(CANCER RESEARCH 58. 4047-4051. September 15. 1998] Advances in Brief Platelet-Type 12-Lipoxygenase in a Human Prostate Carcinoma Stimulates Angiogenesis and Tumor Growth1 Daotai Nie, Gilda G. Hulmán, Timothy Geddes, Keqin Tang, Christopher Pierson, David J. Grignon, and Kenneth V. Honn2 Departments of Radiation Oncology ¡D.N., T. G., K. T., K. V. HJ. Urology ¡G.G. HJ, and Pathology [C. P.. D. J. G., K. V. HJ. Wayne State University School of Medicine, Detroit, Michigan 48202 Abstract Previously, we found a positive correlation between the expression of platelet-type 12-lipoxygenase ( 12-1.OX l and the progression of human prostate adenocarcinoma (PCa; Gao et al., Urology, 46: 227-237, 1995). To determine the role of 12-1.OX in PCa progression, we generated stable 12-LOX-transfected PC3 cells, which synthesize high levels of 12-LOX protein and 12(.S)-hvdroxyeicosatetraenoic acid metabolite. In vitro, 12LOX-transfected PC3 cells demonstrated a proliferation rate similar to neo controls. However, following s.c. injection into athymic nude mice, 12-LOX-transfected PC3 cells formed larger tumors than did the controls. Decreased necrosis and increased vascularization were observed in the tumors from 12-LOX-transfected PC3 cells. Both endothelial cell migra tion and Matrigel implantation assays indicate that 12-LOX-transfected PC3 cells were more angiogenic than their neo controls. These data indicate that 12-LOX stimulates human PCa tumor growth by a novel angiogenic mechanism. Introduction The growth and metastasis of solid tumors are dependent upon the ability of tumor cells to induce angiogenesis (1). Angiogenesis, the formation of new blood vessels from preexisting ones, involves en dothelial cell proliferation, motility, and differentiation. Tumor cells can secrete a variety of angiogenic factors, such as basic fibroblast growth factor and vascular endothelial growth factor, to stimulate angiogenesis (2). Tumor cells also produce angiogenesis inhibitors such as thrombospondin and angiostatin to control angiogenesis (2). The balance between angiogenesis stimulators and inhibitors deter mines the angiogenicity of tumor cells (2). In human PCa,3 the level of vascularization positively correlates with tumor stage (3-5). Inhi bition of angiogenesis by linomide or TNP-470 potently inhibits PCa growth and metastasis by causing necrosis and apoptosis in tumors (6, 7). Although various potential angiogenesis factors have been identified in prostate cancer (8), it is still unclear by which process PCa cells become angiogenic. We have previously detected the ex pression of platelet-type 12-LOX in human PCa and demonstrated a correlation between 12-LOX mRNA expression and pathological stage (9). Platelet-type 12-LOX uses only arachidonic acid as sub strate and forms 12(5)-HETE exclusively (10). Here, we have exam ined the function of 12-LOX on PCa tumor growth. Our data dem- onstrate that 12-LOX has no detectable effect on PCa cell growth in vitro but stimulates PCa tumor growth in vivo. This effect of 12-LOX on tumor growth is closely related to increased angiogenesis. Both in vitro and in vivo angiogenesis assays suggest that PCa cells expressing high levels of 12-LOX are more angiogenic than those expressing no or low levels of 12-LOX. Our results provide a novel function for platelet-type 12-LOX in PCa progression. Materials and Methods Cell Culture. Rat angiogenic endothelial cell line RV-ECT (a gift from Dr. Clement Diglio, Department of Pathology, Wayne State University) was maintained in DMEM with 10% FBS (11). The cells were used between passage numbers 29 and 34. The human prostate carcinoma cell line PC3 was originally purchased from American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 with 10% FBS. All culture reagents were purchased from Life Technologies, Inc. Stable Transfection of PC3 Cells and Characterization. Passage 28 PC3 cells were cotransfected using a Lipofectin reagent (Life Technologies, Inc.) with a pCMV-platelet-type 12-LOX construct (a gift from Dr. Collin Funk, Center for Experimental Therapeutics, University of Pennsylvania; Ref. 10), and pCMV-neo, which encodes a neomycin-resistant protein. PC3 cells transfected with pCMV-neo were used as controls. Transfectants were selected using 1 mg/ml geneticin (G418) in RPMI with 10% FBS and then cloned using a limiting dilution method in 96-well plates. The cloned transfectants were propagated and characterized for 12-LOX mRNA expression by Northern blot and 12-LOX protein expression by Western blot. Human epidermoid carci noma A431 cells that express 12-LOX (12) were used as a positive control. The probe used in Northern blot was the 12-LOX cDNA from pCMV 12-LOX construct. Rabbit 12-LOX polyclonal antibody used in Western blot was purchased from Oxford Biomedicai Inc. (Oxford. MI). Actin antibody was from Amersham (Arlington Heights, IL). The synthesis of 12(5)-HETE by 12-LOX transfectants was determined using a RIA kit from Perspective Diagnostics (Cambridge. MA) according to the manufacturer's instructions. In Vitro Proliferation Assay. To study the growth kinetics of PC3 trans fectants in culture. 2 x 10' cells per well were seeded in 96-well culture plate. The number of viable cells at intervals of 48 h was assessed using an MTS cell proliferation assay kit (Promega Corp, Madison. MI). The /\49(,nmreadings 2-3 h after plating were used as baselines. The number of cells was expressed as the percentage of increase from the Am> nm baselines. Animal Model and Histochemical Studies. A total of 4 X IO6 12-LOXtransfected PC3 cells or neo control cells in 200 /¿Iof HBSS were injected s.c. into the right flank of 4-6-week-old male BALB/c nude mice (obtained from Received 5/20/98: accepted 7/30/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by NIH CA 29997. United States Army Prostate Cancer Research Program DAMD 17-98-1-8502. and the Harper Hospital Development Fund (to K. V. H.). D. N. was supported by a fellowship from the Cancer Research Foundation of American. 2 To whom requests for reprints should be addressed, at Department of Radiation Oncology, 431 Chemistry Building. Wayne State University. Detroit. MI 48202. Phone: (313)577-1018: Fax: (313)577-0798. 3 The abbreviations used are: PCa, prostate adenocarcinoma: 12-LOX, 12-lipoxygen ase; 12(S)-HETE, !2(5)-hydroxyeicosatetraenoic acid; RV-ECT, rat vascular endothelial cells-tube forming; FBS. fetal bovine serum; HPF, high-power field. University of South Florida. Tampa. FL). The resulting tumors were measured using a vernier caliper, and tumor volume was calculated using the formula: (width)2 X length X 0.5 (7). Six to 7 weeks after injection, mice were sacrificed, and the tumors were resected and photographed under an SP SZ-4060 stereomicroscope (Olympus America, Melville, NY). Tumors were fixed in 10% neutral buffered formalin and embedded in paraffin, and sections (5 fxm) were prepared for histology staining. Sections were stained with H&E to examine the presence of necrosis. The assessment of tumor necrotic area was performed for a total of 10 HPFs per tumor using a double-blind approach. CD31 staining was used to assess tumor vascularization. Immunohistochemical staining for CD31 (DAKO Corp.: dilution, 1:20) was performed 4047 Downloaded from cancerres.aacrjournals.org on February 20, 2016. © 1998 American Association for Cancer Research. 12-LIPOXYGENASE. using a standard avidin-biotin complex-immunoperoxidase ANGIOGENESIS. procedure. AND TUMOR GROWTH The slides were counterstained with hematoxylin. The vascularity was assessed qualitatively on the basis of overall vessel organization and quantitatively by microvessel density. A total of 10 fields per tumor were evaluated for both microvessel density and vessel organization. The microvessel density was indicated by the average number of vessels crossing an arbitrary line across one HPF field. The rating of vessel organization was performed according to the following scale: 0, disorganized, staining randomly distributed; 1, inter mediate, vessel-like structures formed; and 2, highly organized, vessels struc 28S _ ,12-LOX r 18S tured and organized. Endothelial Cell Migration Assay. For the cell migration assay, RV-ECT endothelial cells were harvested by trypsinization and resuspended in RPMI with 10% FBS, and 5 X IO5 cells in 0.5 ml were plated on the top chamber of a modified Boyden chamber (Becton Dickinson. Bedford, MA). Then. 1 ml of RPMI-10% FBS medium conditioned from PC3 or various transfectant cul tures or fresh medium with 12(S)-HETE was added in triplicate into the lower chamber. After 4 h of incubation, the cells on the top side of the transwell membrane were removed with cotton swabs. The membrane was then cut out, fixed in a quick-fix solution, double-stained, and mounted for observation and counting. Usually, 12 fields (X100) representing two perpendicular cross-lines B of each membrane were counted. Matrigel Implantation Assay for Tumor Cell-induced Angiogenesis. The Matrgel implantation assay was performed as described by Ito et al. (13) with the following modifications. Matrigel (Becton Dickinson, Bedford, MA: 0.4 ml premixed with 2 X IO6 PC3 12-LOX transfectant or neo control cells) A kPa v 2> 6» ,12-LOX 75- was injected s.c. into nude mice (four mice per group). Mice were sacrificed 12 days after injection and dissected to expose the implants for recording. 33- Results Generation of PC3 Transfectants That Constitutively Synthe size 12-LOX and 12(S)-HETE. To determine the function of 12LOX in PCa progression, PC3 cells were transfected with a platelettype 12-LOX cDNA construct. Stable transfectants were cloned and named the nL series. Several stable transfectants (neo series) isolated from PC3 cells transfected with pCMV-neo were used as controls. Northern blot analyses of transfectant clones show that the levels of 12-LOX mRNA were increased in various nL clones, compared to the neo controls or wild-type PC3 (Fig. 1A). The 12-LOX mRNA levels in various nL clones were higher than in A431, a cell line that constitutively expresses 12-LOX (12). 12-LOX-transfected PC3 cells also had higher levels of 12-LOX protein than neo controls or wildtype PC3, as revealed by Western blot analysis (Fig. ÕB).Among the various clones analyzed, nL-2, nL-8, nL-11, and nL-12 expressed 12-LOX at the highest levels. We also found that 12-LOX-transfected PC3 clones nL-2, nL-8, and nL-12 synthesized 6-10-fold more 12(S)HETE than the neo control or wild-type PC3 cells (Fig. 1C), indicat ing that 12(S)-HETE biosynthesis was greatly enhanced in 12-LOXtransfected PC3 cells. 12-LOX Transfectants Have an in Vivo but not an in Vitro Growth Advantage. In vitro, the growth rates of several 12-LOX transfectant clones were similar to those of neo controls and wild-type PC3 cells (Fig. 2A), with an approximate doubling time of 36 h. However, following s. c. injection into nude mice, 12-LOX-trans fected PC3 cells (nL-2 and nL-12) grew faster and formed larger tumors than did neo controls (neo-cr and neo-a; Fig. 2ß).As shown in Fig. 2C, tumors derived from 12-LOX-transfected PC3 cells were larger than those obtained from neo controls, indicating that 12-LOXtransfected PC3 cells had an in vivo growth advantage compared to neo controls or wild-type PC3 cells. Similar results were obtained with an additional 12-LOX transfectant clone tested (nL-8; data not shown). Assessment of tumor necrosis from H&E-stained tumor sec tions revealed that tumor necrosis was significantly reduced in the tumors derived from 12-LOX-transfected PC3 cells (P < 0.05 by Student's / test), whereas 12.1% of tumor area of neo-cr tumors were necrotic (n = 7; range, 5-35%), only 1.9% of tumor area in the nL-12 300 250 5ï 200 l 150 100 CM O. 50 O Fig. 1. Generalion of PC3 transfectants synthesizing high levels of 12-LOX and 12(5)-HETE. The transfection of PC3 cells and the cloning of stable transfectants were performed as described in "Materials and Methods." A, Northern blot analysis of 12-LOX mRNA levels in various clones of PC3 12-LOX transfectants. Top, blot probed with 12-LOX cDNA; bottom, blot probed with actin cDNA as the loading control. B, Western blot analysis of 12-LOX protein expression in various clones of PC3 12-LOX transfec tants. The blot was probed with a 12-LOX polyclonal antibody and actin antibody. C, 12(S)-HETE levels in various 12-LOX transfectants. The levels of 12(S)-HETE in total cell lysates were measured using RIA and were normalized to cell number and expressed as pg of 12(S)-HETE/1 X IO6 cells. clone was necrotic (n = 8; range, 0-10%). A significant decrease in tumor necrosis was also observed in the tumors derived from 12-LOX transfectants nL-2 and nL-8, compared to neo-a (data not shown), suggesting that the increased tumor growth by 12-LOX transfectants is mainly due to the reduction of tumor necrosis. Increased Angiogenesis in the Tumors from 12-LOX Transfec tants. Because angiogenesis plays an important role in tumor growth by influencing tumor necrosis and apoptosis (2), we studied whether the increased tumor growth by 12-LOX transfectant is angiogenesis 4048 Downloaded from cancerres.aacrjournals.org on February 20, 2016. © 1998 American Association for Cancer Research. 12-LIPOXYGENASE, ANGIOGENESIS, AND TUMOR GROWTH B fe 1600 Fig. 2. 12-LOX transfectants have an in vivo but not in vilro growth advantage. A, growth ki netics of PC3 transfectants in culture. Cell prolif eration of various transfectants was measured as described in "Materials and Methods." Shown here 01234567 Days of Culture are the growth curves of PC3 wild type (O). neo-tr (•),nL-8 (V), and nL-12 (A). Data points, means of six determinations: bars, SE. Other clones such as nL-2 and neo-a also had similar growth kinetics (data not shown). B, growth kinetics of the tumors derived from 12-LOX transfectants and neo con trols. Data points, mean volumes of eight tumors for nL-12 (•)and neo-cr (O). five tumors for nL-2 (D) and neo-a (V), and six tumors for PC3 wild type (T); bars. SE. C. mice with tumors from 12-LOX transfectants or from neo control. Left, three mice with tumors from neo-o~ (arrows): right, three mice bearing tumors from I2-LOXtransfected PC3 cells (nL-12; arrows). neo-a 0 10 20 30 40 50 60 70 Days after Injection nL-12 ! i dependent. We found significant vascularization in tumors derived from 12-LOX-transfected PC3 cells, whereas the neo control tumors showed little vessel penetration (Fig. 3A). Immunostaining with CD31 antibody, which detects the presence of endothelial cells, showed that the vascular networks in tumors derived from nL-12 were sinusoidal in pattern and well developed in structure (Fig. 30, right). In contrast, in neo control tumors, endothelial cells were present but were ran domly distributed and did not form an organized vascular network (Fig. 35, left). There were fewer vessels in neo-tr tumors than in nL-12, as suggested by microvessel density (Fig. 3Q. The assessment of the vessel organization demonstrated that the majority of vessels in the tumors derived from 12-LOX-transfected PC3 cells were highly organized, whereas in those from neo-er, they showed a disorganized to intermediate pattern (Fig. 3D). In tumors derived from nL-2 and nL-8, we also observed a similar increase in angiogenesis when compared to neo-a (data not shown). Increased Angiogenicity of 12-LOX Transfectants. The in creased angiogenesis in the tumors generated from 12-LOX-trans fected PC3 cells raises the question of whether the observed increase in angiogenesis is the cause or a consequence of the increased tumor growth. To address this issue, we first assayed the conditioned culture medium of PC3 12-LOX-transfected PC3 cells or neo controls for their ability to stimulate endothelial cell migration. As shown in Fig. 4A, the medium from the 12-LOX-transfected PC3 cells induced more RV-ECT migration than did the medium from neo controls. Under similar assay conditions, 12(S)-HETE itself also stimulated RV-ECT migration at nanomolar levels (Fig. 4B). The increased angiogenicity of 12-LOX transfectants was confirmed by the Matrigel implantation assay. As shown in Fig. 4C, within 12 days, 12-LOX-transfected PC3 cells (nL-12) in Matrigel induced massive angiogenesis, indicated by the accumulation of blood in the gel, compared to the neo control (neo-o-). The results clearly illustrate that the 12-LOX-transfected PC3 cells are more angiogenic than their neo controls. Discussion Here, we found that the increased expression of 12-LOX in human PCa cells stimulates prostate tumor growth by enhancing their angio genicity. The findings have significant bearing on the regulation of PCa progression because, in patients diagnosed with prostate carci noma, some tumors are extremely malignant, with rapid progression, whereas others are localized and dormant for many years. Exploration of the mechanism underlying the transition from latent to rapidly growing PCa will provide useful information for PCa management. 4049 Downloaded from cancerres.aacrjournals.org on February 20, 2016. © 1998 American Association for Cancer Research. 12-LlPOXYGENASE. neo-oc nL-2 ANGIOOENESIS. B AND TUMOR GROWTH neo-a nL-12 Fig. 3. Increased angiogenesis in the lumors from 12-LOX-transfected PC3 cells. A. tumor morphology. Left, two tumors from neo-a; right. Iwo tumors from 12-LOX-transfected PC3 cells (nL-2). X8. B, CD31 immunostaining. Brawn, positive staining. Left, control tumor. Note the scattered vascular spaces which are randomly distributed and do not form a structured vascular network. Righi, tumor from 12-LOX-transfected PC3 cells. Note the numerous vascular channels showing a highly organized sinusoidal pattern surrounding small nests of tumor cells. X250. C. microvessel density. Column*, microvessel densities, expressed as the average number of vessel-like structures crossing an arbitrary line in one HPF; burs. SE. Note the significant increase in microvessel density in the tumors derived from nL-12 (n = 7) as compared in those of neo-cr (n = 1: *, P < 0.05 by Student's t test). D. organization of intratumoral blood vessels. The vessel organization was scored as described in "Materials and Methods." Columns, mean scores of tumors derived from nL-12 (n = 7) and neo-<r (n = 7); bars. SE (**, P < 0.01 by Student's l test). Our observations here, together with our previous demonstration of the correlation between 12-LOX expression and PCa progression in clinical samples (9), suggest that 12-LOX may play a critical role in the progression of human PCa. The increased tumor growth observed with 12-LOX-transfected PC3 cells is due to the reduction in tumor necrosis as a result of increased angiogenesis. The increased 12-LOX levels in PC3 cells did not confer a growth advantage in vitro, suggesting that 12-LOX overexpression does not have direct effect on PC3 cell growth and that the growth advantage of 12-LOX transfectants in vivo is due to the host environment. This tumor-host interaction based mechanism is supported by the observed increase in angiogenesis in the tumors from 12-LOX-transfected PC3 cells. Because angiogenesis is required for tumor expansion, the lack of or inhibition of angiogenesis has been demonstrated to induce tumor cell necrosis and apoptosis, thereby limiting tumor growth in PCa (2, 6-7). Indeed, histological analysis revealed that the tumors derived from neo controls had increased necrosis, suggesting that it is the insufficient vascularization that limited the growth of the neo control tumors. The increased angiogenesis in the tumors from 12-LOX-trans fected PC3 cells is at least partly due to their increased angiogenicity. 12-LOX-transfected PC3 cells have increased ability to lators and inhibitors of angiogenesis (2). Therefore, it will be interesting to determine how 12-LOX up-regulates the angiogenicity of PCa cells. One explanation is that 12-LOX or 12(S)-HETE may increase the angiogenicity of tumor cells by influencing the expression of angiogenic or angiostatic molecules. An alternative interpretation is that 12(5)-HETE may directly alter the balance in favor of angiogenic factors due to its proangiogenic nature. This is supported by our finding here that 12(S)-HETE stimulated endo thelial cell migration at nanomolar levels and previous reports showing that 12(5)-HETE stimulated endothelial cell proliferation (14), retraction (15), and adhesion and that it increased the surface expression of integrin avß3in both macro- and microvascular endothelial cells (16). It is noteworthy that integrin avß,is pre dominantly associated with angiogenic blood vessels (17) and plays an essential role in human cancer angiogenesis (18). Thus, 12(5)-HETE may directly increase the angiogenicity of PCa cells by stimulating angiogenesis or by eliciting several proangiogenic responses that can be additive or synergistic to effects from other angiogenic factors produced by PCa cells because different factors have their own distinct effects on the process of angiogenesis (19). Studies are ongoing to determine whether increased 12-LOX ex pression in PCa cells influences the gene expression of angiogenic factors and whether 12(S)-HETE can stimulate angiogenesis alone stimulate endothelial cell migration in vitro and neovascularization of Matrigel in vivo, compared to their neo controls. The angiogeor by its additive or synergistic interaction with other putative nicity of tumor cells is controlled by the balance between stimuangiogenic factors. 4050 Downloaded from cancerres.aacrjournals.org on February 20, 2016. © 1998 American Association for Cancer Research. 12-LIPOXYGENASE. ANGIOGENESIS. AND TUMOR GROWTH Acknowledgments Special thanks are directed to Dr. Clement Diglio for providing RV-ECT endothelial cells. We thank Dr. Mohit Trikha and Karoly Szekeres for helpful discussions. We acknowledge the excellent technical support from Homan Kian and Ning Wu. References Conditioned Media 1. Folkman. J.. and Shing, Y. Angiogenesis. J. Biol. Chem.. 267: 10931-10934, 1992. 2. Hanahan. D.. and Folkman. J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell, 86: 353-364. 1996. 3. Vartanian. R. K.. and Weidner. N. EC proliferation in prostatic carcinoma and prostatic hyperplasia: correlation with Gleason's score, microvessel density, and epithelial cell proliferation. Lab. Invest., 73: 844-850. 1995. 4. Wakui. S.. Furusato. M.. Itoh. T.. Sasaki. H.. Akiyama. A.. Kinoshita. I., Asano. K.. Tokuda. T.. Aizawa. S., and Ushigome. S. Tumor angiogenesis in prostatic carcinoma with and without bone marrow metastasis: a morphometric study. J. Pathol., 168: 257-262, 1992. 5. Weidner, N.. Carroll. P. R., Flax, J., Blumenfeld, W., and Folkman, J. Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. Am. J. Pathol.. ¡43:401-409, 1993. 6. Vukanovic, J., and Isaacs, J. T. Human prostatic cancer cells are sensitive to programed (apoptotic) death induced by the antiangiogenic agent linomide. Cancer Res., 55:3517-3520, 1995. 7. Yamaoka. M.. Yamamoto. T., Ikeyama. S.. Sudo. K., and Fujita. T. Angiogenesis inhibitor TNP-470 (AGM-1470) potently inhibits the tumor growth of hormoneindependent human breast and prostate carcinoma cell lines. Cancer Res.. 53: 5233- 12(S)-HETE (nM) / CM Fig. 4. Increased angiogenicity of 12-LOX-transfected PC3 cells. A, stimulation of endothelial cell migration by the conditioned medium from 12-LOX transfectants. The conditioned media were harvested after 24 h of culture and used for migration assay as described in "Materials and Methods." Columns, average numbers of cells migrated per field; bars, SE (**, P < 0.01 by Student's 1 test). B, 12(S)-HETE stimulates endothelial cell migration. The migration assay was performed essentially as described in A except that media with various levels of 12(5)-HETE. instead of the conditioned media, were placed into the lower chamber. Columns, means; bars, SE (**, P < 0.01 by Student's / test). C, induction of angiogenesis in Matrigel by 12-LOX transfectants. Top, three Matrigel implants premixed with 2X10" neo-o- cells. Note the vessel penetration into the gel was minimal, with little blood accumulated in the gel. Bottom, in contrast, the Matrigel premixed with 2 x IO6 12-LOX transfectant (nL-12) demonstrates considerable blood accumulation. One final point concerns the expression of 12-LOX during human PCa progression. If the effects of 12-LOX on PCa tumor growth and angiogenesis just described are of physiological significance, it should be expected that PCa cells express 12-LOX. This has, in fact, been observed in vivo, where the expression of 12-LOX has been positively correlated with tumor stage (9). The question of how 12-LOX expres sion is up-regulated in PCa cells is currently being explored. 5236. 1993. 8. Campbell, S. C. Advances in angiogenesis research: relevance to urological oncology. J. Uro!., ISS: 1663-1674, 1997. 9. Gao, X., Grignon. D. J., Chbihi, T., Zacharek, A.. Chen, Y. Q., Sakr. W., Porter, A. T.. Crissman, J. D., Pontes, J. E., Powell, I. J.. and Honn, K. V. Elevated 12-lipoxygenase mRNA expression correlates with advanced stage and poor differentiation of human prostate cancer. Urology, 46: 227-237, 1995. 10. Funk. C. D.. Furci. L.. and FitzGerald. G. A. Molecular cloning, primary structure. and expression of the human platelet/erythroleukemia cell 12-lipoxygenase. Proc. Nati. Acad. Sci. USA, 87: 5638-5642, 1990. 11. Diglio, C. A.. Liu. W.. Grammas. P.. Giacomelli. F.. and Wiener. J. Isolation and characterization of cerebral resistance vessel endothelium in culture. Tissue Cell. 25: 833-846, 1993. 12. Hagmann. W., Gao, X., Timar, J., Chen. Y. Q.. Strohmaier, A. R., Fahrenkopf. C., Kagawa, D., Lee, M., Zacharek, A., and Honn. K. V. 12-Lipoxygenase in A431 cells: genetic identity, modulation of expression, and intracellular localization. Exp. Cell Res.. 228: 197-205. 1996. 13. Ito, Y., Iwamoto, Y., Tanaka. K.. Okuyama. K.. and Sugioka, Y. A quantitative assay using basement membrane extracts to study tumor angiogenesis in VMYJ. Int. J. Cancer, 67: 148-152, 1996. 14. Tang, D. G., Renaud. C.. Stojakovic, S., Diglio. C. A.. Porter. A., and Honn. K. V. 12(S)-HETE is a mitogenic factor for microvascular ECs: its potential role in angiogenesis. Biochem. Biophys. Res. Commun.. 211: 462-468. 1995. 15. Honn, K. V.. Tang. D. G., Grossi. I.. Duniec, Z. M., Timar, J.. Renaud, C.. Leithauser. M.. Blair. I.. Johnson, C. R., Diglio. C. A., Kimler, V. A.. Taylor, J. D.. and Marne», L. J. Tumor cell-derived 12(5)-hydroxyeicosatetraenoic acid induces microvascular endothelial cell retraction. Cancer Res.. 54: 565-574, 1994. 16. Tang, D. G., Chen, Y. Q., Diglio, C. A., and Honn, K. V. Transcriptional activation of EC integrin av by protein kinase C activator 12(S)-HETE. J. Cell Sci., 108: 2629-2644. 1995. 17. Brooks. P. C.. Clark, R. A. F.. and Cheresh. D. A. Requirement of vascular integrin avßjfor angiogenesis. Science (Washington DC). 264: 569-571, 1994. 18. Brooks. P. C.. Stromblad. S.. Klemke, R.. Visscher. D.. Sarkar, F. H.. and Cheresh. D. A. Antiintegrin av/3, blocks human breast cancer growth and angiogenesis in human skin. J. Clin. Invest., 96: 1815-1822, 1995. 19. Kumar, R.. Yoneda, J.. Bucana. C. D.. and Fidler. I. J. Regulation of distinct steps of angiogenesis by different angiogenic molecules. Int. J. Oncol.. 12: 749-757, 1998. 4051 Downloaded from cancerres.aacrjournals.org on February 20, 2016. © 1998 American Association for Cancer Research. Platelet-Type 12-Lipoxygenase in a Human Prostate Carcinoma Stimulates Angiogenesis and Tumor Growth Daotai Nie, Gilda G. Hillman, Timothy Geddes, et al. Cancer Res 1998;58:4047-4051. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/58/18/4047 Sign up to receive free email-alerts related to this article or journal. 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