8 research outputs found
Small molecules targeting histone H4 as potential therapeutics for chronic myelogenous leukemia
We recently identified a polyamide-chlorambucil conjugate, 1R-Chl, which alkylates and down-regulates transcription of the human histone H4c gene and inhibits the growth of several cancer cell lines in vitro and in a murine SW620 xenograft model, without apparent animal toxicity. In this study, we analyzed the effects of 1R-Chl in the chronic myelogenous leukemia cell line K562 and identified another polyamide conjugate, 6R-Chl, which targets H4 genes and elicits a similar cellular response. Other polyamide conjugates that do not target the H4 gene do not elicit this response. In a murine model, both 1R-Chl and 6R-Chl were found to be highly effective in blocking K562 xenograft growth with high-dose tolerance. Unlike conventional and distamycin-based alkylators, little or no cytotoxicities and animal toxicities were observed in mg/kg dosage ranges. These results suggest that these polyamide alkylators may be a viable treatment alternative for chronic myelogenous leukemia
REPSA Directed Assessment of Native Cleavage Resistance of DNA to Type IIS Restriction Endonucleases and Modification of REPSA for High Temperature Application
We have modified the combinatorial selection method Restriction Endonuclease Protection and Selection Assay (REPSA) to work in high temperature conditions for the discovery of new DNA-binding proteins in thermophiles (HT-REPSA). We utilized Thermus thermophilus (HB-8/ATCC 27634/DSM 579) as a test organism due to its amenable nature in a laboratory setting and current status as a model thermophilic organism. We used a TetR Family (TFR) transcription factor SbtR as the model protein for optimization of HT-REPSA protocols, as data had previously been obtained regarding SbtR physical characteristics and DNA-binding properties. REPSA was conducted until a cleavage resistant species arose after 7 rounds. Massively parallel sequencing of the selected DNAs and bioinformatics analysis yielded a consensus binding sequence of 5\u27-GA(t/c)TGACC(c/a)GC(t/g)GGTCA(g/a)TC, a 20base pair palindromic site comparable to that described in the literature. Taken together, our data provide a proof-of-concept that HT-REPSA can be successfully used to identify the preferred DNA-binding sequences of transcription factors from extreme thermophilic organisms
DNA sequence-specific adenine alkylation by the novel antitumor drug tallimustine (FCE 24517), a benzoyl nitrogen mustard derivative of distamycin.
FCE 24517, a novel distamycin derivative possessing potent antitumor activity, is under initial clinical investigation in Europe. In spite of the presence of a benzoyl nitrogen mustard group this compound fails to alkylate the N7 position of guanine, the major site of alkylation by conventional nitrogen mustards. Characterisation of DNA-drug adducts revealed only a very low level of adenine adduct formation. Using a modified Maxam-Gilbert sequencing method the consensus sequence for FCE 24517-adenine adduct formation was found to be 5'-TTTTGA-3'. A single base modification in the hexamer completely abolishes the alkylation of adenine. Using a Taq polymerase stop assay alkylations were confirmed at the A present in the hexamer TTTTGA and, in addition, in one out of three TTTTAA sequences present in the plasmid utilized. The sequence specificity of alkylation by FCE 24517 is therefore the most striking yet observed for an alkylating agent of small molecular weight
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Identification Of Effective New Drugs Combinations Exploiting The Ability Of trabectedin To Modulate Transcription
Ewing’s sarcoma (ES) is the second most common malignant bone tumor of childhood, characterized by the chimeric protein EWS-FLI1 (hallmark of this pathology), that alters the transcription of different genes. The five-year overall survival of patients with localized disease is approximately 70%; unfortunately the majority of patients with a metastatic disease have a poor prognosis with a five-year overall survival around the 30%. Among the recently registered drugs for the therapy of sarcomas, trabectedin could be of potential great interest as it seems very active in some “translocated sarcomas”.
Trabectedin exerts its antitumor activity with different mechanisms of action.
One of the most important is related to its ability to interfere with DNA repair mechanisms (NER and HR), that cause cell cycle perturbations.
Furthermore trabectedin is able to displace the oncogenic EWS-FLI1 chimera from its target promoters, modulating the transcription of these genes, in ES cells.
Although trabectedin has shown some activity against ES, the overall clinical results indicated only a marginally activity of trabectedin given as single agent in ES.
The thesis is aimed at using the available knowledge on trabectedin mechanism of action to identify some effective combinations.
Since trabectedin induces cell cycle perturbations I speculated that its activity could be increased by checkpoint inhibitors. The studies performed on the combination of trabectedin and the WEE1 inhibitor, AZD-1775, have shown that the inhibition of WEE1 enhances the trabectedin activity, thus the combination is synergic.
Since trabectedin affects the transcription of several genes, I have developed a new approach based on the use of silencing RNA (siRNA) libraries to identify whether the downregulation of some genes was synthetically lethal when ES cells were pretreated with trabectedin. An important part of the thesis was the development, validation and initial application of this approach
Molecular and cellular pharmacology of novel pyrrolo [2,1-c] [1,4] diazepine-based anticancer agents.
The pyrrolo 2,1-c 1,benzodiazepines (PBDs) are a family of naturally occurring antitumour antibiotics which includes anthramycin, DC-81, tomaymycin and sibiromycin. They exert their biological activity through covalent binding to the exocyclic N2 group of guanine in the minor groove of DNA and block transcription in a sequence-specific manner. These PBD monomers span three DNA base pairs and have a preference for binding to purine-G-purine triplets. The PBDs have been used as a scaffold to attach other moieties, leading to novel sequence-selective DNA minor groove alkylating agents. In addition, as part of a rational approach to producing more efficient and selective DNA interstrand crosslinking agents, two PBD monomers have been linked together to form PBD dimers. The research in this thesis is a study of the molecular and cellular pharmacology of several series of novel PBD-containing agents including novel PBD dimers with different linker lengths, PBD-nitrogen mustard conjugates, PBD-polyamide conjugates and C2-aryl PBD monomers. Cytotoxicity in human tumour cell lines, efficiency of DNA interstrand crosslinking in naked linear plasmid DNA, and DNA sequence specificity were assessed. DNA interstrand crosslink formation and repair in cells were also measured. Resulting from this work Q2lQT-exo-unsaturated PBD dimers have been characterised to be highly cytotoxic and efficient in producing interstrand crosslinks both in naked DNA and in cells that are not repaired up to 48 hours. Only two of the PBD-nitrogen mustard conjugates showed some interaction with DNA although several members of this group showed significant cytotoxicity. A PBD-tri-pyrrole conjugate was found to bind preferentially to the sequence 5'-AGATTATC. Novel C2-aryl PBD monomers were shown to bind selectively to 5'-purine-G-purine sequences and demonstrated significant cytotoxicity. In addition, a method utilizing fluorescently end-labelled oligonucleotides was developed and validated to screen libraries of PBD-containing molecules synthesised on beads by combinatorial chemistry. This method allowed the isolation and discrimination of beads containing compounds, which have a high affinity for specific DNA sequences
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Molecular analysis of DNA damage induced by a novel trinuclear platinum complex (BBR 3464)
We evaluated the activity of the trinuclear platinum complex BBR 3464 in two human ovarian carcinoma cell lines (OAW42, A2780) and in their cisplatin-resistant counterparts (OAW42Mer, A2780cp8). An increased cytotoxic potency of BBR 3464 compared to cisplatin was generally observed, an a collateral sensitivity or a very modest cross-resistance to BBR 3464 was found in OAW42Mer and A2780cp8 cell lines, respectively. Loss of mismatch repair proteins (hMLHl, hPMS2) or overexpression of nucleotide excision repair proteins (ERCC1) was not detrimental for the cellular sensitivity to the trinuclear platinum complex. BBR 3464 intracellular accumulation and DNA-bound platinum were consistently higher than those observed with cisplatin. After exposure to BBR 3464 and cisplatin of purified DNA or intact cells, a similar sequence preference of DNA damage was observed. Conversely, interesting differences in the kinetics of formation and removal of DNA lesions at the single-gene (N-ras) level were observed between the two drugs. The interference exerted by BBR 3464 with cell cycle progression and its ability to induce apoptosis were evaluated in OAW42 and OAW42Mer cell lines. Flow cytometric experiments indicated that in the two cell lines BBR 3464 was able to induce a persistent G2M block whereas cisplatin caused an initial accumulation of cells in the S phase followed by an increase in the G2M cell fraction. Exposure to IC50 drug concentrations induced apoptosis in both cell lines. However, the percentage of cells with an apoptotic nuclear morphology was slightly higher after cisplatin than BBR 3464 treatment in OAW42 cells, whereas the opposite pattern was observed in OAW42Mer cells. Degradation of the nuclear lamin B was detected in OAW42 cells after exposure to each drug whereas in OAW42Mer cells the cleavage was only
appreciable after BBR 3464 exposure. In OAW42 cells the mitochondrial membrane potential (É…Ń°mt) was not affected by the two drugs, whereas in the OAW42Mer cell line a marked (É…Ń°mt) reduction was observed only after exposure to BBR 3464. Overall, the results would suggest that the collateral sensitivity to BBR 3464 observed in the OAW42Mer cell line might be attributable to the ability of this drug to modify DNA differently from that of cisplatin and, as a consequence,
to induce different cellular responses to DNA damage such as the triggering of specific apoptotic pathways