Distinct functions for Ras GTPases in the control of proliferation and apoptosis in mouse and human mesangial cells.
In previous work, we have demonstrated that Ras GTPases regulate proliferation in a range of human renal cells. The present work compares human and mouse mesangial cell (HMC and MMC) responses to specific knockdown of Ras genes with antisense oligonucleotides (AS-oligos), and examines the role of the p21 (cip1) and p27 (kip1) cyclin-dependent kinase inhibitors in these responses in mouse cells. HMC and MMC were lipofectin transfected with ras-targeted AS-oligo at 200-400 nM for 18 h followed by growth of cells in 20% serum for 18-72 h. Cell proliferation was assessed with an MTS assay and bromodeoxyuridine (BrdU) uptake. Apoptosis was quantified using nuclear stain with Hoechst 33342 dye. In MMC, Ha-ras AS-oligo caused an increase in apoptosis from <2% to 10-15% of cells after 18 h in serum (P<0.01). Control, Ki-ras and N-ras AS-oligos had minimal effects on apoptosis. BrdU uptake studies showed that BrdU+ve MMC were increased by 20-40% (P<0.05) after Ha-ras AS-oligo at 24 h; other ras AS-oligos were inactive. HMC number was reduced by 40-80% (P<0.01) at 48-72 h by both Ha-ras and Ki-ras AS-oligos. These actions were associated with reductions in BrdU+ve cells. In HMC, the ras AS-oligos did not induce apoptosis. p21(-,-) MMC showed exaggerated apoptotic responses to Ha-Ras AS-oligo. In mouse cells, Ha-Ras expression appears necessary to prevent apoptotic cell death; Ras expression does not appear necessary for cells to progress through the cell cycle. In human cells, Ras does not appear necessary to prevent apoptosis but Ha-Ras and Ki-Ras appear to be required for cell cycle progression.
Hendry
B M
BM
Department of Renal Medicine, King's College London, London, UK. bruce.hendry@kcl.ac.uk
Khwaja
A
A
Qu
Q Y
QY
Shankland
S J
SJ
D000818:Animals; D017209:Apoptosis; D049109:Cell Proliferation; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000032:analysis; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000032:analysis; D004247:DNA / Q000096:biosynthesis; D005920:Glomerular Mesangium / Q000166:cytology*; D006801:Humans; D051379:Mice; D051057:Proto-Oncogene Proteins c-akt / Q000032:analysis / Q000502:physiology; D018631:ras Proteins / Q000502:physiology*
C493956:CDKN1A protein, human; C493957:Cdkn1a protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004247:DNA; D051057:Proto-Oncogene Proteins c-akt; D018631:ras Proteins