Effect of (E)-5-(2-bromovinyl)-2'-deoxyuridine on the growth and viral capsid antigen expression of Epstein-Barr virus-associated tumor (B-95-8) cells transplanted to nude mice

When persistently Epstein-Barr virus (EBV)-infected lymphoblastoid (B-95-8) cells were transplanted subcutaneously or intracerebrally to nude mice of either BALB/c or NIH background, tumors developed, and the tumor cells spontaneously expressed viral capsid antigen (VCA). This model was used to evaluate the in vivo anti-EBV activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), a highly potent and selective antiherpes agent, which was recently shown to inhibit several parameters of EBV infection in vitro. When administered intraperitoneally at 200 mg/kg/day for 4 weeks, or 500 mg/kg/day for 2 weeks, starting immediately after B-95-8 cell inoculation, BVDU effectively reduced tumor growth and VCA expression of either subcutaneously or intracerebrally inoculated B-95-8 cells.     

NK cells use NKG2D to recognize a mouse renal cancer (Renca), yet require intercellular adhesion molecule-1 expression on the tumor cells for optimal perforin-dependent effector function

The NKG2D receptor on NK cells can recognize a variety of ligands on the tumor cell surface. Using a mouse renal cancer (Renca), we show that NKG2D recognition by NK cells was crucial for their ability to limit tumor metastases in vivo in both liver and lungs using perforin-dependent effector mechanisms. However, for the R331 cell line established from Renca, NKG2D recognition and perforin-dependent lysis played no role in controlling liver metastases. R331 cells were also more resistant to perforin-dependent lysis by NK cells in vitro. We therefore used these phenotypic differences between Renca and R331 to further investigate the crucial receptor:ligand interactions required for triggering lytic effector functions of NK cells. Reconstitution of R331 cells with ICAM-1, but not Rae-1gamma, restored NKG2D-mediated, perforin-dependent lysis. Interestingly, R331 cells were efficiently lysed by NK cells using death ligand-mediated apoptosis. This death ligand-mediated killing did not depend on NKG2D recognition of its ligands on tumor cells. This result suggests that the intracellular signaling in NK cells required for perforin and death ligand-mediated lysis of tumor target cell are quite distinct, and activation of both of these antitumor lytic effector functions of NK cells could improve therapeutic benefits for certain tumors.     

The NK-1.1(-) mouse: a model to study differentiation of murine NK cells

The NK-1.1(-) mouse was constructed by weekly injections of monoclonal anti-NK-1.1 antibody from birth through adulthood. Spleen cells from these mice have decreased NK-1.1+ cells and null (Thy-1- and B220-) cells. Their splenic NK activity to YAC targets was low and was not enhanced by IFN-alpha or IFN-beta. Bone marrow (BM) of these NK-1.1(-) mice have normal precursors to NK cells: 1) NK activity could be generated from NK-1.1(-) BM cells cultured in rIL 2 for 5 to 6 days. These cultured BM cells expressed Qa-5, Thy-1, AsGm-1, and NK-1.1 antigens. The precursor cells of these BM cytotoxic cells are NK-1.1-; 2) transfer of BM cells from the NK-1.1(-) mice reconstituted the NK activity of irradiated, NK-depleted recipients. Lymphokine-activated killer cells could also be generated from spleens of these NK-1.1(-) mice. Therefore, the NK-1.1(-) mice were specifically depleted of mature cytotoxic NK cells, but not the NK-1.1- precursors of NK cells. This mouse model is valuable to study ontogeny and physiologic relevance of NK cells.     

Lack of tumor suppression but induced loss of copies of indigenous chromosome 10 in vitro following microcell-mediated transfer of a deleted human der(9)t(X;9) chromosome to Syrian hamster BHK-191-5C cells

We have previously shown that microcell-mediated transfer of a der(9)t(X;9) chromosome, containing an almost complete human chromosome (HSA) 9 derived from the human fibroblast strain GM0705, into the Syrian hamster (Mesocricetus auratus) cell line BHK-191-5C suppressed the anchorage independence and tumorigenicity of the hybrids. Transfer of a normal HSA X did not have any effect on these phenotypes. Although the recipient cell line contained a 1:1 ratio of near-diploid and near-tetraploid cells, all hybrids retaining the der(9) chromosome were near-tetraploid, in contrast to hybrids retaining a normal X chromosome. In the present study, we have generated microcell hybrids by transferring another der(9)t(X;9) chromosome derived from the human fibroblast strain GM01429. This derivative chromosome contained a deletion on the short arm of HSA 9 and was also missing the distal part of the long arm of HSA 9 due to the involvement in a reciprocal (constitutive) translocation of this chromosome with HSA X. Cytogenetic analysis showed that all hybrid clones were near-tetraploid, confirming our previous finding. We also observed that the introduction of the deleted der(9) chromosome forced the hybrids to lose Syrian hamster chromosome 10. A soft agar test and nude mice assay indicated that none of the hybrids was suppressed for either anchorage independent growth or tumor formation. These data suggest that there is an antagonistic relationship between growth-promoting genes and antiproliferative genes. The observed dosage effects of both growth-promoting and growth-suppressing genes indicate that cellular growth may be a quantitative trait.     

The mouse Hoxd13(spdh) mutation, a polyalanine expansion similar to human type II synpolydactyly (SPD), disrupts the function but not the expression of other Hoxd genes.

Polyalanine expansion in the human HOXD13 gene induces synpolydactyly (SPD), an inherited congenital limb malformation. A mouse model was isolated, which showed a spontaneous alanine expansion due to a 21-bp duplication at the corresponding place in the mouse gene. This mutation (synpolydactyly homolog, spdh), when homozygous, causes malformations in mice similar to those seen in affected human patients. We have studied the genetics of this condition, by using several engineered Hoxd alleles, as well as by looking at the expression of Hox and other marker genes. We show that the mutated SPDH protein induces a gain-of-function phenotype, likely by behaving as a dominant negative over other Hox genes. The mutation, however, seems to act independently from Hoxa13 and doesn't appear to affect Hox gene expression, except for a slight reduction of the HOXD13 protein itself. Developmental studies indicate that the morphological effect is mostly due to a severe retardation in the growth and ossification of the bony elements, in agreement with a general impairment in the function of posterior Hoxd genes.     

The mouse chorionic gonadotropin beta-subunit-like (muCG beta l) molecule produced by tumor cells elicits the switch of T-cell immunity response from TH2 to TH1 in mice immunized with DNA vaccine based on rhesus monkey homologous CG beta (rmCG beta)

BACKGROUND: CG beta is expressed not only in placenta, but also in a wide range of tumors. To study DNA vaccine based on xenogeneic CG beta for cancer immuno-therapy, we investigated whether rhesus monkey CG beta (rmCG beta) DNA vaccine could induce protective T-cell responses and humoral responses in mouse. METHODS: We constructed a plasmid containing the rmCG beta coding sequence. Two cloned syngeneic SP2/0 myeloma cell lines that stably express muCG beta l (SP2/0-muCG beta l) and HN (SP2/0-HN) protein were established. Inoculation of these cell lines was made into mice that had been immunized with DNA vaccine. Specific IgG and IgG type were measured by ELISA and the cytokine expression was detected with RT-PCR. To measure the lymphocyte metabolic activity, the MTS assay was used. RESULTS: After injection of SP2/0-muCG beta l into mice that had been immunized with DNA vaccine, a significant increase in the IgG2a specific to the antigen (p < 0.05) and a decrease in the specific IgG1 (p < 0.05) were measured. The expression of T(H)1 but not T(H)2 cytokines, including IFN-gamma and IL-2, were detected in the splenocytes. However, injection of tumor cells expressing irrelevant or mock molecules into immunized mice could not induce these changes. The survival rate of vaccine-immunized mice injected with SP2/0-muCG beta l was as high as 58.3% after 55 days. CONCLUSIONS: The rmCG beta DNA vaccine has proved to be a potential strategy for protection against tumors with homologous molecules. The muCG beta l produced by tumors is able to elicit an immunity switch from T(H)2 to T(H)1 in vaccinated mice.     

The histone deacetylase inhibitor sodium butyrate interacts synergistically with phorbol myristate acetate (PMA) to induce mitochondrial damage and apoptosis in human myeloid leukemia cells through a tumor necrosis factor-alpha-mediated process

Interactions between the histone deacetylase inhibitor sodium butyrate (SB) and phorbol 12-myristate 13-acetate (PMA) were examined in human myeloid leukemia cells (U937 and HL-60). Exposure of U937 cells to 1 mM SB and 1 nM PMA (24 h) markedly induced caspase activation and apoptosis, events accompanied by impaired differentiation induction (e.g., reduced plastic adherence and diminished expression of CD11b) as well as reduced clonogenic survival. The PKC inhibitor GF109203X blocked SB-/PMA-mediated apoptosis. Comparable results were obtained in HL-60 cells. Apoptosis was associated with early procaspase 8 activation and Bid cleavage, accompanied by pronounced mitochondrial damage (e.g., loss of mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c release). Neutralization of endogenous TNFalpha by a human soluble TNF receptor substantially blocked SB-/PMA-induced cytochrome c release and apoptosis. Consistent with this, ectopic expression of a mutant dominant-negative caspase 8 or CrmA resulted in a significant decrease in SB-/PMA-induced apoptosis, whereas Bcl-2 overexpression did not. SB/PMA treatment also triggered a decline in the S and G(2)M populations, and dephosphorylation of p34(cdc2). These results indicate that SB interacts with low concentrations of PMA to induce apoptosis in human leukemia cells and that this process proceeds through a PKC-/TNFalpha-dependent pathway in which procaspase 8 and Bid activation play key roles.