Extracellular vesicles derived from immortalized human natural killer cell line NK3.3 as a novel therapeutic for multiple myeloma.

Introduction: Over the last decade, there have been many advancements in the therapeutic treatment of multiple myeloma (MM), including the use of natural killer (NK) cells. However, despite promising results from clinical trials, there are concerns over the use of NK cell-based therapy. Cells often undergo growth arrest, limiting their experimental utility; donor cells are extremely heterogeneous, resulting in content variability; and patients receiving allogeneic cells are at risk for graft-versus-host disease and/or cytokine release syndrome. Extracellular vesicles (EVs) have emerged as a new natural therapeutic tool. EVs are known to carry cargo derived from the parent cell from which they originate. NK cells play an important role in the innate immune system, targeting and killing tumor cells. This has led many researchers to isolate EVs from NK cells for their cytotoxic potential. Methods: In this study, we isolated EVs from the NK cell line, NK3.3, which was derived from the peripheral blood of a healthy donor. Currently, it is the only normal human NK cell line reported with all the functional characteristics of healthy NK cells. To address the issue of growth arrest, we immortalized NK3.3 cells with lentivirus encoding the catalytic subunit of human telomerase htert (NK3.3-LTV). EVs from these cells were isolated using a modified polyethylene glycol (PEG)-acetate precipitation protocol to simplify processing and increase EV yield. Results and conclusions: We demonstrated that NK3.3-LTV EVs target both sensitive and drug-resistant MM cell lines as well as primary patient MM cells in vitro, decreasing proliferation and inducing apoptotic cell death as well as or better than EVs from non-immortalized cells with no toxicity towards normal cells. This study is the first step towards developing an immunotherapeutic product designed to treat patients with relapsed/refractory MM.

NK3.3-Derived Extracellular Vesicles Penetrate and Selectively Kill Treatment-Resistant Tumor Cells.

Cancer treatments often become ineffective due to the development of tumor resistance, leading to metastasis and relapse. Treatments may also fail because of their inability to access cells deep within the tumor tissue. When this occurs, new therapeutic agents are needed. We previously reported that NK3.3EVs, extracellular vesicles (EVs) derived from the normal human natural killer (NK) cell line, NK3.3, have strong cytotoxic activity against leukemia and breast cancer cell lines, without harming normal cells. Here, we used a three-dimensional (3D) MCF7 breast cancer mammosphere model to reproduce a more physiological environment that NK3.3EVs would encounter in vivo. NK3.3EVs penetrated MCF7 mammospheres, inducing death by apoptosis. We generated an imatinib-resistant K562 chronic myeloid leukemia (CML) cell line to investigate whether NK3.3EVs were able to kill tumor cells resistant to front-line chemotherapy. NK3.3EVs were even more cytotoxic to imatinib-resistant cells than parental cells, inducing apoptosis via caspase-3/-7 activation. The small population of cancer stem cells (CSCs) within tumors also contributes to therapeutic resistance. NK3.3EVs reduced the CSC-like CD34+/CD38- subpopulation in imatinib-resistant and parental K562 cultures and decreased CSC-associated expression of tumor-promoting genes. Our results provide strong evidence that NK3.3EVs may be a potential new immunotherapeutic agent for difficult-to-treat cancers.

Characterization of uridine-cytidine kinase like-1 nucleoside kinase activity and its role in tumor growth.

Uridine-cytidine kinase like-1 (UCKL-1) is a largely uncharacterized protein with high sequence similarity to other uridine-cytidine kinases (UCKs). UCKs play an important role in the pyrimidine salvage pathway, catalyzing the phosphorylation of uridine and cytidine to UMP and CMP, respectively. Only two human UCKs have been identified, UCK1 and UCK2. Previous studies have shown both enzymes phosphorylate uridine and cytidine using ATP as the phosphate donor. No studies have evaluated the kinase potential of UCKL-1. We cloned and purified UCKL-1 and found that it successfully phosphorylated uridine and cytidine using ATP as the phosphate donor. The catalytic efficiency (calculated as kcat/KM) was 1.2 × 104 s-1, M-1 for uridine and 0.7 × 104 s-1, M-1 for cytidine. Our lab has previously shown that UCKL-1 is up-regulated in tumor cells, providing protection against natural killer (NK) cell killing activity. We utilized small interfering RNA (siRNA) to down-regulate UCKL-1 in vitro and in vivo to determine the effect of UCKL-1 on tumor growth and metastasis. The down-regulation of UCKL-1 in YAC-1 lymphoma cells in vitro resulted in decreased cell counts and increased apoptotic activity. Down-regulation of UCKL-1 in K562 leukemia cells in vivo led to decreased primary tumor growth and less tumor cell dissemination and metastasis. These results identify UCKL-1 as a bona fide pyrimidine kinase with the therapeutic potential to be a target for tumor growth inhibition and for diminishing or preventing metastasis.

Extracellular Vesicles From the Human Natural Killer Cell Line NK3.3 Have Broad and Potent Anti-Tumor Activity.

Natural killer (NK) cells are critical mediators of immune function, responsible for rapid destruction of tumor cells. They kill primarily through the release of granules containing potent cytolytic molecules. NK cells also release these molecules within membrane-bound exosomes and microvesicles - collectively known as extracellular vesicles (EV). Here we report the characterization and anti-tumor function of EVs isolated from NK3.3 cells, a well described clonal normal human NK cell line. We show that NK3.3 EVs contain the cytolytic molecules perforin, granzymes A and B, and granulysin, and an array of common EV proteins. We previously reported that the E3 ubiquitin ligase, natural killer lytic-associated molecule (NKLAM), is localized to NK granules and is essential for maximal NK killing; here we show it is present in the membrane of NK3.3 EVs. NK3.3-derived EVs also carry multiple RNA species, including miRNAs associated with anti-tumor activity. We demonstrate that NK3.3 EVs inhibit proliferation and induce caspase-mediated apoptosis and cell death of an array of both hematopoietic and non-hematopoietic tumor cell lines. This effect is tumor cell specific; normal cells are unaffected by EV treatment. By virtue of their derivation from a healthy donor and ability to be expanded to large numbers, NK3.3 EVs have the potential to be an effective, safe, and universal immunotherapeutic agent.

Natural Killer Lytic-Associated Molecule (NKLAM): An E3 Ubiquitin Ligase With an Integral Role in Innate Immunity.

Natural Killer Lytic-Associated Molecule (NKLAM), also designated RNF19B, is a unique member of a small family of E3 ubiquitin ligases. This 14-member group of ligases has a characteristic cysteine-rich RING-IBR-RING (RBR) domain that mediates the ubiquitination of multiple substrates. The consequence of substrate ubiquitination varies, depending on the type of ubiquitin linkages formed. The most widely studied effect of ubiquitination of proteins is proteasome-mediated substrate degradation; however, ubiquitination can also alter protein localization and function. Since its discovery in 1999, much has been deciphered about the role of NKLAM in innate immune responses. We have discerned that NKLAM has an integral function in both natural killer (NK) cells and macrophages in vitro and in vivo. NKLAM expression is required for each of these cell types to mediate maximal killing activity and cytokine production. However, much remains to be determined. In this review, we summarize what has been learned about NKLAM expression, structure and function, and discuss new directions for investigation. We hope that this will stimulate interest in further exploration of NKLAM.

Mice deficient in NKLAM have attenuated inflammatory cytokine production in a Sendai virus pneumonia model.

Recent studies have begun to elucidate a role for E3 ubiquitin ligases as important mediators of the innate immune response. Our previous work defined a role for the ubiquitin ligase natural killer lytic-associated molecule (NKLAM/RNF19b) in mouse and human innate immunity. Here, we present novel data describing a role for NKLAM in regulating the immune response to Sendai virus (SeV), a murine model of paramyxoviral pneumonia. NKLAM expression was significantly upregulated by SeV infection. SeV-infected mice that are deficient in NKLAM demonstrated significantly less weight loss than wild type mice. In vivo, Sendai virus replication was attenuated in NKLAM-/- mice. Autophagic flux and the expression of autophagy markers LC3 and p62/SQSTM1 were also less in NKLAM-/- mice. Using flow cytometry, we observed less neutrophils and macrophages in the lungs of NKLAM-/- mice during SeV infection. Additionally, phosphorylation of STAT1 and NFκB p65 was lower in NKLAM-/- than wild type mice. The dysregulated phosphorylation profile of STAT1 and NFκB in NKLAM-/- mice correlated with decreased expression of numerous proinflammatory cytokines that are regulated by STAT1 and/or NFκB. The lack of NKLAM and the resulting attenuated immune response is favorable to NKLAM-/- mice receiving a low dose of SeV; however, at a high dose of virus, NKLAM-/- mice succumbed to the infection faster than wild type mice. In conclusion, our novel results indicate that NKLAM plays a role in regulating the production of pro-inflammatory cytokines during viral infection.

Reduced inflammation and cytokine production in NKLAM deficient mice during Streptococcus pneumoniae infection.

Streptococcus pneumoniae is a leading cause of pneumonia and a significant economic burden. Antibiotic-resistant S. pneumoniae has become more prevalent in recent years and many pneumonia cases are caused by S. pneumoniae that is resistant to at least one antibiotic. The ubiquitin ligase natural killer lytic-associated molecule (NKLAM/RNF19b) plays a role in innate immunity and studies using NKLAM-knockout (NKLAM-KO) macrophages have demonstrated that NKLAM positively affects the transcriptional activity of STAT1. Using an inhalation infection model, we found that NKLAM-KO mice had a significantly higher lung bacterial load than WT mice but had less lung inflammation. Coincidently, NKLAM-KO mice had fewer neutrophils and NK cells in their lungs. NKLAM-KO mice also expressed less iNOS in their lungs as well as less MCP-1, MIP1α, TNFα, IL-12, and IFNγ. Both neutrophils and macrophages from NKLAM-KO mice were defective in killing S. pneumoniae as compared to wild type cells (WT). The phosphorylation of STAT1 and STAT3 in NKLAM-KO lungs was lower than in WT lungs at 24 hours post-infection. NKLAM-KO mice were afforded some protection against a lethal dose of S. pneumoniae compared to WT mice. In summary, our novel data demonstrate a role for E3 ubiquitin ligase NKLAM in modulating innate immunity via the positive regulation of inflammatory cytokine expression and bactericidal activity.

Bitter Melon Enhances Natural Killer-Mediated Toxicity against Head and Neck Cancer Cells.

Natural killer (NK) cells are one of the major components of innate immunity, with the ability to mediate antitumor activity. Understanding the role of NK-cell-mediated tumor killing in controlling of solid tumor growth is still in the developmental stage. We have shown recently that bitter melon extract (BME) modulates the regulatory T cell (Treg) population in head and neck squamous cell carcinoma (HNSCC). However, the role of BME in NK-cell modulation against HNSCC remains unknown. In this study, we investigated whether BME can enhance the NK-cell killing activity against HNSCC cells. Our results indicated that treatment of human NK-cell line (NK3.3) with BME enhances ability to kill HNSCC cells. BME increases granzyme B accumulation and translocation/accumulation of CD107a/LAMP1 in NK3.3 cells exposed to BME. Furthermore, an increase in cell surface expression of CD16 and NKp30 in BME-treated NK3.3 cells was observed when cocultured with HNSCC cells. Collectively, our results demonstrated for the first time that BME augments NK-cell-mediated HNSCC killing activity, implicating an immunomodulatory role of BME. Cancer Prev Res; 10(6); 337-44. ©2017 AACR.

E3 ubiquitin ligase NKLAM ubiquitinates STAT1 and positively regulates STAT1-mediated transcriptional activity.

Signal transducer and activator of transcription 1 (STAT1) is critically important for the transcription of a large number of immunologically relevant genes. In macrophages, interferon gamma (IFNγ) signal transduction occurs via the JAK/STAT pathway and ends with the transcription of a number of genes necessary for a successful host immune response. The predominant mechanism of regulation of STAT1 is phosphorylation; however, there is a growing body of evidence that demonstrates STAT1 is also regulated by ubiquitination. In this report we show that JAK1 and STAT1 in macrophages deficient in an E3 ubiquitin ligase termed Natural Killer Lytic-Associated Molecule (NKLAM) are hyperphosphorylated following IFNγ stimulation. We found NKLAM was transiently localized to the IFNγ receptor complex during stimulation with IFNγ, where it bound to and mediated K63-linked ubiquitination of STAT1. In vitro nucleofection studies demonstrated that STAT1-mediated transcription was significantly reduced in NKLAM-KO macrophages. There was no obvious defect in STAT1 nuclear translocation; however, STAT1 from NKLAM-KO macrophages had a reduced ability to bind a functional gamma activation DNA sequence. There was also less mRNA expression of STAT1-mediated genes in NKLAM-KO macrophages treated with IFNγ. Our results demonstrate for the first time that NKLAM is a positive regulator of STAT1-mediated transcriptional activity and is an important component of the innate immune response.

Uridine Cytidine Kinase Like-1 Enhances Tumor Cell Proliferation and Mediates Protection from Natural Killer-Mediated Killing.

Uridine cytidine kinase like-1 (UCKL-1) is a largely uncharacterized protein over-expressed in many tumor cells, especially in highly malignant, aggressive tumors. Sequence analysis indicates that UCKL-1 has homology to uridine kinases, enzymes that play a role in DNA and RNA synthesis and that are often up-regulated in tumor cells. Previous studies have shown that UCKL-1 is a substrate for natural killer lytic-associated molecule (NKLAM), an E3 ubiquitin ligase found in NK cell cytolytic granules. Ubiquitination of UCKL-1 by NKLAM leads to its degradation. Increased expression of NKLAM enhances NK-mediated tumoricidal activity. The fact that UCKL-1 is a substrate for NKLAM suggests that UCKL-1 may provide resistance to NK killing in tumor cells. Here we show that UCKL-1 over-expression protects tumor cells from NK killing and enhances tumor survival in vivo. UCKL-1 also has a much broader role, protecting tumor cells from spontaneous and drug-induced apoptosis and increasing tumor cell proliferation. Nuclear factor-kappa B (NF-κB) activity is higher in tumor cells transfected with UCKL-1 compared to control transfected cells, suggesting at least one possible mechanism by which UCKL-1 influences tumor growth and survival.

E3 ubiquitin ligase NKLAM positively regulates macrophage inducible nitric oxide synthase expression.

Stimulated macrophages generate potent anti-microbial reactive oxygen and nitrogen species within their phagosomes. Previous studies have shown that the E3 ubiquitin ligase natural killer lytic-associated molecule (NKLAM) is a macrophage phagosomal protein that plays a role in macrophage anti-bacterial activity. In vivo, NKLAM-knockout (KO) mice produce less nitric oxide (NO) upon exposure to lipopolysaccharide (LPS) than wild type (WT) mice. In vitro, we found that NO production and inducible nitric oxide synthase (iNOS) protein were diminished in LPS-stimulated NKLAM-KO bone marrow-derived and splenic macrophages. Additionally, LPS-stimulated NKLAM-KO macrophages displayed defects in STAT1 tyrosine phosphorylation and production of interferon beta (IFNß). The JAK/STAT pathway is critical for the production of IFNß, which augments iNOS protein expression in mice. iNOS protein expression is also regulated by the transcription factor NFκB, thus we investigated whether NKLAM influences NFκB function. LPS-stimulated NKLAM-KO macrophages showed evidence of delayed nuclear translocation of the NFκB subunit p65. This was associated with a reduction in p65/DNA colocalization. The defect in p65 translocation was independent of IKBα degradation. NKLAM-KO macrophages also expressed less p65 and showed evidence of defective p65 phosphorylation at serine 536. Importantly, LPS-stimulated NKLAM-KO macrophages have diminished NFκB transcriptional activity as assessed by transfection of a luciferase reporter plasmid. Collectively, our data implicate NKLAM as a novel modulator of macrophage iNOS expression.

Natural killer lytic-associated molecule plays a role in controlling tumor dissemination and metastasis.

Natural killer lytic-associated molecule (NKLAM) is an E3 ubiquitin ligase that plays a major role in the cytolytic activity of NK cells. NKLAM is rapidly synthesized and then targeted to the granule membranes of NK cells upon NK activation. Previous studies have shown an essential role for NKLAM in NK killing activity in vitro. These findings were extended to an in vivo model of NK-mediated tumor killing in which NKLAM-deficient knockout (KO) mice injected with B16 melanoma cells were found to have significantly higher numbers of pulmonary tumor nodules than wild-type (WT) mice. To further investigate the role of NKLAM and NK function in tumor immunity in vivo, we utilized additional tumor models to compare tumor development and progression in NKLAM KO and WT mice. Primary tumor growth, dissemination, and metastasis of RMA-S lymphoma cells and E0771 breast cancer cells were evaluated. Both tumor cell lines were stably transfected with constructs that allow expression of green fluorescent protein (GFP), which serves as a tumor-specific marker. Intravenous injection of NK-sensitive RMA-S lymphoma cells resulted in greater dissemination of lymphoma cells in NKLAM KO mice than in WT mice. Lymphoma cells were found in the lymph nodes and bone marrow (BM) of NKLAM KO mice 2 weeks after injection; few detectable tumor cells remained in WT mice. E0771 syngeneic breast cancer cells were injected into the mammary pads of NKLAM KO and WT mice. Primary tumor growth was greater in NKLAM KO than in WT mice. More significantly, there were 4-5-fold more tumor cells in the blood and lungs of NKLAM KO than in WT mice 2 weeks after injection of tumor cells into the mammary pad. These results indicate that NKLAM plays a role in tumor development in vivo, especially in controlling tumor dissemination and metastasis to distant sites.

E3 ubiquitin ligase NKLAM is a macrophage phagosome protein and plays a role in bacterial killing.

Macrophages are a critically important component of the innate and adaptive immune systems. They are equipped with oxidative and non-oxidative mechanisms to kill ingested pathogens. Natural Killer Lytic-Associated Molecule (NKLAM) is an E3 ubiquitin ligase expressed in macrophages and natural killer cells. We show that NKLAM expression in macrophages was enhanced by Toll-like receptor agonists and pro-inflammatory cytokines. Using confocal microscopy, we found that NKLAM colocalized with ingested Escherichia coli. In assays using IgG-opsonized latex beads as targets, we demonstrated that NKLAM translocated to the phagosome early during maturation at a time that coincided with elevated levels of ubiquitinated phagosome proteins. In killing assays with bone marrow-derived macrophages from wild type and NKLAM-deficient mice, we found that NKLAM-deficient macrophages demonstrated less killing of E. coli than wild type macrophages. Collectively, our data show that NKLAM is a novel component of macrophage phagosomes and is involved in macrophage bactericidal functions.

Platelet-activating factor and metastasis: calcium-independent phospholipase A2ß deficiency protects against breast cancer metastasis to the lung.

We determined the contribution of calcium-independent phospholipase A(2)ß (iPLA(2)ß) to lung metastasis development following breast cancer injection into wild-type (WT) and iPLA(2)ß-knockout (iPLA(2)ß-KO) mice. WT and iPLA(2)ß-KO mice were injected in the mammary pad with 200,000 E0771 breast cancer cells. There was no difference in primary tumor size between WT and iPLA(2)ß-KO mice at 27 days postinjection. However, we observed an 11-fold greater number of breast cancer cells in the lungs of WT mice compared with iPLA(2)ß-KO animals (P < 0.05). Isolated WT lung endothelial cells demonstrated a significant increase in platelet-activating factor (PAF) production when stimulated with thrombin [1 IU/ml, 10 min, 4,330 ± 555 vs. 15,227 ± 1,043 disintegrations per minute (dpm), P < 0.01] or TNF-α (10 ng/ml, 2 h, 16,532 ± 538 dpm, P < 0.01). Adherence of E0771 cells to WT endothelial cells was increased by thrombin (4.8 ± 0.3% vs. 70.9 ± 6.3, P < 0.01) or TNF-α (60.5 ± 4.3, P < 0.01). These responses were blocked by pretreatment with the iPLA(2)ß-selective inhibitor (S)-bromoenol lactone and absent in lung endothelial cells from iPLA(2)ß-KO mice. These data indicate that endothelial cell iPLA(2)ß is responsible for PAF production and adherence of E0771 cells and may play a role in cancer cell migration to distal locations.

Impaired NK cytolytic activity and enhanced tumor growth in NK lytic-associated molecule-deficient mice.

NK lytic-associated molecule (NKLAM) is a protein involved in the cytolytic function of NK cells. It is weakly expressed in resting NK cells but upon target cell stimulation or after incubation with cytokines that enhance NK killing, NKLAM mRNA levels increase and protein is synthesized and is targeted to cytoplasmic granule membranes. We have previously shown that NKLAM plays a role in perforin/granzyme-mediated cytolysis in vitro. To further investigate the function of NKLAM in NK cell-mediated cytotoxicity, we generated, by gene targeting, NKLAM-deficient mice. These mice have normal numbers of NK cells and other lymphoid populations in the spleen. They also have no alterations in NK maturation or NK receptor repertoire. NK cells from NKLAM-deficient and WT mice have comparable amounts of perforin, granzyme B, and lysosomal membrane-associated protein 1 (CD107a) in their cytotoxic granules and comparable levels of granule exocytosis are induced by PMA and calcium ionophore A23187. However, NKLAM-deficient NK cells display significantly less NK cytotoxic activity in vitro than WT NK cells. They also secrete less IFN-gamma upon target cell stimulation, In addition, NKLAM-deficient mice exhibit greater numbers of pulmonary metastases after i.v. injection with B16 melanoma cells. These studies indicate that NKLAM-deficient mice have diminished capacity to control tumor metastases and support the role for NKLAM in NK function both in vitro and in vivo.

Downregulation of uridine-cytidine kinase like-1 decreases proliferation and enhances tumor susceptibility to lysis by apoptotic agents and natural killer cells.

Natural killer (NK) cells target and kill tumor cells by direct anti-tumor cytotoxicity. NK lytic-associated molecule (NKLAM) is a protein involved in this cytolytic function. Acting as an E3 ubiquitin ligase, NKLAM binds to and ubiquitinates a novel protein, uridine-cytidine kinase like-1 (UCKL-1), targeting it for degradation. However, UCKL-1's function in tumor cell survival and NK cell cytotoxicity is unknown. UCKL-1's homology to uridine kinases and over expression in tumor cells suggests a role for UCKL-1 in tumor growth and/or survival. We propose that NKLAM and UCKL-1 interact in the tumor cell, where degradation of UCKL-1 leads to increased tumor cell apoptosis. Here we use RNA interference to downregulate UCKL-1 expression in K562 erythroleukemia cells. It was seen that downregulation of UCKL-1 initiated apoptosis and slowed the cell cycle, resulting in lower growth in the small interfering UCKL-1 RNA treated K562 cell culture. In addition, the chemotherapeutic agent staurosporine was seen to be more effective in inducing cell death by apoptosis in UCKL-1 depleted K562 cells compared with controls. We also found that UCKL-1 depleted K562 cells were more susceptible to NK mediated cytolysis than controls. These results indicate a role for UCKL-1 in tumor cell survival and suggest possible therapeutic potential of UCKL-1 inhibitors in cancer treatment.

NK lytic-associated molecule, involved in NK cytotoxic function, is an E3 ligase.

NK lytic-associated molecule (NKLAM) is a protein involved in the cytolytic function of NK cells and CTLs. It has been localized to the cytolytic granules in NK cells and is up-regulated when cells are exposed to cytokines IL-2 or IFN-beta. We report in this study that NKLAM contains a cysteine-rich really interesting new gene (RING) in between RING-RING domain, and that this domain possesses strong homology to the RING domain of the known E3 ubiquitin ligase, Dorfin. To determine whether NKLAM functions as an E3 ligase, we performed coimmunoprecipitation binding assays with ubiquitin conjugates (Ubcs) UbcH7, UbcH8, and UbcH10. We demonstrated that both UbcH7 and UbcH8 bind to full-length NKLAM. We then performed a similar binding assay using endogenous NKLAM and UbcH8 expressed by human NK clone NK3.3 to show that the protein interaction occurs in vivo. Using the yeast two-hybrid system, we identified uridine kinase like-1 (URKL-1) protein as a substrate for NKLAM. We confirmed that NKLAM and URKL-1 interact in mammalian cells by using both immunoprecipitation and confocal microscopy. We demonstrated decreased protein expression and enhanced ubiquitination of URKL-1 in the presence of NKLAM. These data indicate that NKLAM is a RING finger protein that binds Ubcs and has as one of its substrates, URKL-1, thus defining this cytolytic protein as an E3 ubiquitin ligase.

Effect of glutamine on the initiation and promotion phases of DMBA-induced mammary tumor development.

BACKGROUND: Oral glutamine (GLN) has been shown to up-regulate tissue glutathione (GSH), augment natural killer (NK) cell activity, and prevent tumor growth in an implantable breast cancer model (MTF-7). We hypothesized that dietary GLN would likewise antagonize the induction or promotion of tumor formation by 7,12-dimethylbenz[a]anthracene (DMBA) via up-regulation of GSH or augmentation of NK activity. METHODS: At age 55 days, 81 Sprague-Dawley rats were gavaged with a one-time dose of 80 mg/kg DMBA, time 0. Rats were randomized into 3 groups (GLN+DMBA, Freamine [FA]+DMBA, water (H2O)+DMBA), pair-fed chow, and gavaged with 1.0 g/kg/day GLN or isonitrogenous amount of FA or H2O for the indicated times: PreFed (-1 to + 16 weeks), Short-Fed (-1 to + 1 weeks) and PostFed (+ 1 to +16 weeks). After 16 weeks, rats were killed and examined for mammary tumors, blood was assayed for GLN and GSH content, and spleens were assayed for NK cytotoxicity. RESULTS: Over the 4-month study period, there was no significant difference in tumorigenesis between FA and H2O groups, regardless of timing of feeding and amino acid diet, except GLN. In Pre- and PostFed GLN groups, there was no significant difference between groups, but there were significant decreases in tumorigenesis in GLN groups compared with either FA or H2O groups. However, in the Short-Fed group, there was no significant difference in tumorigenesis from the GLN, FA, or H2O groups. CONCLUSIONS: Continuously supplemented GLN significantly reduced DMBA-induced breast cancer growth when compared with the non-GLN-supplemented and Short-Fed supplemental GLN groups. Furthermore, GLN appears to have its primary effect on promotion and not initiation of tumor formation. This decreased tumor formation was associated with significantly higher arterial GLN and GSH levels and NK activity at killing in the GLN+DMBA group. Protein in the presentation of FA did not promote or prevent tumor growth. These data indicate that GLN may be useful in the chemoprevention of breast cancer.