Search

Urokinase-type plasminogen activator (uPA) is a member of the serine protease family and can break down various components of the extracellular matrix to promote growth, invasion, and metastasis of several malignancies including breast cancer. In the current study we examined the role that the DNA methylation machinery might be playing in regulating differential uPA gene expression in breast cancer cell lines. uPA mRNA is expressed in the highly invasive, hormone-insensitive human breast cancer cell line MDA-MB-231 but not in hormone-responsive cell line MCF-7. Using methylation-sensitive PCR, we show that 90% of CpG dinucleotides in the uPA promoter are methylated in MCF-7 cells, whereas fully demethylated CpGs were detected in MDA-MB-231 cells. uPA promoter activity, which is directly regulated by the Ets-1 transcription factor, is inhibited by methylation as determined by uPA promoter-luciferase reporter assays. We then tested whether the state of expression and methylation of the uPA promoter correlates with the global level of DNA methyltransferase and demethylase activities in these cell lines. We show that maintenance DNA methyltransferase activity is significantly higher in MCF-7 cells than in MDA-MB-231 cells, whereas demethylase activity is higher in MDA-MB-231 cells. We suggest that the combination of increased DNA methyltransferase activity with reduced demethylase activity contributes to the methylation and silencing of uPA expression in MCF-7 cells. The converse is true in MDA-MB-231 cells, which represents a late stage highly invasive breast cancer. The histone deacetylase inhibitor, Trichostatin A, induces the expression of the uPA gene in MDA-MB-231 cells but not in MCF-7 cells. This supports the hypothesis that DNA methylation is the dominant mechanism involved in the silencing of uPA gene expression. Taken together, these results provide insight into the mechanism regulating the transcription of the uPA gene in the complex multistep process of breast cancer progression.

Parathyroid hormone (PTH)-related peptide (PTHrP) can modulate the proliferation and differentiation of a number of cell types including osteoblasts. PTHrP can activate a G protein-coupled PTH/PTHrP receptor, which can interface with several second-messenger systems. In the current study, we have examined the signaling pathways involved in stimulated type I collagen and alkaline phosphatase expression in the human osteoblast-derived osteosarcoma cells, MG-63. By use of Northern blotting and histochemical analysis, maximum induction of these two markers of osteoblast differentiation occurred after 8 h of treatment with 100 nM PTHrP-(1-34). Chemical inhibitors of adenylate cyclase (H-89) or of protein kinase C (chelerythrine chloride) each diminished PTHrP-mediated type I collagen and alkaline phosphatase stimulation in a dose-dependent manner. These effects of PTHrP could also be blocked by inhibiting the Ras-mitogen-activated protein kinase (MAPK) pathway with a Ras farnesylation inhibitor, B1086, or with a MAPK inhibitor, PD-98059. Transient transfection of MG-63 cells with a mutant form of Galpha, which can sequester betagamma-subunits, showed significant downregulation of PTHrP-stimulated type I collagen expression, as did inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) by wortmannin. Consequently, the betagamma-PI 3-kinase pathway may be involved in PTHrP stimulation of Ras. Collectively, these results demonstrate that, acting via its G protein-coupled receptor, PTHrP can induce indexes of osteoblast differentiation by utilizing multiple, perhaps parallel, signaling pathways.

Prostate carcinoma is one of the most common malignancies affecting males, resulting in a high rate of morbidity and mortality. This hormone-dependent malignancy is characteristically associated with a high incidence of osteoblastic skeletal lesions. However, osteolytic lesions invariably accompany blastic ones. In the current study, we assessed the role of parathyroid hormone-related peptide (PTHRP), a potent bone-resorbing agent, in contributing to bone breakdown and prostatic skeletal metastasis using a syngeneic rat prostate cancer model. The full-length cDNA encoding rat PTHRP was subcloned as a Hind III insert in the sense orientation into the mammalian expression vector pRc-CMV to generate the expression vector pRc-PTHRP-S. Both control and experimental plasmids were stably transfected into low PTHRP-producing Dunning R3227, Mat Ly Lu rat prostate cancer cells. Following antibiotic selection, monoclonal cell lines expressing the highest amount of PTHRP mRNA and immunoreactive PTHRP were selected as experimental tumor cells for further analysis. Increased PTHRP production by these cells had no significant effect in vitro on the invasive capacity of these cells. Control and experimental cells were inoculated s.c. into the right flank or by the intracardiac (i.c.) route into the left ventricle of inbred male Copenhagen rats. No skeletal metastases occurred after s.c. injection with either cells. In contrast, i.c. inoculation led to lumbar vertebra metastasis and consequent hind-limb paralysis. Furthermore, histological examination of skeletal metastases in experimental animals showed a marked increase in osteoclastic activity. Our results demonstrate that PTHRP can increase osteoclastic osteolysis in the presence of focal osseous prostate cancer metastases and may contribute to the lytic lesions which generally accompany osteoblastic lesions in prostate cancer.

Previous studies have demonstrated that overexpression of urinary plasminogen activator (uPA) in rat prostate cancer cells results in increased skeletal metastases, which are primarily of the osteoblastic variety. The osseous activation induced by the metastases appears to be mediated through the amino terminal fragment (ATF) of uPA, which lacks the catalytic domain and can act as a growth factor for osteoblasts. To explore further the mechanism of action of uPA in bone cells, we evaluated the effects of ATF on modulating the expression of various proto-oncogenes. Human-osteoblast-derived osteosarcoma cells, SaOS2, were treated with graded doses of ATF for 10–120 min, and effects on early response proto-oncogenes were monitored. ATF increased c-myc, c-jun, and c-fos gene expression in a time-dependent manner for up to 60 min, after which mRNA levels fell. The maximum induction was seen in c-fos gene expression, which was found to be dose dependent. This effect of ATF was localized to its growth-factorlike domain. Examination of the half life of these transcripts in the presence of the transcriptional inhibitor actinomycin D demonstrated that ATF does not alter the stability of c-fos mRNA in these bone cells. Nuclear run-off assays indicated that ATF effects were due to stimulation of c-fos gene transcription. An increase in c-fos protein levels was correlated with the augmentation of its mRNA in ATF-treated SaOS2 cells. Pretreatment of SaOS2 cells with the protein tyrosine kinase inhibitor herbimycin and recombinant soluble uPA receptor (uPAR) caused a significant reduction in the ability of ATF to induce c-fos expression. These results demonstrate a novel role for uPA in activating early response proto-oncogenes, in particular c-fos, which plays an important role in bone cell growth and differentiation and may be a key factor in the signal transduction pathway of ATF. J. Cell. Physiol. 172:137–145, 1997. © 1997 Wiley-Liss, Inc.

PTH-related peptide (PTHrP) has been shown to be the major mediator of hypercalcemia of malignancy, but may also exert effects on cell growth and differentiation. The Leydig cell tumor H-500, when implanted in Fischer rats, produces abundant PTHrP and eventually causes the death of the host animal. In the present study we have used antisense RNA technology to block the effects of PTHrP in H-500 Leydig tumor cells in vivo. The full-length rat PTHrP complementary DNA encoding amino acid -36-->141 was subcloned as an EcoRI-BglII insert in the antisense orientation into the mammalian expression vector pRc/CMV to produce the plasmid pRc-PAS. This plasmid was then stably transfected into the H-500 Leydig tumor cells with a Lipofectin reagent. After selection with the neomycin derivative G-418, a stable cell line, H-500-PTHrP-AS, was obtained which showed 80% inhibition of endogenous PTHrP messenger RNA compared to wild-type or vector-only transfected H-500 cells. Conditioned culture medium from these experimental cells showed a marked decrease in PTHrP immunoreactivity and in the ability of the medium to stimulate adenylate cyclase in UMR-106 rat osteosarcoma cells. Furthermore, inhibition of PTHrP production resulted in a significant increase in the doubling time of the H-500 cells. Transfection of the experimental plasmid into Rat-2 fibroblasts, which do not produce PTHrP, had no effect on cell growth. Control and experimental cells were then implanted sc into male Fischer rats. Animals were killed at timed intervals, and their tumor volumes were determined. Experimental animals receiving cells transfected with antisense PTHrP plasmid showed near-normal levels of plasma calcium and decreased expression of tumoral PTHrP messenger RNA. These animals also showed a 30-70% lower tumor volume during the course of the experiment compared to control animals. These studies have demonstrated that PTHrP can play a role as a promoter of tumor growth in vitro and in vivo.

PTHrP is the major pathogenetic factor for hypercalcemia in several malignancies including prostate cancer. In the current study, we have assessed the ability of androgens to regulate PTHrP production in androgen-insensitive human prostate cancer cells PC-3 and cells transfected with androgen receptor (PC-3T). Androgen responsiveness caused a marked decrease in PC-3T cell growth, and treatment of these cells with dihydrotestosterone led to inhibition of PTHrP production. These inhibitory effects were readily reversed by androgen receptor antagonist flutamide. To determine the effect of androgens on tumor growth and PTHrP production in vivo, PC-3 and PC-3T cells were injected into the right flank of male BALB/c nu/nu mice. Animals inoculated with PC-3 and PC-3T cells developed palpable tumors at wk 2 and 4, respectively. Inoculation of PC-3T cells into castrated animals resulted in rapid tumor growth in PC-3T tumors, effects that were reversed in PC-3T tumors grown in castrated hosts. Using PTHrP promoter luciferase reporter, a 30% decrease in luciferase activity was seen following treatment with dihydrotestosterone. These results indicate that PC-3 cell growth correlates inversely with androgen sensitivity and directly with PTHrP production in vitro and in vivo, androgens can regulate PTHrP production, and the androgen effect on PTHrP is mediated at least in part by transcriptional regulation via the androgen receptor.

Urokinase-type plasminogen activator (uPA) is a member of the serine protease family and can break down various components of the extracellular matrix to promote growth, invasion, and metastasis of several malignancies including breast cancer. In the current study we examined the role that the DNA methylation machinery might be playing in regulating differential uPA gene expression in breast cancer cell lines. uPA mRNA is expressed in the highly invasive, hormone-insensitive human breast cancer cell line MDA-MB-231 but not in hormone-responsive cell line MCF-7. Using methylation-sensitive PCR, we show that 90% of CpG dinucleotides in the uPA promoter are methylated in MCF-7 cells, whereas fully demethylated CpGs were detected in MDA-MB-231 cells. uPA promoter activity, which is directly regulated by the Ets-1 transcription factor, is inhibited by methylation as determined by uPA promoter-luciferase reporter assays. We then tested whether the state of expression and methylation of the uPA promoter correlates with the global level of DNA methyltransferase and demethylase activities in these cell lines. We show that maintenance DNA methyltransferase activity is significantly higher in MCF-7 cells than in MDA-MB-231 cells, whereas demethylase activity is higher in MDA-MB-231 cells. We suggest that the combination of increased DNA methyltransferase activity with reduced demethylase activity contributes to the methylation and silencing of uPA expression in MCF-7 cells. The converse is true in MDA-MB-231 cells, which represents a late stage highly invasive breast cancer. The histone deacetylase inhibitor, Trichostatin A, induces the expression of the uPA gene in MDA-MB-231 cells but not in MCF-7 cells. This supports the hypothesis that DNA methylation is the dominant mechanism involved in the silencing of uPA gene expression. Taken together, these results provide insight into the mechanism regulating the transcription of the uPA gene in the complex multistep process of breast cancer progression.

The serine protease urinary plasminogen activator or urokinase (uPA), produced in abundance by many malignancies, plays a key role in tumor cell invasion and metastasis. uPA is localized within the malignant cell milieu via its cell surface receptor [uPA receptor (uPAR)], which is expressed by tumor and tumor-associated cells. In the present study, we have used a syngeneic model of rat breast cancer to directly evaluate the role of uPAR as a diagnostic and therapeutic target in metastatic breast cancer. A polyclonal antibody against the ligand-binding NH(2)-terminal domain of rat uPAR (ruPAR) was developed. This antibody recognizes ruPAR by both immunofluorescence and Western blot analysis. Recombinant ruPAR and ruPAR IgG displaced the binding of (125)I-labeled ruPAR IgG to rat prostate cancer cells (Dunning R3227 Mat Ly Lu) and breast cancer cells (Mat B-III) overexpressing ruPAR (Mat B-III-uPAR). ruPAR IgG also blocked the invasive capacity of these tumor cells in a dose-dependent manner. Mat B-III-uPAR cells were inoculated s.c. into the mammary fat pad of syngeneic female Fischer rats. On day 10 after tumor cell inoculation, animals were injected with (125)I-labeled preimmune or ruPAR IgG and then sacrificed at timed intervals. Maximum (125)I uptake was observed in primary tumors and in tissues commonly affected by tumor metastases (liver, spleen, lungs, and lymph nodes) at 12 h. Injection of (125)I-labeled preimmune or ruPAR IgG into normal non-tumor-bearing animals resulted in minimal basal levels of uPAR expression and established the specificity of the ruPAR IgG. Similar results were obtained by Northern blot and PCR analysis of mRNA isolated from tissues of normal and tumor-bearing animals. To evaluate the effectiveness of this antibody in tumor progression, ruPAR IgG (50-100 microg/day) was injected s.c. for 7 days (day 1-7) at the site of tumor cell inoculation (mammary fat pad), and animals were sacrificed at various time points for evaluation of tumor growth and metastases. Animals receiving ruPAR IgG showed a marked decrease in tumor growth and metastases as compared with control tumor-bearing animals receiving the same dose of preimmune rabbit IgG. Histological analysis of experimental primary tumors showed marked tumor necrosis that was due to increased tumor cell apoptosis as determined by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Together, these studies demonstrate the ability of anti-uPAR antibody to decrease tumor volume and detect the presence of microscopic occult tumor metastases in malignancies where uPA/uPAR play a key role in tumor progression.

We have synthesized and purified recombinant parathyroid hormone related peptide (PTHrP (1-141)) and PTHrP (38-141) using an E. coli system that requires minimal purification. The cDNAs encoding PTHrP (1-141) and PTHrP (35-141) respectively were inserted into the multiple cloning site of the pTrcHis-B bacterial expression plasmid. The PTHrP encoded sequences were thereby fused at their NH2-termini to six histidine residues within the fusion protein. The recombinant plasmids were transfected into E. coli cells and PTHrP synthesis was induced by addition of 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) at 37 degrees C. The recombinant fusion proteins were purified by binding of the histidine residues to a nickel column followed by gradient elusion and dialysis. PTHrP (1-141) was released from its fusion protein by cyanogen bromide cleavage, whereas PTHrP (38-141) was released by enzymatic digestion with enterokinase. This rapid isolation method resulted in pure PTHrP (1-141) and (38-141) as judged by SDS-polyacrylamide gel electrophoresis and NH2-terminal sequence analysis. PTHrP (1-141) stimulated cAMP accumulation and mobilized intracellular calcium ([Ca2+]i) in UMR106 osteoblast-like cells, and stimulated phosphate transport in OK/E renal cells, whereas PTHrP (38-141) was inert in these bioassays. Availability of PTHrP and its NH2-terminally truncated analogue, which lacks the sequence necessary for its hypercalcemic actions, will enable their biological activities to be examined in greater detail.