TOP3A amplification and ATRX inactivation are mutually exclusive events in pediatric osteosarcomas using ALT.

In some types of cancer, telomere length is maintained by the alternative lengthening of telomeres (ALT) mechanism. In many ALT cancers, the α-thalassemia/mental retardation syndrome X-linked (ATRX) gene is mutated leading to the conclusion that the ATRX complex represses ALT. Here, we report that most high-grade pediatric osteosarcomas maintain their telomeres by ALT, and that the majority of these ALT tumors are ATRX wild-type (wt) and instead carry an amplified 17p11.2 chromosomal region containing TOP3A. We found that TOP3A was overexpressed in the ALT-positive ATRX-wt tumors consistent with its amplification. We demonstrated the functional significance of these results by showing that TOP3A overexpression in ALT cancer cells countered ATRX-mediated ALT inhibition and that TOP3A knockdown disrupted the ALT phenotype in ATRX-wt cells. Moreover, we report that TOP3A is required for proper BLM localization and promotes ALT DNA synthesis in ALT cell lines. Collectively, our results identify TOP3A as a major ALT player and potential therapeutic target.

Synthetic lethality of cytolytic HSV-1 in cancer cells with ATRX and PML deficiency.

Cancers that utilize the alternative lengthening of telomeres (ALT) mechanism for telomere maintenance are often difficult to treat and have a poor prognosis. They are also commonly deficient for expression of ATRX protein, a repressor of ALT activity, and a component of promyelocytic leukemia nuclear bodies (PML NBs) that are required for intrinsic immunity to various viruses. Here, we asked whether ATRX deficiency creates a vulnerability in ALT cancer cells that could be exploited for therapeutic purposes. We showed in a range of cell types that a mutant herpes simplex virus type 1 (HSV-1) lacking ICP0, a protein that degrades PML NB components including ATRX, was ten- to one thousand-fold more effective in infecting ATRX-deficient cells than wild-type ATRX-expressing cells. Infection of co-cultured primary and ATRX-deficient cancer cells revealed that mutant HSV-1 selectively killed ATRX-deficient cells. Sensitivity to mutant HSV-1 infection also correlated inversely with PML protein levels, and we showed that ATRX upregulates PML expression at both the transcriptional and post-transcriptional levels. These data provide a basis for predicting, based on ATRX or PML levels, which tumors will respond to a selective oncolytic herpesvirus.

ATRX represses alternative lengthening of telomeres.

The unlimited proliferation of cancer cells requires a mechanism to prevent telomere shortening. Alternative Lengthening of Telomeres (ALT) is an homologous recombination-mediated mechanism of telomere elongation used in tumors, including osteosarcomas, soft tissue sarcoma subtypes, and glial brain tumors. Mutations in the ATRX/DAXX chromatin remodeling complex have been reported in tumors and cell lines that use the ALT mechanism, suggesting that ATRX may be an ALT repressor. We show here that knockout or knockdown of ATRX in mortal cells or immortal telomerase-positive cells is insufficient to activate ALT. Notably, however, in SV40-transformed mortal fibroblasts ATRX loss results in either a significant increase in the proportion of cell lines activating ALT (instead of telomerase) or in a significant decrease in the time prior to ALT activation. These data indicate that loss of ATRX function cooperates with one or more as-yet unidentified genetic or epigenetic alterations to activate ALT. Moreover, transient ATRX expression in ALT-positive/ATRX-negative cells represses ALT activity. These data provide the first direct, functional evidence that ATRX represses ALT.

Alternative lengthening of telomeres in normal mammalian somatic cells.

Some cancers use alternative lengthening of telomeres (ALT), a mechanism whereby new telomeric DNA is synthesized from a DNA template. To determine whether normal mammalian tissues have ALT activity, we generated a mouse strain containing a DNA tag in a single telomere. We found that the tagged telomere was copied by other telomeres in somatic tissues but not the germline. The tagged telomere was also copied by other telomeres when introgressed into CAST/EiJ mice, which have telomeres more similar in length to those of humans. We conclude that ALT activity occurs in normal mouse somatic tissues.

Enhanced isolation of fibroblasts from human skin explants.

Here we describe a method for growing fibroblasts from human skin explants that increases the number of cells obtained by up to two orders of magnitude, thus increasing the amount of material available for research and diagnostic purposes and potentially for cell-based therapies. Explants can be transferred sequentially up to 80 times, if required, at which point the explants appear to be completely depleted of fibroblasts. Utilizing skin samples obtained from 16 donors, aged 18-66 years old, the first 20 transfers produced cultures with lifespan and growth characteristics that were all very similar to each other, but the cultures derived from later transfers had a decreasing replicative capacity. Final cumulative population doublings did not correlate with donor age, but correlated positively with the telomere length at early passage. We also demonstrated that explants can be transduced directly by lentiviral infection, and that cryopreserved tissue can be explanted successfully using this procedure.

MicroRNA-296 is enriched in cancer cells and downregulates p21WAF1 mRNA expression via interaction with its 3' untranslated region.

MicroRNAs (miRNAs) are a class of noncoding small RNAs that act as negative regulators of gene expression. To identify miRNAs that may regulate human cell immortalization and carcinogenesis, we performed comparative miRNA array profiling of human normal and SV40-T antigen immortalized cells. We found that miR-296 was upregulated in immortalized cells that also had activation of telomerase. By an independent experiment on genomic analysis of cancer cells we found that chromosome region (20q13.32), where miR-296 is located, was amplified in 28/36 cell lines, and most of these showed enriched miR-296 expression. Overexpression of miR-296 in human cancer cells, with and without telomerase activity, had no effect on their telomerase function. Instead, it suppressed p53 function that is frequently downregulated during human cell immortalization and carcinogenesis. By monitoring the activity of a luciferase reporter connected to p53 and p21(WAF1) (p21) untranslated regions (UTRs), we demonstrate that miR-296 interacts with the p21-3'UTR, and the Hu binding site of p21-3'UTR was identified as a potential miR-296 target site. We demonstrate for the first time that miR-296 is frequently upregulated during immortalization of human cells and contributes to carcinogenesis by downregulation of p53-p21(WAF1) pathway.

Normal human mammary epithelial cells proliferate rapidly in the presence of elevated levels of the tumor suppressors p53 and p21(WAF1/CIP1).

In normal cells, p53 protein is maintained at low levels, but the levels increase after stress or inappropriate growth signals to coordinate growth arrest or apoptosis. Human mammary epithelial cells (HMECs) are unusual in that they exhibit two phases of growth. The second growth phase, referred to as post-selection, follows a period of temporary growth arrest and is characterized by the absence of p16(INK4a) (also known as CDK4I and p16-INK4a) expression. Previously, we observed that post-selection HMECs have elevated levels of p53. Exogenous p16(INK4a) expression decreased levels of both p53 transcript and protein, and this effect was inhibited by nutlin-3a, indicating that p16(INK4a) can regulate p53 expression by affecting both p53 transcription and Mdm2-dependent degradation of p53. The p53 in post-selection HMECs was wild type and, as expected, increased p53 expression was associated with elevated p21(WAF1/CIP1) and Mdm2 levels; the p53 response to DNA damage seemed normal. Despite elevated levels of wild-type p53 and p21(WAF1/CIP1), post-selection cells grew more rapidly than their pre-selection HMEC precursors. We found that the post-selection HMECs contain a truncated Mdm2 protein (p60), which presumably lacks the p53 ubiquitylation domain. We propose that the increased levels of p53 in post-selection HMECs are due to the presence of an Mdm2 fragment that binds p53 but does not result in its degradation.

Alterations in the p16(INK4a) and p53 tumor suppressor genes of hTERT-immortalized human fibroblasts.

Exogenous expression of the catalytic subunit of telomerase, hTERT, in a normal human foreskin fibroblast cell strain resulted in telomerase activity and an extended proliferative lifespan prior to a period of crisis. Three immortalized cell lines with stably maintained telomere lengths were established from cells that escaped crisis. Each of these cultures underwent a significant downregulation of p16(INK4a) expression due to gene deletion events. One cell line also acquired mutations in both alleles of the p53 tumor suppressor gene. Downregulation of p16(INK4a) and loss of wild-type p53 expression occurred after escape from crisis, so these mutations are most likely not required for immortalization of these cells but rather were selected for during continuous growth in vitro. These findings emphasize the need for caution in the use of hTERT-immortalized cells in studies of normal cell biology or in tissue engineering and the need to monitor for genetic instability and the accumulation of mutations in both the p16(INK4a)/pRb and p53 pathways.

Comparison of human mammary epithelial cells immortalized by simian virus 40 T-Antigen or by the telomerase catalytic subunit.

We directly compared two methods of immortalizing human mammary epithelial cells (HMECs). Cells were transfected with an expression plasmid either for hTERT, the catalytic subunit of telomerase, or for the simian virus 40 (SV40) early region genes. Under standard culture conditions, HMECs were not immortalized by hTERT unless they had spontaneously ceased expression of the p16(INK4a) tumor suppressor gene. Untransfected HMECs had low levels of telomerase expression, and immortalization by both methods was associated with an increase in telomerase activity and prevention of telomere shortening. SV40-induced immortalization was accompanied by aberrant differentiation, loss of DNA damage response, karyotypic instability and, in some cases, tumorigenicity. hTERT-immortalized cells had fewer karyotypic changes, but had intact DNA damage responses, and features of normal differentiation. Although SV40-immortalized cells are useful for studies of carcinogenesis, hTERT-immortalized cells retain more properties of normal cells.