Characterization of autoantigen (p105) in a model of colonic adenocarcinoma in the rat]

Sera from BDIX rats bearing the syngeneic colon tumors PROb or REGb were analysed by Western blotting in order to detect a possible humoral response against the grafted tumor. The PROb clone grows progressively in syngeneic hosts and metastasizes, whereas the REGb clone grows slowly and then is rejected. This model was developed by F Martin and his group in Dijon, France. We observed that rats bearing PROb tumors only develop an antibody response against a water-soluble protein of 105 kDa (p105) which is expressed by both tumor clones. This antibody response has never been detected in rats bearing REGb tumors. The antigen p105 was also expressed by normal adult colon as well as some other foetal or adult tissues. It is also present in extracts from several tumor cell lines including human colorectal carcinoma cell lines. Moreover, the titer of detected antibody at day 30 was inversely correlated with the survival of rats after tumor inoculation, suggesting a possible facilitating role of this antibody response.     

In vivo and in vitro reactivity of rat spleen cells against regressor and progressor colon-cancer cell variants

Previous reports demonstrated that progressor and regressor tumor-cell variants isolated from the same colon carcinoma chemically induced in a BD-IX rat differed in their capacity to induce an immune response. The present study was aimed at analyzing the characteristics of the responses to the regressor REGb and progressor PROb clones. Spleen cells from rats bearing early REGb tumors neutralized PROb cell tumorigenicity in a Winn-type local transfer assay, but responded occasionally to REGb and PROb cells in vitro. However, spleen cells from rats immunized by several injections of REGb and PROb cells strongly proliferated when cultured with PROb or REGb cells. This response was selective for the cell lines generated from the individual tumor at the origin of PROb and REGb lines, was dependent on CD4⁺ spleen cells, and was partially inhibited by an antibody against major histocompatibility complex class-II molecules. Although PROb cells shared tumor-rejection antigen(s) with REGb cells, splenocytes from PROb tumor-bearing rats did not neutralize PROb-cell tumorigenicity in vivo, nor did they proliferate when cultured with PROb or REGb cells in vitro. The unresponsiveness of spleen cells from PROb tumor-bearing rats was not due to the presence of immune suppressive cells, and a defect of antigen-presenting cells was shown to be unlikely. This unresponsiveness was limited to a lymphocyte subpopulation, since spleen cells from tumor-bearing rats responded normally to stimulation by PHA or allogeneic lymphocytes. These results strongly suggest that unresponsiveness of spleen cells from tumor-bearing rats is due to a tumor-specific anergy of the lymphocyte clones able to respond to tumorassociated antigens.     

In vitro proliferative responses of spleen lymphocytes from rats bearing progressive or regressive tumors induced by cell variants of a syngeneic colon carcinoma

From one colonic carcinoma chemically induced in the rat, 2 sublines of tumor cells have been cloned, one (PROb) inducing progressive tumors, the other (REGb) generating tumors that regress a few weeks after s.c. injection into syngeneic hosts. Our study was aimed at comparing cellular immunity between animals bearing PROb or REGb tumors. Spleen cells were first tested for in vitro proliferation in response to mitomycin-treated PROb or REGb cells. Only spleen cells from rats injected with REGb cells proliferated significantly when mixed with PROb or REGb cells. The proliferative response induced by REGb cells was considerably higher than the response to PROb cells. When spleen cells from rats bearing REGb tumors were cultured with a mixture of REGb and PROb cells at various PROb/REGb cell ratios, PROb cells significantly suppressed the strong proliferative response generated by the same number of REGb cells alone. REGb-immune spleen cells, after in vitro stimulation by PROb or REGb cells, were not cytotoxic for either cell variant. REGb-immune spleen cells did not differ in their content of T lymphocytes expressing CD4 or CD8 markers when they were stimulated by PROb or REGb cells in vitro, but REGb cells induced a larger number of activated lymphocytes expressing the IL-2 receptor. Our results indicate that, compared to REGb cells, PROb cells are poorer stimulators of proliferation of tumor-immune spleen cells, and that they are able to suppress the proliferative response induced by REGb cells.     

Specificity of the immune response leading to protection or enhancement by regressive and progressive variants of a rat colon carcinoma

Two cell clones, K12/TRb (PROb) and K12/TSb (REGb), have been isolated from the same serially transplantable tumor, DHD, established from a colon carcinoma chemically induced in the rat. Inoculation of REGb cells gives a tumor which regresses within 4 to 8 weeks and generates immune protection against subsequent injection of the progressive tumor cells, PROb. Inoculation of PROb cells gives a progressive tumor and generates tolerance allowing progressive growth of contralaterally injected REGb cells. Inoculation of REGb cells fully protects the host against growth of a DHD tumor graft, the tumor from which REGb and PROb cells were originally obtained. On the other hand, inoculation of REGb cells does not confer any protection against growth of 4 other syngeneic tumor grafts, DHA, DHB, DHC and DHE. These tumors were obtained from other colonic tumors induced as DHD by 1.2 dimethylhydrazine (DMH). Progressive growth of the tumor induced by inoculation of REGb cells is observed in animals bearing a contralateral DHD tumor, but not in animals bearing tumor from other transplantable lines, DHA, DHB, DHC and DHE. Our results show that immune enhancement of a regressive tumor and the immune protection that it confers constitute specific responses to a tumor-specific transplantation antigen present on a single transplantable colon tumor.     

FAS(CD95) ligand expression by tumor cell variants can be unrelated to their capacity to induce tolerance or immune rejection.

According to the results of in vitro experiments, Fas(CD95) ligand expression by cancer cells might induce apoptosis of activated T cells and contribute to immune tolerance. However, Fas ligand expression had never been explored in vivo in tumor cell models yielding either immune response or tolerance. In the present study, we analyzed the expression and function of Fas ligand in 2 clones of tumor cells originating from the same rat colon carcinoma. REGb cells were immunogenic and yielded tumors that regressed in immunecompetent syngeneic hosts, whereas PROb cells induced active tolerance and yielded progressive tumors. Fas ligand was expressed on the plasma membrane of both REGb and PROb cells, and its cDNA sequencing showed no mutation. However, neither REGb nor PROb cells induced apoptosis of co-cultured Fas-sensitive target cells. Our results show that surface expression of Fas ligand by tumor cells does not always induce killing of adjoining Fas-sensitive cells and that tumor cells may induce a protective immune response or an active tolerance independently of Fas ligand expression.     

Plasminogen receptors on rat colon carcinoma cells.

Cells from rat carcinoma cell lines PROb (giving progressive tumours) and REGb (giving regressive tumours) have cell surface receptors which bind specifically rat plasminogen and plasmin. Affinity for Pg was found to be higher in PROb (Kd = 10(-7) M) than in REGb cells (Kd = 5.10(-7) M) but with a concomitant decrease in the number of binding sites, 0.9 x 10(6)/cell (range from 0.6 to 1.2 x 10(6)) in PROb vs 3.6 x 10(6)/cell (range 1.2 to 6 x 10(6)) in REGb cells. The number and the affinity of binding sites varied in an opposite way in PROb and REGb cells. The difference in affinity parameters was unrelated to the degree of invasiveness of tumour cells in syngenetic rats. Bound plasmin retained its enzymatic activity, which indicates that its binding does not involve the catalytic active site. In cell solubilisates plasminogen receptor appeared as one major band situated in the area of 50-60 kDa.     

Correlation between cell surface oligosaccharides and tissue target-selective adhesion of two rat adenocarcinoma cell lines

We observed that two rat colon adenocarcinoma variants originating from a single parental cell line and differing by their progressive and metastatic capacities in syngeneic BDIX rats differed by their organ distribution after intravenous injections. The PROb cells accumulated in the lung, wherefrom the REGb cells were rapidly cleared. In order to explore the role of cell surface glycoconjugates in organ-specific metastasis, cytofluorometric and histochemical studies using labelled lectins were performed. This revealed that the metastatic variant PROb presented more alpha-L-Fuc(1----2) beta D-Gal-R structures than the regressive nonmetastatic variant REGb. At variance, REGb cells exposed more D-galactosyl and N-acetyl-D-galactosaminyl residues than PROb cells. Monosacharides inhibited specifically cell adhesions on frozen organ sections. L-Fuc and N-acetyl-D-galactosamine (D-GalNAc) most strongly inhibited the adhesion of PROb cells on lungs, whereas D-Gal and D-GalNAc most strongly inhibited that of REGb cells. On the liver, adhesions of both cell lines were inhibited by D-Gal and D-GalNAc. These observations support the involvement of sugar-lectin receptors in the adhesion of these cells to the lungs or liver. The possible involvement of previously described lectins is discussed.     

F11C antigen: a membrane marker able to distinguish two regressive and progressive variants from a rat colon adenocarcinoma.

Cell variants that differ in their tumorigenicity and immunogenicity have been isolated from a BDIX rat colon adenocarcinoma cell line, DHD/K12. One variant, PRO, and the clones derived from it, are poorly immunogenic and induce progressive and metastatic tumors; the other one, REG, and its clones, are highly immunogenic and induce regressive tumors. When looking for a membrane marker distinguishing between PRO and REG lines, we obtained monoclonal hybridomas by immunizing BALB/c mice with PROb or REGb cell clones. Hybridoma F11C, producing an IgM monoclonal antibody (MAb) was able to distinguish between the cell variants on membrane immunofluorescence. All REGb cells strongly express F11C membrane antigen. On PROb cells, F11C antigen expression is weak, as demonstrated by cytofluorimetric analysis, and limited to a fraction of the cell population. The F11C membrane antigen is highly specific for the DHD/K12 cell line and the variants derived from it, but is not expressed on cells dissociated from the DHD transplanted tumor, from which DHD/K12 was established, suggesting that F11C antigen emerged during cell culture. Fluorescence absorption on synthetic oligosaccharides demonstrated that F11C antibody cross-reacts with A type 3, A type 4 and A type 5 chain blood group tetrasaccharides.     

Effects of a single injection of anti-asialo GM1 serum on natural cytotoxicity and the growth of a regressive colonic tumor in syngeneic rats

The REGb tumor cell line is a cloned variant of the DHD-K12 cell line, established from a colon carcinoma chemically induced in the rat. Unlike the parent DHD-K12 cell line, or other clones, which give progressive tumors when inoculated to the syngeneic rat, REGb cells produce tumors which regress in 3 to 5 weeks and never cause metastasis. In order to explore the role of natural killer (NK) cells in REGb tumor regression, each rat was given one injection of anti-asialoGM1 (anti-asGM1) serum, a known inhibitor of NK activity. This injection was done 24 hr before REGb cell challenge. This injection significantly depressed the in vitro cytotoxicity of peripheral blood lymphocytes on REGb cells for 2 weeks. REGb tumors grew larger and regressed later in the treated animals than those in the controls. Furthermore, a progressive or recurrent tumor was observed in 4 out of 10 treated rats, giving lung and/or lymph-node metastases in 2 cases. Immuno-histological study of the cells infiltrating the REGb tumors in control and treated animals showed a decrease number of asGM1+ and OX8+ lymphocytes, presumably NK cells, after anti-asGM1 treatment. An increase in number of macrophages was demonstrated in the progressive tumors of treated animals. These results suggest that NK cells play an important role in the initial stage of the regression TSb tumors in untreated syngeneic rats.     

Cinchonine, a potent efflux inhibitor to circumvent anthracycline resistance in vivo.

Circumvention of multidrug resistance is a new field of investigation in cancer chemotherapy, and safe and potent multidrug resistance inhibitors are needed for clinical use. We investigated several analogues of quinine for their ability to increase anthracycline uptake in resistant cancer cells. Cinchonine was the most potent inhibitor of anthracycline resistance in vitro, and its activity was little altered by serum proteins. Serum from rats treated with i.v. cinchonine produced greater uptake of doxorubicin in cancer cells (DHD/K12/PROb rat colon cells and K562/ADM human leukemic cells) than did serum from quinine-treated rats (ex vivo assay). Cinchonine was more effective than quinine in reducing tumor mass and increasing the survival of rats inoculated i.p. with DHD/K12/PROb cells and treated i.p. with deoxydoxorubicin. Moreover, the acute toxicity of cinchonine in rats and mice was lower than that of other quinine-related compounds. The lower toxicity and greater potentiation of in vivo anthracycline activity produced by cinchonine are favorable characteristics for its use as an anti-multidrug resistance agent in future clinical trials.     

Relationship between sensitivity to natural killer cells and MHC class-I antigen expression in colon carcinoma cell lines.

The sensitivity of colorectal tumors to NK-cell-mediated cytotoxicity and their expression of major histocompatibility complex (MHC) class-I antigens were studied in an attempt to determine whether such antigens play a role in the susceptibility of colorectal tumors to NK-cell lysis. In a rat colon-carcinoma model, 2 clones differing in their sensitivity to NK-cell-mediated cytotoxicity were tested for class-I expression; it was seen that the more sensitive cells (REGb) expressed less class-I products than did the resistant cells (PROb). However, when MHC class-I antigen expression was increased by IFN-gamma treatment, no change in NK-cell lysis was found with the PROb cells, while an increase in cytotoxicity was obtained with the REGb cells. After in vivo or in vitro selection of NK-resistant REGb cells, we observed in the selected cells an important decrease in RT-I class-I antigen expression. Fifteen different human colorectal cell lines were also studied for HLA class-I expression and NK-cell susceptibility, and no quantitative correlation between these 2 features was seen. However, cell lines which were deficient in HLA class-I antigens were more sensitive than class-I-positive cells.     

Microglial cells induce cytotoxic effects toward colon carcinoma cells: Measurement of tumor cytotoxicity with a γ-glutamyl transpeptidase assay

Activated macrophages have been shown to exert cytostatic and cytotoxic effects toward tumor cells via nitric oxide (NO) release. In the CNS, microglial cells are considered to be the main resident population of immune effector cells. In this study, cytotoxic activity of N11, an immortalized murine microglial cell line, toward rat progressive DHD/PROb and regressive DHD/REGb colon carcinoma cells was examined in parallel with NO production. Cytotoxicity was evaluated using a novel method, the γ-glutamyl transpeptidase (γ-GTP) assay, based on the fact that DHD tumor cells expressed high levels of γ-GTP activity, while no γ-GTP activity was found in cells of the monocyte/macrophage lineage. Results showed that activation of N11 cells by interferon-γ plus either lipopolysaccharide or tumor necrosis factor-α induced high amounts of NO release and cytotoxic effects toward DHD/PROb as well as DHD/REGb cells. NO release by activated N11 cells was augmented by addition of tumor cell-conditioned medium. Both NO release by N11 cells and cytotoxicity were blocked by addition of N^G-monomethyl-L-arginine (L-NMA), an inhibitor of NO synthase, suggesting that cytotoxicity was mediated by N11-derived NO. However, in the presence of L-NMA an increased production of interleukin-6 was also observed. In conclusion, in opposition to information obtained with brain-derived endothelial cells, brain-derived microglial cells did not differentiate between progressive and regressive clones of colon carcinoma cells. Our results point to a specific role for both endothelial and microglial cell types in the context of brain metastasis. Microglial cells can be cytotoxic for tumor cells, and this cytotoxicity is mediated by NO. Int. J. Cancer, 70:169–174, 1997. © 1997 Wiley-Liss, Inc.     

Involvement of histo-blood-group antigens in the susceptibility of colon carcinoma cells to natural killer-mediated cytotoxicity.

The susceptibility to natural-killer-cell lysis and expression of histo-blood-group antigens of 2 clones from a rat colon adenocarcinoma, of variants derived from them and of 17 human colon carcinoma cell lines were assessed in an attempt to determine if the major glycosidic tissue antigens of epithelial cells could influence the NK susceptibility of tumor target cells of epithelial origin. The rat REGb clone, which is relatively NK-sensitive, expressed higher levels of precursor structures T and Tn and lower levels of H antigenic determinants than the PROb clone, which displays higher resistance to NK-cell lysis. Cell variants were obtained from these 2 clones; it appeared that whether the cell variants were selected on the basis of expression of a blood-group antigenic determinant or on the basis of altered susceptibility to NK-cell lysis, there was a link between increased resistance and higher expression of cell-surface A and H histo-blood-group antigens, or conversely, between increased sensitivity and higher expression of precursor structures. Similar conclusions were obtained upon study of the human cell lines, since a significant correlation was found between the level of expression of T or Tn antigens and sensitivity to NK-cell lysis. A significant relationship was found between the expression of Lewis antigens and increased resistance to NK-cell-mediated cytotoxicity.     

T-cell co-stimulation by the CD28 ligand B7 is involved in the immune response leading to rejection of a spontaneously regressive tumor

Cell variants from experimental tumors may lose their tumorigenicity or give rise to tumors that regress after a short period of progression in immunocompetent syngeneic animals. Rejection of these tumor cells is often T-cell-dependent. It has recently been reported that, besides the specific signal delivered through the clonogenic receptor, T-cell activation requires a co-stimulatory signal, delivered through its CD28 receptor by B7-1 and/or B7-2 molecules expressed at the surface of the antigen-presenting cells. CTLA4Ig, a fusion molecule that specifically inhibits B7-1 and B7-2 binding to their receptor on T cells, was used to investigate the role of B7 in the spontaneous regression of the tumors induced in syngeneic rats by REGb cells, a regressor cell line established from a chemically induced colon carcinoma. When rats received either 1 or 3 CTLA4Ig injections, REGb tumors grew 3 or 7 times larger than in control animals, respectively. However, in most animals, single or repeated CTLA4Ig injections delayed rather than suppressed REGb tumor rejection. Antibodies to CTLA4Ig appeared in treated rats and could explain this transient effect. Neither REGb cells nor freshly isolated MHC class-11⁺ antigen-presenting cells infiltrating REGb tumors expressed B7, establishing that the target of CTLA4Ig was not located inside the tumor. In contrast, MHC class-II⁺ B7⁺ accessory cells were found in the REGb tumor-draining lymph nodes, suggesting that lymphoid tissue, rather than the tumor itself, was the site of tumor-antigen presentation to tumor-specific T cells. These results establish the role of the B7/CD28 co-stimulation pathway in the control of a spontaneously regressive tumor. © 1996 Wiley-Liss, Inc.     

Possible involvement of TGF beta 1 in the distinct tumorigenic properties of two rat colon carcinoma clones.

The presence in tumors of numerous cytokines suggests that they potentially modulate tumor cell activities and host tissue remodelling. To investigate the possible involvement of transforming growth factor type beta (TGF beta) in the metastatic process of cancer development, we have studied the effect of this factor on two rat colon carcinoma cell lines. These cell clones had been previously tested and selected for their ability to develop metastases in syngenic animals or lack of it. The two cell lines were characterized for their production of TGF beta. Production of active and latent forms of TGF beta 1 in the medium conditioned by the rat colon cancer cells were quantified using a bioassay. The presence of active TGF beta 1 was demonstrated in conditioned medium from the progressive tumor (PROb) cells and significant expression of latent forms of TGF beta 1 were found in the conditioned media from both cell clones. TGF beta 1 slightly inhibited proliferation of PROb cells which had been previously described as moderately differentiated, and significantly stimulated proliferation of the regressive (REGb) cells, described as poorly differentiated. On the basis of our observations, we suggest that this endogenous factor could be involved in autocrine regulation of tumor cell activities and in paracrine regulation of stroma cell and immune responses. Active and/or latent expression of TGF beta 1 by the two rat colon carcinoma cell lines, and their variable responses to the growth factor, strongly suggest that this polypeptide is involved in the regulation of tumorigenic expression of adenocarcinoma cells.     

Autoimmune expression of a cytoplasmic protein p60 in mice bearing metastasizing SV40-transformed tumors

In STU mice bearing metastasizing SV40-transformed 51A-232B-M tumors, an immune response against a cellular 60kDa protein (p60) developed in about 50% of the tumor-bearing animals, in addition to the response against SV40 large T-antigen and cellular protein p53. The anti-p60 auto-immune response could be observed as early as 11 days after tumor challenge and was strictly linked with metastatic spread but was not a prerequisite for metastasis. Anti-p60 antibodies could not be detected in sera of animals bearing metastasizing Rous-sarcoma virus-transformed or methylcholanthrene-induced tumors, or in sera from human cancer patients with clinically confirmed metastatic spread. The anti-p60 auto-antibodies showed a broad cross-reactivity against components of similar size in a great number of cell lines of various species and in normal mouse tissue. The p60 auto-antigen is a cytoplasmic protein which is neither phosphorylated nor glycosylated in vivo. Immunoblotting performed with fresh cell lysates under non-reducing conditions using tumor-bearer sera revealed a diffuse p60 double band, but under reducing conditions only one sharp p60 band was observed. The reaction of p60 with anti-p60 auto-antibodies could be completely blocked by pre-treatment of fresh cell lysates with N-ethylmaleimide or p-chloromercuriphenyl sulfonate, or by oxidation in air prior to immunoblotting, indicating that the anti-p60 autoimmune response was directed against an epitope sensitive to SH-group-blocking reagents. Immunofluorescence studies with tumor-bearer sera showed only a very weak cytoplasmic fluorescence, possibly due to the nature of the p60 SH-groups in situ being masked. Immunoprecipitates with monoclonal antibodies against SV40 large T-antigen and p53 obtained from fresh cell lysates of SV40-transformed tumor cells contained no associated p60 auto-antigen. The p60 auto-antigen was purified from tumor cell homogenates with an enrichment factor of about 2,000; its iso-electric point is at pH 6.8. Determination of the biological half-life of p60 yielded a value of about 28 hr. The p60 auto-antibodies in pools of tumor-bearer sera taken at day 40 after tumor challenge all belonged to the IgG1 subclass.     

In vivo and in vitro invasiveness of a rat colon-cancer cell line maintaining E-cadherin expression: An enhancing role of tumor-associated myofibroblasts

In various cell systems, an inverse relationship was found between expression of E-cadherin, a molecule involved in the Ca²⁺-dependent homophylic cell-to-cell attachment of epithelial cells, and the capacity to invade extracellular matrix gels or normal tissues in vitro., DHD/K12/TRb (PROb) cells, maintained as a cell line derived from a rat colon carcinoma, homogeneously expressed. in vitro immunoreactive E-cadherin, which was functional as shown in cell dissociation-reassociation assays. PROb cells were found to be non-invasive in 3 different assays in vitro., However, tumors resulting from a s.c. injection of PROb cells into syngeneic BD-IX rats were invasive, although PROb cells maintained E-cadherin expression in the tumors. Cells from a freshly dissociated PROb tumor showed, not only PROb cells but also tumor-associated myofibrobfasts and were able to cross a Matrigel-coated filter. PROb tumors were indeed infiltrated by numerous myofibroblasts, mainly located at the invasive edge of the tumor. Cells from an established culture of tumor-infiltrating myofibroblasts were able to confer upon PROb cells invasiveness through Matrigel-coated filter or into chick-heart fragments. PROb cells maintained their capacity to express E-cadherin after myofibroblast-enhanced Matrigel invasion. Tumor-associated myofibroblasts, but not PROb cells, secreted a 72-kDa collagenase that could play a role in tumor-cell invasion. These results strongly suggest that cells from the tumor stroma, and more specifically myofibroblasts, may be involved in the invasiveness of epithelial tumor cells in vivo, even when E-cadherin expression prevents tumor-cell invasiveness in different in vitro assays.     

Epinephrine enhances penetration and anti-cancer activity of local cisplatin on rat sub-cutaneous and peritoneal tumors.

Despite the theoretical advantages of a high local concentration of anti-cancer drugs, local chemotherapy often fails to produce complete and lasting responses in experimental and human solid tumors. Experiments using Patent Blue dye showed that fluids diffused poorly into tumor mass when injected inside or around s.c. rat tumors in rats. In the same way, Patent Blue dye distributed poorly from the peritoneal cavity into the tumor nodules of rats with peritoneal carcinomatosis. The potent vasoconstrictor, epinephrine (1 mg/kg of body weight) was shown to facilitate the penetration of Patent Blue dye into s.c. and peritoneal rat tumors. Platinum concentration evaluated by micro-PIXE in s.c. DHD/K12/ PROb colon tumors or by atomic absorption spectrometry in DHD/K12/PROb peritoneal tumors was 4- to 12-fold higher when epinephrine was added to local cisplatin. Peri-tumoral or intra-tumoral injection of cisplatin (2 mg/kg) alone does not cure s.c. DHD/K12/PROb colon tumors or GV1A1 glioma tumors in BD IX rats. By contrast, a complete and lasting cure of s.c. tumors was achieved regularly and without skin necrosis when epinephrine was added to intra-tumoral or peri-tumoral cisplatin. Rats with peritoneal-tumor nodules 1 to 2 mm in diameter, and insensitive to i.p. cisplatin alone, were cured when the anti-cancer drug was combined with epinephrine. These experimental results could justify clinical trials using a combination of cisplatin and epinephrine in the treatment of locally growing solid tumors.     

An atypical caspase-independent death pathway for an immunogenic cancer cell line

REGb cell line, a highly immunogenic tumor cell variant isolated from a rat colon cancer, yields regressive tumors when injected into syngeneic hosts. We previously demonstrated that REGb tumor immunogenicity was related to the capacity of releasing dead cells in vivo. Also, in vitro, REGb cell monolayers release dead cells, especially when cultured in serum-free medium. In the current study, we show that the release of dead cells results from an atypical death process associating features of necrosis and apoptosis. In spite of features considered as hallmarks of caspase-dependent apoptosis, including chromatin fragmentation and DNA oligonucleosomal cleavage, caspases are not activated and caspase inhibitors are ineffective to prevent REGb cell death. In contrast with a number of other types of cell death, the spontaneous death of REGb cells in culture depends on de novo protein synthesis as this death is blocked by low doses of the mRNA translation inhibitor cycloheximide. This unusual mode of cell death that associates necrotic and apoptotic features could provide optimal conditions for triggering a specific immune response.