Induction of p21WAF1 expression protects HT29 colon cancer cells from apoptosis induced by cryoinjury.
BACKGROUND
Cryotherapy is a method of in situ destruction of tumors by freeze/thaw mechanisms. Cancer cells located in the peripheral zone of the tumor undergoing cryotherapy can die by apoptosis. We hypothesized that p21WAF1 is involved in the mediation of cryotherapy-induced apoptosis.


METHODS
HT29 cells grown on a plate were subjected to -10 degrees C and returned to 37 degrees C for various periods of time. Cells were analyzed by flow cytometry, Western blot, and reverse transcriptase-polymerase chain reaction for determining cell-cycle distribution, p21WAF1 protein expression, and messenger RNA levels, respectively. The p21WAF1 expression in nude mouse tumor xenografts after cryotherapy was examined by immunofluorescence staining. A series of the p21WAF1 promoter cloned into a luciferase reporter vector were transfected into HT29 cells for identifying the response element to cryoinjury. Antisense oligodeoxynucleotide (ODN) was applied to examine the effect of p21WAF1 expression on cryotherapy-induced apoptosis.


RESULTS
Both protein and messenger RNA of p21WAF1 were induced by cryoinjury in cultured cells and tumor xenografts. Deletion analysis of the p21WAF1 promoter revealed that a region from -121 to -95 base pairs was responsible for the activation and that this activation was p53 independent. HT29 cells arrested at the G1 phase after cryoinjury. The cryotherapy-induced apoptotic rate in HT29 cells was increased in the presence of antisense p21WAF1 ODN in comparison to the random ODN.


CONCLUSIONS
Induction of p21WAF1 increases tumor cell survival and may result in recurrences at treated sites after cryotherapy. Combining antisense ODN targeted against p21WAF1 and cryotherapy may improve clinical outcomes in the treatment of colorectal cancer.
Sharma
Rohit R
RR
Department of Surgery, Montefiore Medical Center, 3400 Bainbridge Avenue, Bronx, New York, 10467, USA.
Ravikumar
T S
TS
Raimo
Derreck
D
Yang
Weng-Lang
WL
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D017679:Cryotherapy; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000096:biosynthesis*; D015316:Genetic Therapy / Q000379:methods*; D019073:HT29 Cells; D006801:Humans; D020319:Oligodeoxyribonucleotides, Antisense / Q000627:therapeutic use*
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D020319:Oligodeoxyribonucleotides, Antisense