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Clinical Cancer Research Vol. 6, 213-222, January 2000
© 2000 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Induction of Apoptosis in Oral Cancer Cells by an Anti-bcl-2 Ribozyme Delivered by an Adenovirus Vector1

Sandra A. Gibson, Christopher Pellenz, Robert E. Hutchison, Frederick R. Davey and Edward J. Shillitoe2

Departments of Microbiology and Immunology [S. A. G., C. P., E. J. S.] and Pathology [R. E. H., F. R. D.], State University of New York College of Medicine, Syracuse, New York 13210


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human oral cancer cells may have any of several genetic changes, but the role of the bcl-2 oncogene is relatively unexplored. To find out if this gene plays a significant role and whether it could act as a target for gene therapy of oral cancer, we have examined the effects of an anti-bcl-2 ribozyme on the phenotype of oral cancer cells. A hammerhead ribozyme was designed to cleave the bcl-2 transcript after nucleotide 279 and was confirmed to be effective against a synthetic bcl-2 transcript. A gene encoding the ribozyme was cloned into an adenovirus vector and transferred to the human oral cancer cell lines 686LN, 1483, and Tu183. Over a 6-day period, the growth of each cancer cell line was reduced, whereas growth of the fibroblast cell line FS7 was less inhibited. Inhibition of the oral cancer cells could be attributed to apoptosis, as indicated by the detection of histone-associated DNA fragments in an immunoassay. Northern blots showed no detectable reduction in the level of bcl-2 mRNA of Tu183 cells, but Western blots showed a reduction of Bcl-2 protein at 24 h after infection with the ribozyme-expressing adenovirus vector. The results imply that (a) expression of the bcl-2 oncogene is necessary for the survival of oral cancer cells, (b) the bcl-2 gene transcript presents a target for gene therapy by ribozymes, and (c) an adenovirus vector is a suitable method for transfection of the ribozyme-expressing gene.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human oral cancers show a variety of genetic changes, with different changes in different tumors. Numerous studies have reported mutation and overexpression of the gene for P53 (1) , overexpression of the receptor for epidermal growth factor (2) , and the presence of human papillomavirus DNA (3) .

Another phenotypic change in oral cancer, which has been studied less intensively, is overexpression of the oncogene bcl-2. The proportion of oral cancers in which Bcl-2 can be seen by immunochemical staining has been reported at between 50% and 75% of cases in most studies (4, 5, 6, 7) , although others have found it only rarely (8 , 9) . When present, it is generally more prevalent in the poorly differentiated tumors (4, 5, 6) . In normal oral mucosa, Bcl-2 is not detectable (9) or is expressed only occasionally in the basal cells (10) . Bcl-2 is frequently seen adjacent to oral cancers (4 , 6) and in dysplastic epithelium (6) . There have been no functional studies that explain the role of the Bcl-2 protein in oral cancer, although one study has shown that when oral keratinocytes were transfected with a bcl-2-expression plasmid, their level of differentiation markers was reduced (11) .

Overexpression of Bcl-2 is common in non-oral cancers. In follicular lymphomas, the overexpression is due to a chromosomal translocation, which places the bcl-2 gene under the control of a more active promoter (12) . Overexpression also occurs in some breast cancers (13) , glioblastomas (14) , lung cancers (15) , and colorectal carcinomas (16) . In these tumors, as in oral cancer, the reason for overexpression is unknown because there is no genetic rearrangement. Overexpression of bcl-2 is not a universal feature of cancer because it is not seen in malignant melanomas (17) or medulloblastomas (14) .

The function of the Bcl-2 protein is to block apoptosis in many cell pathways (18) , but not all. It cannot block the apo-ptosis that eliminates autoreactive T cells (19 , 20) or apoptosis of lymphocytes induced by the CD95 "death receptor" pathway (21 , 22) . The Bcl-2 protein does not transform cells by itself, but renders them transformable by other oncogenes such as myc (23) .

When expression of bcl-2 is inhibited, some cancer cells lose their malignant behavior. In lymphoma cells, the expression of bcl-2 can be blocked by antisense RNA, and this modifies the malignant phenotype of the cells (23 , 24) . An anti-bcl-2 ribozyme can cancel the ability of Bcl-2 to block the effects of proapoptotic agents on prostate cancer cells that had previously been transduced with bcl-2 (25) . Recently we showed that anti-bcl-2 oligonucleotides could induce apoptosis in human glioblastoma cell lines.3 In the present study, we have attempted to show whether Bcl-2 is important in the growth of oral cancer cell lines by blocking its function with a specific ribozyme.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Ribozyme.
A hammerhead ribozyme was designed to cleave the transcript of the bcl-2 gene between nucleotides 279 and 280. The ribozyme gene was then synthesized in two forms. One was a single-stranded oligonucleotide with an upstream T7 polymerase-binding site for in vitro synthesis of the ribozyme. The other was a double-stranded oligonucleotide, which was cloned into the eukaryotic expression vector plasmid pMAM-neo (Clontech Laboratories, Palo Alto, CA) adjacent to the MMTV promoter. The gene, together with the promoter and downstream poly(A) sequences, was then subcloned into the EcoRI and BamHI sites of the transfer plasmid pAvS6 (26 , 27) . This replaced the adenoviral tripartite leader and RSV promoter of pAvS6 with the MMTV promoter. The plasmid was then recombined into an adenovirus vector as described previously (27) to yield a virus designated Av1Rz279. The adenovirus was purified by ultracentrifugation on cesium chloride and was determined by titration to contain 109 pfu4 /ml. At the conclusion of experiments, the titer was determined again to ensure accuracy and to confirm that frozen stocks had not lost titer. The design and construction of the ribozyme was described earlier (28) , and it is illustrated in Fig. 1Citation .



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  		Fig. 1. The anti-bcl-2 ribozyme and its target. The open reading frame of the bcl-2 gene is shown with numbers that indicate nucleotide positions (54) . The positions of the Bcl-2 homology domains, BH1 through BH4, and the transmembrane domain, TM, are indicated (42) . The sequence of the target site in the gene transcript is shown and includes the cut site after nucleotide 279. Uppercase letters, the canonical GUC of the target site in the bcl-2 transcript and the conserved nucleotides of the ribozyme.

 
As a control for nonspecific adenoviral toxicity, the adenovirus vector Av1LacZ4 was used. This expresses ß-gal and has been used previously (27 , 29) . As an additional control, a vector was used in one experiment in which the ß-gal sequences had been replaced by the unrelated ribozyme Rz309, which targets a transcript of the human papillomavirus (30) .

Activity of Ribozyme in Vitro.
To confirm that the ribozyme had the potential to cleave the target RNA, a transcript of 19 bp that included the target site was prepared by run-off synthesis from an oligonucleotide, which encoded the target site and included the T7 promoter. T7 polymerase and 32P-labeled dCTP were used. Run-off transcripts of the ribozyme were prepared in the same way. Ribozyme and target transcripts were then mixed in ratios of from 1:0.1 up to 1:20 in the presence of 10 mM MgCl2 in 50 mM Tris buffer at 37°. At intervals, an aliquot was removed from the reaction mixture and examined by gel electrophoresis followed by phosphorimage analysis.

Cells.
The 686LN and 1483 oral cancer cell lines were obtained from Dr. P. Sacks, and the Tu183 cell line was obtained from Dr. G. Clayman (M.D. Anderson Cancer Center, Houston, TX). The human FS7 fibroblast cell line was kindly provided by Dr. R. Dougherty (SUNY, Syracuse, NY).

Infection of Cells.
To measure the ability of an adenovirus vector to transduce each cell type, cells were plated at 1000 cells/well in microtiter plates. The vector Av1LacZ4 was then added at titers of 105, 106, 107, or 108 pfu/ml in a volume of 50 µl. After 48 h, the expression of ß-gal was measured by spectrophotometry. To find the proportion of cells that were transduced, infected cells were fixed in 2% formaldehyde and 0.2% glutaraldehyde in PBS in 96-well plates, stained with X-gal, and counted under x200 magnification.

Expression of Ribozyme in Cells.
To confirm that the adenovirus vector Av1Rz279 did express the ribozyme, 5 x 106 Ad293 cells were plated in 80-cm2 flasks to form a 90% confluent monolayer by the next day. The cells were then infected with 1 ml of virus vector at 108 pfu/ml for 90 min. F-12 medium with 10% fetal bovine serum was added to the cultures and incubated overnight, and total RNA was extracted using the RNAqueous kit (Ambion, Austin, TX). Total RNA was reverse-transcribed with random primers using a reverse transcription-PCR kit (Stratagene, La Jolla, CA). The cDNA was then amplified by a PCR reaction. One primer, designated rib.sense, was designed to bind to the consensus sequence of a ribozyme and was complementary to the rib.core primer, which we used previously (29) . Its sequence was CTGATGAGTCCGTGAGGACGAA. The other primer, pAVS13', was designed to bind to a sequence that was derived from the adenoviral sequence of pAVS6. Its sequence was GCATCACAGGCTGGTTCC. Amplified products were visualized after electrophoresis on a 1% agarose gel to find out whether a fragment of the predicted size of 990 bp was observed. The product of the amplification reaction was also digested with Ase 1 to find out whether it was cut into the predicted sizes of 429 and 561 bp.

Expression of bcl-2 mRNA in Cells.
To examine the expression of bcl-2 mRNA in oral cancer cells, Northern blotting was performed. A 90%-confluent monolayer of Tu183 cells was formed in 80-cm2 flasks by plating 5 x 106 cells. The next day, the cells were infected with 1 ml of virus vector at 1 x 107 pfu/ml for 1.5 h at 37° with gentle agitation every 15 min. Ten milliliters of complete medium were added to each flask, and the cells were allowed to incubate a further 24 h. Poly(A) RNA was isolated with the MicroPoly(A)Pure isolation kit (Ambion) according to the manufacturers’ directions. The amount of poly(A) RNA was measured by UV spectrophotometry (1 A260 nm = 40 µg/µl), and 2 µg of poly(A) RNA were loaded on 1% agarose gel (NorthernMax kit, Ambion). Electrophoresis was performed at 5 V/cm, and RNA was transferred to a BrightStar membrane (Ambion) and UV cross-linked (Fisher Biotech Cross Linker). The membrane was prehybridized for 1 h and then hybridized at 65° for 16 h with a probe. The probe had been transcribed with T3 polymerase from the plasmid SYNBL2SM, which contains the bcl-2 open reading frame (kindly provided by Dr. S. Korsmeyer) and which had been linearized with Nsp1. The Ambion MAXIscript in vitro transcription kit was used, and labeling was accomplished with T7 polymerase and 32P dCTP. The membrane was washed to high stringency as described in the directions for the Ambion NorthernMax kit, was exposed to a Molecular Dynamics phosphorimager cassette, and was examined by phosphorimage analysis.

For detection of actin RNA, the membrane was then prehybridized again for 1 h at 65°. An RNA probe for actin was prepared by transcription from the pTRI-ACTIN HUMAN template (Ambion) and labeled as before. This probe was allowed to hybridize for 16 h, washed to high stringency, exposed again for 1 h to a phosphorimager cassette, and examined by phos-phorimage analysis.

Expression of Bcl-2 Protein in Cells.
To examine the expression of the Bcl-2 protein, Western blotting was performed. Cells were extracted with PBSTDS (Calbiochem-Novabiochem, San Diego, CA), which includes a cocktail of protease inhibitors. As a positive control, the lymphoma cell line SUDHL-6, which expresses high levels of Bcl-2 (31) , was included as well as several concentrations of a recombinant Bcl-2 protein (PharMingen, San Diego, CA). The concentration of Bcl-2 in the recombinant standard was assessed by its electrophoresis on a 12% polyacrylamide gel alongside several concentrations of albumin, followed by staining of the gel with amido black, and interpolation of the standard curve. In this way, we estimated that the concentration of Bcl-2 in the preparation was 30 ng/µl. The total protein concentration of each cell sample was determined by the BCA Protein Assay (Pierce, Rockford, IL), with optical absorbances read at 562 nm. Samples were separated on 12% SDS-PAGE gels, which were loaded with 10 µg of protein/lane, for 3 h. Proteins were then transferred to a nitrocellulose membrane of 0.45-µ pore size (Bio-Rad, Hercules CA) on a semidry electrotransfer unit (Owl Separation Systems, Woburn, MA). Hamster monoclonal antibody to human Bcl-2 (6C8, PharMingen) was incubated with the blot for 2 h at a 1/100 dilution. Biotinylated secondary antibody was reacted with the Vectastain avidin-biotin complex system and stained with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate using the Vectastain avidin-biotin complex-alkaline phosphatase system (Vector Laboratories, Burlingame, CA). Blots were then scanned by a digital scanner, and the scans were examined by computerized densitometry. A standard curve relating the density of each band to the concentration of Bcl-2 was prepared with the use of several dilutions of the recombinant Bcl-2 protein. Parallel gels were blotted and stained with an antiactin antibody (Sigma Chemical Company, St. Louis, MO), and other lanes of these gels were loaded with different concentrations of bovine actin to act as concentration standards.

Apoptosis.
To confirm that the anti-bcl-2-ribozyme induced apoptosis in cancer cells and to determine whether the effect of the ribozyme was specific for cancer cells, FS7 cells and Tu183 cells were grown in 96-well dishes that were seeded with 2 x 103 cells/well. After 24 h, the cells were infected with either Av1Rz279 or with Av1LacZ4, in volumes of 50 µl/well at titers of either 107 or 108 pfu/ml. Some cultures were exposed to camptothecin at 4 µg/ml. Infected cells were harvested after a further 24 h or 6 days, whereas the camptothecin-exposed cells were harvested after 4 h. These time points and concentrations had been determined to be optimal in pilot experiments.

For harvesting of cells, the supernatants were removed and discarded. The cells were then lysed in buffer and added to microtiter plates coated with streptavidin (Boehringer Mannheim Cell Death Detection ELISA-Plus kit). A mixture of anti-histone-biotin and anti-DNA-peroxidase were then added and incubated for 2 h according to the manufacturer’s directions. Nucleosomes were then quantitated by the addition of the substrate, [2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)], and the optical absorbance (A405 nm) was determined in a spectrophotometer. Each assay included a DNA-histone complex that was supplied with the kit as a positive control, and a negative control, which consisted of an incubation buffer in place of the cell lysate.

Growth of Cells.
To find the effect of the anti-bcl-2 ribozyme on viability of cells, their growth was monitored by their ability to metabolize (3–4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide to its blue product, formazan (32 , 33) as before (34) . Cells were plated in 96-well plates at a starting number of 1000 cells/well, and some wells were infected the following day with 50 µl of virus. Cultures were evaluated on the day of infection (Day 0) and on Days 2, 4, and 6. The A560–690 value was used as a measure of cell number. Each experiment was performed with at least 6 wells/group/experiment, and each experiment was performed at least twice. For infection by adenovirus, the concentration of virus varied from 105 to 108 pfu/ml.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Activity of Ribozyme in Vitro.
The target bcl-2 sequences were readily cleaved by exposure to the ribozyme, with increasing efficiency up to a ratio of 10:1 and increasing time up to 60 min. At a ratio of 1:1, >50% of the target was cleaved in 1 h (Fig. 2)Citation . The calculated value of the Kcat/Km ratio was 2.2 x 10-6 nm/liter/min.



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  		Fig. 2. In vitro assays showing the effects of the ribozyme:target ratio and of incubation time on cleavage of a synthetic bcl-2 19-bp target by the anti-bcl-2 ribozyme. The upper half of each panel shows a phosphorimage of a gel, and the lower half shows the percentage of the target that is present as a cleavage product. A, effects of the ribozyme:target ratio when the target and ribozyme were mixed for 60 min. B, effects of incubation time when the ribozyme:target ratio was 20:1.

 
Infection of Cells.
The proportion of cells that could be transduced by an adenovirus vector was determined by examining cells that had been infected with Av1LacZ4. With increasing concentrations of virus, increasing proportions of each cell type showed expression of ß-gal (Table 1)Citation . To obtain staining of 50% of cells, the FS7 and 686LN cells required 107 pfu/ml, the 1483 cells required 106 pfu/ml, and the Tu183 cells required 105 pfu/ml.


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  		Table 1 Proportion of each cell type that expressed ß-gal after infection with Av1LacZ4

 
The expression of ß-gal from infected Tu183 cells was detectable at 12 h after infection with 108 and 107 pfu/ml of virus and at 18 h after infection with 106 pfu/ml. The highest levels of expression were seen with 107 pfu/ml at 24 h, 36 h, and 48 h after infection.

Expression of Ribozyme in Cells.
The reverse transcription-PCR reaction revealed an amplimer of the expected size in cells that had been infected with Av1LacZ4 or Av1Rz279, but not in mock-infected cells. The band was cleaved by the restriction enzymes into two fragments of the expected sizes (not shown). This confirmed that the anti-bcl-2 ribozyme was expressed from the adenoviral vector.

Effect of Ribozyme on Expression of bcl-2 mRNA in Cells.
No differences were detected in the level of bcl-2 mRNA in Tu183 cells that had been infected with either Av1Rz279 or the control vectors Av1LacZ4 or Av1Rz309 (Fig. 3)Citation .



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  		Fig. 3. Northern blot showing the expression of bcl-2 RNA in cells. Tu183 cells were either mock-infected or infected with one of the adenovirus vectors as indicated. mRNA was extracted after 24 h and blotted to a membrane. The blot was probed first with a probe for bcl-2 mRNA and then for actin. The figure is a scan from a phosphorimage.

 
Effect of Ribozyme on Levels of Bcl-2 Protein in Cells.
The Bcl-2 protein was readily detected in the control SUDHL-6 cells by Western blotting, as expected (31) , and the concentration in different experiments varied between 0.5 µg and 1.1 µg/10 µg of cell protein. In Tu183 cells, the level of Bcl-2 varied between 0.1 µg and 0.6 µg/10 µg of cell protein. In FS7 cells, Bcl-2 was not detectable and was therefore present at a lower concentration than the lowest concentration of the standard, which was equivalent to 0.03 µg/10 µg of cell protein (Fig. 4)Citation . In 686LN cells and 1483 cells, there were detectable amounts of Bcl-2, but the apparent concentrations were lower than in SUDHL-6 or Tu183 cells, and the bands were not detected in every preparation.



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  		Fig. 4. Western blots showing expression of Bcl-2 protein in FS-7, SUDHL-6, and Tu183 cells. The Tu183 cells were either mock-infected or infected with Av1Rz279 or Av1LacZ4. Adenovirus vectors were at a concentration of 107 pfu/ml. In the upper half of each panel, some lanes contained recombinant Bcl-2 standards at the concentrations shown. The upper half of each panel was stained with anti-Bcl-2 serum. In the lower half of each panel, actin was used as a standard, and that half was stained with antiactin serum. A, cells that were infected for 6 h. B, cells that were infected for 24 h.

 
When Tu183 cells were infected with the adenovirus vectors, the level of Bcl-2 was unchanged during the first 6 h (Fig. 4A)Citation . After 24 h, the level of Bcl-2 declined in the cells that had been infected with Av1Rz279 but remained the same in cells that had been infected with Av1LacZ4. In the representative experiment shown in Fig. 4BCitation , the level of Bcl-2 was 0.9 µg/10 µg in the presence of Av1LacZ4 and 0.15 µg/10 µg in the presence of Av1Rz279. This was a reduction of 75%. The level of actin was unchanged.

Apoptosis.
After 2 days, the mock-infected Tu183 cells showed an A405 value of 0.02, and FS7 cells showed a value of 0.006. After 6 days, this had risen to 0.22 for Tu183 cells and 0.024 for FS7 cells. These values were considered as representing background levels and were subtracted from the values obtained from virus-infected cells at the same time points.

After 24 h of infection by Av1Rz279, the Tu183 cells showed apoptosis as indicated by a corrected A405 value of 0.49 when a virus titer of 108 pfu/ml was used. This dropped to 0.1 after 6 days (Fig. 5)Citation . When lower concentrations of virus were used, apoptosis was induced with peak values at later times (data not shown). Av1LacZ4 did not induce apoptosis in Tu183 cells at any titer tested at either of the time points used, and neither Av1Rz279 nor Av1LacZ4 induced apoptosis in FS7 cells (Fig. 5)Citation . Camptothecin induced apoptosis in Tu183 cells but with a maximum corrected absorbance value of only 0.031.



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  		Fig. 5. Apoptosis after infection by either Av1Rz279 or Av1LacZ4. Cells were Tu183 (upper panels) or FS7 (lower panels). Apoptosis was detected by an ELISA technique to detect histone-bound DNA. The corresponding value from mock-infected cells was subtracted.

 
Growth of Cells.
When cells were infected with the concentration of adenovirus that transduced 50% of cells, each of the oral cancer cell lines was inhibited by Av1Rz279 but not by Av1LacZ4. FS-7 cells were not inhibited under these conditions by either of the adenovirus vectors (Fig. 6)Citation . When the concentration of the virus vectors was varied from 105 up to 108 pfu/ml and growth inhibition was expressed as a percentage reduction on the sixth day of growth, some toxicity was detectable with each virus vector (Fig. 7)Citation ; under these conditions, Av1Rz279 produced up to 48% inhibition of FS7 cells and up to 98% inhibition of Tu183 cells. Av1Rz279 was always more inhibitory than Av1LacZ4 by about two logarithmic dilutions.



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  		Fig. 6. Growth curves of cells after either mock-infection or infection with Av1Rz279 or Av1LacZ4. Each cell line was infected with the titer of virus that transduced 50% of cells. This was 107 pfu/ml for FS-7 and 686LN cells, 106 for 1483 cells, and 105 for Tu183 cells. Growth was monitored by the (3–4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay.

 


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  		Fig. 7. Percent inhibition of growth of each cell type by different concentrations of adenovirus vectors after 6 days of culture under the conditions indicated for Fig. 6Citation . The arrows on the X-axes indicate the virus titer at which 50% of the cells were transduced.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A role for the Bcl-2 protein in oral cancer has often been suggested because of its visibility in some cases after immunohistochemical staining. However, its role in this disease has yet to be established. In the present study, the Bcl-2 protein was detectable in both the 686LN and 1483 oral cancer cells. In Tu183 oral cancer cells, the level of expression of Bcl-2 was extremely high and was similar to that of the lymphoma cell line SUDHL6 (Fig. 4)Citation . In contrast, the protein could not be detected in the FS7 fibroblasts. These data support a role for Bcl-2 in at least some oral cancers.

When a short synthetic fragment of the bcl-2 transcript was exposed to a ribozyme, it was found that the transcript had a susceptible site for cleavage between nucleotide 279 and 280. The site was found to be extremely sensitive, with >50% of the target cleaved within 1 min (Fig. 2A)Citation . These data suggested that a ribozyme might be used for examining the role of bcl-2 in oral cancer and exploring new therapeutic options. A gene that encoded the ribozyme could be introduced into cells quite readily by the use of an adenovirus vector. However, it was found that the different cells used in this study were transfected at different efficiencies by the adenovirus, and multiple concentrations of adenovirus were therefore used for evaluation of the ribozyme.

When the ribozyme was expressed in Tu183 cells, it led to a marked reduction in the level of the Bcl-2 protein (Fig. 4)Citation , which is consistent with its potent anti-bcl-2 effect. Interestingly, the ribozyme did not produce a detectable reduction in the level of the bcl-2 mRNA in the cells (Fig. 3)Citation . Therefore, although the ribozyme does have catalytic activity, the reduction of the Bcl-2 protein might have been due at least in part to the antisense activity of the flanking arms. It is known that bcl-2 can be inhibited by antisense oligonucleotides in lymphoma, glioma, and melanoma cells and that this affects the malignant phenotype and induces chemosensitivity (23 , 35 , 36) .3 Thus, the relative contributions of antisense and catalytic activities of Rz279 remain to be determined.

The reduction of the level of the Bcl-2 protein was accompanied by the appearance of apoptosis in the Tu183 cells, as shown by the appearance of histone-bound fragments of DNA (Fig. 5)Citation . Apoptosis appeared more rapidly when higher concentrations of virus were used, as might be expected because the marker gene was also expressed sooner with higher levels of virus. By 6 days after infection with Av1Rz279, apoptosis was detected with even the lowest concentration of virus, although by this time, the levels from the higher concentrations had passed their peak and were declining. FS7 cells did not show apoptosis with any concentration of virus at either of the two time points studied, which is consistent with the lack of Bcl-2 protein in these cells.

Consistent with the induction of apoptosis, the expression of the ribozyme affected the growth of the oral cancer cells with less effects on fibroblasts (Figs. 6Citation and 7)Citation . The ribozyme was particularly effective on the Tu183 cells, which are the cells that expressed the highest level of Bcl-2. Interestingly, the MMTV promoter that was used to express the ribozyme was found recently to be relatively weak in oral cancer cells (37) . Thus, even stronger anti-bcl-2 effects could be expected if a stronger promoter was used.

Adenovirus vectors do have some nonspecific inhibitory effects on oral cancer cells, as we have noted before (38) . The nonspecific toxicity is not related to the multiplicity of infection, but rather to the concentration of virus5 , and thus we have not shown the data in terms of multiplicity of infection. The nonspecific effects were easily distinguished from specific effects in four ways: (a) The anti-bcl-2 ribozyme was more inhibitory in cells that expressed the Bcl-2 protein. (b) At the virus concentration, which transduced 50% of cells, the ribozyme-expressing virus was more toxic than the ß-gal-expressing virus in each cell type. (c) The ribozyme-expressing virus induced apoptosis in the Tu183 cells but not in fibroblasts, and the ß-gal-expressing virus did not induce apoptosis in either cell type. (d) The ribozyme-expressing virus reduced the level of the Bcl-2 protein in Tu183 cells, whereas the ß-gal-expressing virus did not.

The present study appears to be the first that shows the use of a ribozyme to modify the transformed phenotype of a tumor cell line that endogenously expresses bcl-2. In an earlier study, an anti-bcl-2 ribozyme did affect the phenotype of a prostate cancer cell line, but the cells had previously been transfected with a bcl-2-expressing plasmid (25) . Furthermore, the target site that was used in the present study, nucleotide 279, does not appear to have been used previously in either antisense or ribozyme studies.

The data from this study suggest that both pro- and antiapoptotic mechanisms are at play in oral cancer cells. The induction of apoptosis by the anti-bcl-2 ribozyme implies that these cells are constitutively expressing a proapoptosis phenotype, which is simultaneously being suppressed by Bcl-2. The origin of the proapoptosis phenotype is unknown, although there are at least three possibilities: (a) Normal epithelial cells are programmed to differentiate, undergo apoptosis and die, and this mechanism might persist in oral cancer. (b) Oral cancer occurs in patients whose cells are inherently susceptible to DNA damage by mutagens (39) , and DNA damage generally leads to apoptosis (18) . (c) When epithelial cells are separated from their substrate, this triggers apoptosis through a mechanism known as anoikis (40) , which can be prevented by overexpression of Bcl-2 (41) . The regulation of apoptosis in oral cancer might involve many other proteins apart from Bcl-2. A large number of pro- and antiapoptotic proteins have been documented in various cell types (18) and could be involved in the regulation of apoptosis in oral epithelium and oral cancer. However, nothing appears to be known about the regulation of apoptosis in oral cancer except that the present data imply that it is a Bcl-2-sensitive pathway that produces the antiapoptotic signal.

If expression of an anti-bcl-2 ribozyme can inhibit the growth of oral cancer cells, this might be useful in cancer therapy. The fact that the ribozyme can be delivered by a viral vector suggests that this is possible. The fact that the fibroblast line FS7 was not inhibited implies that this could be relatively safe. A further advantage of any anti-bcl-2 treatment would be that it might sensitize cells to conventional therapy because Bcl-2 is known to inhibit the anticancer effects of several chemotherapeutic drugs (42 , 36) . It has been shown that levels of Bcl-2 can be reduced by retinoic acid with resulting sensitivity to chemotherapy (43) , and it is known that retinoic acid can reduce the malignant progression of leukoplakia and inhibit development of second primary and recurrent oral cancers (44) . It is thus possible that the protective effects of retinoids occur by their effect on a proapoptotic pathway that involves Bcl-2.

There are other examples of ribozymes that inhibit the malignant phenotype of human cancer cells when delivered by a viral vector. Adenoviruses have been used to deliver ribozymes against ras to bladder and lung cancer cells (45) and against HER-2/neu or pleiotropin to other cancer cells (46) . Retroviruses have been used to deliver ribozymes against ras to malignant melanoma cells (47) and against BCRABL to lymphoid cells (48) . Despite these few examples, most earlier studies of ribozyme expression have used a plasmid expression vector or used target cells that were transfected with the transforming gene. Examples of targets include papillomaviruses (30 , 49) , EBV (50) , epidermal growth factor (51) , and ras (52 , 53) . The present study extends the potential use of virus vectors for the expression of ribozymes in human cancer cells and increases the spectrum of potential targets in oral cancer to include the bcl-2 oncogene.


   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 NIH Grant DE10842 (to E. J. S.). Back

2 To whom requests for reprints should be addressed, at Department of Microbiology and Immunology, SUNY College of Medicine, 750 East Adams Street, Syracuse NY 13210. Back

3 The abbreviations used are: pfu, plaque-forming units; ß-gal, ß-galactosidase. Back

4 Gibson et al., manuscript in preparation. Back

5 Julien, T., Frankel, B., Longo, S., Kyle, M., Gibson, S., Shillitoe, E. J., and Ryken, T. Antisense-mediated inhibition of the bcl-2 gene induces apoptosis in human malignant glioma, submitted for publication, 1999. Back

Received 8/ 2/99; revised 10/21/99; accepted 10/25/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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