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The immortalization process of T cells: with focus on the regulation of telomere length and telomerase activity
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cellular immortalization is a major hallmark of cancer and is a multi-step process that requires numerous cell-type specific changes, including inactivation of control mechanisms and stabilization of telomere length. The telomeres at the chromosome ends are essential for genomic stability, and limit the growth potential of most cells. With each cell division, telomeres are shortened. Short telomeres may induce an irreversible growth arrest stage called senescence, or a growth crisis stage characterized by high genomic instability and cell death. Only very rarely do cells escape from crisis and become immortal, a stage that has been associated with the activation of the telomerase enzyme which can elongate and stabilize the telomeres.

The processes leading to senescence bypass, growth crisis escape and finally immortalization are only beginning to be elucidated. Most of our knowledge of the immortalization process is based on analyses of human fibroblast and epithelial cell cultures immortalized by genetic modification. In this thesis, spontaneously immortalized human T lymphocytes derived from patients with Nijmegen Breakage Syndrome and a healthy individual were used to identify critical events for senescence bypass and immortalization. Genetic analysis showed a clonal progression and non-random genetic changes including the amplification of chromosomal region 2p13-21 as an early event in the immortalization process. Telomere length gradually shortened at increasing population doublings and growth crisis was associated with critically short telomeres. The clone(s) that escaped growth crisis demonstrated a logarithmic growth curve, very short telomeres and, notably, no increase in telomerase activity or expression of the telomerase catalytic gene, hTERT. Instead, upregulation of telomerase activity and telomere length stabilization were late events in T lymphocyte immortalization. Escape from crisis was associated with downregulation of DNA damage response genes and altered expression of cell cycle regulators and genes controlling the cellular senescence program.

These data indicated that a number of layers of regulation are important in the process of immortalization and to provide further mechanistic detail, epigenetic analysis was carried out. Genome wide methylation array analysis identified early and step-wise methylation changes during the immortalization process. Interestingly, applying these findings to tumors of T cell origin revealed commonly methylated CpG sites in transformed cells. Deregulated gene expression of the polycomb complexes may have contributed to the epigenetic changes observed.

Taken together, our analysis of spontaneously immortalized T cell cultures identified several steps in the immortalization process including genetic, epigenetic, gene expression and telomere/telomerase regulatory events, contributing further insights to the complexity of cancer cell immortalization.

Place, publisher, year, edition, pages
Umeå: Umeå university , 2010. , 60 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1347
Keyword [en]
immortalization, senescence, T cells, Nijmegen breakage syndrome, telomere, telomerase, hTERT, generic aberrations, methylation
National Category
Medical and Health Sciences
Research subject
Pathology
Identifiers
URN: urn:nbn:se:umu:diva-33466ISBN: 978-91-7459-007-4 (print)OAI: oai:DiVA.org:umu-33466DiVA: diva2:312884
Public defence
2010-05-21, Betula, Norrlands Universitetssjukhus, Byggnad 6M,, UMEÅ, 09:00 (English)
Opponent
Supervisors
Available from: 2010-04-30 Created: 2010-04-26 Last updated: 2010-04-30Bibliographically approved
List of papers
1. Telomere maintenance and cell cycle regulation in spontaneously immortalized T-cell lines from Nijmegen breakage syndrome patients
Open this publication in new window or tab >>Telomere maintenance and cell cycle regulation in spontaneously immortalized T-cell lines from Nijmegen breakage syndrome patients
2003 (English)In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 287, no 1, 178-189 p.Article in journal (Refereed) Published
Abstract [en]

Nijmegen breakage syndrome (NBS) is a rare genetic instability syndrome associated with a high incidence of lymphoid malignancies. The NBS1 protein has been implicated in telomere biology suggesting that cells from NBS patients might have deficient telomere maintenance capacity. In this study we characterized spontaneously immortalized T-cell lines derived from three NBS patients regarding growth characteristics, telomere biology, expression of cell-cycle regulators, and response to DNA damage to understand the role of NBS1 in the immortalization process. In all the NBS T-cell lines the acquisition of an immortal phenotype was associated with telomere length stabilization, high telomerase activity, and increased mRNA expression of the catalytic subunit of telomerase (hTERT), together with c-myc up-regulation. Our findings provide evidence that telomere length maintenance was intact in the T lymphocytes in the absence of a full-length NBS protein, presumably due to the presence of an alternatively transcribed NBS protein of 70 kDa. Normal protein expression patterns for pRb and p53 in all the immortal lines coincided with altered expression of some cell-cycle proteins as well as with an impaired G1/S arrest after gamma irradiation, despite a seemingly normal p53/p21 pathway. The here described, spontaneously immortalized NBS derived T-cell lines can be useful in future analysis of the biologic effects in the NBS.

Keyword
Nijmegen Breakage syndrome, T lymphocyte, immortalization, telomere biology, cell cycle control
National Category
Cell and Molecular Biology
Research subject
Pathology
Identifiers
urn:nbn:se:umu:diva-33448 (URN)10.1016/S0014-4827(03)00140-X (DOI)12799193 (PubMedID)
Available from: 2010-04-26 Created: 2010-04-26 Last updated: 2013-06-12Bibliographically approved
2. Spontaneously immortalized human T lymphocytes develop gain of chromosomal region 2p13-24 as an early and common genetic event
Open this publication in new window or tab >>Spontaneously immortalized human T lymphocytes develop gain of chromosomal region 2p13-24 as an early and common genetic event
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2004 (English)In: Genes, Chromosomes and Cancer, ISSN 1045-2257, E-ISSN 1098-2264, Vol. 41, no 2, 133-144 p.Article in journal (Refereed) Published
Keyword
Cell Line; Transformed, Chromosome Aberrations, Chromosome Banding, Chromosome Mapping, Chromosomes; Human; Pair 2/*genetics, Chromosomes; Human; Pair 8/genetics, Humans, In Situ Hybridization; Fluorescence, Karyotyping, Neoplasms/genetics, T-Lymphocytes/*physiology
National Category
Cell and Molecular Biology
Research subject
Pathology
Identifiers
urn:nbn:se:umu:diva-15213 (URN)10.1002/gcc.20059 (DOI)15287026 (PubMedID)
Available from: 2008-01-11 Created: 2008-01-11 Last updated: 2010-04-30Bibliographically approved
3. Telomerase upregulation is a postcrisis event during senescence bypass and immortalization of two Nijmegen breakage syndrome T cell cultures
Open this publication in new window or tab >>Telomerase upregulation is a postcrisis event during senescence bypass and immortalization of two Nijmegen breakage syndrome T cell cultures
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2010 (English)In: Aging Cell, ISSN 1474-9718, E-ISSN 1474-9726, Vol. 9, 220-235 p.Article in journal (Refereed) Published
Abstract [en]

Summary Our knowledge on immortalization and telomere biology is mainly based on genetically manipulated cells analyzed before and many population doublings post growth crisis. The general view is that growth crisis is telomere length (TL) dependent and that escape from crisis is coupled to increased expression of the telomerase reverse transcriptase (hTERT) gene, telomerase activity upregulation and TL stabilization. Here we have analyzed the process of spontaneous immortalization of human T cells, regarding pathways involved in senescence and telomerase regulation. Two Nijmegen breakage syndrome (NBS) T cell cultures (S3R and S4) showed gradual telomere attrition until a period of growth crisis followed by the outgrowth of immortalized cells. Whole genome expression analysis indicated differences between pre-, early post- and late postcrisis cells. Early postcrisis cells demonstrated a logarithmic growth curve, very short telomeres and, notably, no increase in hTERT or telomerase activity despite downregulation of several negative hTERT regulators (e.g. FOS, JUN D, SMAD3, RUNX2, TNF-alpha and TGFbeta-R2). Thereafter, cMYC mRNA increased in parallel with increased hTERT expression, telomerase activity and elongation of short telomeres, indicating a step-wise activation of hTERT transcription involving reduction of negative regulators followed by activation of positive regulator(s). Gene expression analysis indicated that cells escaped growth crisis by deregulated DNA damage response and senescence controlling genes, including downregulation of ATM, CDKN1B (p27), CDKN2D (p19) and ASF1A and upregulation of CDK4, TWIST1, TP73L (p63) and SYK. Telomerase upregulation was thus found to be uncoupled to escape of growth crisis but rather a later event in the immortalization process of NBS T cell cultures.

Place, publisher, year, edition, pages
John Wiley & Sons, 2010
Keyword
hTERT, immortalization, senescence, T cell, telomerase, telomere
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-33450 (URN)10.1111/j.1474-9726.2010.00550.x (DOI)000275677000010 ()20089118 (PubMedID)
Available from: 2010-04-26 Created: 2010-04-26 Last updated: 2012-02-29Bibliographically approved
4. Senescence bypass and immortalization of T cell cultures are linked to stepwise DNA methylation changes
Open this publication in new window or tab >>Senescence bypass and immortalization of T cell cultures are linked to stepwise DNA methylation changes
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(English)Manuscript (preprint) (Other academic)
Keyword
T cell, CpG methylation, immortalization, telomerase, gene expression
National Category
Cell and Molecular Biology
Research subject
Pathology
Identifiers
urn:nbn:se:umu:diva-33451 (URN)
Available from: 2010-04-26 Created: 2010-04-26 Last updated: 2010-04-30Bibliographically approved

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