O papel modulador do gene AIRE - capes

468 R.S. Cardoso et al. / Molecular Immunology 43 (2006) 464–472Table 1Frequency (%) of apoptotic and necrotic cells in 4 Gy irradiated and controlFTOCsApoptosisNecrosisStagingStagingControl 15 1.2 ± 0.5 15 4.1 ± 1.516 1.0 ± 0.5 16 3.0 ± 1.017 3.2 ± 1.0 17 5.2 ± 1.520 2.0 ± 1.0 20 2.7 ± 1.0Irradiated 15 1.8 ± 1.0 15 4.1 ± 0.516 1.9 ± 1.0 16 4.2 ± 1.017 3.0 ± 0.5 17 2.3 ± 0.520 2.1 ± 1.0 20 5.8 ± 1.5Mean and standard deviation of three independent determinations. Data analyzedby t-test (P < 0.05).Staging corresponds to development of fetal thymus in culture; 15FTOC = 13 day gestation thymus plus two days in FTOC.genes implicated in cell cycle control, DNA repair, T-celldifferentiation and V(D)J recombination were selected fordiscussion. The complete color heat-map indicating thesix gene clusters is available online at (www.rge.fmrp.up.br/passos/FTOCcluster/9216).Among the genes differentially expressed, we selected 42to discuss from six clusters whose functions are associatedwith cell cycle control, transcription factors, immune regulation,T-cell V(D)J recombination and DNA repair (Table 2).4. Discussion4.1. T-cell receptor and TRBV8.1 rearrangement occursin vitro in FTOCThe IL-7 is a cofactor associated with modulation ofV(D)J recombination of TCR genes (Muegge et al., 1993;Sollof et al., 1997).In this study, we described for the first time the occurrenceof TRBV8.1-BD2.1 V(D)J recombination in vitro in FTOC.The onset of TRBV8.1-BD2.1 V(D)J recombination invitro mimicked the thymus gestation in vivo, demonstratingthat this model system is similar to the in vivo development ofFig. 2. Clustering RNA (cDNA) samples and 9216 genes of each developmentalphase of control and gamma-irradiated FTOCs. The hierarchicalclustering algorithm compared means of the different genes and RNA(cDNA) samples whose standard deviations did not overlapped computingthe dendrogram that assembled all elements into a tree. Complete colorheat-map indicating the repressed, induced and unmodulated genes amongall situation studied is available online at (www.rge.fmrp.usp.br/passos/FTOC/cluster9216).the thymus in inbred mouse strains, as previously describedby our group (Espanhol et al., 2003; Macedo et al., 1999).On the basis of a variety of criteria, FTOC reproducesthe normal thymic development; however, the results can beinfluenced by the length of culture, and mouse strain and age(DeLuca et al., 1995).In addition, it was shown that there are differences intemporal rearrangements at the TRBV locus, including theTRBV8.1 segment, among BALB-c, C57Bl/6 and CBA/Jinbred mouse strains, probably due to the different geneticbackgrounds of the different strains (Espanhol et al., 2003;Macedo et al., 1999; Junta and Passos, 1998).For this reason, the fetuses used in this study were obtainedwithin the age range of 13–15 days of gestation pc, whichpermitted comparisons with the data obtained in vivo withthe BALB-c strain.4.2. Hierarchical clusteringThe expression patterns disclosed several clusters of coexpressedgenes, six of which (clusters 1–6) were selectedfor discussion on the basis of the biological functions of themost relevant genes investigated in this study.Cluster 1 (correlation = 0.81) harbors genes which wereinduced in irradiated cultures. This cluster displayed genesinvolved in cell cycle/cell division and DNA repair. Amongthem, we pinpointed MYB (myeloblastosis oncogene, accessionno. NM010848, chromosome 10), which has a rolein transcriptional activation and in the control of proliferationand differentiation of hematopoietic progenitor cells,and Kirsten rat sarcoma oncogene 2 (KRAS2, accession no.NM021284, chromosome 6). This oncogene belongs to thesmall GTPase superfamily (ras family), whose proteins bindto GDP/GTP and possess intrinsic GTPase activity. Its biologicalfunction is associated with control of the cell cycle.The induction of these oncogenes suggests triggering cell divisionby irradiation in FTOCs.Interestingly, the cell division cycle 42 homolog S. cerevisiaegene (CDC42, accession no. NM009861, chromosome4) which plays a role in GTPase-mediated signal transductionand participates in cytokinesis, presented an expressionprofile similar to that of the KRAS2 oncogene, suggestingthat GTPase activity seems to be required after exposure toionizing radiation.The X-ray repair complementing defective repair in Chinesehamster cells 5 gene (Ku80) (XRCC5, accession no.NM009533, chromosome 1) participates in the mechanismof DNA double-strand break repair (DSB repair) via NHEJ.The induction of the XRCC5 gene suggests that NHEJ mayconstitute an important process of DNA repair in irradiatedFTOCs.Cluster 2 (correlation = 0.71) encompasses genes involvedin the regulation of transcription in T-cells suchas signal transducer and activator of transcription 4 gene(STAT4, accession no. NM011487, chromosome 1), whoseactivities are associated with a transcription factor and