Nicotinamide extends the replicative life span of primary human cells

2006 ◽  
Vol 127 (6) ◽  
pp. 511-514 ◽  
Author(s):  
Chang-Su Lim ◽  
Malcolm Potts ◽  
Richard F. Helm
Keyword(s):  
Life Span ◽  
Human Cells ◽  
Replicative Life Span ◽  
Primary Human Cells

scholarly journals Control of Replicative Life Span in Human Cells: Barriers to Clonal Expansion Intermediate Between M1 Senescence and M2 Crisis

1999 ◽  
Vol 19 (4) ◽  
pp. 3103-3114 ◽  
Author(s):  
J. A. Bond ◽  
M. F. Haughton ◽  
J. M. Rowson ◽  
P. J. Smith ◽  
V. Gire ◽  
...  
Keyword(s):  
Life Span ◽  
Clonal Evolution ◽  
Human Fibroblasts ◽  
Fold Increase ◽  
Clonal Expansion ◽  
Suppressor Gene ◽  
Human Cells ◽  
Cell Type ◽  
Replicative Life Span ◽  
Population Doublings

ABSTRACT The accumulation of genetic abnormalities in a developing tumor is driven, at least in part, by the need to overcome inherent restraints on the replicative life span of human cells, two of which—senescence (M1) and crisis (M2)—have been well characterized. Here we describe additional barriers to clonal expansion (Mint) intermediate between M1 and M2, revealed by abrogation of tumor-suppressor gene (TSG) pathways by individual human papillomavirus type 16 (HPV16) proteins. In human fibroblasts, abrogation of p53 function by HPVE6 allowed escape from M1, followed up to 20 population doublings (PD) later by a second viable proliferation arrest state, MintE6, closely resembling M1. This occurred despite abrogation of p21WAF1 induction but was associated with and potentially mediated by a further ∼3-fold increase in p16INK4a expression compared to its level at M1. Expression of HPVE7, which targets pRb (and p21WAF1), also permitted clonal expansion, but this was limited predominantly by increasing cell death, resulting in a MintE7 phenotype similar to M2 but occurring after fewer PD. This was associated with, and at least partly due to, an increase in nuclear p53 content and activity, not seen in younger cells expressing E7. In a different cell type, thyroid epithelium, E7 also allowed clonal expansion terminating in a similar state to MintE7 in fibroblasts. In contrast, however, there was no evidence for a p53-regulated pathway; E6 was without effect, and the increases in p21WAF1 expression at M1 and MintE7 were p53 independent. These data provide a model for clonal evolution by successive TSG inactivation and suggest that cell type diversity in life span regulation may determine the pattern of gene mutation in the corresponding tumors.

Telomerase Alone Extends the Replicative Life Span of Human Skeletal Muscle Cells Without Compromising Genomic Stability

2003 ◽  
Vol 14 (15) ◽  
pp. 1473-1487 ◽  
Author(s):  
Martha Wootton ◽  
Karen Steeghs ◽  
Diana Watt ◽  
June Munro ◽  
Katrina Gordon ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Life Span ◽  
Human Skeletal Muscle ◽  
Genomic Stability ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Replicative Life Span ◽  
Human Skeletal Muscle Cells

scholarly journals Loss of Ubp3 increases silencing, decreases unequal recombination in rDNA, and shortens the replicative life span in Saccharomyces cerevisiae

2014 ◽  
Vol 25 (12) ◽  
pp. 1916-1924 ◽  
Author(s):  
David Öling ◽  
Rehan Masoom ◽  
Kristian Kvint
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Life Span ◽  
Rna Polymerase Ii ◽  
Ribosomal Dna ◽  
Genetic Evidence ◽  
Wild Type ◽  
Replicative Life Span ◽  
Unequal Recombination

Ubp3 is a conserved ubiquitin protease that acts as an antisilencing factor in MAT and telomeric regions. Here we show that ubp3∆ mutants also display increased silencing in ribosomal DNA (rDNA). Consistent with this, RNA polymerase II occupancy is lower in cells lacking Ubp3 than in wild-type cells in all heterochromatic regions. Moreover, in a ubp3∆ mutant, unequal recombination in rDNA is highly suppressed. We present genetic evidence that this effect on rDNA recombination, but not silencing, is entirely dependent on the silencing factor Sir2. Further, ubp3∆ sir2∆ mutants age prematurely at the same rate as sir2∆ mutants. Thus our data suggest that recombination negatively influences replicative life span more so than silencing. However, in ubp3∆ mutants, recombination is not a prerequisite for aging, since cells lacking Ubp3 have a shorter life span than isogenic wild-type cells. We discuss the data in view of different models on how silencing and unequal recombination affect replicative life span and the role of Ubp3 in these processes.

scholarly journals SIGIRR/TIR-8 is an inhibitor of toll-like receptor signaling in primary human cells and regulates inflammation in models of rheumatoid arthritis

2010 ◽  
Vol 62 (8) ◽  
pp. 2249-2261 ◽  
Author(s):  
Stefan K. Drexler ◽  
Philip Kong ◽  
Julia Inglis ◽  
Richard O. Williams ◽  
Cecilia Garlanda ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Human Cells ◽  
Receptor Signaling ◽  
Toll Like Receptor ◽  
Primary Human Cells

Multiscale porous ceramic scaffolds forin vitroculturing of primary human cells

2012 ◽  
Vol 111 (5-6) ◽  
pp. 262-268 ◽  
Author(s):  
A Finoli ◽  
N Ostrowski ◽  
E Schmelzer ◽  
I Nettleship ◽  
J Gerlach
Keyword(s):  
Porous Ceramic ◽  
Human Cells ◽  
Primary Human Cells

scholarly journals Complex CSF3R Protein Isoform Interactions Dictate Cytokine Responsiveness

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 35-35
Author(s):  
Sara L. Seegers ◽  
Amanda Lance ◽  
Lawrence J Druhan ◽  
Belinda R Avalos
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Splice Variant ◽  
Splice Variants ◽  
Polyclonal Antibodies ◽  
Human Cells ◽  
Dependent Manner ◽  
Epitope Tag ◽  
Proliferative Signaling ◽  
Primary Human Cells

CSF3R, the receptors for granulocyte colony stimulating factor, is a critical regulator of neutrophil production. Multiple CSF3R mRNA transcripts have been identified and are annotated in Genbank. The expression and function of the different CSF3R proteins have not been fully elucidated. We generated antibodies specific for two of the identified and annotated isoforms, V3 and V4. CSF3R-V4 is a truncated variant of V1 with a unique C-terminal 34 amino acids and this variant confers enhanced growth signals. Changes in the ratio of V1:V4 isoforms have been implicated in chemotherapy resistance and relapse of AML. CSF3R-V3 is a variant of V1 with a 27 amino acid insertion between two conserved domains in the cytoplasmic portion of the receptor involved in JAK/STAT activation, termed the box 1 and box 2. CSF3R-V3 produces reduced proliferative signaling in response to G-CSF. When V3 is co-expressed with V1, proliferative signaling is reduced in a concentration dependent manner. In order to generate custom rabbit polyclonal antibodies specific for CSF3R-V3 and CSF3R-V4 we used either a peptide that corresponds to a unique amino acid sequence present only in CSF3R-V3 or a peptide specific for a portion of the C-terminal amino acid sequence unique to the CSF3R-V4 isoform conjugated to an immunogenic carrier protein. These immunogens both produced robust immune responses, and the polyclonal antibodies were subsequently purified from bulk sera. Immunoblot analysis of lysates from Ba/F3 cells expressing CSF3R-V1 (V1), CSF3R-V3 (V3), or CSF3R-V4 (V4) demonstrated that both the custom generated anti-CSF3R-V3 and anti-CSF3R-V4 antibodies were very specific, recognizing only the appropriate CSF3R receptor isoform. All three CSF3R splice variants are recognized by commercially available anti-CSF3R (clone LMM741 to CD114), while the anti-CSF3R-V4 custom antibody and the custom anti-CSF3R-V3 antibody recognizes only the CSF3R-V4 and CSF3R-V3 isoforms, respectively. We next sought to detect the CSF3R receptor isoforms in primary human cells. Using our custom antibodies, we detected for the first time, both the CSF3R-V3 and CSF3R-V4 receptor forms in primary neutrophils isolated from healthy donors. Each of the CSF3R isoforms produce unique signaling, and we hypothesized that the observed differences in G-CSF-dependent signaling is produced by the expression level of each receptor isoform via both homodimerization and by heterodimerization of the receptor splice variant proteins. To investigate the potential for heterodimerization of the CSF3R-V1 with the V3 and V4 isoforms, we generated a CSF3R-V1 with a c-terminal epitope tag and co-expressed this construct with both CSF3R-V3 or CSF3R-V4. Immunoprecipitation with an antibody to the epitope tag (recognizing the V1 variant) followed by immunoblotting with the custom anti-V3 or anti-V4 antibodies demonstrated that both CSF3R-V3 and CSF3R-V4 co-immunoprecipitated with CSF3R-V1, in agreement with our hypothesis that the splice variants form receptor heterodimers. Of note, the CSF3R receptor heterodimers are detected even in the absence of G-CSF, thus demonstrating that CSF3R exist as a preformed receptor dimer in an inactive state. In conclusion, we have generated antibodies that specifically detect the CSF3R-V3 and the CSF3R-V4 receptor proteins. These are the first studies to demonstrate the expression of the CSF3R splice variants at the protein level, in both cell lines and primary human cells. In addition, these are the first studies to demonstrate the formation of heterodimers of the CSF3R splice variants, providing a mechanism for the observed alteration in ligand-dependent signaling produced under conditions of altered splice variant expression. Disclosures Avalos: Juno: Membership on an entity's Board of Directors or advisory committees; Best Practice-Br Med J: Patents & Royalties: receives royalties from a coauthored article on evaluation of neutropenia.

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