PMID- 16061257
OWN - NLM
STAT- MEDLINE
DCOM- 20051006
LR  - 20081121
IS  - 0022-2836 (Print)
IS  - 0022-2836 (Linking)
VI  - 352
IP  - 1
DP  - 2005 Sep 9
TI  - SW480, a p53 double-mutant cell line retains proficiency for some p53 functions.
PG  - 44-57
AB  - During certain types of cellular stress, the p53 tumor suppressor protein binds 
      to DNA and transactivates a variety of genes that regulate critical responses 
      including apoptosis, cell cycle checkpoints, differentiation, and angiogenesis. 
      In addition, functional p53 is known to be required for efficient nucleotide 
      excision repair (NER) of bulky DNA adducts generated through exposure to 
      environmental mutagens such as UV light. Nonetheless, we previously showed that 
      the model p53-mutated human adenocarcinoma strain SW480 is proficient in the 
      removal of UV-induced cyclobutane pyrimidine dimers (CPD) via NER. We undertook 
      the present study to begin probing the molecular basis for this unexpected repair 
      phenotype. Cytogenetic analysis indicated that SW480 is stable at the chromosomal 
      level, i.e. manifests a karyotypic profile very similar to that revealed for this 
      line as far back as 14 years ago. After fluorescence in situ hybridization 
      (FISH), using a probe complementary to the p53 gene, we found that 98% of the 
      SW480 interphase nuclei contains three copies of the gene, later revealed to be 
      localized on intact short arms of three chromosomes 17. DNA sequence analysis 
      further showed that all three p53 copies in SW480 carry two point mutations 
      (R273H and P309S), and levels of the corresponding mutated p53 protein are about 
      20-fold higher than in the closely related p53 wild-type strain LoVo. Using an 
      electrophoretic mobility shift assay (EMSA), we demonstrated that R273H/P309S p53 
      is able to bind with wild-type affinity to its consensus DNA sequence in vitro. 
      Analysis of p21(Cip1/WAF1) expression and in vivo footprinting by 
      ligation-mediated PCR (LMPCR) showed that, in wild-type LoVo cells, an exposure 
      to cellular stress (e.g. UV or ionizing radiation) is necessary for p53 
      activation of the p21(Cip1/WAF1) promoter. In contrast, the R273H/P309S-mutated 
      p53 protein in SW480 constitutively activates p21(Cip1/WAF1) in the absence of 
      stress through an unknown mechanism. A similar phenomenon whereby mutated p53 in 
      SW480 is able to induce NER-related proteins might explain the normal DNA repair 
      phenotype previously observed in this strain. For now we conclude that, in 
      general, results obtained using SW480 as a p53-deficient cell line should be 
      interpreted very cautiously.
FAU - Rochette, Patrick J
AU  - Rochette PJ
AD  - Department of Medical Biology, Faculty of Medicine, Laval University and Unite de 
      Recherche en Genetique Humaine et Moleculaire, Research Center, Hopital 
      St-Francois d'Assise, Centre Hospitalier Universitaire de Quebec, Quebec, Canada 
      G1L 3L5.
FAU - Bastien, Nathalie
AU  - Bastien N
FAU - Lavoie, Josee
AU  - Lavoie J
FAU - Guerin, Sylvain L
AU  - Guerin SL
FAU - Drouin, Regen
AU  - Drouin R
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - Netherlands
TA  - J Mol Biol
JT  - Journal of molecular biology
JID - 2985088R
RN  - 0 (CDKN1A protein, human)
RN  - 0 (Cell Cycle Proteins)
RN  - 0 (Cyclin-Dependent Kinase Inhibitor p21)
RN  - 0 (Tumor Suppressor Protein p53)
SB  - IM
MH  - Cell Cycle Proteins/genetics/metabolism
MH  - *Cell Line, Tumor
MH  - Chromosomes, Human, Pair 17
MH  - Cyclin-Dependent Kinase Inhibitor p21
MH  - DNA Mutational Analysis
MH  - Gene Expression Regulation
MH  - Humans
MH  - In Situ Hybridization, Fluorescence
MH  - Karyotyping
MH  - Promoter Regions, Genetic
MH  - Tumor Suppressor Protein p53/*genetics/*metabolism
EDAT- 2005/08/03 09:00
MHDA- 2005/10/07 09:00
CRDT- 2005/08/03 09:00
PHST- 2004/12/01 00:00 [received]
PHST- 2005/06/08 00:00 [revised]
PHST- 2005/06/09 00:00 [accepted]
PHST- 2005/08/03 09:00 [pubmed]
PHST- 2005/10/07 09:00 [medline]
PHST- 2005/08/03 09:00 [entrez]
AID - S0022-2836(05)00695-9 [pii]
AID - 10.1016/j.jmb.2005.06.033 [doi]
PST - ppublish
SO  - J Mol Biol. 2005 Sep 9;352(1):44-57. doi: 10.1016/j.jmb.2005.06.033.