Caroline O. Oyedeji

A missense mutation in KIT kinase domain 1 correlates with imatinib resistance in gastrointestinal stromal tumors

KIT gain of function mutations play an important role in the pathogenesis of gastrointestinal stromal tumors (GISTs). Imatinib is a selective tyrosine kinase inhibitor of ABL, platelet-derived growth factor receptor (PDGFR), and KIT and represents a new paradigm of targeted therapy against GISTs. Here we report for the first time that, after imatinib treatment, an additional specific and novel KIT mutation occurs in GISTs as they develop resistance to the drug. We studied 12 GIST patients with initial near-complete response to imatinib. Seven harbored mutations in KIT exon 11, and 5 harbored mutations in exon 9. Within 31 months, six imatinib-resistant rapidly progressive peritoneal implants (metastatic foci) developed in five patients. Quiescent residual GISTs persisted in seven patients. All six rapidly progressive imatinib-resistant implants from five patients show an identical novel KIT missense mutation, 1982T→C, that resulted in Val654Ala in KIT tyrosine kinase domain 1. This novel mutation has never been reported before, is not present in pre-imatinib or post-imatinib residual quiescent GISTs, and is strongly correlated with imatinib resistance. Allelic-specific sequencing data show that this new mutation occurs in the allele that harbors original activation mutation of KIT.

Murine kupffer cells and hepatic natural killer cells regulate tumor growth in a quantitative model of colorectal liver metastases

This investigation aimed to develop a biologically relevant murine model of colorectal liver metastases and determine if Kupffer cells (KC) and hepatic natural killer cells (hNKC) regulate tumor growth. The model involves the injection of murine colon adenocarcinoma 26 (MCA 26) tumor cells into the portal vein of female-specific pathogen-free BALB/c mice. Metastases developed in all animals, and the growth was limited entirely to the liver. To determine if KC and hNKC control the development of liver metastases, the in vivo function of these hepatic effector cells was modulated. Tumor growth was quantitated by the uptake of 125I into tumor DNA. Stimulation of the KC and hNKC produced a significant (P < 0.01) dose-dependent decrease in 125I uptake in the liver in both treatment groups, which was associated with a significant improvement in survival (P < 0.05). The in vivo cytotoxic function of the liver was inhibited with an intravenous injection of gadolinium chloride (for KC) or asialo GM1 antiserum (for hNKC). Inhibition of KC and hNKC cytotoxic function led to a significant (P < 0.01) increase in 1251 uptake in the liver and a significant decrease in survival (P < 0.05).

Mechanisms of Kupffer cell cytotoxicity in vitro against the syngeneic murine colon adenocarcinoma line MCA26

We have previously demonstrated that in vivo activation or inhibition of Kupffer cell (KG) cytotoxic function can reduce or enhance, respectively, the hepatic tumor burden in a syngeneic murine colon adenocarcinoma (MCA26) tumor model. In the current study, we have performed in vitro experiments to define the possible mechanisms of KG cytotoxicity against MGA26 cells. Addition of either anti‐tumor necrosis factor (TNF) or anti‐interleukin‐1α (IL‐lα) antisera reduced KG cytotoxicity in coculture against MCA26 targets in a dose‐ dependent fashion; addition of these sera together resulted in approximately additive inhibition, suggesting the existence of parallel pathways for these effector molecules. Nitric oxide as a mediator of cytotoxicity by KCs in coculture with MCA26 cells was evaluated by two approaches. Activated KGs produced detectable levels of nitric oxide; however, activated KC exerted cytotoxicity against MCA26 targets in the absence of exogenous free L‐arginine. Thus, TNF and IL‐1 play major roles in producing murine KG cytotoxicity against MCA26 colon cancer cells in vitro, whereas reactive nitric oxides do not.

Bilirubin inhibits cartilage metabolism and growth in vitro

The potential effect of bilirubin on cartilage metabolism and growth in vitro was studied using embryonic chick and hypophysectomized rat cartilage bioassays, both very sensitive to cartilage growth factors and growth inhibitors. In chick cartilage, 0.25 mmol/L bilirubin caused significant but reversible inhibition of growth. Growth inhibition was due, at least in part, to bilirubin-induced inhibition of [35S]sulfate incorporation into proteoglycans by as little as 0.05 mmol/L bilirubin and to a lesser extent by inhibition of [14C]leucine incorporation into proteins and [3H]uridine incorporation into RNA. The addition of albumin or serum had no effect on bilirubin bioactivity. Thus, bilirubin may have a novel role in the pathophysiology of skeletal growth ratardation associated with some chronic diseases.

Somatomedin Inhibitors in Serum and Liver of Growth Hormone-Deficient Diabetic Rats

Diabetes of moderate severity was induced with streptozotocin in growth hormone (and therefore somatomedin)-deficient rats. Somatomedin inhibitors were identified in the serum and liver perfusate of these animals, as shown by the ability of samples to blunt basal cartilage sulfation and cartilage stimulation by added normal serum. The data suggest that the induction of somatomedin inhibitors is under the influence of insulin and nutrition rather than growth hormone, indicating that their regulation may differ from that of growthpromoting somatomedins. With this model, it is possible to obtain preparations that are relatively free of somatomedins in which to study the properties of somatomedin inhibitors.

Human Kupffer cells are cytotoxic against human colon adenocarcinoma

Colorectal liver metastases are a common clinical problem and require more effective therapy. Kupffer cells (KC) perform many important homeostatic functions within the liver and may also possess the ability to mediate tumor cytotoxicity. We investigated the ability of human KC to mediate cytotoxicity against human colon adenocarcinoma targets (HT 29) in vitro. Unstimulated human KC were cytotoxic against the HT 29 targets at all effector/target ratios tested. This cytotoxicity was increased significantly (p < 0.05) when the KC were stimulated with interferon‐γ and lipopolysaccharide. Human KC produced tumor necrosis factor (TNF), and KC stimulation significantly (p < 0.05) increased secretion of this monokine. The addition of anti‐ TNF antibody to the KC‐HT 29 cocultures completely neutralized all of the available TNF yet cytotoxicity was not affected, suggesting the participation of a membrane‐bound form of TNF or other mechanisms. (SURGERY 1990;l08:400–5).

Bilirubin and Heme as Growth Inhibitors of Chicken Embryos in Ovo

ABSTRACT: The increased morbidity during pregnancies complicated by hematologic or liver disease has generally been attributed to the metabolic abnormalities of the illness itself. Because tetrapyrrole concentrations are elevated in these conditions, we introduced bilirubin or heme (prepared as 10 mM solutions) into the air sac of fertilized chicken eggs to study their effect on the growth of normal chicken embryos. In 9-d eggs, the injection of 0.06 mL heme resulted in significant embryo growth inhibition as indicated by overall wt (91 ± 3% versus control), tibia length (84 ± 2%), tibia wt (81 ± 3%), femur length (88 ± 1%), and femur wt (78 ± 3%); doses greater than 0.10 mL resulted in substantial fetal losses. The injection of 0.06 mL bilirubin into the same-age eggs also resulted in less than normal tibia length (87 ± 2% versus control), tibia wt (75 ± 4%), femur length (91 ± 2%), and femur wt (81 ± 3%); larger doses resulted in more pronounced growth inhibition, but fetal losses were less common than with heme. Older chick embryos (12-d) appeared more resistant to the effects of bilirubin: 0.15 mL bilirubin inhibited only tibia and femur wt; larger doses were required to significantly suppress the other growth parameters. The sameage chicken embryos, however, remained exquisitely sensitive to heme; 0.05 mL resulted in less than normal whole embryo wt (88 ± 2% versus control), tibia length (80 ± 1%), tibia wt (76 ± 1%), femur length (78 ± 1%), and femur wt (77 ± 1%). Substantial fetal loss occurred with heme doses above 0.10 mL as well as among the less mature chicks. Significant inhibition of the growth of long bones (tibia and femur) also occurred when we added 0.05 mM bilirubin or 0.025 mM Hb to bones maintained in organ culture in vitro for 5 d. We conclude that several heme compounds and bilirubin inhibit the growth and survival of normal chick embryos in ovo and in vitro.We suggest that prenatal exposure to high levels of these compounds may have a previously underestimated negative influence on fetal development.

Hematoporphyrin can inhibit the metabolism and growth of embryonic chicken cartilage in vitro

Hematoporphyrin (HP) derivatives have been recognized chiefly as growth inhibitors of neoplastic tissues within the context of tumor phototherapy. We investigated the possibility that HP may also modify the growth potential of rapidly growing nonneoplastic tissues and describe the effect of HP on the metabolism and growth of embryonic chick cartilage in vitro. In the embryonic chick pelvic rudiment prolonged organ-culture bioassay, pelvic rudiment growth was significantly inhibited when HP 0.01 mmol/L was added to the culture medium (after 5 days in organ culture, weight increment in presence of serum + 136% +/- 12% v + 90% +/- 8% in presence of serum plus HP 0.01 mmol/L and + 43% +/- 10% in presence of serum plus HP 0.025 mmol/L, P less than .001). Inhibition of cartilage growth was irreversible if HP was added to the culture medium without serum for 2 days, but partial reversibility was observed when hypophysectomized rat serum was present during that period. Therefore, the protective effect of serum was apparently unrelated to pituitary-dependent growth factors. Binding of HP to albumin did not eliminate its inhibitory effects. Uridine incorporation into RNA and, to a lesser extent, sulfate incorporation into proteoglycans were reduced in cartilage incubated overnight with HP. Our study shows that HP can exert partially reversible inhibition of cartilage metabolism and growth suggesting potentially novel tissue actions for this compound.