Glutathione (GSH) is a major source of reducing equivalents in mammalian cells. To examine the role of GSH synthesis in development and cell growth, we generated mice deficient in GSH by a targeted disruption of the heavy subunit of ␥-glutamylcysteine synthetase (␥GCS-HS tm1 ), an essential enzyme in GSH synthesis. Embryos homozygous for ␥GCS-HS tm1 fail to gastrulate, do not form mesoderm, develop distal apoptosis, and die before day 8.5. Lethality results from apoptotic cell death rather than reduced cell proliferation. We also isolated cell lines from homozygous mutant blastocysts in medium containing GSH. These cells also grow indefinitely in GSH-free medium supplemented with N-acetylcysteine and have undetectable levels of GSH; further, they show no changes in mitochondrial morphology as judged by electron microscopy. These data demonstrate that GSH is required for mammalian development but dispensable in cell culture and that the functions of GSH, not GSH itself, are essential for cell growth.

Prolonged expression of activated ras mutants resulted in both neoplastic transformation and suppression of serum-induced c-fos expression in Rat1 fibroblasts. Expression of other serum-inducible genes, including c-jun and beta-actin, was not suppressed in ras-transformed Rat1 cells, indicating that these effects are specific for c-fos and that growth-factor signal transduction pathways remain essentially intact. Run-on transcription studies indicated that c-fos transcription was blocked at the level of initiation in these cells. Transient transfection studies using 360 bp from the wild-type c-fos promoter as well as a series of mutated c-fos promoter fragments linked to the chloramphenicol acetyltransferase gene indicated that repression of c-fos was mediated by approximately 49 bp immediately upstream of the dyad symmetry element (DSE). Deletion of this region, referred to as the upstream repressor region (URR), restored serum inducibility to the c-fos promoter in ras-transformed cells. In contrast, suppression of c-fos transcription was not affected by either deletion of 240 bp between the DSE and the TATA element or by base-substitution mutations that inactive the ternary complex factor and fos-AP-1-like binding sites. In addition, in vitro competition studies indicated that ras-transformed cells express one or more repressor factors that interact with as-yet-unidentified elements within the c-fos promoter (possibly the URR) and block serum induction of c-fos. These findings suggest that prolonged expression of activated ras results in the activation of one or more as-yet-unidentified proteins that suppress transcription of the c-fos gene by interacting with the URR.