Impact of the hypoxic microenvironment on spermatogonial stem cells in culture.

The stem cell niche plays a crucial role in the decision to either self-renew or differentiate. Recent observations lead to the hypothesis that O2 supply by blood and local O2 tension could be key components of the testicular niche of spermatogonial stem cells (SSCs). In this study, we investigated the impact of different hypoxic conditions (3.5%, 1%, and 0.1% O2 tension) on murine and human SSCs in culture. We observed a deleterious effect of severe hypoxia (1% O2 and 0.1% O2) on the capacity of murine SSCs to form germ cell clusters when plated at low density. Severe effects on SSCs proliferation occur at an O2 tension ≤1% and hypoxia was shown to induce a slight differentiation bias under 1% and 0.1% O2 conditions. Exposure to hypoxia did not appear to change the mitochondrial mass and the potential of membrane of mitochondria in SSCs, but induced the generation of mitochondrial ROS at 3.5% and 1% O2. In 3.5% O2 conditions, the capacity of SSCs to form colonies was maintained at the level of 21% O2 at low cell density, but it was impossible to amplify and maintain stem cell number in high cell density culture. In addition, we observed that 3.5% hypoxia did not improve the maintenance and propagation of human SSCs. Finally, our data tend to show that the transcription factors HIF-1α and HIF-2α are not involved in the SSCs cell autonomous response to hypoxia.

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.