also having a less robust anti-parasite and altered anti-SIV antibody response. CONCLUSIONS/SIGNIFICANCE: These data suggest that infection with both SIV and Plasmodium enhances SIV-induced disease progression and impairs the anti-Plasmodium immune response. These data support findings in HIV/Plasmodium co-infection studies. This animal model can be used to further define impacts of lentivirus and Plasmodium co-infection and guide public health and therapeutic interventions.

The role of TRAIL death receptors in the treatment of hematological malignancies.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising new treatment for the hematological malignancies. TRAIL induces apoptosis by binding to its two death receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2). The extent of apoptosis by TRAIL is tightly regulated by the expression of these receptors and by downstream signaling. Chemotherapeutic agents increase the expressions of DR4 and DR5 on tumor cells through the activation of various transcription factors and there is enhanced killing on combining these agents with TRAIL. In this review, we will discuss the mechanism of TRAIL death receptor-induced apoptosis and the regulation of DR4 and DR5 expression. In particular, we will focus on the regulation of TRAIL death receptor signaling in hematological malignancies and the mechanisms responsible for the sensitization of leukemia and lymphoma cells to TRAIL-induced apoptosis by chemotherapy. Finally, we shall review the clinical data regarding the use of recombinant TRAIL and activating monoclonal antibodies against the TRAIL death receptors in the hematological malignancies.