The Use of Fas Ligand

INTRODUCTION

In the United States prostate cancer (PCa) is the second most frequently diagnosed cancer after skin cancer. When PCa is diagnosed early there are at least four treatment options including radical prostatectomy, brachytherapy, cryotherapy and external beam radiation. None of these approaches are guaranteed curative over a 10-15 year interval. Survival is less than 28% at 15 years and all treatment modalities come associated with the significant morbidity of impotence and incontinence. The prognosis is decidedly worse if on diagnosis, PCa has escaped the capsule and become metastatic. At the present time, treatment options for metastatic disease are chemical or surgical castration, which prolongs life although all patients eventually succumb to the disease if they live long enough. Thus, with the advent of gene therapeutic approaches for treatment of cancer, there is widespread interest in the possibility that this type of science may eventually impact on survival in PCa patients as well as providing improvement in quality of life issues. An excellent wide-ranging review of gene therapy for prostate cancer was recently published [Steiner and Gingrich 2000].

There are a number of different approaches for gene therapy (molecular therapy) for the treatment of prostate cancer presently in clinical trials (www.clinicaltrials.gov) that are wide-ranging in scope. Substantial efforts have been directed toward immunotherapy using cytokine delivery and a variety of vaccine approaches. Other trials have examined the concept of corrective therapy using replacement of a defective gene by the non-defective gene for example, p16, and p53. The p53 approach has been applied, with some efficacy, to other cancers as well, specifically head and neck, ovarian and lung cancer. Recently, studies have demonstrated synergism of this approach with low-dose chemotherapy. In addition, other trials are underway on suicide gene therapy, a promising new area of research based on expressing an enzyme such as the thymidine kinase gene from Herpes simplex, which has the property of being able to metabolize a nontoxic pro-drug into a toxic molecule, which diffuses in the localized area to cause a bystander effect.

Another approach not yet in clinical trials is based on over-expressing apoptosis-inducing genes in prostate cancer such as Fas Ligand, Bax or TRAIL. FasL and TRAIL and perhaps Bax function by inducing a bystander effect that can enhance molecular therapy. The bystander effect is defined as the situation where more cells die than would be predicted by the number of cells transduced. The mechanism for this is not clearly understood but likely is a combination of events from cell-to-cell transfer of toxic molecules, recruitment of immune system components, to production of microvesicles from apoptotic cells that maintain the ability to induce apoptosis [Martinez-Lorenzo 1999]. This bystander effect is especially important because of the current unsatisfactory status of delivery vehicles used in gene therapy trials. Thus, if it were possible to deliver a gene to one cell which, when it died, would kill three additional cells, then delivery to 25% of the tumor (achievable now) would be predictive of a cure. This approach is the focus of this review and covers the use of adenovirus to deliver apoptosis-inducing genes that subsequently kill surrounding cells. FasL and TRAIL take advantage of this potential. It is not yet clear if Bax kills by bystander action.

FAS LIGAND

Fas Ligand (CD95L or APO-1L) is a 40kDa type II membrane protein belonging to the Tumor Necrosis Factor (TNF) family. Its receptor, Fas (CD95 or APO-1) is a 45 kDa type I membrane protein belonging to the TNF/NGF (Nerve Growth Factor) superfamily of receptors [Suda 1994, Takahashi 1994]. Following engagement with its ligand, Fas functions to initiate an apoptotic signal in Fas-bearing cells. This signal originates at the death inducing signaling complex (DISC), which forms just below the cell’s surface on the cytoplasmic domain of Fas. The DISC, in part, is composed of Fas, an adapter molecule (FADD/MORT), and pro-caspase 8 (FLICE/ MACH) [Ashkenazi 1998]. Upon Fas stimulation, FADD and pro-caspase 8 are recruited to Fas enabling pro-caspase 8 to autocatalytically activate itself [Medema 1997]. Active caspase 8, in turn, cleaves and/or activates several downstream substrates including the effector caspases 3 and 7 [Muzio 1997]. These effector caspases are responsible for cleaving vital cellular substrates (for example, RB, PARP, and lamins), which ultimately leads to apoptosis.

Fas is a widely expressed protein found on the plasma membrane in most tissues including prostate. FACS analysis has been used to detect membrane Fas expression on the surface of a number of different human PCa cell lines in vitro [Hyer 2000, Hedlund 1998, Frost 1997, Rokhlin 1997, Uslu 1997]. Membrane Fas expression has also been detected on the surface of both benign [Rokhlin 1997] and malignant human prostate tissue samples [Sasaki 1998] using immuno-histochemistry. In contrast, FasL expression appears to be more tightly regulated on the plasma membrane. Membrane FasL (mFasL) expression has only been detected in immune privileged tissues such as: testis [Bellgrau 1995], retina [Griffith 1995], cornea [Stuart 1997], and in immunological cells (T and NK cells) [Rouvier 1993, Arase 1995, Stalder 1994]. However, several recent reports suggest that mFasL occurs in both normal and malignant prostate, although this data remains controversial. Liu et al. [Liu 1998] detected mFasL expression on the surface of cultured LNCaP cells using FACS analysis. In the same report, they also detected soluble FasL (sFasL) in the culture media of PC-3, DU145, LNCaP cells, and within the intraluminal secretions of normal prostate epithelial cells (human). Soluble FasL is generated by matrix metalloproteinase (MMP) cleavage of membrane bound FasL (mFasL) between a.a. 127 and 128 [Mariani 1995, Kayagaki 1995, Powell 1999]. In contrast to the aforementioned report, Sasaki et al. [Sasaki 1998] was unable to detect mFasL expression in 21 of 21 localized PCa specimens using a similar approach. Cleavage of mFasL by the MMP may explain these discrepancies.

Despite the inconsistencies regarding surface FasL expression in prostate, several experiments have demonstrated that a functional Fas-mediated apoptotic pathway exists in the prostate. This evidence comes both from in vitro and in vivo studies. In vitro, some PCa cell lines (PPC-1, ALVA-31, JCA-1, [Hedlund 1998], PC-3 [Rokhlin 1997]) are sensitive to Fas-mediated apoptosis when challenged with a Fas agonist i.e. anti-Fas antibody or FasL expressing effector cells [Hyer 2000, Rokhlin 1997, Hedlund 1998]. Other PCa cell lines (DU145, ND1, JCA-1 [Rokhlin 1997], PC-3 [Frost 1997, Uslu 1997] were found to be resistant when challenged with a Fas agonist. This resistance, however, was overcome by pretreatment using sub-toxic concentrations of cyclohexamide, cis-diamminedichloroplatinum(II) (CDDP), VP-16, adriamycin (ADR), or camptothecin [Rokhlin 1997, Frost 1997, Uslu 1997, Costa-Pereira 1999]. These chemo-therapeutic drugs have different mechanisms of action, but presumably function to remove a block in the Fas-mediated pathway and allow the death signal to proceed. Interestingly, LNCaP cells were found to remain Fas-resistant even after drug pre-treatment. However, Hyer et al. [Hyer 2000] demonstrated that LNCaP cells were uniformly sensitive to Fas-mediated apoptosis following treatment with a FasL expressing adenovirus. Collectively, these data demonstrate that a Fas-mediated apoptotic pathway is functional in all PCa cells tested to date.

There is also in vivo evidence suggesting a functional Fas-mediated apoptotic pathway present in both rat and mouse prostate models. Suzuki et al. demonstrated in mice that a functional Fas-mediated prostate apoptotic pathway exists. They report both Fas upregulation and prostate gland regression following castration of normal mice [Suzuki 1996]. However, castration of lpr mice [Watanabe-Fukunaga 1992], lacking functional Fas receptor (FasR) due to gene mutation, resulted in no change in prostate gland size [Suzuki 1996]. These data suggest a functional Fas receptor is necessary for castration-induced prostate regression to occur. Similarly, Woolveridge et al. [Woolveridge 1998] reported that Fas upregulation occurred in the rat ventral prostate following castration. Thus, it is likely that the Fas-mediated apoptotic pathway is responsible for human prostate gland regression following castration or hormonal therapy although another paper questions this [de la Taille 1999].

One of the limitations in PCa gene therapy is delivery of the therapeutic gene to every cell in the tumor. FasL gene therapy attempts to address this problem by taking advantage of the “bystander effect.” The bystander effect occurs when the number of apoptotic cells is greater than the number of cells expressing the transgene. Potentially, this can allow for complete regression of a solid tumor without having to deliver FasL to every cell. FasL can initiate the bystander effect three different ways: 1) by remaining associated with the FasL expressing cell, 2) by being released as a soluble form from the FasL expressing cell [Liu 1998, Mariani, 1995], 3) or by being released as a membrane bound form in microvesicles [Martinez-Lorenzo 1999]. It has been shown in vitro that FasL expressing effector cells (K562-FasL cells) can kill the following Fas+ PCa cell lines: ALVA-31, TSU-PR1, PPC-1, and JCA-1 [Hedlund 1999]. In addition, Liu et al. has demonstrated that FasL derived from the media of cultured LNCaP cells was capable of inducing apoptosis in Fas+ Ramos cells [Liu 1998]. It is unclear in the above two studies whether the target cells were dying from sFasL or membrane bound FasL. The role sFasL plays in Fas-mediated apoptosis is controversial. Some reports suggest sFasL stimulates the Fas pathway [Liu 1998], while others contend sFasL inhibits the pathway [Tanaka 1998]. In PCa an in vivo bystander effect has not been demonstrated.

Using FasL to induce apoptosis in PCa is a promising new strategy. Recently it has been shown in vitro, that following transduction with a FasL expressing adenovirus, apoptosis occurs in the following PCa cell lines: LNCaP, PPC-1, TSU-Pr1, DU145, PC-3, JCA-1, and ALVA-31 [Hyer 2000, Hedlund 1999]. Interestingly, adenovirus-mediated FasL delivery was capable of overcoming Fas-resistance in all cell lines determined to be resistant to antibodies with Fas agonistic characteristics. The mechanism whereby virally-expressed FasL overcomes Fas-resistance has not been determined [Hyer 2000]. Adenovirus-mediated FasL delivery has successfully been used to both reduce tumor burden and increased survival in the following human and mouse (in vivo) tumor models: glioma [Ambar 1999], leiomyosarcoma, [Aoki 2000], colon carcinoma [Arai, 1997], and mouse renal carcinoma [Arai, 1997]. Evidence suggests that observed tumor reduction is the result of the following two phenomena: 1) FasL induced apoptosis of Fas bearing cells, and 2) a FasL stimulated immune response. In the colon carcinoma model, elimination of the tumor was mediated exclusively by inflammatory cells [Arai 1997]. FasL expression has also been shown to be a potent chemoattractant for human neutrophils [Ottonello 1999]. However, it is still unclear exactly what role the immune system plays in eliminating FasL expressing tumor cells. With regard to PCa, Hedlund et al. [Hedlund 1999] has demonstrated that TSU-Pr1 cells, which were pre-infected with a FasL containing adenovirus and then subcutaneously injected into nude mice, exhibited reduced tumor potential compared to controls [Hedlund 1999]. However, further studies are necessary to determine the full therapeutic value of FasL as an in vivo PCa gene therapy.

TRAIL

Another member of the TNF family is TNF- related apoptosis inducing ligand (TRAIL, Apo-2). Full length TRAIL is a 32kDa protein identified in 1995 as a novel membrane protein with amino acid similarity to TNF (23%) and FasL (28%) [Wiley 1995]. Like other members of the TNF family, TRAIL can induce receptor-mediated apoptosis by activating the caspase cascade [Kim 2000]. In contrast to FasL, which can cause severe hepatotoxicity and TNF which has been associated with septic shock, TRAIL can induce apoptosis in tumorigenic and transformed cells without adversely affecting normal cells. Safety studies in mice [Walczak 1999] and cynomolgus monkeys [Ashkenzai 1999] indicate that TRAIL is well tolerated in vivo, although some concern has been raised about its toxicity against primary cultures of human hepatocytes [Jo 2000]. The apparent lack of toxicity coupled with the ability to kill a variety of tumor cells in vitro [Kim 2000, Griffith 1998] and in vivo [Walczak 1999, Ashkenzai 1999, Gliniak 1999] has sparked great interest in the potential use of TRAIL as a novel anticancer agent. Although numerous tumor cell lines have been analyzed for susceptibility, receptor status, and mechanism of TRAIL-induced apoptosis, data obtained from prostate cancer cell lines is limited and sometimes contradictory.

Unlike other members of the TNF family, TRAIL is expressed in a wide variety of tissues [Wiley 1995], and it was originally thought that susceptibility to TRAIL may be regulated by restrictive expression of a TRAIL receptor. To date four ubiquitously expressed TRAIL receptors have been identified which raises the question of how normal tissues maintain resistance to TRAIL (see below). The prostate is one of the tissues which express high levels of TRAIL [Wiley 1995] and transcripts for each TRAIL receptor can be detected by RT-PCR in primary cultures of prostate epithelial cells (PrEC) (personal communication, Griffith). Several hypotheses have been developed to explain the mechanism of resistance to TRAIL. TRAIL responses are mediated by a complex receptor system. Of the four TRAIL receptors that have been identified, two DR4/TRAIL-R1 [Pan 1997] and DR5/TRAIL-R2, [Macfarlane 1997, Bodmer 2000] have functional death domains that can bind FADD or FADD-like adaptor molecules thereby initiating the caspase cascade and apoptosis [Kuang 2000]. The two remaining receptors either lack (DcR1/TRAIL-R3) [Pan 1997, Macfarlane 1997, Degliesposti 1997] or have a truncated death domain (DcR2/TRAIL-R4) [Pan 1998, Degliesposti 1997] and are presumed to be decoy receptors. This was based on the observation that many tumor cells lack these receptors and over-expression in TRAIL-sensitive cells resulted in protection from TRAIL-induced apoptosis [Pan 1998, Degliesposti 1997]. Subsequent studies examining numerous cell lines for TRAIL receptor expression, were unable to support this hypothesis because levels of receptors correlated poorly with TRAIL susceptibility [Griffith 1998, Griffith 1998, Leverkus 2000, Mitsiades 2000]. It is important to note that many studies measure levels of TRAIL receptor mRNA which do not necessarily correlate with cell surface expression [Zhang 1999]. A second hypothesis, based on the observation that DcR2 can activate NFkB, suggested that decoy receptors may transduce anti-apoptotic signals [Degliesposti 1997, Jeremias 1998, Jeremias 1998]. A later study found that DR4 and DR5 can also induce NFkB activation without any protective effects [Schneider 1997, Chaudhary 2000, Yamanaka 2000]. It has also been suggested that resistance to TRAIL may be determined by levels of intracellular inhibitors such as FLIP [Kim 2000, Zhang 1999]. It is likely that a combination of TRAIL receptor levels, competing apoptotic and anti-apoptotic signals as well as intracellular levels of various pro- and anti-apoptotic proteins ultimately determine a cell's fate in response to TRAIL.

PrEC have been demonstrated to be resistant to TRAIL by several investigators [Ashkenazi 1999, Griffith 2000], (unpublished observations from our laboratory). In another study, the prostate adenocarcinoma cell lines PC3, Du145, and LNCaP were also found to be resistant to TRAIL-induced apoptosis and resistance did not correlate with TRAIL receptor levels as measured by RT-PCR [van Ophoven 1999]. Unfortunately, data regarding TRAIL susceptibility of PC3 and Du145 prostate cancer cell lines differ between laboratories (Table I). We, and others find that PC3 cells have lower levels of DcR1 and DcR2 transcript levels relative to Du145 and LNCap, and are still sensitive to TRAIL [Griffith 2000, Yu 2000]. Over-expression of DR4 enhances this susceptibility [Yu 2000]. Yu et al. [Yu 2000] also observed Du145 to be sensitive to TRAIL-induced apoptosis which is not in agreement with our findings and observations made by Sun et al. [Sun 2000]. It is possible that these contradictory findings stem from clonal heterogeneity or the different assays used to measure sensitivity to TRAIL.

TRAIL resistant cells can be sensitized by inhibitors of RNA and protein synthesis (actinomycin D, cycloheximide) [Griffith 1998, Thomas 1998], chemotherapeutic agents (cisplatinum, etoposide, doxorubicin) [Kim 2000, Ashkenazi 1999, Gliniak 1999, Keane 1999, Gibson 2000, Nagane 2000] or radiation [Chinnaiyan 2000]. Yu et al. [Yu 2000] who found Du145 and PC3 cells susceptible to TRAIL-induced apoptosis, were unable to further enhance killing by co-treatment with cycloheximide [Yu 2000]. However, in studies in which these cells were found to be resistant to TRAIL, low concentrations of actinomycin D have been shown to convert Du145, LNCaP and PC3 cells to a TRAIL-sensitive phenotype [van Ophoven 1999, Bonavida 1999], indicating that the presence of intracellular inhibitors of apoptosis may mediate resistance. The synthetic retinoid CD437 also acts synergistically with TRAIL by upregulating DR5 [Sun 2000]. Taken together, these results indicate that the TRAIL-mediated apoptotic pathway is intact in human prostate cancer cells but that combination therapy may be required to achieve high levels of killing. We are currently investigating the effect of doxorubicin and TRAIL in a panel of prostate cancer cell lines.

The first in vivo studies in mice bearing mammary or colon cancer xenografts demons-trated that TRAIL administration significantly prolonged survival [Walczak 1999, Ashkenazi 1999, Gliniak 1999]. Furthermore, combination of TRAIL and the camptothecin, CPT-11, resulted in a high proportion of complete tumor regression in TRAIL sensitive tumors and dramatically slowed growth of TRAIL resistant tumors. One of the problems with in vivo use of TRAIL is the high concentration requirement, in part, because soluble TRAIL has a short half-life in plasma (about 32 minutes) [Ashkenazi 1999] and an elimination half-life of less than 5 hours [Walczak 1999]. To improve delivery and better target TRAIL to the tumor site, Griffith et al. [Griffith 2000] developed a TRAIL expressing adenoviral vector. Upon viral infection and production of TRAIL, sensitive targets such as PC3 cells were killed rapidly, whereas resistant targets such as PrEC were unaffected. Interestingly, PrEC still expressed adenovirally derived TRAIL and were able to kill PC3 cells in co-incubation experiments [Griffith 2000]. This suggests that not all tumor cells would have to be infected by the adenovirus as normal cells surrounding the tumor could aid in tumor cell apoptosis, i.e. a bystander effect.

Bax in Prostate Cancer Gene Therapy

Bax is a 21 kDa pro-apoptotic member of the Bcl-2 family of genes which regulate apoptosis [Brady 1998]. The pro-apoptotic proteins: Bax, Bad, Bid, Bcl-XS, Bak, Bik, Bim, and Hrk can form homo or heterodimers with anti-apoptotic proteins such as: Bcl-2, Bcl-XL, Bcl-W, Mcl-1, and A1, through the BH3 (Bcl-2 Homology 3) domain [Kelekar 1998]. It is thought that the ratio of pro- to anti-apoptotic proteins is the determining factor for induction of apoptosis. Bax is primarily found in the cytosol, but when a cell death stimulus, such as removal of growth factors occurs, it translocates to the mitochondrial membrane where it forms a membrane-bound structure through a c-terminal conformational change [Nechushtan 1999] where it becomes associated with the inner membrane adenine nucleotide translocator protein (ANT) and the outer membrane voltage-dependent anion channel (VDAC/Porin) proteins of the permeability transition pore complex (PTP[Marzo 1999, Shimizu 1999]. The a5 and a6 helices of the Bax protein have been determined to have pore-forming ability believed to be involved with the release of cytochrome c and other factors from the mitochondria due to membrane permeability from PTPC opening [Adams 1998]. Once cytochrome c is released, the cell is committed to die either through apoptosis via Apaf-1 (apoptosis-activating factor-1) which complex with cytochrome c and ATP activating Pro-caspase-9, which in turn activates the caspase cascade resulting in apoptotic cell death or through the uncoupling of the respiratory chain resulting in disruption of electron transport, oxidative phosphorylation, and ATP production with reactive oxygen species production leading to necrotic cell death. The anti-apoptotic members of the Bcl-2 family are also believed to act at the mitochondrial membrane to prevent the release of cytochrome c from the mitochondria, thus preventing caspase activation and subsequent cell death [Green 1998]. Bcl-2 has been shown to prevent translocation of Bax to the mitochondrial membrane as well as its homodimerization [Gross1998]. Aberrations in expression and protein levels of the Bcl-2 family have been implicated in many types of malignancy including prostate [Marcelli 2000]. Unscheduled expression of Bcl-2 and other family members seems associated with progression to more advanced stages of cancer and resistance to treatments including chemotherapy and radiation [Srivastava 1999].

Prostate cancer is heterogeneous with respect to genetic mutation, and therefore difficult to target with corrective gene therapy. The progression of prostate cancer from a tumor localized within the prostate capsule to locally-invasive, extracapsular disease and metastasis involves a number of gene mutations for example, loss of p53, mutations in Rb, and an increase in anti-apoptotic Bcl-2 protein [Gjertsen 1999]. Wild type p53 induces Bax gene expression as well as repressing Bcl-2; therefore mutations rendering p53 nonfunctional may also lead to over-expression of Bcl-2 which could alter the ratio of pro-apoptotic to anti-apoptotic genes and lead to the protection of tumor cells from natural apoptotic signals [Pearson 2000]. The promoter regulating expression of Bax has been found to contain p53-binding sites and can, therefore, be trans-criptionally activated by p53 [Ikawa 1999]. Mutations in the Bax gene have also been found associated with breast, colon, and ovarian cancers, and for these reasons, Bax is thought to have tumor suppressor activity [Xiang 2000, Yagi 19898, Ouyang 1998].

Resistance to chemotherapy of prostate cancer is due to its slow growth and perhaps, in part, to increased Bcl-2 protein. Bcl-2 over-expression has also been shown to be associated with the progression of prostate tumors to androgen-independence [Gjertsen 1999]. Recent studies reveal alterations of Bcl-2 levels by chemotherapy, radiation, and herbal therapies. A plant estrogen licochalcone-A found in licorice root was demonstrated to modulate the level of Bcl-2 in prostate cancer in human cell lines, and alter the ratio of Bcl-2/Bax ratio towards apoptosis [Rafi 2000, Ng 2000, Blutt 2000]. The chemotherapeutic drug phenylbutyrate has been shown to repress Bcl-2 as well as upregulate Bax leading to apoptosis in androgen-dependent and androgen-independent cell lines (Ng 2000]. Another study showed that 1,25-Dihydroxyvitamin D3 (calcitriol) induced apoptosis in prostate cancer cells accompanied by a down-regulation of Bcl-2 and Bcl-XL anti-apoptotic genes [Blutt 2000].

Bcl-2 levels and Bcl-2 / Bax ratios may also be useful as predictive of susceptibility to therapy and as a prognostic indicator. Patient biopsies following radiotherapy revealed that Bcl-2 levels were higher in prostatic tumors not responsive to radiation treatment [Mackey 1998]. One mechanism by which chemotherapeutic drugs are thought to act is through activation of kinases such as protein kinase A leading to Bcl-2 hyperphosphorylation which prevents Bcl-2 Bax dimerization [Srivastava 1998]. Cytoplasmic serine/threonine kinases (Raf, Akt) are mutated in several malignancies [Tsatsanis 2000]. Con-currently, it has also been shown that human malignancies and tumor cell lines that have a higher level of the serine/threonine kinase, PKR, have a better prognosis [Jagus 1999].

Tumor suppressor genes may also have an effect on apoptosis by altering Bcl-2 and Bax protein levels. Mutations in the breast cancer susceptibility tumor suppressor gene, BRCA-1, may play a role in prostate cancer through alterations in Bcl-2 and Bax expression [Fan 1998]. Current treatments that involve restoring the dysfunctional regulation of apoptosis in malignancy may prove to be effective at overcoming resistance to therapy due to imbalance of the Bcl-2 family proteins.

Modulation of apoptosis factors in prostate cancer is an attractive approach for cytoreductive gene therapy. Key regulators such as the pro-apoptotic genes Bax, Bak, and Bcl-XS are good targets due to their downstream position in the apoptotic pathway. One study has demonstrated use of anti-sense Bcl-2 oligodeoxynucleotides combined with the chemotherapeutic agent docetaxel and androgen-ablation lengthened the time to androgen-independence [Gleave 1999].

Gene therapy approaches, using delivery of an adenoviral vector containing the Bax gene, have been tested in our laboratory and by others [Lowe 2000, Arafat 2000, Kagawa 2000]. This approach utilizes a prostate-specific probasin promoter to direct expression of the pro-apoptotic Bax to prostate cells, and results in selective induction of apoptosis even in the context of high levels of Bcl-2 characteristic of advanced stage prostate cancer [Lowe 2000]. This approach has many advantages over currently used cytotoxic therapies. Bax is far downstream in the signalling pathway of apoptosis and has been shown to induce apoptosis in cells that both p53-sensitive resistant [Kagawa 2000]. Therefore, despite heterogeneous mutations found within a population of tumor cells, Bax expression provides a universal target for induction of apoptosis. Once cytochrome c is released from the mitochondria via bax overexpression, the cell is committed to die either with or independently of caspase-8 activation. Unlike other genes involved in the modulation of apoptotic response such as caspases, Bax protein is not modified to become activated. Caspases which are translated in the zymogen form must be cleaved to be activated. Bax has an advantage over the BH3-only proapoptotic proteins such as Bid in that it can not only bind and antagonize anti-apoptotic proteins, but also contains regions that allow association with the mitochondrial membrane which can initiate the release of cytochrome c. This makes pro-apoptotic bax an attractive tool for use in cytotoxic gene therapy.

Other strategies are being developed that target the mitochondrial permeability transition pore complex itself as a novel target cancer therapy. Some experimental anticancer drugs are currently under investigation to modulate apoptosis induction through inhibition of specific mito-chondrial proteins involved in opening of the PTPC [Costantini 2000]. Additional novel targets include the ubiquitin/proteasome-dependent pathway for Bax degradation. A study shows that the addition of a proteasome inhibitor increased levels of ubiquitinated forms of Bax protein, and that reconstitution of the proteasome resulted in Bax degradation. This represents another target for anticancer therapy, with the Bcl-2 gene family [Li 2000].

There are still many aspects of the Bcl-2 protein family, and how they interact that require study. It has recently been determined that conformational changes in the c-terminus of Bax are involved in the transition to a membrane-bound protein. Still unresolved are the signals which initiate the conformational change and induce translocation of Bax to the mitochondrial membrane [Suzuki 2000]. Another question yet to be fully understood is the mechanism by which Bcl-2, Bax, Bcl-XL, and other members interact with mitochondrial PTPC to regulate apoptosis. Bax has been shown to associate with both the ANT and VDAC/Porin of the PTPC, but specifics of these interactions are still under investigation [Marzo 1999, Shimizu 1999]. Bcl-2 family members have been shown to have pore-forming abilities, however the mechanisms of interactions involving release of cytochrome c including mitochondrial membrane permeability, and release of other factors remains to be determined as requirements for apoptosis [Green 1998]. Although progress has been made in understanding the regulation of apoptosis induction by Bcl-2 family members; nevertheless, their involvement in cancer progression and resistance to current treatment modalities make them favorable targets for novel approaches for cancer gene therapy.

DELIVERY OF CANCER KILLING GENE BY ADENOVIRUS

There are numerous gene therapy delivery systems including retroviruses, adenovirus, adeno-associated viruses, other viral systems, and liposomes. Retroviral and adenoviral systems are more commonly used in prostate cancer. Since genes that induce apopotosis are capable of killing any cell, this type of gene therapy requires cellular targeting.

Targeting of adenovirus vectors is accomplished by means of an injection directly into the tumor mass. Since large virus particles cannot disperse efficiently throughout solid tissues, only a limited number of cell layers around the injection site get infected [Timme 1998, Kuriyama 2000]. This “built-in” safety feature controls vector spread and allows the use of strong constitutively active promoters and cytotoxic genes for Ad-based tumor therapy. However, if the treatment protocol includes systemic vector delivery, or if there is a concern for significant vector “escape” from the injection site additional safety mechanisms can be utilized to limit cytotoxic effects to tumor cells only. One such approach, known as transcriptional targeting, makes use of tumor or tissue specific promoters to selectively express the transgenes in target cells [Anderson 1999, Walther 1996, Siders 1996]. For example, prostate-specific promoters (such as PSA and probasin) are active only in those cells that express the androgen receptor, and can therefore limit Ad vector-delivered toxic gene expression to cancer cells of prostatic origin [Latham 2000, Lu 2000]. Our laboratory has generated an Ad vector which utilizes a modified probasin promoter, ARR2PB [Rubinchik 2001], to drive expression of the proapoptotic fusion protein FasL-GFP. Using this vector in vitro, we have demonstrated that FasL-GFP expression (and associated apoptosis) occurs only in prostate cancer cells such as LNCaP. However, the transcriptional activity of ARR2PB was found to be significantly lower than that of hCMVie or tetracycline regulated TRE driven constructs [Rubinchik 2001], thus, limiting its potential usefulness. To combat this, we combined prostate specificity of the ARR2PB promoter with high expression level and regulatability of the tet-regulated expression system [Gossen 1992] by developing a vector in which the ARR2PB promoter drives the expression of the tetracycline transcriptional activator (tTA), which in turn induces expression of the FasL-GFP protein under the control of the TRE promoter. In addition to limited dispersion and prostate cell limited expression, this vector contains a third safety feature, which is the ability to suppress, if so desired, expression of apopotosis inducing genes (FasL, Bax, TRAIL) in target cells to very low levels by the addition of sub-therapeutic levels of tetracycline or doxycycline [Gossen 1992, Rubinchik 2000]. The latter approaches should allow for orthotopic adminis-tration of these vectors to human prostate cancer and provide a safety mechanism if liver damage was observed during therapy.

DISCUSSION

An advantage of cytotoxic gene therapy is the lack of necessity of gene integration for therapeutic action. Transient expression of the transgene, as occurs by transduction of cells with adenoviral vectors, is sufficient to activate cytotoxic genes resulting in elimination of the transduced cells. A key aspect in delivery of cytotoxic genes to tumor cells is the selective targeting of specific cells or tissues. A way to overcome inadvertant toxicity is through the use of tissue-specific promoters which limit the expression of the gene-of-interest to a specific cell population.

Presently there are 17 NIH approved gene therapy trials underway for prostate cancer (http://www.clinicaltrials.gov, using the search words prostate, cancer, gene, therapy). Nine of the trials involve manipulation of the immune system, four are suicide gene therapies and the remainder are focused on gene replacement where induction of apoptosis is the likely outcome.

Gene therapy trials in humans began in 1989 at NIH so this translational research effort is still in its infancy. With the promise of the genome project, scientists and clinicians will soon (2002) have access to all human genes. Although much work remains to be done to understand gene function, for example in the context of cancer, the promise of this remains great. We must increase our focus on developing delivery systems to take advantage of the wealth of knowledge now accruing. The delivery of apoptosis inducing genes, particularly ones that exhibit bystander activity as described in this review, is one important component of this activity.

ACKNOWLEDGEMENTS

We would like to thank Janie Nelson for secretarial assistance with this manuscript. Research supported by CA69596 to J.S. Norris.

REFERENCES

Adams, J.M., and Cory, S. (1998) The Bcl-2 Protein Family: Arbiters of Cell Survival. Science, 281: 1322-1326.

Ambar, B.B., Frei K., Malipiero U., Morelli A.E., Castro M.G., Lowenstein P.R. and Fontana A. (1999) Treatment of experimental glioma by administration of adenoviral vectors expressing Fas ligand. Hum Gene Ther, 10: 1641-8.

Anderson, L.M., Swaminathan, S., Zackon, F., Tajuddin, A.K., Thimmapaya, B., and Weitzman, S.A. (1999) Adenovirus-mediated tissue-targeted expression of the HSVtk gene for the treatment of breast cancer. Gene Ther, 6: 854-64.

Aoki, K., Akyurek L.M., San H., Leung K., Parmacek M.S., Nabel E.G. and Nabel G.J. (2000) Restricted expression of an adenoviral vector encoding Fas ligand (CD95L) enhances safety for cancer gene therapy. Mol Ther, 1: 555-65.

Arafat, W.O., Gomez-Navarro, J. Xiang, J., Barnes, M.N., Mahasreshti, P., Alvarez, R.D., Siegal, G.P., Badib, A.O., Buchsbaum, D., Curiel, D.T., and Stackhouse, M.A. (2000) An adenovirus encoding proapoptotic Bax induces apoptosis and enhances the radiation effect in human ovarian cancer. Molecular Therapy, 1: 545-554.

Arai, H., Gordon, D., Nabel, E.G. and Nabel, G.J. (1997) Gene transfer of Fas ligand induces tumor regression in vivo. Proc Natl Acad Sci USA, 94: 13862-7.

Arase, H., Arase N. and Saito T. (1995) Fas-mediated cytotoxicity by freshly isolated natural killer cells. J Exp Med, 181: 1235-8.

Ashkenazi, A. and Dixit V.M. (1998) Death receptors: signaling and modulation. Science, 281: 1305-8.

Ashkenazi, A., Pai, R.C., Fong, S., Leung, S., Lawrence, D.A., Masters, S.A., Blackie, C., Chang, L., McMurtrey, A.E., Hebert, A., DeForge, L., Koumenis, I.L., Lewis, D., Harris, L., Bussiere, J., Koeppen, H., Shahrokh, Z., and Schwall, R.H. (1999) Safety and antitumor activity of recombinant soluble Apo2 ligand. Clinical Investigation, 104: 155-162.

Bellgrau, D., Gold D., Selawry H., Moore J., Franzusoff A. and Duke R.C. (1995) A role for CD95 ligand in preventing graft rejection [see comments] [published erratum appears in Nature 1998 Jul 9;394(6689):133]. Nature, 377: 630-2.

Blutt, S.E., McDonnell, T.J., Polek, T.C., and Weigel, N.L. (2000) Calcitriol-induced apoptosis in LNCaP cells is blocked by overexpression of Bcl-2. Endocrinology, 141: 10-17.

Bodmer, J. (2000) TRAIL receptor-2 signals apoptosis through FADD and caspase-8. Nature Cell Biology, 2: 241-243.

Bonavida, B., Ng, C.P., Jazirehi, A., Schiller, G., and Mizutani, Y. (1999) Selectivity of TRAIL-mediated apoptosis of cancer cells and synergy with drugs: the trail to non-toxic cancer therapeutics. International Journal of Oncology, 15: 793-802

Brady, H., and Gil-Gomez, G. (1998) Molecules in focus. Bax. The pro-apoptotic Bcl-2 family member, Bax. International Journal of Biochemistry and Cell Biology, 30: 647-650.

Chaudhary, P.M., Eby, M.T., Jasmin, A., Kumar, A., Liu, L., and Hood, L. (2000) Activation of the NF-kappa B pathway by Caspase 8 and its homologs. Oncogene, 19: 4451-4460.

Chinnaiyan, A.M., Prasad, U., Shankar, S., Hamstra, D.A., Shanaiah, M., Chenevert, T.L., Ross, B.D., and Rehemtulla, A. (2000) Combined effect of tumor necrosis factor-related apoptosis-inducing ligand and ionizing radiation in breast cancer therapy. Proceedings of the National Academy of Sciences USA, 97: 1754-1759.

Costantini, P., Jacotot, E., Decaudin, D., and Kroemer, G. (2000). Mitochondrion as a novel target of anticancer chemotherapy. Journal of the National Cancer Institute, 92: 1042-1053.

Costa-Pereira, A.P. and Cotter T.G. (1999) Camptothecin sensitizes androgen-independent prostate cancer cells to anti-Fas-induced apoptosis. Br J Cancer, 80, 371-8.

de la Taille, A., Chen, M.W., Shabsigh, A., Bagiella, E., Kiss, A., and Buttyan, R. (1999) Fas antigen/CD-95 upregulation and activation during castration-induced regression of the rat ventral prostate gland. Prostate, 40: 89-96.

Degliesposti, M.A., Dougall, W.C., Smolak, P.J., Waugh, J.Y., Smith, C.A., and Goodwin, R.G. (1997) The novel receptor Trail-R4 induces Nf-Kappa-B and protects against Trail-mediated apoptosis. Yet retains an incomplete death domain. Immunity, 7: 813-820.

Degliesposti, M.A., Smolak, P.J., Walczak, H., Waugh, J., Huang, C.P., Dubose, R.F., Goodwin, R.G., and Smith, C.A. (1997) Cloning and Characterization of Trail-R3, a Novel Member of the Emerging Trail Receptor Family. Journal of Experimental Medicine, 186: 1165-1170.

Fan, S., Wang, J.A., Yuan, R.Q, Ma, Y.X., Meng, Q., Erdos, M.R., Brody, L.C., Goldberg, I.D., Rosen, E.M. (1998) BRCA1 as a potential human prostate tumor suppressor: modulation of proliferation, damage responses and expression of cell regulatory proteins. Oncogene, 16: 3069-3082.

Frost, P., Ng C.P., Belldegrun A. and Bonavida B. (1997) Immunosensitization of prostate carcinoma cell lines for lymphocytes (CTL, TIL, LAK)-mediated apoptosis via the Fas-Fas-ligand pathway of cytotoxicity. Cell Immunol, 180, 70-83.

Gibson, S.B., Oyer, R., Spalding, A.C., Anderson, S.M., and Johnson, G.L. (2000) Increased expression of death receptors 4 and 5 synergizes the apoptosis response to combined treatment with etoposide and TRAIL. Molecular & Cellular Biology, 20: 205-212.

Gjertsen, B.T., Logothetis, C.J., and McDonnell, T.J. (1999) Molecular regulation of cell death and therapeutic strategies for cell death insuction in prostate carcinoma. Cancer and Metastasis Reviews, 17: 345-351.

Gleave ME. Miayake H. Goldie J. Nelson C. Tolcher A. (1999) Targeting bcl-2 gene to delay androgen-independent progression and enhance chemosensitivity in prostate cancer using antisense bcl-2 oligodeoxynucleotides. Urology, 54: 36-46.

Gliniak, B., and Le, T. (1999) Tumor necrosis factor-related apoptosis-inducing ligand's antitumor activity in vivo is enhanced by the chemotherapeutic agent CPT-11. Cancer Research, 59: 6153-6158.

Gossen, M., and Bujard H. (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA, 89: 5547-51.

Green, D.R., and Reed, J.C. (1998) Mitochondria and Apoptosis. Science, 281: 1309-1312.

Griffith, T.S. and Lynch, D.H. (1998) Trail - A molecule with multiple receptors and control mechanism. Current Opinion In Immunology, 10: 559-563.

Griffith, T.S., Anderson, R.D., Davidson, B.L., Williams, R.D., and Ratliff, T.L. (2000) Adenoviral-mediated transfer of the TNF-related apoptosis-inducing ligand/Apo-2 ligand gene induces tumor cell apoptosis. Journal of Immunology, 165: 2886-2894.

Griffith, T.S., Brunner T., Fletcher S.M., Green D.R. and Ferguson T.A. (1995) Fas ligand-induced apoptosis as a mechanism of immune privilege [see comments]. Science, 270: 1189-92.

Griffith, T.S., Chin, W.A., Jackson, G.C., Lynch, D.H., and Kubin, M.Z. (1998) Intracellular Regulation of Trail-Induced Apoptosis in Human Melanoma Cells. Journal of Immunology, 161: 2833-2840.

Gross, A., Jockel, J., Wei, M.C., and Korsmeyer, S.J. (1998) Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO, 17: 3878-3885.

Hedlund, T.E., Duke R.C., Schleicher M.S. and Miller G.J. (1998) Fas-mediated apoptosis in seven human prostate cancer cell lines: correlation with tumor stage. Prostate, 36: 92-101.

Hedlund, T.E., Meech S.J., Srikanth S., Kraft A.S., Miller G.J., Schaack J.B. and Duke R.C. (1999) Adenovirus-mediated expression of Fas ligand induces apoptosis of human prostate cancer cells. Cell Death Differ, 6: 175-82.

Hyer, M.L., Voelkel-Johnson C., Rubinchik S., Dong J. and Norris J.S. (2000) Intracellular fas ligand expression causes fas-mediated apoptosis in human prostate cancer cells resistant to monoclonal antibody-induced apoptosis [In Process Citation]. Mol Ther, 2: 348-58.

Ikawa, S., Obinata, M., and Ikawa, Y. (1999) Human p53-p51 (p53-Related) Fusion Protein: A Potent BAX Transactivator. Japanese Journal Cancer Research, 90: 596-599.

Jagus, R., Joshi, B., and Barber, G.N. (1999) PKR, apoptosis and cancer. International Journal of Biochemistry and Cell Biology, 31: 123-138.

Jeremias, I. and Debatin, K.M. (1998) TRAIL induces apoptosis and activation of NF kappa B. European Cytokine Network, 9: 687-688.

Jeremias, I., Kupatt, C., Baumann, B., Herr, I., Wirth, T., and Debatin, K.M. (1998) Inhibition of Nuclear Factor Kappa-B Activation Attenuates Apoptosis Resistance in Lymphoid Cells. Blood, 91: 4624-4631.

Jo, M, Kim, T.H., Seol, D.W., Esplen, J.E., Dorko, K., Billiar, T.R., and Strom, S.C. (2000) Apoptosis induced in normal human hepatocytes by tumor necrosis factor-related apoptosis-inducing Ligand. Nature Medicine, 6: 564-567.

Kagawa, S., Gu, J., Swisher, S.G., Ji, L., Roth, J.A., Lai, D., Stephens, L.C., Fang, B. (2000) Antitumor Effect of Adenovirus-mediated Bax Gene Transfer on p53-sensitive and p53-resistant Cell Lines. Cancer Research, 60: 1157-1161.

Kayagaki, N., Kawasaki A., Ebata T., Ohmoto H., Ikeda S., Inoue S., Yoshino K., Okumura K. and Yagita H. (1995) Metalloproteinase-mediated release of human Fas ligand. J Exp Med, 182: 1777-83.

Keane, M.M., Ettenberg, S.A., Nau, M.M., Russell, E.K., and Lipkowitz, S. (1999) Chemotherapy augments TRAIL-induced apoptosis in breast cell lines. Cancer Research, 59: 734-741.

Kelekar, A., and Thompson, C.B. (1998) Bcl-2-family proteins: the role of the BH3 domain in apoptosis. Trends in Cell Biology, 8:324-330.

Kim, K.H., Fisher, M.J., Xu, S.Q., and El-Deiry, W.S. (2000) Molecular determinants of response to TRAIL in killing of normal and cancer cells. Clinical Cancer Research, 6: 335-346.

Kuang, A.A., Diehl, G.E., Zhang, J.K., and Winoto, A. (2000) FADD is required for DR4-and DR5-mediated apoptosis - Lack of trail-induced apoptosis in FADD-deficient mouse embryonic fibroblasts. Journal of Biological Chemistry, 275: 25065-25068.

Kuriyama, N., Julin, C.M., Lamborn, K., and Israel, M.A. (2000) Pretreatment with protease is a useful experimental strategy for enhancing adenovirus-mediated cancer gene therapy. Human Gene Ther, 11: 2219-30.

Latham, J.P., Searle, P.F., Mautner, V., and James, N.D. (2000) Prostate-specific antigen promoter/enhancer driven gene therapy for prostate cancer: construction and testing of a tissue-specific adenovirus vector. Cancer Res, 60: 334-41.

Leverkus, M., Neumann, M., Mengling, T., Rauch, C.T., Brocker, E.B., Krammer, P.H., and Walczak, H. (2000) Regulation of tumor necrosis factor-related apoptosis-inducing ligand sensitivity in primary and transformed human keratinocytes. Cancer Research, 60: 553-559.

Li, B., and Dou, Q.P. (2000) Bax degradation by the ubiquitin/proteasome-dependent pathway: involvement in tumor survival and progression. Proceedings of the National Academy of Science, USA. 97: 3850-3855.

Liu, Q.Y., Rubin M.A., Omene C., Lederman S. and Stein C.A. (1998) Fas ligand is constitutively secreted by prostate cancer cells in vitro. Clin Cancer Res, 4: 1803-11.

Lowe, S.L., Rubinchik, S., Honda, Y., McDonnell, T.J., Dong, J-Y., Norris J.S. (2000) Prostate-Specific Expression of Bax Delivered by an Adenoviral Vector Induces Apoptosis in LNCaP Prostate Cancer Cells. Unpublished Data. Submitted to Gene Therapy.

Lu, Y., and Steiner, M.S. (2000) Transcriptionally regulated adenoviruses for prostate-specific gene therapy. World J Urol, 18: 93-101.

Macfarlane, M., Ahmad, M., Srinivasula, S.M., Fernandesalnemri, T., Cohen, G.M., and Alnemri, E.S. (1997) Identification and Molecular Cloning of Two Novel Receptors For the Cytotoxic Ligand Trail. Journal of Biological Chemistry, 272: 25417-25420.

Mackey, T.J., Borkowski, A,. Amin,P., Jacobs, S.C., and Kyprianou, N. (1998) Bcl-2/bax ratio as a predictive marker for therapeutic response to radiotherapy in patients with prostate cancer. Urology, 52: 1085-1090.

Marcelli, M,. Marani, M., Li, X., Sturgis, L., Haidacher, S.J., Trial, J.A., Mannucci, R. Nicoletti, I., and Denner, L. (2000) Heterogeneous Apoptotic Responses of Prostate Cancer Cell Lines Identify an Association Between Sensitivity to Staurosporine-Induced Apoptosis, Expression of Bcl-2 family Members, and Caspase Activation. The Prostate, 42: 260-273.

Mariani, S.M., Matiba B., Baumler C. and Krammer P.H. (1995) Regulation of cell surface APO-1/Fas (CD95) ligand expression by metalloproteases. Eur J Immunol, 25: 2303-7.

Martinez-Lorenzo, M.J., Anel, A., Gamen, S., Monle, N.I., Lasierra, P., Larrad, L., Pineiro, A., Alava, M.A., and Naval, J. (1999) Activated human T cells release bioactive Fas ligand and AP02 ligand in microvesicles. J. Immunology, 163: 1274-1281.

Marzo, I., Brenner, C., Zamzami, N., Jurgensmeier, J.M., Susin, S.A., Vieira, H.L., Prevost, M.C., Xie, Z., Matsuyama, S., Reed, J.C., and Kroemer, G. (1998) Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science, 281: 2027-2031.

Medema, J.P., Scaffidi C., Kischkel F.C., Shevchenko A., Mann M., Krammer P.H. and Peter M.E. (1997) FLICE is activated by association with the CD95 death-inducing signaling complex (DISC). Embo J, 16: 2794-804.

Mitsades, N., Poulaki, V., Tseleni-Balafouta, S., Koutras, D.A., and Stamenkovic, I. (2000) Thyroid carcinoma cells are resistant to FAS-mediated apoptosis but sensitive to tumor necrosis factor-related apoptosis-inducing Ligand. Cancer Research, 60: 4122-4129.

Muzio, M., Salvesen G.S. and Dixit V.M. (1997) FLICE induced apoptosis in a cell-free system. Cleavage of caspase zymogens. J Biol Chem, 272: 2952-6.

Nagane, M., Pan, G.H., Weddle, J.J., Dixit, V.M., Cavenee, W.K., and Huang, H.J.S. (2000) Increased death receptor 5 expression by chemotherapeutic agents in human gliomas causes synergistic cytotoxicity with tumor necrosis factor-related apoptosis-inducing ligand in vitro and in vivo. Cancer Research, 60: 847-853.

Nechushtan, A., Smith, C.L., Hsu, Y., and Youle, R.J. (1999) Conformation of the Bax C-terminus regulates subcellular location and cell death. EMBO, 18: 2330-2341.

Ng, A.Y., Bales, W., and Veltri, R.W. (2000) Phenylbutyrate-induced apoptosis and differential expression of Bcl-2, Bax, p53, and Fas in human prostate cancer cell lines. Analytical & Quantitative Cytology & Histology, 22: 45-54.

Ottonello, L., Tortolina, G., Amelotti, M., and Dallegri, F. (1999) Soluble Fas Ligand is chemotactic for human neutrophilic polymorphonuclear leukocytes. J Immunol, 162: 3601-6.

Ouyang, H., Furukawa, T., Abe, T., Kato, Y., and Horii, A. (1998) The BAX gene, the promoter of apoptosis, is mutated in genetically unstable cancers of the colorectum, stomach, and endometrium. Clin. Cancer Res., 4: 1071-74.

Pan, G.H., Ni, J., Wei, Y.F., Yu, G.L., Gentz, R., and Dixit, V.M. (1997) An Antagonist Decoy Receptor and a Death Domain-Containing Receptor For Trail. Science, 277: 815-818.

Pan, G.H., Ni, J., Yu, G.L., Wei, Y.F., and Dixit, V.M. (1998) Trundd, a new member of the Trail receptor family that antagonizes Trail signaling. FEBS Letters, 424: 41-45.

Pearson AS. Spitz FR. Swisher SG. Kataoka M. Sarkiss MG. Meyn RE. McDonnell TJ. Cristiano RJ. Roth JA. (2000) Up-regulation of the Prospoptotic Mediators Bax and Bak after Adenovirus-mediated p53 Gene Transfer in Lung Cancer Cells. Clinical Cancer Research, 6: 887-890.

Powell, W.C., Fingleton B., Wilson C.L., Boothby M. and Matrisian L.M. (1999) The metalloproteinase matrilysin proteolytically generates active soluble Fas ligand and potentiates epithelial cell apoptosis. Curr Biol, 9: 1441-7.

Rafi MM. Rosen RT. Vassil A. Ho CT. Zhang H. Ghai G. Lambert G. DiPaola RS. (2000). Modulation of bcl-2 and cytotoxicity by licochalcone-A, a novel estrogenic flavonoid. Anticancer Research, 20: 2653-2658.

Rokhlin, O.W., Bishop G.A., Hostager B.S., Waldschmidt T.J., Sidorenko S.P., Pavloff N., Kiefer M.C., Umansky S.R., Glover R.A. and Cohen M.B. (1997) Fas-mediated apoptosis in human prostatic carcinoma cell lines. Cancer Res, 57: 1758-68.

Rouvier, E., Luciani M.F. and Golstein P. (1993) Fas involvement in Ca(2+)-independent T cell-mediated cytotoxicity. J Exp Med, 177: 195-200.

Rubinchik, S., Ding, R., Qiu, A.J., Zhang, F., and Dong, J. (2000) Adenoviral vector which delivers FasL-GFP fusion protein regulated by the tet-inducible expression system. Gene Therapy, 7: 875-885.

Rubinchik, S., Lowe, S, Jia, Z., Norris, J.S., and Dong, J.-Y. (2001) Creation of a new transgene cloning site near the right ITR of Ad5 results in reduced enhancer interference with tissu-specific and regulatable promoters. Gene Therapy, 8: 247-253.

Sasaki, Y., Ahmed H., Takeuchi T., Moriyama N. and Kawabe K. (1998) Immunohistochemical study of Fas, Fas ligand and interleukin-1 beta converting enzyme expression in human prostatic cancer. Br J Urol, 81: 852-5.

Schneider, P., Thome, M., Burns, K., Bodmer, J.L., Hofmann, K., Kataoka, T., Holler, N., and Tschopp, J. (1997) Trail Receptors 1 (Dr4) and 2 (Dr5) Signal Fadd-Dependent Apoptosis and Activate Nf-Kappa-B. Immunity, 7: 831-836.

Shimizu, S., Narita, M., and Tsujimoto. (1999) Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature, 399: 483-488.

Siders, W.M., Halloran, P.J., and Fenton, R.G. (1996) Transcriptional targeting of recombinant adenoviruses to human and murine melanoma cells. Cancer Res, 56: 5638-46.

Srivastava RK. Srivastava AR. Korsmeyer SJ. Nesterova M. Cho-Chung YS. Longo DL. (1998) Involvement of microtubules in the regulation of Bcl-2 phosphorylation and apoptosis through cyclic AMP-dependent protein kinase. Molecular and Cellular Biology, 18: 3509-3517.

Srivastava, R.K., Sasaki, C.Y., Hardwick, J.M., and Longo, D.L. (1999) Bcl-2-mediated Drug Resistance: Inhibition of Apoptosis by Blocking Nuclear Factor of Activated T Lymphocytes (NFAT)-induced Fas Ligand Transcription. The Journal of Experimental Medicine, 190: 253-265.

Stalder, T., Hahn S. and Erb P. (1994) Fas antigen is the major target molecule for CD4+ T cell-mediated cytotoxicity. J Immunol, 152: 1127-33.

Steiner, M.S., and Gingrich, J.R. (2000) Gene therapy for prostate cancer: where are we now? J. Urology, 164: 1121-1136.

Stuart, P.M., Griffith T.S., Usui N., Pepose J., Yu X. and Ferguson T.A. (1997) CD95 ligand (FasL)-induced apoptosis is necessary for corneal allograft survival. J Clin Invest, 99: 396-402.

Suda, T., and Nagata, S. (1994) Purification and characterization of the Fas-ligand that induces apoptosis. Journal Experimental Medicine, 179: 873-879.

Sun, S.Y., Yue, P., and Lotan, R. (2000) Implication of multiple mechanisms in apoptosis induced by the synthetic retinoid CD437 in human prostate carcinoma cells. Oncogene, 19: 4513-4522.

Suzuki, A., Matsuzawa A. and Iguchi T. (1996) Down regulation of Bcl-2 is the first step on Fas-mediated apoptosis of male reproductive tract. Oncogene, 13: 31-7.

Suzuki, M., Youle, R.J., and Tjandra, N. (2000) Structure of Bax: Coregulation of Dimer Formation and Intracellular Localization. Cell, 103: 645-654.

Takahashi, T., Tanaka M., Inazawa J., Abe T., Suda T., and Nagata S. (1994) Human Fas ligand: gene structure, chromosomal location and species specificity. International Immunology, 6: 1567-1574.

Thomas, W.D., and Hersey, P. (1998) Tnf-Related Apoptosis-Inducing Ligand (Trail) Induces Apoptosis in Fas Ligand-Resistant Melanoma Cells and Mediates Cd4 T Cell Killing of Target Cells. Journal of Immunology, 161: 2195-2200.

Timme, T.L., Hall, S.J., Barrios, R., Woo, S.L., Aguilar-Cordora, E., and Thompson, T.L. (1998) Local inflammatory response and vector spread after direct intraprostatic injection of a recombinant adenovirus containing the herpes simplex virus thymidine kinase gene and ganciclovir therapy in mice. Cancer Gene Ther, 5: 74-82.

Tsatsanis, C., and Spandidos, D.A. (2000). The role of oncogenic kinases in human cancer. International Journal of Molecular Medicine, 5: 583-590.

Uslu, R., Borsellino N., Frost P., Garban H., Ng C.P., Mizutani Y., Belldegrun A. and Bonavida B. (1997) Chemosensitization of human prostate carcinoma cell lines to anti-fas- mediated cytotoxicity and apoptosis. Clin Cancer Res, 3: 963-72.

van Ophoven, A., Ng, C.P., Patel, B., Bonavida, B., and Belldegrun, A. (1999) Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) for treatment of prostate cancer: first results and review of the literature. Prostate Cancer & Prostatic Diseases, 2: 227-233.

Walczak, H., Miller, R.E., Ariail, K., Gliniak, B., Griffith, T.S., Kubin, M., Chin, W., Jones, J., Woodward, A., Le, T., Smith, C., Smolak, P., Goodwin, R.G., Rauch, C. T., Schuh, J.C.L., and Lynch, D.H. (1999) Tumoricidal activity of tumor necrosis factor related apoptosis-inducing ligand in vivo. Nature Medicine, 5: 157-163.

Walter, W., and Stein, U. (1996) Cell type specific and inducible promoters for vectors in gene therapy as an approach for cell targeting. J Mol Med, 74: 379-92.

Watanabe-Fukunaga, R., Brannan C.I., Copeland N.G., Jenkins N.A. and Nagata S. (1992) Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature, 356: 314-7.

Wiley, S.R., Schooley, K., Smolak, P.J., Din, W.S., Huang, C.P., Nicholl, J.K., Sutherland, G.R., Smith, T.D., Rauch, C., Smith, C.A., and Goodwin, R.G. (1995) Identification and Characterization of a New Member of the Tnf Family That Induces Apoptosis. Immunity, 3: 673-682.

Woolveridge, I., Taylor M.F., Wu F.C. and Morris I.D. (1998) Apoptosis and related genes in the rat ventral prostate following androgen ablation in response to ethane dimethanesulfonate. Prostate, 36, 23-30.

Xiang, J., et al. (2000) Pro-Apoptotic Treatment with an Adenovirus Encoding Bax Enhances the Effect of Chemotherapy in Ovarian Cancer. Journal of Gene Medicine. 2(2):97-106.

Yagi OK. Akiyama Y. Nomizu T. Iwama T. Endo M. Yuasa Y. (1998) Proapoptotic gene BAX ids frequently mutated in hereditary nonpolyposis colorectal cancers but not in adenomas. Gastroenterology, 114: 268-274.

Yamanaka, T., Shiraki, K., Sugimoto, K., Ito, T., Fujikawa, K., Ito, M., Takase, K., Moriyama, M., Nakano, T., and Suzuki, A. (2000) Chemotherapeutic agents augment TRAIL-induced apoptosis in human hepatocellular carcinoma cell lines. Hepatology, 32: 482-490.

Yu, R., Mandlekar, S., Ruben, S., Ni, J., and Kong, A.N.T. (2000) Tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in androgen-independent prostate cancer cells. Cancer Research, 60: 2384-2389.

Zhang, X.D., Franco, A., Myers, K., Gray, C., Nguyen, T., and Hersey, P. (1999) Relation of TNF-related apoptosis-inducing ligand (TRAIL) receptor and FLICE-inhibitory protein expression to TRAIL-induced apoptosis of melanoma. Cancer Research, 59: 2747-2753.