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Experimental Therapeutics |
Department of Head and Neck Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
ABSTRACT
Human head and neck squamous cell carcinoma (HNSCC) lines infected with a replication-defective Ad5CMV-p53 vector bearing a wild-type human p53 gene were used to examine alterations in the production of proteins implicated in regulating apoptosis. Because HNSCC lines express abundant levels of c-myc, and simultaneous expression of c-myc and p53 is known to trigger apoptosis in other cells, cooperation between these two genes was examined. Surprisingly, levels of c-myc mRNA and protein were rapidly and profoundly suppressed after infection with wild-type p53. Suppression of c-myc using antisense oligodeoxynucleotides (in the absence of p53) was sufficient to trigger apoptosis in Tu-138 cells, raising the possibility that the reduction of c-myc may be involved in at least one of the cell death pathways mediated by p53. Expression of a panel of Bcl-2 homology proteins was also examined in HNSCC lines undergoing p53-mediated apoptosis. No changes in Bcl-2, Bak, or Bcl-xS were found after p53 expression. Increased levels of the apoptosis-accelerating protein Bax were found in HNSCC lines after infection with Ad5CMV-p53. Induction of the apoptosis-inhibiting protein Bcl-xL was observed in Tu-167 cells and may account for the delayed onset of apoptosis in these cells. These studies suggest that multiple pathways may regulate apoptosis after transient overexpression of p53.
INTRODUCTION
Although phase I clinical trials using p53-mediated gene therapy to treat cancers of the lung or head and neck have now been completed (1 , 2) , a current limitation is inefficient delivery of the exogenous gene to target tumor cells. One way to circumvent this problem would be to use combinational or adjuvant therapy that could produce additive or synergistic cytopathic effects. Because the molecular pathway of p53-mediated programmed cell death is poorly understood, selection or optimization of an appropriate cotherapy is difficult. Moreover, some tumors (i.e., certain colon cancers) are relatively resistant to p53-mediated gene therapy (3) . A better understanding of the molecular events leading to cell death in sensitive cells could facilitate development of strategies for overcoming resistance.
Besides overexpression of wild-type p53, a variety of genotoxic agents are capable of inducing apoptosis in tumor cells. Considerable data suggest that all of these agents may operate through the same terminal cell events including activation of caspases (4 , 5) . Although the mechanism of caspase activation in apoptotic cells is controversial, recent evidence suggests that changes in mitochondrial membrane permeability (6) and leakage of cytochrome c (7) may be instrumental. How p53 could trigger such events is also a matter of controversy (8) .
Wild-type p53 functions as either a transcriptional activator or repressor of genes (9) . One candidate gene up-regulated by p53 that has been implicated in apoptosis is Bax (9, 10, 11) . Bax is a member of the Bcl-2 multigene family of homologous proteins that through the formation of hetero- and homodimers with each other seem to regulate apoptosis. Certain members of the Bcl-2 family including Bcl-2 and Bcl-xL inhibit apoptosis, whereas others such as Bax, Bcl-xS, and Bak accelerate or induce apoptosis (11) .
The proto-oncogene c-myc has also been implicated as a regulator of p53-mediated apoptosis (12, 13, 14)
. Paradoxically, c-myc is involved in stimulating both proliferation and apoptosis, depending on the cellular environment. Ectopic expression of c-myc under conditions that induce p53 can lead to apoptosis (12, 13, 14)
. Amplification or overexpression of c-myc occurs in a number of tumor types, including HNSCCs3
(14, 15, 16, 17)
. We have reported previously (18)
that the introduction of exogenous p53 into human HNSCC lines using the Ad5CMV-p53 vector results in apoptosis. Because c-myc has been associated with p53-mediated programmed cell death in lymphomas (19)
and rodent fibroblasts (20)
, it was hypothesized that elevated c-myc expression may also contribute to the apoptotic mechanism in HNSCC lines infected with Ad5CMV-p53. We, therefore, investigated levels of c-myc protein and mRNA in HNSCC lines undergoing Ad5CMV-p53 mediated cell death. In addition, we also looked for alterations in a panel of Bcl-2 homology proteins that had previously been implicated in regulating apoptosis.
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Cells and Reagents.
HNSCC lines Tu-167 and MDA 686LN were established at the Department of Head and Neck Surgery (University of Texas M. D. Anderson Cancer Center, Houston, TX) from patients with cancer of the floor of mouth and base of the tongue, respectively. Human HNSCC line Tu-138 has been described previously (21)
. All of the three HNSCC lines contain mutations in p53, and none of the three express wild-type p53. Cells were maintained in DMEM/F-12 medium supplemented with 10% fetal bovine serum, penicillin, streptomycin, and 2 mM glutamine.
Recombinant Adenovirus Infection.
The replication-defective adenovirus vector Ad5CMV-p53 that contains a human wild-type p53 gene and the control vector (dl312) that does not contain a p53 expression cassette have been described previously (21
, 22)
. Cells were grown in 10-cm dishes to a density of 2–3 x 106 per dish and infected with either Ad5CMV-p53 or dl312 at a MOI equal to 100 (i.e., 2–3 x 108 plaque-forming units) by coculturing with virus at 37°C for 1 h in 3 ml of complete medium. An additional 7 ml of complete medium was then added, and cells were cultured in the continued presence of virus for the lengths of time indicated. Using a recombinant adenovirus vector encoding the detectable green fluorescence marker GFP, we have previously reported that an MOI equal to 100 is more than sufficient to transduce 100% of a cell population for Tu-138 and Tu-167 cells (23)
.
Western Blotting.
Mouse monoclonal anti-p53 and rabbit polyclonal antibodies to Bak, Bax, Bcl-xL/S, Bcl-2, and c-myc were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibody specific for Bcl-xS was obtained from Oncogene Research (Cambridge, MA); mouse monoclonal anti-actin antibody was from Sigma (St. Louis, MO); and horseradish peroxidase-conjugated secondary antibodies were from Transduction Laboratories (Lexington, KY). For infection, 2–3 x 106 cells were plated in 10-cm dishes and infected on the following day with Ad5CMV-p53 or dl312 using 100 MOI. Infected cells were washed in PBS, scraped in ice-cold PBS containing protease and phosphatase inhibitors (1 mM PMSF, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 5 mM EDTA, 0.2 mM orthovanadate, and 10 mM NaF), pelleted, and lysed in ice-cold buffer [50 mM Tris, 150 mM NaCl, 1% NP40, 0.5% deoxycholate, and 0.1% SDS (pH 7.6)] containing protease and phosphatase inhibitors for 20 min at 4°C. Proteins resolved by SDS PAGE(50 µg/lane) were transferred to nitrocellulose filters using a Trans-Blot Semidry electroblotting apparatus (Bio-Rad, Richmond, CA) and analyzed by Western blotting using the enhanced chemiluminescence (ECL, Amersham) nonradioactive detection system as described previously (24)
. Blots were exposed to Hyperfilm (Amersham), and bands were quantitated with a densitometer (Molecular Dynamics Inc., Sunnyvale, CA).
Northern Blotting.
Total RNA was isolated with the TRI reagent (Molecular Research, Cincinnati, OH) according to the supplier’s detailed instructions. RNA (7.5 µg/sample) was fractionated by agarose-formaldehyde gel electrophoresis according to previously described methods (25)
, and transferred to a Hybond-N+positively charged nylon membrane (Amersham). Membranes were hybridized with [
-32P]-labeled probes in Rapid-hyb buffer (Amersham) according to the supplier’s instructions. Blots were exposed to Hyperfilm at -80°C in the absence of intensifying screens, and bands were quantitated by densitometry. Probes complementary to c-myc and GAPDH were prepared by subcloning PCR-amplified products corresponding to portions of each cDNA into the pCR-Script SK+plasmid (Stratagene, La Jolla, CA). PCR was performed after the reverse transcription of total RNA isolated from untreated Tu-138 cells. The sense and antisense primers used to amplify c-myc were 5'-CCCACCACCAGCAGCGACT-3' and 5'-ACTACCTTGGGGGCCTTT-3', respectively, which span an intron to generate a 450-bp amplicon containing sequences from exons 2 and 3. The sense and antisense primers used to amplify GAPDH were 5'-ACGGATTTGGTCGTATTGGG-3' and 5'-ACTAAAACCTCCCTAGAGCG-3', respectively, which amplify a 232-bp region spanning 3 exons. The human Bax cDNA probe was a gift from Dr. Ta-Jen Liu (University of Texas, M. D. Anderson Cancer Center, Houston, TX). Probes were labeled with 50 uCi [-32P]dCTP (3000 Ci/mmol) using a Megaprime DNA labeling kit (Amersham).
Antisense Treatment and Apoptosis Assay.
Tu-138 cells were treated with phosphorothioate-modified [S]ODN sequences using a regimen similar to that described previously (26)
. A 15-mer antisense [S]ODN (5'-AACGTTGAGGGGCAT-3') complementary to the translation initiation region of human c-myc mRNA and a 15-mer scrambled [S]ODN sequence (5'-AAGCATACGGGGTGT-3') containing a "G-quartet" prepared and purified by Genosys Biotechnologies (The Woodlands, TX) were resuspended in distilled deionized water, sterile filtered, and quantitated by measuring absorbance at 260 nm. Tu-138 cells were seeded into triplicate wells of an 8-well Permanox chamber slide (Fisher Scientific, Pittsburgh, PA) at an initial density of 1000 cells/well. The next day, [S]ODNs were added to a concentration of 100 µg/ml, followed by additional doses of 50 µg/ml at 48 h, 72 h, and 96 h from the time of plating. At 48 h after the last dose, slides were stained for apoptotic cells using an Apop Tag direct in situ fluorescein detection kit (Oncor, Gaithersburg, MD) according to the supplier’s instructions. Briefly, slides were rinsed in PBS, fixed in 10% neutral buffered formalin for 5 min, permeabilized with ethanol:acetic acid (2:1) at -20°C for 10 min, and incubated for 1 h with fluorescein-conjugated nucleotide and terminal deoxynucleotide transferase enzyme. Slides were photographed on a Nikon Optiphot microscope (Nikon, Garden City, NY) equipped with an episcopic-fluorescence attachment and Nikon FX-35DX 35 mm camera. To access the efficacy of antisense treatment, 3 x 104 Tu-138 cells were seeded into 60-mm dishes and incubated with [S]ODNs as described; proteins harvested 48 h after treatment were analyzed by Western blotting.
RESULTS
Infection with Ad5CMV-p53 Induces Profound Suppression of c-myc.
Previously we have shown that the infection of HNSCC lines Tu-138 and MDA 686LN with Ad5CMV-p53 induces apoptosis (18)
. We used these cell lines and the HNSCC line Tu-167, which also undergoes apoptosis with Ad5CMV-p53 infection,4
to study expression of apoptosis-related genes after exogenous introduction of wild-type p53. Levels of c-myc protein in Tu-138 cells were examined at 11 h and 18 h postinfection with 100 MOI of either Ad5CMV-p53 or the replication-defective adenovirus control vector (dl312) that does not harbor a p53 gene (Fig. 1)
. Two bands with apparent Mr 65,000 and 62,000 were detected by the anti-c-myc antiserum and probably correspond to Myc-1 and Myc-2, respectively, as cells from a wide range of species produce two c-myc proteins that result from alternative translational initiations of the same mRNA and differ in Mr by a few 1000 (27)
. Alternatively, the doublet detected by Western blotting could represent differentially phosphorylated forms of the same c-myc protein (28)
. Both p65- and p62 myc proteins disappeared in Tu-138 at 11 h after infection with Ad5CMV-p53 (Fig. 1A)
and were drastically reduced at 18 h (Fig. 1B)
compared with mock (no virus) and dl312-infected cells. Although levels of c-myc after 11-h infection with the dl312 control were increased compared with mock-treated cells, no difference between these two controls was observed at the 18-h postinfection time. To control for possible differences in protein loading and transfer, blots were stripped and reprobed with an antibody to actin, and levels of c-myc quantitated by densitometry were adjusted to actin. Levels of p62 myc after 18-h infection with Ad5CMV-p53 were reduced by a range of 55- to 65-fold compared with levels in mock-treated cells and by 48- to 50-fold (range) compared with dl312-infected cells. Levels of p65 myc after 18-h infection with p53 were reduced by 2- to 4-fold compared with dl312 infected cells and by 3- to 4-fold compared with mock-treated cells. Comparable reductions in c-myc protein after infection of Tu-138 with Ad5CMV-p53 have been observed in many other experiments and as late as 24 h after infection (data not shown), at which time a large percentage of Tu-138 cells were already apoptotic (18)
. Expression of exogenously introduced wild-type p53 was confirmed in Ad5CMV-p53-infected cells by Western blotting (Fig. 1)
. Three prominent bands with Mr close to 53,000 were highly up-regulated only in cells infected by Ad5CMV-p53, and these bands probably correspond to different posttranslational modifications of p53.
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. Both p65 and p62 myc were readily visible in mock and dl312-infected cells but were reduced to levels that were barely detectable by Western blotting 18 h after infection with Ad5CMV-p53. No differences in c-myc expression were observed among mock or dl312-infected controls.
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. Bcl-xs and Bcl-2 were undetectable by Western blot analysis in Tu-138 cells, regardless of whether the cells were infected. There was a modest (less than 2-fold) increase of Bak protein at 11 h postinfection with Ad5CMV-p53 (Fig. 3A)
, but by 18 h no differences in Bak levels were apparent (Fig. 3B)
. Levels of Bax after 11 h infection with Ad5CMV-p53 (Fig. 3A)
ranged from 2- to 6-fold higher and from 3- to 5-fold higher, respectively, when compared with mock and dl312 infected cells. Comparable increases (i.e., 2- to 6-fold range) in Bax were observed after 18 h infection with wild-type p53 (Fig. 3B)
and at 22 h or 24 h postinfection (data not shown).
Changes in Bax protein were also examined in Tu-167 cells after Ad5CMV-p53 infection. In preliminary experiments no increases in Bax protein were detected at 18 h after Ad5CMV-p53 infection. Because the onset of apoptosis in Tu-167 occurs at roughly 48 h postinfection,4
a later time point was also examined. At 45 h postinfection, levels of Bax protein were elevated by 2- to 3-fold compared with mock-treated cells, and by 4- to 6-fold compared with dl312-infected cells (Fig. 4)
. Bcl-2 was also undetectable in Tu-167 but increased Bcl-xL protein was observed in this cell line after 45 h of Ad5CMV-p53 infection (Fig. 4)
. Two bands with apparent Mr of approximately 29,000 each were recognized by the anti-Bcl-xL/S antiserum and may correspond to different posttranslational modifications of Bcl-xL. Although not yet demonstrated for Bcl-xL, at least two members of the Bcl-2 gene family are known to be modified by phosphorylation (29
, 30)
. Levels of the slower running Bcl-xL protein after p53 infection were increased by 25- to 30-fold compared with mock-treated cells and by 7- to 10-fold compared with dl312-infected cells. The faster running form of Bcl-xL was induced by an even greater amount. Because the antiserum recognizing Bcl- xL also can react with Bcl-xS (Mr 14,000–21,000), which is derived from alternatively spliced Bcl-x mRNA (11)
, another antiserum that is specific for the shorter form of Bcl-x was used to confirm that the doublet up-regulated in Ad5CMV-p53-infected Tu-167 was in fact Bcl-xL. As no Bcl-xS was detected in Tu-167 regardless of infection status (data not shown), it would appear that both bands of the doublet correspond to Bcl-xL. Expression of Bcl-2 homology proteins in MDA 686LN was not determined.
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. Levels of c-myc mRNA were not reduced by Ad5CMV-p53 infection at 0 h and 3 h. However, at 6 h after infection, c-myc message was reduced by greater than 2-fold when normalized to GAPDH (Fig. 5B)
and continued to decline at the later time points. By 12 h postinfection, c-myc mRNA levels had decreased more than 7-fold compared with dl312 infected cells. The time course of p53 protein production was also examined using protein fractions isolated from the same samples used in Northern analysis (Fig. 5C)
. Exogenously introduced p53 protein first appeared at 6 h post Ad5CMV-p53 infection and continued to increase in level at 9 h and 12 h. Only one prominent band corresponding to p53 increased after Ad5CMV-p53 infection, rather than the three bands usually detected, possibly because protein in this experiment was isolated using the TRI reagent.
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in Tu-138 cells after Ad5CMV-p53 infection. Bands corresponding to the two alternatively spliced species of baxmRNA (31)
were visible by Northern blotting, but only the Mr 21,000 bax protein encoded by the more predominate 1.0 kb mRNA could be detected on Western blotting. The time course of baxmRNA changes paralleled the appearance of p53 protein expression in a manner similar to what was observed for c-myc. By 6 h postinfection, there was approximately a 2-fold increase in the 1.0-kb Bax mRNA, and by 9 h postinfection, there was a 6-fold increase. Levels of baxmRNA remained elevated at 12 h postinfection.
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but were virtually undetectable (less than 0.5% of the population) after treatment with control scrambled ODNs (Fig. 7, C-D)
. The antisense experiment has been performed on two separate occasions with nearly identical results. Western blot analysis confirmed a greater than 90% reduction in c-myc protein levels (by densitometry) after treatment with antisense [S]ODNs (Fig. 7E)
. No detectable changes in Bax protein were found to occur after the c-myc antisense treatment (data not shown).
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Using HNSCC cell lines infected with Ad5CMV-p53 as a model, alterations in the expression of genes implicated previously in the regulation of p53-mediated apoptosis were investigated. Because c-myc amplification and overexpression commonly occur in HNSCC (14, 15, 16, 17) , it was anticipated that coexpression of c-myc and wild-type p53 would induce apoptosis in accordance with the "conflicting signal model" (12) . Unexpectedly, however, levels of c-myc mRNA and protein were rapidly suppressed in HNSCC lines infected with Ad5CMV-p53, and this suppression was sustained even at later time points when the majority of Tu-138 cells were already apoptotic (18) .
Other studies have examined cooperation between c-myc and p53 in triggering apoptosis. Ectopic expression of c-myc using exogenously introduced hormone-responsive gene constructs triggers apoptosis in serum-starved rodent fibroblasts (31) in a manner that has been shown to be dependent on p53 expression (20) . Exogenous introduction of a p53 temperature-sensitive mutant induces apoptosis at the permissive temperature in a v-myc-transformed murine lymphoma (19) , in a human Burkitt’s lymphoma containing the c-myc translocation (32) , and in a rat hepatocellular carcinoma with deregulated c-myc expression (33) .
Few studies have examined c-myc levels during p53-mediated apoptosis in cells that do not have deregulated or ectopic c-myc expression. During the induction of apoptosis using temperature-sensitive p53 mutants, a reduction in c-myc RNA levels has been reported in the murine myeloid leukemic M1 cell line (34) , and a decrease in c-myc protein has been observed in the murine erythroleukemic line DP16–1 (35) . We are unaware of reports that have examined the relationship between c-myc expression and p53-induced apoptosis in human tumors that do not contain translocations of c-myc. Our findings suggest that c-myc expression is similarly regulated in both murine leukemias and human HNSCC after the transient overexpression of p53.
The ability of p53 to repress transcription from the c-myc promoter has been demonstrated in vitro (36) and in cotransfection assays using a reporter gene driven by the c-myc promoter (37) . Repression involves the association between p53 and the TATA Box binding protein, which prevents the latter from forming transcriptional initiation complexes (36) . We observed decreased levels of steady-state c-myc mRNA levels that exactly paralleled the appearance of p53 protein expression in Tu-138, supporting the hypothesis that de novo synthesis of an intermediate is not required for transcriptional repression.
Reduction of c-myc alone using antisense [S]ODNs (in the absence of wild-type p53) induced apoptosis in a proportion of Tu-138 cells, whereas apoptotic cells were virtually undetectable in Tu-138 cells treated with the control scrambled [S]ODNs. Inability to detect cell death in a larger percentage of antisense treated cells could have been caused by an asynchronous apoptotic response to the 6-day treatment regimen, in as much as the terminal deoxynucleotidyl transferase mediated nick end labeling assay can detect apoptotic cells only during a narrow window in time. In agreement with our findings, antisense to c-myc has been reported to induce apoptosis in several other tumor types (26 , 38 , 39) . The ability of c-myc suppression alone to induce apoptosis in HNSCC lines raises the possibility that p53 may trigger programmed cell death in these cells by lowering c-myc levels. This possibility is further supported by the observation that c-myc protein levels dropped well before the onset of apoptosis in Ad5CMV-p53 infected cells, which normally begins at 20 h postinfection in Tu-138 cells and at 48 h in MDA 686LN (18) .
Presently, the role of Bax in p53-mediated apoptosis is controversial (40) . Overexpression of Bax alone can lead to changes in mitochondrial membrane permeability, release of cytochrome c, activation of caspases, and subsequent apoptotic cell death (41) . Recent evidence that cytochrome c can directly activate the caspase cascade (42) provides a link between Bax and crucial events in apoptosis. Overexpression of wild-type p53 leads to increased levels of Bax protein and subsequent apoptosis in murine leukemias (43) as well as in a human glioma (44) and hepatocellular carcinoma (45) . We detected increased Bax expression in two HNSCC lines infected with Ad5CMV-p53 at time points preceding onset of apoptosis, which is consistent with the hypothesis that Bax accelerates or induces programmed cell death in these cells. The magnitude of Bax protein induction that we observed in Tu-138 and Tu-167 cells after infection with Ad5CMV-p53 is comparable to what has been reported in human gliomas undergoing apoptosis after wild-type p53 expression (44) .
In Tu-167 cells, an increase in the apoptosis-inhibiting protein Bcl-xL was also observed along with increased Bax expression. Bcl-xL is known to antagonize Bax function and to inhibit p53-mediated apoptosis (46) . The magnitude of the Bcl-xL increase in Tu-167 was greater than that of Bax; however, the control of apoptosis may depend upon the ratio and the absolute amounts of various Bcl-2 homology proteins (11) , which cannot be determined from Western blots. Zhan et al. (47) reported increased levels of Bcl-xL mRNA and protein in human tumors undergoing radiation-induced apoptosis, and the Bcl-xL up-regulation was found to be strictly dependent on expression of endogenous wild-type p53. It was suggested that Bcl-xL induction may limit the severity of Bax activation in order for cells to recover from radiation-induced damage under some circumstances.
Ryan et al. (35) were able to protect murine DP16 cells from p53-mediated apoptosis by generating double-stable transfectants coexpressing ectopic c-myc and Bcl-2 (35) . As neither gene alone could prevent apoptosis, Ryan’s results argue that p53 can trigger apoptosis through parallel pathways: one involving c-myc reduction and the other being independent of c-myc but regulated by Bcl-2. The reduction in c-myc and the increase in Bax observed in our HNSCC lines undergoing p53-mediated apoptosis would be consistent with such a model. The possibility that p53 can induce two independently regulated apoptotic pathways, has important implications for gene therapy of cancer. It will be of interest to determine whether gene transfer of wild-type p53 can be made more effective by combining it with vectors containing the Bax gene or antisense directed against c-myc.
FOOTNOTES
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 Supported by an American Cancer Society Career Development Award, National Institute of Dental Research 1-P50-DE11906 (93-9); NIH First Investigator Award R29 DE11689-01A1 and Training of the Academic Head and Neck Surgical Oncologist T32 CA60374-3 (G. L. C.); and gifts to the University of Texas Division of Surgery and Anesthesia from Tenneco and Exxon for its Core Lab Facililty, and Cancer Center Core Grant NIH-NCI-CA16672. 
2 To whom requests for reprints should be addressed, at The University of Texas M. D. Anderson Cancer Center, Department of Head and Neck Surgery, 1515 Holcombe Boulevard., Box 69, Houston, TX 77030. Phone: (713) 792-6920; Fax: (713) 794-4662.
3 The abbreviations used are: HNSCC, head and neck squamous cell carcinoma; MOI, multiplicity of infection; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ODN, oligodeoxynucleotide.
Received 8/14/98; revised 10/22/98; accepted 11/13/98.
REFERENCES
-ray response of Bcl-xL depends on normal p53 function. Oncogene, 13: 2287-2293, 1996.[Medline]
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