Carmela P. Morales

Absence of cancer-associated changes in human fibroblasts immortalized with telomerase

The ectopic expression of telomerase in normal human cells results in an extended lifespan, indicating that telomere shortening regulates the timing of cellular senescence. As telomerase expression is a hallmark of cancer, we investigated the long–term effects of forced expression of human telomerase catalytic component (hTERT) in normal human fibroblasts. In vitro growth requirements, cell–cycle checkpoints and karyotypic stability in telomerase–expressing cells are similar to those of untransfected controls. In addition, co–expression of telomerase, the viral oncoproteins HPV16 E6/E7 (which inactivate p53 and pRB) and oncogenic HRAS does not result in growth in soft agar. Thus, although ectopic expression of telomerase in human fibroblasts is sufficient for immortalization, it does not result in changes typically associated with malignant transformation.

Hallmarks of cancer progression in Barrett's oesophagus

CONTEXT Hanahan and Weinberg proposed in 200 that carcinogenesis involves DNA changes that enable cells to:provide their own growth signals, ignore growth-inhibitory signals, avoid apoptosis, replicate without limit, sustain angiogenesis, and invade and proliferate in unnatural locations. The metaplastic cells of Barretts oesophagus are predisposed to develop these cancer hallmarks. STARTING POINT The genetic changes that have been described in Barretts oesophagus can be categorised according to the predominant cancer hallmark affected. For example, M Auvinen and colleagues recently observed abnormalities in the expression of vascular endothelial growth factors (VEGFs) in Barretts oesophagus (J Clin Oncol 2002; 20: 2971-79). These abnormalities can be categorised as those that affect angiogenesis, a process that is essential for the development and progression of tumours. WHERE NEXT? The cancer hallmarks of Barretts oesophagus provide a framework to categorise the genetic abnormalities described and to further understanding of the genetic events that underlie oesophageal carcinogenesis.

Bypass of telomere-dependent replicative senescence (M1) upon overexpression of Cdk4 in normal human epithelial cells

Many stimuli causing ‘stress’ or DNA damage in cells can produce phenotypes that overlap with telomere-based replicative senescence. In epithelial systems, the p16/RB pathway may function as a stress senescence-signaling pathway independent of telomere shortening. Overexpressing cyclin-dependent kinase 4 (Cdk4) in human epidermal keratinocytes and human mammary epithelial cells not only prevents the p16INK4a-associated premature growth arrest due to telomere-independent stress (e.g., inadequate culture conditions), but also bypasses the ensuing telomere-dependent senescence (M1). Overexpressed Cdk4 in epithelial cells induces a dramatic upregulation of p16INK4a and milder upregulation of p53 and p21WAF1, which become unresponsive to UV irradiation. Despite the high levels of these checkpoint factors, Cdk4-overexpressing cells divide in an apparently normal regulated fashion, are able to respond to changes in calcium levels, retain the stem cell phenotype, and fully differentiate and stratify. These results suggest that the differentiation pathways in Cdk4-overexpressing cells remain intact.

Review article: a conceptual approach to understanding the molecular mechanisms of cancer development in Barrett's oesophagus.

Oesophageal adenocarcinoma is one of the most deadly human malignancies. Gastro‐oesophageal reflux disease (GERD) has been established as a strong risk factor for oesophageal adenocarcinoma, and more than 40% of adult Americans experience regular GERD symptoms. GERD can be complicated by oesophagitis, and by replacement of oesophageal squamous mucosa with metaplastic, intestinal‐type epithelium (Barrett’s oesophagus) that is predisposed to malignancy.

Characterisation of telomerase immortalised normal human oesophageal squamous cells

Background and aims: Oesophageal cell lines derived from malignancies have numerous genetic abnormalities and therefore are of limited value for studying the early events in carcinogenesis. Reported attempts to establish normal human oesophageal cell lines either have failed to achieve immortalisation or have achieved it by disrupting important cell functions. We have used telomerase technology to establish normal human oesophageal cell lines. Methods: Endoscopic biopsy specimens of normal oesophageal squamous epithelium were trypsinised, dispersed into single cell suspensions, and cocultivated with ATCC Swiss 3T3 cells. Oesophageal cells were infected with the catalytic subunit of human telomerase (hTERT) using a defective retroviral vector. The integrity of cell cycle checkpoints was tested by measuring p53 response to UV irradiation, and p16 response to infection with H-RasGV12. Expression of a differentiation marker was tested by measuring involucrin response to calcium exposure. Results: Cultures of uninfected oesophageal cells had weak telomerase activity at baseline but exhibited loss of telomerase activity and progressive telomere shortening before undergoing senescence between population doublings (PD) 40–45. In contrast, hTERT infected cells exhibited sustained telomerase activity and stabilisation of telomere length. These cells have reached PD 100 with no diminution in growth rate, while cell cycle checkpoint integrity and involucrin response to calcium exposure have remained intact. Conclusions: By introducing telomerase into normal human oesophageal squamous cells cocultivated with feeder layers, we have established a cell line that retains normal cell cycle checkpoints and normal differentiation markers. This cell line may be useful for studying the early events in oesophageal carcinogenesis.

In situ hybridization for telomerase RNA in routine cytologic brushings for the diagnosis of pancreaticobiliary malignancies

BACKGROUND Brush cytology for the diagnosis of pancreaticobiliary malignancy has an overall sensitivity of 50%. Accurate and specific markers are therefore needed for the optimal evaluation of pancreaticobiliary strictures. Telomerase activity is present in 85% to 90% of all human cancers. We sought to determine the utility of in situ hybridization for telomerase RNA in endoscopic brushings for the diagnosis of pancreaticobiliary malignancy. METHODS Endoscopic brushings from 18 patients with pancreatic or biliary strictures were evaluated by routine cytology and in situ hybridization for telomerase RNA. RESULTS Eight of 18 strictures were malignant. Cytology was positive in 5 patients, whereas telomerase RNA was positive in 6. All malignancies were diagnosed by either cytology or telomerase RNA; however, both studies were positive in only 3. There were no false-positive results by either technique. CONCLUSION The detection of telomerase RNA in endoscopic brushings may be an important adjunct to cytology for cancer diagnosis in pancreaticobiliary strictures.

ADRENAL GLAND SAMPLING DURING TRANSJUGULAR LIVER BIOPSY LEADS TO MISDIAGNOSIS OF HEPATOCELLULAR CARCINOMA

Adrenal Gland Sampling During Transjugular Liver Biopsy Leads to Misdiagnosis of Hepatocellular Carcinoma

Detection of Telomerase by In Situ Hybridization and by the Polymerase Chain Reaction-Based Telomerase Activity Assay

The onset of human cancer typically requires numerous genetic mutations, generally specific for the tissue type from which the cancer originates. Thus, it has been difficult to screen all tumor types for a single mutation. In recent years, telomerase activity has been associated with at least 85% of human malignancies as well as with some lesions considered preneoplastic by traditional cytology (1,2). Telomerase appears to be ubiquitously associated with a wide array of human cancers from a variety of tissue sources. Therefore, detection of telomerase activity relative to human cancer development is likely to be an important and novel method, in combination with cytology, for cancer diagnosis.