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Telomeres are specialized nucleoprotein structures at chromosome ends that undergo dynamic changes after each cell cycle. Understanding the mechanisms of telomere dynamics is critically dependent on the ability to accurately measure telomere length in a cell population of interest. Techniques such as Southern blot, which measures average telomere length, and quantitative fluorescence in situ hybridization (Q‐FISH), which can estimate telomere length in individual chromosomes, are limited in their capacity to determine the distribution of cells with differing telomere lengths in a given cell population.We employed flow‐FISH to determine whether mouse and human cell lines exhibit subpopulations of cells with differing telomere lengths.Our analysis showed that at least one of four analyzed mouse cell lines had two subpopulations of cells with differing telomere lengths. Differences in telomere length between subpopulations of cells were significant, and we term this phenomenon TELEFLUCS (TElomere LEngth FLUctuations in Cell Subpopulations). We also observed TELEFLUCS in 1 of 19 analyzed human nonalternative lengthening of telomere cell lines and in 1 of 2 analyzed human alternative lengthening of telomere cell lines. The existence of cell subpopulations with differing telomere lengths was confirmed by Q‐FISH.Our results underscore the importance of flow‐FISH in telomere length analysis. © 2004 Wiley‐Liss, Inc.

The diagnosis-related group (DRG)-based Medicare prospective payment system pays hospitals a fixed amount for the care of similar patients. The DRG definitions serve as the modifier of payment for Medicare patients. The dependence on these patient definitions raises many questions, among them the reason(s) for observed resource variability within a DRG. Various severity-of-illness measures have been shown to account for some of the resource variability noted within the DRGs. Most severity-of-illness studies to date, however, have not attempted to assess the effect of other known sources of resource variation, such as differing physician practice patterns. The authors examined the ability of severity of illness, as defined by disease staging, and physician practice variation to explain residual intra-DRG variability in length of stay. They demonstrate that physician practice variation accounts for more variance reduction than does severity of illness.

The established mouse cell line NIH 3T3 has been used with considerable success over the past three years as the basis of an in vitro transformation assay for demonstrating the presence of transfectable transforming genes in the DNA of certain human and rodent tumour cells (for review see ref. 1). In the case of the human bladder carcinoma cell lines EJ and T24, this approach has led2–4to the molecular cloning of a transforming gene which is closely related to the rat-derived Harvey sarcoma virus oncogene, v-Ha-ras5,6. A single point mutation, which distinguishes these genes from their normal human homologue (c-Ha-ras1), is thought to be solely responsible for their transforming potential7–10. However, carcinogenesis in both humans and laboratory rodents is a multi-stage process (reviewed in ref. 11) of which the NIH 3T3 cell, already partly transformed, may represent only the penultimate stage. We therefore chose to examine the transforming effects of the EJ oncogene in a hamster fibroblast system originally developed in our laboratory to study stages in carcinogen-induced malignant transformation of normal diploid cells12. We show here that EJ c-Ha-ras-1 lacks complete transforming activity when transfected into normal fibroblasts which have a limited life-span, but can fully transform fibroblasts that have been newly ‘immortalized’ by carcinogens.

BENZO[a]PYRENE (BP) is one of a group of chemically inert carcinogens which, in order to exert their cytotoxic, mutagenic and carcinogenic effects, require metabolic activation by mixed function oxygenases (MFC) to products capable of covalent reaction with cellular macromolecules1–3. More specifically, it has been shown in mouse skin4and various cultured mammalian cells5, that, in the case of DNA, the extent of this reaction correlates with the carcinogenic potency of the compound. The hypothesis that DNA is the important target for this series of hydrocarbons is further supported by evidence that the most carcinogenic members are also the most mutagenic towards hamster cells in vitro1. Here we present evidence that a diol epoxide derivative of BP is a powerful mutagen when applied to mammalian cells, and suggest therefore that this compound is the ultimate carcinogenic metabolite of BP.

Limited lifespan in normal diploid mammalian cells in culture is well documented1–3and there is evidence that it is also a property of normal tissues serially transplanted in vivo4,5. In contrast, in favourable growth conditions, cells derived from tumours or premalignant tissue often exhibit unlimited proliferative potential (immortality) both in vitro and in vivo2,4,6–10. Malignant transformation of diploid cells in culture by carcinogens has been clearly demonstrated in several laboratories2,11–13. However, although it has been frequently reported that establishment in culture is one of many alterations in cell behaviour associated with transformation, the induction of immortality and its role in malignancy have not previously been subjected to serious scientific examination. Here, we show that treatment of Syrian hamster cell cultures with carcinogenic chemicals can induce rare immortal variants, the progeny of which then frequently progress to anchorage independence and malignancy after a further period of growth. In addition, by studying the properties of cell lines that are either immortal or anchorage-independent but not both, we have obtained evidence that immortality, although insufficient by itself, may be a prerequisite for malignant transformation.