The small molecule anti-tumor agent, 5, 6-dimethylxanthenone-4-acetic acid (DMXAA, now called Vadimezan) is a potent macrophage and dendritic cell activating agent that, in the murine system, results in the release of large amounts of cytokines and chemokines. The mechanisms by which this release is mediated have not been fully elucidated. The mitogen-activated protein kinase (MAPK) pathways plays an important role in the regulation of proinflammatory cytokines, such as, TNFα, IL-1β, as well as the responses to extracellular stimuli, such as, lipopolysaccharide (LPS). The results of this study demonstrate that DMXAA activates three members of mitogen-activated protein kinase (MAPK) superfamily, namely p38 MAPK, extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), and c-Jun N-terminal kinases (JNKs) via a RIP2-independent mechanism in murine macrophages. By using selective inhibitors of MAPKs, this study confirms that both activated p38/MK2 pathways and ERK1/2 MAPK play a significant role in regulation of both TNF-α and IL-6 protein production induced by DMXAA at the post-transcriptional level. Our findings also show that Interferon-γ priming can dramatically augment TNF-α protein secretion induced by DMXAA through enhancing activation of multiple MAPKs pathways at the post-transcriptional level. This study expands current knowledge on mechanisms of how DMXAA acts as a potent anti-tumor agent in murine system and also provides useful information for further study on the mechanism of action of this potential anti-tumor compound in human macrophages.
MAPK; post-transcriptional regulation; TNFα; DMXAA; proinflammatory cytokines
Drugs that can rapidly inhibit respiratory infection from influenza or other respiratory pathogens are needed. One approach is to engage primary innate immune defenses against viral infection, such as activating the IFN pathway. In this study, we report that a small, cell-permeable compound called 5,6-di-methylxanthenone-4-acetic acid (DMXAA) can induce protection against vesicular stomatitis virus in vitro and H1N1 influenza A virus in vitro and in vivo through innate immune activation. Using the mouse C10 bronchial epithelial cell line and primary cultures of nasal epithelial cells, we demonstrate DMXAA activates the IFN regulatory factor-3 pathway leading to production of IFN-β and subsequent high-level induction of IFN-β–dependent proteins, such as myxovirus resistance 1 (Mx1) and 2′,5′-oligoadenylate synthetase 1 (OAS1). Mice treated with DMXAA intranasally elevate mRNA/protein expression of Mx1 and OAS1 in the nasal mucosa, trachea, and lung. When challenged intranasally with a lethal dose of H1N1 influenza A virus, DMXAA reduced viral titers in the lungs and protected 80% of mice from death, even when given at 24 hours before infection. These data show that agents, like DMXAA, that can directly activate innate immune pathways, such as the IFN regulatory factor-3/IFN-β system, in respiratory epithelial cells can be used to protect from influenza pneumonia and potentially in other respiratory viral infections. Development of this approach in humans could be valuable for protecting health care professionals and “first responders” in the early stages of viral pandemics or bioterror attacks.
innate immunity; interferon; influenza; pneumonia; bronchial epithelium
Multiple immunotherapy approaches have improved adaptive anti-tumor immune responses in patients with early stage disease; however, results have been less dramatic when treating patients with late stage disease. These blunted responses are likely due to a host of factors, including changes in the tumor microenvironment and systemic immunosuppressive features, which accompany advanced tumor states. We hypothesized that cytoreductive surgery could control these immunosuppressive networks and restore the potency of immunotherapy in advanced disease scenarios.
To test these hypotheses, two representative intratumoral immunotherapies (an adenoviral vector encoding a suicide gene, AdV-tk, or a type-I interferon, Ad.IFNα) were tested in murine models of lung cancer. Cytoreductive surgery was performed following treatment of advanced tumors. Mechanistic underpinnings were investigated using flow cytometry, in vivo leukocyte depletion methods and in vivo tumor neutralization assays.
AdV-tk and Ad.IFNα were effective in treating early lung cancers, but had little anti-tumor effects in late stage cancers. Interestingly, in late stage scenarios, surgical cytoreduction unmasked the anti-tumor potency of both immunotherapeutic approaches. Immune mechanisms that explained restoration in anti-tumor immune responses included increased CD8 T-cell trafficking and reduced myeloid derived suppressor cell populations.
This study demonstrates that surgical resection combined with immunotherapy may be a rational therapeutic option for patients with advanced stage cancer.
Surgical oncology; Immunotherapy; Cancer; Animal model
Nearly 30% of cancer patients undergoing curative surgery succumb to distant recurrent disease. Despite large implications and known differences between primary and recurrent tumors, preclinical adjuvant therapy evaluation frequently occurs only in primary tumors and not recurrent tumors. We hypothesized that well characterized and reproducible models of postoperative systemic recurrences should be used for preclinical evaluation of adjuvant approaches.
We examined traditional animal models of cancer surgery that generate systemic cancer recurrences. We also investigated models of systemic cancer recurrences that incorporate spontaneously metastatic cell lines and surgical resection. For each model, we critiqued feasibility, reproducibility and similarity to human recurrence biology. Using our novel model, we then tested the adjuvant use of a novel systemic inhibitor of TGF-β, 1D11.
Traditional surgical models are confounded by immunologic factors including concomitant immunity and perioperative immunosuppression. A superior preclinical model of postoperative systemic recurrences incorporates spontaneously metastatic cell lines and primary tumor excision. This approach is biologically relevant and readily feasible. Using this model, we discovered that “perioperative” TGF-β blockade has strong anti-tumor effects in the setting of advanced disease that would not be appreciated in primary tumor cell lines or other surgical models.
There are multiple immunologic effects that rendered previous models of postoperative cancer recurrences inadequate. Use of spontaneously metastatic cell lines followed by surgical resection eliminates these confounders, and best resembles the clinical scenario. This preclinical model provides more reliable preclinical information when evaluating new adjuvant therapies.
Surgery; recurrence; models; surgical oncology; concomitant immunity; perioperative immunosuppression; TGF-β
Surgery is the most effective therapy for cancer in the United States, but disease still recurs in more than 40% of patients within 5 years after resection. Chemotherapy is given postoperatively to prevent relapses; however, this approach has had marginal success. After surgery, recurrent tumors depend on rapid neovascular proliferation to deliver nutrients and oxygen. Phosphatidylserine (PS) is exposed on the vascular endothelial cells in the tumor microenvironment but is notably absent on blood vessels in normal tissues. Thus, PS is an attractive target for cancer therapy after surgery. Syngeneic mice bearing TC1 lung cancer tumors were treated with mch1N11 (a novel mouse chimeric monoclonal antibody that targets PS), cisplatin (cis), or combination after surgery. Tumor relapses and disease progression were decreased 90% by combination therapy compared with a 50% response rate for cis alone (P = .02). Mice receiving postoperative mch1N11 had no wound-related complications or added systemic toxicity in comparison to control animals. Mechanistic studies demonstrated that the effects of mch1N11 were associated with a dense infiltration of inflammatory cells, particularly granulocytes. This strategy was independent of the adaptive immune system. Together, these data suggest that vascular-targeted strategies directed against exposed PS may be a powerful adjunct to postoperative chemotherapy in preventing relapses after cancer surgery.
The role of myeloid cells in supporting cancer growth is well established. Most work has focused on myeloid-derived suppressor cells (MDSC) that accumulate in tumor-bearing animals, but tumor-associated neutrophils (TAN) are also known to be capable of augmenting tumor growth. However, little is known about their evolution, phenotype, and relationship to naïve neutrophils (NN) and to the granulocytic fraction of MDSC (G-MDSC).
In the current study, a transcriptomics approach was used in mice to compare these cell types. Our data show that the three populations of neutrophils are significantly different in their mRNA profiles with NN and G-MDSC being more closely related to each other than to TAN. Structural genes and genes related to cell-cytotoxicity (i.e. respiratory burst) were significantly down-regulated in TAN. In contrast, many immune-related genes and pathways, including genes related to the antigen presenting complex (e.g. all six MHC-II complex genes), and cytokines (e.g. TNF-α, IL-1-α/β), were up-regulated in G-MDSC, and further up-regulated in TAN. Thirteen of the 25 chemokines tested were markedly up-regulated in TAN compared to NN, including striking up-regulation of chemoattractants for T/B-cells, neutrophils and macrophages.
This study characterizes different populations of neutrophils related to cancer, pointing out the major differences between TAN and the other neutrophil populations.
The role of chemokines in the pathogenesis of lung cancer has been increasingly appreciated. Monocyte chemoattractant protein–1 (MCP-1, also known as CCL2) is secreted from tumor cells and associated tumor stromal cells. The blockade of CCL2, as mediated by neutralizing antibodies, was shown to reduce tumorigenesis in several solid tumors, but the role of CCL2 in lung cancer remains controversial, with evidence of both protumorigenic and antitumorigenic effects. We evaluated the effects and mechanisms of CCL2 blockade in several animal models of non–small-cell lung cancer (NSCLC). Anti-murine–CCL2 monoclonal antibodies were administered in syngeneic flank and orthotopic models of NSCLC. CCL2 blockade significantly slowed the growth of primary tumors in all models studied, and inhibited lung metastases in a model of spontaneous lung metastases of NSCLC. In contrast to expectations, no significant effect of treatment was evident in the number of tumor-associated macrophages recruited into the tumor after CCL2 blockade. However, a change occurred in the polarization of tumor-associated macrophages to a more antitumor phenotype after CCL2 blockade. This was associated with the activation of cytotoxic CD8+ T lymphocytes (CTLs). The antitumor effects of CCL2 blockade were completely lost in CB-17 severe combined immunodeficient mice or after CD8 T-cell depletion. Our data from NSCLC models show that CCL2 blockade can inhibit the tumor growth of primary and metastatic disease. The mechanisms of CCL2 blockade include an alteration of the tumor macrophage phenotype and the activation of CTLs. Our work supports further evaluation of CCL2 blockade in thoracic malignancies.
tumor immunology; CCL2; lung cancer; mesothelioma; tumor-associated macrophages
Immune surveillance by T helper type 1 (Th1) cells is critical for the host response to tumors and infection, but also contributes to autoimmunity and graft-versus-host disease (GvHD) after transplantation. The inhibitory molecule programmed death ligand-1 (PDL1) has been shown to anergize human Th1 cells, but other mechanisms of PDL1-mediated Th1 inhibition such as the conversion of Th1 cells to a regulatory phenotype have not been well characterized. We hypothesized that PDL1 may cause Th1 cells to manifest differentiation plasticity. Conventional T cells or irradiated K562 myeloid tumor cells overexpressing PDL1 converted TBET+ Th1 cells into FOXP3+ regulatory T cells (TREGS) in vivo, thereby preventing human-into-mouse xenogeneic GvHD (xGvHD). Either blocking PD1 expression on Th1 cells by siRNA targeting or abrogation of PD1 signaling by SHP1/2 pharmacologic inhibition stabilized Th1 cell differentiation during PDL1 challenge and restored the capacity of Th1 cells to mediate lethal xGVHD. PD1 signaling therefore induces human Th1 cells to manifest in vivo plasticity, resulting in a TREG phenotype that severely impairs cell-mediated immunity. Converting human Th1 cells to a regulatory phenotype with PD1 signaling provides a potential way to block GvHD after transplantation. Moreover, because this conversion can be prevented by blocking PD1 expression or pharmacologically inhibiting SHP1/2, this pathway provides a new therapeutic direction for enhancing T cell immunity to cancer and infection.
By inhibiting Nedd8, Tbata suppresses thymic epithelial cell proliferation and thymus size in mice.
Niche availability provided by stromal cells is critical to thymus function. Thymi with diminished function contain fewer stromal cells, whereas thymi with robust function contain proliferating stromal cell populations. Here, we show that the thymus, brain, and testes–associated gene (Tbata; also known as SPATIAL) regulates thymic epithelial cell (TEC) proliferation and thymus size. Tbata is expressed in thymic stromal cells and interacts with the enzyme Uba3, thereby inhibiting the Nedd8 pathway and cell proliferation. Thymi from aged Tbata-deficient mice are larger and contain more dividing TECs than wild-type littermate controls. In addition, thymic reconstitution after bone marrow transplantation occurred more rapidly in Rag2−/−Tbata−/− mice than in Rag2−/−Tbata+/+ littermate controls. These findings suggest that Tbata modulates thymus function by regulating stromal cell proliferation via the Nedd8 pathway.
Since an immuno-inhibitory environment exists within tumors, successful vaccines will likely require additional approaches to alter the tumor microenvironment. Monocyte chemoattractant proteins (such as CCL2) are produced by many tumors and have both direct and indirect immuno-inhibitory effects. We hypothesized that CCL2 blockade would reduce immunosuppression and augment vaccine immunotherapy. Anti-murine-CCL2/CCL12 monoclonal antibodies were administered in three immunotherapy models: one aimed at the HPV-E7 antigen expressed by a non-small cell lung cancer line, one targeted to mesothelin expressed by a mesothelioma cell line, and one using an adenovirus expressing Interferon-α to treat a non-immunogenic, non-small cell lung cancer line. We evaluated the effect of the combination treatment on tumor growth and assessed the mechanism of these changes by evaluating cytotoxic T cells, immunosuppressive cells, and the tumor microenvironment. Administration of anti-CCL2/CCL12 antibodies along with the vaccines markedly augmented efficacy with enhanced reduction in tumor volume and cures of approximately half of the tumors. The combined treatment generated more total intra-tumoral CD8+ T-cells that were more activated and more anti-tumor antigen specific, as measured by tetramer evaluation. Another important potential mechanism was reduction in intratumoral T-regulatory (T-reg) cells. CCL2 appears to be a key proximal cytokine mediating immunosuppression in tumors. Its blockade augments CD8+ T cell immune response to tumors elicited by vaccines via multifactorial mechanisms. These observations suggest that combining CCL2 neutralization with vaccines should be considered in future immunotherapy trials.
CCL2; Cancer immunotherapy; Lung Cancer; Mesothelioma; T-lymphocytes
Vesicular stomatitis virus (VSV) has shown promise as an oncolytic agent, although unmodified VSV can be neurotoxic. To avoid toxicity, a vector was created by introducing the interferon-β (IFN-β) gene (VSV.IFN-β). We conducted this study to determine the ability of VSV.IFN-β to lyse human cancer (mesothelioma) cells and to evaluate the potential of this recombinant virus for clinical translation. Four normal human mesothelial and 12 mesothelioma cell lines were tested for their susceptibility to VSV vectors in vitro. VSV.hIFN-β did not cause cytotoxicity in any normal lines. Only 4 of 12 lines were effectively lysed by VSV.hIFN-β. In the eight resistant lines, pretreatment with IFN-β prevented lysis of cells by VSV.GFP, and VSV infection or addition of IFN-β protein resulted in the upregulation of double-stranded RNA-dependent protein kinase (PKR), myxovirus resistance A (MxA), and 2′,5′-oligo-adenylate-synthetase (2′5′-OAS) mRNA. In the susceptible lines, there was no protection by pretreatment with IFN-β protein and no IFN- or VSV-induced changes in PKR, MxA, and 2′5′-OAS mRNA. This complete lack of IFN responsiveness could be explained by marked downregulation of interferon alpha receptors (IFNARs), p48, and PKR in both the mesothelioma cell lines and primary tumor biopsies screened. Presence of p48 in three tumor samples predicted responsiveness to IFN. Our data indicate that many mesothelioma tumors have partially intact IFN pathways that may affect the efficacy of oncolytic virotherapy. However, it may be feasible to prescreen individual susceptibility to VSV.IFN-β by immunostaining for the presence of p48 protein.
TGF-β blockade significantly slows tumor growth through many mechanisms, including activation of CD8+ T-cells and macrophages. Here, we show that TGF-β blockade also increases neutrophil-attracting chemokines resulting in an influx of CD11b+/Ly6G+ tumor-associated neutrophils (TAN) that are hypersegmented, more cytotoxic to tumor cells, and express higher levels of pro-inflammatory cytokines. Accordingly, following TGF-β blockade, depletion of these neutrophils significantly blunts anti-tumor effects of treatment and reduces CD8+ T-cell activation. In contrast, in control tumors, neutrophil depletion decreases tumor growth and results in more activated CD8+ T-cells intra-tumorally. Together, these data suggest that TGF-β within the tumor microenvironment induces a population of TAN with a pro-tumor phenotype. TGF-β blockade results in the recruitment and activation of TAN with an anti-tumor phenotype.
tumor immunology; immunosuppression; TGFβ; tumor associated macrophages; Tumor associated neutrophils; lung cancer; mesothelioma
Telomere length analysis has been greatly simplified by the quantitative flow cytometry technique flow-FISH. In this method, a fluorescein-labeled synthetic oligonucleotide complementary to the telomere terminal repeat sequence is hybridized to the telomere sequence and the resulting fluorescence measured by flow cytometry. This technique has supplanted the traditional laborious Southern blot telomere length measurement techniques in many laboratories, and allows single cell analysis of telomere length in high-throughput sample formats. Nevertheless, the harsh conditions required for telomere probe annealing (82°C) has made it difficult to successfully combine this technique with simultaneous immunolabeling. Most traditional organic fluorescent probes (i.e. fluorescein, phycoerythrin, etc.) have limited thermal stability and do not survive the high-temperature annealing process, despite efforts to covalently crosslink the antigen-antibody-fluorophore complex. This loss of probe fluorescence has made it difficult to measure flow-FISH in complex lymphocyte populations, and has generally forced investigators to use fluorescent-activated cell sorting to pre-separate their populations, a laborious technique that requires prohibitively large numbers of cells.
In this study, we have substituted quantum dots (nanoparticles) for traditional fluorophores in FISH-flow. Quantum dots were demonstrated to possess much greater thermal stability than traditional low molecular weight and phycobiliprotein fluorophores. Quantum dot antibody conjugates directed against monocyte and T cell antigens were found to retain most of their fluorescence following the high-temperature annealing step, allowing simultaneous fluorescent immunophenotyping and telomere length measurement. Since quantum dots have very narrow emission bandwidths, we were able to analyze multiple quantum dot-antibody conjugates (Qdot 605, 655 and 705) simultaneously with FISH-flow measurement to assess the age-associated decline in telomere length in both human monocytes and T cell subsets. With quantum dot immunolabeling, the mean decrease rate in telomere length for CD4+ cells was calculated at 41.8bp/year, very close to previously reported values using traditional flow-FISH and Southern blotting. This modification to the traditional flow-FISH technique should therefore allow simultaneous fluorescent immunophenotyping and telomere length measurement, permitting complex cell subset-specific analysis in small numbers of cells without the requirement for prior cell sorting.
FISH-flow cytometry; quantum dots; telomere length
Human regulatory T cells inhibit graft-versus-host disease that can occur after tissue transplantation, in part through expression of programmed death ligand 1 and modulation of antigen-presenting cells.
Immunotherapy using regulatory T cells (Treg) has been proposed, yet cellular and molecular mechanisms of human Tregs remain incompletely characterized. Here, we demonstrate that human Tregs promote the generation of myeloid dendritic cells (DC) with reduced capacity to stimulate effector T cell responses. In a model of xenogeneic graft-versus-host disease (GVHD), allogeneic human DC conditioned with Tregs suppressed human T cell activation and completely abrogated posttransplant lethality. Tregs induced programmed death ligand-1 (PD-L1) expression on Treg-conditioned DC; subsequently, Treg-conditioned DC induced PD-L1 expression in vivo on effector T cells. PD-L1 blockade reversed Treg-conditioned DC function in vitro and in vivo, thereby demonstrating that human Tregs can promote immune suppression via DC modulation through PD-L1 up-regulation. This identification of a human Treg downstream cellular effector (DC) and molecular mechanism (PD-L1) will facilitate the rational design of clinical trials to modulate alloreactivity.
Graft-versus-host disease (GVHD) is the most serious complication of bone marrow transplants between individuals (so-called allogenic transplants). The class of suppressor immune cells called regulatory T cells (Tregs) inhibit GVHD by dampening the effects of donor immune cells in the grafted tissue. The cellular and molecular mechanisms involved in this process have not been fully characterized, particularly for human cells. In this study, we report that human Tregs, which we generated from precursor cells ex vivo, express high levels of a cell surface protein called PD-L1 (programmed death ligand-1) that is known to mediate immune suppression. Coculture of these Tregs with allogeneic antigen-presenting cells (APCs), which are known to initiate GVHD, increased, in turn, the amount of PD-L1 on the APCs. The Treg-conditioned APCs were then less able than unconditioned APCs to provoke GVHD in a mouse model of the condition, preventing the death of the animals after transplantation. We found that an antibody against PD-L1 blocked the immunosuppressive effects of Tregs or Treg-conditioned APCs, indicating that this protein is an important part of the molecular mechanism. These findings are potentially important for attempts to modulate immune responses in disease by transplanting T cells into patients.
Locally-produced TGF-β promotes tumor-induced immunosuppression and contributes to resistance to immunotherapy. This paper explores the potential for increased efficacy when combining immunotherapies with TGF-β suppression using the TGF-β type I receptor kinase inhibitor, SM16. Adenovirus expressing IFNβ (Ad.IFNβ) was injected intratumorally once in established subcutaneous AB12 (mesothelioma) and LKR (lung cancer) tumors or intratracheally in a K-ras orthotopic lung tumor model. Mice bearing TC1 (lung cancer) tumors were vaccinated with two injections of adenovirus expressing HPV-E7 (Ad.E7). SM16 was administered orally in formulated chow. Tumor growth was assessed and cytokine-expression and cell populations were measured in tumors and spleens by real time-PCR and flow cytometry. SM16 potentiated the efficacy of both immunotherapies in each of the models and caused changes in the tumor microenvironment. The combination of SM16 and Ad.INFβ increased the number of intratumoral leukocytes (including macrophages, NK cells, and CD8+ cells) and increased the percentage of T-cells expressing the activation marker CD25. SM16 also augmented the anti-tumor effects of Ad.E7 in the TC1 flank tumor model. The combination did not increase HPV-E7 tetramer-positive CD8+ T cells in the spleens, but did induce a marked increase in the tumors. Tumors from SM16-treated mice showed increased mRNA and protein for immunostimulatory cytokines and chemokines, as well as endothelial adhesion molecules, suggesting a mechanism for the increased intratumoral leukocyte trafficking. Blockade of the TGF-β signaling pathway augments the anti-tumor effects of Ad.INFβ immune-activating or Ad.E7 vaccination therapy. The addition of TGF-β blocking agents in clinical trials of immunotherapies may increase efficacy.
tumor immunology; immunosuppression; TGFβ; tumor associated macrophages; cytokines; lung cancer; mesothelioma; tumor vaccine; interferon-β
Adoptive cellular immunotherapy has promise as an approach to eradicate established tumors. However, a significant hurdle in the success of cellular immunotherapy involves recently identified mechanisms of immune suppression on cytotoxic T-cells at the effector phase.
Transforming growth factor-β (TGF-β) is one of the most important of these immunosuppressive factors because it affects both T-cell and macrophage functions. We thus hypothesized that systemic blockade of TGF-β signaling combined with adoptive T-cell transfer would enhance the effectiveness of the therapy.
Flank tumors were generated in mice using the OVA-albumin (OA) expressing thymoma cell line, EG7. Splenocytes from transgenic OT-1 mice (whose CD8 T-cells recognize an immunodominant peptide in OA) were activated in vitro and adoptively transferred into mice bearing large tumors in the presence or absence of an orally available TGF-β receptor-I kinase blocker (SM16).
We observed markedly smaller tumors in the group receiving the combination of SM16 chow and adoptive transfer. Additional investigation revealed that TGF-β receptor blockade increased the persistence of adoptively transferred T-cells in the spleen and lymph nodes, increased numbers of adoptively transferred T-cells within tumors, increased activation of these infiltrating T-cells, and altered the tumor microenvironment with a significant increase in TNF-α and decrease in arginase mRNA expression
We found that systemic blockade of TGF-β receptor activity augmented the anti-tumor activity of adoptively transferred T-cells and may thus be a useful adjunct in future clinical trials.
tumor immunology; immunosuppression; TGFβ; Cytotoxic T-cells; cytokines; adoptive transfer
To investigate the potential role of protein kinase C-δ (PKC-δ) in insulin-like growth factor I receptor (IGF-IR)-mediated cell transformation, an oncogenic gag-IGF-IR β-fusion receptor lacking the entire extracellular domain, which was designated NM1, and a full-length IGF-IR were coexpressed with either wild-type PKC-δ (PKC-δWT) or an ATP-binding mutant of PKC-δ (PKC-δK376R) in NIH 3T3 fibroblasts. While overexpression of PKC-δWT did not affect NM1- and IGF-IR-induced focus and colony formation of NIH 3T3 cells, expression of PKC-δK376R severely impaired these events. In contrast, NM1-mediated cell growth in monolayer was not affected by coexpressing PKC-δK376R. PKC-δWT and PKC-δK376R were constitutively phosphorylated on a tyrosine residue(s) in the NM1- and IGF-IR-expressing cells and were associated with them in an IGF-I-independent manner. Activated IGF-IR was able to phosphorylate purified PKC-δ in vitro and stimulated its kinase activity. Furthermore, the level of endogenous PKC-δ protein was up-regulated through transcriptional activation in response to long-term IGF-IR activation. Taken together, our results demonstrate that PKC-δ plays an important role in IGF-IR-mediated cell transformation, probably via association of the receptor with PKC-δ and its activation through protein up-regulation and tyrosine phosphorylation. Competition with endogenous PKC-δ for NM1 and IGF-IR association by PKC-δK376R is probably an important mechanism underlying the PKC-δK376R-mediated inhibition of cell transformation by NM1 and IGF-IR.