Non-core region modulates interleukin-11 signaling activity: generation of agonist and antagonist variants.

Human interleukin-11 (hIL-11) is a pleiotropic cytokine administered to patients with low platelet counts. From a structural point of view hIL-11 belongs to the long-helix cytokine superfamily, which is characterized by a conserved core motif consisting of four α-helices. We have investigated the region of hIL-11 that does not belong to the α-helical bundle motif, and that for the purpose of brevity we have termed "non-core region." The primary sequence of the interleukin was altered at various locations within the non-core region by introducing glycosylation sites. Functional consequences of these modifications were examined in cell-based as well as biophysical assays. Overall, the data indicated that the non-core region modulates the function of hIL-11 in two ways. First, the majority of muteins displayed enhanced cell-stimulatory properties (superagonist behavior) in a glycosylation-dependent manner, suggesting that the non-core region is biologically designed to limit the full potential of hIL-11. Second, specific modification of a predicted mini α-helix led to cytokine inactivation, demonstrating that this putative structural element belongs to site III engaging a second copy of cell-receptor gp130. These findings have unveiled new and unexpected elements modulating the biological activity of hIL-11, which may be exploited to develop more versatile medications based on this important cytokine.

Gene expression profiling predicts response to temozolomide in malignant gliomas.

Malignant gliomas are highly lethal neoplasms that cannot be cured with currently available therapies. Temozolomide (TMZ) is a recently introduced alkylating agent that has yielded significant benefits and become a key agent in the treatment of high-grade gliomas. However, its survival benefit remains unsatisfactory. Understanding the molecular basis of TMZ sensitivity/resistance is necessary for improving the treatment outcome by devising strategies that are able to circumvent primary drug resistance. We therefore combined the in vitro TMZ response with microarray gene expression data to identify genes that could potentially be used to predict the response of malignant gliomas to TMZ therapy. We first obtained the individual IC50 values for TMZ in seven malignant glioma cell lines (A-172, AM-38, T98G, U-87MG, U-138MG, U-251MG and YH-13) and then identified the genes whose expression correlated most highly with TMZ sensitivity employing a cDNA microarray. We present here a list of the most highly up-regulated and down-regulated genes which may be involved in conferring TMZ sensitivity/resistance in malignant gliomas, although most of the genes have not been implicated as a causal factor in the TMZ response except MGMT. We also demonstrated and confirmed the MGMT methylation status, quantitative MGMT mRNA levels, and MGMT protein expression levels in TMZ resistant glioma cells in vitro. Our results are thus consistent with previous studies and suggest that a dominant mechanism conferring sensitivity/resistance to TMZ exists in malignant glioma cells. Although the present study dose have several limitations, our reported candidate genes could represent not only potential molecular markers for TMZ sensitivity/resistance but also chemotherapy targets. Furthermore, the present study could provide a foundation for alternative therapeutic strategies including novel combination treatments that incorporate additional reagents directed at overcoming resistance to TMZ.

Effect of IFN-beta on human glioma cell lines with temozolomide resistance.

Human interferon-beta (IFN-beta) is known to exhibit pleiotropic biological activities including antitumor effects. On the other hand, temozolomide (TMZ), an oral bioavailable alkylating agent with excellent tolerability, has demonstrated efficacy and has become a key therapeutic agent in patients with malignant gliomas; however, its survival benefit remains unsatisfactory. More recent studies have indicated that there might be favorable therapeutic interactions between IFN-beta and TMZ, although the therapeutic advantages of such a combination have not yet been fully explored. The main aim of the present study was to determine whether an antitumor effect could be potentiated by a combination of IFN-beta and TMZ. The antitumor effect of and cell sensitivity to IFN-beta and TMZ and the synergistic potential of IFN-beta and TMZ in combination were evaluated in six malignant glioma cell lines. Correlations among the MGMT methylation status, quantitative level of MGMT mRNA, MGMT protein expression and the antitumor effect of these agents were also evaluated, since one of the most prominent resistance mechanisms to TMZ involves the DNA repair protein MGMT. The cell growth inhibitory effects of IFN-beta and TMZ on all tumor cell lines were observed in a dose-dependent manner, and the human malignant glioma-derived cell lines differed in their sensitivity to TMZ. The MGMT status, including promoter hypermethylation, quantitative mRNA expression and protein expression, was strongly correlated with TMZ sensitivity. A synergistic cell growth inhibitory effect and down-regulated MGMT mRNA levels were significantly observed when a clinically achievable CNS dose of IFN-beta was combined with TMZ, as compared to treatment with IFN-beta or TMZ alone in TMZ-resistant T98G cells. Furthermore, significant amounts of endogenous IFN-beta protein were detected in TMZ-treated T98G cells by ELISA. These results suggest that the clinical therapeutic efficacy of TMZ might be improved by a combination with IFN-beta in malignant gliomas unmethylated at the MGMT gene. The data provide an experimental basis for future strategies in TMZ chemotherapy, although further studies are needed to determine the detailed role of combined IFN-beta and TMZ chemotherapy in increasing tumor sensitivity.

A possible role of autogenous IFN-beta for cytokine productions in human fibroblasts.

It has been already known that human diploid fibroblasts are able to produce not only high levels of IFN-beta but also various kinds of cytokines by poly rI: poly rC, and some inflammatory cytokines are induced by IFN-beta gene activation. We also obtained similar results. However, in our system, cytokine productions were extremely enhanced by treating the cells with a low dose of type 1 IFN and the priming effects on cytokine productions were blocked by cycloheximide similar to those on IFN-beta productions. Most of cytokines were produced later than IFN-beta and synthesis patterns of their mRNA showed the same phenomena. We made clear that cytokine productions by poly rI: poly rC are mediated by secreted IFN-beta at a protein level using a monoclonal antibody against human IFN-beta. Further, it was shown that intra-cellular IFN-beta which is not secreted might also participate in cytokine productions. Meanwhile, IL-1beta induced various kinds of cytokines in human fibroblasts and production time courses of these cytokines were similar to those of poly rI: poly rC induced cytokines. Although secreted IFN-beta was not detected in IL-1beta stimulated culture, expression of IFN-beta mRNA was augmented. These results showed that priming effects of type 1 IFN on cytokine productions by poly rI: poly rC might not be the direct action, but successive IFN-beta production might be essential in the production processes of other cytokines. Further, it was suggested that inducible IFN-beta might also take part in IL-1beta-induced cytokine productions.

[Research and development of interferons].

After interferon was discovered, it has passed in 50 years. It looks back upon the basic research which contributed to progress of clinical application in the meantime, and development of the various IFNs which made the large-scale clinical test possible.