WILD-TYPE HUMAN P53 AND A TEMPERATURE-SENSITIVE MUTANT INDUCE FAS APO-1 EXPRESSION/

Fas/APO-1 is a cell surface protein known to trigger apoptosis upon sp ecific antibody engagement. Because wild-type p53 can activate transcr iption as well as induce apoptosis, we queried whether p53 might upreg ulate Fas/APO-1. To explore this possibility, we examined human p53-nu ll (H358 non-small-cell lung adenocarcinoma and K562 erythroleukemia) and wild-type p53-containing (H460 non-small-cell lung adenocarcinoma) cell lines. When H358 or H460 cells were transduced with a replicatio n-deficient adenovirus expression construct containing the human wild- type p53 gene but not with vector alone, a marked upregulation (ap pro ximately a three- to fourfold increase) of cell surface Fas/APO-1 was observed by flow cytometry. Similarly, K562 cells stably transfected w ith a plasmid vector containing the temperature-sensitive human p53 mu tant Ala-143 demonstrated a four- to sixfold upregulation of Fas/APO-1 by flow-cytometric analysis at the permissive temperature of 32.5 deg rees C. Temperature-sensitive upregulation of Fas/APO-1 in K562 Ala-14 3 cells was verified by immunoprecipitation and demonstrated to result from enhanced mRNA production by nuclear run-on and Northern (RNA) an alyses. Stably transfected K562 cells expressing temperature-insensiti ve, transcriptionally inactive p53 mutants (His-175, Trp-248, His-273, or Gly-281) failed to upregulate Fas/APO-1 at either 32.5 degrees or 37.5 degrees C. The temperature-sensitive transcription of Fas/APO-1 o ccurred in the presence of cycloheximide, indicating that de novo prot ein synthesis was not required and suggested a direct involvement of p 53. Collectively, these observations argue that Fas/APO-1 is a target gene for transcriptional activation by p53.