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R R Reddel

Publications and source records attributed to R R Reddel.

At least 55 records · Page 3Linked to original sources
PubMed1998

Differential display of mRNA.

Differential display of mRNA (DD) is a technique in which mRNA species expressed by a cell population are reverse transcribed and then amplified by many separate polymerase chain reactions (PCR). PCR primers and conditions are chosen so that any given reaction yields a limited number of amplified cDNA fragments, permitting their visualization as discrete bands following gel electrophoresis. This robust and relatively simple procedure allows identification of genes that are differentially expressed in different cell populations. Here we review DD including some recent modifications, and compare it with other techniques for analyzing differential mRNA expression.

DNA Primers↗
PubMed1997

Normal telomere maintenance in immortal ataxia telangiectasia cell lines.

Telomeres are maintained in germ line cells and immortal cell lines, but shorten with each cell division in most somatic cells. Blood lymphocytes from individuals with ataxia telangiectasia (AT) demonstrate an accelerated rate of telomere shortening and high levels of telomere associations. This accelerated loss of telomeres in somatic cells in AT could be due to either the loss of more telomeric DNA with every cell division or an increased rate of cell division. The gene for AT shares homology with the yeast TEL1 gene, in which mutations result in abnormally shortened telomeres. Thus, mutations in the gene for ataxia telangiectasia may also influence the ability of germ line cells and immortal cell lines to properly maintain telomere homeostasis. To investigate a possible defect of telomere maintenance in AT we have analyzed 8 simian virus 40 (SV40)-immortalized AT cell lines and twelve SV40-immortalized non-AT cell lines for both telomerase activity and telomere length. The results demonstrate that telomere length in AT cells is maintained via telomerase or an alternative (ALT) pathway in a manner indistinguishable from cell lines derived from normal cells. We also investigated telomere dynamics in one telomerase-positive AT cell line by analyzing the changes in the length of a single telomere, and found that this telomere maintained its equilibrium mean length (EML) similar to normal cell lines with stable chromosomes. The combined results show no significant differences between the telomeres of immortal AT and non-AT cell lines, demonstrating that the absence of wild-type ATM does not result in a fundamental defect in telomere maintenance in these cells.

Ataxia Telangiectasia↗
PubMed1997

Telomere dynamics and telomerase activity in in vitro immortalised human cells.

This article reviews the current understanding of the involvement of telomerase in in vitro immortalisation of human cells. In vitro immortalisation with DNA tumour viruses or chemicals usually occurs in two phases. The first stage is an extension of lifespan beyond that at which cells would normally senescence, after which the culture enters a period of crisis. The second stage involves the escape from crisis of a rare cell in the culture, which goes on to proliferate indefinitely. The hypothesis that telomere shortening acts as a signal for senescence and crisis, and that cells need to activate telomerase to survive these states, gained support from early studies examining telomere behaviour and telomerase activity in immortalised cell lines. In many cases, telomeres were found to continue to shorten during the phase of extended lifespan, and no telomerase was detectable. Cells which survived crisis had activated telomerase and had stable or lengthened telomerase. However, it is now clear that this model does not apply to all cell lines. Approximately a quarter of in vitro immortalised cell lines so far examined have no detectable telomerase activity, yet have very long and heterogeneous telomeres. These cell lines have acquired a novel mechanism for lengthening their telomeres, named ALT (Alternative Lengthening of Telomeres). The nature of ALT is not yet understood, but may involve non-reciprocal recombination between telomeres. ALT is not merely a phenomenon of in vitro immortalised cell lines, but has also been found in tumours and tumour-derived cell lines. Furthermore, there are a number of cell lines which have been shown to have low levels of telomerase prior to crisis while telomere shortening is still occurring, and the function of these low levels of telomerase activity is unknown.

Cellular Senescence↗
PubMed1997

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

The gradual loss of DNA from the ends of telomeres has been implicated in the control of cellular proliferative potential. Telomerase is an enzyme that restores telomeric DNA sequences, and expression of its activity was thought to be essential for the immortalization of human cells, both in vitro and in tumor progression in vivo. Telomerase activity has been detected in 50-100% of tumors of different types, but not in most normal adult somatic tissues. It has also been detected in about 70% of human cell lines immortalized in vitro and in all tumor-derived cell lines examined to date. It has previously been shown that in vitro immortalized telomerase-negative cell lines acquire very long and heterogeneous telomeres in association with immortalization presumably via one or more novel telomere-lengthening mechanisms that we refer to as ALT (alternative lengthening of telomeres). 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.

Adult↗
PubMed1997

Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types.

Telomerase is a multicomponent reverse transcriptase enzyme that adds DNA repeats to the ends of chromosomes using its RNA component as a template for synthesis. Telomerase activity is detected in the germline as well as the majority of tumors and immortal cell lines, and at low levels in several types of normal cells. We have cloned a human gene homologous to a protein from Saccharomyces cerevisiae and Euplotes aediculatus that has reverse transcriptase motifs and is thought to be the catalytic subunit of telomerase in those species. This gene is present in the human genome as a single copy sequence with a dominant transcript of approximately 4 kb in a human colon cancer cell line, LIM1215. The cDNA sequence was determined using clones from a LIM1215 cDNA library and by RT-PCR, cRACE and 3'RACE on mRNA from the same source. We show that the gene is expressed in several normal tissues, telomerase-positive post-crisis (immortal) cell lines and various tumors but is not expressed in the majority of normal tissues analyzed, pre-crisis (non-immortal) cells and telomerase-negative immortal (ALT) cell lines. Multiple products were identified by RT-PCR using primers within the reverse transcriptase domain. Sequencing of these products suggests that they arise by alternative splicing. Strikingly, various tumors, cell lines and even normal tissues (colonic crypt and testis) showed considerable differences in the splicing patterns. Alternative splicing of the telomerase catalytic subunit transcript may be important for the regulation of telomerase activity and may give rise to proteins with different biochemical functions.

Alternative Splicing↗
PubMed1997

The telomere lengthening mechanism in telomerase-negative immortal human cells does not involve the telomerase RNA subunit.

According to the telomere hypothesis of senescence, the progressive shortening of telomeres that occurs upon division of normal somatic cells eventually leads to cellular senescence. The immortalisation of human cells is associated with the acquisition of a telomere maintenance mechanism which is usually dependent upon expression of the enzyme telomerase. About one third of in vitro immortalised human cell lines, however, have no detectable telomerase but contain telomeres that are abnormally long. The nature of the alternative telomere maintenance mechanism (referred to as ALT, for Alternative Lengthening of Telomeres) that must exist in these telomerase-negative cells has not been elucidated. It has previously been shown that abnormal lengthening of yeast telomeres may occur due to mutations in the yeast telomerase RNA gene. That this is not the mechanism of the abnormally long telomeres in ALT cell lines was demonstrated by the finding that seven of seven ALT lines have wild-type human telomerase RNA (hTR) sequence, including a novel polymorphism that is present in 30% of normal individuals. We found that two ALT cell lines have no detectable expression of the hTR gene. This shows that the ALT mechanism in these cell lines is not dependent on hTR. Expression of exogenous hTR via infection of these cells with a recombinant hTR-adenovirus vector did not result in telomerase activity, indicating that their lack of telomerase activity is not due to absence of hTR expression. We conclude that the ALT mechanism is not dependent on the expression of hTR, and does not involve mutations in the hTR sequence.

Base Sequence↗
PubMed1997

Immortalized cells with no detectable telomerase activity. A review.

Immortalization of human cells in culture is usually associated with expression of telomerase activity. In some cases, however, no telomerase activity is detectable even though comparison of the terminal restriction fragment (TRF) pattern before and after immortalization shows that lengthening of telomeres has occurred. The extreme heterogeneity in telomere length and the differences in the dynamics of telomere maintenance in telomerase-negative cell lines compared to telomerase-positive cell lines indicate that these cells have utilized one or more alternative mechanisms for lengthening of telomeres (ALT). All telomerase-negative immortalized cell lines examined to date show evidence of ALT activity, consistent with the hypothesis that telomere maintenance either by telomerase or by ALT is required for immortalization. The nature of the ALT mechanism(s) is currently unknown, but studies of telomere dynamics in an ALT cell line containing a marker just proximal to the telomeric sequences show gradual shortening of the telomere followed by rapid elongation. This is consistent with a non-reciprocal recombinational mechanism similar to that found in telomerase-defective mutant yeast strains.

Cell Cycle↗
PubMed1997

Genetic changes associated with immortalization. A review.

Human fibroblasts appear to have three terminal proliferative arrest (TPA) states that act as independent barriers to immortalization. The first of these TPA states is senescence, and several recent studies have shown that abrogation of p53 function permits temporary escape from senescence that ends in a poorly characterized form of arrest (referred to as p53-minus TPA) in which the pRB and p16INK4 genes appear to be involved. Abrogation of the function of both p53 and pRB (or p16INK4) results in continued proliferation until the cells enter crisis. Escape from crisis is always associated with the activation of a telomere maintenance mechanism. We also review evidence for the involvement of other genes in the immortalization process. Immortalization appears to be a complex process involving many genetic changes, not all of which are necessarily related to telomere maintenance.

Animals↗
PubMed1996

Characteristics of tumor cell bioactivity in oncogenic osteomalacia.

Oncogenic osteomalacia is a condition where renal phosphate wasting occurs causing defective mineralisation, in the presence of a tumor. Cultures of cells were established from a hemangiopericytoma resected from a patient with oncogenic osteomalacia. Conditioned media from the cells inhibited phosphate uptake in opossum kidney cells and stimulated of cAMP in rat osteosarcoma cells, a standard parathyroid hormone (PTH)-like assay. This cAMP stimulation was suppressed by the PTH analogue, 3-34 bPTH and also by heat and trypsin treatment of the media. Tests of conditioned media for PTH and parathyroid hormone related protein (PTHrP) immunoreactivity were negative, however, and no hybridisation to probes for PTH, PTHrP or human stanniocalcin was detected in tumor cell RNA on Northern blot. These data support the hypothesis that tumors responsible for oncogenic osteomalacia produce a humoral substance that reduces renal phosphate reabsorption and provide evidence that the factor may act via PTH/PTHrP receptors.

Animals↗
PubMed1996

Molecular cloning and characterization of mouse stanniocalcin cDNA.

In bony fish, stanniocalcin is a glycoprotein hormone thought to be an important regulator of calcium uptake from the aquatic environment. Although stanniocalcin was previously thought to be unique to fish, recent evidence has indicated the existence of a human homologue. To facilitate studies of the function of stanniocalcin in mammals, we have now isolated the mouse stanniocalcin cDNA. This cDNA encodes a predicted protein of the same length as its human counterpart and with a high level of similarity (238/247) amino acids are identical, and five represent conservative changes). As in human, the mRNA is expressed in many mouse tissues, suggesting that mammalian stanniocalcin has a paracrine rather than endocrine role.

Amino Acid Sequence↗
PubMed1996

A highly expressed 81 kDa protein in immortalized mouse fibroblast: its proliferative function and identity with ezrin.

An 81 kDa protein was found to be highly expressed in spontaneously immortalized CD1-ICR mouse fibroblasts, RS-4, as compared to normal fibroblasts. RS-4 cells have a reduced serum requirement and exhibit multilayered growth in vitro but are not tumorigenic. The protein was purified from RS-4 cell extracts and used to obtain a polyclonal antibody that specifically immunoprecipitated an 81 kDa protein from cell lysates. Immunocloning of its cDNA and sequence analysis revealed its identity with ezrin, an F-actin binding protein that is a component of the cortical cytoskeleton. Microinjection of the purified IgG fraction of the anti-p81 antiserum into the cytoplasm of RS-4 cells blocked their entry into S phase suggesting that the protein has a proliferative function. Immunostaining of normal mouse tissues showed that the expression of p81/ezrin was highest in proliferating cell populations. Unlike RS-4, NIH3T3 cells exhibit contact inhibition and express levels of p81/ezrin similar to those of normal fibroblasts. When NIH3T3 cells were transfected with p81/ezrin cDNA they lost contact inhibition and thus resembled RS-4 cells. The study demonstrates a proliferative function of p81/ezrin and suggests its involvement in pathways that negatively regulate contact inhibition.

3T3 Cells↗
PubMed1996

Association of extended in vitro proliferative potential with loss of p16INK4 expression.

This study addresses the question of whether loss of p16INK4 expression contributes to the immortalization of human cells. In vitro immortalization usually proceeds through two phases. In the first phase (lifespan extension), cells continue proliferating and their telomeres continue shortening beyond the point at which normal cells become senescent. In the second phase (immortalization), the cells activate a telomere maintenance mechanism and acquire an unlimited proliferative potential. It has previously been shown that immortalized cells containing viral oncoproteins that bind and inactivate p110RB contain wild-type p16INK4; we therefore examined the p16INK4 status of cell lines that became immortalized in vitro in the absence of these oncoproteins. Three such lines were identified: III-CF/.2A1 and III-CF/E6A2 (both derived from Li-Fraumeni syndrome fibroblasts, probably by spontaneous immortalization) and MePV-231 (normal mesothelial cells transfected with HPV-16 E6/E7 genes that underwent deletion of these genes before immortalization). In each case p16INK4 expression was lost at or before immortalization. Further, a cell strain was identified that had an extended but finite lifespan associated with loss of p16INK4 (and p53) expression. Thus loss of p16INK4 expression was associated with extended in vitro lifespan but was not sufficient for immortalization, even in the absence of wild-type p53.

Carrier Proteins↗
PubMed1996

Genetic differences between the pancytosolic and perinuclear forms of murine mortalin.

To determine the genetic relation between the pancytosolic (uniformly distributed in cytoplasm-p66mot-1) and the perinuclear (p66mot-2) mortalin proteins found in normal and immortal mouse cells, respectively, we initiated the present study using PCR cloning, sequencing, and single nucleotide primer extension analyses. The results indicate that the difference between mot-1 and mot-2 at 2 bp in the open reading frame does not arise by mutations in the immortalized cells. Intriguingly, both mot-1 and mot-2 sequences were detected in genomic DNA from normal (CMEF) and immortal (RS-4) cells derived from the CD1-ICR mouse strain. We extended the analyses to four other strains of mouse and detected both mot-1 and mot-2 sequences in genomic DNA from the Balb/c mouse also. Swiss and C57BL/6 mice exhibited only mot-2 and the C3H He mouse only mot-1. Southern analyses on a mouse strain that showed the presence of only mot-2 (Swiss) and one that showed both mot-1 and mot-2 (CD1-ICR) by PCR revealed that there are structural differences between mot-1 and mot-2 loci. Furthermore, there appears to be a correlation between frequency of spontaneous immortalization of cultured mouse fibroblasts and the mot loci present in the mouse strain from which they were derived.

Animals↗
PubMed1996

An effective elimination of false positives isolated from differential display of mRNAs.

A reverse Northern analysis that effectively eliminates the false positives isolated from differential display of mRNAs (DD) is demonstrated. Preparation of probe by one-step labeling in reverse transcription reaction is found to be more effective and specific as compared to the preparation of probe by random priming of reverse transcribed cDNA pool. Reverse Northern assay of DNA fragments isolated from DD prior to their cloning into plasmid, analysis of multiple fragments on single slot blot, and requirement of RNA only in small amounts as compared to conventional Northern makes the protocol quick, effective, and economic.

3T3 Cells↗
PubMed1996

Chromosomal changes and progressive tumorigenesis of human bronchial epithelial cell lines.

A simian virus 40 (SV40)-transformed human bronchial epithelial cell line, BEAS-2B, underwent progressive changes, including the development of tumorigenicity, during extended in vitro passaging. Karyotypic changes occurred in parallel with the phenotypic changes. For the first 12 passages following viral transformation, there were random karyotypic changes. Immortalization occurred between passages 12 and 21, corresponding with the accumulation of four characteristic abnormal chromosomes-m-1: add(15)(p11.1); m-2: der(8;9)(q10;q10); m-3: add(16)(p13); and m-4: mar4- and the loss of one homolog of chromosomes 8, 15, 16, 21, and 22. With further passaging (from 21 to 63), the acquisition of weak tumorigenicity was observed, accompanied by an increased frequency of cells containing all four common abnormal chromosomes, m-1 through m-4, and missing one normal homolog of chromosomes 8, 15, 16, and 22. Four tumor cell lines (B39-TL, B39-TR, B61-T4 and B61-T7) were established from tumors induced by the injection of these weakly tumorigenic BEAS-2B 39th- and 61st- passage cells into athymic nude mice. One of the cell lines, B39-TL, is significantly more tumorigenic than the others. It is notable that B39-TL showed two specific abnormal chromosomes, del(3p);der(3;15) (q10;q10) and m-6; der(21)t(3;21)(p14.2;p12) inducing deletion of a short arm of chromosome 3. Fluorescence in situ hybridization analysis with a probe for protein tyrosine phosphatase-gamma demonstrated loss of heterozygosity in the 3p14 region. The development of step-wise karyotypic changes in this in vitro carcinogenesis model parallels changes documented in several common human cancers.

Animals↗
PubMed1995

Involvement of RB-1, p53, p16INK4 and telomerase in immortalisation of human cells.

Involvement of the retinoblastoma susceptibility (RB-1), p16INK4, p53 and telomerase genes in immortalisation was examined by determining their status in 15 human cell lines representing four immortalisation complementation groups. No abnormalities of RB-1, p53 and p16INK4 were detected in cell lines containing DNA tumour virus proteins known to bind to the protein products of the RB-1 and p53 genes. In contrast, in all other cell lines from each of the four groups either RB-1 was mutant or p16INK4 protein was undetectable and there were cell lines containing p53 mutations in three of the groups. Telomerase activity was detected in 12/15 lines, including some of the virally immortalised lines and in some lines from each group. Since none of these changes correlated with complementation group, other genetic changes must be required for immortalisation.

Base Sequence↗
PubMed1995

Telomere elongation in immortal human cells without detectable telomerase activity.

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.

Base Sequence↗