Lysophospholipid growth factors in the initiation, progression, metastases, and management of ovarian cancer.

Levels of lysophosphatidic acid (LPA) and lysophosphatidylcholine (LPC) are elevated in the plasma and ascites of ovarian cancer patients, but not in most other tumor types. LPA increases cell proliferation, cell survival, resistance to cisplatin, cell shrinkage, and production of vascular endothelial growth factor, urokinase plasminogen activator, and LPA itself in ovarian cancer cells, but not in normal ovarian surface epithelial cells. PSP24 and members of the endothelial differentiation gene (EDG) family (EDG1, EDG2, EDG4, and EDG7) of G protein-coupled receptors mediate LPA signaling. Ovarian cancer cell lines do not express EDG1 mRNA, have variable EDG2 mRNA and protein levels, and frequently exhibit levels of EDG4 mRNA and protein, suggesting that EDG4 may contribute to the deleterious effects of LPA in ovarian cancer. In contrast, activation of the EDG2 LPA receptor on ovarian cancer cells may lead to apoptosis and counter the effects of other LPA receptors. Thus, the development of agonists and antagonists for the appropriate spectrum of LPA receptors may alter proliferation, apoptosis, or response to therapy of ovarian cancer cells. Indeed, over 60% of all current drugs target the G protein-coupled family of receptors, making the LPA receptor family a "drugable" target. LPC, although not as thoroughly studied, increases cellular proliferation and mediates multiple other functions through unique signaling pathways.

Elevated levels and altered fatty acid composition of plasma lysophosphatidylcholine(lysoPC) in ovarian cancer patients.

Lysophosphatidylcholine (lysoPC), a product of phosphatidylcholine (PC) hydrolysis via phospholipase A activity, has been proposed to activate cells from a number of lineages. Here, we demonstrate that lysoPC levels are significantly elevated (by 43% overall, relative to normal controls) in the plasma of ovarian cancer patients. This does not appear to be common to all cancers as 5 out of 6 leukemia patients tested had markedly lower (less than one-half of normal) plasma lysoPC. In the plasma of ovarian cancer patients, the percentages of palmitoyl- and stearoyl-lysoPC species were significantly higher, whereas oleoyl and particularly linoleoyl-lysoPC were significantly lower than in control subjects. The molar ratios of lysoPC/PC and palmitoyl-lysoPC/linoleoyl-lysoPC were also significantly elevated in the plasma of ovarian cancer patients compared with those of control subjects. Furthermore, the calculated value of plasma (lysoPC/ PC) x (palmitoyl-lysoPC/linoleoyl-lysoPC) was markedly higher in patients compared with controls.

Increased formation of phosphatidylinositol-4-phosphate in human platelets stimulated with lysophosphatidic acid.

Lysophosphatidic acid (LPA, 1-acyl-sn-glycerol 3-phosphate), at a concentration of 1-40 microM, was found to induce the formation of [3H]inositol-labelled phosphatidylinositol-4-phosphate (PIP) without significantly altering the levels of either phosphatidylinositol (PI) or phosphatidylinositol bisphosphate (PIP2) in washed human platelets. Preincubation of platelets with the cyclooxygenase/lipoxygenase inhibitor, BW755C at 100 microM, did not alter the LPA-induced formation of PIP. Activation of platelets with the phorbol ester, phorbol 12-myristate 13-acetate (PMA), elicited a similar response (induction of PIP formation). The specific protein kinase C (PKC) inhibitor, GF109203X (10 microM), completely blocked the effect of PMA but not the LPA-induced generation of PIP. The present results indicate that LPA can induce PIP formation via PI-4-kinase activation, through processes which are independent of the eicosanoid/TxA2 pathway and are not PKC-dependent.

Characterization of an ovarian cancer activating factor in ascites from ovarian cancer patients.

Ascites from ovarian cancer patients contain potent growth-promoting activity toward human ovarian cancer cells both in vitro and in vivo. This activity is associated with rapid increases in cytosolic free calcium ([Ca2+]i) as a consequence of phosphoinositide hydrolysis. In this study, we describe the purification, characterization, and identification of an ovarian cancer activating factor (OCAF) from ascites of ovarian cancer patients. We have isolated OCAF by a combination of solvent extraction, silica gel chromatography, and TLC. Mass spectral analysis, phospholipase sensitivity, and gas chromatographic behavior of purified OCAF indicate that OCAF is composed of various species of lysophosphatidic acid (LPA), including LPAs with polyunsaturated fatty acyl chains (linoleic, arachidonic, and docosahexaenoic acids). However, OCAF is more potent than sn-1 palmitoyl, oleoyl, or stearoyl LPA in increasing [Ca2+]i in ovarian cancer cells. The ability of OCAF to alter [Ca2+]i is sensitive to the effects of lipoxidase, whereas the activity of sn-1 oleoyl, stearoyl, or palmitoyl LPA is not, suggesting that polyunsaturated bonds in the fatty acyl chain of OCAF may account for its increased ability to activate ovarian cancer cells. Furthermore, a sn-2 linoleoyl LPA generated by phospholipase A1 treatment of synthetic phosphatidic acid is much more active than are sn-1 palmitoyl, stearoyl, or oleoyl LPA in increasing [Ca2+]i in ovarian cancer cells. Taken together, these data suggest that the ability of OCAF to increase cellular calcium may reside in the structure and/or location of the fatty acyl chain of LPA. Purified OCAF, at concentrations similar to those present in ascites from ovarian cancer patients, was sufficient to induce proliferation of ovarian cancer cells, as indicated by thymidine incorporation, reduction of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, or colony formation. However, even at optimal concentrations of OCAF, proliferation was lower than that induced by FCS or ascites from ovarian cancer patients, indicating that, although OCAF may be a major regulator of ovarian cancer cells in vivo, it is not the sole mediator present in ascites, and it likely functions in concert with other growth factor activities.

Single-use percutaneous catheters for intraperitoneal P32 therapy.

BACKGROUND: Intraperitoneal (IP) radioactive chromic phosphate (P32) remains investigational in the treatment of patients with ovarian and/or endometrial cancer. Single-use percutaneously placed catheters offer the advantage of therapy without additional surgery. METHODS: Between August, 1986 and October, 1992, 25 patients underwent bedside percutaneous catheter placement under local anesthesia without ultrasonographic or radiologic guidance, using a specialized central venous catheter. RESULTS: Catheter insertion was successful in 22 of 25 patients (88%) with good IP distribution. Of these, 18 of 22 patients (82%) underwent successful catheter placement with one attempt and 4 of 22 (18%) after one to three additional attempts. The technical failure rate was 12%. Multiple catheter placement attempts were associated with an increased incidence of complications (r = 0.63). Bowel entry occurred in 4 of 25 patients (16%) during 5 of 43 attempts at catheter placement (12%) but was without clinical sequelae. The likelihood of bowel entry significantly increased with more than two attempts (P = 0.02). A median of 39 days (range, 7-156 days) elapsed between the preceding laparotomy and catheter insertion. CONCLUSIONS: Percutaneous catheter placement is successful and well tolerated in the majority of patients and should be considered for patients receiving IP P32.

Correlation between platelet aggregation and dephosphorylation of a 68 kDa protein revealed through the use of putative PKC inhibitors.

The efficacy of two structurally and functionally unrelated protein kinase C (PKC) inhibitors, chelerythrine and calphostin C, was assessed in intact human platelets by studying platelet aggregation in response to stimulation with phorbol 12-myristate 13-acetate (PMA) or the thromboxane-A2 mimetic, U46619. Surprisingly, both inhibitors increased aggregation in response to PMA, but decreased aggregation in response to U46619. To further explore this phenomenon, gel electrophoresis of 32P-labelled proteins from PMA- or U46619-stimulated platelets in the presence and absence of the two putative PKC inhibitors was performed. Although neither chelerythrine nor calphostin C proved to be effective PKC inhibitors in intact human platelets, a strong correlation between the dephosphorylation of a 68 kDa protein and the rate of platelet aggregation was observed. From these results, the indiscriminate use of PKC inhibitors in whole platelets is questioned and attention is drawn to the role of protein dephosphorylation in platelet activation. The 68 kDa protein was the major phosphorylated substrate in resting platelets. Okadaic acid increased phosphorylation of this band, indicating active phosphate group turnover under resting conditions.

Mass and fatty acid composition of the 3-phosphorylated phosphatidylinositol bisphosphate isomer in stimulated human platelets.

By high pressure liquid chromatography (HPLC) analysis, the occurrence of radiolabeled 3-phosphorylated phosphoinositides has been well documented in several cell systems, including agonist-stimulated platelets. The actual mass amounts and fatty acid composition of these unique lipids, however, have not been reported to date. In the present study, we report the mass and fatty acid composition of phosphatidylinositol (PI) 3,4-P2 from U46619-stimulated platelets using a thin-layer chromatographic system for the separation of PI 3,4-P2 from PI 4,5-P2. The mass of PI 3,4-P2 in the stimulated platelet was 180 +/- 9.7 pmol/1 x 10(9) platelets (mean +/- S.E., n = 4), representing 9.3% of total phosphatidylinositol bisphosphate (PIP2). Based on HPLC analysis, PI 3,4-P2 in unstimulated platelets represented < 0.5% of total PIP2 (which corresponds to < 7.0 pmol/1 x 10(9) platelets). Fatty acid analysis of this lipid revealed a composition very similar to the conventional polyphosphoinositides (stearic and arachidonic acids accounting for 44.2 and 40.4 mol %, respectively, of the fatty acids). Since PI 3,4-P2 also did not appear to be distinct from the other polyphosphoinositides, in regard to radiolabeling properties, it was concluded that this lipid is unlikely to originate from a unique precursor pool. This conclusion validates the use of HPLC analysis of radiolabeled phosphoinositides for the estimation of PI 3,4-P2 mass in agonist-stimulated platelets. The chromatographic procedure described should prove useful for the mass and fatty acid analysis of PI 3,4-P2 from other cell systems.

Effect of genistein, a tyrosine kinase inhibitor, on U46619-induced phosphoinositide phosphorylation in human platelets.

Recent evidence suggests that the agonist-induced formation of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) via PI and PIP kinases may play an important role in transmembrane signalling. In the present work, the effect of genistein, a specific inhibitor of protein-tyrosine kinase, on phosphoinositide phosphorylation was studied in human platelets stimulated with the endoperoxide analogue, U46619. At 100 microM concentration, genistein, but not the related compounds, flavone and biochanin A, which possess only weak anti-protein-tyrosine kinase activity, significantly inhibited the U46619-induced accumulation of [3H]PIP (by 71%) and [3H]PIP2. These data suggest that phosphoinositide phosphorylation may be regulated, in part, by tyrosine phosphorylation in U46619-stimulated platelets.

The functional heterogeneity of CD8+ cells defined by anti-CD45RA (2H4) and anti-CD29 (4B4) antibodies.

The monoclonal antibodies, anti-2H4(CD45RA), and anti-4B4(CD29), along with UCHL1-(CD45RO), identify reciprocal populations of CD4 cells with distinct suppressor inducer (CD45RA+CD29-CD45RO-) and helper inducer (CD45RA-CD29+CD45RO+) functions. Although the CD8+ population is known to contain precytotoxic, cytotoxic, suppressor, and some natural killer cells, the exact phenotypic identities of these functional CD8 subsets has not been established. In this study, we tried to determine whether these monoclonal antibodies could distinguish functionally distinct subsets of cells within the CD8+ population. For this purpose, whole T cells or fractionated T cells were sensitized with irradiated allogeneic non-T cells for 6 days, following which, CD8+ or CD8+CD11b- cells were isolated and cellular functions such as suppressor, killer precursor, and killer effector activity were assessed. The results showed that both class I-restricted alloantigen-specific killer effector and killer precursor cells belonged to the CD8+CD11b-CD45RA-CD29+ population. Moreover, these killer effector cells expressed the CTL-associated S6F1 molecule, an epitope of the LFA-1 antigen. In contrast, suppressor effector cells belonged to the CD8+CD11b-CD45RA+CD29- cell population. Although the UCHL1 antigen has been reported to define the CD4+CD29+ helper inducer cell, over 90% of allo-activated CD8+ cells expressed this antigen, whereas only 40-60% of these cells expressed either CD45RA or CD29 antigens. These results suggest that anti-CD45RA and anti-CD29 antibodies may provide useful tools for distinguishing between suppressor effector versus killer effector and killer precursor cells within the CD8+CD11b- population.

Effect of albumin-bound DHA on phosphoinositide phosphorylation in collagen stimulated human platelets.

The effect of exogenous albumin-bound docosahexaenoic acid (22:6n-3) (DHA), arachidonic acid (20:4n-6) (AA), and eicosapendaenoic acid (20:5n-3) (EPA) on phosphoinositide metabolism following collagen stimulation was studied using [3H]inositol prelabelled platelets. Collagen stimulation (3 min, 1.8 micrograms/ml) increased the labelling of both phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4,5-biphosphate (PIP2). Of the fatty acids tested, only pre-incubation (2 min) with DHA (20 microM) significantly attenuated the collagen-induced increased PIP and PIP2 labelling; EPA was without effect, while AA enhanced PIP labelling. Forty microM DHA was less effective at attenuating the increased PIP and PIP2 labelling even though this concentration of DHA resulted in greater inhibition of platelet aggregation. Neither concentration of DHA attenuated the increased polyphosphoinositide labelling resulting from stimulation by the endoperoxide analogue U46619, or the phorbol ester, PMA. These data suggest that the effect of DHA on attenuating the increased PIP and PIP2 labelling following collagen stimulation likely occurs before thromboxane receptor occupancy, may not occur at the level of protein kinase C activation, and could be mediated in part via a lessened synthesis of thromboxane A2.

Albumin-bound docosahexaenoic acid and collagen-induced human platelet reactivity.

An in vitro system designed to mimic the effect of various plasma nonesterified (polyunsaturated) fatty acids on platelet function and metabolism was employed. Human platelet aggregation induced by submaximal (1.8 micrograms/ml) collagen stimulation was significantly inhibited by 2 min preincubation with 20 microM albumin-bound docosahexaenoic acid (22:6n-3) (DHA), but not by the other fatty acids tested. [3H]Phosphatidic acid (PA) formation, an indicator of phospholipase C activation following platelet stimulation, was moderately inhibited by eicosapentaenoic acid (20:5n-3), 11,14,17-eicosatrienoic acid (20:3n-3), dihomo-gamma-linolenic acid (20:3n-6), as well as DHA, but not by arachidonic acid (20:4n-6); this inhibition of phospholipase C activation could not explain the differential effect of DHA on platelet aggregation. The decreased production of thromboxane A2 (TxA2), as assessed by [3H]12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) formation, may account for the inhibition of collagen-induced aggregation by 20 microM DHA. Surprisingly, preincubation with 40 microM albumin-bound DHA, even though resulting in greater inhibition of collagen-induced aggregation, had less impact on HHT formation. A small but significant increase in [3H]prostaglandin D2 (PGD2) levels following 3-min collagen stimulation may have contributed to the greater antiaggregatory effect of 40 muM DHA. It is concluded that increased plasma nonesterified DHA may contribute to the dampened platelet activation and altered metabolism following fish oil supplementation of the diet.

Dynamics of adrenocorticotropin (ACTH) secretion in cyclic Cushing's syndrome: evidence for more than one abnormal ACTH biorhythm.

We have studied a 57-yr-old woman with cyclic Cushing's syndrome of apparent pituitary origin who had a predominant cycle of 2-6 days. The patient also demonstrated an abnormal circadian rhythm, with afternoon peaks of plasma ACTH and plasma cortisol. In addition to these abnormal biorhythms, Fourier analysis showed what appeared to be a separate 35-day cycle. After 35 days of consecutive urinary free cortisol measurement, the patient was given cyproheptadine. During therapy with this agent, the urinary free cortisol levels fell dramatically, but cyclic secretion continued, albeit with a diminished amplitude. During general anesthesia for a bilateral adrenalectomy, there was a striking increase in the plasma ACTH level, and the ACTH concentration remained high in both the immediate and late postoperative periods. These observations indicated that stress could overcome cyclic ACTH secretion and that cortisol exerted feedback suppression on ACTH secretion. Although this is the predictable response for classic pituitary-dependent Cushing's syndrome, it is of interest in cyclic Cushing's syndrome, since previous studies of this entity have implied that cortisol secretion is independent of stimulation or feedback.