Mutation of the serine 15 phosphorylation site of human p53 reduces the ability of p53 to inhibit cell cycle progression.

Overexpression of wild-type p53 prevents cells from entering the S phase of the cell cycle. The amino-terminal transactivation region of p53 is phosphorylated by several protein kinases, including DNA-PK, a nuclear serine/threonine protein kinase that in vitro requires DNA for activity. DNA-PK was recently shown to phosphorylate serines 15 and 37 of human p53 (Lees-Miller et al., 1992. Mol. Cell. Biol., 12, 5041-5049). To prevent phosphorylation at these sites, mutants were constructed that changed the codons for serine 15 or serine 37 to alanine codons. Expression of p53-Ala-37 in stably transformed T98G cells blocked progression of the cells into S phase as well as did the expression of wild-type p53. In contrast, p53-Ala-15 was partially defective in blocking cell cycle progression. Several cell clones transformed with the mutant p53-Ala-15 gene expressed normal levels of p53 mRNA but accumulated little or no detectable p53 protein. However, by using a transient expression system driven by a strong cytomegalovirus promoter, we showed that the inability of p53-Ala-15 to fully block cell cycle progression was not due to inadequate levels of expression or to a failure of the mutant protein to accumulate in the nucleus. These results suggest that phosphorylation of Ser-15 may affect p53 function.

Cell cycle effects of microinjected antisense oligodeoxynucleotides to p34cdc2 kinase.

In this study the effect of antisense oligomers targeted against the mRNA transcripts of p34cdc2 kinase on G1 progression into S-phase was examined. For this purpose, antisense, sense, or nonsense oligomers were introduced directly into the cytoplasm of T98G cells grown in monolayer cultures by glass-capillary microinjection. The microinjection of antisense oligomers (but not sense or nonsense oligomers) into growth-arrested cells before serum stimulation inhibited G1 progression into S-phase. This inhibition was correlated with a reduction in the steady-state levels of nuclear p34cdc2 protein. Microinjection of antisense oligomers into cells at 2 and 6 hours after serum stimulation also resulted in a marked inhibition in the ability of cells to enter S-phase. The inhibitory effect decreased when cells were microinjected at 12 hours after serum stimulation. When cells were microinjected at 18 and 24 hours after serum stimulation, only a slight inhibition was observed. As the antisense oligomers were introduced directly into the cytoplasm of cells at each of the time points examined, the observed differences in the inhibitory effects of the antisense oligomers at later times after serum stimulation cannot be explained by differences in uptake. An alternative explanation is that after a certain threshold level of nuclear p34cdc2 protein is reached in late G1 phase; no further increase is necessary, because the cells become committed to enter S-phase. In yeast, p34cdc2 appears to play an important role in the G1/S-phase transition at a control point in late G1 phase called START (reviewed by Lewin). In mammalian cells a control point that could be equivalent to START is the "restriction point" which is defined as the time after which inhibition of protein synthesis fails to block entry into S-phase (reviewed by Pardee). The effects observed with antisense oligomers to p34cdc2 kinase are strikingly similar to what is observed when low concentrations of the drug cycloheximide are added to these cells at different times after serum stimulation; entry into S-phase is significantly inhibited when cycloheximide is added up to 12 hours postimulation. Thus, the results reported in this study are in agreement with the idea that p34cdc2 kinase plays a role in the G1/S phase transition in mammalian cells. Finally, introduction of antisense oligomers directly into the cytoplasm of cells grown in monolayer cultures by glass-capillary microinjection appears to be a viable alternative to simply adding the oligomers to the culture medium.(ABSTRACT TRUNCATED AT 400 WORDS)

Growth arrest induced by wild-type p53 protein blocks cells prior to or near the restriction point in late G1 phase.

Conditional expression of wild-type (wt) p53 protein in a glioblastoma tumor cell line has been shown to be growth inhibitory. We have now more precisely localized the position in the cell cycle where growth arrest occurs. We show that growth arrest occurs prior to or near the restriction point in late G1 phase of the cell cycle. The effect of wt p53 protein on the expression of four immediate-early genes (c-FOS, c-JUN, JUN-B, and c-MYC), one delayed-early gene (ornithine decarboxylase), and two late-G1/S-phase genes (B-MYB and DNA polymerase alpha) was also examined. Of this subset of growth response genes, only the expression of B-MYB and DNA polymerase alpha was significantly repressed. The possibility that decreased expression of B-MYB may be an important component of growth arrest mediated by wt p53 protein is discussed.

Growth suppression induced by wild-type p53 protein is accompanied by selective down-regulation of proliferating-cell nuclear antigen expression.

The p53 gene is a frequent target of mutation in a wide variety of human cancers. Previously, it was reported that conditional expression of wild-type p53 protein in a cell line (GM47.23) derived from a human glioblastoma multiform tumor had a negative effect on cell proliferation. We have now investigated the effect that induction of wild-type p53 protein in this cell line has on the expression of the proliferating-cell nuclear antigen gene. The proliferating-cell nuclear antigen gene encodes a nuclear protein that is an auxiliary factor of DNA polymerase delta and part of the DNA replication machinery of the cell. We show that inhibition of cell cycle progression into S-phase after induction of wild-type p53 protein is accompanied by selective down-regulation of proliferating-cell nuclear antigen mRNA and protein expression.

Negative growth regulation in a glioblastoma tumor cell line that conditionally expresses human wild-type p53.

To investigate the effect that human wild-type p53 (wt-p53) expression has on cell proliferation we constructed a recombinant plasmid, pM47, in which wt-p53 cDNA is under transcriptional control of the hormone-inducible mouse mammary tumor virus promoter linked to the dominant biochemical selection marker gene Eco gpt. The pM47 plasmid was introduced into T98G cells derived from a human glioblastoma multiforme tumor, and a stable clonal cell line, GM47.23, was derived that conditionally expressed wt-p53 following exposure to dexamethasone. We show that induction of wt-p53 expression in exponentially growing cells inhibits cell cycle progression and that the inhibitory effect is reversible upon removal of the inducer or infection with simian virus 40. Moreover, when growth-arrested cells are stimulated to proliferate, induction of wt-p53 expression inhibits G0/G1 progression into S phase and the cells accumulate with a DNA content equivalent to cells arrested in the G0/G1 phase of the cell cycle. Taken together, these studies suggest that wt-p53 may play a negative role in growth regulation.

Growth factor regulated expression of poly(A)+ binding protein messenger RNA.

A 72,000 mol wt protein designated PABP binds to the poly(A)+ track of messenger RNAs with high affinity and has been suggested to play an important role in mRNA metabolism in eucaryotic cells. We have employed a human PABP cDNA probe to study the expression of this gene at the mRNA level in BALB/c3T3 mouse cells under different growth conditions and in exponentially growing HeLa cells throughout the cell division cycle. We describe experiments which establish that in BALB/c3T3 cells the expression of this gene is growth factor regulated. Moreover, the gene behaves like a primary response gene in that its induction in quiescent cells does not require the prior synthesis of other growth factor-regulated proteins. In exponentially growing HeLa cells PABP mRNA is expressed throughout the cell division cycle indicating that the expression of this gene is not limited to a specific phase of the cell cycle.