Establishment and characterization of an in vitro model of acquired resistance to cisplatin in a human testicular nonseminomatous germ cell line.

Clinically, human testicular nonseminomatous germ cell tumors exhibit remarkable sensitivity to platinum-based chemotherapy. To define better the mechanistic basis for this unusual sensitivity, the biochemical determinants of platinum-induced cytotoxicity have been investigated in a human testicular tumor cell line (GCT27) established from a previously untreated patient and in an in vitro derived 5.6-fold cisplatin-resistant stable variant (GCT27cisR). Compared to 12 ovarian and 5 cervical human tumor cell lines, the parent GCT27 line was among the most sensitive to the cytotoxic effects of both cisplatin (dosage producing 50% inhibition, 0.2 microM) and carboplatin (dosage producing 50% inhibition, 2.9 microM), thus reflecting clinical data. A 4-day exposure sulforhodamine B-staining assay was used to determine that GCT27cisR was cross-resistant to carboplatin and iproplatin and the classical bifunctional alkylating agents melphalan and chlorambucil. Partial cross-resistance was observed to tetraplatin, methotrexate, and mitomycin C. No cross-resistance was observed to Adriamycin, etoposide, vinblastine, bleomycin, 1-beta-D-arabinofuranosylcytosine, and 5-fluorouracil. Intracellular cisplatin accumulation across the dose range 2.5-100 microM (for 2 h) was 1.6 +/- 0.39-fold (mean +/- SD) greater for the parent line. There was no significant difference in glutathione levels between the two lines. The acquired resistance line was 1.9-fold more resistant than the parent line to the cytotoxic effects of cadmium chloride. There was no significant difference between the two lines, however, in the total amounts of platinum bound to DNA after cisplatin exposure (25, 50, or 100 microM for 2 h). The removal of total platinum adducts from DNA was significantly faster for GCT27cisR compared to the parent line (half-times of removal, 32 and 67 h, respectively). These data suggest that the abnormal sensitivity of the parent testicular tumor cell line to platinum-containing anticancer drugs may be due predominantly to an inherent defect in the ability of these cells to remove platinum from their DNA. This defect is apparently lost in the acquired resistance counterpart. Reduced intracellular accumulation and increased cytoplasmic concentrations of metallothionein may also contribute, in part, to the acquisition of cisplatin resistance in this model.     

A quantitative analysis of valgus torque on the ACL: a human cadaveric study.

The loads needed to elicit a positive pivot shift test in a knee with an anterior cruciate ligament (ACL) rupture have not been quantified. The coupled anterior tibial translation (ATT), coupled internal tibial rotation (ITR), and the in situ force in the ACL in response to a valgus torque, an inherent component of the pivot shift test, were measured in 10 human cadaveric knee specimens. Using a robotic/universal force-moment sensor testing system, valgus torques ranging from 0.0 to 10.0 Nm were applied in nine increments on the intact and ACL-deficient knee in flexion ranging from 0 degrees to 90 degrees. At 15 degrees of knee flexion, the coupled ATT and ITR were significantly increased in the ACL-deficient knee when compared to the intact knee. Coupled ATT increased a maximum of 291% (6.7 mm, p<0.05), while coupled ITR increased a maximum of 85% (5.1 degrees, p<0.05). At 30 degrees, the increases in coupled ATT and ITR were significant at valgus loads of 3.3 Nm and greater with a maximum increase in coupled ATT of 137% (6.3 mm, p<0.05) and a maximum increase in coupled ITR of 38% (3.6 degrees, p<0.05). At 45 degrees, coupled ATT increased significantly (maximum of 69%, 4.4 mm, p<0.05), but only at torques > or =6.7 Nm. The in situ force in the ACL was less than 20 N for all flexion angles when a torque between 3.3 and 5.0 Nm was applied. Low valgus torque elicited tibial subluxation in the ACL-deficient knee with low in situ ACL forces, similar to a positive pivot shift test. Thus, application of a valgus torque may be suitable to evaluate ACL-deficient and ACL-reconstructed knees, since subluxation can be achieved with minimal harm to the ACL graft. This work is important in understanding one load component needed for the pivot shift examination; further studies quantifying other load components are essential for better comprehension of the in vivo pivot shift examination.