Biomedical subjects

Albert J Fornace

Publications and source records attributed to Albert J Fornace.

At least 19 recordsLinked to original sources

PubMed2006

Regulation of ATM/p53-dependent suppression of myc-induced lymphomas by Wip1 phosphatase.

The ataxia telangiectasia mutated (ATM) kinase is a key tumor suppressor that regulates numerous cell cycle checkpoints as well as apoptosis. Here, we report that ATM is a critical player in the regulation of apoptosis and lymphomagenesis in the presence of c-myc. In turn, deletion of the inhibitory ATM phosphatase, Wip1, results in ATM up-regulation and suppression of Emicro-myc-induced B cell lymphomas. Using mouse genetic crosses, we show that the onset of myc-induced lymphomas is dramatically delayed in Wip1-null mice in an ATM- and p53-, but not p38 MAPK- or Arf-, dependent manner. We propose that Wip1 phosphatase is critical for regulating the ATM-mediated tumor surveillance network.

Animals↗

PubMed2006

Wip1 phosphatase modulates ATM-dependent signaling pathways.

Deletion of Ppm1d, the gene encoding the Wip1 phosphatase, renders cells resistant to transformation and mice resistant to tumor development. Here, we report that deficiency of Wip1 resulted in activation of the ataxia-telangiectasia mutated (ATM) kinase. In turn, overexpression of Wip1 was sufficient to reduce activation of the ATM-dependent signaling cascade after DNA damage. Wip1 dephosphorylated ATM Ser1981, a site critical for ATM monomerization and activation, and was critical for resetting ATM phosphorylation as cells repaired damaged DNA. We propose that the Wip1 phosphatase is an integral component of an ATM-dependent signaling pathway.

Animals↗

PubMed2006

G1/S arrest induced by histone deacetylase inhibitor sodium butyrate in E1A + Ras-transformed cells is mediated through down-regulation of E2F activity and stabilization of beta-catenin.

Tumor cells are often characterized by a high and growth factor-independent proliferation rate. We have previously shown that REF cells transformed with oncogenes E1A and c-Ha-Ras do not undergo G(1)/S arrest of the cell cycle after treatment with genotoxic factors. In this work, we used sodium butyrate, a histone deacetylase inhibitor, to show that E1A + Ras transformants were able to stop proliferation and undergo G(1)/S arrest. Apart from inducing G(1)/S arrest, sodium butyrate was shown to change expression of a number of cell cycle regulatory genes. It down-regulated cyclins D1, E, and A as well as c-myc and cdc25A and up-regulated the cyclin-kinase inhibitor p21(waf1). Accordingly, activities of cyclin E-Cdk2 and cyclin A-Cdk2 complexes in sodium butyrate-treated cells were decreased substantially. Strikingly, E2F1 expression was also down-modulated at the levels of gene transcription, the protein content, and the E2F transactivating capability. To further study the role of p21(waf1) in the sodium butyrate-induced G(1)/S arrest and the E2F1 down-modulation, we established E1A + Ras transformants from mouse embryo fibroblast cells with deletion of the cdkn1a (p21(waf1)) gene. Despite the absence of p21(waf1), sodium butyrate-treated mERas transformants reveal a slightly delayed G(1)/S arrest as well as down-modulation of E2F1 activity, implying that the observed effects are mediated through an alternative p21(waf1)-independent signaling pathway. Subsequent analysis showed that sodium butyrate induced accumulation of beta-catenin, a downstream component of the Wnt signaling. The results obtained indicate that the antiproliferative effect of histone deacetylase inhibitors on E1A + Ras-transformed cells can be mediated, alongside other mechanisms, through down-regulation of E2F activity and stabilization of beta-catenin.

Adenovirus E1A Proteins↗

PubMed2006

Effects of expression of p53 and Gadd45 on osmotic tolerance of renal inner medullary cells.

The response of renal inner medullary (IM) collecting duct cells (mIMCD3) to high NaCl involves increased expression of Gadd45 and p53, both of which have important effects on growth and survival of the cells. However, mIMCD3 cells, being immortalized by SV40, proliferate rapidly, which is known to sensitize cells to high NaCl, whereas IM cells in situ proliferate very slowly and survive much higher levels of NaCl. In the present studies, we have examined the importance of Gadd45 and p53 for survival of normal IM cells in their usual high-NaCl environment by using more slowly proliferating second-passage mouse inner medullary epithelial (p2mIME) cells and comparing cells from wild-type and gene knockout mice. Acutely elevating NaCl (and/or urea) reduces Gadd45a, but increases Gadd45b and Gadd45g mRNA, depending on the mix of NaCl and urea and the rate of increase of osmolality. Nevertheless, p2mIME cells from Gadd45b(-/-), Gadd45g(-/-), and Gadd45bg(-/-) mice survive elevation of NaCl (or urea) essentially the same as do wild-type cells. p53(-/-) Cells do not tolerate as high a concentration of NaCl (or urea) as p53(+/+) cells, but urinary concentrating ability of p53(-/-) mice is normal, as is the histology of inner medullas from p53(-/-) and Gadd45abg(-/-) mice. Thus although Gadd45 and p53 may play roles in osmotically stressed mIMCD3 cells, we do not find that their expression makes an important difference, either for Gadd45 in slower proliferating p2mIME cells or for Gadd45 or p53 in normal inner medullary epithelial cells in situ.

Animals↗

PubMed2006

Tumor susceptibility and apoptosis defect in a mouse strain expressing a human p53 transgene.

Activation of apoptosis is believed to be critical for the role of p53 as a tumor suppressor. Here, we report a new mouse strain carrying a human p53 transgene in the mouse p53-null background. Expression of human p53 in these mice was comparable with wild-type murine p53; however, transactivation, induction of apoptosis, and G(1)-S checkpoint, but not transrepression or regulation of a centrosomal checkpoint, were deregulated. Although multiple functions of p53 were abrogated, mice carrying the human p53 transgene did not show early onset of tumors as typically seen for p53-null mice. In contrast, human p53 in the p53-null background did not prevent accelerated tumor development after genotoxic or oncogenic stress. Such behavior of human p53 expressed at physiologic levels in transgenic cells could be explained by unexpectedly high binding with Mdm2. By using Nutlin-3a, an inhibitor of the interaction between Mdm2 and p53, we were able to partially reconstitute p53 transactivation and apoptosis in transgenic cells. Our findings indicate that the interaction between p53 and Mdm2 controls p53 transcriptional activity in homeostatic tissues and regulates DNA damage- and oncogene-induced, but not spontaneous, tumorigenesis.

Animals↗

PubMed2006

Neural tube development requires the cooperation of p53- and Gadd45a-associated pathways.

BACKGROUND: Numerous genetically engineered mouse models for neural tube defects (NTDs) exist, and some of the implicated proteins are functionally related. For example, the growth arrest and DNA damage-inducible protein Gadd45a and tumor suppressor p53 are functionally similar, and both are involved in neural tube development (Gadd45a- and Trp53-null embryos show low levels of exencephaly). To assess their roles in neural tube development, we generated double-null mice from Gadd45a- and Trp53-null mice, as well as from cyclin-dependent kinase inhibitor (Cdkn1a) (p21)-null and xeroderma pigmentosum group C (XPC)-null mice that do not show spontaneous exencephaly. METHODS: Gadd45a-, Trp53-, Cdkn1a-, and XPC-null mice were crossed to generate several double-null mouse models. Embryos (embryonic day [ED] 16-18) from the single- and double-null crosses were scored for NTDs. RESULTS: Deletion of both Gadd45a and Trp53 in mice increased exencephaly frequencies compared to the deletion of either single gene (34.0% in Gadd45a/Trp53-null compared to 8.4% and 9.1% in the Gadd45a- and Trp53-null embryos, respectively). Furthermore, although deletion of another p53-regulated gene, Cdkn1a, is not associated with exencephaly, in conjunction with Gadd45a deletion, the exencephaly frequencies are increased (30.5% in the Gadd45a/Cdkn1a-null embryos) and are similar to those in the Gadd45a/Trp53-null embryos. Although XPC deletion increased exencephaly frequencies in Trp53-null embryos, XPC deletion did not increase the exencephaly frequencies in Gadd45a-null embryos. CONCLUSIONS: The increased genetic liability to exencephaly in the Gadd45a/Trp53- and Gadd45a/Cdkn1a-null embryos may be related to the disruption of multiple cellular pathways associated with Gadd45a and p53.

Animals↗

PubMed2006

Gadd34 requirement for normal hemoglobin synthesis.

The protein encoded by growth arrest and DNA damage-inducible transcript 34 (Gadd34) is associated with translation initiation regulation following certain stress responses. Through interaction with the protein phosphatase 1 catalytic subunit (PP1c), Gadd34 recruits PP1c for the removal of an inhibitory phosphate group on the alpha subunit of elongation initiation factor 2, thereby reversing the shutoff of protein synthesis initiated by stress-inducible kinases. In the absence of stress, the physiologic consequences of Gadd34 function are not known. Initial analysis of Gadd34-null mice revealed several significant findings, including hypersplenism, decreased erythrocyte volume, increased numbers of circulating erythrocytes, and decreased hemoglobin content, resembling some thalassemia syndromes. Biochemical analysis of the hemoglobin-producing reticulocyte (an erythrocyte precursor) revealed that the decreased hemoglobin content in the Gadd34-null erythrocyte is due to the reduced initiation of the globin translation machinery. We propose that an equilibrium state exists between Gadd34/PP1c and the opposing heme-regulated inhibitor kinase during hemoglobin synthesis in the reticulocyte.

Animals↗

PubMed2005

Hematopoietic cells from Gadd45a- and Gadd45b-deficient mice are sensitized to genotoxic-stress-induced apoptosis.

Gadd45a, gadd45b and gadd45g (Gadd45/MyD118/CR6) are genes that are rapidly induced by genotoxic stress. However, the exact function of Gadd45 proteins in the response of mammalian cells to genotoxic stress is unclear. Here, advantage was taken of gadd45a- and gadd45b-deficient mice to determine the role gadd45a and gadd45b play in the response of bone marrow (BM) cells to genotoxic stress. BM cells from gadd45a- and gadd45b-deficient mice were observed to be more sensitive to ultraviolet radiation chemotherapy (UVC), VP-16 and daunorubicin (DNR)-induced apoptosis compared to wild-type (wt) cells. The increased apoptosis in gadd45a- and gadd45b-deficient cells was evident also by enhanced activation of caspase-3 and poly-ADP-ribose polymerase cleavage and decreased expression of c-inhibitor of apoptotic protein-1, Bcl-2, Bcl-xL compared to wt cells. Reintroduction of gadd45 into gadd45-deficient BM cells restored the wt apoptotic phenotype. Both gadd45a- and gadd45b-deficient BM cells also displayed defective G2/M arrest following exposure to UVC and VP-16, but not to DNR, indicating the existence of different G2/M checkpoints that are either dependent or independent of gadd45. Taken together, these findings identify gadd45a and gadd45b as anti-apoptotic genes that increase the survival of hematopoietic cells following exposure to UV radiation and certain anticancer drugs.

Animals↗

PubMed2005

Inhibition of the ATR/Chk1 pathway induces a p38-dependent S-phase delay in mouse embryonic stem cells.

Ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinases, family members of the PI-3 kinase related proteins, play a key role in checkpoint activation and maintenance of genomic stability following DNA damage. We have used wild type (WT) and p38alpha-deficient mouse embryonic stem (ES) cells to investigate the role of ATR and ATM kinases during embryonic cell cycle. We have found that inhibition of ATR and ATM kinases with caffeine or Chk1 with UCN-01, results in activation of a p38-dependent intra-S-phase checkpoint and activation of apoptosis in ES cells. However, wortmannin at a concentration, that inhibits ATM kinase but not ATR kinase, did not affect cell cycle progression. Furthermore, the presence of caffeine results in activation of p38 kinase, accumulation of p21/Waf1 in a complex with Cdk2 and decrease of Cdk2 kinase activity. In contrast, caffeine-treated p38alpha-/- ES cells show less apoptosis, and fail to trigger an effective S-phase checkpoint and accumulation of p21/Waf1. We conclude that ATR kinase activity is essential for normal cell cycle progression of exponentially proliferating mouse ES cells even in the absence of exogenous DNA damage, and ATR deregulation triggers p38alpha-dependent cell-cycle checkpoint and apoptotic responses.

Animals↗

PubMed2005

Chemical inhibition of Wip1 phosphatase contributes to suppression of tumorigenesis.

Wip1 is an amplified oncogene whose deletion causes a tumor resistant phenotype in mice. These observations provide justification for a search for Wip1 chemical inhibitors as potential anticancer drugs. Here we report a group of Wip1 inhibitors with anticancer properties both in vitro and in vivo. In vitro, inactivation of Wip1 reduces the proliferation rate of breast cancer cell lines and enhances growth inhibition caused by doxorubicin. In vivo, administration of Wip1 inhibitors decreases proliferation of xenograph tumors and tumors developed in MMTV-c-Neu transgenic mice. We propose that these agents may serve as lead compounds for the development of anticancer drugs targeting Wip1 phosphatase.

Adenoviridae↗

PubMed2005

Activating p38 MAPK: new tricks for an old kinase.

Mitogen-activated protein kinases (MAPKs) participate in signaling initiated by a wide variety of extracellular stimuli. MAPKs are most commonly activated by a series of phosphorylation events in which one kinase phosphorylates another, the "MAPK cascade". The cascade concludes with the dual phosphorylation of MAPKs on a conserved Thr-X-Tyr motif. In the case of the p38 MAPK, an exception to this paradigm has been found when signaling via the T cell antigen receptor (TCR). Rather than trigger the MAPK cascade, TCR-mediated stimulation activates proximal tyrosine kinases, which results in the phosphorylation of p38 on a noncanonical activating residue, Tyr-323. This phosphorylation activates p38 to phosphorylate third party substrates as well as its own Thr-Gyl-Tyr motif. Here we discuss the structural and functional implications of this alternative p38 activation pathway, which may provide a new target for tissue-specific pharmacologic inhibition.

Amino Acid Motifs↗

PubMed2005

Deletion of XPC leads to lung tumors in mice and is associated with early events in human lung carcinogenesis.

Chromosome 3p and 1p deletions are among the most frequent genetic changes in human lung cancer and although candidate tumor suppressor genes have been identified in these regions, no causative correlations have been drawn between deletion or mutation of these and lung carcinogenesis. We identify XPC and Gadd45a as genes within each of these regions involved in lung tumor initiation and progression, respectively. One hundred percent of XPC-/- mice develop multiple spontaneous lung tumors with a minority progressing to non-small cell lung adenocarcinoma, occasionally with metastasis to adjacent lymph nodes. Deletion of Gadd45a alone does not lead to increased lung tumors in mice, but coupled with an XPC deletion, it results in lung tumor progression. Analysis of published data indicated allelic loss of XPC in most human lung tumors and allelic loss of Gadd45a in some human lung and other cancer types. Because DNA repair capacity is compromised in XPC+/- cells, it is possible that the loss of a single XPC allele in the human lung might confer a mutator phenotype. Coupled with cigarette carcinogens, decreased DNA repair would lead to additional mutations in genes such as p53 that are frequent targets in lung cancer.

Alleles↗

PubMed2005

Stress-specific signatures: expression profiling of p53 wild-type and -null human cells.

Gene expression responses of human cell lines exposed to a diverse set of stress agents were compared by cDNA microarray hybridization. The B-lymphoblastoid cell line TK6 (p53 wild-type) and its p53-null derivative, NH32, were treated in parallel to facilitate investigation of p53-dependent responses. RNA was extracted 4 h after the beginning of treatment when no notable decrease in cell viability was evident in the cultures. Gene expression signatures were defined that discriminated between four broad general mechanisms of stress agents: Non-DNA-damaging stresses (heat shock, osmotic shock, and 12-O-tetradecanoylphorbol 13-acetate), agents causing mainly oxidative stress (arsenite and hydrogen peroxide), ionizing radiations (neutron and gamma-ray exposures), and other DNA-damaging agents (ultraviolet radiation, methyl methanesulfonate, adriamycin, camptothecin, and cis-Platinum(II)diammine dichloride (cisplatin)). Within this data set, non-DNA-damaging stresses could be discriminated from all DNA-damaging stresses, and profiles for individual agents were also defined. While DNA-damaging stresses showed a strong p53-dependent element in their responses, no discernible p53-dependent responses were triggered by the non-DNA-damaging stresses. A set of 16 genes did exhibit a robust p53-dependent pattern of induction in response to all nine DNA-damaging agents, however.

B-Lymphocytes↗

PubMed2005

Cytokine-driven cell cycling is mediated through Cdc25A.

Lymphocytes are the central mediators of the immune response, requiring cytokines for survival and proliferation. Survival signaling targets the Bcl-2 family of apoptotic mediators, however, the pathway for the cytokine-driven proliferation of lymphocytes is poorly understood. Here we show that cytokine-induced cell cycle progression is not solely dependent on the synthesis of cyclin-dependent kinases (Cdks) or cyclins. Rather, we observe that in lymphocyte cell lines dependent on interleukin-3 or interleukin-7, or primary lymphocytes dependent on interleukin 7, the phosphatase Cdc25A is the critical mediator of proliferation. Withdrawal of IL-7 or IL-3 from dependent lymphocytes activates the stress kinase, p38 MAPK, which phosphorylates Cdc25A, inducing its degradation. As a result, Cdk/cyclin complexes remain phosphorylated and inactive and cells arrest before the induction of apoptosis. Inhibiting p38 MAPK or expressing a mutant Cdc25A, in which the two p38 MAPK target sites, S75 and S123, are altered, renders cells resistant to cytokine withdrawal, restoring the activity of Cdk/cyclin complexes and driving the cell cycle independent of a growth stimulus.

Animals↗

PubMed2005

The autoimmune suppressor Gadd45alpha inhibits the T cell alternative p38 activation pathway.

The p38 MAP kinase (MAPK) is phosphorylated and activated by upstream MAPK kinases. T cells have an alternative pathway in which T cell receptor-activated tyrosine kinase Zap70 phosphorylates p38 on Tyr323. Mice lacking Gadd45alpha, a small p38-binding molecule, develop a lupus-like autoimmune disease. Here we show that resting T cells but not B cells from Gadd45a(-/-) mice had spontaneously increased p38 activity in the absence of 'upstream' MAPK kinase activation. The p38 from resting Gadd45a(-/-) T cells was spontaneously phosphorylated on Tyr323, and its activity was specifically inhibited by recombinant Gadd45alpha in vitro. Thus, constitutive activation of T cell p38 through the alternative pathway is prevented by Gadd45alpha, the absence of which results in p38 activation, T cell hyperproliferation and autoimmunity.

Animals↗

PubMed2005

Alternative p38 activation pathway mediated by T cell receptor-proximal tyrosine kinases.

Signaling-responsive MAP kinases (MAPKs) are key in mediating immune responses and are activated through the phosphorylation of a Thr-X-Tyr motif by upstream MAPK kinases. Here we show that T cells stimulated through the T cell receptor (TCR) used an alternative mechanism in which p38 was phosphorylated on Tyr323 and subsequently autophosphorylated residues Thr180 and Tyr182. This required the TCR-proximal tyrosine kinase Zap70 but not the adaptor protein LAT, which was required for activation of extracellular signal-regulated protein kinase MAPKs. TCR activation of p38 lacking Tyr323 was diminished, and blocking of p38 activity prevented p38 dual phosphorylation in normal T cells but not in B cells. Thus, phosphorylation of Tyr323 dependent on the tyrosine kinase Lck and mediated by Zap70 serves as an important mechanism for TCR activation of p38 in T cells.

Animals↗

PubMed2005

Loss of Gadd45a does not modify the pulmonary response to oxidative stress.

It is well established that exposure to high levels of oxygen (hyperoxia) injures and kills microvascular endothelial and alveolar type I epithelial cells. In contrast, significant death of airway and type II epithelial cells is not observed at mortality, suggesting that these cell types may express genes that protect against oxidative stress and damage. During a search for genes induced by hyperoxia, we previously reported that airway and alveolar type II epithelial cells uniquely express the growth arrest and DNA damage (Gadd)45a gene. Because Gadd45a has been implicated in protection against genotoxic stress, adult Gadd45a (+/+) and Gadd45a (-/-) mice were exposed to hyperoxia to investigate whether it protected epithelial cells against oxidative stress. During hyperoxia, Gadd45a deficiency did not affect loss of airway epithelial expression of Clara cell secretory protein or type II epithelial cell expression of pro-surfactant protein C. Likewise, Gadd45a deficiency did not alter recruitment of inflammatory cells, edema, or overall mortality. Consistent with Gadd45a not affecting the oxidative stress response, p21(Cip1/WAF1) and heme oxygenase-1 were comparably induced in Gadd45a (+/+) and Gadd45a (-/-) mice. Additionally, Gadd45a deficiency did not affect oxidative DNA damage or apoptosis as assessed by oxidized guanine and terminal deoxyneucleotidyl transferase-mediated dUTP nick-end labeling staining. Overexpression of Gadd45a in human lung adenocarcinoma cells did not affect viability or survival during exposure, whereas it was protective against UV-radiation. We conclude that increased tolerance of airway and type II epithelial cells to hyperoxia is not attributed solely to expression of Gadd45a.

Adenocarcinoma↗

PubMed2005

Casein kinase 2- and protein kinase A-regulated adenomatous polyposis coli and beta-catenin cellular localization is dependent on p38 MAPK.

Skin cancer is the most common form of malignancy in the world with epidemic proportions. Identifying the biochemical and molecular mechanisms underlying the events leading to tumors is paramount to designing new and effective treatments that may aid in treating and/or preventing skin cancers. Herein we identify p38 MAPK, along with its positive modulator, Gadd45a, as important regulators of nucleocytoplasmic shuttling of the adenomatous polyposis coli (APC) tumor suppressor. APC normally functions to block beta-catenin from promoting cell proliferation and migration/invasion. Keratinocytes lacking proper p38 MAPK activation, either due to lack of Gadd45a or through the use of p38 MAPK-specific inhibitors, are unable to effectively transport APC into the nucleus. We also show that p38 MAPK is able to directly associate with and modulate both casein kinase 2 (CK2) and protein kinase A (PKA), which promote and block APC nuclear import, respectively. We demonstrate that p38 MAPK is able to not only enhance CK2 kinase activity but also suppress PKA kinase activity. Moreover, lack of normal p38 MAPK activity in either Gadd45a-null keratinocytes or in p38 MAPK inhibitor treated keratinocytes leads to decreased CK2 activity and increased PKA activity. In either case, disruption of APC nuclear import results in elevated levels of free cellular, and potentially oncogenic, beta-catenin. Numerous tumors, including skin cancers, are associated with high levels of beta-catenin, and our data indicate that p38 MAPK signaling, along with Gadd45a, may provide tumor suppressor-like functions in part by promoting APC nuclear localization and effective beta-catenin regulation.

Adenomatous Polyposis Coli↗