Antisense RNA-mediated reduction of p53 induces malignant phenotype in nontumorigenic rat urothelial cells

p53 mutation is commonly associated with high-grade, high-stage human urothelial carcinomas. Recent studies suggest that p53 mutation in low- grade, low-stage bladder carcinomas may be correlated with the progression of the disease. In the present study, we used antisense RNA methodology in vitro to evaluate the significance of the loss of p53 function at an early stage of urinary bladder carcinogenesis. An immortalized nontumorigenic rat urothelial cell line (MYP3) that strongly expresses wild-type (WT) p53 was transfected with a plasmid (pcDL-SR alpha-296) containing a rat WT p53 cDNA in antisense orientation. The transfection resulted in a significant reduction in p53 mRNA expression and protein synthesis, in stimulation of anchorage- dependent growth, and in acquisition of anchorage-independent growth potential. Three such clones, when tested in athymic nude mice, all formed muscle-invasive, high-grade transitional cell carcinomas at s.c. injection sites. When cells were inoculated into an orthotopic site (urinary bladder), one of two antisense transfectants tested formed bulky tumors in the bladder in all seven nude mice and metastases to lungs in three of the seven mice. Analysis of these cells revealed a decrease in the expression of p21 (WAF1, sdi1, or CIP1) and retinoblastoma (Rb) gene product. Phosphorylation of Rb protein was not inhibited when the cells were starved. No significant difference was observed in the expression of p16 protein. In cell cycle analysis, all antisense transfectants tested escaped from G1 arrest by starvation. Furthermore, secretion of interleukin (IL)-6 into culture medium was increased significantly. Treatment with anti-IL-6 antibody suppressed anchorage-dependent growth. This study directly demonstrates that the loss of p53 function at an early stage of urothelial carcinogenesis may result in acquisition of a malignant phenotype by regulating IL-6 production as well as cell cycle related genes.      

Signal transduction by the platelet Fc receptor

We have evaluated the mechanism by which crosslinking human platelet Fc receptor (FcR) for IgG triggers platelet aggregation and the platelet release reaction. Platelet FcR was crosslinked by incubating purified human platelets with anti-FcRII monoclonal antibody and F(ab')2 anti- mouse Ig. The resultant [Ca2+]i increase, monitored by Fura-2 and measured in the absence of extracellular Ca2+, reached a peak of 750 +/- 50 nmol/L. The effects of cyclooxygenase inhibitors, aspirin and indomethacin, and a phospholipase A2 inhibitor, dibromoacetophenone, were examined. Regardless of the inhibitor, at least 25% of the [Ca2+]i increase remained. Thrombin (0.2 U/mL) stimulated an immediate [Ca2+]i increase that reached 1.95 +/- 0.8 mumol/L. The [Ca2+]i increase generated by thrombin was only slightly reduced by these inhibitors. Crosslinking the FcRII of platelets resulted in a fivefold increase in the production of [3H]inositol phosphates, (IP) which, in the absence of extracellular Ca2+ was insensitive to aspirin. The activation of a [Ca2+]i increase along with the measured increases in IP indicate that FcRII crosslinking leads to the activation of phospholipase C (PLC). In contrast to thrombin, platelet activation via FcRII depends to a large extent on arachidonic acid metabolites. However, neither cyclooxygenase nor phospholipase A2 inhibitors completely blocked FcRII-stimulated [Ca2+]i increase. These observations led us to propose that crosslinking of platelet FcRII initially activates PLC.     

The Role of Serotonin (5-HT) in Behavioral Control: Findings from Animal Research and Clinical Implications

The neurotransmitters serotonin and dopamine both have a critical role in the underlying neurobiology of different behaviors. With focus on the interplay between dopamine and serotonin, it has been proposed that dopamine biases behavior towards habitual responding, and with serotonin offsetting this phenomenon and directing the balance toward more flexible, goal-directed responding. The present focus paper stands in close relationship to the publication by Worbe et al. (2015), which deals with the effects of acute tryptophan depletion, a neurodietary physiological method to decrease central nervous serotonin synthesis in humans for a short period of time, on the balance between hypothetical goal-directed and habitual systems. In that research, acute tryptophan depletion challenge administration and a following short-term reduction in central nervous serotonin synthesis were associated with a shift of behavioral performance towards habitual responding, providing further evidence that central nervous serotonin function modulates the balance between goal-directed and stimulus-response habitual systems of behavioral control. In the present focus paper, we discuss the findings by Worbe and colleagues in light of animal experiments as well as clinical implications and discuss potential future avenues for related research.     

Subpopulation heterogeneity in human acute myeloid leukemia determined by monoclonal antibodies

The leukemic population in 63 patients with acute myeloid leukemia (AML) was studied with 15 monoclonal antibodies that detect lineage-related and stage-related antigens on normal hemopoietic cells. Indirect immunofluorescence and fluorescence-activated cell sorting showed that subpopulations of leukemic cells reacted with some or all antibodies, but the percentage of cells reacting with a single antibody varied widely among patients. The composite antigenic phenotype of the various cases, as determined by immunofluorescence assay, did not correlate with the French-American-British morphological classification. Furthermore, some cells in each case failed to express any antigen normally expressed on myelomonocytic precursors from the level of the early CFU-GM to the mature granulocyte or monocyte. In double-fluorescence experiments, the individual cells expressed none, one, or both antigens. These results demonstrate that there is considerable subpopulation heterogeneity in AML. This heterogeneity may considerably limit or complicate the use of monoclonal antibodies for diagnosis, prognosis, and treatment of acute nonlymphocytic leukemia (ANLL).     

VEGFR-3 Neutralization Inhibits Ovarian Lymphangiogenesis,Follicle Maturation,and Murine Pregnancy

Lymphatic vessels surround follicles within the ovary, but their roles in folliculogenesis and pregnancy, as well as the necessity of lymphangiogenesis in follicle maturation and health, are undefined. We used systemic delivery of mF4-31C1, a specific antagonist vascular endothelial growth factor receptor 3 (VEGFR-3) antibody to block lymphangiogenesis in mice. VEGFR-3 neutralization for 2 weeks before mating blocked ovarian lymphangiogenesis at all stages of follicle maturation, most notably around corpora lutea, without significantly affecting follicular blood angiogenesis. The numbers of oocytes ovulated, fertilized, and implanted in the uterus were normal in these mice; however, pregnancies were unsuccessful because of retarded fetal growth and miscarriage. Fewer patent secondary follicles were isolated from treated ovaries, and isolated blastocysts exhibited reduced cell densities. Embryos from VEGFR-3–neutralized dams developed normally when transferred to untreated surrogates. Conversely, normal embryos transferred into mF4-31C1–treated dams led to the same fetal deficiencies observed with in situ gestation. Although no significant changes were measured in uterine blood or lymphatic vascular densities, VEGFR-3 neutralization reduced serum and ovarian estradiol concentrations during gestation. VEGFR-3–mediated lymphangiogenesis thus appears to modulate the folliculogenic microenvironment and may be necessary for maintenance of hormone levels during pregnancy; both of these are novel roles for the lymphatic vasculature.Ovarian neovascularization provides a unique environment in which to study physiological adult vasculogenesis apart from the traditional settings of wound healing and cancer pathologies. Lymphatic circulation plays a central role in fluid, lipid, and cellular transport,¹ and lymphatic vessels are present within the ovary and surround follicles during development and maturation,^2–5 but the importance of the lymphatic vasculature and lymphangiogenesis in the ovary is unclear. Consequently, the potential roles of lymphatic vessels in follicle maturation and pregnancy, and the extent of involvement or even necessity of maternal lymphangiogenesis in reproduction, are undefined. This contrasts with ovarian blood angiogenesis, whose critical roles in follicular nourishment and maturation and in the formation and maintenance of the corpus luteum are well appreciated; indeed, oocyte fertilization, embryonic implantation, uterine expansion, and successful gestation all require blood angiogenesis.^6–8 Lymphangiogenesis, which is often concurrent with blood angiogenesis,⁹ may also play an important role in these processes.Adult blood angiogenesis requires signaling via vascular endothelial growth factor receptor 2 (VEGFR-2), most potently by VEGF ligation.^10,11 In murine ovaries, VEGF expression increases during angiogenic growth phases,¹² and blockade of VEGFR-2 signaling in mice effectively prevents angiogenesis, resulting in a marked decrease in ovarian weight, blood vessel density, and number of corpora lutea, and in infertility.^13–15 Because gonadotropin treatment apparently does not correct these deficiencies,¹⁶ it is likely that follicle maturation and successful pregnancy are highly dependent on VEGFR-2–mediated neovascularization in the ovary.^6,17 Vascularization also occurs in the uterine wall and decidua during pregnancy, and significant disruption of angiogenesis by VEGFR-2 blockade in these tissues after fertilization has been shown to greatly reduce pregnancy success.¹⁸VEGFR-3 is expressed primarily on lymphatic endothelial cells in adult tissue,^19,20 and its signaling, via ligation by VEGF-C or VEGF-D, is necessary for lymphangiogenesis by inducing lymphatic endothelial cell proliferation and migration.^19–23 Blockade of VEGFR-3 signaling using a function-blocking antibody such as mF4-31C1 (ImClone Systems; Eli Lilly, Indianapolis, IN) completely blocks the initiation of new lymphatic vessels in adult mice without affecting pre-existing lymphatic morphology or function and without apparently affecting blood angiogenesis.^18,21,22 The ovary contains a dense lymphatic network that has been morphologically assessed in large rodents.^24–26 Recent studies in which murine ovarian lymphatic vessel expansion was impaired during development found the dams to be infertile as adults.³We investigated VEGFR-3–mediated lymphangiogenesis and the roles of new lymphatic vessels and lymphangiogenesis in female reproduction and found that lymphangiogenesis occurs within the murine ovary during reproductive cycles and folliculogenesis and that VEGFR-3 neutralization prevents viable, full-term pregnancies. Using combined in vivo, ex vivo, and in vitro methods, we examined which aspects of female fertility are influenced by inhibited maternal lymphangiogenesis including oocyte and follicular development and maturation, uterine implantation, and embryonic development. After we had eliminated direct effects on fetal and uterine VEGFR-3–mediated neovascularization, our results suggested that the new ovarian lymphatic vessels specifically modulate follicle development and hormone production, demonstrating a critical and novel role for ovarian lymphangiogenesis in reproduction.     

Activated natural killer cells and interleukin-2 promote granulocytic and megakaryocytic reconstitution after syngeneic bone marrow transplantation in mice

Purified populations of natural killer (NK) cells were obtained from mice with severe combined immune deficiency (SCID). SCID spleen cells were cultured and activated with recombinant human interleukin-2 (rhIL- 2) in vitro. The activated NK cells were then transferred with syngeneic BALB/c bone marrow cells (BMC) and rhIL-2 into lethally irradiated syngeneic recipients to determine their effect on long-term hematopoietic reconstitution. On analysis, the transfer of rhIL-2- activated NK cells along with BMC resulted in significant increases in splenic and BM hematopoietic progenitor cells when compared with those for mice not receiving NK cells. Histologic and flow cytometric analysis showed a marked increase in granulocytic and megakaryocytic lineage cells present in the spleens of the mice receiving activated NK cells. Analysis of the peripheral blood indicated that the transfer of activated NK cells with BMC also significantly improved platelet and total white blood cell counts, with increases in segmented neutrophils. Erythroid recovery was not affected. Finally, lethally irradiated mice receiving activated NK cells and rhIL-2 along with limiting numbers of syngeneic BMC showed a marked increase in survival rate. These results show that the use of populations enriched for activated NK cells after syngeneic BM transplantation (BMT) has a profound enhancing effect on engraftment primarily affecting megakaryocytic and granulocytic cell reconstitution. Therefore, the transfer of activated NK cells and rhIL- 2 may be of clinical use to promote hematopoietic reconstitution after BMT.     

IgMs produced by two acquired immune deficiency syndrome lymphoma cell lines: Ig binding specificity and VH-gene putative somatic mutation analysis [published erratum appears in Blood 1994 Aug 1;84(3):995]

Two B-cell lines, 2F7 and 10C9, were established by single cell cloning from biopsies obtained from two acquired immune deficiency syndrome patients with Burkitt's lymphoma. Representation of the original tumors was verified by demonstration of (1) identical biallelic rearrangement of Ig genes for 2F7 and (2) shared idiotype for 10C9. Both cell lines displayed cell-surface Ig and secreted Ig (IgM lambda for 2F7, IgM kappa for 10C9). IgMs from both cell lines immunoprecipitated actin; in addition, 2F7 IgM lambda immunoprecipitated recombinant human immunodeficiency virus type 1 (HIV-1) gp 160. 2F7 IgM lambda did not react with other autoantigens (double-stranded and single-stranded DNA, actin, bovine serum albumin, IgG), whereas 10C9 IgM kappa reacted with human IgG. The 2F7 IgM heavy-chain variable region (VH) showed a 95% nucleotide homology with a previously sequenced VHIII germline gene, hv3019b9, whereas the 10C9 IgM VH showed a 95% homology with a previously sequenced VHIV germline gene, VH4.21. Use of minimally modified VH genes and demonstration of reactivity with chronically present antigens (ie, actin, HIV-1 gp 160, or human IgG) suggests that B cells in HIV-1-infected individuals proliferating in response to chronic antigenic stimulation may be at increased risk for lymphomagenesis.     

A transmembrane form of annexin XII detected by site-directed spin labeling

Previous studies of the annexin family of Ca²⁺ binding proteins identified a soluble monomer in the absence of Ca²⁺ and a trimer adsorbed on the membrane surface in the presence of Ca²⁺. On the basis of site-directed spin-labeling studies of annexin XII at low pH, we now report a membrane-inserted form of the protein with a dramatically different structure. The data suggest that upon insertion a continuous transmembrane α-helix is reversibly formed from a helix–loop–helix motif in the solution structure. Other regions with similar membrane-insertion potential were identified in the amino acid sequence, and we propose that the corresponding helices come together to form an aqueous pore that mediates the ion channel activity reported for several annexins.     

Stability of "salt bridges" in membrane proteins.

We estimate the free energies of transfer of ionized amino acid side chains in water to both their ion-paired and neutral hydrogen-bonded states in low-dielectric media. The difference between the two free energies corresponds to the proton transfer free energy in a "salt bridge" formed between acidic and basic groups (i.e., lysine and glutamic acid residues). Our approach is to use gas phase proton transfer data, pK values, and experimentally determined solvation energies to estimate the standard state free energy changes involved in transferring amino acid side chains, in both ionized and neutral form, from water (dielectric constant epsilon = 80) to vacuum (epsilon = 1). The familiar expressions for the charging energy of a sphere and dipole are used to interpolate between epsilon = 1 and epsilon = 80. Our results suggest that it costs approximately 10-16 kcal/mol to transfer a salt bridge from water to a medium of epsilon = 2-4, in ionized or neutral form within the resolution of our estimates. The proton transfer energy is thus approximately 0. The tendency of salt bridges to form additional hydrogen bonds in real proteins suggests that the ion pair will be present in most biological systems.