Human DMTF1β antagonizes DMTF1α regulation of the p14(ARF) tumor suppressor and promotes cellular proliferation

The human DMTF1 (DMP1) transcription factor, a DNA binding protein that interacts with cyclin D, is a positive regulator of the p14ARF (ARF) tumor suppressor. Our earlier studies have shown that three differentially spliced human DMP1 mRNAs, α, β and γ, arise from the human gene. We now show that DMP1α, β and γ isoforms differentially regulate ARF expression and promote distinct cellular functions. In contrast to DMP1α, DMP1β and γ did not activate the ARF promoter, whereas only β resulted in a dose-dependent inhibition of DMP1α-induced transactivation of the ARF promoter. Ectopic expression of DMP1β reduced endogenous ARF mRNA levels in human fibroblasts. The DMP1β- and γ-isoforms share domains necessary for the inhibitory function of the β-isoform. That DMP1β may interact with DMP1α to antagonize its function was shown in DNA binding assays and in cells by the close proximity of DMP1α/β in the nucleus. Cells stably expressing DMP1β, as well as shRNA targeting all DMP1 isoforms, disrupted cellular growth arrest induced by serum deprivation or in PMA-derived macrophages in the presence or absence of cellular p53. DMP1 mRNA levels in acute myeloid leukemia samples, as compared to granulocytes, were reduced. Treatment of acute promyelocytic leukemia patient samples with all-trans retinoic acid promoted differentiation to granulocytes and restored DMP1 transcripts to normal granulocyte levels. Our findings imply that DMP1α- and β-ratios are tightly regulated in hematopoietic cells and DMP1β antagonizes DMP1α transcriptional regulation of ARF resulting in the alteration of cellular control with a gain in proliferation.

Ritonavir inhibits intratumoral docetaxel metabolism and enhances docetaxel antitumor activity in an immunocompetent mouse breast cancer model

Docetaxel (Taxotere(®) ) is currently used intravenously as an anticancer agent and is primarily metabolized by Cytochrome P450 3A (CYP3A). The HIV protease inhibitor ritonavir, a strong CYP3A4 inhibitor, decreased first-pass metabolism of orally administered docetaxel. Anticancer effects of ritonavir itself have also been described. We here aimed to test whether ritonavir co-administration could decrease intratumoral metabolism of intravenously administered docetaxel and thus increase the antitumor activity of docetaxel in an orthotopic, immunocompetent mouse model for breast cancer. Spontaneously arising K14cre;Brca1(F/F) ;p53(F/F) mouse mammary tumors were orthotopically implanted in syngeneic mice lacking Cyp3a (Cyp3a(-/-) ) to limit ritonavir effects on systemic docetaxel clearance. Over 3 weeks, docetaxel (20 mg/kg) was administered intravenously once weekly, with or without ritonavir (12.5 mg/kg) administered orally for 5 days per week. Untreated mice were used as control for tumor growth. Ritonavir treatment alone did not significantly affect the median time of survival (14 vs. 10 days). Median time of survival in docetaxel-treated mice was 54 days. Ritonavir co-treatment significantly increased this to 66 days, and substantially reduced relative average tumor size, without altering tumor histology. Concentrations of the major docetaxel metabolite M2 in tumor tissue were reduced by ritonavir co-administration, whereas tumor RNA expression of Cyp3a was unaltered. In this breast cancer model, we observed no direct antitumor effect of ritonavir alone, but we found enhanced efficacy of docetaxel treatment when combined with ritonavir. Our data, therefore, suggest that decreased docetaxel metabolism inside the tumor as a result of Cyp3a inhibition contributes to increased antitumor activity.

p73 regulates basal and starvation-induced liver metabolism in vivo

As a member of the p53 gene family, p73 regulates cell cycle arrest, apoptosis, neurogenesis, immunity and inflammation. Recently, p73 has been shown to transcriptionally regulate selective metabolic enzymes, such as cytochrome c oxidase subunit IV isoform 1, glucose 6-phosphate dehydrogenase and glutaminase-2, resulting in significant effects on metabolism, including hepatocellular lipid metabolism, glutathione homeostasis and the pentose phosphate pathway. In order to further investigate the metabolic effect of p73, here, we compared the global metabolic profile of livers from p73 knockout and wild-type mice under both control and starvation conditions. Our results show that the depletion of all p73 isoforms cause altered lysine metabolism and glycolysis, distinct patterns for glutathione synthesis and Krebs cycle, as well as an elevated pentose phosphate pathway and abnormal lipid accumulation. These results indicate that p73 regulates basal and starvation-induced fuel metabolism in the liver, a finding that is likely to be highly relevant for metabolism-associated disorders, such as diabetes and cancer.

An Intergenic Regulatory Region Mediates Drosophila Myc -Induced Apoptosis and Blocks Tissue Hyperplasia

Induction of cell-autonomous apoptosis following oncogene-induced overproliferation is a major tumor-suppressive mechanism in vertebrates. However, the detailed mechanism mediating this process remains enigmatic. In this study, we demonstrate that dMyc-induced cell-autonomous apoptosis in the fruit fly Drosophila melanogaster relies on an intergenic sequence termed the IRER (irradiation-responsive enhancer region). The IRER mediates the expression of surrounding proapoptotic genes, and we use an in vivo reporter of the IRER chromatin state to gather evidence that epigenetic control of DNA accessibility within the IRER is an important determinant of the strength of this response to excess dMyc. In a previous work, we showed that the IRER also mediates P53-dependent induction of proapoptotic genes following DNA damage, and the chromatin conformation within IRER is regulated by polycomb group-mediated histone modifications. dMyc-induced apoptosis and the P53-mediated DNA damage response thus overlap in a requirement for the IRER. The epigenetic mechanisms controlling IRER accessibility appear to set thresholds for the P53- and dMyc-induced expression of apoptotic genes in vivo and may have a profound impact on cellular sensitivity to oncogene-induced stress.

Expression cloning of a candidate prostate cancer oncogene

Genetic analysis, both karyotyping and comparative genomic hybridization, of prostate cancer cell lines and specimens have revealed multiple areas of concordant increases in DNA content. An increase of DNA in specific regions of the genome in cancer is often associated with the amplification of oncogenes. Based on these observations we have hypothesized that oncogenes are involved in the initiation or progression of prostate cancer. An expression cloning approach was utilized to identify candidate oncogenes in prostate cancer. ^ A full-length, unidirectional cDNA expression library was constructed from DU145 prostate cancer cells. The cDNA library was screened using CP12, a rat prostate epithelial cell line. In soft agarose assays, CP12 (parental or vector transfected) do not form colonies. However, upon the introduction of a number of known oncogenes CP12 becomes anchorage independent in soft agarose. Based on this in-vitro phenotypic shift, a DU145 cDNA library was stably transfected into CP12, and selected for anchorage independence. Two hundred fifty nine anchorage independent clones were isolated. Some colonies contained more than one insert, bringing the candidate oncogene pool to approximately 400. Seven inserts were sequenced at random. Using the sequences obtained, GenBank was screened, and matches were found with p53, PARG1, a mitochondrial ATPase, RNF6, and three unknown genes that mapped to Unigene clusters. As the pool of cDNA inserts appeared promising, overexpressed genes were further selected. From 259 clones, 17 clones were overexpressed more than 6-fold in DU145 compared to Normal Prostate. From the 17 clones, 12 cDNA inserts were strongly expressed in DU145 and were isolated for sequencing. ^ Two of the sequences, 1G6 and 3E9, were identical. Expression of 1G6/2G9/3E9 was tested by RT-PCR. 1G6/2G9/3E9 was not expressed in normal prostate, but was expressed in all prostate cancer cell lines tested as well as six prostate cancer samples. When retransfected into CP12, 1G6/2G9/3E9 induced the formation of foci and anchorage independent colonies. Thus, functional and expression data suggest that 1G6/2G9/3E9 may be a prostate cancer oncogene. ^

Molecular mechanism of PUVA-induced apoptosis in mouse epidermal cells

A combination of psoralens and ultraviolet-A radiation referred to as PUVA, is widely used in the treatment of psoriasis. PUVA therapy is highly effective in killing hyperproliferative cells, but its mechanism of action has not been fully elucidated. Psoralen binds to DNA, and upon photoactivation by UVA, it forms monofunctional adducts and interstrand cross-links. PUVA treatment has been shown to be mutagenic and to produce tumors in animals. In addition, epidemiological studies have reported a 10 to 15 percent increased risk of developing squamous cell carcinoma in individuals treated chronically with PUVA. However, it remains a treatment for skin disorders such as psoriasis because its benefits outweigh its risks. The widespread use of PUVA therapy and its associated cancer risk requires us to understand the molecular mechanisms by which PUVA induces cell death. Immortalized JB6 mouse epidermal cells, p53−/− mice, and Fas Ligand−/− (gld) mice were used to investigate the molecular mechanism by which PUVA kills cells. Treatment of JB6 cells with 10 μg/ml 8-methoxypsoralen followed by irradiation with 20 kJ/m2 UVA resulted in cell death. The cells exhibited morphological and biochemical characteristics of apoptosis such as chromatin condensation, DNA ladder formation, and TUNEL-positivity. PUVA treatment stabilized and phosphorylated p53 leading to its activation, as measured by nuclear localization and induction of p21Waf/Cip1, a transcriptional target of p53. Subsequent in vivo studies revealed that there was statistically significantly less apoptosis in p53 −/− mice than in p53+/+ mice at 72 hours after PUVA. In addition, immunohistochemical analysis revealed more Fas and FasL expression in p53+/+ mice than in p53−/− mice, suggesting that p53 is required to transcriptionally activate Fas, which in turn causes the cells to undergo apoptosis. Studies with gld mice confirmed a role for Fas/FasL interactions in PUVA-induced apoptosis. There was statistically significantly less apoptosis in gld mice compared with wild-type mice 24, 48, and 72 hours after PUVA. These results demonstrate that PUVA-induced apoptosis in mouse epidermal cells requires p53 and Fas/FasL interactions. These findings may be important for designing effective treatments for diseases such as psoriasis without increasing the patient's risk for skin cancer. ^

Integrin mediated growth and apoptosis in human gastric adenocarcinoma

Integrins are important as the primary cell adhesion molecule providing information about the extracellular microenvironment to the interior of the cell to influence cellular behavior such as differentiation, proliferation and apoptosis. Apoptotic death due to loss of adhesion is termed anoikis. In this study we have obtained a parental human gastric adenocarcinoma cell line that yielded two variant lines that had differing responses to lack of adhesion. The STAD.APO cell line undergoes apoptosis when denied adherence and the STAD.ARR cell line enters into cell cycle arrest under the identical suspended conditions. We have shown that cyclin A and cyclin D mRNA and protein are down regulated when cells are denied adherence for 24 hours in tissue culture wells previously coated with poly-HEMA. To test whether cyclin A was able to rescue cells from cell cycle arrest and/or anoikis by overriding the cell cycle machinery we transfected the full length cDNA in to each cell type. Surprisingly we found that anoikis and cell cycle arrest due to suspended conditions was not affected by overexpression of cyclin A protein, but that growth under adhered conditions was reduced compared to vector alone control transfectants. Further, we transfected other cell lines; ST7, gastric cancer, MDA-MB-4.35, breast cancer, and HPB T-cell leukemic and in no case were suspended culturing conditions overcome by cyclin A. This result indicates an additional level of regulation for the cell cycle machinery. Additionally, soluble collagen was shown to be able to save from anoikis and also from cell cycle arrest while the β1 specific mAb 33B6 was only able to save from anoikis. Immunofluorescent studies show that soluble collagen creates clusters of β1 with FAK and also β1 with actin in the STAD.ARR cells but does not in the STAD.APO cells. This result indicates that the phenotypes under suspended conditions between these cell lines may diverge at their requirements for integrin ligation. Additionally we characterized the nature of anoikis by showing cytochrome c release, caspase 3, p21 and p53 activation in STAD.APO cells. Thus, our results have implications in the understanding of integrin biology and neoplastic progression. ^

Mechanisms of apoptosis in bleomycin-induced lung injury: Implications for pulmonary fibrosis

Chronic inflammation leading to pulmonary fibrosis develops in response to environmental pollutants, radiotherapy, or certain cancer chemotherapeutic agents. Studies have shown that several cell types accumulate during the inflammatory process, but little information is known about what actually triggers and stimulates persistent inflammation culminating in fibrosis. As a first step in defining the events that precipitate inflammation in the lung, the biological mechanism(s) mediating apoptosis and cellular targets must be identified. The purpose of this study was to determine the molecular mechanism(s) of bleomycin-induced apoptosis in the lung using mice deficient in genes that we hypothesized to play a key role in apoptosis. Intratracheal administration of bleomycin led to caspase-mediated DNA fragmentation characteristic of apoptosis. The effects of bleomycin were associated with translocation of p53 from the cytosol to the nucleus only in alveolar macrophages that had been exposed to the drug in vivo, suggesting that the lung microenvironment regulated p53 activation. Experiments with a thiol antioxidant (N-acetylcysteine) in vivo and nitric oxide donors in vitro confirmed that reactive oxygen species were required for p53 activation. A specific role for NO was demonstrated in experiments with iNOS−/− macrophages, which failed to demonstrate nuclear p53 localization after in vivo bleomycin exposure. Strikingly, rates of bleomycin-induced apoptosis were at least two-fold higher in iNOS−/− and p53−/− C57BL/6 mice compared to wild-type controls. Laser Scanning Cytometry (LSC) analysis revealed that bleomycin exposure resulted in a 2-fold induction in Fas and FasL expression in wild-type mice but not iNOS−/− or p53−/− mice. Experiments using gld mice confirmed that the Fas/FasL pathway was the primary mechanism of bleomycin-induced apoptosis in the lung. LSC-mediated analysis indicated that bleomycin exposure resulted in a 2-fold induction in Bax expression in iNOS−/− and P53−/− mice but not wild-type mice. Furthermore, LSC analysis revealed that bleomycin exposure induced a 3-fold increase in thrombospondin expression in wild-type mice. However, thrombospondin was not expressed in either the iNOS−/− or p53−/− mice, implicating a thrombospondin-mediated apoptotic cell clearance mechanism in the lung. Together, these results demonstrate that iNOS and p53 positively regulate apoptosis via the Fas/FasL pathway and mediate a novel apoptosis-suppressing pathway in the lung. ^

4HPR-X radiation mechanisms of interactions in non-small cell lung cancer (NSCLC) cells in vitro

Lung cancer is the leading cause of cancer death. However, poor survival using conventional therapies fuel the search for more rational interventions. The objective of this study was to design and implement a 4HPR-radiation interaction model in NSCLC, employing a traditional clinical modality (radiation), a relatively new, therapeutically unexplored agent (4HPR) and rationally combining them based on molecular mechanistic findings pertaining to their interactions. To test the hypothesis that 4HPR sensitizes cells to radiation-induced cell death via G2+M accumulation, we designed a working model consisting of H522 adenocarcinoma cells (p53, K-ras mutated) derived from an NSCLC patient; 4HPR at concentrations up to 10 μM; and X radiation up to 6 Gy generated by a patient-dedicated Phillips RT-250 X ray unit at 250 KV, 15 mA, 1.85 Gy/min. We found that 4HPR produced time- and dose-dependent morphological changes, growth inhibition, and DNA damage-inducing enhancement of reactive oxygen species. A transient G2+M accumulation of cells maximal at 24 h of continuous 4HPR exposure was used for irradiation time scheduling. Our data demonstrated enhanced cell death (both apoptotic and necrotic) in irradiated cells pre-treated with 4HPR versus those with either stressor alone. 4HPR's effect of increased NSCLC cells' radioresponse was confirmed by clonogenic assay. To explore these practical findings from a molecular mechanistic perspective, we further investigated and showed that levels of cyclin B1 and p34cdc2 kinase—both components of the mitosis promoting factor (MPF) regulating the G2/M transition—did not change following 4HPR treatment. Likewise, cdc25C phosphatase was not altered. However, enhanced p34cdc2 phosphorylation on its Thr14Tyr15 residues—indicative of its inactivation and increased expression of MPF negative regulators chk1 and wee1 kinases—were supportive of explaining 4HPR-treated cells' accumulation. Hence, p34cdc2 phosphorylation, chk1, and wee1 warrant further evaluation as potential molecular targets for 4HPR-X radiation combination. In summary, we (1) demonstrated that 4HPR not only induces cell death by itself, but also increases NSCLC cells' subsequent radioresponse, indicative of potential clinical applicability, and (2) for the first time, shed light on deciphering 4HPR-X radiation molecular mechanisms of interaction, including the finding of 4HPR's role as a p34cdc2 inactivator via Thr14Tyr15 phosphorylation. ^

The utility of N5 for gene therapy of human cancer

Cancer is a result of defects in the coordination of cell proliferation and programmed cell death. The extent of cell death is physiologically controlled by the activation of a programmed suicide pathway that results in a morphologically recognizable form of death termed apoptosis. Inducing apoptosis in tumor cells by gene therapy provides a potentially effective means to treat human cancers. The p84N5 is a novel nuclear death domain containing protein that has been shown to bind an amino terminal domain of retinoblastoma tumor suppressor gene product (pRb). Expression of N5 can induce apoptosis that is dependent upon its intact death domain and is inhibited by pRb. In many human cancer cells the functions of pRb are either lost through gene mutation or inactivated by different mechanisms. N5 based gene therapy may induce cell death preferentially in tumor cells relative to normal cells. We have demonstrated that N5 gene therapy is less toxic to normal cells than to tumor cells. To test the possibility that N5 could be used in gene therapy of cancer, we have generated a recombinant adenovirus engineered to express N5 and test the effects of viral infection on growth and tumorigenicity of human cancer cells. Adenovirus N5 infection significantly reduced the proliferation and tumorigenicity of breast, ovarian, and osteosarcoma tumor cell lines. Reduced proliferation and tumorigenicity were mediated by an induction of apoptosis as indicated by DNA fragmentation in infected cells. We also test the potential utility of N5 for gene therapy of pancreatic carcinoma that typically respond poorly to conventional treatment. Adenoviral mediated N5 gene transfer inhibits the growth of pancreatic cancer cell lines in vitro. N5 gene transfer also reduces the growth and metastasis of human pancreatic adenocarcinoma in subcutaneous and orthotopic mouse model. Interestingly, the pancreatic adenocarcinoma cells are more sensitive to N5 than they are to p53, suggesting that N5 gene therapy may be effective in tumors resistant to p53. We also test the possibilities of the use of N5 and p53 together on the inhibition of pancreatic cancer cell growth in vitro and vivo. Simultaneous use of N5 and RbΔCDK has been found to exert a greater extent on the inhibition of pancreatic cancer cell growth in vitro and in vivo. ^

Mechanism of damage sensing and cell killing following the inhibition of nucleotide excision repair by fludarabine in quiescent cells

Inhibition of DNA repair by the nucleoside of fludarabine (F-ara-A) induces toxicity in quiescent human cells. The sensing and signaling mechanisms following DNA repair inhibition by F-ara-A are unknown. The central hypothesis of this project was that the mechanistic interaction of a DNA repair initiating agent and a nucleoside analog initiates an apoptotic signal in quiescent cells. The purpose of this research was to identify the sensing and signaling mechanism(s) that respond to DNA repair inhibition by F-ara-A. Lymphocytes were treated with F-ara-A, to accumulate the active triphosphate metabolite and subsequently DNA repair was activated by UV irradiation. Pre-incubation of lymphocytes with 3 μM F-ara-A inhibited DNA repair initiated by 2 J/m2 UV and induced greater than additive apoptosis after 24 h. Blocking the incorporation of F-ara-A nucleotide into repairing DNA using 30 μM aphidicolin considerably lowered the apoptotic response. ^ Wild-type quiescent cells showed a significant loss in viability than did cells lacking functional sensor kinase DNA-PKcs or p53 as measured by colony formation assays. The functional status of ATM did not appear to affect the apoptotic outcome. Immunoprecipitation studies showed an interaction between the catalytic sub-unit of DNA-PK and p53 following DNA repair inhibition. Confocal fluorescence microscopy studies have indicated the localization pattern of p53, DNA-PK and γ-H2AX in the nucleus following DNA damage. Foci formation by γ-H2AX was seen as an early event that is followed by interaction with DNA-PKcs. p53 serine-15 phosphorylation and accumulation were detected 2 h after treatment. Fas/Fas ligand expression increased significantly after repair inhibition and was dependent on the functional status of p53. Blocking the interaction between Fas and Fas ligand by neutralizing antibodies significantly rescued the apoptotic fraction of cells. ^ Collectively, these results suggest that incorporation of the nucleoside analog into repair patches is critical for cytotoxicity and that the DNA damage, while being sensed by DNA-PK, may induce apoptosis by a p53-mediated signaling mechanism. Based on the results, a model is proposed for the sensing of F-ara-A-induced DNA damage that includes γ-H2AX, DNA-PKcs, and p53. Targeting the cellular DNA repair mechanism can be a potential means of producing cytotoxicity in a quiescent population of neoplastic cells. These results also provide mechanistic support for the success of nucleoside analogs with cyclophosphamide or other agents that initiate excision repair processes, in the clinic. ^

The role of UV radiation in the development of myeloid malignancy in Rag2 knockout mice

Excessive exposure to the UV radiation present in sunlight can lead to the development of skin cancer in humans. Majority of the UV-induced skin tumors in immune-competent mice are highly antigenic in nature. Additionally, they exhibit a high frequency of mutations in the p53 gene, which arise very early in the course of UV radiation and most of them disappear before the development of skin tumors. ^ Initially, this study was to determine whether UV radiation induces skin tumors much earlier in immune deficient Rag2 knockout mice than in immune-competent mice, and if so, compare their antigenic properties and p53 mutation spectra. However, chronic UV irradiation (10 kJ/m2) induced myeloproliferative disease (MPD) as early as 4 weeks in Rag2 knockout mice instead of skin tumors. Conversely, unirradiated Rag2 knockout mice developed MPD at a low frequency, but the frequency increased with the animal's age. Although the UV-irradiated wild type mice (B6129) developed MPD, its frequency was lower and the occurrence much later than the Rag2 knockout mice. ^ This observation led to our new hypothesis that UV irradiation plays a role in the development of MPD in Rag2 knockout mice. After 4 weeks of UV radiation, both histopathology (myeloid:erythroid ratio, number of blast cells) and flow cytometry (mature myeloid, granulocytes and immature cells) demonstrated an increased number of mice affected with the disease in the UV-irradiated Rag2 knockout group than the other groups. ^ We also investigated the role of cytokines and absence of T and B cells in the development of MPD in the Rag2 knockout mice. Results indicated that IL-3 and IL-3Rα chain expression was upregulated in the spleens of the UV-irradiated Rag2 knockout mice (4 weeks). Reconstitution of the Rag2 knockout mice with T and B cells abrogated the UV-accelerated development of MPD. Both histopathology and flow cytometric analysis (mature myeloid cells, granulocytes) showed a decrease in the number of mice affected with the disease in the UV-irradiated, reconstituted group rather than any other group. In summary, this study provides the first experimental evidence that exposure to UV irradiation can lead to the development of MPD in immune deficient mice. ^

Preclinical evaluation of proteasome inhibition for the treatment of prostate cancer

Prostatic carcinoma is the most prevalent cancer detected in men. Bortezomib is the first proteasome inhibitor to undergo clinical trials for several forms of cancer. Although we know this class of agent preferentially kills cancer cells, our knowledge of proteasome inhibition mechanisms of induced death is far from complete. We investigated the effects of bortezomib on the LNCaP-Pro5 (Pro5) and PC-3-Pro4 (Pro4) human prostatic adenocarcinoma cells lines. We showed a reduction in proliferation and an increase in DNA fragmentation, caspase 3 activity, and cell surface phosphatidyl serine exposure. The bortezomib-treated tumors from both cell lines were dramatically reduced, and apoptosis was induced. There was also a reduction in proliferation in the treated tumors from both cells lines. We looked at changes in the levels of the proangiogenic factors VEGF, IL-8 and bFGF in vitro and in vivo. Although there was a reduction in the levels of VEGF produced by the Pro5 cell line and tumor due to bortezomib, no similar observations were made for the other angiogenic factors or in the Pro4 cells. We investigated the effects of bortezomib on p53 in the Pro5 cell line. Bortezomib induced strong stabilization of p53. It did not promote phosphorylation on serines 15 and 24 and p53 remained bound to its inhibitor, mdm2. Nonetheless, confocal microscopy revealed that bortezomib stimulated p53 translocation to the nucleus and enhanced p53 DNA binding, accumulation of p53-dependant transcripts, and activation of a p53-responsive reporter gene. Furthermore, stable transfectants of LNCaP-Pro5 expressing the p53 inhibitor, HPV-E6, displayed reduced bortezomib-induced p53 activation and cell death. Our data shows bortezomib to induce antitumor effects in the human Pro4 and Pro5 prostatic adenocarcinoma cell lines by the direct induction of apoptosis. The drug also causes a reduction in cell proliferation and mean vessel density while modulating the secretion of proangiogenic factors. Although we show that proteasome inhibition stimulates p53 activation via a novel mechanism in Pro5 cells, it is also toxic to p53 null cells as is seen in the Pro4 line. ^

GCN5 maintains genome stability during development

The histone acetyltransferase, GCN5, is essential for survival of mice during embryogenesis. GCN5 null embryos die early during development due to increased apoptosis. We have demonstrated that the increased apoptosis in associated with increased p53 protein levels. Loss of p53 rescues the embryonic apoptosis in the GCN5 null embryos. These results raised the question of what molecular trigger leads to p53 stabilization and cell death in the absence of GCN5. p53 is generally referred to as the gatekeeper of the cell, monitoring cellular responses to DNA damage, genotoxic stress, and other unfavorable conditions in the cell. Therefore, we examined individual cells in wild type and mutant embryos for gross chromosomal aberrations that might trigger a genome integrity checkpoint. Karyotype analysis indicates that approximately 30% of the cells in an E8.5 GCN5 null embryo display chromosomal aberrations, predominantly chromosomal end adhesions and associations. In wild type E8.5 embryos, only 6% of the cells have chromosomal aberrations. Recent data using telomeric FISH demonstrates that cells from GCN5 null embryos have a decreased telomeric signal. Telomere maintenance is essential for maintaining genome integrity. Telomeric defects are associated with loss of chromosomes and chromosomal rearrangements that can lead to detrimental gene fusions involved in many types of cancers. Little is known about the chromatin structures present near the telomeric ends, or whether any of the telomere-associated proteins are subject to post-translational modification such as acetylation. Our results are the first data to demonstrate the involvement of a histone acetyltransferase, GCN5, in maintaining genome integrity through telomere maintenance and/or capping. ^

Characterization of E2F1's oncogenic, apoptotic and tumor-suppressive activities utilizing the K5 mouse model

Though E2F1 is deregulated in most human cancers by mutations of the p16-cyclin D-Rb pathway, it also exhibits tumor suppressive activity. A transgenic mouse model overexpressing E2F1 under the control of the bovine keratin 5 (K5) promoter exhibits epidermal hyperplasia and spontaneously develops tumors in the skin and other epithelial tissues after one year of age. In a p53-deficient background, aberrant apoptosis in K5 E2F1 transgenic epidermis is reduced and tumorigenesis is accelerated. In sharp contrast, K5 E2F1 transgenic mice are resistant to papilloma formation in the DMBA/TPA two-stage carcinogenesis protocol. K5 E2F4 and K5 DP1 transgenic mice were also characterized and both display epidermal hyperplasia but do not develop spontaneous tumors even in cooperation with p53 deficiency. These transgenic mice do not have increased levels of apoptosis in their skin and are more susceptible to papilloma formation in the two-stage carcinogenesis model. These studies show that deregulated proliferation does not necessarily lead to tumor formation and that the ability to suppress skin carcinogenesis is unique to E2F1. E2F1 can also suppress skin carcinogenesis when okadaic acid is used as the tumor promoter and when a pre-initiated mouse model is used, demonstrating that E2F1's tumor suppressive activity is not specific for TPA and occurs at the promotion stage. E2F1 was thought to induce p53-dependent apoptosis through upregulation of p19ARF tumor suppressor, which inhibits mdm2-mediated p53 degradation. Consistent with in vitro studies, the overexpression of E2F1 in mouse skin results in the transcriptional activation of the p19ARF and the accumulation of p53. Inactivation of either p19ARF or p53 restores the sensitivity of K5 E2F1 transgenic mice to DMBA/TPA carcinogenesis, demonstrating that an intact p19ARF-p53 pathway is necessary for E2F1 to suppress carcinogenesis. Surprisingly, while p53 is required for E2F1 to induce apoptosis in mouse skin, p19ARF is not, and inactivation of p19ARF actually enhances E2F1-induced apoptosis and proliferation in transgenic epidermis. This indicates that ARF is important for E2F1-induced tumor suppression but not apoptosis. Senescence is another potential mechanism of tumor suppression that involves p53 and p19ARF. K5 E2F1 transgenic mice initiated with DMBA and treated with TPA show an increased number of senescence cells in their epidermis. These experiments demonstrate that E2F1's unique tumor suppressive activity in two-stage skin carcinogenesis can be genetically separated from E2F1-induced apoptosis and suggest that senescence utilizing the p19ARF-p53 pathway plays a role in tumor suppression by E2F1. ^