M C Hollander

Mammalian genes coordinately regulated by growth arrest signals and DNA-damaging agents.

More than 20 different cDNA clones encoding DNA-damage-inducible transcripts in rodent cells have recently been isolated by hybridization subtraction (A. J. Fornace, Jr., I. Alamo, Jr., and M. C. Hollander, Proc. Natl. Acad. Sci. USA 85:8800-8804, 1988). In most cells, one effect of DNA damage is the transient inhibition of DNA synthesis and cell growth. We now show that five of our clones encode transcripts that are increased by other growth cessation signals: growth arrest by serum reduction, medium depletion, contact inhibition, or a 24-h exposure to hydroxyurea. The genes coding for these transcripts have been designated gadd (growth arrest and DNA damage inducible). Two of the gadd cDNA clones were found to hybridize at high stringency to transcripts from human cells that were induced after growth cessation signals or treatment with DNA-damaging agents, which indicates that these responses have been conserved during mammalian evolution. In contrast to results with growth-arrested cells that still had the capacity to grow after removal of the growth arrest conditions, no induction occurred in HL60 cells when growth arrest was produced by terminal differentiation, indicating that only certain kinds of growth cessation signals induce these genes. All of our experiments suggest that the gadd genes are coordinately regulated: the kinetics of induction for all five transcripts were similar; in addition, overexpression of gadd genes was found in homozygous deletion c14CoS/c14CoS mice that are missing a small portion of chromosome 7, suggesting that a trans-acting factor encoded by a gene in this deleted portion is a negative effector of the gadd genes. The gadd genes may represent part of a novel regulatory pathway involved in the negative control of mammalian cell growth.

Genomic instability in Gadd45a- deficient mice

Gadd45a-null mice generated by gene targeting exhibited several of the phenotypes characteristic of p53-deficient mice, including genomic instability, increased radiation carcinogenesis and a low frequency of exencephaly. Genomic instability was exemplified by aneuploidy, chromosome aberrations, gene amplification and centrosome amplification, and was accompanied by abnormalities in mitosis, cytokinesis and growth control. Unequal segregation of chromosomes due to multiple spindle poles during mitosis occurred in several Gadd45a –/– cell lineages and may contribute to the aneuploidy. Our results indicate that Gadd45a is one component of the p53 pathway that contributes to the maintenance of genomic stability.

Gadd34 Requirement for Normal Hemoglobin Synthesis

ABSTRACT The protein encoded by growth arrest and DNA damage-inducible transcript 34 (Gadd34) is associated with translation initiation regulation following certain stress responses. Through interaction with the protein phosphatase 1 catalytic subunit (PP1c), Gadd34 recruits PP1c for the removal of an inhibitory phosphate group on the α subunit of elongation initiation factor 2, thereby reversing the shutoff of protein synthesis initiated by stress-inducible kinases. In the absence of stress, the physiologic consequences of Gadd34 function are not known. Initial analysis of Gadd34-null mice revealed several significant findings, including hypersplenism, decreased erythrocyte volume, increased numbers of circulating erythrocytes, and decreased hemoglobin content, resembling some thalassemia syndromes. Biochemical analysis of the hemoglobin-producing reticulocyte (an erythrocyte precursor) revealed that the decreased hemoglobin content in the Gadd34-null erythrocyte is due to the reduced initiation of the globin translation machinery. We propose that an equilibrium state exists between Gadd34/PP1c and the opposing heme-regulated inhibitor kinase during hemoglobin synthesis in the reticulocyte.

Genomic Instability in Gadd45a-/- Cells is Coupled with S-Phase Checkpoint Defects

Gadd45a is a p53-regulated gene whose protein product, like p53, is involved in maintenanceof genome stability. Specifically, deletion of Gadd45a leads to extensive aneuploidy as aconsequence of centrosome amplification and subsequent abnormal segregation of chromosomesduring mitosis. S-phase checkpoints were investigated in Gadd45a-/- cells to determine possibledefects contributing to the uncoupling of centrosome duplication and DNA replication. In thepresence of hydroxyurea, Gadd45a-/- mouse embryo fibroblasts show increased centrosomeamplification coupled with loss of a sustained S-phase checkpoint. Gadd45a deletion allowsanother form of genomic instability, gene amplification, when p21 (Cdkn1a gene product) isdeleted also. Gene amplification in Gadd45a-/-p21-/- cells correlated with loss of both G1 andS-phase checkpoints. Multiple conditions of nutrient deprivation failed to prevent DNAsynthesis in Gadd45a-/- cells. Gadd45a is therefore required for proper S-phase control andcheckpoints under multiple conditions of nutrient deprivation. It is proposed that loss of S-phasecontrol may account for both the uncoupling of DNA replication and centrosome duplication,and conferring gene amplification proficiency in cells lacking Gadd45a-/-. This is of particularimportance for solid tumors, which may lack sufficient nutrients yet are unable to elicitcheckpoints preventing genomic instability under these conditions.

Gadd45a acts as a modifier locus for lymphoblastic lymphoma.

Gadd45a−/− and p53−/− mice and cells derived from them share similar phenotypes, most notably genomic instability. However, p53−/− mice rapidly develop a variety of neoplasms, while Gadd45a−/− mice do not. The two proteins are involved in a regulatory feedback loop, whereby each can increase the expression or activity of the other, suggesting that common phenotypes might result from similar molecular mechanisms. Mice lacking both genes were generated to address this issue. Gadd45a−/−p53−/− mice developed tumors with a latency similar to that of tumor-prone p53−/− mice. However, while p53−/− mice developed a variety of tumor types, nearly all Gadd45a−/−p53−/− mice developed lymphoblastic lymphoma (LBL), often accompanied by mediastinal masses as is common in human patients with this tumor type. Deletion of Gadd45a in leukemia/lymphoma-prone AKR mice decreased the latency for LBL. These results indicate that Gadd45a may act as modifier locus for T-cell LBL, whereby deletion of Gadd45a enhances development of this tumor type in susceptible mice. Gadd45a is localized to 1p31.1, and 1p abnormalities have been described in T-cell lymphomas. Related human tumor samples did not show Gadd45a deletion or mutation, although changes in expression could not be ruled out.