Aghdass Rasouli-Nia

Senescence evasion by MCF-7 human breast tumor-initiating cells

IntroductionA subpopulation of cancer cells, tumor-initiating cells, is believed to be the driving force behind tumorigenesis and resistance to radiation and chemotherapy. The persistence of tumor-initiating cells may depend on altered regulation of DNA damage and checkpoint proteins, as well as a reduced propensity to undergo apoptosis or senescence.MethodsTo test this hypothesis, we isolated CD24-/low/CD44+ tumor-initiating cells (as mammospheres) from MCF-7 breast cancer cells grown in adherent monolayer culture, and carried out a comprehensive comparison of cell death and DNA damage response pathways prior to and after exposure to ionizing radiation in mammospheres and monolayer MCF-7 cells. Single and double-strand break repair was measured by single-cell gel electrophoresis. The latter was also examined by phosphorylation of histone H2AX and formation of 53BP1 and Rad51 foci. Apoptosis was quantified by flow-cytometric analysis of annexin V-binding and senescence was analyzed on the basis of cellular β-galactosidase activity. We employed the telomeric repeat amplification protocol to quantify telomerase activity. Expression of key DNA repair and cell cycle regulatory proteins was detected and quantified by western blot analysis.ResultsOur data demonstrate that in comparison to the bulk population of MCF-7 cells (predominantly CD24+/CD44+), the MCF-7 mammosphere cells benefit from a multifaceted approach to cellular protection relative to that seen in monolayer cells, including a reduced level of reactive oxygen species, a more active DNA single-strand break repair (SSBR) pathway, possibly due to a higher level of expression of the key SSBR protein, human AP endonuclease 1 (Ape1), and a significantly reduced propensity to undergo senescence as a result of increased telomerase activity and a low level of p21 protein expression. No significant difference was seen in the rates of double-strand break repair (DSBR) between the two cell types, but DSBR in mammospheres appears to by-pass the need for H2AX phosphorylation.ConclusionsEnhanced survival of MCF-7 tumor-initiating cells in response to ionizing radiation is primarily dependent on an inherent down-regulation of the senescence pathway. Since MCF-7 cells are representative of cancer cells that do not readily undergo apoptosis, consideration of senescence pathways may play a role in targeting stem cells from such tumors.

PATHWAYS OF PROLIFERATION AND ANTIAPOPTOSIS DRIVEN IN BREAST CANCER STEM CELLS BY STEM CELL PROTEIN PIWIL2

Cancer stem cell studies may improve understanding of tumor pathophysiology and identify more effective strategies for cancer treatment. In a variety of organisms, Piwil2 has been implicated in multiple roles including stem cell self-renewal, RNA silencing, and translational control. In this study, we documented specific expression of the stem cell protein Piwil2 in breast cancer with predominant expression in breast cancer stem cells. In patients who were evaluated, we determined that 90% of invasive carcinomas and 81% of carcinomas in situ exhibited highest expression of Piwil2. In breast cancer cells, Piwil2 silencing suppressed the expression of signal transducer and activator of transcription 3, a pivotal regulator of Bcl-X(L) and cyclin D1, whose downregulation paralleled a reduction in cell proliferation and survival. Our findings define Piwil2 and its effector signaling pathways as key factors in the proliferation and survival of breast cancer stem cells.

Integration, Networking, and Global Biobanking in the Age of New Biology.

Scientific revolution is changing the world forever. Many new disciplines and fields have emerged with unlimited possibilities and opportunities. Biobanking is one of many that is benefiting from revolutionary milestones in human genome, post-genomic, and computer and bioinformatics discoveries. The storage, management, and analysis of massive clinical and biological data sets cannot be achieved without a global collaboration and networking. At the same time, biobanking is facing many significant challenges that need to be addressed and solved including dealing with an ever increasing complexity of sample storage and retrieval, data management and integration, and establishing common platforms in a global context. The overall picture of the biobanking of the future, however, is promising. Many population-based biobanks have been formed, and more are under development. It is certain that amazing discoveries will emerge from this large-scale method of preserving and accessing human samples. Signs of a healthy collaboration between industry, academy, and government are encouraging.

Key Issues Related to Cryopreservation and Storage of Stem Cells and Cancer Stem Cells: Protecting Biological Integrity.

Cryopreservation and biobanking of stem cells are becoming increasingly important as stem cell technology and application attract the interest of industry, academic research, healthcare and patient organisations. Stem cell are already being used in the treatment of some diseases and it is anticipated that stem cell therapy will play a central role in future medicine. Similarly, the discovery of both hematopoietic and solid tumor stem cells and their clinical relevance have profoundly altered paradigms for cancer research as the cancer stem cells are considered promising new targets against cancer. Consequently, long-term cryopreservation and banking of normal and malignant stem cells is crucial and will inevitably become a routine procedure that requires highly regulated and safe methods of specimen storage. There is, however, an increasing amount of evidence showing contradictory results on the impact of cryopreservation and thawing of stem cells, including extensive physical and biological stresses, apoptosis and necrosis, mitochondrial injuries, changes to basal respiration and ATP production, cellular structural damage, telomere shortening and cellular senescence, and DNA damage and oxidative stress. Notably, cell surface proteins that play a major role in stem cell fate and are used as the biomarkers of stem cells are more vulnerable to cold stress than other proteins. There are also data supporting the alteration in some biological features and genetic integrity at the molecular level of the post-thawed stem cells. This article reviews the current and future challenges of cryopreservation of stem cells and stresses the need for further rigorous research on the methodologies for freezing and utilizing cancer stem cells following long-term storage.