Cell death and cancer: replacement of apoptotic genes and ... - Nature

Gene Therapy (1998) 5, 728–739  1998 Stockton Press All rights reserved 0969-7128/98 $12.00 http://www.stockton-press.co.uk/gt

REVIEW

Cell death and cancer: replacement of apoptotic genes and inactivation of death suppressor genes in therapy M Favrot1,2, J-L Coll1,2, N Louis1 and A Negoescu1

´ ´ ´ Lung Cancer Research Group, Institut Albert Bonniot, Faculte de Medecine, Universite Joseph Fourier, Grenoble; and 2Department ´ ´ of Tumor Biology, Centre Leon Berard, Lyon, France

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This review provides a critical evaluation of the increasing use of gene therapy in the treatment of malignancies to induce active cell death (ACD, apoptosis). This approach is consistent with the notion that cancer is an anomalous accumulation of cells largely resulting from diminished cell death. The review details the main genes potentially useful for therapy. Among these, p53 has received the majority of the investigators’ attention and provided encouraging results. Even greater hope is offered by newly tried direct inducers of apoptosis, such as bax, bclXS and caspases. Another fruitful direction is the association of apoptosisinducing gene transfer with radio- and chemotherapy,

which are also inducers of ACD. There is a delicate balance between cell gain through mitosis and cell loss in neoplasia because spontaneous apoptosis is widely present in tumors. In fact, the tumor environment favors bystander cell killing which appears to be a fundamental mechanism insofar as the rate of observed cell mortality cannot be accounted for by the known methods of gene transduction with efficiencies far below 100%. We conclude that apoptosis offers a mainstream approach for cancer gene therapy since ACD is highly inducible and only limited gains in malignant cell apoptosis may displace tumors from growth to regression.

Keywords: apoptosis; cancer; gene therapy

Introduction Neoplasia has long been envisioned as a disease of cell proliferation, occurring through enhanced rates of tumor cell division. In fact, there are neoplastic cells which divide more slowly than their benign counterparts but do succeed in producing tumors because of prolonged lifespan. Therefore, a broader image has emerged, perceiving cancer as an anomalous accumulation of cells engendered both by increased proliferation and diminished cell death.1,2 Indeed, among the multiple genetic events associated with tumor development are activation of antiapoptotic oncogenes such as bcl2,3 and inactivation of apoptosis-inducing tumor suppressor genes including p53 (abnormal in more than half of all malignant cell types)4 and bax.5–7 The term apoptosis describes genetically driven, active cell death (ACD). Apoptotic cells undergo separation from their neighbors, membrane blebbing, chromatin condensation, nuclear breakdown into micronuclei and cytolysis into condensed apoptotic bodies.8,9 Yet, viewing cancer from this standpoint may engender an underestimation of tumor cell loss, as shown by cell loss factor determination. Cell production, determined by mitosis quantification, allows the calculation of the potential doubling time (Tp, ie the time in which the cell number would have doubled had there been no cell

´ Correspondence: M Favrot, GRCPVA, Institut Albert Bonniot, Faculte ´ ´ de Medecine, Universite Joseph Fourier, Domaine de la Merci, 38706 Grenoble, France

loss), whereas the actual doubling time (Ta) is given by direct measurements of tumors. These two parameters permit the calculation of the cell loss factor (CLF = (1 − Tp/Ta) × 100).10 In transplantable animal tumors selected for rapid growth CLF exceeds 60%; it is still higher in human cancers, such as melanoma or colorectal carcinoma, and can reach 99% in some pulmonary tumors. Moreover, CLF augments during tumor enlargement.11,12 Cell death accounts for the majority of cancer cell elimination.12 Malignant tumors frequently display necrosis, which is mostly the result of hypoxia. Though conspicuous histologically, necrosis does not satisfactorily account for most of the losses predicted on the basis of CLF.13,14 It is therefore likely that apoptosis, which exists in all tumors,15,16 is responsible for an important part of cell death in neoplasia. Due to the rapidity of the phenomenon (2–5 min for cell condensation and fragmentation, 3 h for apoptotic-body phagocytosis and digestion), strong ACD-induced cell losses are often based on less than 5% of simultaneously visible apoptotic cells within a tissue.17 Moreover, tumor apoptosis occurs unevenly, in bursts. These features presumably precluded the precise quantification of ACD contribution to tumor cell deletion.12,18 The factors responsible for the spontaneous occurrence of ACD in malignant tumors are not unequivocally established.2 They presumably consist of two mechanisms: on the one hand, cell population regulatory mechanisms originating in the host organism, such as mild ischemia, suppression of survival signals originating in nontumor cells or death signals provided by the immune system (the so-called ‘social controls on cell death’19); on the other hand, apoptosis initiated by

Apoptosis–based gene therapy of cancer M Favrot et al

processes intrinsic to malignant cells, as indicated by different rates of ACD found in similar tumors expressing different oncogenes. For example, cancer cells frequently contain overexpressed myc gene family members20 and an inactivated retinoblastoma (rb) gene.21 The Myc protein signals cellular proliferation; yet, in cells lacking other specific mitogenic stimuli Myc induces ACD.22 Analogously, the Rb protein can be either an apoptosis inducer23 or suppressor.24 Taken together, the above data make plausible two assumptions. First, that the margins between tumor growth and regression could be crossed by a relatively minor augmentation in the rate of cell death. Second, that apoptosis, by its intrinsic property of being highly inducible, may provide a therapeutic way to increase cell losses. Indeed, over the last 15 years, a general concept has emerged that all nonsurgical modalities of cancer therapy act primarily via the induction of apoptosis, as do chemotherapy and radiotherapy, but also immunotherapy or hormone ablation.1,25–28 Mechanisms involved in therapyinduced ACD are only partially elucidated.2,27 Yet, a general property which provides the therapeutic window appears to be related to the sensitivity to apoptosis induction, which is generally lower in normal, as opposed to neoplastic cells. Thus, surrounding tissues may pause and repair the damage produced by therapy while tumor cells die through apoptosis. It is currently unclear exactly what determines this ‘threshold’ of apoptosis initiation, probably because multiple factors are likely to modulate the set point.29 Gene therapy, the newest approach for cancer therapeutics, has focused on two broad goals: DNA constructs are transduced to potentiate existing therapy, such as radiotherapy, or to deliver more specifically existing therapy, such as chemotherapy via drug sensitivity genes or immunotherapy; alternatively, transgenes are introduced to revert the genetic profile of malignancy within tumor cells.30,31 The broad spectrum of genetic abnormalities in cell transformation made unexpected the results given by the latter approach. Indeed, it might have been supposed that, to be effective, all the genetic lesions in every tumor cell would have had to be corrected, which is unlikely since vectors available have efficiencies far below 100%. Yet, it appears that correction of a single essential gene abnormality within only a fraction of tumor cells can be sufficient to induce tumor repression. Moreover, both copies of a tumor suppressor gene must be generally inactivated to eradicate its function.32 Therefore, replacement of only one normal copy of the gene in cells with homozygous loss of function can restore tumor suppression.30 Research in this area has recently concentrated on the direct induction of apoptosis through replacement of tumor suppressor genes involved in the cell death process or inactivation of ACD-inhibiting genes. Using others’ and our experience in this domain we will provide a current and critical global evaluation of this field of application of gene therapy.

The main mechanisms of apoptosis in tumor cells suggest candidate genes for cancer therapy A model progressively emerged for apoptosis induction giving the central position in ACD regulation to the

members of the bcl2 gene family.3,33 The bcl2 gene was discovered in a B cell lymphoma where a chromosomal translocation moves it into juxtaposition with trancriptional enhancer elements of the immunoglobulin locus.34 However, as described in various solid tumors, transregulatory mechanisms can also be responsible for the high levels of Bcl2 protein.3,35 Bcl2 heterodimerizes with Bax, a protein of the same family sharing 21% homology with Bcl2. Bax can be considered as the main effector of apoptosis. Its function in active cell death as a homodimer Bax-Bax is opposed by heterodimerization with Bcl2 (Bax–Bcl2). This model was substantiated by results showing that Bax also heterodimerizes with other ACDinhibiting members of the Bcl2 family such as BclXL, Mcl1, A-1;36 it is also corroborated by the correlation between Bcl2 expression and both low spontaneous ACD in tumors and general resistance to cancer therapy.3,5,35,37,38 Conversely, Bax expression is correlated with high incidence of apoptosis in lung and stomach cancers5,39 and low Bax levels are associated with poor response to chemotherapy in breast and ovary tumors.6,7 As recently demonstrated, lack of Bax protein in cancer cells can be the result of mutations in the bax gene.40 BclX is another member of the bcl2 gene family. Due to alternate mRNA splicing, bclX is transcribed into long (bclXL) and short (bclXS) forms. The BclXL protein, like Bcl2, functions as an inhibitor of apoptosis. In contrast, the BclXS protein is a dominant inducer of ACD.41 However, BclXS acts as a monomer and does not show the ability to form heterodimers with other Bcl2 family members.42 The final step of apoptosis induction, termed ‘executioner’ by Martin and Green,43 is represented by proteases of the interleukin-1␤ converting enzyme (ICE) family, which directly generate cell destruction; they have recently been grouped under the term ‘caspases’ for ‘cysteine-aspartic acid specific proteinase’.44 Caspases are most probably stimulated by the Bax-Bax homodimer and the process can be opposed by heterodimerization with Bcl2 (Bax-Bcl2) producing caspase blocking and cell survival.45,46 Recent results indicate that Bax can induce apoptosis independently of dimerization processes47,48 and, furthermore, without caspase activation;49 caspaseindependent execution was also described for another death-inducing homologue of Bax, Bak.50 This would imply that Bax and Bak are not merely inducers of the executioner but, together with caspases, components of it. This model proved useful to explain tentatively the function of most ACD regulators, including those not belonging to the bcl2 family. The first among these is the P53 protein. The mechanisms underlying apoptosis induction by P53 are as yet obscure.51 The wild-type (wt) p53 gene is induced in response to genotoxic stresses, including DNA damage, oxygen deficiency and altered ribonucleotide pools.52 According to the hypothesis put forward by Lane,53 P53 blocks the cell cycle to allow DNA repair. If repair fails, P53 triggers apoptosis. Thus, tumor cells lacking normal P53 will accumulate mutations and chromosome rearrangements leading to selection of highly malignant clones. This ‘guardian of the genome’ model for P53 function,53 while far from representing a complete explanation of P53-dependent ACD, is supported by the lack of normal p53 in more than half of all tumor cell types.4 P53 being the main known downregulator of bcl2, its mutations could result in elevated

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production of Bcl2. In addition, P53 strongly induces the expression of bax.54–56 Thus, p53 appears to be an important regulator of apoptosis through the Bax-Bcl2 balance, and any type of p53 inactivation could abrogate cell death through Bax/Bcl2 dysbalance, as recently confirmed in vivo.57 Accordingly, the survival advantage conferred by the absence of normal P53 in malignant cells may explain their increased drug resistance.4,58,59 Similarly, radiation-induced ACD is P53-dependent and is inhibited by Bcl2 and BclXL.58–63 Finally, overexpression of the murine double minute 2 gene (mdm2), which inhibits P53-directed transcription of target genes,64,65 may induce a multiple drug resistance phenotype in tumor cells expressing wt p53,66 presumably by blocking p53 apoptotic function.67 Caspase activation can also occur directly, without mediation by Bax homodimers. Such a mechanism is best illustrated by the tumor necrosis factor receptor (TNFR) family members, such as Fas (CD95, specific for the Fas ligand) and TNFR1 (which binds TNF␣).68 Upon its activation, Fas recruits FADD (Fas-associated protein with death domain). Similarly, TRADD (TNFR1-associated death domain protein) binds to TNFR1 and connects it to FADD via interaction between death domains. Subsequently, FADD activates FLICE (FADD-like ICE, caspase 8) inducing apoptotic execution.69 Thus, the TNFR family may be a promising therapeutic target since its direct caspase activation is both quick and irreversible.69 This brief presentation of the main pathways of apoptosis for which tumor cells may be deficient, readily suggests candidates for therapy: ACD-inducing genes and antisense constructs aimed to block apoptosis repressors. However, ACD augmentation would be effective against a tumor only if transduced DNA does not enhance the replication rate. This caveat does not apply to p53, which induces either G1 arrest or apoptosis.21 The difficulty may appear within the bcl2 gene family since Bcl2 reduces cell proliferation, and Bax, while antagonizing the cell survival function of Bcl2, also reverts its antiproliferative activity.70,71 Few results are available to resolve this matter. However, the obstacle seems limited when compared with the high apoptotic rates which gene transduction can provide, enhanced, as shown below, by various mechanisms of bystander cell killing.

Wild-type p53-mediated anticancer strategies Due to its widespread alterations in malignant cells and its central position in both proliferation arrest and apoptosis induction, p53 has been the most investigated candidate for apoptotic gene replacement therapy methods. Results provided by these studies are often contradictory. In vitro, with some exceptions, such as neuroepithelioma or pancreatic cell lines, transduced p53 suppressed growth of tumor cells, as described for leukemia and lymphoma, osteosarcoma, melanoma, stomach, colon, ovary, uterus, breast, prostate, bladder, head and neck, and lung cancers.72–96 However, while unequivocally demonstrated in some of these cell types (such as lymphoma, uterine, head and neck carcinoma and osteosarcoma cells) apoptosis was absent from cells originating in pancreatic cancers.79,82–84,97–100 Glioblastoma cells gave contrasted results: no apoptosis80,101 or, at the opposite

extreme, ACD initiation.102,103 The same conflicting results were noted between p53-transfected colon carcinoma cells92,104 and for hepatocarcinoma cells.80,105 Lung tumor cells are one of the most investigated targets for the effects of wt p53 transfection, which contributed to the application to lung cancer of the first clinical administration of p53. In vitro, ACD induction by transfection of wild-type p53 proved variable in these cells. Thus, p53 did not induce in vitro apoptosis of H358 cells, a p53-null human non-small cell lung cancer (NSCLC) line despite a virus-mediated transfection efficiency superior to 80%,106,107 a result contested by Katayose et al108 and Qazilbash et al.96 Alternatively, Fujiwara et al109 reported apoptosis induction in spheroids formed by H322 cells (a human NSCLC line bearing mutated p53) whose growth was inhibited by retroviral wt p53 transfection. In a small cell lung cancer (SCLC) line (N417, mutated p53) wt p53 transfection did induce apoptosis.110 In vivo, transduced p53 generally induced tumor growth retardation in nude mice bearing tumors produced by glioblastoma, osteosarcoma, melanoma, breast, colon, head and neck, and human lung malignant cells. Apoptosis was specifically detected in these tumors. However, in similar conditions, human pancreatic cancer cells produced tumors growing independently of p53 administration and ACD induction was not observed. Prostate cancer cells (148-1PA line, p53-null) implanted in syngeneic mice produced tumors unresponsive to p53 transduction; however, in nude mice, another prostatic line (Tsu-pr1, mutated p53) gave tumors whose growth was strongly retarded by wt p53. The antitumoral effect is largely related to the number of implanted tumor cells and the time allotted for tumor growth before treatment. Thus, head and neck squamous carcinoma cells when implanted in low amounts (2.6 × 106) in nude mice and treated before visible nodule formation produced no tumors, whereas starting with twice as many cells, allowed to produce 1 cm diameter nodules, gave only a 50% reduction in tumor size due to p53 transfection. Analogously, only 25% of the nude mice inoculated intratracheally with 2 × 106 squamous lung cancer cells (H226Br line, mutated p53) developed tumors when intratracheally instilled with wt p53 3 days after cell administration.76,77,80,82–84,93,95,96,103,104,111–115 The above contradictory experimental results suggest several explanations. The first is offered by p53 genotype of the treated cells. Indeed, in most of the studies cited above, growth inhibition and cell death were observed in cell lines defective for the P53 protein (mutated or absent). Yet, Liu et al84 reported cytocidal effect of p53 in head and neck cancer cells although wild-type P53 was expressed. Reciprocally, ACD did not occur after wt p53 transfection in neuroepithelioma, colon or pancreatic cancer cells despite the lack of endogenous wt P53.75,82,116 Surprisingly, in vitro P53-induced ACD seems to occur only above a certain percentage of transfected cells. Thus, p53 adenovirus-transduced glioma cells responded by apoptosis only at transfection efficiencies superior to 40%. Growth arrest and apoptosis were not observed below this proportion of infected cells, though the original status of their p53 gene was defective.117 Another explanation may be the cellular level of transduced wt P53 which dictates the response of the cell. Low levels of P53 result in cycle arrest whereas high levels result in apoptosis;118–120 at still lower levels, insufficient


to cause cell cycle arrest, P53 offers protection against apoptosis.121 Moreover, recent results indicate that cotransfection of wt p53 together with a fusion peptide corresponding to the carboxy-terminal part of P53 activates DNA binding of both wt and mutant P53 resulting in strong induction of apoptosis.122 In any event, with most gene transfer efficiencies below 100%, the antitumoral effect of p53 relies strongly on the ability of transduced cells to induce apoptosis around them, in parental cells.123 This phenomenon was termed ‘by-stander killing’; it consists in the induction of apoptosis and was mainly investigated for suicide genes, in particular, herpes simplex virus thymidine kinase (HSVtk).124,125 In vitro, the phenomenon relies on toxicmetabolite circulation between transfected and nontransfected cells via apoptotic bodies,126,127 gap junctions128 or by diffusion in the culture medium.129 These mechanisms have been proposed for P53-induced bystander apoptosis of H322 and H358 lung cancer cells in vitro.96,106 In vivo, bystander apoptosis may be favored by increased cell– cell interaction130 and by apoptotic-body phagocytosis, stronger in tissues than in culture.18,96,131 Furthermore, bystander killing is boosted in vivo by an immune component which also allows the rejection of distant metastases and resistance to further rechallenges with nontransfected tumor cells.132,133 However, the immune basis of this distant bystander effect is still uncertain and could possibly be the result of either natural killer cells or of a still unknown soluble factor when observed in severe combined immunodeficient (SCID) and nude mice.134–136 These data are strengthened by very recent results showing that therapy-resistant (clonogenic) neoplastic cells fail to produce tumors if surrounded by apoptotic cells. When surrounding cells are not dead, but only cyclearrested, they presumably provide a ‘feeder’ support to the clonogenic cells allowing them to proliferate.137 P53 acts far upstream in the apoptotic pathway so that many other genes are epistatic to it.138 Therefore, apoptosis can be both dependent on, and independent of, P53 status and function.139 Moreover, P53-mediated apoptosis can occur, in certain cell types, in the absence of P53-dependent transcriptional activation of P53-target genes.67,140 P53 is also known to be capable of associating with a plethora of cellular proteins,141 and it is thus plausible that it may exert an ACD regulatory function through many of these factors. For example, Fas/APO1 is a direct transcriptional target of P53, as determined in vitro on colon and NSCLC cell lines. A certain threshold for the effects of P53 on Fas expression might exist in the presence of mutant P53.142,143 Wt p53 is also able to reduce tumor growth and metastases through inhibition of angiogenesis. Wild-type p53transduced glioma cells secrete an effector of yet uncertain structure, termed GD-AIF (glioma-derived angiogenesis inhibitory factor), which strongly neutralizes the angiogenic signals produced by the parental cells, as well as those of basic fibroblast growth factor.144 Moreover, P53 stimulates the in vitro production of thrombospondin I by fibroblasts, a potent inhibitor of vascular development.145 Finally, mutant P53 potentiates the induction of vascular endothelial growth factor (VEGF), an activator of tumor neoangiogenesis.146 Indeed, p53 gene transfer induced growth retardation in a model of human breast cancer cells implanted into nude mice. No evidence of cytotoxicity was detected in treated tumors, but they

exhibited a reduced number of blood vessels.123 Therefore, wt p53 restoration may have therapeutic implications beyond the growth and apoptosis regulation in individual cells and the complex mechanisms involved in the antitumoral effect of P53 in vivo cannot be entirely extrapolated from in vitro results. The abundant experimental data concerning p53 transfection provided a sound basis for clinical applications. To our knowledge, eight clinical protocols have been proposed, involving 72 patients. The first clinical results of wt p53 transfection have been recently reported by Roth and co-workers,147,148 who obtained tumor regression in three patients and tumor stabilization in three others out of nine non-small cell lung cancer (NSCLC)-afflicted humans. The lesions were treated either by bronchoscopic or by percutaneous injections with a retroviral vector containing the p53 gene. ACD induction was strong in seven patients (10.6 ± 2.9 apoptotic cells, versus 1 ± 0.5 for pretreatment biopsies) independently of their response to therapy. The same authors also proposed a clinical protocol involving combined wt p53-antisense ras therapy for NSCLC: out of six patients, one gave a complete response; two, a partial response; and three exhibited stable disease. Habib and coworkers proposed a protocol for p53 transfection in colorectal liver metastases and another one for hepatocellular carcinoma: the first elicited no response; the second induced one complete response, two partial responses, and one minor response in four patients.30 Venook and colleagues proposed a protocol for liver carcinoma and Clayman and co-workers proposed a wt p53 protocol for head and neck squamous cell carcinoma: no data are available about responses obtained in the latter two protocols.30,149

Current evolutions and promising directions to potentiate apoptosis induction through genetic engineering Gene therapy-based enforcement of chemotherapy and radiotherapy As ACD progressively gains a central place within the goals of cancer gene therapy, new mechanisms are devised to increase apoptosis-inducing strategies. One of these mechanisms is P53-mediated sensitization to ACD induced by nongenetic means of cancer therapy, ie drugs and radiation.2,25 Radiation-induced apoptosis is P53-dependent, as demonstrated both in vitro and in vivo.60,63,150 And indeed, wt p53 expression strongly potentiates the ACD-based cytotoxicity of ionizing radiation, as demonstrated in vitro using colon carcinoma cells. Interestingly, wt P53 did not have by itself a cytotoxic effect on these cells which expressed endogenous mutant P53 and responded to wt p53 transfection by mere growth arrest. Similar results have been reported for glioblastoma cells. In vivo, combined p53 and radiation therapy reduced colorectal carcinoma growth within nude mice more efficiently than each of the two methods performed alone.116,151,152 In vitro, expression of wt p53 into cancer cells powerfully enhanced their sensitivity to apoptosis induced by drugs, such as platinum salts and topoisomerase II inhibitors. These results were essentially obtained for glioblastoma, liver and colon cancer cells.116,151,153 Accordingly, mdm2 antisense oligonucleotides, by liberating P53

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activity, increased the in vitro susceptibility of a glioblastoma cell line to cisplatin-induced apoptosis.66 In vivo, experiments performed in nude mice for lung cancer cells indicated that cisplatin plus p53 gene transfer strategy yielded significantly greater apoptosis and tumor growth retardation than wt p53 gene transfer alone. The timing was shown as critical: cisplatin administration simultaneous with or subsequent to p53 gene was less effective than cisplatin-first sequential treatment.107,154 However, important exceptions exist here too. Wild-type p53 gene transduction in a p53-null pancreatic carcinoma cell line did not change its sensitivity to four different types of anticancer drugs, including cisplatin, etoposide, 5-fluorouracil and paclitaxel.82 Moreover, normal p53 function protected fibroblasts against the anticancer agent paclitaxel; in this case, p53-deficient cells stalled in G2/M upon drug action and triggered apoptosis, whereas cells harboring wt p53 traversed through mitosis and escaped ACD.155 Analogously paradoxical results were reported by Fan et al156 for cisplatin and pentoxifilline in breast cancer cells. Furthermore, wt P53 stimulated the promoter of the multidrug resistance gene when transfected into p53-null H358 NSCLC cells as in ovarian or colon carcinoma cell lines.157 Results obtained in vitro and in vivo with combined cisplatin and p53 transfection in lung cancer cells form the basis for the clinical protocol recently proposed by Roth and co-workers.158 This protocol employs two strategies, the first one exploring the adenovirus vector-connected toxicities, and the second consisting of effective wt p53 and cisplatin administration in patients. To emphasize even more the complexity of the mechanisms involved in P53-mediated potentiation of chemotherapy, in vitro incubation of acute myelogenous leukemia cells (wt p53) with p53 antisense oligonucleotides resulted in a 90% reduction of the cell population. When the surviving cells were replated in the absence of the oligonucleotides, a continuing decline in cell number was recorded. Since preclinical trials demonstrated a very low toxicity of these oligonucleotides, a phase I clinical trial was conducted in 16 patients. Though no clinical complete response was obtained, leukemic cell growth was reduced and their sensitivity to apoptosis-inducing agents augmented. Measurement of p53 expression demonstrated a transient decline during the initial treatment with the antisense construct, followed by a four-fold increase. This led the authors to hypothesize that the transient inability of p53 to respond to the stimulatory messages given by drug-induced DNA damage eventually results in p53 overexpression driving cells into ACD.159,160

Cancer gene therapy based on final inductors of apoptosis Conflicting results obtained with p53, a multifunctional gene acting far upstream in the ACD pathway, led to targeting cancer gene therapy toward the final steps of apoptosis induction. Indeed, ACD-induction efficiency might be inversely proportional to the length of the downstream pathway between the function of the transfected gene and cell execution. One of the factors thus considered is Waf1 (Cip1, P21), a known mediator of P53-induced growth arrest and apoptosis.161–163 Waf1 is a cyclin-dependent kinase (cdk) inhibitor which controls cdk-mediated inactivation of Rb

through phosphorylation. Rb inactivation permits DNA synthesis and mitosis which explains the large number of tumors containing a mutated, or deleted rb gene.164 Rb also inhibits apoptosis, presumably because its antiproliferative function facilitates the growth arrest necessary to DNA repair. In the absence of Rb, damaged cells would apoptose after receiving the signal to proceed inappropriately through the cycle.165 However, variable results were obtained by transfecting waf1, because it proved able to act through three mechanisms, ie cell cycle arrest, apoptosis initiation or cell differentiation. In vitro, it initiated ACD in cell lines of osteosarcoma cells, but not in lung H358 cells, melanoma or renal carcinoma cells. In the two latter cell types, it produced morphological changes, such as an increased nuclear:cytoplasmic ratio, and increases in adherence, and growth arrest, consistent with a differentiated phenotype. Results for breast cancer cells are contradictory: cell cycle arrest without ACD or, on the contrary, general apoptosis induction. Interestingly, before ACD induction, the majority of the waf1-transfected breast carcinoma cells displayed a 50 to 100-fold increase in size and persisted as single giant cells for several days without division, while the remainder underwent nuclear division (multiple nuclei) but were unable to complete cytokinesis. Some of these differences may be attributed to the status of p53 because P53 mutants can variably counter one or other of the functions of Waf1. In nude mice, waf1 retarded tumor progression in prostate cancer, melanoma and renal carcinoma cell-induced tumors, despite the lack of detectable apoptosis.77,108,112,166–169 In vitro transduction of another cdk inhibitor, p16INK4 (inhibitor of cdk4), inhibited proliferation of NSCLC cell lines. It also induced ACD if overexpressed together with p53 in liver and colon tumor cell lines, in which p53 alone did not induce apoptosis. Apoptosis dependent upon P16 appeared to be connected to its ability to repress Rb synthesis. In vitro, tumors produced in nude mice with NSCLC lines grew slower after p16 transfection; for liver and colon carcinoma cell-induced tumors, some growth retardation was observed with p16, little effect was seen with p53, but strong inhibition of tumor growth was obtained with the combination of the two genes.170,171 BclXS efficiency as an antitumoral agent has been recently tested. In vitro, its expression consistently induced growth arrest via apoptosis induction in cell lines originating in tumors of breast, stomach, colon and neuroblastoma. In accordance with its probable dimerization-independent apoptotic action, BclXS was strongly efficient even in the presence of high levels of Bcl2 or BclXL. Moreover, BclXS potentiated chemotherapyinduced apoptosis in cultured breast cancer cells. In vivo, BclXS suppressed growth of mammary tumor cells implanted in nude mice via apoptosis induction. Antitumoral activity was greater than predicted from the percentage of transduced cells, which raises the possibility of a BclXS-mediated bystander apoptosis.172–175 Bax in vitro transduction of breast and ovarian cancer cells did not affect viability but increased chemo-, and radio-induced apoptosis.176,177 During the last year, we concentrated upon comparing therapeutic effects of p53, bax and bclXS, in H322 (mutated p53) and H358 (p53null) bronchioloalveolar carcinoma cells.178 These indicated an important advantage for bax in terms of cytotoxicity and terminal deoxynucleotidyl transferase-mediated


dUTP nick end-labeling (TUNEL)-quantified apoptosis over both p53 and bclXS, the latter two providing almost identical effects. Therefore, it is conceivable that transfection-induced Bax expression acts by enriching the cellular pool of Bax-Bax homodimers, which readily turns on caspases and initiates the execution of the cell. Such a mechanism could be more immediate and thus more efficient than ACD induction by P53. This interpretation is supported by the difference we observed in Bax cytotoxicity between H322 cells (about 70% of Bax-expressing cells died) and H358 cells (more than 90% cytotoxicity). Indeed, blot experiments revealed undetectable levels of Bcl2 in H-358 cells as opposed to a notable expression of this protein in H322 cells. Since P53 regulates the BaxBcl2 balance by repressing bcl2 and transactivating bax,54,55 bax-p53 cotransfections would be expected to enhance the apoptotic effect of bax by favoring the liberation of Bax from heterodimers with endogenous Bcl2 and the accumulation of ACD-inducing Bax-Bax homodimers. We were not able to identify such a cytotoxic advantage for bax-p53 cotransfections, possibly because there was little enhancement to be gained beyond the strong effect of bax acting alone. Our results showed poor, if any, bystander killing and growth inhibition by transfected p53 or bax in vitro. Finally, work in progress in our laboratory indicates that the in vivo antitumoral effect of bax is still stronger than that of p53 and that bax delays in vivo tumor growth via bystander mechanisms. The first attempt of tumoricidal therapy based on caspase gene transfection reported that ICE (caspase 1)induced apoptosis in the majority of transduced glioma cells, both in vitro and in vivo, despite their lack of functional P53; yet, caspase-tolerant tumor cells subpopulations could be detected.179 Finally, caspase activation can be obtained through Fas gene transfection, as demonstrated in vitro for glioma cell lines. The Fas/APO1 cDNA expression vector dramatically enhanced cell surface expression of Fas and induced susceptibility to Fas antibody-mediated apoptosis.180 Similarly, TNF␣-transduced lymphoma T cell lines underwent apoptosis. The ACD process was mediated by the released TNF␣ in autocrine and paracrine fashion and promoted a strong bystander killing of parental cells. Interestingly, while leaving unchanged P53 and Bax levels (as expected from its action downstream of these proteins) TNF␣ down-regulated Bcl2 expression, showing that its apoptotic function is complex. In vivo, TNF␣ secreted by in vitro engineered cells implanted in syngeneic mice together with parental tumor cells, induced tumor regression after a short initial growth. However, the effect is likely to depend on immunity since immunosuppression of animals eliminated the ability of TNF␣transduced tumors to regress.181–183

Oncogene inactivation therapy Oncogene inactivation concerns primarily antisense therapy directed against genes which encode signal transduction proteins. These approaches have succeeded in reversing the neoplastic phenotype and dramatically reducing cell proliferation and tumorigenicity. The most extensive application of the methodology is anti-ras therapy.184–187 Anti-raf,188 anti-fos189 and anti-myc190 have also been tried. These antisense procedures generally do not induce apoptosis, though raf is an inhibitor of ACD, pre-

sumably via raf,191 and protects cells against chemotherapy-induced ACD.192 Bcr-abl antisense nucleotides initiate in vitro apoptosis of chronic myelogenous leukemia cells and render these cells susceptible to ACD produced by chemotherapeutic agents. Most likely, the BcrAbl oncoprotein provides an internal stimulus which renders these cells independent of ACD triggers. By reducing this stimulus, when growth factors are not sufficiently available or a cytotoxic signal is present, cells can undergo apoptosis.193–195 In vivo, chronic myelogenous leukemia evolution was strongly retarded and cure was achieved in about 50% of severe combined immunodeficient mice treated with a combination of bcr-abl antisense oligonucleotides and either myc antisense DNA or cytotoxic drugs; apoptosis was demonstrated in these cells.196,197 The main target for survival-gene inactivation was bcl2 and antisense strategies have been developed to lower bcl2 expression. In vitro, they potentiated spontaneous apoptosis and chemotherapy-induced ACD of lymphoma cell lines or of acute myeloid leukemia-patient-derived cells.198–201 In vivo, SCID mice inoculated with lymphoma cells treated with antisense bcl2 before inoculation failed to develop malignancy. The likely explanation is that, by down-regulating bcl2 (about 30% reduction as compared with control) in a cell that is heavily dependent on Bcl2 for survival, the cell is committed to ACD. Even if Bcl2 expression increases after this critical point the cell cannot recover. Thus, antisense-treated cells, although apparently viable when they are injected into the mice, are doomed and tumors do not result.202 Bcl2 antisense strategy formed the basis of a first clinical protocol for non-Hodgkin lymphoma.203 An 18-base antisense oligonucleotide was subcutaneously administered for 2 weeks to nine patients. In two patients, tumors regressed and in two others, the number of circulating lymphoma cells decreased. The eight patients received further chemotherapy, six of them answering by favorable evolution. Encouragingly, the oligonucleotide maximum-tolerated dose was not reached, doses used being just above those effective in animals; no toxic effects were noted, which permits higher doses to be used in the future. This is particularly promising since there was concern that bcl2 antisense might affect cells that normally contain high levels of this protein in connection to their long life span, eg memory B cells or neurons. Yet, these cell types apparently resisted, possibly due to other ACD inhibitors or to limited access of the nucleotides to the brain. Alternatively, enforced expression of anti-apoptotic genes can be used to diminish ACD-based myelosuppression during cancer chemotherapy. Indeed, in vitro experiments demonstrated that bcl2-transduced murine bone marrow cells resist apoptosis induced by both topoisomerase I and II inhibitor cytotoxic drugs.204 Similarly, in vitro experiments demonstrated that bclXL is a strong inhibitor of chemotherapy-induced apoptosis. In addition, this property spans over a wide variety of cells and anticancer agents, its expression conferring a multidrug resistance phenotype.205,206

The incidence of gene transfer techniques on apoptosis induction Despite the central position of apoptosis in the action of almost all gene therapy protocols for cancer, little infor-

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mation is available about the involvement of DNA transfer techniques in ACD initiation. Adenoviruses produce several proteins which are implied in apoptosis regulation in the host cell. The main participants are the early (E) viral proteins E1A, which induce apoptosis by both p53-dependent and p53-independent mechanisms and E4, which initiates ACD independently of p53 expression.207–209 The viral e1B gene encodes two products that inhibit apoptosis, a 55 kDa protein which inactivates P53 and a 19 kDa protein which acts downstream, analogously to Bcl2, to block Bax.210–213 Adenoviruses engineered for gene therapy have generally e1A and e1B deletions to preclude uncontrolled virus replication, but e4 has been mostly left functional and thus can exert its lethal effect.214 Data are lacking to evaluate the impact of adenovirus vector on apoptosis during cancer gene therapy. However, at least one salient application of one of the abovedescribed mechanisms has been tested. An E1B-55 kDadeficient adenovirus, unable to bind and block P53 replicates in, and kills, p53-deficient tumor cells but not cells with functional p53. Tested in vitro on p53-defective glioblastoma, uterine, colon and pancreas carcinoma cells, as well as in nude mice bearing human cervical carcinoma tumors, the defective virus exhibited a strong cytopathic effect specific for tumor cells, inducing complete regression of 60% of implanted tumors.215 Interesting results have also been provided by cationic liposomes (lipofection216), an attractive method since, by not requiring packaging cells, they are adapted to the transfection of highly cytotoxic DNA as ACD-inducing genes. The sequence of phenomena linked to liposomemediated delivery of DNA may influence cell viability. Indeed, recently published data indicate that lipofection specifically induces apoptosis in lymphocytes.217 These results are in agreement with our observations that many NSCLC adherent cells gained apoptotic morphology and became TUNEL-positive upon cationic-lipid delivery. Moreover, underlining its apoptotic nature, cytotoxicity was diminished by transfection of the ACD-inhibiting bclXL gene (Coll, unpublished observations).

Conclusions The above overview presents gene therapy of malignant disease in general as an attempt to induce apoptosis through genetic engineering. This focusing upon ACD is supported by the emerging in vivo and clinical data showing that the efficiency of the approach is accompanied by low general toxicity. Encouraging results obtained with the most used gene, p53, showed that the apoptotic program can be imposed in malignant cells by exogenous DNA constructs. But the variability of upstream ACD initiation also caused therapy to be targeted towards the progressively discovered final steps of apoptosis induction. This approach is based on the hypothesis that a short pathway between gene action and cell execution contributes to achieve reproducibly intense effects. As disclosed by quickly accumulating results, the strong cytocidal efficiency of bax, bclXS and caspase 1 supports this assumption. The same goal is also best attained by the association of ACD-aiming transfection with other methods of therapy, ie radiation and drugs, which are also inducers of apoptosis. Yet, performed either singly or in combination with

other methods, cancer gene therapy can reach only part of the cells forming a tumor and its metastases. Thus, a basic mechanism needed for tumor therapy is bystander apoptosis. The above data point to the counterintuitive fact that the attempt to induce ACD may be favored by a tumoral environment. While mechanisms underlying this peculiarity are far from elucidation, the trend is not totally unexpected. One could argue that, considering cell loss factor values, most cancers meet apoptosis-inducing genes with a sort of ‘spontaneous bystander killing’ around 90% of the newly produced tumor cells. Thus, the titles of two seminal papers may serve to formulate a final conclusion for this review. WT Shier asked:218 ‘Why study the mechanisms of cell death?’. The answer may reside in that, concentrating on killing the 10% surviving cells and using for this purpose the highly regulated mechanisms of active cell death, gene therapy could sustain the hope expressed by DE Fisher:29 ‘Apoptosis in cancer therapy: crossing the threshold’.

Acknowledgements ` This work was supported by the MESR (Ministere de ´ l’Enseignement Superieur et de la Recherche), the ´ ´ FNLCC (Federation Nationale de Lutte Contre le Cancer), the LNCC (Ligue Nationale de Lutte Contre le Cancer), ´ the Fondation pour la Recherche Medicale and the AFLM (Association Franc¸aise de Lutte Contre la Mucoviscidose). N Louis acknowledges financial support from the LNCC and A Negoescu from the COMARES ´ ` (Comite des Maladies Respiratoires de l’Isere). We thank Dr Ruth Griffin-Shea who kindly agreed to revise the English version of this manuscript.

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