Thrombopoietin Secretion by Human Ovarian Cancer Cells

Abstract

The thrombopoietin (TPO) gene expression in human ovary and cancer cells from patients with ovarian carcinomatosis, as well as several cancer cell lines including MDA-MB231 (breast cancer), K562 and HL60 (Leukemic cells), OVCAR-3NIH and SKOV-3 (ovarian cancer), was performed using RT PCR, real-time PCR, and gene sequencing. Human liver tissues are used as controls. The presence of TPO in the cells and its regulation by activated protein C were explored by flow cytometry. TPO content of cell extract as well as plasma of a patient with ovarian cancer was evaluated by ELISA. The functionality of TPO was performed in coculture on the basis of the viability of a TPO-dependent cell line (Ba/F3), MTT assay, and Annexin-V labeling. As in liver, ovarian tissues and all cancer cells lines except the MDA-MB231 express the three TPO-1 (full length TPO), TPO-2 (12 bp deletion), and TPO-3 (116 pb deletion) variants. Primary ovarian cancer cells as well as cancer cell lines produce TPO. The thrombopoietin production by OVCAR-3 increased when cells are stimulated by aPC. OVCAR-3 cell's supernatant can replace exogenous TPO and inhibited TPO-dependent cell line (Ba/F3) apoptosis. The thrombopoietin produced by tumor may have a direct effect on thrombocytosis/thrombosis occurrence in patients with ovarian cancer.

Figure 1

TPO primers' selection. (a, b) Schematic illustration of human TPO gene and mRNA isoforms and selected TPO primers. (a) TPO gene contains 6 exons (E 1–6) and 5 introns (I 1–5). (b) Alternative RNA splicing patterns previously identified for TPO. Horizontal arrows represent the amplified regions by RT-PCR. (c) Primers used for PCR and nested-PCR. First PCR products were used as DNA template for the nested-PCR. TPO-amplified isoforms and their sizes are shown. GADPH PCR was used as control. (d) TaqMan Probes for TPO and GADPH.

Figure 2

TPO gene expression in cultured cells from ascitic fluids of cancer patients. (a) Subject data. (b) Photographs taken of ascitic fluid cells in culture. (c) Analysis of TPO and GADPH gene expression. 2% agarose gel. PCR using F1/R1 primers for TPO gene amplification.

Figure 3

TPO gene expression by cell lines. (a) First PCR analysis of TPO and GADPH gene expressed by various cell lines, ovarian (OVCAR-3 and SKOV-3), breast (MDA-MB231 and MCF7), gastric (AGS, KATO-III), intestinal (LS174T), lung (A549), leukemia (K562), cervical (HELA), and human microvascular endothelial (HMEC-1) cell lines. 2% agarose gel. Normal adult ovary (1 and 2) and liver (1 and 2) tissues served as control. (b) 2% agarose gel pattern of nested-PCR product of TPO: TPO-1 (full length), TPO-2 (12 bp deletion), and TPO-3 (116 bp deletion). Boxes (1, 2) represent bands chosen for sequencing.

Figure 4

Comparison of TPO-3 sequence as well as TPO levels in the soluble cancer cells extracts. (a) Sequences of PCR products extracted from agarose gel bands are shown in the upper line. R2 primer was used for sequencing. Boxes show a C/T 5183 SNP. “…” and “∗” symbols refer to two different alternative splicing sites (12 bp and 116 bp deletions, resp.). A 116 bp deletion characterizing TPO-3 is detected. BLAST reveals no significant mutation. (b) TPO levels in the soluble cancer cells extracts evaluated by enzyme-linked immunosorbent assay (ELISA) for 10⁶ cell/ml (the mean of three experiments) from OVCAR-3 NIH cell line, breast cancer MDA-MB231 cell line (as control), myeloid leukemia K562, promyelocytic leukemia HL60 cell lines, and primary ovarian carcinomatosis (n = 22).

Figure 5

TPO gene expression in the presence or absence of protein C. (a) Quantification of TPO gene expression using TaqMan Probes in different cancer cell lines such as MDA-MB231 (as control), OVCAR-3, SKOV-3, and K562. Nonsignificant results were observed in the presence of protein C (P < 0,05). High TPO gene expression was observed in leukemic K562 cell. Kruskal-Wallis test (^∗∗P < 0,02, ^∗∗∗P < 0,01). Flow cytometry distribution plots are shown. Graphs represent Geometric Fluorescence Mean. (b, c): graphs for MDA-MB231 (b) and OVCAR-3 (c) cells incubated with the secondary antibody alone (orange), labeled with primary and secondary antibodies after incubation without PC or aPC (blue), and with PC (purple) or with aPC (green). (d) TPO-released protein by OVCAR-3. OVCAR-3 were incubated (for 5 hours) without (blue) or with (green) protein transport inhibitor or with only 2nd antibody as control (orange). 2nd antibody GMFI/GMFI sample ratio was calculated for each condition.

Figure 6

Functionality of TPO: viability study of TPO-dependent Ba/F3 cells. (a) The panel shows the distribution of two populations of Ba/F3 cells cultured in the presence of TPO: a nonlabeled population FITC-Annexin-V (viability) and a labeled population FITC-Annexin-V (apoptosis). (b) The graph shows Ba/F3 viability (not stained by FITC-Annexin-V). (c, d) Relative TPO secreted quantity (ng). (P < 0,02). Student's t-test (^∗P < 0,05).