Murine Neuroblastoma Cell Line Research Articles

Interactions of viral proteins with neurotransmitter receptors may protect or destroy neurons.

Virus infection, as a rule, is associated with the development of cytopathic changes in the host cell both in vivo and in vitro. The study of persistent virus infections, however, has led to the recognition that this cytopathic change may evolve slowly and may even be minimal. At the end of the 1970s it became evident that some viruses may alter cell function even without causing any kind of morphological damage to the host cell (Oldstone 1984). During this process some of the differentiated functions of the cell suffered, while the vital functions could remain intact, securing survival of the cell. This phenomenon was first noted in vitro in murine neuroblastoma cell lines exhibiting a disturbance of acetylcholine (ACh) metabolism when persistently infected with lymphocytic choriomeningitis virus (LCMV) (Oldstone et al. 1977). In vivo infection of mice induced disturbance in growth hormone (GH) production and glucose metabolism; however, there were no lytic changes in the GH-producing somatotrophic pituitary cells, although replication of LCMV was restricted to this cell type of the anterior pituitary (Oldstone et al. 1982, 1984; Rodriguez et al. 1983). Another interesting observation emerged from experimental infection of mice with canine distemper virus, which induced severe obesity and other hormonal abnormalities. Cell destruction, however, could not be observed (Lyons et al. 1982).

DR-nm23 expression affects neuroblastoma cell differentiation, integrin expression, and adhesion characteristics.

Nm23 gene family has been associated with metastasis suppression and differentiation. We studied DR-nm23 during neuroblastoma cells differentiation. DR-nm23 expression increased after retinoic acid induction of differentiation in human cell lines SK-N-SH and LAN-5. In several cell lines, overexpression of DR-nm23 was associated with more differentiated phenotypes. SK-N-SH cells increased vimentin expression, increased deposition of collagen type IV, modulated integrin expression, and underwent growth arrest; the murine neuroblastoma cell line N1E-115 showed neurite outgrowth and a striking enhancement of beta1 integrin expression. Up-regulation of beta1 integrin was specifically responsible for the increase in the adhesion to collagen type I-coated plates. Finally, cells overexpressing DR-nm23 were unable to growth in soft agar. In conclusion, DR-nm23 expression is directly involved in differentiation of neuroblastoma cells, and its ability to affects the adhesion to extracellular substrates and to inhibit growth in soft agar suggests an involvement in the metastatic potential of neuroblastoma.

Proteasome activity is critical for the cAMP-induced differentiation of neuroblastoma cells.

1. The ubiquitin-proteasome pathway is involved in a variety of cellular functions in mammalian cells. The role of proteasome, however, in the course of cell differentiation is not well characterized. We hypothesized that proteasome activity might be essential during neuronal cell differentiation. 2. To investigate the role of proteasome during neuronal differentiation, we made use of a murine neuroblastoma cell line (NBP2) that terminally differentiates into mature neurons upon elevation of the intracellular level of adenosine 3',5'-cyclic monophosphate (cAMP). To monitor proteasome activity in NBP2 cells, we integrated an expression cassette for a short-lived green fluorescent protein (d2EGFP) into these cells, which were designated as NBP2-PN25. When NBP2-PN25 cells were treated with a proteasome inhibitor, lactacystin or MG132, a dose-dependent increase in the constitutive levels of d2EGFP expression was detected. 3. We also found that proteasome inhibition by lactacystin during the cAMP-induced differentiation of NBP2-PN25 cells triggered cell death. Both lactacystin and cAMP induction reduced the expression of mRNA for the differentiation-associated genes, such as N-myc and cyclin B1. While cAMP-inducing agents decreased the level of N-myc and cyclin B1 proteins, lactacystin increased the level of these proteins. 4. Our data suggest that a reduced level of N-myc and cyclin B1 proteins is critical to commence differentiation, and this can be blocked by a proteasome inhibitor, leading to cell death. Concomitant induction of differentiation and proteasome inhibition, may, therefore, be potentially useful for the treatment of human neuroblastomas.

A model of bacterial translocation in neuroblastoma-bearing mice

Purpose: The aim of this study was to establish a model of bacterial translocation (BT) in neuroblastoma-bearing mice. Methods: A suspension of 1 × 106 cells of the murine neuroblastoma cell line C1300 was injected subcutaneously into the thighs of 8-week-old female A/J mice, which were then killed after 7, 14, and 21 days. Some of the mice were given 1-μm or 2-μm fluorescein-labeled latex beads in their drinking water for 7 days before being killed. Mesenteric lymph nodes (MLNs) were aseptically removed and cultured for 72 hours at 37°C. Segments of distal ileum were obtained for histologic examination. Samples of venous blood were obtained for laboratory tests. Results: Tumors were found at the injection sites on days 14 and 21 after C1300 injection. Although tumors were not found in 7 days, significantly high number of 1-μm latex beads were detected in MLNs compared with the control, and the number increased with tumor growth. The number of 2-μm latex beads was significantly higher on days 14 and 21. The percentage of mice with MLN cultures positive were significantly higher on day 14, and the percentage increased along with tumor growth. On day 21 after C1300 injection, body weight loss and anemia were observed, and histologic findings of the terminal ileum showed mucosal edema and villous thinning. Serum levels of interleukin (IL)-6 were significantly higher in mice killed 14 and 21 days after injection. Conclusions: The results suggest that BT from the gut to MLNs may occur in neuroblastoma C1300-bearing mice, and it increases along with tumor growth. Even in the early stage of malignancy, particles as small as 1 μm may translocate from the gut to MLNs. J Pediatr Surg 35:1701-1705. Copyright © 2000 by W.B. Saunders Company.

Inhibitory Effect of TNP-470 on Hepatic Metastasis of Mouse Neuroblastoma

TNP-470 is a strong inhibitor of angiogenesis. The present study was designed to determine whether the angiogenesis inhibitor TNP-470 inhibits metastasis of mouse neuroblastoma cells to the liver and thus increases survival. A murine neuroblastoma cell line, C1300, and A/J mice were used in this study. First, to demonstrate the inhibitory effects of TNP-470 on angiogenesis, we quantified the area of angiogenesis on images made with SP-500 image analyzer (Olympus) 7 days after implanting a millipore chamber and compared the areas for the TNP-470-treated mice and control mice. Next, to determine the inhibitory effect of TNP-470 on metastasis of neuroblastoma cells to the liver, we made a murine hepatic metastasis model by implanting C1300 cells (1 x 10(6)) in the spleen of the mice and compared histologic findings, sizes, and weights of the livers of treated mice and control mice 14 days after the beginning of a 7-day infusion of TNP-470 (60 mg/kg). We also compared survival rates using the Kaplan-Meier method. When the angiogenesis inhibitor TNP-470 was infused into mice that received tumor cells, the area of angiogenesis in the TNP-470-treated mice was smaller than that in the control mice (52.5 +/- 6.3 SD vs 94.1 +/- 27.6 mm(2), P < 0.001). After the same treatment in other mice, no histologic evidence of metastasis was found, whereas control mice had countless tumor cell masses. Similarly, the weight of the liver was less in TNP-470-treated mice (0.8 +/- 0.1 g vs 4.5 +/- 0.3 g, P < 0.001). Survival was longer in the TNP-470-treated mice than in controls (80% of treated mice were alive more than 60 days after treatment, whereas all control mice died by Day 20). TNP-470 inhibits metastasis of mouse C1300 neuroblastoma cells to the liver, and thus increases survival. TNP-470 inhibits metastasis by inhibiting angiogenesis.

Mouse peripherin isoforms

Three distinct mRNAs have been shown to be produced by alternative splicing from the unique mouse peripherin gene. They generate three translation products, one major form, Pe–58, and two minor forms, Pe–56 which possess a shorter C-terminal sequence, and Pe–61 in which an additional sequence has been inserted in the central rod domain (Landon et al., 1989, EMBO J. 8, 1719–1726). In this study, the simultaneous occurrence of multiple transcripts in murine nervous tissues and neuroblastoma cell lines was shown by PCR amplification of fragments overlapping the sites of alternative splicing. Recombinant peripherin isoforms were purified from E. coli expressing full-length cDNAs. Rabbit antisera were raised against synthetic peptides mimicking parts of the two C-terminal sequences and of the inserted sequence of Pe–61 and were immunoadsorbed until they became monoreactive. By western blot analysis, the peripherin isoforms were localised in neuroblastoma NB2a cell lysates and detergent insoluble fractions separated by two-dimensional electrophoresis. In addition, each isoform was resolved into several charge variants. At the cellular level, each antibody decorated the filament array of the NB2a cells, suggesting the participation of the minor peripherin isoforms in the intermediate filament network.

The effects of serum depletion and dexamethasone on growth and differentiation of human neuroblastoma cell lines

Background/Purpose: Neuroblastoma is the most solid common extracranial malignancy in childhood. Despite multimodality treatment, high-risk disease continues to carry a poor prognosis. Glucocorticoids have been shown previously to induce differentiation in murine neuroblastoma cell lines, but no such effect has been documented in human neuroblastoma cells. Glucocorticoids are known to be active in the differentiation process of the neural crest. These studies describe the effects of dexamethasone on 6 human neuroblastoma cell lines.Methods: Dexamethasone was added to cultured neuroblastoma cell lines (LA1-5S, LA1-15N, BE[2]S, BE[2]N, SH-EP-1, SH-SY5Y) maintained in media supplemented with either normal serum or charcoal-depleted serum. Proliferation assays were performed, and flow cytometry was used to assess alterations in cell cycle. Cells were closely monitored for morphological changes with serial phase-contrast microscopy. Immunohistochemistry (3F8, NF-1, TRK-A) of cultured cells was used to evaluate differentiation. Glucocorticoid receptor levels was assessed using immunoblotting.Results: Dexamethasone decreased the rate of cellular proliferation in both standard and charcoal-depleted conditions. Flow cytometry showed a G1 accumulation. Increased expression of the differentiation-associated antigens was found in cells cultured in charcoal-depleted media, and a further augmentation was seen with the addition of dexamethasone. In standard media, dexamethasone had no detectable effect on the expression of these antigens. Glucocorticoid receptor expression was found to be comparable in all cell lines.Conclusions: Human neuroblastoma cells are sensitive to the differentiating effects of dexamethasone in an environment of charcoal-depleted serum. This phenomenon may be caused by the existence of growth and mitogenic factors in serum that are inhibiting differentiation.

Intracellular Ca2+ Increase in Neuro-2A Cells and Rat Astrocytes Following Stimulation of Bradykinin B2 Receptor

Murine neuroblastoma cell line Neuro-2A cells and rat brain astrocytes showed a dose-dependent increase in intracellular Ca2+ in response to bradykinin, when assessed by a single cell image analyzing system. The Ca2+ increase in Neuro-2A cells by bradykinin was also examined by a suspension fluorescent assay using fura-2 loading. The Ca2+ increase in both cases was suppressed by a bradykinin B2 receptor antagonist, Hoe 140, but not by a B1 receptor antagonist, des-Arg-Hoe 140, suggesting that the effect occurred via specific B2 receptor activation. RT-PCR for bradykinin B2 receptor mRNA showed that both Neuro-2A cells and the astrocytes expressed B2 receptor mRNA. Binding of [3H]bradykinin to Neuro-2A cells was assessed, and a specific binding constant of 0.75 nM was determined. Furthermore, the increase in [Ca2+]i by bradykinin could be caused by a release of Ca2+ from storage sites in the endoplasmic reticulum, since thapsigargin and U-73122 attenuated the effect of bradykinin in Neuro-2A as well as in astrocytes. These results indicate that both astrocytes and neuroblastoma Neuro-2A cells stimulated by bradykinin could express a bradykinin B2 receptor-mediated intracellular Ca2+ increase leading to signal transduction.

Successful transmission of three mouse-adapted scrapie strains to murine neuroblastoma cell lines overexpressing wild-type mouse prion protein.

Propagation of the agents responsible for transmissible spongiform encephalopathies (TSEs) in cultured cells has been achieved for only a few cell lines. To establish efficient and versatile models for transmission, we developed neuroblastoma cell lines overexpressing type A mouse prion protein, MoPrP(C)-A, and then tested the susceptibility of the cells to several different mouse-adapted scrapie strains. The transfected cell clones expressed up to sixfold-higher levels of PrP(C) than the untransfected cells. Even after 30 passages, we were able to detect an abnormal proteinase K-resistant form of prion protein, PrP(Sc), in the agent-inoculated PrP-overexpressing cells, while no PrP(Sc) was detectable in the untransfected cells after 3 passages. Production of PrP(Sc) in these cells was also higher and more stable than that seen in scrapie-infected neuroblastoma cells (ScN2a). The transfected cells were susceptible to PrP(Sc)-A strains Chandler, 139A, and 22L but not to PrP(Sc)-B strains 87V and 22A. We further demonstrate the successful transmission of PrP(Sc) from infected cells to other uninfected cells. Our results corroborate the hypothesis that the successful transmission of agents ex vivo depends on both expression levels of host PrP(C) and the sequence of PrP(Sc). This new ex vivo transmission model will facilitate research into the mechanism of host-agent interactions, such as the species barrier and strain diversity, and provides a basis for the development of highly susceptible cell lines that could be used in diagnostic and therapeutic approaches to the TSEs.

Recent progress of the research on novel microbial metabolites

Abstract

Differential responses of staurosporine on protein kinase C activity and proliferation in two murine neuroblastoma cell lines

We studied the effect of staurosporine (SSP), a broad-spectrum protein kinase inhibitor, on the levels of protein kinase C (PKC) activity and proliferation in two murine neuroblastoma cell lines, Neuro2a and its clone NB41A3. The PKC activity was examined in whole cell lysate, cytosolic and particulate fractions. A differential response to SSP treatment in the enzyme activity in whole cell lysate and particulate fractions was demonstrated in the two cell lines. The data on proliferation indicated that Neuro2a cells were more sensitive to the SSP treatment with significant inhibition in DNA synthesis in a time course study. Our findings suggest that the data on basal levels of PKC activity in tumors will be of significance in studies using PKC inhibitors as an approach for therapeutic intervention.

Effects of Retroviral-Mediated Herpes Simplex Virus Thymidine Kinase Gene Transfer to Murine Neuroblastoma Cell Lines In Vitro and In Vivo

Selective introduction of genes conferring chemosensitivity on proliferating tumor cells can be used to treat cancer. We investigated the efficacy of retrovirus-mediated gene transfer of the herpes simplex virus thymidine kinase (HSV-TK) gene to murine neuroblstoma cell lines (neuro-2a) in vitro and in vivo. Retrovirus-mediated HSV-TK gene transfer to the neuro-2a cells resulted in sensitivity to ganciclovir (GCV) in vitro. In A/J mice, tumors produced from HSV-TK transduced neuro-2a cells regressed after GCV treatment. Intratumoral injection of recombinant retrovirus expressing HSV-TK gene also inhibited growth of the tumor established in A/J mice. These results demonstrate that HSV-TK gene therapy might be a feasible approach for inhibiting the growth of neuroblastoma.

Activation of Receptor Gene Transcription Is Required to Maintain Cell Sensitization after Agonist Exposure: STUDY ON NEUROTENSIN RECEPTOR

Neurotensin (NT) acts through specific G protein-coupled receptors to induce effects in the central nervous system and periphery. In this study we have shown that in the human neuroblastoma cell line CHP 212, an NT agonist, JMV 449, induced high affinity neurotensin receptor (NTR) gene activation. 125I-NT binding of cells challenged with JMV 449 rapidly decreased then reappeared and subsequently stabilized at 50% of the control values after 48 h of agonist exposure. These receptors, which reappeared at the cell surface, are as active as those found in control cells as demonstrated by Ca2+ mobilization. Furthermore, the tyrosine hydroxylase (TH) gene, a known NT target gene, remained activated after prolonged NT agonist exposure in this cell line. In the murine neuroblastoma cell line, N1E-115, NT did not stimulate NTR gene activation but induced NTR mRNA destabilization after long term agonist exposure. In this cell line, NT binding dropped to 15% of control values and remained at this value after agonist treatment. The TH expression, which was originally activated upon NT agonist exposure, decreased to control values after prolonged agonist exposure. These observations combined with the data obtained from a complementary study with HT-29 cells (Souazé, F., Rostène, W., and Forgez, P. (1997) J. Biol. Chem. 272, 10087-10094) revealed the crucial role of agonist-induced receptor gene transcription in the maintenance of cell sensitivity. A model for G protein-coupled receptor regulation induced by prolong and intense agonist stimulation is proposed.

Expressional down-regulation of neuronal-type nitric oxide synthase I by glucocorticoids in N1E-115 neuroblastoma cells.

Neuronal-type nitric oxide synthase (NOS I) is involved in ischemia-induced brain damage, and glucocorticoids have been reported to protect from brain damage. This prompted us to investigate if the activity or expression of NOS I was influenced by glucocorticoids. We used the murine neuroblastoma cell line N1E-115 as our experimental model. Short-term incubation (30 min) of the N1E-115 cells with dexamethasone (10 nM to 1 microM) or hydrocortisone (100 nM to 10 microM) did not change the enzymatic activity of NOS I. However, the glucocorticoids inhibited NOS I mRNA expression in a concentration-dependent fashion (down to 53.3 +/- 2. 5% of control). In time-course experiments with 100 nM dexamethasone, maximum down-regulation of NOS I mRNA was seen after 24 hr (55.6 +/- 6.3% of control). Similar effects were seen with 10 microM hydrocortisone. The effect of 100 nM dexamethasone was completely reversed by 1 microM of the glucocorticoid receptor antagonist mifepristone. In experiments with actinomycin D (10 microg/ml), the half-life of the NOS I mRNA was determined to be approximately 12 hr and remained unchanged after glucocorticoid incubation. Nuclear run-on analyses indicated that the decrease in NOS I mRNA was the result of a glucocorticoid-induced inhibition of NOS I gene transcription. In Western blots, the 160-kDa NOS I protein band was down-regulated to 68.5 +/- 8.4% of control after an incubation of the N1E-115 cells with 100 nM dexamethasone for 26 hr. Similarly, NO production was down-regulated to 57.8 +/- 8.7% of control. These data demonstrate that glucocorticoids reduce the expression of NOS I without changing its activity.

Effect of lipopolysaccharide on the gene expression of the enzymes involved in tetrahydrobiopterin de novo biosynthesis in murine neuroblastoma cell line N1E-115

The amounts of messenger RNA for three enzymes, namely guanosine triphosphate (GTP) cyclohydrolase I, 6-pyruvoyltetrahydropterin synthase, and sepiapterin reductase, all of which are involved in the de novo biosynthesis of (6 R)- l- erythro-dihydroxypropyl-2-amino-4-hydroxy-5,6,7,8-tetrahydropteridine (BH 4) from GTP, were measured quantitatively in murine neuroblastoma cell line N1E-115 by the competitive polymerase chain reaction (PCR) technique after reverse transcription using a heterologous DNA fragment as an internal standard. Twenty-four hour activation of this cell line with 1 μg/ml lipopolysaccharide resulted in statistically significant increases in the amounts of the messages of all three enzymes. Our data suggest that lipopolysaccharide can activate the intrinsic pathway resulting in the enhanced gene expression of these three enzymes in neuron-derived cells such as N1E-115.

Effects of 1,25-dihydroxyvitamin D 3 on growth of mouse neuroblastoma cells

Epitopes of the 1,25-dihydroxyvitamin D (1,25(OH) 2D 3) receptor have been shown in developing dorsal root ganglia in fetal mice, as well as in cells maintained in culture [Johnson, J.A., Grande, J.P., Windebank, A.J. and Kumar, R., 1,25-Dihydroxyvitamin D 3 receptors in developing dorsal root ganglia of fetal rats, Dev. Brain Res., 92 (1996) 120–124]. To investigate a possible role for 1,25(OH) 2D 3 in neural cell growth and development, a murine neuroblastoma cell line that expresses 1,25(OH) 2D 3 receptors, was treated with 1,25(OH) 2D 3. Treatment with 1,25(OH) 2D 3 resulted in a decrease in cell proliferation, a change in cell morphology, and the expression of protein markers of mature neuronal cells. The decrease in cell proliferation was accompanied by an increase in the expression of nerve growth factor (NGF). Anti-NGF monoclonal antibody added to the growth medium blocked the decrease in cell proliferation caused by 1,25(OH) 2D 3 treatment. Our results show that the sterol hormone 1,25(OH) 2D 3, causes a decrease in the proliferation of mouse neuroblastoma cells through alterations in the expression of NGF.

TNP-470 antiangiogenic therapy for advanced murine neuroblastoma

The finding that angiogenesis plays an important role in the progression and metastasis of malignant tumors has led to the development of several antiangiogenic drugs. The authors report here an examination of the effect of the antiangiogenic agent TNP-470 on the growth, metastases, and survival of two differing murine neuroblastoma cell lines, TBJ and C1300. We found that TNP-470 significantly reduced primary tumor volumes in mice injected with either cell line. In addition, antiangiogenic therapy significantly reduced the size of axillary lymph node metastases in both groups as well as decreased the size of liver metastases in mice receiving TBJ neuroblastoma. TNP-470 treatment also improved animal survival. These data suggest that antiangiogenic therapy retards the growth of primary and metastatic murine neuroblastoma. We speculate that antiangiogenic therapy may be a useful therapeutic modality in the treatment of advanced neuroblastoma once side effects and appropriate dosage requirements are determined.

Development of resting membrane potentials in differentiating murine neuroblastoma cells (N1E-115) evaluated by flow cytometry.

With the aid of a voltage-sensitive oxonol dye, flow cytometry was used to measure relative changes in resting membrane potential (V(m)) and forward angle light scatter (FALS) profiles of a differentiating/differentiated murine neuroblastoma cell line (N1E-115). Electrophysiological differentiation was characterized by V(m) establishment. The (V(m))-time profile was found to be seed cell concentration-dependent for cell densities of less than 2 × 10(4) cells/cm(2). At higher initial cell densities, under differentiating culture conditions, V(m) development commenced on day 2 and reached a steady-state on day 12. The relative distribution of differentiated cells between low and high FALS has been proposed as a potential culture electrophysiological differentiation state index. These experiments offer a general methodology to characterize cultured excitable cells of nervous system origin, with respect to electrophysiological differentiation. This information is valuable in studies employing neuroblastoma cells as in vitro screening models for safety/hazard evaluation and/or risk assessment of therapeutical and industrial chemicals under development.

Tetrahydrobiopterin biosynthesis enhanced by lipopolysaccharide stimulation in murine neuroblastoma cell line N1E-115.

We investigated for the first time the effect of lipopolysaccharide and the signal transduction pathway on the biosynthesis of tetrahydrobiopterin [(6R-L-erythro-1',2'-dihydroxypropyl) -2-amino-4-hydroxy-5,6,7,8-tetrahydropteridine], the cofactor for the enzymatic hydroxylation of the aromatic amino acids, in the murine neuroblastoma cell line N1E-115, which synthesizes tetrahydrobiopterin constitutively. Activation of N1E-115 cells with 1 microgram/ml lipopolysaccharide resulted in statistically significant increases in both intracellular tetrahydrobiopterin contents and the activity (Vmax) of GTP cyclohydrolase I, a rate-limiting enzyme in tetrahydrobiopterin de novo biosynthesis. Following simultaneous addition of the inhibitors of protein tyrosine kinases and GTP-binding proteins into serum-free culture media with lipopolysaccharide, we analyzed the transduction pathway of lipopolysaccharide signal toward the tetrahydrobiopterin biosynthetic system in N1E-115 cells. Our data indicate the following conclusions: (a) Protein tyrosine kinase systems are involved in mediating lipopoly-saccharide signal to tetrahydrobiopterin production, and (b) there may be a cross-talk between GTP-binding protein and the protein tyrosine kinase system in mediating lipopolysaccharide signal. These observations suggest that a neuronal cell such as N1E-115, which barely expresses CD14 on its cell surface, responds to lipopolysaccharide like macrophages and monocytes in the absence of soluble CD14.

Retrovirus-mediated gene transfer of B7-1 and MHC class II converts a poorly immunogenic neuroblastoma into a highly immunogenic one.

The T cell co-stimulatory molecule B7-1 was transduced into a poorly immunogenic murine neuroblastoma cell line (Neuro-2a, N-2a) alone or in combination with MHC class II genes to test the ability of these genes to stimulate antitumor immunity. N-2a cells transduced with B7-1 exhibited reduced tumorigenicity, whereas N-2a cells overexpressing both MHC class II (syngeneic, I-Ak) and B7-1 totally abrogated tumorigenicity. Rejection of I-Ak/B7-1 cells was dependent on both CD4+ and CD8+ T cells. The ability of both vaccines to induce protection against parental N-2a was temporally dependent on the time of secondary N-2a challenge. To investigate the immunity generated by N-2a/B7-1 and N-2a/I-Ak/B7-1 vaccines, we tested the ability of these modified cells to stimulate in vitro the proliferation of syngeneic splenocytes from naive mice. A significant increase in splenocyte proliferation was observed with N-2a/I-Ak/B7-1 cells compared to N-2a cells. We also determined that vaccination with N-2a/I-Ak/B7-1 cells was able to generate cytotoxic T cell responses to unmodified N-2a cells. The introduction of B7-1 and I-Ak into N-2a was able to convert a poorly immunogenic tumor to a highly immunogenic one; however, mice bearing large established unmodified tumors had little response to vaccination with N-2a/I-Ak/B7-1 cells. Our results emphasize the importance of tumor immunogenicity in the treatment of established tumors with MHC class II/B7-1 tumor cell vaccines.