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Telomere analysis of normal and neoplastic hematopoietic cells: studies focusing on fluorescence in situ hybridization and flow cytometry
Umeå University, Faculty of Medicine, Medical Biosciences.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The telomeres are specialized structures at the end of the chromosomes composed of the repeated DNA sequence (TTAGGG)n and specific proteins bound to the DNA. The telomeres protect the chromosomes from degradation and end to end fusions. Due to the end-replication problem, the telomeric DNA shortens every cell division, forcing the cells into senescence at a critical telomere length. This process can be counteracted by activating a specialized enzyme, telomerase, which adds telomeric repeats to the chromosome ends leading to an extended or infinite cellular life span. Telomerase activity is absent in most somatic tissues but is found in germ cells, stem cells, activated lymphocytes and the vast majority of tumor cells and permanent cell lines. Hence, telomerase has been suggested as a target for cancer treatment as malignant cells almost exclusively express the enzyme and in that context telomere length measurements will be of great importance.

Telomere length is traditionally measured with a Southern blot based technique. A new method for telomere analysis of cells in suspension, called flow-FISH, was developed based on fluorescence in situ hybridization using a telomeric peptide nucleic acid (PNA) probe,

DNA staining with propidium iodide and quantification by flow cytometry. Flow-FISH had high reproducibility and the telomere length measurements showed good correlation with Southern blotting results. The flow-FISH technique also allows studies of cells in specific phases of the cell cycle and the replication timing of telomeric, centromeric and other repetitive sequences were analyzed in a number of cells. Like previous studies, centromeres were shown to replicate late in S phase while the telomere repeats were found to replicate early in S phase or concomitant with the bulk DNA, which is opposite to the patterns described in yeast.

In benign immunopurified lymphocytes from tonsils, high telomerase activity was found in germinal center (GC) B cells. This population also had high hTERT mRNA levels and displayed a telomere elongation as shown by flow-FISH and Southern blotting. Combined immunophenotyping and flow-FISH on unpurified tonsil cells confirmed the results.

Chronic lymphocytic leukemia (CLL), the most common leukemia in adults, can be divided into pre-GC CLL, characterized by unmutated immunoglobulin VH genes and worse prognosis, and post-GC CLL, with mutated VH genes and better prognosis. In 61 cases of CLL, telomere length was measured with Southern blotting and VH gene mutation status was analyzed. A new association was found between VH mutation status and telomere length, where cases with longer telomeres and mutated VH genes (post-GC CLL) had better prognosis

than CLL with short telomeres and unmutated VH genes (pre-GC CLL). A larger study of 112 CLL cases was performed using flow-FISH. The same correlation between telomere length and VH mutation status was found but gender seemed to be of importance as telomere length was a significant prognostic factor for the male CLL patients but not in the female group. Age of the patients and spread of disease seemed to affect the prognostic value of VH gene mutation status.

Place, publisher, year, edition, pages
Umeå: Medicinsk biovetenskap , 2003. , 66 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 840
Keyword [en]
Biomedicine, telomere, telomerase, fluorescence in situ hybridization, flow cytometry, flow-FISH, replication timing, chronic lymphocytic leukemia, immunoglobulin gene, prognosis
Keyword [sv]
Biomedicin
National Category
Microbiology in the medical area
Research subject
Medical Cell Biology
Identifiers
URN: urn:nbn:se:umu:diva-76ISBN: 91-7305-444-5 (print)OAI: oai:DiVA.org:umu-76DiVA: diva2:144448
Public defence
2003-05-23, Betula, 6M, Umeå, 09:00
Available from: 2003-06-26 Created: 2003-06-26Bibliographically approved
List of papers
1. Telomere analysis by fluorescence in situ hybridization and flow cytometry
Open this publication in new window or tab >>Telomere analysis by fluorescence in situ hybridization and flow cytometry
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1998 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 26, no 16, 3651-3656 p.Article in journal (Refereed) Published
Abstract [en]

Determination of telomere length is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Fluorescence in situ hybridization (FISH) of telomere repeats has been used to calculate telomere length, a method called quantitative (Q)-FISH, We here present a quantitative flow cytometric approach, Q-FISHFCM, for evaluation of telomere length distribution in individual cells based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA)(3) probe and DMA staining with propidium iodide, A simple and rapid protocol with results within 30 h was developed giving high reproducibility, One important feature of the protocol was the use of an internal cell line control, giving an automatic compensation for potential differences in the hybridization steps. This protocol was tested successfully on cell lines and clinical samples from bone marrow, blood, lymph nodes and tonsils. A significant correlation was found between Southern blotting and Q-FISHFCM telomere length values (P = 0.002), The mean sub-telomeric DNA length of the tested cell lines and clinical samples was estimated to be 3.2 kbp, With the Q-FISHFCM method the fluorescence signal could be determined in different cell cycle phases, indicating that in human cells the vast majority of telomeric DNA is replicated early in S phase.

Place, publisher, year, edition, pages
Oxford: Oxford University Press, 1998
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-5076 (URN)10.1093/nar/26.16.3651 (DOI)000075408200006 ()
Available from: 2003-06-26 Created: 2003-06-26 Last updated: 2017-12-14Bibliographically approved
2. Replication timing of human telomeric DNA and other repetitive sequences analyzed by fluorescence in situ hybridization and flow cytometry
Open this publication in new window or tab >>Replication timing of human telomeric DNA and other repetitive sequences analyzed by fluorescence in situ hybridization and flow cytometry
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2001 (English)In: Experimental Cell Research, ISSN 0014-4827, Vol. 271, 223-229 p.Article in journal (Refereed) Published
Abstract [en]

The replication timing of telomeres seems to differ between species. Yeast telomeres are late replicating, whereas limited data from very few human cell lines have indicated telomere replication throughout S phase. In the present study a series of permanent cell lines and patient samples was investigated using a flow cytometric approach for telomere length determination based on in situ hybridization using peptide nucleic acid probes and DNA staining. This method permits selective analysis of cells in specific phases of the cell cycle without perturbation of the cell cycle machinery. The timing of replication of telomeric C(3)TA(2) and T(2)AG(3) repeats was found to differ between individual samples and could precede or be concomitant with the replication of bulk DNA. Replication of the T(2)AG(3) strand seemed to occur somewhat later than that of the C(3)TA(2) strand in some samples. (GTG)(n) and other repetitive sequences generally showed a replication pattern similar to that of the bulk of DNA with slightly individual differences, whereas centromeric DNA repeats consistently replicated within a short time frame in late S phase. The apparent variability in replication timing seen for telomeric DNA might suggest individual differences in firing of replication origins.

Place, publisher, year, edition, pages
Elsevier, 2001
Keyword
telomere, centromere, repetitive sequence, fluorescence in situ hybridization, flow cytometry, replication, timing
National Category
Cancer and Oncology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-5077 (URN)10.1006/excr.2001.5391 (DOI)000172749500003 ()11716534 (PubMedID)
Available from: 2003-06-26 Created: 2003-06-26 Last updated: 2014-04-14Bibliographically approved
3. Telomerase regulation and telomere dynamics in germinal centers
Open this publication in new window or tab >>Telomerase regulation and telomere dynamics in germinal centers
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2001 (English)In: European Journal of Haematology, ISSN 0902-4441, E-ISSN 1600-0609, Vol. 67, no 5-6, 309-317 p.Article in journal (Refereed) Published
Abstract [en]

Telomere length maintenance, usually executed by telomerase, is a prerequisite for an extended or infinite division potential. Nevertheless most telomerase positive normal cells exhibit telomere shortening. This study details the telomerase expression and telomere dynamics in purified tonsil B cell subsets during the germinal center (GC) reaction. Significant telomere lengthening was observed as naive B cells matured to centroblasts and when centroblasts matured further to centrocytes, resulting in an increase in telomere length of about 4 kbp determined by Southern blotting. Immunopurified cell populations were also studied by fluorescence in situ hybridization and flow cytometry (flow-FISH) confirming that the GC B cells exhibited lengthened telomeres. These data were further verified in unpurified tonsil cells by combining flow-FISH and immunophenotyping using selected surface markers. Centroblasts expressed high levels of telomerase activity, which was increased in centrocytes, whereas resting naive, activated naive and memory B cells were telomerase activity negative. Expression levels of the catalytic subunit (hTERT) RNA paralleled the telomerase activity levels. The unique telomere elongation in GC B cells permits extensive proliferation during the GC reaction and provides the memory cells with a substantial increase in division potential. Understanding the telomere biology of GC cells is important in defining requirements for telomere elongation in vivo, with implications for the normal immune system as well as for lymphomas, and could provide insights into how the division potential of cells can be manipulated in vitro.

Place, publisher, year, edition, pages
Blackwell Munksgaard, 2001
Keyword
germinal center, subpopulations, telomerase activity, catalytic subunit, telomere maintenance
National Category
Hematology
Identifiers
urn:nbn:se:umu:diva-5078 (URN)10.1034/j.1600-0609.2001.00588.x (DOI)000173874400005 ()
Available from: 2003-06-26 Created: 2003-06-26 Last updated: 2017-12-14Bibliographically approved
4. Association between telomere length and V-H gene mutation status in chronic lymphocytic leukaemia: clinical and biological implications
Open this publication in new window or tab >>Association between telomere length and V-H gene mutation status in chronic lymphocytic leukaemia: clinical and biological implications
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2003 (English)In: British Journal of Cancer, ISSN 0007-0920, E-ISSN 1532-1827, Vol. 88, no 4, 593-598 p.Article in journal (Refereed) Published
Abstract [en]

The immunoglobulin V-H gene mutation status can divide B-cell chronic lymphocytic leukaemia (CLL) into two entities with a different clinical course. Cases with unmutated V-H genes, considered to evolve from pregerminal centre (GC) cells, have a worse outcome compared to cases showing mutated VH genes, that is, post-GC derived. Also, telomere length has been reported to be of prognostic significance in CLL. Interestingly, telomerase becomes activated during the GC reaction and an elongation of the telomeres occurs in GC B cells. We performed telomere length and VH gene analysis in a series of 61 CLL cases, in order to investigate if the unique telomere lengthening shown in GC B cells could reflect the telomere status in the two subsets of mutated and unmutated CLL. A novel association was found between VH gene mutation status and telomere length, since significantly shorter telomeres were demonstrated in the unmutated group compared to the mutated group (mean length 4.3 vs 63 kbp). Shorter telomeres also constituted a subgroup with a worse prognosis than cases with longer telomeres (median survival 59 vs 159 months), Furthermore, the I-g gene sequence data revealed that samples with high mutations frequency (> 6%) had long telomeres (similar to 8 kbp). Thus, both the telomere and VH gene mutation status in CLL appear linked, which may reflect the proliferative history of the clonal cells with regard to the GC reaction. (C) 2003 Cancer Research UK.

Place, publisher, year, edition, pages
London: Nature Publishing Group, 2003
Keyword
telomere length, immunoglobulin gene, somatic hypermutation, chronic lymphocytic leukaemia, prognosis
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-5079 (URN)10.1038/sj.bjc.6600763 (DOI)000181535200017 ()
Available from: 2003-06-26 Created: 2003-06-26 Last updated: 2017-12-14Bibliographically approved
5. Telomere length and VH gene mutation status in chronic lymphocytic leukemia: Differences related to gender, age and sample site
Open this publication in new window or tab >>Telomere length and VH gene mutation status in chronic lymphocytic leukemia: Differences related to gender, age and sample site
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Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-5080 (URN)
Available from: 2003-06-26 Created: 2003-06-26 Last updated: 2010-01-13Bibliographically approved

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