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Department of Biology and Therapy of Cancer, Institut National de la Santé et de la Recherche Médicale, Contract Jeune Formation 94-8, Faculty of Medicine, Dijon, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously found that tumor cell clones, which we had established from a chemically induced colon carcinoma growing in an inbred strain rat, constitutively differed in their tumorigenicity and immunogenicity in syngeneic hosts (1, 2). Several clones gave rise to tumors that grew continuously, produced distant metastases, and finally killed their host. These clones were named PRO, as they yielded progressive tumors. Other clones gave rise to tumors that progressed for 2 to 3 weeks, regressed, and completely disappeared. We showed that these clones, named REG for regressive, induced a strong tumor-specific immune response that fully and durably protected animals against a subsequent injection of PRO cells (3). The spontaneous regression of the REG tumors was related to this T cell-dependent immune response, since REG cells gave rise to progressive tumors in nude mice, cyclosporin-treated syngeneic rats (4), or rats in which a growing PRO tumor had induced a tumor-specific immune tolerance (3). These properties made the PRO/REG cell model well-suited for the study of tumor immunity.
In the present study, we show that the tumorigenicity and immunogenicity of the PRO and REG cell lines is related to their ability to undergo apoptosis when cultured in vitro and injected in vivo. Overexpression of the protooncogene bcl-2 was shown to enhance cell survival following growth factor withdrawal (5) and to inhibit the apoptosis induced by a variety of stimuli (6-8). We also show that bcl-2 overexpression decreases REG cell sensitivity to apoptosis and induces REG tumor progression in a syngeneic host. Bcl-2-transfected REG cells remain sensitive to a previously established antitumor immune response. These results suggest that tumor cell apoptosis contributes to the induction of a specific immune response in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The animals used in these experiments were syngeneic BD-IX strain rats that had been bred in our laboratory by brother-sister mating. The regressive variant DHD-K12/TSb (REGb) and the progressive variant DHD-K12/TRb (PROb), as well as other PRO and REG clones used in some experiments, were all established from the DHD tumor, which is a colon adenocarcinoma induced by 1.2-dimethylhydrazine in a BD-IX rat (1). These tumor cells were cultured in a mixture of Ham’s F-10 medium, DMEM, and FBS (10:10:1 v/v, complete culture medium) as described previously (2). For the tumorigenicity assays, 1 x 106 tumor cells in 100 µl of serum-free Ham’s F-10 medium were injected s.c. into the thoracic wall of syngeneic BD-IX rats. Tumor volume was evaluated weekly, using a caliper to measure two perpendicular diameters. Rats bearing tumors that were >6 ml were killed and examined for metastases.
Histologic study of the tumor cell injection site
Animals were killed at 6, 24, or 48 h after PRO or REG cell injection. The site of tumor cell injection was resected and either fixed in formaldehyde (4% in PBS) and embedded in paraffin or embedded in Tissue-Tek (Miles, Elkhart, IN) and snap-frozen in methylbutane that had been cooled in liquid nitrogen. Apoptotic cells were labeled on 5-µM paraffin-embedded sections according to the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)3 procedure (9) using terminal transferase and biotin-16-2'-deoxyuridine-5'-triphosphate from Boehringer (Mannheim, Germany) and streptavidin-peroxidase (Amersham, Les Ullis, France), followed by hemalum staining. Tumor cells on slides stained with hemalum-eosin were distinguished from inflammatory cells according to their size, their large nuclei with oversized nucleoles, and their assembly in nodules. An immunohistochemical study of tumor-infiltrating inflammatory cells was performed in acetone-fixed 5-µM cryostat sections. Mouse mAbs to rat monocytes (ED1), class II MHC (OX-17), CD3 (R7/3), CD4 (W3/25), and CD8 (OX8), as well as an IgG isotype-matched control were obtained from Serotec (Oxford, U.K.). Mouse mAb against rat mature tissue macrophages (Ki-M2R) was a gift of Professor H. H. Wacker (Institute of Pathology; Kiel, Germany). Sections were incubated with mAb and biotinylated sheep Ab to mouse IgG (Amersham), subsequently incubated with streptavidin-peroxidase, and stained with aminoethylcarbazole. In other sections, tumor cells were stained after incubation with a rabbit polyclonal Ab to cytokeratin (Monosan, Uden, The Netherlands).
Cell sensitivity to apoptosis in vitro
The sensitivity of cultured cells to apoptosis was determined
after serum withdrawal from the culture medium. Tumor cells (1 x
106 cells in 1 ml of complete medium) were seeded in the
wells of 24-well flat-bottom microculture plates (Intermed, Nunc,
Roskilde, Denmark) and cultured for 24 h. The wells were gently
washed three times with serum-free Ham’s F-10 medium and cultured in
this medium for 72 h at 37°C in a 5% CO2
atmosphere. At the end of this incubation period, floating cells were
pooled with adherent cells that had been detached with EDTA-trypsin.
The pooled cells were incubated for 18 h at 4°C in HBSS (Life
Technologies, Paisley, U.K.) containing 2% formaldehyde and 10 µg/ml
Hoechst 33258 bisbenzimide fluorochrome (Aldrich-Chemie, Steinheim,
Germany). Next, the percentage of apoptotic cells was determined on an
epifluorescence microscope (DMR, Leica, Wetzlar, Germany) at x400
magnification. Apoptotic cells were identified by chromatin
condensation or fragmentation that clearly distinguished them from
mitotic cells. The percentage of apoptotic cells was determined in
populations of 
400 cells. To compare the cytotoxic effect of
serum-free medium on the various PRO and REG clones, 2 x
104 tumor cells in 200 µl of complete medium were seeded
in wells of 96-well flat-bottom microculture plates (Nunc) and cultured
for 24 h. The medium was subsequently sucked up, and the wells
were washed three times with serum-free medium and filled with either
serum-free or 10% FBS-supplemented medium. Cells were cultured for 14
days at 37°C in a 5% CO2 atmosphere. Serum-free and
FBS-complemented medium was replaced every other day by homologous
fresh medium. At the end of this incubation period, the culture medium
was drained off, the wells were gently washed twice with PBS, and the
cultures were fixed for 15 min with methanol. The plates were allowed
to dry and then stained with 1% crystal violet per well for 30 min at
room temperature. The plates were carefully washed under tap water and
dried, and the residual stain was eluted in 100 µl of 33% acetic
acid in water (v/v). The absorbance of each well was read at 570 nm on
an Anthos scan (Labtec Instruments, Salzburg, Austria). Quadruplicate
wells were used to determine the mean and SD.
Bcl-2 transfection of the REG cell line
REG cells were transfected with pEBS7 expression plasmid, which was either empty or contained cDNA-encoding human bcl-2 (10). pEBS7 plasmid contains the hygromycin B phosphotransferase gene, which is a selectable marker for resistance to hygromycin. Transfections were performed using calcium phosphate precipitation procedures (11). The bcl-2 or control transfectants were selected for 4 wk in complete medium supplemented with 1 mg/ml hygromycin (Sigma, St. Louis, MO). At such a concentration, hygromycin is lethal in 7 days for wild-type (wt) REG cells. Hygromycin-resistant cells were expanded and then cloned by limiting dilution. We selected REG clones with the highest Bcl-2 expression as determined by Western blot analysis. REG cells transfected with empty pEBS7 plasmid were used as a control.
Western blot analysis
After detachment with trypsin and EDTA, subconfluent cultured cells were lysed at 4°C for 30 min in lysis buffer (150 mM NaCl, 1 mM KH2PO4, 1 mM EGTA, 1 mM Na3PO4, 5 mM MgCl2, 0.1 mM PMSF, 0.15 U/ml aprotinin, 1 µg/ml pepstatin, and 10% glycerol) and centrifuged for 15 min at 15,000 x g. The protein concentration was measured in the supernatant using the micro bicinchoninic acid protein assay (Pierce, Asnieres, France). Equal amounts of proteins (50 µg) were separated by SDS-PAGE using a 12% polyacrylamide gel and were electroblotted to a polyvinylidene difluoride membrane (Bio-Rad, Ivry-sur-Seine, France). Blots were blocked with 5% nonfat milk in PBS with 0.1% Tween 20. To control the effects of cell transfection, blots were probed with mouse mAb (1/1000) to human Bcl-2 (Dako, Glostrup, Denmark) or to rat Hsp90 (StressGen, Victoria, Canada). To determine the content of autochthonous proteins, blots were probed with rabbit polyclonal Ab (1/1000) reacting with rat rBcl-2 or Bax (Santa Cruz Biotechnology, Santa Cruz, CA). After three 10-min washes in PBS with 0.1% Tween 20, the blots were incubated with peroxidase-conjugated anti-mouse or anti-rabbit IgG (Amersham). The Amersham enhanced chemiluminescence Western blotting analysis system was subsequently used for protein detection. The results are representative of three separate experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As previously reported (12), PRO and REG cells give rise to
measurable, histologically proven local tumors that progress until 14
to 18 days after s.c. injection. At this point, PRO tumors continue to
progress, while REG tumors regress until their complete disappearance,
which occurs at 4 to 6 wk postinjection. To analyze the reasons for
this different evolution, we studied tumor foci during the 48 h
after a s.c. injection of PRO or REG cells. When examined at 6 h
postinjection, tumor cells from both lines were similarly preserved.
PRO cells remained morphologically normal at 24 and 48 h
postinjection, whereas numerous REG cells demonstrated condensed or
fragmented chromatin and eosinophilic staining of the cytoplasm. These
changes suggested cell apoptosis. Indeed, when the TUNEL assay was
performed on sections of the tumor cell injection site, numerous
apoptotic tumor cells were found in the REG cell foci, whereas
apoptotic cells were absent or rare in the PRO cell foci (Fig. 1
). We also examined inflammatory cells
that infiltrate PRO and REG cell injection sites. These cells were rare
in sections of sites that were resected 6 h after tumor cell
injection but became abundant after 24 and 48 h. Polymorphonuclear
cells were identified at 24 h after tumor cell injection but had
almost entirely disappeared after 48 h. T cells remained rare in
the tumor infiltrate up to 48 h after tumor cell injection. The
most abundant infiltrating cells inside and around PRO and REG cell
foci were round mononuclear cells that were labeled with ED1 mAb, a
marker of rat monocytes and macrophages (13). These ED1-stained cells
were also seen inside and around the blood vessels surrounding the
tumor cell area, suggesting their blood origin. Inflammatory cells did
not express class II MHC molecules at 24 h after tumor cell
injection, whereas a fraction of these cells expressed this marker of
APCs after 48 h. Unlike the mature resident macrophages found in
the normal s.c. tissue, tumor-infiltrating inflammatory cells were not
stained with Ki-M2R mAb. No significant difference was found between
the PRO and REG cell injection sites with regard to the density and the
type of infiltrating inflammatory cells at 24 and 48 h after tumor
cell injection.
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To determine whether the high rate of apoptosis observed in REG
cells relative to PRO cells after s.c. injection was due to intrinsic
properties of the cells or to an early reaction of the host, PRO and
REG cells were compared with regard to their sensitivity to apoptosis
when cultured in vitro as two-dimensional monolayers, with or without
FBS supplementation. The deprivation of growth factors in serum-free
medium is known to induce the apoptosis of sensitive cells. We compared
six PRO clones and four REG clones that were independently obtained
from the same colon cancer cell line and yielded progressive or
regressive tumors, respectively, when injected into syngeneic hosts
(2). The percentage of apoptotic cells was low and nearly the same in
PRO and REG clones when cultured for 3 days in FBS-supplemented medium.
REG clones were more sensitive than PRO clones to apoptosis induced
through a 3-day culture in serum-free medium. The rate of apoptotic
cells, as determined after Hoechst 33258 staining, was higher in the
REG clones than in the PRO clones (Fig. 2A). Numerous apoptotic cells
and cellular debris were observed floating in the medium above the
serum-deprived REG clones. When apoptotic cells and cellular debris
were collected daily for 4 days by centrifugation of the serum-free
culture medium and measured as proteins, they were found to be seven
times more abundant in REG than in PRO cell supernatant. We also
observed that the density of residual adherent cells was strongly
reduced when the four REG clones had been cultured for 14 days in
serum-free medium, whereas this treatment had only a limited effect on
the six PRO clones (Fig. 2B).
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Western blot analysis was performed on REG and PRO cell extracts
using Abs against rat antiapoptotic Bcl-2 and proapoptotic Bax
proteins. REG and PRO cells expressed these endogenous proteins in
comparable amounts (Fig. 3). To induce
Bcl-2 overexpression, REG cells were transfected with either empty or
human bcl-2 cDNA-containing pEBS7 plasmid. Several clones of
hygromycin-resistant transfected cells were obtained. Two clones of REG
cells that had been transfected with bcl-2 cDNA were
selected for their high expression of the human Bcl-2 protein, as
detected by Western blot analysis using a human-specific anti-Bcl-2
Ab (Fig. 4). This expression was checked
to ensure that it remained stable after multiple passages in vitro.
These clones were called REG-bcl2 cells. One of the REG clones that had
been transfected with the empty vector (REG-hygro) was used as control.
REG-bcl2 cells were resistant to the apoptosis induced by
serum-withdrawal (13% apoptotic cells after a 3-day depletion, 95%
confidence interval (c.i.): 4.3%) when compared with REG-hygro cells
(35% apoptotic cells, 95% c.i.: 4.7%) (Fig. 5). We used the TUNEL assay to identify
apoptotic cells on sections of the tumors yielded by transfected REG
cells into syngeneic rats. No apoptotic tumor cells were observed at
the injection site where REG-bcl2 cells had been administered 48 h
before, whereas REG-hygro nodules contained a mixture of apoptotic and
nonapoptotic tumor cells (Fig. 6). wt REG
and REG-hygro cells gave rise to tumors that completely regressed in 3
to 4 wk. In contrast, continuously progressive tumors were observed in
14 of 18 injected rats in three separate experiments with two different
REG-bcl2 clones (Fig. 7). Although
REG-bcl2 tumors grew about twice as slowly as PRO tumors, they
progressed to large volume tumors. Histologically proved metastases to
axillary and mediastinal lymph nodes and several micrometastases to the
lung were found in two rats bearing a voluminous s.c. tumor that were
killed at 126 days after REG-bcl2 cell injection. Furthermore,
fragments from REG-bcl2 resected tumors grew as a progressive tumor
when transplanted into naive syngeneic recipients. Most of the cancer
cells in these REG-bcl2 tumors still expressed human Bcl-2 on
immunohistologically stained sections, demonstrating the in vivo
stability of Bcl-2 overexpression (data not shown).
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To determine whether the progression of REG-bcl2 tumors was due to their resistance to the effector mechanisms of antitumor immunity, REG-bcl2 cells were injected into naive rats and into rats that had been immunized with three monthly injections of 1 x 106 wt REG cells. REG-bcl2 cells gave rise to continuously progressive tumors in five of six naive animals, while no tumors appeared in the six preimmunized rats. These observations indicate that REG-bcl2 cells are rejected by the immune system despite Bcl-2 overexpression, suggesting that the progression of REG-bcl2 tumors is not the consequence of tumor cell resistance to immune rejection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate a possible relationship between apoptosis and the terminal regression and healing of REG tumors, we enhanced REG cell resistance to apoptosis by overexpressing Bcl-2, an apoptosis-suppressing protein. The clones that overexpressed Bcl-2 were more resistant to apoptosis in vivo and in vitro and gave rise to progressive, metastatic, and lethal tumors. Bcl-2 overexpression might either enhance the resistance of tumor cells to an established immune response or decrease the capability of tumor cells to induce this response. Our observation that REG-bcl2 cells were rejected in rats that had been preimmunized against wt REG cells is more in keeping with the second possibility. Bcl-2 transfection reportedly does not protect cells from lysis mediated by CTLs or activated NK cells (16). We have previously shown that PRO cells were also rejected in rats that had been previously immunized against REG cells (3). This finding suggests that the resistance of REG-bcl2 and PRO cells to apoptosis decreases their capability to induce an immune response rather than their sensitivity to the effectors generated by this response.
The induction of a relative resistance to apoptosis after bcl-2 transfection was sufficient to prevent a regression of the REG tumors. Thus, REG-bcl2 cells are fairly similar to PRO cells, which are relatively resistant to apoptosis, are poorly or nonimmunogenic when injected into syngeneic hosts, and are able to give rise to progressive and lethal tumors. PRO and REG cells express similar levels of endogenous Bcl-2 and Bax. Bax is a Bcl-2 family protein that heterodimerizes with Bcl-2, neutralizes its apoptosis-protecting property (17), and can suppress tumorigenesis in other tumor models (18). Thus, wt PRO and REG cells differ in their sensibility to apoptosis through other still undetermined mechanisms.
PRO and REG cells express comparable levels of class I MHC on their plasma membrane but do not express class II MHC nor B7 costimulatory molecules and are not capable of directly presenting their Ags to T cells (data not shown). To induce an immune response, tumor Ags have to be presented to T cells by professional APCs (19), preferentially inside of the lymph nodes or the spleen (20). Unlike PRO cells, apoptosing REG cells release a great amount of cellular debris in the culture medium, and the same release is likely to occur in vivo. This cellular debris is an efficient source of Ag for T cell activation (21-23). The cellular debris of apoptotic cells, called apoptotic bodies, is still enclosed by a cell membrane. However, this membrane differs from that of healthy cells, since phosphatidylserine is aberrantly exposed on its outer leaflet and enhances recognition and internalization by phagocytic cells (24). We found that apoptotic cells and the debris released by serum-deprived REG cells were labeled with FITC-conjugated annexin V, a ligand for surface-exposed phosphatidylserine (data not shown). The phosphatidylserine receptor is more strongly expressed on monocytes than on mature macrophages (24). Thus, apoptotic bodies are likely to be recognized and ingested by the abundant monocytes that infiltrate the tumor cell foci early after cell injection. These monocytes strongly express ED1, a marker of phagolysosomes that is correlated with the phagocytic activity of these cells (25). Inflammatory monocytes and macrophages have the capacity to migrate into the secondary lymphoid tissues (26), in which they can deliver antigenic material from ingested particles to dendritic cells for presentation to T cells (27).
The PRO and REG cell variants have been isolated from the same tumor cell culture. Unlike other nontumorigenic or regressive cell variants (28), REG cells did not result from a mutagenic treatment inducing highly immunogenic protein mutations. Despite their dramatic difference in immunogenicity and tumorigenicity, PRO and REG cells share a similar antigenic profile, since PRO cells are regularly rejected in hosts preimmunized with REG cells (3). The most striking finding of the present study was that resistance to apoptosis through Bcl-2 overexpression was sufficient to restore a progressive and lethal phenotype to tumor cell variants that constitutively yielded immunogenic and regressive tumors in all cases. This finding indicates that tumor cell apoptosis may be an important factor for the triggering of an effective immune response against cancer.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. François Martin, Department of Biology and Therapy of Cancer, Institut National de la Santé et de la Recherche Médicale, Contract Jeune Formation 94-8, Faculty of Medicine, 7 Bd Jeanne d’Arc, 21033 Dijon, France.
3 Abbreviations used in this paper: TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphospate nick-end labeling; wt, wild-type; c.i., confidence interval.
Received for publication February 3, 1998. Accepted for publication April 6, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5 subunit in HT29 colon carcinoma cells suppresses apoptosis triggered by serum deprivation. Exp. Cell Res. 224:208.[Medline]
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