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10.1245/ASO.2005.11.021
Annals of Surgical Oncology 12:743-752 (2005)
© 2005 Society of Surgical Oncology
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Original Article

Induction of p21WAF1 Expression Protects HT29 Colon Cancer Cells From Apoptosis Induced by Cryoinjury

Rohit R. Sharma, MD1, T. S. Ravikumar, MD1,2, Derreck Raimo, MD2 and Weng-Lang Yang, PhD2

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.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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°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, 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.

Key Words: Cryotherapy • Apoptosis • Colon cancer • p21WAF1 • Antisense


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cryotherapy is an effective regional intervention in the treatment of colorectal cancer metastases confined to the liver, especially in patients not considered candidates for curative resection.1,2 In addition to the treatment of liver tumors, cryotherapy has been applied to treat a variety of solid tumors, such as those in the kidney, prostate, and breast.3–5 Cryotherapy causes tumor cell necrosis by the induction of cellular dehydration, the formation of damaging intracellular ice crystals, the alteration of intracellular pH, and the osmotic lysis of cells and from delayed ischemia produced by the disruption of vascular structures.6 Within the iceball, a temperature gradient develops from –170°C at the center (the site of cryogen delivery by the probe) to 0°C at the periphery of the lesion.7 Cells in the central and intermediate zones undergo death by necrosis at temperatures colder than –50°C, but cells in the peripheral zone, where the temperature ranges from 0°C to –40°C, may be only partially damaged.6 Tumor cells located in the peripheral zone that survive cryotherapy contribute significantly to treated site recurrence and subsequent metastasis.2,8

Our previous studies demonstrated that human colon cancer cells could die by apoptosis under sublethal cryoinjury conditions corresponding to the peripheral zone of 0° to –40°C during cryotherapy.9,10 Therefore, the efficacy of cryotherapy will be affected by the differential regulation of apoptosis machinery in cancer cells. In other words, recurrence after cryotherapy may be a function of the presence of cancer cells in the sublethal zone that possess a phenotype resistant to cryotherapy-induced apoptosis. Identification of the cellular events that antagonize cryotherapy-induced apoptosis in these cancer cells is essential for improving their response to cryotherapy.

Expression of p21WAF1, a universal inhibitor of cyclin-dependent kinases, has been found to increase after exposure to a wide variety of stress agents, including genotoxins, oxidants, and metabolic perturbations. p21WAF1 inhibits the activity of cyclin/ cyclin-dependent kinase complexes and consequently plays an important role in the cell-cycle arrest events that accompany exposure to such insults.11 The fundamental purpose of this cell-cycle block is to preserve the fidelity of DNA replication by giving the cell the opportunity to repair damaged DNA before the next cell-cycle phase.12 In addition to its role in checkpoint regulation, there is accumulating evidence that p21WAF1 may have a significant effect on the response to apoptosis induced by cytotoxic agents in cancer cells. For example, p21WAF1 could function as a survival factor by demonstrating that a defective p21WAF1 response can lead human colon cancer cells exposed to DNA-damaging drugs or radiation to undergo apoptosis.13,14 Similarly, ectopic expression of p21WAF1 in RKO colorectal carcinoma cells was found to confer protection against cyclopentenone prostaglandin A2–mediated cell death.15 In contrast, expression of p21WAF1 can also induce apoptosis in some other cell types under certain stress conditions.16 p21WAF1 was originally identified as a downstream target of p53 in response to DNA damage17; however, its expression can also be up-regulated in a p53-independent manner by various stimuli, including transforming growth factor ß,18 progesterone,19 and nerve growth factor.20

In this study, we investigated the role of p21WAF1 in cryotherapy-induced apoptosis in human colon cancer cells. We first examined the effect of sublethal cryotherapy on the expression of p21WAF1 in cultured HT29 human colon cancer cells and in in vivo nude mouse human tumor xenografts. We then analyzed the localization of p21WAF1 in cryotherapy-treated cells. We also identified the response element in the p21WAF1 promoter for its transactivation by cryoinjury. Finally, we tested the effect of inhibiting p21WAF1 expression on cryotherapy-induced apoptosis in HT29 cells by using an antisense oligodeoxynucleotide (ODN) approach.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture
HT29 human colon carcinoma cells obtained from the American Type Culture Collection were cultured in McCoy’s 5A medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (Gemini Bioproducts, Calabasas, CA) and supplemented with penicillin and streptomycin. Cells were maintained in a humidified incubator at 37°C and 5% carbon dioxide.

Sublethal Cryotreatment to Cultured Cells
HT29 cells were subjected to cryotreatment as described previously.9,10 Briefly, cells grown on 60-mm dishes were subjected to a final freezing temperature of –10°C in a cryogenic chamber at a freezing rate of 20°C/min, controlled by a Forma Scientific (Marietta, OH) Model 1010 Controlled Rate Freezing System. After freezing treatment, cells were thawed at room temperature for 30 minutes (assigned as the 0-hour time point), washed with phosphate-buffered saline (PBS) to remove the cryolytic cells, and returned to a 37°C incubator with fresh medium. Floating and adherent cells were harvested together after rewarming at 37°C for various time periods.

In Vivo Cryotreatment
National Cancer Institute (Frederick, MD) nude athymic mice were subcutaneously inoculated with HT29 cells (1 x 106 cells). When tumors had grown to 1 to 2 cm3, they were exposed surgically and subjected to cryotherapy. The cryosurgical instrument used in this study was a Cryogun (Brymill Cryogenic Systems, Ellington, CT), which can produce atomized liquid nitrogen spray with a high liquid content. During cryotherapy, liquid nitrogen (–196°C) was sprayed directly on to one end of the tumor while a thermocouple placed at the opposite end of the tumor monitored the temperature. The operation was stopped immediately once the peripherally placed thermocouple registered –10°C. After thawing at room temperature, the skin was sutured over the tumor, and the mouse was allowed to recover in a warm pocket. Tumors subjected to the same procedure, but without cryotherapy, served as sham controls. Mice were anesthetized with isoflurane during the entire surgical procedure. Tumors were harvested from the mice at 4 hours after cryotherapy. The animal procedure used in this study was approved by the Animal Care and Use Committee of the Albert Einstein College of Medicine.

Flow Cytometric Analysis of Cell Cycle and Apoptosis
The harvested cells were fixed in 75% cold ethanol. The fixed cells were washed with PBS, incubated in PBS containing ribonuclease (100 µg/mL) and propidium iodide (10 µg/mL), and then subjected to a FACScan flow cytometer (Becton Dickinson, San Jose, CA). A total of 10,000 events were collected per sample. Data acquisition and cell-cycle analysis was performed with CellQuest (Becton Dickinson, San Jose, CA) software.

Western Blot Analysis
Cells were lysed on ice for 30 minutes in radio-immunoprecipitation assay buffer (10 mM of Tris-HCl [pH 7.5], 120 mM of NaCl, 1% NP–40, 1% sodium deoxycholate, and .1% sodium dodecyl sulfate) containing a protease inhibitor cocktail (Sigma, St. Louis, MO) and then centrifuged at 10,000 x g for 10 minutes. A Bio-Rad (Hercules, CA) protein assay was used to determine the protein concentration. Protein was electrophoresed on sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk in Tris-Tween–buffered saline buffer (.1% Tween–20, 20 mM of Tris-HCl [pH 7.5], and 140 mM of NaCl). Membranes were incubated with primary antibody against p21WAF1 (Oncogene, San Diego, CA), poly(adenosine diphosphate-ribose) polymerase (PARP; Pharmingen, San Diego, CA), PARP p85 fragment (Promega, Madison, WI), p27KIP1, or actin (Santa Cruz Biotechnology, Santa Cruz, CA), followed by secondary antibody/horseradish peroxidase conjugate (Pierce, Rockford, IL) and detected by using chemiluminescence (Pierce) and autoradiography.

Immunofluorescence Staining
Tumors dissected from mice were fixed with 10% neutral buffered formalin and embedded in paraMn. Tumor sections were permeabilized with .1% Triton X- 100, blocked with 5% bovine serum albumin, and incubated with a mouse monoclonal antibody against p21WAF1 conjugated with fluorescein (Ab-1; Oncogene). This antibody is specific for human p21WAF1 and does not react with mouse p21WAF1. The slides were then mounted with Vectashield mounting medium containing 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA) as a counter-stain. The images were taken by a digital camera with a fluorescent microscope for the same field in both p21WAF1 and DAPI staining.

Cytoplasmic and Nuclear Fractionation
Cells were pelleted and resuspended in buffer containing 10 mM of HEPES/KOH (pH 7.9), 1.5 mM of MgCl2, 10 mM of KCl, .5 mM of dithiothreitol, and a protease inhibitor cocktail. After 15 minutes on ice and centrifugation, the supernatant (cytoplasmic fraction) was collected and stored, and the pellet was resuspended in buffer containing 20 mM of HEPES/KOH (pH 7.9), 25% glycerol, 420 mM of NaCl, 1.5 mM of MgCl2, .2 mM of EDTA, .5 mM of dithiothreitol, and a protease inhibitor cocktail and incubated on ice for 20 minutes. After centrifugation, the supernatant (nuclear fraction) was recovered.

Reporter Gene Assays
A series of deletion mutants of the p21WAF1 promoter cloned into a luciferase reporter gene was previously described.21 The plasmids (5 µg) were transfected into HT29 cells by using Lipofectamine reagent (Invitrogen). Approximately 16 hours after transfection, cells were subjected to cryotreatment at a final sample temperature of –10°C. Cells were then harvested 24 hours later for the reporter assay. Luciferase activity was measured by using the luciferase assay reagent from Promega according to the specification of the manufacturer. As controls, 5 µg of a wild-type p53 expression plasmid (a gift from Dr. A. Levine, The Rockefeller University, New York, NY) was cotransfected with the p21WAF1 promoter constructs into HT29 cells without being subjected to cryotreatment.

Semiquantitative Reverse Transcriptase-Polymerase Chain Reaction Analysis
Total RNA was isolated from HT29 cells at various time points after cryotreatment by using an RNeasy kit (Qiagen, Valencia, CA). The integrity of the isolated RNA was examined by agarose gel electrophoresis. A total of .3 µg of isolated total RNA was subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) analysis by using the Qiagen OneStep RT-PCR kit. The RT reaction was performed at 50°C for 30 minutes, followed by an initial PCR activation step at 95°C for 15 minutes. The PCR conditions used were denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, and extension at 72°C for 1 minute; this was continued for 25 cycles, followed by a final step at 72°C for 10 minutes. ß-Actin was used as an internal control. The number of PCR cycles and amount of input total RNA for each product were determined after definition of the linear exponential portion of the amplification. Primers used for PCR were as follows: p21 forward, 5'-CCCAGTGGA-CAGCGAGCAGC-3'; p21 reverse, 5'-ACT-GCAGGCTTCCTGTGGGC-3'; ß-actin forward, 5'-GGCATCGTGATGGACTCC GG-3'; and ß-actin reverse, 5'-GCTGGAAGGTG GACAGCGA-3'. The PCR products were separated on a 1.6% aga-rose gel and stained with ethidium bromide. Bands were visualized via UV transillumination and recorded by Polaroid (Waltham, MA) photography. Semiquantitative levels of band intensity were determined by scanning densitometry and analysis by ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Antisense ODNs
The antisense phosphorothioate ODN against p21WAF1 (p21AS; 5'-TGTCATGCTGGTCTGCCG GC-3') that is complementary to the 3' end of the noncoding region22 and the random control ODN (RD; 5'-CCGGTGAACGAGCGAGCACA-3') were transfected into cells by using Lipofectamine reagent from Invitrogen. Approximately 24 hours after transfection, cells were subjected to cryotreatment at a final temperature of –10°C and were recovered at 37°C for 4 and 10 hours, followed by Western blot and flow cytometry analyses, respectively.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Induction of p21WAF1 Protein Expression by Cryoinjury in Cultured HT29 Cells
We first examined the protein-expression levels of p21WAF1 in HT29 cells after cryoinjury. The conditions were analogous to those in tumor cells at the peripheral zone of cryoinjury during clinical treatment of tumors. Cells were subjected to a freezing temperature of –10°C, thawed at room temperature for 30 minutes, and returned to a 37°C incubator. Cells were then harvested at different time points after incubation up to 10 hours and analyzed by Western blot. As shown in Fig. 1AGo, the basal levels of p21WAF1 were barely detectable because of the p53 mutation in HT29 cells. The p21WAF1 could be seen at 1 hour, reached its maximum expression at 4 hours, and declined its expression at 8 hours. In contrast, the expression levels of p27KIP1, a cyclin-dependent kinase inhibitor in the same family as p21WAF1, were not altered after cryoinjury.


Figure 1
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  		FIG. 1. Protein expression and localization of p21WAF1 after cryoinjury. HT29 cells were subjected to cryotreatment at –10°C. (A) Total cell lysate was isolated from the cryotherapy-treated cells at the indicated time points after cryotreatment. A total of 50 µg of cell lysate was subjected to Western blot analysis against the indicated antibodies. U, untreated cells; 0 hours, right after thawing at room temperature for 30 minutes. (B) Four hours after cryotreatment, cell lysate was fractionated into nuclear and cytoplasmic portions and subjected to Western blot analysis against the indicated antibodies. Unt., untreated cells; N, nucleus fraction; C, cytoplasmic fraction; PARP, poly(adenosine diphosphateribose) polymerase.

 
Recent studies have shown that p21WAF1 can accumulate in the cytoplasm to block caspase activation. We therefore examined the localization of the cryotherapy-induced p21WAF1 by biochemical cellular fractionation in cultured HT29 cells. Cell lysates were isolated from cells subjected to –10°C followed by a 4-hour rewarming period. After cryotreatment, the induced p21WAF1 was present in both the nucleus and the cytoplasm (Fig. 1BGo). The amount of p21WAF1 in the cytoplasmic fraction was much higher than in the fraction isolated from the nucleus (Fig. 1BGo). PARP was used as a nuclear marker to indicate that the cytoplasmic isolate was not contaminated during fractionation.

Induction of p21WAF1 Protein Expression by Cryoinjury In Vivo
In our previous study, we demonstrated that HT29 cells located at the –10°C region could die by apoptosis when a tumor was subjected to cryotherapy by using a nude mouse tumor xenograft model.10 We further tested whether p21WAF1 could be induced by cryoinjury in tumor xenografts as in culture cells. Four hours after cryotreatment, p21WAF1 expression in the tumor was analyzed by immunofluorescence staining. The nuclear location of these tumor cells was identified by DAPI staining. As in culture cells, p21WAF1 expression was detected in tumor cells located at the far end of the liquid nitrogen–sprayed site, whereas it was undetectable in the untreated tumor cells (Fig. 2Go). By merging the images of p21WAF1 and DAPI staining, we observed that the cryotherapy-induced p21WAF1 was mainly located in the cytoplasm (Fig. 2Go). Taken together, the results from both in vitro and in vivo indicate that cryoinjury can induce p21WAF1 protein expression in the cytoplasm during the early recovery period in HT29 cells.


Figure 2
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  		FIG. 2. Expression of p21WAF1 in mouse tumor xenografts after cryotreatment. Tumor sections from mouse untreated (A) or cryotherapy-treated (B) tumor xenografts at 4 hours after cryotherapy were stained with 4',6-diamidino-2-phenylindole (DAPI; blue) and p21WAF1 (green). FITC, fluorescein isothiocyanate.

 
Activation of p21WAF1 Transcript by Cryoinjury
We next examined whether the induction of p21WAF1 protein expression was regulated at the transcriptional level. After cryotreatment, the total RNAs of the cultured HT29 cells at different time points were isolated, and their p21WAF1 messenger RNA (mRNA) levels were determined by semiquantitative RT-PCR analysis. As shown in Fig. 3AGo, the p21WAF1 mRNA level increased to 2.4-fold at .5 hours and to 4.5-fold at 1 hour and started to decrease at 8 hours after cryotreatment.


Figure 3
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  		FIG. 3. Transcript levels of p21WAF1 after cryoinjury. (A) HT29 cells were subjected to cryotreatment at –10°C. The isolated RNAs were subjected to RT-PCR analysis as described in Methods. The 4-fold normalized transcript levels of p21WAF1 in untreated cells were used as the baseline; data were averaged from two independent experiments. U, untreated cells; H, total RNA isolated from HCT116 cells; M, 100–base pair (bp) DNA marker. (B) Deletion analysis of the p21WAF1 promoter in HT29 cells subjected to cryotreatment at –10°C. p21WAF1 full-length and deletion promoter constructs were transfected into HT29 cells. Luciferase activities were normalized for cellular protein concentration. As a control, a wild-type p53 expression plasmid was cotransfected with the p21WAF1 promoter constructs into HT29 cells without cryo-treatment. TATA represents the p21WAF1 TATA box located 45 bp from the transcription start site (defined as +1). The 5' boundaries of the reporters are indicated to the left of each construct, and the entire constructs shown share the same 3' boundary located at +16 bp downstream of the p21WAF1 transcription-initiation site. S1 and S2 indicate p53 binding sites. The data represent three independent experiments. RLU, relative light units.

 
After identifying that cryostress can activate the transcription of p21WAF1, we then performed the deletion analysis of its promoter by using a series of deletion mutants cloned into a luciferase reporter vector, to identify a region responsible for the activation. The deletion constructs were transfected into HT29 cells and then subjected to cryotreatment at –10°C. As shown in Fig. 3BGo, the luciferase activity of p21–121, which contains just 121 base pairs (bp) of the p21WAF1 5'-flanking sequence, was activated by cryoinjury to a degree similar to that of p21–2300. However, the deletion construct containing p21–94 lost its luciferase activity after cryoinjury. This comparison indicates that the responsiveness to cryoinjury is conferred by sequences between –121 and –95 bp of the p21WAF1 promoter. We also conducted a control experiment by cotransfection of a wild-type p53 expression plasmid with the luciferase reporter plasmid containing the p21WAF1 promoter. As expected, p53 can activate the luciferase activity of the plasmid containing the full-length p21WAF1 promoter (p21–2300), yet it is unable to trigger the luciferase activity in a deletion mutant of the p21WAF1 promoter up to –1200 bp (p21–1200; Fig. 3BGo). This control experiment verified the location of the p53 response elements in the p21WAF1 promoter as described previously23 and confirmed that p21–1200 lacks the two p53 binding sites yet was strongly activated by cryoinjury. These results indicate that cryoinjury activated the transcription of p21WAF1 in a p53-independent pathway, consistent with p21WAF1 induction in HT29 cells with mutant p53.

Cryoinjury Causes G1 Phase Arrest
A well-identified function of p21WAF1 is to regulate cell-cycle progression; therefore, we examined the effect of cryoinjury on cell-cycle distribution. After cryotreatment at –10°C, HT29 cells were harvested at different time points after recovery at 37°C. At 12 hours, 61.11% of the cryotherapy-treated cells were in the G1 phase, whereas only 27.40% of control cells were in the G1 phase (Fig. 4Go). To further confirm the retention of cells at the G1 phase by cryoinjury, we exposed control and cryotherapy-treated cells to nocodazole. Nocodazole causes cell-cycle arrest at the G2/M phase and prevents cells from entering the next G1 phase cycle. Comparing the cell-cycle phase distributions between untreated and cryotherapy-treated cells in the presence of nocodazole makes it easier to determine the effect of cryoinjury on the retention of cells in the G1 phase. We observed a delay in the cryotherapy-treated cells entering the G2/M phase compared with control cells (Fig. 5Go). At 24 hours, 74.56% of control cells were in the G2/M phase, but only 55.22% of cryotherapy-treated cells were in the G2/M phase. These results indicate that cryostress can cause HT29 cells to arrest at the G1 phase.


Figure 4
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  		FIG. 4. Effect of antisense oligodeoxynucleotide (ODN) on cryotherapy-induced p21WAF1 expression. Cells were transfected with p21 antisense or random control ODN before cryotreatment as described in Methods. Four hours after cryotreatment, cell lysate was isolated and subjected to Western blot analysis against p21WAF1 antibody. AS, cells treated with p21 antisense ODN; RD, cells treated with random control ODN.

 

Figure 5
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  		FIG. 5. Effect of cryoinjury on cell-cycle distribution. HT29 cells were subjected to cryotreatment at –10°C. Cells were harvested in the presence or absence of nocodazole (.4 µg/mL) at the indicated time points. Flow cytometry with propidium iodide staining was used for cell-cycle analysis. 0 hours, right after thawing at room temperature for 30 minutes. The data represent three independent experiments.

 
Increased Cryotherapy-Induced Apoptosis by Inhibition of p21WAF1 Expression
The function of p21WAF1 is not only to delay cell-cycle progression, but also to regulate apoptosis in cells exposed to a variety of stressors. We further elucidated the role of p21WAF1 expression in cryotherapy-induced apoptosis by a genetic approach. Because our studies showed that the cryotherapy-induced p21WAF1 was regulated at transcriptional levels, we chose antisense technology to inhibit p21WAF1 production. HT29 cells were transfected with either a p21AS ODN or a random ODN before cryotreatment. As shown in Fig. 4Go, p21AS at a concentration of 400 nM dramatically inhibited the induction of p21WAF1 protein by cryoinjury, whereas the RD showed no alteration in p21WAF1 expression in comparison to control cells without ODN transfection.

Subsequently, we determined the apoptotic rate of these cells by using flow cytometry analysis 10 hours after recovery at 37°C from cryotreatment. The apoptosis rates for p21AS ODN, RD ODN, and nontransfected cells were 31.67%, 14.15%, and 13.91%, respectively, whereas the rate was only 3.60% for the control cells without cryotreatment (Fig. 6AGo). We further confirmed the cryotherapy-induced apoptosis in these ODN-treated cells by using Western blot analysis to detect the cleavage of PARP (Fig. 6BGo). The anti-PARP p85 fragment antibody specifically recognizes the 85-kDa degradation product from 116-kDa PARP, generated by caspase activation during apoptosis.24 The expression of PARP-degraded product was very weak in the control cells, whereas its expression was induced by cryotreatment (Fig. 6BGo). The intensity of 85-kDa PARP in p21AS ODN–treated cells was stronger than that in RD ODN–treated and only cryotherapy–treated cells (Fig. 6BGo). These results indicate that inhibition of p21WAF1 expression can enhance the cryotherapy-induced apoptosis in HT29 cells.


Figure 6
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  		FIG. 6. Effect of p21WAF1 antisense oligodeoxynucleotide (ODN) on cryotherapy-induced apoptosis. Cells were transfected with 400 nM of p21 antisense or random control ODN before cryotreatment as described in Methods. (A) Ten hours after cryotreatment, cells were harvested and subjected to flow cytometric analysis with propidium iodide staining. (B) Four hours after cryotreatment, cell lysate was isolated and subjected to Western blot analysis against 4',6-diamidino-2-phenylindole (PARP) p85 antibody. a, untreated control cells; b, cryotherapy-treated cells; c, cryotherapy-treated cells with p21 antisense ODN; d, cryotherapy-treated cells with random control ODN. The data represent two independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Apoptosis is a tightly regulated process essential to maintaining tissue homeostasis. Acquisition of a phenotype resistant to apoptosis plays an important role in tumorigenesis, because it disrupts the balance between cell proliferation and cell destruction and, in turn, confers the ability of cancer cells to resist various therapeutic interventions. In previous studies, using in vitro–cultured cells and an in vivo mouse model, we identified that apoptosis in human colon cancer cells occurred at sublethal freezing temperatures.9,10 Cryotherapy-induced apoptosis is associated with Bax translocation and cytochrome c release from the mitochondria, followed by activation of caspase 9 and caspase 3.9,10 Furthermore, the induction of apoptosis by cryoinjury does not require a functional p53 protein, and the intensity of cryotherapy-induced apoptosis does not correlate with the functional status of p53.10

To understand how colon cancer cells can escape from apoptosis during cryotherapy, we investigated the potential role of p21WAF1 in HT29 cells responding to cryoinjury. In kinetic analysis, we demonstrated that p21WAF1 expression was markedly induced by cryoinjury at both the protein and mRNA levels in cell culture. The induction of p21WAF1 by cryotherapy was further confirmed in vivo by using a nude mouse tumor xenograft model. We identified that a GC-rich region of the p21WAF1 promoter between –121 and –95 bp is responsible for its activation during cryoinjury. The facts that this region locates outsides of the p53 response elements in the p21WAF1 promoter and that HT29 cells carry mutant p53 indicate that the activation of p21WAF1 expression by cryoinjury is p53 independent. This GC-rich region in the p21WAF1 promoter contains two putative binding sites for Sp1 and one for the AP2 transcription factor.25 Sp1 is a member of a multigene family and binds DNA through C-terminal zinc-finger motifs.26 Sp1 not only acts as a basal transcription factor, but also is involved in the control of transcription after several different stimuli, such as oncogenes,27 antimetabolites,28 growth-stimulation signals,29 and differentiation factors.30 The AP2 consensus binding site is used to mediate the activation of p21WAF1 transcription in human leukemia cells treated with phorbol ester or okadaic acid.31 The exact mechanism of how cryostress induces the binding of Sp1 or AP2 on the p21WAF1 promoter needs to be further elucidated.

We then analyzed the cellular responses of HT29 cells with an increase of p21WAF1 expression after cryoinjury. We found that HT29 cells would arrest at the G1 phase after cryoinjury, which corresponds to a known survival mechanism for cells under stress to repair their damage before progressing through the cell cycle.32 We also demonstrated that inhibition of p21WAF1 expression by antisense ODN could enhance the apoptosis rate induced by cryoinjury in HT29 cells. The requirement for p21WAF1 in protecting cells from apoptosis induced by stress stimuli—such as radiation, chemotherapeutic drugs, and heat shock—has also been demonstrated in colon cancer cells by using isogenic cell lines.13 Taken together, the cellular responses of HT29 cells to cryoinjury are reflected by the activation of p21WAF1 expression.

Another observation is that a large amount of the cryotherapy-induced p21WAF1 protein accumulates in the cytoplasm (besides in the nucleus) after cryoinjury, as shown in the cultured cells and tumor xenografts. A well-established function of p21WAF1 is to inhibit cell-cycle progression. To exert this activity, p21WAF1 has to locate in the nucleus of cells, which has been demonstrated in fibroblasts and epithelial cells.33,34 In contrast, the caspase cascades for triggering apoptosis occur in the cytoplasm.35 Therefore, the cytoplasmic p21WAF1 induced by cryoinjury may interact with the apoptotic machinery to inhibit apoptosis. Indeed, it has been reported that p21WAF1 can associate with procaspase 3 on mitochondria and blocks the cleavage of this proenzyme during Fas-mediated apoptosis.36 Furthermore, during monocytic differentiation of U937 and HL60 cells stimulated by vitamin D3, cytoplasmic p21WAF1 expression is induced and forms a complex with apoptosis signal-regulating kinase 1, which results in inhibition of the stress-activated mitogen-activated protein kinase cascade and acquisition of resistance to various apoptogenic stimuli.37 The mechanism of preventing p21WAF1 translocation into the nucleus is still not clear. Zhou et al.38 demonstrated that Akt-mediated phosphorylation of p21WAF1 at Thr145, in the region of its nuclear localization signal, would cause p21WAF1 to lose its nuclear localization ability; however, this result could not be confirmed by other groups.39

In this study, we have demonstrated that the induction of p21WAF1 expression can protect colon cancer cells from undergoing apoptosis after cryoinjury. Therefore, p21WAF1 induction can be considered a survival mechanism against cryotherapy-induced cell death, thereby increasing the likelihood of recurrence after cryotherapy. To reduce the recurrence, the use of two freeze/thaw cycles for cryoablation has been applied to treat liver tumors in the clinical setting. Although our group has advocated two cycles, many other clinicians (personal observations and meeting discussions) perform only one freeze/thaw cycle. Because addressing recurrences in the clinical setting was the goal of this study, we used one freeze/thaw cycle for our experiments. Several antisense ODNs designed to inhibit the targeted gene expression have entered phase II or III clinical trials for cancer, such as Genasense (Genta Inc., Berkeley Heights, NJ) targeting BCL-2, ISIS 2503 (ISIS Pharmaceuticals, Carlsbad, CA) targeting Ha-ras, and Gem-231 (Hybridon Inc., Cambridge, MA) targeting protein kinase A.40 Our study indicates that a combination of cryotherapy with antisense ODNs targeted against p21WAF1 may be a valuable approach to enhance the overall efficacy of this modality and improve clinical outcomes.


   ACKNOWLEDGMENTS
 
Supported by the Albert Einstein Cancer Center and the Department of Surgery Research Fund. The authors thank Lixin Qi for technical assistance.

Received for publication November 17, 2004. Accepted for publication April 17, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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