MicroRNA-146a controls functional plasticity in γδ T cells by targeting NOD1.

γδ T cells are major providers of proinflammatory cytokines. They are preprogrammed in the mouse thymus into distinct subsets producing either interleukin-17 (IL-17) or interferon-γ (IFN-γ), which segregate with CD27 expression. In the periphery, CD27- γδ (γδ27-) T cells can be induced under inflammatory conditions to coexpress IL-17 and IFN-γ; the molecular basis of this functional plasticity remains to be determined. On the basis of differential microRNA (miRNA) expression analysis and modulation in γδ T cell subsets, we identified miR-146a as a thymically imprinted post-transcriptional brake to limit IFN-γ expression in γδ27- T cells in vitro and in vivo. On the basis of biochemical purification of Argonaute 2-bound miR-146a targets, we identified Nod1 to be a relevant mRNA target that regulates γδ T cell plasticity. In line with this, Nod1-deficient mice lacked multifunctional IL-17+ IFN-γ+ γδ27- cells and were more susceptible to Listeria monocytogenes infection. Our studies establish the miR-146a/NOD1 axis as a key determinant of γδ T cell effector functions and plasticity.

Telomere dysfunction is related to the intrinsic radio-resistance of human oral cancer cells.

Evidence from telomerase-deficient mice strongly suggests that dysfunctional short telomeres affect cellular radio-sensitivity but this idea has yet to be extensively tested in relevant human cancer types such as oral squamous cell carcinomas (OSCCs), which are frequently treated by radiotherapy. The OSCC line BICR7 has low levels of telomerase activity, short telomeres and high levels of telomere dysfunction (judged by a high level of anaphase bridges); whereas the BICR6 line has high levels of telomerase and is more radio-resistant. Ectopic expression of the human TElomerase Reverse Transcriptase (hTERT) reduced telomere dysfunction and increased radio-resistance in BICR7 cells, but not BICR6. Furthermore, the radio-resistance of GM847 cells, which use telomerase-independent mechanisms of telomere maintenance, and of telomerase-negative normal human fibroblasts with long telomeres are similarly unaffected by ectopic expression of telomerase. We tested whether telomere function, as measured by the Anaphase Bridge Index (ABI), was found to be a useful predictor of radio-resistance in a panel of OSCC lines. Using inverse regression analysis, we found a strong inverse relationship between the ABI and radio-resistance (P<0.001), as measured by the Surviving Fraction at 4Gy (SF4). These results suggest that telomerase inhibitors could sensitise a subset of oral SCCs with short telomeres to radiotherapy and for the first time demonstrate that the tumour ABI may assist the selection of cancers that would be suitable for such sensitisation therapy.

Analysis of telomerase activity and telomere function in cancer.

Telomeres are the structures at the ends of chromosomes, composed of repetitive sequences and associated proteins, which cap chromosome ends to maintain genomic stability. These structures are maintained by the enzyme complex telomerase in germ cells and some stem cells, but are absent in the majority of somatic cells. The consequence of this lack of telomerase in normal somatic cells is the shortening of the telomeric repeat, which results in a limited replicative life span. However, in cancer cells, which grow indefinitely, telomerase activity has been detected in a large number of different cancer cell types. This has lead to a great deal of interest in establishing techniques to measure telomerase activity, telomere length, and telomere function in both normal and cancer cells/tissue. Here we describe the TRAP (telomeric repeat amplification protocol) assay, a technique to measure telomerase activity in cells, TRF (terminal restriction fragment length) analysis to estimate telomere length, and both the anaphase bridge index and the frequency of dicentric chromosomes as indicators of telomere dysfunction.

Senescing oral dysplasias are not immortalized by ectopic expression of hTERT alone without other molecular changes, such as loss of INK4A and/or retinoic acid receptor-beta: but p53 mutations are not necessarily required.

Our previous work showed that acquisition of immortality at the dysplasia stage of oral cancer progression was consistently associated with four changes: loss of retinoic acid receptor (RAR)-beta and p16INK4A expression, p53 mutations and activation of telomerase. One atypical dysplasia (D17) that underwent delayed senescence after an extended lifespan showed loss of RAR-beta and p16INK4A/p14ARF expression, but retained functional wild-type p53 and telomerase was not activated. We now demonstrate that retroviral delivery of hTERT results in telomere lengthening and immortalization of D17 without loss of functional wild-type p53 activity. In contrast, the expression of hTERT in two other typical mortal dyplasia cultures (that retain RAR-beta and p16INK4A expression) does not extend their lifespan, even though telomeres are lengthened.

High levels of telomere dysfunction bestow a selective disadvantage during the progression of human oral squamous cell carcinoma.

Human epithelial cells experience multiple barriers to cellular immortality in culture (mortality mechanisms 0, 1, and 2). Mortality mechanism 2 (M2) is termed crisis and involves telomere dysfunction due to lack of telomerase. However, proliferating normal keratinocytes in vivo can express telomerase, so it is unclear whether human squamous cell carcinomas (SCCs), which usually have high telomerase levels, develop from preexisting telomerase-positive precursors or by the activation of telomerase in telomerase-deficient somatic cells. We show that 6 of 29 oral SCCs show characteristics of M2 crisis in vivo, as indicated by a high anaphase bridge index (ABI), which is a good correlate of telomere dysfunction, and that 25 of 29 tumors possess some anaphase bridges. ABIs in excess of 0.2 in the primary tumor showed a decrease in the corresponding lymph node metastases. This suggests that high levels of telomere dysfunction (>0.2) and, by inference, M2 crisis bestow a selective disadvantage on SCCs during progression stages of the disease. Supporting this, SCCs with high levels of telomere dysfunction grow poorly in culture, and the ectopic expression of telomerase corrects this, together with other features of M2 crisis. Our data suggest that a substantial proportion of oral SCCs in vivo ultimately arise from telomerase-deficient keratinocytes rather than putative telomerase-proficient cells in the undifferentiated parts of the epithelium. Furthermore, the presence of significant levels of telomere dysfunction in a high proportion of SCCs at diagnosis but not in the normal epithelium implies that the therapeutic inhibition of telomerase should selectively compromise the growth of such tumors.

Functional evidence for a squamous cell carcinoma mortality gene(s) on human chromosome 4.

Squamous cell carcinoma (SCC) immortality is associated with p53 and INK4A dysfunction, high levels of telomerase and loss of heterozygosity (LOH) of other chromosomes, including chromosome 4. To test for a functional cancer mortality gene on human chromosome 4 we introduced a complete or fragmented copy of the chromosome into SCC lines by microcell-mediated chromosome transfer (MMCT). Human chromosome 4 caused a delayed crisis, specifically in SCC lines with LOH on chromosome 4, but chromosomes 3, 6, 11 and 15 were without effect. The introduction of the telomerase reverse transcriptase into the target lines extended the average telomere terminal fragment length but did not affect the frequency of mortal hybrids following MMCT of chromosome 4. Furthermore, telomerase activity was still present in hybrids displaying the mortal phenotype. The MMCT of chromosomal fragments into BICR6 mapped the mortality gene to between the centromere and 4q23. Deletion analysis of the introduced chromosome in immortal segregants narrowed the candidate interval to 2.7 Mb spanning D4S423 and D4S1557. The results suggest the existence of a gene on human chromosome 4 whose dysfunction contributes to the continuous proliferation of SCC and that this gene operates independently from telomeres, p53 and INK4A.

Telomerase inhibition and the future management of head-and-neck cancer.

Telomeres are tandem repeats of DNA associated with specific proteins. These structures cap eukaryotic chromosomes and maintain the integrity of the chromosome ends. In the germline, telomeres are maintained by the enzyme telomerase, but in normal somatic cells the enzyme's activity is low or undetectable. Human tumours, including squamous-cell carcinoma of the head and neck (SCCHN), need telomerase to maintain telomere function; inhibition of the enzyme can lead to apoptosis. Furthermore, because most tumour cells have very short telomeres, they are more likely to succumb to telomerase inhibition than normal cells. Telomerase is therefore a potential selective anticancer target. The telomere is also involved in the repair of DNA double strand breaks, and telomere dysfunction provokes radiosensitivity. In this review we consider whether manipulation of telomere function may selectively sensitise SCCHN to radiotherapy and discuss the possible pitfalls. We also assess how some conventional treatments may affect the subsequent use of telomerase inhibitors.