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Telomere analysis by fluorescence in situ hybridization and flow cytometry
Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
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1998 (Engelska)Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 26, nr 16, s. 3651-3656Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
Oxford: Oxford University Press, 1998. Vol. 26, nr 16, s. 3651-3656
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:umu:diva-5076DOI: 10.1093/nar/26.16.3651ISI: 000075408200006OAI: oai:DiVA.org:umu-5076DiVA, id: diva2:144443
Tillgänglig från: 2003-06-26 Skapad: 2003-06-26 Senast uppdaterad: 2018-06-09Bibliografiskt granskad
Ingår i avhandling
1. Telomere analysis of normal and neoplastic hematopoietic cells: studies focusing on fluorescence in situ hybridization and flow cytometry
Öppna denna publikation i ny flik eller fönster >>Telomere analysis of normal and neoplastic hematopoietic cells: studies focusing on fluorescence in situ hybridization and flow cytometry
2003 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Umeå: Medicinsk biovetenskap, 2003. s. 66
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 840
Nyckelord
Biomedicine, telomere, telomerase, fluorescence in situ hybridization, flow cytometry, flow-FISH, replication timing, chronic lymphocytic leukemia, immunoglobulin gene, prognosis, Biomedicin
Nationell ämneskategori
Mikrobiologi inom det medicinska området
Forskningsämne
medicinsk cellbiologi
Identifikatorer
urn:nbn:se:umu:diva-76 (URN)91-7305-444-5 (ISBN)
Disputation
2003-05-23, Betula, 6M, Umeå, 09:00
Tillgänglig från: 2003-06-26 Skapad: 2003-06-26 Senast uppdaterad: 2018-01-13Bibliografiskt granskad

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Hultdin, MagnusGrönlund, ElisabethNorrback, Karl-FredrikRoos, Göran

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