Acid ceramidase induces sphingosine kinase 1/S1P receptor 2-mediated activation of oncogenic Akt signaling.

Acid ceramidase (AC) is overexpressed in most prostate tumors and confers oncogenic phenotypes to prostate cancer cells. AC modulates the cellular balance between ceramide, sphingosine and sphingosine 1-phosphate (S1P). These bioactive sphingolipids have diverse, powerful and often oppositional impacts on cell signaling, including the activation status of the oncogenic kinase Akt. Our studies show that AC expression correlates with phosphorylation of Akt in human prostate tumors, and elevation of phosphorylated Akt in tumor versus patient-matched benign tissue is contingent upon AC elevation. Investigation of the mechanism for AC-induced Akt activation revealed that AC activates Akt through sphingosine kinase 1 (SphK1)-derived generation of S1P. This signaling pathway proceeds through S1P receptor 2 (S1PR2)-dependent stimulation of PI3K. Functionally, AC-overexpressing cells are insensitive to cytotoxic chemotherapy, however, these cells are more susceptible to targeted inhibition of Akt. AC-overexpressing cells proliferate more rapidly than control cells and form more colonies in soft agar; however, these effects are profoundly sensitive to Akt inhibition, demonstrating increased dependence on Akt signaling for the oncogenic phenotypes of AC-overexpressing cells. These observations may have clinical implications for targeted therapy as PI3K and Akt inhibitors emerge from clinical trials.

Autophagy is increased in prostate cancer cells overexpressing acid ceramidase and enhances resistance to C6 ceramide.

Acid ceramidase (AC) overexpression has been observed in prostate cancer cell lines and primary tumors, and contributes to resistance to chemotherapy and radiation. The consequence of AC overexpression is the ability to convert ceramide, which is often produced as a proapoptotic response to stress, to sphingosine, which can then be converted to the prosurvival molecule sphingosine-1-phosphate. In addition to their ability to metabolize ceramide produced in response to stress, we show here that prostate cancer cell lines overexpressing AC also have increased lysosomal density and increased levels of autophagy. Furthermore, pretreatment with 3-methyladenine restores sensitivity of these cells to treatment with C(6) ceramide. We also observed increased expression of the lysosomal stabilizing protein KIF5B and increased sensitivity to the lysosomotropic agent LCL385. Thus, we conclude that AC overexpression increases autophagy in prostate cancer cells, and that increased autophagy enhances resistance to ceramide.

HLA class II antigen presentation by prostate cancer cells.

Prostate cancer is the second most commonly diagnosed cancer in men. Recent evidence suggests that reduced expression of target protein antigens and human leukocyte antigen (HLA) molecules is the predominant immune escape mechanism of malignant prostate tumor cells. The purpose of this study was to investigate the prospect of antigen specific immunotherapy against prostate cancer via the HLA class II pathway of immune recognition. Here, we show for the first time that prostate cancer cells express HLA class II proteins that are recognized by CD4+ T cells. Prostate tumor cells transduced with class II molecules efficiently presented tumor-associated antigens/peptides to CD4+ T cells. This data suggests that malignant prostate tumors can be targeted via the HLA class II pathway, and that class II-positive tumors could be employed for direct antigen presentation, and CD4+ T-cell mediated tumor immunotherapy.Prostate Cancer and Prostatic Diseases (2008) 11, 334-341; doi:10.1038/sj.pcan.4501021; published online 16 October 2007.

How much can be concluded from the International Subarachnoid Aneurysm Trial (ISAT)?

Recently published data from the International Subarachnoid Aneurysm Trial (ISAT) shows that for patients enrolled in the trial there is a 7.4% reduction in the incidence of death or dependency at 1 year if they undergo coiling, rather than clipping. Furthermore, extrapolation of longer-term follow-up data for patient mortality appears to suggest that this advantage will be maintained in the longer term. Based on a reassessment of the published data, the authors note: (1) the incidence of rebleeding following treatment is approximately three times higher in the coiled group (p<0.001); (2) the need for aneurysm retreatment is likely to be higher in the coiled group; (3) trends in longer-term mortality data are not a reliable basis for predicting future outcomes of the trial; (4) trends in longer-term morbidity data are more reliable and suggest that the advantage of coiling diminishes with time; (5) The absence of up-to-date published rates of aneurysm retreatment and of longer-term rates of death or dependence makes ISAT extremely hard to interpret. It is far from clear that the early advantage of coiling will be maintained in the future and, hence, longer follow-up is required. Treatment of aneurysms is a continually evolving field and there is currently no other major source of information concerning management of aneurysms. For these reasons the authors recommend the instigation of a national aneurysm registry to prospectively collect data.

International Society for Cell and Gene Therapy of Cancer: 2005 meeting in Shenzhen, China.

The 2005 International Society for Cell and Gene Therapy of Cancer (ISCGT) Congress was held in Shenzhen, China (www.iscgtchina2005.com) from December 9th-11th 2005. Here, we describe a representation of the most seminal presentations providing an overview of the progress in the field of cancer gene therapy including the successful introduction of the first approved gene therapy drug.

Combined therapeutic use of AdGFPFasL and small molecule inhibitors of ceramide metabolism in prostate and head and neck cancers: a status report.

As of January 2005, there were 1020 gene therapy clinical trials ongoing worldwide with 675 or 66.2% devoted to cancer gene therapy. The majority are occurring in the US and Europe (http://www.wiley.co.uk/genetherapy/clinical/). At the present time, to our knowledge there are no trials that employ gene delivery of Fas Ligand (FasL). As an important note, and in contrast to somatic cell therapy trials, there are no reported deaths due to therapeutic vector administration in any cancer gene therapy trial. That said, from our studies and from the published literature, the issue of gene delivery remains the major obstacle to successfully employing gene therapy for cancer treatment. Numerous laboratories are studying this with many different approaches. My co-workers and I have focused on the delivery issue by using various approaches that address tumor targeting and transgene expression. In addition, we are focusing on enhancing tumor cell killing via the bystander effect and through use of small molecules to enhance bystander activity.

FasL gene therapy: a new therapeutic modality for head and neck cancer.

In this study, we investigated the in vitro and in vivo efficacy of Fas ligand (FasL) gene therapy for the treatment of head and neck cancer. Three head and neck squamous cell carcinoma (HNSCC) cell lines (SCC-1, SCC-12, and SCC-14a) were treated with the Fas agonist CH-11, a monoclonal antibody to the Fas receptor, or with a replication-incompetent adenovirus (AdGFPFasL) expressing a modified murine Fas ligand gene fused to green fluorescent protein (GFP). A replication-incompetent adenovirus containing the GFP gene alone was used as a control for viral transduction toxicity (AdGFP). Cell death was quantified using a tetrazolium-based (MTS) assay. Cells were analyzed by flow cytometry to determine the expression of adenoviral and Fas receptors on the surface of the cells. Our results showed that the head and neck cancer cell lines are resistant to cell death induction when treated with the anti-Fas monoclonal antibody CH-11. This resistance can be overcome with AdGFPFasL, which was able to induce cell death in all three cell lines. Apoptosis induction was demonstrated using Western blotting by evaluating poly(ADP-ribose) polymerase, and caspase 9 cleavages. In addition, intratumoral injections of AdGFPFasL into SCC-14a xenografts induced significant growth suppression of tumors, indicating that FasL gene therapy may provide a new efficient therapeutic modality for HNSCC that is worthy of a clinical trial.

Aneurysmal subarachnoid haemorrhage: guidance in making the correct diagnosis.

BACKGROUND: The natural history of untreated aneurysmal subarachnoid haemorrhage carries a dismal prognosis. Case fatalities range between 32% and 67%. Treatment with either surgical clipping or endovascular coiling is highly successful at preventing re-bleeding and yet the diagnosis is still missed. METHODS: Based on the national guidelines for analysis of cerebrospinal fluid for bilirubin in suspected subarachnoid haemorrhage and a review of other available literature this study has compiled guidance in making the diagnosis. CONCLUSION: In patients presenting with a suspected non-traumatic subarachnoid haemorrhage, computed tomography within 12 hours will reliably show 98% of subarachnoid haemorrhage. In patients who present after 12 hours with a negative computed tomogram, formal cerebrospinal fluid spectophotometry will detect subarachnoid haemorrhage for the next two weeks with a reliability of 96%. Between the early diagnosis with the aid of computed tomography and the later diagnosis with the added benefit of spectophotometry in the period where computed tomograms become less reliable, it should be possible to diagnose most cases of subarachnoid haemorrhage correctly.

Angiography postclipping and coiling of cerebral aneurysms.

Postclipping cerebral angiography is generally not practised in the UK. The International Subarachnoid Trial (ISAT) data show that coiling compared favourably with clipping in the early posttreatment phase. We present a 4-year, single unit experience comparing cerebral angiography at 6 months postclipping and postcoiling, defining the proportion of aneurysms in either group, which were incompletely excluded from the cerebral circulation after treatment. There were 4 'dog-ear' remnants (4.6%) in the clipping group of 86 aneurysms, one of which required further surgery. Thirty-one out of 82 (37.8%) coiled aneurysms that underwent check angiography were inadequately excluded from the cerebral circulation at 6 months. Of these, to date, four patients have undergone re-coiling. Although the immediate complications of coiling may be less than those of clipping (ISAT), it seems that the degree and permanence of exclusion of an aneurysm from the cerebral circulation may be more secure with surgery. In summary, the rates of incomplete aneurysmal exclusion from the cerebral circulation, the requirement for reintervention and the requirement for continuing surveillance were all higher in the coiled population than in the clipped population.

Transcription activity in fibroblasts from the adult tight skin (TSK) mouse.

OBJECTIVE: To assess if adult tight-skin mouse (TSK) skin fibroblasts have a parallel increase in transcription of collagen and non-collagen genes. METHODS: One-year-old TSK/+ and +/+ (normal littermate) dermal fibroblasts were transfected (lipotransfection) with plasmid constructs containing Chloramphenicol Acetyl Transferse (CAT) gene, directed by promoters of mouse alpha1(I) alpha2(I) and alpha1(III) collagen genes, and by viral enhancers of Simian virus 40, Rous sarcoma virus and an LTR from a Syrian hamster tumour retrovirus. Syrian hamster derived tumour cell lines MF2B and GRI, and fibroblast cell line 3T3/NIH were used as controls. In some experiments, transfected cells were treated with hormones as transcription activating factors. Mixing experiments of tumour cells and TSK/+ or +/+ fibroblasts were done to study potential inhibitors. RESULTS: Collagen genes promoters failed to induce transcriptional activity in TSK/+ or +/+ fibroblasts, even in the presence of hormone treatment. Mixing experiments did not reveal inhibitor factors acting in these fibroblasts. Viral enhancers induced 2 to 5 times more transcription activity in TSK/+ than in +/+ fibroblasts. CONCLUSION: Increased transcription of viral enhancers and not of collagen genes in adult TSK fibroblasts, suggests the presence of transcription activating factors independent of collagen gene activation.

The tight skin mouse: an animal model of systemic sclerosis.

The search for an animal model of systemic sclerosis (SSc) was tenaciously pursued by E.C. LeRoy. We studied several aspects of the tight skin mouse (Tsk) genetics and pathogenesis under his stimulating influence that contributed to a better understanding of the fibrotic scleroderma-like phenotype of this mouse. The identification of the fibrillin-1 mutation in the Tsk mouse and the characterization of the cellular and molecular pathways leading to Tsk fibrosis by numerous research groups has opened new avenues in the investigation of human SSc. The enigmatic connections between autoimmunity and ECM homeostasis in fibrotic diseases have received extensive attention in this mouse in which a prirmary alteration of a connective tissue microfibrilar protein leads to the reproduction of cellular and autoimmune abnormalities strikingly similar to human SSc. The use of this mouse as a tool to explore anti-fibrotic therapeutic interventions has demonstrated its value in providing useful information on the search for a therapy for this untreatable facet of human disease.

Controlled expression in Klebsiella pneumoniae and Shigella flexneri using a bacteriophage P1-derived C1-regulated promoter system.

The utility of promoters regulated by the bacteriophage P1 temperature-sensitive C1 repressor was examined in Shigella flexneri and Klebsiella pneumoniae. Promoters carrying C1 operator sites driving LacZ expression had induction/repression ratios of up to 240-fold in S. flexneri and up to 50-fold in K. pneumoniae. The promoters exhibited remarkably low basal expression, demonstrated modulation by temperature, and showed rapid induction. This system will provide a new opportunity for controlled gene expression in enteric gram-negative bacteria.

A complex adenovirus vector that delivers FASL-GFP with combined prostate-specific and tetracycline-regulated expression.

Cell-type-restricted transgene expression delivered by adenovirus vectors is highly desirable for gene therapy of cancer, as it can limit cytotoxic gene expression to tumor cells. However, many tumor- and tissue-specific promoters are weaker than the constitutively active promoters and are thus less effective. To combine cell-type specificity with high-level regulated transgene expression, we have developed a complex adenoviral vector. We have placed the tetracycline transactivator gene under the control of a prostate-specific ARR2PB promoter, and a mouse Tnfsf6 (encoding FASL)-GFP fusion gene under the control of the tetracycline responsive promoter. We have incorporated both expression cassettes into a single construct. We show that FASL-GFP expression from this vector is essentially restricted to prostate cancer cells, in which it can be regulated by doxycycline. Higher levels of prostate-specific FASL-GFP expression were generated by this approach than by driving the FASL-GFP expression directly with ARR2PB. More FASL-GFP expression correlated with greater induction of apoptosis in prostate cancer LNCaP cells. Mouse studies confirmed that systemic delivery of both the prostate-specific and the prostate-specific/tet-regulated vectors was well tolerated at doses that were lethal for FASL-GFP vector with CMV promoter. This strategy should be able to improve the safety and efficacy of cancer gene therapy using other cytotoxic genes as well.

Prostate-specific expression of Bax delivered by an adenoviral vector induces apoptosis in LNCaP prostate cancer cells.

In prostate carcinoma, overexpression of the anti-apoptotic gene Bcl-2 has been found to be associated with resistance to therapies including radiation and androgen ablation. Restoring the balance of Bcl-2 family members may result in the induction of apoptosis in prostate cancer cells previously resistant to treatment. To accomplish this, a strategy involving overexpression of the pro-apoptotic gene Bax was executed. The use of cytotoxic genes such as Bax require selective expression of the gene. In this study, we examined the ability of selective expression of Bax protein directed by a prostate-specific promoter to induce apoptosis in human prostate carcinoma. A second-generation adenoviral vector was constructed with the modified prostate-specific probasin promoter, ARR2PB, directing expression of an HA-tagged Bax gene and a green fluorescent protein reporter translated from an internal ribosome entry site (ARR2PB.Bax.GFP). ARR2PB promoter activity is tightly regulated and highly prostate specific and is responsive to androgens and glucocorticoids. The prostate-specific promoter-Bax-GFP transgene cassette was inserted into a cloning site near the right inverted terminal repeat of the adenoviral vector to retain specificity of the promoter. LNCaP cells infected with Ad/ARR(2)PB.Bax.GFP showed high levels of Bax expression 48 h after infection resulting in an 85% reduction in cell viability. Importantly, LNCaP cells stably transfected to overexpress Bcl-2 showed similar patterns of cell death when infected with Ad/ARR(2)PB.Bax.GFP, an 82% reduction in cell viability seen 48 h after infection. Apoptosis was confirmed by measuring caspase activation and using the TUNEL assay. Tissue specificity was evaluated using A549 cells (lung adenocarcinoma), SK-Hep-1 (liver cancer) cells, and Hela (cervical cancer) cells which did not show detectable expression of virally delivered Bax protein or any increase in cell death. Systemic administration of Ad/ARR2PB. Bax.GFP in nude mice revealed no toxicity in liver, lung, kidney, or spleen. This study shows that infection with the second-generation adenovirus, ARR2PB.Bax.GFP, results in highly specific cytotoxicity in LNCaP cells, and that consequent overexpression of Bax in prostate carcinoma, even in the context of high levels of Bcl-2 protein, resulted in apoptosis. These results suggest that a second-generation adenovirus-mediated, prostate-specific Bax gene therapy is a promising approach for the treatment of prostate cancer.

Steroid hormones modulate expression of cytochrome P450 enzymes in male hamster reproductive tract and leiomyosarcomas.

Syrian hamsters treated with estrogen and androgen for 8 months develop leiomyosarcomas in the vas deferens. Metabolism of estrogen by cytochrome P450s (CYPs) produces catechols and reactive oxygen species, and may contribute to tumor formation. To examine this issue, male hamsters were treated with 17 beta-estradiol (E2), testosterone propionate (TP) or both hormones. Reproductive tract tissues from control and treated animals were immunostained with antibodies specific for four CYP enzymes (1A1, 1A2, 1B1 and 3A1/2). Immunoreactive CYP1A1 was not found in the reproductive tract of control or treated animals. In untreated hamsters, CYP1A2 was detected only in principal cells of the caput epididymis. TP alone had no effect, but treatment with E2 induced expression of CYP1A2 in columnar epithelial cells throughout the epididymis and lining of the vas deferens. Treatment with E2 + TP blocked the induction of CYP1A2 seen in surface epithelial cells treated with E2 alone, but not the constitutive expression of this enzyme. Instead, simultaneous exposure to both hormones induced CYP1A2 in basal cells of the epididymis and vas deferens. CYP3A1/2 was not detected in the reproductive tract of control or TP-treated males, but immunostaining was induced in the inner layer of vas deferens smooth muscle by E2, and in all smooth muscle layers by dual hormone treatment. In controls, CYP1B1 was present in smooth muscle lining the epididymis and surrounding the vas deferens and dual hormone treatment increased staining intensity for CYP1B1 in these cells. Immunoreactive CYP1A2 was not detectable in leiomyosarcomas but the enzyme was present in both columnar and basal cells of the vas deferens epithelium adjacent to the tumors. In contrast, tumor cells showed heterogeneous expression of both CYP1B1 and CYP3A1/2. The relationships between hormone treatment, differential CYP expression and tumor formation strengthen our hypothesis that metabolism of estrogen is an important element in this model of hormonal carcinogenesis.

The use of Fas Ligand, TRAIL and Bax in gene therapy of prostate cancer.

Prostate cancer is the second leading cause of cancer death in the United States. Treatment options for confined disease are generally successful in prolonging life but long-term cures (10-15 years) are elusive for the majority of patients. The prognosis for advanced extra-capsular prostate cancer is grim. However, we are now entering the era of gene therapy options for treatment of prostate cancer. The human genome project coupled with genomics and protemics are providing information that will lead to selection of genes for treatment of prostate cancer. The problem is the science of delivery lags behind knowledge of gene function. Thus, it is important to develop therapies that do not require delivery to 100% of tumor cells but which nevertheless kills the entire cancer by virtue of the bystander effect or other means. This review covers the use, in gene therapy, of apoptotic inducing molecules such as Fas Ligand, and TRAIL which are believed to induce bystander killing activity and Bax which also may function in a similar way.

Intracellular Fas ligand expression causes Fas-mediated apoptosis in human prostate cancer cells resistant to monoclonal antibody-induced apoptosis.

Several laboratories have attempted with little success to induce Fas-mediated apoptosis in prostate cancer (PCa) cells, using different external Fas agonists, i.e., anti-Fas antibodies and membrane-bound FasL. The present study confirms these earlier results using the anti-Fas antibody CH-11 in five human PCa cell lines (PPC-1, LNCaP, PC-3, TSU-Pr1, and DU145). However, intracellular murine FasL expression induced Fas-mediated apoptosis in all CH-11-resistant cell lines. Adenovirus (AdGFPFasL(TET)) was used to deliver a Murine FasL-GFP fusion gene into human PCa cells resulting in 70-98% apoptosis at 48 h as determined by the MTS assay. DU145 and PPC-1 cells treated with AdGFPFasL(TET) stained positive for the TUNEL assay, indicating that cell death was via apoptosis. Using immunofluorescent microscopy, Fas and GFPFasL colocalized to the same intracellular compartment. The anti-Fas neutralizing antibody ZB-4 was unable to block AdGFPFasL(TET)-mediated cell death, suggesting that intracellular FasL may ligate Fas within the Golgi and/or endoplasmic reticulum. This is the first evidence suggesting that these two molecules interact prior to cell surface presentation. Collectively, these findings indicate that intracellular GFPFasL expression is superior to CH-11 at inducing Fas-mediated apoptosis in human PCa cells and may allow use of AdGFPFasL(TET) for PCa gene therapy.

Nuclear factor-kappaB/IkappaB signaling pathway may contribute to the mediation of paclitaxel-induced apoptosis in solid tumor cells.

Paclitaxel (Taxol), a naturally occurring antimitotic agent, has shown significant cell-killing activity in a variety of tumor cells through induction of apoptosis. The mechanism by which paclitaxel induces cell death is not entirely clear. Recent studies in our laboratory demonstrated that glucocorticoids selectively inhibited paclitaxel-induced apoptosis without affecting the ability of paclitaxel to induce microtubule bundling and mitotic arrest. This finding suggests that apoptotic cell death induced by paclitaxel may occur via a pathway independent of mitotic arrest. In the current study, through analyses of a number of apoptosis-associated genes or regulatory proteins, we discovered that paclitaxel significantly down-regulated IkappaB-alpha, the cytoplasmic inhibitor of transcription factor nuclear factor-kappaB (NF-kappaB), which in turn promoted the nuclear translocation of NF-kappaB and its DNA binding activity. In contrast, we found that glucocorticoids could antagonize paclitaxel-mediated NF-kappaB nuclear translocation and activation through induction of IkappaB-alpha protein synthesis. Northern blotting analyses demonstrated that the steady-state level of IkappaB-alpha mRNA was not affected by paclitaxel, which suggests that the down-regulation of IkappaB-alpha by paclitaxel is attributable to protein degradation rather than suppression of transcription. Furthermore, through transfection assays, we demonstrated that tumor cells stably transfected with antisense IkappaB-alpha expression vectors remarkably increased their sensitivity to paclitaxel-induced apoptosis. Finally, we found that a key subunit of IkappaB kinase (IKK) complex, IKKbeta, was up-regulated by paclitaxel, which implies that paclitaxel might down-regulate IkappaB-alpha through modulation of IKKbeta activity. All of these results suggest that the NF-kappaB/IkappaB-alpha signaling pathway may contribute to the mediation of paclitaxel-induced cell death in solid tumor cells.