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Interplay among mitochondria and p53 family proteins during apoptosis induced by dna damage - a new strategy for cancer therapy

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Impaired radiotherapy response in NSCLC is clinical problem leading to improper tumour control. We have found that defective signalling from the initial DNA damage to induction of apoptosis is one contributing factor. Thus we have shown that in a NSCLC cell line defective activation of the pro-apoptotic proteins Bak and Bax as well as impeded activation of the stress-activated protein kinases JNK and p38 contribute to apoptotic resistance to low-LET? -Radiation. Irradiation using high Linear Energy Transfer (high-LET) i.e., accelerated ions or radioactive nucleotides emitting? -Particles give rise to more complex DNA-strand breaks compared to low-LET? -Radiation and are currently evaluated for clinical use. We have found that increased apoptotic signalling results from high-LET radiation in NSCLC cells compared to Low-LET radiation suggesting that high-LET radiation might have improved efficacy. Moreover, we report that high and low-LET radiation likely differ in their way to activate the pro-apoptotic Bcl-2 family members Bak and Bax as well as the stress-activated protein kinase (SAPK) c-jun N-terminal kinases (JNK). Potential users of this knowledge is pharmaceutical industries developing DNA damaging sensitizing drugs as well as researches which analyze radio resistance mechanisms or explore cellular responses to different radiation qualities.
This result centers on the identification of a novel smaller form of the ARF tumor suppressor protein. Unlike the regular ARF protein, which is almost exclusively a nuclear protein, this smaller form (smARF) localizes to mitochondria, where it causes loss of mitochondrial membrane potential and slow cell death. This activity of smARF does not depend on the presence of functional p53 in the tumor cells, unlike the regular ARF protein, whose tumor suppressor activity is largely (albeit not exclusively) dependent on its ability to activate p53. The contribution of smARF to tumor cell death and its utility as a new target for activation by anti cancer agents need to be explored.
This work has examined genomic and phenotypic consequences of over expressing p53 family members in a number of cell types including TF-1, Saos2, and HeLa cells. Using microarray and QPCR technologies we have examined genetic consequence of over expression. Cluster analysis of genes induced by p53 but not p73 over a 24h time period identified 1145 genes that were regulated. In contrast, cluster analysis of genes induced by p73 but not p53 over a 24h time period revealed that 175 genes were regulated, demonstrating the enhanced ability of p53 to regulate gene expression. We provide some literature surveys on some of the top regulated genes, which demonstrate an association with apoptosis. In addition to the genomic consequences of over expressing p53 family members and using proprietary survival assays, the functional consequences were also examined. Contrary to expectations, we demonstrated that certain members of the p73 family were anti-apoptotic and protected cells from growth factor induced apoptosis.
To elucidate the individual role of the TAp63a and Np63a isoforms in the development of the epidermis, we generated transgenic mice expressing either TAp63a or Np63a under the control of a keratinocyte specific promoter; we then crossed these mice into a p63-/- background. p63-/- die shortly after birth and among other things lack epidermis. Our data obtained re-introducing the different isoforms show that: - p63-/- epidermis showed very limited and partially differentiated epithelial areas; - Np63a significantly increases the number of epidermal basal cells, thus allowing a higher degree of differentiation; - TAp63a and Np63a have synergistic functions in the formation of the epidermis. Altogether these data strongly suggest a role for Np63a, controlling the proliferation potential in epithelial cells, and are compatible with a role of TA in controlling further differentiation of these cells. In addition we have identified a number of molecular targets of p63 and defined their role in p63 signalling pathways.
We performed a detailed search for factors responsible for resistance of lung cancer cells to treatment. Detailed investigation revealed that although expression of heat-shock proteins was reported to be important for maintaining of resistance of some tumors, level of Hsp72 does not modulate ionizing radiation-induced death of non-small lung carcinomas (NSCLC). In addition, endogenously released Smac was also insufficient to mediate cell death of human lung carcinoma in response to DNA damage. We showed that apoptosis-inducing factor (AIF) determines the chemoresistance of NSCLC. In addition, PKC412 was able to sensitize NSCLC cells to DNA damage. Importantly, one of the isoforms of p53 family proteins, p73-alpha, efficiently repressed drug-induced apoptosis in small-cell lung carcinomas. Altogether, these data demonstrate that mechanism(s) of resistance of lung cancer cells differ from many other types of tumors and that resistance to chemo- and radiotherapy may imply both nuclear and cytoplasmic compartments. Potential users of this knowledge are pharmaceutical industries developing DNA damaging sensitizing drugs as well as researches, which analyze chemo- and radio-resistance mechanisms or explore cellular responses to different treatment qualities.
Dr. Kroemer's team showed that roughly one quarter of resected stage I non-small cell lung carcinomas (NSCLC), (T1N0M0 or T2N0M0), manifested a marked nuclear localization of Apaf-1 (Apaf-1Nuc), as compared to mostly cytoplasmic localization of Apaf-1 found in the majority of tumors (Apaf-1Cyt). Importantly, the subcellular location of Apaf-1 (but not that of p53 or Hsp70) was found to constitute an accurate prognostic factor for overall survival in non-small cell lung carcinoma.
We identified the gene encoding AIF as a potential target for positive regulation by p53. The AIF protein is an important player in apoptosis, through its ability to translocate from its normal mitochondrial location into the cell nucleus, where it can induce DNA fragmentation and cell death. We found that the AIF is positively regulated by p53 under basal conditions, namely even in the absence of overt DNA damage or other types of stress. This is unusual in the sense that most known p53 target genes are selectively induced by p53 in response to genotoxic stresss, whereas such stress has very little effect on AIF levels. We show that AIF transcriptional induction is part of the arsenal that allows p53 to promote apoptosis in response to chemotherapy. Understanding the precise relationship between p53 and AIF may serve to optimize the selective impact of chemotherapy.
Cytochrome P450 (Cyp450) enzymes have the capability of playing key metabolic roles in several aspects of cancer as a consequence of their unusually broad substrate specificities. Cyp450 are also important in the metabolism of anticancer therapeutic drugs, enhancing or diminishing the efficacy of the drugs depending on whether the drug or its metabolites are efficacious. In the current project we examined the Cyp450 profile of approximately 25 Cyp family members over a panel of NSCLC and SCLC cell lines. We identified a number of differences between the various lung cancer cell lines and are examining whether they might be the reason why cells are refractive to chemotherapy.
Impaired radiotherapy response in NSCLC is a clinical problem prohibiting proper tumour control. In the present subject matter the role of Insulin like growth factor 1 (IGF-1R) signalling on radiotherapy responses in non-small cell lung cancer cells are analyzed. Data are presented which show that pharmacological inhibition of IGF-1R signalling increases cell death responses in NSCLC cells through apoptotic cell death. Thus in NSCLC cells IGF-1R signalling is activated in response to radiation through a mechanism involving both PI3K/Akt as well as p38 MAPK kinase signalling. A link between IGF-1R signalling and DNA repair function by KU86 is presented. The invention comprises knowledge that growth factor signalling inhibition can be used as methods for sensitizing NSCLC to radiotherapy. Potential users of this knowledge are oncologists treating lung cancer patients, pharmaceutical industries developing DNA damaging sensitizing drugs and researchers which analyze mechanisms of radio resistance or that develop methods to detect resistance prior to therapy.
We established a model of acquired oxaliplatin resistance derived from the HCT116 oxaliplatin-sensitive cell line (HCT116S) and consisting in two resistant clones (HCT116R1, HCT116R2) and their total or partial revertants (HCT116Rev1 and HCT116Rev2, respectively). Using this cellular model, we explored the contribution of mitochondrial apoptosis and nuclear DNA to oxaliplatin-mediated apoptosis induction and oxaliplatin resistance. We showed that the activity of oxaliplatin is mediated by the induction of Bax/Bak-dependent mitochondrial apoptosis and that oxaliplatin resistance is mediated by a defect in Bax/Bak activation correlating with a reduced loss of the mitochondrial transmembrane potential (DeltaPsim). In addition, we observed that p53 only contributed marginally to oxaliplatin-induced cytotoxicity and was not involved in oxaliplatin resistance. Moreover and surprisingly, depletion of the nucleus in HCT116S cells did not abolish the oxaliplatin-induced DeltaPsim loss indicative of imminent apoptosis. Enucleation abolished the oxaliplatin resistance of HCT116R1 cells, while HCT116R2 cytoplasts conserved their resistant phenotype. Altogether, these data demonstrate that oxaliplatin exerts its cytotoxic effects by inducing mitochondrial apoptosis and that these effects can be initiated by interacting on other cellular structures than nuclear DNA. Resistance to oxaliplatin may imply both nuclear and cytoplasmic compartments.
We have clarified the mechanisms through which p73 induces cell death. These mechanisms include regulation of Bax and PUMA as well as the control of the CD95 pathway. Moreover we have described the different roles of the TA (pro-apoptotic) and DN (anti-apoptotic) isoforms. Finally we have identified a number of p73 interacting molecules that regulate its activity, this molecules include Itch, PIAS-1, Bub-1 and FLASH. Itch is an E3 ubiquitin ligase that specifically binds p73 but not p53 targeting it for degradation. Itch is down regulated in response to DNA damage allowing p73 levels to increase. PIAS-1 is a SUMO E3 ligase that binds and sumoylates p73 decreasing its trascriptional activity in a cell cycle dependent manner. The interaction between Bub-1 and TAp73 explains the poliploidy observed in TAp73 over-expressing tumours. Finally we have shown that FLASH is an essential component of CBs that interacts with p73 and regulates histone transcription and cell cycle progression.
The subject matter of the present invention relates to an in vitro method for the screening of anti-HIV compounds, or for predicting the progression of HIV related diseases, or for determining the efficiency of an anti-HIV-1 treatment based on the measuring of the expression level of Puma and, optionally, on the measuring of the phosphorylated P53 and/or the p38 MAP kinase protein level in circulating white blood cells of a sample from a HIV infected patient, particularly HIV-1. The invention further comprises a method for the diagnostic of HIV infection in a patient and kits for performing these methods.
We have analyzed how different types of DNA damages i.e., ionizing radiation and DNA cross linking platinum agents are detected by the Non-homologous end joining (NHEJ)-and Homologous recombination repair (HRR) systems in vitro and in vivo in tumour cell lines. We have studied the NHEJ DNA double strand break (DSBs) repair efficiency and fidelity in cell extracts from NSCLC cells sensitive and resistant to radiotherapy using an in vitro functional assay using restriction enzyme-generated double strand breaks with different end structures (compatible and incompatible ends) in a pBR322 plasmid. Results show that neither repair efficiency of DNA DSBs nor fidelity of NHEJ correlated with sensitivity to radiation i.e. to SF2 values. Moreover, no correlation between NHEJ key protein levels and SF2 value was observed in either NSCLC or SCLC subtypes. The role of NHEJ components, DNA dependent kinase (DNA-PKcs) and KU86 in cisplatin and oxaliplatin-induced cellular sensitivity has been studied. Results show that mutation of DNA-PKcs or deficiency in KU86 results in increased sensitivity to platinum drugs i.e., cisplatin and oxaliplatin. Moreover the sensitivity to oxaliplatin was higher in DNA-PKcs deficient cells than in cells missing KU86 suggesting that these subunits of DNA-PK may have different function with regard to oxaliplatin-induced cell signalling. The influence of p53 on the HRR protein Rad 51 protein level and sub cellular localisation in response to ionising radiation has been assessed in human colon cancer cell lines deficient or proficient in p53. We found that p53 has a role in Rad 51 clearance post radiation and that nucleoli might be sites of Rad 51 degradation.
RNA interference (RNAi) is a novel target validation technology that has just come to the fore over the last 2-3 years. We have established a validation platform using small interfering RNA (siRNA), to enable us to study the phenotypic consequence of knocking out targets identified by through this collaboration, in compliance with the role for Partners 6 described in the Work Programme. Scientists at EiRx have demonstrated that by using various instructive web sites and commercial algorithms, as much as 90% of siRNA designed in-house cause greater than 60% knockdown of target mRNA. This is also reflected in the protein levels, where western blotting of cell lysates from siRNA treated cells has confirmed that specific target protein is reduced by approximately 70 90% in most cases, 48h post treatment. EiRx also developed a transient transfection method for the delivery of siRNA into cells, with limited toxicity since the overall transfection period is 4 hours. Using a FAM (a fluorescent probe)- labelled siRNA oligonucleotide, the transfection rate for this methodology has been demonstrated to be greater than 90% across a breadth (>80) of cell lines tested to date. As well as developing the siRNA methodology we have also developed a number of assays in order to interrogate the phenotypic consequences of targeting various cellular genes. These assays include apoptotic specific assays such as DNA fragmentation, Annexin binding and mitochondrial depolarisation (JC1), as well as clonagenic and anchorage independent assays. We have also developed siRNA compatible chemotaxis assays. We are now able to use these capabilities to examine the pro and anti-apoptotic role of novel targets in Non Small Cell Lung Cancer chemotherapy resistance.
We have developed a novel series of DNA reactive peptides (DR peptides) that carry groups which alkylate DNA and as such resemble conventional alkylating agents i.e. Melphalan. Our lead DR peptides do, however, circumvent resistance to conventional alkylating agents and in this work we have explored mechanisms behind the efficacy of DR peptides in tumour cell lines. Moreover, we have studied which combinations of DR peptides and chemotherapy agents that have synergistic effects when used in combination. In addition, a candidate drug is now underway to formal pre-IND testing. Results show that DR peptides have improved efficiency compared to the lead substance Melfalan in several human tumours cell lines including lines derived from lung cancer. A synergistic effect between DR peptides and DNA damaging therapy e.g., topo II inhibitors, cyclophosphamide and carboplatin in childhood tumour neuroblastoma. Our data show that lysosomes are involved in DR peptide induced cell death in tumour cell lines as are stress activated protein kinases. Potential users of this knowledge are pharmaceutical industries developing DNA damaging sensitizing drugs and researchers, which analyze chemoresistance mechanisms.
Mitochondrial outer membrane permeabilization is therefore considered a key initiative step in the apoptotic process, although the precise mechanisms regulating this event for long time remained elusive. The permeabilization of the mitochondrial outer membrane and release of intermembrane space proteins, especially, cytochrome c is unequivocally one of the central events of apoptosis. The mechanism by which these intermembrane space proteins are released from mitochondria depends presumably on cell type and the nature of stimuli. It might include the induction of mitochondrial permeability transition, formation of specific pores by Bcl-2 family proteins, volume-dependent MPT-independent mechanisms as well as mediated by caspase-2. The variety of mechanisms of cytochrome c release may explain diversities in the response of mitochondria to numerous apoptotic stimuli in different types of cells. Potential users of this knowledge are researchers in cell death field, oncologists developing new tools to activate cell death machinery via mitochondria and pharmaceutical industries developing specific drugs acting via mitochondria.