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Tumor Biology |
Department of Periodontics/Prevention/Geriatrics [K. M. D., A. J. K., L. K. M.], University of Michigan Comprehensive Cancer Center [K. J. P.], The University of Michigan, Ann Arbor, Michigan 48109-1078; Department of Medicine, McGill University, Montreal, Quebec, Canada H3T 1EZ [J. E. H.]; and Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio 43210 [E. A. G. B., T. J. R.]
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ABSTRACT |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Often, bone metastases grow at a more rapid rate than primary tumors or other metastatic lesions. This has been attributed to interactions between prostate cancer and bone cells (4 , 5) and likely reflects the presence of factors that either promote cell growth or inhibit cell death. However, the factors that influence the preferential spread of prostate tumor cells to the bone and the subsequent stimulation of bone formation are unknown.
PTHrP3 is an autocrine or paracrine factor that binds to receptors on osteoblasts and stimulates bone formation and resorption (6) . Originally described as the factor responsible for humoral hypercalcemia of malignancy (7 , 8) , PTHrP is also produced by many normal cell types during development and adult life (9) . PTHrP has limited homology to PTH at its NH2 terminus and can bind the same receptor as PTH with similar biological activity (6 , 10) . Suggested physiological roles for PTHrP include regulation of calcium transport, keratinocyte differentiation, smooth muscle relaxation, and cartilage development (11) . In the latter instance, complete deficiency of PTHrP in mice leads to abnormal endochondral bone formation and lethal skeletal dysplasia (12 , 13) . PTHrP is secreted in a regulated or constitutive fashion, depending on the cell type (14 , 15) . Additionally, the protein contains a functional NLS (amino acids 87–107) that can target the protein to the nucleus or the nucleolus (16) . Hence, in addition to functioning in a classical autocrine or paracrine manner, PTHrP has the capacity to act in an intracrine manner, bypassing the need for interaction with cell surface receptors.
Many features of PTHrP make it an attractive candidate for influencing prostate carcinoma growth. PTHrP is produced by normal prostate epithelial cells, from which prostate carcinoma arises, and PTHrP is found in the seminal fluid (17) . PTHrP has been immunohistochemically identified in prostate cancer tissue in patients with clinically localized disease (18) , is found in higher levels in prostate intraepithelial neoplasia than in normal prostate epithelium (19) , and is found in higher levels in prostate carcinoma than in benign prostatic hyperplasia (20) . There is also evidence that PTHrP can regulate malignant tumor growth in an autocrine manner in human renal cell carcinoma (21) , enhance breast carcinoma metastasis to bone (22, 23, 24) , and act as an autocrine growth factor for prostate cancer cells in vitro (25) . Recent evidence indicates that expression of nuclear-targeted PTHrP can protect other cell types (i.e., chondrocytes) from apoptosis (16) , bind RNA (26) , or act as a mitogen in vascular smooth muscle cells in vitro (27) ; however, the ability of PTHrP to regulate apoptosis or cell proliferation in prostate carcinoma is unknown. The purpose of this study was to determine the role of PTHrP in the regulation of prostate carcinoma cell and tumor growth.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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. PC-3 and LNCaP cells were obtained from American Type Culture Collection (Manassas, VA). PC-3 MB cells were obtained from Dr. Mark Stearns (University of Pennsylvania, Philadelphia, PA), and AT-2.1, AT-3.1, MAT-Lu, MLL, and GP9F3 cells were a gift from Dr. John Isaacs (Johns Hopkins University, Baltimore, MD). MC3T3-E1 cells were obtained from Dr. M. Kumegawa (Meikai University, Sakado, Japan) via Dr. Renny Franceschi (University of Michigan, Ann Arbor, MI) and maintained as described previously (28)
. All prostate cell lines except DU-145 were maintained in RPMI 1640 (Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 100 units/ml penicillin, 100 µg/ml streptomycin, and 250 µg/ml Gentamicin (Sigma, St. Louis, MO) in a humidified, 5% CO2, 37°C incubator. DU-145 cells were cultured in DMEM-F12 (Life Technologies, Inc.) supplemented as described above. ROS17/2.8 cells (29)
were cultured in DMEM-F12 with 5% serum.
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87-107 is a derivative of PTHrPv3 with a deletion of the coding sequence for the NLS (16)
. These plasmids were a kind gift from Dr. Andrew Karaplis (McGill University, Montreal, Quebec). The pcDNA3.1+ control vector was obtained from Invitrogen.
Cell Transfection.
MLL cells were plated at 10,000 cells/cm2 in 6-well plates and grown to 50–60% confluence. Cells were transfected with 2 µg of control plasmid pcDNA3.1+ (pcDNA-MLL) or PTHrPv3 (PTHrP-MLL) using Lipofectin (Life Technologies, Inc.). The cells were placed under G418 selection (700 µg/ml; Life Technologies, Inc.) for 3 weeks to obtain stable lines.
LNCaP cells were plated at 14,000 cells/cm2 in 60-mm dishes and grown to 70–80% confluence. The cells were transfected with 10 µg of pcDNA3.1+ (pcDNA-LNCaP), PTHrPv3 (PTHrP-LNCaP), or PTHrP87-107 (-LNCaP) using Lipofectin and selected in 500 µg/ml G418. The cells were kept on fibronectin-coated plates (5 µg/ml; Sigma) during selection and subsequent expansion.
Northern Blot Analysis.
RNA was isolated from cells in culture by the guanidinium isothiocyanate method (30)
, and Northern blot analysis was performed as described previously (31)
. Tumor RNA was isolated from tissue flash-frozen in liquid nitrogen. The tissue was homogenized briefly in cold guanidinium isothiocyanate using the Tissue Tearor homogenizer (Dremel, Racine, WI) at setting 4. After homogenization, tumor RNA isolation was carried out as described for tissue culture cells. Total RNA (20 µg) was electrophoresed on 1.2% agarose-formaldehyde gels and transferred to nylon membranes (Duralon U.V.; Stratagene Inc., La Jolla, CA), and UV cross-linked. [32P]dCTP-labeled probes for rat PTHrP (32)
, PTH/PTHrP receptor (R15B; Ref. 33
), or human osteopontin (34)
were made using the Redi-prime kit (Amersham, Arlington Heights, IL). After overnight hybridization followed by washes of increasing stringency, the nylon membranes were exposed to Kodak X-OMAT or Biomax film (Rochester, NY) at -70°C. Quantitation of radioactivity (cpm) was determined using an Instant Imager (Packard Instrument Co., San Diego, CA). Loading was standardized by hybridizing with an 18S rRNA probe (35)
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PTHrP Immunoradiometric Assay.
The INCSTAR intact PTHrP immunoradiometric assay (DiaSorin, Stillwater, MN) was used for the determination of biologically active PTHrP (1–84) in cell culture supernatants and rat plasma. Initially, cell culture supernatants were collected either on ice in borosilicate glass tubes with no additives or on ice in Nichols collection tubes containing protease inhibitors. Because there was no difference in the PTHrP levels detected between the two methods, supernatants were subsequently collected on ice with no additives and stored at -20°C. Rat blood was collected in EDTA (4 mM final concentration) on ice and centrifuged (9000 rpm, 15 min, 4°C). Plasma was transferred to borosilicate glass tubes and frozen. The immunoradiometric assay uses a first antibody specific for PTHrP (1–40) and a second antibody specific for PTHrP (57–80) that is labeled with 125I. The detection limit of the assay is 0.2 pmol/liter. The concentration of PTHrP in experimental samples was determined using human recombinant PTHrP (1–84) standards and calculated using Graph Pad Prism software (Graph Pad Software, Inc., San Diego, CA). All samples were assayed in duplicate.
Proliferation and Apoptosis Assays.
PC-3 and MLL cells were plated at 20,000 cells/cm2 and allowed to attach overnight. Cell culture supernatants were collected every 24 h for 5 days (MLL) or 8 days (PC-3) for PTHrP immunoradiometric assay. DNA levels were determined by collecting the cell layer in 10 mM Tris (pH 7.4), 0.5 M HCl, and 0.1% SDS and measuring Hoechst dye 33258 binding using a Hoefer TKO fluorometer as described previously (36)
. Calculations were done using a standard curve and Graph Pad Instat software (Graph Pad Software, Inc.).
The in vitro proliferation of transfected MLL and LNCaP cell lines was monitored using the MTT colorimetric assay as described previously (37 , 38) with slight modifications on cells seeded in 96-well plates [5000 (MLL) and 6000 (LNCaP) cells/cm2 in triplicate for each day].
Apoptosis was assessed in the three stably transfected LNCaP cell lines described above using the APO-BRDU Flow Cytometry Kit (Phoenix Flow Systems, San Diego, CA). The cells were grown to 60% confluence and then treated for 24 h with 10 nM PMA (Sigma) or vehicle (0.1% ethanol). After treatment, floating and adherent cells were collected, fixed, and processed for flow cytometry following the manufacturer’s protocols. The assay was repeated four times with similar results.
PTHrP Immunohistochemistry.
PTHrP immunohistochemistry was performed as described previously (39, 40, 41)
. Metastatic prostate carcinoma specimens were collected from patients by biopsy or at autopsy, fixed in Bouin’s fixative, routinely processed, and paraffin-embedded. Tissue sections (5 µm) were deparaffinized in xylene and descending concentrations of ethanol and washed in water. Endogenous peroxidase was blocked with 1% H2O2 for 15 min. The slides were washed in PBS (pH 7.4) for 20 min, and nonspecific binding was inhibited by incubating the slides in dilute horse serum for 15 min. The slides were incubated with immunopurified chicken antihuman PTHrP antibody (42)
for 12 h at 4°C and then washed in PBS for 10 min, and the secondary antibody, biotinylated rabbit antichicken IgG (Zymed Laboratories, Inc., San Francisco, CA), was applied for 45 min at room temperature. The slides were washed in PBS for 10 min and then incubated for 15 min with avidin-biotin-horseradish peroxidase complex (Immunopure Ultrasensitive ABC Staining Kit; Pierce Chemical, Rockford, IL), washed for 10 min in PBS, rinsed in 0.5% Triton X-100 and PBS for 30 s, and stained with 0.05% diaminobenzidine tetrachloride (Immunopure DAB; Pierce Chemical) for 7 min. The slides were washed in water for 5 min, counterstained with hematoxylin, dehydrated with ascending concentrations of ethanol and xylene, and coverslipped. Slides stained with no primary antibody or with control egg yolk IgG from nonimmunized chickens were used as negative controls.
Receptor Binding Assay.
PTH/PTHrP receptor binding assays were performed as described previously (38)
on cells grown to confluence in 24-well plates. Each well was incubated with 25,000 cpm of mono-iodinated 125I-labeled [Tyr36] PTHrP (1–36) in addition to varying concentrations of nonradioactive PTHrP (1–36). The cells were incubated for 2 h at 4°C with gentle shaking. The unbound peptides were washed off the cell monolayer twice with HBSS, the cells were lysed with 0.5 M NaOH for 30 min, and the resultant suspension was counted in a scintillation counter.
RT-PCR.
RT-PCR of the PTH/PTHrP receptor was performed using the Gene Amp PCR kit (Roche Molecular Systems, Branchburg, NJ). Total RNA from ROS-17/2.8 or LNCaP cells was isolated using the guanidinium isothiocyanate method (see above). The RT-PCR reaction was performed as described in the manufacturer’s protocols with 1 µg of total RNA, using random hexamers for priming. A negative control reaction, without the addition of reverse transcriptase, was performed for each RNA sample. The PCR reaction was carried out using one-half of the reverse transcriptase product. The primers used for amplification of the receptor were 5'-ACCAATGAGACTCGTGAACGG-3' and 5'-AAGGACAGGAACAGGTGCATG-3', with an amplification product of 167 bp. Annealing was done at 60°C, and the reaction was carried out for 35 cycles. RT-PCR products were analyzed by electrophoresis on a 2% agarose gel, and positive bands were visualized by ethidium bromide staining.
Animal Studies.
Male Copenhagen rats (200–250 g) were obtained from Harlan Sprague Dawley (Indianapolis, IN). Fig. 1
outlines the three experiments performed. To determine the effects of MLL cell transfectants on tumor growth rate and metastasis (LCM study), male Copenhagen rats were injected s.c. in the upper leg with 1 x 105 of the transfected MLL cells described above (10 rats/group). Two-dimensional measurements of the primary tumors were taken daily using calipers. Tumors were removed when they reached a size of 2 cm2 by amputation of the tumor-bearing leg under ketamine (100 mg/kg; Fort Dodge Laboratories, Fort Dodge, IA) and methoxyflurane anesthesia. The tumors were weighed and frozen in liquid nitrogen for RNA isolation. Two weeks after the primary tumor was removed, the animals were sacrificed, plasma was collected and frozen for the PTHrP immunoradiometric assay, and the lungs were fixed in Bouin’s solution (Polysciences Inc., Warrington, PA). The numbers of metastatic lesions in the lung were determined by counting the surface lesions (43)
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All animal experiments were performed using protocols approved by the University of Michigan Animal Investigation Committee.
Statistical Analysis.
The results of the animal experiments were analyzed by unpaired t test with Welch correction using the InStat 3.0 biostatistics program (Graph Pad Software, Inc.). The receptor binding assay and PTHrP immunoradiometric assay were analyzed using the Graph Pad Prism program. All in vitro experiments were repeated at least three times.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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. These cells represent cell lines of human and rat origin and have varying degrees of metastatic potential and androgen dependency, as indicated in Table 1
. PTHrP mRNA could not be detected by Northern blot analysis in two human prostate carcinoma cell lines, namely, LNCaP and DU-145.
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A. The expected sigmoidal-shaped curve was observed in the positive control MC3T3-E1 cells, as described previously (46)
. Competitive binding assays were performed with all cell lines listed in Table 1
and with the three LNCaP-transfected cell lines used for apoptosis studies. Specific binding of PTHrP was not found in the prostate carcinoma cell lines. To determine whether there were low levels of PTH/PTHrP receptor expression in the LNCaP cell line, RT-PCR was performed using receptor-specific primers. Using RT-PCR, a positive signal for PTH/PTHrP receptor mRNA was detected (Fig. 4B)
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. The PTHrP signal in pcDNA-MLL cells was equivalent to that of the parental MLL cell line (data not shown). Additional parameters of the transfected cells were analyzed to determine whether PTHrP overexpression altered other cellular characteristics. Steady-state osteopontin (an adhesion protein thought to be associated with metastatic potential; Ref. 50
) mRNA levels were unchanged in the three cell lines (Fig. 5A)
. The amount of PTHrP secreted by the transfected cells into the cell culture medium was quantitated by radioimmunometric assay to determine whether increased PTHrP mRNA resulted in increased protein production. PTHrP production by PTHrP-MLL cells was significantly higher than that of the pcDNA transfected cells (36 ± 1.6 versus 7.3 ± 2.0, respectively; P < 0.001; Fig. 5B
).
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, and they had growth rates similar to that of untransfected MLL cells. The lack of alteration in proliferation and osteopontin gene expression suggests that if there are any differences in the behavior of the cell lines in vivo, this would be attributable to PTHrP production and not to some artifact of the culture or transfection protocol.
PTHrP Influences Tumor Growth in the Dunning Rat Prostate Adenocarcinoma Model.
To determine whether PTHrP expression affects tumor cell growth, three in vivo experiments were performed using the Dunning rat prostate adenocarcinoma metastasis model (see Fig. 1
for experimental design). For each of these studies, pcDNA-MLL and PTHrP-MLL cells were injected s.c. into the hind leg of male Copenhagen rats. This injection route does not lead to the formation of skeletal metastases. The LCT (105 cells injected) and HCT (2.5 x 105 cells injected) studies evaluated the ability of MLL transfectants (producing low and high levels of PTHrP) to influence primary tumor size at a fixed time (17 days and 14 days, respectively). The LCM study (105 cells injected) evaluated both the growth rate of the primary MLL tumors and the numbers of lung metastases based on a primary tumor of defined size (2 cm2). The HCT study also evaluated the ability of MLL transfectants to metastasize to the lung.
The effects of PTHrP expression on primary tumor size are shown in Fig. 6A
. In both the HCT and LCT studies, primary tumor weight was increased to a greater extent in tumors derived from PTHrP-MLL cells as compared with tumors derived from pcDNA-MLL cells. This trend, which was seen in the LCT study, reached statistical significance when larger numbers of cells were used to establish the primary tumor (HCT study).
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that increased PTHrP expression promotes tumor growth in vivo. To determine whether the level of PTHrP expression had an effect on the metastatic potential of prostate carcinoma cells, the extent of lung metastasis was analyzed 2 weeks after removal of the primary tumor when it reached a fixed size of 2 cm2 (LCM study). There was no significant difference in the average number of lung lesions observed between the two cell lines injected (pcDNA-MLL, 161 ± 36; PTHrP-MLL, 113 ± 27). Similarly, there was no difference in the numbers of lung metastases in the HCT study, although the primary tumors were significantly larger in the PTHrP-MLL group.
The plasma PTHrP level in all rats was also determined after sacrifice. There was no detectable PTHrP in the plasma of uninjected control rats (n = 3), suggesting that the PTHrP measured in treatment rats was attributable to production by the tumor cells. The average plasma PTHrP level (pM) for the HCT studies (in which the rats were sacrificed, and plasma was collected 2 weeks after primary tumor removal) was significantly higher in the animals injected with PTHrP-MLL cells (9.03 ± 2.56; n = 10) than in animals injected with pcDNA-MLL cells (0.67 ± 0.21; n = 8; P < 0.05; Fig. 6A
). In the LCT study, the plasma levels were determined at the time of primary tumor removal. The average value was also significantly higher in the PTHrP-MLL group (3.61 ± 0.78; n = 7) than in the pcDNA-MLL group (0.56 ± 0.19; n = 8; P < 0.01; Fig. 6A
).
In the LCT study, the plasma PTHrP levels were compared with tumor weight to determine whether the larger tumors observed in rats injected with the PTHrP-MLL cells could account for their increased plasma PTHrP levels. The average PTHrP level for animals injected with PTHrP-MLL cells was 0.75 ± 0.08 pmol/g tumor, whereas that for animals injected with pcDNA-MLL cells was 0.12 ± 0.02 pmol/g tumor (P < 0.0001). Therefore, the higher blood levels could not be attributed simply to larger tumors but appear to be dependent on the amount of PTHrP made by the tumor cells. This is supported by the Northern blot analysis of total RNA extracted from the primary tumors (Fig. 6B)
. The steady-state level of PTHrP mRNA, normalized to 18S rRNA, was increased 10-fold in tumors from PTHrP-MLL cells (n = 8) versus tumors from pcDNA-MLL cells (n = 6; P = 0.01). Thus, the tumor cells transfected with PTHrP maintained their ability to produce increased amounts of PTHrP in vivo.
PTHrP Is Expressed by Metastatic Prostate Carcinoma in Vivo.
Primary human prostatic carcinoma is immunohistochemically positive for PTHrP, but little is known of PTHrP expression in bone metastasis. To determine whether prostate carcinoma metastases in bone produce PTHrP, immunohistochemistry was performed on sections from human patients. A polyclonal antibody against PTHrP positively stained the metastatic tumor tissue in all three patient samples evaluated. A representative section is seen in Fig. 7
. Positive staining was specific to the tumor tissue, because the adjacent normal marrow in these samples were negative for PTHrP. Staining was observed throughout the cytoplasm, with intense staining localized at the nuclear envelope (Fig. 7B)
. No staining was observed in samples incubated without primary antibody or incubated with control egg yolk from nonimmunized chickens (data not shown).
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-LNCaP). The cells were treated with PMA to induce apoptosis. It has been demonstrated previously that PMA promotes apoptotic death in LNCaP cells using light microscopy, transmission electron microscopy, and DNA laddering techniques (51)
. Using the APO-BRDU assay to end-label the fragmented DNA indicative of apoptosis, the cells expressing full-length PTHrP were found to have significantly less PMA-induced apoptosis than pcDNA-LNCaP cells (Fig. 8)
. Apoptosis in -LNCaP cells was similar to that in pcDNA.
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-LNCaP cell lines as for the LNCaP parent line (data not shown).
Because Massfelder et al. (27)
have found that full-length PTHrP acts as a mitogen and NLS-deleted PTHrP acts as a growth-supressor in vascular smooth muscle cells, the effect of PTHrP expression on proliferation was tested in the transfected LNCaP cells in vitro. The pcDNA-LNCaP and PTHrP-LNCaP cells exhibited identical rates of growth using the MTT proliferation assay, and no antimitogenic effects were observed in the -LNCaP cells (data not shown).
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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PTHrP was expressed in many, but not all, of the prostate cancer lines examined. PTHrP produced by the cells increased during the proliferative phase, suggesting bioavailability of PTHrP during tumor formation in vivo. There was no apparent correlation between expression of PTHrP and the aggressiveness or androgen dependency of the tumor from which the cell lines were derived. These same cells did not have detectable PTH/PTHrP receptors using Northern blot analysis or binding assays. The detection of transcript by RT-PCR suggests that receptors may be present at a low level, but its physiological relevance is unknown. In a previous study, PTHrP was found to stimulate prostate cancer cell growth in vitro via an autocrine loop (25) ; however, we did not find any alteration in proliferation in vitro when cells overexpressed PTHrP.
Does PTHrP play a role in the establishment or growth of primary tumors? Several lines of evidence from this study support this idea. The first is the observed trend that rat prostate cancer cells which overexpress PTHrP form larger primary tumors than cells producing lower levels of PTHrP. Second, the rate of tumor growth was increased with PTHrP expression in vivo, whereas these same cells did not show an increase in growth rate in vitro. This suggests that PTHrP may be exerting its effects through a paracrine path in vivo or, alternatively, that expression of PTHrP could protect cancer cells from apoptotic stimuli present in the in vivo microenvironment via an autocrine or intracrine mechanism.
The s.c. MLL model was also used to assess the effect of PTHrP expression on lung metastasis. Our results showed that the number of lung metastases was not influenced by the level of PTHrP produced by the tumor cells. This is in agreement with results obtained using the same cell lines in an intracardiac injection study to evaluate metastasis to bone (52) . The incidence of metastasis was not affected by the amount of PTHrP made by the cell line, although osteoclast numbers were increased in the PTHrP-MLL lesions compared to trabeculae from pcDNA-MLL lesions. Similar skeletal metastatic findings were observed by Rabbani et al. (53) .
Studies of LNCaP cell lines in vitro provide other insights into potential mechanisms of PTHrP action. This cell line provides a good model for assessing the effects of PTHrP expression because the parental cell line produces no detectable PTHrP. The present study revealed that expression of full-length PTHrP in this cell line was protective against PMA-induced apoptosis, whereas the expression of NLS-deleted PTHrP in the same cells had no effect on apoptosis. Therefore, PTHrP must reach the nucleus to exert its antiapoptotic effect. These data are in agreement with Henderson et al. (16) , who originally suggested that PTHrP could act as an inhibitor of apoptosis. Because PTHrP expression protects prostate cancer cells from an apoptotic stimulus, PTHrP could give cells a growth advantage during primary tumor formation and/or subsequent metastasis. In these transfected cells, expression of PTHrP did not have a mitogenic effect in vitro, nor did the NLS-deleted PTHrP transfected cells have altered proliferation rates. This is in contrast to what has been observed in similar experiments using vascular smooth muscle cells (23) . However, as Massfelder et al. (27) point out in their vascular smooth muscle study, the cellular response to PTHrP appears to be cell type specific, inhibiting proliferation in some cells and stimulating proliferation in others. Although the present study indicates that PTHrP expression stimulates primary tumor growth, whether this occurs via a specific mitogenic effect in vivo remains to be tested.
In summary, the findings that PTHrP is expressed in many prostate cell lines, in primary and metastatic prostate cancer, and that PTHrP can influence tumor growth in vivo and apoptotic rates in vitro support the hypothesis that this protein plays an important role in the progression of prostate cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Specialized Programs of Research Excellence in prostate cancer P50 CA69568. 
2 To whom requests for reprints should be addressed, at Department of Periodontics/Prevention/Geriatrics, The University of Michigan, 1011 North University Ave., Ann Arbor, MI 48109-1078. Phone: (734) 647-3206; Fax: (734) 763-5503; E-mail: mccauley{at}umich.edu
3 The abbreviations used are: PTHrP, parathyroid hormone-related protein; NLS, nuclear localization sequence; PTH, parathyroid hormone; MLL, MATLyLu; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PMA, phorbol 12-myristate 13-acetate; RT-PCR, reverse transcription-PCR; LCM, low cell metastasis; LCT, low cell tumor; HCT, high cell tumor.
Received 6/ 8/99. Accepted 10/ 4/99.
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REFERENCES |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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signaling blockade inhibits PTHrP secretion by breast cancer cells and bone metastases development. J. Clin. Investig., 103: 197-206, 1999.[Medline]
1 regulates steady-state PTH/PTHrP receptor mRNA levels and PTHrP binding in ROS 17/2.8 osteosarcoma cells. Mol. Cell. Endocrinol., 101: 331-336, 1994.[Medline]
1 on PTH/PTHrP receptor mRNA levels in MC3T3-E1 cells. J. Bone Miner. Res., 10: 1243-1255, 1995.[Medline]
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 R. H. Hastings, F. Araiza, D. W. Burton, L. Zhang, M. Bedley, and L. J. Deftos Parathyroid hormone-related protein ameliorates death receptor-mediated apoptosis in lung cancer cells Am J Physiol Cell Physiol, December 1, 2003; 285(6): C1429 - C1436. [Abstract] [Full Text] [PDF]
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 J. C. Mills, N. Andersson, T. S. Stappenbeck, C. C. M. Chen, and J. I. Gordon Molecular Characterization of Mouse Gastric Zymogenic Cells J. Biol. Chem., November 14, 2003; 278(46): 46138 - 46145. [Abstract] [Full Text] [PDF]
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 J. Qian, M. C. Colbert, D. Witte, C.-Y. Kuan, E. Gruenstein, H. Osinska, B. Lanske, H. M. Kronenberg, and T. L. Clemens Midgestational Lethality in Mice Lacking the Parathyroid Hormone (PTH)/PTH-Related Peptide Receptor Is Associated with Abrupt Cardiomyocyte Death Endocrinology, March 1, 2003; 144(3): 1053 - 1061. [Abstract] [Full Text] [PDF]
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 R. J. MacLeod, N. Chattopadhyay, and E. M. Brown PTHrP stimulated by the calcium-sensing receptor requires MAP kinase activation Am J Physiol Endocrinol Metab, February 1, 2003; 284(2): E435 - E442. [Abstract] [Full Text] [PDF]
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A. Cebrian, A. Garcia-Ocana, K. K. Takane, D. Sipula, A. F. Stewart, and R. C. Vasavada Overexpression of Parathyroid Hormone-Related Protein Inhibits Pancreatic {beta}-Cell Death In Vivo and In Vitro Diabetes, October 1, 2002; 51(10): 3003 - 3013. [Abstract] [Full Text] [PDF]
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V. A. Tovar Sepulveda, X. Shen, and M. Falzon Intracrine PTHrP Protects against Serum Starvation-Induced Apoptosis and Regulates the Cell Cycle in MCF-7 Breast Cancer Cells Endocrinology, February 1, 2002; 143(2): 596 - 606. [Abstract] [Full Text] [PDF]
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 J. C. Mills, A. J. Syder, C. V. Hong, J. L. Guruge, F. Raaii, and J. I. Gordon A molecular profile of the mouse gastric parietal cell with and without exposure to Helicobacter pylori PNAS, November 20, 2001; 98(24): 13687 - 13692. [Abstract] [Full Text] [PDF]
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 V. Richard, M. D. Lairmore, P. L. Green, G. Feuer, R. S. Erbe, B. Albrecht, C. D'Souza, E. T. Keller, J. Dai, and T. J. Rosol Humoral Hypercalcemia of Malignancy : Severe Combined Immunodeficient/Beige Mouse Model of Adult T-Cell Lymphoma Independent of Human T-Cell Lymphotropic Virus Type-1 Tax Expression Am. J. Pathol., June 1, 2001; 158(6): 2219 - 2228. [Abstract] [Full Text] [PDF]
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A. Gujral, D. W. Burton, R. Terkeltaub, and L. J. Deftos Parathyroid Hormone-related Protein Induces Interleukin 8 Production by Prostate Cancer Cells via a Novel Intracrine Mechanism Not Mediated by its Classical Nuclear Localization Sequence Cancer Res., March 1, 2001; 61(5): 2282 - 2288. [Abstract] [Full Text]
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D. E. H. Afar, I. Vivanco, R. S. Hubert, J. Kuo, E. Chen, D. C. Saffran, A. B. Raitano, and A. Jakobovits Catalytic Cleavage of the Androgen-regulated TMPRSS2 Protease Results in Its Secretion by Prostate and Prostate Cancer Epithelia Cancer Res., February 1, 2001; 61(4): 1686 - 1692. [Abstract] [Full Text]
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M. M. Aarts, D. Davidson, A. Corluka, E. Petroulakis, J. Guo, F. R. Bringhurst, J. Galipeau, and J. E. Henderson Parathyroid Hormone-related Protein Promotes Quiescence and Survival of Serum-deprived Chondrocytes by Inhibiting rRNA Synthesis J. Biol. Chem., October 5, 2001; 276(41): 37934 - 37943. [Abstract] [Full Text] [PDF]
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