TY - JOUR
T1 - Differential gene expression between young and senescent, quiescent WI-38 cells
AU - Doggett, David L.
AU - Rotenberg, Mitch O.
AU - Pignolo, Robert J.
AU - Phillips, Paul D.
AU - Cristofalo, Vincent J.
N1 - Funding Information:
Technical assistance in the propagation and maintenance of cell cultures was provided by Jennifer McIlhenney and Sarah O'Brien. This work was supported by National Institutes of Health Grants AG00378 and AG09778.
PY - 1992/9
Y1 - 1992/9
N2 - To investigate age-related changes in gene expression in WI-38 cells, we isolated RNA from young and senescent, quiescent cultures and made subtracted cDNA libraries. Density-arrested cells were incubated in serum-free MCDB-104 for 3 days. RNA was then isolated and subtracted cDNA libraries were made in the phagemid vector pCDM8. Both by picking clones at random from these subtracted libraries and by differential hybridization screening with subtracted cDNA probes from young and senescent cells, we have identified a total of 11 genes for which RNA is expressed differentially in these quiescent young and senescent WI-38 cultures. Two genes, EPC-1 and EPC-A2, with elevated RNA levels in young cells, have sequences which have not previously been identified. Two of the genes with elevated RNA expression in the senescent cells are the mitochodnria-coded genes for NADH dehydrogenase subunit 4 and for cytochrome b. We also identified seven other genes with elevated RNA levels in senescent cells. Three of these, LPC-1, LPC-14 and LPC-24, have been partially sequenced and have not previously been identified. These studies show that density-arrested, serum-deprived, quiescent young and senescent cells express a number of genes differentially. These differences are not growth-dependent, but are age-dependent. Our studies also show that the methods employed here, which include careful regulation of the cell cultures and subtraction of the libraries, result in libraries from which differentially expressed genes can be identified, either by random selection or by differential hybridization screening with subtracted probes.
AB - To investigate age-related changes in gene expression in WI-38 cells, we isolated RNA from young and senescent, quiescent cultures and made subtracted cDNA libraries. Density-arrested cells were incubated in serum-free MCDB-104 for 3 days. RNA was then isolated and subtracted cDNA libraries were made in the phagemid vector pCDM8. Both by picking clones at random from these subtracted libraries and by differential hybridization screening with subtracted cDNA probes from young and senescent cells, we have identified a total of 11 genes for which RNA is expressed differentially in these quiescent young and senescent WI-38 cultures. Two genes, EPC-1 and EPC-A2, with elevated RNA levels in young cells, have sequences which have not previously been identified. Two of the genes with elevated RNA expression in the senescent cells are the mitochodnria-coded genes for NADH dehydrogenase subunit 4 and for cytochrome b. We also identified seven other genes with elevated RNA levels in senescent cells. Three of these, LPC-1, LPC-14 and LPC-24, have been partially sequenced and have not previously been identified. These studies show that density-arrested, serum-deprived, quiescent young and senescent cells express a number of genes differentially. These differences are not growth-dependent, but are age-dependent. Our studies also show that the methods employed here, which include careful regulation of the cell cultures and subtraction of the libraries, result in libraries from which differentially expressed genes can be identified, either by random selection or by differential hybridization screening with subtracted probes.
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U2 - 10.1016/0047-6374(92)90039-G
DO - 10.1016/0047-6374(92)90039-G
M3 - Article
C2 - 1279330
AN - SCOPUS:0026728343
SN - 0047-6374
VL - 65
SP - 239
EP - 255
JO - Mechanisms of Ageing and Development
JF - Mechanisms of Ageing and Development
IS - 2-3
ER -