Induction of differentiation in mouse neuroblastoma cells

In the presence of 1--2% dimethyl sulfoxide (DMSO), mouse neuroblastoma cells are induced to differentiate morphologically as well as electrically. In addition, treatment of neurolbastoma cells with 2% DMSO results in a marked increase in the veratridine-activated K+ or Rb+ efflux. At 4% DMSO, neurite outgrowth is completely repressed and electrical activity is poorly developed. However, at this concentration, the cells have a relatively high resting potential which suggested that membrane components determining passive and active permeability properties are not necessarily under coordinated control. Induction of differentiation by 2% DMSO is also accompanied by an increase in a heavier molecular form of acetylcholinesterase sedimenting at 10.5S. The effect of other agents on the growth and differentiation of neuroblastoma cells is also presented.     

Induction of interferon by polyribonucleotides containing thiopyrimidines

The influence of thioketo substitution in pyrimidine bases of double-stranded polynucleotides on interferon induction was investigated. The stabilizing effect of 2-thioketo substitution was reflected in the increased interferon inducing activity of poly(A-s2U) over that of poly(A-U). Poly(A-s2U) and poly(I)-poly(s2C) were as effective as poly(I)-Poly(C) in rabbit cells. Poly(I)-poly(C) and poly(I)-poly(s2C) were compared in several animal species. No differences in biological effects were observed in rabbits and dogs. In rodents, poly(I)-poly(s2C) was less effective and less toxic.Poly(I)-poly(s2C) was highly resistant against degradation by human serum. Further investigations seem to be justified to elucidate whether this property offers any advantages for the potential clinical utilization of poly(I)-poly(s2C).     

Variant mouse lymphoma cells with modified response to interferon demonstrate enhanced immunogenicity

We have previously developed an experimental model for the xenogenization of malignant lymphoma. From highly tumorigenic S49 mouse lymphoma cells that proliferate in suspension culture (designated T-25), we selected variant clones that grew as an adherent monolayer (designated T-25-Adh) and were non-tumorigenic in syngeneic mice. Furthermore, priming of syngeneic hosts with T-25-Adh cells protected them against subsequent challenges with the tumorigenic T-25 cells. Several lines of evidence have indicated that antigens of an endogenous mouse mammary tumor virus (MMTV) are involved in the immunogenicity of T-25-Adh cells. Since interferon (IFN) is known to affect retroviral assembly and maturation on the cell membrane, we have studied the effects of IFN on endogenous MMTV-related structures, as well as on the immunogenicity of T-25-Adh cells. We observed that mouse alpha and beta interferons affect the morphogenesis of intracellular MMTV-related precursors in the immunogenic T-25-Adh cells, but not in tumorigenic T-25 cells. From T-25-Adh cells we selected variants that were either high responders or low responders to the above-mentioned interferon effect. The high-response variants were significantly more protective against tumorigenic T-25 cells than the low-response variants. Involvement of MMTV-related antigens in the immune response of the host to T-25-Adh cells was further suggested by immunoelectron-microscopical analysis, demonstrating that antisera from mice, immunized with T-25-Adh cells, interacted specifically with cell-surface MMTV budding particles. These findings indicate a novel method for xenogenization of lymphoma cells by IFN. Since endogenous retroviruses are present in all tissues of the mouse, this approach might be applicable to a wide variety of tumors.     

Fidelity levels of DNA polymerases in tumorigenic state cells and serially transplantable tumor cells

The crystal structure of a calcium-bound form of bovine annexin VI has been determined with X-ray diffraction data to 2.9 A by molecular replacement. Six Ca2+ ions were found, five in AB loops, one in a DE loop. Two loops (II-AB, which binds calcium, and V-AB, which does not) have conformations that differ significantly from those in calcium-free, human recombinant annexin VI. There are only small differences between the calci- and the apo-annexin VI in the rest of the molecule. Calcium by itself does not promote a major conformational change.     

Induction of the c-fos gene in the chick brain during visual imprinting

We describe a patient with combined meningococcal septicemia and meningitis, cerebral edema and acute respiratory distress syndrome, in whom we balanced the conflicting carbon dioxide strategies for optimal pulmonary and neurological management using jugular oxygen saturation (SjvO2) monitoring to identify the upper limit of "tolerable" hypercapnia. Our observations suggest that significant acidosis was not well tolerated; however, cautious induction of pH down to 7.32 and an arterial carbon dioxide tension (PaCO2) < 5.9 kPa was tolerated acutely without significant cerebral hyperemia. Moreover, with the development of metabolic compensation and normal pH, higher levels of PaCO2 could be permitted. In similar cerebro-pulmonary circumstances we suggest that these findings warrant consideration. Alternatively, invasive monitoring of SjvO2 could be undertaken so that patient-specific criteria for permissive hypercapnia can be determined.     

Induction of matrix metalloproteinases and collagenolysis in chick embryonic membranes before hatching

In NIH 3T3 fibroblasts and several other cellular systems, ethanol (50-80 mM) was previously shown to greatly enhance the mitogenic effects of insulin particularly in the presence of zinc. Here we report that in NIH 3T3 fibroblasts the combined stimulatory effects of ethanol and insulin on DNA synthesis can be further increased by bombesin both in the absence and presence of zinc. Bombesin also enhanced insulin-plus-ethanol-induced DNA synthesis in mouse Swiss 3T3 and Balb/c 3T3 fibroblasts, but in these cells bombesin was effective only in the presence of zinc. In NIH 3T3 fibroblasts, the potentiating effects of ethanol on insulin-induced DNA synthesis by the zinc-dependent and bombesin-dependent mechanisms were additive. Wortmannin, an inhibitor of phosphatidylinositol 3'-kinase (PI3K), prevented the comitogenic effect of ethanol in the presence of bombesin but not in the presence of zinc. Furthermore, bombesin, but not ethanol, was found to enhance the stimulatory effect of insulin on PI3K activity. Rapamycin, an indirect inhibitor of p70 S6 kinase actions, inhibited the comitogenic effects of ethanol in the presence of both zinc and bombesin. However, only ethanol, but not bombesin, enhanced the stimulatory effect of insulin on p70 S6 kinase activity; this effect of ethanol was zinc-dependent. Neither ethanol nor bombesin enhanced the stimulatory effects of insulin on the phosphorylation (activation) of p38/p42/p44 mitogen-activated protein kinases. The results suggest that in mouse fibroblasts maximal stimulation of DNA synthesis by physiologically relevant concentrations of ethanol occurs if both PI3K and p70 S6 kinase are activated. These data suggest a mechanism by which ethanol may affect growth in affected human tissues during its tumor promoting actions.     

Cytotoxic and interferon gamma-producing activities of gamma delta T cells in the mouse intestinal epithelium are strain dependent

We have analyzed the cytolytic activity of freshly isolated intraepithelial T cells (i-IEL) from the intestines of several different mouse strains in an anti-T-cell receptor monoclonal antibody-mediated redirected lysis assay. The cytolytic activity of gamma delta i-IEL but not that of alpha beta i-IEL was strain dependent. Mouse strains could be divided into high (H), marginal (M), and null (N) strains. The anti-gamma delta T-cell receptor monoclonal antibody-induced interferon gamma production showed the same strain-dependent variability, but the proliferative responses to gamma delta T-cell receptor crosslinking did not show this variability. The N phenotype of gamma delta i-IEL was found to be dominant in (H x N)F1 mice. In radiation bone-marrow chimeras the H/N phenotype was determined by the genotype of the reconstituting bone-marrow-derived cells but was not determined by the genotype of the radioresistant host cells. Analysis of (H x N)F1 backcross animals indicated that at least two genes are involved in determination of the H/N phenotype. One of these genes is major-histocompatibility-complex linked. No difference in the use of the variable region segment of the gamma-chain or delta-chain was seen between the gamma delta i-IEL from H and N strains. Various models that might explain the strain-dependent gamma delta i-IEL phenotypes are discussed.     

Induction and suppression of reagins in the neonatal mouse

Neonatal SWR mice are capable of synthesizing reagins when immunized with a mixture of concanavalin and ovalbumin or a mixture of Bordetella pertussis, alum and ovalbumin. Reaginic antibody-forming cells can be found in the spleen, lymph nodes, bone marrow and Peyer's patches. Tolerance with respect to IgE can be induced by the injection of deaggregated ovalbumin into neonatal mice.     

Action of heavy metal salts on the development of sea urchin embryos and on protein synthesis by the cells of transplantable mouse tumors

Zu, Cu, Cd, Pb and Hg salts blocked the cleavage divisions in the sea urchin embryos and, in lesser concentrations, affected gastrulation and induced postgastrulation defects. The early embryo- and cytotoxic effects of Zn salts appear to be based on the inhibition of protein synthesis. Unithiol exerted a protective effect. All salts under study inhibited protein synthesis in the Ehrlich's ascite carcinoma and ascite hepatoma 22a cells; unithiol and other suldhydryl compounds exerted in this case a good protective effect against Cd salts only.     

Induction of cyclooxygenase-2 expression by peroxisome proliferators and non-tetradecanoylphorbol 12,13-myristate-type tumor promoters in immortalized mouse liver cells

Increased expression of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostaglandin synthesis, has been associated with growth regulation and carcinogenesis in several systems. COX-2 is known to be induced by cytokines and the skin tumor promoter 12-tetradecanoylphorbol-13-myristate (TPA). In the present study, we investigated the effects of several non-TPA-type tumor promoters on COX-2 expression in immortalized mouse liver cells. Specifically, we tested peroxisome proliferators (PPs), which are rodent liver tumor promoters that cause gross alterations in cellular lipid metabolism, the rodent liver tumor promoter phenobarbital, and the skin tumor promoters okadaic acid and thapsigargin. The PPs Wy-14643, mono-ethylhexyl phthalate, clofibrate, ciprofibrate ethyl ester, and eicosatetraynoic acid each caused large increases in COX-2 mRNA and protein, with maximal expression seen approximately 10 h after treatment of quiescent cells. COX-2 expression was also induced by thapsigargin, okadaic acid, and calcium ionophore A23187, but not by phenobarbital or the steroid PP dehydroepiandrosterone sulfate. Induction of COX-2 expression generally resulted in increased synthesis of prostaglandin E2 (PGE2). However, the PPs caused little or no increase in PGE2 levels, and they inhibited serum-induced PGE2 synthesis. Unlike non-steroidal anti-inflammatory drugs, the PPs do not directly inhibit cyclooxygenase enzyme activity in vitro. Thus, PPs regulate prostaglandin metabolism via both positive (COX-2 induction) and inhibitory mechanisms. In summary, the strong induction of COX-2 expression by PPs, thapsigargin, and okadaic acid suggests a possible role for COX-2 in the growth regulatory activity of these non-TPA-type tumor promoters.     

Induction of alpha/beta interferon and dependent nitric oxide synthesis during Chlamydia trachomatis infection of McCoy cells in the absence of exogenous cytokine

The propensity of two Chlamydia trachomatis strains (L2/434/Bu [biovar LGV] and E/DK20/ON [biovar trachoma]) to induce putative host defense responses upon infection of McCoy (mouse) cell cultures was examined. Both strains induced production of alpha/beta interferon and nitric oxide (NO) by McCoy cells. NO synthesis was mediated by the inducible isoform of NO synthase as indicated by the ability of cycloheximide or the arginine analog NG-monomethyl-L-arginine to abolish NO production; the extent of the response was dependent upon the dose of chlamydiae applied. Incubation of McCoy cells with chloramphenicol prior to infection reduced NO production by strain 434 but not by DK20, suggesting that initial chlamydial metabolism was essential to induction by the LGV strain. Antibody inhibition studies indicated that NO synthesis was dependent upon production of alpha/beta interferon and induction via lipopolysaccharide. Overall, our findings show that chlamydiae are capable of the induction of interferon and NO in murine fibroblasts in the absence of exogenous cytokines. However, the role of NO as an antichlamydial effector could not be clearly demonstrated since treatment with an arginine analog, while suppressing NO production, gave no consistent enhancement of infected cell numbers.     

Induction of antitumor immunity by interleukin-2 gene-transduced mouse mammary tumor cells versus transduced mammary stromal fibroblasts

BACKGROUND: Tumor cell-targeted cytokine gene transfer has been used to generate tumor cell vaccines, but this approach is limited by the need to establish and implant live tumor cells. PURPOSE: The purpose of this study was to determine if stromal fibroblasts could be used as an alternative vehicle for delivery of the cytokine interleukin-2 (IL-2) into the tumor microenvironment. We attempted to establish the feasibility of (a) genetic immunotherapy in a mammary tumor system and (b) engineering stromal fibroblasts as well as tumor cells. We compared the effects of tumor cell-mediated and stromal fibroblast-mediated local IL-2 expression on the generation of antitumor immune responses. METHODS: Retroviral vectors containing a human IL-2 gene were used to transduce a mouse mammary tumor line, 4TO7, and an immortalized but nontumorigenic fibroblast line established from syngeneic mammary fatpads. Expression of the IL-2 gene in transduced cells was determined by measuring IL-2 secretion, by RNA-polymerase chain reaction, and by immunochemistry. Groups of 5-12 BALB/c mice were injected with either 4TO7 cells or various doses of IL-2-secreting 4TO7 cells (4TO7-IL-2); tumor growth was monitored. To test whether local IL-2 expression by transduced cells could influence the growth of unmodified tumor cells, we determined tumor development in groups of mice treated with 4TO7 cells co-injected with either 4TO7-IL-2 cells or IL-2-secreting fibroblasts. RESULTS: 4TO7-IL-2 cells induced active immunity able to reject the immunizing tumor and to resist challenge with parental 4TO7 cells on the contralateral side. Mice pretreated with 4TO7-IL-2 were significantly protected compared with untreated control animals or mice pretreated with irradiated 4TO7 cells. The immunity induced by 4TO7-IL-2 cells did not protect against challenge with another subline, 4T1, which was derived from the same spontaneously arising mammary tumor as 4TO7. Co-injection of 4TO7 cells with 4TO7-IL-2 cells reduced tumorigenicity, whereas co-injection of 4TO7 cells with IL-2 secreting fibroblasts did not. CONCLUSION: Our results suggest that induction of anti-tumor immune response by local IL-2 production is most effective when the helper cytokine is secreted by the tumor cell. IMPLICATION: Our studies caution against the use of IL-2 gene-transduced syngeneic stromal cells as an alternative strategy of gene therapy for cancer. However, they may allow study of the mechanisms of tumor antigen recognition and the possible involvement of co-stimulatory signals for effective tumor vaccination by gene-modified cells.     

Odontogenic epithelium induces similar molecular responses in chick and mouse mandibular mesenchyme

Previous observations have shown that, during the initiation phase of odontogenesis, signals from mouse odontogenic epithelium can elicit teeth in non-odontogenic but neural crest-derived mesenchyme isolated from ectopic sites including chick mandibular mesenchyme. In the present study the formation of ectopic tooth buds and dental mesenchyme in chick mandibular mesenchyme was examined using heterospecific recombinations between E11 mouse odontogenic epithelium and stage 23 chick lateral mandibular mesenchyme. Both morphological criteria and chick-specific probes for Msx-1, Msx-2, and Bmp-4 mRNAs were used as markers for early dental mesenchyme. Our results demonstrated that interactions of mouse odontogenic epithelium with chick mandibular mesenchyme induce early changes in the chick mandibular mesenchyme including the appearance of a translucent zone, cell proliferation, and induction of expression of Msx-1, Msx-2, and Bmp-4, which have been shown to be associated with the formation of dental mesenchyme. In addition, tooth bud-like structures that resemble E13 tooth buds in vivo both morphologically and in their patterns of gene expression formed after 6 days in the heterospecific recombinations. The tooth bud-like structures consist of invaginated mouse mandibular epithelium and condensed chick mandibular mesenchyme expressing high levels of Msx-1 and Bmp-4, but undetectable levels of Msx-2. Unlike the induction of Msx-1, Msx-2, and Bmp-4 in the underlying mesenchyme, which is specific for signals derived from odontogenic epithelium, the induction of a translucent zone and cellular proliferation in the underlying mesenchyme may be related to the growth-promoting potential of embryonic epithelia and not be specific to signals derived from the odontogenic epithelium. Similar to mouse odontogenic epithelium, agarose beads soaked in recombinant BMP-4 induced a translucent zone, cellular proliferation, and expression of Msx-1, Msx-2, and Bmp-4 in chick mandibular mesenchyme after 24 hours. These observations together showed that avian mandibular mesenchyme has odontogenic potential that is expressed upon interactions with inductive signals from mouse odontogenic epithelium. Similar to odontogenesis in vivo, formation of dental mesenchyme in chick mandibular mesenchyme is mediated by the activation of Msx-1, Msx-2, and Bmp-4.     

Phenotypic changes in lipopolysaccharide-treated cloned cells derived from transplantable rat malignant fibrous histiocytoma

To investigate a possible phenotypic modulation, MT-8L and MT-9L cells were induced by in vitro culture of undifferentiated MT-8 and fibrohistiocytic MT-9 cells, which had been established from a rat malignant fibrous histiocytoma (MFH), in the medium containing 10 micrograms lipopolysaccharide (LPS)/ml. MT-8L and MT-9L gave greater positive reactions for histiocytic lysosomal markers and showed ultrastructures of histiocytic natures. In MT-8L, alpha-smooth muscle actin-positive myofibroblastic cells also significantly increased in number. MT-8L expressed both histiocytic and myofibroblastic phenotypes. MT-8L-induced tumors consisted mainly of storiform type MFH, differing from undifferentiated sarcoma type induced by MT-8. MT-9L and MT-9 tumors showed a storiform pattern. A phenotypic modulation of MFH cells was easily induced by LPS treatment.     

Expansion in vitro of adult murine hematopoietic stem cells with transplantable lympho-myeloid reconstituting ability

Elucidation of mechanisms that regulate hematopoietic stem cell self-renewal and differentiation would be facilitated by the identification of defined culture conditions that allow these cells to be amplified. We now demonstrate a significant net increase (3-fold, P < 0.001) in vitro of cells that are individually able to permanently and competitively reconstitute the lymphoid and myeloid systems of syngeneic recipient mice when Sca-1(+)lin- adult marrow cells are incubated for 10 days in serum-free medium with interleukin 11, flt3-ligand, and Steel factor. Moreover, the culture-derived repopulating cells continued to expand their numbers in the primary hosts at the same rate seen in recipients of noncultured stem cells. In the expansion cultures, long-term culture-initiating cells increased 7- +/- 2-fold, myeloid colony-forming cells increased 140- +/- 36-fold, and total nucleated cells increased 230- +/- 62-fold. Twenty-seven of 100 cultures initiated with 15 Sca-1(+)lin- marrow cells were found to contain transplantable stem cells 10 days later. This frequency of positive cultures is the same as the frequency of transplantable stem cells in the original input suspension, suggesting that most had undergone at least one self-renewal division in vitro. No expansion of stem cells was seen when Sca-1+TER119- CD34+ day 14.5 fetal liver cells were cultured under the same conditions. These findings set the stage for further investigations of the mechanisms by which cytokine stimulation may elicit different outcomes in mitotically activated hematopoietic stem cells during ontogeny and in the adult.     

Role of gamma interferon in Helicobacter pylori-induced gastric inflammatory responses in a mouse model

The immune responses to Helicobacter pylori infection play important roles in gastroduodenal diseases. The contribution of gamma interferon (IFN-gamma) to the immune responses, especially to the induction of gastric inflammation and to protection from H. pylori infection, was investigated with IFN-gamma gene knockout (IFN-gamma-/-) mice. We first examined the colonizing abilities of eight H. pylori strains with a short-term infection test in order to select H. pylori strains which could colonize the mouse stomach. Only three strains (ATCC 43504, CPY2052, and HPK127) colonized C57BL/6 wild-type mice, although all of the strains except for ATCC 51110 could colonize IFN-gamma-/- mice. The number of H. pylori organisms colonizing the stomach in wild-type mice was lower than that in IFN-gamma-/- mice. Oral immunization with the CPY2052 sonicate and cholera toxin protected against infection with strain CPY2052 in both types of mouse. These findings suggested that IFN-gamma may play a protective role in H. pylori infection, although the degree of its protective ability was estimated to be low. In contrast, in a long-term infection test done to examine the contribution of IFN-gamma to gastric inflammation, CPY2052-infected wild-type mice developed a severe infiltration of mononuclear cells in the lamina propria and erosions in the gastric epithelium 15 months after infection, whereas CPY2052-infected IFN-gamma-/- mice showed no inflammatory symptoms. This result clearly demonstrated that IFN-gamma plays an important role in the induction of gastric inflammation caused by H. pylori infection.