The genetic basis of human keratinocyte immortalisation in squamous cell carcinoma development: The role of telomerase reactivation

Normal human keratinocytes have a finite replicative lifespan which culminates in senescence. Chromosomal telomere length may act as a mediator of replicative senescence, signalling cell cycle arrest in G1 when one or more telomeres become too short. Telomeric attrition in normal keratinocytes may be due to inadequate levels of telomerase activity and possibly also to oxidative damage. In advanced squamous cell carcinoma replicative senescence breaks down to yield immortal variants, in which several dominantly acting genes are functionally compromised, including p53 and the cyclin D-Cdk4/6 inhibitor CDKN2A/p16. The increased activity of both of these proteins would be expected to contribute to the G1 arrest in senescence and we have shown that levels of p16 are dramatically increased in senescent keratinocytes. In addition, two other genes which control a cell cycle G1 checkpoint independently of p53 and pRb appear dysfunctional. These genes are uncloned but map to chromosome 4q and 7q31.1 and appear to represent senescence complementation groups B and D, respectively. In immortal neoplastic keratinocytes, telomerase is strongly upregulated and there is evidence for a suppressor of the enzyme on the short arm of chromosome 3 mapping to 3p21.2-p21.3. We have also mapped the human telomerase RNA gene to 3q26.3 and found it to be overrepresented or amplified in a proportion of squamous cell tumours and cell lines. These observations may explain why isochromosome 3q is so common in human squamous carcinoma. None of these genetic alterations are seen in carcinomas which senesce and suggest that multiple genetic alterations are required for keratinocyte immortality

Mapping of the gene for the mouse telomerase RNA component, Terc, to chromosome 3 by fluorescence in situ hybridization and mouse chromosome painting

No abstract available

Amplification, increased dosage and in situ expression of the telomerase RNA gene in human cancer

Telomere length is maintained by the enzyme, telomerase, which has been linked to cellular immortality and tumour progression, However, the reasons for the high levels of telomerase found in human tumours are unknown, We have mapped the human telomerase RNA gene, (hTR), to chromosome 3q26.3 and show the hTR gene to be amplified in four carcinomas, (2/33 cervix, 1/31 head and neck, 1/9 lung), In addition, increased copy numbers of the hTR locus was also observed in 97% of tumours, By in situ hybridisation, the histological distribution of high levels of hTR expression could be demonstrated in a lung tumour and its metastasis with hTR amplification, These results are the first report of genetic alterations involving a known component of telomerase in human cancer, Indeed, it is also the first report of the amplification of a specific locus within the chromosome 3q region frequently subject to copy number gains in human tumours, In addition, we also show for the first time the histological distribution of the RNA component of telomerase in human tumours

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 tumor

Increased dosage and amplification of the focal adhesion kinase gene in human cancer cells

Focal adhesion kinase (pp125FAK) is present at sites of cell/extracellular matrix adhesion and has been implicated in the control of cell behaviour. In particular, as a key component of integrin-stimulated signal transduction pathways, pp125FAK is involved in cellular processes such as spreading, motility, growth and survival. In addition, a number of reports have indicated that pp125FAK may be up-regulated in human tumour cells of diverse origin, and consequently, a role has been proposed for pp125FAK in the development of invasive cancers. However, to date the mechanisms that lead to elevated pp125FAK expression in tumour cells have not been determined. Here we used in situ hybridization to confirm chromosome 8q as the genomic location of the human fak gene and report that elevation of pp125FAK protein in cell lines derived from invasive squamous cell carcinomas is accompanied by gains in copy number of the fak gene in all cases examined. In addition, we observed increased fak copy number in frozen sections of squamous cell carcinomas. Furthermore, increased dosage of the fak gene was also observed in many cell lines derived from human tumours of lung, breast and colon, including two cell lines Calu3 and HT29, in which fak was amplified. In addition, in an in vitro model for human colon cancer progression there was a copy number gain of the fak gene during conversion from adenoma to carcinoma, which was associated with increased pp125FAK protein expression. Thus, we show for the first time that many cell lines derived from invasive epithelial tumours have increased dosage of the fak gene, which may contribute to the elevated protein expression commonly observed. Although other genes near the fak locus are co-amplified or increased in copy number, including the proto-oncogene c-myc, the biological properties of pp125FAK in controlling the growth, survival and invasiveness of tumour cells, suggest that it may contribute to the selection pressure for maintaining increased dosage of the region of chromosome 8q that encodes these genes

Cloning and characterization of human and mouse telomerase RNA gene promoter sequences

Variation in telomerase activity is correlated with cellular senescence and tumour progression. However, although the enzymatic activity of telomerase has been well studied, very little is known about how expression of telomerase genes is regulated in mammalian cells. We have therefore cloned the promoter regions of the human (hTR), and mouse, (terc), telomerase RNA genes in order to identify the regulatory elements controlling telomerase RNA gene transcription, 1.76 kb encompassing the hTR gene promoter region was sequenced, as was 4 kb encompassing the terc promoter. No significant sequence similarity could be detected in comparisons between human and mouse 5'-regions, flanking the transcribed sequences. However, both the human and mouse telomerase RNA genes are within CpG islands and may therefore be under the regulation of DNA methylation. Transient expression of hTR-reporter gene constructs in HeLa and GM847 cells identified the elements responsible for promoter activity are contained in a 231 bp region upstream of the transcriptional start site, Transient expression of terc-reporter gene constructs in Swiss3T3 and A9 cells identified the elements responsible for promoter activity are contained in a 73 bp region upstream of the transcriptional start site. These studies have implications for novel transcription targeted cancer therapies

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

Evaluation of plasma immunoglobulin G and BW thresholds for predicting preweaning mortality in commercially raised dairy goat kids

The high preweaning mortality rate is a concerning issue for the commercial dairy industry. In this context, early identification of at-risk individuals can be instrumental. To address this, we conducted a prospective cohort study with the objective of evaluating plasma immunoglobulin G concentration (pIgG-24 h) and initial BW (IBW) measured at 1d old in 363 male dairy kids (Saanen) for predicting preweaning mortality under commercial conditions. Receiver operator characteristic (ROC) analysis was used to determine critical thresholds for pIgG-24 h and IBW. Subsequently, areas under the curve (AUC), sensitivity (Se), and specificity (Sp) were examined to assess the accuracy of these thresholds. Multivariable regressions were used to model odds ratios (OR) for mortality, controlling for confounding effects between IBW and pIgG-24 h. The mean (±SD) pIgG-24 h and IBW were 16.4 ± 9.37 g/L and 4.0 ± 0.61 kg. Overall mortality ≤ 14d and ≤42d old was 12% and 21%, respectively. Critical pIgG-24 h thresholds predicting mortality ≤ 14 d and ≤42 d old were < 10.1 g/L (AUC = 0.74, Se = 59%, and Sp = 82%) and <11.4 g/L (AUC 0.70, Se = 53%, and Sp = 77%), respectively. Kids with pIgG-24 h < 10.1 g/L were six times more likely to die ≤ 14 d old [OR; 95% CI (6; 3–12)], and kids with pIgG-24 h < 11.4 g/L were four times more likely to die ≤ 42 d old (4; 2–6). The IBW threshold most linked to mortality ≤ 14 d was <3.95 kg (AUC 0.60, Se = 59%, and Sp = 61%). However, this association became inconclusive after adjusting for pIgG-24 h differences. Conversely, an IBW of <3.0 kg was associated with notably higher mortality odds within both 14 and 42 d, irrespective of pIgG-24 h levels (10; 3–37, and 4; 1–20, respectively), suggesting that kids with an IBW < 3.0 kg face an increased likelihood of dying before 42 d, irrespectively of their IgG levels. While our findings suggest pIgG-24 h < 11.4 g/L and IBW < 3.0 kg as strong indicators of early mortality risks in male dairy kids, these results require further validation for other systems

Molecular changes associated with oral dysplasia progression and acquisition of immortality: Potential for its reversal by 5-azacytidine

This study has identified molecular changes characteristic of early oral cancer progression. We reported previously that acquisition of the immortal phenotype is an early event in oral cancer development (F. McGregor et al., Cancer Res., 57: 3886–3889, 1997); our current data indicate that about half of oral dysplasia cultures are immortal, and this is associated with loss of expression of retinoic acid receptor (RAR)-ß and the cell cycle inhibitor p16ink4a (p16), p53 mutations, and increased levels of telomerase/human telomerase reverse transcriptase mRNA. In contrast, increased expression of the epidermal growth factor receptor, known to be a characteristic of oral cancer, does not occur until after the dysplasia stage in squamous cell carcinomas. Acquisition of invasive properties as judged by an in vitro Matrigel invasion assay also does not occur until the carcinoma stage and is further increased in metastases. Interestingly, one atypical mortal dysplasia with a considerably extended life span has lost expression of RAR-ß and p16, but it still expresses only wild-type p53 (albeit at a higher level than normal) and has not activated telomerase. RAR-ß and/or p16 re-expression can be induced by treatment with 5-aza-2-deoxycytidine (Aza-C) in some immortal dysplasias, and this has been shown to be due to silencing of gene expression by promoter methylation. Aza-C treatment also down-regulated telomerase activity and human telomerase reverse transcriptase mRNA. Interestingly, with one dysplasia, Aza-C was able to reverse its immortal phenotype, as judged by morphological criteria and expression of the senescence-associated acid ß-galactosidase activity during terminal growth arrest; this immortal dysplasia was the only one in which Aza-C treatment not only down-regulated telomerase activity but also induced re-expression of both RAR-ß and p16. The possibility of reversing the immortal phenotype of some dysplasias by Aza-C may be of clinical usefulness