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Induction of p21^WAF1 Expression Protects HT29 Colon Cancer Cells From Apoptosis Induced by Cryoinjury

¹ Department of Surgery, Montefiore Medical Center, 3400 Bainbridge Avenue, Bronx, New York 10467
² Department of Surgery, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461

Correspondence: Address correspondence and reprint requests to: Weng-Lang Yang, PhD; E-mail: wlyang{at}nshs.edu.

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 p21^WAF1 is involved in the mediation of cryotherapy-induced apoptosis.

Methods: HT29 cells grown on a plate were subjected to –10°C and returned to 37°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, p21^WAF1 protein expression, and messenger RNA levels, respectively. The p21^WAF1 expression in nude mouse tumor xenografts after cryotherapy was examined by immunofluorescence staining. A series of the p21^WAF1 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 p21^WAF1 expression on cryotherapy-induced apoptosis.

Results: Both protein and messenger RNA of p21^WAF1 were induced by cryoinjury in cultured cells and tumor xenografts. Deletion analysis of the p21^WAF1 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 p21^WAF1 ODN in comparison to the random ODN.

Conclusions: Induction of p21^WAF1 increases tumor cell survival and may result in recurrences at treated sites after cryotherapy. Combining antisense ODN targeted against p21^WAF1 and cryotherapy may improve clinical outcomes in the treatment of colorectal cancer.

Key Words: Cryotherapy • Apoptosis • Colon cancer • p21^WAF1 • Antisense