Cell cycle checkpoints appear to contribute to an increase in cell survival and a decrease in abnormal heritable genetic changes following exposure to DNA damaging agents. Though several radiation-sensitive yeast mutants have been identified, little is known about the genes that control these responses in cells. Recent studies from our laboratory have demonstrated a close correlation between expression of wild-type p53 genes in human hematopoietic cells and their ability to arrest in G1 phase after certain types ofDNA damage. In the present study, this correlation was first generalized to nonhematopoietic mammalian cells as well. A cause and effect relationship between expression of wild-type p53 and the G, arrest that occurs after y irradiation was then established by demonstrating (1) acquisition of the G, arrest after y irradiation following transection of wild-type p53 genes into cells lacking endogenous p53 genes and (it) loss of the G, arrest after irradiation following transfection of mutant p53 genes into cells with wild-type endogenous p53 genes. A defined role for p53 (the most commonly mutated gene in human cancers) in a physiologic pathway has, to our knowledge, not been reported previously. Furthermore, these experiments illustrate one way in which a mutant p53 gene product can function in a "dominant negative" manner. Participation of p53 in this pathway suggests a mechanism for the contribution of abnormalities in p53 to tumorigenesis and genetic instability and provides a useful model for studies of the molecular mechanisms of p53 involvement in controlling the cell cycle.Transient alterations in cell cycle progression after exposure to various different DNA damaging agents have been observed in many cell types (1-10). These alterations presumably permit optimal repair of damage before the cell reinitiates replicative DNA synthesis (Gi arrest) and/or begins mitosis (G2 arrest) (e.g., ref. 1). Failure to repair DNA damage prior to replicative synthesis or mitosis could result in "fixation" and propagation of mutagenic lesions (11, 12) and could contribute to the progressive accumulation of genomic changes necessary for neoplastic transformation to occur. In yeast, the availability of genetic mutants has led to the identification ofthe RAD9 gene product as a critical factor in the G2 arrest after y irradiation (XRT) (1, 2). However, little is known about the cellular signals required for these cell cycle checkpoints after DNA damage in mammalian cells.We recently began to investigate some of the mechanisms in mammalian cells that control the cell cycle changes in response to DNA damage (13). We found that nonlethal doses of XRT can transiently inhibit replicative DNA synthesis by G, and G2 arrests (in agreement with data from other laboratories; e.g., refs. 1-5) and that levels of the "tumor suppressor" nuclear protein, p53, increase (apparently by a posttranscriptional mechanism) in temporal association with the decrease in replicative DNA synthesis. Inhibition of the rise in p53 protein...

For several human tumour types, allelic loss data suggest that one or more tumour suppressor genes reside telomeric to the p53 gene at chromosome 17p13.1. In the present study we have used a new strategy, involving molecular analysis of a DNA site hypermethylated in tumour DNA, to identify a candidate gene in this region (17p13.3). Our approach has led to identification of HIC-1 (hypermethylated in cancer), a new zinc-finger transcription factor gene which is ubiquitously expressed in normal tissues, but underexpressed in different tumour cells where it is hypermethylated. Multiple characteristics of this gene, including the presence of a p53 binding site in the 5' flanking region, activation of the gene by expression of a wild-type p53 gene and suppression of G418 selectability of cultured brain, breast and colon cancer cells following insertion of the gene, make HIC-1 gene a strong candidate for a tumour suppressor gene in region 17p13.3.

Recent studies have demonstrated imprinting of the human neuronatin (NNAT) gene. NNAT maps to 20q11.2-q12, a region exhibiting loss of heterozygosity in acute myeloid leukemia and myelodysplastic/myeloproliferative disease. To investigate possible epigenetic dysregulation of genes in this region relevant to leukemogenesis, we analyzed methylation of the NNAT gene in normal tissues and in leukemias. We found a differential methylation pattern, typical of imprinted genes, at sites in the CpG island containing NNAT exon 1 in normal pituitary, peripheral blood cells and bone marrow-derived CD34-positive hematopoietic progenitor cells. Substantial or complete loss of the unmethylated NNAT allele was observed in leukemia cell lines and in 20 of 29 (69%) acute myeloid or lymphoid leukemia samples. While most highly expressed in brain, NNAT mRNA was also detected in normal hematopoietic progenitor cells and in leukemia cells exhibiting the normal methylation pattern, although not in hypermethylated leukemia cells. Demethylation by treatment of hypermethylated leukemia cells with 5-aza-2'-deoxycytidine resulted in reactivation of NNAT expression, concomitant with a reversion to the normal methylation pattern. The data demonstrate that hypermethylation of the NNAT locus is a frequent event in both myeloid and lymphoid acute leukemias of childhood. Aberrant hypermethylation of the NNAT locus suggests that the dysregulation of genes at 20q11.2-q12 in leukemia may be the result of epigenetic as well as genetic events.

The temporal relationship between DNA damage and DNA replication may be critical in determining whether the genetic changes necessary for cellular transformation occur after DNA damage. Recent characterization of the mechanisms responsible for alterations in cellcycle progression after DNA damage in our laboratory have implicated the p53 (tumor suppressor) protein in the G, arrest that occurs after certain types of DNA damage. In particular, we found that levels of p53 protein increased rapidly and transiently after nonlethal doses of y irradiation (XRT) in hematopoietic cells with wild-type, but not mutant, p53 genes. These changes in p53 protein levels were temporally linked to a transient GI arrest in these cells. Hematopoietic cells with mutant or absent p53 genes did not exhibit this G, arrest, through they continued to demonstrate a G2 arrest. We recently extended these observations of a tight correlation between the status of the endogenous p53 genes and this G, arrest after XRT and this cell-cycle alteration after XRT was then established by transfecting cells lacking endogenous p53 genes with a wild-type gene and observing acquisition of the G1 arrest and by transfecting cells processing endogenous wild-type p53 genes with a mutant p53 gene and observing loss of the GI arrest after XRT. These observations and their significance for our understanding of the mechanisms of DNA damage-induced cellular transformation are discussed.Transient alterations in cell-cycle progression after DNA damage are well documented. Presumably, these responses permit optimal repair of damage before the cell reinitiates replicative DNA synthesis (G1 arrest) and/or begins mitosis (G2 arrest). If replicative DNA synthesis or mitosis occurred before repair of the damage, then mutagenic lesions could be "fixed" and propagated (1) and could contribute to the progressive increase in genomic changes necessary for neoplastic transformation (Fig. 1). Currently, little is known about the cellular signals required for these cell-cycle check points after DNA damage in mammalian cells. In yeast, the RAD9 gene product appears to be necessary for G2 arrest after damage (2), but the factors required for G1 arrest remain unclear.We recently began to characterize some of the mechanisms that control cell-cycle changes in response to DNA damage in mammalian cells (3). Using hematopoietic cell lines as models, we found that nonlethal doses of y irradiation (XRT) transiently inhibit replicative DNA synthesis via both G1 and G2 arrests. We reasoned that this inhibition of replicative DNA syn- ' thesis after XRT could result from either inhibition of a positive regulator of DNA synthesis or stimulation or a negative regulator. Because the p53 gene product had been demonstrated to be a negative regulator of DNA synthesis (4-7) and because this tumor-suppressor gene is the most commonly mutated gene thus far identified in human cancers, linkage of p53 to this DNA damage-induced inhibition of DNA replication was an attractive possibility.Because the wil...