Control of mammary tumor differentiation by SKI-606 (bosutinib).

C-Src is infrequently mutated in human cancers but it mediates oncogenic signals of many activated growth factor receptors and thus remains a key target for cancer therapy. However, the broad function of Src in many cell types and processes requires evaluation of Src-targeted therapeutics within a normal developmental and immune-competent environment. In an effort to understand the appropriate clinical use of Src inhibitors, we tested an Src inhibitor, SKI-606 (bosutinib), in the MMTV-PyVmT transgenic mouse model of breast cancer. Tumor formation in this model is dependent on the presence of Src, but the necessity of Src kinase activity for tumor formation has not been determined. Furthermore, Src inhibitors have not been examined in an autochthonous tumor model that permits assessment of effects on different stages of tumor progression. Here we show that oral administration of SKI-606 inhibited the phosphorylation of Src in mammary tumors and caused a rapid decrease in the Ezh2 Polycomb group histone H3K27 methyltransferase and an increase in epithelial organization. SKI-606 prevented the appearance of palpable tumors in over 50% of the animals and stopped tumor growth in older animals with pre-existing tumors. These antitumor effects were accompanied by decreased cellular proliferation, altered tumor blood vessel organization and dramatically increased differentiation to lactational and epidermal cell fates. SKI-606 controls the development of mammary tumors by inducing differentiation.

Attenuation of interleukin 2-induced pulmonary vascular leak syndrome by low doses of oral methotrexate.

Pulmonary vascular leak induced in mice by interleukin 2 (IL-2) was attenuated by pretreatment with single or multiple doses of oral methotrexate. Methotrexate also attenuated pulmonary vascular leak when either larger doses of IL-2 or when lymphokine-activated killer (LAK) cells or LAK cells plus IL-2 were administered. Lymphoid infiltrates in the lungs of mice treated with IL-2 and methotrexate were significantly lower. The number of mice surviving treatment with high doses of IL-2 was also significantly increased when these mice were treated with methotrexate. Methotrexate prevented the IL-2-induced increase in the number of splenocytes that were asialo GM1+ but had no effect on Lyt 2+ or L3T4+ cell content. A marginal but significant inhibition in the generation of effector splenocytes that were cytolytic to either YAC or MCA-205 tumor targets was observed in mice treated with methotrexate and IL-2. In vivo studies indicated that methotrexate did not compromise the anti-tumor efficacy of treatment regimens that contained IL-2, LAK cells, or IL-2 and LAK cells. These results demonstrate the potential clinical utility of methotrexate in attenuating pulmonary vascular leak induced by IL-2 without compromising its efficacy. One potential mechanism of action of methotrexate is related to its ability to stimulate the release of adenosine followed by the inhibition of the adhesion of leukocytes to the IL-2-activated endothelium.

Studies evaluating the antitumor activity and toxicity of interleukin-15, a new T cell growth factor: comparison with interleukin-2.

Interleukin-15 is a new cytokine that stimulates the proliferation of T cells and other cells of the immune system. Some of the biological properties of interleukin-15 overlap that of interleukin-2. Using murine models, the present studies have shown that interleukin-15, in vivo, is three to four times more potent than interleukin-2 in generating cytolytic effector splenocytes that lyse YAC target cells. It is approximately one-third as potent as interleukin-2 in inducing specific cytolytic cells that lyse allogeneic target cells. Interleukin-15 is approximately half as potent as interleukin-2 in suppressing pulmonary metastasis induced by MCA-205 tumor cells. The dose of interleukin-15 required to induce pulmonary vascular leak in mice is six times higher than that required for interleukin-2. These results support the view that interleukin-15 exhibits a therapeutic index that is superior to interleukin-2.

Effect of CL 184,005, a platelet-activating factor antagonist in a murine model of Staphylococcus aureus-induced gram-positive sepsis.

Experiments using a murine model of heat-killed Staphylococcus aureus-induced gram-positive bacterial sepsis indicate that the lethal bacterial effects can be prevented if mice are pretreated with CL 184,005, a platelet-activating factor (PAF) antagonist. CL 184,005 was ineffective when administered after bacterial challenge. Plasma of mice pretreated with CL 184,005 contained significantly less tumor necrosis factor (TNF), suggesting that CL 184,005 interferes with TNF synthesis induced by S. aureus. Spleen-associated TNF protein was also decreased by pretreatment with CL 184,005. Although TNF levels were significantly decreased in mice treated with CL 184,005, interleukin-6 levels in serum were significantly increased. Athymic mice were also susceptible to the lethal effects of S. aureus, suggesting that T cells were not involved. When rats rendered hypotensive with S. aureus were treated with CL 184,005, their blood pressure was normalized. Mice treated with enterotoxin B were not protected if they were pretreated with CL 184,005; however, TNF levels in these mice were significantly lower, suggesting that mediators other than PAF and TNF may contribute to the lethal effects of enterotoxin.

Studies on the effect of CL 306,293, a substituted quinoline carboxylic acid, on the clinical disease induced in mice with LP-BM5 virus.

CL 306,293, a substituted quinoline carboxylic acid, is a potent inhibitor of dihydroorotic acid dehydrogenase, an enzyme essential for the biosynthesis of pyrimidines. In mammalian cell culture, the agent exhibits antiproliferative properties that can be reversed by the addition of uridine. CL 306,293 inhibits the development of the clinical disease in a murine model of immunodeficiency induced by a mixture of LP-BM5 retroviruses. In infected mice, the agent prevents the development of hypergammaglobulinemia, lymphadenopathy, splenomegaly and induction of an IL-2 deficiency. The CD4/CD8 ratio and the number of B cells in the lymph nodes are decreased if the infected animals are treated with CL 306,293. CL 306,293 was more efficacious and potent than 3'-azido-3'-deoxythymidine. The beneficial effects of CL 306,293 observed in this model are most probably related to its antiproliferative properties.

Preclinical studies with platelet-activating factor antagonists in models of septic shock.

Since the isolation and elucidation of the structure of platelet-activating factor (PAF) in the late 1970's, several preclinical studies have suggested that PAF is a key mediator of septic shock induced in animals by either endotoxin or by Gram-negative bacteria. A number of PAF antagonists have been sythesized that protect animals from the lethal effects of endotoxin. Some of these antagonists are in early stages of clinical development. The most advanced cadidate is BN 52021, a ginkgolide, that is in Phase II/III clinical trials in patients with septic shock. Preliminary results with BN 52021 indicate that it is efficacious and significantly reduces mortality associated with Gram-negative sepsis. Pivotal trials with BN 52021 aer ongoing. The present review summarizes the biological effects of PAF and the effect of PAF antagonists in animal models of septic shock. The interrelationship of PAF and tumor necrosis factor (another key mediator of septic shock) is also discussed.

Pluronic F 127 liquid sensitizes mice to low doses of Escherichia coli lipopolysaccharide.

BACKGROUND AND METHODS: In murine models of endotoxemia, large amounts of lipopolysaccharide have to be administered to induce mortality. If mice are pretreated with D-galactosamine, the amount of lipopolysaccharide required to induce mortality is significantly lowered. Pluronic F 127 liquid is a relatively non-toxic copolymer that exhibits reverse gelation properties. Thus, it is a liquid at cold temperature and a gel at body temperature. The present studies were performed to ascertain whether the reverse gelation properties of Pluronic F 127 liquid could be used in devising a model of septic shock where a sustained delivery of lipopolysaccharide occurred. In evaluating this model, dose-response studies were conducted with lipopolysaccharide when a) it was administered intraperitoneally in saline or in Pluronic F 127 liquid, and b) it was administered intravenously to mice that had been pretreated with saline or Pluronic F 127 liquid. Mortality was followed for up to 72 hrs. RESULTS: Various doses of Escherichia coli lipopolysaccharide dissolved in saline or in Pluronic F 127 liquid were administered intraperitoneally to mice. The lethal dose of lipopolysaccharide required to kill 50% of the mice (LD50) administered in Pluronic F 127 liquid was approximately ten- to 15-fold less than the values obtained for lipopolysaccharide administered in saline. This decrease in the LD50 of lipopolysaccharide was also observed if the mice were treated intraperitoneally with Pluronic F 127 liquid and challenged 6 hrs later with iv lipopolysaccharide. The concentrations of tumor necrosis factor and interleukin-6 in the plasma were significantly higher when a low dose of lipopolysaccharide was administered to mice that had been pretreated with Pluronic F 127 liquid. While there was no effect on the liver enzymes, Pluronic F 127 liquid caused an increase in the plasma triglycerides. CONCLUSIONS: The data reported in this paper indicate that the LD50 of lipopolysaccharide is significantly decreased if it is administered in Pluronic F 127 liquid or administered to mice that have been pretreated with the Pluronic F 127 liquid. Thus, Pluronic F 127 liquid appears to sensitize mice to low levels of lipopolysaccharide. Unlike the D-galactosamine model, lipopolysaccharide can be administered as late as 6 hrs after treatment with Pluronic F 127 liquid. While the mechanisms by which Pluronic F 127 liquid sensitizes mice is not known, plasma triglycerides were increased in mice treated with this agent, suggesting that tissues responsible for the synthesis and/or degradation of triglycerides play a role in this sensitization process.

Studies of the effect of a platelet-activating factor antagonist, CL 184,005, in animal models of gram-negative bacterial sepsis.

The effect of CL 184,005, a potent and specific platelet-activating factor antagonist, has been examined in a variety of animal models relevant to gram-negative bacterial sepsis. Pretreatment of mice with CL 184,005 protected them from the lethal effects of platelet-activating factor. When rats or primates rendered hypotensive with endotoxin were treated with CL 184,005, blood pressure was normalized. Pretreatment of rats with CL 184,005 protected them from the gastrointestinal lesions induced by endotoxin. Pretreatment of rats and mice with CL 184,005 protected them from the lethal effects of endotoxin. Plasma tumor necrosis factor levels in endotoxin-treated mice were lower when the mice were pretreated with CL 184,005. These observations suggest that CL 184,005 may be potentially useful in the treatment of gram-negative bacterial sepsis, and the agent is undergoing clinical evaluation.

Studies on the homing of Mycobacterium-sensitized T lymphocytes to the synovium during passive adjuvant arthritis.

The migration of intravenously administered adjuvant sensitized T lymphocytes to the knee synovium of recipient rats undergoing passive adjuvant arthritis has been followed. Using fluorescein isothiocyanate (FITC)-labeled adjuvant-sensitized T cells and anticollagen IgG, the present studies demonstrate the presence of fluorescent cells in the inflamed knee synovium of recipient rats undergoing passive arthritis. Proliferation studies indicate that synovial cells from these rats respond to Mycobacterium tuberculosis (MT). Since cross-reactivity between Mycobacterial antigens and cartilage proteoglycans has been previously demonstrated, it is suggested that adjuvant-sensitized T cells that are injected into naive rats migrate to the synovium, proliferate in response to cartilage proteoglycan, and initiate passive arthritis.

M protein deficient streptococcal cell walls can induce acute and chronic arthritis rats.

Cell walls from M+ and M- protein variants of group A streptococci were examined for their arthritogenicity in female Lewis rats. Intraperitoneal administration of both of these sonicated cell wall preparations caused a severe acute and chronic arthritis in recipient rats. Histological evaluation of the hind paw of these rats indicated synovial lining hyperplasia, cell infiltration in the subsynovial space, pannus formation, and erosions of bone and cartilage. Joint pathology was similar in the hind paws of rats immunized with cell walls prepared from either the M+ or the M- protein variants. Cell-mediated immunity was also similar when lymph nodes were exposed to cell walls derived from these two preparations. A recombinant M6 protein from streptococci did not elicit a proliferative response from lymph nodes prepared from arthritic rats. These observations indicate that the M protein that has previously been implicated in auto-immunity does not have a critical role in the pathogenesis of streptococcal cell wall arthritis in rats.

Streptococcal cell wall arthritis. Passive transfer of disease with a T cell line and crossreactivity of streptococcal cell wall antigens with Mycobacterium tuberculosis.

Primary lymph node cells derived from streptococcal cell wall arthritic rats or those derived from adjuvant arthritic rats proliferated in response to cell wall antigens derived from either streptococcal cell walls or those from M. tuberculosis. In addition, two T cell lines have been isolated from lymph nodes of rats during the chronic phase of streptococcal cell wall arthritis. These T cell lines transfered clinical disease to naive syngeneic irradiated recipients, and they proliferated in the presence of cell wall antigens derived from streptococci or antigens derived from Mycobacterium but failed to proliferate in the presence of the 65-kD antigen (containing the sequence TFGLQLELT) derived from Mycobacterium. These observations indicate that T cells play a crucial role in the pathogenesis of streptococcal cell wall arthritis and suggest that antigenic crossreactivity exists between cell walls of group A streptococci and antigens derived from Mycobacterium. The 65-kD Mycobacterium protein is not involved in the observed antigenic crossreactivity.

Studies on the synergy between collagen and adjuvant arthritis in rats.

Intravenous administration of subarthritogenic doses of anticollagen IgG and adjuvant-sensitized spleen cells to syngeneic naive rats induces an erosive arthritis in recipients. The onset of the clinical disease in recipients is rapid and the disease is severe when compared to those recipients receiving cells alone. Immunocytochemical analysis of the knee synovium indicates the accumulation in the adipose tissue of Ia+ (ED1+)macrophages, OX-19+ T lymphocytes, and neutrophils. A large proportion of the lining cells of the proliferative synovium are Ia+. The knee synovium is extremely edematous and contains fibrin. If recipient rats are decomplemented, clinical disease is delayed and the number of mononuclear and polymorphonuclear cells accumulating in the synovium is decreased. Similar results are observed if recipient rats are treated with anti-Ia+ antibody. However, anti-Ia+ treatment does not induce depletion of serum complement.

Con A induction of IgG secretion from a B-lymphoid tumour cell line, A20.

We have examined the effect of concanavalin A (Con A), a conventionally known T-cell mitogen, on the induction of Ig secretion from BALB/c B lymphoid cell lines expressing membrane IgM (X16C8.5, BALENTL 17.7.2 and BCL1) or membrane IgG (A20 and M12.4). A20 tumour cells, but not other tumour cells, responded to Con A by secreting Ig as measured by the reverse plaque-forming cell (PFC) assay. Another typical T-cell mitogen, phytohaemagglutinin (PHA), did not have any effect on the differentiation of A20 tumour cells. The addition of NaN3 abolished the PFC formation, showing that IgG PFC formation of A20 tumour cells was due to the active synthesis of secreted IgG and not due to the shedding of mIgG. The increase in the biosynthesis of secreted IgG from A20 tumour cells by Con A was also observed by polyacrylamide gel electrophoresis. The ability of Con A to induce IgG secretion from A20 tumour cells was abolished by the addition of alpha-methylmannoside, confirming that Con A itself, and not some contaminating material in Con A, induced the maturation of A20 tumour cells. Con A was also shown to induce Ig secretion, but not proliferation, in a subset of normal B cells. Thus, we conclude that Con A does give a differentiation signal to a subset of B cells.

Selective immunomodulation by the antineoplastic agent mitoxantrone. I. Suppression of B lymphocyte function.

Novantrone mitoxantrone, an antineoplastic agent with antiproliferative properties, is under investigation as an immunomodulating agent. The impact of mitoxantrone treatment on B lymphocyte reactivity is presented here. Administered i.p. in H2O at a dose of 0.5 mg/kg, daily for 14 days, mitoxantrone abrogated both the in vivo antibody response (to ovalbumin) and the in vitro plaque-forming cell (PFC) response (to SRC). In addition to the effects on thymus-dependent reactivity, PFC responses to the thymus-independent antigens TNP-LPS and TNP-Ficoll were also inhibited when tested in vivo or in vitro. B cells were identified as a target for the suppressive activity of mitoxantrone by using T cell-replacing factor to reconstitute the in vitro anti-SRC PFC response of a T lymphocyte-depleted spleen cell preparation. LPS-induced B cell mitogenesis was largely inhibited by mitoxantrone treatment. However, depletion of Sephadex G-10-adherent cells significantly restored the proliferative response. Flow cytometric analysis revealed a dramatic decrease in splenic B lymphocyte content. Therefore, mitoxantrone exerted a potent suppressive influence on the humoral immune system through a direct reduction in B cell number augmented by macrophage-mediated inhibition of B cell proliferation.

Selective immunomodulation by the antineoplastic agent mitoxantrone. II. Nonspecific adherent suppressor cells derived from mitoxantrone-treated mice.

Mitoxantrone exerts a potent suppressive influence upon humoral immune responses. The B cell is a likely target for this inhibitory effect, and we have reported evidence supporting this possibility. The impact of mitoxantrone upon T lymphocyte reactivity was assessed as a second mode of action of this novel antineoplastic drug. TH and TS lymphocyte induction were tested in the in vitro anti-sheep erythrocyte response, and a surprising differential effect of mitoxantrone was observed. Helper activity was abrogated and suppressor function was enhanced. In apparent disagreement with this result, mitoxantrone inhibited the in vivo induction of TS cells using trinitrophenylated spleen cells. Macrophages were investigated as potential mediators of these effects upon immunoregulatory function. Replacement of macrophages in mitoxantrone-treated spleen cell preparations by normal adherent cells allowed the induction and complete expression of TH lymphocyte function. Conversely, replacement of mitoxantrone-treated macrophages with normal adherent cells before induction of TS cells failed to generate TS cell function. Thus, TH cells were resistant and TS cells were completely susceptible to mitoxantrone. Furthermore, supplementation of normal TH cell cultures with splenic macrophages from mitoxantrone-treated mice inhibited the induction of helper function. Production of the lymphokines IL 2 and TRF in mitoxantrone-treated mice was normal. This is consistent with the retention of functional TH cells in drug-treated spleens. Macrophages in the spleens of mitoxantrone-treated mice were responsible for the abrogated helper function and the enhanced suppressor activity. Although TS cell induction was directly inhibited by the drug, the effect upon TH cell function was secondary to the action of mitoxantrone-induced suppressor macrophages. Mitogen-stimulated lymphokine production was normal. Thus, mitoxantrone is a selective immunomodulator. The macrophage-mediated suppression of TH cell induction and humoral immunity investigated in spleens from mitoxantrone-treated mice is an intriguing finding that may have significant implications for immunotherapy.