p53 activates expression of HIC-1, a new candidate tumour suppressor gene on 17p13.3.

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.

Absence of WAF1 mutations in a variety of human malignancies.

A newly cloned gene named wild-type p53-activated fragment 1 (WAF1; also known as p21, Pic-1, Cip-1, or SDI1) is directly regulated by p53 and can itself suppress tumor cell growth in culture. Induction of expression of WAF1 may be an important means by which cells with DNA injury arrest their growth to repair DNA or undergo apoptosis. Based on the hypothesis that mutations of this gene may play a role in carcinogenesis, we have studied 351 DNAs from 14 kinds of malignancies, as well as 36 human transformed cell lines, for alterations of WAF1 gene by single-strand conformation polymorphism analysis of polymerase chain reaction amplification of the DNA coding region of the WAF1 gene.

p53 gene mutations are associated with decreased sensitivity of human lymphoma cells to DNA damaging agents.

The present study assessed the role of the p53 tumor suppressor gene in cell cycle arrest and apoptosis following treatment of Burkitt's lymphoma and lymphoblastoid cell lines with gamma-rays, etoposide, nitrogen mustard, and cisplatin. Cell cycle arrest was measured by flow cytometry; p53 and p21Waf1/Cip1 protein levels were measured by Western blotting; cell survival was measured in 72-96-h growth inhibition assays and by trypan blue staining, and apoptotic DNA fragmentation was assessed by either agarose gel electrophoresis or a modified filter elution method. We found that gamma-rays and etoposide induced a strong G1 arrest in the wild-type p53 lines while nitrogen mustard and cisplatin induced relatively little G1 arrest.

p53 tagged sites from human genomic DNA.

The product of the tumor suppressor gene p53 binds to DNA and activates transcription from promoters containing its consensus binding site. This activity has been hypothesized to be responsible for its biological effects. However, the total number and nature of human genomic sites with which p53 can functionally interact is unknown.

Sequence-specific transcriptional activation is essential for growth suppression by p53.

Although several biochemical features of p53 have been described, their relationship to tumor suppression remains uncertain. We have compared the ability of p53-derived proteins to act as sequence-specific transcriptional (SST) activators with their ability to suppress tumor cell growth, using an improved growth-suppression assay. Both naturally occurring and in vitro derived mutations that abrogated the SST activity of p53 lost the ability to suppress tumor cell growth.

WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis.

The tumor growth suppressor WAF1/CIP1 was recently shown to be induced by p53 and to be a potent inhibitor of cyclin-dependent kinases. In the present studies, we sought to determine the relationship between the expression of WAF1/CIP1 and endogenous regulation of p53 function. WAF1/CIP1 protein was first localized to the nucleus of cells containing wild-type p53 and undergoing G1 arrest.

WAF1, a potential mediator of p53 tumor suppression.

The ability of p53 to activate transcription from specific sequences suggests that genes induced by p53 may mediate its biological role as a tumor suppressor. Using a subtractive hybridization approach, we identified a gene, named WAF1, whose induction was associated with wild-type but not mutant p53 gene expression in a human brain tumor cell line. The WAF1 gene was localized to chromosome 6p21.

A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia.

Cell cycle checkpoints can enhance cell survival and limit mutagenic events following DNA damage. Primary murine fibroblasts became deficient in a G1 checkpoint activated by ionizing radiation (IR) when both wild-type p53 alleles were disrupted. In addition, cells from patients with the radiosensitive, cancer-prone disease ataxia-telangiectasia (AT) lacked the IR-induced increase in p53 protein levels seen in normal cells.

Isolation and characterization of the cDNA encoding human DNA methyltransferase.

We have cloned a series of overlapping cDNA clones encoding a 5194 bp transcript for human DNA methyltransferase (DNA MTase). This sequence potentially codes for a protein of 1495 amino acids with a predicted molecular weight of 169 kDa. The human DNA MTase cDNA has eighty percent homology at the nucleotide level, and the predicted protein has seventy-four percent identity at the amino acid level, to the DNA MTase cDNA cloned from mouse cells.

Definition of a consensus binding site for p53.

Recent experiments have suggested that p53 action may be mediated through its interaction with DNA. We have now identified 18 human genomic clones that bind to p53 in vitro. Precise mapping of the binding sequences within these clones revealed a consensus binding site with a striking internal symmetry, consisting of two copies of the 10 base pair motif 5'-PuPuPuC(A/T)(T/A)GPyPyPy-3' separated by 0-13 base pairs.

High expression of the DNA methyltransferase gene characterizes human neoplastic cells and progression stages of colon cancer.

DNA methylation abnormalities occur consistently in human neoplasia including widespread hypomethylation and more recently recognized local increases in DNA methylation that hold potential for gene inactivation events. To study this imbalance further, we have cloned and localized to chromosome 19 a portion of the human DNA methyltransferase gene that codes for the enzyme catalyzing DNA methylation. Expression of this gene is low in normal human cells, significantly increased (30- to 50-fold by PCR analysis) in virally transformed cells, and strikingly elevated in human cancer cells (several hundredfold).

Mechanisms of error discrimination by Escherichia coli DNA polymerase I.

The mechanism of base selection by DNA polymerase I of Escherichia coli has been investigated by kinetic analysis. The apparent KM for the insertion of the complementary nucleotide dATP into the hook polymer poly(dT)-oligo(dA) was found to be 6-fold lower than that for the noncomplementary nucleotide dGTP, whereas the Vmax for insertion of dATP was 1600-fold higher than that for dGTP. The ratio of Kcat/KM values for complementary and mismatched nucleotides of 10(4) demonstrates the extremely high specificity of base selection by DNA polymerase I and is in agreement with results obtained with a different template-primer, poly(dC)-oligo(dG) [El-Deiry, W.

Molecular mechanisms of manganese mutagenesis.

The mechanism by which DNA polymerase discriminates between complementary and noncomplementary nucleotides for insertion into a primer terminus has been investigated. Apparent kinetic constants for the insertion of dGTP and dATP into the hook polymer d(C)194-d(G)12 with Escherichia coli DNA polymerase I (large fragment) were determined. The results suggest that the high specificity of base selection by DNA polymerase I is achieved by utilization of both Km and Vmax differences between complementary and noncomplementary nucleotides.

Stabilization of proteins by guanidination.

Earlier studies have indicated the marked resistance of two pronase endopeptidases to denaturation in high concentrations of urea or guanidine hydrochloride (Siegel, S., and Awad, W. M.