Towards a genomic-based diagnosis.
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Breast Neoplasms↗
Biomedical subjects
J Brugarolas
Publications and source records attributed to J Brugarolas.
Inhibition of cyclin-dependent kinase 2 by p21 is necessary for retinoblastoma protein-mediated G1 arrest after gamma-irradiation.
In mammalian cells, activation of certain checkpoint pathways as a result of exposure to genotoxic agents results in cell cycle arrest. The integrity of these arrest pathways is critical to the ability of the cell to repair mutations that otherwise might compromise viability or contribute to deregulation of cellular growth and proliferation. Here we examine the mechanism through which DNA damaging agents result in a G1 arrest that depends on the tumor suppressor p53 and its transcriptional target p21. By using primary cell lines lacking specific cell cycle regulators, we demonstrate that this pathway functions through the growth suppressive properties of the retinoblastoma protein (pRB) tumor suppressor. Specifically, gamma-irradiation inhibits the phosphorylation of pRB at cyclin-dependent kinase 2-specific, but not cyclin-dependent kinase 4-specific, sites in a p21-dependent manner. Most importantly, we show that pRB is a critical component of this DNA damage checkpoint. These data indicate that the p53 --> p21 checkpoint pathway uses the normal cell cycle regulatory machinery to induce the accumulation of the growth suppressive form of pRB and suggest that loss of pRB during the course of tumorigenesis disrupts the function of an important DNA damage checkpoint.
Animals↗
The cyclin kinase inhibitor p21CIP1/WAF1 limits glomerular epithelial cell proliferation in experimental glomerulonephritis.
BACKGROUND: During glomerulogenesis, visceral glomerular epithelial cells (VECs) exit the cell cycle and become terminally differentiated and quiescent. In contrast to other resident glomerular cells, VECs undergo little if any proliferation in response to injury. However, the mechanisms for this remain unclear. Cell proliferation is controlled by cell-cycle regulatory proteins where the cyclin-dependent kinase inhibitor p21Cip1,WAF1 (p21) inhibits cell proliferation and is required for differentiation of many nonrenal cell types. METHODS: To test the hypothesis that p21 is required to maintain a differentiated and quiescent VEC phenotype, experimental glomerulonephritis was induced in p21 knockout (-/-) and p21 wild-type (+/+) mice with antiglomerular antibody. DNA synthesis (proliferating cell nuclear antigen, bromodeoxyuridine staining), VEC proliferation (multilayers of cells in Bowman's space), matrix accumulation (periodic acid-Schiff, silver staining), apoptosis (TUNEL), and renal function (serum urea nitrogen) were studied on days 5 and 14 (N = 6 per time point). VECs were identified by location, morphology, ezrin staining, and electron microscopy. VEC differentiation was measured by staining for Wilms' tumor-1 gene. RESULTS: Kidneys from unmanipulated p21-/- mice were histologically normal and did not have increased DNA synthesis, suggesting that p21 was not required for the induction of VEC terminal differentiation. Proliferating cell nuclear antigen and bromodeoxyuridine staining was increased 4.3- and 3.3-fold, respectively, in p21-/- mice with glomerulonephritis (P < 0.0001 vs. p21+/+ mice). At each time point, VEC proliferation was also increased in nephritic p21-/- mice (P < 0.0001 vs. p21+/+ mice). VEC re-entry into the cell cycle was associated with the loss of Wilms' tumor-1 gene staining. Nephritic p21-/- mice had increased extracellular matrix protein accumulation and apoptosis and decreased renal function (serum urea nitrogen) compared with p21+/+ mice (P < 0.001). CONCLUSION: These results show that the cyclin kinase inhibitor p21 is not required by VECs to attain a terminally differentiated VEC phenotype. However, the loss of p21, in disease states, is associated with VEC re-entry into the cell cycle and the development of a dedifferentiated proliferative phenotype.
Animals↗
The cyclin kinase inhibitor p21WAF1/CIP1 is required for glomerular hypertrophy in experimental diabetic nephropathy.
BACKGROUND: Diabetic nephropathy is characterized by glomerular hypertrophy. We have recently shown that experimental diabetes mellitus is associated with an increase in glomerular expression of the cyclin kinase inhibitor p21WAF1/CIP1 (p21). Furthermore, in vitro glucose-induced mesangial cell hypertrophy is also associated with an up-regulated expression of p21. In this study, we tested the hypothesis that p21 mediates diabetic glomerular hypertrophy in vivo. METHODS: Experimental diabetes mellitus was induced by streptozotocin in mice in which p21 was genetically deleted (p21 -/-) and in wild-type mice (p21 +/+). Kidney biopsies were obtained from diabetic and control (citrate injected) p21 +/+ and p21 -/- mice at day 60. The tissue was used for morphologic studies of glomerular size (measured by computer image-analysis system), glomerular cellularity (cell count), glomerular matrix expansion (silver stain), apoptosis (TUNEL), and expression of transforming growth factor-beta1 (TGF-beta1) by in situ hybridization. RESULTS: The glomerular tuft area increased 11.21% in diabetic p21 +/+ mice at day 60 compared with control (3329.98 +/- 244.05 micrometer(2) vs. 2994. 39 +/- 176.22 micrometer(2), P = 0.03), and the glomerular cell count did not change in diabetic p21 +/+ mice at day 60 compared with the control. These findings are consistent with glomerular hypertrophy. In contrast, the glomerular tuft area did not increase in diabetic p21 -/- mice at day 60 compared with the control (3544.15 +/- 826.49 vs. 3449.15 +/- 109.65, P = 0.82), nor was there an increase in glomerular cell count (41.41 +/- 13.18 vs. 46.95 +/- 3.00, P = 0.43). Diabetic p21 +/+ mice, but not p21 -/- mice, developed an increase in proteinuria at day 60 compared with the control. Tubular cell proliferation, measured by proliferating cell nuclear antigen immunostaining, was increased in both diabetic p21 +/+ (2.1-fold) and p21 -/- (7.61-fold) mice compared with controls. Glomerular cell apoptosis did not increase in diabetic mice. Although glomerular TGF-beta1 mRNA levels increased in both strains of diabetic mice at day 60, the glomerular matrix did not expand. CONCLUSIONS: Hyperglycemia was associated with glomerular hypertrophy in p21 +/+ mice. Despite the increase in TGF-beta1 mRNA, diabetic p21 -/- mice did not develop glomerular hypertrophy, providing evidence that the cyclin kinase inhibitor p21 may be required for diabetic glomerular hypertrophy induced by TGF-beta1. The loss of p21 increases tubular but not glomerular cell proliferation in diabetic nephropathy. The absence of glomerular hypertrophy appears protective of renal function in diabetic mice.
Animals↗
p21 is a critical CDK2 regulator essential for proliferation control in Rb-deficient cells.
Proliferation in mammalian cells is controlled primarily in the G1-phase of the cell cycle through the action of the G1 cyclin-dependent kinases, CDK4 and CDK2. To explore the mechanism of cellular response to extrinsic factors, specific loss of function mutations were generated in two negative regulators of G1 progression, p21 and pRB. Individually, these mutations were shown to have significant effects in G1 regulation, and when combined, Rb and p21 mutations caused more profound defects in G1. Moreover, cells deficient for pRB and p21 were uniquely capable of anchorage-independent growth. In contrast, combined absence of pRB and p21 function was not sufficient to overcome contact inhibition of growth nor for tumor formation in nude mice. Finally, animals with the genotype Rb+/-;p21(-/-) succumbed to tumors more rapidly than Rb+/- mice, suggesting that in certain contexts mutations in these two cell cycle regulators can cooperate in tumor development.
Animals↗
Involvement of p53 and p21 in cellular defects and tumorigenesis in Atm-/- mice.
Disruption of the mouse Atm gene, whose human counterpart is consistently mutated in ataxia-telangiectasia (A-T) patients, creates an A-T mouse model exhibiting most of the A-T-related systematic and cellular defects. While ATM plays a major role in signaling the p53 response to DNA strand break damage, Atm-/- p53(-/-) mice develop lymphomas earlier than Atm-/- or p53(-/-) mice, indicating that mutations in these two genes lead to synergy in tumorigenesis. The cell cycle G1/S checkpoint is abolished in Atm-/- p53(-/-) mouse embryonic fibroblasts (MEFs) following gamma-irradiation, suggesting that the partial G1 cell cycle arrest in Atm-/- cells following gamma-irradiation is due to the residual p53 response in these cells. In addition, the Atm-/- p21(-/-) MEFs are more severely defective in their cell cycle G1 arrest following gamma-irradiation than Atm-/- and p21(-/-) MEFs. The Atm-/- MEFs exhibit multiple cellular proliferative defects in culture, and an increased constitutive level of p21 in these cells might account for these cellular proliferation defects. Consistent with this notion, Atm-/- p21(-/-) MEFs proliferate similarly to wild-type MEFs and exhibit no premature senescence. These cellular proliferative defects are also rescued in Atm-/- p53(-/-) MEFs and little p21 can be detected in these cells, indicating that the abnormal p21 protein level in Atm-/- cells is also p53 dependent and leads to the cellular proliferative defects in these cells. However, the p21 mRNA level in Atm-/- MEFs is lower than that in Atm+/+ MEFs, suggesting that the higher level of constitutive p21 protein in Atm-/- MEFs is likely due to increased stability of the p21 protein.
Animals↗
Deletion of p21 cannot substitute for p53 loss in rescue of mdm2 null lethality.
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Animals↗
Double indemnity: p53, BRCA and cancer. p53 mutation partially rescues developmental arrest in Brca1 and Brca2 null mice, suggesting a role for familial breast cancer genes in DNA damage repair.
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Animals↗
Transcriptional activation by p53, but not induction of the p21 gene, is essential for oncogene-mediated apoptosis.
The p53 tumor suppressor limits cellular proliferation by inducing either G1 arrest or apoptosis, depending on the cellular context. To determine if these pathways are mechanistically distinct, we have examined the effects of different p53 mutants in p53 null primary mouse embryo fibroblasts. We chose this system as it is highly physiological and ensures that the interpretation of the results will not be confounded by the presence of endogenous p53 or oncoproteins which target p53. Using single cell microinjection assays for both G1 arrest and apoptosis, with loss-of-function and chimeric gain-of-function mutants, we have demonstrated that transcriptional activation is critical for both processes. Replacement of the p53 activation domain with that of VP16, or replacement of the p53 oligomerization domain with that of GCN4, reconstituted both G1 arrest and apoptosis activities. However, despite the importance of transcriptional activation in both processes, the target gene requirements are different. The p21 cyclin-dependent kinase inhibitor, which has been shown to be a direct target of p53 and a component of the radiation-induced G1 arrest response, is dispensable for oncogene-induced apoptosis, suggesting that these two p53-dependent transcriptional pathways are distinct.
Adenovirus E1A Proteins↗
Radiation-induced cell cycle arrest compromised by p21 deficiency.
The protein p21 is a dual inhibitor of cyclin-dependent kinases and proliferating-cell nuclear antigen (PCNA), both of which are required for passage through the cell cycle. The p21 gene is under the transcriptional control of p53 (ref. 5), suggesting that p21 might promote p53-dependent cell cycle arrest or apoptosis. p21 has also been implicated in cell senescence and in cell-cycle withdrawal upon terminal differentiation. Here we investigate the role of p21 in these processes using chimaeric mice composed partly of p21-/- and partly of p21+/+ cells. Immunohistochemical studies of the p21+/+ and p21-/- components of adult small intestine indicated that deletion of p21 has no detectable effect on the migration-associated differentiation of the four principal intestinal epithelial cell lineages or on p53-dependent apoptosis following irradiation. However, p21-/- mouse embryo fibroblasts are impaired in their ability to undergo G1 arrest following DNA damage.
Animals↗