ABSTRACT
Antibiotic-resistant Neisseria gonorrhoeae (Ng) are an emerging public health threat due to increasing numbers of multidrug resistant (MDR) organisms. We identified two novel orally active inhibitors, PTC-847 and PTC-672, that exhibit a narrow spectrum of activity against Ng including MDR isolates. By selecting organisms resistant to the novel inhibitors and sequencing their genomes, we identified a new therapeutic target, the class Ia ribonucleotide reductase (RNR). Resistance mutations in Ng map to the N-terminal cone domain of the α subunit, which we show here is involved in forming an inhibited α4ß4 state in the presence of the ß subunit and allosteric effector dATP. Enzyme assays confirm that PTC-847 and PTC-672 inhibit Ng RNR and reveal that allosteric effector dATP potentiates the inhibitory effect. Oral administration of PTC-672 reduces Ng infection in a mouse model and may have therapeutic potential for treatment of Ng that is resistant to current drugs.
Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/drug effects , Gonorrhea/drug therapy , Pyridines/pharmacology , Ribonucleotide Reductases/metabolism , Allosteric Regulation , Animals , Deoxyadenine Nucleotides/metabolism , Disease Models, Animal , Escherichia coli/drug effects , Female , Gonorrhea/metabolism , Humans , Mice , Mice, Inbred BALB C , Microbial Sensitivity Tests/methods , Neisseria gonorrhoeae/drug effectsABSTRACT
PTC596 is an investigational small-molecule tubulin-binding agent. Unlike other tubulin-binding agents, PTC596 is orally bioavailable and is not a P-glycoprotein substrate. So as to characterize PTC596 to position the molecule for optimal clinical development, the interactions of PTC596 with tubulin using crystallography, its spectrum of preclinical in vitro anticancer activity, and its pharmacokinetic-pharmacodynamic relationship were investigated for efficacy in multiple preclinical mouse models of leiomyosarcomas and glioblastoma. Using X-ray crystallography, it was determined that PTC596 binds to the colchicine site of tubulin with unique key interactions. PTC596 exhibited broad-spectrum anticancer activity. PTC596 showed efficacy as monotherapy and additive or synergistic efficacy in combinations in mouse models of leiomyosarcomas and glioblastoma. PTC596 demonstrated efficacy in an orthotopic model of glioblastoma under conditions where temozolomide was inactive. In a first-in-human phase I clinical trial in patients with cancer, PTC596 monotherapy drug exposures were compared with those predicted to be efficacious based on mouse models. PTC596 is currently being tested in combination with dacarbazine in a clinical trial in adults with leiomyosarcoma and in combination with radiation in a clinical trial in children with diffuse intrinsic pontine glioma.
Subject(s)
Benzimidazoles/pharmacology , Glioblastoma/drug therapy , Leiomyosarcoma/drug therapy , Pyrazines/pharmacology , Tubulin Modulators/pharmacology , Adult , Aged , Aged, 80 and over , Animals , Apoptosis , Benzimidazoles/pharmacokinetics , Cell Proliferation , Female , Glioblastoma/pathology , Humans , Leiomyosarcoma/pathology , Male , Maximum Tolerated Dose , Mice , Mice, Nude , Middle Aged , Prognosis , Pyrazines/pharmacokinetics , Tissue Distribution , Tubulin Modulators/pharmacokinetics , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
There exists an urgent medical need to identify new chemical entities (NCEs) targeting multidrug resistant (MDR) bacterial infections, particularly those caused by Gram-negative pathogens. 4-Hydroxy-2-pyridones represent a novel class of nonfluoroquinolone inhibitors of bacterial type II topoisomerases active against MDR Gram-negative bacteria. Herein, we report on the discovery and structure-activity relationships of a series of fused indolyl-containing 4-hydroxy-2-pyridones with improved in vitro antibacterial activity against fluoroquinolone resistant strains. Compounds 6o and 6v are representative of this class, targeting both bacterial DNA gyrase and topoisomerase IV (Topo IV). In an abbreviated susceptibility screen, compounds 6o and 6v showed improved MIC90 values against Escherichia coli (0.5-1 µg/mL) and Acinetobacter baumannii (8-16 µg/mL) compared to the precursor compounds. In a murine septicemia model, both compounds showed complete protection in mice infected with a lethal dose of E. coli.
Subject(s)
Anti-Bacterial Agents/pharmacology , DNA Topoisomerases, Type II/chemistry , Drug Discovery , Drug Resistance, Multiple, Bacterial/drug effects , Gram-Negative Bacteria/drug effects , Sepsis/drug therapy , Topoisomerase II Inhibitors/pharmacology , Animals , Anti-Bacterial Agents/chemistry , Female , Mice , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Protein Conformation , Pyridines/chemistry , Sepsis/microbiology , Structure-Activity Relationship , Topoisomerase II Inhibitors/chemistryABSTRACT
BMI-1, also known as a stem cell factor, is frequently upregulated in several malignancies. Elevated expression of BMI-1 correlates with poor prognosis and is therefore considered a viable therapeutic target in a number of malignancies including ovarian cancer. Realizing the immense pathologic significance of BMI-1, small-molecule inhibitors against BMI-1 are recently being developed. In this study, we functionally characterize PTC-028, an orally bioavailable compound that decreases BMI-1 levels by posttranslational modification. We report that PTC-028 treatment selectively inhibits cancer cells in clonal growth and viability assays, whereas normal cells remain unaffected. Mechanistically, hyperphosphorylation-mediated depletion of cellular BMI-1 by PTC-028 coupled with a concurrent temporal decrease in ATP and a compromised mitochondrial redox balance potentiates caspase-dependent apoptosis. In vivo, orally administered PTC-028, as a single agent, exhibits significant antitumor activity comparable with the standard cisplatin/paclitaxel therapy in an orthotopic mouse model of ovarian cancer. Thus, PTC-028 has the potential to be used as an effective therapeutic agent in patients with epithelial ovarian cancer, where treatment options are limited. Mol Cancer Ther; 17(1); 39-49. ©2017 AACR.
Subject(s)
Benzimidazoles/pharmacology , Carcinoma, Ovarian Epithelial/drug therapy , Polycomb Repressive Complex 1/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Pyrazines/pharmacology , Animals , Antineoplastic Agents/pharmacology , Carcinoma, Ovarian Epithelial/metabolism , Carcinoma, Ovarian Epithelial/pathology , Cell Line, Tumor , Female , Humans , Mice , Mice, Nude , Polycomb Repressive Complex 1/metabolism , Proto-Oncogene Proteins/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Tumor cells upregulate many cell signaling pathways, with AKT being one of the key kinases to be activated in a variety of malignancies. GSK2110183 and GSK2141795 are orally bioavailable, potent inhibitors of the AKT kinases that have progressed to human clinical studies. Both compounds are selective, ATP-competitive inhibitors of AKT 1, 2 and 3. Cells treated with either compound show decreased phosphorylation of several substrates downstream of AKT. Both compounds have desirable pharmaceutical properties and daily oral dosing results in a sustained inhibition of AKT activity as well as inhibition of tumor growth in several mouse tumor models of various histologic origins. Improved kinase selectivity was associated with reduced effects on glucose homeostasis as compared to previously reported ATP-competitive AKT kinase inhibitors. In a diverse cell line proliferation screen, AKT inhibitors showed increased potency in cell lines with an activated AKT pathway (via PI3K/PTEN mutation or loss) while cell lines with activating mutations in the MAPK pathway (KRAS/BRAF) were less sensitive to AKT inhibition. Further investigation in mouse models of KRAS driven pancreatic cancer confirmed that combining the AKT inhibitor, GSK2141795 with a MEK inhibitor (GSK2110212; trametinib) resulted in an enhanced anti-tumor effect accompanied with greater reduction in phospho-S6 levels. Taken together these results support clinical evaluation of the AKT inhibitors in cancer, especially in combination with MEK inhibitor.
Subject(s)
Antineoplastic Agents/pharmacology , Diamines/pharmacology , Gene Expression Regulation, Neoplastic , Pancreatic Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Tumor Burden/drug effects , Administration, Oral , Animals , Antineoplastic Agents/chemical synthesis , Blood Glucose/metabolism , Cell Line, Tumor , Diamines/chemical synthesis , Drug Evaluation, Preclinical , Drug Synergism , Female , Humans , MAP Kinase Kinase Kinases/antagonists & inhibitors , MAP Kinase Kinase Kinases/genetics , MAP Kinase Kinase Kinases/metabolism , Mice , Mice, SCID , PTEN Phosphohydrolase/antagonists & inhibitors , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Pancreatic Neoplasms/enzymology , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/chemical synthesis , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Pyrazoles/chemical synthesis , Ribosomal Protein S6 Kinases/antagonists & inhibitors , Ribosomal Protein S6 Kinases/genetics , Ribosomal Protein S6 Kinases/metabolism , Signal Transduction , Xenograft Model Antitumor AssaysABSTRACT
The androgen receptor (AR) is a member of a unique class of transcription factors because it contains a ligand-binding domain that, when activated, results in nuclear translocation and the transcriptional activation of genes associated with prostate cancer development. Although androgen deprivation therapies are effective initially for the treatment of prostate cancer, the disease eventually relapses and progresses to castration-resistant prostate cancer (CRPC). Nonetheless, the AR still plays a critical role because late-stage investigational agents that deplete testosterone (abiraterone) or block ligand binding (MDV3100) can still control tumor growth in patients with CRPC. These findings indicate that downmodulation of AR expression may provide a complementary strategy for treating CRPC. In this article, we describe a novel, locked, nucleic acid-based antisense oligonucleotide, designated EZN-4176. When administered as a single agent, EZN-4176 specifically downmodulated AR mRNA and protein, and this was coordinated with inhibition of the growth of both androgen-sensitive and CRPC tumors in vitro as well as in animal models. The effect was specific because no effect on growth was observed with a control antisense oligonucleotide that does not recognize AR mRNA, nor on tumors derived from the PC3, AR-negative, tumor cell line. In addition, EZN-4176 reduced AR luciferase reporter activity in a CRPC model derived from C4-2b cells that were implanted intratibially, indicating that the molecule may control prostate cancer that has metastasized to the bone. These data, together with the continued dependency of CRPC on the AR signaling pathway, justify the ongoing phase I evaluation of EZN-4176 in patients with CRPC.
Subject(s)
Carcinoma/pathology , Oligodeoxyribonucleotides, Antisense/pharmacology , Prostatic Neoplasms/pathology , Receptors, Androgen/genetics , Androgen Antagonists/pharmacology , Androgen Antagonists/therapeutic use , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Carcinoma/drug therapy , Carcinoma/genetics , Cell Line, Tumor , DNA/pharmacology , DNA/therapeutic use , Disease Models, Animal , Down-Regulation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Male , Mice , Mice, Nude , Oligodeoxyribonucleotides, Antisense/therapeutic use , Orchiectomy , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/genetics , Receptors, Androgen/metabolism , Treatment Failure , Xenograft Model Antitumor AssaysABSTRACT
Ribosomal stress is an important, yet poorly understood, mechanism that results in activation of the p53 tumour suppressor. We present a mutation in the ribosomal protein Rpl27a gene (sooty foot ataxia mice), isolated through a sensitized N-ethyl-N-nitrosourea (ENU) mutagenesis screen for p53 pathway defects, that shares striking phenotypic similarities with high p53 mouse models, including cerebellar ataxia, pancytopenia and epidermal hyperpigmentation. This phenocopy is rescued in a haploinsufficient p53 background. A detailed examination of the bone marrow in these mice identified reduced numbers of haematopoietic stem cells and a p53-dependent c-Kit down-regulation. These studies suggest that reduced Rpl27a increases p53 activity in vivo, further evident with a delay in tumorigenesis in mutant mice. Taken together, these data demonstrate that Rpl27a plays a crucial role in multiple tissues and that disruption of this ribosomal protein affects both development and transformation.
Subject(s)
Cerebellar Ataxia/genetics , Ribosomal Proteins/genetics , Tumor Suppressor Protein p53/metabolism , Anemia/genetics , Anemia/metabolism , Animals , Body Weight/genetics , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Cerebellar Ataxia/metabolism , Cerebellar Ataxia/pathology , Disease Models, Animal , Growth Disorders/genetics , Growth Disorders/metabolism , Haploinsufficiency/genetics , Hematopoietic Stem Cells/pathology , Hyperpigmentation/genetics , Hyperpigmentation/metabolism , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Mutagenicity Tests , Phenotype , Ribosomal Proteins/metabolism , Ribosomal Proteins/physiology , Signal Transduction/physiologyABSTRACT
Topoisomerase I inhibitors down-regulate HIF-1α leading to tumor growth inhibition, but only while maintaining sustained levels of drug exposure. EZN-2208, a multi-arm 40 kDa pegylated, releasable SN38-drug conjugate, provides higher, longer lasting exposure of tumors to SN38 in contrast to SN38 that is released from CPT-11. EZN-2208 also consistently has greater antitumor activity than CPT-11 in a variety of solid and hematological tumor models. In this report, the ability of PEG-SN38 to down-regulate HIF-1α and its downstream targets, in a more potent, sustained manner compared with CPT-11 was examined. To do so, U251 glioma xenografts that stably expressed a hypoxia response element-dependent luciferase reporter gene were implanted in mice. After treatment it was found that EZN-2208 induced potent, sustained HIF-1α down-regulation (37% at 48 h and 83% at 120 h) in the tumors, whereas CPT-11 caused only minor, transient HIF-1α down-regulation. In addition, EZN-2208 down-regulated mRNA levels of HIF-1α targeted genes (MMP2, VEGF1, Glut1, Glut3 and TGFß1). Further, western blot analyses of xenograft tumors demonstrated that EZN-2208 had significantly more effect than CPT-11 in down-regulating HIF-1α, VEGF, Glut1 and MMP2 protein levels. Significant down-regulation of HIF-1α and VEGF proteins translated to EZN-2208's superior anti-angiogenic activity compared with CPT-11, confirmed by microvessel density reduction in a chorioallantoic membrane assay and in CD-31 immunohistochemistry studies. Additional studies done with matrigel implants devoid of tumor cells show that EZN-2208 significantly inhibits angiogenesis while CPT-11 has little or no effect. It is concluded that the superior antitumor activity of EZN-2208 compared with CPT-11 is attributed, in part, to an anti-angiogenic effect. Ongoing clinical Phase I and Phase II studies will assess safety and efficacy of EZN-2208.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Camptothecin/analogs & derivatives , Down-Regulation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Glioma/drug therapy , Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis , Neoplasm Proteins/biosynthesis , Neovascularization, Pathologic/drug therapy , Polyethylene Glycols/pharmacology , Animals , Antineoplastic Agents, Phytogenic/pharmacology , Camptothecin/pharmacology , Cell Line, Tumor , Glioma/metabolism , Glioma/pathology , Humans , Irinotecan , Mice , Mice, Nude , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Xenograft Model Antitumor AssaysABSTRACT
Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might have utility as an anticancer agent. Herein we describe our lead optimization of compound 1 toward highly potent and selective PDK1 inhibitors via a structure-based design strategy. The most potent and selective inhibitors demonstrated submicromolar activity as measured by inhibition of phosphorylation of PDK1 substrates as well as antiproliferative activity against a subset of AML cell lines. In addition, reduction of phosphorylation of PDK1 substrates was demonstrated in vivo in mice bearing OCl-AML2 xenografts. These observations demonstrate the utility of these molecules as tools to further delineate the biology of PDK1 and the potential pharmacological uses of a PDK1 inhibitor.
Subject(s)
Antineoplastic Agents/chemical synthesis , Indazoles/chemical synthesis , Morpholines/chemical synthesis , Piperidines/chemical synthesis , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrimidines/chemical synthesis , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Crystallography, X-Ray , Drug Screening Assays, Antitumor , Indazoles/chemistry , Indazoles/pharmacology , Mice , Mice, SCID , Models, Molecular , Molecular Structure , Morpholines/chemistry , Morpholines/pharmacology , Neoplasm Transplantation , Phosphorylation , Piperidines/chemistry , Piperidines/pharmacology , Protein Binding , Pyrimidines/chemistry , Pyrimidines/pharmacology , Pyruvate Dehydrogenase Acetyl-Transferring Kinase , Stereoisomerism , Structure-Activity Relationship , Transplantation, HeterologousABSTRACT
This study aimed to investigate the antitumor and antiangiogenic effects utilizing a novel therapy regimen of metronomic topotecan and pazopanib, a multireceptor tyrosine kinase inhibitor. In vitro (Western blot) and in vivo dose-finding experiments were done following pazopanib therapy in ovarian cancer models. Pazopanib and metronomic (daily) oral topotecan therapy was examined in an orthotopic model of ovarian cancer. Tumor weights, survival, and markers of the tumor microenvironment [angiogenesis (CD31 and pericyte coverage), proliferation (Ki-67), and apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling)] were analyzed by immunostaining following therapy. Pazopanib therapy reduced vascular endothelial growth factor receptor 2 (VEGFR-2) activity in vitro and vivo in a dose-dependent manner. Compared with control mice, pazopanib reduced tumor weight by 28% to 82% (P < 0.01 in the SKOV3ip1 model) and metronomic topotecan reduced tumor weight by 40% to 59% in the HeyA8 (P = 0.13) and SKOV3ip1 (P = 0.07) models. Combination therapy had the greatest effect with 79% to 84% reduction (P < 0.01 for both models). In the SKOV3ip1 and A2780 models, mouse survival was significantly longer (P < 0.001 versus controls) with pazopanib and metronomic topotecan therapy. Pazopanib therapy reduced murine endothelial cell migration in vitro in a dose-dependent manner following VEGF stimulation and decreased tumor microvessel density and pericyte coverage when given in combination with metronomic topotecan. Tumor cell proliferation decreased in all treatment arms compared with controls (P < 0.01 for combination groups) and increased tumor cell apoptosis by 4-fold with combination therapy. Pazopanib therapy in combination with metronomic topotecan therapy showed significant antitumor and antiangiogenic properties in preclinical ovarian cancer models and warrants further investigation as a novel therapeutic regimen in clinical trials. Mol Cancer Ther; 9(4); 985-95. (c)2010 AACR.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Biological Therapy , Ovarian Neoplasms/drug therapy , Pyrimidines/therapeutic use , Sulfonamides/therapeutic use , Topotecan/therapeutic use , Angiogenesis Inhibitors/pharmacology , Angiogenesis Inhibitors/therapeutic use , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Cell Death/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Humans , Indazoles , Kaplan-Meier Estimate , Mice , Models, Biological , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/pathology , Ovarian Neoplasms/blood supply , Ovarian Neoplasms/pathology , Pyrimidines/pharmacology , Receptors, Vascular Endothelial Growth Factor/metabolism , Sulfonamides/pharmacology , Topotecan/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
The insulin-like growth factor-I receptor (IGF-IR) signaling pathway is activated in various tumors, and inhibition of IGF-IR kinase provides a therapeutic opportunity in these patients. GSK1838705A is a small-molecule kinase inhibitor that inhibits IGF-IR and the insulin receptor with IC(50)s of 2.0 and 1.6 nmol/L, respectively. GSK1838705A blocks the in vitro proliferation of cell lines derived from solid and hematologic malignancies, including multiple myeloma and Ewing's sarcoma, and retards the growth of human tumor xenografts in vivo. Despite the inhibitory effect of GSK1838705A on insulin receptor, minimal effects on glucose homeostasis were observed at efficacious doses. GSK1838705A also inhibits the anaplastic lymphoma kinase (ALK), which drives the aberrant growth of anaplastic large-cell lymphomas, some neuroblastomas, and a subset of non-small cell lung cancers. GSK1838705A inhibits ALK, with an IC(50) of 0.5 nmol/L, and causes complete regression of ALK-dependent tumors in vivo at well-tolerated doses. GSK1838705A is therefore a promising antitumor agent for therapeutic use in human cancers.
Subject(s)
Antineoplastic Agents/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrimidines/pharmacology , Pyrroles/pharmacology , Receptor, IGF Type 1/antagonists & inhibitors , Xenograft Model Antitumor Assays , Anaplastic Lymphoma Kinase , Animals , Blood Glucose/metabolism , Cell Proliferation/drug effects , Enzyme Activation/drug effects , Humans , Mice , Phosphorylation/drug effects , Protein-Tyrosine Kinases/metabolism , Receptor Protein-Tyrosine Kinases , Receptor, IGF Type 1/metabolism , Receptor, Insulin/metabolism , Signal Transduction/drug effectsABSTRACT
PURPOSE: Dysregulation of the insulin-like growth factor-I receptor (IGF-IR) signaling pathway has been implicated in the development of many types of tumors, including prostate, colon, breast, pancreatic, ovarian, and sarcomas. Agents that inhibit IGF-IR activity may be useful in treatment of patients with various cancers. EXPERIMENTAL DESIGN: Kinase assays were used to identify a selective small-molecule inhibitor of IGF-IR activity. The effects of this compound on IGF-IR signaling, cell proliferation, and the cell cycle were determined using a panel of cell lines. Antitumor activity was evaluated in human tumor xenografts growing in athymic mice. Inhibition of IGF-IR and the closely related insulin receptor (IR) was measured in vivo, and the effect on glucose metabolism was evaluated. RESULTS: GSK1904529A selectively inhibits IGF-IR and IR with IC(50)s of 27 and 25 nmol/L, respectively. GSK1904529A blocks receptor autophosphorylation and downstream signaling, leading to cell cycle arrest. It inhibits the proliferation of cell lines derived from solid and hematologic malignancies, with multiple myeloma and Ewing's sarcoma cell lines being most sensitive. Oral administration of GSK1904529A decreases the growth of human tumor xenografts in mice, consistent with a reduction of IGF-IR phosphorylation in tumors. Despite the potent inhibitory activity of GSK1904529A on IR in vitro and in vivo, minimal effects on blood glucose levels are observed in animals at doses that show significant antitumor activity. CONCLUSION: GSK1904529A is a promising candidate for therapeutic use in IGF-IR-dependent tumors.
Subject(s)
Antineoplastic Agents/pharmacology , Imidazoles/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacology , Receptor, IGF Type 1/antagonists & inhibitors , 3-Hydroxybutyric Acid/metabolism , Animals , Apoptosis/drug effects , Blood Glucose/metabolism , Blotting, Western , Cell Cycle/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Humans , Imidazoles/metabolism , Male , Mice , Mice, Nude , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , Phosphorylation/drug effects , Pyridines/metabolism , Receptor, IGF Type 1/metabolism , Receptor, Insulin/antagonists & inhibitors , Receptor, Insulin/metabolism , Xenograft Model Antitumor AssaysABSTRACT
The tumor suppressor p53 is important for inhibiting the development of breast carcinomas. However, little is known about the effects of increased p53 activity on mammary gland development. Therefore, the effect of p53 dosage on mammary gland development was examined by utilizing the p53+/m mouse, a p53 mutant which exhibits increased wild-type p53 activity, increased tumor resistance, a shortened longevity, and a variety of accelerated aging phenotypes. Here we report that p53+/m virgin mice exhibit a defect in mammary gland ductal morphogenesis. Transplants of mammary epithelium into p53+/m recipient mice demonstrate decreased outgrowth of wild-type and p53+/m donor epithelium, suggesting systemic or stromal alterations in the p53+/m mouse. Supporting these data, p53+/m mice display decreased levels of serum IGF-1 and reduced IGF-1 signaling in virgin glands. The induction of pregnancy or treatment of p53+/m mice with estrogen, progesterone, estrogen and progesterone in combination, or IGF-1 stimulates ductal outgrowth, rescuing the p53+/m mammary phenotype. Serial mammary epithelium transplants demonstrate that p53+/m epithelium exhibits decreased transplant capabilities, suggesting early stem cell exhaustion. These data indicate that appropriate levels of p53 activity are important in regulating mammary gland ductal morphogenesis, in part through regulation of the IGF-1 pathway.
Subject(s)
Aging , Mammary Glands, Animal/embryology , Morphogenesis , Tumor Suppressor Protein p53/metabolism , Animals , Female , Genes, p53 , Insulin-Like Growth Factor I/metabolism , Mammary Glands, Animal/metabolism , Mice , Mice, Inbred C57BL , Specific Pathogen-Free Organisms , Tumor Suppressor Protein p53/geneticsABSTRACT
The ability of tissues to maintain homeostasis is dependent in part on the function of adult tissue stem cells, which have the capability to self-renew and differentiate into multiple lineages. It has been hypothesized that the ability of stem cells to maintain tissue homeostasis declines functionally with age and that this decline may account for many of the biological phenotypes associated with aging. Recently, tumor suppressors such as p53 have been implicated in both aging and the regulation of stem cell dynamics. Our recent findings suggest that p53 may impact hematopoietic stem cell (HSC) dynamics during mammalian aging. Utilizing mouse models of varying levels of p53 dosage, we have shown that alteration of p53 activity affects stem cell number, proliferation, and functionality with age. Several other recent studies have implicated other tumor suppressors in potential age-related regulation of HSC dynamics as well. These data support a model in which aging is caused in part by a decline in tissue stem cell regenerative function, regulated in part by tumor suppressors.
Subject(s)
Cellular Senescence/physiology , Stem Cells/physiology , Tumor Suppressor Protein p53/metabolism , Animals , Cell Proliferation , Cellular Senescence/genetics , Genes, Tumor Suppressor/physiology , Humans , Stem Cells/cytology , Stem Cells/metabolism , Tumor Suppressor Protein p53/geneticsABSTRACT
A temporal decline in tissue stem cell functionality may be a key component of mammalian aging. The tumor suppressor p53 has recently been implicated as a potential regulator of aging. We examined age-associated hematopoietic stem cell (HSC) dynamics in mice with varying p53 activities. Reduced p53 activity in p53+/- mice was associated with higher numbers of proliferating hematopoietic stem and progenitor cells in old age compared with aged wild-type (p53+/+) mice. We also assessed HSC dynamics in a p53 mutant mouse model (p53+/m) with higher apparent p53 activity than wild-type mice. The p53 hypermorphic (p53+/m) mice display phenotypes of premature aging. Many aged p53+/m organs exhibit reduced cellularity and atrophy, suggesting defects in stem-cell regenerative capacity. HSC numbers from old p53+/m mice fail to increase with age, unlike those of their p53+/+ and p53+/- counterparts. Moreover, transplantation of 500 HSCs from old p53+/m mice into lethally irradiated recipients resulted in reduced engraftment compared with old wild-type p53+/+ and p53+/- HSCs. Thus, alteration of p53 activity affects stem-cell numbers, proliferation potential, and hematopoiesis in older organisms, supporting a model in which aging is caused in part by a decline in tissue stem cell regenerative function.
Subject(s)
Aging , Hematopoietic Stem Cells/cytology , Tumor Suppressor Protein p53/physiology , Animals , Cell Count , Cell Proliferation , Hematopoiesis , Mice , Mice, KnockoutABSTRACT
The p53-induced serine/threonine phosphatase, protein phosphatase 1D magnesium-dependent, delta isoform (PPM1D) (or wild-type p53-induced phosphatase 1 (Wip1)), exhibits oncogenic activity in vitro and in vivo. It behaves as an oncogene in rodent fibroblast transformation assays and is amplified and overexpressed in several human tumor types. It may contribute to oncogenesis through functional inactivation of p53. Here, we show that the oncogenic function of PPM1D is associated with its phosphatase activity. While overexpressed PPM1D may be oncogenic, PPM1D null mice are resistant to spontaneous tumors over their entire lifespan. This cancer resistance may be based in part on an augmented stress response following DNA damage. PPM1D null mice treated with ionizing radiation display increased p53 protein levels and increased phosphorylation of p38 MAP kinase, p53, checkpoint kinase 1 (Chk1), and checkpoint kinase 2 (Chk2) in their tissues compared to their wild-type (WT) counterparts. Male PPM1D null mice show a modest reduction in longevity, reduced serum insulin-like growth factor 1 (IGF-1) levels, and reduced body weight compared to WT mice. The PPM1D null mouse phenotypes indicate that PPM1D has a homeostatic role in abrogating the DNA damage response and may regulate aspects of male longevity.
Subject(s)
Cell Transformation, Neoplastic/genetics , DNA Damage/genetics , Longevity/genetics , Neoplasm Proteins/genetics , Phosphoprotein Phosphatases/genetics , Aging/genetics , Animals , Body Weight/genetics , Cell Transformation, Neoplastic/metabolism , Checkpoint Kinase 1 , Checkpoint Kinase 2 , Insulin-Like Growth Factor I/analysis , Male , Mice , Mice, Knockout , Neoplasm Proteins/metabolism , Phenotype , Phosphoprotein Phosphatases/metabolism , Phosphorylation , Protein Kinases/metabolism , Protein Phosphatase 1 , Protein Phosphatase 2C , Protein Serine-Threonine Kinases/metabolism , Sex Factors , Tumor Suppressor Protein p53/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
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.
Subject(s)
Aging , Genes, p53 , Animals , Cellular Senescence , Disease Models, Animal , Genes, Tumor Suppressor , Genetic Predisposition to Disease , Humans , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Mutation , Phenotype , Tumor Suppressor Protein p53/physiologyABSTRACT
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.
Subject(s)
Gene Targeting/methods , Genes, p53 , Mice, Knockout , Tumor Suppressor Protein p53/metabolism , Aging/physiology , Animals , Gene Transfer Techniques , Genetic Vectors , Genotype , Humans , Mice , Neoplasms/metabolism , Stem Cells/cytology , Stem Cells/physiology , Tumor Suppressor Protein p53/geneticsABSTRACT
Oval cells are bipotential liver stem cells able to differentiate into hepatocytes and bile duct epithelia. In normal adult liver oval cells are quiescent, existing in low numbers around the periportal region, and proliferate following severe, prolonged liver trauma. There is evidence implicating oval cells in the development of hepatocellular carcinoma, and hence the availability of an immortalized oval cell line would be invaluable for the study of liver cell lineage differentiation and carcinogenesis. A novel approach in the generation of cell lines is the use of the p53 knockout mouse. Absence of p53 allows a cell to cycle past the normal Hayflick limit, rendering it immortalized, although subsequent genetic alterations are thought necessary for transformation. p53 knockout mice were fed a choline-deficient, ethionine-supplemented diet, previously shown to increase oval cell numbers in wild-type mice. The oval cells were isolated by centrifugal elutriation and maintained in culture. Colonies of hepatic cells were isolated and characterized with respect to phenotype, growth characteristics and tumorigenicity. Analysis of gene expression by Northern blotting and immunocytochemistry suggests they are oval-like cells by virtue of albumin and transferrin expression, as well as the oval cell markers alpha fetoprotein, M(2)-pyruvate kinase and A6. Injection into athymic nude mice shows the cell lines are capable of forming tumors which phenotypically resemble hepatocellular carcinoma. Thus, the use of p53 null hepatic cells successfully generated immortalized and tumorigenic hepatic stem cell lines. The results presented support the idea that deleting p53 allows immortalization and contributes to the transformation of the oval-like cell lines. Further, the tumorigenic status of the cell lines is direct evidence for the participation of oval cells in the formation of hepatocellular carcinoma.