The hallmarks of cancer.

Oncogenic ras Provokes Premature Cell Senescence Associated with Accumulation of p53 and p16INK4a

The early notion that cancer is caused by mutations in genes critical for the control of cell growth implied that genome stability is important for preventing oncogenesis. During the past decade, knowledge about the mechanisms by which genes erode and the molecular machinery designed to counteract this time-dependent genetic degeneration has increased markedly. At the same time, it has become apparent that inherited or acquired deficiencies in genome maintenance systems contribute significantly to the onset of cancer. This review summarizes the main DNA caretaking systems and their impact on genome stability and carcinogenesis.

Genome maintenance mechanisms for preventing cancer

https://delangelab.rockefeller.edu/pubs/50_Griffith_Cell_99.pdf

Mammalian Telomeres End in a Large Duplex Loop

Telomere Shortening and Tumor Formation by Mouse Cells Lacking Telomerase RNA

Immortalization of human cells is often associated with reactivation of telomerase, a ribonucleoprotein enzyme that adds TTAGGG repeats onto telomeres and compensates for their shortening. We examined whether telomerase activation is necessary for immortalization. All normal human fibroblasts tested were negative for telomerase activity. Thirteen out of 13 DNA tumor virus-transformed cell cultures were also negative in the pre-crisis (i.e. non-immortalized) stage. Of 35 immortalized cell lines, 20 had telomerase activity as expected, but 15 had no detectable telomerase. The 15 telomerase-negative immortalized cell lines all had very long and heterogeneous telomeres of up to 50 kb. Hybrids between telomerase-negative and telomerase-positive cells senesced. Two senescent hybrids demonstrated telomerase activity, indicating that activation of telomerase is not sufficient for immortalization. Some hybrid clones subsequently recommenced proliferation and became immortalized either with or without telomerase activity. Those without telomerase activity also had very long and heterogeneous telomeres. Taken together, these data suggest that the presence of lengthened or stabilized telomeres is necessary for immortalization, and that this may be achieved either by the reactivation of telomerase or by a novel and as yet unidentified mechanism.

Telomere elongation in immortal human cells without detectable telomerase activity.

The gradual loss of DNA from the ends of telomeres has been implicated in the control of cellular proliferative potential1–3. Telomerase is an enzyme that restores telomeric DNA sequences4, and expression of its activity was thought to be essential for the immortalization of human cells, both in vitro and in tumor progression in vivo5. Telomerase activity has been detected in 50–100% of tumors of different types, but not in most normal adult somatic tissues6,7. It has also been detected in about 70% of human cell lines immortalized in vitro and in all tumor-derived cell lines examined to date7. It has previously been shown that in vitro immortalized telomerase-negative cell lines acquire very long and heterogeneous telomeres in association with immortalization8–11 presumably via one or more novel telomere-lengthening mechanisms that we refer to as ALT (alternative lengthening of telomeres)11. Here we report evidence for the presence of ALT in a subset of tumor-derived cell lines and tumors. The maintenance of telomeres by a mechanism other than telomerase, even in a minority of cancers, has major implications for therapeutic uses of telomerase inhibitors.

Evidence for an alternative mechanism for maintaining telomere length in human tumors and tumor-derived cell lines

Telomerase-negative immortalized human cells maintain their telomeres by a mechanism known as alternative lengthening of telomeres (ALT). We report here that ALT cells contain a novel promyelocytic leukemia (PML) body (ALT-associated PML body, APB). APBs are large donut-shaped nuclear structures containing PML protein, telomeric DNA, and the telomere binding proteins human telomere repeat binding factors 1 and 2. Immunostaining showed that APBs also contain replication factor A, RAD51, and RAD52, proteins involved in DNA synthesis and recombination. During immortalization, APBs appeared at exactly the same time as activation of ALT. APBs were found in ALT tumors and cell lines but not in mortal cell strains or in telomerase-positive cell lines or tumors.

https://cancerres.aacrjournals.org/content/canres/59/17/4175.full.pdf

Telomerase-negative Immortalized Human Cells Contain a Novel Type of Promyelocytic Leukemia (PML) Body

Normal cells have a strictly limited growth potential and senesce after a defined number of population doublings (PDs). In contrast, tumor cells often exhibit an apparently unlimited proliferative potential and are termed immortalized. Although spontaneous immortalization of normal human cells in vitro is an extremely rare event, we observed this in fibroblasts from an affected member of a Li-Fraumeni syndrome kindred. The fibroblasts were heterozygous for a p53 mutation and underwent senescence as expected at PD 40. In four separate senescent cultures (A to D), there were cells that eventually recommenced proliferation. This was associated with aneuploidy in all four cultures and either loss (cultures A, C, and D) or mutation (culture B) of the wild-type (wt) p53 allele. Loss of wt p53 function was insufficient for immortalization, since cultures A, B, and D subsequently entered crisis from which they did not escape. Culture C has continued proliferating beyond 400 PDs and thus appears to be immortalized. In contrast to the other cultures, the immortalized cells have no detectable p16INK4 protein. A culture that had a limited extension of proliferative potential exhibited a progressive decrease in telomere length with increasing PD. In the culture that subsequently became immortalized, the same trend occurred until PD 73, after which there was a significant increase in the amount of telomeric DNA, despite the absence of telomerase activity. Immortalization of these cells thus appears to be associated with loss of wt p53 and p16INK4 expression and a novel mechanism for the elongation of telomeres.

Anna Englezou

Papers

Telomere elongation in immortal human cells without detectable telomerase activity.

Evidence for an alternative mechanism for maintaining telomere length in human tumors and tumor-derived cell lines

Telomerase-negative Immortalized Human Cells Contain a Novel Type of Promyelocytic Leukemia (PML) Body

Alterations in p53 and p16INK4 expression and telomere length during spontaneous immortalization of Li-Fraumeni syndrome fibroblasts

The hTERTα Splice Variant is a Dominant Negative Inhibitor of Telomerase Activity