According to the mitochondrial theory of aging, reactive oxygen species (ROS) derived primarily from mitochondria cause cumulative oxidative damage to various cellular molecules and thereby... Show moreAccording to the mitochondrial theory of aging, reactive oxygen species (ROS) derived primarily from mitochondria cause cumulative oxidative damage to various cellular molecules and thereby contribute to the aging process. On the other hand, a pivotal role of the proteasome, as a main proteolytic system implicated in the degradation of oxidized proteins during aging, is suggested. In this study, we analyzed mitochondrial function in dermal fibroblasts derived from biopsies obtained from healthy young, middle-aged, and old donors. We also determined proteasome activity in these cells, using a degron-destabilized green fluorescent protein (GFP)-based reporter protein. We found a significant decrease in mitochondrial membrane potential in samples from aged donors, accompanied by a significant increase in ROS levels. Respiratory activity was not significantly altered with donor age, probably reflecting genetic variation. Proteasome activity was significantly decreased in fibroblasts from middle-aged donors compared with young donors; fibroblasts derived from the oldest donors displayed a high heterogeneity in this assay. We also found intraindividual coregulation of mitochondrial and proteasomal activities in all human fibroblast strains tested, suggesting that both systems are interdependent. Accordingly, pharmacological inhibition of the proteasome led to decreased mitochondrial function, whereas inhibition of mitochondrial function in turn reduced proteasome activity. Show less
Fits, L. van der; Kester, M.S. van; Qin, Y.J.; Out-Luiting, J.J.; Smit, F.; Zoutman, W.H.; ... ; Vermeer, M.H. 2011
The presence of recurrent high-risk mutations in cyclin-dependent kinase inhibitor 2A/cyclin-dependent kinase 4 (CDKN2A/CDK4) among melanoma-prone families suggests that a high-throughput,... Show moreThe presence of recurrent high-risk mutations in cyclin-dependent kinase inhibitor 2A/cyclin-dependent kinase 4 (CDKN2A/CDK4) among melanoma-prone families suggests that a high-throughput, multiplex assay could serve as an effective initial screening tool. To this end, we have developed a multiplex bead-based assay for high-throughput CDKN2A/CDK4 genotyping in the context of familial melanoma. Genomic DNA from 1,603 subjects (1,005 in training set and 598 in validation set) were amplified by multiplex PCR using five CDKN2A/CDK4 primer sets followed by multiplex allele-specific primer extension for 39 distinct germline variants. The products were then sorted and analyzed using the Luminex xMAP system. Genotypes were compared with previously determined sequence data. In the Toronto training cohort, all 145 samples with known variants were detected by the bead assay (100% concordance). Analysis of the 598 samples from the GenoMEL validation set led to identification of 150/155 expected variants (96.77%). Overall, the bead assay correctly genotyped 1,540/1,603 (96.07%) of all individuals in the study and 1,540/1,545 (99.68%) of individuals whose variants were represented in the probe set. Out of a total of 62,517 allelic calls, 62,512 (99.99%) were correctly assigned. The multiplex bead-based assay is an accurate method for genotyping CDKN2A/CDK4 variants and is potentially useful in genotyping low-to-moderate melanoma risk single-nucleotide polymorphisms. Show less
Mycosis fungoide (MF) patients who develop tumors or extracutaneous involvement usually have a poor prognosis with no curative therapy available so far. In the present European Organization for... Show moreMycosis fungoide (MF) patients who develop tumors or extracutaneous involvement usually have a poor prognosis with no curative therapy available so far. In the present European Organization for Research and Treatment of Cancer (EORTC) multicenter study, the genomic profile of 41 skin biopsies from tumor stage MF (MFt) was analyzed using a high-resolution oligo-array comparative genomic hybridization platform. Seventy-six percent of cases showed genomic aberrations. The most common imbalances were gains of 7q33.3q35 followed by 17q21.1, 8q24.21, 9q34qter, and 10p14 and losses of 9p21.3 followed by 9q31.2, 17p13.1, 13q14.11, 6q21.3, 10p11.22, 16q23.2, and 16q24.3. Three specific chromosomal regions, 9p21.3, 8q24.21, and 10q26qter, were defined as prognostic markers showing a significant correlation with overall survival (OS) (P = 0.042, 0.017, and 0.022, respectively). Moreover, we have established two MFt genomic subgroups distinguishing a stable group (0-5 DNA aberrations) and an unstable group (> 5 DNA aberrations), showing that the genomic unstable group had a shorter OS (P = 0.05). We therefore conclude that specific chromosomal abnormalities, such as gains of 8q24.21 (MYC) and losses of 9p21.3 (CDKN2A, CDKN2B, and MTAP) and 10q26qter (MGMT and EBF3) may have an important role in prognosis. In addition, we describe the MFt genomic instability profile, which, to our knowledge, has not been reported earlier. Show less
Primary cutaneous anaplastic large cell lymphoma (C-ALCL) has an indolent clinical course and favorable prognosis. On the contrary, primary cutaneous peripheral T-cell lymphoma not otherwise... Show morePrimary cutaneous anaplastic large cell lymphoma (C-ALCL) has an indolent clinical course and favorable prognosis. On the contrary, primary cutaneous peripheral T-cell lymphoma not otherwise specified (PTL-NOS) shows aggressive clinical behavior. To identify genomic events relevant in the pathogenesis of these cutaneous T-cell lymphomas (CTCLs), we carried out array-based comparative genomic hybridization (CGH) analysis. Simultaneously, gene expression profiling was conducted to gain insight into gene expression programs associated with the different clinical behavior of these CTCLs. C-ALCL was characterized by gains on chromosome 7q and 17q and losses on 6q and 13q. PTL-NOS similarly showed gains on 7q and 17q, but was distinguished by gains on chromosome 8 and loss of a focal overlapping region on 9p21. We identified minimal common regions harboring candidate oncogenes and tumor suppressor genes in C-ALCL and PTL-NOS. Genes with a role in lymphocyte chemotaxis, apoptosis, and proliferation were overrepresented among genes differentially expressed between these lymphomas. C-ALCL showed higher expression of the skin-homing chemokine receptor genes CCR10 and CCR8, which may explain the lower tendency to disseminate to extracutaneous sites. Furthermore, C-ALCL and PTL-NOS showed aberrant expression of distinct genes implicated in apoptosis and proliferation, such as IRF4/MUM1 and PRKCQ, which may account for differences in clinical aggressiveness. Show less
Faaij, C.M.; Annels, N.E.; Ruigrok, G.; Burg, M. van der; Ball, L.M.; Bredius, R.G.; ... ; Lankester, A.C. 2010
