[Cancer Research 59, 1816-1819, April 1, 1999]
© 1999 American Association for Cancer Research
[Cancer Research 59, 1816-1819, April 15, 1999]
© 1999 American Association for Cancer Research
Tumorigenic Conversion Resulting from Inhibition of Apoptosis in a Nontumorigenic HeLa-derived Hybrid Cell Line
Kiyomasa Oka1,
Bing Qi and
Masuo Yutsudo2
Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Suita 565-0871, Japan
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ABSTRACT
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Although tumorigenicity in nude mice is one of the most important transformed phenotypes, its mechanism has been little analyzed. To understand the molecular basis of tumorigenicity, we characterized nontumorigenic CGL1 and tumorigenic CGL4 cell lines, both of which were originated from a common ancestral HeLa-human diploid fibroblast hybrid cell clone and retained a malignant state except tumorigenicity. When injected into nude mice, nontumorigenic CGL1 cells underwent apoptosis, but tumorigenic CGL4 cells did not. In vitro, CGL1 was also less resistant to various apoptotic stimuli than CGL4. These results suggested that inhibition of apoptosis may lead to tumorigenicity. To examine this hypothesis, we introduced antiapoptotic genes into the CGL1 cell line and injected the resulting clones into nude mice. The results showed that the ectopic expression of Bcl-2 or E1B19k, but not of crmA, converted CGL1 cells to tumorigenicity, suggesting strongly that this phenotype may be conferred by evasion of apoptosis.
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Introduction
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Malignant cell transformations are known to be accompanied by various phenotypical alterations, such as morphological change, immortality, anchorage independency, decreased serum requirement, and tumorigenicity in nude mice. Most of these phenotypes seem to be independently regulated, because different genes induce different transformed phenotypes. For example, the c-myc gene immortalizes primary rat embryo fibroblasts but does not induce any malignant phenotypes (1)
. The E7 transforming gene of high-risk types of human papillomavirus renders rodent cell lines anchorage-independent but not tumorigenic, whereas the other transforming gene of the human papillomavirus, the E6 gene, confers tumorigenicity to mouse cell lines (2)
. A human fibrosarcoma cell line, HT1080, which contains an activated N-ras allele, shows anchorage independency and tumorigenicity, but loss of the activated N-ras allele from this cell line results only in loss of anchorage independency, not in that of tumorigenicity (3)
. These facts clearly show that many of the phenotypic alterations associated with malignant cell transformations are caused by different mechanisms.
Among these transformed phenotypes, tumorigenicity has been little analyzed. Here, to investigate the molecular mechanism of tumorigenicity, we used a hybrid-cell system established by Stanbridge et al. (4
, 5)
. They have shown that CGL1, a hybrid cell clone resulting from the fusion of tumorigenic HeLa with normal human fibroblasts, loses tumorigenicity but retains several other transformed phenotypes, such as immortality and anchorage independency. Although the nontumorigenic hybrid cell clone CGL1 is very stable, a tumorigenic segregant, CGL4, appeared very rarely from the common ancestral hybrid cell clone after a long-term culture. CGL1 and CGL4 exhibit similar karyotypes with quasitetraploid chromosomes derived from HeLa and normal fibroblasts except for some chromosomes, including one copy each of both chromosomes 11 and 14 lost in CGL4 (4)
. These cell lines with their nearly identical genetic backgrounds might serve as powerful tools for the analysis of the molecular mechanism of tumorigenicity, because tumorigenicity is dissociated from anchorage independency or other transformed phenotypes in this hybrid cell system.
In this report, we found that the nontumorigenic CGL1 cells underwent apoptosis when injected into nude mice and were less resistant to apoptotic stimuli than CGL4 in vitro. These results suggested that inhibition of apoptosis may lead to tumorigenesis. To investigate this possibility, CGL1 cells were transfected with antiapoptotic genes to confer resistance to apoptosis and then examined for tumorigenicity. The results indicated that inhibition of apoptosis is sufficient to confer tumorigenicity in this cell line.
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Materials and Methods
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Cells.
CGL1 and CGL4 were cultured in DMEM supplemented with 10% FCS.
DNA Transfection.
CGL1 was stably transfected with expression vectors for human Bcl-2 (pCAGGS-bcl-2), E1B19k of adenovirus type 5 (pCMV-E1B19k), and crmA of cowpox virus (pEF-crmA) together with the neomycin-resistance gene using the Lipofectamine reagent according to the protocol of the manufacturer (Life Technologies, Inc., Rockville, MD). To select stable transfectants, the cells were cultured for 2 weeks in medium containing G418 (600 µg/ml).
Assay for CPP32 Activity.
Cells (1 x 105/100 mm dish) cultured with or without serum for 2 days were harvested and assayed for CPP32 activity using the ApoAlert CPP32 Fluorescent Assay kit (Clontech, Palo Alto, CA) according to the manufacturer’s protocol. The assay is based on the specific cleavage of the substrate DEVD-7-amino-4-trifluoromethyl coumarin and measurement of the resulting fluorescence.
Detection of Oligonucleosomal DNA Fragmentation.
Cells (1 x 105/100 mm dish) were plated with or without serum. Three days later, adherent and detached cells were collected and lysed in 100 µl of lysis buffer [10 mM Tris-HCl (pH 7.4), 10 mM EDTA, and 0.5% Triton X-100] for 10 min on ice. The lysate was centrifuged at 14,000 rpm for 30 min. The supernatants were incubated with RNaseA for 1 h at 37°C and then with proteinase K for an additional hour. DNA was precipitated and electrophoresed in 2.5% agarose gel in 0.5X TBE buffer. DNA fragments were visualized by staining with SYBR green.
TUNEL3
Assay.
Tissue samples were fixed in 10% formalin and embedded in paraffin. Tissue sections of 3-µm thickness were deparaffinized, and in situ TUNEL assays were performed with an Apoptag in situ apoptosis detection kit (Oncor, Gaithersburg, MD). Apoptotic cells were visualized by fluorescence microscopy.
Cell Death Assay.
Cells (5 x 104/35 mm dish) were plated in DMEM-10% FCS in the presence or absence of Act D. After a 16-h incubation, cells were harvested and stained with 0.5% trypan blue for 5 min at room temperature. The number of dead cells were counted with a hemocytometer.
Northern Blot Hybridization.
The total RNA was isolated with ISOGEN (Nippon Gene, Tokyo, Japan). Poly(A) RNA purified with Oligo-Tex (Takara, Otsu, Japan) was subjected to 1.2% formaldehyde-agarose gel electrophoresis, blotted onto a nylon filter, hybridized with 32P-labeled probe, and autoradiographed.
Western Blotting Analysis.
Cells were lyzed with RIPA buffer [0.15 M NaCl, 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 100 µg/ml PMSF, and 0.25 TIU/ml aprotinin]. Equal amounts of the cell lysate were run on a 5–20% SDS-polyacrylamide gel and electroblotted onto a polyvinylidene difluoride membrane. The membrane was incubated successively with a mouse monoclonal anti-bcl-2 antibody (clone 7; Transduction Laboratories, Lexington, KY) and a horseradish peroxidase-conjugated anti-mouse IgG (Amersham, Buckinghamshire, England). Protein bands were detected using ECL reagent.
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Results
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Apoptosis Occurred in Nontumorigenic CGL1 Cells Injected into Nude Mice.
Outwardly, when injected into nude mice, nontumorigenic CGL1 cells formed a lump that maintained its mass for several days before suddenly disappearing at 7–10 days after injection, whereas tumorigenic CGL4 cells grew to form a large tumor after an initial latency period. What happens in the nontumorigenic cells in nude mice? To investigate this question, we injected CGL1 and CGL4 cells into nude mice s.c., fixed the lump with formaldehyde at 2, 4, 6, or 9 days, and carried out a TUNEL assay for detection of DNA fragmentation caused by apoptosis (Fig. 1)
. Fig. 1
clearly shows that apoptosis was induced in nontumorigenic CGL1 but not in tumorigenic CGL4 cells. The most prominent fluorescence was observed at 6–9 days in this and in other independent experiments, suggesting that many of the injected nontumorigenic cells died by apoptosis during this period. This result is consistent with the outward appearances described above. It is therefore clear that tumorigenicity is closely correlated to apoptosis.
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Fig. 1. Detection of apoptosis in CGL1 cells injected into nude mice by TUNEL staining. Nude mice were injected s.c. with CGL1 and CGL4 and sacrificed at day 2, 4, 6, or 9. The histological sections were stained with an in situ apoptosis detection kit. x200. The results shown are representative of several independent experiments.
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Nontumorigenic CGL1 Cells Are Less Resistant to Apoptotic Stimuli in Vitro.
Next, we investigated in vitro differences in apoptotic response between nontumorigenic CGL1 and tumorigenic CGL4 cell lines. Although CGL1 cells lost tumorigenicity by fusion with normal cells, they retained anchorage independency and growth ability in low serum media containing 0.2% FCS (5
and Fig. 2a
). However, in serum-free media, CGL1 cells could not grow, but CGL4 cells could (Fig. 2b)
. CGL1 cells did not seem to arrest at the G1 phase of the cell cycle and were exfoliated from the dish shortly after the removal of serum, suggesting that CGL1 might die in serum-free media. Therefore, we supposed that the cells died by apoptosis and examined the activity of CPP32 (caspase-3), which is known to be activated by apoptosis (6)
. Fig. 2c
shows that CPP32 in CGL1, but not in CGL4, was activated by serum withdrawal (a 3-fold increase compared with CGL1 with serum). We also carried out another assay to corroborate the finding that apoptosis was induced. DNA was extracted from cells cultured in serum-free media and analyzed by agarose gel electrophoresis. Fig. 2d
shows that genomic DNA of CGL1 was digested to oligonucleosomal fragments whose occurrence is characteristic in apoptosis. These results indicate that apoptosis was induced in CGL1 by withdrawal of serum (or some type of growth factor).
Further, we examined the sensitivity of these cell lines to other apoptotic stimuli. After CGL1 and CGL4 were treated with Act D (Fig. 3f)
or irradiated by UV light (data not shown), the dead cells were counted by staining with trypan blue. In both cases, the survival rate of CGL1 cells was significantly lower than that of CGL4 cells. Thus, the nontumorigenic CGL1 cell line was less resistant to various apoptotic stimuli than the tumorigenic CGL4 cell line both in vitro and in vivo.
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Fig. 3. Ectopic expression of antiapoptotic genes in CGL1. a, Northern blot analysis of introduced Bcl-2 mRNA in CGL1. b, Western blot analysis of Bcl-2 protein. c and d, Northern blot analysis of adenovirus E1B19k (c) and poxvirus crmA (d) mRNA in CGL1. e, growth abilities in serum-free medium of CGL1 expressing Bcl-2, E1B19k, and crmA. CGL1/Bcl, CGL1/19k, and CGL1/crmA were plated and counted as described in Fig. 2b
.  , CGL1;  , CGL1/Bcl #1;  , CGL1/19k #1; •, CGL1/crmA #1. The other transfected cell clones were also examined, and similar results were obtained. f, resistance to Act D-induced apoptosis of CGL1 expressing antiapoptotic genes. Cells were plated in the presence or absence of Act D, and dead cells were counted 16 h later. , CGL1;  , CGL4; , CGL1/Bcl #1; , CGL1/19k #1; •, CGL1/crmA #1. The other transfected cell clones were also examined, and similar results were obtained. Results shown are representative of at least two experiments.
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Introduction of Antiapoptotic Genes Conferred Tumorigenicity on Nontumorigenic CGL1 Cells.
The results described above suggest that evasion of apoptosis may be indispensable for tumorigenicity. This also leads to the idea that a blocking of apoptosis may endow tumorigenicity to cells that are almost fully transformed but still nontumorigenic. To examine this possibility, we transfected the anchorage-independent, nontumorigenic cell line CGL1 with antiapoptotic Bcl-2 gene (7)
and isolated several cell clones (CGL1/Bcl) that overexpressed Bcl-2 mRNA and protein (Fig. 3, a and b)
. These cell clones were highly resistant to Act D, as was tumorigenic CGL4 (Fig. 3f)
, indicating that the introduced Bcl-2 certainly functioned in the transfected cell clones. These cell clones also gained an ability to grow in serum-free media (Fig. 3e)
, and DNA ladder formation in these cell clones was completely negative (data not shown). Using these cell clones, we examined whether a blocking of apoptosis is sufficient for tumorigenicity. As shown in Table 1
, all CGL1/Bcl clones tested formed tumors, although slowly, in nude mice, suggesting that a critical event in the pathway leading to tumorigenicity may be resistance to apoptosis. The conversion of cells from nontumorigenic to tumorigenic was also brought about by another antiapoptotic gene. We introduced two other antiapoptotic genes, E1B19k (8)
and crmA (9)
, into CGL1 to block apoptosis and isolated several cell clones expressing E1B19k (CGL1/19k) and crmA (CGL1/crmA; Fig. 3, c and d
). The adenovirus E1B19k protein, like the Bcl-2 protein, blocks apoptosis by binding to proapoptotic gene products such as Bax and Bak (10
, 11)
, whereas cowpox virus crmA protein inhibits activity of certain caspases, such as caspase-1 and caspase-8, which act downstream of the Fas/TNFR-activated apoptotic pathway but does not inhibit caspase-3, caspase-6, caspase-7, and caspase-10 (12
, 13)
. Therefore, these genes may exert different effects. Indeed, when these cell clones were treated with Act D, CGL1/19k showed resistance to apoptosis induced by this drug, but CGL1/crmA did not (Fig. 3f)
. These cell clones were next injected into nude mice to examine their tumorigenic abilities. As shown in Table 1
, CGL1/19k cell clones efficiently formed tumors, confirming the results obtained for CGL1/Bcl. However, CGL1/crmA cell clones formed no tumors, indicating that Fas- or caspase-1/8-mediated apoptosis is not be involved in tumorigenicity in this cell system.
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Discussion
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In this report, we showed that apoptosis was induced in the HeLa-derived nontumorigenic hybrid cell line CGL1 when injected into nude mice (Fig. 1)
, and that CGL1 that expressed either of the ectopic antiapoptotic genes, Bcl-2 or E1B19k, formed tumors in these mice (Table 1)
. These results clearly indicate that tumorigenicity is conferred by inhibition of apoptosis in the nontumorigenic cell line CGL1.
To date, there have been many reports suggesting that apoptosis may be involved in malignant transformation. For example, the proapoptotic tumor suppressor gene TP53 is inactivated in various cancers at high frequencies (14)
, and the antiapoptotic gene Bcl-2 is activated and overexpressed through translocation in B-cell follicular lymphoma (15)
. These facts suggest that evasion of apoptosis may be essential for development of cancers. None of these reports, however, indicate which stage of cancer development or which transformed phenotype requires this evasion of apoptosis. But the present results strongly suggest that evasion of apoptosis may be essential for tumorigenicity in nude mice.
We do not know whether the present results will be extendable to other cells. To examine this, we will need appropriate cell lines transformed to a certain state of malignancy, because it is obvious that Bcl-2 does not show any oncogenic activity in normal cells. In non-human cell systems, however, a few reports have suggested the possible involvement of apoptosis in tumorigenicity. MDCK is an untransformed canine cell line that is anchorage dependent and nontumorigenic. When this cell line was transfected with the Bcl-2 gene, the transfectant converted the cell line to anchorage independence and tumorigenicity (16)
. But in this report, the Bcl-2 gene conferred two different phenotypes simultaneously, and apoptosis in the Bcl-2-transfected cells was not examined. In another study, a murine hematopoietic cell line, Ba/F3, transfected with BCR-ABL gene was able to grow in the absence of interleukin 3 and to form tumors in nude mice. In this transformed cell line, Bcl-2 mRNA was up-regulated. When Bcl-2 expression in the transformed cells was suppressed by an antisense Bcl-2 construct, the cells lost tumorigenicity and growth ability in the absence of interleukin 3 (17)
. This result presented evidence that Bcl-2 expression was essential for tumorigenicity but did not exclude the possibility that Bcl-2 was involved only in an early stage of transformation but not in tumorigenicity itself. Moreover, human cells are known to be highly resistant to malignant transformation, and thus the mechanism of transformation of human cells may be considerably different from that of rodent or nonhuman cells. Nevertheless, these earlier reports strongly support the conclusion presented here.
We also showed that sensitivity to in vitro apoptotic stimuli is different between nontumorigenic CGL1 and tumorigenic CGL4 cells. To determine whether this different sensitivity was due to the expression level of endogenous Bcl-2 or other apoptosis-related genes, we examined expression levels of Bcl-2, Bcl-XL, Bcl-XS, and Bax mRNAs and Bcl-2 protein in CGL1 and CGL4 in the presence or absence of serum. However, we could not detect any significant differences (data not shown). The question remains: which apoptotic pathway or stimulus is involved in tumorigenicity in nude mice? Because certain growth factors inhibit apoptosis by inactivating a proapoptotic Bcl-2 family protein, Bad, through the phosphatidylinositol-3-kinase/Akt pathway (18)
, and because tumorigenic CGL4, but not nontumorigenic CGL1, grows in serum (growth factor)-free media, we first suspected that ability to produce a certain growth factor or constitutive activation of the growth factor-mediated antiapoptotic pathway may result in tumorigenicity of the CGL4 cell line. However, this may not be the case, because crmA, in contrast to Bcl-2 and E1B19k, did not confer tumorigenicity to CGL1 (Table 1)
, despite its potential to confer growth ability in the absence of growth factor (Fig. 3e)
. These results also indicate that the crmA-related pathway is not involved in tumorigenicity. In other words, tumorigenicity may not be correlated with the Fas/TNFR-induced pathway, because crmA can inhibit caspases specific to this pathway (12)
. Interestingly, resistance to Act D-induced apoptosis coincided with tumorigenicity (Fig. 3f)
; that is, Bcl-2 and E1B19k rendered CGL1 tumorigenic and resistant to Act D-induced apoptosis. Analyses of this mechanism may present some clue to understanding the nature of tumorigenicity. Apoptosis may also regulate anchorage dependency in certain cells. In these anchorage-dependent cells, the loss of integrin-mediated signal may cause attenuation of the Ras/MAPK pathway and thus triggers apoptosis (19)
. However, because both CGL1 and CGL4 cell lines used in this study are anchorage independent, the integrin-mediated signal pathway may not be associated with tumorigenicity.
Although further studies will be needed to clarify the tumorigenicity-associated apoptotic pathway, we presented here an important result that tumorigenicity was conferred by inhibition of apoptosis in a human cell system.
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ACKNOWLEDGMENTS
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We are grateful to Dr. E. J. Stanbridge for providing the CGL1 and CGL4 cell lines and to Drs. Y. Tsujimoto, T. Yamashita, and S. Nagata for the Bcl-2, E1B19k, and crmA plasmids, respectively.
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 K. O. is a Research Fellow of the Japanese Society for the Promotion of Science. 
2 To whom requests for reprints should be addressed, at Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita 565-0871, Japan.
3 The abbreviations used are: TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; Act D, actinomycin D.
Received 10/19/98.
Accepted 3/ 2/99.
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ABSTRACT
Introduction
