RESUMEN
Applied molecular evolution is a rapidly developing technology that can be used to create and identify novel enzymes that nature has not selected. An important application of this technology is the creation of highly drug-resistant enzymes for cancer gene therapy. Seventeen O(6)-alkylguanine-DNA alkyltransferase (AGT) mutants highly resistant to O(6)-benzylguanine (BG) were identified previously by screening 8 million variants, using genetic complementation in Escherichia coli. To examine the potential of these mutants for use in humans, the sublibrary of AGT clones was introduced to human hematopoietic cells and stringently selected for resistance to killing by the combination of BG and 1,3-bis(2-chloroethyl)-1-nitrosourea. This competitive analysis between the mutants in human cells revealed three AGT mutants that conferred remarkable resistance to the combination of BG and 1,3-bis(2-chloroethyl)-1-nitrosourea. Of these, one was recovered significantly more frequently than the others. Upon further analysis, this mutant displayed a level of BG resistance in human hematopoietic cells greater than that of any previously reported mutant.
Asunto(s)
Antineoplásicos/farmacología , Evolución Molecular Dirigida , Resistencia a Antineoplásicos/genética , Guanina/análogos & derivados , Guanina/farmacología , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Secuencia de Aminoácidos , Carmustina/farmacología , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Estabilidad de Enzimas , Biblioteca de Genes , Terapia Genética , Humanos , Células K562 , Datos de Secuencia Molecular , Mutación/genética , O(6)-Metilguanina-ADN Metiltransferasa/química , O(6)-Metilguanina-ADN Metiltransferasa/genética , Transducción GenéticaRESUMEN
PURPOSE: Accurate assessment of marrow cellularity is necessary for establishing diagnoses and monitoring the effects of treatment in a large number of malignant and nonmalignant pediatric illnesses, and for evaluating sibling donors for transplantation. However, normal values for age-related bone marrow cellularity in pediatric patients have not been well established. This study was designed to better define pediatric normal values for bone marrow cellularity. PATIENTS AND METHODS: A retrospective review of 448 bone marrow core biopsy or clot specimens, including 45 samples from healthy donors, were taken from the posterior iliac crest of patients aged from younger than 1 to 18 years (55% male). All samples were collected and fixed in a standardized fashion. Patients with hematopoietic malignancies and other systemic conditions known to impact marrow cellularity were excluded. RESULTS: The mean cellularity of the entire sample was 65.4%. Cellularity was similar in boys and girls, but varied (p < 0.001) with age. Cellularity was highest in patients younger than 2 years (79.8%), and declined in patients 2 to 4 years old (68.6%) and 5 to 9 years old (59.1%). Cellularity remained stable in older patients (60.1% and 61.1%, respectively, in patients 10 to 14 and 15 to 18 years of age). Adjusting for age and gender, mean cellularity was similar in patients with an underlying nonhematologic malignancy compared to health donors but was roughly 6% higher in patients with hematopoietic disorders. CONCLUSIONS: This study demonstrates that average cellularity during the first two decades of life, using current techniques of marrow collection and standardized analysis, is lower than previously estimated. In addition, cellularity declined with age until the age of 5 years, but was similar thereafter. After adjusting for age, differences according to diagnosis were relatively small.
