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Lawrence A Donehower

Publications and source records attributed to Lawrence A Donehower.

32 records · Page 2Linked to original sources
PubMed2004

Insights into aging obtained from p53 mutant mouse models.

Cancer suppression is an integral component of longevity in organisms with renewable tissues. A number of genes in the mammalian genome function in cancer prevention, and some of these have been directly implicated in longevity assurance. One such longevity assurance gene is the tumor suppressor p53, a transcription factor that is mutated or dysregulated in most human cancers. Early studies have linked p53 to the induction of cellular senescence, whereas recent reports implicate it as a potential regulator of organismal aging. We have shown by gene inactivation studies that loss of p53 function enhances tumor susceptibility and reduces longevity in the mouse. A recent serendipitously generated p53 mutant allele resulted in a hypermorphic version of p53 that displays increased cancer resistance, yet also mediates decreased longevity. The reduced longevity is accompanied by the accelerated onset of a variety of aging phenotypes. These include a 20% decrease in median life span, early osteoporosis, lordokyphosis, organ atrophy, delayed wound healing, and a reduced regenerative response after various stresses. Since the initial characterization of these mutant mice, we have attempted to elucidate the underlying molecular and cellular mechanisms that could be influencing the early aging phenotypes. Molecular studies of the p53 mutant allele product indicate that it induces an increase in p53 activity in both in vitro and in vivo contexts. The age-associated loss of organ cellularity and reduced tissue regenerative responses in the mutant mice are consistent with an accelerated loss of stem cell functional capacity. Our model is that enhanced growth inhibitory activity of p53 produces an earlier loss of the ability of stem cells to produce adequate numbers of progenitor and mature differentiated cells in each organ. Currently, we are performing stem cell functional assays from p53 mutant and wild-type mice to test this model. One challenge for the future will be to find ways to manipulate p53 function to provide increased cancer resistance, yet still enhance overall organismal longevity.

Aging↗
PubMed2003

Cooperativity of p19ARF, Mdm2, and p53 in murine tumorigenesis.

The p19ARF gene product responds to oncogenic stresses by interfering with the inhibitory effects of Mdm2 on p53, thus enhancing p53 activity and its antiproliferative functions. The absence of p19ARF in the mouse leads to early tumor susceptibility, presumably in part due to decreased p53 activity. To examine the tumorigenic cooperativity of p19ARF, Mdm2, and p53 in vivo, p19ARF-deficient mice were crossed first to p53-deficient mice and then to Mdm2 transgenic mice. The progeny were monitored for tumors. Cooperativity between p19ARF and p53 deficiencies in accelerating tumor formation was observed for most genotypes except p53-/- p19ARF-/- mice. p53-/- p19ARF-/- mice had a tumor incidence similar to p53-/- mice. In this context, tumor suppression by ARF appears to be primarily p53 dependent. The majority of the p19ARF+/- tumors deleted the wildtype p19ARF allele, in agreement with the previous studies, suggesting that p19ARF is a classic 'two hit' tumor suppressor. In a p53+/- background, however, all p19ARF+/- tumors retained a wildtype ARF allele and most also retained wildtype p53. In the second cross between p19ARF-deficient and Mdm2 transgenic mice, cooperativity in tumor incidence between Mdm2 overexpression and ARF deficiency was observed, consistent with the role of p19ARF in negatively regulating Mdm2 activity. These experiments further demonstrate in vivo the inter-relationships of the p19ARF-Mdm2-p53 signaling axis in tumor suppression.

Alleles↗
PubMed2003

High susceptibility of nullizygous p53 knockout mice to colorectal tumor induction by 1,2-dimethylhydrazine.

PURPOSE: The susceptibility of male p53 nullizygote (-/-), heterozygote (+/-), and wild-type (+/+) mice to 1,2-dimethylhydrazine (DMH) induction of colon carcinogenesis was investigated. METHODS: In a preliminary short-term experiment, male mice of three genotypes were given s.c. of 20 mg/kg DMH once weekly for 5 weeks. In a medium-term experiment, mice were given weekly s.c. of DMH for 15 weeks. In a long-term experiment, male p53 (+/-) and (+/+) mice were given weekly injections of DMH for 15 weeks, and killed at week 30. RESULTS: In the medium-term experiment, carcinomas were observed in 70% of p53 (-/-) mice, although there were no carcinomas in p53 (+/+) and (+/-) mice. In the long-term experiment, there was no significant difference in incidences of adenomas and carcinomas between p53 (+/+) and (+/-) mice. PCR-single strand conformation polymorphism analysis of exons 5-8 of p53 gene revealed four mutations in one focal atypia, one adenoma, and two carcinomas, out of 56 colonic proliferative lesions in the medium- and long-term experiments. CONCLUSIONS: These results suggest that p53 might not be a direct target of DMH but complete loss of p53 might elevate susceptibility to DMH-induced colorectal carcinogenesis.

1,2-Dimethylhydrazine↗
PubMed2003

Activities of wildtype and mutant p53 in suppression of homologous recombination as measured by a retroviral vector system.

DNA repair of double strand breaks, interstrand DNA cross-links, and other types of DNA damage utilizes the processes of homologous recombination and non-homologous end joining to repair the damage. Aberrant homologous recombination is likely to be responsible for a significant fraction of chromosomal deletions, duplications, and translocations that are observed in cancer cells. To facilitate measurement of homologous recombination frequencies in normal cells, mutant cells, and cancer cells, we have developed a high titer retroviral vector containing tandem repeats of mutant versions of a GFP-Zeocin resistance fusion gene and an intact neomycin resistance marker. Recombination between the tandem repeats regenerates a functional GFP-Zeo(R) marker that can be easily scored. This retroviral vector was used to assess homologous recombination frequencies in human cancer cells and rodent fibroblasts with differing dosages of wild type or mutant p53. Absence of wild type p53 stimulated spontaneous and ionizing radiation-induced homologous recombination, confirming previous studies. Moreover, p53(+/-) mouse fibroblasts show elevated levels of homologous recombination compared to their p53(+/+) counterparts following retroviral vector infection, indicating that p53 is haploinsufficient for suppression of homologous recombination. Transfection of vector-containing p53 null Saos-2 cells with various human cancer-associated p53 mutants revealed that these altered p53 proteins retain some recombination suppression function despite being totally inactive for transcriptional transactivation. The retroviral vector utilized in these studies may be useful in performing recombination assays on a wide array of cell types, including those not readily transfected by normal vectors.

DNA Repair↗
PubMed2003

Development of an HIV-based cDNA expression cloning system.

Expression cloning of cDNAs is a powerful tool with which to identify genes based on their specific functional properties. Here we describe the development of a cDNA library transfer system based on the human immunodeficiency virus type-1 (HIV). This system represents an improvement over current oncoretroviral cDNA expression systems in terms of target cell range and the inclusion of a selectable marker. By use of a simple packaging system, we were able to produce high-titer vector stocks from HIV vector-based cDNA libraries and demonstrate highly efficient cDNA expression cloning in three model experiments. First, HOS TK(-) cells, which are null for thymidine kinase (TK) expression, were transduced with an HIV-based cDNA library derived from primary human foreskin fibroblasts (HFFs) and functionally selected for TK expression. In a second experiment, hypoxanthine guanine phosphoribosyltransferase-1-deficient (HPRT(-)) fibroblasts were transduced with a T cell (PM1) line-derived cDNA library and selected for HPRT expression. Both TK (frequency 1 in 5.0 x 10(4)) and HPRT (frequency 1 in 2.0 x 10(4)) cDNAs were readily isolated from these HIV-based cDNA libraries. As a third example, we demonstrated the ability of this vector system to allow functional cDNA library screens to be performed in primary, mitotically inactive cell types. Using senescent HFFs as a target cell population, we were able to isolate SV40 large T antigen cDNA-containing clones (frequency 1 in 2.5 x 10(4)) based on their ability to overcome the senescence-induced block to cell proliferation. Thus, this system can be used to clone relatively low-abundance cDNAs based upon their expression. Because of the ability of HIV-based vectors to transduce primary and nondividing cells efficiently, this vector system will further broaden the range of cell types in which expression cloning studies can be performed.

Base Sequence↗
PubMed2003

Cyclin D1 repression of peroxisome proliferator-activated receptor gamma expression and transactivation.

The cyclin D1 gene is overexpressed in human breast cancers and is required for oncogene-induced tumorigenesis. Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor selectively activated by ligands of the thiazolidinedione class. PPAR gamma induces hepatic steatosis, and liganded PPAR gamma promotes adipocyte differentiation. Herein, cyclin D1 inhibited ligand-induced PPAR gamma function, transactivation, expression, and promoter activity. PPAR gamma transactivation induced by the ligand BRL49653 was inhibited by cyclin D1 through a pRB- and cdk-independent mechanism, requiring a region predicted to form an helix-loop-helix (HLH) structure. The cyclin D1 HLH region was also required for repression of the PPAR gamma ligand-binding domain linked to a heterologous DNA binding domain. Adipocyte differentiation by PPAR gamma-specific ligands (BRL49653, troglitazone) was enhanced in cyclin D1(-/-) fibroblasts and reversed by retroviral expression of cyclin D1. Homozygous deletion of the cyclin D1 gene, enhanced expression by PPAR gamma ligands of PPAR gamma and PPAR gamma-responsive genes, and cyclin D1(-/-) mice exhibit hepatic steatosis. Finally, reduction of cyclin D1 abundance in vivo using ponasterone-inducible cyclin D1 antisense transgenic mice, increased expression of PPAR gamma in vivo. The inhibition of PPAR gamma function by cyclin D1 is a new mechanism of signal transduction cross talk between PPAR gamma ligands and mitogenic signals that induce cyclin D1.

3T3 Cells↗
PubMed2003

Generation and characterization of p53 mutant mice.

p53 is one of the most well-characterized members of the tumor suppressor gene family. The role of p53 in controlling cellular homeostasis has proven critical, with over half of all human tumors having either lost or mutated p53. The emergence of technology facilitating the ablation of a gene within an animal's genome allowed great advances in the study of p53. The p53 knockout mouse was one of the first of its kind and provided a powerful tool for the study of p53. Production of the p53 knock-out mouse demonstrated the protein's dispensability during embryogenesis, while highlighting its essential role in controlling tumor formation. A variety of p53 mutant models have emerged since the original p53 knock-out mouse, along with improved techniques for regulating gene targeting. This chapter describes the necessary steps and protocols involved in producing a mutant mouse as well as the characterization that follows.

Aging↗
PubMed2002

p53 mutant mice that display early ageing-associated phenotypes.

The p53 tumour suppressor is activated by numerous stressors to induce apoptosis, cell cycle arrest, or senescence. To study the biological effects of altered p53 function, we generated mice with a deletion mutation in the first six exons of the p53 gene that express a truncated RNA capable of encoding a carboxy-terminal p53 fragment. This mutation confers phenotypes consistent with activated p53 rather than inactivated p53. Mutant (p53+/m) mice exhibit enhanced resistance to spontaneous tumours compared with wild-type (p53+/+) littermates. As p53+/m mice age, they display an early onset of phenotypes associated with ageing. These include reduced longevity, osteoporosis, generalized organ atrophy and a diminished stress tolerance. A second line of transgenic mice containing a temperature-sensitive mutant allele of p53 also exhibits early ageing phenotypes. These data suggest that p53 has a role in regulating organismal ageing.

Adipose Tissue↗
PubMed2002

Does p53 affect organismal aging?

The p53 protein plays a critical role in the prevention of cancer. It responds to a variety of cellular stresses to induce either apoptosis, a transient cell cycle arrest, or a terminal cell cycle arrest called senescence. Senescence in cultured cells is associated with augmented p53 activity and abrogation of p53 activity may delay in vitro senescence. Increasing evidence suggests that p53 may also influence aspects of organismal aging. Several mutant mouse models that display alterations in longevity and aging-related phenotypes have defects in genes that alter p53 signaling. Recently, my laboratory has developed and characterized a p53 mutant mouse line that appears to have an enhanced p53 response. These p53 mutants exhibit increased cancer resistance, yet have a shortened longevity and display a number of early aging-associated phenotypes, suggesting a role for p53 in the aging process. The nature of the aging phenotypes observed in this p53 mutant line is consistent with a model in which aging is driven in part by a gradual depletion of stem cell functional capacity.

Aging↗
PubMed2002

Elevated susceptibility of the p53 knockout mouse esophagus to methyl-N-amylnitrosamine carcinogenesis.

Mutations of the p53 tumor suppressor gene constitute one of the most frequent molecular changes in a wide variety of human cancers, including those in the esophagus. Mice deficient in p53 have recently attracted attention for their potential to identify chemical genotoxins. In this study we investigated the susceptibility of p53 nullizygous (-/-), heterozygous (+/-) and wild-type (+/+) mice to methyl-N-amylnitrosamine (MNAN), which specifically induces esophageal tumors in mice and rats. The p53 (+/-) and (+/+) mice were treated with 5 or 15 p.p.m. MNAN in their drinking water for 8 weeks then maintained without further treatment for an additional 7 or 17 weeks, being killed at experimental weeks 15 or 25. An additional group of p53 (-/-) mice were given 5 p.p.m. MNAN for 8 weeks and killed at week 15. At 15 weeks in the 5 p.p.m. groups, squamous cell carcinomas (SCCs) were observed in 10/12 (83.3%) p53 (-/-) and 1/15 (6.7%) p53 (+/-) mice, but in none of the p53 (+/+) mice. In the animals receiving 15 p.p.m., 2/14 (14.3%) p53 (+/-) and 1/11 (9.1%) p53 (+/+) mice developed SCCs. At 25 weeks, the incidence of SCCs was 7/16 (43.8%) and 8/14 (57.1%) in p53 (+/-) mice and 1/13 (7.7%) and 2/10 (20.0%) in p53 (+/+) mice at 5 and 15 p.p.m., respectively. Of the SCCs examined by PCR-single strand conformation polymorphism analysis, 61% (14/23) from p53 (+/-) and 50% (6/12) from p53 (+/+) mice demonstrated mutations in the p53 gene (exons 5-8). These results indicate the order of susceptibility to MNAN-induced esophageal tumorigenesis to be as follows: nullizygotes (-/-) > heterozygotes (+/-) > wild-type (+/+), and provide strong evidence of involvement of p53 mutations in the development of esophageal SCCs.

Animals↗
PubMed2002

Mice deficient for the wild-type p53-induced phosphatase gene (Wip1) exhibit defects in reproductive organs, immune function, and cell cycle control.

The Wip1 gene is a serine/threonine phosphatase that is induced in a p53-dependent manner by DNA-damaging agents. We show here that Wip1 message is expressed in moderate levels in all organs, but is present at very high levels in the testes, particularly in the postmeiotic round spermatid compartment of the seminiferous tubules. We have confirmed that Wip1 mRNA is induced by ionizing radiation in mouse tissues in a p53-dependent manner. To further determine the normal biological function of Wip1 in mammalian organisms, we have generated Wip1-deficient mice. Wip1 null mice are viable but show a variety of postnatal abnormalities, including variable male runting, male reproductive organ atrophy, reduced male fertility, and reduced male longevity. Mice lacking Wip1 show increased susceptibility to pathogens and diminished T- and B-cell function. Fibroblasts derived from Wip1 null embryos have decreased proliferation rates and appear to be compromised in entering mitosis. The data are consistent with an important role for Wip1 in spermatogenesis, lymphoid cell function, and cell cycle regulation.

Animals↗
PubMedDate not supplied

p53: guardian AND suppressor of longevity?

The p53 tumor suppressor gene enhances longevity by inhibiting cancer development. However, Van Heemst et al. in this issue show that polymorphisms in the p53 gene may affect longevity in unexpected ways. A meta-analysis indicates that individuals homozygous for the p53 codon 72 Pro allele instead of the more prevalent Arg allele have a modest increase in cancer incidence. This difference in cancer suspectibility is consistent with molecular studies showing that the p53 Pro polymorphic variant is a less robust anti-proliferative molecule than its Arg counterpart. The most surprising result was obtained in a prospective study of individuals age 85 or older. Despite having a 2.5-fold increased cancer incidence, the p53 codon 72 Pro/Pro individuals exhibited a significant 41% enhanced survival compared to codon 72 Arg/Pro and Arg/Arg individuals. These paradoxical findings suggest that p53 may enhance survival through most of our life span, but may actively suppress longevity during old age. The mechanisms for such duplicity remain unclear, but insights from p53 mutant mouse models may help to sort out the mystery.

Aging↗
PubMedDate not supplied

Visual genotyping of a coat color tagged p53 mutant mouse line.

The p53 knockout mouse has been widely used as a model in cancer research and other applications. Because neither homozygous nor heterozygous mutant p53 mice exhibit an overt phenotype, each animal requires laborious molecular genotyping. Here we describe a new p53 mutant mouse that is tagged with a tyrosinase coat color minigene. On an albino background, heterozygous tyrosinase-tagged p53 mutant mice exhibit a light tan coat color, while homozygous mutants display a darker brown coat color. Thus, by 8-10 days of age, mice with two, one, or no mutant p53 alleles are immediately distinguishable by their coat color, eliminating the time, costs, and errors associated with molecular genotyping. Moreover, the homozygous mutant p53-tyrosinase mice display a tumor incidence and spectrum virtually identical to previous p53 null mouse lines. Thus, tagging targeted mutations with such coat color markers provides a generally applicable genotyping method for embryonic stem cell-derived mice.

Alleles↗