Leukemia 1997 Apr;11 Suppl 3:324-326
We have generated a series of murine erythroleukemia clones that ectopically express a temperature sensitive mutant p53 allele. In many clones, activation of p53 at low temperature resulted in the accumulation of cells in G1 and in apoptosis. Several cytokines including erythropoietin, IL-3 and the ligand for the Kit receptor blocked p53-dependent apoptosis in p53ts-expressing cells at 32 degrees C. Cytokine-treated cells were reversibly arrested in G1 and resumed growth upon return to 37 degrees C. Certain clones exhibited only a G1 arrest in response to p53 activation at 32 degrees C. One of the these clones secreted erythropoietin and another secreted IL-3. We tested the possibility that autocrine secretion of IL-3 played a role in preventing apoptosis and showed that disruption of the autocrine loop by cell dilution or with neutralizing antibodies to IL-3 restored p53-dependent apoptosis at 32 degrees C. Thus, two properties of p53 protein, namely, its ability to arrest cells in G1 and its ability to promote apoptosis could be uncoupled by cytokines acting as survival factors.
PMID: 9209379, UI: 97353121
Radiother Oncol 1996 Sep;40(3):197-223
BACKGROUND AND PURPOSE: Experimental studies have implicated the normal
or "wild type' p53 protein (i.e. WTp53) in the cellular response to ionizing
radiation and other DNA damaging agents. Whether altered WTp53 protein
function can lead to changes in cellular radiosensitivity and/or clinical
radiocurability remains an area of ongoing study. In this review, we describe
the potential implications of altered WTp53 protein function in normal
and tumour cells as it relates to clinical radiotherapy, and describe novel
treatment strategies designed to re-institute WTp53 protein function as
a means of sensitizing cells to ionizing radiation. METHODS AND MATERIALS:
A number of experimental and clinical studies are critically reviewed with
respect to the role of the p53 protein as a determinant of cellular oncogenesis,
genomic stability, apoptosis, DNA repair and radioresponse in normal and
transformed mammalian cells. RESULTS: In normal fibroblasts, exposure to
ionizing radiation leads to a G1 cell cycle delay (i.e. a "G1 checkpoint')
as a result of WTp53 mediated inhibition of G1-cyclin-kinase and retinoblastoma
(pRb) protein function. The G1 checkpoint response is absent in tumour
cells which express a mutant form of the p53 protein (i.e. MTp53), leading
to acquired radioresistance in vitro. Depending on the cell type studied,
this increase in cellular radiation survival can be mediated through decreased
radiation-induced apoptosis, or altered kinetics of the radiation-induced
G1 checkpoint. Recent biochemical studies support an indirect role for
the p53 protein in both nucleotide excision and recombinational DNA repair
pathways. However, based on clinicopathologic data, it remains unclear
as to whether WTp53 protein function can predict for human tumour radiocurability
and normal tissue radioresponse. CONCLUSIONS: Alterations in cell cycle
control secondary to aberrant WTp53 protein function may be clinically
significant if they lead to the acquisition of mutant cellular phenotypes,
including the radioresistant phenotype. Pre-clinical studies suggest that
these phenotypes may be reversed using adenovirus-mediated gene therapy
or pharmacologic strategies designed to re-institute WTp53 protein function.
Our analysis of the published data strongly argues for the use of functional
assays for the determination of WTp53 protein function in studies which
attempt to correlate normal and tumour tissue radioresponse with p53 genotype,
or p53 protein expression.
EMBO J 1996 Aug 15;15(16):4392-4401
In blast cells obtained from patients with acute myelogenous leukemia, p53 mRNA was present in all the samples examined while the expression of p53 protein was variable from patient to patient. Mutations in the p53 gene are infrequent in this disease and, hence, variable protein expression in the majority of the samples cannot be accounted for by mutation. In this study, we examined the regulation of p53 gene expression in human leukemic blasts and characterized the p53 transcripts in these cells. We found control both at the level of RNA abundance and at the level of translation. Four experiments point towards translational control of human p53 gene expression. First, there is no correlation between the level of p53 mRNA and the level of p53 protein expression in blast cells. Second, in two cell lines with similar levels of p53 protein expression but with different levels of p53 mRNA, we find that there is preferential association of p53 mRNA with large polysomes in the cells with less p53 RNA. Third, translation of synthetic human p53 transcripts in cell-free extracts is inhibited by the p53 3'UTR. Fourth, the p53 3'UTR, when present in cis, can repress translation of a heterologous transcript. These observations raise the possibility that human p53 mRNA translation may be regulated in vivo by RNA binding factors acting on the p53 3'UTR.
PMID: 8861966, UI: 97015138
PURPOSE: Changes in wild-type p53 protein function occur in the majority
of human tumors, and may alter genomic stability and the cellular response
to ionizing radiation. Whether oncoproteins can render tumor cells both
radioresistant and metastatic, may have implications for clinical strategies
designed to improve local tumor control. In the studies reported here,
we tested the hypothesis that acquired radioresistance correlates with
metastatic potential within a large panel of transformed rat embryo cell
(REF) lines following transfection with activated H-ras, mutant p53, and
HPV16-E7 alleles. METHODS AND MATERIALS: Rat embryo cells (REF cells) were
transfected using the calcium-phosphate technique with an activated H-ras
gene alone, or in combination with human papillomavirus HPV16-E7 and/or
human or murine mutant p53 sequences. Other rat embryo cell clones expressing
transfected HPV-E7 and activated ras sequences subsequently acquired endogenous
p53 gene mutations during culture in vitro. The relative expression of
p21ras and p53 protein for each REF transformant was determined by Western
blot analysis following transfection. REF clones were phenotypically characterized
at early passage (i.e., passages 5-7) and late passage (i.e., passages
10-20) for their: (a) relative tumor growth rate, and (b) their ability
to undergo spontaneous metastasis following intramuscular injection into
the hind legs of SCID mice. In vivo phenotypic end points were then compared
to previously measured parameters of in vitro radiosensitivity for each
cell line. Additionally, the expression of the cellular protease, plasminogen
activator, was determined for a number of metastatic and nonmetastatic
cell lines. RESULTS: We found no evidence that selected oncogene-transfected
REF transformants that were radioresistant in culture had a greater spontaneous
metastatic potential than nonradioresistant REF transformants. Neither
the level of expression of the p21ras protein nor that of the p53 protein
was correlated with the spontaneous metastatic phenotype when tested at
early passage. The metastatic phenotype appeared to be independent of p53
genotype. The majority of metastatic REF clones tested (7 out of 9 clones)
expressed plasminogen activator following oncogene transfection, in contrast
to nonmetastatic REF transformed cell lines. CONCLUSIONS: Our results suggest
that (a) intrinsic radioresistance does not correlate with spontaneous
metastatic potential in oncogene-expressing REF transformant cell lines,
and (b), novel clinical strategies designed to overcome oncogene-mediated
radioresistance could potentially impact on overall survival, as gains
in local tumor control may not be offset by a greater risk of distant metastasis.