Elisabeth Oelmann

Autocrine interleukin-1 receptor antagonist can support malignant growth of glioblastoma by blocking growth-inhibiting autocrine loop of interleukin-1

In situ hybridization (ISH) of human glioblastoma tissue sections revealed expression of interleukin‐1 (IL‐1)α and/or β and IL‐1 receptor types I and II (IL‐1R I and II) in the majority of cases evaluable. To understand the function of IL‐1‐family members in human glioblastomas, we have studied 6 glioblastoma cell lines. RT‐PCR, ISH, ELISA and 125I‐IL‐1‐binding assays revealed expression of IL‐1 and high‐affinity receptors for human (h)IL‐1 in all but 1 cell line. Using a colony growth assay in semi‐solid media for testing serial plating efficacy (PE, number of colonies per number of cells seeded in %), only the IL‐1R‐negative cell line was not influenced by recombinant human (rh)IL‐1α or ‐β, whereas IL‐1 down‐regulated the self‐renewal of clonogenic cells of the other glioblastomas. Tritiated thymidine uptake was down‐regulated by rhIL‐1 in all cell lines studied. Cell viability remained unchanged by rhIL‐1. Wherever growth modulation by rhIL‐1 was detected, it could be reversed by either soluble IL‐1R I or II or by rhIL‐1 receptor antagonist (ra). IL‐1ra not only was able to reverse rhIL‐1‐induced growth modulation but alone could modulate glioblastoma growth in comparison with control in cell lines producing IL‐1. Our results show the presence of public autocrine loops for IL‐1 leading to growth inhibition in some glioblastomas. To understand these loops, we have studied expression and function of IL‐1ra in glioblastomas. ISH of human glioblastoma tissue sections revealed expression of hIL‐1ra in all 8 cases evaluable. In 4 of 6 cell lines, IL‐1ra was found in the supernatant under constitutive conditions, the IL‐1R‐negative line being among the 2 non‐producers. The other non‐producing cell line, HTB 17, showed expression of hIL‐1R II. Most interestingly, a neutralizing antibody against IL‐1ra down‐regulated growth of IL‐1‐ and IL‐1ra‐producing glioblastoma cells to approx. 30% of the controls. Thus, public autocrine loops for IL‐1 in human glioblastomas exist and result in growth inhibition. An autocrine production of IL‐1‐antagonizing molecules such as IL‐1ra by these tumors can counteract this IL‐1 function and represent a basic escape mechanism supporting malignant growth in some glioblastomas. Int. J. Cancer 71: 1066‐1076, 1997.

Recombinant human stem cell factor does exert minor stimulation of growth in small cell lung cancer and melanoma cell lines

We have previously reported on the stimulation of clonal growth of a glioblastoma cell line by rhSCF (Berdel et al., Cancer Res 1992, 52, 3498-3502). Within an extensive screening programme of haematopoietic growth factor activity on malignant cells, the effects of rhSCF were further tested on the growth of 29 different human cell lines derived from a wide range of solid tumours, among them six lung cancers and five melanomas. RhSCF (0, 1, 10, 100 ng/ml) was tested in a human tumour cloning assay (HTCA) which reliably detects growth modulation of tumour cells by cytokines. Additionally, a tritiated thymidine uptake test was used. Growth of 27 of the 29 cell lines tested was not affected by rhSCF. However, growth of the small cell lung cancer (SCLC) cell line HTB 120 was slightly stimulated (1.5 fold that of controls), and that of the melanoma cell line MeWo was stimulated up to 1.3-fold. This activity was eliminated dose-dependently by the tyrosine kinase inhibitor, genistein. We further analysed the cell lines for expression of the proto-oncogene C-KIT and its ligand SCF. All melanoma and lung cancer cell lines expressed SCF as assessed at the mRNA level. Northern blotting also revealed clear C-KIT mRNA expression in three melanoma (HAS, MeWo, SK-MEL-28), one NSCLC (HTB 53), and four SCLC cell lines (HTB 119, HTB 120, HTB 171, HTB 175). Furthermore, C-KIT protein expression was detected by flow cytometric analysis on the cell surface of MeWo, HTB 119 and HTB 120 cells. Our data indicate that SCF can be operative in growth modulation of non-haematopoietic malignant cells, especially SCLC and melanoma. However, our extensive screening of SCF/tumour cell interaction shows that this interaction is rare and makes potential hazards, such as tumour stimulation upon clinical use of rhSCF in conjunction with chemotherapy in cancer patients, unlikely for the majority of other tumour histologies.

Antiproliferative Effect of Human Interleukin-4 in Human Cancer Cell Lines: Studies on the Mechanism

Interleukin-4 (IL-4) plays an important role in activating the immune system against malignant cells. The human interleukin-4 receptor (hIL-4R) is not only expressed by hematopoietic cells but also on a large number of tissue specimens which include colon, breast and lung carcinomas. In this study we report that rhIL-4 has an antiproliferative effect on 2 out of 3 non-small cell lung carcinoma (NSCLC) cell lines in vitro as measured by human tumor cloning assays (HTCA). In comparison, rhIL-4 had no effect on the growth of small cell lung carcinoma cell lines (SCLC) in vitro. The response towards the cytokine is correlated with expression of at least 1500 high affinity receptors/cell for hIL-4 on the responsive cell lines. Xenotransplanting the human lung tumor cell lines into nude mice followed by 12 days of systemic treatment of the mice with rhIL-4 revealed a significant growth retardation of the IL-4R positive NSCLC cell lines when compared with the controls, whereas the growth of the IL-4R negative SCLC cell lines was unaffected also in vivo. Studies of possible mechanisms involved in the antiproliferative effect of rhIL-4 showed that rhIL-4 does not induce apoptosis or modulation of the transcription factor c myc in the responsive NSCLC cell lines. Additionally, the expression of the epidermal growth factor receptor (EGFR), which is discussed as mediating autocrine/paracrine growth stimulation of NSCLC, is unaffected by rhIL-4. However, we have observed that rhIL-4 inhibited G1-S-phase cell cycle progression. We conclude that rhIL-4 has an antiproliferative effect on the growth of some NSCLC in vitro and in vivo. The mechanisms involved remain to be further elucidated.

Expression of Interleukin-1 and Interleukin-1 Receptors Type 1 and Type 2 in Hodgkin Lymphoma

Signaling through the IL-1-receptor type 1 (IL-1R1), IL-1 is required for initiation and maintenance of diverse activities of the immune system. A second receptor, IL-1R2, blocks IL-1 signal transduction. We studied expression of IL-1beta, IL-1R1, and IL-1R2 in 17 Hodgkin lymphomas (HL) by in situ hybridization (ISH). IL-1beta expressing cells, morphologically consistent with endothelial cells and fibroblasts, occurred in all HL tissues with elevated transcript levels in areas of active fibrosis. Hodgkin and Reed-Sternberg (HRS) cells of all cases expressed low IL-1R1 transcript levels in some tumor cells, and high levels of IL-1R2 in large proportions of HRS cells. Only few bystander cells showed low levels of IL-1R1 and IL-1R2 RNA. Supernatants of 4 out of 7 HL-derived cell lines contained soluble IL-1R2 protein at high levels. HL patient sera carried variably amounts of IL-1R2 protein with significantly increased titers in patients with active disease compared to patients in complete remission and control individuals without HL. Western blots and co-immunoprecipitations showed binding of the IL-1R2 to the intracellular IL-1R-accessory protein (IL-1IRAcP). These data suggest functions of the IL-1R2 as a „decoy-receptor” sequestrating paracrine IL-1 extracellularly and intracellularly by engaging IL-1IRAcP, thus depriving IL1-R1 molecules of their extracellular and intracellular ligands. Expression of IL1-R2 by HRS cells seems to contribute to local and systemic modulation of immune function in HL.

Receptor for platelet-activating factor (PAF) is not detectable by flow cytometry on the surface of myeloid leukemic cells

Flow cytometry was applied to test for platelet-activating-factor receptor (PAF-R) presence on the membranes of acute myeloid leukemia (AML) cells. We have used six human AML cell lines and freshly taken density gradient separated blasts from the bone marrow of ten AML patients covering the majority of French–American–British (FAB) subtypes. Additionally, we have used one histiocytic lymphoma cell line and mature human granulocytes/monocytes as controls. Our results indicate lack of membrane PAF-R on AML of all FAB subtypes tested. This was particularly true for the more mature and differentiated subtypes M4 and M5, including monocytic cell elements, and the promyelocytic M3 AML. In contrast, membrane PAF-R could be easily detected in a histiocytic lymphoma cell line and mature granulocytes/monocytes from peripheral blood used as positive controls. In conclusion, this observation precludes the use of membrane PAF-R as an immunophenotypic marker for AML classification or detection of minimal residual disease.

Megakaryocyte Growth and Development Factor Does Not Stimulate Growth of Lymphomas and Solid Tumors in vitro

The hematopoietic growth factor Mpl ligand was recently cloned [1–3] and characterized as the ligand of a member of the cytokine receptor superfamily. The endogenous Mpl ligand thrombopoietin is the most important regulator for platelet production [4]. Thrombocytopenia following dose-intensive cytotoxic chemotherapy or radiation protocols remains a considerable problem for cancer patients. In animal models among other ligands for Mpl a truncated recombinant Mpl ligand, megakaryocyte growth and development factor (PEG rHu-MGDF), was able to accelerate platelet recovery after myelotoxic therapy without [5, 6] and with hematopoietic stem cell rescue [7]. Since also in humans MGDF stimulates the Megakaryocyte Growth and Development Factor Does Not Stimulate Growth of Lymphomas and Solid Tumors in vitro Fax Communication · Fax-Kommunikation