Clare L. Fasching

Telomere maintenance by recombination in human cells

Telomeres of eukaryotic chromosomes contain many tandem repeats of a G-rich sequence (for example, TTAGGG in vertebrates). In most normal human cells, telomeres shorten with each cell division, and it is proposed that this limits the number of times these cells can replicate. Telomeres may be maintained in germline cells, and in many immortalized cells and cancers, by the telomerase holoenzyme (first discovered in the ciliate Tetrahymena), which uses an RNA subunit as template for synthesis of telomeric DNA by the reverse transcriptase catalytic subunit. Some immortalized human cell lines and some tumours maintain their telomeres in the absence of any detectable telomerase activity by a mechanism referred to as alternative lengthening of telomeres (ALT). Here we show that DNA sequences are copied from telomere to telomere in an immortalized human ALT cell line, indicating that ALT occurs by means of homologous recombination and copy switching.

Progression of colorectal cancer is associated with multiple tumor suppressor gene defects but inhibition of tumorigenicity is accomplished by correction of any single defect via chromosome transfer.

Carcinogenesis is a multistage process that has been characterized both by the activation of cellular oncogenes and by the loss of function of tumor suppressor genes. Colorectal cancer has been associated with the activation of ras oncogenes and with the deletion of multiple chromosomal regions including chromosomes 5q, 17p, and 18q. Such chromosome loss is often suggestive of the deletion or loss of function of tumor suppressor genes. The candidate tumor suppressor genes from these regions are, respectively, MCC and/or APC, p53, and DCC. In order to further our understanding of the molecular and genetic mechanisms involved in tumor progression and, thereby, of normal cell growth, it is important to determine whether defects in one or more of these loci contribute functionally in the progression to malignancy in colorectal cancer and whether correction of any of these defects restores normal growth control in vitro and in vivo. To address this question, we have utilized the technique of microcell-mediated chromosome transfer to introduce normal human chromosomes 5, 17, and 18 individually into recipient colorectal cancer cells. Additionally, chromosome 15 was introduced into SW480 cells as an irrelevant control chromosome. While the introduction of chromosome 17 into the tumorigenic colorectal cell line SW480 yielded no viable clones, cell lines were established after the introduction of chromosomes 15, 5, and 18. Hybrids containing chromosome 18 are morphologically similar to the parental line, whereas those containing chromosome 5 are morphologically distinct from the parental cell line, being small, polygonal, and tightly packed. SW480-chromosome 5 hybrids are strongly suppressed for tumorigenicity, while SW480-chromosome 18 hybrids produce slowly growing tumors in some of the animals injected. Hybrids containing the introduced chromosome 18 but was significantly reduced in several of the tumor reconstitute cell lines. Introduction of chromosome 5 had little to no effect on responsiveness, whereas transfer ot chromosome 18 restored responsiveness to some degree. Our findings indicate that while multiple defects in tumor suppressor genes seem to be required for progression to the malignant state in colorectal cancer, correction of only a single defect can have significant effects in vivo and/or in vitro.

Sustained nontumorigenic phenotype correlates with a largely stable chromosome content during long‐term culture of the human keratinocyte line HaCaT

Altered growth and differentiation and a highly abnormal karyotype are generally believed to be indicators for tumorigenic conversion of human cells. Inactivation of TP53 is supposedly one possible mechanism for accelerated genetic aberrations via reduced control of the genetic integrity. To examine the significance of this functional relationship, we investigated the long‐term development of the spontaneously immortalized human skin keratinocyte line HaCaT, carrying UV‐specific mutations in both alleles of the TP53 tumor suppressor gene. During >300 passages, proliferation, clonogenicity, and serum‐independent growth potential increased. In addition, HaCaT cells gained anchorage independence and at late passages showed reduced differentiation. Karyotypic analysis up to passage 225 revealed a high frequency of translocations and deletions, with a particular increase during passages 30 and 50. Nevertheless, the HaCaT cells remained nontumorigenic when injected subcutaneously, and noninvasive in surface transplants in nude mice. By comparative genomic hybridization, we confirmed the karyotypically identified phase of increased chromosomal aberrations between passages 30 and 50. However, before and thereafter, the CGH profiles of the individual chromosomes were largely unchanged, demonstrating that those translocations—also maintained in later passages—did not cause a gross chromosomal imbalance. Thus, our data suggest that multiple changes often correlated with a “transformed phenotype,” including extensive karyotypic alterations and mutational inactivation of TP53, are well compatible with a nontumorigenic phenotype of the HaCaT cells, and that preserved chromosomal balance may be crucial for this stability during long‐term propagation. Genes Chromosom. Cancer 19:201–214, 1997.

Telomerase-Independent Telomere Length Maintenance in the Absence of Alternative Lengthening of Telomeres–Associated Promyelocytic Leukemia Bodies

Immortal tumor cells and cell lines employ a telomere maintenance mechanism that allows them to escape the normal limits on proliferative potential. In the absence of telomerase, telomere length may be maintained by an alternative lengthening of telomeres (ALT) mechanism. All human ALT cell lines described thus far have nuclear domains of unknown function, termed ALT-associated promyelocytic leukemia bodies (APB), containing promyelocytic leukemia protein, telomeric DNA and telomere binding proteins. Here we describe telomerase-negative human cells with telomeres that contain a substantial proportion of nontelomeric DNA sequences (like telomerase-null Saccharomyces cerevisiae survivor type I cells) and that are maintained in the absence of APBs. In other respects, they resemble typical ALT cell lines: the telomeres are highly heterogeneous in length (ranging from very short to very long) and undergo rapid changes in length. In addition, these cells are capable of copying a targeted DNA tag from one telomere into other telomeres. These data show that APBs are not always essential for ALT-mediated telomere maintenance.

Loss of a putative tumor suppressor locus after gamma-ray-induced neoplastic transformation of HeLa x skin fibroblast human cell hybrids.

The nontumorigenic HeLa x skin fibroblast hybrid cell line, CGL1, can be induced to re-express HeLa tumor-associated cell surface antigen, p75-IAP (intestinal alkaline phosphatase), with resulting neoplastic transformation, by exposure to gamma radiation. This has allowed the human hybrid system to be developed into a quantitative in vitro model for radiation-induced neoplastic transformation of human cells. Recently, several gamma-ray-induced IAP-expressing mutants (GIMs) of the nontumorigenic HeLa x skin fibroblast hybrid CGL1 were isolated and all were tumorigenic when injected subcutaneously into nude mice (Mendonca et al., Cancer Res. 51, 4455-4462, 1991). Control cell lines which were negative for p75-IAP (CONs) were also isolated from irradiated populations, and none were found to be tumorigenic. We have now begun to investigate the molecular basis of radiation-induced neoplastic transformation in this system by studying the potential genetic linkage between p75/IAP expression, tumorigenicity and damage to a putative tumor suppressor locus on fibroblast chromosome 11. Previous analysis of rare spontaneous segregants has indicated that this locus is involved in the regulation of tumorigenicity and in the expression of the HeLa tumor-associated cell surface marker intestinal alkaline phosphatase (p75-IAP) in this system. Therefore, analysis by restriction fragment length polymorphism and chromosome painting have been performed for chromosome 11, and for chromosome 13 as a control, for the p75/IAP-positive GIM and p75/IAP-negative CON cell lines. We report that in five of eight of the GIMs large-scale damage to the fibroblast chromosome 11s is evident (four GIMs have lost one complete copy of a fibroblast chromosome 11 and one GIM has both copies of fibroblast chromosome 11 heavily damaged). None of the CONs, however (0/5), have lost a complete copy of either fibroblast chromosome 11. No large-scale damage to the control chromosome 13s was detected in the GIMs or CONs. The data further suggest that both copies of fibroblast chromosome 11 contain an active locus and that radiation-induced loss of either fibroblast chromosome 11 will result in neoplastic transformation in this system. We conclude that it is the loss of a putative tumor suppressor locus on fibroblast chromosome 11 which is responsible at least in part for radiation-induced neoplastic transformation of these human hybrid cells.

Loss of suppressor loci on chromosomes 11 and 14 may be required for radiation-induced neoplastic transformation of HeLa x skin fibroblast human cell hybrids

We have previously reported a linkage between radiation-induced damage to a putative tumor suppressor locus on fibroblast chromosome 11 and the re-expression of tumorigenicity in a hybrid cell line (HeLa x human skin fibroblast) used to study neoplastic transformation. Further investigation into the molecular basis of radiation-induced neoplastic transformation of the hybrid cell, CGL1, indicates that loss of fibroblast chromosome 11 appears to be necessary but not sufficient for neoplastic transformation. Previous analysis had suggested, though not clearly demonstrated, a possible role for loss of alleles on fibroblast chromosome 14 in the neoplastic transformation of the hybrid cells. Therefore, the status of chromosome 14 in the gamma-ray-induced, neoplastically transformed (GIM) hybrid cell lines and in nontumorigenic control (CON) hybrid cell lines isolated from irradiated populations has been investigated. Chromosome painting and molecular studies using restriction fragment length polymorphisms and tetranucleotide repeat polymorphism analysis were performed. As an additional control, the status of chromosome 12 was also examined. We report that five of the eight GIM cell lines have lost one complete copy of a fibroblast chromosome 14 while only one of the five CON cell lines has lost a complete copy of a fibroblast chromosome 14. No evidence of large-scale loss of chromosome 12 was detected in the GIM or CON cells. The data further suggest that both copies of fibroblast chromosome 14 contain an active tumor suppressor locus and that radiation-induced loss of either fibroblast chromosome 14 is associated with neoplastic transformation in this system. We now conclude that loss of alleles on both fibroblast chromosome 11 and 14 may be required for the radiation-induced neoplastic transformation of these human hybrid cells.

Cys(9), Cys(104) and Cys(207) of simian virus 40 Vp1 are essential for infectious virion formation in CV-1 cells.

Structural studies have implicated Cys(9), Cys(104) and Cys(207) of simian virus 40 (SV40) Vp1 in disulfide bond formation. Recently, we have shown the three cysteines to be essential for disulfide linkage of Vp1 complexes in vitro. Here, the role of the three cysteines was explored during the course of SV40 infection. Single-, double- and triple-mutant Vp1 at Cys(9), Cys(104) and Cys(207) continued to localize to the nuclei of transfected CV-1 cells and to bind DNA, but showed a range of abilities to form plaques. Only mutants containing the Cys(9)-->Ser change showed defects in plaque formation. Single mutants at Cys(9) formed small plaques; mutants at Cys(9). Cys(104), Cys(9). Cys(207) and Cys(9). Cys(104). Cys(207) formed no plaques. All three isolated revertants contained back-mutations at the Vp1 Cys(9) codon. These results further confirm the involvement of the three Vp1 cysteines in protein-protein interactions during virus assembly. Cys(9) is critical for production of wild-type infectious virions, whereas Cys(104) and Cys(207) play secondary roles.

Pictures in Molecular Medicine: Telomeres copying telomeres in human cells

Telomeres consist of repetitive DNA sequences at the ends of chromosomes, which, together with specific binding proteins, form protective chromosomal cap structures. In normal human cells, telomeres shorten with every cell division, and this progressive shortening is ultimately able to limit the number of cell division cycles that can occur. After dividing a limited number of times, cells become incapable of responding to proliferative signals, and become senescent. This finite proliferative capacity of normal cells is a potent barrier to carcinogenesis which cancer cells must breach 1xThe role of senescence and immortalization in carcinogenesis. Reddel, R.R. Carcinogenesis. 2000; 21: 477–484Crossref | PubMedSee all References1. Many cancers contain populations of cells that are immortalized, in that they are capable of apparently unlimited proliferation. In all cases examined to date, immortalization is associated with the activation of a mechanism that prevents the inexorable telomere shortening that occurs with normal cell division2xTelomere maintenance mechanisms and cellular immortalization. Colgin, L.M and Reddel, R.R. Curr. Opin. Genet. Dev. 1999; 9: 97–103Crossref | PubMed | Scopus (138)See all References2. In the majority of cancers, telomeres are maintained by the action of a holoenzyme, telomerase, that replaces eroded telomeric DNA by reverse transcribing the template region of its RNA subunit that is complementary to the telomere repeat sequence. Some cancer cells have very low or undetectable levels of telomerase, but are able to lengthen their telomeres by a mechanism that is referred to as alternative lengthening of telomeres (ALT) 3xEvidence for an alternative mechanism for maintaining telomere length in human tumors and tumor-derived cell lines. Bryan, T.M et al. Nat. Med. 1997; 3: 1271–1274Crossref | PubMedSee all References3.Telomere length dynamics in a telomerase-negative cell line was found to be consistent with a recombinational mechanism of telomere maintenance 4xTelomere dynamics in an immortal human cell line. Murnane, J.P et al. EMBO J. 1994; 13: 4953–4962PubMedSee all References4. To test the hypothesis that telomere–telomere recombination has a role in telomere maintenance in ALT cells, plasmid DNA was targeted into the telomeres of an ALT cell line 5xTelomere maintenance by recombination in human cells. Dunham, M.A et al. Nat. Genet. 2000; 26: 447–450Crossref | PubMed | Scopus (542)See all References5. Telomeres containing plasmid DNA were visualized by fluorescence in situ hybridization (FISH). At early population doubling levels, a small number of different telomeres contained the plasmid tag (Fig. 1Fig. 1a) and after additional population doublings the number of tagged telomeres had increased (Fig. 1Fig. 1b). To confirm that a single tagged telomere can be copied by another chromosome, a human chromosome containing a tagged telomere was propagated in a murine cell line (Fig. 1Fig. 1c) and then transferred into an ALT cell line, which was subsequently observed to have telomeric tags on more than one chromosome (Fig. 1Fig. 1d). These data indicate that telomeres in ALT cell lines are able to be lengthened by copying of other telomeres 5xTelomere maintenance by recombination in human cells. Dunham, M.A et al. Nat. Genet. 2000; 26: 447–450Crossref | PubMed | Scopus (542)See all References5. Identification of the proteins involved in this process is expected to reveal new targets for anticancer therapies.Fig. 1(a) DAPI stained (blue) metaphase chromosomes of early passage ALT cells with few plasmid tagged telomeres, (b) late passage ALT cells with additional tagged telomeres, (c) FITC-painted mouse chromosomes with single human chromosome containing telomere tag, (d) two telomere tagged chromosomes in ALT cell after transfer of single tagged chromosome.View Large Image | Download PowerPoint Slide