Biomedical subjects

Tyler Jacks

Publications and source records attributed to Tyler Jacks.

At least 37 records · Page 2Linked to original sources

PubMed2004

An oncogenic KRAS2 expression signature identified by cross-species gene-expression analysis.

Using advanced gene targeting methods, generating mouse models of cancer that accurately reproduce the genetic alterations present in human tumors is now relatively straightforward. The challenge is to determine to what extent such models faithfully mimic human disease with respect to the underlying molecular mechanisms that accompany tumor progression. Here we describe a method for comparing mouse models of cancer with human tumors using gene-expression profiling. We applied this method to the analysis of a model of Kras2-mediated lung cancer and found a good relationship to human lung adenocarcinoma, thereby validating the model. Furthermore, we found that whereas a gene-expression signature of KRAS2 activation was not identifiable when analyzing human tumors with known KRAS2 mutation status alone, integrating mouse and human data uncovered a gene-expression signature of KRAS2 mutation in human lung cancer. We confirmed the importance of this signature by gene-expression analysis of short hairpin RNA-mediated inhibition of oncogenic Kras2. These experiments identified both a pattern of gene expression indicative of KRAS2 mutation and potential effectors of oncogenic KRAS2 activity in human cancer. This approach provides a strategy for using genomic analysis of animal models to probe human disease.

Adenocarcinoma↗

PubMed2004

Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome.

The p53 tumor suppressor gene is commonly altered in human tumors, predominantly through missense mutations that result in accumulation of mutant p53 protein. These mutations may confer dominant-negative or gain-of-function properties to p53. To ascertain the physiological effects of p53 point mutation, the structural mutant p53R172H and the contact mutant p53R270H (codons 175 and 273 in humans) were engineered into the endogenous p53 locus in mice. p53R270H/+ and p53R172H/+ mice are models of Li-Fraumeni Syndrome; they developed allele-specific tumor spectra distinct from p53+/- mice. In addition, p53R270H/- and p53R172H/- mice developed novel tumors compared to p53-/- mice, including a variety of carcinomas and more frequent endothelial tumors. Dominant effects that varied by allele and function were observed in primary cells derived from p53R270H/+ and p53R172H/+ mice. These results demonstrate that point mutant p53 alleles expressed under physiological control have enhanced oncogenic potential beyond the simple loss of p53 function.

Alleles↗

PubMed2004

Mutation at p53 serine 389 does not rescue the embryonic lethality in mdm2 or mdm4 null mice.

Mdm2 and its homolog Mdm4 inhibit the function of the tumor suppressor p53. Targeted disruption of either mdm2 or mdm4 genes in mice results in embryonic lethality that is completely rescued by concomitant deletion of p53, suggesting that deletion of negative regulators of p53 results in a constitutively active p53. Thus, these mouse models offer a unique in vivo system to assay the functional significance of different p53 modifications. Phosphorylation of serine 389 in murine p53 occurs specifically after ultraviolet-light-induced DNA damage, and phosphorylation of this site enhances p53 activity both in vitro and in vivo. Recently, mice with a serine to alanine substitution at serine 389 (p53S389A) in the endogenous p53 locus were generated. To examine the in vivo significance of serine 389 phosphorylation during embryogenesis, we crossed these mutant mice to mice lacking mdm2 or mdm4. The p53S389A allele did not alter the embryonic lethality of mdm2 or mdm4. Additional crosses to assay the effect of one p53S389A allele with a p53 null allele also did not rescue the lethal phenotypes. In conclusion, the phenotypes due to loss of mdm2 or mdm4 were not even partially rescued by p53S389A, suggesting that p53S389A is functionally wild type during embryogenesis.

Animals↗

PubMed2004

The Rb tumor suppressor is required for stress erythropoiesis.

The retinoblastoma tumor suppressor gene plays important roles in cell cycle control, differentiation and survival during development and is functionally inactivated in most human cancers. Early studies using gene targeting in mice suggested a critical role for pRb in erythropoiesis, while more recent experiments have suggested that many of the abnormal embryonic phenotypes in the Rb null mouse result from a defective placenta. To address this controversy and determine whether Rb has cell intrinsic functions in erythropoiesis, we examined the effects of Rb loss on red cell production following acute deletion of pRb in vitro and under different stress conditions in vivo. Under stress conditions, pRb was required to regulate erythroblast expansion and promote red cell enucleation. Acute deletion of Rb in vitro induced erythroid cell cycle and differentiation defects similar to those observed in vivo. These results demonstrate a cell intrinsic role for pRb in stress erythropoiesis and hematopoietic homeostasis that has relevance for human diseases.

Anemia, Aplastic↗

PubMed2004

Defective apoptosis and B-cell lymphomas in mice with p53 point mutation at Ser 23.

Phosphorylation of the p53 tumor suppressor at Ser20 (murine Ser23) has been proposed to be critical for disrupting p53 interaction with its negative regulator, MDM2, and allowing p53 stabilization. To determine the importance of Ser23 for the function of p53 in vivo, we generated a mouse in which the endogenous p53 locus was targeted to replace Ser23 with alanine. We show that, in mouse embryonic fibroblasts generated from Ser23 mutant mice, Ser23 mutation did not dramatically reduce IR-induced p53 protein stabilization or p53-dependent cell cycle arrest. However, in Ser23 mutant thymocytes and in the developing cerebellum, p53 stabilization following IR was decreased and resistance to apoptosis was observed. Homozygous Ser23 mutant animals had a reduced lifespan, but did not develop thymic lymphomas or sarcomas that are characteristic of p53-/- mice. Instead, Ser23 mutant animals died between 1 and 2 years with tumors that were most commonly of B-cell lineage. These data support an important role for Ser20/23 phosphorylation in p53 stabilization, apoptosis and tumor suppression.

Animals↗

PubMed2004

Susceptibility to astrocytoma in mice mutant for Nf1 and Trp53 is linked to chromosome 11 and subject to epigenetic effects.

Astrocytoma is the most common malignant brain tumor in humans. Loss of the p53 signaling pathway and up-regulation of the ras signaling pathway are common during tumor progression. We have shown previously that mice mutant for Trp53 and Nf1 develop astrocytoma, progressing to glioblastoma, on a C57BL/6J strain background. In contrast, here we present data that mice mutant for Trp53 and Nf1 on a 129S4/SvJae background are highly resistant to developing astrocytoma. Through analysis of F1 progeny, we demonstrate that susceptibility to astrocytoma is linked to chromosome 11, and that the modifier gene(s) responsible for differences in susceptibility is closely linked to Nf1 and Trp53. Furthermore, this modifier of astrocytoma susceptibility is itself epigenetically modified. These data demonstrate that epigenetic effects can have a strong effect on whether cancer develops in the context of mutant ras signaling and mutant p53, and that this mouse model of astrocytoma can be used to identify modifier phenotypes with complex inheritance patterns that would be unidentifiable in humans. Because analysis of gene function in the mouse is often performed on a mixed C57BL/6,129 strain background, these data also provide a powerful example of the potential of these strains to mask interesting gene functions.

Animals↗

PubMed2004

Cre-lox-regulated conditional RNA interference from transgenes.

We have generated two lentiviral vectors for conditional, Cre-lox-regulated, RNA interference. One vector allows for conditional activation, whereas the other permits conditional inactivation of short hairpin RNA (shRNA) expression. The former is based on a strategy in which the mouse U6 promoter has been modified by including a hybrid between a LoxP site and a TATA box. The ability to efficiently control shRNA expression by using these vectors was shown in cell-based experiments by knocking down p53, nucleophosmin and DNA methyltransferase 1. We also demonstrate the usefulness of this approach to achieve conditional, tissue-specific RNA interference in Cre-expressing transgenic mice. Combined with the growing array of Cre expression strategies, these vectors allow spatial and temporal control of shRNA expression in vivo and should facilitate functional genetic analysis in mammals.

Amino Acid Sequence↗

PubMed2004

Cell type-specific effects of Rb deletion in the murine retina.

Certain cells of the human retina are extremely sensitive to loss of function of the retinoblastoma tumor suppressor gene RB. Retinoblastomas develop early in life and at high frequency in individuals heterozygous for a germ-line RB mutation, and sporadic retinoblastomas invariably have somatic mutation in the RB gene. In contrast, retinoblastomas do not develop in Rb+/- mice. Although retinoblastoma is thought to have developmental origins, the function of Rb in retinal development has not been fully characterized. Here we studied the role of Rb in normal retinal development and in retinoblastoma using conditional Rb mutations in the mouse. In late embryogenesis, Rb-deficient retinas exhibited ectopic S-phase and high levels of p53-independent apoptosis, particularly in the differentiating retinal ganglion cell layer. During postnatal retinal development, loss of Rb led to more widespread retinal apoptosis, and adults showed loss of photoreceptors and bipolar cells. Conditional Rb mutation in the retina did not result in retinoblastoma formation even in a p53-mutant background. However, on a p107- or p130-deficient background, Rb mutation in the retina caused retinal dysplasia or retinoblastoma.

Animals↗

PubMed2004

Discrete signaling pathways participate in RB-dependent responses to chemotherapeutic agents.

The retinoblastoma (RB) tumor suppressor has been proposed to function as a key mediator of cell cycle checkpoints induced by chemotherapeutic agents. However, these prior studies have relied on embryonic fibroblasts harboring chronic loss of RB, a condition under which compensation of RB functions is known to occur. Here we utilized primary adult fibroblasts derived from mice harboring loxP sites flanking exon 3 of the Rb gene to delineate the action of RB in the chemotherapeutic response. In this system we find that targeted disruption of Rb leads to little overt change in cell cycle distribution. However, these cells exhibited deregulation of RB/E2F target genes and became aneuploid following culture in the absence of RB. When challenged with both DNA damaging and antimetabolite chemotherapeutics, RB was required for primary adult cells to undergo DNA damage checkpoint responses and loss of RB resulted in enhanced aneuploidy following challenge. In contrast, following spontaneous immortalization and the loss of functional p53 signaling, the antimetabolite 5-fluorouracil (5-FU) failed to induce arrest despite the presence of RB. In these immortal cultures RB/E2F targets were deregulated in a complex, gene-specific manner and RB was required for the checkpoint response to camptothecin (CPT). Mechanistic analyses of the checkpoint responses in primary cells indicated that loss of RB leads to increased p53 signaling and decreased viability following both CPT and 5-FU treatment. However, the mechanism through which these agents act to facilitate cell cycle inhibition through RB were distinct. These studies underscore the critical role of RB in DNA-damage checkpoint signaling and demonstrate that RB mediates chemotherapeutic-induced cell cycle inhibition in adult fibroblasts by distinct mechanisms.

Aneuploidy↗

PubMed2004

Classification of proliferative pulmonary lesions of the mouse: recommendations of the mouse models of human cancers consortium.

Rapid advances in generating new mouse genetic models for lung neoplasia provide a continuous challenge for pathologists and investigators. Frequently, phenotypes of new models either have no precedents or are arbitrarily attributed according to incongruent human and mouse classifications. Thus, comparative characterization and validation of novel models can be difficult. To address these issues, a series of discussions was initiated by a panel of human, veterinary, and experimental pathologists during the Mouse Models of Human Cancers Consortium (NIH/National Cancer Institute) workshop on mouse models of lung cancer held in Boston on June 20-22, 2001. The panel performed a comparative evaluation of 78 cases of mouse and human lung proliferative lesions, and recommended development of a new practical classification scheme that would (a) allow easier comparison between human and mouse lung neoplasms, (b) accommodate newly emerging mouse neoplasms, and (c) address the interpretation of benign and preinvasive lesions of the mouse lung. Subsequent discussions with additional experts in pulmonary pathology resulted in the current proposal of a new classification. It is anticipated that this classification, as well as the complementary digital atlas of virtual histological slides, will help investigators and pathologists in their characterization of new mouse models, as well as stimulate further research aimed at a better understanding of proliferative lesions of the lung.

Animals↗

PubMed2004

Activation of the p53-dependent G1 checkpoint response in mouse embryo fibroblasts depends on the specific DNA damage inducer.

The p53 tumor suppressor protein inhibits proliferation by inducing either cell cycle arrest or apoptosis in response to cellular stresses. Mouse embryo fibroblasts (MEFs) provide a primary cell model system in which to examine both functions of p53. MEFs treated with gamma-rays undergo p53-dependent G1 arrest, while oncogene-expressing MEFs treated with a variety of DNA-damaging agents undergo p53-dependent apoptosis. Although the p53-dependent G1 arrest checkpoint response to gamma-rays in MEFs has been well characterized, the response to other DNA-damaging agents has not. Here, we examine the effects of commonly utilized chemotherapeutics, including doxorubicin, etoposide, and cisplatin, on cell cycle arrest in MEFs, and we define the p53 dependence of these effects. In addition, we examine the response of MEFs to ultraviolet light (UVC), as a representative agent acting by inducing pyrimidine dimers. Although p53 is clearly activated by all the agents examined, as measured by p21 induction, there are surprising differences in the activities of these agents. For example, doxorubicin but not cisplatin can effectively induce a p53-dependent G1 arrest. UVC, in contrast, induces a p53-independent G1 arrest response. Thus, the exact response of cells to DNA damage depends on the specific agent used.

Adenovirus E1A Proteins↗

PubMed2004

RB signaling prevents replication-dependent DNA double-strand breaks following genotoxic insult.

Cell cycle checkpoints induced by DNA damage play an integral role in preservation of genomic stability by allowing cells to limit the propagation of deleterious mutations. The retinoblastoma tumor suppressor (RB) is crucial for the maintenance of the DNA damage checkpoint function because it elicits cell cycle arrest in response to a variety of genotoxic stresses. Although sporadic loss of RB is characteristic of most cancers and results in the bypass of the DNA damage checkpoint, the consequence of RB loss upon chemotherapeutic responsiveness has been largely uninvestigated. Here, we employed a conditional knockout approach to ablate RB in adult fibroblasts. This system enabled us to examine the DNA damage response of adult cells following acute RB deletion. Using this system, we demonstrated that loss of RB disrupted the DNA damage checkpoint elicited by either cisplatin or camptothecin exposure. Strikingly, this bypass was not associated with enhanced repair, but rather the accumulation of phosphorylated H2AX (gammaH2AX) foci, which indicate DNA double-strand breaks. The formation of gammaH2AX foci was due to ongoing replication following chemotherapeutic treatment in the RB-deficient cells. Additionally, peak gammaH2AX accumulation occurred in S-phase cells undergoing DNA replication in the presence of damage, and these gammaH2AX foci co-localized with replication foci. These results demonstrate that acute RB loss abrogates DNA damage-induced cell cycle arrest to induce gammaH2AX foci formation. Thus, secondary genetic lesions induced by RB loss have implications for the chemotherapeutic response and the development of genetic instability.

Animals↗

PubMed2004

Endogenous oncogenic K-ras(G12D) stimulates proliferation and widespread neoplastic and developmental defects.

Activating mutations in the ras oncogene are not considered sufficient to induce abnormal cellular proliferation in the absence of cooperating oncogenes. We demonstrate that the conditional expression of an endogenous K-ras(G12D) allele in murine embryonic fibroblasts causes enhanced proliferation and partial transformation in the absence of further genetic abnormalities. Interestingly, K-ras(G12D)-expressing fibroblasts demonstrate attenuation and altered regulation of canonical Ras effector signaling pathways. Widespread expression of endogenous K-ras(G12D) is not tolerated during embryonic development, and directed expression in the lung and GI tract induces preneoplastic epithelial hyperplasias. Our results suggest that endogenous oncogenic ras is sufficient to initiate transformation by stimulating proliferation, while further genetic lesions may be necessary for progression to frank malignancy.

Animals↗

PubMed2004

Increased sensitivity to UV radiation in mice with a p53 point mutation at Ser389.

Phosphorylation is important for p53 protein stabilization and activation after DNA damage. Serine 389 of p53 is specifically phosphorylated after UV irradiation, whereas gamma radiation activates p53 through a different pathway. To study the in vivo significance of p53 phosphorylation at serine 389, we generated a physiological mouse model in which p53 phosphorylation at serine 389 is abolished by alanine substitution. Homozygous mutant p53.S389A mice are viable and have an apparently normal phenotype. However, cells isolated from these mice are partly compromised in transcriptional activation of p53 target genes and apoptosis after UV irradiation, whereas gamma radiation-induced responses are not affected. Moreover, p53.S389A mice show increased sensitivity to UV-induced skin tumor development, signifying the importance of serine 389 phosphorylation for the tumor-suppressive function of p53.

Animals↗

PubMed2004

Conditional expression of oncogenic K-ras from its endogenous promoter induces a myeloproliferative disease.

Oncogenic ras alleles are among the most common mutations found in patients with acute myeloid leukemia (AML). Previously, the role of oncogenic ras in cancer was assessed in model systems overexpressing oncogenic ras from heterologous promoters. However, there is increasing evidence that subtle differences in gene dosage and regulation of gene expression from endogenous promoters play critical roles in cancer pathogenesis. We characterized the role of oncogenic K-ras expressed from its endogenous promoter in the hematopoietic system using a conditional allele and IFN-inducible, Cre-mediated recombination. Mice developed a completely penetrant myeloproliferative syndrome characterized by leukocytosis with normal maturation of myeloid lineage cells; myeloid hyperplasia in bone marrow; and extramedullary hematopoiesis in the spleen and liver. Flow cytometry confirmed the myeloproliferative phenotype. Genotypic and Western blot analysis demonstrated Cre-mediated excision and expression, respectively, of the oncogenic K-ras allele. Bone marrow cells formed growth factor-independent colonies in methylcellulose cultures, but the myeloproliferative disease was not transplantable into secondary recipients. Thus, oncogenic K-ras induces a myeloproliferative disorder but not AML, indicating that additional mutations are required for AML development. This model system will be useful for assessing the contribution of cooperating mutations in AML and testing ras inhibitors in vivo.

Adenoma↗

PubMed2003

Perp is a mediator of p53-dependent apoptosis in diverse cell types.

The induction of apoptosis by the p53 protein is critical for its activity as a tumor suppressor. Although it is clear that p53 induces apoptosis at least in part by direct transcriptional activation of target genes, the set of p53 target genes that mediate p53 function in apoptosis in vivo remains to be well defined. The Perp (p53 apoptosis effector related to PMP-22) gene is highly expressed in cells undergoing p53-dependent apoptosis as compared to cells undergoing p53-dependent G1 arrest. Perp is a direct p53 target, and its overexpression is sufficient to induce cell death in fibroblasts, implicating it as an important component of p53 apoptotic function. Here, through the generation of Perp-deficient mice, we analyze the role of Perp in the p53 apoptosis pathway in multiple primary cell types by comparing the cell death responses of Perp null cells to those of wild-type and p53 null cells. These experiments demonstrate the involvement of Perp in p53-mediated cell death in thymocytes and neurons but not in E1A-expressing MEFs, indicating a cell type-specific role for Perp in the p53 cell death pathway. In addition, we show that Perp is not required for proliferation-associated functions of p53. Thus, Perp selectively mediates the p53 apoptotic response, and the requirement for Perp is dictated by cellular context.

Animals↗

PubMed2003

Acute mutation of retinoblastoma gene function is sufficient for cell cycle re-entry.

Cancer cells arise from normal cells through the acquisition of a series of mutations in oncogenes and tumour suppressor genes. Mouse models of human cancer often rely on germline alterations that activate or inactivate genes of interest. One limitation of this approach is that germline mutations might have effects other than somatic mutations, owing to developmental compensation. To model sporadic cancers associated with inactivation of the retinoblastoma (RB) tumour suppressor gene in humans, we have produced a conditional allele of the mouse Rb gene. We show here that acute loss of Rb in primary quiescent cells is sufficient for cell cycle entry and has phenotypic consequences different from germline loss of Rb function. This difference is explained in part by functional compensation by the Rb-related gene p107. We also show that acute loss of Rb in senescent cells leads to reversal of the cellular senescence programme. Thus, the use of conditional knockout strategies might refine our understanding of gene function and help to model human cancer more accurately.

Animals↗

PubMed2003

Dynamic regulation of the Ras pathway via proteolysis of the NF1 tumor suppressor.

Mutations in the NF1 tumor suppressor underlie the familial tumor predisposition syndrome neurofibromatosis type I. Although its encoded protein, neurofibromin, functions as a Ras-GTPase activating protein (GAP), nothing is known about how it is normally regulated or its precise role in controlling Ras signaling pathways. We show here that neurofibromin is dynamically regulated by the ubiquitin-proteasome pathway. Degradation is rapidly triggered in response to a variety of growth factors and requires sequences adjacent to the catalytic GAP-related domain of neurofibromin. However, whereas degradation is rapid, neurofibromin levels are re-elevated shortly after growth factor treatment. Accordingly, Nf1-deficient mouse embryonic fibroblasts (MEFs) exhibit an enhanced activation of Ras, prolonged Ras and ERK activities, and proliferate in response to subthreshold levels of growth factors. Thus, the dynamic proteasomal regulation of neurofibromin represents an important mechanism of controlling both the amplitude and duration of Ras-mediated signaling. Furthermore, this previously unrecognized Ras regulatory mechanism may be exploited therapeutically.

3T3 Cells↗