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Experimental Therapeutics |
Laboratory of Signal Transduction [M. W-J., R. K.] and the Departments of Radiation Oncology [A. H-F., W-C. L., Z. F.], Surgery [M. G., R. H., W. D. W. H.],,2 and Pathology [C. C-C.] Memorial Sloan-Kettering Cancer Center, New York, New York 10021
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The predominant mechanism by which radiation kills mammalian cells is the reproductive (also known as clonogenic) death pathway. DNA is the target, and double-stranded breaks in the DNA are regarded as the specific lesions that initiate this lethal response (5 , 6) . Whereas most radiation-induced DNA double-stranded breaks are rapidly repaired by constitutively expressed DNA repair mechanisms, residual unrepaired or misrepaired breaks lead to genetic instability and to increased frequency of mutations and chromosomal aberrations (5, 6, 7) . Lethal mutations or dysfunctional chromosomal aberrations eventually lead to progeny cell death (7 , 8) , usually after several mitotic cycles (9 , 10) .
Radiation can also signal apoptosis, although it appears to be significantly less prevalent than clonogenic cell death (11) . Apoptosis is an inducible death pathway of sequential biochemical events that are constitutively expressed in an inactive form in most, if not all, mammalian cells (12) . A variety of physiological or environmental stresses impact distinct cellular targets to initiate cell type-specific apoptotic signaling pathways (12 , 13) . The various upstream signaling cascades converge downstream to activate a common final caspase-dependent effector mechanism for dismantling the dying cell (12) . Radiation was shown to target either the cell membrane or the nucleus to activate different apoptotic pathways (14, 15, 16, 17, 18) . In endothelial cells, radiation signals the hydrolysis of plasma membrane sphingomyelin, generating the proapoptotic second messenger ceramide via activation of SMase3 (14) . Alternately, in thymocytes, radiation initiates p53-mediated apoptosis via recognition of DNA damage (17 , 18) . Whole body irradiation of p53 and acid SMase knockout mice proved that these death pathways are distinct and independent (15) .
An alternative mechanism to SMase-mediated generation of ceramide in response to stress is a pathway that involves de novo synthesis of ceramide. Ceramide synthesis is catalyzed by the enzyme ceramide synthase, which localizes to the endoplasmic reticulum and the mitochondria (19, 20, 21) . Ceramide synthase, once activated, catalyzes the condensation of sphinganine and fatty acyl-CoA to form dihydroceramide, which is rapidly oxidized to ceramide (19) . Recent studies demonstrated that in different cells, radiation may activate either of these mechanisms for ceramide generation, sometimes activating both within a single cell type (16) .
The pleiotropic nature of death pathways induced by radiation suggests that radiation resistance is likely to be regulated by a variety of mechanisms, each of which is associated with a specific death pathway. Whether radiation resistance of human prostate tumor clones is associated with a single mechanism or a spectrum of mechanisms is unknown. There are only a few immortalized human prostate cell lines because it is unusually hard to establish primary cultures or xenographs from surgical specimens of human prostate tumors, even at short term (22)
. The best characterized lines include the PC-3, DU-145, and LNCaP cell lines established from metastatic human tumor lesions (23, 24, 25)
. In general, these cell lines are among the most radioresistant human tumor cells, as assessed by the clonogenic assay (26, 27, 28, 29)
. However, the dose-survival data do indicate distinct differences between these cell lines, as expressed by the Dq, D0, SF-2, and the linear quadratic 
and 
exponents. There are also differences in the apoptotic response to radiation. Several studies reported lack of apoptosis in PC-3 cells up to 72 h after exposure to doses of 10–30 Gy (30
, 31)
and an incidence of 10–15% apoptosis in DU-145 cells at 72 h after 10–12 Gy (30
, 32)
. However, one study reported 40% apoptosis in PC-3 cells at 72 h after 20 Gy (33)
. This study is also the only study that has thus far reported apoptosis in LNCaP cells, occurring at a rate of 35% at 72 h after exposure to 20 Gy (33)
. Altogether, these observations indicate clone-specific sensitivities of human prostate tumor cells to radiation. These data also suggest that an approach to reduce radiation resistance clinically might require the use of combinations of chemical and biological modifiers to cover a spectrum of resistance mechanisms that may operate in prostate cancer.
In a recent study, we reported that protein kinase C (PKC) activation by TPA induced ceramide synthase activation in LNCaP cells (34) . Ceramide generation was rapid, detectable by 1 h after TPA treatment, and progressive for 12 h. This was followed by a delayed form of apoptosis that reached maximal levels at 48 h. Investigations into the mechanism of TPA-induced ceramide generation revealed that acid and neutral SMase activities were not enhanced. In contrast, TPA induced an increase in ceramide synthase activity that persisted for at least 16 h. Treatment with FB1, a natural competitive inhibitor of ceramide synthase, abrogated both TPA-induced ceramide production and apoptosis. Thus, ceramide synthase activation appears to be required for TPA-induced apoptosis in LNCaP cells.
In the present study, we demonstrate that ceramide synthase activation also appears to be required for radiation-induced apoptosis in LNCaP cells. Whereas LNCaP cells failed to respond to radiation with ceramide generation and apoptosis, pretreatment with TPA converted this pattern, enabling radiation to signal ceramide synthase activation and apoptosis. Furthermore, treatment of nude mice with i.v. TPA before radiation also resulted in a synergistic apoptotic response in orthotopically implanted LNCaP tumors, sensitizing the tumors to the effects of radiation in vivo. This represents the first description of a signaling-based therapy designed to overcome one form of radiation resistance expressed preferentially in human prostate cancer cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-32P]ATP (3000 Ci/mmol) were obtained from DuPont (New England Nuclear). Octyl--D-glucopyranoside was from Calbiochem. C2-ceramide was stored at -20°C and dissolved in DMSO (final concentration, 0.4%) just before each experiment. FB1 was diluted in sterile saline (0.9%) and stored at 4°C until use. TPA was dissolved in DMSO, aliquoted, and stored at -20°C until use.
Cell Cultures, Mice, and Irradiation.
LNCaP cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum. For apoptosis and lipid studies, cells were plated at a density of 5.0 x 105 cells/ml onto either 6- or 12-well plates and treated when 80–95% confluent. Of note, this cell density was significantly greater than the density used in our previous report (34)
. For ceramide synthase assays, cells were plated at a similar density onto 100-mm dishes. Irradiation of cultured cells was carried out at 25°C in a gamma-cell 40 chamber containing two sources of 137Cs (Atomic Energy of Canada) at a dose rate of 100 cGy/min. One hour before irradiation, the culture medium was changed to RPMI 1640 containing 0.2% human serum albumin.
The mice used in this study were 8–10-week-old male Swiss nude (nu/nu) mice obtained from Taconic Farms and housed at the animal core facility of Memorial Sloan-Kettering Cancer Center. This animal housing facility is approved by the American Association for Accreditation of Laboratory Animal Care and is maintained in accordance with the regulations and standards of the United States Department of Agriculture and the Department of Health and Human Services, NIH. For irradiation, mice received whole body irradiation, using a 137Cs irradiator (Shepherd Mark-I, Model 68, SN643) at a dose rate of 270 cGy/min, as described previously (15) .
Cell Cycle Analysis.
Cells were washed with PBS and then trypsinized for 1 min in 37°C. The cells were resuspended in PBS and preserved in cold 70% ethanol. Before analysis, the ethanol was removed, and the cells were resuspended in a propidium iodide solution as described previously (35)
. DNA content was measured with FACScan equipped with a Facstation running CellQuest software (Becton Dickinson). Cell cycle analysis of DNA histograms was performed with Muticycle (Phoenix Flow Systems). A minimum of 1 x 104 cells were counted per sample.
Quantification of Ceramide.
LNCaP cells were prepared for the DG kinase assay as described previously (34)
. Briefly, the cells were detached from the monolayer by gentle pipetting. The cells were pelleted by centrifugation (500 x g 5 min), washed with ice-cold PBS, and extracted with 2 ml of chloroform:methanol:1 N HCl (100:100:1, v/v/v), 540 µl of buffered saline solution [135 mM NaCl, 1.5 mM CaCl2, 0.5 mM MgCl2, 5.6 mM glucose, and 10 mM HEPES (pH 7.2)], and 60 µl of 100 mM EDTA. Lipids in the organic phase extract were dried under N2 and subjected to mild alkaline hydrolysis (0.1 N methanolic KOH for 1 h at 37°C) to remove glycerophospholipids. Samples were re-extracted, and the organic phase was dried under N2. Ceramide was quantified by the DG kinase assay as described (36)
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Ceramide Synthase Assay.
Microsomal membranes were prepared as described previously (34)
. Briefly, LNCaP cells were washed twice with ice-cold PBS, scraped off the plate, and resuspended in 300 µl of homogenization buffer [25 mM HEPES (pH 7.4), 5 mM EGTA, 50 mM NaF, and 10 µg/ml each of leupeptin and soybean trypsin inhibitor]. Cells were disrupted by sonication on ice, and lysates were pelleted at 800 x g for 5 min. The postnuclear supernatant was centrifuged at 250,000 x g for 35 min. The microsomal membrane pellet was resuspended in 0.5 ml of homogenization buffer. Assays of ceramide synthase activity were performed as described previously (34
, 36) . Briefly, microsomal membrane protein (75 µg) was incubated with 2 mM MgCl2, 20 µM fatty acid-free BSA, the indicated concentrations of sphinganine, 70 µ M unlabeled palmitoyl-CoA, and 0.2 mCi of [1-14C]palmitoyl-CoA at 37°C for 1 h. After extraction of the lipids with 2 ml of chloroform:methanol (1:2), radiolabeled dihydroceramide was resolved by TLC using a solvent system of chloroform/methanol/3.5 N ammonium hydroxide (85:15:1, v/v/v). Dihydroceramide was detected by iodine vapor staining based on comigration with commercially available ceramide (Sigma Type III) and quantified by liquid scintillation counting. Under the conditions used, the substrate was not rate-limiting, and the reaction was linear for time and enzyme concentration.
Quantification of Apoptotic Cells.
Morphological changes in the nuclear chromatin of cells undergoing apoptosis were detected by staining with the DNA-binding fluorochrome bis-benzimide trihydrochloride (Hoechst 33258), as described previously (34)
. Briefly, LNCaP monolayers were detached with 0.25% trypsin and 0.02% EDTA in HBSS and combined with the floating population. The cell pellet was washed in PBS, resuspended in 50 µl of 3% paraformaldehyde, and incubated for 10 min at room temperature. The fixative was removed, and the cells were resuspended in 20 ml of PBS containing 8 µg/ml Hoechst 33258. After a 15-min incubation at room temperature, an 8-µl aliquot was placed on a glass slide, and 500 cells were scored for the incidence of apoptotic chromatin changes under an Olympus BH2 fluorescence microscope using a BH2-DMU2UV Dich Mirror Cube filter.
Orthotopic Transplantation of LNCaP Tumors.
The 8–10-week-old male Swiss nude (nu/nu) mice were implanted orthotopically with LNCaP cells. Immediately before tumor implantation, LNCaP cells grown in culture were trypsinized and resuspended in RPMI 1640 with 10% FBS at a concentration of 20 x 106 cells/ml, and viability was determined by trypan blue exclusion. Only single cell suspensions with >90% viability were used for in vivo injection. Mice were anesthetized with pentobarbital (75 mg/kg i.p.), and orthotopic tumor implantation was performed as described previously (37)
. Briefly, a low midline abdominal incision was made with a #15 blade (Bard Parker). The peritoneal cavity was entered by sharply incising the linea alba. The bladder and seminal vesicles were identified and gently raised, thus exposing the dorsal lobes of the mouse prostate. Before intraprostatic injection, the LNCaP cells were resuspended to a final concentration of 2.0 x 107 LNCaP cells/ml culture medium. Either lobe was injected with 0.1 ml using a disposable 26-gauge needle. Proper implantation of cell suspension was indicated by blebbing under the prostatic capsule. Visceral contents were then replaced into the abdominal cavity, and the wound was closed with surgical clips (Autoclip; Becton Dickinson). Mice were monitored during the postoperative period according to animal care facility guidelines. The injected mice were housed (2–3 mice/cage) in a pathogen-free environment, using filtered, laminar air flow hoods in standard vinyl cages with air filter tops. Cages, bedding, and water were autoclaved before use and cared for in accordance with the institutional and NIH guidelines.
Tumors were allowed to grow for approximately 4 weeks, at which time tumor take was assessed by serum PSA determination as described previously (37)
. It has been reported that orthotopically transplanted LNCaP tumors in nude mice produce and secrete PSA that can be detected histochemically in the tumor cells and by radioimmunassays in the mouse serum (37)
. To assess LNCaP tumor take and tumor volume after intraprostatic transplantation, mice were anesthetized with pentobarbital (50 mg/kg i.p.), and phlebotomy was performed by accessing the retro-orbital venous plexus with a microcapillary pipette (Fisher). Serum PSA determinations were performed by radioimmunoassay (Hybritech) according to the recommendations of the manufacturer. Pilot experiments revealed that prostatic tumor weight correlated with serum PSA values (r = 0.952; Fig. 1
). Additional studies demonstrated the PSA doubling time for orthotopically implanted tumors was approximately 10 days (data not shown). This doubling time is markedly prolonged when compared to the doubling time of LNCaP in vitro (24–36 h; data not shown). Using serum PSA determinations as an indicator of tumor size, mice were allocated to experiments when PSA levels ranged between 1.1 and 10.4 ng/ml, corresponding to a tumor weight of 45–100 µg.
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0.08 µg/g mouse body weight). When radiation was delivered, mice were anesthetized as described above and injected with i.v. vehicle or TPA 20 min before irradiation.
Assessment of Apoptotic Cells in Orthotopically Transplanted LNCaP Tumors.
Apoptosis in vivo was assessed by the TUNEL assay, as described previously (39
, 40)
. LNCaP tissue sections from paraffin-embedded blocks (5-µm thick) were adhered to polylysine-treated slides, deparaffinized, and rehydrated. The slides were incubated in 10 mM Tris-HCl (pH 8) for 5 min, digested with 0.1% pepsin, rinsed in distilled water, and treated with 3% H2O2 in PBS for 5 min at 22°C to inactivate endogenous peroxidase. After three washes in PBS, the slides were incubated for 15 min at 22°C in buffer [140 mM sodium cacodylate (pH 7.2), 30 mM Trizma base, and 1 mM CoCl2) and then incubated for 30 min at 37°C in reaction mixture [0.2 unit/µl terminal deoxynucleotidyl transferase, 2 nM biotin-11-dUTP, 100 mM sodium cacodylate (pH 7.0), 0.1 mM DTT, 0.05 mg/ml BSA, and 2.5 mM CoCl2]. The reaction was stopped by transferring the slices to a bath of 300 mM NaCl, 30 mM sodium citrate for 15 min at 22°C. The slides were washed in PBS, blocked with 2% human serum albumin in PBS for 10 min, rewashed, and incubated with avidin-biotin peroxidase complexes. After 30 min at 22°C, cells were stained with the chromogen 3,3' -diamonobenzidine tetrachloride and counterstained with hematoxylin. Nuclei of apoptotic cells appeared brown and granular, whereas normal nuclei stained blue.
Statistical Analysis.
Statistical analysis was performed by Student’s t test and the t test for the correlation coefficient. Linear regression analysis was performed by the method of least squares.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TPA Modulates the Response of LNCaP Cells to Radiation.
Our previous studies demonstrated that TPA induces sequential ceramide synthase activation, ceramide generation, and apoptosis in exponentially growing LNCaP cells (34)
. In those experiments, 1.5 x 105 cells were plated in 6- or 12-well plates, and when they reached 40–50% confluence, the cells were treated with 10 ng/ml TPA, leading to ceramide generation by 3 h and apoptosis by 12 h (34)
. The present studies showed that plateau-phase LNCaP cells exhibited delayed and reduced ceramide generation and apoptosis in response to treatment with 10 ng/ml TPA. Fig. 2
shows initial evidence of ceramide elevation from a baseline of 643 ± 12 to 749 ± 65 pmol/106 cells was detected at 10 h, reaching a level of only 866 ± 44 pmol/106 cells at 16 h. Apoptosis remained undetectable in TPA-treated cells up to 12 h and reached a level of only 18 ± 5% at 24 h. However, exposure of plateau-phase LNCaP cells to both 10 ng/ml TPA and 20 Gy of irradiation resulted in an accelerated and enhanced generation of ceramide and apoptosis. Fig. 2
shows that cells treated with both TPA and radiation already reached a near maximal level of ceramide elevation (from 643 ± 12 to 941 ± 13 pmol/106 cells) by 6 h. Similarly, 18 ± 5% of the cells showed evidence of apoptosis at 12 h. There was a dose-response relation for TPA in sensitizing the cells to apoptosis after irradiation. Doses of 0.5–10 ng/ml TPA were used, and apoptosis was assessed in 1000 cells at 24 h after exposure to 20 Gy. Whereas 1.5 ng/ml TPA given alone had no significant effect on apoptosis (2.1 ± 0.4%), a small but statistically significant synergistic effect (8.4 ± 2.4%) was observed when it was given in combination with radiation (P < 0.05). Higher TPA doses (3.0 and 10.0 ng/ml) induced an apoptotic response even when given alone (6.1 ± 3.3% and 22.9 ± 2.8%, respectively), but apoptosis was significantly enhanced when the cells were also exposed to 20 Gy of irradiation (13.4 ± 1.0% and 36.2 ± 3.8%, respectively; P < 0.05). Because radiation alone did not result in the induction of either cellular ceramide or apoptosis (Fig. 2)
, these data indicate that TPA modulates the resistance of LNCaP cells to irradiation, leading to synergistic effects with the combination. It should be noted that the level of the effect of TPA on LNCaP cells varied with different TPA preparations. Therefore, it was necessary to establish the optimal concentration of TPA required to confer the effects described above for each new batch of TPA.
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shows the Eadie-Hofstee transformation of these data. At 3 h, cells treated with TPA alone manifested only a 34% increase in the Vmax of the reaction (from 116 to 156 pmol/min/mg protein), without a change in the Km. However, there was a significant increase in the Vmax to 232 pmol/min/mg protein when the cells were exposed to the combination of 10 mg/ml TPA and 20 Gy of irradiation. Radiation alone (20 Gy) did not have any discernible effect on ceramide synthase activity. Similar results were observed in three independent experiments. Because the activity of ceramide synthase can currently be measured only by an enzymatic assay in microsomal membrane preparations, there is significant interassay variation, which does not permit the collation of data from different experiments for statistical analysis. However, when all data were normalized to their own controls, a statistically significant increase in the Vmax of ceramide synthase was detected only in the TPA + XRT group (P < 0.05). These data indicate that TPA confers sensitivity to ceramide synthase activation by ionizing irradiation, leading to a synergistic activation of the enzyme by the combination of TPA and radiation. Ceramide synthase activation appeared to represent the only mechanism for the proapoptotic generation of ceramide, because neither radiation, TPA, nor a combination of the two induced the activation of neutral or acidic SMase (data not shown). Baseline activity of neutral and acidic SMase was 122 and 943 pmol/mg protein/min, respectively, and did not change upon cell treatment with TPA, radiation, or the combination of the two.
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shows that treatment of exponentially growing LNCaP cells with 100 µM FB1 reduced the induction of apoptosis in response to TPA at 24 h from 28 ± 1% to 11 ± 1% and reduced the induction of apoptosis in response to TPA with XRT from 42 ± 8% to 20 ± 1%. Concomitantly, this concentration of FB1 reduced the baseline level of ceramide from 566 ± 13 to 74 ± 28 pmol/106 cells and blocked the increase in response to TPA (10 ng/ml) and to TPA plus 20 Gy of XRT by 78% and 60%, respectively (data not shown). These data support the notion that the synergistic effects of TPA and radiation on ceramide generation and apoptosis are mediated via ceramide synthase activation.
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, LNCaP tumors grow as solid neoplasms with few glandular-like structures. TUNEL staining revealed no spontaneous apoptosis. Only incidental apoptosis was manifest at 24 h after treatment with 20 Gy or at other time points from 12–36 h after irradiation (data not shown). TPA induced minimal (1–5%) apoptosis within this time frame. However, in animals treated with TPA followed by 20 Gy, substantial apoptosis (20–25%) was evident. This synergistic effect was detected by 12 h and was manifested for as long as 36 h. Thus, the TPA-mediated sensitization of LNCaP cells to radiation-induced apoptosis observed in vitro was recapitulated in vivo in LNCaP tumors growing orthotopically within the prostate.
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. Although TPA was without effect on the normal rectum, TPA pretreatment almost completely abrogated the damaging effects of radiation on the rectum. Similar protection was observed at all times from 12–36 h. These studies show tissue type-specific transmodulation of apoptosis by TPA.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The effects of TPA on apoptosis in LNCaP cells are conditional. Our previous study (34) examined apoptosis in log-phase cultures of LNCaP cells. Under these conditions, we found LNCaP cells to be quite sensitive to the apoptotic effect of TPA. In the present studies, we explored the apoptotic response of plateau-phase, rather than exponentially growing LNCaP cells, because this culture condition more closely resembles the state of the majority of tumor cells in vivo. Under these conditions, LNCaP cells are more resistant to TPA-induced apoptosis and manifest slower kinetics of ceramide generation and apoptosis.
The effects of TPA on apoptotic responses are pleiotropic (42)
. In most cells, TPA provides antiapoptotic protection. In irradiated endothelial cells, TPA confers such an antiapoptotic effect, at least in part, via the inhibition of SMase-mediated generation of ceramide (14)
. In a few instances, phorbol esters have proven to be proapoptotic. In this regard, EBV-infected Burkitt lymphoma cells display substantial apoptosis in response to phorbol esters, although uninfected Burkitt cells are resistant (43)
. Immature thymocytes also appear to be susceptible to phorbol ester-induced apoptosis (44)
. Although the mechanism by which TPA serves as a proapoptotic agent is unknown, prolonged activation of PKC may play a role. de Vente et al. (45)
overexpressed PKC in MCF-7 breast cancer cells, which conferred phorbol ester-induced cytotoxicity. Furthermore, Powell et al. (46)
demonstrated in LNCaP cells that TPA induced a specific increase in PKC mRNA and prolonged the translocation of this PKC isoform to the membrane before apoptosis. TPA action in LNCaP was also shown to involve retinoblastoma dephosphorylation and G0/G1 arrest (47)
, events reported to occur in response to ceramide treatment in other cell types (48
, 49)
. Whether ceramide synthase activation is upstream of cyclooxygenase 2 or 5-lipoxygenase inhibition (50
, 51)
, regulation of the levels of Bcl-2 family members (52)
, or Fas/Fas-ligand interaction (53)
, events involved in LNCaP apoptosis in response to other stresses will require additional experimentation.
The present studies extend an emerging literature that is defining a role for ceramide synthase in stress signaling. Although originally reported as mediating daunorubicin-induced apoptosis in p388, HL-60, and U937 cells (36
, 54)
, recent studies have demonstrated its involvement in multiple forms of apoptotic damage. In primary cultures of cerebral endothelial cells, the sensitizing effect of cycloheximide to tumor necrosis factor-induced apoptosis appeared to require ceramide synthesis (55)
. Furthermore, FB1 abrogated daunorubicin-induced apoptosis in hen granulosa cells (56)
and in CPT-11-induced apoptosis in L929 cells (57)
. Shimabukuro et al. (58)
proposed that ceramide synthase-mediated apoptosis might be involved in the pathogenesis of type 1 diabetes. These investigators showed that islets from Zucker fatty diabetic rats, which display a defect in leptin receptor signaling, manifested elevated ceramide levels and apoptosis. In response to a challenge with free fatty acids, these islets displayed reduced fatty acid oxidation and markedly increased incorporation into ceramide, accompanied by apoptosis. FB1 blocked both ceramide generation and apoptosis. Furthermore, transfection of the wild-type leptin receptor restored the ability of leptin to reverse these effects (59)
. Ceramide synthesis was also found to be involved in mitochondrial functions associated with apoptosis. Paumen et al. (60)
isolated CPT I in a screen for genes involved in apoptosis after interleukin 3 withdrawal from LyD9 hematopoieitic precursor cells. This enzyme is located in the outer mitochondrial membrane and catalyzes the transfer of long-chain fatty acids into the mitochondria for -oxidation. In the presence of a CPT I inhibitor, treatment of cells with fatty acids such as palmitate, which serve as precursors of de novo ceramide synthesis, led to ceramide generation and apoptosis. FB1 blocked these events. CPT I has also been shown to bind bcl-2, suggesting that these two proteins may regulate each other’s activity (61)
.
The mechanisms of LNCaP resistance to radiation-induced ceramide synthase activation and its modulation by TPA remain unknown. In recent studies, we demonstrated that radiation-induced activation of ceramide synthase may be regulated in some cell types by the gene mutated in ATM (the ATM gene; Ref. 16 ). 2 ,3 Metabolic incorporation of [125I]5-iodo-2'-deoxyuridine, which produces DNA double-stranded breaks, signaled in endothelial cells de novo ceramide synthesis by posttranslational activation of ceramide synthase and apoptosis. Ceramide synthase activation was obligatory, because FB1 abrogated [125I] 5-iodo-2'-deoxyuridine-induced death. X-irradiation yielded similar results. EBV-immortalized B cells from ATM patients exhibited markedly increased radiation-induced ceramide synthase activation, ceramide generation, and apoptosis. Stable transfection of wild-type ATM cDNA reversed these events, whereas antisense inactivation of ATM in normal B cells conferred the ATM phenotype (16) . 2 ,3 These studies demonstrated that one of the functions of ATM is to constrain activation of ceramide synthase, thereby regulating DNA damage-induced apoptosis. Whether LNCaP cells express high levels of the ATM protein or activity and whether TPA converts the LNCaP apoptotic resistance to radiation by affecting ATM are possibilities that require further investigation.
In the present studies, TPA was also found to confer antiapoptotic protection against radiation-induced apoptosis in the rectal mucosa through an unknown mechanism. In a recent study, we reported that lipopolysaccharide-induced apoptosis of intestinal epithelial cells occurs, at least in part, secondary to acid SMase-mediated apoptosis of microvascular endothelial cells in the lamina propria of the intestinal crypts (62) . Inactivation of acid SMase by genetic mutation or i.v. injection of basic fibroblast growth factor, which specifically protects the endothelium against stress-induced apoptosis (39 , 40) , abrogated the apoptotic response in the mucosal epithelial cells (62) . Whether the antiapoptotic effect of TPA on radiation-induced apoptosis in the rectal mucosa is mediated via a similar mechanism remains unknown. However, it should be noted that TPA mimics basic fibroblast growth factor in the inhibition of SMase-mediated apoptosis in irradiated bovine endothelial cells (63) .
The dual action of TPA, which serves as a radiation sensitizer of LNCaP cells while concomitantly protecting the rectal mucosa against radiation-induced damage, provides a potential for clinical use. Clinical data indicate that the failure of XRT to control prostate tumors results, in many patients, from an inability to deliver sufficient radiation doses to overcome tumor clonal radiation resistance. This inability results in part from the need to include parts of the adjacent and overlapping rectum in the treatment fields to avoid the risk of missing portions of the tumor tissue (4 , 64) . The rectum is highly sensitive to radiation, thus restricting the maximal doses to the tumor target to suboptimal levels (4 , 64) . The dual action of TPA on LNCaP and rectal tissue radiosensitivity might provide at least a partial solution to this problem. However, TPA has been shown to act as a tumor promoter on the skin of mice previously initiated with aromatic hydrocarbons, although topical application of TPA alone had no tumorigenic effect (65, 66, 67) . i.v. TPA therapy, at dose levels similar to those used in our study, has recently been used safely in patients with myelocytic leukemia and other malignancies (68 , 69) . However, the long-term effects of such therapy are unknown. The search for non-tumor-promoting biological response modifiers of the apoptotic response to radiation that retain the dual effects of TPA on prostatic versus rectal tissues represents a challenge with an important potential for clinical application in prostate cancer therapy.
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FOOTNOTES |
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1 This work was supported by CaPCURE Foundation award to A. H. F. and a grant to R. N. K. and Z. F. from the PepsiCo Foundation. 
2 To whom requests for reprints should be addressed, at the Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: (212) 639-5868, Fax: (212) 794-3188.
3 The abbreviations used are: SMase, sphingomyelinase; XRT, radiation therapy; TPA, 12-O-tetradecanoylphorbol acetate; FB1, fumonisin B1; DG, diacylglycerol; PSA, prostate-specific antigen; Vmax, maximal velocity; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; PKC, protein kinase C; CPT, carnitine palmitoyltransferase; ATM, ataxia telangiectasia.
Received 1/25/99. Accepted 8/18/99.
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 A. M. Gonzalez-Guerrico and M. G. Kazanietz Phorbol Ester-induced Apoptosis in Prostate Cancer Cells via Autocrine Activation of the Extrinsic Apoptotic Cascade: A KEY ROLE FOR PROTEIN KINASE C{delta} J. Biol. Chem., November 25, 2005; 280(47): 38982 - 38991. [Abstract] [Full Text] [PDF]
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 A. Kaliski, L. Maggiorella, K. A. Cengel, D. Mathe, V. Rouffiac, P. Opolon, N. Lassau, J. Bourhis, and E. Deutsch Angiogenesis and tumor growth inhibition by a matrix metalloproteinase inhibitor targeting radiation-induced invasion Mol. Cancer Ther., November 1, 2005; 4(11): 1717 - 1728. [Abstract] [Full Text] [PDF]
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 G. E. Avila, X. Zheng, X. X. Cui, A. D. Ryan, A. Hansson, J. Suh, A. B. Rabson, R. L. Chang, W. J. Shih, Y. Lin, et al. Inhibitory Effects of 12-O-Tetradecanoylphorbol-13-acetate Alone or in Combination with All-trans Retinoic Acid on the Growth of Cultured Human Pancreas Cancer Cells and Pancreas Tumor Xenografts in Immunodeficient Mice J. Pharmacol. Exp. Ther., October 1, 2005; 315(1): 170 - 187. [Abstract] [Full Text] [PDF]
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J.-P. Truman, N. Gueven, M. Lavin, S. Leibel, R. Kolesnick, Z. Fuks, and A. Haimovitz-Friedman Down-regulation of ATM Protein Sensitizes Human Prostate Cancer Cells to Radiation-induced Apoptosis J. Biol. Chem., June 17, 2005; 280(24): 23262 - 23272. [Abstract] [Full Text] [PDF]
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 T. L. Wadsworth, J. M. Carroll, R. A. Mallinson, C. T. Roberts Jr., and C. E. Roselli Saw Palmetto Extract Suppresses Insulin-Like Growth Factor-I Signaling and Induces Stress-Activated Protein Kinase/c-Jun N-Terminal Kinase Phosphorylation in Human Prostate Epithelial Cells Endocrinology, July 1, 2004; 145(7): 3205 - 3214. [Abstract] [Full Text] [PDF]
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X. Zheng, R. L. Chang, X.-X. Cui, G. E. Avila, S. Lee, Y. P. Lu, Y. R. Lou, W. J. Shih, Y. Lin, K. Reuhl, et al. Inhibitory Effect of 12-O-Tetradecanoylphorbol-13-acetate Alone or in Combination with All-trans-Retinoic Acid on the Growth of LNCaP Prostate Tumors in Immunodeficient Mice Cancer Res., March 1, 2004; 64(5): 1811 - 1820. [Abstract] [Full Text] [PDF]
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S. Altuwaijri, H.-K. Lin, K.-H. Chuang, W.-J. Lin, S. Yeh, L. A. Hanchett, M. M. Rahman, H.-Y. Kang, M.-Y. Tsai, Y. Zhang, et al. Interruption of Nuclear Factor {kappa}B Signaling by the Androgen Receptor Facilitates 12-O-Tetradecanoylphorbolacetate-Induced Apoptosis in Androgen-sensitive Prostate Cancer LNCaP Cells Cancer Res., November 1, 2003; 63(21): 7106 - 7112. [Abstract] [Full Text] [PDF]
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S. L. Scott, J. D. Earle, and P. H. Gumerlock Functional p53 Increases Prostate Cancer Cell Survival After Exposure to Fractionated Doses of Ionizing Radiation Cancer Res., November 1, 2003; 63(21): 7190 - 7196. [Abstract] [Full Text] [PDF]
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A. Erdreich-Epstein, L. B. Tran, N. N. Bowman, H. Wang, M. C. Cabot, D. L. Durden, J. Vlckova, C. P. Reynolds, M. F. Stins, S. Groshen, et al. Ceramide Signaling in Fenretinide-induced Endothelial Cell Apoptosis J. Biol. Chem., December 13, 2002; 277(51): 49531 - 49537. [Abstract] [Full Text] [PDF]
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J. K. Pru, I. R. Hendry, J. S. Davis, and B. R. Rueda Soluble Fas Ligand Activates the Sphingomyelin Pathway and Induces Apoptosis in Luteal Steroidogenic Cells Independently of Stress-Activated p38MAPK Endocrinology, November 1, 2002; 143(11): 4350 - 4357. [Abstract] [Full Text] [PDF]
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L. J. Siskind, R. N. Kolesnick, and M. Colombini Ceramide Channels Increase the Permeability of the Mitochondrial Outer Membrane to Small Proteins J. Biol. Chem., July 19, 2002; 277(30): 26796 - 26803. [Abstract] [Full Text] [PDF]
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Y. Chen, T. DeWeese, J. Dilley, Y. Zhang, Y. Li, N. Ramesh, J. Lee, R. Pennathur-Das, J. Radzyminski, J. Wypych, et al. CV706, a Prostate Cancer-specific Adenovirus Variant, in Combination with Radiotherapy Produces Synergistic Antitumor Efficacy without Increasing Toxicity Cancer Res., July 1, 2001; 61(14): 5453 - 5460. [Abstract] [Full Text] [PDF]
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V. E. Nava, O. Cuvillier, L. C. Edsall, K. Kimura, S. Milstien, E. P. Gelmann, and S. Spiegel Sphingosine Enhances Apoptosis of Radiation-resistant Prostate Cancer Cells Cancer Res., August 1, 2000; 60(16): 4468 - 4474. [Abstract] [Full Text]
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D. K. Perry, J. Carton, A. K. Shah, F. Meredith, D. J. Uhlinger, and Y. A. Hannun Serine Palmitoyltransferase Regulates de Novo Ceramide Generation during Etoposide-induced Apoptosis J. Biol. Chem., March 17, 2000; 275(12): 9078 - 9084. [Abstract] [Full Text] [PDF]
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