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The development of targeted therapies offers new hope for patients affected by incurable cancer. However, multiple challenges persist, notably in controlling tumor cell plasticity in patients with refractory and metastatic illness. Neuroblastoma (NB) is an aggressive pediatric malignancy originating from defective differentiation of neural crest-derived progenitors with oncogenic activity due to genetic and epigenetic alterations and remains a clinical challenge for high-risk patients. To identify critical genes driving NB aggressiveness, we performed combined chromatin and transcriptome analyses on matched patient-derived xenografts (PDXs), spheroids, and differentiated adherent cultures derived from metastatic MYCN nonamplified tumors. Bone marrow kinase on chromosome X (BMX) was identified among the most differentially regulated genes in PDXs and spheroids versus adherent models. BMX expression correlated with high tumor stage and poor patient survival and was crucial to the maintenance of the self-renewal and tumorigenic potential of NB spheroids. Moreover, BMX expression positively correlated with the mesenchymal NB cell phenotype, previously associated with increased chemoresistance. Finally, BMX inhibitors readily reversed this cellular state, increased the sensitivity of NB spheroids toward chemotherapy, and partially reduced tumor growth in a preclinical NB model. Altogether, our study identifies BMX as a promising innovative therapeutic target for patients with high-risk MYCN nonamplified NB.

CIC-DUX4-rearranged sarcoma (CDS) is a rare and aggressive soft tissue tumor that occurs most frequently in young adults. The key oncogenic driver of this disease is the expression of the CIC-DUX4 fusion protein as a result of chromosomal rearrangements. CIC-DUX4 displays chromatin binding properties, and is therefore believed to function as an aberrant transcription factor. However, the chromatin remodeling events induced by CIC-DUX4 are not well understood, limiting our ability to identify new mechanism-based therapeutic strategies for these patients. Here, we generated a genome-wide profile of CIC-DUX4 DNA occupancy and associated chromatin states in human CDS cell models and primary tumors. Combining chromatin profiling, proximity ligation assays, as well as genetic and pharmacological perturbations, we show that CIC-DUX4 operates as a potent transcriptional activator at its binding sites. This property is in contrast with the repressive function of the wild-type CIC protein, and is mainly mediated through the direct interaction of CIC-DUX4 with the acetyltransferase p300. In keeping with this, we show p300 to be essential for CDS tumor cell proliferation; additionally, we find its pharmacological inhibition to significantly impact tumor growth in vitro and in vivo. Taken together, our study elucidates the mechanisms underpinning CIC-DUX4-mediated transcriptional regulation.

Oncogenic fusion proteins generated by chromosomal translocations play major roles in cancer. Among them, fusions between EWSR1 and transcription factors generate oncogenes with powerful chromatin regulatory activities, capable of establishing complex gene expression programs in permissive precursor cells. Here we define the epigenetic and 3D connectivity landscape of Clear Cell Sarcoma, an aggressive cancer driven by the EWSR1-ATF1 fusion gene. We find that EWSR1-ATF1 displays a distinct DNA binding pattern that requires the EWSR1 domain and promotes ATF1 retargeting to new distal sites, leading to chromatin activation and the establishment of a 3D network that controls oncogenic and differentiation signatures observed in primary CCS tumors. Conversely, EWSR1-ATF1 depletion results in a marked reconfiguration of 3D connectivity, including the emergence of regulatory circuits that promote neural crest-related developmental programs. Taken together, our study elucidates the epigenetic mechanisms utilized by EWSR1-ATF1 to establish regulatory networks in CCS, and points to precursor cells in the neural crest lineage as candidate cells of origin for these tumors., (© 2022. The Author(s).)

Synovial sarcoma (SyS) is an aggressive neoplasm driven by the SS18-SSX fusion, and is characterized by low T cell infiltration. Here, we studied the cancer-immune interplay in SyS using an integrative approach that combines single-cell RNA sequencing (scRNA-seq), spatial profiling and genetic and pharmacological perturbations. scRNA-seq of 16,872 cells from 12 human SyS tumors uncovered a malignant subpopulation that marks immune-deprived niches in situ and is predictive of poor clinical outcomes in two independent cohorts. Functional analyses revealed that this malignant cell state is controlled by the SS18-SSX fusion, is repressed by cytokines secreted by macrophages and T cells, and can be synergistically targeted with a combination of HDAC and CDK4/CDK6 inhibitors. This drug combination enhanced malignant-cell immunogenicity in SyS models, leading to induced T cell reactivity and T cell-mediated killing. Our study provides a blueprint for investigating heterogeneity in fusion-driven malignancies and demonstrates an interplay between immune evasion and oncogenic processes that can be co-targeted in SyS and potentially in other malignancies.

Synovial sarcoma (SyS) is an aggressive mesenchymal malignancy invariably associated with the chromosomal translocation t(X:18; p11:q11), which results in the in-frame fusion of the BAF complex gene SS18 to one of three SSX genes. Fusion of SS18 to SSX generates an aberrant transcriptional regulator, which, in permissive cells, drives tumor development by initiating major chromatin remodeling events that disrupt the balance between BAF-mediated gene activation and polycomb-dependent repression. Here, we developed SyS organoids and performed genome-wide epigenomic profiling of these models and mesenchymal precursors to define SyS-specific chromatin remodeling mechanisms and dependencies. We show that SS18-SSX induces broad BAF domains at its binding sites, which oppose polycomb repressor complex (PRC) 2 activity, while facilitating recruitment of a non-canonical (nc)PRC1 variant. Along with the uncoupling of polycomb complexes, we observed H3K27me3 eviction, H2AK119ub deposition and the establishment of de novo active regulatory elements that drive SyS identity. These alterations are completely reversible upon SS18-SSX depletion and are associated with vulnerability to USP7 loss, a core member of ncPRC1.1. Using the power of primary tumor organoids, our work helps define the mechanisms of epigenetic dysregulation on which SyS cells are dependent., (© 2020 Boulay et al.)

Synovial sarcoma is a highly aggressive soft tissue malignancy that often affects adolescents and young adults. It is associated with a unique chromosomal translocation that results in the formation and expression of the fusion gene SS18-SSX, which underlies its pathogenesis. Although SS18-SSX provides a potentially unique therapeutic target, all attempts to neutralise it have been unsuccessful thus far. When complete surgical removal of the tumour fails, therapy is limited to largely ineffective cytotoxic drug regimens. Nevertheless, recent discoveries about the mechanisms of SS18-SSX protein function have provided insight into potential alternative therapeutic strategies. SS18-SSX displays oncogenic activity through protein-protein interactions and participation in chromatin remodelling complexes. This review summarises our current understanding of the function of SS18-SSX and the mechanisms by which it alters the epigenetic landscape of permissive cells to induce transformation and the subsequent development of synovial sarcoma.

Expression of the SS18/SYT-SSX fusion protein is believed to underlie the pathogenesis of synovial sarcoma (SS). Recent evidence suggests that deregulation of the Wnt pathway may play an important role in SS but the mechanisms whereby SS18-SSX might affect Wnt signaling remain to be elucidated. Here, we show that SS18/SSX tightly regulates the elevated expression of the key Wnt target AXIN2 in primary SS. SS18-SSX is shown to interact with TCF/LEF, TLE and HDAC but not β-catenin in vivo and to induce Wnt target gene expression by forming a complex containing promoter-bound TCF/LEF and HDAC but lacking β-catenin. Our observations provide a tumor-specific mechanistic basis for Wnt target gene induction in SS that can occur in the absence of Wnt ligand stimulation.

Cancer stem cells (CSCs) display plasticity and self-renewal properties reminiscent of normal tissue stem cells, but the events responsible for their emergence remain obscure. We recently identified CSCs in Ewing sarcoma family tumors (ESFTs) and showed that they retain mesenchymal stem cell (MSC) plasticity. In the present study, we addressed the mechanisms that underlie ESFT CSC development. We show that the EWS-FLI-1 fusion gene, associated with 85%-90% of ESFTs and believed to initiate their pathogenesis, induces expression of the embryonic stem cell (ESC) genes OCT4, SOX2, and NANOG in human pediatric MSCs (hpMSCs) but not in their adult counterparts. Moreover, under appropriate culture conditions, hpMSCs expressing EWS-FLI-1 generate a cell subpopulation displaying ESFT CSC features in vitro. We further demonstrate that induction of the ESFT CSC phenotype is the result of the combined effect of EWS-FLI-1 on its target gene expression and repression of microRNA-145 (miRNA145) promoter activity. Finally, we provide evidence that EWS-FLI-1 and miRNA-145 function in a mutually repressive feedback loop and identify their common target gene, SOX2, in addition to miRNA145 itself, as key players in ESFT cell differentiation and tumorigenicity. Our observations provide insight for the first time into the mechanisms whereby a single oncogene can reprogram primary cells to display a CSC phenotype.

Overexpression of the polycomb group protein enhancer of zeste homologue 2 (EZH2) occurs in diverse malignancies, including prostate cancer, breast cancer, and glioblastoma multiforme (GBM). Based on its ability to modulate transcription of key genes implicated in cell cycle control, DNA repair, and cell differentiation, EZH2 is believed to play a crucial role in tissue-specific stem cell maintenance and tumor development. Here, we show that targeted pharmacologic disruption of EZH2 by the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep), or its specific downregulation by short hairpin RNA (shRNA), strongly impairs GBM cancer stem cell (CSC) self-renewal in vitro and tumor-initiating capacity in vivo. Using genome-wide expression analysis of DZNep-treated GBM CSCs, we found the expression of c-myc, recently reported to be essential for GBM CSCs, to be strongly repressed upon EZH2 depletion. Specific shRNA-mediated downregulation of EZH2 in combination with chromatin immunoprecipitation experiments revealed that c-myc is a direct target of EZH2 in GBM CSCs. Taken together, our observations provide evidence that direct transcriptional regulation of c-myc by EZH2 may constitute a novel mechanism underlying GBM CSC maintenance and suggest that EZH2 may be a valuable new therapeutic target for GBM management.

Background: A characteristic SYT-SSX fusion gene resulting from the chromosomal translocation t(X;18)(p11;q11) is detectable in almost all synovial sarcomas, a malignant soft tissue tumor widely believed to originate from as yet unidentified pluripotent stem cells. The resulting fusion protein has no DNA binding motifs but possesses protein-protein interaction domains that are believed to mediate association with chromatin remodeling complexes. Despite recent advances in the identification of molecules that interact with SYT-SSX and with the corresponding wild type SYT and SSX proteins, the mechanisms whereby the SYT-SSX might contribute to neoplastic transformation remain unclear. Epigenetic deregulation has been suggested to be one possible mechanism., Methodology/principal Findings: We addressed the effect of SYT/SSX expression on the transcriptome of four independent isolates of primary human bone marrow mesenchymal stem cells (hMSC). We observed transcriptional changes similar to the gene expression signature of synovial sarcoma, principally involving genes whose regulation is linked to epigenetic factors, including imprinted genes, genes with transcription start sites within a CpG island and chromatin related genes. Single population analysis revealed hMSC isolate-specific transcriptional changes involving genes that are important for biological functions of stem cells as well as genes that are considered to be molecular markers of synovial sarcoma including IGF2, EPHRINS, and BCL2. Methylation status analysis of sequences at the H19/IGF2 imprinted locus indicated that distinct epigenetic features characterize hMSC populations and condition the transcriptional effects of SYT-SSX expression., Conclusions/significance: Our observations suggest that epigenetic features may define the cellular microenvironment in which SYT-SSX displays its functional effects.

Cancer stem cells that display tumor-initiating properties have recently been identified in several distinct types of malignancies, holding promise for more effective therapeutic strategies. However, evidence of such cells in sarcomas, which include some of the most aggressive and therapy-resistant tumors, has not been shown to date. Here, we identify and characterize cancer stem cells in Ewing's sarcoma family tumors (ESFT), a highly aggressive pediatric malignancy believed to be of mesenchymal stem cell (MSC) origin. Using magnetic bead cell separation of primary ESFT, we have isolated a subpopulation of CD133+ tumor cells that display the capacity to initiate and sustain tumor growth through serial transplantation in nonobese diabetic/severe combined immunodeficiency mice, re-establishing at each in vivo passage the parental tumor phenotype and hierarchical cell organization. Consistent with the plasticity of MSCs, in vitro differentiation assays showed that the CD133+ cell population retained the ability to differentiate along adipogenic, osteogenic, and chondrogenic lineages. Quantitative real-time PCR analysis of genes implicated in stem cell maintenance revealed that CD133+ ESFT cells express significantly higher levels of OCT4 and NANOG than their CD133- counterparts. Taken together, our observations provide the first identification of ESFT cancer stem cells and demonstration of their MSC properties, a critical step towards a better biological understanding and rational therapeutic targeting of these tumors.

Background: The EWS-FLI-1 fusion protein is associated with 85-90% of Ewing's sarcoma family tumors (ESFT), the remaining 10-15% of cases expressing chimeric genes encoding EWS or FUS fused to one of several ets transcription factor family members, including ERG-1, FEV, ETV1 and ETV6. ESFT are dependent on insulin-like growth factor-1 (IGF-1) for growth and survival and recent evidence suggests that mesenchymal progenitor/stem cells constitute a candidate ESFT origin., Methodology/principal Findings: To address the functional relatedness between ESFT-associated fusion proteins, we compared mouse progenitor cell (MPC) permissiveness for EWS-FLI-1, EWS-ERG and FUS-ERG expression and assessed the corresponding expression profile changes. Whereas all MPC isolates tested could stably express EWS-FLI-1, only some sustained stable EWS-ERG expression and none could express FUS-ERG for more than 3-5 days. Only 14% and 4% of the total number of genes that were respectively induced and repressed in MPCs by the three fusion proteins were shared. However, all three fusion proteins, but neither FLI-1 nor ERG-1 alone, activated the IGF1 promoter and induced IGF1 expression., Conclusion/significance: Whereas expression of different ESFT-associated fusion proteins may require distinct cellular microenvironments and induce transcriptome changes of limited similarity, IGF1 induction may provide one common mechanism for their implication in ESFT pathogenesis.

Ewing's sarcoma family tumors (ESFT) express the EWS-FLI-1 fusion gene generated by the chromosomal translocation t(11;22)(q24;q12). Expression of the EWS-FLI-1 fusion protein in a permissive cellular environment is believed to play a key role in ESFT pathogenesis. However, EWS-FLI-1 induces growth arrest or apoptosis in differentiated primary cells, and the identity of permissive primary human cells that can support its expression and function has until now remained elusive. Here we show that expression of EWS-FLI-1 in human mesenchymal stem cells (hMSC) is not only stably maintained without inhibiting proliferation but also induces a gene expression profile bearing striking similarity to that of ESFT, including genes that are among the highest ESFT discriminators. Expression of EWS-FLI-1 in hMSCs may recapitulate the initial steps of Ewing's sarcoma development, allowing identification of genes that play an important role early in its pathogenesis. Among relevant candidate transcripts induced by EWS-FLI-1 in hMSCs, we found the polycomb group gene EZH2, which we show to play a critical role in Ewing's sarcoma growth. These observations are consistent with our recent findings using mouse mesenchymal progenitor cells and provide compelling evidence that hMSCs are candidate cells of origin of ESFT.

A subset of sarcomas is associated with specific chromosomal translocations that give rise to fusion genes believed to participate in transformation and oncogenesis. Identification of the primary cell environment that provides permissiveness for the oncogenic potential of these fusion genes is essential to understand sarcoma pathogenesis. We have recently shown that expression of the EWS-FLI-1 fusion protein in primary mesenchymal progenitor cells (MPCs) suffices to develop Ewing's sarcoma-like tumors in mice. Because most sarcomas bearing unique chromosomal translocations are believed to originate from common progenitor cells, and because MPCs populate most organs, we expressed the sarcoma-associated fusion proteins FUS/TLS-CHOP, EWS-ATF1, and SYT-SSX1 in MPCs and tested the tumorigenic potential of these cells in vivo. Whereas expression of EWS-ATF1 and SYT-SSX1 failed to transform MPCs, FUS-CHOP-expressing cells formed tumors resembling human myxoid liposarcoma. Transcription profile analysis of these tumors revealed induction of transcripts known to be associated with myxoid liposarcoma and novel candidate genes, including PDGFA, whose expression was confirmed in human tumor samples. MPC(FUS-CHOP) and the previously described MPC(EWS-FLI-1) tumors displayed distinct transcription profiles, consistent with the different target gene repertoires of their respective fusion proteins. Unexpectedly, a set of genes implicated in cell survival and adhesion displayed similar behavior in the two tumors, suggesting events that may be common to primary MPC transformation. Taken together, our observations suggest that expression of FUS-CHOP may be the initiating event in myxoid liposarcoma pathogenesis, and that MPCs may constitute one cell type from which these tumors originate.

Ewing's sarcoma is a member of Ewing's family tumors (EFTs) and the second most common solid bone and soft tissue malignancy of children and young adults. It is associated in 85% of cases with the t(11;22)(q24:q12) chromosomal translocation that generates fusion of the 5' segment of the EWS gene with the 3' segment of the ETS family gene FLI-1. The EWS-FLI-1 fusion protein behaves as an aberrant transcriptional activator and is believed to contribute to EFT development. However, EWS-FLI-1 induces growth arrest and apoptosis in normal fibroblasts, and primary cells that are permissive for its putative oncogenic properties have not been discovered, hampering basic understanding of EFT biology. Here, we show that EWS-FLI-1 alone can transform primary bone marrow-derived mesenchymal progenitor cells and generate tumors that display hallmarks of Ewing's sarcoma, including a small round cell phenotype, expression of EFT-associated markers, insulin like growth factor-I dependence, and induction or repression of numerous EWS-FLI-1 target genes. These observations provide the first identification of candidate primary cells from which EFTs originate and suggest that EWS-FLI-1 expression may constitute the initiating event in EFT pathogenesis.

The present study reports the modulation of basement membrane (BM) components, laminin, entactin, and type IV collagen, expression in prepubertal rat Sertoli cell by the thyroid hormone T3. Immunocytochemical studies of permeabilized Sertoli cells in culture showed that T3 treatment (10[-7] M for 24 h) increased the number of cells staining positive for laminin and/or entactin (from 58 +/- 5.3% to 86.4 +/- 6.5%, P < 0.01). In contrast, a strong inhibition of type IV collagen immunopositivity was observed. Western blot analysis of Sertoli cell-conditioned media indicated that T3 treatment significantly (P < 0.01) increased the level of secreted entactin by 60-65% without affecting the levels of laminin A and B1/B2 chains. Moreover, thyroid hormone treatment of Sertoli cells significantly reduced type IV collagen secretion by 62% (P < 0.05). Slot blot analysis of poly-A RNA demonstrated a significant (P < 0.01) increase in the level of entactin messenger RNA (mRNA) by 140% (P < 0.01) and a 50% reduction of type IV collagen alpha1 chain mRNA after thyroid hormone treatment. No effect of the hormone was observed on the accumulation of the laminin B1 and B2 chain mRNAs in Sertoli cell cultures. These effects cannot be ascribed to changes in the degradation of BM components, because no effect of thyroid hormone was observed on plasminogen activators or metalloproteinase secretion by Sertoli cells. These observations indicate the Sertoli cell as a source of entactin within the testis, demonstrate the ability of T3 to differentially regulate the expression of BM components, and can be regarded as a part of the integrated mechanism by which thyroid hormone affects testicular development and differentiation.

Kurloff cells may represent a major component of NK cell activity in the guinea pig. We have pursued to characterize the mechanism of their action. Using murine target cells, we found Kurloff cell cytotoxicity to be selective for the NK-sensitive YAC-1 target cell, with minimal activity against the NK-resistant P815 target cell. In the presence of PHA, but not ConA, cytotoxicity was markedly augmented against both YAC-1 and P815. While effector-target conjugate formation was observed with YAC-1 cells but not P815 cells in control cultures, it was augmented with both target cell types in cultures with PHA. Pretreatment alone with PHA was ineffective, however. NK cell activity of Kurloff cells was dependent on extracellular Ca++ and entry of Ca++ into the effector cells, as demonstrated by abrogation of cytotoxicity when extracellular Ca++ was chelated with EDTA or EGTA, or following treatment with the Ca++ channel blockers verapamil and diltiazem. Furthermore, inhibition of PKC by H7 resulted in significant reduction of Kurloff cell-mediated NK activity, while pretreatment of effector cells with the PKC activator TPA enhanced NK activity. Kurloff cells could also be stimulated to produce serine esterases by contact with target cells or treatment with phorbol ester and ionophore. Finally, a majority of Kurloff cells, identified by the monoclonal antibody 14D1, reacted with the human NK cell marker CD56. Taken together, these data suggest that Kurloff cells have NK-like characteristics and activity, with target cell selectivity, and that their lytic mechanisms involve influx of extracellular Ca++, PKC activation and serine esterase production.

In the present paper we have shown evidence for a significant increase of type II sPLA2 activity in A-LAK cells. The A-LAK-mediated cytotoxicity against YAC-1 target cells was strongly inhibited by two inhibitors of sPLA2, p-BPB and mepacrine, suggesting the involvement of this enzyme in the lytic mechanism of A-LAK. On the other hand, stimuli such as A23187 ionophore and TPA, which were able to induce in control cells an increased AA release, failed to cause this effect in IL-2-treated cells, suggesting that PLA2 was not active in these cells. Thus, we analyzed the levels of calpactin I, which is considered to be involved in the down-regulation of PLA2 activity. HrIL-2 treatment led to an increased expression of calpactin I at both the RNA and the protein level. A substantial portion of calpactin I was associated with the external surface of A-LAK and was able to exert a strong inhibitory effect on a purified porcine pancreatic PLA2 activity in vitro. Our results suggest that the role of calpactin I could be relevant to regulate PLA2 activity, and to protect the effector cells against a possible toxic effect which this enzyme could exert if present at high levels.

Arachidonic acid (AA) release was observed following T cell receptor (TCR)/CD3 complex cross-linking in different tumor T cell lines as well as on purified peripheral T cells in vivo. Direct measurement of enzymatic activity in vitro of TCR/CD3-stimulated Jurkat cell extracts on labeled vesicle substrates showed that TCR/CD3 cross-linking resulted in AA release from sn-1,2-diacylglycerol (DAG) vesicles, as detected by TLC analysis, suggesting that DAG lipase was activated following TCR/CD3 stimulation and DAG generation. On the contrary, no phospholipase A2 activation was observed in response to TCR/CD3 stimulation, since no lyso-phospholipids were generated in vitro from either phosphatidylcholine or phosphatidylinositol-3,4-bisphosphate, or from phosphatidic acid vesicles. Moreover, the 1-DAG lipase inhibitor RHC80267 completely blocked TCR/CD3-dependent AA release in vitro and in vivo, without effect upon TCR/CD3-dependent inositol-1,4,5-trisphosphate (IP3) generation. Importantly, evidence for further metabolism of released AA was obtained, since synthesis and release of cysteinyl leukotrienes (CLT), but not of leukotriene B4 or cyclooxygenase products, could be detected by radioimmunoassay in different T cell lines and peripheral blood T cells following TCR/CD3 cross-linking. Moreover, HPLC analysis revealed an accumulation of leukotriene E4 in TCR/CD3 stimulated Jurkat cells. This was associated with translocation of 5-lipoxygenase from the cytosol to the cell membranes. Finally, TCR/CD3-mediated CLT production was blocked by MK886, a specific inhibitor of 5-LO translocation and activation. Our data help define a further level in the fate of second messengers generated after TCR/CD3 triggering and suggest that additional mediators can play a role in the context of T cell activation.

Strong and increasing evidence shows that nitric oxide (NO) contributes to immune function, and in particular to 'non-specific host defense'. The aim of the present review was to focus the current understanding of the role of NO as a biochemical effector of L-arginine-dependent cell-mediated immune responses to neoplastic cells in vitro and in vivo. The cytokine-inducible nitric oxide synthase (NOS) seems to mainly be implicated in the cytotoxic activity of almost all the effector cells involved in tumor cell killing. The cytotoxic actions of NO against tumor cells appear to be related mainly to inhibition of several heme-containing enzymes of the mitochondrial electron transport complex and the citric acid cycle.

Several lines of evidence suggest that nitric oxide (NO), generated through nitric oxide synthase (NOS) by cleavage of terminal guanidino nitrogen from L-arginine, mediates tumor cell killing by mononuclear phagocytes. Natural killer (NK) cells are cytotoxic effector cells that lyse a variety of tumor and virus-infected cells in a MHC-unrestricted manner. NK cells cultured with interleukin 2 proliferate and acquire the ability to lyse a wide range of targets, including NK-resistant tumor cells (LAK activity). The present study was designed to investigate whether a NOS pathway exists in fresh or IL-2-activated NK cells and to assess the importance of NO synthesis in their activation and cytotoxic functions. NKR-P1 triggering, which is known to induce NK cell activation and mediate reverse ADCC, was able to induce arginine metabolism with consequent increase of nitrite and citrulline levels. Moreover, stimulated NO synthesis leads to guanylate cyclase activity with consequent cGMP generation. We also report that cytotoxic activities of fresh or IL-2-activated NK cells appear to be dependent on arginine levels in medium. Tumoricidal activity of both these effector cells, assessed against YAC-1 and P815 target cells, respectively, was indeed significantly reduced when cytotoxic assays were performed in arginine-free medium or in the presence of the L-arginine analog L-N-monomethyl-arginine, which inhibits nitroxide formation from L-arginine. Normal levels of cytotoxic activities could be restored by addition of exogenous L-arginine. NO generation by NK and LAK cells, determined as nitrite, citrulline, and cGMP synthesis, correlated well with their cytotoxic activities. Moreover, NOS activity gradually increased during the LAK generation and correlated well with the increasing capability of IL-2-activated NK cells to lyse NK-resistant targets, such as P815.