Interleukin 2 and stimulator lymphoblastoid cells will induce human thymocytes to bind and kill K562 targets.

Human thymocytes cultured in the presence of IL-2 and an irradiated B cell line became cytotoxic to K562 target cells. Thymocytes cultured alone or with only IL-2 exhibited almost no killing, but thymocytes cultured in the presence of stimulator cells alone exhibited low levels of cytotoxic activity. Removal of Fc gamma receptor-bearing cells from the activated thymocyte population almost completely abolished the binding and lytic activity. Separation of thymocytes into Fc microns+ and Fc microns-cells before culturing with IL-2 and stimulator cells revealed that only the Fc microns+ subpopulation developed into K562 killer cells. These findings indicate that modulation of Fc microns to Fc gamma receptors on the thymocyte cell surface is part of the maturation process of this particular subset of cytotoxic cells. Morphologically, most of the activated Fc gamma+ K562-binding cells were large, granulated lymphocytes. Only very few of the round, nongranulated small thymocytes were bound to K562 target cells.

Retinoic acid induces the differentiation of B cell hybridomas from patients with common variable immunodeficiency.

Human-human B cell hybridomas constructed from B lymphocytes of common variable immunodeficiency (CVI) patients and the nonsecreting cell line WIL2/729 HF consistently secrete low levels of Ig and appear to retain a defect characteristic of the CVI patient's B cells. We assessed the differentiative capacity of retinoic acid (RA) on these hybridomas, as well as on hybridomas constructed from normal B cells and from patients with selective IgA deficiency. RA at concentrations varying between 10(-5) and 10(-9) M augmented IgM secretion 4-20-fold from four of four CVI hybridomas tested, but did not affect Ig secretion from normal or IgA-deficiency hybridomas. In support of this elevated Ig secretion, RA enhanced the de novo synthesis of biosynthetically labeled light (kappa) and heavy (mu) Ig (up to 4- and 15-fold, respectively) in the CVI hybridoma line JK32.1. The increase in IgM synthesis/secretion could not be accounted for by RA-induced alteration in the cell cycle. In inducing this increase in IgM production, RA was found to affect two aspects of Ig gene expression: (a) the steady-state levels of heavy and light chain mRNAs were enhanced, and (b) the processing of mu heavy chain transcripts to the secreted mRNA form became favored over the membrane mRNA form. We also show that expression of Leu-17 (CD38), a surface marker that is re-expressed in the late pre-plasma stage of B cell development, was increased by RA from less than 20% to greater than 90% of the total cell population, with a concomitant 4-10-fold augmentation in the mean fluorescence intensity. Changes in both Leu-17 expression and de novo Ig synthesis were prominent by 24 h, but could be observed as early as 8 h after induction. Taken together, our study demonstrates that RA affects a marked alteration in the differentiated state of the CVI hybridoma clones. This finding suggests that retinoids can enhance the functional capabilities of B cells with defects in maturation and support further studies to evaluate their clinical potential in CVI.

Human immunodeficiency virus-1 (HIV-1) gp120 superantigen-binding serum antibodies. A host factor in homosexual HIV-1 transmission.

HIV-1 gp120 is an immunoglobulin superantigen which can bind to preimmune serum Ig. We hypothesize that levels of such preimmune antibodies vary in the population and might affect host resistance or susceptibility to viral transmission. This study tests two predictions: (a) levels of preimmune anti-gpl20 Igs are a polymorphic trait; and, (b) these levels are correlated with resistance or susceptibility to HIV-1 transmission. The first prediction was confirmed in a longitudinal study of a low-risk seronegative population. In this group, levels of both endogenous anti-gpl20 IgM and IgG varied widely, but were characteristic and stable for each individual. The second prediction was addressed in a study of participants of the Multicenter AIDS Cohort Study, in which men "susceptible" and "resistant" to HIV infection were identified based on numbers of sexual partners and eventual seroconversion. Specimens consisted of archival sera obtained > 2 yr before seroconversion. Men in the susceptible population (low-risk seroconverters) were distinguished by low levels of anti-gpl20 IgG. We conclude that the level of preimmune anti-gpl20 IgG is a polymorphic population trait, and low levels are a potentially specific and significant factor in homosexual transmission of HIV infection.

Retinoic acid-induced growth inhibition and morphologic differentiation of human neuroblastoma cells in vitro.

Retinoic acid (RA) induced concentration-dependent morphologic differentiation and growth inhibition in the LA-N-1 human neuroblastoma cell line. Time course studies demonstrated a significant increase in the formation of long neurites in LA-N-1 cultures within 48 hours of RA addition; maximum expression of differentiation occurred at approximately 4 days. This differentiation profile corresponded to a detectable decrease in [3H]thymidine incorporation at 48 hours and complete inhibition of cell growth after 3-4 days. The RA-induced morphologic differentiation and growth inhibition persisted despite removal of the drug. A soft agar assay system showed that RA also inhibited the ability of LA-N-1 cells to form anchorage-independent colonies and induced morphologic differentiation in colonies that did develop. These findings suggest that RA promoted the differentiation of LA-N-1 neuroblastoma cells, resulting in an altered expression of the malignant phenotype.

Modulation of Mr 53,000 protein with induction of differentiation of human neuroblastoma cells.

LA-N-5 human neuroblastoma cells were found to express high levels of an Mr 53,000 cellular tumor antigen (p53), a protein that has been implicated as playing a fundamental role in the control of cell division and differentiation processes in a variety of tumor systems. When LA-N-5 cells are treated with retinoic acid, they undergo growth and morphological, biochemical, and electrophysiological changes that are characteristic of neuronal maturation and a reduction of the malignant phenotype. We find that these retinoic acid-induced changes are accompanied by a marked decrease in the levels of p53 and p53 mRNA. In our study, p53 levels decreased in concert with both morphological differentiation and with inhibition of cellular proliferation in vitro. These results suggest that p53 levels are intimately related to an undifferentiated phenotype in neuroblastoma cells and support studies which relate p53 levels to the malignant phenotype in other tumor systems.

Modulation of intracellular cyclic adenosine monophosphate levels and the differentiation response of human neuroblastoma cells.

We have tested the ability of various compounds to raise intracellular cyclic AMP (cAMP) levels and, either alone or in combination with retinoic acid (RA), to promote differentiation of two "RA-resistant" sublines of LA-N-5 human neuroblastoma cells, designated LA-N-5HP and LA-N-5R9. Direct activation of adenylate cyclase by forskolin and cholera toxin increased intracellular cAMP levels over 10-fold in both cell lines after 1 h of treatment, after which the levels slowly declined for the next 16 to 24 h. After 5 days of continuous treatment, cAMP levels still remained 2- to 7-fold elevated above controls and were accompanied by a decrease in cell proliferation and an increase in neurite outgrowth. All these effects were exaggerated when the agents were combined with phosphodiesterase enzyme inhibitors. Increasing cAMP levels (up to 24-fold) with N6,O2'-dibutyryl cyclic AMP (dbcAMP) or 8-bromo-cAMP also resulted in decreased proliferation and an increase in morphological differentiation. Isoproterenol and epinephrine did not alter cAMP levels and had no discernible biological effects. Of the agents that raised cAMP levels, only dbcAMP caused an increase in acetylcholinesterase activity. This effect was duplicated with sodium butyrate and prostaglandin E1 in the absence of an increase in cAMP. RA promoted differentiation but also had little effect on cAMP levels. Combination treatment of cells with RA plus agents that raised cAMP levels resulted in greater degrees of differentiation than seen with single agent treatments. We conclude that: (a) the cAMP synthetic and degradative pathways are functional in LA-N-5HP and LA-N-5R9 cells; (b) elevation of cAMP is sufficient for inhibiting proliferation and promoting neurite outgrowth from these cells, but is not a necessary condition for inducing differentiation; and (c) elevation of intracellular cAMP potentiates the differentiation-inducing activity of RA.

Differentiation of human neuroblastoma by phenylacetate is mediated by peroxisome proliferator-activated receptor gamma.

Phenylacetate (PA) is a member of a class of aromatic fatty acids that has demonstrated antitumor activity in experimental models and in humans. Previous reports have shown that PA and its analogues can act as ligands for the peroxisome proliferator-activated receptor (PPAR) and thereby regulate certain gene expression through peroxisome proliferator response elements. The role of this activity in the antitumor activity of PA has not been determined. To address this question, we have used the human neuroblastoma cell line LA-N-5, which expresses PPARgamma and can be induced to differentiate with PA and with classical PPARgamma ligands. Our results indicated that the PPARgamma ligands 15-deoxy- prostaglandin J2 and GW1929 as well as PA induced LA-N-5 cells to differentiate to a similar phenotype as evidenced by inhibition of cell proliferation, neurite outgrowth, increased acetylcholinesterase activity, and decreased N-myc gene expression. Furthermore, induction with all of the compounds was accompanied by up-regulation of mRNA levels of the nuclear retinoic acid receptor beta (RARbeta) and specific activation of a reporter gene construct (SVbetaRE-CAT) that contains the canonical RA response element located in the RARbeta promoter. All of the assessed functional and molecular effects of PA on LA-N-5 cells, as well as those of the classical PPARgamma ligands, were inhibited by cotreatment with specific PPARgamma antagonists (GW9662 and/or GW0072). Taken together, these studies have confirmed a role for PPARgamma in neuroblastoma cell biology and indicated that the PPARgamma signaling pathway plays a direct role in the PA-induced differentiation response of this cell type.

Pharmacokinetics of chronically administered all-trans-retinoyl-beta-glucuronide in mice.

After the subcutaneous injection of retinoyl beta-glucuronide (RAG), both RAG and retinoic acid (RA), formed by the hydrolysis of RAG in vivo, achieved peak plasma concentrations within 1-2 h. Thereafter, RA was rapidly cleared from the plasma whereas RAG was eliminated much more slowly. No significant changes were noted in the peak (2 h) plasma levels of RAG for treatment periods up to 56 days (one injection of RAG/day), in the clearance rate of RAG from plasma, or in plasma retinol concentrations. Similarly, no consistent decrease in plasma levels of the RA hydrolysis product was observed. Mice undergoing these long-term chronic treatments with RAG did not show any clinical manifestations of retinoid toxicity. Taken together, our findings that chronic dosing with RAG produces sustained levels of both the parent compound and the RA hydrolysis product, combined with the apparent low toxicity of RAG, suggest that RAG could be a safe and useful alternative to some retinoids which are presently being utilized in the clinic.

Identification and quantitation of intracellular retinol and retinoic acid binding proteins in cultured cells.

Although the mechanism whereby vitamin A mediates normal cell differentiation and inhibits tumor cell proliferation is unknown, intracellular receptor-like proteins for retinol and retinoic acid have been implicated in the molecular action of vitamin A. We have assayed these two binding proteins, cellular retinol binding protein (protein R) and cellular retinoic acid binding protein (protein RA), in the cytosolic fraction of various normal and tumor cells via sucrose density gradient centrifugation and saturation analysis. Employing charcoal separation of bound and free tritiated retinoid, the saturation analysis yields an approximate Kd for ligand binding and an estimate of the number of protein R and protein RA molecules per cell. Unique protein R and protein RA macromolecules sedimenting at 2 S with Kd values of 7-42 nM are detected in murine cells (1 degree epidermal, 3T6 fibroblasts and melanoma) and human neuroblastoma cells. Concentrations of the intracellular binding proteins range from 55 000 to 3 000 000 copies per cell. When one cell line (C-127 mouse mammary) is transformed by bovine papilloma virus, protein RA levels increase from undetectable to 193 000 copies per cell. Assessment of growth inhibition by 10(-6) M retinol or retinoic acid in the culture medium reveals that there exists a partial, but not absolute, correlation between the presence of protein R or protein RA and the antiproliferative effect of the particular retinoid in the tested cell lines. We conclude that the 2 S intracellular binding proteins for the retinoids are present in most vitamin A responsive cells, but may not be essential for biologic actions of the vitamin such as growth inhibition in monolayer culture.

Sodium currents during differentiation in a human neuroblastoma cell line.

The electrophysiological properties of a human neuroblastoma cell line, LA-N-5, were studied with the whole-cell configuration of the patch clamp technique before and after the induction of differentiation by retinoic acid, a vitamin A metabolite. Action potentials could be elicited from current clamped cells before the induction of differentiation, suggesting that some neuroblasts of the developing sympathetic nervous system are excitable. The action potential upstroke was carried by a sodium conductance, which was composed of two types of sodium currents, described by their sensitivity to tetrodotoxin (TTX) as TTX sensitive and TTX resistant. TTX-sensitive and TTX-resistant sodium currents were blocked by nanomolar and micromolar concentrations of TTX, respectively. The voltage sensitivity of activation and inactivation of TTX-resistant sodium current is shifted -10 to -30 mV relative to TTX-sensitive sodium current, suggesting that TTX-resistant sodium current could play a role in the initiation of action potentials. TTX-sensitive current comprised greater than 80% of the total sodium current in undifferentiated LA-N-5 cells. The surface density of total sodium current increased from 24.9 to 57.8 microA/microF after cells were induced to differentiate. The increase in total sodium current density was significant (P less than 0.05). The surface density of TTX-resistant sodium current did not change significantly during differentiation, from which we conclude that an increase in TTX-sensitive sodium current underlies the increase in total current.

Novel expression and function of peroxisome proliferator-activated receptor gamma (PPARgamma) in human neuroblastoma cells.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors that has been shown to play a major role in adipocyte and monocyte/macrophage differentiation. Recent work has also suggested a role for PPARgamma in cell cycle control and/or differentiation of other cell types including breast and lung cancer cells. Using reverse transcription-PCR, we now show for the first time that human neuroblastoma (nb) cells express PPARbeta and -gamma, but not -alpha. Using the LA-N-5 nb cell line, we have determined that the natural PPARgamma ligand 15-deoxy-delta prostaglandin J2, as well as the synthetic PPARgamma agonist GW1929, can stimulate the differentiation of nb cells, as evidenced by the inhibition of cell proliferation, neurite outgrowth, increased acetylcholinesterase activity, and the reduction of N-myc expression. We have also demonstrated that PPARgamma is expressed in primary nb and, furthermore, that the expression of this receptor correlates with the maturational stage of the nb cells. Taken together, these studies have implicated a role for PPARgamma in peripheral nerve cell biology and suggest that the PPARgamma signaling pathway is involved in the regulation of nb cell growth and differentiation.

Effects of 13-cis retinoic acid therapy on human antibody responses to defined protein antigens.

We have evaluated the in vivo effects of 13-cis retinoic acid (13-cis RA) on human antibody responses to immunization with tetanus toxoid (TT) and keyhole limpet hemocyanin (KLH). Subjects with severe cystic acne were immunized with suboptimal doses (10 micrograms) of KLH 7 d and 3 months after starting retinoid therapy (13-cis RA, 1 mg/kg/day for 4 mo). A standard booster immunization with TT was given along with the initial KLH sensitization. A control group of acne patients received identical immunization regimens, but no 13-cis RA. Plasma retinoid levels were evaluated by reverse-phase HPLC and confirmed that blood-level concentrations of 13-cis RA and metabolites in these acne patients reached values previously demonstrated to be immunomodulatory in vitro. The retinoid had no effect on responses to TT as reflected by the characteristics of increased anti-TT IgG levels or the isotype distribution of the antibody. In contrast, the anti-KLH response was significantly enhanced in the 13-cis-RA-treated group. Whereas anti-KLH antibody was detected in only 4 of 13 control subjects after the secondary immunization, 10 of 13 retinoid-treated subjects had measurable levels of anti-KLH IgG (p less than 0.05). Among the responders, no differences were noted in the isotype distribution of anti-KLH antibody. These results showing enhanced anti-KLH responses induced by 13-cis RA therapy represent the first demonstration in humans that in vivo administration of a retinoid can modulate antigen-specific immune responses.

Lymphocyte surface markers and cytotoxicity following cryopreservation.

The effects of cryopreservation (CP) on lymphocyte subpopulation distribution and functional activity in blastogenic and cytotoxicity assays were tested. Peripheral blood lymphocytes (PBL) from 12 healthy human donors were obtained by Ficoll-Hypaque separation. Half of each sample was tested fresh, while the other half was cryopreserved and then thawed and tested the same day. Each sample of CP-PBL was compared to fresh PBL from the same donor in simultaneous assays. Following CP there was a significant reduction in the percentage of E, EA gamma, and EA mu rosette-forming cells with a reciprocal increase in EAC rosette-forming cells. The blastogenic response to alloantigens was stable following CP while blastogenesis in unstimulated control cultures was significantly reduced. Mixed lymphocyte culture (MLC)-induced cell-mediated lympholysis (CML) was consistently and significantly diminished by CP. Cytotoxicity in 4 h chromium release NK (K562), ADCC, and LDCC assays was also significantly diminished by CP. In contrast, cytotoxicity was unaffected in an 18 h cytotoxicity assay against adherent cultured melanoma target cells.

Ultrastructural features of a human neuroblastoma cell line treated with retinoic acid.

This report examines the morphological changes that occur in a line of human neuroblastoma cells (LA-N-5) following treatment with retinoic acid, in vitro. The results demonstrate that retinoic acid induces pronounced differentiation of these cells. Perikarya aggregate into tight clusters and extend long processes that are frequently fasciculated. Growth cones appear at the ends of these processes. Transmission electron microscopy reveals that after 10 days of treatment these long neurites give rise to varicosities which contain clusters of large dense-core vesicles and smaller clear vesicles. After 18 days of treatment the cultures cease to differentiate further. The pattern of neurite outgrowth is very complex by this point and the frequency of growth cones and vesicle-containing varicosities is greatly increased compared with shorter treatments. Most of these varicosities contain a mix of large dense-core vesicles and smaller clear vesicles and in some profiles the clear vesicles are round while in others they are pleomorphic. Despite this increase in the number of vesicle-containing profiles no membrane specializations were seen that resemble mature synapses. The present results demonstrate that retinoic acid can produce morphological changes in these cells in culture, and that these changes closely mimic those of normal differentiating neurons in culture. Considered with previous studies, these findings suggest that this cell line might provide a useful model system for studying neural differentiation.

Increased in vivo mitogenicity of anti-TCR/CD3 monoclonal antibody through reduced interaction with Fcgamma receptors.

Since its initial clinical use in 1980, anti-TCR/CD3 monoclonal antibody (mAb) has been shown to be a potent immunosuppressive agent in the prevention of renal allograft rejections. However, toxic side effects caused by release of cytokines, predominantly from activated CD4+ T-cells, remain a major problem with the use of these reagents. Previous work has shown that this activation is mediated via antibody binding to Fcgamma receptors (FcgammaR) on host effector cells. In the present study, we have demonstrated in an in vivo mouse model that the anti-TCR/CD3 mouse mAb 7D6, as well as that from rat (17A2) and hamster (H57-597), induce a gradual depletion of host CD4+ T-cells without any apparent proliferative effects on the cells. In contrast, when treatment with these mAbs was combined with a mAb (2.4G2) that blocks the low-affinity Fcgamma receptors (FcgammaRII/III), we found that the in vivo actions of the anti-TCR/CD3 mAbs resulted in a significant expansion, rather than depletion, of CD4+ cells. The ability of 2.4G2 to reduce mAb 7D6-FcgammaR interaction was directly demonstrated in an in vitro assay system in which 2.4G2 partially suppressed 7D6-mediated T-cell responses. Taken together, our results have shown that some so-called "nonmitogenic" anti-TCR/CD3 mAbs in fact possess potent activating properties and that their mitogenic potential can be exposed by reducing their interaction with FcgammaR on host effector cells.

Effects of retinoic acid on the in vivo growth of human neuroblastoma cells.

We have investigated the effects of retinoic acid (RA) on the development and growth in nude mice of tumors derived from the human neuroblastoma cell line LA-N-5. When cells were treated with 4 x 10(-6) M RA in vitro there was a marked reduction in the number of mice developing tumors when compared to solvent-treated controls. In vivo treatment with RA reduced tumor formation when the retinoid was given for 5 days before tumor injection and continued for 14 days thereafter. In established tumors, RA inhibited progressive tumor growth. There was no demonstrable effect of RA in vivo on the morphologic phenotype of the tumor cells when these regimens were used. We conclude that oral retinoid administration may prove useful in inhibiting or arresting the growth of neuroblastoma, particularly when there is a small initial tumor burden.

Antiproliferative effect of the garlic compound S-allyl cysteine on human neuroblastoma cells in vitro.

A variety of compounds derived from garlic bulbs have been shown in animal systems to possess anticancer properties. However, little information is available regarding the effectiveness of garlic in the prevention or treatment of human cancers. In the current study, we have assessed the ability of S-allyl cysteine (SAC), a derivative of aged garlic extract, to affect the proliferation and differentiation of LA-N-5 human neuroblastoma cells in vitro. Time-and dose-dependent inhibition of cell grow was observed in cultures treated with SAC for at least 2 days, with a half-maximal response at approximately 600 micrograms/ml. SAC treatment was unable to induce differentiation in neuroblastoma cells as assessed by morphological, biochemical and molecular markers. In addition, SAC was unable to potentiate the effects of retinoic acid and 8-bromo-cyclic AMP, agents known to promote differentiation of LA-N-5 cells. Our results indicate that SAC can inhibit human neuroblastoma cell growth in vitro. However, the apparent inability of this compound to induce differentiation may limit its therapeutic potential.

Interferon-gamma and retinoic acid down-regulate N-myc in neuroblastoma through complementary mechanisms of action.

The N-myc oncogene plays a key role in the biology of neuroblastoma and the differentiation process. N-myc expression is associated with metastatic disease, as well as the undifferentiated state of normal neuroblasts migrating from the neural crest during embryogenesis. Its down-regulation is a pivotal event in the differentiation of neuroblastoma cells by retinoic acid (RA). Our previous work has shown that RA works synergistically with other agents, such as interferon-gamma (IFN-gamma), to down-regulate N-myc expression and induce differentiation. The present study demonstrates that IFN-gamma, like RA, decreases N-myc transcription. However, functional analysis of N-myc upstream regulatory sequences using 5' deletion mutants of a promoter-CAT construct containing germ line sequences from nucleotide position -887 to +151 showed that IFN-gamma and RA act through different sites on the N-myc promoter. In addition to its transcriptional effect, IFN-gamma was also found to shorten the half-life of N-myc mRNA. Taken together, these findings provide a mechanistic basis for the synergistic action of IFN-gamma and RA in inducing neuroblastoma differentiation and a rationale for the possible development of combination differentiation therapy for clinical use.

Human neuroblastoma cell lines as models for the in vitro study of neoplastic and neuronal cell differentiation.

Neuroblastoma is a childhood solid tumor composed of primitive cells derived from precursors of the autonomic nervous system. This neoplasm has the highest rate of spontaneous regression of all cancer types and has been noted to undergo spontaneous and chemically induced differentiation into elements resembling mature nervous tissue. As such, neuroblastoma has been a prime model system for the study of neuronal differentiation and the process of cancer cell maturation. In this paper we review those agents that have been described to induce the differentiation of neuroblastoma, with an emphasis on the effects and possible mechanisms of action of a group of related compounds, the retinoids. With this model system and the availability of subclones that are both responsive and resistant to chemically induced differentiation, fundamental questions regarding the mechanisms and processes underlying cell maturation have become more amenable to in vitro study.

Human monoclonal antibody to a neuroectodermal tumor antigen (OFA-I-2).

Human IgM kappa monoclonal antibody to human tumors of neuroectodermal origin was produced in the spent medium of an Epstein-Barr virus-transformed B-lymphoblastoid cell line, L72. Chemically, the antigen was identified as ganglioside GD2 [Gal NAc beta 1----4 (Neu Ac alpha 2----8 Neu Ac alpha 2---3) Gal beta 1----4 Glc----ceramide]. Twenty-seven mg of pure human IgM were obtained from 10 liters of L72 spent medium using salt and hypotonic precipitation, ultracentrifugation, and Sephacryl-S 300 superfine gel filtration. The monoclonal origin of the antibody was determined by agarose isoelectrofocusing. This human monoclonal antibody may be a particularly useful reagent for immunotherapy trials in cancer patients.