In Vitro Growth Suppression by Adenoviral Transduction of P21 and P16 in Squamous Cell Carcinoma of the Head and Neck a Research Model for Combination Gene Therapy

ORIGINAL ARTICLE In Vitro Growth Suppression by Adenoviral Transduction of p21 and p16 in Squamous Cell Carcinoma of the Head and Neck A Research Model for Combination Gene Therapy

Steven Ross Mobley, MD; Ta-Jen Liu, PhD; J. Michael Hudson, PhD; Gary L. Clayman, DDS, MD

Objectives: To determine the duration of expression Ad5CMV-p16 alone. Cell cycle analysis demonstrated of the cell cycle regulators p21 and p16 and the effect of that the percentage of cells arrested at G1 stage in the these 2 genes both alone and in combination on the cells transduced with Ad5CMV-p16 was similar to that growth of squamous cell carcinoma of the head and in the cells transduced with both Ad5CMV-p21 and neck cell lines in vitro. Ad5CMV-p16. No significant in vivo growth suppression was observed in tumor nodules treated with Experimental Methods and Design: Cells were trans- Ad5CMV-p16. duced via an adenoviral vector with p21 (Ad5CMV- p21), p16 (Ad5CMV-p16), or both. Western blotting was Conclusions: Although p21 and p16 are believed to func- performed to determine duration of expression of the pro- tion as cell cycle regulators of cyclin-dependent ki- tein products of the transduced p21 and p16 genes. In nases, we observed no additive or synergistic effect when vitro growth assays and cell cycle analyses were per- using them in combination. The expression of trans- formed on transduced cells. duced p21 and p16 gene products up to days 9 and 12, respectively, was consistent with the growth suppres- Results: Transduced gene products were detected up sion and cell cycle arrest observed. This work provides to day 12 after infection. Western blotting showed high information on the previously uncharacterized dura- levels of p21 and p16 in transduced cells. Growth sup- tion of p21 and p16 transgene product expression and pression was observed in squamous cell carcinoma of also lends insight into the interaction of these 2 cell the head and neck cell lines transduced with Ad5CMV- cycle regulators in squamous cell carcinoma of the head p21, Ad5CMV-p16, or both, but the combination of p21 and neck. and p16 did not achieve significantly greater growth suppression than that seen in cells transduced with Arch Otolaryngol Head Neck Surg. 1998;124:88-92

ECHNOLOGY in the field of Another potential tumor suppressor molecular biology is devel- gene, p16, has recently been cloned and lo- oping at a rapid pace, pro- calizedonchromosome9p21.6 Thisgenehas viding investigators with demonstrated many characteristics of a tu- increasing opportunities to mor suppressor gene. It is a known inhibi- Tattenuate the growth of cancer cells at the tor of cyclin-dependent kinase 4, whose ac- molecular level. As of June 1996, there were tivity is critical to regulation of normal cell approximately 232 approved gene therapy cycle progression.6 Deletions of p16 have clinical trials worldwide, with 175 having been shown to occur at a high frequency in been initiated.1 These trials are evaluating tumorsandhavebeendetectedin51%ofsqua- the utility of gene therapy in treating a va- mous cell carcinomas of the esophagus.7,8 rietyofcancers,includingbrain,breast,lung, The actions of another tumor and squamous cell carcinoma of the head suppressor gene, p21, have been demon- and neck (SCCHN). Much interest has fo- strated to be transcriptionally regulated cused on the clinical potential of this new downstream by p53.9,10 Like p16, p21 has modality to treat cancer, especially on the the potential to affect the cell cycle by use of tumor suppressor genes. Previous inhibiting a cyclin-dependent kinase (cy- 11 From the Department of Head studies of tumor suppressor gene therapy clin E). Several features suggest that and Neck Surgery, The for SCCHN focused largely on the use of p21 too is a tumor suppressor gene. Cells 2-4 University of Texas M. D. p53. Theactionofp53,asnowunderstood, overexpressing p21 accumulate in G1, Anderson Cancer Center, is to induce cell death (apoptosis) and cause and p21 has been shown to participate in 5 Houston. cell cycle arrest at the G1 checkpoint. G1 checkpoint regulation downstream

ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 124, JAN 1998 88

©1998 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 MATERIALS AND METHODS STATISTICAL ANALYSES The effect of viral infection on cell growth was analyzed CELL LINES by means of a 1-way analysis of variance test. For the days on which the groups exhibited a significant difference, the Tumor cells TU-138 and TU-167 were established from hu- individual means were compared with each other also by man primary SCCHN and have been described else- a 1-way analysis of variance. All statistical analyses were 14 where. TU-138 has a known single-base-pair mutation of performed by means of SigmaStat software (Jandel Corp, p53. TU-167 has a splicing mutation at exon 5 of p53. San Rafael, Calif). RECOMBINANT ADENOVIRUSES WESTERN BLOT ANALYSIS The recombinant adenovirus Ad5CMV-p21 contains the cytomegalovirus promoter, wild-type p21 complemen- Total cell lysates in protein lysis buffer (150-mmol/L so- tary DNA, and SV40 polyadenylation signal in a mini- dium chloride, 1.0% NP-40, 0.5% deoxycholate, 0.1% so- gene cassette inserted into the E1-deleted region of dium dodecyl sulfate, 50-mmol/L Tris [pH, 8.0], phenyl- modified adenovirus type 5 (Ad5). The Ad5CMV-p16 methylsulfonylfluoride, and aprotinin) were obtained. adenoviral vector is similar in construction. Our methods Protein concentration, transfer, and blocking were per- for recombinant viral vector construction have been formed as described elsewhere.2 The membrane was probed described elsewhere.2 with primary antibodies to p16 (rabbit anti–human poly- clonal antibody [Santa Cruz Biotechnologies, Santa Cruz, ADENOVIRAL INFECTION FOR DURATION Calif]), p21 (mouse anti–human monoclonal antibody [On- OF INDUCED p21 AND p16 PROTEIN EXPRESSION cogene Science Inc, Union Dale, NY]) and ␤-actin (mouse anti–human monoclonal antibody as a loading control [Am- ersham Inc, Arlington Heights, Ill]). Secondary antibod- The SCCHN cell line TU-138 was plated and infected at ies were horseradish peroxidase–conjugated goat anti- 30 multiplicity of infection (MOI) of Ad5CMV-p21 or mouse and antirabbit (Amersham Inc). The membranes were Ad5CMV-p16. Total cell protein lysates were obtained on processed and developed according to the manufacturer’s days 1, 2, 3, 6, 8, 9, 12, and 14 and assayed by Western suggestions. blot analysis.

ADENOVIRAL INFECTIONS CELL CYCLE ANALYSIS

Cells infected with the following schema were used for Cell monolayers were trypsinized, washed with cold the in vitro growth assays, cell cycle analyses, and corre- PBS, and fixed by storing in 70% ethanol at −20°C. sponding Western blots. Three adenoviruses were used: These fixed cells were washed twice with PBS before cell Ad5CMV-p21, Ad5CMV-p16, and Ad5CMVPA. cycle analysis. Approximately 1.0ϫ106 cells were resus- Ad5CMVPA is an adenovirus that does not carry a thera- pended in 70-µg/mL RNase A and 5-µg/mL propidium peutic gene and serves as a vector-only control. We used iodide in a final volume of 900 µL of PBS. After gentle 4 treatment groups and 1 mock treatment group. All mixing, cells were incubated in the dark for 30 minutes at treatment groups received 2 treatments with an adenovi- room temperature. Samples were analyzed by means of a rus vector, 48 hours apart, at 50 MOI. In this schema, flow cytometer (EPICS Profile II, Coulter Corp, Miami, after 48 hours, all treatment groups had been exposed to Fla). The proliferative fraction was determined as the per- two 50-MOI viral infections. The treatment groups were centage of cells in S-phase according to the method of as follows: group 1 received Ad5CMVPA/Ad5CMVPA; Baisch et al.15 group 2 received Ad5CMV-p21/Ad5CMVPA; group 3 received Ad5CMV-p16/Ad5CMVPA; and group 4 received Ad5CMV-p21/Ad5CMV-p16. The mock treatment group IN VIVO GROWTH SUPPRESSION received no viral vector infection. The effect of Ad5CMV-p16 on a microscopic residual MEASUREMENT OF TUMOR CELL GROWTH disease model of SCCHN was determined in nude mice. Eight mice were used for both cell lines TU-138 and TU- Tumor cells (TU-138 or TU-167) were seeded into 6-well 167 and the experiment was performed twice. Ex- plates at a density of 2.0ϫ104 cells/well. At specified times periments were reviewed and approved by institutional after adenoviral infection, medium was removed from the committees for both Animal Care and Utilization and wells containing the cells to be counted, and cells were the Biosafety Committee for Recombinant DNA rinsed with phosphate-buffered saline (PBS) and trypsin- Research. ized (0.25%) with scraping to recover all cells. Cells were Subcutaneous flaps were seeded with tumor cells in immediately added to 5 mL of growth medium containing the dorsal flanks of the animals in a manner described 10% serum, pelleted by centrifugation, then resuspended elsewhere.4 Forty-eight hours after tumor cell delivery, in 1 mL of PBS. Growth measurements for a specific day the flaps were infected with Ad5CMV-p16, Ad5CMVPA, were obtained by counting triplicate wells of cells on an or PBS. The mice were observed daily for tumor devel- automated cell counter (Coulter model Zf, Coulter Corp, opment and were killed immediately in cases of exces- Miami, Fla). sive tumor burden or after 12 weeks of observation.

ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 124, JAN 1998 89

β-Actin β-Actin

p16 p21

Figure 1. Duration of expression of transduced p16 (left) and p21 (right). Squamous cell carcinoma of the head and neck cell line TU-138 was infected at 30 multiplicity of infection and total cell protein lysates were obtained on days 1, 2, 3, 6, 8, 9, 12, and 14. Lane numbers correspond to the number of days after adenovirus vector infection.

12345 12345

β-Actin

34 kb 34 kb p21 p21 14 kb p16 p16 14 kb

Figure 2. Western blot analysis. Cellular extracts from squamous cell carcinoma of the head and neck cell lines TU-138 (left) and TU-167 (right) isolated 24 hours after the second infection were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Lane 1 represents mock-infected cells; lane 2, vector-only (Ad5CMVPA) control-infected cells; lane 3; Ad5CMVPA/Ad5CMV-p16–infected cells; lane 4, Ad5CMV-p21/Ad5CMVPA–infected cells; and lane 5, combination infection with Ad5CMV-p21/Ad5CMV-p16.

from p53.11-13 Others have shown that p21 has the abil- in vitro growth assays. Western blotting demonstrated ity to function as a tumor suppressor in both colon and high levels of protein expression of p16 and p21.Wedid prostate cancer cell lines.9,14 not detect any significant basal levels of either p16 or p21 Since p21 and p16 both regulate the cell cycle as in- in mock-infected cells (Figure 2). In addition, sequenc- hibitors of different cyclin-dependent kinases, we sought ing of p16 in our laboratory from both TU-138 and TU- to determine whether combination gene therapy using 167 disclosed no mutations (data not shown). There- both genes would additively or synergistically inhibit the fore, the cell cycle arrest observations in these experiments growth of SCCHN cells. can be attributed to exogenously supplied p16 and p21, which are not in competition with abnormal levels of RESULTS either wild-type or mutant p16. Expression of p21 or p16 was similar whether the genes were delivered alone with DURATION OF EXPRESSION control vector or in combination. Actin controls con- OF TRANSDUCED p21 AND p16 firmed equal protein loading (Figure 2).

To investigate the duration of expression of p21 and p16 IN VITRO GROWTH RATE transgenes in TU-138 and TU-167, after viral infection of OF TRANSDUCED CELLS each of these cell lines, cells were harvested and protein extracts prepared at different times after infection. Western The effects of transduced p21, p16, and both p21 and p16 blot analyses were performed to detect protein levels at dif- on the growth rates of SCCHN were assessed. The vec- ferent times. Western blotting showed high levels of trans- tor Ad5CMVPA served as a control for growth inhibi- duced gene products up to day 9 to 12 after infection. No tion observed in cells treated with viral vector alone. With significant expression was seen by day 14 (Figure 1). the use of the infection schema described above, cell counts were obtained for 1 week after the first infection. TRANSGENE PROTEIN PRODUCT EXPRESSION Marked growth suppression was seen in cells transduced with Ad5CMV-p21/Ad5CMV-p16. However, this Forty-eight hours after the first infection, protein ex- effect was not significantly different from that of the group tracts were obtained for all 5 groups represented in the Ad5CMVPA/Ad5CMV-p16.Ondays5to7(Figure 3),

ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 124, JAN 1998 90

©1998 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 Mock Mock VO/VO VO/VO 20.0 20.0 VO/p16 VO/p16 p21/VO p21/VO p21/p16 p21/p16 15.0 15.0 5 5 10 10 × ×

10.0 10.0 Cell Count, Cell Count,

5.0 5.0

0.0 0.0 0 2 4 68 0 2 4 68 Day Day Figure 3. Inhibition of squamous cell carcinoma of the head and neck cell growth in vitro. Cell lines TU-138 (left) and TU-167 (right) were examined. VO indicates Ad5CMVPA (the vector-only control); p16, Ad5CMV-p16; p21, Ad5CMV-p21. At each indicated time, 3 dishes of cells were trypsinized and counted. The mean of cell counts per triplicate well after infection was plotted against the number of days since first infection; bars indicate SEM.

the mock-infected cells (no viral infection) or the cells infected with vector only exhibited statistically signifi- Cell Cycle Analysis of TU-138 and TU-167* cant differences (PϽ.001) from those cells infected with either p16 alone or the combination of p16 and p21.No TU-138 TU-167 Time After significant growth suppression was seen in the Ad5CMV- Infection, h Vector %G0/1 %S+G2M %G0/1 %S+G2M p21/Ad5CMVPA group or the 2 control groups, mock or 48 V0/V0 73.6 26.4 60.8 39.2 vector-only control (Ad5CMVPA/Ad5CMVPA), for TU- p21/V0 87.3 12.7 84.7 15.3 167 (Figure 3, right). Significant growth suppression 72 Mock 33.9 66.1 35.3 64.7 (PϽ.001) was observed for TU-138 (Figure 3, left) in the V0/V0 47.3 52.7 50.1 49.9 Ad5CMV-p21/Ad5CMVPA group compared with mock- V0/p16 72.9 27.1 62.2 37.8 infected or vector-only infected groups. p21/V0 61.8 38.2 86.3 13.7 p21/p16 78.8 21.2 90.7 9.3 96 V0/V0 57.1 42.9 73.1 26.9 CELL CYCLE ANALYSIS V0/p16 70.0 30.0 87.3 12.7 p21/V0 87.9 12.1 86.3 13.7 To measure the effect of the p16 and p21 transgenes on p21/p16 89.9 10.1 92.3 7.7 cell cycle progression in both TU-138 and TU-167, we performed flow cytometric analyses of infected cells. Flow *VO indicates vector only; mock, no viral infection; p16, p16 infection; cytometry demonstrated that cells transduced with either p21, p21 infection; % G0/1, percentage in stage G0/1;and%S+G2M, percentage in S-phase plus stage G2M. Vector combinations are designated p21, p16, or both became arrested at the G1 stage of the as, for example, p21/p16 (infection with p21 vector followed by infection cell cycle up to 96 hours after infection (Table). At 96 with p16 vector 48 hours later). hours, TU-138 cells transduced with p21 alone were 88% in G1 and cells transduced with p16 alone were 70% in G1, while those cells transduced with both p16 and p21 promise as our understanding of molecular mecha- were 90% in G1. By comparison, at 96 hours, TU-167 cells nisms regulating malignant neoplasms progresses. Clini- transduced with p21 alone were 86% in G1, cells trans- cally, one could hypothesize the theoretical advantage duced with p16 alone were 87% in G1, and cells trans- of treating tumors with 2 genes that in combination have duced with both p16 and p21 were 92% in G1. a greater tumor-suppressive potential than either gene alone. While other authors have examined the effects of IN VIVO MODEL either p21 or p16 in vitro, no one has looked at the potential of these 2 genes to act synergistically.9,14,16 The com- On the basis of the significant in vitro growth suppres- bination of p16 and p21 was chosen because the prod- sion attributable to p16 gene alone, we used a nude mouse ucts of both of these genes are known to function through model for microscopic residual disease to determine the similar mechanisms of cell cycle regulation by inhibi- biological effect of p16 in vivo. No significant growth sup- tion of cyclin-dependent kinases. pression was observed in these studies (data not shown). DURATION OF TRANSGENE COMMENT PRODUCT EXPRESSION Combination gene therapy, which may selectively regu- We have described elsewhere that the adenoviral vector late tumor cell growth, induce apoptosis, or regulate sen- is an effective vehicle for the delivery of exogenous genes.2 sitivity to radiation therapy or chemotherapy, may have To better characterize the efficacy of the p21 and p16 genes

ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 124, JAN 1998 91

©1998 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 in suppressing tumor growth, Western blotting was per- ciety, Atlanta, Ga; grant 1-P50-DE11906 from the Na- formed to confirm the duration of overexpression of these tional Institute of Dental Research, Bethesda, Md; First transgene products. To our knowledge, this had not been Investigator Award R29 DE11689-01A1 from the National previously described and was critical to our understand- Institutes of Health, Bethesda; Training of the Academic Head ing of the duration of action of these cell cycle regula- and Neck Surgical Oncologist grant T32 CA60374-03 (GLC) tors. Infections were performed at only 30 MOI because and cancer center core grant NIH-NCI-CA-6672 from the further increases in MOI resulted in such marked growth National Cancer Institute, National Institutes of Health, suppression that extracts could not be obtained for Bethesda; and a gift to the Division of Surgery and Anes- Ad5CMV-p16 beyond day 6 (unpublished data). West- thesia from Tenneco Inc, Greenwich, Conn, for its core labo- ern blotting of protein extracts from SCCHN cell lines ratory facility. up to 14 days after transduction with either p21 or p16 Presented at the Fourth International Conference on confirmed that high levels of exogenous protein were de- Head and Neck Cancer, Toronto, Ontario, July 28, 1996. tectable up to 12 days after infection. We thank Mary Wang, MS, for technical support, Jose Western blotting of the protein extracts correspond- C. Juarez for his assistance in computer graphics, Dianna ing to the 5 experimental treatment groups used in the B. Roberts, PhD, for her assistance with statistical analy- in vitro growth assay confirmed also that high levels of ses, and Peggy Tinkey, DVM, for veterinary assistance. p21 and p16 could be detected in cells transduced with Reprints: Gary L. Clayman, DDS, MD, The Univer- either p21 or p16 adenoviruses, or a combination of both. sity of Texas M. D. Anderson Cancer Center, Department However, no expression of these gene products was de- of Head and Neck Surgery, 1515 Holcombe Blvd, Box 69, tected in the mock infection or vector-only control groups. Houston, TX 77030 (e-mail: [email protected]. tmc.edu). IN VITRO AND IN VIVO BIOLOGICAL EFFECTS OF AD5CMV-p21 AND AD5CMV-p16 REFERENCES

Moderate growth suppression was seen in cells trans- 1. Anderson, WF. End-of-the-year potpourri. Hum Gene Ther. 1996;7:2201-2202. duced by Ad5CMV-p21. However, more marked growth 2. Liu TJ, Zhang WW, Taylor DL, Roth JA, Goepfert H, Clayman GL. Growth sup- suppression was observed in cell lines transduced with pression of human head and neck cancer cells by the introduction of a wild-type either Ad5CMV-p16 or the combination Ad5CMV-p21/ p53 gene via a recombinant adenovirus. Cancer Res. 1994;54:3662-3667. Ad5CMV-p16. The growth suppression resulting from in- 3. Liu TJ, El-Naggar AK, McDonnell TJ, et al. Apoptosis induction mediated by wild- type p53 adenoviral gene transfer in squamous cell carcinoma of the head and fection with Ad5CMV-p21/Ad5CMV-p16 was similar to neck. Cancer Res. 1995;55:3117-3122. that seen with the group Ad5CMVPA/Ad5CMV-p16. 4. Clayman GL, El-Naggar AK, Roth JA, et al. In vivo molecular therapy with p53 These observations suggest that the growth suppression adenovirus for microscopic residual head and neck squamous carcinoma. Can- seen with combination p21/p16 was not the result of an cer Res. 1995;55:1-6. 5. Weinberg RA. Tumor suppressor genes. Science. 1992;254:1138-1145. additive or synergistic interaction of these 2 genes, but 6. Serrano M, Hannon G, Beach D. A new regulatory motif in cell cycle control caus- rather was attributable principally to the effects of the ing specific inhibition of cyclin D/CDK 4. Nature. 1993;366:704-707. p16 gene alone. 7. Okamoto A, Demetrick DJ, Spillare EA, et al. Mutations and altered expression No significant in vivo growth inhibition by Ad5CMV- of p16INK4 in human cancer. Proc Natl Acad SciUSA. 1994;91:11045-11049. p16 was observed in xenograft nude mouse animal ex- 8. Mori T, Miura K, Aoki T, Nishihira T, Mori S, Nakamura Y. Frequent somatic mutations of the MTS1/CDK41 (multiple tumor suppressor/cyclin dependent ki- periments. This is understandable, since the growth- nase 4 inhibitor) gene in esophageal squamous cell carcinoma. Cancer Res. 1994; suppressive transgene product persisted for only about 12 54:3396-3397. days, which would have allowed at most only a slight de- 9. Eastham JA, Hall SJ, Sehgal I, et al. In vivo gene therapy with p53 or p21 ad- lay in tumor formation. In contrast, our previous studies enovirus for prostate cancer. Cancer Res. 1995;55:5151-5155. have shown that transient expression of p53 inhibits tu- 10. Waldman T, Kinzler KW, Vogelstein B. p21 is necessary for the p53-mediated G1 arrest in human cancer cells. Cancer Res. 1995;55:5187-5190. mor formation by induction of apoptosis, not by cell cycle 11. Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ. The p21 cdk-interacting 3 arrest. This suggests that cell cycle regulators may have protein cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993;75: little utility as a gene intervention strategy alone. How- 805-816. ever, cell cycle regulators may sensitize tumors to other 12. Harper JW, Elledge SJ, Keyomarsi K, et al. Inhibition of cyclin dependent kinases by p21. Mol Biol Cell. 1995;6:387-400. interventions, such as chemotherapy or radiation therapy. 13. Deng C, Zhang P, Harper JW, Elledge SJ, Leder P. Mice deficient in p21 develop

Further basic investigations of gene therapy strategies, alone normally but have defects in G1 checkpoint function. Cell. 1995;82:675-684. or in combination, that may selectively destroy malig- 14. Chen YQ, Cipriano JM, Arenkeil JM, Miller FR. Tumor suppression by p21WAF1. nant neoplasms but spare normal cells, are needed. Cancer Res. 1995;55:4536-4539. 15. Baisch H, Gohde W, Linden, WA. Analysis of PCP-data to determine the fraction of cells in the various phases of cell cycle. Radiat Environ Biophys. 1975;12:31-39. Accepted for publication July 24, 1997. 16. Jin X, Nguyen D, Zhang WW, Kyritsis AP, Roth JA. Cell cycle arrest and inhibi- This study was supported in part by a Career Devel- tion of tumor cell proliferation by the p16INK4 gene mediated by an adenovirus opment Award (grant 93-9) from the American Cancer So- vector. Cancer Res. 1995;55:3250-3253.

ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 124, JAN 1998 92