Radiation-induced upregulation of telomerase activity escapes PI3-kinase inhibition in two malignant glioma cell lines.

Tumor relapse after radiotherapy is a great concern in the treatment of high-grade gliomas. Inhibition of the PI3-kinase/AKT pathway is known to radiosensitize cancer cells and to delay their DNA repair after irradiation. In this study, we show that the radiosensitization of CB193 and T98G, two high-grade glioma cell lines, by the PI3K inhibitor LY294002, correlates with the induction of G1 and G2/M arrest, but is inconsistently linked to a delayed DNA double-strand break (DSBs) repair. The PI3K/AKT pathway has been shown to activate radioprotective factors such as telomerase, whose inhibition may contribute to the radiosensitization of cancer cells. However, we show that radiation upregulates telomerase activity in LY-294002-treated glioma cells as well as untreated controls, demonstrating a PI3K/AKT-independent pathway of telomerase activation. Our study suggests that radiosensitizing strategies based on PI3-kinase inhibition in high-grade gliomas may be optimized by additional treatments targeting either telomerase activity or telomere maintenance.

Detection of circulating carcinoma cells by telomerase activity.

Telomerase has been shown to be a marker of epithelial cancer cells. We developed a method that allows the detection of circulating carcinoma cells in the blood of cancer patients. Circulating epithelial cells are harvested from peripheral blood mononuclear cells by immunomagnetic separation using BerEP4-coated beads. A telomeric repeat amplification protocol (TRAP)-ELISA is then used to measure telomerase in harvested epithelial cells. This method is specific and sensitive as demonstrated by experiments using BerEP4-positive and negative cell lines. Whereas we never found telomerase activity in harvested epithelial cells (HEC) samples from 30/30 healthy donors, we have detected telomerase activity in HEC from 11/15 (73%) patients with stage IIIB or IV non-small cell lung cancer (NSCLC) patients and from 8/11 (72%) stage C or D (Dukes classification) colon cancer patients. This non-invasive method could be of great value as a diagnostic or prognostic marker, or for monitoring cancer progression.

Fibroblast growth factor 2 up regulates telomerase activity in neural precursor cells.

During brain development, neuronal and glial cells are generated from neural precursors on a precise schedule involving steps of proliferation, fate commitment and differentiation. We report that telomerase activity is highly expressed during embryonic murine cortical neurogenesis and early steps of gliogenesis and progressively decreases thereafter during cortex maturation to be undetectable in the normal adult brain. We evidenced neural precursor cells (NPC) as the principal telomerase-expressing cells in primary cultures from E15 mouse embryo cortices. Their differentiation either in neurons or in glial cells lead to a down regulation of telomerase activity that was directly correlated to the decrease of telomerase core protein (mTERT) mRNA synthesis. Furthermore, we show that FGF2 (fibroblast growth factor 2), one of the main regulators of CNS development, induces a dose-dependant increase of both the proliferation of NPC and telomerase activity in primary cortical cultures without affecting the mTERT mRNA synthesis compared to that of glyceraldehyde-3-phosphate dehydrogenase (mGAPDH). Finally, we evidenced that AZT (3'-azido-2', 3'-dideoxythymidine), known to inhibit telomerase activity, blocks in a dose dependant manner the FGF2-induced proliferation of NPC. Altogether, our results are in favor of an important role of telomerase activity during brain organogenesis. Oncogene (2000).

Molecular detection of telomerase-positive circulating epithelial cells in metastatic breast cancer patients.

The detection of circulating tumor cells and micrometastases may have important therapeutic and prognostic implications. Telomerase is a hallmark of cancer and is absent from normal epithelial cells. The aim of this study was to use telomerase activity as a molecular marker for the detection of cancer cells in blood of patients with breast cancer. Blood samples were collected from 25 women with stage IV breast cancer and 9 healthy volunteers. Peripheral blood mononuclear cells were isolated by using Ficoll/Hypaque. Immunomagnetic beads coated with an epithelial-specific antibody (BerEP4) were used to harvest epithelial cells from peripheral blood mononuclear cells. Telomerase activity was detected in harvested epithelial cells (HECs) using two different telomerase-PCR-ELISA methods. HECs from blood samples of 21 of 25 (84%) patients with breast cancer were telomerase positive. Telomerase activity was undetectable in HECs from the nine healthy volunteers, demonstrating the specificity of the association between telomerase activity in HECs and stage IV breast cancer. Thus, determination of telomerase activity in HECs appears to be a sensitive, specific, and noninvasive approach for detecting circulating epithelial cancer cells in patients with metastatic breast cancer. This method could be of great value in monitoring the cancer cell proliferation during chemotherapy. This study should be now extended to patients with early-stage breast cancer to investigate the role of telomerase expression by HECs and to evaluate its prognostic value.

Transplantation of stromal cells transduced with the human IL3 gene to stimulate hematopoiesis in human fetal bone grafts in non-obese, diabetic-severe combined immunodeficiency mice.

The non-obese diabetic-severe combined immunodeficiency (NOD-SCID) mouse is a convenient host for human hematopoietic tissues and cells. Human fetal bone fragments engrafted subcutaneously in NOD-SCID mice sustain human hematopoiesis for several months. MS5 murine bone marrow stromal cells were transfected by electroporation with a plasmid containing the human interleukin-3 gene. As expected, stably transfected hu-IL3-MS5 cells supported human hematopoiesis in vitro more efficiently than MS5 cells. hu-IL3-MS5 cells were then injected intravenously into hu-NOD-SCID mice to test their ability to home to the mouse and/or human bone marrow, and to evaluate the role of hu-IL3 secretion on human hematopoiesis in vivo. hu-IL3 was detected in the mouse serum for up to an observation time of 8 weeks. hu-IL3-MS5 cells engrafted the bone marrow, spleen, liver and lungs of the mice but also the human bone graft. The presence of hu-IL3-MS5 cells in the human bone significantly stimulated local human hematopoiesis. This setting could be used to model the bone marrow homing of intravenously injected stromal cells or stromal cell precursors. The same experimental principle could also be applied in a therapeutic perspective to malignant human bone marrow hematopoiesis.