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Insulin-like growth factors I and II (IGF-I and IGF-II) are growth factors implicated in both normal mammary gland development and breast cancer. We have previously reported on the effects of components of the IGF system on breast epithelial cells. Since data suggests that stromal-epithelial interactions play a crucial role in breast cancer, we have now investigated the mitogenic properties of IGF-I, IGF-II, insulin-like growth factor binding protein-3 (IGFBP-3) and epidermal growth factor (EGF) on human breast stromal cells in primary culture. We show that, under serum-free conditions, stromal cells are stimulated to grow in response to IGF-I and IGF-II in a dose-dependent manner. IGF-I and EGF, a potent stimulator of human breast epithelial cell growth in primary culture and also associated with breast cancer, appear to stimulate stromal cell growth in a synergistic manner. IGFBP-3 does not inhibit the stimulation of growth by IGF-I, or IGF-I plus EGF. However, IGFBP-3 does inhibit the stimulation of growth by IGF-II. In contrast to our previous results with human breast epithelial cells, IGFBP-3 does not have an IGF-independent inhibitory effect on stromal cell growth. This study is the first to address the effects of IGF-I, IGF-II and IGFBP-3 alone and in combination with EGF on human breast stromal cell growth in primary culture. Characterizing the role of the IGF system in both normal breast epithelial cells and stromal cells will aid in our understanding of the mechanisms behind the role of the IGF system in breast cancer.

The effects of 1-O-hexadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-16-OCH3-GPC) and its metabolite 1-O-hexadecyl-2-O-methyl-sn-glycerol (AMG) on the activity of diacylglycerol kinase (DGK) in WEHI-3B cells were investigated. Treatment of WEHI-3B cells with 200 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 5 min leads to the activation of cytosolic DGK without significant effect on microsomal DGK. When these cells were first exposed to 50 μM ET-16-OCH3-GPC for 30 min prior to activation with TPA, the activity of DGK was inhibited by about 70%, as measured by the ability of enzyme to form [32P]phosphatidic acid ([32P]PA). Addition of either ET-16-OCH3-GPC or AMG to the preparation of enzyme in vitro also inhibited 1,2-dioleoyl-sn-glycerol (DG) phosphorylation in the presence of [γ-32P]ATP. The IC50 value for inhibition of cytosolic DGK by ET-16-OCH3-GPC and AMG were about 8.5 and 15 μM, respectively. ET-16-OCH3-GPC also inhibited the ability of guanosine 5′-O-(3-thiophosphate) (GTP-7S) to activate DGK in vitro. The potency of ET-16-OCH3-GPC at 10 μM in inhibiting DGK was greater than that of sphingosine at 50 μM, but less than that of R59022 (a specific DGK inhibitor) at 10 μM. The abilities of ET-16-OCH3-GPC and AMG to inhibit cytosolic DGK in intact WEHI-3B cells and enzyme preparations in vitro suggest that the cytotoxic activity of ether lipids may in part result from interference with this vital enzyme involved in the synthesis of phospholipids from DG and in cell-signaling systems.Key words: diacylglycerol, kinases, second messengers, ether lipids, cancer, WEHI-3B cells.

ABSTRACT: We analyzed the distribution of HLA class II alleles and haplotypes in one Na-Dene (Athabaskan) group from British Columbia (Canada) by PCR amplification of the DRB1, DQA1 and DQB1 second exon sequences. We extended the typing of the DRB1 in an Amerindian group (Penutian) from British Columbia. The presence of the alleles DRB1∗ 0405, ∗0407 and ∗0410 only in Na-Dene and alleles DRB1∗0408, ∗1301∗1302, ∗1304, ∗1305, ∗1502 and ∗1601 only in Amerindians suggests separate origins of these two groups. There were fifteen different DRB1/DQA1/DQB1 haplotypes. One unique haplotype previously reported in Native Americans was found. Thirty-four per cent of Athabaskans presented Native American haplotype DRB1∗1402/DQA1∗0501/DQB1∗0301. In addition, the results of this study are compatible with previous evidence with mitochondrial (mtDNA) polymorphisms indicating that Amerindians and Na-Dene populations derived from different migrations from Asia.