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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.

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.

An examination of the sterols of UTEX 2341, a small (ca. 2μm), nonmotile unicellular marine alga identified as Chlorella minutissima Fott et Novakova, yielded results inconsistent with any of 35 Chlorella strains analyzed previously. UTEX 2341 contained cholesterol as the principal sterol, with 24-methylenecholesterol, fucosterol, and isofucosterol also present; these are not dominant sterols in any other Chlorella species. Presence of eicosapentaenoic acid in UTEX 2341 also contrasted with fatty acids of Chlorella strains analyzed previously. Pigment analysis of UTEX 2341 revealed that it contained chlorophyll a, but not chlorophylls b or c; violaxanthin was the only major xanthophyll pigment. Both lipid and pigment compositions suggest that UTEX 2341 is not a member of the genus Chlorella but, rather, belongs in the Eustigmatophyceae; it may be Nannochloropsis sp. Cells with possible extracellular structures were present at an appreciable percentage of the stationary-phase population studied; centrifuging removed or collapsed these structures. The high cholesterol and polyunsaturated fatty acid contents of UTEX 2341 make it attractive as a potential aquaculturefeed, provided it is, or can be made, digestible.

Sterols were identified and quantified in six marine microalgal strains identified as belonging to the prymnesiophyte genus Isochrysis , several of these strains are in wide use in the commerical mariculture industry. One strain contained only cholesterol, and another contained a complex mixture of Δ 5 -sterols and dihydroxysterols. Two strains contained brassicasterol and two others contained epibrassicasterol as the principal sterol. The strains containing brassicasterol or epibrassicasterol also contained long chain alkenones characteristic of some members of the Prymnesiophyceae; whereas, those without brassicasterol or epibrassicasterol contained no long chain alkenones. These qualitative biochemical differences appear to have taxonomic significance and may be important in the value of these algae as live feeds for rearing marine invertebrates.

1. 1. Sterols were identified from 11 isolates of Tetraselmis , a unicellular Prasinophyte alga used frequently as food in mariculture. 2. 2. The principal sterol in eight isolates was either 24-methylenecholesterol or 24-methylcholesterol; the latter was determined to be campesterol in all cases. 3. 3. Campesterol is the first 24α sterol to be reported in the Prasinophyceae. 4. 4. In the remaining three isolates, cholesterol was the principal sterol with smaller amounts of 24-methylenecholesterol and campesterol present; in two of these strains total sterol approached 3% of dry weight. 5. 5. This is the first report of cholesterol as the principal sterol of a Prasinophyte; the C28 sterols found in Tetraselmis are the dominant sterols in most Prasinophyceae studied to date.

Pavlova gyrans, a unicellular alga of interest as food for oysters, was cultured axenically and examined for sterol composition. Desmethyl monohydroxy sterols, which are frequently seen in algae, made up 40% of the total sterols and were observed primarily in the free sterol fraction. The principal sterols of this group were 5-ergostenol, poriferasterol, and clionasterol, as well as some poriferast-22-enol and poriferastanol. Several “methyl” sterols with unusual structures made up 27% of the total sterols. The principal “methyl sterols” were 4α-methyl ergostanol, 4α-methyl poriferastanol, and 4α-methyl poriferast-22-enol. Methyl sterols were found primarily in the ester fraction. Also observed was a new class of dihydroxysterols composing 33% of the total sterols. These sterols are structurally related to the methyl and desmethyl sterols ofPavlova but contain an extra nuclear hydroxyl which can be acetylated when present on a desmethyl sterol, but which is nonreactive with acetic anhydride in 4α-methyl sterols. None of these sterols were observed in ester form but are concentrated in the acid-hydrolyzable, bound fraction. The unique nature of these sterols suggests potential taxonomic utility.

1. 1. Sterols were identified from eight isolates of five species in the Chromophycota that were cultured axenically and harvested in the stationary phase. 2. 2. Analyses were performed on four strains from the Prymnesiophyceae, two strains from the Cryptophyceae and one from the Bacillariophyceae. Most strains examined contained only one major sterol, 24-methyl-22-dehydrocholesterol. 3. 3. Analysis by capillary GC, HPLC, and in one instance NMR, showed that the two strains provisionally identified as Isochrysis contained brassicasterol (24β-methyl-22-dehydrocholesterol); whereas, all other species examined contained primarily epibrassicasterol (24α-methyl-22-dehydrocholesterol). 4. 4. Stigmasterol (24α-ethyl-22-dehydrocholesterol) accompanied epibrassicasterol in Pleurochrysis carterae. 5. 5. Analyses of C-24 alkyl isomers in these algae may provide useful information concerning their taxonomic placement. 6. 6. The occurrence of both isomers of 24-methyl-22-dehydrocholesterol in oysters is explained by the occurrence of both isomers among algae which are probably dietary sources for oysters.

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.

Dietary sterol is required by the oyster for growth, and sterol is believed to be obtained primarily from dietary phytoplankton. Seven isolates ofChaetoceros and one ofSkeletonema, which are of potential use as oyster food, were analyzed for sterol composition using gas chromatography, high-performance liquid chromatography and gas chromatography/mass spectrometry.Skeletonema and five isolates ofChaetoceros contained cholesterol as their major sterol. Two other isolates ofChaetoceros also contained cholesterol, but 24-methylenecholesterol was the principal sterol. Cholesterol has rarely been reported as the major sterol from phytoplankton. In view of the widespread occurrence ofSkeletonema andChaetoceros in the marine environment, these algae could be an important source of the oyster's cholesterol.

Sterols were analyzed from four phytoplankton strains which are under investigation as possible sources of food for oysters in culture. One strain ofPyramimonas contained only 24-methylenecholesterol as a major sterol component.Pyramimonas grossii, Chlorella autotrophica andDunaliella tertiolecta each contained a complex mixture of C28 and C29 sterols with Δ7, Δ5,7 and Δ5,7,9(11) nuclear double bond systems. Sterols were found both with and without the C-22 side chain double bond. Ergosterol and 7-dehydroporiferasterol were the principal sterols in each of the latter three species, which also contained the rare tetraene sterols, 24-methylcholesta-5,7,9(11),22-tetraen-3β-ol and 24-ethylcholesta-5,7,9(11),22-tetraen-3β-ol.

Sterols were identified from seven species of axenically cultured diatoms which may be used for oyster food. Five species contained 24-methylenecholesterol as the major sterol. Stigmasterol was the principal sterol in Amphora coffaeformis while 24-ethylcholesterol was the major sterol in Navicula pelliculosa . Each of the four species of the order Centrales had 24-methylcholesterol, which was all 22-dihydrobrassicasterol (24β-methyl epimer). The 24-methylcholesterol from Nitzschia brevirostris (order Pennales) was all campesterol (24α-methyl epimer). From this and previously published works on diatom sterols, it appears that sterols from the order Centrales are 24β-oriented and those from the order Pennales are 24α-oriented.

The oyster cannot synthesize sterols from smaller molecules but must obtain them from its diet, which consists of detritus and small organisms, i.e., mostly single-celled algae. Algae differ widely in their effectiveness as oyster food. Small (5 microns) algae which are abundant in sterols and polyunsaturated fatty acids appear to be most effective. Recent studies have shown the occurrence of cholesterol in strains of the unicellular algae Tetraselmis, Chaetoceros and Skeletonema, sometimes in large quantities. In the study reported here, six isolates of a recently constructed algal class, the Eustigmatophyceae, have been examined for sterols and fatty acids by gas chromatography and gas chromatography/mass spectrometry. All strains were shown to contain cholesterol as the principal sterol. Two isolates contained large amounts of total sterol (400-1000 fg/cell), and one (Sticho 0-18) also contained large amounts of eicosapentaenoic acid (20:5n-3). These biochemical characteristics are desirable in a potential food source for oysters.

Indomethacin (20µg.ml−1), a cyclooxygenase inhibitor, and OKY-1581 (50 ug.ml−1), a thromboxane synthetase inhibitor, did not affect contractions of the superfused human lung parenchymal strips induced by histamine, leukotriene C4 (LTC4) and leukotriene D4 (LTD4). Indomethacin but not OKY-1581 reduced by 50% the myotropic activity of leukotriene A4 (LTA4) on the human lung strips. Injections of LTD4 (100 ng; 0.2 n mole) in the circulation of perfused specimens of human lung did not stimulate the release of prostaglandins (PGs) and thromboxanes (TXs) whereas they produced a large release of these icosanoids from perfused guinea-pig lungs. Perfused human lung specimens almost completely inactivated intraarterial injections of PGE2 (50 ng) and released PGs and TXs upon mechanical stimulation. It is concluded that the actions of leukotrienes C4 and D4 in human lung (parenchymal strips and perfused specimens) are not mediated by the release of PGs and TXs.

The metabolism of leukotriene (LT) A4, B4, C4, D4, and E4 was studied using both bioassay and reversed phase high performance liquid chromatography (RP-HPLC) methods. The incubation of 20,000 g supernatants of homogenates of human adult lung with LTA4 and LTC4 for various periods of time produced substances of higher biologic activity than the controls (without incubation) when measured on strips of guinea pig lung parenchyma and ileum. RP-HPLC analyses of the incubation media revealed the formation of LTB4, LTC4, LTD4, and LTE4 from LTA4 and the formation of LTD4 and LTE4 from LTC4. LTD4 was converted to LTE4 whereas LTB4 and LTE4 were not catabolized to an appreciable extent during a 2-h incubation period. Supernatants (20,000 g) of human fetal lung homogenates also contain the enzymatic activities to transform LTC4 into LTD4 and LTE4; however, LTA4 was mainly converted to LTB4 and to products of the nonenzymatic hydrolysis of LTA4 such as the delta 6-trans-LTB4, delta 6 -trans-12-epi-LTB4 and the 5,6-dihyroxyeicosatetraenoic acids; much smaller quantities of the peptidoleukotrienes were formed than in adult lung homogenates.

Desulfitobacterium frappieri PCP-1 was induced for ortho - and para -dechlorinating activities by different chlorophenols. Dehalogenation rates ranging from 25 to 1,158 nmol/min/mg of cell protein were observed according to the chlorophenol tested and the position of the chlorine removed. D. frappieri shows a broad substrate specificity; in addition to tetrachloroethylene and pentachloropyridine, strain PCP-1 can dehalogenate at ortho , meta , and para positions a large variety of aromatic molecules with substituted hydroxyl or amino groups. Reactions of O demethylation and reduction of nitro to amino substituents on aromatic molecules were also observed.