Protein kinase C-dependent effects of 12(S)-HETE on endothelial cell vitronectin receptor and fibronectin receptor.

12(S)-HETE, a lipoxygenase metabolite of arachidonic acid induced a nondestructive and reversible endothelial cell (EC) retraction. 12(S)-HETE induced EC retraction was inhibited by protein kinase C inhibitors calphostin C and staurosporine but not by the protein kinase A inhibitor H8. The role of EC integrins alpha v beta 3 and alpha 5 beta 1 in 12(S)-HETE induced EC retraction was investigated. In confluent EC cultures, alpha v beta 3 is localized to focal adhesions at both the cell body and cell-cell borders and is colocalized with vinculin-containing focal adhesions. In contrast, alpha 5 beta 1 is primarily enriched at the cell-cell borders, demonstrating codistribution with cell cortical microfilaments and extracellular fibronectin. Both receptors were functional in mediating cell-cell or cell-matrix interactions based on the observations that specific antibodies inhibited EC adhesion to intact subendothelial matrix and disrupted the monolayer integrity. 12(S)-HETE induced a multistep, temporally defined redistribution of the alpha v beta 3-containing focal adhesions, leading to an eventual decrease in alpha v beta 3 plaques in the retracted ECs. This effect of 12(S)-HETE was inhibited by calphostin C but not by H8. The alterations of alpha v beta 3-containing focal adhesions preceded the development of EC retraction. 12(S)-HETE also enhanced EC alpha v beta 3 surface expression as revealed by immunofluorescence, flow cytometry, and digitized image analysis. 12(S)-HETE-induced alpha v beta 3 rearrangement (i.e., decreased focal adhesion localization and enhanced surface expression) did not result from altered mRNA transcription (as revealed by semi-quantitative RT-PCR analysis) or protein translation (as revealed by Western blotting). In contrast to its effect on alpha v beta 3, 12(S)-HETE did not demonstrate a temporally related, well-defined effect on the distribution pattern and the surface expression of alpha 5 beta 1, although the cell-cell border staining pattern of alpha 5 beta 1 was disrupted due to EC retraction. It is concluded that 12(S)-HETE-induced decrease of alpha v beta 3 localization to focal adhesions may contribute to the development of EC retraction and that 12(S)-HETE induced increase in alpha v beta 3 surface expression may promote adhesion of inflammatory leukocytes as well as tumor cells to endothelium.

Prostacyclin: a potent antimetastatic agent.

Metastasis is the principal cause of failures to cure human cancers. Prostacyclin is a powerful antimetastatic agent against B16 amelanotic melanoma cells. This effect, which may result from the platelet antiaggregatory action of prostacyclin, is potentiated by a phosphodiesterase inhibitor. Inhibitors of prostacyclin synthesis increase metastasis. Prostacyclin and agents that may increase endogenous prostacyclin production or prolong its activity are suggested as new antimetastatic agents.

Lysosomal cathepsin B: correlation with metastatic potential.

Although lysosomal enzymes are implicated in the processes of tumor invasion and metastasis, their cellular origin within the tumor is unclear. The activity of the lysosomal proteinase cathepsin B is significantly elevated in a variant of the B16 melanoma with high metastatic potential. The cathepsin B activity is localized to the lysosomes of the tumor cells.

Tumor cell-platelet aggregation: induced by cathepsin B-like proteinase and inhibited by prostacyclin.

The ability of tumor cells to metastasize may be related to their ability to promote aggregation of host platelets. The use of inhibitors of cysteine proteinases resulted in parallel inhibition of B16 amelanotic melanoma-induced platelet aggregation and of a cathepsin B activity. The antimetastatic agent prostacyclin inhibited platelet aggregation induced by the tumor cells and by papain, a cathepsin B-mimicking agent.

Inhibition by prostacyclin of the tumor cell-induced platelet release reaction and platelet aggregation.

Prostacyclin was examined for its inhibitory effects on the tumor cell-induced platelet release reaction. Prostacyclin inhibited in a dose-dependent manner tumor cell-induced release of platelet dense granules and alpha-granules concomitant with an inhibition of platelet aggregation. Release was determined by assay of biochemical markers (serotonin for dense granules and beta-thromboglobulin for alpha-granules). A tenfold higher concentration of prostacyclin was required to inhibit completely serotonin release as compared to the concentration required for beta-thromboglobulin release. Correlative ultrastructural studies demonstrated that prostacyclin at doses of over 10 ng/ml inhibited the ultrastructural changes associated with tumor cell-induced platelet shape change and platelet granule release. Platelet aggregates exhibited the retention of granule reservoirs that could potentially be involved in long-term release of biologically active substances.

12(S)-HETE enhancement of prostate tumor cell invasion: selective role of PKC alpha.

BACKGROUND: Prostate carcinoma has become the second most fatal cancer in American men. In Dunning R3327 rat prostate adenocarcinoma cells, elevated invasiveness positively correlates with metastatic potential. However, the mechanism(s) responsible for regulation of tumor cell motility and invasion is poorly understood. We have reported that a lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], augments tumor cell metastatic potential through activation of protein kinase C (PKC). PURPOSE: We proposed to determine the effect of 12(S)-HETE on the motility and invasion of low-metastatic rat prostate AT2.1 tumor cells and the effect of 12(S)-HETE activation of specific PKC isoform(s) in these processes. METHODS: The motility of AT2.1 cells was determined by the colloidal gold phagokinetic track assay and the invasiveness measured as their ability to invade through basement membrane Matrigel-coated filters. Expression of PKC isoforms was determined by Western blotting of the whole cell lysate with isoform-specific anti-PKC antibodies. Cytosol and membrane fractions were prepared and the subcellular distribution of PKC was analyzed by Western blotting and activity assay. The effect of 12(S)-HETE on cell proliferation was examined. Data were analyzed for significance of difference with the two-sampled, two-sided Student's t test. RESULTS: 12(S)-HETE increased the motility and invasion of AT2.1 cells, and this 12(S)-HETE-increased motility and invasion were inhibited by a selective PKC inhibitor, calphostin C, as well as a Ca2 chelator, bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/tetra(acetoxy-methyl)ester. AT2.1 cells expressed the PKC isoforms alpha and delta, and 12(S)-HETE increased the membrane association of PKC alpha but not delta. Further, the motility and invasion of AT2.1 cells were increased by thymelea toxin, a selective activator of PKC alpha over PKC delta. CONCLUSION: 12(S)-HETE augments the invasiveness of AT2.1 cells via selective activation of PKC alpha. IMPLICATIONS: 12(S)-HETE modulation of PKC alpha invasiveness may be an important mechanism of action for the regulation of the invasive potential of rat prostate carcinoma cells, and the 12-lipoxygenase enzyme and/or PKC alpha may serve as key targets for the development of anti-invasive agents useful for combating the spread of prostate cancer.

Platelet enhancement of tumor cell adhesion to subendothelial matrix: role of platelet cytoskeleton and platelet membrane.

Platelet involvement during tumor cell adhesion to subendothelial matrix was examined in vitro. Platelets were subjected to thrombin stimulation and mechanical lysis and examined for their effects on tumor cell adhesion. These treatments altered the platelet ultrastructure and cytoskeletal integrity. Untreated washed rat platelets (WRP) exhibited extensive adhesion to and spreading on substrates and substantially enhanced tumor cell adhesion to the same substrates (i.e., 250% greater than tumor cells without platelets). Thrombin prestimulation of platelets limited platelet adhesion and spreading and platelet facilitation of tumor cell adhesion. Complete mechanical lysis disrupted both the platelet membrane and the cytoskeleton and eliminated the ability of platelets to adhere or to enhance tumor cell adhesion. Partially lysed platelets resembled membrane ghosts and facilitated tumor cell adhesion by a mechanism independent of spreading and cytoskeletal rearrangement. Fractionation studies indicated that platelet cytoskeletal components played a role in the adhesion process. Pretreatment of WRP with cytochalasin A or B dose dependently inhibited microfilament-mediated platelet spreading and platelet-enhanced tumor cell adhesion. Colchicine and vinblastine induced microtubule depolymerization, but they had no observable effect on platelet spreading or platelet-enhanced tumor cell adhesion. It was concluded that platelet-enhanced tumor cell adhesion to subendothelial matrix depends on an intact platelet cytoskeleton and on a platelet membrane component(s) and is mediated by surface contact between platelets and tumor cells. Furthermore, platelet-mediated tumor cell adhesion to subendothelial matrix may involve two mechanisms: one dependent on, and one independent of, platelet spreading and cytoskeletal rearrangement.

Autocrine motility factor induces differential 12-lipoxygenase expression and activity in high- and low-metastatic K1735 melanoma cell variants.

A M(r) 55,000 tumor cell-secreted cytokine has been described which influenced the migration of the producing cells and was called autocrine motility factor (AMF). Activation of the cell surface receptor for AMF (gp78) was shown to stimulate production of a 12-lipoxygenase metabolite of arachidonic acid, 12-(S)-hydroxyeicosatetraenoic acid [12-(S)-HETE], in highly metastatic murine melanoma cells. AMF stimulated the motility of the high-metastatic (K1735-M1) but not the low-metastatic variant (K1735-Cl.11) of the K1735 murine melanoma and increased expression of the 12-lipoxygenase enzyme predominantly in the high-metastatic counterpart. The K1735-M1 cells responded to motile stimulation with increased endogenous 12-(S)-HETE production, and, reciprocally, exogenous 12-(S)-HETE up-regulated surface gp78 and caused gp78 translocation from an intracellular perinuclear pool to tubulovesicles which extended to the cell periphery in the K1735-M1 cells exclusively. These results suggest that differences in AMF responses may be due to alterations in the capacity of low-metastatic cells to transduce signals through 12-lipoxygenase or to involve downstream effector(s) of 12-(S)-HETE after gp78 activation.

Identification of a novel truncated alphaIIb integrin.

Integrin alphaIIb beta3 requires its cytoplasmic tails to participate in tumor cell adhesion, spreading, and migration. Using 3' rapid amplification of cDNA ends, we have amplified two alphaIIb cDNAs from human leukemia, prostate adenocarcinoma, and melanoma cells. One of these is the predicted wild-type alphaIIb cDNA, and the other is a novel truncated alphaIIb variant. This variant is unique in that it lacks the transmembrane and cytoplasmic portions of the alphaIIb light chain. The truncated alphaIIb integrin protein is expressed by human leukemia, prostate adenocarcinoma, and melanoma cells but not by platelets or normal prostate epithelial or normal breast epithelial cells. Tumor cells secrete this protein and deposit it on the extracellular matrix. To our knowledge, this is the first report of a naturally occurring variant of an alpha integrin that lacks the transmembrane and cytoplasmic tail.

Activation of microvascular endothelium by eicosanoid 12(S)-hydroxyeicosatetraenoic acid leads to enhanced tumor cell adhesion via up-regulation of surface expression of alpha v beta 3 integrin: a posttranscriptional, protein kinase C- and cytoskeleton-dependent process.

Tumor cell interaction with endothelial cells is a crucial step leading to organ-selective metastasis. Adhesion of murine B16 amelanotic melanoma cells (B16a) to murine microvascular endothelial cells (CD3) was enhanced, in a dose- and time-dependent manner, by pretreating CD3 cells with 12(S)-hydroperoxyeicosatetraenoic acid [i.e., 12(S)-HETE], a 12-lipoxygenase metabolite of arachidonic acid. The metabolic precursor of 12(S)-HETE, 12-HPETE (12-hydroperoxyeicosatetraenoic acid) also enhanced B16a cell adhesion to CD3 monolayers, whereas other lipoxygenase products, i.e., 5(S), 11(S), and 15(S)-HETEs were ineffective. 12(S)-HETE-enhanced tumor cell adhesion was blocked by treating endothelial cells with antibodies against the alpha v beta 3 complex or against individual subunits but not with antibodies against alpha 5 beta 1. In contrast, neither of these two integrins appeared to be involved in tumor cell adhesion to unstimulated endothelium. Flow cytometric analysis, immunofluorescent labeling, and image analysis indicated that 12(S)-HETE induced a time- and dose-dependent increase in the surface expression of alpha v beta 3 but not alpha 5 beta 1 on CD3 cells. The increased surface expression of alpha v beta 3 on endothelial cells did not result from an increased transcription or translation of alpha v beta 3 message as confirmed by quantitative reverse transcription-polymerase chain reaction, Northern blotting, and quantitative Western blotting. Instead, subcellular fractionation studies revealed an increased translocation of alpha v beta 3 integrins from the cytosolic pool to the membrane fractions. Pretreatment of endothelial cells with several cytoskeleton-disrupting agents (i.e., cycloheximide or acrylamide to disrupt intermediate filament vimentin, cytochalasin D to disrupt microfilaments, colchicine or Nocodazole to disrupt microtubules) abolished the 12(S)-HETE-enhanced alpha v beta 3 surface expression as well as tumor cell adhesion to endothelial cells. Also, pretreatment of CD3 cells with protein kinase C inhibitor calphostin C, but not with protein kinase A inhibitor H8, blocked 12(S)-HETE-enhanced alpha v beta 3 surface expression and tumor cell adhesion. Collectively, these results suggest that eicosanoid 12(S)-HETE modulates tumor cell interaction with endothelium via protein kinase C- and cytoskeleton-dependent up-regulation of the surface expression of alpha v beta 3 integrin.

Frequent loss of expression and loss of heterozygosity of the putative tumor suppressor gene DCC in prostatic carcinomas.

The putative tumor suppressor gene DCC has been shown to be frequently lost or expressed at low levels in colorectal, gastric, pancreatic, and esophageal carcinomas. In the present study, the DCC gene and its mRNA expression in human and rat prostatic carcinoma cells as well as in prostatic carcinoma tissues were examined by reverse transcriptase-polymerase chain reaction and polymerase chain reaction-loss of heterozygosity. The DCC gene was present and expressed in normal prostatic cells. However, its expression was decreased or undetectable in all prostatic carcinoma cells from either humans (4 cell lines) or rats (5 cell lines). In patients, 12 of 14 cases (86%) showed reduced DCC expression and 5 of 11 informative cases (45%) showed loss of heterozygosity at the DCC locus. These results demonstrate that loss of DCC expression and loss of heterozygosity at the DCC locus are a frequent feature of prostatic carcinoma cells.

Effects of prostacyclin on tumor cell-induced platelet aggregation.

Prostacyclin has been evaluated for its ability to inhibit tumor cell-induced platelet aggregation (TCIPA) induced by several rodent tumor lines: B16a (melanoma); 3LL (carcinoma); 15091A (adenocarcinoma); and W256 (carcinosarcoma). Aggregation of human platelets by all four lines was inhibited by prostaglandin I2 (PGI2) in a dose-dependent manner, with complete inhibition observed at 10 ng/ml. However, higher PGI2 concentrations were required to inhibit aggregation of homologous rat platelets induced by W256 cells. Prostacyclin was compared to other icosanoids known to inhibit platelet aggregation and was found to be 100-fold more potent than either prostaglandin E1 or prostaglandin D2 and 1000-fold more potent than its stable nonenzymatic metabolite (6-ketoprostaglandin F1 alpha). Prostaglandin E2 in contrast to prostaglandin E1 and prostaglandin D2, did not inhibit TCIPA; however, both prostaglandin E2 and its enzymatic metabolite (13,14-dihydro-15-ketoprostaglandin E2) prevented PGI2 inhibition of TCIPA. The addition of prostaglandin I2 (100 ng/ml) after initiation of TCIPA (50% of maximum response) resulted in immediate arrest of TCIPA followed by reversal of platelet aggregation. Prostacyclin partially reversed platelet aggregation when added at 100% of maximum response. Platelets enhanced the adhesion of [125I]uridine-labeled W256 cells to plastic culture dishes under both aggregatory and nonaggregatory conditions. Prostacyclin in vitro inhibited platelet-facilitated tumor cell adhesion. These in vitro results demonstrated that PGI2 is a potent inhibitor of TCIPA and of tumor cell adhesion; we suggest that these are possible mechanisms to explain the antimetastatic effects of PGI2 in vivo [Honn, K. V., Cicone, B., and Skoff, A. Science (Wash. D.C.), 212: 1270-1272, 1981].

In vivo characterization of combination antitumor chemotherapy with calcium channel blockers and cis-diamminedichloroplatinum(II).

We have examined nifedipine, a dihydropyridine class calcium channel blocker, for ability to overcome cis-diamminedichloroplatinum(II) (cisplatin) resistance in a murine tumor line variant, B16a-Pt, which we developed for resistance to cisplatin. Nifedipine significantly enhanced the antitumor actions of cisplatin against primary subcutaneous B16a-Pt tumors and their spontaneous pulmonary metastases. We have characterized, in vivo, the pharmacokinetics and dose-response interactions between nifedipine and cisplatin. We now report our studies designed to compare, in vivo, the efficacy of nifedipine and other calcium active compounds including: (a) structurally similar calcium channel blockers (nimodipine, nicardipine) from the dihydropyridine class, (b) structurally different calcium channel blockers from the benzothiazepine (diltiazem) and the phenylalkylamine (verapamil) classes, and (c) calmodulin antagonists (trifluoperazine and calmidazolium) for ability to enhance the antitumor action of cisplatin. Nifedipine was included as the standard or reference compound. In these studies verapamil and diltiazem failed to enhance the antitumor actions of cisplatin as did both calmodulin antagonists. Our findings suggest that nifedipine has a greater degree of specificity for B16a-Pt cells than structurally different calcium channel blockers from other chemical classes (i.e., diltiazem and verapamil), or the two calmodulin antagonists (i.e., trifluoperazine and calmidazolium). We concluded that nifedipine interacts with specific target site(s) which are not accessible by verapamil, by diltiazem, or by the calmodulin antagonists. Surprisingly, the two dihydropyridine class calcium channel blockers, nimodipine and nicardipine, also failed to enhance cisplatin's antitumor actions despite the fact that their specificity and kinetics for binding to the dihydropyridine receptor component of the calcium channel favors them (nimodipine and nicardipine) over nifedipine. Therefore, we postulate that the synergism between cisplatin and nifedipine is independent of the latter's effect on the voltage sensitive, slow inward calcium channel. We suggest that cisplatin cytotoxicity is enhanced by nifedipine's interaction with an as yet unidentified specific "target site," as opposed to nonspecific interactions with the tumor cell plasma membrane or specific interactions with calmodulin or the P-glycoprotein (which is responsible for pleiotropic resistance).

Cathepsin B-like activity in viable tumor cells isolated from rodent tumors.

The cathepsin B-like cysteine proteinase activity which has been implicated in tumor malignancy has been attributed to several cellular sources, including viable tumor cells, necrotic tumor cells, and host-inflammatory cells. We have isolated subpopulations of cells from eight rodent tumors of five histological types, using centrifugal elutriation, and verified the cellular composition of the subpopulations cytologically. Ninety-two % or greater of the cathepsin B-like activity was associated with the isolated fractions containing greater than or equal to 95% tumor cells of 86 +/- 2% (SE) viability (beta fractions). The isolated fractions consisting of necrotic tumor cells and inflammatory cells (alpha fraction) apparently contain a cysteine proteinase inhibitor, since both cathepsin B-like and cathepsin H activities in the beta fraction of B16 amelanotic melanomas could be inhibited by addition of the alpha fraction.

Solid tumor cells express functional "tethered ligand" thrombin receptor.

Previous work demonstrated that alpha-thrombin promoted tumor cell adhesion to endothelium and extracellular matrix as well as enhanced the metastatic capacity of tumor cells. This study was initiated to investigate whether the thrombin effect on tumor cells is mediated through the "tethered ligand" thrombin receptor. RT-PCR analysis using primers based on the human thrombin receptors detected mRNA in human colon adenocarcinoma cells (clone A), whose authenticity was confirmed by Southern hybridization. The presence of thrombin receptor mRNA in rat (W256 carcinosarcoma) and mouse (melanoma) tumor cells was demonstrated by RT-PCR/Southern blotting using species-specific PCR primers. Sequencing of the PCR fragment of clone A cells revealed complete homology with the reported human cDNA sequence. Subsequently, tumor cells derived from three species, i.e., human, rat, and mouse, were found to express the thrombin receptor protein as revealed by immunoblotting using ligand peptide-derived mAb ATAP138, whose reactivity towards the M(r) approximately 66,000, potential thrombin receptor was blocked by preincubating the antibody with the immunogen peptide SFLLRNPNDKYEPF (TRP 14). Finally, peptides TRP 14 and TRP 7 (SFLLRNP), but not TRP 5 (FLLRN), were found to mimic alpha-thrombin in stimulating tumor cell adhesion to fibronectin, suggesting that the thrombin receptors expressed on solid tumor cells are biologically functional.

Loss of heterozygosity of the BRCA1 and other loci on chromosome 17q in human prostate cancer.

A putative tumor suppressor gene, the BRCA1 gene, on chromosome 17q21 has recently been identified and shown to be mutated in breast and ovarian cancers. We have undertaken the present study to explore the possible involvement of the BRCA1 and/or other potential genes on chromosome 17q in prostate cancer. Twenty-three patients were screened by PCR for loss of heterozygosity at five microsatellite loci spanning the region of 17q12-21. One of the loci (i.e., D17S855) studied is intragenic to the BRCA1. Forty-four and 40% of the informative cases showed loss of heterozygosity at the BRCA1 (D17S855) and D17S856 loci, respectively, whereas 10%, 10%, and 11% of the informative cases were positive for loss of heterozygosity at the D17S250, D17S579, and D17S588 loci, respectively. Overall, 52% (11/21) of the informative cases have allelic loss of at least one locus on chromosome 17q. Our data suggest that the BRCA1 and/or other genes within the interval between BRCA1 and D17S856 on 17q21 may be important in the pathogenesis of prostate cancer.

Autocrine motility factor signals integrin-mediated metastatic melanoma cell adhesion and invasion.

The binding of autocrine motility factor (AMF) to its cell surface receptor, gp78, stimulates tumor cell motility. In this report, we provide evidence that stimulation of gp78 by either AMF or a monoclonal antibody to gp78 (3F3A) increases adhesion and spreading of metastatic murine melanoma (B16a) cells on fibronectin. This gp78-regulated increase is mediated by up-regulation of surface alphaIIbbeta3++ and alpha5beta1 integrin receptors. In addition, AMF treatment of B16a cells increased translocation of alphaIIbbeta3 and alpha5beta1 from the cytoplasm to the cell surface. However, alphaIIbbeta3 and alpha5beta1 demonstrate separate and unique staining patterns at the surface of B16a cells in response to stimulation of gp78. Furthermore, stimulation of B16a cells with AMF increased their invasion through Matrigel. This stimulated invasion was inhibited by antibodies to alphaIIbbeta3 but not by antibodies to alpha5beta1. The increased integrin surface expression and function in response to AMF was blocked by N-benzyl-N-hydroxy-5-phenylpentanamide, an inhibitor of 12-lipoxygenase, and calphostin C, an inhibitor of protein kinase C. The results demonstrate that AMF stimulates integrin-mediated B16a cell adhesion, spreading, and invasion, and these events are regulated by a signaling pathway involving 12-lipoxygenases and protein kinase C.

Morphological study of the interaction of intravascular tumor cells with endothelial cells and subendothelial matrix.

Multiple steps or events have been described as essential in the metastatic cascade. Tail vein injection of single cell suspensions was used to study the ultrastructural details of the events involved in the initial arrest and attachment of circulating tumor cells. Lewis Lung Carcinoma (3LL) and a mammary adenocarcinoma (16c) were compared to a previous ultrastructural study of B16 amelanotic melanoma (B16a) detailing morphological events in the initial arrest and attachment of tumor cells in lung. The three murine tumors followed similar steps and varied only slightly in the time sequence of the steps. We observed the following steps: (a) initial arrest of tumor cells was characterized by an intimate tumor endothelial cell contact; (b) platelet activation and aggregation was noted by two minutes. Platelet aggregation continued for 1-4 h until a thrombus formed; (c) after approximately 4 h endothelial cell separation with extension of the tumor cell to the subendothelial matrix was noted; (d) at approximately 24 h the tumor cell associated thrombus dissipated and the attached tumor cells were exposed to a reestablished circulation. (e) mitoses were observed after 24 h with cell division and the development of intravascular tumor nodules; (f) the final step in the extravasation sequence was dissolution of the basement membrane by the attached tumor cells.

A new in vitro model for investigation of tumor cell-platelet-endothelial cell interactions and concomitant eicosanoid biosynthesis.

We have developed a new in vitro model system to examine tumor cell-platelet-endothelial cell interactions under dynamic conditions. Using the same model, we can determine endogenous eicosanoid metabolism and alterations in the prostacyclin-thromboxane A2 balance associated with interactions among tumor cells, platelets, and endothelial cells. The model consisted of cloned rat aortic endothelial cells grown on gelatin microcarrier beads under dynamic conditions (i.e., spinner culture). Interactions of these endothelial cells with platelets (heparinized rat platelet rich plasma) and/or tumor cells (rat Walker 256 carcinosarcoma) were assessed in an aggregometer. Gelatin beads alone or microcarrier grown endothelial cells did not elicit spontaneous aggregation of platelet rich plasma over a time period of 30 min. Microcarrier grown endothelial cells inhibited tumor cell induced platelet aggregation in a dose dependent fashion (i.e., depending on endothelial cell number). The ability of microcarrier grown endothelial cells to inhibit tumor cell induced platelet aggregation depended on endogenous production of prostacyclin. This conclusion is based on the following results: an increased number of microcarrier grown endothelial cells caused a prolongation of the aggregation lag time; an increased number of microcarrier grown endothelial cells caused a proportionate increase in 6-keto-prostaglandin F1 alpha concentration; an increased number of microcarrier grown endothelial cells was inversely correlated with thromboxane A2 production by platelets; indomethacin pretreatment of microcarrier grown endothelial cells caused a decrease in prostacyclin production and therefore overcame the associated inhibition of tumor cell induced platelet aggregation; and the inhibition of tumor cell induced platelet aggregation in the presence of endogenous prostacyclin produced by microcarrier grown endothelial cells was the same as that observed in the presence of exogenous prostacyclin. Scanning electron microscopy of aggregometry samples revealed: little or no platelet or tumor cell adhesion to gelatin beads alone, a low basal adhesion of tumor cells to microcarrier grown endothelial cells, and large aggregates of platelets and tumor cells located primarily at gaps in the monolayer of indomethacin treated microcarrier grown endothelial cells. This new in vitro model provides a method for examining the effects of eicosanoid metabolism by endothelial cells on tumor cell-platelet-endothelial cell interactions under dynamic conditions.

The high affinity alphaIIb beta3 integrin is involved in invasion of human melanoma cells.

Integrins play an important role in mediating tumor cell-extracellular matrix (ECM) and tumor cell-endothelial cell interactions. The integrin alphaIIb beta3 (GPIIb-IIIa) is expressed on the surface of platelets in an inactive state and requires a conformational change to recognize extracellular matrix proteins such as fibrinogen, fibronectin, vitronectin, and others. In this study, we questioned whether human melanoma cells express the alphaIIb beta3 integrin. Reverse transcription-PCR/Southern blotting, Northern blotting, and dot blotting demonstrated the presence of the platelet-type alphaIIb beta3 integrin in human melanoma WM 983B, WM 983A, and WM 35 cells. AP-2, a monoclonal antibody (mAb) to alphaIIb beta3, positively stained two human melanoma specimens, indicating expression of this integrin in vivo. Phorbol 12-myristate 13-acetate and 12(S)-hydroxyeicosatetraenoic acid, two activators of protein kinase C, stimulated adhesion of melanoma cells to immobilized fibronectin and PAC-1, a mAb to alphaIIb beta3. PAC-1 specifically recognizes the conformationally active form of platelet alphaIIb beta3. Phorbol 12-myristate 13-acetate-stimulated adhesion of WM 983B cells to PAC-1 was completely blocked by an RGD peptide, thus providing evidence that tumor cell adhesion to PAC-1 is mediated via the alphaIIb beta3 integrin but not the Fc receptor. Confocal immunofluorescent studies demonstrated that fibronectin-adherent melanoma cells possess an intracellularly localized pool of high-affinity alphaIIb beta3. Invasion of WM 983B cells through fibronectin was stimulated by 12(S)-hydroxyeicosatetraenoic acid, and this stimulated invasion was blocked by the mAb PAC-1. The data suggest that melanoma cells express the high-affinity alphaIIb beta3 integrin, which is involved in tumor invasion.