No abstract

Summary Rapamycin has antiangiogenic activity against tumors. This has been discussed while addressing the problem of cancer in organ transplantation. Here we investigated effective dosing schedules against tumors and angiogenesis. Growth of established CT‐26 colon adenocarcinoma tumors was measured in Balb/c mice treated with total equivalent rapamycin doses (1.5 mg/kg/day) given once a day, once every 3 days, or by continuous infusion. Tumors were most inhibited with continuous rapamycin infusion, and less by bolus dosing. Interestingly, however, continuous dosing produced the lowest rapamycin blood levels (15 ng/ml). As rapamycin‐sensitive p70S6‐kinase intracellular signaling is critical for angiogenesis, p70S6‐kinase activation was measured in endothelial cells by Western blotting. Maximal p70S6‐kinase inhibition occurred from 1–5 ng/ml rapamycin. These same rapamycin concentrations optimally blocked vessel‐sprouting from cultured aortic rings. Therefore, low‐level rapamycin dosing most effectively controls tumors in mice. Importantly, antiangiogenic rapamycin levels are compatible with immunosuppressive doses, supporting its potential use in transplant patients with cancer.

Purpose: Colorectal neoplasms remain a leading cause of cancer-related deaths. A recognized weakness of conventional 5-fluorouracil (5-FU) therapy relates to expression of the intracellular enzyme, thymidine phosphorylase (TP). Although TP promotes 5-FU cytotoxicity, TP-derived 2-deoxy-D-ribose (dRib) counterproductively stimulates tumor angiogenesis. Here, the newly discovered antiangiogenic drug rapamycin was combined with 5-FU to counteract the potential escape mechanism of dRib-induced angiogenesis.Experimental Design: Orthotopic tumor growth was assessed in rapamycin and 5-FU-treated BALB/c mice with TP-expressing CT-26 colon adenocarcinoma cells. To examine liver metastasis, green-fluorescent protein-transfected CT-26 cells were visualized by fluorescence microscopy after intraportal injection. Cell counting and Ki67 staining were used to determine in vitro and in vivo cell expansion, respectively. In vitro angiogenic effects of dRib were assessed with endothelial cell migration and aortic ring assays. Western blotting detected dRib effects on p70/S6 kinase activation.Results: Rapamycin treatment of mice bearing orthotopic tumors inhibited tumor growth more than did 5-FU, and mice treated with both drugs typically developed no tumors. In the liver metastasis assay, combination therapy blocked metastatic expansion of solitary tumor cells. Interestingly, complex drug activities were suggested by tumorcell proliferation being more sensitive to 5-FU than to rapamycin in vitro, but more sensitive to rapamycin in vivo. With regard to angiogenesis, dRib-induced endothelial cell migration and aortic ring formation were completely abrogated by rapamycin, correlating with blockage of dRib-induced p70/S6 kinase activation in endothelial cells.Conclusions: Inhibition of dRib-induced angiogenesis with rapamycin counteracts a potential TP-based escape mechanism for colorectal cancer under 5-FU therapy, introducing a novel, clinically feasible, combination treatment option for this common neoplasm.

Gene transfer into the skin is a promising approach to treat duced within 48 h. This effect was independent of individinherited or acquired dermatological diseases and sysual skin donors and different body areas serving as the temic monogenic deficiencies. For this purpose, the source for keratinocyte isolation. rAAV had no significant efficient and sustained gene delivery into keratinocytes is influence on cell viability, but induced a growth arrest in of critical importance. Recombinant adeno-associated transduced keratinocytes. This growth arrest was overvirus (rAAV) vectors hold the potential to achieve a longcome by replating cells in fresh media. rAAV/GFP-transterm gene transfer into various human organs. In order to duced keratinocytes could be passaged several times, evaluate this potential for skin gene therapy, human keraexpressed GFP for up to 50 days, and passed the transtinocytes were transduced in vitro with rAAV vectors encogene to their daughter cells, suggesting that keratinocyte ding the reporter genes ␤-galactosidase (rAAV/LacZ) or precursor cells were also transduced. Taken together, the green fluorescent protein (rAAV/GFP). Using rAAV/LacZ at results suggest that rAAV is a promising gene transfer a multiplicity of infection (MOI) of five transducing particles vehicle for skin gene therapy. per cell, up to 70% of human keratinocytes were trans-

Outcome in patients with PVT seems to be dependent on pre-operative disease severity. In contrast to compensated liver disease, no influence of PVT on outcome could be found in decompensated liver disease, and should therefore not be considered as a contraindication in LTx.