Induction of cells with cancer stem cell properties from nontumorigenic human mammary epithelial cells by defined reprogramming factors.

Cancer stem cells (CSCs), a small and elusive population of undifferentiated cancer cells within tumors that drive tumor growth and recurrence, are believed to resemble normal stem cells. Although surrogate markers have been identified and compelling CSC theoretical models abound, actual proof for the existence of CSCs can only be had retrospectively. Hence, great store has come to be placed in isolating CSCs from cancers for in-depth analysis. On the other hand, although induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine, concern exists over the inadvertent co-transplantation of partially or undifferentiated stem cells with tumorigenic capacity. Here we demonstrate that the introduction of defined reprogramming factors (OCT4, SOX2, Klf4 and c-Myc) into MCF-10A nontumorigenic mammary epithelial cells, followed by partial differentiation, transforms the bulk of cells into tumorigenic CD44(+)/CD24(low) cells with CSC properties, termed here as induced CSC-like-10A or iCSCL-10A cells. These reprogrammed cells display a malignant phenotype in culture and form tumors of multiple lineages when injected into immunocompromised mice. Compared with other transformed cell lines, cultured iCSCL-10A cells exhibit increased resistance to the chemotherapeutic compounds, Taxol and Actinomycin D, but higher susceptibility to the CSC-selective agent Salinomycin and the Pin1 inhibitor Juglone. Restored expression of the cyclin-dependent kinase inhibitor p16INK4a abrogated the CSC properties of iCSCL-10A cells, by inducing cellular senescence. This study provides some insight into the potential oncogenicity that may arise via cellular reprogramming, and could represent a valuable in vitro model for studying the phenotypic traits of CSCs per se.

Coexistence of alternative lengthening of telomeres and telomerase in hTERT-transfected GM847 cells.

It has been shown previously that some immortalized human cells maintain their telomeres in the absence of significant levels of telomerase activity by a mechanism referred to as alternative lengthening of telomeres (ALT). Cells utilizing ALT have telomeres of very heterogeneous length, ranging from very short to very long. Here we report the effect of telomerase expression in the ALT cell line GM847. Expression of exogenous hTERT in GM847 (GM847/hTERT) cells resulted in lengthening of the shortest telomeres; this is the first evidence that expression of hTERT in ALT cells can induce telomerase that is active at the telomere. However, rapid fluctuation in telomere length still occurred in the GM847/hTERT cells after more than 100 population doublings. Very long telomeres and ALT-associated promyelocytic leukemia (PML) bodies continued to be generated, indicating that telomerase activity induced by exogenous hTERT did not abolish the ALT mechanism. In contrast, when the GM847 cell line was fused with two different telomerase-positive tumor cell lines, the ALT phenotype was repressed in each case. These hybrid cells were telomerase positive, and the telomeres decreased in length, very rapidly at first and then at the rate seen in telomerase-negative normal cells. Additionally, ALT-associated PML bodies disappeared. After the telomeres had shortened sufficiently, they were maintained at a stable length by telomerase. Together these data indicate that the telomerase-positive cells contain a factor that represses the ALT mechanism but that this factor is unlikely to be telomerase. Further, the transfection data indicate that ALT and telomerase can coexist in the same cells.

Alternative lengthening of telomeres in human cells.

Activation of a telomere maintenance mechanism appears to be essential for immortalization. In most human tumors and tumor cell lines, the telomere maintenance mechanism involves the activity of telomerase, a reverse transcriptase holoenzyme that synthesizes telomeric repeat DNA. In some cases, telomere maintenance occurs in the absence of telomerase activity by a mechanism referred to as alternative lengthening of telomeres (ALT). The development of telomere-targeted anticancer therapies will be facilitated by an understanding of the molecular mechanisms of ALT and of the means whereby ALT is repressed in normal cells.

Identification of a novel human mitochondrial D-loop RNA species which exhibits upregulated expression following cellular immortalization.

We report the identification and characterization of a novel human mitochondrial RNA species approximately 0.47 kb long that is transcribed from the mtDNA L-strand and is derived from the D-loop. Its expression increases when human cells become immortal, a key event in tumorigenesis. The RNA is therefore designated IDL (Immortalization-associated D-Loop). Sequence and hybrid cell analyses suggest that the increased level of IDL RNA in immortal cells is due to a recessive change, possibly in the activity of a trans-acting factor that controls IDL RNA expression.

Repression of an alternative mechanism for lengthening of telomeres in somatic cell hybrids.

Some immortalized cell lines maintain their telomeres in the absence of detectable telomerase activity by an alternative (ALT) mechanism. To study how telomere maintenance is controlled in ALT cells, we have fused an ALT cell line GM847 (SV40 immortalized human skin fibroblasts) with normal fibroblasts or with telomerase positive immortal human cell lines and have examined their proliferative potential and telomere dynamics. The telomeres in ALT cells are characteristically very heterogeneous in length, ranging from very short to very long. The ALT x normal hybrids underwent a rapid reduction in telomeric DNA and entered a senescence-like state. Immortal segregants rapidly reverted to the ALT telomere phenotype. Fusion of ALT cells to telomerase-positive immortal cells in the same immortalization complementation group resulted in hybrids that appeared immortal and also exhibited repression of the ALT telomere phenotype. In these hybrids, which were all telomerase-positive, we observed an initial rapid loss of most long telomeres, followed either by gradual loss of the remaining long telomeres at a rate similar to the rate of telomere shortening in normal telomerase-negative cells, or by maintenance of shortened telomeres. These data indicate the existence of a mechanism of rapid telomere deletion in human cells. They also demonstrate that normal cells and at least some telomerase-positive immortal cells contain repressors of the ALT telomere phenotype.

p53 represses SV40 transcription by preventing formation of transcription complexes.

There is now much evidence to suggest that the p53 tumour suppressor protein functions to monitor the integrity of the genome. When DNA damage is detected, p53 suppresses cell growth to allow repair or directs the cell into apoptosis. The mechanism of action of p53 is as yet unclear but recent evidence has accumulated to suggest that p53 might act by regulating gene expression. Consistent with this model, p53 can both activate and repress a number of viral and cellular promoters. p53 has also been shown to bind to the CCAAT-binding Factor and TATA-binding protein (TBP), and there is direct evidence that p53 represses in vitro transcription by preventing TBP from binding DNA. We now provide evidence that p53 can repress transcription from the SV40 promoter by disrupting DNA/protein complexes involving transcription factor Sp1.

p53-dependent activation of the mouse MCK gene promoter: identification of a novel p53-responsive sequence and evidence for cooperation between distinct p53 binding sites.

Transcriptional activation by p53 is dependent on the presence of a specific p53 binding site within control sequences of the target gene. One such target gene is the mouse muscle-specific creatine kinase (MCK) gene, which contains a p53 binding site between promoter residues -3182 and -3133 relative to the transcription start site. This DNA sequence is reported to be sufficient to confer p53-dependent activation on the MCK promoter. In contrast to this finding, evidence from promoter deletion studies suggests that sequences in the MCK promoter other than this p53 binding site also permit p53-dependent activation. To investigate this possibility, we have further examined sequences in the MCK promoter required for transcriptional activation by mouse p53. We report here identification of a second p53-responsive sequence within the MCK promoter. This novel sequence is situated between residues -177 and -81, and can confer p53-dependent, position- and orientation-independent activation on a heterologous promoter. Moreover, this sequence can specifically bind mouse and human p53. By promoter deletion studies, we provide evidence that these two elements cooperate to provide high-level, p53-dependent activation of the MCK promoter.