Gene Ther Mol Biol, Vol 5 page 25-35, 2002

Strategy of sensitizing tumor cells with adenovirus-p53 transfection

Review Article

Jekunen Antti¹, Miettinen Susanna², Mäenpää Johanna³, Kairemo Kalevi⁴

¹Department of Clinical Pharmacology, Helsinki University, and Department of Oncology, Turku University, and Aventis Pharma Finland, Finland. ²Department of Anatomy, Tampere University, Finland. ³Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Tampere University Hospital and Tampere University, Finland. ⁴Department of Nuclear Medicine, Uppsala University Hospital, Sweden

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*Correspondence: Antti Jekunen, MD, PhD, PL96, 00241 Helsinki, Finland; Tel. +358400 755208; Fax. +3589 47638140; e-mail:antti.jekunen@aventis.com

Received: 29 January 2002; accepted: 06 March 2002; electronically published: July 2003

Summary

Loss or malfunction of the p53-mediated apoptotic pathway has been proposed as one mechanism by which tumors become resistant to chemotherapy. While it may be the most frequently mutated gene in human tumor samples, the function of p53 is critical for maintaining the integrity of the cellular genome in its responses to treatment with cytotoxic agents. Intact p53 protein in nuclei of normal cells acts as a transcriptional activator for a group of genes involved in cell cycle arrest, DNA repair and apoptosis. The transfection of adenovirus p53 (adeno-p53) alone has been shown in ovarian cancer cell culture models to inhibit cell growth and to promote apoptosis regardless of the endogenous p53 status of the cells. Both mutant p53 in the tumor cells and the loss of p53 function were associated with resistance to chemotherapeutic agents. There are various reports of at least additive interactions between adeno-p53 and several chemotherapeutic agents in a number of cancers, e.g. bladder cancer, NSCLC, prostate cancer, breast cancer, and ovarian cancer both in vitro and in vivo. The mechanisms of these interactions are unknown, but they may depend on the chemotherapeutic agents used, the targets and critical tissues, and the intracellular signal transduction pathways affected.Results obtained with a speculative treatment regimen consisting of oligonucleotide therapy and p53 transfection suggest that p53 expression in tumor cells may improve their sensitivity to routine chemotherapy, e.g. docetaxel and irinotecan, which are efficacious drugs possessing different modes of action: prevention of depolymerization of tubulin and specific DNA topoisomerase I inhibition, respectively. It is known, however, that even these new agents cannot achieve responses in all tumors, and that in some tumors the efficacy, once established, diminishes along with the treatment. In these cases of resistant tumors or recurrences and relapses, combined treatment with adeno-p53 and chemotherapeutic agents may be an attractive strategy for inhibiting the progression of local cancers. In fact, the ground is ready for a rapid practical development of adeno-p53, which itself causes only minimal side-effects after administration, e.g. injection site rashes and fever, and an immunostimulation that seems to be quite mild and transient in nature. Future cancer therapy strategies may consist of effective chemotherapy coupled to molecular medicine specifically targeting tumor cells. So far, we do not have proper means in molecular medicine for achieving high enough tumor access with any of the current systemic virus vectors having the proper level of selectivity between tumor and normal cells. We have already some clinical experience, however, with intratumoral approaches that ensure the highest possible concentrations inside NSCLC, ovarian cancer and head and neck cancer tumors. It seems that there is clear evidence of good tolerability at non-maximal doses, but unfortunately, only modest activity when the construct is used alone. We review here the published data on the use of adenovirus p53 for sensitizing tumors to chemotherapeutic agents and outline perspectives for the future.

I. Introduction

A. Function of p53

The p53 protein, a nuclear phosphoprotein, is indispensable for genomic integrity and cell cycle control. Its basic function is to control the entry of the cell into the S phase of the cell cycle. p53 extends the time available for DNA repair before S phase entry (Fan et al, 1995). The wild- type gene product regulates cell growth and division negatively. Although not essential for progression of the cell cycle, it is critical as a checkpoint that blocks uncontrolled cell division (Levine, 1992). In the nuclei of normal cells, the intact p53 protein acts as a transcriptional activator for a group of genes involved in cell cycle arrest (p21^cip1/waf1), DNA repair (GADD45), and apoptosis (Bax) (O'Connor et al, 1997; Sugrue et al, 1997; Yin et al, 1997; Carrier et al, 1999). In addition to this, p53 is a potent inducer of programmed cell death (apoptosis) within a cell in which the DNA has been damaged. Normally, the p53 gene is inactive. When, after DNA damage, the normal p53 is activated, the levels of p21, p27, and GADD 45 may become very high (Sherr, 1994). DNA damage in cells induces expression of p53 and interruption of the cell cycle in both G1 and G2 (Chu and DeVita, 2001). If DNA repair is successful, the cell continues its cycle. If repair does not succeed, the cell undergoes apoptosis.

B. Mutation of p53

Mutations in the p53 gene are among the most common genetic alterations observed in human tumor samples (Oren, 1992). The specific cytotoxic treatment, the conditions of treatment, the p53 status, and other elements of cell-cycle regulation may all contribute to the outcome of exposure of a cell to DNA-damaging agents (Chu and DeVita, 2001). p53 can activate an apoptotic response to DNA damage, especially in hematopoietic and lymphoid cells, which often overrides the G1 checkpoint response (Fan et al, 1995). In cell types programmed for apoptosis, loss of p53 function decreases their sensitivity to a wide variety of DNA-damaging agents, while in cell apoptosis, it has been more difficult to establish a clear relationship between p53 gene status and chemosensitivity types of some solid tumors not inherently programmed for (Fan et al, 1995). If the DNA is damaged, the cell with intact p53 function will undergo p53-dependent apoptosis (Chu and DeVita, 2001). In tumor cells with mutated p53, the loss of p53 function, is thought to result in resistance to chemotherapeutic agents (Lowe et al, 1994; Righetti et al, 1996; Blandino et al, 1999). A recent study of ovarian cancer shows that women with tumors having the p53 null mutation have a survival disadvantage over those with p53 missense mutations (Shahin et al, 2000).

II. Evidence of the role of p53 in chemosensitizing

A. p53 and chemotherapeutic agents

Dysregulation of the p53 pathway may lead to drug resistance due to overproduction of the gene products responsible for entry into the S phase and rapid cell growth (Figure 1).

Activation of these genes could theoretically increase the resistance of cells to the following chemotherapeutic agents: methotrexate, 2-chlorodeoxyadenosine, hydroxyurea, fludarabine, cytosine arabinoside, and 5-fluorouracil. Under some experimental circumstances, cell death in response to exposure to DNA-damaging agents may require an intact p53-dependent apoptotic mechanism. Some of the genes that are transcriptionally activated by p53 belong to a class of proteins known to inhibit cyclin-dependent kinases (cdk). p21 forms a complex with proliferating cell nuclear antigen or inhibits cdk’s, e.g. cdk4 (Polyak et al, 1997). Activated p53 can cause a G1 cell cycle arrest by increasing the transcription of the cdk inhibitor p21 (Figure 2), which block cdk4 activity, preventing reitinoblastoma gene product (RB) phosphorylation (Sherr, 1994) and release of E2F blocking the transcription of a number of genes, and inhibiting entry into S phase (Kirsch, 1998). The E2F family of transcription factors bind to the regulatory regions of a number of genes that participate in the synthesis of DNA (Figure 2).

Figure 1. Effect of chemotherapy via p53 pathway. After chemotherapy has induced DNA damage, p53 protein is activated and transcription of many genes is increased, resulting in cell cycle arrest and apoptosis. For apoptotically sensitive cells, genotoxic damage can signal an immediate apoptotic response, while for apoptotically insensitive cells, the primary apoptotic decision point is disabled. Cells that avoid apoptotic or necrotic death after DNA repair can survive and grow. (Kirsch 1998; Brown and Wouters 1999)

These genes include ribonucleotide reductase, dihydrofolate reductase, DNA-dependent RNA polymerase, thymidylate synthase, c-myc, c-fos, and c-myb. Activation of these gene products facilitates the entry of the cell into the S phase.

There is much evidence in support of the idea that a mutation in p53 may lead to resistance to cytotoxic agents. In premenopausal women with node negative breast cancer, it has been shown by immunohistochemistry that p53(+) tumors are less sensitive to treatment with a regimen including 5-fluorouracil, doxorubicin, and cyclophosphamide than p53 (-) tumors. (Clahsen et al, 1998). Under in vitro conditions Koechli et al, have shown that mutant p53 can increase chemoresistance to 5-fluorouracil, cyclophosphamide, and methotrexate (Koechli, 1994). Cisplatin resistance seems to be connected with p53 mutations, and in advanced ovarian cancer, the p53 mutational status is a predictor of the responsiveness to platinum-based chemotherapy (Calvert, 1999). However, there are also reports that apparently disagree with the chemoresistance effect of p53 (Fan et al, 1995; Stal 1995; Hawkins et al, 1996).

Human fibroblasts lacking functional p53 were more sensitive to cisplatin, carboplatin, paclitaxel, nitrogen mustard or melphalan than cells with functional p53 (Hawkins et al, 1996). Similar results, loss of p53 function and the sensitizing effect of cisplatin, have been demonstrated in MCF-7 breast cancer cells and RKO methotrexate, and 5-fluorouracil have been reported in colon cancer cell lines with or without disruption of p53 function by a dominant negative p53 transgene (Fan et al, 1995).

Increased rates of response to cyclophosphamide, patients with breast cancer who were determined to be immunohistochemically p53(+) (Stal,1995).

B. In vitro interactions

Synergy between two chemical agents in vitro is an empirical phenomenon, in which the observed effect of the combination is greater than would be predicted from the effect of each agent working alone. While synergy is not directly measurable in clinical practice, it may predict a favorable outcome when two treatments are combined in vivo and may strongly suggest the presence of synergy in vivo. Nielsen et al, used three-dimensional statistical modeling to evaluate the presence of synergistic, additive, or antagonistic efficacy between adenovirus-mediated p53 gene transfer and paclitaxel in a panel of human tumor cell lines, including those for ovarian, head and neck, prostate, and breast cancer (Nielsen et al, 1998). Cells were either pretreated with paclitaxel 24 h or not, before proliferation was measured 3 days later. Paclitaxel had synergistic or additive efficacy with p53 transfer, independently of whether the cells expressed mutant p53 protein or no p53 protein at all. Cell cycle analysis demonstrated that, prior to apoptotic cell death, p52 transfection arrested cells in the G0/G1 stage, whereas paclitaxel arrested cells in the G2-M stage. When combined, the relative concentrations of the two agents determined the dominant cellular response. The observed synergy remained unexplained; however, some speculations were offered. P53 has been shown to down regulate the expression of the antiapoptotic bcl-2 gene and up regulate the expression of the pro-apoptotic bax gene in other tumor cells (Selter and Montenarh 1994). Thus, p53 and paclitaxel may potentiate each other in stimulating the apoptotic pathway in neoplastic cells (Nielsen et al, 1998). It may also be that paclitaxel increased the number of cells transfected by the adenovirus. Particularly, the concentrations of paclitaxel responsible for increased adenovirus transduction are lower than the concentrations required for microtubule condensation. Moreover, the rate of change in the number of cells transduced by adenovirus appears to be independent of paclitaxel-induced cell death. The authors also determined the efficacy of the combination therapy in vivo. In some instances, it seems that loss of p53 may increase resistance to one agent, while simultaneously increasing sensitivity to another. Bunz et al, (1999) have reported that deletion of p53 in colorectal cancer cell lines maintained the cells that were resistant to 5-fluorouracil, but increased the sensitivity to doxorubicin and radiation in vitro. If the compound exerts it effects by apoptosis, as does 5-fluorouracil, loss of the apoptotic pathway may lead to resistance.

Figure 2. Two examples of cell cycle arrest via p53 activation. P53 mediated cell-cycle arrest is demonstrated with two examples: A) inhibition of cdk4 and cdk2 resulting G1-S and G2-M arrest, respectively. B) p53 activation increases the transcription of the cyclin-dependent kinase (cdk) inhibitor p21. Increase levels of p21 protein prevent cdk’s from phosphorylating their substrates, such as the retinoblastoma protein (RB) and thus block cell-cycle progression from G1 into S phase. (Kirsch 1998; Brown and Wouters 1999)

Recently, a report using isobologram modelling have showed that the combination of adeno-p53 + radiation produced significantly synergistic effects in NSCL cell lines, whereas the combination of docetaxel + adeno-p53 and docetaxel + radiation produced mixed effects ranging between additive and synergistic (Nguyen al., 1996). The three-agent combination also produced significantly synergistic effects.

Brown and Wouters have criticized the sensitizing results obtained in cell cultures. They have pointed out the need for further evidence in relating p53 to the sensitivity of anticancer agents (Brown and Wouters, 1999). Because apoptosis, particularly p53–dependent apoptosis, can occur rapidly after drug exposure, short-term growth rate assays tend to underestimate overall death of cells with mutant p53 or of cells not undergoing apoptosis. This may result in a situation where short-term assays may incorrectly assess overall cell death in tumor cells with different probabilities of undergoing early apoptosis. Thus, results may have a bias toward increased cell death in wild-type p53 cells and decreased cell kill in mutant p53 cells. Results of experiments with normal cells transformed with dominant oncogenes have often been extrapolated to tumor cells, instead of initially using cancer cell models. Transformed normal cells are usually apoptotically more sensitive than cancer cells. Therefore, in sensitizing experiments, both long term clonogenic assays and tumor cell models with solid tumors should be used rather than growth rate assays and transformed normal cells. However, the more widely accepted conclusion drawn from studies conducted in cancer cell lines and tumors of different origin is still that restoration of normal p53 function in tumors restores the apoptotic pathway and leads to an increased response to chemotherapy (Peller, 1998; Ferreira, 1999; Chang, 2000)

C. Transfection of cell cultures with the adenovirus p53 gene construct

Adenovirus vectors have many advantages over other viral and non-viral vectors. Their transfection efficacy is high, in both dividing and resting cells, and they show high expression levels (Hwu, 2001). As adenoviral DNA is not incorporated into the cell genome, expression of the transgene is transient, but adenoviral vectors can be produced at high titers. Introduction of wild-type p53 into tumors with non functional p53 offers a novel strategy for treating cancer, by inducing apoptotic death in neoplastic cells.

Genomic instability accompanied by loss of p53-mediated apoptosis can also lead to therapy resistance. The support for this rationale is that loss of p53 could desensitize cells to the damaging effects of drugs. Normal transgenic hematopoetic cells (Lotem and Sachs, 1993), E1A-expressing transgenic fibroblasts (Lowe et al, 1993), and transformed transgenic fibroblasts (Lowe et al, 1994) were all more resistant to apoptosis following treatment with any of a wide variety of anticancer agents, than were comparable cells from the parental strain of mice, which expressed wild-type p53. Apoptosis seemed to be enhanced in cells that expressed wild-type p53 and were able to trigger their own cell death program.

In cell culture models, adenovirus-mediated p53 gene transfer alone inhibits cell growth and promotes apoptosis, regardless of the endogenous p53 status of the ovarian cancer cells (Santoso et al, 1995). In tumor cells, mutated p53 and also loss of p53 function were associated with resistance to chemotherapeutic agents. There are several reports of at least an additive interaction between adeno-p53 and cisplatin in bladder cancer (Miyake et al, 2000), between adeno-p53 and cisplatin, SN-38 (a metabolite of irinotecan), 5-fluorouracil, taxanes, bleomycin, and cyclophosphamide in NSCLC (Fujiwara et al, 1994) (Horio et al, 2000), and between adeno-p53 and paclitaxel in ovarian cancer (Nielsen et al, 1998). In the ovarian cancer model, enhanced efficacy has been reported in a three-drug combination of adeno-p53, cisplatin, and paclitaxel (Gurnani et al, 1999).

There is some evidence that chemosensitivity can be increased by replacement of the p53 gene. Roth (Roth, 1996) reported that recombinant-adenovirus-mediated transfer of the wild-type p53 gene into several human cells with homozygous deletions of p53 markedly increased cellular chemosensitivity to the major chemotherapeutic drugs. An additive antiproliferative effect was reported in p53null H358 lung cancer cells when cultured with cisplatin for 24 h before transduction with adeno-p53 (Fujiwara et al, 1994). Enhanced apoptosis, detected by DNA fragmentation, was reported for the combination compared with each agent alone.

A viability assay demonstrated that a replication-defective adenovirus encoding the wild-type p53 gene (INGN 201, Introgen Therapeutics, Inc.) suppresses growth and enhances sensitivity to DNA-damaging chemotherapeutic drugs (5-fluorouracil, doxorubicin, cisplatin) in p53-mutant-expressing cell lines (Gjerset and Mercola, 2000). These cells lines represent DLD-1 colon cancer, T47D breast cancer, PC-3 prostate cancer, and T98G glioblastoma. Transfection efficiencies were 60-70%. It seems that restoration of the wild-type p53 to mutant p53-expressing or p53null cells results in marked enhancement of sensitivity to several DNA damaging agents. This enhancement of sensitivity was not observed in two wild-type p53-expressing cell lines, MCF7 and LS174T, suggesting that, in this model, wild-type p53 gene transfer is effective as therapy sensitization only in tumors that have lost wild-type p53 function.

1. Glioma and pancreatic cancer

Somatic gene therapy based on the reintroduction of p53 limits the proliferation of human malignant glioma cells, but is unlikely to induce clinically relevant sensitization to chemotherapy in these tumors. Wild-type p53 failed to sensitize glioma cells to cytotoxic drugs including BCNU, cytarabine, doxorubicin, teniposide, and vincristine. The combined effects of the wild-type p53 gene transfer and drug treatment were less than additive rather than synergistic, suggesting that the intracellular cascades activated by p53 and chemotherapy were redundant. Unexpectedly, forced expression of mutant-p53-modulated drug sensitivity enhanced the toxicity of some drugs but attenuated the effects of others (Trepel et al, 1998). Likewise, in p53-null pancreatic carcinoma cells, wild-type p53 gene transduction had no effect on in vitro chemosensitivity to cisplatin, etoposide, 5-fluorouracil and paclitaxel (Kimura et al, 1997). Moreover, in anaplastic thyroid cancer cells, adeno-p53 increased the sensitivity to doxorubicin with a 10-fold decrease in IC₅₀ values.

2. Hepatocellular cancer

One of the goals of gene therapy for treating cancer is selective expression of cytotoxic gene products in tumor cells. When replication-defective retroviruses were constructed containing p53 cDNA that was transcriptionally regulated by the human hepatocellular-carcinoma-associated alpha-fetoprotein gene transcriptional control elements, the expression of exogenous wild-type p53 from this retroviral vector was limited to the cells producing alpha-fetoprotein. Introduction of wild-type p53 into alpha-fetoprotein positive human hepatocellular carcinoma cells by retroviral infection markedly inhibited their clonal growth in a monolayer and increased the sensitivity of these cells to the chemotherapeutic drug cisplatin (Xu et al, 1996).

3. Ovarian cancer

In cell culture models adenovirus-mediated p53 gene therapy is one way to inhibit cell growth and promotes apoptosis, regardless of the endogenous p53 status of the ovarian cancer cells (Santoso et al, 1995) (Wolf et al, 1999). Adeno-p53 gene transfer, combined with cisplatin, doxorubicin, 5-fluorouracil, methotrexate, or etoposide, inhibited cell proliferation more effectively than chemotherapy alone in head and neck, ovarian, prostate and breast tumor cell lines. Of particular significance, in an ovarian cancer model enhanced efficacy was noted when using the three-drug combination of adeno-p53, cisplatin, and paclitaxel (Gurnani et al, 1999). In human head and neck, ovarian, prostate, and breast cancer cells, low concentrations of paclitaxel also increase the number of cells transduced by recombinant adeno-p53 in a dose-dependent manner (Nielsen et al, 1998). The concentration of paclitaxel responsible for increased adenovirus transduction is lower than that required for microtubule condensation.

4. Breast cancer

Transduction of cells using replication-deficient adenovirus vectors can induce endogenous p53 expression in cells containing the wild-type p53 gene and this response is different from the p53 induction observed after DNA damage (McPake et al, 1999). Lebedeva et al, have examined the effects of a replication-defective adenovirus encoding p53 (INGN 201, Ad5CMV-p53), alone or in combination with the breast cancer therapeutic doxorubicin, in suppressing growth and inducing apoptosis in breast cancer cells in vitro (Lebedeva et al, 2001). They found that whereas in vitro treatment of cells with adeno-p53 reduced ³H-thymidine incorporation by about 90% at 48 hr, cell viability at 6 days was reduced by only about 50% relative to controls. Although apoptosis is detectable in the adeno-p53-treated cultures, these results suggest that a large fraction of adeno-p53-treated cells merely undergo reversible cell cycle arrest. Combined treatment with adeno-p53 and doxorubicin results in a greater than additive loss of viability in vitro and increased apoptosis. These data indicate an additive to synergistic effect of adeno-p53 and doxorubicin for the treatment of primary and metastatic breast cancer.

However, in breast cancer cell lines results without any clear cut link between transfection of p53 and a sensitizing effect have been reported. Two human breast cancer cell lines, MDA-MB-231 and MDA-MB-435, both with p53 mutations, were transduced with adenoviral vectors containing wild-type p53 and the effects on growth were determined by clonogenic assays (Parker et al, 2000). Combining VP-16 and paclitaxel with Ad5CMV-p53 did not consistently or significantly decrease clonogenic survival.

5. Bladder cancer

Combined treatment with Ad5CMV-p53 and cisplatin could be an attractive strategy for inhibiting progression of bladder cancer. In human bladder cancer KoTCC-1 cells, transfer of an adenovirus-mediated p53 gene enhances cisplatin cytotoxicity in vitro, and Ad5CMV-p53 and cisplatin synergistically inhibit growth and metastasis in vivo. Ad5CMV-p53 substantially enhances cisplatin chemosensitivity in a dose-dependent manner, reducing the median IC₅₀ by more than 50%. Furthermore, orthotopic injection of adeno-p53 combined with cisplatin therapy synergistically inhibits growth of subcutaneous KoTCC-1 tumors and the incidence of metastasis (Miyake et al, 2000). In contrast, p21^cip1/waf1 gene therapy had no effect on in vitro or in vivo chemosensitivity to cisplatin (Miyake et al, 1998).

6. Lung cancer

Recombinant adenovirus-mediated transfer of the wild-type p53 gene into monolayer cultures or multicellular tumor spheroids of the human NSCLC cell line H358, in which there is homozygous deletion of p53, markedly increased the cellular sensitivity of these cells to cisplatin (Fujiwara et al, 1994). In a study made by Osaki et al,(Osaki et al, 2000), an alteration in drug chemosensitivity caused by the adenovirus-mediated transfer of the wild-type p53 gene in human lung cancer cells was tested on a human pulmonary squamous cell carcinoma cell line, NCI-H157, and a human pulmonary large-cell carcinoma cell line, NCI-H1299. Based on isobologram data, a supra-additive effect was observed for 5-fluorouracil and SN-38 on NCI-H157 cells. An additive effect was also observed for cisplatin, paclitaxel, bleomycin, and cyclophosphamide on NCI-H157 cells. Cisplatin, paclitaxel, 5-fluorouracil, and SN-38 had an additive effect on NCI-H1299 cells. No drug showed any subadditive or protective effects. These findings suggest that CPT-11 and 5-fluorouracil may be useful as anticancer agents for use in a combination therapy regimen, using wild-type p53 gene transfer. These results indicate that CPT-11, as well as cisplatin, is a candidate for the combination of chemotherapy and gene therapy for NSCLC. Adeno-p53 and DNA-damaging agents, cisplatin, etoposide and CPT-11 showed synergistic effects in NSCLC, but, in contrast had additive effects with antitubulin agents such as paclitaxel and docetaxel (Horio, Hasegawa et al, 2000). Perdomo et al, (Perdomo et al, 1998) have demonstrated that human NSCLC cells having a mutant form of p53 grow faster in vivo than wild-type p53 cell lines and the treatment with cisplatin or radiation does not reduce the size of mutant p53 tumors, although wild-type p53 tumors regress markedly. Apoptosis occurred in mutant p53 cell types only at high cisplatin doses and not at the magnitude detected in wild-type tumors.

III. In vivo evidence of chemosensitization by adenovirus p53

These observations have been extended to in vivo models. Tumors have been treated in vivo with replication-defective p53 adenovirus and chemotherapy. Nguyen et al, have reported convincing in vivo studies, in which p53null H1299 lung tumor xenografts were given i.p. cisplatin before, concurrently with, or after intratumoral adenovirus p53 (Nguyen et al, 1996). The most effective dosing regimen was cisplatin given two days before p53 therapy. Cisplatin and CPT-11 had a significant antitumoral effect on lung cancer H157 cell xenografts of nude mice in vivo. Human head and neck cancer and colon cancer (Gjerset et al, 1997) and prostate cancer (Gjerset and Mercola 2000) in nude mice models in vivo have been found to exhibit a similar sensitization effect with adenovirus plus cisplatin as in studies in vitro. Gjerset et al, demonstrated increased sensitivity to cisplatin cytotoxicity in p53mut T98G glioblastoma and p53 mut H23 small cell lung carcinoma cells transduced with p53 expression vectors one or two days before exposure to cisplatin (Gjerset et al, 1995). These results are consistent with other in vivo studies in animal models showing a combined benefit of p53 and chemotherapy (Badie et al, 1998), (Fujiwara et al, 1994), (Miyake et al, 1998), (Nielsen et al, 1998), (Nguyen et al, 1996). Gjerset and Mercola are convinced that these results support the clinical application of adenovirus p53 combination approaches to tumors expressing mutant p53 (Gjerset and Mercola 2000). Chemosensitization by p53 has also been studied using ex vivo modified cells in an orthotopic model of glioblastoma in Fisher rats (Dorigo et al, 1998).

The combination of p53 with 5-fluorouracil and topotecan has been studied in p53mut SW480 colorectal tumor cells transfected with an inducible p53 construct (Yang et al, 1996). Dose-dependent enhancement of cytotoxicity was observed with these drugs by the concurrent expression of wild-type p53. Increased cytotoxicity has been reported in p53mut SkBr3 mammary tumor cells when transduction with p53 was followed 8 hr later by doxorubicin or mitomycin-C, but not by vincristine (Blagosklonny and El-Deiry 1996).

In the p53 null SK-OV-2 xenograft model of ovarian cancer, a dosing schedule of the p53 therapy that, by itself, had a relatively minimal effect on the tumor burden (16%) caused a much greater decrease in tumor burden (55%) when combined with paclitaxel (Nielsen et al, 1998). Further, in nude mice implanted intraperitoneally with 2774 human ovarian cancer cells (mutated p53), the response to adeno-p53 gene therapy showed significant survival duration, with a survival time greater than that of untreated animals. However, no statistically significant survival advantage was observed between adeno-p53- and adenovirus-bgal-treated mice (von Gruenigen et al, 1998). In another ovarian cancer study using nude mice, the adeno-p53 treatment effectively suppressed the growth of peritoneal tumors and prolonged the survival of the treated group, especially when the tumor burden was small (Kim et al, 1999). Greater combined efficacy was observed in the p53null DU-145 prostate, p53Mut MDA-MB-468 breast, and p53met MDA-MB-231 breast cancer xenograft models in vivo. The authors concluded that their data, taken together, offer the possibility of enhanced antitumor activity with lower than normal doses of paclitaxel and adenovirus p53, when the two drugs are administered in combination (Nielsen et al, 1998). They noted that this could potentially decrease the chemotherapy-induced side effects, increasing the quality of life of the patients and, perhaps, reducing the overall expense of a complete course of cancer treatment.

IV. Clinical results of adenovirus p53 transfection with chemotherapy

The first evidence of the efficacy of p53 gene therapy for cancer was given by a pilot study in which retroviral p53 expression vectors were directly injected into small endobronchial lesions of NSCLC patients (Roth et al, 1996). Tumor regression was noted in three patients out of nine, and tumor growth stabilized in three other patients. The safety and feasibility of the intratumoral injection of adenoviral wild-type p53 expression vectors have been established in NSCLC patients, with clear evidence for transgenic expression, and possibly induction of apoptosis (Swisher et al, 1999; see Table 1). The antitumor activity in this trial was consistent with the activity of retroviral p53 injection in NSCLC patients. Twenty-four patients received intratumor injections of adenovirus p53 and two patients achieved a partial response, while 17 patients achieved stable disease as the best clinical response.

A nonrandomized, phase I, dose-escalating study by Clayman et al expanded these findings into head and neck squamous cell carcinoma (Clayman et al, 1998). Patients with incurable recurrent local or regionally metastatic HNSCC received multiple intratumoral injections of adeno-p53, either with or without tumor resection. P53 expression was detected in tumor biopsies despite antibody responses after injections. prevent the appearance of adeno-p53 in blood and urine. were seen in the study As expected, almost Neither dose-limiting effects nor serious adverse events all the patients developed anti-adenovirus antibodies in the course of treatment, but this immune response did not treatment. The most common treatment-related adverse event was pain at injection site. Other reported adverse events were transient fever, headache, pain, and edema. No evidence of systemic hypersensitivity or allergic reactions was seen, despite the fact that patients received many repeated courses of treatment. In some patients, adenovirus p53 administration led to objective antitumor activity. Two out of 17 patients showed objective tumor regressions greater than 50% and six patients showed stable disease for up to 3.5 months. In addition, one patient showed a complete pathologic response. The median survival for responding patients was 13.6 months, and the overall median survival was 267 days, which is about 60% longer than that reported in chemotherapy trials with a similar patient profile (Schornagel et al, 1995). Of course, it is impossible, for a phase-one study with limited numbers of patients to state anything more than that these results are promising and that further studies are needed, and are underway, to determine the actual role of adenovirus-mediated p53 intratumoral injections as a treatment option for HNSCC. The next step in the development of p53 treament is to include combination therapy with cytotoxic agents.

There is also a negative trial published by Schuller and coworkers (Schuler et al, 2001). Twenty-five patients with non-resectable NSCLC were enrolled in an open-label, multicenter, phase II study of three cycles of chemotherapeutics with intratumoral injection of recombinant adenovirus p53. The main idea of this small study was to compare the isolated responses of a tumor lesion treated by transfer of the adenoviral wild-type p53 gene with a comparable lesion not receiving any injections in patients undergoing first-line chemotherapy for NSCLC. In the 13 patients receiving carboplatin and paclitaxel, there was no obvious difference between the mean response of gene-therapy-treated and the reference lesions. In contrast, the mean regression of the reference lesions in patients treated with cisplatin and vinorelbine was 15%, whereas it amounted to 55% in lesions that were additionally injected with the gene construct.

There was no difference between the responses of lesions treated with p53 gene therapy in addition to chemotherapy (52%) and those of lesions treated with chemotherapy alone (48%). The authors concluded that, in these patients the therapy appears to provide no additional benefit. However, there were several possible shortcomings in the clinical set-up: no injections to the reference lesions, highly restrictive inclusion criteria may result in selection bias, a higher response rate (50%) than is normally achieved in this disease, a chance of having a biologically inactive virus construct, and insufficient spreading of the replication-defective adenoviral vectors within the tumors after only one central intralesional injection.

Recently, attemps have been made to overcome the problem of ineffective vector spreading by administration of replication-competent adenoviruses (Heise, Sampson et al, 1997) and encouraging clinical results have been reported (Khuri, Nemunaitis et al, 2000). There were concerns about the safety, which, however, turned out to be exaggerated. Khuri et al, demonstrated an acceptable safety pattern with no sign of any dissemination to the environment. A Phase II trial of a combination of intratumoral ONYX-015 injection with cisplatin and 5-fluorouracil was carried out with patients having recurrent squamous cell cancer of the head and neck. Only pain at the injection site (45%), mucous membrane disorder (21%), syncope (5%), kidney failure (5%), and anorexia (3%) could not be ruled out as attributable to Onyx-015.

In addition, the injected tumors achieved objective responses at a substantially higher rate (9 of the 11) than the non-injected tumors (3 of the 11) within the same patients. In six patients, the injected tumor responded and the non injected tumor did not respond. The time to tumor progression was also longer for the injected tumors than for the non-injected tumors. There was no correlation between the response and the baseline tumor size, baseline neutralizing antibody titer, p53 gene status, or prior treatment. It was also clear that the efficacy of the intratumoral injection was not prevented by neutralizing antibodies. There has been discussion about whether or not enough evidence about viral replication of ONYX-015 in patients, as along experience based on 190 patients treated by a replication-defective adenovirus demonstrating similar biodistribution (Clayman, El-Naggar et al, 1998; Constenla-Figueiras, Betticher et al, 1999). It may simple be that Taqman real-time polymerase chain reaction technology is not sufficient to prove that viral reproduction is taking place (Yver et al, 2001).

Table 1, Sensitising effect of adenovirus-p53 on chemotherapeutic agents, major clinical treatment results

Disease	Phase	Combination	n	Treatment responses	Reference (first author year)
NSCLC	II	no	24	2 PR, 17 SD¤	(Swisher et al, 1999)
Head & neck	II	no	17	1CR, 2 PR, 6 SD¤	(Clayman et al, 1998)
NSCLC	II	Cisplatin + vinorelbin	25	13 PR*	(Schuler et al, 2001)
Heach & neck	II	Cisplatin +5-FU	11	9 PR*	(Khuri et al, 2000)

(¤) on patients

(*) on measurable lesions

V. Conclusion

Several subsequent studies have confirmed that various malignant cell lines and tumors expressing mutant or deleted p53 are chemoresistant to a wide range of anticancer agents. However, other studies disagree suggesting that cells with impaired p53 function can become sensitized to various anticancer agents. Thus, the relationship between p53 status and chemosensitivity is complex and presumably depends on a number of factors, including the specific cytotoxic stimuli, tissue-specific differences, and the specific cellular context that incorporates the overall genetic machinery and the various intracellular signaling pathways (Chu and DeVita 2001). The relationship between p53 and chemotherapy depends on the chemotherapeutic agents used, the target and the critical tissues, and the intracellular signal transduction pathways affected.

The theoretical basis of the sensitizing effect of chemotherapeutic agents in combination with adenovirus p53 has been presented and so have a number of supportive data. As adenovirus p53 has its own activity, there seems to be a possibility that the cytotoxicity may be enhanced at least in some cell lines by transfer of the gene into the tumor cells. This concept has reached the level of proof in some, although not all, experimental conditions. This leaves a room for doubt, as all spontaneous solid tumors are heterogeneous and there may always remain cell clones that fail to obey the sensitizing principle. It is clear that more evidence is needed to support this principle, especially clonogenic assays and classical interaction studies. Although the in vivo experiments are convincing and strongly positive, it may not be altogether correct to extrapolate these results into clinical practice. There is a relative lack of pharmacokinetic studies and pharmacokinetic interaction studies in adenovirus p53 gene therapy.

Several strategies may be used to develop p53-based anticancer therapies, with the goal of resensitizing tumor cells to conventional chemotherapy (Chang 2000). These include reintroduction of the gene encoding wild-type p53 and methods for restoring normal p53 function to mutant p53. In addition, methods are being developed that target the p53-mdm-2 interaction of using lack of wild-type p53 in tumors to protect normal tissue from the adverse effects of chemotherapy. Replacement of the wild-type p53 by intratumoral transfection has already reached the phase III stage of clinical trials. Transfection of p53 can be combined with radioimmunotherapy as part of a tumor manipulation scheme (Kairemo, Jekunen et al, 1999). Increasing suppressor gene p53 expression in tumor cells improves the sensitivity of the tumor cells to routine chemotherapy. In a variety of tumor types, docetaxel and irinotecan are efficacious drugs with a new mode of action: prevention of depolymerization of tubulin and inhibition of specific DNA topoisomerase I, respectively. But we cannot obtain responses from all tumors, and in some tumors the efficacy, although established, diminished with time. In these cases of resistant tumors or recurrences and relapses, combined treatment with adeno-p53 and chemotherapeutic agents may be an attractive strategy for inhibiting progression of local cancers. It is clear that even a modest change in drug sensitivity may bring some refractory tumors within a range that is treatable with conventional chemotherapy. Future therapy might couple standard cytotoxic agents with new biologic agents that attack specific molecular targets to reregulate the cell-cycle checkpoint.

Human data supporting the effect of sensitizing chemotherapy with adenovirus p53 is still maturing, although we have not found a way to use systemic administration. We know that is s safe to perform intratumoral gene therapy with adenovirus either with a replication non-competent or replication competent vector. As yet, there is no clinical evidence to support a definite conclusion that adenovirus p53 provides a clinically meaningful improvement on conventional chemotherapy. However, it is clear that in some trial set ups it has been possible to demonstrate encouraging results and the possibility of a clinical sensitizing effect of p53 gene therapy on the chemotherapy used when specifically indicated. Intratumoral expression of transgenes and tumor-selective tissue destruction have been documented in phase I and phase II clinical trials of adenovirus p53 mediated gene therapy. However, durable responses and the clinical benefit seen have been limited, with of 10-15% response rates.

The rationale of combining p53 gene therapy with a chemotherapeutic agent in the clinical setting has been noted to be as follows: combinations of agents with different toxicologic profiles can result in increased efficacy without increased overall toxicity, they may thwart the development of resistance to the single agents, they may offer a solution to the problem of heterogeneous tumor cell populations with different drug sensitivity profiles and they allow the physician to take advantage of possible synergies between drugs, resulting in increased anticancer efficacy in patients (Nielsen, Lipari et al, 1998). Several phase III clinical trials with adenovirus p53 therapy in head and neck cancer, NSCLC, and ovarian cancer, will be completed in the near future, and the role of gene therapy may become routine a part of treatment regimens.

Acknowledgments

We would like to thank Aventis Pharma Finland for supporting this work.

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