The introduction of systemic chemotherapy improved significantly the prognosis of osteosarcoma. Despite this success, approximately 30 -40% of patients will relapse. Cytotoxic drugs have been shown to induce apoptosis in the target cells independent of their primary effects. The underlying molecular mechanisms and the intracellular mediators, however, are still largely unknown. Therefore, the purpose of our study was to identify drug-regulated genes in osteosarcoma cells useful as prognostic factors and for the development of new therapeutic strategies. Using suppressive subtractive hybridization (SSH) the gene expression pattern of untreated Saos-2 cells was compared to cells treated with cisplatin, methotrexate and doxorubicin, respectively. We identified 8 genes that are regulated >2-fold in drug-treated osteosarcoma cell lines. Expression of ferritin light chain, rhoA, inosine monophosphatdgehydrogenase II, ribonucleotide reductase M2, pro2000 and pro1859 were increased after drug treatment, whereas prohibitin and ␣-actinin expressions were significantly downregulated. Differential expression of the identified genes was verified by Northern blot analysis of 3 different osteosarcoma cell lines. In addition, the effects on chemosensitivity of 4 selected genes was analyzed by overexpression of recombinant constructs in Saos-2 cells and subsequent quantification of drug-induced apoptosis. Overexpression of prohibitin and rhoA reduced significantly drug sensitivity to approximately 52% and 59% indicating a crucial role in the modulation of drug-induced cell death.
Apoptosis is a key mechanism of the organism that regulates embryogenesis and development, maintains homeostasis of the immune system and removes potentially hazardous cells. A dysregulation of apoptosis signaling may thus disturb the balance of cell survival and cell death, leading to the development of several diseases including cancer. In order to determine whether osteosarcomas display an increased frequency of genetic alterations that affect apoptosis signaling, we analyzed the death domains of the death receptor genes CD95/Fas/Apo1, TNFR1, DR3/Apo3/WSL-1/LARD/TRAMP, DR5/TRAIL-R2/TRICK2/KILLER, DR6 and the complete coding sequences of the death receptor gene DR4/TRAIL-R1 and the genes of the adaptors TRADD and FADD/MORT-1. The investigation included 15 osteosarcoma tumor samples, 3 osteosarcoma cell lines (SAOS-2, HOS and MG63) and peripheral blood from 20 donors as controls. We were able to identify 4 different sequence variations within the DR4 gene located on exons 3, 4, 5 and 10 (death-domain). No alterations have been detected in the other genes or exons investigated. Except the sequence variant affecting exon 4, the alterations were homozygous in 15% of the tumor samples and cell lines, whereas the same alterations found in the control group were heterozygous or even not detectable. Three out of 4 alterations are located in the receptor's extracellular cysteine rich domain, which contains the ligand binding area and 1 on exon 10 coding for the death-domain. They may thus exert influence on ligand-receptor interactions and subsequent apoptosis induction. Our findings suggest that homozygous genetic alterations within the DR4 gene may be implicated in the formation of osteosarcoma.
Mutations in the TRADD gene may contribute to the development of different hematological diseases. The identified mutations demonstrate a putative impact on TRADD signaling and cell survival but may not mainly explain the pathology of the diseases investigated.
Recent studies showed contrasting findings in morphological changes due to competitive soccer in adolescent players (SP). We present a prospective study in 315 consecutive adolescent (10-14 years) male elite SP and 53 healthy matched active controls (CON). All participants underwent a complete transthoracic two-dimensional echocardiography (TTE). The mean age in SP was 12.8 ± 0.65 years compared to 12.6 ± 0.8 years in CON. For all left ventricular (LV) dimensions, mean Z-score values were higher in SP. There was a significant Z-score increase in interventricular septum diastolic diameter (2.47z vs. 1.62z, p < 0.05), left ventricular posterior wall diastolic and systolic diameter (1.15z vs. 0.47z, p < 0.05 and 1.05z vs. − 0.4z, p < 0.05). Athletes had significant greater LV mass indexed for BSA (94 ± 12 g/m 2 vs. 81 ± 13 g/m 2 , p < 0.05). There was no significant difference in LV function or diameters. Conclusion: Our findings suggest that elite soccer training in adolescent male is a type of sport predominantly related to cardiac resistance remodeling. Adolescent SP may develop supernormal left ventricular wall dimensions (+ 2.0 to + 2.5z). If in SP Z-scores, any LV dimension above + 2.5 is measured, primary or secondary cardiomyopathies should be excluded.
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