Articles in PresS. Am J Physiol Cell Physiol (August 13, 2003). 10.1152/ajpcell.00269.2003
R1
1
PARATHYROID HORMONE-RELATED PROTEIN AMELIORATES DEATH RECEPTOR-MEDIATED APOPTOSIS IN LUNG CANCER CELLS
Randolph H. Hastings,1,2,4 Flavio Araiza,1 Douglas W. Burton,1,3,5 Lu Zhang, 1 Maxwell Bedley1 and Leonard J. Deftos1,3,5
Research,1 Anesthesiology,2 and Medicine Services,3 VA San Diego Healthcare System, San Diego, CA 92161; Departments of Anesthesiology4 and Medicine,5 University of California San Diego, La Jolla, CA 92093
Abbreviated title: PTHrP and lung cancer apoptosis
Corresponding Author:
Randolph H. Hastings, M.D., Ph.D. VA Medical Center (125) 3350 La Jolla Village Dr. San Diego, CA 92161-5085 (858) 642-3437, FAX (858) 822-5009 E-mail address:
[email protected]
Copyright (c) 2003 by the American Physiological Society.
R1
2
Abstract PTHrP is expressed in more advanced, aggressive tumors, and may play an active role in cancer progression. This study investigated the effects of PTHrP on apoptosis after UV irradiation, Fas ligation, or staurosporine treatment in BEN human squamous lung carcinoma cells. Cells at 70% confluency were treated for 24 h with 100 nM PTHrP 1-34, PTHrP 38-64, PTHrP 67-86, PTHrP 107-139, or PTHrP 140-173 in media with serum, exposed 30 min to 0.9 mJ/cm2 UV-B and maintained for another 24 h. Caspase 3, 8, and 9 activities increased 5-fold. Pretreatment with PTHrP 1-34 and PTHrP 140-173 ameliorated apoptosis after UV exposure, as indicated by reduced caspase activities, increased cell protein, decreased nuclear condensation and increased clonal survival. Other peptides had no effect on measures of apoptosis. PTHrP 140-173 also reduced caspase activities after Fas ligation by activating antibody, but neither peptide had effects on caspase 3 or 9 activities after 1 µM staurosporine. These data indicate that PTHrP 134 and PTHrP 140-173 protect against death receptor-induced apoptosis in BEN lung cancer cells but are ineffective against mitochondrial pathways. PTHrP contributes to lung cancer cell survival in culture and could promote cancer progression in vivo. The mechanism for the protective effect against apoptosis remains to be determined.
Key words: caspases; cell surface receptors; growth substances
R1
3
INTRODUCTION Parathyroid hormone-related protein (PTHrP) was discovered as a hypercalcemia- inducing factor in squamous cell carcinomas (18). It is expressed in many normal and malignant tissues and has local effects related to growth, skeletal metabolism, and smooth muscle function (24). The amino terminus, PTHrP 1-34, has structural similarities to parathyroid hormone (PTH) 1-34, binds the same receptor, and mimics all of the effects of PTH including hypercalcemia in tissues bearing the receptor. Through alternate gene splicing, PTHrP is expressed in three isoforms of 139, 141 and 173 amino acids that differ in sequence beyond residue 139. PTHrP 1-173 is unique in that expression has only been observed in humans. The three proteins are pro-hormones that undergo post-translational processing to various peptides. Although more is known about the effects and mechanism of action of PTHrP 1-34, midmolecule and carboxy-terminal peptides of PTHrP are also biologically active and have independent effects. For example, PTHrP 109-141 stimulates osteoclast retraction, inhibits osteoclast tartrateresistant acid phosphatase activity, and stimulates cAMP production in osteoblasts(30) . PTHrP 39-94 and smaller included fragments stimulate cAMP production and calcium fluxes in a variety of tissues (34). PTHrP 67-86 inhibits mitogenesis and stimulates the metastatic potential of human breast carcinoma cells (23). Finally, PTHrP 140-173 alters metabolism or orthophosphate in articular chondrocytes (11). PTHrP expression is generally a poor prognostic sign in cancer. Elevated PTHrP levels have been associated with more advanced tumors in lung cancer and breast cancer (15, 35) and may portend a shorter life expectancy in breast cancer, renal cell carcinoma, and lung cancer (16, 17, 21, 28, 35). PTHrP production is a frequent feature of lung cancer cells of all histological types (3), and the degree of expression has been suggested to have implications for stage and/or survival. Hidaka et al. isolated two small cell lung cancer lines from cells obtained at different times from pleural cells of the same
R1
4
patient. Both cell lines expressed PTHrP, but the line isolated at the more advanced stage expressed three-fold greater levels of PTHrP mRNA and secreted five-fold more PTHrP into the culture medium (15). Hiraki et al. found that elevated levels of serum PTHrP measured at the time thatthe cancer first presents were associated with an increased rate of bone metastasis and decreased survival in hypercalcemic lung cancer patients (16). PTHrP might contribute to cancer progression by stimulating cancer cell growth and/or inhibiting apoptosis. PTHrP 1-34 is a growth stimulant in many malignant cell lines, including human lung cancer cells (4, 19), and it inhibits apoptosis in various cells, including growth plate chondrocytes, pancreatic
cells, LNCaP prostate carcinoma cells, MCF-7 breast
carcinoma cells, cerebellar neurons, cytotrophoblasts, and coronary endothelial cells (5, 8, 14, 26, 29). The effects of PTHrP on lung cancer cell apoptosis are unknown. The first goal of this study was to investigate the effects of PTHrP on the sensitivity of BEN human squamous bronchial lung carcinoma cells to apoptosis induced by UV-B irradiation. Ultraviolet radiation was chosen as the stimulus because it caused reproducible degrees of apoptosis in preliminary studies. UV radiation can cause apoptosis through death-receptor or mitochondrial-initiated pathways (2, 31), also known as the extrinsic and intrinsic pathways, respectively. Thus, studies of UV-induced apoptosis may elucidate mechanisms common to lung cancer cell apoptosis in a variety of settings, including DNA damage after chemotherapy or radiation. We initially measured caspase 3 activity in cells treated with peptides spanning the entire PTHrP sequence, since biologic activity has been ascribed to multiple portions of the molecule (11, 13, 23, 24, 30, 34). Two peptides inhibited caspase 3, PTHrP 1-34 and PTHrP 140-173, and were evaluated further for effects on caspase 8 activity, caspase 9 activity, and nuclear fragmentation/condensation. Our second goal was to determine whether these peptides had inhibitory actions on extrinsic or intrinsic apoptosis pathways. Caspase activities were assayed after Fas ligation or staurosporine treatment, stimuli that initiate solely death receptor and
R1
5
mitochondrial pathways, respectively. Finally, we examined the effects of PTHrP 1-34 and PTHrP 140173 on clonal survival of BEN cells after UV exposure.
R1
6
METHODS General Protocols Cell culture. BEN cells (generously provided by TJ Martin) were maintained in RPMI 1640 media supplemented with 10% fetal calf serum and 2 mM glutamine and incubated at 37° C in a humidified incubator with 5% CO2 and 95% air. Cells were plated in 100 mm dishes for experiments involving protein measurements or caspase assays and in NUNC Slide Tek wells for nuclear condensation experiments. Treatments. Cells were treated with 100 nM PTHrP 1-34, PTHrP 38-64, PTHrP 67-86, PTHrP 107-138, or PTHrP 140-173 (Bachem, Torrance, CA) for 24 h before apoptotic stimulus. Peptides were dissolved in growth media. Apoptotic stimuli. Cells were exposed to apoptotic stimuli at 60-70% confluency. Three different treatments were employed: UV-B irradiation, ligation of Fas with 15 µg/ml Jo-2 activating Fas antibody (Pharmingen, San Diego, CA) for 7.5 h, or incubation with 1 µM staurosporine for 48 h. Control cells for the staurosporine experiments received an equal volume of the DMSO vehicle (1:1000). UV-B radiation was administered in a sterile hood at room temperature by a shortwave UV transilluminator (Fotodyne, Inc., New Berlin, WI) placed over the cells for 30 min. The lid of the cluster well plate was left in place. The cells were then allowed to recover for 24 hours at 37oC in a humidified incubator before further processing for apoptotic markers. Total UV dose was 0.9 J/cm2 measured at 310 nm by a UV detection J-meter (UVP, Upland, CA). The probe was placed at an equivalent distance from the UV source and under the cluster plate top to mimic the conditions under which the cells were exposed. Fluorescent microscopy. Fluorescent cell imaging was performed with an Olympus BX60 upright microscope with fluorescent attachments (Olympus America, Melville, NY). Light from a 100-watt
R1
7
mercury lamp was passed through an Omega UV-2E/C filter set for Hoechst 33342 fluorescence or an XF-100-2 filter for Alexa-488 fluorescence. Immunofluorescence. Cells grown on Slide Tek wells were washed with PBS, fixed with 2% formaldehyde/0.25% glutaraldehyde, and permeabilized with 0.2% Triton X-100 in PBS for 10 min. Non-specific protein binding was blocked in 20% FBS, 0.2%% gelatin, 0.01% azide in PBS. Cells were incubated overnight at 4°C with 20 µg IgM/ml CH11, a primary antibody to Fas (Upstate Cell Signaling, Waltham, MA). The next day cells were incubated with a biotinylated anti-mouse IgM followed by strepatvidin-Alexa-488 conjugate, washed in PBS, mounted with Prolong Antifade media (Molecular Probes, Eugene, OR) and examined. Cell survival assays. A clonal survival assay w as used to determine the ultimate effect of PTHrP peptides on cell outcome after UV irradiation. Irradiation was performed as described above, except that the dose was limited to 60 mJ/cm2 UV-B delivered over 2 min. After exposure, cells were trypsinized, counted, and stained for viability by trypan blue exclusion. Cells were then replated in fresh media with 10% FBS at 1000 viable cells/well in 6 well cluster plates. Control cells that were not exposed to UV radiation were also trypsinized and replated at 200 cells/well. PTHrP peptides were present in the media only during the initial 24 h pretreatment period. In other words, adherence and growth after exposure to UV occurred without exogenous PTHrP peptides. After 12 d, plates were washed and stained with Giemsa stain (Searle Diagnostics, Bucks, UK). Two separate observers, unaware of the experimental group, counted colonies of greater than or equal to 50 cells. The two results generally agreed within 5% and were averaged for data analysis. Specific Protocols Effect of PTHrP peptides on UV-induced apoptosis. Caspase 3 activities were compared among BEN cells treated with 100 nM PTHrP 1-34, PTHrP 38-64, PTHrP 67-86, PTHrP 107-138, and PTHrP 140-
R1
8
173 with and without UV-irradiation. The subsequent studies focused on PTHrP 1-34 and PTHrP 140173 because these peptides reduced caspase 3 activity in the initial studies, while the other peptides did not. Caspase 3 was used as the initial marker for apoptosis because it is the downstream executioner caspase that catalyzes the cleavage of many key enzymes and structural proteins during apoptosis, such as poly (ADP-ribose) polymerase, protein kinase C , and lamin A (7). Caspase 8 activity, caspase 9 activity, loss of cell mass, and nuclear condensation were quantified as independent measures of cell death and apoptosis. Effect of PTHrP 1-34 peptides on death receptor- or mitochondria- initiated apoptosis. Caspase 3, caspase 8, and caspase 9 activities were investigated after treating BEN cells with staurosporine to activate the mitochondrial pathway or with activating Fas antibody to initiate the death receptor pathway. Death receptor activation after UV. BEN cells were stained for Fas examined by fluorescent microscopy to investigate whether UV caused clustering of Fas, an event that indicates activation. Cell survival after UV. Clonal survival assays were performed to determine whether the protective effects of PTHrP 1-34 and PTHrP 140-173 against apoptosis translated into increased survival. Measurements Caspase assays. Adherent cells were washed once in PBS, scraped in 500 µL caspase lysis buffer [50mM PIPES/KOH, 2 mM EDTA, 0.1% (w/v) CHAPS, 1mM dithiothreitol (DTT), 1 µM leupeptin and pepstatin A], pooled with the non-adherent cells from the same wells and lysed through sonication (Branson Sonifier, Danbury, CT). Lysates were kept at -20° C until time of assay. Assays were performed in 96 well plates with protein from cell lysates. The mass of cell protein for each sample was
R1
9
adjusted to 20 µg, measured by the Pierce BCA protein assay. Substrates were acetyl-aspartyl -glutamyl -valyl-aspartyl (DEVD)-7-amino-4-methyl coumarin (AMC) for caspase 3, acetyl-isoleucyl -glutamyl threonyl-aspartyl (IETD)-AMC for caspase 8, and acetyl-leucyl -glutamyl -histidyl-aspartyl (LEHD)AMC for caspase 9 (Alexis Biochemicals, Carlsbad, CA). Substrates were diluted to 0.1 mM in caspase assay buffer [50mM HEPES, 100mM NaCl, 0.1% (w/v) CHAPS, 5 mM DTT]. Assays were initiated by adding the substrate to the sample well at 4°C and then heating the plates to 37°C. Fluorescence of AMC cleaved from the substrate was detected on a 96-well multilabel reader (Perkin Elmer, Boston, MA) at excitation and emission wavelengths of 355 nm and 460 nm, respectively, with a 0.5 sec capture window. Fluorescent readings were taken at 30 min intervals, exported to Microsoft Excel, and converted to relative fluorescent units (RFU's) by subtracting the background fluorescence of the reagent blank for each plate in order to compensate for plate-to-plate variability. Activities were calculated as the slope of the plot of RFU versus time over the first 1-2 h, the linear portion of the curve. Nuclear condensation assessment. Nuclear condensation was quantified as described previously (13). Adherent cells in Slide-Tek wells were washed with PBS and fixed with 100% methanol. Nuclei were stained for 15 min with 4 mM Hoescht 33342 dye and imaged with a SPOT 2e Digital Camera (Diagnostic Instruments Incorporated, Sterling Heights, Michigan, USA) at 200x magnification. Images were saved as 12-bit color TIFF files and converted to 8-bit grayscale for image analysis. Nuclear area was quantitated using NIH Image 1.62. BEN cells that were not exposed to UV nor treated with PTHrP were used to establish a lower threshold for the area of nuclei in non-apoptotic cells. Condensed nuclei were defined as those nuclei whose area was less than or equal to the fifth percentile for nuclear areas of normal cells. At least 200 cells were examined per experimental condition and cell isolation and at least 300 cells were evaluated per control group in establishing the cutoff area.
R1
10
Data Analysis Nuclear condensation, caspase activities, cell protein, and cell survival were compared among groups by analysis of variance (ANOVA). Dunnett’s test was used for post hoc pairwise comparisons of individual experimental vs. control values. Significance was accepted when the probability of a type II error was less than 0.05.
R1
11
RESULTS
Effect of PTHrP peptides on UV - induced apoptosis. Effect on caspase activities. On average, UV exposure caused 5 to 6-fold increases in caspase 3 activity compared to non-irradiated cells (Figure 1). Pretreatment with PTHrP 1-34 or PTHrP 140-173 slowed the rate of fluorescence release in irradiated cells and attenuated the increase in activity due to UV by 20-30%. Other PTHrP peptides did not significantly affect caspase 3 activity and none of the peptides had effects in non-irradiated cells. The effects of PTHrP 1-34 and PTHrP 140-173 on caspase 3 activity after UV exposure were dose-dependent (Figure 2). We also investigated effects of PTHrP 1-34 and PTHrP 140-173 pre-treatment on activities of caspase 8 and caspase 9, upstream enzymes in the death receptor and mitochondria-dependent apoptosis cascades, respectively. UV irradiation increased caspase 8 activity by 7-fold and caspase 9 activity by 3 to 4-fold, (Figure 3). PTHrP 1-34 and PTHrP 140-173 reduced the activities of these caspases after UV irradiation to a similar extent compared to their effects on caspase 3. Effect on loss of cell mass. UV exposure caused a 50% decline in cell protein compared to non-irradiated cells (Figure 4). The loss of cell protein was significantly less after PTHrP 1-34 treatment or PTHrP 140-173 treatment. Protein in irradiated cells was increased 15% by pretreatment with either peptide (P < 0.05, N = 6). The effects of PTHrP 1-34 and PTHrP 140-173 were not significantly different. The peptides had no effects on total cell protein in non-irradiated cells. Effects on nuclear condensation. The percentage of cells with condensed or fragmented nuclei increased from 5.0 ± 1% in controls to 11.3 ± 1.8% in UV-irradiated cells (Figure 5). PTHrP 1-34 and PTHrP 140-173 treatment reduced nuclear condensation to 5.6 ± 1.3 and 5.6 ± 0.9% of cells, respectively (P < 0.05), not significantly different from levels in non-irradiated cells. Figure 6
R1
12
demonstrates the nuclear morphology of control cells and cells exposed to UV with and without peptide treatment. Apoptotic bodies and cells with condensed nuclei were present among irradiated cells, but to a lesser degree after PTHrP peptide treatment.
Effect of PTHrP peptides on death receptor- or mitochondria- initiated apoptosis Fas ligation. Activating Fas antibody increased caspase 3 and caspase 8 activities in BEN cells by about 70% (Figure 7). Caspase 9 activities were increased 90 ± 1.5% (P < 0.01, not included in figure). PTHrP 140-173 pretreatment reduced caspase activities in Fas-treated cells by half, to levels similar to those in control cells without Fas activation. Staurosporine treatment. Treating BEN cells with staurosporine for 48 h increased caspase 3 activity by 75-100% (P < 0.01) but did not augment caspase 8 activity compared to the low activity in untreated cells (data not shown). PTHrP 1-34 and PTHrP 140-173 had no effect on caspase 3 activity or caspase 9 activity after staurosporine.
Death receptor activation after UV Irradiation BEN cells underwent immunofluorescent staining to investigate whether UV exposure caused clustering of Fas receptor. Irradiated cells demonstrated dense staining that was primarily localized to the plasma membrane (data not shown). Non-irradiated cells had very faint, diffuse staining.
Effects of PTHrP 1-34 and PTHrP 140-173 on cell survival after UV Irradiation Exposure to UV for 2 minutes resulted in a 95% decrease in colony formation after 12 d (Figure 8). Pretreatment with PTHrP 1-34 and PTHrP 140-173 increased colony survival nearly two- and four-
R1
13
fold, respectively, compared to untreated irradiated cells (*P < 0.05, **P < 0.01 vs. no treatment group & P < 0.05 vs. PTHrP 1-34 group, N=3).
R1
14
DISCUSSION Our first goal in this study was to investigate the effects of PTHrP peptides on the sensitivity of lung cancer cells to apoptosis. We found that PTHrP 1-34 and PTHrP 140-173 protected BEN cells against apoptosis induced by UV-B radiation and Fas ligation. The protection was demonstrated by decreases in activities of caspase 3, caspase 8 and caspase 9 (Figures 1-3, 7), reduced nuclear condensation/fragmentation (Figures 5, 6), increases in cell mass, and improved cell survival (Figure 8) after UV exposure. Our second goal was to begin to explore the mechanisms for these anti-apoptotic effects. Specifically, we wanted to determine the PTHrP effects on extrinsic and intrinsic apoptosis pathways. Since the PTHrP peptides decrease caspase 8 activity and caspase 9 activity, they could be acting on a death receptor apoptosis pathway alone or also on mitochondrial-initiated apoptosis. Caspase 8 is activated after recruitment to trimerized death receptors through binding to adapter proteins, such as FADD (Fas-associated death domain), and serves as the initiator caspase for the extrinsic pathway (25). Caspase 9 is the upstream caspase in the intrinsic pathway and is activated by binding in complex of apoptotic protease activating factor 1 (APAF1), cytochrome C released from mitochondria, and dATP (12). We explored this issue by testing the effects of the peptides on caspase activities after stimuli that exclusively work through one or the other pathway. Staurosporine affects apoptosis predominantly through mitochondria, as demonstrated in BEN cells by lack of stimulation of caspase 8. Neither peptide altered caspase 3 or caspase 9 activities after staurosporine treatment, suggesting that they did not act on intrinsic pathways. We tested extrinsic pathways by ligating Fas with an activating antibody, Jo-2. In contrast to the lack of efficacy after staurosporine, PTHrP 140-173 decreased caspase 8 activity, caspase 9 activity and caspase 3 activity after Fas ligation (Figure 7). Thus, PTHrP 140-173
R1
15
appears to protect BEN cells against apoptosis mediated by the death receptor pathway. Since caspase 8 levels are reduced, the effect appears to be exerted at or upstream of the level of caspase 8. It is not clear why PTHrP 1-34 differed from PTHrP 140-173 in effects against Fasmediated apoptosis. Since both peptides protect against apoptosis after UV irradiation, we expected similar effects versus Fas antibody. PTHrP 140-173 could be more potent than PTHrP 1-34. It exerted greater effects than PTHrP 1-34 at the same dose in the clonal survival assay (Figure 8). Alternatively, the lack of effect versus Fas may reflect differences specific to the stimulus. We found no evidence that PTHrP peptides protect BEN cells against apoptosis mediated solely by mitochondrial mechanisms, but have not completely ruled out this possibility. UV-B radiation can activate extrinsic and intrinsic apoptosis pathways (2, 31). Our data are consistent with stimulation of the extrinsic pathway in BEN cells. The mechanism may be through aggregation of Fas, as we demonstrated with immunofluorescent staining. Clustering of Fas after UV exposure is associated with activation of the death receptor pathway in various cell types (2). PTHrP has been shown to protect against the intrinsic apoptosis pathway in other cells and tissues. In growth plate chondrocytes, PTHrP increases expression of Bcl-2, delaying their maturation and apoptotic cell death (1). MCF-7 breast cancer cells overexpressing wild type PTHrP were more resistant to apoptosis induced by serum starvation than cells transfected with vector control and expressed higher Bcl-2 to Bax and Bcl-XL to Bax ratios (32). Pancreatic islet cells from transgenic mice overexpressing PTHrP under the control of the rat insulin II promoter express three to four-fold higher levels of Bcl-2 mRNA than islet cells from wild type mice (5). Finally, increases in PTHrP expression in coronary endothelial cells improve resistance to apoptosis associated with increases in Bcl-2 levels (29). To our knowledge, these data are the first demonstration that PTHrP can regulate a death receptor
R1
16
apoptosis pathways. These effects were elicited by exogenous peptides and are paracrine in nature. In many cases, PTHrP elicits effects on apoptosis after localization to the nucleus (14, 29, 32), actions that are termed “intracrine” effects. We have not investigated the intracrine effects of PTHrP in lung cancer cells. To our knowledge, regulatory effects of PTHrP 140-173 on apoptosis have not been described previously. This region of the PTHrP molecule has been shown to have biologic activity in only a few other studies. In cultured articular chondrocytes PTHrP 140-173 decreases extracellular orthophosphate levels (11). A receptor for PTHrP 140-173 has not been identified yet. The receptor for PTHrP 1-34, the combined PTH/PTHrP receptor, is a G-protein coupled receptor and can signal through increases in PKA- or PKC-dependent mechanisms (27). The dose response experiments (Figure 2) are evidence that the effects of the peptides on caspase 3 activity are receptor-mediated, although other mechanisms have not been excluded. Since PTHrP 140-173 has similar effects on BEN cell apoptosis as PTHrP 1-34, PTHrP 140-173 might act through a G-protein-coupled receptor as well. In pre-confluent mesenchymal cells, anti-apoptotic effects of PTHrP 1-34 are exerted through increases in cAMP (6). In contrast, PTHrP 1-34 is pro-apoptotic in HEK293 embryonic human kidney cells by acting through a Gqmediated phospholipase C/Ca2+ signaling pathway (33). In alveolar epithelial cells, protective effects against apoptosis are apparently mediated through a PKC-dependent pathway (13). Further work is necessary to establish the signaling mechanisms for PTHrP 1-34 and PTHrP 140-173 in lung cancer cells. Inhibitory effects on apoptosis would be clinically tenuous if PTHrP simply prolonged the interval between stimulus and lung cancer cell demise or changed the mode of death from apoptosis to necrosis. However, clonal survival studies showed that PTHrP peptides protect the cells from death. The data reflect survival, not increased adherence or proliferation, because the cells were exposed to
R1
17
peptides only through the period of UV exposure. The overall protection was small, since 87-94% of the cells died after PTHrP treatment, compared to 97% with no treatment. This result is not inconsistent with the effects on caspase activity or nuclear condensation, because the survival outcome would not be expected a priori to change in proportion to changes in these intermediate measures. The high death rate could also represent necrosis or caspase-independent apoptosis occurring along with caspasemediated apoptosis. This being said, the peptides still increased survival several fold compared to untreated cells. A small increase in survival could make a large difference in the progression of an exponentially expanding tumor. Thus, autocrine protection of PTHrP against apoptosis could contribute to lung cancer survival and increase resistance to chemotherapeutic agents that act through inducing apoptosis. Further work is indicated to investigate how the anti-apoptotic effects of PTHrP 1-34 and PTHrP 140-173 interact with the pathophysiology of lung cancer. PTHrP contributes to tumor progression in some animal models of cancer. For example, overexpression of PTHrP in MatLyLu rat prostate carcinoma cells accelerated growth of tumors in rat hind limbs. PTHrP was found to protect prostate carcinoma cells from apoptosis after phorbol myristate acetate (9). Effects on Fas-induced apoptosis are potentially relevant to lung cancer. Expression of Fas and Fas ligand have been reported to be positive prognostic factors for patients with lung cancer (20), and cis-platinum, topotecan and gemcitabine all induce apoptosis in lung cancer cell lines through death receptor-dependent mechanisms (10). In summary, we have demonstrated UV irradiation causes apoptosis of the BEN lung cancer cell line as indicated by increased activities of caspase 3, caspase 8 and caspase 9, increased nuclear condensation, and decreased cell survival. Pre-treatment with PTHrP 1-34 or PTHrP 140-173 attenuate the apoptotic effects of the radiation and increase cell survival in clonal survival assays. PTHrP 140-173 also attenuates increases in activities of all three caspases after Fas ligation, but neither peptide has
R1
18
effects on caspase 3 or caspase 9 activities after staurosporine-induced apoptosis. These data indicate that the peptides protect BEN cells from apoptosis mediated by death receptor activation at or proximal to the action of caspase 8. UV-induced apoptosis in BEN cells apparently occurs through an extrinsic apoptosis pathway, possibly due to radiation-mediated aggregation of Fas receptor. The mechanism for the effects of the amino-terminal and carboxy-molecule PTHrP peptides on death receptor-mediated apoptosis in lung cancer cells requires further investigation. The clinical significance of these findings is under investigation.
R1
19
Acknowledgements This work was supported by VA Merit Review grants (Hastings and Deftos), NIH grants ES09227 (Hastings) and DK60588 (Deftos), the California Tobacco-Related Disease Research Program Grant Number 10RT-0161, and a grant from the Flight Attendants Medical Research Institute.
R1
20
REFERENCES 1.
Amling M, Neff L, Tanaka S, Inoue D, Kuida K, Weir E, Philbrick WM, Broadus AE, and Baron R. Bcl-2 lies downstream of parathyroid hormone-related peptide in a signaling pathway that regulates chondrocyte maturation during skeletal development. J Cell Biol 136: 205-213, 1997.
2.
Aragane Y, Kulms D, Metze D, Wilkes G, Poppelmann B, Luger TA, and Schwarz T. Ultraviolet light induces apoptosis via direct activation of CD95 (Fas/APO-1) independently of its ligand CD95L. J Cell Biol 140: 171-182, 1998.
3.
Brandt DW, Burton DW, Gazdar AF, Oie HE, and Deftos LJ. All major lung cancer cell types produce parathyroid hormone-like protein: Heterogeneity assessed by high performance liquid chromatography. Endocrinology 129: 2466-2470, 1991.
4.
Burton PBJ and Knight DE. Parathyroid hormone-related peptide can regulate the growth of human lung cancer cells, and may form part of an autocrine TGF- loop. FEBS Letters 305: 228-232, 1992.
5.
Cebrian A, A. Garcia-Ocana, Takane KK, Sipula D, Stewart AF, and Vasavada RC. Overexpression of parathyorid hormone-related protein inhibits pancreatic -cell death in vivo and in vitro. Diabetes 51: 3003-3013, 2002.
6.
Chen HL, Demiralp B, Schneider A, Koh AJ, Silve C, Wang CY, and McCauley LK. Parathyroid hormone and parathyroid hormone-related protein exert both pro- and antiapoptotic effects in mesenchymal cells. J Biol Chem 277: 19374-19381, 2002.
7.
Cohen GM. Caspases: the executioners of apoptosis. Biochem J 326: 1-6, 1997.
8.
Crocker I, Kaur M, Hosking DJ, and Baker PN. Rescue of trophoblast apoptosis by parathyroid hormone-related protein. BJOG 109: 218-220, 2002.
R1 9.
21 Dougherty KM, Blomme EAG, Koh AJ, Henderson JE, Pienta KJ, Rosol TJ, and McCauley LK. Parathyroid hormone-related protein as a growth regulator of prostate carcinoma. Cancer Res 59: 6015-6022, 1999.
10.
Ferreira CG, Span SW, Peters GJ, Kruyt FA, and Giaccone G. Chemotherapy triggers apoptosis in a caspase-8-dependent and mitochondria controlled manner in the non-small cell lung cancer cell line NCI-H460. Cancer Res 15: 7133-7141, 2000.
11.
Goomer RS, Johnson KA, Burton DW, Amiel D, Maris TM, Gujral A, Deftos LJ, and Terkeltaub R. The tetrabasic KKKK 147-150 motif determines intracrine regulatory effects of PTHrP 1-173 on chondrocyte PPi metabolism and matrix synthesis. Endocrinology 141: 4613-4622, 2000.
12.
Green DR and Reed JC. Mitochondria and apoptosis. Science 281: 1309-1312, 1998.
13.
Hastings RH, Quintana RA, Sandoval R, Duey D, Rascon Y, Burton DW, and Deftos LJ. Pro-apoptotic effects of parathyroid hormone-related protein in type II pneumocytes. Am J Respir Cell Mol Biol June 5: Epub ahead of print, 2003.
14.
Henderson JE, Amizuka N, Warshawsky H, Biasotto D, Lanske BM, Goltzman D, and Karaplis AC. Nucleolar localization of parathyroid hormone-related peptide enhances survival of chondrocytes under conditions that promote apoptotic cell death. Molec Cell Biol 15: 4064-4075, 1995.
15.
Hidaka N, Nishimura M, and Nagao K. Establishment of two human small cell lung cancer cell lines: the evidence of accelerated production of parathyroid hormone-related protein with tumor progression. Cancer Letters 125: 149-155, 1998.
16.
Hiraki A, Ueoka H, Bessho A, Segaa Y, Takigawa N, Kiura K, Eguchi K, Yoneda T, Tanimoto M, and Harada M. Parathyroid hormone-related protein measured at the time
R1
22 of first visit is an indicator of bone metastases and survival in lung carcinoma patients with hypercalcemia. Cancer 95: 1706-1713, 2002.
17.
Kamai T, Arai K, Koga F, Abe H, Nakanishi K, Kambara T, Furuya N, Tsujii T, and Yoshida K-I. Higher expression of K-ras is associated with parathyroid hormone-related protein-induced hypercalcaemia in renal cell carcinoma. BJU International 88: 960-966, 2001.
18.
Kemp BE, Moseley JM, Rodda CP, Ebeling PR, Wettenhall RE, Stapleton D, Diefenbach-Jagger H, Ure F, Michelangeli VP, and Simmons HA. Parathyroid hormone-related protein of malignancy: active synthetic fragments. Science 238: 15681570, 1987.
19.
Kikuchi H, Shigeno C, Lee K, Ohta S, Shiomi K, Ideda T, Sone T, Dokoh S, and Konishi J. On the transforming growth factor -like activity of synthetic polypeptides comprising the amino-terminal sequence of human parathyroid hormone-related peptide. Endocrinology 128: 1229-1237, 1991.
20.
Koomagi R and Volm M. Expression of Fas (CD95/APO-1) and Fas ligand in lung cancer, its prognostic and predictive relevance. Int J Cancer 84: 239-243, 1999.
21.
Linforth R, Anderson N, Hoey R, Nolan T, Downey S, Brady G, Ashcroft L, and Bundred N. Coexpression of parathyroid hormone related protein and its receptor in early breast cancer predicts poor patient survival. Clin Cancer Res 8: 3172-3177, 2002.
22.
Luo X, Budihardjo I, Zou H, Slaughter C, and Wang X. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94: 481-490, 1998.
R1 23.
23 Luparello C, Burtis WJ, Raue F, Birch MA, and Gallagher JA. Parathyroid hormonerelated peptide and 8701-BC breast cancer cell growth and invasion in vitro: evidence for growth-inhibiting and invasion-promoting effects. Mol Cell Endocrinol 111: 225-232, 1995.
24.
Martin TJ. Parathyroid hormone-related protein. J Int Med 233: 1-4, 1993.
25.
Muzio MM, Chinnaiyan AM, Kischkel FC, K.O'Rourke, Shevchencko A, Ni J, Scaffidi C, Bretz JD, ZShang M, Gentz R, Mann M, Krammer PH, Peter ME, and Dixit VM. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death--inducing signaling complex. Cell 85: 817-827, 1996.
26.
Ono T, Inokuchi K, Ogura A, Ikawa Y, Kudo Y, and Kawashima S. Activitydependent expression of parathyroid hormone-related protein (PTHrP) in rat cerebellar granule neurons. Requirement of PTHrP for the activity-dependent survival of granule neurons. J Biol Chem 272: 14404-14411, 1997.
27.
Orloff JJ, Reddy D, DePapp AE, Yang KH, Soifer NE, and Stewart AF. Parathyroid hormone-related protein as a prohormone: posttranslational processing and receptor interactions. Endocrine Rev 15: 40-60, 1994.
28.
Pecherstorfer M, Schilling T, Blind E, Zimmer-Roth I, Baumgartner G, Ziegler R, and Raue F. Parathyroid hormone-related protein and life expectancy in hypercalcemic cancer patients. J Clin Endocr Metab 78: 1268-1270, 1994.
29.
Schorr K, Taimor G, Degenhardt H, Weber K, and Schluter K-D. Parathyroid hormone-related peptide is induced by stimulation of
1A-adrenoceptors
and improves
resistance against apoptosis in coronary endothelial cells. Molecu Pharamcol 63: 111-118, 2003.
R1 30.
24 Seitz PK, Zhang R-W, Simmons DJ, and Cooper CW. Effects of C-terminal parathyroid hormone-related peptide on osteoblasts. Mineral Electrolyte Metab 21: 180-183, 1995.
31.
Sitailo LA, Tibudan SS, and Denning MF. Activation of caspase-9 is required for UVinduced apoptosis of human keratinocytes. J Biol Chem 277: 19346-19352, 2002.
32.
Tovar Sepulveda VA, Shen X, and Falzon M. Intracrine PTHrP protects against serum starvation-induced apoptosis and regulates the cell cycle in MCF-7 breast cancer cells. Endocrinology 143: 596-606, 2002.
33.
Turner PR, Mefford S, Christakos S, and Nissenson RA. Apoptosis mediated by activation of the G protein-coupled receptor for parathyroid hormone (PTH)/PTH-related protein. Mol Endocrinol 14: 241-254, 2000.
34.
Wu TL, Vasavada RC, Yang K, Massfelder T, Ganz M, Abbas SK, Care AD, and Stewart AF. Structural and physiologic characterization of the mid-region secretory species of parathyroid hormone-related protein. J Biol Chem 271: 24371-24381, 1996.
35.
Yoshida A, Nakamura Y, Shimizu A, Harada M, Kameda Y, Nagano A, Inaba M, and Asaga T. Significance of the parathyroid hormone-related protein expression in breast carcinoma. Breast Cancer 7: 215-220, 2000.
R1
25
Figure Legends Figure 1. Effect of PTHrP peptides on caspase 3 activity after UV irradiation in BEN cells. UV caused a six-fold increase in caspase 3 activity compared to non-irradiated cells (*P < 0.01). Pretreatment with 100 nM PTHrP 1-34 or PTHrP 140-173 for 24 h before irradiation reduced caspase 3 activity approximately 25% (**P < 0.05). Other PTHrP peptides did not have significant effects and peptides had no effect on non-irradiated cells. Data are mean ± SEM for 7 separate experiments. Figure 2. Dose response for effects of PTHrP 1-34 and PTHrP 140-173 on caspase 3 activity in UV-irradiated cells. Cells were incubated for 24 h with the concentration of PTHrP 1-34 or PTHrP 140-173 indicated on the abscissa, then irradiated and allowed 24 h to recover as described for Figure 1. The amino-terminal and carboxy-terminal PTHrP peptides caused a dose-dependent reduction in caspase 3 activity after UV exposure. (*P < 0.05 vs. no PTHrP peptide treatment, N = 3-5 cell preparations per experimental group). Figure 3. Effect of PTHrP 1-34 and PTHrP 140-173 on caspase 8 and caspase 9 activities after UV irradiation in BEN cells. Caspase 8 and caspase 9 activities were measured in fluorescent assays with IETD-AMC and LEHD-AMC, respectively, as the substrates. Irradiation significantly increased caspase 8 and caspase 9 activities six-fold and three-fold, respectively, above levels in non-irradiated cells (*P < 0.01). PTHrP 1-34 and PTHrP 140-173 significantly reduced caspase activities compared to irradiated cells with no treatment (**P < 0.05 vs. untreated irradiated cells, N = 5). Figure 4. Effect of PTHrP peptides on cell protein after UV. Twenty-four h after UV exposure, adherent and non-adherent BEN cells were lysed in buffer with protease inhibitors for protein measurement by the Bradford method. Irradiation caused a 50% loss in cell protein compared to nonirradiated cells (*P < 0.05). PTHrP 1-34 and PTHrP 140-173 pre-treatment significantly reduced the loss of cell protein (**P < 0.05, N = 6).
R1
26
Figure 5. Effects of PTHrP peptides on nuclear fragmentation/condensation in BEN cells after UV irradiation. BEN cell nuclei were visualized by fluorescent microscopy after staining with Hoescht 33342. Nuclear areas were quantified by image analysis with NIH Image 1.62. UV exposure more than doubled the percentage of cells with condensed nuclei, defined as cells with nuclear areas less than the 5th percentile of non-irradiated cells. Pre-treatment with PTHrP peptides reduced nuclear condensation to control levels indicating significant protection from apoptosis. *P < 0.05 vs. nonirradiated cells, **P < 0.05 vs. irradiated untreated cells, N = 3. Figure 6. Nuclear morphology of BEN cells after UV irradiation and treatment with PTHrP peptides. The figure shows micrographs of non-irradiated (A) and irradiated (B, C, D) BEN cell nuclei stained with Hoescht 33342. The cells received no PTHrP treatment (A, B), or 24 h treatment with PTHrP 1-34 (C) or PTHrP 140-173 (D) before UV exposure. Cells with condensed and/or fragmented nuclei, indicating apoptosis, are present in all groups after UV exposure, but are decreased in cells treated with either PTHrP peptide. Arrowheads point to representative apoptotic bodies or condensed nuclei. The micrographs are representative of the data shown in Figure 5. Figure 7. Effect of PTHrP peptides on caspase activity after Fas ligation. BEN cells were treated with 15 µg/ml activating Fas antibody Jo-2 for 7 h. Caspase activities were measured by fluorescent substrate assay as described in Figure 3. Fas ligation caused significant increases in the activities of caspase 3, and caspase 8 (*P < 0.05), as well as caspase 9 (data not shown). Pretreatment with 140-173 significantly reduced activities compared to Jo-2 treated cells without PTHrP supplementation. (**P < 0.05, N = 3). Peptides had no effect on activities in cells that were not treated with antibody. Figure 8. Effect of PTHrP peptides on BEN cell clonal survival after UV. BEN cells were treated for 24 h with media plus serum alone, 100 nM PTHrP 1-34, or 100 nM PTHrP 140-173. Cells were exposed to 60 µJ/cm2 UV-B over 2 min, trypsinized and replated at 1000 cells/well in six-well cluster
R1 plates in media without PTHrP peptides. Colonies
27 50 cells were counted 12 d later. Colony counts for
untreated cells were less than 5% of counts for non-irradiated cells. PTHrP 1-34 treatment increased colony survival nearly 2-fold, while PTHrP 140-173 caused a 4-fold increase (*P < 0.01 vs. untreated irradiated cells, **P < 0.001 vs. other two groups). The data represent results from three independent experiments and 6-9 wells for each experimental group.
R1
Figure 1
28
R1
Figure 2
29
R1
Figure 3
30
R1
Figure 4
31
R1
Figure 5
32
R1
Figure 6
33
R1
Figure 7
34
R1
Figure 8
35