Enhanced cell motility and invasion of chicken embryo fibroblasts in response to Jun over-expression.

Malignant tumor cells exhibit a number of distinct properties involved not only with deregulated cell proliferation but also enhanced migration and invasion. The Jun oncogene has been well studied in regard to its role in cell proliferation. Many of the target genes deregulated by Jun encode matrix metalloproteases (MMPs) such as MMP1, MMP3 and MMP9. These targets implicate a prominent role for Jun in tumor cell invasion, in addition to its role in growth transformation. To investigate this possibility, we have examined the effect of over-expression of transforming and non-transforming versions of Jun on motility and invasion of chicken embryo fibroblasts (CEFs). We found that over-expression of either form of Jun results in elevated intrinsic cellular motility as well as increased motility in response to several different chemo-attractants, including 3T3-conditioned media, basic fibroblast growth factor, hepatocyte growth factor and Matrigel. The capacity of these cells to invade through Matrigel is also elevated as a result of Jun over-expression. In addition to these effects, CEFs expressing Jun secrete factors that stimulate the motility of a human tongue carcinoma cell line. Our results suggest that Jun plays an important role in the potentiation of cell motility and invasion through multiple mechanisms.

Involvement of Shc in the signaling response of human prostate tumor cell lines to epidermal growth factor.

Autocrine growth factors for the epidermal growth factor receptor (EGFR) have been identified in prostate tumors, implicating a role for EGFR in the progression of prostate cancer. To investigate early signaling mechanisms used by the EGFR in prostate tumor cells, we have characterized the involvement of the Shc (src homology 2/x-collagen related) adapter protein in EGFR signaling in several human prostate tumor cell lines. In androgen-responsive lymph node-prostate cancer (LNCaP) cells and androgen-insensitive PC3, DU145 and PPC-I cells, Shc was identified as one of the most prominent phosphotyrosine proteins to be elevated in response to EGF. Equivalent levels of the 46- and 52-kDa Shc isoforms were detected in all of the tumor cell lines tested. However, levels of the 66-kDa isoform were variable among the cell lines. In all of the tumor cell lines, EGF caused an association between Shc and Grb2, another adapter protein linked to cellular ras activation. Additionally, several phosphotyrosine proteins, including a 115-120-kDa protein in EGF-treated LNCaP cells, co-associated with Shc. The profile of these Shc-associating proteins, however, differed among the tumor cell lines. Our results indicate that Shc is a common downstream element of EGFR signaling in prostate tumor cells and suggest multiple functions for Shc in prostate tumorigenesis.

Cytochrome P-450-dependent HETEs: profile of biological activity and stimulation by vasoactive peptides.

The cytochrome P-450 pathway is capable of metabolizing arachidonic acid to omega- and subterminal hydroxylase metabolites, 16-, 17-, 18-, 19-, and 20-hydroxyeicosatetraenoic acids (P-450 HETEs). We have quantitated, by gas chromatography-mass spectrometry (GC/MS), endogenous HETEs exiting the rabbit isolated perfused kidney elicited by hormonal stimulation. Kidneys were perfused with Krebs-Henseleit solution containing indomethacin (2.8 microM) to prevent further metabolism of HETEs by cyclooxygenase. Phenylephrine (2-3 microM) was added to the perfusate to raise perfusion pressure to approximately 80 mmHg. Angiotensin II (ANG II), arginine vasopressin (AVP), and bradykinin (BK) were injected into the renal artery and perfusates collected throughout the vasoactive response. After addition of an internal standard, deuterated 19-HETE, perfusates were extracted and purified and P-450 HETEs were derivatized for GC/MS analysis. Under basal conditions, 16-, 18-, 19-, and 20-HETEs were released (range: 50-270 pg/ml), 19-HETE being the highest and fivefold greater than 16-HETE, the lowest. Injection of 50 ng ANG II increased by two- to sixfold P-450 HETE release associated with an increase of 40 +/- 11 mmHg in perfusion pressure. An equipressor dose of AVP (50 ng) did not release P-450 HETEs nor did a 5-micrograms dose of the vasodilator peptide BK, which decreased perfusion pressure by 22 +/- 6 mmHg. Authentic 19- and 20-HETE isomers resulted in dose-dependent dilation, as did 18(R)- and 16(R)-HETEs, whereas their enantiomers and 17-HETE isomers were without effect on perfusion pressure. The vasodilator effects of 18(R)- and 16(R)-HETEs, like 20- and 19-HETEs, were inhibited by indomethacin. Furthermore, P-450 HETEs exhibited both regio- and stereoselective inhibition of proximal tubule adenosine triphosphatase (ATPase) activity. The (S) enantiomers of 16- and 17-HETE potently inhibited activity, whereas their (R) isomers and other P-450 HETEs had negligible effects on ATPase activity. The quantity of HETEs released from the kidney, either under basal conditions or when stimulated by ANG II, and their biological profile suggest that subterminal HETEs may participate in renal mechanisms affecting vasomotion and tubular transport.

Renal vasodilator activity of 5,6-epoxyeicosatrienoic acid depends upon conversion by cyclooxygenase and release of prostaglandins.

The 5,6-epoxyeicosatrienoic acid (5,6-EET), a renal vasodilator metabolite of arachidonic acid via cytochrome P450 (P450) requires cyclooxygenase for expression of its vasoactivity as the responses are inhibited by indomethacin and other aspirin-like drugs. We now report on the metabolism of 5,6-EET by rabbit kidneys in order to characterize those metabolites that may account for its vasoactivity. The 5,6-EET was injected close-arterially into the rabbit isolated Krebs-Henseleit perfused kidney, preconstricted with phenylephrine, and the effluent collected throughout the response period. Basal collections, following injection of 100 microliters of vehicle, were made at 20-min intervals before each 5,6-EET injection. Prior to acidic extraction, deuterated 6-keto-prostaglandin (PG) F1 alpha and PGE2 were added as internal standards. The extracts were separated by TLC and prostaglandins were derivatized for gas chromatography-mass spectrometry analysis using a negative ion chemical ionization mode. Injection of 0.5, 1, 5, 10, and 20 micrograms of 5,6-EET (n = 4) resulted in dose-related decreases in perfusion pressure of 6 +/- 2, 12 +/- 4, 21 +/- 4, 26 +/- 4, and 27 +/- 7 mm Hg, respectively. Basal perfusates contained 6-keto-PGF1 alpha and PGE2, levels of which were increased by 2-fold or more by 5,6-EET. Perfusates, collected during 5,6-EET administration, also contained 5-hydroxy-PGI1 and 5,6-epoxy-PGE1, cyclooxygenase metabolites of 5,6-EET. This is the first report of the recovery and identification of these 5,6-EET metabolites from an intact organ. Since the responses to 5,6-EET are endothelial-dependent, we also studied the profile of eicosanoids formed following incubation of 5,6-EET with cultured bovine pulmonary endothelial cells. Endothelial cells metabolized 5,6-EET to products with a similar radioactive profile on reverse-phase high pressure liquid chromatography compared to kidney perfusates. We compared the vasodilator activity of 5,6-epoxy-PGE1 and 5-hydroxy-PGI1, chemically synthesized by us from PGE2 and PGF2 alpha, respectively, with PGE2 and PGI2 in the rabbit kidney. The 5,6-epoxy-PGE1 was equipotent to PGE2 as a vasodilator. The ED50 values for 5,6-EET, 5,6-epoxy-PGE1, and PGE2 were 4.69, 0.43, and 0.42 nmol, respectively. Although PGI2 was a potent vasodilator (ED50, 0.24 nmol), 5-hydroxy-PGI1 was devoid of activity. Thus, the cyclooxygenase-dependent vasoactivity of 5,6-EET in the rabbit kidney has two components: release of vasodilator prostaglandins, PGE2 and PGI2, and metabolism of 5,6-EET to a prostaglandin analog, 5,6-epoxy-PGE1.