Retinoblastoma RB94 Enhances Radiation Treatment of Head and Neck Squamous Cell Carcinoma (original) (raw)

Cancer Therapy: Preclinical| June 02 2008

1Department of Otorhinolaryngology-Head and Neck Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania;

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Sidrah M. Ahmad;

1Department of Otorhinolaryngology-Head and Neck Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania;

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Guoyan Li;

2Department of Otolaryngology-Head and Neck Surgery, University of Maryland School of Medicine, Baltimore, Maryland;

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David A. Bray, Jr;

3Bray Plastic Surgery Medical Center, Inc., Torrance, California;

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Koichiro Saito;

1Department of Otorhinolaryngology-Head and Neck Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania;

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Daiyou Wang;

1Department of Otorhinolaryngology-Head and Neck Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania;

4Department of Oral and Maxillofacial Surgery, College of Dental Medicine, Guangxi Medical University, Nanning, Guangxi, P.R. of China; and

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Uwe Wirtz;

5Department of Technology and Product Development, Titan Pharmaceuticals, Inc., South San Francisco, California

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Sunil Sreedharan;

5Department of Technology and Product Development, Titan Pharmaceuticals, Inc., South San Francisco, California

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Bert W. O'Malley, Jr;

1Department of Otorhinolaryngology-Head and Neck Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania;

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Daqing Li

1Department of Otorhinolaryngology-Head and Neck Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania;

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Requests for reprints: Daqing Li, Department of Otorhinolaryngology-Head and Neck Surgery, University of Pennsylvania School of Medicine, CRB145, 415 Curie Boulevard, Philadelphia, PA 19104. Phone: 215-746-2953; Fax: 215-573-1934; E-mail: lidaqing@mail.med.upenn.edu.

Received: October 03 2007

Revision Received: January 09 2008

Accepted: January 27 2008

Online ISSN: 1557-3265

Print ISSN: 1078-0432

American Association for Cancer Research

2008

Clin Cancer Res (2008) 14 (11): 3514–3519.

Article history

Received:

October 03 2007

Revision Received:

January 09 2008

Accepted:

January 27 2008

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Abstract

Purpose: To assess whether adenovirus-mediated retinoblastoma 94 (Ad-RB94) transgene expression enhances efficacy of radiation therapy (XRT) of human head and neck squamous cell carcinoma (HNSCC).

Experimental Design: The HNSCC cell lines (JHU006 and JHU012) were treated in vitro and in a nude mouse xenograft model with Ad-RB94, Ad-DL312 control vector, or untreated as mock control. Cell viability and tumor growth were evaluated and combined RB94/XRT antitumor activity was analyzed by measuring DNA double-strand breaks, apoptosis-associated early DNA fragmentation, and levels of RB-regulated cell cycle progression E2F1 transcription factor.

Results: Ad-RB94/XRT resulted in significant HNSCC cell growth inhibition compared with XRT alone or Ad-RB94 alone in vitro and caused significant tumor regression compared with XRT alone and Ad-DL312/XRT in JHU006 and with XRT alone, Ad-DL312/XRT and Ad-RB94 alone in JHU012 in vivo. Neutral comet analysis revealed that DNA damage was significantly elevated in cells treated with Ad-RB94 alone and Ad-RB94/XRT. Tumors treated with Ad-RB94 alone showed a striking increase in early apoptosis DNA fragmentation, and DNA fragmentation was further enhanced with XRT. In addition, levels of E2F1 were up-regulated by Ad-RB94/XRT combination, whereas Ad-RB94 alone did not affect E2F1 levels and XRT alone led to down-regulation of E2F1.

Conclusions: A potent antitumor effect has been observed after Ad-RB94/XRT combination treatment in HNSCC xenograft tumors. Enhanced tumor regression correlated with increased apoptosis. Ad-RB94 treatment enhances the efficacy of XRT through tumor cell sensitization by arresting the cells at the radiation-sensitive G2-M cell cycle and via E2F1 up-regulation.

External beam radiation therapy (XRT) with or without an adjuvant therapy has great merits for organ preservation during cancer therapy. Organ preservation is essential for maintaining quality of life, especially in head and neck cancer. Innovative approaches to enhance the antitumor effects of XRT could lead to reduction of the dose of XRT and thus minimize the damage to exposed healthy tissues. Recent exciting developments in gene therapy have offered new hope for enhancing antitumor effects of XRT (1, 2).

One candidate gene for sensitizing cancer cells to XRT is the retinoblastoma (RB) tumor suppressor gene, encoding the 110-kDa, 928–amino acid nuclear phosphoprotein pRB. The RB protein binds to members of the cell cycle progression essential transcription factor family, E2F, thereby suppressing the transcriptional activity of E2F and inhibiting G1-S cell cycle progression (3, 4). On mitogenic stimulation, pRB is thought to be phosphorylated by cyclin-dependent kinase 2, 4, and 6. This hyperphosphorylation of pRB releases E2F, which then actively transcribes genes involved in G1- and S-phase cell cycle progression (3, 4).

In addition to its role in cell cycle regulation, pRB is also involved in DNA damage responses, DNA repair, and DNA replication and plays a protective role against apoptosis and differentiation, all of which may contribute to its function as a tumor suppressor (3, 4). Studies have shown that gene therapy using wild-type RB causes cessation of tumor cell growth in vitro as well as in vivo in some cancers (5, 6). However, wild-type pRB has limited efficacy as a tumor growth suppressor due to its rapid phosphorylation and inactivation in RB-positive and some RB-negative tumor cells.

A NH2-terminal truncated RB protein of 94 kDa (pRB94), lacking 112–amino acid residues, has been found to have greater efficacy than wild-type pRB in tumor suppression (5, 6). pRB94 was reported to remain in a hypophosphorylated state and showed a significantly greater half-life than the wild-type pRB (5, 6). We have shown previously that adenovirus-mediated RB94 (Ad-RB94) gene transfer significantly inhibits human head and neck squamous cell carcinoma (HNSCC) tumor growth in vitro and in vivo (7). Transgene expression of RB94 arrests tumor cells in the G2-M phase, inhibits telomerase activity, and induces apoptosis and suppresses tumor growth.

XRT also causes tumor regression in head and neck cancer animal models and tumor cells are more sensitive to radiation during cellular division. RB94 transgene expression in HNSCC tumor cells arrests the cells in the G2-M phase and therefore provides a rational for a therapeutic basis for adjuvant therapy with radiation, as tumor cells are most sensitive to radiation in this cell cycle phase.

The present study investigates whether Ad-RB94 combined with external beam XRT can enhance therapeutic outcome for the treatment of HNSCC and how it might accomplish the potent antitumor effect through cellular and molecular mechanisms.

Materials and Methods

Cell lines. The human HNSCC cell lines JHU006 and JHU012 were used for the described experiments. These cell lines originated from human tumor explants and have been well characterized and are known to express wild-type pRB (7). Cells were propagated in RPMI 1640 with 10% fetal bovine serum and 1% Penicillin and Streptomycin at 37°C in 5% CO2.

Animals. Institutional guidelines regarding animal experimentation/care were followed for the nude mice (athymic, BALB/c nu/nu).

Irradiation. Cells and animals were exposed to X-ray with 2 Gy at 24 h postviral infection using a Seifert X-ray unit (Rich. Seifert & Co.). This X-ray unit was operated at 250 kVp, 15 mA with 0.5 mm copper plus 1.0 mm aluminum added filtration (Half Value Layer 1.56 mm copper) with the average dose rate of 2 Gy/min.

The bodies of the animals excluding the tumor region were shielded during X-ray exposure.

Construction of recombinant adenoviral vectors. The construction of the replication-defective adenoviral vector containing the RB94 gene under transcriptional control of the human cytomegalovirus promoter (Ad-RB94) has been described previously (7). The replication-defective control adenovirus not carrying the RB94 gene, Ad-DL312 (E1a region deleted), was obtained from Dr. Tom Shenk (Princeton University). Viruses were amplified and plaque-purified. Titers were determined by standard plaque assay.

Cell growth in vitro. Triplicate samples of 3 × 103 log-phase JHU012 or JHU006 cells were plated in 96-well tissue culture plates and allowed to adhere overnight. Medium was removed and cells were incubated with either Ad-RB94 or Ad-DL312 or mock-treated with PBS at a multiplicity of infection of 1:10 in 40 μL medium for 4 h, after which 160 μL medium was added. After 24 h, cells were treated with ionizing radiation at a dose of 2 Gy for the combination groups. Cells were incubated at 37°C and inspected daily for 5 days. Cell growth was determined by adding 20 μL MTT (5 μg/mL) to each well and incubating for an additional 4 h at 37°C. Supernatant was discarded and 150 μL DMSO was added. Absorbance was determined by spectrophotometry using a wavelength of 570 nm with 630 nm as a reference.

Neutral comet assay. JHU006 and JHU012 tumor cells were grown overnight as a monolayer under 2 mL medium in 24-well tissue culture plates. The cells were transfected with 8.5 × 107 plaque-forming units of either Ad-RB94 or Ad-DL312 in 50 μL volumes. Xenografts were transfected at a multiplicity of infection of 1:5 for 4 h. The cells were incubated at 37°C. Combination treatment groups were irradiated with 2 Gy. The cells were harvested after 48 h for assessment of DNA damage using the neutral comet assay, which was adapted from techniques described by Olive et al. (8). The slides were examined under a microscope (Eclipse 80i, Nikon) at 40-fold magnification and images of 25 to 50 randomly distributed cells per sample were captured using a digitized imaging system.

Quantification of DNA double-strand breaks. Comet measurements and quantitative analysis were done using imaging analysis software from Scion obtained from a public domain. This program allows for automatic measurement of the tail moment or the product of the tail length and the percentage of DNA in the tail. The tail length is defined as the distance beginning at the edge of the head to the end of the tail. The calculation of the percentage of DNA in the tail required several steps. First, the area covered by the whole comet was multiplied by the mean comet intensity as a measure of the DNA in the entire comet (DNAC). Then, the DNA content of the head (DNAH) was measured in a similar fashion by calculating the product of the area of the head and the mean head intensity. The DNA content of the tail (DNAT) was derived by subtracting the DNA content of the head from the DNA content of the tail: DNAT = DNAC - DNAH. The percentage of DNA in the tail was calculated as (DNAT / DNAT + DNAH) × 100%. Then, the tail moment, which indicates the level of DNA double-strand break (DSB) damage, was calculated by multiplying the tail length and percentage of DNA in the tail. After measuring the tail moment of 25 to 50 tumor cells, a mean tail moment (MTM) was calculated for each sample group and graphically displayed.

Xenograft tumors in vivo. Six-week-old female mice were anesthetized by i.p. injection of 6 to 10 mg tribromoethanol with depth of anesthesia determined by toe pinch. Mice were then injected s.c. in the left flank with 1 × 107 JHU006 or JHU012 cells suspended in 100 μL HBSS. Ten days following injection, animals were reanesthetized with tribromoethanol. Skin flaps were raised and tumors were exposed. Internal measurements were done in three dimensions using calipers. A total of 60 mice (30 mice with each cell line) were divided into 6 groups. Tumors were then injected using a 100 μL Hamilton syringe and a 26 gauge needle with 8.5 × 107 plaque-forming units of Ad-RB94 or Ad-DL312 in 50 μL volume. Skin incisions were closed with 4-0 silk suture. Twenty-four hours after injection, 3 of the 6 groups were irradiated with 2 Gy. External tumor measurements were done over the skin in two dimensions using calipers every 2 days after treatment to check the dynamic tumor growth. Twelve days following irradiation, mice were sacrificed and residual tumor mass was measured internally in three dimensions and harvested for immunohistochemical study.

Apoptosis detection. The ApopTag Peroxidase In situ Apoptosis Detection Kit and protocols (Millipore) were used to detect early DNA fragmentation associated with apoptosis in tumors extracted from treated mice. The DNA strand breaks are detected by enzymatically labeling the free 3′-OH termini with modified nucleotides. These new DNA ends that are generated on DNA fragmentation are typically localized in morphologically identifiable nuclei and apoptotic bodies. Tumor samples were fixed in formalin and embedded in paraffin using standard procedures. Tissue samples were cut in 7 μm sections and slides were viewed by microscopy. Four randomly selected views under a microscope (Eclipse 80i, Nikon) at 200-fold magnification were digitally recorded for each tumor and all cells stained positive for apoptosis within the view were counted with the assistance of IP Lab software (Scanalytics).

E2F1 immunohistochemistry. The effect of Ad-RB94 transfection on E2F1 levels was evaluated in tumors excised from the treated mice. Immunohistochemistry was done using VECTASTAIN Elite ABC kit (Vector Laboratories). For the immunohistochemistry of E2F1, frozen sections were cut 8 μm thick, air-dried for 4 h, and fixed in cold acetone for 10 min. Sections were then incubated with normal blocking serum for 20 min and incubated with primary antibody for 1.5 h according to the manufacturer's instructions. Rabbit polyclonal antibody to human E2F1 antigen (GeneTex) was used at a dilution of 1:50. After incubation with diluted biotinylated secondary antibody solutions, sections were incubated with VECTASTAIN Elite ABC reagent for 30 min.

Subsequently, sections were incubated with 3,3′-diaminobenzidine tetrahydrochloride-H2O2 solution for visualization and counterstained with hematoxylin. Four randomly selected views under a microscope (Eclipse 80i, Nikon) at 200-fold magnification were digitally recorded for each tumor. All E2F1 antigen-positive cells within the field of view were counted with the assistance of IP Lab software (Scanalytics).

Statistical analysis. Mann-Whitney analysis was applied using STATMOST (Detaxion Software) to determine statistical significance.

Results

Cancer cell growth inhibition in vitro. Triplicate samples of HNSCC cell lines JHU006 or JHU012 were transfected with Ad-RB94, Ad-DL312, or mock treated. After 24 hours, cells were treated with ionizing radiation at a dose of 2 Gy. Cells were harvested and counted from day 1 to day 6. Cell growth curves of JHU006 and JHU012 cell lines were plotted (Fig. 1A and B). In multiple trials, combination treatment with Ad-RB94 and XRT resulted in greater inhibition of cell growth than XRT alone, Ad-DL312 with XRT, or Ad-RB94 alone. In JHU006, cell growth diminution appeared to begin on day 2 and was sustained through day 6 after treatment with Ad-RB94 and XRT. In JHU012, combination therapy resulted in marked cell regression between days 3 and 6. Control groups, which included mock treatment and Ad-DL312, grew steadily by logarithmic growth.

Fig. 1.

Combination of Ad-RB94 transfection and XRT suppresses tumor cell growth in vitro. Tumor cell growth curve analysis in vitro of JHU006 (A) and JHU012 (B) in six different treatment groups. Cells were transfected with Ad-RB94, Ad-DL312, or no treatment with or without XRT. Mean viable cell numbers were assessed for 6 consecutive days as absorbance of their MTT stain values. Control groups, which included no treatment and Ad-DL312 alone, grew steadily by logarithmic growth. Combination treatment with Ad-RB94 and XRT resulted in greater inhibition of cell growth than XRT alone, Ad-Dl312 with XRT, or Ad-RB94 alone.

Fig. 1.

Close modal

DNA DSB induction. Cells treated with Ad-RB94 and the combination of Ad-RB94 and XRT gave rise to the formation of a “comet” in a neutral comet assay, characterized by a brightly fluorescent head and a tail, indicative of DNA DSB. Cells from the control groups (no treatment and Ad-DL312) exhibited minimal tail formation in both cell lines. The MTM, indicative of DNA DSB, was calculated. MTM was significantly increased in tumor cells treated with Ad-RB94 and XRT compared with cells treated with XRT alone (JHU006: 21,717.9 versus 7,524.1, P < 0.01; JHU012: 15,122.9 versus 7,427.8, P < 0.01) or Ad-RB94 alone (JHU006: 21,717.9 versus 17,230.0, P < 0.01; JHU012: 15,122.9 versus 11,663.6, P < 0.01) (Fig. 2A and B). These results show that, indeed, Ad-RB94 transfection augments radiation-induced DNA damage in JHU006 and JHU012 cells. Furthermore, the level of DNA damage correlates with the degree of cytotoxicity observed by growth curve analysis in both cell lines.

Fig. 2.

Combination of Ad-RB94 transfection and XRT inducts DNA DSB in vitro. Columns, MTM indicative of DNA DSB in JHU006 (A) and JHU012 (B) cells at 48 h (multiplicity of infection of 5). Cells from the control groups (no treatment and Ad-DL312) show minimal tail formation in both cell lines, whereas cells treated with Ad-RB94 have marked tail migration. MTM was most elevated in tumor cells that were simultaneously treated with Ad-RB94 and XRT and significantly above the controls.

Fig. 2.

Close modal

Tumor growth inhibition in vivo. Nude mice carrying established JHU006 and JHU012 tumors were treated with Ad-RB94, Ad-DL312, or mock treatment. After 24 hours, half of the mice were treated with ionizing radiation at a dose of 2 Gy. External tumor measurements were done over the skin in two dimensions every 2 days after treatment to check the dynamic tumor growth. At the time of the treatment and 12 days following treatment, internal tumor measurements were done in three dimensions to evaluate true antitumor effect.

Tumor growth curves of JHU006 and JHU012 cell lines measured in two dimensions were plotted (Fig. 3 A and B). In JHU006, tumor growth diminution appeared to begin on day 4 and was sustained through day 12 after treatment with Ad-RB94 and XRT. In JHU012, combination therapy resulted in marked cell regression between days 8 and 12. Control groups, which included mock treatment and Ad-DL312, grew steadily by logarithmic growth in both cell lines.

Fig. 3.

Combination of Ad-RB94 transfection and XRT suppresses tumor cell growth in vivo. HNSCC JHU006 and JHU012 xenograft tumors were propagated in the flanks of nude mice after s.c. injection and tumors were treated with Ad-RB94, Ad-DL312, or no treatment with or without XRT. External tumor measurements were done over the skin in two dimensions every 2 d after treatment to check the dynamic tumor growth. At the time of the treatment and 12 d following treatment, internal tumor measurements were done in three dimensions. Tumor cell growth curve in vivo of JHU006 (A) and JHU012 (B) in six different treatment groups showed that control groups, which included no treatment and Ad-DL312 alone, grew steadily by logarithmic growth. Combination treatment with Ad-RB94 and XRT resulted in greater inhibition of tumor growth than XRT alone, Ad-DL312 with XRT, or Ad-RB94 alone. True tumor volume of JHU006 (C) and JHU012 (D) in six different treatment groups showed that treatment of XRT alone, Ad-DL312 with XRT, Ad-RB94 alone, and combination treatment of Ad-RB94 with XRT resulted in tumor growth suppression when compared with control groups (no treatment and Ad-DL312) in both cell lines. The combination Ad-RB94/XRT treatment had the greatest efficacy in reducing tumor size, with statistically significant differences when compared with XRT alone and Ad-DL312 with XRT in JHU006 and compared with XRT alone, Ad-DL312 with XRT, and Ad-RB94 alone in JHU012.

Fig. 3.

Close modal

True tumor volume of JHU006 and JHU012 cell lines 12 days after treatment (Fig. 3C and D) showed that treatment of XRT alone, Ad-DL312 with XRT, Ad-RB94 alone, and combination treatment of Ad-RB94 with XRT resulted in tumor growth suppression when compared with control groups (no treatment and Ad-DL312) in both cell lines. The greatest tumor suppression occurred when Ad-RB94 gene therapy was combined with XRT in both cell lines. The combination Ad-RB94/XRT treatment had the greatest efficacy in reducing tumor size, with statistically significant differences when compared with the controls. For the JHU006 cell line, comparison with mock treatment (623.7 versus 48.7 mm3, P < 0.01), Ad-DL312 treatment (646.7 versus 48.7 mm3, P < 0.01), XRT alone (208.3 versus 48.7 mm3, P < 0.05) and Ad-DL312 with XRT (180.8 versus 48.7 mm3, P < 0.05) yielded statistically significant differences. Similarly, for the JHU012 cell line, a statistical difference was observed in comparison with mock treatment (635.6 versus 31.7 mm3, P < 0.01), Ad-DL312 treatment (557.3 versus 31.7 mm3, P < 0.01), XRT alone (177.3 versus 31.7 mm3, P < 0.01), Ad-DL312 with XRT (180.7 versus 31.7 mm3, P < 0.01), and RB94 alone (150.0 versus 31.7 mm3, P < 0.01). There are no statistical differences between “no treatment” and “Ad-DL312” or “XRT alone” and “Ad-DL312/XRT” in either JHU006 or JHU012 cell line.

Apoptotic early DNA fragmentation in vivo. An apoptosis detection kit was used to detect early DNA fragmentation that is associated with apoptosis. The apoptotic index was found to be significantly higher in the Ad-RB94/XRT-treated tumors than in controls (Fig. 4A and B). For the JHU012 cell line, comparison with mock treatment (27.8 versus 108.2 cells, P < 0.01) and Ad-DL312 treatment (30.4 versus 108.2 cells, P < 0.01) resulted in a statistically significant difference. For the JHU006 cell line, comparison with mock treatment (18.25 versus 74.2 cells, P < 0.05), Ad-DL312 treatment (22.6 versus 74.2 cells, P < 0.05), XRT alone (31.8 versus 74.2 cells, P < 0.05), and Ad-RB94 alone (39.4 versus 74.2 cells, P < 0.05) revealed a statistically greater apoptotic index for the combination treatment group.

Fig. 4.

Combination of Ad-RB94 transfection and XRT induces apoptosis in vivo. An apoptosis detection kit (ApopTag Peroxidase In situ Apoptosis Detection Kit) was used to detect early DNA fragmentation that is associated with apoptosis in HNSCC JHU006 and JHU012 xenograft tumors. The apoptotic index was evaluated with four randomly selected views under a microscope at 200-fold magnification. Apoptotic cell number in JHU006 (A) and JHU012(B). Tumors of both cell lines exhibit a significant increase in early apoptotic DNA fragmentation after transfection with Ad-RB94/XRT combination when compared with control groups (no treatment and Ad-DL312).

Fig. 4.

Close modal

E2F1 expression in vivo. E2F1 is the key target in the RB pathway. It is a transcriptional activator of genes that regulate G1-S cell cycle transition and has a trigger function for apoptosis induction. Thus, we examined E2F1 levels in tumor cells by immunohistochemistry following various treatments. The number of E2F1-positive cells in Ad-DL312- or Ad-RB94-treated tumors did not show a significant difference when compared with the mock treatment group. However, when compared with the mock treatment, the number of E2F1-positive cells was found to be significantly lower in XRT-treated (JHU006: 6.8 versus 54.5 cells, P < 0.01; JHU012: 3.0 versus 36.0 cells, P < 0.05) and Ad-DL312/XRT-treated (JHU006: 12.6 versus 54.5 cells, P < 0.05; JHU012: 5.0 versus 36.0 cells, P < 0.05) groups. Furthermore, the E2F1-positive cells were significantly higher in the Ad-RB94/XRT-treated group (JHU006: 112.8 versus 54.5 cells, P < 0.05; JHU012: 68.6 versus 36.0 cells, P < 0.05) when compared with mock treatment (Fig. 5A and B). These results indicate that E2F1 expression is down-regulated after XRT but not with Ad-RB94 or Ad-DL312 gene therapy. Unexpectedly, the combination of Ad-RB94 and XRT, but not the combination of Ad-DL312 and XRT, led to up-regulation of E2F1 levels; this may consequently trigger G1-S cell cycle progression and apoptosis induction.

Fig. 5.

Combination of Ad-RB94 transfection and XRT lead to up-regulation of E2F1 levels in vivo. E2F1, which may trigger G1-S cell cycle progression and apoptosis induction, was evaluated with E2F1 immunohistochemistry. E2F1-positive cell number was calculated from four randomly selected views under a microscope at 200-fold magnification in JHU006 (A) and JHU012 (B) cell-derived tumors. A greater number of E2F1-positive cells were observed in the Ad-RB94/XRT combination group compared with no treatment group, whereas smaller number of E2F1-positive cells were observed in XRT or Ad-DL312 with XRT groups.

Fig. 5.

Close modal

Discussion

We showed that Ad-RB94 gene transfer in combination with XRT significantly suppresses HNSCC tumor growth both in vitro and in vivo. We have reported previously that JHU006 and JHU012 tumor cells expressed pRB94 after Ad-RB94 transfection and resulted in cell cycle arrest in the G2-M phase; furthermore, we had reported that Ad-RB94 transfection inhibited telomerase activity, induced apoptosis, and suppressed tumor cell growth. Cells in the Ad-DL312 treatment or mock treatment groups expressed cellular wild-type pRB only and arrested in the G1-S phase of the cell cycle (7). Another group studied Ad-RB94 gene therapy in bladder cancer and immortalized urothelial cells and showed induction of apoptosis associated with caspase-dependent pathways, induction of telomere erosion and chromosomal crisis, and suppression of tumor growth (9). It has been reported that Ad-RB94 gene therapy in pancreatic cancer cells results in antiproliferative effects, apoptosis induction, and S-G2 cell cycle arrest in tumor cells (10). The common factor of these findings was the cell cycle progression through the G1 phase and the shift of the cell cycle arrest to G2 after Ad-RB94 gene therapy. This is advantageous for treatment with XRT because the cells arrested in G2-M phase have greater sensitivity to radiation than the ones arrested in other phases. For this reason, Ad-RB94 gene therapy was hypothesized to be an effective adjuvant therapy with radiation. Our findings suggest that the transgene expression of RB94 in tumor cells may result in G2-M cell cycle arrest and therefore enhance the therapeutic outcome of XRT.

The results of the current study show that combination therapy with Ad-RB94 and XRT results in additive or synergistic tumor growth suppression both in vitro and in vivo. Unlike wild type pRB, NH2-terminal truncated pRB94 may not bind and thereby does not suppress E2F1 expression. One study indicated that a short deletion in the NH2 terminus resulted in a loss of E2F binding (11), and based on the critical role of E2F cell cycle progression, transcription factors suggested that mutations in the NH2 terminus compromise RB tumor suppression function in RB knockout mice (12). Some reports have, however, shown that the NH2 terminus is dispensable for pRB cell growth suppression function, suggesting another role of pRB in tumor suppression other than E2F1 binding (13).

It has been reported that the adenovirus E4-6/7 proteins block the association of E2F with wild-type pRB by binding the E2F MB domain (12) and expression of E4-6/7 results in diminishing E2F suppression. The NH2 terminus of the wild-type pRB may suppress E4-6/7 expression or affect the binding function of E4-6/7. By contrast, due to the NH2-terminal truncation; pRB94 fails to affect E4-6/7 and therefore does not affect E2F1 expression levels. The mechanism of E2F1 up-regulation in combined Ad-RB94 and XRT remains unclear and requires further studies.

We would furthermore like to point out that the two therapeutic components, that is, XRT and Ad-RB94, activate both the intrinsic and the extrinsic apoptosis pathways through induction of DNA damage (XRT) and increase of active E2F1 levels (RB94); thus, synergistic apoptosis induction is plausible. Altogether, we propose the following tumor cell growth inhibition mechanism: Ad-RB94 transfection, unlike the wild-type pRB, does not lead to suppression of free active E2F1 and hence does not arrest the cell cycle in the G1-S phase but arrests the cell cycle in the G2-M phase. As a result, sensitivity to XRT is increased. XRT induces DNA damage, including DSB. Ad-RB94 gene therapy alone also induces DNA DSB; furthermore, telomerase inactivation and suppression of cell proliferation are induced (7). Apoptosis is induced by the intrinsic pathway that is activated by XRT and the RB94-mediated increase in E2F1 levels (14). Concurrently, the extrinsic pathway of apoptosis may also be activated by the loss of E2F1 repression (15). Therefore, after Ad-RB94/XRT combination treatment, additional or synergistic induction of apoptosis can be expected with a potent effect on tumor suppression.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant support: National Institute of Dental and Craniofacial Research/NIH from R01 DE 14562 and The Flight Attendant Medical Research Institute.

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