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CD40 activation in epithelial ovarian carcinoma cells modulates growth, apoptosis, and cytokine secretion

¹ CRC Institute for Cancer Studies, University of Birmingham Medical School, Birmingham B15 2TA, UK
² Department of Histopathology, Heartlands Hospital, Bordesley Green East, Birmingham B9 5SS, UK

Correspondence to:
Dr L S Young, Cancer Research UK, Institute for Cancer Studies, University of Birmingham Medical School, Birmingham B15 2TA, UK;
L.S.Young{at}bham.ac.uk

Background/Aims: CD40, a member of the tumour necrosis factor (TNF) receptor family, is expressed on a variety of haematopoietic cells and is crucial in orchestrating both humoral and cellular immune responses. CD40 is also expressed on some carcinoma cells, where its function remains largely unknown. This study investigated the effects of CD40 ligation on ovarian carcinoma cell growth and apoptosis and on cytokine production, in addition to the role of the NF-B and JNK signalling pathways.

Methods: CD40 expression was measured in epithelial ovarian carcinoma (EOC) biopsies by immunohistochemistry and in EOC cell lines by flow cytometry. To examine the effects of CD40 ligation on cell growth recombinant soluble CD40 ligand was used to stimulate EOC cell lines and growth was measured by MMT assays. Cytokine production was measured by enzyme linked immunosorbent assays interleukin 8 (IL-8) gene transcription was estimated by means of reverse transcription polymerase chain reaction. The integrity of the CD40 signalling pathway in those cell lines that did not produce cytokines in response to CD40 ligation was assessed by the detection of the transcription factor NF-B by an electrophoretic mobility shift assay. To investigate the defect in the NF-B pathway the phosphorylation status of IB was determined by an antibody specific to phosphorylated IB and dissociation of the IB–p65 complex was assessed by co-immunoprecipitation.

Results: CD40 is expressed in primary ovarian carcinoma biopsies and EOC cell lines. CD40 ligation resulted in growth inhibition in most of these carcinoma cell lines and was also found to promote apoptosis, with this last effect only being evident in early passage EOC cells. CD40 ligation also induced significant IL-6 and IL-8 production in most of the EOC cell lines examined and it was confirmed for IL-8 that this effect was regulated at the transcriptional level. NF-B activation in response to CD40 ligation was found in three of the EOC cell lines and specific defects in the CD40 induced NF-B pathway were identified in two cell lines. However, CD40 engagement induced JNK activation in all the EOC cell lines.

Conclusions: These data suggest that the CD40 pathway is functional in ovarian carcinoma cells and highlight the need for further studies to provide insight into the role of CD40 in the carcinogenic process and the possible exploitation of this pathway for novel therapeutic approaches.

Keywords: CD40; cytokines; ovarian carcinoma

Abbreviations: CD40L, CD40 ligand; CHX, cycloheximide; ECL, enhanced chemiluminescence; ELISA, enzyme linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; EOC, epithelial ovarian carcinoma; FCS, fetal calf serum; HIGM, X-linked hyper-IgM syndrome; HIV-LTR, human immunodeficiency virus long terminal repeat; HRP, horseradish peroxidase; IKK, inducible IB kinase; IL, interleukin; JNK, c-jun N-terminal kinase; NIK, NF-B inducing kinase; PBS, phosphate buffered saline; rsCD40, recombinant soluble CD40 ligand; RT-PCR, reverse transcription polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TBS, Tris buffered saline; TNF, tumour necrosis factor; TRAF, TNF receptor associated factor

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