Differential Gene Expression following DHX36/G4R1 Knockout Is Associated with G-Quadruplex Content and Cancer.

G-quadruplexes (G4s) are secondary DNA and RNA structures stabilized by positive cations in a central channel formed by stacked tetrads of Hoogsteen base-paired guanines. G4s form from G-rich sequences across the genome, whose biased distribution in regulatory regions points towards a gene-regulatory role. G4s can themselves be regulated by helicases, such as DHX36 (aliases: G4R1 and RHAU), which possess the necessary activity to resolve these stable structures. G4s have been shown to both positively and negatively regulate gene expression when stabilized by ligands, or through the loss of helicase activity. Using DHX36 knockout Jurkat cell lines, we identified widespread, although often subtle, effects on gene expression that are associated with the presence or number of observed G-quadruplexes in promoters or gene regions. Genes that significantly change their expression, particularly those that show a significant increase in RNA abundance under DHX36 knockout, are associated with a range of cellular functions and processes, including numerous transcription factors and oncogenes, and are linked to several cancers. Our work highlights the direct and indirect role of DHX36 in the transcriptome of T-lymphocyte leukemia cells and the potential for DHX36 dysregulation in cancer.

The RNA helicase DHX36-G4R1 modulates C9orf72 GGGGCC hexanucleotide repeat-associated translation.

GGGGCC (G4C2) hexanucleotide repeat expansions in the endosomal trafficking gene C9orf72 are the most common genetic cause of ALS and frontotemporal dementia. Repeat-associated non-AUG (RAN) translation of this expansion through near-cognate initiation codon usage and internal ribosomal entry generates toxic proteins that accumulate in patients' brains and contribute to disease pathogenesis. The helicase protein DEAH-box helicase 36 (DHX36-G4R1) plays active roles in RNA and DNA G-quadruplex (G4) resolution in cells. As G4C2 repeats are known to form G4 structures in vitro, we sought to determine the impact of manipulating DHX36 expression on repeat transcription and RAN translation. Using a series of luciferase reporter assays both in cells and in vitro, we found that DHX36 depletion suppresses RAN translation in a repeat length-dependent manner, whereas overexpression of DHX36 enhances RAN translation from G4C2 reporter RNAs. Moreover, upregulation of RAN translation that is typically triggered by integrated stress response activation is prevented by loss of DHX36. These results suggest that DHX36 is active in regulating G4C2 repeat translation, providing potential implications for therapeutic development in nucleotide repeat expansion disorders.

Yin Yang 1 contains G-quadruplex structures in its promoter and 5'-UTR and its expression is modulated by G4 resolvase 1.

Yin Yang 1 (YY1) is a multifunctional protein with regulatory potential in tumorigenesis. Ample studies demonstrated the activities of YY1 in regulating gene expression and mediating differential protein modifications. However, the mechanisms underlying YY1 gene expression are relatively understudied. G-quadruplexes (G4s) are four-stranded structures or motifs formed by guanine-rich DNA or RNA domains. The presence of G4 structures in a gene promoter or the 5'-UTR of its mRNA can markedly affect its expression. In this report, we provide strong evidence showing the presence of G4 structures in the promoter and the 5'-UTR of YY1. In reporter assays, mutations in these G4 structure forming sequences increased the expression of Gaussia luciferase (Gluc) downstream of either YY1 promoter or 5'-UTR. We also discovered that G4 Resolvase 1 (G4R1) enhanced the Gluc expression mediated by the YY1 promoter, but not the YY1 5'-UTR. Consistently, G4R1 binds the G4 motif of the YY1 promoter in vitro and ectopically expressed G4R1 increased endogenous YY1 levels. In addition, the analysis of a gene array data consisting of the breast cancer samples of 258 patients also indicates a significant, positive correlation between G4R1 and YY1 expression.

The DEAH-box RNA helicase RHAU binds an intramolecular RNA G-quadruplex in TERC and associates with telomerase holoenzyme.

Guanine-quadruplexes (G4) consist of non-canonical four-stranded helical arrangements of guanine-rich nucleic acid sequences. The bulky and thermodynamically stable features of G4 structures have been shown in many respects to affect normal nucleic acid metabolism. In vivo conversion of G4 structures to single-stranded nucleic acid requires specialized proteins with G4 destabilizing/unwinding activity. RHAU is a human DEAH-box RNA helicase that exhibits G4-RNA binding and resolving activity. In this study, we employed RIP-chip analysis to identify en masse RNAs associated with RHAU in vivo. Approximately 100 RNAs were found to be associated with RHAU and bioinformatics analysis revealed that the majority contained potential G4-forming sequences. Among the most abundant RNAs selectively enriched with RHAU, we identified the human telomerase RNA template TERC as a true target of RHAU. Remarkably, binding of RHAU to TERC depended on the presence of a stable G4 structure in the 5'-region of TERC, both in vivo and in vitro. RHAU was further found to associate with the telomerase holoenzyme via the 5'-region of TERC. Collectively, these results provide the first evidence that intramolecular G4-RNAs serve as physiologically relevant targets for RHAU. Furthermore, our results suggest the existence of alternatively folded forms of TERC in the fully assembled telomerase holoenyzme.

G4 resolvase 1 tightly binds and unwinds unimolecular G4-DNA.

It has been previously shown that the DHX36 gene product, G4R1/RHAU, tightly binds tetramolecular G4-DNA with high affinity and resolves these structures into single strands. Here, we test the ability of G4R1/RHAU to bind and unwind unimolecular G4-DNA. Gel mobility shift assays were used to measure the binding affinity of G4R1/RHAU for unimolecular G4-DNA-formed sequences from the Zic1 gene and the c-Myc promoter. Extremely tight binding produced apparent K(d)'s of 6, 3 and 4 pM for two Zic1 G4-DNAs and a c-Myc G4-DNA, respectively. The low enzyme concentrations required for measuring these K(d)'s limit the precision of their determination to upper boundary estimates. Similar tight binding was not observed in control non-G4 forming DNA sequences or in single-stranded DNA having guanine-rich runs capable of forming tetramolecular G4-DNA. Using a peptide nucleic acid (PNA) trap assay, we show that G4R1/RHAU catalyzes unwinding of unimolecular Zic1 G4-DNA into an unstructured state capable of hybridizing to a complementary PNA. Binding was independent of adenosine triphosphate (ATP), but the PNA trap assay showed that unwinding of G4-DNA was ATP dependent. Competition studies indicated that unimolecular Zic1 and c-Myc G4-DNA structures inhibit G4R1/RHAU-catalyzed resolution of tetramolecular G4-DNA. This report provides evidence that G4R1/RHAU tightly binds and unwinds unimolecular G4-DNA structures.

Role of the amino terminal RHAU-specific motif in the recognition and resolution of guanine quadruplex-RNA by the DEAH-box RNA helicase RHAU.

Under physiological conditions, guanine-rich sequences of DNA and RNA can adopt stable and atypical four-stranded helical structures called G-quadruplexes (G4). Such G4 structures have been shown to occur in vivo and to play a role in various processes such as transcription, translation and telomere maintenance. Owing to their high-thermodynamic stability, resolution of G4 structures in vivo requires specialized enzymes. RHAU is a human RNA helicase of the DEAH-box family that exhibits a unique ATP-dependent G4-resolvase activity with a high affinity and specificity for its substrate in vitro. How RHAU recognizes G4-RNAs has not yet been established. Here, we show that the amino-terminal region of RHAU is essential for RHAU to bind G4 structures and further identify within this region the evolutionary conserved RSM (RHAU-specific motif) domain as a major affinity and specificity determinant. G4-resolvase activity and strict RSM dependency are also observed with CG9323, the Drosophila orthologue of RHAU, in the amino terminal region of which the RSM is the only conserved motif. Thus, these results reveal a novel motif in RHAU protein that plays an important role in recognizing and resolving G4-RNA structures, properties unique to RHAU among many known RNA helicases.

Prevalence of methicillin-resistant Staphylococcus aureus in ambulances in southern Maine.

OBJECTIVE: To determine whether methicillin-resistant Staphylococcus aureus (MRSA) could be found in ambulances in a predominantly rural state. METHODS: Samples were obtained from specified areas in 51 ambulances in southern Maine. These samples were tested on mannitol salt agar containing 4 microg/mL oxacillin. Resulting colonies were gram-stained and tested for the presence of catalase and coagulase. RESULTS: Of the 51 ambulances tested, 25 (49%) had at least one area positive for MRSA contamination. CONCLUSIONS: A significant number of ambulances operating in southern Maine have MRSA contamination, and ambulances may represent an important reservoir for the transmission of potentially serious infections to patients and EMS personnel. There was no statistical difference between the service types (fire-based vs. non-fire-based) or annual call volume. There was, however, a statistically significant lower rate of contamination in services that provided paid, 24-hour coverage versus those that did not.

G-Quadruplex Helicase DHX36/G4R1 Engages Nuclear Lamina Proteins in Quiescent Breast Cancer Cells.

G-quadruplexes (G4s) are nucleic acid structures found enriched within gene regulatory sequences. G4s control fundamental cellular processes, including replication, transcription, and translation. Proto-oncogenes are enriched with G4 sequences, while tumor-suppressor genes are depleted, suggesting roles for G4s in cell survival and proliferation. Specialized helicases participate in G4-mediated gene regulation via enzymatic unwinding activity. One such enzyme, DHX36/G4R1, is the major G4-helicase and is a master regulator of G4-DNAs and mRNAs. G4-resolution promotes the expression of proproliferative genes; as such, DHX36/G4R1 promotes cell proliferation. Little is known about how DHX36/G4R1 itself is regulated in nondividing cells. We hypothesized that DHX36/G4R1 protein binding partners are altered when a cell transitions from a dividing to a quiescent state. We found that DHX36/G4R1 co-purifies with a distinct set of proteins under quiescent conditions, which may represent a novel complex that regulates DHX36/G4R1 during cell cycle transitions and have implications for development and cancer.

Breast tumor cells isolated from in vitro resistance to trastuzumab remain sensitive to trastuzumab anti-tumor effects in vivo and to ADCC killing.

An understanding of model systems of trastuzumab (Herceptin) resistance is of great importance since the humanized monoclonal antibody is now used as first line therapy with paclitaxel in patients with metastatic Her2 overexpressing breast cancer, and the majority of their tumors has innate resistance or develops acquired resistance to the treatment. Previously, we selected trastuzumab-resistant clonal cell lines in vitro from trastuzumab-sensitive parental BT-474 cells and showed that cloned trastuzumab-resistant cell lines maintain similar levels of the extracellular Her2 receptor, bind trastuzumab as efficiently as the parental cells, but continue to grow in the presence of trastuzumab and display cell cycle profiles and growth rates comparable to parental cells grown in the absence of trastuzumab (Kute et al. in Cytometry A 57:86-93, 2004). We now show that trastuzumab-resistant and trastuzumab-sensitive cells both surprisingly display trastuzumab-mediated growth inhibition in athymic nude mice. This demonstrates that resistance developed in vitro is not predictive of resistance in vivo. The observation that in vitro resistant cells are sensitive to trastuzumab in vivo could be explained by antibody dependent cellular cytotoxicity (ADCC). Therefore, both parental and trastuzumab-resistant cells were assayed for ADCC in real time on electroplates with and without trastuzumab in the presence of a natural killer cell line (NK-92), and granulocyte or mononuclear cellular fractions isolated from human peripheral blood. Mononuclear cells and NK-92 cells were more effective in killing both parental and trastuzumab-resistant cells in the presence of trastuzumab. Both trastuzumab-resistant cells and trastuzumab-sensitive cells showed similar susceptibility to ADCC despite displaying divergent growth responses to trastuzumab. The granulocyte fraction was able to kill these cells with equal efficacy in the presence or absence of trastuzumab. These results support a model of trastuzumab tumor cell killing in vivo mediated primarily by ADCC from the mononuclear fraction of innate immune cells and suggest that in the clinical setting not only should changes in signaling transduction pathways be studied in acquired tumor resistance to trastuzumab, but also mechanisms by which tumors impede immune function should be evaluated.

A Novel Mechanism for Zika Virus Host-Cell Binding.

Zika virus (ZIKV) recently emerged in the Western Hemisphere with previously unrecognized or unreported clinical presentations. Here, we identify two putative binding mechanisms of ancestral and emergent ZIKV strains featuring the envelope (E) protein residue asparagine 154 (ASN154) and viral phosphatidylserine (PS). Synthetic peptides representing the region containing ASN154 from strains PRVABC59 (Puerto Rico 2015) and MR_766 (Uganda 1947) were exposed to neuronal cells and fibroblasts to model ZIKV E protein/cell interactions and bound MDCK or Vero cells and primary neurons significantly. Peptides significantly inhibited Vero cell infectivity by ZIKV strains MR_766 and PRVABC59, indicating that this region represents a putative binding mechanism of ancestral African ZIKV strains and emergent Western Hemisphere strains. Pretreatment of ZIKV strains MR_766 and PRVABC59 with the PS-binding protein annexin V significantly inhibited replication of PRVABC59 but not MR_766, suggesting that Western hemisphere strains may additionally be capable of utilizing PS-mediated entry to infect host cells. These data indicate that the region surrounding E protein ASN154 is capable of binding fibroblasts and primary neuronal cells and that PS-mediated entry may be a secondary mechanism for infectivity utilized by Western Hemisphere strains.

Risk perception and water purification practices for water-borne parasitic infections in remote Nepal.

This study assesses water-borne infection risk perception and water boiling habits in a remote Sankhuwasava region of Nepal using a brief interview-style questionnaire. All subjects were aware of the risks associated with drinking unpurified water, but a majority (65%) reported they did not boil water regularly, and almost 60% of villagers interviewed had history of infection despite their boiling practices. In contrast to reports from other communities in Nepal, risk awareness was sufficient in this region. Water boiling alone did not confer protection. Future efforts should target sanitation, screening, and other sources of contamination.

Adenosine kinase inhibition promotes survival of fetal adenosine deaminase-deficient thymocytes by blocking dATP accumulation.

Thymocyte development past the CD4(-)CD8(-) stage is markedly inhibited in adenosine deaminase-deficient (ADA-deficient) murine fetal thymic organ cultures (FTOCs) due to the accumulation of ADA substrates derived from thymocytes failing developmental checkpoints. Such cultures can be rescued by overexpression of Bcl-2, suggesting that apoptosis is an important component of the mechanism by which ADA deficiency impairs thymocyte development. Consistent with this conclusion, ADA-deficient FTOCs were partially rescued by a rearranged T cell receptor beta transgene that permits virtually all thymocytes to pass the beta-selection checkpoint. ADA-deficient cultures were also rescued by the adenosine kinase inhibitor 5'-amino-5'-deoxyadenosine (5'A5'dAdo), indicating that the metabolite responsible for the inhibition of thymocyte development is not adenosine or deoxyadenosine, but a phosphorylated derivative of an ADA substrate. Correction of ADA-deficient FTOCs by 5'A5'dAdo correlated with reduced accumulation of dATP, implicating this compound as the toxic metabolite. In ADA-inhibited FTOCs rescued with a Bcl-2 transgene, however, dATP levels were superelevated, suggesting that cells failing positive and negative selection continued to contribute to the accumulation of ADA substrates. Our data are consistent with dATP-induced mitochondrial cytochrome c release followed by apoptosis as the mechanism by which ADA deficiency leads to reduced thymic T cell production.

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1.

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are >375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.

Microglial content-dependent inhibitory effects of calcitonin gene-related peptide (CGRP) on murine retroviral infection of glial cells.

C57BL/6 (B6) mice develop peripheral neuropathy post-LP-BM5 infection, a murine model of HIV-1 infection, along with the up-regulation of select spinal cord cytokines. We investigated if calcitonin gene-related peptide (CGRP) contributed to the development of peripheral neuropathy by stimulating glial responses. An increased expression of lumbar spinal cord CGRP was observed in vivo, post-LP-BM5 infection. Consequently, in vitro CGRP co-treatments led to a microglial content-dependent attenuation of viral loads in spinal cord mixed glia infected with selected doses of LP-BM5. This inhibition was neither caused by the loss of glia nor induced via the direct inhibition of LP-BM5 by CGRP.

Mutational Dissection of Telomeric DNA Binding Requirements of G4 Resolvase 1 Shows that G4-Structure and Certain 3'-Tail Sequences Are Sufficient for Tight and Complete Binding.

Ends of human chromosomes consist of the six nucleotide repeat d[pTTAGGG]n known as telomeric DNA, which protects chromosomes. We have previously shown that the DHX36 gene product, G4 Resolvase 1 (G4R1), binds parallel G-quadruplex (G4) DNA with an unusually tight apparent Kd. Recent work associates G4R1 with the telomerase holoenzyme, which may allow it to access telomeric G4-DNA. Here we show that G4R1 can tightly bind telomeric G4-DNA, and in the context of the telomeric sequence, we determine length, sequence, and structural requirements sufficient for tight G4R1 telomeric binding. Specifically, G4R1 binds telomeric DNA in the K+-induced "3+1" G4-topology with an apparent Kd = 10 ± 1.9 pM, a value similar as previously found for binding to unimolecular parallel G4-DNA. G4R1 binds to the Na+-induced "2+2" basket G4-structure formed by the same DNA sequence with an apparent Kd = 71 ± 2.2 pM. While the minimal G4-structure is not sufficient for G4R1 binding, a 5' G4-structure with a 3' unstructured tail containing a guanine flanked by adenine(s) is sufficient for maximal binding. Mutations directed to disrupt G4-structure similarly disrupt G4R1 binding; secondary mutations that restore G4-structure also restore G4R1 binding. We present a model showing that a replication fork disrupting a T-loop could create a 5' quadruplex with an opened 3'tail structure that is recognized by G4R1.

Mechanisms of apoptosis in developing thymocytes as revealed by adenosine deaminase-deficient fetal thymic organ cultures.

Adenosine deaminase (ADA) catalyzes the conversion of adenosine and deoxyadenosine to inosine and deoxyinosine, respectively. ADA-deficient individuals suffer from severe combined immunodeficiency and are unable to produce significant numbers of mature T or B lymphocytes. This occurs as a consequence of the accumulation of ADA substrates or their metabolites. dATP is a candidate toxic metabolite because its concentration in RBCs of ADA-deficient patients correlates with the severity of disease. Murine fetal thymic organ culture (FTOC) under ADA-deficient conditions can be used as a model system to investigate the biochemical mechanism responsible for the inhibition of thymopoiesis. In ADA-deficient FTOCs initiated at day 15 of gestation, thymocyte development was arrested at the CD4(-)CD8(-)CD44(lo)CD25(+) to CD4(-)CD8(-)CD44(lo)CD25(-) transition. Apoptosis appeared to be involved because the cultures could be rescued by the pan-caspase inhibitor zVADfmk, a Bcl-2 transgene, or deletion of apoptotic protease activating factor-1. As in ADA-deficient patients, dATP was also elevated in ADA-deficient FTOCs. dATP levels were normalized and thymocyte development was rescued in cultures treated with an inhibitor of adenosine kinase, the enzyme that phosphorylates deoxyadenosine to dAMP. zVADfmk also prevented the accumulation of dATP in ADA-deficient FTOCs, suggesting that deoxyadenosine was derived from thymocytes undergoing apoptosis as a consequence of failing the beta selection checkpoint. In contrast, dATP levels remained elevated in ADA-deficient FTOCs with fetal thymuses from Bcl-2 transgenic mice. These data suggest that thymocyte apoptosis as a consequence of failing developmental checkpoints involves one or more caspases that are not regulated by Bcl-2.

Public health considerations associated with molluscan aquaculture systems: Human viruses.

The documentation of several recent outbreaks of human virus diseases associated with the consumption of shellfish has reiterated the threat posed by these agents to the shellfish industry. This article reviews pertinent outbreaks, identifies principal viral agents involved, and delineates systems which may be at greatest risk. The results of two recent laboratory studies which sought to define environmental factors that contribute to virus accumulation by shellfish are also discussed. First, the accumulation of environmentally significant levels of feces-associated and monodispersed poliovirus by oysters (Crassostrea virginica) and clams (Mercenaria mercenaria) was investigated. The results of this study suggested that virus accumulation by mollusks may not be significant when water column concentrations are below ⋍0.01 plaque-forming units (PFU) per milliliter. The second study focused on the relative contributions of undisturbed sediments versus those in the water column in the accumulation of viruses by epifaunal and infaunal shellfish (C. virginica and M. mercenaria). Viruses were found to be most efficiently accumulated when suspended in the water column.

The role of beliefs on learning about homosexuality in a college course.

The present research investigated how personal beliefs about homosexuality influence learning in a college course. We tested students in introductory psychology over material on the science of homosexuality by Simon LeVay (2010). All students reported information about their typical academic habits and the extent to which homosexuality was consistent with their beliefs and values. The results showed that students' personal beliefs were related to academic behaviors (e.g., reading assignments, skipping class) and retention of the course material. The results also showed that students' recall of course material six weeks later was predicted by the extent to which they reported studying information that is inconsistent with their beliefs for an exam and then forgetting it. Students who reported the material to be inconsistent with their beliefs engaged in selective forgetting of the material on homosexuality. The results provide evidence that personal beliefs can reduce the retention of belief-inconsistent information in a college course.

Interactions between immunity, proliferation and molecular subtype in breast cancer prognosis.

BACKGROUND: Gene expression signatures indicative of tumor proliferative capacity and tumor-immune cell interactions have emerged as principal biology-driven predictors of breast cancer outcomes. How these signatures relate to one another in biological and prognostic contexts remains to be clarified. RESULTS: To investigate the relationship between proliferation and immune gene signatures, we analyzed an integrated dataset of 1,954 clinically annotated breast tumor expression profiles randomized into training and test sets to allow two-way discovery and validation of gene-survival associations. Hierarchical clustering revealed a large cluster of distant metastasis-free survival-associated genes with known immunological functions that further partitioned into three distinct immune metagenes likely reflecting B cells and/or plasma cells; T cells and natural killer cells; and monocytes and/or dendritic cells. A proliferation metagene allowed stratification of cases into proliferation tertiles. The prognostic strength of these metagenes was largely restricted to tumors within the highest proliferation tertile, though intrinsic subtype-specific differences were observed in the intermediate and low proliferation tertiles. In highly proliferative tumors, high tertile immune metagene expression equated with markedly reduced risk of metastasis whereas tumors with low tertile expression of any one of the three immune metagenes were associated with poor outcome despite higher expression of the other two metagenes. CONCLUSIONS: These findings suggest that a productive interplay among multiple immune cell types at the tumor site promotes long-term anti-metastatic immunity in a proliferation-dependent manner. The emergence of a subset of effective immune responders among highly proliferative tumors has novel prognostic ramifications.

Understanding key assay parameters that affect measurements of trastuzumab-mediated ADCC against Her2 positive breast cancer cells.

Use of the antibody trastuzumab to kill HER2+ breast cancer cells is an attractive therapy because of its specificity and minimal adverse effects. However, a large fraction of HER2+ positive patients are or will become resistant to this treatment. No other markers are used to determine sensitivity to trastuzumab other than HER2 status.Using the xCELLigence platform and flow cytometry, we have compared the ability of mononuclear cells (MNCs) from normal and breast cancer patients to kill different breast cancer cell lines in the presence (i.e., ADCC) or absence of trastuzumab. Image analysis and cell separation procedures were used to determine the differential contribution of immune cell subsets to ADCC activity. The assay demonstrated that ADCC activity is dependent on the presence of trastuzumab, the level of HER2 expression on the target, and the ratio of MNCs to tumor cells. There is a wide range of ADCC activity among normal individuals and breast cancer patients for high and low HER2-expressing tumor targets. Fresh MNCs display higher ADCC levels compared with cryopreserved cells. Natural killer cells display the highest ADCC followed by monocytes. T cells and B cells were ineffective in killing. A major mechanism of killing of tumor cells involves insertion of granzyme B and caspase enzymes via the antibody attached MNCs.