Birgit Reufi

Studies on the role of recombinant human erythropoietin in the growth regulation of human nonhematopoietic tumor cells in vitro

SummaryRecombinant human (rh) erythropoietin (EPO) is attracting increasing interest as an agent for treating cancer-related anemia. Thus, we have tested the effects of rhEPO on the clonal growth of 22 different cell lines derived from a wide range of human solid tumors (head and neck 3, lung 2, breast 2, stomach 1, colorectal 3, hepatocellular 1, pancreas 1, ovary 1, choriocarcinoma 1, osteogenic sarcoma 1, glioblastoma 2, neuroblastoma 1, prostate 1, renal 2) in vitro. RhEPO (dose range 0.01–100 U/ml) caused no significant and reproducible stimulation of clonal growth as measured by a capillary modification of the human tumor cloning assay in agar in any of the cell lines tested. In particular, there was no sensitivity for rhEPO of those cell lines which were shown to be responsive to interleukin-3 and GM-CSF. On the other hand, there were no growth inhibitory effects of rhEPO on the cell lines of this study. Finally, neutralizing anti-human EPO antibody had no effect on the clonal growth of two kidney carcinoma cell lines, making autocrine growth regulation by hEPO in these lines unlikely.

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.

Culturing human umbilical cord blood: a comparison of mononuclear vs CD34+ selected cells.

We compared UCB mononuclear cells (MNC) with CD34+ selected cells in a serum-free static culture system. Cell number proliferation of MNCs was inferior to CD34+ selected cells. MNCs, however, showed a substantial increase from 0.94% CD34+ cells on day 0 to 5.8% on day 7, whereas in the CD34+selected samples the CD34+ cell content declined continously from 62.2% on day 0 to 27.7% on day 7. The number of CFU-GM increased during culture of both cell fractions. Here, only the MNCs showed a substantial increase in clonogenicity on day 7 and day 14 to 11.1- and 4.1-fold input, respectively. This expansion of the CD34+ progenitor cell pool in the MNCs fraction was at least in part attributable to T cells, since the physical abrogation of T cells blocked this effect. Refeeding and reseeding of cells on day 7 had stimulating effects especially on the CD34+ cells, where cell number proliferation increased from 16.3-fold without to 58.1-fold on day 14. Also, we could find sporadic chromosomal aberrations in four of 100 metaphases examined after 7–20 days of ex vivo expansion. The significance of this observation needs to be clarified in a larger series.

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.

The efficiency of tumor cell purging using immunomagnetic CD34 + cell separation systems

Immunomagnetic separation with anti-CD34 monoclonal antibodies and paramagnetic microbeads has been used to enrich hematopoietic stem cells from human bone marrow (BM) or mobilized peripheral blood mononuclear cells (PBMNC). The introduction of this technique also constitutes a new principle of tumor cell purging. The efficiency in terms of purging tumor cells from PBMNC was evaluated in seven different experiments. Mobilized (chemotherapy and G-CSF) PBMNC were collected from patients with solid tumors (n = 6) and multiple myeloma (n = 1) by leukapheresis using an automated MNC separation system and contaminated with 1% (n = 5) or 10% (n = 2) tumor cells from different epithelial cell lines being CD34-negative. The cell mixture was sensitized with anti-CD34 (9C5) antibodies and sheep anti-mouse IgG1 paramagnetic microspheres and enriched for CD34+ cells using an Isolex 50 magnetic separator. Purity of CD34+ cells was studied by flow cytometry (FACScan) and tumor cell depletion was evaluated by comparative human tumor cloning assays (HTCA) containing methylcellulose and agar. We achieved a median purity of CD34+ cells of 85.9% (range 69.8–92.9%) and a median yield of 48.1% (range 21.0–85.2%). From these data in each case the estimated log depletion of tumor cells was calculated and compared with the experimentally achieved (HTCA) log depletion (log Δ depletion = log experimental depletion − log calculated depletion). In our experiments we achieved a median depletion of 2.75 log (range 1.55–3.69 log). When corrected for CD34+ cell yield of each experiment we observed a median ‘yield corrected depletion’ of 2.38 log (range 1.48–3.15 log). The following Δ depletion values were obtained: +0.32 log (HTB 129, breast), +0.21 log (HTB 26, breast), +0.04 log (HTB 26) for experiments with higher experimental depletion, and −0.23 log (HTB 26), −0.9 log (HTB 26, PBMNC from patient with multiple myeloma), −0.82 log (HTB 131, breast) and −1.66 log (HTB 131) for lower depletion efficacy than calculated. These data suggest that depletion may depend on specific cell surface characteristics of tumor cells. Moreover, plasma factors (eg paraprotein) may also have some impact. In summary, the Isolex 50 provides a high purity of CD34+ cells and depletion of tumor cells was efficient. However, calculated and experimental purging efficiencies are not necessarily identical.

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.

Lack of therapeutic effects of platelet activating factor antagonists in WEHI-3B leukemia, human xenotransplanted colorectal and lung cancer and Lewis-lung tumor in vivo

Four new antagonists of platelet activating factor (PAF) from two different chemical classes (imidazoisoquinolines: SDZ 62-434, SDZ 63-135, SDZ 62-759; imidazopiperidines: SDZ 62-293) were tested for in vivo therapeutic activity in various tumor models including the murine myelomonocytic leukemia WEHl-3B, xenografts of human colon (HTB 38) and lung (HTB 119) cancer cell lines and the murine Lewis-lung tumor. After intraperitoneal (i.p.) injection of 1 x 10(3), 5 x 10(3) and 1 x 10(4) WEHl-3B cells into Balb/c mice, the drugs were given per os (p.o.) or i.p. over 6-14 days. Drug doses were pushed to exceed the lethal dose for 10% of the animals (LD10) and ranged from 1 to 100 mg/kg daily for p.o. treatment and from 1 to 75 mg/kg daily for i.p. treatment. In the xenotransplants and the Lewis-lung tumor experiments, PAF antagonists were given i.p. to nude Balb/c and C57 Black mice after intracutaneous (i.c.) tumor cell inoculation. None of the four compounds induced reproducible prolongation of life span, significant numbers of long term survivors, reduction of tumor size, or delay of tumor growth in any of the therapeutic models. Oral SDZ 62-759 had some activity in experiments in which there was slow WEHl-38 tumor growth in the controls. Toxicity of equivalent drugs doses was higher in the i.p. than in the p.o. schedules.

Peripheral Blood Progenitor Cell Mobilization with Dexa-Beam/G-CSF, Ether Lipid Purging, and Autologous Transplantation After High-Dose CBV Treatment: A Safe and Effective Regimen in Patients with Poor Risk Malignant Lymphomas

High-dose chemotherapy followed by autologous peripheral blood progenitor cell transplantation (PBPCT) is increasingly applied in patients with relapsed, poor risk malignant lymphomas. Different strategies for progenitor cell mobilization using cytoreductive chemotherapy, hematopoietic growth factors, or both have been described. We studied the safety and efficacy of a modified DexaBEAM regimen (dexamethasone, BCNU [carmustine], etoposide, ara-C, melphalan) followed by granulocyte-colony stimulating factor (G-CSF) that was administered in order to minimize any residual disease and to obtain a sufficient amount of progenitor cells in the autografts. Until now, 16 patients at poor risk (8 with Hodgkins disease, 8 with non-Hodgkins lymphoma) entered the study. All the 12 patients with measurable disease at study entry responded to DexaBEAM. Median time of subsequent leukopenia (leukocytes < 1.000/microL) was 6 days (range 5-8 days). Peak numbers of CD34+ hematopoietic progenitor cells appeared in the peripheral blood after a median of 20 days (range 18-22 days) after onset of therapy. At that time, peripheral mononuclear cells were collected for autografting. Thereafter, the leukapheresis products were frozen until the day of transplantation, either unpurged in the case of Hodgkins disease or purged with the ether lipid edelfosine in cases of non-Hodgkins lymphoma. After high-dose chemotherapy with the CBV regimen (cyclophosphamide, BCNU, etoposide) the patients received their autografts, followed again by G-CSF treatment. A stable hematopoietic recovery was reached with granulocytes > 2.000/muL within 11 days (range 8-17 days), and platelets > 50.000/microL within 15 days (range 10-31 days), respectively, without significant differences between the purged and unpurged transplants. After a median follow-up of 28 months (range 1-40 months) 7 patients are alive without signs of recurrent disease, while 1 patient has died due to acute treatment related toxicity. Three patients had refractory disease, and 5 have relapsed of whom 4 have died. In summary, the DexaBEAM/G-CSF/CBV strategy appears to be safe and effective for salvage treatment in patients with poor risk malignant lymphomas.

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