Fibrinopeptide A and the phosphate content of fibrinogen in venous thromboembolism and disseminated intravascular coagulation

Concentrations of plasma fibrinopeptide A (FPA) were measured by radioimmunoassay in 50 patients with venous thromboembolism or disseminated intravascular coagulation or both. A consistent discrepancy was observed in values obtained with two anti-FPA antisera. Analysis of extracts from plasma of these patients by high-performance liquid chromatography (HPLC) revealed the presence of a phosphorylated and an unphosphorylated form of the A peptide. Differences in concentrations of FPA measured with the two antisera could be accounted for by their different reactivity with phosphorylated FPA (FPA-P). The differences were abolished by treatment with alkaline phosphatase. A good correlation was observed between the FPA-P content of free A- peptide material and of fibrinogen in plasma as determined by HPLC (r = .88, P less than .001, n = 11). In patients with elevated FPA levels, the mean FPA-P content of fibrinogen was significantly higher (P less than .002, n = 13) than in patients with normal FPA levels (n = 8) and in healthy controls (n = 14). Phosphorus in fibrinogen did not correlate with fibrinogen degradation products or fibrinogen levels and became normal on adequate anticoagulation. Therefore, blood-clotting activation may lead to a high phosphate content of fibrinogen and of free FPA in plasma.     

Modulation of implantation-associated integrin expression but not uteroglobin by steroid hormones in an endometrial cell line

In order to test the hypothesis that integrin and uteroglobin (UG) expression in cultured endometrial cells are affected by hormone treatment, Ishikawa-CH endometrial cancer cells were cultured and exposed to oestradiol or oestradiol and progesterone regimens and assayed using immunohistochemistry. We evaluated the intensity of immunohistochemical staining for the integrin monomers alpha(v) and beta1, the dimers alpha(v)beta3 and alpha(v)beta6, and for the secretory protein uteroglobin under various experimental conditions. Cells grown in control media stained positively for the integrin monomers alpha(v) and beta1, the dimer alpha(v)beta3, and for UG. Oestradiol and sequential oestradiol/progesterone reversibly suppressed staining for the dimer alpha(v)beta3. Hormone treatment had no effect on the staining of the beta1 and alpha(v) monomers or UG. The alpha(v)beta6 dimer antibody did not stain under any experimental treatment conditions. These data indicate that expression of the integrin complex alpha(v)beta3 is reversibly suppressed by oestradiol in Ishikawa cells and that these cells may be a good model for studying hormone-driven molecular changes in endometrium.      

Viral delivery of P450 reductase recapitulates the ability of constitutive overexpression of reductase enzymes to potentiate the activity of mitomycin C in human breast cancer xenografts

Indolequinones such as mitomycin C (MMC) require enzymatic bioreduction to yield cytotoxic moieties. An attractive approach to overcome the potential variability in reductive bioactivation between tumors is to exploit specific enzyme-bioreductive drug combinations in an enzyme-directed gene therapy (GDEPT) approach. To this end, human breast cancer cell lines (T47D, MDA468, and MDA231) that overexpress either DT-diaphorase (DTD) or NADPH:cytochrome P450 reductase (P450R) have been developed. Cytotoxicity of MMC was evaluated in the panel of cell lines following aerobic or anoxic exposure in vitro. DTD and/or P450R overexpression sensitized cells to MMC in air with no further increase in the cytotoxicity of MMC under anoxia. The most profound effect was seen in the MDA468 cells, where a 27-fold increase in potency was observed for MMC in the DTD-overexpressing cell line. The MMC sensitization achieved through DTD and P450R overexpression in MDA468 cells was maintained in vivo. Xenografts established from the clonal lines exhibited significant tumor control following MMC treatment (treated/control [T/C] 17% and 51% for DTD and P450R xenografts, respectively) that was not seen in wild-type tumors (T/C 102%). Delivery of a clinically relevant adenoviral vector encoding P450R to MDA468 wild-type tumors yielded comparable P450R activity to that seen in the P450R clonal xenografts and resulted in greater MMC sensitization (T/C 46%). The model systems developed will facilitate the identification of novel indolequinone agents that are targeted toward a specific enzyme for bioactivation and are consequently of potential use in a GDEPT approach.     

Hyaluronan-dependent motility of B cells and leukemic plasma cells in blood, but not of bone marrow plasma cells, in multiple myeloma: alternate use of receptor for hyaluronan-mediated motility (RHAMM) and CD44

We investigated the ability of blood B cells, bone marrow (BM) plasma cells, and terminal leukemic plasma cells (T-PCL) from patients with multiple myeloma (MM) to migrate on extracellular matrix proteins. Hyaluronan (HA), but not collagen type I, collagen type IV, or laminin, promoted migration of MM blood B cells, as determined by time-lapse video microscopy. Between 13% and 20% of MM blood B cells migrated on HA with an average velocity of 19 micron/min, and greater than 75% of MM blood B cells exhibited vigorous cell movement and plasma membrane deformation, as did circulating T-PCL and extraskeletal plasma cells from patients with MM. In contrast, plasma cells obtained from BM of patients with MM lacked motility on all substrates tested and did not exhibit cell membrane protrusions or cellular deformation. MM blood B cells and MM plasma cells from all sources examined expressed the HA- binding receptors receptor for HA-mediated motility (RHAMM) and CD44. On circulating MM B cells, both RHAMM and CD44 participated in HA- binding, indicating their expression ex vivo in an activated conformation. In contrast, for the majority of BM plasma cells in the majority of patients with MM, expression of RHAMM or CD44 was not accompanied by HA binding. A minority of patients did have HA-binding BM plasma cells, involving both RHAMM and CD44, as evidenced by partial blocking with monoclonal antibodies (MoAbs) to RHAMM or to CD44. Despite HA binding by both RHAMM and CD44, migration of MM blood B cells on HA was inhibited by anti-RHAMM but not by anti-CD44 MoAbs, indicating that RHAMM but not CD44 mediates motility on HA. Thus, circulating B and plasma cells in MM exhibit RHAMM- and HA-dependent motile behavior indicative of migratory potential, while BM plasma cells are sessile. We speculate that a subset(s) of circulating B or plasma cells mediates malignant spread in myeloma.     

Induction of NK activity in large granular lymphocyte leukemia: activation with anti-CD3 monoclonal antibody and interleukin 2

Large granular lymphocyte (LGL) leukemia is a rare disease characterized by clonal expansion of LGL associated with chronic neutropenia, multiple auto-antibodies, and occasionally polyarthritis. We studied cell surface antigen expression and functional activity of leukemic LGL from ten such patients. Using two-color flow cytometric analysis, we found that leukemic LGL from all ten patients expressed the CD3 and HNK-1 markers, while cells from only four patients expressed IgG Fc receptors (FcR). The LGL leukemic cells had little or no NK activity (defined as MHC-nonrestricted cytotoxicity against K562 target cells); however, NK activity could be induced in leukemic LGL by in vitro treatment with as little as 0.05 microgram/mL of anti-CD3 monoclonal antibody. Cell sorting experiments demonstrated that NK activity was induced in CD3+ leukemic LGL (either CD3+, HNK-1+ or CD3+, FcR+) with anti-CD3 monoclonal antibody but not in normal CD3+, FcR- T cells. Treatment with purified interleukin 2 (IL 2) also caused direct activation of some CD3+ leukemic LGL. Despite induction with anti-CD3 MAb or IL 2, activated leukemic LGL did not proliferate or express high density IL 2 receptors detectable by cell sorter analysis. Treatment with alpha interferon had minimal effect on NK activity of LGL leukemic cells. These results suggest that leukemic LGL may provide a useful model for examining the signals required for LGL maturation and activation.     

Platelet storage: changes in cytosolic Ca2+ actin polymerization and shape

Platelets gradually lose their disc shape during storage. The authors studied simultaneous changes in platelet cytosolic Ca2+ (Cai) and the polymerization state of actin as related to the shape. Platelet concentrates were stored under blood bank conditions for up to 10 days. Aliquots were removed and analyzed as follows: platelet Cai and increments in Cai induced by adenosine diphosphate (ADP) were determined by fluorescence of fura-2-loaded cells; loss of disc shape was determined by differences in light scattering intensity induced by stirring; and the ratio of globular and total actin (G/T) of platelets in plasma was determined by a modification of the DNase inhibition assay. Globular actin was found to be 86 +/- 3% of total actin in freshly drawn platelets suspended in plasma. The following changes occurred during storage: G/T in platelet concentrates increased from 63 +/- 5 (day 0) to 74 +/- 2% in the first 24 hours then fell to 33 +/- 6% by day 10. The percent discoid platelets also increased from day 0 to day 1 then fell in the ensuing days. There was an initial drop in Cai from day 0 to day 1, after which Cai increased on days 3 and 6. Globular actin polymerization during storage closely correlated with the change in percent discs (r = 0.95). During 6 days of storage Cai was highly correlated with shape change (r = 0.97) and to a lesser extent (r = 0.87) with the ratio of globular actin. The authors conclude that actin polymerization, shape, and Ca2+ change in a related fashion during storage.     

Proliferation of human malignant hematopoietic cells in immunodeficient mice: suppression by antibody to pluripotent K-562 leukemia cells involves direct cytolysis and effector cells

Six human hematopoetic cell lines were successfully heterotransplanted into athymic (nude) and asplenic-athymic (lasat) neonatal mice. The tumors arising from leukemia and lymphoma cells could then be serially transplanted into adult nude mice. Seven days after the fourth serial mouse passage, each mouse was treated with goat immune gamma globulin against K-562 cells. One control group was treated similarly, but with nonimmune (normal) gamma globulin, while another control group was not treated. The goat gamma globulin was not toxic for nude and lasat mice, and the immune, but not the normal, gamma globulin suppressed local subcutaneous growth of myelosarcomas, lymphosarcomas, and Burkitt lymphoma cells. On the other hand, the growth of lung, breast, and prostatic carcinomas and a melanoma of human origin were not altered by the immune gamma globulin. Since suppression of cell growth occurred equally well in decomplemented mice, a complement-mediated cytotoxicity apparently cannot be considered as responsible for the abrogation. The Fab fragment of the immunoglobulin did not suppress the growth of the myelosarcomas. We conclude that antibody suppression of the in vivo proliferation was specific for malignant hematopoietic cells and that the Fc portion of IgG is necessary for in vivo cytolysis of leukemia cells. The most probable mechanisms are direct antibody cytolysis and antibody-dependent macrophage-mediated cytotoxicity.     

Expression of pertussis toxin adenosine diphosphate-ribosyltransferase in a T-cell hybridoma reduces metastatic capacity

T-cell hybridomas are highly metastatic, and their in vitro invasiveness correlates with metastatic capacity. Invasion is blocked by pertussis toxin (PT), which adenosine diphosphate (ADP)-ribosylates G1-proteins, and we have provided evidence that the PT-sensitive signal stimulates leukocyte function-associated antigen-1 (LFA-1)-mediated adhesion required for invasion. PT pretreatment of TAM2D2 T-cell hybridoma cells reduced metastasis, but only to a limited extent. In the present study, we have transfected the cDNA of the PT ADP- ribosyltransferase S1 subunit into TAM2D2 cells to abrogate G1-protein function permanently. We report here a substantial reduction in the metastatic capacity of two transfectants, S05 and S09, in which 88% and 95% of the G1-proteins was ADP-ribosylated. Two-thirds of the mice injected with S09 cells were tumor-free. Metastasis to the liver was almost completely prevented and less metastases were formed in the spleen and kidneys. Metastasis formation by S05 cells in liver and spleen was much reduced, but in lymph nodes and peritoneal tissues, metastases occurred with a frequency similar to that of controls. We conclude that G1-proteins play an important role in T-cell hybridoma metastasis. We propose that the reduction in metastasis is due to diminished entry of tumor cells from the blood into tissues.