The zinc transporter ZIP8 is highly expressed in T cells derived from human subjects. T cell ZIP8 expression was markedly up-regulated upon in vitro activation. T cells collected from human subjects who had received oral zinc supplementation (15 mg/day) had higher expression of the activation marker IFN-gamma upon in vitro activation, indicating a potentiating effect of zinc on T cell activation. Similarly, in vitro zinc treatment of T cells along with activation resulted in increased IFN-gamma expression with a maximum effect at 3.1 microM. Knockdown of ZIP8 in T cells by siRNA decreased ZIP8 levels in nonactivated and activated cells and concomitantly reduced secretion of IFN-gamma and perforin, both signatures of activation. Overexpression of ZIP8 by transient transfection caused T cells to exhibit enhanced activation. Confocal microscopy established that ZIP8 is localized to the lysosome where ZIP8 abundance is increased upon activation. Loss of lysosomal labile zinc in response to activation was measured by flow cytometry using a zinc fluorophore. Zinc between 0.8 and 3.1 microM reduced CN phosphatase activity. CN was also inhibited by the CN inhibitor FK506 and ZIP8 overexpression. The results suggest that zinc at low concentrations, through inhibition of CN, sustains phosphorylation of the transcription factor CREB, yielding greater IFN-gamma expression in T cells. ZIP8, through control of zinc transport from the lysosome, may provide a secondary level of IFN-gamma regulation in T cells.

Many proteins are associated with the outer layer of the cell membrane through a posttranslationally added glycosyl phosphatidylinositol (GPI) anchor. The functional significance of this type of protein linkage is unclear, although it results in increased lateral mobility, sorting to the apical surface of the cell, reinsertion into cell membranes, and possibly cell signaling. Here evidence is presented that GPI-linked proteins can undergo intermembrane transfer in vivo. GPI-linked proteins expressed on the surface of transgenic mouse red blood cells were transferred in a functional form to endothelial cells in vivo. This feature of GPI linkage may be potentially useful for the delivery of therapeutic proteins to vascular endothelium.

We characterize a line of transgenic pigs that express the human complement-regulatory proteins human CD59 and human decay-accelerating factor. These genes, under the control of heterologous promoters, are expressed in a variety of organs, including the vasculature of the heart, kidney, and liver. We demonstrate that moderate levels of these gene products are sufficient to protect peripheral blood cells from human or baboon complement. Using pig to baboon heterotopic heart transplants, we show that expression of these proteins is sufficient to block the complement-mediated damage that is the hallmark of such xenografts, when nontransgenic organs are used. These results indicate that there is significant species specificity of intrinsic complement regulatory protein function. This specificity is evident in transgenic organs in which low levels of human CD59 and human decay-accelerating factor expression significantly effect the humoral immune response that causes xenograft rejection. This result suggests that transgenic organs with high levels of human complement-regulatory protein expression will be sufficient to alleviate the humoral immunological barriers that currently block the use of xenogeneic organs for human transplantation.

We explored the nature of the tumor-initiating cell in osteosarcoma, a bone malignancy that predominately occurs in children. Previously, we observed expression of Oct-4, an embryonal transcriptional regulator, in osteosarcoma cell cultures and tissues. To examine the relationship between Oct-4 and tumorigenesis, cells from an osteosarcoma biopsy (OS521) were stably transfected with a plasmid containing the human Oct-4 promoter driving a green fluorescent protein (GFP) reporter to generate the transgenic line OS521Oct-4p. In culture, only f24% of the OS521Oct-4p cells were capable of activating the transgenic Oct-4 promoter; yet, xenograft tumors generated in NOD/SCID mice contained f67% GFP + cells, which selectively expressed the mesenchymal stem cellassociated surface antigens CD105 and ICAM-1. Comparison of the tumor-forming capacity of GFP-enriched and GFPdepleted cell fractions revealed that the GFP-enriched fractions were at least 100-fold more tumorigenic, capable of forming tumors at doses of <300 cells, and formed metastases in the lung. Clonal populations derived from a single Oct-4/GFP + cell were capable of forming tumors heterogeneous for Oct-4/GFP expression. These data are consistent with the cancer stem cell model of tumorigenesis in osteosarcoma and implicate a functional link between the capacity to activate an exogenous Oct-4 promoter and tumor formation. This osteosarcoma tumor-initiating cell appears highly prolific and constitutes a majority of the cell population in a primary xenograft tumor, which may provide a biological basis for the particular virulence of this type of cancer.

Cancer cells with the surface marker profile CD44+/CD24- have previously been described to possess cancer stem cell-like properties. This manuscript evaluates those properties in ovarian cancer cell lines. The proportion of CD44+/CD24- cells corresponded to the clinical aggressiveness of each ovarian cancer cell line histologic subtype. CD44+/CD24- cells demonstrated enhanced progressive differentiation as well as showing a 60-fold increase in Matrigel invasion in both SKOV3 and OV90 cell lines (p < 0.001 each) compared to other phenotypes. CD44+/CD24- demonstrated significant resistance to all chemotherapy agents used in all cell lines, with a 71-93 % increase in resistance compared with baseline. Using a threshold of 25 % CD44+/CD24- ovarian cancer cells found in ascites, patients with >25 % CD44+/CD24- were significantly more likely to recur (83 vs. 14 %, p = 0.003) and had shorter median progression-free survival (6 vs. 18 months, p = 0.01). In conclusion, the CD44+/CD24- phenotype in ovarian cancer cells demonstrate cancer stem cell-like properties of enhanced differentiation, invasion, and resistance to chemotherapy. This CD44+/CD24- phenotype correlates to clinical endpoints with increased risk of recurrence and shorter progression-free survival in patients with ovarian cancer.