IL-21 induces in vivo immune activation of NK cells and CD8(+) T cells in patients with metastatic melanoma and renal cell carcinoma.

PURPOSE: Human interleukin-21 (IL-21) is a class I cytokine previously reported in clinical studies on immune responsive cancers. Here we report the effects of systemic IL-21 therapy on the immune system in two phase 1 trials with this novel cytokine. EXPERIMENTAL DESIGN: Recombinant IL-21 was administered by intravenous bolus injection at dose levels from 1 to 100 microg/kg using two planned treatment regimens: thrice weekly for 6 weeks (3/week); or once daily for five consecutive days followed by nine dose-free days (5 + 9). The following biomarkers were studied in peripheral blood mononuclear cells (PBMC) during treatment: phosphorylation of STAT3, alterations in the composition of leukocyte subsets, ex vivo cytotoxicity, expression of effector molecules in enriched CD8(+) T cells and CD56(+) NK cells by quantitative RT-PCR, and gene array profiling of CD8(+) T cells. RESULTS: Effects of IL-21 were observed at all dose levels. In the 5 + 9 regimen IL-21 induced a dose dependent decrease in circulating NK cells and T cells followed by a return to baseline in resting periods. In both CD8(+) T cells and CD56(+) NK cells we found up-regulation of perforin and granzyme B mRNA. In addition, full transcriptome analysis of CD8(+) T cells displayed changes in several transcripts associated with increased cell cycle progression, cellular motility, and immune activation. Finally, cytotoxicity assays showed that IL-21 enhanced the ability of NK cells to kill sensitive targets ex vivo. CONCLUSIONS: IL-21 was biologically active at all dose levels administered with evidence of in vivo NK cell and CD8(+) T cell activation.

Augmenting tumor sensitivity to topotecan by transient hypoxia.

We examined how the effect of topotecan is modulated by transient hypoxia in three different tumor lines, Lewis lung carcinoma (LLC), U87 human glioblastoma and DMS273 human small cell lung cancer. Four groups of tumor bearing mice were treated with saline or a single dose of topotecan, immediately followed by 6-h or 72-h exposure to a hypoxic environment (10% O(2)) or normal air. Topotecan + hypoxia resulted in significantly greater suppression of tumor growth than normoxic topotecan or hypoxia alone. Correspondingly, the sensitivity of LLC cells to topotecan in a clonogenic survival assay was significantly higher under hypoxia. This effect of hypoxia was not a general phenomenon, since the tumor growth inhibitory effect of the alkylating agent cisplatin was not changed by hypoxic environment. In a parallel series of in vitro experiments, cell cultures were exposed to hypoxia (0.1% or 0.7% O(2)) in a hypoxic chamber or normoxia for 24 h. We found a dose-dependent downregulation of HIF-1alpha by topotecan (30-270 nM). The hypoxic upregulation of Glucose transporter-1 and VEGF secretion to the culture medium was inhibited by the addition of topotecan, while doses up to 270 nM had no effect on VEGF under normoxia. VEGF protein levels in tumors were also reduced by topotecan. These data show that the effect of topotecan is increased by transient hypoxia, and this may be a direct effect on the ability of cells to survive under hypoxia as well as an antiangiogenic effect, mediated through the HIF-1 inhibitory effect of topotecan.

Regulation of YKL-40 expression during genotoxic or microenvironmental stress in human glioblastoma cells.

YKL-40 is a 40 kDa secreted glycoprotein belonging to the family of 'mammalian chitinase-like proteins', but without chitinase activity. YKL-40 has a proliferative effect on fibroblasts, chondrocytes and synoviocytes, and chemotactic effect on endothelium and vascular smooth muscle cells. Elevated YKL-40 levels are found in serum of patients with diseases characterized by inflammation, fibrosis and tissue remodeling. Several studies have reported that high serum YKL-40 levels in patients with cancer are associated with poor prognosis. YKL-40 expression is strongly elevated in serum and biopsy material from glioblastomas patients. We investigated the expression of YKL-40 in three human malignant glioma cell lines exposed to different types of stress. Whereas a polymerase chain reaction transcript was detectable in all three cell lines, only U87 produced measurable amounts of YKL-40 protein. In U87, hypoxia and ionizing radiation induced a significant increase in YKL-40 after 24-48 h. The hypoxic induction of YKL-40 was independent of HIF1. Etoposide, ceramide, serum depletion and confluence all led to elevated YKL-40. Inhibition of p53 augmented the YKL-40 expression indicating that YKL-40 is attenuated by p53. In contrast, both basic fibroblast growth factor and tumor necrosing factor-alpha repressed YKL-40. These are the first data on regulation of YKL-40 in cancer cells. Diverse types of stress resulted in YKL-40 elevation, which strongly supports an involvement of YKL-40 in the malignant phenotype as a cellular survival factor in an adverse microenvironment.

Characterisation of cytotoxicity and DNA damage induced by the topoisomerase II-directed bisdioxopiperazine anti-cancer agent ICRF-187 (dexrazoxane) in yeast and mammalian cells.

BACKGROUND: Bisdioxopiperazine anti-cancer agents are inhibitors of eukaryotic DNA topoisomerase II, sequestering this protein as a non-covalent protein clamp on DNA. It has been suggested that such complexes on DNA represents a novel form of DNA damage to cells. In this report, we characterise the cytotoxicity and DNA damage induced by the bisdioxopiperazine ICRF-187 by a combination of genetic and molecular approaches. In addition, the well-established topoisomerase II poison m-AMSA is used for comparison. RESULTS: By utilizing a panel of Saccharomyces cerevisiae single-gene deletion strains, homologous recombination was identified as the most important DNA repair pathway determining the sensitivity towards ICRF-187. However, sensitivity towards m-AMSA depended much more on this pathway. In contrast, disrupting the post replication repair pathway only affected sensitivity towards m-AMSA. Homologous recombination (HR) defective irs1SF chinese hamster ovary (CHO) cells showed increased sensitivity towards ICRF-187, while their sensitivity towards m-AMSA was increased even more. Furthermore, complementation of the XRCC3 deficiency in irs1SF cells fully abrogated hypersensitivity towards both drugs. DNA-PKcs deficient V3-3 CHO cells having reduced levels of non-homologous end joining (NHEJ) showed slightly increased sensitivity to both drugs. While exposure of human small cell lung cancer (SCLC) OC-NYH cells to m-AMSA strongly induced gammaH2AX, exposure to ICRF-187 resulted in much less induction, showing that ICRF-187 generates fewer DNA double strand breaks than m-AMSA. Accordingly, when yeast cells were exposed to equitoxic concentrations of ICRF-187 and m-AMSA, the expression of DNA damage-inducible genes showed higher levels of induction after exposure to m-AMSA as compared to ICRF-187. Most importantly, ICRF-187 stimulated homologous recombination in SPD8 hamster lung fibroblast cells to lower levels than m-AMSA at all cytotoxicity levels tested, showing that the mechanism of action of bisdioxopiperazines differs from that of classical topoisomerase II poisons in mammalian cells. CONCLUSION: Our results point to important differences in the mechanism of cytotoxicity induced by bisdioxopiperazines and topoisomerase II poisons, and suggest that bisdioxopiperazines kill cells by a combination of DNA break-related and DNA break-unrelated mechanisms.

Differential regulation of VEGF, HIF1alpha and angiopoietin-1, -2 and -4 by hypoxia and ionizing radiation in human glioblastoma.

We examined how ionizing radiation (IR) delivered under either severe hypoxia (< 0.1% O2) or normoxia affects the expression of hypoxia inducible factor 1alpha (HIF-1alpha) and the angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietins 1, 2 and 4 in U87 human glioblastoma cells. IR was delivered as single doses of 0, 2, 5, 10 and 20 Gy after 6-hr hypoxic incubation and in normoxic controls. Irradiation at any dose did not affect the cellular protein levels of any of the angiopoietins, whereas hypoxia led to increasing levels of both angiopoietin-4 and angiopoietin-2. Levels of angiopoietin-1 protein were unaltered throughout the observation period. A dose-dependent increase in levels of secreted VEGF in the medium occurred after IR at doses from 5-20 Gy. In hypoxic cells, 20 Gy IR induced an additional significant increase in VEGF relative to nonirradiated hypoxic control cells with elevated baseline VEGF levels induced by hypoxia. HIF-1alpha and glucose transporter-1 (Glut-1) were not correspondingly upregulated by IR. Blocking HIF-1alpha by antisense treatment induced a reduced baseline VEGF at normoxia, while the relative upregulation of VEGF by IR was unaffected. These data provide evidence that VEGF is upregulated by IR by mechanisms independent of HIF-1 transactivation.