Emerging evidence to support the use of endothelial progenitor cells (EPCs) for angiogenic therapies or as biomarkers to assess cardiovascular disease risk and progression is compelling. However, there is no uniform definition of an EPC, which makes interpretation of these studies difficult. Although hallmarks of stem and progenitor cells are their ability to proliferate and to give rise to functional progeny, EPCs are primarily defined by the expression of cell-surface antigens. Here, using adult peripheral and umbilical cord blood, we describe an approach that identifies a novel hierarchy of EPCs based on their clonogenic and proliferative potential, analogous to the hematopoietic cell system. In fact, some EPCs form replatable colonies when deposited at the singlecell level. Using this approach, we also identify a previously unrecognized population of EPCs in cord blood that can achieve at least 100 population doublings, replate into at least secondary and tertiary colonies, and retain high levels of telomerase activity. Thus, these studies describe a clonogenic method to define a hierarchy of EPCs based on their proliferative potential, and they identify a unique population of high proliferative potentialendothelial colony-forming cells (HPPECFCs) in human umbilical cord blood.
Several protein kinases from diverse eukaryotes known to perform important roles in DNA repair have also been shown to play critical roles in telomere maintenance. Here, we report that the human telomere-associated protein TRF2 is rapidly phosphorylated in response to DNA damage. We find that the phosphorylated form of TRF2 is not bound to telomeric DNA, as is the ground form of TRF2, and is rapidly localized to damage sites. Our results suggest that the ataxia-telangiectasia-mutated (ATM) protein kinase signal-transduction pathway is primarily responsible for the DNA damage-induced phosphorylation of TRF2. Unlike DNA damageinduced phosphorylation of other ATM targets, the phosphorylated form of TRF2 is transient, being detected rapidly at DNA damage sites postirradiation, but largely dissipated by 2 hours. In addition, we report that the phosphorylated form of TRF2 is present at telomeres in cell types undergoing telomere-based crisis and a recombination-driven, telomerase-independent, alternative lengthening of telomeres (ALT) pathway, likely as a consequence of a telomere-based DNA damage response. Our results link the induction of TRF2 phosphorylation to the DNA damage-response system, providing an example of direct cross-talk via a signaling pathway between these two major cellular processes essential for genomic stability, telomere maintenance, and DNA repair.
Cancer-associated fibroblasts (CAFs) are one of the most prominent cell types in the stromal compartment of the tumor microenvironment. CAFs support multiple aspects of cancer progression, including tumor initiation, invasion, and metastasis. The heterogeneous nature of the stromal microenvironment is attributed to the multiple sources from which the cells in this compartment originate. The present study provides the first evidence that cancer stem cells (CSCs) are one of the key sources of CAFs in the tumor niche. We generated CSC-like cells by treating mouse induced pluripotent stem cells with conditioned medium from breast cancer cell lines. The resulting cell population expressed both CSC and pluripotency markers, and the sphere-forming CSC-like cells formed subcutaneous tumors in nude mice. Intriguingly, these CSC-like cells always formed heterogeneous populations surrounded by myofibroblast-like cells. Based on this observation, we hypothesized that CSCs could be the source of the CAFs that support tumor maintenance and survival. To address this hypothesis, we induced the differentiation of spheres and purified the myofibroblast-like cells. The resulting cells exhibited a CAF-like phenotype, suggesting that they had differentiated into the subpopulations of cells that support CSC self-renewal. These findings provide novel insights into the dynamic interplay between various microenvironmental factors and CAFs in the CSC niche.
Several lines of evidence suggest that defects in telomere maintenance play a significant role in the initiation of genomic instability during carcinogenesis. Although the general concept of defective telomere maintenance initiating genomic instability has been acknowledged, there remains a critical gap in the direct evidence of telomere dysfunction in human solid tumors. To address this topic, we devised a multiplex PCR-based assay, termed TAR (telomereassociated repeat) fusion PCR, to detect and analyze chromosome end-to-end associations (telomere fusions) within human breast tumor tissue. Using TAR fusion PCR, we found that human breast lesions, but not normal breast tissues from healthy volunteers, contained telomere fusions. Telomere fusions were detected at similar frequencies during early ductal carcinoma in situ and in the later invasive ductal carcinoma stage. Our results provide direct evidence that telomere fusions are present in human breast tumor tissue and suggest that telomere dysfunction may be an important component of the genomic instability observed in this cancer. Development of this robust method that allows identification of these genetic aberrations (telomere fusions) is anticipated to be a valuable tool for dissecting mechanisms of telomere dysfunction.breast cancer | retrotransposon D efects in telomere maintenance have been suggested to play significant roles in the initiation of genomic instability via breakage-fusion-bridge cycles and aneuploidy, which are associated with the development of human cancers, including breast cancer (1, 2). A critical function of the telomere is to disguise the chromosome end from being recognized as a double-strand break, to prevent aberrant chromosomal end joining and recombination events. Cells disguise telomeric DNA by encapsulating or "capping" the chromosome ends with several telomere-associated proteins and unique telomere-specific structures (3). In healthy cells, telomere length is highly regulated in a tissue-and cell typespecific manner and is dependent on mitotic turnover rate, telomerase activity, and telomerase-associated factors (4, 5).Several lines of evidence from mouse and human systems suggest that defects in telomere maintenance play an important role in the development of cancer (1, 2). Induction of telomere dysfunction by deficiency in the telomerase RNA component (mTER) in a p53 mutant mouse background results in significant levels of breast adenocarcinomas and colon carcinomas (6-8). Telomere dysfunction also has been reported in a human mammary epithelial cell culture model (9). In this model, late-passage human mammary epithelial cell escape a stress-associated senescencelike barrier and acquire genomic alterations, including telomere fusions (9). In addition, clinical studies have shown that a significant proportion of normal breast luminal cells and ductal carcinoma in situ (DCIS) tissues have shortened telomeric DNA lengths when assayed by telomere FISH (10). Several studies have reported that anaphase bridges, possibly formed...
Protein kinases of the phosphatidylinositol 3-kinase-like kinase family, originally known to act in maintaining genomic integrity via DNA repair pathways, have been shown to also function in telomere maintenance. Here we focus on the functional role of DNA damage-induced phosphorylation of the essential mammalian telomeric DNA binding protein TRF2, which coordinates the assembly of the proteinaceous cap to disguise the chromosome end from being recognized as a double-stand break (DSB). Previous results suggested a link between the transient induction of human TRF2 phosphorylation at threonine 188 (T188) by the ataxia telangiectasia mutated protein kinase (ATM) and the DNA damage response. Here, we report evidence that X-ray-induced phosphorylation of TRF2 at T188 plays a role in the fast pathway of DNA DSB repair. These results connect the highly transient induction of human TRF2 phosphorylation to the DNA damage response machinery. Thus, we find that a protein known to function in telomere maintenance, TRF2, also plays a functional role in DNA DSB repair.Telomeres act as protective caps to disguise the chromosome end from being recognized as a DNA double-strand break (DSB) and play other important roles in maintaining genomic integrity (2,21,26). Telomere capping dysfunction resulting in genomic instability is likely a major pathway leading to human cancers and other age-related diseases (8,27).An increasing number of proteins known to play important roles in DNA repair have also been found to be critical for telomere maintenance (6). Specifically, phosphatidylinositol (PI) 3-kinase-like kinase family members, such as ataxia telangiectasia mutated protein kinase (ATM) and the DNA-dependent protein kinase catalytic subunit in mammals, originally known to act in maintaining genomic stability via DNA repair pathways, have been shown to be important in telomere maintenance (1,4,7,9,10,16,25). Previous reports indicate that ATM is required for the DNA damage-induced phosphorylation of two major telomere-associated proteins in mammals, human TRF1 and TRF2 (16,28). The specific molecular roles played by the DNA damage-induced phosphorylation of TRF1 and TRF2 in telomere maintenance and/or DNA repair are unclear and under active investigation. We previously reported that upon DNA damage, human TRF2 was rapidly and transiently phosphorylated at threonine 188 (T188) (28). Here, we report that X-ray-induced phosphorylation of human TRF2 at T188 plays a functional role in the fast pathway of DNA DSB repair. MATERIALS AND METHODSConstruction of TRF2 mutants. Site-directed mutagenesis was performed using Stratagene's QuikChange kit. The MluI and NheI sites were introduced into primer sequences in order to subclone wild-type human TRF2 (TRF2 WT ) into pTRE2Hyg (Clontech, CA). Nucleotide sequences of the constructs were checked and confirmed by sequencing both strands. The TRF2 cDNA was provided by Titia de Lange (Rockefeller University, New York, NY). Cell culture and transfection. HT1080 Tet-Off, advanced MCF7 Tet-Off, and S...
SummaryDuring aging, chromosome ends, or telomeres, gradually erode or shorten with each somatic cell division. Loss of telomere length homeostasis has been linked to age-related disease. Remarkably, specific environmental assaults, both physical and psychological, have been shown to correlate with shortened telomeres. However, the extent that genetic and/or environmental factors may influence telomere length during later stages of lifespan is not known. Telomere length was measured in 686 male US World War II and Korean War veteran monozygotic (MZ) and dizygotic (DZ) twins (including 181 MZ and 125 DZ complete pairs) with a mean age of 77.5 years (range 73-85 years). During the entire process of telomere length measurement, participant age and twin status were completely blinded. White blood cell mean telomere length shortened in this elderly population by 71 base pairs per year ( P < < < < 0.0001). We observed no evidence of heritable effects in this elderly population on telomere length maintenance, but rather find that telomere length was largely associated with shared environmental factors ( P < < < < 0.0001). Additionally, we found that individuals with hypertension and cardiovascular disease had significantly shorter telomeres ( P = = = = 0.0025 and 0.002, respectively). Our results emphasize that shared environmental factors can have a primary impact on telomere length maintenance in elderly humans.
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