Glutamine prevents DMBA-induced squamous cell cancer.

The etiology of oral squamous cell carcinoma has been linked to environmental carcinogens, such as activated aromatic heterocyclic radicals and epoxides. Our previous work on implantable and 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast cancer showed that oral glutamine (GLN) inhibited tumor growth possibly through stimulation of host - and selective inhibition of tumor glutathione (GSH). This finding was associated with up-regulation of NK cell activity, decreased IGF-1 and TGF-beta in the circulation and downregulation of PI-3K/Akt antiapoptotic signaling in tumors. The present study was designed to investigate the effect of topically applied GLN on DMBA-induced hamster buccal pouch squamous cell carcinoma. Histopathological alterations in buccal pouches were studied by light microscopy. GLN and GSH levels in blood and buccal mucosa were determined using specific enzyme assays. The protein expression of bax, bcl-2 and PARP was determined by western blotting. H-ras and p53 genes were examined for presence of mutations using direct DNA sequencing. Fourteen weeks after DMBA application none of the GLN-supplemented animals developed tumors, while all of the control animals had well developed squamous cell carcinomas. The inhibition of DMBA-carcinogenesis by GLN application was associated with increased arterial GLN and GSH, elevated buccal mucosa GSH as well as induction of bax and PARP, and inhibition of bcl-2. H-ras and p53 were wild type. The results from this study in combination with our previous data suggest that the chemopreventive effects of GLN are exerted by enhancing the antioxidant status of the body and activation of apoptosis.

Reduced expression of plakoglobin correlates with adverse outcome in patients with neuroblastoma.

Plakoglobin and its homologue beta-catenin are cytoplasmic proteins that mediate adhesive functions by interacting with cadherin receptors and signaling activities by interacting with transcription factors. It has been suggested that plakoglobin can suppress tumorigenicity whereas beta-catenin can act as an oncogene. We investigated the correlation between the expression pattern of N-cadherin, beta-catenin, and plakoglobin and tumor behavior in primary tumors of 20 neuroblastoma patients of all stages and in 11 human neuroblastoma cell lines. N-cadherin and beta-catenin were detected in 9 of 11 and 11 of 11 cell lines, respectively, whereas plakoglobin was undetectable or severely reduced in 6 of 11 cell lines. Tumor cells from 16 of 20 patients expressed N-cadherin and 20 of 20 patients expressed beta-catenin at levels similar to those of normal ganglion cells. Plakoglobin was undetectable in 9 of 20 tumors. Plakoglobin deficiency in the primary tumors was significantly associated with adverse clinical outcome. Five of the patients with plakoglobin-negative tumors died whereas four patients are alive without evident disease. In contrast, all patients with plakoglobin-positive tumors are alive; 2 of 11 are alive with the disease and 9 of 11 are alive without evident disease. These results suggest that down-regulation of plakoglobin may be of prognostic value for neuroblastoma patients as predictor of poor outcome.

Amplification of immunological functions by subcutaneous injection of intermediate-high dose interleukin-2 for 2 years after autologous stem cell transplantation in children with stage IV neuroblastoma.

BACKGROUND: Immunotherapy given post-autologous stem cell transplantation may eliminate residual tumor cells escaping the conditioning protocol. METHODS: Five children suffering from stage IV neuroblastoma were treated by recombinant interleukin-2 (IL-2) post-autologous peripheral blood stem cell transplantation. The patients' peripheral mononuclear cells were monitored for CD3+ and CD56+ levels, their proliferative response and killing of various cell lines targets. RESULTS: An increase in the level of total lymphocytes, mainly due to expansion of T cells, and enhanced proliferative response to phytohemaglutinin were observed. Elevated cytotoxicity against K562 and neuroblastoma target cells was detected in four patients and against K562 targets in one patient. Toxicity included mild thrombocytopenia, and fever in four patients and mild to moderate encephalopathy which necessitated withdrawing one patient from the protocol. Three of five patients studied are alive today, one of them whose IL-2 was stopped, is in relapse. Two patients have died. CONCLUSIONS: Immunotherapy with s.c. intermediate-high dose IL-2 is feasible and results in expansion of T cells and in stimulation of killing activity against several targets including in some cases, neuroblastoma tumor cells.

Expression of wild-type p53 and Bcl-2 family genes oscillates with recurrent remission and relapse in an unusual case of low-grade lymphoma.

Downregulation of apoptosis has been proposed as a mechanism of clonal expansion in low-grade B cell neoplasms. We have previously described an unusual case of CD5+ B cell lymphoma characterized by cycles of leukemic phase alternating with spontaneous remission. In the present study, we examined the involvement of apoptosis-related proteins in the progression of this cyclic lymphoma ex vivo. During the leukemic phases, the clonal cells were activated blasts expressing elevated levels of wild-type (wt) p53, Bcl-2, Bcl-x(L), and Bax, while Bak expression increased during the decline of lymphocytosis. Bax heterodimerized with Bcl-2 but not with Bcl-x(L). The anti-apoptotic Bcl-2/Bax heterodimers peaked during early leukemic phases and declined during regression. The elevation in Bcl-2, Bcl-x(L) and Bax expression during early leukemic phases seems to result from cell activation since a similar increase was induced by activating the remission phase leukemic cells in culture. The data suggest that wt p53, Bcl-x(L), and Bcl-2/Bax heterodimers support the accumulation of activated leukemic cells during the leukemic phases, while Bax and Bak may be involved in their decline during regression.

Neoplastic cell activation and proliferative response to CD40-ligand characterize recurrent leukemic bouts in an unusual case of low grade lymphoma.

Spontaneous fluctuations in activity of low-grade B cell lymphomas are common but not understood. An explanation may be offered by studying an atypical SLL/CLL case characterized by recurrent cycles of leukemic phase alternating with spontaneous remission (1). During remissions, residual IgMkappa+ leukemic cells exhibited resting phenotype, low proliferative response to CD4O-ligand and delayed apoptosis. In contrast, the acute phase counterparts were phenotypically activated, underwent rapid apoptosis in culture and proliferated extensively in response to membrane-anchored CD40-ligand. Transient bursts of serum TNFalpha and IL-10 preceded the acute phases, which were characterized by the co-existence of CD40-ligand+ T lymphocytes and lymphoma cells in the bone marrow. Based on ex-vivo and in-vitro data, we suggest that changes in the lymphoma milieu affect the neoplastic cell activation status, rate of proliferation in response to activated T cells and rate of apoptosis. These responses may underlie both the induction and spontaneous regression of the acute phases in this unique lymphoma. Our findings raise the possibility that part of this mechanism may have evolved during transformation of indolent common CLL to its more aggressive form.

Biopsia radioquirúrgica mamaria / Radiosurgical biopsy of breast

Se estudiaron prospectivamente las mujeres sometidas a biopsia radioquirúrgica con marcación preoperatoria con alambre fino en el Hospital Base de Valdivia entre julio de 1997 y junio de 1998. La serie corresponde a 21 mujeres, con edad promedio de 48,3 años, en las cuales producto del tamizaje mamográfico se detectaron 13 microcalcificaciones sospechosas (12 agrupadas y 1 ramificada), 5 nódulos espiculados y 3 nódulos asociados a microcalcificaciones. La instalación del alambre fino fue exitosa en todos los casos y en 20 pacientes la lesión mamográfica fue extraída totalmente en la primera resección. El estudio histopatológico reveló carcinoma en 9 (42,9 por ciento) casos y lesiones benignas en 12 (57,1 por ciento). De los 9 carcinomas 7 fueron invasores y 2 in situ. No se registraron complicaciones relacionadas con la marcación preoperatoria con el alambre fino flexible ni con el procedimiento quirúrgico

Thrombin promotes platelet-mediated melanoma cell adhesion to endothelial cells under flow conditions: role of platelet glycoproteins P-selectin and GPIIb-IIIA.

We investigated the role of platelets in human melanoma cell (line 397) interaction with vascular endothelial cells (ECs) under flow conditions. The ability of the tumour cells to adhere to the EC monolayer was significantly reduced by application of flow at a shear rate of 250 s(-1). A 2.2-fold increase in tumour cell adhesion to ECs under flow was observed upon addition of thrombin receptor agonist peptide (TRAP)-activated platelets but not resting platelets. A similar increase (2.5-fold) in tumour cell adhesion to ECs under flow was observed when the tumour cells were incubated with resting platelets on thrombin-treated ECs. However, thrombin treatment of the ECs alone had no effect on tumour cell adhesion in the absence of platelets. The enhancement of tumour cell adhesion to ECs by TRAP-activated platelets was virtually abolished by blockade of the platelet glycoproteins P-selectin and GPIIb-IIIa by monoclonal antibodies. Blockade of P-selectin also inhibited the direct adhesion of TRAP-activated platelets to ECs, but did not affect the interaction of the tumour cells with platelets immobilized on subendothelial extracellular matrix (ECM). Blockade of GPIIb-IIIa inhibited both platelet-EC and platelet-tumor cell interactions. Our results indicate that tumour cell adhesion to the endothelium under flow is enhanced by platelets under conditions that allow platelet adhesion to ECs. Inhibition studies suggest that activated platelet adhesion to ECs is mediated by P-selectin and GPIIb-IIIA, and tumour cell adhesion to EC-bound platelets--mainly by GPIIb-IIIa.

Platelets mediate tumor cell adhesion to the subendothelium under flow conditions: involvement of platelet GPIIb-IIIa and tumor cell alpha(v) integrins.

The aim of our study was to explore the role of platelets and their specific integrin receptors in mediating the interaction of 4 human tumor cell lines (3 melanoma and 1 carcinoma) with the extracellular matrix (ECM) under static and arterial flow conditions. Under static conditions, all 4 cell lines adhered to the ECM. The adhesion capacity of all 4 cell lines was virtually abolished by application of flow during incubation with the ECM. Under static conditions, tumor cell adhesion was not affected by adding platelets to the cell suspension and was slightly reduced by pre-coating the ECM with platelets prior to the addition of tumor cells. In contrast, under flow conditions, platelets significantly increased tumor cell adhesion to the ECM, the enhancing effect being more pronounced when platelets were pre-incubated with the ECM prior to the addition of tumor cells than when incubated simultaneously with the cells. Platelet-mediated tumor cell adhesion under flow was markedly inhibited by blockade of the platelet GPIIb-IIIa or of the tumor cell alpha(v) integrins. Platelets of a Glanzmann thrombastenia (GT) patient were unable to support tumor cell adhesion to the ECM under flow. Our results suggest that the interaction of tumor cells with subendothelium-bound platelets under flow conditions is mediated by platelet GPIIb-IIIa and by tumor cell alpha(v) integrins independently of the nature of the beta subunit.

Cloning of the murine lymphocyte function-associated molecule-1 alpha-subunit and its expression in COS cells.

The lymphocyte function-associated molecule 1 (LFA-1, CD11a/CD18) is an integrin that mediates adhesion of immune cells by interaction with two members of the Ig superfamily, ICAM-1 and ICAM-2. LFA-1 consists of an alpha subunit (Mr = 180,000) and a beta subunit (Mr = 95,000). We report here the isolation and expression of the murine alpha subunit cDNA (GenBank accession no. M60778). The deduced sequence comprises a 1061 amino acid extracellular domain, a 29 amino acid transmembrane region, and a 50 amino acid cytoplasmic domain. It has a 72% amino acid identity with its human counterpart and 34% identity with the murine Mac-1 alpha subunit. The murine LFA-1 alpha subunit could be expressed on the cell surface of a fibroblastoid cell line, COS, by cotransfection with either the human or murine beta subunit cDNA.

Lymphokine-activated killer (LAK) cells: interferon-gamma synergizes with interleukin-2 to induce LAK cytotoxicity in homogeneous leukemic preparations.

Lymphokine-activated killer (LAK) cells are generated by the incubation of lymphocytes with high levels of interleukin-2 (IL-2). We report here that interferon-gamma (IFN-gamma) acts synergistically with low levels of IL-2 to promote LAK differentiation in peripheral blood lymphocytes as well as in homogeneous T acute lymphocytic leukemic cells exhibiting LAK precursor reactivity. No augmentation of LAK response was observed with IFN-alpha-2, IFN-beta-1, and IFN-beta-2/IL-6. The synergism between IL-2 and IFN-gamma was expressed in the ability of activated lymphocytes to lyse natural killer resistant cell line targets and surgically removed melanoma cells. The augmented LAK response due to IFN-gamma does not reflect up-regulation of the high-affinity IL-2 receptors consisting both of alpha and beta subunits, since expression of the alpha (Tac) subunit on the responding leukemic cells was not increased by IFN-gamma. The observed IFN-gamma/IL-2 synergism in the induction of monoclonal LAK precursors suggests that a single precursor cell responds to both IFN-gamma and IL-2 and that different mechanisms underlie the basal IL-2-mediated LAK response and its enhancement by IFN-gamma.

T cell activation: independent induction of killing activity and interleukin 2 secretion in cytolytic hybridomas.

Memory-like cytotoxic T lymphocytes (CTL) hybridomas exhibiting inducible killing activity and IL2 production were used to analyze the anamnestic response of CTL. Four activating agents were examined; anti-Thy-1 monoclonal antibody G7, staphylococcal enterotoxin B, interferon (IFN)-alpha/beta and IFN-gamma. These agents seemed to affect CTL activities in three distinct ways. Anti-Thy-1 monoclonal antibody, like specific antigen, was found to be a potent inducer of specific killing and IL2 production, whereas staphylococcal enterotoxin B induced IL2 production, but not cytolytic activity. On the other hand, IFN-alpha/beta and IFN-gamma effectively stimulated cytotoxicity without inducing IL2 production. The independent triggering of specific killing and IL2 secretion in the monoclonal cytolytic hybridomas suggests that in CTL distinct signals stimulate killing activity and IL2 production. The results also suggest that IFN-alpha/beta and IFN-gamma trigger the cytolytic program through an alternative activation pathway which does not involve the T cell receptor.

Resistance of cytolytic lymphocytes to perforin-mediated killing. Induction of resistance correlates with increase in cytotoxicity.

CTL and NK cells cultured in vitro are known to produce a cytolytic pore-forming protein (PFP, perforin) localized in their cytoplasmic granules. Using purified perforin, we showed here that both cloned CTL and primary killer cell populations, including allospecific CTL, NK/lymphokine-activated killer cells, and MHC-non-restricted CTL, were more resistant to perforin-mediated killing than other lymphocyte populations and cell types. Similar results were obtained with both murine and human cytolytic lymphocyte populations. Resistance of killer cells to perforin correlated in general with their cytolytic capability. Thus, cells that have acquired competence to kill after stimulation with Con A, IL-2, or leukocyte-conditioned medium, were also the more resistant cells. IL-2-independent CTL lines and hybridomas derived in our laboratories could be triggered to become cytotoxic and perforin resistant by short-term stimulation with various cytokines, indicating that the acquisition of resistance to perforin-mediated lysis was independent of cell proliferation. Activation of one IL-2-independent CTL line with IL-2 also resulted in enhanced production of perforin and in enhanced serine esterase activity. The acquisition of cell resistance to perforin by these IL-2-independent cell lines after activation with stimulatory reagents was independent of protein and RNA neosynthesis: emetine, cycloheximide, and actinomycin D, while effectively blocking the incorporation of [35S]methionine into cell proteins, did not affect the induced increase in perforin resistance.

Induction of c-ets and c-fos gene expression upon antigenic stimulation of a T cell hybridoma with inducible cytolytic capacity.

Expression of cellular oncogenes was studied in a T cell hybridoma that undergoes cytolytic activation when stimulated by specific antigen or by anti-Thy-1 antibody. The activation occurs without induction of hybridoma proliferation, providing a model to examine oncogene expression during functional differentiation of lymphocytes. We found that c-fos and c-ets-1 mRNAs were transiently induced at high levels in the hybridoma 30 min and 4 h after stimulation, respectively. c-myc and c-ets-2 oncogenes were constitutively expressed in the hybridoma and their mRNA levels were unaffected during 4 h of stimulation, although c-myc expression was reduced in the later stage of stimulation. Inhibitors of T cell activation, cyclosporin A and anti-LFA-1 antibody, blocked the induction of c-fos and c-ets-1 mRNAs without reducing the levels of c-myc and c-ets-2. The results indicate that the functional activation of the CTL hybridoma is associated with induction of c-fos and c-ets-1 genes.

Interleukin 2 induces human acute lymphocytic leukemia cells to manifest lymphokine-activated-killer (LAK) cytotoxicity.

Lymphokine-activated killer cells (LAK) were originally distinguished from natural killers (NK) and cytotoxic T lymphocytes. Recently, however, IL 2-activated NK cells were suggested as the major source of LAK reactivity in human peripheral blood (PBL). Because certain T cell acute lymphoblastic leukemia (T-ALL) cells are phenotypically similar to LAK precursors, we have asked whether these leukemic cells can be induced toward LAK-cytotoxicity and express NK reactivity before stimulation. Five out of seven T-ALL preparations were induced by IL 2 to kill target cells. The cytotoxicity of the leukemic-LAK cells resembled that of normal LAK effectors as they lysed efficiently the NK-resistant target Daudi, as well as fresh human sarcoma, carcinoma, and renal cancer cells but not normal PBL. The ALL-LAK precursors phenotype was T3-, T4-, T8-, and T11+, similar to most normal LAK precursors. In contrast to normal PBL that generated LAK effectors when their proliferation was inhibited, the irradiated, nonproliferating T-ALL leukemic cells did not respond to IL 2. Therefore, the T-ALL LAK cytotoxicity was attributed to the leukemic cells rather than to residual normal lymphocytes. The IL 2-responding T-ALL cells did not express autonomous NK type cytotoxicity, suggesting that they reflect LAK precursors of non-NK origin. The homogeneous leukemic preparations with inducible LAK cytotoxicity described herein provide a model system for studying normal LAK cells.

Rearrangement and transcription of a T-cell receptor beta-chain gene in different T-cell subsets.

The functionally defined sets of T lymphocytes--helper T cells, cytotoxic T cells, and suppressor T cells--were examined for the possible involvement of a recently identified T-cell receptor beta gene locus in receptor formation. Since gene rearrangements are required for functional gene expression, cloned T-cell lines from each of the groups were surveyed for the expression of unique gene rearrangements. In addition, cell lines that showed gene rearrangements were further tested for the expression of the mature 1.2- to 1.3-kilobase mRNA transcribed from a productive gene rearrangement. The results of such experiments show that helper and cytotoxic T cells may use a common beta chain of the receptor, whereas suppressor cells do so rarely, if at all.

Antigen/mitogen induced cytolytic activity and IL-2 secretion in memory-like CTL-hybridomas.

Memory-like monoclonal CTL hybridomas, derived from fusion of the AKR thymoma BW5147 with secondary CTL generated in vivo or in MLC cultures, have been used to study the mechanism whereby antigen/mitogen induces anamnestic CTL responses. Specifically, we have asked whether induction of cytolytic activity can be promoted by an antigenic/mitogenic signal without involvement of IL-2 receptors, IL-2, or other extrinsic factors. We have found that antigen/lectin alone can trigger the cytolytic potential of the hybridomas and induce IL-2 secretion. Pure IL-2 and conditioned medium were ineffective inducers of cytotoxicity. Moreover, IL-2 receptors were not detected on the hybrid cells before and after antigenic stimulation, demonstrating that expression of IL-2 receptors and induction of specific killing activity are not genetically linked. Non-activated and activated cells conjugated with target cells equally well, suggesting that induction of cytolytic activity involves a post target cell binding step. Close linkage between cytotoxicity and IL-2 secretion has been observed: induction of killing was consistently associated with IL-2 secretion and stimulation of both activities could be blocked by Cyclosporin A. IL-2 was secreted by the CTL hybrids as early as 3 h following stimulation. We propose that the immediate supply of IL-2 by such memory CTL enhances antigenic response of other, IL-2-dependent T cells.

Mechanism of action of Cyclosporin A: inhibition of lymphokine secretion studied with antigen-stimulated T cell hybridomas.

We have employed bifunctional T cell hybridomas, which can be stimulated to secrete lymphokine(s) and lyse specific target cells, to analyze the effect of Cyclosporin A (CsA) on T cell helper and effector functions. We report here the effects of CsA on antigen- and lectin-induced lymphokine secretion. We have found that a pharmacologic level of CsA (10 ng/ml) blocks antigen- and lectin-driven interleukin 2 (IL 2) secretion without affecting cell proliferation. In addition, one monoclonal hybridoma that is induced by concanavalin A to secrete colony stimulating factors (CSF) as well as IL 2 is concomitantly blocked by CsA for production of IL 2 and CSF. Because the hybridomas grow constitutively and are devoid of functional IL 2 receptors, they permit analysis of the kinetics of the inhibitory response. We have shown that CsA blocks not only stimulation of lymphokine secretion but also ongoing IL 2 production, probably by interfering with the effective interaction of receptor and antigen. Thus, blocking of IL 2 secretion from preactivated cells by CsA occurs by 1 to 2 hr, the time required to stop IL 2 production by removal of Ag/Lectin stimulator. The results are consistent with a mechanism of action of CsA on T cells that involves a direct interference of CsA with binding of Ag to Ag-receptor and results in blocking of induction and active secretion of multiple lymphokines.

Memory CTL-hybridoma: a model system to analyze the anamnestic response of cytolytic T lymphocytes.

We have previously described monoclonal CTL-hybridomas growing continuously in culture in the absence of a known antigenic stimulus or growth-promoting factor(s), exhibiting specific anti-H-2Db killing activity in vitro after a distinct 2-hr lag period before the onset of lysis. Here we provide evidence indicating that the hybridoma Md 26.15 represents "memory" CTL, and we examine the mechanism whereby they respond to antigen/mitogen. The brief period (2 to 3 hr) required for stimulation suggests that expression of potentiated effector function(s) after stimulation requires no cell replication. The frequency of effector hybridoma cells capable of specific binding to target cells before and after stimulation is the same, as determined in a direct hybridoma-target cell conjugation test, although cytolytic activity is markedly enhanced. On the other hand, kinetic and cell dispersion assays indicate that the potentiation of cytolytic activity can be attributed to shortening of the lag period of killing as well as to a small enhancement in the recycling ability of cytolytic effectors. We suggest that shortening of the lag period involves activation of potential effectors, which is manifested at a post-binding stage of effector-target interaction. The CTL hybridoma described herein provides a unique model system for studying CTL reactivation.

Blocking by cyclosporine of antigen-induced maturation and lymphokine secretion by cytotoxic T lymphocyte hybridomas.

We have investigated the effect of cyclosporin (Cys) on the maturation of cytotoxic T lymphocytes (CTL), and on the induction of interleukin-2 (IL-2) secretion using two bifunctional CTL hybridomas. Both hybridomas can be stimulated with allogeneic cells to secrete IL-2 and to specifically kill target cells. Cys, at 10-50 ng/ml, eliminates the induction of both functions (secretion and lysis). Although maturation of both specific and lectin-mediated killing by the hybrid cells exhibits high sensitivity to Cys, the actual killing of target cells by previously activated cells is less affected. Our results suggest that pharmacological levels of Cys directly interfere with the antigen-responsiveness of helper-independent cytotoxic T cells, as represented by these hybridomas, and prevent their maturation.