Signaling mechanisms responsible for lysophosphatidic acid-induced urokinase plasminogen activator expression in ovarian cancer cells

Lysophosphatidic acid (LPA) enhances urokinase plasminogen activator (uPA) expression in ovarian cancer cells; however, the molecular mechanisms responsible for this event have not been investigated. In this study, we used the invasive ovarian cancer SK-OV-3 cell line to explore the signaling molecules and pathways essential for LPA-induced uPA up-regulation. With the aid of specific inhibitors and dominant negative forms of signaling molecules, we determined that the G(i)-associated pathway mediates this LPA-induced event. Moreover, constitutively active H-Ras and Raf-1-activating H-Ras mutant enhance uPA expression, whereas dominant negative H-Ras and Raf-1 block LPA-induced uPA up-regulation, suggesting that the Ras-Raf pathway works downstream of G(i) to mediate this LPA-induced process. Surprisingly, dominant negative MEK1 or Erk2 displays only marginal inhibitory effect on LPA-induced uPA up-regulation, suggesting that a signaling pathway distinct from Raf-MEK1/2-Erk is the prominent pathway responsible for this process. In this report, we demonstrate that LPA activates NF-kappaB in a Ras-Raf-dependent manner and that blocking NF-kappaB activation with either non-phosphorylable IkappaB or dominant negative IkappaB kinase abolished LPA-induced uPA up-regulation and uPA promoter activation. Furthermore, introducing mutations to knock out the NF-kappaB binding site of the uPA promoter results in over 80% reduction in LPA-induced uPA promoter activation, whereas this activity is largely intact with the promoter containing mutations in the AP1 binding sites. Thus these results suggest that the G(i)-Ras-Raf-NF-kappaB signaling cascade is responsible for LPA-induced uPA up-regulation in ovarian cancer cells.     

Species-specific DNA sequences for identification of somatic hybrids between Lycopersicon esculentum and Solanum acaule

Summary Species-specific highly repeated DNA sequences can be used to screen the progeny of protoplast fusions combining different species. Such probes are easy to clone and can be detected by fast methods, e.g., hybridization to total genomic DNA. Furthermore, due to their high copy number, hybridization signals are strong and represent more than one locus, unlike isozymes or resistance markers. After cloning and screening for species-specific DNA sequences we characterized the highly repeated DNA sequences of the solanaceous species Solanum acaule and Lycopersicon esculentum var. gilva. DNA sequencing and hy ridization revealed a prominent, tandemly arranged satellite DNA repeat of 162 bp in Lycopersicon esculentum and a different satellite repeat of 183 bp, also tandemly organized, in Solanum acaule. Each repeat is absent in the respective other species. Therefore, we have used these DNA repeats as markers to distinguish regenerated interspecific somatic hybrids from the respective fusion partners. These hybrids were clearly identified by Southern hybridization and dot-blot assays to the respective ³²P-labelled satellite DNA.     

Analysis of the equine lymphocyte antigen system by Southern blot hybridization

Fourteen Standardbred horses homozygous for one of six equine lymphocyte antigen (ELA) specificities (A1, A3, A4, A5, A6, or A10) were analyzed by Southern blot hybridization using DNA probes derived from the mouse major histocompatibility complex (MHC). Total genomic DNA from peripheral lymphocytes was digested with the restriction enzymes Hind III, Pvu II, or Eco RI. Twenty-three to thirty-three bands were generated for individual horses with the class I cDNA probe. The resulting band patterns revealed 12-14 nonpolymorphic fragments, which is consistent with the highly conservedQa/Tla genes seen in other species. The remaining 10–19 bands displayed significant polymorphism; no two animals had identical band patterns when studied with all three enzymes. The polymorpism was markedly decreased between animals of the same ELA serotypes. Unique bands were identified in both Al horses and all four A6 animals. Pvu II digestions of lymphocyte DNA were hybridized with mouse MHC class II probes. A cDNA probe for theE gene revealed only a single nonpolymorphic band. In contrast, a cDNA probe for theH-2 A locus displayed three to five strong bands in each animal with polymorphism that was most pronounced between horses of different ELA serotypes. Genomic DNA probes forAandE genes both revealed multiple polymorphic bands. However, cross-hybridization between these two probes prevented distinction betweenA andE equivalent loci. The reduced polymorphism evident within ELA specificities is consistent with the concept that the equine lymphocyte antigen system includes two families of closely linked MHC genes.     

Functional characterization of the H-2Dp gene product

A clone containing the D ^p gene was used to transform L cells. The D^p product expressed was identified by two-dimensional gel electrophoreis and flow cytometry. The D^p product expressed by the L cells was recognized by D^P-specific flow cytometry. The D^p product expressed by the L cells was recognized by D^p-specific but not K^p-specific killer T cells. This killing was inhibited by monoclonal antibodies specific for D^p but not K^p or K^k antigens. Similarly, lymphocytic choriomeningitis virus (LCMV) killer T cells from B 10.P mice were able to kill LCMV-infected L12a cells, but not LCMV-infected Ltk⁺. Again only D^p monoclonal antibodies could inhibit this killing.     

Cell-mediated immune response to syngeneic UV induced tumors: I. The presence of tumor associated macrophages and their possible role in the in Vitro generation of cytotoxic lymphocytes

A primary in vitro sensitization system employing a chromium release assay was utilized to investigate reactivity of murine spleen cells toward syngeneic ultraviolet (uv) light induced fibrosarcomas. These tumors are immunologically rejected in vivo when implanted into normal syngeneic mice but grow progressivly when implanted into syngeneic mice that had previously been irradiated with subcarcinogenic levels of uv light. Following appropriate sensitization, spleen cells from both normal and uv irradiated mice are capable of developing cytotoxic lymphocytes in vitro against the uv induced tumors. It was subsequently discovered that in situ uv induced tumors all contained macrophages of host origin that became demonstrable only after enzymatic dissociation of the tumor tissue. These macrophages were immunologically active in vitro as their presence in the stimulator cell population was necessary to achieve an optimum anti-tumor cytotoxic response following in vitro sensitization. Anti-tumor reactivity generated by mixing spleen cells and tumor cells in the absence of tumor derived macrophages could be greatly enhanced by the addition of normal syngeneic peritoneal macrophages. When in vitro anti-tumor reactivity of spleen cells from normal and uv treated mice was compared under these conditions we again found no significant difference in the magnitude of the responses. In addition, the cytotoxic cells generated in response to uv induced tumors appeared to be highly cross reactive with respect to their killing potential. Cross reactive killing was observed between all uv induced tumors tested as well as with a syngeneic benz[a]pyrene (BP) induced fibrosarcoma. No cytotoxicity was observed against normal syngeneic PEC's even through these cells were shown to be susceptible to lysis by anti-H-2^k effector cells. It was concluded that: (a) A significant number of host-derived macrophages are present in uv tumor tissue. (b) These macrophages are important for the in vitro generation of tumor specific cytotoxicity. (c) Spleen cells from uv treated mice are capable of recognizing and responding against uv tumor associated antigens in vitro. Cytotoxic effector cells generated in response to uv induced tumors appear to have specificity for tumor associated antigens (TAA) present on all uv tumors tested as well as a syngeneic BP induced tumor. The relationship between in vivo and in vitro reactivity against uv tumors is discussed.     

Morphinan alkaloids in Papaver bracteatum: Biosynthesis and fate

The known metabolic pathway for hydrophenanthrene alkaloids in Papaver somniferum has been examined for occurrence in P. bracteatum, a species reported to contain thebaine but no codeine or morphine. 1,2-Dehydro-reticulinium-[3-¹⁴C] chloride and (±)-reticuline-[3-¹⁴C] were fed to P. bracteatum plants and both were incorporated, the former into reticuline and thebaine and the latter into thebaine, suggesting that thebaine biosynthesis is the same in the two species. Studies of the natural abundance of morphinan alkaloids in P. bracteatum and the results from feeding codeinone-[16-³H] and codeine-[16-³H] indicate that this species can reduce codeinone to codeine but can not perform either of the demethylations to produce codeinone or morphine. Fed thebaine-[16-³H] was substantially metabolized but not by pathways that involved demethylations to either oripavine or northebaine.     

Probucol therapy overcomes the reproductive defect in CTP: phosphocholine cytidylyltransferase beta2 knockout mice

The synthesis of phosphatidylcholine (PtdCho), the major phospholipid in mammalian cells, is regulated by the CTP:phosphocholine cytidylyltransferase (CCT). Loss of the CCTbeta2 isoform expression in mice results in gonadal dysfunction. CCTbeta2(-/-) females exhibit ovarian tissue disorganization with progressive loss of follicle formation and oocyte maturation. Ultrastructure revealed a disrupted association between ova and granulosa cells and disorganized Golgi apparati in oocytes of CCTbeta2(-/-) mice. Probucol is a cholesterol-lowering agent that stimulates the uptake and retention of lipids carried by lipoproteins in peripheral tissues. Probucol therapy significantly lowered both serum cholesterol and PtdCho levels. Probucol therapy increased fertility in the CCTbeta2(-/-) females 100%, although it did not completely correct the phenotype, the morphological abnormalities in the knockout ovaries or itself stimulate CCT activity directly. These data indicated that a deficiency in de novo PtdCho synthesis could be complemented by altering the metabolism of serum lipoproteins, an alternative source for cellular phospholipid.