Sister Chromatid Exchange Formation in Mammalian Cells Is Modulated by Deoxyribonucleotide Pool Imbalance

1999 ◽  
Vol 25 (2) ◽  
pp. 101-108 ◽  
Author(s):  
Nicholas C. Popescu
Keyword(s):  
Sister Chromatid ◽  
Sister Chromatid Exchange ◽  
Mammalian Cells

Telomeric Recombination in Lymphocytes Implicates ALT, an Alternative Mechanism for Telomere Length Maintenance, in Normal Human Hematopoietic Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1332-1332
Author(s):  
Rodrigo T. Calado ◽  
Solomon A. Graf ◽  
Neal S. Young
Keyword(s):  
Telomere Length ◽  
Sister Chromatid ◽  
Hela Cells ◽  
Peripheral Blood ◽  
Sister Chromatid Exchange ◽  
Mammalian Cells ◽  
K562 Cells ◽  
Peripheral Blood Lymphocytes ◽  
Blood Lymphocytes ◽  
Telomeric Probe

Abstract Telomeres are the very ends of chromosomes and protect the genome from recombination, end-to-end-fusion, and recognition as damaged DNA. Telomeres are eroded with each cell division, eventually reaching such critically short length as to cause cell cycle arrest, apoptosis, or genomic instability. In most highly proliferative cells, including hematopoietic stem cells and T lymphocytes, telomere attrition is countered by telomere extension by telomerase reverse transcriptase complex. The majority of cancer cells also express telomerase, which maintains telomere length and allows indefinite cell proliferation. However, about 10% of tumors maintain telomere length in the absence of telomerase by mechanisms collectively termed alternative lengthening of telomeres (ALT). ALT mainly acts through asymmetrical exchange of telomeric material between chromosomes or sister chromatids, producing one daughter-cell with short telomeres and a limited life-span and its sister with long telomeres and higher proliferative capacity. To date, ALT has only been reported in cancer cells or through genetic engineering of mammalian cells. Here we investigated whether ALT mechanisms were active in hematopoietic cells using chromosome orientation fluorescent in situ hybridization (CO-FISH). In standard FISH, a telomeric probe produces fours signals per chromosome, one at each end of the two chromatids. Using CO-FISH, the newly synthesized DNA strand is fragmented by BrdU incorporation and UV light exposure and then digested by exonucleases. In CO-FISH, a telomeric probe produces two signals only, one at each end of the chromosome; in the presence of telomeric recombination, the telomeric signal is split, generating more than two signals per chromosome. Peripheral blood lymphocytes from three healthy volunteers, normal human fibroblasts, K562 cells, telomerase-positive HeLa cells (known to be negative for ALT),and telomerase-negative VA13 cells (known to be positive for ALT) were investigated for telomeric sister chromatid exchange (t-SCE); at least 20 metaphases per cell type were examined. Cultured peripheral blood lymphocytes and VA13 cells both showed increased levels of telomeric sister chromatid exchange in comparison to the other cells (P=0.0001): telomeric probe generated 2.62±0.11 telomeric signals/chromosome in lymphocytes; 2.23±0.04 in VA13 cells; 2.09±0.01 in HeLa cells; 2.02±0.01 in K562 cells; and 2.02±0.01 in human skin fibroblasts. Staining incorporated-BrdU over 24 hours and evaluation of “harlequin” chromosomes point to a similar rate of genomic sister chromatid exchange in lymphocytes, VA13 cells, and HeLa cells, suggesting that high chromatid exchange is confined to the telomeric region. A physical association between promyelocytic leukemia protein (PML) and telomeres is characteristic of some ALT-positive cells, but confocal microscopy failed to co-localize the telomeric probe and anti-PML monoclonal antibody in peripheral blood lymphocytes, suggesting that t-SCE in lymphocytes is not mediated by PML. This is the first demonstration of ALT activation in normal mammalian cells. ALT may be activated in peripheral blood lymphocytes as a complementary mechanism to maintain telomere length, and may explain the differences in age-related telomere shortening observed between lymphocytes and granulocytes.

scholarly journals Chromosome instability associated with human alphoid DNA transfected into the Chinese hamster genome.

1988 ◽  
Vol 8 (9) ◽  
pp. 3611-3618 ◽  
Author(s):  
M W Heartlein ◽  
J H Knoll ◽  
S A Latt
Keyword(s):  
Sister Chromatid ◽  
Dna Sequences ◽  
Cho Cells ◽  
Sister Chromatid Exchange ◽  
Mammalian Cells ◽  
Chromosome Structure ◽  
Chromosome Instability ◽  
Chinese Hamster ◽  
Alphoid Dna ◽  
Sister Chromatid Exchange Frequency

Repetitive DNA sequences have been implicated in the mediation of DNA rearrangement in mammalian cells. We have tested this hypothesis by using a dihydrofolate reductase (DHFR) expression vector into which candidate sequences were inserted. DHFR- Chinese hamster ovary (CHO) cells were transfected with this vector, the amplification of which was then selected for by methotrexate (MTX) exposure. Cells transfected with the vector alone (and resistant to 0.02 or 1.0 microM MTX) or with a poly(dG-dT) insert (and resistant to 0.05 or 1.0 microM MTX) showed little change in chromosome aberrations or sister chromatid exchange frequencies. In contrast, transfection of DHFR- CHO cells with a vector containing either of two distinct 0.34-kilobase human alphoid DNA segments (and selection to 0.05 to 10.0 microM MTX) showed an approximately 50% increase in chromosome number and marked changes in chromosome structure, including one or two dicentric or ring forms per cell. The sister chromatid exchange frequency also increased, to more than double the frequency of that in cells transfected without insert or those containing poly(dG-dT). In situ hybridization of one 0.34-kilobase insert in some cells suggested clustering of homologous sequences in structurally abnormal recipient CHO cell chromosomes. The approach described provides an introduction to a unique means for a coordinate molecular and cytological study of dynamic changes in chromosome structure.

Chromosome instability associated with human alphoid DNA transfected into the Chinese hamster genome

1988 ◽  
Vol 8 (9) ◽  
pp. 3611-3618
Author(s):  
M W Heartlein ◽  
J H Knoll ◽  
S A Latt
Keyword(s):  
Sister Chromatid ◽  
Dna Sequences ◽  
Cho Cells ◽  
Sister Chromatid Exchange ◽  
Mammalian Cells ◽  
Chromosome Structure ◽  
Chromosome Instability ◽  
Chinese Hamster ◽  
Alphoid Dna ◽  
Sister Chromatid Exchange Frequency

Repetitive DNA sequences have been implicated in the mediation of DNA rearrangement in mammalian cells. We have tested this hypothesis by using a dihydrofolate reductase (DHFR) expression vector into which candidate sequences were inserted. DHFR- Chinese hamster ovary (CHO) cells were transfected with this vector, the amplification of which was then selected for by methotrexate (MTX) exposure. Cells transfected with the vector alone (and resistant to 0.02 or 1.0 microM MTX) or with a poly(dG-dT) insert (and resistant to 0.05 or 1.0 microM MTX) showed little change in chromosome aberrations or sister chromatid exchange frequencies. In contrast, transfection of DHFR- CHO cells with a vector containing either of two distinct 0.34-kilobase human alphoid DNA segments (and selection to 0.05 to 10.0 microM MTX) showed an approximately 50% increase in chromosome number and marked changes in chromosome structure, including one or two dicentric or ring forms per cell. The sister chromatid exchange frequency also increased, to more than double the frequency of that in cells transfected without insert or those containing poly(dG-dT). In situ hybridization of one 0.34-kilobase insert in some cells suggested clustering of homologous sequences in structurally abnormal recipient CHO cell chromosomes. The approach described provides an introduction to a unique means for a coordinate molecular and cytological study of dynamic changes in chromosome structure.

Effect of lead chromate on chromosome aberration, sister-chromatid exchange and DNA damage in mammalian cells in vitro

1980 ◽  
Vol 77 (2) ◽  
pp. 157-163 ◽  
Author(s):  
George R. Douglas ◽  
R.D.L. Bell ◽  
C.E. Grant ◽  
J.M. Wytsma ◽  
K.C. Bora
Keyword(s):  
Dna Damage ◽  
Chromosome Aberration ◽  
Sister Chromatid ◽  
Sister Chromatid Exchange ◽  
Mammalian Cells ◽  
Lead Chromate
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