AK-5 Cells Research Articles

The Ischemia-Responsive Protein 94 (Irp94) Activates Dendritic Cells through NK Cell Receptor Protein-2/NK Group 2 Member D (NKR-P2/NKG2D) Leading to Their Maturation

Tumor recognition and killing, the uptake of released immunogenic substrate, and the generation of immunity are crucial aspects of dendritic cell (DC)-mediated antitumor immune response. In the context of direct tumoricidal activity, we have recently shown NK cell receptor protein-2 (NKR-P2)/NK group 2 member D (NKG2D) as a potent activation receptor on rat DCs. The activation of DCs with agonistic anti-NKR-P2 mAb, the binding of soluble NKR-P2 to the AK-5 tumor, and DC maturation with fixed AK-5 cells led us to identify a putative NKR-P2 ligand on the AK-5 cell surface. In this study we have shown that the AK-5 tumor-derived ischemia-responsive protein-94 (Irp94, a 110 kDa Hsp family member) acts as a functional ligand for NKR-P2 on DCs and enhances Irp94-NKR-P2 interaction-dependent tumor cell apoptosis via NO. Surface expression of Irp94 was also found on tumors of diverse origin in addition to AK-5. Furthermore, the Th1-polarizing cytokine IL-12, produced from Irp94-ligated BMDCs, augments NK cell cytotoxicity. Irp94-NKR-P2 interaction drives the maturation of BMDCs by up-regulating MHC class II, CD86, and CD1a and also induces autologous T cell proliferation, which displays a crucial state of DCs for adaptive antitumor immune response. These functional properties of Irp94 reside in the COOH terminus subdomain but not in the NH2 terminus ATPase domain of Irp94. We also show the involvement of PI3K, ERK, protein kinase C, phosphatases, and NF-kappaB translocation as downstream mediators of DCs activation upon NKR-P2 ligation with Irp94. Our studies demonstrate for the first time a novel role of a 110-kDa heat shock protein (Irp94) as a ligand for NKR-P2 on DCs, which in turn executes both innate and adaptive immunity.

Activation of NFκB and Ub-proteasome pathway during apoptosis induced by a serum factor is mediated through the upregulation of the 26S proteasome subunits

We have been investigating differential gene expression associated with apoptosis in AK-5 cells (a spontaneously regressing rat histiocytoma) and have observed catalytic subunits beta 7 and alpha 5 of the 26S proteasome and ubiquitin to be upregulated during apoptosis induced by a variety of agents. The observed elevation in gene expression was parallel to a comparable increase in the cytosolic protein expression of the proteasome and ubiquitin and a markedly amplified increase in the proteasome activity. Inhibition of the increase in gene expression resulted in the inhibition of the rise in the proteasome activity subsequently leading to an inhibition of apoptosis. Similarly, pretreatment with proteasome inhibitors, MG132 and lactacystin, resulted in a significant inhibition of apoptosis pointing to the requirement of a highly active protein degradation machinery during apoptosis. The apoptosis inhibitory effect of the proteasome inhibitors involved an inhibition of the activation of various initiator and effector caspases but was independent of any changes in the mitochondrial membrane depolarization and cytochrome c release associated with apoptosis. Inhibition of proteasome activity or its upstream PI3 kinase activity inhibited NFkappaB translocation thereby suppressing apoptosis, which highlights the requirement of NFkappaB activation for completion of apoptosis in AK-5 cells. Hence, the apoptosis associated induction of the Ub-proteasome pathway components and the proteasome activity suggests that the proteasome, in its capacity as an efficient protein degradation complex, plays an important role in the successful execution of apoptosis.

Differential gene expression during apoptosis induced by a serum factor: Role of mitochondrial F0-F1 ATP synthase complex

The number of genes that are up regulated or down regulated during apoptosis is large and still increasing. In an attempt to characterize differential gene expression during serum factor induced apoptosis in AK-5 cells (a rat histiocytoma), we found subunit 6 and subunit 8 of the transmembrane proton channel and subunit alpha of the catalytic core of the mitochondrial F(0)-F(1) ATP synthase complex to be up regulated during apoptosis. The increase in the expression levels of these subunits was concomitant with a transient increase in the intracellular ATP levels, suggesting that the increase in cellular ATP content is a result of the increase in the expression of ATP synthase subunits' gene and de novo protein synthesis. Depleting the cellular ATP levels with oligomycin inhibited apoptosis significantly, pointing to the requirement of ATP during apoptosis. Caspase 1 and caspase 3 activity and the loss of mitochondrial membrane potential were also inhibited by oligomycin during apoptosis in these cells, suggesting that the oligomycin induced inhibition of apoptosis could be due to inhibition of caspase activity and inhibition of mitochondrial depolarization. However, cytochrome C release during apoptosis was found to be completely independent of intracellular ATP content. Besides the ATP synthase complex genes, other mitochondrial genes like cytochrome C oxidase subunit II and III also showed elevated levels of expression during apoptosis. This kind of a mitochondrial gene expression profile suggests that in AK-5 cells, these genes are upregulated in a time-linked manner to ensure sufficient intracellular ATP levels and an efficient functioning of the mitochondrial respiratory chain for successful completion of the apoptotic pathway.

Role of STAT3 and NFκB signaling in the serum factor-induced apoptosis in AK-5 cells

AK-5, a rat histiocytoma, is rejected in about 70% of the syngeneic animals when injected subcutaneously. The sera from the tumor rejecting animals possess a potent factor, referred to as serum factor (SF) that induces apoptosis in AK-5 tumor cells. In the present study, we show that treatment with SF or JAK/STAT inhibitors AG490 and Piceatannol induces apoptosis to a similar extent in BC-8 (a single cell clone of AK-5) cells. Our results demonstrate downregulation of a transcription factor, STAT3, as a critical regulator of SF-induced apoptosis in BC-8 cells. SF treatment enhanced the activity of NFκB, another transcription factor that regulates both pro- and antiapoptotic genes. The enhanced NFκB activity resulted in the elevation of TRAIL and its receptor DR4, both known to induce apoptosis. Activation of death receptors in turn enhances caspase-8 activity and stimulates the downstream pathways regulating BC-8 cell apoptosis. SF induced apoptosis in BC-8 cells mediated through downregulation of STAT3 and elevated NFκB activity is abrogated by treatment with MAPK inhibitors—PD98059 and SB203580. Our studies therefore indicate that modulation of MAPK activity plays a central role in SF-induced death signaling pathways in BC-8 cells.

Suppression of macrophage function in AK-5 tumor transplanted animals: role of TGF-β1

Transforming growth factor-beta 1 (TGF-β1), a multifunctional cytokine secreted by various cell types has been implicated in diverse physiologic and pathophysiologic functions, including immunological, inflammatory, and neoplastic processes. AK-5 tumor cells when injected intraperitoneally grow as ascites, causing 100% mortality. Peritoneal macrophages cocultured with AK-5 cells, in vitro showed specific suppression of tumoricidal function. We report that AK-5 cells secrete the latent form of TGF-β1, which is converted to its active homodimeric form, causing suppression of the secretion of cytocidal molecule by macrophages thereby inhibiting their cytotoxic capabilities. When macrophages are cocultured with AK-5 cells in the presence of antibodies against TGF-β1 or the receptor II for TGF-β, there is significant recovery in the secretion of nitric oxide and macrophage cytotoxic potential. These results suggest a major role for TGF-β produced by the tumor cells, in the immune escape mechanisms adopted by the AK-5 tumor cells.

Effect of C-terminal deletion of P53 on heat induced CD95 expression and apoptosis in a rat histiocytoma.

Tumor suppressor gene product p53 in its wild-type conformation, is an effector of apoptosis. A rat histiocytic tumor, AK-5 which has a rearranged and mutated p53 gene undergoes apoptosis upon heat shock through surface expression of CD95 receptor. DNA sequence analysis of p53 gene from tumor cells revealed a deletion of 'C' at nucleotide position 942 and an addition of 'A' at position 1055. Deletion of one nucleotide caused premature termination of p53 protein which resulted in shorter p53 protein with an altered sequence from amino acids 315 to 341. Altered p53 was unable to protect BC-8, a single cell clone of AK-5 cells from apoptosis upon heat shock. BC-8 cells transfected with a wild-type p53gene (3B4 cells) were resistant to heat induced apoptosis and did not show the expression CD95 death receptor. Inhibition of p53 expression by using antisense oligo induced apoptosis upon heat shock in 3B4 cells. Similarly, inhibition of CD95 expression by antisense oligo inhibited heat induced apoptosis in BC-8 cells. In addition, cell cycle regulatory molecules, cdc2 and cdk2 are differentially regulated in a non-cell cycle dependent manner in these tumor cells. These results, in view of lack of heat shock response in BC-8 cells suggest a complex interaction between p53, CD95 and hsp70 which determines the fate of the cell. In the absence of functional p53, CD95 appears to be an effector of apoptosis in BC-8 cells.

Target cell induced activation of NK cells in vitro: cytokine production and enhancement of cytotoxic function.

This study examines the effect of fixed AK-5 tumour cells on rat NK cells. Co-culture of NK cells with fixed tumour cells augmented the cytotoxicity of NK cells against NK-sensitive targets, YAC-1 and AK-5, and induced the secretion of IFN-gamma by NK cells. Antibody against IFN-gamma suppressed the anti-tumour activity of NK cells, whereas the addition of T cells during co-culture enhanced this activity. However, macrophages and B cells had no significant effect when present during co-culture with NK cells. All the inducible cytotoxicity was contained within the NK (CD161+) and NKT (CD3+, CD161+) subsets of lymphocytes. However, in the presence of T cells, the cytolytic potential of NKT cells was higher than that of NK cells alone. The augmentation of cytotoxic activity of NK cells by AK-5 cells in presence of T cells was dependent on IL-2 and IFN-gamma secretion. NK cell activation was blocked by specific antibodies to IL-2 and IFN-gamma in the presence of T cells. Interaction between fixed AK-5 cells with NK and T cell populations induced the expression of Fas-L and perforin in NK cells. These data demonstrate that fixed AK-5 cells initiated cytokine synthesis by NK cells, and the enhanced cytotoxic activity in the presence of T cells was induced as a consequence of the products secreted by activated T lymphocytes. The present observations reflect the possible interactions taking place in vivo after the transplantation of AK-5 tumour in animals. They also suggest direct activation of NK cells after their interaction with the tumour cells.

Induction of apoptosis in AK-5 tumor cells by a serum factor from tumor rejecting animals: cytochrome c release independent of Bcl-2 and caspases.

The ability to selectively induce apoptosis in tumor cells is the prime goal in cancer immunotherapy and aims at identifying potential molecular targets, regulating this process. Here we show that the sera from the animals which had spontaneously rejected the AK-5 tumor (a rat histiocytoma) had an effective and potent ability to counteract and kill tumor cells by inducing apoptosis, with a high degree of specificity. Apoptosis induced by the serum factor involved the activation of caspases and cytochrome c release to the cytosol. A reduction in mitochondrial transmembrane potential (Delta psi(m)) occurred considerably later than cytochrome c translocation. The anti-apoptotic protein Bcl-2 and the pancaspase inhibitor zVAD-fmk did not prevent cytochrome c release, but completely blocked the reduction in Delta psi(m), DNA fragmentation and apoptosis. Cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition (MPT) pore had no effect on cytochrome c release and apoptosis mediated by serum factor in AK-5 cells, suggesting that apoptosis was independent of MPT. Taken together these results suggest that the serum factor in conjunction with the immune cells may be participating in the efficient rejection of the tumor in syngeneic hosts and Delta psi(m) disruption but not cytochrome c release, is a critical and decisive event to trigger apoptotic cell death induced by the serum factor in AK-5 tumor cells.

Activated Macrophages Migrate to the Subcutaneous Tumor Site via the Peritoneum: A Novel Route of Cell Trafficking

Spontaneous regression of AK-5 tumor in syngeneic hosts reported earlier involves the interplay of Th1-type cytokines and cell-mediated immunity. Upon subcutaneous transplantation of AK-5 cells, there was accumulation of immune cells in the peritoneum, of which macrophages were the predominant type and were found to be in a hyperactive state. They released macrophage-derived tumoricidal mediators like NO, O−2, and ONOO− which exhibited potent cytotoxic activity against AK-5 cells in vitro. Interestingly, there was a dramatic disappearance of these hyperactive cells from the peritoneal cavity which correlated well with the onset of tumor regression at the subcutaneous site. Direct labeling of these cells in the peritoneum by the tracking dye PKH26 showed their migration to the tumor site. Similarly, frozen tumor sections when scanned under confocal microscope clearly exhibited fluorescent macrophages embedded into the tumor. Immunohistochemical sections of these intratumoral macrophages showed nitrotyrosine residues, indicating their contribution in the free-radical-mediated AK-5 cell death, thereby leading to successful tumor remission. These observations suggest a directional migration of the hyperactivated peritoneal population to the tumor site. We have also confirmed the influx of macrophages and other immune cells into the peritoneum after sc transplantation of Meth A tumor cells in Balb/c mice. Our studies suggest a role for the peritoneal compartment in imparting appropriate stimulus to the immune cells prior to their participation in the antitumor immune response. These studies suggest a novel route of macrophage trafficking via the peritoneum.

Regulation of CD40L expression on natural killer cells by interleukin-12 and interferon gamma: its role in the elicitation of an effective antitumor immune response.

Effector cell functions are regulated by a number of positive signals for the mediation of antitumor immunity. The CD40 and CD40 ligand (CD40L) interaction has been implicated in the generation of effective cell-mediated and humoral immune responses, where cytokines have been shown to play a significant role in the expression of these molecules. Our earlier studies have shown that spontaneous regression of a rat histiocytoma transplanted s.c. is mediated by CD8 + CD3- NK cells. The CD40-CD40L mediation during tumor regression was of interest. Tumor-transplanted animals showed enhanced expression of CD40L on natural killer (NK) and T cells, when compared to cells from normal animals. CD40 expression on AK-5 tumor cells was also induced after s.c. transplantation. Administration of anti-(interleukin-12) (anti-IL-12) and anti-(interferon gamma) (anti-IFNgamma) antibodies in tumor-bearing animals showed down-regulation of the expression of CD40L on NK and T cells with simultaneous inhibition of cytotoxic acitivity of NK cells, cytokine release and the production of antitumor antibody. Naive NK cells, when co-cultured with fixed AK-5 cells, were induced to express CD40L. CD40L expression modulated the immune response exerted by NK cells, in part by the activation of nuclear factor kB (NF-kB). Furthermore, the signaling via CD40L through the use of anti-CD40L antibody promoted the in vitro activation of cytotoxic as well as NF-kappaB binding activity in NK cells from tumor-transplanted animals. These observations demonstrate that the expression of CD40L by the effector cells is regulated by IL-12 and IFNgamma, and could effectively modulate the NK-cell-mediated immune response during the regression of AK-5 tumor.

Interleukin-2-induced nitric oxide synthase and nuclear factor-kappaB activity in activated natural killer cells and the production of interferon-gamma.

We have previously shown that inducible nitric oxide synthase (iNOS) was up-regulated in natural killer (NK) cells when AK-5 tumour cells were transplanted subcutaneously into syngeneic Wistar rats. This study was designed to investigate the role of interleukin (IL)-2 during the induction of iNOS and to understand the subsequent events involved in NK cell activation. There was up-regulation of iNOS expression when naïve NK cells were cultured in the presence of recombinant IL-2. These NK cells produced a higher nitrite content and possessed cytotoxic activity against YAC-1 and AK-5 tumour cells. Induction of iNOS enhanced nuclear factor (NF)-kappaB binding activity in IL-2 activated NK cells, which was confirmed using L-NAME, an NO synthesis inhibitor. Addition of L-NAME along with rIL-2 significantly blocked NF-kappaB activity and also down-regulated the production of NO and the cytotoxic activity of NK cells. Furthermore, injection of anti-IL-2 antibody in subcutaneous tumour transplanted animals abrogated significantly the expression of iNOS and NF-kappaB activity, leading to reduced NO production and cytotoxic activity of NK cells against YAC-1 and AK-5 cells. In addition, the expression of interferon (IFN)-gamma by NK cells was also inhibited in anti-IL-2 antibody injected animals compared with the control animals. Finally, there was enhanced tumour growth and delayed regression in anti-IL-2 injected animals compared with control animals.

Target-cell-induced anergy in natural killer cells: suppression of cytotoxic function.

Our earlier studies have demonstrated that natural killer (NK) cells are the effectors that participate during the spontaneous regression of AK-5 tumour in syngeneic hosts. We have shown that the tumour cells are killed by necrosis and apoptosis. In this study, we have examined the induction of functional anergy in NK cells following coculture with fixed AK-5 tumour cells at high ratio. NK cells, upon coculture with fixed AK-5 cells (1:1 ratio), showed loss of cytotoxic function against both AK-5 (antibody-dependent cell cytotoxicity) as well as YAC-1 targets. The response of these cells to the activation by recombinant interleukin-2 and recombinant interferon gamma was poor. Induction of tumour necrosis factor alpha (TNFalpha) secretion was observed after coculture of NK cells with fixed AK-5 cells. The cocultured cell supernatant inhibited the cytotoxic activity of NK cells, which was partially restored with anti-TNFalpha antibody. In addition, NK cells, after treatment with fixed tumour cells showed overexpression of the Fas receptor. We have also observed induction of apoptosis in cocultured NK cells. These studies suggest that the fixed tumour cells (antigen) at high ratio are able to suppress NK cell function as well as induce death in NK cells.

Induction of Nitric Oxide Production by Natural Killer Cells: Its Role in Tumor Cell Death

It has been recognized that natural killer (NK) cells destroy AK-5 tumor cells, largely by cytolysis and apoptosis. The objective of this study was to elucidate the existence and the role of nitric oxide (NO) during this killing. The target cell killing ability of NK cells was associated with an increased production of NO with higher expression of inducible nitric oxide synthase. In part, the production of NO was confirmed by significant increase in cell lysis in the presence of l-arginine and attenuation of cell lysis, DNA fragmentation, and apoptosis by Nω-nitro-l-arginine methyl ester (L-NAME). An increased oxidation of intracellularly trapped dichlorofluorescein was observed in NK cells, which was effectively prevented by L-NAME. Exposure of AK-5 cells to chemically generated NO also induced DNA fragmentation in AK-5 cells. Further evidence for the involvement of NO in apoptosis was provided by the inhibition of specific cleavage of PARP and activation of CPP32 by L-NAME. Increased production of NO with simultaneous enhancement of the cytotoxic activity of NK cells from sc tumor-transplanted animals has been implicated in tumor regression when compared to the ip tumor-bearing animals. Overall, these observations suggest an important role for NO during NK cell-mediated apoptosis and lysis of AK-5 cells.

Role of IFN-gamma produced after intraperitoneal transplantation of AK-5 cells in the induction of Fas ligand expression by tumor cells leading to immune evasion.

AK-5 tumor cells expressed Fas-L on their surface after intraperitoneal transplantation in syngeneic animals. Fas-L expression by AK-5 cells is involved in the killing of the effector cells. Thus, the tumor has developed an escape mechanism from immune attack. In the present study, we showed that Fas-L expression on AK-5 cells is regulated by interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), as injection of antibodies against IFN-gamma downregulated the expression of Fas-L by tumor cells as determined by immunostaining and Northern hybridizations. Fas-L present on the tumor cells is biologically functional, as it induced DNA fragmentation in Fas+ YAC-1 cells. We have also shown shedding of Fas-L in cell-free ascitic fluid from tumor-bearing animals. These observations suggest that such cytokines as IFN-gamma and TNF-alpha play an important role in regulating the expression of Fas-L by AK-5 cells.

Interaction between B.7 and CD28 costimulatory molecules is essential for the activation of effector function mediating spontaneous tumour regression.

The spontaneous regression of a rat histiocytoma, AK-5, is mediated by activated natural killer cells through antibody-dependent cellular cytotoxicity. In addition to the Fc-FcR interaction between the target and the effector cells demonstrated previously, we show the participation of costimulatory molecules B7 and CD28 in the efficient killing of the tumour cell. Blockade of the costimulatory interaction in vivo using anti-CD28 led to increased tumour growth and a suppressed cytokine response. Anti-CD28 antibody administration in vivo also diminished the cytotoxic potential of NK cells against AK-5 cells in vitro. Our studies also demonstrate the expression of B7.1 and B7.2 antigen on AK-5 tumour cells. The cytotoxic activity of natural killer cells was significantly inhibited when the effector/target cells were cultured in the presence of antibodies raised against B7.1, B7.2 and CD28. Administration of anti-CD28 in vivo also affected the efficiency of the formation of effector/target conjugates in vitro. Similarly, anti-CD28 injections affected expression of the adhesion molecules LFA 1 and ICAM 1 by splenocytes. Administration of anti-B7.1 and B7. 2 antibodies in AK-5 tumour-bearing animals showed a differential response. The cytotoxicity of natural killer cells was significantly inhibited after anti-B7.2 administration, suggesting the preferential participation of B7.2 molecules in vivo. These observations suggest an important role for B7-CD28 interaction in AK-5 tumour regression.

Antitumor activity of curcumin is mediated through the induction of apoptosis in AK-5 tumor cells

Curcumin, the yellow pigment of turmeric ( Curcuma longa), used commonly as a spice, has been shown to possess anticarcinogenic activity. Curcumin inhibited AK-5 tumor growth and induced apoptosis in AK-5 cells. Curcumin induced apoptosis is mediated through the activation of caspase-3, which is specifically inhibited by the tetrapeptide Ac-DEVD-CHO. In addition, curcumin induced tumor cell death is caused through the generation of reactive oxygen intermediates which is inhibited by N-acetyl- l-cysteine. Our studies suggest that the apoptotic process induced by curcumin is the mechanism mediating AK-5 tumor cell death.

Effect of cis-unsaturated fatty acids on the activity of protein kinases and protein phosphorylation in macrophage tumor (AK-5) cells in vitro

Cis-unsaturated fatty acids (c-UFAs) have been shown to be capable of decreasing the survival of macrophage tumor (AK-5) cells in vitro. This cytotoxic action of c-UFAs was found to be associated with an increase in free radical generation and lipid peroxidation process and a simultaneous decrease in cellular anti-oxidants such as superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione reductase, glutathione and vitamin E. In the present study, it was observed that c-UFAs such as gamma linolenic acid (GLA), arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can activate phospholipase C (PLC) and enhance diacylglycerol formation; all the fatty acids except alpha linolenic acid (ALA) increased the binding of phorbol dibutyrate acetate (PDBu) suggesting translocation of protein kinase C (PKC) and at the same time these fatty acids (especially GLA, AA, EPA and DHA) also enhanced PKC activity. AA, EPA and DHA decreased the activity of protein kinase A (PKA) both in the cytosol and particulate fractions whereas ALA and GLA enhanced the PKA activity in the particulate fractions; all the fatty acids except ALA reduced cyclic AMP levels and an enhanced phosphorylation of about 13 proteins of the nuclear fraction and about eight proteins of the plasma membrane fraction was noted in c-UFA treated AK-5 cells in vitro. These results suggest that c-UFAs can alter the activities of second messenger systems such as diacylglycerol and protein kinases and can phosphorylate both plasma membrane and nuclear proteins which are likely to be components of NADPH oxidase. Based on these results, it is suggested that fatty acids may mediate their cytotoxic action in part by modulating the expression of PKC. Activated PKC may then intensify the pro-oxidant state by augmenting NADPH oxidase, so inducing superoxide anion generation which may ultimately lead to cytolysis.

Depletion of the Natural Killer Cell Population in the Peritoneum by AK-5 Tumor Cells Overexpressing Fas-Ligand: A Mechanism of Immune Evasion

AK-5 tumor kills 100% hosts when injected ip and only 20–30% of animals die when the tumor cells are transplanted sc. Seventy percent of animals regress the subcutaneous tumor and exhibit total immunity against subsequent challenges of AK-5 cells by either route. Initially the 100% killing in ip-injected animals was attributed to the rapid growth of the tumor cells in the peritoneum thereby not giving enough time for the host immune system to mount an antitumor response. In the present report we have demonstrated overexpression of Fas-L by day 3 and day 4 ascitic tumor cells which depletes the peritoneum of Fas+lymphocytes. In addition the effector cells from the ascites are ineffective in inducing cytotoxicity against tumor cellsin vitro.However, splenocytes from animals bearing ip tumors possessed cytotoxicity against YAC-1 as well as AK-5 cells. We have also shown the presence of soluble Fas-L in the ascitic fluid, which induced DNA fragmentation in Fas+Jurkat cells. Fas-L present in the tumor cells is able to induce apoptosis in activated lymphocytes and Jurkat cells, suggesting retention of its biological function. These studies implicate Fas-L in the depletion of the effector cell number and inhibition of their functional activity. They also suggest differential regulation of Fas-L expression by the tumor cells depending upon the site of transplantation.

Molecular cloning and characterization of a tumor rejection antigen from rat histiocytoma, AK-5.

We have cloned and sequenced a tumor rejection antigen from a highly immunogenic rat histiocytoma, AK-5. Recombinant antigen induced an antibody response in syngeneic hosts, and the immunized animals showed significant resistance to AK-5 tumor challenge, where growth of the tumor was retarded. Autologous anti-AK-5 antibody, as well as the specific monoclonal antibodies raised against whole AK-5 cells, recognized the recombinant protein. Similarly, recombinant antigen was able to neutralize antitumor antibody in a complement-mediated lysis assay. Antibodies raised against the fusion protein recognized a 60-kDa protein from AK-5 cell extracts. A cDNA sequence analysis revealed significant homology with mouse thymus (81%), lymph node (83%), and human brain (71%) cDNAs; however, the function of these genes is not known. These observations suggest the recombinant protein to be a strong candidate for the tumor rejection antigen that is involved in the spontaneous regression of the AK-5 tumor in syngeneic hosts.

Cytokine-induced production of NO by macrophages induces apoptosis and immunological rejection of AK-5 histiocytic tumor.

The immunological rejection of the AK-5 histiocytoma in syngeneic hosts involves the participation of NK cells and the upregulation of Th1 type cytokine response. The tumor cells are killed by necrosis and apoptosis. We have studied the role of host peritoneal macrophages in tumor regression. Activated macrophages from tumor- bearing animals produce cytokines like IL-1 beta, TNF-alpha, IL-12 and free radicals like nitric oxide during tumor regression. IL-12 and IFN-gamma played a crucial role in the induction of NO production by the host macrophages, since administration of anti IL-12 and anti IFN-gamma antibodies in AK-5 tumor-bearing animals suppressed NO production by the macrophages. Similarly the cytotoxic activity of the host macrophages which is dependent on NO production was also affected in antibody injected animals. These studies indicate an important role for cytokines in the activation of host macrophages which in turn produce nitric oxide that is involved in the induction of apoptosis in AK-5 cells, leading to the regression of the tumor.