Telomere dynamics in a human cancer cell line

Telomere maintenance is thought to be essential for immortalization of human cancer cells to compensate for the loss of DNA from the ends of chromosomes and to prevent chromosome fusion. We have investigated telomere dynamics in the telomerase-positive squamous cell carcinoma cell line SCC-61 by marking the ends of chromosomes with integrated plasmid sequences so that changes in the length of individual telomeres could be monitored. Despite having very short telomeres, SCC-61 has a relatively stable genome and few telomere associations. The marked telomeres in different SCC-61 clones have similar mean lengths which show little change with increasing time in culture. Thus, each marked telomere is maintained at a specific length, which we term the equilibrium mean length (EML). The Gaussian distribution in the length of the marked telomeres demonstrates that telomeres continuously fluctuate in length. Consistent with this observation, the mean lengths of the marked telomere in subclones of these cell lines initially differ, but then gradually return to the EML of the original clone with increasing time in culture. The analysis of a clone with two marked telomeres demonstrated that changes in telomere length can occur on each marked telomere independently or coordinately on both telomeres. These results suggest that the short telomeres in many tumor cell lines do not result from an inability to properly maintain telomeres at a specific length.     

Telomere instability in a human tumor cell line expressing a dominant-negative WRN protein

Werner Syndrome (WS) is an autosomal recessive disease characterized by premature aging and chromosome instability. The protein involved in WS, WRN, is a RecQ-type helicase that also has exonuclease activity. WRN has been demonstrated to bind to a variety of other proteins, including RPA, DNA-PKcs, and TRF2, suggesting that WRN is involved in DNA replication, repair, recombination, and telomere maintenance. In culture, WS cells show premature senescence, which can be overcome by transfection with an expression vector containing the gene for the catalytic subunit of telomerase. However, telomerase expression does not eliminate chromosome instability in WS cells, which led to the proposal that telomere loss is not the cause of the high rate of chromosome rearrangements in WS cells. In the present study, we have investigated how a WRN protein containing a dominant-negative mutation (K577M-WRN) influences the stability of telomeres in a human tumor cell line expressing telomerase. The results demonstrate an increased rate of telomere loss and chromosome fusion in cells expressing K577M-WRN. Expression of K577M-WRN results in reduced levels of telomerase activity, however, the absence of detectable changes in average telomere length demonstrates that WRN-associated telomere loss results from stochastic events involving complete telomere loss or loss of telomere capping function. Thus, telomere loss can contribute to chromosome instability in cells deficient in WRN regardless of the expression of telomerase activity.     

Telomere dynamics in an immortal human cell line.

The integration of transfected plasmid DNA at the telomere of chromosome 13 in an immortalized simian virus 40-transformed human cell line provided the first opportunity to study polymorphism in the number of telomeric repeat sequences on the end of a single chromosome. Three subclones of this cell line were selected for analysis: one with a long telomere on chromosome 13, one with a short telomere, and one with such extreme polymorphism that no distinct band was discernible. Further subcloning demonstrated that telomere polymorphism resulted from both gradual changes and rapid changes that sometimes involved many kilobases. The gradual changes were due to the shortening of telomeres at a rate similar to that reported for telomeres of somatic cells without telomerase, eventually resulting in the loss of nearly all of the telomere. However, telomeres were not generally lost completely, as shown by the absence of polymorphism in the subtelomeric plasmid sequences. Instead, telomeres that were less than a few hundred base pairs in length showed a rapid, highly heterogeneous increase in size. Rapid changes in telomere length also occurred on longer telomeres. The frequency of this type of change in telomere length varied among the subclones and correlated with chromosome fusion. Therefore, the rapid changes in telomere length appeared occasionally to result in the complete loss of telomeric repeat sequences. Rapid changes in telomere length have been associated with telomere loss and chromosome instability in yeast and could be responsible for the high rate of chromosome fusion observed in many human tumor cell lines.     

Telomere instability and cancer

Telomeres are required to preserve genome integrity, chromosome stability, nuclear architecture and chromosome pairing during meiosis. Given that telomerase activity is limiting or absent in most somatic tissues, shortening of telomeres during development and aging is the rule. In vitro, telomere length operates as a mechanism to prevent uncontrolled cell growth and therefore defines the proliferation potential of a cell. In vitro, in somatic cells that have lost proliferation control, shortening of telomeres becomes the main source of genome instability leading to genetic or epigenetic changes that may allow cells to become immortal and to acquire tumor phenotypes. In vivo, mice models have indisputably shown both the protective and the promoting role of very short telomeres in cancer development. In humans, although telomere shortening and other types of telomere dysfunction probably contribute to the genome instability often detected in tumors, the specific contributions of such instability to the development of cancer remain largely undetermined.     

Telomere instability in a human tumor cell line expressing NBS1 with mutations at sites phosphorylated by ATM

Nijmegen breakage syndrome (NBS) is an autosomal genetic disease demonstrating a variety of phenotypic abnormalities, including premature aging, increased cancer incidence, chromosome instability, and sensitivity to ionizing radiation. The gene involved in NBS, NBS1, is part of the MRE11/RAD50/NBS1 (MRN) complex that also includes MRE11 and RAD50, which is involved in DNA repair and cell cycle regulation in response to DNA damage. The MRN complex is also involved in telomere maintenance, as demonstrated by the shortened telomeres in NBS primary human fibroblasts and the association of NBS1 with the telomere-binding protein TRF2. To learn more about how a deficiency in telomere maintenance might contribute to chromosome instability in NBS, we have investigated the stability of telomeres in two telomerase-positive human tumor cell clones, BNmt-On and BNmt-Off, expressing an inducible NBS1(S278A/S343A) gene containing mutations at serines 278 and 343 phosphorylated by ATM. The results demonstrate an increased rate of telomere loss in both clones following expression of NBS1(S278A/S343A). The absence of detectable changes in average telomere length suggests that NBS1-associated telomere loss results from stochastic events involving complete telomere loss or loss of telomere capping function. The recombination events associated with telomere loss were found to be similar to those shown previously to result in breakage/fusion/bridge cycles, suggesting that telomere loss can contribute to chromosome instability in NBS1-deficient cells. Telomere loss showed no correlation with radiosensitivity or radioresistant DNA synthesis, demonstrating that NBS1(S278A/S343A) promotes telomere loss through a separate pathway from these other phenotypes associated with NBS.     

Alpha-fetoprotein receptors in a human breast cancer cell line

Evidence is presented for the existence of specific receptors for alpha-fetoprotein on the surface of MCF-7 human breast cancer cells. At 4 degrees C, the binding of alpha-fetoprotein to these cells displayed a biphasic saturation curve. Scatchard analysis revealed the presence of at least two binding sites with dissociation constants of 4.5 X 10(-9) M (2,000 sites/cell) and 1.3 X 10(-8) M (135,000 sites/cell), respectively. Binding was inhibited by 85% in the presence of a 5,000-fold excess of unlabeled alpha-fetoprotein and by 50% with the same excess of serum albumin. Competition by other serum proteins was not significant. At 37 degrees C, alpha-fetoprotein was endocytosed and the uptake curve reached a plateau after 3-4 hours of incubation.     

Telomere dynamics and genome stability in the human pancreatic tumor cell line MIAPaCa-2

Telomeres are specialized structures found at the ends of eukaryotic chromosomes serving as guardians of genome stability. In normal cells telomeres shorten with each cell division, but immortal cells undergoing multiple divisions constantly have to maintain telomere lengths above a critical level. This is accomplished either through expression of telomerase or the alternative recombination pathway (ALT). In the present study, we analyzed telomere dynamics of the telomerase positive human pancreatic tumor cell line MIAPaCa-2. The cells demonstrated genomic instability with a high frequency of chromosomal aberrations resulting in differences between individual karyotypes within the same cell population. The telomeres were short when compared with normal human fibroblasts, and about 39% of the chromosome ends did not have detectable telomere repeats as demonstrated by PNA-FISH. In many cases telomere signals were missing even when sister chromatids were strongly labeled. In addition, we used an internal PNA probe specific for the X chromosome, present in a single copy in these cells, in order to follow telomere dynamics on individual chromatids. High heterogeneity in telomere signals among individual X chromosomes as well as between their sister chromatids suggested sudden and stochastic loss or gain of telomere repeats. Such constant genomic instability often results in apoptosis and death of a fraction of cells present in the culture at all times. We discuss possible molecular mechanisms that may explain this observed telomere heterogeneity and possible adaptive repair mechanisms by which these cells maintain their chromosomes in order to survive such extreme and permanent genomic instability.     

Glycolaldehyde induces apoptosis in a human breast cancer cell line

Activated phagocytes employ myeloperoxidase to generate glycolaldehyde, 2-hydroxypropanal, and acrolein. Because alpha-hydroxy and alpha,beta-unsaturated aldehydes are highly reactive, phagocyte-mediated formation of these products may play a role in killing bacteria and tumor cells. Using breast cancer cells, we demonstrate that glycolaldehyde inactivates glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and Cu,Zn superoxide dismutase, suppresses cell growth, and induces apoptosis. These results suggest that glycolaldehyde might be an important mediator of neutrophil anti-tumor activity.     

Telomere dysfunction in genome instability syndromes

Telomeres are nucleoprotein complexes located at the end of eukaryotic chromosomes. They have essential roles in preventing terminal fusions, protecting chromosome ends from degradation, and in chromosome positioning in the nucleus. These terminal structures consist of a tandemly repeated DNA sequence (TTAGGG in vertebrates) that varies in length from 5 to 15 kb in humans. Several proteins are attached to this telomeric DNA, some of which are also involved in different DNA damage response pathways, including Ku80, Mre11, NBS and BLM, among others. Mutations in the genes encoding these proteins cause a number of rare genetic syndromes characterized by chromosome and/or genetic instability and cancer predisposition. Deletions or mutations in any of these genes may also cause a telomere defect resulting in accelerated telomere shortening, lack of end-capping function, and/or end-to-end chromosome fusions. This telomere phenotype is also known to promote chromosomal instability and carcinogenesis. Therefore, it is essential to understand the interplay between telomere biology and genome stability. This review is focused in the dual role of chromosome fragility proteins in telomere maintenance.     

Cytogenetic study of a cell line of human penile cancer

A cell line of penile cancer from a 60-year-old Ugandan black patient has been studied by the authors. Transmission and scanning electron microscopy showed a large number of blebs and microvilli at cell surface; desmosomes were evident at TEM. Cytogenetic investigation (R-, C-, Nor-banding) showed the frequent presence of some markers: del(1p),del(1q),iso(3q),der(4),del(8p),11q+, t rob(13;14), 14p+, t rob(21;21). The epidemiology, geographical distribution, and aetiological role of human papilloma virus type 16 and herpes simplex type 2 are discussed.     

Telomere dysfunction and chromosome instability

The ends of chromosomes are composed of a short repeat sequence and associated proteins that together form a cap, called a telomere, that keeps the ends from appearing as double-strand breaks (DSBs) and prevents chromosome fusion. The loss of telomeric repeat sequences or deficiencies in telomeric proteins can result in chromosome fusion and lead to chromosome instability. The similarity between chromosome rearrangements resulting from telomere loss and those found in cancer cells implicates telomere loss as an important mechanism for the chromosome instability contributing to human cancer. Telomere loss in cancer cells can occur through gradual shortening due to insufficient telomerase, the protein that maintains telomeres. However, cancer cells often have a high rate of spontaneous telomere loss despite the expression of telomerase, which has been proposed to result from a combination of oncogene-mediated replication stress and a deficiency in DSB repair in telomeric regions. Chromosome fusion in mammalian cells primarily involves nonhomologous end joining (NHEJ), which is the major form of DSB repair. Chromosome fusion initiates chromosome instability involving breakage-fusion-bridge (B/F/B) cycles, in which dicentric chromosomes form bridges and break as the cell attempts to divide, repeating the process in subsequent cell cycles. Fusion between sister chromatids results in large inverted repeats on the end of the chromosome, which amplify further following additional B/F/B cycles. B/F/B cycles continue until the chromosome acquires a new telomere, most often by translocation of the end of another chromosome. The instability is not confined to a chromosome that loses its telomere, because the instability is transferred to the chromosome donating a translocation. Moreover, the amplified regions are unstable and form extrachromosomal DNA that can reintegrate at new locations. Knowledge concerning the factors promoting telomere loss and its consequences is therefore important for understanding chromosome instability in human cancer.     

Activation of N-ras in a human melanoma cell line.

DNA isolated from cell line Mel Swift, a human melanoma cell line, transforms NIH3T3 cells. Southern blot analysis of DNA from secondary foci revealed conserved 8.8- and 7.8-kilobase EcoRI fragments which hybridized with a human repetitive sequence clone, blur 8. The activated transforming gene was identified as N-ras, and the 8.8-kilobase EcoRI fragment from a secondary transformant was cloned. Synthetic 17-mer oligonucleotides which spanned either the normal codon 61 (CAA) or a mutant codon 61 (AAA) were used for hybridization. Cloned N-ras from melanoma cell line Mel Swift hybridized to the mutant (AAA) oligonucleotide. From this we predicted a glutamine-to-lysine substitution in amino acid 61, a change confirmed by conventional sequencing of the first and second exons of N-ras from cell line Mel Swift. Transfection experiments showed that only those recombinant clones with the mutation in position 61 were biologically active.     

Chromosome studies on a human liposarcoma cell line.

Numerical and structural chromosome analysis of a human retroperitoneal liposarcoma cell line maintained under standard cell culture conditions revealed a very stable hypodiploid mode. If the cells were not trypsinized for several generations, a near-triploid stemline, which was generally a duplication of the hypodiploid mode, emerged. Some chromosomes appeared to be relatively stable pairs (1, 2, 7, 9, and 12), but most had "lost" one homolog or both (4 and 21) or were rearranged into "new" marker chromosomes. Quantitation of the genetic material showed a loss of 12.0 +/- 3.7% per spread. Only one characteristically long marker chromosome, which is present in every cell, could be identified with certainty as a translocation between chromosomes 4 and 11. Several of the marker chromosomes showed interstitial negatively staining regions with the trypsin-Giemsa method.     

The loss of a single telomere can result in instability of multiple chromosomes in a human tumor cell line

Spontaneous telomere loss has been proposed as an important mechanism for initiating the chromosome instability commonly found in cancer cells. We have previously shown that spontaneous telomere loss in a human cancer cell line initiates breakage/fusion/bridge (B/F/B) cycles that continue for many cell generations, resulting in DNA amplification and translocations on the chromosome that lost its telomere. We have now extended these studies to determine the effect of the loss of a single telomere on the stability of other chromosomes. Our study showed that telomere acquisition during B/F/B cycles occurred mainly through translocations involving either the nonreciprocal transfer or duplication of the arms of other chromosomes. Telomere acquisition also occurred through small duplications involving the subtelomeric region of the other end of the same chromosome. Although all of these mechanisms stabilized the chromosome that lost its telomere, they differed in their consequences for the stability of the genome as a whole. Telomere acquisition involving nonreciprocal translocations resulted in the loss of a telomere on the donor chromosome, which consequently underwent additional translocations, isochromosome formation, or complete loss. In contrast, telomere acquisition involving duplications stabilized the genome, although the large duplications created substantial allelic imbalances. Thus, the loss of a single telomere can generate a variety of chromosome alterations commonly associated with human cancer, not only on a chromosome that loses its telomere but also on other chromosomes. Factors promoting telomere loss are therefore likely to have an important role in generating the karyotype evolution associated with human cancer.     

Bombesin stimulates the in vitro growth of a human gastric cancer cell line

Bombesin (BBS) and its mammalian equivalent, gastrin-releasing peptide (GRP) exhibit diverse biological functions, including that of a neurotransmatter, a regulator of gastrointestinal hormone release, and a trophic factor for various normal and neoplastic tissues. Bombesin stimulates the growth of normal cells of the stomach, pancreas, and bronchial epithelium as well as cells in breast cancer, gastrinoma, and small cell lung cancer. The purpose of this study was to determine whether BBS regulates the growth of a human gastric cancer cell line (SIIA) in vitro, and if so, to examine the mechanisms of signal-transduction that are involved. We found that BBS stimulated the growth of SIIA cells in vitro. The GRP receptor antagonists, BIM 26189 and BIM 26226, had no effect on growth of SIIA cells. Although these antagonists blocked the BBS-induced increase of [Ca²⁺]_i, they failed to block the growth-stimulatory effect of BBS. BBS stimulated intracellular tyrosine phosphorylation of multiple proteins, with a predominant protein of apparent molecular weight of 125 kDa. Inhibition of intracellular tyrosine kinases by tyrphostin blocked the growth-stimulatory effect of BBS on SIIA cells. These results indicate that BBS exerts its trophic effect on SIIA cells through a receptor(s) linked to tyrosine kinase pathway, but not to the phospholipase C (PLC) pathway. © 1994 Wiley-Liss, Inc.     

Telomere higher-order structure and genomic instability

Telomeres, nucleoprotein complexes at the end of eukaryotic chromosomes, have vital roles in chromosome integrity. Telomere chromatin structure is both intricate and dynamic allowing for a variety of responses to several stimuli. A critical determinant in telomere structure is the G-strand overhang. Facilitated by telomeric proteins, the G-strand overhang stabilizes telomere higher-order assemblies most likely by adopting unusual DNA structures. These structures influence activities that occur at the chromosome end. Dysfunctional telomeres induce signals resulting in cell growth arrest or death. To overcome telomere dysfunction, cancer cells activate the DNA polymerase, telomerase. The presence of telomerase at the telomere may establish a particular telomeric state. If the chromosome ends of cancer and normal cells exist in different states, cancer-specific telomere structures would offer a unique chemotherapeutic target.     

Characterization of glycoprotein ligands for P-selectin on a human small cell lung cancer cell line NCI-H345.

P-selectin (CD62P) is a cell adhesion molecule expressed on stimulated endothelial cells and on activated platelets. It interacts with PSGL-1 (P-selectin glycoprotein ligand-1; CD162) on leukocytes and mediates recruitment of leukocytes during inflammation. P-selectin also binds to several types of cancer cells in vitro and facilitates growth and metastasis of colon carcinoma in vivo. Here we show that P-selectin, but not E-selectin, binds to NCI-H345 cells, a cell line derived from a human small cell lung cancer. EDTA or P7 (a leukocyte adhesion blocking mAb to P-selectin), but not PL5 (a leukocyte adhesion blocking mAb to PSGL-1), can inhibit this binding. P-selectin affinity chromatography can precipitate a approximately 110-kDa major band and a approximately 220-kDa minor band from [3H]-glucosamine-labeled NCI-H345 cells. No expression of PSGL-1 protein and mRNA can be detected in NCI-H345 cells. Taken together, these results suggest that NCI-H345 cells express glycoprotein ligands for P-selectin that are distinct from leukocyte PSGL-1.     

Radiation-induced damage to DNA in drug- and radiation-resistant sublines of a human breast cancer cell line.

An Adriamycin-resistant subline of a human breast cancer cell line, MCF-7 ADRR, has been shown to exhibit radioresistance associated with an increase in the size of the shoulder on the radiation survival curve. In the present study, damage to DNA of MCF-7 sublines WT and ADRR by 60Co gamma radiation was measured by filter elution techniques. The initial amount of DNA damage, measured by both alkaline and neutral filter elution, was lower in ADRR cells, suggesting that these cells are resistant to radiation-induced single- and double-strand DNA breaks. In the case of double-strand breaks the difference between WT and ADRR cells was significant only at the lower radiation doses studied (up to 100 Gy). In cells depleted of glutathione (GSH) by L-buthionine sulfoximine (BSO) treatment, ADRR cells were sensitized to radiation-induced DNA damage, while WT cells were unaffected. The rate of repair of single- and double-strand DNA breaks following radiation was the same for both sublines, and repair of radiation damage was not affected by BSO treatment in either cell line. The relative resistance of ADRR cells to initial DNA damage by radiation is the only difference so far detected at the molecular level which reflects radiation survival, and it is possible that other factors are involved in the resistance of ADRR cells to killing by radiation. Sensitization of ADRR cells to radiation-induced DNA damage by GSH depletion, although not likely to involve inhibition of GSH-dependent detoxification enzymes per se (irradiation was done at 4 degrees C), suggests that at the molecular level radioresponse in this subline is related to maintenance of GSH/GSSG redox equilibrium.