RESUMO
The mouse PancChip, a microarray developed for studying endocrine pancreatic development and diabetes, represents over 13,000 cDNAs. After computationally assigning the cDNAs on the array to known genes, manual curation of the remaining sequences identified 211 novel transcripts. In microarray experiments, we found that 196 of these transcripts were expressed in total pancreas and/or pancreatic islets. Of 50 randomly selected clones from these 196 transcripts, 92% were confirmed as expressed by qRT-PCR. We evaluated the coding potential of the novel transcripts and found that 74% of the clones had low coding potential. Since the transcripts may be partial mRNAs, we examined their translated proteins for transmembrane or signal peptide domains and found that about 40 proteins had one of these predicted domains. Interestingly, when we investigated the novel transcripts for their overlap with noncoding microRNAs, we found that 1 of the novel transcripts overlapped a known microRNA gene.
Assuntos
Ilhotas Pancreáticas/metabolismo , Análise em Microsséries/métodos , Pâncreas/metabolismo , Proteínas/genética , Animais , Clonagem Molecular , Biologia Computacional/métodos , DNA Complementar , Diabetes Mellitus Tipo 1/genética , Perfilação da Expressão Gênica , Genoma , Camundongos , Camundongos Endogâmicos NOD , Transcrição GênicaRESUMO
Reactivation of mutant p53 is likely to provide important benefits for treatment of chemotherapy- and radiotherapy-resistant tumors. We demonstrate here that the maleimide-derived molecule MIRA-1 can reactivate DNA binding and preserve the active conformation of mutant p53 protein in vitro and restore transcriptional transactivation to mutant p53 in living cells. MIRA-1 induced mutant p53-dependent cell death in different human tumor cells carrying tetracycline-regulated mutant p53. The structural analog MIRA-3 showed antitumor activity in vivo against human mutant p53-carrying tumor xenografts in SCID mice. The MIRA scaffold is a novel lead for the development of anticancer drugs specifically targeting mutant p53.
Assuntos
Maleimidas/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Separação Celular , DNA/química , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Glutationa Transferase/metabolismo , Humanos , Camundongos , Camundongos SCID , Microscopia de Fluorescência , Modelos Químicos , Mutação , Transplante de Neoplasias , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica , Tetraciclina/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismoRESUMO
The tumor suppressor p53 inhibits tumor growth primarily through its ability to induce apoptosis. Mutations in p53 occur in at least 50% of human tumors. We hypothesized that reactivation of mutant p53 in such tumors should trigger massive apoptosis and eliminate the tumor cells. To test this, we screened a library of low-molecular-weight compounds in order to identify compounds that can restore wild-type function to mutant p53. We found one compound capable of inducing apoptosis in human tumor cells through restoration of the transcriptional transactivation function to mutant p53. This molecule, named PRIMA-1, restored sequence-specific DNA binding and the active conformation to mutant p53 proteins in vitro and in living cells. PRIMA-1 rescued both DNA contact and structural p53 mutants. In vivo studies in mice revealed an antitumor effect with no apparent toxicity. This molecule may serve as a lead compound for the development of anticancer drugs targeting mutant p53.
