Transcriptional networks and cellular senescence in human mammary fibroblasts.
Senescence, the molecular program that limits the finite proliferative potential of a cell, acts as an important barrier to protect the body from cancer. Techniques for measuring transcriptome changes and for modulating their expression suggest that it may be possible to dissect the transcriptional networks underlying complex cellular processes. HMF3A cells are conditionally immortalized human mammary fibroblasts that can be induced to undergo coordinated senescence. Here, we used these cells in conjunction with microarrays, RNA interference, and in silico promoter analysis to promote the dissection of the transcriptional networks responsible for regulating cellular senescence. We first identified changes in the transcriptome when HMF3A cells undergo senescence and then compared them with those observed upon replicative senescence in primary human mammary fibroblasts. In addition to DUSP1 and known p53 and E2F targets, a number of genes such as PHLDA1, NR4A3, and a novel splice variant of STAC were implicated in senescence. Their role in senescence was then analyzed by RNA silencing followed by microarray analysis. In silico promoter analysis of all differential genes predicted that nuclear factor-kappaB and C/EBP transcription factors are activated upon senescence, and we confirmed this by electrophoretic mobility shift assay. The results suggest a putative signaling network for cellular senescence.
Hardy
K
K
Ludwig Institute for Cancer Research, University College School of Medicine, London W1W 7BS, United Kingdom.
Mansfield
L
L
Mackay
A
A
Benvenuti
S
S
Ismail
S
S
Arora
P
P
O'Hare
M J
MJ
Jat
P S
PS
D018797:Cell Cycle Proteins / Q000235:genetics; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000235:genetics* / Q000502:physiology; D016895:Culture Media, Serum-Free; D004268:DNA-Binding Proteins / Q000235:genetics; D054638:Dual Specificity Phosphatase 1; D024202:Electrophoretic Mobility Shift Assay; D005260:Female; D005347:Fibroblasts / Q000378:metabolism*; D005786:Gene Expression Regulation; D020868:Gene Silencing; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics; D042361:Mammary Glands, Human / Q000166:cytology; D046228:Microarray Analysis; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics; D010749:Phosphoprotein Phosphatases / Q000235:genetics; D011401:Promoter Regions, Genetic; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases / Q000235:genetics; D034622:RNA Interference; D012326:RNA Splicing; D011987:Receptors, Steroid; D011988:Receptors, Thyroid Hormone; D012190:Retroviridae / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D018797:Cell Cycle Proteins; D016895:Culture Media, Serum-Free; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D016328:NF-kappa B; C490412:NR4A3 protein, human; D009419:Nerve Tissue Proteins; C491923:PHLDA1 protein, human; D011987:Receptors, Steroid; D011988:Receptors, Thyroid Hormone; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; C103302:STAC protein, human; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C517019:DUSP1 protein, human; D054638:Dual Specificity Phosphatase 1; D017027:Protein Tyrosine Phosphatases