Heather Ramsey

Update on Late Relapse of Germ Cell Tumor: A Clinical and Molecular Analysis

PURPOSE Analysis of patients with late relapse (LR) of germ cell tumor (GCT) with reports on clinical characteristics, outcomes, and molecular and cytogenetic features. PATIENTS AND METHODS Eighty-three patients evaluated at Indiana University from 1993 through 2000 for relapse of GCT more than 2 years from initial therapy were reviewed. Available specimens were investigated for expression of the transcription regulator FoxD3 and apurinic/apyrimidinic endonuclease and the presence of chromosome 12 abnormalities. RESULTS Median interval from initial presentation to LR was 85 months. Forty-three of 49 LR patients who underwent surgery were rendered disease free (NED), and 20 (46.5%) remain continuously NED. Thirty-two patients received chemotherapy, but only six (18.8%) obtained a complete remission. Five of these patients remain continuously NED after chemotherapy alone, including three who were chemotherapy naïve. Eighteen of these 32 patients were successfully rendered NED by postchemotherapy surgery, and 12 remain continuously NED. Two patients continue on observation with no treatment for their LR. Overall, 69 of the 81 treated patients (85.2%) ultimately achieved an NED state, and 38 (46.9%) remain continuously NED with median follow-up from LR therapy of 24.5 months (range, 1 to 83 months), whereas nine other patients are currently NED after therapy for subsequent relapses. Because of the small numbers of specimens tested, we were unable to draw any definitive conclusions from the molecular and cytogenetic analyses. CONCLUSION GCT patients require lifetime follow-up. At the time of LR, surgical resection alone remains our preferred therapy.

Redox Regulation of the Embryonic Stem Cell Transcription Factor Oct‐4 by Thioredoxin

Oct‐4 is a transcriptional regulator required to maintain the totipotentiality of embryonic stem (ES) cells. Downregulation of its activity is required for proper differentiation of the blastocyst during uterine implantation. Uterine implantation and subsequent vascularization increase oxygen exposure of the developing embryo, thereby altering the intracellular reduction‐oxidation status. We tested whether Oct‐4 could be regulated by these changes in reduction‐oxidation status. We found that Oct‐4 DNA binding was exquisitely sensitive to abrogation by oxidation but that the DNA binding of another ES cell transcription factor, FoxD3, was much less sensitive to oxidation. The reducing enzyme Thioredoxin (but not Ape‐1) could restore DNA‐binding activity of Oct‐4. Thioredoxin was less effective at restoring the DNA‐binding ability of FoxD3. It was also found that Thioredoxin (but not Ape‐1) could physically associate with cysteines in the POU domain of Oct‐4. Finally, overexpressing normal Thioredoxin increased the transcriptional activity of Oct‐4, while overexpressing a mutant Thioredoxin decreased the transcriptional activity of Oct‐4. These data imply that ES cell transcription factors are differentially sensitive to oxidation and that Thioredoxin may differentially regulate ES cell transcription factors.

The human set and transposase domain protein Metnase interacts with DNA Ligase IV and enhances the efficiency and accuracy of non-homologous end-joining

Transposase domain proteins mediate DNA movement from one location in the genome to another in lower organisms. However, in human cells such DNA mobility would be deleterious, and therefore the vast majority of transposase-related sequences in humans are pseudogenes. We recently isolated and characterized a SET and transposase domain protein termed Metnase that promotes DNA double-strand break (DSB) repair by non-homologous end-joining (NHEJ). Both the SET and transposase domain were required for its NHEJ activity. In this study we found that Metnase interacts with DNA Ligase IV, an important component of the classical NHEJ pathway. We investigated whether Metnase had structural requirements of the free DNA ends for NHEJ repair, and found that Metnase assists in joining all types of free DNA ends equally well. Metnase also prevents long deletions from processing of the free DNA ends, and improves the accuracy of NHEJ. Metnase levels correlate with the speed of disappearance of gamma-H2Ax sites after ionizing radiation. However, Metnase has little effect on homologous recombination repair of a single DSB. Altogether, these results fit a model where Metnase plays a role in the fate of free DNA ends during NHEJ repair of DSBs.

Down-regulation of the myeloid homeobox protein Hex is essential for normal T-cell development

The haematopoietic homeobox gene Hex (also called Prh) is expressed in myeloid cells and B cells but not T cells. To investigate whether Hex levels might play a role in myeloid versus T‐cell development, two types of transgenic mouse lines were constructed, each with ectopic expression of Hex in T cells (CD11a/Hex and Lck/Hex). Both these types of transgenic mouse had the same defects in T‐cell maturation, indicating that proper T‐cell development may be dependent not just on the up‐regulation of lymphoid‐specific transcriptional regulators but also on the co‐ordinated down‐regulation of myeloid‐specific transcriptional regulators such as Hex. In addition, Hex over‐expression significantly increased myeloid progenitor cycling, which may explain its role in retrovirally induced murine leukaemia.