The reprogramming of differentiated cells to pluripotent cells (induced pluripotent stem (iPS) cells) is known to be an inefficient process. We recently reported that cells with short telomeres cannot be reprogrammed to iPS cells despite their normal proliferation rates, probably reflecting the existence of 'reprogramming barriers' that abort the reprogramming of cells with uncapped telomeres. Here we show that p53 (also known as Trp53 in mice and TP53 in humans) is critically involved in preventing the reprogramming of cells carrying various types of DNA damage, including short telomeres, DNA repair deficiencies, or exogenously inflicted DNA damage. Reprogramming in the presence of pre-existing, but tolerated, DNA damage is aborted by the activation of a DNA damage response and p53-dependent apoptosis. Abrogation of p53 allows efficient reprogramming in the face of DNA damage and the generation of iPS cells carrying persistent DNA damage and chromosomal aberrations. These observations indicate that during reprogramming cells increase their intolerance to different types of DNA damage and that p53 is critical in preventing the generation of human and mouse pluripotent cells from suboptimal parental cells.

The mechanisms involved in the reprogramming of differentiated cells into induced Pluripotent Stem (iPS) cells by Oct4, Klf4 and Sox2 (3F) remain poorly understood 1 . The Ink4/Arf tumour suppressor locus encodes three potent inhibitors of proliferation, namely p16 Ink4a , p15 Ink4b and Arf, which are basally expressed in differentiated cells and upregulated by aberrant mitogenic signals 2-4 . We show here that the locus is completely silenced in iPS cells, as well as in embryonic stem (ES) cells, acquiring the epigenetic marks of a bivalent chromatin domain, and retaining the ability to be reactivated upon differentiation. Cell culture conditions during reprogramming enhance the expression of the Ink4/Arf locus, further highlighting the importance of silencing the locus to allow proliferation and reprogramming. Indeed, the 3F together repress the Ink4/Arf locus soon after their expression and concomitant with the appearance of the first molecular markers of stemness. This downregulation also occurs in cells carrying the oncoprotein large-T, which functionally inactivates the pathways regulated by the Ink4/Arf locus, thus implying that the silencing of the locus is intrinsic to reprogramming and not the result of a selective process. Genetic inhibition of the Ink4/Arf locus has a profound positive impact on the efficiency of iPS generation, increasing both the kinetics of reprogramming and the number of emerging iPS colonies. In murine cells, Arf, rather than Ink4a, is the main barrier to reprogramming through activation of p53 and p21; whereas, in human fibroblasts, INK4a is more important than ARF. Finally, organismal aging upregulates the Ink4/Arf locus 2,5 and, accordingly, reprogramming is less efficient in cells from old organisms, but this defect can be rescued by inhibiting the locus with an shRNA. All together, we conclude that the silencing of Ink4/Arf locus is rate limiting for reprogramming, and its transient inhibition may significantly improve the generation of iPS.The Ink4/Arf tumour suppressor locus encodes three important tumour suppressors that activate two critical anti-proliferative pathways, namely, the Rb and p53 pathways, whose activation prevents the propagation of aberrant cells, either by apoptosis or senescence (see scheme in Supplementary Fig. S1) 4 . Briefly, the paralogs p16 Ink4a and p15 Ink4b bind and inhibit the cyclin D-dependent kinases Cdk4 and Cdk6, which in turn are important to relieve the cell-cycle inhibitory activity of the Rb tumour suppressor. On the other hand, Arf

Telomere shortening is associated with organismal aging. iPS cells have been recently derived from old patients; however, it is not known whether telomere chromatin acquires the same characteristics as in ES cells. We show here that telomeres are elongated in iPS cells compared to the parental differentiated cells both when using four (Oct3/4, Sox2, Klf4, cMyc) or three (Oct3/4, Sox2, Klf4) reprogramming factors and both from young and aged individuals. We demonstrate genetically that, during reprogramming, telomere elongation is usually mediated by telomerase and that iPS telomeres acquire the epigenetic marks of ES cells, including a low density of trimethylated histones H3K9 and H4K20 and increased abundance of telomere transcripts. Finally, reprogramming efficiency of cells derived from increasing generations of telomerase-deficient mice shows a dramatic decrease in iPS cell efficiency, a defect that is restored by telomerase reintroduction. Together, these results highlight the importance of telomere biology for iPS cell generation and functionality.

Head and neck squamous cell carcinoma (HNSCC) is a leading cause of cancer mortality worldwide. Recent reports have associated a subset of HNSCC with high-risk human papillomaviruses (HPVs), particularly HPV16, the same subset of HPVs responsible for the majority of cervical and anogenital cancers. In this study we describe a mouse model for HPV-associated HNSCC that employs mice transgenic for the HPV16 oncogenes E6 and E7. In these mice, E6 and E7 induce aberrant epithelial proliferation and, in the presence of a chemical carcinogen, they increase dramatically the animal's susceptibility to HNSCC. The cancers arising in the HPV16-transgenic mice mirror the molecular and histopathological characteristics of human HPV-positive HNSCC that distinguish the latter from human HPV-negative HNSCC, including overexpression of p16 protein and formation of more basaloid cancers. This validated model of HPV-associated HNSCC provides the means to define the contributions of individual HPV oncogenes to HNSCC and to understand the molecular basis for the differing clinical properties of HPV-positive and HPV-negative human HNSCC. From this study, we identify minichromosome maintenance protein 7 (MCM7) and p16 as potentially useful biomarkers for HPV-positive head and neck cancer.head and neck squamous cell carcinoma (HNSCC) ͉ human papillomavirus 16 (HPV16) ͉ minichromosome maintenance protein 7 (MCM7) ͉ p16 ͉ transgenic H ead and neck cancers are the sixth most common malignancy in the world (1), and recent advances in treating them are not reflected in improved survival. Head and neck squamous cell carcinoma (HNSCC) is etiologically associated with tobacco use; alcohol consumption; and, in Southeast Asia, chewing of betel nut (2-6). However, more recent studies have implicated high-risk human papillomaviruses (HR-HPVs), the same viruses recognized as the causative agents of cervical and most other anogenital malignancies, in the etiology of a subset of HNSCC (7).HR-HPV DNA has been detected in Ϸ20% of HNSCC, particularly in malignancies of the oropharynx, where Ϸ50% of the cancers have been found to harbor viral DNA. HPV16, the type associated with the majority of cervical carcinomas, is the HR-HPV linked with the overwhelming majority of HPVpositive HNSCC (95%) (8-10). Other HR-HPV genotypes have been detected, including HPV18, HPV31, and HPV33. Viral oncogenes E6 and E7, the papillomaviral genes considered largely responsible for the onset as well as persistence of cervical cancer, have been detected in both integrated and extrachromosomal HPV genomes in HNSCC (11). E6 and E7 are best known for their ability to bind and inactivate the tumor suppressors p53 and pRb (12)(13)(14)(15)(16)(17), and these respective properties have been associated with their oncogenic properties (18,19). Consistent with E6 and E7 contributing to HPV-positive HNSCC is the absence of genetic or epigenetic alterations in the p53 and pRb pathways in HPV-positive HNSCC, in stark contrast to what is observed in HPV-negative HNSCC (11). Furthermore, di...

Infection with high-risk human papillomaviruses (HPV) and in particular the expression of the viral proteins E6 and E7 have been associated with the etiology of a subset of head and neck squamous cell cancer (HNSCC). However, the individual consequences of E6 and E7 expression in an in vivo model have not been examined in these tissues. We have used transgenes that direct expression of the HPV16 E6 and E7 proteins to the head and neck tissues of mice to dissect the contribution of these proteins to head and neck carcinogenesis. We report here that E7 is the major transforming oncogene in HPV-associated HNSCC, whereas E6 is more likely to play a secondary role in contributing to later stages of carcinogenesis. Furthermore, a conditional deletion of Rb, a prominent target for E7, in the same tissues did not recapitulate all E7-mediated phenotypes. Although our results do not preclude an important role for the E7-pRb interaction, they highlight the importance of pRb-independent functions of E7 in head and neck carcinogenesis. [Cancer Res 2007;67(24):11585-93]