Polymorphisms in MDM2 and TP53 Genes and Risk of Developing Therapy-Related Myeloid Neoplasms.

One of the most severe complications after successful cancer therapy is the development of therapy-related myeloid neoplasms (t-MN). Constitutional genetic variation is likely to impact on t-MN risk. We aimed to evaluate if polymorphisms in the p53 pathway can be useful for predicting t-MN susceptibility. First, an association study revealed that the Pro variant of the TP53 Arg72Pro polymorphism and the G allele of the MDM2 SNP309 were associated with t-MN risk. The Arg variant of TP53 is more efficient at inducing apoptosis, whereas the Pro variant is a more potent inductor of cell cycle arrest and DNA repair. As regards MDM2 SNP309, the G allele is associated with attenuation of the p53 apoptotic response. Second, to evaluate the biological effect of the TP53 polymorphism, we established Jurkat isogenic cell lines expressing p53Arg or p53Pro. Jurkat p53Arg cells presented higher DNA damage and higher apoptotic potential than p53Pro cells, after treatment with chemotherapy agents. Only p53Pro cells presented t(15;17) translocation and del(5q). We suggest that failure to repair DNA lesions in p53Arg cells would lead them to apoptosis, whereas some p53Pro cells, prone to cell cycle arrest and DNA repair, could undergo misrepair, generating chromosomal abnormalities typical of t-MN.

A sequential methodology that allows apoptotic cell sorting and FISH analysis in human testicular cells.

The objective of this study was to develop a methodology that permits the detection and separation of apoptotic cells in human testicular tissue and their subsequent cytogenetic analysis by fluorescence in situ hybridization (FISH). The sequential methodology consisted of five steps: 1) enzymatic disaggregation of testicular tissue, 2) specific staining of apoptotic cells, 3) cell sorting by flow cytometry, 4) cell fixation, and 5) FISH. Enzymatic disaggregation yielded cell counts that ranged from 1.7x10(5) to 5x10(6) cells, and viability values greater than 72%. The apoptotic (mean ± SD: 22% ± 5.3%) and viable (45.5% ± 7.3%) populations were identified and selected by flow cytometry and demonstrated purity values ranging between 62% and 100%. The paraformaldehyde fixation of the selected fractions resulted in cell loss values of less than 10%. The application of three treatments before FISH (membrane permeabilization, elimination of cytoplasmic components, and re-fixation of the sample) resulted in hybridization frequencies of greater than 98%. In both selected fractions, cells of all spermatogenic stages and Sertoli cells were identified. The methodology developed has enabled the preparation of a cellular suspension with optimal viability and counting, the efficient selection of the apoptotic population, and its analysis by cytogenetic techniques. The application of this methodology in testicular cells should help establish whether there is a direct relationship between chromosome anomalies and apoptosis.