Genetic and epigenetic changes in p21 and p21B do not correlate with resistance to doxorubicin or mitomycin and 5-fluorouracil in locally advanced breast cancer.
OBJECTIVE
The cyclin-dependent kinase inhibitor p21 acts as a main executor of p53-induced growth arrest. Recently, a second transcript, p21B, was found to code for a protein expressing proapoptotic activity. We investigated p21 and p21B for mutations and epigenetic silencing in locally advanced breast cancers treated with doxorubicin or 5-fluorouracil/mitomycin and correlated our findings with treatment response and TP53 status.


METHODS
We used reverse transcription-PCR to analyze p21/p21B mutation status in 73 breast cancer samples. The p21 promoter region was sequenced and analyzed for hypermethylations by methylation-specific PCR. In addition, a selection of patients were analyzed for mutations in the p21B promoter.


RESULTS
The p21 gene was neither mutated nor silenced by promoter hypermethylation in any of the tumors examined. One patient harbored a novel p21 splice variant in addition to the wild-type transcript. We observed two base substitutions in the p21 transcript, C93A and G251A, each affecting six patients (8.2%). The G251A variant had not been reported previously. In 12 patients (16.4%), we observed a novel base substitution, T35C, in p21B. All three base substitutions were observed in lymphocyte DNA and therefore considered polymorphisms. The polymorphisms did not correlate with p21 staining index, treatment response to doxorubicin or 5-fluorouracil/mitomycin, or TP53 status.


CONCLUSIONS
Our findings do not suggest that genetic or epigenetic disturbances in p21 or p21B cause resistance to doxorubicin or mitomycin/5-fluorouracil in breast cancer. Future studies should assess potential associations between these novel polymorphisms and breast cancer risk.
Staalesen
Vidar
V
Department of Medicine, Section of Oncology, Haukeland University Hospital, Bergen, Norway.
Leirvaag
Beryl
B
Lillehaug
Johan Richard
JR
Lønning
Per Eystein
PE
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D019175:DNA Methylation; D017931:DNA Primers / Q000737:chemistry; D004317:Doxorubicin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm; D005472:Fluorouracil / Q000494:pharmacology*; D020868:Gene Silencing; D006801:Humans; D007150:Immunohistochemistry; D008214:Lymphocytes / Q000378:metabolism; D016685:Mitomycin / Q000494:pharmacology*; D008957:Models, Genetic; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D016283:Proto-Oncogene Proteins p21(ras) / Q000096:biosynthesis* / Q000235:genetics*; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D020668:rap GTP-Binding Proteins / Q000096:biosynthesis*
D000903:Antibiotics, Antineoplastic; D000964:Antimetabolites, Antineoplastic; D017931:DNA Primers; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D016685:Mitomycin; D012313:RNA; D004317:Doxorubicin; C065562:RAP2B protein, human; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D020668:rap GTP-Binding Proteins; D005472:Fluorouracil